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I Volume 33 Advances in Genetics m Incorporating Molecular Genetic Medicine Edited by Jeffrey C. Hall Jay C. Dunla...

Author: Jeffrey C. Hall | Jay C. Dunlap | Theodore Friedmann | Francesco Giannelli

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I

Volume 33

Advances in Genetics m

Incorporating Molecular Genetic Medicine

Edited by

Jeffrey C. Hall

Jay C. Dunlap

Department of Biology Brandeis University Waltham, Massachusetts

Department of Biochemistry Dartmouth Medical School Hanover, New Hampshire

Associate Editors Theodore Friedmann Department of Pediatrics Center for Molecular Genetics School of Medicine University of California, San Diego La Jolla, California

Francesco Giannelli Division of Medical and Molecular Genetics United Medical and Dental Schools of Guy’s and St. Thomas’ Hospital London Bridge, London SEI 9RT United Kingdom

Academic Press San Diego New York Boston London Sydney Tokyo Toronto

This book is printed on acid-free paper. @ Copyright 0 1995 by ACADEMIC PRESS, INC. All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher.

Academic Press, Inc.

A Division of Harcourt Brace & Company 525 B Street, Suite 1900, San Diego, California 92101-4495 United Kingdom Edition published by Academic Press Limited 24-28 Oval Road. London NW I 7DX International Standard Serial Number: 0065-2660 International Standard Book Number: 0-12-017633-5

PRINTED IN THE UNlTED STATES OF AMERICA 95

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Contributors Numbers in parentheses indicate the pages on which the authors’ contributions begin.

Jamel Chelly, Institut Cochin de G6nCtique Mol6culaire, INSERM Unit6 129, CHU Cochin-Port-Royal, 75014 Paris, France (233) Bjorn Dahlbiick, Department of Clinical Chemistry, University Hospital, S-20502 Malmo, Sweden (135) Peter Gruss, Department of Molecular Cell Biology, Max Planck Institute for Biophysical Chemistry, D-37077 Gottingen, Germany (255) Terry J. Hassold, Department of Genetics, Case Western Reserve University, Cleveland, Ohio 44106-4955 (101)

Patricia A. Jacobs, Wessex Regional Genetics Laboratory, Salisbury District Hospital, Salisbury, Wiltshire SP2 SBJ, United Kingdom (101) Robin J. Leach, Department of Cellular and Structural Biology, The University of Texas Health Science Center at San Antonio, San Antonio, Texas 78284 (63) Anthony P. Monaco, Imperial Cancer Research Fund Laboratories, Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford OX3 9DU, United Kingdom (233) Peter O’Connell, Department of Pathology, The University of Texas Health Science Center at San Antonio, San Antonio, Texas 78284 (63) Thomas D. Petes, Department of Biology and Curriculum in Genetics, University of North Carolina, Chapel Hill, North Carolina 27599-3280 (41) Patricia J. Pukkila, Department of Biology and Curriculum in Genetics, University of North Carolina, Chapel Hill, North Carolina 27599-3280

(41) Roland G. Roberts, Division of Medical and Molecular Genetics, United Medical and Dental Schools, London SE19RT, United Kingdom (177)

Alan J. Schafer, Department of Genetics, University of Cambridge, Cambridge CB2 3EH, United Kingdom (275) Edward T. Stuart, Department of Molecular Cell Biology, Max Planck Institute for Biophysical Chemistry, D-37077 Gottingen, Germany (255)

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Contributors

Kathleen L. Triman, Department of Biology, Franklin and Marshall College, Lancaster, Pennsylvania 17604 (1) Yoshifumi Yokota, Department of Molecular Cell Biology, Max Planck Institute for Biophysical Chemistry, D-37077 Gottingen, Germany (255)

Preface With Volume 33 of Advunces in Genetics we are pleased to continue the increased coverage of the field of human genetics initiated in the last volume. As ever, our job as editors is to identify and promote both breadth and quality of coverage within the general area of genetics. The tenets of genetics inform research into all biological systems, and the current volume exemplifies this, with coverage of organisms extending from bacteria to fungi to humans, and topics ranging from the genetics of 16s ribosomal RNA structure and function to the series of topical reviews on the genetics of human pathologies and dysmorphologies. As always, our goals for the Advunces in Genetics series remain the identification of emerging problems in genetics as they coalesce and the recruitment and promotion of contributions that are at the same time comprehensive and comprehensible, informed and informative, critical, insightful, and readable. We continue to believe that, despite today’s trend toward the breathless minireview, there remains a place in the realm of scholarship for the wellwritten, thoughtful, and broadly cast overview. We trust that the contents of the present volume justify this belief. Jeffrey C. Hall Jay C. Dunlap

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I

Mutational Analysis of 16s Ribosomal RNA Structure and Function in ischerichia coli

Kathleen 1. Triman

Department of Biology Franklin and Marshall College Lancaster, Pennsylvania 17604

1. INTRODUCTION The ribosome is responsible for the translation of the genetic code in all living organisms. The structural complexity of the ribosome presents an obstacle to the definition of the molecular mechanism of ribosome action. The Eschen'chia coli ribosome is the best-characterized system in which translation has been studied at the molecular level. The E. coli ribosome is a (70s) complex of RNA and protein composed of two subunits, the large (50s) subunit and the small (30s) subunit. The 50s subunit is composed of two RNA species, 23s ribosomal RNA and 5s ribosomal RNA, and 31 ribosomal proteins. The 30s subunit is composed of one species of RNA, 16s ribosomal RNA, and 21 ribosomal proteins (Sl, S2, etc.; see Riley, 1993, and references therein). Evidence from both biochemical and genetic approaches suggests that ribosomal RNA plays a functional role in the process of translation (reviewed in Nomura, 1987; Dahlberg, 1989; Noller et al., 1990; Leclerc and Brakier-Gingras, 1990; Firpo and Dahlberg, 1990; Noller, 1991, 1993; see also relevant chapters in Nierhaus et al., 1993, and in Zimmermann and Dahlberg, 1994). Genetic approaches have proved useful for the identification of new aspects of ribosomal RNA structure and function that are not accessible to study by biochemical methods alone. This review outlines genetic strategies designed to improve our understanding of the structure and function of 16s ribosomal RNA in E. coli. The general approach has been to investigate the effects of mutations introduced into rRNA genes.

Advances in Genelics, Yo/. 33 Copyright 0 1995 by Academic Press. Inc. All rights of reproduction in any form reserved

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Kathleen L. Triman

11. METHODS OF DETECTION OF rRNA MUTANTS IN €sclrerlchla coli Genetic analysis of the structure and function of ribosomal RNA has proved difficult, because ( i ) expression of rRNA genes is essential and (ii) there are seven copies of the rRNA genes in the E. coli genome (Lindahl and Zengel, 1986; Riley, 1993). Both of these challenges have been met by the use of plasmids containing a single copy of one of the seven operons (rrnA, rrnB, m C , m D , rmE, rrnG, and m H ) found in the genome.

A. Plasmid expression of rRNA mutations Plasmid pKK3535, a derivative of pBR322, is a high copy number plasmid containing the intact m B operon (Brosius et al., 1981a,b,c). Plasmid pLC7-21 is a recombinant plasmid that contains m H on a ColEl vehicle (Sigmund and Morgan, 1982). Other plasmids contain a copy of an rRNA operon under the control of an inducible promoter/operator, such as bacteriophage lambda pL (Gourse et al., 1985), permitting conditional rRNA expression in strains containing the temperature-sensitive c1857 repressor (Jacob et al., 1987; Thomas et al., 1988; Powers and Noller, 1990). Appropriate bacterial host strains can be used to maintain plasmids containing deleterious mutations at low copy number (O’Connor et al., 1992). Plasmids provide the opportunity to manipulate rRNA genes directly and, in some cases, to control expression of manipulated rRNA genes. The general genetic approach to the study of ribosomal RNA structure and function in E. coli has involved mutagenesis of plasmid rRNA genes (reviewed in De Stasio et al., 1988; Leclerc and Brakier-Gingras, 1990; Tapprich et al., 1990b).

1. In vivo expression Transformed cells can be grown under conditions in which both chromosomally encoded rRNA and plasmid-encoded rRNA are expressed. Mutations introduced by site-directed mutagenesis into plasmid rRNA genes may confer an altered growth phenotype demonstrating a dominant effect in transformed cells (e.g., Stark et a!., 1982; Zwieb and Dahlberg, 1984; Montandon et al., 1986; Jacob et al., 1987). Plasmid-derived rRNA containing a dominant mutation presumably interferes with the normal function of chromosomally encoded rRNA. In extreme cases, the defect may be a dominant lethal mutation, the expression of which causes cell death (e.g., Thomas et al., 1988; Powers and Noller, 1990; De Stasio and Dahlberg, 1990; Jemiolo et al., 1991; Santer et al., 1990). A special class of dominant mutations is represented by the conditional

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dominant which confers mutant growth properties, for example, at low temperature but not at higher temperatures (Dammel and Noller, 1993). Sigmund et al. (1988) selected a number of antibiotic resistance mutations in rmH by chemical mutagenesis of plasmid pLC7-21 and the use of media containing antibiotics. The detection of recessive rRNA mutations was made possible by the introduction of two of these selectable antibiotic resistance markers into plasmid rRNA. Plasmid pSTL102 (Triman et al., 1989) was constructed from pKK3535 by introduction of a spectinomycin resistance allele (C to U change at position 1192) into the 16s rRNA gene and an erythromycin resistance allele (A to G change at position 2058) into the 23s rRNA gene (Sigmund et al., 1984; Morgan et al., 1988). Mutations leading to loss of function of the cloned 16s rRNA gene cause loss of spectinomycin resistance (Spcr), and mutations in the cloned 23s rRNA gene will affect erythromycin resistance (Eryr). Erythromycin resistance can be used to control against transcriptional defects when 16s rRNA mutants are being sought. Recessive 16s rRNA mutant growth phenotypes can be detected only under conditions that select for spectinomycin resistance, whereas dominant mutant growth phenotypes can be detected in the absence of spectinomycin. Stark et al. (1982) developed a maxicell procedure for expression of plasmid-coded rRNA in the complete absence of host-coded rRNA synthesis. Maxicells are derived from strains of E. coli unable to repair UV light-damaged DNA; ribosomes isolated from maxicells containing mutagenized plasmids can be analyzed for the effects of specific mutations on rRNA processing, proteinrRNA interaction, and subunit assembly (Dahlberg, 1986; Jemiolo et al., 1988). Hui and DeBoer (1987) developed a unique system involving the use of specialized ribosomes. This in vivo system involves expression of mutant rRNA that also contains an altered anti-Shine-Delgarno sequence (e.g., 5’ GGAGG). When rRNA containing this altered sequence is transcribed and assembled into ribosomes, it can translate only specifically engineered mRNAs containing the complementary Shine-Delgarno sequence (e.g., 5’ CCUCC).

2. In vitro expression In vitro expression of mutant rRNA has been facilitated by the construction of plasmids in which the promoters normally used for transcription of the rrnB operon are replaced with a promoter for T7 RNA polymerase (Steen et al., 1986; Krzyzosiak et al., 1987; Melancon et al., 1987; Powers and Noller, 1991). These plasmids provide (i) a “silent” copy of the 16s rRNA gene to avoid potential deleterious physiological effects of mutant rRNA genes, and (ii) the opportunity for expression of mutant 16s rRNA in vitro using T7 RNA polymerase. Plasmid constructs containing DNA fragments corresponding to spe-

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cific regions of the 16s rRNA sequence under the control of the promoter of bacteriophage T7 polymerase have also been generated. In vitro transcription from these constructs produces synthetic RNA fragments suitable for use in filter-binding assays to study RNA-protein interactions or in conformational studies (e.g. , Cormack and Mackie, 1991; Dragon and Brakier-Gingras, 1993; Mougel et al., 1993; Weitzmann et al., 1993).

3. Allele-specific structural probing of plasmid-derived 16s rRNA Rapid chemical and enzymatic probing methods have permitted the identification of residues in rRNA that interact with ribosomal proteins, tRNA, elongation factors, and antibiotics (Noller et al., 1990; Noller, 1991). Biochemical characterization of mutant ribosomes in vitro has been hindered, however, by the fact that ribosomes isolated from cells are heterogeneous, containing both mutant plasmid-derived rRNA and wild-type chromosomally derived rRNA. Powers and Noller (1993a) have addressed this problem by constructing derivatives of plasmid pSTLlO2, each carrying one of four specific mutations, priming sites, that allow for selective probing of mutant ribosomes. Each of the mutations, introduced into a phylogenetically variable region of 16s rRNA, is phenotypically silent (Powers and Noller, 1993a). These specific priming site mutations are presented in the appropriate section of this chapter according to their localization in 16s rRNA.

6. Introduction of mutations

1. Random mutagenesis Sigmund and Morgan (1982) treated cells containing plasmid pLC7-21 in viw with methanesulfonic acid ethyl ester, plated them on media containing antibiotics, and succeeded in isolating a number of antibiotic-resistance mutations in rmH (Sigmund et d.,1988). These mutations included the C to U change at position 1192 of 16s rRNA that confers spectinomycin resistance (Sigmund et al., 1984). Treatment of plasmid pSTL102 with hydroxylamine yielded a number of mutants containing G to A or C to U alterations in rRNA (Douthwaite et al., 1985;Triman et al., 1989; Mori et al., 1990; Allen and Noller, 1991; Dammel and Noller, 1993). In each case mutants were identified among transformants containing mutagenized DNA by the particular growth phenotype associated with introduction of a specific alteration. The most convenient methods of detection of randomly introduced mutations involve (i) selection or screen for growth on plates containing antibiotics that specifically target rRNA [e.g., spectinomycin,

1. Mutational Analysis of 16s Ribosomal RNA

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erythromycin, or streptomycin (Cundliffe, 1987, 1990)]; or (ii) selection or screen for growth at extreme temperatures outside the optimum range for E. coli [e.g., 26 or 42°C (Ingraham, 1987)l. Identification of randomly introduced mutations requires a mapping technique, such as restriction fragment exchanges between mutant and wildtype plasmids, in order to ( i ) limit the region of DNA to be subjected to sequence analysis and (ii) rule out the presence of one or more secondary mutations.

2. Site-directed mutagenesis Methodologies for construction of deletion mutations, transition mutations, or oligonucleotide-directed mutations of 16s rRNA have been described in a review by Tapprich et al. (1990a).

a. Mutagenesis targeted to regions of 16s rRNA

Gourse et al. (1982) isolated the first site-directed rRNA mutants by limited exonuclease Bal-31 digestion from selected restriction sites in a plasmid. These mutants included some containing deletions at one of seven positions corresponding to bases 614, 704, 1384, and 1504 in 16s rRNA. Using a plasmid with a deletion between bases 822 and 874 in 16s rRNA, Zwieb and Dahlberg (1984) produced bisulfite mutations in plasmids; of 33 possible cytosine residues available for modification within the single-stranded region of the heteroduplex, only 5 were found to be altered. The inability to get base changes at other positions suggested that single alterations at particular positions could severely affect the formation of a functional ribosome (Dahlberg, 1986). Plasmid constructs containing specific DNA fragments corresponding to regions of 165 rRNA sequence under the control of the promoter of phage T7 polymerase have been utilized to obtain synthetic RNA fragments suitable for mutational analysis of RNA-protein complex formation and filter-binding assays. Examples of interactions defined by this strategy include the 16s rRNA binding sites for ribosomal proteins S7 (Dragon and Brakier-Gingras, 1993), S8 (Mougel et al., 1993), and S20 (Cormack and Mackie, 1991).

b. Mutagenesis targeted to a specific 16s rRNA base

The use of M13 constructs (e.g., Makosky and Dahlberg, 1987; Bonny et al., 1991; O’Connor et al., 1992) or phagemids derived from Bluescript (Stratagene) vectors permits preparation of 16s rDNA in single-stranded form for oligonucleotide-directed mutagenesis (e.g., Melancon et al., 1990; Powers and Noller, 1991; Pinard et al., 1993). The 16s rRNA gene can be manipulated in the niutagenesis plasmid and then transplanted into an expression vector.

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111. MUTATIONAL ANALYSIS OF 16s rRNA STRUCTURE AND FUNCTION Mutational analysis of the promoter region of the E. coli rrnB operon has been carried out by a number of groups. These studies of the control of expression of rRNA are beyond the scope of this chapter but the details can be found in the published work of Gaal et af. (1989), Zacharias et al. (1989, 1990, 1991), Leirmo and Course (1991), and Ross et al. (1993).

A. Secondary structure of 16s rRNA Figure 1.1 illustrates the higher-order structure diagram for E. coli 16s rRNA (from Gutell, 1993a, with permission). The 16s rRNA molecule is subdivided into three major structural domains and one minor domain by three sets of longrange base-paired interactions: (i) the 5' major domain contains residues 26557 and is defined by the 27-37/547-556 helix; (ii) the central domain contains residues 564-912 and is defined by the 547-5701880-886 helix; and (iii) the 3' major domain contains residues 926-1391, the 3' minor domain contains residues 1392-1541, and these two domains are defined by the 926-933/13841392 helix (Noller and Nomura, 1987). Examples of the results of mutational analysis of 16s rRNA structure and function presented in this chapter are organized according to the structural domain in which particular mutations are located.

1. Genetic evidence in support of secondary structure base pairings Seven recessive 16s rRNA alterations have been identified, each of which disrupts pairing at positions involved in G-C base pairs and results in the loss of expression of spectinomycin resistance in pSTLlO2 (Triman et al., 1989). These mutations were isolated following random mutagenesis of pSTLlO2, namely in vitro treatment of plasmid DNA with hydroxylamine. Transformants containing mutagenized plasrnid were screened for those which had lost spectinomycin resistance. The sites of mutation were localized to small regions of the 16s gene by restriction fragment exchange and identified by DNA sequence analysis. In every case, the mutation was 'a single base change (G to A or C to U) at a position that is base paired in the secondary structure of 16s rRNA. In two mutants (G359A or G146A) the alteration leads to unconditional sensitivity to the antibiotic spectinomycin (Spcs phenotype), whereas in five mutants (ClSSU, G350A, G538A, G1292A, or C1293U) the loss of resistance occurs only at elevated temperatures (Spct~phenotype). Five of the alterations replace

1. Mutational Analysis of 16s Ribosomal RNA

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Figure 1.1. Higher-order structure diagram for Eschrrichia coli 165 rRNA; reproduced by permission of R. R. Gutell and Oxford University Press (Fig. 1, Gutell, 1993a, p. 3052).

G-C base pairs with A / C mismatches; two G-C base pairs are replaced with G/U pairs. It was hypothesized that the mutant phenotypes observed might result from significant decreases in the predicted thermodynamic stability of some part of the 16s rRNA structure. The temperature sensitivity could he due to disruption of the RNA

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Kathleen 1. Triman

structure itself or to disruption of RNA-protein interactions. All but one (A538) of the base changes are located outside regions of 16s rRNA implicated in ribosome function. However, all the base changes are located in or near sites that interact with ribosomal proteins during in vitro assembly (Stem et al., 1989). For example, A1292 and U1293 are in the region that interacts with ribosomal protein S7, A350 is in the region that interacts with S16, and U153 is in the region that interacts with S20. The growth pattern for each of the temperaturesensitive mutants is characterized by a lag of about two cell doublings between the time of temperature shift and the time at which growth ceases. This growth pattern implies that the structural defects in these mutants result in impairment of ribosome assembly. Mutations affecting ribosome assembly are predicted to be recessive if they do not interfere with the function of normal ribosomes. The distribution of plasmid-encoded versus chromosomally encoded 16s rRNA can be measured by using cDNA analysis to quantitate rRNA containing U1192 (plasmid-derived) versus that containing C1192 (chromosomal) (Sigmund et al., 1988; Triman et al., 1989). Substantial levels (50-75%) of mutant U1192-containing rRNA were detected in 70s and polysome fraction particles, even from cells grown at the restrictive temperature. We concluded that mutant 16s rRNA is assembled into ribosome-like particles at 42"C, even though these ribosome-like particles do not interfere with the function of normal ribosomes. We do not know the nature of the presumed assembly defects in the ribosome-like particles formed in these mutants. A Spcs growth phenotype is observed in cells transformed with pSTL102 containing the G to A change at 359 (i.e., C52/A359; Triman et al., 1989). Introduction of a second-site compensatory alteration (i.e., U52-A359) resulted in restoration of spectinomycin resistance to the level normally found in pSTLlO2 transformants (M. McEvoy, K. Triman, and H. F. Noller, unpublished results). The growth phenotype observed in transformants containing the compensatory C to U change alone (i.e., U52/G359) is only slight sensitivity to spectinomycin. The growth phenotypes of pSTLlO2 transformants containing either C or U at 52 and either G or A at 359 in the absence of spectinomycin are indistinguishable. Restoration of spectinomycin resistance to the level conferred by plasmid pSTL102 in constructs containing the recessive 16s alteration (3590 to A) and the compensatory change (52C to U) that is predicted to permit base pairing at the position of the primary mutation provides evidence that the Spcs phenotype is attributable to the disruption of the base pair. Likewise, the phenotype conferred by plasmids carrying the compensatory change in the absence of the primary mutation is consistent with the presence of a weak G / U interaction. We have not yet demonstrated that the Spcs phenotype conferred by A146 or the Spct~phenotype conferred by U153, A350, A1292, or U1293 in 16s rRNA is the result of destabilization of the G-C base pair in which each

1. Mutational Analysis of 16s Ribosomal RNA

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nucleotide is involved. We have initiated mutagenesis experiments involving the introduction of compensatory second-site alterations that result in the restoration of base pairing in these mutants. Our hypothesis is that replacement of an A / C mismatch or G/U pair with an A-U base pair might result in restoration of normal expression of the Spcr marker. Alternatively, the failure of an A-U base pair to restore normal expression of the Spcr marker might indicate that the specific identity of nucleotides at the position of the original G-C base pair must be maintained to preserve some other aspect of ribosomal structure. Recessive 16s rRNA mutations isolated by random hydroxylamine mutagenesis of plasmid pSTL102 and detected by loss of expression of its spectinomycin-resistance marker may prove useful in the identification of unanticipated features of 16s rRNA structure.

B. Mutations upstream of the 16s rRNA coding region Among the mutants detected in screens for pSTL102 tranformants exhibiting a Spcts phenotype were three that contained unique mutations upstream of the 16s rRNA coding region. These mutations, located at -13, -30, and -59 relative to the 5’ terminus of mature 16s rRNA, abolish spectinomycin resistance at elevated temperatures (Mori et al., 1990). The sites of mutation are downstream from the promoter region, PIP2, from the sites (Box A,B, and C) involved in transcriptional antitermination, and from the RNAse 111 processing site. Processing of the 5’ end of 16s rRNA from the mutant operons appears to be normal at both restrictive and permissive temperatures as determined by primer extension (Mori et al., 1990). We were intrigued by the possibility that these mutations affect ribosome assembly and sought second-site revertants to each of them in order to identify structural elements that include the mutated posit ions. Plasmid DNA containing each of the upstream mutations was treated with hydroxylamine, subsequently used to transform cells, and the transformants were screened for the ability to grow in the presence of spectinomycin at elevated temperatures. Several general suppressors were found (-48A, -62T, and -63T) which suppress more than one of the original mutations. In addition, two of the original mutations (-13A and -30A) were recovered as reciprocal suppressors to one another. Surprisingly, a suppressor of the mutation at -13 was identified at position 21 within 16s rRNA. Subsequently, alterations in the upstream region at positions -114, -55, and -5 that suppress the cold-sensitive dominant mutation in 16s rRNA at position 23 have been identified (Dammel and Noller, 1993; U23 is discussed under Section 111,C). These suppression patterns provide evidence for transient formation of structure involving interactions between these regions in the 16s precursor. Some C to T transition mutations, located between 19 and 45 nucle-

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otides upstream of the mature 16s rRNA 5’ end, have pronounced effects on the growth phenotype of mutant cells and on ribosomal subunit assembly (Theissen et al., 1990). Detailed characterization of ribosomes from these mutants provided evidence that the rRNA leader sequence is important for the correct structure formation and biogenesis of functionally active 30s ribosomes, despite the fact that it is not part of the ribosomal particles formed (Theissen et al., 1993; Wagner et al., 1993).

C. Mutations in the 5’ major domain of 16s rRNA 1. Central pseudoknot region (9-13/21-25 and 17-19/916-918) Comparative sequence analyses of 16s rRNA have shown that there are three potential pseudoknot structures each involving an interaction of bases within a hairpin loop with bases external to the hairpin; the central pseudoknot is formed by regions (9-13/21-25 and 17-19/916-918 (Gutell and Woese, 1990). Brink et al. (1993a,b) studied the requirement of the potential formation of the central pseudoknot for the function of the 30s subunit by introducing mutations at positions 18 and 917, respectively. The effect of changing the base-paired residues (C18 and G917) on ribosome activity was studied in vivo using the specialized ribosome system developed by Hui and DeBoer (1987). Mutation of C18 to an A, G, or U resulted in a dramatic reduction in translational activity of ribosomes, whereas introduction of complementary mutations at positions 18 and 917 resulted in the formation of fully active ribosomes (Brink et al., 1993a,b). Brink et al. (1993a,b) demonstrated that 30s subunits containing a disrupted pseudoknot are not capable of forming 70s ribosomal complexes. A point mutation in 165 rRNA, a C to U transition at position 23, results in the conversion of the Gll-C23 base pair in the 5‘ terminal helix to a G-U pair and confers dominant cold sensitivity (Dammel and Noller, 1993). Isolation of second-site suppressors, including GllA, G15A, and C-5U (at a position upstream within the 16s rRNA precursor sequence), led to the suggestion that these nucleotides might be involved in a competing RNA secondary structure resulting from pairing of an upstream precursor sequence and a sequence found in one strand of the 5’ mature helix (Dammel and Noller, 1993). Although the basis for the dominance of the C23U mutation is not yet understood, the analysis of this mutation illustrates the importance of the use of conditional mutations and second-site suppressors to define alternative conformations. Pinard et al. (1993) reported the isolation of two mutations, U to A or U to C at position 13 in 16s rRNA, as part of a mutagenesis study to investigate the possible involvement of this position in alternative pairing with position 914. The suggestion had been made that a portion of the 5’ helix (positions 12-

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16) might alternatively pair with positions 911-915 to form a pseudoknot structure (Leclerc and Brakier-Gingras, 1990; Leclerc et al., 1991a). Although the mutations at position 13 did not affect cell growth, they did reduce the capacity of ribosomes to bind streptomycin. Thus, there is evidence for involvement of the 5' helix in the binding of streptomycin. Double mutants containing compensatory changes at 914, however, did not restore the ability of mutant ribosomes containing alterations at position 13 to bind streptomycin (Pinard et al., 1993). Further discussion of streptomycin binding to the 915 region appears under Section E.

2. 530 Loop Bases in the 530 stem-loop region have been shown to be protected from chemical probes by tRNA (Noller, 1991). Mutational analysis of the 530 loop has led to demonstrations that ( i ) a C to A change at position 523 (Melancon et al., 1988) and a C to U change at position 525 (Powers and Noller, 1991) each confer streptomycin resistance; (ii) dominant lethality of base subsitutions at G530 is due primarily to mutant ribosomes blocking a crucial step in protein synthesis after translational initiation (Powers and Noller, 1990); (iii) a higherorder structural interaction, a pseudoknot, involving residues 524-526 and 505-507 (Gutell and Woese, 1990) is required for proper functioning of the ribosome (Powers and Noller, 1991); (iv) ribosomes carrying the U525 (Powers and Noller, 1991) streptomycin-resistance mutation or the (2523 mutation (Leclerc et al., 1991b) have a reduced affinity for streptomycin; (v) frameshift enhancement results from mutations in the 530 loop (OConnor et al., 1992); (vi) mutant ribosomes containing G530A are impaired in in vitro EF-Tudependent binding of aminoacyl-tRNA to the ribosomal A site (Powers and Noller, 199313); and (vii) ribosomes reconstituted from in vitro transcripts of mutant 16s rRNA containing G530U are deficient in making the first dipeptide from a natural mRNA when tested in an in vitro protein synthesis assay (M. Santer et al., 1993). The genetic dissection of the 530 stem-loop region by site-directed mutagenesis has led to the suggestion that conformational changes in this region of 16s rRNA play an essential role in the selection for tRNA by the ribosome (Powers and Noller, 1994).

3. 5' Domain fragments Cormack and Mackie (1991) constructed a series of substitution and deletion mutations in a plasmid encoding the first 617 residues of 16s rRNA under the control of the bacteriophage T7 promoter. Labeled RNAs derived by transcription of the mutant 16s rRNAs were assayed for their ability to bind S20 protein

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using gel filtration and sucrose gradient centrifugation and for their ability to compete with control transcripts for S20. The results of these studies defined a minimum binding site for S20 within residues 1-402 of 16s rRNA and identified specific structural elements critical for binding (Cormack and Mackie, 1991). A fragment of 16s rRNA corresponding to most of the 5’ domain (residues 1-526) prepared by in vitru runoff transcription was incubated with a mixture of 30s proteins and demonstrated to form a discrete 16s particle containing only four of the proteins: S4, S16, S17, and S20 (Weitzmann et d.,

1993).

D. Mutations in the central domain of 16s rRNA 1. Mutations targeted to the central domain A number of groups reported the isolation and characterization of mutations following mutagenesis procedures targeted to the central domain of 16s rRNA. These mutations include those generated following Bal-3 1 treatment of either SmaI or BglII cut plasmid DNA (Gourse et d., 1982; Stark et ul., 1982, 1984): (i) a spontaneous 16-base insertion between residues 614 and 615, (ii) deletions of up to five bases at position 614, (iii) deletion of nucleotide 615, (iv) a 770base deletion from positions 615-1384, (v) deletions from positions 657-718 or 693-72 1, and (vi) deletion of either 29 or 60 bases beginning with position 704. Central domain mutations were also isolated by Zwieb and Dahlberg (1984) following bisulfite mutagenesis targeted to the region of plasmid pKK3535 corresponding to positions 822-874 in 16s rRNA; mutations included one or two base changes at positions 839-841, 867, or 876. Maxicell analysis of these central domain mutations revealed effects on ribosomal protein binding (S6, S8, S15, and S18 are known to bind to the central domain of 16s rRNA), rRNA processing, and subunit assetnbly. Powers and Noller ( 1993a) introduced mutations into nonconserved portions of the 620 helix in 16s rRNA in order to create a priming site for the conserved 530 region. All of the mutations resulted in deleterious phenotypes when expressed in plasmid pSTLlO2, despite the fact that Watson-Crick pairing was maintained in each of the new constructs. It appears that the identity of particular bases in the 620 helix must be maintained in order to preserve unidentified structural features of this region.

2. Site-directed mutagenesis of the binding site for S8 (588-605/633-651) Gregory and Zimmermann (1986) utilized both bisulfite and oligonucleotidedirected mutagenesis to define the binding site for ribosomal protein S8. Mutations isolated included (i) C to U changes at one of the following positions: 618,

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13

624, 631, 634, 643, or 651; (ii) G to A changes at either position 627 or 645; and (iii) each of the possible sequence alterations at positions 642 or 643. Among these alterations, the mutations at 627, 642, and 643 were found to reduce the affinity of ribosomal protein S8 for its binding site (Gregory et ul., 1988). Mougel et al. (1993) have reported the results of S8 binding studies using fragments of 16s rRNA generated from plasmid expression of DNA corresponding to positions 588-653; a minimal RNA binding site was defined and crucial residues were identified within this region by site-directed mutagenesis.

3. Mutations in a highly conserved single-strand region (790 loop) Prescott and Dahlberg (1990) demonstrated that a C to G mutation at position 726 in 16s rRNA affected both proper assembly of the 30s subunit and the correct functioning of the ribosome. The presence of the 7260 mutation caused the following changes in protein synthesis: (i) induction of some of the heatshock proteins, (ii) altered levels in the expression of wild-type proteins, and (iii) the synthesis of novel peptides (Prescott and Dahlberg, 1990). Further characterization of mutant ribosomes containing 726G revealed effects of the alteration on both mRNA binding and translation-termination (Prescott and Goringer, 1990). Tapprich et al. (1989) reported the isolation and characterization of a G to A change at position 791 in 16s rRNA. Mutant 30s subunits containing 791A exhibited reduced affinity for 50s subunits and a decreased ability to bind initiation factor 3 (Tapprich et al., 1989, 1990b). Santer et al. (1990) reported the isolation and characterization of mutations at position 792 in 16s rRNA. Base changes at this position also affected the assembly of 70s ribosomes.

4. Priming site mutation for structural probing of the 690 region and the 790 loop Powers and Noller ( 1993a) introduced a phenotypically silent mutation consisting of six single-base alterations in the region 838-854 to create a unique sequence that permits allele-specific priming. The priming site allows structural probing of the 690 region and the 790 loop in 16s rRNA.

E. Mutations in the 3’ major domain of 16s rRNA 1. Mutations targeted to the 3’ major domain A number of groups reported the isolation of mutations in the 3’ major domain. Among the mutations reported were those generated by exonuclease Ba131 digestion of SmaI cut plasmid DNA, including deletions of up to five bases

14

Kathleen L. Trlman

starting at position 1384 or 1385 (Gourse et al., 1982; Stark et al., 1982). Single base deletions at these positions were demonstrated in maxicells to severely retard both precursor rRNA muturation and 30s subunit assembly (Dahlberg, 1986).

2. Mutations in the conserved 900 region Montandon et al. (1986) introduced a C to U change at position 912 in 16s rRNA and demonstrated that this alteration confers streptomycin resistance. Frattali et al. (1990) reported the results of deletion of 912 (lethal), and alteration of C912 to A (no effect on growth or streptomycin sensitivity) or to G (confers streptomycin resistance). Bonny et al. (1991) extended the mutational analysis of this region by introducing alterations at 914 (A to C) and a double mutation at positions 912 and 888 (C to T:G to A). They demonstrated that mutations in the 912-915 region can confer resistance toward the killing effect of streptomycin (Bonny et al., 1991). Leclerc et al. (1991a) reported the effects of alterations at 913 (A to G) and 915 (A to G); mutant ribosomes containing either alteration were resistant to the stimulation of misreading and to the inhibition of protein synthesis by streptomycin. Both of these effects correlated with a decreased binding of the drug, confirming that mutations in the 915 region alter the interaction between the ribosome and streptomycin (Leclerc et al., 1991b). Powers and Noller (1991) also demonstrated that ribosomes carrying the U912 resistance mutation have a reduced affinity for streptomycin.

3. Mutations at methyl-modified sites Jemiolo et al. (1991) reported the isolation and characterization of mutations at three bases, 966, 967, and 1207, that are sites for post-transcriptional modification by methylation. They constructed a deletion mutation at 967 (lethal) and three substitution mutations at G966 (phenotypically silent), and either G to C or G to U at 1207 (dominant lethals) (Jemiolo et al., 1991). These results demonstrate the importance of certain methyl-modified residues for protein synthesis and cell viability. Previous experiments by Krzyzosiak et al. (1987) and Melancon et al. (1987) had shown that 30s subunits assembled in vitro from synthetic unmethylated 16s rRNA are about 60-70% as active in protein synthesis under the direction of an artificial or natural messenger as 30s subunits assembled with natural 16s rRNA (Cunningham et al., 1990b; Leclerc and Brakier-Gingras, 1990).

4. Priming site mutation for structural probing of the 900 region Powers and Noller ( 1993a) introduced a phenotypically silent mutation consisting of six single base alterations between residues 1006 and 1022 and creating a

1. Mutational Analysis of 16s Ribosomal RNA

15

unique sequence that allows allele-specific structural probing of the conserved 900 region as well as the 960 loop and position 926.

5. Mutations in helix 34 Helix 34 contains residues 1046-1067/1189-1211; perturbations in the region have been implicated in decoding of translational stop signals during the termination of protein synthesis (Goringer et at., 1991; Brown et at., 1993). The initial observations that deletion of a single base in this region, (21054, increased readthrough of some UGA termination codons (Murgola et al., 1988, 1990) and in certain circumstances, readthrough of UAG and UAA terminations codons (Prescott e t al., 1991a) support involvement of helix 34 in translation termination. A series of mutations within helix 34 have now been isolated and characterized including 1054 C to A, G, or U (Hanfler et al., 1990), 1192 C to U, A, or G (which confers spectinomycin resistance) (Sigmund et al., 1984; Makosky and Dahlberg, 1987; Bilgin et al., 1990), 1199 U to C, and 1202 U to C (Goringer et al., 1991). Helix 34 contains tandem conserved UCA sequences (bases 1199-1204) complementary to UGA; a double mutant ((21199 (21202) was found to be lethal (Tapprich et al., 1990b). Analysis of these mutations revealed that the ability of certain mutations to affect UGA-dependent termination correlated with the ribosomes’ ability to reduce suppressor tRNA activity (Prescott et al., 1991b; Prescott and Kornau, 1992). Recently, Brown et al. (1993) investigated the effect of alterations at position 1192 on binding of polypeptide release factors and in vitro termination as well as the effects of spectinomycin binding on termination reactions and presented evidence for a functional interaction between position 1192 and bases in the decoding site at the base of helix 44 (positions 1409-141 1/1491-1489).

6 . Mutations in the S7 binding site The localization of the binding domain of ribosomal protein S7 has been defined by a variety of biochemical methods, including cross-linking, nuclease protection, and protein footprinting experiments (Dragon and Brakier-Gingras, 1993). One class of the biochemical evidence for this interaction, a cluster of strongly protected nucleotides in the region, can be interpreted either as (i) demonstration of direct RNA-protein 5 7 contact or (ii) demonstration of conformational change caused by interaction of S7 with a more remote region of the RNA (Powers e t d., 1988). Genetic dissection of the roles of specific nucleotides in the 3’ major domain complements the data available from biochemical analysis. Dragon and Brakier-Gingras ( 1993) have constructed plasmids containing (i) the sequence corresponding to the 3’ major domain of 16s rRNA (nucleotides 926-1393) or ( i i ) the lower half of the 3’ major domain plus the 3’ minor

16

Kathleen L. Triman

domain of 16s rRNA (nucleotides 926-986/1219-1542, and (iii) a T7 promoter. Deletion derivatives of these constructs were generated using in vitro mutagenesis and RNA fragments were produced from the mutant plasmids by in vitro runoff transcription with T7 polymerase. Results from S7-binding experiments with each of the RNA fragments defined minimum structures containing the major determinants for the interaction between 16s rRNA and protein S7, the 1304-1308/1329-1333 helix, and the 1351-1371 hairpin (Dragon and BrakierGingras, 1993).

F. Mutations in the 3‘ minor domain of 16s rRNA 1. Mutations targeted to the 3’ minor domain A number of groups have reported the isolation and characterization of mutations in the 3’ minor domain of 16s rRNA. Among these mutations are some deletions generated by exonuclease Bal-31 treatment of HaeII cut plasmid DNA (Zwieb et d., 1986), and single base alterations generated by bisulfite mutagenesis targeted to the region 1385-1505 (Jemiolo et al., 1985; Meier et d., 1986) or by site-directed mutagenesis (Jacobet al., 1987; Hui and deber, 1987; Krzyzosiak et d., 1987; reviewed in Zimmermann et al., 1990, and in Cunningham et ul., 1993).

2. Mutations in the 1400 region Two single-stranded regions in the 3’ minor domain show extensive sequence conservation (Noller, 1993); both 1394-1408 and 1492-1505 have been implicated in tRNA binding and other protein synthesis functions (Noller, 1991). The nucleotide C 1400 and the conserved single-stranded regions are often referred to collectively as the “decoding region” (Zimmermann et d., 1990; Noller, 1993). Phenotypes observed in mutants containing alterations in this region range from lethal (C to U transitions at positions 1395 and 1407 and deletion of C1400; Thomas et ul., 1988) to mildly deleterious (C to U transitions at positions 1399, 1402, and 1404) or neutral (C to U transitions at positions 1388, 1389, 1397, and 1400; Jemiolo et al., 1985; Thomas et al., 1988). a.

C1399

Rottmann et al. (1988) constructed double mutants in which C1399 was converted to A and G1401 was changed to either U or C; cells carrying these mutations were viable only when the mutant rRNA was expressed from the lambda PL promoter; these mutants were shown to be defective in subunit association. Likewise, Cunningham et d. (1992a) demonstrated that single changes U1401 and C1401 were defective in subunit association.

1. Mutational Analysis of 16s Ribosomal RNA

17

b. C1400

Replacement of C14OO by U, A, or G did not markedly inhibit tRNA binding or peptide synthesis as measured in vitro (Denman et al., 1989a,b). Assembly defects were most pronounced by the change of C1400 to G (Krzyzosiak et al., 1987). Mutation of C1400 to any other base only moderately affected a set of in vitro protein synthesis partial reactions (Cunningham et al., 1992a). Deletion of C1400, however, created a dominant lethal phenotype (Thomas et al., 1988). Hui et d. (1988) showed that replacement of C1400 with G or A was quite deleterious in vivo.

c. G1401:C1501 tertiary base pair Mutations of G1401, or of C1501 to G, inactivate all in vitro functions of the ribosome (Cunningham et al., 1992a,b). However, the double mutant, C1401: G1501, with the base pair reversed, was demonstrated to have nearly full activity for tRNA binding (Cunningham et al., 1992b). The double mutant C1401: G1501 was subsequently shown to be able to form 70s initiation complexes but unable to form the first peptide bond; toeprinting assays revealed that the double mutant had lost the capacity to bind elongator tRNAs to the P site (Ringquist et al., 1993).

d. C1404:G1497 and G1405:C1496 tertiary base pairs Cunningham et al. (1993) found evidence for the importance of these two additional tertiary base pairs for tRNA binding. When either base pair was broken binding activity was severely inhibited but could be recovered when base pairing was restored (Cunningham et al., 1993). e. C1409-G1491 base pair De Stasio and Dahlberg (1990) studied the effects of 11 different single and double mutations at the base-paired secondary structure at 1409-1491. Mutations disrupting Watson-Crick base pairing (e.g., C-C and C-U) produced resistance to paramomycin as well as most other aminoglycosides in vioo and showed a loss of drug-dependent protection of nucleotides 1408 and 1494 in vitro (De Stasio et al., 1989). Mutations that substituted unpaired purines at these positions proved to be lethal (De Stasio and Dahlberg, 1990). O'Connor et al. (1991) transformed a series of restrictive and nonrestrictive protein S12 mutant strains with plasmids containing 1409G-l491C, 1409C-l491U, or 1409C1491C; these rRNA mutations affected the responses of these strains to paramomycin and streptomycin providing evidence for a direct interaction between protein S12 and the 1409-1491 region. Zimmermann et d.(1990) reviewed the effects of various deletion and point mutations in the 1409-1491 penultimate helix; there is evidence that certain bases within this helix are critical for

18

Kathleen L. Trirnan

interaction between the 30s and 50s subunits of the ribosome (Zwieb et al., 1986; Meier et al., 1986; Rottmann et al., 1988).

f. 1469

A C to U substitution at position 1469 in 16s rRNA suppresses streptomycin dependence and causes increased translational error frequencies (Allen and Noller, 1991). Although mutations in ribosomal proteins S4 and S5 causing increased miscoding can compensate for the restrictive phenotypes of streptomycin-dependent mutations in protein S12, none of the these proteins have assembly effects in the 1469 region of 16s rRNA; it is likely that there is no direct interaction between U1469 and any of the proteins implicated in translational fidelity (Allen and Noller, 1991).

g. 1505 suppressor mutation

The lethal phenotype associated with C to U transitions at positions 1395 and 1407, as well as the deletion of C1400, was suppressed intragenically by replacement of G1505 with A, C, or U (Thomas et al., 1988; Zimmermann et d., 1990).

3. Anti-Shine-Delgarno region A single base mutation, C1538U, was constructed by Jacob et d. (1987) and demonstrated to be lethal when expressed from the normal promoters of rRNA operons in a high copy number plasmid. This mutation is located in the (antiShine-Delgarno) region of 16s rRNA that interacts with the Shine-Delgarno region of mRNA and alters the base pairing between these regions. Expression of this mutation from the conditional bacteriophage lambda P, promoter permitted the isolation of cells with functional ribosomes containing mutant 16s rRNA; the presence of mutant ribosomes resulted in severe cell growth retardation and drastic alterations in the synthesis of many proteins (Jacob et al., 1987). Hui and DeBoer (1987) altered the anti-Shine-Delgamo sequence in 165 rRNA from 5‘CCTCC to 5’GGAGG or 5’CACAC to compensate for and permit pairing with altered Shine-Delgamo sequences contained in a single mutated mRNA species. This experimental system, the specialized ribosome system, provided a means to study the effects of 16s rRNA alterations that would otherwise be lethal to the cell, as well as in v i m evidence for the base-pairing interactions between mRNA and rRNA. Weiss et al. (1988) demonstrated frameshifting effects of disruption of this pairing interaction at position 1538 and correction of these effects by the restoration of pairing. Yamagishi et al. (1987) also demonstrated effects of mutation in the anti-Shine-Delgamo region on regulation of FWA and tRNA expression.

1. Mutational Analysis of 16s Ribosomal RNA

19

4. Priming site mutations for structural probing of the 1400 and 1500 regions Powers and Noller ( 1993a) have introduced two phenotypically silent mutations, one containing four single base alterations in the 1450-1466 region and the other containing four single base alterations in the 1514-1530 region, to permit allele-specific structural probing in the 1400 and 1500 regions, respectively.

IV. CONCLUSIOWS One objective of this chapter was to outline genetic strategies designed to improve our understanding of the structure and function of 16s ribosomal RNA in E. coli. A second objective has been to attempt to tabulate the effects of mutations introduced into 16s rRNA. I t is useful to consider these mutational effects in light of the conservation data that have emerged from comparative analysis of 16s-like rRNA sequences (e.g., see Ofengand et al., 1993). Table 1.1 contains a list of 228 highly conserved bases in 16s rRNA based on Gutell’s comparative analysis of 5 1 representative sequences from eubacteria (20 sequences), archaebacteria (1 1 sequences), and eukaryotes (20 sequences) (Gutell, 1992, 1993b,c; Gutell et al., 1994). Table 1.2 contains a fairly comprehensive list of 16s rRNA single base mutations isolated and characterized since 1986. Mutations reported prior to 1986 were extensively reviewed by De Stasio et al. (1988) and are not included in Table 1.2. Likewise, multiple base mutations, such as deletions or insertions, were not included in this list. Work is currently in progress to expand the list of mutations from Table 1.2 into a database that will be accessible on the Internet (Triman, 1994). Considered together, these tables represent goals for future work (Table 1.1) and a progress report (Table 1.2) for the mutational analysis of 16s rRNA. Mutational analysis of 16s ribosomal RNA structure and function has proven to be a powerful approach to the study of the role of this RNA in the process of translation. There is also great promise in two novel genetic approaches to the study of 16s rRNA: (i) inactivation of as many as four chromosomal rrn operons in E. coli by insertion-deletion mutagenesis using antibiotic resistance cassettes (Condon et al., 1992, 1993), and (ii) the introduction of antibiotic resistance mutations into the single chromosomal rRNA operon of Halobacterium halobium (e.g., Mankin et al., 1992; Mankin, 1994). Knowledge of the process of translation is also being advanced rapidly as a result of the mutational analyses of ribosomal proteins (e.g., Ryden-Aulin et al., 1993; Traut et al., 1993; Wu et at., 1993), elongation factor (e.g., Tubulekas

20

Kathleen L. Triman Table 1.1. List of 228 Conserved Nucleotidesa

13U -

37u 109A 244U 323U 346G 3570 388G 509A 521G 531U 571U 676A 720C 7696 788U 801U 816A 892A 909A 92 1U 942G 956U 969A 1052U 1073U 1lO2A 1209C 1227A 1316G 1339A 1379G 1394A 1406U 1494G 1500A 1512U

1% -

51A ll2G 246A 3266 347G 362G 389A 515G 522C 532A 573A 695A 725G 1756 791G 802A 820U 8986 911U 9226 944G 958A 972C 1053G 1085U llllA 1213A 1230C 1318A 1341U 1382C

16A 54C 119A 251G 327A 351G 364A 394G 516U 527(; 533A 574A 696A 727G 781A 792A 804U 865A 899C

- -

1395(= 1495U 15170

924C 946A 959A 981U 1090u 11870 12216 1235U 1319A 13476 138% 1396A 1468A 1496C 1502A 1518A

17U 55A 149A 282A 329A 352C 368U 397A 517G 528C 536C 581G 704A 729A 782A 794A 81OC 8856 900A 915A 9256 950U 960U 983A 1055A 1093A 1191A l222G 1237C 1333A 1348U 1390U

21G 56U 151A 318G 342C 355c 372C 499A 519C 529G 565U 583A 714G 732C 7866 795C 814A 88944 907A 919A 926G 951G 9636 984C 1057G 1095U 1199U 1223C 1238A 1337G 1349A 1391U 1398A

- -

1397(= 1403C 1483A 14976 1504G

-

1492A 1498U 153OG

35G 57G 160A 322C 344A 356A 375u 505G 520A

530G

566G 664G 715A 759A 787A 796C 815A 891U 908A 920U 934c 9546 964A 1048G 1058G 1lOlA 1206G 1226C 1315U 13386 1373G 13926 1405G 1493A 1499A 1506U 1531A

“Positions at which single base alterations have been introduced are indicated by boldface and underlining (see Table 1.2). The bases tabulated here are universally conserved among the 16s-like rRNAs in all three primary kingdoms (see Fig. 1 in Noller, 1993;

Gutell, 1992).

21

1. Mutational Analysis of 16s Ribosomal RNA Table 1.2. Single and Double Mutations in 16s Ribosomal RNA Position.

Alteration

Phenotypeb,c

11

G to A

U23 suppressorb

13a

U to A or C

15.

U 13A/A914U U 13C/A914G 0 to A

Reduction of both streptomycininduced misreading and streptomycin binding. No suppression of U13 effects. No suppression of U13 effects. Moderate U23 suppressorb

18

C to A, G, or U

23

c to u

Dramatic reduction in translational activity6 Translational activity restoredb Translational activity restoredb Translational activity restoredb Cold-sensitive dorninantb

146 153 189

C to A c to u A 189GIA190G

(With U1192) Spcs recessiveb (With U1192) Spcrs recessiveh No effect on S20 binding.

190

A190GIA189G

No effect on S20 binding

250

A250GIG251A

Abolished S20 binding

25 1

G251A/A250G

Abolished S20 binding

32 1

A to C or G

Abolished S20 binding

A321ClG322U

Abolished S20 binding

A321GlG322A

Abolished S20 binding

C to A

Abolished S20 binding

G322AlA32 1G

Abolished S20 binding

G322UIA321C

Abolished S20 binding

323

u to G

Abolished S20 binding

332

G to A

Abolished S20 binding

350

G to A

(With U1192) Spc" recessiveh

C 18AIG917U c1EGG9 17c C 18U/G917A

322

Reference Dammel and Noller (1993) Pinard et al. (1993) Pinard et d.(1993) Pinard et al. (1993) Dammel and Noller (1993) Brink et al. (1993a,b) Brink et al. (1993a,h) Brink et al. (1993a,h) Brink et al. (1993a,b) Dammel and Noller (1993) Triman et a!. ( 1989) Triman et al. (1989) Cormack and Mackie (1991) Cormack and Mackie (1991) Cormack and Mackie (1991) Cormack and Mackie (1991) Cormack and Mackie (1991) Cormack and Mackie (1991) Cormack and Mackie (1991) Cormack and Mackie (1991) Cormack and Mackie (1991) Cormack and Mackie (1991) Cormack and Mackie (1991) Cormack and Mackie (1991) Triman et al. (1989) (continues)

22

Kathleen 1. Triman

Table 1.2.-Cuntinued Reference

352

C352U/A353G

No effect on S20 binding<

Cormack and Mackie

353

A353GiC352U

No effect on S20 binding'

Cormack and Mackie

359 505

G to A G to u

(With U1192) Spch recessiveb (With U1192) streptomycinr at elevated temperature; cold-sensitive growth on ampicillin; Spcc'b (With U1192) streptomycinsb

Triman et al. (1989) Powers and Noller

(With U1192) lethal under control of natural promaterb (With U1192) streptomyciwb

Powers and Noller

(With U1192) U525 suppresses severe growth defect of A506; A506 suppresses the weak growth defect of U525; strep-

Powers and Noller

(1991) (1991)

G505C/G506C

(1991) Powers and Noller

(1991) 506

G to A G506C/G505C G506A/C525U

507

c to u C507U/G524A

5 17'1

AG G to A, C, or U G5 17U/U534G

523

A to C

524

G to A G524A/C507U

525

c to u

tomycinbh (With U1192) reduced growth at elevated temperatures on ampicillin; streptomycinrb (With U1192) reduced growth at elevated temperatures; s t r e p tomycinr; slight stimulation of growth at elevated temperatures in the presence of streptomycinh Increased level of translational errorsh Increased level of translational errors; G5 17A had most deleterious effect on cell growthb Increased level of translational errod Streptomycin'; impaired binding of streptomycinb (With 1192) lethal under natural promoterb (With U1192) reduced growth at elevated temperatures; streptomycinr; slight stimulation of growth at elevated temperatures in the presence of streptomycinh Streptomycinr; (With U1192) slightly Spcc~b

(1991) Powers and Noller

(1991) (1991)

Powers and Noller

(1991) Powers and Noller

(1991)

O'Connor et al.

(1992) O'Connor et al.

(1992) O'Connor et al.

(1992) Melancon et al. (1988); Leclerc et

al. (1991b) Powers and Noller

(1991) Powers and Noller

(1991)

Powers and Noller

(1991)

23

1 . Mutational Analysis of 16s Ribosomal RNA Table I .2.-Continued Posit ion L1 525

Alteration C525UIG506A

C525GlC526G 526

C to A C526AIG505U C526GlC52 5G

527.1

G to U

528.

C

529.

530i1

G

to

to

G

u

G to A

G to U 53 1

U to G

534

U

to G or

C

U534G/G517U 538

G to A

Phenotypeh ' (With U1192) U525 suppresses severe growth defect of A506; A506 suppresses the weak growth defect of U525; strep tomycin'h Lethalh (With U1192) reduced cell growth on ampicillin; Spcsh (With U1 192) suppression of U505 effects; streptomycinfh Lethal', Little or no effect on cell o r inducible P-galactosidase production. Mutant rRNA found significantly only in free 30s ribosomesh Little or no effect on cell or inducible P-galactosidase production. Affects growth and protein synthesis only when representing 30% or more of the total rRNAh Highly deleterious effect on cell growth; depression o f inducible P-galactosidase production. Leads to irreversible cessation of growth when incorporated into less that 25% of the polysome ribosomes" Dominant lethal. h Impaired interaction between ribosomes and ternary complex; EF-Tu-ribosome interaction affected. Dominant 1ethal.b Impaired A-site function' No effect o n growth or protein synthesis when incorporated into 50% or more of the ribosomes in the polysome fractionh Little effect on cell growth or translational fidelityb Increased level of translational errorsh (With U1192) Spc" recessiveh

Reference Powers and Noller (1991)

Powers and Noller (1991) Powers and Noller (1991) Powers and Noller (1991) Powers and Noller (1991) U V. Santer et al. 1993a,b)

U V. Santer et al. 1993a,b)

U. V. Santer et al. (1993a,b)

Powers and Noller (1993b, 1994)

M. Santer (1993) M. Santer (1993)

et

al.

et

al.

OConnor et al. (1992) OConnor et al. (1992) Triman et al. (1989) (continues)

24

Kathleen 1. Mman

Table 1.2.-Cmtinued Positions 595

Alteration

AA

A to U

Phenotypeb,c Drastic reduction in S8 binding'

597

G597ClC643G

598

U to A

599

U598AIA642U C to G C599GlG639C

No effect on S8 binding. Suppression of effects of 643G on S8 binding. Drastic reduction in S8 binding. Drastic reduction in 58 binding' Drastic reduction in S8 binding. No effect on S8 binding or cell

618

c to u

No effect on S8 binding or cell

c to u

No effect on S8 binding or cell gr0wthb.c

627

G to A

63 1

c to u

Reduced S8 binding; decreased growth rateb.c

No effect on S8 binding or cell gr0wthb.c

634

c to u

No effect on S8 binding or cell gr0wthb.C

639

G639ClC599G

640

AA A to U

64 1

u to c

642

AA

643

U to A

Mougel et al. (1993) Mougel et al. (1993) Mougel et al. (1993) Mougel et al. Mougel et al. Mougel et al. Mougel et al.

(1993) (1993) (1993) (1993)

gr0wthb.c growth b,c

624

Reference

No effect on S8 binding or cell gr0wthb.c Drastic reduction in S8 binding' Drastic reduction in S8 binding. Drastic reduction in S8 binding. No effect on 58 binding or cell gr0wthb.C Reduced growth rate; drastic reduc, tion in S8 bindingb,c

Gregory and Zimmermann (1986); Gregory et al. (1988) Gregory and Zimmermann (1986); Gregory et a[. (1988) Gregory and Zimmermann (1986); Gregory et al. (1988) Gregory and Zimmermann (1986); Gregory et al. (1988) Gregory and Zimmermann (1986); Gregory et al. (1988) Mougel et al. (1993) Mougel et d. (1993) Mougel et al. (1993) Mougel et al. (1993) Mougel et al. (1993) Gregory and Zimmermann (1986); Mougel et al.

(1993)

A to U

Drastic reduction in S8 binding'

Gregory and Zimmermann (1986); Mougel er al.

A642UiU598A

Drastic reduction in S8 binding' Reduced S8 binding; decreased growth rateb,c

C to G C643GlG597C

Drastic reduction in S8 binding. Suppression of effects of 643G on S8 bindingc

Mougel et al. (1993) Gregory and Zimmermann (1986); Gregory et al. (1988) Mougel et al. (1993) Mougel et al. (1993)

c to u

(1993)

25

1. Mutational Analysis of 16s Ribosomal RNA Table I.Z.-Continued

Positions 645

Alteration

G to A

65 1

c to u

726

C to G

7910

G to A

792~

A to G, C, or U

814~

888 912

G888AIC9 12U AC C to A C to G

c to u

Phenotypeb,c

No effect o n S8 binding or cell growthb.c No effect o n S8 binding or cell growthb,' Temperature-sensitive cell growth; decreased levels of S2 and S21 in 30s subunits; altered levels of normal proteins; novel proteins including heat-shock proteinsh Reduced association between 30s and 50s subunits; decreased IF3 binding and protein synthesishmc Reduced association between 30s and 50s subunits and reduced protein synthesis. (A792C associated with loss of IF3 binding)bp' (With U1192) growth rate and rate of protein synthesis decreased; recessiveb Streptomycinrh Dominant lethalb No effect o n cell growthh Decreased cell growth rate; low level streptomycinrb Streptomycinrb

Reference Mougel et al. (1993) Mougel et al. (1993) Prescott and Dahlberg (1990)

Tapprich et al.

(1989)

M. Santer et al. (1990)

McLaughlin et al. ( 1988) Bonny et al. (1991) Frattali et al. (1990) Frattali et al. (1990) Frattali et al. (1990) Montandon et al. (1986); Bonny et

al. (1991)

913

914"

9150

C9 12UlG888A A to G

A to C A to G or U A914GiU13A A914UlU 13C A to G

Streptomycinrb Binding of streptomycin decreased; both streptomycin-induced misreading and streptomycininduced inhibition of protein synthesis decreased. Streptomycinrb Reduction of both streptomycininduced misreading and streptomycin binding' No suppression of U13 effects. No suppression of U13 effects. Binding of streptomycin decreased; both streptomycin-induced mis. reading and streptomycininduced inhibition of protein synthesis decreased.

Bonny et at. (1991) Leclerc et al. (1991a)

Bonny et al. (1991) Pinard et al (1993)

Pinard et at. (1993) Pinard et al. (1993) Leclerc et al. (1991a)

(continues)

26

Kathleen L. Triman

Table 1.2.-Continued Positiona

Alteration

Phenotypeh,c

Reference

980

G917AIC18U G917CIC18G 0 9 17U/C18A G to A, C, or U AC C to A, G, o r U C to A or G

Brink et al. (1993a,h) Brink et al. (1993a,b) Brink et al. (1993a,h) Jemiolo et al. ( 1991 ) Jemiolo et al. (1991) Jemiolo et al. (1991) U. V. Santer et al. (1991)

1054~

AC

Translational activity restoredh Translational activity restoredb Translational activity restoredh No effect on cell growth ratell Dominant lethalh No effect on cell growth rateh (With U1192) A980 has only a small effect o n growth; G980 decreases growth rate dramatically in the presence of Spc.b UGA suppressionh

917 966 967

C to A, G, or U

Only C1054G resulted in significant reduction of horh cellular growth rate and ability of ribosomes to stop specifically at UGAh Spectinomycin'h

1192

C to A, G, or U

1 199~1

1207 1292 1293 1388

to c u1199c/u1202c to c U1202C/U 1199C G to C or U G to A c to u c to u

Increased U G A readthroughh Dominant lethalh Increased U G A readthroughb Dominant lethalb Dominant lethalh (With U1192) Spccb recessiveh (With U1192) Spcts recessiveb No effect on growth rateb

1389

c to u

No effect on growth rateb

1395~1

c to u

Dominant 1ethal.h Partial activity<

13970

c to u

No effect on growh rateh

AC

Impaired in initiation of translation=

1202

u

u

Murgola et al. (1988, 1990); Goringer et af. (1991); Prescott et al. (1991a) Hanfler et al. (1990)

Sigmund et al. (1984); Makosky and Dahlberg (1987); Bilgin et d. (1990) Goringer et al. ( 1991) Goringer et al. (1991) Goringer et al. (1991) Goringer et al. (1991) Jemiolo et al. (1991) Triman et al. (1989) Triman et al. (1989) Jemiolo et d.(1985); Thomas et af. (1988) Jemiolo et d. (1985); Thomas et al. (1988) Thomas et d. (1988); Zimmermann et af. (1990) Cunningham et al. ( 1990a) Jemiolo et d.(1985); Thomas et af. (1988) Denman et al. (1989a,b)

27

1. Mutational Analysis of 16s Ribosomal RNA

Table 1.2.-Continued Position.

Alteration

13980

AA

1399d

C

1400~

to

A

c to u

Reference

Impaired in initiation of translation' Lethal under natural promoter" Slight reduction in cellular growth rateh

Denman et al. (1989a,b) Rottmann et d.(1988) Jerniolo et al. (1985); Thomas et al. (1988) Meier et nl. (1986); Thomas et al. (1988); Rottmann et al. (1988) Meier et nl. (1986); Thomas et al. (1988); Rottmann et al. (1988) Thomas et al. (1988); Denman et al. (1989a,b); Zimmermann et al. (1990) Hui et al. (1988); Thomas et al. (1988); Denman et al. (1989a,b) Hui et al. (1988), Thomas et al. (1988); Denman et al. (1989a,h) Jemiolo et al. (1985); Hui et al. (1988); Thomas et al. (1988); Denman et al. (1989a,b) Thomas et al. (1988); Rottmann et al. (1988); Denman et al. (1989a,b) Thomas et al. (1988); Rottmann et al. (1988); Cunningham et al. (1992a) Rottmann et al. (1988); Cunningham et ul. (1992a)

C1399A/G1401C

Lethal under natural promoter. Ribosomes are totally inactiveh

C1399A/G1401U

Lethal under natural promoter. Severe impairment of ribosome functionh

AC

Dominant lethalh; Initiationdependent protein synthesis blocked.

C to A

Lethal under natural promoter; inhibited ribosomal activity", no inhihition in vitru

c to G

Inhibited ribosomal activityh; no inhibition in vitro

c to u

140Iu

Phenotypeb,L

AG

G

to

No effect o n ribosomal activitvh,<

Lethal under natural promoterh; all ribosomal functions blocked.

A

G to C or U

Lethal under natural promoterh; all ribosomal functions tested in protein synthesis partial reactions were blocked. Disrupted tRNA binding and p l y peptide synthesis; all ribosomal functions tested in protein synthesis partial reactions were blocked'

(continues)

28

Kathleen 1. Triman

Table 1.2.-Cmtinued Position0

Alteration

G1401UIC1399A

Phen0typeb.c

Reference

Lethal under natural promoter. Ribosomes are totally inactiveh

Meier et al. (1986); Thomas et al. (1988); Rottmann et af. (1988) Meier et al. (1986); Thomas et al. (1988); Rottmann et al. (1988) Cunningham et al. (1992b); Ringquist et al. (1993) Jemiolo et al. (1985); Thomas et al. (1988) Denman et al. (1989a,b) Jemiolo et al. (1985); Thomas et al. ( 1988) Cunningham et al. (1993) Cunningham et al. (1993) Cunningham et al. (1993) Cunningham et al. (1993) Cunningham et al. (1993) Cunningham et al. (1993) Thomas et al. (1988); Zimmermann et al. ( 1990) De Stasio et a1 (1989) De Stasio et d. (1989) Hui et al. (1988) Hui et al. (1988); De Stasio et al. (1989) De Stasio et al. (1989) De Stasio et al. (1989) Hui et al. (1988) Meier et al. (1986); Rottmann et al. (1988)

Lethal under natural promoter; severe impairment of ribosome functionb Impaired A-site function; enhanced tRNA fMet selectivity'

c to u

Slight reduction in cellular growth rat@

AC

c to u

Initiation-dependent protein synthesis blocked. Slight reduction in cellular growth rat& inhibited tRNA binding'

c to (3

Inhibited tRNA bindingc

C 1404G/C 1496G

Inhibited tRNA bindingc

C1404G/G 1497c

Restored tRNA bindingc

G to c

Inhibited tRNA bindingc

G 1405C/C1496G

Restored tRNA bindingc

C1405CIG1497C

Inhibited tRNA bindingc

1407a

c to u

Dominant Lethalb

1409

C to C or A

Lethalb No drug resistanceb Ribosome activity reducedb Ribosome activity reducedb

14024

1404a

1409

c to u

C1409A/C1411G C1409A/G1491U

1411 1416

C1409GIG1491C C1409U/G1491A C 1411G/C1409A

G to

u

No drug resistanceb No drug resistanceb Ribosome activity reducedb Reduced association between 30s and 50s subunits

29

1. Mutational Analysis of 16s Ribosomal RNA

Table 1.2.-Continued Positiona

Alteration

Phenotypeh,'

Reference Jerniolo et al. (1985); Allen and Noller (1991) Hui et al. (1988) De Stasio et al. (1989) De Stasio et at. (1989)

1469

c to u

Slow growth rate; ribosomal ambiguity phenotypeb,'

1489 1491

G1489C/G 1491U G to A G to c or u

G 1491A/C1409G G 149lC/C1409G G 1491U / C 1409A

Ribosome activity reducedh Lethalb Paramomycinr, neomycinr, kanamycinr, tobramycin: gentamicinr, apramycin: hygromycinr"; higher order structure reanangementC No drug resistanceb No drug resistanceb Ribosome activity reducedb

G 1491U/G 1489C c to G

Ribosome activity reducedb Inhibited tRNA binding'

C1496GK1404G

Inhibited tRNA binding

C 1496GiG1405C

Restored tRNA binding

G to c

Inhibited tRNA binding.

G 1497C/G 1405C

Inhibited tRNA binding

G 1497C/C1404G

Restored tRNA bindingc

1498.

U to G, C, or A

1501.

c to G

U1498G strongly inhibited y e t Val formation; enhanced tRNA 'Met tRNA selectivity< Disrupted tRNA binding and polypeptide synthesis' Enhanced tRNA ,Met selectivityc

14960

1497~

C1501G/G 1401c 1505.

0 to A, C, or U

Suppresses AC1400, C1395U, and C1407Ub.'

15120

U to C or G

U512G no effect on initiation complex formation; U512C enhanced tRNA (Met binding and selectivity' No effect on initiation complex formation'

u 1 5 12c/c1524u

De Stasio et al. (1989) De Stasio et al. (1989) Hui et d. (1988); De Stasio er al. (1989) Hui er al. (1988) Cunningham et al. (1993) Cunningham er al. (1993) Cunningham et al. (1993) Cunningham et al. (1993) Cunningham et al. (1993) Cunningham et al. (1993) Ringquist et aL (1993); Cunningham et al. (1993) Cunningham et al. (1992b) Cunningham et al. (1992b); Ringquist etal. (1993) Thomas et al. (1988); Zimmermann et ai. (1990) Ringquist et al. (1993)

Ringquist et al. (1993) (continues)

Kathleen L. Riman

30 Table 1.2.-Continued Positionu

Alteration

Phenotypebsc

Reference

1518

A to C, G , or U

1519

A to C, G, or U

1523

G to A

Cunningham et al. (1990b) Cunningham er al. (1990b) Ringquist er al. (1993)

1524

c1524u/u1512c

1535-1540

CCTCC to

Little effect on rihosome assembly or function' Little effect on rihosome assemhly o r function' No effect on initiation complex formationc No effect on initiation complex formationc Lethal under natural promoter; preferentially translates mRNA containing mutated complementary Shine-Delgarno sequence& Lethal under natural promoter; preferentially translates mRNA containing mutated complementary Shine-Delgarno sequenceb Dominant lethal under natural promoter; decreased complementarity to Shine-Delgarno region produces lower level protein synthesish Suppresses RFZ mRNA frameshift mutationh

CACAC

1535-1540

CCTCC to GGAGG

1538

c to u

c to G

Ringquist et al. (1993) Hui and DeBoer (1987)

Hui and D e b e r (1987)

Jacoh et al. (1987)

Weiss et al. (1988)

CLHighly conserved; see Table 1.1 hln viva.

and Hughes, 1993; Weijland et al., 1993)) initiation factor (e.g., Spurio et al., 1993), release factor (e.g., Tate et al., 1993), tRNA (e.g., Atkins et al., 1993), mRNA (e.g., De Smit and Van Duin, 1994; Saito and Nomura, 1994), 23s rRNA (e.g., Melancon et al., 1992; Douthwaite et al., 1993; OConnor and Dahlberg, 1993; Saarma et al., 1993), and translational fidelity (Parker, 1992). The common theme of all of these kinds of studies is the remarkable power of molecular tools for genetic manipulation of RNA and protein to uncover unanticipated features of structure and function. There are, however, some hints about limitations for the future success of genetic approaches to the study of rRNA structure and function. Lewicki et al. (1993) reported that (i) 85% of 23s rRNA transcribed from a plasmid by T7 RNA polymerase became degraded, (ii) 50s particles containing the intact plasmidaderived 23s rRNA did not enter the pool of active ribosomes, and (iii) the particles with plasmid-derived 23s rRNA were inactive in protein synthesis

1. Mutational Analysis of 16s Ribosomal RNA

31

in uitro. Reduction of the rate of T7 RNA polymerase transcription to a rate comparable to that of host RNA polymerase permitted the formation of active subunits, emphasizing the importance of the coupling of rRNA transcription and ribosomal assembly in vivo (Lewicki et al., 1993). Ambiguous results from the traditional site-specific mutagenesis approaches with Hepatitis delta virus RNAs that assume multiple conformers (Gottlieb et al., 1994) demonstrate the difficulties that might be encountered in regions of rRNA that undergo alternate foldings. The most powerful mutagenesis approach involves the isolation of second-site suppressor mutations. For example, Allen and Noller (1991) identified an unanticipated single base substitution (U1469) in 16s ribosomal RNA that suppresses a streptomycin-dependent ribosomal protein S 12 mutation by using random mutagenesis and selection for streptomycin-independent growth. Likewise, Dammel and Noller (1993) isolated second-site suppressors of the dominant cold sensitivity conferred by the U23 mutation in 16s ribosomal RNA by random mutagenesis. Ultimately, we can look forward not only to the identification of additional 16s ribosomal RNA mutations that suppress ribosomal protein mutations and/or 16s rRNA mutations, but also to the identification of mutations in proteins and factors involved in translation that suppress ribosomal RNA mutations. We can also expect the search for suppressors to lead to the identification of specific interactions between 16s rRNA and 23s rRNA.

Acknowledgments This work was supported by the NSF (MCB-9315443) and grants from the Dean of Franklin and Marshall College. I thank Harry Noller and the NSF (Research Opportunity Award Supplement to DMB-9005016) for support during my 1993 visit to the Noller lab. This chapter was inspired by the Vassar Ribosomal RNA Symposia hosted by David Jemiolo in 1991, Albert Dahlberg in 1992, and Me1 Santer in 1993. I am grateful to Lea Brakier-Gingras, Albert Dahlberg, Robin Gutell, David Jemiolo, Alexander Mankin, Harry Noller, James Ofengand, Me1 Santer, William Tapprich, and Robert Zimmermann for communicating results prior to publication. I am particularly grateful to Nicole Harner and Steven Vavoulis for their help with the preparation of the manuscript and to Lea Brakier-Gingras, Harry Noller, James Ofengand, and Me1 Santer for their comments on the manuscript.

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Kathleen L. l i m n

Bilgin, N., Richter, A. A., Ehrenberg, M., Dahlberg, A. E., and Kurland, C. G. (1990). Ribosomal RNA and protein mutants resistant to spectinomycin. EMBO J. 9735-739., Bonny, C., Montandon, P.-E., Marc-Martin, S., and Stutz, E. (1991). Analysis of streptomycinresistance of Escherichiu coli mutants. Biochim. Biophys. A c w 1089213-2 19. Brink, M. F., Verbeet, M. P., and DeBoer, H. A. (1993a). Formation of the central pseudoknot in 165 rRNA is essential for initiation of translation. EMBO J. 12:3987-3996. Brink, M. F., Verbeet, M. P., and DeBoer, H. A. (1993b). The central pseudoknot connecting the three major domains in 16s rRNA is required for translational initiation. In “The Translational Apparatus: Structure, Function, Regulation and Evolution” (K. H. Nierhaus, F. Franceschi, A. R. Subramanian, V. A. Erdmann, and B. Wittmann-Liebold, eds.), pp. 371-374. Plenum, New York. Brosius, J., Dull, T. J., Sleeter, D. D., and N o h , H. F. (1981a). Gene organization and primary structure of a ribosomal RNA operon from Escherichia coli. J. Mol. Bid. 148:107-127. Brosius, J., Palmer, M. L., Kennedy, J. P., and Noller, H. F. (1981b). Complete nucleotide sequence of a 16s ribosomal RNA gene from Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 75:4801-4805. Brosius, J., Ullrich, A., Raker, M. A., Gray, A., Dull, T. J., Gutell, R. R., and Noller, H. F. (1981~).Construction and fine mapping of recombinant plasmids containing the rmB ribosomal RNA operon of E. coli. Plasmid 6:112-118. Brown, C. M., McCaughan, K. K., and Tate, W. P. (1993). Two regions of the Escherichia coli 16s ribosomal RNA are important for decoding stop signals in polypeptide chain termination. Nuckic Aclds Res. 21:2109-2115. Condon, C., Philips, J., Fu, 2.-Y., Squires, C., and Squires, C. L. (1992). Comparison of the expression of the seven ribosomal RNA operons in Eschmichia coli. EMBO J. 11:4175-4185. Condon, C., French, S., Squires, C., and Squires, C. L. ( 1993). Depletion of functional ribosomal RNA operons in Escherichia coli causes increased expression of the remaining intact copies. EMBO J. 12:4305-4315. Cormack, R. S., and Mackie, G. A. (1991). Mapping ribosomal protein S20-16s rRNA interactions by mutagenesis. J. Bid. Chem. 266:18525-18529. Cundliffe, E. (1987). On the nature of antibiotic binding sites in ribosomes. Biochimie 69:863-869. Cundliffe, E. (1990). Recognition sites for antibiotics within rRNA. In “The Ribosome: Structure, Function, and Evolution” (W. E. Hill, A. Dahlberg, R. A. Garrett, P. B. Moore, D. Schlessinger, and J. R. Warner, eds.), pp. 479-490. American Society for Microbiology, Washington, Dc. Cunningham, P. R., Weitzmann, C. I., Negre, D., Sinning, J. G., Frick, V., Nurse, K., and Ofengand, J. (1990a). In vitro analysis of the role of rRNA in protein synthesis: Site-specific mutation and methylation. In “The Ribosome: Structure, Function, and Evolution” (W. E. Hill, A. Dahlberg, R. A. Garrett, P. B. Moore, D. Schlessinger, and 1. R. Warner, eds.), pp. 243252. American Society for Microbiology, Washington, Dc. Cunningham, P. R., Weitzmann, C. J., Nurse, K., Masurel, R., Van Kinippenberg, P. H., and Ofengand, J. (1990b). Site-specific mutation of the conserved m:Am;A residues of E. coli 16s ribosomal RNA. Effects on ribosome function and activity of ksgA methyltransferase. Biochim. Biophys. Acra 1050: 18-26. Cunningham, P. R., Nurse, K., Weitzmann, C. J., Negre, D., And Ofengand, 1. (1992a). (31401: A keystone nucleotide at the decoding site of Eschm’chiu coli 30s ribosomes. Biochemistry 3 1~7629-7637. Cunningham, P. R., Nurse, K., Bakin, A., Weitzmann, C. J., Pflumm, M., and Ofengand, J. (1992b). Interaction between the two conserved single-stranded regions at the decoding site of small subunit ribosomal RNA is essential for ribosome function. Biochemistry 31:12012-12022. Cunningham, P. R., Nurse, K., Weitzmann, C. J., and Ofengand, J. (1993). Functional effects of

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1. Mutational Analysis of 16s Ribosomal RNA ~

~~

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Gourse, R. L., Stark, M. J. R., and Dahlberg, A. E. (1982). Site-directed mutagenesis of ribosomal RNA: Construction and characterization of deletion mutants. J. Mol. Bid. 159:397-416. Gourse, R. L., Takehe, Y., Sharrock, R. A., and Nomura, M. (1985). Feedback regulation of rRNA and tRNA synthesis and accumulation of free ribosomes after conditional expression of rRNA genes. Proc. Natl. Acad. Sci. U.S.A. 82:1069-1073. Gregory, R. J., and Zimmermann, R. A. (1986). Site-directed mutagenesis of the binding site for ribosomal protein S8 within 16s ribosomal RNA from Escherichia cob. Nuckic Acids Res. 145741-5776. Gregory. R. J., Cahill, P. B. F., Thurlow, D. L., and Zimmermann, R. A. (1988). Interaction of Escherichia coli protein S8 with binding sites in ribosomal RNA and messenger RNA. J. Mol. Bid. 204:295-307. Gutell, R. R. (1992). Evolutionary characteristics of 16s and 23s rRNA structures. In “The Origin and Evolution of the Cell” (H. Hartman and K. Matsuno, eds.), pp. 243-309. World Scientific Pub. Co., River Edge, NJ. Gutell, R. R. (1993a). Collection of small subunit (16s- and 16s-like) ribosomal RNA structures. Nuckic Acids Res. 21:3051-3054. Gutell, R. R. (1993b). Comparative studies of RNA: Inferring higher-order structure from patterns of sequence variation. Cum. Opin. Strut. Biol. 3:313-322. Gutell, R. R. ( 1 9 9 3 ~ ) The . simplicity behind the elucidation of complex structure in ribosomal RNA. In “The Translational Apparatus: Structure, Function, Regulation and Evolution” (K. H. Nierhaus, F. Franceschi, A. R. Subramanian, V. A. Erdmann, and B. Wittmann-Liebold, eds.), pp. 477-488. Plenum, New York. Gutell, R. R., and Woese, C. R. (1990). Higher order structural elements in ribosomal RNAs: Pseudoknots and the use of non-canonical pairs. Proc. Natl. Acd: Sci. U.S.A. 87:663-667. Gutell, R. R., Larsen, N., and Woese, C . R. (1994). Lessons from an evolving rRNA: 16s and 23s rRNA structures from a comparative perspective. Microbiol. Rev. 58:lO-26. Hanfler, A , , Kleuvers, B., and Goringer, H. U. (1990). The involvement of base I054 in 16s rRNA for UGA stop codon dependent translational termination. Nuckic Acids Res. 185625-5632. Hui, A., and DeBoer, H. A. (1987). Specialized ribosome system: Preferential translation of a single mRNA species by a suhpopulation of mutated ribosomes in Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 84:4762-4766. Hui, A. S., Eaton, D. H., and DeBoer, H. A. (1988). Mutagenesis at the mRNA decoding site in the 16s ribosomal RNA using the specialized ribosome system in Escherichia coli. EMBO J. 7:4383-4388. Ingraham, J. (1987). Effect of temperature, pH, water activity, and pressure on growth. In Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology” (F. C. Neidhardt et al., eds.), Val. 2, pp. 1543-1554. American Society for Microbiology, Washington, DC. Jacob, W. F., Santer, M., and Dahlberg, A. E. (1987). A single base change in the Shine-Delgarno region of 16s rRNA of Escherichia coli affects translation of many proteins. Proc. Natl. Acad. Sci. U.S.A. 844757-4761. Jemiolo, D. K., Zwieb, C., and Dahlberg, A. E. (1985). Point mutations in the 3’ minor domain of 16s rRNA of E. coli. Nucleic AcidF Res. 13:8631-8643. Jemiolo, D. K., Steen, R., Srark, M. J. R., and Dahlberg, A. E. (1988). Analysis of plasmid-coded ribosomal RNA maxicell techniques. In “Methods in Enzymology” (H. Noller and K. Moldave, eds.), Vol. 164, pp. 691-706. Jemiolo, D. K., Taurence, J. S.. and Giese, S. (1991). Mutations in 16s rRNA in Escherichia coli at methyl-modified sites: (3966, G967, and (31207. Nuckic Acids Res. 19:4259-4265. Krzyzosiak, W., Denman, R., Nurse, K., Hellmann, W., Bouhlik, M., Gehrke, C. W., Agris, P. F., and Ofengand, J. (1987). In vitro synthesis of 16s rihosomal RNA containing single base changes and assembly into a functional 30s ribosome. Biochrmrstry 26:2353-2364.

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Leclerc, D., and Brakier-Gingras, L. (1990). Study of the function of Escherichiu coli rihosomal RNA through site-directed mutagenesis. Biochem. Cell Bid. 68:169-179. Leclerc, D., Melancon, P., and Brakier-Gingras, L. (l991a). Mutations in the 915 region of Escherichia coli 16s ribosomal RNA reduce the hinding of streptomycin to the ribosome. Nuckic Acids Res. 193973-3977, Leclerc, D., Melancon, P., and Brakier-Gingras, L. (199 lh). The interaction between streptomycin and ribosomal RNA. Biochimie 73:1431-1438. Leirmo, S., and Course, R. L. (1991). Factor-independent activation of Escherichia coli rRNA transcription. J. Mol. Biol. 220:555-568. Lewicki, 8. T. U., Margus, T.; Remme, J., and Nierhaus, K. H. (1993). Coupling of rRNA transcription and ribosomal assembly in vivo. J. Mol. Biol. 231:581-593. Lindahl, L., and Zengel, J. M. (1986). Ribosomal genes in Escherichia coli. Annu. Rev. Genet. 20:29 7-326. Makosky, P. C., and Dahlberg, A. E. (1987). Spectinomycin resistance at site 1192 in 1 6 s ribosomal RNA of E. coli: A n analysis of three mutants. Biochimie 69:885-889. Mankin, A. S. (1994). Selection of spontaneous and engineered mutations in the rRNA genes in halophilic archaea. In “Archaea: A Laboratory Manual” (F. T. Rohb, ed.), Cold Spring Harbor Lab., Cold Spring Harbor, NY (in press). Mankin, A. S., Zyrianova, 1. M., Kagramanova, V. K . , and Garrett, R. A. (1992). Introducing mutations into the single-copy chromosomal 23s rRNA gene of the archaeon Halobacterium halobium by using an rRNA operon-hased transformation system. Proc. Natl. Acad. Sci. U.S. A. 89:6535-653Yt McLaughlin, J.. Reyes; B., Bennett-Guerrero, E., and Santer, M. (1988). Mutations at position 814 of 1 6 s rRNA of E. coli. J. Cell Biol. 107:332a. Meier, N., Gqringer, H. U., Kleuvers, B., Scheihe, U., Eherle, J., Szymkowiak, C., Zacharias, M., and Wagner, R . (1986). The importance of individual nucleotides for the structure and function of rRNA molecules in E. coli: A mutagenesis study. FEBS Lett. 204:89-95. Melancon, P.,Gravel, M., Boileau, G., and Brakier-Gingras, L. (1987). Reassembly of active 30s ribosomal subunits with an unmethylated in v i m transcribed 1 6 s rRNA. Biochem. Cell Biol. 65: 1022-1030. Melancon, P.,Lemieux, C., and Brakier-Gingras, L. (1988). A mutation in the 530 loop of Escherichia coli 16s ribosomal RNA causes resistance to streptomycin. Nuckic Acids Res. 16:963 1-9639. Melancon, P., Leclerc, D., and Brakier-Gingras, L. (1990). A deletion mutation at the 5’ end of Escherichia coli 1 6 s ribosomal RNA. Biochim. Biophys. Acta 1050:98-103. Melancon, P., Tapprich, W. E., and Brakier-Gingias, L. (1992). Single-base mutations at position 2661 of Escherichia coli 23s rRNA increase efficiency of translational proofreading. 1. Bacteriol. 174:7896-7901. Montandon, P. E., Wagner, R., and Stutz, E. (1986). E. coli. rihosomes with a C912 to U base change in the 165 rRNA are streptomycin resistant. EMBO J. 5:3705-3708. Morgan, E. A . , Gregory, S.T., Sigmund, C. D., and Borden, A. (1988). Antibiotic resistance mutations in Escherichia coli ribosomal RNA genes and their uses. NATO AS1 Ser., SeT. H H14:43-53. Mori, H . , Damrnel, C., Becker, E., Triman, K., and Noller, H. F. (1990). Single base alterations upstream of the E. coli 16s rRNA coding region result in temperature-sensitive 16s rRNA expression. Biochim. Biophys. Ac& 1050:323-327. Mougel, M., Allmang, C., Eyermann, F. Cachia, C., Ehresmann, B., and Ehresmann, C. (1993). Minimal 1 6 s rRNA binding site and role of conserved nucleotides in Escherichia coli ribosomal protein S8 recognition. Eur. J. Biochem. 215:787-792. Murgola, E. I., Hijazi, K. A . , Goringer, H. U., and Dahlberg, A. E. (1988). Mutant 1 6 s rihosomal ~

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RNA: A novel codon-specific translational suppressor. Proc. Natl. Acud. Sci. U. S. A. 85:41624165. Murgola, E. I., Dahlberg, A. E., Hijazi, K. A., and Tiedeman, A. A. (1990). rRNA and codon recognition: The rRNA-mRNA base-pairing model of peptide chain termination. In “The Ribosome: Structure, Function, and Evolution” (W. E. Hill, A. Dahlberg, R. A. Garrett, P. B. Moore, D. Schlessinger, and J. R. Warner, eds.), pp. 402-407. American Society for Microbiology, Washington, DC. Nierhaus. K. H., Franceschi, F., Subramanian, A. R., Erdmann, V. A., and Wittmann-Liebold, B. (1993). “The Translational Apparatus.” Plenum, New York. Noller, H. F. (1991). Ribosomal RNA and translation. Annu. Rev. Biochem. 60:191-227. Noller, H. F. (1993). On the origin of the ribosome: Coevolution of subdomains of tRNA and rRNA. In “The RNA World” (R. F. Gesteland and J. F. Atkins, eds.), pp. 137-156. Cold Spring Harbor Lab., Cold Spring Harbor, NY. Noller, H. F., and Nomura, M. (1987). Ribosomes. In Escherichia coli and SdmoneUa typhimurium: Cellular and Molecular Biology” (F. C. Neidhardt, ed.) Vol 1, pp. 104-125. American Society for Microbiology, Washington, DC. Noller, H. F., Moazed, D., Stem, S., Powers, T., Allen, P., Robertson, 1. M., Weiser, B., and Triman, K. (1990). Structure of rRNA and its functional interactions in translation. In “The Rihosome: Structure, Function, and Evolution” (W. E. Hill, A. Dahlberg, R. A. Garrett, P. B. Moore, D. Schlessinger, and J. R. Warner, eds.), pp. 73-92. American Society for Microbiology, Washington, DC. Nomura, M. (1987). The role of RNA and protein in ribosome function: A review of early reconstitution studies and prospects for future studies. Cold Spring Harbor Symp. Quant. Bid. 52653663. OConnor, M., and Dahlberg, A. E. (1993). Mutations at U2555, a tRNA-protected base in 23s rRNA, affect translational fidelity. Prm. Natl. Acad. Sci. U.S.A. 909214-9218. OConnor, M., De Stasio, E. A., and Dahlberg, A. E. (1991). Interaction between 16s ribosomal RNA and ribosomal protein S12: Differential effects of paromomycin and streptomycin. Biochimie 73: 1493-1500. O’Connor, M., Goringer, H. U., and Dahlberg, A. E. (1992). A ribosomal ambiguity mutation in the 530 loop of E. coli 165 rRNA. Nuckic Acids Res. 204221-4227. Ofengand, J., Bakin, A., and Nurse, K. (1993). The functional role of conserved sequences of 16s ribosomal RNA in protein synthesis. In “The Translational Apparatus: Structure, function, Regulation and Evolution” (K.H. Nierhaus, F. Franceschi, A. R. Subramanian, V. A. Erdmann, and B. Wittmann-Liebold, eds.), pp. 489-500. Plenum, New York. Parker, J. (1992). Variations in reading the genetic code. In “Transfer RNA in Protein Synthesis” (D. L. Hatfield, B. 1. Lee, and R. M. Pirtle, eds.), pp. 191-267. CRC Press, Boca Raton, FL. Pinard, R., Payant, C., Melancon, P., and Brakier-Gingras,L. (1993). The 5’ proximal helix of 16s rRNA is involved in the binding of streptomycin to the ribosome. FASEB J. 7:173-176. Powers, T., and Noller, H. F. (1990). Dominant lethal mutations in a conserved loop in 16s rRNA. PTOC.Natl. A d . Sci. U.S.A. 87:1042-1046. Powers, T., and Noller, H. F. (1991). A functional pseudoknot in 16s ribosomal RNA. EMBO]. 102203-2214. Powers, T., and Noller, H. F. (1993a). Allele-specific structure probing of plasmid-derived 16s ribosomal RNA from Escherichiu cob. Gene 123:75-80. Powers, T., and Noller, H. F. (1993b). Evidence for functional interaction between elongation factor Tu and 16s ribosomal RNA. Proc. Narl. Acud. Sci. U.S.A. 90:1364-1368. Powers, T., and Noller, H. F. (1994). The 530 loop of 165 rRNA: A signal to EF-Tu?Trends Genet. 10227-31.

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Powers, T.,Changchien, L.-M., Craven, G. R., andNoller, H. F. (1988). Probing the assembly of the 3’ major domain of 16s ribosomal RNA: Quaternary interactions involving ribosomal proteins S7, S9 and S19. J. Mol. Biol. 200:309-319. Prescott, C. D., and Dahlberg, A. E. (1990). A single base change at 726 in 16s rRNA radically alters the pattern of proteins synthesized in uivo. EMBO J. 9:289-294. Prescott, C. D., and Goringer, H. U. (1990). A single mutation in 16s rRNA that affects mRNA binding and translation-termination. Nuckic Acids Res. 18:5381-5386. Prescott, C. D., and Kornau, H.-C. (1992). Mutations in E. cob 16s rRNA that enhance and decrease the activity of a suppressor tRNA. Nuckic Acids Res. 20:1567-1571. Prescott, C. D., Krabben, L., and Nierhaus, K. (1991a). Ribosomes containing the C1054-deletion mutation in E. coli 16s rRNA act as suppressors at all three nonsense codons. Nuckic Acids Res. 195281-5283. Prescott, C. D., Kleuvers, B., and Goringer, H. U. (1991b). A rRNA-mRNA base pairing model for UGA-dependent termination. Biochimie 73:1121- 1129. Riley, M. (1993). Functions of the gene products of Escherichia coli. Minobiol. Rew. 57:862-952. Ringquist, S., Cunningham, P., Weitzmann, C . , Formenoy, L., Pleij, C., Ofengand, J., and Gold, L. (1993). Translational initiation complex formation with 30s ribosomal particles mutated at conserved positions in the 3‘minor domain of 16s RNA. J. Mol. Bid. 234:14-27. Ross, W., Gosink, K. K., Salomon, J., Igarashi, K., Zou, C., Ishihama, A., Severinov, K., and Course, R. L. (1993). A third recognition element in bacterial promoters: DNA binding by the alpha subunit of RNA polymerase. Science 262:1407-1413. Rottmann, N., Kleuvers, B., Atmadja, J., and Wagner, R. (1988). Mutants with base changes at the 3’-end of the 16s RNA from Escherichia coli: Construction, expression and functional analysis. Eur. J. Biochem. 177:81-90. Ryden-Aulin, M., Shaoping, Z., Kylsten, P., and Isaksson, L. A. (1993). Ribosome activity and modification of 16s RNA are influenced by deletion of ribosomal protein S20. Mol. Microbiol. 7:983-992. Saarma, U., Lewicki, B. T.U., Margus, T., Nigul, S., and Remme, J. (1993). Analysis of mutations in the 23s rRNA. In “The Translational Apparatus: Structure, Function, Regulation and Evolution” (K. H. Nierhaus, F. Franceschi, A. R. Subramanian, V. A. Erdmann, and B. WittmannLiebold, eds.), pp. 163-172. Plenum, New York. Saito, K., and Nomura, M. (1994). Post-transcriptional regulation of the str operon in Escherichia coli: Structural and mutational analysis of the target site for translational repressor S7. 1. Mol. Biol. 235:125-139. Santer, M., Bennett-Guerrero, E., Byahatti, S., Czarnecki, D., OConnell, D., Meyer, M., Khoury, J., Cheng, X., Schwartz, I., and McLaughlin, J. (1990). Base changes at position 792 of Escherichiucoli 16s rRNA affect assembly of 70s ribosomes. Proc. Natl. Acad. Sci. U.S.A. 87:37003704. Santer, M., Santer, U., Nurse, K., Bakin, A., Cunningham, P., Zain, M., O’Connell, D. O., and Ofengand, J. (1993). Functional effects of a G to U base change at position 530 in a highly conserved loop of Escherichia coli 16s RNA. Biochemistry 325539-5547. Santer, U. V., Dahlberg, A. E., and Santer, M. (1991). Mutations at position 980 of 16s rRNA of E. cob. J. Cell Biol. 115:72a. Santer, U. V., Tate, D., Canfield, S., Kansil, S., and Santer, M. (1993a). Base changes at position 529, but not 527 or 528, in 16s rRNA are highly deleterious for growth and protein synthesis. FASEB J. 7:A1093. Santer, U. V., Cekleniak, J. A., and Santer, M. (1993b). The 530 loop of 16s ribosomal RNA: Each base confers its own phenotype. Mol. Biol. Cell 4:420a. Sigmund, C . D., and Morgan, E. A. (1982). Erythromycin resistance due to a mutation in a ribosomal RNA operon of Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 79:5602-5606.

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Sigmund, C. D., Ettayebi, M., and Morgan, E. A. (1984). Antibiotic resistance mutations in 16s and 23s ribosomal RNA genes of Eschelichia cob. Nucleic Acids Res. 1.24653-4663. Sigmund, C. D., Ettayebi, M., Borden, A., and Morgan, E. A. (1988). Antibiotic resistance mutations in ribosomal RNA genes of Escherichia cob. In “Methods in Enzymology” (H. Noller and K . Moldave, eds.), Vol. 164, pp. 673-690. Academic Press, San Diego, CA. Spurio, R., Severini, M., La Teana, A., Canonaco, M. A., Pawlik, R. T., Gualeni, C. O., and Pon, C. L. (1993). Novel structural and functional aspects of translational initiation factor IF2. In “The Translational Apparatus: Structure, Function, Regulation and Evolution” (K. H. Nierhaus, F. Franceschi, A. R. Subramanian, V. A. Erdmann, and B. Wittmann-Liebold, eds.), pp. 241-252. Plenum, New York. Stark, M. J. R., Course, R. L., and Dahlberg, A. E. (1982). Site-directed mutagenesis of ribosomal RNA: Analysis of ribosomal RNA deletion mutants using maxicells. 1. Mol. Biol. 159417-439. Stark, M. J. R., Gregory, R. J., Course, R. L., Thurlow, D. L., Zwieb, C., Zimmermann, R. A., and Dahlberg, A. E. (1984). Effects of site-directed mutations in the central domain of 16s ribosomal RNA upon ribosomal protein binding, RNA processing and 30s subunit assembly. J. Mol. Biol. 178:303-322. Steen, R., Jemiolo, D. K., Skinner, R. H., Dunn, J. J., and Dahlberg, A. E. (1986). Expression of plasmid-coded mutant ribosomal RNA in E. cub: Choice of plasmid vectors and gene expression systems. Prog. Nucleic Acid Res. Mol. Biol. 33:l-18. Stem, S.,Powers, T., Changchien, L.-M., and Noller, H. F. (1989). RNA-protein interactions in 30s ribosomal subunits: Folding and function of 16s rRNA. Science 244:783-790. Tapprich, W. E., Goss, D. J., and Dahlberg, A. E. (1989). Mutation at position 791 in Escherichia coli 16s ribosomal RNA affects processes involved in the initiation of protein synthesis. Proc. Natl. A d . Sci. U S .A. 86:4927-493 1. Tapprich, W. E., Goringer, H. U., De Stasio, E. A., and Dahlberg, A. E. (1990a). Site-directed mutagenesis of E. cofi rRNA. In “Ribosomes and Protein Synthesis: A Practical Approach” ( 0 . Spedding, ed.), pp. 253-271. Oxford Univ. Press, New York. Tapprich. W. E., Goringer, H. U., De Stasio, E. A., Prescott, C., and Dahlberg, A. E. (1990b). Studies of ribosome function by mutagenesis of Escheiichiu coli rRNA. In “The Ribosome: Structure, Function and Evolution” (W. E. Hill, A. Dahlberg, R. A. Garrett, P. B. Moore, D. Schlessinger, and J. R. Warner, eds.), pp. 236-242. American Society for Microbiology, Washington, DC. Tate, W. P., Adamski, F. M., Brown, C . B., Dalphin, M. E., Gray, J. P., Horsfield, J. A., McCaughan, K. K., Moffat, J. G., Powell, R. J . , Timms, K. M., andTrotman, C. N. A. (1993). Translational stop signals: evolution, decoding for protein synthesis and recoding for alternative events. In “The Translational Apparatus: Structure, Function, Regulation and Evolution” (K. H. Nierhaus, F. Franceschi, A. R. Subramanian, V. A Erdmann, and B. Wittmann-Liebold, eds.), pp. 253-262. Plenum, New York. Theissen, G., Eberle, J., Zacharias, M., Tobias, L., and Wagner, R. (1990). The tLstructure within the leader region of the E. coii ribosomal RNA operons has post-transcriptional functions. Nuckic Acids Res. 18:3893-3901. Theissen. G., Thelen, L., and Wagner, R. (1993). Some base substitutions in the leader of an Escherichia coli ribosomal RNA operon affect the structure and function of ribosomes; evidence for a transient scaffold function of the rRNA leader. J. Mol. Biol. 233:203-218. Thomas, C. L., Gregory, R. I., Winslow, G . , Muto, A., and Zimmermann, R. A. (1988). Mutations within the decoding site of Escherichia coli 16s rRNA: Growth rate impairment, lethality and intragenic suppression. Nuckic Acids Res. 16:8129-8146. Traut, R. R., Oleinikov, A. V.. Makarov, E., Jokhadze, G., Perroud, B., and Wang, B. (1993). Structure and function of Escherichin coli ribosomal protein L7/L12: Effect of cross-links and deletions. In “The Translational Apparatus: Structure, Function, Regulation and Evolution”

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(K. H. Nierhaus, F. Franceschi, A. R. Suhramanian, V. A. Erdmann, and B. Wittmann-Liehold, eds.), pp. 521-532. Plenum, New York. Triman, K. (1994). The 16s Ribosomal RNA Mutation Database (16SMDB). Nucleic Acids Res. 22:3563-3565. Triman, K., Becker, E., Dammel, C . , Katz, J., Mori, H., Douthwaite, S., Yapijakis, C., Yoast, S., and Noller, H. F. (1989). Isolation of temperature-sensitive mutants of 16s rRNA in Escherichia cob. J. Mol. Biol. 209:645-653. Tubulekas, I., and Hughes, D. (1993). A single amino acid substitution in elongation factor Tu disrupts interaction hetween the ternary complex and the ribosome. J. Bactenol. 175:240-250. Wagner, R., Theissen, G., and Zacharias, M. (1993). Regulation of ribosomal RNA synthesis and control of rihosome formation in E. coli. In “The Translational Apparatus: Structure, Function, Regulation and Evolution” (K. H. Nierhaus, F. Franceschi, A. R. Suhramanian, V. A. Erdmann, and B. Wittmann-Liebold, eds.), pp. 119-130. Plenum, New York. Weijland, A , , Harmark, K., Anhorgh, P. H., and Parmeggiani, A. (1993). Specific functions of elongation factor Tu, a molecular switch in protein hiosynthesis, as studied by site-directed mutagenesis. In “The Translational Apparatus: Structure, Function, Regulation and Evolution” (K. H. Nierhaus, F. Franceschi, A. R. Suhramanian, V. A. Erdmann, and B. Wittmann-Liehold, eds.), pp. 295-304. Plenum, New York. Weiss, R. B., Dunn, D. M., Dahlberg, A. E., Atkins, J. F., and Gesteland, R. F. (1988). Reading frame switch caused hy hase-pair formation between the 3’ end of 16s rRNA and the mRNA during elongation of protein synthesis in Escherichia coli. EMBO J. 7:1503-1507. Weitzmann, C. J., Cunningham, P. R., Nurse, K., and Ofengand, J. (1993). Chemical evidence for domain assembly of the Escherichia coli 30s rihosome. FASEB ./. 7:177-180. Wu, H., Wower, I., and Zimmermann, R. A. (1993). Mutagenesis of ribosomal protein S8 from Eschericha coli: Expression, stability, and RNA-hinding properties of S8 mutants. Biochemistry 32:4761-4768. Yamagishi, M., DeBoer, H. A., and Nomura, M. (1987). Feedback regulation of rRNA synthesis: A mutational alteration of the anti-Shine-Delgamo region of the 1 6 s rRNA gene abolishes regulation. J. Mol. Biol. 198:547-550. Zacharias, M., Goringer, H. U., and Wagner, R. (1989). Influence of the G C G C discriminatory motif introduced into the ribosomal RNA P2- and fa^ promoter on growth-rate control and stringent sensitivity. EMBO J. 8:3357-3363. Zacharias, M., Goringer, H. U., and Wagner, R. (1990). The signal for growth rate control and stringent sensitivity in E. coli is not restricted to a particular sequence motif within the promoter region. Nucleic Acids Res. 18:6271-6275. Zacharias, M., Theissen, G., Bradaczek, C., and Wagner, R. (1991). Analysis of sequence elements important for the synthesis and control of ribosomal RNA in E. coli. Biochimir 73:699-712. Zimmermann, R. A , , and Dahlherg, A. E. (1994). “Ribosomal RNA: Structure, Evolution, Gene Expression and Function in Protein Synthesis.” CRC Press, Boca Raton, FL (in press). Zimmermann, R. A , , Thomas, C. L., and Wower, J. (1990). Structure and function of rRNA in the decoding domain and at the peptidyltransferase center. In “The Ribosome: Structure, Function and Evolution” (W. E. Hill, A. Dahlberg, R. A. Garrett, P. B. Moore, D. Schlessinger, and J. R. Warner, eds.), pp. 33 1-347. American Society for Microbiology, Washington, DC. Zwieb, C., and Dahlherg, A. E. (1984). Point mutations in the middle of 16s ribosomal RNA of E. coli produced by deletion loop mutagenesis. Nuclric Acids Res. 12:4361-4375. Zwieh, C., Jemiolo, D. K., Jacob, W. F., Wagner, R., and Dahlherg, A. E. (1986). Characterization of a collection of deletion mutants at the 3‘-end of 16s rihosomal RNA of Escherichia coli. Mol. Gen. Genet. 203:256-264.

Meiotic Sister Chromatid Recornbination Thomas 0. Petes and Patricia J. Pukkila Department of Biology and Curriculum in Genetics University of North Carolina Chapel Hill, North Carolina 27599-3280

1. INTRODUCTION Meiotic recombination occurs after DNA synthesis. Meiotic recombination is usually analyzed by detecting new linkage relationships between two or more heterozygous markers (Figure 2. la). Sister chromatid exchanges are invisible to this type of analysis, since exchange between two identical DNA molecules will not alter linkage relationships (Figure 2.lb). In this chapter, we discuss the genetic and cytogenetic evidence that sister chromatid meiotic recombination events occur; our discussion will emphasize genetic results obtained with Saccharomyes cerevisiue. We compare various properties (frequency, effects of various mutations, etc.) of sister chromatid exchange with “classical” recombination events. Section V concerns an issue that we call the “sister chromatid paradox,” the finding that meiotic crossovers (except for those that involve the rRNA gene cluster) appear to preferentially involve nonsister rather than sister chromatids. Two types of recombination are discussed in this chapter, crossovers and gene conversions. Distinctions between these types of exchange are most easily made in organisms in which all four products of meiosis can be analyzed. In tetrads derived from a diploid with linked markers A and B on one homologue and u and b on the other, a single crossover between the markers results in two spores with markers in the parental configurations (AB and ab) and two markers with markers in the recombinant configurations (Ab and uB) (Figure 2.la). Each marker examined individually shows 2:2 segregation within the tetrad. Gene

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a

7

I

B

Figure 2.1. Sister- and nonsister chromatid crossovers. Centrorneres are indicated by ovals. (a) A single crossover between the A and B loci on nonsister chrornatlds results in two chromosomes with markers in the parental configuration and two chromosomes with markers in the recombinant configurations. (b) A single crossover between sister chromatids results in four chromosomes with markers in the original parental configuratlons.

conversion is the nonreciprocal transfer of information from one chromosome to the other. For example, if sequences derived from the A allele replace those derived from the a allele, 3A: la segregation would be observed. For purposes of this chapter, several points are relevant (reviewed by Petes et al., 1991). First, conversion occurs at every locus, although the frequency of conversion in unselected loci varies from about 1 to 50%. Second, for classical recombination events, about 40% of conversion events are associated with crossovers of flanking DNA. Third, conversion events in S . cerevisae usually show parity (3A:la tetrads equal to 1A:3a tetrads). Fourth, the amount of DNA transferred during a conversion event varies between a few bp to over 10 kb, with an average amount of about 1 kb. Fifth, most meiotic gene conversion in yeast is a consequence of the asymmetric transfer of one strand from one allele to the other, resulting in formation of a heteroduplex. If the heteroduplex contains a mismatch, repair of this mismatch can result in conversion. For example, if the A allele donates a strand to the a allele, and the resulting mismatch in the heteroduplex is corrected to A, then a conversion tetrad of the class 3A: la will be observed. Failure to repair the mismatch will result in a postmeiotic segregation (PMS) event. Finally, as is discussed under Section 11, although most studies have been done with conversion events that involve single-copy genes located on homologous chromosomes, gene conversion can also occur between repeated genes on one chromosome or between genes duplicated on nonhomologous chromosomes. In this chapter, we discuss both sister chromatid conversion and crossover events.

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II. GENETIC AND PHYSICAL ANALYSES OF MEIOTIC SISTER CHROMATID RECOMBINATION A. Unequal sister chromatid crossovers in ribosomal DNA Two types of genetic approaches have been used to study sister chromatid recombination in eukaryotes: detection of unequal sister chromatid exchanges between repeated genes on the chromosome and use of circular DNA molecules. One example of the first approach was the use of recombinant DNA procedures and the yeast transformation system to insert a selectable yeast gene (LEU2) into the rRNA gene cluster (Petes, 1980; Szostak and Wu, 1980). In S. cerevisiae, this gene cluster contains about 150 tandemly repeated 9-kb units, located on chromosome XU. Although the repeats within one haploid strain usually have the same DNA sequence, differences have been detected between different haploids. For example, although most strains of yeast have repeats with seven EcoRI sites per repeat (form I rDNA), strains with only six sites per repeat (form I1 strains) have been identified (Petes et al., 1978). Diploids were constructed in which one homologue contained form I repeats with an insertion of LEU2 and the other homologue had form I1 repeats with a LEU2 insertion (Petes, 1980). The diploid was also homozygous for leu2 mutations at the normal location of this gene. When this diploid was sporulated, although most tetrads segregated 4 Leu+:O Leu- spores, about 10% of the tetrads segregated 3 Leu+:l Leu-spores. Physical analysis of DNA isolated from the Leu+ spores in tetrads with one Leu- spore showed that one of the Leu+ spores had a duplication of the LEU2 insertion. In addition, the spore that had the LEU2 duplication had the same type of rDNA (form I or form 11) as the spore that had lost the insertion. This result strongly indicates that the insertions are redistributed among the spores by unequal sister chromatid crossover events (Figure 2.2a). If the crossovers involved rRNA gene clusters located on homologous chromosomes, the spores that had lost or duplicated LEU2 insertions would contain mixtures of form I and form I1 rDNA (Figure 2.2b); such spores were not detected in the 10 tetrads examined. Thus, unequal sister chromatid crossovers at this locus occur more frequently than crossover events that involve nonsisters. Although the frequency of unequal crossovers detected in this experiment was lo%, this estimate of unequal exchange is a minimal estimate, since only those exchanges occurring between the misaligned insertions could be detected. It is likely that unequal sister chromatid crossovers occur within the rDNA in most tetrads. In contrast, meiotic crossover events involving rRNA gene clusters on nonsister chromatids are relatively infrequent. In diploids with one form I cluster and one form I1 cluster, only 5% of the tetrads had spores with mixtures of form I and form I1 genes (Petes and Botstein, 1977; Petes, 1979).

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a

b

I Figure 2.2. Crossovers within the tandem array of ribosomal RNA genes in yeast (Petes, 1980). White or stippled boxes represent individual rRNA gene repeats; only 9 of the approximately 100 repeats are shown. The white boxes indicate one class of rRNA gene (form I) and the stippled boxes indicate another (form 11). These two types of repeats can be distinguished by restriction analysis. The black boxes represent an insertion of the yeast

LEU2 gene. (a) Unequal sister-strand crossover. The crossover results in one spore with form I repeats without an insertion (Leu- spore) and a second spore with form I repeats with two insertions (Leu+ spore). In addition, two Leu+ spores would have form I1 repeats with a single LEU2 insertion in each. (b) Crossover between insertions on nonsister chromatids. Such a crossover would result in two spores with mixtures of form 1 and form I1 repeats, one Leu- spore with no LEU2 insertions, and one Leu+ spore with two LEU2 insertions. The other two Leu+ spores would have a single LEU2 insertion and contain either form I or form I1 rDNA.

Compared to “classical” (nonsister chromatid) recombination in other regions of the yeast genome, recombination between rRNA genes is suppressed about 100fold. As discussed under Section 11, B, the apparent preference for sister chromatid crossovers relative to nonsister chromatid crossovers appears unique to the rRNA genes.

6. Unequal exchange between duplicated genes A number of related systems have been developed for the analysis of unequal recombination events between duplicated genes; when these exchanges occur between repeats located on one chromosome, they are termed “intrachromosomal” events. Since a large number of these experiments have been done with quite similar results, we restrict detailed discussion to a few experiments. Jackson and Fink (1985) constructed a diploid yeast strain homozygous for a structural alteration of chromosome III in which tandem copies of a 19-kb

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Figure 2.3. Unequal crossovers between duplicated HIS4 genes. Th e duplicated sequences are indicated by brackets, and the broad dark line shows plasmid sequences. The white (his4C)and black (his4A)rectangles represent mutant his4 genes. (a) Unequal crossover between nonsister chromatids. The crossover would result in the following types of spores: ( 1 ) his4C genotype, one copy of plasmid sequences; (2) his4A genotype, no plasmid sequences; (3) His- phenotype, His+ papillations as a consequence of mitotic gene conversion between his4A and his4C genes, two copies of plasmid sequences; and (4) his4A genotype, one copy of plasmid sequences. (b) Unequal sister chromatid crossover. The crossover would result in the following types of spores: ( 1 and 2) his4C genotype, one copy of plasmid sequences; (3) his4A genotype, no plasmid sequences; and (4) his4A phenotype, two copies of plasmid sequences.

direct repeat were separated by bacterial plasmid sequences (Figure 2.3). This duplication contained the HIS4 gene. One homologue contained repeats with a his4A mutation and the other homologue contained repeats with a his4C mutation. This diploid was induced to undergo meiosis, and tetrads were analyzed. The genotype of each spore was analyzed by mating to appropriate tester strains (to determine whether the spores contained his4A or his4C alleles) and by looking for the ability of the spore to revert from the His- to His+ phenotype. Spores that contain one or more copies of his4A or one or more copies of his4C revert at low frequencies, whereas spores with a copy of his4A and a copy of his4C revert to His+ frequently (as a consequence of mitotic recombination between the duplicated genes). In addition, the spores were analyzed by colony hybridization or by Southern analysis to determine whether they contained one, two, or no copies of the plasmid sequences that separated the duplications.

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At a frequency of 17%, Jackson and Fink (1985) found tetrads with the patterns expected for unequal crossing over between the duplicated nonsister chromatids; one example of such a tetrad is shown in Figure 2.3a. The frequency of unequal crossing over between sister chromatids was 1.2%, about 14-fold lower than that between nonsisters. An example of one such tetrad is shown in Figure 2.3b. In a similar experiment with a different repeat (Maloney and Fogel, 1987), unequal crossovers between nonsisters was 5-fold higher than that between unequal sister chromatids. As diagrammed in Figure 2.3b, a reciprocal unequal sister chromatid crossover redistributes the repeats among the spores, but the total number of repeats is conserved in the tetrad. Jackson and Fink (1985) found one tetrad (in 686) that had lost one copy of the repeat. An intrachromatid crossover could generate a tetrad with a net loss of a repeat, since the circular plasmid formed by intrachromatid crossing over would not have had a chromosomal replication origin and would be lost. The rarity of this class of event suggests that crossovers between nonsisters is strongly preferred to both intrachromatid and sister chromatid crossovers. Strains with a deletion of one copy of a duplication could also result from gene conversion as a consequence of repair of a gap (Maloney and Fogel, 1987; Rothstein et al., 1987; Fasullo and Davis, 1987; Schiestl et al., 1988) or by repair using a single-strand annealing pathway (Fishman-Lobe11 et al., 1992). Tetrads with non-reciprocal gains of repeats also represent gene conversion events, presumably by a mechanism related to that causing the deletion (Maloney and Fogel, 1987). Meiotic gene conversion events between duplicated genes that are not associated with loss of a repeat have also been investigated. One system involved a diploid in which one chromosome had duplicated LEU2 genes (one wild-type and one mutant allele separated by plasmid sequences) and the other homologue had one wild-type LEU2 gene (Klein and Petes, 1981). Most (96%) of the tetrads derived from this diploid had 4 Leu+ and no Leu- spores with two of the Leu+ spores having two copies of LEU2 and two having one copy. Six tetrads (of 306) had the properties expected for intrachromosomal gene conversion: 3 Leu+: 1 Leu- spores in which one Leu+ spore and the Leu- spore had two copies of LEU2, and the other two Leu+ spores had one copy of LEU2. Four tetrads had the pattern expected for gene conversion between nonsisters: 3 Leu+:l Leusegregation, with two of the Leu+ spores containing two copies of LEU2, one of the Leu+ spores containing one copy, and the Leu- spore containing one LEU2. Thus, the frequency of intrachromosomal gene conversion was similar to the frequency of classical gene conversion. Since these conversion events were not associated with crossing over, it is unclear whether the transfer of sequences during intrachromosomal conversion involved intrachromatid or unequal sister chromatid pairing. Similar high frequencies of intrachromosomal gene conver-

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sion have been seen in a number of other studies (Klein, 1984;Jackson and Fink, 1985; Wagstaff et al., 1985; Maloney and Fogel, 1987; Gottlieb et al., 1989). High frequencies of intrachromosomal conversion were found even when the duplicated genes were far apart (greater than 100 kb) on the chromosome (Lichten et al., 1987). Approximately 40% of classical gene conversion events are associated with crossing over of flanking markers (reviewed by Petes et al., 1991). In the experiment described previously, none of the six tetrads with intrachromosomal gene conversion had associated crossovers. In a larger sample size of intrachromosomal convertants (40), Klein (1984) found no associated crossovers. These results suggest that intrachromosomal conversion events are qualitatively different from interchromosomal conversion events (reviewed by Fink and Petes, 1984). This conclusion is also supported by experiments in which intrachromosomal recombination between repeated genes is induced by a double-strand break made in vivo in one copy of the repeat (Ray et al., 1988). In the diploid used in this experiment, one homologue had closely linked mutant copies of ADE4 separated by sequences containing TRPl and a yeast origin of DNA replication. One of the mutant genes had an insertion of the target site for the HO endonuclease, and the other copy had a restriction site alteration. The other homologue had a single ade4 gene with a restriction site alteration that distinguished it from the other mutant ade4 genes. Double-strand breaks were stimulated in the gene with the HO target site by using a galactose-inducible HO gene. Conversion events were detected by the formation of wild-type ADE4 spores. Intrachromatid conversion events associated with a crossover would be expected to produce a plasmid containing the TRPl gene. Unequal sister chromatid conversion associated with a crossover would result in loss of the TRPf marker from one spore and a duplication of TRPl in the sister spore. Of 48 tetrads with an intrachromosomal gene conversion, only one was associated with intrachromatid crossing over and one was associated with unequal sister chromatid crossing over. Another interesting feature of the data in these experiments is that the double-strand breaks induced by HO cleavage preferentially (sevenfold preference) underwent gene conversion with the sister chromatids rather than with the nonsisters. In a similar study involving a different method of inducing HO cleavage, double-strand breaks were repaired with no apparent preference for sister or nonsister interactions (J. Haber, personal communication). If doublestrand breaks induced by HO resemble the “natural” lesion that initiates meiotic exchange, these results suggest that there is no preference for nonsister over sister chromatid interactions for gene conversion events, but conversion events between sister chromatids are not often associated with crossovers.

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C. Recombination within circular chromosomes 1. Genetic studies Meiotic crossovers between sister chromatids in yeast were also studied using circular (ring) chromosomes (Haber et al., 1984) based on similar analyses of Drosophila and maize. A diploid strain was constructed that had one circular and one linear homologue of chromosome III. The circular derivative was marked with a URA3 insertion, and the linear derivative was marked with MAL2. A single crossover between a linear and circular chromosome would be expected to generate a dicentric linear chromosome. If this dicentric chromosome was not broken during meiotic segregation, a spore with a Ura+ Mal+ phenotype would be generated; this phenotype might be expected to be mitotically unstable because of the dicentric nature of the chromosome. Such spores were detected. In addition, Haber et al. (1984) found tetrads with two viable spores in which the surviving spores were Mal+ Ura-. These tetrads have the properties expected as a consequence of a single meiotic crossover between the circular sister chromatids, resulting in a dicentric circle. The frequency of observed sister strand exchanges between rings (0.2 per tetrad) in the ring/linear heterozygote was eight-fold less than the frequency of observed exchange between nonsister chromatids (1.7 per tetrad).

2. Physical studies Recombination between sister chromatids of circular chromosomes has also been examined by gel electrophoresis of whole chromosomes (Game et al., 1989). As discussed previously, sister chromatid crossovers between two circular chromosomes would result in a double circle. Although double circles could also be produced by certain combinations of multiple nonsister interactions (Game et al., 19891, calculations of the expected distribution of molecules in various multimeric classes suggested that there was about one sister chromatid crossover per chromosome per meiosis. Since the level of nonsister crossovers was about 2.8 per chromosome, this result indicated that the sister chromatid crossovers were suppressed only three-fold relative to nonsister exchanges.

D. Physical assay of recombination intermediates Two studies provide additional physical evidence relevant to the issue of sister strand recombination (Schwacha and Kleckner, 1994; Collins and Newlon, 1994). In these studies, DNA was isolated from meiotic cells and analyzed by two dimensional gel electrophoresis. DNA from the two homologues contained heterozygous restriction sites in order to distinguish sister and nonsister recombination intermediates. Joint molecule formation between two DNA strands,

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resulting in X-shaped structures, was detected by their aberrant migration in the two-dimensional gel system (Bell and Byers, 1979; Brewer and Fangman, 1987). Joint molecule formation between sister chromatids, in both studies, was at least 10-fold less frequent than that observed for nonsister chromatids. This observation can be interpreted as indicating that the distinction between sister and nonsister chromatid crossovers is made at an early stage of the exchange process, although (as discussed later) this interpretation is difficult to resolve with the observation that there is no apparent difference in the frequency of sister and nonsister gene conversion.

E. Competition between sister and nonsister chromatid recombination Wagstaff et al. (1985) examined intrachromosornal recombination in haploid (spoJ3) strains undergoing meiosis. Using strains with a selectable marker integrated in the ribosomal DNA, they found that 2% of the dyads had undergone an unequal crossover event deleting the marker; this frequency is somewhat less than that observed for unequal crossovers of the same type in diploid strains (Petes, 1980). In haploid strains containing a HIS4 duplication, however, a different result was found. About 30% of the dyads had segregation patterns indicating intrachromosornal recombination. Of 49 recombinant dyads, 24 were unequal sister chromatid crossovers, 17 were intrachromatid crossovers (or conversion events deleting one repeat nonreciprocally), and 5 were intrachromosoma1 conversion events unassociated with crossing over. In diploid strains with the same HIS4 duplication, the frequency of intrachromosomal recombination events was considerably less (Jackson and Fink, 1985); about 1% of the tetrads had undergone intrachromosornal gene conversion and about 1.2% had undergone intrachromosornal crossovers. The simplest interpretation of these studies is that sister and nonsister chromatid recombination events compete for some rate-limiting recombination factor (Wagstaff et al., 1985). Three caveats to this conclusion should be noted. First, since the haploid and diploid strains used in the two studies were not isogenic, an effect of the genetic background on intrachromosomal recombination, independent of the ploidy, cannot be ruled out. Second, the spo13 mutation may influence the frequency of recombination. Third, no competition between sister and nonsister chromatid recombination is observed in the rRNA genes. In this regard, it is interesting that the level of sister chromatid crossovers within the repeated ribosomal RNA genes is extremely high (Petes, 1980), whereas the level of nonsister exchange is extremely low (Petes and Botstein, 1977; Petes, 1979). It is possible that sister chromatid exchange among the ribosomal RNA genes occurs prior to “normal” recombination (Petes, 1980). By this model, sister chromatid exchange would inhibit nonsister exchange, but nonsister chromatid exchange could not inhibit sister chromatid exchange.

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F. Contrast between sister chromatid exchange and exchange involving repeats on nonhomologous chromosomes Although frequent meiotic recombination events between closely linked duplicated sequences are expected, meiotic recombination in yeast is also frequently observed between repeated genes located on nonhomologous chromosomes (Jinks-Robertson and Petes, 1985, 1986; Lichten et al., 1987); this type of recombination has been termed “heterochromosomal.” The frequencies of heterochromosomal gene conversion are similar to that observed for classical recombination. Unlike intrachromosomal conversion events, these events show the same association with crossovers as classical conversion events (JinksRobertson and Petes, 1986; Lichten et al., 1987). Since the duplications in these experiments were small (2-5 kb), extensive sequence homology is not necessary in order to resolve a gene conversion event as a crossover. Therefore, the lack of crossovers observed in intrachromosomal gene conversion is not a consequence of the size of the duplication. The genetic control of heterochromosomal recombination appears to be similar to that observed for classical exchange (Steele et al., 1991). Strains containing rud50, spoll, or hop1 have reduced levels of both types of recombination. In contrast, as discussed under Section 111, the hop1 mutation affects meiotic intrachromosomal exchange less significantly than exchange between homologues (Hollingsworth and Byers, 1989), and the r&O mutation does not reduce unequal sister strand meiotic crossovers in the rRNA gene tandem array (Gottlieb et al., 1989).

111. GENETIC CONTROL OF SISTER CHROMATID RECOMBINATION One method of detecting potential mechanistic differences between sister chromatid and nonsister chromatid meiotic recombination is to examine the effects of different mutations on the two processes. Since mutations that eliminate classical meiotic recombination generally lead to chromosome nondisjunction and spore inviability, these studies usually require special methods of analysis (reviewed in Petes et al., 1991). One method involves shifting cells into medium that promotes sporulation, but returning the cells to medium promoting vegetative growth before the cells are committed to spore formation (return-to-growth protocol). This method was based on the observation that commitment to meiotic levels of exchange precedes commitment to meiotic chromosome disjunction (Sherman and Roman, 1963; Esposito and Esposito, 1974). The second method involves the use of the spol3 mutation, a mutation causing bypass of the reductional division of meiosis (Klapholz and Esposito, 1980). Since meiotic recombination appears to be required for accurate chromosome disjunction only

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at the reductional division, Rec- spoJ3/spoJ3 diploids form viable diploid spores. In addition, haploid spoi3 Rec- strains that express both a and a information form viable haploid meioric products (Wagstaff et al., 1982; Malone, 1983). As expected, some mutations [such as spoJJ (Wagstaff er al., 1985), met4 (Menees and Roeder, 1989)] eliminate both classical recombination and intrachromosomal events (gene conversion and unequal crossovers). Such mutations may represent blocks early in meiosis prior to the initiation of recombination, or defects in enzymes that catalyze steps common to all types of exchange (for example, strand transfer). Other mutations, such as merJ (Engebrecht and Roeder, 1989), reduce (about 10-fold) but do not eliminate both classical and intrachromosomal recombination. Such mutations identify genes that encode products that stimulate, but are not absolutely required for, meiotic exchange. Alternatively, these mutations suggest the possibility of multiple pathways of generating meiotic recombinants. Some mutations have different effects on intrachromosomal and classical recombination. For example, the hop1 mutation reduces classical recombination approximately 10-fold, but has little effect on intrachromosomal gene conversion or intrachromosomal events leading to loss of a duplication (Hollingsworth and Byers, 1989); in other strain backgrounds, however, intrachromosoma1 recombination was reduced by hopJ, although recombination between homologs was reduced more severely (N. Kleckner, personal communication). The mutation redl has effects similar to those observed with hopJ (Rockmill and Roeder, 1990). Since both mutations result in failure to form normal synaptonema1 complexes, these observations suggest that these complexes are not required for certain types of intrachromosomal recombination. Some mutations have different effects on different types of intrachromosomal exchange. Mutations in rad50 greatly reduce classical meiotic recombination and intrachromosomal recombination events involving duplicated HIS4 sequences (both gene conversion and unequal sister chromatid crossovers), but have no effect on unequal crossovers within the rRNA gene tandem array (Gottlieb er al., 1989). Mutations in the sir2 gene result in 10-fold elevated levels of unequal crossing over within the rRNA genes, but have no obvious effect on intrachromosomal recombination involving duplicated HIS4 genes (Gottlieb and Esposito, 1989); thus, the wild-type SIR2 gene product, which is required for silencing expression and recombination of the silent mating type cassettes (reviewed by Herskowitz et al., 1992), also represses recombination within the rRNA gene tandem array. A mutation in redl reduced (12-fold) the frequency of gene conversion between 3-kb duplications containing HIS4 sequences, but had no effect on a 13 kb duplication containing HIS4 (Rockmill and Roeder, 1990). It is not clear whether these types of differences reflect an effect of the sequence, spacing, chromosomal context, transcriptional activity of the repeats, or some more subtle factor. However, these results indicate that it

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may be dangerous to make general conclusions about the effects of mutations on intrachromosomal recombination without examining more than one type of repeat. Mutations in topoisomerase or putative topoisomerase genes lead to elevated levels of intrachromosomal mitotic exchange for some classes of repeats (Christman et al., 1988; Wallis et al., 1989; Kim and Wang, 1989; Aguilera and Klein, 1990). In the only experiment in which the effects of such mutations were examined in meiosis, the topJ mutation had no effect on unequal crossing over in the rRNA gene tandem array (Christman et al., 1988).

IV. CYTOLOGICAL ANALYSES OF SISTER CHROMATID EXCHANGE DURING MEIOSIS It is clear from the previous discussion that sister chromatid exchange can be detected using genetic tools, but since sister chromatids are by definition genetically identical, this detection requires specialized circumstances. In contrast, as Taylor (1958) first showed, sister chromatid exchanges can be detected readily using cytological methods. Taylor (1965) was also the first to show that these techniques could be used to analyze patterns of chromatid breakage and rejoining and relate these to chiasma formation. Ideally, physical methods could be used to distinguish sister chromatids along their length throughout meiotic prophase, so that exchanges involving sister chromatids, exchanges involving homologous chromatids, and chiasmata could simply be recorded. In practice, the available methods fall short of the desired resolution, and some ambiguity remains in interpreting the results. Since DNA replicates in a semiconservative fashion, methods to distinguish between sister chromatids rely on the segregation of differentially marked chromatids after two rounds of replication. One useful method relies on the fact that DNA that contains the base analogue 5bromodeoxyuridine (BRdU) has altered staining patterns (Perry and Wolff, 1974). In particular, chromatids whose DNA contains BRdU in only one of the two chains of the double helix show dark staining when treated with the fluorochrome Hoechst 33258 and the purple Giemsa stain, while chromatids whose DNA contains BRdU in both chains of the double helix stain less intensely. Thus, to obtain differential staining of sister chromatids, one applies BRdU during two consecutive S phases (the premeiotic S phase, as well as during the S phase that immediately precedes it) as illustrated in Figure 2.4a. Using this method, bivalents can be scored directly, so that each chiasma and the pattern of chromatid breakage and rejoining around the chiasma can be observed. In the very first application of this technique to meiotic chromosomes (Tease, 1978)) anomalies in the staining pattern were observed.

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b

53

x-

C

d

Figure 2.4. Differential staining of sister chromatids. Centromeres are indicated by ovals. Solid bars indicate darkly stained chromatids, open bars indicate lightly staining chromatids. In b-d, the bivalents are redrawn in the “open-cross”configurationthat is seen typically in late diplotene. In this configuration, sister chromatids remain associated, homologous centromeres lie on opposite sides of the bivalent, and no chromatids overlap. (a) Differential labeling of sister chromatids after two rounds of DNA replication in the presence of BRdU. DNA strands with no base analogues are indicated using solid lines and DNA strands with BRdU incorporated are indicated using dashed lines. T h e gonial S phase (1) and the premeiotic S phase (2) are indicated. (b) Results of crossing over between two similarly labeled chromatids. (c) Results of crossing over between two differentially labeled chromatids. The chromatids with label switches are not adjacent. (d) Results of crossing over plus sister chromatid exchange. The chromatids with label switches are adjacent.

Tease and Jones (1978) went on to show that the anomalies could be explained if sister chromatid exchange occurs in association with crossing over. They restricted their attention to 326 monochiasmate bivalents of the grasshopper Locusta migratoria in late diplotene-diakinesis where chromosome condensation was optimal for observing the labeling patterns in the differentially stained chromatids. A t this stage, there appears to be considerable homologue repulsion, so that most bivalents lie in an “open cross” configuration (i.e., the sister

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chromatids lie side by side and do not lie on top of each other). They observed that 159 bivalents lacked visible label switches near the chiasma (as expected if the crossover had occurred between similarly stained chromatids) as illustrated in Figure 2.4b. In contrast, 136 bivalents showed chromatids with label switches (one light portion and one dark portion on each) plus two parental chromatids. The position of the label switch in these 136 bivalents corresponded with the position of the chiasma in every case, confirming that chiasma are the result of breakage and rejoining, and sister chromatids remain associated on both sides of the chiasma (there is no “terminalization” of the chiasma by changes in chromatid pairing partners) as illustrated in Figure 2 . 4 ~ .In addition, they observed 95 label switches in the 326 bivalents which bore no obvious relationship to the chiasma position. They concluded that these were the result of sister chromatid exchange. Finally, they observed 3 1 bivalents in which there were two chromatids with a reciprocal label switch, but the chromatids with label switches lay adjacent to each other in the “open cross” configuration (Figure 2.4d). Such bivalents cannot be the result of a single crossover. Tease and Jones (1978) concluded that these bivalents arose from a crossover associated with a sister chromatid exchange. Their additional studies (Jones and Tease, 1981) confirmed the initial findings. Among a total of 1283 monochiasmate bivalents, 150 exhibited an associated sister chromatid exchange. Such events could result in segregations in which it would appear that two “recombinant” chromatids (chromatids with label switches) would proceed through metaphase attached to the same centromere. Thus, in this material, 25% of the sister chromatid exchanges occurred in association with a chiasma. Additional studies have revealed label switches that could only have arisen from sister chromatid exchange (Taylor, 1965; Jones and Craig-Cameron, 1969; Church and Wimber, 1969; Peacock, 1970; Craig-Cameron and Jones, 1970; Jones, 1971), and label switches consistent with the coincident occurrence of sister chromatid exchange and crossing over have also been seen in mammals (Allen, 1979; Kanda and Kato, 1980). It is important to assess if such exchanges are likely to be a feature of meiosis in all systems. For example, it is known that the labeling regimes employed can induce sister chromatid exchange (Kato, 1974). I t is also possible that these exchanges are not meiotic. Due to the labeling regimes employed, it is usually not possible to determine if any particular sister chromatid exchange arose during meiosis or during the previous cell cycle. However, if all sister chromatid exchanges arise as a consequence of the labeling regimes, either mitotically or meiotically, the frequent association between chiasma and sister chromatid exchange that has been observed in several systems is difficult to explain. When these cytological studies are compared to the genetic studies using yeast, several common features are apparent. It is clear that information transfer during meiosis between sister chromatids is common in all these sys-

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terns. It is also clear that the enzymatic machinery that carries out the required breakage and rejoining reactions must be able to distinguish between sister chromatids and nonsister chromatids. This conclusion stems from the observation that the frequency and distribution of sister chromatid exchanges are not equivalent to those between homologues in any system.

V. THE SISTER CHROMATID PARADOX: HOW ARE SISTER CHROMATIDS DISTINGUISHED FROM NONSISTER CHROMATIDS? Crossovers between nonsister chromatids result in chiasmata that can be detected cytologically (Figure 2.4). Chiasmata are essential for proper chromosome disjunction, and it is clear that both their frequency and their distribution are carefully regulated during meiosis. Since homologous DNA sequences on sister chromatids would be expected to be closer within the cell than homologous regions on nonsister chromatids, one might expect that a lesion on a chromatid would be repaired (generating a recombination event) more frequently from its sister than its nonsister. This expectation is in fact met for the rRNA gene tandem array and for DNA damage induced in mitotic cells (Kadyk and Hartwell, 1992).However, such a preference would not lead to chiasmata formation between homologues. Alternatively, if proximity is irrelevant, one might expect that a recipient chromatid would find a homologue twice as often as it would find its sister. This expectation appears to be met for interactions that lead to gene conversion, but not for interactions that lead to crossovers. Instead, there is often greater than a twofold preference for nonsister chromatid crossovers. The mechanisms utilized to distinguish between sister and nonsister chromatids are likely to be fundamental to the mechanism of chiasmata formation. The relevant data (excluding the rDNA results) can be summarized by the following statements: (i) sister chromatid and intrachromatid crossovers are suppressed relative to classical crossovers, although they do occur; (ii) intrachromosomal gene conversion events (sister chromatid and/or intrachromatid) occur as frequently as conversion events between homologues; (iii) meiosisspecific DNA structures that have some of the properties expected for intermediates in recombination are recovered primarily between nonsister chromatids rather than between sister chromatids; (iv) intrachromosomal gene conversion and crossovers occur as often in the absence of a synaptonemal complex as in its presence; and (v) gene conversion events between small (2 kb) regions of homology on nonhomologous chromosomes are often associated with crossovers. For purposes of discussion, we assume that (i) the initiating lesion is a doublestrand break, (ii) the initiating event occurs after DNA replication, (iii) the probability of initiating the break within one pair of sisters is the same as the probability of initiating the break on the other pair, and (iv) the broken chro-

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matid (recipient chromatid) is repaired by interacting (recombining) with an unbroken chromatid (donor chromatid). Below, we discuss whether the distinction between sister and nonsister crossovers represents a late or early step in recombinat ion. If the preference for nonsister crossovers reflects a late step in recombination, then we assume that heteroduplex formation occurs without any distinction between sister and nonsister chromatids. If the heteroduplex involves sister or intrachromatid interactions, the resulting intermediate is preferentially resolved as a noncrossover. According to this view, meiotic gene conversion events between repeated genes on one chromosome should occur at frequencies similar to conversion events involving homologues, but these conversion events would not be associated with crossovers. The model also predicts that conversion events involving similar-sized repeats on nonhomologous chromosomes should have the same association with crossovers as classical conversions. We can imagine several ways in which the late distinction between sisters and nonsisters could be made. For example, the distinction may be made by directing which strands are cut in order to resolve the heteroduplex junction connecting the chromatids. The resolution of the intermediate may be guided by torsional stress differences between intrachromosomal and interchromosomal interactions. Alternatively, sister chromatids might cross over only rarely because of a constraint involving the synaptonemal complex. The role of the complex in recombination has been quite controversial (reviewed by Petes et al., 1991). Kinetic studies indicate that synaptonemal complexes are formed after doublestrand breaks but before production of mature recombinant molecules (Padmore et al., 1991). According to this view, after double-strand breakage, heteroduplex is formed between the broken chromosome and either the sister or nonsister chromatids. Because of pairing constraints imposed by the synaptonemal complex, however, only those heteroduplexes that form between nonsisters are efficiently resolved as crossovers. If the selection against sister chromatid interactions is made at the stage of resolving the recombination intermediate, one might expect that early recombination intermediates would be observed to involve both sister and nonsister chromatids. Schwacha and Kleckner (1994) and Collins and Newlon (1994) have reported meiosis-specific DNA structures that have the properties expected for early recombination intermediates. Although some of these intermediates had the size and properties expected for joint molecules between sister chromatids, these were only 10% of the joint molecules. It is certainly possible that the model is inadequate and neither joint molecules nor heteroduplexes typically form between sister chromatids. Instead, such interactions may occur between homologues and also (to explain the observed conversion patterns) between duplicated sequences present on a single chromatid. Such intrachromatid intermediates would be preferentially resolved as noncrossovers.

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The distinction between sister and nonsister chromatids might be made at an early stage. For example, only one of each pair of sister chromatids might be capable of recombinational interactions (Schwacha and Kleckner, 1994). To explain the data, we suggest that both the ability to act as a donor and also the ability to act as a recipient would need to be regulated. In addition, if one of the two sisters is specifically tagged for recombination interactions, the tag could not extend continuously over a long (greater than 50 kb) region of the chromosome. When patterns of double crossovers were examined in yeast, the proportions of 2:3:4-strand double crossovers were approximately 1:2: 1, as though the interaction among nonsister chromatids is random (Hawthorne and Mortimer, 1960; Fogel and Furst, 1967). Although the physical distances between markers used in these experiments were unknown, if recombination was restricted to one of each pair of sisters for a long distance along the chromosome, then an excess of two-strand double crossovers would have been observed. What mechanism could restrict recombination to one member of each pair of sister chromatids? One possibility involves the conservative segregation of DNA-binding proteins (nucleosomes or sequence-specific binding proteins) to one of the two sister chromatids following DNA replication. Since the presence of transcription factors has been shown to be required for activity of a recombination hot spot near HIS4 (White et al., 1991, 1993), it is tempting to consider that the partition of these factors to one of the sisters may be relevant in distinguishing their recombinational properties. Although such a mechanism may be important in localizing the double-strand break to one of the two sisters, the binding of transcription factors does not inhibit the ability of a chromatid to act as a substrate in the repair of a double-strand break, since strains that are heterozygous for alterations in transcription factor binding sites show half the wild-type levels of recombination (White et al., 1991, 1993). If the pair of chromatids that was unable to bind transcription factors was incapable of interacting with the broken chromatid derived from the other pair of sisters, the heterozygous strains would have no recombination activity. Although these considerations indicate that it is unlikely that conservative segregation of transcription factors is involved in distinguishing sisters for recombination activity, conservative segregation of other sequence-specific proteins or of nucleosomes may be relevant. It would be worthwhile to determine whether histone mutations affect the ratio of sister to nonsister chromatid recombination. There are two more radical models that are mentioned briefly. First, it is possible that conversions and crossovers involve different intermediates. According to this view, gene conversions would arise from a class of short-lived heteroduplex DNA intermediates. These would form frequently between both sister and nonsister chromatids, but the invading strands would not be ligated. Such heteroduplex interactions could be readily reversible, and might escape detection by the methods of Schwacha and Kleckner (1994) and Collins and

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Newlon (1994). These intermediates could result in gene conversions (and rare exchanges) involving both sister and nonsister chromatids. A second class of longer-lived joint molecules would be restricted to occur between homologues and could result in crossovers. Second, it is possible that double strand breaks are essential for chiasma formation, but are not intermediates in the formation of crossovers. One version of this model is that such breaks (never more than one per sister) serve to "mark" the chromatids that cannot serve as recipients in heteroduplex DNA formation. The breaks might also serve as essential topological swivels to permit efficient sister chromatid decatenation and local separation, since joint molecule formation involving two pairs of catenated sister chromatids would be difficult. Such a model would explain the correlation between double-strand breaks and recombination levels while also providing a simple explanation for the puzzling observation that the psoralen-dependent joint molecules studied by Schwacha and Kleckner (1994) contained neither broken nor recombinant single strands. It should be pointed out that the issue of whether both members of a pair of sister chromatids can receive a double-strand break is separable from the issue of preferential crossovers with nonsister chromatids. Relevant information about the ability of both sister chromatids to be broken in one meiosis is provided by studies of hot spots for recombination. The most informative of such studies utilize heterozygous mutations that yield high levels of PMS. In yeast, PMS events are observed as spore colonies in which the two alleles (for example, A and a) segregate postmeiotically; such colonies are generated as a consequence of heteroduplex formation between the two alleles and failure to repair the resulting mismatch (Petes et al., 1991). Thus, the frequency of PMS events reflects the frequency of recombinational interactions. In most tetrads with PMS events, single-sectored colonies of two types are observed: 5A:3a tetrads (two A, one sectored A/a, and one a spore colonies) or 3A:5a tetrads (one A, one sectored A/a, and two a spore colonies). Such tetrads are likely to represent the transfer of a single strand from one allele to the other. At lower frequencies, tetrads with two-sectored colonies are observed: aberrant 6A:2a (two A and two sectored A/a colonies), aberrant 2A:6a (two sectored Ala and two a colonies), and aberrant 4:4 (one A, two sectored A/a, and one a colony) tetrads. Assuming the chromatid with the double-strand break receives information from unbroken chromatid, one can explain these patterns as follows: aberrant 6:2 (both a sisters broken), aberrant 2:6 (both A sisters broken), and aberrant 4:4 (one A and one a sister broken). If chromatids are broken at random and if the broken chromatids interact only with nonsisters, then the expected ratio of aberrant 6:2 plus aberrant 2:6 to aberrant 4:4 tetrads is about 1:l. In most studies (Nag et al., 1989; Nag and Petes, 1990; Alani et al., 1994), the tetrads of the aberrant 6:2 and 2:6 classes are observed less frequently than expected. For example, Nag et al. (1989) found 18 aberrant 4:4 tetrads and 6 tetrads of the aberrant 6:2 and 2:6

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classes. Although Alani et al. (1994) suggest that the excess aberrant 4:4 tetrads represent single double-strand breaks that generate symmetric heteroduplexes, we prefer the aIternative explanation that (usually) only one of the two sister chromatids is broken during meiotic recombination. This explanation is consistent with the observation that the frequency of aberrant 4:4 tetrads is similar to that expected if these events reflect two independent events of heteroduplex formation (Nag et al., 1989; Detloff and Petes, 1992). Thus, this line of experimental evidence supports the conclusion that the two sister chromatids may be different in their abilities to receive a double-strand break, consistent with the suggestion of Schwacha and Kleckner (1994). These data, however, do not demonstrate that the sister chromatids are different in their abilities to interact with a broken chromatid, a necessary condition in order to achieve a preference for nonsister chromatid crossovers. In summary, two types of models are consistent with most of the available genetic information. In one model, heteroduplex formation occurs without preference for nonsister or sister chromatids, but resolution as a crossover occurs primarily when the interaction involves nonsister chromatids. In the second model, only one of each pair of sisters is capable of recombinational interactions. Neither of these models explicitly addresses the observation that crossovers in the rEWA genes preferentially involve sister rather than nonsister chromatids. Depending on which model is preferred, one could explain this reversal of the normal chromatid preference by qualitative differences in the types of heteroduplexes formed between rRNA genes (influencing the resolution of the heteroduplex). Alternatively, if the synaptonemal complex had a role in restricting crossovers to nonsister strands, its absence in the rRNA gene cluster (Giroux et al., 1989) might result in a preference for sister chromatid interactions. Further progress in understanding sister chromatid recombination may require the isolation and characterization of mutants that affect the ratio of sister and nonsister crossovers.

Acknowledgments We thank Sue links-Robertson, Gareth Jones, Nancy Kleckner, and Carol Newlon for their useful comments. The work from our laboratories included in this chapter was supported by the ACS and NIH (T.D.P.) and the ACS and NSF (P.J.P.).

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Hollingsworth, N. M., and Byers, B. (1989). HOP1: A yeast meiotic pairing gene. Genetics 121:445-462. Jackson, J. A., and Fink, G. R. (1985). Meiotic recomhination between duplicated genetic elements in Saccharomyces cerevisiae. Genetics 109:303-332. Jinks-Robertson, S.,and Petes, T. D. (1985). High frequency meiotic gene conversion between repeated genes on non-homologous chromosomes in yeast. Proc. Natl. Acad. Sci. U.S.A. 82:3350-3354. Jinks-Robertson. S., and Petes, T. D. (1986). Chromosomal translocations generated by high frequency meiotic recombination between repeated yeast genes. Genetics 114:73 1-752. Jones, G. H. (1971). The analysis of exchanges in tritium-labelled meiotic chromosomes 11. Stethophyma grossum. Chromosoma 34:367-382. Jones, G. H., and Craig-Cameron, T. (1969). Analysis of meiotic exchange by tritium autoradiography. Nature (London) 223:946-947. Jones, G. H., and Tease, C . (1981). Meiotic exchange analysis by molecular labelling. Chromosomes Today 7:114-125. Kadyk, L. C., and Hartwell, L. H. (1992). Sister chromatids are preferred over homologs as substrates for recombinational repair in Saccharomyces cerevisiae. Genetics 132:387-402. Kanda, N., and Kato, H. (1980). Analysis of crossing over in mouse meiotic cells by BRdU labelling technique. Chromosoma 78:113-121. Kato, H. (1974). Possible role of DNA synthesis in formation of sister-chromatid exchanges. Nature (London) 252:739-741. Kim, R. A., and Wang, J. C. (1989). A subthreshold level of DNA topoisomerases leads to the excision of yeast rDNA as extrachromosomal rings. Cell (Cambndge, Mass.) 57:975-985. Klapholz, S., and Esposito, R. E. (1980). Recombination and chromosome segregation during the single division meiosis in spol2-J and spoJ3-J diploids. Genetics 96:589-611. Klein, H. L. (1984). Lack of association between intrachromosomal gene conversion and reciprocal exchange. Nature (London)310:748-753. Klein, H. L., and Petes, T. D. (1981). lntrachromosomal gene conversion in yeast. Nature (London) 289: 144-1 48. Lichten, M., Ports, R. H., and Haber, J. E. (1987). Meiotic gene conversion and crossing-over hetween dispersed homologous sequences occurs frequently in Saccharomyces cerevisiae. Genetics 115:233-246. Malone, R. E. (1983). Multiple mutant analysis of recomhination in yeast. Mol. Gen. Genet. 189405-41 2. Maloney, D., and Fogel, S. (1987). Gene conversion, unequal crossing-over and mispairing at a non-tandem duplication during meiosis of Saccharomyces cerevisiae. Cum. Genet. 12: 1-7. Menees, T. M., and Roeder, G. S. (1989). ME14, a yeast gene required for meiotic recombination. Genetics 123:675-682. Nag, D. K., and Petes, T. D. (1990). Genetic evidence for preferential strand transfer during meiotic recombination in yeast. Genetics 125:753-761. Nag, D. K., White, M. A., and Petes. T. D. (1989). Palindromic sequences in heteroduplex DNA inhibit mismatch repair in yeast. Nature (London) 340:3 18-320. Padmore, R., Cao, L., and Kleckner, N. (1991). Temporal comparison of recomhination and synaptonemal complex formation during meiosis in S. cerevisiae. Cell (Cambridge, Mass. ) 66: 1239- 1256. Peacock, W. J. (1970). Replication, recombination and chiasmata in Goniaea australasiae. Genetics 65: 593-6 17. Perry, P., and Wolff, S. (1974). New Giemsa methods for the differential staining of sister chromatids. Nature (London) 251:156-158. Petes, T. D. (1979). Meiotic mapping of yeast ribosomal DNA on chromosome XJI. J. Bacteriol. 138:185-192.

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Petes, T. D. (1980). Unequal meiotic recombination within tandem arrays of yeast ribosomal DNA genes. Cell (Cambndge, Mass. ) 19:765-774. Petes, T. D., and Botstein, D. B. (1977). Simple Mendelian inheritance of the reiterated ribosomal DNA of yeast. Proc. Natl. Acad. Sci. U.S.A. 74:5091-5095. Petes, T. D., Hereford, L. M., and Skryabin, K. G. (1978). Characterization of two types of rihosomal RNA genes. J. Bacteriol. 134:295-305. Petes, T. D., Malone, R. E., and Syrnington, L. S. (1991). Recombination in yeast. In “The Molecular Biology of the Yeast Saccharomyces” [ J. R. Broach, J. R. Pringle, and E. W. Jones, eds.), Vol. 1, pp. 407-521. Cold Spring Harbor Lab., Cold Spring Harbor, NY. Ray, A , , Siddiqi, I., Kolodkin, A. L., and Stahl, F. W. (1988). Intra-chromosomal gene conversion induced by a DNA double-strand break in Sarcharomyces cerevisiae. J. Mol. Biol. 201:247-260. Rockmill, B., and Roeder, G. S. (1990). Meiosis in asynaptic yeast. Genetics 126563-574. Rothstein, R., Helms, C., and Rosenberg, N. (1987). Concerted deletions and inversions are caused by mitotic recombination between delta sequences in Saccharumyces cerewtsiue. Mol. Cell. Biol. 7: 1198- 1207. Schiestl, R. H., lgarashi, S., and Hastings, P. J. (1988). Analysis of the mechanism for reversion of a disrupted gene. Genetics 119:237-247. Schwacha, A., and Kleckner, N. (1994). Identification of joint molecules that form frequently between homologs but rarely between sister chromatids during yeast meiosis. Cell (Cambndge, Mass.) 76:51-63. Sherman, F., and Roman, H. (1963). Evidence for two types of allelic recombination in yeast. Genetics 48:253-261. Steele, D. F., Morris, M. E., and Jinks-Robertson, S. (1991). Allelic and ectopic interactions in recombination-defective yeast strains. Genetics 127:53-60. Szostak, 1. W., and Wu, R. (1980). Unequal crossing-over in the ribosomal DNA of Saccharomyces cerewisiae. Nature (London) 284:426-430. Taylor, J, H. (1958). Sister chromatid exchanges in tritium-labeled chromosomes. Genetics 43:5 15529. Taylor, J. H. (1965). Distribution of tritium-labeled DNA among chromosomes during meiosis. J. Cell Biol. 2557-67. Tease, C. (1978). Cytological detection of crossing-over in BUdR substituted chromosomes using the fluorescent plus Giemsa technique. Nature (London) 272:823-824. Tease, C., and Jones, G. H. (1978). Analysis of exchanges in differentially stained meiotic chromosomes of Locusta migraroria after BUdR-substitution and FPG staining. Chromosomn 69:163-178. Wagstaff, J. E., Klapholz, S., and Esposito, R. E. (1982). Meiosis in haploid yeast. Proc. Natl. Acad. Sci. U.S.A. 79:2986-2990. Wagstaff, J. E., Klapholz, S., Waddell, C. S., Jensen, L., and Esposito. R. E. (1985). Meiotic exchange within and between chromosomes requires a common Rec function in Saccharomyces cerewisiae. Mol. Cell. Biol. 5:3532-3544. Wallis, J. W., Chrebet, G., Brodsky, G., Rolfe, M., and Rothstein, R. (1989). A hyperrecornbination mutation in S. cerewisiae identifies a novel eukaryotic topoisomerase. Cell (Cambridge, Mass.) 58:409-419. White, M. A,, Wierdl, M., Detloff, P., and Petes, T. D. (1991). DNA-binding protein RAP1 stimulates meiotic recombination at the HIS4 locus in yeast. Proc. Natl. Acad. Sci. U.S.A. 88:9755-9759. White, M. A., Dominska, M., and Petes, T. D. (1993). Transcription factors are required for the meiotic recombination hotspot at the HIS4 locus in Saccharomyces cerewisiae. Proc. Natl. Acad. Sci. U.S.A. 90:6621-6625.

Mapping of Mammalian

Genomes with Radiation (Goss and Harris) Hybrids Robin J. Leach* and Peter O’Connellt Departments of ‘Cellular and Structural Biology, and +Pathology The University of Texas Health Science Center at San Antonio San Antonio, Texas 78284

1. INTRODUCTION Somatic cell genetic techniques have provided the resources for mapping many genes in the human genome. A wide variety of hybrids have been utilized for this purpose, including (i) whole cell hybrids which are obtained by fusing interspecific cells (Harris and Watkins, 1965), (ii) whole cell hybrids or “framework hybrids” constructed from samples containing well-defined translocation breakpoints (Ruddle, 1973), (iii) hybrids containing human chromosome fragments constructed by chromosome-mediated gene transfer (McBride and Ozer, 1973), (iv) microcell hybrids constructed by microcell-mediated chromosome transfer (Fournier, 1981), and (v) radiation hybrids constructed by irradiation of cells carrying human chromosomes with subsequent fusion to a rodent cell. Of the types of somatic cell hybrids mentioned, only radiation hybrids allow the clear determination of a linear order of markers for mapping of a chromosome. Radiation hybrid mapping offers two major advantages over genetic or meiotic mapping: first, it allows placement of both polymorphic markers and nonpolymorphic markers o n the same map, and, second, it has much higher resolution. Thus, radiation hybrid maps bridge the gap between genetic maps and physical maps constructed using yeast artificial chromosomes or by pulsedfield gel electrophoresis (Cox et al., 1990; Walter and Goodfellow, 1993). This

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chapter describes the use of radiation hybrids for constructing high-resolution maps of the human genome.

II. KEY ADVAWCES IN SOMATIC CELL GENETICS Three technologic advances in the late 1960s and early 1970s expedited the mapping of genes to specific human chromosomes. The first advancement was the ability to construct intertypic hybrids between human X mouse cells and human x hamster cells using inactivated Sendai virus and, later, polyethylene glycol (Harris and Watkins, 1965; Yerganian and Nell, 1966; Pontecorvo, 1975). It was observed by Weiss and Green (1967) that the human chromosomal complement in many human x mouse hybrids was unstable and would segregate with passage in culture, thus leaving interspecific hybrids with a reduced human chromosomal complement. The second advancement was the development of selective media allowing the rapid isolation of specific hybrid cells. The most notable of these was the development of HAT (hypoxanthine, aminopterin, thymidine) selection which selectively kills cells deficient in either hypoxanthine phosphoribosyl transferase (HPRT) or thymidine kinase (TK)while allowing the growth of phenotypically wild-type cells (Littlefield, 1964). In addition to the HAT selection, “back” selections have been developed which allow for the isolation of TKdeficient or HPRT-deficient cell lines. The HAT selection system was rapidly implemented for the isolation of hybrid cells. The final major advancement was the ability to identify the chromosomal content in the hybrids through improved chromosome banding methods (Caspersson et al., 1969). These improved banding methods have made it possible to precisely identify the human components within each hybrid. Taken together, these advancements greatly expedited the mapping of genes to specific human chromosomes. By 1973, approximately 50 genes had been assigned to specific human chromosomes (Ruddle, 1973). Mapping of these human genes, however, required that the gene be expressed in the rodent background. The genes identified either had an electrophoretic variant, distinguishing the human and rodent isozymes expressed in the hybrid cell lines, or had the ability to complement a defined mutant in a rodent cell. In addition, the majority of these genes were not known to be polymorphic in humans so their relative order on the chromosome could not be determined by genetic or meiotic mapping. Moreover, regional localization of the genes in the human chromosomes was difficult and relied heavily on the identification of translocations which segregate with selectable genes. Thus, it became apparent that another means of mapping was needed to provide information for the ordering of genes on a chromosome.

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111. EXPERIMENTS OF GOSS AND HARRIS A. Preliminary experiments of Goss and Harris Pontecorvo (1971) was the first to suggest the use of irradiation to reduce the complexity of somatic cell hybrids. It has been observed that in both intraspecific and interspecific hybrids the chromosomes segregate; however, there were no known predictors of which chromosomes were lost. In order to target a specific genome or parental line for segregation, Pontecorvo proposed pretreating one of the parental lines with X-irradiation or y-irradiation, rendering it vulnerable for preferential loss. In the majority of his experiments, Pontecorvo fused hamster and mouse cell lines after treating one with sublethal doses of irradiation, and obtained the desired segregation pattern in the hybrids. However, no hybrids were obtained in his experiments with lethal doses of irradiation (1600 rads). Goss and Harris (1975) were able to extend Pontecorvo’s approach to mapping the human genome. They found that with larger quantities of cells they were able to obtain both mouse x human and hamster x human hybrids after lethal doses of irradiation. Higher doses of irradiation broke the chromosomes into smaller fragments suitable for high-resolution mapping. In their experiments, human diploid male lymphocytes were irradiated with varying doses of y rays and subsequently fused to hamster cells deficient for HPRT. The cell population was plated at low density and after 24 hr, the cells were refed with medium containing HAT. The HAT-sensitive HPRT-deficient hamster cells were killed and the lymphocytes failed to attach to the fusion flasks. Thus, after removing the medium, the only cells remaining in the flasks were the human x hamster hybrids that were HAT resistant. A total of four fusions were performed by Goss and Harris, one each at 0, 1, 2 , and 4 krad doses. Since the hybrids were isolated by HAT selection, all of the hybrids contained the human HPRT gene, which is located on the human X chromosome. To ensure that each hybrid was of independent origin, each hybrid was isolated from separate flasks. The hybrid cells were tested for the presence of HPRT and three other human X-linked genes: phosphoglycerate kinase (PGKl), glucose-6-phosphate dehydrogenase (G6PD), and a-galactosidase (GLA). The human forms of all of these enzymes are distinguishable from the hamster forms using standard electrophoretic techniques. From their experiments, Goss and Harris demonstrated that the cotransfer frequencies of these markers with the HPRT locus decreased with increasing doses of y-radiation. They argued that the most likely explanation for this observation was that these genes were separated from the HPRT gene by radiation-induced chromosomal breaks. It was highly unlikely that the absence of the enzyme activities was a result of actual gene mutation, since the probability of mutation is too infre-

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quent to account for the large loss of enzyme activity observed after radiation in the experiments. Furthermore, once these genes were separated from the HPRT locus, they would be lost since there would be no selective pressure maintaining them in the cell population. Goss and Harris defined this phenomenon as radiation-induced segregation of syntenic genetic loci. Based on the data generated from the four fusions, the order for these four genes on the human X chromosome was determined. The presented order was consistent with that derived from studies involving spontaneous translocations. In addition to determining the map order for these four genes, Goss and Harris presented the basic theory by which the relative distance between loci could be determined. They defined a segregation event as any event that disrupts the linkage of two genetic loci. The number of segregation events was shown to be proportional to the dose of radiation delivered. Events that segregate one locus from another occur by breaking the DNA between the two loci. The further apart two loci are on a chromosome, the more likely a radiation event will break the DNA between them, resulting in the markers being carried on different chromosomal fragments. Using these principles, they calculated a relative distance between the three loci tested and the HPRT locus by estimating the frequency of breakage between the loci. However, a map containing distances between adjacent markers was not presented in these preliminary experiments. .

B. Subsequent experiments by Goss and Harris Two years later, in the “Journal for Cell Science,” Goss and Harris published two serial papers describing their methods in more detail. The first paper extended their previous report on mapping the human X chromosome (Goss and Harris, 1975) and further characterized the hybrids with other methods of analysis (Goss and Harris, 1977a). The second article described the construction of a new radiation hybrid panel from a human diploid fibroblast line irradiated and fused to an HPRT-deficient mouse cell line. These new hybrids were analyzed from the presence of human genes from both human X chromosome and human chromosome 1 (Goss and Harris, 1977b). In their first paper, Goss and Harris (1975) assumed that the loss of X-linked markers from their panel of hybrids was the result of X-chromosome breakage, with subsequent loss of genes no longer linked to HPRT. In order to test this assumption, each hybrid was examined for the presence of genes not present on the human X chromosome, i.e., indophenol oxidase A (renamed superoxide dismutase 1, mitochondrial; SOD1) and indophenol oxidase B (renamed superoxide dismutase 3 , mitochondrial; SOD2) which map to human chromosomes 21 and 6, respectively. These markers were not linked to the HPRT locus; thus, they serve as models for X-linked genes whose linkage with the HPRT locus has been disrupted. Since the hybrids had been constructed

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from a lymphoblastoid line derived from a male, only one X chromosome could be present in each cell. SOD1 and SOD2 would be expected to have higher frequencies since they were on autosomes and thus each hybrid contained two copies of these genes at the time of fusion. Therefore, the gene frequencies were adjusted to determine the true retention frequency per single copy. Based on this analysis, it was demonstrated that nonselected genes were present in the hybrids with relatively low retention frequencies. Karyotypic analysis was performed on a subset of the hybrid lines. In hybrids obtained from unirradiated lymphocytes, a single copy of the human X chromosome was observed. In contrast, no human chromosome could be identified in any hybrids produced by fusion with highly irradiated lymphocytes. This was consistent with the theory that chromosomal breakage followed by segregation was responsible for the pattern of X-linked markers observed in the hybrids. Further experiments were undertaken in an attempt to demonstrate that linkage to the HPRT chromosome played a role in the retention of X-linked genes. Ten of the original hybrids were grown in medium containing 6-thioguanine, a hypoxanthine analogue. Only hybrids which have lost the human HPRT gene can grow in this medium. These hybrids are referred to as backselectants, presumably arising from the segregation of the human portion of the X chromosome carrying the HPRT gene. Goss and Harris observed that only 1 of 16 unselected X-linked genes originally present in these 10 original hybrids was retained in the backselectants. These data supported the hypothesis that the majority of the X-linked genes are maintained in the original hybrids by virtue of their linkage to the directly selected HPRT gene. In this later publication, Goss and Harris (1977a) presented a map of the X chromosome. To determine the relative distance between loci, they applied the same reasoning that was presented in their original manuscript, and extended this to multiple loci by defining the distance between loci as additive. Thus, this map contained the relative distance between all four genes used to map the X chromosome. The second publication in the “Journal of Cell Science” (Goss and Harris, 1977b) described how radiation hybrid panels could be used to map all human chromosomes, independent of whether the human chromosome of interest was selectively retained in the hybrids. To demonstrate these mapping methods, human chromosome 1 was chosen because it was the largest chromosome and thus has the most widely spaced syntenic human loci. As stated previously, Goss and Harris constructed a new panel of radiation hybrids using an HPRT-deficient mouse cell line fused to irradiated human fibroblasts. The resulting hybrids were screened with a total of eight chromosome 1 loci, the four X-linked enzymes used in their two previous publications, plus two other enzymes (one assigned on human chromosome 9 and one assigned on human chromosome 11). Their data were analyzed using the methods developed for the human X

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chromosome in the previous work (Goss and Harris, 1975,1977a). The frequency of cotransfer of loci in a syntenic group can be used to derive a gene map. Since many of the markers tested were not linked, i.e., these markers were known to be on different chromosomes, it was clear that the coretention of two genes in any one hybrid was not necessarily a direct measurement of the cotransfer frequencies of those genes. To derive the relationship between gene retention in the hybrids and cotransfer, they assumed that the retention of any one human chromosome fragment in a hybrid was independent of the retention of all other fragments. To simplify their calculations, they also assumed that all fragments had the same probability of being retained, regardless of their sequence. More recent data from other laboratories have demonstrated that these two assumptions do not interfere with the final gene order determined from the analysis (see Section VII). Analysis of data from chromosome 1 by Goss and Harris resulted in a map of the chromosome which was completely consistent with the order predicted by cytogenetic analysis. Their human X chromosome data analysis provided a map with the same order and relative distance as that obtained from their first panel of radiation hybrids. In addition, analysis of the hybrids with two nonsyntenic autosomal loci indicated no spurious linkage between these loci and either the X chromosome or chromosome 1. A total of 100 hybrids were utilized to create their maps and they proposed that this set should be sufficient to make maps of all human chromosomes. In summary, the works of Goss and Harris provided a strong foundation for gene mapping with radiation hybrids. They not only developed the methodology for constructing the hybrids, but they also provided the foundations for statistical analysis, which has subsequently been expanded by numerous researchers. Their work was instrumental in developing this valuable technique; however, because of the low density of markers available at the time that these landmark experiments were undertaken, these techniques were virtually unused until the late 1980s.

IV. EXTENSION OF GOSS AND HARRIS' EXPERIMENTS A. Additional experiments using whole genome radiation hybrid panels The radiation hybrids produced by Goss and Harris were again utilized in 1979 to

localize the gene for human phosphoribosyl pyrophosphatase synthetase 1 (PRPS1) to the long arm of the X chromosome between HPRT and GLA (Becker et al., 1979). Human and hamster forms of PRPSl could not be distinguished by electrophoresis; therefore, the human PRPS 1 was detected by specific

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immunochemical inactivation of the enzyme using the concentrated immunoglobulin G fraction of serum from immunized rabbits. This allowed the localization of this gene despite the fact that the PRPSl protein had no detectable polymorphism between species. The radiation hybrid panel has previously been used to localize the gene for PGKl on the X chromosome (Goss and Harris, 1975). In 1985, the radiation hybrid panel was reutilized to localize one of the PGK pseudogenes on the human X chromosome (Willard et al., 1985). The PGKl gene was first localized based on its activity in the hybrids; however, once the gene was isolated and used as a molecular probe on Southern blots (Southern, 1975), a complex multigenic family was observed. To localize the PGK gene family members, a panel of hybrids including both framework hybrids and radiation hybrids were used. In all the previous experiments with the radiation hybrid panel, the hybrids had never been evaluated for specific genomic sequences. Therefore, in order to utilize the panel, Willard et al. (1985) isolated DNA from the cell lines and simultaneously reassayed the hybrids for the other X-linked loci (GLA, G6PD, and PGK1). This was an important step since many of the fragments in the radiation hybrids are not selectively maintained and thus are lost in culture over time. From this work, Willard et al. (1985) localized a PGK pseudogene to the region proximal to the functional PGK gene. Walter et al. (1994) recently described the construction of a human x hamster hybrid panel known as whole genome irradiation and fusion gene transfer radiation hybrids (WH-RHs). Walter et al. (1994) describe the isolation of a panel of 44 hybrids isolated from a fusion between thymidine kinase-deficient A23 hamster cells and human fibroblasts irradiated with 3000 rads. To test for the presence of chromosome-specific markers on the WHRHs panel, polymerase chain reaction (PCR; Saiki et al., 1985)-based markers were used. A total of 40 chromosome 14-specific markers were screened through the WH-RH panel. Their resulting map integrated previously published maps and localized 9 new markers. From these experiments, Walter et al. (1994) suggested that a single panel of 100-200 WH-RHs would be sufficient for constructing maps of the whole genome.

B. Use of radiation for gene enrichment in hybrids Cirullo et al. (1983) had sought to find a means other than transfection for isolating hybrids with small amounts of human DNA. These hybrids would be useful for isolating a gene of interest. Specifically, they were interested in isolating two human tRNA synthetases. For both of these genes, there existed Chinese hamster ovary thermolabile mutants which were complemented by the human homologues. Inspired by the work of Goss and Harris, interspecific hybrids constructed by fusing the thermolabile mutants with primary lympho-

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cytes were lethally irradiated and subsequently refused to the original temperature-sensitive Chinese hamster mutant, and secondary hybrids were isolated. The original hybrids each contained one or a few different human chromosomes, and the secondary hybrids retained small fragments of human genomic DNA including the selected gene. These hybrids were subjected to irradiation, refusion, and selection. The final tertiary hybrids had less than 0.1% of human genomic DNA, thus achieving the desired goal of a hybrid suitable for gene isolation.

C. Chromosome-specific radiation hybrid panels In the late 1980s and in 1990, three groups of investigators published additional panels of radiation hybrids (Graw et al., 1988; Benham e t al., 1989; Cox et al., 1989, 1990). The one unifying factor to all of these panels was that they were all constructed from human x rodent hybrid lines and not diploid human cells. Graw et al. (1988) used the methods described by Cirullo and coworkers (1983) to construct a panel of hybrids containing the selectable gene phosphoribosylglycinamide (GAR) synthetase, which is located on human chromosome 2 1. Unlike their predecessors, however, these authors used these hybrids to regionally localize genes linked to GAR synthetase. Goodfellow and co-workers (Benham et al., 1989) generated a panel of radiation hybrids using a human-hamster hybrid containing a single human X chromosome as a donor. This hybrid was y-irradiated and fused to a TK-deficient hamster cell line with no selection placed on the human component. A total of 29 hybrid clones were isolated and characterized. The motivation for constructing these hybrids was twofold: first, the hybrid panel was considered a useful resource for high-resolution mapping over short distances. Interestingly, Goodfellow had previously utilized chromosome-mediated gene transfer methods (CMGT; McBride and Ozer, 1973) for generating hybrids with small fragments of human X chromosome (Pritchard and Goodfellow, 1987). Benham e t al. (1989) compared the quality of the fragments transferred into radiation hybrids to fragments transferred into hybrids by CMGT. They observed that radiation hybrids show no evidence for rearrangement, unlike CMGT hybrids. Second, the hybrids were seen as a valuable asset for the isolation of genes and sequences from this region of the X chromosome. However, Benham e t al. (1989) did not appreciate the potential of radiation hybrids as mapping tools, stating that radiation hybrids are “. . . not particularly well suited to high-resolution mapping because of the occurrence of multiple human fragments within a recipient line.” Cox and colleagues also brought about a renewed interest in radiation hybrid mapping. In 1989 they published an article on the development of a radiation hybrid panel in the region of the Huntington disease locus on human

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chromosome 4 (Cox et al., 1989). Since they had no hybrid with a selectable maker in this region, they irradiated a Chinese hamster-human hybrid cell line containing an intact chromosome 4 and fused it to GM459, a near-diploid Chinese hamster cell line deficient in HPRT. Seventy-two hybrids were isolated and analyzed. They discovered that the unselected human DNA fragments were retained at high frequencies in the hybrids. The retention frequencies (i.e., the proportion of radiation hybrid cell lines that contain a given marker) ranged from 30 to 60%. This was an important observation since it implied that radiation hybrids provide a means for isolating human chromosome segments that lack selectable markers. In 1990, Cox and co-workers published a key article which described in detail the methods for radiation hybrid mapping. The authors constructed a map of human chromosome 2 1 using 14 DNA markers. Once again, they had created a RH panel without selecting for the human DNA. This paper expanded the statistical principles needed for construction of a radiation hybrid map and for determining the likelihood of one order versus alternative orders. In addition, the programs used to construct the map were made available to other investigators. Cox et at. (1990) were able to compare their radiation hybrid map with the physical maps prepared by pulsed-field gel electrophoresis. Their general conclusion was that the distance estimates deduced by radiation hybrid mapping were directly proportional to physical distances. Moreover, no evidence for hot spots of X-ray breakage on human chromosome 21 was detected. These results once again demonstrated the effectiveness of radiation hybrid mapping for constructing maps of human chromosomes. Since these publications, a flurry of articles have emerged describing the generation of chromosome-specific radiation hybrid panels. A summary of the panels and their general characteristics are presented in Table 3.1. Over 30 different panels have been developed, with each characterized using chromosomespecific markers. Furthermore, numerous hybrid panels under development have been described at human chromosome workshops. For example, at the First International Workshop on Human Chromosome 8, a total of 4 panels were reported (Woods et al., 1993). However, only 1 of these panels has been published to date (Sapru et al., 1994). It appears that radiation hybrid mapping has become a generally utilized means for constructing high-resolution maps of human chromosomes.

V. CONSTRUCTIOW OF RADIATION HYBRIDS To construct radiation hybrids, the donor cells are irradiated with lethal doses of either y rays or X rays to induced double-strand breaks. In general, the X-ray

Nl

Table 3.1. Chromosome-Specific Radiation Hybrid Panels

HC

Parental lines

Species

3 3(W 4 4(5P 4

A9(neo3/t)-5 X A9 GM7297 X B14-150 9TK x GM459 HHW661 x UCW113 HD113.2B x CHTG49

Hu-M X M Hu-Ha x CH Hu-CH X CH Hu-CH X CH Hu-M X CH

4 5(4P 5(4)b 6 6 8 9 9 9 10 11

HHW416 x Ucw113 HHW661 x UCW113 HHW661 x UCWl13 1-7 X RJK459 R2llBl x A23 or W3GH 706-B6 clone 17 X ATS49tg 640-63a12 X W3GH PK87-9 X CHTG49 640-63a12 X CHTG49 762-8A X W3GH J1-11 X CHOKl

Hu-CH Hu-CH Hu-CH Hu-Ha Hu-Ha Hu-Ha Hu-Ha Hu-Ha Hu-Ha Hu-CH Hu-Ha

11

J1

Hu-Ha x Ha

X

380-6

CH CH X CH x CH x CH X Ha x CH x CH x CH X CH x CH X

X

Dose (krad)

yor X

X

Retention No. RH

(%)

No. loci

2-8 2.2-25 6 6.5 0.5-8

96 86 72/10 109/22& 12

3 1-96~ 4-29 30-60 22-28 8-92~

25

6.5 6.5 6.5 7

X X Y

10 40 1-8 4-8 50 4-8

1011134. 1091226. 109126& 65/93c 4140. 11147~ 23 531250. 61-W 28 12

21-27 16-28 15-21 up to 60 up to 1ooa 9-82 4-48 9-100" 0-1wa 4-54 up to 100.

15 13 18 15 21 47 19 17 21 9 56

X

9

102

21-39

16

Y

X Y Y

Y Y Y Y X Y X Y

40

7

7 11 10

Reference Tamari et al. (1992) Siden et al. (1992) C o x et al. (1 989) Altherr et aL (1992) Doucette-Stamm et ai. (1991) Winokur et al. (1993) Warrington et al. (1991) Warrington et al. (1992) Zoghbi et al (1991) Ragoussis et al. (1991) Sapru et al (1994) Florian et al. (1991) Jackson et al (1992) Henske et al (1992) Goodfellow et al (1990) Rose et al. (1990); Glaser et al (1990) Richard et al. (1991)

11 11 12 16 17 17 17 18 19 21 21 21 21 22 X

X X

X(3P

J 1 X 380-6 J1 X 380-6 M28 X Wg3-h RJ83.1FT X RJKM F’CTBA1.8 X W3gH or RAG 7AE-4 X GM459 7AE-4 X GM459 Xll-4A-ld-F-e X Don/a3 2OXP3542-1-4 X UV20 and 2OXP3542-1-4 X Wg3h 153-E9A x adeC 153-E7BX x adeC CHG3 X GM459 CHG3 X GM459 EYEF3A6 X 380-6 C12D X W3GH GM0616 x A23 GM0616 x A23 GM7297 X 814-150

Hu-Ha X Ha Hu-Ha X Ha Hu-M X CH Hu-Ha x Ha Hu-M X C H o r M Hu-R X CH Hu-R X CH Hu-CH x CH Hu-CH x Ha Hu-CH Hu-CH Hu-CH Hu-CH Hu-Ha Hu-CH Hu-CH Hu-CH Hu-Ha

x CH

CH X CH x CH X Ha x CH X CH x CH X CH X

X X

X Y

X Y Y Y Y

9 9 40 7 10 8 3-6 7 5

102 100 17/6@ 233 38/1m 76 44/6lC 98/108. 19/83c

18-29 14-24 12-59 8-83 8-45 44-67 31-60 6-65 NA.

30 32 10 38 22 22 35 91 27

Richard et al. (1993a) Richard er al. (1993h) Sinke et d.(1992) Ceccherini et d (1992) Black et al. (1993) Abel et al. (1993) O’Connell et al. (1994) Francke et al. (1994) Brook et al. (1992)

3 8-10

10/18< 5 103 103 85/13OC 29 67 67 86

NAa NAa 32-59 32-59 17-42 NA 22-70 22-70 4-29

7

Craw et al. (1988) Craw et al. (1988) Cox et al. (1990) Burmeister et al. (1991) Frazer et al. (1992) Benham et al. (1989) Gorski et al. (1992b) Gorski et d (1992a) Siden et al. (1992)

a

8 8 6, 20, 50

8 8 2.5-25

7

14 28 18 5 14 32 22

Note. Hu, human; CH, Chinese hamster; Ha, hamster; M, mouse; R, rat; Hc, human chromosome; RH, radiation hybrid; NA, not available. aHybrids directly selected for human DNA component. hA(B), donor line carried A:B translocation, markers scored on chromosome A. cNo. hybrids analyzed/No. hybrids isolated.

4

w

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Robin J. leach and Peter O’Connell

Figure 3.1. Production of radiation hybrids. A donor cell line is irradiated with lethal doses of X ray or y rays. The donor cell line usually contains a selectable marker. The irradiated cells are fused to a recipient cell line with either polyethylene glycol (PEG) or inactivated Sendai virus. The fused population is placed in selective media and hybrids are isolated.

doses can be given in a shorter amount of time than y rays. Thus, the higher dosage hybrids (e.g., 40-50 krad) have all been constructed with X rays (see Table 3.1). During the exposure, the cells are placed in ice-cold medium to minimize the amount of DNA repair (Goss and Harris, 1975). The irradiated cells are subsequently fused with the unirradiated recipient cells using either polyethylene glycol or inactivated Sendai virus (See Figure 3.1). The recipient cells usually have a mutation which is exploited during the selection of the hybrids. Alternatively, if there is a positive selectable marker (e.g., neo) in the donor line, this selectable marker can be utilized to obtain hybrids but is biased for the retention of the marker locus (Doucette-Stamm et al., 1991). After fusion, the mixed population is placed in medium containing the selective reagent(s). A large number of the radiation hybrid panels have utilized HAT selection. In the majority of these cases, the complementing gene (i.e., hypoxanthine phosphoribosyl transfer gene or thymidine kinase gene) is coded in the rodent genome of the donor hybrid. Thus, the human component in these hybrids is not selectively maintained. This is the most ideal situation for constructing a radiation hybrid map because it allows relatively even retention frequencies for all markers. In the case in which the selectable marker is carried on the human chromosome, retention frequencies approach 100% near the selected locus, thus distorting the map in this area. For hybrids in which the human component is not selectively retained, there is concern over the stability of the human fragments in the radiation hybrids. When these hybrids are tested for the presence of human sequences, it

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is vital that all results be obtained from the same cell population or DNA isolation. Clones produced using high-dosage irradiation lose their human DNA component with prolonged culture or repeated freezing and thawing (Walter and Goodfellow, 1993). Glaser et al. (1990) noted that this instability was observed even when the human DNA was attached to a hamster chromosome. Recent experiments have further demonstrated the long-term instability of human chromosome fragments in radiation hybrids; Sapru et al. (1994) isolated 20 subclones from one of their radiation hybrids. These were tested for the markers that were present in the parental cell line. The clones showed extensive genetic heterogeneity, with only one of the subclones retaining the entire complement of human chromosome sequences present in the original line. The retention rates for the individual PCR-based markers ranged from 5 to loo%, implying that the losses from the subclonal populations were not random. This genetic heterogeneity is an important consideration when evaluating the human sequences in a radiation hybrid panel.

A. Species considerations In experiments described by Goss and Harris (1977a,b), human cells were fused to both hamster and mouse cell lines. Since these experiments, however, most radiation hybrid panels have been constructed using a only hamster recipient cell line (see Table 3.1). In addition to the use of hamster cells as recipients, the majority of chromosome-specificpanels have utilized human-hamster hybrids as donors. The human-hamster x hamster hybrids are consider more useful than other rodent backgrounds since it is believed that hamster hybrids are relatively stable in a near-tetraploid state (Glaser et al., 1990). Numerous investigators have observed that viable hybrids are not produced with high frequencies with every combination of donor and recipient cell line. Cox et al. (1990) have reported success in using a different recipient Chinese hamster cell line after encountering problems with particular crosses. Abel et al. (1993) described problems with two different pairs of donor X recipients when attempting to construct radiation hybrids for human chromosome 17. Specifically, they had difficulty in obtaining hybrids when fusing two different monochromosomal hybrids to two different species of recipient cells (hamster and mouse). From these reports, it is clear that the relative yields for production of a radiation hybrid panel cannot be easily predicted, and improvement in yield must be determined empirically.

B. Dosage effects and retention frequencies Radiation-induced chromosome exchanges involve two distinct steps: breakage of the two strands and subsequent reunion in the new configuration (Heddle,

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Robin J. leach and Peter O’Connell

1965). In radiation hybrids, the irradiation is utilized to both kill the donor line and to induce chromosomal breaks producing hybrids with the desired fragment size. Siden et al. (1992) constructed radiation hybrids using a series of doses: 2.5, 5, 12.5, and 25 krads. By increasing the irradiation treatment from 5 to 25 krads, they observed a 5- to 10-fold reduction in the size of the fragments, as well as a dramatic reduction in the retention frequencies from 27 to 3%. The optimal dosage chosen to construct a panel of radiation hybrids is dependent upon the intended use of the lines. When panels have been constructed for use in making maps of human chromosomes, low dosages result in decreased resolution of the maps, while higher dosages increase the resolution. However, with higher doses there is a significant problem with retention frequencies. At very high doses (greater than 10,000 rads) no significant linkage is observed between loci due to extensive fragmentation and loss. The maximum amount of information is obtained when the retention frequency of 50%. Higher-dosage hybrids which carry a small fragment of DNA from a region of biological interest have been used for constructing recombinant DNA libraries and DNA probes (Florian et al., 1991). When a high-dosage hybrid contains multiple fragments of human DNA, it can be reduced to a single fragment through subcloning, but this requires that the fragments are carried as different chromosomes. Another approach for reducing the complexity of a hybrid containing more than one fragment utilizes fusion of the radiation hybrid with a normal cell line to obtain segregating subpopulations. This method was used by Altherr et al. (1992) to isolate a region from human chromosome 4p. Sixteen secondary hybrids were isolated, with 1 containing the region of interest without other contaminating human DNA sequences. It is generally believed that breakage along the chromosome, as well as the rejoining of the broken ends, is a random process (Heddle, 1965). However, stabilization of a fragment in the hybrid requires the rejoining of the fragment with elements needed for replication and stable mitotic segregations. The preferential retention of the centromere in radiation hybrids has been observed in a number of radiation hybrid panels (Benham et al., 1989; Goodfellow et al., 1990; Lawrence et al., 1991; Ceccherini et al., 1992; Gorski et al., 199213; Sinke et al., 1992; Abel et al., 1993; O’Connell et al., 1994; Francke et al., 1994). This retention may reflect the inherent stability of centromeres in somatic cell hybrids. Goss and Harris (197713) did not see the centromeric effect in their original experiments with human chromosome 1. They observed no significant difference between the retention frequencies of proximal and distal loci. However, Goss and Harris only analyzed the hybrids with a total of eight markers for human chromosome 1 in their experiments. The preferential retention near the centromere was first noted when a much higher density of markers was used in

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the analysis of hybrid panels and especially when markers associated with the centromeric alphoid repeat were used (Goodfellow et al., 1990; Gorski et al., 199213; Francke et al., 1994). An alternate explanation for the high centromeric retention frequencies was proposed by Francke et at. (1994). Since the centromeric heterochromatic region containing the alphoid repeats may comprise a large amount of DNA (as much as 8 Mb for chromosome 18), retention of fragments from any part of this region could give a positive hybridization signal. Thus, they propose that the alphoid repeat should not be considered a single copy locus. However, this cannot explain all the data, since single copy markers adjacent to the alphoid region are often retained with higher frequencies (OConnell et al., 1994). Theoretically, another region that could be retained preferentially in radiation hybrids is the telomere. In experiments by Altherr et al. (1992), who studied the Huntington's disease region of human chromosome 4, one locus which is known to map within 300 kb of the telomere was analyzed. Their data demonstrated that DNA fragments very near the telomeres were as equally likely to be retained in radiation hybrids as other loci. In summary, retention frequencies and dosage are important variables to consider when utilizing any radiation hybrid panel. To increase the level of resolution for a radiation hybrid panel, the panel can be constructed with higher doses of radiation. However, with higher doses of radiation, the retention frequencies decrease. If the retention frequencies become too low, no significant linkage will be obtained from the hybrid panels. An alternate method to increase map resolution is to increase the number of hybrids used in the analysis. Thus, it is clear that an important balance among all these variables must be sought in order to obtain a useful radiation hybrid panel. The roles of retention frequencies, dosage, and number of hybrids in constructing a radiation hybrid map are further described under Section VII.

VI. ANALYSIS OF HUMAN COMPONENT IN RADIATION HYBRIDS A. Scoring radiation hybrids for single-copy human sequences Radiation hybrids have been used to map a wide range of markers. Goss and Harris (1975) used isozymes to characterize the human complement of their hybrids. Antibodies were used by Becker et al. (1979) to detect a human-specific isozyme of PRPSl, whereas Ragoussis et al. (199 1) utilized antibodies to immunoselect hybrids expressing HLA class I heavy chains. Southern blot analysis was first used with radiation hybrids to map genes which were not expressed in the hybrids (Willard et al., 1985). Southern blot analysis continues to be used for many chromosome-specific panels (Benham et al., 1989; Cox et al., 1989;

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Robin J. Leach and Peter O’Connell

Figure 3.2. PCR analysis of a chromosome 3-specific radiation hybrid panel. The marker D3S1305 was amplified in 100-ng aliquots of each radiation hybrid and results from one-half of the panel are shown. A PCR product indicates that the marker is present in the hybrid (e.g., for the upper row, the interpretation is: +---+--++++-). M is a size standard marker line, HUM is human genomic DNA positive control, FH(3)5 is a mouse X human somatic cell hybrid specific for chromosome 3 positive control (this line is the irradiated donor for the hybrid panel), GM459 is the hamster recipient negative control, and FTO-28 is a mouse negative control line. Water is a negative control for the PCR assay.

Goodfellow et al., 1990; Cox et al., 1990; Zoghbi et al., 1991; Burmeister et al., 1991; Frazer et al., 1992; Tamari et al., 1992; Siden et al., 1992; Sinke et al., 1992; Ceccherini et al., 1992; Brook et al., 1992; Gorski et al., 1992b). Recently, investigators have used PCR-based markers almost exclusively. Figure 3.2 demonstrates the scoring of radiation hybrids using PCR-based markers. Francke et al. (1994) screened a radiation hybrid panel for human chromosome 18 with 90 PCR-based markers and reserved Southern blot analysis for determining the presence of the repetitive centromeric marker D18Z1. Some investigators have utilized many different types of markers for screening their panels. For example, Florian et al. (1991) screened their radiation hybrid panel of human chromosome 9 with isozymes, PCR-based markers, and DNA probes using Southern blot analysis. As described (see Section V), radiation hybrids in which the human DNA is not physically linked to a selectively retained sequence will lose human DNA sequences with passage in culture. Since segregation occurs with each

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passage, each radiation hybrid contains a heterogeneous mixture of human DNA sequences. This heterogeneity was clearly illustrated by Sapru et al. (1994), who isolated subclones from a radiation hybrid and demonstrated the wide range of retention frequencies for the different markers. Because of the problem of heterogeneity, the analysis of the hybrids for the presence or absence of markers can be problematic, especially when hybrids are screened using different methods (e.g., Southern blot analysis versus PCR analysis). For example, Francke et al. (1994) compared their data obtained by scoring a portion of their panel using two separate detection methods: Southern blot hybridization and PCR. From the analysis of 66 clones with 14 markers, they observed a 4% difference using these two methods. For each discrepancy, the cell line had scored negative by Southern blot analysis but was positive by PCR analysis. These results demonstrate the greater sensitivity of PCR for scoring hybrid, and the heterogeneous nature of the radiation hybrids.

B. Other types of analysis Fluorescence in situ hybridization (FISH) has become an increasingly popular method for analyzing the content of somatic cells (Pinkel et al. , 1986). Using labeled total human genomic DNA as a probe permits the detection of humanspecific chromosomes or fragments in rodent backgrounds (Durnam et al., 1985). In radiation hybrid panels, FISH analysis using labeled total human DNA has been used to determine the number and relative size of human fragments carried in hybrids (see Figure 3.3A). For example, Siden et al. (1992) screened eight hybrids and demonstrated the presence of one to eight human DNA fragments per cell. The number of fragments appeared to be independent of the irradiation dose used to generate the hybrids. However, the results of FISH analysis were not in complete agreement with those of Southem blot analysis performed on the same hybrids. In one clone, the FISH analysis demonstrated a single human fragment, but the marker analysis clearly showed that at least two different fragments of the human X chromosomes were present. These observations may be explained by undetected heterogeneity in the clone or retention of discontiguous pieces of DNA fragments as a single fragment in the

hybrid.

FISH has been used as a screening procedure to identify hybrids containing human DNA. Sinke et al. (1992) screened 60 radiation hybrids by FISH and identified 12 human DNA-containing hybrids which were subsequently used for marker analysis. Francke et al. (1994) screened 31 of 98 radiation hybrid clones by FISH and identified 17 with sizable fragments of human DNA. Nine of these 17 hybrids revealed more than one pattern of integration indicating heterogeneity. When these FISH data were compared to the typing results from 90

Figure 3.3. (A) Biotin-labeled total human DNA was hybridized to a metaphase spread from a human X rat X hamster radiation hybrid containing portions of human chromosome 17.

Three human chromosomal fragments were detected in this cell line. (8)DNA from human X rat X hamster radiation hybrid, containing portions of human chromosome 17, was IRS-PCR amplified and hybridized to a normal human metaphase spread. One fragment was detected in this radiation hybrid.

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PCR-based markers, the majority of the results were in agreement. However, 3 hybrids were positive for several PCR-based markers and yet were negative by

FISH.

Philippe et ul. (1993) utilized the FISH method on radiation hybrids to study the type of rearrangements which occur in radiation hybrids. Their hybrids were constructed by irradiating a human-hamster cell line containing human X chromosomes and fusing them to a mouse cell line. Thus, donor and recipient rodent lines were from different species. From this model system, they demonstrated that the majority of the interspecific rearrangements occurred between the human and hamster chromosomes, the these chromosomes were rarely associated with mouse DNA. Hamster-mouse chromosomal rearrangements were commonly seen in their hybrids; however, human fragments integrating into mouse chromosomes were observed in only a small percentage of analyzed mitoses. Another common technique utilized for rapidly evaluating the human DNA content in hybrids is a method called interspersed repetitive sequencepolymerase chain reaction (IRS-PCR; Nelson et d., 1989; Ledbetter et d., 1990). This method has been utilized in two ways: first, as a rapid screen for identifying radiation hybrids carrying human sequences, and second, as a method for preparing a FISH probe to determine the origins of the human content in hybrids. The second method is also referred to as “reverse” FISH and was first described by Lengauer et d. (1990). In reverse FISH, IRS-PCR is performed on the radiation hybrid and the PCR products are subsequently used as a probe for hybridizing to normal human metaphase spreads (see Figure 3.3B). Francke et al. ( 1994) used reverse FISH to characterize the hybrid panel described previously. Their FISH results showed an excellent correlation with the marker profile for the individual hybrids. The only exceptions were observed in regions with only a few detectable PCR-based markers. Apparently these regions were too small to generate a sufficient reverse-FISH probe. IRS-PCR has been used as a preliminary screening method for many radiation hybrid panels. For example, Warrington et al. (1991) isolated a total of 226 radiation hybrids and screened them for the presence of human DNA by IRS-PCR. Based on this analysis, 109 human DNA-containing hybrids were identified and utilized to construct the radiation hybrid map. To determine whether this screening affects the final radiation hybrid map, 26 of the IRSPCR-negative hybrids were tested for the presence of four different loci. Only one clone was found to contain any of the four loci, and it contained only one locus. Therefore, some IRS-PCR-negative hybrids do indeed contain human DNA. However, the authors claimed that these hybrids would not affect the radiation hybrid map order but would increase the distances between some markers. In addition, IRS-PCR products from radiation hybrids have been used

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Robin J. Leach and Peter O’Connell

to directly screen cosmid and yeast artificial chromosome libraries (Monaco et

al., 1991; Francis et al., 1994). This eliminates the need for purification, cloning, and analysis of individual IRS-PCR products and provides a rapid method for identifying cloned sequences from regions of interest.

VII. STATISTICAL ANALYSIS FOR RADIATION HYBRID DATA Somatic cell hybrid panels have been extensively used for gene localization studies. Framework somatic cell hybrid panels are collections of cytogenetic breakpoints arising from chromosome-specific translocations and deletions (terminal or interstitial). When a marker is tested, the pattern of the presence (+) or absence (-) across the panel defines a cytogenetic placement; those markers with the same pattern of and - are colocalized in the same cytogenetic “bin.” Ordering of the bins is carried out either by the ordering of the known cytogenetic breakpoints, or by minimization of the obligate breakpoints under the assumption that the majority of the rearranged chromosomes arise from a single breakage event. These analyses are relatively straightforward and have traditionally been carried out manually, although analysis packages are available (Aston and Chakravarti, 1992). The resolution of this type of panel is a function of the number and spacing of the cytogenetic breakpoints, and markers colocalized in a bin cannot be ordered. Radiation hybrid panels address this resolution problem by increasing the number of breakpoints as a function of radiation dose. The donor cell line can be either diploid human cells or a chromosome-specific somatic cell hybrid. The panel of radiation hybrid cell lines can be assayed for retention of specific markers or loci, and the distance between loci can be calculated by the concordance or discordance of marker retention across the panel. The likelihood for the occurrence of a radiation-induced breakpoint between two markers is assumed to be a function of the distance between two markers; therefore, marker coretention is evidence of linkage. The first issue in the design of a radiation hybrid mapping experiment is the number of hybrids required to achieve optimal resolution. This problem has been reviewed by Lunetta and Ebehnke (1994). They calculated the resolving power of radiation hybrid panels of varying size as a function of retention frequency. Retention frequency is the total number of radiation hybrids retaining a given marker divided by the total number of radiation hybrids tested with the marker. Retention frequency for most radiation hybrid panels varies between 0.2 and 0.5 according to the source of the donor and recipient lines in the fusion experiment and the region of the chromosome under study. Lunetta and Ebehnke (1994) showed that the worst-case scenario of a 100-member hybrid panel with 0.2 retention frequency had a 90% probability to uniquely order

+

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(greater than 1000:1 maximum likelihood against next-best order) six randomly spaced markers across a 170 centiRay (cRay) distance. The best-case scenario of a 100-hybrid panel with 0.5 retention frequency had a 90% probability to order eight or nine markers across this distance with the same odds. These results, and results determined empirically by a number of mapping efforts, suggest a radiation hybrid panel size of 90-100 lines is adequate for most mapping experiments (see Table 3.1). It is useful to begin with approximately 130 radiation hybrid lines; this gives the researcher flexibility to discard undesirable lines, i.e., duplicate clones, lines with whole chromosomes, lines with no human DNA, and lines that cannot be clearly scored. This permits the selection of a final radiation hybrid panel of minimum size and maximum resolving power. Interpretation of linkage is complicated by the occurrence of multiple fragments of the same chromosome in a given radiation hybrid. Multipoint analysis can determine the order of markers across the map. Due to the complicated patterns of fragment retention and the number of comparisons needed to evaluate the data, statistical radiation hybrid data analysis packages are used to construct the radiation hybrid map. A number of approaches for analysis of radiation hybrid data have been developed such as those that combine two-point analysis results to build a multipoint map (Falk, 1991, 1992; Wilson, 1992; Cox et al., 1990) and multipoint methods (Green, 1992; Lawrence and Morton, 1992; Boehnke et al., 1991). The multipoint methods most commonly utilized arrive at an order either by minimum breakpoint methods or by maximum likelihood methods. Rather than review all of these methods, we confine our discussion to a widely used and comprehensive radiation hybrid analysis package developed by Boehnke et ul. (1991), designated RHMAP.

A. Radiation hybrid data The protocol for scoring markers on a radiation hybrid panel is a critical step in building the map. Markers are scored as either present (+) or absent (-), which are completely informative; thus, false positives and false negatives distort the map. Ambiguous data can be entered as unknown (?). Testing of the markers is carried out either by Southern blot analysis or, more commonly, by visual inspection of ethidium bromide-stained PCR products from sequence-tagged site (STS) markers. The problem with scoring many markers across the hybrid panel is variation in the relative sensitivity of the markers tested. For instance, a STS marker with suboptimal primers may score negative in hybrid lines for which a fully linked and well-optimized STS marker scores positive; therefore, the former marker will not show linkage to its proper position on the map. These problem markers show abnormally high or low retention frequencies in the initial two-point analysis. We try to avoid selecting such markers as anchor points in initial radiation hybrid map construction.

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Another variable is marker typing strategy. Repeated typing of a single marker-radiation hybrid pair may distort the map because of ascertainment bias. One option to avoid problems is to type markers in parallel and enter data for hybrids that are inconsistent between the two runs as unknowns. This approach is useful provided the number of inconsistencies is low. As with any mapping approach, careful checking of data entry is essential. The initial analysis of the map data identifies regions that may require further examination of data (i.e., isolated positives or negatives).

6. Two-point analysis The first phase of analysis is a pair by pair test of each marker against all other tested markers, or two-point analysis. For example, Table 3.2 shows typing data for four chromosome 8 loci tested in a 97-member chromosome 8-specific radiation hybrid line (T. Lewis and R. Leach, unpublished panel). These data were used for the two-point analysis shown in Tables 3.3A and 3.3B. Table 3.3A indicates the computed retention frequencies for the loci tested. Table 3.3B shows the calculated recombination frequency (e), distance (D), as well as a logarithm of the odds (LOD) score. The value of 6 is determined by summing the number of breakpoints between two markers and dividing that sum by the total number of hybrids tested adjusted for the retention frequency (Cox et al., 1990). The mapping function, D = - In( 1 - O), is analogous to Haldane’s no interference mapping function (Haldane, 1919). The units for D are cRays (100 cRays = I Ray) (Cox et al., 1990). The LOD score evaluates the significance of map distance estimates between two markers. The likelihood of obtaining the observed data for the pair of markers, assuming that the frequency of breakage is 8, is divided by the likelihood assuming the two markers are not linked (8 = 1) and converted to a logarithm expressed as a LOD score. As in meiotic mapping, a LOD score of 3.0 is considered as significant evidence for linkage. The two-point analysis can be used to estimate retention frequencies, distances between markers, and connectivity of the map and can identify linkage groups subject to multipoint analysis. Good estimation of the retention frequencies permits selection of the most appropriate model for subsequent multipoint analysis. Use of smaller linkage groups to simplify the initial multipoint analysis is important for several reasons: multipoint analyses are computationally intensive, with N!/2 possible orders to consider, and inclusion of an unlinked markers in multipoint runs reduces the effectiveness of the analysis.

C. Multipoint analysis Four analysis strategies exist for multipoint radiation hybrid data analysis. First, it is possible to test user-specified orders (from evaluation of the two-point

3. Mapplng ol Mammalian Genomes with Radiation Hybrids Table 3.2. Radiation Hybrid Data for Four Chromosome 8 Loci

(continues)

85

86

Robin J. Leach and Peter O’Connell Table 3.2.-Continued Radiation hybrid No.

44 45 46 47 4a 49 50 51 52 53 54 55 56 57 58 59

Retention status data

D8S341

+

-

-

-

-

-

60 61

-

62 63

-

64

65 66

67 68 69 70

71 72 73 74 75 76 77

78 79 a0 a1 a2 a3 a4 85 86

a7

+ -

+ +

-

-

-

-

+

-

-

+

KW298

D8S323

~asi9a

3. Mapping of Mammalian Genomes with Radiation Hybrids

87

Table 3.2.-Continued Radiation hybrid No.

Retention status data

D8S341

KW298

88 a9 90 91 92 93 94 95 96 97 Note.

D8S323

D8S198

-

+ + +

+ , Marker present; -,

marker absent.

analysis), but this is impractical for large numbers of markers, as N!/2 possible solutions exist for any group of N markers. It is best to consider clusters of markers that show strong evidence of linkage from the two-point analysis. Three options for automated ordering of marker clusters are provided. The most conservative approach is the branch-and-bound method. This method can either accept a user-specified candidate order or establish a machine-generated trial order that is as optimal as possible. As new loci are added one at a time, the program either identifies suboptimal new orders that require more breaks (and eliminates consideration of any possible order that includes that partial order) or identifies a superior solution. In practice, a list of best orders that differ by a userspecified number of breaks is stored. A related approach is designated stepwise ordering, which operates by the same logic but uses partial orders. Once again, computational efficiency is achieved by eliminating all potential orders that include any partial order requiring significantly more breaks than the current Table 3.3A. Locus Retention Probabilities for Four Chromosome 8 Loci (RH2PT) Locus

Typed

P(type4

Retained

P( retained)

D8S34 1 KW298 D8S32 3 D8S198 Total

97 97 97 97 388

1.000 1 .ooo 1.000 1.000 1.000

21 21 18 18 78

0.216 0.216 0.186 0.186 0.201

Table 3.3B. Maximum LOD Scores and Breakage Probability and Distance Estimates for Four Chromosome 8 Loci (RH2PT) LOD

Locus 1

Locus 2

Both typed

D8S341 D8S341 D8S341

KW298 D8S323 D8S198

KW298 KW298 D8S323

Unequal retention

Equal retention

+/+

P(BR)

Dist.

Score

P(BR)

Dist.

Score

9 12 15

12 9 6

0.547 0.673 0.865

0.792 1.118 2.004

3.774 1.964 0.356

0.587 0.674 0.867

0.862 1.121 2.014

3.835 1.944 0.347

4 6

7 9

14 I2

0.352 0.481

0.435 0.481

7.522 4.829

0.353 0.481

0.435 0.657

7.481 4.797

4

4

14

0.273

0.655

9.914

0.257

0.297

9.258

-I-

-/+

+/-

97 97 97

67 67 64

9 9 12

D8S323 D8S198

97 97

72 70

D8S198

97

75

Note. P(BR) and Dist. are equal to the breakage probability and distance in Rays under the unequal and equal models of fragment retention. LOD score is the logarithm of the odds (see text).

3. Mapping of Mammalian Genomes with Radiation Hybrids

89

best partial candidate order. This method is analogous to the build option in the meiotic linkage analysis program. CRIMAP (Barker et al., 1987). This method works best when a good candidate starting map is available and when new markers can be added based on some knowledge of their position (e.g., from two-point analysis). Finally, a simulated annealing approach is available which starts with an order of the markers in question and evaluates new orders based on inversion of blocks of loci. The program accepts any new order that significantly reduces the number of breaks required and rejects any new order that increases the number of breaks. Nearly all possible inversions of the order are considered, and a list of the best orders encountered is kept. The RHMAP package offers two complementary criteria for evaluating marker orders, a minimum breaks criterion and a maximum likelihood criterion. The minimum breaks analysis (RHMINBRK) is computationally efficient and can rapidly select a series of candidate orders. However, RHMINBRK cannot estimate the recombination fraction or enumerate how much better one potential order is versus a second potential order. The maximum likelihood criterion (RHMAXLIK) is computationally intensive and substitutes probabilistic models for radiation hybrid mapping data rather than minimization of radiation-induced breakpoints. In addition, the RHMAXLIK package provides a number of fragment-retention probability models to correct estimates of recombination if differences in fragment retention occur across the chromosome. The provisional map orders and retention frequencies determined from the two-point analysis should be considered in selecting the best fragment-retention model for the maximum likelihood analysis. For example, Table 3.4 shows the minimum breaks analysis for the four chromosome 8 loci considered previously. This analysis was carried out with the stepwise analysis with machine-generated candidate orders. The six best orders are shown ranked by the number of breaks required for each order. Table 3.5 shows the maximum likelihood analysis of the same chromosome 8 data. The orders are ranked by the relative odds against the best-supported order. Also shown are the estimates of D (in Rays) between each Table 3.4. List of Minimum Obligate Break Locus Orders for Four Chromosome 8 Loci (RHMINBRK)

Rank

Breaks

1 2 3 4 5 6

37 41 44 46 47 47

Order

D8S341 D8S341 D8S341 D8S341 KW298 D8S341

KW298 KW298 D8S323 D8S198 D8S341 D8S323

D8S323 D8S198 D8S198 D8S323 D8S323 KW298

D8S198 D8S323 KW298 KW298 D8S198 D8S198

Table 3.5. Most Likely Locus Orders for Four Chromosome 8 Loci (RHMAXLIK)

b&wo

Rank

1

2 3 4 5 6

likelihood difference

Odds

Breaks

O.oo00 2.6848 3.4368 4.5236 5.5377 6.2737

1.0 484.0 2734.3 33385.5 344888.5 1877811.7

37 41 46 44 47 47

Locus order

D8S341 D8S341 D8S341 D8S341 -298 D8S341

KW298 W298 D8S198 D8S323 D8S341 D8S323

D8S323 D8S198 D8S323 D8S198 D8S323 KW298

D8S198 D8S323 KW298 KW298 D8S198 D8S198

0.808 0.809 1.995 1.108 0.809 1.121

0.434 0.654 0.305 0.304 1.117 0.435

0.307 0.307 0.431 0.649 0.307 0.652

1.549 1.770 2.731 2.061 2.234 2.213

3. Mapping of Mammalian Genomes with Radiation Hybrids

91

344888.5 D8S341-

u

D8S198

1877811.7 Figure 3.4. A radiation hybrid map of 4 loci on human chromosome 8. Distances are shown in dose-specific centrirays. Odds are given against the inversion of adjacent loci, i.e., the likelihood of order AEXD vs the likelihood of ACBD, ABCD vs BACD for the left end of the map, ABCD vs ABDC at the right end of the map.

locus for each possible order. These data were calculated with the stepwiseordering option under an equal retention model (retention = 0.201) from Table

3.3A.

Figure 3.4 summarizes these analyses into a map of the four chromosome 8 loci showing the best order and the odds against local inversion. Other possible orders exist (e.g., the third- and fourth-ranked orders from Table 3 . 5 ) , but they are not well supported and it is difficult to represent them in a single linear order.

D. The problem of influential hybrids Once trial orders have been established by the analysis methods, examination of the data will identify certain influential hybrids that are unusual either by containing large numbers of breaks under the best order or by demonstrating isolated + or - data under the best order. These may represent errors in the initial scoring of the markers, and rechecking of the data usually identifies some simple errors. It is not clear how helpful retesting of individual marker-hybrid combinations is as these tests have a strong ascertainment bias. Our approach is to repeat the entire set of hybrids with a problem marker and make a decision as to whether the disputed data point is valid or questionable. A more conservative approach is to repeat all the markers through the problem hybrids, but this is impractical with large numbers of markers. Generally, we retain data that cannot be edited on the basis of rechecking the duplicate typings. In some cases, we drop a hybrid that yields extremely large numbers of tiny fragments if the data suggest that the hybrid itself or the DNA preparation was suboptimal. In summary, the most efficient strategy for generating a robust radiation hybrid map is the use of a careful design of data capture protocols to ensure the maximum confidence in the data. Two-point analysis strategies can then be utilized to assess retention frequency, to evaluate the extent of coverage of the chromosome segment with the markers tested, and to identify marker clusters with trial orders. Multipoint analysis can refine those trial orders, but it is most

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Robin J. leach and Peter OConnell

efficient to subdivide the problem into the clusters of markers and order the clusters, then order and orient the ordered clusters. The minimum breaks criteria offer the fastest solution to generating trial orders, and it is best to carry out this step first to generate and integrate the partial orders determined before proceeding to the maximum likelihood analysis. The trial orders generated and retention frequencies estimated by two-point analysis should be used to select the most appropriate fragment-retention model for the maximum likelihood analysis. The final stage of map assembly should reassess exceptional marker data and the retention model.

VIII. RADIATION HYBRID MAPS IN OTHER MAMMALIAN SPECIES To date, three radiation hybrid panels have been described for mapping a portion of the mouse genome (Hunter et al., 1991; Ollmann et al., 1992; Sefton et d., 1992). The panels were all constructed for high-resolution mapping of regions of interest. These experiments are described briefly. The panel constructed by Hunter et al. (1991) was utilized to map mouse chromosome 1 in the region of Rmc-1, a gene encoding a cellular receptor for MCF class murine retroviruses. A total of 292 hybrids were generated by fusing a pSV2gtp marked mouse-hamster hybrid carrying a portion of mouse Chromosome 1, 72gpt, with a hygromycin B-marked Chinese hamster lung fibroblast, Chyg. The donor cells were exposed to 3-9 krads of y-irradiation and the fused population was placed in selective medium for recovery of the gtp gene (Gazdar e t al., 1977). In order to identify the hybrids carrying the Rmc-1 gene, Hunter e t al. (1991) screened the hybrids with a mouse repetitive element, Tu96, to demonstrate the presence of mouse-specific sequences in hamster cell background. In addition, hybrids were assayed for their suspectibility to MCF infection. Nineteen hybrids were selected from the pool to generate a fine structure map of the region of interest. No statistical analysis or retention frequencies were determined on this hybrid panel. The radiation hybrid panel by Ollmann e t al. (1992) was constructed to map the region around the mouse agouti locus located on mouse chromosome 2. The parent lines for their fusions were ADCT-25, a mouse X Chinese hamster ovary hybrid which carries a 2:16 translocated chromosome, and GM459, an HPRT-deficient hamster fibroblast line used for human x hamster radiation hybrid panels (Cox et al., 1989). The donor hybrid was exposed to 8000 rads of X-irradiation. A total of 86 hybrid were isolated. The radiation hybrid panel was analyzed with eight markers from mouse chromosome 2 and a map was constructed. The resolution of this map was approximately 40-fold higher than that of the meiotic map despite the fact that the average retention of single-copy

3. Mapping of Mammalian Genomes with Radiation Hybrids

93

mouse DNA sequences in the hamster background was only 12%. This is substantially lower than those of similar experiments using human radiation hybrid panels with comparable doses of radiation (Cox et al., 1989). The third radiation hybrid mapping paper for mapping the mouse genome was published by Sefton et at. (1992). Two panels were constructed to precisely map the X-inactivation region in the mouse genome. In these experiments, mouse-hamster hybrid HYBX, which retains the mouse X chromosome in a hamster background, was X-irradiated with 10 (10K) or 50 krads (50K) and subsequently fused to A23, a TK-deficient Chinese hamster cell line. The resulting fused population was placed in HAT, yielding only hybrids which had retained the HYBX-derived thymidine kinase gene without selection of the mouse genome. The 10K radiation hybrids were screened with 17 different markers and the retention frequencies ranged from 5 to 30%. The 50K radiation hybrids were screened with a total of 22 X-specific markers and the retention frequencies ranged between 0 and 50%. However, the overall average frequency was much lower for the 50K panel than for the 10K panel. The two panels of radiation hybrids proved complementary for mapping the X chromosome. The 10K panel was used to map the terminal region of the X chromosome, while the 50K region allowed the orientation of tightly linked sequences in the central region of the X chromosome near the X-inactivation center. These panels of mouse x hamster radiation hybrids appear to complement the existing interspecies meiotic mapping panels that have been extensively utilized for mapping the mouse genome (Davisson and Roderick, 1989). In addition, the radiation hybrids provide valuable resources for obtaining cloned sequences from regions of high biological interest.

IX. SUMMARY AND FUTURE DIRECTION Radiation hybrid mapping has become a means for constructing high-resolution maps of the human genome. In addition, radiation hybrids have recently been implemented for mouse mapping and have been found to have a higher level of resolution compared to that of interspecies meiotic mapping panels. In general, radiation hybrids prepared from human diploid cells have not been used because of the success of chromosome-specifichybrid panels. However, there has recently been a renewed interest in mapping with whole genome radiation hybrids (Walter and Goodfellow, 1993). There are clear advantages and disadvantages to the two different types of radiation hybrid panels. One advantage of the chromosome-specific panels is their reduced complexity. Since the human component in the hybrids is derived from a single chromosome or a reduced number of chromosomes, the hybrid can

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Robin J. Leach and Peter O’Connell

be used as a resource for chromosome-specific marker isolation. Using methods, such as IRS-PCR, to obtain the human sequences has greatly improved their usefulness. Moreover, the hybrids are easier to analyze with markers from multigenic families due to their reduced complexity. There are also numerous disadvantages to chromosome-specificpanels. First, the donor hybrid containing the “intact” human chromosome may actually contain deletions or small rearrangements in the human chromosome that are not detectable by routine analysis. In these cases, these altered regions will produce inappropriate maps. Second, the production of these chromosomespecific panels has spawned a “cottage industry.” For example, numerous laboratories have prepared multiple panels for the human X chromosome, as well as human chromosomes 4 and 17 (see Table 3.1). One reason for the production of duplicate panels is related to the logistics of distributing hybrids or Dias. Since the hybrids are heterogeneous, it is not ideal to distribute hybrids to other laboratories for preparing chromosome maps because the hybrids change with time in culture. In order to combine the data generated from multiple laboratories, it is necessary to generate large quantities of DNA for all the analyses; unfortunately, this is not always feasible. In addition, hybrid data gathered from multiple panels of radiation hybrids using the same markers cannot be merged unless the panels are made with the same dosage of radiation. As radiation hybrid mapping has become more popular, numerous laboratories have expressed an interest in gaining access to hybrid panels. Since many laboratories work with markers distributed throughout the genome, it is difficult for these investigators to obtain a panel for every chromosome. Thus, there is a renewed interest in total genome hybrid panels because they can be used without a piori knowledge of a marker’s location. Whole genome radiation hybrids have numerous advantages. The human donor lines can be primary cells which are known to be euploid. Since a single panel covers the whole genome, an estimated 100 hybrids are necessary for obtaining a map of each chromosome. It has been proposed that a universal panel be constructed and widely distributed; such a panel is currently being propagated (announced at 1994 Mapping and Sequencing Meeting at Cold Spring Harbor). This panel will allow data generated in many laboratories to be combined. Although the whole genome panel is a wonderful mapping resource, it will not provide the luxury of obtaining cloned regions of interest, as does the single chromosome panel. In addition, the diploid nature of the donor lines must be taken into consideration using new statistical models. With the rapid increase in the number of genetic markers, the radiation hybrids first envision by Goss and Harris (1975) have now become important tools for the mapping of the human genome.

3. Mapping of Mammalian Genomes with Radiation Hybrids

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Acknowledgments We thank Charles Leach and Tracey Lewis for their critical comments and suggestions. We also thank Tracey Lewis for her unpublished data, David Cox and Michael Boehnke for the RHMAP analysis programs, and Kai Yu for assistance with the analysis. We appreciate the help of Debbie Montoya, Vladimir Pekkel, and Morris Lewis for their assistance with figures and the manuscript, and Michael Siciliano for Fig. 38. The work was supported in part by Grant HG00470 from the NIH.

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Isolation of DNA markers from a region between incontinentia pigmenti 1 (IP1) X-chromosomal translocation breakpoints by a comparative PCR analysis of a radiation hybrid subclone mapping panel. Genomics 14:649-656. Gorski, J. L., Boehnke, M., Reyner, E. L., and Burright, E. M. (1992b). A radiation hybrid map of the proximal short arm of the human X chromosome spanning incontinentia pigmenti 1 (IP1) translocation breakpoints. Genomics 14:657-665. Goss, S. J., and Harris, H. (1975). New methods for mapping genes in human chromosomes. Nature (London) 255:680-684. Goss, S. J., and Harris, H. (1977a). Gene transfer by means of cell fusion. I. Statistical mapping of the human X-chromosome by analysis of radiation-induced gene segregation. J. Cell Sci. 215: 17-37. Goss, S. J., and Harris, H. (1977b). Gene transfer by means of cell fusion. 11. The mapping of 8 loci on human chromosome 1 by statistical analysis of gene assortment in somatic cell hybrids. J. Cell Sci. 25:39-57. Graw, S., Davidson, J., Gusella, J., Watkins, P., Tanzi, R., Neve, R., and Patterson, D. (1988). Irradiation-reduced human chromosome 21 hybrids. Somatic Cell Mol. Genet. 14:233-242. Green, P. (1992). Construction and comparison of chromosome 21 radiation hybrid and linkage maps using CRI-MAP. Cytogenet. Cell Genet. 59:122-124. Haldane, J. B. S. (1919). The combination of linkage values, and the calculation of distance between the loci of linked factors. J. Genet. 8:299-309. Harris, H., and Watkins, J. F. (1965). Hybrid cells derived from mouse and man: Artificial heterokaryons of mammalian cells from different species. Nature (London) 205:640-646. Heddle, J. A. (1965). Randomness in the formation of radiation-induced chromosome aberrations. Genetics 52:1329-1334. Henske, E. P., Ozelius, L., Anderson, M. A,. and Kwiatkowski, D. J. (1992). Aradiation-reduced hybrid cell line containing 5 Mb/17 cM of human DNA from 9q34. Genomics 13:841-844. Hunter, K., Housman, D., and Hopkins, N. (1991). Isolation and characterization of irradiation fusion hybrids from mouse chromosome 1 mapping Rmc-1, a gene encoding a cellular receptor for MCF class murine retroviruses. Somatic Cell Mol. Genet. 17:169-183. Jackson, C . L., Britt, D. E., Graw, S. L., Potts, A., Santoro, K., Buckler, A. J., Housman, D. E., and Mark, H. F. L. (1992). Construction and characterization of radiation hybrids for chromosome 9, and their use in mapping cosmid probes on the chromosome. Somatic Cell Mol. Genet. 18:285-301. Lawrence, S., and Morton, N. (1992). Physical mapping by multiple painvise analysis. Cytogenet. Cell Genet. 59:107-109. Lawrence, S., Morton, N. E., and Cox, D. R. (1991). Radiation hybrid mapping. Proc. Natl. Acad. Sci. U.S.A. 88:7477-7480. Ledbetter, S. A., Nelson, D. L., Warren, S. T., and Ledbetter, D. H. (1990). Rapid isolation of DNA probes within specific chromosome regions by interspersed repetitive sequences (IRS) PCR. Genomics 6:475-481. Lengauer, C., Riethman, H., and Cremer, T. (1990). Painting of human chromosomes with probes generated from hybrid cell lines by PCR with Alu and L1 primers. Hum. Genet. 86:l-6. Littlefield, J. W. (1964). Selection of hybrids from matings of fibroblasts in oitro and their presumed recombinants. Science 145:709-710. Lunetta, K. L., and Boehnke, M. (1994). Multipoint radiation hybrid mapping: Comparison of methods, sample size requirements, and optimal study characteristics. Genomics 21:92-103. McBride, 0. W., and Ozer, H. L. (1973). Transfer of genetic information by purified metaphase chromosomes. Proc. Natl. Acad. Sci. U.S.A. 70:1258-1262. Monaco, A. P., Lam, V. M. S., Zehetner, G., Lennon, G. G., Douglas, C., NizetiC, D., Good-

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The Origin of Numerical Chromosome Abnormalities Patricia A. Jacobs

Wessex Regional Genetics Laboratory Salisbury District Hospital Salisbury, Wiltshire SP2 8BJ United Kingdom

Terry J. Hassold Department of Genetics Case Western Reserve University Cleveland, Ohio 44106-4955

1. INTRODUCTION Until the late 1950s the human chromosome number was thought to be 48, as first reported by von Winiwarter in 1912, on the basis of observations of meiotic cells from sections of human testes. Von Winiwarter’s observations were supported by a number of other authors over the subsequent years and it was not until the seminal publication of Tjio and Levan in 1956 based on observations of dividing fetal lung fibroblasts that the correct number of 46 was established. Tjio and Levan’s observations were quickly confirmed by Ford and Hamerton (1956) using direct preparations of human testicular material. However, the realization that monosomy and trisomy involving a whole chromosome were compatible with survival to birth and associated with significant human morbidity did not occur until 3 years later. In 1959 an additional small acrocentric autosome was shown to be the cause of Down’s syndrome (Lejeune et al., 1959), an additional sex chromosome the cause of Klinefelters syndrome (Jacobs and Strong, 1959), and a missing sex chromosome the basis of Turner’s syndrome (Ford et al., 1959). These first observations of the cytogenetic basis of human disease ushered in the modem era of clinical genetics. The three syndromes shown in 1959 to be caused Advances in Genelico, Vol. 33 Copyright 0 1995 by Academic Press. Inc. All rights of reproduction in any form reserved.

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by additional or missing chromosomes were already well documented as clinical entities. However, the subsequent year saw the description of previously unrecognized syndromes caused by an additional chromosome, notably Edward’s syndrome which is due to an additional chromosome 18 (Edwards et uI., 1960) and Patau’s syndrome which is caused by an additional chromosome 13 ( Patau et al., 1960). These were but the first of many previously unrecognized syndromes identified as clinical entities on the basis of their causal chromosome abnormality. While it subsequently became apparent that the only complete monosomy compatible with life was that of the X chromosome and the only complete trisomies compatible with survival to term were those involving the sex chromosomes and autosomes 13, 18, and 21, many partial monosomies and partial trisomies are now known to be the cause of a variety of different congenital abnormalities and syndromes. These partial monosomies and trisomies can either arise de novo during gametogenesis in one or the other parent or result from aneuploid segregation in a parent carrying a balanced structural rearrangement. In 1963 Carr published the first cytogenetic study of spontaneously aborted fetal material and showed that a remarkably high proportion were associated with chromosome abnormalities that were incompatible with survival to term. Many subsequent studies confirmed Carr’s observations and it is now generally agreed that approximately 50% of all pregnancies that survive long enough to be clinically recognized and that are subsequently spontaneously aborted prior to the 24th to 28th week of gestation have an abnormal chromosome constitution. The great majority of chromosome abnormalities in spontaneous abortions are numerical, with X chromosome monosomy being responsible for about 20% of the aberrations and trisomy of both the sex chromosomes and the autosomes are responsible for no less than 50%. Thus, monosomy and trisomy are one of the most common causes of morbidity and mortality in our species.

II. THE INCIDENCE OF MONOSOMY AND TRISOMY In the 1970s a number of centers undertook cytogenetic testing of large series of unselected newborns and the results of these studies have been summarized by Hook and Hamerton (1977) and Jacobs et al. (1992). The frequencies of the different classes of abnormalities were very similar in all studies and the results showed 0.023% of all births to have a 45,X constitution, 0.162% to have an additional sex chromosome, and 0.142% to have an additional autosome. Thus, some 0.3% of all newborns are monosomic or trisomic for a whole chromosome. A number of cytogenetic studies of spontaneous abortions and stillbirths were summarized by Jacobs and Hassold (1987). These showed 8.6% of spontaneous abortions and 0.25% of stillbirths to have a 45,X constitution, while no fewer

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Table 4.1. Frequency of Chromosome Abnormalities Abnormality (%) Population

45,X

Trisomic

Polyplaid

Structural

Spontaneous abortions Stillbirths Livebirrhs All clinically recognized pregnancies"

8.60 0.25 co.01 1.30

26.8 3.8 0.3 4.3

9.8 0.6 -

2.0 0.4 0.6

1.5

0.8

0.3

5.8

Probability of survival to birth (%)

0

62.0

Other

0.7 0.6 0.02 0.15 11.5

Total abnormal

41.9 5.65 0.93 8.05 6.0

OAssurning 15% spontaneous abortions and 1% stillbirths.

than 26.8% of spontaneous abortions and 3.8% of stillbirths were trisomic with one, or occasionally two, additional chromosomes (Table 4.1). The distribution of monosomies and trisomies by specific chromosome in the newborn and in early and late pregnancy losses is shown in Table 4.2 together with their probability of survival to birth. A number of characteristics are shown from this Table. First, the great difference in the incidence of trisomy for different chromosomes even among spontaneous abortions, ranging from the absence of trisomies of chromosomes 1 and 19 to the observation that 7.5% of spontaneous abortions have an additional chromosome 16, which makes trisomy 16 the most common trisomy in our species. While some of the variation in frequency among different chromosomes is undoubtedly due to differential selection at different stages of pregnancy, it must also reflect different frequencies of occurrence of nondisjunction among different chromosomes. Second, the enormous amount of selection that occurs prior to birth with trisomies only for the sex chromosomes and autosomes 13, 18, and 21, being compatible with live birth. Third, even among the monosomies and trisomies compatible with live birth, there is a great deal of selective intrauterine mortality with only 0.3% of 45,X conceptuses, 3% of trisomy 13, 5% of trisomy 18, and 22% of trisomy 21 pregnancies surviving to birth. Because of their very abnormal phenotypes, it is understandable why so few conceptuses with these abnormalities survive to term for the autosomal trisomies, but it is not at all clear why so many 45,X conceptuses, with their relatively normal phenotypes when live born, succumb during embryonic and fetal life. Even two of the three sex chromosome trisomies have an increased intrauterine mortality with only 55% of the 47,XXY and 70% of the 47,XXX conceptions surviving to birth. In contrast, a 47,XYY karyotype has never been recorded in a spontaneous abortion or stillbirth making it the only numerical chromosome abnormality that is not selected against prior to birth.

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Table 4.2. Monosomics and Trisomics: Frequency and Probability of Survival to Birth Probability of survival to birth

Population Chromosome

1 2 3 4

5

6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

Spontaneous abortions

Stillbirths

Livebirths

0 0 0 0 0 0

1.1 0.3 0.8 0.1 0.3 0.9 0.8 0.7 0.5 0.1 0.2 1.1 1.0 1.7

0.005 -

1.1 0.1 0.5

xxx XYY xo

7.5 0.1 1.1 0.6 2.3 2.7 1.1 0.8 0.2 0.1 8.6

0.25

0.01 0.12 0.02 0.05 0.05 0.05 <0.01

Total

34.7

4.25

0.3

Mosaic trisomy Double trisomy

XXY

(%)a

-

-

0.4 0.3

-

0 0 0 0 0

2.8 0 0 0 0 5.4 0 22.1 0 9.0 0 55.3 70.0 100.0 0.3 4.6

aAssuming 15% spontaneous abortion and 1% stillbirth.

The fourth characteristic to note from Table 4.2 is the absence of all monosomies with the exception of that for the X chromosome. Yet for every gamete formed with an additional chromosome there should be a complementary one with a missing chromosome which would, upon fertilization, result in a monosomic conceptus. The absence of monosomies from even the earliest cytogenetically studied human pregnancies suggest that such conceptuses are lethal prior to the establishment of a clinically recognized pregnancy. It has been shown in the mouse that nullisomic gametes are indeed formed with the ex-

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pected frequency but that the resulting monosomic conceptions die at a very early stage of pregnancy (Gropp and Winking, 1981). It seems reasonable to assume that a similar situation exists in man and that autosomal and Y chromosome monosomy, together with the most lethal trisomies, are responsible for a significant proportion of very early pregnancy loss.

111. THE ETIOLOGY OF MONOSOMY AND TRISOMY A very large number of factors, both extrinsic, such as ionizing radiation, oral contraceptives, fertility drugs, and smoking, and intrinsic, such as thyroid autoimmunity, decreased parental HLA heterogeneity, persistent nucleolar associations, and some types of cytogenetic heteromorphisms, have been invoked in the etiology of numerical chromosome abnormalities in general and trisomy 21 in particular (reviewed in Hassold and Jacobs, 1984). However, the only factor that is unequivocally known to be of importance in the etiology of trisomy is increased maternal age.

A. Maternal age The relationship between increased maternal age and Down syndrome was demonstrated by Penrose (1933) over 25 years before the chromosome basis of the syndrome was known. The increase in trisomy 21 is moderate at young maternal ages, doubling from approximately 0.05% of live births at age 20 to 0.1% at age 30, but thereafter the increase is much steeper, to 0.25% at age 35, 0.9% at age 40, and to over 3% at age 45. Penrose and others concluded that there were at least two components to the age effect, one independent of maternal age and the other dependent on maternal age. Spontaneous abortions are an excellent source of material for the study of the effects of parental age on monosomy X and trisomies, and a number of such studies have been published (Hassold et at. , 1984; Hassold and Chiu, 1985; Risch et al., 1986; Morton et at., 1988) which, together with data from liveborn sex chromosome trisomies (Court Brown et at., 1969), are in general agreement that trisomy for all chromosomes is associated with increased maternal age. However, the effect is different for different chromosomes, being more pronounced for the small chromosomes and less pronounced for the large chromosomes. Furthermore, for at least two chromosomes, 16 and 2, the effect seems to be linear having no maternal age-independent component. Thus, it is clear that increased maternal age is an extremely important factor in the etiology of trisomy for most, if not all, chromosomes but that both the magnitude and the nature of the maternal age effect vary among different chromosomes. Kajii and Ohama (1979) and Warburton et al. (1980) suggested, on the

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basis of data from spontaneous abortions, that monosomy X was associated with a reduced maternal age. However, these observations were not confirmed by Mathur et al. (1991) or Lorda-Sanchez et al. (1992a), whose studies were based mainly on liveborn individuals.

6. Paternal age Paternal age has been implicated in the etiology of trisomy but the evidence is contradictory. Stene et al. (1977) reported a significant increased risk of Down syndrome in fathers over age 55 and a similar effect was reported by Matsunaga et al. (1978) and Erickson and Bjerkedal (1981). Subsequently, Stene reported an increased risk of trisomy 21 in fathers over age 41 based on an analysis of amniocentesis data (1981); however, this effect was not substantiated in a larger series of amniocentesis data (Stene et al., 1981). Many other studies of Down syndrome have failed to find any link between trisomy 21 and an increased paternal age (e.g., Roth et al., 1983; Roecker and Heuther 1983) and the weight of evidence suggests that increased paternal age is not an important factor in the etiology of Down syndrome. However, as paternal nondisjunction is responsible for only a very small proportion of human trisomics (Sherman et at., 1991; Petersen et al., 1993; Fisher et al., 1993), a definitive answer to the effect of paternal age should be forthcoming when sufficient cases in which the nondisjunctional event is known to involve a paternal meiosis are available for analysis.

IV. PARENTAL ORIGIN The parental origin of the single chromosome in monosomy and the additional chromosome in trisomy can be established by following the segregation of a trait that is inherited in a simple Mendelian fashion and that is on the chromosome in question. Such a trait may be a gene, a cytogenetic heteromorphism, or a molecular polymorphism, and the study of all three types of traits has contributed to our knowledge of the parental origin of aneuploidy in man. The first extensive use of a gene to determine the origin of chromosome abnormalities was that of the X-linked blood group gene Xg used to determine the origin of numerical and structural abnormalities of the X chromosome. Sanger et al. (1977) reported their results of Xg typing in 424 45,X females in which they found the single X to be of maternal origin in 77% and of paternal origin in 23%. The same authors reported the results of typing 566 47,XXY males in which they showed 33% to have resulted from a nondisjunctional error in spermatogenesis and 67% from an error of oogenesis. Unfortunately, as it is not possible to discriminate between one, two, or three doses of the Xg antigen, it is not possible to determine the parental origin of the additional X chromo-

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some in 47,XXX females. However, the distribution of the Xga antigens among 47,XXX females suggested that the majority arose as a consequence of an error of oogenesis. There are stable, heritable alterations in size and shape of the heterochromatic regions of certain chromosomes and such cytogenetic characteristics can, in favorable circumstances, be used to determine the parental origin of additional chromosomes. In 1970 a case of Down syndrome was described in which the origin of the additional chromosome 21 was determined by the observation of two copies of a relatively rare cytogenetic heteromorphism inherited from the mother (de Grouchy, 1970), and since then the cytogenetic heteromorphisms of many families with a Down syndrome child have been examined. Even when the analysis of cytogenetic heteromorphisms does not reveal the exact parental origin of the additional chromosome, it often provides some useful information and a mathematical theory was developed by Jacobs and Morton (1977) for the use of this information in maximum likelihood analysis of the paternal origin of trisomies. Large amounts of data have been published in which cytogenetic heteromorphisms were used to determine both the parent and cell division (see Section VII, B) of trisomies, especially those involving chromosome 21. However, subsequent analysis of parental origin using more easily and reliably interpreted molecular polymorphisms has demonstrated that many of the studies using cytogenetic heteromorphisms were flawed (Lorber et al., 1992), in all probability because of overinterpretation of very subtle differences, many of which are at the limit of resolution of the light microscope. The recent definition of a very large number of molecular polymorphism, including restriction fragment length polymorphisms and VNTR repeat polymorphisms detected by the analysis of Southern blots and CA repeat polymorphisms detectable by the polymerase chain reaction, has revolutionized the analysis of the origin of chromosome abnormalities. First, they can be used to determine the origin of aneuploidy involving any chromosome, not just those carrying a favorable gene or cytogenetic heteromorphisms. Second, the number of available polymorphisms is so great that, providing DNA is available from the monosomic or trisomic conceptus and both parents, the parental origin can be unambiguously determined in all cases.

V. CELL DIVISION OF ORIGIN The vast majority of monosomies and trisomies involving whole chromosomes are de now events arising from chromosomally normal parents. Thus, the aneuploid is likely to result from an error of cell division occurring during formation of the egg or sperm or alternatively at a very early postzygotic division of the embryo. However, while it is possible to determine the cell division of error

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giving rise to a trisomy because the results of the error are available for study, it is impossible to determine the cell division that results in a monosomy because only the “normal” chromosome is present and the source of the abnormality must be inferred as involving the chromosome of the parent who is not represented in the monosomy. While this is a reasonable inference concerning the parent of origin, no inference is possible concerning the cell division at which the nonexistent chromosome was lost. Thus, studies of the cell division at which aneuploidy arise are restricted to trisomies. Errors arising during gametogenesis could theoretically involve a premeiotic mitotic division of the oogonia or spermatagonia or either the first or second meiotic divisions of the oocyte or spermatocyte, while errors arising at an early division of the zygote must involve a postzygotic mitotic (PZM) error. Errors involving a premeiotic mitotic division cannot be differentiated from a meiotic error because the fate of the additional chromosome depends on its paring and segregational behavior at the subsequent meiotic divisions. However, it is possible, at least in theory and increasingly in practice, to differentiate between a first and a second meiotic division error and between a meiotic and a postzygotic mitotic error. In order to discriminate between a first and second meiotic division error it is necessary to have a centromere polymorphism that is heterozygous in the parent whose chromosome is present in two copies in the trisomy. Such a polymorphism will be nonreduced in a trisomy resulting from an error of the first meiotic division but will be reduced to homozygosity in a trisomy resulting from an error of the second meiotic division or of a PZM error (Table 4.3). If an informative centromere polymorphism is not available in the analysis of a given trisomy, a polymorphism situated close to the centromere can be used to determine the cell division of error. If recombination in the nondisjoined chromosomes is the same as that in normal bivalents, an error of classification of the cell division of nondisjunction whose magnitude is proportional to the genetic distance from the centromere of the polymorphism under study will result. If, however, recombination in the nondisjoined chromosomes deviates

Table 4.3. Cell Division of Nondisjunctional Error Status of polymorphism Cell division

Centromeric

Proximal

Distal

Nonreduced Nonreduced Reduced Reduced

Nonreduced Nonreduced Reduced Reduced

Nonreduced Reduced Nonreduced Reduced

MI Nullichiasmate Chiasmate

MI1

PZM

4. The Origin of Numerical Chromosome Abnormalities

109

from that in a bivalent undergoing normal disjunction, the magnitude of the error is more difficult to quantify. The study of the pattern of segregation of polymorphic markers along the whole length of the nondisjoined chromosome enables the distinction to be made between a second meiotic division and a postzygotic mitotic division. In the former, the centromere marker will be reduced and polymorphisms near the centromere will also tend to be reduced, while those situated more distally will tend to be nonreduced. In contrast, in PZM errors all polymorphisms are reduced (Table 4.3). The study of the pattern of segregation of polymorphic markers along the length of the chromosome also allows the construction of a genetic map of the nondisjoined bivalent. A comparison of the nondisjunctional genetic map with the appropriate sex-specific normal genetic map makes it possible to determine whether or not aberrant recombination is associated with nondisj unction.

VI. 45,X ANEUPLOIDY A number of studies of liveborns, therapeutic and spontaneous abortions, and stillbirths with a 45,X constitution have been undertaken using X-linked molecular polymorphisms to determine the parental origin of the single X chromosome. These studies are summarized in Table 4.4. As can be seen, the parental origin of the single X chromosome was similar in cases presenting as abortions and those presenting as livebirths, suggesting that the parental origin of the X chromosomes does not affect the survival of the 45,X conceptus. Hassold et al. (1988), in a study based largely on spontaneous abortions, showed the 45,X(P) class, i.e., those in whom the error involved a maternal chromosome, to have a significantly lower maternal age than the 45,X(M) class, thus suggesting a basis for the lower maternal age reported in spontaneously aborted 45,X fetuses (Kajii and Ohama, 1979; Warburton et al., 1980). However, in a subsequent series also based largely on spontaneous abortions, Hassold et at. (1992) found no difference in maternal age between the 45,X(M) and 45,X(P) classes and this was also the conclusion of Mathur et al. (1991), Loughlin et al. (1991), and Lorda4anchez et al. (1992a) based largely on their analysis of livebom 45,X patients. Thus, all studies are in general agreement that some 80% of apparent nonmosaic 45,X individuals result from an error involving the paternal sex chromosome, while 20% involve the maternal X chromosome-proportions very similar to those reported by Sanger et a[. (1977) analysis using the Xga blood group. There also appeared to be no significant difference in either paternal or maternal age between the two classes of 45,X conceptions (Lorda-Sanchez e t al., 1992a; Hassold et al., 1992). However, these observations are based on relatively small numbers, especially of the 45,X(P) class, and further analysis of larger

110

Patricia A. Jacobs and Terry J. Hassold Table 4.4. The Parental Origin of the Single X Chromosome in Nonmosaic 45,X Conceptuses Origin of X chromosome Population Liveborns

Total livebom Therapeutic abortions Spontaneous abortions

Total abortions

Paternal

Maternal

Reference

0

3 18 12 20 12 40 105

Villamar et al. (1990) Mathur et al. (1991) Loughlin er at. (1991) Jacobs et al. (1990) Hassold et al. (1992) Lorda-Sanchez et al. (1992a)

5

Cockwell et al. (1991) Hassold et al. (1992) Lorda-Sanchez et al. (1992a) Cockwell et al. (1991) Loughlin et al. (1991)

7

0 9 3 8 27 2 8 2 0 0 12

38 5

2

4

54

All conceptions Total %

39 20

159 80

series is necessary before the question of the effect of parental age on the 45,X genotype is finally resolved.

VII. 47,XXY AND 47,XXX ANEUPLOIDY A. Parental origin We have studied a series of 47,XXY and 47,XXX individuals using molecular polymorphisms to determine the parental origin of the additional sex chromosome and our results are summarized in Table 4.5. As can be seen, almost 50% of Klinefelters have two paternal sex chromosomes and 50% have two maternal X chromosomes, while among 47,XXX females only 10% have received two copies of a paternal X, the remaining 90% have two maternal X chromosomes.

B. Cell division of origin A centcomere polymorphism pBam X9 (Willard et al., 1986; Jacobs et al., 1988) is available for the X chromosome and it is therefore possible to determine the

111

4. The Origin of Numerical Chromosome Abnormalities

Table 4.5. X Chromosome Aneuploidy: Parent and Cell Division of Error ~

~

~

~

~~

Paternal Karyotype

Total

MI

MI1

PZM

?

Total

MI

MI1

PZM

66

66 100

-

-

-

44

70

16 25

13

-

76 -

3

-

5

-

-

-

2 100

45

-

29 66

8 18

7

1

-

16

-

66 97

-

2

3

121

73 68

24

10

14 -

47,XXY NO.

%

47,xxx No.

5

% Total

No. %

Maternal

71 -

-

3

3

-

-

22

9

?

cell division of the additional sex chromosomes, and the results of such studies are shown in Table 4.5. All 47,XXY males of paternal origin must have arisen as the result of an error of the first meiotic division (MI). This is therefore a very common segregational error in male meiosis. It was possible to determine the cell division of error of only two of the five paternally derived 47,XXX cases and both appeared to be due to a PZM error. Thus, it appears that paternal MI1 (pat MII) errors involving the X chromosome are extremely rare in human male meiosis. The cases of both 47,XXY and 47,XXX conceptuses in which the additional X chromosome is of maternal origin and the cell division of error has been established are also shown in Table 4.5. As can be seen, 68% were attributable to an error of maternal MI (mat MI), 22% to an error of mat MII, and 9% to a PZM error involving the maternal X chromosome. Thus, errors involving mat MI are the most common cause of sex chromosome aneuploidy involving female gametogenesis but a substantial minority are the result of a MI1 or PZM error.

C. Recombination in nondisjoined sex chromosomes Human sex chromosome aneuploidy is unique among all human aneuploids studied to date in showing a very high proportion attributable to errors involving the paternal sex chromosome. In 47,XXY males of paternal origin the error must have occurred at the first paternal meiotic division and their high frequency suggests that the XY bivalent is particularly prone to undergo nondisjunction. In normal male meiosis a single chiasma is formed in the XY pairing or pseudoautosomal region. The absence or loss of this single chiasma might well result in XY univalency or desynapsis and consequently to nondisjunction at anaphase

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Patricia A. Jacobs and Terry J. Hassold

I. In a study of recombination in the pseudoautosomal region of 41 47,XXY males of paternal origin Hassold et al. (1991a) found crossing over in only 6 of 39 informative cases. These data were subsequently used to generate a nondisjunctional genetic map of the pseudoautosomal region which was found to be very significantly shorter than the normal male map of this region. Thus, it appears that the great majority of paternally derived 47,XXY males are the result of a meiosis in which the X and Y chromosomes do not recombine. Studies of recombination in X chromosomes involved in maternal nondisjunction have been undertaken by MacDonald et al. (1994) and LordaSanchez et al. (199213). Among the 73 cases attributable to error of mat MI, MacDonald et al. observed three different mechanisms of origin; approximately 30% were associated with complete absence of recombination (nullichiasmate), approximately 24% were associated with a normal number but an abnormal distribution of exchanges (perturbed recombination), while approximately 45% were associated with a normal number and distribution of recombination events (normochiasmate). Lorda-Sanchez studied 9 cases of 47,XXY males attributable to a mat MI error and found 8 to lack recombination. However, this study had relatively few informative markers and if more markers had been studied it is possible that some of the cases would have shown recombination. Nonetheless, the results agree with MacDonald et al.’s in finding the absence of recombination to be an important factor in the genesis of mat MI nondisjunction. Among the cases with perturbed recombination, sectional analysis showed that there was a significant excess of pericentromeric recombination offset by a significant reduction in recombination in proximal Xp and Xq and distal Xq. The distribution of recombinant events was also studied in the 23 cases attributable to mat MI1 (MacDonald et al., 1994). Surprisingly, analysis of the nondisjunctional MI1 genetic map showed it to be significantly shorter than the standard map. In addition, sectional analysis showed an excess of pericentromeric recombination and a significant decrease in recombination in proximal Xp and distal Xp and Xq. Furthermore, all nondisjunctioned MI1 seemed to be associated with perturbed recombination. As recombination occurs during the prophase of MI, the observation of an abnormal distribution of recombinational events associated with MI1 nondisjunction implies that events which occur during MI can affect the orderly segregation of chromatids during MIL Thus, the detailed analysis of recombination in the nondisjoined maternal X chromosomes implied there are at least five different mechanisms by which this can happen (Table 4.6): (i) nullichiasmate MI nondisjunction, (ii) perturbed recombination leading to nondisjunction at mat MI, (iii) perturbed recombination leading to nondisjunction at mat MII, (iv) mat MI nondisjunction associated with a normal number and distribution and exchange of events, and (v) the nondisjunction of the maternal X chromosome at a PZM division.

113

4. The Origin of Numerical Chromosome Abnormalities

Table 4.6. X Chromosome Aneuploidy: Maternal Age No. of Category

cases

Paternal origin (all) Maternal origin (all) Maternal MI Maternal MI1 Maternal PZM Maternal MI nullichiasmate Maternal MI normochiasmate Maternal MI perturbed recombination

58 97 58 20

7

18 26 14

P vaiue 27.4(0.7). 29.3(0.7) 30.7(0.9) 26.9( 1.4) 29.0(2.9) 31.1(1.3) 32.2( I . 4) 27.5(1.8)

1 co.01 <0.8 <0.6 <0.02 <0.01 <0.8

Control.

D. Parental age Studies of parental age in individuals with an additional X chromosome were first published in 1969 (Court Brown et al., 1969), when it was shown that both the 47,XXY and the 47,XXX individuals were associated with an increased maternal age. Studies of parental age in cases in which the parental cell division and mechanism of origin of the additional chromosome are known have shown that maternal, but not paternal, nondisjunction is associated with an increased maternal age and that this increase is found only in those cases attributable to a mat MI error, those cases resulting from a MI1 error having a normal maternal age (MacDonald et al., 1994; Lorda-Sanchez et al., 1992b). Furthermore, MacDonald et d. found that among the maternal cases only those attributable to nullichiasmate or normochiasmate nondisjunction were associated with an increased maternal age, while in mat MI nondisjunction associated with perturbed recombination the maternal age was not elevated (Table 4.6). Among the 58 cases attributable to a paternal nondisjunctional event MacDonald et al. found no evidence for an increased paternal age, whereas Lorda-Sanchez et al. (1992b) did find a significant increased paternal age in 23 cases attributable to pat MI nondisjunction. Further studies will show whether this is a chance finding or of biological significance.

VIII. 47,XYY ANEUPLOIDY The presence of an additional Y chromosome among male conceptuses is a relatively common event (Table 4.2). It is obvious that the additional chromo-

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Patricia A. Jacobs and Terry J. Hassold

some must be of paternal origin and the error leading to the 47,XYY genotype can only arise at pat MI1 or at a PZM division as there is evidence that spermatogonia and/or spermatocytes with an additional Y chromosome are selected against during gametogenesis resulting in 47,XYY males having sperm with an essentially normal sex chromosome constitution (Melnyk et al., 1969). Both pat MI1 and PZM nondisjunction involving a paternal chromosome are relatively rare events for the X chromosome and most if not all autosomes, and it is of considerable interest to know the precise origin of the additional chromosome in 47,XYY males. There are three plausible mechanisms for the origin of the additional Y chromosome which can, in principle, be differentiated from one another by the study of the segregation of polymorphic markers situated at the distal tip of the pseudoautosomal region. The first mechanism is pat MI1 nondisjunction following a normochiasmate MI division. This would result in Y chromosomes heterozygous for a distal pseudoautosomal marker and homozygous for proximal pseudoautosomal markers in all informative cases. The second mechanism is pat MI1 nondisjunction following a nullichiasmate MI division. This would result in the Y chromosomes being entirely homozygous and identical to those of the father in all cases. The third mechanism is a PZM error following normal meiosis, in which case the Y chromosomes would be entirely homozygous but, as the result of a single pseudoautosomal exchange, the distal pseudoautosomal loci would be paternally derived 50% of the time and maternally derived 50% of the time. Thus, it is relatively easy, given DNA from an XYY individual and his parents, to detect a pat MI1 error following a normal pat MI division, but it is only possible to discriminate between a pat MI1 error following a nullichiasmate MI division and a PZM error by establishing phase of the paternal pseudoautosomal loci which requires grandpaternal DNA. Furthermore, even when phase of the paternal pseudoautosomal loci is known it is only possible to discriminate between the two mechanisms in 50% of the cases. We have studied the segregation of distal pseudoautosomal probes, DXYS20 and DXYSl4, in five 47,XYY individuals and their parents. In three individuals the error was clearly attributable to a pat MI1 error following a chiasmate MI division, while in two it was the result of a PZM error or an MI1 error following a nullichiasmate MI. While these numbers are very small they do show that more than one mechanism is involved in the genesis of 47,XYY males.

IX. SEX CHROMOSOME POLYSOMY Rare individuals with four or even five sex chromosomes have been described and studies of the parental origin of the additional chromosomes in such individuals using the Xga blood group and molecular polyrnorphisms have shown that in

4. The Origin of Numerical Chromosome Abnormalities

115

the great majority all additional chromosomes come from the same parent (Race and Sanger, 1969; Hassold et al., 1990; David et al., 1992). Furthermore, it is well established that the parental age of such individuals is not elevated (Lorda. has been shown that 47,XXY and 47,XXX individuals Sanchez et al., 1 9 9 2 ~ ) It can result from perturbed recombination occurring at mat MI or at mat MI1 and that such perturbed recombination is not associated with an increased maternal age (MacDonald et al., 1994). Thus, it is reasonable to suggest that perturbed recombination in mat MI, and perhaps also pat MI, leads to nondisjunction resulting in secondary oocytes and spermatocytes with all four sex chromosome chromatids instead of the normal two. Segregation of these four chromatids at MI1 can occur in a number of ways and the potentially viable results of segregation include a 48,XXXX or 48,XXXY individual with three maternal X chromosomes, a 48,XXXY or 48,XXYY individual with three paternal sex chromosomes, a 49,XXXXX or 49,XXXXY individual with four maternal X chromosomes, and a 49,XXXYY individual with four paternal sex chromosomes. All these types of sex chromosome polysomy have been reported and it is therefore clear that MI nondisjunction followed by MI1 nondisjunction of the sex chromosomes occurs during both maternal and paternal gametogenesis, although such occurrences must be rare.

X. TRISOMY 21 While trisomy 21 is not the most frequent autosomal trisomy in man, it is the one that is found most commonly in the liveborn population and has been a focus of research for over 100 years. The chromosome basis of the syndrome was recognized in 1959, and subsequently it was realized that some chromosome staining techniques revealed morphological variations of chromosome 2 1 which were inherited in a Mendelian fashion. These observations set the stage for a number of publications in which the parent and cell division of origin of the additional chromosome 2 1 were determined by following the inheritance of chromosome heteromorphisms from parents to trisomy 21 offspring. T h e first description of such a parental origin was that of de Grouchy (1970) who described a Down’s syndrome patient who had inherited two copies of one maternal heteromorphism. As chromosome heteromorphisms are all at the centromere or very close to the centromere on the p arm and because very little recombination is thought to occur on the short arm, at least as observed in male meiosis and extrapolated to female meiosis, informative hetermorphisms provide information not only on the parent but also on the cell division of origin of the additional chromosome. Using cytogenetic heteromorphisms, the origin of the additional chromosome in Down syndrome was studied in over 1500 families in a variety of laboratories (e.g., Dagna-Bricarelli et al., 1989). These studies

116

Patricia A. Jacobs and Terry J. Hassold

concluded that most cases of trisomy 21 resulted from a nondisjunctional error at mat MI but that errors at mat MII, pat MI, and pat MI1 also occurred and accounted for approximately one-third of all cases. These results were found in virtually all studies and applied both to the majority of trisomy 21 conceptuses that were spontaneously aborted and the minority that were liveborn. However, these observations raised a number of problems. First, there appeared to be an increased maternal age effect in nondisjunctional events associated with an additional paternal chromosome (Ayme and Lippman-Hand, 1982). Second, numerous observations suggested that nondisjunction might be associated with age-related reduced or absent recombination at female meiosis (e.g., Sherman et al., 1991, 1994), a hypothesis difficult to reconcile with a paternal nondisjunctional event. However, the use of the more reliably scored (although as yet less advantageously positioned) molecular polymorphisms has led to the general recognition that there were a number of systematic errors in scoring the cytogenetic heteromorphisms, errors which are largely overcome by the use of molecular markers (Lorber et al., 1992).

A. Parental origin The study of segregation of chromosome 21 polymorphic molecular markers in parents and their trisomy 21 conceptuses has shown that the additional chromosome is maternal in origin in approximately 91% of patients and paternal in only 9% (Sherman et al., 1991; Antonarakis et al., 1992). These figures are significantly different from the 75-80% maternal nondisjunction rate suggested by studies of cytogenetic heteromorphisms. The difference presumably is due to overenthusiastic interpretation of marginal differences in heteromorphisms. That is, if almost all nondisjunctional events are maternal, random errors in classification of parental origin will increase the apparent frequency of paternal nondisjunction.

B. Cell division of origin Unfortunately there is, as yet, no molecular marker for the centromere of chromosome 21; thus, all studies of the cell division of origin rely on markers in the pericentromeric region of the chromosome, whether they are heteromorphisms or molecular polymorphisms. If pericentric recombination in nondisjoined chromosomes is similar to that in normal chromosomes, the error in assigning cell division is quantifiable and probably no more than 5-10% (Jabs et al., 1991). If the pericentromeric recombination is reduced in nondisjoined chromosomes by comparison with normal chromosomes, the error rate will be even less as there will be very little recombination between the centromere and the peri-

4. The Origin of Numerical Chromosome Abnormalities

117

centromeric markers. However, if there is an excess of pericentric recombination, the error rate in apportioning cell division of nondisjunction will be difficult to quantify and may be considerable. Despite these caveats, a number of studies have been made in which the cell division of origin of the additional chromosome 21 has been inferred (Table 4.7) and the cell division of origin of over 500 trisomy 21 patients with an additional chromosome 21 of maternal origin has been reported. There is general agreement that some 75% are the result of a mat MI error, 22% of a mat MI1 error, and 3% of a PZM error involving a maternal chromosome 21. Among 35 cases with an additional paternal chromosome, 27 were meiotic in origin (7 as.the result of an MI error and 15 as the result of an MI1 error), while in 5 the cell division at which the error occurred was not known. The remaining 8 were compatible with a PZM error involving the paternal chromosome 21 (Petersen et al., 1993). Thus, not only are the relative proportions of paternal and maternal nondisjunction involving chromosome 21 very different, but the relative contribution of the three different stages of nondisjunction to trisomy 21 is also very different between the two classes of parental origin (Table 4.7).

C. Recombination and nondisjunction of chromosome 21 It was first shown by Warren et al. (1987) that reduced recombination was associated with maternal nondisjunction of chromosome 21 and this was confirmed by Sherman et al. (1991) who showed that a significant proportion of chromosome 2 1 nondisjunction involved chromosomes that were nullichiasmate. A more recent analysis in which the material was divided by inferred division of origin (Sherman et al., 1994) found that, among nondisjunction of maternal origin, the nondisjunctional genetic map of trisomies due to mat meiotic errors was very significantly shorter than the normal map. In contrast, the length of the MI1 nondisjunctional map was not significantly different from that of the normal map. Detailed analysis of the mat MI nondisjunctional map suggested an overall reduction in map length with an excess of cases with both complete absence of recombination and with only a single recombination, and a concomitant deficiency of cases with two or more recombinational events. Sectional analysis of the MI nondisjunctional map showed the distribution of recombinant events to be significantly different from that of the normal map. This is consistent with altered recombination along the length of the map rather than a complete absence of recombination in a proportion of cases and normal recombination in the remainder. Among the mat MI cases that showed a recombinational event, the reduction in recombination appeared to be restricted to proximal 21q with recombination in distal 21q actually increasing. In contrast. no difference was found in the number or distribution of

Table 4.7. Trisomy 21, Trisomy 16, and Trisomy 18: Parent and Cell Division of Error Paternal Chromosome

21 No. %

Maternal

Total

MI

MI1

PZM

?

Total

MI

MI1

PZM

?

Reference

24 9

9 3

15 6

-

-

240 91

173 66

58 22

9 3

-

-

2 4

-

59 96

16 29

35 62

3 5

Sherman et al. (1994) and Sherman (unpublished observations) Fisher et al. (1993) and Jacobs (unpublished observations)

-

-

58 100

54 100

-

-

4

-

-

-

18 2 4

-

%

I6 No.

-

-

No.

%

-

5 -

Hassold et al. (1991b) and Hassold (unpublished observations)

4. The Origin of Numerical Chromosome Abnormalities

119

recombinant events between the mat MI1 nondisjunctional map and the normal map, suggesting that aberrant recombination does not play a role in mat MI1 nondisjunction involving chromosome 2 1. Unfortunately, to date there is an insufficient number of cases of nondisjunction of paternal meiosis to allow an analysis of the effect, if any, of aberrant recombination on paternal nondisjunction.

0 . Parental age and sex ratio All molecular studies of trisomy 21 show that increased maternal age is restricted to those cases of maternal origin, and the maternal age of the few cases of paternal origin is significantly lower. Among the cases of paternal origin considered as a group there was no obvious increase in paternal age, but the mean paternal age of the seven cases associated with pat MI nondisjunction was 31.1 years, 4 years higher than that associated with pat MI1 nondisjunction (Petersen et al., 1993). These data suggest that nondisjunction at pat MI involving chromosome 21 may be associated with an increased paternal age. A similar suggestion of increased paternal age associated with pat MI nondisjunction involving the XY bivalent has been made (Lorda-Sanchez et al., 1992b), but no effect of paternal age on XY nondisjunction was found in another study involving a larger number of patients (MacDonald et al., 1994). It has been well established that at all stages of gestation there is an increased sex ratio associated with trisomy 2 1. A recent analysis of sex ratio by mechanism of origin of the additional chromosome 21 demonstrated the abnormally high sex ratio to be restricted to those cases in which the additional chromosome 2 1 was of paternal origin and, furthermore, to those cases in which the additional chromosome arose as a result of an error of a pat MI or pat MI1 division but not of a PZM division (Petersen et al., 1993). These data suggest that some abnormality associated with trisomy of chromosome 21 in paternal meiosis affects the orderly segregation of the sex chromosome. In the two large studies of the parent and cell division of origin of the additional chromosome 2 1, an increased maternal age was found to be associated with both mat MI and mat MI1 nondisjunction; indeed, the maternal ages of the two classes were not significantly different from one another (Antonarakis, 1993; Sherman et at., 1994). In a detailed analysis of the maternal age effect among trisomy 2 1 conceptuses due to mat meiotic errors, Sherman et al. ( 1994) found a very significant correlation between the number of recombinant events in the nondisjoined chromosome and maternal age, with the number of recombinational events in older mothers being significantly less than that in younger mothers. In contrast, no association was found between maternal age and recombination in nondisjunction attributable to mat MI1 errors. Therefore, re-

120

Patricia A. Jacobs and Terry J. Hassoid

duced recombination appears to be a very significant contributor to trisomy 21 of mat MI origin for women of all ages, especially for older women.

XI. TRISOMY 18 A. Parental origin Trisomy for chromosome 18 is the second most common autosomal trisomy among livebirths although only some 5% survive to birth, with the great majority of trisomy 18 conceptuses presenting as spontaneous abortions or stillbirths. Recent studies of the parental origin of trisomy 18, both those lost during fetal life and those liveborn, have shown about 95% to have an additional maternal chromosome, while only 5% have an additional paternal chromosome 18 (Fisher e t al., 1993).

B. Cell division of origin Unfortunately, as in the case of chromosome 21, there is no polymorphic centromere marker available for chromosome 18. Thus, the cell division at which the nondisjunctional error took place has to be inferred from the status of pericentromeric markers. However, because chromosome 18 is submetacentric it is possible to use information from pericentromeric markers on both sides of the centromere to determine the status of the centromere and the cell division at which the nondisjunction occurred. If such markers are concordant and nonreduced it implies an MI error and if they are concordant and reduced an MI1 or PZM error is implied, the latter two possibilities being distinguished by the status of polymorphic markers along the length of the chromosome. Assignment of cell division of origin based on closely flanking concordant markers is likely to be very accurate as it will only be scored incorrectly if there is a double crossover event involving the two nondisjoined chromatids-a very unlikely occurrence. In cases in which closely flanking pericentromeric markers are discordant with one reduced and one nonreduced, the cell division at which the nondisjunction occurred cannot be inferred. However, such cases are rare when markers closely flanking the centromere are used. In a recent study of 61 trisomy 18 patients, 56 were informative for both parent and cell division of origin of the additional chromosome 18 (J. M. Fisher et al., unpublished observations) (Table 4.7). In both cases which had an additional paternal chromosome and in three cases which had an additional maternal chromosome, the additional chromosome 18 resulted from a PZM error. Among the remaining 5 1 cases which had an additional maternal chromosome, 16 resulted from an error at mat MI and 35 from an error at mat MIL The

4. The Origin of Numerical Chromosome Abnormalities

121

substantial excess of MI1 compared to MI errors is unexpected and contrasts with the situation for chromosomes 21 and the X.

C. The effect of recombination Among the 16 cases attributable to a mat MI error studied by J. M. Fisher et al. (unpublished observations), at least 5 had 10 or more informative probes, all of which were nonreduced. Such an observation is compatible with nullichiasmate nondisjunction in these 5 cases. The number of nullichiasmate nondisjunctions expected by chance is less than 0.5 and therefore it is likely that approximately one-third of all mat MI nondisjunctional events involving chromosome 18 are nullichiasmate. When the nullichiasmate cases are excluded, the nondisjunctional MI and MI1 maps of chromosome 18 are not significantly different in length from the normal map. Furthermore, sectional comparisons between the normal and trisomic meiotic maps show no significant differences in the distribution of recombinant events suggesting that, with the exception of the nullichiasmate class, abnormal recombination does not play a significant role in nondisjunction involving chromosome 18.

D. Parental age The parental age of the only two cases that were paternally derived was unremarkable. However, the maternal age of the cases which had received an additional maternal chromosome 18 was significantly increased over the control. Furthermore, MI, MII, and PZM errors all showed an increased maternal age, although this was only formally significant for the mat MI1 class. It seems reasonable to assume that the increased maternal age would have reached significance for the MI class had there been more cases, whereas the increase in the PZM class is a chance event due to small numbers. Thus, on the basis of current evidence it appears that trisomy 18 is almost entirely attributable to errors of maternal meiosis, with about twice as many MI1 as MI errors. Approximately one-third of mat MI errors are attributable to nullichiasmate nondisjunction, but recombination appears to be normal in the remaining MI errors and in the MI1 errors. Increased maternal age appears to be associated with both mat MI and MI1 errors.

XII. TRISOMY 16 The most common trisomy in our species is that involving chromosome 16, which occurs in over 1% of conceptions that survive long enough to be clinically recognized although virtually all trisomy 16 conceptuses are spontaneously

122

Patricia A. Jacobs and Terry J. Hassold

aborted. It has been recognized for some time that all trisomy 16 conceptuses are associated with increased maternal age-there being no maternal ageindependent component (Morton et al., 1988; Risch et al., 1986). The frequency of trisomy 16 increases linearly with age and it is its relatively high frequency at young maternal ages that accounts for its prevalence among all clinically recognized conceptions (Risch et al., 1986). Data are available on the parental origin of 62 trisomy 16 conceptuses and in all of them the additional chromosome was found to be maternal in origin (Hassold et al., 1991b; T. J. Hassold, unpublished observations). Chromosome 16 has a very polymorphic centromere marker (D16Z2) which enables the cell division of origin of the additional chromosome to be determined in the great majority of cases. Among the 58 trisomy 16 conceptuses studied with this probe 54 were informative and all were found to be due to an error of the mat MI division (Table 4.7). Thus, the very great majority, if not all, of trisomy for chromosome 16 is attributable to a mat MI error. Analysis of recombination in the nondisjoined chromosome is not yet complete but preliminary data suggest that trisomy 16 is associated with a general reduction in recombination, as among the nondisjoined bivalents there appears to be an excess with zero or one recombinational events and a concomitant absence of nondisjoined bivalents with three or more exchanges. Furthermore, the reduction appears to be restricted to the pericentromeric regions, with the distal short and long arms having apparently normal levels of recombination. Thus, mat MI nondisjunction of chromosome 16 is similar to MI nondisjunction of chromosome 2 1 by being associated with age-related reduced recombination, especially in the proximal regions.

XIII. TRISOMY 13, 14, 15, AND 22 A relatively small number of observations have been made on nondisjunction involving the large acrocentric chromosomes, 13, 14, and 15, and the small acrocentric chromosome, 22 (Zaragoza et al., 1994). The great majority of such studies have utilized material from spontaneous abortions although the information on trisomy 13 also includes livebirths (Table 4.8). In all, some 12% have an additional paternal chromosome, a figure somewhat higher than that for chromosome 21 and considerably higher than that seen in trisomies 16 and 18. However, the cell division of origin of the additional paternal chromosome was not determined. Among the 64 cases of maternal origin the cell division was determined by the segregation of the most centromeric informative polymorphism. This analysis inferred a cell division of origin in 25 cases, with 17 of mat MI origin and 8 of mat MI1 origin, a ratio of mat M1:mat MI1 similar to that seen for chromosome 2 1. As yet there are few data available on the effect of aberrant recombination on nondisjunction involving these chromosomes. Data on paren-

123

4. The Origin of Numerical Chromosome Abnormalities

Table 4.8. Trisomy 13, Trisomy 14, Trisomy 15, and Trisomy 22: Parent and Cell Division of Error Paternal Chromosome

13 14 15 22

Total

3 2 2 2

MI 1

-

-

Maternal

MI1

PZM

?

Total

MI

MI1

PZM

?

1

-

-

1

22 10 1

4

4

1 1

17 2 8 11

2 1

-

-

1 2

1

3

7

5

6

5

-

2

-

tal age are very sparse but there appears to be no significant difference between nondisjunction at MI or MI1 and in both categories maternal age appears to be elevated, which is also similar to the situation for chromosome 21. In summary, the very small amount of data available on acrocentric chromosomes, other than the data on chromosome 2 1, suggests that the proportion of paternal to maternal nondisjunction and the proportion of mat MI to mat MI1 errors are very similar to those seen for chromosome 21. It may be that both the large and the small acrocentric chromosomes have common mechanisms underlying nondisjunction and Robinson et al. (1993) have suggested an effect of reduced recombination in nondisjunction of chromosome 15 based o n analysis of 27 patients with maternal uniparental disomy.

XIV. COMPARISON OF MECHANISMS OF ORIGIN OF TRISOMY There is considerably more information available on the mechanism of origin of trisomies involving the X chromosome and autosomes 16, 18, and 21 than for any other chromosome. It is salutary to compare the information for these four types of trisomy and this is summarized in Table 4.9. As can be seen, there must be at least three different mechanisms of nondisjunction involving the paternal chromosome and seven involving the maternal chromosome. Nondisjunction involving the paternal chromosome is unknown for chromosome 16 and it is rare for chromosomes 18 and 21. Furthermore, when it does occur, it not infrequently involves a postzygotic mitotic division. The one exception to the rarity of paternal meiotic nondisjunction is the X chromosome in which pat MI nondisjunction of the XY bivalent is common and frequently involves a nullichiasmate bivalent. Thus, the XXY situation is unique among trisomies in that approximately 50% result from a paternal nondisjunctional event. The four chromosomes show surprisingly disparate patterns with respect to the seven observed types of segregational error involving the maternal chromosome. The X, 16, and 21 all have a predominance of MI nondisjunction. In

Table 4.9. Nondisjunction: Summary Maternal

MI

Paternal Mechanism

MI

MI1

PZM

Nullichiasmate

Maternal age

N ...

N

N

-

-

-

..T

-

-

X

16 18 21

RR

PR

Normochiasmate

PR

Normochiasmate

PZM

t

N ..

..t

N

..

N

-

-

T -

-

-

... ... -

Note. RR, reduced recombination; PR, perturbed recombination; N , normal; ' observed, infrequent; * * frequent; * * ' very frequent.

MI1

-

-

T , increased; -,

not observed

... .. -

-

4. The Origin of Numerical Chromosome Abnormalities

125

both chromosomes 16 and 21 MI nondisjunction is associated with a marked reduction in recombination and all chromosome 16 nondisjunction may be attributable to aberrant recombination at MI. In contrast, chromosome 21 has, in addition, a considerable proportion of normochiasmate mat MI1 nondisjunction as well as a small number of PZM errors. MI nondisjunction of the X chromosome is almost equally divided among nullichiasmate nondisjunction, normochiasmate nondisjunction, and nondisjunction associated with perturbed recombination with a pericentromeric excess. However, in contrast to chromosomes 16 and 21, the X chromosome shows no evidence of a general reduction, as opposed to a complete absence, of recombination associated with MI nondisjunction. Chromosome 18 is unique in having the majority of nondisjunctional events attributable to normochiasmate mat MI1 errors. This type of nondisjunction is also important for chromosome 2 1, where it is responsible for about onefourth of all maternal errors. Mat MI1 nondisjunction also represents about onefourth of maternal errors of the X chromosome but, in the case of the X, the nondisjoined chromatids show an excess of recombination in the pericentromeric region. The X and chromosomes 18 and 2 l have a small proportion of PZM nondisjunction, while trisomy 16 never appears to result from either a mat MI1 or a PZM error. Thus, it is clear that the seven different types of maternal nondisjunctional events associated with trisomy differ markedly in their relative importance among the four chromosomes that have been most extensively studied. However, the effect of increased maternal age seems to involve the same mechanisms irrespective of the chromosome involved. Thus, of the seven types of maternal nondisjunction, four, namely mat MI nullichiasmate nondisjunction, mat MI errors associated with reduced recombination, and mat MI and mat MI1 normochiasmate nondisjunction, are all associated with a marked increase in maternal age, while three, namely mat MI and mat MI1 errors associated with perturbed recombination and PZM errors, are not associated with increased maternal age. The reason for the surprisingly wide diversity of nondisjunctional mechanisms, especially involving maternal meiosis, is currently unknown.

XV. THE FREQUENCY OF DlSOMlC GAMETES FOR CHROMOSOMES X, 16, 18, AND 21 The frequency of the different classes of disomic gametes can be calculated based on the frequency of trisomic conceptuses among all clinically recognized pregnancies and the proportion of such conceptuses due to the different types of meiotic nondisjunctional mechanisms. These calculations are based on two simplifying assumptions: (i) that the frequency of trisomies among all clinically

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recognized pregnancies is an accurate reflection of their frequency at conception, and (ii) that there is no gametic selection operating against disomic gametes. The first assumption is almost certainly untrue, at least for those trisomies associated with a very severe phenotype leading to abortion in the first trimester of pregnancy, while the second may well be valid. The results of such a calculation for chromosomes X, 16, 18, and 21 are given in Table 4.10 with the XXY and XXX classes of X chromosome aneuploidy shown separately. As can be seen, the predicted frequency of sperm with an XY constitution is very similar to the predicted frequency of sperm that are disomic for chromosome 21, while no sperm are predicted to be disomic for the X chromosome or for autosomes 16 and 18, although presumably such abnormalities do occur, if only rarely. Among ova, 0.08% are predicted to be disomic for the X, 0.17% to be disomic for chromosome 18, 0.4% to be disomic for chromosome 21, and an astonishing 1.13% to be disomic for chromosome 16. Among disomic ova about 85% are attributable to an MI error and 15% to an MI1 error. Thus, even among these four chromosomes there is a more than 14-fold difference in the predicted frequency of disomic ova. It is unlikely that much, if any, of this variation is attributable to selective mortality prior to the time the trisomic pregnancy becomes clinically recognizable because the most severely affected fetuses are those with trisomy 16 and the least affected are those with an additional X. Early intrauterine mortality would therefore be expected to operate against trisomy 16 fetuses to a greater extent than XXY or XXX fetuses, a situation which would increase rather than decrease the differences in frequency among disomic ova. It is impossible to dismiss the possibility that there may be windows of development, prior to the pregnancy becoming clinically recognizable, that are particularly “at risk” for certain otherwise relatively normal genotypes, e.g., XXX. However, this possibility is not intuitively attractive and it seems likely that the large differences in the predicted frequency of the different classes of disomic gametes are a reflection of real differences in the frequency with which they occur. Data on the observed frequency of chromosome abnormalities in human sperm are available from two sources: (i) from direct observations of sperm chromosomes obtained by the fertilization of hamster eggs with human sperm, the so-called “humster technique”; and (ii) from estimates of the frequency of disomic sperm obtained from the direct microscopic observation of chromosome-specific probes using fluorescence in situ hybridization (FISH). A summary of the studies of sperm chromosome complements observed using the humster technique was recently published (Jacobs, 1992). In this review the frequency of XY sperm over all studies was 0.07%, the frequency of XX sperm was 0.02%,the frequency of disomic 16 sperm was 0.06%, and the frequency of disomic 21 sperm was 0.1%. With the exception of the XY class, in which

Table 4.10. Predicted Frequencies of Disomic Gametes

Chromosome abnormality

XXY

xxx +16 +18 +21

Frequency among all

Parental origin of error

(%I

CRC

Proportion due to meiotic errors (%)

Predicted frequency of disomic gametes

VQ)

Proportion due to Mat M1 and Mat MI1 errors (YO)

Predicted frequency of disomic ova (Yo)

(%I

Pat

Mat

Pat

Mat

Sperm

Ova

Mat MI

Mat MI1

Mat MI

Mat MI1

0.08

46 10 0 3 8

54 90

100

97 97

0.04 0

0.04 0.04

26

0.03

0.01

0.03

0.01

100

0 0

0

1.13

74 78 100

0

1.13

0

0 0.03

0.17 0.40

31

69 25

0.05

0.12

0.30

0.10

0.05 1.13 0.18

0.45

97 92

0

77

100

97 97

75

22

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Patricia A. Jacobs and Terry J. Hassold

the predicted and observed categories are in relatively good agreement, all these observations are in considerable excess of the predicted frequencies of such sperm. Estimates of disomy based on sperm FISH studies are similarly higher than the predicted values, again with the exception of the XY class. For example, in one recent study the frequency of XY sperm was 0.07%, the frequency of XX sperm was 0.02%, the frequency of disomic 16 sperm was 0.13%, and the frequency of disomic 18 sperm was 0.04% (Williams et aI., 1993; T. J. Hassold, unpublished observations). The reason for the differences between the humster and FISH studies and the predicted values is not altogether clear. It may be that some classes of disomic sperm are at a selective disadvantage by comparison with normal sperm, or that trisomic fetuses are much more likely to abort spontaneously than is currently appreciated. Alternatively, it may be that the results from the humster and FISH studies do not accurately reflect the in vivo situation; after all, the former technique relies on interspecific in vitro fertilization and the latter on analysis of the number of “spots” in sperm nuclei. Currently there is little basis for discriminating between these or other explanations for the discrepancy; nevertheless, in the absence of other information, we think it prudent to use the gametic studies (e.g., FISH sperm studies) to answer questions related to differences in frequency of aneuploidy (e.g., differences among chromosomes and differences among sperm donors) than to answer questions requiring exact estimates of the frequency of aneuploidy. Data on the observed frequency of potentially disomic ova are available from a number of studies of the chromosomes of human oocytes and these were recently reviewed by Jacobs ( 1992). However, there are many difficulties associated with the accurate observation of this very technically difficult material. Furthermore, the oocytes are observed at mat MI1 and thus the number of chromosomes seen reflects only events occurring at mat MI and can be compared only with the predicted frequency of disomic ova resulting from an MI error. The best estimate of the observed frequency of ova with an additional X chromosome arising from an MI division is 0.03%, the frequency of disomic 16 ova is 0.26%, and the frequency of disomic 2 1 ova is 0.78%. These figures are clearly in rather poor accord with the predicted frequencies of 0.06, 1.13, and 0.3%, respectively. This is most likely due to the inaccurate estimate of the chromosome number and morphology in this type of suboptimal material. While the meaningfulness of these results is uncertain, one recent cytogenetic study of oocytes is notable for providing a novel hypothesis of human nondisjunction. In a series of studies, Angell and her colleagues (1994) observed that most numerical abnormalities seen at MI1 involve chromatids, not whole chromosomes. From this observation, Angell et al. suggested that the most common source of human nondisjunction is premature centromere division at MI, i.e., for one chromosome the MI is equational rather than reductional. If

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these observations are confirmed, they may provide a basis for the variation in chromosome-specific rates of maternal nondisjunction. For example, the tendency to predivide may be chromosome specific due to the extensive variation among chromosomes in the types and distribution of centromere-related repetitive DNA. Additionally, “chromatid” nondisjunction may be important in the aetiology of maternal age-dependent trisomy, as Angel1 et al. (1994) identified an age effect in association with these abnormalities.

XVI. CONCWSIONS Humans appear to be unique in their very high frequency of numerical chromosome abnormalities. The recent development of large numbers of molecular probes for all human chromosomes has provided the tools to enable the mechanisms underlying the high frequency of aneuploidy to be investigated. Initial observations have concentrated on a small number of chromosomes, mainly those that are most frequently associated with aneuploidy. These investigations have shown a very wide diversity of mechanisms underlying numerical chromosome abnormalities. Furthermore, the relative importance of the different mechanisms differs widely among chromosomes. With the exception of the 45,X, 47,XXY, and 47,XW aneuploids, nondisjunction rarely involves a paternal chromosome and for the autosomes the proportion due to nondisjunction of a maternal chromosome ranges from 90 to 100%. Among the trisomies resulting from an error at maternal meiosis, a number of different mechanisms have been observed. These include errors of mat MI and mat MI1 in which the nondisjoined chromosomes/chromatids show normal recombination, and errors of mat MI and mat MI1 in which the nondisjoined chromosome/chromatids show abnormal recombination. Abnormal recombination includes total absence of recombination, reduced recombination, and abnormally high levels of pericentromeric recombination. The welldocumented effects of increased maternal age appear to be associated with normochiasmate nondisjunction, irrespective of whether it occurs at MI or MIL Increased maternal age is also associated with absent or reduced recombination, but not with an excess of pericentromeric recombination. These recent results are surprising in a number of ways: ( i ) the sheer diversity of mechanisms associated with nondisjunction, (ii) the observation that at least two classes of nondisjunction associated with abnormal recombination also show a strong maternal age effect. As recombination occurs during fetal life it is difficult to envisage errors of recombination playing a role in age-related nondisjunction (see MacDonald et al., 1994, for discussion). For aberrant recombination to play a role in age-related nondisjunction, there must be a mechanism that results in oocytes with abnormal recombination being ovulated with

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increased frequencies at advanced maternal ages. Two such mechanisms have been postulated. The production line hypothesis suggests that germ cells become committed to meiosis sequentially in fetal life and are released as mature ova in the sequence in which they entered meiosis, and that the number of exchanges is fewer in ova laid down late in fetal life, resulting in an increased number of univalents and thus aneuploid offspring in old females. The second mechanism suggested to account for the maternal age effect is the depleted oocyte hypothesis. This postulates that as women age there is a decreasing number of antralstage follicles per cycle and thus an increased likelihood of ovulating suboptimal oocytes, which may include those with aberrant recombination. Reduced recombination at advanced maternal age is an essential component of the production line hypothesis and, while not an essential component of the depleted oocyte hypothesis, can be accommodated by it. Recent evidence suggests that one or both of these mechanisms may be important in nondisjunction of human chromosomes; (iii) the observation that increased maternal age plays a role in nondisjunction occurring at the second meiotic division, a cell division that is initiated at ovulation and completed after fertilization; and (iv) the observation that perturbed recombination, which must occur during fetal life, can affect the orderly segregation of chromosomes not only at MI but also at MIL Clearly we are only at the outset of the quest to understand nondisjunction, one of the most important causes of morbidity and mortality in our species.

Acknowledgments We thank the Wellcome Trust who provided support for P.A.J., and the National Institutes of Health who provided Grant HD21341 and Contract HD92709 to T.J.H. during the writing of this chapter. We also record our gratitude to Judy Gladding for her secretarial assistance during the preparation of the manuscript.

References Angell, R. R., Xian, J., Keith, J., Ledger, W., and Baird, D. T. (1994). First meiotic division abnormalities in human oocytes-mechanism of trisomy formation. Cytogenet. Cell Genet. 65: 194-202. Antonarakis, S. E. (1993). Human chromosomes-21-genome mapping and exploration, circa 1993. Trends Genet. 9:142-148. Antonarakis, S. E., Petersen, M. B., McInnis, M. G., Adelsherger, P. A., Schinzel, A. A., Binkert, F., Pangalos, C., Raoul, O., Slaugenhaupt, S.A., Hafez, M., Cohen, M. M., Roulson, D.,Schwartz, S., Mikkelsen, M., Tranehjaerg, L., Greenherg, F., Hoar, D. I., Rudd, N. L., Warren, A. C., Metaxotou, C., Bartsocas, C., and Chakravarti, A. (1992). The meiotic stage of nondisjunction in trisomy-2 1-determination hy using DNA pulymorphisms. Am. 1. Hum. Genet. 50544-550. Ayme, S., and Lippman-Hand. A. (1982). Maternal-age effect in aneuploidy: Does altered emhryonic selection play a role! Am. J Hum. Genet. 34558-565.

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Carr, D. H. (1963). Chromosome studies in abortuses and stillborn infants. Lancet 2:603-606. Cockwell, A., MacKenzie, M., Youings, S., and Jacobs, P. (1991). A cytogenetic and molecular study of series of 45,X fetuses and their parents. J. Med. Genet. 28:151-155. Court Brown, W. M., Law, P.,and Smith, P. G. (1969). Sex chromosome aneuploidy and parental age. Ann. Hum. Genet. 33:l-14. Dagna-Bricarelli, F., Pierluigi! M., Landucci, M., Arslanian, A., Coviello, D. A., Ferro, M. A., and Strigini, P. (1989). Parental age and the origin of trisomy 2 1. Hum. Genet. 82:20-26. David, D., Marques, R. A., Carreiro, M. H., Moreira, I., and Boavida, M. G. (1992). Parental origin of extra chromosomes in persons with X-chromosome tetrasomy. J. Med. Genet. 29: 595-596. de Grouchy, I. (1970). 2lp-Maternal en double exemplaire chez un trisomique 21. Ann. Genet. 1352-55. Edwards, J. H., Harnden, D. G . , Cameron, A. H., Crosse, V. M., and Wolf, 0. H. (1960). A new trisomic syndrome. Lancet 9:787-789. Erickson, J. D., and Bjerkedal, T. (1981). Down syndrome associated with father’s age in Norway. J. Med. Genet. 18:22-28. Fisher, J. M., Harvey, J. F., Lindenbaum, R. H . , Boyd, P. A., and Jacobs, P. A. (1993). Molecular studies of trisomy 18. Am. J. Hum. Genet. 52:1139-1144. Ford, C. E., and Hamerton, 1. L. (1956). The chromosomes in man. Nature (London) 178: 1020- 1023. Ford, C. E., Jones, K. W., Poiani, P. E., de Almeida, J. C., and Briggs, J. H. (1959). A sex chromosome anomaly in a case of gonadal dysgenesis (Turner’s Syndrome). Lancet 1:711-713. Gropp, A . , and Winking, H. (1981 ). Robertsonian translocations: Cytobgy, meiosis, segregation patterns and biological consequences of heterozygosit. Symp. 2001. SOC. London 47:141-181. Hassold, T. J . , and Chiu, D. (1985). Maternal age-specific rates of numerical chromosome abnormalities with special reference to trisomy. Hum. Genet. 7O:ll-17. Hassold, T. J., and Jacobs, P. A. (1984). Trisomy in man. Ann. Rev. Genet. 18:69-97. Hassold, T. 1.. Warburton, D., Kline, I., and Stein, Z. (1984). The relationship of maternal age and trisomy among spontaneous abortions. Am. J. Hum. Genet. 36:1349-1356. Hassold, T. I., Benham, F., and Leppert, M. (1988). Cytogenetic and molecular analysis of sexchromosome monosomy. Am. ]. Hum. Genet. 42:534-541. Hassold, T. I., Pettay, D., May, K., and Robinson, A. (1990). Analysis of non-disjunction in sex chromosome tetrasomy and pentasomy. H. Gent. 85:648-650. Hassold, T. J., Sherman, S. L., Pettay, D., Page, D. C., and Jacobs, P. A. (1991a). XY-chromosome nondisjunction in man is associated with diminished recombination in the pseudoautosomal region. Am. J. Hum. Genet. 49:253-260. Hassold, T. J., Pettay, D., Freeman, S. B., Grantham, M., and Takaesu, N. (1991b). Molecular studies of non-disjunction in trisomy 16. J. Med. Genet. 28:159-162. Hassold, T. I., Pettay, D., Robinson, A , , and Uchida, I. (1992). Molecular studies of parental origin and mosaicism in 45,X-conceptuses. Hum. Genet. 89:647-652. Hook, E. B., and Hamerton, J. L. (1977). The frequency of chromosome abnormalities detected in consecutive newborn studies-differences between studies-results by sex and by severity of phenotypic involvement. In “Population Cytogenetics, New York State Department of Health, Birth Defects Institute, Symposium (1975), Albany, New York, October 1975” (E. B. Hook and I. H. Porter, eds.), pp. 63-79. New York: Academic Press. Jabs, E. W., Warren, A. C . , Taylor, E. W., Colyer, C. R., Meyers, D. A . , and Antonarakis, S. E. (1991). Alphoid DNA polymorphisrns for chromosome 21 can be distinguished from those of chromosome 13 using probes homologous to both. Genornics 9:141-146. Jacobs, P. A. (1992). The chromosome complement of human gametes. Oxford Reu. Reprod. Bid. 14:47-72.

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Jacobs, P. A., and Hassold, T. J. (1987). In “Proceedings of the 7th International Congress, Berlin 1986” (F. Vogel and K. Sperling, eds.), pp. 223-244. Springer-Verlag, Berlin. Jacobs, P. A., and Morton, N. E. (1977). Origin of human trisomies and polyploids. Hum. Hered. 27:59-72. Jacobs, P. A., and Strong, J. A. (1959). A case of human intersexuality having a possible XXY sexdetermining mechanism. Nature (London) 183:302-303. Jacobs, P. A., Bacino, C., Hassold, T., Morton, N. E., Keston, M., and Lee, M. (1988). A cytogenetic study of 47,XXY males of known origin and their parents. Ann. Hum. Genet. 52~319-325. Jacobs, P. A., Betts, P. R., Cockwell, A. E., Crolla, J. A., MacKenzie, M. J., Robinson, D. O., and Youings, S. A. (1990). A cytogenetic and molecular reappraisal of a series of patients with Turner’s syndrome. Ann. Hum. Genet. 54209-223. Jacobs, P. A., Browne, C., Gregson, N., Joyce, C., and White, H. (1992). Estimates of the frequency ot chromosome abnormalities detectable in unselected newborns using moderate levels of banding. J. Med. Genet. 29:103-108. Kajii, T , and Ohama, K. (1979). Inverse maternal age effect in rnonosomy X. Hum. Genet. 5 12147-151. Lejeune, J., Gautier, M., and Turpin, R. (1959). Etudes des chromosomes somatique de neuf enfants mongoliens. C.R. Hebd. Seances Acad. Sci., Ser. D 248:1721-1722. Lorber, B. J., Grantham, M., Peters, J., Willard, H. F., and Hassold, T,J. (1992). Nondisjunction of chromosome-21-comparisons of cytogenetic and molecular studies of the meiotic stage and parent of origin. Am. J. Hum. Genet. 51:1265-1276. Lorda-Sanchez, I., Binkert, F., Maechler, M., and Schinzel, A. (1992a). Molecular study of 45,X conceptuses: Correlation with clinical findings. Am. J. Med. Genet. 42:487-490. Lorda,Sancher, I., Binkert, F., Maechler, M., Robinson, W. P., and Schinzel, A. A. (1992b). Reduced recombination and paternal age effect in Klinefelter syndrome. Hum. Genet. 89: 524-530. Lorda-Sanchez, I., Binkert, F,, Hinkel, C.G., Moser, H., Rosenkranz, W., Maechler, M., and Schinzel, A. (19924. Uniparental origin of sex chromosome polysomies. Hum. Hered. 42: 193-197. Loughlin, S. A. R., Redha, A., McIver, J., Boyd, E., Carothers, A., andconnor, J. M. (1991). Analysis of the origin of Turner’s syndrome using polymorphic DNA probes. J. Med. Genet. 28:156-158. MacDonald, M., Hassold, T., Harvey, J., Wang, L. H., Morton, N. E., and Jacobs, P. (1994). The origin of 47,XXY and 47,XXX aneuploidy. Hum. Mol. Genet. 3:1365-1371. Mathur, A., Stekol, L., Schatr, D., Maclaren, N. K., Scott, M. L., and Lippe, B. (1991). The parental origin of the single X-chromosome in Turner Syndrome-lack of correlation with parental age or clinical phenotype. Am. J. Hum. Genet. 48:682-686. Matsunaga, E., Tonomura, A., Oishi, H., and Kikuchi, Y. (1978). Reexamination of paternal age effect in Down’s syndrome. Hum. Genet. 40259-268. Melnyk, J., Thompson, H., Rucci, A. J., Vanasek, F., and Hayes, S. (1969). Failure of transmission of the extra chromosome in subjects with 47,XYY karyotype. Lancet 2:797-798. Morton, N. E., Jacobs, P. A., Hassold, T., and Wu, D. (1988). Maternal age in trisomy. Ann. Hum. Genet. 52:227-235. Patau, K. A., Smith, D. W., Therman, E. M., Inhorn, S. L., and Wagner, H. P. (1960). Multiple congenital anomaly caused by an extra autosome. Lancet 1:790-793. Penrose, L. S. (1933). The relative effects of paternal and maternal age in mongolism. J. Genet. 27:219-224. Petersen, M. B., Antonarakis, S. E., Hassold, T. J., Freeman, S. B., Sherman, S. L., Avramopoulos, D., and Mikkelsen, M. (1993). Paternal nondisjunction in trisomy 21-excess of male patients. Hum. Mol. Genet. 2:1691-1695.

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Race, R. R., and Sanger, R. (1969). Xg and sex chromosome abnormalities. Br. Med. Bull. 25: 99-103. Risch, N., Stein, Z., Kline, J., and Warburton, D. (1986). The relationship between maternal age and chromosome size in autosomal trisomy. Am. J. Hum. Genet. 39:68-78. Robinson, W. P., Fkmasconi, F., Mutirangura, A., Ledbetter, D. H., Langlois, S.,Malcolm, S., Morris, M. A., and Schinzel, A. A. (1993). Nondisjunction of chromosome-15-origin and recombination. Am. J. Hum. Genet. 53:740-75 1. Roecker, G., and Huether, C. (1983). An analysis for paternal age effect in Ohio’s Down syndrome births, 1970-80. Am. J. Hum. Genet. 35:1297-1306. Roth, M.-P., Feingold, J., Baumgarten, A., Bigel, P., and Stoll, C. (1983). Re-examination of paternal age effect in Down’s syndrome. Hum. Genet. 63:149-152. Sanger, R., Tippett, P., Gavin, J., Teesdale, P., and Daniels, (3. L. (1977). Xg groups and sex chromosome abnormalities in people of northern European ancestry: An addendum. J. Med. Genet. 14:210-213. Sherman, S. L., Takaesu, N., Freeman, S. B., Grantham, M., Phillips, C., Blackston, R. D., Jacobs, P. A., Cockwell, A. E., Freeman, V., Uchida, I., Mikkelsen, M., Kurnit, D. M., Buraczynska, M., Keats, B. J., Hassold, T. J., Grantham, M., and Freeman, V. (1991). Trisomy 2 1: Association between reduced recombination and non-disjunction. Am. J. Hum. Genet. 49:608-620. Sherman, S. L., Petersen, M. B., Freeman, S. B., Hersey, J., Pettay, D., Taft, L., Frantzen, M., Mikkelsen, M., and Hassold, T. J. (1994). Non-disjunction of chromosome 21 in maternal meiosis I: Evidence for a maternal-age dependent mechanism involving reduced recombination. Hum. Mol. Genet. 3:1529-1535. Stene, J., Fischer, G., Stene, E., Mikkelsen, M., and Petersen, E. (1977). Paternal age effect in Down’s syndrome. Ann. Hum. Genet. 40299-306. Stene, J., Stene, E., Stengel-Rutkowski, S., and Murken, J.-D. (1981). Paternal age and Down’s syndrome. Data from prenatal diagnosis (DFG). Hum. Genet. 59: 119-124. Tjio, J. H., and Levan, A. (1956). The chromosome number of man. Heredim 42:l-6. Villamar, M., Fernandez, E., Ayuso, C . , Ramos, C., and Benitez, J. (1990). Study of the parental origin of sexual aneuploidy in ten families using RFLPs. Ann. Genet. 33:29-31. von Winiwarter, H. (1912). Etudes sur la spermatoginkse humaine. Arch. Bid. 27:97-189. Warburton, D., Kline, J., Stein, Z., and Susser, M. (1980). Monosomy X; A chromosomal anomoly associated with young maternal age. Lancet 1:167-169. Warren, A. C., Chakravarti, A., Wong, C., Slaugenhaupt, S.A., Halloran, S. L., Watkins, P. C., Metaxotou, C., and Antonarakis, S. E. (1987). Evidence for reduced recombination on the nondisjoined chromosomes 21 in Down Syndrome. Science 237:652-654. Willard, H. F., Wave, J. S., Skolnick, M. H., Schwartz, C. E., Powers, V. E., and England, S. B. (1986). Detection of restriction fragment length polymorphisms of the centromeres of human chromosomes using chromosome-specific alpha satellite DNA: Implications for the development of centromere-based genetic linkage maps. Proc. Natl. Acad. Sci. U.S.A. 83:5611-5615. Williams, B. J., Ballenger, C. A., Malter, H. E., Bishop, F., Tucker, M., Zwingman, T. A., and Hassold, T. J. (1993). Non-disjunction in human sperm-results of fluorescence in situ hybridization studies using two and three probes. Hum. Mol. Genet. 2:1929-1936. Zaragoza, M., James, R. S..Jacobs, P. A., Rogan, P.,Sherman, S., and Hassold, T. (1994). Nondisjunction of human acrocentric chromosomes. Hum. Genet. 94:411-417.

I

Thrombophilia: The Discovery of Activated Protein C Resistance Bjorn Dahlback Department of Clinical Chemistry University Hospital, Malmo S-20502 Malmo, Sweden

1. INTRODUCTION Blood coagulation is a powerful defense system which ensures that injury to the vessel wall does not to lead to excessive blood losses (Davie et al., 1991; Furie and Furie, 1988; Stenflo and Dahlback, 1994). The importance of the system is illustrated by the severe bleeding disorders which affect individuals with deficiencies of coagulation factors, the most common being a deficiency of factor VIII (Hemophilia A ) or factor IX (Hemophilia B) (Sadler and Davie, 1994). The extremely efficient coagulation system also poses a threat to the organism, as uncontrolled clotting leads to occlusion of the vascular system. Careful regulation of the reactions of the coagulation cascade is therefore a prerequisite for health and several anticoagulant mechanisms operate under physiological conditions to ensure blood fluidity.

A. Inhibition of coagulation by antithrombin 111 Most enzymes formed during the coagulation process are inhibited by the serine protease inhibitor antithrombin 111 (AT 111) (Lane et al., 1992; Broze and Tollefsen, 1994). The effect of AT I11 is stimulated by heparin-like substances which are exposed on the vascular endothelium. Genetic defects in the gene for AT 111, resulting in reduced plasma levels of the protein or loss of its function, are associated with a lifelong imbalance between pro- and anticoagulant forces and increased risk of thrombosis. AT 111deficiency, which was the first genetic defect found to be associated with impaired anticoagulation and venous thrombophilia, Advances in Genetics, Val. 33 Copyright 0 1995 by Academic Press, Inc. All rights of repraduction in any form reserved

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is a rare genetic defect which is found only in a few percent of patients with thrombosis (Lane et al., 1992; Dahlback, 1994b; Broze and Tollefsen, 1994).

6. Regulation of coagulation by the protein C system During the past 20-30 years we have discovered additional anticoagulant mechanisms and genetic defects which are associated with increased risk of thrombosis. This chapter focuses on a physiologically important natural anticoagulant system which is commonly referred to as the protein C system (Figure 5.1) (Esmon, 1992; Dahlback and Stenflo, 1994; Dahlback, 1994a, 1995). This system regulates blood coagulation by cleaving and inhibiting the activated forms of coagulation cofactors V and VIlI (Va and VIIIa) (Figure 5.2). Protein C, which is the key component of the system, is vitamin K dependent and circulates in plasma as a zymogen to a serine protease. The protein C system is activated in response to intravascular thrombin generation. Thrombin, which

Procoagulant A

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Figure 5.1. A simplified scheme showing most of the reactions of blood coagulation and those of the protein C anticoagulant system. The scheme emphasizes the balance between the proand anticoagulant mechanisms of thromhin and the specificity of APC. 'Ihe reactions leading to the formation of factor XIa have not been included in the coagulation cascade nor are the feedback activation of factors V, VII, and VIII by factor Xa shown. APC degrades factors Va and VIIIa when they are bound to phospholipid. T h e binding of protein S to C4b-binding protein results in inhibition of its anticoagulant properties. TF denotes tissue factor which triggers the reactions involving factor VII.

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5. Thrombophilia

P1-U A1

A2

t t tm Fv B

A3 C1C2

t t ---. Al A2

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Figure 5.2. Schematic models of factor VIII and factor V. Factor VIII and factor V are homologous and hoth molecules contain multiple modules; three A modules, one B region, and two C modules (Kane and Davie, 1988; Fay, 1993). Thrombin cleaves three peptide bonds in both procofactors, as indicated by the arrows. In factor Va, the A1-A2 containing heavy chain and the A3-Cl-CZ composed of light chain form a calcium-dependent complex. In factor VIII, thrombin cleaves a peptide bond between A1 and A2 and factor VIlla is therefore a heterotrimeric complex composed of A l , A2, and A3-ClC2. In factor VIII, the small peptide region between 1649 and 1690, which is cleaved off by thrombin, contains the binding site for the von Willebrand factor. Activation fragments are derived from the central B region and they have no known function. APC inactivates factors VlIIa and Va cleaving multiple bonds as indicated. In factors Va and VIIIa, the cleavages at Arg506 and Arg526, respectively, correlate with cofactor Inactivation.

functions as a procoagulant enzyme in the extravascular space, has the potential to attain anticoagulant properties intravascularly. Binding of thrombin to thrombomodulin (TM), a high-affinity receptor on the surface of intact endothelial cells, is associated with modulation of thrombin function. Procoagulant properties are lost and anticoagulant properties are gained because thrombin bound to T M efficiently activates protein C. Activated protein C (APC)mediated cleavage and inactivation of factor Va and factor VIIIa is potentiated by another vitamin K-dependent plasma protein, protein S, which, unlike protein C, is not a zymogen to a serine protease. Deficiencies of protein C or protein S are associated with functional impairment of anticoagulation which, due to the resulting imbalance between pro- and anticoagulant forces, yield lifelong hypercoagulability and increased risk of thrombosis (reviewed in Dahlback, 1994a, 1995; Dahlback and Stenflo, 1994). In cohorts of thrombosis patients, protein C or protein S deficiencies are found together in 5-10% of the patients (Dahlback, 1994b). Until recently, these deficiencies, together with AT 111 deficiency and dysfibrinogenemia, were

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the only known inherited pathogenetic risk factors of thrombosis. As thrombosis often is familial, this suggested unknown genetic risk factors to be involved in the pathogenesis of thrombosis.

C. APC resistance and venous thrombosis Recently, I discovered a previously unrecognized mechanism for familial thrombophilia which is characterized by an inherited poor anticoagulant response to APC (Dahlback et ul., 1993). This APC resistance, which is the most common genetic abnormality associated with thrombosis yet discovered, is caused by a single point mutation in the gene for factor V, which predicts replacement of arginine at position 506 (Arg506) in the APC cleavage site with a glutamine (Gln) (Bertina et al., 1994; Dahlback, 1994b;Greengard e t ul., 1994b; Voorberg et ul., 1994; Zoller and Dahlback, 1994). The APC resistance is associated with a lifelong hypercoagulable state because the mutated factor Va is less sensitive to degradation by APC than normal factor Va (Bertina et al., 1994; Sun et ul., 1994; Dahlback, 199413). As a result, affected individuals are at 5- to l0dfold higher risk of developing thrombosis than those without the mutation. Section 11, although having its main emphasis on the history of APC resistance, also discusses the physiology of the protein C anticoagulant system and the key observations which have advanced our knowledge in this field.

II. PROTEIN C-A VITAMIN K-DEPENDENT PROTEIN WITH ANTICOAGULANT PROPERTIES In 1976, Johan Stenflo reported on the isolation and characterization of a fifth vitamin K-dependent protein. Because it was recovered in pool C from an ionexchange chromatography, he called it protein C. To fully understand the rapid development of the knowledge of the protein C system we must go further back in history.

A. Identification of a vitamin K-dependent modification of glutamic acid residues In the late 1960s, Johan Stenflo started his Ph.D. thesis work in the laboratory of Professor Carl-Bertil Laurell, at Malmo General Hospital with Per Olof Ganrot as supervisor. Laurell had among other things developed the Laurell rocket technique and the crossed immunoelectrophoresis method. In an effort to improve laboratory monitoring of antivitamin K therapy, Ganrot and Nihlen (1968) had, using an antiserum against prothrombin, applied the rocket technique to plasma from patients on oral anticoagulation and made the surprising observation that the plasma levels of prothrombin did not drop to the expected

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low levels during oral anticoagulation. Using the crossed immunoelectrophoresis technique, it was then observed that the prothrombin which was present in plasma of orally anticoagulated patients did not bind calcium in a normal fashion (Stenflo, 1970; Stenflo and Ganrot, 1972). Johan Stenflo’s task was to elucidate the molecular difference between normal prothrombin and the prothrombin which was formed during antivitamin K therapy. Stenflo’s work led to the identification and characterization of the vitamin K-dependent amino acid, y-carboxy glutamic acid (Gla) in 1974 (Stenflo et al., 1974). Vitamin K is required for the postribosomal y-carboxylation of the first 9-12 glutamic acid residues in the vitamin K-dependent proteins and oral anticoagulant therapy with vitamin K antagonists leads to inhibition of this reaction (reviewed in Stenflo and Dahlback, 1994). Prothrombin and the other vitamin K-dependent proteins do not undergo the modification when synthesized during vitamin-K deficiency and are secreted with regular glutamic acid residues (Furie and Furie, 1990; Stenflo and Dahlback, 1994). The Gla residues are crucial for calcium binding to the Gla modules and in the presence of calcium these modules attain a conformation which binds negatively charged phospholipid membranes. In the past few years, the enzyme which is involved in the vitamin K-dependent y-carboxylation has been identified and steps have been taken to unravel the molecular mechanisms involved in the calcium and phospholipid binding of the Gla module (Wu et al., 1991; Stenflo and Dahlback, 1994). One might ask why the Gla residue had not been previously described. One reason is that the Gla residues are decarboxylated during acid hydrolysis. However, they are stable during basic hydrolysis which has made it possible to determine the Gla content in proteins. This formed the basis for identification of other vitamin K-dependent proteins, such as protein C and protein S.

B. Isolation and characterization of protein C The work which led to the structural characterization of the Gla residues in prothrombin involved purification of large quantities of normal prothrombin. During this work, Stenflo observed an unknown protein which eluted in the third pool of an ion-exchange chromatography. He purified the protein to homogeneity, found it to contain Gla residues, and thus to be vitamin K dependent (Figure 5 . 3 ) (Stenflo, 1976). The vitamin K-dependent proteins known at the time, factor VII, factor IX, factor X, and prothrombin, were all zymogens to serine proteases and protein C was soon found to be similar in this respect as it was possible to activate protein C by trypsin, thrombin, and by an enzyme present in the venom from Russel’s viper (Kisiel et al., 1977; Esmon et al., 1976). The activated form of protein C was soon found to have anticoagulant properties and Walter Kisiel and his colleagues (1977) reported that it prolonged the activated partial thromboplastin time (APTT) because it degraded factor Va. Shortly thereafter, factor VIIIa was also found to be a substrate for APC

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Serine protease Figure 5.3. Schematic model of protein C demonstrating the modular composition. Human protein C contains a vitamin K-dependent Gla module (Gla), two epidermal growth factor (EGF)-like modules, and a serine protease module (Dahlback and Stenflo, 1994). The arrow indicates the site for thrombin (Ha) cleavage, which leads to activation of protein C.

(Vehar and Davie, 1980).The rapid elucidation of the function of protein C was facilitated by the realization that APC was identical to autoprothrombin IIa, which was an anticoagulant activity described in 1960 by Walter Seegers and colleagues in Detroit (Seegers ei al., 1976). Originally, autoprothrombin IIa was believed to be derived from prothrombin and it was identified after thrombin treatment of a prothrombin preparation, which in retrospect obviously also contained protein C. However, in the early 1970s, Eva Marciniak (1972) had shown that the precursor of autoprothrombin IIa was distinct from prothrombin. Shortly after reporting on the identification and characterization of the Gla residue, Stenflo spent a year as a Fogarthy postdoctoral fellow in John Suttie’s laboratory in Madison, Wisconsin. During this time he performed experiments on the activation of protein C together with Charles T. (Chuck) Esmon, who was a postdoctoral fellow in the laboratory. In retrospect, this collaboration was crucially important for the field. A few years later, Esmon was involved in the discovery of thrombomodulin.

111. THROMBOMODULIN-A

MODULATOR OF THROMBIN FUNCTION

Although it was known that thrombin could activate protein C , most people had doubts about the physiological significance of this because protein C activation by thrombin alone was quite inefficient. The identification of thrombomodulin, by Chuck Esmon and White Owen, is one of the greatest discoveries of this field and is in itself a very interesting story. Esmon and Owen were old friends from the time they had spent together in the laboratory of Craig Jackson in St. Louis. Esmon had moved to Oklahoma City, whereas Owen was working in Iowa City. After his postdoctoral period in Suttie’s laboratory, Esmon contin-

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ued to work on the biological properties of protein C, whereas Owen was interested in the interaction between AT 111 and the vascular wall. In his laboratory, Owen had established a so-called Langendorff heart preparation model for studies of the interaction between AT 111 and the capillary endothelium. During a visit to Owen’s laboratory, Esmon and Owen performed an experiment in which they perfused the Langendorff heart preparation with a combination of thrombin and protein C. During circulation through the capillary bed, protein C was activated, but only if protein C and thrombin were coinfused or if thrombin was infused prior to protein C. From these results, Esmon and Owen realized that a component present on the surface of endothelial cells bound thrombin and functioned as cofactor to thrombin in the activation of protein C (Owen and Esmon, 1981). Shortly thereafter, they purified the thrombin cofactor from rabbit lung and found it to be a membrane protein which bound thrombin with high affinity in a reaction which led to a change in the specificity of thrombin from a pro- to an anticoagulant enzyme (Figure 5.4) (Esmon et al., 1982). Due to the ability of this protein to modulate

Figure 5.4. Schematic model of thromhomodulin (TM) demonstrating molecular interactions occurring during protein C activation. Thrombin (T) bind to a high-affinity binding site located in the fifth EGF-like module (Sadler ec al., 1993). The fourth EGF-like module of T M is required for interaction with protein C. The anionic exosite of thrombin is denoted by multiple plus signs. The glycosaminoglycan (GAG) side chain of TM is important for expression of full anticoagulant activity of T M and required for stimulation of AT 111-dependent inhihition of hound thrombin.

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the function of thrombin, they named it thrombomodulin. Thrombomodulin, which is present on essentially all endothelial cells, is of course a key component in the protein C system and has been the subject of intensive research. The reader is referred to recent reviews for a detailed description of the structural and functional properties of thrombomodulin (Esmon, 1992; Dahlback and Stenflo, 1994).

IV. PROTEIN S-A VITAMIN K-DEPENDENT COFACTOR TO ACTIVATED PROTEIN C In the late 1970s, Fred Walker worked as postdoctoral fellow in the laboratory of Esmon on the degradation of bovine factor Va by APC. After leaving Esmon’s laboratory, Walker continued his work on the functional properties of APC and made the observation that APC was more efficient in plasma than in a highly purified system. This suggested to him that a plasma component enhanced the activity of APC. He isolated the component and found it to be identical to protein S (Walker, 1980), a vitamin K-dependent plasma protein which was originally isolated and characterized in 1977 by Richard DiScipio, who was at the time a Ph.D. student in Earl Davie’s laboratory (Figure 5.5) (DiScipio et al., 1977). Walker’s work suggested that protein S to functioned as a cofactor to APC in the degradation of factor Va. He found the cofactor function of protein S to depend on the ability of APC and protein S to form a 1:l stoichiometric complex on the surface of negatively charged phospholipid (Walker, 1981). The concept that protein S is an important component of the protein C anticoagulant system gained further support in the mid 1980s by the identified association between protein S deficiency and inherited thrombosis (Schwarz et al., 1984; Comp et al., 1984; Comp and Esmon, 1984).

m

1

p .

Gla TSR EGF,

Figure 5.5. Schematic model of protein S. Protein S is composed of the following modules: A Cia module followed by a thrombin-sensitive region, four EGF-like modules, and a C-terminal half which is homologous to sexual hormone-binding globulin (SHBG). T h e thrombin-sensitive region and the first EGF domain are important for APC cofactor activity, whereas C4BP bind to one or more sites in the SHBG-like region. Adapted from Dahlback et nl. (1986).

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A. Molecular mechanism of the APC cofactor activity of protein S It has been noted in many laboratories that the APC cofactor activity of protein S is weak, particularly in purified systems (Bakker e t al., 1992; Dahlback, 1986). Thus, its presence is only associated with an approximate two fold increased rate of APC-mediated degradation of factor Va. There are at least two possible explanations for the lack of correlation between the strong association between protein S deficiency and thrombosis on the one hand and the weak APC cofactor activity of protein S on the other. One is that protein S is not directly functioning as an APC cofactor and that its anticoagulant activity is mediated through a different mechanism, the other is that yet another component is required for expression of maximum APC activity. There are reports in the literature to suggest that indeed both of these possibilities are at hand. First, some background information. In the prothrombinase complex, factor Xa and factor Va form a 1:l stoichiometric complex on the surface of negatively charged phospholipid vesicles (Mann e t al., 1990). It was shown early on that factor Va in the fully assembled prothrombinase complex was less sensitive to APC than membrane-bound factor Va alone, which was due to protection of factor Va by found factor Xa (Suzuki e t al., 1983). In 1988, Susan Solymoss and her colleagues reported that protein S has the ability to abrogate the protective effect of factor Xa on factor Va, i.e., that protein S makes the substrate factor Va available for proteolysis. Philip Fay and co-workers found a similar effect of protein S on factor IXa-mediated protection against APC cleavage and inactivation of factor VIIIa (Regan et al., 1994). Recently, it was discovered in our laboratory that in a purified factor VIIIa degradation system, the APC cofactor function of protein S, is stimulated by intact factor V (Shen and Dahlback, 1994). This suggests that protein S and factor V function in synergy as APC cofactors.

B. APC-independent anticoagulant activity of protein S Another mode of anticoagulant action of protein S, which is independent of APC, has also been suggested. Several groups have shown that protein S inhibits prothrombin activation in systems using purified components (Heeb e t al., 1993, 1994; Hackeng et al., 1994). The mode of action of this APC-independent anticoagulant action of protein S is not known, but it is believed to be dependent on the ability of protein S to interact directly with both factor Xa and factor Va. It is not known whether there are mechanisms which regulate the APCindependent anticoagulant activity of protein S and the physiological significance of this activity remains to be elucidated.

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V. INTERACTION BETWEEN PROTEIN S AND C4b-BINDING PROTEIN A. Two forms of protein S in plasma A n observation which I made in the late 1970s suggested that protein S had functions outside the protein C anticoagulant system and its physiological role was rather complex. The observation was that protein S in human plasma circulates in two forms, as free protein and in a high-molecular-weight complex with the complement regulatory protein, C4b-binding protein (Dahlback and Stenflo, 1981). As this protein-protein interaction in itself is interesting and of potential physiological importance, I briefly outline the events that led to its identification. During my Ph.D. training, which started in 1977 with Johan Stenflo as supervisor, one of the tasks I had was to purify protein C from human plasma. In one of several different purification procedures that I performed, a highly purified vitamin K-dependent protein was obtained which turned out to be identical to human protein S (DiScipio et al., 1977). Antibodies against protein S were raised in rabbits and with the aim of measuring protein S in different patient groups, 1 tried to measure plasma protein S using the Laurell rocket technique. The initial results of this exercise were discouraging. The rockets did not look like any of the nice rockets that were being run in Laurell's laboratory at the time. After consulting with Laurell, who was the head of the laboratory, I subjected human plasma to analysis by crossed immunoelectrophoresis using antibodies against protein S. This exercise revealed that plasma contained two forms of protein S. One of the forms in plasma corresponded in position to that of purified protein S, while the other had a very different migration rate in the first-dimension agarose gel electrophoresis, which was part of the crossed immunoelectrophoresis technique. Laurell suggested to me that the shape of this second immunoprecipitate indicated that protein S was present in a complexed form in plasma. This stimulated me to try to purify the complexed form as it was obvious that this would lead to the identification of the complexing partner and possibly to elucidation of an important functional role of protein S.

1. Purification of the complexed form of protein S I found that both forms of protein S were adsorbed to barium citrate, which for many years had been used as a first step in the purification of vitamin K-dependent proteins. The barium citrate precipitate was dissolved in EDTA and this solution was subjected to fractionated ammonium sulfate precipitation. The vitamin K-dependent proteins were recovered in the 40-70% ammonium sulfate fractions, but the complexed form of protein S could not be found in this fraction. The complexed form of protein S turned out to be present in the 0-40

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% ammonium sulfate fraction. The addition of 40% ammonium sulfate to the barium citrate eluate had traditionally been used as a means to remove remaining barium because insoluble barium sulfate is formed upon the addition of ammonium sulfate. To my acknowledge it had not previously been observed that one of the major protein in the barium citrate eluate was precipitated in this step. The dominating protein in the 0-40% ammonium sulfate reaction was of very high molecular weight. After reduction, a major 70-kDa species was observed on polyacrylamide gel electrophoresis, which suggests the protein to be composed of multiple identical subunits linked by disulfide bridges. On nondenaturing agarose gel electrophoresis, another technique which was refined by Laurell, the protein migrated very slowly to a position in the y-zone. This protein-species reacted with the antiserum against protein S, which suggested that indeed it represented the complexed form of protein S. To elucidate the identity of the high-molecular-weight protein S-binding protein, the protein complex was further purified by anionic-exchange chromatography, heparinsepharose chromatography, and gel filtration chromatography. The protein S was found to remain firmly, but noncovalently, bound to the high-molecularweight component throughout the purification procedure. The purified protein complex was subjected to aminoterminal amino acid sequencing, but the obtained sequence did not help me to reveal the identity of the protein.

2. Identification of the protein S complexing protein as C4b-binding protein As is often the case, serendipity solved the problem. I attended a lecture on the biology of the complement system which was given by Anders Sjoholm who was working in the department of microbiology in Lund. After his lecture, I approached him with the question whether “my” protein could possibly be involved in the complement system. After describing the characteristics of the protein to Sjoholm and showing him some of the results in the laboratory, he suggested that the protein could be identical to C4b-binding protein. C4BP had just been identified as an important regulator of the classical complement pathway and Sjoholm fortunately had access to a C4BP antiserum. Exchange of reagents led to the identification of the “protein S-binding protein” as C4BP. I t was of course very exciting that a vitamin K-dependent protein in plasma formed a tight complex with one of the regulatory proteins of complement as this suggested a direct link between the regulations of the coagulation and complement systems. The importance of the observation was readily realized by Professor Emeritus Jan Waldenstrom, who had been head of the department of Internal Medicine at Malmo General Hospital and my teacher at medical school. As a member of the National Academy of Science in the United States, Waldenstrom communicated the protein S-C4BP paper to the “Proceed-

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ings of the National Academy of Science” and it was published in 1981 (Dahlback and Stenflo, 1981). The protein S-C4BP interaction has been a major theme in my research and the interested reader is referred to reviews describing this topic in more detail (Dahlback, 1991; Dahlback and Stenflo, 1994).

B. Molecular structure of C4b-binding protein To provide the basis for understanding the discussion on different types of protein S deficiency, I briefly discuss the subunit composition of the C4BP molecule. During 1982 and 1983, I spent 1 year as a Fogarthy postdoctoral fellow

in the laboratory of Hans J Muller-Eberhard, who was at the time head of the department of Immunology at Scripps Clinic and Research Foundation in La Jolla. Muller-Eberhard’s group had for a long time been one of the leading research teams studying the molecular mechanisms of the complement system. In Muller-Eberhard’slaboratory, I met Craig Smith who had developed a sample preparation technique, the so-called pleated sheet method, for high-resolution electron microscopy of purified proteins. I learned the technique from Craig Smith and applied it to studies of C4BP. C4BP turned out to have a most unusual molecular shape as it appeared in the electron microscope as a spider with seven extended tentacles, each of which was formed by a 70-kDa subunit (now referred to as the a-chain) (Figure 5.6) (Dahlback et al., 1983). Each a-chain contains a binding site for C4b. In addition, I observed that the C4BP

Figure 5.6. Molecular model C4b-binding protein. Electron microscopy of C4BP revealed an octopus-like structure, with the dtstance from the center of the molecule to the peripheral end of an extended tentacle 330 8, and tentack diameter 30 A (Dahlback et al., 1983). C4BP contains two types ofsubunits, seven a-chains and one @-chainlinked by disulfide bridges involving cysteine residues located in the COOH-terminal ends of the chains. Each a-chain binds a C4b, whereas the @-chainbinds protein S. Adapted from Hillarp and Dahlback (1990).

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molecule contained a small distinct subunit which appeared to bind protein S. Although the limited resolution of the technique did not allow a more detailed elucidation of the three-dimensional structure of C4BP and of its hypothetical protein S-binding subunit, the results convinced me that C4BP indeed must have a separate subunit which binds protein S. The isolation and characterization of this subunit turned out to be challenging and for many years the subunit was elusive.

1. Identification of the P-chain in C4b-binding protein After returning to the department of Clinical Chemistry in Malmo, I established my own laboratory and was joined by Andreas Hillarp who wrote his Ph.D. thesis with me. His project was to elucidate the molecular basis of the protein SC4BP interaction and to identify a protein S-binding subunit. Andreas Hillarp’s work was successful and he managed to identify, purify, and structurally characterize a previously unrecognized 45-kDa subunit, now referred to as the C4BP p-chain (Hillarp and Dahlback, 1988, 1989; Hillarp et a!., 1989). Both a- and P-chains belong to a family of proteins composed of multiple, internally homologous, tandemly arranged modules denoted short consensus repeats (SCR), complement control repeats (CCP), or Sushi repeats (Hillarp, 1991). The a-chain has eight SCRs, whereas the shorter @chain contains three copies of the protein modules. In addition, both chains contain nonrepeat C-terminal regions, each of which has two cysteine residues. During synthesis of C4BP, six to eight a-chains and one @-chainare disulfide linked through these cysteines which results in the formation of the spider-like C4BP molecule. The availability of cDNA clones for both chains has made it possible to conclusively demonstrate the protein S binding site to be located in the P-chain and several lines of evidence suggest the first SCR to be involved (Hardig et af.,1993; Fernandez and Griffin, 1994; Hillarp et al., 1994).

VI. THROMBOEMBOLISM ASSOCIATED WITH DEFICIENCIES OF PROTEIN C OR PROTEIN S A. Protein C deficiency The elucidation of the anticoagulant properties of protein C and protein S stimulated severai research groups to measure the plasma concentrations of these proteins in patients with thrombosis. The first identified thrombosis patient with heterozygous protein C deficiency was reported by John H. Griffin and coworkers in 1981. A few years later, the severe neonatal thromboembolism which is caused by homozygous protein C deficiency was described (Seligsohn et al., 1984; Marlar and Neumann, 1990). Since then, a large number of protein C

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deficiencies have been reported from many different laboratories and the genetic defects have been determined in many of the cases (Reitsma et al., 1993).

1. Different types of protein C deficiency There are two types of protein C deficiency described: type I, representing the majority of cases, is a true protein deficiency, whereas type 11 is a functional defect with normal or slightly low plasma concentrations of protein C (Dahlback and Stenflo, 1994). In most cohorts of thrombosis patients, protein C deficiency is found in less than 5% of the patients (Malm et al., 1992; Tabernero et al., 1991; Heijboer et al., 1990). In the families of these protein C-deficient patients, a clear association between the protein C defect and an increased risk of thrombosis has been found. From the number of known protein C cases with thrombosis in the Netherlands it was estimated that the prevalence of protein C deficiency associated with thrombosis in the population was 1:16,000 (Broekmans and Conard, 1988). Therefore, it was very surprising when Joseph Miletich and his colleagues in St. Louis reported that they had found the prevalence of protein C deficiency in healthy blood donors to be 1:250 (Miletich et al., 1987). Even more troubling was the observation that thrombosis did not appear to be particularly common in families of these protein C-deficient individuals. Adding further to the paradox, the same mutation was found in a thrombosisprone protein C-deficient family and in a protein C-deficient family in which thrombosis was not particularly common (Reitsma et al., 1991, 1993). Protein C-deficient families without thrombosis have been referred to as clinically recessive. In its homozygous form, the clinically recessive protein C deficiency is associated with purpura fulminance. Heterozygous protein C deficiency associated with thrombosis has been called clinically dominant protein C deficiency (Bertina, 1988). It has been speculated that additional genetic defects influence the thrombosis risk in protein C-deficient individuals and that the presence or absence of such defects determine whether protein C deficiency is clinically dominant or clinically recessive (Miletich et at., 1993). In support of this concept, Rogier Bertina and colleagues recently reported that Dutch thrombosisprone protein C-deficient patients have a significantly higher prevalence of APC resistance than that observed in the general population (Koeleman et al., 1994). As discussed under Section VI, B, it is indeed becoming more and more obvious that thrombosis is the result of a combination of circumstantial and genetic risk factors and that thrombosis patients often carry more than one genetic risk factor (Dahlback, 199413; Zoller et al., 1994).

B. Protein S deficiency The first cases with protein S deficiency were described in 1984 (Comp and Esmon, 1984; Comp et al., 1984; Schwarz et al., 1984). In one of these early

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reports is was demonstrated that the patients were only deficient in the free form of protein S, whereas total protein S was essentially normal (now referred to as type 111) (Comp et al., 1984). Several reports have confirmed the original observation that protein S-deficient patients may have a selective deficiency of free protein S (reviewed in Dahlback and Stenflo, 1994). Other protein S-deficient patients present with decreased levels of both free and complexed forms of protein S (type I). The molecular difference between type I and type 111has for a long time remained elusive. Recently, we have found coexistence of both types in several families. This, together with careful determination of plasma concentrations of different isoforms of C4BP and protein S, has suggested the two types of protein S deficiency to be phenotypic variants of the same genotype (Zoller et al., 1995a). The explanation is that under normal conditions, the molar plasma concentration of protein S exceeds that of P-chain-containing C4BP (C4BPP+) by approximately 30-40% (Griffin et al., 1992; Garcia de Frutos et al., 1994). Due to a high affinity of the interaction between protein S and C4BPp-t also in citrated plasma, essentially all C4BPP+ molecules are complexed with protein S, with free protein S being the remaining molar surplus of protein S. In protein S deficiency, the decreased plasma levels of protein S are reflected in distinctly low concentrations of free protein S, whereas there is an overlap in concentrations of total protein S between those with and those without protein S deficiency. From this follows that some individuals with protein S deficiency have normal total protein S, whereas all have low free protein S (Zoller et at., 1995a).

1. The protein S gene The protein S gene, which is located on chromosome 3, contains 15 exons and spans more than 75 kb (Schmidel et al., 1990; Ploos van Amstel et al., 1987, 1990; Edenbrandt et al., 1990; Long et al., 1988; Watkins et al., 1988). In addition, the human genom contains a protein S pseudogene, which is more than 90% identical in nucleotide sequence to the expressed protein S gene. These features have made it difficult to determine the genetic defects in protein S-deficient patients and so far few reports describing protein S gene defects have been published (Schmidel et al., 1991; Ploos van Amstel et al., 1989; Reitsma et

al., 1994).

2. Problems with functional protein S assays Several different types of functional assays for protein S have been devised. However, it has been observed that these assays lack specificity, which became particularly obvious in a multicenter study which compared the performance of functional assays from four different laboratories (Boyer-Neumann et ai., 1993). Recently, it has been shown that several of the patients which were originally classified as type I1 protein S deficiency have no protein S gene defect but

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instead suffer from inherited APC resistance (Faioni e t al., 1993). The reason for the misclassification is that the Arg506 to Gln mutation in the factor V gene affects functional protein S assays.

C. Critical evaluation of laboratory investigation of thromboembolism During the mid 1980s, Johan Malm, who was working on his Ph.D. thesis in my laboratory, performed a large study (439 patients and 100 controls) which was aimed at critically evaluating the laboratory parameters which were routinely used in the evaluation of thrombosis patients (Malm et al., 1992). The results were quite disappointing but set the groundwork for future research. Johan Malm found that less than 10% of the patients could be diagnosed as having a welldefined genetic defect, such as deficiency of AT 111, protein C, or protein even though 40% of the patients indicated positive family histories of thrombosis. This indicated that there were one or more genetic defects which we could not detect. The study also evaluated a series of fibrinolytic parameters which had been used extensively for many years. The basis for using these laborious methods was that several studies had suggested that up to 30% of thrombosis patients had low fibrinolytic activity either due to high levels of plasminogen activator inhibitors or to a deficient release of tissue plasminogen activator (Nilsson et al., 1985; Wiman et al., 1985). To our disappointment we found that, although there were some statistical differences in fibrinolytic activity after venous occlusion between patients and controls, measurements of fibrinolytic components were not useful for the evaluation of individuals patients. Like other researchers, we found the fibrinolytic activity to correlate inversely with the concentrations of plasminogen activator inhibitor I (PAI-I). PAI-I is one of the acute phase reactants and accordingly the fibrinolytic response to venous occlusion in a given patient fluctuated from one time to another. It could therefore not be concluded whether the poor fibrinolytic activity was present before the thrombotic episode and had contributed to the development of the thrombosis or if low fibrinolysis was the result of the disease. Thus, even though the results of this very labor-intensive study were rather disappointing, they opened my mind to the possibility that other genetic defects were involved in thrombophilia and that they were there to be found.

s,

VII. DISCOVERY OF ACTIVATED PROTEIN C RESISTANCE A. Unexpected behavior of patient plasma in functional protein C assay initiated the research During the mid 1980s, I was performing basic research on protein S, CqBP, and factor V at the department of Clinical Chemistry and at the same time I

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was a consultant at the coagulation unit at Malmo General Hospital. I used to take a tour every day and check with the technicians to see if they had problems which I could possibly help them solve. One day in the fall of 1986 one of the technicians, Mrs. Ing-Marie Persson, asked me about some strange results she had observed when performing an in-house functional protein C assay. The assay included barium citrate adsorption of the vitamin K-dependent proteins, elution of the proteins with citrate, activation of protein C with a thrombin-thrombomodulin mixture, and finally determination of the anticoagulant activity of the patient’s protein C in normal plasma using an APTT reaction (Hickton et al., 1986). She observed that when plasma from a particular patient with thrombosis was tested, different dilutions of the barium citrate eluate yielded different protein C values. At low dilutions, the sample from the patient gave low values for protein C, whereas at high dilutions the protein C activity of the patient sample was essentially normal. There was of course a number of possible explanations for this behavior and I will describe in detail the work which finally led to the elucidation of the molecular defect in the patient (Dahlback et al., 1993), I begin with a few words about the patient. He was a middle-aged man with multiple episodes of thrombosis, the first thrombotic event occurring at the age of 19. He was referred to the coagulation unit by Dr. Magnus Carlsson at the Ryhovs hospital in Jonkoping, Sweden. Apart from the strange response in the protein C assay, all other tests included in the thrombosis evaluation were normal. Thus, he had normal plasma levels of AT 111, protein C antigen, and protein S. Routine coagulation tests, such as APTT, thrombin, and prothrombin times, were also normal and there were no indications of the presence of coagulation inhibitory lupus anticoagulants. This was the information which was known to us when we started elucidating the possible mechanisms for the strange response in the protein C assay. In the work that was to follow, Mrs. Persson performed many of the initial experiments. I might also add that the project at this stage did not involve any of the people in my research laboratory who all worked on more basic aspects of the protein C anticoagulant system. Of course, I did not expect that the outcome of the experiments would be so important and that they would have such an impact on our understanding of the pathogenesis of thromboembolic diseases. The work was mainly initiated out of curiosity and by the challenge to explain the unexpected behavior of an individual plasma sample in the functional protein C assay.

B. Possible mechanisms for the APC resistance Initially, we observed that the patient’s plasma was lipemic and our first idea was that lipid in some way may have affected one or more steps in the complicated functional protein C assay. To test this possibility, plasma was treated with the

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detergent Triton X-100 prior to the barium citrate absorption to avoid any interference of lipids in the assay. However, this exercise did not affect the outcome of the assay.

1. No APC inhibitor in APC resistance The second hypothesis we tested was that the patients plasma contained an inhibitor to activated protein C, either congenital or acquired. To investigate the possibility of a plasma protein C inhibitor we needed a more direct assay. Therefore, instead of testing for the function of the patient's protein C, we used his plasma to test the anticoagulant response to highly purified normal APC. A n APTT system was used and it became obvious that APC, when added to the patient plasma, did not yield the expected prolongation of clotting time (Figure 5.7). Repeated experiments on new plasma samples yielded the same pattern of resistance to APC. The possibility that the patient plasma contained an acquired inhibitor of immunoglobulin type was tested in several different ways, but we finally concluded that the patient plasma did not contain an inhibitor to APC. Thus, when the patient plasma was subjected to absorption of the different immunoglobulin fractions, the resistance to APC remained in the unabsorbed fraction. Moreover, gel filtration chromatography of the patient plasma and testing of all fractions for APC-inhibitory activity failed to detect any inhibitor. The results of this experiment indicated that, in the patient plasma, not only inhibitors of immunoglobulin type, but also other types of APC inhibitors, such as a mutated a-1 antitrypsin, or any other kind of serine protease

I 0

1

2

3

4

5

APC (uglml) Figure 5.7. APC resistance in a thrombosis patient. APC, when added to an APTT reaction, prolongs the clotting time of control plasma (0).In contrast, plasma from a middleaged man with recurrent thrombosis did not respond to the anticoagulant activity of AF'C (0).Modified from Dahlback et d. (1993).

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inhibitor were absent. Moreover, measurement of protein C activity in the patient plasma with a snake venom-based chromogenic assay yielded normal values for protein C, which indicated that there was no inhibitor of the active site of APC present in the plasma. This was further corroborated by an observed normal half-life of APC activity in the patient plasma, as measured by a chromogenic synthetic substrate assay (Dahlback et al., 1993).

2. APC resistance not caused by protein S deficiency The next possibility we tested was that the patient had functional protein S deficiency. A series of experiments ruled out this possibility, e.g., addition of normal protein S to the patient plasma failed to correct the defect. Moreover, it was observed that the patient plasma was resistant to the anticoagulant action of a combination of bovine APC and bovine protein S. As bovine APC, due to it species specificity, is not potentiated by human by human protein S,this experiment provided further support for the conclusion that the patient did not suffer from functional protein S deficiency. At this stage, the project was temporarily discontinued because I was invited by Dr. Fletcher B. Taylor Jr. to spend a 6-month period as a Greenberg visiting scholar at the Oklahoma Medical Research Foundation in Oklahoma City and this coincided with an end of my term as a consultant at the coagulation unit. After returning to Malmo in 1990, I was appointed head of the laboratory section of the coagulation unit at the Malmo General Hospital. Already in 1989, I had been given a position as full professor of blood coagulation research at the university of Lund. The joint appointment with the hospital provided a good basis for me to continue the work on the APC-resistant patient and also made it possible to test other thrombosis patients for the APCresistance defect. To obtain new samples from the patient, I contacted Dr. Magnus Carlsson in Jonkoping in the fall of 1990. A new series of experiments was initiated and in retrospect it is obvious that some of the results we obtained at this stage initially led me in a wrong direction, i.e., when the results relate to which gene and which mutation is responsible for the APC resistance. On the other hand, without these experimental results I would not have discovered the anticoagulant functions of intact factor V. So, one has to take the bad news with the good news and enjoy the progress that has indeed been made in this field since 1990.

3. Mutations in APC substrates as possible causes of APC resistance One day, it occurred to me that the explanation for the poor anticoagulant response could actually be that one of the substrates for APC, factors Va and

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VIIIa, was insensitive to APC. To investigate this possibility, we tested the effect of APC on the patient plasma in a functional factor VIII assay, which was based on the use of chromogenic synthetic substrates. Diluted plasma was incubated with increasing concentrations of APC, phospholipid, factor IXa, factor X, and calcium and the amount of factor Xa formed was measured with a synthetic substrate. The first experiment clearly showed that APC was less efficient in inhibiting factor VIII from the patient plasma than factor VIII from plasma of a normal control. This led me to suspect that the patient’s factor VIII was insensitive to APC and that this was caused by a mutation in the gene for factor VIIIa affecting one of the regions which encodes the APC cleavage sites. We also tried an experimental approach which aimed at elucidating whether the patient’s factor Va was resistant to APC. In this case we used a dotting-based assay in which the patients plasma was incubated together with increasing concentrations of APC, phospholipid, factor Xa, and calcium. The clotting time was measured and we observed prolongation of the clotting time, which at the time did not seem very different from that of a normal control. The combination of the results obtained in the factor VIIIa- and factor Va-based assays suggested to me that factor VIIIa rather than factor Va was the problem. We now know that this is not correct and that in fact it is the other way around. How can this be explained? There are several reasons. One is that the first patient was only heterozygous for the factor V gene mutation causing APC resistance (B. Dahlback, unpublished observation) even though his plasma response to APC was very poor and closely similar to that seen in an individual with homozygous factor V gene mutation. I believe that in addition to the factor V gene mutation, the patient and several of his family members have an additional genetic defect, which is yet to be characterized. This defect contributes to the severity of the APC resistance and also to the thrombotic tendency. As the patient was heterozygous for the factor V gene mutation, half of the factor Va molecules were normal and they were degraded in a normal fashion by APC. In a factor Xa assay, the addition of APC to a heterozygous plasma leads to a prolongation of the clotting time which is approximately half of normal. As the patient plasma was severely APC resistant in an APTT-based assay, I expected a similarly severe APC resistance in the factor Va-based assay, if indeed factor V was the problem. If the first patient had been homozygous for the factor V gene mutation, the patient’s factor V would have been completely APC resistant and I would probably have drawn the correct conclusion at this time. What then explains that factor VIII from the patient was less readily degraded than factor VIII from a control? This might be caused by the factor V gene mutation, because mutated factor Va is not degraded by APC. In the patient’s sample, this leads to stabilization of the prothrombinase complex (factor Xa, factor Va, phospholipid, and calcium), which is formed during the incubation time, and the rates of thrombin generation in the patient sample are higher

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than those in a control. The generated thrombin activates both factor VIII and V and in the patient sample, the concentration of APC substrate will be much higher than that in the control incubation. Such a mechanism may thus explain the apparent lower APC response in the factor VIIIa-based assay on plasma from a patient having the factor V gene mutation which causes APC resistance. From more recent experiments we now know that factor V, but not factor Va, serves as an APC cofactor together with protein S (Shen and Dahlback, 1994). The increased concentrations of thrombin which are generated in the patient sample will thus lead to a secondary loss of APC cofactor activity of mutated factor V. Using these results as a basis, my working hypothesis was that a factor VIII gene mutation caused the APC resistance and that this mutation changed one of the APC cleavage sites in factor VIIIa. I was thrilled and fascinated by this, because this would be the first case in which a factor VIII gene mutation caused thrombophilia rather than hemophilia. During one of my telephone conversations with Dr. Magnus Carlsson in the fall of 1990, I asked him about the patient’s family history and the possibility that any of the proband’s male relatives had experienced thrombosis. I remember how excited I was when Dr. Carlsson told me that the patient’s older brother and a maternal uncle also had suffered multiple episodes of thrombosis. This, together with the results of the factor VIII assay, indicated that this was consistent with X-linked thrombophilia caused by a factor VIII gene mutation. Since it was close to January 1991, the time to submit abstracts to the ISTH meeting in Amsterdam, and because I was excited about the idea of a factor VIII gene defect causing thrombophilia, I submitted an abstract which was accepted as an oral presentation (Dahlback and Carlsson, 1991). The presentation generated a great deal of interest, but everyone thought that this was a curiosity from one of the remote Scandinavian countries and, in retrospect, it is obvious that my presentation did not have a major impact. A t the time of the presentation, we had not yet had a chance to investigate whether the relatives demonstrated a similar laboratory defect and furthermore, we had not had a chance to investigate whether the patient’s factor VIII gene indeed was mutated.

4. APC resistance not

caused by factor VIII gene mutation

In the summer and early fall of 1991 we obtained both plasma and whole blood samples from the patient’s family and we were now ready to hunt down the factor VIII gene mutation. There were two possible cleavage sites in factor VIII for APC at position 336 and 526. Only the 336 site was known from the literature. In Amsterdam, I met and talked to Dr. Philip Fay, who had given an excellent presentation on the structure-function relationships of factor VIII, and he informed me about his results which demonstrated the 526 cleavage site to be crucially important for APC degradation of factor VIIIa. The recently described

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polymerase chain reaction (PCR) technique made it possible to investigate whether one or the other of these sites were mutated in the factor VIII gene from the patient. It was a real disappointment to find that the nucleotide sequences in regions encoding positions 336 and 526 were completely normal in the patient. We also performed a linkage study using polymorphic markers for the factor VIII gene and found that the patient and his affected brother had inherited different factor VIII genes from the mother. This of course made is less likely that the gene defect was to be found in the factor VIII gene. At the time, we did not perform the same exercise with the gene for factor V. One reason was that there were no intron sequences available (required for primers to the PCR), even though the intron-exon boundaries had been reported (Cripe et al., 1992). Moreover, no polymorphic sites were known in the factor V gene. Thus, a PCR-based approach similar to that used for the factor VIII gene was not an easily performed task at the time. These problems, together with the clotting data which indicated that the proband’s factor V could be essentially normally inhibited, were the reasons why I did not focus more of my attention to the possibility that mutation of the APC cleavage sites of factor V was the cause of APC resistance.

5. Involvement of novel APC cofactor in APC resistance? In the fall of 1991, after excluding the possibility of a factor VIII gene mutation, I really did not know in what direction to take the work. 1 thought that I had investigated every possible cause for the APC resistance and I could still not give Mrs. Persson an explanation for the strange results she had observed in the functional protein C assay. During my summer vacation in 1992 I decided to go through all the data we had on APC resistance in order to find out if I had overlooked a possible cause of the APC resistance. Being more relaxed than usual it was also a good time to come up with some new ideas. Indeed, a new idea occurred to me while driving my oldest daughter to a gymnastics camp 80 km north of Malmo. During this car trip it suddenly occurred to me that most of the data were compatible with a deficiency of a previously unrecognized cofactor to APC. I remember how excited I was with this idea and decided to start working on the APC resistance again. In September 1992, I summarized the results of the first family with APC resistance in a paper which was published in “Proceedings of the National Academy of Science, U S A in February 1993 (Dahlback et al., 1993). As evident from that paper, we had examined almost every possible cause of APC resistance, except for a factor V gene mutation changing one of the APC cleavage sites, although the published paper discussed the possibility of this being a potential mechanism of APC resistance. The conclusion at the time was

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that we had found a novel mechanism of familial thrombophilia which possibly could be caused by a defect in a previously unrecognized APC cofactor. This was, in a way, both right and wrong, because work to follow demonstrated intact factor V to be an APC cofactor and APC resistance to be due to a mutation in the factor V gene (Bertina et al., 1994; Greengard et al., 199413; Voorberg et al., 1994; Zoller and Dahlback, 1994). However, what came as a surprise to me was that APC resistance in more than 90% of cases is caused by the same point mutation (Bertina et al., 1994; Zoller et al., 1994). When the paper was completed it was, of course, very important to decide how and where to present the data. After careful consideration, I came to the conclusion that “Proceedings of the National Academy of Science, USA” was a good choice. It was obvious to me that APC resistance was going to be very important and a major issue for years to come. However, my concern at the time was that clinically oriented journals may not understand the significance of the work at this early stage. I was concerned that they would regard the report as interesting, but that more work was going to be required in order to show the general importance of the defect. In retrospect, I am convinced that the decision was right. In order to publish the paper in “Proceedings of the National Academy of Science, USA,” I again approached Professor Emeritus Jan Waldenstrom. He read the paper with great interest but asked me whether this was just a single rare case. I informed him that my new collaborator, Dr. Peter Svensson, had just found two additional families with APC resistance. He urged me to include those family studies in the paper, which I did. Professor Waldenstrom also made a very interesting remark. He thought it quite strange that almost all family members demonstrated APC resistance and that this raised questions about whether the defect was truly inherited as an autosomal dominant trait. We now know that most of the family members carry the factor V gene mutation in heterozygous form (B. Dahlback, unpublished observation). At the time of submission of the first paper, I realized that I would have at most 1 year to finish the purification of the hypothetical APC cofactor I1 and to characterize the molecular defect of APC resistance. This estimate turned out to be correct.

VIII. APC RESISTANCE AS A BASIS FOR THROMBOEMBOLIC DISEASE As I have previously mentioned, I was appointed head of the laboratory section at the coagulation unit at Malmo General Hospital at the end of 1990. The first major change which I introduced at the laboratory was to discontinue the laborintensive fibrinolytic assays, which included venous occlusion tests on 2 consecutive days. The background for this decision was the study that John Malm and I had performed in which we demonstrated that the fibrinolytic techniques were

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not useful in evaluating individual patients (Malm et al., 1992) and since we had no active research in the fibrinolytic field, it was of course not possible to continue with these tests. The problem was that we now had almost nothing to test for and little to offer physicians who referred thrombosis patients for evaluation. Occasionally, the laboratory screening led to the identification of patients with lupus anticoagulants or deficiencies of AT 111, protein C, or protein S, but in more than 90% of thrombosis patients referred to our unit, we found nothing wrong. In a situation in which up to 40% of the patients indicated that they had positive family histories of thrombosis, this was of course very frustrating.

A. Development of APC-resistance test In the summer of 1991, I initiated a line of research which would pay off richly. We established an easy screening technique for APC resistance in the laboratory. In a series of preliminary experiments, increasing concentrations of APC were added to patient plasma and the clotting time was measured in an APTT reaction. Initially, the results really did not look very encouraging and the range of anticoagulant response in normal controls was very wide. However, as plasma samples from several of the members of the first family consistently demonstrated a low anticoagulant response to APC, I was encouraged to continue the work. At this point, we also tested a few samples from thrombosis patients and some of those turned out to respond poorly to APC. This convinced me that a screening assay, which was based on the APCIAPTT approach, could be developed. What made the assay procedure really easy was that we found it possible to include the APC directly in the calcium chloride solution, which is the final reagent in the APTT reaction. The APC-resistance test turned out to be very sensitive to different influences, such at the choice of the APTT regent, the APC concentration, the sample handling, and the instrumentation, but when all those variables were kept under strictly standardized conditions, the assay worked well.

6. Screening of thrombosis patients for APC resistance In September 1991, the APC-resistance test was ready for use in the routine laboratory and we started to run it on all samples that arrived to the laboratory. There was a great variation in APC-dependent prolongation of clotting time seen among the examined samples but most importantly, we did find new cases with very low APC responses, which encouraged me to continue using the assay. However, it was difficult to convince my clinical colleagues about the merits of the test, as they thought it showed pathological results too often. They argued that it could hardly be true that almost half of the thrombosis patients could have the defect. I had to ask myself whether the APC-resistance situation was

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similar to that of the fibrinolytic system, the assays of which we had just discontinued performing for reasons discussed previously. However, there was one major and important difference between the APC-resistance test and the assays for fibrinolytic components. APC resistance was an inherited defect and could thus also be detected in family members without thrombosis. This convinced me to continue with the APC-resistance test. A major problem at the time was distinguishing between a normal and a pathological APC resistance as several apparently healthy individuals were also found to be APC resistant. We now know that approximately 10% of the Swedish population has the factor V gene mutation which causes APC resistance, but this was of course not easily envisioned in the early days of the assay. In September 1992, a young medical doctor, Peter Svensson, began work in the clinical section of the coagulation unit. He was enthusiastic about the work on APC resistance which was going on in the laboratory and we agreed that his job was to evaluate the first year’s results with the APC-resistance test. He had to go through the medical records of all patients that had been examined during the year. In the study population, he included patients in whom the diagnosis of thrombosis had been objectively verified. Using this criteria, he found 104 patients and their APC-resistance test results were compared with those of normal controls. For the presentation of the data, we found it useful to convert the clotting data into APC ratios (clotting time obtained with the calcium chloride containing APC divided by the clotting time measured in the presence of calcium chloride without APC). When the data were plotted, it became obvious that there was a bimodal distribution of APC ratios in the patient population (Figure 5.8). Approximately 40% of the patients were in the “low APC ratio” group, whereas around 7% of controls were in the “low APC ratio” group. Plasma mixing experiments suggested that the APC-resistant patients suffered from the same or similar defects (Svensson and Dahlback, 1994).

C. APC resistance-An

inherited risk factor of thrombosis

Already started in September 1991, APC-resistant patients had been invited to participate in family studies which aimed at elucidation of the inherited nature of the APC resistance. During the following years, many APC-resistant families were found; indeed, new families were discovered every week. When data from 177 relatives to 34 index patients were analyzed it became clear that APC resistance in most cases was inherited as an autosomal dominant trait. The results of a Kaplan-Meier analysis demonstrated family members with low APC ratios to have higher risk of thrombosis than those with high APC ratios (Figure 5.9). The results of the analysis were very exciting and it became obvious to everybody in the coagulation units that APC resistance was important and was there to stay.

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st I

0 0

I C

Control

Patients

Figure 5.8. APC resistance in thrombosis patients. Approximately 40% of patients were below the cut off APC ratio of 2, whereas the corresponding value in controls was 7%. The difference in APC ratios between thrombosis patients and controls was highly significant (P < 0.0001). Adapted from Svensson and Dahlback (1994).

The result of the thrombosis study were first presented in preliminary form at the German Thrombosis and Haemostasis (GTH) meeting in Badgastein in February 1993, then at an ETRO meeting in Paris during the spring, and later during the summer at the International Society of Thrombosis and Haemostasis meeting in New York (Svensson and Dahlback, 1993a). The response among other scientist in the field was amazing and many wanted to test their patients for APC resistance. To make such testing possible, I had, in the fall of 1991, initiated a collaboration with Dr. Steffen Rosen and colleagues at Chromogenix (former Kabi diagnostica). The people at Chromogenix were really very excited about the APC resistance and the test became available in May 1993. It is interesting to note that after the GTH meeting, a multicenter evaluation of the APC-resistance test kit was initiated. The study, which included more than 20 laboratories, was performed during the spring of 1993 and later published (Ros6n et al., 1994). It is interesting to look back on how quickly the knowledge on APC resistance spread and that the results gained general acceptance early on. My close collaboration with Chromogenix helped to spread the news about APC resistance. In many countries Chromogenix people contacted scientist in their respective countries in February 1993 and informed them about the new discovery and that APC resistance according to preliminary results obtained in my

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80 n W

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Age (years) Figure 5.9. APC resistance is a risk factor for thrombosis. (A) Thrombosis-free survival curves of APC-resistant (n = 104) and non-APC-resistant (n = 107) individuals from 34 families. (B) Results after exclusion of the 34 probands and 2 persons with protein S deficiency. Differences in survival curves are highly significant in both A and B. Modified from Svensson and Dahlback (1994).

laboratory was a very prevalent cause of thrombophilia. This triggered intense activity in many laboratories and homemade APC-resistance tests were quickly established. However, as the date for the deadline for abstract submission to the New York meeting had already passed, my group was the only one which presented data on APC resistance at the ISTH meeting that summer. We had a total of five presentations on APC resistance and several of them were oral presentations (Svensson and Dahlback, 1993a,b; Johansson et al., 1993; Dahlback and Carlsson, 1993; Dahlback, 1993).

D. Other laboratories confirm APC resistance to be a common defect in thrombosis Scientists in several different laboratories quickly confirmed our results. In a collaborative study between the laboratories of John Griffin and Bruce Evatt,

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APC resistance was identified in 60% of a small group of patients with severe thrombophilia which was unexplained by other mechanisms. Their paper on the topic was submitted to “Blood” 2 weeks after the New York meeting, accepted within 5 days, and published in October 1993 (Griffin et al., 1993). To me, since I had worked on the story since 1986, this was indeed amazingly quick. In Leiden, the work on APC resistance also took off very quickly. The scientists in Leiden have a long-standing interest in the genetic mechanisms of thrombosis and for the purpose of studying this in detail they had initiated a large case control study in 1988, the so-called Leiden thrombophilia study. Between the years 1988 and 1992 they had collected information on close to 500 thrombosis patients and the same number of controls. When they had established the APC-resistance test in their laboratory they had the golden opportunity to quickly examine their patients and controls. I became aware of their study during the summer meeting in New York and, in September, Frits Rosendaal, while visiting my laboratory in Malmo, informed me that they had submitted their work to the “Lancet” a few days before his trip to Malmo. The paper was very quickly accepted and published in December (Koster et al., 1993). The paper was important for establishing the general consensus that APC resistance indeed is an important cause of thrombosis. In the Leiden study, 2 1% of patients and 3% of controls were found to have APC resistance and the risk for thrombosis contributed by APC resistance was calculated to be approximately 7. The paper was accompanied by an editorial comment by Tuddenham (1993). The information given by Frits Rosendaal was shocking to me. It was already September and we had not yet started to write our paper. Because I had the information about the Leiden paper on a Friday, I decided to use the weekend to write the manuscript on our study. Fortunately, all the statistical calculations had been done and I just had to do the writing. This was indeed a hectic weekend, but by Monday the paper was essentially ready to go. I was quite proud of the paper, and together with Peter Svensson decided to send it to the “New England Journal of Medicine.” This was of course a play with high stakes as I knew that the review process for this journal was very difficult. It really was! Well, the paper was finally accepted, but not published until February 1994 (Svensson and Dahlback, 1994). It was accompanied by an interesting editorial written by Kenneth Bauer (1994), who would later that year publish his own study on an interesting family with multiple family members having APC resistance due to homozygous factor V gene mutation (Greengard et al., 1994a; Hajjar, 1994). Let me now return to the question of what the molecular mechanism of APC resistance is and how the conclusion was reached that the condition was due to a defect in the factor V gene.

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IX. ELUCIDATION OF THE MOLECULAR BASIS OF APC RESISTANCE Serendipity continued to play a role for the success of our work. To establish a normal range of the APC-resistance test, samples were drawn from the staff at the department and one of the female technicians was found to have pronounced APC resistance. In the APC-resistance test, the addition of APC to her plasma was essentially without effect and the APC resistance found in her plasma was even more pronounced than that of the original patient. The technician was 45 years old and had no personal or family history of thrombosis (her family was small, however). In repeated samples, severe APC resistance was consistently demonstrated and she appeared to have the same defect as the first patient because a 1:l mixture of plasmas from the two individuals was equally APC resistant as any of the two individual plasmas. An inherited nature of her APC resistance was apparent because plasma from her mother demonstrated severe APC resistance. We now know that both the daughter and her mother are homozygous for the factor V gene mutation (B. Dahlback, unpublished observation). The availability of the severely APC-resistant plasma was of paramount importance for the elucidation of the molecular defect of APC resistance.

A. Isolation of novel APC cofactor One of my working hypotheses was that APC resistance was due to a defect in a previously unrecognized cofactor. This conclusion appeared to gain support when we found a crude fraction of normal plasma to correct the APC resistance, whereas a corresponding fraction of APC-resistant plasma was without effect (Dahlback, 1993). The activity was tentatively given the name APC cofactor 11. It was obvious that purification of this factor would lead to the identification of the genetic defect which caused APC resistance and together with Mrs. Bergisa Hildebrand, a technician with whom I have worked for many years, I embarked upon a protein purification exercise which began in September 1992. This type of work was something we were both acquainted with as we had previously devised protein purification procedures for factor V, protein S, protein C, C4bbinding protein, and many other coagulation and complement proteins. For the successful purification of an unknown protein, an assay which measures the activity of interest is of the utmost importance. The availability of severely APC-resistant plasma made such an assay possible. Fractions from different kinds of chromatographies were added to the APC-resistant plasma and tested for their ability to correct the APC resistance. A large number of different purification steps were tried and we soon had devised a potential purification procedure which involved barium citrate absorption (removed the vitamin K-de-

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pendent proteins), fractionated precipitation with polyethylene glycol 6000 (PEG 6000), anionic-exchange chromatography, ammonium sulfate precipitation, and finally gel filtration chromatography. We noticed that the activity which corrected the APC resistance was labile and that the yield of the final product increased when calcium chloride and an abundance of serine protease inhibitors were added throughout the purification. Early during this work it occurred to us that the protein we were about to purify behaved just like coagulation factor V, a labile protein which we had isolated from human plasma in the late 1970s (Dahlback, 1980).

B. Identification of factor V as the novel APC cofactor The difficult problem was to prove that the protein which expressed APC cofactor I1 activity indeed was factor V and not a contaminating protein. The final proof of the identity between APC cofactor I1 and factor V was obtained when APC cofactor I1 activity was found to bind to a monoclonal antibody column against human factor V and that both factor V and APC cofactor I1 activities were recovered from the column after elution of bound proteins with high pH. At the time, my interpretation of the results was that factor V functioned as an APC cofactor and I believed that the molecular defect of APC resistance was caused by a mutation in the factor V gene which affected this activity (Dahlback and Hildebrand, 1994). This was of course an unorthodox interpretation of the factor V function but it was supported by other results. An important observation which convinced me that factor V indeed functions as APC cofactor was that one of our monoclonal antibodies against factor V, when added directly to plasma, inhibited APC-mediated prolongation of clotting time without affecting the procoagulant activity of factor V (Dahlback and Hildebrand, 1994). In addition, in a more recent study by Shen Lei, a Chinese postdoctoral fellow in my laboratory, additional evidence for the role of factor V as APC cofactor was obtained. In a factor VIIIa degradation system using purified components, she demonstrated factor V to express APC cofactor activity and to work in synergy with protein S (Shen and Dahlback, 1994). The ability of factor V to correct the APC resistance did not elucidate the true molecular mechanism, but suggested one or more mutations in the factor V gene to be involved in the pathogenesis of APC resistance. At the GTH meeting in February 1993 I had indicated that APC resistance could be corrected by a protein fraction of normal plasma. This was presented in more detail at the International Society of Thrombosis and Haemostasis meeting at New York in July 1993 (Dahlback, 1993). At that time I was convinced that factor V was involved but I could not disclose it because I had not yet had time to write the paper. After the summer, I decided it was time to publish the results. Professor Jan Waldenstrom and the “Proceedings of the National Academy of

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Science” were also a natural choice for this paper. Professor Waldenstrom was very enthusiastic to learn that factor V was involved in APC resistance and be indicated that he found particular pride in communicating the paper. The paper was submitted in September, accepted after a few months, and published in February 1994 (Dahlback and Hildebrand, 1994).

C. Linkage of APC resistance to the factor V gene Our results had shown that the genetic defect associated with APC resistance was found in the gene for factor V. In order to link the APC-resistance phenotype to the factor V gene, we needed both a suitable family with APC resistance and appropriate polymorphic markers in the factor V gene. One of my collaborators, Bengt Zoller, was studying families with protein S deficiency as part of his Ph. D. training and it turned out that one of his families, apart from having protein S deficiency, also had independent inheritance of APC resistance. The family was suitable for linkage and the results indicated that the APC-resistance phenotype was indeed tightly coupled to the factor V gene, as demonstrated by a LOD score of 3.9 (Zoller and Dahlback, 1994). We had started searching for the causative mutation when I went to Snowbird in Utah during the Easter holiday to participate in a small meeting on genetic approaches to study familial thrombosis.

D. APC resistance caused by a single factor V gene mutation At the meeting, Rogier M. Bertina from Leiden presented their data on the molecular genetics of APC resistance. During the fall of 1993 they had reached the conclusion that the factor V gene was probably involved. The approach they had taken was to mix APC-resistance plasma with coagulation factor deficiency plasmas. Their basic idea was that APC resistance was not caused by a deficiency of a new APC cofactor, but that it was caused by a defect in one of the coagulation factor genes. They found APC resistance to be corrected by all plasmas except those deficient in factor V. Using a large family, which had independent inheritance of protein C deficiency and APC resistance, they could demonstrate linkage between the APC-resistance phenotype and an extragenic polymorphism. Within a very short time, they had also identified a mutation in the factor V gene which was a likely candidate for the causative mutation. A G to A transition at position 1691 predicted replacement of Arg at position 506 with a Gln (Bertina et al., 1994). As Arg506 is known to be located in one of the APC cleavage sites, it was expected that the mutation would lead to APCresistant factor Va. They had found that factor V activated with factor Xa was resistant to inactivation by APC, whereas surprisingly they reported that mutated factor Va, activated by thrombin, was sensitive to inactivation by APC. In

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a more recent study, this conclusion was challenged when thrombin-activated mutated factor Va was also shown to be resistant to APC (Sun et al., 1994). Obviously, more work is needed to clarify this important question. After returning from the Snowbird meeting, I contacted William Kane at Duke University and obtained the required factor V gene intron sequences from him. Appropriate primers were designed and a PCR/restriction enzyme cleavage-based assay was devised. This assay was used on Zoller‘s APC-resistant family and the Arg506 to Gln mutation was found to be present in the genetically related APC-resistant family members. It was interesting to note that four individuals who were married into the family also had APC resistance. Two of them had the Arg506 to Gln mutation in the factor V gene, whereas the other two individuals did not. Obviously, the factor V gene mutation is not the only cause for APC resistance even though it is clearly the dominating cause. The identification of the Arg506 to Gln mutation in our family was included in a paper which was published in the “Lancet” in June 1994 (Zoller and Dahlback, 1994). During May and June 1994, two other groups also published papers on the identification of the same factor V gene mutation in patients with APC resistance (Voorberg et al., 1994; Greengard et al., 1994b). This really illustrates the power of molecular biology techniques for identification of genetic defects, once it is known which gene to investigate. The report by Bertina and colleagues appeared in “Nature” in the May 5, 1994 issue and was the subject of a very interesting editorial written by Philip Majerus (1994). In their study, the Leiden group found a perfect correlation between the presence of APC resistance and the factor V gene mutation. Except for a few patients with borderline APC ratios, their APC-resistant patients all had the factor V gene mutation. The majority of course in heterozygous form, but also some with homozygosity. The individuals with homozygous factor V gene mutation had lower APC ratios than those with heterozygous mutation.

E. Family studies of APC resistance and factor V gene mutation In our laboratory, we had access to 308 individuals from 50 families with inherited APC resistance and they were investigated for the presence of the factor V gene mutation. The mutation was found in 47 of the 50 families and in more than 90% of the cases with APC resistance. There was a limited number of cases with APC resistance in whom the mutation could not be identified, providing further support to the idea that some other genetic defect may also cause APC resistance. Everybody in the coagulation unit was very excited and the work was completed within a few weeks. The paper on this study was submitted to “Journal of Clinical Investigation” in the beginning of June and luckily accepted (Zoller et al., 1994). This study was important from the point of view that it demonstrated that even individuals with homozygous factor V gene mutation may live

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,

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Figure 5.10. Thrombosis-free survival curves in heterozygous and homozygous APC resistance. (A) The probahility to he free from thrombotic events at a certain age is presented in the Kaplan-Meier analysis for normal (n = 146), heterozygous (n = 144), and homozygous (n = 18) individuals. (B) The same analysis after exclusion of the 33 APCresistant prohands and 18 protein S-deficienr family members. Adapted from Zoller er al. (1994).

healthy lives without thrombosis. Around 40% of the homozygous cases had not had thrombosis at the age of 50 (Figure 5.10). However, it should be kept in mind that they have a lifelong increased risk of thrombosis and that thrombosis may occur at older age. In any event, it is interesting to note that homozygous factor V gene mutation is not associated with the same severe thrombotic disease as homozygous deficiency of protein C or protein S (Greengard et al., 1994a; Hajjar, 1994).

F. Molecular mechanism of APC resistance After the identification of the factor V gene mutation I reevaluated the idea that a defect in the cofactor function of factor V was the cause of APC resistance. It was, at that time, a good hypothesis and also led to the identification of the APC cofactor function of factor V (Dahlback and Hildebrand, 1994; Shen and Dahlback, 1994). However, it is now obvious that APC resistance is not directly caused by a deficiency of the APC cofactor function of factor V, even though the mutation indirectly leads to a loss of factor V-dependent APC cofactor activity because the resulting hypercoagulable state is associated with increased thrombin generation and activation of factor V; the APC cofactor activity of factor V is only associated with intact factor V and is not associated with factor Va. What then is the molecular mechanism of the APC resistance? It is

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becoming evident that the Arg506 to Gln mutation in factor V is associated with resistance of mutated factor Va for APC, even though some conflicting results have been reported (Bertina et al., 1994; Sun et a!. 1994). Mutated factor Va incorporated in the prothrombinase complex is, in the presence of APC, more stable than normal factor Va and this will result in an increased rate of thrombin generation. Thrombin feedback activates the coagulation system by cleaving and activating both factors VIII and V. As a result, the rate of activation of the coagulation system increases and APC is unable to provide sufficient control of the coagulation process.

X. HIGH PREVALENCE OF APC RESISTANCE IN THE POPULATION The factor V gene mutation causing APC resistance is highly prevalent in the general population. In most countries in northern Europe the mutation is found in approximately 5% of the population. In Sweden, the mutation is even more common and at least 10% of the population are affected. The hypercoagulable state, which is the result of the APC resistance, is lifelong and heterozygosity of the factor V gene mutation is associated with a 5- to 10-fold increased risk of thrombosis (Bertina et al., 1994; Svensson and Dahlback, 1994; Dahlback, 1994b). Homozygous individuals have a 50- to 100-fold increased risk of thrombosis, but, importantly, even homozygous cases may never suffer from thrombosis.

A. APC resistance combined with other anticoagulant protein deficiencies Some interesting conclusions can be drawn from the observed high population frequency of the Arg506 to Gln mutation in the factor V gene. In a country such as Sweden, which has an allelic frequency of approximately 5%, homozygosity is expected in 0.25% of the population. Approximately 10% of individuals with other genetic or acquired diseases are also expected to have the factor V gene mutation. Thus, 10% of individuals with deficiency of protein C or protein S are also expected to carry the factor V gene mutation. That individuals with such combined genetic defects suffer from a much higher risk of thrombosis has been shown for both protein C deficiency and protein S deficiency (Koeleman et al., 1994; Zoller et al., 1995b). Combinations of genetic and environmental risk factors also increase the risk of thrombosis. Thus, individuals with one or more genetic defects are expected to have a high incidence of thrombosis in association with surgery, immobilization, pregnancy, and oral contraception. In support of such a concept, we have found a very high incidence of APC resistance in women with a history of thrombosis in association with pregnancy (60%) and

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oral contraception (30%) (Hellgren et al., 1995). Recently, it was also shown that the combination of oral contraception and APC resistance is associated with a 34-fold increased risk of thrombosis (Vandenbroucke et al., 1994). Even higher risk is associated with combinations of oral contraception and homozygosity for the factor V gene mutation.

B. APC resistance in association with other diseases I t will be of considerable interest to determine whether APC resistance is associated with arterial thrombosis. We have found a few cases of young individuals with myocardial infarction who carry the factor V gene mutation in homozygous form (Holm et al., 1994). Future work will also reveal whether heterozygosity of the APC-resistance mutation, possibly in combination with other risk factors such as smoking, increases the risk of arterial thrombosis. It is also noteworthy that 5 1 0 % of individuals with other diseases, such as diabetes, autoimmune diseases, inflammatory states, and infections, are expected to have the mutation. APC resistance may affect the clinical outcome of these diseases, even though disturbances of the coagulation reactions are not primarily involved in the pathogenesis of the diseases.

C. APC resistance during evolution The high prevalence of the factor V gene mutation in the general population raises the question of whether there have been beneficial effects of the mutation during evolution (Majerus, 1994; Hajjar, 1994). It has been suggested that the hypercoagulable state, which is the result of the factor V gene mutation, has been advantageous for fertility, e.g., for egg implantation in the uterus (Majerus, 1994). The hypercoagulable state and the associated reduced bleeding tendency may also have been beneficial in association with traumatic injury, menstruation, and after delivery.

XI. CONCLUDING REMARKS The physiologically important protein C anticoagulant system has been unraveled during the past 20 years and the beautiful and ingenious mechanisms of the system have amazed spectators of this scientific field. The elucidation of the transformation act, from pro- to anticoagulant enzyme, that thrombin undergoes when it binds to thrombomodulin on the surface of intact endothelium was particularly fascinating and one of the highlights of the field. It is not surprising that disturbances of the delicate balance between pro- and anticoagulant mechanisms cause thrombosis. However, it has been surprising to learn that inherited

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APC resistance, which is caused by a single mutation in the factor V gene, is the underlying pathogenetic risk factor in so many thrombosis patients. The APCresistance story has once again demonstrated that nature indeed performs the best experiments and that observations made in single patients may still lead to the unraveling of important pathophysiological mechanisms. Inherited APC resistance together with deficiencies of protein C, proand antithrombin account for up to 60-70% of cases with familial tein thrombophilia. Elucidation of genetic defects involved in the remaining unexplained 3040% is a challenge for the future. New assays detecting functional abnormalities may, together with genetic linkage analysis in thrombosis-prone families, lead to the identification of new pathogenic mechanisms.

s,

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organization of the active human protein S gene PSa and its pseudogene PSb: Duplication and E ~ I wing during primate evolution. Biochemistry 29:7853-7861. Regis., L. M., Lamphear, B. J., Walker, F. J., and Fay, P. J. (1994). Factor IXaprotectsfactor VIIIa from activated protein C: Factor IXa inhibits activated protein C-catalyzed cleavage of factor VIIIa at Arg562.J. Biol. Chem. 269:9445-9452. Reitsma, P. H., Poort, S. R., Allaart, C. F., Briet, E., and Bertina, R. M. (1991). The spectrum of genetic defects in a panel of 40 Dutch families with symptomatic protein C deficiency type I: Heterogeneity and founder effects. Blood 78:890-894. Reitsma, P. H., Poort, S. R., Bernardi, F., Gandrille, S.,Long, G. L., Sala, N., and Cooper, D. N. (1993). Protein C deficiency: A database of mutations. Thromb. Haemostasis 69:77-84. Reitsma, P. H., Ploos van Amstel, H. K., and Bertina, R. M. (1994). Three novel mutations in five unrelated subjects with hereditary protein S deficiency type I. J. Clin. Inwest. 93:486-492. Rosin, S., Johansson, K., Lindherg, K., and Dahlback, B. (1994). Multicenter evaluation of a kit for activated protein C resistance on various coagulation instruments using plasmas from healthy individuals. Thromb. Huemostasis 72:255-260. Sadler, J. E., and Davie, E. W. (1994). Hemophilia A,, hemophilia B., and von Willebrand disease. In “The Molecular Basis of Blood Diseases.” G.Stamatoyannopoulos, W. Nienhuis, P. W. Majerus, and H. Varmus, eds.), 2nd ed., pp. 657-700. Saunders, Philadelphia. Sadler, J. E., Lentz, S. R., Sheehan, J. P., Tsiang, M., and Wu, Q. (1993). Structure-function relationships of the thrombin-thromhomodulin interaction. Haemostasis 23(Suppl. 1): 183- 193. Schmidel, D. K., Tatro, A. V., Phelps, L. G., Tomczak, J. A., and Long, G.L. (1990). Organization of the human protein S gene. Biochemistry 29:7845-7852. Schmidel, D. K., Nelson, R. M., Broxson, E. H., Comp, P. C., Marlar, R. A., and Long, G. L. (1991). A 5.3-kb deletion including exon XI11 of the protein S a gene occurs in two protein S-deficient families. Blood 77:551-559. Schwan, H. P.,Fischer, M., Hopmeier, P.,Batard, M. A., and Griffin, J. H. (1984). Plasma protein S deficiency in familial thrombotic disease. Blood 64:1297- 1300. Seegers, W. H., Novoa, E., Henry, R. L., and Hassouna, H. 1. (1976). Relationship of ‘new’ vitamin K-dependent protein C and ‘old’ autoprothrombin 11-A. Thrutnb. Res. 8543-552. Seligsohn, U., Berger, A,, Abend. A , , Rubin, L., Attias, D., Zivelin, A., and Rapaport, S. I. (1984). Homozygous protein C deficiency manifested by massive thrombosis in the newhom. N. Engl. J. Med. 310559-562. Shen, L., and Dahlback, B. (1994). Factor V and protein S as synergistic cofactors to activated protein C in degradation of factor V11Ia. J. Biol. Chem. 269:18735-18738. Solymoss, S., Tucker, M. M., and Tracy, P. B. (1988). Kinetics of inactivation of membrane-bound factor V by activated protein C. 1. Biol. Chem. 263:14884-14890. Stenflo, J. ( 1970). Dicoumarol-induced prothrombin in bovine plasma. Acta Chem. Scad. 24:3762-3763. Stenflo, J. (1976). A new vitamin K-dependent protein. J. Biol. Chem. 251:355-363. Stenflo, J., and Dahlhack, B. (1994). Vitamin K-dependent proteins. In “The Molecular Basis of Blood Diseases” (G. Stamatoyannopulos, A. W. Nienhuis, P. W. Majerus, and H. Varmus, eds.), 2nd ed. pp. 565-598. Saunders, Philadelphia. Stenflo, J., and Ganrot, P. (1972). Vitamin K and the biosynthesis of prothrombin. 1. Identification and purification of a Dicoumarol-induced abnormal Prothrombin from bovine plasma. J. Biol. Chem. 247:8 160-8 166. Stenflo, J., Femlund, P., Egan, W., and Roepstorff, P. (1974). Vitamin K dependent modifications of glutamic acid residues in. Proc. Natl. Acad. Sci. U.S.A. 71:2730-2733. Sun, X., Evatt, B., and Griffin, J. H. (1994). Blood coagulation factor Va abnormality associated with resistance to activated protein C in venous thrombophilia. Blood 83:3 120-3 125. ~

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Suzuki, K., Stenflo, I., Dahlhack, B., and Teodorson, B. (1983). Inactivation of human coagulation factor V hy activated protein C. J. Biol. Chem. 258:1914-1920. Svensson, P. J., and Dahlback, 8. (1993a). Novel mechanism for thrombosis characterized hy poor anticoagulant response to activated protein C constitutes a major cause of thrombophilia. Thromb. Hmmostasis 69:999 (ahstr.). Svensson, P. J., and Dahlhack, B. (1993h). Twenty novel families with thrombophilia and inherited resistance to activated protein C. Thromb. Hmmostatis 69:1252 (abstr.). Svensson, P. J., and Dahlhack, 8. (1994). Resistance to activated protein C as a basis for venous thrombosis. N. Engl. 1. Med. 330:s 17-52 1. Tahernero, M. D., Tomas, J. F., Alberca, I., Orfao, A., Borrasca, A. L., and Vicente, V. (1991). Incidence and clinical characteristics of hereditary disorders associated with venous thrombosis. Am. J. Hematul. 36:249-254. Tuddenham, E. G. D. (1993). Thrombophilia: A new factor emerges from the mists (comments). Lancet 342: 1501-1502. Vandenbroucke, J. P., Koster, T., Bri@t,E., Reitsma, P. H., Bertina, R. M., and Rosendaal, F. R. (1994). Increased risk of venous thromhosis in oral-contraceptive users who are carriers of factor V Leiden mutation. Lancet 344:1453-1457. Vehar, G. A., and Davie, E. W. (1980). Preparation and properties of bovine factor VIII (antihemophilic factor). Biochemistry 19:401-410. Voorberg, J., Roelse, I., Koopman, R., Biiller, H., Berends. F., ten Care, J. W., Mertens, K., and van Mourik, J. A. (1994). Association of idiopathic thromhoembolism with single point mutation at Arg506 of factor V. Lancet 343:1535-1536. Walker, F. J. (1980). Regulation of activated protein C by a new protein. J. Biol. Chem. 255: 5521-5524. Walker, F. J. (1981). Regulation of activated protein C by protein S, the role of phospholipid in factor Va inactivation. J. Biol. Chem. 256:11128-11131. Watkins, P. C . , Eddy, R., Fukushima, T., Byers, M. G., Cohen, E. H., Dackowski, W. R., Wydro, R. M., and Shows, T. B. (1988). The gene for protein S maps near the centromere of human chromosome 3. Blood 71:238-241. Wiman, B., Ljungherg, B., Chmielewska, I., Urden, G., Blomhack, M., and Johnsson, H. (1985). The role of the fihrinolytic system in deep vein thrombosis. J. Lab. Clin. Med. 105:265-270. Wu, S. M., Cheung, W. F., Frazier, D.,and Stafford, D. W. (1991). Cloning and expression of the cDNA for human y-glutamyl carboxylase. Science 254: 1634-1636. Zoller, B., and Dahlbick, B. (1994). Linkage between inherited resistance to activated protein C and factor V gene mutation in venous thrombosis. Lancet 343:1536-1538. Zdler, B., Svensson, P. I., He, X., and Dahlback, 8 . (1994). Identification of the same factor V gene mutation in 47 out of 50 thrombosis-prone families with inherited resistance to activated protein C. J. Clin. Inwest. 94:2521-2524. Zoller, B., Garcia de Frutos, P., and Dahlback, B. (1995a). Evaluation of the relationship between protein S and C4b-binding protein isoforms in hereditary protein S deficiency demonstrating type I and type Ill deficiencies to be phenotypic variants of the same genetic disease. Blood (in press). Zdler, B.. Berntsdotter, A., Garcia de Frutos, P., and Dahlback, B. (1995b). Resistance to activated protein C as an additional genetic risk factor in hereditary deficiency of protein S. Blood (in press).

Dystrophin, Its Gene, and the Dystrophinopathies Roland G. Roberts Division of Medical and Molecular Genetics United Medical and Dental Schools London SE1 9RT,United Kingdom

1. INTRODUCTION Prompted by the prevalence and severity of Duchenne and Becker muscular dystrophy (DMD and BMD), the process of elucidation of their etiology has unearthed a wealth of information concerning the dystrophin gene and its product. The gene itself, with its absurd size and extraordinary complexity of structure and transcriptional regulation, has posed a challenge to geneticists at every turn; its unusual mutational spectrum shaping novel strategies for prenatal and carrier diagnosis. The protein has provided a window into a previously unsuspected system of membrane-associated proteins and will ultimately reveal the molecular basis of an essential biological function.

II. DUCHENNE AND BECKER MUSCULAR DYSTROPHY A. The nature of the disease in muscle 1. Gross phenotype In the 19th century, a French physician named Duchenne described 13 patients affected with a characteristic muscle disorder (Duchenne, 1868). The dystrophy which now bears his name (DMD) is estimated to affect 1 in 3000 live male births. The disease is generally first noticed between the ages of 2 and 5 years (Dubowitz, 1978;Jennekens et al., 1992))presenting initially as a “waddling”gait or difficulty in climbing stairs. Motor milestones are often delayed, and the child Advances in Genetics, Yo/. 33 Copyright 0 1995 hy Academic Press, Inc. All rights of reproduction in any form resewed.

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appears to adapt his behavior to cope with the encroaching muscle weaknessthe Gowers sign is a classic example of this (unable to raise himself from the floor using only his legs, the patient “climbs” up his own thighs using his hands). The calves are often the site of pronounced pseudohypertrophy and muscular pain, resulting in a characteristic toe gait. Serum creatine phosphokinase (CPK) activity is grossly elevated (up to 50 times normal) very early in life (Ebashi et al., 1959), constituting a useful diagnostic feature. Progressive loss of strength in the legs results in confinement to a wheelchair between 7 and 12 years of age (a child is classified as “intermediate” if he retains the ability to walk after the age of 12, and as a “Becker muscular dystrophy” case if he can still walk after the age of 14). The transition from mobility to loss of ambulation may be gradual or sudden, and sometimes appears to be precipitated by immobilization for unrelated reasons. Weakness of the arms occurs later than that in the legs, commencing proximally and extending to distal muscles. After confinement to a wheelchair, patients tend to develop contractures of leg and arm muscles, followed by progressive scoliosis. Death usually occurs in the early twenties from severe respiratory insufficiency (due to intercostal weakness) or acute myocardial insufficiency (see Section 11,C). Becker muscular dystrophy (BMD; Becker and Kiener, 1955) shares many features with DMD but has a much milder course. Diagnosis is generally before the age of 25 but patients are often able to walk well into adulthood. As the incidence is approximately one-third that of DMD (Bushby et al., 1991), the longer life expectancy results in approximately equal total prevalences for the two diseases.

2. Muscle pathology The visible sequence of events in DMD muscle has been well characterized. The myofibres undergo cycles of necrosis and regeneration in which necrosis ultimately wins out, and fibrosis occurs where regeneration has failed. A number of light and electron microscopic studies of pathological muscle have been aimed at identifying morphological aberrations which may precede (and therefore contribute to) the onset of necrosis. Mokri and Engel (1975) found that -4% of (nonnecrotic) myofibrils had “delta lesions”-triangular-shaped rarefactions of the myofibre contents. Electron microscopy showed that regions of the sarcolemma1 membrane associated with the delta lesions were disrupted or absent, although the overlying basement membrane was intact. In some instances, reduplication of the basal lamina was apparent, perhaps representing an attempt to repair the defect. Carpenter and Karpati (1979) also noted this focal disruption of the sarcolemma as a prelude to any overt signs of necrosis. As necrosis set in, cells

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were observed to lose their plasma membrane entirely, although the basal lamina was maintained. Occasionally, necrosis only affected a segment of the myofiber, the remainder having a normal appearance with well-ordered myofibrils. All necrotic cells contained at least one macrophage (invading through the basal lamina) whose cytoplasm grew darker with the progression of necrosis. Tubes of basal lamina were observed which contained dense deposits of collagen fibrils instead of a myofiber, presumably indicating where a necrotic myofiber has failed to be replaced and fibrosis has occurred. Occasional reduplication of the basal lamina, similar to that reported by Mokri and Engel, was also seen. Comparison of the proportion of cells observed at various stages suggested that the interval between plasma membrane breakdown and onset of necrosis was short (a matter of hours). The interval between onset of necrosis and macrophage invasion must also be much shorter than that observed in, for example, experimental ischemia, probably because the continuous necrosis in DMD muscle results in a much higher macrophage “presence.” Thus, microscopic studies seem to suggest that sarcolemmal membrane is specifically weakened; attempts are made to repair the breaks which occur in the membrane but often the lesion precipitates loss of the entire membrane and subsequent necrosis. For some reason, regeneration appears unable to compensate for the loss of muscle cells; net reduction in cell number occurs and collagen is deposited in the empty basal lamina tubes. Other studies support a primary defect in the sarcolemma. Mokri and Engel (1975) showed that DMD membranes exhibited focal permeability to peroxidase (also observed by Bonilla et at., 1978, using concanavalin Aperoxidase conjugate). Schotland et al. (1980) performed freeze-fracture studies on the plasma membrane of normal and DMD muscle. The appearance of both sides of the membrane was dramatically altered. Particles observed in normal membranes were reduced to 30% (outer face) and 50% (inner face) abundance in dystrophic membranes. The distribution of remaining particles was less uniform, resulting in large featureless patches. It is possible that the missing particles correspond to the dystrophin-associated glycoprotein complexes discussed under Section V,A, and that their loss removes constraints on the distribution of other transmembrane proteins. Capaldi et al. (1984) studied the binding of Ricinus communis agglutinin-I (a D-galactose-specific lectin) to normal and DMD sarcolemma. Normal sarcolemma was heavily stained by the lectin, as was the basal lamina in both normal and dystrophic muscle. The sarcolemma in DMD samples, although clearly visible, was devoid of stain. This is consistent with the loss of specific membrane glycoproteins in DMD (see Section V). The effects of such weakness manifest at the cellular level in DMD and mdx (dystrophic mouse model, see Section II,D) myotubes as a high [Ca2+], (Turner et at., 1988) resulting from increased “open probability” of CaZ+ leak channels (Fong et at., 1990) and increased abundance of stretch-inactivated

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Ca2+ channels (Franc0 and Lansman, 1990). Measurement of the sensitivity of muscle fibers to osmotic shock (Menke and Jockusch, 1991) showed that mdx fibers are significantly more shock sensitive than wild-type fibers and that this variation is related to the absence of dystrophin rather than the high proportion of regenerated myotubes. These results strongly suggest that muscle cell death in DMD and mdx results from increased fragility of the sarcolemmal membrane, which predisposes it to contraction-induced tearing (Karpati and Carpenter, 1986) and/or increased Ca2+ influx. The latter may result in the activation of intracellular proteases (Turner et al., 1988) or phospholipase A, which may dismantle the sarcolemma (Jackson et al., 1984). The relative importance to membrane disruption of mechanical stress and increased intracellular Ca2+ has been examined by Petrof et al. (1993) who compared the effect of number of activations (i.e., time-averaged CaZ+ levels) and extent of contractions (i.e., degree of mechanical stress) on membrane damage in various normal and mdx mouse muscles. Mechanical stress was found to be the major determinant, and mdx tissue sustained at least four-fold more damage than normal tissue during the regimes used. Further factors contributing to cell death may be the loss of other components of the dystrophin-associated glycoprotein complex which are not involved directly in interactions with the basal lamina (see Section V,A).

B. Mental retardation The issue of mental retardation in the muscular dystrophies has been beset by controversy and vogue. The phenomenon was first noted in the 19th century by workers such as Duchenne and Gower, but a number of studies in the 1940s and 1950s found no significant differences in rates of mental retardation between normal and dystrophic individuals. Later studies showed a tendency to explain the difference as a natural consequence of disability (due to social insularity, loss of education, or emotional problems) or as a result of socioeconomic factors. Comparative studies using control patients with severely disabling spinal muscular atrophy (Worden and Vignos, 1962; Kozicka et al. , 1971; Florek and Karolak, 1977) or postpoliomyelitis (Michal, 1972) suggested that intelligence quotient (IQ) reduction in DMD patients was specific to this condition and not a feature of severe physical handicap in general, while several studies have been based on comparisons with unaffected sibs (Worden and Vignos, 1962; Cohen et al., 1968; Prosser et al., 1969), which presumably control for socioeconomic factors. Much work [see the 18 studies reviewed by Karagan (1979)lfrom the 1960s onwards has firmly established mental retardation as a feature of a substantial proportion of DMD patients. The literature consensus is that approximately one-third of DMD patients are found to be mentally retarded (IQ < 75; cf. < l% of the general population). This could be explained as being due to a partially penetrant effect

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which causes mental retardation or predisposes the brain to damage by an independent factor. Prosser et al., however, report a correlation between IQs of patients and normal sibs, presenting their data as general downshift of IQ of =l standard deviation as a result of DMD. Cohen et al. (1968) and Ogasawara (1989) found a high concordance between the IQs of affected sibs. These results would be better explained by a fully penetrant effect which reduces the IQ by a more or less fixed amount. The mental deficiency is nonprogressive and its degree appears to be fixed from an early age (Dubowitz, 1965; Prosser et al., 1969). Recent studies show that, rather than being global, the nature of the disability seems to be specific to tasks such as verbal reasoning and sentence comprehension (Anderson et al., 1988; Billard et al., 1992). This is consistent with the restricted pattern of expression of dystrophin in the brain (see Section 111)C). Several groups have noted electroencephalogram abnormalities in a high proportion of patients, correlating with severity of mental impairment (Kozicka et al., 1971; Florek and Karolak, 1977). The IQ of carrier females, even those with exceptionally high CPK values, is normal (Prosser et af., 1969). Interestingly, the reproductive fitness of males with BMD (0.12) is much lower than that of males with limb girdle muscular dystrophy (0.98) despite the comparable degrees of physical handicap in these disorders (PassosBueno and Zatz, 1991). Ir is possible that milder psychological effects of the dystrophin mutation may include a personality disorder which might render BMD males less able to acquire a partner. Mental retardation per se, however, is rare in BMD boys, and its incidence probably does not differ significantly from that of the general population.

C. Pathology in other tissues The complexity of the DMD phenotype

becomes more apparent each year, and although its principal features seem to reflect the gross expression pattern of the main dystrophin isoforms, some of the smaller C-terminal isoforms are very widely expressed (see Section IV,B). It may therefore be that in the -10% of boys in which these proteins are defective (Roberts et al., 1994)) the disorder is truly a multisystem disorder with the milder manifestations masked by the severity of the skeletal myopathy.

1. Cardiac muscle Cardiomyopathy is severe in most DMD patients and is a significant cause of mortality. This seems to result from both ventricular dilation (due in turn to dystrophic changes in the myocardium not unlike those in skeletal muscle) and a conduction defect (Perloff, 1984). Over half of the cases of DMD have akinetic

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or dyskinetic areas on echocardiogram, of whom about 40% have ventricular dilation (de Kermadec et al., 1994). Furthermore, two-thirds of the cases of BMD have electrocardiogram anomalies (mostly right ventricular, with left ventricular involvement in older patients) (Melacini et al., 1993). Expression of dystrophin in heart muscle is apparent during embryonic development a short time before expression in skeletal muscle becomes apparent. It differs somewhat both temporally-the heart muscle becomes contractile before the appearance of dystrophin (Buckingham et al., 1992), whereas these events are essentially simultaneous in skeletal muscle-and spatially-cardiac dystrophin is concentrated in the T tubules at the Z line (Klietsch et ul., 1993), rather than being uniformly distributed over the sarcolemma. Specific isoforms of dystrophin are also expressed in the cardiac Purkinje fibers (Bies et al., 1992a). Interestingly, the cardiac phenotype can occasionally appear in isolation. Towbin et al. (1993) reported cosegregation of X-linked dilated cardiomyopathy (XDCM) with markers within the dystrophin gene. Dystrophin protein in cardiac, but not in skeletal, muscle was found to be abnormal in affected individuals. Since then, there have been a number of reports in which a defect in the dystrophin gene has affected cardiac function but has left skeletal muscle wholly (Muntoni et al., 1993) or partially (Yoshida et ul., 1993) unscathed. The characterized mutations abolish the muscle-specific promoter, probably without removing either the brain- or Purkinje-specific promoters, Presumably one or both of these elements is capable of directing transcription in skeletal muscle (hence rescuing the phenotype) but not in cardiac muscle.

2. Smooth muscle Although there is substantial expression of dystrophin in smooth muscle, and dystrophic changes have been noted in the muscle of the esophagus, stomach, and intestine, the effects of dystrophin loss on this tissue are poorly studied. Anecdotal reports describe gastrointestinal symptoms ranging from a feeling of bloating to life-threatening acute gastric dilatation. Barohn et al. (1988) found gastric-emptying times in DMD boys to be approximately three times longer than in wild-type controls. Korman et al. (1991), on the other hand, demonstrated that orocecal transit times in DMD patients do not differ from those of normal individuals (as this parameter largely depends on small intestine transit time, it is not inconsistent with the gastric phenotype).

3. Retina Pillers et al. (1989) described a patient with a contiguous gene syndrome comprising DMD, glycerol kinase deficiency, congenital adrenal hypoplasia, and a

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form of congenital stationary night blindness which was then believed to be &and Island eye disease (now known to map closer to the centromere). It has since been demonstrated that, rather than being an unusual phenotype in a patient with an unusual genotype, this phenomenon (now termed Oregon eye disease) is shared with almost all DMD patients (Pillers e t al., 1993; Sigesmund e t at., 1994). Ninety percent of all cases of DMD and BMD have a reduced or absent scotopic b-wave on their electroretinogram, representing a severe reduction in the response of retinal neurons to light stimulation of dark-adapted (but not light-adapted) rods and cones. These clinical findings are consistent with immunohistochemical work, which shows clear expression in the outer plexiform layer of the retina (Miike e t al., 1989; Pillers e t al., 1993), specifically in the postsynaptic regions of rod and cone synapses (Schmitz e t al., 1993).

D. Animal models Bulfield et al. (1984) described a potential mouse model for DMD. Although the mdx mouse has an extremely benign myopathy, with symptoms only apparent after 1 year of age (indeed, the disorder was only recognized by its effect on serum pyruvate kinase levels), initially casting doubt on its homology with DMD, Hoffman e t al. (1987a) found dystrophin protein to be absent from both DMD and m d x muscle, while Sicinski et at. (1989) identified a nonsense mutation in the dystrophin gene of the mdx mouse. With genetic homology confirmed, the reasons for the phenotypic disparity have been a subject of much debate. Interestingly, Stedman e t al. (1991) found marked pathological effects in the m d x diaphragm and related this to work rate. Hence, the basal work rate of the murine diaphragm is approximately five times that of the human diaphragm. The power output of the limb muscles of the caged mouse is almost negligible, while human limb muscles have to raise a heavy body against gravity. If the fundamental deficiency in the dystrophinopathies is one of resistance to workinduced injury (see Section I,A), then the specific phenotype in a given animal should match the work distribution pattern. A second dystrophinopathic mouse strain, m d x 3 C v , has been found (Cox et al., 1993) to have a splicing defect which is expected to disrupt all dystrophin isoforms [see Section IV,B and compare 16 human mutations reviewed in Roberts et al. (1994)l. Other animal models include cats (Carpenter et a]., 1989; Gaschen et al., 1992) and dogs (Kornegay et al., 1988). The latter have phenotypes very similar to DMD, with the onset at 10 weeks of muscular weakness, and a stiff, shuffling gait. They also have eating difficulties and in later stages suffer severe spinal curvature. A splice site mutation in exon 8 has been shown to be responsible for the disease in golden retrievers (Sharp e t al., 1992).

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111. THE DYSTROPHIN GENE A. Identification of the gene Duchenne (1868) described his disease as predominantly affecting boys, and this can perhaps be regarded as the first step in mapping the gene (to & of the human genome). Linkage was excluded with color blindness, G6PD, and Xg blood group (Emery et al., 1969; Zatz et al., 1974; Race and Sanger, 1975). Further localization was not achieved until a report of a reciprocal Xp21;11q13 translocation in a girl affected with DMD (Greenstein et al., 1977). A number of reports of other X;autosome translocations in DMD females followed [x;21 (Verellen et al., 1978), X;3 (Canki et al., 1979), and X;1 (Lindenbaum et a l , 1979)l. A detailed cytogenetic study of nine reported translocations (Boyd and Buckle, 1986) revealed an apparent spread of breakpoints between Xp21.1 and Xp21.2, perhaps genuinely reflecting the large size of the locus. Linkage studies first bore fruit in 1982 as a restriction fragment length polymorphism (RFLP) for the Xp21-Xp22.3 probe RC8 was found to be linked to DMD at a distance of -10 cM (Murray et al., 1982). Further probes in Xp21 were isolated and shown to cosegregate with DMD and BMD (L1.28, 15 CM (Davies et al., 1983); C7, 10 cM (Dorkins et al., 1985); 754, 10 cM (Hoflcer et d., 1985)l. Dorkins et al. (1985) showed by linkage analysis that probes C7 and 754 flank the DMD gene, consistent with their localization ascertained from X;autosome translocations. Franke et al. (1985) described a patient, BB, with a complex syndrome of chronic granulomatous disease, McLeod erythrocyte phenotype (mild hemolytic anemia and acanthocytosis), retinitis pigmentosa, mild mental retardation, and DMD. Cytogenetic examination showed that band Xp21 was shortened, with an absence of the light subband Xp21.2. Screening of the patient’s DNA with existing X-linked probes showed that 754 was deleted, but B24, L1, M2C, and OTC (also in Xp21) were not. DNA from patient BB was used as a subtractive “driver” in a phenol-enhanced reassociation experiment (Kunkel et al., 1985) to select for sequences within the deleted region. Four of the clones obtained (pERT87, pERT84, pERT145, pERT55) were absent from the genome of BB. Monaco et al. (1985) found pERT87 to be deleted in 9% of affected boys tested. A larger survey of 1346 affected males (Kunkel et al., 1986) found 6.5% to have deletions of pERT87. One of the DMD female translocation events (Verellen et al., 1978; Verellen-Dumoulin et al., 1984) was found to juxtapose Xp21 sequences with the distal end of the chromosome 21 rDNA cluster (Worton et al., 1984). This serendipity was exploited by Ray et al. (1985), who used a probe from the 28s rRNA gene to isolate a clone from a somatic cell hybrid containing one of the translocation products. Part of this clone (XJ-1.1)recognized a TaqI RFLP tightly linked to DMD and was deleted from the genomes of a number of affected boys.

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Both groups performed chromosome walks from their initial probes in an attempt to isolate coding sequences (see Figure 6 . 1 ~ ) Monaco . et al. (1986) reported the expansion of a set of contiguous phage clones (a “contig”) around pERT87 to 220 kb (locus DXS164, now known to contain exons 8-21 of the dystrophin gene). Subclones of this contig were used as probes on Northern blots and zoo blots to test for transcription and evolutionary conservation. Two subclones, pERT87-25 and pERT87-4, recognized a variety of mammalian DNAs and were used to screen a mouse genomic library. Comparison of murine and human sequences showed both regions to contain candidate exons. pERT87-4 (which lies in intron 15) failed to give signals on Northern blots, but pERT8725 (which contains exon 19) weakly detected a large (“16 kb”) transcript in fetal skeletal muscle RNA. pERT87-25 was used to isolate clones from a fetal skeletal muscle cDNA library. One of the resulting clones, cDNA5 (1 kb in length), hybridized to eight distinct sites across 120 kb of the DXS164 contig, leading to the prediction that the entire genomic locus might extend across 1000-2000 kb. In the meantime, Burghes et al. (1987) had extended the cloned region surrounding XJ-1.1 (DXS206) until it joined DXS164 (creating a 350-kb contig). Two subclones near the end of DXS206 (XJ-10.2 and XJ-10.3, containing exons 9 and 8, respectively) recognized clones from a muscle cDNA library. The initial genomic clone, XJ-1.1, was found to lie at the middle of a 105-kb intron. Screening of a fresh cDNA library with cDNA5 followed by a number of “walks” resulted in the compilation of clones corresponding to the entire 14kb transcript (Koenig et al., 1987). Preliminary sequencing projects (a total of 4.3 kb) showed strong homology between mouse and human clones (Hoffman et al., 198713;Koenig et al., 1987). The cDNA recognized a large number of restriction fragments in genomic DNA, at least one of which was deleted in over 50% of patients (Koenig et al., 1987).

B. Structure of the gene in normal individuals In a paper describing the complete cloning of the dystrophin cDNA (Koenig et

al., 1987), it was shown that hybridization of the full-length clone to human genomic DNA digested with HindIII yielded 65 distinguishable bands. As there are only five HindIII sites within the cDNA sequence, this implied a minimum of 60 exons. The approximate order of these fragments, with a number of ambiguities, was established by cDNA hybridization to deletion patient DNA and genomic contig DNA from the walks surrounding pERT87, pERT84, J-Bir, J-MD, and 1-47 (Monaco et al., 1987). Large-scale mapping of the gene (van Ommen et al., 1986; Kenwrick et al., 1987; Burmeister et al., 1988; Meitinger et at., 1988; den Dunnen et al., 1989) using pulsed-field gel electrophoresis (PFGE) has placed limits on the distances between a number of intragenic landmarks and has led to an estimate of 2.4 Mb for the total size of the gene. This is, to date, the largest gene ever characterized, and contains the largest intron known

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Figure 6.1. Structure of the dystrophin gene. (a) Exon structure. The bold open box represents the (muscle-specific)dystrophin gene. Vertical lines within this box represent exons, constrained by their presence in genomic Sfil fragments (den Dunnen et al., 1989) and YAC clones (Coffey et al., 1992). and numbered according to Robem et al. (1993a). Arrows indicate promoters (see Section 111,C). Distance along the X-chromosome (musclespecific promoter at 0) marked in kilobases (kb). (b) Coarse physical map. The horizontal line represents the X chromosome, along which are displayed a selection of genomic landmarks (bars on top of line) and the Sfil restriction sites (bars below lines). (c) Stippled boxes show the extent of historical genomic cloning projects (see Section IILB).

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[brain intron 1 is 400 kb in size (Boyce et al., 1991)l. Figure 6.1 summarizes the results of PFGE analysis, together with the positions of genomic probes, the extents of previous genomic cloning projects in the dystrophin gene and the distribution of exons, as refined by our analysis of yeast artificial chromosomes clones (see Coffey et al., 1992; Roberts et al., 1992a; compare also Monaco et al., 1992). Exon boundaries and intron sequences for exon 4 and exons 10-21 were determined (Monaco et al., 1988) by sequencing genomic subclones with exon-specific primers. Similarly, Malhotra et at. ( 1988) determined boundaries for exons 1- 10 and established the distribution of exons 3-9 within the 150-kb genomic contig surrounding DXS206 (XJ1.1). Boundaries of exons 44, 45, and 48 were established by Chamberlain et al. (1988), while Koenig et al. (1989), documented the boundaries ofexons 22, 31-33,35-37,41,43,45,47-51,5355, and 60, also established by sequencing of subclones of genomic phage clones. The final number and boundary sequences of the remaining exons (exons 23-30, 34, 38-40, 56-79) have been determined using vectorette PCR (Roberts et al., 1992a, 1993a; Figure 6. la). Knowledge of the relationship between exon boundaries and translational reading frame has been used to examine the genotype/phenotype relationship in patients with gross rearrangements (see Section VI,B). The genomic structure of a gene can often shed light on its evolutionary history. It is interesting that, despite the sparse distribution of most of the exons, the cysteine-rich domain is encoded in its entirety by three exons (E65-E67) which lie within 6 kb, and that the first half of the carboxy-terminal domain (the syntrophin-binding region) is encoded by a single 14-kb cluster of seven exons (E68-E74). Such a “modular” structure is often observed in vertebrate genes (cf. Gilbert, 1978), probably reflecting the selective advantage of maintaining the genomic integrity of sets of exons which contribute to a single stable protein domain. The genetic map of the gene is as unusual as its physical counterpart. The flanking markers 754 (DXS84) and C7 (DXSZS), which lie less than 7 Mb apart, were found by Dorkins et al. (1985) to map 11 and 12 cM from the DMD disease locus, respectively (giving a recombination frequency of 3.5 cM/Mb). Recombination between intragenic markers and the disease locus has variously been estimated to be between <1 (Donald eta!., 1988; Mulley et al., 1988) and 5 or 6% (Fischbeck et al., 1986; Hejtmancik et al., 1988; Kunkel et al., 1986). A literature survey by Chen et al. (1989) suggested a recombination frequency of almost 3% between the intragenic loci DXS206 (XJ1.1) and DXS270 (JBir), which are =600 kb apart. All these studies were based on DMD/BMD families and are therefore open to the challenge that recombination rates in mutated chromosomes might not be representative (Davies et al., 1986). Using novel markers at each end of the dystrophin gene, however, Abbs et al. (1990) and

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Oudet et al. (1991) measured the true intragenic recombination rate to be 1012% in normal pedigrees. The relationship between genetic and physical maps therefore changes from 2 cM/Mb proximal to the gene to 4.8 cM/Mb within the gene, then reverts to = l cM/Mb distal to the gene [the average for the X chromosome is 1.2 cM/Mb (Drayna and White, 1985)l. Oudet et al. (1992) have since shown that the recombination events occur predominantly in two hot spots located between exons 1 and 8 and exons 44 and 51. In these regions the recombination rate is extraordinarily high, rising to 14 cM/Mb between exons 44 and 45. The correspondence between hot spots for meiotic recombination and for deletion breakpoints (see Section V1,A) suggests that the two phenomena may be causally linked.

C. Regulation of gene expression The dystrophin gene is expressed in a spatially and temporally regulated manner from at least five distinct promoters (Figure 6. lb). The highest level of transcription is in skeletal muscle, where the transcript represents 0.02-0.1% of RNA species, while lower levels are present in smooth muscle and brain [5 and 1% of the skeletal muscle abundance, respectively (Chelly et al., 1988)l. Nude1 et al. (1988) used RNase protection assays to show that the high level of skeletal muscle transcription is not present in cultured myoblasts and is only induced during cell fusion and myotube formation. This property is shared with a large number of muscle-specific proteins which are coordinately induced at myogenesis (for example, the myosin subunits and enzymes such as creatine kinase). Such regulation has been associated with trans-acting factors of the basic helix-loop-helix family [such as MyoD, myogenin, and myf-5 (reviewed by Weintraub et al., 1991)l. In order to investigate the basis of this regulation, Klamut et al. (1989) isolated a cosmid containing genomic sequences surrounding the first exon of the skeletal muscle dystrophin transcript. A subclone containing 850 bp of the upstream region was sequenced and examined for previously characterized regulatory elements. Standard basal transcription elements were present; a TATA box at -24 and an Spl-binding GC box at -61. Note that nucleotide positions have been revised in view of the demonstration (Klamut et al., 1990) that the transcriptional start site is 37 bp 5’ of the start of the sequence published by Koenig et al. (1988)-this is more consistent with the usual position of the TATA box. In addition to the basal elements, several well-known musclespecific elements (Sternberg et al., 1988; Nikovits et d., 1986; Mar and Ordahl, 1988; Buskin and Hauschka, 1989) were found, including a copy of the CArG box (CC(A/T),GG) and the MCAT heptamer (CATTCCT), and three potential binding sites for MEF-l, one of which resembles the consensus site for members of the MyoD family of transcriptional activators. Klamut et d. (1990)

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found that a construct containing only the 3‘-most 144, bp ot the promoter was sufficient to drive myotube-specific transcription of a chloramphenicol acetyltransferase (CAT) gene when transfected into a range of cell types. This strongly implicates the MEF-1 site and the M-CAT motif in regulation of dystrophin gene transcription. The high levels of dystrophin transcript in cultured myotubes compared with those of skeletal muscle (Lev et al., 1987) suggest that at the time of induction, the gene may be strongly upregulated to provide an initial stock of dystrophin with which to construct the new cytoskeletal network (see Section IV, D). After this, transcriptional activity would be reduced to levels sufficient for network “maintenance.” Nude1 et al. (1989) and Feener et al. (1989) showed that the 5’ end of the dystrophin transcript differs between brain and muscle. Genomic sequences adjacent to brain exon 1 constitute a promoter capable of directing transcription of a reporter gene in transfected cells (Ebyce et al., 1991; Makover et al., 1991). The brain promoter lies approximately 100 kb 5’ of the muscle promoter and lacks any currently recognized regulatory elements (even the TATA box). Chelly et al. (1990a) found that the brain promoter is specifically activated in cultured neuronal cells, while astroglial cells use only the muscle promoter (at a low and possibly nonfunctional level). The pattern of high-level expression from the “brain” promoter has been localized to pyramidal neurons in the cerebral cortex and to the hippocampus (Lidov et al., 1990; Gbrecki et al., 1992). A transcript expressed in the cerebellar Purkinje cells was recognized using probes complementary to exons 2, 3, or 4, but not the muscle or brain exons 1 (Gbrecki et al., 1992). A novel Purkinje cell-specific exon 1 was characterized and shown to map between muscle exon 1 and exon 2. Although the promoter has yet to be characterized, the 5’ untranslated region is highly conserved between human and mouse. The three different classes of transcript described previously are all approximately 14 kb in length, differing primarily with respect to the identity of their first exons. Bar e t al. (1990) described a 6.5-kb transcript present in many tissues (e.g., liver, brain, lung, testis, but not skeletal muscle) capable of protecting probes encoding the C-terminal domains, but not the N-terminal or repeat domains, in an RNase assay. Further characterization of this species (Lederfein et al., 1992) showed it to comprise sequence corresponding to exons 63-79 (with exons 71 and 78 alternatively spliced), prefixed with a novel first exon (c. 140 bp). These attributes have also been ascribed to transcripts of the more expected sizes of 4.5 (Hugnot et al., 1992) or 4.8 kb (Blake et al., 1992). It is now apparent (Lederfein et al., 1993) that these discrepancies result from anomalous migration on different electrophoresis systems, and that all of these reports concern a single 4.6-kb transcript. Lederfein et al. (1993) showed this promoter to be of a structure associated with housekeeping genes, with a number

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of Spl binding sites. A further distal transcript, 5.2 kb in size and expressed from a promoter about 1 kb 5’ of exon 56, was found specifically in peripheral nerve (Byers et al., 1993). Apart from the promoter regions, a number of other features of the dystrophin gene suggest a potential regulatory role. Striking conservation of parts of the dystrophin 3’ untranslated region between man and chicken [82% homology over 456 bp near the polyadenylation signal (Lemaire et al., 1988)] must surely be due to some functional constraint. Such high conservation in a 3’ untranslated region has been reported in the case of c-fos in which sequences are involved in regulating transcript half-life (Rahmsdorf et al., 1987). Interestingly, Nigro et al. (1994) noted a mutation (of unknown pathogenicity) a short distance 3’ of the natural dystrophin termination codon in a DMD patient. The muscle-specific 5’ untranslated region, on the other hand, shows no significant homology between human and chicken (Lemaire et al., 1988), and only 80% between human and mouse (Koenig et al., 1987). This is in contrast to the 99% identity of the rat and human brain-specific 5‘ untranslated regions (Feener et al., 1989). Thus, the brain 5’ untranslated region is expected to have functional significance, either in the regulation of stability or translation of the transcript or as part of the brain-specific promoter. The sheer size of the dystrophin gene, and the evolutionary conservation of this apparently inconvenient size (Mandel, 1989), suggest that this too may constitute a regulatory feature. The in vivo rate of transcriptional elongation by RNA polymerase I1 has been estimated to be 20-25 nucleotides per second (Ucker and Yamamoto, 1984); this implies that a single act of transcription would take up to 24 hr, a period which may lead to interference by cell division. Tennyson et al. (1993) have since directly shown that transcription of the dystrophin gene takes more than 16 hr. A complex tissue-specific pattern of alternative splicing is observed in the 3’ end of the gene (see Section IV,B). Alternative splicing can be stringently regulated by the interaction of lineage- or stage-specific trans-acting factors with constitutive cis-acting elements which lie adjacent to or within the alternatively spliced exons (Inoue et al., 1990; Breitbart and Nadal-Ginard, 1987), masking default splice sites or favoring distinct hnRNA secondary structures (d’Orval et al., 1991).

IV. THE DYSTROPHIN PROTEIN A. Structure from homology Convergent evolution at the level of amino acid sequence is extremely rare; hence, weak similarities between primary structures can indicate strong sim-

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Figure 6.2. Possihle structure of the dystrophin dimer. A model for the dystrophin homodimer (length = 125 nm) compiled from various sources, principally Koenig and Kunkel (1990). Individual repeat units shown in inset are hased on Yan et U L (1993). See text (see Section IV,A) for details.

ilarities between both tertiary structures and functions. In addition, the degree to which amino acid sequence has been conserved through evolution can serve as an indication as to the strength of functional constraints on sequence variation. Homology searches using the newly isolated dystrophin amino acid sequence revealed a number of illuminating similarities (Hammonds, 1987; Davison and Critchley, 1988; Koenig et al., 1988). The protein could be divided into four domains with distinct affinities (Figure 6.2). The N-terminal domain (amino acids 1-252) bears a strong similarity to the N-terminal domains of a-actinin (Hammonds, 1987) and P-spectrin (Byers et al., 1989). a-Actinin is involved [via its N-terminal domain (Mimura and Asano, 1987)] in the cross-linking of F-actin to form networks or bundles, while spectrins have been shown to associate with actin and integral membrane proteins in a wide range of cell types. The homology is strongest in regions

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directly implicated in actin binding (Kuhlman et al., 1992), and this domain has indeed been shown to bind F-actin in vitro (Way et al., 1992; Hemmings et al., 1992) and to confer cytoskeletal localization on an a-actinin fusion protein (Hemmings et al., 1992). The central domain (amino acids 253-3 112) comprises approximately 75% of the protein. This was first reported to consist of 26 copies of a 109residue repeat motif, bearing 10-25% identity to each other (Davison and Critchley, 1988; Koenig et al., 1988). Similar repeats are found in a number of rod-like cytoskeletal proteins, including a-actinin and the spectrins. These were predicted by Speicher and Marchesi (1984) to form triple-helical domains. The initial phasing of the dystrophin repeats (Koenig et al., 1988) was revised by Koenig and Kunkel (1990) on the basis of detailed sequence analysis [including comparison with the chicken sequence (Lemaire et al., 1988)] and proteolytic data. The resulting redefinition of the repeated motif tallies well with the revised phasing of Drosophila melanogaster a-spectrin by Winograd et al. (1991). In the latter work, repeat units with different phasings were expressed as fusion proteins and tested for structural integrity by protease digestion and circular dichroism measurement. Similar approaches have since been used to confirm this phasing directly on a dystrophin repeat motif (Kahana e t al., 1994). With the revised phasing it was evident that the central domain actually comprised only 24 spectrin repeats, interspersed with regions of no homology to spectrin. The recent crystal structure of a repeat unit from Drosophila a-spectrin (Yan et al., 1993) confirms predictions that each unit consists of three a-helices, each with four or five heptad repeats of hydrophobic amino acids. These are separated by proline-rich turns and bend back on each other to form compact coiled-coil structures (Figure 6.2, inset). Like the other known proteins which bear such motifs, they probably confer upon dystrophin a rigid, extended structure, estimated to be approximately 125 nm in length. Cross et ul. (1990) stressed that the dystrophin repeats adhere much more loosely to a consensus than do those of its relatives, and they proposed a variation on the spectrin model which suggests a degree of elasticity. Four stretches of sequence within the central domain bear no homology to the spectrin family and are proline rich and protease sensitive (Koenig and Kunkel, 1990). These are conserved in the chicken and mouse and are postulated to form flexible hinge regions (Figure 6.2). They lie between the N-terminal domain and repeat 1 (amino acids 253-327), between repeats 3 and 4 (amino acids 668-717), between repeats 19 and 20 (amino acids 2424-2470), and between repeat 24 and the cysteine-rich domain (amino acids 3041-3112). This latter hinge region is more highly conserved, being almost identical in utrophin/DRP and invertebrate protodystrophin. A further 16-amino acid nonhomologous protease-sensitive region lies between repeats 15 and 16.

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The cysteine-rich domain (amino acids 3113-3270) shows weak homology to the C-terminal domain of chicken and Dictyostelium a-actinins, although residues involved in Ca2+ coordination in the a-actinin EF hand-like Ca2+-binding motif are not conserved. Thus, the dystrophin molecule up to amino acid 3270 resembles an elongated member of the spectrin family (Dictyostelium a-actinin has 4 repeats, while Dosophila a-spectrin has 20). Suzuki et al. (1392, 1994) demonstrated that the cysteine-rich domain binds to the transmembrane protein P-dystroglycan in a specific and Caz+-insensitive manner (it also binds a 97-kDa protein which may represent the unprocessed dystroglycan precursor: see Section V, A); this probably represents the principal site of interaction of dystrophin with the membrane. The remainder of the protein (amino acids 3271-3685) was found not to show significant homology with any known protein sequence. Sequence conservation is extremely high and, at over 95% identity with the chicken protein (Lemaire et al., 1988), is strongly indicative of a site of intermolecular interaction. This region is also present in the dystrophin homologues utrophinlDRP, protodystrophin, and the 87K tyrosine kinase substrate (see Section IV,C), and contains a conserved leucine zipper motif (amino acids 35593594). The C-terminal two-thirds of this domain (amino acids 3443-3685) has been shown to bind members of the syntrophin family and protein A0 (Suzuki et al., 1994). a-Actinin associates into antiparallel homodimers (Wallraff et al., 1986) mediated by the extended repeat domain (Mimura and Asano, 1987). This, together with the positions of the hinge regions and the membrane association of the dystrophin protein (see below) led Koenig and Kunkel (1990) to propose a model of a membrane-associated dystrophin network. In this scheme, dystrophin molecules form antiparallel homodimers via repeats 4- 19 (Figure 6.2). The free terminal domains then associate, either directly or via common receptor molecules (see Section V,A), with other dystrophin homodimers. This would form a flexible “chicken wire” lattice of hexagons (=75 nm on each side; see Figure 6.3). Although electronmicroscopy studies of isolated dystrophin molecules suggest the existence of side-by-side dimers, end-to-end dimers and tetramers, and side-by-side staggered tetramers (Murayama et al., 1990; Sat0 et al., 1992-pending the possibility that the species observed were collagen VII rather than dystrophin), this remains the only direct evidence for dystrophin oligomerization. It is possible that the existence of significant myopathy in BMD patients with short in-frame deletions in the rod domain (see Section VI,A) constitutes evidence for antiparallel homodimerization (subtle length changes in the “rod” would be expected to be more structurally disruptive in an antiparallel homodimeric arrangement than in a parallel dimeric or monomeric arrangement).

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bornpiex

Figure 6.3. Structural model of the dystrophin network. A model for a hexagonal lattice of dystrophin (Koenig and Kunkel, 1990) and its association with the sarcolemmal membrane via the glycoprotein complex (Campbell and Kahl, 1989).

B. Protein isoforms Although the sequence described by Koenig et al. represented the principal muscle transcript, it soon became apparent (Nudel et al., 1989; Feener et al., 1989) that the structure of both ends of the dystrophin transcript varied in a tissue-specific manner. Use of alternative first exons is expected to result in the replacement of the first 11 amino acids (M-L-W-W-E-E-V-E-D-C-Y-) of the muscle dystrophin protein [M-dystrophin (Koenig et al., 1988)] with 3 amino acids (ME-D-) in the case of the cortical brain protein (C-dystrophin; Nudel et al., 1989) or 7 amino acids (M-S-E-V-S-S-D-) in the case of the cerebellar Purkinje cell-specific protein (P-dystrophin; G6recki et al., 1992). It is possible, however, that this alteration of protein sequence is of little functional significance and is an incidental by-product of the need for differential regulation of expression in brain and muscle (see Section 111,C). The distal transcripts of the gene encode small proteins corresponding to the C-termini of the larger isoforms. The message transcribed from intron 62 encodes a 71-kDa protein (Dp71, also known as G-dystrophin and apodystrophin- 1) comprising half of the hinge region 4, the cysteine-rich domain, and the C-terminal domain, prefixed with a short novel sequence [M-R-E-HL-K-G- (Lederfein et al., 1992)]. The peripheral nerve-specific transcript encodes a larger 116-kDa protein (Dp116, also known as S-dystrophin and apodystrophin-2) comprising the last 2.5 spectrin repeats followed by the remaining C-terminal domains with a novel 10-residueN-terminus [M-L-H-R-K-T-YH-V-K- (Byers et al., 1993)l. D p l l 6 has been localized to the Schwann cell membrane and is particularly concentrated at the nodes of Ranvier. Although

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both Dp71 and Dp116 constitute potential ligands for the membrane glycoprotein complex in cell types in which full-length dystrophin isoforms are absent, any attempt to propose a function for Dp71 and Dp116 will have to be reconciled with the apparently simple phenotypes of at least 19 DMD and BMD patients whose mutations would disrupt translation of one or both isoforms (Roberts e t al., 1994). Feener e t al. (1989) used RT-PCR to perform a systematic study of the structure of dystrophin transcripts in human skeletal muscle, smooth muscle, and brain. Bies e t al. (199213) performed a parallel study in mouse tissues. Although a rich variety of alternative splicing events was observed in the portion of the transcript encoding the C-terminal domain, only those forms which have been observed in more than one organism are discussed. In both skeletal muscle and brain several events involve the removal of one or more exons which leave the translational reading frame intact. Thus, the 39-nucleotide exon 7 1 is missing from a proportion of transcripts in muscle and brain, which would result in a protein with an interstitial deletion of amino acids 3409-3421. This transcript has been observed in human, mouse, and invertebrates (Roberts e t al., 1995). Loss of the adjacent 66-nucleotide exon 72 has been observed in mouse, chicken, and Xenopus. Brain tissue and cardiac Purkinje fibers of mouse and human also contain transcripts lacking 330 nucleotides corresponding to exons 7 1-74 [in-frame deletion of amino acids 34093518 (Bies et at., 1992a,b)]. Coordinated splicing of these four exons may be aided by their tight clustering in the gene (Roberts e t al., 1992a). This latter isoform would lack most of the region implicated in syntrophin binding, and so should differ functionally from the muscle isoform. Perhaps the most interesting alternative splicing event involves the developmentally regulated loss of the 32-nucleotide exon 78 from many brain and skeletal muscle transcripts (Feener e t al., 1989; Bies e t al., 199213). The proportion of transcripts lacking exon 78 decreases markedly during ontogeny (Bies e t al., 199213). This deletion shifts the translational reading frame so as to bring into register a conserved novel open reading frame which lies in a normally untranslated region of exon 79. The predicted protein has a hydrophobic 32amino acid stretch in place of its normal C-terminal 14 residues. Kunkel e t al. (199 1) reported that antibodies raised against a synthetic peptide corresponding to part of this novel sequence detected a dystrophin isoform, predominantly in smooth muscle-containing tissues such as gut and uterus.

C. Relatives of dystrophin 1. UtrophidDRP As outlined under Section IV,B, dystrophin was found to share a number of attributes (domains which interact with the membrane and with the cyto-

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skeleton, separated by an extended rod) with members of the spectrin superfamily. These similarities were dwarfed by the discovery of a striking homologue of dystrophin which is widely expressed in human tissues (Love et al., 1989; Tinsley et al., 1992). This protein, encoded by a 900-kb gene (Pearce et al., 1993) in the region 6q24, has a gross domain structure identical throughout its length (except for limited deletions in the areas of hinges 1 and 3 and repeat 15) to that of dystrophin and has been variously termed utrophin, dystrophin-related protein (DRP) and DMD-like (DMDL) protein. No disorder has yet been associated with the utrophin/DRP gene; its almost ubiquitous pattern of expression suggests that its absence may be incompatible with life [although 6924 is syntenic with the region of mouse chromosome 10 which contains the dy myopathy locus, expression of this protein is normal In affected mice (Love et al., 1991)l. The utrophin/DRP gene is very widely expressed. Transcripts are found in liver, intestinal smooth muscle, skeletal muscle, testis, kidney, placenta, and fetal heart (Love et al., 1991), although it has been suggested (e.g., Matsumura et al., 1993) that this apparent ubiquity might be due to its known expression in vascular endothelial cells. In normal skeletal muscle utrophin/DRP is specifically localized at the neuromuscular junction in a domain slightly larger than that occupied by the acetylcholine receptors (Ohlendieck et al., 1991a). In general, the skeletal muscle expression pattern is reciprocal to that of dystrophin, being upregulated and localized throughout the sarcolemma in denervated, regenerating, and myopathic tissue. Its distribution in brain is highly specific, but distinct from that of dystrophin, being concentrated in the vasculature, the choroid plexus, pia mater, and endfeet of astrocytes at the blood-brain barrier (Khurana et al., 1992). UtrophinIDRP has been shown to bind to the same, or similar, proteins and glycoproteins as dystrophin (Matsumura et al., 199210; see Section V,A); however, a different isoform of syntrophin is probably involved in this complex (Adams et d., 1993), and components of the “sarcoglycan” complex are not expressed in nonmuscular tissues. The possibility that utrophin/DRP is the prototypic species, of which dystrophin is a recently evolved, specialized, muscle-specific isoform, is discussed under section IV,C,2.

2. Protodystrophin ,

Dystrophins from such distantly related vertebrates as man and Torpedo still show a far greater similarity to each other than either does to human utrophin/DRP. Concluding from this that the dystrophin/utrophin family probably predates the vertebrate radiation, we have characterized the C-terminal domains of dystrophins and utrophins from both vertebrates and invertebrates (Roberts et

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al., 1995). While the vertebrates examined all express molecules with obvious affinity to either the dystrophin or the utrophin branches, protochordates (seasquirt and amphioxus) appear to have a single “protodystrophin” which is equally related to both vertebrate branches. This suggests that a gene duplication event occurred in an amphioxus-like ancestor of the vertebrates, enabling one copy to fulfill a novel role. It is evident that the rate of divergence of dystrophins is approximately one-fourth that of utrophins and protodystrophins, perhaps implying that utrophin fulfills an “ancestral” role, while dystrophin has acquired specialized functions which further constrain its sequence.

3. 87K tyrosine kinase substrate An 87K protein found in association with nicotinic acetylcholine receptors (nAChRs) in the postsynaptic membranes of Torpedo electric organ is tyrosine phosphorylated in response to the nAChR-aggregating signal delivered by nervederived agrin (Wagner et al., 1993). Other work has suggested that, in these membranes, the 87K protein is also found in a complex with syntrophin (58K; see Section V,A) and dystrophin (Butler et al., 1992). Expression cloning of the cDNA encoding the 87K protein (Wagner et al., 1993) reveals it to be a distant relative (27% amino acid identity) of the dystrophin cysteine-rich and C-terminal domains, with an additional 150-residue C-terminus containing potential phosphorylation targets. Sequence likely to represent a human homologue has been isolated as an expressed sequence tag.

0. Subcellular localization The initial study of the dystrophin protein (Hoffman et al., 1987c) on Western blots of subcellular fractions of skeletal muscle tissue seemed to show that dystrophin was enriched in the heavy microsomal fraction. This fraction contains the triads and sarcoplasmic reticulum and includes such triad-associated proteins as (Ca2+, MgZ+)-ATPase, calsequestrin, and receptors for rhyanodine and dihydropyridine. Immunohistochemical approaches (Bonilla et al., 1988; ZubrzyckaGaam et al., 1988; Arahata et al., 1988) yielded very different results. Transverse sections yielded a thin but clear layer of uniform immunostaining on the sarcolemma, with no intracellular staining. Bonilla et al. reconciled their results with those of Hoffman et al. ( 1 9 8 7 ~ by ) pointing out that physical arrangement of the external sarcolemma and the sarcotubular system would result in strong immunocytochemical staining of the former and apparent enrichment of the latter in subcellular fractions. Zubrzycka-Gaarn et al. (1988), on the other hand, obtained strong staining of tubular structures using an antibody against tubular

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CaZ+-ATPase, and ascribed the results of Hoffman et al. ( 1 9 8 7 ~to ) the presence of sarcolemmal vesicles in the heavy microsomal fraction. It is now accepted that most dystrophin is associated with the sarcolemma rather than the triads. All workers demonstrated absent or reduced staining in DMD muscle, discontinuous staining in BMD muscle, and normal staining in muscle from individuals with various other myopathies. Occasional dystrophin-positive fibers are observed in DMD muscle sections and have been termed “revertants” (Hoffman et al., 1990). It is considered more likely, however, that these are not true genetic revertants, but are the transient effect of aberrant splicing events. Electron microscopy using immunogold conjugates (Watkins et al., 1988) also showed localization to the inner face of the sarcolemma, and occasionally revealed a periodicity of one-tenth that of the sarcomeres, consistent with the 125-nm estimate for the length of the dystrophin rod domain. A more detailed study (Cullen et al., 1990) of both longitudinal and transverse sections of muscle (using gold-conjugated antibodies raised against dystrophin repeats 8 and 9) showed that this periodicity is statistically significant, with a modal nearest-neighbor distance of 120 nm and smaller peaks at 240 and 360 nm (with an estimated labeling efficiency of 62%). This part of the protein was estimated to lie 15 nm from the membrane surface. Similar studies using an antibody to the C-terminus (Cullen et al., 1991) showed that this domain lies against the membrane surface and showed a similar periodicity. A sarcolemmal localization for dystrophin suggests that it may be involved in strengthening the sarcolemmal membrane and/or in mediating its association with the myofibrillar cytoskeleton (via the potential actin-binding domain of dystrophin) and with the basal lamina (via transmembrane proteins). More support for a major structural role for dystrophin is provided by the finding that although the protein has an apparently low abundance (0.002%) in total muscle extracts, it represents 2% of the sarcolemmal protein (Ohlendieck et al., 1991b) and 5% of the sarcolemmal cytoskeleton (Ohlendieck and Campbell, 1991). This is comparable to the density of brain spectrin in brain membranes (Bennet et al., 1982). The function of dystrophin in neuronal tissue is less clear. Expression of the mRNA and protein has been demonstrated in the brain and is abolished in DMD patients. Lidov et al. (1990) showed that dystrophin is particularly enriched in Western blots of the cerebellum and cerebral cortex (although this is complicated by the presence of vascular smooth muscle). Immunofluorescence clearly labels soma and dendrites of cerebellar Purkinje cells and of cerebral cortical pyramidal neurons in a punctate fashion. Immunogold electron microscopy confirms a strictly postsynaptic localization in the cerebellum. The absence of dystrophin from Purkinje cell axons is reminiscent of the subcellular restriction of dystrophin to neuromuscular junctions and acetylcholine receptor-rich membranes in the Torpedo californica electric organ (Chang et al., 1989).

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V. DYSTROPHIN-ASSOCIATED PROTEINS AND THEIR DEFICIENCIES A. Structure of the dystrophin-glycoprotein complex Campbell and Kahl (1989) observed that dystrophin could be strongly selected from solubilized muscle membrane preparations using wheat germ agglutinin and showed that this was due to a specific interaction between dystrophin and an integral membrane glycoprotein complex (dystrophin-glycoprotein complex, DGC). Further studies (Ervasti et al., 1990) demonstrated that the 18s DGC comprises proteins with M, of 156, 50, 43, 35, 59, and 25 kDa, together with dystrophin itself. Of these, the first four bound wheat germ agglutinin and concanavalin A, and are therefore glycoproteins. In parallel, a Japanese group (Yoshida and Ozawa, 1990) characterized the same complex with similar results (the correspondence between Campbell and Ozawa nomenclature is as follows: 156DAG/156DAG; 50DAG/A2; 43DAG/A3a, A3b doublet; 35DAG/A4; 59DAP/Ala, A l b , A l c triplet; 25DAP/A5; in addition, the Ozawa group recognized protein A0 of '94 kDa). Antibodies against the 156DAG and 50DAG/A2 components specifically stain sarcolemmal membrane in skeletal muscle preparations. Application of the anti-156DAG antibodies to Western blots of muscle membrane protein samples revealed a reduction in signal of 8590% in m d x or DMD muscle. The reduction in levels of an integral membrane protein consequent to the loss of an associated cytoskeletal protein is reminiscent of the reduction of erythrocyte glycophorin C (an integral membrane protein) in hereditary elliptocytosis, which results from a deficiency in the cytoskeletal protein band 4.1 (Alloisio et al., 1985). Generation of antibodies specific for the 59DAP/A1, 43DAG/A3a, and 35DAG/A4 components enabled a more detailed study of the structure of the DGC (Ervasti and Campbell, 1991). All proteins were present in the complex in equal stoichiometry. Attempted alkaline extraction (which removes peripheral membrane proteins) removed only dystrophin and the 156DAG and 59DAP/A1 components, suggesting that the remainder were all integral membrane proteins. Correspondingly, treatment of the native complex with a hydrophobic radioactive photolabel resulted in labeling of the 50DAGiA2, 43DAG/A3a, 35DAG/A4, and 25DAP/A5 proteins. These data suggest a model of the DGC (see Figure 6.4) in which the transmembrane proteins form a link between the cytoskeleton (dystrophin, 59DAP/A1, AO) and the extracellular matrix (156DAG, merosin, agrin).

1. Dystroglycan and the basal lamina By screening expression libraries with antibodies specific for 43DAG, Ibraghimov-Beskrovnaya et al., (1992) isolated a cDNA clone capable of encod-

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Figure 6.4. Model of dystrophin-glycoprotein interaction. Proposed arrangement of subunits in the DAG complex (compiled from many sources; see Section V,A). Top, extracellular; bottom, intracellular.

ing a 97-kDa protein (895 amino acids) with homology to no known protein. They demonstrated that this 97-kDa precursor, after proteolytic cleavage and extensive glycosylation (two-thirds of the M,of the 156DAG), gives rise to both the extracellular 156DAG and the transmembrane 43DAG/A3a subunits of the DGC, which were renamed a- and P-dystroglycan, respectively. Both mRNA and protein were detected in a wide range of tissues, although the degree of glycosylation of the latter varied. Although mRNA levels were unaffected in m d x skeletal muscle, levels of P-dystroglycan were dramatically reduced in muscle but not in other tissues. The human dystroglycan gene has been characterized (Ibraghimov-Beskrovnayaet d., 1993) and shown to comprise two exons in chromosomal region 3p2 1. a-Dystroglycan was found to bind strongly and specifically in a Ca2+dependent manner to merosin, the muscle-specific isoform of laminin, a major component of the extracellular matrix (Sunada et al., 1994). Thus, i t may be important for the interaction between cells from a variety of tissues and their respective extracellular environments. Ibraghimov-Beskrovnayaet al. noted that the protein recognized by the a-dystroglycan-specific antibodies in nonmuscle tissues may be identical to the brain glycoprotein, cranin (Smalheiser and Schwartz, 1987). Cullen et al. (1994) used immunogold labeling to show that P-dystroglycan has a similar periodic distribution on the sarcolemma to dystrophin and colocalizes with the C-terminal domain of the latter. As dystroglycan is expressed in tissues in which full-length dystrophin is only present at low levels, presumably it must have to associate with a different

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cytoskeletal protein. The widely expressed Dp71 isoform of dystrophin (Bar et

al., 1990; see Section IV,B) and utrophin/DRP (see Section IV,C) are strong candidates for this role; indeed, association of utrophin/DRP with components of the dystrophin-associated glycoprotein complex has been demonstrated (Matsumura et al., 199213). It would therefore be interesting to examine the expression of dystroglycan in nonmuscle tissues of DMD boys whose mutation should specifically disrupt translation of Dp71 (Roberts et al., 1994). In addition to binding merosin, a-dystroglycan has recently been shown to be the principal receptor for agrin in Torpedo electric organ (Bowe et al., 1994) and mammalian neuromuscular junction (Campanelli et al., 1994; Gee et al., 1994). Agrin is a ligand (containing a merosin-like G-domain) produced by motorneurons which has been implicated in inducing the postsynaptic clustering of acetylcholine receptors (AChRs). This constitutes strong evidence for a role for the dystroglycan-utrophin/DRP complex in agrininduced AChR clustering in the neuromuscular junction, but must be reconciled with the additional presence of dystroglycan (complexed with dystrophin) over the entire sarcolemmal surface.

2. Other integral membrane proteins The remaining integral membrane components of the DGC (50DAGlA2, A3b, 35DAG/A4, 25DAP/A5) as yet have no attributable function, although the severe myopathy resulting from their specific loss (Matsumura et al., 1992a; Yamanouchi et al., 1994; see Section V, B) suggests that they are in themselves essential for normal muscle function. Yoshida et al. (1994) have shown that the detergent n-octyl-P-D-glucoside can solubilize the 50DAG/A2, A3b, and 35DAG/A4 subunits as an integral subcomplex, separable from dystroglycan. This distinct structure, together with the restriction of expression of components of the subcomplex to skeletal and cardiac muscle (Roberds et al., 1993; Mizuno et al., 1994), has led to the provisional name of iisarcoglycancomplex.” This complex associates with dystroglycan but does not appear to interact directly with either dystrophin or the components of the basal lamina. The gene encoding the 50DAG/A2 subunit has been cloned (Roberds et al., 1993) and shown to encode a novel 41-kDa transmembrane protein, renamed “adhalin,” 75% of which forms an N-glycosylated extracellular domain.

3. Cytoplasmic components The cytoplasmic or cytoskeletal components of the DGC include dystrophin, a heterogeneous group of proteins called 59DAP/Al or syntrophins, and AO, which may be identical to the 87K tyrosine kinase substrate (see Section IV,C).

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Dystrophin is described in detail under Section V,A and has been shown to associate with the membrane-bound portion of the DGC predominantly through a specific interaction between its cysteine-rich domain and the intracellular domain of P-dystroglycan (Suzuki et al., 1992). The syntrophins and A0 bind directly to the C-terminal domain of dystrophin (Suzuki et al., 1994) and do not appear to interact significantly with other components of the DGC. Yamamoto et al. (1993) have classified the syntrophins into acidic (a’) and basic (p-) forms on the grounds of their strikingly different isoelectric points. Genes encoding several members of the syntrophin family have been cloned. Adams et al. (1993) isolated cDNAs encoding two distinct novel mouse proteins sharing 50% identity at the amino acid level. Syntrophin-1, expressed at high levels in skeletal muscle, has a low predicted PI and is 94% identical to a rabbit sequence described by Yang et al. (1994), and 59% identical to a single characterized Torpedo syntrophin isoform. Syntrophin-2, which is more ubiquitously expressed, is 50% identical to syntrophin- 1 and, although incomplete, probably has a high PI. A human-expressed sequence tag is 82% identical to mouse syntrophin-2 and maps to chromosome 16. Ahn et al. (1994) characterized a human basic syntrophin, P-A1, which is equally related to syntrophin- 1 and syntrophin-2 (-50% identity), mapping to chromosomal region 8q23-24.

B. Disorders of the dystrophin-glycoprotein complex The function of dystrophin seems to be inextricably bound to that of the DGC as a whole, and indeed the loss of the entire DGC secondary to the loss of dystrophin in DMD presents the possibility that the dystrophinopathies result more directly from a defect in the membrane complex. It has therefore come as little surprise that components of the DGC other than dystrophin have been implicated in inherited myopathies. It is also interesting that only musclespecific components (dystrophin, adhalin, merosin), and not ubiquitous components or isoforms (dystroglycan, utrophin/DRP), have been found to be involved in skeletal myopathies. Severe childhood autosomal recessive muscular dystrophy (SCARMD) is a disorder which is phenotypically indistinguishable from DMD or severe BMD (Gardner-Medwin, 1980), but which has an autosomal recessive mode of inheritance and normal levels of dystrophin protein. It is estimated to account for less than 5% of all DMD-like cases in most populations, but has a high incidence (probably greater than that of DMD) in North African countries where consanguinity is common (Ben Hamida et al., 1983). Matsumura et al. (1992a) showed that 50DAG/A2 (adhalin) was specifically missing from muscle of Algerian SCARMD patients, and while there was a slight reduction in the abundance of 35DAGiA4 and A3b, all other DGC proteins appeared to be

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unaffected. A study of 243 Japanese myopathic muscle biopsies reveals specific adhalin deficiency in 2% of the cases (Koga et al., 1994). Although a study of Tunisian families (Ben Othmane et al., 1992) showed strong linkage between SCARMD and markers in 13q12, Passos-Bueno et al. (1993) found that in the more ethnically heterogeneous Brazilian population, a genetically distinct locus must predominate. The human adhalin gene has since been mapped to 17q1221.3, and missense mutations have been detected in non-African cases of SCARMD (Campbell and Sunada, 1994). I t has been proposed that the chromosome 13 locus encodes a protein required for the assembly of the sarcoglycan complex. The cardiomyopathic hamster (NSJ-my/my) also seems to have a specific deficiency of adhalin in its skeletal and cardiac muscle (Yamanouchi et al., 1994) and may represent a legitimate animal model for SCARMD. The dystrophia muscularis (dy) mouse was once considered as an animal model for DMD and was recently reexamined on the basis of synteny between the mouse dy locus (mouse chromosome 10) and the human utrophin/DRP gene [human 6424 (Love et al., 1991)l. It has now been shown (Sunada et al., 1994) that the muscle-specific M-chain of merosin (a ligand for a-dystroglycan; see above) maps to the same region of mouse chromosome 10, and that merosin expression is severely disrupted in the peripheral nerve and skeletal and cardiac muscle of dyldy mice. Merosin deficiency is also suspected to be the primary defect in “occidental” congenital muscular dystrophy in humans.

VI. MUTATIONAL SPECTRA AND MOLECULAR ETIOLOGY Mutational spectra of genetic disorders are often highly idiosyncratic, reflecting peculiarities of underlying mutational mechanisms, the biology of the protein product, or population history. The dystrophin gene is no exception, and although the mutational spectrum has yet to be fully defined, >95% of mutations which have been characterized form an extremely unusual pattern. The mutational spectra of DMD and BMD not only convey information regarding the biology of the dystrophin protein, but determine the nature of diagnostic strategies (see Section V,II).

A. Mutational spectra and distribution in DMD and BMD Characterization of the mutational spectra of these disorders has largely been driven by advances in knowledge of the gene structure and in methods for mutation analysis. The first mutations to be described were those visible at a cytogenetic resolution [X;autosome translocations and extensive deletions (Verellen et al.,

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1978; Franke et al., 1985)]; these not only predated the identification of the gene but were instrumental to its discovery (see Section 111,A). With the advent of the intragenic pERT87 probes, gross deletions in the dystrophin gene of affected individuals were detected in 6.5% of DMD patients (Kunkel et al., 1986). Further characterization of the gene enabled studies by Koenig et al. (1987) (using cDNA probes) and den Dunnen et al. (1987) (using FlGE and a number of genomic probes) to increase this figure to ~ 5 5 %of DMD patients and located a region of substantial clustering of such deletions. This clustering was further associated with a single intron (intron 44) in which endpoints of deletions lay in 16% of cases (Wapenaar et al., 1987). A large-scale analysis by den Dunnen et al. (1989) showed that 60% of all DMD mutations are gross deletions (i.e., comprising one or more exons), 5% are gross duplications (see also Hu et al., 1990), and the remainder are presumably point mutations or small rearrangements. Gross rearrangements are found in a somewhat higher proportion of BMD patients (75-85%). In the 4 years following the publication of the cDNA sequence (Koenig et d . , 1988) reports appeared describing many hundreds of rearrangement mutations (Monaco et aI., 1988; Malhotra et al., 1988; Hodgson et al., 1989; Gillard et al., 1989; den Dunnen et al., 1989, and many subsequent papers). Despite this, only one point mutation had been described, its position narrowed down by the size of the residual protein on a Western blot (Bulman et al., 1991). This state of affairs arose largely on account of the size and complexity of the gene, lack of knowledge of its exon structure (a requirement for DNA-based mutation screening), and the efficacy of indirect diagnosis. Since then, however, screening techniques have evolved which are capable of addressing a gene of this size (see Section VII,B), the entire gene structure has been established (Roberts et al., 1993a), and the ultimate weakness of indirect diagnosis has left a legacy of pedigrees which are refractory to analysis (see Section VI1,A). A review of published and unpublished dystrophin gene mutations written at the end of 1993 (Roberts et al., 1994) was able to survey 70 mutations, and the number is climbing exponentially. As yet, however, no study has taken a large set of patients and screened them exhaustively in order to establish the true mutational spectra of these disorders. A substantial contribution to the “DMD/BMD population by autosomal phenocopies (see Section V,B) remains possible. The mutational spectra are presented in Figure 6.5, and their implications for the biology and diagnosis of DMD and BMD will be discussed under Sections VI,B, VI,C, and VI1,B. The distribution of rearrangement mutations is extremely nonrandom (Figure 6.6a) with two “hot spots”; one in the 5’ end (exons 2-20, with most breakpoints occurring in the large introns 1 and 7) and one two-thirds of the way down the gene (exons 45 to 53, with most breakpoints occurring in introns 44, 45, 47, and 50). This distribution has been described in many different popula-

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Figure 6.5. Spectra of mutations in DMD and BMD. Almost all large and small rearrangements and splice site mutations in DMD patients result in a shift in the translational reading frame

of the transcript. Almost all rearrangements and splice site mutations in BMD patients leave at least a subset of transcripts with an intact (hut interstitially deleted) open reading frame. Based on gross rearrangement data from many sources and point mutation data reviewed in Roherts et al. (1994).

tions, although it has been suggested that subtle variations are present even between different European populations (Danieli et al., 1993). The behavior of the 170-kb intron 44 is of particular interest. Although this appears as a mutagenic focus on the scale of the entire gene (Wapenaar et al., 1987), on closer inspection (Blonden et al., 1991) the breakpoints are found to be spread rather evenly throughout the intron. The hot spot therefore seems to result from a regional attribute, rather than necessarily being the function of a specific sequence motif. Oudet et al. (1992) demonstrated that meiotic recombination occurs within intron 44 at a rate 12-fold higher than the genome average. This correlation also is obtained over a broader area, in that 50% of deletion events occur between exons 44 and 50, as do 50% of recombination events. Unequal meiotic recombination or mitotic sister chromatid exchange become immediate suspects, but any proposed mechanism must account for three further unusual features of the spectrum of rearrangement mutations. First, there is a strong polarity to deletions extending from the intron 44 hot spot in that the vast majority extend in a distal (3’) direction. Second, there is a 10-fold excess of deletions over duplications in the gene as a whole, rising to >20-fold in the distal hot spot region (Hu et al., 1990). Most conventional mutational mechanisms are expected to generate deletions and duplications with equal frequency. Third, 80% of the cases of demonstrable germline mosaicism have proximal deletion, only 20% have a deletion in the distal hot spot (Passos-Bueno et al., 1992); this provides an almost even distribution of deletions in mosaic cases. It has been suggested that this results from distal deletion occurring later in gonadal development, giving rise to smaller clones which rarely manifest as cases of

Figure 6.6. Distribution of mutations in the dystrophinopathies. (a) Distribution of gross rearrangements: percentage of gross deletions (vertical axis) in which each exon (horizontal axis) is lost. Data compiled by S. Abbs. (b) Distribution of point mutations (after Roberts et al., 1994) showing exon structure of transcript: (a) mutations characterized by screening of entire coding region, ( 0 )mutations characterized by screening of <20 exons. (c) Relationship between protein domain structure, isoforms, and mutation distribution.

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germline mosaicism. From these observations it is evident that a mechanism of unusual temporal and spatial properties makes a major contribution to the mutational spectrum of DMD and BMD. The distribution of the known point mutations is presented in Figure 6.6b. Although the distribution of most of the mutations is biased by the selective screening strategies used, those characterized in our laboratory (black circles in Figure 6.6b) are the product of comprehensive screening of the entire coding region, and therefore may more closely represent the true distribution. It is apparent that, unlike gross rearrangement mutations, point mutations are evenly distributed throughout the gene.

B. Genotype/phenotype correlations-Gross

rearrangements

The original suspicion that DMD and BMD might be manifestations of mutations in the same gene (Becker and Kiener, 1955) seemed justified as first DMD (Davies et al., 1983) and then BMD (Kingston et al., 1984) were shown to be linked to markers in Xp21 (Goodfellow et al., 1985). This was confirmed by the discovery that gross deletions associated with both disorders encompassed overlapping regions (Kunkel et al., 1986; Monaco et al., 1987; Hart et al., 1987). Although the predominance in the literature of rearrangement mutations has until recently restricted genotype/phenotype correlation studies to a rather coarse resolution, the important “frameshift hypothesis” was established which holds to this day. As there was no obvious correlation between the size and location of the deletions and the severity of the phenotype of the patient [for example, a BMD patient lacking 46% of the dystrophin coding sequence has been reported (England et al., 1990), while deletions of single exons can cause DMD], Monaco et al. (1988) set out to explain this apparent inconsistency in terms of the effect of the genomic rearrangement on the translational reading frame of the putative mutant mRNA. The relationship between the exon-intron junctions was established for exon 4 and exons 10-21 in the DXS164 locus. Analysis of three DMD and three BMD patients whose deletions lay within this region showed that the conjectured transcripts from the genes in the DMD patients would have translation frameshifts, resulting in premature termination of translation, while transcripts from the BMD genes would continue in-frame 3‘ of the deleted region. Thus, it would be expected that the dystrophin protein in DMD patients would be truncated shortly after the point of deletion, while the protein in BMD patients would only suffer an interstitial deletion corresponding to the deleted region. These predictions were tested by Hoffman et al. (1988), who found that the dystrophin protein was generally absent or grossly reduced in abundance in DMD muscle, and present at >40% of normal abundance (95% of cases) but usually with reduced size (65% of cases) in BMD muscle.

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The frameshift hypothesis gained support as more exon boundaries were identified, enabling larger numbers of patients with gross deletions (Malhotra et al., 1988; Koenig et al., 1989; Gillard et al., Gilgenkrantz et al., 1989) and duplications (Hu et al., 1990) to be analyzed. Koenig et al. (1989) found that in 92% of patients the phenotype/genotype correlation was in agreement with the frameshift hypothesis. The exceptions consist largely of (i) BMD and intermediate patients deleted for exons 3-7 (Malhotra et al., 1988), (ii) various patients in whom a deletion breakpoint lies in introns 43 or 44 (Gillard et al., 1989; Koenig et al., 1989; Hodgson et al., 1989; Norman et al., 1990), (iii) DMD patients with extremely large in-frame deletions [e.g., patient DL66.6 in den Dunnen et al. (1989), who is deleted for exons 13-60], (iv) patients with myalgia and severe cramps who have in-frame deletions in the region of exons 10-44 (Ekggs et al., 1991), and (v) a single patient with an in-frame deletion of exons 35-44 who has mild cramps and elevated serum CPK levels (Ekggs et al., 1991). Categories iv and v may be explained by assuming that domains encoded by these regions are of little functional significance. The variable phenotypes of patients deleted for exons 3-7 await explanation, although Chelly et al. (1990b) showed that only a small amount of alternatively spliced transcript is present, and Winnard et al. (1994) have strong evidence that the predominant translation product is initiated from a downstream methionine codon in exon 8. Patients in category ii may be explained in part by the tendency of exon 44 to be alternatively spliced (Roberts et al., 1991). Thus, in the vast majority of cases, empirical findings fit the hypothesis. As the frameshift hypothesis is based on an extrapolation from gene structure to transcript structure, it is reassuring that studies of patient transcript structure (Chelly et al., 1990b; Roberts et al., 1991) yield results largely in line with its predictions. The reasoning on which the hypothesis was originally founded involves a number of testable assumptions: (i) interstitial gene rearrangements do not significantly affect transcription or transcript processing, i.e., the distinction between a “Duchenne” transcript and a “Becker” transcript is made only by the translation apparatus, although premature failure of translation may result in subsequent transcript instability; see Section VI,C; (ii) truncation at virtually any point in the protein results in a product of severely compromised function and/or stability-implicit in this is that the carboxy-terminal domain is of particular importance; (iii) interstitial deletion of virtually any part of the protein results in a product which retains some function but is qualitatively or quantitatively deficient. Exceptions to the hypothesis may represent failure of any one of these three assumptions. Specifically, rearrangements may affect splicing of an adjacent exon (Roberts et al., 1991), truncation very close to the carboxy-terminus may be less detrimental (Love et al., 1990), and interstitial deletion in the carboxy-terminal domain may abrogate protein function. Although DMD is relatively phenotypically homogeneous, there is a

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wide range of severity within the BMD spectrum. Beggs et al. (1991) showed that in-frame deletions which removed all or part of the N-terminal domain resulted in a severe BMD phenotype (onset before the age of 10 years). Deletions in the rod domain are associated with varying phenotypes, with more severe phenotypes at the C-terminal end and very mild phenotypes in the central portion (see also Norman et al., 1990). These variable phenotypes show no obvious correlation with the number of remaining repeat units being an integer or noninteger. It is possible that if, like or-actinin, dystrophin forms antiparallel homodimers, then the degree of disruption of the dimerization process (and in turn the phenotypic severity) may be influenced by the symmetricality of the deletion about the midpoint of the dimerization domain. Complete removal of the muscle-specific promoter results in a BMD phenotype (Boyce et al., 1991) or cardiomyopathy (see Section II,C), suggesting that BMD can arise through solely quantitative effects on the dystrophin protein, and that promoter mutations are unlikely to be involved in DMD.

C. Genotype/phenotype correlations-point

mutations

The recent increase in reports of point mutations in the dystrophin gene (e.g., Bulman et al., 1991; Roberts et al., 1992b; Prior et al., 1993a; Lenk et al., 1993) has at last allowed examination of genotype/phenotype correlations to be extended to the entire mutational spectrum (Roberts et al., 1994). Perhaps surprisingly for such a highly conserved gene with an apparently essential C-terminal domain, almost all cases of DMD are associated with mutations which are expected to disrupt the translational reading frame (see Figure 6.5); missense mutations represent <2% of all DMD mutations. DMD mutations, which comprise nonsense mutations, small frameshifting rearrangements, and frameshifting splice site mutations, are distributed throughout the gene from exon 2 to exon 76, with no correlation between position of the mutation and phenotypic severity (Figure 6.6b). In light of this evidence, one is forced to an interesting conclusion (Roberts et al., 1994), which is that >98% of DMD mutations involve an essentially homogeneous null genotype and phenotype, and that the simplest explanation for this is that regardless of how much of the open reading frame is left intact, premature translational termination at any point will result in almost complete lack of a protein product. This phenomenon is well known in other systems (McIntosh et al., 1993), and appears to result from a translational “vetting” system in which the translocation of a nascent mRNA from the nucleus depends on the successful translation of an (unknown) element near the 3’ end (Baserga and Benz, 1992; Belgrader et al., 1993; Cheng and Maquat, 1993). Translational termination at any point upstream of this element will result in degradation of the mRNA, probably triggered by removal of the 5’ cap (Muhlrad

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and Parker, 1994). Indeed, the level of dystrophin gene transcripts is known to be very low (<5% of wild-type) in the muscle of most DMD patients (OronziScott et al., 1988; Chelly et al., 1990b). This has disappointing implications for the use of DMD mutations in the functional dissection of the dystrophin protein in that >98% of all mutations can convey no information regarding the particular importance of any domain, C-terminal or otherwise. One is left with a handful of potentially informative mutations, including large in-frame deletions (Chelly et al., 1990b; Hoffman et al., 1991) and missense mutations (Prior et al., 1993b). These are expected (or known-Chelly et al., 1990b) to have high levels of transcript, and probably express a protein whose function is profoundly defective. Approximately half of BMD point mutations are splice site mutations which result in in-frame exon skipping or cryptic splice site usage-these therefore are analogous in their effect to in-frame single-exon deletions (Roberts et al., 199313). The remainder appear to be missense mutations, predominantly in the N-terminal or C-terminal domains, although the pathogenic status of none of the reported mutations has been confirmed (reviewed in Roberts et al., 1994). The connection between mental retardation and various dystrophin mutations is still unclear. If one accepts the view that some (30-50%) DMD boys are mentally retarded while the remainder are essentially unaffected, one might expect the nature of individual mutations to be one of the factors which determines whether mental deficiency occurs. Intrafamilial concordance is then seen as evidence that given mutations have a uniform effect on IQ. If, however, one takes the position (Prosser et al., 1969) that DMD boys have mental abilities which are poorer than those of their unaffected sibs by a fixed amount (and are therefore all mentally affected), then all mutations which cause DMD (and therefore abolish production of functional dystrophin) are expected to cause mental impairment to a similar degree. Intrafamilial concordance merely reflects background IQ concordance between sibs, minus a fixed quantity for dystrophin deficiency. A problem with differentiating between these two situations is that almost all studies of patients with known mutations provide a simple yes/no datum for mental retardation, often based on informal assessment or school performance, rather than providing results of psychometric tests (papers describing detailed psychometry, on the other hand, rarely include molecular data). Searches for genotype/phenotype correlation have been relatively fruitless. Rapaport er al. (1991) claimed that 70% of patients deleted for exon 52 have mental retardation, but these findings have not been supported elsewhere. It has been suggested that disruption of the Dp71 transcript (see Section IV,B), which is highly expressed in the brain, may be connected with mental retardation. This appears to be borne out by the results of Lenk et at. (1993), who found a predominance of mental retardation in six out of seven patients with point mutations in the Dp71 region (exons 63-79; Figure 6 . 6 ~ ) .In our laboratory, however, there appears to be no simple association between mental phenotype

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and position of point mutation (Roberts et al., 1992b; Gardner et al., 1995). Furthermore, despite the fact that the Dp71 region is only affected by about 1% of deletions, the prevalence of mental retardation in deletion patients does not differ greatly from that in nondeletion patients.

VII. CARRIER AND PRENATAL DIAGNOSIS IN DMDlBMD As is the case with most other lethal or severely disabling genetic diseases for which no cure is available, carrier and prenatal diagnosis of DMD and BMD are performed with a view to prevention. Only families with a previous case of the disease are referred for diagnosis. Samples, usually in the form of venous blood, are obtained from key family members, particularly from the proband, and DNA or RNA is extracted for analysis. Carrier diagnosis provides a risk of carrier status for female relatives, on which information they can decide whether or not to request prenatal diagnosis during pregnancy (or indeed whether to try for a family at all). A prospective mother with a high carrier risk will generally opt for prenatal diagnosis, in which fetal genotype is sampled via either cultured amniotic cells or chorionic villus tissue. Risks of procedure-related miscarriage attend both of these sampling techniques, so a mother at low but nonzero carrier risk must balance the costs and probabilities of the possible outcomes. The fetal sample is first sexed cytogenetically. Female fetuses are assigned zero risk while male fetal samples are further analyzed for their genotype at the dystrophin locus using direct diagnosis (analysis of the disease mutation) or indirect diagnosis (analysis of a linked marker). In either case, the result of a prenatal diagnosis will take the form of a risk probability to the fetus, o n the basis of which the parents must decide (with the aid of a trained genetic counselor) whether to have the pregnancy terminated or whether to continue to term. Strategies for prenatal and carrier diagnosis of genetic disease reflect the peculiarities of the gene in question. In the case of the dystrophin gene, its high mutation rate, its high intragenic recombination frequency, its immense size and complexity, and the unusual preponderance of deletion mutations have all shaped the strategies applied to DMD and BMD.

A. Strengths and limitations of indirect diagnosis Direct diagnosis of DMD and BMD, while desirable, is not always possible. In the 60% of cases which involve gross deletions (den Dunnen et al., 1989; see Section VI, A), although the mutation is easily identifiable, carrier diagnosis is complicated by the presence of a normal dystrophin gene in the female relatives, while in the 35% of cases which involve point mutations, the difficulty of identifying such small changes in such a large gene has until recently rendered

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direct diagnosis on unfeasible proposition. Hence, carrier diagnosis in 60% of families (see Section VI1,C) and carrier and prenatal diagnosis in 40% of families rely heavily on indirect approaches. Indirect diagnosis involves monitoring the pattern of inheritance of a linked marker in the presenting family and is founded on the assumption that the disease mutation cosegregates with the marker throughout the family. The strength of this assumption, and hence the accuracy of diagnosis, is limited by the frequency of recombination between marker(s) and mutation and by lack of knowledge of the origin of mutation. These two points are of particular importance in the case of DMD and BMD in which the frequencies of intragenic crossover and of new mutation are so high. In addition to these considerations, indirect diagnosis is also limited in its applicability by the informativity of available markers in a given family. The frequency of recombination within the gene has been measured at 12% (see Section III,B), which tallies with a reported 6% recombination rate between intragenic markers and the mutation (Fischbeck et al., 1986). Thus, cosegregation of disease and marker allele will occur in only 95% of meioses. This limitation can be circumvented by analyzing flanking markers (Cole et al., 1988), which enables the detection of recombination events within the gene. Initially, RFLPs detected by Southern blot were used (e.g., Kunkel et al., 1986); these were labor intensive and their diallelic nature limited the number of pedigrees for which each marker was informative. Although the assays were simplified by conversion to a PCR format (Roberts et al., 1989), these loci were ultimately superseded by the more informative multiallelic microsatellite markers (Weber and May, 1989). Highly informative (heterozygosity 3 0 % ) markers are available near the promoter (Feener et al., 1991) and within the distal deletion hot spot (Clemens et al., 1991). Although there are no highly informative markers distal to exon 50, intragenic recombination in the 3' region occurs in only 0.6% of meioses (Oudet et al., 1992), and only 10% of all mutations lie 3' of exon 50. Knowledge of the origin of mutation in DMD/BMD families is limited by the fact that one-third of cases represent new mutations. In the remaining cases the mutation is highly likely to be of grandparental or great-grandparental origin. Thus, inheritance of the same haplotype as the proband need not entail inheritance of the mutation. These fundamental problems with indirect diagnosis are compounded by the poor diagnostic value of clinical tests of carriership (see Section VI1,C).

B. Direct diagnosis The shortcomings of indirect diagnosis are circumvented by direct diagnosis, in which the causative mutation is defined and used as a diagnostic marker. This

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~

strategy has been highly successful in the prenatal and carrier diagnosis of disorders which result from mutations in small genes. Green et al. (1989) for example, amplified individual exons of the factor IX gene from hemophilia B patients. These products were screened for mutations using chemical mismatch analysis and then sequenced. The accumulated data have contributed to a nationwide database of factor IX mutations, which can subsequently be used for prenatal or carrier diagnosis in the families analyzed (Giannelli et al., 1991). These data are also extremely useful for the biological dissection of the gene product. For once, the peculiarities of the dystrophin gene are in favor of the clinical geneticist. Sixty percent of mutations are readily detectable through the absence from the patient's genome of one or more exon of the gene; moreover, the clustering of these mutations means that assay of only 23% of the exons can reveal >98% of deletions (Abbs et al., 1991). Gross deletions were initially detected by the failure of genomic probes or cDNA clones to hybridize to Southern blots of (HindIII-digested) patient DNA (e.g., Monaco et al., 1985; den Dunnen et al., 1989). With the advent of PCR, several groups have designed multiplexed reactions containing primers which direct amplification of a selection of commonly deleted exons (Chamberlain et at., 1988, 1989; Beggs et al., 1990; Abbs et al., 1991). Deletion of an exon results in its failure to be amplified. These assays are rapid, nonradioactive, and use small amounts of patient DNA. Such assays now form the frontline of attack for most laboratories involved in DMD/BMD diagnosis. Gross duplications can be detected by the presence of junction fragments on Southern blots of PFGE or (occasionally) conventional gels (den Dunnen et af., 1989). Under optimal conditions they are also apparent as increased dosage on Southern blots or multiplexed PCRs (Abbs and Bobrow, 1992). In many ways, the problems associated with duplication detection are the same as those discussed under Section VII,C in connection with carrier diagnosis of deletions. Due to the size and complexity of the gene and the inaccessibility of the transcript, the detection of point mutations on a diagnostic basis has only recently become a possibility. Strategies for the characterization of point mutations have fallen into two fundamentally different approaches. First, Chelly et al. (1988) recognized that the dystrophin gene is ectopically transcribed, albeit at extremely low levels, in such readily accessible tissues as peripheral blood lymphocytes. This made possible the amplification of dystrophin gene cDNA obtained from reverse transcription of lymphocyte RNA (Roberts et al., 1991) and subsequent mutation screening of the amplification products using chemical cleavage analysis (Roberts et at., 199213). Recently, from realization that most DMD point mutations disrupt translation, Roest et al. (1993) developed an assay truncated peptide product based on in vitro transcription and translation-a pinpoints the position of the mutation, Second, the determination of the

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exon structure of the dystrophin gene (Roberts et al., 1993a) and acquisition of flanking intron sequences has opened the door for more conventional approaches. Thus, exons are amplified singly or in multiplex from genomic DNA and are screened for mutations using a simple method such as heteroduplex analysis (Prior et al., 1993a) or single strand conformation polymorphism analysis (Lenk et al., 1993). After localization and sequencing of point mutations, it can be advantageous, particularly in larger pedigrees, to establish simple dedicated PCR assays for future prenatal and carrier diagnosis (Yau et al., 1993). A radically novel approach to prenatal diagnosis has been proposed by Sancho et al. (1993) in which myogenesis is induced in cultured amniocytes by transfection with a construct encoding the myogenic transcription factor MyoD. The ability to produce dystrophin is then assayed immunocytochemically, obviating the need to know the mutation.

C. Carrier diagnosis In two-thirds of all cases of DMD and BMD the disease is sporadic with no family history; segregation analysis is usually uninformative, and it is difficult to estimate carrier risk for these women (Kaariainen et al., 1990; Hodgson et al., 1989). Furthermore, the high mutation rate means that in half of these sporadic cases (a third of all cases) the disease is due to a new mutation and the mother is not a carrier (Zatz et al., 1977). Analysis of serum CPK levels (Sibert et al., 1979) has been used; this relies on phenotypic expression in the heterozygote and can give variable results. The results can be used in conjunction with family data to yield Bayesian probabilities rather than definitive diagnoses. Direct diagnostic schemes described under Section VII,B for the analysis of patients with gross deletions are complicated in carrier females by the presence of the normal chromosome, which would mask the result from the defective chromosome. Carrier diagnosis (and duplication detection in patients) is therefore usually performed by dosage analysis using either Southern blotting (Mao and Cremer, 1989) or PCR (Abbs and Bobrow, 1992). This involves comparison of the intensity of specific bands in different samples. However, band intensity is dependent on a number of experimental factors, and so carries a degree of uncertainty which many laboratories find is unacceptable in clinical practice. Recently, electrophoresis of fluorescently labeled multiplex PCR products in an automatic sequencing machine has been shown in blind trials to provide accurate and reproducible results in carrier diagnosis of deletions (Yau et al., 1995). Many approaches aim to avoid quantitative analysis by assaying a qualitative attribute of the deletion event. In 17% of duplication and deletion patients (den Dunnen et al., 1989) the breakpoint is sufficiently close to a non-

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deleted exon that it lies within the restriction fragment detected by a cDNA probe on a Southern blot-this results in a band of altered mobility on a Southern blot which will be visible in a carrier female. The frequency of detection of such diagnostic “junction fragments” can be increased significantly by the (technically demanding) use of pulsed-field gel electrophoresis (den Dunnen et al., 1989; Chen et al., 1989). Junction fragments provide unequivocal identification of the mutation in carriers. The advantages of such a qualitative diagnostic feature have been paralleled in the development of a method of carrier detection based on the amplification across the deleted region of ectopic dystrophin transcripts (Roberts et al., 1990b). The deletion event can also remove an informative polymorphic locus, revealing the inheritance of the mutation in a female carrier as an unexpected hemizygosity. This approach is becoming more widely used as markers in the distal deletion hot spot have been identified (Clemens et al., 1991), but it still requires an appropriate family structure.

VIII. QUESTIONS FOR THE FUTURE-FROM DISEASE PHENOTYPE

PROTEIN DEFECT TO

Despite the wealth of knowledge which has accumulated, we still lack a clear picture of the in vivo function of dystrophin and of the route by which impairment of this function leads to the characteristic phenotypes of BMD and DMD. A number of pressing problems remain.

1. Dystrophin is considered by many to perform a purely structural role, forming a reinforcing scaffold which contributes to the mechanical strength of the sarcolemmal membrane. Its membrane-bound site of attachment is also associated with the extracellular matrix, suggesting that dystrophin may be needed to localize the “bosses” which pin the myotube to its environment. This simple picture can be accounted for by the spectrin-like regions of dystrophin and the transmembrane dystroglycan link, and ascribes no function to the “sarcoglycan” subcomplex and the C-terminal domain of dystrophin with its attendant subunits. Indeed, the fact that SCARMD can reproduce most of the phenotypic features of DMD while leaving the supposed transmembrane link intact might suggest that these mechanical properties are irrelevant to the issue of DMD etiology. 2. As discussed above, most subunits of the DGC have yet to be associated with a function. Do they have enzymatic activity? Are they involved in transduction of extracellular stimuli? Do they sense and signal changes in the membrane, such as mechanical or osmotic stress? Does proposed phosphorylation of DGC subunits play a role in this? How can

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the complete loss of a complex of up to 10 highly conserved proteins be compatible with almost normal muscle function over several years? 3. Genes encoding the muscle-specific subunits of the DGC constitute candidate disease loci for heritable myopathies-are genes encoding more ubiquitously expressed subunits likely to be involved in multisystem disorders or is their disruption incompatible with life? 4. The complex is expressed in many tissues which lack full-length dystrophin protein. What substitutes for dystrophin as its ligand? Dp71, Dp116, or utrophin/DRP? What is the function of the complex in these tissues and what is the function of the ancestral protodystrophin and proto-DGC in invertebrates? 5. Complete loss of dystrophin occurs in both DMD humans and the m d x mouse. The function of skeletal muscle in the former is severely compromised, while the latter is virtually asymptomatic. Why? The mdx diaphragm exhibits a pathology reminiscent of DMD (Stedman et al., 1991). Does this imply that the damage incurred by DMD muscle is predominantly work related and that the difference in work rate of the skeletal musculature might be the sole reason for the phenotypic differences between DMD and mdx and for the specific pattern of pathology in each of these disorders? 6. What is the relationship between DMD and BMD? What aspect of normal dystrophin function is lost in BMD, and what aspect of function retained in BMD is lost in DMD-are the differences merely quantitative [a number of BMD mutations exist which are expected to have no qualitative effect on the gene product (Roberts et al., 1994)]?What is the molecular basis of the broad range of BMD phenotypic severity? 7. The cysteine-rich and C-terminal domains of dystrophin are highly conserved between vertebrates and invertebrates, yet missense mutations which affect these regions have not been found to cause DMD. Will missense mutations be more prevalent in the less-studied BMD cases? Can DMD only be caused by null mutations? 8. What is the role of dystrophin in the central nervous system? Does its loss predispose to mental retardation by independent factors or does it per se cause a reduction of IQ with full penetrance? 9. What is the functional significance of the multiple isoforms of dystrophin? Is there no special extramuscular phenotype in the many DMD patients in whom Dpl16 and Dp7l expression is disrupted, or is this feature masked by the severity of the myopathy?

It is my hope that readers of this chapter will soon be in a position to pencil in the answers to many of these hanging questions, as it can only be to the advantage of those individuals who suffer the effects of life without dystrophin.

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Acknowledgments I am the recipient of a Medical Research Training Fellowship. My work is also supported by the Muscular Dystrophy Group of Great Britain and Northern Ireland and the Generation Trust. I am indebted to Professor Martin Bobrow for his direction and interest, and to Koshie Boye for support beyond the call of duty.

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Murayama, T. et al. (1990). Molecular shape of dystrophin purified from rabbit skeletal muscle myofihrils. Proc. Jpn. Acad. Ser. B 66:96-99. Murray, J. M., Davies, K. E., Harper, P. S., Meredith, L., Mueller, C. R., and Williamson, R. (1982). Linkage relationship of a cloned DNA sequence on the short arm of the X chromosome to Duchenne muscular dystrophy. Nature (London) 300:69-7 1. Nigro, V., Politano, L., and Nigro, G. (1994). Identification of point mutations in the dystrophin gene by multiple SSCP. Muscle Nerve, Suppl. 1:S37. Nikovits, W., Jr., Kuncio, G., and Ordahl, C. P. (1986). The chicken fast skeletal troponin I gene: Exon organization and sequence. Nucfeic Acids Res. 14:3377-3390. Norman, A. M., Thomas, N. S. T., Kingston, H. M., and Harper, P. S. (1990). Becker muscular dystrophy: Correlation of deletion type with clinical severity. J. Med. Genet. 27:236-239. Nude], U., Robzyk, K., and Yaffe, D. (1988). Expression of the putative Duchenne muscular dystrophy gene in differentiated myogenic cell cultures and in the brain. Nature (London) 331635-638. Nude], U., Zuk, D., Einat, P., Zeelon, E., Levy, Z., Neuman, S.,and Yaffe. D. (1989). Duchenne muscular dystrophy gene product is not identical in muscle and brain. Nature (London) 337: 76-78. Ogasawara, A. (1989). Similarity of IQs of siblings with Duchenne progressive muscular dystrophy. Am. 1. Ment. Retard. 93:548-550. Ohlendieck, K., and Campbell, K. P. (1991). Dystrophin constitutes five percent of membrane cytoskeleton in skeletal muscle. FEBS Lett. 283:230-234. Ohlendieck, K., Ervasti, J. M., Matsurnura, K., Kahl, S. D., Leveille, C. J , , and Campbell, K. P. (1991a). Dystrophin-related protein is localized to neuromuscular junctions of adult skeletal muscle. Neuron 7:499-508. Ohlendieck, K., Ervasti, J. M., Snuok, J. B., and Campbell, K. P. (1991b). Dystrophinglycoprotein complex is highly enriched in isolated skeletal muscle sarcolemma. J. Cell Bid. 112:135-148. Oronzi-Scott, M., Sylvester, J. E., Heiman-Patterson, T., Shi, Y.-J., Fieles, W., Stedman, H., Burghes, A., Ray, P., Worton, R., and Fischheck, K. H. (1988). Duchenne muscular dystrophy gene expression in normal and diseased human muscle. Science 239:1418-1420. Oudet, C., Heileg, R., Hanauer, A., and Mandel, J.-L. (1991). Non-radioactive assay for new microsatellite polymorphisms at the 5’ end of the dystrophin gene, and estimation of intragenic recombination. Am. J. Hum. Genet. 49:311-319. Oudet, C., Hanauer, A., Clemens, P., Caskey, T., and Mendell, J. L. (1992). Two hotspots of recombination in the DMD gene correlate with deletion prone regions. Hum. Mol. Genet. 1:599-603. Passos-Bueno, M. R., and Zatz, M. (1991). Reproductive fitness and frequency of new mutations in Becker muscular dystrophy: Implications for genetic risk estimates. J Med. Genet. 28:286-288. Passos-Bueno, M. R., Bakker, E., Kneppers, A. L., Takata, R. I., Rapaport, D., den Dunnen, J. T., Zatz, M., and van Ommen, G. J. (1992). Different mosaicism frequencies for proximal and distal Duchenne muscular dystrophy (DMD mutations indicate difference in etiology and recurrence risk. Am. J. Hum. Genet. 51:1150-1155. Passos-Bueno, M. R., Oliveira, J. R., Bakker, E., Anderson, R. D., Marie, S. V., Vainzof, M., Roberds, S., Campbell, K. P., and Zatz, M. (1993). Genetic heterogeneity for Duchenne-like muscular dystrophy (DLMD) based on linkage and 50 DAG analysis. Hum. Mol. Genet. 2:19451947. Pearce, M., Blake, D. J., Tinsley. J. M., Byth, B. C., Campbell, L., Monaco, A. P., and Davies, K. E. (1993). The utrophin and dystrophin genes share similarities in genomic structure. Hum. Mol. Genet. 2:1765-1772. Perloff, J. K. (1984). Cardiac rhythm and conduction in Duchenne’s muscular dystrophy: A prospective study of 20 patients. J. Am. Coll. Cardio. 3:1263-1268.

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Anthony P. Monaco Imperial Cancer Research Fund Laboratories Institute of Molecular Medicine John Radcliffe Hospital Headington, Oxford OX3 9DU United Kingdom

Jamel Chelly Institut Cochin de Genetique Moleculaire INSERM Unit6 129 CHU Cochin-Port-Royal 75014 Paris, France

1. COPPER HOMEOSTASIS Copper is a heavy metal ion essential for the activity of a variety of enzymes in the body. These include cytochrome oxidase in the electron transport chain, superoxide dismutase for free radical detoxification, tyrosinase and dopamine P-hydroxylase for production of melanin and catecholamines, respectively, lysyl oxidase for cross-linking collagen and elastin, and an unknown enzyme responsible for the cross-linking of keratin. In excess, copper is a very toxic ion, therefore an efficient regulation of its metabolism is required. Copper homeostasis, for the most part, is a balance between absorption from dietary sources across the intestine and excretion in the bile, since urinary excretion is negligible. The liver plays a pivotal role in copper homeostasis since most intestinally absorbed copper is received there and the bitiary excretion of copper is intimately connected to the liver. Menkes and Wilson diseases are examples of genetic disorders in which copper homeostasis is defective leading to copper deficiency and toxicity, respectively. Albumin is the major carrier of copper to the liver after transport across Advances in Genetics, Vol. 33 Copyright 0 1995 hy Academic Press. Inc.

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the intestine and uses the N-terminal tripeptide Asp- Ala-His in human for copper binding (Danks, 1989). Ceruloplasmin is the major copper carrier (>95%) in the blood, synthesized mostly in the liver, secreted into the serum, and can bind six atoms of copper. It is thought to have ferrous oxidase activity as well as possible roles in tissue angiogenesis, coagulation, and acute phase reactions to inflammation, infection, and injury where it could potentially act as an antioxidant. The ferrous oxidase activity of ceruloplasmin may be important for the oxidation of ferrous iron to the ferric form which can then be released from ferritin and transferred to transferrin. Recently, a duplication or rearrangement in the 5’ end of the ceruloplasmin gene has been associated with dominantly inherited epilepsy in epileptic mice with enhanced expression of liver ceruloplasmin mRNA and oxidase activity (Garey et al., 1994). In human, the ceruloplasmin gene maps to chromosome 3q23-q25 and recently mutations have been associated with systemic hemosiderosis (Yoshida et al., 1995). Metallothionein is thought to bind excess heavy metals, including copper, inside the cell and can be upregulated upon heavy metal overload. Recently, a gene involved in the transport of copper across the plasma membrane has been identified in yeast (Dancis et al., 1994). This protein, called CTRl, is a multispanning membrane protein that contains a methionine- and serine-rich domain with 11 copies of the sequence Met-X-X-Met, also found in many bacterial proteins involved in copper transport. Surprisingly, mutants in CTRl were also found to have a ferrous ion uptake deficiency, indicating a close relationship between copper and iron transport. Askwith et a!. (1994) showed that the ferrous ion uptake deficiency was secondary and due to aberrant activity of the copper-containing protein, FET3, which had a cytosolic ferrooxidase activity. Ferrous iron is transported inside the cell by a ferrous transporter (not yet identified) and then oxidized to the ferric form by the copper-requiring FET3 protein, thus keeping it within the cell. There is sequence similarity between the FET3 ferrous oxidase and ceruloplasmin and other enzymes (laccases, ascorbate oxidase) with multicopper oxidase activity (Askwith et al., 1994). With the identification of human or animal homologues of CTRl it should be interesting to see if copper transport across the plasma membrane in mammalian cells utilizes a similar mechanism. For example, a copper deficiency in swine has shown profound alterations in iron homeostasis resulting in anemia (Lee et al., 1968). However, little is known about the transport of copper inside mammalian cells to the enzymes which require copper for their activity. Clues to this process have recently come from the isolation of the genes responsible for Menkes disease and Wilson disease (Table 7.1). These genetic disorders exhibit copper deficiency and copper toxicity phenotyes, respectively, yet genes responsible for these disorders encode very similar copper-transporting P-type ATPases.

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7. Menkes and Wilson Diseases Table 7.1. Comparison of Menkes Disease and Wilson Diseased Menkes disease Location Clinical

Lab findings

Cultured cells Defect

Xq13 .H/recessive Onset a t birth Cerebral degeneration/MR Abnormal hair and facies Hypopigmentation Bone changedcutis laxa Arterial rupturelthrombosis Hypothermia Death <3 years Decreased serum C u Decreased serum ceruloplasmin Increased intestinal/kidney C u Decreased liver C u Increased Cu accumulation Decreased Cu release Placental/Intestinal Cu transport Deficiency of Cu-dependent enzymes

Treatment

No effective treatment

Animal models Gene product

Mottled mouse (Mo) Cu-binding P-type ATPase

Expression Mutation

All tissues except liver 16% deletions, remainder probably point mutations

Wilson disease 13q14.3/recessive Onset late childhood Liver disease Loss of coordination Involuntary movements Dysarthria Kayser-Fleischer rings (cornea)

Decreased serum C u Decreased serum ceruloplasmin Increased urinary Cu Increased liver Cu Normal in most patients Biliary C u excretion

Cu incorporation into holocerulo, plasmin Chelating agents: penicillamine, zinc salts LEC rat Cu-binding P-type ATPase (60% identity with MNK) Liver, kidney, and placenta Point mutations Small deletion

aAdapted from Chelly and Monaco (1993).

This chapter discusses the clinical aspects of both diseases, the steps of positional cloning taken to isolate and characterize the genes responsible for each disease, and animal models which have been implicated.

II. MENKES AND WILSON DISEASES: CLINICAL FINDINGS For a detailed review of the clinical picture of Menkes and Wilson diseases the reader is referred to Danks (1989) and for Menkes disease to Horn et ul. (1992). Some of the distinctive clinical, biological, and molecular features of these diseases are listed in Table 7.1.

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A. Menkes disease Menkes disease was first described by J. H. Menkes and associates (1962) with reference to the peculiar “steely” hair, focal cerebral and cerebellar degeneration, and sex-linked recessive inheritance. The major clinical findings are severe mental retardation, seizures, growth retardation, characteristic facies, skeletal changes, variable hypothermia, peculiar depigmented kinky hair (pili torti), and susceptibility to infections. The pathological findings include widespread arterial tortuosity and lumen changes such as fragmentation of the internal elastic lamina and thickening of the intima. Death usually occurs before the age of 3 years. The incidence of Menkes syndrome was originally estimated to be 1/35,000 (Danks et al., 1972) but more recent estimates have shown it to be a very rare disorder with 1/298,000 liveborn babies affected (Tmnesen et al., 1991). There is some clinical variability of patients with Menkes disease with less severe forms being reported (Procopis et al., 1981; Gerdes et al., 1988; Westman et al., 1988). The mildest form is occipital horn syndrome with defects in connective tissue manifesting in bladder diverticula, inguinal hernias, skin laxity and hyperelasticity, and several skeletal abnormalities with the most prominent being occipital horn-like exostoses (Peltonen et al., 1983). Occipital horn syndrome was proposed to be allelic to Menkes syndrome due to similarities in copper findings (see below) and connective tissue abnormalities. Recently, Horn et al. (1994) completed a survey of 428 Menkes patients and divided the clinical forms into six subdivisions based on criteria of mental retardation, neurological symptoms, life span, and response to parenteral copper therapy (see below). The major biochemical defect in Menkes disease is copper deficiency which was discovered by Danks et al. (1972). They found low levels of serum copper and ceruloplasmin and demonstrated a defect in the intestinal absorption of copper. The copper deficiency would have its largest impact on the activity of enzymes requiring copper and many of the clinical and pathophysiological features of Menkes disease can be understood as secondary deficiencies of copper requiring enzymes (see Horn et d., 1992). For example, many of the connective tissue and vascular manifestations of the disease are most likely due to a deficiency in the copper-requiring enzyme lysyl oxidase which is responsible for the cross-linking of collagen and elastin. The study of cells in culture from Menkes patients showed an increase in copper accumulation over a 20-hr period when compared to normal control fibroblasts (Horn, 1976). Prenatal diagnosis of pregnancies at risk for Menkes disease involved testing for copper accumulation in amniotic fluid cells (Horn, 1976). Over 250 prenatal diagnoses and 220 postnatal diagnoses have been performed by the Denmark group using 64-Cu uptake (Horn et al., 1992). Detailed studies of copper uptake into Menkes disease and normal lymphoid

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cells in culture have shown that both initial uptake and efflux rates are normal (Herd et al., 1987). This suggests that the copper accumulation defect does not involve the transport of copper across the plasma membrane but is most likely due to a disturbance of intracellular transport and incorporation into copperrequiring enzymes. It has also been demonstrated that the induction of metallothionein by copper in both muscle cells and fibroblasts from Menkes patients is higher than that of control cells (Herzberg et al., 1990). In general, copper therapy has not been shown to alter the course of the disease and the neurological findings present at birth are most likely not reversible. However, a recent review has indicated that subcutaneous administration of copper-histidine had some benefit with respect to neurological symptoms in two patients treated at less than 1 month of age (Sarkar et al., 1993).

B. Wilson disease Wilson disease or hepatolenticular degeneration was originally described by Wilson in 1912. Wilson disease is inherited as an autosomal recessive disorder and was shown in the 1940s to be due to excess copper accumulating in the liver and lenticular nucleus of the brain. Most patients present with liver disease or neurologic symptoms, but other presenting symptoms can occur (Danks, 1989). The liver disease usually manifests itself between the ages of 8 and 16 years with symptoms such as acute jaundice, vomiting, and malaise. Neurologic symptoms are rare before the age of 12 years and can include dysarthria, deterioration of coordination of voluntary movements, production of involuntary movements, and problems with posture and muscle tone (Danks, 1989). Other problems include disorders of bones, joints, and renal stones. The main clinical diagnostic signs are hepatic cirrhosis and Kayser-Fleischer corneal rings due to copper accumulation. The incidence of Wilson disease has been estimated from 1/ 35,000 in Sardinia to 1/50,000-1/100,000 in Melbourne (Danks, 1989). Laboratory findings in Wilson disease include increased urinary copper and decreased serum copper. Scheinberg and Gitlin (1952) first described a deficiency of ceruloplasmin in patients with Wilson disease. In general, Wilson disease has two major disturbances of copper homeostasis, a reduction in copper accumulation into ceruloplasmin and in biliary excretion of copper. Liver biopsy to detect increased copper content is often performed, especially in patients with a history of chronic liver disease. In contrast to Menkes disease, the accumulation of 64-Cu in cultured cells from patients with Wilson disease is not a reliable test for the disease (Danks, 1989). However, despite the lack of knowledge about the primary defect, an effective therapy using chelating agents, such as penacillamine, for the copper toxicity in Wilson disease was introduced by Walshe in 1956 and is still the main treatment (Walshe, 1973).

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111. MOLECULAR GENETICS A. Menkes disease 1. Linkage analysis In families segregating X-linked Menkes disease, linkage analysis was performed with increasing levels of refinement for the disease location (Figure 7.1). A proximal short-arm localization was suggested in the first DNA polymorphic studies using restriction fragment length polymorphisms (RFLPs) as genetic markers (Wienker et al., 1983). Horn et al. (1984) showed a cytogenetic C-banded polymorphism of the X chromosome centromere that was tightly linked in five Danish families segregating Menkes disease suggesting a pericentromeric location for the disease locus. The most recent genetic analysis using 11 RFLPs from the X chromosome and multipoint linkage analysis gave the best location between markers in Xq12 to Xq21 (Tonnesen et al., 1992).

DXS56

breakpoint region

[

A !

PGKl

Figure 7.1. Schematic drawing depicting the increased levels of refinement for localizing the Menkes disease gene on the human X chromosome. XIST is X-inactive specific transcript gene and PGKI is phosphoglycerate kinase gene 1.

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2. Chromosome rearrangements Genetic linkage analysis had narrowed the Menkes disease locus between Xq12 and q21 (Figure 7.1). Considering the number of deletions of the Xq21 region in male patients that do not exhibit Menkes disease, the location was therefore suggested to be in Xq12-q13.3. The most convincing evidence for the location of the Menkes disease gene was a female Menkes disease patient with an X; autosome translocation and the karyotype (46,X,t(X;2)(913;q32.2) (Kapur et a!. , 1987). The translocation in Xq13 most likely disrupted the Menkes disease gene and nonrandom inactivation of the normal X chromosome in this female patient gave rise to the disease. Molecular analysis of the translocation chromosome in somatic cell hybrids placed the breakpoint in the subregion Xq13.3 proximal to the gene for phosphoglycerate kinase (PGKI) and distal to a cluster of anonymous DNA markers (DXS128, DXS56, DXS356, DXS171, DXS441, DXS325, DXS347) (Verga et at., 1991). This localization was confirmed in two other Menkes disease patients who exhibited cytogenetic rearrangements of the Xq13.3 region (Figure 7.1). One male patient with Menkes disease had an insertion of the long-arm segment Xq13.3-$1.2 into the short arm of his X chromosome at band Xpll. 4 giving a karyotype 46,XY, ins(X)(pll.4;ql3.3qZl. 2) (Turner et at., 1992a). It was assumed that the breakpoint in Xq13.3 involved in the insertion disrupted the Menkes disease gene. A recent case of a second female patient with Menkes disease and an X; 1 translocation breakpoint reinforced the cytogenetic evidence that the Menkes disease gene is located in Xq13.3 (Beck et al., 1994).

3. Positional cloning The isolation of genes for inherited diseases based only on their chromosome location without prior knowledge of the defective protein is termed “positional cloning” (Collins, 1992). Positional cloning of the gene responsible for Menkes disease was based mostly on the information provided from the cytogenetic rearrangements in Xq13.3 associated with the disease (Figure 7.1). The study by Verga et al. (1991) showed that the X;2 translocation breakpoint had occurred in Xq13.3 near the gene for PGK1. Turner et at. (1992b) isolated cloned DNA from this region in yeast artificial chromosomes (YACs) and constructed a contig of overlapping YACs covering 1.0 Mb of DNA including the loci for PGKl and DXS56 (Figure 7.1). Using the large YAC inserts as probes in fluorescence in situ hybridization (FISH) after suppression of repetitive sequences, Tumer et al. (1992b) showed that both the X;2 translocation breakpoint in the female Menkes disease patient and the Xq13.3 insertion breakpoint in the male Menkes disease patient were located in a region of about 100 kb within the overlapping

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region of YACs positive for DXS56 and PGK1. Similarly, using the same FISH technique of YAC clones to metaphase spreads of patient chromosomes, Mercer et al. (1993) localized the X;1 translocation breakpoint within 300 kb of PGK1. The location of the chromosome rearrangements to within a 100-kb region near PCKl (Figure 7.1) set the stage for three groups to isolate expressed sequences that were part of the gene responsible for Menkes disease (Vulpe et al., 1993a; Chelly et al., 1993; Mercer et al., 1993). All three groups chose to subclone the YACs, which spanned the chromosome rearrangements, into smaller insert bacteriophage clones. Small genomic clones surrounding the translocation and insertion breakpoints were identified and cDNA clones isolated. Vulpe and colleagues (1993a) used two independent methods to find the Menkes disease gene. They isolated a 100-kb Sjil fragment from the YAC and hybridized it after suppression of repetitive sequences to a placental cDNA library. A 3-kb cDNA clone was identified that turned out to be mostly 3’ untranslated sequence. They also used the small insert phage subclones from the YAC in a technique to amplify exons based on their surrounding consensus splice sites after expression in COS cells. The method termed “exon amplification” (Buckler et al., 1991) resulted in the isolation of two potential exons that were then used to isolate more cDNA clones. Chelly et al. (1993) and Mercer et al. (1993) both hybridized single-copy probes near the chromosome breakpoints directly to kidney cDN A libraries to isolate cDNA clones that were candidates for the Menkes disease gene. The cDNA clones were hybridized to Northern blots by all three groups and shown to detect an 8.5-kb transcript in poly-A+ RNA from heart, brain, lung, muscle, kidney, pancreas, placenta, and fibroblasts. Notably, there was little or no mRNA detected in liver, a tissue which does not accumulate copper in Menkes disease (Darwish et d., 1983). When RNA isolated from fibroblast cell lines from Menkes patients was tested, both Vulpe et al. (1993a) and Mercer et al. (1993) found little or no expression of the Menkes candidate gene in most samples. Proof that the cDNA clones isolated were part of the Menkes disease gene came from deletions detected by Chelly et al. (1993) in 16 out of 100 unrelated Menkes disease patients. These deletions had breakpoints that in some cases were nonoverlapping and spread across the genomic exon-containing fragments. Vulpe et al. (1993a) also found genomic deletions in 4 out of 26 unrelated Menkes patients. Recently, Tumer et al. (1994) performed a first trimester prenatal diagnosis of Menkes disease based on direct detection of a partial gene deletion. Point mutations in nondeletion cases detected using RTPCR of Menkes gene has recently been reported for both Menkes and occipitalhorn syndrome patients (Das et al., 1994, 1995; Kaler et al., 1994). The genomic organization of the Menkes disease gene has been determined and contains 23 exons spanning approximately 150 kb (Tumer et al., 1995).

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4. Sequence and protein analysis Sequences near the 5' end of the Menkes disease gene were shown to contain six repeated motifs of about 30 amino acids that contained the almost invariant sequence GMTCXXC. This motif was very similar to putative heavy metalbinding domains found in bacterial genes conferring resistance to heavy metals such as mercury and cadmium. However, while most of the bacterial heavy metal resistance genes had one copy of this motif, the Menkes disease gene had six copies that were spaced fairly evenly over the first 630 amino acids. Vulpe et al. (1993a) had isolated overlapping cDNA sequences from the entire Menkes disease gene and predicted an open reading frame of 4500 bp encoding a protein of 1500 amino acids. Protein sequence homology searches showed that the gene for Menkes disease had significant similarity to a family of cation-transporting ATPases of the P type (Figure 7.2). This family has several transmembrane

r I I

ATP

BINDING

c c

I -y? -@:*A

I V'

I I

Figure 7.2. Schematic drawing depicting the copper transporting P-type ATPase of Menkes and Wilson diseases. There are six copper-binding motifs near the N terminus followed by four transmembrane domains, a phosphatase (TGEA) domain, a transduction (CPC) domain in transmembrane segment 6, an aspartyl phosphorylation residue, the conserved motifs in the ATP-binding domain (TGDN . . . PSHK . . . VGDG(VI1)NDSPAL), followed by two final transmembrane domains.

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domains and uses an aspartate residue that is transiently phosphorylated by ATP to transport cations through the membrane. The Menkes disease gene is thought to contain eight potential transmembrane domains (Vulpe et al., 1993b). The closest relatives to the human Menkes ATPase are the copper-exporting ATPases from Enterococcus h i m operon CopAB (Odermatt et al., 1993). The CopA gene is thought to be involved in the uptake of copper and the CopB gene in the efflux of copper from the gram-positive bacterium. A recent review of the homology of the human Menkes disease gene and bacterial cation transporting ATPases can be found in Silver et al. (1993).

B. Wilson disease 1. Linkage analysis and linkage disequilibrium Genetic linkage studies of families segregating Wilson disease in an autosomally recessive inheritance pattern showed linkage on chromosome 13 to the red cell enzyme, esterase D (Frydman et al., 1985; Bonn6-Tamir et al., 1986). Further analysis using RFLP-detecting DNA markers from chromosome 13 narrowed the location of Wilson disease locus to 13q14.3 (Figure 7.3; Bowcock et al., 1987, 1988; Yuzbasiyan-Gurkan et al., 1988). Multipoint linkage analysis was used to position the Wilson disease locus between the markers D13S31 and D13S59 in a region of approximately 1.6 cM (Farrer et al., 1991). D13S31 was located only 0.4 cM proximal to the Wilson disease locus and showed allelic association in both Dutch families and Canadian families of northern European origin, indicating its closeness to the gene (Houwen et al., 1992; Thomas et al., 1993). More genetic markers of short tandem repeat polymorphisms (microsatellites) have been isolated in the region from YAC clones positive with D13S31 so that the combination of genetic markers has a higher predictive value for the disease (Stewart et a[., 1993).

2. Positional cloning Genetic linkage studies narrowed the Wilson disease region to 13q14 near the marker D13S31 (Figure 7.3). New probes have been isolated from this region using Alu-PCR of radiation hybrids containing 13914 and from flow-sorted chromosome 13 libraries (Bull et al., 1993a). These markers, as well as the existing markers D13S31 and D13S59, were used to construct a long-range physical map of genomic DNA in the region covering about 4 Mb (Bull and Cox, 1993). The region has also been isolated in overlapping YACs and cosmid clones covering over 2 Mb (Figure 7.3; Petrukhin et al., 1993; Bull et al., 1993b). Several groups have isolated a series of new microsatellite genetic markers across the region from YAC and cosmid clones and used them to study

243

7. Menkes and Wilson Diseases YACs P - 12 11.2 13

11 1

D13SZ94

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13ql4.2

D13S31

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12 1 12.2

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12.3

D13S25 14.1 14 2

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D13S133 D13S59

D13S296

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D13S133 D13S297

f

013.5298

D13S119 136114.3

D13S301

Wilson disease gene (ATP7B)

Dl35299

'

1 1I

D13S300 Dl3559

Figure 7.3. Schematic drawing indicating the increased levels of refinement for localizing the Wilson disease gene on human chromosome 13. The order of markers on the YAC contig is taken from Petrukhin et al. (1993). RBI is the retinoblastoma gene 1.

linkage disequilibrium and haplotypes in Wilson disease families from Canada, America, Sardinia, the Middle East, East Asia, and Russia (Petrukhin et al., 1993; Thomas et al., 1994; Bowcock et al., 1994). The allelic association and haplotype analysis indicated a central region of several 100 kb where the disease gene was located. At this point, the positional cloning of the Wilson disease gene could have taken the more traditional approach of searching for expressed sequences via cDNA library screening or exon amplification using genomic subclones from the 13q14 critical region. In contrast to Menkes disease, there were no chromosome rearrangements that pinpointed exactly where the disease gene was disrupted, although linkage disequilibrium and haplotype analysis had narrowed the region quite significantly. Because of the similarity between the two diseases exhibiting a basic defect in copper homeostasis, it was suggested by Vulpe et al. (1993a) that a similar copper-transporting ATPase expressed in liver, where the Menkes disease gene was notably not expressed, could be responsible for Wilson disease. This hypothesis turned out to be correct and two groups isolated the

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Wilson disease gene based on low-stringency hybridization of the copper-binding motif of the Menkes disease gene (Bull et al., 199310; Yamaguchi et d., 1993). Bull et al. (199333) hybridized the Menkes disease heavy metal-binding motif to YAC and cosmid clones from the critical region and then used a PCR-based cDNA selection strategy to isolate liver cDNA clones that hybridized at low stringency with the heavy metal-binding sequence. Yamaguchi et al. (1993) hybridized the heavy metal-binding sequences from the Menkes disease gene directly to a liver cDNA library at low stringency and isolated positive clones. In contrast, Tanzi et al., (1993) used a degenerate oligonucleotide corresponding to a novel heavy metal-binding motif in the AP region of the amyloid P-protein precursor to isolate cDNA clones from a brain cDNA library. Interestingly, they isolated a 3.5-kb cDNA clone with strong homology to the human Menkes disease gene, although the AP-binding motif was not actually present in the predicated protein of the Wilson disease gene since it is out of frame. The cDNA clones isolated by all three groups with strong homology to the Menkes disease gene were then shown to map in the correct region of 13q14. To prove that they were good candidate genes, the coding sequence was analyzed for mutations and four disease-specific mutations were found by Petrukhin et al. (1993), including two frameshifts and two transversions that were closely correlated to the Wilson disease haplotypes. Bull et d. (199313) found a 7-bp deletion in one Wilson disease patient that resulted in a shift of the open reading frame. Recently, Thomas et al. (1995) have characterized a spectrum of 23 mutations in Wilson disease patients that provide an explanation for the wide phenotypic variation seen in the disease. The cDNA clones were found to hybridize to a 7.5-kb transcript in polyA+ RNA from liver, kidney, and placenta. Analysis of liver RNA from two Wilson disease patients showed the absence or marked reduction in transcript levels (Yamaguchi et al., 1993).

3. Sequence and protein analysis The full-length cDNA sequence (Bull et al., 1993b) predicted a 1411-amino acid protein with strong similarity to other cation-transporting P-type ATPases (Figure 7.2). The best homology is with the Menkes disease gene with 54% amino acid identity overall and higher levels of homology (>78%) in specific domains (phosphatase, transduction-phosphorylation,ATP binding). The official gene names have now been designated ATP7A for the Menkes disease gene and ATP7B for the Wilson disease gene. They both contain six heavy metalbinding domains with 65% homology to each other. There was a small hydrophobic region in the fourth copper-binding domain of ATP7B which was suggested by Tanzi et al. (1993) to potentially cross the membrane. Tanzi et d. (1993) found only five heavy metal-binding motifs in their brain cDNA clone compared to six found in the liver cDNAs of Bull et al. (199313). Further analysis

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using PCR-based mRNA amplification of polyA+ RNA from liver and brain showed differences due to alternative splicing of exons 6, 7, 12, 13, and 17 that were specific to brain mRNAs (Petrukhin et al., 1994). All the exon:intron borders of the ATP7B gene have been sequenced and it is distributed in 21 exons with an alternatively spliced 3’ end in exon 22 (Petrukhin et al., 1994; Thomas et

al., 1995).

IV. ANIMAL MODELS A. Menkes disease and mottled mice The mouse model of Menkes disease, mottled, was first described by Fraser et al. (1953) as variegation of light and dark coat color in female heterozygous mice and lethality in male mutant mice. The mottled coat color in female heterozygous mice was important evidence for the hypothesis put forth by Lyon (1961) for X-chromosome inactivation. There are a number of mottled alleles with differing phenotypes and severity (Davisson, 1987). The best characterized are mottled (Mo), which is male lethal in utero, mottled brindled (Moh), which is severe and lethal by 14 days postpartum, mottled dappled (Mode), which is male lethal in utero and has skeletal defects, mottled viable-brindled (Movh), which has reduced viability and is sterile, and mottled blotchy (Mobb), which has connective tissue defects but is viable and sterile. The mottled mice were first shown to have a defect in copper metabolism with decreased brain levels of copper and abnormal accumulation of copper in intestinal cells suggesting a lack of transport across the gut into the blood stream and of incorporation into copper-requiring enzymes (Hunt, 1974). Rowe et al. (1974) also showed that, similar to Menkes disease, mottled mice had defects in cross-linking collagen and elastin giving rise to connective tissue and arterial abnormalities. The copper transport defect seen in cultured cells from Menkes disease patients was also shown in cultured mottled mouse cells with accumulation of copper (Sayed et al., 1981; Packman, 1987). However, the copper accumulation in hepatocytes from both Menkes patients and mottled brindled mice was shown to be normal (Darwish et al., 1983). Therefore, mottled represents a good mouse model for Menkes disease. The mottled locus was mapped very close to Pgkl on the mouse X chromosome in the conserved linkage group from the androgen receptor to collagen 4A5 (Brown et d.,1993). Interspecific backcross analysis and pulsedfield gel electrophoresis using human Menkes disease cDNA clones as hybridization probes enabled George et al. (1994) to position the mouse locus, Mnk, proximal to and within 150-200 kb of mouse Pgkl. Both Levinson et al. (1994) and Mercer et al. (1994) isolated the complete coding region of the Menkes

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disease gene in mice and showed that it has a 4473-bp open reading frame encoding a predicted 1491-amino acid protein. The nucleotide sequence in the open reading frame is 88% identical and predicts an 89% identical protein with strong homology to the E. hirae CopA and CopB genes. The most striking difference between the human and mouse Menkes sequence is an 8-amino acid deletion just before the fifth heavy metal-binding domain. George et al. (1994) were unable to find any structural alterations, such as deletions or insertions, in nine mottled mutants at the Mnk locus. Two other groups (Levinson et al., 1994; Mercer et al., 1994) reported that the mottled dappled mutant had an internal deletion of the Mnk genomic locus and no expression of Mnk mRNA by Northern blot analysis. Both groups found that the mottled blotchy mouse had two larger transcripts by Northern blot analysis indicating a likely defect in RNA splicing. Although the RNA defects seen by these two groups provide quite convincing evidence that mottled is indeed due to defects at the Mnk locus, Reed and Boyd (1994) pointed out that the genomic DNA alterations described in mottled dappled are also seen in other strains of mice. George et al. (1994) were unable to detect any genomic rearrangements in mottled dappled and the altered size-restriction fragments seen by Levinson et al. (1994) and Mercer et al. (1994) appear to be due to restriction fragment length variations present in laboratory strains of mice (Reed and Boyd, 1994). More recently, Das et al. (1995) have characterized a splice-donor mutation in mottled blotchy that confirmed the RNA splicing defect giving rise to the phenotype.

B. Wilson disease and LEC rats The toxic milk mouse and the Bedlington terrier were previously considered to be potential animal models for Wilson disease due to copper abnormalities, especially in the liver. In toxic milk mice, copper deficiency is seen in pups fed milk from their mother yet copper accumulation in the liver with hepatic copper toxicosis is seen in pups fed milk from normal female mice (Rauch, 1983; Biempica et al., 1988). Most Bedlington terriers have copper accumulation in the liver but normal levels of ceruloplasmin in blood and no neurological effects (Hardy et al., 1975; Su et al., 1982). More recently, inbred Long-Evans Cinnamin (LEC) rats have been studied as a potential animal model for Wilson disease. LEC rats have an autosoma1 recessive inheritance of hepatitis, with eventual development of hepatocellular carcinoma after 1 year (Sasaki et al., 1985). Li et al. (1991a,b) have shown increased copper in the liver of LEC rats and decreased levels of serum copper and ceruloplasmin. Further studies of ceruloplasmin in LEC rats showed normal levels of mRNA and protein by Northern and Western blot analyses, but no radioactive copper accumulation (Yamada et al., 1993; Kojimahara et al., 1994). Using a monoclonal antibody to the active site of ceruloplasmin, (i.e., it

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could block its ferroxidase activity) Hiyamuta and Takeichi (1993) showed that LEC rats had no hepatocyte staining for active ceruloplasmin. Using a different assay involving quantitation on polyacrylamide gel electrophoresis of ceruloplasmin lacking ferroxidase activity, Kojimahara et al. (1994) showed that the inactive form of ceruloplasmin was predominant in LEC rats. These studies of ceruloplasmin in the LEC rat suggested that it was a very good animal model for Wilson disease. With the cloning of the Wilson disease gene it was possible to directly test whether the LEC rat has a mutation in the homologous rat gene. Yamaguchi et al. (1994) isolated a rat cDNA clone for the human Wilson disease gene and showed by Northern blot analysis that it was riot expressed in LEC rat tissues, including liver, kidney, spleen, and stomach. Wu et al. (1994) also cloned rat cDNAs for the entire coding region of the human Wilson disease gene and showed that there was approximately 80% amino acid sequence identity between the rat and human genes. There was a deletion, however, of the fourth heavy metal-binding motif in the predicted rat protein sequence. Using the rat cDNAs and RT-PCR of polyAf liver RNA they detected a partial deletion in the Atp7b gene in LEC rats. The deletion removes at least 900 bp of the coding region near the 3' end of the gene including the ATP-binding domain and extends downstream of the gene. The LEC rat seems to be a deletion mutation which decreases the transcript level and would be predicted to produce a truncated protein. Therefore, the LEC rat is a good model for Wilson disease and should provide insights into the pathophysiology of the disease as well as the role of the Atp7b gene in copper transport.

V. FUTURE CONSIDERATIONS The molecular understanding of Menkes and Wilson disease genes by positional cloning has unraveled an interesting new family of human copper transporters of the P-type ATPase. Although only two have been isolated, there could possibly be many more that are involved in the transport of other heavy metals besides copper as seen in bacteria. In S. cerevisiae, a plasma membrane copper transporter has been isolated (Dancis et al., 1994) and a yeast copper-transporting P-type ATPase (ccc2) homologous to the human Menkes and Wilson diseases has been identified (Beeler et al., 1994). These yeast genes should be important for understanding the functional relationship of the various proteins involved in copper transport across the plasma membrane and within the cell. Recently, the Menkes-Wilson disease gene homology in yeast (ccc2) has been shown to be required for the export of copper from the cytosol into an extracytosolic compartment, and ccc2 provides copper to a ceruloplasminlike oxidase required for iron uptake (Yuan et al., 1995).

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It remains to be seen where in the cell the Menkes and Wilson disease proteins are localized and how they function to transport copper to the enzymes that require it for their activity. Antibodies specific for each protein will be used to pinpoint the subcellular localization. It will also be interesting to see if the same cell in a tissue can express both genes simultaneously and this could be assessed using RNA in situ hybridization or specific antibodies in immunohistochemical studies. Other outstanding questions are whether these genes can provide copper-transporting function when overexpressed in cells in which they are not normally transcribed. Full-length coding cDN As cloned in expression vectors for each gene and transfected into COS cells and cell lines from patients should be used. Studies on cell lines from patients with Menkes disease or from mottled mice will reveal if it is possible to complement the mutant defect by transfecting the Menkes disease gene and also see if the Wilson disease gene can complement the copper-accumulation defect as well. Similar experiments could also be done for Wilson disease and LEC rat cell lines. Hopefully, the information gained from such experiments will benefit the diagnosis and therapy of patients with these inherited diseases.

Acknowledgments We thank Drs. Yvonne Boyd and Francesco Giannelli for helpful comments on the manuscript.

References Askwith, C., Eide, D., Van Ho, A., Bernard, P. S., Li, Liangtao, Davis-Kaplan, S., Sipe, D. M., and Kaplan, J. (1994). The FET3 gene of S. cerewisiae encodes a multicopper oxidase required for ferrous iron uptake. CeU (Cambridge, Mass.) 76:403-410. Beck, J., Enders, H., Schliephacke, M., Buchwld-Saal, M., and Turner, Z. (1994). X;1 translocation in a female Menkes patient: Characterization by fluorescence in situ hybridization. Clin. Genet. 46:295-298. Beeler, T., Fu, D., and Dunn, T. (1994). Identificationof an S. cerewisiae copper transporting P-type ATPase which is the homolog of the human Menkes’ and Wilson’s genes. FASEB J. 8:A1454. Biempica, L., Ranch, H., Quintana, N., and Stemlieb, I. (1988). Morphological and chemical studies on a murine mutation (toxic milk mice) resulting in hepatic copper toxicosis. Lab. huest.

59:500-508.

Bonn&-Tamir, B., Farrer, 1.A., Frydman, M., and Kannaane, 1.H. (1986). Evidence for linkage between Wilson’s disease and esterase D in three kindreds: Detection of linkage for an autosomal recessive disorder in the family study method. Genet. Epidemiol. 3:201-209. Bowcock, A. M., Farrer, L. A., Cavalli-Sfona, L. L., Hebert. J. M., Kidd, K. K., Frydman, M., and Bonn6-Tamir, B. (1987). Mapping of the Wilson disease locus to a cluster of linked polymorphic markers on chromosomes 13. Am. J. Hum. Genet. 41:27-35. Bowcock, A. M. Farrer, L. A., Hebert, J. M., Agger, M., Stemlieb, I., Scheinberg, I. H., Buys, C. H. C. M., Scheffer, H.,Frydman, M., Chajek-Saul, T., Bonn6-Tamir, B.,and Cavalli-Sfona, L. L. (1988). Eight closely linked loci place the Wilson disease locus within 13q14-qZl. Am. J. Hum. Genet. 43:664-674.

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Bowcock, A. M., Tomfohrede, J., Weissenbach, J., Bonn&-Tamir,B., St George-Hyslop, P., Giagheddu, M., Cavalli-Sforza, L. L., and Farrer, L. A. (1994). Refining the position of Wilson disease by linkage disequilibrium with polymorphic microsatellites. Am. J. Hum. Genet. 54:7987. Brown, S. D. M., Avner, P., Boyd, Y., Chapman, V., Rastan, S., Sefton, L., Thomas, J. D., and Herman, G. E. (1993). The mouse X chromosome. Mamm. Genome 4:269-281. Buckler, A. J., Chang, D. D., Craw, S. L., Brook, J. D., Haber, D. A., Sharp, P. A., and Housman, D. E. (1991). Exon amplification: A strategy to isolate mammalian genes based on RNA splicing. Proc. Natl. Acad. Sci. U.S.A. 88:4005-4009. Bull, P. C., and Cox, D. W. (1993). Long range restriction mapping of 13q14.3 focused on the Wilson disease region. Genomics 16693-598. Bull, P. C., and Cox, D. W. (1994). Wilson disease and Menkes disease: New handles on heavymetal transport. Trends Genet. 10:246-252. Bull, P. C., Barwell, J. A., Hannah, H. T.-L., Pautler, S. E., Higgins, M. J., Lalande, M. J., and Cox, D. W. (1993a). Isolation of new probes in the region of the Wilson disease locus, 13q14.214.3. Cyrogenet. CeU Genet. 64:12-17. Bull, P. C . , Thomas, G. R., Rommens, 1. M., Forbes, J. R., and Cox, D. W. (1993b). The Wilson disease gene is a copper transporting P-type ATPase similar to the Menkes gene. Nat. Gener. 5:327-337. Chelly, J., and Monaco, A. P. (1993). Cloning the Wilson disease gene. Nat. Genet. 5:317-318. Chelly, J., Turner, Z., Tonnesen, T., Petterson, A., Ishikawa-Brush, Y., Tommerup, N., Horn, N., and Monaco, A. P. (1993). Isolation of a candidate gene for Menkes disease that encodes a potential heavy metal binding protein. Nar. Genet. 3:14-19. Collins, F. S. (1992). Positional cloning: Let’s not call it reverse anymore. Nat. Gener. 1:3-6. Dancis, A., Yuan, D. S., Haile, D., Askwith, C., Eide, D., Moehle, C., Kaplan, J., and Klausner, R. D. (1994). Molecular characterization of a copper transport protein in S. cerevisiae: An unexpected role for copper in iron transport. Cell (Cambridge, Mass.) 76:393-402. Danks, D. M. (1989). Disorders of copper metabolism. In “The Metabolic Basis of Inherited Disease” (C. Scrives, A. L. Beaudet, W. S. Sly, and D. Valle, eds.), 6th ed., pp. 141 1-1431. McGraw-Hill, New York. Danks, D. M., Campbell, P. E., Stevens, B. J., Mayne, V., and Cartwright, E. (1972). Menkes’ kinky hair syndrome-an inherited defect in copper absorption with widespread effects. Pediatrics 50:188-201. Danvish, H. M., Hoke, 1. E., and Ettinger, M. J. (1983). KineticsofCu(l1) transport and accumulation by hepatocytes from copper-deficient mice and the brindled mouse models of Menkes disease. J. Bid. Chem. 258:13621-13626. Das, S., Levinson, B., Whitney, S., Vulpe, C., Packman, S., and Gitschier, J. (1994). Diverse mutations in patients with Menkes disease often lead to exon skipping. Am. J. Hum. Genet. 55:883-889. Das, S., Levinson, B., Vulpe, C., Whitney, S., Gitschier, J., and Packman, S. (1995). Similar splicing mutations of the Menkes/mottled copper transporting ATPase gene in occipital horn syndrome and blotchy mice. Am. J . Hum. Genet. 56:570-576. Davisson, M. T. (1987). X-linked genetic homologies between mouse and man. Genomics 1:213227. Farrer, L. A., Bowcock, A. M., Hehert, 1. M., Bonn&-Tamir,B., Sternlieb, I., Giagheddu, M., St. George-Hyslop, P., Frydman, M., Ldssner, J., Demelia, L., Carcassi, C., Lee, R., Ekker, R., Bale, A. E., Donis-Keller, H., Scheinherg, I. H., and Cavalli-Sfona, L. L. (1991). Predictive testing for Wilson’s disease using tightly linked and flanking DNA markers. Neurology 41:992999. Fraser, A. S., Sobey, S., and Spicer, C. C. (1953). Mottled, a sex-modified lethal in the house mouse. J. Genet. 51:217-221.

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Frydman, M., Bonnk-Tamir, B., Farrer, L. A., Conneally, P. M., Magazanik, A., Ashbel, S., and Goldwitch, Z. (1985). Assignment of the gene for Wilson disease to chromosome 13: Linkage to the esterase D locus. Proc. Natl. Acad. Sci. U.S.A. 82:1819-1821. Carey, C. E., Schwarzrnan, A. L., Rise, M. L., and Seyfried, T. N. (1994). Ceruloplasmin gene defect associated with epilepsy in EL mice. Nat. Genet. 6:426-43 1. George, A. M., Reed, V., Glenister, P., Chelly, J., Turner, Z., Horn, N., Monaco, A. P., and Boyd, Y. (1994). Analysis of Mnk, the murine homologue of Menkes’ disease in normal and mottled mice. Genomics 22:27-35. Cerdes, A. M., T@nnesen,T.,Pergament, E., Sander, C., Baerlocher, K. E., Wartha, R., Guttler, F., and Horn, N. (1988). Variability in clinical expression of Menkes’ syndrome. Eur. J. Pediatr. 148:132-135. Hardy, R. M., Stevens, 1. B., and Stowe, C. M. (1975). Chronic progressive hepatitis in Bedlington terriers associated with elevated liver copper concentrations. Minn. Vet. 15:13-24. Herd, S. M., Camakaris, J., Christifferson, R., Wookey, P., and Danks, D. M. (1987). Uptake and efflux of copper-64 in Menkes’-disease and normal continuous lymphoid cell lines. Biochem. 3. 247:341-347. Herzberg, N. H., Wolterman, R. A , , van der Betg, G. J., Barth, P. G., and Bolhuis, P. A. (1990). Metallothionein in Menkes’s disease: Induction in cultured muscle cells. J. Neurol. Sci. 100:5056. Hiyamuta, S., and Takeichi, N. (1993). Lack of copper binding site in ceruloplasmin of LEC rats with abnormal copper metabolism. Biochm. Biophys. Res. Commun. 197: 1140-1 145. Horn, N. (1976). Copper incorporation studies on cultured cells for prenatal dlagnosis of Menkes’ disease. Lancet 1:1156-1158. Horn, N., Stene, J., Mollekaer, A. M., and Friedrich, U. (1984). Linkage studies in Menkes disease: The Xg blood group system and C banding of the X chromosome. Ann. Hum. Genet. 48:16 1- 172. Horn, N., Tonnesen, T., and Turner, 2. (1995). Menkes disease: An X-linked neurological disorder of the copper metabolism. Brain Pathol. 2:351-362. Horn, N., Tonnesen, T., and Turner, Z. (1995). Variability in clinical expression of an X-linked copper disturbance, Menkes disease. In “Metals and Genetics” (B. Sarkar, ed.). Dekker, New York (in press). Houwen, R. H. J., Berger, R.. Cox, D. W., and Buys, C. H. C. M. (1992). Allelic association for Wilson disease-D13S31. J,. Hepatol. 16:S15. Hunt, D. M. (1974). Primary defect in copper transport underlies mottled mutants in the mouse. Nature (London) 249:850-854. Kaler, S. G., Gallo, L. K., Proud, V. K., Percy, A. K., Mark, Y., Segal, N. A., Goldstein, D. S., Holmes, C. S.,and Gahl, W. A. (1994). Occipital horn syndrome and a mild Menkes phenotype associated with splice site mutations at the MNK locus. Nature Genet. 8:195-202. Kapur, S., Higgins, J. V., Delph, K., and Rogers, B. (1987). Menkes syndrome in a girl with X-autosome translocation. Am. J. Med. Genet. 26:503-5 10. Kojimahara, N., Nakabayshi, H., Shikata, T., and Esumi, M. (1994). Defective copper binding to apo-ceruloplasmin in rat model and patients with Wilson’s disease. Liver (in press). Lee, R. L., Nacht, S.,Lukens, 1. H., and Cartwright, G. E. (1968). Iron metabolism in the copperdeficient swine. J. Clin. Invest. 47:2058-2069. Levinson, B., Vulpe, C., Eider, B., Martin, C., Verley, F., Packman, S., and Gitschier, J. (1994). The mottled gene is the mouse homologue of the Menkes disease gene. Nut. Genet. 6:369-373. Li, Y., Togashi, Y., Sato, S., Emoto, T., Kang, 1.-H., Takeichi, N., Kobayashi, H., Kojima, Y., Une, Y., and Uchino, I. (1991a). Spontaneous hepatic copper accumulation in LEC rats with hereditary hepatitis: A model of Wilson’s disease. 3. Clin. Invest. 87:1858-1861. Li, Y.,Togashi, Y., and Takeichi, N. (1991b). Abnormal copper accumulation in the liver of LEC rats: A rat form of Wilson’s disease. In “The LEC Rat: A New Model for Hepatitis and Liver

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Cancer” (M. Mori, M. C. Yoshida, and N. Takeichi, eds.), pp. 122-132. Springer-Verlag, Hong Kong. Lyon, M. F. (1961). Gene action in the X-chromosome of the mouse (Mus mwculw L.). Nature (London) 190:3 72-3 73. Menkes, J. H., Alter, M., Steigleder, G. K., Weakley, D. R., and Sung, 1. H. (1962). A sex-linked disorder with retardation of growth, peculiar hair and focal cerehral and cerebellar degeneration. Pediatrics 29:764- 779. Mercer, J. F. B., Livingston, J., Hall, B., Paynter, J. A., Chandrasekharappa, S., Lockhart, P., Grimes, A., Bhave, M., Siemieniak, D., and Glover, T. W. (1993). Isolation of a partial candidate gene for Menkes disease hy positional cloning. Nat. Gmer. 3:20-25. Mercer, 1. F. B., Grimes, A., Anibrosini, L., Lockhart, P., Paynter, J. A., Dierick, H., and Glover, T. W. (1994). Mutations in the murine homologue of the Menkes gene in dappled and blotchy mice. Nat. Genet. 6:374-378. Odermatt, A., Suter, H., Krapf, R., and Solioz, M. (1993). Primary structure of two P-type ATPases involved in copper homeostasis in Enterococcus hirae. J. Bid. Chem. 268:12775- 12779. Packman, S. (1987). Regulation of copper metabolism in the mottled mouse. Arch. Dermntol. 123:1545- 1547. Peltonen, L., Kuivaniemi, H., Palotie, A , , Horn, N., Kaitila, I., and Kivirikko, K. L. (1983). Alterations of copper and collagen metabolism in the Menkes’s syndrome and a new subtype of Ehlers-Danlos syndrome. Biochemistry 22:6156-6163. Petrukhin, K., Fischer, S. G., Pirastu, M., Tanzi, R. E., Chernov, I., Devoto, M., Brzustowicz, L. M., Cayanis, E., Vitale, E., Russo, J . J., Matseoane, D., Ekwkhgalter, B., Wasco, W., Figus, A. L., Loudianos, J., Cao, A , , Sternlieb, I., Evgrafov, O., Parano, E., Pavone, L., Warburton, D., Ott, J., Penchaszadeh, G . K., Scheinberg, I. H., and Gilliam, T. C. (1993). Mapping, cloning and genetic characterization of the region containing the Wilson disease gene. Nat. Genet. 5:338-343. Petrukhin, K., Lutsenko, S., Chernov, I., Ross, B. M., Kaplan, J. H., and Gilliam, T. C . (1994). Characterization of the Wilson disease gene encoding a P-type copper transporting ATPase: Genomic organisation; alternative splicing; and structure/function predictions. Hum. Mol. Genet. 3:1647-1656. Procopis, P., Camakaris, J . , and Danks, D. M. (1981). A mild form of Menkes’ steely hair syndrome. J. Pediatr. 98:97-99. Rauch, H. (1983). Toxic milk, a new mutation affecting copper metabolism in the mouse. J. Hered. 74: 141- 144. Reed, V., and Boyd, Y. (1994). RFLVs in mottled dappled alleles. Not. Genet. 8:11-12. Rowe, D. W., Goodwin, E. B., Martin, G. R., Sussman, M. D., Grahn, D., Faris, B., and Franzblau, C. (1974). A sex-linked defect in the cross-linking of collagen and elastin associated with the mottled locus in mice. J. Exp. Med. 139:180-192. Sarkar, B., Lingertat-Walsh, K., and Clarke, J. T. R. (1993). Copper-histidine therapy for Menkes disease. J. Pediatr. 123:828-830. Sasaki, M., Yoshida, M. C., Kagami, K . , Dempo, K., and Mori, M. (1985). Spontaneous hepatitis in an inbred strain of Long-Evans rats. Rat News Lett. 14:4-6. Sayed, A. K., Edwards, J. A., and Bannerman, R. M. (1981). Copper metabolism of cultured fibroblasts from the brindled mouse (gene symbol Mohr). PTOC.Soc. Exp. Bid. Med. 166:153156. Scheinberg, 1. H., and Gitlin, D. (1952). Deficiency of ceruloplasmin in patients with hepatolenticular degeneration (Wilson’s disease). Science 116:484-485. Silver, S., Nucifora, G . , and Phung, L. T. (1993). Human Menkes X-chromosome disease and the staphylococcal cadmium-resistance ATPase: A remarkable similariw in protein sequences. Mol. Microbiol. 10:7-12. Stewart, E. A , , White, A , , Tomfohrde, J . , Oshorne-Lawrence. S., Prestridge, L., Bonn6-Tamir, B.,

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Scheinberg, I. H., St. George-Hyslop, P., Giagheddu, M., Kim, J.-W., Seo, J. K., Lo, H.-Y., Ivanova-Smolenskaya,I. A., Limborska, S. A., Cavalli-Sforza, L. L., Farrer, L. A., and Bowcock, A. M. (1993). Polymorphic microsatellites and Wilson disease (WD). Am. J. Hum. Genet. 53~864-873. Su, L.-C., Ravanshad, S., Owen, C. A., Jr., McCall, J. T., Zollman, P. E., and Hardy, R. M. (1982). A comparison of copper-loading disease in Bedlington terriers and Wilson's disease in humans. Am. J. Physiol. 243:G226-G230. Tanzi, R. E., Petrukhin, K., Chernov, I., Pellequer, J. L., Wasco, W., Ross, B., Romano, D. M., Parano, E., Pavone, L., Brzustowicz, L. M., Devoto, M., Peppercorn, I., Bush, A. I., Sternlieb, I., Pirastu, M., Gusella, J. F., Evgrafov, O., Penchaszadeh, 0. K., Honig, B., Edelman, 1. S., Soares, M. B., Scheinberg, I. H., and Gilliam, T. C. (1993). The Wilson disease gene is a copper transporting ATPase with homology to the Menkes disease gene. Nat. Genet. 5:344-350. Thomas, G. R., Forbes, J. R., Roberts, E. A., Walshe, J. M., and Cox, D. W. (1995). The Wilson disease gene: spectrum of mutations and their consequences. Nature Genet. 9:210-217. Thomas, G. R., Roberts, E. A., Rosales, T. O., Moroz, S. P., Lambert, M. A., Wong, L. T. K., and Cox, D. W. (1993). Allelic association and linkage studies in Wilson disease. Hum. Mot. Genet. 2: 1401- 1405. Thomas, 0. R., Bull, P. C., Roberts, E. A., Walshe, J. R., and Cox, D. W. (1994). Haplotype studies in Wilson disease. Am. 1. Hum. Genet. 54:71-78. Tannesen, T., Kleijer, W. M., and Horn, N. (1991). Incidence of Menkes disease. Hum. Genet. 86:408-410. Tcinnesen, T., Peterson, A., Kruse, T. A., Gerdes, A.-M., and Horn, N. (1992). Multipoint linkage analysis in Menkes disease. Am. J. Hum. Genet. 50:1012-1017. Turner, Z., Tommerup, N., Tmnesen, T., Kreuder, J., Craig, I. W., and Him, N. (1992a). Mapping of the Menkes locus to Xq13.3 distal to the X-inactivation center by an intrachromosoma1 insertion of the segment Xq13.3-q21.2. Hum. Gene. 88:668-672. Turner, Z., Chelly, J., Tommerup, N., Ishikawa-Brush, Y., Tginnesen, T., Monaco, A. P., and Horn, N. (1992b). Characterization of a 1.0 Mb YAC contig spanning two chromosome breakpoints related to Menkes disease. Hum. Mol. Genet. 1:483-489. Turner, Z., Tannesen, T., Bijhmann, J., Marg, W., and Horn, N. (1994). First trimester prenatal diagnosis of Menkes disease by DNA analysis. J. Med. Genet. 31:615-617. Turner, Z., Vural, B., Tcinnesen, T., Chelly, J., Monaco, A. P., and Horn, N. (1995). Characterization of the exon structure of the Menkes disease gene using vectorete PCR. Genomics 26 (in press). Verga, V., Hall, B. K., Wang, S., Johnson, S., Higgins, J. V., and Glover, T. W. (1991). Localization of the translocation breakpoint in a female with Menkes syndrome to Xq13.2-q13.3 proximal to PGK-1. Am. J. Hum. Genet. 48:1133-1138. Vulpe, C., Levinson, B., Whitney. S., Packman, S., and Gitschier, J. (1993a). Isolation of a candidate gene for Menkes disease and evidence that it encodes a copper-transportingATPase. Nat. Genet. 3:7-13. Vulpe, C., Levinson, B., Whitney, S., Packman, S., and Gitschier, J. (1993b). Isolation of a candidate gene for Menkes disease and evidence that it encodes a copper-transportingATPase. (Correction) Not. Genet. 3:273. Walshe, J. M. (1973). Copper chelation in patients with Wilson disease. Q. J. Med. 42:441. Westman, J. A., Richardson, D. C., Rennert, 0. M., and Morrow, G. (1988). Atypical Menkes' steely hair disease. Am. 1. Med. Genet. 30:853-858. Wienker, T. F., Wieacker, P., Choke, H. J., Horn, N., and Ropers, H.-H. (1983). Evidence that Menkes locus maps on proximal Xp. Hum. Genet. 65:72-73. Wilson, S. A. K. (1912). Progressive lenticular degeneration: A familial nervous disease associated with cirrhosis of the liver. Brain 34:295-509.

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Edward T. Stuart, Yoshifumi Yokota, and Peter Gruss Department of Molecular Cell Biology Max Planck Institute for Biophysical Chemistry D-37077 Cattingen, Germany

1. INTRODUCTION The mechanisms that underlie cellular growth control are multifaceted. By conventional wisdom, there must be a link between the mechanisms that control cell growth and those that result in the deregulation of cell growth. Cancer arises by a multistep process that results in the loss of cell growth control (Nowell, 1976; Vogelstein and Kinzler, 1993). On the contrary, embryonic development requires an exquisite control of cell growth and differentiation so that the complex structures and cell types that constitute the final organism may be formed. It is therefore reasonable to hypothesize that those genes that encode proteins which are involved in the developmental process may, in certain circumstances, be involved in the initiation or progression of cancer. To date, many genes have been identified which are implicated in the control of development and have also been identified to be misappropriately expressed in neoplastic cells. The Wnt family of genes, for example, encodes glycoproteins that are involved in the genesis of certain structures in the developing central nervous system (CNS); however, Wnt genes were originally identified as protooncogenes due to their activation by preferential proviral insertion by the MMTV in murine mammary tumors (Gavin et al., 1990; McMahon and Bradley, 1990; Kostriken and Weisblat, 1992; Nusse and Varmus, 1992). Here we present an overview of current observations concerning two groups of genes which are intimately involved in embryonic development and have been postulated to be involved in a number of different types of cancer.

Advances in Genetics, Vol. 33 Copyright 0 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.

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First, the PAX genes that encode nuclear transcription factors and have been shown to be involved in the development of many tissues and organs, predominantly the CNS; and second, the HOX family that also encodes nuclear transcription factors which have been suggested to be one of the master controllers of embryonic development.

II. PAX GENES IN DEVELOPMENT AND NEOPLASIA The PAX family of genes encodes nuclear transcription factors that are thought to play an important role in embryonic development (Chalepakis et al., 1992, 1993; Gruss and Walther, 1992; Fritsch and Gruss, 1993; Noll, 1993; Stuart et al., 1994). The PAX family consists of nine members each of which share a common motif termed the paired box (Figure 8.1) (Stapleton et al., 1993). The paired box is 128 amino acids in length and displays DNA-binding properties (Chalepakis et al., 1992, 1993; Stuart et al., 1994). The nine PAX genes are grouped into four classes according to the structural similarity and homology of the proteins they encode (Walther et al., 1991). The first class is represented by PAXI and PAX9 which contain the paired domain and a conserved region of 8 amino acids termed the octapeptide. The second class is represented by PAX3 and PAX7 which, besides the paired domain and octapeptide, contain a pairedtype homeodomain. Containing the paired domain, octapeptide, and a partial homeodomain, PA=, PAX.5, and PAX8 represent the third class. Finally, the fourth class consists of PAX4 and PAX6 which contain the paired domain and the homeodomain but lack the octapeptide. The paired domain is a DNAbinding region as is the full homeodomain. The function of any of the octapeptide motifs is as yet unknown. In general, PAX genes encode mRNA that ranges from 3.0 to 5.0 kb (Stuart et al., 1994), the exception being PAX5 whose mRNA is 9.5-10.0 kb in length (Adams et al., 1992). The respective proteins vary from 360 to 480 amino acids in length. Structurally, the paired domain consists of three a-helices, two of which are located near the carboxy end and the other is located in proximity to the amino end of the domain. PAX proteins are presumed to be nuclear transcription factors because they are localized in the nucleus (Dressler and Douglass, 1992) and are able to bind DNA in vitro (Chalepakis et al., 1991, 1994; Goulding et al., 1991; Adams et al., 1992; Dressler and Douglass, 1992; Zannini et al., 1992; Czerny et al., 1993). The Dosophila paired protein (prd) can bind to a sequence in the even-skipped promoter e5 (Treisman et al., 1989, 1991). The sequences bound by prd include the ATTA motif, which is recognized by the homeodomain, and sequences further downstream presumably recognized by the paired domain. Using this sequence it has been demonstrated that PAX proteins have a large (20-24 base

GENE

CHROMOSOMAL LOCATION MOUSE

HUMAN

PAXl

2

PAX2

19

log25

PAX3

1

2q35

PAX4

6

7

PAX5

4

9P13

PAX6

2

llp13

PAX7

4

1p33.2

PAX8

2

2q12q14

PAX9

12

14q12q13

STRUCTURE

ASSOCIATEDMUTANTS MOUSE

ASSOCIATED CANCER

HUMAN

Undulated

Wilms' tumor

Splotch

Waardenburgs syndrome

Rhabdomyosarcoma

Pleomorphic xanthroastrocytoma? Glioblastoma

Small eye

Aniridia

Rhabdomyosarcoma

Wilms' tumor

Figure 8.1. The PAX gene family. The PAX genes are all located at independent loci in the mouse and human. T h e four classes of PAX genes are depicted by the shading of the paired domain. Mouse mutants, human mutations, and cancers associated with PAX genes are shown on the right. Abbreviations: PD, paired domain; OCT, octapeptide, HD, homeodomain.

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pair) recognition sequence. PAX proteins, which contain the homeodomain and paired domain, e.g., PAX3, also recognize the ATTA motif and a downstream motif characterized by the GTYMC core motif. Currently, little is known about the regulators of PAX genes or target genes for PAX proteins. Functional target sequences for class 111 have been identified. PAX5 was originally identified as a B cell-specific transcription factor and potentially regulates the CD19 gene which encodes a B cell-specific surface protein (Kozmik et al., 1992). Its paralog in the sea urchin, TSAP, regulates two pairs of nonallelic histone genes, H2A-2 and H2B-2 (Barberis et al., 1989). PAX8, which is expressed in the thyroid, binds to and regulates the thyroperoxidase and thyroglobulin genes (Zannini et al., 1992).

111. PAX GENES ARE IMPLICATED IN THE CONTROL OF DEVELOPMENT In general, murine PAX genes are expressed during embryogenesis with distinct spatial and temporal patterns beginning between Days 8 and 9.5 postcoitum (pc). A common feature of PAX genes is that they are expressed in the CNS and/or the paraxial mesoderm and its derivatives. PAXZ-8 are expressed in the developing and the adult CNS. PAXJ expression in the CNS has not been demonstrated at any stage. The neuroanatomical regions of PAX gene expression have been previously described in detail and the reader is referred to Stoykova and Gruss (1994) for further details. Those PAX genes which, in addition to the paired domain, encode a full homeodomain (PAX3, PAX6, and PAX7) are expressed before the onset of differentiation (Day 8-8.5 pc). In contrast, PAX genes which lack a full homeodomain (PAXZ, PAXS, and PAX8) begin to be expressed during neural differentiation (Day 10 pc). Apart from the CNS, PAX genes are expressed in a number of different structures. PAX1 is expressed from Day 9 pc in the segmented prevertebral column and later in the sternum and thymus (Deutsch et al., 1988). PAW is expressed in the condensing mesenchyme of the developing kidney, its early epithelial derivatives, and in the collecting duct epithelium (Dressler et al., 1990; Dressler and Douglass, 1992). PAX2 is required for the earliest phase of mesenchyme-to-epithelium conversion during kidney development (Phelps and Dressler, 1993) and is repressed upon terminal differentiation of the renal epithelium. PAX2 is also expressed in the Wolfian duct, the ureter, and the developing eye and ear. Both PAX3 and PAX7 are expressed in the somites of Days 9 and 10 pc embryos. Also, at this stage PAX3 is expressed in the undifferentiated mesenchyme of both fore- and hindlimb. A t Day 12 pc, the expanding myotome but not the dermatome still expresses PAX7. AT later stages, PAX7 is still expressed

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in the myotome-derived intercostal muscles and the skeletal muscles of the trunk. In contrast, PAX3 is not expressed in the mesoderm or its derivatives after Day 11 pc. The expression of PAX7 is related to myogenesis (Jostes et al., 1991) and should exhibit a cell lineage-restricted expression similar to PAX1 expression in sclerotome-derived cells. At Day 13 pc PAX3 and PAX7 are expressed in the nasal pits. The expression of PAX6 extends to all structures of the developing eye and nose (Walther and Gruss, 1991). PAX5 is expressed in many tissues involved in B cell differentiation, such as the spleen and lymph node, and in the pre-B, pro-B, and mature B cells. PAX5 expression is also seen in the adult testis (Adams et al., 1992). PAX8 is expressed in the developing kidney but at a later stage than PAX2, i.e., at more differentiated stages (Poleev et al., 1992). PAX8 is also expressed in the thyroid (Zannini et al., 1992) and in the placenta (Kozmik et al., 1993).

IV. WHY PAX GENES COULD BE CONSIDERED ONCOGENES The classification of transcription factors as protooncogenes is not straightforward. Inappropriate expression of a transcription factor in neoplastic cells does not necessarily imply that it is a protooncogene. However, there is increasing evidence to suggest that PAX genes should be considered protooncogenes for two reasons: first, they are able to transform cells in culture and form tumors in mice, and second, they have been demonstrated to be inappropriately expressed in a number of unrelated tumors.

V. AN IN YIVO MODELpFmX-INDUCED TUMORIGENESIS The initial observations suggesting that PAX genes could be considered protooncogenes were based on experiments which demonstrated that PAX genes could transform cells in culture and that these cells could form tumors in nude mice (Maulbecker and Gruss, 1993b). When PAX cDNAs are expressed under the control of the CMV promoter/enhancer in NIH3T3 mouse fibroblasts or 208F rat fibroblasts, the cells overcome their contact inhibition and grow in foci. These cells also demonstrate the ability to grow in increasing concentrations of soft agar demonstrating that their growth has become independent of the need of a substratum. Injection of cells which stably express PAX proteins into nude mice results in the formation of tumors at the site of injection. Morphologically these tumors are well vascularized and histologically are similar to spindle cell fibrosarcomas. Although

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metastasis was not demonstrated because tumor-bearing animals were sacrificed a short time after tumor formation was evident, it is possible that these tumors may demonstrate metastatic potential. Such observations would require a more in-depth investigation ideally involving tumor induction in a well-vascularized organ preferably on the arterial side of the capillary bed thus negating the artifactual observation of metastasis due to the lack of a capillary bed between the site of injection and site of metastasis.

VI. PAX GENE EXPRESSION IN PRIMARY HUMAN TUMORS The involvement of putative protooncogenes in tumor initiation or progression requires the identification of altered expression patterns in primary tumors, i.e., expression as an altered protein form or activation of expression where there should normally be no expression. Due to the controversy surrounding the identification of alterations within cell lines cultured from tumors, i.e., alterations in gene expression due to culture artifacts, it is now clear that studies of the role of a protooncogene in a tumor type must also include primary tumor samples. This has two advantages: first, it directly identifies the protooncogene in the original tumor, and second, it affords the investigator the possibility to identify in vzvo targets or cooperating pathways for the gene of interest. The major drawback, however, is that in general the quantity of material for investigation is extremely limited and thus the investigator must either have good knowledge of what to look for or must have an appreciable level of good fortune.

VII. PAX AND BRAIN CANCERS With the exception of PAX1 and PAX9, expression in the CNS is a salient feature of the PAX gene family. Tumors of the CNS thus represent a logical starting point from which the investigation of PAX genes in human tumors can proceed. Astrocytomas represent the most common form of human brain cancer and may be divided into two main categories based on the overall prognosisdiffuse astrocytomas comprise nearly 75% of all human astrocytomas and are prognostically unfavorable, whereas nondiffuse astrocytomas, i.e., pilocytic astrocytomas, pleomorphic xanthroastrocytomas, and subependymal giant cell astrocytomas, are prognostically more favorable due to their slow growth rate. Clinically, diffuse astrocytomas occur in three different states of malignancy: astrocytomas which display low malignancy (WHO grade 1-11), anaplastic astrocytoma (medium malignancy, WHO grade III), and glioblastoma multiforme (highly malignant, WHO grade IV). The common feature of diffuse astro-

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cytomas is their tendency to progress to higher forms of malignancy such that patients presenting with a low grade of tumor will inevitably represent in the future with a more malignant form of the tumor which in all cases results in death. Currently, little is understood about the complex mechanisms that underlie this progression to higher forms of malignancy (Mikkelsen et al., 1991). Cytogenetic analysis of tumors has led to the identification of some of the molecules involved in astrocytoma progression (Bigner et al., 1986, 1988; Rey et al., 1987; James et al., 1988; Collins and James, 1993). The most frequent cytogenetic abnormalities thus far identified in astrocytomas are the presence of double-minute bodies and polysomy of chromosome 7, varying abnormalities concerning chromosome 9p, and loss of chromosome 10 and chromosome 17. Although the molecular basis for the involvement of chromosome 10 has not as yet been reported, alterations Concerning chromosome 17 have been suggested to involve the tumor suppressor gene p53. Inactivation of p53 has been observed in many tumor types and has been suggested to be one of the initial requirements for tumor initiation. Indeed, most cases of p53-involved tumorigenesis are due to a mutant form of the protein which generally results from point mutagenesis within its evolutionarily conserved exons, and such alterations have been reported for glioblastomas (James et al., 1989; Fults et al., 1992; Reifenberger et al., 1993). The abnormalities Concerning chromosome 7 led to the observation that the epidermal growth factor receptor (EGFR) is amplified in many cases of glioblastoma multiforme (Humphrey et al., 1988; Wong et al., 1992). Normally located on chromosome 7q, the EGFR has been demonstrated to be highly expressed, often as a truncated mutant. The truncated protein appears to lack the extracellular ligand-binding domain, whereas the intracellular catalytic domain appears to be normal. As the EGFR is a member of the tyrosine kinase family of growth factor receptors, it is likely that such a deregulation would be a strong stimulus to cell growth in a manner similar to the viral and cellular counterparts of the src gene. Of great interest are the chromosomal abnormalities concerning 9p. Many candidate genes appear to be clustered on 9p, specifically the bands 9p139p22 (Figure 8.2). Originally, the interferon cluster ( 9 ~ 2 2was ) demonstrated to be deleted in many medium- and high-grade astrocytomas. However, recent studies have assigned a putative tumor suppressor gene p16 (CDI(N2) to 9p21 (Kamb et al., 1994). This locus is frequently rearranged in a number of different cancers, e.g., glioblastoma, melanoma, etc., and was assumed to harbor a tumor suppressor gene. Although there is currently much controversy over the relevance of p16 in primary tumors as it has been suggested to be an artifact of cell culturing techniques, there is great interest in its potential as a tumor suppressor gene. To add to the complexity of the status of 9p in glioblastomas, PAX5 is

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23

''

TYRP

IFN a,P

REGIONS ASSOCIATED

CDKNZ (~16)

WITH SPECIFIC CANCERS

GGTB2 PAX5

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Figure 8.2. Chromosomal loci of genes identified on chromosome 9p13-ter. TYRP, tyrosinaserelated protein; IFN, interferon; CDKNZ, cyclin-dependent kinase inhibitor-2; GGTBZ, glycoprotein-4-~-galactosyltransferase-2; GALT, galactose- 1-phosphate uridylyltransferase.

inappropriately expressed in astrocytomas and its expression correlates with increasing malignancy (Stuart et al., 1995). In normal brain PAX6 is the only PAX gene expressed in the telencephalon. However, PAX5 is expressed weakly in low-grade astrocytomas and strongly in many highly malignant glioblastomas originating in the forebrain. The fact that Southern analysis of tumor genomic DNA could not identify consistent rearrangements indicates that the deregulated PAX5 expression is not a result of large-scale chromosomal changes but may be due to (if the loss of up- or downstream sequences which influence its transcription or (ii) activation of transcription by another factor which itself is either activated or inactivated during the progression to glioblastoma multiforme. In those tumors which were shown to express PAXS, certain populations of cells within the tumor highly expressed PAX5 mRNA, whereas other

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areas either did not express or weakly expressed the mRNA. In addition, those areas which expressed PAX5 in each case expressed Myc, lun, Fos, or the EGFR either singularly or in combination. This would in part support the theory that the most malignant portion of a tumor influences the prognosis. Alternatively, in a nondiffuse astrocytoma, pleomorphic xanthroastrocytorna (WHO grade HI), whose prognosis is much more favorable, PAX5 was absent. PAX4, however, was highly expressed in the nondiffuse tumor but not at all in the diffuse tumors. This suggests that PAX5 may indeed be biologically relevant in these tumor types. The identification of a biological role for a transcription factor requires the discovery of its target genes and also those genes for which it is a target. Although expression of PAX5 coincided in every case with expression of the EGFR, a direct interaction between these two genes could not be demonstrated. Treatment of primary murine astrocyte cultures with EGF does not increase PAX5 expression nor does the PAX5 protein bind to the EGFR promoter (E. T. Stuart, C . Kioussi, and P. Gruss, unpublished observations).

VIII. WILMS’ TUMOR A RENAL CARCINOMA REMINISCENT OF EARLY RENAL DEVELOPMENT Nephroblastoma or Wilms’ tumor is the most common solid pediatric tumor and is also associated with aniridia, mental retardation, and urogenital malfunctions -collectively termed the WAGR syndrome. Wilms’ tumor is characterized by the incomplete differentiation of mesenchymal stem cells into the normal epithelial components of the nephron (Hastie, 1993). Wilms’ tumor involves a number of genes including the tumor suppressor gene WTI, PAXZ, and PAX8. WTl encodes a nuclear transcription factor which binds DNA via its four zinc fingers (Rauscher et al., 1990). In a similar fashion to the identification of PAX proteins in cancers, the identification of the WTJ gene is the first step in a chain of events which requires the identification of upstream regulators of its transcription and downstream target genes before its usefulness as a potential target gene for therapy can be considered. Thus far some target genes have been proposed. The first suggested binding site for the WT1 protein was identified as being similar to that recognized by the early growth response-l gene product (EGR-1) (Rauscher et al., 1990). EGR-1, also known as Krox 24, is a serum-inducible nuclear protein which also contains zinc fingers and binds to the consensus sequence 5’-GGAGCGGGGGCG-3’. The identification of a consensus DNA-binding sequence aids the identification of potential target genes for the transcription factor, and thus far this sequence has been identified in the genes which encode TGF-P1, CSF-1, EGR-1, PDGFA chain, IGF-11, IGF- 1 receptor, and, interestingly, PAX2 (Rauscher, 1993).

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WTl is also capable of physically interacting with p53 which appears to be of importance for the proper functioning of WTl in regulating transcription (Maheswaran et ul., 1992). The identification of WTJ as the gene most likely to be responsible for Wilms’ tumor was the result of cytogenetic studies which identified the rearranged locus involved in these tumors as llp13. The llp13 locus also harbors the PAX6 gene; however, PAX6 has not been implicated in this tumor type although it is involved in aniridia, which itself is part of the WAGR syndrome (Ton et al., 1991). The identification of the WTJ locus at llp13 and its intragenic deletions leading to premature translational termination before zinc finger transcription in patients with Wilms’ tumor constitute the basis for the supposition of its involvement in this tumor type (Haber et al., 1990; Pelletier et al., 1991). Although it is likely that a number of genes are involved in the genesis of Wilms’ tumor, such as PAW and PAX8 (Dressler and Douglass, 1992; Poleev et al., 1992), loss of heterozygosity of the Wilms’ tumor suppressor gene locus appears to be one of the consistently observed features and thus is probably one of the primary factors required for Wilms’ tumor formation. The tumor morphology is suggestive of a developmental malfunction (Dressler and Douglass, 1992). PAW has been demonstrated to be required for mesenchyme to epithelium progression and the presence of the PAX2 protein has been demonstrated in primary Wilms’ tumors (Dressler and Douglass, 1992). Similarly, PAX8, which is also expressed in the developing kidney, has been demonstrated to be expressed in Wilms’ tumor (Poleev et al., 1992).

IX. CHROMOSOMAL REARRANGEMENTS IN RHABDOMYOSARCOMA INVOLVE PAX LOCI Rhabdomyosarcoma is a pediatric tumor originating in striated muscle. A common chromosomal rearrangement occurring in rhabdomyosarcoma involves the PAX3 locus (Barr et aI., 1993). The translocation t(2;13)(q35;q14) results in the translocation of a part of the PAX3 locus, which normally resides at 2q35, and its expression as a fusion protein. This fusion protein has been characterized as being composed of the 5’ region of the wild-type PAX3 protein fused to a portion of a novel forkhead gene termed FKHR (Galili et al., 1993) or ALV (Shapiro et al., 1993). The PAX3 portion of the protein retains the paired domain, octapeptide, and the paired-type homeodomain. The forkhead portion retains only 60% of the forkhead domain along with its wild-type 3’ region. Therefore, the fusion protein retains the PAX3 DNA-binding domains but the incomplete forkhead domain suggests that this component is no longer functional as an intact forkhead domain is required for DNA binding in normal circumstances (Lai et al., 1990). Although it is yet to be reported whether this

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fusion protein retains its DNA-binding ability, one must presume that the inappropriate expression of the PAX3 DNA-binding domains would be sufficient to influence transcription. As PAX3 is thought to have a negative regulatory influence on transcription, the inhibition of gene expression could be the functional pathway to neoplasia in this cancer type if indeed PAX3 is involved. A further investigation of rhabdomyosarcomas has identified a subset of these tumors which, in contrast to those previously reported, demonstrate a translocation of PAX7 t(1;13)(p36;q14) instead of the PAX3 locus (Davis et al., 1994). Although these tumors are a minor subset of rhabdomyosarcomas, the fascinating feature of these tumors is that a portion of the PAX7 locus is again translocated to exactly the same site as PAX3. This again results in a potential fusion protein in which the DNA-binding domains of wild-type PAX7 are retained and exactly the same amino acid residue is targeted for fusion within the forkhead domain. As PAX3 and PAX7 are members of the same subclass of PAX proteins, this finding would suggest that there must be a dominant mechanism that preferentially joins these PAX loci to this particular forkhead locus. Although it is to early to substantiate this theory, if proven to be true, this tumor type would represent an extremely useful model for the investigation of the mechanisms that underlie chromosomal rearrangements.

X. HOMEOBQR: GENES ARL INVOLVED IN EARLY DEVELOPMENTAL PROCESSES Initially identified in h-osophih developmental regulatory genes, the homeodomain is a 60-amino acid motif, elements of which are conserved in the products of the gap, pair-rule, segment polarity, homeotic selector, and maternal effect genes (Gehring, 1987). Based on their homology to Drosophila developmental control genes, many murine and human homeodomain-containing genes have been isolated. The foremost murine family of homeobox-containing genes are the HOX genes, and other genes which encode more divergent homeodomains which have Drosophila homologs include PAX (paired) (described above), En (engrailed; Joyner et al., 1985; Joyner and Martin, 1987), Eux (even skipped; Bastian and Gruss, 1990; Bastian et al., 1992), Cdx (caudal; Meyer and Gruss, 1993), Six (sine oculis; G. Oliver, A. Maihlos, and P. Gruss, unpublished data), Prox (prospero; Oliver et al., 1993) andgoosecoid (Blum et al., 1992; Gaunt et al., 1993; Izpisfia-Belmonte et al., 1993; Niehrs et al., 1994). In both mouse and human the HOX genes are arranged in clusters (Figure 8.3). There are four clusters, each found on different chromosomes: in the mouse the clusters are located on chromosomes 6, 11, 15, and 2 and in the human the clusters are located on chromosomes 7, 17, 12, and 2. One of the distinguishing characteristics of the HOX family is that there is a physical

Edward T. Stuart ef al.

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Figure 8.3. The HOX genes are clustered but differ in their temporal and spatial expression domains and their responsiveness to retinoic acid.

correlation between the location of a gene in its cluster and its expression along the anterior/posterior axis. Thus, genes located at the 3’ end of the cluster are the earliest expressed and have the most anterior expression domain. The temporal and spatial aspects of expression shift later and more posterior as the 5’ region of the cluster is reached (Kessel and Gruss, 1990; Krumlauf, 1994). In addition, retinoic acid has been shown to induce HOX gene expression and, indeed, those genes located at the 3’ region of the cluster are more sensitive than those at the 5’ region in accordance with the temporal expression pattern (Kessel and Gruss, 1991). The homeodomain binds to DNA sequences characterized by the 5’-TAAT-3’ core motif. The majority of the reports concerning the mode of homeodomain binding have used the Drosophila antennapedia homeodomain (which differs by only one amino acid from the human HOX-A7 homeodomain). The antennapedia homeodomain contains three helices folded into a tight globular structure. The three helices and the spacers between them have distinct functions: the third helix acts as the recognition helix and sits in the major groove, and the first and second helices are aligned in an antiparallel arrangement above the DNA and interact with the DNA backbone. Additional contacts to the minor groove are made by the flexible N-terminal arm (Gehring et al., 1994).

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XI. HOMEOB00(-CONTAINING GENES AND THEIR PUTATIVE ROLE IN CANCER The presumed position of homeobox genes in the developmental hierarchy suggests that they may be extremely important players in the genesis of many neoplasias (Blatt, 1990; Castronovo e t al., 1994). Being early promoters of development implies that they would influence the transcription of a wide range of genes either directly (downstream target genes) or indirectly (subsequent target genes for those directly influenced).

XII. AN IN YIYO MODEL FOR HOX-INDUCED TUMORIGENESIS Many examples of putative homeobox gene involvement in leukemia suggest that these genes are involved in acute pediatric cases. To determine whether deregulated homeobox genes may alone be sufficient to perform such roles, a number of different members of the murine HOX family as well as other homeobox-containing genes were tested for their ability to induce transformation of cells in culture and form tumors in vivo (Maulbecker and Gruss, 1993a). Expression of HOXa-I, a-5, a-7, b-7, and c-8 under the control of the CMV promoter/enhancer leads to the transformation of NIH3T3 mouse fibroblasts and 208F rat fibroblasts. Cells stably transfected with these HOX genes grow in foci and have the ability to overcome contact inhibition, growing in increasing concentrations of soft agar. However, the different HOX genes did not produce consistently similar abilities in both these assays. HOXa-5-expressing cells were unable to grow in 1.2% soft agar, whereas the other HOX-expressing cells were able to indicating a weaker transforming potential for this gene. Injection of HOX-expressing cells into nude athymic mice resulted in tumor formation within 2 weeks with the exception of HOXa-5, which failed to induce tumors in some cases which supports the earlier observations concerning is weaker transforming potential. Histologically, the tumors resembled poorly differentiated spindle cell sarcomas and were well vascularized, showing recruitment of blood vessels and the establishment of a tumor microcirculation. Metastasis was not evident due to the short period of growth in the animal although initial signs of infiltration into striated muscle near the site of injection were observed.

XIII. HOMEOBOX GENES IN LEUKEMIA One of the notable examples of homeobox gene involvement in cancer is leukemia. Although homeobox genes are widely expressed throughout embryonic development, their expression is preferentially restricted to mitotically active

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tissues in the adult. Therefore, inhibition of differentiation appears to be one of their functions. The development of myeloid leukemia can be associated with blocks in differentiation and it has been demonstrated that expression of HOXb-8 is able to disrupt interlukin-6-induced myeloid differentiation. After treatment of cells which stably express HOXb-8, interleukin-6-induced changes in levels of c-myc, c-myb, and c-fos were attenuated, whereas c-jun and junB were unaffected (Blatt et al., 1992). Moreover, induction of HOXb-8 expression by proviral insertion into bone marrow cells results in a strongly leukemogenic phenotype. In this study, the transforming ability of HOXb-8 was demonstrated to be associated with the interlukin-3 pathway such that HOXb-8 impeded the interlukin-3-driven terminal differentiation of myeloid cells (Perkins et al., 1990). Further evidence for HOXb-8 involvement in myeloid leukemia is provided by the findings that in murine myeloid leukemia cells WEHI-3B, HOXb-8 is constitutively expressed due to a chromosomal rearrangement which inserts an intracisternal A particle upstream of HOXb-8 (Blatt et al., 1988), and that alone HOXb-8 is capable of producing fibrosarcomaas in nude mice (Aberdam et d., 1991). Apart from HOX genes, another homeobox-encoding gene has been identified by virtue of its involvement in pre-B cell leukemias-a specific subset of childhood acute lymphoblastic leukemias (pre-B ALL). In pre-B cell leukemias a consistent chromosomal translocation, t( 1;19), involves the E2A gene which encodes the enhancer-binding transcription factors E 12 and E47 that bind the immunoglobulin-k gene E box motif. E2A is located on chromosome 19 and, after rearrangement with chromosome 1, a fusion protein is produced which is composed of two-thirds of the E2A gene fused to a protein derived from chromosome 1. The novel chromosome 1-derived gene, termed prl or PBX-I, was demonstrated to encode a homeodomain-containing transcription factor. Therefore, the translocation results in a fusion protein which retains the binding capacity of @I as well as the putative leucine zipper of E2A. However, prl is normally expressed in a restricted manner and fusion with E2A would result in a transcriptional deregulation of prl (Kamps et al., 1990; Nourse et d., 1990). Further proof of the oncogenic potential of this fusion transcript has been provided by the observation that it causes acute myeloid leukemia in mice. The cDNA encoding the fusion protein was introduced into mouse marrow progenitors using a retroviral vector and these cells, when introduced into irradiated mice, developed leukemia within 3-8 months (Kamps and Baltimore, 1993).

XIV. HOMEOBQX GENES IN MHER CANCERS Apart from leukemia, homeobox gene expression has been identified in other tumor types such as breast, colon, rectal, gastric, lung, renal, and testicular

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cancer (Wewer et ul., 1990). Colorectal cancer is one of the most frequent human malignancies arising from benign adenomatous polyps which later progress to adenocarcinomas. Such progression is typical for the multistep nature of cancer. Although some of the genes involved have been identified, primarily familial adenomatous polypsis (Leppert et ul., 1987) and deleted in colon cancer (Stanbridge and Cavanee, 1989; Stanbridge, 1990; Fearon et al., 1990; Fearon and Vogelstein, 1990; Weinberg, 1993), there are many more molecular changes yet to be identified. Homeobox-containing genes display an altered expression pattern in colorectal cancer (DeVita et ul., 1993). In both normal and tumor tissue, HOXu-I3 and HOXb-4 were expressed equally, whereas HOXb-7 and HOXd-I J transcription was increased in primary tumors and their metastases.

XV. CONCWSIONS AND FUTURE PROSPECTS In this chapter we presented a synopsis of the involvement of PAX and HOX genes in cancer. The involvement of nuclear transcription factors which direct embryogenesis in cancer is now starting to be recognized as a potentially very fruitful area for cancer researchers. Genes which are involved in early embryogenesis are evidently positioned so that they are upstream of a multitude of genes which they influence either directly or indirectly via a combination of downstream target genes. The fact that the transcription of these genes must be strictly controlled so that they can influence cell growth and differentiation in a defined manner implies that when this mechanism goes awry, the consequences will be profound. In embryogenesis, alterations in development-controlling proteins lead to identified mutants both in mouse and man. However, activation of an embryonic developmental cascade later in life will lead to disorganization of the cell cycle and may lead to neoplasia. We discussed observations concerning PAX and HOX genes in different types of cancer, demonstrating that altered expression of both of these genes can be a result of gross chromosomal rearrangements or, alternatively, the basis of misappropriate expression is not yet understood. In the case of both PAX and HOX, at present it is difficult to elaborate on the role of these proteins in the multistep process which is cancer. The upstream regulators of PAX genes are not well understood. The relevant downstream target genes of PAX and HOX are likewise elusive. In the case of PAX genes, it is likely that their role in cancer lies either in cooperation with known oncogenes as PAX genes are unable to transform primary fibroblast cultures, or their downstream target genes will be directly involved in regulation of the cell cycle. Of surprise is the paucity of cancers which can be directly linked to HOX genes. As master controllers of development the array of target genes must be large and divergent. This could indeed be one possible explanation for their lack of involvement, i.e., that they

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themselves are a large family with a larger set of target genes and therefore functional redundancy between the genes is probable. In conclusion, in the future the number of cancers which involve PAX and HOX genes will no doubt increase and the identification of up- and downstream genes associated with these gene families will lead to new breakthroughs in many areas such as devel. opment, cancer research, and cell cycle control.

Acknowledgments The financial support of the Max-Planck Society and The Mildred Scheel foundation is gratefully acknowledged. Y.Y. was supported by grants from The Alexander von Humboldt foundation and The Human Frontiers Science Program.

References Aberdam, D., Negreanu, V., Sachs, L., and Blatt, C. (1991). The oncogenic potential of an activated Hox-2.4 homeobox gene in mouse fibroblasts. Mol. CeU. Bid 11(1):554-557. Adams, B., Dorfler, P., Aguzzi, A., Kozmik, Z., Urbinek, P., Maurer-Fogy, I., and Busslinger, M. (1992). Pax-5 encodes the transcription factor BSAP and is expressed in B lymphocytes, the developing CNS, and in adult testes. Genes Dew. 6:1589-1607. Barberis, A , , Superti-Furga, G., Vitelli, L., Kemler, I., and Busslinger, M. (1989). Developmental and tissue-specific regulation of a novel transcription factor of the sea urchin. Genes Dev. 3:663675. Barr, F. G., Galili, N., Holick, I., Biegel, J. A., Rovera, G., and Emanuel, B. S. (1993). Rearrangement of the PAX3 paired hox gene in the paediatric solid tumor alveolar rhabdomyosarcoma. Nat. Genet. 3:113-117. Bastian, H., and Gruss, P. (1990). A murine even-skipped homologue, Ewx 1 , is expressed during early embryogenesis and neurogenesis in a biphasic manner. EMBO J. 9(6):1839-1852. Bastian, H., Gruss, P., Duboule, D., and Izpisda-Belmonte, J. C. (1992). The murine ewen-skippedlike gene Evx-2 is closely linked to the Hex-4 complex, but is transcribed in the opposite direction. Mamm. Genome 3:241-243. Bigner, S. H., Mark, J., Bullard, D. E., Mahaley, M. S., Jr., and Bigner, D. D. (1986). Chromosomal evolution in malignant human gliomas starts with specific and usually numerical deviations. Cancer Genet. Cytogenet. 22:121-135. Bigner, S. H., Mark, J., Burger, P. C., Mahaley, M. S., Jr., Bullard, D. S., Muhlbaier, L. H., and Bigner, D. D. (1988). Specific chromosomal abnormalities in malignant human gliomas. Cancer Res. 48:405-4 11. Blatt, C. (1990). The betrayal of homeo box genes in normal development: The link to cancer. CanceT Cells 6(2):186-189. Blatt, C. D., Aberdam, D., Schwartz, R., and Sachs, L. (1988). DNA rearrangement of a homeobox gene in myeloid leukaemic cells. EMBO J. 7(13):4283-4290. Blatt, C., Lotem, J., and Sachs, L. (1992). Inhibition of specific pathways of myeloid cell differentiation by an activated Hox-2.4 homeobox gene. Cell GTowth Differ. 3:671-676. Blum, M., Gaunt, S.J., Cho, K. W. Y., Steinbeisser, H . , Blumberg, B., Bittner, D., and De Robertis, E. M. (1992). Gastrulation in the mouse: The role of the homeobox gene goosecoid. CeU (Cambndge, Mass.) 69: 1097-1106. Castronovo, V., Kusaka, M., Chariot, A., Gielen, J., and Sobel, M. (1994). Homeobox genes:

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Potential candidates for the transcriptional control of the transformed phenotype and invasive phenotype. Biochem. Pharmacol. 47( 1):137-143. Chalepakis, G., Fritsch, R., Fickenscher, H., Deutsch, U., Goulding, M., and Gruss, P. (1991). The molecular basis of the undulatedil‘ax-l mutation. Cell (Cambridge, Mas. ) 6 6 8 7 3 4 8 4 . Chalepakis, G . , Tremblay, P., and Gruss, P. (1992). Pux genes, mutants and molecular function. J. Cell Sci. 16(Suppl.):61-67. Chalepakis, G., Stoykova, A., Wijnholds, J., Tremblay, P., and Gruss, P. (1993). Pax: Gene regulators in the developing central nervous system. I. Neurobiol. 24( 10):1367-1384. Chalepakis, G., Goulding, M., Read, A., Strachan, T., and Gruss, P. (1994). The molecular basis of splotch and Waardenburg Pax-3 mutations. Proc. Nutl. Acad. Sci. U.S.A. 91:3685-3689. Collins, V. P., and James, C. D. (1993). Gene and chromosomal alterations associated with the development of human gliomas. FASEB J. 7:926-930. Czerny, T., Schaffner, G., and Busslinger, M. (1993). DNA sequence recognition by Pax proteins: Bipartite structure of the paired domain and its binding site. Genes Deu. 7:2048-2061. Davis, R. I., DCruz, C. M., Lovell, M. A., Biegel, J. A . , and Barr, F. G. (1994). Fusion of PAX7 to FKHR hy the variant t( l;l3)(p36;q14) translocation in alveolar rhabdomyosarcoma. Cancer Res. 54:2869-2872. Deutsch, U., Dressler, G. R., and Gruss, P. (1988). Pax!, a member o f a paired box homologous murine gene family, is expressed in segmented structures during development. Cell (Cambndge, Mas.)53:617-625. DeVita, G., Barha, P., Odartchenko, N.,Givel, J. C., Freschi, G., Bucciareli, G., Magli, M. C., Bonicelli, E., and Cillo, C. (1993). Expression of homeohox-containing genes in primary and metastatic colorectal cancer. Eur. J. Cancer 29(A):887-893. Dressler, G. R., and Douglass, E. C. (1992). Pax-2 is a DNA-binding protein expressed in embryonic kidney and Wilms tumor. Proc. Natl. Acud. Sci. U. S. A. 89: 1179-1 183. Dressler, 0. R., Deutsch, U., Chowdhury, K., Nornes, H. O., and Gruss, P. (1990). P a d , a new murine paired-box-containing gene and its expression in the developing excretory system. Dewlopment (Cumbndge, UK) 109:787-795. Fearon, E. R., and Vogelstein, B. (1990). A genetic model for colorectal tumorigenesis. Cell (Cambndge, Mass.) 61:759-767. Fearon, E. R.,Cho, K. R., Nigro, J. M., Kern, S. E., Simons, J. W., Ruppert, J. M., Hamilton, S. R., Preisinger, A. C., Thomas, G., Kinzler, K. W., and Vogelstein, B. (1990). Identification of a chromosome 18q gene that is altered in colorectal cancers. Science 247:49-56. Fritsch, R., and Gruss, P. (1993). Murine paired box containing genes. In “Cell-cell Signalling in Vertebrate Development” (E. Robertson, F. R. Maxfield, and H. J. Vogel, eds.), pp. 229-245. Academic Press, San Diego, CA. Fults, D., Brockmeyer, D., Tullous, M. W., Pedone, C. A., and Cawthon, R. M. (1992). p53 mutation and loss of heterozygosity on chromosomes 17 and 10 during human astrocytoma progression. Cancer Res. 52:674-679. Galili, N., Davis, R. j., Fredericks, W. J., Mukhopadhyay, S., Rauscher, F. J., Emanuel, B. S., Rovera, G., and Barr, F. G. (1993). Fusion of a forkhead domain gene to PAX3 in the solid tumor alveolar rhahdomynsarcoma. Nat. Genet. 5:230-235. Gaunt, S.J., Blum, M., and De Robertis, E. M. (1993). Expression of the mouse goosecoid gene during mid-embryogenesis may mark mesenchymal cell lineages in the developing head, limbs and body wall. Development (Cnmbndge, UK) 117:769-778. Gavin, B. J., McMahon, J. A., and McMahon, A. P. (1990). Expression of multiple novel Wnt-i tint-] -related genes during fetal and adult mouse development. Genes Deu. 35:43-54. Gehring, W. 1. (1987). Homeo boxes in the study of development. Science 236: 1245-1252. Gehring, W. J., Qian, Y. Q., Billeter, M., Furukuho-Tokunaga, K., Schier, A. F., R.-P. D., Affolter, M., Otting, G., and Wiithrich, K. (1994). Homeodomain-DNA recognition. Cell (Cambridge, Mass.) 78:211-223.

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I

Sex Determination and Its Pathology in Man

Alan J. Schafer

Department of Genetics University of Cambridge Cambridge CB2 3EH United Kingdom

The study of mammalian sexual development has led to the recognition of three sequential processes: establishment of chromosomal sex at fertilization, the development of the undifferentiated gonads into testes or ovaries, and the subsequent differentiation of internal ducts and external genitalia as a result of endocrine functions associated with the type of gonad present. Development of the sexual phenotype is the result of complex interactions of genetic, cellular and hormonal signals which participate in a cascade of events required to generate the male or female phenotype. The phenotypic differences between males and females are readily apparent and are often taken as absolute. However, aberrations can occur at many points during development and can lead to a discrepancy between the karyotypic sex and the phenotypic sex (sex reversal). The reversal can be complete, with chromosomal males developing fully as females or chromosomal females developing as phenotypic males. In some cases features of both sexes develop partially or wholly and an individual presents with ambiguous genitalia. Major contributions to our current understanding of sex determination and differentiation in humans have come from studies of patients with abnormal sexual phenotypes. Many nonlethal aberrations occur which disrupt sexual development and the variety of distinct phenotypes suggests that they may represent mutations in many of the loci important in sex determination. Abnormalities are frequently diagnosed due to observation of anomalous external Advances in Genetics, Val. 33 Copyright 0 1995 by Academic Press, Inc. All rights of repraduction in any form reserved.

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genitalia at birth, by the appearance of somatic irregularities which accompany some sex chromosome aberrations, or as a result of postpubertal anomalies (including infertility). Advances in cytogenetic and molecular techniques have complemented clinical studies and the investigation of karyotype-phenotype correlations continues to be crucial in the identification of genes involved in sex determination and in furthering our understanding of the pathology of sex reversal.

A. Biology of sexual development Pioneering studies of sexual development performed by Jost (1947, 1953) and lost et al. (1973) demonstrated that the dimorphic sexual phenotype in mammals is mediated by the testes via hormonal secretions. Key experiments involved the in utero gonadectomy of embryonic rabbits at a developmental stage prior to the differentiation of the internal and external genitalia. Ovariectomy of female embryonic rabbits led to a normal female phenotype, showing that organogenesis of female sex characteristics proceeds in the absence of ovaries. Castration of male fetal rabbits before a critical point also resulted in animals which developed internally and externally as females. These experiments indicated that the presence of testes prevented the genital tract from becoming a female and imposed male phenotypic development on the embryo. lost showed that the testes mediated these events by repression of the presumptive female ducts and by secretion of a masculinizing hormone (Jost et al., 1973). The experiments made clear that sex determination is controlled by the specification of male gonadal development and are the basis for the distinction between sex determination-the control of events leading to the development of gonadal (primary) sex-and sex differentiation-subsequent development of the genital tract in response to endocrine events associated with the type of gonad present. The focus of this chapter is on the molecular pathology of sex determination. The developmental events involved in the formation of the human sexual phenotypes are illustrated in Figure 9.1. The ovaries and testes arise from the genital ridge, which contains a bipotential (or indifferent) gonad composed of three cell types: cortical and medullary cells derived from the mesonephric region, and germ cells which arise extragonadally in connection with endoderm and migrate to the site of the gonad primordia (Byskov, 1986; Wylie and Heasman, 1993). During early mammalian development, two pairs of ducts form from discrete primordia. The Wolffian ducts are progenitors of the male accessory structures: the epididymis, vas deferens, and seminal vesicle. Female accessory structures (the uterus, fallopian tubes, and vagina) develop from the Mullerian ducts. The fetal testes play a decisive role in controlling the differentiation of the genital ducts. Testicular differentiation occurs earlier than ovarian differentiation, so

Figure 9.1. Overview of sexual development in humans. XX and XY indicate chromosomal sex. AMH, anti-Mullerian hormone; DHT, dihydrotestosterone. Solid lines indicate direct cellular lineage. Dashed lines represent hormone action on target tissues (and do not necessarily correlate with the time line). See text for details.

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the first sexually dimorphic developmental event observed is the cellular and structural organization of the developing testes. Ovaries at the same stage of development do not undergo these obvious changes. In developing testes, Sertoli cells differentiate from the cortex, a process which is probably induced by the Y chromosome (Palmer and Burgoyne, 1991). Sertoli cells produce antiMiillerian hormone (AMH, also called Miillerian inhibiting substance or Miillerian inhibitory factor) which causes regression of the Miillerian ducts, thereby blocking development of the female accessory structures (Jost et al., 1973). Leydig cells arise from the medulla and probably require the presence of Sertoli cells. The primary function of Leydig cells is the production and secretion of testosterone, which masculinizes the genital tract. Testosterone stimulates the Wolffian ducts to differentiate into the internal male organs and local conversion into dihydrotestosterone mediates the masculinization of the urogenital sinus and external genitalia. The ability of testes to differentiate in the absence of germ cells demonstrates that the signals for testicular differentiation are expressed in the somatic cells and emphasizes the importance of Sertoli cells in testis formation (Grumbach, 1968; McLaren, 1985). The development of the ovaries is more subtle than that of the testes, with definitive evidence of a differentiated ovary appearing in humans at 3 months gestation when the oogonia enter meiosis. The cortex of the indifferent gonad becomes the area of ovogenesis, supplying primitive oogonia with follicle cells while the medulla supplies interstitial cells. Ovarian differentiation requires the presence of germ cells. In the absence of inhibition, the Miillerian ducts develop into the internal female structures. The lack of masculinizing hormones leads to regression of the Wolffian ducts and to female development of the external genitalia. Upon examination of the sexual development pathways, it is apparent that mutations occurring in genes at different levels in the cascade can lead to varying degrees of sex reversal. A complete failure of testis determination will result in the development of a female phenotype, while mutations resulting in partial testicular development can give rise to varying degrees of masculinization.

II. MOLECULAR GENETICS OF SEX DETERMINATION A. The chromosomal basis of sex determination Sexual dimorphism in humans is obvious, but the source of the differences is not. Mendel (1865) quite reasonably speculated that determination of the sexual phenotype may be the result of heredity and segregation, a view elaborated upon following the “rediscovery” of Mendel’s work (Strasburger, 1900; Bateson and

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Saunders, 1902; Castle, 1903). Although a sex chromosome was first visualized more than 100 years ago (Henking, 1891), the establishment of a chromosomal basis for sex determination required the association of chromosomes with genetic information and the distribution of Mendelian traits (Sutton, 1903; Bridges, 1916). Through cytological studies on insects, it became clear that there were constant differences in the chromosome content of male and female cells (Doncaster, 1914). In certain species, one sex was seen to have a chromosome (called an accessory chromosome) which lacked a pairing partner. This chromosomal configuration was referred to as “XO.”In other insect species, both sexes had the same number of chromosomes, but the cells of one of the sexes contained a chromosome pair which was unequal in size and shape (heteromorphic). The other sex had the larger of the heteromorphic pair (the X chromosome) present in two copies and lacked the smaller (Y) chromosome. These observations provided a model for chromosome dependent sex determination. The sex with heteromorphic chromosomes would be heterogametic, producing one type of gamete containing an X chromosome and another type containing a Y chromosome. (In species with an accessory chromosome, either the accessory chromosome or no sex chromosome would be present.) The sex of an offspring would depend on the combination of sex chromosomes it received from its parents and would thus be determined by the type of gamete donated by the heterogametic parent. In addition to establishing the heterogametic parent as being sex determining, the model accounted for the observation that, in most cases, half of the offspring were female and the other half were male. Early observations of human cells revealed humans to be a species in which the male has one chromosome less than the female. In studies of germ cells, all ova were seen to contain an X chromosome, while only half of spermatozoa contained an X chromosome (Guyer, 1910; von Winiwarter, 1912). These observations led to the conclusion that “. . . man belong(s) to the class with two sorts of spermatozoa and with all eggs alike in respect of their chromosomes. . .” (Doncaster, 1914). Coupled with observations of sex-limited inheritance patterns, it was concluded that in humans the male is sex determining. This invalidated a well-developed theorem of the time which held that human females were sex determining and that male-determining and femaledetermining ova were alternately discharged from right and left ovaries, respectively (Dawson, 1909). A chromosomal mechanism of sex determination was first elucidated in studies of Drosophila. Breeding experiments which resulted in aberrant numbers of sex chromosomes relative to autosomes showed that the Drosophila Y chromosome is not important in primary sex determination, but rather it is the ratio of X chromosomes to autosomes. Normally, female flies are XX (1:l X chromosome:autosome ratio) and males are XY (1:2 X chromosome:autosome ratio).

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XXY flies are female (1:l ratio), XO flies are male (1:2 ratio), and triploid XXY flies are intersex (2:3 ratio) (Bridges, 1921). The observations of X dosage as the genetic determinant in Drosophila and the apparent dispensability of the Y chromosome led to the belief that XY sex chromosome pair was a primitive system from which the XO (accessory chromosome) system was derived (Crew, 1933).

The X chromosome dosage mechanism of sex determination in insects was held as a model for mammalian sex determination, so the observation of a Y chromosome in humans by Painter (1921) did not implicate it as having an important role in sex determination. This study also reported the human diploid chromosome number as 46 or 48. In subsequent studies the number was established as 48 (Evans and Swezy, 1929) and would remain unchanged for more than 25 years. Following advances in mammalian cell culture and cytogenetic techniques, Tjio and Levan (1956) observed 46 human chromosomes and asserted this to be the true number. The dogma of 48 chromosomes in humans was so generally accepted at the time that in studies of human liver cells conducted by the researchers Hansen-Melander, Melander and Kulander, the finding of 46 chromosomes in the liver cells resulted in their temporary abandonment of the experiments (Tjio and Levan, 1956). The necessity of the Y chromosome was debated; however, its presence was taken by some to imply that it must have a function (Stem, 1957). The improvement in cytogenetic techniques of the 1950s also led to the elucidation of the mechanism of chromosomal sex determination in humans. In 1959, several papers were published which made it clear that the sex chromosomes functioned differently in humans and mice than in Drosophila. The finding of a 45,X female (Ford et al., 1959b) and male development in a 47,XXY individual (Jacobs and Strong, 1959) were key observations indicating that in the absence of a Y chromosome the human sexual phenotype was female, and that the Y chromosome is a dominant inducer of the male phenotype. The dominance of the Y chromosome was further demonstrated with the description of a 48,XXY +21 karyotype in a man with both Klinefelter and Down syndromes (Ford et al., 1959a) and reports of 48,XXXY and 49,XXXXY individuals with an unmistakably male phenotype (Barr et al., 1959; Fraccaro et d . , 1960; Fraccaro and Lindsten, 1960). These observations, along with the demonstration of female development of XO mice (Russell et al., 1959; Welshons and Russell, 1959) and XXY male mice (Cattanach, 1961; Russell and Chu, 1961), firmly established the role of the Y chromosome in initiating the development of a male phenotype in mammals. The concept of the XY system as being more primitive than the XO system was subsequently revised and remnants of a dosagedependent sex-determining mechanism may still be extant in mammalian sex determination (Marshall Graves and Reed, 1993; see Sections II,D,4 and

III,A,8).

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Although the Y chromosome clearly carried at least one genetic determinant involved in directing the undifferentiated gonad to develop as a testis (Ford, 1970), it was uncertain if the Y chromosome contained all of the genes necessary for male development or if a single, activating locus was Y specific and other genes involved in testis formation were located on different chromosomes. The nature of the determinant was unknown: it might have been a single gene, multiple genes, or noncoding regulatory sequences. In humans, the Y chromosome signal responsible for induction of testis formation became referred to as the testis determining factor, or TDF. Regardless of the composition of TDF, its localization to the Y chromosome provided a focus for investigation of a fundamental component of the testis determination pathway and stimulated intensive research aimed at identifying TDF.

B. Early candidates for the testis determining factor 1. Histocornpatability Y antigen A male-specific antigen was first detected in experiments in which female inbred mice rejected skin transplants from isogenic males (Eichwald and Silmser, 1955). The male-specific expression indicated that the Y chromosome contained the antigen structural gene or a gene(s) regulating antigen expression. Additional detection systems for histocompatability-Y (H-Y) antigen which did not require isogenic strains and transplantation were developed, one serologically based assay and another relying on cell-mediated cytotoxicity. The H-Y antigen phenotype was found to be dependent upon the method that was used, suggesting the existence of multiple male-specific antigens and that different genes were detected by each assay. The antigens are thus referred to as transplantation antigen (H-Yt), serological antigen (H-Ys), and cytotoxic assay detected (HYc), although the cytotoxic assay probably detects the same antigen as that which is involved in transplantation rejection (Wiberg, 1987). H-Ys is sometimes referred to as SDM (serologically detected male antigen). Several features of H-Ys led to the proposition that it was the Y chromosome factor responsible for male differentiation (Wachtel et al., 1975). In addition to male-specific expression, the antigen was shown to be evolutionarily conserved in animals with heterogametic sex determination (and only detected in the heterogametic sex of those animals). Examination of H-Ys in human XY females with Y deletions and XX males containing Y chromosome translocations showed that in some cases H-Ys expression was concordant with the male phenotype (Ohno et al., 1979; Wachtel, 1983), but discordancies were also reported. Some XY females with dysgenetic gonads were lacking H-Ys antigen (Ghosh et al., 1978), but not all phenotypically normal males were H-Ys positive (Teyssier et al., 1983) and the presence of testicular tissue was not always associ-

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ated with H-Ys expression (Haseltine et al., 1984). The lack of H-Ys in XX male mice carrying a minimal testis determining portion of the Y chromosome conclusively removed it as a candidate for TDF (McLaren, 1988; Goldberg et id., 1991). H-Ys may be involved in other aspects of male sexual development. Normal XY testicular cells dissociated and treated with H-Y antibody have been shown to reaggregate into ovarian follicle-like structures (Ohno et al., 1978; Zenzes et al., 197813) and H-Ys induces formation of seminiferous tubules in organ cultured fetal bovine ovaries (Ohno et al., 1979). Transgenic mice chronically expressing AMH have been observed to have seminiferous tubules (Behringer et al., 1990) and both AMH and H-Ys are secreted by Sertoli cells (Zenzes et al., 1978a), prompting speculation that AMH and H-Ys are related. Crossreactivity of H-Y antisera with AMH has been shown (Muller and Wachtel, 1991), suggesting that they are the same or share a common epitope; however, this proposition is qualified by indications that H-Y antisera cross-reacts with some other proteins (Lau et al., 1989; Su et al., 1992). H-Y antigen as defined by the T cell cytotoxicity assay (H-Yc) maps to a different region of the Y chromosome than H-Ys and is distinct from TDF as shown by discordance between H-Yc expression and sexual phenotype in XX male mice, human XX males with translocated Y chromosome sequences, and XY females with Y chromosome deletions (McLaren et al., 1984; Simpson et al., 1987). The observation of male mice which do not express H-Yc and have a failure in spermatogenesis led to speculation that H-Yc is involved in spermatogenesis (Burgoyne et al., 1986), but the detection of H-Yc in azoospermic and oligospermic human males with deleted Y chromosomes excludes H-Yc as being the Y chromosome azoospermia factor(s) (Simpson e t at., 1993). Recently, a mouse Y chromosome gene has been cloned which maps to the human Y chromosome deletion interval containing H-Yc. The gene is evolutionarily conserved on the Y chromosome of eutherians and metatherians, and is a good candidate for the H-Yc gene (Agulnik et al., 1994).

2. Banded krait minor (Bkm) satellite DNA In snakes and birds, females are the heterogametic sex and their sex chromosome pair is termed ZW. Males have a ZZ sex chromosome pair. Repetitive DNA sequences isolated from the banded krait snake were found to be concentrated on the W chromosome in association with heterochromatin with a low abundance on autosomal chromosomes. The DNA sequences, consisting primarily of GATA repeats, were isolated from density gradients as a “satellite” band partitioned away from the principle genomic DNA, and so were termed Bkm satellite DNA (Singh et al., 1981). The repeats were found to be phylogenetically conserved in animals ranging from mammals to insects and were present in the

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heterogametic sex of many species of reptiles, birds, and mammals. Bkm sequences were found clustered in the sex-determining region of the mouse Y chromosome, leading to the hypothesis that they functioned as sex-determining sequences (Jones and Singh, 1981). When used as a probe on a strain of mice which bore some offspring as XX males, Y chromosome Bkm sequences were shown to be located o n one of the X chromosomes of the XX male mice (Singh and Jones, 1982). This explained the sex reversal of these XX mice as being the result of a submicroscopic transfer of the Y chromosome sex-determining region onto one of the X chromosomes. Although this demonstrated close linkage to TDF, in humans Bkm sequences were found to be spread throughout the genome and not concentrated in the sex-determining region and therefore were unlikely to be involved in sex determination (Kiel-Metzger et al., 1985).

C. Cloning of the testis determining factor 1. Structure of the Y chromosome The human Y chromosome is a small acrocentric chromosome approximately one-third the size of the X chromosome. The short arm (Yp) contains approximately 13 megabasepairs (Mb) of DNA, the long arm (Yq) contains 46 Mb. Cytogenetically, the chromosome consists of a euchromatic region and a heterochromatic region (Figure 9.2). The heterochromatic region contains highly repeated sequences located on distal Yq which display a distinctive fluorescence when stained with quinacrine. Apart from the centromere, the remainder of the chromosome is euchromatic. During male meiosis, the sex chromosomes do not

Figure 9.2. An ideogram of a G-banded human Y chromosome. PAR1 is the Yp pseudoautosomal region and is the region of cytologically discernible X-Y pairing. A second pseudoautosomal region (PARZ) located on the tip of Yq is not indicated.

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pair across their entire length as do other chromosome pairs; rather, the pairing is ordinarily limited to the distal portion of the short arms of X and Y. Pairing is variable though, and can extend into the long arm of the Y chromosome (Chandley et al., 1984). Chromosome pairing has long been equated with regions of shared genetic homology (Muller, 1918) and the unpaired regions were interpreted as containing X and Y chromosome nonhomologous regions involved in the sex-determining function of these chromosomes. Citing observations of partial pairing of sex chromosomes in Drosophila and Melandrium, Crew (1933) described the pairing regions as consisting of homologous genic constitution and maintained that “the production of gametes qualitatively different in respect of the genes corresponding to the characters of sex is made possible by the existence of a mechanism which prevents crossing-over of the mutated and heterologous portions of the sex chromosomes.” Observations of crossing over in male rat meioses provided evidence that the pairing segments of mammalian sex chromosomes were regions of homology (Koller and Darlington, 1934). The sex chromosome pairing regions were predicted to behave with regard to recombination and inheritance in the same manner as autosomal chromosomes, and these portions of the X and Y chromosome were termed “pseudoautosomal” regions (PAR) (Burgoyne, 1982). Studies of the inheritance of polymorphic pseudoautosomal markers have demonstrated that transfer of homologous DNA sequences does occur between the pairing regions (Cooke et al., 1985; Simmler et al., 1985; Rouyer et al., 1986). A sharp transition in DNA sequence homology occurs at the boundary between the Yp pseudoautosomal region and the Y unique sequences (Ellis et al., 1989) and TDF was expected to reside in the Y-unique sequences lying outside of the pseudoautosomal region. An additional, smaller pseudoautosomal region has recently been identified at the ends of the long arms of the X and Y chromosomes. No genes have been identified in this region and its role in pairing is unknown. Genes which are exclusive to the Y chromosome are by definition male specific and might be predicted to play a role in male development. Not surprisingly, the functions of the few genes cloned from Y-unique regions have been shown to be, or are proposed to be, involved in testis formation and function (Schafer, 1994).

2. Deletion mapping of the Y chromosome The sublocalization and cloning of TDF was achieved as the result of establishing correlations between sex chromosome abnormalities and sexual phenotype. Initial observations of the absence of an entire sex chromosome were made in 45 ,X Turner syndrome patients. These individuals display normal female external and internal genitalia but are infertile due to the presence of dysgenetic “streak” gonads. Other salient features are short stature, sexual infantilism at

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puberty, short IVth metacarpals, multiple pigmented naevi, shield chest, and congenital heart disease (Grumbach and Conte, 1992). These individuals may be viewed as lacking an X chromosome or as lacking a Y chromosome. From the viewpoint of being X deficient, the implications are that some X chromosome genes are needed in a double dose for normal ovarian and normal somatic development. Viewed as being Y chromosome deficient, the absence of TDF results in female development, and the somatic stigmata result from the absence of genes common to the X and Y chromosomes which are normally required in a double dose. Many Y chromosome deletions resulting in female development in 46,XY individuals are not accompanied by the somatic Turner features, demonstrating that the loci responsible for these phenotypes are distinct from TDF. Initially, Y chromosome rearrangements were used to map the location of TDF to the Y chromosome short arm. Two phenotypically female individuals were reported to have a single normal X chromosome along with an isochromosome for the long arm of Y (Yq duplication with the loss of Yp) (Jacobs and Ross, 1966). The absence of the Y short arm and the sex-reversed phenotype in these patients implicated Yp as containing the male-determining region. Over the next 15 years several hundred Y chromosome structural anomalies were documented and examined for phenotype-karyotype correlations (Davis, 1981). The compiled data convincingly demonstrated that TDF was located on the short arm of the Y chromosome and that most of the long arm could be excluded from containing male determining factors, although some studies suggested that a factor(s) influencing testis formation may be in the pericentric region of the long arm. Further progress came from the investigation of 46,XX males. These subjects are seemingly paradoxical, developing as phenotypic males in the presence of a normal female 46,XX karyotype (de la Chapelle et ul., 1964). The simplest explanation for the sex reversal is the presence of Y chromosome sequences (including TDF), either as a result of mosaicism or due to the submicroscopic presence of Y sequences in all cells. Efforts to detect cryptic sex chromosome mosaicism, for instance, involving 46,XY or 46,XXY cells, showed this to be an unlikely mechanism of sex reversal in the majority of patients. Insight into a cause of XX sex reversal came via studies of the inheritance patterns of the X-specific blood group antigen Xg. In two families, XX males were discovered who had not inherited their fathers’ Xg allele. FergusonSmith proposed ( 1966) that an aberrant meiotic recombination event between the paternal X and Y chromosomes resulted in the X chromosome losing the paternal Xg allele and acquiring a male-determining gene horn the Y chromosome (Figure 9.3). Direct evidence supporting the “X-Y interchange’’model was found in expression studies of the cell surface protein 12E7, the product of the pseudoautosornal gene M E 2 (Goodfellow et al., 1983; Goodfellow et al., 1986). An XX male was identified as expressing a Y-linked gene controlling 12E7

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Figure 9.3. X-Y interchange. Normal meiotic recombination results in the exchange of homologous sequences between the pseudoautosomal regions (PAR) of the X and Y chromosomes. Aberrant recombination can occur, extending beyond the regions of X-Y homology and resulting in the reciprocal exchange of nonhomologous chromosome sequences. If the abnormal interchange includes the Y chromosome testis determining factor (TDF), the resultant X chromosome will carry TDF, be testis determining, and give rise to an XX male. T h e Y chromosome, having lost its testis determining function, can result in an XY female. In the diagram, the interchange also results in the transfer of the X-specific locus Xg to the Y chromosome. T h e size of the pseudoautosomal region is exaggerated for clarity.

expression, but failing to express his father’s allele for Xg (de la Chapelle et al., 1984). Further support for the X-Y interchange model was provided in flow cytometry studies of chromosomes of XX males showing one enlarged X chromosome, and high resolution chromosome banding of XX males which revealed the transfer of some distal Yp material to Xp (Madan, 1976; Evans et al., 1979; Magenis et al., 1982; Ferguson-Smith, 1988). The development of DNA probes provided a means to directly test the Y DNA content in XX males. Submicroscopic Y sequences were found on an X chromosome in XX males and X-Y interchange was confirmed (Guellaen et al., 1984). The aberrant transfer of Y sequences (including the sex-determining region) to the X chromosome by X-Y interchange has subsequently been shown to be responsible for the great majority of XX males (Affara et al., 1986; Anderson et al., 1986; Muller et al., 1986; Buckle et al., 1987; Petit et al., 1987; Palmer et al., 1989). The application of molecular techniques to evaluate deletions of the Y chromosome in XY females and the Y chromosome sequences present in XX males led to the cloning of TDF. Deletion maps based on the genomes of these individuals were constructed by several laboratories and TDF was mapped to the most distal portion of the Y-unique region of the short arm of the Y chromosome, adjacent to the pseudoautosomal boundary (Page, 1986; Vergnaud et al.,

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1986; Affara et al., 1987; Miiller, 1987; Page et al., 1987a). Meiotic mapping positioned the gene MIC2 as the most proximal pseudoautosomal locus known (Goodfellow et al., 1986) and the meiotic map was integrated with the deletion maps by long-range physical mapping (Pritchard et al., 1987). Probes from the MIC2 region were used to map an area extending from the pseudoautosomal region into the adjoining Y-unique region, spanning the pseudoautosomal boundary. Within the Y-unique region, a CpG-rich region similar to ones often associated with genes was identified.

3. Zinc finger Y gene (ZFY) A 140-kb interval thought to contain the testis determining factor was defined by aligning the Y-specific DNA present in an XX male with a Y chromosome deletion of the same region in an XY female (Page et al., 1987b). In a chromosome walk across the region, sequences associated with the CpG island near the pseudoautosomal boundary showed evolutionary conservation and were used to identify a gene encoding a protein with multiple “zinc finger” domains, a characteristic of a class of proteins which bind DNA in a sequence-specificmanner and regulate transcription. Based on its position and structural similarity to regulatory genes, ZFY was proposed as a candidate for TDF (Page, 1988). Some characteristics of ZFY did not agree with predictions for TDF. Contrary to expectations, ZFY was found to have a homologous locus on the human X chromosome (ZFX), so models of ZFY as TDF were derived to allow for homologs in males and females (Page, 198713). Comparison of the ZFY and ZFX predicted protein sequences showed that they were highly similar (SchneiderGadicke et al., 1989). The ZFX gene was found to escape X inactivation in humans, implying that a double dose of what appeared to be the same gene was expressed in both males (ZFY and ZFX) and in females (2 doses of ZFX) (Schneider-Gadicke et al., 1989; Palmer et al., 1990). Furthermore, expression of the two genes was not limited to tissue in the gonad lineage; rather, transcripts were present in a wide range of adult and fetal human tissues (Page et al., 198710; Lau and Chan, 1989; Palmer et al., 1990). Studies in the mouse showed the presence of two Y chromosome genes related to ZFY; Zfy-J and Zfy-2 (Mardon et al., 1989; Nagamine et at., 1989). Zfy-2 was not present in XXSxr’ (XXSxrb) male mice which carried a small sexreversing translocation of the testis determining region. From this it was inferred that Zfy-2 was not important in mouse testis development (Page, 1988). Zfy-2 expression was not found in the developing male gonad, and although Zfy-1 expression was found in fetal male gonads at the time of overt testis differentiation, the expression was dependent upon the presence of germ cells (Koopman et al., 1989). As testes can form in the absence of germ cells, this was used as an

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argument against a role for Zfy-1 in sex determination. More recent studies, however, have shown expression of Zfy-1 in mice lacking germ cells (Mastrangelo et al., 1994; Zambrowicz et al., 1994). Investigations of marsupials, which have a Y-dominant male sex determination like eutharians, showed that ZFX and ZFY were not on the sex chromosomes, but were autosomal (Sinclair et al., 1988). These findings strongly implied that ZFY was not TDF. Genetic evidence contradicting the assignment of ZFY as TDF was provided with the description of XX males with Y chromosome sequences but lacking ZFY (Palmer et al., 1989). It would be highly improbable to have X-Y interchange unrelated to the sex reversal in these individuals, so the absence of ZFY implied that it was not TDF. The range of expression and the structure of ZFY and ZFX implicate them in a general role in transcriptional regulation, but their function is currently unknown.

4. Sex-determining region Y gene (SRY) The discovery of Y-specific sequences in XX males lacking ZFY imposed new limits on the location of TDF, placing it in a 35-kb region adjacent to the pseudoautosomal boundary. Using probes from this region a single-copy malespecific sequence was found which was evolutionarily conserved (Sinclair et al., 1990). Sequence analysis revealed an open reading frame and the gene was termed SRY in humans and Sry in mouse (Gubbay et al., 1990). Mouse Sry was shown to map to the mouse Sxr’ (Sxrb) chromosome which contained the minimal male-determining region of the mouse Y chromosome known at the time (Gubbay et d., 1990) and was located within an 11-kb deletion of the mutant Ytdv-ml chromosome which had lost its sex-determining function (Lovell-Badge and Robertson, 1990). The marsupial Y chromosome is sex determining and contains SRY, as predicted for TDF (Foster et al., 1992). Subsequent studies of the XY female used to define the region from which ZFY was cloned showed that she had a second, previously undetected deletion which removed SRY (Page et al., 1990). SRY has many of the predicted properties of TDF and considerable evidence has accumulated equating SRY with TDF. a. Gene structure Analysis of the SRY open reading frame revealed a predicted protein of 223 amino acids which contained a highly conserved 79-amino acid region with homology to a DNA-binding motif referred to as the HMG-box. This binding domain had been previously defined based on sequence similarity between the chromatin-associated protein HMG- 1 and the nucleolar transcription factor UBF (Jantzen et al., 1990) and was subsequently found in other DNA-binding proteins including several transcription factors (Nerr, 1992). Using the SRY

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HMG-box as a probe in genomic library screens, a subfamily of closely related genes was identified. Members of this family were defined as those encoding a region with 60% or greater amino acid similarity to the SRY HMG-box motif and were called SOX (SRY-box related) genes (Gubbay et al., 1990; Denny et al., 1992a; Goodfellow and Lovell-Badge, 1993). Comparison of the SRY protein coding regions outside of the HMG-box of human, mouse, and marsupial SRY revealed no significant homology (Foster et al., 1992). Alignment of the nonHMG-box regions of several primate species showed poor conservation and many amino acid changes (Whitfield et at., 1993). This suggests that SRY regions outside of the HMG-box may not have an important function or that sequence changes have been driven by strong evolutionary forces (Hurst, 1994).

b. Mouse expression The testis determining factor must exert its influence prior to the overt differentiation of testes, and any candidate for TDF is expected to be expressed at some stage in the developing indifferent gonad. Sry expression has been investigated in developing mice and was shown to be consistent with a role in testis determination. In mice, gonads developing from the genital ridge show sexually dimorphic differentiation at 12.5 days postcoitum (dpc). Using reverse transcriptionPCR (RT-PCR) on mouse embryos, Sry expression was detected in genital ridge at 10.5, 11.5, and 12.5 dpc, and no expression was found in any other embryonic tissue tested. In mice lacking germ cells, the genital ridge still expressed Sry, demonstrating that embryonic expression occurs in the somatic portion of the genital ridge (Koopman et al., 1990). In adult male mice, Sry expression is detected exclusively in the testes, primarily in pre- and postmeiotic germ cells (Koopman et al., 1990; Rossi et al., 1993). Sry does not have a cell autonomous role in testis, as the Sry-deleted Ytdv-ml chromosome is transmissible through sperm in chimeric animals (LovellBadge and Robertson, 1990). No testicular rote for Sry has been proposed, and the mouse transcript exists predominantly as an unusual nonpolyadenylated circular message which does not appear to be translated (Cape1 et al., 199313). Sry circles have been detected in pre- and postmeiotic germ cells and in adult testicular interstitial cells which are not Leydig cells (Zwingman et al., 1994). This unexpected transcript structure may reflect a form of control in testes, but might also be a consequence of the mouse Sry inverted repeat genomic structure (Gubbay et al., 1992), making an important role in testicular function questionable. The detection of linear and circular Sry transcripts in preimplantation embryos has been reported, commencing as early as the two-cell stage (Zwingman et al., 1993; Boyer and Erickson, 1994), but currently there is no evidence for a function at this stage of development.

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c. Human expression

The human SRY locus is a single exon gene with multiple clustered transcription-initiation sites (Behlke et al., 1993; Clepet et al., 1993; Su and Lau, 1993). In an RT-PCR study of SRY expression, transcripts were detected in all fetal male and several adult male tissues tested (Clepet et al., 1993). The RTPCR assay is highly sensitive, but only semiquantitative, so the relative abundance in each tissue is not known. Northern blot analysis detected SRY expression in adult testis and in tissue culture cell lines of hepatic and testis origin. In contrast to the mouse, a circular SRY transcript was not evident in adult human testis.

d. Protein function

The presence of the HMG-box in SRY predicted that the protein would bind to DNA. In vitro binding assays demonstrated sequence-specific binding of SRY to the recognition sites for the HMG-box proteins TCF-1 (AACAAAG) (van de 1991). Wetering and Clevers, 1992) and LEF-1 (NCAAAG) (Travis et Alteration of the core sequences reduced binding efficiency (Nasrin et al., 1991). The SRY DNA binding domain homology to that of TCF-1 and LEF-1 is about 3 0 4 0 % and other members of the HMG family also bind the same or similar sequences (Nasrin et al., 1991; Sugimoto et al., 1991; Denny et al., 1992b). No homology between protein sequences outside of the HMG-box is seen between different members of the HMG-box family. Using oligonucleotide selection, an optimal consensus target sequence of AITAACAAT was defined which had a fivefold greater affinity than the LEF-l/TCF-1 site (Harley et al., 1994). The range of proteins which bind to the same sequences indicates that the in vitro binding sites may not be the same as the in vivo sites and care must be taken when interpreting interactions based on in vitro binding or predicted binding sites. In addition to sequence-specific binding, the nuclear localization of the SRY protein (Poulat et al., 1995) and the demonstration of activation activity of a glutamine/histidine-rich region from the mouse protein are consistent with a role in regulation of transcription (Dubin and Ostrer, 1994). In vitro experiments have shown that sequences similar to the optimal SRY binding site are located in the promoter sequences of P450 aromatase (Haqq et al., 1993), of AMH (Haqq et al., 1993; Harley and Goodfellow, 1994), and of the insulin response element-binding protein IRE-ABP (AlexanderBridges et al., 1992), and that these elements are bound by SRY at high affinity. Each of these promoters was tested on the basis of sequence similarity with the SRY binding site; moreover, P450 aromatase and AMH have differential regulation during sexual development. P450 aromatase catalyzes conversion of testosterone to estradiol during female gonadal development and is downregulated in males. AMH induces regression of the Miillerian ducts during male development, is expressed in Sertoli cells, and initiates in the mouse at 12.5 dpc,

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overlapping with Sry expression. The mouse AmhlSry expression overlap raised the possibility that SRY directly activates AMH. Experiments using mouse Sertoli cell nuclear extracts have shown the presence of a factor which binds to the AMH promoter, but at a different site than the one which is bound by recombinant SRY and Sry (Shen et al., 1994). A cell line derived from the differentiating genital ridge of male rat embryos used in cotransfection experiments demonstrated SRY-dependent activation of constructs containing the AMH promoter (Haqq et al., 1994). Mutation of SRY at isoleucine 68 (a site of partial side chain intercalation) abolished its ability to induce transcription of the AMH reporter. However, mutation of the AMH promoter SRY in vitro binding site did not abrogate the activation, indicating that the effect is indirect and requires at least one other factor. Experiments have shown an interaction between SRY and fra-J (Cohen et al., 1994). The fra- J gene encodes a component of the transcription factor AP- 1 and is expressed during many developmental processes including spermatogenesis. An SRY consensus binding site in the fra-J promoter is bound in vitro by SRY. Reporter constructs containing native fra- J promoter elements including the SRY binding site were trans-activated by SRY, suggesting that the downstream effects of SRY may include activation of transcription factor AP-1. In addition to sequence specificity, other properties of SRY binding have been observed. As had been shown for the HMG-box of the transcription factor LEF-I, binding by SRY induced a bend in the DNA to which it was bound (Ferrari et al., 1992; Giese et al., 1992). This characteristic is compatible with a model of transcriptional regulation which involves contortion of DNA such that sequences (and any bound factors) separated by some linear distance are brought together to interact and exert their effect. Another characteristic is the in vitro binding of SRY to cruciform DNA (Ferrari et al., 1992). The meaning and relevance of this activity is currently unknown.

e. Mutations in sex-reversed patients

A n expectation for TDF was that mutations in its protein sequence would result in sex reversal. Examination of SRY sequence in XY females has identified more than 20 mutations in the protein coding sequences (references in Hawkins, 1993; Iida et al., 1994; Poulat et al., 1994; Tajima et al., 1994; Schmitt-Ney et al., 1995). All but one of these point mutations and microdeletions lie within the 237 base pairs coding for the DNA-binding domain. Some of the SRY mutations cause drastic alterations in the predicted protein by generating frameshifts or termination codons and others cause an amino acid change in positions conserved across species. In several instances, only the DNA-binding domain has been examined for mutations, so the clustering of mutations reported may be biased, but the importance of this region in the protein function is clear. In vitro binding assays using recombinant SRY with HMO-box mutations corresponding to those present in XY females have shown reductions in or a loss of sequence-

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specific DNA binding (Nasrin et al., 1991; Harley et al., 1992; Jager et al., 1993; Schmitt-Ney et al., 1995). The single mutation found in the SRY protein coding sequences which occurs outside of the DNA-binding domain is located 3' to the HMG-box. The mutation is unlikely to effect binding, indicating that this portion of the protein has other functional importance. One XY female has been shown to have a deletion of sequences 1.8 kb 5' to SRY but does not interrupt the transcription unit (McElreavey et al., 1992b). This deletion may interfere with SRY expression levels or patterns and may define DNA control elements. Similarly, deletions of mouse Y chromosome sequences outside of, and at a considerable distance away from Sry have been found which are sex reversing and the lack of Sry expression observed is presumably due to interference with the proper regulation of Sry (Cape1 et al., 1993a). SRY-induced DNA bending may be an important part of the protein function. Studies of DNA binding and bending using SRY mutations found in XY females have identified a mutation which causes a 100-fold reduced binding but with unchanged DNA-bending properties (Pontiggia et al., 1995). A second XY female mutation shows only slightly reduced DNA binding but has greatly altered bending properties. Taken together, these data suggest that the DNAbinding and DNA-bending activities of SRY can be decoupled. Another prediction for TDF is that its presence can cause XX male sex reversal. Most XX males have Y chromosome sequences which are evident either cytogenetically or by DNA marker analysis (Guellaen et al., 1984; Petit et al., 1987). All but one XX males with Y-specific sequences have been found to contain SRY. This correlation indicates a causative role of SRY in these cases of XX sex reversal.

f. Transgenic mice A direct test of the equivalence of SRY and TDF is to assess whether its presence in an XX genetic background (and in the absence of other Y chromosome sequences) can induce testis formation. This experiment was performed by generating transgenic mice using a 14-kb fragment of the murine Y chromosome containing Sry (Koopman et al., 1991). This large fragment was used in the hope that it would encompass essential regulatory elements; no additional genes were detected in the fragment by sequence analysis (Gubbay et al., 1992). Using this DNA segment XX transgenic mice were generated which showed phenotypically normal male internal and external genitalia along with small testes. Examination of the testes revealed histological features similar to those of testes in human XX males: lacking in spermatogenesis, with degenerating germ cells. The mice exhibited normal male sexual behavior when caged with female mice. Failure of some XX transgenic mice to develop as males was presumably due to position effects of the integration site of the transgene or due to mosaicism. The sex-reversed phenotype in the absence of other Y chromosome sequences dem-

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onstrates that Sry is the only Y chromosome gene required for testis determination, and that other genes involved in producing the male phenotype are located on the X chromosome or on autosomes. The temporal and tissue-specific regulation of the Sry transgene indicates control by upstream genes which are present in both XX and XY individuals. Transgenic mice were also made using the human SRY gene, but sex reversal was not observed despite expression of the transgene in the genital ridge (Koopman et al., 1991). The mouse and human gene sequences differ significantly outside of the DNA-binding domain, and these sequence differences may reflect functional differences. Although both proteins show specific binding to the same DNA sequence in oitro, close examination of binding properties has shown some differences between SRY and Sry HMG-box interactions, with the mouse protein showing a higher sequence specificity and more extensive minor groove contact than human SRY (Giese et al., 1994). The insufficiency of the human gene to sex-reverse mice may be a result of these differences or may be a result of variance in other aspects of the protein. Arguments have been put forth that sex determination is a result of a growth-promoting effect of the Y chromosome (Mittwoch, 1992, 1993). Subtle differences in growth rates of XX and XY mouse embryos have been observed in pregonadal tissue as well as in the embryo proper, first visible at the preimplantation stage. In CD1 mice, the growth effects have been shown to be due to the CD1 Y chromosome (Burgoyne, 1993), but no evidence directly links the growth difference to sex determination. Zwingman et al. (1993) suggested that expression of Sry or Zfy in preimplantation embryos might play a role in growth. An alternative explanation for Y chromosome growth effects is that the Y chromosome is an attractor for "selfish" growth factors which act as sex ratio distorters: in animals with multiple zygotic implantations, larger male embryos might outcompete their smaller embryonic sisters for available resources (Hurst, 1994). Experiments by Thomhill and Burgoyne (1993) have shown in 10.5 dpc mouse embryos that the slower growth of XX embryos is the result of the paternal X chromosome retarding development, and that the larger size of the XY embryo is neither necessary nor sufficient for testis determination. Further experiments should resolve whether the observed growth differences between mouse XX and XY embryos are developmentally relevant. Genetic and molecular data have established that SRY can be equated to TDF. Biochemical data have provided insight into how it may function, but the identities of genes which interact directly upstream and downstream with SRY are currently unknown.

D. Other genes involved in gonad determination The Sry-induced male development of XX transgenic mice demonstrated that Sry is the sole Y chromosome gene required for testis determination. The occur-

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Genital

Regression

Miillerian Duct

Ovary Theca Cells

Female Internal Genitalia

Figure 9.4. Molecular events in human sex determination. The genes S F - I , WTI, SRY, SOX9, and the locus DSS are shown at their known or putative position in the sex determination pathway. AMH, anti-Mullerian hormone; DHT, dihydrotestosterone.

rence of XX male humans with SRY as their only detected Y chromosomederived gene indicates that this is true in humans as well (Sinclair et al., 1990; Jager et al., 1990; Fukutani et al., 1993). Gonad differentiation undoubtedly involves many genes, with SRY acting as a differential in a complex reaction system which is encoded on chromosomes other than the Y. Many XY sex reversals cannot be accounted for by mutations in SRY and some XX males are not due to the presence of SRY (see Sections III,A and III,B,3). Some of these individuals are presumed to have mutations in an X chromosome or autosomal gene involved in testis determination. These observations are consistent with sex determination systems in Dosophila and Caenorhabditis (Hodgkin, 1990; Parkhurst and Meneely, 1994), in which most genes involved in sex determination are located on autosomes rather than on sex chromosomes. The roles of other genes known to be involved in mammalian gonad formation are described below and their potential or known position in the sex determination pathway is shown in Figure 9.4.

1. WTl The WI gene was isolated as the result of positional cloning experiments which identified an oncogene on human chromosome 11 involved in the etiology of the childhood kidney cancer, Wilms tumor (Call et al., 1990; Rose et al., 1990). The gene was originally localized by examining chromosomal deletions in children with WAGR syndrome (characterized by Wilms tumor, aniridia,

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genitourinary abnormalities, gonadoblastoma, and mental retardation) (van Heyningen and Hastie, 1992). The deletions presumably affected genes in addition to WTI which contributed to the diverse phenotypes, although mutations in a single gene could be responsible for several or all of the phenotypes. In mouse, the gene (designated WT-I) is expressed at 9 dpc in the genital ridge of both males and females prior to overt gonad sexual differentiation. Expression in mature gonads is restricted to the Sertoli cells of the testis and to the homologous female ovarian granulosa cells (Pritchard-Jones et al., 1990; Pelletier e t al., 1991c; Armstrong e t al., 1992). The WTI gene was found to be expressed primarily in the kidney and the gonads of the developing human fetus (Pritchard-Jones et al., 1990). This pattern of expression suggested that there might be a connection between the WTI gene and genital anomalies. Support for this was provided with the description of two patients with Wilms tumor and genital abnormalities which had constitutional heterozygous deletions that resulted in a truncation of the WTI gene (Pelletier et al., 1991b). Wilms tumor is also found in association with Denys-Drash syndrome (DDS) (Denys et al., 1967; Drash et al., 1970). Other predominant features of DDS are renal failure and gonadal and genital abnormalities (Mueller, 1994). Individuals with a normal male karyotype usually have ambiguous or female external genitalia and often have dysgenetic gonads. The nephropathy seen in these children occurs at the site of highest expression of WTI during kidney formation (Pritchard-Jones et al., 1990). These associations made a compelling argument that WT1 mutations might be present in DDS. The first reported mutations in Denys-Drash syndrome were heterozygous mutations which clustered in the zinc-finger region of the WTI protein (Pelletier et al., 1991a), the domain responsible for the DNA-binding activity of the WTI transcription factor. All subsequently identified WTI mutations in DDS have been located in this region (Mueller, 1994) and mutations have been found in WTI in the majority of DDS patients tested (references in Mueller, 1994; Bardeesy e t al., 1994; Nordenskjold e t al., 1994). Nearly 40 individuals with DDS have been shown to have WTI mutations. In each case, the mutations are heterozygous and are likely to disrupt or remove DNA binding, but leave the rest of the protein intact (Hastie, 1992; Mueller, 1994). This has led to the hypothesis that the mutations function as tramdominant gain of function or “dominant negative” mutations which exert their effect due to an altered function of the mutant protein. Disruption of the WT-I gene in transgenic mice demonstrated its importance in gonad formation (Kreidberg et al., 1993). Mice heterozygous for WT-I null mutations developed normally, but the presence of homozygous null alleles resulted in a failure of kidney and gonad development, with subsequent embryonic death. The first stage of gonadal development in normal mice is a thickening of epithelium on the coelomic surface of the urogenital ridge at Day 11 of gestation, leading to the formation of a prominent gonadal component at

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Day 12. In homozygous null WT-J mice, the epithelium thickening was sharply reduced and further gonad development was aborted. Although the chromosomal sex of the embryos was not determined, presumably both XX and XY embryos were examined. The WT-J mutation did not interfere with normal germ cell migration into the primordial gonad. The expression patterns and mutation phenotype of WT-1 suggest that it exerts its effects upstream of Sry and is likely to be necessary for commitment and maintenance of gonadal tissue.

2.

sox9

Campomelic dysplasia (CD) is a congenital bone and cartilage malformation syndrome which is frequently accompanied by XY sex reversal (Hovmoller et al., 1977). Approximately three-fourths of the karyotypic males born with CD show genital malformations and some of the cases examined histologically exhibit gonadal dysgenesis, indicating that the gene mutated in these individuals plays a part in testis formation (Houston et al., 1983). No evidence for mutation of SRY was found in five XY female CD patients examined (Ebensperger et al., 1991). Chromosomal translocations in sex-reversed CD patients were used to map a human autosomal XY sex-reversal locus (SRAI) associated with the campomelic dyplasia locus (CMPDJ) to human chromosome 17q24.3-q25.1 (Tommerup et al., 1993). A candidate gene for CMPDJ and SRAJ was identified by association with a chromosome 17 translocation breakpoint in a CD XY female (Foster et al., 1994). This gene, SOX9, had been isolated from a testis cDNA library in a screen for genes sharing homology to the SRY HMG-box (conserved DNAbinding domain) and was used as a marker in the breakpoint cloning. Due to its proximity to the breakpoint, a role for SOX9 in CD and sex reversal was investigated by analysis of SOX9 sequences in CD patients without chromosomal aberrations. Heterozygous mutations were found in an XX female and in XY female sex-reversed CD patients, demonstrating that mutations in the gene are responsible for both sex reversal and CD (Foster et al., 1994). No evidence of mutation of both SOX9 alleles has been found, suggesting that the phenotypes are the result of haploinsufficiency and adding to an expanding list of congenital anomalies which result from the loss of one copy of a gene (Fisher and Scambler, 1994). In independent experiments, SOX9 was screened for mutations in CD patients on the basis of the expression pattern and positional location of the mouse sox-9 gene (Wright et al., 1995) and heterozygous mutations were found in four CD XY females (Wagner et al., 1994). In situ hybridization experiments showed SOX9 expression in developing bone of 7 week human embryos, in the area of the rete testis and seminiferous tubules of an 18 week human male fetus, and in developing mouse skeleton as well as a few nonskeletal mouse tissues (Wagner et al., 1994; Wright et al., 1995). Northern analysis of SOX9 expression showed transcripts in fetal brain, liver, and kidney. Adult expression appeared to be greatest in testes and was present in nearly all adult tissues tested

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with the exceptions of leukocytes, spleen, and thymus (Foster et al., 1994; Wagner et al., 1994). The widespread distribution of SOX9 expression may be indicative of expression in connective and support cells, which are a component of all tissues, or may imply a more general cellular role. The relationship between translocation breakpoints and the SOX9 gene in CD patients is currently unresolved. The breakpoints which have been mapped relative to SOX9 are located at a minimum of 50 kb from the apparent 5' end of the transcriptional start site and do not appear to be clustered (Foster et al., 1994; Wagner et al., 1994). Although the entire open reading frame of SOX9 has been isolated, some sequences of the transcript remain unidentified and it is possible that 5' exons were interrupted by the chromosome breaks. Alternatively, the translocations may exert position effects induced by sequences brought adjacent to the region containing SOX9 or may interrupt SOX9 control elements. The SOX9 HMG-box amino acid sequence is 71% similar to the SRY HMG-box and exhibits specific binding in vitro to the same DNA target sequences as SRY (V. R. Harley and P. N. Goodfellow, personal communication). A glutamine and proline rich region similar to that found in some transcriptional activators is present in the C-terminal end of the protein. These features suggest that SOX9 is likely to function as a transcription factor. The similarity of the SOX9 gene to SRY might suggest a priuri a role in sex determination. In fact, many members of the SOX gene family had previously been isolated, including ones with greater HMG-box similarity to SRY than SOX9 (Gubbay et al., 1990; Denny et al., 1992a,b; Coriat et al., 1993; Farr et al., 1993; Goze et al., 1993; StevanoviC et al., 1993; van de Wetering and Clevers, 1993; van de Wetering et al., 1993; Wright et al., 1993). Examination of sequences outside of the HMG box of the SOX genes revealed high divergence and no significant similarity to SRY. Expression patterns have been investigated in some of these SOX genes and are suggestive of diverse roles (Denny et al., 1992b; Farr et al., 1993; StevanoviC et al., 1993; van de Wetering et al., 1993), but none (other than SOX9) have been implicated in sex determination. Therefore, involvement of SOX9 in sex determination was unexpected. The association of SOX9 with the development of two diverse tissues suggests that other SOX genes, including those which have known expression patterns, should be carefully assessed for a role in sex determination. Further characterization of SOX9 expression patterns and targeted mutations in transgenic animals should provide insight into the role of SOX9 in gonad differentiation.

3. Steroidogenic factor 1 (SF-1)IFtz-FI Experiments using mouse adrenocortical cells have shown that the orphan nuclear receptor, SF-1, acts as a key regulator of enzymes involved in steroid production, including the sex hormones. In the adult mouse, SF-1 is expressed

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in all primary steroidogenic tissue: adrenal cortex, testicular Leydig cells, ovarian theca, granulosa cells, and corpus luteum (Ikeda et at., 1993). Isolation of genomic clones resulted in the discovery that, by alternative promoter usage and splicing, the same structural gene encodes both SF-1 and the previously identified embryonal long terminal repeat binding protein, ELP. SF-1 and ELP show similarity to the Drosophila orphan protein FTZ-F1, particularly in their shared zinc-finger domain, so the mouse ELP/SF-1 locus was designated Ftz-FI (Ikeda et

al., 1993).

Surprisingly, studies of the developing mouse showed SF-1 expression in the urogenital ridge of male and female mice at 9-9.5 dpc, the earliest stage of organogenesis of gonads, and continuing through 12.5 dpc, when the first sex differences appear in the developing structure of the gonad of males. SF-1 expression was found to persist beyond 12.5 dpc in males, but was extinguished in females (Ikeda et al., 1994). Furthermore, SF-1 was shown to be expressed in fetal Sertoli cells. This indicated that in addition to regulating steroid hydroxylases, SF-1 might play a role in early gonadal differentiation. Targeted disruption of Ftz-FI resulted in homozygous null mice which survived normally in utero but died postnatally by Day 8, probably as a result of adrenocortical insufficiency (Luo et al., 1994). Both XX and XY animals lacked adrenals and gonads, and both XX and XY mice developed female internal genitalia. Examination of the developing gonads showed that abnormal gonadal development occurred at the stage where sex differences become manifest. The 10.5 dpc genital ridges in Ftz-FI null homozygotes were similar to those of wild type. At 12 dpc, the genital ridge was markedly attenuated in Ftz-FI-disrupted mice, with cells appearing to undergo apoptosis, and by 12.5 dpc the genital ridge was nearly absent. Primordial germ cells did not require Ftz-FI for migration and survival in early stages of gonadal development and, although their numbers diminished following gonadal degeneration in Ftz-FI null mice, it was probably due to the abnormal structural environment. Recent experiments have demonstrated that SF-1 is likely to be a regulator of anti-Mullerian hormone (Shen et al., 1994). Promoter sequences of the mouse Amh gene which could support transcription in Sertoli cells were shown to bind a protein present in mouse Sertoli cell nuclear extracts. The site bound by the Sertoli protein contained a consensus sequence for nuclear receptor binding and was bound by recombinant mouse SF-1 in uitro. No protein interactions were observed at the SRY in vitro binding site located in the Amh promoter and recombinant SRY protein did not compete with the Sertoli protein for binding to the nuclear receptor consensus sequence. Examination of Arnh and SF-1 expression showed parallel sexually dimorphic expression patterns in embryonic and postnatal gonads. Collectively, these data indicate that SF-1 regulates Amh and suggest a pivotal role for SF-1 in sex determination. Experiments using an embryonic rat genital ridge cell line have failed to demonstrate transac-

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tivation of human AMH reporter constructs with mouse SF-1, and SF-1 exerted a repressive effect on SRY-induced activation of the AMH reporters (Haqq et al., 1994). SF-1 may act as a transcriptional repressor in the context of the cells used or the different SF-I activity may reflect species differences in the DNA sequences and cells used. SF-1 is regulated to assume male-specific expression as a result of testis formation induced by SRY, but it is unlikely that SRY directly controls SF-I. SF-1 expression begins before SRY is detected and peaks and continues well after SRY transcript is no longer detected. The similarity of onset of expression in mice of SF-1 (at 9-9.5 dpc) and WT-1 (at 9 dpc), and the similar phenotype of gonadal degeneration in transgenic disruption experiments, is suggestive of their activities functioning in a common pathway. Studies of WT-1 expression in F t p FJ disrupted mice and Ftr-F J expression in WT- I -disrupted mice should reveal if they act independently or coordinately. In humans, XY females with gonadal dysgenesis can result from W T I mutations, making SF-1 a prime candidate for investigation of human primary sex reversal.

4. Dosage-sensitive sex reversal (DSS) In 1978, the first indication of an X-specific gene involved in human sex determination was provided by the identification of a family with an apparent X-linked mode of inheritance of 46,XY gonadal dysgenesis (German et al., 1978). Further evidence for the presence of a locus on Xp involved in sex determination was provided when cytogenetic studies of two sex-reversed sisters revealed the presence of an X chromosome short arm interstitial duplication and an apparently normal Y chromosome (Bernstein et al., 1980a,b). Similar duplications accompanied by female or ambiguous genitalia have subsequently been reported (Bardoni et al., 1993; Arn et al., 1994; Ogata and Matsuo, 1994). Investigation of the extent of Xp duplications in several patients showed that they overlapped and did not share common breakpoints, suggesting that duplication, rather than disruption of a gene in this region is responsible for sex reversal. The male phenotype of 47,XXY individuals develops in the presence of two X chromosomes, one of which is inactivated, leading to the hypothesis that the duplicated X chromosome causes XY sex reversal by expressing a double dose of a gene normally subject to X inactivation. Screening of XY females with an intact SRY gene and without cytogenetic anomalies detected a submicroscopic duplication which localized the locus, DSS, to a 160-kb region (Bardoni et al., 1994). This region had previously been the focus of studies of the contiguous gene deletion syndrome composed of an X-linked cytomegalic form of adrenal hypoplasia congenita (AHC), glycerol kinase deficiency, and Duchenne muscular dystrophy. Sequences from the AHC-DSS critical interval were examined and a single CpG island was identified. Adjacent sequences, which

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showed evolutionary conservation, were used to isolate cDNAs from fetal adrenal and adult testis libraries. Sequencing of the cDNAs revealed a gene, DAX-1 (DSS-AHC critical region on the X), which encodes a new member of the nuclear hormone receptor family (Zanaria et al., 1994). The gene was examined in AHC patients and point mutations were found, implicating DAX-I as the AHC gene (Zanaria et ai., 1994; Muscatelli et at., 1994). Adrenal hypoplasia congenita is an inherited disorder of adrenal gland development. The common derivation of the adrenals and gonads from mesenchymal elements and their shared steroidogenic properties promotes the idea of DAX-I being both the adrenal hypoplasia congenita gene and DSS. However, the mutations found in DAX-I associated with adrenal hypoplasia did not result in sex reversal, and the analysis of 30 XY females did not reveal any rearrangements of DAX-I (Zanaria et ai., 1994). Overexpression of DSS would be predicted to cause XY sex reversal, and such regulatory mutations will likely be difficult to identify. A direct test of the role of DAX- J in XY sex reversal will be the production of transgenic mice carrying an additional copy of rhe gene.

5. Chromosomal deletions associated with sex reversal Chromosomal aberrations continue to be important in identifying genes involved in sex determination. Sex reversal has been reported in a number of subjects with terminal deletion of 9p (Bennett et al., 1993) and in individuals with terminal deletion of 1Oq (Wilkie et al., 1993). In each case, the cytogenetically observed distances between the breakpoints makes it unlikely that they are interrupting a common gene, suggesting that the deletion results in the uncovering of heterozygous mutations or that monosomy of a critical gene (or genes) is responsible for the phenotype.

111. ABNORMALITIES OF HUMAN SEX DETERMINATION AND PRIMARY SEX REVERSAL As described previously, genetic and molecular studies of primary (gonadal) sexreversal syndromes have been instrumental in the identification and cloning of genes involved in sex determination. This relationship is complementary; investigations of the biology of these genes have helped to classify and to explicate primary sex-reversal syndromes. Below is a discussion of these syndromes and a description of our current understanding of their etiology. Sexual development following the establishment of gonadal sex involves the hormonally regulated differentiation of the internal and external genitalia dependent upon the presence or absence of testes. Defects in fetal endocrinology during sexual differentiation can result in sex reversal (Grumbach

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and Conte, 1992). Individuals with a 46,XX karyotype accompanied by ovaries and ambiguous genitalia are diagnosed as female pseudohermaphrodites, with masculinization of the genitalia resulting from exposure to androgenic steroids at specific stages of embryonic and fetal development (New, 1992). Male pseudohermaphroditism is the diagnosis given to individuals with a 46,XY karyotype and normal testes who exhibit incomplete or absent masculinization due to errors in the production of masculinizing hormones, or as a result of target tissue insensitivity to these hormones (Kupfer et al., 1992). A detailed description of sex differentiation anomalies which do not result from primary gonadal failure is beyond the scope of this chapter.

A. XY females Errors occurring in the normal testis determination pathway result in ovarian development in 46,XY embryos. Defects may occur at different points along the developmental cascade and male sexual development, being actively induced, affords many opportunities for mishap. In XY females, the presence of a single X chromosome rather than of two X chromosomes results in degeneration of the ovaries (gonadal dysgenesis). The resulting gonads often appear as white fibrous streaks, devoid of follicles and normal germ cells, and are endocrinologically inert. Occasionally, small nests of a few hundred cells, believed to be abnormal XY germ cells, are seen within the ovarian stroma. These cells are prone to neoplastic transformation and may develop into gonadoblastomas, from which may arise a variety of malignancies, including dysgerminoma and choriocarcinoma (Scully, 1970). Due to the propensity of 46,XY patients with gonadal dysgenesis to develop gonadal tumors, prophylactic removal of the streak gonads is indicated (Gourlay et al., 1994). The term “pure gonadal dysgenesis” was originally used to describe the presence of bilateral streak gonads in phenotypic women lacking the extragonadal defects of Turner syndrome and is now used as a synonym for “complete gonadal dysgenesis,” which is characterized by the complete lack of functioning gonadal tissue and the histological absence of testicular tissue (Berkovitz, 1992). Phenotypically female patients with an 46,XY karyotype exhibiting complete gonadal dysgenesis are considered to exhibit “Swyer syndrome” (Swyer, 1955) or “Swyer-James syndrome” (Guidozzi et al., 1994). Not all 46,XY females exhibit complete gonadal dysgenesis. The sexreversed phenotype of XY females depends upon the severity of the impairment and on the level in the developmental cascade at which a defect occurs. Partial testicular determination may occur, resulting in gonads which contain some testicular tissue, but reductions in AMH and testosterone production lead to varying degrees of failure of Miillerian duct regression and Wolffian duct differentiation. As a result, the majority of newborns with “partial gonadal dysgenesis” (or “mixed gonadal dysgenesis”) present with ambiguous genitalia. The

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phenotype is highly variable and dependent upon the amount of testosterone produced. Testicular development can be inhibited unilaterally, resulting in ipsilateral development of female internal genitalia. Individuals with a 45,X/46,XY chromosomal constitution exhibit clinical features of 46,XY partial gonadal dysgenesis (Robboy et al., 1982).

1. XY females lacking SRY Submicroscopic alterations of the Y chromosome removing the sex-determining region are surprisingly uncommon considering that this type of mutation can arise in the derivative Y chromosome foIlowing X-Y interchange (Figure 9.3). High in uteio lethality is seen in 45,X Turner syndrome, possibly as a result of haploinsufficiency of a gene or genes common to the sex chromosomes. The underrepresentation of interchange 46,XY (SRY-) females relative to the occurrence of 46,XX (SRY+) females may be due to a loss of these genes during X-Y interchange. In some cases in which the loss of SRY is accompanied by deletion of material from the Y chromosome short arm, somatic features of Turner syndrome are present. Y-autosome translocations are a second mechanism by which SRY can be lost from the Y chromosome (Page et al., 1990). Dysgenetic gonads are invariably present in SRY-negative XY females (Disteche et al., 1986; Blagowidow et al., 1989; Levilliers et al., 1989) and patients are usually taller than the average female due to the effect of a Y chromosome unique or pseudoautosomal gene (Ferguson-Smith, 1991; Ogata et al., 1992).

2. XY females with SRY mutations Mutations involving SRY have been found in approximately 10-15% of XY females tested. In some studies, only the DNA binding domain has been analyzed for mutations, but in many patients the entire gene has been tested, with no mutations found. All XY females with SRY mutations have been found to have complete gonadal dysgenesis (references in Hawkins, 1993; Iida et al., 1994; Poulat et al., 1994; Tajima et al., 1994). In studies of XY females with a histological diagnosis of complete gonadal dysgenesis, the frequency of SRY mutation found was higher than those of previous investigations, indicating that some patients which have been classified as complete gonadal dysgenesis may have partial gonadal dysgenesis or may not have primary sex reversal (Berkovitz et al., 1991; Hawkins et al., 1992; Vilain et al., 1993). Mutations in SRY lead to ovaries with the same gonadal phenotype as 45,X Turner patients. One explanation for the gonadal dysgenesis found in Turner patients is that genes located on the X chromosome are needed in a double dose for normal ovarian formation and the absence of a secondary X

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chromosome results in haploinsufficiency. The genes required for ovarian development which are missing in Turner syndrome patients are likely to be X specific and not shared by the X and Y chromosomes, otherwise normal ovarian development would be expected in XY females with SRY mutations. The normal extragonadal development of XY females with SRY mutations suggests that even though SRY is expressed in many tissues, its function is not crucial for normal development of somatic tissues. Individuals with gonadal dysgenesis and no detected SRY mutations may have mutations in regions of the gene which have not been examined or may be mosaic for a mutation which cannot be detected from a single tissue sample. Given the large percentage of unresolved XY sex reversals, it is likely that many cases result from mutations in X chromosome or autosomal genes involved in sex determination.

3. Familial XY gonadal dysgenesis associated

with mutations in SRY

Investigation of SRY sequences has resulted in the identification of inherited sequence variants in SRY in seven families (Berta et al., 1990; Hawkins et al., 1992; Vilain et al., 1992; Affara et al., 1993; Jager et al., 1993; Tajima et at., 1994, Schmitt-Ney et al., 1995). In several cases where the transmission does not appear to be from a mosaic father, recombinant SRY protein corresponding to the mutations have been tested for DNA binding using in vitro assays (Nasrin et al., 1991; Harley et al., 1992; Jager et al., 1993). Whereas all de now0 mutations tested have lost sequence-specific DNA binding, the inherited mutations vary between greatly reduced binding and normal binding. The loss of binding in some familial cases strongly implies that the sequence changes are conditionally sex-reversing mutations and not neutral polymorphisms. The basis of the variability in sex reversal is not known. One possibility is that in cases of reduced binding, SRY is near a threshold of functional activity and slight fluctuations in expression or activity make the difference between effect or lack of effect. A n alternative explanation is that polymorphic alleles which interact with SRY have forms which can and forms which cannot assuage the SRY mutation.

4. XY sex reversal associated with Denys-Drash syndrome The primary feature of DDS, progressive nephropathy, usually appears in the first year of life, with the onset of Wilms tumor occurring at a similar time. Although most cases of DDS have been diagnosed in patients with an XY karyotype, the vast majority appear at birth as phenotypically normal female or have ambiguous external genitalia. It is most common for the internal genitalia

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~~

~

to be inappropriate for the external genitalia, and the gonads of DDS patients are often dysgenetic, appearing as streak gonads, underdeveloped testicular tissue (immature, infantile, or rudimentary), or both testicular and ovarian tissue (Mueller, 1994). Analysis of mutations in the WTJ gene of individuals with DDS has resulted in the identification of mutations in the majority of cases examined, but no association of a particular mutation with any class of phenotypes has emerged. In fact, a wide variety of genital and gonadal phenotypes are present in patients with a common mutation, 394Argto Trp, which constitutes nearly half of the Denys-Drash WTJ mutations identified. Some of these patients exhibit ambiguous genitalia, others female external genitalia. Some have both Miillerian and Wolffian structures, others have neither. In some cases the gonads are streak, while one individual had a normal left testis and a dysgenetic right gonad. In addition, the same 394Arg to Trp mutation has been reported in tumor and constitutional DNA from a patient with sporadic Wilms tumor and no urogenital abnormalities (Akasaka e t al., 1993). Patients with different WTf mutations exhibit a spectrum of phenotypes similar to that found accompanying the 394Arg to T rp mutation. An understanding of the relationship between WTI mutations and the degree of abnormal sexual development awaits the identification of molecules which interact with the normal and mutant proteins. A single familial case of WTf mutation in DDS has been identified. An affected child was found to have a mutation which is present as a constitutionally heterozygous mutation in the patient’s phenotypically normal father (Coppes et al., 1992). The difference in phenotypes between the parent and child may be the result of either the father being a somatic mosaic for the mutation or due to incomplete penetrance of the WTf allele. Genomic imprinting of the WTf gene is unlikely to play a role, as maternally and paternally transmitted alleles have been shown to be similarly expressed (Little e t al., 1992). Examination of WTJ sequences in 27 parents of 14 patients showed no indication of mutation, suggesting that the majority of WTI mutations in DDS are de novo (Mueller, 1994). The XY sex reversal associated with WTJ mutations in DDS patients makes it a candidate for involvement in other syndromes involving anomalous sexual development, but recent studies have been unable to establish such a connection. Frasier syndrome has many similarities to DDS including streak gonads, pseudohermaphroditism, and renal failure (Moorthy et al., 1987). Examination of exons 8 and 9 of the WTJ gene (where the majority of WTJ mutations in DDS have been found) in three Frasier patients detected no mutations suggesting that the two syndromes have distinct molecular origins (Poulat et al., 1993). It is possible that mutations responsible for Frasier syndrome in these individuals occurred in untested portions of the WTI gene. To investigate a potential role of WTI mutations in the etiology of genital anomalies which

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occur in the absence of kidney dysfunction, the gene was examined in 12 XY females with various genital abnormalities and normal kidneys (Clarkson et al., 1993). No W T I mutations were detected, although the screening procedure used could not absolutely exclude the presence of mutations.

5. XY sex reversal associated with campomelic dysplasia 46,XY patients afflicted with the congenital skeletal malformation disease CD exhibited a wide range of sexual phenotypes, including normal male, female with testicular tissue, female with dysgenetic gonads, and normal female with ovaries containing oocytes. There have been no reports of 46,XX females with campomelic dysplasia and gonadal abnormalities. No correlation is seen between the extent of the skeletal defects and the 46,XY phenotype, although the CD phenotype is usually less severe in patients with chromosomal translocations (Mansour, 1994). The severity of the skeletal defects usually results in neonatal death. Mutations in the SRY-related gene SOX9 have been reported in seven XY female CD patients and four mutations have been reported in subjects known to have ovaries containing follicles (Foster et al., 1994; Wagner et al., 1994). The limited number of SOX9 mutations currently identified does not provide insight into a relationship between mutation type and the degree of sexual abnormalities. However, comparison of mutations in XY females and of mutations in XY males is suggestive of a relationship between the type of mutation and the presence or absence of sex reversal. In the seven reported XY females, each patient had a mutation which is predicted to be severe, resulting in truncation of the protein. A mutation found in an additional XY female is also predicted to severely effect the SOX9 protein via disrupted transcript splicing, while mutations identified in two XY male CD patients are amino acid substitutions (C. Kwok, P. A. Weller, and P. N. Goodfellow, personal communication). Each mutation causes CD, but a severe mutation of the SOX9 protein may be necessary to elicit both XY sex reversal and skeletal malformation. An alternative possibility is that different regions of the protein are involved in the bone function and the gonadal function. Both of the nonsex-reversing mutations in the XY males are located in the DNA-binding domain and do not lead to truncation or disruption of other regions of the protein, while the sex-reversing mutations each result in the loss of the carboxy terminal region of the SOX9 protein. It is possible that SOX9 mutations may cause XY sex reversal without the skeletal abnormalities of CD. If the severity of the mutation dictates whether sex reversal occurs, it would be unlikely to find mutations in SOX9 unaccompanied by CD. If different domains of the protein are important in different functions, XY females resulting from SOX9 mutations without CD should exist. Analyses of XY females without bone malformations are required to determine if

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SOX9 mutations can cause sexual development abnormalities without inducing skeletal defects.

6. Familial XY gonadal dysgenesis with unknown etiology Familial cases of gonadal dysgenesis with inheritance patterns consistent with an X-linked trait, sex limited autosomal dominance and autosomal recessive have been described (Chemke et al., 1970; Simpson, 1976; Moreira-Filho et al., 1979; Simpson et al., 1981). The mode of transmittance rules out SRY as being responsible, and the gonadal dysgenesis is not associated with other phenotypes, as in the case of WTI (Denys-Drash syndrome) and SOX9 (campomelic dysplasia). It is possible that WTI or SOX9 is involved, but as yet mutations in these genes exclusively causing sex reversal have not been identified. The location and nature of the gene or genes responsible for these familial cases are unknown.

7. XY females with embryonic testicular regression The term “embryonic testicular regression” is used to describe a group of disorders characterized by a 46,XY karyotype, variable masculinization of internal and external genitalia, and the absence of testicular or ovarian tissue on one or both sides at birth (Coulam, 1979). The adult phenotype ranges from complete male differentiation with congenital anorchia to female differentiation with upper vagina, Miillerian derivatives, partial Wolffian structures, and lack of secondary sexual development. The phenotype has been interpreted to suggest that testes form initially, but degenerate at a critical stage of embryonic development. Observations by Marcantonio and colleagues (1994) imply that in some cases gonadal tissue was intrinsically abnormal at the time of testicular regression, and they suggest that testicular regression sequence is part of the clinical spectrum of 46,XY gonadal dysgenesis. The occurrence of embryonic testicular regression in several members of a family suggests a genetic basis for the condition, with inheritance patterns implicating the involvement of an X chromosome gene (or autosomal-dominant sex-limited trait) (Fechner et al., 1993). The observation of an affected individual with first-cousin parents, and segregation analysis of a large number of cases, favors autosomal recessive inheritance (Rosenberg et al., 1984).

8. XY gonadal dysgenesis associated with chromosomal aberrations Chromosomal deletions of 9p and 1Oq and duplications of Xp have been described in XY sex-reversed patients. Six cases have been reported of deletion of the chromosome 9 short arm associated with XY gonadal dysgenesis, of which four were associated with familial chromosomal translocations (Bennett et al.,

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1993). In the one patient examined histologically, the gonads exhibited complete dysgenesis. Deletions of 9p accompanied by ambiguous genitalia have also been reported (Young et al., 1982; Huret et al., 1988). Deletions of 1Oq have primarily been associated with male pseudohermaphroditism, but in one case a deletion was accompanied by the apparent absence of gonads and the absence of endocrinologically active testicular tissue. XY sex reversal has been found in several patients with a duplication of part of the short arm of the X chromosome. A locus implicated in the etiology of the sex reversal, DSS, has been localized to a 160-kb region of Xp (Bardoni et al., 1994). In subjects with a Y chromosome and the partially duplicated X chromosome, sexual phenotypes of normal female and ambiguous external genitalia occur. In some of the cases, the gonads have been examined histologically, revealing mixed gonadal dysgenesis. A gene cloned from the duplicated region (DAX-I ) has been implicated in the etiology of congenital adrenal hyperplasia and put forth as a candidate for DSS (Zanaria et al., 1994). Analysis of XY females has failed to detect rearrangements of DAX-I and it is currently unknown if DAX-1 mutations can cause XY sex reversal. Regardless of the function of DAX-I , observations can be made about the role of DSS in sex determination. Deletions which remove the DSS region do not impair testis formation, so DSS is not required in male development (Bardoni et al., 1993). Two copies of DSS result in formation of ovaries. This suggests that DSS is normally necessary for ovarian development and may be repressed in XY individuals to allow testis formation. If this is correct, ovaries would not develop in XX individuals with homozygous DSS null mutations, and perhaps some XX males arise from mutations in DSS. The dosage effect of DSS may reflect past function in an ancestral dosedependent sex-determination mechanism. A role as a switch, in sex determination may have been supplanted by a dominant mechanism of sex determination and is now uncovered only in abnormal circumstances.

9. Other syndromes associated with XY gonadal dysgenesis In addition to the syndromes described above, 46,XY gonadal dysgenesis has been associated with multiple congenital anomalies (genitopalatocardiac syndrome) (Simpson, 1989), with Smith-Lemli-Opitz syndrome (Bialer et al., 1987), with Frasier syndrome (Moorthy et al., 1987), with short-rib (polydactyly) syndrome type IV (Beemer-Langer) (Cideciyan et al., 1993) and with X-linked (Y thalassemia/mental retardation (ATR-X syndrome) (Gibbons et al., 1991). The pathogenesis of gonadal dysgenesis in these syndromes is not known and could be due to pleiotropic effects of a single gene or the result of chromosomal aberrations affecting multiple loci. It is also possible that the observed sex reversal may be secondary to other developmental anomalies present in the syndrome. Structural abnormalities may interfere with the timing of the SRY

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signal or with important cell-cell interactions, disrupting induction of the male phenotype and leading to female development.

B. XX males Clinically and endocrinologically, XX males are similar to individuals with 47,XXY Klinefelter syndrome. Both present a male phenotype and postpubertally have small azoospermic testes, gynecomastia, and varying degrees of testosterone deficiency. Unlike Klinefelter syndrome, there is no tendency toward mental retardation, the skeletal proportions and height are normal, and the karyotype is 46,XX. The patients are infertile due to breakdown of the germinal epithelium in the presence of two X chromosomes, which are unable to sustain spermatogenesis. 46,XX sex reversal occurs in about 1 in 20,000 human males (de la Chapelle, 1981). The origin of the male phenotype in XX individuals can be theorized to occur as a result of (i) cryptic sex chromosome mosaicism; (ii) the presence of Y chromosome sequences including SRY in all cells, carried as a translocation to the X chromosome or an autosome; or (iii) a mutation in an autosomal or X-linked gene in the testis-determining pathway. The majority of XX males have inherited Y chromosome sequences, including SRY, translocated to the X chromosome by aberrant recombination during meiosis. Some cases of males with a 45,X karyotype have been reported (Lo Curto et al., 1974; Forabosco et al., 1977; de la Chapelle et al., 1986; Gal et aE., 1987; Munke et al., 1988). In each instance, Y chromosome sequences translocated to autosomal chromosomes have ultimately been found. The variety of associated abnormalities which appear in these patients are not usually present in 45,X gonadal dysgenesis and are almost certainly due to the alteration of the autosomes involved in the translocations.

1. Cryptic mosaicism In a few cases of XX sex reversal, XXY cells have been identified, usually as the result of investigation of a large number of cells. Miro and co-workers (1978) found Y-specific staining in 1 oral mucosal cell of 400 in one XX male and a single XXY cell out of 500 lymphocytes examined in a different XX male. These data and similar findings in other laboratories have led to the estimation that XX/XXY mosaicism could account for sex reversal in up to 17% of all XX males (Miro et al., 1978).

2. XX males with translocated Y chromosome sequences The presence of Y chromosome sequences on the X chromosome resulting from X-Y interchange can be demonstrated in about 80% of XX males (Ferguson-

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Smith, 1966; Affara et al., 1986; Page et al., 1987a; Petit et al., 1987; FergusonSmith et al., 1990b). Patients with Y sequences have all tested positive for SRY, with a single exception (Ferguson-Smith et al., 1990a). The amount of Y DNA present is variable, ranging from less than 40 kb (Sinclair et al., 1990) to over 75% of the Y chromosome short arm (Ferguson-Smith and Affara, 1988). Examination of XX males with and without Y sequences has revealed differences in the phenotypes of the two groups. Although exceptions occur, the presence of Y sequences generally results in a more masculinized phenotype. Those without Y sequences are likely to have incomplete masculinization and ambiguous genitalia (Ferguson-Smith et al., 1990a; Boucekkine et al., 1994; Wachtel, 1994). Correlations between the amount of Y chromosome sequence present and the phenotype have been sought, and some interesting variants have been observed.

1. Most Y sequence-positive XX males have no disability apart from their infertility. They are of average height for normal females (below average for males), have normal intelligence, and have no ambiguity of the external genitalia (Ferguson-Smith et al., 1990a). The Y sequences transferred by X-Y interchange usually include the proximal genes ZFY and RPS4Y (Page et al., 1987a; Fisher et al., 1990). 2. Extensive translocation of most of the small arm of the Y chromosome to the X chromosome is observable by cytological examination. In these instances, the height is above average for normal male (corresponding to the height of Klinefelter patients), suggesting that a Y chromosome locus for stature has been transferred in the X-Y interchange (Ferguson-Smith, 1991; Ogata et al., 1992). 3. A few Y sequence-positive XX males have lost more X chromosome sequence than Y sequence gained by X-Y interchange. An X-linked locus for short stature may have been lost in a patient with below average height (as in Turner syndrome) (Ferguson-Smith, 1991). Rare cases of mental handicap also seen in these patients may result from the involvement of an X-linked gene associated with mental retardation (Ballabio et d., 1989). 4. X-Y interchanges involving transfer of small regions of the Y chromosome have been reported. Three patients have been described with X chromosomes carrying SRY, but not the proximal genes ZFY and RPS4Y. Two of the subjects have hypospadias and the other has bilateral cryptorchidism (Palmer et ul., 1989). One of the hypospadic individuals has a sibling with XX true hermaphroditism, i.e. , both ovarian and testicular tissue are present, accompanied by ambiguous genitalia. The incomplete development of the genitalia in the XX males suggests the loss of determinants close to SRY, or, more plausibly, is explained by altered SRY expression. In XX individuals who have inherited larger regions of the Y chromosome (and do not usually have ambiguous

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genitalia), the additional Y sequences may act as a buffer protecting SRY in its new chromosomal position. Shorter Y sections inserted into the X chromosome might thus be sensitive to positional effects or to the spread of X-inactivation from adjacent X sequences. Such an effect has been shown to occur in mice carrying the Sxr (Sma) chromosome, an X chromosome containing translocated Y chromosome sequences which include the sex-determining region (McLaren and Monk, 1982). The phenotype of XXSxra mice can be female, male, or (rarely) hermaphroditic due to the variable extent of inactivation spreading into the Y chromosome sequences from adjacent X chromosome sequences. 5. An XX male has been described with inherited proximal Y chromosome short-arm sequences, but without Y chromosome sequences from the testis determining region, including SRY, ZFY, and RPS4Y. The patient has perineal hypospadias and an undescended left testicle (Ferguson-Smith et al., 1990a). No Y-linked coding regions have been identified. The sex-reversed phenotype in this individual may be the result of disruption of an X-linked gene by the translocated Y chromosomes sequences. 6 . One XX male has been described with Wolf-Hirschhorn syndrome, which is characterized by severe growth retardation, mental defect, microcephaly, “Greek helmet” facies, and closure defects, but is not usually associated with aberrations of sexual development. Karyotyping revealed that Y chromosome sequences, including SRY, were present on the tip of the short arm of one of the X chromosomes (Coles et al., 1992). In addition, material originating from the short arm of the paternal X chromosome was translocated to chromosome 4, replacing sequences normally present at the end of the chromosome 4 short arm. Deletions of 4p have previously been associated with Wolf-Hirschom syndrome (Gusella et al., 1985). The probable explanation for the two phenotypes in this patient is that they originate from two separate chromosomal rearrangement events: an aberrant X-Y interchange giving rise to the sex reversal and an X;4 translocation resulting in a deletion of 4p which produced the Wolf-Hirschorn syndrome.

3. Y sequence-negative XX males About 20% of XX males have no detectable Y sequences when analyzed with a range of Y-specific probes, including SRY (Palmer et al., 1989; Numabe et al., 1992). Most of the Y sequence-negative patients have ambiguous genitalia (Ferguson-Smith et al., 1990a; bucekkine et nl., 1994; Wachtel, 1994), although the male phenotype can be complete (Vilain et al., 1994). No evidence of a molecular defect has been found to explain the sex reversal, and the

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phenotypic heterogeneity among subjects who lack SRY suggests that there is genetic heterogeneity in this group (Fechner et al., 1993). The simplest model to explain male development in XX individuals is that a gene downstream of SRY and normally quiescent or inactive in the absence of SRY gains constitutive activity via mutation and initiates the testis development pathway. A more complex model which postulates that loss of function mutations at recessive alleles leads to expression of male characteristics in XX males has also been described (McElreavey et al., 1993).

C. True hermaphrodites True hermaphrodites contain both ovarian and testicular gonadal tissue in the same or opposite gonads. The most common gonad present is the ovotestis: at least one is found in approximately 80% of true hermaphrodites (van Niekerk and Retief, 1981; Ramsay et al., 1988). In about half of the cases, the ovotestes is unilateral, accompanied by a testis or ovary on the other side. Less frequently, the gonads are lateral, with a testis on one side and an ovary on the other, or bilateral, with testicular and ovarian tissue on both sides, usually in the form of ovotestes. The testes and testicular portion of ovotestis in true hermaphrodites are similar to the testes in XX males, containing only Sertoli cells and no evidence of spermatogenesis. The ovarian portions contain normal-looking follicles and several instances of children borne to XX true hermaphrodites demonstrate that the ovarian tissue can be functional (Williamson et al., 1981). The differentiation of the internal ducts usually follows that of the gonads. Bilateral ovotestes are usually accompanied by bilateral tubes; in cases of a unilateral testis and ovotestis, the Mullerian ducts are suppressed on the side with the testis (Jones et al., 1965). Adult patients are of normal female stature and normal intelligence and the external genitalia may appear male or female but most often are ambiguous. Most true hermaphrodites patients are reared as males because of the size of the phallus. At puberty, virilization occurs, extensive breast development often ensues, and menses commence in more than half of the patients. The question of how male development occurs in the absence of a Y chromosome in XX males also applies to XX true hermaphrodites. Moreover, equivalent explanations can be applied to XX hermaphrodites: mosaicism (and chimerism), the presence of translocated Y sequences, or mutation of an Xlinked or autosomal sex-determination gene.

1. Mosaicism and chimerism Analysis of cytogenetic studies of 195 true hermaphrodite patients found that 60% had a 46,XX karyotype, 12% had a 46,XY karyotype, 13% were 46,XX/46,XY chimeras, and the remainder were sex chromosome mosaics (van

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Niekerk and Retief, 1981). Mosaicism arises from errors in meiosis and mitosis. Postzygotic nondisjunction in an XXY embryo will produce a 46,XX/47,XXY mosaic and the presence of an active Y chromosome in only a portion of the cells can lead to hermaphroditic development. XX/XY chimerism is usually the consequence of double fertilization of an ovum and its polar body (Gartler et al., 1962). The resulting individual is composed of the cells of two embryos: a mixture of testis-determining and ovarydetermining cells. The sexual phenotype depends upon the distribution of the cells in the developing gonad. Experiments with XX/XY chimeric mice have shown a preponderance of normal male development, suggesting that testis organization can be induced by a locally diffusible molecule controlled by Sry, but able to affect XY and XX cells alike (McLaren, 1984).

2. XX true hermaphrodites with translocated Y sequences Few true hermaphrodites have Y-specific sequences. A variety of reports have described the presence of a small portion of the Y chromosome, including SRY, on one of the X chromosomes of XX true hermaphrodites (Page, 1986; Jager et d.,1990; Nakagome et al., 1991; McElreavey et al., 1992a). In these cases, random X chromosome inactivation may have resulted in an analogous situation to XX/XY chimeras, with different sets of cells either being testis determining (due to SRY expression) or ovarian determining (due to absence of SRY expression). A different mechanism producing the same effect has been reported in a 46,XY true hermaphrodite who experienced a postzygotic SRY mutation resulting in gonads which contain a mixture of normal SRY and mutant SRY cells (Braun et al., 1993).

3. Y sequence-negative XX true hermaphrodites In the majority of true hermaphrodites, there is no evidence for mosaicism, chimerism, or translocated Y chromosome sequences. The karyotype is normal 46,XX and the Y chromosome sex-determining region is not present (Palmer et al., 1989;Jager et al., 1990; Berkovitz et d.,1992;Tho et al., 1992). Presumably, the masculinization in these subjects is caused by mutation of an X chromosome or an autosomal gene.

D. Familial occurrences of M males and M true hermaphrodites A number of families with XX males, XX hermaphrodites, or both have been reported (Fraccaro et al., 1979; Ramsay et al., 1988). Affected individuals which have been examined are Y sequence negative. The clinical and endocrinological

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phenotypes of XX males with sexual ambiguity can be remarkably similar to those of XX true hermaphrodites, and careful histological examination of the gonads may be required to classify a patient (Abbas et al., 1990). This similarity, coupled with the appearance of XX males and true hermaphrodites in the same kindred, is compelling evidence that the two conditions are closely related (Berger et al., 1970; Palmer et al., 1989; Pereira et al., 1991). An attractive explanation for the familial occurrence of XX males and XX hermaphrodites is that an X-linked gene normally regulated by SRY is mutated to become constitutively active. In each cell, the mutated X chromosome would be accompanied by a normal X chromosome, so random X inactivation will result in different cell populations. Those with the mutant chromosome inactivated would act as normal feminizing cells in gonadal tissue, and those with the normal chromosome inactivated would be masculinizing when present in gonadal tissue. The overall phenotype would depend upon the proportion and distribution of each cell type in the developing gonad. This model could account for many of the familial cases of XX males and XX true hermaphrodites, but in some instances autosomal inheritance is implicated (Kasdan et al., 1973;Skordis et al., 1987). A common genetic defect may be responsible for the differing XX sex reversal in members of the same family, but the mechanism leading to the development of testes in some individuals and the development of testicular and ovarian tissue in others remains to be determined.

E. Familial agonadism There have been two reports of primary agonadism (lacking detectable gonadal tissue) in sisters with a normal female external phenotype. In each report, one sister had a 46,XY karyotype and the other a 46,XX karyotype. In one sibling pair, the phenotype was complex, consisting of pulmonary hypoplasia, agonadism, omphalocele/diaphragmaticdefect, and dextrocardia (termed PAGOD syndrome) (Kennerknecht et al., 1993). The other pair of sisters exhibited hypoplastic Mullerian derivatives and gonadal agenesis without any other somatic abnormalities. The sisters were born to a consanguineous marriage, suggesting the involvement of an autosomal recessive mutation, although an X-linked dominant pattern of inheritance has not been excluded (Mendonca et al., 1994). The conserved DNA-binding region of the SRY gene showed no mutations in this case and the agonadal phenotype is not consistent with the phenotype seen in patients with SRY, WTI, or SOX9 mutations. Mice with SF-1 or WT-I homozygous null mutations are agonadal, but the mutations are also lethal. It seems likely that the defect in these individuals occurs in a sexdetermination gene required for induction or maintenance of gonadal tissue prior to sexual differentiation, and the failure of gonad formation leads to a female phenotype. Nothing further is known about the nature or location of the mutations in these patients.

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Over the past few years, significant progress has been made in the identification and cloning of genes involved in sex determination. T h e advances have primarily resulted from the interdisciplinary analysis of individuals with altered or abnormal sexual development. This in turn has clarified the molecular basis for the wide variety of sex-reversal syndromes. The demarcation between sex determination and sex differentiation is becoming less distinct, particularly with the discovery of a gene (SF-I) which is involved at multiple levels of sexual development: in gonadal genesis, AMH activation, and steroidogenesis. Many of the genes currently known to be involved in sex determination also have critical functions in other developmental processes: SF-1 in adrenal gland development, WT-1 in kidney formation, and SOX9 in bone morphogenesis. These diverse roles emphasize the developmental complexities of embryonic differentiation and provide a n insight into the intricacies of sex determination which await elucidation.

Acknowledgments I thank 1. R. Hawkins, P. N. Goodfellow, and Y. D. Ramkissoon for thoughtful discussion and reading of the manuscript, and M. Schafer for editorial assistance. I am grateful to Y. D. Ramkissoon for preparation of Figures 1 and 4, and to L. S.Whitfield for Figure 3.

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Ahnormalities chromosomal, 101- 130 aneuploidy

45,X, 109-1 10 47,XXX, 110-113 47,XXY, 110-1 13 47,XYY, 113-1 14 cell division origin, 107-109 disomic gamete frequencies, 125-1 29 monosomy etiology, 105-106 frequency, 102-105 overview, 101-102 parental origin, 106-107 sex chromosome polysomy, 1 14- 115 trisomy

13, 14, 15, 16, 18, 21, 22,

122-123 122-123 122-123 121-122 120-121 115-120 122-123 etiology, 105-106 frequency, 102-105 mechanisms, 123- 125 sex determination, 300-313 familial agonadism, 3 13 hermaphrodites, 31 1-313 XX males, 308-313 XY females, 301-308 Activated protein C, see Protein C Agonadism, familial, 3 13 Aneuploidy, chromosome numerical abnormalities

45,X, 109-110 47,XXX, 110-113 47,XXY, 110-113 47,XYY, 113-114 Antithrombin 111, 135-136

Cancer development, 255-270 future research directions, 269-270 homeohox genes cancer role, 267 developmental processes, 265-266 expression cancers, 268-269 leukemia, 267-268 tumorigenesis, in viw model, 267 paired-box genes embryonic development role, 256-259 expression brain cancers, 260-263 primary human tumors, 260 rhabdomyosarcoma, 264-265 Wilms’ tumor, 263-264 oncogene classification, 259 tumorigenesis, in wivo model, 259-260 Cardiac muscle, pathology in muscular dystrophy, 181-182 C4h-binding protein, interactions with protein S in thrombophilia, 144-147 Chromatids, see Sister chromatid exchange Chromosomes circular, meiotic recombination, sister chromatid exchange, 48 mapping, see Genome mapping meiotic recombination, sister chromatid exchange circular chromosomes, 48 nonhomologous chromosome repeats,

50 numerical abnormalities, 101-130 aneuploidy

45,X, 109-110 47,XXX, 110-1 13 47,XXY, 110-113 47,XYY, 113-114 cell division origin, 107-109 disomic gamete frequencies, 125-129

33 1

332 monosomy etiology, 105-106 frequency, 102-105 overview, 101-102 parental origin, 106-107 sex chromosome polysomy, 114-115 trisomy 13, 122-123 14, 122-123 15, 122-123 16, 121-122 18, 120-121 21, 115-120 22, 122-123 etiology, 105-106 frequency, 102-105 origin mechanisms, 123-125 Y chromosome deletion mapping, 284-287 structure, 283-284 Capper homeostasis, 233-248 characteristics, 233-235 Menkes disease clinical findings, 235-237 future research directions, 247-248 molecular genetics, 238-242 mottled mouse model, 245-246 Wilson disease clinical findings, 235, 237-238 future research directions, 247-248 Long-Evans Cinnamin rat model, 246-247 molecular genetics, 242-245

Defects, see Abnormalities Dystrophin, 177-216 dystrophin-associated proteins, 199-203 future research directions, 215-216 gene characteristics, 184-190 expression regulation, 188-190 identification, 184-185 structure, 185-188 muscular dystrophy carrier diagnosis, 211-215 direct diagnosis, 212-214 indirect diagnosis, 21 1-212 disease characteristics, 177-183 animal models, 183

Index cardiac muscle, 181-182 mental retardation, 180-181 muscles, 177-180 retina, 182-183 mutational distribution, 203-207 prenatal diagnosis, 21 1-214 mutational spectra, 203-21 1 genotypelphenotype correlations gross rearrangements, 207-209 point mutations, 209-21 1 muscular dystrophy distribution, 203207 protein Characteristics, 190-198 isoforms, 194- 195 related forms, 195-197 87K tyrosine kinase substrate, 197 protodystrophin, 196-197 utrophinlDRP, 195-196 structure, 190-193 subcellular localization, 197-198

Embryonic development, see Neoplasia E s c h h i a cob, 16s ribosomal RNA mutational analysis, 1-31 conserved nucleotides, 19, 20 detection methods, 2-5 mutation introduction, 4-5 plasmid expression, 2-4 double mutations, 19, 21-30 limitations, 30-31 second-site suppressor mutations, 3 1 single mutations, 19, 21-30 structure-function analysis, 6- 19 3' major domain mutations, 13-16 3' minor domain mutations, 16-19 5' major domain mutations, 10-12 central domain mutations, 12-13 secondary structure, 6-9 upstream mutations, 9- 10

Familial agonadism, 313 Fluorescence in situ hyhridization, radiation hybrid genome mapping, human component analysis, 79-80

333

Index Genetic defects, see Abnormalities Genetic hybrids, see Radiation hybrids Genome mapping, radiation hybrids, 63-94 construction methods, 71-77 dosage effects, 75-77 retention frequencies, 75-77 species considerations, 75 future directions, 93-94 Goss and Harris experiments, 65-68 extensions, 68-71 chromosome-specific hybrid panels, 7071 hybrid gene enrichment, 69-70 whole genome experiments, 68-69 human component analysis, 77-82 fluorescence in situ hybridization, 79-80 interspersed repetitive sequencepolymerase chain reaction, 81-82 single-copy sequence scoring, 77-79 mouse genome maps, 92-93 somatic cell genetics, 64 statistical analysis, 82-94 data collection, 83-84 influential hybrids, 91-92 multipoint analysis, 84-91 two-point analysis, 84 Gonads sex-determining genes, 293-300 dosage-sensitive sex reversal, 299-300 sex-reversing chromosomal deletions, 300 SOX9 gene, 296-297 steroidogenic factor 1, 297-299 WTI gene, 294-296 testis determining factor candidates, 281-283 cloning, 283-293 sex-determining Y gene, 288-293 Y chromosome deletion mapping, 284-287 structure, 283-284 zinc finger Y gene, 287-288 Goss and Harris experiments, radiation hybrid genome mapping extensions, 68-71 chromosome-specific hybrid panels, 70-

71 hybrid gene enrichment, 69-70 whole genome experiments, 68-69

later experiments, 65-66 preliminary experiments, 66-68

Harris, see Goss and Harris experiments Heart, see Cardiac muscle Hermaphrodites, sex determination, 31 1-313 Homeobox genes, in neoplasia cancer role, 267 developmental processes, 265-266 expression cancers, 268-269 leukemia, 267-268 future research directions, 269-270 overview, 255-256 tumorigenesis, in uiwo model, 267 Homeostasis, see Copper homeostasis Hybrids, see Radiation hybrids

Interspersed repetitive sequence-polymerase chain reaction, radiation hybrid genome mapping, human component analysis, 81-82

Leukemia, homeobox gene expression, 267268 Long-Evans Cinnamin rat, Wilson disease model, 246-247

Mapping, see Genome mapping Meiotic recombination, sister chromatids, 4159 cytological analysis, 52-55 genetic analysis, 43-50 chromosome repeats, 50 circular chromosomes, 48 duplicated gene exchange, 44-47 nonsister chromatid competition, 49 ribosomal DNA crossovers, 43-44 genetic control, 50-52 nonsister chromatid comparison, 55-59 recombination intermediates, 48-49

334 Menkes disease, 233-248 clinical findings, 235-237 copper homeostasis, 233-235 future research directions, 247-248 molecular genetics, 238-242 mottled mouse model, 245-246 Mental retardation, muscular dystrophy, 180181 Muscular dystrophy carrier diagnosis, 211-215 direct diagnosis, 212-214 indirect diagnosis, 211-212 disease characteristics, 177-183 animal models, 183 cardiac muscle, 181-182 mental retardation, 180-181 muscles, 177-180 retina, 182-183 mutational distribution, 203-207 prenatal diagnosis, 211-214 Mutational analysis dystrophin, 203-21 1 genotype/phenotype correlations gross rearrangements, 207-209 point mutations, 209-21 I muscular dystrophy distribution, 203-207 16s ribosomal RNA, 1-31 conserved nucleotides, 19, 20 detection methods, 2-5 mutation introduction, 4-5 plasmid expression, 2-4 double mutations, 19, 21-30 limitations, 30-3 1 second-site suppressor mutations, 3 1 single mutations, 19, 21-30 structure-function analysis, 6-19 3’ major domain mutations, 13-16 3’ minor domain mutations, 16-19 5’ major domain mutations, 10-12 central domain mutations, 12-13 secondary structure, 6-9 upstream mutations, 9-10

Neoplasia, 255-270 future research directions, 269-270 homeobox genes cancer role, 267

Index developmental processes, 265-266 expression cancers, 268-269 leukemia, 267-268 tumorigenesis, in vim model, 267 paired-box genes embryonic development role, 256-259 expression brain cancers, 260-263 primary human tumors, 260 rhabdomyosarcoma, 264-265 Wilms’ tumor, 263-264 oncogene classification, 259 tumorigenesis, in viw model, 259-260

Paired-box genes, in neoplasia embryonic development role, 256-259 expression brain cancers, 260-263 primary human tumors, 260 rhabdomyosarcoma, 264-265 Wilms’ tumor, 263-264 future research directions, 269-270 oncogene classification, 259 overview, 255-256 tumorigenesis, in vim model, 259-260 Plasmids, 16s ribosomal RNA detection, 2-4 Polymerase chain reaction, see Interspersed repetitive sequence-polymerase chain reaction Protein C, in thrombophilia activation, 142-143 anticoagulant properties, 138-140 deficiency effects, 147-148 overview, 136-138 resistance discovery, 150-157 elucidation, 163-168 overview, 138 prevalence, 168-169 thromboembolic disease, 157- 162 Protein S, in thrombophilia C4b-binding protein interactions, 144147 deficiency effects, 148-150 protein C activation, 142-143 Protodystrophin, characteristics, 196- 197

Index Radiation hybrids, genome mapping, 63-94 construction methods, 7 1-77 dosage effects, 75-77 retention frequencies, 75-77 species considerations, 75 future directions, 93-94 Goss and Harris experiments, 65-68 extensions, 68-71 chromosome-specific hybrid panels, 7071 hybrid gene enrichment, 69-70 whole genome experiments, 68-69 human component analysis, 77-82 fluorescence in situ hybridization, 79-80 interspersed repetitive sequencepolymerase chain reaction, 81-82 single-copy sequence scoring, 77-79 mouse genome maps, 92-93 somatic cell genetics, 64 statistical analysis, 82-94 data collection, 83-84 influential hybrids, 91-92 multipoint analysis, 84-91 two-point analysis, 84 Recombination, see Meiotic recomhination Retardation, relationship to muscular dystrophy, 180-181 Retina, pathology in muscular dystrophy, 182183 Rhabdomyosarcoma, paired-box gene expression, 264-265 Ribosomal RNA, 16S, mutational analysis, 131 conserved nucleotides, 19, 20 detection methods, 2-5 mutation introduction, 4-5 plasmid expression, 2-4 double mutations, 19, 21-30 limitations, 30-31 second-site suppressor mutations, 31 single mutations, 19, 21-30 structure-function analysis, 6-19 3' major domain mutations, 13-16 3' minor domain mutations, 16-19 5' major domain mutations, 10-12 central domain mutations, 12-13 secondary structure, 6-9 upstream mutations, 9-10 RNA, see Ribosomal RNA

335

Sex determination, 275-314 abnormalities, 300-3 13 familial agonadism, 3 13 hermaphrodites, 31 1-313 XX males, 308-313 XY females, 301-308 molecular genetics, 278-300 chromosomal basis, 278-281 gonad-determining genes, 293-300 dosage-sensitive sex reversal, 299300 sex-reversing chromosomal deletions, 300 SOX9 gene, 296-297 steroidogenic factor 1, 297-299 WTJ gene, 294-296 testis determining factor candidates, 281-283 cloning, 283-293 sex-determining Y gene, 288-293 Y chromosome analysis, 283-287 zinc finger Y gene, 287-288 overview, 2 75-2 78 Sister chromatid exchange, meiotic recombination, 41-59 cytological analysis, 52-55 genetic analysis, 43-50 chromosome repeats, 50 circular chromosomes, 48 duplicated gene exchange, 44-47 nonsister chromatid competition, 49 ribosomal DNA crossovers, 43-44 genetic control, 50-52 nonsister chromatid comparison, 5559 physical assay, recombination intermediates, 48-49 Smooth muscle, pathology in muscular dystrophy, 182 SOX9 gene, gonad determination, 296297 Statistical analysis, radiation hybrid genome maps, 82-94 data collection, 83-84 influential hybrids, 91-92 multipoint analysis, 84-91 two-point analysis, 84 Steroidogenic factor 1, gonad determination, 297-299

336

Index

Testis, see Gonads; Sex determination Thrombophilia, 135-170 overview, 135-138 antithrombin 111, 135-136 coagulation inhibition, 135-136 coagulation regulation, 136-138 protein C system, 136-138 venous thrombosis, 138 protein C activation, 142- 143 anticoagulant properties, 138-140 deficiency effects, 147-148 overview, 136-138 resistance discovery, 150-157 elucidation, 163-168 overview, 138 prevalence, 168-169 thromboembolic disease, 157-162 protein S C4b-binding protein interactions, 144-

147

deficiency effects, 148-150 protein C activation, 142-143 thromboembolism activated protein C resistance, 157-162 protein C deficiency, 147-148 protein S deficiency, 148-150 thrombomodulin, 140-142 Tissue development, see Neoplasia

Trisomy, chromosome numerical abnormalities

13, 14, 15, 16, 18, 21, 22,

122-123 122-123 122-123 121-122 120-121 115-120 122-123 etiology, 105- 106 frequency, 102-105 mechanisms, 123-125 Tumors, see Cancer development Tyrosine kinase, characteristics, 197

Utrophin/DRP, characteristics, 195-196

Wilmd tumor, paired-box gene expression,

263-264

Wilson disease, 233-248 clinical findings, 235, 237-238 copper homeostasis, 233-235 future research directions, 247-248 Long-Evans Cinnamin rat model, 246-

247

molecular genetics, 242-245 WTI gene, gonad determination, 294-296

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