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No Longitudinal Mitochondrial DNA Sequence Changes in HIV-infected Individuals With and Without Lipoatrophy

ORTIZ, M., et al.

Abstract

The potential for mitochondrial (mt) DNA mutation accumulation during antiretroviral therapy (ART), and preferential accumulation in patients with lipoatrophy compared with control participants, remains controversial. We sequenced the entire mitochondrial genome, both before ART and after ART exposure, in 29 human immunodeficiency virus (HIV)–infected Swiss HIV Cohort Study participants initiating a first-line thymidine analogue–containing ART regimen. No accumulation of mtDNA mutations or deletions was detected in 13 participants who developed lipoatrophy or in 16 control participants after significant and comparable ART exposure (median duration, 3.3 and 3.7 years, respectively). In HIV-infected persons, the development of lipoatrophy is unlikely to be associated with accumulation of mtDNA mutations detectable in peripheral blood.

ORTIZ, M., et al . No Longitudinal Mitochondrial DNA Sequence Changes in HIV-infected

Individuals With and Without Lipoatrophy. The Journal of Infectious Diseases , 2011, vol. 203, no. 5, p. 620-624

DOI : 10.1093/infdis/jiq106 PMID : 21227914

Available at:

http://archive-ouverte.unige.ch/unige:13239

Disclaimer: layout of this document may differ from the published version.

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B R I E F R E P O R T

No Longitudinal Mitochondrial DNA Sequence Changes in

HIV-infected Individuals With and Without Lipoatrophy

Milla´n Ortiz,1,a Estella S. Poloni,3,a Hansjakob Furrer,5 Helen Kovari,6 Raquel Martinez,1 Mireia Arnedo,1,11 Luigia Elzi,7 Enos Bernasconi,9 Pietro Vernazza,10 Bernard Hirschel,4 Matthias Cavassini,2 Bruno Ledergerber,6Huldrych F. Gu¨nthard,6Amalio Telenti,1Philip E. Tarr,8 and the Swiss HIV Cohort Study

1Institute of Microbiology, University Hospital Center and University of Lausanne;

2Infectious Diseases Service, University Hospital, Lausanne;3Laboratory of Anthropology, Genetics and Peopling History, Department of Anthropology, University of Geneva;4Infectious Diseases Service, University Hospital, Geneva;5Clinic for Infectious Diseases, Bern University Hospital and University of Bern, Bern;6Division of Infectious Diseases and Hospital Epidemiology, University Hospital Zurich and University of Zurich, Zurich;7Infectious Diseases Service, University Hospital;

8Infectious Diseases Service, Kantonsspital Bruderholz, University of Basel, Basel;

9Infectious Diseases Service, Ospedale Regionale, Lugano;10Infectious Diseases Service, Kantonsspital, St Gallen, Switzerland; and11Retrovirology and Viral Immunopathology Laboratory, Hospital Cli´nic-Institut d'Investigacions Biome`diques August Pi i Sunyer, University of Barcelona, Barcelona, Spain

The potential for mitochondrial (mt) DNA mutation accu- mulation during antiretroviral therapy (ART), and prefer- ential accumulation in patients with lipoatrophy compared with control participants, remains controversial. We se- quenced the entire mitochondrial genome, both before ART and after ART exposure, in 29 human immunodeficiency virus (HIV)–infected Swiss HIV Cohort Study participants initiating a first-line thymidine analogue–containing ART regimen. No accumulation of mtDNA mutations or deletions was detected in 13 participants who developed lipoatrophy or in 16 control participants after significant and comparable ART exposure (median duration, 3.3 and 3.7 years, re- spectively). In HIV-infected persons, the development of lipoatrophy is unlikely to be associated with accumulation of mtDNA mutations detectable in peripheral blood.

Lipoatrophy is an important and poorly reversible complication associated with human immunodeficiency virus (HIV) infection and antiretroviral therapy (ART), particularly the thymidine- analogue nucleoside reverse-transcriptase inhibitors, stavudine and zidovudine. The pathogenesis of lipoatrophy is in- completely understood. A widely implicated mechanism is mi- tochondrial dysfunction, which is associated with HIV infection and thymidine-analogue treatment [1].

Several studies now suggest a genetic predisposition to lipoatrophy, but results have not been uniform regarding, for example, an association with certain mitochondrial DNA (mtDNA) haplogroups [2–4]. A related question is the po- tential for mtDNA genomic damage, possibly induced by thymidine analogues, to contribute to lipoatrophy [5, 6]. The accumulation of mtDNA point mutations has been experi- mentally linked to premature aging [7], and advancing age is an unexplained risk factor for lipoatrophy in HIV-infected persons [8]. Martin et al [5] recorded the accumulation during ART of multiple mutations in mtDNA extracted from the peripheral blood. The mtDNA mutations were preferen- tially seen in patients who developed lipoatrophy compared with control participants without lipoatrophy. However, the lipoatrophy case patients had more intensive ART exposure than the control patients; therefore, it remained possible that lipoatrophy or mtDNA mutations might have developed with prolonged ART exposure in the participants classified as controls [5]. In a second study with longitudinal mtDNA samples, McComsey et al [6] observed no accumulation of mtDNA mutations in peripheral blood samples, but the me- dian duration of follow-up was only 13 months.

This study was therefore conducted to reassess the value of mitochondrial genome sequencing in HIV-infected participants with and without lipoatrophy, and with significant and com- parable ART exposure. We assessed a potential correlation be- tween the development of lipoatrophy and the accumulation of mtDNA mutations in peripheral blood.

METHODS

Study participants were enrolled in the Swiss HIV Cohort Study (SHCS; http://www.shcs.ch), which involves a standardized clinical evaluation of fat loss and fat accumulation every 6 months. Participants gave written, informed consent for ge- netic testing. The SHCS Genetics Project was approved by the ethics committees of participating centers. Participants were male and self-identified as white, to minimize the influence

Received 25 August 2010; accepted 23 November 2010.

Potential conflicts of interest: none reported.

aM.O. and E.S.P contributed equally to the study.

Reprints or correspondence: Philip E. Tarr, MD, Infectious Diseases Service, 4101 Kantonsspital Bruderholz, University of Basel, Basel, Switzerland (philip.tarr@unibas.ch) and Dr. Amalio Telenti, Institute for Microbiology, University of Lausanne, 1011 Lausanne-CHUV, Switzerland (amalio.telenti@chuv.ch).

The Journal of Infectious Diseases 2011;1–5

ÓThe Author 2011. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail:

journals.permissions@oup.com 1537-6613/2011/05-0001$15.00 DOI: 10.1093/infdis/jiq106

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of sex and of population structure on mtDNA sequences.

Each participant started a first-line zidovudine- or stavudine- containing combination ART regimen after 1 April 1998, had a cell sample stored prior to ART initiation, and had no fat loss recorded prior to ART. Case patients developed significant fat loss (patient/physician agreement;>2 body sites), consistently recorded at >4 subsequent, biannual SHCS visits, without reversion to no fat loss. To avoid excluding participants who rapidly developed lipoatrophy, we required no minimum du- ration of ART. Controls had no fat loss for>3 years after ART initiation.

We permitted modifications of ART components other than zidovudine or stavudine. We excluded participants with ART interruption for>30 days as well as those with initial immune reconstitution followed by a CD4 cell decline to,200 cells/lL, to exclude significant complications (eg, tuberculosis or lym- phoma) as confounding factors for fat loss. To exclude ART nonadherence or active viral replication as a confounder for the presence or absence of lipoatrophy, all participants had HIV RNA counts of ,400 copies/mL starting >1 year after ART initiation.

The treating HIV physician confirmed in writing the presence or absence of lipoatrophy and interviewed each participant re- garding his ancestry. We assessed the value of clinical lipoa- trophy classification by quantitative assessment of total and regional fat mass by dual-energy x-ray absorptiometry (DEXA;

Hologic QDR-4500W) in a subset of study participants. DEXA provided quantitative assessments of total body mass, peripheral (sum of arm and leg fat) and total fat mass, and the ratio be- tween peripheral and total fat mass to adjust for differences in body weight [9].

Sequencing and Mutational Screening of mtDNA

We extracted mtDNA from frozen leukocyte pellets. We se- quenced the entire genomic (16569 bp) mtDNA at 2 time points: prior to any ART exposure, and after lipoatrophy di- agnosis (cases) or after>3 years of ART (controls). Polymerase chain reaction (PCR) amplification and direct forward and re- verse sequencing was performed by Macrogen, using BigDye Terminator sequencing chemistry on a 3730XL DNA Sequencer (Applied Biosystems). Primers and PCR conditions were as published elsewhere [10]. ABI PRISM SeqScape software (ver- sion 2.6; Applied Biosystems) was used to analyze the electro- pherogram files. All sequences were analyzed without knowledge of sample classification as case or control. We performed quality checks of mtDNA sequences as described in the supplementary material. We aligned mtDNA sequences and compared them with the revised Cambridge reference sequence (available at http://jid.oxfordjournals.org/) using SeqScape and further manual check. We classified variant positions relative to this reference sequence either as phylogenetically informative (useful to assign a particular sequence to a mtDNA haplogroup) or

uninformative [11]. Among the latter class, we distinguished mutations occurring in the mtDNA coding region, which have the potential to be nonsynonymous, from those occurring in the noncoding control region. We used 3 large databases to identify known sequence variants in humans: PhyloTree (http://

www.phylotree.org), MITOMAP (http://www.mitomap.org), and mtDB (http://www.genpat.uu.se/mtDB/). We made classi- fications into haplogroups using the phylogenetic tree of global human mtDNA variation by van Oven and Kayser [12], avail- able at PhyloTree.

RESULTS

We included 13 case patients with lipoatrophy and 16 control patients without lipoatrophy, whose characteristics are shown in Table 1. Lipoatrophy was first noted in case patients after a median ART duration of 2 years (interquartile range [IQR], 1.5–3.3 years). Of the 6 case patients and 4 control patients that underwent DEXA, the cases had higher body weight, total mass, and total fat, and slightly more peripheral fat than the controls, but despite this, their peripheral to total fat ratio was decreased, consistent with lipoatrophy [9]. However, these dif- ferences were not statistically significant in this small study population (Table 1).

The entire mtDNA was sequenced from samples taken before ART and after a median ART duration of 3.3 years in cases and 3.7 years in controls. Comparison of each participant’s mtDNA sequences before and during ART revealed no point mutations, no insertions or deletions, and no evidence of point hetero- plasmy in any samples (Supplementary Table 1).

Participants belonged to the main mtDNA haplogroups H, U, K, J, and L (13, 7, 2, 6, and 1 participant, respectively). Thus, H and U were most frequent haplogroups, followed by J and K, findings that are consistent with the European origin of the participants (see Supplementary Results), whereas 1 participant carried an L haplogroup (L0a2a2a). By pooling certain hap- logroups (ie, U and K; J and L), a formal test of homogeneity showed that this simplified haplotype distribution did not de- viate from 3 previously published western European regional samples (P5.55,P5.40, andP5.14, respectively; see Sup- plementary Results). A slight excess of haplogroup J (21%) was observed in the study population, compared with 10%, 9%, and 6%, respectively, in the regional European samples.

Case participants who belonged to mtDNA haplogroups H, U, K, J, and L numbered 6, 4, 1, 2, and 0, respectively, and control participants numbered 7, 3, 1, 4, and 1. No formal comparison of the frequency distribution of haplogroups be- tween cases and controls was feasible because of limited sample size. Single haplogroup frequency comparison indicated that the frequency of haplogroup H did not differ between cases and controls (P..05), and haplogroup J was more frequent among

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Table 1. Clinical Characteristics of 13 Case Patients With Lipoatrophy and 16 Control Patients Without Lipoatrophy Who Had Previously Initiated a First-line thymidine analogue–containing Antiretroviral Therapy Regimen

Lipoatrophy cases (n513) Controls without lipoatrophy (n516) P1

Age at pre-ART mtDNA sample, median (IQR), years 51.8 (46.4–62.8) 45.1 (39.4–55) .16

Age at ART initiation, median (IQR), years 53.2 (46.4–62.8) 45.2 (39.6–55.1) .12

CD4 cell count at ART initiation, median (IQR), cells/lL 206 (142–224) 178 (117–238) .90

HIV RNA at ART initiation, median (IQR), copies/mL 77,000 (32,000–93,000) 176,000 (97,000–218,000) .03

Body weight at ART initiation, median (IQR), kg 72 (67–78) 70 (64–74) .41

Body mass index at ART initiation, median (IQR), kg/m2 23 (21.7–26.1) 22.5 (21.8–25.2) .46

Initial ART regimen NRTI components: ZDV13TC (n511),

d4T1DDI (n51), d4T13TC (n51);

Third agent: efavirenz (n56), nelfinavir (n54), lopinavir/ritonavir (n52), indinavir (n51)

NRTI components: ZDV13TC (n512), ZDV13TC1ABC (n51), d4T1DDI (n52), d4T13TC (n51);

Third agent: efavirenz (n55), nelfinavir (n54), lopinavir/ritonavir (n54), indinavir (n52), saquinavir/ritonavir (n51)

.81 .50

Age at during-ART mtDNA sample, median (IQR), years 56 (49.8–66.1) 48.8 (43.6–58.3) .18

Cumulative ART exposure at during-ART mtDNA sample, median (IQR), years

3.3 (2.5–4.1) 3.7 (3.4–3.8) .16

Body site(s) of fat loss Face (n59), arms (n512), legs (n511),

buttocks (n53)

No fat loss

Fat accumulation n59 n55

Hyperlactatemia, peak level, symptoms/acidosis n51, 2.6 mmol/L, asymptomatic n51, 5.9 mmol/L, asymptomatic

Peripheral neuropathy n52 (attributed to vitamin B12 deficiency in 1

participant)

n51 (attributed to diabetic neuropathy)

Pancreatitis None None

DEXA, number of participants 6 4

dAge at DEXA, median (IQR), years 59.3 (53.7–66.9) 51.4 (44.8–59.9) .41

dTotal mass, median (IQR), kg 72 (60.8–72.3) 67.4 (63.4–68.3) .21

dTotal fat, median (IQR), kg 15.3 (11.6–18.9) 10.3 (9.5–11.9) .41

dPeripheral fat, median (IQR), kg 4.9 (4.3–6.7) 4.6 (4–5.2) .54

dPeripheral/total fat ratio, mean % 37.9 41.4 .54

NOTE.Abbreviations: ART, antiretroviral therapy; mtDNA, mitochondrial DNA; IQR, interquartile range; DEXA, dual-energy x-ray absorptiometry

1Pvalues of tests for equality of distribution in lipoatrophy cases versus controls without lipoatrophy. All comparisons performed applying the Mann-Whitney U test except for initial ART regimens, which were compared with thev2homogeneity test (collapsed categories: zidovudine vs stavudine, and efavirenz vs nelfinavir vs other).

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controls, whereas haplogroup U was slightly more common among cases (bothP..05).

We also compared mtDNA sequences with the revised Cambridge reference sequence. As expected, numerous variant mtDNA positions or small insertions or deletions were in- dentified. Among 90 phylogenetically uninformative variant positions (43 in the coding region, 47 in the noncoding control region), 86 are known to be polymorphic in the human pop- ulation, thus providing no signal of a possible association with lipoatrophy (see Supplementary Results). Among the 4 variants not previously recorded as polymorphic, only 1 (a single tran- sition at mtDNA position 7962 in a lipoatrophy case bearing haplogroup H) was nonsynonymous.

DISCUSSION

Using whole mitochondrial genome amplification and direct sequencing, we observed no accumulation of mtDNA mutations or deletions in a comprehensive mtDNA sequence analysis of an HIV-infected study population that included 13 case partic- ipants with lipoatrophy and 16 control participants without lipoatrophy. In both cases and controls, mtDNA was sequenced twice: first while participants were ART-naı¨ve and again after substantial (median,>3.3 years) and comparably extensive ex- posure to thymidine analogue–containing ART. This finding argues against the accumulation of mtDNA mutations as a ma- jor driver behind the development of lipoatrophy. In addition, no signal was detected when comparing mtDNA haplogroups in cases and controls, or when comparing mtDNA sequences with the revised Cambridge reference mtDNA sequence.

The accumulation of multiple mtDNA mutations over time is the central tenet of the mitochondrial hypothesis of aging that has been experimentally validated in the mtDNA ‘‘mutator mouse’’ model [7]. However, the link between mtDNA damage and metabolic complications in HIV-infected individuals re- mains circumstantial. Previously, 2 groups have assessed the mitochondrial genome for longitudinal mtDNA changes during ART. In the study by Martin et al [5], mtDNA mutations ac- cumulated during follow-up in 1 of 11 patients without lipoa- trophy and in 4 of 5 patients with lipoatrophy. However, patients without lipoatrophy had a shorter mean ART duration (1.9 years) than the that of the lipoatrophy case patients (3.5 years). McComsey et al [6] recorded the longitudinalreversionof heteroplasmic mtDNA sequence variants to homoplasmic ones in no participant with lipoatrophy and 2 participants without lipoatrophy. However, this occurred in the hypervariable mtDNA control region, the median duration of follow-up in this study was only 13 months, and the authors expressed concern that the method used to detect any mtDNA mutations (tem- poral temperature gradient gel electrophoresis [TTGE] of the PCR products) may have been insufficiently sensitive.

Moreover, in both studies [5, 6], all recorded mtDNA mu- tations were heteroplasmic. He et al [13] recently reported the use of massively parallel sequencing-by-synthesis approaches to statistically quantify heteroplasmy. A high level of heteroplasmy was seen in normal participants, with variable frequencies among tissues. Thus, mtDNA heteroplasmy may not represent an important end point in lipoatrophy genetic studies, unless a significant heteroplasmy increase were detected preferentially after ART exposure, and in lipoatrophy participants compared with patients without lipoatrophy.

Our study is limited by sample size to detect mtDNA hap- logroup associations and by clinical diagnosis of lipoatrophy.

However, we included participants only if significant lipoa- trophy was consistently present during follow-up, case or con- trol status was confirmed in writing by the treating HIV physician, and by DEXA assessment of the study criteria in a subset of participants. Another limitation of studies of the mtDNA genome in HIV-infected persons is the source of mtDNA, which is extracted from the most accessible tissue (ie, peripheral blood). As with mtDNA depletion, if and to what extent mtDNA sequences in blood correlate with mtDNA se- quences in affected tissue (ie, subcutaneous fat) is unclear [14].

The mitochondrial toxicity hypothesis of lipoatrophy [1] has evolved from a hypothesis centered on polymerase-cto a more complex picture including the differential effects of ART in different tissue compartments. For example, recently, Payne et al [15] identified mtDNA variants in muscle biopsies, but point mutations were only found in cytochrome c oxidase–deficient fibers (ie, cells with mitochondrial dysfunction).

In conclusion, our findings, in conjunction with the reports by Martin et al [5] and McComsey et al [6], suggest that lip- oatrophy genetic studies based on peripheral blood mtDNA specimens may not permit the detection of possible mtDNA changes in particular tissue types or cell populations. Sub- cutaneous fat biopsy samples seem best suited to evaluate a possible role for mtDNA genomic variation in the patho- genesis of lipoatrophy. Patient acceptance limits the feasibility of fat biopsies, particularly in the setting of a longitudinal study, and the paucity or lack of fat tissue in lipoatrophy patients may be a problem. In contrast, peripheral blood samples are suffi- cient to identify potentially lipoatrophy-predisposing single nucleotide polymorphisms in nuclear encoded genes, including TNF -238G . A, APOC3, hemochromatosis gene variants, FAS, AR Beta-2, HLA-B*4001, and potentially lipoatrophy- predisposing mtDNA haplogroups [2–4].

Funding

This work was supported by the Swiss National Science Foundation (SNF 3345-062041) and was financed within the framework of the Swiss HIV Cohort Study (SHCS project 513).

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Acknowledgments

We thank the patients participating in the SHCS for their commitment, the study nurses and study physicians for their invaluable work, the SHCS laboratories for their commitment, and the SHCS data center for data management.

The members of the Swiss HIV Cohort Study are M. Battegay, E.

Bernasconi, J. Bo¨ni, H.C. Bucher, Ph. Bu¨rgisser, A. Calmy, S. Cattacin, M. Cavassini, R. Dubs, M. Egger, L. Elzi, M. Fischer, M. Flepp, A. Fontana, P. Francioli (President of the SHCS, Centre Hospitalier Universitaire Vaudois, CH-1011 Lausanne), H. Furrer (Chairman of the Clinical and Laboratory Committee), C. Fux, M. Gorgievski, H. Gu¨nthard (Chairman of the Scientific Board), H. Hirsch, B. Hirschel, I. Ho¨sli, Ch. Kahlert, L. Kaiser, U. Karrer, C. Kind, Th. Klimkait, B. Ledergerber, G. Martinetti, B. Martinez, N. Mu¨ller, D. Nadal, M. Opravil, F. Paccaud, G. Pantaleo, A. Rauch, S. Regenass, M. Rickenbach (Head of Data Center), C. Rudin (Chairman of the Mother and Child Substudy), P. Schmid, D. Schultze, J. Schu¨pbach, R. Speck, P. Taffe´, A. Telenti, A. Trkola, P. Vernazza, R. Weber, and S. Yerly.

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2. Hulgan T, Tebas P, Canter JA, et al; and AIDS Clinical Trials Group 384 and A5005s Study Teams. Hemochromatosis gene polymorphisms, mitochondrial haplogroups, and peripheral lipoatrophy during anti- retroviral therapy. J Infect Dis2008; 197:858–66.

3. Nasi M, Guaraldi G, Orlando G, et al. Mitochondrial DNA hap- logroups and highly active antiretroviral therapy-related lipodys- trophy. Clin Infect Dis2008; 47:962–8.

4. Hendrickson SL, Kingsley LA, Ruiz-Pesini E, et al. Mitochondrial DNA haplogroups influence lipoatrophy after highly active antiretroviral therapy. J Acquir Immune Defic Syndr2009; 51:111–6.

5. Martin AM, Hammond E, Nolan D, et al. Accumulation of mito- chondrial DNA mutations in human immunodeficiency virus-infected patients treated with nucleoside-analogue reverse-transcriptase in- hibitors. Am J Hum Genet2003; 72:549–60.

6. McComsey G, Bai RK, Maa JF, Seekins D, Wong LJ. Extensive inves- tigations of mitochondrial DNA genome in treated HIV-infected subjects: Beyond mitochondrial DNA depletion. J Acquir Immune Defic Syndr2005; 39:181–8.

7. Trifunovic A, Wredenberg A, Falkenberg M, et al. Premature ageing in mice expressing defective mitochondrial DNA polymerase. Nature 2004; 429:417–23.

8. Bernasconi E, Boubaker K, Junghans C, et al. Abnormalities of body fat distribution in HIV-infected persons treated with antiretroviral drugs:

The Swiss HIV Cohort Study. J Acquir Immune Defic Syndr2002;

31:50–5.

9. van der Valk M, Casula M, Weverlingz GJ, et al. Prevalence of lipoa- trophy and mitochondrial DNA content of blood and subcutaneous fat in HIV-1-infected patients randomly allocated to zidovudine- or sta- vudine-based therapy. Antivir Ther2004; 9:385–93.

10. Maca-Meyer N, Gonza´lez AM, Larruga JM, Flores C, Cabrera VM.

Major genomic mitochondrial lineages delineate early human ex- pansions. BMC Genet2001; 2:13.

11. Chinnery PF, Howell N, Andrews RM, Turnbull DM. Mitochondrial DNA analysis: Polymorphisms and pathogenicity. J Med Genet1999;

36:505–10.

12. van Oven M, Kayser M. Updated comprehensive phylogenetic tree of global human mitochondrial DNA variation. Hum Mutat 2009;

30:E386–94.

13. He Y, Wu J, Dressman DC, et al. Heteroplasmic mitochondrial DNA mutations in normal and tumour cells. Nature2010; 464:610–4.

14. Shikuma CM, Hu N, Milne C, et al. Mitochondrial DNA decrease in subcutaneous adipose tissue of HIV-infected individuals with periph- eral lipoatrophy. AIDS2001; 15:1801–9.

15. Payne B, Price A, Chinnery P. Accumulated molecular damage and long-term ART [poster 729]. Program of the 17th Conference on Retroviruses and Opportunistic Infections (San Francisco, CA).

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1

Supplementary Material (online only):

No longitudinal mitochondrial DNA sequence changes in HIV-infected individuals with and without lipoatrophy

Millán Ortiz

1 *

, Estella S. Poloni

2 *

, Hansjakob Furrer

3

, Helen Kovari

4

, Raquel Martinez

1

, Mireia Arnedo

1, 5

, Luigia Elzi

6

, Enos Bernasconi

7

, Pietro Vernazza

8

, Bernard Hirschel

9

, Matthias Cavassini

10

,

Bruno Ledergerber

4

, Huldrych F. Günthard

4

, Amalio Telenti

1

, Philip E. Tarr

11

, and the Swiss HIV

Cohort Study

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2

Supplementary Methods

mtDNA sequence quality checks. To avoid artificial generation of length variation and point

mutations produced by Taq DNA polymerase during PCR, a proof-reading polymerase was used for amplification (TaKaRa LA Taq™). To further rule out amplification artifacts when potential

heteroplasmy, length variation or point mutations between pre-ART and on-ART sequences from the same patient were observed, a second independent PCR product was generated with different primers followed by forward and reverse sequencing. Screening for the presence of heteroplasmy

(simultaneous presence of more than one mtDNA haplotype within the cells) was performed using SeqScape: a nucleotide position was considered heteroplasmic if a secondary peak of more than 30%

of the height of the primary peak was present and confirmed with >1 forward and 1 reverse sequencing reactions.

Supplementary Results

Comparison of mtDNA sequences of the study population to the rCRS [S1]. Of the 43

variants in the coding region, only 8 translate in a non-synonymous change with respect to the rCRS (i.e. transitions at nps 5493 in NADH dehydrogenase gene subunit 2, 7962 in COX cytochrome c oxidate subunit II, 12557, 13359 and 13759 in NADH dehydrogenase subunit 5, 14831, 15257 and 15326 in Cytochrome b (bold in Supplemental Table 1). All these transitions but one (i.e. T7962C) are included among known polymorphic positions.

Comparison of mtDNA haplogroup distributions. Estimated haplogroup frequencies in

European populations were found in [S2-S7]. To compare the frequency distributions of haplogroups

in our study population with that of European regional populations, data from [S5] (and references

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3

therein) were used. The distribution of haplogroups reported in Table 2 could not be readily used in this comparison, because of the small sample size of our study population. However, by pooling certain haplogroups (i.e. U and K; J and L), a formal test of homogeneity showed that this simplified haplotype distribution did not deviate from that of previously published European samples (a Swiss+Austrian sample, P=0.55; a German sample, P=0.40; and a French+Italian sample, P=0.14;

data from [S5] and references therein).

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4

Supplemental Table 1.

Mutations relative to the revised Cambridge reference sequence (rCRS)

1

observed in the 58 completely sequenced mitochondrial genomes of the study participants and classification into haplogroups.

ID Before ART

/ On ART Haplogroup

2

Mutations relative to rCRS used to classify sequences into haplogroups (phylogenetically informative mutations)

Phylogenetically

uninformative mutations in the coding region (nps 577-16023)

3

Phylogenetically

uninformative mutations in the control region (nps 16024-576)

LA cases

1 Before ART H1c 263, 477, 750, 1438, 3010, 4769, 8860, 15326 6671, 9266 199, 292, 315.1C, 16519

On ART H1c 263, 477, 750, 1438, 3010, 4769, 8860, 15326 6671, 9266 199, 292, 315.1C, 16519 2 Before ART H5 263, 456, 750, 1438, 4769, 8860, 15326, 16304 10410, 13725, 14831, 15930 207, 315.1C, 16311

On ART H5 263, 456, 750, 1438, 4769, 8860, 15326, 16304 10410, 13725, 14831, 15930 207, 315.1C, 16311

3 Before ART H7 263, 750, 1438, 4769, 4793, 8860, 15326 11440, 12280

121, 146, 309.1C, 309.2C, 315.1C, 573.1C, 573.2C, 16051, 16290, 16519

On ART H7 263, 750, 1438, 4769, 4793, 8860, 15326 11440, 12280

121, 146, 309.1C, 309.2C, 315.1C, 573.1C, 573.2C, 16051, 16290, 16519

4 Before ART 4 H11'12 195, 263, 750, 1438, 4769, 8860 2159, 13095 315.1C, 16093, 16368, 16519

On ART 5 H11'12 195, 263, 750, 1438, 4769, 8860 2159, 13095, 15257, 15326 315.1C, 16093, 16368, 16519

5 Before ART H11'12 195, 263, 750, 1438, 4769, 8860, 15326, 16311 7962 315.1C, 523d, 524d, 16278,

16519

On ART H11'12 195, 263, 750, 1438, 4769, 8860, 15326, 16311 7962 315.1C, 523d, 524d, 16278, 16519

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5 Supplemental Table 1 (continued).

6 Before ART H37 263, 750, 1438, 3531, 4769, 8860, 15326 930, 4703 309.1C, 315.1C, 16519

On ART H37 263, 750, 1438, 3531, 4769, 8860, 15326 930, 4703 309.1C, 315.1C, 16519

7 Before ART U3a'a26

73, 150, 200, 263, 750, 1438, 1811, 2294, 2706, 4703, 4769, 6518, 7028, 8860, 9266, 10506, 11467, 11719, 12308, 12372, 13934, 14139, 14766, 15326, 15454, 16343, 16390

867, 2010, 10352 185, 189, 315.1C, 523d, 524d, 16311, 16519

On ART U3a'a26

73, 150, 200, 263, 750, 1438, 1811, 2294, 2706, 4703, 4769, 6518, 7028, 8860, 9266, 10506, 11467, 11719, 12308, 12372, 13934, 14139, 14766, 15326, 15454, 16343, 16390

867, 2010, 10352 185, 189, 315.1C, 523d, 524d, 16311, 16519

8 Before ART U4a2

73, 195, 263, 310, 499, 750, 1438, 1811, 2706, 4646, 4769, 5999, 6047, 7028, 8818, 8860, 11332, 11467, 11719, 12308, 12372, 14620, 14766, 15326, 15693, 16356

13359 16519

On ART 7 U4a2

73, 195, 263, 310, 499, 750, 1438, 1811, 2706, 4646, 4769, 5999, 6047, 7028, 8818, 8860, 11332, 11467, 11719, 12308, 12372, 14620, 14766, 15326, 15693, 16356

13359 16519

9 Before ART U4b1b

73, 146, 152, 195, 263, 499, 750, 1438, 1811, 2706, 4646, 4769, 5999, 6047, 7028, 7705, 8860, 11332, 11339, 11467, 11719, 12308, 12372, 13528, 13565, 14620, 14766, 15326, 15693, 16356

309.1C, 315.1C, 524.1A, 524.2C, 16265C, 16519

On ART U4b1b

73, 146, 152, 195, 263, 499, 750, 1438, 1811, 2706, 4646, 4769, 5999, 6047, 7028, 7705, 8860, 11332, 11339, 11467, 11719, 12308, 12372, 13528, 13565, 14620, 14766, 15326, 15693, 16356

309.1C, 315.1C, 524.1A, 524.2C, 16265C, 16519

(12)

6 Supplemental Table 1 (continued).

10 Before ART U4a1a8

73, 195, 263, 499, 750, 961, 1438, 1811, 2706, 4646, 4769, 5999, 6047, 7028, 8818, 8860, 11332, 11467, 11719, 12308, 12372, 12937, 14620, 14766, 15326, 15693, 16134, 16356

315.1C, 524.1A, 524.2C, 16167, 16519

On ART U4a1a8

73, 195, 263, 499, 750, 961, 1438, 1811, 2706, 4646, 4769, 5999, 6047, 7028, 8818, 8860, 11332, 11467, 11719, 12308, 12372, 12937, 14620, 14766, 15326, 15693, 16134, 16356

315.1C, 524.1A, 524.2C, 16167, 16519

11 Before ART K1c29

73, 146, 263, 750, 1189, 1438, 1811, 2706, 3480, 4769, 7028, 8860, 9006, 9055, 9698, 10398, 10550, 11299, 11467, 11719, 12308, 12372, 14002, 14040, 14167, 14766, 14798, 15326, 16224, 16311, 16320

309.1C, 315.1C, 16519

On ART K1c29

73, 146, 263, 750, 1189, 1438, 1811, 2706, 3480, 4769, 7028, 8860, 9006, 9055, 9698, 10398, 10550, 11299, 11467, 11719, 12308, 12372, 14002, 14040, 14167, 14766, 14798, 15326, 16224, 16311, 16320

309.1C, 315.1C, 16519

12 Before ART J1c2c1

73, 185, 188, 222, 228, 263, 295, 462, 489, 750, 1438, 2706, 3010, 4216, 4769, 7028, 8860, 10398, 10685, 11251, 11719, 12612, 13281, 13708, 13933, 14766, 14798, 15326, 15452A, 16069, 16126

146, 315.1C, 524.1A, 524.2C, 16086, 16519

On ART J1c2c1

73, 185, 188, 222, 228, 263, 295, 462, 489, 750, 1438, 2706, 3010, 4216, 4769, 7028, 8860, 10398, 10685, 11251, 11719, 12612, 13281, 13708, 13933, 14766, 14798, 15326, 15452A, 16069, 16126

146, 315.1C, 524.1A, 524.2C, 16086, 16519

(13)

7 Supplemental Table 1 (continued).

13 Before ART 10 J2a'a211

73, 150, 195, 263, 295, 489, 750, 1438, 2706, 4216, 4769, 6671, 7028, 7476, 8860, 10398, 10499, 11002, 11251, 11377, 11719, 12570, 12612, 13708, 14766, 15257, 15326, 15452A, 16069, 16126

5493, 13759, 15097 207, 309.1C, 315.1C, 524.1A, 524.2C

On ART J2a'a211

73, 150, 195, 263, 295, 489, 750, 1438, 2706, 4216, 4769, 6671, 7028, 7476, 8860, 10398, 10499, 11002, 11251, 11377, 11719, 12570, 12612, 13708, 14766, 15257, 15326, 15452A, 16069, 16126

5493, 13759, 15097 207, 309.1C, 315.1C, 524.1A, 524.2C

Controls

1 Before ART H 263, 750, 1438, 4769, 8860, 15326 2098, 10993, 11896 315.1C, 16189, 16221, 16519

On ART H 263, 750, 1438, 4769, 8860, 15326 2098, 10993, 11896 315.1C, 16189, 16221, 16519

2 Before ART H3d 73, 263, 750, 1438, 4769, 6776, 8860, 15326 4248 309.1C, 315.1C, 16262, 16519

On ART H3d 73, 263, 750, 1438, 4769, 6776, 8860, 15326 4248 309.1C, 315.1C, 16262, 16519

3 Before ART H5 263, 456, 750, 1438, 4769, 8860, 15326, 15355,

16304 6731, 8682 309.1C, 315.1C

On ART H5 263, 456, 750, 1438, 4769, 8860, 15326, 15355,

16304 6731, 8682 309.1C, 315.1C

4 Before ART H6a1b 239, 263, 750, 1438, 3915, 4727, 4769, 8860, 9380,

10589, 15326, 16362, 16482 4164 204, 309.1C, 309.2C, 315.1C,

16193, 16219

On ART H6a1b 239, 263, 750, 1438, 3915, 4727, 4769, 8860, 9380,

10589, 15326, 16362, 16482 4164 204, 309.1C, 309.2C, 315.1C,

16193, 16219 5 Before ART H10a'a112 263, 750, 1438, 4216, 4769, 8860, 14470A, 15326,

16114 309.1C, 315.1C, 16519

On ART H10a'a112 263, 750, 1438, 4216, 4769, 8860, 14470A, 15326,

16114 309.1C, 315.1C, 16519

(14)

8 Supplemental Table 1 (continued).

6 Before ART H31 146, 195, 263, 750, 1438, 4769, 7930T, 8860,

10771, 15326 315.1C, 16519

On ART H31 146, 195, 263, 750, 1438, 4769, 7930T, 8860,

10771, 15326 315.1C, 16519

7 Before ART H31 146, 195, 263, 750, 1438, 4769, 7930T, 8860,

10771, 15326 315.1C, 16519

On ART H31 146, 195, 263, 750, 1438, 4769, 7930T, 8860,

10771, 15326 315.1C, 16519

8 Before ART U2e2

73, 152, 263, 508, 750, 1438, 1811, 2706, 3720, 3849, 4769, 5390, 5426, 6045, 6152, 7028, 8860, 10876, 11467, 11719, 12308, 12372, 13020, 13734, 14766, 15326, 15907, 16051, 16129C, 16189, 16362

1719, 4553, 4736, 8473, 9708, 12557, 15891

189, 217, 315.1C, 16092, 16183d, 16193.1C, 16519

On ART U2e2

73, 152, 263, 508, 750, 1438, 1811, 2706, 3720, 3849, 4769, 5390, 5426, 6045, 6152, 7028, 8860, 10876, 11467, 11719, 12308, 12372, 13020, 13734, 14766, 15326, 15907, 16051, 16129C, 16189, 16362

1719, 4553, 4736, 8473, 9708, 12557, 15891

189, 217, 315.1C, 16092, 16183d, 16193.1C, 16519

9 Before ART U5a1a113

73, 263, 750, 1438, 1700, 2706, 3197, 4769, 5495, 7028, 8860, 9477, 11467, 11719, 12308, 12372, 13617, 14766, 14793, 15326, 15924, 16256, 16270, 16399

315.1C

On ART U5a1a113

73, 263, 750, 1438, 1700, 2706, 3197, 4769, 5495, 7028, 8860, 9477, 11467, 11719, 12308, 12372, 13617, 14766, 14793, 15326, 15924, 16256, 16270, 16399

315.1C

(15)

9 Supplemental Table 1 (continued).

10 Before ART U5b2c14

73, 150, 263, 723, 750, 1438, 1721, 2706, 3197, 4769, 7028, 7768, 8860, 9477, 11467, 11719, 12308, 12372, 13017, 13617, 13637, 14182, 14766, 15326, 16192, 16270

3861, 5836, 10262 309.1C, 315.1C

On ART U5b2c14

73, 150, 263, 723, 750, 1438, 1721, 2706, 3197, 4769, 7028, 7768, 8860, 9477, 11467, 11719, 12308, 12372, 13017, 13617, 13637, 14182, 14766, 15326, 16192, 16270

3861, 5836, 10262 309.1C, 315.1C

11 Before ART K1a

73, 263, 497, 750, 1189, 1438, 1811, 2706, 3480, 4769, 7028, 8860, 9055, 9698, 10398, 10550, 11299, 11467, 12308, 12372, 14167, 14766, 14798, 15326, 16224, 16311

11719 150, 152, 207, 315.1C, 16137, 16519

On ART K1a

73, 263, 497, 750, 1189, 1438, 1811, 2706, 3480, 4769, 7028, 8860, 9055, 9698, 10398, 10550, 11299, 11467, 12308, 12372, 14167, 14766, 14798, 15326, 16224, 16311

11719 150, 152, 207, 315.1C, 16137, 16519

12 Before ART J1c3

73, 185, 228, 263, 295, 462, 489, 750, 1438, 2706, 3010, 4216, 4769, 7028, 8860, 10398, 11251, 11719, 12612, 13708, 13934, 14766, 14798, 15326, 15452A, 16069, 16126

315.1C

On ART J1c3

73, 185, 228, 263, 295, 462, 489, 750, 1438, 2706, 3010, 4216, 4769, 7028, 8860, 10398, 11251, 11719, 12612, 13708, 13934, 14766, 14798, 15326, 15452A, 16069, 16126

315.1C

(16)

10 Supplemental Table 1 (continued).

13 Before ART J1c6

73, 185, 228, 263, 295, 462, 489, 750, 1438, 2706, 3010, 4025, 4216, 4769, 7028, 8860, 10398, 11251, 11719, 12612, 13708, 14766, 14798, 15326, 15452A, 16069, 16126

315.1C

On ART J1c6

73, 185, 228, 263, 295, 462, 489, 750, 1438, 2706, 3010, 4025, 4216, 4769, 7028, 8860, 10398, 11251, 11719, 12612, 13708, 14766, 14798, 15326, 15452A, 16069, 16126

315.1C

14 Before ART J1c6

73, 185, 228, 263, 295, 462, 489, 750, 1438, 2706, 3010, 4025, 4216, 4769, 7028, 8860, 10398, 11251, 11719, 12612, 13708, 14766, 14798, 15326, 15452A, 16069, 16126

315.1C, 16292

On ART J1c6

73, 185, 228, 263, 295, 462, 489, 750, 1438, 2706, 3010, 4025, 4216, 4769, 7028, 8860, 10398, 11251, 11719, 12612, 13708, 14766, 14798, 15326, 15452A, 16069, 16126

315.1C, 16292

15 Before ART J2b115

73, 150, 152, 263, 295, 489, 750, 1438, 2706, 4216, 4769, 5633, 7028, 7476, 8860, 10172, 10398, 11251, 11719, 12612, 14766, 15257, 15326, 15452A, 15812, 16069, 16126, 16193

6830A, 8095 315.1C, 16195, 16221,

16242A, 16319, 16357, 16526

On ART J2b115

73, 150, 152, 263, 295, 489, 750, 1438, 2706, 4216, 4769, 5633, 7028, 7476, 8860, 10172, 10398, 11251, 11719, 12612, 14766, 15257, 15326, 15452A, 15812, 16069, 16126, 16193

6830A, 8095 315.1C, 16195, 16221,

16242A, 16319, 16357, 16526

(17)

11 Supplemental Table 1 (continued).

16 Before ART L0a2a2a

93, 152, 189, 204, 207, 236, 247, 263, 750, 769, 825A, 1018, 1048, 1438, 2245, 2706, 2758, 2885, 3516A, 3594, 4104, 4312, 4586, 4769, 5147, 5231, 5442, 5460, 5603, 5711, 6185, 6257, 7028, 7146, 7256, 7521, 8281-8289d, 8428, 8460, 8468, 8566, 8655, 8701, 8860, 9042, 9347, 9540, 9545, 9554, 9755, 9818, 10398, 10589, 10664, 10688, 10810, 10873, 10915, 11143, 11172, 11176, 11641, 11719, 11914, 12007, 12705, 12720, 13105, 13116, 13276, 13506, 13650, 14308, 14755, 14766, 15136, 15326, 15431, 16148, 16172, 16187, 16188G, 16189, 16223, 16311, 16320

5096 315.1C, 523d, 524d, 16230,

16343, 16519

On ART L0a2a2a

93, 152, 189, 204, 207, 236, 247, 263, 750, 769, 825A, 1018, 1048, 1438, 2245, 2706, 2758, 2885, 3516A, 3594, 4104, 4312, 4586, 4769, 5147, 5231, 5442, 5460, 5603, 5711, 6185, 6257, 7028, 7146, 7256, 7521, 8281-8289d, 8428, 8460, 8468, 8566, 8655, 8701, 8860, 9042, 9347, 9540, 9545, 9554, 9755, 9818, 10398, 10589, 10664, 10688, 10810, 10873, 10915, 11143, 11172, 11176, 11641, 11719, 11914, 12007, 12705, 12720, 13105, 13116, 13276, 13506, 13650, 14308, 14755, 14766, 15136, 15326, 15431, 16148, 16172, 16187, 16188G, 16189, 16223, 16311, 16320

5096 315.1C, 523d, 524d, 16230,

16343, 16519

1 GenBank accession NC_012920. All mutations are transitions unless specified (transversion: the base follows immediately the position; deletion: indicated by "d"; insertion:

a decimal and the base inserted follow the position).

2 Mitochondrial genomes were classified into haplogroups using PhyloTree.org [S8, S9].

3 Non-synonymous mutations are shown in bold; mutations not reported in MITOMAP, PhyloTree or mtDB [S9] are shown in italics.

4 No sequencing results for 434 bp from position 15233 to 15666.

(18)

12

5 No sequencing results for 304 bp from position 15720 to 16023.

6 This sequence shares all defining states of haplogroup U3a2 but the transition at 11050.

7 No sequencing results for 86 bp from position 316 to 401.

8 This sequence shares all defining states of haplogroup U4a1a but the U4a1-defining transition at 152.

9 This sequence shares all defining states of haplogroup K1c2 but the K1c-defining transition at 152 and deletion at 498.

10 No sequencing results for 308 bp from position 15701 and 16008.

11 This sequence shares all defining states of haplogroup J2a2 but the transition at position 15679.

12 This sequence shares all defining states of haplogroup H10a1 but the transition at 14548.

13 This sequence shares all defining states of haplogroup U5a1a1 but the U5a1-defining transition at 15218.

14 This sequence shares all defining states of haplogroup U5b2c but the insertion after 960 (960.1C) and the transition at 16249.

15 This sequence shares all defining states of haplogroup J2b1 but the J-defining transition at 13708.

(19)

13

Supplementary References

S1. Andrews RM, Kubacka I, Chinnery PF, et al. Reanalysis and revision of the Cambridge reference sequence for human mitochondrial DNA. Nat Genet 1999; 23: 147.

S2. Torroni A, Bandelt HJ, D'Urbano L, et al. mtDNA analysis reveals a major late Paleolithic population expansion from southwestern to northeastern Europe. Am J Hum Genet 1998;62:1137-1152.

S3. Macaulay V, Richards M, Hickey E, et al. The emerging tree of West Eurasian mtDNAs: a synthesis of control-region sequences and RFLPs. Am J Hum Genet 1999;64:232-249.

S4. Richards M, Macaulay V, Hickey E, et al. Tracing European founder lineages in the Near Eastern mtDNA pool. Am J Hum Genet 2000;67:1251-1276.

S5. Helgason A, Hickey E, Goodacre S, et al. mtDna and the islands of the North Atlantic: estimating the proportions of Norse and Gaelic ancestry. Am J Hum Genet 2001;68:723-737.

S6. Pereira L, Richards M, Goios A, et al. High-resolution mtDNA evidence for the late-glacial resettlement of Europe from an Iberian refugium. Genome Res 2005;15:19-24.

S7. Pliss L, Tambets K, Loogvali EL, et al. Mitochondrial DNA portrait of Latvians: towards the understanding of the genetic structure of Baltic-speaking populations. Ann Hum Genet 2006;70:439-458.

S8. van Oven M, Kayser M. Updated comprehensive phylogenetic tree of global human mitochondrial DNA variation. Hum Mutat 2009; 30: E386-394.

S9. PhyloTree.org (mtDNA tree Build 8 of 21 March 2010). Available at: http://www.phylotree.org. Accessed 31 May 2010. MITOMAP, a human mitochondrial genome database. Available at:

http://www.mitomap.org. Accessed 31 July 2010. mtDB - Human Mitochondrial Genome Database.

Available at: http://www.genpat.uu.se/mtDB/. Accessed 31 July 2010.

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