Highly active antiretroviral therapy (HAART) markedly decreases AIDS progression among HIV-infected individuals.1 However, mitochondrial toxicity resulting from the use of specific antiretrovirals used as part of HAART has been linked to several adverse effects including lipodystrophy, peripheral neuropathy, hepatic steatosis, myopathy, cardiomyopathy, pancreatitis, bone marrow suppression, and lactic acidosis.2-6 Nearly all of these adverse effects resemble clinical symptoms seen in inherited mitochondrial diseases,7,8 suggesting that host mitochondrial genotype may play a role in their development. This hypothesis is supported by previous studies of patients receiving antiretroviral therapy for whom mitochondrial DNA (mtDNA) haplogroup T was overrepresented among patients with peripheral neuropathy in the AIDS Clinical Trials Group cohort9,10 and in 5 patients with haplogroup J who had higher median limb fat change posttherapy compared with other haplogroups.11
One of the most common clinical pathologies associated with HAART is lipoatrophy, a physically disfiguring mitochondriotoxicity that occurs in 13%-63% of patients on HAART.12-17 Associated primarily with the thymidine analogue nucleoside reverse transcriptase inhibitors (NRTIs), zidovudine (AZT) and stavudine (d4T), lipoatrophy is the loss of subcutaneous fat from the face, extremities, and buttocks.18 The distinctive sunken cheeks and wasted appearance can have profound social and psychological impacts for affected persons and can lead to decreased therapy adherence.19-22 Lipoatrophy has also been observed as a prelude to other health risks such as hypertension23 and coronary heart disease.24
Lipoatrophy is caused by a combination of cellular mechanisms including inhibition of mitochondrial gamma polymerase, depleted mtDNA, acquired mtDNA mutations, and oxidative stress.8,25-27 Because mitochondria are critical for energy production and for control of cellular apoptosis, disruption of mitochondrial processes has serious metabolic consequences. Through oxidative phosphorylation (OXPHOS), mitochondria convert calories to adenosine triphosphate (ATP), release heat that maintains body temperature, and generate reactive oxygen species (ROS). Many mitochondrial diseases occur when energy production drops below the energetic threshold for a given process in the cell.28-31 NRTI-induced mtDNA depletion disrupts OXPHOS, likely causing energy deprivation.32 Compromised ATP production in turn may lower fat production because ATP is needed for triglyceride synthesis in adipocytes.33 Further, mitochondrial perturbation and oxidative stress can result in the release of apoptosis-inducing factors causing apoptosis of adipocytes and consequent peripheral fat loss.26,34
The mitochondrial genome encodes 13 proteins that participate in OXPHOS. Variation in these polypeptides may influence energy production efficiency, ROS generation, and levels of apoptosis.35 Mitochondrial variation has been associated with climate adaptation,28,36 susceptibility to neurodegenerative disease,37-40 energy deficiency disease,41,42 longevity,43-45 sperm motility,46,47 sprint performance,48 and microbial infection.49 In a previous study, we also showed that specific mtDNA genotypes are associated with AIDS progression in untreated HIV-infected patients.50
Hence, we sought to determine whether the host mtDNA genotype was associated with propensity for development of lipoatrophy in HIV-infected patients on HAART in the Multicenter AIDS Cohort Study (MACS). We determined the mtDNA haplogroup of 410 male European American patients who had been assessed for lipoatrophy by clinical examination of fat in the limbs and buttocks and investigated whether severity of fat loss was associated with mtDNA genotype.
The MACS is a United States-based ongoing prospective study of HIV-1 infection in adult (aged 18-70 years) men who have sex with men in Baltimore, Chicago, Pittsburgh, and Los Angeles.51 This study focused on men who self-reported as “white.” White Hispanic men were not included due to different genetic background.
Details of Atrophy Assessment
The severity of peripheral atrophy was quantified by a standardized physical exam assessment scale which used mild, moderate, and severe gradations for each of the affected body areas (arms, legs, face, and buttocks). Mild was recorded for atrophic changes that were evident to the MACS clinician upon close inspection; moderate was recorded for changes that were evident without close inspection; and severe atrophy was recorded for atrophic changes that were evident to a nonmedical person by casual observation. Specific anatomic features noting presence of moderate or greater atrophy were prominence of the nasolabial folds, hollowing of the cheeks (sunken cheeks), peripheral venous prominence, and apparent bony landmarks. Physical exams were performed during the HAART era between 1999 and 2006.
DNAs were extracted from immortal lymphoblastoid B-cell lines for each patient. Six haplotype-tagging single-nucleotide polymorphisms (SNPs) were used initially to classify individuals as mitochondrial macro-haplogroups N, M, and L groups. Haplogroups within the western European (N) subset were further parsed using SNPs in the mitochondrial haplogrouping using candidate functional variants approach as described in Hendrickson et al.50 The SNPs used in this study to identify the major haplogroups and subgroups within H are shown in Figure 1. Based on the hierarchical nature of the human mtDNA tree, we devised a strategy for identifying haplotypes by subdividing the samples using highly conserved polymorphic sites located at key haplogroup branch points. Genotyping was performed using TaqMan Assays-by-DesignSM. Thermocycling conditions were an initial 95°C hold for 3 minutes, followed by 30 cycles of 92°C for 15 seconds, and 56°-62°C annealing for 1 minute depending on primer specificity. Haplogroups were compared against the remaining haplogroups in statistical analyses. Rare, loosely associated haplogroups R*, HV* and JT* were excluded from individual analyses but included in controls.
Associations between mitochondrial haplogroup and severity of lipoatrophy were assessed with proportional odds logistic regression.52,53 All analyses were performed with SAS version 9.1 (SAS Institute, Inc, Cary, NC). Sensitivity seemed to vary between the measures of atrophy on arms, legs, and buttocks; therefore, we analyzed each lipoatrophy assessment separately. Further, we attempted an analysis of the average of these values; however, the score test was significant suggesting it violated the model assumptions, likely due to small cell size in the uppermost levels of severity.54 We used backward selection to test environmental variables to include in our proportional odds logistic regression models. Age at HAART initiation (P = 0.03), body mass index (BMI) at the time of lipoatrophy assessment (P < 0.0001), and AZT and d4T use (both P < 0.0001) were all significant at the P ≤ 0.05. In a previous study, cumulative exposure to NRTIs was associated with decreases in BMI and body circumference over 5 years of follow-up among HIV-infected men in the MACS cohort,55 therefore, we used a continuous variable to account to the number of visits (6-month intervals) before assessment at which a patient was taking either d4T or AZT.
HIV-1-infected Caucasian men have an increased prevalence of lipoatrophy56; therefore, the European American men on HAART in the MACS represent a high-risk group. We successfully genotyped 536 patients who self-identified as “white” and had a western European, or “N”, mitochondrial macro-haplogroup. Individuals who had L or M macro-haplogroups (found in Africa and East Asia) were excluded from the study, and those within the N macro-haplogroup were further parsed into haplogroups H, T, IWX, J, T, V, and U. A complete clinical data set for all variables used in the final analyses of lipoatrophy and mitochondrial haplogroup association was available from 410 of these patients.
Clinical characteristics of study participants are shown in Table 1. Age at HAART initiation, BMI at the time of lipoatrophy assessment, and cumulative AZT and d4T exposure were all significantly associated with an increased incidence of lipoatrophy, consistent with previous studies33 (Table 2). Age was only strongly significant for atrophy in the legs according to our models, but because of its known importance in previous studies, we included it in all models. We also evaluated whether tenofovir, which has been reported to cause lipodystrophy in a small percentage of patients,57 or nelfinavir were associated with lipoatrophy but found no associations between their use and lipoatrophy in the MACS.
Mitochondrial haplogroup H was strongly associated with significant increases in extremity lipoatrophy (arms: P = 0.007, odds ratio (OR) = 1.77, 95% confidence interval (CI) = 1.17 to 2.69; legs: P = 0.03, OR = 1.54, 95% CI = 1.03 to 2.31) (Table 3). We also observed a trend for increased lipoatrophy in the closely related V haplogroup (P = 0.07, OR = 2.59, 95% CI = 0.93 to 7.26). The phylogenetic tree of the major haplogroups is shown in Figure 1. In contrast, weak significance suggesting a protective effect against lipoatrophy was observed with haplogroup T (P = 0.05). No significant associations were observed for haplogroup J, which is closely related to T, however, ORs were consistently protective.
Because BMI is a confounding variable during atrophy assessment but is also biologically related to atrophy, we repeated the analysis without BMI as a covariate in the model. Results were generally the same but with slightly weaker associations observed. The association between haplogroup H and increased arm atrophy remained significant (P = 0.021, OR = 1.60, 95% CI = 1.07 to 2.38), but associations with buttock and leg lipoatrophy became nonsignificant (P values of 0.15 and 0.08, respectively). The association between haplogroup T and buttock lipoatrophy diminished to borderline significance (P = 0.07). All other results were nonsignificant.
Haplogroup H is composed of 6 distinct subhaplogroups (H1-H6), which are separated by SNPs 3010 G > A in 16S rRNA (noncoding), 1438 A > G in the 12S gene (consensus), 6776 C > T in the cytochrome oxidase I (synonymous), 4024 G > A in NADH dehydrogenase 1 (ND1) (T240A), 4336 C > T in TQ (tRNA glutamine) and 3915 A > G in NDI (synonymous) as shown in Figure 1B. The haplogroups defined by 3010, 4336, and the H* (the remaining unclassified H mtDNA) haplogroup demonstrated significant associations with lipoatrophy in the same direction as the H haplogroup. The other haplogroups occurred infrequently (<4%) in our sample; therefore, any lack of association may simply be a consequence of their low prevalence and lack of power (for genotypes with frequency ∼4%, power is only 13% for a OR = 1.5 at α = 0.05).
Lipoatrophy and lipoaccumulation may arise via different mechanisms because they represent extremes in metabolism and are often independent.22 In our patients, the presence of lipoaccumulation in the back of the neck, known as a dorsocervical fat pad or “buffalo hump,” was correlated with lipoatrophy with a Pearson correlation coefficient of 0.2 (P < 0.0001). We saw a trend for T to be protective against the presence of buffalo hump (P = 0.06, OR = 0.30, 95% CI = 0.09 to 1.04), but no other statistically significant associations were observed.
We examined the genetic association between 6 major European mtDNA haplogroups and clinical severity of lipoatrophy in 410 patients receiving HAART in the MACS cohort. We found a significant association between haplogroup H and increased risk for lipoatrophy among men graded on lipoatrophy presence and severity in legs, arms, and buttocks. We also observed a borderline significant association between the presence of haplogroup T and protection against lipoatrophy. In the context of previous studies of fat accumulation conducted in the AIDS Clinical Trials Group cohort, we did not observe a significant association between the presence of haplogroup J and protection against fat loss as reported by Hulgan et al11; however, ORs in our study suggest that J may be protective against lipoatrophy.
We recently investigated the relationship between mitochondrial haplogroups and rate of progression to AIDS in untreated patients infected with HIV, where we observed an association between the J haplogroup and accelerated AIDS progression and associations between certain U haplogroups and IWX and progression to disease.50 Although the effects of HIV virus and drugs would likely influence mitochondrial function through different mechanisms, it was important to determine whether mtDNA haplogroups associated with disease progression in untreated patients were later affiliated with adverse events in patients on HAART. In patients on HAART in the present study, no significant associations were found between J and lipoatrophy despite the strong association between J and accelerated AIDS progression in untreated patients. Further, we found strong associations between haplogroup H and increased risk of lipoatrophy, however, we saw no overall association between haplogroup H and progression in untreated patients, and one group within H (H3, which contains a mutation 6776 C > T) was found to be protective against AIDS progression and death in transfusion patients. These data suggest that the risk of drug toxicities associated with different mitochondrial haplogroups in patients on NRTIs is independent of associations between mitochondrial haplogroups and disease progression in untreated patients.
Although the mechanism by which lipoatrophy develops during NRTI exposure has not been elucidated, one of the proposed mechanisms is that the release of apoptosis-inducing factors by damaged mitochondria cause apoptosis of adipocytes which leads to peripheral fat loss.26,34 Variation in mtDNA among haplogroups may influence energy production efficiency, ROS generation, and levels of apoptosis. H in particular is thought to be tightly coupled to the production of ATP and consequently generates more ROS, whereas J and T are partially uncoupled, thus produce less ATP and therefore less ROS.35 In patients with the H haplogroup, oxidative stress and subsequent apoptosis could be exacerbated by increased baseline ROS production compared with other mitochondrial haplogroups. Further, oxidative stress has also been proposed as a principle mechanism operative in NRTI-related mitochondrial toxicity that leads to mtDNA depletion and subsequent mitochondrial energetic deficiencies.27 Again, the threshold effect of increased ROS production in the tightly coupled H haplogroup may worsen this effect. On the other hand, haplogroups J and T, which are uncoupled, had less atrophy, consistent with this hypothesis. However, the consistency in the protection of the T haplotype against both lipoatrophy and the presence of “buffalo hump” (P = 0.07) seems to support the hypothesis that fat loss and accumulation are part of a single syndrome,58,59 and a better understanding of the pathology of NRTI-related lipodystrophy and additional genetic studies are needed to elucidate a mechanistic relationship between mitochondrial haplogroup and altered fat distribution.
One other potential explanation for the observed associations involves consideration of the strong phylogeographic structure of mitochondrial haplogroups. Because of this phenomenon, it is possible that the associations observed in our study are correlated with background nuclear genetic effects that are distinctive between geographically separated populations. However, population stratification analysis using 304 autosomal markers in a previous study50 and ongoing work in our laboratory based on a genome scan did not reveal significant geographic structure in the mitochondrial haplogroups associated with lipoatrophy. Regardless, it will be important to repeat these associations in populations of different ethnic background.
Our results would no longer be significant if a conservative Bonferoni correction was performed, however, we did see more significant associations than would be expected by chance, which implies that mitochondrial genotype has at least a moderate effect on lipoatrophy. We also realize that lipoatrophy is difficult to assess, and therefore, the results could potentially mask a stronger relationship. An indication that this may be the case is the very strong association between low BMI and severe atrophy. This inverse correlation may reflect the slowed diagnosis of moderate to severe atrophy in those with very high BMIs and hence very high peripheral fat depots.
Although many mechanisms are likely involved in the development of mitochondrial dysfunction and subsequent lipoatrophy, this study demonstrates that mitochondrial haplogroup may be an important genetic factor in the development of lipoatrophy associated with NRTI treatment in HIV-infected persons.
The MACS includes the following-Baltimore: The Johns Hopkins University Bloomberg School of Public Health: Joseph B. Margolick (principal investigator), Haroutune Armenian, Barbara Crain, Adrian Dobs, Homayoon Farzadegan, Joel Gallant, John Hylton, Lisette Johnson, Shenghan Lai, Ned Sacktor, Ola Selnes, James Shepard, and Chloe Thio; Chicago: Howard Brown Health Center, Feinberg School of Medicine, Northwestern University, and Cook County Bureau of Health Services: John P. Phair (principal Investigator), Joan S. Chmiel (coprincipal investigator), Sheila Badri, Bruce Cohen, Craig Conover, Maurice O'Gorman, David Ostrow, Frank Palella, Daina Variakojis, and Steven M. Wolinsky; Los Angeles: University of California, UCLA Schools of Public Health and Medicine: Roger Detels (principal investigator), Barbara R. Visscher (coprincipal investigator), Aaron Aronow, Robert Bolan, Elizabeth Breen, Anthony Butch, Thomas Coates, Rita Effros, John Fahey, Beth Jamieson, Otoniel Martínez-Maza, Eric N. Miller, John Oishi, Paul Satz, Harry Vinters, Dorothy Wiley, Mallory Witt, Otto Yang, Stephen Young, and Zuo Feng Zhang; Pittsburgh: University of Pittsburgh, Graduate School of Public Health: Charles R. Rinaldo (principal investigator), Lawrence Kingsley (coprincipal investigator), James T. Becker, Robert L. Cook, Robert W. Evans, John Mellors, Sharon Riddler, and Anthony Silvestre; Data Coordinating Center: The Johns Hopkins University Bloomberg School of Public Health: Lisa P. Jacobson (principal investigator), Alvaro Muñoz (coprincipal investigator), Keri Althoff, Christopher Cox, Gypsyamber D'Souza, Stephen J. Gange, Elizabeth Golub, Janet Schollenberger, Eric C. Seaberg, and Sol Su. Mational Institutes of Health: National Institute of Allergy and Infectious Diseases: Robin E. Huebner; National Cancer Institute: Geraldina Dominguez; National Heart, Lung, and Blood Institute: Cheryl McDonald. Website located at http://www.statepi.jhsph.edu/macs/macs.html. We also thank Mike Malasky and Mary McNally of the Laboratory of Genomic Diversity Genotyping Core and Holli Hutcheson who was involved in the initial genotyping of patients.
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