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Molecular identification of HLA-B75 serotype markers by qPCR: A more inclusive pharmacogenetic approach before carbamazepine prescription - PubMed

Molecular identification of HLA-B75 serotype markers by qPCR: A more inclusive pharmacogenetic approach before carbamazepine prescription

Kanoot Jaruthamsophon et al. Clin Transl Sci. 2024 Jun.

Abstract

Genetic screening for HLA-B*15:02 before prescribing carbamazepine is standard practice to prevent severe cutaneous adverse reactions in Asian populations. These reactions are associated not only with this allele but also with closely related HLA-B75 serotype markers-HLA-B*15:11 and HLA-B*15:21-which are prevalent in several Asian countries. However, a reliable method for identifying HLA-B75 serotype markers is still not available. We developed an in-house quantitative PCR (qPCR) for HLA-B75 screening and validated it using 303 anonymized DNA samples. Due to inadequate quality control, the qPCR results for 11 samples were excluded. We analyzed the sensitivity and specificity of the test using 93 HLA-typed samples. The concordance between the qPCR method and an established screening method was assessed using 199 HLA-screened samples tested for HLA-B*15:02 at Songklanagarind Hospital, Songkhla, Thailand. All discordant results were confirmed by Sanger sequencing. The qPCR method demonstrated a sensitivity of 100% (95% confidence interval = 83.16%-100.00%) and a specificity of 100% (95% confidence interval = 95.07%-100.00%). Concordance analysis revealed a 96.5% agreement between methods (192/199; 44 positive and 148 negative results). All discordant results were due to HLA-B75 markers not being HLA-B*15:02 (two samples with HLA-B*15:11 and five samples with HLA-B*15:21). In conclusion, this qPCR method could be useful for identifying HLA-B75 carriers at risk of carbamazepine-induced reactions in Asian populations where carriers of HLA-B*15:02, HLA-B*15:11, or HLA-B*15:21 are common.

© 2024 The Author(s). Clinical and Translational Science published by Wiley Periodicals LLC on behalf of American Society for Clinical Pharmacology and Therapeutics.

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Conflict of interest statement

The authors declared no competing interests in this work.

Figures

FIGURE 1
FIGURE 1

Primer and probe binding sites on exon 2 of HLA‐B gene. The primer binding sites are highlighted in blue. The probe binding site and HLA‐B75 serotype markers are highlighted in yellow. The alignment was obtained from the IMGT/HLA database.

FIGURE 2
FIGURE 2

Quantitative PCR result and Sanger sequencing result. (a) qPCR results of samples with and without HLA‐B75 marker. Blue lines display the signal from the B75‐FAM probe. Green lines display the signal from ACTB‐VIC probe. Samples with a Cq of ACTB‐VIC of >30 were excluded from the analysis. (b, c) Confirmatory Sanger sequencing result of samples with HLA‐B*15:11 (b) and HLA‐B*15:21 (c).

FIGURE 3
FIGURE 3

Test validation results. The performance of qPCR in identifying HLA‐B75 serotype markers was evaluated using 303 samples, of which 11 samples were excluded due to failed control. The qPCR results were compared with the HLA‐B genotype in 93 samples and HLA‐B*15:02 screening result in 199 samples. AS‐PCR‐DDB, allele‐specific PCR and direct dot‐blot hybridization.

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