SUN-167 Advancing The Genetic Understanding Of Differences Of Sex Development Through Whole Genome Sequencing

Abstract Disclosure: G. Atlas: None. J. Van den Bergen: None. G. Robevska: None. K. Bell: None. E. Tucker: None. C. Hanna: None. M.A. O'Connell: None. T.Y. Tan: None. A.H. Sinclair: None. K. Ayers: None. Aims: Currently a genetic diagnosis is found for up to 40-50% for individuals with a difference of sex development (DSD), indicating that there are likely other genomic aetiologies that need to be considered. This study aimed to improve the rates of genetic diagnoses for a cohort of individuals with DSD using whole genome sequencing (WGS), and develop insights into the utility of WGS for rare disease cohorts. Methods: Individuals with severe forms of DSD such as 46,XY gonadal dysgenesis, who were undiagnosed after previous genomic testing (whole exome sequencing or targeted gene panel) were identified from our laboratory group’s biobank. Previous data were reanalysed with an updated gene list, including undertaking an analysis of copy number variants (CNVs) using a locally developed CNV-caller. WGS (Illumina DNA, PCR-free with ∼30x post alignment 2x150bp NovaSeq 6000 sequencing) was performed for 76 individuals, including 27 affected probands and their family members. Analysis and variant curation were undertaken, focusing on 138 diagnostic and candidate genes (and their surrounding genomic regions, as well as introns) known to be associated with DSD. Identified variants (likely pathogenic and pathogenic) were confirmed with Sanger Sequencing, and functional work undertaken to assist with classification. Results: Previously unrecognised genetic diagnoses were identified in 4/28 (14%) of individuals. CNV analysis revealed an individual with a WT1 deletion, not previously identified through standard analysis of whole exome sequencing, and WGS yielded a cohort of 3 individuals with loss-of-function variants in PPP1R12A. Both WT1 and PPP1R12A are genes with extragonadal associations, and identification of these variants explains other features (such as tracheoesophageal fistula in PPP1R12A) or leads to targeted screening (WT1 deletion, with risk of nephropathy/Wilms tumour). Additionally, WGS identified variants of interest in 2/27 (7%) including a variant of uncertain significance in DAAM2 and variant in a potential enhancer of NR5A1, a key DSD gene. This variant was identified in a family with two affected individuals (sisters with 46,XY gonadal dysgenesis). For this family, functional studies using luciferase assays showed a reduction in activity in the variant compared to wild type. Conclusion: The benefit of reanalysis of genomic data with new technologies and new gene lists is shown, with an additional 21% having a finding of interest. This work has identified the activity of an enhancer upstream of NR5A1, with reduced NR5A1 expression being a proposed reason for gonadal dysgenesis in these individuals. Analysis of this first cohort of WGS has led to insights on looking at regions upstream of key DSD genes, which will guide future analysis approaches for WGS in rare disease. Presentation: Sunday, July 13, 2025