A Novel Allele-Specific PCR Protocol for the Detection of the HLA-C*03:02 Allele, a Pharmacogenetic Marker, in Vietnamese Kinh People

Abstract

Background

Allopurinol, a common anti-hyperuricemia drug, is well known as an inducer of severe cutaneous adverse drug reactions (SCARs). One of the most well-defined risk factors of allopurinol-induced SCARs is the presence of polymorphic alleles of human leukocyte antigen (HLA) genes, such as HLA-B*58:01 and HLA-C*03:02 alleles. There is no commercial test or published in-house protocol for the specific detection of the HLA-C*03:02 allele. In this article, we established for the first time a simple allele-specific (AS) PCR method to identify HLA-C*03:02 allele carriers, and at the same time, determine their zygosities.

Methods

A two-step AS-PCR protocol, using four primer sets, was designed to specifically amplify and differentiate the HLA-C*03:02 allele from 17 other HLA-C alleles found in Vietnamese people. The protocol was validated with PCR-sequencing-based typing (SBT) of 100 samples of unknown genotypes.

Results

The PCR protocol can detect the HLA-C*03:02 allele and determine the zygosity. The results of this protocol were highly consistent with those of the SBT (ĸ = 0.98, p < 0.001). Regarding the specific detection of the HLA-C*03:02 allele, the PCR protocol had a sensitivity of 100% (95% CI: 91.61–100%) and specificity of 98.3% (95% CI: 90.9–99.7%). The protocol was used to determine the distribution of the HLA-C*03:02 allele in 810 unrelated Vietnamese Kinh people, 14.2% of which were HLA-C*03:02 carriers, the allele frequency was 7.5%.

Conclusion

A novel AS-PCR protocol with a sensitivity of 100% for the detection of the HLA-C*03:02 allele was established. The protocol can be used for personalized treatment with allopurinol in order to minimize the risk of SCARs in Vietnamese people as well as in other Asian populations with similar genetic characteristics.

Keywords:

• severe cutaneous adverse reactions
• HLA-C*03:02
• allopurinol
• HLA genotyping
• AS-PCR

Abbreviations

AF, allele frequency; AS, allele specific; DRESS, drug reactions with eosinophilia and systemic symptoms; HLA, human leukocyte antigen; SBT, sequencing-based typing; SCAR, severe cutaneous adverse drug reactions; SJS, Steven-Johnson syndrome; SNP, Single nucleotide polymorphism; SSO, sequence specific oligonucleotide; TEN, toxic epidermal necrolysis.

Acknowledgments

The authors would like to thank Dr. Duong Tuan Linh (National Institute of Nutrition) for his valuable supports and comments on the research.

Author Contributions

All authors made substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data; took part in drafting the article or revising it critically for important intellectual content; agreed to submit to the current journal; gave final approval of the version to be published; and agree to be accountable for all aspects of the work.

Disclosure

The authors report no conflicts of interest for this work.

Additional information

Funding

This study was funded by Vietnam Ministry of Health, Grant# 4694/QD-BYT.