汉族人群中22个HLA-Cw等位基因全长序列单核苷酸多态性分析  

Single nucleotide polymorphisms in 22 HLA-Cw alleles in Chinese Han population

作　　者：曾健强[1] 徐筠娉[1] 王大明[1] 高素青[1] 邹红岩[1] 邓志辉[1] 

机构地区：[1]深圳市血液中心,深圳市组织配型与免疫遗传重点实验室,深圳518035

出　　处：《遗传》2010年第5期473-479,共7页Hereditas（Beijing)

基　　金：深圳市科技计划项目(编号:200802116);广东省科技计划项目(编号:2008B030301277);广东省自然科学基金项目(编号:9451803501004124)资助

摘　　要：为探讨HLA-Cw(Human leukocyte antigen-Cw)基因全长序列分子遗传多态性,文章对28个HLA-Cw基因型已知的汉族个体样本,采用长距离PCR技术和高保真性的Pfu酶,扩增HLA-Cw基因全长序列4.5kb,进行分子克隆和单倍体测序。采用群体遗传学研究方法分析了HLA-Cw等位基因全长序列中各亚区的单核苷酸多态性。结果表明:在28个样本中共检测出22种等位基因,序列均已提交GenBank和国际IMGT/HLA数据库并获得了认可;其中Cw*0706、Cw*030301、Cw*140201的全长序列为首次报道,尤其是Cw*0706内含子序列的获得,能够重新设计对该等位基因测序分型的引物,避免测序分型中可能对这一等位基因的漏检。将28个样本的56条单倍体序列用Clustal软件进行序列排比,输入到DnaSP4.0进行多态性分析,共发现244个SNPs,10处插入/缺失多态性。对HLA-Cw等位基因各亚区多态性的分析,发现第4内含子及以前并没有受到关注的第5外显子受到平衡选择的作用,在进化中受到了选择压力,预示着它们在免疫系统的进化过程中可能扮演着重要的角色。To analyze the molecular genetic polymorphism of full-length HLA-Cw gene,a total of 28 samples with known genotypes from Chinese Han population were amplified by long-range PCR using high-fidelity Pfu polymerase.A fragment 4.5 kb in length of HLA-Cw gene was subjected to cloning and haplotype sequencing.The single nucleotide polymorphisms（SNPs） in all segments of the whole region of HLA-Cw gene were analyzed.As a result,we detected 22 different HLA-Cw alleles in 28 samples,all of which were submitted to GenBank and the IMGT/HLA Database.Among the 22 HLA-Cw alleles,the intronic sequences of Cw＊030301,Cw＊0706 and Cw＊140201 were firstly elucidated.The novel intronic sequence and the SNPs information may help to design allele-specific primers for accurate sequence-based typing（SBT） and to avoid allele dropout events in SBT test.We aligned all the diploid sequences using ClustalX program and imported them into Dnasp4.0 to calculate polymorphism in all coding-and non-coding regions.We found 244 SNPs and 10 insertion/deletions（Indels）.According to the analysis of polymorphism level,phylogenetic trees and frequency spectrum,we proposed that the evolution of intron 4 and exon 5 was under balancing selection.Selection on these segments indicated that they may be functionally important in evolution of HLA-Cw gene.The full-length sequences obtained and related SNPs information can be used as resources of markers for high-resolution typing,complex diseases association studies and human evolution.

关 键 词：人类白细胞抗原 HLA-Cw基因 基因组全长序列 克隆和测序分析 单核苷酸多态性 

分 类 号：R394[医药卫生—医学遗传学]