PROJECT SUMMARY/ABSTRACT Bladder (urothelial) cancer has a substantial inherited component, with an estimated heritable fraction of approximately 30%. Genome-wide association studies document an estimate of familial relative risk for urothelial cancer similar to that seen in familial bladder cancer registries (1.37 vs 1.69). Highly penetrant cancer susceptibility genes such as those in the mismatch- repair pathway (eg, MSH2, MSH6) account for only a small fraction of genetic susceptibility to urothelial cancer. There are also numerous reports of multiple-case urothelial cancer kindreds that have not yet been systematically studied using modern sequencing methodologies. Using bait capture next-generation sequencing assays of paired somatic and germline DNA, we showed that that 20% of urothelial cancer patients have moderate- to highly-penetrant known predisposition genes. Approximately 70% of these genes are in DNA damage response gene pathways, genes that predispose for a broad spectrum of non-urothelial cancers. Preliminary data also identified a novel mechanism of bladder cancer susceptibility in a nucleotide excision repair pathway. This project seeks to identify novel urothelial cancer susceptibility genes by genotyping DNA from >480 kindreds demonstrating familial urothelial cancer, ~275 patients with early (≤ age 45) and extremely early (≤ age 30) disease onset, and a selected subset of over 1000 patients in whom urothelial cancer is a component of multiple primary cancers of which one is urothelial cancer. We will utilize next-generation sequencing assays of paired somatic and germline DNA to identify loss of heterozygosity in bladder tumors, as well as interrogation of germline genomes and exomes from familial and early-onset cases to discover putative bladder cancer susceptibility genes. We will assess candidate genes that may contribute to hereditary urothelial cancer in the above-mentioned kindred studies, genes in pathways governing DNA repair, and genes identified in prior genome-wide association studies. For both the discovery and validation phases, we will utilize pre-formed cohorts of 3,000 cases and 3,000 controls for genetic and genetic epidemiologic studies at MSK, augmented by cases provided by national and international collaborators, including a highly informative subset ascertained by NCI investigators. To inform the discovery of putative urothelial cancer susceptibility genes, we will pursue functional characterization of deleterious genetic mutations identified by the above- mentioned genetic epidemiologic approaches.