Refining the Role of PMS2 in Lynch Syndrome
Refining the Role of PMS2 in Lynch Syndrome
Background and aim The majority of mismatch repair (MMR) gene mutations causing Lynch syndrome (LS) occur either in MLH1 or MSH2. However, the relative contribution of PMS2 is less well defined. The aim of this study was to evaluate the role of PMS2 in LS by assessing the pathogenicity of variants of unknown significance (VUS) detected in the mutational analysis of PMS2 in a series of Spanish patients.
Methods From a cohort of 202 LS suspected patients, 13 patients showing loss of PMS2 expression in tumours were screened for germline mutations in PMS2, using a long range PCR based strategy and multiplex ligation dependent probe amplification (MLPA). Pathogenicity assessment of PMS2 VUS was performed evaluating clinicopathological data, frequency in control population and in silico and in vitro analyses at the RNA and protein level.
Results Overall 25 different PMS2 DNA variants were detected. Fourteen were classified as polymorphisms. Nine variants were classified as pathogenic: seven alterations based on their molecular nature and two after demonstrating a functional defect (c.538-3C>G affected mRNA processing and c.137G>T impaired MMR activity). The c.1569C>G variant was classified as likely neutral while the c.384G>A remained as a VUS. We have also shown that the polymorphic variant c.59G>A is MMR proficient.
Conclusions Pathogenic PMS2 mutations were detected in 69% of patients harbouring LS associated tumours with loss of PMS2 expression. In all, PMS2 mutations account for 6% of the LS cases identified. The comprehensive functional analysis shown here has been useful in the classification of PMS2 VUS and contributes to refining the role of PMS2 in LS.
Heterozygous germline mutations in either one of the mismatch repair (MMR) genes are responsible for Lynch syndrome (LS). Although MLH1 and MSH2 are the most frequently mutated genes in LS, the contribution of PMS2 is less well defined, representing 1–13.7% of all MMR gene mutations. The analysis of PMS2 is especially complex due to the presence of multiple pseudogenes. Fourteen of these pseudogenes share a high homology with the PMS2 5' end, and the pseudogene PMS2CL has strong homology to PMS2 exons 9 and 11–15. In order to avoid spurious amplification of pseudogenes, long range (LR) PCR strategies have improved the yield of mutational analysis. When patients are selected for mutation analysis on the basis of negative PMS2 staining in tumours, the reported mutation rate varies considerably (0–62%), likely linked to differences in the selection criteria and in the strategy used for genetic analysis.
Most of the reported mutations identified in LS suspected patients are truncating and classified as pathogenic. However, a significant number (30%) are classified as variants of unknown significance (VUS). The assessment of pathogenicity is difficult and requires the integration of clinical, pathological, and functional data (reviewed by Rasmussen). While functional analyses of MMR variants are frequently performed for MLH1 and MSH2 genes, the impact of PMS2 VUS has been incompletely analysed for a limited number of cases. Studies have been done at mRNA level, on protein expression, and on protein interaction. Also, site directed mutagenesis of residues located in the PMS2 ATPase domain has been performed in function–structure studies, and the impact of PMS2 VUS in other processes such as apoptosis has been explored.
The aim of this study was to evaluate the contribution of PMS2 in LS by assessing the pathogenicity of VUS detected in the mutational analysis of PMS2 in a series of Spanish patients with LS associated tumours showing loss of PMS2 expression. For PMS2 VUS classification, a strategy was followed which integrates frequency in controls, family history and tumour pathology data with a variety of in silico and in vitro analyses at RNA and protein level.
Abstract and Introduction
Abstract
Background and aim The majority of mismatch repair (MMR) gene mutations causing Lynch syndrome (LS) occur either in MLH1 or MSH2. However, the relative contribution of PMS2 is less well defined. The aim of this study was to evaluate the role of PMS2 in LS by assessing the pathogenicity of variants of unknown significance (VUS) detected in the mutational analysis of PMS2 in a series of Spanish patients.
Methods From a cohort of 202 LS suspected patients, 13 patients showing loss of PMS2 expression in tumours were screened for germline mutations in PMS2, using a long range PCR based strategy and multiplex ligation dependent probe amplification (MLPA). Pathogenicity assessment of PMS2 VUS was performed evaluating clinicopathological data, frequency in control population and in silico and in vitro analyses at the RNA and protein level.
Results Overall 25 different PMS2 DNA variants were detected. Fourteen were classified as polymorphisms. Nine variants were classified as pathogenic: seven alterations based on their molecular nature and two after demonstrating a functional defect (c.538-3C>G affected mRNA processing and c.137G>T impaired MMR activity). The c.1569C>G variant was classified as likely neutral while the c.384G>A remained as a VUS. We have also shown that the polymorphic variant c.59G>A is MMR proficient.
Conclusions Pathogenic PMS2 mutations were detected in 69% of patients harbouring LS associated tumours with loss of PMS2 expression. In all, PMS2 mutations account for 6% of the LS cases identified. The comprehensive functional analysis shown here has been useful in the classification of PMS2 VUS and contributes to refining the role of PMS2 in LS.
Introduction
Heterozygous germline mutations in either one of the mismatch repair (MMR) genes are responsible for Lynch syndrome (LS). Although MLH1 and MSH2 are the most frequently mutated genes in LS, the contribution of PMS2 is less well defined, representing 1–13.7% of all MMR gene mutations. The analysis of PMS2 is especially complex due to the presence of multiple pseudogenes. Fourteen of these pseudogenes share a high homology with the PMS2 5' end, and the pseudogene PMS2CL has strong homology to PMS2 exons 9 and 11–15. In order to avoid spurious amplification of pseudogenes, long range (LR) PCR strategies have improved the yield of mutational analysis. When patients are selected for mutation analysis on the basis of negative PMS2 staining in tumours, the reported mutation rate varies considerably (0–62%), likely linked to differences in the selection criteria and in the strategy used for genetic analysis.
Most of the reported mutations identified in LS suspected patients are truncating and classified as pathogenic. However, a significant number (30%) are classified as variants of unknown significance (VUS). The assessment of pathogenicity is difficult and requires the integration of clinical, pathological, and functional data (reviewed by Rasmussen). While functional analyses of MMR variants are frequently performed for MLH1 and MSH2 genes, the impact of PMS2 VUS has been incompletely analysed for a limited number of cases. Studies have been done at mRNA level, on protein expression, and on protein interaction. Also, site directed mutagenesis of residues located in the PMS2 ATPase domain has been performed in function–structure studies, and the impact of PMS2 VUS in other processes such as apoptosis has been explored.
The aim of this study was to evaluate the contribution of PMS2 in LS by assessing the pathogenicity of VUS detected in the mutational analysis of PMS2 in a series of Spanish patients with LS associated tumours showing loss of PMS2 expression. For PMS2 VUS classification, a strategy was followed which integrates frequency in controls, family history and tumour pathology data with a variety of in silico and in vitro analyses at RNA and protein level.
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