Abstract 812: A novel model for functional profiling of germline <i>SDHA</i> variants associated with cancer
Authors: Jason Kent, Lillian Klug, Michael Heinrich
Journal: Cancer Research (2021)

Abstract The functional inactivation of the metabolic enzyme complex succinate dehydrogenase (SDH) is a well-validated cause of multiple human cancers. Germline inheritance of a loss-of-function (LOF) SDHA mutation can result in SDH-deficiency is associated with a high lifetime risk of developing one or more SDH-deficient cancers. There are no highly effective medical therapies for advanced unresectable or metastatic SDH-deficient cancer, but most are curable if resected at an early stage. Early detection is therefore critical. However, despite knowing that loss of enzyme activity leads to tumor development, it has been difficult to determine which variants disrupt enzyme activity and contribute to cancer risk. There are currently over 600 SDHA missense reported in ClinVar, yet over 90% are considered variants of uncertain significance (VUS) due to a lack of functional evidence. Nearly 1% of the population carries a SDHA missense VUS and more are discovered every day with the increased availability of genetic testing modalities (clinical and direct-to-consumer). Only those carrying SDHA variant known to be LOF (pathogenic) can be recommended for enhanced tumor surveillance or genetic counseling. Currently, without an adequate model to determine the functional consequence of SDHA variants, an individual who carries SDHA VUS may have a significant lifetime risk of cancer, but are unlikely to benefit from early detection. In order to address this critical need, we sought to develop a novel human cell line model of SDHA-deficiency that would allow us to interrogate the functional consequences of SDHA VUS. Using CRISPR-Cas9 technology, we created a clonal HAP1 SDHA knockout cell line. Genetic inactivation of SDHA was verified by sequencing and immunoblotting, and resulted in complete loss of SDH activity. To investigate the utility of this model for characterizing the functional consequences of SDHA variants, we performed complementation experiments with SDHA variants with known functional consequences by lentiviral transduction. SDHA expression was confirmed by quantitative RT-PCR and immunoblotting and SDH activity was measured from isolated mitochondria using a commercial kit. As expected, expression of wild-type SDHA or known no-effect (benign) SDHA variants restores SDH-activity, whereas expression of known LOF (pathogenic) SDHA variants fails to restore enzyme activity. Using this model, we can functionally characterize SDHA VUS and obtain the evidence needed to reclassify their clinical significance. This model will lead to the discovery of novel pathogenic SDHA variants and help identify those at-risk for developing SDH-deficient cancers. Citation Format: Jason Kent, Lillian Klug, Michael Heinrich. A novel model for functional profiling of germline SDHA variants associated with cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 812.