Epigenetic inactivation of a cluster of genes flanking MLH1 in microsatellite-unstable colorectal cancer.

Biallelic promoter methylation and transcriptional silencing of the MLH1 gene occurs in the majority of sporadic colorectal cancers exhibiting microsatellite instability due to defective DNA mismatch repair. Long-range epigenetic silencing of contiguous genes has been found on chromosome 2q14 in colorectal cancer. We hypothesized that epigenetic silencing of MLH1 could occur on a regional scale affecting additional genes within 3p22, rather than as a focal event. We studied the levels of CpG island methylation and expression of multiple contiguous genes across a 4 Mb segment of 3p22 including MLH1 in microsatellite-unstable and -stable cancers, and their paired normal colonic mucosa. We found concordant CpG island hypermethylation, H3-K9 dimethylation and transcriptional silencing of MLH1 and multiple flanking genes spanning up to 2.4 Mb in microsatellite-unstable colorectal cancers. This region was interspersed with unmethylated genes, which were also transcriptionally repressed. Expression of both methylated and unmethylated genes was reactivated by methyltransferase and histone deacetylase inhibitors in a microsatellite-unstable colorectal carcinoma cell line. Two genes at the telomeric end of the region were also hypermethylated in microsatellite-stable cancers, adenomas, and at low levels in normal colonic mucosa from older individuals. Thus, the cluster of genes flanking MLH1 that was specifically methylated in the microsatellite-unstable group of cancers extended across 1.1 Mb. Our results show that coordinate epigenetic silencing extends across a large chromosomal region encompassing MLH1 in microsatellite-unstable colorectal cancers. Simultaneous epigenetic silencing of this cluster of 3p22 genes may contribute to the development or progression of this type of cancer.

MLH1 germline epimutations as a factor in hereditary nonpolyposis colorectal cancer.

BACKGROUND & AIMS: Hereditary nonpolyposis colorectal cancer (HNPCC) is caused by heterozygous germline sequence mutations of DNA mismatch repair genes, most frequently MLH1 or MSH2. A novel molecular mechanism for HNPCC has recently been suggested by the finding of individuals with soma-wide monoallelic hypermethylation of the MLH1 gene promoter. In this study, we determined the frequency and role of germline epimutations of MLH1 in HNPCC. METHODS: A cohort of 160 probands from HNPCC families who did not harbor germline sequence mutations in the mismatch repair genes were screened for methylation of the MLH1 and EPM2AIP1 promoters by combined bisulfite and restriction analyses. Allelic expression and family transmission of MLH1 were determined using polymorphisms in intron 4 and the 3' untranslated region. RESULTS: One of 160 individuals had monoallelic MLH1 hypermethylation in peripheral blood, hair follicles, and buccal mucosa, indicative of a soma-wide alteration. Monoallelic transcription of the paternal MLH1 allele was shown using a heterozygous expressed polymorphism within the 3' untranslated region. The hypermethylated allele was maternally transmitted, however, the mother and siblings who inherited the same maternal homologue were unmethylated at MLH1, suggesting the epimutation arose as a de novo event. CONCLUSIONS: Germline MLH1 epimutations are functionally equivalent to an inactivating mutation and produce a clinical phenotype that resembles HNPCC. Inheritance of epimutations is weak, so family history is not a useful guide for screening. Germline epimutations should be suspected in younger individuals without a family history who present with a microsatellite unstable tumor showing loss of MLH1 expression.