ORIGINAL RESEARCH
Microbes, Infection and Chemotherapy ISSN: 2789 - 4274 https://doi.org/10.54034/mic.e1855
CCR5-∆32, CCR2-64I, SDF1-3'A, and IFNλ4 rs12979860 and rs8099917 gene polymorphisms in individuals with HIV-1, HIV/HTLV-1, and HIV/HTLV-2 in São Paulo, Brazil
Adele Caterino-de-Araujo1,*, Karoline R. Campos1,2, Emylenne C. Cabral-de-Oliveira 1, Ana Kelly S. Rodrigues1, Rafael X. Silva1, Bruna V. Azevedo1, Rosa M. N. Marcusso3
1Instituto Adolfo Lutz, Centro de Imunologia, Laboratório de Pesquisa em HTLV, São Paulo, SP, Brasil.
2Instituto Adolfo Lutz, Centro de Respostas Rápidas, Laboratório Estratégico, São Paulo, SP, Brasil.
3Instituto de Infectologia Emilio Ribas, Serviço de Neurologia Clínica, Ambulatório de HTLV, São Paulo, SP, Brasil
Corresponding author: E-mail:adele.caterino@ial.sp.gov.br
Orcid ID: *https://orcid.org/0000-0003-0155-6580
Submitted: april 14, 2023
Reviewed: may 12, 2023
Approved: may 30, 2023
Abstract
Background. Chemokine and chemokine-receptor polymorphisms have been associated with protection against HIV infection and delayed progression to AIDS, whereas polymorphisms in IFNλ4 (formerly IL28B) have been associated with human T-lymphotropic virus 1 (HTLV-1)-associated myelopathy (HAM) development. Evolutionary selection against ancestral genes differs among human populations, resulting in varying risks of acquiring and developing viral diseases. Methods. DNA samples from 434 patients infected with HIV-1 and/or co-infected with HTLV-1/-2, and samples from 74 HIV and HTLV non-infected individuals from São Paulo, Brazil, were divided into five groups: HIV-naïve, n=160; HIV-ART, n=180; HIV/HTLV-1, n=53; HIV/HTLV-2, n=41; and control, n=74. These samples were analyzed for CCR5-∆32 deletion, CCR2-64I, SDF1-3'A, and IFNλ4 rs12979860 and rs8099917 single nucleotide polymorphisms using PCR and PCR-RFLP techniques. These polymorphisms' genotype and allele frequencies were calculated and compared among groups using logistic regression analysis. Results. All polymorphism profiles described in the literature were detected in this study. The wild-type genotype predominated in all genes analyzed except for IFNλ4 rs12979860. Statistical differences in allele frequencies among groups were detected in the CCR5 and CCR2 genes, with a high frequency of ∆32 in HIV-naïve vs. HIV-ART (OR 2.45, P=0.037) and a minus mutant allele A (CCR2-64I) in HIV-naïve vs. HIV/HTLV-1 (OR 1.90, P=0.048), HIV-ART vs. HIV/HTLV-1 (OR 2.62, P=0.003), and HIV/ART vs. HIV/HTLV-2 (OR 2.42, P=0.016). Conclusions. The polymorphism profiles detected in the study groups corroborate the profiles described in racial admixed populations. High CCR2-64I mutant allele frequencies were detected in HIV/HTLV-1/-2 co-infected individuals, and CCR5-∆32 showed predictive value for ART initiation.
Keyword: HIV-1, HTLV-1/2, chemokine, chemokines receptors, IFNλ4, polymorphisms.
Introduction
Studies have highlighted the importance of individual genetic backgrounds in disease outcomes by reporting the influence of genetic markers on the host immune response against viral infections, such as human immunodeficiency virus 1 (HIV-1), hepatitis C virus (HCV), and human T-lymphotropic virus 1 (HTLV-1). Evolutionary selection against ancestral genes varies across human populations(1-2), resulting in a varied risk of acquiring and developing such viral diseases(3-9).
Chemokine and chemokine-receptor polymorphisms have been associated with protection against HIV infection and progression to AIDS (CCR5-∆32, CCR2-64I, SDF1-3'A), but their frequencies vary by regions/populations(10-12).
A homozygous mutation in the HIV-1 CCR5 co-receptor (deletion of 32 nucleotides in CCR5 gene; CCR5-∆32 polymorphism) blocks the entry of HIV-1 R5 strains into cells, whereas heterozygosity delays this infection(4,5).
A single nucleotide polymorphism (SNP) in the CCR2 HIV-1 co-receptor gene (CCR2-64I; nucleotide transition G>A at codon 64, encoding isoleucine (ATC) instead of valine (GTC)) has been associated with protection against HIV and other inflammatory disease progressions(13,14). This is due to its ability to dimerize with CXCR4 and/or CCR5, reducing CXCR4 and CCR5 levels in peripheral blood mononuclear cells (PBMC) and delaying the progression to seroconversion in HIV-infected donors carrying this mutation(15).
Similarly, high concentrations of the chemokine SDF1 block or delay HIV entry by binding to the HIV co-receptors CCR5 and CXCR4. The G>A nucleotide change in the SDF1 gene (SDF1-3'A polymorphism) has been associated with high production of this chemokine and conferred protection against HIV infection(6,16).
Polymorphisms in other protein genes, formerly known as IFNL3/IL28B and presently as IFNλ4 (nucleotide transition C>T in rs12979860 and T>G in rs8099917), in patients with hepatitis C have been associated with reduced HCV clearance and poor response to PEG-IFNα treatment(7,17,18). Interestingly, the same polymorphisms in patients infected with HTLV-1 are associated with developing HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP)(19-21).
The frequencies and functions of such polymorphisms are widely known, predominantly for HIV and HCV infections and HIV/HCV coinfections(22,23); however, there is a lack of studies on HIV/HTLV coinfections. The present study aimed to determine the frequencies of these polymorphisms in HIV/HTLV-1 and HIV/HTLV-2 co-infected patients from São Paulo, Brazil, and to compare them with the frequencies obtained in HIV mono-infected, naïve, and antiretroviral therapy (ART) patients, along with HIV and HTLV-1/2 non-infected individuals from the same geographical region.
Methods
Study design, population, samples, and groups for analysis
This study was conducted as an anonymous, cross-sectional, and descriptive study using DNA samples previously used for the molecular diagnosis, characterization, and surveillance of HTLV-1 and HTLV-2 in individuals infected with HIV in São Paulo, Brazil(24-27). DNA samples were obtained from patients attending the AIDS/STD Reference and Training Center in São Paulo (CRT DST/AIDS-SP) and the HTLV Research Laboratory of the Immunology Department of Instituto Adolfo Lutz (IAL) in São Paulo, SP. Blood samples were collected from 2013 to 2016, separated into plasma and peripheral blood leukocytes (PBL), divided into aliquots, and stored at -80 °C for subsequent use. We randomly selected available DNA samples from 340 HIV-1 mono-infected patients, 94 HIV/HTLV-1/2 co-infected patients, and 74 HIV and HTLV-1/2 seronegative individuals to search for polymorphism in chemokine, chemokine receptors, and IFNL4 genes and grouped these samples into five groups for analyses: HIV-naïve, 160 HIV-infected patients without ART; HIV-ART, 180 HIV-infected patients on ART, matched for sex/age and time of HIV infection with HIV-naïve patients; HIV/HTLV-1, 53 patients infected with HIV and HTLV-1; HIV/HTLV-2, 41 patients infected with HIV and HTLV-2; control, 74 individuals HIV and HTLV-1/2 seronegative (20 from the staff and 54 from the laboratory routine). Patient characteristics (age and sex) and color/race data were obtained from interviews, medical records (CRT DST/AIDS-SP), and routine medical requests (IAL). Unfortunately, the color/race data was unavailable for HIV-and/or HTLV-infected individuals and controls since this characteristic is not included in routine medical requests. Thus, this variable was unavailable in 38 HIV-naïve, 23 HIV-ART, 30 HIV/HTLV-1, and 26 HIV/HTLV-2 patients, as well as for all individuals in the control group (Table 1).
Polymorphism analyses were conducted at the HTLV Research Laboratory (IAL). Informed consent was obtained from patients and controls. The study was approved by the Ethics Committee for Research of IAL (Ministry of Health protocol numbers CAAE #55837316.0.0000.0059 and #52493316.1.0000.0059). The data were analyzed anonymously.
Table 1 Characteristics of the study groups employed for the evaluation of polymorphisms in CCR∆532, CCR2-64I, SDF1-3'A, and IFNλ4 rs12979860 and rs8099917 genes
| Groups | ||||||
|---|---|---|---|---|---|---|
| Variables | HIV-Naïve n=160 | HIV-ART | HIV/HTLV-1 | HIV/HTLV-2 | Control | P value |
| n=180 | n=53 | n=41 | n=74 | |||
| Gender, n (%) | ||||||
| Male | 131 (81.9) | 135 (75.0) | 30 (56.6) | 21 (51.2) | 31 (41.9) | < 0.001b |
| Female | 29 (18.1) | 45 (25.0) | 23 (43.4) | 20 (48.8) | 43 (58.1) | |
| Age, years (mean, 95% CI) | ||||||
| Male | 34.9 (33.3–36.6) | 36.4 (34.7–38.1) | 46.9 (43.2–50.6) | 48.9 (45.6–52.2) | 40.7 (33.8–47.6) | < 0.001c |
| Female | 38.6 (34.9–42.4) | 42.6 (40.1–45.2) | 46.7 (41.8–51.6) | 49.0 (45.4–52.7) | 33.2 (28.1–38.3) | |
| P value | 0.067 d | < 0.001 d | 0.941 d | 0.883 d | 0.069 d | |
| Color/race, n (%)a | ||||||
| White | 72 (59.0) | 100 (63.7) | 10 (43.5) | 6 (40.0) | - | 0.139b |
| Black and pardum | 48 (39.4) | 54 (34.4) | 11 (47.8) | 9 (60.0) | ||
| Amerindian | 1 (0.8) | 0 | 1 (4.35) | 0 | ||
| Japanese | 1 (0.8) | 3 (1.9) | 1 (4.35) | 0 | ||
DNA Extraction
Genomic DNA was extracted and purified from PBL using the Roche MagNA Pure RLC Robot Instrument (Mannheim, Germany) with the LC MagNA Pure Nucleic Acid Isolation Kit I (Roche Diagnostics, Mannheim, Germany) according to the manufacturer's instructions and eluted with 100 µL of PCR-grade water. The extracted DNA was aliquoted into four vials for subsequent analysis and thawed once.
Characterization of CCR5-∆32, CCR2-64I, SDF1-3'A, and IFNλ4 rs12979860 and rs8099917 polymorphisms
Table 2 presents the sequences of primers and restriction enzymes used in the CCR5-∆32, CCR2-64I, SDF1-3'A, and IFNλ4 rs12979860 and rs8099917 gene polymorphism analyses, the fragment size of each genotype, and the authors' reference number.
For CCR5-∆32 genotype characterization, the PCR reaction mixture contained 20 pmol of each primer, 2.5 X GoTaq colorless master mix (Promega), 2.5 µL of genomic DNA, and H2O in a total volume of 25 µL. The cycling amplification consisted of 5 cycles at 94 °C for 1 min, 55 °C for 1 min and 72 °C for 1.5 min; 35 cycles at 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 45 s, followed by 1 cycle of 72 ºC for 10 min. A 10 µL aliquot of each 25 µL PCR reaction mixture was run on a 3 % agarose gel, electrophoresed, and the appropriately sized products were visualized under UV illumination after staining with SYBR™ Safe DNA Gel Stain (Invitrogen)(28) (Table 2).
The PCR conditions for CCR2-64I were as follows: 10 pmol of each primer, 2.5 X GoTaq colorless master mix (Promega), 2.5 µL of genomic DNA, H2O, in a total volume of 25 µL. The cycling conditions were as follows: 94 °C for 10 min, followed by 40 cycles at 94 °C, 56.5 °C, and 72 °C for 1 min at each temperature, followed by 72 °C for 7 min. For the CCR2-64I mutation detection, the amplified product was digested with the BsaBI restriction endonuclease (New England, BioLabs Inc.) at 72 °C for 4 h, and then the PCR amplification product was run on a 2 % agarose gel electrophoresis stained with SYBR™ Safe DNA Gel Stain (Invitrogen)(14). For SDF1-3'A polymorphism detection, the PCR mix was the same as for CCR2-64I, except that 20 pmol of each primer was used. The cycling scheme consisted of 1 cycle at 94 °C for 10 min, followed by 35 cycles at 94 °C, 55 °C, and 72 °C for 30 s at each temperature, followed by 72 °C for 7 min. After amplification, the PCR product was digested with the restriction endonuclease MspI (New England, BioLabs Inc.) at 37 °C for 4 h, and then run on a 3% agarose gel electrophoresis stained with SYBR™ Safe DNA Gel Stain (Invitrogen)(29). The size products in base pairs (bp) for different CCR2 and SDF1 genotypes are presented in Table 2. The bands of 23 bp in CCR2-64I genotypes GA and AA, and bands of 30 bp in IFNλ4 rs12979860 genotypes CT and TT were not visualized in agarose gel electrophoresis.
Regarding the polymorphisms in IFNλ4 (IL28B) rs12979860 and rs8099917, the PCR reactions consisted of 5 µL of genomic DNA, 1 U of Platinum® Taq DNA polymerase (Invitrogen, São Paulo, Brazil), 200 µM of each deoxynucleoside triphosphate (GE Healthcare, Little Chalfont, UK), 2.5 mM of MgCl2 (Invitrogen), PCR buffer (20 mM Tris–HCl, pH 8.4; 50 mM KCl) and 10 pmol of each primer in a total volume of 50 µL. The reaction for rs12979860 amplification was enhanced by the addition of 5% dimethyl sulfoxide. The annealing temperature was 59 °C for rs12979860 and 57.5 °C for rs8099917, and the thermal cycling scheme was as follows: 94 °C for 3 min, followed by 40 cycles at 94 °C for 30 s, 59 or 57.5 °C for 30 s, 72 °C for 1 min, and 72°C for 7 min. The amplicon corresponding to rs12979860 was digested with Hpy166II, and rs8099917 was digested with BsrDI (both from New England BioLabs Inc.). Incubations were performed at 37 °C (Hpy166II) and 65 °C (BsrDI) for 2 h, and the products of digestion with Hpy166II were separated through 3 % agarose gel electrophoresis for 2 h, and for BsrDI through 1.5 % agarose gel electrophoresis for 1.5 h (protocols adapted from Moreira et al.)(30). The product sizes are presented in Table 2.
Table 2.
PCR primers and restriction enzymes employed for the evaluation of polymorphisms in CCR532, CCR2-64I, SDF1-3'A, and IFNλ4 (IL28B) rs12979860 and rs8099917 genes
| Polymorphism | Primer | Sequence 5’- 3’ | Tm (oC) | Direction | Product size (bp) | Restriction enzyme, time, temperature | Recognition site | Genotype | Fragment (bp) | Ref |
|---|---|---|---|---|---|---|---|---|---|---|
| CCR5∆32 | P1 (2975) | AAC AGA TCT CAA AAA GAA GGT CT | 55 | Sense | 189 | NA | NA | wt/wt | 189 | 28 |
| P2 (2976) | CAT GAT GGT GAA GAT AAG CCT CAC A | Antisense | wt/∆32 | 189 + 157 | ||||||
| ∆32/∆32 | 157 | |||||||||
| CCR2-64I | CKR2IAF | ATTG TGG GCA ACA TGA TGG | 56,5 | Sense | 183 | BsaBI, 4 h, 60 oC | GATNN^NNATC | G/G | 183 | 14 |
| CCR264IR | CTG TGA ATA ATT TGC ACA TTG C | Antisense | G/A | 183 + 165 + 23 | ||||||
| A/A | 165 + 23 | |||||||||
| SDF1-3’A | SDF1 F | CAG TCA ACC TGG GCA AAG CC | 55 | Sense | 302 | MspI, 4 h, 37 oC | C^CGG | G/G | 202 + 100 | 29 |
| SDF1 R | AGC TTT GGT CCT GAG AGT CC | Antisense | G/A | 302 + 202 + 100 | ||||||
| A/A | 302 | |||||||||
| IFNλ4 rs12979860 | IL28B-860F | AGC AGG ACA GAT TGG CAA AG | 59 | Sense | 694 | Hpy166II, 2 h, 37 oC | GTN^NAC | C/C | 509 + 185 | 30 |
| IL28B-860R | CAC AAT TCC CAC CAC GAG AC | Antisense | C/T | 509 + 185 + 155 + 30 | ||||||
| T/T | 509 + 155 + 30 | |||||||||
| IFNλ4 rs8099917 | IL28B-917F | CTG GAA CAA ATC GTC CCA AT | 57,5 | Sense | 496 | BsrDI, 2 h, 65 oC | GCAATG^NN | T/T | 496 | 30 |
| IL28B-917R | TTC CTT TAG GCC TGT GGA TG | Antisense | T/G | 496 + 272 + 224 | ||||||
| G/G | 272 + 224 |
Statistical Analyses
Differences in the characteristics of individuals regarding sex and race/color were evaluated using the Chi-square (X2) of the Pearson test, and age was evaluated using the nonparametric Kruskal-Wallis test (for three or more groups) and Mann-Whitney U test (for two groups). Allele frequency was determined using the formula f = (1 × h + 2 H) / 2 N, where h is the number of heterozygotes, H is the number of homozygotes, and N is the total number of individuals. Differences in genotype and allele frequencies among groups were evaluated using the X2 of the Pearson test and logistic regression univariate analysis by calculating the odds ratio (OR) and 95% confidence interval (CI). The level of significance was set at P < 0.05. Data were analyzed using SPSS® Statistics 29 (Statistical Package for the Social Sciences, 29.0), (Statistical Software: IBM, NY, USA).
Results
Table 1 shows the characteristics of the study groups with statistically significant differences. For instance, the HIV-naïve and HIV-ART groups included more males than females, while the HIV/HTLV-1 and HIV/HTLV-2 groups included more black/pardum individuals. Regarding age, HIV/HTLV-1 and HIV/HTLV-2 co-infected patients were older, regardless of being male or female. In the HIV-ART group, women were older than men (P< 0.001). The comparison of ages between the two groups revealed statistically significant differences: HIV-naïve vs. HIV-ART (P=0.048); HIV-naïve vs. HIV/HTLV-1 and HIV/HTLV-2 (both P< 0.001); HIV-ART vs. HIV/HTLV-1 and HIV/HTLV-2 (both P< 0.001); HIV-ART vs. control (P=0.017); and HIV/HTLV-1 and HIV/HTLV-2 vs. control (both P< 0.001). No differences in age were detected between the HIV/HTLV-1 vs. HIV/HTLV-2 (P=0.312) groups or the HIV-naïve vs. control (P =0.408) groups.
This study detected all the polymorphism profiles described in the literature (Figure 1). The wild-type genotype was prevalent in all genes analyzed except for IFNλ4 rs12979860. The genotypes and allele frequencies of the polymorphisms are shown in Table 3. Preliminary analyses of the polymorphisms in CCR5 showed differences in genotype frequencies (P=0.036) and high ∆32 alleles in HIV-naïve, HIV/HTLV-2, and control groups, but without significant differences among them (P=0.154). In CCR2 polymorphism analysis, differences in genotype (P=0.011) and allele frequencies (P< 0.001) were detected, and more allele A mutants were identified in the HIV/HTLV-1 and HIV/HTLV-2 groups compared to the control group. No difference in genotype and allele frequencies in SDF1 and IFNλ4 rs12979860 and rs8099917 were detected (all P >0.05, Table 3).
Figure 1. Electrophoretic representative patterns of all genotypes related to CCR5∆32, CCR2-64I, SDF1-3'A, and IFNλ4 rs12979860 and rs8099917 polymorphisms detected in the present study
Legend: M: molecular size marker (100 bp Ladder) except in CCR5 (50 bp Ladder); CCR5 – WT: wt/wt wild genotype, WT∆32: wt/∆32 polymorph genotype, ∆32: ∆32/∆32 polymorph genotype; CCR2 – GG: wild genotype, GA and AA polymorph genotypes; SDF1- GG: wild genotype, GA and AA polymorph genotypes; IFNλ4 rs12979860 – CC: wild genotype, CT and TT polymorph genotypes; IFNλ4 rs8099917 – TT: wild genotype, TG and GG polymorph genotypes.
Table 3. Genotypic and allelic frequencies of CCR5∆32, CCR2-64I, SDF1-3'A, and IFNλ4 rs12979860 and rs8099917 genes polymorphisms according to the study groups
| HIV-Naïve | HIV-ART | HIV/HTLV-1 | HIV/HTLV-2 | Control | x2 P value |
|
|---|---|---|---|---|---|---|
| CCR5D32 | n=156 | n=155 | n=52 | n=34 | n=71 | |
| n (%) | n (%) | n (%) | n (%) | n (%) | ||
| Genotype | ||||||
| wt/wt | 137 (87.8) | 148 (95.5) | 50 (96.2) | 30 (88.24) | 63 (88.7) | |
| wt/∆32 | 19 (12.2) | 6 (3.9) | 1 (4.6) | 3 (8.82) | 8 (11.3) | 0,036 |
| ∆32/∆32 | 0 | 1 (0.6) | 1 (4.6) | 1 (2.94) | 0 | |
| Allele | ||||||
| wt | 0,939 | 0,974 | 0,971 | 0,926 | 0,934 | |
| ∆32 | 0,061 | 0,026 | 0,029 | 0,074 | 0,056 | 0,154 |
| CCR2-64I (G>A) | n=122 | n=157 | n=48 | n=35 | n=74 | |
| n (%) | n (%) | n (%) | n (%) | n (%) | ||
| Genotype | ||||||
| G/G | 97 (79.5) | 131 (83.5) | 31 (64.5) | 23 (65.7) | 67 (90.5) | |
| G/A | 22 (18.0) | 25 (15.9) | 15 (31.3) | 11 (31.4) | 7 (9.5) | 0,011 |
| A/A | 3 (2.5) | 1 (0.6) | 2 (4.2) | 1 (2.9) | 0 | |
| Allele | ||||||
| G | 0,885 | 0,914 | 0,802 | 0,814 | 0,953 | |
| A | 0,115 | 0,086 | 0,198 | 0,186 | 0,047 | < 0.001 |
| SDF1-3’A (G>A) | n=104 | n=145 | n=45 | n=33 | n=69 | |
| n (%) | n (%) | n (%) | n (%) | n (%) | ||
| Genotype | ||||||
| G/G | 68 (65.4) | 98 (67.6) | 31 (68.9) | 22 (66.7) | 48 (69.6) | |
| G/A | 34 (32.7) | 45 (31.0) | 14 (31.1) | 9 (27.3) | 21 (30.4) | 0,565 |
| A/A | 2 (1.9) | 2 (1.4) | 0 | 2 (6.0) | 0 | |
| Allele | ||||||
| G | 0,817 | 0,831 | 0,844 | 0,803 | 0,848 | |
| A | 0,183 | 0,169 | 0,156 | 0,197 | 0,152 | 0,907 |
| INFλ4/IL28B SNP rs12979860 (C>T) | n=142 | n=158 | n=28 | n=20 | n=71 | |
| n (%) | n (%) | n (%) | n (%) | n (%) | ||
| Genotype | ||||||
| C/C | 62 (43.7) | 81 (51.3) | 11 (39.3) | 9 (45.0) | 33 (46.5) | |
| C/T | 57 (40.1) | 59 (37.3) | 13 (46.4) | 4 (20.0) | 31 (43.7) | 0,131 |
| T/T | 23 (16.2) | 18 (11.4) | 4 (14.3) | 7 (35.0) | 7 (9.8) | |
| Allele | ||||||
| C | 0,637 | 0,699 | 0,625 | 0,55 | 0,683 | |
| T | 0,363 | 0,301 | 0,375 | 0,45 | 0,317 | 0,23 |
| IFNλ4/IL28B SNP | n=143 | n=158 | n=28 | n=20 | n=74 | |
| rs8099917 (T>G) | n (%) | n (%) | n (%) | n (%) | n (%) | |
| Genotype | ||||||
| T/T | 92 (64.3) | 106 (67.1) | 20 (71.4) | 14 (70.0) | 54 (73.0) | |
| T/G | 47 (32.9) | 48 (30.4) | 7 (25.0) | 4 (20.0) | 18 (24.3) | 0,64 |
| G/G | 4 (2.8) | 4 (2.5) | 1 (3.6) | 2 (10.0) | 2 (2.7) | |
| Allele | ||||||
| T | 0,808 | 0,823 | 0,839 | 0,8 | 0,851 | |
| G | 0,192 | 0,177 | 0,161 | 0,2 | 0,149 | 0,823 |
Using logistic regression analysis, statistically significant differences among groups were confirmed only in the CCR5 and CCR2 genes (Table 4), and all analyses are presented in the Supplementary Table. Briefly, higher ∆32 allele frequencies were detected in the HIV-naïve, HIV/HTLV-2, and control groups than in the HIV-ART group, with statistical significance only when comparing HIV-naïve vs. HIV-ART (P=0.037). However, a tendency towards significance was observed between the HIV-ART and HIV/HTLV-2 groups (P=0.061), and no difference was observed between the HIV-ART and control groups (P=0.112). Interestingly, although ∆32 allele frequencies differ between HIV/HTLV-1 and HIV/HTLV-2 (0.029 and 0.074, respectively), no significant difference was detected using regression analysis (P=0.189). Regarding CCR2, high allele A mutant frequencies were detected in the HIV/HTLV-1 (0.198) and HIV/HTLV-2 (0.186) groups, a more modest frequency in the HIV-naïve group (0.115), and low frequencies in the HIV-ART (0.086) and control groups (0.047). However, significant differences were detected between HIV-naïve vs. HIV/HTLV-1 (P=0.048), HIV-naïve vs. control (P=0.028), HIV-ART vs. HIV/HTLV-1 (P=0.003), and HIV-ART vs. HIV/HTLV-2 (P=0.016). All other genes displayed similar percentages of allele frequencies among groups without significant differences, except for a tendency towards significance when IFNλ4 rs12979860 T allele was compared between HIV-ART and HIV/HTLV-2 (P=0.059).
Table 4 CCR5∆32 and CCR2-64I gene polymorphisms that were significant predictors of HIV infection/progression using logistic regression univariate analysis in the study groups
| Gene locus | Allele frequency | Genotype frequency | OR (95% CI) | |||
|---|---|---|---|---|---|---|
| n (%) | n (%) | -/- n (%) | P value | |||
| CCR5D32 | wt | D32 | wt/wt | wt/D32 | D32/D32 | |
| HIV-naïve(n=156) | 0,939 | 0,061 | 137 (87.8) | 19 (12.2) | 0 | 2.45 (1.06–5.68) |
| HIV-ART (n=155) | 0,974 | 0,026 | 148 (95.5) | 6 (3.9) | 1 (0.6) | 0,037 |
| CCR2-64I | G | A | GG | GA | AA | |
| HIV-naïve(n=122) | 0,885 | 0,115 | 97 (79.5) | 22 (18.0) | 3 (2.5) | 1.90 (1.01–3.60) |
| HIV-HTLV-1 (n=48) | 0,802 | 0,198 | 31 (64.5) | 15 (31.3) | 2 (4.2) | 0,048 |
| HIV-naïve(n=122) | 0,885 | 0,115 | 97 (79.5) | 22 (18.0) | 3 (2.5) | 2.61 (1.11–6.14) |
| Control (n=74) | 0,953 | 0,047 | 67 (90.5) | 7 (9.5) | 0 | 0,028 |
| HIV-naïve(n=122) | 0,885 | 0,115 | 97 (79.5) | 22 (18.0) | 3 (2.5) | 2.61 (1.11–6.14) |
| Control (n=74) | 0,953 | 0,047 | 67 (90.5) | 7 (9.5) | 0 | 0,028 |
| HIV-ART (n=157) | 0,914 | 0,086 | 131 (83.5) | 25 (15.9) | 1 (0.6) | 2.62 (1.38–4.97) |
| HIV-HTLV-1 (n=48) | 0,802 | 0,198 | 31 (64.5) | 15 (31.3) | 2 (4.2) | 0,003 |
| HIV-ART (n=157) | 0,914 | 0,086 | 131 (83.5) | 25 (15.9) | 1 (0.6) | 2.42 (1.18–4.98) |
| HIV-HTLV-2 (n=35) | 0,814 | 0,186 | 23 (65.7) | 11 (31.4) | 1 (2.9) | 0,016 |
| HIV-HTLV-1 (n=48) | 0,802 | 0,198 | 31 (64.5) | 15 (31.3) | 2 (4.2) | 4.97 (2.00–12.35) |
| Control (n=74) | 0,953 | 0,047 | 67 (90.5) | 7 (9.5) | 0 | < 0.001 |
| HIV-HTLV-2 (n=35) | 0,814 | 0,186 | 23 (65.7) | 11 (31.4) | 1 (2.9) | 4.59 (1.74–12.11) |
| Control (n=74) | 0,953 | 0,047 | 67 (90.5) | 7 (9.5) | 0 | 0,002 |
Discussion
The heterogeneous population residing in the city of São Paulo (Southeastern Brazil) reflects the high frequency of racial inter-mixing over time, firstly between Amerindians, Europeans and Africans, and subsequently with migrants from other regions of the country and elsewhere. Thus, the ethnicity of this population varies greatly, and although it should be necessary to use more objective methods to evaluate the patients' ethnic background to measure ancestry, the genetic polymorphism results obtained in the present study corroborate the polymorphisms disclosed in an admixed population. The present study uses self-reported and phenotypic racial features to define race/color.
The age and sex of the study groups were variable (Table 1). The HIV/HTLV-1/2 co-infected patients had (i) high age, (ii) approximately equal proportions of males and females (except when compared with the control group), and (iii) a majority of black/pardum color/race individuals. These characteristics have been extensively analyzed in previous studies(24,25,31), corroborating the characteristics of HTLV-1/2 patients in Brazil(32-34).
Concerning CCR5-∆32 frequencies, the mutant allele is common in North America and Europe, with heterozygote frequencies ranging from 10 to 20 % in Caucasians and 2 to 5 % in the Middle East and Indian populations(4,10). This allele is not found in aboriginal populations outside Eurasia, and its presence elsewhere probably represents European gene flow into local populations(10). This is the case in Brazil, where the population is predominantly of European ancestry with significant contributions from African and Amerindian gene pools. Since the ∆32 alleles have not been identified in Africans or Amerindians, the presence of the CCR5-∆32 in Brazilians can be attributed to European immigration. In effect, one study conducted in Belém, PA (Northern Brazil), showed ∆32 allele frequencies of 3.04 % in the general population, 0.75 % in Afro-descendants, and 0 % in Amerindians and Japaneses(35), and 2.7 and 2.2 % in HIV positive and negative individuals, respectively(36). In Salvador, BA, among blood donors, the ∆32 alleles were detected at a frequency of 2.6 %, while in HIV-infected individuals without ART, it was 4.4 %(37). German-descendant blood donors from Joinville, SC (Southern Brazil) had a ∆32 allele frequency of 6.5 %, while among Tiriyó and Waiampi Amerindian tribes from the Amazon River basin (North region), this allele has not been found(37). In Belo Horizonte, MG (Southeast region), among unrelated healthy individuals, the ∆32 allele frequency was 5.3 %, while in blood donors screened for HTLV infection, the frequencies varied: 7.0 % in HTLV-1 seronegative, 5.7 % in HTLV-1 serum indeterminate, 4.1 % in HTLV-1 seropositive asymptomatic carriers, and 0% in HAM(38). In patients with colorectal cancer from São Paulo, SP, the ∆32 allele frequency was 5.2 %(38). A systematic review and meta-analysis of 30 different healthy populations from the north-northeast (N-NE) and south-southeast (S-SE) regions of Brazil found ∆32 allele frequencies ranging from 0 to 10 %, with an average of 3 and 4 % in the N-NE and S-SE populations, respectively (P=0.002)(39). Considering these data, the ∆32 allele frequencies detected in the present study (2.9 to 7.4%,Table 3) agree with the frequencies detected in mixed-race populations living in Brazil and with European ancestry in São Paulo.
Regarding the functional consequences of CCR5-∆32 polymorphism in HIV-1 infected patients, one study from Salvador showed a minor HIV viral load (VL) and in vitro partial resistance to R5-HIV-1 strains in the PBMC of heterozygote individuals(37). In Rio Grande, RS (Southern Brazil), among HIV-infected patients followed for an average of 6.4 years, the presence of CCR5-∆32 was associated with a reduction in the risk of CD4+ T-cell depletion and an increased risk of death after AIDS diagnosis(40). In São Paulo, ∆32 allele frequencies of 3.8 and 5.5% were detected in HIV-1-infected and healthy individuals, respectively. Multivariate regression analysis identified an association between CCR5-D32/CCR2-V64I polymorphisms and positive CD4+ T cell recovery after ART(41). Herein, although the cross/sectional design did not allow concluding protection against HIV infection and delayed progression to AIDS, the higher ∆32 allele frequency in HIV-naïve (6.1 %) compared to HIV-ART (2.6 %) (OR 2.45, P=0.0037, (Table 4) confirms the prognostic value of the ∆32 allele for ART (remembering that both groups are matched for sex, age, and time of HIV acquisition). Notably, a tendency toward a statistical difference was detected when comparing the HIV-ART vs. HIV/HTLV-2 groups (OR=2.99, P=0.061, Supplemental Table). Although both groups received ART, HIV/HTLV-2 co-infected subjects were older and more likely to have HIV and HTLV-2 infections than HIV-ART subjects(24,25). Furthermore, HTLV-2 in HIV-infected subjects has been associated with delayed progression to AIDS and death(42), lower HIV VL, and higher CD4+ T cell counts compared to HIV/HTLV-1 co-infected subjects(43). Thus, the ∆32 allele could be another protective factor that delayed HIV disease progression in HIV/HTLV-2 co-infected subjects.
Regarding the CCR2-64I polymorphism, these genetic variants are observed in almost all populations studied. The mutant (G>A) allele frequencies are the highest (approximately 35 %) in Africa and Asia, decrease in Northern Europe, in Caucasian-Americans, and are low in Pacific Islander populations(11), contrasting with CCR5-∆32 polymorphism distribution(10). In Brazil, the CCR2-64I allele frequencies vary according to the population analyzed, reaching 14 % in HIV non-infected subjects from Salvador (80 % of which are African descendants), 18 % in German descendants from the Joinville Regional Hemocenter, 26 % in Tiriyó and 30 % in Waiampi Indian tribes from Amazonia(44). In Belém, PA, the CCR2-64I frequencies were 16.08 % in the general population, 22.95 % in Afro-descendants, 13.27 % in Amerindians, 24 % in Japanese(35), 5.4 % in HIV-infected, and 12.5 % in HIV uninfected subjects, the last data suggesting a protective effect of such gene polymorphism in HIV acquisition(36). In the present study, differences in CCR2-64I frequencies were detected, with emphasis on HIV/HTLV co-infected patients who presented the highest frequencies (19.8 % in HIV/HTLV-1 and 18.6 % in HIV/HTLV-2, Table 3), correlating with the percentage detected in Afro-descendants from Northern Brazil(35), and the black/pardum color/race of this group of patients(24). In contrast, the lowest frequency of CCR2-64I was detected in the control group (4.7 %, Table 3), specifically in the group where colors/races were not disclosed. The majority were probably white or Caucasian, similar to most employees of the IAL laboratory.
Studies on CCR2-64I polymorphism and HIV outcome are scarce in Brazil; one study of 6.4 years follow-up from Rio Grande, RS, found 11.3 % CCR2-64I allele frequency and the association of this polymorphism with a reduced risk for developing AIDS(40). In São Paulo, SP, although the frequencies of CCR2-64I did not differ between HIV-infected (14 %) and HIV-uninfected (11 %) subjects, the presence of both CCR2-64I/CCR5-∆32 polymorphisms was associated with positive recovery of CD4+ T cells after ART(41). Unfortunately, herein, it was not possible to associate CCR2-64I with HIV disease progression but rather with the diversity of the genetic backgrounds of individuals residing in São Paulo. In fact, as mentioned above, the highest frequencies of such polymorphism were detected among HIV/HTLV-1/2 co-infected individuals (Table 3), just the groups with more black/pardum individuals (Table 1). However, it is important to emphasize that these groups of patients had more age and consequently more years of retrovirus infections (more than 20 years of HIV infection), as previously described(24).
In Brazil, in the SDF-1-3'A polymorphism, the mutant 3'A allele frequencies vary according to region/population: in the North, it was detected in 22.33 % of healthy individuals from Belém, 15.33 % of Afro-descendants, 22.74 % of Amerindians, 26 % of Japaneses(35), 15.4 % of HIV-infected, and 21 % of HIV uninfected subjects(36), as well as 24 % in Tiriyó and 5 % in Waiampi Indian tribes(16). In other regions, it was detected in 17 % of healthy subjects from Salvador, 21 % from Joinville(37), 20.7 % of HIV-infected patients from Rio de Janeiro(40), and 14 % of HIV-infected patients from São Paulo(41). These frequencies agree with the frequencies detected in this study in HIV-infected and HIV/HTLV-co-infected individuals (15.6 to 19.7 %, Table 3). In addition, an association between SDF1-3'A/CCR2-64I polymorphisms and a minor risk for developing AIDS was detected in a study conducted in Rio de Janeiro(40), but the same result was not observed in a study conducted in São Paulo(41). Notably, one longitudinal study of at least 6 years in HIV-infected patients from the Amazon region found an association of the 3'A allele variant with a marked loss of CD4+ T lymphocytes and high plasma VL(45), emphasizing the need of more studies for this issue in Brazil.
Regarding the polymorphisms in IFNλ4 rs12979860 (C>T) and rs8099917 (T>G) and HAM development, one study of Spanish HTLV-1 infected subjects (12 with HAM and 29 asymptomatic carriers) associated IL28B rs12979860 polymorphisms CT/TT with HAM and high HTLV-1 proviral load (PVL)(46). In contrast, another study conducted in São Paulo with 112 unrelated Brazilian subjects (81 HTLV-1 asymptomatic carriers, 24 with HAM/TSP, and 7 with Adult T cell leukemia/lymphoma) did not support these associations; neither the homozygote TT nor the heterozygote CT mutations nor the combination genotypes (TT/CT) were associated with high PVL and HAM/TSP(47). Similarly, no significant differences were observed in Belém, PA, when comparing 26 HAM/TSP patients, 53 asymptomatic carriers, and 300 seronegative healthy subjects(48). In contrast, two studies from São Paulo associated IL28B rs8099917 genotype GG with HAM/TSP(20,21); the first included 229 subjects (136 HTLV-1 asymptomatic carriers and 93 HAM/TSP)(20), and the other 247 subjects (160 HTLV-1 asymptomatic carriers and 87 HAM/TSP)(21). In the present study, there were no differences in IFNλ4 rs12979860 and rs8099917 polymorphisms frequencies among groups, nor the case of HAM in HIV/HTLV co-infected patients(24). But, a tendency toward a statistical difference was detected when comparing the frequencies of IFNλ4 rs12979860 polymorphism in HIV-ART vs. HIV/HTLV-2 groups (OR=1.90, P=0.059, Supplemental Table). Curiously, the HIV-ART group showed the highest percentage of IFN‐λ4 rs12979860 wild-type genotype (CC), while the HIV/HTLV-2 had the highest percentage of the mutant genotype (TT). Recently, one study of 30 years follow-up conducted with a cohort of 402 HIV‐1‐infected subjects from São Paulo disclosed an association of IFN‐λ4 rs12979860 wild-type genotype (CC) with higher mortality rate compared to CT and TT genotypes and with an increased probability of death from AIDS (P =0.01)(49). The authors suggested that this effect may be related to higher baseline plasma HIV VL and altered immune reconstitution, associated with lack of interferon λ expression in that population(49). Unfortunately, we do not know the HIV infection disease outcome of patients from the present study, but taking into account the IFNλ4 rs12979860 polymorphism results, we can hypothesize worse disease outcomes in HIV-ART and better outcome in HIV/HTLV-2 co-infected patients also on ART.
Despite the limitations of the present study regarding its cross-sectional design and the lack of some information that does not allow firm conclusions, the results support the use of CCR5-∆32 polymorphism as a predictive marker for ART initiation in individuals without universal access to this treatment. In addition, the results show the presence of all polymorphism profiles described in the literature in the studied groups (Figure), corroborating data from inter-mixed racial populations residing in São Paulo and elsewhere. High CCR2-64I mutant allele frequencies were detected in HIV/HTLV co-infected patients, and this finding deserves more investigation. Finally, well-designed longitudinal studies are needed to know the effects of the polymorphisms detected in the present study on the clinical outcome of HIV-infected and HIV/HTLV co-infected individuals.
Author Contribution Statement
The authors confirm their contribution to the paper as follows: study conception, design and supervision: ACA and KRC; data collection: ECCO, AKSR, RXS and BVA; analysis and interpretation of results: ACA, KRC and RMNM; draft manuscript preparation: ACA, KRC, ECCO, AKSR, RXS, BVA and RMNM. All authors reviewed the results and approved the final version of the manuscript. All authors agreed to be responsible for all aspects of the work to ensure the accuracy and integrity of the published manuscript.
Ethics statement
The authors declare that the published work reflects an investigation and analysis carried out truthfully and completely. The study was approved by the Ethics Committee for Research of IAL (Ministry of Health protocol numbers CAAE #55837316.0.0000.0059 and #52493316.1.0000. 0059). The data were analyzed anonymously.
Conflicts of interest
The authors declare no conflict of interest.
Funding
This study was supported by grants from: Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP; grant 2016/03654-0 ACA, and scholarship to BVA 2016/01666-0); Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES; scholarship to KRC, grant 001); Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq; grant 302661/2015-8 to ACA, scholarship to ECCO 142983/2016-0, and RXS 122800/2017-6); Fundação de Desenvolvimento Administrativo (FUNDAP; scholarship to AKSR 2017); and Instituto Adolfo Lutz (IAL; CTC 106D/2012 and 62H-2015). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Availability of data
Available from the corresponding author upon request.
Acknowledgment
The authors are indebted to Wong Kuen Alencar (in Memorium) from CRT DST/AIDS-SP for patient attendance.
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