Figure 4. In vitro cell-free mismatch repair assay confirms loss of function proteins translated from MLH1c.1A>G p.(Met1?) alleles.
A) Alleles generated by PCR including the full-length variant allele and an allele carrying a deletion of 34 amino acids as anticipated from MLH1-GFP fusion construct assays. B) Sequence confirmed alleles. C) 35S-Methionine labelled proteins derived for the cell-free assay. The p.Met1Val allele was unable to produce a detectable protein. D) A protein lacking amino acids 1–34 reduces MMR activity to a level equivalent to known pathogenic missense mutation, MLH1:p.Gly67Arg.