罕见弱表达D-变异体的遗传背景分析和家系调查

发布时间：2019-01-04 14:19

【摘要】：背景:Rh血型系统是所有血型系统中最复杂的血型系统,包含了 50多种抗原,在胎母免疫和新生儿胎儿溶血病(HDFN)中,是最具临床价值的血型系统。RHD、RHCE基因编码决定了 Rh血型抗原。D--变异体是Rh血型系统中一种非常罕见的Rh基因变异型,其红细胞膜上只有D抗原,而C、c、E、e均不能正常表达。本文对D--变异体综合进行RHD、RHAG、RHCE基因、血清学以及家系分析。目的:研究D--变异体的分子遗传背景,以期能够发现新的Rh血型变异体发生机制,同时初步探索了中国人RHAG血型系统中RHAG高频抗原,以及RhD、RhCE抗原表达的影响因素。方法:1.研究对象实验样本来源于甘肃省血液中心的1个D--个体样本及家系成员(其弟弟,父亲,母亲),所有实验样本均是经过血液中心法定检测项目的合格血液。2.实验方法(1)血清学Rh表型检测:分别用盐水试管法、微柱凝胶卡法和间接抗人球蛋白试验鉴定D--个体样本及家系成员RhD、C、c、E和e抗原。(2)序列特异性PCR法(PCR-SSP)检测RHCE基因:设计4对序列特异性引物,根据体系内扩增产物的有或无来判断RHCE基因型。(3)测序:依据RHD、RHCE基因序列的特异性,分别扩增这3个基因的10个外显子,确认产物条带符合后将扩增产物送公司纯化测序,测序序列与标准基因序列进行比对。(4)RHD合子型分析:采用双管PCR方法扩增融合Rh盒子和RHD基因第一外显子,通过扩增产物的条带一次性判断3种RHD合子型。(5)家系分析:通过RHCE基因测序结果结合RHD合子型分析绘制D--个体的家系谱图。结果:1.血清学测定个例为弱D--表型;2.RHC 基因测定结果为DC-表型;3.RHD、RHAG、基因编码区第1-10外显子测序,结果与标准序列一致。RHAG基因未观察到形成Oldeide和Duclos-Like抗原的碱基变异,亦未见形成高频抗原Duclos的RHAG316CG突变,所有序列均未见杂合峰;4.RHCE基因编码区第1-10外显子测序,发现该个例第3-5外显子缺失,其它外显子正常,第1-2外显子为RHC基因型,确定个体携带RHCE(e)-D(3-5)-CE(e)等位基因,因此血清学检测为D--表型,而基因检测为DC-表型;5.先证者父母和其弟的RHAG、RHD基因编码区序列与先证者完全一致,父母Rh血清学表型分别为DCCee和DccEE,父亲的PCR-SSP和测序结果一致,母亲PCR-SSP结果DCcEE与测序结果一致,而与血清学结果不符(DccEE),弟弟血清学表型、PCR-SSP和测序结果均与先证者一致。结合RHD合子型分析结果,该家系父母分别为DCe/DC-和DcE/DC-基因型,先证者和兄弟为DC-/DC-。结论:1.RHCE(e)-D(3-5)-CE(e 等位基因形成D--表型并稳定遗传;2.RHAG Duclos高频抗原在中国人群中需要观察更多标本才能认可。3.本研究未发现RHAG和RHD基因编码区变异。
[Abstract]:Background: the Rh blood group system is the most complex blood group system of all blood groups. It contains more than 50 antigens. It is the most clinically valuable blood group system in fetal immunity and neonatal fetal hemolytic disease (HDFN). RHD, RHCE gene encodes Rh blood group antigen. D- variant is a very rare variant of Rh gene in Rh blood group system. The RHD,RHAG,RHCE gene, serology and pedigree analysis of D-variant were studied. Objective: to study the molecular genetic background of D- variant in order to find out the new mechanism of Rh blood group variant, and to explore the high frequency antigen of RHAG and the factors influencing the expression of RhD,RhCE antigen in Chinese RHAG blood group system. Methods: 1. The experimental samples were collected from a D- individual sample and a family member (brother, father, mother) from the blood center of Gansu province. All the samples were qualified blood from the blood center. 2. Methods (1) serological Rh phenotype detection: D- individual samples and family members RhD,C,c, were identified by saline tube test, microcolumn gel calorie assay and indirect antihuman globulin test, respectively. E and e antigens. (2) sequence-specific PCR assay (PCR-SSP) for detection of RHCE genes: design 4 pairs of sequence-specific primers to determine RHCE genotypes according to the presence or absence of amplified products in the system. (3) sequencing: based on RHD, The specificity of RHCE gene sequence, 10 exons of the three genes were amplified, the products were confirmed to match the bands, and the amplified products were sent to the company for purification and sequencing. (4) RHD zygotypic analysis: the fusion Rh box and the first exon of RHD gene were amplified by double-tube PCR. (5) Family analysis: RHCE gene sequencing and RHD zygote analysis were used to draw the family pedigree of D- individuals. Results: 1. The results of serological analysis were weak D- phenotype, 2.RHC gene was DC- phenotype. 3. RHD-RHAG, exon 1-10 of gene coding region, was sequenced and the results were consistent with the standard sequence. The RHAG gene did not observe the base mutation to form Oldeide and Duclos-Like antigens, nor did RHAG316CG mutation to form high frequency antigen Duclos. No heterozygous peak was found in all sequences. The sequence of exon 1-10 in the coding region of 4.RHCE gene showed that the deletion of exon 3-5 was found in this case, the other exons were normal, and exon 1-2 was RHC genotype. RHCE (e) D (3-5)-CE (e) alleles were identified in individuals, so serological tests showed D- phenotypes, while genes were detected as DC- phenotypes. 5. The sequence of RHAG,RHD gene coding region of parent and younger brother of proband was identical with that of proband. The serological phenotype of parent Rh was the same as that of DCCee and DccEE, father, and the result of DCcEE from mother PCR-SSP was consistent with the result of sequencing. The results of PCR-SSP and sequencing were consistent with those of the proband. Combined with the results of RHD zygote analysis, the parents of the family were DCe/DC- and DcE/DC- genotype, and the proband and brother were DC-/DC-.. Conclusion: 1.RHCE (e) D (3-5)-CE (e alleles form D- phenotypes and be stably inherited, and 2.RHAG Duclos high frequency antigens need to be observed in more samples in order to be recognized. No variation in the coding region of RHAG and RHD genes was found in this study.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R457.11

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