Development of the High Resolution Melt (HRM) method to detect the HLA-B*58:01 allele in order to prevent allopurinol-associated drug hypersensitivityTools Nawoor, Anusha Devi (2022) Development of the High Resolution Melt (HRM) method to detect the HLA-B*58:01 allele in order to prevent allopurinol-associated drug hypersensitivity. PhD thesis, University of Nottingham.
AbstractThe HLA-B*58:01 allele was identified as a genetic marker for allopurinol-induced severe cutaneous adverse drug reactions (SCARs) in gout patients. Malaysia has a high frequency of 10.4% of the HLA-B*58:01 allele in allopurinol-induced SCARs patients. However, the strength of association of the HLA-B*58:01 allele to allopurinol-induced SCARs still need to be further validated in Malaysian pharmacogenetics studies. This project aims to develop a new, cost-effective, user-friendly and rapid method of screening for this allele by using the High Resolution Melt (HRM) method. The HRM method was used for its ability of reference curve-based targeted genotyping, where a positive control’s melt curve is used as a reference for screening of unknown samples. A gout cohort (n=145) and a healthy volunteers cohort (n=145), matched for age, gender and ethnicity, were used for the HRM screening. The HRM method showed a sensitivity of 0%, specificity of 57%, positive predictive value of 0% and negative predictive value of 57%, due to the presence of significant limiting factors. Several significant limitations were met in this study, starting with the slow sample collection, low number of SCARs samples, positive control’s heterozygosity, low primer specificity and the high level of polymorphism in the HLAB*58:01 allele. Moreover, the Sanger sequencing and NGS methods were used to validate the HRM method and delve into the complexity of the HLA-B alleles’ role in Malaysians. The newer theory of the presence of numerous HLA-B alleles as pharmacogenetic markers in populations was also investigated. HLA-B*58:01 was seen as a strong genetic marker in mild allopurinol-induced hypersensitivities and SCARs by Sanger sequencing. HLA-B*58:01 positive samples identified by Sanger sequencing had high frequencies of 32.1% for two different alleles; HLA-B*58:01:01 and HLA-B*35:01:01. Moreover, healthy volunteer samples showed high frequencies of 28.5% for HLA-B*58:01:01 and HLA-B*35:01:01 alleles. Hence, all these aforementioned HLA-B alleles are identified as potential pharmacogenetic markers in Malaysia. Next Generation Sequencing (NGS) was performed on 6 gout samples and 3 Malaysian specific SNPs (rs11423052, rs151341211 and rs9279154) were identified for the HLA-B*58:01 allele. Future studies need to focus on SNPs amplification in order to fully exploit the HRM method’s strengths as a screening method.
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