Peripheral neuropathy of any grade was reported in 147 (29%) genetic study participants during ACTG 384, 108 (73%) of whom had been randomized to receive ddI plus d4T, and 39 (27%) to receive ZDV plus 3TC (P < 0.001). Sixty-one participants (42%) developed at least grade 2 peripheral neuropathy. Participants who developed peripheral neuropathy had lower baseline CD4 T-cell counts (P = 0.003), higher baseline plasma HIV-1 RNA concentrations (P = 0.004), and were older at study entry (P = 0.0001) than those who did not develop peripheral neuropathy (Table 1). The proportion of study participants who developed peripheral neuropathy did not differ by race or sex (P > 0.3 for each). Among white participants, those who developed peripheral neuropathy were older (P = 0.01) and more likely to have been randomized to a d4T plus ddI treatment arm (P = 0.007) than those who did not. They also tended to have higher baseline HIV RNA and lower CD4 T-cell counts, but these comparisons were not significant (P > 0.1 for both).
We attempted to characterize the polymorphisms that define nine European mitochondrial haplogroups in all study participants. Because mitochondrial haplogroups for African–Americans are not well characterized, we focused on haplogroups that have been described among Caucasians in Europe and North America [16,19]. Approximately 95% of whites could be assigned to known European haplogroups (Table 2). Six (2.3%) did not have a polymorphism pattern characteristic of any of these groups, and one or more polymorphic sites were unresolved in seven (2.7%). Haplogroup frequencies among whites were similar to those previously reported , with the signature polymorphism for haplogroup T present in 10%.
Of the nine European mitochondrial haplogroups, only haplogroup T was seen with greater frequency in participants with peripheral neuropathy (Table 2). Among white participants, 17.1% of those who developed peripheral neuropathy belonged to haplogroup T compared to 6.7% of those who did not develop peripheral neuropathy (OR, 2.8; 95% CI, 1.1–7.1; P = 0.03; Fig. 2a). This relationship was strongest among the 137 whites randomized to receive ddI plus d4T. In this subgroup, 10 of 48 (20.8%) who developed peripheral neuropathy belonged to haplogroup T, while four of 89 (4.5%) who did not develop peripheral neuropathy belonged to this haplogroup (OR, 5.4; 95% CI, 1.4–25.1; P = 0.009; Fig. 2b). We used an intent-to-treat analysis, but some participants had switched from the initially randomized NRTI prior to documented peripheral neuropathy. Results were similar when this analysis was repeated based on NRTI prescribed at the time closest to documented peripheral neuropathy (data not shown).
To identify independent predictors of peripheral neuropathy in white study participants, we used a multivariate logistic regression model that included age, sex, randomization arm (ddI plus d4T or ZDV plus 3TC, as well as blinded EFV, NFV, or both), baseline CD4 T-cell count, baseline plasma HIV RNA concentration, and mitochondrial haplogroup T (Table 3). Factors significantly associated with development of peripheral neuropathy in this model were randomization to receive ddI plus d4T (OR, 2.57 versus ZDV plus 3TC; 95% CI, 1.37–4.83; P = 0.003), older age at randomization (OR, 1.05 per year; 95% CI, 1.02–1.09; P = 0.005), and mitochondrial haplogroup T (OR, 2.89; 95% CI, 1.17–7.13; P = 0.02).
In this study, we observed that white ACTG 384 participants who belonged to mitochondrial haplogroup T were at increased risk of developing peripheral neuropathy during antiretroviral therapy, particularly if they had been randomized to receive ddI plus d4T. By multivariate analysis, randomization to receive ddI plus d4T, older age, and belonging to mitochondrial haplogroup T were independent predictors of peripheral neuropathy. To our knowledge, this is the first study to identify a possible genetic predictor of peripheral neuropathy during antiretroviral therapy, and the first to characterize mitochondrial haplogroups among HIV-infected individuals.
There is considerable evidence that peripheral neuropathy and other NRTI toxicities involve inhibition of mitochondrial DNA polymerase-γ, ultimately leading to mitochondrial dysfunction, impaired oxidative phosphorylation, and tissue injury [4,24,25]. Incubation of T-lymphoblastoid cell lines with NRTI results in mitochondrial DNA depletion, morphologic changes of mitochondria, and increased lactic acid production [26–29]. In a rabbit model of drug-induced peripheral neuropathy, mitochondria in neuronal cells from animals exposed to the dideoxynucleoside zalcitabine (ddC) exhibited structural changes . Among HIV-infected individuals with ddC-induced peripheral neuropathy, mitochondria in peripheral nerves were morphologically abnormal, and mitochondrial DNA content was decreased . Importantly, the tendency of NRTI to cause peripheral neuropathy and other putative mitochondrial toxicities approximates their in vitro affinities for mitochondrial DNA polymerase-γ (ddC > ddI and d4T > other NRTI) . In addition, there are in vitro and animal data supporting effects of NRTI on mitochondria independent of mitochondrial DNA polymerase-γ [31,32]. Clinical manifestations of some inherited disorders of mitochondrial function and oxidative phosphorylation resemble NRTI-associated toxicities, and can include neuropathies and other neurodegenerative phenotypes [12,13]. These observations provide rationale for a link between mitochondrial genetics and NRTI-induced peripheral neuropathy.
Mitochondrial haplogroups have been most extensively used to understand prehistoric human migrations . Only recently has the potential relevance of functional differences between mitochondrial haplogroups to human health and disease been appreciated. Studies in northern Italy  and Finland  have identified greater longevity among persons belonging to mitochondrial haplogroup J. In a recent age- and sex-matched case–control study involving persons of European ancestry, Parkinson disease was significantly less frequent among persons belonging to haplogroups J or K . A similar observation has been reported in a Finnish population . Parkinson disease is believed to result, at least in part, from mitochondrial impairment, free radical-induced oxidant injury, and neurodegeneration , similar to NRTI-associated peripheral neuropathy. Molecular mechanisms underlying functional differences between haplogroups are unknown.
The polymorphisms that define mitochondrial haplogroups are homoplasmic, and their functional implications are likely to be subtle, contributing to susceptibility to additional genetic or epigenetic factors, such as drugs or environmental toxins. These differ from somatic mutations that accumulate in mitochondrial DNA during the lifetime of an individual, resulting in heteroplasmic mixtures of mutant and wild-type DNA that are present in variable proportions of mitochondria and are often tissue specific . Heteroplasmy can also directly affect expression of rare mitochondrial diseases in which the proportion of mutant mitochondrial DNA in a given tissue can determine the clinical phenotype .
There are possible explanations for a relationship between mitochondrial haplogroup T and peripheral neuropathy. Although aberrations in mitochondrial DNA can impair energy production , haplogroup T has not been previously associated with mitochondrial dysfunction or disease. In addition, the signature haplogroup T polymorphism at position 13368, located in the ND5 region of the mitochondrial gene encoding complex I respiratory chain subunits, does not change an amino acid is therefore unlikely to directly alter mitochondrial function. However, polymorphisms that change amino acids have been described in other mitochondrial diseases and are reported in persons belonging to the T haplogroup . Such polymorphisms may affect the efficiency of oxidative phosphorylation to an extent that is not apparent under basal conditions, but is unmasked by exposure to additional stressors, such as NRTI. Importantly, mitochondrial polymorphisms that influence susceptibility to toxic exposures could plausibly explain variations in clinical phenotypes resulting from both mitochondrial DNA polymerase-γ dependent and independent mechanisms.
There are limited previous data to support an association between mitochondrial haplogroups, NRTI, and susceptibility to drug neurotoxicity. There are reports of HIV-infected persons with Leber hereditary optic neuropathy, a rare cause of inherited blindness that typically occurs in the third decade of life and is associated with one of three mitochondrial DNA mutations in a majority of cases . Not all persons with one of these mutations lose vision, however, suggesting that additional genetic or epigenetic factors are necessary for disease expression. In all six cases of Leber hereditary optic neuropathy reported in HIV-infected persons, the atypically late onset of visual loss occurred during NRTI therapy [41–45]. With respect to associations between mitochondrial haplogroups and drug toxicity in general, one study suggested that cisplatin-induced mitochondrial ototoxicity may be more frequent among persons belonging to European haplogroup J . These reports support the concept that exposure to drugs, and NRTI in particular, can precipitate clinical mitochondrial dysfunction in genetically predisposed individuals.
The relationship between mitochondrial haplogroup T and peripheral neuropathy must be validated in independent cohorts. In addition, because African–American haplogroups have not been well defined, our ability to identify potential genetic effects in this study was limited to whites. Similar analyses must be extended to African–American and other non-European haplogroups. Peripheral neuropathy is a complex phenotype, and it is possible that this outcome was misclassified in some subjects. However, if a diagnosis of NRTI-associated peripheral neuropathy was assigned when it was not present, or not recorded when present, this should have biased our results toward finding no effect. Peripheral neuropathy caused by HIV infection rather than NRTI exposure was unlikely in this study, since study participants received potent antiretroviral therapy and most had excellent virologic responses . The sample size was too small to assess haplogroup differences based on severity of peripheral neuropathy.
Current guidelines recommend avoiding combined use of ddI and d4T because of increased toxicity , but these and other NRTI are still widely prescribed. Because of its ease of generic production and co-formulation with other antiretroviral drugs, d4T is a particularly important antiretroviral agent in resource limited settings . As the AIDS epidemic has increasingly affected persons of non-European ancestry, it is critical to define relationships between mitochondrial haplogroups, peripheral neuropathy, and other NRTI toxicities in these populations. At present, there are no reliable clinical predictors of NRTI-associated peripheral neuropathy, and available therapies are of limited efficacy . Identifying individuals at greatest risk could further influence prescribing practices to avoid this complication. In addition, the results of the present study may guide biochemical investigations to elucidate functional mechanisms that underlie the relationship between mitochondrial haplogroup T and NRTI-associated peripheral neuropathy.
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Other members of the ACTG 384 team were M. Fischl (Miami University), M. Dube (Indiana University), C. Pettinelli (National Institutes of Health), R. Delapenha (Howard University), B. Putnam (University of Colorado), M. Klebert (Washington University), M. Testa (Harvard School of Public Health), A. Chiesi, C. Tomino (Istituto Superiore de Sanita), S. Deeks (University of California, San Francisco), T. Nevin (Social & Scientific Systems), J. Levin, V. French, O. Fennell (Adult AIDS Clinical Trials Group Community Constituency Group), M. Stevens, R. Grosso, B. Dusak, S. Hodder (Bristol-Myers Squibb), J. Tolson, C. Brothers (GlaxoSmithKline), R. Leavitt (Merck), D. Manion, N. Ruiz, K. Morrissey (DuPont Pharmaceuticals), M. Becker, B. Quart (Agouron), C. Jennings (Northwestern University), L. Gedeon, S. Dascomb, M. Cooper, M. Murphy, K. Blakelock (Frontier Science and Technology Foundation), A. Doolan (Massachusetts General Hospital).
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