KIR2DS4基因分型方法建立及新等位基因KIR2DS4^(*)016的鉴定

Establishment of sequence-based typing assay for KIR2DS4 gene and identification of a new allele KIR2DS4^(*)016

目的建立稳定的KIR2DS4基因测序分型法并研究KIR2DS4在中国汉族人群中等位基因多态性。方法采用PCR序列特异性引物(sequence specific primer,SSP)分析法对222名随机中国汉族个体基因组中的KIR2DS4基因进行阴阳性初筛鉴定,对KIR2DS4阳性的标本采用高保真、长片段PCR测序分型技术(PCR-sequence based typing,PCR,PCR-SBT)对KIR2DS4基因的第4、5外显子的片段进行扩增、测序及分型。结果建立的PCR-SBT方法成功扩增出KIR2DS4基因的第4、5外显子序列,长度达3.2 kb。探究中国汉族人群中KIR2DS4等位基因型及频率。检出KIR2DS4阳性个体为209个,阳性率为94.1%。测序分型共发现12种基因型和7种等位基因,6种已知的等位基因及其检出频率为:KIR2DS4^(*)00101/011(180次,81.1%),KIR2DS4^(*)010(53次,23.9%),KIR2DS4^(*)004(34次,15.3%),KIR2DS4^(*)003(15次和6.8%),KIR2DS4^(*)006(2次,0.9%)以及KIR2DS4^(*)015(1次,0.5%)。发现并鉴定一个新等位基因KIR2DS4^(*)016,与其最相近的等位基因KIR2DS4^(*)010区别在第5外显子,KIR2DS4^(*)010的第5外显子是22 bp缺失型,而KIR2DS4^(*)016的第5外显子为完整型。该新等位基因在受检人群中被发现3次,频率为1.4%,并非罕见等位基因,该等位基因序列已向GenBank(登录号:KC414890)及IPD-KIR数据库(提交号:IWS40001804)提交,并获得世界卫生组织HLA系统命名因子委员会的命名。结论本文中我们成功建立了KIR2DS4基因4、5外显子测序分型方法,并对其等位基因多态性进行了探究,发现了新的KIR2DS4等位基因型,丰富了中国汉族人群KIR2DS4基因多态性数据。

Objective To establish a reliable sequence-based typing method for KIR2DS4 and study its allele polymorphism in Chinese Han population.Methods Using PCR-SSP method to detect the positive or negative of KIR2DS4 gene in 222 random Chinese Han individuals,and then using the method of high fidelity and long-fragment PCR-SBT to amplified,sequence and genotype the exons 4 and 5 of KIR2DS4 positive individuals.Results We successfully amplify the fragment with 3.2 kb length contains exons 4 and 5 of KIR2DS4 and detected the KIR2DS4 allele frequency in Chinese Han population.209 KIR2DS4 positive individuals were detected,and the positive rate is 94.1%.By sequence-based typing,we identified 12 genotypes and 7 alleles of KIR2DS4.The 6 known alleles and their detection frequency is as follows:KIR2DS4^(*)00101/011(180,81.1%),KIR2DS4^(*)010(53,23.9%),KIR2DS4^(*)004(34,15.3%),KIR2DS4^(*)003(15 and 6.8%),KIR2DS4^(*)006(2,0.9%)and KIR2DS4^(*)015(1,0.5%).In this study,we found a new allele,KIR2DS4^(*)016,with the difference in exon 5 comparing its most similar allele KIR2DS4^(*)010.In the exon 5 of KIR2DS4^(*)010,there is a 22bp-deletion,while the exon 5 of KIR2DS4^(*)016 is normal.This is not a rare allele because it was detected 3 times in studied population and with the frequency of 1.4%.The sequence of the new allele sequence has been submitted to GenBank(accession no.:KC414890)and the IPD-KIR database(submission no.:IWS40001804),and was nominated by WHO nomenclature committee for HLA system.Conclusion In this study,a sequence-based typing method for KIR2DS4 was established,and the polymorphism data of KIR2DS4 in Chinese Han population was enriched by studying the allele polymorphism and new allele.