Mitochondria are essential organelles that provide the eukaryotic cells with energy via oxidative phosphorylation and regulate cellular survival via control of apoptosis. A physiological consequence of oxidative phosphorylation is the generation of reactive oxygen species (ROS), which can be produced at high levels when there is a mitochondrial respiratory chain dysfunction. Mitochondria have its own genome, the mitochondrial DNA (mtDNA), and variations in mtDNA sequence have been associated with several disorders as cancer, sepsis, diabetes, and degenerative diseases.1 Some polymorphic variants of mtDNA, acquired throughout human evolution history, have subdivided the human population into a number of discrete mitochondrial haplogroups, which are defined on the basis of specific polymorphisms scattered throughout the mtDNA. In European whites, 4 mtDNA cluster or major haplogroups (HV, U, JT, and IWX), and several main haplogroups have been identified (H, V, pre-V, J, T, Uk, W, X, I, etc).2
In HIV infection, mtDNA haplogroups have been related to AIDS progression, lipodistrophy, metabolic alterations, and liver fibrosis progression.3–8 Mitochondria may play a key role in the immune response against HIV infection and systematic mitochondrial dysfunction is evident during AIDS progression. A mtDNA depletion, increased ROS production, deficiency of antioxidant enzymes, and increased oxidative damage have been described among HIV-infected patients with accelerated disease.9 In addition, HIV proteins have been implicated in the destabilization of mitochondrial membrane to promote the targeted depletion of key immune cells, which assists in HIV evasion and contributes to the characteristic global immunodeficiency observed during AIDS progression.9
The aim of our study was to determine whether mtDNA haplogroups are associated with the clinical pattern of AIDS progression in patients infected with HIV and without antiretroviral treatment (ART).
PATIENTS AND METHODS
We carried out a cross-sectional study on 469 HIV-infected patients from the Cohort of the Spanish HIV Research Network (CoRIS),10 Cohort of Long Term Non-Progressors (LTNP), and its associated HIV Biobank.11 Additionally, 171 healthy blood donors [HIV, hepatitis C virus (HCV), and hepatitis B virus–negative subjects] from the “Centro de Transfusión de la Comunidad de Madrid” participated as a control group. The study was conducted in accordance with the Declaration of Helsinki. Participants gave informed genetic consent for the study.
HIV-infected patients were classified in 3 groups according to the clinical pattern of AIDS progression10: (1) 154 LTNP patients who were defined as subjects with asymptomatic HIV infection more than 10 years after seroconversion and always CD4+ ≥500 cells per cubic millimeter and RNA viral load ≤10,000 copies per milliliter, (2) 233 typical or moderate progressors (MP) patients who had at least 2 years of follow-up without ART with an average decrease of 50–100 CD4+ per cubic millimeter per year, and (3) 82 rapid progressors (RP) patients who had 2 or more CD4+ T-cell measurements below 350 per cubic millimeter within 3 years after seroconversion without ART and/or who had AIDS or AIDS-related death.
The clinical and epidemiological data provided by patients were included in the cohort of adult with HIV infection of the AIDS Research Network (CoRIS) launched in 2004. CoRIS is an open cohort, multicenter cohort of patients newly diagnosed with HIV infection at the hospital or treatment center, more than 13 years of age, and naive to ART. Each participating patient signed an informed consent form. The programme was approved by the institutional review boards of the participating hospitals and centers (the cohort has been described in detail elsewhere).10 The information is subject to internal quality controls; once every 2 years, information on 10% of the cohort is audited by an externally contracted agency.10
Samples from patients were kindly provided by the HIV Biobank integrated in the Spanish AIDS Research Network (RIS).11 Samples were processed after current procedures and frozen immediately after their reception. All patients participating in the study gave their informed consent and protocols were approved by institutional ethical committees.
Total DNA was extracted from peripheral blood with Qiagen columns (QIAamp DNA Blood Midi/Maxi; Qiagen, Hilden, Germany). DNA samples were sent to the Spanish National Genotyping Center (http://www.cegen.org/) to genotype 15 polymorphisms in mtDNA. Genotyping was performed by using Sequenom’s MassARRAY platform (San Diego, CA) using the iPLEX Gold assay design system.
Haplogroups within the European (N macrohaplogroup) were further parsed with single nucleotide polymorphisms in the Mitochondrial Haplogrouping by the Candidate Functional Variants approach described by Hendrickson et al.6 The subjects were classified in the most common European haplogroups as described in our previous study.5 Haplogroups that could not be assigned to any of the European haplogroups by their single nucleotide polymorphisms combination were designated as “others.”
Categorical data and proportions were analyzed by using the χ2 test or Fisher exact test as required. Kruskal–Wallis and Mann–Whitney tests were used to compare data among independent groups when the dependent variable was continuous.
The 3 patient groups (LTNP, MP, and RP) may be considered as 3 different and very distinct progression paths for the natural history of HIV infection. Thus, the clinical pattern of AIDS progression (clinical outcome) was scored as an ordinal variable as follows: 1, non-AIDS progression or LTNP patients; 2, moderate AIDS progression or MP patients; and 3, rapid AIDS progression or RP patients. The association of mtDNA haplogroups in HIV-infected patients with the clinical pattern of AIDS progression was tested by Gamma correlation coefficient (values between −1 and +1), a nonparametric test for measuring the association between ordinal data. Furthermore, this association was also studied by ordinal logistic regression, assuming that the relationship between each pair of outcome groups is the same and the coefficient that describes the relationship between the lowest versus all higher categories of the response variable is the same as those that describes the relationship between the next lowest category and all higher categories.
All tests were 2-tailed, and P values <0.05 considered statistically significant. Statistical analysis was performed by SPSS 15.0 software (SPSS Inc, Chicago, IL).
Characteristics of the Study Population
The control group and all HIV-infected patients had similar age (42 vs 42.6 years, respectively) and count of males [123 (71.9%) vs 367 (78.2%), respectively]. However, when HIV-infected patients were classified according to clinical pattern of AIDS progression, LTNP group had higher values than MP and RP groups of age, percentage of HIV acquired by intravenous drug users (IDUs), or homosexual transmission; but LTNP group had the lowest percentage of males (Table 1). Also, it is to note that the median year of HIV diagnosis in LTNP was 1990, whereas MP and RP groups were 2005 and 2008, respectively.
For the first HIV marker data collected, LTNP group had higher values of CD4+ T cells and lower values of HIV viral load than MP and RP groups (Table 1). Besides, RP group had the lowest values of CD4+ T cells and the highest values of viral load (Table 1). When we analyzed the evolution of HIV markers according to clinical pattern of AIDS progression in these patients without ART (see Figure S1, Supplemental Digital Content, http://links.lww.com/QAI/A391), LTNP group had an excellent performance of CD4+ T cells and HIV viral load, whereas RP group had the worst evolution of theses HIV markers.
mtDNA Haplogroups and Clinical Pattern of AIDS Progression
HIV-infected patients and healthy control group had a similar percentage of European haplogroups (Fig. 1A). However, the distribution of European haplogroups in HIV-infected patients according to clinical pattern of AIDS progression was different (Fig. 1B). We found a negative ordinal correlation for cluster HV (γ = −0.167, P = 0.034) and haplogroup H (γ = −0.186, P = 0.020; Fig. 1B). Thus, the pattern of rapid AIDS progression tended to be lower when patients had haplogroups HV and H. In addition, LTNP group had higher frequency than RP group of cluster HV (50.6% vs 36.6%, P = 0.053) and haplogroup H (46.8% vs 31.7%, P = 0.036; Fig. 1B).
We also analyzed the influence of mtDNA haplogroups on clinical pattern of AIDS progression by adjusted ordinal logistic regression. The chance of not having an AIDS progression was 1.45 [95% of confidence interval (CI) = 1.02 to 2.05, P = 0.035] times greater in patients with cluster HV and 1.51 (95% CI = 1.06 to 2.18, P = 0.021) times greater in patients with haplogroup H. However, we only found significant values for haplogroup H (odds ratio = 1.52, 95% CI = 1.01 to 2.32, P = 0.049) after adjusting the ORL by gender, age at HIV infection, IDU, and HCV infection.
In this study, we examined the genetic association of 4 European mtDNA cluster (HV, U, JT, and IWX) and 8 haplogroups (H, V, pre-V, J, T, I, W, and X) with 3 clinical pattern of AIDS progression in HIV-infected patients of 2 Spanish large cohorts (CoRIS and LTNPs). Our results showed that cluster HV and haplogroup H were more frequent in LTNP patients than in RP patients, and these 2 haplogroups were statistically associated with the clinical pattern of non-AIDS progression. These 3 definitions of clinical patterns of AIDS progression involved no ART, although patients of MP and RP groups could be subsequently treated. That is, there are 3 patterns of evolution of the natural history of HIV infection. Moreover, our results showed that the distribution of mtDNA haplogroups across our HIV-infected patients were similar to data found by other authors in white population with HIV infection.3,6,8
The molecular mechanism underlying the functional differences between mitochondrial haplogroups may play a role in HIV progression. Mitochondrial haplogroup H has demonstrated to have a higher activity in the electron transport chain, producing more quantity of adenosine triphosphate (ATP) and ROS than other haplogroups of less energy efficient, such as haplogroup J.12 These differences in energy efficiency and production of ROS may be related to the association of haplogroups with the decline of CD4+ T cells and AIDS progression, lipodystrophy, metabolic alterations, and liver fibrosis progression in HIV-infected patients.3–6,8
Moreover, our data partially confirm the findings of Hendrickson et al,6 which reported an association between the uncoupled haplogroups J and U (lower ATP production and ROS) with increased AIDS progression, whereas the more tightly coupled haplogroup H (higher ATP production and ROS) was associated with decreased AIDS progression and death in naive HIV-infected patients.6 In our study, we found that patients with haplogroups HV and H (higher ATP and ROS production) had reduced odds of rapid AIDS progression. However, there may be a contradiction at this point because haplogroup H may produce more ROS,12 increasing the oxidative damage in the immune system during disease progression.13 In this regard, increased generation of ROS accompanied with an increase of antioxidant defenses during HIV infection has been described.14,15 Furthermore, higher rates of ROS production may lead to an upregulation of antioxidant defenses without causing severe immune damage,16 which may contribute to maintain a good immune function, ensuring a good control of HIV replication and, in turn, decreasing oxidative stress and apoptosis.17,18 Besides, it is possible that the degree of energy efficiency could have a larger impact on the pathophysiology of HIV infection than the generation of ROS, allowing patients with haplogroups HV and H to have slower AIDS progression than patients with other haplogroups.
Note that there were other significant associations with LTNP group in this article. The significant difference in age and years of HIV diagnosis was obviously expected for the LTNP group. However, the very high proportion of IDU and HCV infection (which are, of course, highly correlated with each other) in the LTNP group was unexpected and very strong. A similar, though less extreme, relationship between HIV controllers and IDU has been reported by Boufassa et al.19 There is no clear explanation for this finding. It is conceivable that homosexual men, as a group, may be treated earlier than IDU patients, making them ineligible as LTNP. We cannot either exclude that the route of infection might play a role.
This study has several limitations that must be taken into account for the correct interpretation of the data: (1) the study design is cross-sectional and the number of patients is low for a genetic epidemiology study, (2) the profound differences among groups regarding to age, sex, HCV infection, and IDU might hide other unmeasured confounders that might introduce a bias selection, and (3) the study was performed over European haplogroups and the AIDS epidemic affects more to people of non-European descent. It is critical to define relationships between mitochondrial haplogroups and AIDS progression in these other populations.
In conclusion, the presence of cluster HV and haplogroup H was associated with a clinical pattern of non-AIDS progression. These data suggest that mtDNA haplogroups might play a significant role in AIDS progression during HIV infection.
The authors particularly acknowledge the patients in this study for their participation and to the HIV Biobank integrated in the RIS and collaborating centers for the generous gifts of clinical samples used in this work. The HIV Biobank, integrated in the RIS, is supported by Instituto de Salud Carlos III, Spanish Health Ministry (RD06/0006/0035) and Fundación para la investigación y prevención del SIDA en España. This study would not have been possible without the collaboration of all the patients, medical and nursery staff, and data managers who have taken part in the project (see Appendix). The CoRIS is funded by the Instituto de Salud Carlos III through the Red Temática de Investigación Cooperativa en SIDA (RIS C03/173). The authors thank the Spanish National Genotyping Center for providing the single nucleotide polymorphism genotyping services (http://www.cegen.org). We also 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.
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Centers and investigators involved in CoRIS:
Executive committee: Juan Berenguer, Julia del Amo, Federico García, Félix Gutiérrez, Pablo Labarga, Santiago Moreno y María Ángeles Muñoz.
Fieldwork, data management and analysis: Paz Sobrino Vegas, Victoria Hernando Sebastián, Belén Alejos Ferreras, Débora Álvarez, Susana Monge, Inmaculada Jarrín, Adela Castelló.
BioBanco HIV: M Ángeles Muñoz-Fernández, Isabel García-Merino, Coral Gómez Rico, Jorge Gallego de la Fuente y Almudena García Torre.
Hospital General Universitario de Alicante (Alicante): Joaquín Portilla Sogorb, Esperanza Merino de Lucas, Sergio Reus Bañuls, Vicente Boix Martínez, Livia Giner Oncina, Carmen Gadea Pastor, Irene Portilla Tamarit, Patricia Arcaina Toledo.
Hospital Universitario de Canarias (Santa Cruz de Tenerife): Juan Luis Gómez Sirvent, Patricia Rodríguez Fortúnez, María Remedios Alemán Valls, María del Mar Alonso Socas, Ana María López Lirola, María Inmaculada Hernández Hernández, Felicitas Díaz-Flores.
Hospital Carlos III (Madrid): Vicente Soriano, Pablo Labarga, Pablo Barreiro, Pablo Rivas, Francisco Blanco, Luz Martín Carbonero, Eugenia Vispo, Carmen Solera.
Hospital Universitario Central de Asturias (Oviedo): Victor Asensi, Eulalia Valle, José Antonio Cartón.
Hospital Clinic (Barcelona): José M. Miró, María López-Dieguez, Christian Manzardo, Laura Zamora, Iñaki Pérez, Mª Teresa García, Carmen Ligero, José Luis Blanco, Felipe García-Alcaide, Esteban Martínez, Josep Mallolas, José M. Gatell.
Hospital Doce de Octubre (Madrid): Rafael Rubio, Federico Pulido, Silvana Fiorante, Jara Llenas, Violeta Rodríguez, Mariano Matarranz.
Hospital Donostia (San Sebastián): José Antonio Iribarren, Julio Arrizabalaga, María José Aramburu, Xabier Camino, Francisco Rodríguez-Arrondo, Miguel Ángel von Wichmann, Lidia Pascual Tomé, Miguel Ángel Goenaga, Mª Jesús Bustinduy, Harkaitz Azkune Galparsoro.
Hospital General Universitario de Elche (Elche): Félix Gutiérrez, Mar Masiá, Cristina López Rodríguez, Sergio Padilla, Andrés Navarro, Fernando Montolio, Yolanda Peral, Catalina Robledano García.
Hospital Germans Trías i Pujol (Badalona): Bonaventura Clotet, Cristina Tural, Lidia Ruiz, Cristina Miranda, Roberto Muga, Jordi Tor, Arantza Sanvisens
Hospital General Universitario Gregorio Marañón (Madrid): Juan Berenguer, Juan Carlos López Bernaldo de Quirós, Pilar Miralles, Jaime Cosín Ochaíta, Isabel Gutiérrez Cuellar, Margarita Ramírez Schacke, Belén Padilla Ortega, Paloma Gijón Vidaurreta, Ana Carrero Gras, Teresa Aldamiz-Echevarría Lois y Francisco Tejerina Picado.
Hospital Universitari de Tarragona Joan XXIII, IISPV, Universitat Rovira i Virgili (Tarragona): Francesc Vidal, Joaquín Peraire, Consuelo Viladés, Sergio Veloso, Montserrat Vargas, Miguel López-Dupla, Montserrat Olona, Alba Aguilar, Joan Josep Sirvent, Verónica Alba, Olga Calavia.
Hospital Universitario La Fe (Valencia): José López Aldeguer, Marino Blanes Juliá, José Lacruz Rodrigo, Miguel Salavert, Marta Montero, Eva Calabuig, Sandra Cuéllar.
Hospital Universitario La Paz (Madrid): Juan González García, Ignacio Bernardino de la Serna, José Ramón Arribas López, María Luisa Montes Ramírez, Jose Mª Peña, Blanca Arribas, Juan Miguel Castro, Fco Javier Zamora Vargas, Ignacio Pérez Valero, Miriam Estébanez, Silvia García Bujalance.
Hospital de la Princesa (Madrid): Ignacio de los Santos, Jesús Sanz Sanz, Ana Salas Aparicio, Cristina Sarriá Cepeda.
Hospital San Pedro-CIBIR (Logroño): José Antonio Oteo, José Ramón Blanco, Valvanera Ibarra, Luis Metola, Mercedes Sanz, Laura Pérez-Martínez.
Hospital San Pedro II (Logroño): Javier Pinilla Moraza
Hospital Universitario Mutua de Terrassa (Terrassa): David Dalmau, Angels Jaén Manzanera, Mireia Cairó Llobell, Daniel Irigoyen Puig, Laura Ibáñez, Queralt Jordano Montañez, Mariona Xercavins Valls, Javier Martinez-Lacasa, Pablo Velli, Roser Font.
Hospital de Navarra (Pamplona): María Rivero, Marina Itziar Casado, Jorge Alberto Díaz González, Javier Uriz, Jesús Repáraz, Carmen Irigoyen, María Jesús Arraiza.
Hospital Parc Taulí (Sabadell): Ferrán Segura, María José Amengual, Eva Penelo, Gemma Navarro, Montserrat Sala, Manuel Cervantes, Valentín Pineda.
Hospital Ramón y Cajal (Madrid): Santiago Moreno, José Luis Casado, Fernando Dronda, Ana Moreno, María Jesús Pérez Elías, Dolores López, Carolina Gutiérrez, Beatriz Hernández, María Pumares, Paloma Martí.
Hospital Reina Sofía (Murcia): Alfredo Cano Sánchez, Enrique Bernal Morell, Ángeles Muñoz Pérez.
Hospital San Cecilio (Granada): Federico García García, José Hernández Quero, Alejandro Peña Monje, Leopoldo Muñoz Medina, Jorge Parra Ruiz.
Centro Sanitario Sandoval (Madrid): Jorge Del Romero Guerrero, Carmen Rodríguez Martín, Teresa Puerta López, Juan Carlos Carrió Montiel, Cristina González, Mar Vera.
Hospital Universitario Santiago de Compostela (Santiago de Compostela): Antonio Antela, Arturo Prieto, Elena Losada.
Hospital Son Espases (Palma de Mallorca): Melchor Riera, Javier Murillas, Maria Peñaranda, Maria Leyes, Mª Angels Ribas, Antoni Campins, Concepcion Villalonga, Carmen Vidal.
Hospital Universitario de Valme (Sevilla): Juan Antonio Pineda, Eva Recio Sánchez, Fernando Lozano de León, Juan Macías, José del Valle, Jesús Gómez-Mateos.
Hospital Virgen de la Victoria (Málaga): Jesús Santos González, Manuel Márquez Solero, Isabel Viciana Ramos, Rosario Palacios Muñoz.
Hospital Universitario Virgen del Rocío (Sevilla): Pompeyo Viciana, Manuel Leal, Luis Fernando López-Cortés, Mónica Trastoy.