[Molecular biologic clarification of Br alloantigens in human platelets and its application in DNA typing]

Alloimmunization against the platelet alloantigen 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 amino acid substitution at position 505 on the mature glycoprotein Ia which is associated with the two serologically defined Br phenotypes. To establish DNA-based genotyping for the Br system we elucidated the genomic organization of the GPIa gene adjacent to the polymorphic base. Using PCR of blood cell DNA we have identified a 144 bp exon encoding the Br polymorphic base. A PCR primer based on the 3' intron sequence of this exon in combination with an exon-primer was used to amplify a 274 bp fragment of the GPIa gene. Restriction analysis using the endonuclease Mnl I leads to a Br-specific restriction fragment length polymorphism (RFLP) which perfectly correlates with serological phenotyping.