洛阳地区无偿献血者Rh血型临床相关高频抗原分布调查

目的:准确估计洛阳地区Rh血型系统Rh表型频率和估算Rh单倍型频率。方法:回顾性统计356041例献血者的RhD检测数据,采用血清学检测10373例RhD阳性献血者的CcEe抗原,Rh基因频率和单倍型频率的计算采用方根法〔1〕。结果:洛阳地区献血者中RhD阳性率为99.54%,其中弱D检出率约为0.02%;阴性率为0.46%,其中Del检出率约为0.03%。Rh抗原基因和单倍型频率为:D:0.9324,d:0.0676,DCe:0.6154,DcE:0.2704,dce:0.0515,Dce:0.0343,dCe:0.0122,DCE:0.0132,dcE:0.0023,DCE:0.0006。弱D以表型Dc-cEe、DCcee为主,Del型以表型DccEe、DCCee为主,RhD阴性献血者表型以dccee、dCcee为主。结论:精确地估算出了献血者中RhD抗原阴性分布频率,并准确计算出RhDCcEe抗原的基因频率和Rh单倍型频率,对一些稀有的Rh弱D型,Del型其D抗原弱表达与C、E抗原有关联。需要制定标准的弱D定型方法和合理的输血策略,来减少抗球蛋白试验对弱D抗原的漏检。     

南京地区RhD阴性个体RHD基因多态性及与RhC抗原表达关系的研究

目的：研究南京地区表型为RhD阴性汉族人群中,RHD基因外显子1～10的多态性以及D基因外显子与C抗原表达之间的关系。方法：随机抽取血清学为RhD阴性的RhCC/Cc标本116例,Rhcc标本117例,PCR-SSP方法检测RHD外显子1、3、4、5、6、7、9、10和RHD内含子2、4v,10以及RHCE内含子2、4。结果：在116例血清学为CC/Cc的RhD阴性标本中,RHD外显子完整者有70例,占60.35%;25例（21.55%）RHD外显子完全缺失;而另有21例（18.10%）RHD外显子部分完整,其中的20例为RHD1-CE（2-9）-D10杂化基因;1例含有D1,Cin2,D9,D10,Din10基因。而在117例血清学为cc的RhD阴性标本中,6例（5.13%）RHD外显子完整;110例（94.02%）RHD外显子完全缺失;1例（0.85%）除D1缺失外,其他RHD2～10外显子均有。结论：南京地区汉族人群RHD基因表现出明显的多态性。cc表型的RHD基因外显子全部缺失的比率（94.02%）为CC/Cc的（21.55%）4倍多,而CC/Cc的RHD外显子完整的比率（60.35%）约为cc的（5.13%）12倍。RHD1-CE（2-9）-D10杂化基因均为CC或Cc表型,在RhD阴性表型中还有少数RHD基因变异型有待研究。     

新疆RhD阴性及其表型特点调查分析

目的:进一步了解新疆地区汉族、维吾尔族、哈萨克族、回族、柯尔克孜族RhD阴性及其表型分布情况。方法:收集新疆维吾尔自治区人民医院2017-11—2018-10随机体检及住院患者28 681例,进行RhD抗原检测,同时对RhD阴性样本进行E、c、C、e抗原鉴定,统计各民族RhD血型抗原及RhD阴性样本表型分布情况并计算其基因频率。结果:初筛RhD阴性抗原频率汉族0.74%、维吾尔族5.92%,哈萨克族1.74%、回族0.53%、柯尔克孜族1.25%;D变异型汉族0.14%、维吾尔族0.37%,哈萨克族0.25%、回族0.05%、柯尔克孜族0;Del占初筛的比例汉族15.71%、维吾尔族5.38%,哈萨克族8.33%、回族0、柯尔克孜族0;基因组合体频率dce维吾尔族哈萨克族汉族,dCe汉族哈萨克族维吾尔族,dcE汉族哈萨克族维吾尔族,dCE哈萨克族汉族维吾尔族;RhD阴性样本E,c,C,e抗原及表型基因频率分布均具有各自的特点,且符合Hardy-Weinberg平衡(P0.05)。结论:本次研究发现新疆地区各民族Rh血型系统具有不同特点,汉族、维吾尔族、哈萨克族、回族、柯尔克孜族人群RhD抗原阴性者均高于其他省份,D变异型及Del型也具有自己的特点,今后可进一步研究,丰富本地区输血学资料,为临床输血提供参考依据。     

内皮型一氧化氮合酶基因多态性与东北汉族人群原发性高血压的相关性

目的通过病例-对照分析研究探讨东北汉族人群中内皮型一氧化氮合酶(eNOS)基因多态性与原发性高血压(EH)的关系。方法本项目主要基于黑龙江省的2次现场流行病学调查研究,总共纳入受试者2208人,其中正常血压组1046人,EH组1162例。通过Sequenom MassArry平台对eNOS基因单核苷酸多态性(SNP)rs1800780,rs2070744,rs891512,rs7830和rs3918227进行基因分型。采用SPSS 20.0软件用于统计基因型和等位基因频率的组间差异,并使用SHEsis软件进行SNP位点Hardy-Weinberg平衡检验和单倍型分析。结果 rs1800780在正常血压组显示Hardy-Weinberg不平衡,其余4个SNP基因型分布在两组中均显示Hardy-Weinberg平衡,因此在后续分析中排除rs1800780。4个SNP的基因型分布和等位基因频率在两组间差异无统计学意义(P0.05);单倍型分析显示,与正常血压组相比,EH组单倍型GCGC(rs891512-rs2070744-rs7830-rs3918227)频率显著增高(1.7%比0.6%,χ~2=8.634,P=0.003,OR=2.834,95%CI1.372～5.856)。结论东北汉族人群中,单个eNOS基因SNP与EH无关,但携带单倍型GCGC(rs891512-rs2070744-rs7830-rs3918227)与EH相关。     

温州地区献血人群Rh血型抗原的分布

目的：掌握温州地区献血人群Rh血型抗原系统的分布规律及基因频率,为Rh血液的储备和临床疑难配血患者的血液供应提供理论依据。方法：采用白瓷板法对165041名献血者血液标本进行RhD抗原初筛,可疑者用盐水试管法进行鉴定;用间接抗人球蛋白试验对初筛RhD阴性的血液标本进行确认,同时用盐水试管法对RhC、c、E和e抗原的检测。结果：RhD阴性率为0.38%,D基因频率为0.9384,d基因频率为0.0616;RhD阴性献血人群的表型分布规律为ccdee〉Ccdee〉CCdee〉ccdEe〉CcdEe〉CCdEe〉ccdEE=CcdEE,cde的基因频率最高;Rh（D）阳性献血人群的表型分布规律为CCDee〉CcDE〉ccDE〉CcDee〉ccDee〉CCDE,组合体基因频率分布规律为CDe〉cDE〉cDe〉CDE。结论：本地区RhD阴性率符合我国汉族人群分布情况,RhD阳性献血人群的表型分布有地域性的特点,从而为建立和完善本地区更安全,快速的有效的临床输血提供了一定的理论基础资料。     

中国汉族人群NOD2、IRGM、ATG16L1和STAT4基因多态性与克罗恩病的相关性研究

背景：克罗恩病（CD）的病因和发病机制尚未完全阐明,近年国外研究发现NOD2、IRGM、ATG16L1、STAT4基因突变与CD相关。目的：分析NOD2、IRGM、ATG16L1、STAT4基因多态性与中国汉族人群CD发病的相关性。方法：连续纳入2007年1月～2010年1月苏州市立医院中国汉族CD患者66例,66名健康体检者作为正常对照,以PCR联合基因测序检测4种基因相应单核苷酸多态性（SNP）位点的基因型,分析各基因型和等位基因频率。结果：CD组和正常对照组NOD2基因rs2066842位点、IRGM基因rs13361189位点、ATG16L1基因rs2241880位点和STAT4基因rs7574865位点基因型和等位基因频率分布均符合Hardy-Weinberg遗传平衡定律,两组间4种基因相应SNP位点的基因型和等位基因频率差异均无统计学意义。结论：NOD2、IRGM、ATG16L1和STAT4基因多态性与中国汉族人群CD发病不相关。     

江夏地区人群RhD(-)血型系统基因频率的分布

目的:探讨RhD阴性血型基因及其MN、P血型基因在江夏地区人群中的分布频率。方法:采用血清学方法对江夏区42 940例住院患者及健康体检者进行RhD、C、c、E、e、M、N、P1抗原表现型检测。结果:共检出RhD阴性136例,其中ccee 76例(50.88%),CCee 10例(7.35%),Ccee 42例(30.88%),ccEe 6例(4.41%),CcEe 2例(1.47%)。从而得出RhD阴性者中C基因频率为0.235 3,c基因频率为0.764 7,E基因频率为0.029 4,e基因频率为0.706,基因组合体频率为cde=0.042,Cde=0.012 6,cdE=0.001 7,CdE=0,并对M、N及P1抗原进行了分布调查,其中M型34例(25%),N型64例(47.06%),MN型38例(27.94%),P1型50例(36.76%)。结论:RhD阴性表型按频率由高到低依次为deeccdeeCcdeeCCdEeccdEeCc,由单倍型来看cde是江夏地区RhD阴性中最常见单倍型,Cde次之,结果比较符合中国汉族RhD阴性血型中Rh单倍型和基因频率分布状况。同时对RhD阴性血的MN及P系统有初步的了解。     

AGTRLl基因变异与原发性高血压

目的探讨AGTRLl基因多态性及单倍型与原发性高血雎的相关关系。方法选取上海地区汉族人群原发性高血压家系248家共1042人为研究样本，应用TaqMan MGB荧光探针定量PCR技术，检测各样本．4GTRLl基因启动子区rs10501367和rs7119375基因多态性分型。应用FBAT软件预测陔两个多态性位点可能组成的单倍型．并对多态位点及单倍型与原发性高血压的关系进行分析。结果所选位点基因型频率在研究人群中的分布均符合Hardy—Weinberg遗传平衡定律：应用FBAT（显性模型遗传模式）统计分析结果显示rs10501367、rs7119375及其组成的单倍型G—G与原发性高血联相关。其中rs7119375的G等位基因（Z=2．390，P=0．017）和rs10501367的G等位基因（Z=2．177．P=0．030）可以由亲代下传给患病子代。结论位于AGTRLl基因启动子区的多态性位点与上海地区汉族人群原发性高血乐存在相关关系，单倍型G—G可能对高血压的发病有贡献．     

细胞间黏附分子-1基因469K/E多态性与心肌梗死的关系探讨

目的探讨细胞间黏附分子-1(ICAM-1)基因K469E多态性与中国汉族人群心肌梗死(MI)发病率的关系.方法采用巢式PCR技术对206例(MI89例)进行ICAM-1基因K469E多态性检测分析.结果基因型频率符合Hardy-Weinberg平衡,MI组和对照组KK、KE、EE基因型频率分别是37.1%、43.8%、19.1%和27.4%、38.5%、34.2%;等位基因K、E频率分别是59.00%、41.00%和46.58%、53.42%,两组差异有统计学意义.回归分析显示,K等位基因、吸烟增加了MI发病的危险,并对MI的发生具有协同作用.结论中国汉族人群存在ICAM-1基因K469E多态性,ICAM-1基因469K/E多态性与MI的发病有关,K等位基因可能为其独立危险因素之一.     

山东地区汉族人群KIR基因多态性分析

目的 分析山东地区汉族人群杀伤细胞免疫球蛋白样受体(KIR)基因多态性及基因型和单倍型多态性,为进一步研究KIRs与疾病的关系奠定基础.方法 采用序列特异性引物PCR法(PCR-SSP)对412例山东地区无血缘关系的汉族健康志愿者进行KIR基因频率检测及基因型和单倍型分析.结果 ①KIR基因频率:可检测到目前已知的18种KIR基因;所有个体均检测到3个框架基因(2D14、3DL2、3D13)以及KIRZ,其基因频率均为100%.3DP1、2DL3、2DL1、3DL1和2DS4基因较为常见,频率分别为99.03%、98.79%、98.79%、98.79%、96.84%;而2DL2、2DS2、3DP1v基因频率较低.②KIR基因型频率:共检出基因型28种,以AJ(2,2)、AF(1,2)型最常见,其次为AH(5,2)、G(4,5)、M(1,5);另外有11种基因型在自人中尚未见报道.③KIR基因单倍型频率:共检出单倍型16种,最常见的是单倍型2,频率为46.36%;其次为单倍型1,频率为25.61%.结论 山东地区汉族人群有其独特的KIRs基因频率、基因型频率和单倍型频率分布;本研究可为进一步研究KIRs与疾病的相关性提供依据.     

Molecular analysis of blood group Rh transcripts from a rGr variant

The Rh blood group antigens D, Cc and Ee are encoded by two related genes, RHD and RHCE . The RhG antigen (Rh12) is associated with the expression of RhC and/or RhD, except in rare variant red cells. Here we have determined the molecular basis of G expression in the absence of D and C in the r^Gr phenotype. Nucleotide sequence analysis revealed that the r  ^G allele resulted either from a segmental DNA exchange between part of exon 2 of the RHce gene and the equivalent region of the RHCE or RHD genes or from a crossing over between positions nt150 and nt178 of the RHce and RHCe genes. The predicted protein encoded by the hybrid r  ^G gene ( c-C-e or c-D-e ) carries Ile60, Ser68 and Ser103 (as C and D polypeptides); any of these positions appear to be critical in the formation of the G antigen. In addition, Cys16 was found to be important in the phenotypic expression of C.     

A two-locus gene conversion model with selection and its application to the human RHCE and RHD genes

A two-locus gene conversion model with selection is developed. Under the joint action of selection, mutation, gene conversion, recombination, and random genetic drift, approximate formulas for the expectations of the moments of allele frequencies and the expected amounts of variation within and between two loci are obtained by a diffusion method assuming relatively strong selection. It is shown that the pattern of allelic variation is mainly determined by the balance between gene conversion and selection, because these two mechanisms act in opposite directions. As an application of the theoretical results, the human RHCE and RHD genes are considered. The very high level of amino acid divergence between the two genes is observed only in a short region around exon 7. It is known that exon 7 encodes amino acids that characterize the difference between the RHCE and RHD antigens. The observed pattern of DNA variation in this region is consistent with the selection model developed in this article, suggesting that strong selection might be working to maintain the RHCE/RHD antigen variation in the two-locus system. The selection intensity is estimated on the basis of the theoretical result.     

Molecular basis of the rare gene complex, DIVa(C)-, which encodes four low-prevalence antigens in the Rh blood group system

Background Over 40 years ago, an unusual Rh phenotype denoted DIVa(C)‐ was identified in a case of fatal haemolytic disease of the newborn in the third child of Madame Nou. Her RBCs expressed a partial D, weak C and four low‐prevalence Rh antigens: Go^a (RH30), Rh33 (RH33), Riv (RH45) and FPTT (RH50). The purpose of this study was to determine the molecular basis associated with this rare DIVa(C)‐ complex. Material and Methods Blood samples were from three donors previously identified as carrying the DIVa(C)‐ haplotype. Molecular analyses were performed by standard methods. Results The three donors were heterozygous for RHD and RHD*DIVa.2, and all carried a compound hybrid allele at the RHCE locus. This hybrid RHCE allele contained exons 2 and 3 from RHD*DIVa.2 and exon 5 from RHD [RHCE*CE‐DIVa.2(2‐3)‐CE‐D(5)‐CE] and is in cis to RHD*DIVa.2. The RHCE allele on the in trans chromosome differs between the donors and is RHCE*cE in donor 1, RHCE*ce (254C, 733G) in donor 2 and RHCE*ce in donor 3. Conclusions The RHD*DIVa.2 encodes the Go^a antigen, whereas the compound hybrid allele most likely encodes Rh33, Riv and FPTT. The weakly expressed C antigen on RBCs with the DIVa(C)‐ phenotype could be encoded by exons 2 and 3 from RHD*DIVa.2 in the compound hybrid. This is the first report of RHD*DIVa.2 being involved in a hybrid gene at the RHCE locus. As only one example of anti‐Riv has been described, our molecular analysis and findings provide a tool by which to predict Riv expression.     

RHD Gene Polymorphisms among RhD-Negative Chinese in Taiwan

Background and Objective: The rare occurrence of anti-D-associated hemolytic disease of the newborn among Chinese is attributable in part to the existence of the weak D phenotype D_el among apparently RhD-negative individuals. While exciting advances in the molecular genetics of the Rh blood group have been noted in recent years, the genomic structure of the D_el phenotype has seldom been studied in the literature. We try to explore the genomic structure of the RhD gene among apparently Rh-negative Chinese in Taiwan in this study. Methods: Genomic DNA from 230 samples of apparently RhD-negative Chinese was studied using four polymerase chain reaction (PCR)-based RhD typing methods. These PCR methods amplified RHD and RHCE genes at exons 4, 5, 7 and 10. All nuclieotides responsible for exofacial amino acid differences between RhD and RhCeEe peptides, including amino acids 169, 170, 172, 223, 226, 233, 238, 350, 353, and 354, were contained in these amplified DNA segments. Southern blot analysis using RHD cDNA fragments as probes was performed. Results: According to the serological study, 155 samples (67.4%) were genuinely RhD-negative and 75 samples (32.6%) were of the D_el phenotype. Successful amplifications for RHD sequences were possible in all 75 D_el samples using four PCR methods. Apparently, all D_el individuals carried an intact RHD gene. While 145 individuals of 155 genuinely Rh-negative (63.0% of apparently RhD-negative individuals) had total deletion of their RHD genes, 10 individuals (4.3% of apparently RhD-negative individuals) were shown to have a preserved 3′ noncoding region of the RHD exon 10 and a gross deletion of RHD exons 4–10. Conclusions: Three classes of RhD-negative polymorphisms among Chinese in Taiwan were observed. These included D_el with grossly intact RHD and weak RhD expression, genuinely RhD-negative with total deletion of the RHD gene. A molecular study is warranted to clarify the mechanism responsible for the weak RHD gene expression in D_el individuals.     

Clinically relevant RHD-CE genotypes in patients with sickle cell disease and in African Brazilian donors

BackgroundAs a consequence of the homology and opposite orientation of RHD and RHCE, numerous gene rearrangements have occurred in Africans and resulted in altered RH alleles that predict partial antigens, contributing to the high rate of Rh alloimmunisation among patients with sickle cell disease (SCD). In this study, we characterised variant RH alleles encoding partial antigens and/or lacking high prevalence antigens in patients with SCD and in African Brazilian donors, in order to support antigen-matched blood for transfusion.ResultsThe distributions of RHD and RHCE alleles in donors and patients were similar. We found RHCE variant alleles inherited with altered RHD alleles in 25 out of 168 patients (15%) and in 22 out of 280 (7.8%) African Brazilian donors. The RHD and RHCE allele combinations found in the population studied were: RHD*DAR with RHCE*ceAR; RHD*weak D type 4.2.2 with RHCE*ceAR, RHD*weak D type 4.0 with RHCE*ceVS.01 and RHCE*ceVS.02; RHD*DIIIa with RHCE*ceVS.02. Thirteen patients and six donors had RHD-CE genotypes with homozygous or compound heterozygous alleles predicting partial antigens and/or lacking high prevalence antigens. Eleven patients were alloimmunised to Rh antigens. For six patients with RHD-CE genotypes predicting partial antigens, no donors with similar genotypes were found.DiscussionKnowledge of the distribution and prevalence of RH alleles in patients with SCD and donors of African origin may be important for implementing a programme for RH genotype matching in SCD patients with RH variant alleles and clinically significant Rh antibodies.     

Identification of RHCE and KEL alleles in large cohorts of Afro-Caribbean and Comorian donors by multiplex SNaPshot and fragment assays: a transfusion support for sickle cell disease patients

To lower the alloimmunization risk following transfusion in blacks, we developed two genotyping assays for large-scale screening of Comorian and Afro-Caribbean donors. One was a multiplex SNaPshot assay designed to identify ce(s) (340), ceMO/AR/EK/BI/SM, ce(s) , ce(s) (1006) and KEL*6/*7 alleles. The other was a multiplex fragment assay designed to detect RHD, RHDψ and RHCE*C and 455A>C transversion consistent with (C)ce(s) Type 1 and DIII Type5 ce(s) . Variant RHCE*ce alleles or RH haplotypes were detected in 58·69% of Comorians and 41·23% of Afro-Caribbeans. The ce(s) allele, (C)ce(s) Type 1, and DIII Type 5 ce(s) haplotypes were identified respectively in 39·13%, 14·67% and 4·88% of Comorians and 32·23%, 5·28% and 1·76% of Afro-Caribbeans. Genotypes consistent with partial D, C, c and/or e antigen expression were observed in 26·08% of Comorians and 14·69% of Afro-Caribbeans. No homozygous genotype corresponding to the RH:-18, -34, and -46 phenotypes were found. However, over 50% of genotypes produced low-prevalence antigens at risk for negative recipients, i.e., V, VS, JAL, and/or KEL6. One new variant RHCE*ce(s) (712) allele was identified. This is the first determination of variant RHCE and KEL allele frequencies. Results indicate the most suitable targets for molecular assay screening to optimize use of compatible blood units and lower immunization risk.     

Serotype Switching in a Partially Deleted RHD Gene

We have studied the RH genes in donors with the RhD-negative haplotype dCe^s. In contrast to the usual arrangement of genes in RhD-negative individuals, where the lack of antigen expression is due to deletion of the entire RHD gene, we find that the dCe^shaplotype includes an RHD gene with an internal deletion. Moreover, there appear to be no 5' sequences characteristic of RHC suggesting that the RhC antigen may be encoded by a truncated RHD or a recombinant RHD/CE gene in these dCe^s/dce genomes.     

武汉地区人群血小板特异性基因(HPA-1～17)的多态性分析

目的:研究武汉地区人群HPA-1~17基因的多态性及其表达频率,建立HPA基因型资料库。方法:采用序列特异性引物-聚合酶链反应(SSP-PCR)对284名健康的已加入中华骨髓库的血小板捐献者HPA基因进行分型,计算基因型频率、基因频率。结果:武汉地区健康血小板捐献者中检测出HPA-a基因中的1a~17a基因;各基因独立的分布频率中,HPA-1a(98.77%)、2a(97.01%)、3a(59.68%)、4a(99.82%)、5a(99.82%)、6a(98.42%)、15a(49.47%),HPA-7a~14a、16a和17a均为100%。仅检测出HPA-b基因中HPA-1b(1.23%)、2b(2.99%)、3b(40.32%)、4b(0.18%)、5b(0.18%)、6b(1.58%)、15b(50.53%),未检测出HPA-7b~14b、16b和17b。文中调查和分析了HPA基因组合型及其频率,发现武汉地区HPA基因有28种组合型,其中仅有3种基因组合型频率10%(44%),另外25种基因组合型的频率均9%(56%)。在与国内外不同地区人群HPA基因多态性分布的比较分析中发现,武汉地区人群中HPA基因频率与上海、成都地区人群没有差异性,与美国、英国、欧洲人群有较有明显差异,而与日本人群的差异较小。结论:HPA-3、15系统具有多态性,在随机血小板输注中,供受者HPA-3、HPA-15系统不配合的机会分别为36.54%、37.50%,是HPA配合性输注关注重点。HPA基因多态性研究数据有利于指导地区性血小板供者库库容的设计,配合临床开展选择适合性血小板输注具有重要意义。     

Molecular basis of weak D in Taiwanese

Two genes, RHD and RHCE, encode the antigens of the RH blood group system. The weak D phenotype is caused by many different RHD alleles encoding aberrant RhD proteins, resulting in distinct serologic phenotypes and anti-D immunization. We analyzed seven weak D phenotypes excluding D^el, using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and direct sequencing methods to detect the changes of all ten RHD exons. The results show that there are four types of weak D in Taiwanese: one case each for CGG to CAG mutation at codon 10, GTG to ATG mutation at codon 174, and GTG to GAG mutation at codon 270, and four cases for GGT to GAT mutation at codon 282. In conclusion, we present the first data of a molecular basis of weak D in Taiwanese, which suggest a clinically relevant potential for anti-D immunization and may improve transfusion strategy in weak D Taiwanese patients.