Development of a HLA-B*58:01 Allele Screening System for Allopurinol-Induced Severe Cutaneous Adverse Reactions Detection

Abstract

The HLA-B*58:01 allele has been identified as a genetic marker in the fatal allopurinol-induced severe cutaneous adverse reactions (SCARs) such as Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN). However, the screening of the HLA-B*58:01 allele prior to allopurinol prescription has not been implemented in hospitals, mostly due to the absence of an inexpensive and rapid screening method. In this work, we have developed a HLA-B*58:01 allele screening system which is able to give results in one and a half hour and is cheaper than any other screening instruments available. The system uses the High-Resolution Melt (HRM) method and generates difference melt curves proportional to a specific DNA's sequence. A positive screening result will be shown if the HLA-B*58:01 allele is detected in unknown samples screened. Twelve HRM experiments were performed to optimize the system and six final optimized experiments are shown in this article, along with their difference melt curves. The system was successfully benchmarked against the Illumina Eco Real Time machine and results were also validated by Sanger sequencing. The system was also compared against other HLA screening methods available on the market. We propose that the implementation of this HLA-B*58:01 screening system in hospitals will help to decrease the number of SCARs cases and eventually eliminate them in the long run. Patients will thus be prescribed allopurinol only if they test negative for the HLA-B*58:01 gene and those who test positive will be given alternative drugs, ultimately saving lives, time of treatment and overall medical cost.

Keywords

• Polymerase chain reaction
• high resolution melting
• HLA-B*58:01 allele
• severe cutaneous adverse reactions (SCARs)
• difference melt curve