复合杂合突变导致遗传性蛋白C缺陷症- -家系调查

摘要:目的调查复合 杂合突变导致遗传性蛋白C缺陷症家系的临床特征与基因突变情况,并分析其基因型与临床表型的关系。方法采集先证者及其家系成员(3代5人)外周静脉血，检测蛋白C活性、蛋白S活性和抗凝血酶活性等指标以明确表型 诊断。采用PCR对先证者PROC基因所有外显子及侧翼序列进行扩增,PCR产物纯化后进行直接测序。运用ClustalX-2. 1-win软件分析突变的保守性;使用在线生物信息学软件预测突变的致病性。结果家系中有4人 存在遗传性蛋白C缺陷症,先证 者临床表现为肺栓塞和下肢深静脉血栓栓塞,其他家系成员无明显的血栓形成事件表现。基因测序发现先证者PROC基因第7号外显子存在c.541T>G 杂合错义突变(p. Phe181Val)和c.577-579delAAG杂合缺失突变( p. Lys192deletion),其父亲为 c.541T>G突变杂合子，母亲和妹妹为c.577-579delAAG 突变杂合子。保守性分析显示Phe181和Lys192在同源物种间均高度保守,生物信息学软件预测该2个突变位点均为有害突变。结论该遗传性蛋白 C缺陷症家系存在c.541T>G杂合错义突变 和e.577-579delAAG 杂合缺失突变,该复合杂合突变可能引起先证者蛋白C活性明显降低,从而导致反复深静脉血栓栓塞和肺栓塞。     

蛋白C基因C64W和F139V突变致蛋白C缺陷的分子机制研究

目的研究蛋白C（PC）基因C64W、F139V和K150缺失突变（K150d）致PC缺陷症的分子机制。方法构建PC基因野生型和突变体表达质粒（PCwt、PC C64W、PC F139V、PC K150d）并瞬时转染至COS-7细胞或CHO细胞，进行体外表达试验和细胞免疫荧光染色；用荧光实时PCR（real-time PCR）检测转染细胞PC mRNA表达量的改变；蛋白降解抑制实验检测突变蛋白在细胞内的降解途径；内糖苷酶-H（Endo H）酶切试验检测PC翻译后侧链糖化修饰。结果PC C64W未从转染细胞内分泌且在细胞内逐渐降解，PC F139V仅部分从细胞内分泌，大部分未分泌并在细胞内逐渐降解，PC K150d突变体蛋白的分泌未受突变的明显影响。real-time PCR结果显示，与野生型PC mRNA相比，突变体PC mRNA不降低；蛋白降解抑制实验显示，PC C64W和PC F139V通过蛋白酶体途径进行细胞内降解。Endo H酶切和转染细胞荧光染色显示，PC C64W和PC F139V主要位于前高尔基体。结论分泌障碍和细胞内降解是PC C64W和PC F139V导致PC缺陷症的原因，PC K150d对突变体蛋白的分泌无明显影响。     

蛋白C、蛋白S的研究与应用

蛋白C(protein C,PC)抗凝系统是机体内重要的抗凝系统,主要由PC、蛋白S(protein S,PS)、蛋白C抑制物(protein C inhibitor,PCI)和血栓调节蛋白(thrombomodulin,TM)组成[1],在血液凝固和纤溶过程起着重要的平衡作用.PC是PC系统最主要的组成成分,是体内重要的抗凝因子,其抗凝活性占全血的20%-30%,体外只要达到0.2μg/ml即可引起明显的抗凝效应,直接影响凝血与抗凝血机制的平衡[1,2],而PC的生理功能又与PS密切相关,故我们综合有关资料对PC抗凝系统中的PC、PS做一介绍.     

血浆蛋白C和蛋白S在静脉血栓栓塞患者体内的表达及意义

目的测定静脉血栓栓塞(VTE)患者血浆中蛋白C(PC)、蛋白S(PS)的活性表达,探讨其联合检测在临床中的意义。方法选取2014年1月至2015年6月该院收治的VTE患者175例为研究组,另选取同期健康体检者40例为对照组,均检测血浆PC、PS的活性表达并将两组结果进行比对分析。结果研究组血浆PC、PS的活性表达低于健康对照组[PC(99.03±35.63)%vs.(112.06±19.21)%;PS(98.51±36.17)%vs.(110.4±14.57)%],差异有统计学意义(P0.05)。研究组PC、PS活性异常的检出率高于健康对照组[PC(19.4%)vs.(0.0%);PS(25.7%)vs.(0.0%)],差异有统计学意义(P0.05)。结论血浆PC及PS活性在VTE患者血清中低表达,检测其水平变化对VTE的预防、诊断及治疗有指导意义。     

蛋白C、蛋白S活性和活化蛋白C反应性的参考值测定

临床上检测蛋白Ｃ(ｐｒｏｔｅｉｎＣ ,ＰＣ)、蛋白Ｓ(ｐｒｏｔｅｉｎＳ ,ＰＳ)和活化蛋白Ｃ(ａｃｔｉｖａｔｅｄｐｒｏｔｅｉｎＣ ,ＡＰＣ)反应性 ,进行血栓性疾病的监测已越来越重要。但其检测值受仪器、试剂的选择和试剂质量的影响 ,本文检测了 80例健康成人的ＰＣ、ＰＳ活性和ＡＰＣ反应性 ,建立了本室的参考值 ,现报道如下。1　材料与方法1 1　检测标本　体检健康成人 80例 ,年龄 16～ 68岁 ,男 3 8例 ,女 4 2例。按不同年龄段分为三组 ,一组 3 0岁以下 ,2 8例 ;二组 3 0～ 4 9岁 ,2 8例 ;三组 5 0岁以上 ,2 4例。每份标本取静脉血 1 8…     

一例PROC基因复合杂合突变致遗传性蛋白C缺乏症患儿的临床诊治

遗传性蛋白C缺乏症(hereditary protein C deficiency,HPCD)主要表现为罕见的新生儿期暴发性紫癜,疾病进展迅速,死亡率高,诊治困难,早期识别和正确诊治可显著改善预后.本文报道1例PROC基因复合杂合突变所致的新生儿HPCD,患儿出生后24 h内即出现反复皮肤紫癜,6月龄前辗转就诊于国内多...     

两个终止密码子导致的遗传性蛋白C和蛋白S缺陷症

目的:研究1个蛋白C(PC)和1个蛋白S(PS)缺陷症家系的表型诊断和基因特征。方法:PC活性(PC:A)和PS活性(PS:A)用发色底物法测定;PC抗原(PC:Ag)和PS抗原(PS:Ag)用ELISA方法测定。用PCR扩增PC和PS基因各个外显子及其侧翼序列,用直接测序法检测突变点。利用逆转录PCR(RT-PCR)分析PCmRNA水平变化。同时利用蛋白印迹分析血浆中PC含量的变化。结果:先证者1的PC:A为49%,PC:Ag为1.34mg/L,基因检测发现PC基因9号外显子的8831有G→A杂合无义突变,导致Trp372Stop;先证者2的PS:A为29%,PC:Ag为8.3mg/L,14号外显子Gln522(CAG)→Stop(TAG)。结论:G8831A杂合突变引起Trp372Stop,其可导致遗传性PC缺陷症;Gln522Stop可导致遗传性PS缺陷症。     

脑梗死患者血浆活化蛋白C抵抗、蛋白C和蛋白S活性的测定

活化蛋白Ｃ抵抗 (ａｃｔｉｖａｔｅｄｐｒｏｔｅｉｎＣｒｅｓｉｓｔａｎｃｅ,ＡＰＣＲ)是目前已知的静脉血栓形成的最高危因素[1] ,80 %的ＡＰＣＲ是因子Ｖ (ＦＶ)基因点突变 (即Ｌｅｉｄｅｎ突变 )引起的遗传性ＡＰＣＲ ,另一部分ＡＰＣＲ则是由各种获得性原因导致的无ＦＶ基因突变的获得性ＡＰＣＲ。ＡＰＣＲ与动脉血栓形成的关系目前仍有争论。蛋白Ｃ( ｐｒｏｔｅｉｎＣ ,ＰＣ)、蛋白Ｓ(ｐｒｏｔｅｉｎＳ ,ＰＳ)是机体ＰＣ抗凝系统的重要组成部分 ,它们量或质的缺陷 (包括先天性和获得性 )都将导致机体抗凝功能的减退或丧失 ,与血栓形成…     

糖尿病足患者血浆活化蛋白C抵抗、蛋白C和蛋白S活性的测定

目的探讨PC、PS活性的变化在糖尿病足形成中的作用和临床意义,以及APCR与糖尿病足形成的关系。方法采用Dahlback法对50例健康成年人(对照组)及60例糖尿病足患者(DF1、DF2组)进行APCR阳性率测定,同时测定受检者蛋白C(PC,)和蛋白S(PS)。结果糖尿病足(DF)患者血浆PC/PS活性下降与对照组有显著性差异。结论血浆PC/PS活性检测对糖尿病足患者的诊断、估计预后和疗效观察有参考价值,且APCR阳性者有动脉血栓发生的倾向。     

Co-segregation of the PROS1 locus and protein S deficiency in families having no detectable mutations in PROS1.

Inherited deficiency of protein S constitutes an important risk factor of venous thrombosis. Many reports have demonstrated that causative mutations in the protein S gene are found only in approximately 50% of the cases with protein S deficiency. It is uncertain whether the protein S gene is causative in all cases of protein S deficiency or if other genes are involved in cases where no mutation is identified. The aim of the current study was to determine whether haplotypes of the protein S gene cosegregate with the disease phenotype in cases where no mutations have been found. Eight protein S-deficient families comprising 115 individuals where previous DNA sequencing had failed to detect any causative mutations were analyzed using four microsatellite markers in the protein S gene region. Co-segregation between microsatellite haplotypes and protein S deficiency was found in seven of the investigated families, one family being uninformative. This suggests that the causative genetic defects are located in or close to the protein S gene in a majority of such cases where no mutations have been found.     

PROC,PROCR and PROS1 polymorphisms,plasma anticoagulant phenotypes,and risk of cardiovascular disease and mortality in older adults: the Cardiovascular Health Study

Summary. Background and objectives: Genes encoding protein C anticoagulant pathways are candidates for atherothrombotic and other aging‐related disorders. Methods: Using a tagSNP approach, and data from the Cardiovascular Health Study (CHS), we assessed associations of common polymorphisms of PROC, PROS1 and PROCR with: (i) plasma protein C, soluble protein C endothelial receptor (sEPCR) and protein S levels measured in a subsample of 336 participants at study entry; and (ii) risk of incident clinical outcomes [coronary heart disease (CHD), stroke, and mortality] in 4547 participants during follow‐up. Secondarily, we explored associations between plasma protein C, protein S and sEPCR levels and other candidate genes involved in thrombosis, inflammation, and aging. Results: The PROCR Ser219Gly polymorphism (rs867186) was strongly associated with higher sEPCR levels, explaining 75% of the phenotypic variation. The PROCR Ser219Gly variant was also associated with higher levels of circulating protein C antigen. An IL10 polymorphism was associated with higher free protein S levels. The minor alleles of PROC rs2069901 and PROS1 rs4857343 were weakly associated with lower protein C and free protein S levels, respectively. There was no association between PROCR Ser219Gly and risk of CHD, stroke, or mortality. The minor allele of another common PROCR tagSNP, rs2069948, was associated with lymphoid PROCR mRNA expression and with increased risk of incident stroke and all‐cause mortality, and decreased healthy survival during follow‐up. Conclusions: A common PROCR variant may be associated with decreased healthy survival in older adults. Additional studies are warranted to establish the role of PROCR variants in ischemic and aging‐related disorders.     

Protein Z, protein S levels are lower in patients with thrombophilia and subsequent pregnancy complications

OBJECTIVE: We posit that low levels of protein S (PS) and protein Z (PZ) contribute to adverse pregnancy outcome (APO). PATIENTS: We evaluated 103 women with subsequent normal pregnancy outcome (NPO), 106 women with APO, and 20 women with thrombophilia (TP). METHODS: We compared first trimester (1st TRI) PZ levels in 103 women with NPO, 106 women with APO, and in 20 women with TP. We compared plasma levels of PZ and free PS antigen during the second (2nd TRI) and third trimesters (3rd TRI) of pregnancy in 51 women with APO and 51 matched women with NPO. RESULTS: The mean 1st TRI PZ level was significantly lower among patients with APO, compared to pregnant controls (1.81 +/- 0.7 vs. 2.21 +/- 0.8 microg mL(-1), respectively, P < 0.001). Of patients with known TP, those with APO had a tendency for lower mean PZ levels compared to those TP women with NPO (1.5 +/- 0.6 vs. 2.3 +/- 0.9 microg mL(-1), respectively, P < 0.0631). There was a significant decrease in the PZ levels in patients with APO compared to NPO (2nd TRI 1.5 +/- 0.4 vs. 2.0 +/- 0.5 microg mL(-1), P < 0.0001; and 3rd TRI 1.6 +/- 0.5 vs. 1.9 +/- 0.5 microg mL(-1), P < 0.0002). Protein S levels were significantly lower in the 2nd and 3rd TRIs among patients with APO compared to patients with NPO (2nd TRI 34.4 +/- 11.8% vs. 38.9 +/- 10.3%, P < 0.05, respectively; and 3rd TRI 27.5 +/- 8.4 vs. 31.2 +/- 7.4, P < 0.025, respectively). CONCLUSIONS: We posit that decreased PZ and PS levels are additional risk factors for APO.     

急性白血病患者蛋白Ｃ系统检测的临床意义

目的 探讨急性白血病（ＡＬ）患者蛋白Ｃ（ＰＣ）系统的改变及其与分型、出血情况和预后的关系。方法 运用ＥＬＩＳＡ或发色底物法对９３例ＡＬ患者血浆ＰＣ活性和抗原（ＰＣ：Ａ和ＰＣ：Ａg）、血栓调节蛋白（TM）及蛋白S（PS）水平进行了检测,结果 治疗血浆TM水平显著升高,ＰＣ：Ａg水平低于正常,PC：A和PS水平与正常对照组无显著差异;缓解后除PC：A和急性淋巴细胞白血病患者ＰＣ：Ａg增高外余均恢复至正常范围内。上述指标与因程度无关,全反式维甲酸治疗组PC：A和TM有所升高,在三氧化二砷治疗组未发现上述现象,治疗前后TM升高,治疗前PS降低者预后较差,其中治疗前PS和治疗后TM是决定患者无复发生存时间独立的预后因素,治疗后TM是决定患者总生存时间独立的预后因素。结论 AL发病过程中存在PC系统的激活,并随病情的好转而基本改善。PC系统并非是决定出血程度的关键因素,但TM升高及PS消耗与患者的预后密切相关。     

血浆蛋白C及蛋白S在肺栓塞患者血清中的表达及意义

目的：测定肺栓塞患者血浆蛋白C及蛋白S的表达,探讨其联合检测在临床中的意义。方法：肺栓塞患者132例为观察组,体检正常成人132例为正常组,均检测血清中血浆蛋白C及蛋白S的表达,分析其不同预后与血浆蛋白C及S的差异及二者间的相关性。结果：与正常组比较,观察组患者血清中血浆蛋白C及蛋白S均呈明显低表达,其中死亡病例更低于好转病例,好转病例明显低于治愈病例（P〈0.05,0.01）;线性相关分析,血清中血浆蛋白C与蛋白S的表达呈正相关。结论：血浆蛋白C及蛋白S在肺栓塞患者血清中均呈低表达,且与预后有关,早期检测血浆蛋白C及蛋白S可能对判断肺栓塞的病情及指导治疗有重要价值。     

一个新的PROC基因突变的检出及其致病机制的研究

目的:鉴定一个遗传性蛋白C缺乏症患者的基因突变,并对该突变的致病机制进行研究。方法:采用血液基因组提取试剂盒获取遗传性蛋白C缺乏症患者的DNA,用聚合酶链反应(PCR)法扩增其PROC基因所有外显子及侧翼序列并测序,应用Chromas软件分析并检出基因突变;将带有突变位点的PROC基因c DNA序列克隆到载体后转染至HEK293细胞,采用定量PCR法检测突变基因m RNA的表达水平。结果:检出一个PROC基因的新突变[c.740G>A(p.Trp247*)],并在细胞水平证明突变基因m RNA的表达水平出现下调。结论 :PROC基因无义突变的m RNA可能通过无义介导的m RNA降解途径快速降解,从而导致遗传性蛋白C缺乏症。     

Genetic abnormalities of the protein C system: shared risk factors in young adults with migraine with aura and with ischemic stroke?

Migraine, particularly migraine with aura (MA), may be a risk factor for ischemic stroke (IS). The reasons for this association are unknown. We investigated the presence of genetic abnormalities of the protein C system in 83 MA patients, 31 IS patients, and 124 healthy controls, all aged under 45 years. We found an increased frequency of activated protein C resistance due to Arg5O6Gln factor V mutation, and of protein S deficiency in both disorders, with figures higher than those reported in the general population and significantly different from those found in controls. These prothrombotic genetic abnormalities may be shared risk factors in IS and MA, and may play a role in increasing the risk of cerebrovascular disease in migraineurs.     

北京地区400例甲型H1N1流感患者流行病学及临床分析

目的 了解北京地区400例新型甲型H1N1流感患者的流行病学和临床特征,总结规律.进一步指导临床诊治.方法 2009年5-12月期间,收治400例甲型H1N1流感确诊病例.主要采用描述性流行病学方法对患者资料进行回顾性分析,并运用单因素方差分析的方法对结果进行检验.结果 患者以青年和儿童人群为主,47.0%的患者有明确...