American Journal of Hematology 82:242–244 (2007)
Gene Frequencies of Human Platelet Alloantigens in Bahraini Arabs Abeer M. Al-Subaie,1 Iman K. Al-Absi,1 Khadija Al-Ola,2 Sarra Saidi,3 Naglaa A. Fawaz,4 and Wassim Y. Almawi1* 1
Department of Medical Biochemistry, College of Medicine and Medical Sciences, Arabian Gulf University, Manama, Bahrain 2 Department of Pediatrics, Salmaniya Medical Complex, Manama, Bahrain 3 Faculty of Pharmacy, University of Monastir, Monastir, Tunisia 4 Faculty of Medicine, King Faisal University, Dammam, Saudi Arabia
Human platelet antigens (HPA) are implicated in the pathophysiology of certain hematological disorders, and as varied distribution of HPA-1 alleles and genotypes were reported for different countries and ethnic populations, we determined the distribution of HPA-1, -2, -3, -4, and -5 alleles, genotypes and haplotypes for 194 healthy Bahraini subjects by polymerase chain reaction with sequence specific primers. The distribution of the HPA polymorphisms was in Hardy-Weinberg equilibrium. Allele frequencies of 0.76 and 0.24 (HPA-1a and -1b), 0.77 and 0.23 (HPA-2a and -2b), 0.57 and 0.43 (HPA-3a and -3b), 0.93 and 0.07 (HPA-4a and -4b), and 0.86 and 0.13 (HPA-5a and -5b) were seen. With the exception of HPA-3a/a (30.4%), the frequencies of homozygous HPA1a/a (56.8%), 2a/a (60.1%), 4a/a (87.2%), and 5a/a (75.7%) were higher than those of heterozygous (a/b) or homozygous (b/b) variants. Our results provide basic information for further studies of the HPA system polymorphism, which in turn will be instrumental in understanding and treating C 2006 Wiley-Liss, Inc. immune-mediated platelet disorders. Am. J. Hematol. 82:242–244, 2007. V Key words: human platelet antigens; PCR-SSP; haplotype; hematological disorders
INTRODUCTION
Human platelets alloantigens (HPA) are complex of platelet membrane glycoproteins with cell-bound antigens [1]. HPAs are polymorphic, with many biallelic variants identified [1], and single nucleotide substitution within HPAs yields the a and b alleles [2]. These were implicated in the pathogenesis of pathological conditions, including ischemic stroke and cerebrovascular diseases. The distribution of HPA genotypes is geographically and ethnically restricted. The prevalence of HPA5b and HPA-2b is high among Africans [3] but very low among Asians [4,5], while the frequency of HPA-1b is higher among Caucasians [6] and Berbers [7] than Asians [4,5]. In so far as data on the distribution of HPA polymorphisms among Arabs are scanty, the present study investigated the diversity in HPA alleles, genotypes, and haplotypes in Bahrain, an island kingdom located in the Arabian Gulf. This study provides basic information for further studies of the HPA diversity and will serve as reference for anthropological studies, and for studies of associations between HPA polymorphisms and disease. C 2006 Wiley-Liss, Inc. V
MATERIALS AND METHODS Study Subjects
Study subjects comprised 194 Bahraini healthy subjects (102 males, 92 females, mean age: 27.7 ± 15.2 years). Participants were considered healthy according to clinical history and laboratory examinations, including complete blood count and hemoglobin electrophoresis. Exclusion criteria included subjects with a personal and familial history of hemoglobinopathies and thrombophilias, and non-Arab origin. All participants were asked to sign a consent form after the pur*Correspondence to: Wassim Y. Almawi, Department of Medical Biochemistry, College of Medicine & Medical Sciences, Arabian Gulf University, PO Box 22979, Manama, Bahrain. E-mail:
[email protected] Received for publication 7 June 2006; Accepted 10 July 2006 Published online in 7 December 2006 Wiley InterScience (www. interscience.wiley.com). DOI: 10.1002/ajh.20769
Brief Report: HPA1-5 Gene Polymorphism in Bahrain TABLE I. HPA Allele and Genotype Frequencies Allele frequency HPA HPA-1 HPA-2 HPA-3 HPA-4 HPA-5
a
Genotype frequency
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DISCUSSION Hardy–Weinberg
A
B
A/A
A/B
B/B
x2
P valueb
0.760 0.767 0.568 0.932 0.861
0.240 0.233 0.432 0.068 0.132
0.568 0.601 0.304 0.872 0.757
0.385 0.331 0.514 0.122 0.209
0.047 0.068 0.176 0.000 0.027
0.467 0.810 0.401 0.179 1.040
0.495 0.368 0.527 0.672 0.308
a
Study subjects comprised 194 Bahraini healthy subjects. Pearson’s x2 test.
b
pose of the study was explained, and after all institutional ethics requirements were met. HPA Polymorphism Genotyping
Total genomic DNA was extracted from the peripheral blood mononuclear leukocytes by the Amersham GFX column methods, according to manufacturer’s specification (Amersham, Buckinghamshire, UK). DNA was dissolved in RNAase-free water, and stored at 48C pending analysis. HPA polymorphism was done by the polymerase chain reaction with sequence specific primers, as previously described [2]. Amplified products were subjected to agarose gel electrophoresis. Allele frequencies were determined using HLAStat-2000 software, and the frequencies of the most frequent haplotypes were determined by the maximum likelihood method. The odds ratio (O.R.) and 95% confidence interval were also determined. Analysis was performed with SPSS version 13.0 for Windows statistical package.
RESULTS HPA Polymorphisms Analysis
The distribution of HPA-1, -2, -3, -4, and -5 was in Hardy-Weinberg equilibrium among study subjects (Table I). Allele frequencies of 0.76 and 0.24 were seen for HPA-1a and -1b, 0.77 and 0.23 for HPA-2a and -2b, 0.57 and 0.43 for HPA-3a and -3b, 0.93 and 0.07 for HPA-4a and -4b, and 0.86 and 0.13 for HPA5a and -5b (Table I). With the exception of homozygous HPA-3a/a (30.4%), the frequencies of homozygous HPA-1a/a (56.8%), 2a/a (60.1%), 4a/a (87.2%), and 5a/a (75.7%) genotypes were higher than heterozygous (a/b) or homozygous (b/b) variants (Table I). The most significant HPA haplotypes encountered were HPA 1a-2a-3a-4a-5a (haplotype frequency ¼ 0.33) and HPA 1a-2a-3b-4a-5a (haplotype frequency ¼ 0.20).
In view of their association with hematological disorders, coupled with their differential world distribution, we investigated the prevalence of HPA-1(5 in Bahrain. To avoid epidemiologic bias, non-Arab Bahrainis (Iranians or recently-naturalized Bahrainis) were excluded. The varied prevalence of the HPA polymorphisms was consistent with previous reports documenting the geographical heterogeneity in their distribution [3,5,6]. The prevalence of HPA-1b (24%) and HPA-2b (23%) were high among Bahrainis, compared with Africans [3], Asians [5,8], and Europeans [6], which was comparable to rates established for Saudi Arabia [9], Tunisia [10], and North African Berbers [7], suggesting that Arabian Peninsula-North Africa is focus of HPA-1b. The prevalence of HPA-3b (43%) was less selective, as prevalence rates among Bahrainis were comparable to Asians [5,8] and Europeans [6]. Although virtually absent from Europeans [6] and Asians [5,8], HPA-4b was present in lower frequency (7%) in Bahrain, and in neighboring Saudi Arabia [9]. Although the highest frequency of HPA-5b was reported in Africa (18.2–26.8%; 3) and Italy (23%), the frequency of HPA-5b in Bahrain (13%) was compared with Saudi Arabia [9] and UK [6], but higher than those seen in Asians, where 2–5% frequencies were reported [5,8]. Although the prevalence of HPA-3b and HPA-4b in Bahrain overlap those observed for several ethnic groups, our results indicate that HPA-1b and 2b alleles are common among Bahrainis. The frequency of HPA-5b among Bahrainis appears to be closer to northern African and Saudi frequencies. Although implications of our findings remain speculative at this stage, they add up to the existing body of literature on HPA system diversity and will aid in determining the risk of hematological disorders in the population studied.
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