Multiplex Ligation-Dependent Probe Amplification (MLPA) detects deletions and duplications of single and multiple exons within a given gene (PMID: 20938835). It is a semi-quantitative technique to determine relative copy number using a multiplex PCR-based reaction. Only hybridized and ligated adjacent probe oligonucleotides are amplified by PCR, thus specific for the sequence of interest. This test involves a copy number analysis of the PMS2 gene.

• Diagnostic testing for Individuals with Lynch syndrome or a family history of Lynch syndrome
• Familial testing for individuals with a family history of known copy number changes in the PMS2 gene

Lynch syndrome (LS, MIM# 120435; formerly known as HNPCC, Hereditary Nonpolyposis Colorectal Cancer) is the most common heritable colorectal carcinoma (CRC) syndrome and is responsible for 2% to 4% of all CRC cases in the Western world. Heterozygous germline mutations in one of the mismatch repair (MMR) genes MSH2 (MIM# 609309), MLH1 (MIM# 120436), MSH6 (MIM# 600678), or PMS2 (MIM# 600259) are responsible for Lynch syndrome (PMID: 20301390). Another cause of Lynch syndrome is the deletion of the 3’ part of EPCAM, leading to constitutional epigenetic silencing of the downstream MSH2 gene. The estimated contribution of the different genes to Lynch syndrome is 15-40% for MLH1, 20-40% for MSH2, 12-35% for MSH6, 5-25% for PMS2 and <10% for EPCAM (PMID: 20301390).

The PMS2 gene provides instructions for making a protein essential to repairing DNA. This protein helps fix errors made when DNA is copied (replication) in preparation for cell division. The PMS2 protein joins with MLH1 to form a protein complex, which coordinates the activities of other proteins that repair errors made during DNA replication. Genetic testing for mutations in PMS2 is greatly complicated by numerous unprocessed PMS2 pseudogenes and segmental duplications containing unspliced segments of the gene (PMID: 8586419). There are 14 pseudogenes containing exons 1 to 5 in various compositions on the long arm of chromosome 7, and one highly homologous copy (PMS2CL) encompassing the 3' end of PMS2, that is, exon 9 and exons 11 to 15, embedded in a 100 kb inverted duplicon, 700 kb centromeric of PMS2 on chromosomal 7p. Among the defects in the PMS2 gene are deletions and duplications of complete exons, which are usually missed by standard sequence analysis. The MLPA technique can detect most of these deletions and duplications except for CNV in the PMS2 exon 12-15 region and therefore complements sequence analysis of the PMS2 gene.

MLPA-based assay for the detection of deletions/duplications in the PMS2 gene. The MRC-Holland reagent kits and the Applied Biosystem 3730xl DNA Analyzer instrument generate fragment analysis data.