Identification of candidate genes predisposing to familial colorectal cancer by germline whole exome sequencing

Abstract

Abstract Colorectal cancer (CRC) is the third most common cause of cancer-related death worldwide. Approximately 5% of CRC is attributable to an inherited high-penetrance pathogenic variant in a known CRC-predisposing gene, often associated with a family history of CRC, a young onset of CRC and/or colonic polyposis. Identification of an inherited genetic variant predisposing to CRC in an individual with CRC enables predictive testing in blood relatives, and implementation of risk reduction strategies for individuals with the causative variant within the family. In individuals with familial or young onset CRCs/colonic polyposis in whom a causative germline variant in a known CRC-predisposing gene has not been identified, it is hypothesised that novel CRC-predisposing genes harbouring rare high risk germline variants remain to be discovered.

Germline whole exome sequencing (WES) offered a novel and comprehensive approach to identifying potential pathogenic variants in candidate CRC-predisposing genes. This thesis used WES of germline DNA in individuals with unexplained CRC and a high a priori risk of an inherited predisposition to CRC, to prioritise candidate CRC-predisposing genes that were subsequently re-sequenced in a validation cohort of young-onset CRC cases.

A discovery cohort was recruited comprising 54 individuals who met one or more of the following criteria: CRC and a family history of CRC (n=29), a diagnosis of CRC before 40 years of age (n=20), or a diagnosis of 20 or more bowel polyps (n=10). No known germline predisposition to CRC had been identified in these individuals through clinical testing. Rare (minor allele frequency of <0.5%) loss-of-function (nonsense, frameshift or essential splice site) variants or missense variants predicted to be deleterious using a Combined Annotation Dependent Depletion C Score of >10 were prioritised for analysis. Three individuals with pathogenic variants in known CRC-predisposing genes were identified and excluded from further analyses.

In the remaining 51 individuals, four separate analysis approaches were used to prioritise candidate CRC-predisposing genes containing prioritised variants: 1) genes with variants in multiple families, 2) genes with a shared variant in multiple family members, 3) genes within known CRC pathways, and 4) genes intolerant of loss-of-function variants. Forty-five candidate CRC-predisposing genes were prioritised using these analyses. Segregation analysis of potentially pathogenic variants within a family (n=1) and loss-of-heterozygosity studies in tumour tissue (n=4) were performed but hampered by lack of availability of samples.

Re-sequencing of the 45 candidate CRC-predisposing genes in a validation cohort of 333 individuals with unexplained young onset CRC identified seven genes (WNT10A, NEDD4, MS4A15, TNFRSF10B, LRRK2, INPP5E, UFL1) with a loss-of-function variant in 1 or more individuals. None of the seven genes harboured LoF variants in >1% of the validation cohort, indicating that a novel gene likely accounts for very few individuals with unexplained CRC and a high a priori risk of an inherited predisposition to CRC, pointing to a very high degree of genetic heterogeneity in such cases. Future studies will require very large numbers of cases and controls to confirm the association of these genes with CRC predisposition.