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INTRODUCTION
In Thailand, the demographic Thai populations include the central, northeastern, northern, and southern Thais. The northern Thai population is one of the major subgroups of ethnic Thais and has close cultural and historic connections with the populations of southern Chinese, Myanmar and Laos. A previous study on the HLA system in 5 ethnic Thai populations recruited from central, northeastern, and northern Thailand demonstrated that the HLA-DRB1*15:02 allele appeared to be dominant in Thais; however, the HLA-DRB1*15:01 and the HLA-DRB1*09:01 alleles were relatively more common in northern Thais than other ethnic Thais [1]. This evidence supports the hypothesis that the heterogeneous southern populations of East Asia are genetically derived from the population of Southeast Asia [2, 3].
Human neutrophil antigens (HNA) are polymorphic structures located in the membranes of neutrophils [4]. Currently, 5 HNA systems (HNA-1 to HNA-5), have been characterized [5, 6]. In the HNA-1 system, 3 antigens, HNA-1a, HNA-1b and HNA-1c, are located on the neutrophil low-affinity Fc-γ-IIIb receptor (CD 16) glycoprotein [7]. Because Fc-γ-IIIb is the most immunogenic glycoprotein on the neutrophil membrane, antibodies to these antigens are frequently implicated in alloimmune and autoimmune neutropenia and transfusion-related acute lung injury (TRALI) [8-12].
The Fc-γ-IIIb receptor is encoded by the FCGR3B gene and occurs in 3 polymorphic forms representing the human neutrophil antigens, that is, HNA-1a, HNA-1b, and HNA-1c, which are encoded by the FCGR3B*1, FCGR3B*2, and FCGR3B*3 alleles, respectively [13]. In addition to their clinical significance, the FCGR3B allele frequencies could be used as an additional marker in population studies [14-16]. Moreover, the HNA genetic system of Thai populations has not yet been adequately studied. Therefore, this study aimed to determine the FCR3B*1, FCGR3B*2, and FCGR3B*3 allele frequencies encoding HNA-1a, HNA-1b, and HNA-1c, respectively, in Thai blood donors by using a PCR sequence-specific primer (PCR-SSP) technique to compare the allele frequencies with those previously reported in other populations.
METHODS
1. Study population
Peripheral venous blood was collected in EDTA-anticoagulated vacutainer from 800 unrelated healthy Thai blood donors. Five hundred samples, that is, 300 samples from our previous study [17] and an additional 200 samples, were from the National Blood Centre, Thai Red Cross Society, Bangkok. In addition, 300 samples were from the Blood Bank, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand. Written informed consent was obtained from each subject. This study was approved by the Committee on Human Rights Related to Research Involving Human Subjects, Thammasat University, Pathumtani, Thailand. Genomic DNA was extracted from peripheral blood samples using the Genomic DNA extraction kit (REAL Genomics, RBCBioscience, Taipei, Taiwan) and by a salting out method, as previously described [18], and was then stored at -20℃ until use for genotyping.
2. FCGR3B DNA amplification using PCR-SSP
Known HNA-1a, HNA-1b, and HNA-1c DNA samples were provided by Dr. Núria Nogués, Laboratori d'Immunohematologia, Banc de Sang i Teixits, Passeig Taulat, Barcelona, Spain. A PCR-SSP technique was performed as previously described [9] with some modifications. The sequences of the primers used for HNA genotyping are shown in Table 1. The specific primers were similar to those previously described [4, 9]. Briefly, 2 µL of genomic DNA (50 ng/µL) was amplified in a total volume of 10 µL with 0.5 µM of HNA-1a forward (F) and reverse (R) primers for FCGR3B*1 genotyping, 0.5 µM of HNA-1b (F) and (R) primers for FCGR3B*2 genotyping, and 0.5 µM of HNA-1c (F) and (R) primers for FCGR3B*3 genotyping. The human growth hormone (HGH) gene was coamplifed using 0.125 µM HGH (F) primer and 0.125 µM HGH (R) primer and was used as an internal control. PCR was performed with 5 µL of DreamTaq DNA polymerase (Thermo Fisher Scientific Inc., Glen Burnie, MD, USA) consisting of 2X DreamTaq Green buffer, 0.4 mM of each of the dNTPs, and 0.4 mM MgCl2 in a G-STORM GS1 thermal cycler (Gene Technologies Ltd., Somerton, UK). The cycling parameters for the PCR program were as follows: 1 cycle of 300 sec at 95℃; 30 cycles each of 30 sec at 95℃, 60 sec at 57℃, and 30 sec at 72℃; and final extension for 5 min at 72℃. The product was then kept at 4℃ until use for analysis. After amplification, the PCR products were analyzed on a 2.0% agarose gel using 1X Tris borate ethylenediaminetetraacetate (TBE) buffer containing SYBR Green I nucleic acid gel stain (Invitrogen, Grand Island, NY, USA) and were visualized under ultraviolet (UV) illumination (Fig. 1).
3. Statistical analysis
Gene frequencies were calculated by gene counting as previously described [4, 19]. The Chi-square test was used to evaluate whether the observed genotype frequencies were in agreement with the expected ones under Hardy-Weinberg equilibrium. Genotype and allele frequencies of FCGR3B*1, FCGR3B*2, and FCGR3B*3 were compared between different populations by using the Chi-square test. P values of less than 0.05 were considered statistically significant.
RESULTS
The FCGR3B genotypes and allele frequencies among different populations are shown in Table 2. For the central Thai blood donors, the FCGR3B*1 allele was most frequently detected (0.548 [548/1,000]), followed by FCGR3B*2 (0.452 [452/1,000]), with FCGR3B*3 being the least frequent (0.004 [4/1,000]). FCGR3B*1/FCGR3B*2 was the most frequent genotype (321/500), followed by FCGR3B*1/FCGR3B*1 (112/500) and FCGR3B*2/FCGR3B*2 (63/500). On the other hand, only 2 alleles, FCGR3B*1 and FCGR3B*2, were found in the northern Thai population, with allele frequencies of 0.677 (406/600) and 0.323 (194/600), respectively. In this population, FCGR3B*1/FCGR3B*1 was the most frequent genotype (140/300), followed by FCGR3B*1/FCGR3B*2 (126/300) and FCGR3B*2/FCGR3B*2 (34/300). We did not find any HNA-1 null subjects among the 800 samples investigated in the present study. Moreover, the FCGR3B locus was in Hardy-Weinberg equilibrium in Thai populations. The genotype frequency distribution of the central Thai population was also compared with that of the northern Thai population and other studies previously reported [9, 14, 16, 20, 21]. The FCGR3B*1 and FCGR3B*2 allele frequencies of the northern Thais did not differ from those of the Taiwanese and Japanese populations [9, 14]. Interestingly, the FCGR3B*1 and FCGR3B*2 allele frequencies were significantly different from those observed in the central Thai, German, Turkish, Tunisian, and Korean populations (P<0.01) [16, 20, 21].
DISCUSSION
In this study, the FCGR3B alleles encoding the HNA-1a, -1b, and -1c antigens were analyzed by the in-house PCR-SSP technique, because the commercial kit for HNA-1a, HNA-1b, and HNA-1c genotyping is expensive and is not available in Thailand. Although the PCR-SSP technique is not suitable for high-throughput genotyping, it is widely used in routine laboratory examinations for both HLA and HNA genotyping because of its simplicity. Moreover, in order to simultaneously read the HNA-1 genotyping results, the same PCR conditions were used for each FCGR3B allele detection, which makes this technique less time consuming than the PCR-SSP technique previously reported [9]. Additionally, the cost of this in-house PCR-SSP technique is approximately only 1 US dollar per test.
It was found that the FCGR3B*1 allele was the most common allele in 800 Thai blood donors. This finding confirms that the FCGR3B*1 allele is more frequent than the FCGR3B*2 allele in Asian populations, Afro-Brazilians, and Native Americans [4, 6, 7, 9, 13, 14]. On the other hand, the FCGR3B*1 and FCGR3B*2 allele frequencies in the northern Thai population were significantly different from those in the central Thai and Korean populations [20]. The FCGR3B*3 allele, which is considered to be a rare allele in Asian populations [9], was found only in central Thai blood donors, similar to the findings of a previous study [17]. This allele was present in 9.09% of Tunisian blood donors and only in 5% of European populations [16].
HNA-1a individuals with the FCGR3B*1/FCGR3B*1 genotype are reported to be more susceptible to idiopathic pulmonary fibrosis, whereas HNA-1b individuals with the FCGR3B*2/FCGR3B*2 genotype have a higher susceptibility to periodontal diseases and chronic periodontitis because of differing reactivity to opsonised bacteria [22]. Therefore, diseases associated with HNA-1 antigens found in the northern Thai population are different from those for the central Thai population. Further studies are required on the association of HNA-1 and disease susceptibility. Additionally, a higher frequency of the FCGR3B*1 allele and a lower frequency of the FCGR3B*2 allele were found in northern Thai blood donors compared with the central Thai population, suggesting that northern Thais would be more susceptible to HNA-1a alloimmunization than the central Thais, similar to other Asian populations [14, 20].
Our results demonstrate that the FCGR3B*1 and FCGR3B*2 allele frequencies significantly differ between the central and northern Thai populations. However, the allele frequencies in the northern Thai population are closely related to those for the Taiwanese and Japanese populations. In addition, further studies on HNA-3, HNA-4, and HNA-5 gene frequencies would be beneficial in order to develop a database of HNA characteristics in Thai populations.
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