广东人抗活化蛋白C及FV Leiden突变的检测

为了解活化蛋白Ｃ抵抗（ＡＰＣＲ）以及凝血因子Ｖ Ｌｅｉｎｄｅｎ（ＦＶ Ｌｅｉｄｅｎ）突变在广东人群及血栓性疾病患者中发生的情况,用序列特异性引物ＰＣＲ（ＰＣＲ－ＳＳＰ）方法检测１３０名广东籍正常人ＦＶ Ｌｅｉｄｅｎ突变,其中５７名正常人用ＡＰＣ－ＡＰＴＴ方法检测了ＡＰＣＲ。结果有３名正常人ＡＰＣＲ阳性。但所有的样本均未发现ＦＶ Ｌｅｉｄｅｎ突变,ＡＰＣＲ阳性是否与其他未知的基因缺陷有关,有待进一步     

一例FⅤ Leiden基因突变所致的抗活化的蛋白C现象

了解抗活性化的蛋白Ｃ在我国人群和血栓性疾患患者中发生情况。方法：用活化的蛋白－ＡＰＴＴ，我聚酶链反应及限制性内切酶分析，ＰＣＲ－ＳＳＰ及ＤＮＡ序列分析，对２８例正常人及１８例血栓性疾患患者进行ＡＰＣ测定及ＦⅤＬｅｉｄｅｎ突变杂合子；３例血栓性疾患患者ＡＰＣ－ＳＲ低于正常，但均无ＦⅤＬｅｉｄｅｎ突变。     

活化蛋白C抵抗物（APCR）蛋白C（PC）蛋白S（PS）在人群中的发生率

用加与不加活化蛋白Ｃ（ＡＰＣ）的两个白陶土部分凝血活酶时间试验来测定人血浆中的ＡＰＣＲ，并对１００例正常人ＡＰＣＲ的发生率进行检测，结果表明，其发生高达４％，男女无差异。本高是对蛋白Ｃ（ＰＣ），蛋白Ｓ（ＰＳ）的发生率进行了检测，检出一例ＰＣ缺乏患者，被检人群未见ＰＳ异常。     

上海地区深静脉血栓形成患者抗活化的白蛋C及FV Leiden突变的初…

应用抗活化的蛋白Ｃ（ＡＰＣＲ）试验、多聚酶链反应－限制性内切酶长度多态性（ＰＣＲ－ＲＦＬＰ）分析及ＤＮＡ序列分析对上海地区深静脉血栓形成（ＤＶＴ）患者ＡＰＣＲ及ＦⅤ Ｌｅｉｄｅｎ突变进行调查。正常人群正常ＡＰＣＲ敏经值（ｎ－ＡＰＣ－ＳＲ）的５％百分位数为０．７５，在被调查的３１例ＤＶＴ患者中有５例患者的ｎ－ＡＰＣ－Ｒ低于此值，ＤＶＴ患者ＡＰＣＲ的发生率为１６％，且ＤＶＴ患者与正常人的ＡＰＣＲ发生率     

上海地区深静脉血栓形成患者抗活化的蛋白C及FⅤLeiden突变的初步调查

应用抗活化的蛋白Ｃ（ＡＰＣＲ）试验、多聚酶链反应－限制性内切酶长度多态性（ＰＣＲ－ＲＦＬＰ）分析及ＤＮＡ序列分析对上海地区深静脉血栓形成（ＤＶＴ）患者ＡＰＣＲ及ＦⅤＬｅｉｄｅｎ突变进行调查。正常人群正常ＡＰＣＲ敏感比值（ｎ－ＡＰＣ－ＳＲ）的５％百分位数为０．７５，在被调查的３１例ＤＶＴ患者中有５例患者的ｎ－ＡＰＣ－Ｒ低于此值，ＤＶＴ患者ＡＰＣＲ的发生率为１６％，且ＤＶＴ患者与正常人的ＡＰＣＲ发生率有显著差异（Ｐ＜０．０５）。用ＰＣＲ－ＲＦＬＰ检查了１４１例排除ＤＶＴ的个体和３５例ＤＶＴ患者的Ｌｅｉｄｅｎ突变情况，发现所有样本的ＰＣＲ－ＲＦＬＰ均显示了同样的代表野生型基因型的电泳条带，未发现Ｌｅｉｄｅｎ型突变。ＡＰＣＲ试验阳性的２例ＤＶＴ患者之多聚酶链反应（ＰＣＲ）产物测序结果进一步证实了实验中ＰＣＲ反应和ＭｎｌＩ酶切反应的特异性。结果说明ＡＰＣＲ现象可能是中国人群ＤＶＴ发病的一个危险因素；但我们与西方调查者实验结果的差别，说明在不同种族中，ＤＶＴ的致病机制可能不同     

抗磷脂血栓综合征与获得性抗活化的影响C现象

了解抗磷脂抗体（ＡＰＡ）,抗活性化的蛋白Ｃ（ＡＰＣＲ）与抗磷脂血栓综合征（ＡＰＬ－Ｔ）的关系,进一步探讨ＡＰＬ－Ｔ血栓发生机制。方法以ＥＬＩＳＡ检测ＡＣＡ（ＩｇＭ,ＩｇＡ）;ＡＰＴＴ检测ＬＡ;ＡＰＴＴ＋／－ＡＰＣ检测ＡＰＣＲ。结果２０例患者符合ＡＰＬ－Ｔ诊断。根据病因分类,１００例ＳＬＥ患者中１４例为继发性ＡＰＬ－６：１６例“原因不明”血栓与习惯性流产患者中６例为原发性ＡＰＬ－Ｔ。根据抗体分类,２     

静脉及心脑血栓性疾病抗活化的蛋白C的研究

为探讨抗活化的蛋白Ｃ（ＡＰＣＲ）及其基因型在中国人血栓性疾病的发病情况，用活化的部分凝血活酶时间（ＡＰＴＴ）以加和不加活化的蛋白Ｃ（ＡＰＣ）的比值（ＡＰＣ－ＳＲ）检测ＡＰＣＲ；用多聚酶链反应扩增因子Ⅴ第１０外显子（包含５０６位密码子）的２２０ｂｐ片段，再用限制性内切酶ＭｎｌⅠ消化检测其基因型。共检测了４６例静脉血栓和下肢动脉血栓，５８例心、脑梗塞，７４例冠心病及４０名正常人的ＡＰＣＲ及其基因型。结果表明血栓性疾病患者与正常人的ＡＰＣＲ无明显差异，亦没有发现与ＡＰＣＲ相关的基因型，提示ＡＰＣＲ很可能不是中国人血栓性疾病常见相关病因     

聚合酶链反应用于进行性脊髓性肌萎缩的诊断

目的了解儿童期发作的进行性脊髓性肌萎缩（ＳＭＡ）患者的运动神经元存活基因（ＳＭＮ）的缺失，探讨聚合酶链反应限制性片段长度多态性（ＰＣＲＲＦＬＰ）技术用于检测ＳＭＡ疾病的诊断价值。方法应用ＰＣＲＲＦＬＰ技术对１０例拟诊为ＳＭＡ患者、６个家系的２０例非ＳＭＡ成员及３０例正常人的ＳＭＮ基因外显子７和８进行了检测。结果１０例ＳＭＡ可疑患者中９例（９０％）有ＳＭＮ基因缺失，其中仅外显子７或８缺失各为１例。家系其他成员及对照组均无ＳＭＮ端粒基因缺失。结论用ＰＣＲＲＦＬＰ法对高度可疑ＳＭＡ的病例进行诊断，具有较高敏感性和特异性，简便易行     

维甲酸和三氧化二砷下调NB4细胞组织因子表达的作用机制

目的 研究全反式维甲酸（ＡＴＲＡ）和三氧化二砷（Ａｓ２Ｏ３）下调ＮＢ４细胞组织因子（ＴＦ）表达的作用机制。方法 用放线菌酮抑制蛋白合成和用放线菌素Ｄ阻断ＲＮＡ合成后,用逆转录－降合酶链反应（ＲＴ－ＰＣＲ）检测ＡＴＲＡ对ＮＢ４细胞ＴＦｍＲＮＡ转录的影响。将含有ＰＭＬ－ＲＡＲα融合蛋白全部编码序列的重组逆转录病毒质粒转染Ｕ９３７细胞,以转染空载体的Ｕ９３７细胞为对照,用ＥＬＩＳＡ方法检测ＡＴＲＡ和Ａｓ     

三种方法在检测Leber‘s视神经萎缩mt—DNA突变的比较

比较ＭＳＰ－ＰＣＲ、ＳＳＣＰ、ＲＦＬＰ在检测Ｌｅｒｂｅｒ’ｓ视神经萎缩（ＬＨＯＮ）线粒体ＤＮＡ（ｍｔ－ＤＮＡ）１１７７８突变中的优缺点。７７例受试者，分别用ＭＳＰ－ＰＣＲ、ＳＳＣＰ、ＲＦＬＰ法检测ｍｔ－ＤＮＡ１１７７８突变。被ＭＳＰ－ＰＣＲ检出的４８例阳性者同时也被ＳＳＣＰ检出，另外ＳＳＣＰ还检出６例杂合性突变；ＭＳＰ－ＰＣＲ和ＳＳＣＰ检测阳性的９例患者，再用ＲＦＬＰ（ＭａｅⅢ）检测也得出一致结果     

静脉血栓栓塞症患者活化蛋白C抗性及凝血因子V活性和基因多态性研究

本研究旨在观察静脉血栓栓塞患者凝血因子V(Factor V,F V)活性改变,检测活化蛋白C抗性(activated protein C resistance,APCR)和凝血因子V基因F V Leiden、F V Cambridge、F V Hong Kong、F V Asp79His和F V I359T多态性.对95例静脉血栓栓塞患者(其中下肢深静脉血栓79例,肺栓塞4例,下肢深静脉血栓合并肺栓塞12例)和95例正常对照者.应用限制性内切酶片段长度多态性测定FV Leiden、F V Cambridge和F V Hong Kong多态性,用MassARRAYTM技术检测F V Asp79His、F V I359T多态性.以一期法和校正的APTT试验分别对其中的65例静脉血栓栓塞患者和60例正常对照者行凝血因子V活性水平和活化蛋白C抵抗检测.结果表明静脉血栓栓塞患者血浆凝血因子V活性水平(108.03%±28.29%)高于对照组(95.17%±29.75%),差异有显著性统计学意义(P=0.008);静脉血栓栓塞组活化蛋白C抵抗阳性13例(20.0%),对照组3例(5.0%),二组比较,有统计学意义(P=0.012).二组均未发现FV Leiden、F V Cambridge、F V Hong Kong、FV Asp79His和FV I359T多态性.结论凝血因子V活性升高和活化蛋白C抵抗是静脉血栓栓塞的危险因素,但APCR与F V Leiden、F V Cambridge、F V HongKong、F V Asp79His和F V I359T多态性无关.     

Type and location of venous thromboembolism in patients with factor V Leiden or prothrombin G20210A and in those with no thrombophilia

BACKGROUND: Patients with factor (F) V Leiden or the prothrombin G20210A polymorphism are at increased risk of developing deep vein thrombosis (DVT). On the other hand, the risk of developing pulmonary embolism (PE) appears to be low in carriers of FV Leiden, perhaps because of a lower tendency to develop iliofemoral DVT than non-carriers. For prothrombin G20210A, data are scanty and controversial. METHODS: The clinical manifestations (isolated DVT, DVT and PE, and isolated PE), the extension of DVT, and the presence of transient risk factors were retrospectively investigated in 115 patients with heterozygous FV Leiden, 87 with prothrombin G20210A and 200 with no thrombophilia marker. RESULTS: Isolated symptomatic PE was less prevalent in patients with FV Leiden (6%) than in those with prothrombin G20210A (21%) and no thrombophilia (23%) (P > 0.0001). The rate of distal DVT was higher in patients with no thrombophilia (16% vs. 7% for FV Leiden and 6% for prothrombin G20210A) (P = 0.02). No difference in the incidence of PE from distal and proximal DVT, the extension of proximal DVT and the type of transient risk factors for venous thromboembolism (VTE) was found in the three groups. Patients with prothrombin G20210A had a younger age at their first VTE (24 years, P < 0.0001) and a higher rate of DVT accompanying PE (P = 0.04) than those with FV Leiden or no thrombophilia. CONCLUSIONS: Carriers of prothrombin G20210A, unlike those of FV Leiden, have an increased risk of developing isolated PE. This difference was not explained by a different rate of distal DVT, extension of proximal DVT, or distribution of transient risk factors in the two groups. Patients with prothrombin G20210A have more severe clinical manifestations than those with FV Leiden or no thrombophilia.     

Increased hemostasis potential persists in women with previous thromboembolism with or without APC resistance

Summary.  Background : Activated thrombin generation and depressed fibrinolysis due to the presence of activated protein C (APC) resistance with or without factor (F)V Leiden mutation are associated with development of deep venous thrombosis (DVT). Objective : A better understanding of the mechanism behind the risk of recurrence of DVT, using our new, recently developed assay of overall hemostasis potential (OHP). Patients and methods : Levels of OHP, as well as APC resistance and FV Leiden mutation, were determined in 88 women (cases) who had previously experienced DVT in connection with pregnancy, and in 25 young healthy individuals (controls). Clotting time and clot lysis time were also investigated. Results : OHP levels in the patients were increased compared with the controls. In the cases with APC resistance and the Leiden mutation this imbalance in hemostasis potential was more severe than in those without. The group with the more severe imbalance had shorter clotting times and longer clot lysis times. Conclusions : A procoagulant state perseveres in patients with a history of pregnancy-related DVT, even after the symptomatic phase is over. The mechanisms behind such an imbalance in overall hemostasis are enhanced thrombin generation and depressed fibrinolysis. These findings may underscore the need for thromboprophylaxis to prevent recurrence of thromboembolism in risk situations.     

正常人和血栓性疾病患者抗活化的蛋白C现象和FⅤLeiden的研究

目的：研究抗活化的蛋白Ｃ（ＡＰＣＲ）现象和凝血因子Ⅴ基因突变（ＦⅤＬｅｉｄｅｎ）在我国健康人群中的发生情况及在血栓病发病机制中的作用。方法：用活化部分凝血活酶时间（ＡＰＴＴ）为基础的ＡＰＣＲ法检测了武汉健康对照组３２名、武汉血栓病患者３８例和德国洪堡血栓病患者１０６例，并对武汉１２例及洪堡１０６例血栓病患者进行了ＦⅤ基因分析。结果：ＡＰＣＲ发生率正常对照组为３．１％，武汉病例组为７９％，洪堡病例组为２２．６％；前两组间无显著差异（Ｐ＞０．０５），洪堡病例组显著高于前两组（Ｐ＜０．０５），其中４５．８％患者（１１例）存在ＦⅤＬｅｉｄｅｎ。结论：ＡＰＣＲ和ＦⅤＬｅｉｄｅｎ的发生存在着地区、种族差异，在中国人中，ＦⅤＬｅｉｄｅｎ可能不是引起ＡＰＣＲ的主要原因。     

The factor V Glu1608Lys mutation is recurrent in familial thrombophilia

BACKGROUND: Co-inheritance of heterozygous factor V deficiency with FV Leiden enhances the activated protein C resistance (APCR) associated with this mutation, resulting in pseudo-homozygous APCR. The role of FV deficiency in modulating thrombotic risk in this rare condition is poorly understood. METHODS AND RESULTS: We have identified in thrombophilic patients with FV deficiency a novel FV gene mutation (c. 4996G>A), predicting the Glu1608Lys substitution in the A3 domain. The heterozygous mutation was detected in three unrelated patients, two carriers of the FV Leiden mutation, and one of the FVHR2 haplotype. The Glu1608Lys change was also present in two subjects with mild FV deficiency, and absent in 200 controls. The FV1608Lys carriers showed reduced mean FV activity (42% +/- 12%) and antigen (53% +/- 18%) levels and, in Western blot analysis, reduced amounts of intact platelet FV. The restriction fragment length polymorphism (RFLP) study identified two haplotypes underlying the mutation, which suggests that it is recurrent. In heterozygous subjects the amount of FV1608Lys mRNA in white blood cells was similar to that produced by the counterpart alleles (FVWt or FVHR2). Recombinant FV1608Lys (rFV1608Lys), detected by Western blot in the conditioned medium, was indistinguishable from rFVWt and FV antigen and activity were found to be respectively 44% +/- 20% and 13% +/- 4% of rFVWt. CONCLUSIONS: Our data indicate that FVGlu1608Lys predicts a CRM (plasma)/CRMred (cell culture) FV deficiency, and may contribute to thrombophilia in carriers of FV Leiden and FVHR2 haplotype via a pseudo-homozygosity mechanism. Our findings help to define the molecular bases of FV deficiency and thrombophilia.     

抗磷脂蛋白抗体、活化蛋白C抵抗与深静脉血栓形成的关系研究

目的探讨抗磷脂蛋白抗体（APA）、活化蛋白C抵抗（APCR）与深静脉血栓形成（DVT）之间的关系及其临床意义。方法选取100例DVT患者和100例正常对照者,分别采用ELISA法检测ACA（IgG,IgM,IgA）,APTT法检测LA,APTT＋／-APC法检测APCR。PCR-限制性片断长度多态性方法（PCR—RFLP）检测FV Leiden变异。结果DVT组患者APA、APCR的总阳性率分别为34％、4％,与对照组相比差异显著;APA阳性组APCR阳性率（8．82％）明显高于APA阴性组（1．51％）,4例APCR阳性患者均未检测到FV Leiden突变。4例APCR阳性患者中有3例APA阳性。并能被血小板磷脂纠正。结论抗磷脂抗体、活化蛋白C抵抗与深静脉血栓形成密切相关,抗磷脂蛋白抗体及其产生获得性APCR是高凝状态和深静脉血栓形成的重要原因。     

Venous thromboembolism: implications for gene-based diagnosis and technology development

Venous thromboembolism is an hypercoagulable state that frequently reflects a complex interplay between inherited, acquired and environmental factors. The overall incidence of venous thromboembolism, which increases with age, is approximately 1:1000 in the US and Western Europe. In addition to known risk factors such as pregnancy and cancer, genetic variants can also increase the venous thromboembolism risk. Once such genetic variant, FV Leiden is characterized by single-point mutation and has been found in approximately 20% of idiopathic venous thromboembolism cases. The discovery of FV Leiden unleashed an increased interest in the genetics of venous thromboembolism as well as other cardiovascular diseases. Because FV Leiden was not only defined by only one common single nucleotide polymorphism but was also widely prevalent, impetus for the development of novel mutation detection methodologies and platforms for DNA analysis in both the clinical and research laboratory was greatly accelerated. An overview of this technology and its relationship to the genetics of venous thromboembolism is reviewed.     

Prothrombotic conditions, oral contraceptives, and the risk of ischemic stroke

BACKGROUND: The role of inherited prothrombotic conditions, including factor V Leiden (FV G1691A), prothrombin G20210A, and the methylenetetrahydrofolate reductase (MTHFR) C677T genotype, in the pathogenesis of ischemic stroke is not well established. The effects of these factors may be potentiated by the use of oral contraceptives, analogous to observations in venous thrombosis. METHODS: Patients (n = 193) were women aged 20-49 years with ischemic stroke. Controls (n = 767) were women without arterial thrombosis stratified for age, calendar year of the index event, and residence. The relative risk of ischemic stroke was estimated with unconditional logistic regression, adjusted for stratification variables. FINDINGS: Factor V Leiden and MTHFR 677TT were more common in patients than in controls [odds ratio (OR): 1.8; 95% confidence interval (CI): 0.9-3.6 respectively OR: 1.5; 95% CI: 0.9-2.6]. The frequency of prothrombin G20210A was similar in cases and controls. Carriers of FV Leiden using oral contraceptives had a 11.2-fold (95% CI: 4.3-29.0) higher risk of ischemic stroke than women without either risk factor. Women with MTHFR 677TT using oral contraceptives had a 5.4-fold (95% CI: 2.4-12.0) higher risk than women without these risk factors. INTERPRETATION: These data suggest that carriers of FV Leiden or MTHFR 677TT who use oral contraceptives have an increased risk of ischemic stroke. When these findings are confirmed, a cost-effectiveness analysis should indicate whether ischemic stroke could be prevented with genetic testing before the start of oral contraceptives.     

凝血因子V基因编码区单核苷酸多态性与静脉血栓栓塞症的相关性分析

目的研究凝血因子Ⅴ（FⅤ）基因编码区单核苷酸多态性与静脉血栓栓塞症的关系。方法检测静脉血栓患者及正常对照者血浆中的FⅤ的活性（FⅤ：C）（一期法）及FⅤ抗原（FⅤ：Ag）（ELISA法），用Premier5软件设计5对与静脉血栓症相关的基因多态性（Asp79His、Arg306The、Arg306Gly、Arg506Gln、Ile359The和His1299Arg）引物，进行PCR扩增、产物纯化、限制性片段长度多态性聚合酶链反应（PCR-RFLP）检测。对所发现的多态性用直接测序法证实，并对含多态性的标本用直接测序法进行FⅤ基因全部外显子编码区的筛查。结果静脉血栓组（VTE组）FⅤ：C（106．9±28．0）％，FⅤ：Ag（110．4±33．3）％；对照组FⅤ：C（102．4±30．9）％，FⅤ：Ag（102．1±24．1）％。VTE组FⅤ：Ag明显高于对照组（P〈0．05），FⅤ：C两者之间差异无明显统计学意义。VTE组和对照组均无Asp79His、Arg306The、Arg306Gly、Arg506Gln和Ile359The多态性，5例静脉血栓栓塞症患者及3名正常人含有His1299Arg多态性。5例静脉血栓栓塞症患者同时伴有Met1736Val多态性，其中3例尚含Asp2194Gly多态性。结论FⅤ含量增高与静脉血栓栓塞症相关，静脉血栓栓塞症与Asp79His、Arg306The、Arg306Gly、Arg506Gln和Ile359The多态性基本无关；His1299Arg在VTE组中的分布频率高于对照组，但差异无统计学意义。Hisl299Arg与Metl736Ⅴal和As02194Gly多态性在人群中有连锁不平衡特性。     

Genetic determinants of hereditary thrombophilia in pathogenesis of venous thrombosis

AIM: To study the role of genetic determinants of hereditary thrombophilia in pathogenesis of various clinical manifestations of venous thrombosis in the citizens of the North-West Region of Russia. MATERIAL AND METHODS: Mutations of the genes of factor V (FV Leiden), prothrombin (G20210-A) and polymorphism C677-T in the gene of methylentetrahydrofolate reductase (MTHFR) were detected using polymerase chain reaction (PCR) with a following restriction analysis of PCR product in 183 patients with venous thrombosis (115 with isolated thrombosis of the deep veins and 68 with thromboembolism of the pulmonary artery). RESULTS: It was established that mutation FV Leiden is a significant risk factor of deep vein thrombosis in the legs and postthrombotic disease, but this mutation is weakly associated with pulmonary artery thromboembolism (PAT). An essential PAT risk factor is carriage of the variant prothrombin G20210A. CONCLUSION: Determination of prothrombotic genotypes is a key factor of treatment efficacy and prevention of life-threatening thromboembolic complications.