Molecular genetic studies of colorectal cancer

Colorectal cancer (CRC) is one of the leading causes of morbidity and mortality due to cancer in the Western countries. Epidemiological studies have shown that at least 20-30% of CRCs have a potentially identifiable genetic cause. Inherited forms of CRC with Mendelian dominant inheritance have been a major focus of study and many high-penetrance susceptibility genes have been identified, such as APC, hMLH1, hMSH2. However these high-penetrance susceptibility genes can only account for 3%-5% of all CRCs, thus additional, so far unknown, genes for CRC predisposition remain to be identified. Recent evidence has shown that a large proportion of genetic variation in CRC risk is probably due to a combination of a large number of common alleles with low penetrance and shared environmental factors. So in order to plot the accurate genetic-risk profiles of CRC for future population-based diagnosis and prevention programs, it is as equally important to identify low penetrance alleles as to identify high penetrance alleles. In this thesis, we evaluated the contribution of, hMLH3, APC and MYH as either high- or low- penetrance susceptibility genes for CRC in the Swedish population by the approaches of mutation screening and association study. hMLH3, is a newly identified DNA Mismatch Repair (MMR) gene interacting with hMLH1 and suggested to be a candidate gene for HNPCC. We screened 70 index patients suggestive of a genetic predisposition for germline mutations in the gene, and found one frameshift and 11 missense variants in 16 index patients (23%). The only frameshift mutation found, 885delG, seemed to segregate very well with cancer in the family as a monogenic high-penetrance allele. All the missense variants identified were not shown to segregate with cancer in the families, so hMLH3 mutations were not important as a major predisposing factor for familial CRC. However, in one family, we observed a co-segregation of one hMLH3 missense variant and one hMSH2 missense variant with the disease phenotype, suggesting that hMLH3 could work as a low-penetrance predisposing gene for CRC by working together with other low-penetrance genes in an additive or multiplicative manner. (Paper I) The recent finding that one missense variant in APC (11307K) led to an increased CRC risk in Askenazi Jews raises the possibility that there should exist other similar predisposing variants in this gene in different populations. We tested this possibility by performing the mutation screening of APC in cohorts of Swedish hereditary CRCs, sporadic CRCs and normal controls. Our results showed that some APC variants other than 11 307K could act as low-penetrance alleles for CRC, for example, the variant 3´UTR 8636C>A. This variant was found to have an increased CRC risk of around two fold-a risk comparable to that of I1307K-though the association studies had statistically significant results only at the borderline (Paper II). In addition, the 3´UTR 8636C>A variant and the APC locus it implicates were incidentally found to be associated with autism spectrum disorder (ASD), substantiated by several follow-up association studies with statistically significant results. (Paper IV). MYH plays a significant role in the repair of mutations caused by 8-oxo-7,8-dihydroxy-2-deoxyguanosine (8-oxoG), one of the most stable product of oxidative DNA damage. Biallelic germline mutations of this gene have been shown to predispose to a proportion of multiple colorectal adenomas and cancer. We screened 84 unrelated Swedish non-FAP and non-HNPCC familial CRCs for germline mutations in the gene to evaluate the contribution of MYH mutations in CRC with few polyps. None of the cases was found to carry any pathogenic sequence change, indicating that MYH mutations are not likely to account for familial CRCs in the absence of multiple adenomas. However, we could demonstrate that the two most common pathogenic variants Y165C and G382D found in Caucasians existed in the Swedish population as well, and both of them appeared to confer a slightly increased CRC risk in heterozygosity. Additional candidate low-penetrance alleles for CRC were also identified at the position of amino acid 423 (R423Q, R423P, and R423R). Whether mutations at position 423 have pathological relevance needs to be further studied. (Paper III) Recently, there is increasing evidence to indicate that hereditary and sporadic MSI cancers evolve through different pathways. We wondered if microarray technology can help differentiate these two tumors into two distinct entities. We transcriptionally profiled six HNPCC and seven sporadic MSI CRCs using Affymetrix Microarray HG-U95Av2 chips. Unsupervised hierarchical clustering analysis and principle component analysis (PCA) failed to distinguish hereditary MSI tumors from its sporadic counterparts, indicating that the molecular difference between these two phenotypes, if any, is insignificant. In addition, a gene called Tight Junction Protein 3 (TJP3), a novel member of the MAGUK protein family found at the cell tight junction, was shown to be downregulated by about 2 fold in transcription in a small subset of HNPCC tumors with suggested bad prognosis compared with other samples, and in a MSI cell line with high metastatic potential compared with its isogenic cell line with low metastatic potential. Therefore, TJP3 would be a metastasis-associated gene in CRCs with MSI, which warrants further functional characterization. (Paper V