血友病B及血管性血友病分子发病机制研究

【作者】 张媛；

【导师】 杨林花；

【作者基本信息】 山西医科大学 ， 内科血液病学， 2009， 博士

【摘要】 研究目的血友病B(hemophilia B, HB)是凝血因子Ⅸ(factorⅨ, FⅨ)基因突变引起血浆FⅨ量的缺乏或质的缺陷所导致的一种X连锁隐性遗传性出血性疾病,在男性中的发病率约为1/30000,是一种严重危害人民健康的疾病。由于目前尚缺乏针对本病的根治措施,故开展基因诊断及携带者检出无疑是防止新的患儿出生、阻断有害基因传递、提高人口素质的一个有效手段。本研究旨在从基因水平对HB患者及携带者做出诊断,探讨HB的分子发病机制。方法1.采集3个无血缘关系的HB家系先证者及成员外周静脉血,提取基因组DNA。2.选择距离FⅨ2cM内的6个STR位点,采用多重PCR及荧光标记引物扩增各STR位点片段,检测先证者及其家系成员6个STR位点的基因多态性,进行家系遗传连锁分析。3.根据FⅨ基因序列,利用Primer 5软件共设计8对引物,采用PCR法对先证者及可疑携带者FⅨ基因8个外显子及其侧翼序列进行扩增,运用双脱氧链终止法在ABI 3700测序仪上对PCR产物进行测序,利用Chromas软件将测序结果与正常序列进行比对,寻找基因突变。结果1.联合6个STR位点对3个HB家系进行检测,根据先证者的基因型,共发现9名可疑携带者,她们均有一条与先证者同源的X染色体。但基因测序证实,家系1、3可疑携带者的FⅨ基因中并不存在与先证者相同的基因缺陷,结合先证者的临床和病史特点,考虑其基因缺陷可能由自发性突变产生。家系2可疑携带者的FⅨ基因中发现了与先证者相应位点相同的杂合突变,STR位点基因多态性检测结果与基因测序结果一致,说明先证者的基因缺陷遗传自其母亲。2.利用PCR法及基因测序技术,在3例先证者的FⅨ基因中均发现了相应的错义突变:家系1先证者外显子6发现G22119A(侧翼序列的剪切位点)点突变,家系2先证者外显子2发现G7932C(Glu8Asp)点突变,家系3先证者外显子8发现T32685C(Cys336Arg)点突变。G22119A发生在外显子6侧翼序列的剪切位点,使FⅨ不能被正常剪切,从而影响其正常生理功能。T32685C发生在外显子8,该部位编码丝氨酸蛋白酶催化区,存在FⅨ与FⅧ的结合位点,可能是由于氨基酸的改变影响了FⅨ蛋白在细胞内的合成与分泌,导致FⅨ功能缺陷。G7932C发生在外显子2,导致Glu突变为Asp,影响了FⅨ与Ca2+依赖性磷脂的结合。结论1. FⅨ基因缺陷是HB的分子发病机制。2.联合多个STR多态性位点对HB家系携带者进行间接诊断,是一种简便、有效、快捷的方法;但是,对于无家族史的散发家系来说,通过遗传连锁分析做出诊断时仍有可能出现误差。3.基因测序技术是诊断HB患者及携带者最直接、最精确的方法之一。研究目的血管性血友病(von Willebrand disease, vWD)是由于血管性血友病因子(von Willebrand factor, vWF)基因突变引起血浆中vWF数量减少或质量异常所导致的一组遗传性出血性疾病,呈常染色体遗传,发病率较高,约为10/10万。患者可有轻度或中度皮肤黏膜出血、鼻衄、胃肠道出血及外伤后出血不止等症状,女性常有月经过多。本研究从3例vWD患者的临床及基因诊断出发,探讨基因型与表现型的相关性,了解vWF蛋白结构与功能的关系,研究基因突变对vWF表达量及功能的影响,旨在从分子水平阐明vWD的发病机制。方法1.采集3例vWD患者外周静脉血,进行血浆vWF:Ag、FⅧ:C测定及RIPA实验和vWF多聚物分析,明确疾病性质及亚型。2.提取患者基因组DNA,应用PCR法扩增患者vWF基因全部52个外显子及其侧翼序列,产物经测序分析寻找突变位点。3.采用基于PCR的定点突变技术构建携带特定突变位点的突变型vWF质粒。4.以野生型质粒为对照,在COS-7细胞中表达突变型质粒,测定转染细胞上清液及裂解液中的vWF:Ag并进行统计学分析。结果1.与正常混合血浆比较,3例患者vWF:Ag、FⅧ:C、RIPA均不同程度降低。2.多聚物分析显示,患者1血浆vWF多聚物缺如,患者2 vWF大中分子量多聚物消失,患者3 vWF多聚物分布及形态正常。3.通过基因测序分析,在3例患者的vWF基因中均发现了相应的杂合性错义突变:患者1外显子37存在C6424T(L2142F)突变;患者2外显子28存在C4738G(L1580V)突变;患者3外显子26存在C3467T(T1156M)突变,其中C6424T(L2142F)在以往国际公开文献中未见报道。4.体外表达实验显示,与转染野生型pSVvWF细胞表达上清中的vWF:Ag比较,pSVvWF3467(T1156M)、pSVvWF4738(L1580V)及pSVvWF6424(L2142F)表达上清中的vWF:Ag均不同程度降低,差异有显著性(P值均<0.01)。与转染野生型pSVvWF细胞裂解液中的vWF:Ag比较,pSVvWF3467及pSVvWF4738裂解液中的vWF:Ag无明显变化(P值均>0.05);pSVvWF6424裂解液中的vWF:Ag显著降低,差异有显著性(P值<0.01)。共转染实验显示,pSVvWF3467(T1156M)+野生型pSVvWF表达上清中的vWF:Ag降低,裂解液中的vWF:Ag增加,差异有显著性(P值<0.01)。结论1. vWF基因突变是导致vWD发生的分子病理机制。2. T1156M突变具有显性抑制效应,影响了正常vWF的分泌;L1580V突变通过改变vWF的空间构象导致其对血浆中的vWF裂解酶异常敏感,属于GroupⅡ突变;L2142F突变导致vWF表达量降低,但降低程度与患者临床症状及实验室检查结果不符,推测患者为复合杂合子,其vWF基因的内含子区域或调控序列中可能存在其他未被发现的致病性突变。更多还原

【Abstract】 ObjectiveThe hemophilia B (HB), which is caused by the mutations in the factorⅨgene, is known as an X-linked recessive disease and occurs in about 1:30000 male live births. At present, due to lacking of the eradicative therapy, the gene diagnosis and detection of the carriers, which is an effective method to prevent the infant patients to be born, block the transmission of harmful gene and improve the population quality, should be actively carrying out. The aim of this study is to diagnosis the propositi and carriers in the gene level and explore the molecule pathogenesy of HB.Methods1. The three unrelated HB families gave informed consent to be included in the study. The genome DNA were collected from each propositus and the family member.2. The polymorphisms of the six STR loci were detected by polymerase chain reaction with multiple reaction system and the fluorescently-labeled primers.3. For the propositi and doubtful carriers, all regions of FⅨgene, including all exons and the flanking sequences, were amplified by PCR using the primer sequences which were devised with Primer five. The products of PCR were sequenced by the dideoxy chain termination using ABI 3700 sequencer, the trial effect was compared with normal sequences of FⅨgene using Chromas for finding the mutations.Results1. 9 doubtful carriers were been found through the allele analysis. But confirmed by the direct sequencing, the doubtful carriers of the first and third family did not have the same gene defects as the propositi, the gene defects were spontaneous mutations. To the second family’s carrier, the heterozygous mutation was found in the corresponding locus of the propositus, the gene defect of the propositus was transmitted from his mother.2. 3 missense mutations were identified in FⅨgene of the propositi when compared with normal sequence. G22119A was identified in exon 6 of the first family’s propositus, it existed in the shearing situs of the flanking sequences, affected the normal physiologic function of FⅨ. G7932C (Glu8Asp) was identified in exon 2 of the second family’s propositus, it impacted FⅨbinding with phospholipids. T32685C (Cys336Arg) was identified in exon 8 of the third family’s propositus, it affected possibly the synthesis and secretion of FⅨintracellular.Conclusions1. The defect of FⅨgene is the molecular pathogenesis of HB.2. Combination analysis of multiple STR loci could be an effective and simple method for indirect diagnosis of the carriers in the HB family. However, there is possible to misdiagnose with the genetic linkage analysis for the families without the family history of HB.3. Gene sequencing is one of the straightest and rigorousest method for the diagnosis of HB. ObjectiveThe von Willbrand disease, which is known as a common inherited bleeding disorder with the prevalence about 10/100000, is caused by genetic defects in the von Willebrand factor (vWF), resulting in quantitative deficiencies or qualitative abnormalities of vWF. The patients have the symptoms of mucocutaneous bleeding, hemorrhinia, gastrointestinal bleeding and unremitting bleeding after injury, female suffers menorrhea often. The aim of this study is to explore the molecular pathogenesis of vWD, investigate the functional and quantitive changes of vWF protein resulting from gene mutations, research the relationship between phenotype and genetype.Methods1. The blood samples were collected from the three unrelated patients with vWD. Through detecting the vWF:Ag, FⅧ:C, RIPA and vWF multimers to determine the diagnosis and the subtype of vWD.2. The genome DNA were collected from each patient. All regions of vWF gene, including fifty-two exons and the flanking sequences, were amplified by PCR. The products of PCR were sequenced by the dideoxy chain termination, the trial effects were compared with normal sequences of vWF gene using Chromas for finding the mutations.3. The expression vectors, carrying the mutations found in the vWF gene of the patients, were constructed by site-directed mutagenesis. 4. Cos-7 cells were transfected with pSVvWF3467, pSVvWF4738, pSVvWF6424 and wild type plasmid. The recombinant proteins and normal vWF were detected by ELISA and analysed by SPSS 13.0.Results1. Comparing with pooled plasma, vWF:Ag、FⅧ:C、RIPA of the patients were decreased.2. The vWF multimers assay disclosed that the vWF multimers of the first patient was absence completely, the high molecular weight multimers was disappeared in the plasma of the second patient, the distribution and structure of vWF was normal in the plasma of the third patient.3. The heterozygous missense mutations were identified in vWF gene of the patients. C6424T (L2142F) mutation was identified in exon 37 of the first patient, C4738G (L1580V) mutation was identified in exon 28 of the second patient, C3467T (T1156M) mutation was identified in exon 26 of the third patient. The first one, a novel mutation, was not reported previously in the international literature.4. The expression experiment in vitro revealed that the antigen levels of vWF T1156M, vWF L1580V and vWF L2142F in media were lower than wild type vWF. In the corresponding transfected Cos-7 cells lysates, the antigen levels of vWF T1156M and vWF L1580V were similar to the level of wild type vWF, however, the antigen level of vWF L2142F was 32.7% of wild type vWF.Conclusions1. The defect of vWF gene is the molecular pathogenesy of vWD.2. The T1156M substitution had a definite effect on the level of vWF expression and secretion in vitro experiments, being substantially less than the secretion of wild type VWF. The space conformation of vWF was changed by the L1580V mutation, the change leaded that the protein had increased sensitiveness with the vWF-cleavring protease. The L2142F mutation resulted in a decreased expression of the recombinant vWF protein, however, the decreased degree is not consistent with the clinical symptom and the laboratory examination report of the patient. The result supposed that the patient might be with a complex heterozygote, there might be an undiscovered morbigenous mutation in the introns or the regulation sequence.更多还原