Two novel DAX1 gene mutations in Chinese patients with X-linked adrenal hypoplasia congenita: clinical, hormonal and genetic analysis

Mutations in the DAX1 gene result in X-linked congenital adrenal hypoplasia (AHC). Affected boys usually present with primary adrenal failure in early infancy or childhood and hypogonadotropic hypogonadism (HH) at puberty. This paper describes the clinical, hormonal, radiological, and genetic characteristics of 2 Chinese patients with X-linked AHC. Primary adrenal insufficiency occurred in the 2 patients during their childhood and HH was recognized at puberty. Genomic DNA was extracted from their peripheral blood leukocytes and coding sequence abnormalities of the DAX1 gene were assessed by PCR and direct sequencing analysis. Genetic analysis of the DAX1 gene revealed 2 novel mutations c.572-575 dupGGGC, p.Thr193Gly,fs,205X and c.773- 774 dupCC, p.Ser259Pro,fs,264X in exon 1, causing frameshifts and yeilding premature stop codons at 205 and 264, respectively. This study identifies 2 novel mutations in the DAX1 gene which can further expand the mutation database and benefit patients in the diagnosis and treatment of AHC.     

Hypogonadotropic hypogonadism as a presenting feature of late-onset X-linked adrenal hypoplasia congenita.

Mutations in the orphan nuclear receptor DAX-1 cause X-linked adrenal hypoplasia congenita. Affected boys usually present with primary adrenal failure in early infancy or childhood. Impaired sexual development because of hypogonadotropic hypogonadism becomes apparent at the time of puberty. We report adult-onset adrenal hypoplasia congenita in a patient who presented with hypogonadism at 28 yr of age. Although he had no clinical evidence of adrenal dysfunction, compensated primary adrenal failure was diagnosed by biochemical testing. Semen analysis showed azoospermia, and he did not achieve fertility after 8 months of treatment with gonadotropins. A novel Y380D DAX-1 missense mutation, which causes partial loss of function in transient gene expression assays, was found in this patient. This case demonstrates that partial loss-of-function mutations in DAX1 can present with hypogonadotropic hypogonadism and covert adrenal failure in adulthood. Further, an important role for DAX-1 in spermatogenesis in humans is confirmed, supporting findings in the Dax1 (Ahch) knockout mouse.     

Identification of a novel missense mutation that is as damaging to DAX-1 repressor function as a nonsense mutation

Mutations in the DAX-1 (NROB1) gene result in X-linked congenital adrenal hypoplasia (AHC) and hypogonadotropic hypogonadism. The clinical presentation is usually as adrenal insufficiency in early life, with hypogonadotropic hypogonadism detected at the time of expected puberty. In this study we identified mutations in the DAX-1 gene of two patients with AHC. One mutation, Y399X, resulted in a premature stop codon and was associated with loss of Leydig cell responsiveness to human chorionic gonadotropin. The second, L297P, was a missense mutation, and human chorionic gonadotropin responsiveness was maintained. Kindred analysis established that the mutations had been inherited from the proband's mothers. The L297P has not been described previously and occurs within a highly conserved binding motif (LLXLXL). Transient transfection assays demonstrated that both mutations resulted in a severe loss of DAX-1 repressor activity. Immunohistochemical analysis of testicular tissue obtained from an affected sibling of the subject with the Y399X mutation, who had died with adrenal failure as a neonate, showed normal testicular morphology and expression of DAX-1, steroidogenic factor-1, and anti-Mullerian hormone protein. These data extend the clinical and molecular information on DAX-1 mutations, confirm normal testicular development at the neonatal stage, and illustrate variability in Leydig cell function.     

DAX1 and X-linked adrenal hypoplasia congenita: clinical and molecular analysis in five patients

OBJECTIVE: Mutations in the gene coding for the orphan nuclear receptor DAX1 cause X-linked adrenal hypoplasia congenita (AHC). Affected boys usually present with primary adrenal failure in early infancy or childhood. Impaired sexual development due to hypogonadotropic hypogonadism becomes manifest at the time of puberty. Moreover, evidence from Dax1 knockout mice and a limited number of patients with AHC, suggests that mutations in DAX1 may directly cause abnormalities in spermatogenesis. The aim of this study was to characterize clinically and genetically five patients with AHC. DESIGN: DNA sequencing analysis, endocrine testing, testicular ultrasound and semen analysis with 1-year follow-up after gonadotropin treatment. METHODS: We report on five men with classic AHC manifestations. Genomic DNA was extracted from patients' peripheral blood leukocytes and the coding region, splice sites, and promoter (-240 bp) region of DAX1 were directly sequenced. RESULTS: Three known and two novel mutations were detected in the DAX1 coding sequence in these patients. Semen analysis was performed in four of the five patients and showed azoospermia. Twelve-month treatment with gonadotropins did not restore fertility in these patients. All patients showed a normal testicular Doppler ultrasound, in contrast with that observed in Dax1-deficient mice, which display abnormalities in the rete testis. CONCLUSIONS: These cases further expand the number of DAX1 mutations reported in the literature, as well as our clinical knowledge of this rare disease.     

Clinical and genetic heterogeneity of congenital adrenal hypoplasia due to NR0B1 gene mutations

Introduction X‐linked adrenal hypoplasia congenita (AHC) is a rare disorder caused by mutations or complete deletion of the NR0B1 gene that encodes the DAX‐1 protein, an orphan member of the nuclear receptor superfamily. AHC is characterized by adrenal insufficiency in infancy and early childhood. Later, hypogonadotropic hypogonadism (HH) manifests as pubertal failure. Patients and methods We evaluated the clinical, endocrine and molecular characteristics of 12 AHC patients from 5 families diagnosed between 1984 and 2007 in Israel. Results Most of the boys (10/12) presented with signs of adrenal insufficiency such as salt wasting and failure to thrive during the neonatal period. Aldosterone deficiency usually preceded cortisol deficiency requiring early mineralocorticoid therapy. Serum cortisol levels in the first weeks of life varied from very low to high levels (<2·76 to >1776 nmol/l). Five boys showed signs of precocious sexual development during infancy and childhood, including enlargement of the penis and testes. In four patients the initial diagnoses were erroneous. Molecular analysis of the NR0B1 gene identified point mutations in six patients including a novel splice site mutation in one patient and his family (IVS1‐1G→C). Contiguous gene deletion was found in six patients from two families who manifested impaired mental development. Conclusions In X‐linked AHC caused by different molecular defects in NR0B1 gene, the clinical spectrum of the disease is quite variable and precocious sexual development is a prominent feature. Genetic testing is indicated in boys presenting with salt‐wasting with or without cortisol deficiency if congenital adrenal hyperplasia has been ruled out.     

Four Japanese patients with adrenal hypoplasia congenita and hypogonadotropic hypogonadism caused by DAX-1 gene mutations: mutant DAX-1 failed to repress steroidogenic acute regulatory protein (StAR) and luteinizing hormone beta-subunit gene promoter activity

Mutations of DSS (dosage sensitive sex reversal)-AHC critical region on the X chromosome, gene 1 DAX-1(NROB1)] results in X-linked adrenal hypoplasia congenita (AHC) and hypogonadotropic hypogonadism (HHG). Here we report four Japanese patients with AHC and HHG caused by the mutations of the DAX-1 gene. All patients manifested adrenal crisis at early childhood. Three patients did not show any pubertal sign and were diagnosed as having HHG. One patient manifested spontaneous pubertal development at 17 years of age. Nevertheless, his puberty did not develop further and his gonadotropin and testosterone levels decreased thereafter. Therefore, he was also diagnosed as having HHG. We performed testicular biopsy in another patient with HHG. Histological examination demonstrated Sertoli cell hypoplasia and no sperm formation in the seminiferous tubules. Molecular analysis demonstrated two novel point mutations (V269D and L278R) in two patients. Transient transfection assays showed that all these mutations (V269D, L271X, L278R, and Q395X) abolished the repression activity to both StAR and LHbeta gene promoter activation. In conclusion, we reported patients with AHC and HHG caused by the loss of function mutations of the DAX-1 gene.     

Adrenal hypoplasia congenita - an uncommon reason of primary adrenal insufficiency

Adrenal hypoplasia congenita (AHC) is a rare inherited condition characterised by primary adrenal failure and hypogonadotropic hypogonadism. Most cases arise from mutations in the NR0B1 gene (Xp21.3), which encodes an orphan nuclear receptor DAX-1. A 20-year-old patient was recently diagnosed with AHC. Adrenal failure had been recognized and treated since his infancy. During adolescence, gradual decrease in growth velocity and low body mass were noted. Lack of puberty and skeletal immaturity were observed. Serum DHEA-S and testosterone were undetectable. Low gonadotropin levels failed to rise after stimulation. Neither dysfunction of the somatotropic nor pituitary-thyroid axis was found and no hypothalamo-pituitary pathology was visible on MRI. Androgen replacement therapy induced the development of secondary sexual characteristics, remarkably improved patient's growth and advanced his bone age. NR0B1 mutation screening revealed nucleotide transversion C > A, resulting in premature stop codon (Y399X). Same mutation was previously identified in a Scottish family, however, phenotypic differences suggest the role of additional factors modifying the disease course. Although it does not change therapeutic strategy, accurate molecular diagnosis allows genetic counselling in family members. Autoimmunity remains the major cause of adrenal failure; however, other rare conditions should always be considered.     

Sibling cases of Addison's disease caused by DAX-1 gene mutations

We report two sibling cases of Addison's disease without any evidence of sexual precocity, adrenal hyperplasia, or autoimmune disease. The diagnosis of primary adrenocortical insufficiency was made at the age of 5 in the younger brother and at the age of 18 in the elder brother. The younger brother had been inactive during infancy and had diffuse skin pigmentation without abnormal external genitalia, while the elder brother had been healthy until the age of 17 when he noticed skin pigmentation and small testes. Both boys had delayed puberty due to hypogonadotropic hypogonadism. The diagnosis of adrenal hypoplasia congenita (AHC) was established by genetic analysis of DAX-1 gene (dosage-sensitive sex reversal-adrenal hypoplasia gene on the X chromosome, gene 1) with the same single frameshift mutation (305delG). However, yet-uncharacterized epigenetic, nongenetic and/or genetic factors other than the DAX-1 gene may be responsible for the differential onset of AHC in these sibling cases.     

DAX-1基因新的移码突变导致一例迟发型先天性肾上腺发育不良症

本研究发现1例先天性肾上腺发育不良症和低促性腺激素性性腺功能减退症患者DAX-1基因第一外显子处993delC新型移码突变,导致终止密码子提前59个氨基酸出现,其母亲、大姐及二姐均为杂合突变,父亲及哥哥为正常野生型.患者糖皮质激素替代治疗后,症状改善,但ACTH未被抑制.

Abstract：

A novel hemizygous frameshift mutation in exon1of DAX-1 gene (993delC) was found in a patient with late-onset adrenal hypoplasia congenita and hypogonadotropic hypogonadism.This mutation led the stop codon to appear in advance of 59 amino acids.His mother and two sisters were the carriers of this hemizygous mutation while his father and brother were wild-type.After glucocorticoid hormone replacement therapy, the clinical symptom was improved, but the level of ACTH was not suppressed.     

Sex determination: a 'window' of DAX1 activity.

Traditionally, DAX1 was considered an 'anti-testis' gene because DAX1 duplications in XY individuals cause male-to-female sex reversal: dosage-sensitive sex reversal (DSS). In DSS, two active DAX1 genes on one X chromosome can abrogate testis formation. By contrast, mutations and deletions of DAX1 cause adrenal hypoplasia congenita (AHC). Although AHC patients develop testes, gonadal defects include disorganized testis cords and hypogonadotropic hypogonadism, which is not completely restored with gonadotropin or androgen therapy. Recent evidence of XY sex reversal in Dax1-deficient mice strongly supports a role for Dax1 as a 'pro-testis' gene. Therefore, perhaps DAX1/Dax1 acts within a 'window' of activity, outside of which testis formation does not occur. Here, we discuss the function and possible mechanisms of DAX1 action in male gonadogenesis.     

Are human male patients with DAX1/NR0B1 mutations infertile?

DAX-1 stands for Dosage sensitive sex-reversal, Adrenal hypoplasia congenital (AHC), on the X chromosome. DAX-1 mutations usually cause primary adrenal insufficiency or congenital adrenal hypoplasia in early childhood and hypogonadotropic hypogonadism (MIM # 300200). DAX-1 protein is necessary to maintain normal spermatogenesis. In humans, male fertility has been studied in few patients carrying DAX-1 mutations. Cases of azoospermia have been reported, as well as unsuccessful gonadotropin treatments. The clinician should be informed that TESE–ICSI technique carries a potential hope to father non-affected children, as shown in this review.     

Late-onset X-linked adrenal hypoplasia (DAX-1, <Emphasis Type="Italic">NR0B1</Emphasis>): two new adult-onset cases from a single center

Purpose

DAX-1 (NR0B1) is an orphan nuclear receptor, which plays a critical role in development and regulation of the adrenal gland and hypothalamo–pituitary–gonadal axis. Mutations in NR0B1 lead to adrenal hypoplasia congenita (AHC), hypogonadotropic hypogonadism (HH) and azoospermia in men. Presentation is typically with adrenal insufficiency (AI) during infancy or childhood. To date only eight cases/kindreds are reported to have presented in adulthood.

Methods

We describe two new cases of men with DAX-1 mutations who presented in adulthood and who were diagnosed at a large University Hospital.

Results

Case 1 presented with AI at 19 years. At 38 years he was diagnosed with HH. Detailed history revealed a brother diagnosed with AI at a similar age. Sequencing of the DAX-1 (NR0B1) gene revealed a heterozygous c.775T?>?C substitution in exon 1, which changes codon 259 from serine to proline (p.Ser259Pro). Case 2 was diagnosed with AI at 30 years. Aged 37 years he presented with HH and azoospermia. He was treated with gonadotropin therapy but remained azoospermic. Testicular biopsy showed maturational arrest and hypospermatogenesis. Analysis of the NR0B1 gene showed a heterozygous c.836C?>?T substitution in exon 1, resulting in a change of codon 279 from proline to leucine (p.Pro279Leu). This change alters the structure of the repression helix domain of DAX-1 and affects protein complex interactions with NR5A family members.

Conclusions

We describe two missense mutations within the putative carboxyl-terminal ligand binding domain of DAX-1, presenting with AHC and HH in adulthood, from a single center. DAX-1 mutations may be more frequent in adults than previously recognized. We recommend testing for DAX-1 mutations in all adults with primary AI and HH or impaired fertility where the etiology is unclear.