A modified PCR-RFLP genotyping method demonstrates the presence of the HPA-4b platelet alloantigen in a North American Indian population

The diallelic HPA-4 (Pen/Yuk) platelet alloantigen system is polymorphic in Asian populations and accounts for the majority of cases of neonatal alloimmune thrombocytopenia in Japan. At the molecular level, the HPA-4a/4b dimorphism is associated with an arginine/ glutamine substitution at amino acid 143 of the gene encoding platelet glycoprotein IIIa. Unlike the five other major diallelic human platelet antigen systems (HPA-1, -2, -3, -5, and -6), the nucleotide substitution corresponding to the HPA-4 antigen system does not involve a common naturally occurring restriction enzyme site. This paper describes a new genotyping method for HPA-4 (polymerase chain reaction–restriction fragment length polymorphism [PCR-RFLP]) that involves restriction enzyme digestion of PCR-amplified genomic DNA using a modified PCR primer to create an artificial Taq I restriction site that is present in the HPA-4a but not in the HPA-4b DNA sequence. The HPA-4 PCR-RFLP method was validated by testing a reference panel of 10 known HPA-4 genotyped Japanese individuals. Thus, genotyping by PCR-RFLP can now be performed for all six major HPA systems. Using the HPA-4 PCR-RFLP genotyping method, we determined a frequency of 2.9 percent for the HPA-4b allele in a North American Indian population. This finding indicates the importance of the HPA-4 antigen system as a potential cause of alloimmune thrombocytopenia in American Indians.