Functional Characterization of a Genetic Variant in the 5' UTR of APC 1B Promoter in a Familial Adenomatous Polyposis Family.

Pathogenic germline variants in the APC gene result in familial adenomatous polyposis (FAP) which can escalate into colon cancer. Standard clinical testing failed to identify pathogenic variants in a 4-generation FAP family. We identified and assessed co-segregation of a 5' untranslated region (UTR) variant, NM_001127511.3 (APC) c.-40G>A (GRCh37 chr5:112043375) that creates a potential out-of-frame AUG start codon. The segregation odds of pathogenicity for the APC c.-40G>A variant are 159:1. Translation initiation confidence values for all possible AUGs in the 5' UTR created by a single nucleotide substitution were calculated using PreTIS online tool. The -40G>A variant scored the highest possible confidence value. To test -40G>A as an initiating methionine, we created reporter constructs consisting of the entire 5' UTR and first 81 bases of APC driving luciferase. When the -40G>A variant was present, luciferase activity was decreased to 14%-25% of the wild-type construct. When the premature start codon created by the -40G>A variant was in-frame with luciferase, we observed luciferase activity from this de novo false start site. Combined, our evidence supports classification of APC c.-40G>A to likely pathogenic, presumably through squelching of the canonical AUG start codon. More importantly, it underlines the feasibility and importance of clinical laboratories to screen noncoding regions.