直接测序法用于四川骨髓库汉族人群HLA-C等位基因分布的研究  

Study of HLA-C distribution in Chinese Sichuan Han population using PCR-SBT

作　　者：夏玲[1,2] 王珏[2] 罗玫[2] 纪欣[2] 杨刘[2] 陈雪黎[2] 陈强[2,3] 

机构地区：[1]中国医学科学院北京协和医学院研究生院,北京100000 [2]中国医学科学院北京协和医学院输血研究所 [3]四川省脐带血造血干细胞库

出　　处：《中国输血杂志》2010年第8期596-601,共6页Chinese Journal of Blood Transfusion

摘　　要：目的建立HLA-C等位基因通用引物直接测序法,研究中华骨髓库四川分库(简称四川骨髓库)中川籍汉族人群HLA-C等位基因分布。方法在四川骨髓库已获HLA-A/B/DRB1高分辨分型结果的600余份川籍汉族街头无关自愿捐献者标本中,随机选择244份标本,采用PCR-SBT对HLA-C位点测序分型,通用引物测序后产生的模棱两可分型结果,分别采用加测相应外显子和组特异性测序方法确认;获得所有标本确切的高分结果后,计算HLA-C各等位基因分布频率,并与其他人群比较。结果 244份标本中直接出HLA-C位点高分辨结果的比例为27.87%(68/244),须加测外显子1、5、6和7的为61.07%(149/244),须组特异性测序的为47.54%(116/244);共检出HLA-C等位基因25个,其中等位基因频率>10%的3个:C*01∶02、C*07∶02和C*03∶04,等位基因频率>1%的12个,累计频率95.69%;四川汉族人群HLA-C等位基因的分布与中国南方人群最为接近,与美国高加索人群和黑人的差异最大。另外,发现了1例新等位基因C*06∶45,它与同源性最高的C*06∶02在第187位碱基存在1个点突变:G>T,使密码子39由GAC>TAC,导致编码的氨基酸由天门冬氨酸(Asp)>酪氨酸(Tyr);此位点在此之前尚未发现过碱基突变。结论建立了针对HLA-C基因第2—4外显子的通用引物直接测序法,并提出了模棱两可结果的解决策略,所测标本均获得确切高分结果;四川汉族人群中HLA-C等位基因呈现多样性并存在自身特点。Objective To investigate the allelic distribution of HLA-C loci in Han individuals from Sichuan branch of Chinese Marrow Donor Program（CMDP） using PCR-SBT.Methods A total of 244 peripheral blood DNA samples,with known HLA-A/B/DRB1 results,were randomly chosen from Sichuan branch of CMDP.Exons 2,3 and 4 of each sample were sequenced using PCR-SBT.Additional exon sequencing and haplotype sequencing were performed to resolve the allele ambiguities.Allele frequencies of HLA-C in Sichuan area were calculated and compared with different populations.Results Of the 244 samples,68 showed conclusive typing results.As to the remaing inconclusive results,149（61.07%） and 116（47.54%） samples needed additional exon sequencing and haplotype sequencing,respectively.All allele ambiguities were resolved during the subsequent testing,and a total of 24 different HLA-C alleles and a novel allele were identified.The most common alleles,with the allele frequency〉 10%,were C^＊01∶02（21.93%）,C^＊07∶02（18.65%） and C^＊03∶04（13.11%）.And 12 kinds of allele were detected with the allele frequency 〉1%,which comprised 95.69% of the total alleles.Comparison of the HLA-C distribution with different populations showed that little difference was observed between Sichuan Han and southern Chinese population,whereas there were significant differences between Sichuan Han and Eastern European American /African American populations.In additional,a novel HLA-C allele C^＊06∶45 was found in our study.This novel allele differs from the closet matching allele C^＊06∶02 by a non-synonymous nucleotide substitution at the first position of codon 39（GAC〉TAC）,resulting in an amino acid change from Asp to Tyr.Interestingly,no substitution at this conserved codon has ever been found before.Conclusion A reliable HLA-C SBT technique,from exon 2 through exon 4,was established in our study.In additional,strategies to resolve the allele ambiguities were also described.The HLA-C allele frequencies we obtained indicate that

关 键 词：HLA-C等位基因 四川汉族 中华骨髓库 直接测序 通用引物测序 组特异性测序 C^＊06∶45 新等位基因 

分 类 号：R457.11[医药卫生—治疗学]