Development and Validation of a Real-Time PCR Assay for HLA-A*31:01 Genotyping in Pharmacogenetic Screening
HLA-A*31:01 Genotyping by Real-Time PCR
Keywords:
HLA-A*31:01, Real-time PCR, Carbamazepine, Severe adverse drug reactions (SADRs), PharmacogeneticsAbstract
Carbamazepine (CBZ) is a widely prescribed anticonvulsant for the treatment of epilepsy, trigeminal neuralgia, and bipolar disorder. Although therapeutically effective, CBZ can trigger severe adverse drug reactions (SADRs), including life-threatening severe cutaneous adverse reactions (SCARs). Robust pharmacogenomic evidence has linked CBZ-induced SADRs to specific human leukocyte antigen (HLA) alleles, particularly HLA-B*15:02 (along with other alleles in the HLA-B75 serotype group) and HLA-A*31:01. While clinical testing for HLA-B*15:02/B75 is widely accessible in Thailand, HLA-A*31:01 genotyping remains unavailable in many settings, leaving a critical gap in comprehensive pharmacogenetic risk assessment. This gap is of particular concern because HLA-A*31:01 is associated with a broader spectrum of CBZ hypersensitivity and is more prevalent in populations where HLA-B*15:02 is uncommon. To close this limitation, we developed and validated a real-time PCR assay for HLA-A*31:01 to complement existing genotyping services. Clinical validation was performed on 198 genomic DNA samples with known HLA-A genotypes, including 14 HLA-A*31:01-positive and 184 negative samples. The assay demonstrated high sensitivity with a reliable detection range of 0.78–200.00 ng DNA/reaction and revealed 100% concordance with the reference method. Its simplicity, speed, and compatibility with standard laboratory workflows highlight its clinical utility. This assay represents a significant step toward safer, personalized CBZ therapy by enabling comprehensive, preemptive screening for SADR-associated HLA alleles.
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