There remains a significant unmet medical need to improve the genetic diagnosis of Differences of Sex Development (DSDs). DSDs are common, with an estimated frequency of 1%. Patients suffer a long diagnostic odyssey, in part due to uncoordinated approaches, and half never receive a definitive diagnosis. Even with whole exome sequencing (WES), the diagnostic yield is still low, especially in 46,XY gonadal dysgenesis which is currently only 30% (Reyes et al., 2023).
Given this impasse, new concepts, approaches and integration of genome-wide technologies are needed to improve the diagnostic yield of DSD [3, 4]. We propose a new pipeline to discover causative DSD genes incorporating innovative structural genome mapping techniques (WES, optical genome mapping, WGS), transcriptomic/ epi-genomics databases and patient disease models. The identified novel DSD variant/ candidate genes will be further validated using cellular model and knock out mouse model. Our lab has successfully generated NT2/D1-Cas 9 cell line expressing stable Cas9 enzyme and has successfully established a protocol to reprogram the patient fibroblasts into the Sertoli-like cells. Using CRISPR to knock in the candidate variants into the NT2/D1-Cas9 cells together with the Sertoli-like cells transdifferentiated from DSD patients will allow us to perform gene expression and cell phenotyping analysis including cell proliferation, migration, adherence, and polarity, on mutant cells. The most promising DSD candidate gene will be selected to generate knockout mice to investigate the gonadal phenotype. In conclusion, with these new approaches, we expect to identify novel DSD gene which will contribute to improving the DSD diagnostic yield.