RNA-Based Mutation Analysis Identifies an Unusual MSH6 Splicing Defect and Circumvents PMS2 Pseudogene Interference

Medical Genomics Laboratory, Department of Genetics, University of Alabama at Birmingham, Birmingham, AlabamaCommunicated by Riccardo FoddeHeterozygous germline mutations in one of the mismatch repair (MMR) genes MLH1, MSH2, MSH6, andPMS2 cause hereditary nonpolyposis colorectal cancer (HNPCC) or Lynch syndrome, a dominantly inheritedcancer susceptibility syndrome. Recent reports provide evidence for a novel recessively inherited cancersyndrome with constitutive MMR deficiency due to biallelic germline mutations in one of the MMR genes.MMR-deficiency (MMR-D) syndrome is characterized by childhood brain tumors, hematological and/orgastrointestinal malignancies, and signs of neurofibromatosis type 1 (NF1). We established an RNA-basedmutation detection assay for the four MMR genes, since 1) a number of splicing defects may escape detection bythe analysis of genomic DNA, and 2) DNA-based mutation detection in the PMS2 gene is severely hampered bythe presence of multiple highly similar pseudogenes, including PMS2CL. Using this assay, which is based ondirect cDNA sequencing of RT-PCR products, we investigated two families with children suspected to sufferfrom MMR-D syndrome. We identified a homozygous complex MSH6 splicing alteration in the index patients ofthe first family and a novel homozygous PMS2 mutation (c.182delA) in the index patient of the second family.Furthermore, we demonstrate, by the analysis of a PMS2/PMS2CL ‘‘hybrid’’ allele carrier, that RNA-basedPMS2 testing effectively avoids the caveats of genomic DNA amplification approaches; i.e., pseudogenecoamplification as well as allelic dropout, and will, thus, allow more sensitive mutation analysis in MMRdeficiency and in HNPCC patients with PMS2 defects. Hum Mutat 29(2), 299–305, 2008.