Infection, Genetics and Evolution 21 (2014) 418–423

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Infection, Genetics and Evolution

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Influence of HLA-G polymorphisms in human immunodeficiency virusinfection and hepatitis C virus co-infection in Brazilian and Italianindividuals

1567-1348/$ - see front matter � 2013 Published by Elsevier B.V.http://dx.doi.org/10.1016/j.meegid.2013.12.013

⇑ Corresponding author. Address: Laboratory of Immunogenetics, Institute ofBiosciencies, Department of Genetics, UFRGS, Av. Bento Gonçalves – 9500, Campusdo Vale, PO Box 15053, 91501970 Porto Alegre, RS, Brazil. Tel.: +55 51 3308 6740;fax: +55 51 3308 7311.

E-mail address: jabchies@terra.com.br (J.A.B. Chies).

G.K. da Silva a, Priscila Vianna a, Tiago Degani Veit a, Sergio Crovella b, Eulalia Catamo c,Elvira Alicia Aparicio Cordero a, Vanessa Suñé Mattevi d, Rosmeri Kuhmmer Lazzaretti e, Eduardo Sprinz e,Regina Kuhmmer e, José Artur Bogo Chies a,⇑a Federal University of Rio Grande do Sul, Brazilb Department of Genetics, Federal University of Pernambuco, Brazilc Immunogenetics Laboratory, University of Trieste, Italyd Federal University of Health Sciences of Porto Alegre, Brazile Hospital de Clínicas de Porto Alegre, Brazil

a r t i c l e i n f o

Article history:Received 30 October 2013Received in revised form 9 December 2013Accepted 12 December 2013Available online 2 January 2014

Keywords:HLA-GPolymorphismHIVHCVEthnicity

a b s t r a c t

Objective: This study aimed to investigate the role of Human Leukocyte Antigen (HLA)-G in the suscep-tibility to HIV-1 infection through the analysis of the HLA-G 30 untranslated region (UTR) polymorphisms14 bp insertion/deletion (rs66554220) and +3142C>G (rs1063320).Design: We analyzed 582 HIV-1 infected patients and 626 uninfected individuals from Brazil and Italy ina case-control study.Methods: HLA-G polymorphisms were genotyped using PCR, PCR-RFLP assays or direct sequencing. Allanalyses were stratified by ethnicity. Genotypic, allelic and diplotypic frequencies were comparedbetween HIV-1 infected subjects and controls using Chi-square or Fischer exact tests. Also, haplotypic fre-quencies were estimated using MLocus software.Results: African-derived HIV-infected individuals presented a higher frequency of the 14 bp insertionallele as compared to non-infected individuals (0.468 versus 0.373, respectively; pBonf = 0.010). A higherfrequency of the 14 bp insertion +3142G (insG) haplotype (0.456 versus 0.346, p < 0.001) and the insG/insG diplotype (OR = 1.88, 95%CI = 1.08–3.23, p = 0.021) was observed among African-derived patientsas compared to uninfected controls. Also, we observed a higher frequency of the ins/ins genotype amongAfrican-derived HIV patients co-infected with HCV (OR = 2.78, 95%CI = 1.20–6.49, p = 0.008).Conclusions: Our data point out to an increased frequency of alleles and genotypes associated with lowHLA-G expression among African-derived patients, suggesting a potential role for HLA-G in the suscepti-bility to HIV-1 infection and HCV co-infection in those individuals.

� 2013 Published by Elsevier B.V.

1. Introduction

The Acquired Immune Deficiency Syndrome (AIDS) becameworldwide known after the 1980’s, when the first epidemic signalsappeared, and until now, there is no cure or effective vaccine forthis disease (Mindel and Tenant-Flowers, 2001). Without treat-ment, life-expectancy of patients diagnosed with AIDS is aroundtwo years. Interestingly, humans show a remarkable variation invulnerability to HIV infection and progression to AIDS, probably

due to genetic and immunologic factors. HIV transmission dependsboth on viral factors of infectivity as well as on host susceptibility(Fellay et al., 2007).

There are two different HIV types, HIV-1 and HIV-2, being HIV-1the more prevalent type in Brazil (Apetrei et al., 2007; Cohen et al.,2008). Infectivity depends on HIV-1 concentration, virus presenceand upon the number of infected cells in body fluids. However,‘‘HIV dependency factors’’ comprising viral and cellular require-ments for HIV-1 transmission remain poorly understood (Cohenet al., 2008). By 2012, there were 35.3 million people living withHIV worldwide. In that year, 2.3 million new cases were reportedand 1.6 million individuals died from the disease (UNAIDS/WHO,2013). In Brazil, until June 2012, approximately 656 thousandcases of the disease had been reported, since the first case in

G.K. da Silva et al. / Infection, Genetics and Evolution 21 (2014) 418–423 419

1980 (Saúde, 2009). Resistance to HIV infection reflects a combina-tion of genetic factors, innate and acquired immune resistance.Among the genetic factors associated with HIV resistance, homozy-gosis of the delta 32 allele of the CCR5 gene is by far the best known(Cohen et al., 2008).

The human immune system involves a complex array of mech-anisms that distinct pathogens or toxins from host cells. This dis-crimination is essential to allow the host to eliminate the injurywithout damaging its own tissues. It is well established that highlypolymorphic molecules encoded by genes of the Major Histocom-patibility Complex (MHC) play a key role in immune system func-tions. The Human Leukocyte Antigen (HLA)-G is a non-classicalHLA class Ib molecule able to suppress immune responses, contrib-uting to immune escape and tolerance (Carosella et al., 1999). Thisimmunomodulatory molecule was first characterized by itsexpression at maternal–fetal interface, protecting the fetus againstthe maternal immune system (Larsen et al., 2010; Ober et al., 2003;Vianna et al., 2007). Over the years, emerging studies have shownthe relevance of the HLA-G molecule in pathologic conditions, suchas transplant rejection, autoimmunity, cancer, viral infection andinflammation (Larsen and Hviid, 2009; Menier et al., 2009; vander Ven et al., 2000; Veit and Chies, 2009). The importance ofHLA-G in the immune system is denoted by its capacity to inhibitimmune responses in several levels, acting in different immune celltypes. The immnunossupressive HLA-G activity is of interest tosome pathogens, since they might use these same mechanisms toescape immune surveillance (Favier et al., 2007). It was already de-scribed that Human Immunodeficiency Virus (HIV) infection leadsto increased HLA-E and HLA-G expression, suggesting a strategy forcontrolling HLA-G expression taking advantage of its specific im-mune tolerance function (Lozano et al., 2002; Tripathi andAgrawal, 2007). However, little is known about the influence ofHLA-G in susceptibility to HIV infection.

The HLA-G gene encodes seven protein isoforms, including fourmembrane-bound (G1–G4) and three soluble molecules (G5–G7),generated by alternative splicing. Its coding region displays aquite low allelic polymorphism, with only 16 protein variants de-scribed to date. However, its 50 regulatory region and its 30

untranslated (UTR) regions are highly polymorphic. The 30 UTRseems to play an important role in regulating HLA-G expression(Menier et al., 2009). Two polymorphisms located at this regionseem to play a role in this regulation: a 14 bp insertion/deletionpolymorphism (rs66554220) and a single nucleotide polymor-phism (SNP) rs1063320. The 14 bp insertion/deletion polymor-phism was shown to play a role in alternative splicing and wasalso associated with different levels of HLA-G in plasma. Tran-scripts from the 14 bp insertion allele are able to undergo anadditional splicing step which removes 92 bases, including the re-gion where the 14 bp indel is located, yielding more stable tran-scripts in vitro (Rousseau et al., 2003). Although the 14 bpinsertion (ins) allele was associated with this more stable subpop-ulation of HLA-G mRNAs, in vivo studies showed that this allelealso presents a decreased HLA-G expression, a phenomenon thatwas called the ‘‘14 bp paradox’’ (Chen et al., 2008; Veit and Chies,2009). The presence or absence of the 14 bp also alters the set ofmicroRNAs (miRNAs) that are capable to binding that locus(Castelli et al., 2009) thus influencing RNA turnover and miRNA-mediated repression of translation. The C/G SNP located less than200 bp away from the 14 bp polymorphic site, at position +3142(rs1063320), is also thought to influence miRNA binding. In thispolymorphic site, the G allele favors the targeting of three miR-NAs (miR-148a, 148b and 152) to the binding site and thereforedirects a reduction on the HLA-G expression (Tan et al., 2007).Both polymorphisms are in linkage disequilibrium (Castelliet al., 2010; Tan et al., 2007).

Some opportunistic pathogens play important roles during HIVinfection, altering the immune system balance. The hepatitis Cvirus (HCV) is a major cause of chronic liver disease worldwideand is frequently associated to HIV infections, sharing routes of vir-al transmission. HCV infections affect approximately 15–30% ofHIV patients (Fregni et al., 2013). HCV co-infection accelerates pro-gression to death in HIV positive patients as well as impairs the im-mune reconstitution (Koziel and Peters, 2007). Polymorphicvariants of the HLA-G have already been associated to HCV infec-tion in sickle cell disease patients (Cordero et al., 2009).

In the present work, we evaluated the 14 bp insertion/deletionand the +3142C>GHLA-G polymorphisms in samples from HIV-in-fected patients and uninfected individuals from Southern Braziland Italy seeking a possible association of these polymorphismswith HIV infection and HCV co-infection.

2. Methods

2.1. Patients and controls

We evaluated two distinct HIV-infected cohorts comprising pa-tients which were consecutively enrolled at the South BrazilianHIV Cohort (SOBRHIV) in Porto Alegre, capital of Brazil’s southern-most state (Dabis et al., 2005) and from north eastern Italy, en-rolled at the laboratory of Virology, Istituto di Ricovero e Cura aCarattere Scientifico (IRCCS) ‘Burlo Garofolo’, Trieste (Italy) (Segatet al. 2009). The inclusion criteria for the patients group were:(1) asymptomatic HIV-infected individuals on High activity Anti-Retrovirus Therapy (HAART) for, at least, one year (prescribedaccording to Brazilian guidelines at that time) (Saúde, 2009); (2)an HIV-1 RNA load <50 copies/mL, determined by the technicalVersant HIV-1 RNA 3.0 assay/bDNA automation system in 340bDNA Analyzer (Bayer, Germany); and (3) older than 18 years.The exclusion criteria were: (1) pregnancy, (2) present use of drugsthat could be associated to body changes such as corticosteroids oranabolic steroids and (3) mental illness.

The sample comprised 582 HIV-infected individuals, 343 menand 239 women. Brazilian patients were classified either as Euro-pean or African-derived according to phenotypic characteristicsof individuals and ethnicity data of parents/grandparents reportedby the participants, filling an appropriate inventory. The group ofItalian patients was evaluated as a different confirmatory cohort.All Italian patients were European Caucasian. The issue arisen onthe skin color-based classification criteria that is used in Brazil iswell documented and has been already assessed by our group inprevious studies (Santos et al., 2010; Vargas et al., 2006; Veitet al., 2009). From the Brazilian patients, 237 were classified asEuropean-derived and 171 as African-derived.

The control group was formed by 626 uninfected individuals,234 European-derived and 309 African-derived from SouthernBrazil, and 83 individuals from Italy. The inclusion criteria for con-trols were: good health, age between 18 and 65 years, weigh over50 kg. The exclusion criteria were: fever, cold or flu, pregnancy,puerperium, people who underwent surgical procedures, vaccina-tion or tattoos in the last six months and use of illicit drugs. Also,positivity to hepatitis, HIV, HTLV, Chagas disease or malaria wasconsidered as exclusion factors in the control group.

The demographic characteristics of both groups are described inTable 1. All patients and controls participating in this study gavetheir written informed consent and the protocol was approvedby the Hospital de Clínicas de Porto Alegre Committee on Ethicsin Research and by the IRCCS Burlo Garofolo. DNA was obtainedfrom peripheral blood, collected with EDTA and purified througha salting-out procedure as previously described (Lahiri and Nurn-berger, 1991).

Table 1Demographic characteristics of the study group.

Characteristics HIV-infected individuals(n = 582)

Control group(n = 626)

Gender (males/females) 343/239 362/142*

Age (years ± S.D. andmin–max)

43.88 ± 9.73 (16–74) 39.97 ± 10.39(15–68)

EthnicityBrazilian European-

derived237 234

Brazilian African-derived 171 309Italian 174 83HCV co-infection (n,%) 133/567 (23.5%) NA

NA – not applicable.* Missing data.

420 G.K. da Silva et al. / Infection, Genetics and Evolution 21 (2014) 418–423

2.2. Genotyping of HLA-G gene polymorphisms

The 14 bp insertion/deletion and +3142 polymorphisms weregenotyped through Polymerase Chain Reaction–Restriction Frag-ment Length Polymorphism (PCR–RFLP) assay, and different reac-tions were performed for each polymorphism, as previouslydescribed by our group (Cordero et al., 2009). The results for the14 bp insertion/deletion polymorphism were analyzed by electro-phoresis in a 6% polyacrilamide gel whereas the results for the+3142 C>G polymorphism were analyzed by electrophoresis in a1.5% agarose gel, both stained with ethidium bromide and visual-ized under UV light. The Italian cohort was evaluated by directsequencing and some samples from the Brazilian cohort were alsosequenced in order to control for genotyping quality.

2.3. Statistical analyses

HLA-G genotypic distribution was determined by direct count-ing. Genotypic frequencies were compared to Hardy–Weinbergexpectations using Chi-Square tests. HLA-G genotypic and allelicfrequencies were compared between patients and controls usingthe Chi-square-test and Fischer exact test, respectively. Haplotypefrequencies were estimated with the MLocus software, which usesan expectation maximization algorithm (Long, 1999). Bonferronicorrection for multiple comparisons was applied when p-valueswere significant. The significance level was set at a = 0.05 (two-tailed). All statistical analyses were performed with SPSS 15.0(SPSS Inc., Chicago, IL) and WinPepi 10.0 (Abramson, 2004).

3. Results

3.1. HLA-G polymorphisms and disease susceptibility

All analyses were performed by subgrouping individualsaccording to their ethnic origin due to differences in HLA-G poly-morphic allelic frequencies between European and African-derivedpopulations (Larsen and Hviid, 2009). Concerning the Brazilian co-hort, the European-derived group was composed by 234 unin-fected individuals and 237 HIV-infected patients whereas theAfrican-derived group was composed by 309 uninfected and 171HIV-positive individuals. The Italian cohort (ethnically classifiedas Caucasian) was composed by 83 uninfected individuals and174 HIV-positive individuals.

All groups had their genotypic frequencies compared to Hardy–Weinberg expectations. No significant differences were observedconcerning both the 14 bp insertion/deletion and +3142 C>G poly-morphisms (data not shown). We then compared genotypic andallelic frequencies of the 14 bp insertion/deletion polymorphismbetween patients and controls (Table 2). A significant differencewas observed in the distribution of genotype frequencies between

African-derived patients and controls (pBonf = 0.026), with lowerfrequency of homozygotes for the 14 bp deletion allele (del/del)in infected subjects with respect to non-infected individuals(0.269 versus 0.394, respectively; residual p = 0.006). Also, a higherfrequency of the ins allele was observed in African-derived patientsas compared to uninfected ethnically matched controls (0.468 ver-sus 0.373, respectively; pBonf = 0.010; Table 2). No significant differ-ences in genotype or allelic frequency distributions were observedbetween patients and controls from the Brazilian European-de-rived (p = 0.391 and 0.207, respectively) or the Italian (EuropeanCaucasian) groups (p = 0.915 and 0.705 respectively, Table 2).

When comparing the genotypic frequencies of the +3142 C>Gpolymorphism in the African-derived group, a higher frequencyof the G/G genotype was observed in patients as compared to unin-fected subjects (0.427 versus 0.332) although this difference do notreached statistical significance (p = 0.107). The frequency of the Gallele was increased in patients, as compared to controls (0.649versus 0.578, respectively) but the difference was not significantafter correcting for multiple comparisons (pBonf = 0.078; Table 2).Again, no significant differences in genotype or allelic frequencieswere observed between patients and controls from the Euro-pean-derived and Italian groups concerning this polymorphism(Table 2).

3.2. HLA-G haplotypes and disease susceptibility

At the haplotype level, we observed a higher frequency of theinsG haplotype in African-derived HIV-infected individuals as com-pared to healthy ethnically-matched individuals (0.456 versus0.346; residual p < 0.001; Table 3). Diplotype frequencies betweenpatients and controls were compared in order to further investi-gate the risk conferred by the insG allele, revealing an increasedfrequency of the insG/insG diplotype in patients (0.199 versus10.4, residual p = 0.006 – Table 3). The associated OR to the riskconferred by the insG/insG diplotype was 1.88 (95%CI = 1.08–3.23, p = 0.021). No significant differences were observed betweenhaplotype or diplotype frequencies between patients and controlsfrom the European-derived (p = 0.412 and 0.913, respectively) andItalian groups (0.497 and 0.839, respectively).

3.3. HLA-G polymorphisms and HCV co-infection

In order to evaluate the possible influence of HLA-G polymor-phisms on clinical features, hepatitis C virus (HCV) co-infectionwas also evaluated (Table 4). Among European-derived individuals43 (19%) were co-infected with HCV (HIV+/HCV+) and 185 did notpresent HCV co-infection (HIV+/HCV�). Comparing these individu-als with respect to the two studied HLA-G polymorphisms, geno-typic and allelic frequencies were similar (Table 4). The Italiansample showed similar characteristics as compared to the Euro-pean-derived group, with 30 (17%) HIV+/HCV+ and 144 HIV+/HCV� patients. Again, no significant differences were observed be-tween HIV+/HCV+ and HIV+/HCV� patients in the genotypic andallelic frequencies of both polymorphisms (Table 4).

Among African-derived individuals, 60 (36%) were HIV+/HCV+and 105 patients were HIV+/HCV�. Remarkably, the frequency ofco-infected patients in this group was about the double as com-pared to the European-derived and Italian groups (0.364 versus0.189 and 0.172, p < 0.001). Regarding the 14 bp polymorphism,no statistically significant differences were observed concerningallelic frequencies between the HIV+/HCV+ and HIV+/HCV� pa-tients (ins allele: 0.525 versus 0.429; p = 0.108). However, an in-creased frequency of the ins/ins genotype in HIV+/HCV+individuals was observed as compared to HIV+/HCV� patients(0.317 versus 0.143; residual p = 0.008). The relative risk of HCVinfection associated with this genotype among HIV patients was

Table 2Genotypic and allelic frequencies of 14 bp and +3142 polymorphisms in HIV + individuals and healthy controls, according to ethnicity.

European-derived African-derived Italian

Control freq (n) HIV+ freq (n) Control freq (n) HIV+ freq (n) Control freq (n) HIV+ freq (n)

Genotypes 14 bp n = 235 n = 237 n = 310 n = 171 n = 83 n = 174Del/del 0.340 (80) 0.380 (90) 0.394 (122)a 0.269 (46)a 0.289 (24) 0.305 (53)Ins/del 0.472 (111) 0.477 (113) 0.468 (145) 0.526 (90) 0.506 (42) 0.511 (89)Ins/ins 0.187 (44) 0.143 (34) 0.139 (43) 0.205 (35) 0.205 (17) 0.184 (32)X2 p value (patients vs. controls) 0.391 0.013

pBonf = 0.026a residual p = 0.0060.915

Alleles n = 470 n = 474 n = 620 n = 342 n = 166 n = 348Del 0.577 (271) 0.618 (293) 0.627 (389) 0.532 (182) 0.542 (90) 0.560 (195)Ins 0.423 (199) 0.382 (181) 0.373 (231) 0.468 (160) 0.458 (76) 0.440 (153)Fisher p value (patients vs. controls) 0.207 0.005

pBonf = 0.0100.705

Genotypes + 3142 n = 235 n = 237 n = 262 n = 171 n = 83 n = 172C/C 0.200 (47) 0.219 (52) 0.176 (46) 0.129 (22) 0.157 (13) 0.198 (34)C/G 0.523 (123) 0.553 (131) 0.492 (129) 0.444 (76) 0.518 (43) 0.517 (89)G/G 0.277 (65) 0.228 (54) 0.332 (87) 0.427 (73) 0.325 (27) 0.285 (49)X2 p value (patients vs. controls) 0.469 0.107 0.664

Alleles n = 470 n = 474 n = 524 n = 342 n = 166 n = 344C 0.462 (217) 0.496 (235) 0.422 (221) 0.351 (120) 0.415 (69) 0.456 (157)G 0.538 (253) 0.504 (239) 0.578 (303) 0.649 (222) 0.585 (97) 0.544 (187)Fisher p value (patients vs. controls) 0.298 0.039

pBonf = 0.0690.394

X2 test was used to compare genotypic frequencies and Fisher test was used to compare allele frequencies between patients and controls; analysis of residues was performedin order to indicate the genotypes responsible by the statistical difference observed; Bonferroni correction was applied when p < 0.05.

Table 3Haplotypic and diplotypic frequencies estimated considering both polymorphisms together, between patients and controls, according to ethnicity.

European-derived African-derived Italian

Control freq (n) HIV+ freq (n) Control freq (n) HIV+ freq (n) Control freq (n) HIV+ freq (n)

Haplotypes n = 470 n = 474 n = 627 n = 342 n = 166 n = 350Del/C 0.451 (212) 0.491 (233) 0.391 (245) 0.339 (116) 0.423 (70) 0.446 (156)Del/G 0.125 (59) 0.127 (60) 0.236 (148) 0.193 (66) 0.125 (21) 0.117 (41)Ins/C 0.011 (5) 0.004 (2) 0.027 (17) 0.012 (4) 0 (0) 0.013 (5)Ins/G 0.413 (194) 0.378 (179) 0.346 (217)⁄ 0.456(156)⁄ 0.452 (75) 0.424 (148)

X2 p = 0.412 X2 p = 0.005⁄Residual p < 0.001

Fisher’s p = 0.497

Diplotypes n = 235 n = 237 n = 259 n = 171 n = 83 n = 172DelC/DelC 0.191 (45) 0.215 (51) 0.170 (44) 0.117 (20) 0.157 (13) 0.186 (32)DelC/DelG 0.136 (32) 0.152 (36) 0.166 (43) 0.111 (19) 0.096 (8) 0.105 (18)DelG/DelG 0.013 (3) 0.013 (3) 0.062 (16) 0.041 (7) 0.036 (3) 0.017 (3)DelC/InsG 0.383 (90) 0.397 (94) 0.297 (77) 0.327 (56) 0.422 (35) 0.401 (69)DelG/InsG 0.085 (20) 0.076 (18) 0.166 (43) 0.187 (32) 0.084 (7) 0.093 (16)InsG/InsG 0.179 (42) 0.139 (33) 0.104 (27)⁄ 0.199 (34)⁄ 0.205 (17) 0.174 (30)Other 0.013 (3) 0.008 (2) 0.035 (9) 0.018 (3) 0.0 (0) 0.023 (4)

X2 p = 0.913 X2 p = 0.042⁄Residual p = 0.006

X2 p = 0.839

X2 test was used to compare haplotypic and diplotypic frequencies between patients and controls; alternatively, Fisher’s exact test was applied in the presence of one or moreempty cells; analysis of residues was performed in order to indicate the haplotypes or diplotypes responsible by the statistical difference observed; Bonferroni correction wasapplied when p < 0.05.

G.K. da Silva et al. / Infection, Genetics and Evolution 21 (2014) 418–423 421

2.78 (95%CI = 1.20–6.49). Regarding the +3142 C>G polymorphism,no significant differences were observed between HIV+/HCV+ andHIV+/HCV� patients concerning genotypic or allelic frequencies(p = 0.887 and 0.811, respectively, Table 4).

4. Discussion

The HIV infection has been extensively studied. However, thevirus ability to infect cells and trick the immune system remainsa puzzle for researchers. Although HIV initially induces an appar-ently appropriated immune response, it later evades the immunesystem allowing a successful infection (Tripathi and Agrawal,2007). HLA-G is an immunomodulatory molecule capable to sup-press immune responses and, since it could be involved in HIV es-cape from the immune surveillance, this feature has attracted

attention of researchers. Several works suggested that there is anincreased expression of HLA-G after HIV infection (Lajoie et al.,2009; Lozano et al., 2002; Tripathi and Agrawal, 2007) but the realinvolvement of this molecule on virus infection remains elusive.

In the present work we investigated the association betweenthe 14 bp insertion/deletion and the +3142 G/C HLA-G gene poly-morphisms with HIV infection. The evaluations were performedin patients and ethnic-matched controls from Southern Braziland in a confirmatory cohort of HIV+ patients from Italy. We ob-served an increased frequency of the genotypes related to lowHLA-G expression in African-derived-HIV-infected as comparedto uninfected individuals, suggesting that low HLA-G expressionalleles may play a role in HIV infection.

Previous studies have reported an association of HLA-G geno-types and HIV vertical transmission risk in Brazilian children. A

Table 4Genotypic and allelic frequencies of 14 bp and +3142 polymorphisms in HIV + individuals with or without HCV co-infection, according to ethnicity.

European-derived African-derived Italian

HIV+ HCV� freq (n) HIV+ HCV+ freq (n) HIV+ HCV� freq (n) HIV+ HCV+ freq (n) HIV+ HCV� freq (n) HIV+ HCV+ freq (n)

Genotypes 14 bp n = 185 n = 43 n = 105 n = 60 n = 144 n = 30Del/del 0.394 (73) 0.372 (16) 0.286 (30) 0.267 (16) 0.278 (40) 0.433 (13)Ins/del 0.476 (88) 0.442 (19) 0.571 (60) 0.417 (25) 0.528 (76) 0.433 (13)Ins/ins 0.130 (24) 0.186 (8) 0.143 (15)⁄ 0.317 (19)⁄ 0.194 (28) 0.133 (4)

X2 p = 0.632 X2 p = 0.024 (pBonf=0.048)⁄Residual p = 0.008

X2 p = 0.233

Alleles n = 370 n = 86 n = 210 n = 120 n = 288 n = 60Del 0.632 (234) 0.593 (51) 0.571 (120) 0.475 (57) 0.541 (156) 0.650 (39)Ins 0.368 (136) 0.407 (35) 0.429 (90) 0.525 (63) 0.459 (132) 0.350 (21)

Fischer p = 0.537 Fischer p = 0.108 Fischer p = 0.153

Genotypes+3142 n = 185 n = 43 n = 105 n = 60 n = 142 n = 30C/C 0.227 (42) 0.209 (9) 0.124 (13) 0.150 (9) 0.176 (25) 0.300 (9)C/G 0.551 (102) 0.535 (23) 0.438 (46) 0.417 (25) 0.507 (72) 0.567 (17)G/G 0.222 (41) 0.256 (11) 0.438 (46) 0.433 (26) 0.317 (45) 0.133 (4)

X2 p = 0.885 X2 p = 0.887 X2 p = 0.081

Alleles n = 370 n = 86 n = 210 n = 120 n = 284 n = 60C 0.503 (186) 0.477 (41) 0.343 (72) 0.358 (43) 0.430 (122) 0.583 (35)G 0.497 (184) 0.523 (45) 0.657 (138) 0.642 (77) 0.570 (162) 0.417 (25)

Fischer p = 0.720 Fischer p = 0.811 Fischer p = 0.033PBonf = 0.066

X2 test was used to compare genotypic frequencies and Fisher test was used to compare allele frequencies between patients and controls; analysis of residues was performedin order to indicate the genotypes responsible by the statistical difference observed; Bonferroni correction was applied when p < 0.05.

422 G.K. da Silva et al. / Infection, Genetics and Evolution 21 (2014) 418–423

protective effect conferred by the 14 bp deletion allele was ob-served and it was hypothesized that the 14 bp deletion, associatedto a higher HLA-G expression, could contribute to create a local Th2immune response, decreasing the production of pro-inflammatorycytokines, which were associated to an increased risk of mother-to-child HIV-1 transmission (Fabris et al., 2009; Segat et al.,2009). Our observations are in agreement with these data and en-dorse a more general view of the influence of 30 UTR polymor-phisms not only in vertical, but also in horizontal HIV transmission.

The fact that our findings are restricted to patients of a particu-lar ethnic origin deserves some considerations. Studies evaluatingthe influence of the 14 bp polymorphism in HIV horizontal trans-mission showed different results according to the population eval-uated (Aghafar et al., 2012; Segat et al., 2010). In European-derivedpopulations, no association was observed. However, among AfricanAmericans a protective effect of the 14 bp del/del genotype was ob-served (Aghafar et al., 2012). These studies support our findingsand endorse the importance of controlling for ethnicity in associa-tion studies. Studies that investigated the influence of other HLA-Galleles in HIV infection susceptibility in women from Italy, Zimba-bwe and Kenya produced controversial results: this fact empha-sizes the need of further investigations on the influence of HLA-Ggenetic variants in disease susceptibility in populations with differ-ent genetic backgrounds (Matte et al., 2004; Segat et al., 2010; Turket al., 2013).

We also investigated the influence of HLA-G polymorphisms inHIV-positive individuals co-infected with HCV. To our knowledge,this is the first work to evaluate the influence of HLA-G and HCVco-infection in HIV patients. In African-derived (but not in Euro-pean-derived) individuals we observed an increased frequency ofthe ins/ins genotype among HIV-infected patients. This genotypeis related to lower HLA-G levels in normal physiological conditions.It is important to point out the high prevalence of HCV co-infectionin this population, strongly suggesting the existence of ethnicity-specific disease modifying factors that could predisposeAfrican-derived subjects to an increased risk of HCV co-infection.In a previous work evaluating sickle cell disease (SCD) patients,we have described that the +3142 G allele, previously associatedto lower HLA-G expression, was associated to HCV infection (Cor-dero et al., 2009). Our findings are consistent with this previous

study and may reflect the fact that individuals with low HLA-Gexpression alleles could be more susceptible to viral infections, atleast in situations characterized by an imbalanced immune system,such as in HIV infection and SCD.

In conclusion, our results suggest a possible role for the HLA-Gmolecule in modulating immunity against HIV and HCV infectionin African-derived individuals. It will be important that futurestudies do not neglect the interaction between genetic markersfrom both pathogen and host, giving special attention to the hostethnic background.

Acknowledgments

The present work was supported by CNPq (Conselho Nacionalde Desenvolvimento Científico e Tecnológico) grant 306349/2011-6 and FAPERGS (Fundação de Amparo à Pesquisa no RioGrande do Sul), grant 1015166.

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