Identifying germline genetic alterations or environmental factors associated with bilateral germ cell tumor (GCT).
Authors: Gleeson Jack Patrick , Harrold Emily , Bromberg Maria , Patel Zalak , Knezevic Andrea , Thomas Tinu , Ravichandran Vignesh , Tran Christina , Silber Deborah , Carver Brett Stewart , Matulewicz Richard , McHugh Deaglan Joseph , Bajorin Dean F. , Bosl George J. , Funt Samuel Aaron , Joseph Vijai , Stadler Zsofia Kinga , Feldman Darren R. 
Published: June 2022

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Background: Germline genomic alterations and/or environmental factors are proposed as critical to GCT development given the significant rates of bilateral and familial cases. A number of low-moderate risk loci for GCT have been identified (Litchfield, Eur Urol 2018 & Pluta, Nat Comm 2021), predominantly by analysing unilateral GCT, with a polygenic heritability model proffered. We postulate bilateral GCT represents an extreme phenotype with higher frequency of loss of function germline genomic alterations and/or pre-disposing environmental exposures. Methods: Bilateral GCT pts who consented to a prospective clinically annotated GCT DNA registry were included for analysis. The registry integrates self-reported questionnaires on environmental exposures, relevant medical history and germline whole exome sequencing (WES). Pathogenicity of variants identified by WES were classified using the algorithm Pathogenicity of Mutation Analyzer (PathoMAN) [Ravichandran, Genet Med 2019]. Aggregated WES results were curated to identify disruptive or deleterious variants affecting the same genes as the most significant individual variants in the Litchfield series. A < 1% minor allele frequency threshold was set for candidate variants, likely benign/benign variants were excluded . Results: Of 43 bilateral cases with germline WES results, median age at first diagnosis was 32.1 years (range 18-73), 9 tumors were synchronous and 34 metachronous with median interval between diagnosis of 76.4 months for metachronous cases. Tumor histology was seminoma in 52 and nonseminoma in 34. 39 of 43 (91%) pts completed questionnaires with 35 (90%) reporting White race, 2 Hispanic/Latino, 1 Asian, and 1 Peruvian. 13 (33%) reported prior smoking, 21 (54%) prior marijuana use, and 6 (15%) other illicit drug use. 6 (15%) pts reported undescended testis and 1 (3%) hypospadias. 24/43 (45%) patients harbored ≥1 variant within 15 Litchfield genes (60%). 43 distinct variants were identified: 33 (77%) nonsynonymous missense alterations, 7 (16%) intron variants, 1 inframe deletion in STH3TC1, and 1 initiator and 1 stop codon variant in DEFB132, which plays a role in binding spermatozoa. Multiple bilateral patients harbored variants in MPDZ (n = 7), EHBP1L1 (n = 6), SKIV2L (n = 5), FAM160A2 (n = 3), ADAMTS18 (n = 3), JMJD4 (n = 3), ABCC4 (n = 3), R3HCC1 (n = 2), MLXIP (n = 2) and VPS16 (n = 2). Conclusions: In this bilateral GCT cohort, we identified several alterations in candidate susceptibility genes from the Litchfield series, whereas no convincing causative recurrent environmental exposures were observed. Our results support a polygenic model of GCT pathogenesis and show that analyzing enriched cohorts such as bilateral GCT may aid understanding of GCT pathogenesis. Additional analysis of the specific variants is required to further assess pathogenicity, elucidate their role and association with bilateral GCT.