The past fifteen years has seen significant progress in our understanding of the molecular basis of childhood adrenal disorders. More than twenty single gene disorders have now been reported that can affect adrenal function in infancy or childhood, and a genetic diagnosis should be attainable in well over 50% of individuals with these conditions (2
). Obtaining a precise biochemical and genetic diagnosis can have important consequences for treatment, predicting prognosis, investigating possible associated features, and for counseling the individual and family so that the risk of other family members being affected can be assessed accurately.
The identification and characterization of DAX1 as the cause of X-linked primary adrenal hypoplasia congenita in 1994 has had significant implications for diagnosis of individuals and families with this condition. An association with disordered puberty means that the majority of boys with X-linked AHC will need pubertal induction and long-term sex steroid replacement. It also seems likely that an intrinsic defect in spermatogenesis, which may be present in humans as well as mice, results in a worse fertility prognosis for individuals with X-linked AHC compared to young men with isolated idiopathic HH (8
The true population prevalence of DAX1 mutations is not currently known. The prevalence of congenital adrenal hypoplasia is often quoted as being 1:12,500, following the 13-year study of infant autopsies at the Royal Women's Hospital, Melbourne, Australia (1959-1971) by Laverty et al (41
). However, there was no sex-bias in cases (6/11 male) and only one male infant had cytomegalic histological changes consistent with the X-linked form of AHC. Furthermore, in a 20-year review of primary adrenal insufficiency in children (0-18 years) presenting to Sainte-Justine Hospital, Montreal (1981-2001) and reported by Perry et al, X-linked AHC due to a DAX1 mutation was found in only one boy (1/103) (42
). Congenital adrenal hyperplasia was diagnosed in 74/103 children (71.8% of the population) and has an estimated occurrence of 1:16,630 (42
). Taken together, these studies suggest that X-linked AHC might occur in anywhere between 1:140,000 and 1:1,200,000 children (or between 1:70,000 and 1:600,000 males). However, extreme caution is needed in interpreting these data, as the number of boys with X-linked AHC in each cohort was extremely small (n=1).
Here, we have focussed on children and adults with primary adrenal insufficiency of unknown etiology, where common causes of adrenal failure such as steroidogenic defects (e.g. 21-hydroxylase deficiency) and metabolic disorders (e.g., X-linked adrenoleukodystrophy) had been excluded. We show that DAX1 mutations are a relatively frequent cause of adrenal failure in phenotypic boys (46
,XY) referred to us with potential primary adrenal hypoplasia. DAX1 mutations were found in over half of individuals studied (37/64, 58%), and in all eight (100%) cases where there was a family history of adrenal failure or unexpected death in males (consistent with an X-linked inheritance pattern) together with a history of arrested or absent puberty. Thus, detailed questioning about family history that could reveal any insight into possible adrenal disease is important. Furthermore, when no such family history was obtained, and when the individual was prepubertal at the time of assessment, it was still possible to detect DAX1 mutations in a substantial proportion of cases (20/44, 45%). If boys with additional features (e.g., low birth weight, skeletal abnormalities) and those with transient forms of adrenal failure were omitted from analysis, the proportion of boys with DAX1 changes found rose to 68% (19/28). Thus, mutational analysis of DAX1 may be worthwhile in any male infant presenting with salt-losing adrenal failure, where steroidogenic disorders (e.g. 21-hydroxylase deficiency, P450 oxidoreductase deficiency), metabolic conditions (e.g Wolman syndrome, Zellweger syndrome), multisystem syndromes (e.g., IMAGe) and adrenal hemorrhage have been excluded, and in the older child where – in addition – autoimmune endocrine disorders (e.g. APECED, autoimmune polyglandular syndrome 2, isolated autoimmune Addison's disease), syndromic ACTH resistance syndromes (e.g., Triple A), infection or iatrogenic causes of adrenal failure are not present.
The 37 DAX1 mutations and deletions detected had a similar distribution to those reported in the literature to date, although we did identify relatively few contiguous gene deletion syndromes compared to isolated deletions of NR0B1
, and a significant proportion of missense mutations () (3
). The contiguous gene deletion syndromes were diagnosed before the onset of signs and symptoms of muscular dystrophy in both the cases shown. Although none of our cohort has evidence of deletion of the IL1RAPL1
gene telomeric to NR0B1
, which is associated with developmental delay, it is important to be aware of this potential association when a child with a Xp21 deletion seems to be failing to reach developmental milestones (44
). The nonsense and frameshift mutations in our cohort were located throughout the NR0B1
gene, with relatively few “hotspots” that could help to focus sequencing strategies. Missense mutations in DAX1 do tend to cluster in certain regions of the ligand-like binding domain, in highly conserved amino acids (35
No SF1 mutations were found in the cohort of boys (n=27) who did not have abnormalities in DAX1. These findings suggest that mutations in SF1 are unlikely to be a frequent cause of an adrenal-only phenotype, with normal male sex development. Other candidate genes for this group include potential regulators of adrenal development, that are emerging from studies of gene expression or transgenic mice (e.g. ACD
, CITED2, PBX1
). Several of our patients had phenotypic features consistent with a variant of the IMAGe syndrome, but the molecular basis of this condition is at present unknown (37
). None of these children were found to harbor changes in DAX1 or SF1.
Analysis of our cohort of 46,XY individuals with adrenal failure and gonadal dysgenesis/impaired androgenization failed to reveal any DAX1 mutations, although there is evidence from studies of transgenic mice that Dax1 may function to support testis development at early stages of embryogenesis (9
). SF1 mutations were found in only two patients, both of whom had 46,XY complete gonadal dysgenesis and persistent Müllerian structures (27
). These findings are consistent with the hypothesis that gene dosage effects of SF1 are important, and that gonadal (testicular) development is more sensitive to loss of SF1 function than adrenal development in humans (48
). Thus, if severe adrenal failure is present due to an SF1 mutation, it is likely that there will be significant underandrogenization, whereas less severe disruption of SF1 can result in partial gonadal dysgenesis/impaired androgenization and normal adrenal function (31
). It is possible that mutations in the SF1 promoter or non-coding sequences, or in related target genes or co-factors, might be identified in those individuals where no SF1 mutations were found. Furthermore, abnormalities in the early stages of steroidogenesis (e.g. steroidogenic acute regulatory protein, CYP11A) might present as complete adrenal failure and impaired androgenization, but without Müllerian structures. The adrenal glands in children with these conditions may not always be enlarged.
Our reports of a late-onset form of X-linked AHC presenting with primary adrenal failure in young adulthood in three men (19
), as well as potential female phenotypic expression of DAX1-related phenotypes (8
) led to studies of DAX1 and SF1 in a cohort of 29 men and women who had Addison disease of unknown etiology. Thus, we hypothesized that milder forms of adrenal hypoplasia congenita might account for a subset of patients with these phenotypes. However, no mutations were found. Although the number of patients was small, these findings suggest that mild forms of adrenal hypoplasia are unlikely to be found in patients with adult-onset “Addison disease of unknown etiology” in the absence of at least partial hypogonadotropic hypogonadism in males (DAX1) or impaired androgenization (SF1).
Taken together, this study shows that mutations in DAX1 are a relatively frequent cause of primary adrenal hypoplasia, even in the absence of a positive family history of adrenal failure or unexpected death in males, or a personal history of abnormal puberty. In contrast, although SF1 mutations are emerging as a cause of 46,XY gonadal dysgenesis in patients with normal adrenal function, SF1 mutations causing adrenal failure are rare and are likely to be associated with significant underandrogenization and gonadal dysfunction in 46,XY individuals. Genetic analysis of DAX1 is now offered by a number of clinical laboratories worldwide. The prevalence of DAX1 changes identified in our series might warrant having a low threshold to undertake this analysis. Although pursuing a commercial approach can be relatively expensive, it may well be worthwhile if such an approach was able to prevent recurrent hypoglycemia, a severe salt-losing crisis or even death in a presymptomatic brother, male relative, or following a future pregnancy (34
). However, it is also important that appropriate counseling is available for the individual and family throughout their interactions with health care services over the years, that appropriate and timely translation of care from pediatric to adult services is established, and that the clinical and research communities work together to determine the molecular basis of disorders of adrenal development when no changes in DAX1, SF1 or other candidate genes are found.