A Comprehensive Strategy for Accurate Mutation Detection of the Highly Homologous PMS2

J Mol Diagn. 2015 Sep;17(5):545-53. doi: 10.1016/j.jmoldx.2015.04.001.


Germline mutations in the DNA mismatch repair gene PMS2 underlie the cancer susceptibility syndrome, Lynch syndrome. However, accurate molecular testing of PMS2 is complicated by a large number of highly homologous sequences. To establish a comprehensive approach for mutation detection of PMS2, we have designed a strategy combining targeted capture next-generation sequencing (NGS), multiplex ligation-dependent probe amplification, and long-range PCR followed by NGS to simultaneously detect point mutations and copy number changes of PMS2. Exonic deletions (E2 to E9, E5 to E9, E8, E10, E14, and E1 to E15), duplications (E11 to E12), and a nonsense mutation, p.S22*, were identified. Traditional multiplex ligation-dependent probe amplification and Sanger sequencing approaches cannot differentiate the origin of the exonic deletions in the 3' region when PMS2 and PMS2CL share identical sequences as a result of gene conversion. Our approach allows unambiguous identification of mutations in the active gene with a straightforward long-range-PCR/NGS method. Breakpoint analysis of multiple samples revealed that recurrent exon 14 deletions are mediated by homologous Alu sequences. Our comprehensive approach provides a reliable tool for accurate molecular analysis of genes containing multiple copies of highly homologous sequences and should improve PMS2 molecular analysis for patients with Lynch syndrome.

Publication types

  • Research Support, Non-U.S. Gov't
  • Validation Study

MeSH terms

  • Adenosine Triphosphatases / genetics*
  • Adult
  • Base Sequence
  • Colorectal Neoplasms, Hereditary Nonpolyposis / genetics*
  • DNA Copy Number Variations
  • DNA Mutational Analysis / methods*
  • DNA Repair Enzymes / genetics*
  • DNA-Binding Proteins / genetics*
  • Female
  • Gene Dosage
  • Germ-Line Mutation*
  • High-Throughput Nucleotide Sequencing
  • Humans
  • Middle Aged
  • Mismatch Repair Endonuclease PMS2
  • Molecular Sequence Data
  • Multiplex Polymerase Chain Reaction / methods*
  • Polymerase Chain Reaction / methods


  • DNA-Binding Proteins
  • Adenosine Triphosphatases
  • PMS2 protein, human
  • Mismatch Repair Endonuclease PMS2
  • DNA Repair Enzymes