X-linked adrenal hypoplasia congenita and hypogonadotropic hypogonadism: Identification and in vitro study of a novel small indel in the NR0B1 gene

Abstract

Abstract. DAX1 is an orphan nuclear receptor that has a key role in the development and function of the adrenal and reproductive axes. Mutations in NR0B1, the gene encoding DAX1, result in X-linked adrenal hypoplasia congenita (AHC) and hypogonadotropic hypogonadism (HHG). A Chinese pedigree with X-linked AHC and HHG was investigated in the present study. Sequence analysis identified a novel small indel variant, c.195_207delinsTG, in the NR0B1 gene. To determine the effect of this variant on DAX1 expression, reverse-transcription quantitative PCR and western blot assays were performed. The mRNA expression levels in carriers of mutant NR0B1 were significantly reduced (62% decrease) compared to those in individuals with wild-type NR0B1 (WT). The c.195_207delinsTG mutation was demonstrated to lead to various truncated DAX1 proteins, including the C-terminal truncated DAX1, which was only detected in the cytoplasm, and the N-terminal truncated DAX1, which was present in the cytoplasm and nucleus. A luciferase assay was then performed to assess the repressor function of DAX1 in modulating steroidogenic factor 1 (SF-1)-mediated transactivation. WT DAX1 significantly suppressed the SF-1-mediated promoter activity of the steroidogenic acute regulatory protein by 35.5±1.9%. In contrast to other known pathogenic mutations which abolish the repressor function of DAX1, the c.195_207delinsTG mutant proxkduced a higher repressor activity, demonstrating a 49.9±2.6% reduction of promoter activity. These findings suggested that the mutation of NR0B1 in X-linked AHC with HHG enhanced the function of DAX1 to repress SF-1 activation, while DAX1 is expected to have additional roles in the pathological mechanism. Introduction X-linked adrenal hypoplasia congenita (AHC) is a rare genetic disorder of adrenal gland development, characterized by absence or near absence of the permanent zone of the adrenal cortex (1). Patients with this condition usually present with primary adrenal failure, including salt-wasting, hyperpigmentation, failure to thrive, reduced serum cortisol and aldosterone and increased plasma adrenocorticotropic hormone (ACTH) (2). The majority of patients present with symptoms within the first two months of their life, whereas the remainder present later in childhood (3,4). The condition is lethal if left untreated with appropriate steroid hormones (5). Besides adrenal insufficiency, hypogonadotropic hypogonadism (HHG) is a frequent feature of X-linked AHC. It is usually recognized during adolescence by the absence or interruption of normal pubertal development (6,7).X-linked AHC was originally mapped to Xp21 and the nuclear receptor sub-family 0, group B, member 1 (NR0B1) was subsequently identified by positional cloning as the gene responsible for X-linked AHC and HHG (2,(8)(9)(10)(11). The NR0B1 gene consists of two exons and encodes a 470-amino acid protein termed dosage-sensitive sex-reversal, adrenal hypoplasia congenital critical region on the X chromosome, protein 1 (DAX1) (12). DAX1 is an orphan member of the ...

Nonsense variant of NR0B1 causes hormone disorders associated with congenital adrenal hyperplasia

“…3 ). As expected, wild-type DAX-1 suppressed the relative luciferase activity, which was in accordance with the finding of a previous report 21 . Intriguingly, mutant DAX-1 significantly improved luciferase activity compared to wild-type DAX-1, suggesting that this NR0B1 variant impaired the repressor function of DAX-1 during transcriptional regulation.…”

“…The loss of DAX-1 function triggered by this NR0B1 variant caused disordered expression of genes involved in energy homeostasis and steroidogenesis, which may lead to abnormal adrenal development and finally CAH. These findings are in line with previous reports in this field 21 , 23 , 26 .…”

“…The regulatory role of NR0B1 in modulating transcription was monitored using the StAR-luc plasmid (luciferase reporter plasmid) following the method outlined in a previous report 21 . Briefly, seeded cells were treated with 20 ng StAR-luc, 0.5 ng pRL-SV40, and 60 ng WT or mutant NR0B1 .…”

Nuclear Receptor Gene Variants Underlying Disorders/Differences of Sex Development through Abnormal Testicular Development

“…Puberty starts spontaneously in some patients with NR0B1 variants but is usually arrested before completion [ 45 ]. Small penile size in adulthood, cryptorchidism, and gynecomastia due to hypogonadism are occasionally present [ 99 , 100 ].…”