Validation of PMS2 germline variants in patients with hereditary cancer syndromes

Background.  Next generation sequencing (NGS) has achieved unprecedented development in recent years. The Sanger sequencing method is actively used to validate variants identified using NGS. The use of the Sanger method is necessary when the identified variant is localized in a gene that has several pseudogenes. The presence of pseudogenes complicates the detection of pathogenic variants in protein-coding regions. The  PMS2 gene, associated with Lynch syndrome, has a large number of pseudogenes  PMS2P1-PMS2P14 and PMS2CL, so additional methodological approaches are required to verify the identified pathogenic variants.

Aim. Testing a validation protocol for identified  PMS2 gene pathogenic variants, taking into consideration the presence of numerous pseudogenes, which exhibit a high percentage of homology with the regions where the validated variants are found.

Methods. The article presents two cases with identified PMS2 gene pathogenic germline variants: chr7:5982823C>T (rs267608172) and chr7:6003716del (hg38).

Results. We developed an algorithm using nested PCR primers to validate the chr7:5982823C>T variant ( PMS2 gene, exon 12) identified by whole-genome sequencing. In the first round, a long fragment containing exons 9-12 is amplified; in the second round, a target fragment suitable for Sanger sequencing is amplified. To validate the chr7:6003716del variant (PMS2 gene, exon 4), the primers were selected in a special way: the differences in the nucleotide sequence of the gene and the pseudogene were located at the 3’-end of the forward primer. As a result, further Sanger sequencing uses the target fragment without pseudogene contamination.

Conclusions.  Specialized algorithms for Sanger sequencing of the PMS2 gene are necessary to ensure that highly homologous pseudogene sequences do not interfere with reading the functional gene sequence.

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