Abstract
Lynch Syndrome (LS) is the most common hereditary cancer syndrome. Heterozygous loss-of-function variants in PMS2 are linked to LS type 4. While these variants are not directly cancer causing, reduced PMS2 function results in the accumulation of somatic variants and increased cancer risk over time due to DNA mismatch repair dysfunction. It is reasonable that other types of genetic variation that impact the expression of PMS2 may also contribute to cancer risk. The Kozak sequence is a highly conserved translation initiation motif among higher eukaryotes and is defined as the nine basepairs upstream of the translation start codon through the first four bases of the translated sequence (5’-GTTGCATCCATGG-3’; human PMS2 NM_000535.7). While Kozak sequence variants in PMS2 have been reported in ClinVar in suspected hereditary cancer patients, all variants upstream of the translation start site are currently classified as variants of undetermined significance (VUSs). We hypothesized that variants significantly disrupting the Kozak sequence of PMS2 would decrease PMS2 protein expression, contributing to increased cancer risk over time. Using a dual luciferase reporter plasmid and site-directed mutagenesis, we generated the wild-type human PMS2 and all the ClinVar VUSs within the PMS2 Kozak sequence. Besides the c.1A>C variant, which is already known to be pathogenic, we implicate six additional variants as ACMG/AMP pathogenic supporting (PP) variants and classify nine as benign supporting (BP). In summary, we present a method for the classification of human PMS2 Kozak sequence variants that can contribute to the re-classification of VUSs identified in patients.