The study cohort consisted of four-hundred and sixty-four (n=464) Bantu-speaking South Africans made up of healthy subjects (n=163) and HIV/AIDS patients (n=301) undergoing efavirenz-based treatment for at least six months. The subjects were recruited from Gauteng and Cape Town. Written informed consent was obtained and each participant provided demographic information such as 1) their ethnic group, 2) health status, 3) dietary habits, 4) smoking habits, and 5) home language were captured using a questionnaire. The study was approved by the Research Ethics Committee of the Faculty of Health Sciences at the University of Cape Town and the University of Witwatersrand Human Research Ethics Committee, Gauteng, South Africa and was performed in accordance with the guidelines of the Helsinki Declaration of 2008.
Two blood samples were obtained for DNA extraction and plasma efavirenz levels, respectively. DNA isolation was performed according to the method adapted from Gustafson et al., or the GenEluteTM Blood Genomic DNA Kit (Sigma-Aldrich, St. Louis, MO, USA) was used when blood sample volumes were limited. Steady state efavirenz plasma levels were available for 137 of the 301 HIV/AIDS patients and were collected 12–16 hours post-dose. Efavirenz concentrations were determined by the use of LC/MS/MS (API 4000 triple quadrupole MS/MS Applied Biosystems, South Africa) according to the method by Chi et al.,
Selection of SNPs and genotyping methods used
Three SNPs in NR1I2 [GenBank: AF364606] and a further three SNPs in NR1I3 [GenBank: BC069626.1] were investigated in this study. The six SNPs were selected based on previous reports of high minor allele frequencies in African-American and other African populations. SNPs were genotyped using either SNaPshot mini-sequencing or the PCR-RFLP method designed for NR1I2 rs2472677C>T (Additional file
1: Table S1).
PCR amplification was performed using the following conditions: initial denaturation at 94°C for 3 min, followed by 40 cycles of denaturation at 94°C for 30s, annealing at the specific temperature for each SNP for 30s, primer extension at 72°C for 20-45s depending on the primer sets and final extension at 72°C for 10 min. A “MyCycler Thermal cycler” (Bio-Rad, Hercules, USA) was used and the PCR reaction contained the following reagents; 50–100 ng of genomic DNA, 1X Green GoTaq Flexi Reaction Buffer (Promega Corporation, Madison, USA), 0.20 mM of each of the deoxynucleotide triphosphates (dNTPs) (Bioline, London, UK), 1.5 mM MgCl2 (Promega Corporation, Madison, USA), 40 pmol of the forward and reverse primers (Integrated DNA Technologies, Inc., Coralville, USA), 1U of GoTaq Flexi DNA Polymerase (Promega Corporation, Madison, USA). PCR amplification was followed by digestion using 3U Hpy188I (New England BioLabs, Inc., Ipswich) in the presence of 1X NEBuffer 4 (New England BioLabs, Inc., Ipswich) when genotyping for the NR1I2 rs2472677C>T polymorphism.
Five separate PCR amplification reactions were carried out. PCR products (5 μl) were then pooled for SNaPshot genotyping. The pooled PCR products were cleaned using 1.5U shrimp alkaline phosphatase (Fermentas Life Sciences, Burlington, Canada) and 2U ExonucleaseI (Fermentas Life Sciences, Burlington, Canada) to remove unincorporated primers and dNTPs. SNaPshot single base extension was performed using the “GeneAmp® PCR System 9700 version 3.08” (Applied Biosystems, Carlsbad, USA) under the following conditions; denaturation at 96°C for 10s, followed by 25 cycles of primer annealing at 50°C for 5s and primer extension at 60°C for 30s. To the 1 μl ABI Prism® SNaPshot™ Multiplex Kit (Applied Biosystems, California, USA), primers (Integrated DNA Technologies, Inc., Coralville, USA) for the pooled PCR products were added. The clean-up reaction was repeated using 1U shrimp alkaline phosphatase. An ABI 3130xl Genetic Analyzer (Applied Biosystems, Carlsbad, USA) was used for capillary electrophoresis and GeneMapper© Software version 4.1 (Applied Biosystems, Carlsbad, USA) was used to analyse results.
Identification of novel SNPs
The NR1I2 and NR1I3 DNA binding domains (DBDs) were sequenced in 32 of the 301 HIV/AIDS patients to search for novel SNPs. The sequencing reaction used the ABI Prism® BigDye® Terminator Cycle Sequencing v3.1 Kit (Applied Biosystems, Carlsbad, CA, USA), which included 1 μl Terminator mix and 1X Sequencing buffer, together with the PCR fragment, and 1 μM of the forward or reverse primer. Analysis of the sequencing data was performed using BioEdit Sequence Alignment Editor v7.0.0. The novel SNPs were assessed for functional significance with the Functional Analysis of Novel SNPs (FANS) program (
 and ESE finder v3.0.
Statistical analyses were performed using the Graphpad Prism statistical program (Version 5, GraphPad Software Inc., San Diego, CA), Statistica v10.0 (StatSoft, USA) and Phase v2.1
[17–19]. Pearson’s χ
2-test and Fisher’s exact test was used to compare the genotype and allele frequencies between the healthy participants and the HIV/AIDS patients as well as the allele frequencies in the South Africans to those of other populations with results in literature. The SHEsis statistical program was used for linkage disequilibrium (LD, D’ and r2)
[20, 21] analysis and Phase v2.1 for inferring of NR1I2 and NR1I3 haplotypes. Statistical significance was defined as P <0.05 and all statistical tests were performed two tailed.