陕西汉族初筛RhD阴性孕妇中D基因多态性研究

Study on D Gene Polymorphism in RhD Negative Pregnant Women of the Han Nationality in Shaanxi Province

【目的】探讨陕西汉族初筛RhD阴性孕妇中D基因多态性。【方法】选择2018年1月至2020年3月在本院定期产检的54例陕西汉族初筛RhD阴性孕妇。采用微柱凝胶卡检测RhD血型,采用抗人球蛋白凝胶卡法进行间接抗球蛋白试验(IAT),采用Rh分型卡鉴定RhCE抗原分型,采用吸收放射试验检测Del血型,采用序列特异性聚合酶链反应(PCR-SSP)鉴定标本的Del血型基因型,采用D-Screen试剂的反应格局表鉴定部分DⅥ3型,针对10个RHD基因外显子的特异性引物进行PCR扩增并测序。【结果】54例初筛RhD阴性孕妇中,单克隆(IgG+IgM)抗D-抗体IAT试验检出3例阳性,经鉴定为D变异型;酸放散法检出阳性18例,除上述3例D变异型外,其余15例均为Del表型,36例D阴性标本。D变异型与Del表型均未产生抗-D,36例D阴性标本中2例产生抗-JKb合并抗-cE,7例产生抗-D(其中1例产生抗-C合并抗-D)。RhCE分型结果显示:D阴性血型以ccee为主,Del表型以Ccee为主,无ccEe与ccee。3例D变异型中,D-Screen检出2例部分DⅥ3型,剩余1例经RHD基因测序分析,结果显示为弱D15型,RHD基因存在c.845G>(p.Gly282Asp)纯合突变。PCR-SSP检出15例阳性,与酸放散法检出阳性的例数一致,15例Del表型均携带RHD*01EL.01。15例Del表型中,合子型D+/D+1例,且其基因型为RHD*01EL.01/01EL.01;合子型D+/D-14例,且其基因型均为RHD*01EL01/01N.01,占93.33%。【结论】54例陕西汉族初筛RhD阴性孕妇中D基因结构存在复杂的多态性,变异等位基因D阴性血型以ccee为主,Del表型以Ccee为主。

【Objective】To investigate the D gene polymorphism in RhD negative pregnant women in the Shaanxi Han nationality.【Methods】From January 2018 to March 2020,54 Han women in Shaanxi who were RhD negative during the initial screening were selected for regular prenatal examination in our hospital.RhD blood group was detected by microcolumn gel card;Indirect antiglobulin test(IAT)was performed by anti human globulin gel card method;RhCE antigen typing was identified using Rh typing cards;Del blood group was detected by absorption radiation test;Sequence specific polymerase chain reaction(PCR-SSP)was used to identify the Del blood group genotype of the specimens;The reaction pattern table of D-Screen reagent was used to identify some types of DⅥ3.PCR amplification and sequencing were performed on 10 specific primers for RHD gene exons.【Results】Of the 54 pregnant women who were RhD negative in the initial screening,3 were positive for monoclonal(IgG+IgM)anti D antibody IAT test and identified as D variant.Acid diffusion method detected 18 positive cases,except for the above 3 cases of D variant,the remaining 15 cases were all Del phenotypes,and 36 cases were D negative samples.Neither the D variant nor the Del phenotype produced anti D,and 2 of the 36 D negative specimens produced anti JKb combined with anti CE,while 7 produced anti D(1 case of them produced anti C combined with anti D).The RhCE typing results showed that the D negative blood group was mainly ccee,the Del phenotype was mainly Ccee,and there were no ccEe and ccee.Among the 3 cases of D variant,D-Screen detected 2 cases of partial DⅥ3 type,and the remaining 1 case was identified as weak D15 type by RHD gene sequencing analysis.The RHD gene had a homozygous mutation of c.845G>(p.Gly282Asp).PCR-SSP detected 15 positive cases,consistent with the number of positive cases detected by acid diffusion method.The 15 Del phenotypes all carried RHD*01EL.01.Among the 15 Del phenotypes,there were D+/D+1 cases with a zygotic genotype of RHD*01EL.01/01EL.01;There were 14 cases of homozygous type D+/D-14,and their genotype was RHD*01EL01/01N.01,accounting for 93.33%.【Conclusion】There are complex polymorphisms in the structure of D gene in 54 cases of RhD negative pregnant women in the Han nationality of Shaanxi province.The variant allele D negative blood group is mainly ccee,and the Del phenotype is mainly Ccee.