Endocrine Abstracts

Background: Genetic causes of Differences of Sex Development (DSD) are identified in only approximately one third of individuals using standard-of-care exome sequencing, leaving a majority without a molecular diagnosis. Variants in non-coding regulatory regions and complex structural variants (SVs) are likely contributors to this diagnostic gap, yet are systematically excluded and still very challenging due to our limited molecular understanding outside of protein-coding sequence effects.

Methods: We analyzed genome sequencing data from over 120 individuals using short-read and long-read sequencing technologies. While more cost intensive, long-read sequencing provides improved discovery of SVs and haplotype-resolved methylation states, enabling investigation of epigenetic contributions to DSD. We implemented computational pipelines for singleton and trio analysis. Identified genetic variants were scored for potential disease relevance, filtered against population databases and cross-referenced with clinical databases to prioritize likely disease-causing alterations. To functionally investigate regulatory variants in genes implicated in sex development, we additionally developed experimental saturation mutagenesis using massively parallel reporter assays (MPRA), targeting promoters and enhancers of multiple DSD-relevant loci.

Results: We developed tier-based reports for short (SNV/InDels) and long sequence variants (SVs). TALOS-based prioritization of short variants identified high-confidence causative variants in established DSD genes including WT1, SF1 (NR5A1), ATRX, HSD17B3, and SRD5A2, as well as syndromic candidates PROKR2 and PPP1R12A. This highlights several coding variants missed in prior analyses. We are currently analyzing potential SV candidates and new non-coding mechanisms of disease shared across individuals. Complementary MPRA experiments are being applied to systematically map the transcriptional effects of regulatory variants across promoters and enhancers of key sex-development genes, enabling nucleotide-resolution functional maps for interpretation of candidate variants. In an initial set of experiments, we have mapped promoter motifs of the Androgen Receptor across three cell-types.

Conclusions: To increase diagnostic yield, shorten the diagnostic journey and to enable a genetically informed counseling of DSD patients, genetic analyses need to extend to the whole genome, and specifically include non-coding variant and SV interpretation. High-throughput functional assays substantially expand our ability to interpret pathogenic variation in regulatory sequences.