The CD4+ T cell is the primary cellular target of HIV, and a continuous loss of CD4+ T cells leads to immunodeficiency, opportunistic diseases, and death.1,2 However, there is substantial interindividual variability in the rate and extent of progression to AIDS, much of which is secondary to host genetic heterogeneity.3 Furthermore, there are other factors (in addition to host genetics) that may contribute to this interindividual variability such as immune function, viral genetic variability, health care, and coinfections, and others.3
In HIV infection, mitochondria may be a key in the immune response against HIV infection,4 and mitochondrial dysfunction might play a role during AIDS progression.5–7 A mitochondrial DNA (mtDNA) depletion, increased reactive oxygen species production, deficiency of antioxidant enzymes, and increased oxidative damage have been described among HIV-infected patients with accelerated disease.8 In addition, HIV proteins have been implicated in the destabilization of mitochondrial membrane promoting the targeted depletion of key immune cells and HIV evasion, which contribute to the characteristic global immunodeficiency observed during AIDS progression.8
Mitochondria have its own genome, the mtDNA, which coding for only 13 proteins that are vital to mitochondrial function.9 However, approximately 1500 additional nuclear-encoded mitochondrial proteins (NEMPs) have been identified as responsible of the machinery for energy and redox function of the mitochondria.9 A high number of genes related to NEMPs have been identified as host factors involved in HIV infection and AIDS progression.4,10,11 Moreover, Hendrickson et al6 found that single nucleotide polymorphisms (SNPs) of 2 genes related to NEMPs, acyl coenzyme A (CoA) synthetase medium-chain family member 4 (ACSM4) on chromosome 12 and peroxisomal D3, D2-enoyl-CoA isomerase (PECI) on chromosome 6, were associated with AIDS progression.
The aim of our study was to determine whether ACSM4 and PECI SNPs are associated with the clinical pattern of AIDS progression in antiretroviral treatment (ART)-naive HIV-infected patients of 2 Spanish large cohorts [Cohort of the Spanish HIV Research Network (CoRIS) and Cohort of Long-term Nonprogressors (LTNP)].
PATIENTS AND METHODS
We carried out a cross-sectional study on 454 HIV-infected patients from CoRIS,12 Cohort of LTNP, and its associated HIV Biobank.13 Additionally, 154 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.
HIV-infected patients were classified in 3 groups according to the clinical pattern of AIDS progression [LTNP, moderate progressors (MP), and rapid progressors(RP)]7: (1) 146 LTNP patients, who were defined as subjects with asymptomatic HIV infection over 10 years after seroconversion and always CD4+ ≥500 cells per cubic millimeter and RNA-viral load ≤10,000 copies per milliliter; (2) 228 typical or 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; (3) 80 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. These 3 definitions of clinical patterns of AIDS progression involved no ART, although patients of MP and RP groups could be subsequently treated. Thus, 3 patients groups may be considered as 3 different progression patterns for the natural history of HIV infection.
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 and multicentre cohort of patients newly diagnosed with HIV infection at the hospital or treatment center, more than 13 years, and naive to ART. Each participating patient signed an informed consent form. The information was subject to internal quality controls; once every 2 years, information on 10% of the cohort is audited by an externally contracted agency.12 The program was approved by the institutional review boards of the participating hospitals and centers (the cohort has been described in detail elsewhere).12 Furthermore, this study was approved by the Research Ethic Committee of the Instituto de Salud Carlos III (ISCIII) and the study was conducted in accordance with the Declaration of Helsinki.
Samples from patients were kindly provided by the HIV BioBank integrated in the Spanish AIDS Research Network.13 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 (CeGen; http://www.cegen.org/) for genotyping of PECI (rs629362 and rs584585) and ACSM4 (rs7961991 and rs7137120) by the iPLEX Gold assay on MassARRAY system (Sequenom Inc., San Diego, CA).
Categorical data and proportions were analyzed by using the χ2 test or Fisher exact test as required. Mann–Whitney test were used to compare data among independent groups when the dependent variable was continuous. For genetic association study, logistic regression was used to compare the clinical patterns of AIDS progression according to a dominant genetic model, which was the model that best fit to our data. The reference category was the LTNP group and the goodness of fit of models was evaluated by Akaike information criterion value and Bayesian information criterion. Logistic regressions were adjusted by gender, age at HIV diagnosis, intravenous drug users (IDU), and HCV infection. All statistical tests were performed with the Statistical Package for the Social Sciences (SPSS) 19.0 software (IBM Corp, Chicago, IL). Graphics were generated by GraphPad PRISM 6.01 (GraphPad Software, Inc, La Jolla, CA). All P values were 2 tailed and statistical significance was defined as P < 0.05.
Hardy–Weinberg equilibrium was assessed by a χ2 test, considering equilibrium when P > 0.05. In addition, pair-wise linkage disequilibrium (LD) analysis was computed to detect the intermarker relationship using the standardized D′ and r2 values. The association test between haplotypes and AIDS progression was performed by multivariate logistic regression adjusted by the covariates above cited. These analyses were performed by using SNPstat software (IDIBELL, Catalan Institute of Oncology, Barcelona, Spain; http://bioinfo.iconcologia.net/SNPStats_web).14 The Functional Analysis and Selection Tool for Single Nucleotide Polymorphism (FastSNP; Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan; http://fastsnp.ibms.sinica.edu.tw/) was used for the prediction of the functional effect of SNPs.15
Table 1 shows the epidemiological and clinical characteristics of 454 HIV-infected patients (146 LTNPs, 228 MPs, and 80 RPs). LTNP group was older at HIV diagnosis and at the moment of inclusion in the study and had higher percentages of IDU and HCV infection (P < 0.05). Besides, LTNP group had lower percentages of men and homosexual HIV transmission than MP and RP groups (P < 0.05). Moreover, 154 healthy blood donors (Control group) were included who had 43.3 years old and 70.1% were men.
Distribution of NEMPs Polymorphisms
ACSM4 and PECI polymorphisms were in Hardy–Weinberg equilibrium (P > 0.05) for healthy controls and HIV-infected patients. They fulfilled the minimum allele frequency >0.05 and less than 5% of missing values were found for all samples.
Similar values of allelic and genotypes frequencies were found for ACSM4 (rs7961991 and rs7137120) and PECI (rs629362 and rs584585) in healthy controls and HIV-infected patients (see Table S1 and S2, Supplemental Digital Content, http://links.lww.com/QAI/A458). Moreover, LD was high for ACSM4 SNPs (D′ = 0.995, r2 = 0.950) and PECI SNPs (D′ = 0.999, r2 = 0.995). The distribution of both ACSM4 and PECI haplotypes were also similar in healthy controls and in HIV-infected patients (see Table S3, Supplemental Digital Content, http://links.lww.com/QAI/A458).
By analyzing these polymorphisms in silico, we found that rs7961991 is classified as intronic enhancer (FastSNP database).
NEMPs Polymorphisms and AIDS Progression
For ACSM4 polymorphisms, the frequency of both rs7137120 AG (Fig. 1A) and rs7961991 CT (Fig. 1B) genotypes were higher in RP group than in LTNP group. However, the frequency of both rs7137120 GG (Fig. 1A) and rs7961991 TT (Fig. 1B) genotypes were lower in RP group than in LTNP group. For PECI polymorphisms, no significant differences were found among groups (Figs. 1C, D).
Taking as reference the LTNP group, the adjusted logistic regression shows that rs7137120 AA/AG genotypes (Fig. 1E) and rs7961991 CC/CT genotypes (Fig. 1F) had higher odds of having a rapid AIDS progression [odds ratio (OR) = 3.21 (P = 0.014) and OR = 3.60 (P = 0.009), respectively]. Additionally, the ACSM4 haplotype integrated for both rs7961991 A and rs7137120 C alleles had higher odds of having a rapid AIDS progression [OR = 2.85; 95% confidence interval = 1.28 to 6.25; P = 0.010]. For PECI polymorphisms, no significant associations were found (Figs. 1F, H).
ACSM4 is a member of acyl CoA synthetase family, which catalyzes the initial step in fatty acid metabolism, activating the fatty acid with CoA to produce acyl-CoA, particularly in mitochondria and endoplasmic reticulum membranes.16 In our study, both rs7137120 AA/AG and rs7961991 CC/CT genotypes (ACSM4 gene) were associated with a rapid AIDS progression. Our data might have relevance on host natural resistance/sensitivity against AIDS progression in subjects never receiving ART.
The rs7961991 polymorphism is located at intron 12–13 of the ACSM4 gene. In our study, patients carrying CC/CT genotypes of rs7961991 had higher odds of rapid AIDS progression than TT carriers. These findings are consistent with a previous article published by Henrickson et al who showed an association between rs7961991 and AIDS progression. However, the mechanism of action of this SNP is still unknown. With the aim of a better understanding of its role, we analyzed its possible functional effect in silico. By using FastSNP software,15 this SNP seems to be involved in the modulation of gene expression as intronic enhancer. Therefore, this polymorphism, as putative enhancer, could be influencing the ACSM4 expression, affecting the activation of fatty acid and consequently, the acetyl CoA production through fatty acids oxidation. According to this, the association between ACSM4 polymorphism and AIDS progression could reflect the influence of rs7961991 on mitochondrial energetics. In 2 recent articles, Wu et al4,17 analyzed the genomic basis of HIV interactions with the cellular transcriptome of both CD4+ and CD8+ T cells, finding that metabolic pathways (components of oxidative phosphorylation, tricarboxylic acid cycle, amino acid metabolism, and fatty acid metabolism) were upregulated, as a transcriptional signature evolved by mitochondria dysfunction in HIV disease progression. In a similar manner, modifications in ACSM4 expression could also be linked to alterations in mitochondrial function and therefore HIV disease progression. As observed in silico, ACSM4 SNPs could be influencing ACSM4 gene expression, and it could be the mechanism responsible for mitochondrial dysfunction and HIV disease progression. However, further studies would be interesting to evaluate the functional implication of ACSM4 SNPs in gene expression and its influence on the metabolic and mitochondrial/oxidative phosphorylation pathways by transcriptomic studies as carried out by Wu et al. On the other hand, it has been described that many regulatory elements might act regulating the expression of neighboring genes, even when they have function unrelated.18 Thus, the effect of rs7961991 on AIDS progression might be due to its influence on expression of CD163 and CD163L1 genes, which are located close to ACSM4 gene and, thus, near to this polymorphism. Burdo et al19,20 have described that plasma levels of soluble CD163 correlated AIDS progression. Thus, a possible explanation of the association between rs7961991 polymorphism and AIDS progression could be the effect of rs7961991 on the modulation of CD163 expression.
Significant association was also found for rs7137120, where AA/AG carriers had higher odds of rapid AIDS progression than GG carriers. The mechanism of action of rs7137120 is also unknown. This polymorphism is located over 1.3-kb upstream from ACSM4 gene. Regarding its possible role, as multiple cisregulatory elements located upstream control the quantitative and spatiotemporal gene expression,21 rs7137120 polymorphism might also play a key role in the modulation of ACSM4 or neighboring genes expression. On the other hand, many regions of the genome tend to be inherited together as a unit and thus polymorphisms located in that areas will be in high LD. It occurs with rs7961991 and rs7137120. Therefore, rs7137120 could indirectly reflect the association between rs7961991 and AIDS progression. However, it is important to take into account that it is difficult to establish the specific polymorphism casually associated with the disease. In fact, it is possible that some other SNP in LD with rs7137120 and/or rs7961991 could be the true causal variable.22 However, although causal SNP is unknown, these SNPs could be useful as important risk markers of AIDS progression.
In this study, we have corroborated the association between ACSM4 polymorphisms and AIDS progression, but no significant association was found for PECI polymorphisms, both previously described by Hendrickson et al6 Apparently, the results of PECI polymorphisms seem to go in the opposite direction to the Hendrickson et al analysis. However, it is very difficult to have a clear idea of this because the OR values were very small and the confidence intervals were very wide. Moreover, it may be possible that this lack of significant association between PECI polymorphisms and AIDS progression may be due to low sample size, which may influence on statistical significance level when the effects of the variable studied are not large. Complex human diseases are under the control of many genes that contribute each one of them with modest individual effects and only big effects would be detected in small populations. Moreover, our results are difficult to compare directly with the study of Hendrickson et al because the differences in the populations necessitated different study designs (cross-sectional design vs. longitudinal design). Our study included subjects from cohorts defined by disease progression rates; thus, the analyses used these categories rather than a “time-to-event” analysis. Furthermore, the profound differences among groups, regarding to age, sex, HCV infection, and IDU, might hide other unmeasured confounders, which might introduce a bias selection.
With the aim of investigating whether previous genome-wide association studies (GWAS) had described an association of ACSM4 and PECI polymorphisms with AIDS progression, we performed a query by SNP in several GWAS database [GWAS DB (https://gwas.biosciencedbc.jp/) and NHGRI (http://www.genome.gov/gwastudies/)]. However, a significant association of these studied polymorphisms with AIDS progression has only been described by Hendrickson et al6 It could be due to the lack of inclusion of these SNPs in GWAS used, genotyping problems, or studies investigating different comparison groups or different ethnic. Additionally, both ACSM4 and PECI polymorphisms have also not been associated with any other disease.
This study has other limitations that must be also taken into account. Our study was performed over European population and the AIDS epidemic affects more to people of non-European descent. It is critical to define relationships of ACSM4 (rs7961991 and rs7137120) and PECI (rs629362 and rs584585) polymorphisms and AIDS progression in these other populations because the frequencies of these SNPs in nonwhite populations are different (see reference SNP: http://www.ncbi.nlm.nih.gov/projects/SNP/). Moreover, in a recent article in these same patients, we have identified mtDNA haplogroups associated with AIDS progression.7 In the present study, we have explored the interaction between mtDNA haplogroups and NEMP polymorphisms, but we did not find any significant values (data not shown). Also be noted that there are other host genetic factors that influence disease progression (eg, known HLA types, CCR5 mutations), but we have not controlled in this study.
In conclusion, the presence of 2 SNPs at ACSM4 was associated with rapid AIDS progression. In the ART era when HIV treatment is recommended for all persons infected with HIV, our results may help to understand HIV pathogenesis and identifying differences among groups of untreated HIV-1–infected patients with different clinical evolution, which could generate hypotheses for mechanisms leading to delay onset of AIDS in people living with HIV.
The authors want to particularly acknowledge the HIV HGM BioBank integrated in the Spanish AIDS Research Network and collaborating Centers for the generous gifts of clinical samples used in this study. The HIV HGM BioBank, integrated in the Spanish AIDS Research Network, is supported by Instituto de Salud Carlos III, Spanish Healt Ministry (grant number 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 RIS Cohort (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 (CeGen) for providing the SNP genotyping services (http://www.cegen.org). The authors 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.
Executive committee: Juan Berenguer, Julia del Amo, Federico García, Félix Gutiérrez, Pablo Labarga, Santiago Moreno y María Ángeles Muñoz-Fernández.
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, Almudena García Torre, José Luis Jiménez Fuentes.
Centers and Investigators Involved in CoRIS Participating Centers
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.