Collection of Samples and DNA Isolation. Forty-three different ethnic, endogamous, Indian populations without any known history of HIV-1 infection were selected on a random basis from all over India (Table 1), which were then subjected to the present study. Ethnic and regional bias within the studied population was minimized by excluding subjects outside the castes and tribes of particular regions of India. Fresh blood samples from 1,882 healthy, unrelated individuals were collected from 15 different states of India after approval from institutional ethical and biosafety committees and written consent from the individuals concerned. Genomic DNA was isolated from 10 mL EDTA treated venous blood samples using the standard proteinase K digestion and phenol-chloroform extraction method [27].

Amplification of the CCR5-∆ 32 Regions. The primers to amplify the target segment for the 32 bp deletion of the CCR5 gene (spanning from position 3245 to 3685 with reference to NCBI sequence ID no. U95626) were designed using Gene Tool software (BioTools Inc., Edmonton, Alberta, Canada). The sequences of the primers were: 5’-GCT GTC GTC CAT GCT GTG TTT-3’ (forward primer) and 5’-CAA CCT GTT AGA GCT ACT GCA ATT-3’ (reverse primer), and these were commercially synthesized by Bioserve Biotechnologies India Pvt. Ltd., Hyderabad, Andhra Pradesh, India. The polymerase chain reaction (PCR) was carried out in 20 µL reaction volume using 10X PCR buffer (Applied Biosystems Corporation, Foster City, CA, USA), 25 mM MgCl₂ (Applied Biosystems Corporation), 5 mM dNTP mix (Eppendorf, Hamburg, Germany), 5 pM each of forward and reverse primers, 2 units of DFS (DNA free sensitive)-Taq DNA polymerase (Bioron GmbH, Ludwigshafen, Germany) and 40 ng of genomic DNA. Amplification was carried out for 35 cycles in an Eppendorf Mastercycler EP (Eppendorf), each consisting of denaturation at 95°C for 30 sec, annealing at 62.6°C for 30 sec and extension at 72°C for 30 sec. Pre denaturation and final extension were performed at 95°C for 10 min and 72°C for 10 min, respectively.

Amplified PCR products from wild type and homozygous mutant type individuals were expected as single bands of 441 and 409 bp, respectively. The PCR products from heterozygous individuals was expected to contain two fragments of 441 and 409 bp lengths. The PCR products were electrophoresed (Figure 1b) on a 4% Tris-acetate-EDTA agarose gel (Sigma-Aldrich Corporation, St. Louis, MO, USA) and visualized by staining with ethidium bromide along with 50 and 100 bp ladders (New England BioLabs, Ipswich, MA, USA) under an UV-transilluminator.

Genotyping for the CCR5-∆32 Polymorphism. To reconfirm the results observed on gel for CCR5-∆32 polymorphism, PCR products were sequenced directly on both strands as described elsewhere [27] using 25 ng of PCR product and 5 pM of forward primer, sequencing buffer and big dye (ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit; Applied Biosystems Corporation) and the volume adjusted to 5.0 µL/reaction. Cycle sequencing was carried out in an Eppendorf Mastercycler EP (Eppendorf) employing 30 cycles at 95°C for 10 sec, 55°C for 5 sec, and 60°C for 4 min. Extended products were purified by absolute alcohol precipitation followed by washing with 70% alcohol. Purified samples were dissolved in 50% Hi-Di™ formamide (Applied Biosystems Corporation) and analyzed in an ABI PRISM® 3700 automated DNA Analyzer (Applied Biosystems Corporation). Sequences were assembled using Auto Assembler sequence assembly software (Applied Biosystems Division, Perkin Elmer, Foster City, CA, USA) with a reference sequence and any type of heterogeneity was checked manually and was noted down (Figure 1c and 1d).

Latitude-Wise Distribution of the CCR5-∆32 Allele in India. To examine the trend in variation of CCR5-∆32 allele frequencies from North to South in India, we plotted (Figure 2) ∆32 allele frequencies (percentage) against latitude (in degrees), reported in Table 1, for each geographical site of origin of the study population. To further verify our results we also plotted graphs (Figure 3) between mean allelic frequencies of CCR5-∆32 between North and South India and the significance was checked by the χ2 test.

Statistical Analyses. The data obtained was then subjected to statistical analyses. Genotypes observed for CCR5-∆32 were categorized into wild type, and heterozygous or homozygous mutant for each population. Allele frequencies of CCR5 (p) and CCR5-∆32 (q) were calculated using [(2H+h)/2N] formula where H, h and N represented the number of homozygous individuals, heterozygous individuals and the sample size of each population, respectively. The genotype distribution was tested for agreement with the Hardy-Weinberg equilibrium. The value of χ2 with associated p value for significance was calculated.