African-based CCR5 single-nucleotide polymorphism associated with HIV-1 disease progression

The CC chemokine receptor 5 (CCR5), a seven transmembrane G-coupled protein, is one of the major co-receptors on CD4 T lymphocytes used for cell entry during HIV-1 infection [1–3]. A 32 base pair (bp) deletion (CCR5Δ32), resulting in a defective CCR5 protein, was identified as reducing the risk of HIV-1 infection in the homozygous state, while offering partial resistance to infection or reducing the rate of disease progression to AIDS and death in the heterozygous state [4–7]. This polymorphism has been reported to occur at allelic frequencies of 0.092 in Caucasians, and is extremely rare in populations of African ethnic origin [4,7,8]. In a previous study [9], we found CCR5Δ32 to be absent (0/268 alleles) in our African population (predominantly Xhosa from central African descent) and present at an allele frequency of 0.03 (6/196 alleles) in our coloured population (mixed ancestral descent, including San, Khoi, African negro, Madagascan, Javanese and European origin) from South Africa. A number of mutations have been identified in the promoter and coding regions of CCR5, which may influence gene regulation or protein function. Two African-based single nucleotide polymorphisms (SNP) in the coding region of CCR5 have been identified by ourselves and others, namely P35 [9] and A335V [10]. Although a recent study of a large Ugandan population showed no association between these polymorphic sites and either HIV-1 infection or the rate of disease progression (P35 occurred at an allelic frequency less than 0.01 within this population) [11], African-based studies have been limited to date. We used these SNPs to identify possible disease association in two African-based populations residing in the Western Cape of South Africa. In an African setting, where clinic attendance is hampered by factors such as vast distances, economic burdens and a lack of patient education, it is very difficult to classify patients accurately into appropriate groups according to disease progression. Out of a study cohort of 1035 HIV-seropositive individuals, 76 (7%) could be classified into 35 normal (22 African, nine coloured, four Caucasian), 22 slow (seven African, eight coloured, seven Caucasian) and 19 fast progressors (11 African, five coloured, three Caucasian). Normal progressors were defined as individuals with a progressive loss in CD4 cell counts, who developed AIDS-related symptoms and co-infections within 10 years after HIV-1 infection. All received clinical monitoring for a minimum period of 5 years. Approximately 5% of HIV-1-infected individuals remain asymptomatic for more than 10 years after seroconversion with stable CD4 cell counts, and are known as long-term non-progressors (LTNP) [12,13], whereas others, namely the fast progressors, develop AIDS within 2–5 years after seroconversion. The criteria used to determine slow and fast progression in this study is based primarily on the length of time the patient remained asymptomatic after seroconversion, as defined in Table 1. Only 17% (13/76) had received some form of antiretroviral treatment.Table 1: Clinical classification of the 22 slow progressors and the 19 fast progressors included in this study.All individuals were screened for the P35 and A335V polymorphisms using the CCR5-denaturing gradient gel electrophoresis (DGGE) assay previously described by our group for fragments B and F, respectively. In addition, all slow progressors were screened for the CCR5Δ32 using CCR5–DGGE fragment D primer set and conditions [9]. Manual allele counting was used for calculating allele frequencies, and statistical significance was determined using Fisher's exact test for 2 × 2 contingency tables (InStat version 3.0). None of the 14 Caucasians analysed were found to have either P35 or A335V and therefore, because of the African origin of the markers, were excluded from further calculations. We first described the SNP occurring at codon 35 and resulting in a silent mutation as an African-based polymorphism, occurring at a significantly increased allelic frequency in coloured HIV-seropositive individuals compared with HIV-seronegative controls, whereas similar frequencies were observed within infected versus non-infected Africans [9]. Increasing our study population we found no statistically significant differences between infected (17/164 alleles, 0.104) and non-infected (13/196 alleles, 0.066) coloured individuals (P = 0.2511). In addition, no statistically significant differences were found when comparing slow versus fast and slow versus normal progressors. When comparing allelic frequencies of the codon 335 polymorphism, which results in a conservative amino acid change from an alanine to a valine, statistically significant differences were observed between both slow (4/30 alleles) versus fast (0/32 alleles) progressors (P = 0.0491) and slow versus normal (1/62 alleles) progressors (P = 0.0374) within the African-based population groups (African and coloured). The CCR5-A335V SNP was found to occur homozygously in a single African woman who, remaining asymptomatic 7 years after infection, was classified as a ‘possible’ LTNP with a CD4 cell count of 450 cells/mm3 and recent (past year) antiretroviral treatment. Two slow progressors, one ‘true’ LTNP coloured man (asymptomatic 14 years after infection; CD4 cell count 610 cells/mm3) and one ‘possible’ LTNP coloured woman (asymptomatic 6 years after infection, CD4 cell count 914 cells/mm3), were heterozygous for this SNP, neither having received any previous antiretroviral treatment. All slow progressors were screened for the CCR5Δ32 mutation so as to exclude its effect on disease progression. Two of the 15, one coloured woman remaining asymtomatic 10 years after infection (CD4 cell count 728 cells/mm3) and one coloured man remaining asymtomatic 8 years after infection (CD4 cell count 1171 cells/mm3), were heterozygous for the CCR5Δ32 mutation. Neither presented with the CCR5-A335V SNP. In this study, we show a statistically significant association between the CCR5-A335V SNP and decreased disease progression from HIV infection to AIDS within individuals of African ethnic origin residing in the Western Cape of South Africa. The variable penetrance observed for this protective association, together with previous findings that this polymorphic marker results in a functional response similar to that of wild-type CCR5 [14], suggests that A335V does not contribute directly in slowing disease progression. We therefore hypothesize that this genetic marker, in combination with other weaker genetic events, collectively acts in slowing disease progression and may ultimately contribute to providing an individual HIV susceptibility risk profile for sub-Saharan Africans.