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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 7. Lewis W, Day BJ, Copeland WC. Mitochondrial toxicity of NRTI anti- TABLE 3. Multivariate Logistic Regression Analyses of Factors viral drugs: an integrated cellular perspective. Nat Rev Drug Discov.
8. Hulgan T, Haas DW, Haines JL, et al. Mitochondrial haplogroups and peripheral neuropathy during antiretroviral therapy: an adult AIDS clinical trials group study. AIDS. 2005;19:1341–1349.
9. Chen YS, Torroni A, Excoffier L, et al. Analysis of mtDNA variation in African populations reveals the most ancient of all human continent-speci fic haplogroups. Am J Hum Genet. 1995;57:133–149.
10. Behar Doron M, van Oven M, Rosset S, et al. A Copernican reassessment of the human mitochondrial DNA tree from its root. Am J Hum Genet.
,18.5 vs. 18.5– 0.93 (0.10–8.43) 0.948 11. Herrnstadt C, Elson JL, Fahy E, et al. Reduced-median-network analysis of complete mitochondrial DNA coding-region sequences for the major African, Asian, and European haplogroups. Am J Hum Genet. 2002;70: 12. van Oven M, Kayser M. Updated comprehensive phylogenetic tree of global human mitochondrial DNA variation. Hum Mutat. 2009;30: 13. Anderson-Mann S. Phylogenetic and Phylogeographic Analysis of Afri- can Mitochondrial DNA Variation [thesis]. Leeds, England: Universityof Leeds; 2006.
14. Gonder MK, Mortensen HM, Reed FA, et al. Whole-mtDNA genome sequence analysis of ancient African lineages. Mol Biol Evol. 2007;24: ndings including nerve conduction velocity and/or intra- 15. Tishkoff SA, Gonder MK, Henn BM, et al. History of click-speaking epidermal nerve fiber density), possibility of undiagnosed pre- populations of Africa inferred from mtDNA and Y chromosome genetic ART neuropathy and lack of information on the role of other variation. Mol Biol Evol. 2007;24:2180–2195.
factors associated with stavudine-induced PN such as poly- 16. Canter JA, Haas DW, Kallianpur AR, et al. The mitocondrial pharma- cogenomics of haplogroup T: MTND2*LHON4917G and antiretroviraltherapy-associated peripheral neuropathy. Pharmacogenomics J. 2007;8:71–77.
17. National Kidney Foundation. K/DOQI clinical practice guidelines for chronic Kidney disease: evaluation, classification, and stratification. Am We report a significant association between L02a with increased risk of PN and a protective effect of L2a in Malawians 18. Ramos A, Santos C, Alvarez L, et al. Human mitochondrial DNA com- on stavudine-based ART. Although it is unlikely that in our plete amplification and sequencing: a new validated primer set that pre-vents nuclear DNA sequences of mitochondrial origin co-amplification.
setting subhaplogroups can be introduced as biomarkers for Electrophoresis. 2009;30:1587–1593.
tailoring antiretroviral drugs to individual patients in the near 19. Ramos A, Santos C, Barbena E, et al. Validated primer set that prevents future, our findings help better understand the mitochondrial nuclear DNA sequences of mitochondrial origin co-amplification: a revi- toxicopathology of NRTI’s and if confirmed by other studies sion based on the New Human Genome Reference Sequence (GRCh37).
may improve drug selection for standard regimens at population 20. Accetturo M, Santamaria M, Lascaro D, et al. Human mtDNA site-specific level and lead to better precision medication.
variability values can act as haplogroup markers. Hum Mutat. 2006;27:965–974.
21. Andrews RM, Kubacka I, Chinnery PF, et al. Reanalysis and revision of the Cambridge reference sequence for human mitochondrial DNA. Nat We thank the ART clinic team at Queen Elizabeth Central Hospital and all participants in the study and University of 22. Grasbon-Frodl EM, Kösel S, Sprinzl M, et al. Two novel point mutations Cape Town, Pharmacogenetics group for teamwork.
of mitochondrial tRNA genes in histologically confirmed Parkinson dis-ease. Neurogenetics. 1999;2:121–127.
23. Ban M, Elson J, Walton A, et al. Investigation of the role of mitochon- drial DNA in multiple sclerosis susceptibility. PLoS One. 2008;3:e2891.
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3. Berger AR, Arezzo JC, Schaumburg HH, et al. 2’,3’-dideoxycytidine 25. Cherry CL, Affandi JS, Imran D, et al. Age and height predict neuropathy (ddC) toxic neuropathy: a study of 52 patients. Neurology. 1993;43: risk in patients with HIV prescribed stavudine. Neurology. 2009;73: 4. Cui L, Locatelli L, Xie M-Y, et al. Effect of nucleoside analogs on 26. Keswani SC, Pardo CA, Cherry CL, et al. HIV-associated sensory neu- neurite regeneration and mitochondrial DNA synthesis in PC-12 Cells.
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27. Anderson PL, Kakuda TN, Lichtenstein KA. The cellular pharmacology 5. Bailey CM, Kasiviswanathan R, Copeland WC, et al. R964C Mutation of of nucleoside- and nucleotide-analogue reverse-transcriptase inhibitors and DNA Polymerase {gamma} imparts increased stavudine toxicity by its relationship to clinical toxicities. Clin Infect Dis.2004;38:743–753.
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Ó 2013 Lippincott Williams & Wilkins J Acquir Immune Defic Syndr  Volume 63, Number 5, August 15, 2013 29. Kamerman PR, Wadley AL, Cherry CL. HIV-associated sensory neuropathy: 33. Canter JA, Haas DW, Kallianpur AR, et al. The mitochondrial phar- risk factors and genetics. Curr Pain Headache Rep. 2012;16:226–236.
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