OR11-02 Functional Characterization Of Sdhb Variants Informs Clinical Classification And Reveals Genotype-phenotype Associations

Abstract Disclosure: S. Lee: None. L. Needleman: None. J. Park: None. R. Schugar: None. J.P. Annes: None. Pathogenic mutations in the succinate dehydrogenase complex, particularly in the SDHB gene, predispose individuals to hereditary pheochromocytoma and paraganglioma (hPPGL). However, more than 80% of missense SDHB variants are classified as variants of uncertain significance (VUS) in ClinVar. Consequently, numerous families harboring an SDHB VUS are at elevated oncologic risk, but do not benefit from early detection protocols. As SDHB pathogenicity is caused by impaired SDH enzymatic activity, functional evaluation may help resolve VUS classification. Hence, we developed a functional biochemical assay using CRISPR/Cas9-engineered SDHB-knockout HEK293 cells to assess succinate/fumarate ratios by LC-MS/MS as a proxy for SDH enzymatic activity. Loss of SDHB led to a marked elevation in the succinate/fumarate ratio, which was restored by transfection of wild-type SDHB. Expression of 35 classified variants (9 benign, 26 pathogenic) clustered into low and high succinate/fumarate ratio groups, validating the assay’s ability to resolve SDHB variant functionality. To improve interpretability, we converted succinate/fumarate ratios into SDH % activity, which was used for the logistic regression modeling of known variants. Using the trained model, P(path) and OddsPath values were calculated to assign ACMG/AMP functional evidence strength (BS3/PS3). Compared to the computational predictor REVEL, our assay provided superior discrimination between benign and pathogenic variants. This model was applied to 33 VUSs, including 16 Stanford patient alleles and three causative of Leigh syndrome. Twenty-three VUSs demonstrated wild-type-like activity (BS3), while 10 exhibited marked SDH dysfunction and were assigned PS3strong. Following ACMG/AMP guidelines, mock clinical interpretation that included our functional assay recategorized 78% of SDHB VUSs (16 likely benign and 10 likely pathogenic). Notably, all SDHB VUSs affecting highly conserved cysteine residues responsible for Fe-S cluster coordination showed complete enzymatic loss, confirming the critical function of these domains. Interestingly, recessive mitochondrial disease-associated SDHB variants (Leighs) were not reliably distinguished from wild-type-like variants, indicating these variants are not causative for hPPGL. Finally, pathogenic variants with relatively lower succinate/fumarate ratios than PPGL-predominant variants were associated with increased occurrence of head and neck paragangliomas (HNPGL; r2=0.45; p=0.0001). Logistic modeling identified a metabolic threshold predictive of tumor location; hence, SDH dysfunction severity influences location-specific tumor risk. Together, these data establish a robust, quantitative platform for SDHB variant classification with immediate clinical implications for risk assessment and tumor surveillance. Presentation: Saturday, July 12, 2025