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Published on: March 29, 2017
PMS2 gene mutational analysis: direct cDNA sequencing to circumvent pseudogene interference
Katharina Wimmer1, Annekatrin Wernstedt
1Division of Human Genetics, Medical University Innsbruck, Peter-Mayr-Strasse 1, Innsbruck, 6020, Austria, katharina.wimmer@i-med.ac.at.
Abstract:
The presence of highly homologous pseudocopies can compromise the mutation analysis of a gene of interest. In particular, when using PCR-based strategies, pseudogene co-amplification has to be effectively prevented. This is often achieved by using primers designed to be parental gene specific according to the reference sequence and by applying stringent PCR conditions. However, there are cases in which this approach is of limited utility. For example, it has been shown that the PMS2 gene exchanges sequences with one of its pseudogenes, named PMS2CL. This results in functional PMS2 alleles containing pseudogene-derived sequences at their 3'-end and in nonfunctional PMS2CL pseudogene alleles that contain gene-derived sequences. Hence, the paralogues cannot be distinguished according to the reference sequence. This shortcoming can be effectively circumvented by using direct cDNA sequencing. This approach is based on the selective amplification of PMS2 transcripts in two overlapping 1.6-kb RT-PCR products. In addition to avoiding pseudogene co-amplification and allele dropout, this method has also the advantage that it allows to effectively identify deletions, splice mutations, and de novo retrotransposon insertions that escape the detection of most DNA-based mutation analysis protocols.
Insights
Mutation analysis of the PMS2 gene is challenging due to its pseudogene, PMS2CL. Direct cDNA sequencing effectively distinguishes between PMS2 and PMS2CL, enabling accurate detection of various mutations.
Area of Science:
- Genetics
- Molecular Biology
- Bioinformatics
Background:
- Highly homologous pseudogenes can interfere with accurate gene mutation analysis, particularly when using PCR-based methods.
- The PMS2 gene and its pseudogene, PMS2CL, share homologous sequences, complicating differentiation based on standard reference sequences.
- Functional PMS2 alleles can contain pseudogene-derived sequences, and nonfunctional PMS2CL alleles can contain gene-derived sequences, rendering standard mutation detection difficult.
Purpose of the Study:
- To address the challenge of distinguishing the PMS2 gene from its highly homologous pseudogene, PMS2CL.
- To develop and validate a method for accurate mutation analysis of the PMS2 gene, overcoming limitations of DNA-based approaches.
- To improve the detection of various genetic alterations, including deletions, splice mutations, and retrotransposon insertions.
Main Methods:
- Utilized direct cDNA sequencing based on the selective amplification of PMS2 transcripts.
- Employed two overlapping 1.6-kb reverse transcription PCR (RT-PCR) products for comprehensive transcript coverage.
- Focused on preventing pseudogene co-amplification and allele dropout during the analysis.
Main Results:
- Successfully circumvented the limitations of distinguishing PMS2 from PMS2CL by using direct cDNA sequencing.
- Achieved selective amplification of PMS2 transcripts, avoiding co-amplification of the PMS2CL pseudogene.
- Demonstrated the method's capability to identify deletions, splice mutations, and de novo retrotransposon insertions.
Conclusions:
- Direct cDNA sequencing is a robust method for accurate mutation analysis of the PMS2 gene in the presence of homologous pseudogenes.
- This approach overcomes the challenges posed by sequence exchange between PMS2 and PMS2CL, enabling reliable genetic analysis.
- The described RT-PCR strategy enhances mutation detection sensitivity, identifying alterations missed by conventional DNA-based methods.
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