A biallelic pathogenic variant in the OGDH gene results in a neurological disorder with features of a mitochondrial disease

Abstract

2-Oxoglutarate dehydrogenase (OGDH) is a rate-limiting enzyme in the mitochondrial TCA cycle, encoded by the OGDH gene. α-Ketoglutarate dehydrogenase (OGDH) deficiency was previously reported in association with developmental delay, hypotonia, and movement disorders and metabolic decompensation, with no genetic data provided. Using whole exome sequencing, we identified two individuals carrying a homozygous missense variant c.959A>G (p.N320S) in the OGDH gene. These individuals presented with global developmental delay, elevated lactate, ataxia and seizure. Fibroblast analysis and modeling of the mutation in Drosophila were used to evaluate pathogenicity of the variant. Skin fibroblasts from subject # 2 showed a decrease in both OGDH protein and enzyme activity. Transfection of human OGDH cDNA in HEK293 cells carrying p.N320S also produced significantly lower protein levels compared to those with wild-type cDNA. Loss of Drosophila Ogdh (dOgdh) caused early developmental lethality, rescued by expressing wild-type dOgdh (dOgdh^WT ) or human OGDH (OGDH^WT ) cDNA. In contrast, expression to the mutant OGDH (OGDH^N320S ) or dOgdh carrying homologous mutations to human OGDH p.N320S variant (dOgdh^N324S ) failed to rescue lethality of dOgdh null mutants. Knockdown of dOgdh in the nervous system resulted in locomotion defects which were rescued by dOgdh^WT expression but not by dOgdh^N324S expression. Collectively, the results indicate that c.959A>G variant in OGDH leads to an amino acid change (p.N320S) causing a severe loss of OGDH protein function. Our study establishes in the first time a genetic link between an OGDH gene mutation and OGDH deficiency.

Keywords: OGDH; TCA cycle; alpha-ketoglutarate dehydrogenase deficiency; genetic disease.

FIGURE 1

FLAIR imaging MRI from patient # 2. A, White arrows = widening of the Sylvian fissure indicating cortical atrophy; black arrows = high FLAIR signal in the lentiform nuclei, internal, and external capsule. B, White arrows = simplified gyral pattern in frontal lobes; black arrows = cystic changes in the basal ganglia

FIGURE 2

OGDH variant in silico analysis. A, A pedigree of two affected probands, three unaffected siblings and parents show segregation of the homozygous OGDH variant c.959A>G (p.Asn320Ser) in subject 1 and subject 2. B, The p.Asn320Ser substitution alters the evolutionarily conserved Asn residues in multiple species including Homo sapiens and Drosophila. C, A schematic of protein domains of OGDH and position of Asn320Ser mutation. D, Homology model of OGDH shows the Asn320 residue contributes to the stability of the α-helix which is connected to the loop carrying histidine 311 and arginine 312 that is required for thiamine pyrophosphate (TPP) binding

FIGURE 3

The p.N320S variant in OGDH impairs the protein stability and activity of OGDH. A, Western blots and quantification for protein levels of OGDH, DLST, ATP5A, and actin in protein extracts from subject 2 cultured fibroblasts and those from two healthy controls. (n = 10). B, WES capillary electrophoresis-based protein assays detected the expression of Flag-tagged OGDH expression and GFP expression in HEK293 cells. The OGDH protein levels were normalized to GFP. (n = 6). C and D, The activities of OGDH and complex I were measured from total cell extracts from subject 2 cultured fibroblasts and control fibroblasts 1 (OGDH activity, n = 6; complex I activity, n = 5). E, Confocal micrographs of subject 2 cultured fibroblasts and control fibroblasts 1. ATP5A antibody labels mitochondria (green) and DAPI labels nuclei (blue). Scale bars, 20 μm. Error bars indicate SEM. P values were calculated using Student’s t test. ***P < .001

FIGURE 4

Biallelic OGDH variant impairs the in vivo function of dOgdh in Drosophila. A, A schematic of transgene expression mediated by dOgdh-T2A-Gal4 allele. A T2A-Gal4 cassette consisting of a splice acceptor (SA), a linker (L), a ribosomal skipping T2A peptide (T2A), and a Gal4 coding sequence, and a polyadenylation signal (pA), was inserted to a coding intron of dOgdh genomic locus by RMEC. B, Expression of dOGDH in adults and larvae. dOGDH is highly expressed in ventral nerve cord (VNC) in larva, and head in adult. C, The lethality caused by dOgdh loss (homozygous for dOgdh-T2A-Gal4) was rescued by OGDH^WT or dOgdh^WT expression, but not by cDNA carrying the patient variant (OGDH^N320S or dOgdh^N324S). D, dOgdh knockdown in neurons exhibited progressive defects in climbing ability, which was fully rescued by expressing dOgdh^WT, but not by dOgdh^N324S. Error bars indicate SEM (n = 3). P values were calculated using Student’s t test. ***P < .001. N.S. indicates not statistically significant