一例凝血因子Ⅷ1680A→G点突变

为了研究在无内含子２２倒位的甲型血友病中的ＦⅧ基因的其他遗传性点突变。应用变性梯度凝胶电泳（ＤＧＧＥ）、单链构象多态性（ＳＳＣＰ）对５１例无内含子２２倒位的甲型血友病患者的ＦⅧ基因外显子１４３′侧进行研究。结果发现一例遗传性点突变，密码子１６８０ＴＡＴ（酪）→ＴＧＴ（半胱）的突变。阐明甲型血友病的点突变有助于了解ＦⅧ蛋白各功能域的精细功能，为甲型血友病家系携带者检测和产前诊断提供一种有价值的方法     

一例凝血因子Ⅷ1680A→C点突变

为了研究在无内含子２２倒位的甲因友病中的ＦⅧ基因的基他遗传性点突变。应用变性梯度凝胶电泳（ＤＧＧＥ）、单链构象多态性（ＳＳＣＰ）对５１例无内含子２２倒位的甲型血友病患者的ＦⅧ基因外显子１４３＇侧进行研究。结果发现一例遗传性点突变，密码子１６８０ＴＡ（酷）→ＴＧＴ（半胱）的突变。阐明甲型血友病的点突变有助于了解ＦⅧ蛋白各功能域的精细功能，为甲型血友病家系携带者检测和产前诊断提供一种有价值的方法。     

长距离PCR检测重型血友病A病人凝血因子Ⅷ基因倒位

为了筛查山东省重型血友病A(hemophiliaA，HA)凝血因子Ⅶ基因倒位的患并检出携带，采用长距离DNA扩增(LD-PCR)方法，以0．6％琼脂糖凝胶电泳技术，检测临床上确诊的55例重型HA患及其家系成员中是否存在凝血因子Ⅷ(FⅧ)基因倒位。电泳出现11kb带，示凝血因子Ⅷ基因倒位；12kb带，示非倒位；这两条带同时出现为凝血因子Ⅷ基因倒位携带。结果表明：55例无亲缘关系的重型HA患中，发现22例患(或家系成员)有凝血因子Ⅷ基因倒位，占重型HA患的40%；15个家系中查出基因倒位携带5名。结论：运用LD-PCR技术可以准确、简便、快速地检测重型血友病A患是否存在凝血因子Ⅷ基因倒位。     

血友病A患者凝血因子Ⅷ抑制物筛查及分析

本研究通过检测重型血友病A(hemophilia A,HA)患者凝血因子Ⅷ(FⅧ)抑制物,探讨抑制物阳性患者基因突变情况.选取58例一期法检测FⅧ:C均小于1% HA患者,以APTT法为基础进行FⅧ抑制物筛查,筛查阳性者用Bethesda法进行FⅧ抑制物定量分析.以基因组DNA为模版,对抑制物阳性患者FⅧ12、14、16外显子进行基因扩增,PCR产物通过直接测序检测突变情况.结果表明:58例HA患者中4例(6.9%)抑制物检测为阳性,FⅧ12、14、16外显子基因均未发现基因突变.结论:本组病例HA患者抑制物阳性率低于国外文献报道,抑制物产生的原因有待于进一步研究.     

注射用重组人凝血因子Ⅷ治疗血友病A的护理

目的探讨注射用重组人凝血因子Ⅷ(拜科奇)治疗血友病A的护理方法和要点。方法通过对7例血友病A患者31次使用注射用重组人凝血因子Ⅷ进行治疗时,予心理护理、药物配制、用药观察以及健康教育等护理干预。结果 7例血友病A患者31次使用注射用重组人凝血因子Ⅷ治疗,均未见不良反应。结论在使用注射用重组人凝血因子Ⅷ治疗中,加强药物及患者的护理,可减少不良反应,提高患者的治疗依从性与治疗效果。     

成年人及儿童重型血友病A患者凝血因子Ⅷ的预防性应用指南

本指南以文献为依据,以专家共识为基础,介绍在重型血友病A的儿童和成年人患者中预防性使用凝血因子Ⅷ(FⅧ)的作用.指南就预防性使用FⅧ的起始时机、预防方案的选择、治疗过程中的监测、突发出血时的处理,以及患者和家属的教育等问题进行了讨论.     

FⅧ His99Arg突变导致血友病A的分子发病机制研究

目的 探讨1例临床出血表现与凝血因子Ⅷ活性(FⅧ:C)检测结果明显不符的血友病A患者表型、基因型诊断及其分子发病机制。方法 凝血、抗凝及纤溶相关指标检测,一步法及发色底物法检测FⅧ:C,ELISA法检测FⅧ的抗原(FⅧ:Ag)。活化部分凝血活酶时间(APTT)纠正实验筛查FⅧ抑制物;PCR扩增先证者F8基因的所有外显子及其侧翼序列,利用直接核苷酸测序法进行基因序列分析。针对先证者的基因突变位点,对其家系成员进行相应外显子基因检测;定点突变法构建B亚基缺失(760aa～1639aa)的人FⅧHis99Arg和His99Ala两种突变的表达质粒(pRC/RS Ⅴ-BDhFⅦcDNA),两种突变质粒分别瞬时转染HEK293T细胞,测定细胞上清液中的FⅧ:C和细胞上清液及裂解液中的FⅧ:Ag。结果 先证者APTT 延长,一步法及发色底物法检测的F Ⅷ:C均低于1.0％,血浆FⅧ:Ag为120.0％。APTT纠正试验阴性,其他凝血、抗凝及纤溶相关检测均未见异常。诊断为交叉反应阳性(CRM+)的血友病A。患者F8基因直接核苷酸测序发现3号外显子存在28828A→G突变,导致His99Arg氨基酸改变,其母亲该位点为杂合突变。体外表达研究结果显示细胞上清液中His99Arg突变蛋白FⅧ:Ag及FⅧ:C分别为野生型的180.0％及5.8％,His99Ala突变蛋白FⅧ:Ag及F Ⅷ:C分别为野生型的45.0％和20.0％。Western blot显示,His99Arg突变质粒表达上清液中FⅧ蛋白含量较野生型明显增高,而His99Ala突变质粒表达上清液FⅧ蛋白含量较野生型减少。提示His99Arg为CRM+血友病A突变而His99Ala为CRM-血友病A突变。结论 体外检测发现His99Arg和His99Ala两种FⅧ能够表达,但常规凝血检测Hi99Arg突变FⅧ基本无功能,而His99Ala凝血功能也降低。     

单中心成人重型血友病A低/中剂量凝血因子Ⅷ预防治疗30例疗效分析北大核心CSCD

目的评估低/中剂量凝血因子Ⅷ(FⅧ)预防治疗在成人重型血友病A患者中的疗效。方法纳入在南京大学医学院附属南京鼓楼医院接受低/中剂量FⅧ预防治疗的30例成人重型血友病A患者(低剂量预防组20例、中剂量预防组10例),对预防治疗前、后的年出血次数(ABR)、年关节出血次数(AJBR)、靶关节数量、血友病功能独立性评分(FISH)、生活质量评分及健康状况评分(SF-36)进行回顾性分析、比较。结果中位随访48个月。与预防前按需治疗时相比,低、中剂量预防治疗组ABR、AJBR、靶关节数量均明显降低(P<0.05),且中剂量预防治疗组改善优于低剂量预防治疗组(P<0.05)。低、中剂量预防治疗后与按需治疗时相比,患者的FISH评分、生活质量评分及SF-36评分均明显改善(P>0.05),但低剂量预防组和中剂量预防组之间以上指标差异无统计学意义(P>0.05)。结论低、中剂量FⅧ预防治疗较按需治疗可明显降低重型血友病A患者出血频率,延缓关节病变的进展,提高患者生活质量。中剂量预防组临床出血改善方面优于低剂量预防组。     

血友病A患者产生凝血因子Ⅷ抑制物的治疗相关危险因素

血友病A的治疗以凝血因子替代治疗为主.替代治疗目前存在的主要问题为血友病患者输注FⅧ制剂后产生针对FⅧ的中和性抗体,其发生率国外各家报道差异较大,危险因素包括患者相关因素(疾病严重程度、患者的种族、基因型和免疫反应)和治疗相关因素(替代治疗的起始时间、种类、剂量和方式),本综述主要阐述了国内外治疗相关危险因素的研究现状.     

血友病A家庭治疗46例经验总结

血友病A是一种X染色体连锁遗传性出血性疾病。男性血友病患病率约1/10000,我国约有男性患者120000人,血友病A约占85%[1]。患者因凝血因子Ⅷ(FactorⅧ,FⅧ)缺乏,终生有自发性或轻微外伤后出血倾向。软组织血肿和关节积血可导致剧烈疼痛、严重的跛行性关节病,颅内出血易导致死亡。血友病A目前虽然无法根治,但及时、足量输注FⅧ能有效的预防和治疗出血,减轻患者痛苦,降低致残率。即使是血浆中FⅧ活性(FⅧ∶C)<1%的重型患者,如果自儿童起就获得较好的治疗,成年后完全可以和正常人一样生活、工作。在医疗机构指导下开展家庭治疗是实现这一目…     

Most factor VIII B domain missense mutations are unlikely to be causative mutations for severe hemophilia A: implications for genotyping

Summary. Background & Objective: The factor VIII (FVIII) B domain shares very little amino acid homology with other known proteins and is not directly necessary for procoagulant activity. Despite this, missense mutations within the B domain have been reported in patients with hemophilia A. Given that the B domain is dispensable for secretion and function of FVIII, we hypothesized that these mutations should not be causative of hemophilia A in these patients. Methods: Plasmid vectors containing B domain missense mutations that were reported to be associated with moderate/severe hemophilia A (T751S, D826E, V993L, H1047Y, T1353A, N1441K, L1462P, E1579D, A1591S, P1641L and S1669L) were analyzed for their effect on synthesis and secretion compared with FVIII wild‐type (WT) following transient transfection into COS‐1 and CHO cells in vitro. Further, H1047Y, N1441K and E1579D mutants were expressed in vivo in a hemophilia A mouse model by hydrodynamic tail‐vein injection. Results: FVIII activity and antigen levels for all mutants expressed into the conditioned media of COS‐1 and CHO cells were similar to FVIII WT. Also, plasma expression of these mutants was similar to FVIII WT in hemophilia A mice. An in vivo tail clip bleeding assay also demonstrated that blood loss from hemophilia A mice expressing FVIII WT, H1047Y, N1441K and E1579D was similar. Conclusions: We conclude that most missense mutations within the FVIII B domain would be unlikely to lead to severe hemophilia A and that the majority of such missense mutations represent polymorphisms or non‐pathologic mutations.     

A homozygous missense mutation in human KLOTHO causes severe tumoral calcinosis

Familial tumoral calcinosis is characterized by ectopic calcifications and hyperphosphatemia due to inactivating mutations in FGF23 or UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 3 (GALNT3). Herein we report a homozygous missense mutation (H193R) in the KLOTHO (KL) gene of a 13-year-old girl who presented with severe tumoral calcinosis with dural and carotid artery calcifications. This patient exhibited defects in mineral ion homeostasis with marked hyperphosphatemia and hypercalcemia as well as elevated serum levels of parathyroid hormone and FGF23. Mapping of H193R mutation onto the crystal structure of myrosinase, a plant homolog of KL, revealed that this histidine residue was at the base of the deep catalytic cleft and mutation of this histidine to arginine should destabilize the putative glycosidase domain (KL1) of KL, thereby attenuating production of membrane-bound and secreted KL. Indeed, compared with wild-type KL, expression and secretion of H193R KL were markedly reduced in vitro, resulting in diminished ability of FGF23 to signal via its cognate FGF receptors. Taken together, our findings provide what we believe to be the first evidence that loss-of-function mutations in human KL impair FGF23 bioactivity, underscoring the essential role of KL in FGF23-mediated phosphate and vitamin D homeostasis in humans.     

B cell–activating factor modulates the factor VIII immune response in hemophilia A

Inhibitors of factor VIII (FVIII) remain the most challenging complication of FVIII protein replacement therapy in hemophilia A (HA). Understanding the mechanisms that guide FVIII-specific B cell development could help identify therapeutic targets. The B cell–activating factor (BAFF) cytokine family is a key regulator of B cell differentiation in normal homeostasis and immune disorders. Thus, we used patient samples and mouse models to investigate the potential role of BAFF in modulating FVIII inhibitors. BAFF levels were elevated in pediatric and adult HA inhibitor patients and decreased to levels similar to those of noninhibitor controls after successful immune tolerance induction (ITI). Moreover, elevations in BAFF levels were seen in patients who failed to achieve FVIII tolerance with anti-CD20 antibody–mediated B cell depletion. In naive HA mice, prophylactic anti-BAFF antibody therapy prior to FVIII immunization prevented inhibitor formation and this tolerance was maintained despite FVIII exposure after immune reconstitution. In preimmunized HA mice, combination therapy with anti-CD20 and anti-BAFF antibodies dramatically reduced FVIII inhibitors via inhibition of FVIII-specific plasma cells. Our data suggest that BAFF may regulate the generation and maintenance of FVIII inhibitors and/or anti-FVIII B cells. Finally, anti-CD20/anti-BAFF combination therapy may be clinically useful for ITI.     

一种新的突变引起遗传性凝血因子Ⅴ缺乏症

目的 研究一个遗传性凝血因子Ⅴ(FⅤ)缺乏症家系的基因缺陷。方法 采用比浊法测定凝血酶原时间、凝血酶时间、活化的部分凝血活酶时间；采用凝血因子活性测定法(一步法)和BAELISA法对先证及其家系成员血浆FⅤ活性和抗原进行测定；采用PCR及DNA序列测定技术，对FⅤ基因组DNA中25个外显子和5′非翻译端的序列进行了：PCR扩增，PCR产物回收纯化后直接测序，并经T／A克隆测序对所发现突变进行证实。结果 先证血浆FⅤ严重缺乏，FⅤ活性为1％，FⅤ抗原为1．54％。基因研究显示为复合杂合子，其基因组DNA第4外显子675位核苷酸发生单个碱基A缺失，呈杂合状态，该致病基因来自先证母亲，与来自父方的另外一条等位基因的未知缺陷，共同导致先证血浆FⅤ严重缺乏。结论发现一种新的突变675delA，该缺失引起移码，导致转录提前终止，引起遗传性FⅤ缺乏症。     

Safety and pharmacokinetics of a recombinant factor VIII with pegylated liposomes in severe hemophilia A

Summary.  Background:  BAY 79-4980 is a sucrose-formulated recombinant factor VIII (rFVIII-FS) combined with pegylated liposomes to prolong activity. Objectives:  To investigate the safety, tolerability, bioavailability, pharmacokinetics and pharmacodynamics of a single administration of BAY 79-4980 compared with standard rFVIII-FS in patients with severe hemophilia A. Methods:  This randomized, double-blind study consisted of two crossover substudies comparing two doses of liposomal rFVIII-FS with standard rFVIII-FS. Males (12–60 years) with severe hemophilia A received a single infusion of standard rFVIII-FS (35 IU kg⁻¹) followed by a single infusion of BAY 79-4980 (13 or 22 mg kg⁻¹ pegylated liposomes) or vice versa , with 12 observation days and a 2-day washout period between treatments. Results:  Twenty-six subjects were enrolled at two centers. No serious adverse events were reported. Transient increases in complement C3a, but not CH50, were seen in subjects receiving both the low- and high-liposome-dose BAY 79-4980. Mild transient elevations of total and low-density lipoprotein cholesterol were observed. There were no clinically significant differences in clotting or laboratory parameters or in pharmacokinetic behavior between BAY 79-4980 and standard rFVIII-FS. The number of subjects with spontaneous bleeds on days 1–14 postinfusion was low, and group comparisons were inconclusive. Conclusions:  Single-dose administration of BAY 79-4980 is well tolerated in patients with severe hemophilia A. Plasma pharmacokinetics of FVIII cannot explain the extended protection from bleeding observed previously with BAY 79-4980. Further studies of efficacy and long-term safety of chronic administration are planned.     

A novel missense mutation responsible for factor VII deficiency in research Beagle colonies

BACKGROUND: Canine factor VII (cFVII) deficiency, an autosomal recessive trait originally identified in research Beagles, is associated with a mild to moderate bleeding tendency. OBJECTIVE: Our aim was to identify and characterize the mutation causing cFVII deficiency. METHODS: In order to sequence the coding regions of the cFVII gene, we cloned the cFVII cDNA. Genomic DNA and plasma from FVII-deficient Beagles and obligate carriers were utilized. RESULTS: In all FVII-deficient dogs, we identified a single causative G to A missense mutation in exon 5, encoding the second epidermal growth factor-like domain, resulting in substitution of glycine 96 by glutamic acid, with plasma FVII coagulant activity of 相似文献