The rate of progression of HIV-1 disease exhibits a remarkable variation among different individuals. Many host genetic factors are now known to affect disease progression rates, especially polymorphisms in genes encoding chemokine receptors [1–4]. One member of this receptor family, CCR5, is the co-receptor for the most commonly transmitted strains of HIV-1 [5–9]. A deletion of 32 nucleotides in the CCR5 gene (denoted CCR5-Δ32) results in a truncated protein that is not expressed on the cell surface; individuals homozygous for this deletion have an absolute resistance to infection by CCR5-using (R5) HIV-1 variants [10–14], although infection by CXCR4-using (X4) strains can occur infrequently [15–17]. Heterozygotes for CCR5-Δ32 do not resist HIV-1 infection but progress more slowly (by approximately 2 years) to AIDS [10, 11,14,18–20]. Another mutation in a closely linked chemokine receptor gene, CCR2, also affects HIV-1 disease progression. This mutation, denoted CCR2-64I, is a G to A substitution that results in a replacement of valine with isoleucine at position 64 of the CCR2 protein . The homozygous CCR2-64I/64I phenotype has no effect on HIV-1 infection, but is associated with a delayed progression to disease. Heterozygotes for CCR2-64I do not progress more slowly to AIDS [21,22]. As the valine to isoleucine substitution is a conservative change in a protein that is rarely used as a HIV-1 co-receptor, this was an unexpected finding; indeed, no effects of the 64I substitution on CCR2 function have been found . Further analysis of the CCR5-CCR2 gene family showed that CCR2-64I was in strong linkage with a C to T mutation approximately 12 kb downstream in the regulatory region of the CCR5 gene (CCR5-59653T). This suggested that the CCR5-59653T mutation (or another unrecognized one with a similar degree of linkage) may be the one that affects HIV-1 disease progression, although this is not yet proven . The precise degree of linkage between CCR2-64I and CCR5-59653T has also not yet been established: initially, the association between CCR2-64I and CCR5-59653T was thought to be absolute , but exceptions have recently been reported . We address this issue here.
The samples studied include many of those in which we previously determined the distribution of CCR5-Δ32[25–28] and a series of further populations obtained as part of epidemiological surveys.
To genotype the three polymorphisms (CCR5-Δ32, CCR2-64I and CCR5-59653T) in each sample from all 54 populations, we used spectral genotyping . The spectral genotyping assays for CCR5-Δ32 and CCR2-64I have been described previously [22,29]. To genotype the CCR5-59653C/T polymorphism, we designed a new assay based on the general operational principle described previously . One molecular beacon, labeled with 6-carboxyfluorescein (FAM), recognizes the wild-type CCR5-59653C allele (position 59653 of GenBank file U95626) the other, labeled with hexachlorofluorescein (HEX), is for the CCR5-59653T genetic variant (C at position 59653). Their nucleotide sequences were respectively: FAM-5′-C C G C G G T T T G C C A A A T G T C T T C T A T C G C G G- 3′-DABCYL and HEX-5′-CCGCG GTTTGCAAAT ATCTTCTAT CGCGG-3′-DABCYL, where DAB CYL is the quencher 4-(4′-dimethylaminophenylazo) benzoic acid and underlined sequences indicate the complementary sequences forming the hairpin structure. The primers were 5′-CAGAAGAGCTGAGA CATCCGT-3′ and 5′-CATTCCAAACTGTGACC CTTTCC-3′. The real-time PCR conditions and data analysis methods have been described previously [22,29]. Each 50 μl PCR contained 0.5–5.0 μg of genomic DNA, 0.25 μM of each molecular beacon, 0.5 μM of each primer, 0.25 mM dATP, 0.25 mM dCTP, 0.25 mM dGTP, 0.25 mM dTTP, 2.5 U AmpliTaq Gold DNA polymerase (PE Biosystems, Foster City, California, USA), 50 mM KCl, 3.5 mM MgCl2, and 10 mM Tris–HCl (pH 8.3). Forty-five cycles of amplification (denaturation, 94°C for 15 s; annealing, 55°C for 30 s; polymerization 72°C for 30 s) were performed in a spectrofluorometric thermal cycler (ABI 7700; PE Biosystems). Those samples that had previously been typed for CCR5-Δ32 by methods involving PCR and agarose gel electrophoresis all yielded identical genotypes when the spectral genotyping assays were used. The high sensitivity of the spectral genotyping method allowed for some individuals to be genotyped who could not be typed by the original procedure.
The geographical distribution of CCR5-Δ32 is now well characterized. It is present at high frequencies (∼12–15%) in populations of northern European origin, and decreases in frequency in a southeast cline towards the Mediterranean [25–28]. Outside Europe and Northern America, it is seen at low frequencies (2–5%) in the Near East and India, and is absent elsewhere apart from isolated occurrences that are probably the result of recent European gene flow (i.e., into North America). The restricted distribution of CCR5-Δ32 indicates that the mutation arose relatively recently in human history [30,31]. The distributions of CCR2-64I and CCR5-59653T have been less thoroughly studied, although these alleles are more common in African–American and Hispanic populations than they are in Caucasian–Americans, in contrast with CCR5-Δ32[21,22,24]. Here we examine in detail the frequencies of CCR2-64I, CCR5-59653T and their combined haplotype in a globally distributed set of populations, including many that we have previously typed for CCR5-Δ32. We also include data on the distribution of CCR5-Δ32 in an additional set of populations.
Table 1 shows the distribution of CCR5-Δ32, CCR2-64I and CCR5-59653T worldwide. Individuals carrying the CCR2-64I and CCR5-59653T alleles can be seen in most of the world's populations. The contrast between the distribution of CCR5-Δ32 and that of CCR2-64I and CCR5-59653T is striking. The Europe-centered distribution of CCR5-Δ32 reported previously is upheld by the new data presented here, and it differs greatly from the globally ubiquitous presence of CCR2-64I and CCR5-59653T (Fig. 1). The frequencies of these genetic variants are highest (> 35%) in sub-Saharan African populations, but they are also observed in Asian populations. Slightly lower frequencies are present in Europeans and Caucasian–Americans, and the lowest are found in Pacific Islander populations.
Another striking feature of the distribution of CCR2-64I and CCR5-59653T is the close association between the allele frequencies of these two polymorphisms. Table 1 shows the congruence between the genotypes for these alleles; i.e., the number of times that an individual was either heterozygous or homozygous for both CCR2-64I and CCR5-59653T. Individuals who were only informative for one of the genotypes were excluded from the congruence calculations. In many populations the association is absolute, indicating a strong degree of linkage between the two polymorphisms. A total of 3341 individuals were genotyped for both polymorphisms: where the congruence was not absolute, in 33 cases an individual had a CCR2-64I allele but not a CCR5-59653T one (0.99%), and in 70 cases an individual had a CCR5-59653T allele but lacked CCR2-64I (2.09%). These observations are consistent with the genealogy proposed by Mummidi et al. in which the CCR5-59653T polymorphism arose first, and a later mutation on a CCR5-59653T chromosome gave rise to the CCR2-64I allele . The slightly greater prevalence of the CCR5-59653T chromosome reflects its ancestral status, while the low frequency of chromosomes bearing solely CCR2-64I is consistent with it being produced from a CCR2-64I/CCR5-59653T chromosome by recombination with a normal chromosome. Such a recombination event would also produce a chromosome carrying CCR5-59653T alone, which would account for some of the occurrences of this chromosome. Calculation of congruence between CCR5-Δ32 and either CCR2- 64I or CCR5-59653T showed no instances of crossing-over between these polymorphisms.
The degree to which the genotype frequencies at each locus agree with Hardy–Weinberg equilibrium (HWE) expectations is also shown in Table 1. This was determined at the regional level, and not for individual populations, as many of the individual samples were too small to allow the agreement with HWE to be calculated accurately. Agreement with HWE (P > 0.05) was seen in 16 out of 18 comparisons. In some cases, the expected values obtained for some genotype frequencies were so low as to introduce bias into the chi-square calculation , which may explain the disagreement seen at CCR5-Δ32 in Asian populations. The other exception (CCR5-59653T in the Americas) was due to an observed excess of homozygotes and a deficit of heterozygotes. The reason for this is not clear, but instead of reflecting an aspect of population structure, this could be a Type I error; a probability threshold of 0.05 would result in HWE being falsely rejected in one out of every 20 tests performed. We conclude that there is little or no selective or other effect of CCR2-64I or CCR5-59653T that is sufficient to perturb the genotype frequencies.
The global distribution of both CCR5-59653T and CCR2-64I indicates that these mutations are much older than CCR5-Δ32. The restricted distribution of CCR5-Δ32 has lead to the suggestion that it arose very recently; age estimates based on flanking STR haplotype analysis have ranged from 750 to 2500 years, although a precise date has yet to be generally accepted [30,31]. Certainly the CCR5-59653T and CCR2-64I mutations are much older than this. Their presence in virtually all of the world's populations would imply that these mutations arose prior to the dispersal of modern Homo sapiens from an African ancestral population more than 100 000 years ago. As human populations have only become exposed to HIV-1 within the past two or three generations at most, any resistance to HIV-1 disease progression conferred by any of these mutations cannot explain their persistence at polymorphic frequencies. It is possible for polymorphisms that have no selective advantage or disadvantage to persist for long periods of time, in which case their frequencies fluctuate randomly due to the stochastic processes of genetic drift. Under these circumstances, the allele frequencies would be expected to fluctuate between populations over a great range, from total absence in some populations to fixation in others. The majority of allele frequencies for CCR5-59653T and CCR2-64I are within a narrow range (10–30%); the persistence of this frequency in such a wide range of populations suggests that random genetic drift is not responsible for the pattern seen. The only exceptions to this are seen in the populations of Oceania, but these populations are known to have undergone a dramatic reduction in size in the recent past that has drastically affected their genetic composition in other ways .
It may be that one or other of the CCR5-59653T and CCR2-64I polymorphisms confers a selective advantage against other, unknown infectious agents. The precise nature of the interactions between chemokines, their receptors and the other components of the chemotactic response to infection is not well understood. The ability of the mutations described here to delay the progression of HIV-1 infection and disease implies a potential for the conferral of a selective advantage against other infectious organisms that activate these components of the immune response. As more chemokine receptor variants become identified and their roles characterized in detail, then our understanding of both the mechanism of HIV-1 infection and the long-term effects of natural selection in human populations will continue to improve.
The authors thank the many researchers who provided the samples for our original study of CCR5-Δ32, and to C. Christodoulou and A. Hatzakis for additional samples.
1. Moore JP. Coreceptors: implications for HIV pathogenesis and therapy.
Science 1997, 276: 51 –52.
2. Berger EA, Murphy PM, Farber MT. Chemokine receptors as HIV-1 coreceptors: Roles in viral entry, tropism, and disease.
Ann Rev Immunol 1999, 17: 657 –700.
3. Fauci AS. Host factors and the pathogenesis of HIV-induced disease.
Nature 1996, 384: 529 –534.
4. O'Brien TR, Goedert JJ. Chemokine receptors and genetic variability: another leap in HIV research.
J Am Med Assoc 1998, 279: 317 –318.
5. Alkhatib G, Combadiere C, Broder CC. et al
. CC CKR5: a RANTES, MIP-1alpha, MIP-1beta receptor as a fusion cofactor for macrophage-tropic HIV-1.
Science 1996, 272: 1955 –1958.
6. Choe H. et al
. The beta-chemokine receptors CCR3 and CCR5 facilitate infection by primary HIV-1 isolates.
Cell 1996, 85: 1135 –1148.
7. Deng HK, Unutmaz D, KewalRamani VN, Littman DR. Expression cloning of new receptors used by simian and human immunodeficiency viruses.
Nature 1997, 388: 296 –300.
8. Doranz BJ, Rucker J, Yi Y. et al
. A dual-tropic primary HIV-1 isolate that uses fusin and the beta-chemokine receptors CKR-5, CKR-3, and CKR-2b as fusion cofactors.
Cell 1996, 85: 1149 –1158.
9. Dragic T, Litwin V, Allaway GP. et al
. HIV-1 entry into CD4+ cells is mediated by the chemokine receptor CC-CKR-5.
Nature 1996, 381: 667 –673.
10. Dean M, Carrington M, Winkler C. et al
. Genetic restriction of HIV-1 infection and progression to AIDS by a deletion allele of the CKR5 structural gene.
Science 1996, 273: 1856 –1862.
11. Huang Y, Paxton WA, Wolinsky SM. et al
. The role of a mutant CCR5 allele in HIV-1 transmission and disease progression.
Nature Med 1996, 2: 1240 –1243.
12. Liu R, Paxton WA, Choe S. et al
. Homozygous defect in HIV-1 coreceptor accounts for resistance of some multiply-exposed individuals to HIV-1 infection.
Cell 1996, 86: 367 –377.
13. Samson M, Libert F, Doranz BJ. et al
. Resistance to HIV-1 infection in caucasian individuals bearing mutant alleles of the CCR-5 chemokine receptor gene.
Nature 1966, 382: 722 –725.
14. Ioannidis JP, O'Brien TR, Rosenberg PS, Contopoulos-Ioannidis DG, Goedert JJ. Genetic effects on HIV disease progression.
Nature Med 1998, 4: 536. 536.
15. O'Brien TR, Winkler C, Dean M. et al
. HIV-1 infection in a man homozygous for CCR5Δ32.
Lancet 1997, 349: 1219. 1219.
16. Theodorou I, Meyer L, Magierowska M, Katlama C, Rouzioux C. HIV-1 infection in an individual homozygous for CCR5Δ32.
Lancet 1997, 349: 1219 –1220.
17. Biti R, French R, Young J, Bennetts B, Stewart G. HIV-1 infection in an individual homozygous for the CCR5 deletion allele.
Nature Med. 1997, 3: 252 –253.
18. Michael NL, Chang G, Louie LG. et al
. The role of viral phenotype and CCR-5 gene defects in HIV-1 transmission and disease progression.
Nature Med 1997, 3: 338 –340.
19. Misrahi M, Teglas J-P, N'Go N. et al
. CCR5 chemokine receptor variant in HIV-1 mother-to-child transmission and disease progression in children.
J Am Med Assoc 1998, 279: 277 –280.
20. Zimmerman PA, Buckler-White A, Alkhatib G. et al
. Inherited resistance to HIV-1 conferred by an inactivating mutation in CC chemokine receptor 5: Studies in populations with contrasting clinical phenotypes, defined racial background, and quantified risk.
Mol Med 1997, 3: 23 –36.
21. Smith MW, Dean M, Carrington M. et al
. Contrasting genetic influence of CCR2 and CCR5 variants on HIV-1 infection and disease progression.
Science 1997, 277: 959 –965.
22. Kostrikis L, Huang X, Moore JP. et al
. A chemokine receptor CCR2 allele delays HIV-1 disease progression and is associated with a CCR5 promoter mutation.
Nature Med 1998, 4: 350 –353.
23. Lee B, Doranz BJ, Rana S. et al
. Influence of the CCR2-V64I polymorphism on human immunodeficiency virus type 1 coreceptor activity and on chemokine receptor function of CCR2b, CCR3, CCR5, and CXCR4. J Virol 1998, 72: 7450 –7458.
24. Mummidi S, Ahuja S, Gonzalez E. et al
. Genealogy of the CCR5 locus and chemokine system gene variants associated with altered rates of HIV-1 disease progression.
Nature Med 1998, 4: 786 –793.
25. Martinson JJ, Chapman NH, Rees DC, Liu YT, Clegg JB. Global distribution of the CCR5 gene 32-basepair deletion.
Nature Genet 1997, 16: 100 –103.
26. Christodoulou C, Poullikas M, Neumann AU, Kostrikis LG. Low frequency of CCR5Δ32 allele among Greeks in Cyprus.
AIDS Res Hum Retroviruses 1997, 13: 1373 –1374.
27. Nasioulas G, Dean M, Koumbarelis E. et al
. Allele frequency of the CCR5 mutant chemokine receptor in Greek Caucasians.
J Acquir Immune Defic Syndr Hum Retrovirol 1998, 17: 181 –182.
28. Lucotte G, Mercier G. Distribution of the CCR5 gene 32-bp deletion in Europe.
J Acquir Immune Defic Syndr Hum Retrovirol 1998, 19: 174 –177.
29. Kostrikis LG, Tyagi S, Mhlanga MM, Ho DD, Kramer FR. Spectral genotyping of human alleles.
Science 1998, 279: 1228 –1229.
30. Stephens JC, Reich DE, Goldstein DB. et al
. Dating the origin of the CCR5-Delta32 AIDS-resistance allele by the coalescence of haplotypes.
Am J Hum Genet 1998, 62: 1507 –1515.
31. Libert F, Cochaux P, Beckman G. et al
. The DCCR5 mutation conferring protection against HIV-1 in Caucasian populations has a single and recent origin in Northeastern Europe.
Hum Mol Genet 1998, 7: 399 –406.
32. Weir BS. Genetic Data Analysis.
Sunderland MA: Sinauer; 1990.
33. Flint J, Boyce AJ, Martinson JJ, Clegg JB. Population bottlenecks in Polynesia revealed by minisatellites.
Hum Genet 1989, 83: 257 –263.