The familial occurrence of adrenal cortical insufficiency due to adrenal hypoplasia, with a pattern of inheritance consistent with X-linkage, is well recognized.7,26–39 Most patients present within the first days, weeks, or months of life, but occasionally affected individuals with family histories compatible with X-linked AHC may develop acute adrenal insufficiency as late as 10 years,27,34 and, in one family of two brothers and their maternal uncles, between 17 and 32 years of age.26 The two brothers who we originally described with AHC, GKD, and a dystrophic myopathy,40,41 who we now know to be completely deleted for DAX1,42,43 did not present with acute adrenal insufficiency until 33 months and 6 years of age. The younger boy died at 33 months, 12 hours into an episode of gastroenteritis following 2 days after surgery for esotropia, and his older brother presented 11 months later, at 6 years of age, 29 hours into an episode of gastroenteritis, with dehydration, acidemia, hyponatremia, and hyperkalemia. In a series of 18 patients from 16 families, among the 17 who presented clinically, the range in age for first acute symptoms was 1 week to 3 years, with a median age of 3 weeks.44 In this series, the younger of two brothers with a deletion of DAX1 and GK presented with salt wasting at 1 month, and the older boy was diagnosed at 3 years of age with a similar episode. Peter et al. reviewed other reports of delayed-onset and intrafamilial variability,44 but the explanation for why some patients have a significantly longer period of residual function from their hypoplastic adrenal cortices remains unknown.
The hypoplastic adrenals with X-linked AHC evidence cytomegalic pathologic changes with absence or near-absence of the permanent zone, and cortical disorganization with nodular formations of eosinophilic cells in the residual cortex.7–13,29–31,45,46 Occasionally, in families with pedigrees consistent with X-linked inheritance, no adrenal tissue could be identified, despite thorough autopsy, including a careful search for adrenal vascular supply.28,33,36,47 This suggests adrenal aplasia.
HH is known to be associated with the X-linked form of AHC.11,32,34,35,37,38,44,48–66 While some investigators suggested that there was a distinct locus telomeric to the AHC gene,67,68 with the cloning of the gene responsible for X-linked AHC, DAX1 (see below), intragenic mutations in this gene were found to be responsible for HH.59,60 The data were contradictory regarding whether the HH in patients with DAX1 mutations was hypothalamic or pituitary in origin,56,57 until the controversy was resolved by showing that the HH in these patients had mixed hypothalamic and pituitary origins.62,63 This explanation of the HH in these patients fit well with the observation that DAX1 is expressed in the hypothalamus and the pituitary.69 The “mini-puberty” of infancy appears to be normal in affected boys, suggesting loss of functional integrity over time or differences in the mechanisms responsible for hypothalamic-pituitary-gonadal axis regulation in infancy and adolescence.44,70,71 Despite the usual association of X-linked AHC and HH, four male cousins, whose maternal grandmothers were sisters, had androgenic precocity of varying degrees, one with nonprogressive virilization at birth, and the other three with advanced growth and/or skeletal maturation and elevated testosterone levels.36
The majority of patients with AHC are not recognized clinically until they develop an adrenal crisis. Some may be misdiagnosed with congenital adrenal hyperplasia (CAH)44 (E.R.B. McCabe, unpublished finding). In a series of 18 patients from one center, 16 presented with salt-wasting, one had an initial hypoglycemic seizure, and one was treated presymptomatically, based on the results of prenatal diagnostic testing.44 Acute deterioration may be preceded by nonspecific features, such as feeding difficulties, vomiting, poor weight gain, and dehydration. Hyperpigmentation resulting from excessive proopiomelanocortin production is usually present and may be quite profound, with the increased keratinocyte melanin giving a coal-black coloration to the skin, except on the palms and soles.72 Wheezing, which may be attributed initially to asthma, may represent a mild form of Addisonian crisis in patients with AHC.73
Neurologic abnormalities and developmental delay may be observed in patients with AHC. Progressive high-frequency hearing loss beginning at about 14 years of age has been observed in three brothers.58 Among those who present later, personality changes may be seen.32,53 Seizures are often seen at presentation or as a consequence of Addisonian crises, and acute adrenal insufficiency may also result in other neurologic sequelae and developmental abnormalities. Bilateral infantile striatal necrosis has been observed in AHC.74
AHC may be due to deletion of DAX1, as part of an Xp21 contiguous gene syndrome (Chap. 97), or it may be an isolated disorder, due to intragenic mutations within the DAX1 gene. Patients with large deletions will have complex phenotypes depending on the other loci involved, most often the GK and Duchenne muscular dystrophy ( DMD ) loci along with the DAX1 gene. There appears to be one or more mental retardation (MR) loci telomeric to DAX1 that may be deleted in some patients (reviewed in Chap. 97). The observation of developmental delay in patients with isolated AHC from acute episodes of adrenal insufficiency may make it difficult to determine the etiology of the MR in individual patients. However, the observations of MR in female carriers for deletions in this region75 and of a patient with MR and an interstitial deletion telomeric to and not involving DAX1,76 support the presence of one or more MR loci independent of AHC. In addition, a patient with a contiguous gene syndrome involving DAX1, GK, and DMD, who also has agenesis of the corpus callosum, suggests the localization of a gene for this midline defect to Xp22.11 to p21.1.77
Acute presentation with suspected adrenal crisis frequently will require parenteral glucose, saline, and glucocorticoid. The mineralocorticoid activity of a glucocorticoid, such as cortisol sodium succinate (Solu-Cortef), often will be adequate for acute management. Clinical status, blood glucose, electrolytes, and hydration must be monitored closely. If the electrolytes do not respond, the only mineralocorticoid available at this time is fludrocortisone (Florinef), which is an oral preparation. Therefore, if the patient will not tolerate oral medication, the dose of parenteral glucocorticoid should be increased to take advantage of its mineralocorticoid activity. For example, 20 mg of Solu-Cortef has the same effect on sodium retention as approximately 0.1 mg of Florinef or 1 mg of deoxycorticosterone (DOC) acetate (see Chap. 159). The patient's electrolyte and water balance must be closely monitored to prevent hypernatremia, overhydration, and pulmonary edema.
Maintenance therapy involves physiological replacement doses of a glucocorticoid, such as hydrocortisone, and titration of the mineralocorticoid fludrocortisone (Florinef). Increased sodium chloride intake may also be required. During times of stress, such as significant intercurrent illnesses, the dose of glucocorticoid should be increased three to four times. The dosage should be 5 to 10 times maintenance for surgeries and significant traumas, and these should be managed in centers where a pediatric endocrinologist is available. Education of the parents, patients, and primary care physicians is extremely important, because deaths in diagnosed patients occur when steroid doses are subtherapeutic, particularly during times of stress. Parenteral Solu-Cortef may be provided for home use with appropriate education. Dosages must be increased appropriately to accommodate the growth of the child, without overdosing. A medical information bracelet or necklace should be used by the patient.
HH should be anticipated in patients with AHC, and treated with testosterone for initiation of secondary sexual characteristics.
Future directions in therapy for AHC, as well as other disorders of the adrenal cortex, may include gene therapy and stem-cell therapy. A promoter that would provide tissue specific expression, alone or in combination with other promoter elements to enhance expression efficiency in the adrenal cortex could be used to drive DAX1 expression in an in vivo gene therapy vector. If the DAX1 promoter is shown to be sufficiently specific and efficient, then it may be a candidate for a gene therapy vector designed to target adrenal cortical expression of DAX1 or other adrenal disease genes.78 If DAX1 dimerization is required for normal function, then the possibility of a dominant negative effect by endogenously expressed mutant protein must be considered.79 Alternative targeting strategies, such as introducing proteins or smaller molecule ligands that would interact with a specific receptor or docking molecule on the surface of the targeting vector, would also be possible.80–85 Identification of one or more adrenal cortical stem cells24,25 would open the possibility of stem-cell therapy for AHC and other adrenal disorders.
My initial screen for a patient suspected of having AHC includes plasma ACTH, which is consistently elevated in AHC, whereas cortisol may be within the normal range; serum triglyceride level and/or urinary organic acids prepared by the solvent extraction method, for evaluation of GKD by pseudohypertriglyceridemia and glyceroluria, respectively; and a serum creatine phosphokinase (CPK) level to rule out DMD or Becker muscular dystrophy (BMD). With this assessment, one will determine whether additional work-up for AHC is indicated and whether the AHC is part of the most common form of the Xp21 contiguous gene syndrome, complex GKD (cGKD), involving the AHC, GKD, and DMD loci (see Chap. 97).
If the ACTH level is elevated and there is evidence of GKD and/or DMD/BMD, then it is most likely that this patient has AHC. For isolated AHC, other work-up may be indicated. In contrast to CAH due to 21-hydroxylase deficiency, in which urinary 17-ketosteroids and pregnanetriol, and plasma cortisol precursors and adrenal androgens typically are elevated (see Chap. 159), these compounds usually will be low or normal in patients with AHC. The patient with AHC will not have a normal cortisol response to an ACTH stimulation test.
Molecular genetic diagnosis of DAX1 is also possible. The DAX1 gene consists of only two exons and is easily sequenced (see section on Genetics, below). It should be noted, however, that not all individuals with a clinical diagnosis of AHC will have mutations in DAX1.44,60 In our own experience, the number of samples referred with an AHC diagnosis but no DAX1 mutation detectable by sequencing has been significant.
Imaging studies may be helpful to attempt to evaluate adrenal size. The normal position of the adrenal gland within the perirenal fat, and the potential for aberrant locations for adrenals, may make it difficult to interpret a study that does not detect normal adrenals. The imaging results must be interpreted in the context of the other clinical and molecular investigations.
If the patient comes to autopsy, the weights of the adrenal glands will be low compared with the normal range for age.86–89 The histologic appearance of the adrenals from an individual with the X-linked cytomegalic form is characteristic, with disorganization of the cortex and large eosinophilic cells (see “Introduction and Historical Perspective” above).
Prenatal diagnostic testing may use a variety of approaches. Maternal plasma estriol levels are low in pregnancies of a fetus with AHC.8,44,47,90–93 If there is a family history of a DAX1 deletion, then fluorescence in situ hybridization (FISH) may be used to determine whether the fetus is at risk for AHC.43 Mutation analysis is well established for identification of affected individuals (see “Genetics” below); however, to our knowledge, this approach has not been used for prenatal diagnosis at this time.
AHC was originally mapped to Xp21 by clinical characterization of patients who had deletions that also involved GKD and DMD/BMD (reviewed in Chap. 97). Not all of the deletions result in loss of the DAX1 coding region. Patient TM, who has AHC, GKD, and DMD,42 is not deleted for DAX1,59 and has a telomeric breakpoint that is estimated to be approximately 100 kb centromeric of the DAX1 coding sequence (Guo and McCabe, unpublished observations). The observations in this patient indicate that deletions in this region may lead to position effects on DAX1 expression.
A series of 18 patients with AHC in 16 families ascertained clinically by a German group gives insight into the approximate proportion of individuals with DAX1 deletions, intragenic mutations, or no detectable gene alteration.44 Seven families had large deletions, four involving AHC, GKD, and DMD (only two of which were analyzed), one with AHC and GKD, and two with deletion of DAX1. Seven families exhibited intragenic mutations, five with frameshift and two with nonsense mutations. One family with isolated AHC had no DAX1 mutation detected, and another did not have mutation analysis performed.
In a review of intragenic DAX1 mutations, we tabulated 42 mutations from 48 families, to determine the types of mutations responsible for AHC and to identify the positions of single amino acid changes in a DAX1 structural model.94 Fourteen new mutations were identified in that report, with the other 28 described previously.43,59,60,62,64,65,70,95,96 One nonsense mutation (W235X) was found in three families, and one frameshift mutation (1292delG), two nonsense mutations (W171X and Y91X), and one in-frame deletion (V269del) were observed in two families each. To our knowledge, the families with identical mutations were unrelated, and we confirmed this by showing substantial differences between the mitochondrial D-loop sequences97 in the two families with the frameshift mutation. The 23 frameshift and 12 nonsense mutations were distributed throughout the DAX1 protein, but all seven of the mutations resulting in single amino acid changes (six missense mutations and one single codon in-frame deletion) mapped to the C-terminal half of DAX1 in the region of similarity with the LBDs of other members of the nuclear hormone receptor superfamily78 (Fig. 167-1). When the seven single amino acid changes were mapped to a three-dimensional model of the DAX1, putative LBD developed by homology with the known structures of rat α1 thyroid hormone receptor98 and human retinoid-X receptor RXRα,99 they all clustered in the hydrophobic core of the LBD (Fig. 167-2). These seven mutations all involved amino acids that were identical in human DAX1 and the homologous murine protein, adrenal hypoplasia congenita, mouse homologue of DAX1 (Ahch), and were predicted to provide a major disruption of receptor folding, dimerization, and overall DAX1 function.94 Subsequent to this tabulation of DAX1 mutations, additional reports that include intragenic mutations have been published,44,100–103 and two new missense mutations have been reported, W291C and K382N.103 Both of these missense mutations map to the same hydrophobic core of the putative LBD as those in our report proposing the model.94
Diagram tabulating 42 intragenic mutations within the DAX1 protein as more specifically identified elsewhere.94 Although the frameshift and nonsense mutations were distributed throughout the protein molecule, the mutations resulting in single amino acid changes were localized to the C-terminal half of the protein in the putative ligand-binding domain (LBD). (From Zhang et al.94 Used by permission.)
Model of the DAX1 putative LBD hydrophobic core showing the residues altered by mutations involving single amino acids that cause AHC.94 The model was generated by homology with the solved structures of rTRα1 98 and hRXRα.99 (From Zhang et al.94 Used by permission.)
Gonadal mosaicism for a DAX1 mutation has been described (Fig. 167-3), which has important implications for genetic counseling.94 The matriarch of a three-generation family had a carrier daughter and an affected son with a 23 bp frameshift deletion in DAX1, but she did not carry this mutation in her blood. The results were confirmed on independent blood samples redrawn from each of the family members, and two additional buccal brushings from the matriarch did not identify a mutation in her by PCR. The matrilineal relationships within the family were affirmed by mitochondrial D-loop sequencing.97 Based on the observation of gonadal mosaicism in this family, if a boy with AHC and a DAX1 mutation is the son of a noncarrier female, she must be counseled that there is the possibility that she may have other offspring with this mutation, either affected males or carrier females, despite the absence of a detectable mutation in the genomic DNA from her somatic cells.
Gonadal mosaicism in a family with a 23 bp frameshift deletion.94 DNA sequencing data were confirmed by allele-specific oligonucleotide (ASO) hybridization of PCR amplification products. The mother (I-1) of a carrier daughter (II-1) and an affected son (II-2) did not carry the deletion. B = blank control and N = normal individual. (From Zhang et al.94 Used by permission.)
While AHC associated with DAX1 mutations is clearly an X-linked disease, early experience showed that girls may occasionally be affected.104 In a family with AHC and GKD, a 7.5-month-old girl was hypoglycemic and unarousable after a nap. She had a large amount of glycerol in her urine and hypoplastic adrenals at autopsy. Based on this, as well as experience with Lyonization in other X-linked disorders, one should counsel a family that a pregnancy with a female carrier fetus has a low but not impossible risk for developing AHC, and one may wish to consider a plasma ACTH level test for such girls in the neonatal period.
DAX1 is an unusual member of the nuclear hormone receptor superfamily with closest resemblance to other superfamily members in the C-terminal portion coding for the putative LBD.43,59,78,105 The deduced amino acid sequences of the porcine homologue, pDAX1, and the murine homologue, Ahch, show 79 percent and 66 percent identity, and 80 percent and 69 percent similarity, respectively, with the human sequence66,106–109 (Fig. 167-4). The N-terminal half of DAX1, which differs strikingly from most other nuclear hormone receptors,78 does not include a canonical zinc finger structure, but does contain cysteine residues that are located appropriately for interaction with zinc ions (Fig. 167-5). All ten of these cysteine residues are conserved in the human, porcine, and murine deduced sequences.66
Comparison of the human DAX1 deduced amino acid sequence with its porcine and murine homologues and with the closely related nuclear hormone receptor, SHP. (Adapted from Vilain et al.66 Drawn by M. Patel.)
Putative zinc finger domain in the N-terminal half of DAX1. The interaction of the zinc ions was drawn arbitrarily with residues 41 and 107, but the interaction could be with cysteines 41 or 43 and 107 or 109. (From Guo et al.43 Used by permission.)
The closest relative to DAX1 is the small heterodimer partner (SHP),110 which also lacks a conventional DBD, but has one of the 3.5 N-terminal repeats found in DAX1. SHP interacts with several other nuclear hormone receptors to serve as a negative regulator in receptor-dependent signaling pathways.
DAX1 is expressed in the human hypothalamus, pituitary, fetal and adult adrenals, ovaries and testes,59,69 and in these same tissues in the developing mouse.108 In situ hybridization in fetal mouse shows that the mouse DAX1 homologue, Ahch , is expressed in the adrenals on day 12.5 days post coitum (dpc), one day following the development of the adrenal primordium from the urogenital ridge, and expression persists.108 In the gonads of male and female mouse fetuses, Ahch is expressed initially at 11.5 dpc, which corresponds to the first expression of Sry and the initial appearance of differentiation in the testes. Subsequently, ovarian expression of Ahch persists at similar levels, but testicular expression is reduced considerably.
The tissue profile of expression for Ftz-F1 is similar to Ahch , but the temporal profiles differ.111,112 Ftz-F1 is the murine homologue of steroidogenic factor 1 (SF1), a nuclear receptor that regulates expression of a number of steroidogenic genes. Ftz-F1 is expressed in the ventromedial nucleus of the hypothalamus, the anterior pituitary, the adrenal cortex, and the gonads of both sexes. However, Ftz-F1 appears 3.5 days earlier than Ahch, at 9 dpc in the urogenital ridge. Development of the ventromedial nucleus and expression of LH and follicle stimulating hormone (FSH) are disrupted in Ftz-F1 knockout mice, and these animals completely lack adrenal tissue.113,114 These observations led to the hypothesis that SF1 and DAX1 may act in a concerted fashion during the normal development of the hypothalamic-pituitary-gonadal axis.115
There are conflicting data regarding a hypothetical interaction between SF1 and DAX1. The identification of potential SF1 response elements (REs) in similar positions in the human DAX1 95,105 and murine Ahch promoters108 is consistent with the temporal pattern of expression suggesting that SF1 would act upstream of DAX1. SF1 binds specifically to this RE.105 Transfection experiments have been contradictory. Transfection with a reporter construct containing various portions of the DAX1 promoter in an adrenal cortical cell line that normally expresses DAX1 and SF1 showed the importance of the SF1 RE, and cotransfection with an SF1-expressing vector stimulated DAX1 promoter expression in the presence of the SF1 RE.116 Transfections in adrenal cortical cells that do not normally express DAX1 or in Leydig-derived cells, however, suggested that the SF1 RE was not required for DAX1 expression.111 DAX1 expression was not impaired in SF1 knockout mice,111 leading to the conclusion that DAX1 and SF1 interact in a common developmental pathway, but that SF1 is not required for the expression of DAX1. These contradictory observations may reflect tissue-dependent differences in the regulation of DAX1 expression, perhaps with the involvement of additional factors in various tissues.66
DAX1 has the structure of a transcription factor,43,59,78 and its nearest relative identified to date is a negative regulator in other nuclear receptor signaling pathways.110 DAX1 also acts as an inhibitory regulator. Coexpression of DAX1 and SF1 inhibits transactivation mediated by SF1.117 This does not appear to involve DAX1-SF1 heterodimerization. Although residues in the N-terminal portion of DAX1 interact directly with SF1, the heterodimerization does not alter SF1 binding to DNA containing the SF1 RE. A C-terminal portion of DAX1, however, contains a transcriptional-silencing domain.117,118 Naturally occurring C-terminal mutations and single amino acid changes (R267P and V269del) reduce transcriptional silencing.117,119 Silencing by the C-terminal DAX1 residues varies with promoter and cell type, suggesting involvement of additional factors, the abundance of which may vary with the cell type.119 One such factor that is involved is the nuclear receptor corepressor (N-CoR), which is recruited by DAX1 to SF1.118 DAX1 transcriptional repression is mediated by DAX1 binding to DNA hairpin structures that are found in the human steroidogenic acute regulatory protein (StAR) and murine Dax1 promoters, and DAX1 decreases StAR RNA expression and steroidogenesis in murine Y-1 adrenocortical tumor cells.120 The close proximity of the SF1 and DAX1 REs in the Dax1 and StAR promoters suggests that DAX1 alters SF1 binding through an allosteric inhibition mechanism.120