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

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*3303 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*3303 allele. OBJECTIVE: This study aimed to design a nested AS-PCR protocol for detecting and distinguishing diplotype genotype of the HLA-A*3303 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*3303 allele and the HLA-A*3303 diplotype genotypes. Comparison with the Sanger sequencing method showed an absolute agreement (κ = 1.00, p < 0.001). The nested ASPCR 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*3303 allele distribution in 810 unrelated Vietnamese Kinh people, showing a frequency of HLA-A*3303 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*3303 allele detection was reported. The protocol can be applied for the stratification of patients at SCAR risks with various drugs.