Identification of a canine model of pyruvate dehydrogenase phosphatase 1 deficiency.

Exercise intolerance syndromes are well known to be associated with inborn errors of metabolism affecting glycolysis (phosphorylase and phosphofructokinase deficiency) and fatty acid oxidation (palmitoyl carnitine transferase deficiency). We have identified a canine model for profound exercise intolerance caused by a deficit in PDP1 (EC 3.1.3.43), the phosphatase enzyme that activates the pyruvate dehydrogenase complex (PDHc). The Clumber spaniel breed was originated in 1760 by the Duc de Noailles, as a hunting dog with a gentle temperament suitable for the 'elderly gentleman'. Here we report that 20% of the current Clumber and Sussex spaniel population are carriers for a null mutation in PDP1, and that homozygosity produces severe exercise intolerance. Human pyruvate dehydrogenase phosphatase deficiency was recently characterized at the molecular level. However, the nature of the human mutation (loss of a single amino acid altering PDP1 activity) made it impossible to discern the role of the second phosphatase isoform, PDP2, in the deficient phenotype. Here we show that the null mutation in dogs provides a valuable animal model with which to study the effects of dysregulation of the PDHc. Knowledge of the molecular defect has allowed for the institution of a rapid restriction enzyme test for the canine mutation that will allow for selective breeding and has led to a suggested dietary therapy for affected dogs that has proven to be beneficial. Pharmacological and genetic therapies for PDP1 deficiency can now be investigated and the role of PDP2 can be fully characterized.

Novel mutations in dihydrolipoamide dehydrogenase deficiency in two cousins with borderline-normal PDH complex activity.

We have diagnosed dihydrolipoamide dehydrogenase (DLD) deficiency in two male second cousins, who presented with markedly different clinical phenotypes. Patient 1 had a recurrent encephalopathy, and patient 2 had microcephaly and lactic acidosis. Their presentation is unusual, in that the DLD subunit deficiency had little effect on pyruvate dehydrogenase complex activity, but caused a severe reduction in the activities of other enzymes that utilize this subunit. We have identified two mutations in the DLD gene in each patient. The second cousins have one novel mutation in common resulting in a substitution of isoleucine for threonine (I47T), which has not been previously reported in the literature. Patient 1 has a second mutation that has been reported to be common in the Ashkenazi Jewish population, G229C. Patient 2 has a second mutation, E375K, which has also been previously reported in the literature. Enzyme kinetic measurements on patient fibroblasts show that under certain conditions, one heteroallelic mutation may have a higher K(m). This may account for the differing clinical phenotypes. These findings have important repercussions for other patients with similar clinical phenotypes, as DLD activity is not normally measured in cases with normal PDHc activity.

Pyruvate dehydrogenase phosphatase deficiency: identification of the first mutation in two brothers and restoration of activity by protein complementation.

CONTEXT: Pyruvate dehydrogenase phosphatase (PDP) deficiency has been previously reported as an enzymopathy, but the genetic basis for such a defect has never been established. OBJECTIVE: The aim of this study was to identify the cause of the defect in two patients who presented with PDP deficiency. PATIENTS: We studied two brothers of consanguineous parents who presented with neonatal hypotonia, elevated lactate, and less than 25% native pyruvate dehydrogenase complex (PDHc) activity in skin fibroblasts compared with controls. The activity of the complex could be restored to normal values by preincubation of the cells with dichloroacetate or by treating cell extracts with calcium. RESULTS: These two individuals were found to be homozygous for a 3-bp deletion in the coding sequence of the PDP isoform 1 (PDP1), which removes the amino acid residue leucine from position 213 of the protein. A recombinant version of this protein was synthesized and found to have a very reduced (<5%) ability to activate purified PDHc. Reduced steady-state levels of PDP1 in the patient's fibroblasts coupled with the low catalytic activity of the mutant PDP1 resulted in native PDHc activity being reduced, but this could be corrected by the addition of recombinant PDP1 (wild type). CONCLUSION: We have identified mutations in PDP1 in two brothers with PDP deficiency and have proven that the mutation is disease-causing. This is the first demonstration of human disease due to a mutation in PDP1.

Deficiency of pyruvate dehydrogenase caused by novel and known mutations in the E1alpha subunit.

Pyruvate dehydrogenase (PDH)-complex deficiency (OMIM 312170) is a clinically heterogeneous disorder, with phenotypes ranging from fatal lactic acidosis (LA) in the newborn to chronic neurological dysfunction. To date, over 80 different mutations have been identified in the PDHA1 gene in patients with PDH complex deficiency, which are thus thought to contribute to the PDH deficient phenotype. We have identified 14 additional patients with total PDH complex deficiency, all of whom were found to contain mutations within the PDHA1 gene (E(1)alpha subunit). The mutations include both missense mutations and duplications. Eight of these patients had novel mutations, and the remaining had mutations that have been identified previously in PDH complex deficient patients, with residual fibroblast activity ranging from 2.4 to 69% of control values. The nature of these mutations illustrates the variability in phenotype for a given gene defect, with intermittent ataxia being the mildest presentation, Leigh syndrome being the most common and severe neonatal LA the most severe.

Respiratory chain analysis of skin fibroblasts in mitochondrial disease.

NADH:ubiquinone dehydrogenase (complex I) deficiency can be diagnosed from cultured skin fibroblasts using a number of methods, the most commonly used is a linked assay of rotenone-sensitive complex I + III activity (NADH:cytochrome c reductase). Because of interference from diaphorases, this method requires either the isolation of mitochondria (or at least partial purification). For a suitable mitochondrial preparation from skin fibroblasts, this requires the culturing of 4-20 individual 100mm tissue culture plates, depending on the purity of preparation required. These assays are therefore time-consuming, and do not assist in a rapid diagnosis. There is also no clear demarkation between the normal range of activity and the deficient range since mild mutations can produce only partial decreases in complex I activity. Equally, assaying patient cells that do not have a specific deficiency may prove to be time-wasting in the process of providing a quick, definitive clinical diagnosis. The lactate/pyruvate ratio of fibroblasts has been used to indicate the extent of respiratory chain involvement, as cells with a metabolic defect usually produce more lactate with an increased ratio from 25:1 to much higher values [Methods Enzymol. 264 (1996) 454]. This measurement may not always be conclusive, as the values can fluctuate as a result of culture medium, cell passage number, cell number and viability. In this report, we evaluate the use of pyruvate oxidation measurements from whole cells prepared from a single plate of cultured fibroblasts as an alternative to lactate/pyruvate ratios, or other methods both direct and indirect as indicators of the extent of respiratory chain involvement and the possibility of a defect within complex I. Whole cell 2-14C pyruvate oxidation appears to indicate the presence of a complex I defect in patients compared to normal controls more reliably than L/P ratios, but this has some puzzling exceptions.