The nuclear receptor subfamily 0 group B member 1 (NR0B1) gene encodes DAX1, an orphan nuclear receptor that plays a critical role in the development and differentiation of the adrenal gland and hypothalamus-pituitary-gonadal axis (1). It is predominantly expressed in the adrenal cortex, hypothalamus, pituitary gland, and gonads (testis and ovary). DAX1 has been proposed to serve as a transcription factor involved in the development of the adrenal cortex and pituitary gonadotropes (2). More than 200 mutations have been reported in NR0B1, most of which are nonsense or frameshift mutations that result in premature truncation of the protein (3). Defective NR0B1 leads to X-linked adrenal hypoplasia congenita (AHC), resulting in a failure to develop the permanent adult adrenal cortex (4). Patients with NR0B1 mutations may present with severe salt wasting in infancy or have a more insidious onset during childhood. AHC rarely presents with adrenal failure later in adulthood, and late-onset primary adrenal insufficiency (AI) is often accompanied by hypogonadotropic hypogonadism (HH) and azoospermia (5–7). In this study, we report a novel mutation in NR0B1 that leads to late-onset AHC and failure of pubertal development.

X-linked AHC is an X-linked recessive inherited disease that typically affects males and presents as primary AI in early infancy or childhood, and HH at puberty with impaired spermatogenesis. Approximately 60% of male patients with AHC develop AI within 2 months after birth, whereas 40% do so between the age of 1 to 9 years. Recently, various cases of late-onset forms of X-linked AHC have been reported. In this study, we report a case of late-onset X-linked AHC and HH caused by a novel variant of NROB1. The proband presented with AI at 17 years of age and a lack of development of secondary sexual characteristics during puberty. Sequencing of NR0B1 revealed a c.1034delC frameshift variant in two affected males in the kindred. The diagnosis of X-linked AHC and HH was established based on the clinical presentation, laboratory tests, and molecular analysis. According to the family history, two other male members of the family had presented with similar manifestations; one (II-3) was diagnosed with Addison’s disease (AD) and male infertility at the age of 30 years and had been treated with prednisone, whereas the other (II-5), who had also been diagnosed with AD at the age of 33 years, succumbed to adrenal crisis at the age of 35 years. This indicates an X-linked mode of inheritance, as was confirmed by the results of the genetic analysis presented herein. In addition, no evident secondary causes of AD were identified, thus allowing us to exclude its presence.

NR0B1 consists of two exons and spans a 4.96 kb region at Xp21.2. It encodes a 470-amino-acid member of the nuclear hormone receptor superfamily. Most of the coding sequence is found in exon one (9, 10), which encodes the N-terminal domain and part of the C-terminal domain of the protein NR0B1. Exon two encodes the remaining part of the C-terminal domain. According to the statistical data of the Human Gene Mutation Database (http://www.hgmd.cf.ac.uk/ac/index.php, accessed on 20180630), a total of 252 mutations have been found in NR0B1. Truncating mutations such as nonsense or frameshift mutations have been identified throughout the gene. In contrast, missense changes have tended to cluster in the carboxyl terminus of the protein, corresponding to the putative LBD. Several cases of changes in the domain such as p.Ser259Pro, p.Pro279Leu, p.Ile439Ser, p.Tyr380Asp, and p.Gln305Hisfs*67 have been associated with a milder or late-onset phenotype (11). In functional studies of Tyr380Asp and Ile439Ser mutants, the partial loss of NR0B1 transcriptional repression was consistent with the late-onset phenotype (12). The p.Pro345Argfs*27 mutation is a novel frameshift mutation within the LBD that introduces premature termination of the NR0B1 protein, thus resulting in a truncated one. All the affected males in the family exhibited a late-onset phenotype. These findings suggest that the truncated NR0B1 protein produced by this mutation may retain part of its function. Further studies are needed to elucidate the function of p.Pro345Argfs*27.

The same variant was identified in another male family member (II-5), who had presented with a dysfunction of the adrenal cortex and had deceased owing to adrenal crisis. Although the dysfunction of his gonadal axis had occurred at an older age compared to that of the proband, it was more severe. His impairment involved the hypothalamus and pituitary, whereas the impairment of the proband primarily involved the hypothalamus. This suggests that, despite possessing the same genetic variant, there are differences in clinical manifestations, even in patients of the same pedigree. Liu also reported a nonsense variant change in exon 1 (c.192C>G) in two male siblings presenting with precocious puberty and late-onset HH but exhibiting different clinical manifestations (13).

There was no evident correlation between the NR0B1 variant type and the clinical presentation. Literature search revealed 11 reports of adult-onset AHC (6, 7, 11, 14–20). The reported cases of adult-onset AI and hypogonadotrophic hypogonadism associated with mutations of NR0B1 gene are summarized in Supplementary Table S1 . Of the 11 reports, three (p.Gln37X in one, p.Trp39X in two kindreds) are amino-terminal nonsense mutations, whereas eight (p.Tyr378Cys, p.Leu386Phe, p.Ser259Pro in two kindreds, p.Pro279Leu, p.Gln305Hisfs*67 (c.915delG), p.Tyr380Asp, p.Ile439Ser) are mutations distributed throughout the carboxyl LBD. The mutation described in this study also falls in the “hotspot.” Most patients presented with AI as the initial symptom followed by HH (or not); however, two cases presented with HH first, and further endocrine investigations confirmed AI. Among these variants, p.W39X was identified in two kindreds; the proband reported by M. Guclu et al. (17) presented with delayed puberty and small size of testes as the initial symptoms, whereas the other proband, reported by M. L. Raffin-Sanson et al., presented with fatigue, sore throat, and dizziness, which are consistent with AI (6). Moreover, the p.Ser259Pro mutation was also found in two families (11, 18), wherein both probands presented with AI initially, however, hypogonadism was absent in the case reported by C. M. Oh et al. (18). The reproductive phenotypes were variable in these patients, ranging from azoospermia to spontaneous fertility, even within the same family, as shown by M. C. C. Vargas et al. (20), referring to a family with spontaneous fertility and a variable spectrum of reproductive phenotypes, in which all males presented a distinct degree of testosterone deficiency and fertility phenotypes, ranging from a variable degree of hypogonadism, oligoasthenoteratozoospermia to spontaneous fertility. Additionally, the same variant can cause diverse phenotypes within one kindred as reported in a family with the p.Trp39X variant; the proband was diagnosed with AI at the age of 19 years but exhibited a preserved hypothalamic–pituitary–gonadal axis; in contrast, his nephew experienced AI crisis at the age of 2 weeks (6). The underlying mechanism remains obscure. The mutation by itself as well as the repressive role of NR0B1 in steroidogenic factor-1 (SF1/Ad4BP, NR5A1) may be involved in this mechanism. DAX1 is expressed in tissues involved in steroid hormone production and reproductive function (21). This expression pattern overlaps significantly with that of another nuclear receptor, SF-1 (22). Previous studies have suggested that DAX1 is a negative regulator that represses SF-1-mediated transactivation of various genes involved in steroidogenesis (23). The loss of this inhibitory property in the LBD of DAX1 in the NR0B1 variant has been demonstrated to be responsible for the pathology of X-linked AHC and HH (24–30).

In addition, the proband’s serum triglyceride (TG) level was significantly high, which can be related to a low testosterone level. The plausible mechanism for the significant association between TG and testosterone levels in men is that low testosterone levels promote insulin resistance (31) by predisposing to visceral obesity, leading to a dysregulation of fatty acid metabolism, which in turn promotes insulin resistance (32). As reported previously, the relative risk of death in Swedish patients with AD is more than two-fold higher than that in the background population, primarily because of cardiovascular disease (CVD); elevated triglycerides are one of the CVD risk factors (33). Combining the dietary habits as well as no family history of hyperlipidemia makes it difficult to explain the common diseases of lipid metabolism. Therefore, whether or not the patient has a deletion of the glycerol kinase (GK) gene should be considered. The GK gene is located in Xp21.3, in a critical region of approximately 50–250 kb, close to the genomic coordinate of the AHC gene, and located distal to the Duchenne muscular dystrophy gene (34). Genomic analysis confirmed that the GK gene was unaffected, excluding combined glycerol kinase deficiency. Moreover, the serum VLCFA level was normal; therefore, adrenoleukodystrophy (ALD), another X-linked AI disorder, was also excluded. ALD is associated with primary testicular failure and characterized by primary AI and demyelination of the central or peripheral nervous system. The disease results from impaired transport of VLCFA into peroxisomes for beta-oxidation, which results in elevated levels of VLCFA in plasma and tissues (35).

In summary, we identified a novel frameshift variant of NROB1. This finding is important for expanding our knowledge of the phenotype and genotype of X-linked AHC. Functional studies of AHC patients are necessary to elucidate the biological effects of the identified mutation on the development and function of the adrenal gland and the hypothalamic–pituitary–gonadal axis.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: https://ngdc.cncb.ac.cn/, HRA002312.

Ethical review and approval were not required for the study on human participants in accordance with the local legislation and institutional requirements. Written informed consent was obtained from the patient prior to his inclusion in the study.

JX designed and led this study. FZ and MZ searched the literature and drafted the manuscript. XD collected patient information, recruited the patient. YL analyzed the data. JX revised the manuscript. All authors read and approved the final manuscript.

This work was supported by grants from the National Natural Science Foundation of China (No. 81770736).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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