Application and insights of targeted next-generation sequencing in a large cohort of 46,XY disorders of sex development in Chinese

46,XY disorders of sex development (46,XY DSD) are characterized by incomplete masculinization of genitalia with reduced androgenization. Accurate clinical management remains challenging, especially based solely on physical examination. Targeted next-generation sequencing (NGS) with known pathogenic genes provides a powerful tool for diagnosis efficiency. This study aims to identify the prevalent genetic variants by targeted NGS technology and investigate the diagnostic rate in a large cohort of 46,XY DSD patients, with most of them presenting atypical phenotypes. Two different DSD panels were developed for sequencing purposes, targeting a cohort of 402 patients diagnosed with 46,XY DSD, who were recruited from the Department of Urology at Children’s Hospital, Zhejiang University School of Medicine (Hangzhou, China). The detailed clinical characteristics were evaluated, and peripheral blood was collected for targeted panels to find the patients’ variants. The clinical significance of these variants was annotated according to American College of Medical Genetics and Genomics (ACMG) guidelines. A total of 108 variants across 42 genes were found in 107 patients, including 46 pathogenic or likely pathogenic variants, with 45.7%(21/46) being novel. Among these genes, SRD5A2, AR, FGFR1, LHCGR, NR5A1, CHD7 were the most frequently observed. Besides, we also detected some uncommon causative genes like SOS1, and GNAS. Oligogenic variants were also identified in 9 patients, including several combinations PROKR2/FGFR1/CYP11B1, PROKR2/ATRX, PROKR2/AR, FGFR1/LHCGR/POR, FGFR1/NR5A1, GATA4/NR5A1, WNT4/AR, MAP3K1/FOXL2, WNT4/AR, and SOS1/FOXL2. The overall genetic diagnostic rate was 11.2%(45/402), with an additional 15.4% (62/402) having variants of uncertain significance. Additionally, trio/duo patients had a higher genetic diagnostic rate (13.4%) compared to singletons (8.6%), with a higher proportion of singletons (15.1%) presenting variants of uncertain significance. In conclusion, targeted gene panels identified pathogenic variants in a Chinese 46,XY DSD cohort, expanding the genetic understanding and providing evidence for known pathogenic genes’ involvement. 46,XY disorders of sex development (46,XY DSD) are conditions where individuals don’t fully develop male genitalia due to reduced androgen hormones. Diagnosing these conditions based only on physical exams is difficult. This study used advanced genetic testing called targeted next-generation sequencing (NGS) to identify common genetic variations in a large group of 46,XY DSD patients, many of whom had unusual symptoms. We examined 402 patients with DSD and a 46,XY karyotype, focusing on 142 candidate genes related to sex development. We found genetic variations in 107 patients, including 45 that were likely responsible for their condition. Some of these variations were new discoveries. The most commonly affected genes were SRD5A2, AR, FGFR1, LHCGR, NR5A1, CHD7. We also found that some patients had variations in multiple genes, suggesting complex genetic causes. Overall, we were able to diagnose 11.2% of patients based on our genetic testing, with another15.4% having uncertain results. Patients tested as a trio or duo (with their parents) had a higher diagnosis rate than those tested alone. This study helps expand our understanding of the genetic factors behind 46,XY DSD in the Chinese population. Two gene panels were designed, comprising 142 and 271 candidate genes associated with sex development, to sequence 402 patients with 46,XY DSD. A total of 108 variants across 42 genes were identified in 107 patients, with 46 classified as pathogenic or likely pathogenic, including several novel variants. SRD5A2, AR, FGFR1, LHCGR, NR5A1, CHD7 were among the most frequently observed genes with variants. The overall genetic diagnostic rate was 11.2%, with an additional 15.4% of patients having variants of uncertain significance, and oligogenic variants were detected in 9 patients, suggesting complex genetic interactions in 46,XY DSD. Patients with trio/duo genetic information had a higher genetic diagnostic rate (13.4%) compared to singletons (8.6%), underscoring the importance of parental genetic data. Phenotypic variability and potential genetic heterogeneity underscore the complexity of 46,XY DSD, highlighting the necessity for further research and multi-center collaboration.