Pathology (1997), 29, pp. 392-398 PLATELET ANTIGEN ALLELE FREQUENCIES IN AUSTRALIAN ABORIGINAL AND CAUCASIAN POPULATIONS ZHENJUN CHEN*, SUSAN LESTERt. BARRY BOETTCHERt AND JAMES MCCLUSKEY* Department of Microbiology and Immunology, The University of Melbourne, Victoria*, Transplant Services, Australian Red Cross Blood Service, Adelaide, South Australiat, and University of Newcastle, New South Wales:f:, Australia Summary We have applied genotyping methods of PCR-SSOP and PCR-RFLP to three, bi-allelic platelet specific antigen sys- tems HPA-1 (Pia), HPA-3 (Bak) and HPA-5 (Br). This com- bination of techniques offers flexibility for high volume or rapid typing. The phenotype and genotype frequencies of alleles from the three systems differ significantly between the Yuendumu Australian Aboriginals (Wailbri) and Aus- tralian Caucasians. The major differences are the very low frequencies of HPA-1b and HPA-3b in Yuendumu Aborigi- nals which are potentially relevant to platelet transfusion in patients of Australian Aboriginal descent. Key words: Platelet antigen, genotyping, PCR-SSOP, PCR-RFLP, Aus- tralian Aboriginals. Abbreviations: HPA; human platelet-specific antigen; NAIT, neonatal alloimmune thrombocytopenia; PTP, post transfusion purpura; PTR, platelet transfusion refractoriness; RFLP, restriction fragment length poly- morphism; SSOP, sequence specific oligonucleotide probe; SSP, sequence specific primers. Accepted 14 May 1997 INTRODUCTION Human platelet specific antigens (HPA) are principally comprised of five bial1elic systems conventionally called PIa (or Zw), Ko (or Sib), Bak, Yuk (or Pen) and Br (Table 1). There are essentially three clinical conditions caused by alloantibodies directed towar:ds platelet alloantigens for which platelet typing and/or matching is of benefit. Neonatal alloimmune thrombocytopenia (NAIT) Although rare, this is a well-defined feto-maternal platelet incompat- ibility. NAIT occurs with a frequency of one to two cases per 1000 neonates, the worst complication being an intra- cerebral hemorrhage (in about 10% of affected neonates) which may occur in utero. In the Caucasian population about 80% of NAIT cases are caused by HPA-1a anti- bodies, 15% by HPA-5b antibodies and 5% are due to other platelet specific antibodies.! Post transfusion purpura (PTP) This is a rare adverse transfusion reaction affecting mainly women OVer the age of 50 who have been preimmunized by pregnancy and/or blood transfusions. Over 90% of cases in the Caucasian population are due to HPA-la antibodies, while the re- maining cases are predominantly due to HPA-Ib, HPA-3a, HPA-3b antibodies and very rarely, HPA-5b antibodies. 2 TABLE 1 Terminology and DNA polymorphism for three platelet antigen systems Alternative Platelet Polymorphism System Terminology Glycoprotein cDNA Sites HPA-la ZW a PL A1 GP IIIa 196: CTG (Leu 33 ) HPA-1b Zwt': PL A2 CCG (Pro 33 ) HPA-3a Bak a , Lek a GP lIb 2622: ATC (Ile 843 ) HPA-3b Bak b , Lek b AGC (Ser 843 ) HPA-5a Brb, Zav b , Hc b GGPla 1648: GAG (Glu 5O ,) HPA-5b Br", Zav a , Hc' AAG (L ys 505) Platelet transfusion refractoriness (PTR) This is a serious problem in patients dependent upon long-term platelet support. The alloantibodies involved are mainly those di- rected against HLA class I antigens, but platelet-specific alIoantibodies are involved in about 10% of cases. Anti- HPA-I b is the most frequent antibody specificity in these cases. Typing and matching for three platelet specific antigen systems, HPA-l, HPA-3 and HPA-5 specificities, would therefore be sufficient for most clinical problems associ- ated with platelet specific antibodies. However serological typing of platelets requires well characterised typing sera (which are generally not readily available) and sufficient platelets to carry out the typing reaction which can be a problem for thrombocytopenic patients. By contrast, platelet antigen genotyping is not . limited by these prob- lems. The DNA polymorphisms responsible for HPA-1, HPA-3 and HPA-5 alloantigen systems have now been identified 3 ,4,5 (Table 1). In each case a single nucleotide substitution results in an amino acid change that appears to control immunogenic epitopes. A variety of DNA typing techniques involving PCR (polymerase chain reaction) amplified DNA have already been applied to typing some of the platelet antigen sys- tems. These techniques include: PCR-RFLP (restriction fragment length polymorphism),6,7 PCR-SSOP (sequence specific oligonucleotide probes)8,9 and PCR-SSP (sequence specific primers ).10,11 We have refined the PCR-SSOP and PCR-RFLP assays for the genotyping of HPA-l, HPA-3 and HPA-5 platelet alloantigen systems. We report here a comparison of the phenotype and genotype frequencies from a sample of Australian Caucasian blood donors and a sample of Yuen- dumu (Wailbri) Australian Aboriginals. The Yuendumu 0031-3025/97/040392-7 © 1997 Royal College of Pathologists of Australasia Pathology (1997), 29, pp. 392-398 PLATELET ANTIGEN ALLELE FREQUENCIES IN AUSTRALIAN ABORIGINAL AND CAUCASIAN POPULATIONS ZHENJUN CHEN*, SUSAN LESTERt. BARRY BOETTCHERt AND JAMES MCCLUSKEY* Department of Microbiology and Immunology, The University of Melbourne, Victoria*, Transplant Services, Australian Red Cross Blood Service, Adelaide, South Australiat, and University of Newcastle, New South Wales:f:, Australia Summary We have applied genotyping methods of PCR-SSOP and PCR-RFLP to three, bi-allelic platelet specific antigen sys- tems HPA-1 (Pia), HPA-3 (Bak) and HPA-5 (Br). This com- bination of techniques offers flexibility for high volume or rapid typing. The phenotype and genotype frequencies of alleles from the three systems differ significantly between the Yuendumu Australian Aboriginals (Wailbri) and Aus- tralian Caucasians. The major differences are the very low frequencies of HPA-1b and HPA-3b in Yuendumu Aborigi- nals which are potentially relevant to platelet transfusion in patients of Australian Aboriginal descent. Key words: Platelet antigen, genotyping, PCR-SSOP, PCR-RFLP, Aus- tralian Aboriginals. Abbreviations: HPA; human platelet-specific antigen; NAIT, neonatal alloimmune thrombocytopenia; PTP, post transfusion purpura; PTR, platelet transfusion refractoriness; RFLP, restriction fragment length poly- morphism; SSOP, sequence specific oligonucleotide probe; SSP, sequence specific primers. Accepted 14 May 1997 INTRODUCTION Human platelet specific antigens (HPA) are principally comprised of five bial1elic systems conventionally called PIa (or Zw), Ko (or Sib), Bak, Yuk (or Pen) and Br (Table 1). There are essentially three clinical conditions caused by alloantibodies directed towar:ds platelet alloantigens for which platelet typing and/or matching is of benefit. Neonatal alloimmune thrombocytopenia (NAIT) Although rare, this is a well-defined feto-maternal platelet incompat- ibility. NAIT occurs with a frequency of one to two cases per 1000 neonates, the worst complication being an intra- cerebral hemorrhage (in about 10% of affected neonates) which may occur in utero. In the Caucasian population about 80% of NAIT cases are caused by HPA-1a anti- bodies, 15% by HPA-5b antibodies and 5% are due to other platelet specific antibodies.! Post transfusion purpura (PTP) This is a rare adverse transfusion reaction affecting mainly women OVer the age of 50 who have been preimmunized by pregnancy and/or blood transfusions. Over 90% of cases in the Caucasian population are due to HPA-la antibodies, while the re- maining cases are predominantly due to HPA-Ib, HPA-3a, HPA-3b antibodies and very rarely, HPA-5b antibodies. 2 TABLE 1 Terminology and DNA polymorphism for three platelet antigen systems Alternative Platelet Polymorphism System Terminology Glycoprotein cDNA Sites HPA-la ZW a PL A1 GP IIIa 196: CTG (Leu 33 ) HPA-1b Zwt': PL A2 CCG (Pro 33 ) HPA-3a Bak a , Lek a GP lIb 2622: ATC (Ile 843 ) HPA-3b Bak b , Lek b AGC (Ser 843 ) HPA-5a Brb, Zav b , Hc b GGPla 1648: GAG (Glu 5O ,) HPA-5b Br", Zav a , Hc' AAG (L ys 505) Platelet transfusion refractoriness (PTR) This is a serious problem in patients dependent upon long-term platelet support. The alloantibodies involved are mainly those di- rected against HLA class I antigens, but platelet-specific alIoantibodies are involved in about 10% of cases. Anti- HPA-I b is the most frequent antibody specificity in these cases. Typing and matching for three platelet specific antigen systems, HPA-l, HPA-3 and HPA-5 specificities, would therefore be sufficient for most clinical problems associ- ated with platelet specific antibodies. However serological typing of platelets requires well characterised typing sera (which are generally not readily available) and sufficient platelets to carry out the typing reaction which can be a problem for thrombocytopenic patients. By contrast, platelet antigen genotyping is not . limited by these prob- lems. The DNA polymorphisms responsible for HPA-1, HPA-3 and HPA-5 alloantigen systems have now been identified 3 ,4,5 (Table 1). In each case a single nucleotide substitution results in an amino acid change that appears to control immunogenic epitopes. A variety of DNA typing techniques involving PCR (polymerase chain reaction) amplified DNA have already been applied to typing some of the platelet antigen sys- tems. These techniques include: PCR-RFLP (restriction fragment length polymorphism),6,7 PCR-SSOP (sequence specific oligonucleotide probes)8,9 and PCR-SSP (sequence specific primers ).10,11 We have refined the PCR-SSOP and PCR-RFLP assays for the genotyping of HPA-l, HPA-3 and HPA-5 platelet alloantigen systems. We report here a comparison of the phenotype and genotype frequencies from a sample of Australian Caucasian blood donors and a sample of Yuen- dumu (Wailbri) Australian Aboriginals. The Yuendumu 0031-3025/97/040392-7 © 1997 Royal College of Pathologists of Australasia