MSI analysis of mESCs expressing <i>Msh6</i> variants.

<p>To quantify the level of MSI, a (G)₁₀-<i>neo</i> slippage reporter was introduced into variant mESCs. Spontaneous DNA polymerase slippage events on the (G)₁₀ repeat that are not corrected can bring the <i>neo</i> in frame, rendering cells Geneticin-resistant. Slippage rates (the emergence of a Geneticin-resistant cell per cell division) of VUS expressing cells are compared to the MMR-proficient <i>Msh6</i>^<i>+/-</i> cell line and MMR-deficient <i>Msh6</i> ^{<i>V397E/-</i>}, <i>Msh6</i> ^{<i>L448P/-</i>}, <i>Msh6</i> ^{<i>G1137S/-</i>}, and <i>Msh6</i> ^{<i>R1332Q/-</i>} pathogenic controls. Statistical differences were calculated using one-tailed, unpaired t-test with Welch’s correction. Asterisks indicate values significantly higher than those of the MMR-proficient <i>Msh6</i>^<i>+/-</i> control: *<i>P</i><0.05; **<i>P</i><0.01; ***<i>P</i><0.001; ****<i>P</i><0.0001.</p

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Identification of pathogenic <i>MSH6</i> VUS.

<p>The genetic screen was used to analyze (A) 18 VUS selected from literature and the InSiGHT database as well as (B) 8 VUS identified in patients from two medical centers in the Netherlands. Variants are displayed according to their amino acid number and change in men and mice. The ‘Nucleotide change’ column presents the one or two base alteration introduced by the LMOs. If antisense-oriented LMOs did not give rise to 6TG-resistant colonies encoding the mutation of interest, the screen was repeated with sense-oriented LMOs (lower row where two rows are present for the variant). The InSiGHT classification of each variant is indicated: 4, likely pathogenic; 3, uncertain; 2, likely not pathogenic; NA, not available. The bars in the ‘Fraction of 6TG-resistant colonies carrying mutation’ column represent the 18 6TG-resistant colonies that were analyzed for the presence of the planned mutation: the white segments represent LOH events; the light grey segments represent background colonies that maintained the <i>Msh6</i>⁺ allele but did not encode the planned mutation; the dark grey segments display the fractions of colonies that maintained the <i>Msh6</i>⁺ allele and encoded the mutations of interest.</p

MNNG-induced mutagenesis in mESCs expressing <i>Msh6</i> variants.

<p>Variant MSH6 expressing mESCs were exposed to MNNG and the number of cells that consequently acquired mutations in <i>Hprt</i> quantified [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006765#pgen.1006765.ref043" target="_blank">43</a>]. <i>Hprt</i>-defective mESCs were identified by long-term exposure to a high dose of 6TG (10 μg/ml). The spontaneous (-) and MNNG induced (+) mutation frequency was compared to MMR-proficient <i>Msh6</i>^<i>+/-</i> mESCs and MMR-deficient <i>Msh6</i> ^{<i>V397E/-</i>}, <i>Msh6</i> ^{<i>L448P/-</i>}, <i>Msh6</i> ^{<i>G1137S/-</i>}, and <i>Msh6</i> ^{<i>R1332Q/-</i>} pathogenic controls. The statistical differences between MNNG-treated <i>Msh6</i>^<i>+/-</i> mESCs and MNNG-treated variant cell lines was calculated using a one-tailed, unpaired t-test with Welch’s correction. Asterisks indicate values significantly higher than those of the MNNG-treated MMR-proficient <i>Msh6</i>^<i>+/-</i> control: *<i>P</i><0.05; **<i>P</i><0.01.</p

Distinguishing pathogenic <i>MSH6</i> variants from polymorphisms.

<p>(A) Five known non-pathogenic variants and (B) four pathogenic mutations tested in the proof of principle study. Variants are annotated according to their amino acid change and location in men and mice. The nucleotide change was first introduced by antisense-oriented LMOs. If no 6TG-resistant colonies encoding the mutation appeared, the screening protocol was repeated with sense-oriented LMOs (lower row where two rows are present). The fourth column presents the InSiGHT classification of each variant where 5 is pathogenic, 3 is uncertain and 1 is not pathogenic. At variance with the InSiGHT classification, a previous study demonstrated variant G1139S is pathogenic and L1087R is not pathogenic [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006765#pgen.1006765.ref025" target="_blank">25</a>]. The bars in the ‘Fraction of 6TG-resistant colonies carrying mutation’ column represent the 18 6TG-resistant colonies that were investigated further. The white portions represent colonies in which the <i>Msh6</i>⁺ allele was lost by LOH; the light grey portions illustrate the fraction of background colonies that apparently survived 6TG selection but maintained the <i>Msh6</i>⁺ allele without the planned mutation; the dark grey portions represent the fractions of colonies that maintained the <i>Msh6</i>⁺ allele and encoded the mutation of interest.</p

MMR capacity of <i>Msh6</i>^{<i>G565R/-</i>} mESCs.

<p>The MMR activity of <i>Msh6</i>^{<i>G565R/-</i>} mESCs was investigated using two assays. (A) 6TG survival assay. The colony-forming capacity of <i>Msh6</i>^{<i>G565R/-</i>} mESCs as well as MMR-deficient <i>Msh6</i>^<i>-/-</i> and MMR-proficient <i>Msh6</i>^<i>+/-</i> and <i>Msh6</i>^<i>+/+</i> cells was determined in response to increasing doses of 6TG. (B) MSI in the <i>Msh6</i>^{<i>G565R/-</i>} mESCs was investigated using the (G)₁₀-<i>neo</i> slippage reporter. The slippage rate (the emergence of a Geneticin-resistant cell per cell division) in <i>Msh6</i>^{<i>G565R/-</i>} cells was compared to the rate in MMR-proficient <i>Msh6</i>^<i>+/-</i> and MMR-deficient <i>Msh6</i>^{<i>V397E/-</i>} control cell lines. Statistical differences were calculated using one tailed, unpaired t-test with Welch’s correction. **** indicates significantly higher than the mismatch repair proficient <i>Msh6</i>^<i>+/-</i> control: <i>P</i><0.0001.</p

Western blot analysis of mESCs expressing <i>Msh6</i> variants.

<p>MSH6, MSH2 and γ-TUBULIN levels were analyzed in whole cell lysates. MSH6 and MSH2 levels in the variant cells lines were quantified with respect to the protein levels seen in <i>Msh6</i>^<i>+/-</i> mESCs.</p

Rare variants in <i>LRP6</i> in three cases.

<p>(A) Distribution of missense <i>LRP6</i> variants identified in the CRC discovery cohort (red dots). Somatic <i>LRP6</i> mutations identified in colorectal tumors [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005880#pgen.1005880.ref024" target="_blank">24</a>] are indicated with grey (missense) and black (protein truncating) dots. Structural domains include the β-propeller domains that are used to form the receptor complex (pink bars), and the transmembrane domain (purple). (B) 3D protein structure of the β-propeller domains of LRP6 with the positions of the identified missense variants in red. Insets show conservation of the region in which the missense variants (indicated with the red box) are located with, underneath, close ups of the local 3D protein structure with mutant (red) and wild-type (green) residues. The mutant residue at position 239 is predicted to disturb the protein structure (project HOPE; <a href="http://www.cmbi.ru.nl/hope/" target="_blank">http://www.cmbi.ru.nl/hope/</a>). The mutant residue at position 789 is much smaller than the wild-type residue and may disturb the binding of Dickkopf-1. Residue 867 is located on the surface of the protein and the mutant residues are not expected to disturb protein structure, but may influence protein binding. (C) Immunofluorescence analyses of LRP6 wild-type and mutant proteins showing similar subcellular localizations. (D) LRP6 protein expression levels normalized to β-actin are similar between wild-type and mutant LRP6. (E) TOPflash analyses of wild-type and mutant LRP6 to determine their effects on the WNT signaling pathway. Luciferase activity was normalized to control and wild-type constructs. Both p.N789S and p.T867A mutants reveal a significant increase in activation compared to the wild-type LRP6 protein. Experiments were performed three times in triplicate. **<i>P</i> <0.001; error bars represent the standard error of the mean.</p

Variants in candidate CRC susceptibility genes identified by whole-exome sequencing and targeted re-sequencing.

<p>Variants in candidate CRC susceptibility genes identified by whole-exome sequencing and targeted re-sequencing.</p

Study design, variant filtering and candidate gene prioritization.

<p>Whole-exome sequencing was performed on germline DNA of 55 early-onset CRC cases. The exome data were first filtered for quality and frequency, followed by filtering for protein truncating and highly conserved missense variants. Next, we removed all known loss-of-function-tolerant genes from this list and searched for known and novel CRC predisposing gene variants.[<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005880#pgen.1005880.ref012" target="_blank">12</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005880#pgen.1005880.ref013" target="_blank">13</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005880#pgen.1005880.ref057" target="_blank">57</a>] An additional filtering was applied to identify genes that were affected by two or more potentially pathogenic variants and to remove genes that are frequently affected by protein-truncating or highly conserved missense variants in healthy controls. The remaining set of recurrent variants was filtered for (i) genes recurrently affected by protein truncating variants; (ii) cancer driver genes in CRC [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005880#pgen.1005880.ref023" target="_blank">23</a>]; (iii) genes identified as CRC susceptibility genes in mice [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005880#pgen.1005880.ref029" target="_blank">29</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005880#pgen.1005880.ref030" target="_blank">30</a>] and involved in cancer-related KEGG pathways [hsa04310 (WNT signaling), hsa04350 (TGF-beta signaling), hsa03430 (mismatch repair), hsa03410 (base excision repair), hsa03420 (nucleotide excision repair), map03450 (non-homologous end-joining), hsa03460 (Fanconi anemia), and hsa05200 (pathways in cancer)] (iv) genes identified in CRC GWAS studies [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005880#pgen.1005880.ref005" target="_blank">5</a>–<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005880#pgen.1005880.ref007" target="_blank">7</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005880#pgen.1005880.ref032" target="_blank">32</a>]. Genes that remained after these filter steps were selected for re-sequencing in a replication cohort of 174 CRC cases. CRC: colorectal cancer; VUS: variant of unknown significance.</p