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Avoidance of pseudogene interference in the detection of 3' deletions in PMS2
Cecily P Vaughn1, Kimberly J Hart, Wade S Samowitz
1ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, Utah 84108, USA.
Abstract:
Lynch syndrome is characterized by mutations in the mismatch repair genes MLH1, MSH2, MSH6, and PMS2. In PMS2, detection of mutations is confounded by numerous pseudogenes. Detection of 3' deletions is particularly complicated by the pseudogene PMS2CL, which has strong similarity to PMS2 exons 9 and 11-15, due to extensive gene conversion. A newly designed multiplex ligation-dependent probe amplification (MLPA) kit incorporates probes for variants found in both PMS2 and PMS2CL. This provides detection of deletions, but does not allow localization of deletions to the gene or pseudogene. To address this, we have developed a methodology incorporating reference samples with known copy numbers of variants, and paired MLPA results with sequencing of PMS2 and PMS2CL. We tested a subset of clinically indicated samples for which mutations were either unidentified or not fully characterized using existing methods. We identified eight unrelated patients with deletions encompassing exons 9-15, 11-15, 13-15, 14-15, and 15. By incorporating specific, characterized reference samples and sequencing the gene and pseudogene it is possible to identify deletions in this region of PMS2 and provide clinically relevant results. This methodology represents a significant advance in the diagnosis of patients with Lynch syndrome caused by PMS2 mutations.
Insights
Accurate Lynch syndrome diagnosis requires identifying mutations in mismatch repair genes like PMS2. This study introduces a new method combining MLPA and sequencing to overcome pseudogene interference, improving deletion detection in PMS2.
Area of Science:
- Genetics
- Molecular Biology
- Cancer Genomics
Background:
- Lynch syndrome is a hereditary cancer predisposition caused by mutations in mismatch repair genes.
- Accurate diagnosis of Lynch syndrome is crucial for patient management and cascade screening.
- Pseudogenes, particularly PMS2CL, complicate the detection of deletions in the PMS2 gene, a key gene in Lynch syndrome.
Purpose of the Study:
- To develop and validate a novel methodology for accurate detection and localization of 3' deletions in the PMS2 gene.
- To overcome the challenges posed by the PMS2CL pseudogene in identifying PMS2 deletions.
- To improve the diagnostic yield for Lynch syndrome patients with PMS2 mutations.
Main Methods:
- Development of a multiplex ligation-dependent probe amplification (MLPA) kit with probes for both PMS2 and its pseudogene PMS2CL.
- Integration of MLPA results with targeted sequencing of PMS2 and PMS2CL.
- Utilization of reference samples with known copy numbers of variants for accurate interpretation.
Main Results:
- Successfully identified eight unrelated patients with various deletions in PMS2 exons (9-15, 11-15, 13-15, 14-15, and 15).
- The combined MLPA and sequencing approach accurately localized deletions to the PMS2 gene, distinguishing them from pseudogene variants.
- Demonstrated the clinical utility of the new methodology in characterizing previously unidentified or incompletely characterized mutations.
Conclusions:
- The developed methodology, combining specialized MLPA and sequencing with reference samples, enables precise identification of PMS2 deletions.
- This approach significantly advances the diagnostic capabilities for Lynch syndrome caused by PMS2 mutations.
- Accurate genetic testing for PMS2 deletions is essential for effective Lynch syndrome diagnosis and patient care.
