Allele specific polymerase-chain reaction for the detection of GYP.Mur allele

Abstract

Glycophorin gene hybrid GYP(B-A-B) encodes a variant glycophorin, GP.Mur, that bears several immunogenic epitopes. Antibodies against GP.Mur have been implicated in cases of hemolytic transfusion reaction and hemolytic disease of the newborn. (1) In Hong Kong and many other Southeast Asia regions, the GP.Mur phenotype is distributed at a relatively high incidence rate. (2) Despite its significance, currently, a simple and specific method of detecting GP.Mur is nonexistent.

This project aims to develop a fast and direct PCR-ASP method that specifically targets the GYP.Mur allele. Allele specific primers were tailored to discriminate GYP.Mur from other glycophorin alleles with respect to single nucleotide polymorphism of the corresponding nucleotide sequences. Polymerase chain reaction protocols were optimized using this primer set and an internal control primer set that amplifies the Human Growth Hormone (HGH) gene. Samples with GP.Mur status screened by serology beforehand were analyzed using this novel PCR design. The results were confirmed by PCR and DNA sequencing approach described by Hsu et al. (3)

A total of 202 untransfused samples from Chinese individuals were screened by undifferentiated in-house Miltenberger antibody(ies). Eleven of which were serologically reactive. The reactive samples and another 11 non-reactive random samples were investigated using the PCR-ASP method. However, nine out of the 11 serologically reactive samples and one of 11 non-reactive samples were found to be GP.Mur-positive by the PCR-ASP. All of 10 GP.Mur positive samples, which included the serologically false negative sample, were verified to carry the GYP.Mur sequence by DNA sequencing. The 2 serologically false positive samples were confirmed as GP.Mur-negative with the PCR-ASP for GYP(B-A-B) allele published by Hsu et al.

The PCR-ASP method is confirmed to be quick and specific for detection of GYP.Mur allele. It is more reliable than serology phenotyping with undifferentiated Miltenberger anti-sera.