DAX1 Mutations Research Articles

Active hypothalamic-pituitary-gonadal axis in an infant with X-linked adrenal hypoplasia congenita

To evaluate the hypothalamic-pituitary-gonadal axis in an infant with adrenal hypoplasia congenita, we measured the serum levels of testosterone and performed a luteinizing hormone–releasing hormone stimulation test. The diagnosis was made because of the presence of a mutation, A300V, in the DAX-1 gene. The results demonstrated an active hypothalamic-pituitary-gonadal axis, with adult-level testosterone of 266 ng/dl on day 0, and maintenance of testosterone concentration in the 100 to 250 ng/dl range for 140 days as expected. The luteinizing hormone–releasing hormone test was compatible with an active pituitary gland with a luteinizing hormone peak of 13.1 IU/L and a follicle-stimulating hormone of 5.0 IU/L. We conclude that the DAX-1 mutation does allow a normal reproductive axis at birth. We speculate that sometime between infancy and puberty this mutation in the DAX-1 gene leads to an inability to activate the reproductive axis from its childhood suppression; thus puberty will not develop in this infant. (J Pediatr 1997;130:488-91)

Inherited disorders of the gonadotropin hormones

Inherited disorders of the pituitary gonadotropins, LH and FSH, are rare. No mutations of the common α-subunit gene have been described. A single case of an FSHβ gene mutation has been reported. This mutation consisted of a two nucleotide deletion that caused a frameshift of codons 61–86 followed by premature termination. A homozygous patient with this mutation presented with primary amenorrhea and infertility. Serum FSH levels were low and LH levels were elevated. A postmenopausal heterozygous relative had subnormal FSH and LH and it was postulated that the mutant FSHβ subunit may have impaired gonadotrope function. Only a single example of an LHβ gene mutation has been described. This case was reported in a male who failed to undergo puberty, had elevated immunoreactive LH, but low bioactive LH and low testosterone. The LHβ gene is a member of the CGβ LHβ gene cluster that resides on chromosome 19q. No rearrangements or deletions were observed and there was a homozygous substitution of Gln 54 with Arg. The substituted Gln residue is conserved in each of the glycoprotein hormone β-subunits. Recombinant mutant LH was expressed in CHO cells and was shown to be immunologically active, but it did not bind to the LH receptor, explaining the absence of bioactivity. This finding suggests that Gln 54 is either a contact site for the receptor or that the mutation alters the conformation of LH to prevent binding to the receptor. The serum LH bio/immuno (B/I) ratio in heterozygotes was 50% of control samples, consisent with normal production and stability of the mutant hormone in vivo. Male heterozygotes exhibited slightly reduced testosterone and only one of four was fertile. Female heterozygotes had regular menses and were fertile. A polymorphic variant of LH has been reproted. The variant is prevalent in Finland (24% heterozygotes) and several cases have been reported in Japan. The LH variant consists of two amino acid substitutions (W8R; I15T) that correspond to residues normally found in CGβ. The I15T substitution may introduce a glycosylation site. The variant LH has increased bioactivity, but a reduced serum half-life. It is unclear whether the LH variant is of clinical significance aside from altering immunoactivity in some assays. In addition to gonadotropin mutations, defects in gonadotrope viability (SF-1; DAX-1 mutations) and in GnRH secretion (Kallmann syndrome: SF-1; DAX-1) can also lead to hypogonadotropic hypogonadism (Fig. 1). As noted in other talks, the LH-R and FSH-R are also targets for mutations. Thus, genetic defects have now been identified at each level of the H-P-G axis.

Adrenal hypoplasia congenita with hypogonadotropic hypogonadism: evidence that DAX-1 mutations lead to combined hypothalmic and pituitary defects in gonadotropin production.

Adrenal hypoplasia congenita (AHC) is an X-linked disorder that typically presents with adrenal insufficiency during infancy. Hypogonadotropic hypogonadism (HHG) has been identified as a component of this disorder in affected individuals who survive into childhood. Recently, AHC was shown to be caused by mutations in DAX-1, a protein that is structurally similar in its carboxyterminal region to orphan nuclear receptors. We studied two kindreds with clinical features of AHC and HHG. DAX-1 mutations were identified in both families. In the JW kindred, a single base deletion at nucleotide 1219 was accompanied by an additional base substitution that resulted in a frameshift mutation at codon 329 followed by premature termination. In the MH kindred, a GGAT duplication at codon 418 caused a frameshift that also resulted in truncation of DAX-1. Baseline luteinizing hormone (LIT), follicle-stimulating hormone (FSH), and free-alpha-subunit (FAS) levels were determined during 24 h of frequent (q10 min) venous sampling. In patient MH, baseline LH levels were low, but FAS levels were within the normal range. In contrast, in patient JW, the mean LH and FSH were within the normal range during baseline sampling, but LH secretion was erratic rather than showing typical pulses. FAS was apulsatile for much of the day, but a surge was seen over a 3-4-h period. Pulsatile gonadotropin releasing hormone (GnRH) (25 ng/kg) was administered every 2 h for 7 d to assess pituitary responsiveness to exogenous GnRH. MH did not exhibit a gonadotropin response to pulsatile GnRH. JW exhibited a normal response to the first pulse of GnRH, but there was no increase in FAS. In contrast to the priming effect of GnRH in GnRH-deficient patients with Kallmann syndrome, GnRH pulses caused minimal secretory responses of LH and no FAS responses in patient JW. The initial LH response in patient JW implies a deficiency in hypothalamic GnRH. On the other hand, the failure to respond to pulsatile GnRH is consistent with a pituitary defect in gonadotropin production. These two cases exemplify the phenotypic heterogeneity of AHC/HHG, and suggest that DAX-1 mutations impair gonadotropin production by acting at both the hypothalamic and pituitary levels.

Genomic sequence of the DAX1 gene: an orphan nuclear receptor responsible for X-linked adrenal hypoplasia congenita and hypogonadotropic hypogonadism.

The gene responsible for X-linked adrenal hypoplasia congenita, DAX1, encodes a member of the nuclear hormone receptor superfamily. We sequenced 8851 bp that contained the DAX1 genomic region. The DAX gene was composed of two exons and one 3.4-kilobase intron. Putative TATA and GC boxes and a putative steroidogenic factor 1 response element were present in the 5'-flanking region. Two potentially polymorphic short tandem repeats were identified. The first exon encoded two putative novel zinc finger motifs within a putative DNA binding domain and part of the ligand binding domain, and the second exon encoded the remainder of the ligand binding domain. Although the putative DNA binding domain of DAX1 does not contain substantial sequence similarity to other nuclear hormone receptor superfamily members, the putative ligand binding domain had remarkable similarity to other family members. Single-strand conformational polymorphism analysis permitted identification of three new mutations in DAX1. In conclusion, single-strand conformational polymorphism analysis facilitates identification of mutations in the DAX1 gene, and the short tandem repeats may permit linkage analysis in families in which mutations are not yet identified. We speculate that DAX1 may be the most primitive member of the nuclear hormone receptor superfamily identified in mammals.

Diagnosis of X-linked Adrenal Hypoplasia Congenita by Mutation Analysis of the DAX1 Gene

To develop a rapid diagnostic approach to individuals with the X-linked cytomegalic form of adrenal hypoplasia congenita (AHC) and hypogonadotropic hypogonadism (HH) due to mutations in DAX1, a new member of the nuclear hormone receptor gene superfamily. Molecular genetic diagnostic investigations of individuals with AHC and their relatives included polymerase chain reaction amplification of DAX1 for identification of intragenic mutations and fluorescence in situ hybridization with a cosmid containing the DAX1 gene for evaluation of larger deletions. Families that had males affected with AHC were evaluated for mutations involving the DAX1 gene. DAX1 mutations were identified in four families that had males affected with AHC. Two apparently independent pedigrees had an identical frame-shift mutation due to a single base pair deletion, and a third had a larger deletion involving the entire DAX1 locus. The fourth family was evaluated by fluorescence in situ hybridization for prenatal diagnosis, and both the DAX1 locus and the contiguous glycerol kinase region were deleted. Molecular genetic and molecular cytogenetic techniques represent rapid and complementary approaches to the diagnosis of mutations in the DAX1 gene responsible for AHC and the associated HH. Specific diagnosis of the cause of adrenal insufficiency in these boys permits anticipatory management of the HH and prenatal counseling for parents of the affected child and other members of their families.

Mutations in the DAX-1 gene give rise to both X-linked adrenal hypoplasia congenita and hypogonadotropic hypogonadism.

Adrenal hypoplasia congenita (AHC) is an X-linked disorder characterized by primary adrenal insufficiency. Hypogonadotropic hypogonadism (HHG) is frequently associated with this disorder but is thought not to be caused by the low adrenal androgen levels due to adrenal hypoplasia. It is uncertain whether there are two distinct yet physically linked genes responsible for AHC and HHG or a single gene responsible for both diseases. AHC can occur as a part of a contiguous deletion syndrome together with Duchenne muscular dystrophy (DMD) and/or glycerol kinase deficiency (GKD). From the analysis of deletions, the following gene order has been deduced: Xpter-AHC-GKD-DMD-cen. An AHC critical region of 200-500 kilobases has been defined by physical mapping and partially overlaps with a 160-kilobase dosage-sensitive sex (DSS) reversal critical region. The DAX-1 (DSS-AHC critical region on the X, gene 1) gene was isolated and found to encode a new member of the nuclear hormone receptor family. Here we report that DAX-1 is deleted in 14 patients and point mutations were found in the coding region in DNA from 12 unrelated individuals. All AHC patients over 14 years old and with only point mutations in DAX-1 were also diagnosed with HHG, confirming that the DAX-1 gene is responsible for both X-linked AHC and HHG. But in four sporadic cases and a single familial case, no point mutations were found, suggesting genetic heterogeneity or differential expression of DAX-1.