凝血因子Ⅷ基因及其基因突变的研究

本拟对甲型血友病ＦⅧ基因及其蛋白质结构和功能，ＦⅧ基因突变类型作一综述。     

凝血因子Ⅷ制剂及其临床应用

本文主要介绍了凝血因子Ⅷ制剂冷沉淀和浓缩物的制备方法和性质特点,临床应用适应症、输注剂量、使用方法、注意事项和不良反应及预防,仅供同道参考。     

10个甲型血友病家系FⅧ基因突变分析

目的 分析10个家系中甲型血友病(hemophilia A,HA)患者及女性疑似携带者FⅧ基因突变,并指导产前诊断.方法 应用聚合酶链反应(polymerase chin reaction,PCR)、变性高效液相色谱技术(denaturing high performance liquid chromatogramphy,DHPLC)和DNA测序技术对10个家系中8例HA患者、12名女性疑似携带者的FⅧ基因进行突变检测,并应用St14(DXS 52)、13(CA)n、EX18/BclⅠ3个遗传标记位点对HA家系进行连锁分析.产前诊断检测已知突变位点.针对未见报道的新突变,用限制性内切酶进行分析,同时与100名表型正常的无关个体进行比较,排除多态性.结果 (1)在10个家系中发现5例错义突变、3例移码突变、2例无义突变和2个单核苷酸多态性(single nucleotide polymorphism,SNP)位点.错义突变c.878A＞G、c.1015A＞G和c.6870G＞T,移码突变c.1282delA、c3072_3073insT和c.4880_4881insA以及SNP位点c.5000 G＞A均为国际血友病网站和人类突变数据库未记载的突变或多态.在100名正常人中未检测到错义突变c.878A＞G、c.1015A＞G和c.6870G＞T.(2)在12名女性疑似携带者中,基困水平确诊9例为HA携带者,3名为正常人.(3)遗传连锁分析为4个家系提供了X风险染色体的有效信息.(4)产前诊断结果显示2例胎儿正常、1例HA携带者和1例HA患者.结论 发现c.878 A＞G、c.1015A＞G、c.6870G＞T、c.1282delA、c.3072 3073insT及c.4880_4881insA共6种能引起甲型血友病的新突变;PCR、DHPLC和DNA测序技术可有效检测甲型血友病患者基因突变,而联合限制性内切酶分析和遗传连锁分析能快速筛查HA携带者,进行有效的产前诊断.

Abstract：

Objective To identify the F Ⅷ gene mutations of patients and suspected female carriers in 10 Hemophilia A (HA) families, and to guide the prenatal diagnosis. Methods PCR, denaturing high performance liquid chromatogramphy(DHPLC) and DNA sequencing technologies were applied to screen the FⅧ gene of 8 HA patients and 12 suspected female carriers in the 10 families. Linkage analysis was performed by using St 14 (DXS 52), intron 13 (CA)n and EX18/Bcl Ⅰ of the FⅧ gene in the HA families.In prenatal diagnosis, we screened the same mutation found in the patients. PCR-restriction fragment length polymorphism was applied to detect the new missense mutations of F Ⅷ gene in 100 unrelated healthy individuals to exclude the possibility of polymorphism. Results (1) Five missense mutations, 3 frameshift mutations, 2 nonsense mutations and 2 single nucleotide polymorphism(SNP) were identified in 10 the HA families. Among them, c. 878A＞G, c. 1015A＞G, c. 6870G＞T, c. 1282delA, c. 3072_3073insT, c. 4880_4881insA and c. 5000G＞A were novel mutations or polymorphism. No missense mutations c. 878A＞G, c.1015A＞G and c. 6870G＞T, were found in the 100 healthy unrelated controls. (2) Nine suspected female carriers were confirmed at the gene level. (3) X risk chromosome could be determined in 4 HA families by genetic linkage analysis. (4) Among the four fetuses for prenatal diagnosis, 2 were normal, 1 was carrier and the remaining 1 was a patient. Conclusion Six novel mutations, i. e. , c. 878A＞G, c. 1015A＞G, c.6870G＞T, c. 1282delA, c. 3072_3073insT and c. 4880_4881insA, were identified in this study. PCR,DHPLC and DNA sequencing could be used to screen the gene mutations of HA patients, to carry out carrier detection and prenatal diagnosis of HA families efficiently, by combining with restriction endonuclease analysis and genetic linkage analysis.     

应用PCR技术对凝血因子Ⅷ基因内HindⅢ和BelⅠ多态性的研究

在甲型血友病携带者诊断和产前诊断中．目前多采用PCR／RFLPs分析，酶解彻底性问题是酶谱分析的关键。在本研究中，我们应用PCR技术选择扩增凝血因子Ⅷ（FⅧ）基因内含HindⅢ和BclⅠ多态性酶切位点的特异片段，酶解后分析它们的多态信息量（PIC）。结果HindⅢ的PIC为0．385，BclⅠ为0．365，而且两者存在高度连锁不平衡关系。其中HindⅢ扩增片段中含两个酶切位点，一个是固定的，可作为内参照，另一个是多态性，因有自身对照，酶解就不存在不彻底问题，结果准确可靠。在30名受检女性中，HindⅢ杂合率为43．3％，Bcll为40％；15个家系中有4个既可用Bcll又可以用HindⅢ多态位点进行分析，对其中8名可疑携带者进行基因分析诊断出基因携带者3个。我们的研究表明，HindⅢ多态性酶切位点是PCR／RFLPs方法中值得首先考虑的一个酶切位点。     

应用PCR技术对凝血因子Ⅷ基因内HindⅢ和BclⅠ多态性的…

关甲型血友病携带者诊断和产前诊断中，目前多采用ＰＣＲ／ＲＦＬＰ。酶解彻底性问题是酶谱分析的关键。在本研究中，我们应用ＰＣＲ技术选择入增凝血因子Ⅷ（ＦⅧ）基因内含ＨｉｎｄⅢ和ＢｃｌⅠ多态性酶切位点的特异片段，酶解后分析它们的多态信息量（ＰＩＣ）。结果ＨｉｎｄⅢ的ＰＩＣ为０．３８５，ＢｃｌⅠ为０．３６５，面且两者存在高速连锁不平衡关系。其中ＨｉｎｄⅢ扩增片段中含两个酶切位点，一个是固定的，可作为内参照     

中国人血友病甲患者FⅧ基因突变研究

为研究血友病甲发病的分子机理，应用聚合酶链反应（ＰＣＲ）结合限制性内切酶ＴａｑⅠ酶切分析和ＰＣＲ结合变性梯度凝胶电泳（ＰＣＲ－ＤＧＧＥ），分别研究了７４名中国血友病甲患者ＦⅧ基因外显子１８、２２～２４、２６和外显子８与１４的３′端的基因突变情况，结果检测到一例基因突变，该突变位于第２４号外显子２２０９位密码子，ＣＧＡ－ＴＧＡ，导致了终止密码子的产生。ＰＣＲ－ＤＧＧＥ发现２例基因突变，分别位于外显子８的３４９位密码子，ＧＡＴ－ＧＡＧ，导致Ａｓｐ３４９Ｇｌｕ和外显子１４的１６８９位密码子，ＣＧＣ－ＴＧＣ，使Ａｒｇ１６８９Ｃｙｓ。血友病甲基因突变的研究将有助于开展遗传咨询和产前诊断工作。     

Mutation screening of the F Ⅷ gene in 10 hemophilia A families

Objective To identify the F Ⅷ gene mutations of patients and suspected female carriers in 10 Hemophilia A (HA) families, and to guide the prenatal diagnosis. Methods PCR, denaturing high performance liquid chromatogramphy(DHPLC) and DNA sequencing technologies were applied to screen the FⅧ gene of 8 HA patients and 12 suspected female carriers in the 10 families. Linkage analysis was performed by using St 14 (DXS 52), intron 13 (CA)n and EX18/Bcl Ⅰ of the FⅧ gene in the HA families.In prenatal diagnosis, we screened the same mutation found in the patients. PCR-restriction fragment length polymorphism was applied to detect the new missense mutations of F Ⅷ gene in 100 unrelated healthy individuals to exclude the possibility of polymorphism. Results (1) Five missense mutations, 3 frameshift mutations, 2 nonsense mutations and 2 single nucleotide polymorphism(SNP) were identified in 10 the HA families. Among them, c. 878A＞G, c. 1015A＞G, c. 6870G＞T, c. 1282delA, c. 3072_3073insT, c. 4880_4881insA and c. 5000G＞A were novel mutations or polymorphism. No missense mutations c. 878A＞G, c.1015A＞G and c. 6870G＞T, were found in the 100 healthy unrelated controls. (2) Nine suspected female carriers were confirmed at the gene level. (3) X risk chromosome could be determined in 4 HA families by genetic linkage analysis. (4) Among the four fetuses for prenatal diagnosis, 2 were normal, 1 was carrier and the remaining 1 was a patient. Conclusion Six novel mutations, i. e. , c. 878A＞G, c. 1015A＞G, c.6870G＞T, c. 1282delA, c. 3072_3073insT and c. 4880_4881insA, were identified in this study. PCR,DHPLC and DNA sequencing could be used to screen the gene mutations of HA patients, to carry out carrier detection and prenatal diagnosis of HA families efficiently, by combining with restriction endonuclease analysis and genetic linkage analysis.     

Mutation screening of the F Ⅷ gene in 10 hemophilia A families

Objective To identify the F Ⅷ gene mutations of patients and suspected female carriers in 10 Hemophilia A (HA) families, and to guide the prenatal diagnosis. Methods PCR, denaturing high performance liquid chromatogramphy(DHPLC) and DNA sequencing technologies were applied to screen the FⅧ gene of 8 HA patients and 12 suspected female carriers in the 10 families. Linkage analysis was performed by using St 14 (DXS 52), intron 13 (CA)n and EX18/Bcl Ⅰ of the FⅧ gene in the HA families.In prenatal diagnosis, we screened the same mutation found in the patients. PCR-restriction fragment length polymorphism was applied to detect the new missense mutations of F Ⅷ gene in 100 unrelated healthy individuals to exclude the possibility of polymorphism. Results (1) Five missense mutations, 3 frameshift mutations, 2 nonsense mutations and 2 single nucleotide polymorphism(SNP) were identified in 10 the HA families. Among them, c. 878A＞G, c. 1015A＞G, c. 6870G＞T, c. 1282delA, c. 3072_3073insT, c. 4880_4881insA and c. 5000G＞A were novel mutations or polymorphism. No missense mutations c. 878A＞G, c.1015A＞G and c. 6870G＞T, were found in the 100 healthy unrelated controls. (2) Nine suspected female carriers were confirmed at the gene level. (3) X risk chromosome could be determined in 4 HA families by genetic linkage analysis. (4) Among the four fetuses for prenatal diagnosis, 2 were normal, 1 was carrier and the remaining 1 was a patient. Conclusion Six novel mutations, i. e. , c. 878A＞G, c. 1015A＞G, c.6870G＞T, c. 1282delA, c. 3072_3073insT and c. 4880_4881insA, were identified in this study. PCR,DHPLC and DNA sequencing could be used to screen the gene mutations of HA patients, to carry out carrier detection and prenatal diagnosis of HA families efficiently, by combining with restriction endonuclease analysis and genetic linkage analysis.     

FVⅢ基因的分子遗传学研究

对若干个不相关个体的FⅧ基因进行4个基因内的RFLPs(BclI,HindⅢ,Xbal Ⅰ,Msp,Ⅰ）及几个基因外的RFLPs(TaqⅠ、BstxⅠ,PstⅠ,AccⅠ）的研究,计算了FⅧ基因内多态位点的杂合子频率,在催含子22处,发现了两个额外的XbalⅠ多态位点,在FⅧ基因外的DXS52区域,用探针Stl4-1检测到两个TaqⅠ多态系统,并计算了杂合子频率,同时观察到在BclⅠ、XbalⅠ、MspⅠ间存在不同程度的连锁不平衡。     

血友病B的凝血IX因子基因突变研究进展

血友病Ｂ是由于凝血因子Ⅸ突变所造成的严重的出血性疾病。本文综述了近年来关于Ⅸ因子基因突变的研究进展，包括Ⅸ因子各功能区突变的分析，以及突变的热点，频率，奠基者效应等方面的研究，并且揭示Ⅸ因子的突变主要是由于内源过程造成的。     

脑血栓患者凝血因子Ⅴ基因突变的研究

本文采用单链构型多态分析检测３０例脑血栓患者凝血因子Ⅴ基因Ａ２区和ＤＮＡ片段（此处含活性蛋白Ｃ结合位点），结果发现６例脑血栓患者有不同于正常的基因突变，提示凝血因子Ⅴ基因突变是脑血栓发病的主要风险之一。     

用长距离PCR技术检测江西籍重型血友病甲Ⅷ因子基因倒位基因

目的用长距离PCR（LD-PCR）技术检测江西籍重型血友病A（hemophiliaA,HA）有无Ⅷ因子基因倒位。方法用Bigg′s一期法检测血浆凝血因子FⅧ活性（FⅧ：C）,LD-PCR方法,以0.6%琼脂糖凝胶电泳技术,检测55例江西籍重型HA进行凝血因子Ⅷ（FⅧ）倒位基因,电泳出现11kb带,示FⅧ基因倒位;12kb带,示非FⅧ倒位,这两条带同时出现者为FⅧ倒位基因携带者。结果检测30例无亲缘关系的重型HA患者中,发现11例患者（或家属成员）有FⅧ倒位基因,占重型HA患者的36.7%;9家系中查出基因倒位携带者4名。结论LD-PCR技术结合血浆FⅧ：C可快速、准确检测重型血友病FⅧ基因倒位。     

应用PCR／RFLPs对甲型血友病携带者的诊断

应用ＰＣＲ／ＲＦＬＰｓ对甲型血友病携带者的诊断石奇珍，吕联煌，张学敏甲型血友病是一种最常见的Ｘ连锁隐性遗传性出血性疾病，是由凝血因子Ⅷ基因缺陷所致［１］。发病几乎全为男性，女性为携带者，其诊断可通过测定ＦⅧ组分进行判断，但有一定误诊率［２］。我们应用...     

凝血因子Ⅸ基因新发错义突变致乙型血友病一例家系分析

目的:分析一例乙型血友病(HemophiliaB,HB)家系的遗传学病因。方法:采用凝固法检测先证者及其家系成员凝血酶原时间(PT)、活化部分凝血酶时间(APTT)、凝血酶时间(TT),免疫比浊法检测D-二聚体浓度;采用血液分析仪检测血红蛋白(Hb)含量,发色底物法检测凝血因子Ⅸ(F9)活性,采用PCR-Sanger测序法分析HB的关键致病基因F9基因序列并进行突变分析。结果:先证者Hb含量降低,凝血功能示APTT延长,D-二聚体增高,F9活性明显下降;先证者母亲及女儿APTT及F9活性分别有不同程度的延长和降低,其父亲、妹妹和哥哥的APTT及F9活性正常。测序结果显示该家系先证者携带F9基因半合子错义突变c.289TG(p.Cys97Gly),为首次发现的变异位点,先证者母亲及女儿各携带杂合突变。结论:F9基因第4外显子c.289TG(p.Cys97Gly)半合子变异可能为HB致病性变异。     

10例血友病A患者的FⅧ因子基因内含子22倒位分析

１０例血友病Ａ患者的ＦⅧ因子基因内含子２２倒位分析吴竞生，陈云弟，王寅文，陈美珏，王祖贻，潘理明，汪健，丁浩血友病Ａ（ＨＡ）是凝血因于Ⅷ（ＦⅧ）基因缺陷所致的Ｘ－连锁隐性出血性遗传病。ＦⅧ因子基因突变类型众多，１９９３年发现约一半重型ＨＡ是由ＦⅧ基因...     

PCR法分析甲型血友病Ⅷ因子基因HindⅢ多态性位点检测携带者

目的探讨ＰＣＲ法对甲型血友病Ⅷ因子基因ＨｉｎｄⅢ多态性位点分析检测携带者。方法用ＰＣＲ扩增６个甲型血友病患者家系和２０７条无亲缘关系的Ｘ染色体的Ⅷ因子基因１９内含子，用ＨｉｎｄⅢ酶切进行Ａｍｐ－ＲＦＬＰｓ分析。结果查明ＨｉｎｄⅢ多态性位点频率为０．２９，根据Ｈａｒｄｙ－Ｗｅｉｎｂｅｒｇ定律计算妇女杂合子频率为０．４１，证明这个指数对检测甲型血友病携带者和进行产前诊断具有足够的信息量。研究的６个家系中有２个有信息（３３％）。结论为临床检测携带者和产前诊断提出有效的检测ＨｉｎｄⅢ多态性新途径     

FⅧ基因倒位与血友病甲

最近发现F Ⅷ基因的内含子22倒位是约半数重型血友病甲(HA)患者的共同分子缺陷。由F Ⅷ基因内含子22内的F ⅧA基因拷贝与F Ⅷ基因上游约500kb处的两个FⅧA基因拷贝之一发生同源重组在Xq28所引入的大片段。DNA倒位把F Ⅷ基因分成两半,从而导致重型HA。倒位可以通过Southern印迹杂交技术检测。这一发现明确了一类HA的分子机制,并且为这类HA的携带者检测和产前诊断提供了一个直接方法,具有重要的应用价值。     

凝血因子V基因多态性与静脉血栓形成

静脉血栓形成(venous thrombosis，VT)在发达国家年发病率1‰。且随年龄增长呈递增现象，儿童的发病率1／10万，老年人接近1％”。。VT多发生在下肢深静脉，与其相关的主要并发症是血栓后遗综合征及致死性的肺动脉栓塞，发生率分别是20％”。及1‰～‰。我国尚缺乏大规模血栓流行病学的调查资料。从近些年我科收治的病人来看，VT     

慢性肺心病急性发作期患者治疗前后血浆凝血因子Ⅷ和D-二聚体水平的变化

慢性肺心病急性发作期患者的血液高凝状态和微小血栓形成[1]可进一步加重肺心病症状。D-二聚体是交联后纤维蛋白被纤溶酶降解的特异标志物之一[2],其与凝血因子Ⅷ相关抗原(FⅧR:Ag)和凝血因子Ⅷ活性(FⅧ:C)等联合检测可较好反映机体凝血/纤溶功能。本文观察34例慢性肺心病急性发作期病人治疗前后上述指标的血浆水平变化,并分析其在病情诊断、疗效观察及预后判断中的作用。1资料与方法1.1对象慢性肺心病病人34例,均为2003年7月~2005年7月收治的住院患者,男20例,女14例,平均年龄67.43±6.74岁,按照1977年全国肺心病会议的诊断标准,经X光、…