Establishment of a high-resolution melting assay for detection of GYP*Mur allele zygosity.

BACKGROUND: is very likely to identify GP.Mur, this method cannot reliably distinguish the underlying GP or confirm the zygosity of GP.Mur, which is relevant for transfusion safety. We, therefore, developed and validated a polymerase chain reaction with high-resolution melting (PCR-HRM) assay for distinguishing homozygous GYP*Mur from heterozygous GYP*Mur/GYPB and homozygous GYPB genotypes. MATERIALS AND METHODS: An HRM assay targeting exon 3 of GYP(B-A-B) hybrid genes was designed and validated using 186 Mi(a+) samples with known sequencing data. This assay was subsequently applied to 211 DNA samples from Thai blood donors, including Mi(a+) and Mi(a-) samples. RESULTS: HRM analysis showed high concordance with sequencing data in 184 of 186 validated samples. Discrepant cases were attributable to rare sequence variations that altered melting profiles. Among Mi(a+) samples not previously sequenced, 81.45% were classified as GYP*Mur/GYPB, 3.97% as GYP*Mur/GYP*Mur, 12.58% as GYP*Thai/GYPB, and 2% as other clusters, which were identified as rare variants by sequencing. All Mi(a-) samples were consistently classified as homozygous GYPB. DISCUSSION: Our PCR-HRM assay can rapidly and reliably discriminate homozygous GYP*Mur from heterozygous GYP*Mur/GYPB and homozygous GYPB genotypes. Rare or unexpected variants can be identified when DNA quality is well controlled, and appropriate quality controls are applied. Accordingly, this method may serve as a practical first-line screening tool to support transfusion safety, particularly in populations with a high prevalence of hybrid GP.