Next-generation sequencing reveals genetic landscape in 46, XY disorders of sexual development patients with variable phenotypes.

Disorders of sexual development (DSD) are rare congenital conditions in which chromosomal, gonadal, or anatomical sex is atypical. Currently, less than 20% of patients receive an accurate genetic diagnosis. Targeted next-generation sequencing, consisting of 33 candidate genes and 47 genes involved in sexual differentiation and development, was performed on 70 46, XY DSD patients. Functional assays were performed to evaluate the expression and transcriptional activity of one reported and nine novel mutations of NR5A1. In total, 113 mutations, including 86 novel and 27 reported sites in 40 genes, were identified in 52 patients. Among them, 37 mutations from 19 genes were first identified in 46, XY DSD patients, including EGF, LHX9, and CST9. Nine patients displayed biallelic mutations, 12 had mutations in sex chromosome genes and 14 had monoallelic mutations in NR5A1, BMP4, and WT1. Higher frequency mutations were identified in AR, SRD5A2, and NR5A1. Six missense, one frameshift, and one three-nucleotide deletion mutations of NR5A1 were shown to impair the transactivation ability with an altered nuclear aggregation of p.T29K and p.N44del variants. Multiple genetic mutations were identified in 33 of the 70 patients. The targeted sequencing panel provides an efficient method for the etiological diagnosis of 46, XY DSD patients and expands the candidate genes and inherited patterns.