A Pathogenic L2HGDH Variant Impairs Mitochondrial Targeting and Enzyme Function in L-2-Hydroxyglutaric Aciduria: Clinical and Functional Evidence from Two Affected Siblings.

Background: L-2-hydroxyglutaric aciduria (L2HGA) is a rare autosomal recessive neurometabolic disorder caused by biallelic loss-of-function variants in the L-2-hydroxyglutarate dehydrogenase () gene, leading to accumulation of L-2-hydroxyglutarate in the brain and other tissues. While various variants have been reported, the pathogenic mechanism of specific variants remains unclear. In this study, we aimed to investigate the molecular consequences of the c.905C>T p.(Pro302Leu) variant, identified in two siblings with typical symptoms of L2HGA, by analyzing its effects on protein localization and enzymatic activity in a cell model.

Methods: HA-tagged wild-type and p.(Pro302Leu) mutant constructs were overexpressed in HEK293T cells. We assessed protein expression, subcellular localization, and enzymatic activity using Western blot analysis, immunofluorescence microscopy, and a specific enzyme assay measuring 2,6-dichloroindophenol (DCIP) reduction to assess L2HGDH enzymatic activity.

Results: Western blotting showed that wild-type exists primarily in the processed, mature mitochondrial form, whereas the p.(Pro302Leu) mutant remained largely in the unprocessed precursor form. Immunofluorescence and differential centrifugation revealed that wild-type protein localized to mitochondria, while the mutant protein accumulated in the cytoplasm in a diffuse or punctate pattern. Enzyme activity assays demonstrated that the mutant retained <30% of wild-type activity.

Conclusions: These findings indicate that the p.(Pro302Leu) variant leads to aggregation of mislocalized protein, thereby impairing L2HGDH function rather than decreasing enzymatic function. This study provides clinical and molecular evidence supporting the pathogenicity of this previously reported mutation and highlights the importance of mitochondrial import for enzyme functionality in L2HGA.