English abstract
The acquired immunodeficiency syndrome (AIDS) is a major global health concern caused by infection with human immunodeficiency virus (HIV). A growing body of evidence supports that the genetic makeup of an individual may influence the HIV/AIDS status and the extent of immune recovery following treatment. C-C motif chemokine receptor-5 (CCR5) is a co-receptor for the R5 strain of HIV-1. A number of polymorphisms were identified within the promoter and coding regions of the CCR5 gene, some of which have been found to affect the protein expression, receptor function and modulate HIV-1 disease progression. Because of this prominent role, variations in this gene have been under differential pressures. Although several CCRS polymorphisms were shown to vary widely in their distribution among different ethnic populations, there has been no study addressing the potential variants of the CCR5 gene in the Omani population. The present study aimed to describe the CCR5 gene and identify the polymorphic sites that exist within the CCR5 gene in Omanis and compare the genetic diversity patterns with other populations. Another aim of the project was to investigate the presence of any association between a particular CCR5 haplotype allele and AIDS pathogenic markers as well as the degree of CD4+ T cell recovery in AIDS patients on combination antiretroviral therapy (CART). Peripheral blood samples were collected from 115 Omani adults, and genomic DNA was extracted to amplify a continuous region of around 4.67 kb of the CCR5 gene by polymerase chain reaction. The amplified DNA was then sequenced to identify the polymorphic sites in the CCR5 gene and the known mutation CCR2V641. New variants were confirmed using the TOPO TA cloning protocol. Bioinformatics tools were used to analyze the nucleotide variants. Furthermore, the distribution of the detected variants was examined and compared with the previously published data. Arlequin v.3,5 package was used to estimate heterozygosity and calculate pairwise linkage disequilibrium between pairs of nucleotide variants. Haplotypes were constructed using Dna$P v5 and POPTREE software was used to generate the phylogenetic tree. Relevant information about the patient's status was retrieved from the electronic database of the Sultan Qaboos University Hospital. Finally, AIDS parameters were assessed for association; mean age, nadir CD4* T cell count, average CD4+ T cell count, average CD8+ T cell count and average viral load among the different groups of AIDS patients on CART. These parameters were also evaluated for any association with CCR5 haplotypes. Four new indels were detected out of 32 variable positions, -2973A/-, -2894A/-, -2827TA/- and -2769T/-, and all were located in the 5'UTR. Furthermore, two new mutations, -2248G/A and +658A/G were observed for the first time; the -2248G/A was detected in the Intron I region in one individual and +658A/G in the coding region of the CCR5 in another individual. In silico analysis showed that the novel variations in the 5'UTR could affect the transcription factor binding sites. Moreover, the results demonstrated that the following four minor alleles were common: CCR5 25547 and CCR5-2086G occurred at a frequency of 49% and 46%, respectively, and CCR5-24594 and CCR5-2135C both existed at a frequency of 36%. These alleles had moderate heterozygosity levels indicating that they were under balancing selection. However, the widely known allele, CCR5432, was relatively rare in our sample population. Eleven human haplogroups (HH) were constructed, four of which were common: HHC (46%), HHE (20%), HHA (14%) and HHF*2 (12%). AIDS
patients were grouped based on their average CD4* T cell count and viral load levels into three different groups: immunological responders (IR), immunological non responders (INR) and viremic group. Only average CD4* T cell count was significantly higher among IR compared to INR (P = 0.005), but no significant relation with the viremic group was observed. The carriers of the HHC allele had significantly high average CD4+ T cell and CD8+ T cell counts (P = 0.022 and 0.045 respectively), compared to individuals who do not carry the HHC allele. The effect of HHC was independent and remained significant even after adjusting for all the other CCR5 haplotypes. This suggests a possible protective role played by this allele on HIV-1 disease course. The results also showed that HHE was significantly associated with lower average viral load levels (P = 0.007), indicating a protective role of the HHE. Omanis have distinct variations of the CCR5 gene, allele distribution and associations with HIV/AIDS parameters compared to other ethnic populations. These findings indicate that the CCR5 is an important genetic factor that may contribute to the HIV-1 replication and cell-mediated immunity that may ultimately affect the HIV/AIDS status of the Omani AIDS patients. Furthermore, this information can be used to develop guidelines that provide early treatment interventions for HIV-1 infected Omani patients at risk of developing immune recovery failure following CART.
