脐血HLA-A位点PCR-SBT分型的研究

HLA-A gene polymorphism detected by high-resolution sequence-based typing in 547 cord blood units

目的建立HLA-A位点等位基因的PCR-SBT高分辨分型方法,探讨DNA测序技术在脐血库样本HLA分型中的应用价值。方法利用PCR产物直接测序,对广州脐血库保存的547份脐血样本进行HLA-A位点2、3、4外显子的序列分析,由分型结果得出基因频率,与中华(上海)骨髓库北方人群、上海地区人群及德国白种人进行比较。结果采用PCR-SBT分型方法并结合分析软件确定了全部样本的HLA-A基因型,广州地区人群HLA-A等位基因以A*110101(30.8%)最为常见,其后依次是A*24020101/02L(16.18%)、A*0207(11.88%)、A*3303(9.42%)。A*110101在广州汉族人群中出现的频率明显高于中华(上海)骨髓库北方人群,而A*010101、A*3001明显低于后者;在HLA-A2亚型人群中,A*020101在广州、上海两地汉族人群中的频率明显低于德国白种人,而广州汉族人群中A*020101与A*0206均明显低于上海汉族人,但A*0203明显高于后者。结论基于核酸序列测定的HLA分型技术能够直接、准确、快速地进行高分辨分型,将有助提高无亲缘关系供者脐血移植的临床效果。改进实验条件、升级分型软件,可以降低试剂成本和节约时间。

Objective To estabhsh a HLA-A alleles typing method with PCR-SBT and to investigate its application in HLA gene typing of cord blood units in Cord Blood Bank. Methods DNA samples of 547 cord blood units from Guangzhou Cord Blood Bank were extracted with QIAampTM Blood kits to assure their purity （A260/280 from 1.6-1.8）, then the sequences of HLA-A loci exon 2, 3, 4 fragments were analyzed by direct sequencing of PCR products with ABI 3100 automatic sequencer and dedicated software. The gene frequencies, which represent HLA-A gene polymorphism in Guangzhou Han population, were compared to those in China （Shanghai） Bone Marrow Donor Registry （ n = 3736）. The distribution of HLA-A2 alleles of these cord blood units was also compared to those of Shanghai Han population （ n = 45 ） and German Caucasians （ n = 116 ） . Results HLA-A high-resolution genotypes of 547 samples were assigned successfully by PCR-SBT method. A total of 28 different HLA-A alleles were detected. The most frequent alleles were A ＊ 110101 （GF = 30.80% ）, A ＊ 24020101/ 02L （GF = 16.18 % ）, and A ＊ 0207 （GF = 11.88 % ）, followed by A ＊ 3303 （GF = 9.42 % ）, A ＊ 0203 （GF = 7.95%）, and A ＊ 020101 （GF= 6.40%）. The frequencies of following alleles, A ＊ 1102, A ＊ 0206, A ＊ 3001, A ＊ 2601, A＊ 310102, A＊ 03010101/02N, A＊ 29010101/02N, were all higher than 1%. The homozygous alleles include A ＊ 110101, A ＊0203, A ＊ 0206, A ＊ 0207, A ＊ 24020101/02L, A ＊ 2601 and A ＊ 3303. The frequencies of HLA-A ＊ 110101 allele was significantly higher than those in China （Shanghai） Bone Marrow Donor Registry, while the frequencies of A ＊ 010101 and A ＊ 3001 alleles were significantly lower. In HLA-A2 allele groups, there were a significant decrease in frequency of A ＊ 020101 in two Chinese Han populations of Guangzhou and Shanghai than that in German Caucasians, while the frequencies of A ＊ 020101 and A ＊ 0206 were significantly lower and A ＊ 0203 was significantly higher in Guangzhou than those in Shanghai. Conclusion PCRSBT method could determine HLA genotype in high-resolution level accurately and rapidly. The cost and time were less with the improvement of experimental systems and update of analysis software. Our results of HLA-A gene polymorphism in Guangdong Chinese Han population, especially the HLA-A2 allele distribution, would be of benefit for some genetic-associated study also forensic identifications.