<Emphasis Type="Italic">FCGR3B</Emphasis> gene frequencies and<Emphasis Type="Italic"> FCGR3</Emphasis> variants in a Chinese population from Zhejiang Province

The human neutrophil antigens HNA-1a, -1b and -1c play an important role in immune neutropenia. The frequencies of the coding FCGR3B genes were determined in different populations. New FCGR3B variants were also found in some populations. This study investigated the FCGR3B gene frequencies and FCGR3 variants in a Chinese population compared with the results of Northern Germans and African Blacks (Uganda). Our results show that the gene frequencies in 413 healthy Chinese individuals from Zhejiang Province were 0.565 for FCGR3B*1, 0.430 for FCGR3B*2 and 0.00 for FCGR3B*3. The genotype frequency of FCGR3B_null was 0.48% (2/413). Sequencing of FCGR3 revealed that in seven out of 19 Chinese individuals, cloned and sequenced DNA fragments that exhibited variants caused by single nucleotide exchanges at one or more of the polymorphic positions 141, 147, 227, 266 and 277 in exon 3 also existed in this Chinese population. From the present study, it is concluded that the FCGR3B*1 gene is more frequent in a Chinese population from Zhejiang Province than the FCGR3B*2 gene, and the FCGR3B*3 gene seems to be absent, which is in contrast to studies in the white populations. Gene variants caused by single nucleotide exchanges were found in addition to the well-known forms, but the reason for this remains unclear.     

Fcgamma receptor gene polymorphisms in Japanese patients with systemic lupus erythematosus: contribution of FCGR2B to genetic susceptibility

OBJECTIVE: Human low-affinity Fcgamma receptors (FcgammaR) constitute a clustered gene family located on chromosome 1q23, that consists of FcgammaRIIA, IIB, IIC, IIIA, and IIIB genes. FcgammaRIIB is unique in its ability to transmit inhibitory signals, and recent animal studies demonstrated a role for FcgammaRIIB deficiency in the development of autoimmunity. Genetic variants of FcgammaRIIA, IIIA, and IIIB and their association with systemic lupus erythematosus (SLE) have been extensively studied in various populations, but the results were inconsistent. To examine the possibility that another susceptibility gene of primary significance exists within the FcgammaR region, we screened for polymorphisms of the human FCGR2B gene, and examined whether these polymorphisms are associated with SLE. METHODS: Variation screening of FCGR2B was performed by direct sequencing and polymerase chain reaction (PCR)-single-strand conformation polymorphism methods using complementary DNA samples. Genotyping of the detected polymorphism was done using genomic DNA, with a specific genotyping system based on nested PCR and hybridization probing. Association with SLE was analyzed in 193 Japanese patients with SLE and 303 healthy individuals. In addition, the same groups of patients and controls were genotyped for the previously known polymorphisms of FCGR2A, FCGR3A, and FCGR3B. RESULTS: We detected a single-nucleotide polymorphism in FCGR2B, (c.695T>C), coding for a nonsynonymous substitution, Ile232Thr (I232T), within the transmembrane domain. The frequency of the 232T/T genotype was significantly increased in SLE patients compared with healthy individuals. When the same patients and controls were also genotyped for FCGR2A-131R/H, FCGR3A-176V/F, and FCGR3B-NA1/2 polymorphisms, FCGR3A-176F/F showed significant association. Two-locus analyses suggested that both FCGR2B and FCGR3A may contribute to SLE susceptibility, while the previously reported association of FCGR3B was considered to be secondary and derived from strong linkage disequilibrium with FCGR2B. CONCLUSION: These results demonstrate the association of a new polymorphism of FCGR2B (I232T) with susceptibility to SLE in the Japanese.     

Correlation of platelet FcγRIIA polymorphism in refractory idiopathic (immune) thrombocytopenic purpura

FcγRIIA, a low affinity receptor for IgG, is a polymorphic molecule: FcγRIIA-HH131, FcγRIIA-HR131 and FcγRIIA-RR131. This polymorphism influences the efficiency of the receptor to bind with IgG2. Recent reports on altered distribution amongst individuals with heparin-induced thrombocytopenia (HIT) prompted us to examine the FcγRIIA polymorphism in a cohort of patients with refractory idiopathic (immune) thrombocytopenic purpura (ITP), in whom severe disease had required them to undergo splenectomy. 29 post splenectomy ITP individuals and 61 normal controls were investigated. Polymerase chain reaction (PCR) and a Southern blotting technique were used to determine the FcγRII polymorphism. Although the distribution of the allotypes of FcγRIIA in the control population was similar to that found in other European studies of Caucasian populations (15 (25%) HH131; 35 (57%) HR131; 11(18%) RR131), the patient group was skewed towards the RR131 allotype which has least efficiency for IgG2 binding (3 (10%) HH131; 12 (42%) HR131; 14 (48%) RR131; P  < 0.005). These findings suggest that FcγRIIA polymorphisms may be implicated in the pathophysiology of ITP or may be responsible for modulating the immune response in this heterogenous autoimmune disease.     

Platelet activation induced by a murine monoclonal antibody directed against a novel tetra-span antigen

MAb 14A2.H1 identifies a novel low-abundance platelet surface antigen, PETA-3, which is a member of the tetra-span (TM4) family. This MAb brings about platelet aggregation and mediator release, which is completely inhibitable by prostaglandin E₁, and partially inhibitable by aspirin and ketanserin. Platelet activation by MAb 14A2.H1 is dependent on interaction with both the platelet Fc receptor, FcγRII, and the specific antigen as it was prevented by either a blocking MAb to FcγRII (IV.3) or F(ab')₂ fragments of 14A2.H1. The extent of platelet activation by the antibody varied considerably between donors, and is believed to reflect the polymorphism of FcγRII. Subaggregating concentrations of 14A2.H1 synergized with other platelet agonists, ADP, adrenaline, collagen and serotonin, indicating signalling via a pathway distinct from these activators. Synergy was also blocked by MAb rv.3, or F(ab')₂ fragments of 14A2.H1. The similar low copy number of PETA-3 and FcγRII in the platelet membrane (approximately 1000/platelet). together with the dependence on FcγRII for activation by MAb 14A2.H1, suggests that PETA-3 may be a component of the FcγRII signal transducing complex in platelets.     

Sequence-tagged connectors: A sequence approach to mapping and scanning the human genome

The sequence-tagged connector (STC) strategy proposes to generate sequence tags densely scattered (every 3.3 kilobases) across the human genome by arraying 450,000 bacterial artificial chromosomes (BACs) with randomly cleaved inserts, sequencing both ends of each, and preparing a restriction enzyme fingerprint of each. The STC resource, containing end sequences, fingerprints, and arrayed BACs, creates a map where the interrelationships of the individual BAC clones are resolved through their STCs as overlapping BAC clones are sequenced. Once a seed or initiation BAC clone is sequenced, the minimum overlapping 5' and 3' BAC clones can be identified computationally and sequenced. By reiterating this "sequence-then-map by computer analysis against the STC database" strategy, a minimum tiling path of clones can be sequenced at a rate that is primarily limited by the sequencing throughput of individual genome centers. As of February 1999, we had deposited, together with The Institute for Genomic Research (TIGR), into GenBank 314,000 STCs ( approximately 135 megabases), or 4.5% of human genomic DNA. This genome survey reveals numerous genes, genome-wide repeats, simple sequence repeats (potential genetic markers), and CpG islands (potential gene initiation sites). It also illustrates the power of the STC strategy for creating minimum tiling paths of BAC clones for large-scale genomic sequencing. Because the STC resource permits the easy integration of genetic, physical, gene, and sequence maps for chromosomes, it will be a powerful tool for the initial analysis of the human genome and other complex genomes.     

慢性乙型肝炎免疫清除期患者HBsAg基因多态性

目的 研究慢性乙型肝炎免疫清除期患者乙型肝炎病毒表面抗原(HBsAg)基因多态性.方法 设计特异性引物,自10例慢性乙型肝炎免疫清除期患者血清中扩增S基因片段,TA克隆法克隆到T载体中,随机选择克隆测序.结果 共20个克隆被测序,20个克隆S蛋白发现12个不同位点的32次变异.主要集中在T细胞表位,B细胞表位及a决定簇.结论 慢性乙型肝炎免疫清除期患者存在HBsAg变异,可能仍然存在HBsAg免疫耐受.     

Cloning and stable maintenance of 300-kilobase-pair fragments of human DNA in Escherichia coli using an F-factor-based vector.

A bacterial cloning system for mapping and analysis of complex genomes has been developed. The BAC system (for bacterial artificial chromosome) is based on Escherichia coli and its single-copy plasmid F factor. It is capable of maintaining human genomic DNA fragments of greater than 300 kilobase pairs. Individual clones of human DNA appear to be maintained with a high degree of structural stability in the host, even after 100 generations of serial growth. Because of high cloning efficiency, easy manipulation of the cloned DNA, and stable maintenance of inserted DNA, the BAC system may facilitate construction of DNA libraries of complex genomes with fuller representation and subsequent rapid analysis of complex genomic structure.     

Polymorphisms in inflammatory cytokines and Fcgamma receptors in childhood chronic immune thrombocytopenic purpura: a pilot study

Inflammatory cytokines and low-affinity Fcgamma receptor (FcgammaR) polymorphisms were investigated in 37 children with chronic immune thrombocytopenic purpura (cITP) and 218 controls. Genotype analysis included common variants in the regulatory regions of cytokines, TNF, LTA, IL1RN, IL1A, IL1B, IL4, IL6 and IL10, and structural variants of the low affinity FcgammaRs, FCGR2A, FCGR3A and FCGR3B. Associations were observed for TNF (P = 0.0032), LTA (P = 0.019), FCGR3A (P = 0.038) and FCGR3B (P = 0.0034). Two combinations of genotypes (TNF and FCGR3A; P = 0.0003, and LTA and FCGR3B; P = 0.011) were significantly associated with cITP. These results provide preliminary evidence that variant genotypes of FcgammaRs and cytokines contribute to cITP pathogenesis.     

乙型肝炎病毒表面抗原一级结构多态性的初步研究

目的 研究慢性乙型肝炎患者体内乙型肝炎病毒（HBV）表面抗原一级结构的多态性。方法 设计特异性引物，自7例慢性乙型肝炎患者血清中扩增S基因全长或全基因组片段，TA克隆法克隆到T载体中，随机选择克隆测序。结果 共20个克隆被测序。20个克隆全S蛋白的总一致率仅为32．0％，13株全长为401氨基酸残基的克隆氨基酸一致效率为82．5％。患者血清中发现2株克隆编码截短型表面抗原中蛋白，在前S1或前S2的免疫决定区或可能的肝细胞结合部位均发现缺失突变。结论 慢性乙肝患者体内存在HBV准种群，病毒编码的截短型表面抗原中蛋白可为HBV诱导原发性肝癌提供一种途径，应加强对HBsAg一级结构多态性的研究。     

弓形虫致密颗粒蛋白GRA8截短型片段在原核中的表达

目的 构建弓形虫GRA8原核重组表达质粒，分析其表达状况。 方法 采用PCR技术扩增GRA8及其截短型片段的基因序列，经克隆后，亚克隆至原核表达载体中，构建GRA8及其截短型片段的重组表达质粒，分析GRA8的表达；将各重组菌进行诱导，将裂解上清用谷胱甘肽-琼脂糖亲合层析法和SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)纯化目的蛋白，免疫印迹法(Western blotting)分析纯化蛋白的活性。 结果 GRA8基因被正确插入原核表达质粒中，原核重组表达质粒在大肠埃希菌JM109中表达GRA8的水平低，几乎无完整GRA8的表达，截短型GRA8经谷胱甘肽-琼脂糖亲合层析法和SDS-PAGE获得纯化。纯化的截短型GRA8能被弓形虫感染兔血清识别。 结论 GRA8的原核重组表达质粒表达GRA8的水平低，纯化的截短型GRA8具一定的抗原反应性。     

Polymorphic restriction endonuclease sites linked to the HLA-DR alpha gene: localization and use as genetic markers of insulin-dependent diabetes.

Polymorphic restriction endonuclease sites within the HLA-DR alpha gene have been defined, localized, and used as genetic markers in the analysis of susceptibility to insulin-dependent diabetes mellitus (IDDM). Hybridization of Bgl II-digested human genomic DNA with a cDNA clone for the HLA-DR alpha chain (pDR alpha-1) has revealed three allelic restriction fragment lengths: 3.8 kilobase pairs (kb), 4.2 kb, and 4.5 kb. Hybridization of EcoRV-digested human genomic DNA with the same probe has revealed two allelic polymorphic restriction fragment lengths: 9.2 kb and 13.0 kb. By analysis of double digests of genomic DNA from individuals homozygous for each of the allelic variants, the polymorphic restriction sites were found to be clustered near the 3' end of the HLA-DR alpha gene. The observed correlations of DR alpha Bgl II restriction site variants with serologically determined DR specificities suggest linkage disequilibrium between the DR alpha and DR beta loci. The 3.8-kb fragment is correlated with the DR1 type (Pc = 4.4 X 10(-4)); and the 4.2-kb fragment, with a subset (B8,DR3) of the DR3 type (Pc = 5.1 X 10(-4)) and with the DR6 type. The segregation pattern of HLA-DR alpha polymorphic Bgl II restriction fragments was analyzed in six IDDM families. The observed association of IDDM with the Bgl II 4.2-kb DR alpha restriction variant is higher than with existing serological markers and supports the utility of this approach in elucidating IDDM inheritance.     

Mago nashi:一个日本血吸虫性别决定基因的发现与鉴定

目的　运用免疫学方法筛选日本血吸虫 (中国大陆株 )童虫cDNA文库 ,寻找血吸虫新基因 ,并克隆和鉴定一个日本血吸虫决定性别的Magonashi样蛋白基因。方法　用混合的血吸虫感染者血清免疫学筛选日本血吸虫童虫cDNA文库 ,随机挑取阳性重组克隆进行测序 ,以获得血吸虫基因 ,并通过BLAST程序进行比较及同源性分析 ,根据分析结果设计合成引物 ,用PCR法扩增出日本血吸虫Magonashi样蛋白基因 (SiM)编码序列 ,将其克隆入pGEM T载体和 pBKCMV载体 ,用双酶切、以质粒为模板进行PCR扩增和测序进行鉴定。结果　本研究共挑取 7个阳性克隆 ,通过测序获得了 5个有表达序列标签(ExpressedSequenceTag .EST)价值的序列 ,并进行了同源性分析 ;用PCR法扩增出大小为 4 4 1bpSjM开放阅读框 ,重组质粒pGEM SjM和 pBKCMV SjM经双酶切均可获得一条为PCR产物一致的DNA片段。 结论　本实验获得了 5个有EST价值的序列 ,并成功地克隆了其中SjM编码基因。     

Sequences variations in 5'-flanking region of ABO gene and correlation with ABO alleles in the indigenous Chinese

Background and Objectives   5'-flanking sequences of the ABO gene play important role in the regulation of gene expression, but polymorphism of 5'-flanking sequence of ABO gene is rarely known. Here, we further characterize the molecular genetic basis and ABO allele-related polymorphism of the 5'-untranslated regions (5'-UTR) of the human ABO gene.
Materials and Methods   Collecting blood samples from 72 blood donors in Hangzhou, China, we analysed sequences of exons 6 and 7 of ABO gene and amplified an enhanced segment of 43 bp repeats in the 5'-UTR. Subsequently, we selected 25 homozygotes [of genotypes A101/A101 (two), A102/A102 (seven), B101/B101 (seven) and O01/O01 (nine)] and five heterozygotes [A102/O01 (two), B101/O01 (two) and O01/O105 (one)] for sequencing 5 -kb amplicons spanning the 5'-UTR and partial exon 1 of the ABO gene. We sequenced the amplicons bidirectionally and, when pertinent, analysed selected haplotypes by cloning.
Results   As a result, we identified 11 new polymorphic sites (10 point mutations, one 8-bp deletion) in the 5'-UTR of the A102, B101 and O01 alleles of common ABO phenotypes. Five A102 alleles carry four tandem repeats of a 43-bp minisatellite unit that deviated from previous reports.
Conclusion   The results revealed the DNA polymorphisms in the 5'-UTRs correlated with the common ABO alleles. Elucidation of the diversity of the 5'-UTRs is an important supplement to existing methods for increasing our understanding of the molecular basis of the ABO blood group system.     

Fcγ receptor gene polymorphisms in Japanese patients with systemic lupus erythematosus: Contribution of FCGR2B to genetic susceptibility

Objective

Human low‐affinity Fcγ receptors (FcγR) constitute a clustered gene family located on chromosome 1q23, that consists of FcγRIIA, IIB, IIC, IIIA, and IIIB genes. FcγRIIB is unique in its ability to transmit inhibitory signals, and recent animal studies demonstrated a role for FcγRIIB deficiency in the development of autoimmunity. Genetic variants of FcγRIIA, IIIA, and IIIB and their association with systemic lupus erythematosus (SLE) have been extensively studied in various populations, but the results were inconsistent. To examine the possibility that another susceptibility gene of primary significance exists within the FcγR region, we screened for polymorphisms of the human FCGR2B gene, and examined whether these polymorphisms are associated with SLE.

Methods

Variation screening of FCGR2B was performed by direct sequencing and polymerase chain reaction (PCR)‐single‐strand conformation polymorphism methods using complementary DNA samples. Genotyping of the detected polymorphism was done using genomic DNA, with a specific genotyping system based on nested PCR and hybridization probing. Association with SLE was analyzed in 193 Japanese patients with SLE and 303 healthy individuals. In addition, the same groups of patients and controls were genotyped for the previously known polymorphisms of FCGR2A, FCGR3A, and FCGR3B.

Results

We detected a single‐nucleotide polymorphism in FCGR2B, (c.695T>C), coding for a nonsynonymous substitution, Ile232Thr (I232T), within the transmembrane domain. The frequency of the 232T/T genotype was significantly increased in SLE patients compared with healthy individuals. When the same patients and controls were also genotyped for FCGR2A‐131R/H, FCGR3A‐176V/F, and FCGR3B‐NA1/2 polymorphisms, FCGR3A‐176F/F showed significant association. Two‐locus analyses suggested that both FCGR2B and FCGR3A may contribute to SLE susceptibility, while the previously reported association of FCGR3B was considered to be secondary and derived from strong linkage disequilibrium with FCGR2B.

Conclusion

These results demonstrate the association of a new polymorphism of FCGR2B (I232T) with susceptibility to SLE in the Japanese.

     

Cross-reactive cytotoxic T lymphocytes against a HIV-1 p24 epitope in slow progressors with B*57

OBJECTIVES: To determine whether CD8 T lymphocytes from HIV-1-infected patients expressing B*5701 and B*5703 show broad cross-reactivity against different variants of a conserved p24 epitope, which might account for the good prognosis of HIV-1-infected individuals with HLA-B*57. DESIGN: B*5701+ and B*5703+ were recruited from Nairobi, Kenya and from Oxford, UK. All patients had been HIV positive for at least 8 years and could be categorized as slow progressors. METHODS: CD8 cytotoxic T cell clones were generated from B*5701+ and B*5703+ donors and tested for their ability to recognize clade variants of an index p24 epitope in standard cytolytic assays. Cross-reactive responses in freshly isolated peripheral blood mononuclear cells (PBMC) were assessed by interferon-gamma (IFNgamma) production and tetramer binding. RESULTS: Broad cross-clade reactivity for both cytolysis and tetramer binding was observed in CD8 T cell clones from patients harbouring the index epitope sequence. Patterns of cross-reactivity were similar in freshly isolated PBMC but varied between individuals in terms of strength and breath of responses generated. One common variant induced an unusual response with tetramer binding but often failed to induce IFNgamma production, and another was a weak stimulator of both IFNgamma and cytolytic activity. CONCLUSION: B*5701+ and B5703+ donors demonstrate broad functional cross-reactivity to both common and rare variants of a dominant p24 epitope, which could be relevant to the association of B*57 alleles with slow progression to AIDS.     

溶藻弧菌热休克蛋白70基因真核表达载体的构建及鉴定

目的构建溶藻弧菌热休克蛋白70(HSP70)真核表达载体pcDNA3.1。方法提取溶藻弧菌基因组DNA,PCR扩增HSP70基因片段,克隆至TA载体,通过PCR、酶切及测序鉴定后,将HSP70基因片段用限制性内切酶切下,克隆至真核表达载体pcDNA3.1,构建pcDNA3.1重组质粒,通过PCR、酶切及序列分析对HSP70基因pcDNA3.1重组质粒进行鉴定。结果扩增出1 914bp的溶藻弧菌HSP70基因片段,并成功构建溶藻弧菌HSP70真核表达载体pcDNA3.1。结论成功构建了溶藻弧菌HSP70真核表达载体pcDNA3.1,为HSP70基因疫苗研制奠定了基础。     

A组β-溶血性链球菌DNase B基因序列的克隆与序列分析

目的　克隆A组β -溶血性链球菌 (GroupAStreptococcus,GAS)DNaseB基因序列。 方法　根据genBank中登录的多种DNA酶B的序列 ,进行同源性分析 ,设计相对保守的针对DNaseB基因的引物 ,以链球菌基因组为模板进行PCR扩增 ,目的片段经TA克隆至pMD18-T质粒后测定其核酸序列 ,然后进行序列的查询与比对。结果　成功克隆出A组链球菌DNaseB基因全长序列共 810bp ,其中包括中 12 8bp的信号肽编码区和 6 81bp的成熟肽编码区 ,以及 5’端非编码区的一个T。结论　A组 β-溶血性链球菌DNaseB基因序列的成功克隆 ,为对其深入研究打下坚实基础     

Sequence conservation in kringle IV-type 2 repeats of the LPA gene

We have studied the homology of repeating kringle IV-type 2 (K IV-type 2) elements of the LPA gene. Two K IV-type 2 genomic polymerase chain reaction (PCR) fragment libraries were constructed, one from an individual with high and one from an individual with low Lp(a) lipoprotein level. Only minor K IV-type 2 repeat length heterogeneity was observed. Sequence analysis data from the cloned K IV-type 2 repeats revealed a high degree of LPA sequence conservation in exons as well as in introns both within and between the two libraries. This sequence conservation of the IV-type 2 kringles is in agreement with our previously reported results of simultaneous 'batch' DNA sequence analyses of all the K IV-type 2 repeats from single individuals. Sequence data from the clones, combined with genomic DNA sequencing, revealed that the K IV-type 2 reading frame of exons 1 and 2 are extended into the conserved flanking introns by 519 base pairs (bp) and 312 bp, respectively. The theoretical coding capacity of the exon 1 extended open reading frame (ORF I) is three times larger (173 amino acids, aa) than the translated exon 1, and that of the extended open reading frame of exon 2 (ORF II) is about twice (104 aa) the length of exon 2. A central portion of the intron separating exons 1 and 2 also exhibited a high degree of sequence conservation, with the exception of a polymorphic CA repeat. Within the 61 K IV repeat clones analysed, 19 different CA repeat patterns with 12-18 CA dinucleotide repeats were observed. A comparison between the 37 clones from the individual with high Lp(a) lipoprotein level and the 24 clones from the individual with low Lp(a) lipoprotein level, revealed that seven of the CA repeat variants were present in both clone libraries. The observed high level of sequence conservation in K IV-type 2 exons and introns matches relevant areas of the plasminogen gene, and our findings fit with recent K IV-type 2 duplications and evolutionary selection pressure theories, although gene conversion events could also explain the findings. DNA sequences within K IV-type 2 appeared to have no influence on Lp(a) lipoprotein level.