This panel includes both sequencing and deletion/duplication (CNV) analysis of the coding regions of the PMS2 gene. PMS2 gene sequencing is greatly complicated by numerous unprocessed PMS2 pseudogenes. For regions that might have pseudogene interference, long-range polymerase chain reaction (LR-PCR) followed by nested PCR and Sanger sequencing is used to accurately detect variants in the PMS2 gene. For regions without pseudogenes, PCR is used directly to amplify targeted regions followed by Sanger sequencing. Deletion/duplication (CNV) analysis is completed via multiplex ligation-dependent probe amplification (MLPA). All variants are classified according to American College of Genetics and Genomics (ACMG) guidelines.

Pathogenic mutations in the PMS2 gene are associated with Lynch syndrome, or hereditary nonpolyposis colorectal cancer, which is a hereditary cancer syndrome characterized by an increased risk of colorectal cancer, as well as other types of cancer, and is caused by mutations in genes involved in DNA mismatch repair. Individuals with Lynch syndrome have a significantly increased risk of developing colorectal cancer at an earlier age compared to the general population. Colorectal cancer may occur in the 20s or 30s, although the average age of onset is around 45 years. Lynch syndrome is associated with an increased risk of other cancers, including endometrial, ovarian, stomach, small intestine, urinary tract, pancreatic, and bile duct. Individuals with Lynch syndrome is typically have a strong family history of cancer, particularly colorectal and endometrial cancer, across multiple generations. Individuals with Lynch syndrome require regular cancer surveillance and management to detect cancers at an early, more treatable stage.

PMS2

PMS2 long-range PCR was performed to capture the genomic sequences for the real PMS2 gene from this individual’s genomic DNA to prevent the pseudogene from being co-amplified. Next-generation sequencing (NGS) was performed on an Illumina system with 100 base pair paired-end reads. A base is considered to have sufficient coverage at 20X and an exon is considered fully covered if all coding bases plus three nucleotides of flanking sequence on either side are covered at 20X or more. Low coverage regions, if any, are limited to ~1% or less of the nucleotides in the test unless a pathogenic variant explaining the phenotype is discovered. A list of these regions is available upon request. Alignment to the human reference genome (hg19) is performed and annotated variants are identified in the targeted region. Variants are called at a minimum coverage of 8X and an alternate allele frequency of 20% or higher. Indels and Single nucleotide variants (SNVs) are confirmed by Sanger sequence analysis before reporting at the director's discretion. This assay cannot detect variants in regions of the exome that are not covered, such as deep intronic, promoter, and enhancer regions or long repetitive regions. Copy number variation (CNV) analysis was assessed using MLPA. This analysis cannot determine the location or orientation of a duplication. This assay is not designed to detect mosaicism; possible cases of mosaicism may be investigated at the discretion of the laboratory director.