Genetic etiology of hereditary colorectal cancer: new mechanisms and advanced mutation detection techniques

Abstract

The human DNA mismatch repair (MMR) system functions to repair mispaired bases in DNA that result from DNA replication errors and thereby prevents the accumulation of mutations due to such replication errors. Hereditary nonpolyposis colorectal cancer (HNPCC), the most common form of inherited colon cancer, is associated with loss of MMR function. HNPCC is an autosomal dominant predisposition to early-onset colorectal cancer, often associated with an increased risk of extracolonic malignancies. Tumors are characterized by a high frequency of replication errors in short tandem repeats, known as microsatellite instability (MSI). The increased risk of developing cancer in HNPCC patients is nearly always due to an inherited mutation in a MMR gene, in most cases MSH2 or MLH1. Inactivation of the remaining copy of the gene by somatic genetic alteration results in a mutator phenotype. The most prevalent types of inherited genetic alterations in HNPCC are point mutations, deletions, insertions and genomic deletions. This thesis reports two novel types of germline genetic alterations of MMR genes in HNPCC patients. The first one involves methylation of the promoter region of the MLH1 gene. In one out of 14 HNPCC cases with high microsatellite instability (MSI-H) and mutation-negative for MMR genes, DNA hypermethylation of one allele was detected in DNA isolated from blood. In the MSI-H tumor from this case, the unmethylated MLH1 allele was eliminated by loss of heterozygosity and the methylated allele was retained. This biallelic inactivation resulted in loss of expression of MLH1 in the tumor as confirmed by immunohistochemistry. This suggests a novel mechanism of tumorigenesis that nonetheless fits the two-hit tumor suppressor gene inactivation hypothesis (Knudson hypothesis). The second novel genetic alteration involves single nucleotide polymorphisms (SNPs) in the regulatory areas of the MSH6 gene. Two SNPs were identified in the consensus sequence of two Sp1 transcription factor binding sites of the MSH6 promoter, resulting in altered promoter activity. Functional analysis showed that the MSH6 promoter contains seven functional putative Sp1 transcription factor binding sites, each bind Sp1 and Sp3 and all contribute to promoter activity. Analysis of the two mutant sites showed that the changes eliminated the Sp1 binding sites and were no longer bound by Sp1 and Sp3 transcription factors, resulting in reduced MSH6 expression at both the mRNA and protein level. In addition, the promoter with the two mutant sites was more sensitive to silencing by DNA methylation in vitro. This suggests that the polymorphic allele could be a preferential target of de novo methylation, resulting in transcriptional silencing of the gene.