Mitochondrial DNA Subhaplogroups L0a2 and L2a
Modify Susceptibility to Peripheral Neuropathy in
Malawian Adults on Stavudine Containing Highly
Elizabeth Kampira, MSc,*† Johnstone Kumwenda, FRCP,‡ Joep J. van Oosterhout, MD, PhD,‡§
independently associated with reduced risk of PN (odds ratio,
Background: Peripheral neuropathy (PN) is one of the main
0.39; 95% confidence interval, 0.16 to 0.94; P = 0.036).
toxicities associated with stavudine. Genetic variants in mitochondrialDNA (mtDNA) haplogroups have been associated with increased risk
Conclusions: Genetic variation in mtDNA confers differential
of developing PN in European non-Hispanic and black patients on
risk of developing PN in patients on stavudine containing ART
stavudine containing antiretroviral therapy (ART). We investigated
mtDNA haplogroups and their role in susceptibility to stavudine-
Key Words: stavudine, mtDNA, subhaplogroup, peripheral neurop-
induced peripheral in Malawian patients on ART.
Method: Two hundred and fifteen adults on stavudine containing
(J Acquir Immune Defic Syndr 2013;63:647–652)
regimens were recruited from the ART clinic at Queen Elizabeth CentralHospital, Blantyre, into a cross-sectional study to investigate the effectsof genetic variants in mtDNA of individuals in relation to responseto treatment. Patients were categorized according to whether or not
they had developed PN after a minimum of 6 months on stavudine
Peripheral neuropathy (PN) is one of the most common
containing ART. Whole mtDNA coding regions of each patient were
neurological complication associated with HIV infection,
sequenced, and CD4 count, viral load, and creatinine were determined.
occurring in up to 35% of the patients.1,2 PN may result from
The mtDNA variation was correlated with clinical characteristics.
HIV infection itself or from the neurotoxic side effects ofantiretroviral drugs, especially nucleoside reverse transcriptase
Results: Fifty-three (25%) of the participants developed PN after
inhibitors (NRTIs).1,3 Among NRTIs, stavudine and didanosine
starting stavudine containing ART. Mitochondrial DNA subha-
have the highest propensity to cause PN. Cui et al4 showed that
plogroup L0a2 was independently associated with increased risk of
stavudine inhibits neurite regeneration; however, the mecha-
PN in a multivariate model (odds ratio, 2.23; 95% confidence
nism of neurotoxicity is not fully established. Phosphorylated
interval, 1.14 to 4.39; P = 0.019), and subhaplogroup L2a was
molecules of NRTIs in the mitochondrial matrix inhibit theactivity of mitochondrial polymerase gamma by competingwithdeoxyribonucleotide triphosphatesfor the binding site and
Received for publication January 29, 2013; accepted April 10, 2013.
then as they incorporate into the growing mitochondrial DNA
From the *Division of Human Genetics, Faculty of Health Sciences, University
(mtDNA) strands by causing inhibition in the synthesis of
of Cape Town, Cape Town, South Africa; and †Department of Pathology,
mtDNA thus prematurely terminating chain elongation.5,6 In
‡Department of Medicine, and §Malawi-Liverpool Wellcome Trust ClinicalResearch Programme, College of Medicine, University of Malawi, Malawi.
addition to mtDNA polymerase gamma inhibition, mitochon-
Presented at the Wellcome Trust 6th Meeting of the Directors of the African
dria dysfunction could be the result of increased mtDNA poly-
Institutions Initiativein Accra, November 28, 2012, Ghana.
morphisms and oxidative stress caused by NRTI that may have
Funded by Welcome Trust and Department for International Development
adverse effect on mitochondrial structure and function.7 It has
(DFID) through Health Research Capacity Strengthening Initiative,
recently been suggested that polymorphisms in mitochondrial
Southern Africa Consortium for Research Excellence, Medical ResearchCouncil of South Africa, the National Research Foundation of South
genes may explain variations in the response to antiretroviral
The authors have no conflicts of interest to disclose.
The evolution of the human mtDNA is characterized by
Correspondence to: Collet Dandara, PhD, Division of Human Genetics,
the emergence of distinct lineages or haplogroups. These
Faculty of Health Sciences, University of Cape Town, Observatory 7995,South Africa (e-mail: collet.dandara@uct.ac.za).
haplogroups are characterized by specific sets of haplotypes or
Copyright 2013 by Lippincott Williams & Wilkins. Unauthorized reproduc-
single nucleotide polymorphisms (SNPs).9 For example, the
tion of this article is prohibited. This is an open-access article distributed
mutations m.5147 G.A, m.5711 A.G, m.6257 G.A, and
under the terms of the Creative Commons Attribution-NonCommercial-
m.8460 A.G describe subhaplogroup L0a2. Many of the
NoDerivatives 3.0 License, where it is permissible to download and share
described haplogroups are characteristic of different populations
the work provided it is properly cited. The work cannot be changed in anyway or used commercially.
and/or ethnic groups.10,11 The mtDNA lineage L characteristically
J Acquir Immune Defic Syndr Volume 63, Number 5, August 15, 2013
J Acquir Immune Defic Syndr Volume 63, Number 5, August 15, 2013
FIGURE 1. Phylotree indicating thebranching of the mtDNA L majorgroup in Africa populations.
defines African populations and is divided into 2 branches, L0
recruited into a cross-sectional study. The patients completed
and L1-612 (see Fig. 1). The distribution and frequency of these
a structured questionnaire that collected demographic infor-
mtDNA L subhaplogoups within Africa seem to have distinctive
mation, medical history, and ancestry of each participant up to
patterns in different geographic regions or ethnic groups.11,13–15
their grandparent’s level. Pregnant women, patients on tuber-
For example, more than 90% of Khoisan group of South Africa
culosis treatment, persons who experienced PN before ART
carry the haplogroup L0, whereas this haplogroup is found in
initiation and those who had missed their medication in the
past 3 days were excluded from participation. Participants had
Analysis of mitochondrial subhaplogroups among
to be on stavudine containing regimen for at least 6 months.
European patients enrolled in AIDS clinical Trials Group studies
The protocol was approved by the College of Medicine
showed that individuals in haplogroup T had increased risk of
Research Ethics Committee of the University of Malawi and
developing PN during ART.8,16 The distribution and frequencies
the Human Research Ethics Committee of the University of
of L subhaplogroups in Africa varies widely between geographic
Cape Town. All participants gave written informed consent.
regions and ethnic groups.11,15 Although some genetic studies on
The study conformed to the declarations of Helsinki 2008.
African have focused on mtDNA, this has mainly been aimed atunraveling demographic phenomena related to the settlement ofpopulations and ethnic groups on the continent, whereas informa-
tion on the association of subhaplogroups with risk of drug tox-
History and physical examinations were performed at
icities is very sparse and completely lacking among Malawians
enrollment. Stavudine-associated PN was defined as a history
and other African populations. Our aim was therefore to investi-
of characteristic symptoms of numbness, dysesthesia, and pain
gate the association of mtDNA subhaplogroups with development
in the feet and legs that had started after initiation of ART.2 The
of PN among adult Malawians on stavudine containing ART.
glomerular filtrate rate was estimated with the US K-DOQIgroup method.17 A sample for CD4 cell count (FACSCount
flow cytometer; Beckton Dickinson, Franklin Lakes, NJ) and
HIV-1 RNA (Amplicor HIV Monitor, version 1.5; RocheDiagnostic Systems, Basel, Switzerland) was collected.
Blood samples for DNA extraction were collected in
Unrelated HIV/AIDS patients from an ART cohort at
ethylenediaminetetraacetic acid-coated tubes and were kept at
Queen Elizabeth Central Hospital in Blantyre, Malawi, were
220°C until DNA extraction. DNA was isolated by use of
Ó 2013 Lippincott Williams & Wilkins
J Acquir Immune Defic Syndr Volume 63, Number 5, August 15, 2013
a GenElute Blood Genomic DNA Kit (Sigma-Aldrich, St
Louis, MO) according to the manufacturer’s protocol. The
We enrolled 215 maternally unrelated adult (according
mtDNA for each of the 215 samples was amplified in 9 par-
to family history information) ART patients. All were
tially overlapping fragments using the primers reported by
Malawian Bantu speakers, 132 (61%) were women and all
Ramos et al.18,19 Each time mtDNA templates were amplified
were on stavudine and lamivudine containing first-line ART
in a total volume of 25 mL in a reaction that consisted of ·1
for at least 23 months, median duration of 25 months (range,
green GoTaq reaction buffer, 200 mM of deoxyribonucleotide
23–29). Two (1%) patients had severely elevated serum cre-
triphosphates, 1.0 mM of MgCl2, 0.4 mmol of each primer, 0.5
atinine levels of 5.4 and 6.0 mg/dL with an estimated glo-
U of Taq DNA polymerase, and 20 ng of DNA were per-
merular filtrate rate of 15.4 mL/min/1.73 m2 and 10.3 mL/
formed in GeneAmp PCR System 9700 by Life technologies
min/1.73 m2, respectively. One percent and 3% of patients
(New York, NY). The polymerase chain reaction (PCR) pro-
were current smokers and alcoholics, respectively. Fifty-three
grammes each consisted of an initial denaturation step at 94°
(25%) patients had PN. Table 1 provides further details of
C for 5 minutes, followed by 35 cycles of denaturation at 94°
C for 1 minute, and annealing and extension step at 57–64.4°C, 72°C for 40 seconds, and 2.5 minutes, respectively, witha final extension step of 5 minutes at 72°C.18 PCR products
were purified using the exonuclease and shrimp alkaline
All 215 samples were successfully sequenced and 143
positions showed nucleotide differences when compared withbases on the rCRS.21 Of these 143 mutations, 134 (94%) have
been reported in specific L subhaplogroups before; 7 (5%) of
Instead of targeting confirmed informative SNPs, we
the mutations, m.3579 A.G, m.3606 A.G, m.5090 T.C,
sequenced whole mtDNA coding region to search for any
m.10463 T.C, m.12192 G.A, m.13104 A.G, and m.15038
novel SNPs in this Malawian population because of the known
G.A, with frequencies of 2%, 2%, 6%, 1%, 3%, 7%, and
genetic diversity in African populations. Through the series of
9%, respectively, are being reported in an African population
9 PCR fragments and use of forward and reverse primers and
for the first time but have been observed previously in non-
additional internal primers all samples (n = 215) were
African populations.12,22,23 Of the few remaining changes,
sequenced from nucleotide position 577–15953 of the mtDNA
2 (1%), m. 12769 G.A with a frequency of 2% and
according to the revised Cambridge reference sequence (rCRS)
m.14612 G.A with a frequency of 4%, were novel and are
Locus NC_012920.1. Capillary electrophoresis for sequencing
not on either MITOMAP or Phylotree. The 134 SNPs were
reactions was run on an ABI PRISM 3130 · l Genetic Ana-
then used to construct haplogroups and subhaplogroups
lyzer (Applied Biosystems, Foster City, CA). Sequences were
according to software provided by van Oven and Kayser.12
aligned to the rCRS for the human mitochondrion Locus
Major L haplogroups (L0–L3) were identified in the study
NC_012920.1 and assembled using Lasergene 10 Core Suite
population and were further characterized into 9 subha-
software supplied by DNASTAR package (Madison, WI).
plogroups, namely L0a1, L0a2, L0d, L0f, L0k, L1c,
After assembly of the sequences, mutations (polymorphisms)
L2a, L3d, and L3e (Table 2). Subhaplogroup L0a2 had the
were determined as nucleotide differences when compared
highest frequency (28%), whereas L3d (2%) was the least
with the Cambridge reference sequence.
common. We did not observe haplogroups L4, L5, and L6and subhaplogroup L1b that have been reported in other Afri-can populations.14,24 Two mitochondrial subhaplogroups were
associated with the presence of PN (see Table 2). In a multivar-
iate logistic regression model, the L0a2 subhaplogroup was an
We classified mtDNA mutations into haplogroups accord-
independent risk factor for PN (odds ratio, 2.23; 95% confi-
ing to the databases of van Oven and Kayser12 www.phylotree.
dence interval, 1.14 to 4.39; P = 0.019) (Table 3). On the other
org/ and Accetturo et al.20 Stata for windows software (version
hand, the presence of the L2a subhaplogroup was associated
SE/11, 4905; Stata Corp, College Station, TX) was used for
with reduced risk for PN (odds ratio, 0.39; 95% confidence
statistical analysis. Subhaplogroup frequencies were compared
interval, 0.16 to 0.94; P = 0.036). The mutations, m.5147
between participants presenting with and without PN using the
G.A, m.5711 A.G, m.6257 G.A, and m.8460 A.G,
To assess the relationship between independent varia-
bles [sex, body mass index (BMI), age, duration on ART,CD4 count, viral load, estimated glomerular filtrate rate, and
subhaplogroups] and the presence of PN, univariate logistic
The study was undertaken to investigate the role of
regression model was performed. Variables, which showed
mtDNA subhaplogroups in the susceptibility to stavudine-
a degree of association with PN (P , 0.1), were included in
induced PN in HIV/AIDS patients from Malawi. We
multivariate logistic regression models, where 1 subhaplogroup
observed that there was no relationship between gender and
was included in the absence of other subhaplogroups. Odds
the risk of PN. The role of age on PN is in contrast to our
ratios are reported with 95% confidence intervals and a P value
earlier findings that reported there was association between
of ,0.05 was considered significant.
age and risk of PN.2 Unlike in other studies where height has
Ó 2013 Lippincott Williams & Wilkins
J Acquir Immune Defic Syndr Volume 63, Number 5, August 15, 2013
TABLE 1. Patient Characteristics and PN Diagnosis
*Data are expressed as N (%) except for age unless otherwise noted. eGFR calculated using US K-DOQI group method. IQR, interquartile range; eGFR, estimated glomerular filtrate rate.
been associated with increased risk for PN, this was not the
bility is that we overlooked the diagnosis of type II diabetes
case in the Malawi cohort.25 In antiretroviral naive patients,
in many obese patients; however, in a cohort that included
PN has been reported to be more common with CD4 counts
many patients from the current study, we found that diabetes
,200 cells per microliter and HIV-1 RNA .10,000 copies
mellitus was very uncommon.2 Another explanation is that
per milliliter.26,27 In our study, all patients were on ART for at
the studies that found an association, low BMI with PN
least 23 months and the vast majority showed good control of
mainly included patients who were not on ART.28,30 After
HIV replication, which likely explains why we did not
starting ART, the prevalence of malnutrition steadily
observe an association of CD4 and HIV-1 RNA with PN.
decreased and the pathogenesis and risk factors of PN are
In some studies, the risk of PN was associated
likely to be different. In ART patients, high BMI has been
with malnutrition28,29; therefore, our finding that high BMI
identified as a risk factor for high lactate syndromes and
(.25 kg/m2) with borderline significance (P = 0.055) in
patients experiencing PN could be remarkable. One possi-
The mtDNA variation has been used in human
phylogeography in association with population genealogyand also in studies trying to define the risk mtDNA poly-
TABLE 2. The Association of mtDNA Subhaplogroups With
morphisms in human disease.11,20,31,32 Previous studies have
demonstrated that European populations with haplogroup T
are more susceptible to developing stavudine-associated
PN compared with other haplogroups.8,33 A study conducted
in blacks of African origin showed that subhaplogroup L1c
was associated with increased susceptibility to developing
stavudine-associated PN,34 whereas in this Malawi popula-
tion, 2 subhaplotypes that, L0a2 and L2a, seem to be the
This is the first study to be carried out within the
indigenous Africans with known demographic information.
However, our study has several limitations that include a small
sample size, which makes it difficult to determine the effectsof subhaplogroups with low frequencies (eg, L3d with 2%),
Values given in bold indicate significant differences.
a weakness in the objective assessment of PN (eg, clinical
Ó 2013 Lippincott Williams & Wilkins
J Acquir Immune Defic Syndr Volume 63, Number 5, August 15, 2013
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TABLE 3. Multivariate Logistic Regression Analyses of Factors
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Abstract Die Effektivität osteopathischer Behandlungen bei autonomen somatoformen Störungen Gesa Diedrichsen Somatoforme autonome Störungen sind gekennzeichnet durch Symptome eines vegetativ innervierten Organs. Das Ziel dieser Arbeit ist es herauszufinden, ob es Studien gibt, die sich mit auseinandersetzen, deren Validität zu bewerten und eine Aussage zum derzeitigen Forschungssta