Background: Heterozygous mutations in the DNA mismatch repair gene, PMS2 contribute to Lynch syndrome. Carrier individuals are at increased risk of developing cancers, including colorectal and endometrial cancers. Biallelic mutations in PMS2 can cause constitutional mismatch-repair-deficiency syndrome, which is characterized by childhood onset malignancy, including brain cancers, hematologic malignancies, colorectal cancers, and multiple intestinal polyposis. Molecular analysis of the PMS2 gene is complicated by the presence of a large family of highly homologous sequences in other genomic regions and frequent gene conversion between the active gene and pseudogenes. Although the combination of multiplex ligation-dependent probe amplification (MLPA) assay and Sanger sequencing can largely overcome this problem, copy number variations (CNVs) at the 3′ end cannot be unambiguously differentiated when gene conversion is involved. Here we described a novel approach that combines long range PCR (LR-PCR), next generation sequencing (NGS), and MLPA to identify sequence and copy number changes of the PMS2 gene.

Method: A comprehensive workflow incorporating MLPA, LR-PCR, and NGS was carefully designed for sequence and CNV analyses of the PMS2 gene. MLPA was used to detect CNVs in PMS2 and PMS2CL. In order to distinguish mutations in the active gene from changes in the pseudogene, the active gene was specifically enriched by LR-PCR, followed by whole gene NGS analysis to detect point mutations and CNVs. CNVs can be further confirmed by MLPA. Allele dropout is overcome by capture-based NGS.

Results: Using the comprehensive workflow described above, we have identified CNVs in all coding exons of the PMS2 gene: exonic deletions (E2-E9, E5-E9, E8, E10, E14), exonic duplications (E11-E12), whole gene deletion (E1-E15), and a nonsense mutation, S22X. We identified an exon 14 deletion (c.2276-113_c.2445+1596del) in the active gene that could not be differentiated originally by the combination of MLPA and Sanger sequencing due to a gene conversion event in the 3′ region between PMS2 and PMS2CL, however, with LR-PCR followed by NGS, the deletion was unequivocally identified. Breakpoint analysis of multiple samples with exon 14 deletions revealed that frequent exon 14 deletions were mediated by homologous Alu sequences. We also identified a potential LINE1 sequence mediated intragenic duplication flanking exons 11 and 12.

Conclusion: The combined strategies of long range PCR, MLPA, and deep coverage NGS ensure accurate and unambiguous identification of a wide spectrum of mutations, including point mutations, small indels, and copy number changes, in addition to the determination of breakpoints. The novel strategy of comprehensive workflow provides a reliable tool for NGS based accurate molecular analysis of genes containing multiple copies of highly homologous sequences such as PMS2.

Citation Format: Jianli Li, Hongzheng Dai, Yangming Feng, Jia Tang, Stella Chen, Xia Tian, Elizabeth Gorman, Eric S. Schmitt, Jing Wang, Victor Wei Zhang, Lee-Jun Wong. A novel strategy for accuurate and unambiguous mutation detection of the highly homologous PMS2 gene. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5264. doi:10.1158/1538-7445.AM2015-5264