中国汉族RhD血型呈阴性无偿献血者RHD基因的分子生物学研究

Molecular biology study of RHD gene in Chinese Han voluntary blood donors with RhD-negative blood type

目的 探讨RhD血型呈阴性无偿献血者的RHD基因结构,并且对1例新发现的RHD基因突变进行分子生物学分析.方法 采用随机数字表法选择2016 2017年于山东省血液中心参加无偿献血的85例RhD血型呈阴性献血者为研究对象.采用实时荧光定量（RQ）-PCR方法对85例献血者DNA样本的RHD基因第7号外显子进行检测.采用多重PCR-序列特异性引物（SSP）方法对献血者DNA样本的RHD基因第3～7和9号外显子进行检测.并且采用荧光标记的Sanger双脱氧链终止法对表达RHD基因第7号外显子的献血者DNA样本的RHD基因进行测序.本研究遵循的程序符合2013年修订版《世界医学会赫尔辛基宣言》的要求,与受试者签署临床研究知情同意书.结果 ①本研究85例RhD血型呈阴性献血者中,30例献血者的DNA样本存在RHD基因第7号外显子,55例献血者的DNA样本RHD基因第7号外显子缺失.②30例DNA样本存在RHD基因第7号外显子的献血者中,29例献血者的DNA样本存在RHD基因的第3～7和9号外显子;1例DNA样本存在RHD基因第3、4、5、7和9号外显子,但是缺失第6号外显子.55例DNA样本RHD基因第7号外显子缺失献血者的RHD基因第3～7和9号外显子均缺失.③30例DNA样本存在RHD基因第7号外显子的献血者中,29例献血者DNA样本的RHD基因第9号外显子上均存在1个c.1227G＞A点突变.多重PCR-SSP检测结果显示,缺失RHD基因第6号外显子的1例献血者DNA样本,在RHD基因第6外显子上存在1个c.933C＞A的无义突变,该突变产生1个终止密码子,导致该例献血者RHD基因多重PCR-SSP与DNA测序结果不一致.结论 RHD基因缺失是引起中国汉族人群RhD阴性表型的主要分子机制.RHD基因c.1227G＞A突变是Del表型的主要特征.1个新发现的RHD基因c.933C＞A突变,导致RhD阴性表型产生.

Objective To explore the RHD gene structure of Chinese Han voluntary blood donors with RhD-negative blood type,and to conduct a molecular biological analysis of a newly discovered RHD gene mutation.Methods From 2016 to 2017,a total of 85 Chinese Han voluntary blood donors who participated in blood donation in Shandong Blood Center were selected as the subject by randomized digital table.Real-time fluorescence quantitative （RQ）-PCR method was used to detect exon 7 of RHD gene in DNA samples from 85 blood donors.The multiplex PCR-sequence specific primer （SSP） method was used to detect exons 3 to 7 and 9 of RHD gene in DNA samples from these 85 blood donors.The RHD gene in DNA samples from 30 blood donors whose DNA samples expressed RHD gene exon 7 were sequenced using a fluorescently labeled Sanger dideoxy chain termination method.The procedure of this study is in accordance with the requirements of the revised World Medical Association Declaration of Helsinki in 2013.Informed consent was obtained from each blood donor.Results ① In 85 blood donors with RhD-negative blood type,DNA samples of 30 donors had exon 7 of RHD gene,and of 55 blood donors had deletion of exon 7 of RHD gene.② Among the 30 donors with RHD gene exon 7,the DNA samples of 29 donors had exons 3 to 7 and 9 of the RHD gene.Exon 3,4,5,7 and 9 of RHD gene were present in DNA sample of one donor,but exon 6 was deleted.DNA samples from 55 donors without RHD gene exon 7,were deletion of RHD gene exons 3 to 7 and 9.③ Among the 30 blood donors with RHD gene exon 7,29 DNA samples had one c.1227G〉A point mutation on RHD gene exon 9.There was a non-sense mutation in c.933C〉A on RHD gene exon 6 in one blood donor＇s DNA sample with deletion of the exon 6 of RHD gene.This mutation produced a stop codon,resulting in inconsistent multiplex PCR-SSP and DNA sequencing results of RHD gene in this blood donor.Conclusions The deletion of RHD gene is the main molecular mechanism that causes the RhD-negative phenotype of Chinese Han people.The RHD gene c.1227G〉A mutation is a major feature of Del phenotype.A new c.933C〉A mutation in the RHD gene results in a RhD-negative phenotype.