Localization of the Br Polymorphism on a 144 bp Exon of the GPIa Gene and Its Application in Platelet DNA Typing

Abstract

Alloimmunization against the human platelet alloantigen system Br (HPA-5) is the second most common cause of neonatal alloimmune thrombocytopenia (NAIT) in Caucasian populations. We have recently shown that a single base polymorphism at position 1648 on platelet mRNA coding for GPIa results in an aminoacid substitution at position 505 on the mature GPIa which is associated with the two serological defined Br phenotypes. Since DNA-typing of platelet alloantigens offers possibilities for useful clinical applications, we designed genomic DNA-based restriction fragment length polymorphism (RFLP) typing for Br alloantigens. To establish this technique we analyzed the genomic organization of GPIa adjacent to the polymorphic base. Using the polymerase chain reaction (PCR) of blood cell DNA we have identified two introns (approximately 1.7 and 1.9 kb) flanking a 144 bp coding sequence of the GPIa gene encompassing the polymorphic base 1648. Based on the intron sequence, a PCR primer was constructed to amplify a 274 bp fragment which was used for allele-specific RFLP to determine the Br genotypes. The results of RFLP analysis using MnlI endonuclease obtained from 15 donors (2 Bra/a, 2 Bra/b and 11 Brb/b) correlate perfectly with serological typing by monoclonal antibody-specific immobilization of platelet antigens (MAIPA) assay.