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1.
Biochim Biophys Acta Mol Basis Dis ; 1865(9): 2257-2266, 2019 09 01.
Article in English | MEDLINE | ID: mdl-31075491

ABSTRACT

Mutations in the gene triosephosphate isomerase (TPI) lead to a severe multisystem condition that is characterized by hemolytic anemia, a weakened immune system, and significant neurologic symptoms such as seizures, distal neuropathy, and intellectual disability. No effective therapy is available. Here we report a compound heterozygous patient with a novel TPI pathogenic variant (NM_000365.5:c.569G>A:p.(Arg189Gln)) in combination with the common (NM_000365.5:c.315G>C:p.(Glu104Asp)) allele. We characterized the novel variant by mutating the homologous Arg in Drosophila using a genomic engineering system, demonstrating that missense mutations at this position cause a strong loss of function. Compound heterozygote animals were generated and exhibit motor behavioural deficits and markedly reduced protein levels. Furthermore, examinations of the TPIArg189Gln/TPIGlu104Asp patient fibroblasts confirmed the reduction of TPI levels, suggesting that Arg189Gln may also affect the stability of the protein. The Arg189 residue participates in two salt bridges on the backside of the TPI enzyme dimer, and we reveal that a mutation at this position alters the coordination of the substrate-binding site and important catalytic residues. Collectively, these data reveal a new human pathogenic variant associated with TPI deficiency, identify the Arg189 salt bridge as critical for organizing the catalytic site of the TPI enzyme, and demonstrates that reduced TPI levels are associated with human TPI deficiency. These findings advance our understanding of the molecular pathogenesis of the disease, and suggest new therapeutic avenues for pre-clinical trials.


Subject(s)
Anemia, Hemolytic, Congenital Nonspherocytic/pathology , Carbohydrate Metabolism, Inborn Errors/pathology , Triose-Phosphate Isomerase/deficiency , Triose-Phosphate Isomerase/metabolism , Alleles , Amino Acid Sequence , Anemia, Hemolytic, Congenital Nonspherocytic/genetics , Animals , Base Sequence , Carbohydrate Metabolism, Inborn Errors/genetics , Catalytic Domain , Child, Preschool , Dimerization , Disease Models, Animal , Drosophila/metabolism , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Female , Fibroblasts/cytology , Fibroblasts/metabolism , Humans , Mutation, Missense , Pedigree , Protein Stability , Sequence Alignment , Triose-Phosphate Isomerase/genetics
2.
Mol Genet Metab ; 126(4): 439-447, 2019 04.
Article in English | MEDLINE | ID: mdl-30683556

ABSTRACT

Seizures are a feature not only of the many forms of epilepsy, but also of global metabolic diseases such as mitochondrial encephalomyopathy (ME) and glycolytic enzymopathy (GE). Modern anti-epileptic drugs (AEDs) are successful in many cases, but some patients are refractory to existing AEDs, which has led to a surge in interest in clinically managed dietary therapy such as the ketogenic diet (KD). This high-fat, low-carbohydrate diet causes a cellular switch from glycolysis to fatty acid oxidation and ketone body generation, with a wide array of downstream effects at the genetic, protein, and metabolite level that may mediate seizure protection. We have recently shown that a Drosophila model of human ME (ATP61) responds robustly to the KD; here, we have investigated the mechanistic importance of the major metabolic consequences of the KD in the context of this bioenergetics disease: ketogenesis, reduction of glycolysis, and anaplerosis. We have found that reduction of glycolysis does not confer seizure protection, but that dietary supplementation with ketone bodies or the anaplerotic lipid triheptanoin, which directly replenishes the citric acid cycle, can mimic the success of the ketogenic diet even in the presence of standard carbohydrate levels. We have also shown that the proper functioning of the citric acid cycle is crucial to the success of the KD in the context of ME. Furthermore, our data reveal that multiple seizure models, in addition to ATP61, are treatable with the ketogenic diet. Importantly, one of these mutants is TPIsugarkill, which models human glycolytic enzymopathy, an incurable metabolic disorder with severe neurological consequences. Overall, these studies reveal widespread success of the KD in Drosophila, further cementing its status as an excellent model for studies of KD treatment and mechanism, and reveal key insights into the therapeutic potential of dietary therapy against neuronal hyperexcitability in epilepsy and metabolic disease.


Subject(s)
Diet, Ketogenic , Glycolysis , Mitochondrial Encephalomyopathies/diet therapy , Seizures/prevention & control , Animals , Dietary Supplements , Disease Models, Animal , Drosophila , Drosophila Proteins/genetics , Ketone Bodies/administration & dosage , Mitochondrial Encephalomyopathies/complications , Mitochondrial Proton-Translocating ATPases/genetics , Seizures/diet therapy , Seizures/etiology , Triglycerides/administration & dosage
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