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Metabolic disorders and hypersensitivities affect tolerability and impact adherence to highly active antiretroviral therapy (HAART). A representative 2% agarose gel of restriction digestion products with MspI for ?482 of APOC3 promoter region genotyping. A representative 2% agarose gel of restriction digestion products with FokI for APOC3 ?455 genotyping. Repeated measures of analysis of APOC3 genotypes of study participants with CD4 and viral load parameters. Observed and expected allele and genotype frequencies of APOC3 promoter region in South African subjects determined by the Hardy-Weinberg method.
These enzymes recognise specific sequences (called restriction sites) of DNA and digest (cut) the DNA at that specific site.
IntroductionThe clinical benefit of highly active antiretroviral therapy (HAART) is tempered with adverse drug events that have emerged as a major concern for long-term successful management of HIV infection [1]. SequencingSequencing of a subset (n = 12) of amplified products of the APOC3 promoter region was carried out to corroborate the RFLP results.
Hardy-Weinberg Equilibrium is not applicable if there are less than five events in the genotypic analysis. Frequencies of C-482 and T-455 polymorphisms in APOC3 were determined by restriction fragment length polymorphism analysis. One band indicates the absence of a restriction site; three bands indicate the presence of a restriction site.
Values are only for 53 patients for whom viral load and CD4+ cell count measurements were available.
POB is financially supported by the National Research Foundation, National Department of Health, and University of Venda.
Although HAART is implicated in the development of such disorders, there is considerable inter-individual variability in patient outcomes in terms of drug disposition, drug efficacy and adverse events.
No analysis was done for APOC3 ?482CT and TT genotypes as very few individuals were identified with these genotypes. In addition, factors such as host genetics and ethnicity are thought to play a significant role [1,5,6,7]. Apolipoprotein C3 gene is involved in transport and clearance of chylomicron remnants, and very low and high density lipoproteins from the bloodstream. There was no significant difference in the increase in CD4+ cell count irrespective of genotypes.


The human APOC3 is mapped to chromosome 11q23, closely linked to APOA1 and APOA4 genes [10].
Significant increases in CD4+ cell count were observed in males and females considering the ?455C genotype, but not in males for the ?455T genotype. The presence of polymorphisms at the insulin responsive element on the promoter region leads to over-expression of the protein [11]. Viral load decreases were significant with the ?455C and ?482C genotypes irrespective of gender. The APOC3 ?482 and ?455 promoter polymorphisms have reduced affinity for nuclear transcription factors mediating insulin responses associated with insulin resistance.
A change from thymine to cysteine at position ?455 is associated with increased triglyceride levels, a risk factor for cardiovascular disease. The apparently high prevalence of APOC3 T-455CC genotype needs confirmation with a larger samples size and triglyceride measurements to support screening of patients to pre-empt HAART associated lipid disorders.
Dyslipidemia and lipodystrophy occur in some HIV-infected patients despite similar drug exposures and comparable demographic, immunologic and virologic characteristics.
However, non nucleoside reverse transcriptase inhibitors (NRTIs), such as stavudine and didanosine, exhibit a number of safety concerns that limit their clinical use and these drugs are associated with the development of peripheral neuropathy, dyslipidemia and lactic acidosis [15].Pathogenesis of a number of drug hypersensitivity reactions involves restricted presentation of drugs to major histocompatibility complex (MHC) molecules prior to T cell presentation [16]. Statistical AnalysesThere was no significant difference in the increase in CD4+ cell count and decrease in plasma viral load among patients irrespective of APOC3 ?482 (CC, CT and TT) and APOC3 ?455 (TT, TC and CC) genotypes. Using the Tukey’s Studentized Range Test at the 0.05 confidence level, significant increases in CD4+ cell count were observed over 6 months in males and females for the APOC3 ?455C genotype, while significant increases in CD4+ cell count was observed among females and not in males for the APOC3 ?455T genotype. Alleles of MHC and their frequencies in different racial groups are implicated in the development of hypersensitive reactions in patients on abacavir and nevirapine within the first six weeks of treatment [17,18,19].
Viral load decreases were significant for both males and females when the APOC3 ?455C genotype was considered.
Although low frequencies of HLA-B*57:01 have been observed in other African populations, data on the prevalence of this allele in South African populations, particularly in the northern region with a different ethnicity is scanty. However, there was a significant range decrease in males and not in females with the APOC3 ?455T genotype.
Furthermore, there are several reports on the associations of variants of the APOC3 promoter region with the development of metabolic disorders [20], and the link of HLA-B*5701 allele with Abacavir hypersensitivity but little is known about the genetics of the population in northern South Africa in this regard. For the ?482C genotypes there were significant range increases in CD4+ cell counts and range decreases in viral load irrespective of gender at the 0.05 confidence level.
APOC3 Promoter Region Amplification and RFLP ResultsAmplified products were obtained for all the subjects, but RFLP results were available for 199 samples (n = 206).


For T-455 site (FokI), TT (homozygous, wild), TC (heterozygous), CC (homozygous) (Figure 1 and Figure 2).
Study Population and DNA ExtractionThe study population comprised a cohort of black South African patients recruited from two AIDS treatments sites in northern South Africa. The experiments were undertaken with the understanding and written consent of each subject, and that the study conforms to the Code of Ethics of the World Medical Association (Declaration of Helsinki). Details on the study demographics, sample collection and genomic DNA extraction procedures have been described [37]. Genotyping of APOC3 Promoter RegionAmplified DNA was purified with a Qiagen DNA purification kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. Digestion products were resolved on 2% Ethidium bromide-stained agarose gels (Invitrogen, Carlsbad, CA, USA). Product sizes of 146 and 92 bp were expected for the wild type genotypes, and a 238 bp product for the variant type allele at the C-482 MspI restriction site.
For the T-455 FokI restriction site, product sizes of 238, 134 and 104 bp for the wild type allele, four bands for a heterozygous allele and a 238 bp product for the variant allele were expected. Twelve representative DNA fragments were selected according to restriction profiles and sequenced on both strands with primers used in the PCR reaction on a BigDye Terminator Cycle Sequencer.
Test sequences and wild type reference sequences were aligned with the BioEdit programme [38] for identification of nucleic acid variations. Statistical AnalysesData was analyzed using the SAS statistical analysis package (SAS Version 8, SAS Institute Inc., Cary, NC, USA) and the student t-test.
Analysis of variance (ANOVA) and Tukey’s test were used for multiple comparisons; Mauchly’s sphericity test was used for covariance determination (available in SAS statistical analysis package version 8). The sample-size dependent standard error of alleles was calculated in terms of 95% confidence intervals of the estimates. Chi-square goodness-of-fit was used to verify the agreement of the observed genotype frequencies with those expected under the Hardy-Weinberg equilibrium [39].



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