A novel nested allele-specific PCR protocol for the detection of the HLA-A*33:03, a SCAR-associated allele, in Vietnamese people

Tran Thu Ha Pham,¹ Quang Binh Tran,² Chonlaphat Sukasem,^3,4,5 Van Dinh Nguyen,^6,7 Chi Hieu Chu,⁸ Thi Quynh Nga Do,⁹ Ngoc Phuong Mai Tran,⁹ Hai Ha Nguyen,¹⁰ Thanh Huong Phung¹

¹ Hanoi University of Pharmacy, Hanoi, Vietnam
² National Institute of Nutrition, Hanoi, Vietnam
³ Division of Pharmacogenomics and Personalized Medicine, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Thailand
⁴ Laboratory for Pharmacogenomics, Somdech Phra Debaratana Medical Center (SDMC), Ramathibodi Hospital, Bangkok, Thailand
⁵ The Thai Severe Cutaneous Adverse Drug Reaction (THAI-SCAR) research group, Thailand
⁶ Allergy and Clinical Immunology Unit, Internal Medicine Department, Vinmec Times City International Hospital, Vinmec Healthcare System, Hanoi, Vietnam
⁷ College of Health Sciences, VinUniversity, Hanoi, Vietnam
⁸ Center of Allergology and Clinical Immunology, Bach Mai Hospital, Hanoi, Viet Nam
⁹ National Institute of Hygiene and Epidemiology, Hanoi, Vietnam
¹⁰ Institute of Genome Research, Vietnam Academy of Science and Technology, Hanoi, Vietnam

Abstract

Background: Severe cutaneous adverse drug reactions (SCARs) are rare but deadly drug reactions with severe damages to patients. One of the most well-known SCARs risk factors is the human leukocyte antigen (HLA) genes polymorphism. Among the HLA polymorphic alleles, the HLA-A*33:03 allele has been found in association with SCARs induced by various drugs, especially in Asian people. There has not been any report on the specific detection protocol of the HLA-A*33:03 allele.
Objective: This study aimed to design a nested AS-PCR protocol for detecting and distinguishing diplotype genotype of the HLA-A*33:03 allele.
Methods: A nested allele-specific (AS)-PCR protocol with four primer sets was designed. The method was compared with the Sanger sequencing method on 100 samples of unknown genotypes of unrelated Vietnamese people.
Results: The nested AS-PCR method could identify the HLA-A*33:03 allele and the HLA-A*33:03 diplotype genotypes. Comparison with the Sanger sequencing method showed an absolute agreement (ĸ = 1.00, p < 0.001). The nested AS-PCR protocol had a sensitivity of 100% (95%CI: 92.13-100%) and a specificity of 100% (95%CI: 93.51-100%). The protocol was used for the determination of HLA-A*33:03 allele distribution in 810 unrelated Vietnamese Kinh people, showing a frequency of HLA-A*33:03 carriers of 19.6% and an allele frequency of 10.55%.
Conclusions: A novel nested AS-PCR method with a hundred-percent sensitivity and a specificity for the HLA-A*33:03 allele detection was reported. The protocol can be applied for the stratification of patients at SCAR risks with various drugs.
Key words: SCAR, HLA-A*33:03, nested AS-PCR, protocol, Vietnam