Identification of novel germline mutations in hereditary colorectal cancer patients and characterization of somatic alterations in their tumors

Colorectal cancer has been reported as the third leading cause of cancer related death in the world. About 5-10% of colorectal cancers are due to an inherited predisposition. This thesis focuses on investigating the prevalence of large genomic rearrangements and other types of germline mutations in novel cancer susceptibility genes in two major hereditary colorectal cancer syndromes, hereditary nonpolyposis colorectal cancer (HNPCC) and familial adenomatous polyposis (FAP). Furthermore, the second somatic mutations were characterized in the tumors from DNA mismatch repair (HNPC) and APC gene mutation carriers (FAP) to address the mechanism(s) of tumorigenesis in these two syndromes. All these investigations aim to understand tumor initiation and progression in hereditary colorectal cancer syndromes in order to enable early and reliable presymptomatic diagnosis of a person at increased risk and offer optimal medical management to prevent cancer. HNPCC is an autosomal dominantly inherited cancer predisposition syndrome caused by germline mutations in DNA mismatch repair (MMR) genes. Prescreening methods are routinely applied to detect MMR gene sequence alterations, but inevitably miss large genomic rearrangements. Here, two novel PCR-based methods to study gene dosage were introduced in 35 MLH/MSH2 HNPCC patients in whom no mutation could be identified by conventional screening methods. These methods are QMPA (quantitative multiplex PCR amplification) and MLPA (multiplex ligation dependent probe amplification). Three patients were found to carry large deletions by QMPA and MLPA. In 1 patient, however, QMPA yielded a false positive result. Both methods, QMPA and MLPA appear to be of comparable sensitivity albeit with different specificity. Since the QMPA technique is difficult to set up and to standardize the PCR conditions, the MLPA assay is better suited to routinely search for large genomic rearrangements. The investigations subsequently continued to detect the frequency and nature of LOH as second, somatic event in tumors from MLH/MSH2 germline deletion carriers. MLPA technique was applied to analyze 18 cancer specimens from two independent sets of Swiss and Finnish MLH1/MSH2 deletion carriers. Results revealed that somatic deletions identical to the ones in the germline occur frequently (55%) in CRCs and that this type of loss of the wild type allele is also present in extracolonic HNPCC associated tumors. Chromosome specific marker analysis implies that loss of the wild type allele predominantly occurs through locus restricted recombination events, i.e. gene conversion, rather than mitotic recombination or deletion of the respective gene
locus. The same investigation was carried on a 31 years old colorectal cancer patient who carries de novo mutation (c.666dupA) in the MLH1 gene. The tumor analysis of this patient showed a similar somatic mutation mechanism to the large genomic deletion carriers. Prior to our analysis of the somatic hits in the attenuated form of familial adenomatous polyposis (AFAP), earlier investigations had shown that in classical FAP the "two hits" in the APC (Adenomatosis polyposis coli) gene are not occurring randomly but are in fact interdependent. AFAP is clinically characterized by fewer than 100 adenomatous polyps in the colorectum and presents with a milder phenotype compared to classical FAP. APC mutations in AFAP patients are typically located in the very 5’ and 3’ gene regions as well as in the alternatively spliced region of exon 9. In a collaborative effort we investigated the somatic alterations in 235 tumors of 35 AFAP patients. Adenomas of AFAP patients were often found to actually exhibit ‘three hits’ at the APC gene that mostly result in loss of the allele carrying the germline APC mutation. We assume that this actually leads to an optimization of the beta-catenin level, hence positively regulating the Wnt signal. Recently, bi-allelic germline mutations in the base excision repair gene MutY homologue (MYH) have been associated with an autosomal recessively inherited predisposition to multiple colorectal adenomas. They are also referred to as MYH-associated polyposis (MAP). Here, we assessed the prevalence of MYH germline alteration in 79 unrelated polyposis patients in whom no APC mutation could be detected. The aims of the study were i) to assess the MYH mutation carrier frequency among Swiss APC mutation negative patients and (ii) to identify phenotypic differences between MYH mutation carriers and APC / MYH mutation negative polyposis patients. dHPLC and direct genomic DNA sequencing were applied to screen for mutation. Overall, 7 biallelic and 9 monoallelic MYH germline mutation carriers were identified. 1 out of 10 classical polyposis and 6 out of 35 attenuated polyposis patients carried biallelic MYH alterations, 2 of which represent novel gene variants (p.R 171q and p.R 231H). On the basis of our finding and earlier reports, MYH mutation screening should be considered if all of the following criteria are fulfilled: (1) presence of classical or attenuated polyposis coli, 2) absence of a pathogenic APC mutation, and 3) a family history compatible with an autosomal recessive mode of inheritance.