Genetic Variants That Predispose to DNA Double-Strand Breaks in Lymphocytes From a Subset of Patients With Familial Colorectal Carcinomas.
OBJECTIVE
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.
Cancer Biology Program, Fox Chase Cancer Center, Philadelphia, Pennsylvania; Molecular Therapeutics Program, Fox Chase Cancer Center, Philadelphia, Pennsylvania.
Sanjeevani
S
Arora
Cancer Biology Program, Fox Chase Cancer Center, Philadelphia, Pennsylvania.
Hong
H
Yan
Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania.
Iltaeg
I
Cho
Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania.
Hua-Ying
HY
Fan
Institute for Personalized Medicine, Fox Chase Cancer Center, Philadelphia, Pennsylvania.
Biao
B
Luo
Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, Massachusetts.
Xiaowu
X
Gai
Inova Translational Medicine Institute, Inova Health System, Falls Church, Virginia.
Dale L
DL
Bodian
Inova Translational Medicine Institute, Inova Health System, Falls Church, Virginia.
Joseph G
JG
Vockley
Biostatistics, Fox Chase Cancer Center, Philadelphia, Pennsylvania.
Yan
Y
Zhou
Biostatistics, Fox Chase Cancer Center, Philadelphia, Pennsylvania.
Elizabeth A
EA
Handorf
Molecular Therapeutics Program, Fox Chase Cancer Center, Philadelphia, Pennsylvania; Biostatistics, Fox Chase Cancer Center, Philadelphia, Pennsylvania.
Brian L
BL
Egleston
Molecular Therapeutics Program, Fox Chase Cancer Center, Philadelphia, Pennsylvania.
Mark D
MD
Andrake
Genomics, Fox Chase Cancer Center, Philadelphia, Pennsylvania.
Emmanuelle
E
Nicolas
Molecular Therapeutics Program, Fox Chase Cancer Center, Philadelphia, Pennsylvania; Kazan Federal University, Kazan, Russia.
Ilya G
IG
Serebriiskii
Cancer Biology Program, Fox Chase Cancer Center, Philadelphia, Pennsylvania.
Timothy J
TJ
Yen
Medical Oncology, Fox Chase Cancer Center, Philadelphia, Pennsylvania; Clinical Genetics, Fox Chase Cancer Center, Philadelphia, Pennsylvania.
Michael J
MJ
Hall
Molecular Therapeutics Program, Fox Chase Cancer Center, Philadelphia, Pennsylvania. Electronic address: erica.golemis@fccc.edu.
Erica A
EA
Golemis
Cancer Biology Program, Fox Chase Cancer Center, Philadelphia, Pennsylvania; Medicine, Fox Chase Cancer Center, Philadelphia, Pennsylvania. Electronic address: greg.enders@fccc.edu.
Greg H
GH
Enders
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D014408:Biomarkers, Tumor / Q000235:genetics* / Q000378:metabolism; D016022:Case-Control Studies; D015179:Colorectal Neoplasms / Q000235:genetics* / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D019295:Computational Biology; D053903:DNA Breaks, Double-Stranded; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism; D004260:DNA Repair; D045643:DNA Repair Enzymes / Q000235:genetics / Q000378:metabolism; D030541:Databases, Genetic; D005090:Exodeoxyribonucleases / Q000235:genetics / Q000378:metabolism; D059472:Exome; D005260:Female; D005787:Gene Frequency; D055785:Gene Knockdown Techniques; D020022:Genetic Predisposition to Disease; D014644:Genetic Variation; D042822:Genomic Instability; D045325:HCT116 Cells; D040941:Heredity; D006657:Histones / Q000378:metabolism; D006801:Humans; D008297:Male; D008875:Middle Aged; D009153:Mutagens / Q000494:pharmacology; D010641:Phenotype; D010766:Phosphorylation; D000075223:Poly-ADP-Ribose Binding Proteins; D053484:RecQ Helicases / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013601:T-Lymphocytes / Q000187:drug effects / Q000276:immunology / Q000378:metabolism / Q000473:pathology*; D014162:Transfection; D015854:Up-Regulation; D000071657:Werner Syndrome Helicase
D014408:Biomarkers, Tumor; C468783:H2AX protein, human; D006657:Histones; D009153:Mutagens; D000075223:Poly-ADP-Ribose Binding Proteins; D005090:Exodeoxyribonucleases; D004265:DNA Helicases; C078673:ERCC6 protein, human; D053484:RecQ Helicases; C098785:WRN protein, human; D000071657:Werner Syndrome Helicase; D045643:DNA Repair Enzymes
Colon Cancer; Genomic Instability; Hereditary Cancer; Tumorigenesis