Distribution Of CCR-5 Δ 32,CCR2-64I,and SDF-1-3′A Alleles Among Jordanians

Abstract

Entry of HIV virus into cells is mediated by chemokine receptors. Genetic variations in chemokine receptors have been shown to modulate susceptibility to HIV infection and disease course. In this study, the frequencies of CCR5 (CCR5-Δ32), CCR2 (CCR2-64I), and SDF-1 (SDF-1-3′) gene polymorphisms were determined in a Jordanian population. A total of 540 subjects were randomly selected from different regions of Jordan (South, Middle, and North). Six individuals were found to carry the CCR5-Δ32 allele (0.6%) and only in the heterozygous genotype. The frequencies of CCR2-64I and SDF1-3′A were 17.5% and 34.2%, respectively. In addition, no significant difference in the distribution of the examined polymorphisms among different regions of Jordan was detected. In conclusion, the CCR5-Δ32 allele is rare, whereas the CCR2-64I and SDF1-3′A alleles are common among Jordanians.

Introduction

V ariations in susceptibility to infections are well known among individuals of the same population. This has been attributed to differences between individuals regarding genetic background and/or environmental factors such as diet and life style.^1
–3 Genetic variations in certain chemokines and their receptors have been reported to be responsible for resistance to HIV-1 infection, delayed progression to AIDS, response to the treatment, and mother-to-child transmission.⁴

Chemokine receptors are proteins found on the cell surface.⁵ The entry of HIV-1 virus to the host cells is primarily mediated by the CD4 receptor and at least one coreceptor from multi-CC and CXC chemokine receptors.⁶ Previous studies have identified the CCR5 chemokine receptor as the coreceptor for macrophage-tropic strain (R5)^5,7,8 and CXCR4 chemokine receptor for the X4 strain.⁸ In addition, in vitro studies showed that CCR2, CCR3, CCR8, and CCR9 chemokine receptors may act as coreceptors.⁷

Among the polymorphisms that modulate HIV infection is deletion of 32 bp in the coding region of the CCR5 receptor (CCR5-Δ32, rs333). The homozygous genotype of the CCR5-Δ32 allele gives strong resistance against R5 strains. On the other hand, the heterozygous genotype gives limited resistance and/or delays the progression to AIDS.^7,9
–11 Another important variation that modulates HIV infection is CCR2-64I polymorphism (rs1799864). Individuals with one or two copies of the 64I variant have reduced risk for progression to AIDS after infection. This risk reduction is 58% in the first 4 years after HIV seroconversion, followed by a 22% reduction in the next 4 years with no reduction after that.^7,10 Finally, the SDF-1-3′A polymorphism (Rs1801157) at the 3′ untranslated region of the SDF-1 gene, which codes for the unique ligand of the CXCR4 receptor, has been shown to be associated with delayed progression to AIDS in homozygous mutant individuals,^7,10,12 and prenatal transmission of HIV-1 in heterozygous pregnant women.¹³

The above mutations have been widely studied and their global distribution was found to be variable among different populations. For example, the CCR5-Δ32 is highly prevalent in Europeans (ranges from 10% to 20%) and Ashkenazi Jews (20.93%).^10,14 The aim of the present study was to determine the frequencies of CCR5-Δ32, CCR2-64I, and SDF-1-3′A polymorphisms in the Jordanian population.

Materials and Methods

Subjects

A total of 540 healthy Jordanian subjects were randomly selected from all the geographic areas of Jordan. Subjects were divided into three groups according to their region: (1) South, with 176 subjects, includes Al-Aqaba, Ma'an, Al-Tafehla, and Al-Karak provinces, (2) Middle, with 212 subjects, includes Amman, Zarqa, Al-Balqa, and Madaba provinces, and (3) North, with 152 subjects, includes Irbid, Ajloun, Jerash, and Al-Mafraq provinces. Written informed consents were obtained from all participants in accordance with the requirements of the Institutional Review Board (IRB) of the Jordan University of Science and Technology.

DNA extraction

The genomic DNA isolation kit (Promega, Madison, WI) was used to extract DNA from EDTA blood samples as described by the manufacturer. The concentration of extracted DNA was measured using a spectrophotometer. DNA samples were stored at −20°C until used.

Genotyping of genes polymorphisms

To analyze the CCR-5Δ32 polymorphism, a polymerase chain reaction (PCR) was carried out to amplify the target DNA using the following pair of primers: forward 5′-ACC AGA TCT CAA AAA GAA GGT CT-3′ and reverse 5′-CAT GAT GGT GAA GAT AAG CCT CAC A-3′ as previously described¹⁰ in a 25 μl reaction that contains GoTaq green master mix (Promega), 0.5 μM of each primer, 5 ng of genomic DNA, and nuclease free water. The reaction mix was subject to 35 cycles: initial denaturation at 94°C for 3 min, denaturation at 94°C for 1 min, annealing at 55°C for 1 min, extension at 72°C for 1 min, and final extension at 72°C for 10 min. The PCR product was then separated on 3% agarose gel stained with ethidium bromide (Sigma-Aldrich Corp.) and visualized under UV light. Positive samples were sequenced as previously described¹⁵ using an ABI Prism 3.1 automated sequencer (Applied Biosystems, Foster City, CA).

For genotyping of CCR2-64I, the PCR restriction fragment length polymorphism method (PCR-RFLP) was used as previously described¹⁰ using FokI restriction enzyme (Fermentas, Germany). The digested product was separated on 2% agarose gel stained with ethidium bromide and visualized under UV light; the wild-type allele remained as one undigested 379-bp fragment while the mutant allele gave two fragments of 164 bp and 215 bp.

SDF1 was also genotyped by using the PCR-RFLP method as described previously¹⁰ using the MspI restriction enzyme (Fermentas, GmbH, St. Leon-Rot, Germany). The wild-type allele gives two fragments of 205 bp and 106 bp while the mutant allele remains as one undigested 311-bp fragment.

Statistical analysis

Data analysis was carried out using the statistical package for social studies (SPSS version 17.0, Chicago, IL). Genotype distribution and allele frequencies were evaluated using the Chi square test. A p value<0.05 was considered to be statistically significant.

Results

A total of 540 subjects volunteered in the study from different regions of Jordan. The distribution of subjects was 36.5% from the south, 39.3% from the middle, and 28.3 from the north of Jordan. Of the 540 participants, 100% were born in Jordan and 51.8% were males. The mean age of sample was 30.5±8.5 years. No significant differences in the demographic variables were observed among groups from different regions (data not shown).

Table 1 shows genotypes and allele frequencies of the CCR5-Δ32 polymorphism in the studied sample. The frequency of the CCR5-Δ32 allele was 0.6% and was present in the heterozygous genotype only. In addition, no significant difference was found in the distribution of this variant among different geographic regions of Jordan (Table 1). To confirm the deletion, all heterozygote samples were sequenced. A 100% correlation was noticed between the results of the two methods. The CCR5-Δ32 genotypic distribution was found to be consistent with Hardy–Weinberg equilibrium (p=0.955).

Table 1.

Frequencies of Genotypes and Alleles of the CCR5-Δ32 Polymorphism in the Studied Sample

	CCR5: +/+		CCR5: +/Δ32		CCR5: Δ32/Δ32		CCR5: + allele		CCR5: Δ32 allele
Geographic area	N	%	N	%	N	%	N	%	N	%
Middle	209	98.6	3	1.4	0	0	421	99.3	3	0.7
South	175	99.4	1	0.6	0	0	351	99.7	1	0.3
North	150	98.7	2	1.3	0	0	302	99.3	2	0.7
Total	534	98.9	6	1.1	0	0	1074	99.4	6	0.6
p value						0.702^a				0.703^b

Calculated p value for genotype frequency.

Calculated p value for allele frequency.

Person chi square test.

Genotypes and allele frequencies of the CCR2-64I polymorphism among Jordanians are illustrated in Table 2. The G/G genotype refers to the homozygous wild-type genotype (CCR2+/+) while G/A and A/A are for the heterozygous (CCR2 +/64I) and homozygous (CCR2 64I/64I) genotypes, respectively. The frequency of the G allele among Jordanians was 82.5% compared to 17.5% for the A allele. When data were analyzed according to different regions, the A allele was more abundant in the middle region (20.8%) compared to other regions (15.1–15.8%). However, the difference in the distribution of this variant among different regions was not statistically significant (p>0.05, Table 2). In Hardy–Weinberg assay, both alleles were in equilibrium in the total population (p=0.550).

Table 2.

Frequencies of Genotypes and Alleles of the CCR2-64I Polymorphism in the Studied Sample

	G/G		G/A		G/A		G allele		A allele
Geographic area	N	%	N	%	N	%	N	%	N	%
Middle	128	60.4	80	37.7	4	1.9	336	79.2	88	20.8
South	128	72.7	43	24.4	5	2.8	229	84.9	53	15.1
North	107	70.4	42	27.6	3	2.0	256	84.2	48	15.8
Total	363	67.2	165	30.6	12	2.2	891	82.5	189	17.5
p value						0.058^a				0.075^b

Calculated p value for genotype frequency.

Calculated p value for allele frequency.

Person chi square test.

The SDF1-3′A polymorphism is highly enriched in the Jordanian population with a frequency of 34.2% of the mutant A allele and 65.8% of the G wild-type allele (Table 3). The allelic frequencies of this variant are similar in the different regions of Jordan (range: 33.5–35.5%, p>0.05). Similar to other variants, the distribution of SDF1-3′A genotypes is consistent with Hardy–Weinberg equilibrium in the total population.

Table 3.

Frequencies of Genotypes and Alleles of the SDF1-3′A Polymorphism in the Studied Sample

	G/G		G/A		A/A		G allele		A allele
Geographic area	N	%	N	%	N	%	N	%	N	%
Middle	86	40.6	110	51.9	16	7.5	282	66.5	142	33.5
South	54	30.7	119	67.6	3	1.7	227	64.5	125	35.5
North	55	36.2	92	60.5	5	3.3	202	66.4	102	33.6
Total	195	36.1	321	59.4	24	4.4	711	65.8	369	34.2
p value						0.10^a				0.811^b

Calculated p value for genotype frequency.

Calculated p value for allele frequency.

Person chi square test.

Discussion

Following the reporting the CCR5-Δ32, CCR2-64I, and SDF1-3′A polymorphisms and their roles in HIV infection, progression to AIDS, and treatment response, a number of studies were carried out to find their frequencies worldwide.¹⁶ Therefore, this study came to comprehensively investigate the frequencies of these polymorphisms in Jordan.

The polymorphism CCR5-Δ32, which has a protective effect against HIV in homozygous mutant form and a delaying effect in heterozygous form, is enriched in Europe.¹⁷ In Arabic countries, the CCR5-Δ32 allele frequency is very low. For example, the reported prevalence of CCR5-Δ32 is <1% in Syria and was absent in Lebanon.^5,18,19 In countries such as Bahrain, Iraq, Kuwait, and Saudi Arabia, the prevalence is approximately equivalent with a distribution of CCR5-Δ32 between 2% and 3%.⁵ The results of the current study showed that the frequency of CCR5-Δ32 in Jordan is 0.6%. In a previous report that considered 50 Jordanians, CCR5-Δ32 was not detected in the Jordanian population.¹⁹ Our results rank Jordan with Egypt with 0.6%⁵ at the lowest frequencies among Arabic countries. Thus, the result presented in this study did not reveal any deviation from what was reported in Arabic countries.

The enrichment of the CCR5-Δ32 allele in European populations suggests that this polymorphism most likely originated in Europe and then spread globally under natural positive selection.¹⁷ Based on this hypothesis, Lebanon, Syria, and Jordan are supposed to have high frequencies compared to other Arabic populations since the admixture with European was very high in these countries. The fact that the CCR5-Δ32 allele is rare in Lebanon, Syria, and Jordan, provides evidence against this hypothesis. The unequal CCR-Δ32 global distribution suggests other unidentified protective mutations in the CCR5 gene among populations in which the CCR-Δ32 allele is absent or has a very low frequency. Identification of such variants will be of great importance both scientifically and clinically.

The chemokine receptor CCR2, on the other hand, modulates HIV infection by delaying disease progression rather than affecting the initial HIV transmission. Although this polymorphism, which substitutes Val with Ile (CCR2-64I), does not change the CCR2 structure and function, both homozygous and heterozygous states are associated with the delay in disease progression. The allele is distributed all over the world and is never inherited together with CCR5-Δ32 because they occur on separate haplotypes.²⁰ The distribution of the CCR2 polymorphism in Arabic countries including Jordan is still lacking. In the present study the frequency of the CCR2-64I allele among Jordanians was 17.5%. This is higher than that reported in Bahrain (8.9%) and is close to that reported in Asians (25%).²⁰

The third gene in this study is the SDF-1, which codes for the ligand of the orphan CXCR4 chemokine receptor.²¹ The effect of this variant occurs during the advanced stages of AIDS when the virus shifts strains from R5 to X4. During disease progression, the X4 strain utilizes CXCR4 as the main coreceptor and the excess amount of SDF-1 competes with the virus on the receptors,^10,22 thus delaying its effect. Analysis of the allele frequencies in our sample revealed that the prevalence of SDF-1-3′A among Jordanians is 34.2%. In other populations such as Brazilian, Hungarian, Chinese, Polish, and Indian, the distribution of this variant ranges between 14% and 40%.^23

–28 Thus, distribution of this variant in the Jordanian population is within the range described in other countries.

Jordan is a small country located in Southwest Asia with total population of 6.13 million as of the 2010 census. The population is predominantly Arab (98%) and is originally from Jordan and Palestine with roughly equal percentages. Since Jordanians and Palestinians share a ommon origin, we do not expect a significant change in the population homogeneity due to migration of Palestinians to Jordan in 1948. The participants of the current study were randomly recruited from different parts of Jordan irrespective of their parental origin. The results did not show significant variations in the allelic and genotypic distribution of the examined polymorphisms among different regions of Jordan (South, Middle, and North). Thus, these results support the homogeneity of the Jordanian population.

It is worth mentioning that the role of the CCR5-Δ32, CCR2-64I, and SDF1-3′A polymorphisms in HIV resistance and progression to AIDS might vary among different populations. For example, a positive association between the CCR5-Δ32 polymorphism and HIV resistance and/or progression to AIDS has been reported in Italian and Australian populations.^29,30 On the other hand, lack of association with this polymorphism has been reported in Polish and Spanish populations.^31,32 Similarly, an association between the CCR2-64I polymorphism and progression to AIDS has been shown in Brazilian and Chinese populations^23,33 but not in Georgian and Kenyan populations.^34,35 For SDF1-3′A, controversial associations with this polymorphism hves been reported.³⁶ Thus, associations between examined polymorphisms and HIV infection and progression of the disease might have a population-specific component, being affected by the population-specific gene pool as well as by gene–environment interactions.

In conclusion, the CCR5-Δ32 allele is rare whereas the CCR2-64I and SDF1-3′A alleles are common among Jordanians. The role of these variants in HIV infection and progression to AIDS in the Jordanian population needs more investigation.

Footnotes

Acknowledgment

This project was supported by grant 38/2010 from the Deanship of Research at the Jordan University of Science and Technology.

Author Disclosure Statement

No competing financial interests exist.

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