Detection of allelic imbalance in MLH1 expression by pyrosequencing serves as a tool for the identification of germline defects in Lynch syndrome.

Lynch syndrome is an autosomal dominant cancer susceptibility syndrome characterized by the early development of microsatellite unstable colorectal, endometrial and other cancers. Lynch syndrome is caused by germline heterozygous loss-of-function sequence mutations within the mismatch repair genes MLH1, MSH2, MSH6 or PMS2. Some individuals with Lynch syndrome have constitutional epimutations, characterized by promoter methylation and transcriptional inactivation of a single allele in normal somatic tissues, while others lack identifiable pathogenic changes in the germline. We hypothesized that analysis of the relative levels of allelic expression of MLH1 would assist in the identification of cryptic pathogenic defects of MLH1 in five presumed Lynch syndrome cases whose tumours demonstrated MLH1 loss, but whose causative mutation remained unidentified. We exploited the common benign c.655A>G SNP (rs1799977) within MLH1 exon 8 to distinguish between the two genetic alleles in heterozygous individuals and to study their transcriptional activity, using quantitative pyrosequencing assays. In one of the five patients we detected loss of expression of one allele and deletion of the other allele in the tumour, prompting renewed germline screening. A novel intronic splice mutation was subsequently identified, which resulted in loss of an entire exon from the transcript. This pyrosequencing assay also proved useful in demonstrating the gradual reversal of a constitutional MLH1 epimutation during lymphoblastoid cell culture, suggesting this defect may not be stably maintained in immortalized cells. Our findings illustrate that the study of allelic behaviour can complement conventional molecular analyses by providing new insight into the genetic or epigenetic mechanisms underlying disease.