Germline Pathogenic Variants in Homologous Recombination and DNA Repair Genes in an Asian Cohort of Young-Onset Colorectal Cancer

Abstract

Background: Growing evidence suggests a role for cancer susceptibility genes such as BRCA2 and PALB2 in young-onset colorectal cancers. Using a cohort of young colorectal cancer patients, we sought to identify and provide functional evidence for germline pathogenic variants of DNA repair genes not typically associated with colorectal cancer.

Methods: We recruited 88 patients with young-onset colorectal cancers seen at a general oncology center. Whole-exome sequencing was performed to identify variants in DNA repair and colorectal cancer predisposition genes. Pathogenic BRCA2 and PALB2 variants were analyzed using immunoblot and immunofluorescence on patient-derived lymphoblastoid cells.

Results: In general, our cohort displayed characteristic features of young-onset colorectal cancers. Most patients had left-sided tumors and were diagnosed at late stages. Four patients had familial adenomatous polyposis, as well as pathogenic APC variants. We identified 12 pathogenic variants evenly distributed between DNA repair and colorectal cancer predisposition genes. Six patients had pathogenic variants in colorectal cancer genes: APC (n = 4) and MUTYH monoallelic (n = 2). Another six had pathogenic variants in DNA repair genes: ATM (n = 1), BRCA2 (n = 1), PALB2 (n = 1), NTHL1 (n = 1), and WRN (n = 2). Pathogenic variants BRCA2 c.9154C>T and PALB2 c.1059delA showed deficient homologous recombination repair, evident from the impaired RAD51 nuclear localization and foci formation.

Conclusion: A substantial portion of pathogenic variants in young-onset colorectal cancer was found in DNA repair genes not previously associated with colorectal cancer. This may have implications for the management of patients. Further studies are needed to ascertain the enrichment of pathogenic DNA repair gene variants in colorectal cancers.

Figure 1.

A) Variants identified in this study. B) Locations of pathogenic DNA repair variants. Red represents frameshift mutations; blue represens missense mutations. BRCA2 c.440A>G (p.Q147R) is a VUS included in the functional studies. CRC = colorectal cancer; VUS = variants of uncertain significance.

Figure 2.

A) Nuclear localization of RAD51 expressed as a ratio of nuclear (N) to cytoplasmic (C) RAD51 levels. Cells with BRCA2 c.9154C>T, PALB2 c.1059delA, and BRCA2 c.8945_8946delAA show impaired RAD51 nuclear localization at the first hour posttreatment, whereas those with BRCA2 c.440A>G displayed normal RAD51 nuclear localization. Triplicates were performed for patients with pathogenic variants. Three healthy controls were used and duplicates were done per control. BRCA2 c.8945_8946delAA was included as a positive control. Independent t test was used to compare RAD51 nuclear localization between variants and healthy controls. A single asterisk (*) refers to P < .05, a double (**) to P < .005. B) Representative blot showing changes in nuclear and cytoplasmic RAD51 levels at 1 hour and 6 hours following etoposide treatment. In the healthy controls, nuclear RAD51 was higher than the cytoplasmic RAD51 following treatment. In contrast, the nuclear RAD51 remained similar to the cytoplasmic RAD51 in BRCA2 c.9154C>T, PALB2 c.1059delA, and BRCA2 c.8945_8946delAA. DMSO = dimethyl sulfoxide.

Figure 3.

A) Immunofluorescence analysis of RAD51 foci formation (represented as green foci) following etoposide treatment. Compared to healthy controls, RAD51 foci formation was impaired for BRCA2 c.9154C>T, PALB2 c.1059delA, and BRCA2 c.8945_8946delAA. B) Percentage of cells with more than 5 RAD51 foci. Impaired RAD51 foci formation was noted for BRCA2 c.9154C>T, PALB2 c.1059delA, and BRCA2 c.8945_8946delAA at 6 hours following treatment. Triplicates were performed for patients with pathogenic variants. Duplicates were done per healthy control. BRCA2 c.8945_8946delAA was included as a positive control. Independent t test was used to compare RAD51 nuclear localization between variants and healthy controls. A single asterisk (*) refers to P < .05; a double (**) refers to P < .005. DMSO = dimethyl sulfoxide.