HPA-1-28w基因分型检测技术体系的建立和广西瑶族、汉族人群HPA-1-28w基因多态性研究

Establishment of the HPA-1-28w genotyping technique system and the study of HPA-1-28w polymorphism in Yao and Han populations in Guangxi,China

目的建立HPA-1-28w基因分型检测技术体系并用以研究广西瑶族和汉族人群的血小板抗原(HPA)-1-28w基因多态性。方法通过设计序列特异性引物和优化PCR反应条件,摸索HPA-1-28w基因分型检测技术体系,其组成包括前期已建立的HPA-1-17w多重PCR-SSP技术和新研发的HPA-18-28w PCR-SSP方法 2部分组成。使用10例已预先测序确定HPA-18-28w基因型和20例已预先使用商品试剂盒检测HPA-18-21w基因型的本研究广西瑶族标本,对新研发的HPA-18-28w PCR-SSP方法做验证。应用所建立的HPA-1-28w基因检测技术对广西地区无血缘关系的1424名健康瑶族人和908名健康汉族人做HPA-1-28w基因分型和多态性分析。结果所建立的HPA-1-28w基因分型技术系统,不仅对HPA-18-28w基因的分型结果与对验证用标本的测序分型结果完全一致,而且对HPA-18-21w基因的分型检测结果与商品试剂盒的分型结果完全一致。广西瑶族人群的HPA各等位基因频率分别为:HPA-1a 0.998 2、1b 0.001 8,2a 0.874 6、2b 0.125 4,3a 0.661 5、3b 0.338 5,5a 0.996 8、5b 0.003 2,6a 0.9786、6bw 0.021 4,15a 0.407 7、15b 0.592 3;汉族人群HPA各等位基因频率分别为:HPA-1a 0.998 3、1b 0.001 7,2a0.957 6、2b 0.042 4,3a 0.532 5、3b 0.467 5,4a 0.999 4、4b 0.000 6,5a 0.984 0、5b 0.016 0,6a 0.989 0、6bw 0.011 0,15a 0.534 1、15b 0.465 9;广西瑶族和汉族人群的HPA-7-14w、HPA-16-28w基因均以aa纯合子形式存在。在本组广西瑶族人群中发现2例罕见的HPA-6bb纯合子基因型个体,而在汉族人群中发现了1例中国人群中罕见的HPA-4ab基因型个体。结论建立了完善的HPA-1-28w PCR-SSP基因分型检测技术体系,并成功应用于广西瑶族和汉族人群的HPA-1-28w基因筛查和分型研究;广西瑶族、汉族人群HPA基因频率分布在HPA-2,-3,-5,-15系统和HPA-6w等均不同(P<0.05),藉此有助于了解广西瑶族和汉族人群HPA的免疫学特点。

Objective To develop a genotyping technique system for human platelet antigens （HPAs）-1 to-28w,and to study on the gene polymorphism of HPA-1 to-28w in Chinese Guangxi Yao and Han population with the developed genotyping technique system.Methods The HPA-1-28w DNA-based genotyping technique system was established by designing sequence-specific primers and optimizing PCR reaction conditions.The technology system consisted of HPA-1-17w multiPCR-SSP technology,which was established and reported earlier,and the newly developed HPA-18-28w PCR-SSP technology method.The effectiveness of the HPA-18-28w PCR-SSP genotyping technique was evaluated by using the samples that had been previously identified the HPA-18-28w genotype by sequencing （10 samples of Guangxi Yao population） or by commercial genotyping kit （20 samples of Guangxi Yao population）.A total of 1 424 and 908 unrelated and random healthy individuals from Yao and Han ethnic groups were studied for HPA-1-28w polymorphism by the developed technique.Results The genotyping results of HPA-18-28w for the validation samples by our developing technique were consistent with both of the results of sequencing genotyping and commercial kit＆#39;genotyping.The HPA allele frequencies in Guangxi Yao population were HPA-la 0.998 2,lb 0.001 8,2a 0.874 6,2b 0.125 4,3a 0.661 5,3b 0.338 5,5a 0.996 8,5b 0.003 2,6a 0.978 6,6bw 0.021 4,15a 0.407 7,15b 0.592 3.The HPA allele frequencies in Guangxi Han population were HPA-la 0.998 3,lb 0.001 7,2a 0.957 6,2b 0.042 4,3a 0.532 5,3b 0.467 5,4a 0.999 4,4b 0.000 6,5a 0.984 0,5b 0.016 0,6a 0.989 0,6bw 0.011 0,15a 0.534 1,15b 0.465 9.In Guangxi Yao and Han population,the only homozygous a/a genotype was detected in HPA-7-14w and HPA-16-28w.The rare HPA-6b/6b genotype was found in two individuals of Guangxi Yao population,and HPA-4a/4b genotype that was rare in Chinese was found in one individual of Han ethnicity.Conclusion In this study,the HPA-1-28w genotyping technique system has been established.The genotyping technique system can be applied to HPA-1-28w genotyping research and screening.The results showes that the frequency distribution of HPA-1-28w gene in Guangxi Yao and Han population is significant in HPA-2,-3,-5,-15 system and HPA-6w （P〈0.05）.The data will help to understand the immunological characteristics of HPA in Yao and Han populations in Guangxi,China.