D‐2‐hydroxyglutaric aciduria Type I: Functional analysis of D2HGDH missense variants

Abstract

D‐2‐hydroxyglutaric aciduria Type I (D‐2‐HGA Type I), a neurometabolic disorder with a broad clinical spectrum, is caused by recessive variants in the D2HGDH gene encoding D‐2‐hydroxyglutarate dehydrogenase (D‐2‐HGDH). We and others detected 42 potentially pathogenic variants in D2HGDH of which 31 were missense. We developed functional studies to investigate the effect of missense variants on D‐2‐HGDH catalytic activity. Site‐directed mutagenesis was used to introduce 31 missense variants in the pCMV5‐D2HGDH expression vector. The wild type and missense variants were overexpressed in HEK293 cells. D‐2‐HGDH enzyme activity was evaluated based on the conversion of [2H4]D‐2‐HG to [2H4]2‐ketoglutarate, which was subsequently converted into [2H4]L‐glutamate and the latter quantified by LC‐MS/MS. Eighteen variants resulted in almost complete ablation of D‐2‐HGDH activity and thus, should be considered pathogenic. The remaining 13 variants manifested residual activities ranging between 17% and 94% of control enzymatic activity. Our functional assay evaluating the effect of novel D2HGDH variants will be beneficial for the classification of missense variants and determination of pathogenicity.

Structure, substrate specificity, and catalytic mechanism of human D-2-HGDH and insights into pathogenicity of disease-associated mutations

“…However, as the activity assays were carried out using the cell lysates of HEK293 cells overexpressing the wild-type and mutant D-2-HGDH proteins, the measured activities seem to be inaccurate probably due to inaccurate measurement of the enzyme’s concentration caused by interference of other endogenous proteins. For instance, the activity of wild-type D-2-HGDH was determined to be 1.73 nmol/min/mg 43 , which is 3 orders of magnitude lower than the activity (2.01 μmol/min/mg) determined in this work (Fig. 3a ).…”

“…Truncated variants are usually deemed as pathogenic, whereas the functional roles of missense variants and their pathogenicity remain elusive. A previous functional study of the missense variants associated with type I D-2-HGA showed that compared to the wild-type D-2-HGDH, 18 mutants have severely impaired activity (<6%) and 13 mutants have substantially reduced activity (17%–94%) 43 . However, as the activity assays were carried out using the cell lysates of HEK293 cells overexpressing the wild-type and mutant D-2-HGDH proteins, the measured activities seem to be inaccurate probably due to inaccurate measurement of the enzyme’s concentration caused by interference of other endogenous proteins.…”

“…Type I D-2-HGA is caused by deficient D-2-HGDH, and genomic analyses of type I D-2-HGA patients have identified 42 variants in the D2HGDH gene, of which 31 are missense variants and 11 are truncated variants 43 . In addition, genomic analyses of DLBCL patients have also identified 4 missense variants in D2HGDH 30 .…”

2-Hydroxyglutarate in Cancer Cells

“…The specific degradation of r -2HG to 2OG proceeds via r -2HG-dehydrogenase (EC 1.1.99.39) ( 94 ) localized to the mitochondrial matrix ( 1 ), whereas the s -enantiomer is catalyzed by the cytosolic plus mitochondrial enzyme of EC 1.1.99.2. A deficiency of r -2HG-dehydrogenase causes type-I glutaric academia ( 107 , 129 , 161 ). Since r -2HG-dehydrogenase accepts electrons from electron transfer flavoprotein:ubiquinone oxidoreductase (ETF:QOR) ( 161 ), deficiencies of this oxidoreductase also lead to a similar disease, glutaric acidemia type-II.…”

Mitochondrial diseases: expanding the diagnosis in the era of genetic testing

“…Patients with biallelic L2HGDH variants have exclusive neurological findings of psychomotor retardation, cerebellar ataxia, macrocephaly, and epilepsy [356] . Recessive and rare dominant D2HGDH variants in the chiral configuration enzyme, D-2hydroxyglutarate dehydrogenase induce D-2-hydroglutaric acid to 2-ketoglutarate, and patients present with epilepsy, hypotonia, and psychomotor retardation [357] .…”