Next-generation Sequencing For The Diagnosis Of 46,XY Disorders Of Sexual Development Patients

Disorders of sexual development(DSD)are rare conditions with abnormal sex determination and gender differentiation.There exists divergence of changes in chromosomal mutation,gonadal or genitalia abnormalities in DSD patients and the etiology of 46,XY DSD is the most intricate in this disorders.Here,we used next-generation sequencing combined with a targeted 80 gene panel to diagnose a cohort of 70 46,XY DSD patients who mainly presented with microphallus,variable degrees of hypospadias,and cryptorchidism at the genetic level and the variants identified by next-generation sequencing were confirmed using Sanger sequencing.For patients identified with NR5A1 gene mutations,clinical analysis and in vitro assays were performed.In total,113 mutations,including 86 novel and 27 reported sites in 40 genes,were identified in 52 patients.37 mutations were identified in 19 potential disease-causing genes which were not reported in 46,XY DSD patients.42.86%(30/70)of patients were diagnosed.In additional,47.14%(33/70)of patients were found to harbor multiple genetic mutations.All NR5A1 variants were shown to impair the transactivation ability,with almost no protein expression of variants p.E148 fs X295 and p.C283*.The phenomenon of altered aggregation in nucleus by p.T29 K and p.N44 del variants was observed with confocal laser scanning microscopy,which revealed abnormal activation in chromatin.The targeted sequencing panel provides an efficient method for the etiological diagnosis of46,XY DSD patients.The study revealed genetic architecture in the Chinese 46,XY DSD patients for the first time and expanded the candidate genes.For some atypical 46,XY DSD patients,a potential digenic or oligogenic pattern may underlie the pathological process.The establishment of such patterns of molecular diagnosis has important implications for the selection of long-term treatment strategy,gender assignment or genetic counseling.