Adrenal disease in childhood: clinical and molecular aspects (Endocrine Development Vol. 2)

............................ Adrenal Disease in Childhood. Clinical and Molecular Aspects

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Endocrine Development Vol. 2

Series Editor

M.O. Savage, London

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Adrenal Disease in Childhood Clinical and Molecular Aspects

Volume Editors

Ieuan A. Hughes, Cambridge Adrian J.L. Clark, London

41 figures, 4 in color, and 9 tables, 2000

............................ Ieuan A. Hughes, MD, FRCP, FRCPCH, F Med Sci Department of Paediatrics University of Cambridge Addenbrooke’s Hospital Cambridge, UK

Adrian J.L. Clark, DSc, FRCP Departments of Endocrinology St Bartholomew’s and the Royal London School of Medicine and Dentistry West Smithfield, London, UK Library of Congress Cataloging-in-Publication Data Adrenal disease in childhood: clinical and molecular aspects / volume editors, Ieuan A. Hughes, Adrian J.L. Clark p.; cm. – (Endocrine development; vol. 2) Includes bibliographical references and index. ISBN 3805570155 (hardcover: alk. paper) 1. Adrenal glands – Diseases – Molecular aspects. 2. Children – Diseases – Molecular aspects. I. Hughes, I.A. II. Clark, Adrian J.L. III. Series. [DNLM: 1. Adrenal Gland Diseases – genetics – Child. 2. Adrenal Gland Diseases – metabolism – Child. WK 700 A2415 2000] RJ420.A27 A37 2000 618.92€45–dc21 00–044393

Bibliographic Indices. This publication is listed in bibliographic services, including Current ContentsÔ and Index Medicus. Drug Dosage. The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any change in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher. Ó Copyright 2000 by S. Karger AG, P.O. Box, CH–4009 Basel (Switzerland) www.karger.com Printed in Switzerland on acid-free paper by Reinhardt Druck, Basel ISSN 1421–7082 ISBN 3–8055–7015–5

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Contents

VII Foreword Savage, M.O. (London) IX Preface Hughes, I.A. (Cambridge); Clark, A.J.L. (London) 1 SF-1 and DAX-1 in Adrenal Development and Pathology Achermann, J.C.; Jeffs, B.; Jameson, J.L. (Chicago, Ill.) 24 Defects of Adrenocorticotropin Action on the Adrenal Franklin, J.L.; Swords, F.; Huebner, A, Elias, L.L.K.; Clark, A.J.L. (London) 37 Proteins Involved in Mitochondrial Cholesterol Transport Stocco, D.M.; Reinhart, A.J. (Lubbock, Tex.); Miller, W.L. (San Franciso, Calif.) 63 Biochemistry and Genetics of Human P450c17 Miller, W.L.; Auchus, R.J. (San Franciso, Calif.) 93 21-Hydroxylase Deficiency Defects and Their Phenotype Acerini, C.L.; Hughes, I.A. (Cambridge) 112 Defects in Aldosterone Biosynthesis Peter, M. (Kiel/Boltenhagen); Sippell, W.G. (Kiel) 134 X-Linked Adrenoleukodystrophy Ga¨rtner, J. (Du¨sseldorf) 150 Cushing Syndrome and Addison Disease Stratakis, C.A. (Bethesda, Md.) 174 Subject Index

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Foreword

The second volume of the series Endocrine Development represents a model of clinical and basic science interaction. Professor Ieuan Hughes and Professor Adrian Clark have brought together an impressive list of world authorities who have contributed to a volume in which there is a timely emphasis on the latest molecular findings in the principal genetic adrenal disorders. These genetic defects are described in the context of the clinical phenotypes with which they are associated. There are very few published texts on paediatric adrenal disease and this volume is remarkable in pinpointing the chief molecular advances. Key areas of adrenal pathology are covered, including disorders seldom described in detail such as aldosterone synthetic defects, adrenoleukodystrophy, ACTH resistance, Cushing syndrome and Addison’s disease. This book achieves the aim of the series, which is to communicate scientific advances in a clinical context. It will be of benefit to both the clinical and the molecular scientist and to all those studying the developmental biology of endocrine disease. Martin O. Savage London, August 2000

VII

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Preface

Compiling a series of books on Endocrine Development implies an emphasis on changing patterns of function and disease throughout the human lifespan. Nowhere is this better illustrated in the field of endocrinology than by considering the role of the adrenal glands in this context. Here is a suprarenal-sited amalgam of two embryologically distinct glands which continue to function almost totally independent of each other in postnatal life. The cortical component is the subject of review for this second volume in the series masterminded by Martin Savage. A further unique developmental aspect of this endocrine gland is the dramatic involution of the fetal cortex and sequential functional maturation of the adult cortical gland characteristic of higher primates. It was 16 years ago when Karger Publishers last produced a series book on adrenal disease in childhood. In that relatively short period, the content is almost unrecognisable compared with what is portrayed in this present volume. The diseases, however, have not changed. The congenital adrenal hyperplasias continue to be a challenge to endocrinologists in diagnosis and management and eponymous references to Cushing and Addison will surely always target the mind to the adrenal glands. Biochemical pathways figured highly 16 years ago but there was no reference to any gene known to be relevant to adrenal function. Even for that common condition in paediatric endocrine practice, 21-hydroxylase deficiency, it was only by linkage studies that the gene which would subsequently be termed CYP21 was found to be closely associated with the HLA complex. Now every major enzymatic step of clinical relevance in cortisol, aldosterone and adrenal androgen biosynthesis has been characterised by molecular studies of the cognate genes. This current

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volume illustrates, for example, the advances in knowledge about cholesterol transport mechanisms and how a defect in a specific transport protein explains cholesterol accumulation in Prader syndrome (congenital lipoid adrenal hyperplasia), rather than what had assumed to be a defect in the cytochrome P450 side chain cleavage enzyme. The study of rare genetic disorders provides valuable insight as to how complex biological pathways may function; this volume is peppered with suitable examples, ranging from the role of SF-1 and DAX-1 in formation of the adrenal glands to genes involved in adrenal tumorigenesis. Much about the understanding of the molecular mechanisms of adrenal disease which is relevant for clinical practice is covered by the authors. The reader will also find in this book a possible answer as to what determines the onset of adrenarche and what may be the true function of the most abundant steroid produced by the adrenals, dehydroepiandrosterone. Those questions, however, deserve a separate treatise to do the subjects justice. The pace of change in molecular and cell biology will undoubtedly render this volume in certain respects dated within a few years. Nevertheless, all is currently and much will remain, of great importance in the investigation and management of adrenal disease. The Editors express thanks to Martin Savage for giving us the privilege to bring together a group of co-authors who are the leading experts in their respective fields and to Hermann Frei for his support with the editing process. Ieuan Hughes Adrian Clark August 2000

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Hughes IA, Clark AJL (eds): Adrenal Disease in Childhood. Clinical and Molecular Aspects. Endocr Dev. Basel, Karger, 2000, vol 2, pp 1–23

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SF-1 and DAX-1 in Adrenal Development and Pathology John C. Achermann, Baxter Jeffs, J. Larry Jameson Division of Endocrinology, Metabolism, and Molecular Medicine, Northwestern University Medical School, Chicago, Ill., USA

During development, the adrenal gland undergoes a distinct series of morphological and functional changes that vary considerably among species. In higher primates, for example, the fetal adrenal cortex lacks the typical adult pattern of zonation, but is composed rather of a large inner fetal zone (FZ) (90%) surrounded by an outer definitive zone (DZ). The FZ grows rapidly during fetal life, but involutes around the time of birth, and the mature adult gland develops. The typical pattern of zonation becomes complete following development of the zona reticularis at adrenarche. Rodents have a predominant ‘X’ zone during early life; it regresses at puberty in males or following the first pregnancy in females. Adrenarche does not occur in non-primates. The mechanisms involved in these processes of adrenal growth and remodelling are poorly understood at present, as are the factors that regulate them. Some insight into the factors involved in adrenal development in humans has been provided by studies of children or families with adrenal hypoplasia. For example, the adrenal hypoplasia seen in children with congenital adrenocorticotropin (ACTH) deficiency (e.g. anencephaly) or mutations in the ACTH receptor [see chapter by Franklin et al., this vol.] underscores the importance of ACTH in adrenal development. Primary familial forms of congenital adrenal hypoplasia also exist, and can have autosomal-recessive or X-linked modes of inheritance. The autosomal recessive form has a ‘miniature adult’ adrenal phenotype and its etiology is not understood. The X-linked ‘cytomegalic’ form of adrenal hypoplasia congenita (AHC) is well described. This condition was shown to result from mutations in the orphan nuclear receptor, DAX-1, in 1994 [1, 2] (fig. 1). These findings raised the possibility that nuclear receptors, or related transcription factors, could play a crucial role in human adrenal

Fig. 1. Experimental landmarks in SF-1 and DAX-1 research over the last decade.

development and disease. Indeed, the orphan nuclear receptor steroidogenic factor-1 (SF-1) was known to affect adrenal development in mice, and the first SF-1 mutation causing adrenal failure in humans has recently been reported [3] (fig. 1). This chapter will review our current knowledge about the roles of SF-1 and DAX-1 in adrenal disease, and will speculate on future directions basic and clinical research in this area may take.

Steroidogenic Factor-1 Identification of the Gene Encoding SF-1 (FTZF1) The existence of a common ‘steroidogenic factor’ was originally proposed following the discovery of a number of similar regulatory elements in the proximal promoter region of the cytochrome P450 steroid hydroxylase gene family [4, 5]. These elements contained variations on an AGGTCA DNA sequence motif suggesting that a shared protein could regulate their transcription. This protein was termed ‘steroidogenic factor-1’. The murine cDNA encoding SF-1 was cloned from an adrenal cDNA library using a probe

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corresponding to the DNA-binding domain (DBD) of a related orphan receptor, the retinoid X receptor b [6]. The bovine equivalent (adrenal 4-binding protein, Ad4BP) was selected from an adrenal cDNA library using the partial sequence of a protein purified from bovine adrenal extracts [7]. The proteins encoded by either cDNA increased the promoter activity of the steroid hydroxylases and, thus, represented the proposed SF-1 factor. The mouse gene was mapped to chromosome 2 and termed Ftz-F1, since it resembles the Drosophila orphan nuclear receptor, fushi tarazu factor 1 (FTZ-F1) [8, 9]. The human homologue, FTZF1, was mapped to chromosome 9q33 and contains 7 exons spanning 30 kb of genomic DNA [10–12]. Structure of SF-1 SF-1 is a 461 amino acid protein that shares structural similarities with many members of the nuclear receptor superfamily [13]. These critical regions possess significant, often absolute, sequence conservation among mammalian species [6, 7, 12] and include a two zinc finger DBD, an ‘A’ box (or FTZF1 box), a hinge region, and an activation function-2 (AF-2) domain (fig. 2, upper panel). The first zinc finger of the SF-1 DBD contains a proximal (‘P’) box, which is implicated in the recognition of DNA-binding sites [14, 15]. This region confers specificity to nuclear receptors in the regulation of target genes. The second zinc finger contains a distal (‘D’) box, which is believed to determine the appropriate spacing of these DNA-binding site motifs. The ‘A’ box may also influence DNA binding by recognizing specific nucleotides proximal to the AGGTCA response element in target genes of SF-1. This interaction may help stabilize monomeric binding by SF-1 [16, 17]. The hinge region and the AF-2 domain are both likely to be involved in transcriptional activation [7, 18]. SF-1 Expression The expression pattern of SF-1, in rodents and humans, is consistent with its critical role in adrenal development, steroidogenesis and gonadal differentiation (fig. 3). In the mouse, Sf-1 is first expressed in the urogenital ridge at embryonic day 9 (E9) [19]. This region subsequently develops into the adrenal cortex, bipotential gonad and mesonephros. In the developing adrenal gland, Sf-1 is transcribed in the adrenal primordium at E11 and localizes to the adrenal cortical cells from E13 into adulthood [20, 21]. Indeed, the expression of Sf1 in the primordium prior to the onset of steroidogenic competence strongly supports a role for SF-1 in regulating adrenal development per se [22]. Sf-1 is also expressed in the developing gonad, where it interacts with transcription factors such as Wilms tumor-1 (WT-1), DAX-1, SRY and SOX9 in the process of male sex determination and testis formation (fig. 3). Following

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Fig. 2. Upper panel. Schematic representation of SF-1. Lower panel. The SF-1 DNAbinding domain contains two zinc fingers. The first human SF-1 mutation (G35E) involves the last amino acid of the ‘P’ box. Adapted with permission from Achermann et al. [3].

gonadal determination (E13), Sf-1 is expressed in the developing testis and persists into adult life [19, 23]. In Sertoli cells, Sf-1 regulates Mu¨llerian inhibiting substance (MIS) expression leading to regression of Mu¨llerian structures in males [24, 25]. In Leydig cells, Sf-1 regulates steroidogenesis and testosterone biosynthesis. Sf-1 is not expressed in the developing rodent ovary, but reappears in the granulosa and theca cells at the onset of folliculogenesis [26], suggesting a key role in the regulation of ovarian steroidogenesis. Sf-1 also plays an important role in the normal development and function of the hypothalamicpituitary gonadal axis. It is expressed in the anterior pituitary from E13.5 and adult gonadotropes thereafter [27, 28]. Surprisingly, Sf-1 is also expressed in the ventral proencephalon (E11.5) [23] and ventral medial hypothalamus (VMH) [27, 28]. The functional significance of its expression in these sites is unclear [29], but it appears to play a role in gonadotropin-releasing hormone (GnRH) secretion (see below). Recently, data have become available for the expression of SF-1 during human development [30]. SF-1 is first detectable in the adrenal primordium at Carnegie stage (CS) 15, 33 days post-ovulation (dpo). This precedes slightly

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Fig. 3. Overview of the roles of SF-1 and DAX-1 in adrenal and gonadal development. WT-1>Wilms tumor-1; MIS>Mu¨llerian-inhibiting substance. Reproduced with permission from the publishers and the authors [74].

the onset of expression of P450scc. SF-1 colocalizes with steroidogenic enzymes in the developing fetal adrenal cortex. Although its role in the differentiation and zonation of the mature adult cortex in primates is unknown, SF-1 is clearly expressed in all layers of the mature gland where it presumably plays an ongoing role in the regulation of steroidogenesis [31]. Function of SF-1 SF-1 regulates a variety of genes involved in sex determination and differentiation, reproduction, and steroidogenesis (table 1). In the adrenal gland, these target genes are involved in multiple steps of steroidogenesis, from the early transport of cholesterol into the mitochondria (e.g. steroidogenic acute regulatory protein, StAR) to the final production of mineralocorticoids and glucocorticoids [32, 33]. SF-1 also stimulates ACTH receptor expression, providing an additional mechanism by which it may regulate adrenal development and function [34, 35]. SF-1 regulates gene expression by binding to specific response elements on the promoters of target genes. These DNA-binding sites contain variations

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Table 1. Putative target genes of SF-1 involved in sexual differentiation, reproduction and steroidogenesis Gene

Reference

Sexual Differentiation WT-1 DAX-1 MIS MIS-R

[91] [87] [24] [97]

Reproduction GnRH-R a-GSU LHb Oxytocin Prolactin-R LEY I-L (INSL3) Inhibin a

[103] [105] [108, 109] [110] [111] [112] [114]

Gene

Reference

Steroidogenesis ACTH-R StAR P450scc 3b-HSD 21-Hydroxylase 11b-Hydroxylase 17a-Hydroxylase P450aldo Aromatase

[34, 35] [32, 33] [95, 96] [98] [99] [100] [101, 102] [104] [106, 107]

Metabolism HDL-R

[113]

WT-1>Wilms tumor-1; MIS>Mu¨llerian-inhibiting substance; GnRH> gonadotropin-releasing hormone; a-GSU>glycoprotein hormone a subunit; LHb>luteinizing hormone b subunit; LEY I-L>Leydig cell-specific insulinlike peptide; ACTH>adrenocorticotropin; StAR>steroidogenic acute regulatory protein; P450scc>cytochrome P450 side chain cleavage enzyme; 3bHSD>3b-hydroxysteroid dehydrogenase; HDL>high-density lipoprotein. The suffix ‘R’ indicates the ligand receptor.

of the AGGTCA DNA sequence motif. SF-1 is believed to bind to these response elements as a monomer. This feature is atypical for nuclear receptors, since the majority require either homodimerization or heterodimerization with a partner such as the retinoid X receptor (RXR). SF-1 may recruit coactivators such as steroid receptor coactivator-1 (SRC-1) to regulate gene expression [18], but it remains unclear whether the presence of a specific ligand is required. Although oxysterols were proposed as a candidate SF-1 ligand [36], subsequent experiments demonstrated that 25-hydroxycholesterol does not substantially enhance transcriptional regulation by SF-1 [37]. Additional reports suggest that phosphorylation events may be important in regulating SF-1 activity. Hammer et al. [38] showed that SF-1 mediated transcription is regulated by phosphorylation of a single serine residue (Ser203). This phosphorylationdependent activation appears to be regulated by the mitogen-activated protein kinase (MAPK) signaling pathway. Further studies will help determine the

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Table 2. Human and murine (XY) consequences of mutations in SF-1 and DAX-1 SF-1

DAX-1

mouse knockout human (G35E) homozygous heterozygous

mouse knockout hemizygous

Adrenal

agenesis

failure

fetal zone retained failure

Testis

absent

dysgenetic

hypogonadal

Male sexual differentiation

XY sex reversal

XY sex reversal normal

normal

Mu¨llerian structures

present

present

absent

absent

GnRH

deficient

intact

intact

deficient

Gonadotropins

deficient

intact

intact

deficient

Spermatogenesis

absent

absent

impaired

impaired

human hemizygous

hypogonadal

mechanisms by which SF-1 plays a pivotal role in the regulation of cell-specific gene transcription. Mouse Ftz-F1 Knockout Models Several groups have used a targeted gene disruption strategy in embryonic stem cells to create Sf-1 (FtzF1) knockout mice [39–42]. Despite different targeting strategies, strikingly similar phenotypic features have been seen. Mice homozygous for the gene deletion (–/–) had adrenal and gonadal agenesis, male-to-female sex reversal, and persistence of Mu¨llerian structures in males (table 2). Whilst some evidence of early urogenital ridge formation was seen in Sf-1 knockout embryos (E10.5), the progenitor cells rapidly regressed through apoptosis (E11.5–12). SF-1 is obligatory, therefore, for both adrenal and gonadal development. Shortly after birth, mice showed evidence of adrenal failure, but could be rescued by steroid replacement therapy. Decreased levels of gonadotropins and GnRH receptor transcripts were detected in the gonadotropes of knockout animals, and a virtual absence of the VMH was observed [40, 43]. Gonadotrope function could be restored by GnRH treatment, suggesting that Sf-1 deficiency does not result in an irreversible loss of function of these anterior pituitary cells [43]. Human SF-1 (FTZF1) Mutation We recently reported the first human SF-1 mutation in a patient with primary adrenal failure, XY sex reversal and persistent Mu¨llerian structures

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[3]. The patient, a phenotypic female, presented in vascular collapse at 17 days of age (sodium=110 mmol/l, potassium 8.3 mmol/l). She exhibited signs of primary adrenal insufficiency during the first 2 weeks of life (hypoglycemia, jaundice, weight loss, malaise and pigmentation) and had inappropriately low cortisol (325 nmol/l) and aldosterone (520 pmol/l) concentrations in the context of her clinical condition. She improved dramatically after treatment with steroids. Primary adrenal insufficiency was confirmed during reevaluation three weeks later (cortisol, 34 nmol/l; ACTH 360 pmol/l) and she was maintained on glucocorticoid and mineralocorticoid replacement throughout childhood. Her karyotype was found to be XY and 20,22-desmolase deficiency or StAR deficiency were considered likely diagnoses. Further investigations before the induction of puberty confirmed primary adrenal insufficiency. There was no cortisol response to exogenous ACTH administration. Pituitary gonadotropins responded to GnRH stimulation but there was no testosterone response to exogenous human chorionic gonadotropin. Laparotomy revealed normal Mu¨llerian structures and streak-like gonads containing poorly differentiated tubules and connective tissue. Puberty was induced with oestrogens and menstruation occurred after the introduction of cyclical progestogen, confirming the presence of a functional uterus. Direct sequencing revealed a heterozygous G35E mutation in the ‘P’ box of the SF-1 DNA-binding domain (fig. 2, lower panel). This mutation was created in an expression vector by site-directed mutagenesis to examine the functional effects of the amino acid substitution. Mutant SF-1 protein showed abnormal binding to target genes and impaired transactivation as predicted from its site in the ‘P’ box. This mutation was not present in either parent, who were phenotypically normal. Sequencing of the remainder of FTZF1 (including promoter and intron-exon boundaries) and related genes (DAX1, SRY, StAR) failed to reveal any further mutations. Indeed, the de novo nature of this heterozygous mutation might be predicted, since it causes infertility and therefore cannot be transmitted. The phenotype of this patient with a heterozygous point mutation in SF-1 is much less severe than that seen in homozygous Sf-1 (– /–) knockout mice, who have complete adrenal agenesis (table 2). In contrast, heterozygous Sf-1 (– /+) knockout mice with deletion of one allele of Sf-1 are reportedly normal, though further studies are in progress to detect more subtle defects. Although dominant negative activity by the human mutant SF-1 protein could account for this difference, we have been unable to demonstrate such an effect by using in vitro studies. An alternative hypothesis is that SF-1 acts in a dosagedependent manner, which has a more pronounced effect on adrenal development in humans compared to mice. Since SF-1 regulates many of the genes involved in steroidogenesis, haplo-insufficiency or a reduced dosage of func-

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Fig. 4. The classic inheritance pattern of X-linked AHC. Affected males (III7, III8, IV2, IV3 and IV4) are indicated by shading. Reproduced with permission from Habiby et al. [62].

tional SF-1 could have a cumulative effect on each step of steroid production. This could be sufficient to cause the clinically significant adrenal failure seen. Mice may be more resistant to abnormalities of adrenal development; the Ahch (Dax1) knockout model described below also does not appear to have significantly impaired adrenal function [44]. Indeed, mice may also be more resistant to dose dependent mechanisms of sex-reversal, as suggested by studies of DAX-1 overexpression [45], WT-1 mutation [46], and the case of SF-1 here. Further reports of human SF-1 mutations and functional studies of their effects are needed to address this issue. However, this case confirms that SF-1 plays a key role in adrenal development and function in humans, and FTZF1 should be considered a candidate gene in children with primary adrenal failure, XY sex-reversal and a uterus.

DAX-1 X-Linked Adrenal Hypoplasia Congenita X-linked adrenal hypoplasia congenita (AHC) is a rare, potentially lifethreatening disorder of adrenal gland development, first described in 1948 [47]. Affected boys typically present with primary adrenal failure, shortly after birth or during childhood. The mature adult zone of the adrenal cortex fails to develop; large vacuolated ‘cytomegalic’ cells resembling fetal adrenocortical cells are seen [48]. The failure of pubertal development in AHC patients kept alive with adrenal steroids led to the recognition that hypogonadotropic hypogonadism (HHG) is also an integral feature of the condition [49]. The X-linked inheritance of AHC is demonstrated in the pedigree shown in figure 4.

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Fig. 5. Upper panel. The gene encoding DAX-1 is located on the short arm of the X chromosome, telomeric to the genes responsible for Duchenne muscular dystrophy (DMD) and glycerol kinase deficiency (GKD) and encompassed by the AHC and dosage-sensitive sex reversal (DSS) loci. Lower panel. Schematic representation of DAX-1. The amino-terminal domain of DAX-1 has a unique structure containing three and a half copies of a 66–67 amino acid repeat motif. The carboxy-terminal domain has structural similarities with the ligandbinding domain (LBD) of several nuclear receptors. Putative transcriptional silencing domains are shown by black bars. Adapted with permission from Zanaria et al. [1].

Identification of the Gene Encoding DAX-1 (AHC) In a subset of patients, X-linked AHC can occur as part of a contiguous gene deletion syndrome in association with glycerol kinase deficiency (GKD), Duchenne muscular dystrophy (DMD), ornithine transcarbamylase deficiency and mental retardation (fig. 5, upper panel) [50]. This observation enabled a critical region of Xp21 (250–500 kb) containing the AHC locus to be mapped using a yeast artificial chromosome (YAC) contig strategy [51]. This region of the short arm of the X chromosome overlaps a 160 kb locus associated with dosage-sensitive sex reversal (DSS) [52]. In DSS, duplications of this part of Xp21 cause genetic males (XY) to undergo incomplete sex reversal [52]. In 1994, the gene DAX-1 (Dosage-sensitive sex-reversal, AHC, on the X-chromosome, gene 1) was cloned, and mutations in it were shown to cause X-linked AHC and HHG [1, 2]. Numerous mutations in DAX-1 have now been reported in families with this condition (fig. 6). DAX1 also appears to be the gene responsible for DSS, as overexpression of Dax-1 can induce sex reversal in male mice [45]. Structure of DAX-1 DAX-1 is a 470 amino acid protein encoded by a 5-kb gene, which consists of two exons (fig. 5, lower panel). The amino-terminal domain of this protein possesses a unique structure consisting of a 66–67 amino acid repeat motif. This structure does not resemble any known DNA-binding domain, but has

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Fig. 6. Naturally occurring DAX-1 mutations.

been proposed to regulate the transcription of target genes by binding to hairpin loop structures in DNA (see below) [53]. In contrast, the carboxyterminal region of DAX-1 has sequence homology to the ligand binding domain (LBD) of several nuclear receptors, including the small heterodimer partner (SHP), the orphan receptor TR4 and chicken ovalbumin upstream promoter transcription factor (COUP-TF). This domain appears to be involved in transcriptional control by DAX-1 [54, 55]. DAX-1 Expression DAX-1 is expressed at each level of the hypothalamic-pituitary-adrenal/ gonadal axis, and its pattern of expression closely follows that of SF-1. In the mouse, Dax-1 transcripts can first be detected in the urogenital ridge at E10.5 (fig. 3). The cells that develop into the adrenal primordium and subsequent three layers of the adrenal cortex express Dax-1 from E12.5. This expression continues throughout adult life [56, 57]. The detection of Dax-1 expression in the proencephalon at E11.5 and in the VMH and anterior pituitary gonadotropes thereafter [56] is consistent with a role in gonadotropin production. Finally, the differentiating gonad also expresses Dax-1 until E12, after which there is a rapid decline in its expression in the testis, but continued expression in the ovary [56, 57]. This pattern supports a role for Dax-1 in gonadal differentiation, either as an ovarian determining gene or as a repressor of testicular development. The latter hypothesis is supported by the XY sexreversal seen with DAX-1 duplication in humans [52] and overexpression in mice [45], along with the fact that ovarian development is successfully maintained in Ahch (Dax1) knockout mice [44] and in a woman who is homozygous for a DAX1 gene mutation [58] (fig. 3). In humans, DAX-1 transcripts appear at least 33 dpo in the gonadal ridge and adrenal primordium [59]. DAX-1 continues to be expressed in the

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developing fetal adrenal gland [59] and is present in the adult adrenal cortex [60]. DAX-1 is also expressed in the testis, hypothalamus and pituitary gland, and at low levels in the adult ovary [60]. Human DAX-1 Mutations Excluding the rare cases of gene deletion, over 50 different mutations in DAX-1 have been reported to date in more than 60 families with X-linked AHC (fig. 6). The majority of these mutations are frameshift or nonsense mutations that result in premature truncation of the DAX-1 protein [1, 2, 58, 61–78]. The limited number of missense mutations reported are all located in the putative LBD [2, 67, 68, 71, 75, 79–82]. Although the incidence of congenital adrenal hypoplasia has been quoted as 1:12,500 [83], the true incidence of X-linked AHC due to DAX-1 gene mutations is probably considerably less than this [73]. Boys with DAX-1 mutations classically present with signs and symptoms of primary adrenal insufficiency shortly after birth (60%) or during childhood (40%) [74]. The median age at presentation is three weeks. Individuals presenting in the first months of life often demonstrate failure to thrive, poor weight gain, vomiting, prolonged jaundice, hyperpigmentation and, ultimately, shock. Hyponatremia and hyperkalemia are evident on biochemical testing and hypoglycemia often occurs. Following appropriate resuscitation, lifelong mineralocorticoid and glucocorticoid replacement is required. Investigations of adrenal function are consistent with primary adrenal failure with elevated ACTH and plasma renin activity, and diminished cortisol, aldosterone and 17-hydroxyprogesterone concentrations. A normal basal cortisol result during stress or prior to the onset of symptoms does not exclude impaired adrenal reserve [68, 82]. An elevation in 11-deoxycortisol has been documented in some children with X-linked AHC, perhaps reflecting persistent fetal adrenal activity [73, 82]. Children who survive the first two months of life often have a more insidious onset of disease in childhood. Signs and symptoms may be subtle, and include nausea, salt-craving, weight loss, hyperpigmentation and hypotension. Presentation may be triggered by a stressful event, such as illness or an operation. This apparently bimodal pattern of presentation may reflect underlying age-related changes in mineralocorticoid production and sensitivity, sodium and fluid intake, and counter-regulatory responses [74]. Nevertheless, the vast majority of patients with X-linked AHC present before the age of ten years. At present there appears to be little correlation between the type of mutation and age at presentation or diagnosis [74]. Indeed, loss of the 11-carboxyterminal amino acids of DAX-1 is sufficient to cause a clinically severe phenotype [66]. Further, age at presentation may vary between sibling pairs with

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the same DAX-1 mutation, suggesting that other genetic and epigenetic factors may be important [74]. Although ‘early presentation’ or ‘late presentation’ appears to be a feature in certain families, the younger brother is often diagnosed at an earlier age, probably reflecting increased awareness of the subtle signs and symptoms of adrenal failure by families and physicians [82]. The association of HHG with X-linked AHC is now clear, but the age at which it becomes clinically evident is variable. For example, several boys with DAX-1 mutations have been reported to have undescended testes at birth [2, 62], whereas normal HPG activity in infancy has been documented in other cases [67, 72, 73, 82]. Indeed, spontaneous onset of puberty has been described in several patients with DAX-1 mutations, although failure to progress beyond the early stages of puberty soon becomes evident [75, 84]. Pubertal failure due to HHG should be considered the norm in boys with this condition, and treatment with sex steroids should be initiated. Attempts to induce puberty or fertility using pulsatile administration of GnRH has generally produced disappointing results [62, 70, 77, 78, 81]. This may reflect the functional importance of DAX-1 at both the hypothalamic and pituitary levels [62]. Recently, gonadotropins have been used in an effort to stimulate testosterone production and to induce spermatogenesis [78, 81]. Short-term [62–64, 66, 69, 74, 79] as well as prolonged [70, 78, 81] administration of human chorionic gonadotropin usually results in a significant testosterone response, suggesting that this aspect of Leydig cell function is intact, although in one case a response was only obtained following concomitant administration of FSH [77]. Despite this, the limited data available suggest that it may be difficult to induce spermatogenesis using exogenous gonadotropins in patients with DAX-1 mutations [78, 81]. DAX-1 may therefore have a direct gonadal effect that impairs spermatogenesis in humans, as reported in Ahch (Dax1) knockout mice (below) [44]. Recently, several cases have been reported which have widened the phenotypic spectrum of patients with DAX-1 gene mutations. In one report, a woman with HHG, but normal ovarian development and normal adrenal function, was found to be homozygous for a DAX-1 mutation through gene conversion [58]. Two hemizygous males in this kindred had the classic phenotype of X-linked AHC, including adrenal failure. However, this family was also atypical in that fertility was preserved in one hemizygous male. In another report, a man who first presented with mild adrenal failure and incomplete HHG in his twenties was found to have a missense mutation in DAX-1, which caused partial loss of function in transcription assays [81]. Finally, extreme delayed puberty has been found in heterozygous female carriers of DAX-1 mutations in one family [78]. These reports prompted us to sequence the DAX-1gene in over 100 patients with familial and sporadic forms of HHG and delayed

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puberty [85]. No mutations were found, suggesting that DAX-1 mutations are unlikely to be a common cause of reproductive disorders in the absence of adrenal failure. Following the identification of DAX-1 mutations, genetic screening and counseling should be offered to families. The proband’s brothers and sisters should be screened for hemizygous and heterozygous mutations respectively, and analysis should be offered to the mother’s siblings and their children. As an example, we recently discovered a DAX-1 mutation in the 8-month-old asymptomatic younger brother of a boy with adrenal failure [82]. Despite normal basal cortisol concentrations, he had biochemical evidence of compensated primary adrenal failure and impaired adrenal reserve following ACTH stimulation. Such individuals highlight the importance of screening those at risk. Prenatal or early postnatal analysis may be desired in some cases. The Mouse Ahch (Dax1) Knockout Model and Spermatogenesis We have recently created Ahch (Dax1) knockout mice as a model for X-linked AHC in humans [44]. A ‘Cre-loxP’ targeting strategy was required as Dax-1 appears essential for embryonic stem (ES) cell survival and because mutations in Dax-1 cause infertility in males (ES cells are XY-derived). Initial development of the fetal and adult cortical zones is similar in male Dax-1 mutant mice and wild-type littermates, and normal zona glomerulosa and fasciculata development are seen in the adult knockout mice. However, the fetal X-zone fails to regress as expected following sexual maturation. This persistence of fetal tissue resembles the cytopathological changes observed in human AHC patients (table 2). Whilst patients with X-linked AHC require steroid treatment, serum corticosterone levels are normal in the knockout model. This finding suggests that murine Dax-1 is required for the initiation of fetal adrenal degeneration but that it is not vital for the formation of the adult cortex or steroidogenesis. This could reflect species-specific differences in the processes that control adrenal development and function. Further, this observation contrasts with the adrenal agenesis seen following disruption of the murine Sf-1 gene (table 2), suggesting that DAX-1 may act downstream of SF-1 in adrenal development. Male Dax1 knockout mice are hypogonadal and infertile, despite having sufficient testosterone production for the formation of male internal and external genitalia. Their testes show progressive seminiferous tubule degeneration and loss of germ cells. Dax-1 may therefore play an intrinsic role in spermatogenesis, since gonadotropin concentrations appear normal in the knockout model. Indeed, Dax-1 is expressed in Sertoli cells during rat spermatogenesis [86]. The Dax1 knockout may therefore represent a good model for examining the role of Dax-1 in spermatogenesis.

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Function of DAX-1 The coexpression of DAX-1 with SF-1 in the gonadal and adrenal axes (above), and the adrenal failure seen in patients with mutations in these genes (table 2) [2, 3], suggest that DAX-1 and SF-1 might interact in a common pathway of adrenal and gonadal development and function. However, many of the mechanisms underlying this interaction remain unclear. It is tempting to speculate that DAX-1 acts downstream of SF-1 in a regulatory cascade. For example, the promoters of both the human [87] and mouse [88, 89] DAX1/ Ahch genes contain SF-1 binding sites, and SF-1 can upregulate DAX-1 expression in vitro [88–90]. However, data regarding down-regulation of Dax-1 expression in Sf-1 knockout mice are equivocal [56, 89], so direct evidence for the regulation of Dax-1 by Sf-1 remains inconclusive. The majority of data available suggest that DAX-1 acts as a repressor of gene transcription in a diverse range of in vitro gene expression assays. For example, DAX-1 has been shown to inhibit basal transcriptional activity [54] and SF-1-mediated transactivation [53, 54, 91, 92], and DAX-1 represses the activity of putative target genes such as StAR, P450scc, 3b-hydroxysteroid dehydrogenase [53, 93], and LHb [94]. An example of the effect of wild-type DAX-1 on SF-1-mediated transcription is shown in figure 7. This repression of gene transcription by DAX-1 is consistent with the XY sex reversal seen in association with overexpression of Ahch/DAX1 in mice [45] and humans [52], where DAX-1 is thought to antagonize the actions of SRY in gonadal development. However, a repressor role for DAX-1 in the adrenal and reproductive axes is more difficult to understand. A variety of mechanisms have been proposed to explain this repression of gene transcription by DAX-1. Zazopoulos et al. [53] have suggested that DAX-1 has a direct action on gene transcription by binding to hairpin loop structures in the promoters of certain target genes such as StAR. Elimination of this DAX-1-binding site results in loss of DAX-1 inhibition. However, since DAX-1 also inhibits genes without such hairpin structures, other mechanisms also must be important; these may involve protein-protein interactions. Evidence for an interaction between DAX-1 and SF-1 has been obtained from protein pull-down assays [54] and two-hybrid approaches [92]. In addition, a transcriptional silencing domain has been localized to the carboxy-terminus of DAX-1 in a region homologous to the AF2 domain of other nuclear receptors (fig. 5) [54, 55]. This region is lost in patients who have frameshift or nonsense (stop codon) mutations (fig. 6). Mutant DAX-1 proteins corresponding to these naturally occurring mutations show impaired inhibition in transient gene expression studies (the frameshift mutation 702delC is shown as an example in fig. 7). The location of this silencing domain raises the possibility that there is a natural ligand for DAX-1, which could alter its

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Fig. 7. Transient gene expression assay to assess the functional interactions of SF-1 and DAX-1. SF-1 stimulates the transcription of a reporter gene containing luciferase. This SF-1mediated transcriptional activation is repressed by the presence of wild-type (WT) DAX-1. This repression is lost when DAX-1 protein containing the naturally occurring 702delC mutation is expressed instead of WT. Adapted with permission from Reutens et al. [74].

functional properties. This process could also involve the recruitment of corepressors, such as NCoR, to the DAX-1 LBD [92], or DAX-1 might interfere with the recruitment of coactivators to other factors such as SF-1. Investigation of naturally occurring missense mutations may be useful in defining critical domains for DAX-1 function and interaction in future.

Future Directions The importance of SF-1 and DAX-1 in adrenal development and function is well established. However, many questions remain unanswered about their specific roles and putative interactions. For example, the processes regulating the development and zonation of primate and rodent adrenals are poorly understood, and many other transcription factors, signaling pathways and humoral elements are likely to be involved in this complex cascade. Nevertheless, SF-1 and DAX-1 clearly play a pivotal role in this process.

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The identification of additional patients with mutations in SF-1 will help to clarify its role in adrenal and gonadal development. Such patients could potentially have milder or more severe phenotypes, depending on the nature of the mutation and its effects on SF-1 structure and function. Similarly, patients with less typical forms of AHC may help identify critical regions of the DAX-1 protein, or other genetic factors that influence disease presentation. Identifying DAX1 mutations in asymptomatic siblings may enable us to study the role of DAX-1 in human fetal zone regression or adult zone development, prior to the onset of steroid therapy. Although the mechanisms of adrenal development may differ between rodents and primates, important information can be obtained from transgenic and knockout models. Targeting specific mutations into transgenic animals rather than knocking out entire genes may allow different functional domains to be characterized. Creating double knockouts of SF-1 and DAX-1 may help to determine their relative roles in the developmental cascade, as well as to explore cumulative gene dosage effects.

Acknowledgments This work received funding from the National Cooperative Program for Infertility Research and was supported by NIH Grants U54-HD-29164, PO1 HD-21921 and GCRC grant MO1-RR-00048. J.C.A. received fellowship support from the Endocrine Fellows Foundation. B.J. holds a Wellcome Trust International Prize Travelling Research Fellowship (Grant 056375).

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Lynch JP, Lala DS, Peluso JJ, Luo W, Parker KL, White BA: Steroidogenic factor 1, an orphan nuclear receptor, regulates the expression of the rat aromatase gene in gonadal tissues. Mol Endocrinol 1993;7:776–786. Halvorson LM, Kaiser UB, Chin WW: Stimulation of luteinizing hormone beta gene promoter activity by the orphan nuclear receptor, steroidogenic factor-1. J Biol Chem 1996;271:6645–6650. Keri RA, Nilson JH: A steroidogenic factor-1 binding site is required for activity of the luteinizing hormone beta subunit promoter in gonadotropes of transgenic mice. J Biol Chem 1996;271:10782– 10785. Wehrenberg U, von Goedecke S, Ivell R, Walther N: The orphan receptor SF-1 binds to the COUPlike element in the promoter of the actively transcribed oxytocin. J Neuroendocrinology 1994;6: 1–4. Hu Z, Zhuang L, Guan X, Meng J, Dufau ML: Steroidogenic factor-1 is an essential transcription activator for gonad-specific expression of promoter I of the rat prolactin receptor gene. J Biol Chem 1997;272:14263–14271. Zimmerman S, Schwarzler A, Buth S, Engel W, Adham IM: Transcription of the Leydig insulinlike gene is mediated by steroidogenic factor-1. Mol Endocrinol 1998;12:706–713. Lopez D, Sandhoff TW, McLean MP: Steroidogenic factor-1 mediates cyclic 3€,5€-adenosine monophosphate regulation of the high-density lipoprotein receptor. Endocrinology 1999;140:3034–3044. Ito M, Park Y, Weck J, Mayo KE, Jameson JL: Synergistic activation of the inhibin alpha promoter by steroidogenic factor-1 and cAMP. Mol Endocrinol 2000, in press.

J. Larry Jameson, MD, PhD, Endocrinology, Metabolism and Molecular Medicine, Northwestern University Medical School, 303 East Chicago Avenue, Tarry Building 15–709, Chicago, IL 60611 (USA) Tel. +1 (312) 503–0469, Fax +1 (312) 503–0474, E-Mail [email protected]

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Hughes IA, Clark AJL (eds): Adrenal Disease in Childhood. Clinical and Molecular Aspects. Endocr Dev. Basel, Karger, 2000, vol 2, pp 24–36

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Defects of Adrenocorticotropin Action on the Adrenal F. Swords, J.L. Franklin, A. Huebner, L.L.K. Elias, A.J.L. Clark Molecular Endocrinology Laboratory, Departments of Endocrinology, St Bartholomew’s and the Royal London School of Medicine and Dentistry, London, UK

Normal Action of ACTH Adrenocorticotropin (ACTH) is a 39 residue peptide secreted by the corticotroph cells of the anterior pituitary. It is synthesised as part of a larger precursor peptide, pro-opiomelanocortin, which also encodes c-MSH and b-endorphin. ACTH normally circulates at concentrations of =5 – 80 pg/ml, and its primary site of action is the ACTH receptor (ACTH-R). This receptor, also known as the melanocortin 2 receptor (MC2-R), is a G protein-coupled receptor with considerable sequence similarity to the other four members of the melanocortin receptor subfamily, and weaker homology to the cannabinoid receptor [1]. The other four closely related members of this family act as receptors for the a- and c-melanocyte stimulating hormone (a, c-MSH) as summarised in table 1. The ACTH-R is primarily expressed on cells of the adrenal cortex, although expression is also found in adipocytes [2–4]. The ACTH receptor is highly selective for ACTH peptides containing the first 18 residues of the peptide. ACTH1–24 appears to be as biologically active as ACTH1–39. The receptor does not respond to peptides truncated to less than ACTH1–17 at the C-terminus, including all the MSHs [5, 6]. The role of ACTH in stimulating cAMP accumulation was one of the earliest such hormone signalling events described [7]. This action, mediated by activation of aS-containing heterotrimeric G proteins, remains an undisputed mode of signalling by this receptor. However, there is also good evidence for activation of calcium influx at low ACTH concentrations by this receptor

Table 1. Biological and pharmacological characteristics of the melanocortin receptors Receptor

Ligand preference

Distribution

Clinical/biological relevance

MC1-R

a-MSH?ACTH?c-MSH

melanocytes

functionally variant receptors in human red hair/pale skin and in multiple animal coat colour variants

MC2-R

ACTH only

adrenal cortex, adipocytes

homozygous defects cause familial glucocorticoid deficiency

MC3-R

c-MSH?a-MSH/ACTH

brain, placenta, macrophages

evidence for role in macrophage margination, possible role in appetite and/or sex drive

MC4-R

a-MSH?ACTH?c-MSH

brain

apetite regulation, deletion in humans or mice associated with obesity

MC5-R

a-MSH?ACTH?c-MSH

widely distributed

deletion in mice associated with defective exocrine gland function

[8], and recent models propose that ACTH acts via adenylate cyclase to stimulate cAMP accumulation which in turn directly inhibits a membrane potassium channel, leading to membrane depolarization and calcium entry, probably through T-type calcium channels [9]. These pathways are summarised in figure 1. Recent work also implicates an additional very rapid role of ACTH in inhibiting guanyl cyclase which in turn inhibits phosphodiesterase type 2 in adrenal glomerulosa cells. This leads to a transient increase in cAMP. As cAMP accumulates, however, this will stimulate this phosphodiesterase which then counteracts the cAMP-generating action of adenylate cyclase [10]. The exact actions resulting from these immediate intracellular signalling events are not well understood. Activation of PKA will result in phosphorylation of a number of target proteins, including the cAMP response element binding protein (CREB). These phosphorylation events are probably responsible, either directly or indirectly, for alteration in the level of expression of several genes including the steroidogenic enzymes CYP11A, 17, 21, 11B1 and the StAR protein [11]. Others have demonstrated stimulation of expression of several of the immediate early response genes [12, 13], as well as less rapid induction of ACTH-R, MC5-R [14, 15], HDL and LDL receptor genes [16]. Thus, the process of transmitting a signal from the ACTH molecule in the circulation to the steroidogenic enzymes is a multi-component process in which each component has an essential role. Defects could theoretically occur at each of these steps and cause a syndrome of ACTH resistance. However, many of these steps are common to several other signalling activities, and

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25

Fig. 1. Mechanism of action of ACTH. Schematic representation of the established pathways of ACTH signalling through adenylate cyclase to activate protein kinase A, and the less established pathways by which cAMP inhibits a potassium channel leading to membrane depolarization and consequent calcium influx through a T-type calcium channel.

thus inherited defects in these might be expected to result in a considerably less tissue restricted syndrome. A good example of this point is that defects in the GaS subunit should, in a homozygous form, cause ACTH resistance. However, heterozygous gene defects cause pseudohypoparathyroidism, characterised primarily by PTH resistance. It is likely that homozygous defects of this gene are incompatible with life.

ACTH Resistance ACTH resistance is a rare disorder defined as a failure of the adrenal cortex to respond to ACTH in the presence of an otherwise normal gland. The usual evidence for the preservation of the gland is the ability to secrete mineralocorticoids under the control of angiotensin II. ACTH-resistant syndromes are inherited as recessive disorders known by various terms including familial, isolated, congenital or inherited glucocorticoid deficiency or hereditary unresponsiveness to ACTH [17–19]. In the remainder of this chapter we will use the term familial glucocorticoid deficiency (FGD). As described below, some cases of ACTH resistance occur as part of a group of syndromes known

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as the triple A syndrome, and in these cases the other features may be the dominant presenting feature [20, 21]. These syndromes have recently been reviewed in depth [19].

Clinical Presentation The initial diagnosis of ACTH resistance may occur in infancy or later. In general, cases presenting in infancy will often have had a history of neonatal hypoglycaemia followed after several months by the observation that the child is excessively pigmented. Occasionally, pigmentation is commented on shortly after birth. Neonatal jaundice may also be an early feature indicative in these cases of glucocorticoid deficiency. Often, an excessively severe response to comparatively minor infective illnesses will result in the investigations that establish the diagnosis. In other cases children may present after several years of age with a severe infective illness, hypoglycaemia or pigmentation. Occasionally, children may become severely ill with overwhelming infection, sometimes accompanied by hypoglycaemia. In such cases it may be impossible to reverse these effects of glucocorticoid deficiency with inevitably fatal consequences. Any of these features should prompt measurement of the plasma cortisol, which will often be subnormal, but not necessarily undetectable. Plasma ACTH will be very high, often exceeding 1,000 pg/ml. Evidence for the relative normality of the renin, angiotensin, aldosterone axis will be found by demonstrating normal electrolytes, renin and aldosterone. Some disturbance of these parameters is occasionally seen [22]. The diagnosis can usually be confirmed using a short Synacthen test which will show an inadequate early cortisol response to exogenous ACTH. These clinical and diagnostic features are summarised in table 2.

Aetiology As discussed above, the ACTH signal has several components, many of which are common to other systems, and thus unlikely to cause isolated ACTH insensitivity. Defects in the biological activity of ACTH, the receptor for ACTH, and possibly in some of the adrenal specific targets of PKA phosphorylation can theoretically provide an explanation for the syndrome. Other explanations include defects in the development of the adrenal cortex or degeneration of the adrenal cortex. In 1993, we reported the first case of a homozygous point mutation in the ACTH receptor gene in a patient with FGD [23]. This was shown to segregate with the disease in the index family,

Defects of ACTH Action

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Table 2. Clinical and diagnostic features of ACTH resistance Presenting clinical features Hypoglycaemia – often neonatal, but may occur in later childhood Neonatal jaundice Increased skin pigmentation Increased susceptibility to infection Frequent and recurrent infection Increased susceptibility to atopy Supporting clinical features Sibling with identical diagnosis Tall stature Diagnostic biochemical features 9 a.m. plasma cortisol – undetectable, subnormal or low normal Plasma ACTH – grossly elevated Renin normal Aldosterone normal Electrolytes normal Short Synacthen test impaired response Supporting biochemical features 17a-Hydroxyprogesterone Very-long-chain fatty acids

normal normal

to be absent in the normal population and to be present in other cases of FGD. We and others subsequently reported a variety of other mutations in this gene in patients with FGD, and these are summarised in figure 2 [24–30]. Some of these mutations introduce premature stop codons into the receptor sequence resulting in a markedly disrupted receptor structure. It is highly likely that these molecules would not function as a receptor and hence result in the disease. In the case of missense mutations this needs to be demonstrated using some sort of functional assay for the mutant receptor. Traditional approaches in which the gene is transfected into a heterologous cell line and its functional properties tested have not been successful in the case of the ACTH receptor. Several groups have used a range of transfection techniques in a variety of cell lines and failed to obtain effective expression in the absence of background. However, Naville et al. [27] have used the Cloudman M3 cell line with some success, although results are partly complicated by the presence of an endogenous melanocortin receptor in these cells. Our own approach has been to use an adrenocortical-derived cell line, the Y6 cell line, that lacks any endogenous ACTH receptor [31]. This approach reveals a range of effects of different mutations including loss of high-affinity ligand binding, loss of max-

Swords/Franklin/Huebner/Elias/Clark

28

Fig. 2. Mutations detected in the ACTH receptor in patients with FGD. A two-dimensional plot of the putative ACTH receptor structure is shown with the various mutations associated with ACTH insensitivity shown. Note P27R is a polymorphism.

imal activation of the adenylate cyclase pathway, and a shift of the doseresponse curve to the right (fig. 3).

FGD Disease Heterogeneity Only about half of all cases of FGD have mutations in the ACTH receptor coding region, and we have not yet identified promoter region mutations in any case without a coding region defect. Furthermore, we have shown that in some of these cases the disease does not segregate in families with the ACTH receptor locus [32, 33]. These cases therefore result from defects in other genes. No obvious alternative candidates stand out at present, and it seems likely that a genetic linkage approach will be the most appropriate route to identification of this gene. A particularly interesting observation is that it appears that patients with ACTH receptor defects are significantly taller as a group than the normal population, whereas those cases of FGD without ACTH receptor mutations are of appropriate height for the population [19]. Furthermore, there is some evidence suggesting that some of these patients have a characteristic facial phenotype and a large head, and some discordance of bone maturation, all

Defects of ACTH Action

29

Fig. 3. Dose-response curves of wild-type and mutant ACTH receptors. Receptors were expressed by stable transfection in Y6 cells, and cAMP dose responses to ACTH[1–24] were determined. See Elias et al. [31], for details.

features that are reminiscent of Sotos syndrome [34, 35]. In some cases it appears that the introduction of glucocorticoid replacement therapy allows the growth rate to revert to normal [30]. It seems probable that this growth difference which is not readily explained by our current endocrine understanding may provide a clue as to the aetiology of normal receptor FGD.

Treatment Patients presenting with FGD may be extremely ill as a result of infection and/or hypoglycaemia complicated by convulsions. Each of these features requires vigorous management, and there is still a significant mortality and longterm morbidity associated with these complications of glucocorticoid deficiency at the time of presentation. In addition, the underlying hormonal defect must be corrected, which can usually be achieved with replacement doses of hydrocortisone. Mineralocorticoid replacement is not required, and failure to maintain a normal electrolyte balance despite glucocorticoid replacement should call the diagnosis into question. The usual advice given to any patient taking glucocorticoid replacement therapy applies to patients with FGD, notably in relation to increasing steroid dosage during illness, trauma or surgery. Parents who have given birth to one child affected with FGD can be advised that they have a 25% chance of having another affected child, which should be noted at the time of any subsequent delivery. The possibility that existing children also have this syndrome should be considered. Affected children should be normally fertile and will have only a minimal risk of bearing affected offspring as a result of non-consanguineous parentage.

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Table 3. Clinical features of triple A syndrome Alacrima (absence of tears) Achalasia of the cardia – may extend into gastric atonia and more extensive gut hypotonia: present in D75% cases ACTH resistance – usually develops after birth and includes mineralocorticoid deficiency in 10% Neurological defects including Autonomic neuropathies – present in ~30% patients Sensorimotor peripheral neuropathies Clumsiness, ataxia, Parkinsonism, mental retardation, optic atrophy Hyperkeratosis of palms and soles – present in D20% patients

Triple A Syndrome Clinical Features The features of this disorder are summarised in table 3 and have been reviewed in Clark and Weber [19]. Usually, the ACTH-resistant component of this disease is not the first to become apparent, but may be the most prominent. Alacrima (absence of tears) is often the first symptom, probably being present from birth, but may not be noted. The degree and time of onset of the achalasia and the neurological disturbance is highly variable. In some cases the adrenal component of this disease may become evident in the second or third decade or even later [36]. Other than the possibly late onset, the clinical features of the ACTH resistance are similar to those of FGD. However, in 10% of cases significant mineralocorticoid deficiency may develop as well [21]. This variable presentation of symptoms in this syndrome may make diagnosis very difficult. Apparently distinct entities such as the achalasia-alacrima syndrome have been described, but are probably always manifestations of the triple A syndrome [37]. Marked phenotypic variation can exist between affected siblings, e.g. [36], indicating modification of the adverse effect of the primary genetic disorder. Such modification may be the result of unlinked modifier genes or evidence of environmental modifiers. If the latter is the case, the prospect of therapeutic manipulation of the symptoms to beneficial effect is raised. Aetiology Several groups have established that this syndrome is not associated with ACTH receptor mutations, nor is it genetically linked to this locus [19, 26]. No other obvious candidate genes are apparent, and no suitable animal models of this disease exist. We therefore undertook a genome search approach to identify the locus for the triple A syndrome gene. Initially using eight large and well

Defects of ACTH Action

31

Fig. 4. Location of the triple A gene on chromosome 12. Linkage analysis indicates that the triple A gene is located on the long arm of chromosome 12 between the markers D12S368 and D12S1586, a region that contains the keratin type II gene cluster, but no other strong candidate genes for this disorder.

characterised unrelated pedigrees with several affected members, we undertook linkage studies with 345 polymorphic markers scattered evenly throughout the human genome. Significant linkage was found to a region on the long arm of chromosome 12 (12q13), close to the keratin type II gene cluster [38] (fig. 4). This syndrome appears to be genetically homogeneous, and there is evidence that patients with a partial phenotype previously characterised as the achalasiaalacrima syndrome also result from defects at this locus [39]. Unfortunately, the disease locus lacks any very obvious candidate genes at this time, although new

Swords/Franklin/Huebner/Elias/Clark

32

genes are being identified and mapped continuously. Furthermore, it is very difficult to predict what type of defective gene product is likely to result in this syndrome in view of its rather bizarre collection of features.

ACTH Constitutive Activity and Hypersensitivity Since prolonged and excessive stimulation of the adrenal by ACTH (e.g. in Cushing’s disease) often leads to marked bilateral adrenal hyperplasia, it would appear theoretically possible that a constitutively active ACTH receptor could mimic this effect. Germ line activating mutations might result in bilateral adrenal hyperplasia, whereas a new mutation in an adrenocortical cell might result in adrenal adenoma formation. There is evidence from several other G-protein-coupled receptors (e.g. gonadotropin, thyrotropin, parathyroid hormone, calcium-sensing receptors) that such germ line or spontaneous mutations can be pathogenic. Moreover, there is evidence that the related MC1 (a-MSH) receptor can be activated by missense mutations giving rise to certain mouse coat colour mutants [40]. However, examination of a number of different adrenal tumours and hyperplastic lesions by two groups failed to identify such activating mutations in the case of the ACTH-R [41, 42]. This may simply be a case of insufficient numbers, or it may be that the phenotype that results from such mutations is not as significant as that predicted. Some evidence that this is the case comes from our recent functional characterisation of a series of missense mutations found in the germ line in patients with FGD. Three of these clinically inactivating mutations exhibited enhanced basal activity when expressed in the Y6 cell system [31]. One of these mutations was exactly analogous to one identified in an activating mutation of the MC1-R that resulted in an abnormal mouse pigmentation phenotype [40]. Evidence that an alternative mechanism of receptor activation might exist comes from a Japanese patient who appeared from clinical characterisation to have increased sensitivity of the ACTH-R. A heterozygous mutation was found in the germ line DNA by DNA sequencing, and although this has not been tested in any expression system to date, it was postulated that this receptor might be excessively sensitive to ACTH [43, 44].

Conclusion Resistance to ACTH action results from at least three distinct autosomal recessive genetic defects. ACTH receptor mutations have been identified by

Defects of ACTH Action

33

several groups as one of the defects causing FGD, and functional analysis of the defects in this receptor is consistent with ACTH resistance. Other cases of FGD are the result of defects in another autosomal gene of unknown function and genomic location. The triple A syndrome also results from defects in an unidentified gene, although the genetic locus has been mapped to the long arm of chromosome 12. It is to be hoped that genetic analysis of affected families and candidate gene studies will eventually allow identification of the triple A syndrome gene. For this reason, it is highly desirable that new cases can be subjected to genetic analysis, since, as is often the case in identifying disease genes, a single patient bearing a specific small deletion may ultimately be the catalyst that is needed to identify the genetic cause.

References 1

2 3

4 5

6

7 8 9

10

11

12 13

Cone RD, Lu D, Koppula S, Vage DI, Klungland H, Boston B, Chen W, Orth DN, Pouton C, Kesterson RA: The melanocortin receptors: Agonists, antagonists, and the hormonal control of pigmentation. Recent Prog Horm Res. 1996;51:287–317. Mountjoy KG, Robbins LS, Mortrud MT, Cone RD: The cloning of a family of genes that encode melanocortin receptors. Science 1992;257:1248–1251. Cammas FM, Pullinger GD, Barker S, Clark AJL: The mouse ACTH receptor gene: Cloning and characterization of its promoter and evidence for a role for the orphan nuclear receptor SF-1. Mol Endocrinol 1997;11:867–876. Boston BA, Cone RD: Characterization of melanocortin receptor subtype expression in murine adipose tissues and in the 3T3-L1 cell line. Endocrinology 1996;137:2043–2050. Ramachandran J, Chung D, Li CH: Adrenocorticotropins XXXIV. Aspects of structure activity relationships of the ACTH molecule. Synthesis of a heptadecapeptide amide, an octadecapeptide amide and a nonadecapeptide amide possessing high biological activities. J Am Chem Soc 1965; 87:2696–2708. Kapas S, Cammas FM, Hinson JP, Clark AJL: Agonist and receptor binding properties of adrenocorticotropin peptides using the cloned mouse ACTH receptor expressed in a stably transfected HeLa cell line. Endocrinology 1966;137:3291–3294. Haynes RC: The activation of adrenal phosphorylase by the adrenocorticotropic hormone. J Biol Chem 1958;233:1220–1222. Kojima I, Kojima K, Rasmussen H: Role of calcium and cAMP in the action of adrenocorticotrophin on aldosterone secretion. J Biol Chem 1985;260:4248–4256. Enyeart JJ, Mlinar B, Enyeart JA: T-type Ca2+ channels are required for adrenocorticotropinstimulated cortisol production by bovine adrenal zona fasciculata cells. Mol. Endocrinol 1993;7: 1031–1040. Cote M, Payet MD, Rousseau E, Guillon G, Gallo-Payet N: Comparative involvement of cyclic nucleotide phosphodiesterases and adenylyl cyclase on adrenocorticotropin-induced increase of cyclic AMP in rat and human glomerulosa cells. Endocrinology 1999;140:3594–3601. Lehoux JG, Fleury A, Ducharme L: The acute and chronic effects of adrenocorticotropin on the levels of messenger ribonucleic acid and protein of steroidogenic enzymes in rat adrenal in vivo. Endocrinology 1998;139:3913–3922. Clark AJL, Balla T, Jones MR, Catt KJ: Stimulation of early gene expression by angiotensin II in adrenal glomerulosa cells: Roles of calcium and protein kinase C. Mol Endocrinol 1992;6:1889–1898. Kimura E, Sonobe MH, Armelin MCS, Armelin HA: Induction of FOS and JUN proteins by adrenocorticotropin and phorbol ester but not by 3€5€ cyclic AMP derivatives. Mol Endocrinol 1993;7:1463–1471.

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Mountjoy KG, Bird IM, Rainey WE, Cone RD: ACTH induces up-regulation of ACTH receptor mRNA in mouse and human adrenocortical cell lines. Mol Cell Endocrinol 1994;99:17–20. Liakos P, Chambaz EM, Feige JJ, Defaye G: Expression of ACTH receptors (MC2-R and MC5-R) in the glomerulosa and fasciculata reticularis zones of bovine adrenal cortex. Endocr Res 1998;24: 427–432. Heikkila P, Arola J, Liu J, Kahri AI: ACTH regulates LDL receptor and CLA-1 mRNA in the rat adrenal cortex. Endocr Res 1998;24:591–593. Shepard TH, Landing BH, Mason DG: Familial Addison’s disease. Am J Dis Child 1959;97: 154–162. Migeon CJ, Kenny FM, Kowarski A, Snipes CA, Spaulding JS, Finkelstein JW, Blizzard RM: The syndrome of congenital adrenocortical unresponsiveness to ACTH: Report of six cases. Pediatr Res 1968;2:501–513. Clark AJL, Weber A: Adrenocorticotropin insensitivity syndromes. Endocr Rev 1998;19:828–843. Allgrove J, Clayden GS, Grant DB, Macaulay JC: Familial glucocorticoid deficiency with achalasia of the cardia and deficient tear production. Lancet 1978;i:1284–1286. Grant DB, Barnes ND, Dumic M, Ginalska-Malinowska M, Milla PJ, von Petrykowski W, Rowlatt RJ, Steendijk R, Wales JHK, Werder E: Neurological and adrenal dysfunction in the adrenal insufficiency/alacrima/achalasia (3A) syndrome. Arch Dis Child 1993;68:779–782. Davidai G, Kahana L, Hochberg Z: Glomerulosa failure in congenital adrenocortical unresponsiveness to ACTH. Clin Endocrinol 1984;20:515–520. Clark AJL, McLoughlin L, Grossman A: Familial glucocorticoid deficiency caused by a point mutation in the ACTH receptor. Lancet 1993;341:461–462. Tsigos C, Arai K, Hung W, Chrousos GP: Hereditary isolated glucocorticoid deficiency is associated with abnormalities of the adrenocorticotropin receptor gene. J Clin Invest 1993;92:2458–2461. Weber A, Toppari J, Harvey RD, Klann RC, Shaw NJ, Ricker AT, Nanto-Salonen, Bevan JS, Clark AJL: Adrenocorticotropin receptor gene mutations in familial glucocorticoid deficiency: Relationships with clinical features in four families. J Clin Endocr Metab 1995;80:65–71. Tsigos C, Arai K, Latronico AC, DiGeorge AM, Rapaport R, Chrousos GP: A novel mutation of the adrenocorticotropin receptor (ACTH-R) gene in a family with the syndrome of isolated glucocorticoid deficiency, but no ACTH-R abnormalities in two families with the triple A syndrome. J Clin Endocr Metab 1995;80:2186–2189. Naville D, Barjhoux L, Jaillard C, Faury D, Despert F, Esteva B, Durand P, Saez JM, Begeot M: Demonstration by transfection studies that mutations in the adrenocorticotropin receptor gene are one cause of the hereditary syndrome of glucocorticoid deficiency. J Clin Endocr Metab 1996;81: 1442–1448. Wu SM, Stratakis CA, Chan CHY, Hallermeier KM, Bourdony CJ, Rennert OM, Chan WY: Genetic heterogeneity of adrenocorticotropin (ACTH) resistance syndromes: Identification of a novel mutation of the ACTH receptor gene in hereditary glucocorticoid deficiency. Mol Genet Metab 1998;64:256–65. Slavotinek AM, Hurst JA, Dunger D, Wilkie AO: ACTH receptor mutation in a girl with familial glucocorticoid deficiency. Clin Genet 1998;53:57–62. Elias LLK, Huebner A, Metherell L, Canas A, Warne GL, Manca Bitti ML, Cianfarani S, Clayton PE, Savage MO, Clark AJL: Tall stature in familial glucocorticoid deficiency. Clin Endocrinol, in press. Elias LLK, Weber A, Pullinger GD, Mirtella A, Clark AJL: Functional characterization of naturally occurring mutations of the human adrenocorticotropin receptor: Poor correlation of phenotype and genotype. J Clin Endocrinol Metab 1999;84:2766–2770. Weber A, Clark AJL: Mutations of the ACTH receptor gene are only one cause of familial glucocorticoid deficiency. Hum Mol Genet 1994;3:585–588. Naville D, Weber A, Genin E, Durand P, Clark AJL, Begeot M: Exclusion of the ACTH receptor (MC2R) locus in some families with ACTH resistance, but no mutations of the MC2R coding sequence (FGD type 2). J Clin Endocrinol Metab 1998;83:3592–3596. Sotos JF, Dodge PR, Muirhead D, Crawford JD, Talbot NB: Cerebral gigantism in childhood. N Engl J Med 1964;271:109–116.

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Cole TRP, Hughes HE: Sotos syndrome. J Med Genet 1990;27:571–576. Moore PSJ, Couch RM, Perry YS, Shuckett EP, Winter JSD: Allgrove syndrome: An autosomal recessive syndrome of ACTH insensitivity, achalasia and alacrima. Clin Endocrinol 1991;34:107–114. Haverkamp F, Zerres K, Rosskamp R: Three sibs with achalasia and alacrimia: A separate entity different from triple-A syndrome. Am J Med Genet 1989;34:289–291. Weber A, Wienker TF, Jung M, Easton D, Dean HJ, Heinrichs C, Reis A, Clark AJL: Linkage of the gene for the triple A syndrome to chromosome 12q13 near the type II keratin gene cluster. Hum Mol Genet 1996;5:2061–2066. Weber A, De Vroede M, Wienker TF, Jansen M: Clinical variability and molecular genetics in a family with triple A syndrome. Horm Res 1997;48(S2):191. Robbins LS, Nadeau JH, Johnson KR, Kelly MA, Roselli-Rehfuss L, Baack E, Mountjoy KG, Cone RD: Pigmentation phenotypes of variant extension locus alleles result from point mutations that alter MSH receptor function. Cell 1993;72:827–834. Light K, Jenkins PJ, Weber A, Perrett C, Grossman A, Boscaro M, Asa SL, Clayton RN, Clark AJL: Are activating mutations of the ACTH receptor involved in adrenal cortical neoplasia? Life Sci 1995;56:1523–1527. Latronico AC, Reincke M, Mendonca BB, Arai K, Mora P, Allolio B, Wajchenberg BL, Chrousos GP, Tsigos C: No evidence for oncogenic mutations in the adrenocorticotropin receptor gene in human adrenocortical neoplasms. J Clin Endocrinol Metab 1995;80:875–877. Hiroi N, Yakushiji F, Shimojo M, Watanabe S, Sugano S, Yamaguchi N, Miyachi Y: Human ACTH hypersensitivity syndrome associated with abnormalities of the ACTH receptor gene. Clin Endocrinol 1998;48:129–134. Clark AJL: Receptor hypersensitivity: A new phenomenon? Clin Endocrinol 1998;48:135–136.

A.J.L. Clark, DSc, FRCP, Molecular Endocrinology Laboratory, Departments of Endocrinology, St Bartholomew’s and the Royal London School of Medicine and Dentistry, West Smithfield, London EC1A 7BE (UK) Tel. +44 171 601 7445, Fax +44 171 601 8468, E-Mail [email protected]

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Proteins Involved in Mitochondrial Cholesterol Transport Douglas M. Stocco a, Adam J. Reinhart a, Walter L. Miller b a

b

Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, Tex., and Department of Pediatrics and The Metabolic Research Unit, University of California, San Francisco, Calif., USA

The intracellular movement of cholesterol is carefully regulated in most cells, although many of the mechanisms involved in this regulation are not well understood. The regulated movement of cholesterol occurs amongst the specialized organelles in the cell such as the plasma membrane, Golgi, lysomes, peroxisomes and endoplasmic reticulum. Most of the intracellular cholesterol can be found in the plasma membrane where it serves to affect membrane fluidity but it is not static and a constant cycling of this sterol occurs. A great deal of work has been performed in an effort to elucidate the nature of intracellular cholesterol transport and movement and cannot adequately be reviewed here. Rather, this review will attempt to focus on another highly specialized type of cholesterol transport which occurs in a highly specialized group of cells in the body. These are the steroidogenic cells of the body and in these cells the importance of cholesterol is readily seen since it serves as the substrate for the synthesis of all steroid hormones. Since steroid hormones make up a very important class of regulatory molecules which are synthesized mainly in the adrenal, testis, ovary and placenta (with smaller amounts being synthesized in the brain), details concerning the movement and delivery of their substrate within steroidogenic cells becomes of major importance. Without the delivery of cholesterol to the appropriate intracellular location, no steroid synthesis is possible. This is critical when one considers that the adrenal gland produces glucocorticoids which serve to regulate carbohydrate metabolism and stress and also produces mineralocorticoids which regulate salt balance and maintain blood pressure. The sex steroids estrogen and progesterone are

synthesized in the ovary and placenta and function to maintain secondary sex characteristics and are also essential for reproductive function in the female. Another sex steroid, testosterone, is synthesized by the testicular Leydig cells and is responsible for maintaining reproductive function and secondary sex characteristics in the male. The biosynthesis of steroids begins with the cleavage of a six carbon unit from the 27 carbon cholesterol molecule to form the first steroid synthesized, the 21 carbon pregnenolone. This reaction is catalyzed by the cytochrome P450 side chain cleavage enzyme (P450scc), which is part of the cholesterol side chain cleavage enzyme system (CSCC) which is located on the matrix side of the inner mitochondrial membrane [1–4]. Thus, at least in steroidogenic cells, the mitochondrion represents an important delivery site for intracellular cholesterol. For many years the trophic hormone induced increase in activity of the P450scc enzyme in converting cholesterol to pregnenolone was considered to be the rate-limiting step in steroidogenesis. However, it soon became clear that the activity of the P450scc enzyme was not rate-limiting in this process [5]. Rather, it was discovered that in order to initiate and sustain steroidogenesis a constant supply of the substrate for steroid biosynthesis, cholesterol, must be available within the cell and second, a mechanism must exist for the delivery of this cholesterol to the site of cleavage in the inner mitochondrial membrane where the P450scc enzyme resides. Given adequate intracellular cholesterol supplies, two separate but equally important processes must occur. First, mobilization of cholesterol from cellular stores such as lipid droplets or other cellular membranes to the outer mitochondrial membrane and second, the transfer of this cholesterol from the outer to the inner mitochondrial membrane [6–8]. The factors and processes responsible for the mobilization of cholesterol to the outer mitochondrial membrane in steroidogenic cells are thought to involve changes in cellular architecture and putative transport proteins but their mechanisms of action are not well understood, and at any rate will not be discussed in any detail here. As stated above, it became clear that the true rate-limiting step effected by hormone stimulation was the delivery of cholesterol from the outer to the inner mitochondrial membrane and to the P450scc [9, 10–13]. Addition of hydroxylated analogs of cholesterol, which can readily diffuse across the mitochondrial membranes to the P450scc, to steroidogenic cells resulted in high levels of steroids being formed in the absence of hormone stimulation [14–17]. This indicated that the P450scc was fully active in unstimulated cells and that the lack of substrate cholesterol for cleavage prevented the production of pregnenolone and other downstream steroids. The major barrier in the translocation of cholesterol to the P450scc was the aqueous space between the outer and inner mitochondrial membranes through which the hydrophobic choles-

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terol does not quickly pass [18–20], and therefore, cannot provide sufficient substrate for the rapid and large increase in steroid production observed. Therefore, it was clear that stimulation of steroidogenesis required a mechanism which rapidly resulted in the transport of this steroid precursor across this barrier. This review will focus on what is currently understood about the mechanism involved in the movement of cholesterol from the outer mitochondrial membrane to the inner mitochondrial membrane.

What Is Required for Intramitochondrial Cholesterol Transport? One of the first and most fundamental observations concerning steroidogenesis was that acute steroid production in response to hormone stimulation had an absolute requirement for the synthesis of new proteins. The first of such studies were performed by Ferguson [21, 22] who demonstrated that the acute stimulation of corticoid synthesis in adrenal glands by ACTH was sensitive to the protein synthesis inhibitor puromycin. Notably, this observation was made prior to the understanding that the rate-limiting step in steroidogenesis was the delivery of cholesterol to the P450scc enzyme. Garren and coworkers also conducted studies which clearly demonstrated that steroidogenesis in adrenal tissue was highly dependent upon the synthesis of new proteins in response to ACTH treatment [23–26]. Following these observations many similar studies were confirmatory of the need for de novo protein synthesis in the hormone regulated, acute production of steroids [9, 27–37]. Simpson and Boyd [38] determined that the cycloheximide sensitive step in this process was located in the mitochondria, but, importantly, Arthur and Boyd [39] noted that protein synthesis inhibitors had no effect on the activity of the P450scc itself. These observations were quickly followed by studies which demonstrated that inhibition of protein synthesis had no effect on the increased delivery of cellular cholesterol to the outer mitochondrial membrane, but that the delivery of this substrate from the outer membrane to the inner mitochondrial membrane was completely inhibited by cycloheximide [35, 40]. As a result, the precise site of the cycloheximide-inhibited regulation had been pinpointed, namely, the transfer of cholesterol to the P450scc enzyme in the inner mitochondrial membrane. The observation that de novo protein synthesis was indispensable for the acute production of steroids in response to hormone stimulation has also been made more recently in several different steroidogenic tissues [41–46]. The effort to identify and characterize this acute regulatory protein(s) has been ongoing since the early observations of Ferguson and Garren and their colleagues. Several candidates have emerged from these efforts. This review

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will attempt to summarize the studies which have been performed on these candidates.

Sterol Carrier Protein 2 One of the proteins suggested to be involved in the acutely regulated intracellular transfer of cholesterol is the sterol carrier protein 2 (SCP2). SCP2 is a 13-kD protein that has been found in high abundance in liver as well as in various steroidogenic tissues. Also known as nonspecific lipid transfer protein, the role of this protein in liver and adrenal cells has previously been reviewed by Vahouny et al. [47, 48]. SCP2 has been demonstrated to transfer cholesterol from lipid droplets to mitochondria in a 1:1 ratio and is capable of stimulating steroid production in isolated adrenal mitochondria [49–52]. Also, SCP2 can mediate the transfer of the phospholipids phosphatidylcholine and phosphatidylethanolamine from vesicles to mitochondria which were isolated from rat liver [53]. SCP2 can transport newly synthesized cholesterol from the endoplasmic reticulum to the plasma membrane in human fibroblasts, indicating that SCP2 may be a general cholesterol transporter within the cell [54]. SCP2 is also able to alter the distribution of cholesterol between kinetic domains in small unilamellar vesicles [55]. It can also bind sterols with an affinity which was consistent with the characteristics of a lipid transfer protein which could act as an aqueous carrier or perhaps at a membrane surface to enhance sterol desorption [56]. Thus, SCP2 is able to bind and transfer sterols between compartments within the cell, a task which would seem to be compatible with steroid regulation in the cell. To support a role in the transfer of cholesterol to the mitochondria in steroidogenesis, it was shown that coexpression of SCP2 with cholesterol side chain cleavage enzyme and adrenodoxin in COS 7 cells resulted in a 2.5-fold increase in steroid production over that seen with expression of the steroidogenic enzyme system alone [57]. Furthermore, treatment of adrenal cells with anti-SCP2 antibody resulted in an inhibition of steroid production [58]. The gene encoding SCP2 gives rise to two transcripts; one encodes a 58-kD protein, SCPx and the other a 15.3-kD protein, SCP2, which is processed to yield a mature 13-kD polypeptide. Both SCPx and SCP2 have carboxy terminal peroxisome targeting sequences and both have been localized to peroxisomes, the sites of cholesterol biosynthesis [59–63]. The synthesis of SCP2 is under the regulation of ACTH in the adrenal; however, this regulation only occurred after chronic hormone stimulation [64]. Nevertheless, while stimulation of adrenal cells with ACTH had no acute effect on SCP2 levels in rat testicular Leydig cells, acute stimulation with LH appeared to result in a rapid redistribu-

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tion of SCP2 within the cell from the cytosol to the mitochondria [65, 66]. This intracellular redistribution of SCP2 has also been observed in rat corpus luteum in response to estrogen stimulation [67]. While regulation of SCP2 by cyclic AMP analogs in the ovary was demonstrated, it was also shown to be regulated in a similar manner in a nonsteroidogenic granulosa cell line, thereby indicating that its regulation is not obligatorily coupled to steroidogenesis [68]. In summary, the data indicate that SCP2 is associated with the intracellular trafficking of cholesterol. One possible exception may be in the intracellular trafficking of lysosomal cholesterol which was found to be unaffected in Zellweger syndrome fibroblasts which totally lack SCP2 [69]. The evidence suggesting that SCP2 is able to effectively transfer cholesterol to the inner mitochondrial membrane and the P450scc in response to hormone stimulation is not convincing. Furthermore, the observations that SCP2 levels do not rapidly change in response to either acute stimulation or to treatment with cycloheximide have excluded this protein as the putative acute regulatory protein as defined by Ferguson and Garren. However, SCP2 may function to maintain sterol movement within the cell to the mitochondria in support of steroidogenesis, perhaps by affecting the utilization of peroxisome-derived cholesterol [70].

Steroidogenesis Activator Polypeptide The steroidogenesis activator polypeptide (SAP) was originally found and described by Pederson and Brownie [71]. SAP was first purified as a 2.2-kD peptide from rat adrenal cells [71, 72], but was later determined to be a 30 amino acid (3.2 kDa) peptide when it was purified from rat Leydig tumor cells [73]. This peptide was found to be present only in steroidogenic cells, its level was acutely increased by trophic hormone stimulation and this increase was prevented by cycloheximide [74–76], characteristics consistent with those of the putative regulatory protein. Addition of SAP to isolated mitochondria was able to increase steroid production by 4- to 5-fold in a dose-dependent manner indicating that SAP may play a role in cholesterol transfer within this organelle [71, 72]. Further characterization of SAP demonstrated that it was nearly identical to the carboxy terminal of a minor heat-shock protein known as glucose-related protein 78 (GRP 78) [77, 78]. Given the reported characteristics of this small peptide, it must be considered as a potential component of the intramitochondrial cholesterol transport system. However, at this writing, there have been no recent studies reported on the further characterization of this polypeptide and thus its role in steroidogenesis remains unknown.

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Peripheral Benzodiazepine Receptor Benzodiazepines are a family of compounds which have been widely used pharmacologically as anticonvulsant and antianxiety agents. The primary action of these compounds is exerted through binding to the benzodiazepine receptor, which is expressed in highest concentrations in the central nervous system (CNS). Diazepam, a benzodiazepine, was found to bind to receptors expressed in the CNS with nanomolar affinity. Later it was found that benzodiazepine compounds exert their pharmachological effect through binding to and modulating the function of the GABA receptor in the CNS [79]. In more extensive surveys of diazepam binding it was found that there was a separate class of benzodiazepine receptor which was expressed in peripheral tissues and was named the peripheral benzodiazepine receptor (PBR) [79–81]. PBR has been shown to be expressed in many tissues, and is expressed in high levels in steroidogenic tissues such as the testis and the adrenal cortex [80, 81]. Subcellular localization studies have shown that PBR is expressed primarily on the outer mitochondrial membrane in steroidogenic tissues [82]. Several investigators have suggested a link between PBR, cholesterol transport and steroidogenesis. Marc and Marselli [83] reported that diazepam affects plasma corticosterone levels in the rat, and subsequent studies have reported effects on serum testosterone levels [84, 85]. More recent studies have shown that PBR may influence steroid production by its involvement in cholesterol transport from the outer to the inner mitochondrial membrane [86, 87]. Although it is clear that PBR is involved in cholesterol transport, to date despite intensive research efforts, the exact role that PBR may play in the transport of cholesterol from cytosolic stores to the inner mitochondrial membrane remains an enigma. The mitochondrial PBR is an 18-kD protein which forms a complex with the 34-kD voltage-dependent anion carrier (VDAC) as well as the inner mitochondrial adenine nucleotide carrier protein (ADC) [82, 88–93]. A functional benzodiazepine binding site requires a complex consisting of both the 18-kD PBR and the VDAC molecules. Detailed studies have suggested that the PBR molecules in the mitochondrial membrane form clusters of four to six molecules. Following a 15-second hCG treatment a dramatic rearrangement of the PBR molecules in the mitochondrial membrane occurs, resulting in the formation of clusters consisting of as many as 25 PBR molecules [94]. These data lead to the hypothesis that hormone stimulation induced the formation of large clusters of PBR molecules at contact sites between the inner and outer mitochondrial membrane. This large complex may possibly function as a pore, allowing the transfer of cholesterol to the inner membrane at this contact point.

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The PBR has been found to play a part in the transfer of cholesterol from the outer to the inner mitochondrial membrane. The first evidence supporting this conclusion came in 1990 when Krueger and Papadopolous [87] demonstrated that PBR agonists could increase steroidogenesis in Y1 adrenocortical cells, although to much lesser extent than ACTH. ACTH-induced stimulation of Y1 cells was cycloheximide-sensitive, while the more modest PBR-mediated increase did not require new protein synthesis. Furthermore, it was found that the PBR agonist PK11195 increased pregnenolone production in isolated mitochondria, and the effect was increased in the presence of cholesterol. In the same study it was shown that PK11195 slightly increased cholesterol accumulation in the inner mitochondrial membrane, and the effect was magnified when mitochondria were pretreated with cholesterol. Since this initial report, a number of additional research papers have been published which support these findings. For example, it was found that in mitochondria isolated from rat liver that different PBR ligands differed in their effectiveness to stimulate cholesterol transport to the inner mitochondrial membrane [86]. A recent discovery concerning PBR’s potential role in cholesterol transport is the identification of a putative cholesterol recognition/interaction site on PBR [95]. This recognition site consists of a leucine or valine, then 2–6 residues followed by tyrosine, then an arginine or lysine 2–6 residues away (L/V-(X)1-5-Y-(X)1-5-R/K). This consensus sequence has been found in the mouse, rat human and bovine PBR sequences as well as a number of other proteins which interact with cholesterol such as P450scc, StAR and apolipoprotein A-I [95]. In steroidogenic tissues, stimulation with trophic hormone results in the rapid translocation of cholesterol from the cytoplasm to the inner mitochondrial membrane, where it is used as a substrate for the P450scc enzyme. As previously stated, this translocation of cholesterol is the rate-limiting step of steroidogenesis, and requires de novo protein synthesis. However, it has been reported by Papadopoulos and colleagues that stimulation of MA-10 cells with hCG induces the formation of a higher affinity PBR site on the mitochondria which in turn results in the rapid translocation of cholesterol to the inner membrane [96]. In support of this finding, it was found that R2C cells, which constitutively synthesize steroids, contain only the high-affinity PBR receptor [97]. PBR levels are not known to be acutely regulated by trophic hormone or cAMP, therefore, if PBR does participate in the acute regulation of steroidogenesis, it must be through a hormone-induced change in the receptor or an endogenous ligand which in turn is hormonally regulated. One such ligand is an 8.2-kD protein known as the diazepam-binding inhibitor (DBI), which has been shown to stimulate Leydig cell steroidogenesis [98]. The DBI is secreted by Leydig and Sertoli cells and is present in interstitial fluid of the

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testis [98]. Furthermore, the DBI was capable of displacing the binding of a known PBR ligand, suggesting that it interacts with PBR [98]. At a concentration of 10Ö7 M, the DBI was shown to stimulate Leydig cell steroidogenesis by approximately 3-fold [98]. This 3-fold increase is somewhat low when it is considered in the context of the near 150-fold increase in steroid production in response to 50 ng/ml hCG treatment shown in the same report. Interestingly, the potent PBR antagonists flunitrazepam and PK11195, did not antagonize the effect of DBI on steroid production [98], which would suggest that DBI does not work through binding to PBR as an endogenous ligand. Papadopoulos and others have suggested that the PBR is directly involved in the transfer of cholesterol to the inner mitochondrial membrane and is the key regulator of steroidogenesis. However, the available evidence indicates that while it is involved in this process, and may be requisite for steroid production, it probably is not the key regulator of steroidogenesis. An alternative role of PBR in steroidogenesis may be more related to mitochondrial function than steroidogenesis per se. For example, it has been shown that steroidogenesis requires a mitochondrial electrochemical gradient and ATP [99]. If the electrochemical gradient is disrupted using the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP), steroidogenesis is inhibited at the level of steroidogenic acute regulatory (StAR) protein-mediated cholesterol transport to the mitochondrial membrane [110]. Because PBR forms a complex with VDAC and the adenine nucleotide carrier protein, and is reported to form channel-like clusters, it may function in some capacity to maintain or regulate certain aspects of mitochondrial function such as the electrochemical gradient or some component in the electron transport chain. If PBR functions in this capacity, its disruption or inhibition would result in deleterious effects in steroidogenesis without being directly involved in the transport of cholesterol to the inner mitochondrial membrane, and thus steroid production. Also, it may be that PBR is one of the components on the outer mitochondrial membrane with which the StAR protein (see below) interacts during the course of regulated cholesterol transfer. In summary, the available evidence indicates that PBR plays a critical role in the transfer of cholesterol to the inner mitochondrial membrane, but it is likely that this role is a passive role and not a regulatory one.

Steroidogenic Acute Regulatory Protein Another protein candidate for the acute reulator is the steroidogenic acute regulatory or StAR protein. A protein identical to what we now know to be StAR was initially described by Orme-Johnson and colleagues as an ACTH-

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induced 30-kD phosphoprotein in hormone-treated rat and mouse adrenocortical cells, and as an LH-induced protein in rat corpus luteum cells and mouse Leydig cells [41–44, 101–104]. StAR has also been characterized in hormonestimulated MA-10 mouse Leydig tumor cells by Stocco and colleagues [45, 46, 105–109]. This protein was found to be localized in the mitochondria and consisted of several forms of a newly synthesized 30-kD protein. In addition to the 30-kD family of mitochondrial proteins, a 37-kD precursor form of these proteins was also detected [43, 46]. The 37-kD precursor contained N-terminal amino acid sequences which served to target the protein to the mitochondrial and facilitate its import and processing by mitochondria. A number of studies have demonstrated correlations between the synthesis of steroids and the synthesis of the 30-kD protein in steroidogenic cells [41–45, 101–103, 107–109]. Even though many positive correlations were made, a direct cause-and-effect relationship between 30-kD protein expression and steroidogenesis was lacking, and it was necessary to clone the 30-kDa protein to unequivocally prove a function for it in steroidogenesis. The cDNA containing the 37-kD precursor protein was successfully cloned from MA-10 cells in 1994 [110]. When compared with other sequences in the database both the nucleic acid sequence and protein sequence were found to be unique indicating the 37-kD protein represented a novel protein. Importantly, transient transfection experiments demonstrated that expression of the cDNA-derived protein in MA-10 mouse Leydig tumor cells resulted in a significant increase in steroid production in the absence of hormone stimulation [110]. Transient transfection of COS-1 cells with the cDNA for the 37-kD protein also resulted in a severalfold increase in the conversion of cholesterol to pregnenolone [8, 111, 112]. As a result of these observations, the protein was named the StAR protein [110]. StAR has an indispensable function in acutely regulated steroidogenesis and its role in this regulation appears to be at the level of the mediation of the transfer of cholesterol from the outer to the inner mitochondrial membrane [113, 114]. An early model of its proposed action hypothesized that in response to trophic hormone stimulation StAR was rapidly synthesized in the cytosol as a 37-kD precursor and became quickly associated with the mitochondria. During the import and processing of StAR contact sites between the inner and outer membranes were formed, and these contact sites served as the conduit for cholesterol to transfer to the inner membrane [7, 8, 43, 46]. It soon became clear, however, that this model was incorrect when it was demonstrated that N-terminal truncations of the StAR protein that removed as many as 62 amino acids had no inhibitory effect on steroid production when transfected into COS-1 cells [115]. These observations were also confirmed by Wang et al. [114] who illustrated that incubation of isolated mitochondria

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with lysate containing StAR protein whose N-terminus was truncated by 47 amino acids displayed no decrease in cholesterol transfer from the outer to inner mitochondrial membrane or in pregnenolone biosynthesis. These observations were also corroborated in a completely in vitro system by Arakane et al. [116] in which it was demonstrated that bacterially produced StAR protein lacking the first 62 N-terminal amino acids was able to fully support steroidogenesis in isolated mitochondria. Surprisingly, truncation of the C-terminus by 28 amino acids resulted in a complete loss of steroid production [112, 114, 115]. These observations indicate that it is the C-terminal region of the StAR protein that functions in cholesterol transfer, and could have perhaps been predicted when one considers that all mutations in congenital lipoid adrenal hyperplasia, a disease in which intramitochondrial cholesterol transport is severely compromised (see below), have been shown to be in the carboxyterminal 40% of the StAR protein. That some of these mutations are single point mutations indicates the powerful role that this region of the molecule must play in cholesterol transfer [117]. Indeed, recent studies by Bose et al. [118] have shown that mutations in the StAR protein that cause lipoid CAH result in protein folding alterations in the StAR protein as assessed by several physical parameters. Recently it has been demonstrated that StAR can act as a sterol transfer protein and that the function of the StAR protein may be to enhance desorption from one sterol-containing membrane to another [119]. In this model, StAR is specifically directed to the mitochondria via its N-terminus and upon its arrival at the outer mitochondrial membrane the C-termminus produces alterations in the outer mitochondrial membrane that in some manner results in the transfer of cholesterol from the outer to the inner membrane. Interestingly, the transfer of cholesterol to heat-treated mitochondrial or microsomes by purified StAR protein was specific in that identical experiments employing phosphatidylcholine failed to show transfer of this phospholipid. This would be particularly pertinent to the situation found in steroidogenic mitochondria in which the desorption of cholesterol from the sterol-rich outer membrane to the sterol-poor inner membrane [120] would serve to enhance greatly pregnenolone synthesis by the P450scc enzyme. A recent review article contains a thorough discussion of this model [121]. While the mechanism of action of the StAR protein is still unknown, it is becoming increasingly clear that cholesterol transfer requires that it interact, at least transiently, with as yet unknown components such as proteins, lipids and/or other factors on the outside of the outer mitochondrial membrane and produce alterations which result in cholesterol transfer. The positive identification of such factors has so far proven to be quite elusive [121]. However, it appears to be clear that the action of StAR in promoting cholesterol transfer

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to the inner mitochondrial membrane occurs as a result of its direct interaction with the outer surface of the mitochondria and not through an intermediary [119]. This was demonstrated through the use of recombinant StAR protein which when added directly to purified mitochondria was shown to increase pregnenolone production. To date, studies designed to identify StAR-interacting proteins have utilized the yeast two-hybrid assay system with StAR as bait, coimmunoprecipitation of StAR expressing COS cell lysates and binding assays using radiolabeled StAR protein incubated with isolated mitochondrial [121] and [Wang and Miller, unpubl. results]. None of these approaches has yet identified a legitimate StAR-binding partner. Perhaps, as recently speculated [119], StAR can stimulate cholesterol transfer either as a result of few very high-affinity stable interactions with the outer mitochondrial membrane which are difficult to detect because of their low number, or as a result of transient interactions which would also be difficult to detect because of their fleeting nature. Also, in light of a substantial amount of evidence indicating a role for the peripheral benzodiazepine receptor in cholesterol mobilization to the P450scc, perhaps this outer mitochondrial membrane protein will prove to be one of the factors with which StAR interacts to promote cholesterol transfer [122]. In an effort to determine how the StAR protein might interact with the outer mitochondrial membrane, Miller and colleagues showed that StAR can form a molten globule in the pH 3.5–4.0 range [123]. The significance of this observation is that as a result of the proton pump which operates within the mitochondria, the pH microenvironment surrounding this organelle may be acidic. If this were the case, the StAR molecule may undergo a conformational shift, forming an extended structure and increasing the flexibility of the linker region located between the N-terminus and the biologically active C-terminus while interacting with the outer mitochondrial membrane. As the transition to a molten globule occurs, this structural change may serve to either lower the energy required to open the StAR structure further, possibly exposing a cholesterol channel or it may prolong the interval with which StAR can reside on the outer membrane thus allowing increased transfer of cholesterol during this period. In addition, partial proteolysis experiments at different pH values showed that the domain comprising residues 63-192 was relatively protease-resistant, and hence presumably tightly folded, whereas the biologically essential carboxyterminal 193-285 domain was very protease-sensitive, and hence probably the principal contributor to the molten globule state. These findings suggested a model in which the mitochondrial leader peptide (residues 1-62) targets StAR to the mitochondrion, then the tightly folded 63-192 domain acts as a “pause-transfer” sequence, permitting the biologically active 193-285 domain to have a longer

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residency time to interact with the outer mitochondrial membrane, thus facilitating StAR’s action [123]. Much remains to be determined about the manner in which StAR effects cholesterol transfer to the inner mitochondrial membrane. In this regard, the identification of the components with which StAR interacts on the outer mitochondrial membrane and the nature of this interaction becomes of critical importance in understanding its mechanism of action. The identification of these putative binding partners has thus far remained elusive and their characterization and the description of their role in cholesterol transfer can be considered as perhaps the most interesting and important undertaking in this field.

Congenital Lipoid Adrenal Hyperplasia: The StAR Knockout of Nature Studies of StAR protein are inexorably lined with studies of congenital lipoid adrenal hyperplasia, which is the disease caused by absence of StAR activity. Congenital lipoid adrenal hyperplasia was described as an autosomalrecessive disorder of adrenal and testicular steroidogenesis between 1955 and 1962 [124–126], although several earlier cases appeared as autopsy reports [127]. These patients appeared to lack all mineralocorticoids, glucocorticoids, and sex steroids as they had very low or unmeasurable serum and urinary steroids. Thus the disease was characterized by a severe, often fatal, saltwasting crisis in infancy and female external genitalia in both sexes [127, 128]. Because these patients appeared to produce no steroid hormones and because their adrenals accumulated the steroid precursor, cholesterol, it was inferred that lipoid CAH was caused by a mutation in the enzyme system that converts cholesterol to pregnenolone (then mistermed ‘20,22 desmolase’). Work between 1965 and 1973 showed that ‘20,22 desmolase’ was P450scc which catalyzes the sequential 20-hydroxylation, 22-hydroxylation, and 20,22 bond scission on a single catalytic site [129], but this was not generally appreciated until the 1980s. In 1972, Degenhart et al. [130] concluded that lipoid CAH was due to a specific lesion in 20a-hydroxylase, as mitochondria from affected tissues could convert 20a-hydroxycholesterol, but not cholesterol to pregnenolone. This observation was eventually explained when Lin et al. [112] showed that soluble hydroxysterols could bypass the action of StAR. However, in 1977 Koizumi et al. [131] reported that affected tissues appeared to lack spectroscopically detectable P450scc, which appeared to confirm the notion that the disorder was in ‘20,22 desmolase’ just as the more common forms of CAH were due to mutations in other steroidogenic P450 enzymes.

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Kirkland et al. [132] reported that first long-term survival in an 8-yearold, and in 1984 Hauffa et al. [128] described a patient surviving to adulthood. This latter report was of particular significance as it provided the first compilation of existing case reports, finding the expected 1:1 ratio of affected males to females but pointing out three unexpected findings: first, the disease appeared to be common in Japan; second, most patients have low but detectable steroids in infancy that disappeared by 1–2 years of age; and third, the age of onset of a salt-losing crisis tended to be somewhat later than in infants with CAH due to 21-hydroxylase deficiency. The clinical picture of lipoid CAH as a severe block in the synthesis of all steroids was then questioned by two reports from Japan. In 1988, Tanae et al. [133] described three 46,XX patients with lipoid CAH who appeared to undergo spontaneous pubertal development, and in 1994 Matsuo et al. presented a preliminary report of a large series of Japanese patients, finding that only 16 of 63 were 46,XX, and that all five 46,XX patients over age 13 had undergone spontaneous breast development and menses and had normal estradiol levels [134]. Thus, the steroidogenic block in lipoid CAH appeared to affect the testis but not the ovary. The genetic study of lipoid CAH began with a demonstration that the P450scc gene was grossly intact in three affected patients [135]. The major surprise, however, came in 1991 when Miller’s laboratory cloned completely normal P450scc cDNA from affected tissue thus ruling out P450scc as a potential cause of lipoid CAH [136]. Hypothesizing that a defect in a factor that transports cholesterol to P450scc could also explain the findings in lipoid CAH, they examined SCP-2, GRP-78, endozepine, and its presumed receptor (PBR) as well as adrenodoxin and adrenodoxin reductase, but found all to be normal [136, 137]. They also considered that the family of proteins termed pp30, pp32 and pp37 (which are now known to be StAR) might be responsible, but could not study this in 1991. The demonstration that P450scc mutations did not cause lipoid CAH was unexpected, and doubted. Pang and coworkers reported a rabbit model of lipoid CAH in which both copies of the P450scc gene had undergone spontaneous deletions [138, 139]. As the phenotypic, clinical, and histologic findings in these animals were very similar to those in children with lipoid CAH, it appeared clear that P450scc mutations should be a cause of lipoid CAH, even if the 2 patients studied by Miller et al. [136] had no mutations. However, clinical studies showed that the P450scc system can function normally in lipoid CAH patients, as the affected placenta continues to make pregnenolone and progesterone [140]. Furthermore, several other groups subsequently confirmed the observation that the P450scc gene was intact in those patients [141–143]. Thus, the cause of lipoid CAH remained mysterious until the cloning of StAR [110].

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Human StAR was cloned in 1995, and found to be expressed in the adrenals and gonads but not in the placenta [111], as predicted by the clinical studies [140] of the factor causing lipoid CAH. Analysis of StAR cDNA from three patients revealed StAR mutations in lipoid CAH [112]. Perhaps even more important than finding that StAR mutations cause lipoid CAH, this seminal report of Lin et al. [112] demonstrated two crucial features of the biology of StAR. First, by transfecting nonsteroidogenic cells with a fusion protein for the P450scc system [144] and with normal or mutant StAR, this study showed that StAR increased steroidogenesis 6-fold, but that there was a low (14%) level of StAR-independent steroidogenesis in both untransfected control cells and those transfected with the StAR mutants. Second, this paper compared the substrate activity of the LDL cholesterol found in serum with that of 20a-hydroxycholesterol, which is known to be freely diffusable into mitochondria, and thus provides an index of maximal cellular steroidogenic capacity [145]. When incubated with 20a-hydroxycholesterol, pregnenolone secretion from the COS-1 cells depended only on the presence of the P450scc system, and was maximal with or without StAR [112]. This experiment, which recapitulated Degenhart’s experiment from 1972 [130], provided the definitive proof that StAR functioned to increase the availability of cholesterol as substrate for the P450scc enzyme system. Subseqent studies confirmed and refined these observations with both 20a-hydroxycholesterol and 22R-hydroxycholesterol for all StAR mutations found in lipoid CAH to date [111, 146–149]. Thus we not only identified StAR as the cause of lipoid CAH, but also established that StAR functioned to increase the flow of cholesterol into mitochondria and provided the first evidence for StAR-independent steroidogenesis [112]. Despite this demonstration that StAR mutations caused lipoid CAH in two families [112], one had to consider whether other factors might cause the same phenotype. Furthermore, as the clinical presentation of lipoid CAH was variable, correlations between genotype and phenotype were of particular interest. The cloning and complete sequencing of the human StAR gene facilitated this, especially as the gene was small and had only 7 exons [150]. Tee et al. [146] studied a 46,XY patient who had an initial onset of a saltlosing crisis at 8 weeks of age, who had low but measurable amounts of mineralocorticoids. Genetic analysis revealed a StAR gene splicing mutation that resulted in the production of a small amount of normally spliced StAR mRNA, and hence a small amount of StAR protein, accounting for the somewhat later age of presentation [146]. A multinational survey of 21 patients in 18 families from 11 countries then found that all but one patient had detectable StAR mutations [117]. The 15 mutations identified were found throughout the StAR gene, but most were in exons 5, 6 or 7 and all of the

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amino acid replacement mutations were in this C-terminal region [117]. This study confirmed that most Japanese and Korean patients carried the Q258X mutation as suggested earlier [112], identified a new genetic isolate of Palestinian Arabs carrying the mutation R192L, and provided genetic diagnostic procedures for ten mutations. Transfection studies with the amino acid replacement mutants identified showed that A218V and L275P appeared to retain a modest amount of activity. These were found in a compound heterozygous patient who survived without steroid replacement therapy until 4 months of age. Thus, as in the patient with the splicing mutation [146], partial activity was correlated with a late onset of clinical signs of salt loss. Despite this partial activity, all of these patients had wholly female external genitalia, suggesting that the fetal testis was more severely affected than the fetal adrenal. Consideration of these clinical findings in correlation with the observation [112] that steroidogenic cells have both StAR-dependent and StAR-independent steroidogenesis led to the formulation of the two-hit model of lipoid CAH [117] which explained these observations and the reports of spontaneous feminization in 46,XX patients [133, 134].

The Two-Hit Model of Lipoid CAH Because the transfection studies showed that StAR-independent steroidogenesis proceeds at about 14% of the StAR-inducible level [111, 112, 117, 146], Bose et al. [117] hypothesized that StAR mutations initially eliminate only the StAR-induced steroidogenesis that occurs in response to tropic hormone stimulation by ACTH, angiotensin II, or gonadotropins. This loss of StARdependent steroidogenesis is the ‘first hit’ of the two-hit model. In the absence of StAR-dependent steroidogenesis, the low levels of StAR-independent steroidogenesis persist, but are insufficient to suppress tropic hormone secretion. High levels of tropic hormones then induce intracellular cAMP, which induces the genes for intracellular cholesterol biosynthesis, and the gene for LDL receptor, promoting uptake of extracellular cholesterol. With time, this accumulated cholesterol (or its autooxidation products) becomes toxic to the cell, disrupting residual StAR-independent steroidogenesis. This loss of StARindependent steroidogenesis is the second hit. This two-hit model [117] accounts for all the clinical observations in lipoid CAH. The fetal testis makes abundant steroids beginning very early in gestation, and hence is affected very early, so that 46,XY patients have normal female external genitalia. The fetal zone of the adrenal makes very large amounts of DHEA and DHEA-S, which are normally inactivated to estriol by the placenta and are not needed for fetal physiology [151]. Because the fetal zone is highly active, these cells in lipoid

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CAH are also rapidly destroyed, resulting in nearly unmeasurable placental estrogens [140]. The definitive zone of the adrenal normally makes very little steroid. Normal fetal blood concentrations of cortisol are very low [152, 153] and glucocorticoids are not needed by the fetus, as shown by the normal development of fetuses with CAH, adrenal hypoplasia, or glucocorticoid receptor mutations [151]. However, the fetal adrenal can make steroids and respond to tropic hormones. Bose et al. noted that most lipoid CAH patients who experienced a very early salt-losing crisis were born in hot, arid climates [117, 147]. The cells that are precursors to zona glomerulosa cells normally do not need to make aldosterone during pregnancy, as the placenta handles the fetal requirements for salt and water balance. However, in hot, arid climates, mild chronically compensated hypovolemia in the pregnant mother apparently can stimulate the fetal renin-angiotensin system. This stimulates the fetal cells that develop into the zona glomerulosa, accelerating the accumulation of cholesterol and the consequent loss of StAR-independent aldosterone production. Thus these children experienced an earlier onset of a salt-wasting crisis. The same logic explains the spontaneous feminization at the appropriate age for puberty in affected 46,XX female patients [117, 147]. Unlike the fetal testis, the fetal ovary makes no steroids and does not express the genes for the steroidogenic enzymes [154]. Thus, although the fetal ovarian granulosa and theca cells carry affected StAR mutations (the first hit), they remain undamaged by the accumulation of cholesterol until they begin to undergo gonadotropin stimulation at the onset of puberty. Human preovulatory granulosa cells do not express StAR [155] and hence do not express the first hit in lipoid CAH. Because gonadotropins recruit and stimulate individual follicles, rather than the whole ovary, the recruited follicle will produce small amounts of estrogen by StARindependent steroidogenesis until the accumulation of cholesterol in response to tropic stimulation (especially the LH surge) destroys steroidogenic capacity, preventing normal luteinization and progesterone production. Monthly cycles of follicular recruitment proceed normally as the cyclicity is hypothalamic, not ovarian. Thus each month a previously undamaged follicle is recruited and produces estrogen (but very little progesterone) by StAR-independent steroidogenesis, leading to breast development and cyclical vaginal bleeding. However, as the model would predict that the follicle cannot luteinize and produce progesterone, these cycles probably are anovulatory. Several recent studies confirm the two-hit model. Nakae et al. found StAR mutations in 19 Japanese lipoid CAH patients, again finding that the Q258X mutation predominates [149]. This single mutation accounts for about 70% of Japanese lipoid CAH alleles, with an estimated carrier rate of 1 in 300 Japanese persons. Similarly, 10 of 10 affected Korean alleles carried Q258X [156]. One of Nakae’s patients was a compound heterozygote carrying the

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mutation M225T, which retained 44% of activity in vitro. This 46,XX patient is the only one described to date to have some virilization of the external genitalia, consistent with a less severe defect in fetal testicular steroidogenesis. She had normal basal and ACTH-stimulated cortisol values in infancy, but eventually developed glucocorticoid and mineralocorticoid deficiency and had a salt-wasting crisis at 10 months of age [149], thus following the course predicted by the two-hit model. Another report described an affected adolescent 46,XX patient who underwent spontaneous feminization at 11.5 years, identifying a novel StAR mutation, and pointing out that medroxyprogesterone treatment should be used in these patients to prevent the formation of ovarian cysts [147]. Fujieda et al. [148] measured sex steroids and gonadotropins at weekly intervals in two affected 46,XX patients, finding normal estradiol and unmeasurable progesterone levels, high LH/FSH ratios, and ovarian cysts suggesting the presence of anovulatory cycles as predicted by the two-hit model. Finally, Caron et al. [157] reported the creation of a StAR gene knockout in mice. These mice had enlarged adrenals with lipid deposits, female external genitalia irrespective of chromosomal sex, and could be maintained with steroid replacement. The histologic appearance of the tissues and clinical course of the animals was exactly analogous to that seen in lipoid CAH patients. Although the findings in pubertal 46,XX female mice were not described, these data were interpreted as confirming the two-hit model [157]. The mechanism of StAR-independent steroidogenesis remains unclear and perhaps one example of this is the human placenta [158] which makes steroid hormones but lacks StAR. While it is possible that StAR-independent steroidogenesis is totally passive, a search for other StAR-like factors has revealed the interesting candidate MLN-64 [159]. This protein, originally identified in breast cancer tissue, has a carboxy terminus with a similar amino acid sequence to that of StAR. Comparison of the MLN-64 sequence from Candida elegans with the human and mouse MLN-64 and StAR sequences suggests that the most highly conserved residues are those that are mutated in lipoid CAH patients [159]. This, together with the limited number of C-terminal StAR amino acid replacements found in lipoid CAH, suggests that many hypothetical StAR amino acid replacements will be clinically silent. Finally, some continue to question whether mutations of P450scc or other factors could also cause a lipoid CAH phenotype. The lack of an identified StAR lesion in one of Bose’s patients [117] suggests this, as do the rabbits with spontaneous P450scc deletions [138, 139]. It is attractive to hypothesize that a mutation in SF-1, a transcription factor required for adrenal development might cause adrenal hypoplasia, as it does in SF-1 knockout mice [160], and might be clinically mistaken for lipoid CAH. However, only one heterozygous SF-1 mutation has been described to date [161], suggesting that the

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homozygous SF-1-deficient phenotype may be incompatible with term gestation. Whatever the disorder in Bose’s patient [117], we believe that no patient will be found with homozygous P450scc mutations, because progesterone is required to maintain pregnancy by suppressing uterine contractility. In the rabbit and goat, the maternal corpus luteum provides this progesterone throughout pregnancy, so that a homozygous P450scc mutation in the placenta remains compatible with normal term delivery, as in the P450scc-deficient rabbit [138, 139]. However, in human pregnancy the maternal corpus luteum provides progesterone only during the first 2 months of pregnancy, then a ‘luteo-placental shift’ occurs between 6 and 8 weeks gestation [162]. Human maternal ovariectomy before 6 to 8 weeks causes spontaneous abortion, but ovariectomy thereafter does not [162]. If a human fetus carried a homozygous mutation for P450scc, adrenodoxin or adrenodoxin reductase, the placenta would be unable to assume the production of progesterone required by the luteo-placental shift, and spontaneous abortion would result [163]. Thus, if factors other than StAR mutations can cause the lipoid CAH phenotype, they remain to be identified.

Acknowledgments The authors would like to thank the support of NIH HD17481 to D.M.S. and DK37922, DK42154 and HD34449 to W.L.M.

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Hapala I, Kavecansky J, Butko P, Scallen TJ, Joiner CH, Schroeder F: Regulation of membrane cholesterol domains by sterol carrier protein-2. Biochemistry 1994;33:7682–7690. Colles SM, Woodford JK, Moncecchi D, Myers-Payne SC, McLean LR, Billheimer JT, Schroeder F: Cholesterol interaction with recombinant human sterol carrier protein-2. Lipids 1995;30:795– 803. Yamamoto R, Kallen CB, Babalola GO, Rennert H, Billheimer JT, Strauss JF: Cloning and expression of a cDNA encoding human sterol carrier protein 2. Proc Natl Acad Sci USA 1991;88: 463–467. Chanderbhan RF, Kharroubi AT, Noland BJ, Scallen TJ, Vahouny GV: SCP2: Further evidence for its role in adrenal steroidogenesis. Endocr Res 1986;12:351–360. Keller GA, Scallen TJ, Clarke D, Maher PA, Krisans SK, Singer SJ: Subcellular localization of sterol carrier protein-2 in rat hepatocytes: Its primary localization to peroxisomes. J Cell Biol 1989; 108:1353–1361. van Amerongen A, van Noort M, van Beckhoven JRCM, Rommerts FFG, Orly J, Wirtz KWA: The subcellular distribution of the nonspecific lipid transfer protein (sterol carrier protein 2) in rat liver and adrenal gland. Biochim Biophys Acta 1989;1001:243–248. Fujiki Y, Tsuneoka M, Tashiro Y: Biosynthesis of nonspecific lipid transfer protein (sterol carrier protein 2) on free polyribosomes as a larger precursor in rat liver. J Biochem 1989;106:1126–1131. Mendis-Handagama SMLC, Zirkin BR, Scallen TJ, Ewing LL: Studies on peroxisomes of the adult rat Leydig cell. J Androl 1990;11:270–278. Ohba T, Rennert H, Pfeifer SM, He Z, Yamamoto R, Holt JA, Billheimer JT, Strauss JF III: The structure of the human sterol carrier protein x/sterol carrier protein2 gene (SCP2). Genomics 1994; 24:370–374. Trzeciak WH, Simpson ER, Scallen TJ, Vahouny GV, Waterman MR: Studies on the synthesis of SCP-2 in rat adrenal cortical cells in monolayer culture. J Biol Chem 1987;262:3713–3720. van Amerongen A, Demel RA, Westerman J, Wirtz KWA: Transfer of cholesterol and oxysterol derivatives by the non-specific lipid transfer protein (sterol carrier protein 2): A study on its mode of action. Biochim Biophys Acta 1989;1004:36–43. van Noort M, Rommerts FFG, van Amerongen A, Wirtz KWA: Regulation of SCP2 in the soluble fraction of rat Leydig cells. Kinetics and possible role of calcium influx. Mol Cell Endocrinol 1988; 56:133–138. McLean MP, Puryear TK, Khan I, Azhar S, Billheimer JT, Orly J, Gibori G: Estradiol regulation of sterol carrier protein-2 independent of cytochrome P450 side-chain cleavage expression in the rat corpus luteum. Endocrinology 1989;125:1337–1344. Rennert H, Amsterdam A, Billheimer JT, Strauss JF III: Regulated expression of sterol carrier protein 2 in the ovary: A key role for cyclic AMP. Biochemistry 1991;47:11280–11285. Johnson WJ, Reinhart MP: Lack of requirement for sterol carrier protein-2 in the intracellular trafficking of lysosomal cholesterol. J Lipid Res 1994;35:563–573. Pfeifer SM, Furth EE, Ohba T, Chang YJ, Rennert H, Sakuragi N, Billheimer JT, Strauss JF III: Sterol carrier protein 2: A role in steroid hormone synthesis? J Steroid Biochem Mol Biol 1993;47: 167–172. Pedersen RC, Brownie AC: Cholesterol side-chain cleavage in the rat adrenal cortex: Isolation of a cyloheximide-sensitive activator peptide. Proc Natl Acad Sci USA 1983;80:1882–1886. Pedersen RC: Polypeptide activators of cholesterol side-chain cleavage. Endocr Res 1984;10:533–561. Pedersen RC, Brownie AC: Steroidogenesis activator polypeptide isolated from a rat Leydig cell tumor. Science 1987;236:188–190. Pedersen RC: Steroidogenesis activator polypeptide (SAP) in the rat ovary and testis. J Steroid Biochem 1987;27:731–735. Mertz LM, Pedersen RC: Steroidogenesis activator polypeptide may be a product of glucose regulated protein 78 (GRP78). Endocr Res 1989;15:101–115. Frustaci J, Mertz LM, Pedersen RC: Steroidogenesis activator polypeptide (SAP) in the guinea pig adrenal cortex. Mol Cell Endocrinol 1989;64:137–143. Mertz LM, Pedersen RC: The kinetics of steroidogenesis activator polypeptide in the rat adrenal cortex. J Biol Chem 1989;264:15274–15279.

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Li X, Warren DW, Gregoire J, Pedersen RC, Lee AS: The rat 78,000 dalton glucose-regulated protein (GRP78) as a precursor of the rat steroidogenesis-activator polypeptide (SAP): The SAP coding sequence is homologous with the terminal end of GRP78. Mol Endocrinol 1989;3:1944–1952. Verma A, Snyder SH: Peripheral type benzodiazepine receptors. Annu Rev Pharmacol Toxicol 1989; 29:307–322. Papadopoulos V: Peripheral-type benzodiazepine/diazepam binding inhibitor receptor: Biological role in steroidogenic cell function. Endocr Rev 1993;14:222–240. Gavish M, Katz Y, Bar-Ami S, Weizman R: Biochemical, physiological, and pathological aspects of the peripheral benzodiazepine receptor. J Neurochem 1992;58:1589–1601. Anholt RR, Pedersen PL, De Souza EB, Snyder SH: The peripheral-type benzodiazepine receptor. Localization to the mitochondrial outer membrane. J Biol Chem 1986;261:576–583. Marc V, Marselli PL: Effect of diazepam on plasma corticosterone levels in the rat. J Pharm Pharmacol 1969;21:784–786. Cook PS, Notelovitz M, Kalra PS, Kalra SP: Effect of diazepam on serum testosterone and the ventral prostate gland in male rats. Arch Androl 1979;3:31–35. Arguelles AE, Rosner J: Diazepam and plasma-testosterone levels (letter). Lancet 1975;ii:607. Tsankova V, Magistrelli A, Cantoni L, Tacconi MT: Peripheral benzodiazepine receptor ligands in rat liver mitochondria: Effect on cholesterol translocation. Eur J Pharmacol 1995;294:601–607. Krueger KE, Papadopoulos V: Peripheral-type benzodiazepine receptors mediate translocation of cholesterol from outer to inner mitochondrial membranes in adrenocortical cells. J Biol Chem 1990; 265:15015–15022. McEnery MW, Dawson TM, Verma A, Gurley D, Colombini M, Snyder SH: Mitochondrial voltagedependent anion channel. Immunochemical and immunohistochemical characterization in rat brain. J Biol Chem 1993;268:23289–23296. McEnery MW, Snowman AM, Trifiletti RR, Snyder SH: Isolation of the mitochondrial benzodiazepine receptor: Association with the voltage-dependent anion channel and the adenine nucleotide carrier. Proc Natl Acad Sci USA 1992;89:3170–3174. Parola AL, Stump DG, Pepperl DJ, Krueger KE, Regan JW, Laird HED: Cloning and expression of a pharmacologically unique bovine peripheral-type benzodiazepine receptor isoquinoline binding protein. J Biol Chem 1991;266:14082–14087. Sprengel R, Werner P, Seeburg PH, Mukhin AG, Santi MR, Grayson DR, Guidotti A, Krueger KE: Molecular cloning and expression of cDNA encoding a peripheral-type benzodiazepine receptor. J Biol Chem 1989;264:20415–20421. Antkiewicz-Michaluk L, Mukhin AG, Guidotti A, Krueger KE: Purification and characterization of a protein associated with peripheral-type benzodiazepine binding sites. J Biol Chem 1988;263: 17317–17321. Garnier M, Dimchev AB, Boujrad N, Price JM, Musto NA, Papadopoulos V: In vitro reconstitution of a functional peripheral-type benzodiazepine receptor from mouse Leydig tumor cells. Mol Pharmacol 1994;45:201–211. Vidic B, Boujrad N, Papadopoulos V: Hormone-induced changes in the topography of the mitochondrial peripheral-type benzodiazepine receptor. Scanning 1995;17:V34–V35. Li H, Papadopoulos V: Peripheral-type benzodiazepine receptor function in cholesterol transport. Identification of a putative cholesterol recognition/interaction amino acid sequence and consensus pattern. Endocrinology 1998;139:4991–4997. Boujrad N, Gaillard JL, Garnier M, Papadopoulos V: Acute action of choriogonadotropin on Leydig tumor cells: Induction of a higher affinity benzodiazepine-binding site related to steroid biosynthesis. Endocrinology 1994;135:1576–1583. Garnier M, Boujrad N, Ogwuegbu SO, Hudson JR Jr, Papadopoulos V: The polypeptide diazepambinding inhibitor and a higher affinity mitochondrial peripheral-type benzodiazepine receptor sustain constitutive steroidogenesis in the R2C Leydig tumor cell line. J Biol Chem 1994;269:22105–22112. Garnier M, Boujrad N, Oke BO, Brown AS, Riond J, Ferrara P, Shoyab M, Suarez-Quian CA, Papadopoulos V: Diazepam binding inhibitor is a paracrine/autocrine regulator of Leydig cell proliferation and steroidogenesis: Action via peripheral-type benzodiazepine receptor and independent mechanisms. Endocrinology 1993;132:444–458.

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King SR, Stocco DM: ATP and a mitochondrial electrochemical gradient are required for functional activity of the steroidogenic acute regulatory (StAR) protein in isolated mitochondria. Endocr Res 1996;22:505–514. King SR, Liu Z, Soh J, Timberg R, Orly J, Stocco DM: Effects of disruption of the mitochondrial electrochemical gradient on steroidogenesis and the Steroidogenic Acute Regulatory (StAR) protein. J Steroid Biochem Mol Biol 1999;69:143–154. Pon LA, Orme-Johnson NR: Acute stimulation of steroidogenesis in corpus luteum and adrenal cortex by peptide hormones: Rapid induction of a similar protein in both tissues. J Biol Chem 1986;261:6594–6599. Pon LA, Hartigan JA, Orme-Johnson NR: Acute ACTH regulation of adrenal corticosteroid biosynthesis: Rapid accumulation of a phosphoprotein. J Biol Chem 1986;261:13309–13316. Pon LA, Epstein LF, Orme-Johnson NR: Acute cAMP stimulation in Leydig cells: Rapid accumulation of a protein similar to that detected in adrenal cortex and corpus luteum. Endocr Res 1986; 12:429–446. Alberta JA, Epstein LF, Pon LA, Orme-Johnson NR: Mitochondrial localization of a phosphoprotein that rapidly accumulates in adrenal cortex cells exposed to adrenocorticotropic hormone or to cAMP. J Biol Chem 1989;264:2368–2372. Stocco DM, Kilgore MW: Induction of mitochondrial proteins in MA-10 Leydig tumour cells with human choriogonadotropin. Biochem J 1988;249:95–103. Stocco MR, Chaudhary LR: Evidence for the functional coupling of cAMP in MA-10 mouse Leydig tumor cells. Cell Signal 1990;2:161–170. Stocco DM: Further evidence that the mitochondrial proteins induced by hormone stimulation in MA-10 mouse Leydig tumor cells are involved in the acute regulation of steroidogenesis. J Steroid Biochem Mol Biol 1992;43:319–333. Stocco DM, Ascoli M: The use of genetic manipulation of MA-10 Leydig tumor cells to demonstrate the role of mitochondrial proteins in the acute regulation of steroidogenesis. Endocrinology 1993; 132:959–967. Stocco DM, King S, Clark BJ: Differential effects of dimethylsulfoxide on steroidogenesis in mouse MA-10 and rat R2C Leydig tumor cells. Endocrinology 1995;136:2993–2999. Clark BJ, Wells J, King SR, Stocco DM: The purification, cloning, and expression of a novel LH-induced mitochondrial protein in MA-10 mouse Leydig tumor cells: Characterization of the steroidogenic acute regulatory protein (StAR). J Biol Chem 1994;269:28314–28322. Sugawara T, Holt JA, Driscoll D, Strauss JF III, Lin D, Miller WL, Patterson D, Clancy KP, Hart IM, Clark BJ, Stocco DM: Human steroidogenic acute regulatory protein: Functional activity in COS-1 cells, tissue-specific expression, and mapping of the gene to 8p11.2 and a pseudogene to chromosome 13. Proc Natl Acad Sci USA 1995;92:4778–4782. Lin D, Sugawara T, Strauss JF III, Clark BJ, Stocco DM, Saenger P, Rogol A, Miller WL: Role of steroidogenic acute regulatory protein in adrenal and gonadal steroidogenesis. Science 1995;267: 1828–1831. Cherradi N, Rossier MF, Vallotton MB, Timberg R, Friedberg I, Orly J, Wang XJ, Stocco DM, Capponi AM: Submitochondrial distribution of three key steroidogenic proteins (steroidogenic acute regulatory protein, p450 side-chain cleavage and 3b-hydroxysteroid dehydrogenase isomerase enzymes) upon stimulation by intracellular calcium in adrenal glomerulosa cells. J Biol Chem 1997; 272:7899–7907. Wang XJ, Liu Z, Eimerl S, Weiss AM, Orly J, Stocco DM: Effect of truncated forms of the steroidogenic acute regulatory (StAR) protein on intramitochondrial cholesterol transfer. Endocrinology 1998;139:3903–3912. Arakane F, Sugawara T, Nishino H, Liu Z, Holt HA, Pain D, Stocco DM, Miller WL, Strauss JF III: Steroidogenic acute regulatory protein (StAR) retains activity in the absence of its mitochondrial import sequence: Implications for the mechanism of StAR action. Proc Natl Acad Sci USA 1996;93: 13731–13736. Arakane F, Kallen CB, Watari H, Foster JA, Sepuri NBV, Pain D, Stayrook SE, Lewis M, Gerton GL, Strauss JF III: The mechanism of action of steroidogenic acute regulatory protein (StAR): StAR acts on the outside of mitochondria to stimulate steroidogenesis. J Biol Chem 1998;273:16339–16345.

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Bose HS, Sugawara T, Strauss JF III, Miller WL: The pathophysiology and genetics of congenital lipoid adrenal hyperplasia. N Engl J Med 1996;335:1870–1878. Bose H, Baldwin MA, Miller WL: Incorrect folding of steroidogenic acute regulatory protein (StAR) in congenital adrenal hyperplasia. Biochemistry 1998;37:9768–9775. Kallen CB, Billheimer JT, Summers SA, Staybrook SE, Lewis M, Strauss JF III: Steroidogenic acute regulatory protein (StAR) is a sterol transfer protein. J Biol Chem 1998;273:26285–26288. Martinez F, Strauss JF III: Regulation of mitochondrial cholesterol metabolism; in Bittman R (ed): Subcellular Biochemistry. Cholesterol: Its Functions and Metabolism in Biology and Medicine. New York, Plenum Press, 1997, vol 28, pp 205–234. Kallen CB, Arakane F, Christenson LK, Watari H, Devoto L, Strauss JF III: Unveiling the mechanism of action and regulation of the steroidogenic acute regulatory protein. Mol Cell Endocrinol 1998;145:39–45. Papadopoulos V: Peripheral-type benzodiazepine/diazepam binding inhibitor receptor: Biological role in steroidogenic cell function. Endocr Rev 1993;14:222–240. Bose HS, Whitall RM, Baldwin MA, Miller WL: The active form of the steroidogenic acute regulatory protein, StAR, appears to be a molten globule. Proc Natl Acad Sci USA 1999;96: 7250–7255. Prader A, Gurtner HP: Das Syndrom des Pseudohermaphroditismus masculinus bei kongenitaler Nebennierenrindenhyperplasie ohne Androgenuberproduktion (adrenaler Pseudohermaphroditismus masculinus). Helv Paediatr Acta 1955;10:397–412. Prader A, Siebenmann RE: Nebenniereninsuffizienz bie kongenitaler Lipoidhyperplasie der Nebennieren. Helv Paediatr Acta 1957;12:569–595. Prader A, Anders CJPA: Zur Genetik der kongenitalen Lipoidhyperplasie der Nebennieren. Helv Paediatr Acta 1962;17:285–289. Miller WL: Congenital lipoid adrenal hyperplasia: The human gene knockout of the steroidogenic acute regulatory protein. J Mol Endocrinol 1997;17:227–240. Hauffa BP, Miller WL, Grumbach MM, Conte FA, Kaplan SL: Congenital adrenal hyperplasia due to deficient cholesterol side-chain cleavage activity (20,22 desmolase) in a patient treated for 18 years. Clin Endocrinol 1985;23:481–493. Miller WL: Molecular biology of steroid hormone synthesis. Endocr Rev 1988;9:295–318. Degenhart HJ, Visser KHA, Boon H, O’Doherty NJD: Evidence for deficiency of 20 alpha cholesterol hydroxylase activity in adrenal tissue of a patient with lipoid adrenal hyperplasia. Acta Endocrinol 1972;71:512–518. Koizumi S, Kyoya S, Miyawaki T, Kidani H, Funabashi T, Nakashima H, Nakanuma Y, Ohta G, Itagaki E, Katagiri M: Cholesterol side-chain cleavage enzyme activity and cytochrome P450 content in adrenal mitochondria of a patient with congenital lipoid adrenal hyperplasia (Prader disease). Clin Chim Acta 1977;77:301–306. Kirkland RT, Kirkland JL, Johnson CM, Horning MG, Librik L, Clayton GW: Congenital lipoid adrenal hyperplasia in an eight-year-old phenotypic female. J Clin Endocrinol Metab 1973;36:488–496. Tanae A, Miki Y, Hibi I: Pubertal presentation in patients with congenital lipoid adrenal hyperplasia (Prader’s syndrome). Acta Paediatr Jpn 1988;30(suppl):236–238. Matsuo N, Tsuzaki S, Anzo M, Ogata T, Sato S: The phenotypic definition of congenital lipoid adrenal hyperplasia: Analysis of the 67 Japanese patients (abstract). Horm Res 1994;41(suppl):106. Matteson KJ, Chung B, Urdea MS, Miller WL: Study of cholesterol side chain cleavage (20,22 desmolase) deficiency causing congenital lipoid adrenal hyperplasia using bovine-sequence P450scc oligodeoxyribonucleotide probes. Endocrinology 1986;118:1296–1305. Lin D, Gitelman SE, Saenger P, Miller WL: Normal genes for the cholesterol side chain cleavage enzyme, P450scc, in congenital lipoid adrenal hyperplasia. J Clin Invest 1991;88:1955–1962. Lin D, Chang YJ, Strauss JF III, Miller WL: The human peripheral benzodiazepine receptor gene: Cloning and characterization of alternative splicing in normal tissues and in a patient with congenital lipoid adrenal hyperplasia. Genomics 1993;18:643–650. Pang S, Yang XH, Wang M, Tissot R, Nino M, Manaligod J, Bullock LP, Mason JI: Inherited congenital adrenal hyperplasia in the rabbit: Absent cholesterol side-chain cleavage cytochrome P450 gene expression. Endocrinology 1992;131:181–186.

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Yang X, Iwamoto K, Wang M, Artwohl J, Mason JI, Pang S: Inherited congenital adrenal hyperplasia in the rabbit is caused by a deletion in the gene encoding cytochrome P450 cholesterol side-chain cleavage enzyme. Endocrinology 1993;132:1977–1982. Saenger P, Konari Z, Black SM, Compagnone N, Mellon SH, Fleischer A, Abrams CAL, Shackleton CHL, Miller WL: Prenatal diagnosis of congenital lipoid adrenal hyperplasia. J Clin Endocrinol Metab 1995;80:200–205. Sakai Y, Yanase T, Okabe Y, Hara T, Waterman M, Takayanagi R, Haji M, Nawata H: No mutation in cytochrome P450 side chain cleavage in a patient with congenital lipoid adrenal hyperplasia. J Clin Endocrinol Metab 1994;79:1198–1201. Fukami M, Sato S, Ogata T, Matsuo N: Lack of mutations in P450scc gene (CYP11A) in six Japanese patients with congenital lipoid adrenal hyperplasia. Clin Pediatr Endocrinol 1995;4:39–46. Okuyama E, Nishi N, Onishi S, Itoh S, Ishii Y, Miyanaka H, Fujita K, Ichikawa Y: A novel splicing junction mutation in the gene for the steroidogenic acute regulatory protein causes congenital lipoid adrenal hyperplasia. J Clin Endocrinol Metab 1997;82:2337–2342. Harikrishna JA, Black SM, Szklarz GD, Miller WL: Construction and function of fusion enzymes of the human cytochrome P450scc system. DNA Cell Biol 1993;12:371–379. Toaff ME, Schleyer H, Strauss JF III: Metabolism of 25-hydroxycholesterol by rat luteal mitochondria and dispersed cells. Endocrinology 1982;111:1785–1790. Tee MK, Lin D, Sugawara T, Holt JA, Guiguen Y, Buckingham B, Strauss JF III, Miller WL: T-A transversion 11 bp from a splice acceptor site in the gene for steroidogenic acute regulatory protein causes congenital lipoid adrenal hyperplasia. Hum Mol Genet 1995;4:2299–2305. Bose HS, Pescovitz OH, Miller WL: Spontaneous feminization in a 46,XX female patient with congenital lipoid adrenal hyperplasia caused by a homozygous frame-shift mutation in the steroidogenic acute regulatory protein. J Clin Endocrinol Metab 1997;82:1511–1515. Fujieda K, Tajima T, Nakae J, Sageshima S, Tachibana K, Suwa S, Sugawara T, Strauss JF III: Spontaneous puberty in 46,XX subjects with congenital lipoid adrenal hyperplasia. J Clin Invest 1997;99:1265–1271. Nakae J, Tajima T, Sugawara T, Arakane F, Hanaki K, Hotsubo T, Igarashi N, Igarashi Y, Ishii T, Koda N, Kondo T, Kohno H, Nakagawa Y, Tachibana K, Takeshima Y, Tsubouchi K, Strauss JF III, Fujieda K: Analysis of the steroidogenic acute regulatory protein (StAR) gene in Japanese patients with congenital lipoid adrenal hyerplasia. Hum Mol Genet 1997;6:571–576. Sugawara T, Lin D, Holt JA, Martin KO, Javitt NB, Miller WL, Strauss JF III: The structure of the human steroidogenic acute regulatory (StAR) protein gene: StAR stimulates mitochondrial cholesterol 27-hydroxylase activity. Biochemistry 1995;34:12506–12512. Miller WL: Steroid hormone biosynthesis and actions in the materno-feto-placental unit. Clin Perinatol 1998;25:799–817. Beitens IZ, Bayard F, Ances IG, Kowarski A, Migeon CJ: The metabolic clearance rate, blood production, interconversion and transplacental passage of cortisol and cortisone in pregnancy near term. Pediatr Res 1973;7:509–519. Kari MA, Raivio KO, Stenman U-H, Voutilainen R: Serum cortisol, dehydroepiandrosterone sulfate, and sterol-binding globulins in preterm neonates: Effects of gestational age and dexamethasone therapy. Pediatr Res 1996;40:319–324. Voutilainen R, Miller WL: Developmental expression of genes for the steroidogenic enzymes P450scc (20,22 desmolase), P450c17 (17alpha-hydroxylase/17,20 lyase) and P450c21 (21-hydroxylase) in the human fetus. J Clin Endocrinol Metab 1986;63:1145–1150. Kiriakidou M, McAllister JM, Sugawara T, Strauss JF III: Expression of steroidogenic acute regulatory protein (StAR) in the human ovary. J Clin Endocrinol Metab 1996;81:4122–4128. Yoo HW: Molecular defect of the steroidogenic acute regulatory protein (StAR) gene in Korean patients with congenital lipoid adrenal hyperplasia (abstr 510). Horm Res 1997;48:S2:107. Caron K, Soo S-C, Wetsel W, Stocco D, Clark B, Parker K: Targeted disruption of the mouse gene encoding steroidogenic acute regulatory protein provides insights into congenital lipoid adrenal hyperplasia. Proc Natl Acad Sci USA 1997;94:11540–11545. Mellon SH: Neurosteroids: Biochemistry, modes of action, and clinical relevance. J Clin Endocrinol Metab 1994;78:1003–1008.

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Watari H, Arakane F, Moog-Lutz C, Callen CB, Tomasetto C, Gerton GL, Rio MC, Baker ME, Strauss JF III: MLN64 contains a domain with homology to the steroidogenic acute regulatory protein (StAR) that stimulates steroidogenesis. Proc Natl Acad Sci USA 1997;94:8462–8467. Luo X, Ikeda Y, Parker KL: A cell-specific nuclear receptor is essential for adrenal and gonadal development and sexual differentiation. Cell 1994;77:481–490. Achermann JC, Ito M, Hindmarsh PC, Jameson JL: A mutation in the gene encoding steroidogenic factor-I causes XY sex reversal and adrenal failure in humans (letter). Nat Genet 1999;22:125–126. Csapo AI, Pulkkinen MO: Indispensibility of the human corpus luteum in the maintenance of early pregnancy: Luteectomy evidence. Obstet Gynecol Surv 1978;83:69–81. Miller WL: Why nobody has P450scc (20,22 desmolase) deficiency (letter). J Clin Endocrinol Metab 1998;83:1399–1400.

Douglas M. Stocco, PhD, Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, TX 79430 (USA) Tel. +1 (806) 743-2505, Fax +1 (806) 743-2990, E-Mail [email protected]

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Hughes IA, Clark AJL (eds): Adrenal Disease in Childhood. Clinical and Molecular Aspects. Endocr Dev. Basel, Karger, 2000, vol 2, pp 63–92

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Biochemistry and Genetics of Human P450c17 Walter L. Miller, Richard J. Auchus Department of Pediatrics and The Metabolic Research Unit, University of California, San Francisco, Calif., USA

P450c17 – The Qualitative Regulator of Steroidogenesis As discussed in the chapter on the steroidogenic acute regulatory protein (StAR), the conversion of cholesterol to pregnenolone is the quantitative regulatory step in steroidogenesis. Although the large variety of steroid hormones made in the adrenal would suggest that no single enzyme could be a qualitative regulator, inspection of figure 1 shows that P450c17 is the qualitative regulator of steroidogenesis, determining which class of steroid will be produced. When P450c17 is absent, such as in the zona glomerolosa, the products are C21 17-deoxysteroids such as aldosterone. When the 17a-hydroxylase activity of P450c17 is present, C21 17-hydroxysteroids such as cortisol are produced. When both the 17a-hydroxylase and 17,20-lyase activities of P450c17 are present, C19 sex steroids are produced. All steroid hormones are made from the pregnenolone produced by P450scc, thus the presence or absence of each of the activities of P450c17 directs this prenenolone toward its final metabolic pathway. While all cytochrome P450 enzymes can catalyze multiple reactions on a single active site, P450c17 is the only one described to date in which these multiple activities are differentially regulated by a physiologic process. Thus, the study of P450c17 is central to the understanding of all P450-catalyzed reactions, not just to the biosynthesis of steroid hormones.

17a-Hydroxylase and 17,20-Lyase – One Enzyme or Two? The isolation and chemical characterization of steroids in the first half of the 20th century showed that there were both 17-deoxy and 17-hydroxy

Fig. 1. Major pathways of human steroidogenesis. Cholesterol is converted to pregnenolone by P450scc and facilitated by the StAR protein, serving as the quantitative regulator of steroidogenesis, while P450c17 serves as the qualitative regulator. Top line: In the absence of P450c17, steroidogenesis is limited to progesterone (e.g. in human placenta), corticosterone (e.g. in rat adrenal zona fasciculata), and aldosterone (adrenal zona glomerulosa). Middle line: When the 17a-hydroxylase activity, but not the 17,20 lyase activity of P450c17 is present in the human adrenal zona fasciculata, cortisol is produced. Bottom line: When both the 17a-hydroxylase and 17,20 lyase activities of P450c17 are present 19-carbon precursors of sex steroids are produced. Human P450c17 catalyzes the 17a-hydroxylation of the D5 steroid pregnenolone, and of the D4 steroid progesterone, equally well, but the 17,20 lyase activity exhibits a 100:1 preference for 17OH pregnenolone over 17OH progesterone. Thus, essentially all human sex steroids are made from DHEA.

C21 steroids, indicating that one or more enzymes had to exist that catalyzed steroid 17a-hydroxylation. Similarly, it was clear that C19 androgenic steroids were derived from C21 steroids indicating that a 17,20-lyase enzyme had to exist. The 1950s brought an early knowledge of genetics and the one geneone enzyme hypothesis. Because everyone thought each enzyme catalyzed only one activity, and even named the enzymes for these activities, it was generally assumed that the 17a-hydroxylase and 17,20-lyase had to be two distinct enzymes encoded by two distinct genes.

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Fig. 2. Human serum concentrations of DHEA-sulfate (DHEAS) as a function of age. The fetal adrenal makes large quantities of DHEAS, which are evident at birth. As the fetal zone of the adrenal involutes, DHEAS concentrations plummet in infancy and remain low until the onset of adrenarche at about age 8. DHEAS concentrations reach maximal values around age 25 then decline slowly with advancing age, returning to childhood levels in the elderly (adrenopause). Note that the abcissa is shown on a logarithmic scale. Data redrawn from Orentreich et al. [3].

Clinical observations in children were consistent with this two-enzyme model. The adrenals of young children make virtually no DHEA or other C19 steroids until the onset of adrenarche and the development of the adrenal zona reticularis (fig. 2). Adrenarche begins at about age 8 to 10, is independent of the gonads or gonadotropins and continues until after the end of puberty, as serum DHEA and DHEA-S concentrations are highest between ages 25–30 years [1–3]. This profound increase in adrenal DHEA synthesis proceeds without changes in the synthesis of cortisol or the secretion of ACTH, and thus appears to involve a selective turning-on of 17,20-lyase activity, while 17a-hydroxylase activity remains essentially unchanged. Consistent with the two enzyme view, in 1972 Zachmann et al. [4] described a 46,XY male pseudohermaphrodite who had normal urinary excretion of 17-hydroxycorticosteroids (17OHCS), increased urinary pregnanetriol (the metabolite of 17a-hydroxyprogesterone, 17OHP), no response of urinary testosterone to gonadal stimulation with hCG, and a normal response of urinary 17OHCS to ACTH. Incubation of the patient’s biopsied testicular tissue with DHEA or D4-androstenedione produced testosterone, but incubation of tissue with pregnenolone, progesterone, 17a-hydroxypregnenolone and 17OHP produced no testosterone. Thus the patient was identified as having ‘Steroid 17,20desmolase deficiency: a new cause of male pseudohermaphroditism’. It was

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concluded that 17a-hydroxylase and 17,20-lyase were two distinct proteins, and this view became the accepted dogma, as ‘confirmed’ by additional reports of isolated 17,20-lyase deficiency [5–11]. However, between 1981 and 1983, Nakajin and Hall [12–15] and others [16, 17] reported the heretical observation that the 17a-hydroxylase and 17,20-lyase activities of neonatal procine testis were catalyzed by a single enzyme, but the biochemistry and endocrinology communities disregarded this work. The addition of cytochrome b5 to these purified prepartations dramatically increased only the 17,20-lyase activity [18], and protein sequencing data suggested that adrenal and testicular P450c17 were distinct proteins [19]. The cloning of bovine [20] and human [21] P450c17 cDNAs and the single human P450c17 gene [22] led to the definitive confirmation of Hall’s radical view. Zuber et al. [20] reported the cloning of bovine P450c17 reverse transcribed from an enriched fraction of mRNA that had been size-selected by sucrose gradient centrifugation. Chung et al. [21] first purified and micro-sequenced porcine P450c17, obtained a small porcine cDNA, and used this to clone human adrenal and testicular P450c17 cDNA. Zuber et al. [23] then expressed the bovine cDNA in transfected COS-1 cells, showing that this single protein conferred both 17a-hydroxylase and 17,20lyase activities to these nonsteroidogenic cells, proving that a single protein catalyzed both activities. Chung et al. [21] cloned identical cDNAs from the human adrenal and testis, proving that a single form of P450c17 was responsible for the varied ratios of 17a-hydroxylase and 17,20-lyase activities seen in the various zones of the adrenal and in both the ovary and testis. Thus, the genetic data proved that there was only one P450c17 enzyme that catalyzed both activities in all steroidogenic tissues, but it was unclear how the ratio of these activities could be different in different cell types or at different times in development.

Mechanism of Catalysis P450c17 is a typical type II P450 enzyme located in the endoplasmic reticulum, as distinct from the type I P450 enzymes found in mitochondria and bacteria. To catalyze each activity, P450c17 must receive two electrons from NADPH via the flavoprotein P450 oxidoreductase (OR) during its catalytic cycle [24, 25]. Like all microsomal P450s, P450c17 receives these electrons in two discrete one-electron steps, with substrate and molecular oxygen binding between these transfers of electrons (fig. 3). By receiving these electrons, P450c17 can catalyze both the 17a-hydroxylation and 17,20-lyase activity with both D5 steroids (pregnenolone, 17OH pregnenolone) and D4 steroids (progesterone, 17OH progesterone). However, the relative efficiencies of these

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Fig. 3. The P450 catalytic cycle. The cycle begins when substrate displaces a water molecule bound to the heme iron. Substrate binding lowers the redox potential of the heme sufficiently to allow its reduction, first by the transfer of one electron from OR, thus lowering the oxidation state of the heme iron from the ferric (Fe3+) to the ferrous (Fe2+) state. Dioxygen binds to the Fe2+ center, followed by two protons. The transfer of the second electron generates water and the active oxygenating species, probably a ferryl (Fe4+) oxene. A ferryl oxene mechanism is generally accepted for the 17a-hydroxylase reaction, and we propose a similar mechanism for the 17,20 lyase reaction (see fig. 7). Unproductive cycles generate reduced forms of O2 such as or H2O2 or superoxide (OÖ 2 ), and release unreacted substrate. The rearrangements and reactions that follow the transfer of the second electron are vary rapid, allowing little or no time for redox partners to dissociate prior to catalysis.

four reactions varies tremendously, depending on the nature of the available electron-donating redox partners and on amino acid sequence differences in the P450c17 from different mammalian species. Although the catalytic cycle of P450c17 uses the same initial steps whether pregnenolone, progesterone, or their 17a-hydroxylated derivatives are substrates, P450c17 catalyzes at least three fundamentally distinct chemical transformations. First, the 17a-hydroxylase reaction is a typical P450-mediated hydroxylation, believed to proceed via an ‘oxygen rebound’ mechanism involving a ferryl oxene as the active oxygenating species [26]. Human P450c17 catalyzes the 17a-hydroxylation of D5 pregnenolone and of D4 progesterone with very similar efficiencies [27]. Human P450c17 also 16a-hydroxylates progesterone, DHEA, and possibly other steroids, presumably by the same mechanism [28, 29]. Second, human P450c17 converts 17a-hydroxysteroids to C19 steroids by the 17,20-lyase reaction, in which a carbon-carbon bond is oxida-

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tively cleaved to yield the ketosteroid and acetic acid. Human P450c17 catalyzes the 17,20-lyase reaction less efficiently than the 17a-hydroxylase reaction, and more importantly, the 17,20-lyase reaction is nearly two orders of magnitude more efficient with D5 17OH pregnenolone than with D4 17OH progesterone [27]. This discrepancy results from the combination of a 10-fold higher Km and a 10-fold slower Vmax for 17OH progesterone compared to 17OH pregnenolone [27, 30]. Kinetic constants obtained for the four principal reactions using His-tagged modified P450c17 purified from Escherichia coli [30] and native microsomal P450c17 expressed in yeast [27] are remarkably consistent, confirming differences in both affinity and turnover rates for the 17,20-lyase substrates. Thus, although rat P450c17 can convert 17-hydroxyprogesterone to D4 androstenedione, this reaction is nearly nonexistent in human tissues. Third, in the presence of b5 an alternate pathway of pregnenolone metabolism forms androst-5,16-diene-3b-ol via another carbon-carbon bond cleavage reaction [30]. In pigs, this third pathway forms an andiene pheromone of some biological importance [31], the role of the corresponding D5,16-diene in human beings is not known. Finally, small amounts of other products such as 17ahydroxy, C19 steroids are formed by P450c17 in some species, but these activities for the human enzyme are not well characterized.

Factors Regulating 17,20-Lyase Activity: Redox Partners The proof that 17a-hydroxylase and 17,20-lyase activities were catalyzed by the same enzyme shifted attention to the mechanism of catalysis of P450c17, and especially the roles of the electron transfer proteins OR and b5, collectively referred to as ‘redox partners’. Hall and colleagues showed that increasing the ratio of (rat) OR to (porcine) P450c17 increased the 17,20-lyase activity in a reconstitution assay using purified, detergent-solubilized proteins [32]. Consistent with this, Hall also found a higher molar ratio of OR to P450c17 in bovine testis, where virtually all steroids undergo both 17a-hydroxylation and 17,20 bond scission, but there was a lower ratio of OR to P450c17 in bovine adrenal, which primarily produces 17-hydroxy, C21 steroids [32]. Similarly, increasing the ratio of OR to human P450c17 in transfected COS-1 cells increases the ratio of 17,20 lyase to 17a-hydroxylase activity [28]. Cytochrome b5 has been proposed as an alternate to OR for electron transfer to P450c17, particularly for the 17,20-lyase reaction. Addition of purified rabbit liver b5 to porcine P450c17 with rat OR increased the 17,20lyase activity [18], and both rat [33] and pig [30] liver b5 augment the 17,20lyase activity of recombinant, modified human P450c17. We showed that the stimulatory effect of b5 on the 17,20-lyase activity of wild-type P450c17 was

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maintained when the human forms of P450c17 and OR were coexpressed in yeast microsomes with and without human b5 [27, 34], demonstrating that the influence of b5 is not limited to reconstituted assay systems. The requirement of b5 for maximal 17,20-lyase activity requires at least a stoichiometric amount of b5, but very high molar ratios inhibit catalytic activity, probably by competing with P450c17 for electron transfer from OR [27]. In fact, as the abundance of OR is reduced, the inhibitory effect of b5 occurs at lower molar ratios of b5 to P450c17 and eventually obscures any stimulatory effect (fig. 4). Indeed, apo-b5, which lacks a heme and therefore cannot participate in electron transfer reactions, stimulates 17,20-lyase activity just as well as holo-b5, but does not inhibit at higher molar ratios, providing additional evidence that b5 acts not as an electron donor to P450c17 directly but as an allosteric facilitator of the P450c17 • OR catalytic complex [27]. This effect of b5 on 17,20-lyase activity was not seen in transfected COS-1 cells [28], presumably because b5 is already expressed in these cells at quantities sufficient to stimulate 17,20-lyase activity. In contrast, results in both transfected COS-1 cells [34] and yeast microsomes [27] demonstrate that raising the amount of human OR increased both 17ahydroxylase and 17,20-lyase activities, but a differential effect on these two activities has been difficult to demonstrate in these systems.

Factors Regulating 17,20-Lyase Activity: Serine Phosphorylation In searching for possible mechanisms by which a cell might differentially regulate the 17a-hydroxylase and 17,20-lyase activities of P450c17, we considered the possibility that P450c17 might undergo post-translational modification. Radiolabeling studies of COS-1 cells transfected with a human P450c17 expression vector or of untransfected human adrenal NCI-H295 cells, which express all of the human steroidogenic enzymes [35] showed that human P450c17 is a phosphoprotein [36]. This phosphorylation is rapidly induced by cAMP, occurs on serine and threonine but not on tyrosine residues, and increases the 17,20-lyase activity of P450c17 [36]. Conversely, dephosphorylation of microsomes containing P450c17 ablates 17,20-lyase activity without reducing 17a-hydroxylase activity, and without affecting steroid binding to the enzyme [36]. The mechanism by which serine phosphorylation promotes 17,20-lyase activity is not known, but the phenomenology of this observation is quite similar to that described above in the redox partner experiments. Serine phosphorylation of P450c17 probably increases its affinity for OR or b5, or both, thus effectively increasing the flow of electrons to the P450. In this fashion, all of the observed data are consistent with a single mechanism for regulating 17,20-lyase activity through a cAMP-regulated serine/threonine

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Fig. 4. Cytochrome b5 exerts an allosteric effect to stimulate the 17,20 lyase activity of human P450c17. Activity was examined in microsomes prepared from human adrenal tissue (triangles) or from yeast expressing human P450c17 and coexpressing high (squares), low (circles) or very low (diamond) amounts of human OR. The high, low, and very low OR contents are 223, 33 and 12 nmol of cytochrome c reduced · minÖ1 · mg proteinÖ1, respectively. Top: Stimulation with holo-b5 maximizes lyase activity at a b5:P450c17 molar ratio of 30:1, after which further increases in b5 inhibit the reaction. However, when OR content is low the inhibitory phase begins at lower b5:P450c17 ratios, and when OR is nearly absent, only the inhibitory action of b5 is observed. Middle: by contrast, apo-b5 stimulates 17,20 lyase activity at all concentrations, and shows no inhibitory phase, whereas coincubation with cytochrome c produces only an inhibitory phase (bottom). Thus, the action of b5 to stimulate 17,20 lyase activity is unrelated to its ability to transfer electrons, and in fact the electrontransfer capacity of small cytochromes inhibits lyase activity. Data from Auchus et al. [27].

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Fig. 5. Regulation of 17,20-lyase activity by the interaction of P450c17 with its redox partners. The FAD moiety of P450 oxidoreductase (OR) picks up electrons from NADPH (not shown) and transfers them to its FMN moiety. The FAD and FMN groups are in distinct protein domains connected by a flexible protein hinge (H). The FMN domain approaches the redox partner binding site of the P450, shown as a concave region. The active site containing the steroid lies on the side of the plane of the heme ring (Fe) opposite from the redox partner binding site. Cytochrome b5 allosterically facilitates the interaction between OR and P450c17 to favor 17,20-lyase activity. Phosphoserine residues (P) also promote 17,20lyase activity, probably by fostering the interaction of P450c17 with OR or b5 or both.

kinase that phosphorylates P450c17 which in turn increases the efficiency with which it can receive electrons from the existing pool of electron donors (fig. 5).

Modeling of Human P450c17 To understand the basis for its remarkable substrate specificity and catalytic versatility, we sought to understand the tertiary structure of human P450c17. Unfortunately, all efforts to crystallize P450c17, even when it has been modified and rendered soluble in E. coli, have been unsuccessful. Thus, substantial work has been directed at building computer-graphic models of eukaryotic P450 enzymes based on the crystal structures of several prokaryotic

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P450s, notably P450cam [37], P450terp [38] and P450BMP [39]. This has led to highly successful models of P450arom [40] and P450 2B4 [41]. In the case of P450c17, three groups reported similar models based on P450cam between 1990 and 1997, all of which featured a bi-lobed substrate binding pocket in which the steroid was presumed to have the D-ring and C-17 carbon over the heme with A-ring in one lobe of the pocket for the hydroxylase reaction, then moving the A-ring to the other lobe for the 17,20 lyase reaction [42–44]. Because models are only computational predictions, they must be subjected to very stringent testing before they can be believed. Only one group tested this model by site-directed mutagenesis, and few of the model’s predictions were confirmed experimentally [43]. More recent modeling efforts based on the x-ray crystal structure of P450BMP have proven more robust [45, 46]. Our model uses a combination of the P450BMP-template, amino acid alignment procedures based on regions of predicted secondary structure rather than on computationally predicted amino acid sequence similarities, and makes extensive use of molecular dynamics simulations at 300 ºK using a Cray 3TE supercomputer [46, 47]; the resulting final hydrated model has a total free energy of –1.8¶104 kcal/mol, which is very similar to the free energy of the P450BMP crystal structure itself (–1.6¶104 kcal/ mol) [46], and is an order of magnitude more stable than the apparently next-best model [45]. Furthermore, the model has ‘passed’ a wide variety of computational tests of its bond angles, energies and distances, and its coordinates have been accepted by the structural database (RCSB 001146) [46]. Unlike the previous models, our current model accommodates only a single orientation for all of the steroid substrates and reactions (fig. 6). This result suggests that the two principal P450c17 reactions proceed not via grossly different substrate orientations and chemistries, but rather via similar mechanisms; we have proposed that both the hydroxylase and lyase reactions proceed through a ferryl oxene mechanism [46]. Isotopic labeling studies are consistent with mechanisms involving the nucleophilic attack of a ferryl peroxide on the C-20 carbonyl of the hydroxysteroid, forming a covalent enzyme-steroid intermediate whose decomposition yields the observed products [48]. However, these studies do not exlude mechanisms that involve the same ferryl oxene used in the hydroxylase reactions, in which a steroid hydroxyl radical at C-17 fragments directly without a covalent intermediate (fig. 7). Indeed, recent experiments demonstrate that little free H2O2 is formed during catalysis [49] and that H2O2 itself cannot reconstitute 17,20-lyase activity [46], findings that cast doubt on the ferryl peroxide mechanisms. Finally, in addition to being energetically favorable and yielding excellent scores in various model evaluation programs, our current model also accurately predicts the activities of all known natural and synthetic site-directed mutants of human P450c17.

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6a

6b

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6c Fig. 6. Positioning of steroid substrates in the active site of P450c17. Each panel shows ˚ ellipse of the a sagittal section of a small portion of the protein molecule within a 10 A steroid substrates; most of the protein atoms in front of and behind the steroid are not shown to clarify the location of the steroid (shown in black) above the iron atom of the heme group. a Pregnenolone sits in the active site with the hydrogen on carbon 17 (H17) above the ferryl oxene (OFe) of the heme ring. The steroid is positioned with the aid of a hydrogen bond between its C-3 hydroxyl hydrogen (HO3) and the carbonyl oxygen of glycine 95 [O(Gly95)]. b The hydrogen bond between the C-3 hydroxyl hydrogen and glycine 95 is also seen with 17a-hydroxypregnenolone in the active site; in this case, the 17a-hydroxyl hydrogen (HO17) is the overwhelmingly favored site of reactivity. c When the D4 steroid progesterone is the substrate, a hydrogen bond forms between its carbonyl oxygen (O3) and the amide hydrogen of glycine 95 [HN(Gly95)]. 17a-Hydroxyprogesterone is a very poor substrate, and is not shown. Images were generated using the neon option of the MidasPlus graphics program from our human P450c17 model structure (http://www.rcsb.org; PDB ID number 2c17).

Few efforts to develop pharmacological inhibitors of human P450c17 have been reported [50]. Nonspecific inhibitors of human P450s, such as aminoglutethimide and ketoconazole, have been used clinically, but there are no FDA-approved selective inhibitors of P450c17 as is the case for aromatase [51]. It was thought that medroxyprogesterone acetate (MPA) and megestrol

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Fig. 7. Proposed ferryl oxene mechanism for the 17,20 lyase reaction. The ferryl oxene abstracts the hydrogen from the hydroxyl on carbon 17 of 17OH-pregnenolone (I), generating a C-17 hydroxyl radical (O•) and a heme (ferric) hydroxyl (II). This unstable radical spontaneously degrades into DHEA (III) and an acetyl radical (H3C-C•>O), while the ferric hydroxyl loses its hydroxyl group (IV). Reaction with the heme hydroxyl yields acetic acid (V), returning the heme to its resting state (VI), where it then binds a water molecule (not shown).

acetate (MEG) both inhibit human P450c17 because MPA and MEG bind with high affinity to rat testis P450s [52], but MPA minimally inhibits human P450c17 at concentrations up to 100 lM [53], and MEG has no effect on P450c17 activity at 100 lM [unpubl. obs.]. It was reported that human P450c17 metabolized dexamethasone [54], but this metabolism is probably performed by a renal P450 enzyme. Using recombinant human P450c17, we recently showed that dexamethasone is not a substrate and is only a very weak competitive inhibitor of human P450c17 (Ki>87 lM ), a property which cannot account for any known therapeutic effect of dexamethasone [53].

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Physiology P450c17 is the qualitative regulator of steroidogenesis because its activities determine which class of steroid is made in each cell type (fig. 1). In the human adrenal zona glomerulosa, human placenta, and rat adrenal, where P450c17 is absent [55], only 17-deoxy, C21 steroids such as progesterone, corticosterone, and mineralcorticoids can be made. These cells reflect the physiology of 17ahydroxylase deficiency, where patients have mineralcorticoid-induced hypertension and sexual infantilism. In the human adrenal zona fasciculata, the 17ahydroxylase activity of P450c17 permits the biosynthesis of cortisol, the principal human glucocorticoid. In the absence of 17a-hydroxylase activity patients do not have glucocorticoid deficiency because in this case corticosterone provides this activity, just as it does in rodents, whose adrenal glands do not express P450c17 [55]. In the adrenal zona reticularis and in the gonads, the high ratio of 17,20-lyase to 17a-hydroxylase activities results in the synthesis of C19 precursors of sex steroids. DHEA is not an ‘adrenal androgen’ as it does not bind to and activate the androgen receptor, but it can undergo peripheral conversion to testosterone in extraglandular tissues. Thus, the types of steroids made by a given steroidogenic cell or tissue are determined by the activities of P450c17 present; by contrast, the total quantity of steroids is mainly modulated by StAR (acutely) and P450scc (chronically) [56–58]. As it is clear that P450c17 catalyzes both 17a-hydroxylase and 17,20 lyase activities, the overwhelming predominance of the hydroxylase activity in the zona fasciculata presents a biochemical conundrum: How does the cell stop the reaction after the hydroxylase activity? This conundrum is further accentuated when one considers the physiological evidence that the ratio of lyase to hydroxylase activity not only differs among cell types, but can be developmentally regulated within a single cell type. That cell type is the adrenal zona fasciculata/ reticularis, and the regulation is evidenced in the phenomena of adrenarche and adrenopause (fig. 2). Adrenarche is the rise in the adrenal secretion of DHEA and DHEAS that begins at about age 8 and reaches maximal levels at age 25–30 when the secretion of these C19 steroids exceeds that of cortisol. As adult life progresses their secretion slowly decreases, reaching early childhood levels in persons over 70 (adrenopause) [3]. Meanwhile, the secretion of cortisol and ACTH (adjusted for body surface area) changes little with age. Adrenarche is a phenomenon unique to human beings, chimpanzees, and possibly some other, large old world monkeys [59–61]; hence, no convenient animal model of adrenarche is available. Adrenarche is independent of puberty, gonads, and gonadotropins [2], and its timing appears to correlate roughly with the expansion of the adrenal zona reticularis [62] and with the rise in serum concentrations of

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IGF-I. However, the cellular and biochemical events responsible for adrenarche remain unclear. Our studies of the regulation of 17,20-lyase activity and the work of others in the cell biology of the zona reticularis have begun to elucidate the probable mechanisms of adrenarche [63]. In addition to the role of redox partners in regulating 17,20-lyase activity [27, 28, 64, 65], serine/threonine phosphorylation of P450c17 preferentially increases 17,20-lyase activity [36]. A developmentally programmed rise in P450c17 phosphorylation could contribute to adrenarche. Furthermore, b5 appears to be more abundant in the zona reticularis [66, 67], although its abundance relative to P450c17 itself is not known. A 30:1 molar ratio of b5 to P450c17 maximizes 17,20-lyase activity [27, 34]. If the preadrenarchal adrenal gland has low molar ratios of b5 to P450c17, and the adrenarchal reticularis achieves high ratios, then b5 abundance could be a defining feature of adrenarche [63]. Region-specific expression of b5, combined with low abundance of 3b-hydroxysteroid dehydrogenase type II (3bHSDII) in the zona reticularis [62, 68, 69] would drive steroids down the D5 pathway to DHEA [63]. Substantially less work has been directed towards the basis of adrenopause. It seems likely that some of the factors that drive adrenarche would reverse themselves during adrenopause, but there are no direct experimental data on the cell biology of the adrenal in aging. Furthermore, the physiologic role of adrenarche, adrenopause, and DHEA are still debated, but accumulating evidence suggests that DHEA and DHEAS are important for developing neuronal connections in the fetal brain [70] and numerous studies indicate that DHEA supplementation in the elderly can have salutory effects on immune function, memory, and sense of well-being [71, 72]. Nearly 80% of testosterone in women derives from peripheral conversion of DHEA(S) [73], suggesting that adrenal DHEA is an important precursor of anabolic steroids in normal women.

Genetics Human P450c17 is encoded by a single gene, formally termed CYP17, that is located on chromosome 10q24.3 [74–76]. This gene encodes a single species of mRNA that is expressed in the adrenals and gonads (testicular Leydig and ovarian theca cells) [21] but (unlike the rodent) not in the placenta or ovarian granulosa cells [55]. The 6.5-kb gene has been sequenced in its entirety, showing it is divided into 8 exons having an organization that is very similar to the gene for P450c21 (steroid 21-hydroxylase) [22]. Amino acid sequence alignments and computational predictions of secondary structure

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Fig. 8. Deletions and insertions causing major disruptions of P450c17. The bar indicates the normal 508 amino acid protein with certain functionally important regions indicated. The terms ‘del’ and ‘dup’ refer to nucleotide deletions and duplications, with the corresponding nucleotide numbers from the cDNA sequence. The open box designates a D0.5-kb DNA substitution mutation [78]. All these mutations cause complete absence of all 17a-hydroxylase/ 17,20 lyase enzymatic activity.

also suggest that the P450c17 and P450c21 proteins have very similar folding and 3-dimensional conformation, despite sharing only 28.9% amino acid sequence identity [21, 22, 43, 46, 77]. In addition to the detailed genetic knowledge of the gene for P450c17, advanced computer-graphic modelling of the P450c17 protein [46] based on the x-ray crystal structure of P450-BMP [39] have permitted us to analyze the disease-causing mutations of P450c17 at genetic, protein-structural, and enzymologic levels.

Deletions, Premature Truncations, Frameshifts and Splicing Errors Several deletions encompassing part, but not all of the gene for P450c17 have been described (fig. 8). The largest deletion reported involves the substitution of 518 bp (comprising most of exon 2 and part of exon 3) with 469 bp

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of unknown DNA, thus disrupting the protein near its very beginning and causing complete 17a-hydroxylase deficiency [78]. The first P450c17 mutation reported, and possibly the most common, is the duplication of the sequence CATC following Ile479 [79]. This 4-bp duplication, which was originally observed in Canadian Menonites of Frisian ancestry [80] and has been subsequently found in at least 6 Dutch Frieslander families [81], leaves 95% of the protein unaffected (including the heme-binding region) and creates a P450c17 mutant whose sequence is altered only in its last 25 residues and is truncated 3 residues prematurely, yet is wholly devoid of enzymatic activity. The crucial nature of the carboxy-terminus of P450c17 is also shown by the complete absence of activity in the 9-bp in-frame deletion of residues Asp487, Ser488 and Phe489 in a patient from Thailand [82]. Because the amino acid sequences of the extreme carboxy end of P450c17 and P450c21 are wholly different, these genetic and enzymologic studies did not tell why this region of the protein is so important or what function it serves. However, our model of human P450c17 suggests why the enzyme is so sensitive to alterations in its carboxy-terminus [46]. After the most carboxy-terminal helix (the L-helix) found in the structures of all P450 enzymes examined [83], the polypeptide chain completes the final two pairs of strands of two b-sheets before returning to the protein surface and terminating. In forming these b-sheet strands, these last residues fold down from the protein surface into the protein core to a point above the heme, forming the ‘top’ of the substrate-binding pocket before turning back on its itself and exiting the protein core [46]. Thus, the last 48 residues of P450c17 are involved in an extended b-sheet structure that forms a scaffold for a region of the protein critical for proper substrate binding and subsequent catalysis. Thus, the Frisian mutation altering the reading frame after Ile479 [79], the Thai mutation deleting residues 487 to 489 [82], and the mutant Gln461Kstop [84], which all retain the heme-binding site, disrupt or lack this critical stretch of residues required for activity. Mutation delTG300,301 changes the reading frame and alters the codon usage beginning within exon 5 [85], and mutation 7bp dup 120 interferes with the reading frame from exon 2 onward [86]. The mutation Trp 17Kstop was found in a homozygous patient [87] and in a heterozygous patient [88]. Both mutations Glu194Kstop and Arg239Kstop represent separate alleles in a single patient with complete 17a-hydroxylase deficiency [89]. These 3 early truncations delete the heme-binding region as well as residues important for substrate and redox partner binding; hence, these mutations are not informative for structure/function studies. In the compound heterozygote bearing the Trp17Kstop mutation, the other allele was a G to T substitution at nucleotide +5 in intron 2 [88]. This mutation disrupted a splice donor site, and exon 2 of this allele is deleted

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during RNA processing (‘exon skipping’), which was demonstrated by RTPCR of testicular RNA [88]. The loss of exon 2 causes a frameshift after amino acid 99 with premature termination at codon 175, and complete loss of activity. An analogous G to A substitution at position +5 of intron 7 in a homozygous patient results in the excision of exon 7 during RNA processing, as proven by mingene expression studies in transfected COS-1 cells [90]. The excision of exon 7 alters the reading frame of exon 8, which changes residues 381 onward and causes premature termination at codon 410, upstream from the heme-binding region that is required for activity. Another patient was homozygous for the deletion of a G in codon 438 [91]. The mutant gene encodes a protein with the Gly-Pro-Arg-Ser-Cys-Ile motif at residues 438–443 in the heme binding region converted to Asp-Leu-Ala-Pro-Val-Stop, which destroys all enzymatic activity. Lastly, an extreme example of premature termination occurs in the ATGKATC substitution that eliminates the initiating methionine codon at position 1 of a patient with complete 17a-hydroxylase deficiency [92].

Amino Acid Substitutions – Combined 17a-Hydroxylase/ 17,20-Lyase Deficiency Amino acid substitution mutations are ‘site directed mutagenesis experiments of nature’, that provide some of the clearest insights into the structure/ function relationships of P450c17 (fig. 9). Two reported substitution mutations appear to involve defective heme binding. The mutation His373Leu lacks all activity, and, when expressed in E. coli, lacks the classical P450 difference spectrum, providing excellent evidence that the protein does not bind heme [93]. However, computer modeling studies predict that His373 does not lie near the heme itself [46]. It is quite possible that mutation His373Leu introduces a substantial structural change elsewhere in the protein that subsequently abolishes heme binding. The mutation Arg440His [94] lies in the heme-binding region two residues away from Cys442, which donates the axial sulfhydryl group to the heme iron. By analogy with structures of bacterial P450 enzymes, the positively charged guanidinium group on this Arg should neutralize the negative charge on one of the two propionic acid groups of the heme moiety, so that substitutions of this arginine should impair heme binding. The analogous mutation of Arg435 in the aromatase enzyme (P450arom) causes severe aromatase deficiency as well [95]. The homozygous mutation Ser106Pro has been found in two apparently unrelated patients from Guam [96]. This mutation introduces a helix-breaking proline into the B€-helix, 6 residues removed from Ile112, which forms a

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Fig. 9. Mis-sense mutations and in-frame mutations of P450c17. Amino acid deletions are designated D, and insertions are designated ins, followed by the three-letter amino acid designation and number. Other symbols as in figure 8.

lateral boundary of the substrate-binding pocket [46]. Hence, P450c17 is quite sensitive to perturbations in this region. In fact, the more conservative replacement of Ser106 with Thr, the corresponding residue found in rainbow trout P450c17 [97], also abolishes most enzymatic activity [28]. Slightly further to the amino terminus are Gly90 and Arg96, which are altered in the mutants Gly90Asp [98] and Arg96Trp [99]. These residues flank strand 2 of b-sheet 1, and Gly95, found in this b-strand, participates directly in substrate binding. During molecular dynamics simulations of D5 and D4 substrates bound to our model of P450c17, the 3b-hydroxyl and 3-keto groups of these substrates, respectively, form hydrogen bonds to the carbonyl group (3b-hydroxy) or the amide hydrogen (3-keto) of Gly95 (fig. 3) [46]. Therefore, it is likely that the 3 mutants Ser106Pro, Arg96Trp and Gly90Asp all primarily impair substrate binding. Two additional mutations cause partial loss of both 17a-hydroxylase and 17,20-lyase activities. A 46, XY patient with ambiguous genitalia was a compound heterozygote for the mutations Tyr64Ser and insIle112 [100]. Whereas the insIle112 mutation is devoid of measurable activity, the Tyr64Ser mutation

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retains about 15% of wild-type 17a-hydroxylase and 17,20-lyase activities [100]. A second, less severe mutation, Pro342Thr, has been described in a 46, XY patient with the premature truncation Arg 239Kstop at the other allele. This patient had ambiguous genitalia despite about 20% residual activity at this allele [101]. The structural consequences of mutations Tyr64Ser and Pro342Thr are more subtle than the more severe perturbations described for the mutations causing complete 17a-hydroxylase deficiency. Tyrosine 64 lies in b-sheet 1, which is involved in substrate binding and membrane attachment. Since serine is considerably less hyrdrophobic than tyrosine, this substitution might interfere with attachment and/or hydrophobic packing interactions. Proline 342 starts the J€ helix, a section of the protein that participates in redox partner interactions [39]. Because proline residues can adopt a range of geometries unique among the amino acids, any substitution involving proline can be catastrophic. In the case of Pro342Thr, however, it appears that the substituted threonine can accommodate a reasonably similar geometry to compensate for the loss of proline. Two other in-frame mutations that wholly ablate 17a-hydroxylase activity merely add or delete a single residue. The mutant insIle112, in which the ATC codon is repeated in-frame, is completely inactive [100]. Although Ile112 is quite distant from the C-terminal b-sheet region in linear sequence, Ile112 is predicted to form a different portion (a lateral edge) of the substrate-binding pocket. Consequently, the insertion of an extra residue at this site disrupts the dimensions of the substrate-binding pocket. By contrast, the mutant DPhe53/54 excises one of the two adjacent phenylalanine residues at positions 53 and 54 [102]. These residues line the hyrdrophobic cleft between the b-sheet and the a-helical domains of P450c17 and may comprise part of the substrate access channel; the absence of one of these Phe residues eliminates most, but not all 17a-hydroxylase and 17,20-lyase activities [103]. Consequently, this DPhe53/54 mutation, is associated with a milder clinical phenotype. This mutation has been described in four apparently unrelated Japanese patients [104] suggesting it is due to a genetic founder effect.

Mutations Causing Isolated 17,20-Lyase Deficiency Several cases of putative isolated 17,20-lyase deficiency were reported on clinical grounds in the 1970s and 1980s [4–11]. The proof that 17,20-lyase activity was catalyzed only by P450c17 led to studies of P450c17 in such patients. However, when the P450c17 of one of these initial patients [8] was studied, the results suggested that isolated 17,20-lyase deficiency could not

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exist [84]. One of the patient’s two alleles contained the Gln461Kstop mutation, and the other contained the substitution Arg496Cys [84]. Although this child was initially reported to have ‘isolated 17,20-lyase deficiency’ [8], the diagnosis was revised to ‘partial, combined 17a-hydroxylase/17,20-lyase deficiency’ when the patient was restudied as an adult [105]. When the mutant proteins were studied in transfected cells, the Gln461Kstop mutant was inactive as predicted, while the Arg496Cys mutant retained =10% of both 17a-hydroxylase and 17,20-lyase activities [84]. Although Arg496 is only 13 residues from the carboxy-terminus, the Arg496Cys mutation eliminates over 90% of enzymatic activity. Arg496 appears to be the last residue in the tandem-b-sheet tongue that forms the roof of the substrate-binding pocket [46], so that its mutation results in a substantial but incomplete loss of activity. This patient was the first patient with presumed isolated 17,20-lyase deficiency to be studied at a molecular genetic level, which showed that the patient could not have an isolated deficiency of 17,20-lyase activity. These sobering results raised the question of whether isolated 17,20-lyase deficiency truly existed. Dr. Berenice Medonc¸a studied the steroid secretion of two 46,XY patients from rural Bahia, Brazil who had clinical and laboratory evidence of isolated 17,20-lyase deficiency. These patients, from two unrelated but consanguineous families, had genital ambiguity, normal 17OHCS excretion rates, but markedly reduced C19 steroid production. Each patient was homozygous for an amino acid substitution, either Arg347His or Arg358Gln (fig. 10) [106]. When studied in transfected cells, these mutants retained the capacity to hydroxylate pregnenolone and progesterone, but neither mutant could convert 17OHpregnenolone to DHEA unless an excess of both OR and b5 was supplied by co-transfection [34]. When studied in yeast microsomes, we found a trace of 17,20-lyase activity, and the ability of b5 to augment 17,20-lyase activity was reduced. Competition experiments conclusively demonstrated that 17OHpregnenolone binds to the mutant enzymes with an affinity equivalent to that of the wild-type enzyme, proving the surprising observation that these mutations did not affect substrate access or substrate binding [34, 106]. Isolated 17,20-lyase deficiency does indeed exist, but this selective loss of 17,20-lyase activity does not result from mutations in the enzyme’s active site. To understand the biochemical and biophysical basis of these extraordinary mutations in P450c17, we again turned to computational chemistry for insight. Arginines 347 and 358 are located near or at the carboxy-termini of the J€ and K helices, in a region of the protein where the presence of highly conserved residues and lack of length discrepancies permit confident structural modeling on the P450BMP template [46]. Both Arg347 and Arg358 lie beneath the heme ring on the surface of P450c17 that interacts with redox partners; the mutations Arg347His and Arg358Gln neutralize some of the positive

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Fig. 10. Amino acid substitutions causing isolated 17,20-lyase deficiency. The mutations at Arg347, Arg358 and Phe417 shown in solid boxes were identified in patients; the other mutations shown in dotted boxes were created in vitro. Symbols as in figures 8 and 9.

charges predicted to lie on this surface [106]. Curiously, the neutralization of the corresponding arginines 346 and 357 of rat P450c17 had been studied previously by site-directed mutagenesis; these mutants (Arg346Ala in particular) also exhibited preferential loss of 17,20-lyase activity [107] and a similar result was obtained for the Arg347Ala mutation in human P450c17 [43]. These residues were targeted because they were located in a contiguous series of amino acids (346-369) believed at that time to comprise a consensus steroidhormone binding site [77]. Modifications in this region, therefore, were believed to alter substrate binding and perhaps change the selectivity of P450c17 chemistry. By contrast, the computer modeling studies indicated that alterations in positive charges on the enzyme’s surface – rather than in a discrete string of residues – caused the isolated loss of 17,20-lyase activity. This suggested that neutralization of other positive charges in the redox-partner binding surface, no matter where they are located in the linear sequence of P450c17, should also preferentially impair 17,20-lyase activity. Accordingly, neutralization of other basic residues on this surface such as lysine 89 with the Lys89Asn mutant, increased the hydroxylase/lyase ratio 3-fold [46]. Analogously, alanine

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substitution of lysines 83, 88, 91, and of arginine 126 also increased the hydroxylase/lyase ratio by 2- to 4.5-fold [108]. Given the wide distribution of these residues in the linear sequence of P450c17 but their congregation in the redox-partner binding site, the evidence implicating this surface as essential to 17,20-lyase activity is compelling. One other reported P450c17 mutation causing isolated 17,20-lyase deficiency is the substitution Phe417Cys [109]. Although Phe417 is not a component of redox-partner binding surface itself, the aromatic side chain participates in hydrophobic packing interactions that help to form one edge of this surface [46]. Hence, the mutation Phe417Cys appears to disrupt the surface geometry, rather than the surface charge, to disrupt redox-partner interactions, but this proposal has not been studied directly. Finally, a male pseudohermaphrodite has been described with congenital methemoglobinemia due to a mutation in the gene for cytochrome b5 [110]. It is tempting to speculate that the cause of this patient’s undermasculization was due to low (but not absent) 17,20-lyase activity with consequent fetal testosterone deficiency, due not to a P450c17 mutation but rather the loss of b5, the cofactor protein that stimulates 17,20lyase activity. Unfortunately, a thorough endocrinologic evaluation of this patient, including circulating steroid homone concentrations, has not been published. If true, this case would prove the physiologic importance of b5 in P450c17 chemistry.

Broader Implications of P450c17 Studies The study of P450c17 has proven that 17a-hydroxylase and 17,20-lyase are two activities catalyzed by one enzyme and has shown that the regulation of electron flow to P450c17 regulates the ratio of these two activities. These studies may have broad implications beyond the elucidation of the molecular genetics of 17a-hydroxylase deficiency and isolated 17,20-lyase deficiency, and even beyond the understanding of adrenarche. We believe that P450c17 holds the key to the understanding of the polycystic ovary syndrome (PCOS), which affects about 5% of women of reproductive age, and which often begins as premature and exaggerated adrenarche. PCOS is a heterogeneous disorder characterized by hirsutism, virilism, hyperandrogenism, menstrual irregularities, chronic anovulation, obesity, insulin resistance, acanthosis nigricans, and high concentrations of LH and ovarian cysts [111, 112]. It seems likely that hyperandrogenism and insulin resistance are the primary lesions, and that the other findings are secondary events [113]. The hyperandrogenism in women with PCOS is of both ovarian and adrenal origin [114–117]. The adrenal hyperandrogenism of PCOS resembles an exaggerated form of adrenarche, and

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girls with premature adrenarche are more likely to develop PCOS [118, 119]. A gain-of-function disorder in serine/threonine phosphorylation of P450c17 could account for such increases in both adrenal and ovarian androgen secretion, and an earlier age of adrenarche [36]. The hyperinsulinism and insulin resistance of PCOS is at the level of insulin receptor signal transduction [120–122]. Serine phosphorylation of the b chain of the insulin receptor interferes with the tyrosine phosphorylation of the receptor that normally follows binding of insulin [123–127]. Furthermore, about 50% of PCOS women have insulin receptors in their fibroblasts that are 3.7-fold hyperphosphorylated [128]. Thus it would seem that a hyperactive kinase resulting from a single gain-of-function mutation may catalyze serine hyperphosphorylation of both P450c17 and the b chain of the insulin receptor, thus accounting for both the hyperandrogenism and insulin resistance of PCOS with a single molecular lesion.

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Kagimoto M, Winter JS, Kagimoto K, Simpson ER, Waterman MR: Structural characterization of normal and mutant human steroid 17-a-hydroxylase genes: Molecular basis of one example of combined 17-a-hydroxylase/17,20 lyase deficiency. Mol Endocrinol 1988;2:564–570. Kagimoto K, Waterman MR, Kagimoto M, Ferreira P, Simpson ER, Winter JSD: Identification of a common molecular basis for combined 17a-hydroxylase/17,20 lyase deficiency in two Mennonite families. Hum Genet 1989;82:285–286. Imai T, Yanase T, Waterman MR, Simpson ER, Pratt JJ: Canadian Mennonites and individuals residing in the Friesland region of The Netherlands share the same molecular basis of 17a-hydroxylase deficiency. Hum Genet 1992;89:95–96. Fardella CE, Zhang LH, Mahachoklertwattana P, Lin D, Miller WL: Deletion of amino acids Asp487-Ser488-Phe489 in human cytochrome P450c17 causes severe 17a-hydroxylase deficiency. J Clin Endocrinol Metab 1993;77:489–493. Hasemann CA, Kurumbail RG, Boddupalli SS, Peterson JA, Deisenhofer J: Structure and function of cytochromes P450: A comparative analysis of three crystal structures. Structure 1995;2:41–62. Yanase T, Waterman MR, Zachmann M, Winter JSD, Simpson ER, Kagimoto M: Molecular basis of apparent isolated 17,20-lyase deficiency: Compound heterozygous mutations in the C-terminal region (Arg(496)KCys, Gln(461)KStop) actually cause combined 17a-hydroxylase/17,20-lyase deficiency. Biochem Biophys Acta 1992;1139:275–279. Monno S, Mizushima Y, Toyoda N, Kashii T, Kobayashi M: A new variant of the cytochrome P450c17 (CYP17) gene mutation in three patients with 17a-hydroxylase deficiency. Ann Hum Genet 1997;61:275–279. Yanase T, Sanders D, Shibata A, Matsui N, Simpson ER, Waterman MR: Combined 17ahydroxylase/17,20 lyase deficiency due to a 7-basepair duplication in the N-terminal region of the cytochrome P45017a (CYP17) gene. J Clin Endocrinol Metab 1990;70:1325–1329. Yanase T, Kagimoto M, Matsui N, Simpson E, Waterman MR: Combined 17a-hydroxylase/17,20 lyase deficiency due to a stop codon in the N-terminal region of 17a-hydroxylase cytochrome P-450. Mol Cell Endocrinol 1988;59:249–253. Suzuki Y, Nagashima T, Nomura Y, Onigata K, Nagashima K, Morikawa A: A new compound heterozygous mutation (W17X, 436+5GKT) in the cytochrome P450c17 gene causes 17 alphahydroxylase/17,20-lyase deficiency. J Clin Endocrinol Metab 1998;83:199–202. Rumsby G, Skinner C, Lee HA, Honour JW: Combined 17 a-hydroxylase/17,20-lyase-lyase deficiency caused by heterozygous stop codons in the cytochrome P450c17a-hydroxylase gene. Clin Endocrinol 1993;39:483–485. Yamaguchi H, Nakazato M, Miyazato M, Kangawa K, Matsukura S: A 5€-splice site mutation in the cytochrome P450 steroid 17a-hydroxylase gene in 17a-hydroxylase deficiency. J Clin Endocrinol Metab 1997;82:1934–1938. Oshiro C, Takasu N, Wakugami T, Komiya I, Yamada T, Eguchi Y, Takei H: Seventeen a-hydroxylase deficiency with one base pair deletion of the cytochrome P450c17 (CYP17) gene. J Clin Endocrinol Metab 1995;80:2526–2529. Satoh J, Kuroda Y, Nawata H, Yanase T: Molecular basis of hypokalemic myopathy caused by 17a-hydroxylase/17,20-lyase deficiency. Neurology 1998;51:1748–1751. Monno S, Ogawa H, Date T, Fujioka M, Miller WL, Kobayashi M: Mutation of histidine 373 to leucine in cytochrome P450c17 causes 17a-hydroxylase deficiency. J Biol Chem 1993;268:25811– 25817. Fardella CE, Hum DW, Homoki J, Miller WL: Point mutation Arg440 to His in cytochrome P450c17 causes severe 17a-hydroxylase deficiency. J Clin Endocrinol Metab 1994;79:160–164. Conte FA, Grumbach MM, Ito Y, Fisher CR, Simpson ER: A syndrome of female pseudohermaphrodism, hypergonadotropic hypogonadism, and multicystic ovaries associated with missense mutations in the gene encoding aromatase (P450arom). J Clin Endocrinol Metab 1994;78:1287–1292. Lin D, Harikrishna JA, Moore CCD, Jones KL, Miller WL: Missense mutation Ser106KPro causes 17a-hydroxylase deficiency. J Biol Chem 1991;266:15992–15998. Sakai N, Tanaka M, Adachi S, Miller WL, Nagahama Y: Rainbow trout cytochrome P450c17 (17a-hydroxylase/17,20 lyase) cDNA cloning, enzymatic properties and temporal pattern of ovarian P450c17 mRNA expression during oogenesis. FEBS Lett 1992;301:60–64.

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Yanase T: 17a-Hydroxylase/17,20 lyase defects. J Steroid Biochem Mol Biol 1995;53:153–157. LaFlamme N, Leblanc J-F, Mailloux J, Faure N, Labrie F, Simard J: Mutation R96W in cytochrome P450c17 gene causes combined 17a-hydroxylase/17,20 lyase deficiency in two French Canadian patients. J Clin Endocrinol Metab 1996;81:264–268. Imai T, Globerman H, Gertner JM, Kagawa N, Waterman MR: Expression and purification of functional human 17a-hydroxylase/17,20 lyase (P450c17) in Escherichia coli. J Biol Chem 1993;268: 19681–19689. Ahlgren R, Yanase T, Simpson ER, Winter JSD, Waterman MR: Compound heterozygous mutations (Arg239KStop, Pro342KThr) in the CYP17 (P450-17a) gene lead to ambiguous external genitalia in a male patient with partial combined 17a-hydroxylase/17,20 lyase deficiency. J Clin Endocrinol Metab 1992;74:667–672. Yanase T, Kagimoto M, Suzuki S, Hashiba K, Simpson ER, Waterman MR: Deletion of a phenylalanine in the N-terminal region of human cytochrome P-45017a results in the partial combined 17a-hydroxylase/17,20-lyase deficiency. J Biol Chem 1989;264:18076–18082. Yanase T, Simpson ER, Waterman MR: 17a-Hydroxylase/17,20 lyase deficiency: From clinical investigation to molecular definition. Endocr Rev 1991;12:91–108. Miura K, Yasuda K, Yanase T, Yamakita N, Sasano H, Nawata H, Inoue M, Fukaya T, Shizuta Y: Mutation of cytochrome P-45017 alpha gene (CYP17) in a Japanese patient previously reported as having glucocorticoid-responsive hyperaldosteronism: With a review of Japanese patients with mutations of CYP17. J Clin Endocrinol Metab 1996;81:3797–801. Zachmann M, Kenpken B, Manella B, Navarro E: Conversion from pure 17,20 desmolase to combined 17,20-desmolase/17a-hydroxylase deficiency with age. Acta Endocrinol 1992;127: 97–99. Geller DH, Auchus RJ, Mendonc¸a BB, Miller WL: The genetic and functional basis of isolated 17,20 lyase deficiency. Nature Genet 1997;17:201–205. Kitamura M, Buczko E, Dufau ML: Dissociation of hydroxylase and lyase activities by site-directed mutagenesis of the rat P450-17a. Mol Endocrinol 1991;5:1373–1380. Lee-Robichaud P, Akhtar ME, Akhtar M: Control of androgen biosynthesis in the human through the interaction of Arg347 and Arg358 of CYP17 with cytochrome b5. Biochem J 1998;332:293–296. Biason-Lauber A, Leiberman E, Zachmann M: A single amino acid substitution in the putative redox partner-binding site of P450c17 as cause of isolated 17,20 lyase deficiency. J Clin Endocrinol Metab 1997;82:3807–3812. Giordano SJ, Kaftory A, Steggles AW: A splicing mutation in the cytchrome b5 gene from a patient with congenital methemoglobinemia and pseudohermaphrodism. Hum Genet 1994;93:568–570. Conway GS, Honour JW, Jacobs HS: Heterogeneity of the polycystic ovary syndrome: Clinical, endocrine and ultrasound features in 556 patients. Clin Endocrinol 1989;30:459–470. Franks S: Polycystic ovary syndrome: A changing perspective. Clin Endocrinol 1989;31:87–120. Dunaif A: Hyperandrogenic anovulation (PCOS): A unique disorder of insulin action associated with an increased risk of non-insulin-dependent diabetes mellitus. Am J Med 1995;98(suppl 1A): 33S–39S. Lachelin GCL, Barnett M, Hopper BR, Brink G, Yen SSC: Adrenal function in normal women and women with the polycystic ovary syndrome. J Clin Endocrinol Metab 1979;62:840–848. Lucky AW, Rosenfield RL, McGuire J, Rudy S, Helke J: Adrenal androgen hyperresponsiveness to ACTH in women with acne and/or hirsutism: Adrenal enzyme defects and exaggerated adrenarche. J Clin Endocrinol Metab 1986;62:840–848. Iban˜ez L, Potau N, Zampolli M, Prat N, Gussinye´ M, Saenger P, Vicens-Calvet E, Carrascosa A: Source localization of androgen excess in adolescent girls. J Clin Endocrinol Metab 1994;79:1778– 1784. Ehrmann DA, Barnes RB, Rosenfield RL: Polycystic ovary syndrome as a form of functional ovarian hyperandrogenism due to dysregulation of androgen secretion. Endocr Rev 1995;16: 322–353. Iban˜ez L, Potau N, Virdis R, Zampolli M, Terzi C, Gussinye´ M, Carrascosa A, Vicens-Calvet E: Postpubertal outcome in girls diagnosed of premature pubarche during childhood: Increased frequency of functional ovarian hyperandrogenism. J Clin Endocrinol Metab 1993;76:1599–1603.

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Oppenheimer E, Linder B, DiMartino-Nardi J: Decreased insulin sensitivity in prepubertal girls with premature adrenarche and acanthosis nigricans. J Clin Endocrinol Metab 1995;80:614–618. Dunaif A, Segal K, Futterweit W, Dobrjansky A: Profound peripheral insulin resistance, independent of obesity, in the polycystic ovary syndrome. Diabetes 1989;38:1165–1174. Dunaif A, Segal DR, Shelley DR, Green G, Dobrjansky A, Licholai T: Evidence for distinctive and intrinsic defects in insulin action in polycystic ovary syndrome. Diabetes 1992;41:1257–1266. Ciaraldi TP, El-Roeiy A, Madar Z, Reichart D, Olefsky JM, Yen SCC: Cellular mechanisms of insulin resistance in polycystic ovarian syndrome. J Clin Endocrinol Metab 1992;75:577–583. Bollag G, Roth R, Beaudoin J, Mochley-Rosen D, Koshland D Jr: Protein kinase C directly phosphorylates the insulin receptor in vitro and reduces its protein-tyrosine kinase activity. Proc Natl Acad Sci USA 1986;83:5822–5824. Stadtmauer L, Rosen OM: Increasing the cAMP content of IM-9 cells alters the phosphorylation state and protein kinase activity of the insulin receptor. J Biol Chem 1986;261:3402–3407. Rapuano M, Rosen OM: Phosphorylation of the insulin receptor by a casein kinase I-like enzyme. J Biol Chem 1991;266:12902–12907. Takayama S, White MF, Kahn CR: Phorbol ester-induced serine phosphorylation of the insulin receptor decreases its tyrosine kinase activity. J Biol Chem 1988;263:3440–3447. Chin JE, Dickens M, Tavare JM, Roth RA: Overexpression of protein kinase C isozymes a, bI, k and e in cells overexpressing the insulin receptor. Effects on receptor phosphorylation and signaling. J Biol Chem 1993;268:6338–6347. Dunaif A, Xia J, Book C-B, Schenker E, Tang Z: Excessive insulin receptor serine phosphorylation in cultured fibroblasts and in skeletal muscle. J Clin Invest 1995;96:801–810.

Prof. Walter L. Miller, Bldg. MR-IV, Room 209, University of California, San Francisco, San Francisco, CA 94143-0978 (USA) Tel. +1 415 476 2598, Fax +1 415 476 6286, E-Mail [email protected]

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21-Hydroxylase Deficiency Defects and Their Phenotype Carlo L. Acerini, Ieuan A. Hughes Department of Paediatrics, University of Cambridge, Addenbrooke’s Hospital, Cambridge, UK

Congenital adrenal hyperplasia (CAH) is a family of disorders characterised by enzyme defects in the steroidogenic pathways that lead to the biosynthesis of cortisol, aldosterone and androgens (fig. 1). The relative decrease in cortisol production, acting via the classic negative feedback loop, results in the increased secretion of ACTH from the anterior pituitary gland and to subsequent hyperplasia of the adrenals. All forms of CAH are inherited in an autosomal recessive manner, and their clinical manifestation is determined by the effects produced by the particular hormones that are deficient and by the excess production of steroids unaffected by the enzymatic block. Deficiency of the 21-hydroxylase enzyme is the most common form of CAH, accounting for over 90% of cases [1]. 21-Hydroxylase is a cytochrome P450 enzyme that catalyses the penultimate step in cortisol biosynthesis, the conversion of 17-hydroxyprogesterone (17-OHP) to 11-deoxycortisol (fig. 1). Cortisol deficiency results in the corticotrophin (ACTH)-induced accumulation of substrate precursors such as 17-OHP, and to increased concentrations of the adrenal androgens, androstenedione and testosterone. The same enzyme is also required for mineralocorticoid production, with deficiency leading to impaired synthesis of aldosterone. Congenital adrenal hyperplasia due to deficiency of the 21-hydroxylase enzyme arises as a result of deletions or deleterious mutations in the active gene (CYP21) located on chromosome 6p. Many different mutations of the CYP21 gene have been identified causing varying degrees of impairment of 21-hydroxylase activity that result in a spectrum of disease expression [2, 3]. CAH can be classified according to symptoms and signs and to age of presentation. The ‘classic’ type includes a severe ‘salt-wasting’ form which usually presents with acute adrenal crisis in early infancy, and a ‘simple virilizing’ form in which patients demonstrate masculinization of the external geni-

Fig. 1. Adrenal and gonadal steroidogenesis. StAR>Steroidogenic autoregulatory protein; SCC>(cholesterol) side chain cleavage enzyme; 3b-HSD>3b-hydroxysteroid dehydrogenase activity/D5-D4 isomerase; 17b-HSD>17b-hydroxysteroid dehydrogenase.

talia in females or virilization in early life in males. Finally, there is a ‘nonclassic’ or ‘late onset’ form that presents in females with signs and symptoms of mild androgen excess at or around the time of puberty. The incidence of this disorder has been reported to be in the region of 1 in 10,000–17,000 in western Europe and the USA, with an overall worldwide figure of approximately 1 in 14,000 births [4]. Unusually high incidences are seen in selected populations such as the Yupik Eskimos of Alaska where rates are in the region of 1 in 280–490 [5]. A ‘founder effect’ is thought to be operating in this population, due to a high rate of inbreeding and the presence of common single defect of the CYP21 gene (intron 2 splice mutation) [6].

Genetics of 21-Hydroxylase Deficiency A genetic basis of CAH due to 21-hydroxylase deficiency was first suspected in the 1950s when family studies suggested an autosomal-recessive

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Fig. 2. Schematic map of the HLA gene region on the short arm of chromosome 6. CYP21P is the inactive 21-hydroxylase pseudogene, CYP21 is the active 21-hydroxylase gene. C4A and C4B are genes encoding complement component 4.

pattern of inheritance [7]. Subsequent genetic linkage studies demonstrated that the locus for the 21-hydroxylase gene was tightly associated with the HLA major histocompatibility complex (MHC) located on the short arm of chromosome 6 [8, 9]. This relationship was useful in the ascertainment of 21hydroxylase phenotypes as certain HLA antigens were shown to be associated with particular clinical forms of 21-hydroxylase, although it was not entirely reliable [4]. It was with the identification of the cytochrome P450 involved in 21-hydroxylase activity [10] that later isolation and sequencing of the CYP21 gene and its inactive pseudogene (CYP21P) was finally achieved [11, 12]. The two genes are found to reside within the class III region of the HLA MHC, immediately adjacent to two other genes encoding the fourth component of complement, C4A and C4B (fig. 2). Sequencing reveals that CYP21 and CYP21P are each composed of 10 exons, and are located approximately 30 kb apart. Although they share 96–98% homology in their coding regions, a number of deleterious mutations in CYP21P renders any transcripts inactive [13]. All enzymatic action is therefore derived solely from the CYP21 gene [14, 15], and CYP21 expression has been shown to be stimulated ACTH and by cAMP [16].

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Fig. 3. Haplotype demonstrating differing number and structure of C4 and 21-hydroxylase gene units within the population. Haplotypes 3 and 10 contain no active CYP21 genes D ‘null mutations’. Adapted with permission from Wedell [3].

The presence of highly homologous, tandemly arranged genes, with alternating C4 and CYP21 pairs, appears to provide an opportunity for frequent misalignment and unequal crossing over between sister chromatids during meiosis. This can lead to complete gene duplication in one chromatid and deletion in the other. The most frequent deletion involves a 30-kb region of DNA spanning the first five to seven exons of CYP21, all of C4B and the 3€ portion of CYP21P [17]. The deletion of the CYP21 gene on both chromosomes leads to complete absence of 21-hydroxylase activity. The precise frequency with which deletions cause clinical 21-hydroxylase deficiency is not clear, but abnormal numbers of CYP21 genes are found frequently in the normal population and in patients with disease [18] (fig. 3). The frequency of gene deletions causing 21hydroxylase deficiency has variously been reported to range from 8 to 38% and is highest in northern European populations [17, 19–21]. Gene conversion is thought to be another mechanism that results in mutations of the CYP21 gene. This involves the nonreciprocal transfer of deleterious gene sequences from CYP21P to the CYP21 gene, thus inactivating the latter [22, 23]. Gene conversion is also thought to be the result of mispairing of chromosomes followed by recombination during meiosis. Large gene conversions in which C4B/CYP21 tandem sequences are completely replaced by a

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Fig. 4. Schematic representation of 21-hydroxylase (CYP21) gene structure and location of the nine most common mutations of the gene. Numbers in the boxes indicate exon number. Numbers in brackets indicate percentage frequency of occurrence. Data derived from Wedell [3].

C4A/CYP21P tandem have been found in up to 15% of cases with 21-hydroxylase deficiency [24]. Nine other smaller gene conversion mutations of CYP21 have also been described [3], of which the most common is a point mutation in intron 2 (fig. 4). Together with complete gene deletions, gene conversion mutations account for approximately 95% of all cases of 21-hydroxylase deficiency. The remaining 5% of cases are due to rare populationspecific mutations which have arisen de novo and not through interaction with CYP21P. Approximately 13 such rare mutations have been characterised to date (table 1) [3].

Clinical Aspects of 21-Hydroxlase Deficiency ‘Classic’ CAH The ‘salt-wasting’ form of 21-hydroxylase deficiency accounts for at least 70% of cases of CAH [5]. This condition is associated with severe 21-hy-

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Table 1. Spectrum of CYP21 gene mutations and their frequencies (adapted with permission from Wedell [3]) Mutation

Frequency %

Deletion/large gene conversion Pseudogene-derived mutations 12 splice Ile172Asn Val281Leu Arg356Trp Gln318stop Pro30Leu E3 del 8bp Ile236Asn Val237Glu Met239Lys Leu307insT

32 AA/CCKAG ATCKAAC GTCKTTG CGGKTGG CAGKTAG CCGKCTG ATCKAAC GTCKGAG ATGKAAG ins T

Intron 2 Exon 4 Exon 7 Exon 8 Exon 8 Exon 1 Exon 3 Exon 1 Exon 1 Exon 1 Exon 7

H

27 20 6 3 2 2 1 1 =1 =1

Combinations of the above Rare mutations Pro453Ser Arg483pro Arg483GGtoC

CCCKTCC CGGKCCG CGKC

Unique mutations

(10 different mutations)

Exon 10 Exon 10 Exon 10

=1 =1 =1 =3

droxylase deficiency, with little or no production of either cortisol or aldosterone and is characterized by severe salt wasting and acute adrenal crises in the absence of treatment. Cortisol deficiency results in maximal activity of the hypothalamic-pituitary adrenal axis and to increased secretion of androgens. The hypersecretion of androgens has different effects on males and females. In the female fetus, it causes a variable degree of posterior fusion of the labia majora, as well as hypertrophy of the clitoris. The enlarged phallus is often tethered down by chordee and may be hidden by a hood of redundant skin. The posterior labial fusion can in addition result in the formation of a urogenital sinus, i.e. a single common opening shared by the vagina and the urethra. The external opening for this is typically located at the base of the phallus. In males, testicular androgens cause normal masculinization of the external genitalia, so the additional source of adrenal androgens rarely has an effect in the newborn. In females with ‘classic’ CAH, the diagnosis is therefore usually suspected in a newborn infant presenting with ambiguous

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Table 2. Causes of salt-wasting in the newborn period Gastrointestinal losses Gastroenteritis Pyloric stenosis Renal losses Preterm infant Acute pyleonephritis Posterior urethral values Renal dysplasia Congenital adrenal hypoplasia Congenital adrenal hyperplasia StAR/SCC deficiency 3bHSD 21-Hydroxylase deficiency Congenital hypoaldosteronism CMO-type I deficiency CMO-type II deficiency Pseudohypoaldosteronism StAR>Steroidogenic auto regulatory protein; SCC> (cholesterol) side chain cleavage enzyme; 3b-HSD>3bhydroxysteroid dehydrogenase activity/D5-D4 isomerase; CMO>corticosterone methyl oxidase.

external genitalia; rarely, masculinization can be so extreme that appearances resemble those of a normal male, apart from the absence of palpable gonads. In such cases, the genetic sex can be readily established by performing a karyotype. Pelvic ultrasonography undertaken to visualise the internal genitalia can be misleading, as absence of a sonographically detectable uterus may be due to a retroflexed uterus which can be easily missed. In affected males, the genitalia are usually normal at birth, although there can be pigmentation secondary to the excess secretion of melanocyte-stimulating hormone (MSH). Nevertheless, this is an unreliable sign, particularly in children with darker skins. Affected males are therefore more at risk of developing the symptoms and signs of acute ‘salt-wasting’ crisis which, in the absence of treatment, readily progresses to cardiovascular collapse and death. Infants usually develop this complication between the 4th and 15th days of life [25], often presenting with non-specific signs such as poor feeding, vomiting, diarrhoea and weight loss. These clinical signs can be misinterpreted due to more common causes (table 2), and a high index of suspicion is needed to avoid the misdiagnosis of a male infant affected with ‘classic’ CAH.

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Despite the 21-hydroxylase deficiency, patients with the ‘simple virilizing’ forms of CAH are able to produce sufficient quantities of cortisol and aldosterone and therefore do not develop symptoms due to salt wasting or adrenal crises. Most females therefore present with ambiguous genitalia at or around the time of birth. In mild cases, masculinization may be minimal and can go unnoticed until the onset of early pubic hair development, usually before the age of 2 years. Males are invariably not diagnosed until later childhood when they present with premature pubic hair, penile development and signs of accelerated skeletal maturation. Other diagnoses, with which it may be difficult to discriminate clinically, include virilizing adrenal tumours, certain gonadal tumours and premature adrenarche. Confirmation of a diagnosis of ‘classic’ CAH cannot be achieved by clinical means alone and relies on the demonstration of characteristic biochemical abnormalities. In the ‘salt-wasting’ form of CAH, the aldosterone deficiency results in hyponatraemia, hyperkalaemia and metabolic acidosis. These are not specific findings and can cause diagnostic confusion with children presenting with more common causes of renal tubular dysfunction, such as acute pyleonephritis. Other causes of congenital adrenal hyperplasia as well as disorders of aldosterone production and action, such as congenital hypoaldosteronism and pseudohypoaldosteronism, can present with similar clinical features and serum electrolyte profiles (table 2). In ‘classic’ CAH, regardless of severity, age and sex of the infant, elevated plasma 17-OHP and 21-deoxycortisol levels and increased urinary adrenocorticosteroid metabolites are the characteristic hormonal abnormalities that confirm the diagnosis [26, 27]. Raised plasma testosterone levels can be a useful marker of CAH, but are not specific to the type of enzyme deficiency. Levels are increased in females with ‘classic’ CAH of all ages, but concentrations in untreated males during the first 6 months of life may appear within the normal range characterised by the increase in testicular testosterone seen at this age [28]. Non-Classic CAH The non-classic forms of CAH are associated with the mildest degree of 21-hydroxylase deficiency and therefore cause small changes in steroidogenesis. The absence of masculinization of the genital tract in females reflects the low level of adrenal androgen secretion. The condition manifests only in females, usually at puberty, since the effects of gonadal testosterone mask any clinical expression in males. In childhood, the condition may present with virilization, which can be potentially confused with conditions such as premature adrenarche and virilizing adrenal tumours. The affected adolescent female can manifest clinical patterns resembling either the polycystic ovarian syndrome (PCOS) (i.e. virilization and menstrual irregularities), hirsituism alone, or a

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cryptic form (i.e. no symptoms) in which the diagnosis is made fortuitously [29]. Clinical features do not distinguish patients with 21-hydroxylase deficient ‘non-classic’ CAH from those with PCOS [30]. Plasma testosterone and DHEAS concentrations are no different from levels observed in patients with other causes of ovarian hyperandrogenism; DHEAS levels may be normal in patients with ‘non-classic’ CAH [30, 31]. The diagnosis of non-classic CAH therefore depends on demonstrating increased basal or stimulated plasma 17OHP levels [31, 32]. However, there are problems with the interpretation of unstimulated 17-OHP values. There is an overlap between the stimulated 17-OHP concentrations in patients with ‘non-classic’ CAH and in subjects heterozygous for ‘classic’ CAH (population frequency 1 in 50) [33]. Heterozygotes for 21-hydroxylase deficiency are thought to be asymptomatic, although there is evidence that they may account for some patients presenting with premature adrenarche. In a recent study, 38% of cases with premature adrenarche were found to be heterozygous for one of nine different mutations of the CYP21 gene [34]. Newborn Screening The clinical diagnosis of ‘classic’ 21-hydroxylase deficiency can be missed in the newborn period, despite the apparently high potential for symptomatic diagnosis. Mild or even severe virilization is not reliably identified at delivery or during the neonatal periods in girls. Up to 30% of cases in one report were either overlooked or incorrectly identified as boys [35]. Boys are at particular risk of remaining undiagnosed until they present in adrenal crisis. An early study reported a ratio of 2.8:1.0 for affected females over affected males with the diagnosis of ‘salt-wasting’ CAH [36]. More recently, this ratio has been reported to be 1.8:1.0, suggesting that boys with undiagnosed ‘classic’ CAH may be dying in infancy [35]. Newborn screening by measurement of 17-OHP from filter paper dried blood spots has been introduced as a method for the early detection of cases with CAH [37]. The initial pilot studies of this method suggested that early diagnosis and treatment of affected, but symptom free, boys could be achieved [38]. However, with mass screening programmes a number of practical problems have emerged. The need for quick and efficient processing of blood spot samples and correct interpretation of results are problems which remain unresolved [5]. The accurate measurement of 17-OHP in neonates can be a problem because of fetal adrenocortical secretion of steroids which cross-react with the 17-OHP immunoassay [39]. Furthermore, 17-OHP levels decline with gestational maturity and may be considerably higher in preterm and sick neonates [40]. Falsepositive results reported in several newborn screening programmes are invariably confined to preterm infants [5, 41]. More rarely, false-negative 17-OHP results can also occur [42]. Attempts to improve the specificity of newborn screening

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for CAH by employing two-stage screening have proved costly and laborious, and not entirely successful [43]. Birth weight and gestational age adjusted criteria for the interpretation of 17-OHP values have been introduced to reduce the frequency of false-positive results seen in low birth weight and preterm infants [41]. Molecular Genetic Diagnosis Allele-specific PCR techniques are available that allow rapid and reliable genotyping for the common mutations of CAH from any tissue sample [44, 45]. Selective amplification of CYP21 without contamination by CYP21P sequences can be achieved by taking advantage of an 8-bp deletion in exon 3 of CYP21P; allele-specific primers have been designed which can discriminate between mutant and wild-type sequences by differences in their respective 3€ ends. Another method using non-radioactive Southern blotting and direct DNA sequencing differentiates between functional and non-functional genes, allowing for rapid analysis of the structure of CYP21 and which can identify up to 99% of all the known mutations [46]. These techniques do not screen for the rarer mutations of the CYP21 gene; these can be characterised by the more time-consuming process of direct DNA sequencing [47]. Since the majority of index cases with CAH are compound heterozygotes, direct sequencing of the entire CYP21 gene locus is recommended for complete detection of mutations. Mutational analysis is available for the purposes of genetic counselling and for the evaluation of future pregnancies in a couple who already have an affected child with CAH. Mutational analysis may have a role to play in neonatal CAH screening programs, by using CYP21 genotyping in conjunction with 17-OHP blood spot measurements. Adjunctive testing by selectively genotyping neonates with borderline 17-OHP values has recently been shown to be useful in the diagnosis of new cases [48, 49]. Blood spots applied to filter paper cards can be successfully used both for the measurement of 17-OHP and for DNA analysis [49]. However, this method of screening may not gain universal favour [50], as the delay between initial screening with 17-OHP and later genotyping (approximately 8–12 days), often exceeds the interval necessary for the identification and treatment of the neonate with salt-wasting CAH. Furthermore, in newborn infants with borderline 17-OHP levels at screening, clinical assessment and measurement of baseline and ACTH stimulated 17-OHP levels may be more useful and cheaper to distinguish between the variant forms of CAH due to 21-hydroxylase deficiency [40]. Molecular Genetics – Genotype/Phenotype Relationships Molecular diagnostic techniques rely on the assumption that specific mutations of CYP21 gene consistently give rise to the same form of CAH expression.

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Fig. 5. The relationship between genotype and phenotype in the common mutations of the CYP-21 gene. Phenotype graded according to clinical severity.

In this disorder genotype and phenotype generally correlate well with a clear relationship between clinical disease severity and the type of CYP21 mutation (fig. 5) [3, 51, 52]. CYP21 mutations can be grouped into 3 categories according to enzyme activity, as measured by in vitro mutagenesis and expression studies [53, 54]. The first group consists of null mutations, which include whole gene deletions or mutations that completely inactivate the gene. They result in absent enzyme activity and produce the most severe form of CAH with salt wasting and prenatal virilization of the external genitalia in females. The intron 2 splice mutation is a frequently detected mutation which involves a change in the normal polymorphic sequence at nucleotide 655 from C or A to G (655C/AKA) that alters premessenger RNA splicing [22]. This mutation is regarded as a slightly less severe variant in this group. It is usually associated with severe prenatal virilization of female genitalia and salt-wasting symptoms, but a small number of patients are not salt-wasters. A gene dosage effect may explain in this situation as it has been noted that salt-wasters are invariably homozygous for the intron 2 splice mutation rather than compound heterozygotes (e.g. intron 2 splice/deletion) [3]. Another explanation may be the production of small, yet sufficient quantities, of normally spliced mRNA by ‘leaky transcription’ that results in some functional enzyme activity [55, 56]. The second group mainly consists of missense mutations such as the Ile172Asn mutation, which is usually associated with severe prenatal virili-

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zation and no salt wasting in 90% of the cases. The Ile172Asn mutant enzyme has only 2–11% of the activity of the wild-type CYP21 enzyme in converting 17-OHP to 11-deoxycortisol [53]. Again, a gene dosage effect can be observed with the homozygous Ile172Asn genotype producing a less severe non-saltwasting state than the compound heterozygous (e.g. Ile172Asn/deletion) genotype. The final group contains examples such as the Val281Leu and Pro30Leu mutations that result in enzymes with 20–60% of normal activity [54, 57]. The Val281Leu mutation is not usually associated with prenatal virilization of the female genitalia, but instead presents with mild, late onset signs, such as the development of acne and hirsutism in adulthood. The Pro30Leu mutation is also associated with the non-classical form of CAH, but produces more severe signs, including clitoromegaly, when compared to the Val281Leu mutation [57]. As a general rule, patients who are compound heterozygotes for two different CAH mutations have a phenotype compatible with the less deleterious mutant allele [2]. The relationship between genotype and blood 17-OHP levels is less robust. Null CYP21 mutations are clearly associated with severely elevated 17-OHP levels in the newborn period with values usually above 500 nmol/l [48]. Concentrations of this steroid are more variable in patients with other mutations and do not concur with the severity of the phenotype (fig. 6). The intron 2 splice and Ile172Asn mutations may be associated with only modestly raised 17OHP values of 150–200 nmol/l but can nevertheless result in a range of disease severity from salt-wasting to non-classical CAH [48]. There are other well-documented examples where the predicted CAH phenotype does not always concord with the genotype. In a recent report of a kindred with ‘classic CAH’, phenotypic variance ranged over two generations from mild virilization to salt wasting. This was despite all affected members being compound heterozygotes for intron 2 splice and exon-4 point mutations in the CYP21 gene [58]. There are also perplexing reports of families with a history of ‘classic’ CAH, in whom antenatal screening had identified diseasecausing mutations in female probands, but who were subsequently born with normal female external genitalia [58, 59]. This occurred despite prenatal treatment with steroids having been withheld. One of the affected female infants born with normal external genitalia subsequently developed a ‘salt-wasting’ crisis in the neonatal period when steroid replacement therapy was temporarily withdrawn [59]. The ‘leaky transcription’ hypothesis associated with the intron 2 splice mutation is invoked as an explanation for such phenotypic variance. Nevertheless, other, as yet unidentified factors or genes may also contribute towards the variable expression of the CAH phenotype [60]. Another possible explanation is the presence of fluctuating intra-adrenal concentrations of progesterone, which may competitively inhibit the mutant enzyme

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Fig. 6. Neonatal screening values of 17-OHP in relation to CYP21 genotype group. Box plots show median values together with 10th, 25th, 75th, and 90th percentiles. Circles represent maximum and minimum values in each group. The dashed line indicates the cutoff limit for a positive test (75 mmol/l). The four groups D Null, I2 splice, Ile172Asn, Val281Leu, and false positives were significantly different from each other. Reproduced with permission from Nordenstrom et al. [48].

for its main substrate, 17-OHP [61]. Pseudo-substrate inhibition of other steroidogenic enzymes by accumulated 21-hydroxylase precursors has also been proposed as a mechanism which may explain phenotypic variance in some cases of CAH [58]. Furthermore, polymorphisms in several genes important for steroidogenesis and hormone action could lead to subtle differences in androgen responsiveness [62, 63] and thus to differences in phenotypic expression in non-null mutation genotypes. Other as yet unidentified genes or transcription factors could also be involved in the determination of 21-hydroxylase activity. This is highlighted in the report of a family with a history of ‘classic’ CAH in whom the typical biochemical features of 21-hydroxylase deficiency were not associated with any identifiable mutations of CYP21, despite extensive sequencing of the gene and its adjacent promoter regions [64]. The mechanism of inheritance of CAH may be more diverse than previously thought, as around 1% of disease causing CYP21 mutations appear to arise de novo, with no evidence of a mutation in one or either of the parents [3]. One explanation for this finding may be the presence of specific germline mutations of the CYP21 gene. De novo mutations in the gene are generated in a relatively high proportion of cells (in the order of 1 in 103–106) in sperm derived from normal subjects [65]. A high frequency (20%) of apparent germ-

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line mutations has been reported in Japanese families with CAH due to 21hydroxylase deficiency [66]. Another possible explanation relates to uniparental disomy, described as a genetic mechanism that can result in the expression of autosomal-recessive inherited disorders [67]. There is a recent report of ‘classic’ CAH inherited by this mechanism [68]. Although these examples are likely to be rare, it is important they be identified so that genetic counselling to families can be as complete as possible. Treatment. Pre-natal mutational analysis performed in early gestation is now possible and forms the basis of pre-natal treatment programmes. First reported in 1984, early administration of glucocorticoids to pregnant mothers has been shown to reduce the degree of virilization in affected female fetuses [69, 70]. The prevention strategy aims to identify the female fetus with the most severe form of CAH; confirmation of the CYP21 gene mutation in a female fetus is needed to avoid unnecessary treatment. As fetal adrenal steroidogenesis starts as early as the 6th week gestation, treatment of at-risk pregnancies has to be started several weeks before a diagnosis of CAH can be satisfied. This approach means that 7 of 8 fetuses are potentially treated unnecessarily until prenatal diagnosis is completed. Dexamethasone is the formulation of glucocorticoid used in the prenatal treatment since this steroid crosses the placenta unmetabolised from the maternal to the fetal circulation. Doses used hitherto (generally around 20 mg/kg/day) have caused severe effects in some mothers, particularly rapid weight gain and cutaneous signs of hypercorticolism. In a recent study from Sweden, a significant number of treated women indicated that they would not wish to repeat the procedure in a subsequent pregnancy [71]. Treatment-related side effects are generally not apparent in children exposed to his form of glucocorticoid therapy, although there were isolated reports of abnormalities in some of the Swedish children. It is in the longer term that reservations have been expressed about the safety of prenatal dexamethasone treatment of CAH [72]. However, evidence to generate concern is based on reduced birth weight and later, elevated blood pressure using a rat model [73], and from the epidemiological studies of Barker et al. [74]. There is a clear need for prospective multi-centre studies of prenatal CAH treatment using an agreed treatment and monitoring protocol. These studies must extend long-term postnatally, with detailed anthropometric and psychometric testing, together with the recruitment of appropriate controls. It has been suggested that early determination of genotype to predict disease severity may assist in the planning of therapy, with consequent improvements in patient outcome [2, 3]. There is a tendency to overtreat mildly affected cases of CAH from birth because the degree of severity is not apparent. Certain genotypes predict a low risk of salt loss; in these patients accelerated growth may not be increased until after 18 months of age [75]. Consequently, glucocor-

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ticoid dosages could be kept to a minimum from the outset and adjusted subsequently according to other clinical and biochemical parameters. Establishing a genotype that is associated with a completely non-functional enzyme may be of value when considering alternative options for managing the female patient who is difficult to control by medical means alone [76, 77]. Thus, if adrenalectomy is to become a realistic planned option for CAH management it will be necessary to demonstrate the presence of null alleles in such patients. Other treatment options recently proposed for CAH are based on exploiting the underlying pathophysiology that results in the expression of disease. These include the use of experimental multi-drug regimens consisting of low-dose glucocorticoid and mineralocorticoid plus a combination of either an anti-androgen (e.g. flutamide) and an aromatase inhibitor (e.g. tesotolactone) [78] or carbenoxolone (an 11b-hydroxysteroid dehydrogenase inhibitor) [79]. Whether these therapeutic regimens ultimately prove to be successful will partly depend on the careful selection of patients, added by an improved understanding of the relationship between genotype and the resulting pathophysiology.

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White PC, New MI, Dupont B: Congenital adrenal hyperplasia. N Engl J Med 1987;316:1519–1524. Speiser PW, Dupont J, Zhu D, Serrat J, Buegeleisen M, Tusie-Luna M-T, Lesser M, New MI, White PC: Disease expression and molecular genotype in congenital adrenal hyperplasia due to 21-hydroxylase deficiency. J Clin Invest 1992;90:584–595. Wedell A: Molecular genetics of congenital adrenal hyperplasia (21-hydroxylase deficiency): Implications for diagnosis, prognosis and treatment Acta Paediatr 1998;87:159–164. Pang S, Wallace MA, Hofman L, Thuline HC, Dorche C, Lyon IC, Dobbins RH, Kling S, Fujieda K, Suwa S: Worldwide experience in newborn screening for classical congenital adrenal hyperplasia due to 21-hydroxylase deficiency. Pediatrics 1988;81:866–874. Pang S, Clark A: Congenital adrenal hyperplasia due to 21-hydroxylase deficiency: Newborn screening and its relationship to the diagnosis and treatment of the disorder. Screening 1993;2:105–139. Speiser PW, New MI, Tannin GM, Pickering D, Yang SY, White PC: Genotype of Yupik Eskimos with congenital adrenal hyperplasia due to 21-hydroxylase deficiency. Hum Genet 1992;88:647–648. Childs B, Grumbach MM, Van Wyk JJ: Virilizing adrenal hyperplasia: A genetic and hormonal study. J Clin Invest 1956;35:213–222. Dupont B, Oberfield SE, Smithwick EM, Lee TD, Levine LS: Close genetic linkage between HLA and congenital adrenal hyperplasia (21-hydroxylase deficiency). Lancet 1977;2:1309–1312. Levine LS, Zachmann M, New MI, Prader A, Pollack MS, O’Neill GJ, Yang SY, Oberfield SE, Dupont B: Genetic mapping of the 21-hydroxylase-deficiency gene within the HLA linkage group. N Engl J Med 1978;299:911–915. Kominami S, Ochi H, Kobayashi Y, Takemori S: Studies on the steroid hydroxylation system in adrenal cortex microsomes: Purification and characterization of cytochrome P-450 specific for steroid C-21 hydroxylation. J Biol Chem 1980;255:3386–3394. Carroll MC, Campbell RD, Porter RR: Mapping of steroid 21-hydroxylase genes adjacent to complement component C4 genes in HLA, the major histocompatibility complex in man. Proc Natl Acad Sci USA 1985;82:521–525.

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Rumsby G, Avey CJ, Conway GS, Honour JW: Genotype-phenotype analysis in late onset 21hydroxylase deficiency in comparison to classic forms. Clin Endocrinol 1988;48:707–711. Dacou-Voutetakis C, Dracopoulou M: High incidence of molecular defects of the CYP21 gene in patients with premature adrenarche. J Clin Endocrinol Metab 1999;84:1570–1574. Thilen A, Larsson A: Congenital adrenal hyperplasia in Sweden 1969–1986. Acta Paediatr 1990; 79:168–175. Murtaza L, Sibert JR, Hughes I, Balfour IC: Congenital adrenal hyperplasia: A clinical and genetic survey. Are we detecting male salt-losers? Arch Dis Child 1980;55:622–625. Pang S, Hotchness J, Drash AL, Levine LS, New MI: Microfilter paper method for 17-alphahydroxyprogesterone radioimmunoassay; its application for rapid screening for congenital adrenal hyperplasia. J Clin Endocrinol Metab 1977;45:1003–1008. Pang S, Clark A: Newborn screening, prenatal diagnosis, and prenatal treatment of congenital adrenal hyperplasia due to 21-hydroxylase deficiency. Trends Endocrinol Metab 1990;July/Aug: 300–306. Donohoue PA, Parker K, Migeon CJ: Congenital adrenal hyperplasia; in Scriver CR, Beaudet AL, Sly WS, Valle D (eds): The Metabolic and Molecular Bases of Inherited Diseases. New York, McGraw Hill, 1995, vol II, pp 2929–2966. Murphy JF, Joyce BG, Dyas J, Hughes IA: Plasma 17-hydroxyprogesterone concentrations in ill newborn infants. Arch Dis Child 1983;58:532–534. Allen DB, Hoffman GL, Fitzpatrick P, Laessig R, Maby S, Slyper A: Improved precision of newborn screening for congenital adrenal hyperplasia using weight-adjusted criteria for 17-hydroxyprogesterone levels. J Pediatr 1997;130:128–133. Balsamo A, Cacciari E, Piazzi S, Cassio A, Bozza D, Pirazzoli P, Zappulla F: Congenital adrenal hyperplasia: Neonatal mass screening compared with clinical diagnosis only in Emilia-Romanga region of Italy, 1980–1995. Pediatrics 1996;98:362–367. Therrell BLJ, Berenbaum SA, Manter-Kapanke V, Simmank J, Korman K, Prentice L, Gonzalez J, Gunn S: Results of screening 1.9 million Texas newborns of 21-hydroxylase deficient congenital adrenal hyperplasia. Pediatrics 1998;101:583–590. Wedell A, Luthman H: Steroid 21-hydroxylase deficiency: Two additional mutations in salt-wasting disease and rapid screening of disease-causing mutations. Hum Mol Genet 1993;2:499–504. Day D, Speiser PW, White PC, Baranay F: Detection of steroid 21-hydroxylase alleles using a multiplexed ligation detection reaction and gene-specific PCR. Genomics 1995;29:152–162. Krone N, Roscher AA, Schwarz HP, Braun A: Comprehensive analytical strategy for mutation screening in 21-hydroxylase deficiency. Clin Chem 1998;44:2075–2082. Wedell A, Ritzen EM, Haglund Stengler B, Luthman H: Steroid 21-hydroxylase deficiency: Three additional mutated alleles and establishment of phenotype-genotype relationships of common mutations. Proc Natl Acad Sci USA 1992;89:7232–7236. Nordenstrom A, Thilen A, Hagenfeldt L, Larsson A, Wedell A: Genotyping is a valuable diagnostic complement to neonatal screening for congenital adrenal hyperplasia due to steroid 21-hydroxylase deficiency (see comments). J Clin Endocrinol Metab 1999;84:1505–1509. Fitness J, Dixit N, Webster D, Torresani T, Pergolizzi R, Speiser PW, Day DJ: Genotyping of CYP21, linked chromosome 6p markers, and a sex-specific gene in neonatal screening for congenital adrenal hyperplasia. J Clin Endocrinol Metab 1999;84:960–969. Root AW: Neonatal screening for 21-hydroxylase deficient congenital adrenal hyperplasia – The role of CYP21 analysis (editorial; comment). J Clin Endocrinol Metab 1999;84:1503–1504. Wedell A, Thilen A, Ritzen EM, Stengler B, Luthman H: Mutational spectrum of the steroid 21-hydroxylase gene in Sweden: Implications for genetic diagnosis and association with disease manifestation. J Clin Endocrinol Metab 1994;78:1145–1152. Jaaskelainen J, Levo A, Voutilainen R, Partanen J: Population-wide evaluation of disease manifestation in relation to molecular genotype in steroid 21-hydroxylase (CYP21) deficicney: Good correlation in a well-defined population. J Clin Endocrinol Metab 1997;82:3293–3297. Higashi Y, Hiromasa T, Tanae A, Miki T, Nakura J, Kondo T, Ohura T, Ogawa E, Nakayama K, Fujii Kuriyama Y: Effects of individual mutations in the P-450(C21) pseudogene on the P-450(C21) activity and their distribution in the patient genomes of congenital steroid 21-hydroxylase deficiency. J Biochem Tokyo 1991;109:638–644.

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Tusie Luna MT, Traktman P, White PC: Determination of functional effects of mutations in the steroid 21-hydroxylase gene (CYP21) using recombinant vaccinia virus. J Biol Chem 1990;265:20916–20922. Schulze E, Scharer G, Rogatzki A, Priebe L, Lewicka S, Bettendorf M, Hoepffner W, Heinrich UE, Schwabe U: Divergence between genotype and phenotype in relatives of patients with the intron 2 mutation of steroid-21-hydroxylase. Endocr Res 1995;21:359–364. Witchel SF, Bhamidipati DK, Hoffman EP, Cohen JB: Phenotypic heterogeneity associated with the splicing mutation in congenital adrenal hyperplasia due to 21-hydroxylase deficiency (see comments). J Clin Endocrinol Metab 1996;81:4081–4088. Tusie Luna MT, Speiser PW, Dumic M, New MI, White PC: A mutation (Pro-30 to Leu) in CYP21 represents a potential nonclassic steroid 21-hydroxylase deficiency allele. Mol Endocrinol 1991;5: 685–692. Chin D, Speiser PW, Imperato-McGinley J, Dixit N, Uli N, David R, Oberfield SE: Study of a kindred with classic congenital adrenal hyperplasia: Diagnostic challenge due to phenotypic variance. J Clin Endocrinol Metab 1998;83:1940–1945. Quercia N, Chitayat D, Babul-Hirji R, New MI, Daneman D: Normal external genitalia in a female with classic congenital adrenal hyperplasia who was not treated during embryogenesis. Prenat Diagn 1998;18:83–85. Wilson RC, Mercado AB, Cheng KC, New MI: Steroids 21-hydroxylase deficiency: Genotype may not predict phenotype. J Clin Endocrinol Metab 1995;80:2322–2329. Dickerman Z, Grant DR, Faiman C, Winter JS: Intraadrenal steroid concentrations in man: Zonal differences and developmental changes. J Clin Endocrinol Metab 1984;59:1031–1036. Raivio T, Huhtaniemi I, Anttila R, Siimes MA, Hagenas L, Nilsson C, Pettersson K, Dunkel L: The role of luteinizing hormone-beta gene polymorphism in the onset and progression of puberty in healthy boys. J Clin Endocrinol Metab 1996;81:3278–3282. Rebbeck TR, Kantoff PW, Krithivas K, Neuhausen S, Blackwood MA, Godwin AK, Daly MB, Narod SA, Garber JE, Lynch HT, Weber BL, Brown M: Modification of BRCA1-associated breast cancer risk by the polymorphic androgen-receptor CAG repeat. Am J Hum Genet 1999;64:1371–1377. Nimkarn S, Cerame BI, Wei J-Q, Dumic M, Zunec R, Brkljacic L, Skrabic V, New MI, Wilson RC: Congenital adrenal hyperplasia (21-hydroxylase deficiency) without demonstrable genetic mutations. J Clin Endocrinol Metab 1999;84:378–381. Tusie Luna MT, White PC: Gene conversions and unequal crossovers between CYP21 (steroid 21-hydroxylase) and CYP21P involve different mechanisms. Proc Natl Acad Sci USA 1995;92: 10796–10800. Tajima T, Fujieda K, Nakayama K, Fujii Kuriyama Y: Molecular analysis of patient and carrier genes with congenital steroid 21-hydroxylase deficiency by using polymerase chain reaction and single strand conformation polymorphism. J Clin Invest 1993;92:2182–2190. Spence JE, Periaccante RG, Greig GM, Willard HF, Ledbetter DH, Hejtmancik JF, Pollack MS, O’Brien WE, Beaudet AL: Uniparental disomy as a mechanism for human genetic disease. Am J Hum Genet 1988;42:217–226. Lopez-Gutierrez AU, Riba L, Ordonez-Sanchez ML, Ramirez-Jimenez S, Cerrillo-Hinojosa M, Tusie-Luna MT: Uniparental disomy for chromosome 6 results in steroid 21-hydroxylase deficiency: Evidence of different genetic mechanisms involved in the production of the disease. J Med Genet 1998;35:1014–1019. David M, Forest MG: Prenatal treatment of congenital adrenal hyperplasia resulting from 21-hydroxylase deficiency. J Pediatr 1984;105:799–803. Forest MG, David M, Morel Y: Prenatal diagnosis and treatment of 21-hydroxylase deficiency. J Steroid Biochem Mol Biol 1993;45:75–82. Lajic S, Wedell A, Bui TH, Ritzen EM, Holst M: Long-term somatic follow-up of prenatally treated children with congenital adrenal hyperplasia. J Clin Endocrinol Metab 1989;83:3872–3880. Seckl JR, Miller WL: How safe is long-term prenatal glucocorticoid treatment? JAMA 1997;277: 1077–1079. Benediktsson R, Lindsay RS, Noble J, Seckl JR, Edwards CR: Glucocorticoid exposure in utero: New model for adult hypertension [published erratum appears in Lancet 1993;341:572] (see comments). Lancet 1993;341:339–341.

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Barker DJ, Gluckman PD, Godfrey KM, Harding JE, Owens JA, Robinson JS: Fetal nutrition and cardiovascular disease in adult life (see comments). Lancet 1993;341:938–941. Thilen A, Woods KA, Perry LA, Savage MO, Wedell A, Ritzen EM: Early Growth is not increased in untreated moderately severe 21-hydroxylase deficiency (see comments). Acta Paediatr 1995;84: 894–898. Van Wyk JJ, Gunther DF, Ritzen EM, Wedell A, Cutler GB Jr, Migeon CJ, New MI: The use of adrenalectomy as a treatment for congenital adrenal hyperplasia. J Clin Endocrinol Metab 1996; 81:3180–3190. Gunther DF, Bukowski TP, Ritzen EM, Wedell A, Van Wyk JJ: Prophylactic adrenalectomy of a three-year-old girl with congenital adrenal hyperplasia: Pre- and postoperative studies. J Clin Endocrinol Metab 1997;82:3324–3327. Laue L, Merke DP, Jones JV, Barnes KM, Hill S, Cutler GB Jr: A preliminary study of flutamide, testolactone, and reduced hydrocortisone dose in the treatment of congenital adrenal hyperplasia. J Clin Endocrinol Metab 1996;81:3535–3539. Irony I, Cutler GB: Effect of carbenoxolone on plasma renin activity and hypothalamic-pituitaryadrenal axis in congenital adrenal hyperplasia due to 21-hydroxylase deficiency. Clin Endocrinol 1999;51:285–291.

Dr. Carlo L. Acerini, Department of Paediatrics, University of Cambridge, Addenbrooke’s Hospital, Hills Road, Cambridge, CB2 2QQ (UK) Tel. +44 1223 336865, Fax +44 1223 336996, E-Mail [email protected]

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Defects in Aldosterone Biosynthesis M. Peter a, b, W.G. Sippell a a b

Division of Paediatric Endocrinology, Department of Paediatrics, University of Kiel, and Sanitas Ostseeklinik Boltenhagen, Germany

Signs of disturbed mineralocorticoid production are present in most of the patients with adrenal steroid biosynthesis defects. Dependent on the mutant enzyme and the degree of reduction of enzymatic activity, symptoms and signs of mineralocorticoid overproduction (17a-hydroxylase and 11b-hydroxylase deficiencies) or mineralocorticoid deficiency (lipoid CAH, 3b-hydroxysteroid dehydrogenase, 21-hydroxylase and aldosterone synthase deficiencies) may occur. In this chapter we will focus on steroid biosynthesis defects leading to disturbed aldosterone secretion. Congenital adrenal hyperplasia (CAH), the inherited inability to synthesise cortisol, usually presents with signs of androgen excess such as masculinisation of female external genitalia. Many patients also develop signs and symptoms of aldosterone deficiency, including hyponatraemia, hyperkalaemia and hypovolaemia. However, by the early 1950s, it was recognised that a small percentage of patients with congenital adrenal hyperplasia develop hypertension rather than mineralocorticoid deficiency and that the hypertension in such patients responds to glucocorticoid replacement. Eberlein and Bongiovanni [1] determined that most of these patients suffer from a distinct metabolic defect, steroid 11b-hydroxylase deficiency, whereas patients without hypertension usually have 21-hydroxylase deficiency. A third group of patients exists which, however, exhibit signs and symptoms of mineralocorticoid excess without masculinisation. These patients suffer from a distinct enzymatic defect, 17ahydroxylase deficiency [2]. On the other hand, a small group of patients do have aldosterone deficiency without any disturbances in cortisol and androgen biosynthesis. In the early 1960s, the diagnosis of a selective mineralocorticoid deficiency was established by urinary steroid metabolite determinations utilising improved laboratory

methods such as gas chromatography. In 1964, Visser and Cost [3] and Ulick et al. [4] were the first to suggest a biosynthetic defect with autosomal-recessive inheritance, causing selective hypoaldosteronism due to deficient 18-hydroxylation of corticosterone [5] and deficient oxidation of 18-hydroxycorticosterone, respectively. In the early 1970s, Ulick [6] suggested that the two biochemically different forms of selective aldosterone deficiency be termed corticosterone methyloxidase (CMO) deficiency type I and type II. In 1996, the nomenclature was changed to aldosterone synthase deficiency type I and type II, since it was clear that one single P450 enzyme, termed aldosterone synthase, catalyses all three steps of the terminal aldosterone biosynthesis [7]. In both aldosterone synthase deficiency types, aldosterone biosynthesis is impaired, while corticosterone of zona glomerulosa origin, under the primary control of the renin angiotensin system, is produced in excess. The two defects differ biochemically in that 18-hydroxycorticosterone is deficient in aldosterone synthase deficiency type I, but overproduced in aldosterone synthase deficiency type II.

Aldosterone and Cortisol Biosynthesis Metabolic Pathways of Aldosterone and Cortisol Biosynthesis Steroid hormones are not stored in the tissues where they are produced. Therefore, the steroid producing cells have to synthesise variable amounts of steroids according to the actual needs, e.g. adequate stress response. The acute stimulation of steroidogenesis in the adrenals and gonads is triggered by tropic hormones (for example, ACTH and LH) which induce generation of cAMP with subsequent activation of phosphorylation and gene transcription by the cAMP-dependent protein kinase A. Steroidogenic acute regulatory (StAR) protein mediates the rapid increase in pregnenolone synthesis stimulated by tropic hormones [8]. Aldosterone is synthesised in the zona glomerulosa of the adrenal cortex by the action of the transport protein steroidogenic acute regulatory (StAR) protein and by the subsequent action of four enzymes (fig. 1). Cholesterol desmolase (or side chain cleavage enzyme; P450scc), 21-hydroxylase (P450c21), and aldosterone synthase (P450c11Aldo) are cytochrome P450 (CYP) enzymes which are membrane-bound heme-containing proteins. The fourth enzyme, 3b-hydroxysteroid dehydrogenase (3b-HSD II), is a member of the short-chain dehydrogenase family [9, 10]. The first two steps of aldosterone biosynthesis (from cholesterol to progesterone) are identical to those required for the biosynthesis of cortisol in the zona fasciculata and are mediated by the same enzymes in both zones (fig. 1). However, the synthesis of cortisol requires 17a-hydroxylation of pregnenolone

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Fig. 1. Biosynthesis of aldosterone and cortisol in the adrenal zona glomerulosa and fasciculata and of sex steroids in the adrenal zona reticularis and in the gonads.

by 17a-hydroxylase (P450c17), which is in the adrenals only expressed in the zona fasciculata [11]. The terminal step in cortisol biosynthesis is the 11bhydroxylation of 11-deoxycortisol which is catalysed by 11b-hydroxylase (P450c11) which is mainly expressed in the zona fasciculata. In contrast, aldosterone synthase is normally expressed in the zona glomerulosa only [12]. Thus, the specific patterns of expression of these two enzymes ensure that aldosterone and cortisol are synthesised appropriately. Enzymes The conversion of cholesterol to pregnenolone is mediated by the side chain cleavage enzyme (P450scc) which is localised in the inner mitochondrial membrane. P450c17 is a 521 amino acids protein and belongs to the family of cytochrome P450 enzymes [13]. 3b-Hydroxysteroid-dehydrogenase (3b-HSD II) and 17a-hydroxylase (P450c17) are both key enzymes in steroid biosynthesis which are involved in adrenal and gonadal synthesis of mineralocorticoids, glucocorticoids and sex steroids. They are localised in the endoplasmatic reticulum. 3b-HSD II is a 372 amino acids protein and belongs to the family of the short chain dehydrogenases, whose electron acceptor is NAD+. 3b-HSD has 3b-hydroxy-

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steroid dehydrogenase activity as well as D4/D5-isomerase activity and can catalyse the reverse reactions with NADH as electron donor [14]. P450c17 is a 508 amino acids protein and belongs to the family of cytochrome P450 enzymes [15]. P450c17 has in vitro both 17a-hydroxylase and 17,20-lyase activities [16]. P450c17 catalyses the conversion of pregnenolone to 17-OH-pregnenolone and of progesterone to 17-OH-progesterone (>17ahydroxylase activity). P450c17 also catalyses an oxidative cleavage of the 17,20carbon-carbon bond (>17,20-lyase activity). This reaction converts 17-OHpregnenolone to dehydroepiandrosterone and 17-OH-progesterone to androstenedione. Pregnenolone is a good substrate, whereas progesterone is a poor substrate for P450c17. Relative to 17a-hydroxylase activity, the 17,20-lyase activity of P450c17 is more strongly expressed in the gonads than in the adrenals which explains why the main products of steroid biosynthesis are found in the gonads. The conversion of progesterone to 11-deoxycorticosterone and of 17-OHprogesterone to 11-deoxycortisol is mediated by 21-hydroxylase (P450c21) which is localised in the endoplasmatic reticulum. P450c21 is a 494 amino acids protein and belongs to the family of cytochrome P450 enzymes [17]. The two 11b-hydroxylase isoenzymes that are responsible for terminal cortisol (P450c11) and aldosterone (P450c11Aldo) biosynthesis in humans are mitochondrial cytochromes P450, located in the inner mitochondrial membrane on the matrix side. Both have 503 amino acid residues, but a signal peptide is cleaved to yield the mature protein of 479 residues [18]. The sequences of both proteins are 93% identical. P450c11 and P450c11Aldo differ in 35 amino acids [19]. Genes Encoding Enzymes Involved in Mineralocorticoid Biosynthesis The gene encoding StAR was first cloned in mice MA-10 Leydig cells [20]. The human StAR gene was cloned by Sugawara et al. [21] and has been mapped to chromosome 8p11.2. The gene spans 8 kb and consists of seven exons and six introns. It encodes a 285 amino acid protein [22]. The StAR protein sequence is highly conserved (80–90% homology) among different species (human, mouse, rat, hamster, cow, sheep and pig). A StAR pseudogene has been mapped to chromosome 13 which is not expected to encode a functional StAR protein. The StAR gene is expressed in adrenal, testis and ovary. StAR mRNA is not detectable in brain or placenta, organs known to produce steroids [21]. The placenta and brain express a protein (MLN-64) with considerable homology to StAR that can stimulate steroidogenesis [23]. Six different genes encoding 3b-HSD have been identified in the human genome and are localised on the short arm of chromosome 1 (1p11-13); however, only two of them are translated. 3b-HSD isoenzymes show a tissue-

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specific pattern of expression. The HSD3B1 gene encoding 3b-HSD type 1 is expressed in placenta, skin and adipose tissue, whereas the HSD3B2 gene encoding 3b-HSD type 2 is expressed in adrenals and gonads. HSD3B1 and HSD3B2 consist of 4 exons of which exon 1 is untranslated [24]. CYP17 is a single copy gene and was cloned from testis [25] and adrenal [26]. It is localised on the long arm of chromosome 10 (10q24.3). Eight exons are spread over 6.7 kb of genomic DNA. The gene encoding 21-hydroxylase is localised on the short arm of chromosome 6 (6p21.3). An active (CYP21B) and an inactive (CYP21A) gene are situated within the gene cluster encoding major HLA histocompatibility complex and complement factor C4 [27]. The active CYP21B and the pseudogene CYP21A consist of 10 exons spread over 3.1 kb. Both genes have a homology of 98% in exon sequence and of 96% in intron sequence. The pseudogene carries a number of deleterious mutations causing this gene not being translated [17, 28]. In humans, the two 11b-hydroxylase isoenzymes are encoded by two genes which are located on the long arm of chromosome 8 [29, 30]. CYP11B1 is the gene encoding 11b-hydroxylase (P450c11) and CYP11B2 is that encoding aldosterone synthase (P450c11Aldo). Both genes are located approximately 40 kb apart. Each gene contains 9 exons spread over 7 kb. The nucleotide sequences of both genes are 93% identical in coding sequences and about 90% identical in introns. Together with CYP11A, the gene encoding cholesterol desmolase, CYP11B1 and CYP11B2 are grouped into a single family within the cytochrome P450 gene superfamily [31]. CYP11B1 expression is primarily controlled by ACTH, which acts through a specific G-protein-coupled receptor to increase levels of cAMP. CYP11B2 is regulated mainly by angiotensin II and potassium. The promoter region of both genes is strikingly different, underlining the fact that both genes are differently regulated on the transcriptional level.

Adrenal Disorders Caused by Mineralocorticoid Deficiencies 17a-Hydroxylase Deficiency Pathophysiology and Frequency of 17a-Hydroxylase Deficiency. Steroid 17-hydroxylase deficiency is an autosomal recessively inherited genetic disorder affecting both the adrenal cortex and the gonads and is caused by mutations in the CYP17 gene. Due to deficient 17a-hydroxylase activity, pregnenolone and progesterone are not efficiently converted to cortisol and sex steroids. Concomitantly, there is overproduction of 17-deoxysteroids in the aldosteroneproducing pathway. Among them, DOC and its metabolites, notably 19-NorDOC, can cause hypertension [32].

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Data regarding the frequency of 17a-hydroxylase deficiency in the general population have not yet been published. Cases of 17a-hydroxylase deficiency have been observed all over the world. Since the first description by Biglieri et al. [2] in 1966, at least 125 cases have been reported. Approximately 1% of patients with congenital adrenal hyperplasia suffer from 17a-hydroxylase deficiency [33]. Clinical Presentation of 17a-Hydroxylase Deficiency. Females affected with classic 17a-hydroxylase deficiency are born with normal female external genitalia. Normal prepubertal internal genitalia are present. Genetic males show female external genitalia and rudimentary or absent structures of Wolffian ducts. The testes may be located intraabdominally or in the inguinal canal. In classic 17a-hydroxylase deficiency, patients of either sex present as phenotypic females with primary amenorrhoea and lack of spontaneous puberty [33, 34]. Overproduction of 17-deoxysteroids in the aldosterone-producing pathway (DOC and its metabolites) cause hypertension. There is only little knowledge about the time of onset and the natural course of hypertension in 17-hydroxylase deficiency patients. Most patients are diagnosed in late pubertal age when they present with primary amenorrhea and lack of pubertal development. At this time, most patients are hypertensive. Six patients have been reported in the literature to be normotensive at least at the time of diagnosis. Moreover, some patients with hypertension were already diagnosed in early childhood because of defects of sexual differentiation. On the other hand, still others were diagnosed only at or after their fourth decade of life. In addition, the degree of hypertension varies from mild to severe among all these patients. Besides the patients’ age and the severity of the enzyme defect, there seem to be unknown factors influencing the time of onset and the severity of hypertension in this disorder [33]. Clinical evidence of glucocorticoid insufficiency is unusual in patients with 17a-hydroxylase deficiency and adrenal crises usually do not occur. Nonclassical less severe forms of 17a-hydroxylase deficiency have been described in which affected 46,XY individuals have ambiguous genitalia, with or without glucocorticoid-responsive hypertension [35]. A few patients with isolated 17,20lyase deficiency presenting with ambiguous genitalia have been reported [36]. Hormonal Findings in 17a-Hydroxylase Deficiency. Due to deficient 17ahydroxylation, pregnenolone and progesterone are not efficiently converted to 17-OH-pregnenolone and 17-OH-progesterone, respectively. This leads to decreased adrenal cortisol and gonadal sex steroid production. Decreased cortisol production leads via poor feedback control to increased ACTH secretion. This stimulates the adrenal to overproduction of steroid precursors prior to the block, mainly 17-deoxysteroids. Because 11-deoxycorticosterone and 19-Nor-DOC are mineralocorticoid agonists, plasma renin activity is sup-

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pressed. It is of interest that most reported cases in the literature have low plasma aldosterone. In a recent report [33], only 17 of 122 cases of 17a-hydroxylase/17,20-lyase deficiency exhibiting increased plasma aldosterone levels were reported from the literature, while the majority of cases had clearly decreased aldosterone secretion. Today, the most reasonable explanation for hypoaldosteronism in patients with 17a-hydroxylase deficiency is that the high secretion of DOC and its potent mineralocorticoid metabolite, 19-Nor-DOC, leads to sodium retention and increase of circulating blood volume [32]. This causes a suppression of plasma renin activity with diminished production of angiotensin II and aldosterone. Other explanations for decreased aldosterone secretion such as a concomitant deficiency of 18-hydroxylase and/or 18-dehydrogenase are less likely because in most cases aldosterone normalised during dexamethasone therapy [37]. However. it is still difficult to explain elevated aldosterone plasma levels together with suppressed plasma renin activity. It has been speculated that the more severe the deficiency of 17a-hydroxylase activity, the more active is the corticosterone methyl oxidase, resulting in an increased production of aldosterone. Diagnostic Recommendations for 17a-Hydroxylase Deficiency. The diagnosis might be suspected in genetic males presenting with female or ambiguous genitalia at birth. The diagnosis of 17a-hydroxylase deficiency has to be taken in consideration in the differential diagnosis of primary amenorrhea and high blood pressure in combination with suppressed renin. The specific hormonal diagnosis of 17a-hydroxylase deficiency can be made by detecting high plasma levels of 17-deoxysteroids (11-deoxycorticosterone and corticosterone) in the presence of low 17-hydroxylated steroids (17-OH-progesterone, 11-deoxycortisol and cortisol) or elevated urinary excretion of metabolites of 17-deoxysteroids. An ACTH test is not always necessary in patients with classic 17a-hydroxylase deficiency. In addition, low renin, hypokaliaemic metabolic alkalosis, absent synthesis of sex steroids, and elevated levels of LH and FSH are observed. Molecular Genetics of 17a-Hydroxylase Deficiency. Mutations in the CYP17 gene cause 17a-hydroxylase deficiency. The first mutation in a patient with classic 17a-hydroxylase deficiency was a premature stop mutation in the N-terminal region of P450c17 [38]. Subsequently, approximately 25 different mutations in the CYP17 gene have been identified including amino acid substitutions, stop mutations and frame shift mutations (fig. 2). The amino acid substitutions are located in regions of known functional importance of the enzyme. Transfection experiments with mutant CYP17 constructs have shown that these mutations diminish or abolish enzymatic activity. The majority of known mutations have been identified in patients with classic 17a-hydroxylase deficiency. Some of the mutations seem to differently influence 17a-hydroxylase

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Fig. 2. Schematic representation of the genomic structure of the human CYP17 gene and positions of mutations reported to date. Exons are represented by boxes; black boxes demarcate the coding regions.

activity and 17,20-lyase activity, respectively (for example DF53) [33, 39]. For two point mutations identified in patients with isolated 17,20-lyase deficiency (R347H and R358Q), it has been shown in transfection experiments that the mutant enzymes have only 5% of 17,20-lyase activity but 65% of 17a-hydroxylase activity of the wild-type P450c17 enzyme. Using computer models of P450c17, it has been shown that the interaction between the enzyme itself and its redox partner, oxido reductase, is impaired in isolated 17,20-lyase deficiency [40, 41]. Treatment and Management of 17a-Hydroxylase Deficiency. Cortisol deficiency is replaced by glucocorticoid administration which reduces ACTH secretion. In addition, the ACTH dependent excess of aldosterone precursors (mainly DOC) will be suppressed by this treatment. Oral hydrocortisone or dexamethasone (in patients who have already reached their final height) is recommended. Clinical evidence of glucocorticoid insufficiency is unusual in patients with 17a-hydroxylase deficiency and adrenal crises usually do not occur, thus increased dosage in illness or stress is not recommended. Additional antihypertensive treatment may be required if hypertension has been of long duration before treatment. Drugs which can be used comprise spironolactone, amiloride and calcium channel blockers. All patients with classical 17a-hydroxylase deficiency are reared as females regardless of their karyotype. Sex assignment is a critical issue for all 46,XY newborns with ambiguous external genitalia. To avoid any doubt about the

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gender of rearing it is necessary that proper diagnosis is made within a short period after birth. The correction of ambiguous genitalia should be performed by an experienced paediatric surgeon, including early gonadectomy in 46,XY patients who are reared as girls. Replacement therapy with sex steroids is necessary in all patients and includes estrogens and androgens dependent on the sex of rearing. 11b-Hydroxylase Deficiency Pathophysiology and Frequency of 11b-Hydroxylase Deficiency. Autosomal recessively inherited defects in 11b-hydroxylase are caused by mutations in the CYP11B1 gene. Due to deficient adrenal 11b-hydroxylase activity, 11deoxycortisol (DOC) and 11-deoxycorticosterone (compoundS) are not efficiently converted to cortisol and corticosterone. This causes deficient cortisol production and increased ACTH secretion which lead to adrenal hyperplasia and marked androgen excess and virilisation. In addition, elevated mineralocorticoid precursors cause low renin hypertension. Data regarding the frequency of 11b-hydroxylase deficiency in the general population are sparse. The frequency of 21-hydroxylase deficiency in the general population is about 1/14,000 live births [42]. Approximately 5% of patients with congenital adrenal hyperplasia suffer from 11b-hydroxylase deficiency. Thus, an incidence for 11b-hydroxylase deficiency of approximately 1/250,000 can be assumed [43]. A large number of cases has been reported in Israel among Jewish immigrants from Morocco. The incidence in this relatively inbred population has been estimated to be 1/5,000–1/7,000 live births [44]. Clinical Presentation of 11b-Hydroxylase. Females affected with classic 11b-hydroxylase deficiency are born with masculinisation of their external genitalia. This is caused by oversecretion of adrenal androgens during embryonic and fetal development. In contrast to the external genitalia, the gonads and the internal genital structures are normal. Rapid somatic growth in childhood, accelerated skeletal maturation leading to premature closure of the epiphyses, and short adult stature are signs of postnatal androgen excess in both sexes. Additionally, affected children may have premature development of sexual and body hair and acne. Patients with nonclassic 11b-hydroxylase deficiency are born with normal genitalia and present with signs and symptoms of androgen excess as children. Adult women may present with hirsutism and oligomenorrhoea. However, only a small percentage of women with hirsutism and hyperandrogenic oligomenorrhoea have nonclassic 11b-hydroxylase deficiency [45–47]. Approximately two thirds of patients with classic 11b-hydroxylase deficiency present with elevated blood pressure. Most of the affected neonates do not have hypertension and suppressed plasma renin activity, however hyperten-

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sion often develops within the first years of life [43, 48]. If hypertension is not well controlled under treatment, left-ventricular hypertrophy and retinopathy as well as death from cerebrovascular apoplexy may occur. Patients with nonclassic 11b-hydroxylase deficiency have normal blood pressure. Rare cases of classic 11b-hydroxylase deficiency who presented with signs of mineralocorticoid deficiency before treatment have been reported. The mechanism by which this occurs is not fully understood [43, 49]. Hormonal Findings in 11b-Hydroxylase Deficiency. Due to deficient 11bhydroxylation, 11-deoxycortisol(s) and DOC are not efficiently converted to cortisol and corticosterone, respectively. Decreased cortisol production leads via poor feedback control to increased ACTH secretion. This stimulates the zona fasciculata to overproduction of steroid precursors prior to the block. These precursor steroids are excreted in the urine as tetrahydro metabolites (THS and THDOC), but the greater part of the massively elevated 11-deoxycortisol and its precursor 17-OH-progesterone is shunted into the androgen pathway, resulting in marked androgen excess and virilisation. Because 11deoxycorticosterone and certain metabolites, e.g. 19-Nor-DOC, are mineralocorticoid agonists, plasma renin activity is suppressed and levels of aldosterone are low even though the ability to synthesise aldosterone is not impaired [50]. In contrast to carriers of 21-hydroxylase deficiency alleles, approximately 80% of whom have elevated 17-OH-progesterone levels after ACTH, the findings in carriers of 11b-hydroxylase deficiency alleles are inconsistent. Two reports did not find any hormonal changes in these individuals [51, 52], whereas our group demonstrated increased responses of plasma 11-deoxycortisol and 11-deoxycorticosterone in the short term ACTH test in family members heterozygous for the R448H mutation using highly specific hormone assays after extraction and chromatography [53]. Diagnostic Recommendations for 11b-Hydroxylase Deficiency. The diagnosis might be suspected in genetic females presenting with ambiguous genitalia at birth or in apparently normal male infants with unpalpable gonads. The specific hormonal diagnosis of 11b-hydroxylase deficiency can be made by detecting high basal or ACTH-stimulated plasma levels of 11-deoxycorticosterone and 11-deoxycortisol. An ACTH test is not always necessary in patients with classic 11b-hydroxylase deficiency because they have greatly elevated plasma levels of 11-deoxycorticosterone and 11-deoxycortisol even in the basal state in the presence of low cortisol. Some patients may have selective elevation of 11-deoxycorticosterone or 11-deoxycortisol [43]. The diagnosis can also be made by detecting increased excretion of tetrahydro-metabolites of these compounds in a 24-hour urine sample [43, 54]. The correct diagnosis of 11bhydroxylase deficiency may be missed if the mild-to-moderate elevations of

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17-OH-progesterone, which are regularly found in 11b-hydroxylase deficiency, lead to the diagnosis of 21-hydroxylase deficiency or if 11-deoxycorticosterone and 11-deoxycortisol are not specifically measured [55]. Prenatal Diagnosis and Treatment in Pregnancies at Risk for 11b-Hydroxylase Deficiency. Classic 11b-hydroxylase deficiency can be prenatally diagnosed by measuring 11-deoxycorticosterone and 11-deoxycortisol in the amniotic fluid [56–58]. This approach has been made in pregnancies known to be at risk because of the prior birth of an affected child. It is also possible to diagnose this disorder prenatally by mutational analysis of DNA obtained from amniocentesis or chorionic villus biopsies [59, 60]. This approach is, however, limited to families at risk, because the mutations are widely distributed within the CYP11B1 gene. In addition to genetic counselling, prenatal treatment can be offered to the families in order to prevent masculinisation of affected female fetuses. The same rationale and recommendations for prenatal treatment as in CAH due to 21-hydroxylase deficiency [61] are valid for 11b-hydroxylase deficiency. Results of prenatal dexamethasone treatment in affected 11b-hydroxylase deficiency fetuses have been reported at two conferences (79th Annual Meeting of the Endocrine Society 1997, Minneapolis, USA, and Conference on the Unborn Child 1998, Tampa, USA) and in one original paper [62]. It is still an experimental type of treatment and both the physical and psychological development of the children has to be evaluated in long-term follow-up studies [63]. Molecular Genetics of 11b-Hydroxylase Deficiency. The first mutation described in patients with the classical form was a single base exchange in codon 448 leading to an amino acid substitution Arg-448-His. Arg-448 is adjacent to Cys-450, which is the fifth ligand of the heme iron atom [64]. Subsequently, more than 30 different mutations in the CYP11B1 gene have been identified (fig. 3). The mutations are located in regions of known functional importance of the enzyme. Transfection experiments with mutant CYP11B1 constructs have shown that these mutations abolish enzymatic activity [59, 60, 65–72]. To date, there is only one published study in patients with nonclassic 11bhydroxylase deficiency. Joehrer et al. [47] detected mutations in two of three patients with nonclassic 11b-hydroxylase deficiency, one patient being a compound heterozygote for N133H and T319M, whereas the other carried a nonsense mutation (Y423X) on one allele and a missense mutation P42S on the other. The three missense mutations P42S, N133H and T319M reduced 11b-hydroxylase activity in vitro to 15, 17 and 37% of wild-type activity, respectively [47]. The same authors, however, were not able to detect CYP11B1 mutations in hirsute women with mildly elevated levels of 11-deoxycortisol.

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Fig. 3. Schematic representation of the genomic structure of the human CYP11B1 gene and positions of mutations reported to date. Exons are represented by boxes; black boxes demarcate the coding regions, and open boxes represent the non-coding regions.

We made the same observation in five hirsute female adolescents and adults with mildly elevated 11-deoxycortisol levels after ACTH stimulation. No close correlation between phenotype and genotype can be observed in classic and nonclassic 11b-hydroxylase deficiency. In the group of patients from Israel, all carrying the same homozygous R448H missense mutation, significant variations in the severity of hypertension, the degree of virilisation and the plasma levels of 11-deoxycorticosterone and 11-deoxycortisol were observed [44]. Treatment and Management of 11b-Hydroxylase Deficiency. Cortisol deficiency is replaced by glucocorticoid administration which reduces ACTH secretion and adrenal androgen production. In addition, the ACTH-dependent excess of aldosterone precursors (mainly DOC) will be suppressed by this treatment. Oral hydrocortisone is recommended since it is identical to the physiological glucocorticoid. A total of 15–25 mg/m2/day are given in 3 doses with approximately 50% in the early morning. An immediate 3- to 5-fold increased dosage in illness or stress is important. Additional antihypertensive treatment may be required if hypertension has been of long duration before treatment. Drugs which can be used comprise spironolactone, amiloride and calcium channel blockers.

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For all girls born with virilised genitalia sex assignment is a critical issue. To avoid any doubt about the gender of rearing it is necessary that the correct diagnosis is made within a short period after birth. The correction of ambiguous genitalia should be performed by an experienced paediatric surgeon. Aldosterone Synthase Deficiency Pathophysiology and Frequency of Aldosterone Synthase Deficiency. Aldosterone synthase deficiency is an autosomal recessively inherited disorder caused by mutations in the CYP11B2 gene. Due to deficient adrenal zona glomerulosa aldosterone synthase activity, 11-deoxycorticosterone is not efficiently converted to aldosterone. Insufficient aldosterone secretion leads to decreased sodium resorption from and potassium secretion into the urine. Precise data regarding the frequency of selective hypoaldosteronism and its two biochemical types in the general population are not available. It is generally assumed that aldosterone synthase deficiency type II is more frequent than aldosterone synthase deficiency type I. This is mainly due to the observation of Ro¨sler [73] who published data of 21 patients of Iranian Jewish origin with selective aldosterone deficiency due to aldosterone synthase deficiency type II. This preponderance of aldosterone synthase deficiency type II deficiency is also reflected in the overall published case reports [5, 6, 74–82]. In contrast to this, our material which is the largest series of unrelated patients diagnosed strictly by the same methodology, indicates a comparable frequency of both types of aldosterone synthase deficiency [83, 84]. Clinical Presentation of Aldosterone Synthase Deficiency. All affected children present with frequent vomiting, failure to thrive, and severe, lifethreatening salt loss in the first weeks of life. In most of the published clinical case reports no differences in the severity of clinical signs between young infants with aldosterone synthase deficiency type I and aldosterone synthase deficiency type II have been observed [5, 6, 74–82]. Ro¨sler [73] has shown that in each affected individual the clinical severity of the disease decreases with age. Adolescents and adults may show only the abnormal steroid pattern which persists throughout life [83]. Hormonal Findings in Aldosterone Synthase Deficiency. The typical steroid profile in patients with aldosterone synthase consists of low to undetectable aldosterone plasma levels and elevated mineralocorticoid precursor levels prior to the block (corticosterone and 11-deoxycorticosterone). Decreased aldosterone production leads to increased renin secretion via poor feedback control. Glucocorticoid biosynthesis is undisturbed, but due to stress cortisol might be elevated in untreated patients in a salt-losing state. Using our method of plasma multisteroid analysis by RIA after extraction and automated Sephadex LH-20 multi-column chromatography [86],

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we diagnosed 30 sporadic cases of congenital isolated hypoaldosteronism [82–85]. Using specific steroid determinations, the plasma level of 18-hydroxycorticosterone distinguishes between aldosterone synthase deficiency type I (where it is decreased or low-normal) and aldosterone synthase deficiency type II (where it is markedly elevated). The clearest distinguishing parameter between the two aldosterone synthase deficiency types reflecting impaired 18-hydroxylation is the ratio of plasma corticosterone/18-hydroxycorticosterone, which is elevated (?40) in aldosterone synthase deficiency type I and decreased (=10) in aldosterone synthase deficiency type II. The ratio of plasma 18-hydroxycorticosterone/aldosterone can also discriminate between the two aldosterone synthase deficiency variants (type I =10; type II ?100). The disadvantage of this ratio is that plasma aldosterone levels in these patients are often at or even below the lower limit of detection [83]. Diagnostic Recommendations for Aldosterone Synthase Deficiency. For practical reasons, we recommend the following procedure for the diagnosis of aldosterone synthase deficiency in young infants. All salt-wasting neonates should be examined carefully. In female newborns with ambiguous genitalia and in all male neonates first of all, 21-hydroxylase deficiency should be excluded by measuring basal plasma 17-hydroxyprogesterone (RIA after extraction). Secondly, basal plasma cortisol, aldosterone, and renin (PRA or active renin concentration) should be determined. Highly specific methods (RIA after extraction and chromatography) for the determination of plasma steroids (particularly for aldosterone) are necessary in the early life period. Many direct RIA methods yield far too high results in neonates and young infants, due to vast amounts of interfering steroids from the fetoplacental unit and/or the still active fetal adrenal cortex. In children with low or undetectably low plasma aldosterone and elevated renin, corticosterone should be determined next. A high ratio of corticosterone over aldosterone leads to the diagnosis of aldosterone synthase deficiency. The ratios of 18-hydroxycorticosterone/aldosterone and corticosterone/18-hydroxycorticosterone are necessary for further differentiation of aldosterone synthase deficiency types I and II. An ACTH test is not necessary for the diagnosis. The diagnosis can also be made by capillary gas chromatography of a 24-hour urine sample [87] which is, however, difficult to collect in neonates. Molecular Genetics of Aldosterone Synthase Deficiency. The molecular basis of aldosterone deficiency has been elucidated in patients with different genetic backgrounds (fig. 4). In type I deficiency, five mutations of the CYP11B2 gene have been described, all except one completely abolishing the activity of aldosterone synthase: Three patients of a consanguineous Amish kindred have a deletion

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Fig. 4. Schematic representation of the genomic structure of the human CYP11B2 gene and positions of mutations reported to date. Exons are represented by boxes; black boxes demarcate the coding regions, and open boxes represent the non-coding regions. The mutations V35delTGCTC, E198D plus V386A, E255X, R384P and L461P have been identified in patients with aldosterone synthase deficiency type I. The mutations R181W and V386A (double homozygous) and T185I (homozygous), the homozygous deletion R173, and the compound heterozygous mutations T318M and T372D1nt have been described in patients with aldosterone synthase deficiency type II.

of 5 nucleotides in exon 1 of CYP11B2, resulting in a frameshift to form a stop codon in the same exon [88]. A boy of a nonconsanguineous German family was identified as having an R384P amino acid substitution [89]. Another single-point mutation CTG (leucine) to CCG (proline) has been identified in a Turkish patient [90]. In 2 of the 3 patients with congenital hypoaldosteronism originally published by Visser and Cost in 1964, we recently identified a homozygous single base exchange (G to T) in codon 255 (GAG), causing a premature stop codon E255X (TAG) [85]. Interestingly, we found the same mutation in a Turkish family with aldosterone synthase deficiency type I [91]. One study in an individual carrying two homozygous mutations, E198D in exon 3 and V386A in exon 7, reported results for the hormonal phenotype suggesting aldosterone synthase deficiency type I (18-hydroxylase deficiency) which were inconsistent with the in vitro transfection data which indicated aldosterone synthase deficiency type II (18-oxidase deficiency) [92]. Aldosterone synthase deficiency type II is frequently observed among Jews of Iranian origin. All affected individuals are homozygous for two missense mutations in CYP11B2, R181W in exon 3 and V386A in exon 7. Individuals

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homozygous for either mutation alone have no clinical symptoms [93, 94]. Zhang et al. [95] reported a patient with a typical clinical and hormonal picture of aldosterone synthase deficiency type II; direct sequencing of PCR products showed that the mother’s allele contributed R181W and the deletion/ frameshift mutation T372D1nt, while the father’s allele contributed T318M and V386A. Two further CYP11B2 mutations have been published, a single amino acid deletion of codon 173 in exon 3 [82] and one single amino acid substitution T185I in exon 3 [96]. Fardella et al. [97] described a gene conversion event (between CYP11B2 and CYP11B1) in 3 of 4 unrelated P450c11Aldo alleles from 2 unrelated patients with aldosterone synthase deficiency type II, by which exons 3 and 4 of the CYP11B2 gene encoding P450c11Aldo were changed to the sequence of the nearby CYP11B1 gene which encodes the related enzyme P450c11. This conversion resulted in a mutant P450c11Aldo protein carrying three changes: Asp-141KGlu, Lys-151KAsn, Ile-248KThr. Expression systems containing these mutants all retained normal 18-oxidase activity of P450c11Aldo, indicating that the detected gene conversion event is associated with, but does not cause, aldosterone synthase deficiency type II [97]. In the group of patients we have collected over the past decade, we identified a wide spectrum of missense and nonsense mutations in aldosterone synthase deficiency type I as well as aldosterone synthase deficiency type II patients [82, 85, 89, 91, 96]. However, we found 3 patients with aldosterone synthase deficiency type II who did not carry any mutation in their entire CYP11B2 alleles [91]. In vitro studies have shown a total loss of 18-hydroxylase activity of P450c11Aldo in almost all aldosterone synthase deficiency type I alleles [85, 88– 90]. However, one study reported inconsistent results between the hormonal phenotype (type I>18-hydroxylase deficiency) and the in vitro transfection study (type II>18-oxidase activity) [92]. In aldosterone synthase deficiency type II, the R181W mutation in CYP11B2 does not impair 11b-hydroxylase activity, while it markedly decreases 18-hydroxylase activity and abolishes 18-oxidase activity. The V386A mutation in CYP11B2 causes a small but consistent reduction in 18-hydroxylase activity [94]. The two CYP11B2 mutations identified in a patient with aldosterone synthase deficiency type II (T372D1nt and T318M) by Zhang et al. [95] showed no measurable enzyme activity in transfection experiments. The two variants of aldosterone synthase deficiency are caused by different mutations within the same gene. On the basis of the data published to date, it is not possible to explain the two different hormonal phenotypes observed in patients with congenital hypoaldosteronism on the molecular level. Further molecular studies on a larger number of aldosterone-deficient patients are necessary to elucidate the structure/function relationship of the enzyme

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P450c11Aldo. Other factors besides P450c11Aldo might be involved in the genesis of the distinctive phenotypes of aldosterone synthase deficiency types I and II. The fact that some patients with congenital hypoaldosteronism do not have mutations in their CYP11B2 alleles [91, 97] raises the question as to whether other enzymes, also involved in mineralocorticoid biosynthesis in humans, may be defective in this disorder. The homologous P450 enzymes aldosterone synthase and 11b-hydroxylase differ in 35 amino acids. Amino acid residues have been identified in a recent study using transfection experiments with cDNAs which encode hybrids between the highly homologous cytochrome P450 enzymes, P450c11 (11b-hydroxylase) and P450c11Aldo (aldosterone synthase), determining the different catalytic activities of both enzymes. Efficient 18-hydroxylation requires a glycine residue at position 288 and subsequent sufficient 18-oxidation requires an alanine at position 320 [98]. Treatment and Management of Aldosterone Synthase Deficiency. Patients recover fully under treatment with an oral mineralocorticoid. Replacement therapy with 9a-fluorocortisol (fludrocortisone) in doses of 100–250 lg/m2/ day is recommended, at least during early infancy and childhood. In addition, generous sodium supplementation may be given. Continued mineralocorticoid replacement therapy after childhood is not always necessary, as shown by the clinical observation that compensatory extra-renal salt-conserving mechanisms mature with age. The same natural course of the disease is observed in many patients with pseudohypoaldosteronism [73].

Acknowledgements The authors thank Mrs. Gisela Hohmann, Susanne Olin, and Sabine Stein for expert technical assistance. This work was supported by grants Pe 589/1-1 and Pe589/1-2 from the Deutsche Forschungsgemeinschaft (DFG) and by an additional grant from the Fritz-ThyssenStiftung. We also thank Mrs. Joanna Voerste for linguistic editing of this manuscript.

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Prof. Wolfgang G. Sippell, MD, Division of Paediatric Endocrinology, Department of Paediatrics, University of Kiel, Schwanenweg 20, D–24105 Kiel (Germany) Tel. +49 431 597 1626, Fax +49 431 597 1675, E-Mail [email protected]

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X-Linked Adrenoleukodystrophy Jutta Ga¨rtner Department of Paediatrics, Heinrich Heine University, Du¨sseldorf, Germany

History In 1923, Siemerling and Creutzfeldt [1] described the first patient who developed a bronzed skin at the age of 4 years, followed by progressive neurological dysfunction leading to death at the age of 7 years. His postmortem examination showed adrenal atrophy and extensive demyelination in the central nervous system. In 1963, Fanconi et al. [2] revealed the X-linked recessive inheritance mode, and in 1970 Blaw [3] introduced the name ‘adrenoleukodystrophy’ (ALD) based on the striking association of a leukodystrophy with adrenal insufficiency. Only a few years later, Igarashi et al. [4] and Powers and Schaumberger [5] described the characteristic tissue accumulation of saturated very long chain fatty acids in adrenal cells of these patients. This observation led to the assumption that X-linked ALD is an inborn error of fatty acid metabolism. In 1986, Lazo et al. [6] suggested that the impaired fatty acid oxidation is due to deficient function of the peroxisomal matrix enzyme lignoceroyl-CoA synthetase. Since purification of the peroxisomal lignoceroyl-CoA synthetase proved difficult, Mosser et al. [7] used a positional cloning approach and isolated the gene in 1993. It came as a surprise that the gene encodes a peroxisomal membrane protein with homology to the ATP-binding cassette (ABC) transporter proteins, rather than to the expected lignoceroyl-CoA synthetase. This protein, now referred to as adrenoleukodystrophy protein (ALDP), belongs to the same protein family as the cystic fibrosis protein and the multidrug resistance protein [8].

Clinical Phenotypes X-linked adrenoleukodystrophy (ALD; McKusick 300100) is the most common inherited peroxisomal disorder. The estimated incidence ranges from

Fig. 1. Pedigree showing the extreme phenotypic variability in a family with X-linked adrenoleukodystrophy.

one in every 15,000 to one in every 100,000 males [9–11]. The current clinical classification of the wide range of phenotypic manifestations has as criteria the age of onset, the organs involved, and the rate of progression of neurological symptoms. There are at least six distinct types ranging in decreasing order of severity from the childhood cerebral form to asymptomatic persons. The various clinical phenotypes commonly occur within the same kindred (fig. 1). Epidemiological analyses of the relative phenotype frequencies revealed that the childhood cerebral form is the most common in France, the United States and Canada, whereas adrenomyeloneuropathy (AMN) is the most frequent phenotype in the Netherlands [9, 11]. Childhood Cerebral ALD The childhood cerebral form is the most severe phenotype. Patients seem unaffected until the age of 2–10 years, when there is onset of adrenal insufficiency and progressive neurological dysfunction [12, 13]. Frequent initial symptoms include emotional lability, hyperactive behavior, school failure, impaired auditory discrimination and difficulties in vision. After onset of symptoms the course is rapidly progressive, leading to an apparently vegetative state within 2–4 years and to death at varying intervals thereafter.

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Adolescent Cerebral ALD Patients with the adolescent cerebral form usually develop initial symptoms between ages eleven and twenty one [12, 13]. Clinical symptoms and deterioration resemble those of the childhood cerebral form. Adult Cerebral ALD The adult cerebral form occurs in patients with an age of onset beyond 21 years [12, 13]. The clinical symptoms and the rate of progression resemble those of the childhood cerebral form. These patients are often misdiagnosed as schizophrenia or other psychiatric disorders. Adrenomyeloneuropathy (AMN ) The age at onset of AMN is the second to fourth decade of life. The disease mainly involves the spinal cord and presents with slowly progressive stiffness and weakness of legs, impaired vibration sense, sphincter disturbances and impotence [12, 13]. Adrenal insufficiency is present in two thirds of the patients. Cerebral changes develop in approximately half of the patients, and then the course of the illness resembles that of the other cerebral ALD forms. In patients without cerebral involvement, intellectual functions may be entirely preserved and life expectancy appears normal. AMN is often misdiagnosed as multiple sclerosis or familial spastic paraparesis. Addison Only 10–20% of ALD patients have primary adrenal insufficiency without evidence of nervous system involvement [12, 13]. These patients are at high risk of eventually developing AMN. At present, the oldest described Addison-only patient is 78 years old [12]. Asymptomatic Some patients with the genetic defect of ALD are free of adrenal insufficiency and neurologic disability despite the presence of highly elevated saturated very long chain fatty acid levels. These patients are still at high risk of eventually developing adrenal insufficiency and/or neurologic symptoms [12, 13]. At present, the oldest described asymptomatic males are in their 60s [13]. ALD Heterozygotes More than half of the women who are heterozygous for ALD have neurologic involvement most likely due to nonrandom X inactivation favouring the mutant allele in heterozygous cells [12–14]. The mean age of onset is in the fourth decade. Except for milder clinical symptoms and a slower rate of

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progression, the clinical course of symptomatic ALD heterozygotes resembles that of AMN patients. Most ALD heterozygotes with paraparesis had previously been diagnosed as having multiple sclerosis. Cerebral involvement and adrenal insufficiency are relatively rare.

Pathological Phenotypes Nervous System Postmortem studies of the central nervous system in the cerebral forms of ALD show that the cortex is usually intact but that the myelin is replaced by grey to brown firm, translucent tissue [15]. These demyelinating lesions are confluent, often bilaterally symmetric and show in the majority of patients a caudorostral progression. They exhibit marked loss of oligodendrocytes and myelinated axons with reactive astrogliosis. In addition, diffuse infiltration and large perivascular cuffs of mononuclear cells, mostly lymphocytes, are a characteristic feature of the cerebral forms of ALD and resemble those observed in multiple sclerosis. Pathological changes in isolated AMN as well as in symptomatic ALD heterozygotes are restricted to the spinal cord. The loss of axons and myelin is greatest in the lumbar corticospinal, cervical gracile, and dorsal spinocerebellar tracts. The inflammatory response is absent or less prominent than in the cerebral forms. In all forms of ALD peripheral nerve involvement is less severe. Sural and peroneal nerves may display a loss of myelinated fibres with endoneurial fibrosis. Adrenal Glands and Testes The adrenal glands show a marked atrophy of the cortex which is progressive [5]. The cells are striated because of lamellate lipid inclusions. There is no accumulation of inflammatory cells and the medulla is preserved. Postpubertal males have lipid inclusions in Leydig cells similar to those observed in adrenocortical cells. Changes in seminiferous tubules are non-specific and include tubular atrophy, vacuolation of Sertoli cells and loss of germ cells.

Biochemical Phenotypes Abnormally high levels of saturated very long chain fatty acids (VLCFA; carbon chains longer than 22 atoms) are the principal biochemical abnormality in patient tissues and body fluids [16, 17]. The VLCFA that accumulate derive from both the diet and endogenous synthesis. They are saturated and unbranched, and involve mainly those with a carbon chain length of 24 (tetraco-

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Fig. 2. Topological model of the adrenoleukodystrophy protein as predicted by hydropathy analyses of the deduced amino acid sequence. The protein has six putative transmembrane domains. The predicted nucleotide-binding fold is indicated as loop containing an ATP site.

sanoic acid, C24:0) and 26 (hexacosanoic acid, C26:0). This accumulation has a specific pattern. Although some degree of excess is present in nearly all tissues, the most striking increases are in brain white matter, adrenal cortex and testis. The biochemical defect of increased VLCFA levels in ALD patients is localised to the level of lignoceroyl-CoA synthesis, a step in the peroxisomal b-oxidation of VLCFA [6]. Aside from defective oxidation of VLCFAs, peroxisome function and structure are normal.

Primary Gene Defect As early as 1981, Migeon et al. [14] found close linkage of the X-linked ALD locus to the glucose-6-phosphate dehydrogenase (G6PD) gene and to the DXS52 marker in the Xq28 region. In 1993, Mosser et al. [7] identified the responsible gene for ALD by positional cloning. The ALD gene comprises 10 exons spanning approximately 21 kb. The cDNA is 2750 bp and detects a 4.2-kb transcript on RNA blots. The encoded 75-kD protein consists of 745 amino acids and is homologous to members of the ATP-binding cassette (ABC) transporter protein family. Proteins of this family are involved in transport across cellular and subcellular membranes of a wide variety of ligands ranging in size from ions to proteins [8]. The amino-terminal half of the ALD protein (ALDP) is hydrophobic with six putative transmembrane domains, the carboxy-terminal half is hydrophilic and contains the Walker A and B sequence motifs characteristic for nucleotide-binding folds (fig. 2). In many

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eucaryotic ABC transporters these structural components are repeated in tandem so that a single polypeptide has four domains, two hydrophobic and two hydrophilic. Therefore, ALDP should be considered a half ABC transporter. Our sequencing data show that ALDP is highly homologous to three other members of the ABC transporter protein family, namely the ALD related protein (ALDR), the 70-kD peroxisomal membrane protein (PMP70) and the PMP70 related protein (P70R) [18–20]. Thus, there are at least four different half ABC transporters in the mammalian peroxisome membrane. The existence of this collection of half ABC transporters in the peroxisomal membrane raises the possibility of formation of functional heterodimeric transporters by combinatorial assembly as shown in the yeast peroxisomal system. Pxa1p and Pxa2p are yeast homologues of the mammalian peroxisomal half ABC transporters and are located in the yeast peroxisome membrane. These proteins assemble to form a complete ABC transporter functioning in transporting long chain fats (C16–18) or fatty acyl CoAs into the peroxisomal matrix [21, 22]. Disruption of Pxa1p or Pxa2p, or both, result in impaired peroxisomal b-oxidation.

Patient Genotypes Several studies have shown that virtually all ALD patients have mutations in the ALD gene, and more than 200 mutant alleles have been identified and characterised (reviewed by Ga¨rtner et al. [23]). Although the nature and distribution of these changes over the functional ALDP domains are highly heterogeneous, there is a tendency to clustering close to and in the nucleotidebinding fold (fig. 3). The most common mutation is the AG deletion at nucleotide 1801 and 1802 in twenty families resulting in a frameshift at amino acid 472. The majority of ALD gene mutations result in the complete loss of immunologically detectable ALDP, suggesting they either destabilise the protein or interfere with targeting of the protein to the peroxisomal membrane. Transfections with wild-type ALD cDNA led to the reappearance of immunoreactive material in ALD patient cells, and to normalisation of the capacity to metabolise saturated very long chain fatty acids [24, 25]

Aetiology and Pathogenesis It is now established that alterations in the ALD gene do indeed represent the primary defect in X-linked ALD. Given that the gene product is a putative half ABC transporter in the peroxisomal membrane, mutations in the ALD

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Fig. 3. Distribution of mutations in the ALD gene. The boxes represent the coding region of 472 amino acids and the putative functional domains (TMS>transmembrane segment; EAA>fatty acid binding motif; PTS>peroxisomal targeting signal). The mutation data analyses from 227 families disclose 51% missense mutations, 22% frameshifts, 10% nonsense mutations, 7% deletions over 0.5 kb, 4% insertions or deletions smaller than 0.5 kb, 4% polymorphisms and 2% splice site changes.

gene have an indirect effect on the peroxisomal b-oxidation of very long chain fatty acids. Based on the peroxisomal yeast proteins Pxa1p and Pxa2p, the ALD protein may heterodimerize in the peroxisomal membrane to form a functional transporter for activated very long chain fatty acids (fig. 4). Alternatively, the primary function of the ALD protein may involve transport of cofactors required for the peroxisomal b-oxidation or for the association of very long chain fatty acids with the peroxisomal membrane. Mutations in the ALD gene may impair the putative peroxisomal import function of the encoded protein by affecting protein stability, ATP sites or substrate binding. Western blot and immunofluorescence analyses have shown that the majority of ALD mutations results in the complete loss of detectable ALD protein [23]. More than two thirds of mutations described are within functional protein domains like the nucleotide-binding domain, the transmembrane segments, the possible fatty acid binding domain or the peroxisomal targeting signal (fig. 3). Considering the importance of ATP binding and hydrolysis for transport, mutations within this region may impair transport function by abolishing ATPase activity or by altering ATP binding affinity. Mutations within the transmembrane segments or the possible fatty acid binding motif may alter protein function by direct interaction with the primary substrate binding sites or by a change in substrate specificity or affinity. It is tempting to imagine a model of the tertiary structure of the ALD protein, in

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Fig. 4. Hypothetical model for the mechanism of transport of activated very long chain fatty acids across the peroxisomal membrane. Using energy transduced from the ATP binding site, the substrates are transported from the cytosol into the peroxisomal matrix through a channel like structure.

which the widely separated functional relevant amino acids in the primary structure are part of the same pore, channel or ATP site involved in transport of peroxisomal matrix components (fig. 4). However, the extreme variability in clinical phenotype, even in individuals in the same family with the same genotype (fig. 1), indicates that other factors strongly influence the phenotype. The distribution of phenotypes within ALD families is consistent with a model in which a highly polymorphic genetic modifier plays a major role in determining phenotypic severity [26]. Although there is no doubt that mutations in the ALD gene result in impairment of peroxisomal b-oxidation and accumulation of saturated very long chain fatty acids in ALD patient cells and body fluids, it is uncertain whether the saturated very long chain fatty acid excess per se plays a role in pathogenesis. While the adrenal dysfunction appears to be directly attributable to the accumulation of VLCFA, the pathogenesis of the central nervous system pathology seems to be more complex [27]. Brain inflammatory reactions have been described as the additional characteristic feature in patients with the severe cerebral forms; these reactions are less common or absent in the slowly progressive or stationary ALD forms [13]. Thus, the excess of saturated very long chain fatty acids may alter the structure or function of the cell membrane and modulate the severity of the immunologic cellular responses.

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Fig. 5. Diagnostic flow chart for X-linked adrenoleukodystrophy.

Diagnostic Tools The initial diagnosis of X-linked ALD relies on the clinical presentation, brain imaging and biochemical analyses of very long chain fatty acids (fig. 5). Clinical Presentation The most common initial clinical symptoms suggestive of X-linked ALD are in the order of decreasing frequency behavioral changes, intellectual deterioration, impaired vision, impaired hearing, speech difficulty, handwriting changes, gait abnormality, seizures and limb weakness [13]. Imaging Techniques Magnetic resonance imaging scans of the brain are obtained as part of the evaluation of clinically suggestive patients. Those with the cerebral form of the disease show characteristic white matter lesions. In the majority of cases, these lesions are symmetric and involve the corpus callosum and the periventricular parietooccipital white matter (fig. 6).

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Fig. 6. T2-weighted magnetic resonance imaging scan of the brain in a 5 year old boy with the childhood cerebral form of X-linked adrenoleukodystrophy. The characteristic demyelinating lesions are symmetric and localised in the occipito-parietal lobes.

Biochemical Assays The impairment of peroxisomal b-oxidation and the accumulation of saturated very long chain fatty acids in tissues and body fluids of patients is pathognomonic for X-linked ALD. Analyses of the plasma very long chain fatty acid levels, including lignoceric acid (C24:0), hexacosanoic acid (C26:0) and their ratios to behenic acid (C22:0), are used to confirm the diagnosis in patients suspected to suffer from the disease [55]. In addition, the majority of patients has clinical or laboratory evidence of adrenal insufficiency and the adrenal function should be assessed in all cases. The presence of adrenal insufficiency is suggestive of X-linked ALD because it is not a feature of other leukodystrophies from which the disease has to be differentiated. Mutation Analyses Since the discovery of the ALD gene in 1993 [7], more than 150 different gene alterations have been identified in patients with this disorder. Knowledge

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Table 1. Therapeutic approaches in X-linked adrenoleukodystrophy Traditional pharmacology Steroids ‘Lorenzo’s oil’ (glyceryl trioleate, glyceryl trierucate) Immunosuppression (b-interferon, immunoglobulins) Others (pentoxifyline, thalidomide) Genetic approaches Allogenic bone marrow transplantation Autologous bone marrow transplantation Pharmacological gene therapy

of the mutation causing ALD in a given family permits diagnosis of disease or heterozygote status by direct DNA based mutation analyses techniques. According to Moser et al. [13] only 80% of obligate heterozygous women had increased concentrations of plasma saturated very long chain fatty acids. Mutation analyses will be surely helpful in these female carriers and ensure accurate genetic counselling. Prenatal Diagnosis Saturated very long chain fatty acids are readily quantified in fetal material, including amniotic fluid, cultured amniotic fluid cells and chorionic villus samples. These biochemical analyses have been successfully performed in more than 200 pregnancies [13]. However, misdiagnoses have been reported. Two affected male fetuses had normal chorionic villus cell saturated very long chain fatty acid profiles [28, 29]. DNA based mutation detection techniques have now provided a quite facile and very accurate tool to identify the genotype of a fetus at risk.

Therapeutic Approaches The increasing activity in the field of molecular genetics and the better understanding of disease pathogenesis promote the attempts at devising effective therapies. Table 1 summarises the therapeutic approaches in X-linked ALD. At present, steroid replacement for adrenal insufficiency is the only effective and readily available therapy. In contrast, traditional pharmacological approaches including Lorenzo’s oil and immunosuppression are of little, if any benefit. Other specific therapies are under evaluation including bone marrow transplantation, gene replacement and pharmacological gene therapy.

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Symptomatic Therapy Although symptomatic therapy does not correct the basic genetic defect, the patient’s current status often ameliorates. In the early disease stage characterised by subtle intellectual and behavioural changes, patients mainly need the assistance of parents, teachers and psychologists. As the disease progresses, major concerns are an increased muscle tone, changes in the sleep wake cycle and bulbar muscle dysfunction. In most patients adequate nutrition has to be maintained by gastrostomy feeding. Dietary Therapy A dietary therapy designed to restrict the intake of very long chain fatty acids was initiated in 1980, after the observation that orally administered labelled hexacosanoic acid accumulated in the brain of a terminally ill patient with childhood cerebral ALD [30]. However, this diet failed to lower plasma concentrations of saturated very long chain fatty acids. The normalisation of the plasma concentration of saturated very long chain fatty acids was achieved when the dietary restriction was combined with the oral supplementation of glyceryl trioleate (GTO) and glyceryl trierucate (GTE), presumably by inhibiting the endogenous fatty acid elongation system [31]. The 4:1 mixture of GTO and GTE oils is often referred to as ‘Lorenzo’s oil’ in recognition of the patient Lorenzo Odone whose parents initiated the development of this therapeutic approach. Therapeutic trials with GTO-GTE oil have been conducted worldwide involving more than 500 patients. The results have been disappointing. The diet failed to halt the neurologic progression and did not improve the endocrine dysfunction in patients with childhood cerebral ALD, AMN or symptomatic heterozygous women [11, 32–34]. Immunosuppression and Other Drug Therapies The extent and severity of white matter changes in the cerebral form of disease seem to correlate with the brain inflammatory response mediated by as yet unknown cytokins or immune mechanisms [13]. All therapeutic trials conducted to modify the inflammatory response did not reveal a relevant clinical benefit from beta interferon, cyclophosphamide, cyclosporin, immunoglobulins, pentoxifylline, and thalidomide [13, 35, 36]. Bone Marrow Transplantation Colonisation of the brain by cells of the monocyte macrophage system provide the rationale for the use of bone marrow transplantation in X-linked ALD [37]. The bone marrow derived hematopoietic cells can enter the central nervous system and undergo transformation to microglial cells. Thus the donor cells, containing the ability to degrade very long chain fatty acids or to provide

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the favorable modifier substance to prevent demyelination, serve as an exogenous source of corrective factors. The permanent engraftment of the bone marrow cells provides a continuously renewable source of corrective factors that may halt and even possibly reverse the brain pathology in X-linked ALD. The first bone marrow transplants in patients with childhood cerebral ALD in an advanced stage were unsuccessful [38]. The procedure was reevaluated in an 8-year-old boy with early neurologic involvement [39]. His non-identical twin was the donor. Two years after transplant, the neurologic deficits and the white matter lesions had disappeared. His cognitive function was equal to that of the twin. The plasma concentration of saturated very long chain fatty acids had normalised on a regular diet. Bone marrow transplantation has now been used to treat more than 100 ALD patients [40, 41]. More than half of the patients who survived did improve or stabilise. About 30% of patients died during or shortly after the procedure of graft-vs.-host disease, immunosuppression or rapidly progressive neurologic disease. Although bone marrow transplantation has a relatively high mortality risk, at this time it provides the only permanent cure when successful. Bone marrow transplantation seems to be an appropriate treatment method for those patients who show evidence of a very early cerebral involvement and for whom a well matched donor is available. Perspective of Gene Therapy The therapeutic success of bone marrow transplantation in some patients with X-linked ALD has shown that the disease can be cured by replacing the patient’s defective hematopoietic stem cells with genetically normal stem cells from another individual. The high risk of allogeneic bone marrow transplantation and the often futile search for well matched donors provide the impetus for the development of somatic gene therapy. One possible strategy is bone marrow ablation followed by autologous transplantation with genetically corrected hematopoietic cells of the patient’s own bone marrow. Preliminary experiments showed that virally mediated transfer of cDNA encoding ALDP restored peroxisomal b-oxidation metabolism in X-linked ALD patient fibroblasts and hematopoietic stem cells [25, 42]. A new approach, pharmacological gene therapy, has also shown promise in the treatment of X-linked ALD. This strategy uses drugs to increase the expression of genes that are functionally related to the disease gene and can complement its function. b-Oxidation of lignoceric acid (C24:0) is deficient in patient fibroblasts. Several groups have shown that the peroxisomal b-oxidation defect can be restored by expression of either the ALD protein or one of the other peroxisomal ABC transporter proteins, namely the PMP70, ALDP or ALDR protein [25, 43]. This effect of restoring peroxisomal b-oxidation could

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also be achieved by 4-phenylbutyrate treatment of X-linked ALD patient cells and knockout mice [44]. In X-linked ALD mice 4-phenylbutyrate treatment for 4 weeks resulted in a substantial reduction of C24:0 and hexacosanoic acid (C26:0) in brain and adrenal tissue when compared to untreated animals. Furthermore, the same treatment for 6 weeks resulted in a complete correction of C24:0 and an 80% reduction of C26:0 in these tissues. The nearly normalization of peroxisomal b-oxidation is most likely due to increased ALDR protein expression. Treatment with 4-phenylbutyrate promoted peroxisome proliferation and enhanced the expression of the ALDR protein. An international prospective study evaluating the efficacy of 4-phenylbutyrate and related compounds (e.g. lovastatine) in X-linked ALD patients is presently ongoing and will hopefully provide an answer in the near future. References 1 2

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Priv.-Doz. Dr. med. Jutta Ga¨rtner, Zentrum fu¨r Kinderheilkunde, Heinrich Heine-Universita¨t, Moorenstrasse 5, D–40225 Du¨sseldorf (Germany) Tel. +49 211 81 16184/17687, Fax +49 211 81 18757, E-Mail [email protected]

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Hughes IA, Clark AJL (eds): Adrenal Disease in Childhood. Clinical and Molecular Aspects. Endocr Dev. Basel, Karger, 2000, vol 2, pp 150–173

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Cushing Syndrome and Addison Disease Constantine A. Stratakis Unit on Genetics and Endocrinology, Developmental Endocrinology Branch, National Institute of Child Health and Human Development, Bethesda, Md., and Divisions of Pediatric Endocrinology and Genetics, Department of Pediatrics, Georgetown University, Washington, D.C., USA

Despite the relatively early description of the anatomy of the adrenal glands by Bartolomeo Eustachius in 1563 [1], the function and diseases of the ‘glandulae renis incumbentes’ remained unknown until Dr. Addison’s report of three patients with anemia and adrenal disease in 1861 [1, 2]. Adrenal tumors were not recognized and studied until much later [2]. The 20th century provided us with a wealth of information on adrenocortical disease, but it wasn’t until the unabated advance of human genetics over the last two decades that light was shed into its molecular pathogenesis. This is particularly true for two of the most common presentations of adrenocortical disease in childhood: Cushing syndrome and Addison disease caused by adrenocortical tumors and autoimmune dysfunction, respectively. This chapter reviews the recent advances in molecular understanding, clinical diagnosis and treatment of (i) adrenocortical tumors, including cancer, leading to Cushing syndrome, and (ii) Addison’s disease.

Cushing Syndrome Adrenocortical Tumors Causing Cushing Syndrome Incidence and Epidemiology. Even though single, benign, adrenocortical adenomas account for the vast majority of adrenal causes of Cushing syndrome in both children and adults, and malignant tumors of the cortex account for only 0.05–0.2% of all cancers, malignancy is far more frequent in children with adrenocortical causes of Cushing syndrome. For adrenocortical cancer,

a bimodal age distribution has been reported, with the first peak occurring before age of 5 years, and the second in the fourth to fifth decade [5–9]. In all published series, females predominate, accounting for 65–90% of the reported cases. Several studies have shown a left-sided prevalence in all adrenocortical tumors; however, others have reported a right-sided preponderance. In approximately 2–10% of the patients, adrenal cancer is found bilaterally [3]. Overall, there appears to be a higher prevalence of adrenocortical carcinoma among patients with incidentally discovered adrenal masses than in the general population, although numerical estimates vary widely in the literature [10]. In children, any adrenocortical mass associated with Cushing syndrome would be suspicious for cancer. In adults, among the radiologically detectable masses, independent of size, one in 1,500 lesions may be an adrenal carcinoma [3]. Using the 5 cm cut-off as the most commonly accepted criterion for clinical investigation of an adrenal tumor [3, 11], carcinoma may be found in as many as 7% of the patients with adrenal lesions (21 of 311 patients in a recent study) [12]. In some areas of the world, higher incidence of adrenal cancer, especially in children, has been documented. This is particularly true for Southern Brazil, where environmental mutagens have been postulated as the relevant pathogenic event [13]. In these areas, evaluation of incidentally discovered adrenal masses appears to be necessary for lesions smaller than 5 cm in adults and of any size in children. Although the incidence of adrenal indidentalomas appears to be higher in some familial neoplasia syndromes like multiple endocrine neoplasia type-1 (MEN 1) [14] and familial adenomatous polyposis (FAP) [15], it is unclear whether this finding is accompanied by a higher predisposition to adrenal cancer. Among children with incidentally discovered adrenal masses, carcinoma may be more prevalent and, once more, the cut-off criterion of 5 cm does not apply [16]. In a recent series of 11 patients (3 boys, 8 girls) with a medial age of 7 years, three histologically proven carcinomas were less than 5 cm in their maximal width [17]. Fortunately, most pediatric patients with adrenal carcinoma present with a hormonal syndrome, which makes their detection easier and leads to their early surgical resection and medical treatment [16–18]. Other features including different genetic background and better prognosis differentiate adrenocortical carcinoma in children from that in adults (see below). Molecular Genetics of Adrenal Tumors. The genetic background of adrenocortical tumors remains poorly characterized despite recent advances in the molecular understanding of adrenal function [19]. Adrenocortical hyperplasia is a polyclonal process but carcinomas are monoclonal lesions [20], indicating that genetic changes at specific loci in the genome are needed for adrenal tumori-

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Fig. 1. A schematic diagram of the possible process of adrenocortical carcinogenesis, based on existing knowledge and analogy to what has been proposed for colon and other cancers. Adrenocortical hyperplasia is a polyclonal process, whereas both single adenomas and adrenal carcinoma are monoclonal processes. The progression to malignancy is accompanied by TP53 gene changes and loss-of-heterozygosity (LOH) of its chromosome 17 locus, amplification of several oncogenes and additional chromosomal and other genetic changes, as demonstrated recently by comparative genomic hybridization (CGH) analysis of adrenocortical tumors [19, 54, 55]. The question marks (?) represent the uncertainty as to whether these molecular processes are continuous (as, for example, in colon tumorigenesis) or follow independent pathways in the various adrenocortical lesions (as is, most likely, the case in thyroid tumorigenesis).

genesis [19] (fig. 1). Investigations have focused on obvious candidates, such as the corticotropin (ACTH) receptor (the MC2R gene) [21, 22] and molecules that participate in its signalling pathway, including the guanine-nucleotide binding protein (G-proteins) subunits Gsa and Gia2 [23, 24]. Although mutations have not been found, loss-of-heterozygosity (LOH) of the MC2R gene locus on the short arm of chromosome 18 (18p11.2) was frequent in carcinomas but not in adenomas [22, 25], suggesting that, perhaps, LOH of this gene participates in the dedifferentiation process leading to adrenocortical carcinogenesis. The Gsa gene (the Gsp proto-oncogene) has not been found mutated in adrenal cancer [23], but patients with McCune-Albright syndrome who bear somatic mutations of this gene, do develop benign adrenal lesions [26]. A fraction of adrenal carcinomas harbor mutations of the gene that codes for Gia2 [24]. Other candidate genes that have been investigated in adrenocortical carcinomas include those coding for aldosterone synthase (the CYP11B2 gene) and 21-hydroxylase (the CYP21B gene) [25, 27], and for aldosterone-producing carcinomas, the angiotensin-II type-1 (AT-1) receptor gene [28]. The MEN 1 gene, menin, and the FAP gene, APC, have also been investigated as possible candidates [29–31] because patients with MEN1 and FAP do get adrenal tumors, which, however, are mostly benign and nonfunctional [14, 15].

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Cytokines and growth factors and their receptors, which may be expressed eutopically or ectopically in adrenocortical tissue, have recently been implicated in carcinogenesis [19]. Expression of the major histocompatibility class-II (MHC-II) antigens in adrenocortical tissue correlates with adrenocortical cell differentiation [32]. The expression of both transforming growth factor-a (TGFa) and epidermal growth factor receptor (EGFR) [33] is markedly elevated in carcinomas (unlike adenomas) and synaptophysin and other neuroendocrine markers are ‘inappropriately’ expressed in adrenocortical cancer [34]. The unexpected presence of proteins with neuroendocrine and other functions in adrenal cancer follows a pattern similar to that observed in benign adrenocortical hyperplasias [35], although in cancer it seems to occur in a wider scale [19, 34]. It is also worth noting that cortisol-producing adrenocortical carcinomas often respond to dexamethasone administration with a ‘paradoxical’ rise of their glucocorticoid production [36], a feature that is almost universally present in primary pigmented adrenocortical disease (PPNAD), a benign, bilateral hyperplasia of the adrenal cortex [37, 38]. The expression of a variety of other factors has been investigated in adrenocortical carcinomas, including A103 and inhibin A [39]. Recent immunohistochemical analysis of cell cycle-related proteins such as p27, a cell cycle inhibitory protein, and Ki-67, a proliferation marker, indicated their possible values in predicting the biologic behavior of adrenocortical carcinoma [40]. Telomerase, an RNAdependent DNA polymerase that extends the ends of chromosomes by synthesizing the oligonucleotide repeat TTAGGG and serves as a marker for cellular immortality, appears to be present in adrenal carcinomas but not in adenomas [41]. A number of other genes and chromosomal abnormalities have been implicated in adrenocortical tumorigenesis, including loci on chromosomes 11 and 17. These include the genes coding for p53 (TP53) (on 17p13.1), p57 (on 11p15.5) (KIP2), and the insulin-like growth factor type-II (IGF-II) (on 11p15.5) [42–46]. LOH of the chromosome 17 locus of the gene that codes for p53 in tumors from patients with Li-Fraumeni syndrome (LFS), led to the identification of germline TP53 mutations in this genetic condition [46]. However, LFS patients develop adrenal cancer rarely [47]: An analysis of 475 tumors in 91 families with LFS revealed that breast cancer, bone and softtissue sarcoma, and brain tumors are most frequent, whereas adrenal cancer developed in only 1% of the patients [48]. In sporadic cancer, TP53 mutations may be present in approximately 30–50% of all lesions but p53 expression does not correlate with prognosis and it is rarely seen in monoclonal but highly differentiated tumors [49–51]. The latter finding suggests that TP53 mutations in sporadic cancer are a late event in the process of carcinogenesis, suggesting that other genetic events precede and may even predispose to TP53

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mutations in adrenal cancer [44–52]. These include mutations or other alterations in genes on chromosomes 2 and 17 (loci 2p16 and 17q22–24, respectively) and the menin locus (11q13) [19, 29, 30, 53]. The roles of tumor suppressor genes APC and inhibin are also being investigated [15, 19]. Comparative genomic hybridization (CGH) is a molecular cytogenetic technique, which allows a genome-wide screening of tumor DNA to identify chromosomal gains and losses. Regions of gains may contain dominantly acting oncogenes, while tumor suppressor genes may map to deleted regions [54, 55]. One important advantage of CGH is that frozen or paraffin-embedded samples can be evaluated because only tumor DNA – not cells in culture – is required for the analysis. In a recent CGH study, 8 adrenal carcinomas and 14 adenomas from adult patients were investigated [54]. The most common genetic aberrations in carcinomas were gains of chromosomes 4 and 5 and losses of chromosomes 11 and 17. CGH was also used to investigate genetic events leading to adrenal tumor formation in children from a region in southern Brazil (Curitiba), which, along with the state of Sa˜o Paulo, has the highest incidence of these tumors worldwide [55]. These patients did not have a diagnosis of any genetic syndromes; they were mostly female and below 4 years of age, and their tumors had better prognosis than adults with comparable stages of tumor development [13]. These features suggested that particular genetic events, germline or somatic, may be associated with tumorigenesis in this population [55]. Indeed, CGH showed that 8 of the 9 tumors exhibited copy number gain of a segment of chromosome 9 corresponding to cytogenetic band 9q34 (fig. 2), suggesting that gene(s) in this locus may play a role in the molecular process leading to adrenal cancer in these patients [55]. Overall, chromosomal gains were far more common than losses in ACT; this finding has been confirmed by other investigators and may be related to (the assumed but not proven) involvement of a number of oncogenes, such as N-ras and others, in the process of adrenocortical tumor development [55, 56]. Among the deletions, the most frequently lost chromosome region was 2q22-q34, a locus that corresponds to that of the human inhibin a-subunit, which has been investigated in other tumors [55, 57]. Loss of other areas of chromosome 2 has also been shown in adrenal tumors [53]. The most frequently lost chromosomal region was the area around the 17p TP53 locus [54], underlining the significant role of p53 in the control of adrenocortical oncogenesis. Clinical Presentation and Pathology. Benign adrenocortical adenomas leading to Cushing syndrome in childhood are easily diagnosed, once the diagnosis of adrenocorticotropin (ACTH)-independent syndrome has been established by appropriate testing [19] and there is computed tomography (CT scan) or magnetic resonance imaging (MRI) proof of an adrenocortical tumor. However, several patients with adrenal cancer present with no detectable

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Fig. 2. Chromosome 9 comparative genomic hybridization (CGH) profiles from three representative tumors investigated in reference 55. In the top panel, the CGH profile was normal; in the middle panel, the profile showed a gain of 9q22qter and an amplification of 9q34; in the tumor shown in the bottom panel, CGH showed an amplification of 9q34. The five vertical lines on the right side of the chromosome ideograms reflect different values of the fluorescence ratio between the tumor DNA and the normal DNA. The values are 0.5, 0.75, 1, 1.25 and 1.5, from left to right. The middle line represents a 1:1 ratio and reflects a balance between the tumor DNA and the normal control DNA. The ratio profile (curve) was computed as a mean value of at least 8 metaphase spreads. For each case, a representative chromosome 9 hybridized with tumor DNA is shown to the left of the left of the ideogram. Notice that the regions of chromosomal gain correlate to an increased hybridization intensity. Chromosomal regions showing an amplification (9q34 for the tumors in the middle and lower panels) show further increase in the signal intensity as compared to the rest of the chromosome.

hormonal abnormality [58], although often inactive steroid precursors, such as pregnenolone, 17-hydroxypregnenolone, and 11-deoxycortisol, or their metabolites can be found in the circulation or the urine, respectively [16]. Occasionally, adrenocortical carcinomas secreting deoxycorticosterone or corticosterone cause hypokalemic alkalosis in the absence of hypercortisolism [59]. These findings follow the recent recognition that a significant number of incidentalomas are associated with hormonal abnormalities in adult patients [60–62]. Generally, however, hormone-secreting adrenocortical carcinomas are very inefficient in producing active hormones such as cortisol, and about 50% of them will have attained palpable size by the time they produce an endocrine

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syndrome [63, 64]. Cushing syndrome is present in 30–40% of patients with adrenal cancer. Virilization is present in 20–30% of patients with a functional carcinoma, in addition to Cushing syndrome or as an isolated manifestation. Feminization, hyperaldosteronism, hypoglycemia, nonglucocorticoid-related insulin resistance and polycythemia are other endocrine or paraneoplastic phenomena that may accompany Cushing syndrome in patients with adrenocortical cancer [3, 16, 19]. Other symptoms include abdominal pain, fullness, or an incidentally discovered abdominal or adrenal mass [65–70]. A palpable mass is present in approximately half of these patients at the time of diagnosis [16, 19]. In a significant proportion of these patients, metastatic disease may cause symptoms before a primary diagnosis is established [16, 63]; this is more common in adult patients. Local invasion, which is found in 20% of the patients at the time of diagnosis, commonly involves the kidneys and inferior vena cava [10, 16]. Metastatic disease may be found in the retroperitoneal lymph nodes, lungs, liver, or bone [65–70]. Adrenocortical carcinomas are clearly distinguished histologically from benign adenomas: Cells are frequently characterized by numerous mitoses, scant cytoplasm, and pleomorphism [71]. Areas of necrosis and hemorrhage within the tumor are common. Finally, local invasion almost always characterizes adrenocortical carcinomas [63, 72]. Treatment. The treatment of all primary adrenal tumors is surgical [16, 19, 73]. If complete resection of an adrenocortical carcinoma can not be achieved, as much as possible of the tumor should be removed. Solitary recurrences or metastases should also be removed surgically [74]. Long-term disease-free status has been produced by complete resection of adrenocortical carcinoma, whereas long-term remissions have followed surgical resection of hepatic, pulmonary, or cerebral metastases [10, 73, 75]. Therapy with o,p€-DDD (mitotane) is initiated either as an adjuvant to surgical treatment or for patients with inoperable cancer [16, 19, 63, 76–78]. o,p€-DDD is given at maximally tolerated oral doses (up 10 g/m2/day). It ameliorates the endocrine syndrome in approximately two-thirds, whereas tumor regression or arrest of growth has been observed in as many as onethird of the patients. Mean survival does not appear to be altered, although there are patients with unresectable carcinomas who achieved long-term survival. The side effects include nausea, vomiting, diarrhea, skin reactions, and neurologic manifestations, primarily lethargy, somnolence, dizziness, and muscle weakness. Other chemotherapeutic agents such as cisplatin, 5-fluorouracil, suramin, doxorubicin and etoposide might be useful [79–84]. Other steroid synthesis inhibitors (aminoglutethimide, metyrapone, trilostane, ketoconazole) or glucocorticoid antagonists (RU 486) may be used for the treatment of Cushing syndrome in patients with inoperable cancer [3, 16]. Patients taking

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mitotane may develop hypoaldosteronism or hypocortisolism, and fludrocortisone or hydrocortisone should be added as needed. Radiation therapy is occasionally helpful for palliation of metastases. The prognosis of adrenal carcinoma is generally poor, with a mean survival of approximately 18 months, although children do generally better than adult patients [16–18]. With aggressive surgical therapy, the mean survival increases to 48 months; survival as long as 10 years has been described for some patients receiving vigilant monitoring and aggressive surgery for local recurrences or metastases [71–78, 81–83]. Cures have been achieved for patients operated on at the early stages of the cancer, when the tumor was still encapsulated [3, 16, 81–83]. Benign, Bilateral Adrenocortical Diseases Leading to Cushing Syndrome Introduction on Primary Pigmented Adrenocortical Disease (PPNAD ). In recent years, two primary adrenal disorders affecting the adrenal cortex have been implicated in the pathogenesis of ACTH-independent Cushing syndrome [37, 84]. Primary pigmented adrenocortical disease (PPNAD), also known as ‘micronodular adrenal disease’, is a congenital disorder, which, in the majority of the reported cases, is associated with Carney complex. The complex is a form of a multiple endocrine neoplasia (MEN) syndrome that affects the adrenal cortex and other endocrine glands, and is associated with abnormal pigmentation of the skin and mucosae, myxomas, and other neoplasms [84]. Massive macronodular adrenocortical disease, is another form of bilateral adrenal hyperplasia, which leads to Cushing syndrome but is not associated with any other clinical findings and has only rarely been reported in young patients [37]. Other forms of bilateral adrenocortical hyperplasia, distinct from PPNAD and not always associated with hypercortisolism, include the lesions of the adrenal glands described in patients with the McCune-Albright and MEN1 syndromes. Those where briefly reviewed above; in the following paragraphs, we will focus on PPNAD and Carney complex. (1) Features of PPNAD: In case reports dating back as early as 1949, children and young adults were described with pituitary-independent CS and a unique type of adrenal pathology in common: a bilateral form of adrenal hyperplasia, characterized by multiple, small, pigmented, adrenocortical nodules that were surrounded by internodular cortical atrophy [84–88]. Various names were given to this peculiar lesion, the most common of which was ‘micronodular adrenal disease’ [86]; this was later replaced by ‘primary pigmented nodular adrenal disease’ (PPNAD), a term that was first coined by Dr. J.Aidan Carney in 1984 [87]. PPNAD may occur independently or, more commonly, as part of the complex of ‘spotty skin pigmentation, myxomas and endocrine overactivity’

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or Carney complex, which was described in 1985 [88–90]. This syndrome also encompasses several familial cases of cutaneous and cardiac myxomas associated with lentigines and blue nevi of the skin and mucosae, which have been described under the acronyms NAME (for nevi, atrial myxoma, myxoid neurofibromata, and ephelides) and LAMB (for lentigines, atrial myxoma, mucocutaneous myxoma, blue nevi) syndromes [91, 92]. In PPNAD, the glands are most commonly normal-sized or small and peppered with black or brown nodules set in a cortex that is usually atrophic [90]. This atrophy is pathognomonic and reflects the autonomous function of these nodules and the suppressed levels of pituitary ACTH. Despite their small size (less than 6 mm), the nodules are visible with CT scan or MRI of the adrenal glands, most likely because of the surrounding atrophy [93]. The combination of atrophy and nodularity gives the glands an irregular contour, which is distinctly abnormal and diagnostic, especially in younger patients with CS (fig. 3). Occasionally, one or both of the glands may be larger and harbor adenomas with a calcified center, while macronodules larger than 10 mm may be present in older patients [38]. Patients with PPNAD often present with a variant of Cushing syndrome called ‘atypical’ (ACS) [94], which is characterized by an asthenic, rather than obese, body habitus. This phenotype is caused by severe osteoporosis, short stature, and severe muscle and skin wasting. ACS was recognized as early as 1956 and has since been described in several cases of patients with the syndrome [94–97]; only recently, however, was this condition associated with PPNAD [38]. A recent review of the literature indicated that almost all the reported cases of ACS could be attributed to PPNAD, including that of a patient who presented 27 years after unilateral adrenalectomy [38]. Patients with ACS tend to have normal or near-normal 24-hour urinary-free cortisol (UFC) production, but this is characterized by the absence of the normal circadian rhythmicity of cortisol [38, 98, 99]. Occasionally, normal cortisol production is interrupted by days or weeks of hypercortisolism, which gives rise to a yet another variant called ‘periodic Cushing syndrome’ (PCS) [98]. PCS is frequently found in children and adolescents with PPNAD [98, 99]. In both ACS and PCS, as well as in classic Cushing syndrome, caused by PPNAD, paradoxical increase of UFC and/or 17-hydroxy-corticosteroids (17-OHS) is seen during the second phase (high dose dexamethasone administration) of the Liddle’s test [99]. This feature may be useful diagnostically for PPNAD [38, 99]; it reflects, perhaps, a tendency that these nodules have for increased responsiveness to other steroids [100]. PPNAD only rarely is present isolated. At the National Institutes of Health (NIH), where vigorous screening for Carney complex has been instituted under a research protocol, among 19 patients treated for PPNAD the

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Fig. 3. Adrenal manifestations of Carney complex: A woman with Cushing syndrome is shown in (a); in addition to lentigines, other pigmented skin lesions on the face and the trunk are present. A young boy with periodic Cushing syndrome (described in reference 98) appears in (b). The irregular contour of adrenal glands with PPNAD is shown in (c) using computed tomography scanning [37, 38, 93, 99]. Sections of a left adrenal gland with primary pigmented nodular adrenocortical disease appear in (d ); an arrow points to one of the characteristic multiple pigmented nodules.

years 1968–1998, only one patient, a 32-year-old (who was diagnosed with PPNAD in her mid-twenties) did not have the complex [37] according to published criteria [101]. Thus, fewer than 10% of patients with PPNAD represent isolated forms of the disease; most of these patients have Carney complex, a syndrome that has a well defined, but extremely variable phenotype. (2) Carney Complex: Among the individual components of Carney complex, the cardiac myxoma is the one most responsible for the significant morbidity and mortality associated with the syndrome. This tumor occurs often at multiple sites (affecting any or all cardiac chambers), at a relatively young age,

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and is equally distributed between the sexes [102]. The cutaneous myxomas have a predilection for the eyelids and external ear canals, although they may affect any part of the skin [103, 104]. Mammary myxoid tumors may also occur at multiple sites and be bilateral; even the clinically ‘normal’ breast of patients with the complex commonly shows microscopic foci of myxomatous masses, which can be identified by MRI [105, 106]. The centrofacial spotty pigmentation of Carney complex involves the vermilion border of the lips and the conjunctiva. The pigmented spots (‘lentigines’) may be tan, irregularly shaped and poorly outlined, or small, sharply delineated, and dark brown to black. The conjunctival pigmentation typically affects the lacrimal caruncle and the conjunctival semilunar fold, and may involve the sclera. Five to 10% of patients with Carney complex have one or more intraoral pigmented spots, and the female external genitalia are commonly heavily pigmented. Blue nevi (the usual type, as well as the exceptionally rare epithelioid type) and combined and common junctional, dermal, and compound nevi, as well as cafe-au-lait spots also occur in the syndrome [107, 108]. About 10% of patients with Carney complex have a GH-secreting pituitary adenoma that results in acromegaly [90, 101]. Although most of the known patients with this condition had macroadenomas, a number of recently investigated cases show that abnormal 24-hour GH secretion can precede the development of a pituitary tumor in the complex. The disorder, therefore, provides the unusual opportunity for prospective screening of affected patients without clinical acromegaly. In one such case, serial measurements of GH or somatomedin C, or both, became progressively abnormal over several years; recently, a pituitary mass was identified on computed tomography, and partial hypophysectomy revealed minute foci of a GH-producing adenoma. Endocrine involvement in Carney complex also includes three types of testicular tumors: large-cell calcifying Sertoli cell tumor (among the rarest of testicular neoplasms), adrenocortical rests, and Leydig cell tumor [109]. About one-third of affected male patients have these masses. The large-cell calcifying Sertoli cell tumor, a bilateral, multicentric, and benign neoplasm, may secrete estrogens and cause precocious puberty, gynecomastia, or both [109]. Finally, three new components of the syndrome have been identified: psammomatous melanotic schwannoma, epithelioid blue nevus, and ductal adenoma of the breast [90, 101, 110–112]. Because thyroid follicular neoplasms, both benign and malignant, have been found in a number of patients, thyroid involvement is also considered a component of the syndrome [113]. Genetics of Carney Complex and PPNAD. Like the other MEN syndromes, Carney complex is inherited in an autosomal-dominant manner. Sporadic cases constitute approximately half of the known patients [101]. Interestingly,

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parent-of-origin effects in the inheritance of the disease have been observed [114]. A genetic locus was determined for Carney complex by linkage analysis of polymorphic markers from likely areas of the genome [101]. Positive lod scores were obtained for nine markers on the short arm of chromosome 2, identifying an approximately 4 centiMorgan (cM)-long area in the cytogenetic band 2p16 (CNC locus), which is likely to contain the gene(s) responsible for the complex [101]. Several lines of evidence indicate that the PPNAD and/or Carney complex gene(s) may be involved in the preservation of chromosonal stability in several human cell types. Indeed, the formation of telomeric associations and dicentric chromosomes is a frequent feature of fibroblasts derived from the myxoid tumors excised from patients with Carney complex [115–117]. Also, a recent study found similar features in cultured in vitro adrenocortical cells derived from PPNAD nodules [118]; microsatellite analysis of the tumors excised from patients with Carney complex confirmed the significant genomic instability that accompanies tumorigenesis in this syndrome [118]. Numerous areas of loss or gain of heterozygosity, and/or deletions, involving all 22 autosomal chromosomes were found. These changes did not include the CNC locus on chromosome 2p16, a finding that suggests that alterations of the heterozygosity of the responsible gene(s) may not be necessary for oncogenesis in this condition. Although mutations of the gsp proto-oncogene were not present in Carney complex tumors [119], and the locations of several genes that code for components of the guanine nucleotide-binding proteins (G-proteins) were excluded by linkage analysis [101], it seems likely that the gene(s) responsible for this condition may participate in G-protein -controlled or -related signalling systems. Recently, a family with Carney complex that does not map to chromosome 2p16 was reported [120]. This finding has been confirmed by our laboratory in another large kindred that had a number of recombinant genotypes with the first locus [121]. Thus, it appears likely that genetic heterogeneity exists in the syndrome. Indeed, a major locus was identified recently on chromosome 17 (17q22-24) [122]. It remains to be seen what the contribution is of each one of the 2 loci to the genetics of PPNAD and/or Carney complex. Treatment. Our current recommendation for all patients with proven PPNAD is bilateral adrenalectomy, including patients with atypical or periodic forms of Cushing syndrome [37, 38, 98]. However, as more families with Carney complex are screened, more patients are found to have biochemical features of PPNAD without any signs of Cushing syndrome [99]. For these patients with minimal, if any, metabolic effects of the abnormally regulated cortisol secretion by the diseased glands, medical treatment may be warranted.

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4a

4b

4c

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Addison Disease Introduction Most of the genetic causes of adrenal insufficiency (AI) (adrenal hypoplasia congenita, StAR deficiency, corticotropin unresponsiveness, adrenoleukodystrophy) are reviewed elsewhere in this book and need not concern us here. Adrenal hemorrhage, iatrogenic, surgical and medical, and infectious causes of AI are also beyond the scope of this review. In the following paragraphs, we will focus on autoimmune destruction of the adrenal glands, which represents the most common cause of AI in older children and adults. In this disease, the size of the adrenal glands gets progressively smaller, the shape retracted and the cortex, in particular, thinner. Fibrous tissue surrounds islets of functioning tissue and extensive lymphocytic infiltration is present (fig. 4) [123]. In the pediatric patient, clinical AI usually presents after the first 2 years of life along with other autoimmune diseases in the form of 2 polyglandular autoimmune syndromes: multiple endocrine abnormalities types -1 and -2 (MEA-1 and MEA-2) (table 1) [124]. In the most comprehensive series of patients with autoimmune Addison disease ever published, 295 patients were analyzed [125]. Addison disease occurred in the context of MEA-1 in association with chronic mucocutaneous candidiasis and/or acquired hypoparathyroidism and the age of onset was predominately in childhood or in the early adult years. MEA-1 was also frequently associated with chronic active hepatitis, malabsorption, juvenile onset pernicious anemia, alopecia and primary hypogonadism, whereas insulin-dependent diabetes and/or autoimmune thyroid disease were infrequent. Addison disease in the context of MEA-2 was associated with insulindependent diabetes and/or autoimmune thyroid disease, had a later but variable age of onset, and it occurred predominately in females. The association of HLA-B8, HLA-DR3 and HLA-DR4 with MEA-2, but not with MEA-1 [126], further confirmed the different clinical and genetic nature of these two syndromes, which account for almost all pediatric and approximately 50% of adult cases of autoimmune Addison disease. MEA-1 MEA-1 is also known as autoimmune polyendocrinopathy-candidiasisectodermal dystrophy or APECED syndrome. It is a rare autosomal-recessive Fig. 4. Panels (a), (b) and (c) represent progressive magnifications [(¶3), (¶100) and (¶400), respectively] of the right adrenal gland of a patient with autoimmune Addison disease; diffuse lymphocytic infiltrates and destruction of the normal architecture of the gland with gradual replacement by fibrous tissue are noted. Slides courtesy of Dr. J.A. Carney, Mayo Clinic.

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Table 1. Clinical manifestations of the MEA-1 and -2 syndromes (with prevalence in %)* Disorder

MEA-1 %

MEA-2 %

Hypoparathyroidism Mucocutaneous candidiasis Adrenal insufficiency Gonadal failure Thyroid disease Insulin-dependent diabetes Hypopituitarism Diabetes insipidus Vitiligo Malabsorption Alopecia Pernicious anemia Hepatitis Myasthenia gravis, thrombocytopenia and other autoimmune disorders

89 75 60 45 12 1 =1 =1 =5 25 20 16 9 not known

not present not present 100 50 70 50 not present =1 =5 not present =1 =1 not present D1

* Modified from references 123, 125 and 143.

disorder [127]. APECED patients usually have at least two out of three main symptoms: Addison’s disease, hypoparathyroidism and chronic mucocutaneous candidiasis (table 1) [128, 129]. Patients may also develop other organ specific autoimmune disorders leading to gonadal failure, pancreatic (b-cell) deficiency, gastric (parietal cells) dysfunction, hepatitis and thyroiditis. Other, less common, clinical manifestations include ectodermal dystrophy, affecting the dental enamel and nails, alopecia, vitiligo and corneal disease (keratopathy) [128–130]. MEA-1 usually occurs in early childhood but new, tissue-specific symptoms may appear throughout lifetime [128–130]. Immunologically, the main finding in APECED patients is the presence of autoantibodies against the affected organs, including those against steroidogenic enzymes (P450scc, P450c17 and P450c21) in patients with Addison’s disease [130], glutamic acid decarboxylase in patients with diabetes [131] and the enzymes aromatic L-amino acid decarboxylase and P4501A2 in patients with liver disease [132, 133]. Addison disease is usually the third clinical disorder to develop in patients with APECED. As with the other manifestations of the syndrome, there is a wide variability of age of onset, from 6 months to 41 years with a peak around 13 years of age [127]. Addison disease develops in 60–100% of patients with

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APECED and may be preceded by months or years of detectable adrenal cortex autoantibodies [126–130]. APECED is more common in certain genetically isolated populations. In Finland, the incidence has been estimated to be 1:25,000 [134] and in Iranian Jews 1:9,000 [135]. APECED is also relatively common among Sardinians (1:14,400) and in Northern Italy [134]. Based on linkage analysis in Finnish APECED families, the locus for APECED gene was mapped to chromosome 21q22.3 [134]. Recently, the gene responsible for this disease was cloned [136, 137]: it is a novel gene encoding a predicted 545 amino acid protein, which was named AIRE (autoimmune regulator). It contains two plant homeodomain (PHD)-type zinc finger motifs and a newly described putative DNAbinding domain SAND, a proline rich region, and three LXXLL motifs [136, 137], all suggestive of a transcription regulator. AIRE is expressed in thymus, lymph node and fetal liver [136], tissues that have important role in the maturation of immune system and development of immune tolerance. These findings together with the immunologic deficiency in APECED patients suggest that AIRE may have an important role in the control of immune recognition and may function as a transcription factor or as transcriptional coactivator. To date, several mutations in the AIRE gene have been described in APECED patients [136–138]. A common Finnish mutation, R257X, was shown to be responsible for 82% of Finnish APECED cases [136, 137]. R257X is also the predominant Northern Italian APECED mutation [138] and, in addition, has been detected in other patients of diverse origin, because it occurs on different haplotypes with closely linked markers suggesting several independent mutational events [136–138]. Another common mutation, a deletion of 13 nucleotides (1094–1106del), has been detected in several patients of different ethnic origin and on different haplotypes [138]. Finally, another nonsense mutation, R139X, is the major mutation among Sardinian APECED patients [138], whereas an additional 5 different mutations have been described in individual kindreds. APECED was the first autoimmune disease that was molecularly characterized (the familial Mediterranean fever gene was cloned at about the same time) and was found to be caused by a defect in a single gene. The protein product of AIRE gene not only has the features of a potent transcriptional regulator but it has also, in the meantime, been localized to nuclear body-like structures of cell nuclei, which appear to be involved in the regulation of transcription, oncogenesis and differentiation of cells [139, 140]. Most recently the mouse Aire gene was cloned and fully characterized [141, 142], a development, which is likely to lead to the generation of a mouse APECED model and, perhaps, new treatments for this disorder.

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MEA-2 MEA-2 is a far more common disease than MEA-1; Addison disease is the cardinal manifestation of MEA-2, with a 100% of the patients with this disease developing AI. Thyroiditis (atrophic or in the form of Hashimoto or Graves disease) and insulin-dependent diabetes are also common but the nonendocrine manifestations (so common in MEA-1) are rare (alopecia, vitiligo) or nonexistent (hepatitis, malabsorption) (table 1) [143]. Approximately half of the cases are familial and inherited in an autosomal dominant manner; as in other autoimmune disorders, women are affected 1.8 times more than men [143, 144]. AI is the first manifestation of MEA-2 in more than 50% of the patients, usually between the ages of ages of 20–40 years. However, pediatric cases of the disease have also been reported [143, 144]. Adrenal autoantibodies, in particular those against 21-hydroxylase [145], measured by either immunoprecipitation or immunofluorescence [146], are considered a sensitive indicator of the disease; their levels seem to correlate to disease activity [147]. Hypogonadism is more frequent in MEA-2 than MEA-1 and is almost exclusively restricted to ovarian failure; it may occur before AI, thus, women or adolescent girls with premature ovarian failure should be screened for Addison disease [148]. The etiology of MEA-2 is unlear; both polygenic and monogenic causes have been implicated but, other than its association with the HLA-B8, HLADR3 and HLA-DR4 alleles [144], little is known. Prognosis and Treatment in MEA-1 and MEA-2 Syndromes In both MEA-1 and MEA-2, autoimmune destruction of the adrenal glands causes permanent damage in adrenocortical function (although lowlevel steroid hormone production may be present for several years after the onset of disease) [123, 124, 143, 144]. The screening of first degree relatives of patients with familial Addison disease for AI (by an ACTH stimulation test) is essential; recommendations vary from annual surveys to every 2–3 years [144]. Treatment for patients with established Addison disease is recommended with hydrocortisone (10–15 mg/m2/day) and fludrocortisone (Florinef Ô ) 0.1 to 0.3 mg/day; salt supplementation at 1–3 g/day may be needed for infants and toddlers with the disease [149].

Acknowledgments We wish to thank Dr. Aidan J. Carney (Mayo Clinic) for providing us with valuable insight into primary pigmented adrenocortical disease and allowing us to use the histologic slide presented in figure 4 from a patient with Addison disease. We also would like to thank Dr. Karen Winer (National Institutes of Health) for allowing us to examine the molecular etiology of MEA-2 is some of her patients.

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Uibo R, Aavik E, Peterson P, Perheentupa J, Aranko S, Pelkonen R, Krohn K: Autoantibodies to cytochrome P450scc, P450c17 and P450c21 in autoimmune polyglandular disease type I and II and in isolated Addison’s disease. J Clin Endocrinol Metab 1994;78:323–328. ˚ , Miettinen A: Antibodies Tuomi T, Bjo¨rses P, Falorni A, Partanen J, Perheentupa J, Lenmark A to glutamic acid decarboxylase and insulin dependent diabetes in patients with APS1. J Clin Endocrinol Metab 1996;81:1488–1493. Husebye ES, Gebre-Medhin G, Tuomi T, Perheentupa J, Ladin-Olsson M, Gustafsson J, Rorsman F, Ka¨mpe O: Autoantibodies against aromatic acid decarboxylase in autoimmune polyendocrine syndrome type I. J Clin Endocrinol Metab 1997;82:147–150. Clemente MG, Obermayer-Straub P, Meloni A, Strassburg CP, Aragino V, Tukey RH, de Virgilis S, Manns MP: Cytochrome P450 1A2 is a hepatic autoantigen in autoimmune polyglandular syndrome type 1. J Clin Endocrinol Metab 1997;82:1353–1361. Aaltonen J, Bjo¨rses P, Sandkuijl L, Perheentupa J, Peltonen L: An autosomal locus causing autoimmune disease: Autoimmune polyglandular disease type I assigned to chromosome 21. Nat Genet 1994;8:83–87. Zlotogora J, Shapiro MS: Polyglandular autoimmune syndrome type I among Iranian Jews. J Med Genet 1992;29:824–826. Nagamine K, Peterson P, Scott HS, Kudoh J, Minoshima S, Heino M, Krohn K, Lalioti MD, Mullis PE, Antonarakis SE, Kawasaki K, Asakawa S, Ito F, Shimizu N: Positional cloning of the APECED gene. Nat Genet 1997;17:393–398. Finnish-German APECED Consortium: An autoimmune disease, APECED, caused by mutations in a novel gene featuring two PHD-type zinc-finger domains. Nat Genet 1997;17:399–403. Heino M, Scott HS, Chen Q, Peterson P, Maebpaa U, Papasavvas MP, Mittaz L, Barras C, Rossier C, Chrousos GP, Stratakis CA, Nagamine K, Kudoh J, Shimizu N, Maclaren N, Antonarakis SE, Krohn K: Mutation analyses of North American APS-1 patients. Hum Mutat 1999;13:69–74. Bjorses P, Aaltonen J, Horelli-Kuitunen N, Yaspo ML, Peltonen L: Gene defect behind APECED: A new clue to autoimmunity. Hum Mol Genet 1998;7:1547–1553. Rinderle C, Christensen HM, Schweiger S, Lehrach H, Yaspo ML: AIRE encodes a nuclear protein co-localizing with cytoskeletal filaments: Altered sub-cellular distribution of mutants lacking the PHD zinc fingers. Hum Mol Genet 1999;8:277–290. Wang CY, Shi JD, Davoodi-Semiromi A, She JX: Cloning of Aire, the mouse homologue of the autoimmune regulator (AIRE) gene responsible for autoimmune polyglandular syndrome type 1 (ASP1). Genomics 1999;55:322–326. Blechschmidt K, Schweiger M, Wertz K, Poulson R, Christensen HM, Rosenthal A, Lehrach H, Yaspo L: The mouse Aire gene: Comparative genomic sequencing, gene organization, and expression. Genome Res 1999;9:158–166. Leshin M: Polyglandular autoimmune syndromes. Am J Med Sci 1985;290:77–88. Riley WJ: Autoimmune polyglandular syndromes. Horm Res 1992;38(suppl 2):9–15. Seissler J, Schott M, Steinbrenner H, Peterson P, Scherbaum WA: Autoantibodies to adrenal cytochrome P450 antigens in isolated Addison’s disease and autoimmune polyendocrine syndrome type II. Exp Clin Endocrinol Diabetes 1999;107:208–213. Betterle C, Volpato M, Pedini B, Chen S, Smith BR, Furmaniak J: Adrenal-cortex autoantibodies and steroid-producing cells autoantibodies in patients with Addison’s disease: Comparison of immunofluorescence and immunoprecipitation assays. J Clin Endocrinol Metab 1999;84:618–622. Laureti S, De Bellis A, Muccitelli VI, Calcinaro F, Bizzarro A, Rossi R, Bellastella A, Santeusanio F, Falorni A: Levels of adrenocortical autoantibodies correlate with the degree of adrenal dysfunction in subjects with preclinical Addison’s disease. J Clin Endocrinol Metab 1998;83:3507–3511. Betterle C, Volpato M: Adrenal and ovarian autoimmunity. Eur J Endocrinol 1998;138:16–25. Baker JR Jr: Autoimmune endocrine disease. JAMA 1997;278:1931–1937. Constantine A. Stratakis, MD, DSc, Chief, Unit on Genetics and Endocrinology, UGEN, NICHD, NIH, Building 10, Room 10N262, 10 Center Dr. MSC1862, Bethesda, MD 20892-1862 (USA) Tel. +1 (301) 4964686/4021998, Fax +1 (301) 4020574, E-Mail [email protected]

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Subject Index

Addison disease etiology 163 multiple endocrine abnormality type 1 163–166 type 2 163, 166 prognosis and treatment of autoimmune diseases 166 Adrenal development adrenocorticotropin role 1 DAX-1 role, see DAX-1 definitive zone 1 fetal zone 1 steroidogenic factor-1 role, see Steroidogenic factor-1 Adrenal tumor, see Cushing syndrome Adrenarche overview 76, 77 P450c17 role 77 Adrenocorticotropin adrenal development role 1 receptors and signal transduction 24, 25 synthesis and processing 24 Adrenocorticotropin resistance, see also Familial glucocorticoid deficiency, Triple A syndrome clinical presentation 27 receptor constitutive activity 33 hypersensitivity 33 mutations 27–29, 33, 34 syndromes 26, 27

Adrenomyeloneuropathy, see X-linked adrenoleukodystrophy AIRE, mutations in multiple endocrine abnormality type 1 165 Aldosterone, biosynthesis 93, 94, 113–115 Aldosterone synthase deficiency clinical presentation 124 diagnosis 125 frequency 124 homology with 11b-hydroxylase 128 hormonal findings 124, 125 management and treatment 128 mutations type I disease 125, 126 type II disease 126, 127 pathophysiology 124 types 113, 124 APECED syndrome, see Multiple endocrine abnormality type 1 Benzodiazepine receptor, see Peripheral benzodiazepine receptor Bone marrow transplantation, X-linked adrenoleukodystrophy patients 145, 146 Carney complex, see Primary pigmented adrenocortical disease Cholesterol, mitochondrial transport cycloheximide inhibition studies 39 functions MLN-64 53

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peripheralbenzodiazepinereceptor 42–44 steroidogenesis activator polypeptide 41 steroidogenic acute regulatory protein 44–54 sterol carrier protein 2 40, 41 steroid synthesis role 37–39 Cholesterol desmolase, see P450scc Comparative genomic hybridization, adrenocortical tumor analysis 154 Congenital adrenal hyperplasia classification 93, 94 gene defects, overview 112, 113 genotype/phenotype relationships 102–106 heredity 93–95, 105, 106 11b-hydroxylase deficiency, see 11b-Hydroxylase 21-hydroxylase deficiency, see CYP21 incidence 94 P450c17 deficiency, see P450c17 steroidogenesis pathways, overview 93, 94 treatment based on genotype 106, 107 Congenital lipoid adrenal hyperplasia clinical features 48 heredity 48, 49 historical perspective of gene mutations 48, 49 P450scc mutation analysis 48, 53, 54 steroidogenic acute regulatory protein deficiency clinical features 48 discovery of gene defects 49 mutation types 50–53 two-hit model 51–53 Cortisol, biosynthesis 93, 94, 115 Cushing syndrome adrenocortical tumors clinical presentation 154–156 epidemiology 150, 151 molecular genetics 151–154 pathology 156 treatment 156, 157 primary pigmented adrenocortical disease Carney complex 157, 159–161 clinical presentation 158 genetics 160, 161 isolated form 158, 159

Subject Index

nomenclature 157, 158 pathology 158 treatment 161 CYP17, see P450c17 CYP21 congenital adrenal hyperplasia classic salt-wasting congenital adrenal hyperplasia, clinical features and diagnosis 98–100 genotype/phenotype relationships 102–106 newborn screening 17-hydroxyprogesterone levels 101, 102 molecular genetics 102 non-classic congenital adrenal hyperplasia 100, 101 treatment based on genotype 106, 107 gene chromosomal duplication and deletion 96 conversion 96, 97 locus 95, 116 mutation germline mutations 105, 106 missense mutations 104 null mutations 103 types and frequencies 97, 98 structure 95, 116 steroidogenesis role 93, 115 DAX-1 expression in development 11, 12 functions 15, 16 gene identification 10 knockout mouse 14, 17 mutation human phenotypes 12–14, 17 screening 14 X-linked adrenal hypoplasia congenita 1, 9, 13 structure 10, 11 DHEA-S, aging effects on serum concentration 65, 76 Familial glucocorticoid deficiency heterogeneity 29, 30, 33, 34 treatment 30

175

11b-Hydroxylase deficiency clinical presentation 120, 121 diagnosis 121, 122 frequency 120 hormonal findings 121 management and treatment 123 mutations 122, 123 pathophysiology 120 genes 116 homology with aldosterone synthase 128 steroidogenesis role 115 17a-Hydroxylase, see P450c17 21-Hydroxylase, see CYP21 17-Hydroxyprogesterone, levels in congenital adrenal hyperplasia 101, 102, 104 3b-Hydroxysteroid dehydrogenase activities 114, 115 genes 115 steroid synthesis 113, 114 tissue distribution 115, 116 Lipoid congenital adrenal hyperplasia, see Congenital lipoid adrenal hyperplasia 17, 20-Lyase, see P450c17 MC2R, loss of heterozygosity in adrenocortical tumors 152 Micronodular adrenal disease, see Primary pigmented adrenocortical disease Mitochondrial cholesterol transport, see Cholesterol, mitochondrial transport MLN-64, steroidogenesis role 53 Multiple endocrine abnormality type 1 Addison disease association 164, 165 AIRE mutations 165 clinical features 163, 164 epidemiology 165 pathology 163 prognosis 166 treatment 166 Multiple endocrine abnormality type 2 Addison disease association 166 clinical features 163, 164, 166 etiology 166 pathology 163

Subject Index

prognosis 166 treatment 166 P450c17 activities identified in single protein 63–66 adrenarche role 76, 77 catalytic mechanism 66–68, 115 deficiency clinical presentation 117 diagnosis 118 frequency 117 historical perspective of disease role 63–66 hormonal findings 117, 118 pathophysiology 116 treatment 119, 120 gene locus 77, 116 structure 77, 78, 116 17, 20-lyase activity regulation phosphorylation 69, 71, 86 redox partners 68, 69 mutations deletions 78, 79 frameshift 79, 80 17, 20–lyase activity mutations 82–85, 118, 119 point mutations affecting both activities 80–82, 118 truncation 79 polycystic ovary syndrome role 85, 86 regulation of activity ratio 73, 75 steroidogenesis regulation 63, 73 structure modeling of human enzyme 71–73, 78 P450c21, see CYP21 P450scc cholesterol delivery, see Cholesterol, mitochondrial transport congenital lipoid adrenal hyperplasia mutation analysis 48, 53, 54 steroid synthesis role 38 p53, mutation in adrenocortical tumors 153, 154 Peripheral benzodiazepine receptor cholesterol interaction site 43

176

transport in mitochondria 42–44 diazepam binding 42 mitochondrial function regulation 44 protein interactions 42, 44 regulation of expression 43, 44 Polycystic ovary syndrome insulin resistance 86 P450c17 role 85, 86 Primary pigmented adrenocortical disease Carney complex 157, 159–161 clinical presentation 158 genetics 160, 161 isolated form 158, 159 nomenclature 157, 158 pathology 158 treatment 161 Steroidogenesis activator polypeptide, cholesterol transport 41 Steroidogenic acute regulatory protein cholesterol transport in mitochondria mechanism 46 protein interactions 46–48 truncation studies 45, 46 deficiency in congenital lipoid adrenal hyperplasia clinical features 48 discovery of gene defects 49 mutation types 50–53 two-hit model 51–53 gene identification 45, 50, 115 structure 115 leader sequence 47, 48 mitochondrial association 44, 45, 47, 48 precursor 45 tissue distribution 115 Steroidogenic factor-1 adrenal development role 2 expression in development 3–5 functions 5, 6 gene identification 2, 3 knockout mouse 7, 17, 53 mutation and human phenotype 7–9, 17 phosphorylation 6, 7 structure 3

Subject Index

Sterol carrier protein 2 cholesterol transport 40, 41 expression 40, 41 gene transcripts 40 lipid specificity 40 Triple A syndrome clinical features 31 etiology 31–34 X-linked adrenoleukodystrophy clinical phenotypes Addison only 136 adrenomyeloneuropathy 136 asymptomatic 136 cerebral forms adolescent 136 adult 136 childhood 135 heterozygotes 136, 137 overview 134, 135 diagnosis clinical presentation 142 fatty acid analysis 143 imaging 142 mutation analysis 143, 144 prenatal diagnosis 144 etiology 139–141 gene identification 134, 138 mutations 139 protein structure and function 138–140 structure 138 genotype/phenotype relationships 141 historical perspective 134 pathogenesis 141, 142 pathological phenotypes 137 treatment bone marrow transplantation 145, 146 dietary therapy 145 gene therapy prospects 146, 147 immunosuppression 145 overview 144 symptomatic therapy 145 biochemical phenotypes 137, 138

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