Saturation functional assays of MUTYH resolve clinical variants of uncertain significance

Variants of uncertain significance (VUS) limit the actionability of genetic testing. A prominent example is MUTYH, a base excision repair factor associated with polyposis and colorectal cancer, which has a pathogenic variant carrier rate approaching 1 in 50 individuals in some populations. To systematically interrogate variant function in MUTYH, we coupled deep mutational scanning with a DNA repair reporter containing its lesion substrate, 8OG:A. Our variant-to-function map covers >97% of all possible MUTYH point variants (n=10,941) and achieves 100% accuracy classifying pathogenicity of known clinical variants (n=247). Leveraging a large clinical registry, we observe significant associations with colorectal polyps and cancer, with more severely impaired missense variants conferring greater risk. We recapitulate known functional differences between pathogenic founder mutations and highlight sites of complete missense intolerance, including residues that intercalate DNA and coordinate essential Zn2+ or Fe-S clusters. This map provides a resource to resolve the 1,122 existing clinical missense VUS in MUTYH and demonstrates a scalable strategy to interrogate other clinically relevant DNA repair factors. Overall design: To measure missense variant impacts, MUTYH cDNA was split into 10 tiles and each underwent saturation mutagenesis. HEK293 MUTYH KO cells were transduced with each library, then transfected with an 8OG:A DNA repair construct. Repair-positive (GFP+) and negative (GFP-) cells were isolated, and the MUTYH library was deeply sequenced from each.