Genotyping of Platelet Alloantigens by DNA Sequencing in Pakistani Population

Introduction: Single-nucleotide polymorphism (SNP) in human platelet antigens (HPAs) glycoproteins leads to alloimmunizations and platelet disorders such as posttransfusion purpura, neonatal alloimmune thrombocytopenia, and refractoriness to platelet transfusion. To study the prevalence in a particular ethnic group, genomic DNA is used to genotype HPAs. Detection of these polymorphisms is imperative to identify the risk of alloimmunization and the provision of HPAs. Current study was planned to determine the frequency of HPAs in the Pakistani population of blood donors. Methodology: Genomic DNA was extracted from blood samples of 300 randomly selected platelet donors from five major cities of Pakistan (Islamabad, Peshawar, Karachi, Quetta, and Mirpur). This study was approved by the ethical committee of Shaheed Zulfiqar Ali Bhutto Medical University, Islamabad, Pakistan. Prior informed consent was taken from all the participants. Sequence-specific primers for platelets glycoprotein genes were designed using Primer 3 online software. The distinct targets were amplified through PCR. Amplified PCR products were eluted from the gel after electrophoresed, purified and sequenced. All the sequences and data obtained were analyzed through SPSS version 25. Results: Genotyping of samples showed that among the subjected HPA systems, HPA-1, HPA-5, HPA-7w, HPA-19w, and HPA-21w systems were found to have both a and b alleles in the Pakistani population while only aa genotype was found in HPA-4, HPA-6w, HPA-8w, HPA-10w, HPA-11w, HPA-16w, and HPA-23w. The frequency of HPA-1a was 0.9333 and HPA-1b was 0.0666, HPA-5a was 0.8033 and HPA-5b was 0.1966, HPA-7wa was 0.98 and HPA-7wb was 0.02, HPA-19wa was 0.95 and HPA-19wb was 0.05 and HPA-21wa was 0.9866 and HPA-21wb was 0.0133. Among the analyzed HPAs, the mismatch probability was higher in HPA-5 while it was lower in HPA-21w. Conclusion: HPA-4b, HPA-6b, HPA- 8b, HPA-10b, HPA-11b, HPA-16b and HPA-23b were absent. No homozygosity was found in the remaining genotyped HPAs. Our study suggests that it is necessary to establish HPA screening sites in blood banks to have HPA typed donor registry providing compatible therapeutic platelets to all unimmunized patients. Our data will be useful to understand and better treat the alloimmune-mediated platelet disorders. Key words: Alloantigens, Genotyping, Sequencing, Platelets, Platelet alloantigens