RNA-based mutation analysis identifies an unusual MSH6 splicing defect and circumvents PMS2 pseudogene interference

Area of Science:

• Genetics
• Molecular Biology
• Oncology

Background:

• Lynch syndrome, caused by heterozygous mismatch repair (MMR) gene mutations, is a dominant cancer susceptibility syndrome.
• A novel, recessively inherited MMR-deficiency (MMR-D) syndrome presents with childhood brain tumors and malignancies, linked to biallelic MMR gene mutations.
• Accurate genetic testing for MMR genes is essential for diagnosing these conditions.

Purpose of the Study:

• To develop and validate an RNA-based mutation detection assay for MMR genes (MLH1, MSH2, MSH6, PMS2).
• To address limitations of DNA-based testing, particularly for PMS2 due to pseudogenes and potential splicing defects.
• To investigate families with suspected MMR-D syndrome.

Main Methods:

• Established an RNA-based mutation detection assay using direct cDNA sequencing of RT-PCR products for four key MMR genes.
• Applied the assay to two families with children exhibiting symptoms suggestive of MMR-D syndrome.
• Analyzed a carrier of a PMS2/PMS2CL "hybrid" allele to assess assay performance.

Main Results:

• Identified a homozygous complex MSH6 splicing alteration in the first family's index patients.
• Discovered a novel homozygous PMS2 mutation (c.182delA) in the second family's index patient.
• Demonstrated that the RNA-based assay effectively avoids pseudogene coamplification and allelic dropout, enhancing PMS2 mutation analysis.

Conclusions:

• The RNA-based assay provides a sensitive and accurate method for detecting MMR gene mutations, including complex splicing alterations and PMS2 defects.
• This assay improves diagnostic capabilities for both MMR-deficiency (MMR-D) syndrome and Lynch syndrome (HNPCC), especially when PMS2 pseudogenes complicate genomic DNA analysis.
• The developed assay offers a significant advancement in identifying genetic causes of hereditary cancer syndromes.