Abstract 4739: Genetic predisposition to DNA double strand break repair defect defines a new class of familial colon cancer
Authors: arora sanjeevani , Yan Hong , Cho Iltaeg , Fan Hua-Ying , Luo Biao , gai xiaowu , bodian dale , vockley joe , zhou yan , handorf elizabeth , andrake mark , nicolas emmanuelle , Serebriiskii Ilya , yen tim , hall michael , enders greg , golemis erica 
Published: August 2015

Abstract
Colorectal cancer (CRC) is the second most common fatal cancer in the U.S., with ∼ 30% cases familial (FCRC). Genetic predispositions have only been defined for 25% of FCRC (including Lynch syndrome, Familial Adenomatous Polyposis (FAP), and MutYH polyposis). For the genetically undefined group (uFCRC), understanding of disease pathogenesis is limited, and the absence of defined defects in relevant pathways that could support molecular diagnosis results in under- and over-screening. We hypothesized that uFCRC would arise distinctly from moderately penetrant germline defects in repair of DNA double-strand breaks (DSBs). Such defects in DSB repair would lead to a moderate level of constitutional genomic instability (CGI) that could be assayed from patient peripheral blood lymphocytes (PBLs). We further hypothesized that such DNA lesions would often be heterozygous, associated with haploinsufficiency or dominant-negative effects, and would impair protein interactions important for DNA repair.
We combined exome sequencing with biological assays to screen a rich resource of uFCRC clinical samples from the Fox Chase Cancer Center Risk Assessment Program. Exome sequencing of PBLs from patients (N = 25) identified a mean of 1.4 rare variants/patient that were 1) strongly predicted to damage protein function using multiple functional predictors (SIFT, Polyphen-2, Provean, and MutationAssessor), 2) involved in pathways that suppress DSBs, and 3) have not been previously implicated in CRC. Further, many of the variant genes were commonly mutated in CRCs and other cancers in TCGA database. Comparison of PBLs from patients and age- and sex- matched controls revealed increased DSB levels (basal and with DNA damaging agents) in 18/25 patients vs. 1/25 normal controls (P = 0.001, AUC = 0.85). Next, knockdown of variant genes in HCT116 CRC cells increased DSB formation with or without damaging agents.
As a proof-of-concept, we studied one patient in detail who had candidate disease-causing variants in 2 DNA repair genes, ERCC6 and WRN. Both variants were dysfunctional in biochemical tests. We established patient-derived EBV-lines and observed, at baseline and with several DNA damaging agents, elevated levels of γH2AX foci and larger nuclear comets, indicative of DSBs. This phenotype could be rescued by expression of wild type WRN or ERCC6 and conversely induced by knockdown in CRC cells. Further, the patient derived lines exhibited reduced expression of WRN and ERCC6, implicating haploinsufficiency of these genes in the phenotype observed.
Our data support the hypothesis that defects in DSB repair genes cause moderate-level CGI that commonly presents as uFCRC. Our studies identify novel genetic subsets of FCRC, and could potentially predict familial risk and improve screening and diagnosis.
Citation Format: sanjeevani arora, Hong Yan, Iltaeg Cho, Hua-Ying Fan, Biao Luo, xiaowu gai, dale bodian, joe vockley, yan zhou, elizabeth handorf, mark andrake, emmanuelle nicolas, Ilya Serebriiskii, tim yen, michael hall, greg enders, erica golemis. Genetic predisposition to DNA double strand break repair defect defines a new class of familial colon cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4739. doi:10.1158/1538-7445.AM2015-4739