High Frequency of HPA-2B Allele in Brazilian Apheresis Blood Donors Detected by Sequence Specific PCR.

Background: Human platelet antigens (HPA), mainly HPA-1 and HPA-2, play a significant role in alloimmune thrombocytopenic syndromes. Accurate HPA typing is important for the diagnosis and therapy of these patients. HPA gene frequencies vary in different populations. Thus, it is relevant to determine HPA genotype in distinct populations. The purpose of this study was to perform HPA-1,-2 genotyping in apheresis Brazilian blood donors.

Material and Methods: Genomic DNA was prepared from whole blood of 57 unrelated repeated apheresis Brazilian blood donors using a commercial DNA isolation kit (Qiagen,. GmbH, Germany). Sequency specific PCR (SSP-PCR) was performed amplifying HPA-1A/HPA-1B and HPA-2A/2B genes using a pair of primers for each allele as described elsewhere (HPA-1A ACT TAC AGG CCC TGC CTC T; HPA-1B ACT TAC AGG CCC TGC CTC C; HPA-1AS AGC CGG AGT GCA ATC CTC TG; HPA-2A CCC CCA GGG CTC CTG AC; HPA-2B CCC CCA GGG CTC CTG AT and HPA-2AS GCC AGC GAC GAA AAT AGA GG). A fragment of the human growth hormone gene (HGH) served as internal positive control. PCR was carried out in a final volume of 20 μL, containing 0.2 mg of genomic DNA, 0.2 mM of each dNTP, 5% of glycerol, 1.5 units of platinum Taq polymerase (Invitrogen, Brazil) in the buffer supllied, 5.0 mM MgCl₂ and 10.0 pmoles of each primer. Fragments of 196 bp derived from HPA-1A/1B mutation and of 241 bp for HPA-2A/2B were separated for 90 minutes at 102V using 0.5 μg.mL⁻¹ ethidium bromide-stained 2.0% agarose gels and visualized in a UV light apparatus (Eagle Eye II, EUA).

Results: The genotype frequencies presented 77.2% of HPA-1A/1A, 19.3% of HPA-1A/1B and 3.5% of HPA1B/1B; for HPA-2 the frequencies were 56.1% of HPA-2A/2A, 35.1% of HPA-2A/2B and 8.8% HPA-2B/2B. The gene frequencies in apheresis blood donors are 0.87 for HPA-1A, 0.13 for HPA-1B, 0.74 for HPA-2A and 0.26 for HPA-2B.

Conclusion: In this studied population we found high gene frequency homozygous HPA-2B when compared with other data already published. (