HLA-B*15:02 screening in epileptic patients using a high resolution melting-real time PCR (HRM-QPCR) method

ABSTRACT

Background: The HLA-B*1502 polymorphism in epileptic patients is known to be associated with carbamazepine-induced Stevens-Johnson syndrome (SJS). The prevalence of HLA-B*1502 polymorphism seemed to be ethnic-specific with a higher frequency of HLA-B*1502 in Asian compared to the Europeans. This study was performed to determine the frequency of the HLA-B*1502 polymorphism in epileptic patients at the Chancellor Tuanku Muhriz Hospital-UKM Medical Centre (HCTM-UKMMC) using high resolution melting-real time PCR (HRM-QPCR) method. Methods: We performed a fast and effective in-house high resolution melting-real time polymerase chain reaction method and compared it with the conventional multiplex-PCR method. The specificity and sensitivity of each test were also determined using DNA from saliva. Results: Using the conventional multiplex-PCR approach for screening, 25 out of 64 (39.1%) epileptic patients were positive for HLA-B*1502. However, using the HRM-QPCR technique, 24/64 (37.5%) of the patients were positive. The one patient who tested positive by the multiplex-PCR but negative using the HRM-QPCR turned out to be negative by DNA sequencing. The HRM-QPCR and DNA sequencing showed 100% sensitivity and specificity. The multiplex-PCR showed 100% sensitivity and 98.4% specificity compared to both HRM-QPCR and DNA sequencing. The HRM-QPCR is also more cost-effective (<$16.40 USD/test) and less time-consuming when compared to the multiplex-PCR ($25.15 USD/test).Conclusion: Our result suggested that multiplex PCR, HRM-QPCR and Sanger sequencing can be used for detection of HLA-B*1502. However, a qualitative method such as multiplex PCR should be confirmed with other quantitative methods such as HRM-QPCR and Sanger sequencing.