Germline mutations in known colorectal cancer (CRC) predisposing genes accounts for less than half of the genetic cause of hereditary CRC cases. The absence of mutations in the remaining patients suggests that other yet unknown CRC predisposing genes might exist. However, recently studies with large cohorts probably discounts the existence of major unknown genes and it is thought that rare or even family private variants and genes may contribute to familial CRC. Thus, the aim of our study was to identify novel CRC susceptibility genes by whole exome sequencing (WES) of a family presenting hereditary CRC. The studied family fulfilled Amsterdam I criteria and appears to segregate a dominant cancer syndrome with a high risk of colon and rectal cancer, with four affected members in three generations. The index patient’s tumor was microsatellite stable and positive for the four mismatch repair proteins at immunohistochemistry. Genetic testing of the proband was negative for the following genes: MLH1, MSH2, MSH6, PMS2, PMS1, TP53, CHEK2. We performed WES in three family members: two with rectal cancer (onset 26 and 56 years) and one with colon cancer (onset 63 years). Using a functional-based variant prioritization, we identified six novel or very rare (MAF<0.002) candidate variants segregating with the phenotype. Two variants were loss-of-function stop codon variants in LSG1 and RRH genes. Three variants were missense (ARID1A, MC1R and SPEN), and three were synonymous (KIT, POLE and XAB2), all occurring in cancer-related genes. The identified KIT variant (c.1983C>A) is located 8 base pairs from the 3’ end of exon 13, and is predicted it to disrupt an exonic splicing enhancer by in silico tools (ESR finder and Human Splice Finder), possibly affecting the splice of this gene. Pyrosequencing of the KIT variant in tumor cDNA revealed an allelic imbalance between mutated and wild type alleles, with the mutated allele being three times more expressed. Transcript analysis using a customized heteroduplex enrichment-based method identified a novel KIT transcript in the index patient’s tumor, presenting an exon skipping event of exon 12. The resulting transcript (c.1775_1879del) is predicted to produce an in-frame deletion of 35 amino acids (p.Gly592_Pro627delinsAla) at the beginning of the protein kinase domain. The functional impact of this transcript and its relevance for CRC predisposition will be further evaluated. Moreover, experiments evaluating allelic imbalance, loss of heterozygosity and alternative spliced transcripts for the other identified variants are ongoing. Our results provide a set of putative CRC predisposing genes and underpin that WES of highly informative CRC families, ascertained on the basis of highly selected phenotypes and followed by functional validation, are likely to continue to prove an effective strategy for gene discovery.

Citation Format: Giovana T. Torrezan, Anna Smirnova, Felipe C. Silva, Erika M. Santos, Renan Valieris, Jorge E. de Souza, Samuel Aguiar, Benedito M. Rossi, Sandro J. de Souza, Dirce M. Carraro. Identification of putative cancer susceptibility genes through whole exome sequencing of a family presenting hereditary colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4281. doi:10.1158/1538-7445.AM2017-4281