Large hepatic mitochondrial DNA deletions associated with l-lactic acidosis and highly active antiretroviral therapy

Fatal l-lactic acidosis occurred in an AIDS patient during stavudine-containing highly active antiretroviral therapy. l-Lactic acidosis was attributed to mitochondrial dysfunction, and was associated with large deletions (49–83% of the mitochondrial genome) of most of the hepatic mitochondrial DNA, including deletions in genes for nicotinamide adenine dinucleotide (reduced form) dehydrogenase, cytochrome oxidase and adenosine triphosphatase. This 64-year-old male AIDS patient was admitted with prominent extrapyramidal signs, unsteady gait and recurrent falls. HIV had been diagnosed 2 years previously after a severe episode of herpes zoster. Therapy with stavudine, lamivudine, and saquinavir had been commenced 12 months before the current presentation. Other medications included acyclovir, cotrimoxazole, clarithromycin, itraconazole for secondary cryptococcal disease suppression, sodium valproate, mexiletine, and sertraline. His neurological signs resolved with the cessation of sertraline. Severe post-herpetic neuralgia worsened on day 14, and was treated with indomethacin 200 mg twice a day. Within 48 h of indomethacin administration the patient developed severe epigastric pain and vomiting. He became obtunded, hypotensive and developed Kussmaul respirations and signs of peritonism. He was acidotic (pH 7.29) and hyperkalaemic (potassium = 7.8 mmol/l). A necrotic pancreas was found at laparotomy. Continuous venovenous haemodialysis against bicarbonate was required to control hyperkalaemia. The plasma l-lactate concentration increased to 28 mmol/l (normal < 2.5 mmol/l) despite these interventions. Multi-organ dysfunction was not present. The patient maintained elevated cardiac output, low systemic vascular resistance with inappropriately low oxygen consumption, and a respiratory quotient of 1 (carbon dioxide : oxygen consumed). The patient died 72 h postoperatively when treatment was withdrawn. Plasma bicarbonate concentrations were low for at least 2 months before death (12–19 mmol/l, normal 23–32 mmol/l), compatible with subclinical lactic acidosis. Immediate post-mortem specimens of blood, skeletal muscle and liver were obtained for mtDNA analysis. MtDNA was amplified by polymerase chain reaction [1]. Amplified products were analysed using restriction fragment length polymorphisms and selected sequencing. Depletion analysis was performed using a modification of Arnaudo et al. [2]. Specific DNA analysis for mutations associated with myoclonus epilepsy-ragged red fibres, mitochondrial encephalomyopathy–lactic acidosis–stroke-like episodes, and neurogenic muscle weakness, ataxia, retinitis pigmentosa were performed on amplified mtDNA using appropriate polymerase chain reaction primers [3]. There was no evidence of point mutations classically associated with myoclonus epilepsy-ragged red fibres, mitochondrial encephalomyopathy–lactic acidosis–stroke-like episodes, or neurogenic muscle weakness, ataxia, retinitis pigmentosa/Leigh's disease in blood, muscle, liver or pancreas. No evidence of mtDNA depletion was detected in any patient samples or in age-matched controls. No detectable mtDNA deletions were present in peripheral blood leukocytes. However, a small fraction of skeletal muscle mtDNA existed as a 3500 base pair (bp) form (79% deleted). In contrast to blood and muscle mtDNA, most of the hepatic mtDNA (normal size 16 569 bp) existed as one of six distinct deleted forms. The sizes of mtDNA forms present (percentage of mtDNA deleted in parenthesis) were: (i) 8500 bp (49%); (ii) 7600 bp (54%); (iii) 6900 bp (59%); (iv) 6100 bp (63%); (v) 3400 bp (80%); and (vi) 2900 bp (83%). Significant functional impairment of oxidative phosphorylation would be expected with such extensive hepatic mitochondrial damage because five of the six forms had deletions including nicotinamide adenine dinucleotide (reduced form) dehydrogenase, cytochrome oxidase and adenosine triphosphatase genes, based on restriction fragment length polymorphism analysis and selected sequencing (data not shown). We hypothesize that our patient acquired hepatic mtDNA deletions, possibly on a background of pre-existing subclinical hepatic mitochondrial disease, during the course of antiretroviral therapy. The mutagenic effects of nucleoside analogues, such as stavudine, are thought to be mediated via the inhibition of DNA polymerase-γ, which is essential for mitochondrial replication [4]. Indomethacin, an uncoupler of mitochondrial oxidative phosphorylation, may have produced acute decompensation in the setting of marginally functional hepatic mitochondria [5]. Paul B. Bartleya Lorraine Westacottb Robert J. Bootsc Margaret Lawsonb Julia M. Potterb Valentine J. Hylandb Marion L. Woods IIa