1个重型遗传性凝血因子Ⅶ缺陷症家系分子遗传分析

凝血因子Ⅶ(FⅦ)在肝脏中合成并以单链酶原的形式分泌到血浆中,活化的FⅩ(FⅩa)、FⅨa及FⅦa(自身)可将FⅦ在Arg 152-Ile153肽键处裂解成轻链(152AA)和重链(254AA),使其变成FⅦa。在血管损伤后,FⅦa与组织因子(TF)结合形成复合物(FⅦa/TF),激活FⅩ、FⅨ等,在内源及     

1例遗传性凝血因子Ⅶ缺陷症患者的家系表型及基因突变分析

目的 分析1例遗传性凝血因子Ⅶ(FⅦ)缺陷症的家系表型及基因突变情况.方法 选取先证者及其父母、妹妹为研究对象,分析该家系表型及基因突变情况.①家系表型分析:在IL-ALC TOP700血凝分析仪上采用凝固法检测先证者及其父母亲、妹妹的凝血酶原时间(PT)、PT纠正试验、国际标准化比值(INR)、凝血酶原活动度(PTA...     

遗传性凝血因子Ⅶ缺陷症家系基因型与临床表型的关系

目的:探讨2例遗传性凝血因子Ⅶ(Coagulation factor Ⅶ,FⅦ)缺陷症家系基因型与临床表型的关系。方法:用DNA直接测序法对先证者及其家庭成员FⅦ基因的全部外显子及其侧翼、启动子和3’非翻译区进行分析,寻找基因突变;将含插入突变序列克隆入pMD18-T TA克隆载体中,对所得两条染色体相应序列分别测序,以确定不同突变在染色体上的分布。用等位基因特异的聚合酶链反应(ASPCR)方法证实测序所发现的突变。结果:家系1先证者在8号外显子上发现一个杂合基因突变:11514C>T,导致Thr(ACG)359 Met(ATG)氨基酸替换,该突变来自其母亲;其父亲为11496G>A改变,导致Arg(CGG)353 Gln(CAG)杂合多态性。家系2先证者发现了三种杂合多态性:即—323插入10个核苷酸(—323P0/P10)、73G>A(G73A)及Arg 353 Gln均来自先证者的母亲和外祖父,三种多态性均位于先证者同一条染色体上。家系其他成员的基因型为野生型。AS-PCR证实了先证者及其家系成员的基因突变。结论:两个家系先证者的FⅦ缺陷症基因型与其临床表型无明显相关性。     

1个遗传性凝血因子Ⅴ缺陷症家系表型与基因分析

目的:对1个遗传性凝血因子Ⅴ(FⅤ)缺陷症家系进行表型与基因分析,探讨其分子发病机制。方法:通过检测1例FⅤ缺陷患者及其家系的凝血酶原时间(PT)、活化部分凝血活酶时间(APTT)、纤维蛋白原(FIB)、血浆FⅤ活性(FⅤ∶C)和血浆FⅤ抗原(FⅤ∶Ag)等凝血指标进行表型诊断,用DNA直接测序法分析患者及其家系成员FⅤ基因的全部外显子及侧翼、5’和3’非翻译区,发现突变位点用反向测序予以证实。结果:患者的PT和APTT均明显延长,分别为18.3s和44.9s,其FⅤ∶C为26.0%,FⅤ∶Ag为20.3%。患者FⅤ基因第22外显子上发现67868C→T的杂合突变,导致2031Glnstop;同时在患者FⅤ基因第10和16外显子上发现2个多态性(SNP)位点,分别为Arg485Lys和Met1736Val;患者的母亲、二弟、三弟、儿子、侄儿同样存在上述的基因杂合突变和SNP,其FⅤ∶C和FⅤ∶Ag也均有不同程度下降;患者的父亲和女儿为正常野生型,其FⅤ∶C和FⅤ∶Ag均在正常水平。结论:FⅤ基因第22外显子上67868C→T杂合突变,导致2031Glnstop与Arg485Lys纯合多态性的协同作用是该遗传性FⅤ缺陷症家系FⅤ水平降低的主要原因。     

2种F10基因新突变导致凝血因子Ⅹ缺陷症的分子发病机制研究

目的:探讨1个凝血因子Ⅹ(FⅩ)缺陷症家系的分子发病机制。方法:对先证者及家系成员凝血、抗凝及纤溶功能筛查以及凝血因子活性及抗原含量检测进行表型诊断;以Western Blotting检测血浆中FⅩ抗原含量和分子量大小;以中和试验检测FⅩ的抑制物。以PCR方法对F10基因所有外显子及侧翼序列和5’端非翻译区进行扩增,产物纯化后直接测序进行基因诊断;构建F10基因突变表达质粒,瞬时转染HEK293T细胞,测定表达产物的FⅩ促凝活性(FⅩ:C)和FⅩ抗原含量(FⅩ:Ag)。结果:先证者FⅩ:C和FⅩ:Ag分别为A和Asp368del。Asp368del体外表达显示FⅩ:C和FⅩ:Ag分别为(0.52±0.04)%和(85.9±5.0)%,为CRM+突变。结论:F10基因双重杂合突变IVS5+1G>A和Asp368del导致该家系遗传性FⅩ缺陷症。剪接位点突变IVS5+1G>A导致内含子无法正常剪接,影响FⅩ正常表达。Asp368del突变蛋白能够正常表达,但功能降低。     

心电图不典型的Brugada综合征家系调查及初步分子遗传学研究

目的 在1个Bruagada综合征病人发现特殊的心电图表现：胸前导联ST段呈上斜形抬高，凸面向上，无明显J波，无右束支阻滞(RBBB)。对其家系进行了临床调查及SCN5A基因(编码心脏电压门控Na^ 通道蛋白a亚单位基因)突变检测，分析其临床及分子遗传学特征。方法 家系临床调查包括收集所有15个家系成员的病史资料及进行常规体格检查，12导联心电图、超声心动图、X光胸片检查等。采用PCR-单链构象多态性技术(SSCP)结合DNA序列测定证实，对病人SCN5A的全部28个外显子进行突变检测。结果 所有成员均无器质性心脏病的证据，先证者有晕厥病史，并被记录到频发极短配对间期的室性早搏、多形室性心动过速及心室颤动，而其他家族成员均无晕厥或猝死病史，先证者的1个儿子及侄儿与其静息心电图表现相似。基因突变检测未能在病人的SCN5A基殷中发现遗传缺陷。结论 发现了一种特殊的Bruagada心电图模式，对目前Bruagada综合征诊断标准提出质疑，并提示存在遗传不均一性，SCN5A可能不是惟一的致病基因。     

一个三组妹同患P450c17α缺陷家系的分子遗传学研究

研究１个三组妹同患Ｐ４５０ｃ１７α缺陷家系的分子遗传学机制。方法采用聚合酶链反应－单链构象多态性,限制性内切酶酶切及自动测序等方法检测患者家系中ＣＹＰ１７基因的突变情况。结论１个三组妹同时患病的中国人Ｐ４５０ｃ１７α缺陷的家系是由于ＣＹＰ１７基因的复合杂合突变所致。     

内啡肽基因变异所致遗传性出血性毛细血管扩张症家系的遗传学分析

目的探讨ENG基因变异所致遗传性出血性毛细血管扩张症(hereditary hemorrhagic telangiectasia, HHT)一家系的遗传学特征。方法选取在大理大学第一附属医院就诊的一个HHT家系3代共17人为研究对象。收集该HHT家系先证者的临床资料及家系患病情况。应用全外显子组测序技术对先证者进行疑似致病基因筛选, 应用Sanger测序进行家系验证。结果先证者及母亲有反复鼻出血、皮肤黏膜毛细血管扩张表现, 先证者及其母亲、女儿和表弟胸部增强CT提示存在不同程度的肺动静脉畸形。全外显子测序结果显示先证者携带ENG基因c.579₅99del非移码缺失突变, Sanger测序显示其母亲、女儿和表弟携带相同的变异。结论 ENG基因c.579₅99del突变可能是导致该家系HHT的遗传学基础。     

JAK2 V617F及exon12突变阴性的红细胞增多患者遗传性红细胞增多症相关基因变异分析

目的 分析JAK2 V617F及exon12突变阴性的红细胞增多患者遗传性红细胞增多症相关基因变异情况。方法 回顾性纳入2021年6月～2022年11月于我院就诊、JAK2 V617F及exon12突变筛查结果为阴性且无继发性红细胞增多原因的红细胞增多症患者31例,收集其一般资料、实验室检查结果,分析遗传性红细胞增多症相关基因变异情况。结果 在31例红细胞增多症患者中,2例(6.45%)检测到EPO基因变异,分别为NM＿000799.4:c.401C>T p.Thr134Ile和NM＿000799.4:c.-36C>A。在受检患者中,检测到PHD2和SH2B3基因共4个单核苷酸多态性(SNP)位点,按发生率由高至低依次为SH2B3基因rs3184504、rs78894077、PHD2基因rs186996510及SH2B3基因rs140649197,其中rs78894077在本研究中的检出率显著高于数据库中人群变异频率,且Mutation Taster及PolyPhen-2软件预测该变异可能有害。结论 在JAK2 V617F及exon12突变阴性的红细胞增多症患者中发现两个新...     

17α羟化酶17，20碳链裂解酶缺陷症家系分子诊断研究

目的 对17α羟化酶17,20碳链裂解酶缺陷症(17OHD)1个家系4人进行分子诊断。方法 对2013年南京军区福州总医院收治的17OHD患者及其母亲、小姨和妹妹全血中抽提基因组DNA,设计8对引物,采用PCR扩增产物直接测序方法进行基因突变分析。结果 先证者的临床表现符合17OHD,其家族中未有其他病例。先证者序列分析表明,其CYP17A1基因第8外显子存在9个碱基(GACTCTTTC)缺失,导致第487~489位氨基酸缺失,使P450c17酶完全失活,从而导致17OHD。其母亲和小姨为该位点杂合缺失,妹妹未检测到同一缺失。结论 建立了基于CYP17A1基因突变分析的分子诊断方法,可以对17OHD进行准确的分子诊断,并可为遗传咨询及其产前诊断奠定基础。     

Molecular biology and clinical manifestation of hereditary factor VII deficiency

Inherited factor VII (FVII) deficiency is a rare autosomal recessive disorder. Mutations and polymorphisms of the FVII gene were characterized in more than 40 unrelated patients with FVII deficiency. Among the 29 different mutations, the most frequent were Ala294 Val, Ala294Val;404delC, IVS7+7, and Val281 Phe. Four novel mutations (IVS2+1G>C, Arg247 Cys, Glu265 Lys, Asp343 His) were detected. The relationships between genotypes of mutations and polymorphisms of the FVII gene, FVII deficiency, and clinical phenotype were investigated. Homozygosity of the Phe4 Leu, IVS4+1G>A, Cys135 Arg, Ala244 Val, and Ala294 Val;404delC and the double heterozygosity of Tyr68 Cys / IVS3-1G>A, Val252 Met / IVS2+5G>T, Val281 Phe / Cys135 Arg, Ala294 Val / Val281 Phe, Ala294 Val;404delC / Val281Phe, Ala294 Val;404delC / Arg152 stop, Ala294Val;404delC / Gln(-35) stop, Ala294 Val / Val252 Met, Ala294 Val / Gly156 Asp, and Thr359 Met / Asp242 His were related to clinical symptoms. Double heterozygotes for Arg247 Cys / IVS2+1G>C, Ala206 Thr / Pro303 Arg, Leu(-20) Pro / Val252 Met as well as IVS7+7 /Ala294 Val, IVS7+7 /Ala206 Thr, and IVS7+7 / Met298 Ile were asymptomatic. The clinical symptomatology is rather poor in correlation with the FVII activity. Concerning the clinical phanotype, a correlation seems to exist between specific mutations and clinical symptoms.     

Molecular analysis of Polish patients with factor VII deficiency

We analyzed the mutations in patients from 10 Polish kindreds with a bleeding diathesis due to factor VII deficiency. Patients from eight families had plasma levels of factor VII coagulant activity (VII:C) and factor VII antigen (VII:Ag) that were less than 4% of normal. The coding sequence of the factor VII gene was amplified from genomic DNA by polymerase chain reaction (PCR). Sequencing demonstrated a C to T transition at position 10798 resulting in Ala294Val, a G to A transition at 10976 resulting in Arg353Gln, and a single bp deletion at 11125 to 11128 causing a frameshift mutation in the triplet encoding amino acid 404. Homozygosity for the three sequence alterations was confirmed with the restriction enzymes AvaII and MspI and allele specific PCR, respectively. A homozygous patient from a ninth family with levels of VII:C and VII:Ag of 4% and 17%, respectively, had Ala294Val and the frameshift mutation, but not Arg353Gln. Investigation of a homozygous patient from a tenth kindred with VII:C and VII:Ag of 11% and 47%, respectively, demonstrated Ala294Val and Arg353Gln, but not the frameshift mutation. Based on the above data, we conclude that the frameshift mutation in the codon for amino acid 404 is associated with marked reductions in VII:C, Arg353Gln can decrease plasma levels of factor VII in the presence of other mutations in the factor VII gene, and Ala294Val results in a dysfunctional factor VII molecule.     

Hip surgery in a patient with severe factor VII deficiency

We describe the clinical history of a 35-year-old woman with severe cross-reacting-material-negative factor VII deficiency. Because of a severe hemophilic arthropathy of the hips, reconstructive surgery was indicated. After advice was obtained from 11 clinical hemostasis experts it was decided to use plasma for factor VII replacement during and after surgery and to use pneumatic boots to prevent thrombosis. Volume overload was treated with plasmapheresis and diuretics. The operation was carried out without abnormal blood loss and the patient recovered without thromboembolic complications. We conclude that continuous infusion of plasma is a suitable way to replace factor VII provided adequate venous access is available for plasmapheresis.     

Recombinant factor VIIa prophylaxis in a patient with severe congenital factor VII deficiency

Use of recombinant factor VIIa (rFVIIa, NovoSeven in patients with congenital FVII deficiency has been reported for the prophylactic management of surgical bleeding and for the treatment of acute bleeding episodes. Because of its short half-life, the use of rFVIIa on a regular prophylactic regimen has not been routinely adopted. In this report, we describe our successful experience with rFVIIa prophylaxis in preventing recurrent target joint bleeding in a severely FVII-deficient adolescent.     

Occurrence of hereditary leaky red cell syndrome and partial coagulation factor VII deficiency in a Spanish family.

A Spanish family was found to have the coexistence of a hereditary haemolytic syndrome associated with excessively leaky RBC membrane to sodium (Na+) and potassium (K+) cations and a partial coagulation factor VII deficiency. Haemolysis was mild in the propositus and the RBC membrane leak included a marked increase in passive permeability to Na+ and K+. This was associated with an increase in active Na+,K(+)-pump activity and in the ouabain-resistant fluxes: Na+, K(+)-cotransport and Na+, Li(+)-countertransport. Factor VII deficiency was of 50% and no clinical expression of the coagulation deficiency was observed. The family study revealed slightly abnormal RBC membrane cationic fluxes only in the father and decreased coagulation factor VII activity of 67% in the mother. Both parents were clinically and haematologically normal. It is suggested that the propositus has inherited the abnormal gene for leaky RBC syndrome from the father and the partial coagulation factor VII deficiency from the mother.     

The use of activated recombinant coagulation factor VII during haemarthroses and synovectomy in a patient with congenital severe factor V deficiency

Factor V deficiency is a rare hereditary bleeding disorder. Currently, FV concentrates are not available, and the treatment of spontaneous bleeding or bleeding associated with invasive procedures is transfusion of fresh frozen plasma (FFP). However, FFP transfusion can lead to the development of inhibitor to FV, and is associated with several potential transfusion reactions including allergic reactions. We report a patient with congenital severe FV deficiency with repeated haemarthroses of a shoulder joint, and progressively severe allergic reactions to FFP transfusions. In addition, the patient also developed acute pulmonary oedema. Activated recombinant coagulation factor VII (rFVIIa) was used as an alternative haemostatic agent to FFP. We describe the use of rFVIIa in this patient during haemarthroses, synovectomy, and physiotherapy.     

Lack of bleeding in patients with severe factor VII deficiency

Factor VII deficiency, although rare, is now recognized as the most common autosomal recessive inherited factor deficiency. It is usually considered to be associated with bleeding only in the severely affected subject and heterozygotes (>10%) are not considered at risk. The general recommendation for surgery is to achieve a FVII level in excess of 15% (0.15 1U/mL). We present three cases of severe factor VII deficiency, each of whom appeared hemostatically competent based on clinical history. Subject 1 is a 33 year-old African-American female with a baseline FVII of <1%, who had a fractured tibia requiring open reduction with internal fixation without any FVII replacement and subsequently underwent successful laparoscopic knee surgery with a factor VII level measured at 6%. Subject 2 is a 58 year-old African-American female with a factor VII level of 9% who underwent an elective left total hip replacement without any factor replacement and had no excessive bleeding, but who sustained a pulmonary embolism postoperatively. Subject 3 is a 19-year-old African-American male with a baseline FVII of 1% with a history of active participation in football without noticeable injury and who underwent an emergent appendectomy without bleeding. These three cases represent individuals with the severe form of FVII deficiency who did not exhibit excessive bleeding when challenged with surgical procedures. The clinical history would appear the most valuable tool in predicting the likelihood of bleeding in these patients, and we suggest that the presumption that all patients with severe FVII deficiency should receive replacement therapy before surgical procedures may not be valid in all cases.     

Characterization of a Cys329Gly mutation causing hereditary factor VII deficiency

We have previously reported a homozygous Cys329Gly mutation in a Chinese patient with factor VII (FVII) deficiency. Others have found a heterozygous Cys329Gly mutation in the F7 gene from patients of three different pedigrees. However, none of the reports included the expression and characterization of the mutant FVII in vitro. To investigate the effect of Cys329Gly on FVII function, we carried out transient transfections of baby hamster kidney cells (BHK-21) with a mutant FVII construct and compared the results to those obtained using a wild-type FVII construct and vector control. The results demonstrate that the level of FVII:Ag secreted into the medium by transfected BHK-21 cells with mutant construct was not affected, but the coagulation activity of the mutant FVII was undetectable. We conclude that Cys329 is critical to FVII coagulation, and the replacement of cysteine 329 by glycine leads to the loss of coagulation activity in the patients, possibly the molecular basis for FVII deficiency in the patients.