Suspected Lynch syndrome associated MSH6 variants: A functional assay to determine their pathogenicity

Lynch syndrome (LS) is a hereditary cancer predisposition caused by inactivating mutations in DNA mismatch repair (MMR) genes. Mutations in the MSH6 DNA MMR gene account for approximately 18% of LS cases. Many LS-associated sequence variants are nonsense and frameshift mutations that clearly abrogate MMR activity. However, missense mutations whose functional implications are unclear are also frequently seen in suspected-LS patients. To conclusively diagnose LS and enroll patients in appropriate surveillance programs to reduce morbidity as well as mortality, the functional consequences of these variants of uncertain clinical significance (VUS) must be defined. We present an oligonucleotide-directed mutagenesis screen for the identification of pathogenic MSH6 VUS. In the screen, the MSH6 variant of interest is introduced into mouse embryonic stem cells by site-directed mutagenesis. Subsequent selection for MMR-deficient cells using the DNA damaging agent 6-thioguanine (6TG) allows the identification of MMR abrogating VUS because solely MMR-deficient cells survive 6TG exposure. We demonstrate the efficacy of the genetic screen, investigate the phenotype of 26 MSH6 VUS and compare our screening results to clinical data from suspected-LS patients carrying these variant alleles

Unexpected moves: a conformational change in MutSα enables high-affinity DNA mismatch binding

The DNA mismatch repair protein MutSα recognizes wrongly incorporated DNA bases and initiates their correction during DNA replication. Dysfunctions in mismatch repair lead to a predisposition to cancer. Here, we study the homozygous mutation V63E in MSH2 that was found in the germline of a patient with suspected constitutional mismatch repair deficiency syndrome who developed colorectal cancer before the age of 30. Characterization of the mutant in mouse models, as well as slippage and repair assays, shows a mildly pathogenic phenotype. Using cryogenic electron microscopy and surface plasmon resonance, we explored the mechanistic effect of this mutation on MutSα function. We discovered that V63E disrupts a previously unappreciated interface between the mismatch binding domains (MBDs) of MSH2 and MSH6 and leads to reduced DNA binding. Our research identifies this interface as a 'safety lock' that ensures high-affinity DNA binding to increase replication fidelity. Our mechanistic model explains the hypomorphic phenotype of the V63E patient mutation and other variants in the MBD interface

Functional Analysis in Mouse Embryonic Stem Cells Reveals Wild-Type Activity for Three <i>Msh6</i> Variants Found in Suspected Lynch Syndrome Patients

<div><p>Lynch syndrome confers an increased risk to various types of cancer, in particular early onset colorectal and endometrial cancer. Mutations in mismatch repair (MMR) genes underlie Lynch syndrome, with the majority of mutations found in <i>MLH1</i> and <i>MSH2</i>. Mutations in <i>MSH6</i> have also been found but these do not always cause a clear cancer predisposition phenotype and <i>MSH6</i>-defective tumors often do not show the standard characteristics of MMR deficiency, such as microsatellite instability. In particular, the consequences of <i>MSH6</i> missense mutations are challenging to predict, which further complicates genetic counseling. We have previously developed a method for functional characterization of <i>MSH2</i> missense mutations of unknown significance. This method is based on endogenous gene modification in mouse embryonic stem cells using oligonucleotide-directed gene targeting, followed by a series of functional assays addressing the MMR functions. Here we have adapted this method for the characterization of <i>MSH6</i> missense mutations. We recreated three <i>MSH6</i> variants found in suspected Lynch syndrome families, MSH6-P1087R, MSH6-R1095H and MSH6-L1354Q, and found all three to behave like wild type MSH6. Thus, despite suspicion for pathogenicity from clinical observations, our approach indicates these variants are not disease causing. This has important implications for counseling of mutation carriers.</p> </div

Functional analysis of <i>Msh6</i>^<i>mut/-</i> heterozygous ESC lines.

<p>(<b>A</b>) Black bars show the average percentage of unstable microsatellites (left Y-axis) as measured in 96 colonies for two different mononucleotide markers. Error bars show standard errors, measured over three independent clones per cell line. Grey bars show the average number of 6-TG resistant colonies per 10⁶ plated cells (right Y-axis). Error bars show standard errors, measured over three independent clones per cell line. (<b>B</b>) Survival of mutant and control cell lines exposed to MNNG. Error bars show standard errors from three independent experiments.</p

Generation of <i>Msh6</i>^<i>mut/-</i> heterozygous ESC lines.

<p>(<b>A</b>) Southern blot analysis of the <i>Msh6</i>^<i>mut/-</i> and control cell lines, showing loss of one of the <i>Msh6</i> alleles in <i>Msh6</i>^<i>mut/-</i> cells. (<b>B</b> and <b>C</b>) Western blot analysis of mutant <i>Msh6</i> homozygous and heterozygous cell lines and controls. Whole cell lysates were analyzed for the presence of MSH6 and MSH2. γ-tubulin was used as a loading control. ‘-’ indicates a knockout allele. The relative percentages of MSH6 levels are indicated.</p

Generation of homozygous <i>Msh6</i> mutant ESC lines.

<p>Sequence analysis of (<b>A</b>) <i>Msh6</i>^<i>+/+</i>, <i>Msh6</i>^<i>PR/+</i> and <i>Msh6</i>^<i>PR/PR</i> genomic DNA, (<b>B</b>) <i>Msh6</i>^<i>+/+</i>, <i>Msh6</i>^<i>RH/+</i> and <i>Msh6</i>^<i>RH/RH</i> genomic DNA and (<b>C</b>) <i>Msh6</i>^<i>+/+</i>, <i>Msh6</i>^<i>LQ/+</i> and <i>Msh6</i>^<i>LQ/LQ</i> genomic DNA. Single letter amino acid codes are given below the sequence. (<b>D</b>) Whole cell lysates were analyzed for MSH6 and MSH2. γ-Tubulin was used as a loading control. ‘-‘ indicates a knockout allele. The relative percentages of MSH6 levels are indicated.</p

Functional analysis of <i>Msh6</i>^{<i>mut/mut</i>} ESC lines.

<p>(<b>A</b>) Black bars show the average percentage of unstable microsatellites (left Y-axis) as measured in 96 colonies for two or three different dinucleotide markers. Error bars show standard errors, measured over two to six independent clones per cell line. The grey bars show the average number of 6-TG-resistant colonies per 10⁶ plated cells (right Y-axis). Error bars show standard errors, measured over three to six independent clones per cell line. (<b>B</b>) Targeting efficiencies are shown in mutant and control cell lines for the 100% homologous (black bars) and the 99.4% homologous (white bars) <i>Rb</i> targeting constructs. Targeting efficiencies in <i>Msh2</i>^<i>+/+</i><i>Msh2</i>^<i>-/-</i> and <i>Msh6</i>^<i>-/-</i> ESCs are taken from de Wind et al. [10,18] and shown as controls. (<b>C</b>) Survival of mutant and control cell lines exposed to MNNG (n=2-6). (<b>D</b>) Survival of mutant and control cell lines exposed to 6-TG (n=2-5). Error bars show standard errors from independent experiments.</p

Oligonucleotide-directed mutagenesis screen to identify pathogenic Lynch syndrome-associated MSH2 DNA mismatch repair gene variants

textabstractSingle-stranded DNA oligonucleotides can achieve targeted base-pair substitution with modest efficiency but high precision. We show that "oligo targeting" can be used effectively to study missense mutations in DNA mismatch repair (MMR) genes. Inherited inactivating mutations in DNA MMR genes are causative for the cancer predisposition Lynch syndrome (LS). Although overtly deleterious mutations in MMR genes can clearly be ascribed as the cause of LS, the functional implications of missense mutations are often unclear. We developed a genetic screen to determine the pathogenicity of these variants of uncertain significance (VUS), focusing on mutator S homolog 2 (MSH2). VUS were introduced into the endogenous Msh2 gene of mouse embryonic stem cells by oligo targeting. Subsequent selection for MMR-deficient cells using the guanine analog 6-thioguanine allowed the detection of MMR-abrogating VUS. The screen was able to distinguish weak and strong pathogenic variants from polymorphisms and was used to investigate 59 Msh2 VUS. Nineteen of the 59 VUS were identified as pathogenic. Functional assays revealed that 14 of the 19 detected variants fully abrogated MMR activity and that five of the detected variants attenuated MMR activity. Implementation of the screen in clinical practice allows proper counseling of mutation carriers and treatment of their tumors

Mutants of the tumour suppressor p53 L1 loop as second-site suppressors for restoring DNA binding to oncogenic p53 mutations: structural and biochemical insights

International audienceTo assess the potential of mutations from the L1 loop of the tumour suppressor p53 as second-site suppressors, the effect of H115N and S116M on the p53 “hot spot” mutations has been investigated using the double mutant approach. The effects of these two mutants on the p53 “hot spots” in terms of thermal stability and DNA binding were evaluated. The results show that: (1) mutants H115N and S116M are thermally more stable than wild-type p53; (2) H115N but not S116M is capable of rescuing the DNA binding of one of the most frequent p53 mutants in cancer, R248Q, as shown by binding of R248Q/H115N to gadd45; (3) the double mutant R248Q/H115N is more stable than wild-type p53; (4) the effect of H115N as a second-site suppressor to restore DNA-binding activity is specific to R248Q but not to R248W; (5) molecular dynamics simulations indicate that R248Q/H115N has conformation similar to wild type p53, which is distinct from that of R248Q. These findings could be exploited in designing strategies for cancer therapy to identify molecules that could mimic the effect of H115N in restoring function to oncogenic p53 mutants

Oligonucleotide-directed mutagenesis screen to identify pathogenic Lynch syndrome-associated MSH2 DNA mismatch repair gene variants

Single-stranded DNA oligonucleotides can achieve targeted base-pair substitution with modest efficiency but high precision. We show that "oligo targeting" can be used effectively to study missense mutations in DNA mismatch repair (MMR) genes. Inherited inactivating mutations in DNA MMR genes are causative for the cancer predisposition Lynch syndrome (LS). Although overtly deleterious mutations in MMR genes can clearly be ascribed as the cause of LS, the functional implications of missense mutations are often unclear. We developed a genetic screen to determine the pathogenicity of these variants of uncertain significance (VUS), focusing on mutator S homolog 2 (MSH2). VUS were introduced into the endogenous Msh2 gene of mouse embryonic stem cells by oligo targeting. Subsequent selection for MMR-deficient cells using the guanine analog 6-thioguanine allowed the detection of MMR-abrogating VUS. The screen was able to distinguish weak and strong pathogenic variants from polymorphisms and was used to investigate 59 Msh2 VUS. Nineteen of the 59 VUS were identified as pathogenic. Functional assays revealed that 14 of the 19 detected variants fully abrogated MMR activity and that five of the detected variants attenuated MMR activity. Implementation of the screen in clinical practice allows proper counseling of mutation carriers and treatment of their tumors