PubMed · 1729036
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M Jorge, R Giniger. 1992. Defining the hypoxic threshold.. https://doi.org/10.1097/00003246-199201000-00033
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The peptide nucleic acid (PNA)-directed PCR clamping technique was modified and applied to the detection of mitochondrial DNA mutations with low heteroplasmy. This method is extremely specific, eliminating false positives in the absence of mutant molecules, and highly sensitive, being capable of detecting mutations at the level of 0.1% of total molecules. Moreover, the reaction can be multiplexed to identify more than one mutation per reaction. Using this technique, the levels of three point mutations, the tRNA(Leu(UUA)) 3243 mutation causing mitochondrial encephalopathy, lactic acidosis and stroke-like episodes (MELAS); the tRNA(Lys) 8344 mutation causing myoclonic epilepsy and ragged red fibers (MERRF); and the nucleotide position 414 mutation adjacent to the control region promoters, were evaluated in human brain and muscle from individuals of various ages. While none of the mutations were detected in brain samples from individuals ranging in age from 23 to 93, the 414 mutation could be detected in muscle from individuals 30 years and older. These data demonstrate that the 3243 and 8344 mutations do not accumulate with age to levels greater than 0.1% in brain and muscle. By contrast, the 414 mutation accumulates with age in normal human muscle, though not in brain. The reason for the striking absence of the 414 mutation in aging brain is unknown.
HISTORY AND CLINICAL PRESENTATION: A severely ill 84-year old patient, known to suffer from a chronic congestive heart failure, was admitted to the emergency room because of increasing dyspnea. His blood pressure was stable (125/80), his heart rate normal (66/min), the respiratory rate, however, was elevated at 35/min and he exhibited substantial peripheral edema. There were no signs indicating an infectious process. EXAMINATIONS: Blood gas analysis yielded a marked, respiratorically compensated metabolic acidosis with a base excess of -8.6. The chest roentgenography showed a markedly dilated heart without pulmonary-vascular congestion or infiltrates. The serum lactate was substantially increased at 11.4 mmol/l (0.6-2.44). TREATMENT AND COURSE: A cardiovascular beriberi syndrome was suspected and 100 mg of thiamine administered i.v. Within hours the clinical picture improved significantly. Two hours after administration of thiamine serum lactate concentration was 5.7 mmol/l, about 12 hours later the patient suffered no longer from dyspnea and the lactate was at a normal level. CONCLUSION: Thiamine deficiency as a cause for an elevated serum lactate or lactate acidosis with or without cardiovascular failure may not be overlooked. Without adequate treatment it may end lethally, although it can be treated easily, effectively and without side effects. The potential frequency of thiamine deficiency among the normal population even in our society may be under-estimated due to the particular properties of this vitamin.
Defects in oxidative metabolism may be caused by mutations either in nuclear genes or in mitochondrial DNA (mtDNA). We tested the hypothesis that recombinant adeno-associated virus (rAAV) could be used to complement mtDNA mutations. AAV vector constructs were designed to express the reporter gene encoding green fluorescent protein (GFP), fused to a targeting presequence that directed GFP to be translocated into mitochondria. These vectors mediated expression of mitochondrial-localized GFP, as indicated by fluorescence microscopy and electron microscopy, in respiring human embryonic kidney 293 cells and nonrespiring mtDNA-deficient (rho 0) cells. However, when sequences encoding hydrophobic segments of proteins normally encoded by mtDNA were inserted between the presequence and GFP, mitochondrial import failed to occur. In similar experiments, a fusion was created between pyruvate dehydrogenase (PDH) E1 alpha subunit, a nuclear-encoded mitochondrial gene with its own targeting presequence, and GFP. With this construct, expression of GFP was observed in mitochondria in vitro and in vivo. We conclude that the hydrophobicity of mtDNA-encoded proteins limits their ability to be transported from the cytoplasm. However, rAAV-based gene therapy may hold promise for gene therapy of PDH deficiency, the most common biochemically proven cause of congenital lactic acidosis.