Research Interests
Unclassified variants of MMR genes
 Rob Dekker |
 Hellen Houlleberghs |
 Eva Wielders |
 Kamila Wojciechowicz-Grzadka |
The non-polyposis form of familial colon cancer, HNPCC, is caused by inherited defects in genes that are essential for post-replicative DNA mismatch repair (MMR). The absence of MMR leaves replication errors unnoticed, which may cause mutational activation of proto-oncogenes or inactivation of tumor suppressor genes and development of cancer.
We have recapitulated this human cancer predisposition syndrome in mice by disrupting the central MMR genes Msh2, Msh6 and Msh3 (de Wind et al., 1995, 1998, 1999). The products of these genes form heterodimeric protein complexes that recognize mismatches in DNA: the MSH2/MSH6 dimer (MutSα) recognizes base.base mismatches and single unpaired nucleotides; MSH2/MSH3 (MutSβ) preferentially targets small unpaired loops of two to five nucleotides. Mice homozygous mutant for either Msh2 or Msh6 were equally sensitive to development of lymphomas and epithelial tumors of the skin and uterus. Also intestinal tumors developed, but only in Msh2- or compound Msh3/Msh6-deficient mice. This indicates that some tumor types (lymphoid, uterine, skin tumors) ensue from mutations that are specifically suppressed by MutSα, while intestinal carcinogenesis requires genetic alterations that are suppressed by MutSα and MutSβ. We envision that also in humans, defects in MSH6 may predispose to cancer without an overt manifestation of intestinal cancer.
In addition to correction of DNA replication errors, the MMR machinery has two other activities: suppression of recombination between homologous but not-identical DNA sequences and induction of cell death in response to certain types of DNA damage, in particular methylation at the O6 position of guanine. The significance of each of these MMR functions for tumor suppression is unclear.
Besides mutations that are overtly pathogenic (large deletions, frameshifts, stop codons), allelic variants of MMR genes have been found with less obvious consequences. Usually these are missense mutations that affect only a single amino acid. We have developed a pipeline to study such “variants of uncertain significance”(VUS) or “unclassified variants” (UV). Briefly, we use oligo targeting to recreate the variant at the endogenous locus in mouse ESC. Cells expressing only the variant allele are subjected to a series of functional tests to assess the capacity of the variant allele to sustain MMR. We have thus far studied four MSH2 VUS and found one (MSH2-P622L) to be pathogenic and three (MSH2-Y103C, MSH2-G322D, MSH2-M688I) to be polymorphic (Wielders et al., 2011). The outcome of these experiments is of direct relevance to clinical counseling of mutation carriers. Moreover, we expect that certain amino acid alterations in MSH2 or MSH6 may cause uncoupling of MMR functions and may therefore be helpful in revealing which of these functions is critical to suppression of cancer.