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

The antigens in the MNS blood group system, presented on glycophorin (GP), are regulated by the GYPA, GYPB, and GYPE genes. Some hybrid GP can express Mi^a antigen, the most common being GP.Mur. Of potential clinical significance in transfusion practice, GYP*Mur homozygotes can produce anti-JENU because they lack expression of the high-frequency JENU antigen. Although serological typing using anti-Mi^a 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.

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.

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.

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.