Genetic Steroid Disorders

By Maria I. New

This is a comprehensive book addressing steroid disorders from hormonal, genetic, psychological, and surgical perspectives. It is meant to educate adult and pediatric endocrinologists, clinical geneticists, genetic counselors, reproductive endocrinologists, neonatologists, urologists, and psychoendocrinologists. It will assist these specialists in the diagnosis and treatment of steroid disorders. The book is written for postgraduate and faculty-level physicians. The content consists of steroid disorders, genetic bases for the disorder and case presentations of each disorder.

• Provides a common language for professionals to discuss and diagnose genetic steroid disorders
• Includes the very latest details on genetic tests and diagnoses
• Offers a strong understanding of the molecular basis for the diseases and therefore correct diagnosis and treatment of steroid disorders
• Presents insight into which medications to use based on the genetic makeup of a patient
• Teaches the best strategies and most effective use of genetic information in the patient counseling setting

• Language: English
• Publisher: Academic Press
• Release date: Aug 22, 2013
• ISBN: 9780123914675

Maria I. New

Dr. New received her Bachelors degree from Cornell University and her Doctor of Medicine degree from the University of Pennsylvania, where she was awarded the Distinguished Graduate Award. She was Chairman of Pediatrics at Weill Medical College of Cornell University from 1980 to 2002 and Founding Director of its Children’s Clinical Research Center, where she also served as Chief of Pediatric Endocrinology from 1964 to 2002. Dr. Maria New is Professor of Pediatrics, Professor of Genetics and Genomic Sciences, and Director of the Adrenal Steroid Disorders Program at Mount Sinai School of Medicine in New York City. She is also serving as Associate Dean for Clinical Research at the Florida International University Herbert Wertheim College of Medicine. Former president of the Endocrine Society, Dr. New has edited or co-edited 12 medical textbooks, published more than 600 peer-reviewed papers and served as editor-in-chief of the Journal of Clinical Endocrinology and Metabolism. She has trained more than 100 young physician-scientists who have become chiefs of pediatric endocrinology and leaders in their field. Her research, clinical work and teaching have taken her around the world. In 2005 and 2006, she led genetics research expeditions to Siberia in collaboration with the School of Medicine, St. Petersburg University, Russia. Dr. New’s contributions have been recognized by her being selected as one of the few pediatricians in the National Academy of Sciences. She has received numerous honors including: the Robert H. Williams Distinguished Leadership Award; the Rhone-Poulenc Rorer Clinical Investigator Award from the American Endocrine Society; the 1996 Dale Medal, the highest award given by the British Endocrine Society; and the 2003 Fred Conrad Koch Award, the highest award given by the American Endocrine Society. In 2010, she received the Van Wyk Prize, the highest award given in pediatric endocrinology. She has conducted pioneering research in the area of Congenital Adrenal Hyperplasia, a term used to describe a family of monogenic autosomal recessive disorders of steroidogenesis in which enzymatic defects result in impaired synthesis of cortisol by the adrenal cortex. In addition, Dr. New discovered a new form of hypertension, Apparent Mineralocorticoid Excess, which opened a new field of receptor biology. She was also the first to describe Dexamethasone-Suppressible Hyperaldosteronism, another form of low-renin hypertension. In 1999, she reported what may be the first example of a transcription factor defect in human beings.

Book preview

1

Introduction

Maria I. New,    Department of Pediatric Endocrinology, Mount Sinai School of Medicine, New York, NY, USA

Abstract

This book demonstrates that each steroid disorder causing both clinical and biochemical abnormalities in patients now has a genetic basis. The genes for each step in steroidogenesis have been mapped and cloned, and the mutations in the gene causing the disorder have been described. In addition, the structural biology of the protein resulting from the mutation in the gene has been reported for many of the disorders.

Keywords

steroid disorders; clinical abnormalities; biochemical abnormalities; steroidogenesis

The history of steroid disorders is very old. The first published report of a cadaver with ambiguous genitalia whose sex was changed from female to male was given to me by one of my mentors, Alfred Bongiovanni [1]. The cadaver was a male who was found at autopsy to have ovaries, uterus, and Fallopian tubes, and the adrenals were extremely large. These findings were considered by the dissector, de Crecchio, to be wondrous and mysterious. This publication by de Crecchio is considered by many to be the first report of a female with congenital adrenal hyperplasia raised as a male.

In a later publication [2], I speculated that the story of a female pope [3,4], Pope Joan, referred to in various publications [5,6], could have been a female virilized by congenital adrenal hyperplasia who presented herself as a male and became a pope. However, after much reading I concluded that the story of Pope Joan was a legend and not history because the case was reported only 400 years after her presumed death in 800 A.D. While it was clearly possible to have a written report in 800 A.D., nothing about Pope Joan appeared until 400 years after her death (she was killed by a crowd who witnessed the birth of her child while in procession from St. Peters to the Lateran). This subject has been treated by several authors as an interesting fact [7]. Indeed, one of the tarot cards is of Pope Joan.

However, when I studied the Old Testament and was taught the history of the Jews by the great historian, Salo Wittmayer Baron, I realized that the ancient pedigree demonstrated consanguinity (Fig. 1.1). Congenital adrenal hyperplasia is an autosomal recessive disorder, which occurs more frequently in consanguineous families. Abraham’s wife, Sarah, was his niece. She was the daughter of his dead younger brother, Haran. Sarah was infertile and did not bear Abraham a son until she was 99 years old. This history could be construed as a family with possible non-classical steroid 21-hydroxylase deficiency, as the features of impaired fertility and consanguinity are frequently observed in families with this deficiency.

FIGURE 1.1 Pedigree of Abraham and Sarah. Source: the Old Testament of the Bible.

As time went on, steroid endocrinology made frequent and important advances (Fig. 1.2). Early studies of steroid disorders investigated steroid metabolites in the urine, and later used serum hormone levels to identify the disorder. Thereafter, steroid disorders benefited greatly from the advent of molecular biology.

FIGURE 1.2 Advances in steroid endocrinology.

Indeed, this book demonstrates that each steroid disorder causing both clinical and biochemical abnormalities in patients now has a genetic basis. The genes for each step in steroidogenesis have been mapped and cloned, and the mutations in the gene causing the disorder have been described. In addition, the structural biology of the protein resulting from the mutation in the gene has been reported for many of the disorders.

The authors of the chapters herein are pioneers and experts in the various genetic disorders presented in this book. They are not the sole contributors to this field, but they are my teachers and I owe much of what I have learned to them. I wish to thank all the great scholarly scientists who made this book possible.

I wish to thank the NIH and the Genesis Foundation for the support of my research.

Finally, I owe a great debt of gratitude to my primary mentor, Dr. Ralph Peterson, who is an unsung hero of steroid endocrinology and who inspired me to develop this book.

Maria I. New MD

Editor-in-Chief

References

1. de Crecchio, Luigi. Sopra un caso di apparenzi virili in una donna. Morgagni. 1865;7:154–188.

2. New MI. Ancient History of Congenital Adrenal Hyperplasia. In: Ghizzoni L, Cappa M, Chrousos G, Loche S, Maghnie M, eds. Pediatric Adrenal Diseases, Endocr Dev. Basel: Karger; 2011;202–211.

3. D’Onofrio Cesare. La papessa Giovanna: Roma e papato tra storia e leggenda. Romana Società Editrice 1979;286.

4. New MI, Kitzinger ES. Pope Joan: A recognizable syndrome. J Clin Endo Metab. 1993;76:3–13.

5. de Mailly, Jean. Chronica Universalis Mettensis (1254) the fable of Pope Joan first appears in written form. In: In: Monumenta Germaniae Historica. Hannover 1879;502–526. Scriptores vol. 24.

6. Polanus Fr Martin. Chronicon Pontificum et Imperatum, AD 1265. In: Pardoe R, Pardoe D, eds. The Female Pope. Wellingborough, England. Crucible: The Mystery of Pope Joan; 1988.

7. Boccaccio Giovanni. De Claris Mulieribus 1350. In: Litterarischer Verein. 1995;341.

Chapter 2

Adrenal Development

Yewei Xing∗, John C. Achermann† and Gary D. Hammer∗∗,    ∗Department of Internal Medicine, University of Michigan, 109 Zina Ptcher Pl, Ann Arbor, MI 48109, USA, †Clinical and Molecular Genetics Unit, UCL Institute of Child Health, 30 Guilford Street, London, WC1N 1EH, UK, ∗∗University of Michigan, Millie Schembechler Professor of Adrenal Cancer, Director, Endocrine Oncology Program, Director, Center for Organogenesis, 109 Zina Pitcher Place, 1528 BSRB, Ann Arbor, MI 48109-2200, USA

Abstract

The adrenal glands comprise two distinct endocrine organs: the inner medulla and the outer cortex. The inner medulla is made up of neuroectodermal cells derived from the neural crest and produces the catecholamine hormones norepinephrine and epinephrine, which are crucial for stress responses. The outer cortex is derived from the mesoderm and synthesizes steroid hormones that are essential to maintain fluid and electrolyte balance, modulate intermediary metabolism and regulate inflammatory processes. Steroidogenesis in the adrenal cortex is mainly regulated by trophic hormones controlled by the hypothalamus–pituitary endocrine axes. Adrenal organogenesis and development of adult steroidogenesis are carefully orchestrated by action of a number of gene products. Although the pattern of development differs somewhat in diverse primates, the same genes appear to regulate the basic developmental program in all mammalian species. Most basic laboratory research is done in mice, in which prenatal development occurs within a compressed period of approximately 19 days and in which adrenals at birth are considerably less developed than in their human counterparts. This chapter describes the contributions of genes responsible for the proper development of the adrenal cortex, as well as how an understanding of adrenal gland disease provides novel fundamental insights into the regulation of adrenal development and steroidogenesis.

Keywords

adrenal development; homeostasis; SF1 (NR5A1); ACTH; adrenal hypoplasia

Introduction

The adrenal glands comprise two distinct endocrine organs: the inner medulla and the outer cortex. The inner medulla is made up of neuroectodermal cells derived from the neural crest and produces the catecholamine hormones norepinephrine and epinephrine, which are crucial for stress responses. The outer cortex is derived from the mesoderm and synthesizes steroid hormones that are essential to maintain fluid and electrolyte balance, modulate intermediary metabolism and regulate inflammatory processes. Steroidogenesis in the adrenal cortex is mainly regulated by trophic hormones controlled by the hypothalamus–pituitary endocrine axes [1]. Adrenal organogenesis and development of adult steroidogenesis are carefully orchestrated by action of a number of gene products. Although the pattern of development differs somewhat in diverse primates, the same genes appear to regulate the basic developmental program in all mammalian species. Most basic laboratory research is done in mice, in which prenatal development occurs within a compressed period of approximately 19 days and in which adrenals at birth are considerably less developed than in their human counterparts. This chapter describes the contributions of genes responsible for the proper development of the adrenal cortex, as well as how an understanding of adrenal gland disease provides novel fundamental insights into the regulation of adrenal development and steroidogenesis.

Adrenal Organogenesis

Adrenal gland organogenesis can be divided into three discrete histological phases. In the initial phase (28–30 days past conception [dpc] in humans, embryonic day (E) 10.0 in mice), the adrenogonadal primordium (AGP) is first distinguished by expression of the essential transcription factor SF1/Sf1 (Ad4BP, NR5A1) [2–5]. By 44 dpc in humans (E10.5 in mice), the AGP separates into two distinct tissues, the adrenal primordial and the gonadal primordial tissues. This process is accompanied by migration of neural crest cells through the fetal cortex to establish the medulla and formation of a mesenchymal capsule around the fetal cortex (48–52 dpc in humans, E11.5–E12.5 in mice), which represents the formation of fetal adrenal gland [6] (Fig. 2.1). In the second phase, as encapsulation progresses (beginning the 20th week of gestation in humans), the formation of the adult cortex (or so-called definitive zone) is initiated [7,8]. The human fetal zone histologically regresses at birth, while the mouse fetal zone (X zone) regresses during puberty in males or at the time of first pregnancy in females [9–11]. The third phase represents the homeostatic phase during adult life, when the adrenal gland is maintained by stem/progenitor cell repopulation throughout the lifespan. Each of the three phases will be detailed individually as below (Fig. 2.1).

FIGURE 2.1 Overview of developmental stages of the adrenal gland.

(A) Different stages and ages for mice adrenal organ development. (B) Representative symbols for different tissues in the adrenal gland. (C) Lineage of different types of cells in the adrenal gland during development. Modified with permission from Wood MA, et al. Fetal adrenal capsular cells serve as a progenitor cell niche for steroidogenic and stromal adrenocortical cell lineages in mice. Development.

Fetal Adrenal Gland

The early precursor population of both the adrenal cortex and the gonads comprises the AGP, a population of cells located in the coelomic epithelium. The AGP can be detected as early as embryonic day 11.5 (E11.5) in rats, E10.0 in mice or 28–30 dpc in humans, by expression of steroidogenic factor 1 or adrenal 4 binding protein (Sf1, Ad4BP, NR5A1; hereafter Sf1) [2], which is essential for adrenal development and a key regulator of steroidogenic pathway gene expression [3,4,12]. The AGP first appears as a thickening of the coelomic epithelium between the urogenital ridge and the dorsal mesentery. Each AGP contains a mixed population of adrenocortical and somatic gonad progenitor cells. Sf1-positive AGP cells delaminate from the epithelium and invade the underlying mesenchyme of the intermediate mesoderm. The majority of these cells migrate dorsolaterally to form the gonadal anlagen, whereas a subset of the AGP that expresses higher levels of Sf1 migrates dorsomedially to form the adrenal anlagen, settling ventrolateral to the dorsal aorta [2,13].

At about 48 dpc in humans (E11.5–E12.5 in mice), neural crest cells migrate from the dorsal midline just lateral to the neural tube to the area where the adrenal cortex is developing [6,14] and differentiate to form the catecholamine-producing chromaffin cells of the adrenal medulla, which persist as discrete islands scattered throughout the adrenals until birth. Meanwhile, the adrenal gland starts to separate from surrounding mesenchyme and becomes encapsulated by the formation of a fibrous layer overlying the developing cortical cells. The whole process is largely complete by about 52 dpc (E14.5 in mice) [7].

Transition from a Fetal to a Definitive Adrenal Cortex

Rapid growth of the fetal adrenal cortex begins from the first developmental stage. For the human fetal adrenal gland, the increase in the weight of the gland is mainly caused by the enlargement of the fetal zone (FZ), while the outer neocortex zone does not change significantly in size. At this stage, the human fetal adrenal produces large amounts of the steroid dehydroepiandrosterone (DHEA), which is then converted by the placenta to estrogens that are necessary for the maintenance of normal pregnancy [10,15]. By the 20th week of gestation, a new functional zone referred to as the definitive zone (which later develops into adult zona glomerulosa (ZG) and zona fasciculata (ZF)) is identified. Throughout the fetal period, the size of the definitive zone remains constant, and the fetal zone constitutes the majority of the gland. According to Johannisson, the fetal adrenal is one of the largest organs in humans at term (0.2% of the total body weight, almost one-third of the size of the kidney), with 80% of the gland composed of fetal zone cells [16].

In the mouse, proliferating cells are observed in a scattered pattern throughout the adrenal gland up to day E13.5 [17,18]. At later time points, these proliferating cells assemble in the periphery of the adrenal gland [19]. The prenatal adrenal cortex is composed of fetal adrenal cells surrounded by a second group of cells that develop to form a densely packed structure, the definitive (adult) cortex. The hypothesis that adrenal precursor cells of the fetal zone give rise to the definitive/adult cortex is supported by experiments performed by Zubair et al. using a FAdE-cre mice model [20]. As mentioned before, Sf1 is the critical factor for proper adrenal organogenesis and is required for steroidogenic function in both the fetal and adult adrenal cortex. Zubair et al. identified a fetal adrenal enhancer (FAdE) that directs Sf1 expression solely in the fetal cortex. During fetal adrenal development, a transcription complex containing the homeobox protein PKNOX1 (Prep1), homeobox gene 9b (Hox) and pre B cell leukemia transcription factor 1 (Pbx1) initiates fetal zone expression of SF1 by binding to the FAdE region, which is later maintained through autoregulation by Sf1 itself [20]. This enhancer is not utilized to activate Sf1 expression in the definitive cortex. However, by breeding a Rosa26 mouse with a transgenic mouse harboring a Cre-recombinase gene driven by a basal Sf1 promoter and FAdE enhancer, investigators were able to lineage-trace the fate of fetal adrenal cells during the development process. This study shows that control of Sf1 expression through the FAdE is only active before E14.5, at which time the fetal cortex begins to regress; however, all the adult cortex cells are derived from FAdE-expressing cells of the fetal adrenal. On the contrary, the adrenal capsule and the medulla were not reported as containing FAdE-derived cells. Moreover, using tamoxifen-inducible FAdE-cre mice, definitive cortex staining is only observed when tamoxifen is administered early in embryogenesis (E11.5–E12.5). Sequentially later administration of tamoxifen results in only fetal zone inner expression of LacZ. If given after E14.5 or after birth, no LacZ-positive cells can be observed, which is consistent with the absence of FAdE activity at later stages. These results suggest that the fetal cortex gives rise to the definitive/adult cortex.

Following birth, substantial remodeling of the adrenal gland occurs: the chromaffin cell islands coalesce to form a rudimentary medulla; fetal cortical cells regress; and the adult cortex begins to differentiate into zones. Studies in humans show that the fetal zone regresses by cell apoptosis; the number of apoptotic nuclei in the inner fetal zone increases with advancing gestation and is maximal during the first postnatal month. The fetal zone completely disappears by the third postnatal month in humans [11,21]. In conjunction with fetal zone regression, the definitive zone of the adrenal cortex forms discrete functional compartments (the outer ZG and the inner ZF). The most inner zona reticularis (ZR) arises around the age of 6–8 years and growth accelerates until puberty. Like the FZ, the ZG produces DHEAs and is thus hypothesized by some to originate from residual fetal zone cells after birth. Zonation is only completed around age 12 years, with the final differentiation of ZG to ZF and ZR of the gland [22]. While a similar developmental process occurs in rodents, the mouse adrenal cortex does not contain a ZR and does not produce DHEAs. It does contain an inner region of eosinophilic cells, called the X-zone [23,24] that degenerates by apoptosis at puberty in males and after the first pregnancy in females [25,26]. Recent studies suggest that the X-zone is the fetal adrenal zone [20].

Adrenal Homeostasis and Stem Cells

After the functional adult zones are established, they are maintained throughout life by stem cells or progenitor cells located in the adrenal gland (reviewed in references [27–29]). Ever since the 1950s, numerous studies have provided evidence that cells from the capsule/subcapsular region of the adrenal gland grow centripetally inward to repopulate the adrenal cortex (reviewed in Kim et al., 2009 [28]). Several studies show that most proliferating cells are located in the outer layer of the mature adrenal gland [19,30,31], suggesting the stem/progenitor cells reside in or directly under the capsule of the cortex. However, all the above studies have been primarily conducted using histology and proliferation markers. In 2010, new genetic data emerged to support this hypothesis. Three laboratories independently provided evidence that the sonic hedgehog (Shh) signaling pathway is essential for adrenal gland development and maintenance [32–34]. An established factor that is involved in the development of vertebral organ systems and regulation of both embryonic stem cells and adult tissue stem cells, Shh was shown to be present in the adrenal gland at E11.5, primarily in the subcapsular region of the adrenal cortex. It co-localizes with Sf1 in cortical cells of the subcapsular region but not in differentiated ZG or ZF cells, which express both Sf1 and markers of fully differentiated steroidogenic cells (i.e. Cyp11b1, Cyp11b2). Mice in which Shh is ablated (specifically in Sf1-expressing cells) revealed marked adrenal hypoplasia, decreased proliferation, and a depleted capsule. On the other hand, observations in a tissue-specific Shh-knockout mouse show that, despite a decrease in size, the adrenal glands maintain proper zonation, which suggests that Shh does not have a role in the initiation of differentiation. Together, these data imply that the Shh pathway is actively involved in proliferation and maintenance of the adrenal cortex. Lineage-tracing studies show that descendents of Shh-expressing cells do express adrenocortical differentiation markers (Cyp11b1 and Cyp11b2), suggesting that Shh-positive cells may serve as progenitor cells for the adrenal cortex. Further studies provide evidence that the adrenal capsule could be the adrenocortical stem/progenitor cell niche by focusing on a downstream activator of the hedgehog pathway, Gli1 [32–34]. In contrast to Shh-expressing cells, Gli1-expressing cells locate specifically in the adrenal capsule and do not express Sf1. This subpopulation of cells is capable of giving rise to Sf1-expressing, differentiated adrenocortical cells. Whether capsular Gli1-positive, Sf1-negative cells or subcapsular Shh-positive, Sf1-positive cells (or both populations) serve as adrenal stem/progenitor cells and what the specifics of the relationship between those two cell population is remains unclear [29]. Further studies are needed to determine the exact mechanism of Shh signaling to the Gli1-positive cells and what factors might regulate Shh signaling in the adrenal gland. These studies have been in agreement with cell migration models of adrenocortical zonation, which propose that a precursor population differentiates first into ZG cells and then changes its phenotype as it migrates centripetally into the ZF and the ZR. Studies using chimeric animals and analyses of the expression pattern of a steroid 21-hydroxylase (CYP21)-β-galactosidase transgene have been interpreted to support this cell migration model that is important for adrenal homeostasis [35,36]. As cells reach the medullary boundary (ZR) an increased frequency of cell death is observed [21,37–39]. Whether these two different experimental observations that cells of both the Sf1-positive fetal zone and cells of the Sf1-negative capsule give rise to Sf1-positive adult adrenocortical cells reflect two temporally distinct lineages of the definitive cortex or reflect a singular developmental and homeostatic mechanism of adrenal growth is unclear.

Molecular Mechanisms That Regulate Adrenal Development

As mentioned above, adrenal development is a highly orchestrated process, controlled by numbers of autocrine, paracrine and endocrine factors at different locations and different stages. The following section will focus on talking about the function of known factors that interplay in adrenal organogenesis and homeostasis.

Hormonal Regulation of Adrenal Development

Adrenocorticotropic Hormone (ACTH)

As a major component in the hypothalamic–pituitary–adrenal axis, ACTH is a 39-amino acid peptide secreted from the anterior pituitary gland under the control of corticotropin-releasing hormone (CRH) [40]. By binding to the transmembrane receptor MC2R, ACTH exerts its effect mainly by activating downstream Ras/MEK/ERK signaling pathways [41]. It has been established that, although during the first trimester of human pregnancy adrenal growth does occur independently of ACTH, after about week 15 of gestation ACTH starts to play an essential role in the morphological and functional development of the adrenal gland [42]. Part of its functions is suggested to be through the stimulation of locally produced growth factors such as insulin-like growth factor 2 (IGF2) and fibroblast growth factor beta (FGFβ) [10,15]. During development, as the outer definitive zone of the cortex begins to emerge, ACTH participates in the regulation of steroidogenesis, cell differentiation, and cell growth [43–45].

Insulin-like Growth Factor 2 (IGF2)

Both IGF1 and IGF2 are mitogens expressed in the adrenal gland. Upon binding to the dimeric/heterodimeric cell surface receptors (insulin receptor [IR] or IGF receptor 1 [IGF1R]), IGFs can induce autophosphorylation of the intracellular part of the receptor, which leads to activation of two downstream signaling pathways, Ras/MEK/ERK and PI3K/AKT [46,47]. Target genes participate in many cellular processes, including cell cycle activation. Although both IGF1 and IGF2 are present during the process of adrenal development, IGF2 is generally considered to play a key role in early fetal development [48–50]. In support of the above statement, infusion of IGF2 does cause a significant increase in adrenocortical growth during embryogenesis. In adulthood, a switch in expression levels makes IGF1 the dominant IGF in the adrenal where it functions as a regulator for postnatal growth maintenance. Although IGF2 levels are much lower in adults compared to the fetal adrenal cortex, its expression is restricted to the capsular region, which coincides with the stem/progenitor location in the gland [48]. Furthermore, IGFs, along with FGF, have been shown to be essential factors for stem cell niche [51,52], supporting a potential role of IGF2 in adrenal stem/progenitor cell maintenance as well.

Adrenal Steroids

As the major products from the adrenal glands, adrenal steroids are generally considered to exert their endocrine effects by working on other organs. However, a study by Gummow et al. suggested that glucocorticoids can also stimulate an intra-adrenal negative feedback loop via activation of the dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1 (Dax-1) expression [53]. A glucocorticoid-dependent synergy between Sf1 and the glucocorticoid receptor (GR) leads to activation of Dax-1, while ACTH stimulation disrupts the formation of this complex by abrogating SF1 binding to the Dax-1 promoter. These data indicate that, instead of the being considered solely as a product, steroids may also play an important role in adrenal regulation.

Transcriptional Regulation of Adrenal Development

Sf1

Sf1 (Nr5a1) is a 462-amino acid orphan nuclear receptor, which is required for adrenal and gonadal development and regulates a large group of adrenal and gonadal target genes. In the mouse, Sf1 is first expressed in the urogenital ridge at E9 and subsequently in the adrenal primordium at E11, and adrenocortical cell at E13 [54,55]. Homozygous deletion of Sf1(–/–) in mice results in adrenal agenesis and death shortly after birth owing to steroid deficiency, while heterozygous mice (Sf1+/–) exhibit smaller adrenal glands and significantly decreased steroid production and steroidogenic gene expression, suggesting a dose-dependent effect of Sf1 on adrenal development and differentiation (steroidogenesis) [56,57]. Similar phenotypes are reported in human patients bearing mutations in the SF1 gene [58,59].

As discussed above, different enhancers can regulate the activity of Sf1 at individual developmental stages, providing a switch mechanism in organogenesis and zonation. FAdE directs Sf1 expression solely in the fetal cortex. During fetal adrenal development, Prep1, Hox and Pbx1 initiate fetal zone expression of Sf1 by binding to the FAdE region, which is later maintained through Sf1 autoregulation [20]. Lineage-tracing studies using the FAdE enhancer show that control of Sf1 expression through the FAdE is only active before E14.5. After that, Sf1 is maintained by the action of a proposed definitive adult adrenal enhancer (DAdE) and definitive zones start to form. However, all the adult cortex cells are derived from FAdE-expressing cells of the fetal adrenal, while the capsule and the medulla do not show a FAdE origin. Sf1 regulates the transcription of a vast array of genes involved in sex determination and differentiation (WT1, DAX1, AMH, AMHR), reproduction (GNRHR, GSUA, LHB, FSHR, oxytocin, PRLR, INSL3, inhibin alpha, Oct3/4), steroidogenesis (ACTHR, STAR, CYP11, CYP19, Akr1b7, etc.), and metabolism (HDLR, SHP, SRB1, SCP2) by direct/indirect binding to their promoters. Depending on the co-activators/co-repressors with which it associates, Sf1 can exert a diverse range of effects on steroidogenesis and development. Additionally, Sf1 activity can also be regulated by several forms of post-translational modification, such as phosphorylation [60–64], acetylation, and SUMOylation [65–69]. All of these factors make Sf1 a fascinating yet complicated mediator of adrenal development and homeostatic maintenance.

Dax-1

The orphan nuclear receptor Dax-1 (Nr0b1) was first cloned as the gene responsible for X-linked cytomegalic adrenal hypoplasia congenita. Its expression in the adrenal gland is enriched in the subcapsular region, suggesting a potential function of Dax-1 in adrenal maintenance. As detailed below, DAX-1-deficient patients classically exhibit histologic adrenal hypoplasia and resultant adrenal insufficiency [70,71]. However, some studies also demonstrate the presence of a hyperfunctional period before hypoplasia, which may be explained by the fact that Dax-1 represses adrenocortical steroidogenesis (or differentiation) by blocking Sf1 activity [72]. Research results from the Morohashi and Parker groups further indicate that Dax-1 may act as the repressor for FAdE activity in the adrenal gland during fetal-to-adult transition [20]. All the above data support the hypothesis that loss of adrenal function in DAX-1-deficient patients is caused by a depletion of an aging adrenocortical progenitor reserve [73–75]. Combined with the recent finding that Dax-1 is highly expressed in the mouse embryonic stem cells, while knockdown of this gene results in increased differentiation, it is reasonable to propose that Dax-1 plays an important role in maintenance of stem/progenitor cell pluripotency.

Accordingly, the regulation of Dax-1 expression is predicted to be a dynamic process balancing progenitor renewal and adrenocortical differentiation/steroidogenesis. Dax-1 transcription in the adrenal gland is activated by Sf1 in cooperation with paracrine Wnt signaling together with glucocorticoids synthesized in the differentiated adult cortex [53]. Conversely, ACTH, the well-established glucocorticoid stimulator, has been shown to remove Sf1 complexes completely from the Dax-1 promoter, thus leading to effective shutdown of Dax-1 transcription. This process would be predicted to be permissive to the response of the Sf1-positive progenitor cells to ACTH and the subsequent initiation of steroidogenesis [53]. In mice embryonic stem cells, Dax-1 has also been proven to be activated by luteinizing hormone releasing hormone (LRH) efficiently [76].

Wnt/β-catenin

The Wnt/β-catenin signaling pathway has been intensively studied for its role in embryonic development, stem cell maintenance, and cell fate determination in many tissues [77–79]. As in a number of cancers, β-catenin activating mutations have been identified in a subset of sporadic adrenocortical adenomas and carcinomas [80–82]. Wnt ligands signal by binding to the Frizzled cell surface receptor, which, upon activation, will disrupt the cytoplasmic complex composed by adrenomatous polyposis coli (APC), Axin, GSK3β, and β-catenin. Once released from the complex, β-catenin (instead of being degraded by ubiquitin) will move into the nucleus in the non-phosphorylated form and activate downstream target genes as a transcription factor. Studies performed in the mouse adrenal revealed that β-catenin expression and activity is present early in the fetal cortex. However, by E18.5, with the emergence of new definitive cortex, β-catenin is restricted to the subcapsular region [83]. Studies performed by Kim et al. employed cre-lox technology to ablate β-catenin specifically in Sf1-expressing cells of the adrenal cortex [84]. In mice expressing a high level of the Sf1-cre transgene, adrenal aplasia is found and mice are embryonic lethal. Careful examination of these mice showed that normal adrenal development continued until E12.5, when adrenal failure became evident precisely when few definitive/adult cortical cells emerged between the coalescing capsule and the fetal zone. Intriguingly, in mice bearing a low expressing Sf1-cre transgene, which continued to express β-catenin in half of the adrenocortical cells, adrenal development progressed normally. However, as the mice aged (i.e. 30 weeks), a progressive cortical thinning and a decreased steroidogenic capacity was identified [83]. This progressive failure of the cortex is hypothesized to be because of the loss of adrenocortical progenitor cells. In support of the role of the Wnt/β-catenin signaling pathway in maintenance of stem/progenitor cells in the adrenal gland, overactivation of this pathway is frequently observed in adrenocortical carcinomas [80–82,85,86]. Although the exact mechanism of Wnt/β-catenin signaling remains unknown, the fact that β-catenin and Sf1 can directly activate Dax-1 suggests that Dax-1 may be a critical mediator of Wnt action in the adrenal cortex.

Shh/Gli

Sonic hedgehog (Shh), along with Desert hedgehog (Dhh) and Indian hedgehog (Ihh), in mammals are the three members of an evolutionarily conserved protein family. Shh has been shown to play important roles in embryonic development, adult stem cell maintenance, and cancer [87–90]. Shh acts by binding to its receptor Patched1 (Ptch1), and subsequently releasing the inhibition on the seven transmembrane domain protein Smoothened (Smo). Activation of Smo changes the ratio of Gli repressor and activator forms, allowing for variable downstream effects proportional to the magnitude of the Hh signal.

Shh starts to express early in rodent adrenal glands. In the adrenal cortex of embryonic mice, Shh mRNA expression can be detected as early as E12.5, in the peripheral adrenocortical cells [87–90] and is maintained in the same pattern throughout embryogenesis [32,33,87,89] and adulthood [32]. However, the major ligand in the adrenal, Gli1, is expressed primarily in the capsule. As mentioned above, lineage-tracing studies using an inducible cre system demonstrated that in adult mice, both Shh- and Gli-positive cells can give rise to differentiated adrenocortical cells and thus are considered potential candidates for stem/progenitor cells in the adrenal gland. Research on Shh mutant adrenals reported defects in both the capsule and cortex. The mutant capsule was thinner with less proliferation, while the cortex has many fewer cortical cells but normal zonation compared to normal controls. Considering the fact that Shh signals to Gli-positive cells, it has been speculated that Shh in the subcapsular region activates Gli signaling in the capsule cells, that serve to maintain an adrenal progenitor pool in or under the capsule, thereby regulating the process of adrenocortical homeostasis.

Others

Wilm’s tumor1 (Wt1) and Cited2 play an important role by upregulating Sf1 expression at the stage of separation of the AGP into gonadal and adrenal regions. While both genes are involved in development of adrenals and gonads, they are critical factors for adrenal specification [14].

Pbx. In the fetal adrenal gland, Sf1 expression is regulated by a fetal adrenal-specific enhancer (FAdE) located in the fourth intron of Sf1. Transgenic assays revealed that the activation of FAdE requires binding of a Hox–Pbx1–Prep1 complex to a site in the FAdE and that maintenance of FAdE-dependent Sf1 expression over time is effected in an autoregulatory manner by Sf1 itself [20].

Inhibin α is an atypical member of the TGFβ family of signaling ligands. Under physiological conditions, it is present in the cortex but not the medulla, with an inner zone-specific pattern. Inhibin α is expressed in both adrenocortical carcinomas and benign adrenocortical adenomas. Following gonadectomy, the adrenal cortex of inhibin-null (Inha–/–) mice undergoes profound remodeling secondary to aberrant luteinizing hormone (LH)-dependent proliferation and gonadal differentiation of subcapsular adrenocortical progenitor cells. Further studies demonstrated that LH signaling specifically upregulates expression of TGFβ2 in the subcapsular region of the adrenal cortex, leading to aberrant Smad3 activation in Inha–/– adrenal glands [91,92]. A switch from predominant expression of Gata6 (endogenous to the adrenal cortex) to Gata4 (defines cellular identity in the ovary) in the Inha–/– adrenal [93] drives both ovarian theca and granulosa cell lineages in the adrenal, suggesting a role of inhibin α in the maintenance of adrenal versus gonadal fate of the adrenocortical progenitor cells. Binding of the synergized β-catenin/Sf1 complex to the promoter region can stimulate inhibin α gene expression in rat [94].

Pod1 is a transcription factor with the basic helix–loop–helix (bHLH) motif, and has been shown to play crucial roles in cell fate determination and differentiation in a variety of tissues, including the gonads. Loss of Pod1 results in increased expression of Sf1 and an increased number of fetal Leydig cells [95–97]. Although its function in the adrenal cortex remains elusive, the finding that expression of Pod1 is restricted to the adrenal capsule [98] and is significantly decreased in adrenal tumors (Lotfi and Hammer, unpublished data), suggests a regulatory role in the adrenal cortex.

Telomerase is a reverse transcriptase that adds DNA sequence repeats to the 3’ end of DNA strands in the telomere regions. It carries its own RNA molecule and uses it as a template when elongating telomeres, which are shortened after each replication cycle. Telomerase is expressed in embryonic stem cells, allowing cells to divide repeatedly during organogenesis. In adults, telomerase is only present in cells that need to divide regularly (e.g. in the immune system), but not in normal resting cells. In accordance with previous findings on adrenal stem/progenitor cell location, the RNA component of telomerase is exclusively found under the capsular region of the adult adrenal, suggesting the presence of active telomerase in proliferating progenitor populations [99,100]. Moreover, the cellular senescence of this population resulting from the loss of function of the mouse tpp/acd gene (adrenocortical dysplasia – a component of the telomere capping complex, shelterin) is rescued in the absence of P53, albeit at the expense of adrenocortical carcinoma [101], indicating a critical role of telomere protection in the maintenance of adrenocortical stem/progenitor cells. Indeed, adrenocortical carcinomas display significantly enhanced telomerase activity compared to benign adrenocortical tumors [102], making telomerase a potential marker for malignancy in adrenal tumor diagnosis [103]. Further study indicates that hormone levels (estrogen) can inhibit telomerase activity and thus reduce cell proliferation in the adrenal gland in mice [104].

All the factors involved in adrenal gland development have been summarized in Table 2.1.

TABLE 2.1

Summary of Factors Involved in Adrenal Development, their Name, Location and Proposed Function

Adrenal Diseases

Underdevelopment of the adrenal glands in humans results in a clinical condition known as adrenal hypoplasia. Adrenal hypoplasia is a potentially life-threatening disorder which can present with adrenal failure soon after birth, or more insidiously throughout childhood or even in young adulthood [105]. Appropriate assessment, diagnosis, and treatment are essential.

Adrenal hypoplasia can be divided into three broad categories: (1) secondary adrenal hypoplasia caused by disruption of ACTH synthesis, processing and/or release by the pituitary corticotrope cells; (2) ACTH resistance and related conditions (also known as familial glucocorticoid deficiency; FGD); and (3) primary adrenal insufficiency (sometimes called adrenal hypoplasia congenita"; AHC) due to defects in development and function of the adrenal gland itself [Fig. 2.2; Table 2.2]).

FIGURE 2.2 Overview of the hypothalamic–pituitary–adrenal axis showing the different types of adrenal hypoplasia.

POMC, Pro-opiomelanocortin; ACTH, adrenocorticotropin; DHEA, dehydroepiandrosterone; FGD, familial glucocorticoid deficiency; AHC, adrenal hypoplasia congenita; CRF, corticotropin-releasing factor; CLAH, congenital lipoid adrenal hyperplasia; NNT, nicotinamide nucleotide transhydrogenase. Modified with permission from Lin L, Achermann JC. Inherited adrenal hypoplasia: not just for kids! Clin Endocrinol 2004;60:529–537. Copyright 2004 Blackwell Publishing Ltd.

TABLE 2.2

Overview of Genetic Causes of Adrenal Hypoplasia and Related Conditions

Aldo, aldosterone; MPHD, multiple pituitary hormone deficiency; SOD, septo-optic dysplasia; ACTH, adrenocorticotropin; def., deficiency; HH, hypogonadotropic hypogonadism; FGD, familial glucocorticoid deficiency; CLAH, congenital lipoid adrenal hyperplasia; N, within the normal range; AHC, adrenal hypoplasia congenita; IUGR, intrauterine growth restriction;

∗mineralocorticoid insufficiency or apparent hyponatremia seen in a proportion of cases.

Modified with permission from Lin L, Achermann JC. Inherited adrenal hypoplasia: not just for the kids! Clin Endocrinol 2004; 60: 529–37. Copyright 2004 Blackwell Publishing Ltd.

Secondary Adrenal Hypoplasia

As outlined above, ACTH plays an important role in the tropic stimulation of the adrenal gland during development. ACTH is synthesized in the corticotrope cells of the anterior pituitary gland in response to hypothalamic corticotropin-releasing factor (CRF) stimulation. In humans, the mature secreted form of ACTH is a 39-amino acid peptide that is cleaved from the larger precursor molecule, pro-opiomelanocortin (POMC), along with other small peptides including α- and β-melanocyte stimulating hormone (MSH) and β-endorphin [106].Consequently, any defect in the development or function of the corticotropes or in the synthesis, processing, and release of ACTH can result in impaired adrenal development and secondary adrenal hypoplasia.

Most children with secondary adrenal hypoplasia present with features of glucocorticoid insufficiency, such as hypoglycemia, prolonged jaundice, and even collapse. Some of these effects (such as hypoglycemia) are exacerbated if the child has multiple pituitary hormone deficiencies. Salt loss is unusual as angiotensin II, the principal drive to adrenal aldosterone production, is unaffected. However, reduced free water clearance owing to cortisol deficiency can produce a tendency to dilutional hyponatremia. Thus, the diagnosis of secondary adrenal hypoplasia is supported by finding low or inappropriately low serum concentrations of ACTH in the presence of glucocorticoid deficiency, a lack of clinical hyperpigmentation, and associated features in certain cases (Table 2.2). In contrast, most children with ACTH resistance or primary adrenal hypoplasia have very high circulating ACTH concentrations.

Multiple Pituitary Hormone Deficiencies (MPHD)

Impaired ACTH production as part of a multiple (or combined) pituitary hormone deficiency (MPHD) can occur in disorders of hypothalamopituitary development that affect corticotrope function (e.g. septo-optic dysplasia, pituitary hypoplasia) or more global disorders affecting brain development (e.g. anencephaly, holoprosencephaly). In general, corticotropes are the anterior pituitary cell lineage that are most resistant to developmental defects or even external insults such as radiotherapy. Consequently, children with corticotrope dysfunction are likely to have deficiencies in other pituitary hormones, such as thyroid-stimulating hormone (TSH), growth hormone (GH) and the gonadotropins (luteinizing hormone [LH] and follicle-stimulating hormone [FSH]). Clinical features can therefore include profound hypoglycemia (owing to ACTH and GH deficiencies), prolonged jaundice (owing to ACTH and TSH deficiencies), and signs of congenital hypogonadotropic hypogonadism such as a small penis and undescended testes, or postnatal growth failure. Other neurodevelopmental defects such as absent septum pellucidum or optic nerve hypoplasia may be present. Posterior pituitary dysfunction with antidiuretic hormone (ADH, vasopressin) insufficiency can occur in some situations, and might only become apparent following glucocorticoid replacement, so expert management of potential neonatal panhypopituitarism and careful monitoring and investigation is required, especially after glucocorticoid replacement is started.

Several single gene disorders causing MPHD have been described in recent years. These are summarized in Table 2.2 and have been discussed in detail elsewhere and are beyond the scope of this chapter [107]. Most of these genes encode transcription factors that play a key role in pituitary development and in the specification and expansion of different pituitary cell lineages, such as HESX1, OTX2, GLI2, LHX3, LHX4 and SOX3. These genes can be disrupted by specific point mutations, deletions or copy number variants and, in some cases, associated features such as septo-optic dysplasia (HESX1) or cervical spine anomalies and neck rigidity (LHX3) can be present.

As corticotrope function is relatively robust compared to other pituitary hormones, ACTH insufficiency may not be clinically or biochemically apparent when the child originally presents, but may develop progressively with time. Therefore, careful monitoring and vigilance of individuals with MPHD is needed if ACTH insufficiency is not present at the time of the original diagnosis, and reaching a molecular diagnosis can be important in predicting whether ACTH insufficiency might develop. One example where this can occur is when MPHD is due to mutations in the transcription factor PROP1 (Prophet of Pit-1). ACTH deficiency was not originally described as part of this phenotype. However, longer term follow-up data suggest that progressive ACTH deficiency may occur in adulthood in a subset of patients with PROP1 mutations [108]. These studies demonstrate how it is important to undertake detailed long-term follow-up of patients with pituitary disorders for the development of additional hormone deficiencies.

Isolated ACTH Deficiency

Isolated ACTH insufficiency is a rare condition that can be caused by mutations in the T-box factor, TPIT (TBX19). This condition is inherited in an autosomal recessive fashion and was described more than a decade ago [109]. TPIT is a transcription factor that regulates the synthesis of POMC specifically in corticotropes, but not in other POMC-producing cells in the body such as the skin and hypothalamus. Consequently, disruption of TPIT function produces defects in the synthesis of POMC and ACTH in the pituitary gland alone. Individuals with TPIT mutations usually present with hypoglycemia and prolonged jaundice owing to severe, early-onset isolated ACTH insufficiency, and sudden death in the first few weeks of life is reported [110]. As POMC production in the skin and hypothalamus are spared, features such as red hair or obesity are not seen. Furthermore, TPIT mutations are unusual when isolated ACTH deficiency first presents in childhood [111]. The molecular basis of this later-onset form of isolated ACTH deficiency is poorly understood.

Disorders in POMC Synthesis and Release

As described above, the mature ACTH peptide is cleaved from POMC together with other short peptides such as β-endorphin and MSH. These peptides are important in regulating appetite and weight in the hypothalamus as well as stimulating pigmentation of the hair and skin through their effects on the melanocortin type 1 receptor (MC1R). Consequently, deletions or mutations of the POMC gene cause obesity, red hair, and pale skin [112,113]. Some of these features, such as pale skin and red hair, may be less pronounced in patients from different ancestral backgrounds [114]. Individuals who have naturally dark hair may have auburn roots as the predominant feature.

ACTH synthesis from POMC is not only affected by changes in the POMC gene itself, but can also result from defects in POMC cleavage owing to disruption of the enzyme prohormone convertase-1 (PC1, also known as proprotein convertase, subtilisin/kexin-type, 1/PCSK1). This enzyme is involved in the cleavage of many different pre-prohormones in the brain and gut, so that clinical features of this condition include obesity, hypogonadism, hypoglycemia, and persistent malabsorptive diarrhoea [115,116]. Abnormalities in ACTH processing caused by defects in PC1 are a rare cause of secondary adrenal failure. However, rare variants in PCSK1 have been associated with an increased risk of obesity [117].

ACTH Resistance and Related Syndromes

The tropic effects of ACTH stimulation of the adrenal gland are mediated by the melanocortin-2 receptor (MC2R, ACTH receptor) and subsequent downstream signaling pathways. ACTH resistance can occur because of defects in the ACTH receptor (MC2R, FGD type 1) or the MC2R accessory protein (MRAP, FGD type 2) (Table 2.2) [118]. Non-classic congenital lipoid adrenal hyperplasia, caused by partial loss of function of the steroidogenic acute regulatory protein (StAR), may also mimic these conditions. Biochemical features similar to ACTH resistance are also found in related causes of adrenal insufficiency, such as Triple A syndrome (alacrima, achalasia, Addison; also known as Allgrove syndrome and caused by defects in ALADIN/AAAS), or in the more recently reported forms of adrenal failure owing to disruption of MCM4 or NNT. However, these conditions may also affect cellular development, function and damage, so the underlying mechanism or mechanisms of these conditions is currently unclear.

Familial Glucocorticoid Deficiency Type 1 (FGD1): MC2R

FGD is a rare autosomal recessive disorder characterized by early-onset severe cortisol deficiency and high plasma ACTH concentrations. Mineralocorticoid levels are usually normal and salt loss does not usually occur. Symptoms of glucocorticoid deficiency, such as hypoglycemia, prolonged jaundice, and failure to thrive, usually appear in the neonatal period or early childhood [118]. Hyperpigmentation usually develops in the first few weeks of life because of the effects of elevated ACTH stimulation on the MC1R in the skin, or caused potentially by higher levels of POMC cleavage peptides such as MSH. Older children can present with recurrent infections, hyperpigmentation, lethargy, and collapse.

The ACTH receptor (officially known as MC2R) was originally cloned in the early 1990s and was the most obvious potential cause of FGD. Mutations in this receptor were first described by Clark and colleagues and termed FGD1 [119]. To date, more than 30 different changes in this receptor that cause loss-of-function have been described [120]. These variants tend to be missense mutations resulting in substitutions of single amino acids throughout the receptor, which affect ligand binding, transmembrane domain structure, or signal transduction. More severely disruptive nonsense or frameshift changes in the MC2R can sometimes be associated with transient hyponatremia for various reasons, and these patients might be misdiagnosed as having primary adrenal hypoplasia [121]. However, long-term mineralocorticoid replacement is not generally needed [122]. Some patients with FGD1 may also be found to have tall stature at presentation [123]. The molecular mechanism of this feature is not fully understood. Hydrocortisone replacement can arrest this increased growth.

Familial Glucocorticoid Deficiency Type 2 (FGD2): MRAP

As mutations in MC2R were only found in approximately 25% of patients with FGD it soon became apparent that FGD was likely be a heterogeneous condition caused by several underlying mechanisms. By studying SNP-array genotyping and homozygosity mapping in a family of three affected siblings with FGD of unknown etiology, Metherell and colleagues were able to define a relevant candidate region at chromosome 21q22.1 which was likely to contain a novel gene responsible for this condition. Thirty different genes were found in this region, but only one of them was expressed strongly in the human adrenal gland [124]. This gene encoded a novel single transmembrane domain protein. This protein was shown to be involved in trafficking the MC2R from the endoplasmic reticulum to the cell membrane, and was therefore termed melanocortin 2 receptor accessory protein (MRAP) [124,125]. Mutations in the gene encoding MRAP were found in approximately 25% of patients with FGD, leading to the categorization of this condition as FGD type 2 (FGD2). To date, nine different mutations in MRAP that severely disrupt the protein function have been described.

Children with MRAP mutations tend to present in a similar fashion to those with FGD1, but tall stature is not a common feature of the condition [118]. This may reflect the fact that most patients with MRAP mutations present at a younger age. Milder disruptive changes in MRAP can present with delayed-onset adrenal failure at an older age [126]. At the molecular level, naturally occurring mutations in MRAP have been shown to impair trafficking of the ACTH receptor (MC2R) to the cell surface [127].

Non-classic Congenital Lipoid Adrenal Hyperplasia (CLAH): STAR

Steroidogenic acute regulatory protein (StAR) plays a key role in the transfer of cholesterol into the mitochondrial membrane, which is needed for the initial stages of steroid synthesis in the adrenal glands and gonads. Classic disruption of STAR protein function results in congenital lipoid adrenal hyperplasia (CLAH), whereby steroidogenic cells not only have reduced steroid production but also undergo destruction due to accumulation of free cholesterol secondary to ACTH stimulation (the two-hit hypothesis) [128]. Children with CLAH typically present with severe salt-losing adrenal failure in the later neonatal period. Children with a 46,XY karyotype have female-appearing genitalia, but no uterus and a failure of puberty owing to the lack of testicular androgen synthesis. However, it is now emerging that milder disruption of the STAR protein can result in relatively preserved Leydig cell function but impaired cortisol production (non-classic CLAH) [129]. Boys with this condition may have normal genitalia or mild hypospadias, and present in childhood with progressive glucocorticoid insufficiency [129,130]. Mineralocorticoid production is intact or only minimally affected, Therefore, these children, including 46,XX girls with delayed onset adrenal failure, can be misdiagnosed as having ACTH resistance. The point changes in the protein often involve residues involved in cholesterol binding and result in partial loss of function in assay systems (e.g. p.R182C). Leydig cell dysfunction may occur with time, so careful monitoring into puberty and adulthood is needed. The presenting features of non-classic CLAH have led some authors to consider this FGD3.

Triple A Syndrome

Triple A syndrome (achalasia–Addisonianism–alacrima syndrome; Allgrove syndrome) is a rare autosomal recessive disorder characterized by ACTH-resistant adrenal failure, alacrima, and achalasia of the esophageal cardia [131]. Progressive central, peripheral, and autonomic neurological defects are commonly seen as well as skin changes in some patients [132]. This is a very heterogeneous condition clinically as only one or two of the three classic features may be present, even within the same kindred, so careful history-taking and examination may be needed to identify other affected family members. Achalasia is often the first feature [133]. Adrenal failure is a key feature of the condition and in some series isolated glucocorticoid deficiency is seen in approximately 80% of the cases, often developing in the first decade of life. Mineralocorticoid deficiency is reported in 15% of cases, making primary adrenal hypoplasia a differential diagnosis. Histologically, the adrenal glands have preserved ZG with atrophic ZF and ZR.

The gene responsible for Triple A syndrome was found following the mapping of the disease locus to chromosome 12q13 [134,135]. Mutations in AAAS were subsequently identified in more than 80% of individuals and families with Triple A syndrome. The protein encoded by AAAS was termed ALADIN and contains a WD repeat domain. Initial studies suggested that this protein might be involved in protein–protein interactions and could mediate the assembly of multimolecular complexes, such as the nuclear pore complex [136]. In addition, the ferritin heavy chain protein was reported to interact with ALADIN following a bacterial two hybrid screen, suggesting a role for ALADIN in protection from oxidative damage [137]. The exact function of ALADIN and the mechanisms that lead to the ACTH-resistant adrenal phenotype remain largely unknown, but could involve reduced import of antioxidant proteins, making the cells more liable to undergo oxidative stress.

Adrenal Failure Due to Replication Pathway Defects: MCM4

Adrenal failure associated with short stature, natural killer cell deficiency and increased chromosomal instability was first reported in a kindred from Ireland in 2008 [138]. Using exome sequencing, the genetic basis of this condition has recently been reported by several groups and found to result from a mutation in maintenance-deficient 4 homolog (MCM4) [139–141]. MCM4 forms part of the MCM2–7 complex that acts as the replicative helicase and is required for DNA replication and genome stability. The exact basis of the adrenal disorder associated with this condition is unclear, but it usually emerges gradually during childhood. Data from mouse studies have suggested that MCM proteins may affect the growth and differentiation of adrenal progenitor cells into steroidogenic tissue [142]. At present this syndrome has only been described in patients of Irish ancestry, though it remains to be seen if variants of this are more widespread in other populations.

Adrenal Failure Due to Oxidative Stress: NNT

Another recently characterized condition that presents with ACTH resistance-like features is caused by mutations in nicotinamide nucleotide transhydrogenase (NNT) [143]. Patients tend to present with isolated glucocorticoid deficiency in the first 4 years of life and harbor changes throughout the NNT gene. NNT encodes an enzyme that forms part of the inner mitochondrial membrane, which uses energy from the mitochondrial proton gradient to generate NADPH. NADPH is required to regenerate reduced glutathione, which allows glutathione peroxidases to detoxify reactive oxygen species (ROS). A failure of ROS detoxifixation in adrenal steroidogenic cells can lead to oxidative stress and cell damage.

Primary Adrenal Hypoplasia

Adrenal hypoplasia congenita (AHC), also known as congenital adrenal hypoplasia, is a disorder of adrenal development resulting in primary adrenal insufficiency. This condition usually presents with severe salt-losing primary adrenal failure in early infancy or childhood, although milder, delayed onset, forms of the condition exist. The most common form of AHC is X-linked congenital adrenal hypoplasia caused by disruption of the nuclear receptor DAX1 (NR0B1) (Table 2.2). Rarer autosomal forms of adrenal hypoplasia can occur with mutations in steroidogenic factor-1 (SF1, NR5A1), or as part of syndromes such as IMAGe (CDKN1C) or SeRKAL (WNT4). Therefore, the inheritance pattern and associated or syndromic features might help point to a specific diagnosis. In a significant proportion of cases of primary adrenal hypoplasia the underlying cause is not known.

X-linked Adrenal Hypoplasia: DAX1 (NR0B1)

X-linked AHC results from mutations in the nuclear receptor DAX1 (NR0B1). This condition is the most prevalent form of primary adrenal hypoplasia reported to date (the key role that DAX1 plays in adrenal development has been discussed earlier). X-linked AHC was first described in the literature by the pathologist Sikl in 1948 in an infant with hyperpigmentation and small adrenal glands who died at 33 days of age. This condition was termed cytomegalic adrenal hypoplasia because of the presence of cytomegalic cells, which are typical of fetal-zone adrenal tissue. The X-linked pattern of inheritance of AHC started to become apparent in the 1960s as more detailed pedigree studies were performed. The introduction of steroid replacement therapy reduced mortality rates and allowed boys with this condition to survive; with this came the discovery that hypogonadotropic hypogonadism and a failure to progress through puberty was also a key feature of this condition.

The gene responsible for X-linked AHC was localized to the short arm of the X-chromosome (Xp21.3) following the discovery that X-linked AHC could occur as part of a contiguous gene deletion syndrome together with glycerol kinase deficiency (GKD), ornithine transcarbamylase deficiency (OTC) and Duchenne muscular dystrophy (DMD). By studying patients with different deletions in this region, a more precise locus for X-linked AHC was defined and in 1994 the gene for this condition, DAX1 (AHCH, NR0B1), was reported [71–74]. DAX1 is an abbreviation for Dosage-sensitive sex reversal-Adrenal hypoplasia congenita critical region on the X chromosome 1, as duplication of this region/gene in 46,XY individuals results in testicular dysgenesis (sex reversal). The identification of point mutations in DAX1 in individuals and families with X-linked AHC conclusively established that DAX1 is the causative gene for this condition [74].

DAX1 (officially called NR0B1) encodes an atypical nuclear receptor consisting of 470 amino acids. The carboxyl terminus of DAX1 has a region that resembles the ligand-binding domain of nuclear receptors, although no known ligand for DAX1 has been identified. The amino terminus of DAX1 has an atypical region consisting of approximately 3.5 repeats of an LXXLL-containing sequence (Fig. 2.3A). DAX1/NR0B1 is expressed in the adrenal gland, gonad, and central reproductive axis during development and postnatal life. These expression patterns are consistent with the role of this transcription factor in the development and function of the adrenal and reproductive systems.

FIGURE 2.3 (A). Schematic of DAX-1 (NR0B1) showing the atypical amino terminal repeat motif structure and a carboxyl terminal region that resembles a ligand-binding