Identification of pathogenic MSH6 VUS.
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 Msh6+ allele but did not encode the planned mutation; the dark grey segments display the fractions of colonies that maintained the Msh6+ allele and encoded the mutations of interest.