Background: A genetic diagnosis is essential for patients with disorders of sex development (DSD) in order to provide appropriate counseling and sex assignment. Therefore, a molecular diagnosis was made for DSD patients who were seen at our institute during 2002-2020. Methods: Molecular diagnosis was performed using: polymerase chain reaction (PCR) for the SRY gene in 46,XX DSD patients; Sanger sequencing of the SRD5A2 and AR genes in 46,XY DSD patients; and next generation sequencing (NGS) of a 46-gene panel in patients without definite diagnoses. Patients with hypogonadotropic hypogonadism and 21-hydroxylase-deficient congenital adrenal hyperplasia were excluded. Results: A total of 150 patients with 46,XY DSD underwent genetic testing for the SRD5A2 gene (n=142) or the AR gene (n=121) or both (n=114). Eighteen patients had homozygous or compound heterozygous mutation in the SRD5A2 gene and 13 patients had hemizygous mutation in the AR gene. One hundred and two patients (46,XY DSD, n=92; 46,XX DSD, n=10) had DNA testing for NGS. Seven patients with 46,XY DSD were found to carry pathogenic or likely pathogenic variants in DSD-related genes: heterozygous variants in the WT1 gene (n=3); heterozygous variants in the SRD5A2 gene (n=2); and hemizygous variant in the MAMLD1 gene (n=2). Thirty-three patients were found to carry only variants of unknown significance (VUS) in DSD-related genes. Two out of six patients with 46,XX ovotesticular DSD were found to carry an SRY gene (one detected by SRY PCR and one by NGS). Conclusion: In our cohort, 5α-reductase type 2 deficiency and androgen insensitivity syndrome were common causes of 46,XY DSD, followed by monoallelic mutations in the WT1, SRD5A2 and MAMLD1 genes.
INTRODUCTION:NR5A1 variants are a common cause of 46,XY disorders of sex development (DSD) and are associated with a wide spectrum of clinical phenotypes. However, genotype-phenotype correlations and the underlying pathogenic mechanisms remain incompletely understood. In this study, we characterized 27 novel NR5A1 variants identified in Chinese patients with 46,XY DSD and investigate their pathogenic mechanisms. METHODS:In silico tools were used to predict the functional impact of novel NR5A1 variants. Functional analyses were performed in HEK293 cells. Protein level of coding missense variants was assessed by Western blotting, subcellular localization was examined by immunofluorescence, and transcriptional activity was evaluated using dual-luciferase reporter assays with pGL4.11-CYP11A1 and pGL4.11-CYP17A1 promoters. RESULTS:27 patients carrying novel NR5A1 variants exhibited a broad phenotypic spectrum, with external masculinization scores ranging from 0 to 9. Among the 27 variants, missense variants were the most frequent (9/27, 33.3%). Functional validation was performed for the 9 missense variants. Western blot analysis showed markedly reduced protein level for variants located in the DBD, A-box, and LBD, whereas variants within the AF-2 domain showed increased protein level. Subcellular localization analysis demonstrated preserved nuclear localization for all variants. Transcriptional assays revealed that variants in the DBD and LBD generally exhibited reduced transactivation activity, while variants in other domains showed greater variability in transcriptional activity. CONCLUSION:No clear genotype-phenotype correlation was identified. Functional assays alone could not explain the wide phenotypic variability observed in individuals carrying novel NR5A1 variants; however, variants located within the A-box or AF-2 domains appeared to display greater functional tolerance.
BACKGROUND:The anuran testis is a heterogeneous organ composed of germline and somatic cells mainly derived from the coelomic epithelium (Sertoli cells) and mesonephros (Leydig cells), which together regulate spermatogenesis and testicular architecture. SUMMARY:We provide an integrative overview of the developmental origin, structure, and function of somatic cells in evolutionary primitive and advanced anurans, with comparisons to other vertebrates. Initially bipotential gonad differentiates into testes by forming seminiferous cords and tubules composed of gonocytes and Sertoli cells, and a peritubular compartment containing Leydig cells. A distinctive feature of anurans is the continued proliferative capacity of Sertoli cells and their ability to form cysts in which spermatogenesis occurs within adult seminiferous tubules. Although key genes involved in testis differentiation are broadly conserved across vertebrates, their timing and functional roles often differ in anurans, where gonadal development is strongly influenced by steroid hormones, a feature typical of anamniotes. KEY MESSAGES:Vertebrate testes are homologous and share a basic organizational plan, differing mainly in cystic spermatogenesis in anamniotes versus tubular spermatogenesis in amniotes. While somatic cell lineages are largely conserved, anurans exhibit increased complexity in molecular regulatory pathways and remain understudied with respect to the developmental origins of specific cell populations.
INTRODUCTION:Delayed puberty in males requires differentiation between self-limited delay and congenital hypogonadotropic hypogonadism (CHH), including Kallmann syndrome (KS). CASE PRESENTATION:We report an adolescent male presenting with delayed puberty and anosmia, consistent with KS. Hormonal evaluation confirmed CHH, and MRI demonstrated olfactory bulb agenesis. Targeted next-generation sequencing identified two heterozygous variants in functionally related genes: a pathogenic nonsense variant in PROK2 and a missense variant in PROKR2 classified as a variant of uncertain significance. Given their ligand-receptor relationship, oligogenic analysis was performed. The variant combination was prioritized as highly likely disease-causing by ORVAL/VarCoPP and further supported by OLIDA-based interpretation, providing objective evidence for digenic inheritance. Parental segregation confirmed trans inheritance. CONCLUSION:To our knowledge, this is the first report with objective support for the high likelihood of the existence of a digenic etiology in a patient with CHH, based on the use of OLIDA, which provides a standardized framework for the interpretation of oligogenic combinations. These findings highlight the contribution of multilocus variation to CHH and underscore the need for standardized approaches to interpret oligogenic mechanisms in clinical practice.
