Predictive functional assay-based classification of PMS2 variants in Lynch syndrome

Abstract

The large majority of germline alterations identified in the DNA mismatch repair (MMR) gene PMS2, a low-penetrance gene for the cancer predisposition Lynch syndrome, represent variants of uncertain significance (VUS). The inability to classify most VUS interferes with personalized healthcare. The complete in vitro MMR activity (CIMRA) assay, that only requires sequence information on the VUS, provides a functional analysis-based quantitative tool to improve the classification of VUS in MMR proteins. To derive a formula that translates CIMRA assay results into the odds of pathogenicity (OddsPath) for VUS in PMS2 we used a set of clinically classified PMS2 variants supplemented by inactivating variants that were generated by an in cellulo genetic screen, as proxies for cancer-predisposing variants. Validation of this OddsPath revealed high predictive values for benign and predisposing PMS2 VUS. We conclude that the OddsPath provides an integral metric that, following the other, higher penetrance, MMR proteins MSH2, MSH6 and MLH1 can be incorporated as strong evidence type into the upcoming criteria for MMR gene VUS classification of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP).

Keywords: DNA mismatch repair; Lynch syndrome; PMS2; diagnostic assessment; functional analysis-based classification; variants of uncertain significance.

Figure 1

Flow diagram of the complete in vitro mismatch repair activity (CIMRA) assay. The variant of interest is produced by a PCR procedure, expressed in a coupled transcription‐translation kit and then heterodimerized with MLH1 protein, expressed from wild‐type MLH1 cDNA. Then the functional activity of the variant heterodimer is assessed in a complementation assay containing a substrate containing a T.G mismatch with a linked fluorescent group (star), and MLH1/PMS2‐deficient cell extract. Repair of the mismatch recreates a Hin1II‐cleavable restriction endonuclease site, resulting in the generation of a fluorescent diagnostic fragment (green arrow) that is visualized by capillary electrophoresis. Following calibration of the assay results against independently classified variants, the relative abundance of this diagnostic fragment is translated into an odds of pathogenicity for the variant PMS2/MLH1 heterodimer (Drost, Koppejan, et al., ; Drost, Lützen, et al., 2013).

Figure 2

A genetic screen to identify inactivating variants in Pms2. Experimental steps to generate Pms2‐inactivating variants in mouse embryonic stem cells (mESCs), to be used as a proxy for predisposing variants. (a) A Pms2‐heterozygous (Pms2^+/−) cell line is treated with N‐ethyl‐N‐nitrosourea to induce random substitution mutations. (b) Cells that have undergone loss of heterozygosity (LOH) at Pms2 (LOH, Pms2^−/−), cells with an inactivating mutation in Hprt (Hprt^−/−), and cells that have acquired an inactivating mutation in the wild‐type allele of Pms2 (Pms2M/−) are selected by their tolerance to 6‐TG. (c) Cells with an Hprt‐inactivating mutation are eliminated by culture in hypoxanthine‐aminopterin‐thymidine (HAT)‐supplemented medium. (d) The inactivating missense substitutions in the remaining clones without LOH (LOH clones are screened against by intragenic Pms2 PCR) are identified by cDNA sequence analysis. These inactivating substitution variants are subsequently included in the calibration and validation set.

Figure 3

Frequency and location of genetic screen‐derived inactivating variants conserved between murine Pms2 and human PMS2A schematic representation of the PMS2 protein showing conserved ATP binding and MLH1 interactions domains and the endonuclease active site. The position and frequency of Pms2‐inactivating mutations (in red) identified in the genetic screen are plotted on the schematic.

Figure 4

Mismatch repair activity in the complete in vitro mismatch repair activity (CIMRA) assay of PMS2 variants used for calibration and validation of the assay. Relative repair efficiencies for human PMS2 variants used in the calibration and validation of the CIMRA assay. Variants are colored according to their ClinVar classification and their identification as inactivating in the genetic screen. The PMS2 p.S46I variant was included in every assay as a mismatch repair‐deficient pathogenic control and p.G197G as a wild‐type control (Borràs et al., 2013) and are colored black. Bars represent ± Standarderror of the mean (SEM) of three independent experiments.

Figure 5

Odds of pathogenicity of PMS2 variants The odds of pathogenicity (OddsPath) for PMS2 variants used in the calibration and validation of the complete in vitro mismatch repair activity (CIMRA) assay plotted against the average relative repair efficiency from the CIMRA assay. The odds of pathogenicity (OddsPath) for each PMS2 variant was derived using the leave‐one‐out procedure. Each variant is colored according to the computational prediction of pathogenicity (Prior‐P) (see Table 1; B. A. Thompson et al., 2013). Four variants for which the bioinformatics‐based Prior‐P and the CIMRA assay‐based OddsPath were strongly discordant are indicated separately in the figure.