Gene frequencies of human platelet alloantigens in Kelantan, Malaysia.

The gene for the ß 3 (GPIIIa) subunit of the integrin α IIb ß 3 (GPIIb-IIIa) present on the platelet membrane contains the platelet antigen alleles (PlA) in a homozygous form (Pl/) or the heterozygous or polymorphic form (Pl/) in humans. This GPIIIa subunit serves as a receptor for adhesive proteins (viz. fi brinogen and von Willebrand factor) and is critical for platelet aggregation. The structural basis of Pl/ polymorphism arises from a single base difference, a thymidine or a cytosine, which leads to an amino acid difference at position 33 in the mature ß 3 subunit: a leucine in Pl or a proline in Pl. Till 1996, signifi cant work on the single nucleotide polymorphisims (SNPs) present in the PlA system, especially the alleles in the gene for GPIIIa, had not been undertaken in detail and correlated with platelet behaviour. It was Weiss et al who identifi ed the heterozygous (polymorphic) Pl/ allelic state as a signifi cant risk factor leading to platelet hyperadhesion and hence to thrombotic events like myocardial infarction and unstable angina, particularly in the younger Caucasian males. Since the initial report that the Pl polymorphism is a cardiovascular risk factor, many population-based studies had been done at many centres to determine the natural occurrence of Pl/ polymorphism amongst the various ethnic groups. Searching the literature indicated that no previous studies had been done on the Pl and Pl allele frequencies in the Malaysian population. We conducted a study on a sample, which included 160 Kelantan residents who were apparently healthy and unrelated to one another. Their ages were between 15 and 50 years, with a mean age of 33 years. There were 150 Malays, of which 20 were males and 130 females. The study also included ten Chinese patients as the cohort of the Chinese population in Kelantan is small. Using 2 ml of venous blood, isolation of DNA was performed with a DNA isolation kit. (QIAmp Blood Kit, Qiagen, Research Biolabs, Kuala Lumpur, Malaysia). The genotypes were determined using allele-specifi c polymerase chain reaction (PCR) amplifi cation. Briefl y, exon 2 of the human platelet glycoprotein IIIa (ITGB3) gene was amplifi ed using primers that amplify a 244 base pair fragment of genomic DNA that contains the polymorphic site responsible for the amino acid substitution causing a given platelet alloantigen. Two PCR procedures were undertaken to achieve alloantigen genotyping of an individual using the Allele Specifi c Oligonucleotide (ASO) technique. One, using one allele-specifi c primer with the consensus primer, is used to identify that corresponding allele, and the second allele is identifi ed in a reaction using the complementary allele-specifi c primers. Thus, in homozygous individuals, amplifi cation occurred only when the corresponding specifi c primer is present in the reaction solution. In heterozygous individuals, amplifi cation occurred in both reactions. The results of PCR amplifi cation viewed by gel electrophoresis are shown in Fig. 1. We used the single proportion Pocok’s formula for arriving at the sample size and the confi dence level.