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

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.