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