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

J Etzler1, A Peyrl, A Zatkova

  • 1Department of Medical Genetics, Medical University Vienna, Vienna, Austria.

Human Mutation
|November 22, 2007
PubMed

Insights

A novel RNA-based assay detects genetic mutations in mismatch repair (MMR) genes, crucial for diagnosing rare MMR-deficiency (MMR-D) syndrome and hereditary nonpolyposis colorectal cancer (HNPCC). This method improves accuracy for PMS2 gene mutations.

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.

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