Introduction: Germline mutations in succinate dehydrogenase subunit B (SDHB) have been well recognised for their association with development of phaeochromocytoma (PC) and paraganglioma (PGL). SDHB immunohistochemistry, expression signature, and more recently succinate:fumarate measurement by LC/MS-MS, have been shown to functionally corroborate pathogenic SDHB variants. However, pathogenic validation of many SDHB variants has been incomplete particularly when tumour tissue is not available. We have developed (1) and refined an in vitro SDH assay as an adjunct for pathogenic classification of SDHB variants.
Objective: To assess an in vitro SDH activity assay for determining functional consequences of germline SDHB missense mutations.
Methods: Sixteen germline SDHB missense variants were selected for in vitro study. These variants encompass a spectrum of pathogenic mutations found in our genetic testing laboratory, some of which are reported in the Exome Aggregate Consortium (ExAC) and/or LOVD. HEK293 cells were transfected with either wild-type or mutant GFP-tagged SDHB constructs (generated by site-directed mutagenesis). SDH activity of complexes containing wild-type or mutant SDHB was then assessed using GFP-pulldown followed by colourimetric analysis.
Results: SDH activitys of complexes containing clinically relevant mutant SDHB were significantly lower than the wild-type control (p<0.05) with the exception of p.Ile127Ser and p.Pro197Arg. For those SDHB variants reported in ExAC, SDH activity was positively correlated with allelic frequency (R2=0.803, p<0.05), such that more common SDHB variants resulted in less severe SDH dysfunction.
Conclusion: Functional assessment of SDHB mutation through use of SDH activity in vitro is a promising method for distinguishing pathogenic mutations from benign variants. Some pathogenic SDHB variants appear to retain SDH activity in vitro, and the mechanism for SDH deficiency in those cases warrants further study.
(1) Kim et al Endocrin Rel Cancer 2015;22:387-97