Abstract 6285: A novel functional cell line model for high throughput assessment of SDHA VUS for pathogenicity.

Abstract Pathogenic (P), loss-of-function (LOF) SDHA germline mutations are associated with an increased risk of developing GI stromal tumors, paraganglioma/pheochromocytoma, or renal cell carcinoma. Although the penetrance of SDHA germline mutations appears less than for other SDH complex members, international guidelines recommend surveillance of germline pathogenic mutation carriers for early detection of SDH mutant cancers using a combination of laboratory testing, routine medical examinations, and periodic imaging. The number of SDHA VUS far exceeds the number of mutations that are classified as benign (B), likely benign (LB), likely pathogenic (LP), or pathogenic (P). As of November 2025, of the 1531 missense SDHA mutations in ClinVar, 1412 (92%) are VUS. We previously generated a CRISPR KO Hap1 cell line with integration of a single genomic copy of a landing pad cassette. Using targeted recombination of individual SDHA variants and measurement of SDHA enzymatic activity measurements, we determined that 19/20 control P/LP variants met the criteria for PS3strong evidence and 16/20 control benign B/LP variants met the criteria for BS3supporting (6 variants) or BS3moderate (10 variants). Using this model we functionally profiled 21 VUS and combined the weight of evidence with pre-existing population frequency data and computational predictions. Using this functional data, we were able to definitively classify 17/21 VUS as either LB (1) or LP (16) with only 4 remaining as VUS. Despite the utility of this model to functional profile and classify individual VUS, the throughput of this system is too limited to meaningful profile large numbers of VUS. Notably, these cell lines have metabolically adapted to culture conditions, and we cannot select against loss-of-function (LOF) variants. To enable metabolic selection against LOF variants, we developed a novel system where parental HAP1 cells with intact genomic SDHA are stably “pre-rescued” by lentiviral SDHA cDNAs that have mutation of the PAM and sgRNA binding sites for our most efficient SDHA sgRNA. The cDNAs encode for variants of interest (WT, VUS, control B and P variants). Stably expressing cells are then transduced with an SDHA sgRNA that edits the genomic SDHA gene but not the cDNA transgene. Parental or empty vector cells are nearly eliminated after transduction of SDHA sgRNA due to efficient editing of the essential genomic SDHA gene, whereas SDHA WT cDNA transduced cells are minimally depleted at 10-14 days after transduction. To date we have profiled 18 P and 18 B control variants in both systems and had 100% agreement between the assay systems. Overall, we profiled 79 variants in our depletion assay system and compared the results to those from our landing pad system and found excellent agreement with a r2=0.89. This system is suitable for deep mutational scanning (DMS) as LOF variants are efficiently depleted. Pilot DMS experiments are currently in progress. Citation Format: Michael C. Heinrich, Christine Robbins, Ajia Town, . A novel functional cell line model for high throughput assessment of SDHA VUS for pathogenicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 6285.