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Single Droplet Digital Polymerase Chain Reaction for Comprehensive and Simultaneous Detection of Mutations in Hotspot Regions
Published on: September 25, 2018
Long-range PCR facilitates the identification of PMS2-specific mutations
Mark Clendenning1, Heather Hampel, Jennifer LaJeunesse
1Human Cancer Genetics Program, Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio, USA.
Human Mutation
|April 19, 2006
Summary
Detecting mutations in the PMS2 gene is crucial for Lynch syndrome diagnosis. A new PCR-based method overcomes challenges posed by PMS2 pseudogenes, enabling accurate mutation identification in hereditary cancer patients.
Area of Science:
- Genetics
- Molecular Biology
- Oncology
Background:
- Mutations in the DNA mismatch repair gene PMS2 are linked to Lynch syndrome, a hereditary cancer predisposition.
- PMS2 pseudogenes complicate direct sequencing, hindering accurate mutation detection.
- Previous genetic screening efforts for Lynch syndrome have been limited by technical challenges in PMS2 analysis.
Purpose of the Study:
- To develop and validate a novel method for accurate PMS2 gene sequencing.
- To overcome the challenge of homologous PMS2 pseudogenes for reliable mutation analysis.
- To screen patients with Lynch syndrome-like features for PMS2 mutations.
Main Methods:
- Utilized long-range PCR to preferentially amplify the PMS2 gene over its pseudogenes.
- Employed exon-specific amplification from diluted long-range products for clean sequencing.
- Screened 41 patients (30 colorectal, 11 endometrial) with PMS2-deficient tumors and no MLH1 mutations.
Main Results:
- The novel method successfully generated clean PMS2 sequences without pseudogene contamination.
- Identified 10 novel and 17 known PMS2 sequence changes in the patient cohort.
- Detected putative pathogenic mutations in 11 of 41 families, including five novel mutations.
Conclusions:
- The developed long-range PCR and exon-specific amplification method is effective for PMS2 mutation detection.
- This approach facilitates feasible and desirable PMS2 mutation screening in selected Lynch syndrome patients.
- Accurate PMS2 mutation analysis is critical for diagnosing hereditary nonpolyposis colorectal cancer (Lynch syndrome).
