新等位基因人类白细胞抗原B＊15：133的鉴定及其原核表达

背景：国际上至2010-04共报告人类白细胞抗原4633个等位基因,中国自2000年至少发现200个新的等位基因,由于资金和时间有限,大部分新等位基因未作血清分型、家系研究以及进一步的功能性研究。目的：在健康志愿者人群中发现新等位基因,并对其进行家系研究,同时构建重链胞外区多肽的原核细胞表达载体,鉴定其在原核细胞中是否表达。方法：利用PCR-SSO和PCR-SBT技术,对人群的人类白细胞抗原进行筛选。使用血清学和SBT技术对拥有新等位基因的供者进行家系分析。用分子克隆的方法构建表达载体转染原核细胞,运用Western-blot鉴定是否表达。结果与结论：发现一个新等位基因与人类白细胞抗原B＊15：18：01在第419位相差一个核苷酸,C-〉T,即Codon116位TCC-〉TTC,导致氨基酸由丝氨酸改变成苯丙氨酸。最终确定命名为人类白细胞抗原B＊15：133。血清学表达B71（70）抗原。发现此供者的新等位基因来自于母亲。表达载体pET30a（＋）-B＊15：133在原核细胞中表达的多肽可被抗HIS标签单克隆抗体特异性识别。结果表明,使用分子生物学技术在大样本的筛查下发现了新等位基因人类白细胞抗原B＊15：133,利用分子克隆的方法成功构建表达载体pET30a（＋）-B＊15：133,并在原核细胞中有大量高纯度蛋白表达。     

中国人群人类白细胞抗原A、B和DRB1基因测序分型中模棱两可等位基因的鉴别

背景:人类白细胞抗原基因测序分型中,当某些等位基因间的差异碱基位于测序范围外时,无法得到清晰的等位基因结果.目的:分析中国人群人类白细胞抗原A,B,DRB1基因测序分型中模棱两可等位基因的分布规律,探讨其在中华骨髓库大样本人类自细胞抗原A、B、DRB1基因的测序分型中的解决方案.设计、时间及地点:采用随机抽样方法对研究样本进行人类白细胞抗原A、B、DRB1基因的测序分型,并对其中的模棱两可等位基因进行验证性实验,于2006-01/2008-06在深圳市血液中心完成.材料:658名深圳骨髓库汉族供者乙二胺四乙酸盐抗凝血5 mL.方法:采用聚合酶链式反应-测序分型方法对658名深圳骨髓库供者的人类白细胞抗原A、B和DRB1基因进行高分辨分型,并采用针对相应位点的高分辨聚合酶链式反应-序列特异性引物方法对其中由于碱基差异位于检测区外而产生的模棱两可等位基因进行鉴别.主要观察指标:模棱两可等位基因的分布及确认.结果:658份标本中,人类白细胞抗原A、B和DRB1三个基因座的模棱两可等位基因分别为9个(2种)、140个(5种)、406个(8种),占等位基因总数的14.06%(565/3 948).高分辨聚合酶链式反应-序列特异性引物鉴定结果显示,中国报道的DRB1*1401被全部确认为DRB1*1454;12例B*0705/B*0706中,有1例被确认为B*0706,其他13种等位基因的鉴定结果分别为A*6801、A*7402、B*2705、B*3501、B*4402、B*5801、DRB1*0101、DRB1*0406、DRB1*0803、DRB1*1101、DRB1*1201、DRB1*1302和DRB1*1502.结论:在中华骨髓库大样本人类白细胞抗原基因的测序分型中可以应用直接鉴别的方法区分模棱两可等位基因,但在临床移植前的高分辨确认试验中则需要采用实验方法进行精确分型.     

新等位基因HLA-A~*11:97与HLA-B~*44:127的确认

背景:近几年来,随着分子生物学的发展及人类白细胞抗原分型技术的提高,人类白细胞抗原新等位基因不断被发现。目的:识别并确认两个新的人类白细胞抗原等位基因。方法:应用PCR-SBT、GSSP及单链测序基因分型技术对两份中华骨髓库供者样本进行人类白细胞抗原A,B,DRB1位点进行基因分型,对新等位基因进行DNA测序并与已知同源性最高的等位基因型进行序列比对。结果与结论:两个样本各有一个位点与目前已知的相应人类白细胞抗原A、B等位基因序列均不一致,样本1与其同源性最高的A*11:01:01的差异表现在第2外显子区域中的193G>T,194C>T,导致第41位密码子由丙氨酸变为亮氨酸,样本2与其同源性最高的B*44:02比较差异表现在第2外显子区域中第320位碱基由G>A,导致编码的第83位密码子由精氨酸变为组氨酸。说明两个样本分别为人类白细胞抗原A和B位点的新等位基因,并已被WHO人类白细胞抗原因子命名委员会正式命名为人类白细胞抗原A*11:97和B*44:127。     

汉族人群中新等位基因人类白细胞抗原-DPA1~*01:11的鉴定

背景:在群体遗传学研究中,发现一个样本人类白细胞抗原-DPA1位点结果异常,而国际上与人类白细胞抗原-DPA1等位基因相关的报道较少。目的:鉴定一个人类白细胞抗原-DPA1*01相关的新变异等位基因。方法:在群体遗传学研究中,采用PCR产物直接测序分型方法,对人类白细胞抗原-DPA1基因第2外显子进行双向测序。对出现异常分型结果的样本,进行分子克隆和单倍体测序。结果与结论:发现一个样本的人类白细胞抗原-DPA1位点结果异常,经分子克隆和单倍体测序,检出了一个正常的人类白细胞抗原-DPA1*01:03和一个DPA1*01相关的新变异等位基因,该新等位基因的序列与DPA1*01:03的序列最接近,其差异是在第二外显子区域的242位碱基发生A>G的替换,并导致了相应的第50位密码子由CAA>CGA,编码的氨基酸残基由谷氨酰胺变成精氨酸。该等位基因为人类白细胞抗原新的等位基因,已被世界卫生组织人类白细胞抗原因子命名委员会正式命名为人类白细胞抗原-DPA1*01:11(提交序列号HWS10015443)。     

基因组序列分析HLA新等位基因B*3818内含子和外显子同时突变

目的 分析HLA新等位基因B*3818的基因组序列.方法 克隆测序方法 对PCRSBT中发现的1个未知HLA-B等位基因的基因组序列进行双向全长测序,并采用微量淋巴细胞毒方法 鉴定该等位基因编码分子的抗原特异性,通过群体调查和家系分析了解该等位基因的群体分布频率和单倍型组成情况.结果 新等位基因HLA-B*3818(序列注册号为FJ561482)与B*380201在第4外显子和第5内含子同时存在1个核苷酸的差异.第4外显子的第660位碱基由C变为A,导致第196个密码子由GAC变为GAA,相应氨基酸由天门冬氨酸变为谷氨酸,与IMGT/HLA中的HLA-B等位基因的序列进行对比,该突变为新的单核苷酸多态性位点.第5内含子的第2133位碱基由A变为C,除此之外,B*3818的内含子序列与B*380101、B*380201和B*3814相同.该新HLA等位基因的血清学特异性为B38,其在中国汉族人群中的分布频率小于0.000 5,家系调查结果 表明携带该新等位基因的单倍型为A*030101-CW*010201-B*3818-DRB1-1312-DQB1*060101.结论 新等位基因HLA-B*3818的内含子和外显子同时存在变异,研究新等位基因的基因组序列可为HLA基因相关研究和应用提供更多的序列信息.     

ABO*BEL.11 等位基因突变致红细胞B 抗原弱表达的家系血型血清学及分子机制研究

目的 探讨由ABO*BEL.11 等位基因导致的ABO 正反定型不符样本及家系的血清学和分子生物学特性,研究其遗传方式。方法 常规血型血清学方法和吸收放散试验鉴定样本的ABO 血型表型;PCR 方法扩增ABO 基因7 个外显子及其侧翼内含子,对扩增产物进行直接测序和克隆测序分析,并对先证者的亲属进行家系调查。结果 先证者血型血清学结果为B弱;测序显示:存在ABO*B.01/O.01.01 杂合且伴c.586C/T 突变;克隆测序:c.261delG,297A>G,c.526C>G,c.586T>C,c.657C>T,c.703G>A,c.796C>A,c.803G>C 和c.930G>A 共9 个突变,基因型结果为ABO*BEL.11/O.01.01。家系调查显示先证者父亲、母亲、妻子和女儿血型血清学结果分别为B 型、O 型、A 型及B 弱,其中先证者父亲及女儿的ABO 基因存在ABO*BEL.11 等位基因。结论 ABO*B.01 等位基因上c.586T>C 的突变产生ABO*BEL.11 等位基因,从而导致红细胞上B 抗原的弱表达,且能够稳定遗传突变。     

中国汉族人群人类白细胞抗原新等位基因A*k1155的确认

背景:在国内发现的新等位基因中,大部分都是先采用人类白细胞抗原分型低分辨方法进行分型,如发现反应格局异常后,再进一步用基因测序或基因克隆方法进行确认,这样有可能导致一些新等位基因末被发现.基因测序分型方法被认为是人类白细胞抗原分型的金标准,它可得出精确的核苷酸序列.目的:直接采用基因测序分型方法对中华骨髓库标本进行检测,识别和确认中国汉族人群中新发现的等位基因.方法:应用中国造血干细胞捐献者测序结果可疑为新等位基凼的重采血样5 mL,采用基因测序分型技术,发现一样本HLA-A位点的杂合结果HLA-A*030101/A*110101在外显子上有1个位置与数据库不符,为了区别哪一个为新等位基因,采用基因克隆(TOPOTA Cloning)技术对两个等位基因1-8外显子进行DNA双向测序,发现1个与HLA-A*110101序列相近的新等位基因,分析两者核苷酸序列的差异.并将测序结果与已知人类白细胞抗原等位基因序列比较分析.结果与结论:通过克隆分离杂合等位基因,再进行测序确认发现新的序列与A*110101相比,在第2外显子第330碱基位置上C→G,密码子86AAC→AAG,发生了氨基酸的改变,由天冬酰氨变为赖氨酸.提示该等位基因为新的HLA-A等位基因,2009-10-31被世界卫生组织人类白细胞抗原因子命名委员会正式命名为HLA-A*1155.     

4例人类白细胞抗原罕见等位基因的结果分析

背景：虽然国内与国外关于新等位基因的报道较多,但是,因某些等位基因首次发现较晚,而后却再次被检测到,并且等位基因频率很低,此类相关文章报道甚少,这些等位基因往往被忽视。目的：检测与确认4例临床移植配型供受者携带的罕见等位基因。方法：采用快速 DNA提取试剂盒从全血样本中提取基因组DNA,经人类白细胞抗原基因商品化测序分型试剂盒检测和SSP高分辨试剂盒验证,得到人类白细胞抗原高分辨分型结果。结果与结论：被检测到的4个罕见等位基因分别为B*27：15、B*51：39、C*07：66和C*08：22。对于以上4个等位基因虽被美国骨髓库列为罕见等位基因,但在中国可能并不罕见。事实证明,被美国骨髓库列为罕见等位基因的C*08：22为中国汉族人群常见等位基因。     

四川骨髓库汉族人群HLA-B~* 15等位基因分布

目的采用聚合酶链反应-序列分析基础上的HLA分型(polymerase chain reaction-sequence based typ-ing,PCR-SBT)方法,研究中国造血干细胞捐献者资料库(以下简称为中华骨髓库,CMDP)四川分库中四川籍汉族人群HLA-B*15组等位基因的分布特点。方法从四川骨髓分库中汉族人群中/低分辨分型HLA-B*15阳性样本中按不同血清学特异性分层抽取107例样本,应用PCR-SBT技术进行测序分型,获得四川籍汉族人群B*15组高分辨分型结果。应用方根法计算HLA-B*15各等位基因频率,同时与其他人群资料进行比较。结果共检测到16种已知HLA-B*15等位基因和1例未知等位基因。本研究中检出的16种等位基因有:B*15010101(B62),B*1502(B75),B*1503(B72),B*1505(B62),B*1507(B62),B*151101(B75),B*1512(B76),B*1513(B77),B*1517(B63),B*1518(B71),B*1525(B62),B*1527(B62),B*1529(B15),B*1532(B62),B*1546(B72),B*1558(B62)。其中以B*1502(36.2%)最常见,其次为B*15010101(21.6%),B*151101(9.5%),B*1518(6.9%),B*1525(6.0%),这5种等位基因占B*15等位基因家族的80%。在B*15等位基因家族中,表达B75抗原的等位基因B*1502和B*151101共占45.7%,但表达B62抗原的B*15等位基因多态性最强,共检出7种等位基因。此外,本研究发现1例未知等位基因,该等基因序列与目前所有已知的B*15或B*46等位基因序列均不相同,在外显子3的116位处存在1个点突变C->T。结论研究表明在四川籍汉族人群中HLA-B*15等位基因水平表现出丰富的多态性和特有的分布特征。     

DNA测序确认人类白细胞抗原新等位基因A*0330及二例家系调查分析

人白细胞抗原(HLA)作为人类的1个遗传标记,具有多样性和种族特异性[1].随着中华骨髓库志愿捐献者日益增多,不断有新的等位基因在中国人中被发现[2-6],但同时在同一地区发现数例相同新等位基因并且做家系调查的较少.我们在1例骨髓移植患者常规HLA分型中发现的新等位基因,经DNA测序,2007年5月31日被WHO人白细胞抗原因子委员会命名为HLA-A*0330(ID号:HWS10004714-EF602747).随后在造血干细胞自愿捐献者中又发现1例,经DNA测序确认与首例患者等位基因相同.我们对2个家系进行调查分析,现报告如下.     

Evolution of the O alleles of the human ABO blood group gene

BACKGROUND: To date, at least 40 different alleles O have been characterized on the basis of exon 6 and exon 7 sequences but not always for intron 6. STUDY DESIGN AND METHODS: Among 415 individuals, from four continents (Africa, Europe, South America, and Asia), studied for exon 6 and exon 7 sequences, we selected 46 individuals (of respective phenotypes O [39], AB [3], B [3], or A [1]) for sequencing 1800-bp amplicons spanning exon 6, intron 6, and exon 7. The amplicons were characterized either by direct sequencing or after cloning when required. RESULTS: We defined 14 new intron 6 O allele sequences, including four recombinant alleles. Based on sequence comparison, a phylogenetic network was constructed. It confirmed recombinant allele origins and that most O alleles are derived by point mutations from the two worldwide distributed alleles O01 and O02. CONCLUSION: Allele O phylogenetic analysis suggests that the most frequent silencing mutation (deletion of a G in exon 6) appeared once in human evolution in the ancient O02 allele lineage and that allele O01 resulted from an interallele exchange between O02 and A101. Assuming constancy of evolutionary rate, diversification of the representative alleles of the three human ABO lineages (A101, B101, and O02) was estimated at 4.5 to 6 million years ago.     

Structural basis for red cell phenotypic changes in newly identified, naturally occurring subgroup mutants of the human blood group B glycosyltransferase

BACKGROUND: Four amino-acid-changing polymorphisms differentiate the blood group A and B alleles. Multiple missense mutations are associated with weak expression of A and B antigens but the structural changes causing subgroups have not been studied. STUDY DESIGN AND METHODS: Individuals or families having serologically weak B antigen on their red cells were studied. Alleles were characterized by sequencing of exons 1 through 7 in the ABO gene. Single crystal X-ray diffraction, three-dimensional-structure molecular modeling, and enzyme kinetics showed the effects of the B allele mutations on the glycosyltransferases. RESULTS: Seven unrelated individuals with weak B phenotypes possessed seven different B alleles, five of which are new and result in substitution of highly conserved amino acids: M189V, I192T, F216I, D262N, and A268T. One of these (F216I) was due to a hybrid allele resulting from recombination between B and O(1v) alleles. The two other alleles were recently described in other ethnic groups and result in V175M and L232P. The first crystal-structure determination (A268T) of a subgroup glycosyltransferase and molecular modeling (F216I, D262N, L232P) indicated conformational changes in the enzyme that could explain the diminished enzyme activity. The effect of three mutations could not be visualized since they occur in a disordered loop. CONCLUSION: The genetic background for B(w) phenotypes is very heterogeneous but usually arises through seemingly random missense mutations throughout the last ABO exon. The targeted amino acid residues, however, are well conserved during evolution. Based on analysis of the resulting structural changes in the glycosyltransferase, the mutations are likely to disrupt molecular bonds of importance for enzymatic function.     

Identification of 12 novel RHD alleles in western France by denaturing high-performance liquid chromatography analysis

BACKGROUND: Unlike the standard RHD+ or RHD- alleles, serologic determination of weak or partial D alleles is often not clear-cut. Most importantly, rare weak D alleles, not typed by serology, are prone to alloimmunization when transfused with D+ blood. Although more than 100 RHD variants have currently been reported, many more rare alleles probably remain to be identified. STUDY DESIGN AND METHODS: To identify novel unusual RHD alleles, genomic DNA samples were collected from 333 blood donors or recipients in western France. All displayed ambiguity for D phenotype as determined by routinely used serologic reagents and analyzed by means of denaturing high-performance liquid chromatography (DHPLC) analysis in parallel with direct sequencing. RESULTS: For the first time it has been established that a reliable DHPLC-based approach potentiates the rapid screening of the entire RHD gene-coding sequence. In so doing, a total of 12 novel RHD alleles were identified. Except for the null allele that is in trans with a Weak D type 4 allele, the predicted effects of the other new alleles on gene expression correlated well with the discrepant routine D phenotype results. In particular, the carrier of the p.Leu214Phe missense mutation developed alloanti-D antibodies after transfusion of D+ blood. CONCLUSION: The identification of 12 novel RHD alleles represents a significant addition to the known repertoire of unusual RHD variants and, at the same time, serves to deepen our understanding of the molecular basis of weak and partial D. The accurate molecular typing of RHD alleles would allow to reduce the alloimmunization risk.     

Origin and functions of human natural killer cells

Summary Recent data have substantially modified our view of natural killer cells. Although maturation of natural killer cells occurs in the absence of a functional thymus, we have shown that clonogenic precursors capable of differentiating into mature CD3−16+56+ natural killer cells exist in CD3−4−8−16− populations isolated from human thymus. Analysis of peripheral bloodderived natural killer clones showed that they can lyse normal cells (e.g., phytohemagglutinin-induced blasts) isolated from some individuals. Importantly, natural killer clones isolated from single individuals displayed different patterns of cytolytic activity against a panel of normal allogeneic cells. These data suggested the existence of a natural killer cell repertoire. A number of observations have revealed that the expression of given HLA class I alleles protects target cells from lysis by different groups of natural killer clones. Evidence has been gained by genetic analysis of the determinants responsible for susceptibility/resistance to lysis by natural killer clones together with analysis, as target cells, of HLA-defective variants or HLA transfectants. Thus, natural killer cells were found to express a clonally distributed ability to recognize HLA class I alleles. The selection of new monoclonal antibodies directed against members of a novel family of natural killer specific p58 molecules allowed the identification of the putative natural killer receptors for different MHC class I alleles. Firstly, a correlation was established between the expression of given p58 molecules (e.g., EB6 and GL183) and the class I alleles recognized. Secondly, anti-p58 monoclonal antibodies restored the natural killer-mediated lysis of class I-protected cells. A similar effect was obtained by inducing modulation of p58 surface molecules with anti-p58 monoclonal antibodies. The implications of these receptor/ligand interactions in the physiopathological behavior of natural killer cells are discussed.     

Gorilla class I major histocompatibility complex alleles: comparison to human and chimpanzee class I.

14 gorilla class I major histocompatibility complex (MHC) alleles have been isolated, sequenced, and compared to their counterparts in humans and chimpanzees. Gorilla homologues of HLA-A, -B, and -C were readily identified, and four Gogo-A, four Gogo-B, and five Gogo-C alleles were defined. In addition, an unusual Gogo class I gene with features in common with HLA-A and its related pseudogene, HLA-H, is described. None of the gorilla alleles is identical or even closely related to known class I alleles and each encodes a unique antigen recognition site. However, the majority of polymorphic substitutions and sequence motifs of gorilla class I alleles are shared with the human or chimpanzee systems. In particular, elements shared with HLA-A2 and HLA-B27 are found in Gogo-A and -B alleles. Diversity at the Gogo-B locus is less than at the Gogo-A locus, a trend the opposite of that seen for HLA-A and -B. The Gogo-C locus also appears to have limited polymorphism compared to Gogo-A. Two basic Gogo-C motifs were found and they segregate with distinctive sets of HLA-C alleles. HLA-A allels are divided into five families derived from two ancient lineages. All chimpanzee A alleles derived from one of these lineages and all gorilla alleles derive from the other. Unlike chimpanzee Patr-A alleles, the Gogo-A alleles do not clearly partition with one of the HLA-A families but have similarities with two. Overall, gorilla class I diversity appears from this sampling to show more distinctions from class I HLA than found for chimpanzee class I.     

Genetic polymorphism of the third component of human complement (C''3)

Polymorphism of human C′3 has been defined by prolonged agarose electrophoresis of fresh serum. At least four, and probably five, alleles have been identified by the electrophoretic mobilities of gene products. Inheritance of three alleles, F₁ F, and S, is consistent with the autosomal condominant type. The inheritance of S₁ is probably codominant and that of F_0·8 is not known. Of the 15 phenotypes predicted by these alleles, eight have been observed.     

Molecular genetics of human major histocompatibility complex: clinical applications

Techniques in molecular biology have proved to be very useful in the study of the biology and genetics of the MHC. In particular we have been able to study the Class II gene region in more detail. It is very important because most of the clinical interest of MHC has been focused on the Class II: Certain Class II alleles are strongly associated with autoimmune diseases, and Class II molecules play a crucial role in the interactions between the cells in immune response. DNA polymorphism studies (RFLP) have shown us that polymorphism of MHC genes seems to be considerable. Other close associations between diseases and Class II markers than using serological HLA typings have been found. Molecular cloning of the Class III genes has allowed us to analyse the molecular basis of the complement protein deficiencies, which are often associated with autoimmune diseases. Furthermore, cloning of the genes coding for steroid 21-hydroxylase has revealed the reason for the linkage between CAH and HLA, and has given a possibility for prenatal diagnosis of this disease at the DNA level.     

中国人群HLA-DRB1＊1454等位基因和HLA-DRB1第3外显子鉴定意义

目的:在中国人群中鉴定人类白细胞抗原(human leukocyte antigen,HLA)DRB11454等位基因,分析供受者HLA-DRB1第3外显子DNA序列信息对器官移植、细胞移植及人类遗传学研究的重要现实意义.方法:应用聚合酶链反应-直接测序分型方法对58例等待造血干细胞移植供受者进行人类白细胞抗原测序分型,1 268例广东地区随机健康供者采用聚合酶链式反应-序列特异寡核苷酸探针反向杂交法进行HLA-DRB1中高分辨基因分型,部分模棱两可结果采用聚合酶链式反应-序列特异引物PCR-SSPHLA-DRB14高分辨方法分型法.结果:在1 268例广东地区随机健康人群中检出HLA-DRB11403,1406,1410,1412,1418,1425和1454等位基因,HLA-DRB1401/1434/1454等位基因模棱两可结果的8例样本被确认为HLA-DRB11454等位基因,DRB11454等位基因可能是广东人群最为常见的HLA-DRB114 基因. 中国人群HLA-DRB114第3外显子存在多态性.结论:中国人群HLA-DRB11454等位基因确认了DRB1等位基因第3外显子多态性的存在,进一步明确了开展中国汉族人群及少数民族HLA-DRB1第3外显子检测的意义.     

The genetic polymorphism of the fourth component of human complement: methodological aspects and a presentation of linkage and association data relevant to its localization in the HLA region

The C4 polymorphism in man has been studied by immunofixation electrophoresis, crossed immunoelectrophoresis, and functional detection after agarose gel electrophoresis. It has so far not been possible to reveal this polymorphism by isoelectric focusing and functional detection of C4 bands. Three common alleles and one less frequently occuring allele have been identified. In a small population sample studied by all the different techniques and verified by family segregation, the following gene frequencies have been found: C4F: 0.46, C4S: 0.32, C4F1: 0.20, and C4M: 0.02. By linkage and association studies in a family material it has been shown that a structural C4 locus is situated in the HLA region of chromosome 6 very close to the HLA-B and Bf loci.