广州地区RhD阴性献血者Del表型及基因型分析  

Analysis of Del phenotype and genotype in RhD-negative blood donors in Guangzhou area

作　　者：黄伯泉[1] 贾双双[1] 温机智[1] 陈景旺 罗广平[1] 姬艳丽[1] HUANG Boquan;JIA Shuangshaung;WEN Jizhi;CHEN Jingwang;LUO Guangping;JI Yanli(Guangzhou Blood Center,The key Medical Laboratory of Guangzhou,Guangzhou 510095,CHINA)

机构地区：[1]广州血液中心广州市血液安全重点实验室,广东广州510095

出　　处：《中国输血杂志》2024年第8期859-865,共7页Chinese Journal of Blood Transfusion

基　　金：广州市科技计划项目(2023A03J0996);广州市医学重点学科(2021-2023)项目。

摘　　要：目的研究广州地区RhD阴性献血者D放散型(Del)分布、表型及基因型以了解本地区DEL血型分子生物学背景。方法对2021年11月1日—2022年6月30日期间广州地区献血者采用盐水法初筛为RhD阴性血液进行间接抗人球蛋白试验(IAT)血清学确认、采用RhCE分型卡确定RhCE表型,对1146例确认RhD阴性所有C+(n=459)以及随机抽取部分C-标本(n=175)进行吸收放散(Del)筛查(共634例);提取Del阳性标本DNA进行高分辨熔解曲线分析法(HRM)实时荧光PCR检测RHD基因c.1227位点基因情况;对HRM检测RHD^(*)1227位点无突变标本采用限制性片段多态性聚合酶链反应(PCR-RFLP)扩增后产物作PstⅠ酶切,进行电泳分析RHD基因合子型;sanger法进行RHD基因进行外显子测序(exon 1-10),采用SeqMan软件分析基因突变情况;采用第3代单分子测序技术对可疑Del阳性标本进行RHD全基因分析。结果634例确认RhD阴性标本吸收放散试验结果显示Del表型占36.1%(229/634),占总RhD确认阴性的20%(229/1146);229例DEL的RhCE分型分别为Ccee 181例,CCee 40例,CcEe 7例,ccEe 1例,HRM结合RHD盒子型分析结果显示170例RHD基因为RHD^(*)1227A/01N.01、32例为RHD^(*)1227A/1227G,26例为RHD^(*)1227A/1227A、6例为RHD^(*)1227G/1227G(其RHD基因测序结果显示1例为弱D12型、4例为D-和1例RHD^(*)01EL.02)。结论广州地区献血者DEL个体基因型以RHD^(*)1227A/01N.01为主且其RhCE表型均为C+的特征;HRM可作为常规筛查“亚洲型”DEL血型基因的分子生物学方法。Objective To investigate the distribution,phenotype and genotype of D-elute type(Del)in blood donors with RhD negative blood in Guangzhou,so as to understand the molecular biological background of DEL blood group in this area.Methods During the period from November 1,2021 to June 30,2022,the RhD-negative blood initially screened by saline method was confirmed by indirect anti-human globulin test(IAT)serology,and RhCE phenotype was determined by RhCE typing card.A total of 1146 RhD-negative samples,including all RhD-negative samples with RhCE C+(n=459)and a randomly selected subset of RhCE C-(n=175),were subjected to adsorption-elution(Del)screening(a total of 634 samples).DNA from Del-positive samples was extracted for real-time fluorescent PCR detection of the RHD gene c.1227 locus using high-resolution melting curve analysis(HRM).For samples without mutations detected at the RHD^(*)1227 locus by HRM,restriction fragment length polymorphism polymerase chain reaction(PCR-RFLP)was performed to amplify the product which was subsequently digested with Pst I enzyme and analyzed by electrophoresis to determine RHD gene haplotypes.Sanger sequencing was performed for exon sequencing(exon 1-10)of the RHD gene,and gene mutations were analyzed using SeqMan software.Suspected Del-positive samples were subjected to RHD whole gene analysis using third-generation single-molecule sequencing technology.Results Among the 634 confirmed RhD-negative samples,229(36.1%)displayed Del phenotype,accounting for 20%(229/1146)of the total confirmed RhD-negative samples.The RhCE phenotypes of the 229 DEL cases were as follows:Ccee in 181 cases,CCee in 40 cases,CcEe in 7 cases,and ccEe in 1 case.HRM combined with RHD haplotype analysis showed that there were 170 cases with RHD gene as RHD^(*)1227A/01N.01,32 cases with RHD gene as RHD^(*)1227A/1227G,26 cases with RHD gene as RHD^(*)1227A/1227A,and 6 cases with RHD gene as RHD^(*)1227G/1227G(sequencing results included 1 case of weak D type 12,4 cases of D-,and 1 case of RHD^(*)01EL.02).Conclu

关 键 词：无偿献血者 RHD阴性 “亚洲型”DEL RhCE表型 高分辨熔解曲线法(HRM) 

分 类 号：R457.11[医药卫生—治疗学]