Post genome wide association studies in testicular germ cell tumors

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The Testicular Cancer Consortium (TECAC) is the only international collaborative group whose goal is to understand the genetic susceptibility to testicular germ cell tumors (TGCT). The incidence of TGCT is highest among men of European ancestry, and the most common cancer affecting men aged 15-45. TECAC and its members have conducted successful genome-wide association studies (GWAS) of TGCT; our latest analysis has identified 22 novel susceptibility alleles bringing the total validated risk markers to 68, accounting for 43% of heritability. Our studies have revealed the critical role of variation affecting essential pathways of male germ cell development and maturation, sex determination, chromosomal segregation, and DNA maintenance in TGCT susceptibility. We propose three complementary aims to deepen our discovery of inherited variation of susceptibility to TGCT, results of which will refine our understanding of the biology of TGCT and male germ cell development, provide insights into inherited variation predisposing to genomic instability, and improve our ability to identify patients at highest risk of disease. In Aim 1, we will identify rare and common variants (individual and gene burden) using whole exome (WES) approaches followed by independent validation. We will conduct de novo WES on extant biosamples from 1000 and 1000 men with and without TGCT, respectively, and assemble existing WES data from 2066 men with TGCT from TECAC members for comparison to genomically matched (1:4) unaffected men from the Penn Medicine and UK Biobanks. To address data heterogeneity, WES data from all sources will be called together using a common pipeline. Fifty genes and 500 SNPs will be selected for validation in an independent set of extant biosamples from 5000 and 5000 men with and without TGCT, respectively, all with existing genome-wide genotyping. In Aim 2, we will conduct a transcriptome-wide association study (TWAS), preceded by the largest GWAS study to date in 15,847 men with TGCT and 27,178 (230,610 with deCODE) men without TGCT. We will use a customized version of FUSION to perform the TWAS, annotated with expression data derived from TGCT cell lines, fetal and adult germ cells, GTEx, and single cell sequencing of germ cells. In Aim 3, after in silico assessment and prioritization, 20 top loci/genes will be evaluated in TGCT in vitro models using siRNA and CRISPR to assess over-expression and knockdown effects on morphology, proliferation, chromosomal abnormalities, and cisplatin sensitivity. We will select candidate genes from our past genetic association efforts (11 preliminarily prioritized) and from novel loci/genes found in our WES (Aim 1), GWAS and TWAS (Aim 2) efforts. Our renewal builds upon our accomplishments and paves the way to identify novel susceptibility loci through three highly cohesive aims. Our findings will lead to further ground-breaking insights into the biology and genetic etiology of TGCT and will provide data needed to identify men at greatest need for surveillance, the optimal way to decrease serious TGCT treatment-related morbidity.