HIV/Hepatitis Coinfection Unit, Microbiology National Center, Carlos III Health Institute, Madrid, Spain
Correspondence: Dr Salvador Resino, Centro Nacional de Microbiología, Instituto de Salud Carlos III (Campus Majadahonda), Carretera Majadahonda- Pozuelo, Km 2.2, 28220 Majadahonda (Madrid), Spain. Tel: +34 918 223 266; fax: +34 918 223 269; e-mail: firstname.lastname@example.org
Mitochondria are multifunctional organelles with a key role in the innate immune response against viral infections. Mitochondrial DNA (mtDNA) haplogroups have been related to AIDS progression and CD4 T-cell recovery in HIV-infected patients, and to a delay in the development of liver fibrosis in HIV/hepatitis C virus (HCV)-coinfected patients. We performed a study to investigate whether mtDNA haplogroups may be associated with HCV treatment response in HIV/HCV-coinfected patients on pegylated interferon (pegIFN) plus ribavirin (RBV).
We performed a retrospective study in 304 patients who completed a course of HCV therapy. mtDNA polymorphisms were genotyped using Sequenom's MassARRAY platform. The interleukin-28B (IL-28B) polymorphism (rs12980275) was genotyped using the GoldenGate® assay. Sustained virological response (SVR) was defined as an undetectable HCV viral load at week 24 after the end of treatment. The statistical analysis was carried out using on-treatment data.
The SVR rates were 52.6% (160 of 304) for all patients, and 37.8% (46 of 201) for patients with HCV genotype 1 or 4 vs. 81.4% (83 of 102) for patients with HCV genotype 2 or 3 (P < 0.001). No significant associations were found between mtDNA haplogroup and SVR when all patients were included in the analysis and when patients were stratified by HCV genotype (i.e. those with genotypes 1/4 and 2/3 analysed separately) or IL-28B rs12980275 genotype.
European mtDNA haplogroups were not related to HCV treatment response in HIV/HCV-coinfected patients on pegIFN-α/RBV therapy.
HIV and hepatitis C virus (HCV) coinfection is a common scenario because the two viruses share the same transmission routes . The consequence of HCV infection is a worse prognosis in HIV/HCV-coinfected patients . Pegylated-interferon-α plus ribavirin (pegIFN-α/RBV) continues to be a cornerstone of the treatment of HCV infection in HIV/HCV-coinfected patients . The rate of sustained virological response (SVR) to pegIFN-α/RBV in HIV/HCV-coinfected patients varies between 27 and 40%, depending on various host-related and virus-related factors . To date, the main determinants of response to pegIFN-α/RBV identified include HCV genotype, serum HCV RNA level, stage of liver fibrosis, and single nucleotide polymorphisms in and around the interleukin-28B (IL-28B) gene . However, an unexplained variability in treatment outcome still remains, suggesting that other host genetic factors may influence the permanent clearing of HCV.
Mitochondria are multifunctional organelles with a key role in the innate immune response against viral infections , and mitochondrial dysfunction is evident during HIV infection  and HCV infection [6, 7]. Variations in mitochondrial DNA (mtDNA) have been directly associated with susceptibility to several diseases . Many of these mtDNA mutations have been acquired throughout human history, subdividing the human population into a number of discrete haplogroups, which are defined on the basis of specific mitochondrial single nucleotide polymorphisms . These mtDNA haplogroups have been associated with susceptibility to several disorders such as cancer, sepsis, diabetes and degenerative diseases [8, 10]. Furthermore, mtDNA haplogroups have been related to AIDS progression, lipodistrophy, and metabolic alterations in HIV-infected patients [11-13], and liver fibrosis progression in HIV/HCV-coinfected patients .
Here, we performed a study to investigate whether mtDNA haplogroups may be associated with HCV treatment response in HIV/HCV-coinfected patients on pegIFN-α/RBV therapy.
Patients and methods
We carried out a retrospective study in 304 HIV/HCV-coinfected patients who completed a course of pegIFN-α/RBV therapy from October 2000 to June 2010 at two hospitals in Madrid, Spain [146 from Hospital General Universitario Gregorio Marañón (HGUGM) and 158 from Hospital Carlos III (HCIII)]. The study was approved by the Institutional Review Board and the Research Ethic Committee of the Instituto de Salud Carlos III (ISCIII). This study was conducted in accordance with the Declaration of Helsinki and patients gave their written consent to participate in the study.
The criteria for starting HCV antiviral treatment were as follows. The inclusion criteria were European White ethnicity, chronic hepatitis C, being naïve to HCV treatment, no clinical evidence of hepatic decompensation, detectable HCV RNA by polymerase chain reaction, being negative for hepatitis B virus surface antigen, CD4 lymphocyte count > 200 cells/μL, and being on stable combination antiretroviral therapy (cART). The exclusion criteria were active opportunistic infections, active drug or alcohol addiction, and other concomitant diseases or conditions. Furthermore, we included only patients who fulfilled the HCV treatment and who had an available DNA sample for genotyping of the mtDNA haplogroups. From 323 patients who met the criteria described above, three patients were excluded because we were unable to genotype the mtDNA polymorphisms to determine their mitochondrial haplogroups. Additionally, to make the study sample more uniform, we excluded 16 patients who were not of the European ‘ ‘N' ’ mitochondrial macro-haplogroup, which is ancestral to almost all European and many Eurasian haplogroups.
The following information was obtained from medical records: age, gender, HIV transmission category, weight, height, nadir CD4 T-cell count, cART, HCV genotype, HCV viral load (HCV RNA), baseline CD4 T cell count, and baseline HIV viral load (HIV RNA).
Liver fibrosis was assessed by different methods, depending on the hospital. At HGUGM, liver biopsy was used , and the fibrosis score was estimated following the criteria established by the METAVIR Cooperative Study Group : F0, no fibrosis; F1, portal fibrosis; F2, periportal fibrosis or rare portal-portal septa; F3, fibrous septa with architectural distortion but with no obvious cirrhosis (bridging fibrosis); and F4, definite cirrhosis. At HCIII, transient elastometry (FibroScan®; Echosens, Paris, France) was used , and liver stiffness values ≤ 7.0, between 7.1 and 9.4, between 9.5 and 12.4, and ≥ 12.5 were considered to correspond to METAVIR scores F0−F1, F2, F3 and F4, respectively .
Total DNA was extracted from peripheral blood using Qiagen columns (QIAamp DNA Blood Midi/Maxi; Qiagen, Hilden, Germany). DNA samples were sent to the Spanish National Genotyping Center (CeGen; http://www.cegen.org/). Genotyping was performed with Sequenom's MassARRAY platform (Sequenom, San Diego, CA) using the iPLEX® Gold assay design system. Additionally, the IL-28B polymorphism (rs12980275) was genotyped using the GoldenGate® assay with VeraCode® Technology (Illumina Inc., San Diego, CA).
Individuals within the European N macro-cluster were further separated into the most common European major haplogroups or clusters (HV, IWX, U and JT) and haplogroups (H, V, pre-V, J, T, I, W and X) according to 14 polymorphisms in the mtDNA.
Treatment regimens included were pegIFN-α 2a or 2b at standard doses (180 μg/week or 1.5 μg/kg/week, respectively) plus weight-adjusted RBV dosing (1000 mg/day for patients weighing < 75 kg and 1200 mg/day for patients weighing ≥ 75 kg). Patients with HCV genotype 1 or 4 received either 48 or 72 weeks of treatment, and patients with HCV genotype 2 or 3 were treated for 24, 48 or 72 weeks, according to virological response at week 4. Early stopping rules were applied for subjects with suboptimal virological response at weeks 12 and 24.
The HCV RNA viral load was measured by quantitative polymerase chain reaction (qPCR) (COBAS® AMPLICOR HCV test; Roche Diagnostic Systems, Branchburg, NJ; and COBAS AmpliPrep/COBAS TaqMan HCV test); results were reported in international units per millilitre (IU/mL), with a lower limit of detection of 10 IU/mL. An SVR was defined as an undetectable serum HCV RNA level (< 10 IU/mL) at week 24 after the end of the treatment. Patients who did not fulfill SVR criteria were considered as nonresponders.
The statistical analysis was carried out by on-treatment analysis of observed data, meaning that those patients who prematurely interrupted pegIFN-α/RBV treatment because of adverse events, who discontinued the study or who were lost to follow-up were excluded from the analysis.
The statistical analysis was performed with spss 15.0 (SPSS Inc., Chicago, IL). Statistical significance was defined as P < 0.05. Categorical data and proportions were analysed using the χ2 test or Fisher′s exact test. The Mann−Whitney test was used to compare continuous variables among independent groups. Logistic regression analyses were performed to investigate the association between mtDNA haplogroup and HCV therapy response. All logistic regression tests were adjusted by gender, age, HCV RNA values (patients with HCV RNA values ≥500 000 IU/mL vs. patients with HCV RNA values <500 000 IU/mL), HCV genotype (genotype 1/4 vs. genotype 2/3), IL28B rs12980275 polymorphism, and significant liver fibrosis (F≥2).
Table 1 shows the clinical and epidemiological characteristics of 304 HIV/HCV-coinfected patients on HCV therapy. Overall, the median age was 42 years and 76% of the patients were male. At baseline, the median CD4 count was 460 cells/μL, 72.4% of patients had HIV RNA < 50 HIV-1 RNA copies/mL, 82.9% were on cART, 66.1% had HCV genotype 1/4, 70.4% had an HCV RNA ≥ 500 000 UI/mL and 65.8% had significant liver fibrosis (F ≥ 2). High HCV RNA load (≥ 500 000 UI/mL), HCV genotype 1/4, and unfavourable IL-28B genotypes (rs12980275 AG/GG) were more frequent in nonresponder patients (P < 0.05).
Table 1. Main baseline epidemiological and clinical characteristics of HIV/hepatitis C virus (HCV)-coinfected patients on HCV antiviral therapy
The haplogroups Pre-V, V, I, X and W had frequencies of ≤ 5% and were therefore included in broader clusters to minimize type I errors in statistical analyses. Thus, the genetic association tests were performed on the major haplogroups or clusters HV, U, JT and IXW and on the haplogroups H, J and T.
The SVR rates were 52.6% (160 of 304) for all patients, and 37.8% (46 of 201) for patients with HCV genotype 1/4 vs. 81.4% (83 of 102) for patients with HCV genotype 2/3 (P < 0.001). When patients were stratified by mtDNA haplogroup, patients with haplogroup IXW had the lowest SVR rate among patients with HCV genotype 1/4 (29%), but we did not find significant differences compared with the other mtDNA haplogroups.
Figure 1 shows the adjusted likelihoods of achieving SVR according to mtDNA haplogroup. No significant associations were found between mtDNA haplogroup and SVR when all patients were included in the analysis and when they were stratified by HCV genotype (those with genotypes 1/4 and 2/3 analysed separately) (Fig. 1). Moreover, an additional subanalysis in which patients were stratified by IL-28B rs12980275 genotype (favourable vs. unfavourable) was performed, and there was no significant association between mtDNA haplogroup and SVR (data not shown).
To the best of our knowledge, this is the first report about the influence of mtDNA haplogroups on HCV treatment response. However, we did not find any association between mtDNA haplogroup and treatment response to pegIFN-α/RBV in HIV/HCV-coinfected patients.
Mitochondria are essential organelles that provide eukaryotic cells with energy via oxidative phosphorylation and regulate cellular survival via control of apoptosis, playing a key role in the innate immune response against virus infection . mtDNA haplogroups have been related to AIDS progression [12, 19] and CD4 T-cell recovery in HIV-infected patients , and to a delay in the development of liver fibrosis in HIV/HCV-coinfected patients . Contrary we expected, our data suggest that there is not significant association between mtDNA haplogroup and SVR in HIV/HCV coinfected patients on pegIFN-α/RBV therapy.
It is possible that this lack of association may be a consequence of the characteristics of our patients who started HCV therapy. However, this cohort has been used by us in other studies in which we have obtained significant results [21, 22]. We cannot rule out the possibility that the lack of an association was a consequence of the lack of statistical power to detect a small difference in outcome vs. no biological effect on outcome, regardless of genetic background. In this study, we had a limited/reduced sample size, which may have impaired our ability to detect less robust associations. In a simulation performed a posteriori (http://www.imim.cat/ofertadeserveis/software-public/granmo/) for two independent subgroups, namely patients with haplogroup H (SVR rate 57%) and those with other haplogroups (SVR rate 49.7%), we verified that, accepting an alpha risk of 0.05, we only achieved a statistical power of 25% to detect significant differences. A different issue is clinical relevance. Overall, the differences among SVR rates were not clinically relevant (< 10%) for the more prevalent haplogroups. However, haplogroup IXW is a very minor haplogroup among White Europeans (< 5%), and therefore it would be very difficult to achieve a large enough sample size to detect significant differences. In any case, if these reasons for an association not being found were valid, this would imply that the association between mtDNA haplogroup and SVR is rather weak.
IL-28B polymorphisms are already being used as a predictive marker of the response to pegIFN-α/RVB therapy in clinical practice . Although the rs12979860 polymorphism is more likely to be correlated with SVR in the Caucasian population , we have recently shown a strong association of four IL-28B polymorphisms (rs12980275, rs8099917, rs7248668 and rs11881222) with SVR in this cohort of HCV/HIV-coinfected patients . In the current study we used rs12980275, which is in high linkage disequilibrium with rs12979860 and has exhibited a very similar association with SVR .
This study has other limitations which must be taken into account for the correct interpretation of our data. First, the study design is retrospective. Secondly, we could not include a group of HCV-monoinfected patients to confirm our results for these patients. Thirdly, the results obtained may have been influenced by the tolerability of IFN/RBV, as only persons completing therapy were included in the analyses. Fourthly, our study was performed in patients with European haplogroups and our results are not generalizable to other racial/ethnic groups.
In conclusion, our data suggest that European mtDNA haplogroups were not related to HCV treatment response in HIV/HCV-coinfected patients on pegIFN-α/RBV therapy.
The authors thank the Spanish National Genotyping Center (CeGen) for providing SNP genotyping services (http://www.cegen.org). We also acknowledge the patients for their participation in this study and the Centro de Transfusión of Comunidad de Madrid for providing healthy donor blood samples.
Conflicts of interest: The authors do not have any commercial or other association that might pose a conflict of interest.
Financial disclosure: This work has been supported by grants from the Instituto de Salud Carlos III (grants PI08/0738 and PI11/00245 to SR; PI08/0928 and PI11/01556 to JB; and PI11/00870 to JMB), Fundación para la Investigación y la Prevención del Sida en España (FIPSE) (grant 361020/10 to JB), Spanish Network for AIDS Research (grant ISCIII-RETIC RD12/0017), and Fundación para la Investigación y la Educación en SIDA (FIES). MGF, MGA and MAJS are supported by grants from the Instituto de Salud Carlos III (CM09/00031, CM08/00101 and CM10/00105, respectively).
Contributions to authorship: JB, VS, JC, JCL and PM participated in patient selection, collection of samples and acquisition of data. MGF, AFR, MAJS, MGA, JMB, CR and NR participated in sample preparation, DNA isolation and genotyping pre-procedure, and contributed to critical revision of the manuscript. SR supervised the study. MGF and SR performed all statistical analysis, and interpretation of the data. JB and VS participated in conceiving the study concept and design and made important contributions to the draft written by SR and MGF. All authors revised and approved the final version of the manuscript.