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

Sanjeevani Arora, Hong Yan, Iltaeg Cho, Hua Ying Fan, Biao Luo, Xiaowu Gai, Dale L. Bodian, Joseph G. Vockley, Yan Zhou, Elizabeth Handorf, Brian L. Egleston, Mark Andrake, Emmanuelle Nicolas, Ilya G. Serebriiskii, Timothy J. Yen, Michael J. Hall, Erica A. Golemis, Greg H. Enders

Research output: Contribution to journalArticlepeer-review

29 Scopus citations

Abstract

BACKGROUND & AIMS: DNA structural lesions are prevalent in sporadic colorectal cancer. Therefore, 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 with 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 γ(phosphorylated)histone2AX (γH2AX) after treatment with DNA damaging agents, compared with T cells from controls (P < .001). Exome sequence analysis identified a mean 1.4 rare variants per 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. A detailed analysis of immortalized patient-derived B cells that contained variants in the Werner syndrome, RecQ helicase-like gene (WRN, encoding T705I), and excision repair cross-complementation group 6 (ERCC6, encoding N180Y) showed reduced levels of these proteins and increased DSBs, compared with 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 the 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.

Original languageEnglish
Pages (from-to)1872-1883.e9
JournalGastroenterology
Volume149
Issue number7
DOIs
StatePublished - Dec 1 2015

Keywords

  • Adult
  • Aged
  • Aged, 80 and over
  • Biomarkers, Tumor/genetics
  • Case-Control Studies
  • Colorectal Neoplasms/genetics
  • Computational Biology
  • DNA Breaks, Double-Stranded
  • DNA Helicases/genetics
  • DNA Repair
  • DNA Repair Enzymes/genetics
  • Databases, Genetic
  • Exodeoxyribonucleases/genetics
  • Exome
  • Female
  • Gene Frequency
  • Gene Knockdown Techniques
  • Genetic Predisposition to Disease
  • Genetic Variation
  • Genomic Instability
  • HCT116 Cells
  • Heredity
  • Histones/metabolism
  • Humans
  • Male
  • Middle Aged
  • Mutagens/pharmacology
  • Phenotype
  • Phosphorylation
  • Poly-ADP-Ribose Binding Proteins
  • RecQ Helicases/genetics
  • Sequence Analysis, DNA
  • T-Lymphocytes/drug effects
  • Transfection
  • Up-Regulation
  • Werner Syndrome Helicase

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