Mutations in the mitochondrial DNA (mtDNA) acquired throughout human history have subdivided the human population into a number of discrete mitochondrial clades or haplogroups, which are defined on the basis of specific mitochondrial single nucleotide polymorphisms (mtSNPs) scattered throughout the mitochondrial genome [1,2]. In European whites, four mtDNA cluster or major haplogroups (HV, U, JT, and IWX) and several main haplogroups have been identified (H, V, J, T, Uk, W, X, I, etc.) .
Mitochondria are critical for energy production and naturally are involved in several metabolic pathways related to metabolic disorders . Additionally, the production of reactive oxygen species (ROS) as a toxic by-product during mitochondrial dysfunction may play a central role in a wide range of disorders such as cancer, AIDS, sepsis, diabetes, and degenerative diseases, which are mtDNA haplogroup associated . Thus, it has been suggested that some of these haplogroup-associated polymorphisms could act as risk factors or protective factors for these disorders and the progression of these diseases [5–8].
Coinfection with hepatitis C virus (HCV) is common among HIV-infected patients because both viruses share the same routes of transmission. Chronic hepatitis C in HIV-infected patients is characterized by a higher rate of fibrosis progression, cirrhosis, and end-stage liver disease compared with HCV-monoinfected patients [9,10]. HCV infection may lead to mitochondrial dysfunction due to increased lipoperoxidation, which may lead to the depletion of mtDNA, leading to an increased production of ROS possibly contributing to enhanced liver damage by HCV . There is evidence for mitochondrial damage from HIV itself and following therapy with nucleoside analogue reverse transcriptase inhibitors. HIV virulence factors have shown to cause mitochondrial damage in vitro and may induce cellular apoptosis via a mitochondrial pathway . Moreover, antiretroviral drugs may also cause mitochondrial dysfunction in the liver (decreased activity of respiratory complex I, but increased activity of respiratory complex IV) and mtDNA depletion in HIV patients, adding to any mitochondrial damage caused by chronic hepatitis C infection . In addition, certain drugs such as thymidine analogues are potent inhibitors of mitochondria DNA polymerase-γ , and thus more likely to inflict liver damage than other drugs .
We hypothesized that HIV infection, HCV liver disease, and mitochondrial DNA polymorphisms are three interrelated factors that might be associated with progression of liver disease. Consequently, we carried out the present study in order to explore whether mtDNA haplogroups had any influence on liver fibrosis progression in HIV/HCV coinfected patients.
Patients and methods
We carried out a cross-sectional study on HIV/HCV coinfected patients that underwent a liver biopsy at Hospital Gregorio Marañón (Madrid, Spain) between September 2000 and November 2008 that had had a DNA sample collected and frozen on the day of the liver biopsy. Liver biopsies were performed on patients who were potential candidates for anti-HCV therapy. The eligibility criteria for anti-HCV therapy included absence of prior hepatic decompensation, CD4+ cell count more than 200 cells/μl, stable antiretroviral therapy or no need for antiretroviral therapy, absence of active opportunistic infections, no active drug addiction, and absence of other concomitant diseases or conditions that contraindicate interferon with ribavirin therapy. From our cohort of 361 HIV/HCV coinfected patients with liver biopsy data, 243 patients had a DNA sample collected and frozen and 231 of these 243 patients had an European ‘N’ mitochondrial macrohaplogroup that is ancestral to almost all European and many Eurasian haplogroups .
Additionally, 126 healthy blood donors (HIV-negative and HCV-negative individuals) from the Centro de Transfusión of the Comunidad de Madrid participated as a control group. The study was conducted in accordance with the Declaration of Helsinki. All patients gave their written consent for the liver biopsy and the Institutional Ethics Committee approved the study.
Clinical and laboratory data
On the day of the liver biopsy, the following information was obtained from medical records: age, sex, risk category, weight, height, Centers for Disease Control and Prevention (CDC) clinical category, nadir CD4 T-cell count, prior antiretroviral therapy, antiretroviral treatment at the time of liver biopsy, and total time on HAART. Patients were questioned in relation to alcohol consumption. We considered the consumption of greater than 50 g of alcohol per day for at least 12 months as a high intake. Height and weight were determined at baseline, and BMI was calculated as the weight in kilograms divided by the square of the height in meters. The degree of insulin resistance was estimated for each patient using the homeostatic model assessment (HOMA) method: fasting plasma glucose (mmol/l) times fasting serum insulin (mU/l) divided by 22.5 . As in other studies, insulin resistance was considered to be altered when the HOMA score was 3.8 or higher .
HIV infection was documented in all the patients by ELISA and western blot assay. All patients tested positive for specific HCV antibodies and had detectable serum HCV-RNA as assessed by PCR. The HCV viral load was measured by PCR (Cobas Amplicor HCV Monitor Test, Branchburg, New Jersey, USA) and the results were reported in international units per milliliter. The HCV genotype was determined by hybridization of biotin-labeled PCR products to oligonucleotide probes bound to nitrocellulose membrane strips (INNO-LiPA HCV II, Innogenetics, Ghent, Belgium).
Liver biopsy and fibrosis
Liver biopsies were performed on an outpatient basis following the recommendations of the Patient Care Committee of the American Gastroenterological Association . All liver biopsies were performed by the same physicians (J.B. and P.M.) with a suction needle (HISTO-CUT 16G, Sterylab Srl. Milano, Milan, Italy). Ultrasound was routinely used to determine the percutaneous biopsy site. The liver tissue sections were fixed in formalin, embedded in paraffin, and stained by hematoxylin–eosin, Mason's trichrome, and Perls’ iron. The samples were evaluated by a pathologist (E.A.) who was unaware of the patients’ clinical or laboratory data.
Liver fibrosis was estimated following the criteria established by the METAVIR Cooperative Study Group . Fibrosis was scored as follows: F0, no fibrosis; F1, portal fibrosis; F2, periportal fibrosis or rare portal–portal septa; F3, fibrous septa with architectural distortion; no obvious cirrhosis (bridging fibrosis); and F4, definite cirrhosis.
In each patient, the fibrosis progression rate (FPR) was calculated dividing the fibrosis stage (0–4) by the estimated duration of HCV infection in years. For example, for a patient with fibrosis stage 2 (F2) and a 10-year duration of HCV infection, the FPR is 0.2. We selected a cut-off of 0.1 (near the 75th percentile) to classify the patients as previously used in another article . Duration of HCV infection was calculated assuming that HCV infection was acquired the first year needles were shared.
Mitochondrial DNA genotyping
Genomic DNA was extracted from peripheral blood using Qiagen columns (QIAamp DNA Blood Midi/Maxi; Qiagen, Hilden, Germany). The recovered DNA was quantified by spectrophotometry (Tecan Infinite 200 PRO Microplate Reader; Tecan Group Ltd, Mannedorf, Switzerland), diluted to a stock concentration of 50 ng/μl, and stored at −70°C. An aliquot of each patient's DNA was amplified (GenomiPhi V2. DNA Amplification Kit; GE Healthcare, Piscataway, New Jersey, USA) and sent to have the mtDNA genotyped at the Spanish National Genotyping Centre (CeGen, http://www.cegen.org/) with Sequenom's MassARRAY platform (San Diego, California, USA) using the iPLEX Gold assay design system.
Our study included European Spanish within the N macrohaplogroup that is ancestral to almost all European and many Eurasian haplogroups . Haplogroups within the western European (N) subset were further parsed with SNPs in the mitochondrial haplogrouping by the candidate functional variants approach described by Hendrickson et al.. Individuals who were not within the N macrohaplogroup (found in Africa and east Asia) were excluded from the study.
The analysis of variance test was used to compare the means between groups. Categorical data and proportions were analyzed using the χ2-test or Fisher's exact test as required. All tests were two tailed with P values less than 0.05 considered significant. Statistical analysis was performed by SPSS 15.0 software (SPSS Inc., Chicago, Illinois, USA).
Logistic regression analyses were performed to analyze the possible effect of the mtDNA haplogroups on the development of liver fibrosis [advanced fibrosis (F≥3) and cirrhosis (F4)] and FPR. When odds ratios (ORs) were calculated, the reference categories were all other haplogroups. The variables used in the adjusted model were sex, age, BMI, nadir CD4 T-cell counts, undetectable HIV-RNA, time on HAART, current HAART with thymidine analogues (zidovudine, didanosine, stavudine), HCV-RNA at least 850 000 IU/ml, HCV-genotype 1, insulin resistance (HOMA ≥ 3.8), and high alcohol intake.
Characteristics of the patients
Table 1 shows the clinical and epidemiological characteristics of 231 HIV/HCV coinfected patients who self-identified as ‘white’ and had a western European, or ‘N’ mitochondrial macrohaplogroup.
We did not find significant differences in the frequencies of mtDNA haplogroups between HIV/HCV coinfected patients and healthy control group, except for haplogroups Uk and X, which were less prevalent in HIV/HCV coinfected patients (Table 2). Genetic association tests were performed on the major haplogroups HV, U, JT, and IXW and on the haplogroups H, V, Uk, J, T, I, X, and W. However, the haplogroups Pre-V, Uk, I, X, and W had absolute frequencies of less than 10 and were, therefore, included in broader haplogroups to minimize type I errors.
Influence of mitochondrial DNA haplogroup on liver fibrosis
The frequency of HIV/HCV coinfected patients with F3 or F4 had a linear downward trend with statistical significance within major haplogroup HV and haplogroup H (Fig. 1). Haplogroup U had a high frequency of patients with cirrhosis, but the P value was not significant.
Table 3 shows the significant associations that exist for the mitochondrial haplogroups HV, H, and U with hepatic fibrosis stage and FPR according to univariate logistic regression analyses. Specifically, the major haplogroup HV was significantly associated with reduced odds of advanced fibrosis (F≥3), cirrhosis (F4), and high fibrosis progression rate (FPR ≥ 0.1). Within the major haplogroup HV, haplogroup H also had decreased odds of advanced fibrosis, cirrhosis, or high FPR. We also found a significant association with increased odds of cirrhosis (F4) in the closely related major haplogroup U (Table 3).
Figure 2 shows the association between mitochondrial haplogroups and liver fibrosis adjusted for epidemiological, clinical, and virological characteristics. Taking F0–F1–F2 as the reference category, we found that the adjusted OR of having F3–F4 decreased significantly in haplogroups HV and H. This effect was not found in haplogroup U (Fig. 2a). When F<4 [indicating the fibrosis stages that are different from F4 (F0, F1, F2, and F3)] was taken as the reference category, we found that the adjusted OR of having F4 also decreased significantly in haplogroups HV and H, and increased in haplogroup U (Fig. 2b). Finally, when FPR of 0.1 was taken as the reference category, we found that the adjusted OR of having an FPR more than 0.1 decreased significantly in haplogroups HV and H. This effect was not found in haplogroup U (Fig. 2c)
In this study, we examined the genetic association between major European mitochondrial DNA haplogroups and liver fibrosis in 231 HIV/HCV coinfected patients. We found that the mitochondrial major haplogroup HV and the haplogroup H were strongly associated with reduced odds of advanced fibrosis, cirrhosis, and fibrosis progression, whereas the major haplogroup U was strongly associated with an increased odds of cirrhosis. To the best of our knowledge, this is the first study to identify an association between mtDNA haplogroups and liver fibrosis in chronic hepatitis C patients, and also the first to characterize mitochondrial haplogroups among HIV/HCV coinfected patients.
mtDNA haplogroups have been most extensively used to understand prehistoric migrations and human evolution . A recent study in European population has shown that in general the most frequent haplogroups were H (41%) and U (21%). Less frequent haplogroups were J and T, each with a frequency of 8%. Frequencies of other haplogroups (V, Pre-V, Uk, W, X, and I) did not exceed 5% . Our results showed that the distribution of mtDNA haplogroups across our HIV/HCV coinfected patients corresponded to that found by other authors across the entire European population  and the white population with HIV infection [7,23]. However, we found a low frequency of haplogroups Uk and X among HIV/HCV coinfected patients with respect to our healthy controls of similar age and sex. We believe that these differences could be due to chance, however, because the absolute frequencies of these haplogroups were very low in both our HIV/HCV coinfected patients and healthy controls.
Only recently has the potential relevance of mitochondrial haplogroup to human health and disease been considered. However, the molecular mechanisms underlying functional differences between haplogroups are still unknown [5,24]. In HIV-negative patients, the association of European mtDNA haplogroups with the evolution of infectious diseases is contradictory [6,25,26]. Haplogroup H has been associated with increased survival rates in individuals with sepsis , but other studies failed to show an association of haplogroup with risk of infection or survival [25,26]. mtDNA haplogroups have been previously studied in HIV-infected patients [7,8,23,27,28] showing an association of mtDNA haplogroup with HIV disease progression and some side-effects of antiretroviral therapy such as lipodystrophy, peripheral neuropathy, and neuroretinal disorder. Of note, one of these studies found an association of haplogroups J and U5a with increased AIDS progression, whereas haplogroup H3 was associated with decreased AIDS progression and death in naive HIV-infected patients .
To the best of our knowledge, mtDNA haplogroup studies have not been carried out on patients with chronic hepatitis C, either HCV monoinfected or HIV/HCV coinfected. In our study, after adjusting for epidemiological, clinical, and virological characteristics related to HIV/HCV coinfection, uncoupled haplogroup U (lower ATP and ROS production) was associated with cirrhosis. Pertaining to haplogroup U might accelerate liver fibrosis, thus enhancing the effects of mtDNA depletion, disruption of oxidative phosphorylation complexes, antioxidant enzyme deficiency, and apoptosis . In contrast, the more tightly coupled haplogroups HV and H (higher ATP and ROS production) were associated with reduced odds of advanced liver fibrosis and cirrhosis in our patients. Haplogroups HV and H might have increased ATP and ROS production, possibly enhancing innate immunity and contributing to slower disease progression . At this point, we are confronted with a contradiction in our data, as haplogroup H may produce more ROS and may increase the oxidative damage in liver, but has lower odds of fibrosis in our patients of haplogroups HV and H. However, only an increased generation of ROS together with decreased antioxidant defenses promotes the development and progression of hepatic complications during HCV infection . It also is possible that higher rates of ROS production may lead to, first, an upregulation of antioxidant defenses , to such an extent as ROS do not cause severe liver damage, and, second, a good immune function delaying the process of fibrosis in HIV/HCV coinfected patients , and ensuring good control of HIV and HCV replication, which may in turn decrease ROS production and apoptosis [32,33]. Besides, it is possible that the degree of energy efficiency could have a larger impact than the generation of ROS in the pathophysiology of chronic hepatitis C, allowing that patients with haplogroups HV and H had slower progression than patients with haplogroup U.
This study has several limitations which must be taken into account for the correct interpretation of the data: the study design is cross-sectional; the number of patients is low for a genetic epidemiology study; we did not include HCV-monoinfected patients; and the study was performed with European haplogroups, and AIDS and chronic hepatitis C epidemics have increasingly affected persons of non-European ancestry. It is critical to define relationships between mitochondrial haplogroups and metabolic disturbance in these other populations.
In conclusion, the mtDNA haplogroups HV and H were associated with slower fibrosis progression and the haplogroup U was associated with faster fibrosis progression in HIV/HCV coinfected patients, suggesting that mtDNA haplogroup may play a significant role in liver fibrogenesis in chronic hepatitis C patients.
Conflicts of interest
This work has supported by grants from Instituto de Salud Carlos III (Ref. PI08/0738 to S.R.; Ref. FIS 06/1030 to Y.C.; and Ref. ISCIII-RETIC RD06/006 and PI08/0928 to J.B.), Fundación Alicia Koplowitz (Grant 2008 to J.B.) and Fundación para la Investigación y la Prevención del Sida en España (FIPSE) (Ref. 36443/03 and 361020/10 to J.B.).
M.G.-F. is supported by a grant of Instituto de Salud Carlos III (CM09/00031). M.G.-A. is supported by a grant of Instituto de Salud Carlos III (CM08/00101).
J.B. is supported by a grant from the ‘Programa de Intensificación de la Actividad Investigadora en el SNS’ (I3SNS).
M.G.-A., J.B., and S.R. performed the study concept and design.
J.B., D.M., J.C.L., J.C., and P.M. performed the acquisition of data.
M.G.-A., M.G.-F., E.A., Y.C., and S.R. performed the analysis and interpretation of data.
M.G.-A., J.B., and S.R. drafted the manuscript.
M.G.-A., J.B., Y.C., and S.R. performed the critical revision of the manuscript for important intellectual content.
M.G.-A. and S.R. performed the statistical analysis.
Administrative, technical, or material support was provided by M.G.-F., M.G.-A., and S.R.
J.B and S.R. supervised the study.
The authors’ acknowledge the patients in this study for their participation and the Centro de Transfusión of Comunidad de Madrid for the healthy donor blood samples provided.
There are no conflicts of interests.
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