Abstract

Abstract Purpose: SDHA mutations are the most common cause of SDH-deficient GIST. Enhanced cancer surveillance of individuals carrying a known pathogenic germline SDHA mutation has the potential to detect early-stage tumors, allowing for improved patient outcomes. However, more than 95% of the > 1,000 SDHA missense variants listed in ClinVar are variants of uncertain significance (VUS). Our ability to interpret the significance of SDHA variants must improve before genetic sequencing can be utilized to its full potential. Experimental Design: SDHA variants were introduced into a clonal SDHA-knockout cell line via Bxb1-mediated recombination. SDH Activity and SDHA abundance were determined for each variant and logistic regression analysis was used to derive functional evidence for clinical variant interpretation. Results: Our analysis revealed that cancer-associated SDHA missense variants can be clearly distinguished from non-cancer variants according to the extent of SDH dysfunction caused. As such, SDH activity data can be used to predict cancer pathogenicity with strong performance metrics, exceeding those of computational prediction tools. From these data, we obtained functional evidence for clinical variant interpretation from 21 of 22 assayed VUS, with 19 in favor of cancer pathogenicity and two against pathogenicity. Lastly, simulating the addition of our functional evidence with limited pre-existing evidence allowed for 18 of 22 variants to be reclassified. Conclusions: We describe a novel pipeline for investigating the functional consequences of SDHA missense variants. In total, we characterized 72 variants, developed criteria for obtaining functional evidence, and demonstrated the potential of this evidence for clinical variant interpretation.