Whole exome sequencing analysis of novel rare variants in susceptibility and unprecedented genes of hereditary colorectal cancer
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High-penetrance mutations lead to predisposition to colorectal cancer (CRC), comprising up to 5% of all colorectal cancer cases, e.g., defects in the DNA mismatch (MMR) genes causing Lynch syndrome. With the advent of next-generation sequencing, the scope of classification of hereditary colorectal cancer has been broadened by better understanding of molecular mechanisms and discoveries of new high-penetrance hereditary genes involved in the disease and a high number of candidate genes for hereditary colorectal cancer. Even so, the cause of pathogenesis of hereditary colorectal cancer in many cases remains unsolved. Identification and characterisation of rare pathogenic variants in known and novel genes may elucidate the enigmatic cause of cases unexplained by the conventional classification of hereditary colorectal cancer. Based on the knowledge that approximately 85% of disease-causing mutations occur in the protein coding region, i.e., the exome, this study aimed to discover and identify novel rare variants in established colorectal cancer susceptibility genes and in unprecedented genes that could be proposed as candidate genes for hereditary colorectal cancer, using whole-exome sequencing (WES) analysis approach. Germline DNA samples were obtained from 48 Australian participants diagnosed with colorectal cancer or whose condition fulfils the Amsterdam II Criteria for Lynch syndrome, including 16 individuals comprising 8 families whilst 32 individuals are unrelated. No germline MMR mutations were found in the samples by Sanger sequencing. All VCF files of 48 samples generated from whole-exome sequencing data were annotated using Filtus, a filtering tool combined with various prediction tools including FATHMM, SIFT, LRT, MutationAssessor, M-CAP, MutationTaster, and PROVEAN. To find novel rare pathogenic variants in MMR genes causing Lynch syndrome, variants were filtered out with a MAF<0.01. From 1,22,770 unique variants in 17,959 genes of raw data, 11,431 variants in 7,218 genes were collected within the range of a MAF<0.01. And 9 rare variants in MMR genes were identified. In other CRC susceptibility genes, 13 unique variants were detected. And these 22 variants were assessed for their pathogenicity and classified according to the ACMG/AMP variant classification guidelines. A nonsense mutation, c.2228C>G p.(Ser743Ter) in MSH2 and a missense variant, c.1A>G p.(Met1Val) in PMS2 were classified as class 5, pathogenic variants. A non-frameshift deletion variant, c.3334_3336del p.(Arg1112del) in MSH6 and a missense variant, c.614A>C p.(Gln205Pro) in PMS2 were identified as class 4, likely pathogenic variants in MMR genes, whereas no pathogenic variants were found in other known CRC susceptibility genes. 12 of 13 variants in other CRC susceptibility genes were classified as class 3, variants of uncertain significance whilst a synonymous variant was identified as class 2, a likely benign variant. To discover novel rare pathogenic variants in genes, this study focused on protein truncating variants including splice site, nonsense, frameshift indel variants with a MAF<0.001. 104 protein truncating variants were collected with the stringent filters applied to Filtus. Among 16 individuals comprising 8 families, 4 novel rare variants were identified in genes not previously reported to be associated with hereditary CRC in four different families; FBN3, KIAA1109, TUBB8, and LAMA4. Among genes harbouring protein truncating mutations, this study prioritised genes involved in development of disease with a loss-of-function mutation. TUBB8 has been reported to be involved in disease with overexpression whereas other three genes may have the pathogenic potential of developing a disease with loss of function. Based on the function of the three genes and their association with other diseases albeit insufficient information documented, this study suggests FBN3, KIAA1109, and LAMA4 as potential candidate genes for hereditary CRC. However, further studies are required to reveal the association between the genes and the development of hereditary CRC. After functional studies, the surviving candidate genes among them may contribute to the colorectal cancer comprehensive panel and to better risk assessment and therapeutics of hereditary colorectal cancer.