Two integrated and highly predictive functional analysis-based procedures for the classification of MSH6 variants in Lynch syndrome

Abstract

Purpose: Variants in the DNA mismatch repair (MMR) gene MSH6, identified in individuals suspected of Lynch syndrome, are difficult to classify owing to the low cancer penetrance of defects in that gene. This not only obfuscates personalized health care but also the development of a rapid and reliable classification procedure that does not require clinical data.

Methods: The complete in vitro MMR activity (CIMRA) assay was calibrated against clinically classified MSH6 variants and, employing Bayes' rule, integrated with computational predictions of pathogenicity. To enable the validation of this two-component classification procedure we have employed a genetic screen to generate a large set of inactivating Msh6 variants, as proxies for pathogenic variants.

Results: The genetic screen-derived variants established that the two-component classification procedure displays high sensitivities and specificities. Moreover, these inactivating variants enabled the direct reclassification of human variants of uncertain significance (VUS) as (likely) pathogenic.

Conclusion: The two-component classification procedure and the genetic screens provide complementary approaches to rapidly and cost-effectively classify the large majority of human MSH6 variants. The approach followed here provides a template for the classification of variants in other disease-predisposing genes, facilitating the translation of personalized genomics into personalized health care.

Keywords: DNA mismatch repair; Lynch syndrome; MSH6; functional analysis-based classification; variants of uncertain significance.

Fig. 1. Outline, calibration and validation of the complete in vitro mismatch repair activity (CIMRA) assay.

(a) Outline of the CIMRA assay. (b) Relative repair efficiencies for MSH6 missense variants from the InSiGHT database, classified based on clinical criteria alone. Variants are ranked according to their mean CIMRA assay activity. The p.G1139S variant is included in every experiment as a (technical) repair-deficient control. Variants are colored according to their International Agency for Research on Cancer (IARC) classification (see figure for legend). Bars represent mean ± S.E.M. of >3 experiments. (c) Regressions of the CIMRA assay training values against odds in favor of pathogenicity. The y-axes of these graphs display probability of pathogenicity rather than Log(odds in favor of pathogenicity) to emphasize sigmoid calibration bounded at probabilities of 1.00 and 0.00. (d) Relative repair efficiencies for InSiGHT/ClinVar database-derived (likely) benign MSH6 missense variants (blue bars) as determined in the CIMRA assay. Variants are ranked according to their mean CIMRA assay activity. Bars represent mean ± S.E.M. of >3 experiments. MMR mismatch repair, PCR polymerase chain reaction, WT wild type.

Fig. 2. A genetic screen for inactivating missense variants in Msh6.

(a) Pipeline to generate mouse embryonic stem (mES) cell lines that carry inactivating Msh6 missense variants. I. A mES cell line, heterozygous for Msh6 (Msh6^+/−), is subjected to mutagenic treatment with ENU. II. Cells that have acquired 6-TG tolerance, by loss of heterozygosity at Msh6 (Msh6^+/−), owing to an ENU-induced inactivating variant at a critical residue in the monoallelic Msh6 gene (Msh6^M/−), or by an inactivating variant at the monoallelic Hprt gene (Hprt^-) are selected using two brief 6-TG selections. III. The (unwanted) Hprt-deficient clones are eliminated by culture in HAT-supplemented medium. IV. Inadvertent clones that have lost the wild-type Msh6 allele by loss of heterozygosity (LOH) (rather than by a missense substitution) are excluded by using an allele-specific polymerase chain reaction (PCR). V. The inactivating substitution in the remaining clones is identified by sequence analysis. A “reverse diagnosis catalog” is compiled that lists inactivating substitutions at Msh6 as a proxy for pathogenic human variants (Fig. 4). (b) Representation of all inactivating missense substitutions in Msh6, identified in this screen. The top and bottom panels show alignments of the mismatch binding and ATPase domains of human and mouse Msh6, highlighting residues that were mutated in the genetic screen (orange boxes). Phe-X-Glu: mismatch-contacting loop. The middle panel displays all other inactivating Msh6 substitutions. Numbers reflect amino acid numbering of mouse Msh6. (c) Microsatellite instability (MSI) analysis. The size of each sphere is proportional to the relative number of subclones with the indicated mBAT-37 PCR fragment length. All mutants from the validation panel display MSI (p < 0.05 compared with the Msh6^+/− line). (d) Tolerance of Msh6 mutant mES cell lines to the methylating drug N-methyl-N′-nitro-N-nitrosoguanidine. Bars represent mean ± S.E.M. *p < 0.05, **p < 0.01, ***p < 0.001 (one-tailed Student’s t test) compared with the parental Msh6^+/− line.

Fig. 3. Mechanism of mismatch repair (MMR) deficiency of Msh6 alleles identified in the genetic screen.

(a) Western blot analysis of total lysates from (Msh6-variant) ES cells. Pcna serves as a loading control. In addition to Msh6 we also probed for Msh2. Since Msh2 stability depends on Msh6, Msh2 proteins levels are a surrogate marker for Msh6 protein stability. (b) Binding of control and Msh6-variant proteins to a G·T mismatch within a double-stranded oligonucleotide probe in an electrophoretic mobility shift assay. Bars represent mean ± S.E.M. of >3 experiments. (c) Adenosine triphosphate (ATP)-induced mismatch release of Msh6-variant proteins in an electrophoretic mobility shift assay. ATP (0.5 mM) was added after allowing proteins to bind to the probe. Bars represent mean ± S.E.M. For the purpose of clarity, all (-) ATP reactions are normalized to 1 and all (+) ATP reactions are normalized to their respective (-) ATP reactions. Bars represent mean ± S.E.M. of >3 experiments. (d) Msh6-mutant proteins deficient for ATP-induced release (Fig. 3c) were challenged with higher amounts of ATP in an electrophoretic mobility shift assay. ATP, in various concentrations, was added after allowing proteins to bind to the probe. Bars represent mean ± S.E.M. For the purpose of clarity, all (-) ATP reactions are normalized to 1 and all (+) ATP reactions are normalized to their respective (-) ATP reactions. Bars represent mean ± S.E.M. of >3 experiments. (e) Relative repair efficiencies, as determined in the complete in vitro MMR activity (CIMRA) assay, for human MSH6 missense variants, corresponding to inactivating murine variants identified in the genetic screen. Variants are ranked according to their mean CIMRA assay activity. Bars represent mean ± S.E.M. of >3 experiments/variant. The human, not the mouse, numbering of the variants is shown. The numbers at the bottom of the figure indicate the International Agency for Research on Cancer (IARC) class for every variant resulting from our calibrated two-component classification (Table S5).

Fig. 4. Independent assessment of complete in vitro MMR activity (CIMRA) assay reproducibility.

MSH6 variants and controls were tested for CIMRA assay activity in different centers worldwide (see legend in figure). The MSH6 p.G1139S variant is included in every experiment as a repair-deficient control.^, Bars represent mean ± S.E.M. of 3–4 experiments. Numbers below the diagrams indicate the International Agency for Research on Cancer (IARC) classification. LUMC Leiden University Medical Center, QIMR QIMR Berghofer Medical Research Institute, WT wild type.