人类白细胞抗原新等位基因A*2636的确认  

Confirmation of human leukocyte antigen novel allele A*2636

作　　者：钮泽雅[1] 王艺芳[1] 杜广有[1] 张伯伟[1] 

机构地区：[1]河南省红十字血液中心,河南省郑州市450012

出　　处：《中国组织工程研究与临床康复》2009年第18期3471-3474,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

摘　　要：背景:目前中华骨髓库主要采用分辨率较高的聚合酶链反应-寡核苷酸探针分型反向杂交荧光微珠法检测人类白细胞抗原HLA-A、B、DRB1,该方法在提高分辨率的同时,有些检测结果难以一次性确定,可采用DNA测序技术确认。目的:识别和确认一个人类白细胞抗原新等位基因。设计、时间及地点:以DNA为观察对象的开放性实验。常规初检聚合酶链反应-寡核苷酸探针分型反向杂交荧光微珠法于2007-11在河南省红十字血液中心中华骨髓库河南分库人类白细胞抗原组织配型实验室完成,测序实验于2008-02在戴诺生物技术(北京)有限公司人类白细胞抗原实验室完成。材料:中国造血干细胞捐献者初检结果可疑为新基因的重采血样5mL。方法:采用聚合酶链反应-寡核苷酸探针分型反向杂交荧光微珠法进行常规人类白细胞抗原分型,发现A位点反应格局异常,提示有2个无法解释的假阳性探针反应(10FP、88FP)和1个假阴性探针反应(58FN)、不能正常判读给出确切结果时,采用DNA测序技术测定人类白细胞抗原A位点外显子2,3,4(Exon2,3,4)的核苷酸序列,并与已知相近等位基因进行序列对比分析。主要观察指标:测序结果与已知人类白细胞抗原等位基因序列的比较分析。结果:聚合酶链反应-寡核苷酸探针分型反向杂交荧光微珠法基因分析结果显示,受检者人类白细胞抗原基因分型为A*02xx,26xx(10FP、88FP58FN),B*15(75),55xx,DRB1 08xx,11xx,A位点反应格局异常,提示有2个无法解释假阳性反应*(10FP、88FP),有1个假阴性探针反应(58FN),不能指定为任何人类白细胞抗原A位点等位基因。对本例人类白细胞抗原A基因第2,3,4外显子双向之直接测序结果分析显示:A*02010101,A*260101292S/C。分析测序结果峰图,292位置应为C,分析软件提示该基因改变在A26new一侧。该序列与人类白细胞抗原A*260101进行比对,同源性99%,在第2外显子区域292�BACKGROUND： The polymerase chain reaction-specific oligonucieotide probes reverse hybridization fluorescent method has been extensively applied to detect human leukocyte antigen （HLA）-A, B, and DRB1 in China Marrow Donor Program （CMDP） at the moment. The method can improve resolution, but some detection result of samples that hide part of novel genes is ambiguous. The easiest method that confirms the susceptible sample is DNA sequencing. OBJECTIVE： To recognize and confirm a HLA novel allele. DESIGN, TIME AND PLACE： An open experiment was assigned DNA as observational subject. The polymerase chain reaction-specific oligonucleotide probes reverse hybridization fluorescent method was performed at HLA Tissue Matching Laboratory of Henan Provincial Red Cross Blood Center, Henan Branch Bank of CMDP in November 2007. And sequencing experiments were performed at HLA Laboratory of DYNAL biological technology （Beijing） Co., LTD. in February 2008. MATERIALS： Blood resamples of 5 mL were from China hematopoietic stem cells donors who were suspected to have new gene. METHODS： HLA typing was analyzed regularly by polymerase chain reaction-specific oligenucleotide probes reverse hybridization fluorescent method. It is found that abnormal results of one sample appear in HLA-A site. It is suggested that two unknown false-positive reaction （10FP, 88FP） and one false-negative reaction （58FN） were presented, and the exact results could not be read out. DNA sequencing based technology （SBT） were used to determine the nucleotide sequence of HLA-A gene exons 2, 3, 4, and then a contrastive analysis of the close known allele was performed. MAIN OUTCOME MEASURES： The comparison between the sequencing result and the known HLA allele sequence. RESULTS. The polymerase chain reaction-specific oligonucleotide probes reverse hybridization fluorescent method genetic analysis results showed that the gene type of client HLA was A ＊ 02xx, 26xx （10FP, 88FP, 58FN）, B ,15 （75）, 55xx, DRB1 ＊08

关 键 词：人类白细胞抗原 新等位基因 序列分析 

分 类 号：R318[医药卫生—生物医学工程]