STUDY OF VARIOUS METHODS FOR DETECTING HLA-B* 57:01 ALLELE IN PLHIV IN EASTERN UP, INDIA.

Abstract

Intro: Abacavir is known to cause fatal hypersensitivity reactions (HSR) in 5-8% of patients. This HSR is linked to the HLA-B*57:01 allele. The gold standard methodology for detecting the HLA-B*57:01 allele is the sequence-based method, which is difficult and expensive in programmatic conditions. HLA complex P5 gene (HCP5) shows single nucleotide polymorphism (SNP). In different populations HCP5-G (minor) allele has been shown to be 99.9% predictive of the presence of the HLA-B*57:01 allele, with a perfect Linkage Disequilibrium (LD). Therefore, in this study, we have compared different methods like direct PCR followed by nested PCR, HCP-5 PCR, and a sequence- based method to develop a simple and cost-effective method for detection of the HLA-B*57:01 allele. Methods: We collected whole blood cells from 366 PLHIV in the EDTA vials and buffy coat was separated by centrifugation followed by DNA isolation by QIAamp DNA Blood Mini Kit (QIAGEN, Hilden, Germany). Direct PCR followed by nested PCR and HCP5 PCR were used to detect HLA-B*57:01 allele and a sequence- based technique was used to confirm the results. Findings: Out of 366 samples, 25 (6.83%) were found positive for HLA-B*57 gene by direct PCR. All positive samples were subsequently analysed for HLA- B*57:01 using Nested PCR and sequenced and were found to be positive. All 366 subjects were also tested for the T/G Polymorphism (rs2395029) by PCR. 25 patients were found to be positive for (T/G) heterozygous (mutant) and they were also positive by nested PCR and Sequencing and suggests 100% LD between HCP5 and HLA-B*57:01 allele. Discussion: This is the first research in eastern UP that uses nested PCR followed by direct PCR and HCP5 for HLA-B*5701 allele detection. The outcome of these two methods was confirmed by SBT. Conclusion: we concluded that detection of HCP5 gene by PCR is a simple and cost-effective way of identifying the HLA-B*57:01 in Indian population.