Mitochondria play an important role in energy production and different metabolic pathways (ie, biosynthesis of cholesterol, phospholipids).1,2 Human mitochondria have their own genome that is inherited maternally and, over millennia, single nucleotide polymorphism in the mitochondria genome have emerged. The combinations of these polymorphisms define the mitochondrial haplotypes, generally called haplogroups.3 Mitochondrial haplogroups have a well-defined phylogeny that has been used to map and understand prehistoric human migrations and genetic differences among populations. Nine major haplogroups (H, T, U, V, W, X, J, I, and K) and 4 major phylogenetic clusters (HV, KU, JT, and IWX) have been identified within European populations.4,5 The most prevalent haplogroup in Europe is haplogroup H or cluster HV, representing approximately 40% of the European population.6
Several studies have shown possible associations between haplogroups and various diseases including metabolic and inflammatory diseases such as arthritis but also infectious diseases such as HIV/AIDS and hepatitis C.1,2,6–8 In the case of hepatitis C virus infection (HCV), there are very limited data available, performed only on HIV/HCV-coinfected patients. Associations have been suggested between haplogroups H and HV with slower fibrosis progression.7 However, these results need to be further explored and confirmed in other clinical cohorts.
In this context, the aim of this study was to evaluate the impact of mitochondrial DNA (mtDNA) haplogroups on the outcome of HCV infection and liver fibrosis in both HCV-monoinfected and HIV/HCV-coinfected patients in clinical follow-up at 2 reference hospitals in the Northwest of Spain.
All HCV-infected patients attending for clinical follow-up at 2 hospitals in the Northwest of Spain (Complexo Hospitalario Universitario de A Coruña and Complexo Hospitalario Universitario de Ferrol) between September 2014 and January 2015 were recorded. Epidemiological, clinical, and virological data were retrospectively recorded using a data sheet collection specifically designed for this study.
The most common European mtDNA haplogroups were determined using a single-base extension assay using a standardized protocol previously described elsewhere.9 Briefly, genomic DNA was extracted from peripheral blood mononuclear cells using conventional methods (QIAamp DNA blood midi/maxi; Qiagen, Hilden, Germany). Six specific primers were designed to amplify, in a single multiplex reaction, the mtDNA fragments containing each of the informative single nucleotide polymorphisms that characterize the 6 major European mtDNA haplogroups (H, V, K, U, T, and J). The polymorphic sites analyzed in this study (m.7028c>t, m.14766 t>c, m.10394a>g, m.4580g>a, m.12308a>g, and m.4216c>t) have been previously reported.
Liver stiffness measurements were performed through transient elastography (FibroScan; EchoSense, Paris, France) and expressed in kilopascals. Fibroscan results measured in kilopascals were converted into the metavir scale as follows: F0-F2 <9.5 kPa; F3 ≥9.5 and ≤12.5 kPa; and F4 >12.5 kPa (cirrhosis).10
Statistical analysis was performed using Statistical Package for the Social Sciences software (SPSS 19.0, Chicago, IL). A descriptive analysis was performed, presenting data as mean ± SD for quantitative variables, frequencies, and percentages for qualitative variables. Liver stiffness values were compared in monoinfected and HIV/HCV-coinfected patients and according to mtDNA haplogroups, using Mann–Whitney and Kruskal–Wallis test, respectively. Prevalence of liver cirrhosis was also compared among clusters of mtDNA haplogroups with the χ2 test for the univariate analysis. Finally, a multivariate logistic regression model was performed to identify those factors independently associated with cirrhosis.
The study was approved by the regional ethics committee (register code 2014/568). All patients included in the study have signed the informed consent.
Characteristics of the Study Population
A total of 367 HCV and HIV/HCV-coinfected patients were recorded. From these, 5 patients without Caucasian ethnicity and 10 patients in which mtDNA haplogroup could not be determined were excluded. Therefore, the final analysis was performed in 352 patients. The majority was male patients (73.6%) with a mean age of 49.95 ± 9.15 years. Fifty-eight percent of patients were HIV/HCV-coinfected, and the main route of transmission was injected drug use (76%). The mean of years since HCV diagnosis was 15.21 ± 7.8. The most prevalent HCV genotypes were G1a 136 (40.7%), G1b 72 (21.5%) followed for G3 66 (19.8%), G4 53 (15.9%), and G2 7 (2.1%). The median fibroscan values at the time of the study was 8.80 kPa (interquartile range: 6.3–14.6 kPa) without differences between HCV-monoinfected and HIV/HCV-coinfected patients (9 vs. 8.56 kPa, P = 0.434). 29.2% of the study population had cirrhosis (>12.5 kPa) and 4% hepatic decompensation.
Distribution of European mtDNA Haplogroups
The distribution of the European mtDNA haplogroups was as follows: H 177 (50.3%), U 44 (12.5%), T 31 (8.8%), Others (I, W, X, M) 34 (9.7%), J 28 (8%), K 21 (6%), V 8 (2.3%), and SVH 9 (2.6%). For the subsequent analysis, patients were pooled into the main clusters of mtDNA haplogroups defined according to the human mitochondrial phylogenetic tree with the following prevalence: 194 (55.1%) HV (H+V+SHV+H10), 65 (18.5%) KU, 59 (16.8%) TJ, and 34 (9.7%) Others (I, X, W).
The prevalence of HCV genotypes (G1, G3, and G4) among the major clusters of mtDNA haplogroups was: 53.6%, 63.5%, and 75.7% for patients belonging to cluster HV; 19%, 11.5%, 8.1% for cluster KU; 16.3%, 17.3%, 10.8% for cluster JT; and 11.1%, 7.7%, 5.4% for cluster Others, respectively. Overall, there were no differences between the HCV genotypes distribution and the major clusters of mtDNA haplogroups. However, a higher prevalence of patients infected with HCV genotype 4 was observed in patients belonging to cluster HV compared with those infected with HCV genotype 1 (64.4% vs. 53.2%, P < 0.014).
Association Between mtDNA Haplogroups and Liver Fibrosis
The median liver stiffness values (kPa) were determined among the European mtDNA haplogroups, having patients belonging to the haplogroup V and Others (I, W, X), presented higher values (16.1 vs. 11.3, respectively) (Fig. 1a). Subsequently, the prevalence of liver cirrhosis (>12.5 kPa) was evaluated among clusters of mtDNA haplogroups based on the degree of liver fibrosis. Interestingly, and after adjusting for the time since HCV diagnosis, a higher proportion of patients with cirrhosis belonging to haplogroup V and Others (I, W, X) were observed compared with those belonging to cluster H and SHV (47.4% vs. 25.3%, P = 0.009) (Fig. 1b).
A multivariate analysis was performed to identify those factors associated with cirrhosis. The analysis was adjusted by age, gender, HCV diagnosis time, HCV genotype, and HIV coinfection. The cluster Others and V was identified as a risk factor for the development of liver fibrosis (odds ratio (OR) = 2.429; 95% CI = 1.086 to 5.433; P = 0.031) together with advanced age (OR = 1.076; 95% CI = 1.04 to 1.113; P < 0.001). Conversely, HCV genotype 4 was identified as a protective factor for the development of cirrhosis (OR = 0.363; 95% CI = 0.149 to 0.884) (Fig. 2).
This study evaluates the impact of mtDNA haplogroups on the outcome of liver fibrosis in 146 HCV-monoinfected and 206 HIV/HCV-coinfected patients in clinical follow-up at 2 reference hospitals in the Northwest of Spain. The mtDNA haplogroup H was the most prevalent (177 patients, 50.3%) in this population. We found an association between mtDNA and liver fibrosis. Interestingly, the cluster Others and V were recognized as risk factors for the development of liver fibrosis. Conversely, those patients infected with HCV genotype 4 strains would have a lower risk for liver fibrosis progression.
Several studies have shown associations between mtDNA haplogroups and various diseases including metabolic and inflammatory diseases such as arthritis but also infectious diseases. This is the case of HIV/AIDS for which the mtDNA haplogroups H and/or HV have been associated with a lower risk of progression to AIDS among HIV-infected patients. However, the number of studies in these fields are very limited and especially in the case of HCV. To our knowledge, there is only one study published by García-Álvarez et al,8 in which they examined the association between major European mtDNA haplogroups and liver fibrosis in 231 HIV/HCV-coinfected patients.
Similar to our data, they also recognized haplogroup H as the most prevalent. These figures are also in agreement with other results published in European population.11 Moreover, they found that both haplogroup H and cluster HV were associated with reduced odds of advanced fibrosis, cirrhosis and fibrosis progression, whereas the haplogroup U was associated with an increased odds of cirrhosis. Our results also found an association between the mtDNA haplogroup H with lower levels of liver fibrosis and risk of cirrhosis. However, the haplogroups related with higher levels of liver fibrosis and cirrhosis were the cluster Others and V. These differences might be due to the heterogeneity of the study populations, study design, and methodology of both studies. Indeed, in this study, we included both HCV-monoinfected and HIV/HCV-coinfected patients instead of only HIV/HCV-coinfected, there are differences in the prevalence among haplogroups U (12.5% vs. 25.1%) and Others (9.7% vs. 4.3%) between both populations, and we assessed liver stiffness using transient elastography instead of liver biopsy.
Moreover, HCV genotype 4 was recognized as a protective factor for the development of cirrhosis compared with genotype 1. Several studies have associated HCV genotype 3 with accelerated fibrogenesis and an increased risk of developing hepatocellular carcinoma relative to other HCV genotypes.12 However, the impact of HCV genotype 4 infected patients on the fibrosis progression has been assessed in few studies with controversial results.13,14 Although some longitudinal studies did not find any association in the fibrosis progression among HCV genotypes, other transversal cohort studies have observed higher rates of fibroscan values. Of note, these transversal studies were performed among HCV-infected patients harboring other infections such as HIV or Schistosoma mansoni, both infections identified as risk factors of fibrosis progression.15
Current therapeutic options for the treatment of HCV infection, highly effective and safe, present a favorable scenario to treat and cure all patients chronically infected with HCV. However, there are limitations, mainly due to economic restrictions. HCV treatment guidelines have been established in which patients HCV treatment needs to be prioritized.16,17 Moreover, recent discoveries have highlighted the influence of host genomics on HCV outcomes and progression of liver diseases.18,19 Therefore, advances in the identification of viral and host factors influencing the progression of liver fibrosis are needed for the selection of HCV-infected patients with higher risk of progression for treatment prioritization. Hence, those patients belonging to haplogroups Others (I, W, X) or V and infected with HCV genotype 1 variants might be the candidates for treatment initiation even in those patients who present low levels of liver fibrosis (F0-F1, metavir scale).
There are some limitations in this study that must be taken in consideration for the correct interpretation of the results obtained. This is a cross-sectional study, and the number of patients belonging to mtDNA haplogroups different from the most prevalent (H) is low for a genetic epidemiology study. However, these data must be of useful to optimize the need and urgency to start HCV treatment, at least in our population.
In conclusion, this study demonstrated an association between liver fibrosis and mtDNA European haplogroups in a large cohort of HCV-monoinfected and HIV/HCV-coinfected patients. The mtDNA haplogroups cluster Others and V had higher risk for the development of cirrhosis while haplogroup H had lower. In addition, patients infected with HCV genotype 4 presented lower levels of fibrosis and lower risk of cirrhosis than those infected with HCV genotype 1. Therefore, these findings might be useful for prioritization of HCV treatment, especially for F0-F1 patients for whom there is no urgency for treatment.
The authors would like to thank to Biobank of A Coruña (SERGAS) for providing us the technical, ethical, and legal advice necessary for the development of our research.
1. DiMauro S, Schon EA. Mitochondrial respiratory-chain diseases. N Engl J Med. 2003;348:2656–2668.
2. Wallace D. A mitocondrial paradigm of metabolic and degenerative diseases, aging, and cancer: a dawn for evolutionary medicine. Annu Rev Genet. 2005;39:359–407.
3. Cann RL, Stoneking M, Wilson AC. Mitochondrial DNA and human evolution. Nature. 1987;325:31–36.
4. Torroni A, Huoponen K, Francalacci P, et al. Classification of European mtDNAs from an analysis of three European populations. Genetics. 1996;144:1835–1850.
5. van Oven M, Kayser M. Updated comprehensive phylogenetic tree of global human mitocondrial DNA variation. Hum Mutat. 2009;30:E386–E394.
6. Rego-Pérez I, Fernandez-Moreno M, Soto-Hermida A, et al. Mitochondrial genetics and osteoarthritis. Front Biosci (Schol Ed). 2013;5:360–368.
7. Hendrickson S, Hutcheson HB, Ruiz-Pesini E, et al. Mitochondrial DNA haplogroups
influence AIDS progression. AIDS. 2008;22:2429–2439.
8. García-Álvarez M, Guzmán-Fulgencio M, Berenguer J, et al. European mitocondrial DNA haplogroups and liver fibrosis
in HIV and hepatitis C virus coinfected patients. AIDS. 2011;25:1619–1626.
9. Rego-Pérez I, Fernández-Moreno M, Fernández-López C, et al. Mitochondrial DNA haplogroups
: role in the prevalence and severity of knee osteoarthritis. Arthritis Rheum. 2008;58:2387–2396.
10. Intraobserver and interobserved variations in liver biopsy interpretation in patients with chronic hepatitis C. The French METAVIR Cooperative Study Group. Hepatology. 1994;20:15–20.
11. Vidrová V, Tesarová M, Trefilová E, et al. Mitochondrial DNA haplogroups
in the Czech population compared to other European countries. Hum Biol. 2008;80:669–674.
12. Probst A, Dang T, Bochud M, et al. Role of hepatitis C virus genotype 3 in liver fibrosis
progression-a systematic review and meta-analysis. J Viral Hepat. 2011;18:745–759.
13. Kamal SM, Nasser IA. Hepatitis C genotype 4: what we know and what we don't yet know. Hepatology. 2008;47:1371–1383.
14. Abdel-Ghaffar TY, Sira MM, Naghi SE. Hepatitis C genotype 4: the past, present, and future. World J Hepatol. 2015;7:2792–2810.
15. Kamal S, Madmar M, Bianchi L, et al. Clinical, virological and histopathological features: long-term follow-up in patients with chronic hepatitis C coinfected with S. mansoni. Liver. 2000;20:281–289.
16. Recommendations for testing, managing, and treating hepatitis C. AALD & IDSA. Available at: http://hcv.guidelines.org
. Accessed August 22, 2015.
18. Rembeck K, Lagging M. Impact of IL28B, ITPA and PNPLA3 genetic variants on therapeutic outcome and progression of hepatitis C virus infection. Pharmacogenomics. 2015;16:1179–1188.
19. Poveda E, Wyles D, Morano L, et al. News on HIV-HCV coinfection: update from the 2015 GEHEP conference. AIDS Rev. 2015;17:231–237.