Genetic Variants That Predispose to DNA Double-Strand Breaks in Lymphocytes From a Subset of Patients With Familial Colorectal Carcinomas

Abstract

BACKROUND & AIMS DNA structural lesions are prevalent in sporadic colorectal cancer, so we proposed that gene variants that predispose to DNA double-strand breaks (DSBs) would be found in patients with familial colorectal carcinomas of an undefined genetic basis (UFCRC). METHODS We collected primary T cells from 25 patients with UFCRC and matched patients without colorectal cancer (controls) and assayed for DSBs. We performed exome sequence analyses of germline DNA from 20 patients with UFCRC and 5 undiagnosed patients with polyposis. The prevalence of identified variants in genes linked to DNA integrity was compared to that of individuals without a family history of cancer. The effects of representative variants found to be associated with UFCRC was confirmed in functional assays with HCT116 cells. RESULTS Primary T cells from most patients with UFCRC had increased levels of the DSB marker γH2AX following treatment with DNA damaging agents, compared to T cells from controls (P<.001). Exome sequence analysis identified a mean 1.4 rare variants/patient that were predicted to disrupt functions of genes relevant to DSBs. Controls (from public databases) had a much lower frequency of variants in the same genes (P<.001). Knockdown of representative variant genes in HCT116 CRC cells increased γH2AX. Detailed analysis of immortalized patient-derived B cells, which contained variants in the Werner syndrome, RecQ helicase-like gene (WRN, encoding T705I), and excision repair cross-complementation group 6 (ERCC6, encoding N180Y), revealed reduced levels of these proteins and increased DSBs, compared to B cells from controls. This phenotype was rescued by exogenous expression of WRN or ERCC6. Direct analysis of the recombinant variant proteins confirmed defective enzymatic activities. CONCLUSIONS These results provide evidence that defects in suppression of DSBs underlie some cases of UFCRC; these can be identified by assays of circulating lymphocytes. We specifically associated UFCRC with variants in WRN and ERCC6 that reduce capacity for repair of DNA DSBs. These observations could lead to a simple screening strategy for UFCRC, and provide insight into the pathogenic mechanisms of colorectal carcinogenesis.

Implication of DNA repair genes in Lynch-like syndrome

“…Moreover, statistical inference indicates that the association is significant as the overall mutational burden of putatively pathogenic variants in our LLS patients was high whereas in the general population is low. Of note, several recently published studies have identified likely pathogenic variants in other genes that maintain DNA integrity, including NTHL1, FAN1, and MCM9 in cases with familial CRC with unknown genetic basis [25][26][27][28] and mutations in BLM in cases with earlyonset CRC [29].…”

Synthetic Lethal Targeting of Mitotic Checkpoints in HPV-Negative Head and Neck Cancer

“…Such an effect might explain the markedly greater activity of the drug combination in the longer-term clonogenic assays than the shorter cell viability assays. To address this possibility, we analyzed levels of γ-H2AX, a phosphorylated histone mark associated with unrepaired DNA breaks [27], in cells treated with vehicle, single drugs, or the pre:ada combinations for 72 h (Figure 4a and Figure S3a). This showed a significant increase in phospho-H2AX signal in cells treated with the drug combination versus single drugs, even at low overall drug concentrations.…”

POLD1: Central mediator of DNA replication and repair, and implication in cancer and other pathologies

“…Families bearing the p.S478N variant in POLD1/p125 develop microsatellite stable, chromosomal unstable colorectal adenocarcinoma and/or oligopolyposis with high penetrance and dominant inheritance ( Palles et al, 2013 , Valle et al, 2014 ). We have described this variant in a patient with familial colorectal cancer, but not bearing mutations in canonical genes associated with risk ( Arora et al, 2015 ). A POLD1 p.L474P proofreading domain variant has also been identified in a patient with hereditary non-polyposis colorectal cancer and deemed pathogenic by evidence from co-segregation, in silico predictions of functionality and functional assay in yeast.…”