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The adrenal gland has two embryologically, anatomically and functionally distinct subunits, the cortex and medulla (1). The adrenal cortex secretes steroid hormones, including glucocorticoids, mineralocorticoids and androgens (1). The glucocorticoid cortisol is secreted by the cells of the intermediate zona fasciculata. Its secretion is tightly regulated by hypothalamic corticotropin-releasing hormone (CRH) and vasopressin (AVP) and by pituitary adrenocorticotropin hormone (ACTH), in a cascade that has been called the hypothalamic-pituitary-adrenal axis (2). Glucocorticoids are crucial for the maintenance of metabolic, cardiovascular and immune homeostasis and for the integrity of several central nervous system functions. The mineralocorticoid aldosterone is produced by the outer adrenal zona glomerulosa (1). This steroid helps to maintain water and electrolyte homeostasis and its secretion is under the principal control of the renin-angiotensin axis and only weakly influenced by ACTH. Adrenal androgens with 19 carbon atoms, such as dehydroepiandrosterone (DHEA) and its sulfate (DHEA-S) and androstenedione, are secreted by the inner zona reticularis and are under the control of ACTH (1).

ADRENOCORTICOTROPIN INSENSITIVITY SYNDROMES

Familial isolated glucocorticoid deficiency is a form of potentially lethal hereditary unresponsiveness to ACTH that manifests as primary adrenal insufficiency, usually without mineralocorticoid deficiency (3,4). Affected children commonly present within the first 2 years of life with hyperpigmentation, recurrent hypoglycemia, chronic asthenia and failure to thrive. Typically, they have deficient production of cortisol and adrenal androgens, in the presence of markedly elevated ACTH levels, while renin and aldosterone levels are usually normal and responsive to activation of the renin-angiotensin axis. In some cases, the isolated glucocorticoid deficiency is accompanied by alacrima (lack of tears) and achalasia of the esophagus, a triad called the triple A syndrome (4,5). Clinical awareness of these syndromes is of considerable prognostic and therapeutic importance.

Isolated Glucocorticoid Deficiency (IGD)

Familial isolated glucocorticoid deficiency is a rare autosomal recessive disorder that manifests as primary adrenal insufficiency, usually without mineralocorticoid deficiency (2-9), that can be lethal, if unsuspected. Indeed, amongst over fifty published cases, eighteen have died as a result of the disease.

From the first description of the syndrome in 1959 (6), it became apparent that affected individuals suffer from a form of hereditary unresponsiveness to ACTH. Affected children commonly present within the first 2 to 3 years of life with hyperpigmentation, recurrent hypoglycemia that can lead to convulsions or coma, chronic asthenia and failure to thrive. Typically, they have deficient production of cortisol and adrenal androgens, in the presence of markedly elevated ACTH levels. Furthermore, circulating cortisol levels, which are undetectable in the majority of cases, do not increase to short or prolonged stimulation with pharmacological doses of ACTH. Aldosterone and adrenal androgen responses to ACTH are also lost in these patients (9), suggesting that the underlying unresponsiveness to ACTH is generalized. Renin and aldosterone levels are usually normal, however, and respond appropriately to activation of the renin-angiotensin axis, whether that is orthostasis, salt restriction, or furosemide-induced diuresis. Treatment of hereditary isolated glucocorticoid deficiency consists of glucocorticoid replacement therapy. Affected subjects achieve normal growth and development with steroid replacement and live an otherwise normal life.

Histological postmortem studies of the adrenal glands in affected patients has revealed that the ACTH-dependent zonae fasciculata and reticularis , which normally represent 90% of the cortical thickness, are extremely atrophic, reduced to a narrow band of fibrous tissue (6,7). In contrast, the angiotensin II-dependent zona glomerulosa is relatively well preserved, suggesting that the defect is limited to the ACTH-dependent zonae of the adrenal cortex. This isolated nature of the defect pointed towards a receptor defect, rather than an intracellular transduction abnormality or an abnormality in adrenocortical development, as the locus for the disease.

The ACTH receptor (melanocortin receptor 2, MC2) together with the other melanocortin receptors form a distinct subfamily of the G-protein coupled receptors (10). Each of the melanocortin receptors, including the ACTH receptor, couples to Gs and adenylyl cyclase but displays a unique pharmacological profile for activation by the different melanocortin peptides (11). The ACTH receptor exhibits an absolute specificity for ACTH. It consists of 297 aminoacids and is encoded by an intronless gene mapped in the distal end of chromosome 18 (18p11.2) (12). Northern blot analysis has revealed ACTH receptor mRNA only in the adrenal cortex (10). In situ hybridisation has actually demonstrated that the receptor is expressed in all three adrenocortical zonae (10). The ACTH receptor gene promoter also contains 3 steroidogenic factor-1 (SF-1) binding sites, binding to which appears to be permissive for the cAMP response (13).

Upon binding with ACTH, the receptors become active, and in turn activate the heterotrimeric G-protein complex, which subsequently activates adenylase cyclase (14). This enzyme catalyzes cyclic AMP generation, which results in stimulation of protein kinase A and the release of the activated catalytic subunit. This, in turn, stimulates cholesterol ester hydrolase, the enzyme responsible for the conversion of cholesterol esters to cholesterol. Cholesterol is then transported inside the mitochondria for side-chain cleavage and the subsequent steroidogenesis steps. In addition to the direct effect on steroidogenesis, ACTH also has an important trophic effect on the adrenal cortices. Thus, ACTH excess produces adrenal hyperplasia, and, conversely, ACTH deficiency causes atrophy.

The etiological involvement of the ACTH receptor gene in familial glucocorticoid deficiency has been proposed since the first description of the syndrome in the late 50's (6), but it was only after the cloning of the ACTH receptor gene in 1992 (10) that this hypothesis could be tested. More than 15 point mutations and frameshift mutations have been reported as homozygote or compound heterozygote mutations in different pedigrees with isolated glucocorticoid deficiency (15-22). These mutations are scattered throughout the ACTH receptor molecule and affect all aspects of receptor function as outlined in Table 1.

Hetrozygotes carrying the R201X and S120R mutants showed the presence of normal cortisol responses with exaggerated ACTH responses, suggestive of a subclinical mild resistance to ACTH action (13). A similar exaggerated ACTH response was observed in a heterozygote for the L192fs mutation but not in heterozygotes for some of the other mutations tested (I44M, R128C, S74I) (18).

Mutations within the coding region of the ACTH receptor gene have not been found in all clinically defined cases of hereditary isolated glucocorticoid deficiency. Naville et al. (24) and Weber et al. (25) have described 4 and 13 such families, respectively, which were clinically indistinguishable from those in whom mutations were identified. Furthermore, using pairs of polymorphic dinucleotide repeats that are localized in the same region of chromosome 18, to which the human ACTH receptor gene has been mapped (18p11.2), they demonstrated no apparent linkage between the disease and the ACTH receptor gene in the majority of these families. These findings suggest that the etiology of isolated glucocorticoid deficiency might be heterogenous and that gene(s) other than that of the ACTH receptor might produce the same phenotype. Reverse genetics and linkage analysis might provide the means to localize the putative gene(s).

Triple A Syndrome (MIM 231550)

A subset of the patients with hereditary unresponsiveness to ACTH, in addition to hypocortisolism, develops alacrima (lack of tears) and achalasia of the esophagus (leading to difficulty in swallowing) (26-28). This constellation of symptoms is referred to as triple A syndrome, first described in 1978 by Allgrove et al. (26). Low tear production, usually present from early infancy, may be confirmed with the Shirmer's test, while esophageal dysmotility can be demonstrated by barium swallow and/or endoscopic examination. In some cases the diagnosis of achalasia may preceed the diagnosis of cortisol deficiency. Occasional patients may also develop variable degree of mineralocorticoid deficiency. More recently, it has become apparent that progressive and variable neurologic impairment that involves both central and peripheral neurons is also frequently associated with the triple A syndrome (29). Neurological defects may include autonomic and peripheral neuropathy, ataxia and mental retardation and may thus result in a severely disabling disease.

It had been originally proposed that the ACTH receptor may also be defective in the triple A syndrome, but no mutations were found in the entire coding region of this gene in several families with the triple A syndrome (16, 20). The first big step towards resolving genetic etiology of the triple A sydrome was the chromosomal localization by linkage analysis of the gene responsible for the triple A syndrome to an 6cM area in chromosome 12 (31). Most recently, homozygote or compound heterozygote mutations were found in the AAAS gene on 12q13 in families with the triple A syndrome (32,33). AAAS codes for the WD-repeat containing protein ALADIN (alacrima-achalasia-adrenal insufficiency-neurologic disorder (32). Screening in the available cohorts of patients with triple A syndrome worldwide, revealed that the IVS14+1G A splice donor mutation is the most common AAAS mutation. In the Puerto Rican and Middle Eastern/southern European populations the frequent presence of this mutation is the result of a founder effect. New mutations have been found, however, in ethnically diverse or mixed populations (34). It is of note that no AAAS mutations have been found in several families with isolated glucocrticoid deficiency with normal ACTH receptor gene tested, suggesting that isolated glucocorticoid deficiency without MC2R gene mutations is not a forme fruste of the triple A syndrome (34). More information on the effect of the individual genetic defects will have to wait until the function of the AAAS gene is clarified. Currently, ALADIN is postulated to involved either in cytoplasmic trafficking, like other proteins with WD repeats, or in peroxisomal activities (32).

X-LINKED CONGENITAL ADRENAL HYPOPLASIA

Congenital X-linked adrenal hypoplasia is a rare disorder characterized by primary adrenal insufficiency and hypogonadotrophic hypogonadism (35). This disease was originally included in contiguous deletion syndrome of the X chromosome. The gene responsible for this disorder, DAX-1 (for dosage-sensitive sex reversal critical region at the adrenal hypoplasia locus on the X chromosome) was recently identified to be located on the short arm of the X chromosome (Xp21) (36). DAX-1 gene encodes a 470-aminoacid member of the nuclear receptor superfamily with an unknown ligand (orphan receptor). This orphan nuclear hormone receptor has a novel DNA-binding domain that has a unique structure consisting of a 66-67 aminoacid repeat motif that does not resemble the classic zinc finger DNA binding domain of these receptors (36). The DAX-1 gene is expressed not only in the adrenal gland but also in most of the reproductive tissues such as hypothalamus, pituitary gonadotropic cells and gonads, playing important roles in the development and functions of both the adrenal glands and reproductive tissues (37).

More than 60 different mutations in DAX-1 have been reported in more than 70 individuals with X-linked congenital adrenal hypoplasia (38). Missence mutations in DAX-1 gene have been identified in the C-terminal ligand-binding domain, whereas frameshift or nonsense mutations have been described in the N-terminal domain, the majority being frameshift or nonsense resulting in premature truncation of the DAX-1 protein (39).

Patients with DAX-1 mutations develop primary, complete adrenal insufficiency, such as salt-wasting and hypoglycemic convulsions in infancy or childhood, as a result of a failure of formation of the adrenal cortex (40). Plasma cortisol and aldosterone are low and do not respond to exogenous ACTH administration. At the present time, because of early diagnosis and treatment of adrenal failure, an increasing number of patients with AHC survive. Hypogonadotropic hypogonadism, however, arises later in life and is recognized as a universal feature of this syndrome in patients who are treated with adrenal steroids and survive beyond childhood (41). It presents as pubertal delay possibly due to defective production of hypothalamic GnRH and impaired responsiveness of pituitary gonadotropes to GnRH. Indeed, pulsatile GnRH has proved ineffective in inducing puberty in these patients (42). Nevertheless, spontaneous onset of puberty, but with incomplete pubertal development, has been reported. More than 10% of the patients have bilaterally undescended testes at birth (42).

Administration of HCG stimulates testosterone concentrations into the normal range in most patients. However, exogenous gonadotropins are ineffective in inducing spermatogenesis, probably reflecting a direct effect of DAX-1 on Sertoli cell function (43). Interestingly, an I496S missense mutation in DAX-1 was recently found in a man who presented with incomplete hypogonadotropic hypogonadism in his twenties and mild adrenal failure. Functional studies showed that this mutation caused a partial loss of DAX-1 function in a variety of gene expression essays. Thus, partial loss-of-function mutations in DAX-1 can present with hypogonadotropic hypogonadism and covert adrenal failure in adulthood (44).

Female heterozygotes for DAX-1 mutations may present with delayed puberty but normal fertility. However, one homozygous female case was recently reported, showing hypogonadotropic hypogonadism, but apparently had normal ovarian development and normal adrenal function (45). In addition, remarkable discrepancy exists in genotype-phenotype relations. Thus, the same mutation resulted in two brothers with the complete syndrome, an unaffected grandfather, and an aunt with only hypogonadotropic hypogonadism (45).

PRIMARY ADRENAL INSUFFICIENCY CAUSED BY SF-1 MUTATIONS

SF-1 (Steroidogenic Factor-1), also called adrenal 4-binding protein (Ad4BP) is an orphan nuclear receptor, formally isolated as a pivotal factor of tissue-specific expression of cytochrome p450 steroid hydroxylases, which are essential for the synthesis of steroid hormones (46, 47). SF-1 is expressed in adrenal glands and gonads, being essential for the development and function of these organs and sexual differentiation; it is also expressed in the pituitary gland and hypothalamus, contributing to the differentiation of pituitary primordial cells into gonadotropes (48). In the adrenal glands, SF-1 regulates not only levels of cytochrome p450 steroid hydroxylases, but also modulates the expression of 3b-hydroxysteroid dehydrogenase, StAR and the ACTH receptor. The SF-1 gene is located on chromosome 9q33.

Two cases of SF-1 gene mutations have been described (49,50). The first mutation was reported in a 46, XY phenotypic female, who presented with primary adrenal failure during the first weeks of life with low circulating cortisol and aldosterone and high ACTH. Although the karyotype of the patient was 46XY, she retained normal Mullerian structures and streak-like gonads, containing poorly differentiated seminiferous tubules and connective tissue (49). The patient had a heterozygote two codon replacement in exon 3 of one of the SF-1 gene, causing substitution of glycine at amino acid 35 by glutamate in the DNA-binding domain of the protein, abolishing its DNA-binding activity. Pituitary gonadotropins responded to GnRH, but testosterone did not respond to exogenous HCG administration, suggesting defective gonadal function. After introduction of estrogen and progesterone, the uterus grew and regular menstruation occurred. From this case, it seems that SF-1 is essential for sex determination, steroidogenesis and reproduction.

The second heterozygote SF-1 mutation was recently described in a 27-month-old 46,XX female with normal external genitalia and adrenal failure (50). Although her pubertal development remains to be seen in the future, impaired ovarian development and/or steroidogenesis would be expected.