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1.
J Thromb Haemost ; 14(9): 1725-35, 2016 09.
Article in English | MEDLINE | ID: mdl-27344059

ABSTRACT

UNLABELLED: Essentials Von Willebrand disease IIC Miami features high von Willebrand factor (VWF) with reduced function. We aimed to identify and characterize the elusive underlying mutation in the original family. An inframe duplication of VWF exons 9-10 was identified and characterized. The mutation causes a defect in VWF multimerization and decreased VWF clearance from the circulation. SUMMARY: Background A variant of von Willebrand disease (VWD) type 2A, phenotype IIC (VWD2AIIC), is characterized by recessive inheritance, low von Willebrand factor antigen (VWF:Ag), lack of VWF high-molecular-weight multimers, absence of VWF proteolytic fragments and mutations in the VWF propeptide. A family with dominantly inherited VWD2AIIC but markedly elevated VWF:Ag of > 2 U L(-1) was described as VWD type IIC Miami (VWD2AIIC-Miami) in 1993; however, the molecular defect remained elusive. Objectives To identify the molecular mechanism underlying the phenotype of the original VWD2AIIC-Miami. Patients and Methods We studied the original family with VWD2AIIC-Miami phenotypically and by genotyping. The identified mutation was recombinantly expressed and characterized by standard techniques, confocal imaging and in a mouse model, respectively. Results By Multiplex ligation-dependent probe amplification we identified an in-frame duplication of VWF exons 9-10 (c.998_1156dup; p.Glu333_385dup) in all patients. Recombinant mutant (rm)VWF only presented as a dimer. Co-expressed with wild-type VWF, the multimer pattern was indistinguishable from patients' plasma VWF. Immunofluorescence studies indicated retention of rmVWF in unusually large intracellular granules in the endoplasmic reticulum. ADAMTS-13 proteolysis of rmVWF under denaturing conditions was normal; however, an aberrant proteolytic fragment was apparent. A decreased ratio of VWF propeptide to VWF:Ag and a 1-desamino-8-d-arginine vasopressin (DDAVP) test in one patient indicated delayed VWF clearance, which was supported by clearance data after infusion of rmVWF into VWF(-/-) mice. Conclusion The unique phenotype of VWD2 type IIC-Miami results from dominant impairment of multimer assembly, an aberrant structure of mutant mature VWF and reduced clearance in vivo.


Subject(s)
Mutation , von Willebrand Disease, Type 2/genetics , von Willebrand Factor/genetics , Adult , Aged , Animals , Deamino Arginine Vasopressin/chemistry , Endoplasmic Reticulum/metabolism , Female , Genes, Dominant , Genes, Recessive , Genotype , Humans , Male , Mice , Mice, Inbred C57BL , Middle Aged , Phenotype , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , von Willebrand Disease, Type 2/metabolism , von Willebrand Factor/metabolism
2.
Thromb Haemost ; 112(1): 96-108, 2014 Jul 03.
Article in English | MEDLINE | ID: mdl-24598842

ABSTRACT

The bleeding disorder von Willebrand disease (VWD) is caused by mutations of von Willebrand factor (VWF), a multimeric glycoprotein essential for platelet-dependent primary haemostasis. VWD type 2A-associated mutations each disrupt VWF biosynthesis and function at different stages, depending on the VWF domain altered by the mutation. These effects cause considerable heterogeneity in phenotypes and symptoms. To characterise the molecular mechanisms underlying the specific VWF deficiencies in VWD 2A/IIC, IID and IIE, we investigated VWF variants with patient-derived mutations either in the VWF pro-peptide or in domains D3 or CK. Additionally to static assays and molecular dynamics (MD) simulations we used microfluidic approaches to perform a detailed investigation of the shear-dependent function of VWD 2A mutants. For each group, we found distinct characteristics in their intracellular localisation visualising specific defects in biosynthesis which are correlated to respective multimer patterns. Using microfluidic assays we further determined shear flow-dependent characteristics in polymer-platelet-aggregate formation, platelet binding and string formation for all mutants. The phenotypes observed under flow conditions were not related to the mutated VWF domain. By MD simulations we further investigated how VWD 2A/IID mutations might alter the ability of VWF to form carboxy-terminal dimers. In conclusion, our study offers a comprehensive picture of shear-dependent and shear-independent dysfunction of VWD type 2A mutants. Furthermore, our microfluidic assay might open new possibilities for diagnosis of new VWD phenotypes and treatment choice for VWD patients with shear-dependent VWF dysfunctions that are currently not detectable by static tests.


Subject(s)
Blood Platelets/physiology , von Willebrand Disease, Type 2/genetics , von Willebrand Factor/metabolism , Dimerization , HEK293 Cells , Humans , Microfluidics , Molecular Dynamics Simulation , Mutation/genetics , Phenotype , Protein Structure, Tertiary/genetics , Shear Strength/physiology , von Willebrand Disease, Type 2/classification , von Willebrand Factor/genetics
4.
J Thromb Haemost ; 10(5): 951-8, 2012 May.
Article in English | MEDLINE | ID: mdl-22429825

ABSTRACT

BACKGROUND: von Willebrand disease (VWD) is caused by mutations in von Willebrand factor (VWF) that have different pathophysiologic effect in causing low plasma VWF levels. Type 1 VWD includes quantitative plasma VWF deficiency with normal VWF structure and function. OBJECTIVES: We report three novel type 1 VWF mutations (A1716P, C2190Y and R2663C) located in different VWF domains that are associated with reduced secretion and reduced formation of elongated Weibel-Palade body (WPB)-like granules. METHODS: Transient expression of recombinant mutant full-length VWF in 293 EBNA cells was performed and secretion, collagen binding and GpIb binding assessed in comparison with wild-type VWF. Expression was also examined in HEK293 cells that form WPB-like granules when transfected with wild-type VWF. RESULTS: Laboratory results and multimer analysis of plasma VWF was compatible with type 1 VWD. Expression experiments demonstrated slightly reduced VWF synthesis and drastically impaired secretion upon homozygous expression. In HEK293 cells, homozygous expression of A1716P and C2190Y VWF variants failed to form elongated WPB-like granules, while R2663C was capable of WPB-like granules. Heterozygous expression of VWF variants had a negative impact on wild-type VWF with a reduction in elongated WPB-like granules in co-transfected cells. CONCLUSIONS: Our results demonstrate that homozygous and heterozygous quantitative VWF deficiency caused by missense VWF mutations in different VWF domains can be associated with inability to form endothelial WPB-like granules.


Subject(s)
Weibel-Palade Bodies/metabolism , Weibel-Palade Bodies/pathology , von Willebrand Disease, Type 1/pathology , von Willebrand Factor/metabolism , Collagen/metabolism , Down-Regulation , Fluorescent Antibody Technique , Genetic Predisposition to Disease , HEK293 Cells , Heterozygote , Homozygote , Humans , Microscopy, Confocal , Mutation, Missense , Phenotype , Platelet Glycoprotein GPIb-IX Complex/metabolism , Protein Binding , Protein Multimerization , Transfection , von Willebrand Disease, Type 1/blood , von Willebrand Disease, Type 1/genetics , von Willebrand Factor/genetics
5.
J Thromb Haemost ; 9(5): 945-52, 2011 May.
Article in English | MEDLINE | ID: mdl-21362127

ABSTRACT

BACKGROUND: Type 3 von Willebrand disease (VWD) is an autosomal recessive bleeding disorder, characterized by virtually undetectable plasma von Willebrand factor (VWF) and consequently reduced plasma factor VIII levels. Genetic mutations responsible for type 3 VWD are very heterogeneous, scattered throughout the VWF gene and show high variability among different populations. METHODS: Twenty-five severe VWD patients were studied by direct sequencing of the 51 coding exons of the VWF gene. The total number of VWD type 3 families in Hungary is 24, of which 23 were investigated. RESULTS: Fifteen novel mutations were identified in 31 alleles, five being nonsense mutations (p.Q1238X, p.Q1898X, p.Q1931X, p.S2505X and p.S2568X), four small deletions and insertions resulting in frame shifts (c.1992insC, c.3622delT, c.5315insGA and c.7333delG), one a large partial deletion (delExon1-3) of the 5'-region, four candidate missense mutations (p.C35R, p.R81G, p.C295S, p.C623T) and one a candidate splice site mutation (c.1730-10C>A). Six previously described mutations were detected in 17 alleles, including the repeatedly found c.2435delC, p.R1659X and p.R1853X. Only one patient developed alloantibodies to VWF, carrying a homozygous c.3622delT. CONCLUSION: We report the genetic background of the entire Hungarian type 3 VWD population. A large novel deletion, most probably due to a founder effect, seems to be unique to Hungarian type 3 VWD patients with high allele frequency. In contrast to previous reports, none of the five patients homozygous for the large partial deletion developed inhibitors to VWF. This discrepancy raises the possibility of selection bias in some of the reports.


Subject(s)
von Willebrand Disease, Type 3/genetics , von Willebrand Factor/genetics , Adolescent , Adult , Child , Female , Gene Deletion , Genotype , Humans , Hungary , Isoantibodies/chemistry , Isoantibodies/genetics , Male , Models, Genetic , Mutation , Mutation, Missense , Registries , Surveys and Questionnaires
6.
J Thromb Haemost ; 8(9): 2011-6, 2010 Sep.
Article in English | MEDLINE | ID: mdl-20586924

ABSTRACT

BACKGROUND: von Willebrand disease (VWD) type Normandy (VWD 2N) is caused by mutations at the factor (F)VIII-binding site of von Willebrand factor (VWF), located in the D'and D3 domains on the N-terminus of mature VWF. The R854Q mutation is the most frequent cause of this phenotype. OBJECTIVES: We report the characterization of a homozygous VWD 2N mutation, R854W, detected in a patient with a severe VWD phenotype. METHODS: The plasma VWF phenotype was studied, transient expression of recombinant mutant full-length VWF in 293 EBNA cells was performed, and the results were compared with those obtained with wild-type (WT) VWF. Furthermore, expression was also examined in HEK293 cells, which form Weibel-Palade body-like granules when transfected with WT VWF. RESULTS: The multimer analysis of plasma VWF showed the lack of the typical triplet structure, with the presence of the central band only, and a relative decrease in the high molecular mass multimers. Homozygous expression of recombinant R854W VWF resulted in normal amounts of cellular VWF, but with a severe reduction in secretion into the medium. Severe reductions in FVIII binding to R854W VWF, glycoprotein Ib binding activity and collagen binding of secreted W854 VWF was observed, and reproduced the phenotypic parameters of plasma VWF. In HEK293 cells, homozygous R854W VWF failed to form Weibel-Palade body-like granules. CONCLUSIONS: Our results demonstrate that a homozygous R854W mutation in the D' domain of VWF induces impaired secretion and activity of the protein, thereby explaining the severe phenotype of the patient.


Subject(s)
Mutation , von Willebrand Diseases/genetics , von Willebrand Factor/genetics , von Willebrand Factor/metabolism , Adult , Blood Coagulation , Cell Line , DNA Mutational Analysis , Female , Heterozygote , Homozygote , Humans , Microscopy, Confocal/methods , Microscopy, Fluorescence/methods , Phenotype , Recombinant Proteins
7.
J Thromb Haemost ; 7(12): 2096-105, 2009 Dec.
Article in English | MEDLINE | ID: mdl-19817991

ABSTRACT

BACKGROUND: To avoid pathological platelet aggregation by von Willebrand factor (VWF), VWF multimers are regulated in size and reactivity for adhesion by ADAMTS13-mediated proteolysis in a shear flow dependent manner. OBJECTIVE AND METHODS: We examined whether tensile stress in VWF under shear flow activates the VWF A2 domain for cleavage by ADAMTS13 using molecular dynamics simulations. We generated a full length mutant VWF featuring a homologous disulfide bond in A2 (N1493C and C1670S), in an attempt to lock A2 against unfolding. RESULTS: We indeed observed stepwise unfolding of A2 and exposure of its deeply buried ADAMTS13 cleavage site. Interestingly, disulfide bonds in the adjacent and highly homologous VWF A1 and A3 domains obstruct their mechanical unfolding. We find this mutant A2 (N1493C and C1670S) to feature ADAMTS13-resistant behavior in vitro. CONCLUSIONS: Our results yield molecular-detail evidence for the force-sensing function of VWF A2, by revealing how tension in VWF due to shear flow selectively exposes the A2 proteolysis site to ADAMTS13 for cleavage while keeping the folded remainder of A2 intact and functional. We find the unconventional 'knotted' Rossmann fold of A2 to be the key to this mechanical response, tailored for regulating VWF size and activity. Based on our model we discuss the pathomechanism of some natural mutations in the VWF A2 domain that significantly increase the cleavage by ADAMTS13 without shearing or chemical denaturation, and provide with the cleavage-activated A2 conformation a structural basis for the design of inhibitors for VWF type 2 diseases.


Subject(s)
ADAM Proteins/metabolism , Protein Denaturation , Protein Stability , von Willebrand Factor/metabolism , ADAMTS13 Protein , Adhesiveness , Humans , Hydrolysis , Mutation , Protein Binding , Protein Conformation , Protein Multimerization , Stress, Mechanical , von Willebrand Factor/genetics
8.
J Thromb Haemost ; 7(8): 1304-12, 2009 Aug.
Article in English | MEDLINE | ID: mdl-19566550

ABSTRACT

BACKGROUND: Candidate von Willebrand factor (VWF) mutations were identified in 70% of index cases in the European study 'Molecular and Clinical Markers for the Diagnosis and Management of type 1 von Willebrand Disease'. The majority of these were missense mutations. OBJECTIVES: To assess whether 14 representative missense mutations are the cause of the phenotype observed in the patients and to examine their mode of pathogenicity. METHODS: Transfection experiments were performed with full-length wild-type or mutant VWF cDNA for these 14 missense mutations. VWF antigen levels were measured, and VWF multimer analysis was performed on secreted and intracellular VWF. RESULTS: For seven of the missense mutations (G160W, N166I, L2207P, C2257S, C2304Y, G2441C, and C2477Y), we found marked intracellular retention and impaired secretion of VWF, major loss of high molecular weight multimers in transfections of mutant constructs alone, and virtually normal multimers in cotransfections with wild-type VWF, establishing the pathogenicity of these mutations. Four of the mutations (R2287W, R2464C, G2518S, and Q2520P) were established as being very probably causative, on the basis of a mild reduction in the secreted VWF or on characteristic faster-running multimeric bands. For three candidate changes (G19R, P2063S, and R2313H), the transfection results were indistinguishable from wild-type recombinant VWF and we could not prove these changes to be pathogenic. Other mechanisms not explored using this in vitro expression system may be responsible for pathogenicity. CONCLUSIONS: The pathogenic nature of 11 of 14 candidate missense mutations identified in patients with type 1 VWD was confirmed. Intracellular retention of mutant VWF is the predominant responsible mechanism.


Subject(s)
Mutation , von Willebrand Factor/genetics , Animals , COS Cells , Chlorocebus aethiops , Humans , Mutant Proteins , Mutation, Missense , Phenotype , Protein Multimerization , Transfection , von Willebrand Diseases/genetics , von Willebrand Factor/metabolism
9.
Hamostaseologie ; 29(2): 158-60, 2009 May.
Article in English | MEDLINE | ID: mdl-19404520

ABSTRACT

UNLABELLED: Molecular genetic testing for factor VIII (FVIII) mutations is indicated in haemophilia A since determination of FVIII activity cannot reliably identify female carriers. Given the large number of FVIII mutations the identification of novel mutations is not uncommon. Since amino acid polymorphisms of FVIII are rare, missense mutations in patients with haemophilia A which are not found in the normal population are considered as causative in general practice when no other mutation can be detected by complete FVIII gene sequencing. We report a novel rare missense variant (P2311S) in a haemophilia A family that was mistakenly considered as pathogenic leading to amniocentesis, prenatal diagnosis and influenced the peripartal management of the putatively affected child. Subsequently, we identified the novel causative mutation V197G in the family's index case which could be detected neither in the neonate nor in his mother. CONCLUSION: This case emphasizes the necessity to establish the molecular diagnosis in the family's index case and to perform expression studies of novel mutations to prove their causative nature.


Subject(s)
Factor VIII/genetics , Genetic Counseling , Hemophilia A/genetics , Mutation, Missense , Female , Genetic Vectors , Humans , Male , Pedigree , Sequence Deletion
10.
Hamostaseologie ; 29(2): 143-8, 2009 May.
Article in English | MEDLINE | ID: mdl-19404524

ABSTRACT

UNLABELLED: We have prospectively evaluated the biologic response to desmopressin (DDAVP) in 28 children with type 2 von Willebrand disease (VWD) in correlation with the phenotype and the molecular defect of VWF. The diagnosis of VWD type 2 was mainly based on VWF functional parameters and/or an aberrant VWF multimer pattern. Seventeen different mutations were identified (6 of them novel). No response with respect to the functional parameters VWF:RCo and/or VWF:CB was seen in patients with severe abnormality of the VWF multimer pattern. One patient with VWD type 2A phenotype IIC Miami did not respond with respect to VWF:CB, but showed a good response of VWF:Ag and FVIII:C as expected. Interestingly he showed a persistently high level of VWF:Ag and FVIII:C up to 4 hours after DDAVP infusion. Patients with minor alterations of multimer structure and particular mutations responded well to DDAVP, whereas patients with normal multimer structure but a defect in platelet dependent functional parameters did not respond with VWF:RCo. CONCLUSION: Children with VWD type 2 show a variable response to desmopressin depending on the mutation that correlates with the functional defect and the presence or absence as well as the half-life of large VWF multimers. Our data emphasize the usefulness of DDAVP testing even in patients with VWD type 2, possibly with the exception of VWD type 2B.


Subject(s)
Deamino Arginine Vasopressin/therapeutic use , von Willebrand Diseases/drug therapy , Child , Hemostatics/therapeutic use , Humans , Mutation , Phenotype , von Willebrand Diseases/genetics , von Willebrand Factor/genetics
11.
J Thromb Haemost ; 6(10): 1729-35, 2008 Oct.
Article in English | MEDLINE | ID: mdl-18665926

ABSTRACT

BACKGROUND: We studied 24 Hungarian patients from 23 unrelated families to identify the genetic background of the entire type 3 von Willebrand disease (VWD) population in this country. The current report focuses on the molecular characterization of a novel large deletion. RESULTS: A large partial deletion (delExon1-3) of the 5'-region of the von Willebrand factor gene (VWF) was detected in 12/48 alleles (25% of all type 3 alleles). The 5'-deletion breakpoint is located in the untranslated region between VWF and CD9, whereas the 3' breakpoint is in intron 3 of VWF. Analysis of the breakpoints showed Alu Y and Alu SP repetitive sequences at the ends of the deletion, suggesting that a recombination event caused the subsequent loss of the 35-kb fragment. DelExon1-3 was not found in any of the other screened populations. CONCLUSION: We report a large novel deletion including exons 1, 2 and 3 of VWF commonly causing type 3 VWD in the Hungarian population. This mutation, probably caused by an Alu-mediated recombination event, and subsequently distributed in Hungary by a founder effect, seems to be unique to Hungarian patients with a high allele frequency. Together, delExon1-3 and 2435delC make up 37.5% of the genetic defects in Hungarian patients with VWD type 3. This offers a rational approach to molecular testing of relevant families in Hungary.


Subject(s)
Gene Deletion , von Willebrand Diseases/genetics , von Willebrand Factor/genetics , 5' Flanking Region , Alu Elements , Exons , Family , Founder Effect , Gene Frequency , Humans , Hungary , Introns , von Willebrand Diseases/classification , von Willebrand Diseases/epidemiology
12.
Blood Cells Mol Dis ; 41(2): 219-22, 2008.
Article in English | MEDLINE | ID: mdl-18485763

ABSTRACT

BACKGROUND: Von Willebrand disease type 3 VWD is an autosomal-recessively inherited severe bleeding disorder with a homogeneous phenotype on the basis of very heterogeneous genotypes. Many different molecular defects have been reported to date. We tried to assess the molecular background of Indian and Greek patients with VWD type 3 by doing a complete VWF gene screen in all index patients. MATERIALS AND METHODS: We investigated 21 unrelated Indian and six Greek patients with type 3 VWD. Mutation screening was done by PCR and direct sequencing of the coding VWF exons 2-52 including flanking intron sequences. RESULTS: The diagnosis of VWD type 3 could be confirmed by the detection of null alleles or two mutations each in 22 patients. In one patient only one heterozygous mutation was identified. In four patients no mutations were identified for unknown reasons. Most of the defects cause null alleles. Eight patients had homozygous nonsense mutations - R1659X (6 patients), W553X (1 patients) and L1267X (1 patient); 2 patients were compound heterozygous - R324X/R373X and N318K/Q565X; 3 patients had small insertions - 3259insT, 3737insCC and 7173insT; 2 patients had small deletions - 3938delG and 1381delG; 2 patients had a duplication of 8 bp (duplAGTGTGGA) in exon 28 and a missense mutation (R273W) in exon 7; one patient had a heterozygous mutation K1794E (second mutation not identified); 5 patients had gene conversions between VWF and its pseudogene (117 bp to 335 bp in length corresponding to the 5' end of exon 28). The mutations as part of the gene conversion were - S1263P, P1266L, V1279I, Q1311X, A1317, I1343V, V1360A, and F1369I. CONCLUSION: VWD type 3 is caused by a broad variety of mutations distributed over the entire VWF sequence. As expected most mutations cause null alleles (16/23). The most common molecular defects found were gene conversions and R1659X in exon 28.


Subject(s)
Mutation , von Willebrand Diseases/genetics , von Willebrand Factor/genetics , Adolescent , Adult , Child , Child, Preschool , DNA Mutational Analysis , Female , Genetic Testing , Greece , Humans , India , Male
13.
Br J Haematol ; 130(5): 752-8, 2005 Sep.
Article in English | MEDLINE | ID: mdl-16115133

ABSTRACT

von Willebrand disease (VWD), the most common inherited bleeding disorder, is very heterogeneous, both in its phenotype and genotype. One particular molecular mechanism of VWD is due to recombination events between the true gene and its pseudogene on chromosome 22. We assessed the frequency and extension of such events in 50 multi-ethnic index patients with severe VWD type 3 and in five index patients with VWD type 2M Vicenza. One additional unclassified patient had been diagnosed with possible VWD in Russia solely on a clinical basis. Gene conversions, previously thought to be rare events, were identified in >10% of our study population: in six multi-ethnic patients with severe VWD type 3, in one patient with VWD type 2M Vicenza and the Russian patient was finally diagnosed with VWD type 2B New York/Malmoe. Our results suggest a significant contribution of this particular molecular mechanism to the manifestation of VWD. The location of the gene conversions, their extension and their occurrence as homozygous, compound heterozygous or heterozygous mutations determines the resulting phenotype.


Subject(s)
Gene Conversion , Mutation, Missense , von Willebrand Diseases/classification , von Willebrand Factor/genetics , Cells, Cultured , DNA Mutational Analysis , Ethnicity , Germany , Greece , Haplotypes , Humans , India , Phenotype , Platelet Aggregation , Ristocetin/pharmacology , Russia , von Willebrand Diseases/ethnology , von Willebrand Diseases/genetics
14.
Hamostaseologie ; 24(1): 65-70, 2004 Feb.
Article in German | MEDLINE | ID: mdl-15029275

ABSTRACT

As hallmark of TTP, generalized hyaline thrombi in the patient's microcirculation is known. These thrombi are composed of platelets and VWF. A severe defect of the VWF cleaving protease (VWF-CP) was found in all known patients with the inherited form of TTP. In contrary, although a severe deficiency of VWF-CP is specific for the acquired form, too, only a fraction of these patients is characterized by a severe deficiency. In most patients with a severe deficiency autoantibodies directed against VWF-CP is detectable in plasma. However, many patients with acquired TTP do not show any severe deficiency. Because treatment differs in inherited and acquired forms and as persistance of autoantibodies during clinical remission is of prognostic value, the determination of the activity of VWF-CP and of antibodies against VWF-CP are important parts in the workup of patients with TTP. In all methods for the determination of the activity of VWF-CP the first step is proteolysis of a specific substrate for the protease. In the second step the activity of the protease is measured by analysis of the residual VWF multimers, by the generation of specific fragments, by using the residual VWF:CB or VWF:RCo as marker of the loss of multimers or with help of specific monoclonal antibodies. In less than 30 min the cone and plate(let) aggregometer helps to distinguish between different forms of thrombotic microangiopathies. While adhesion and aggregation of platelets from a healthy person are clearly enhanced after addition of a small amount of plasma from a TTP patient, both characteristics are weakened by plasma from patients with other forms of thrombotic microangiopathy (dilution effect). Molecular genetics are established methods in the differentiation between inherited and acquired forms of TTP in those cases without autoantibodies against VWF-CP.


Subject(s)
Purpura, Thrombotic Thrombocytopenic/diagnosis , ADAM Proteins , ADAMTS13 Protein , Diagnosis, Differential , Humans , Metalloendopeptidases/genetics , Platelet Aggregation , Purpura, Thrombotic Thrombocytopenic/blood , von Willebrand Factor/genetics
15.
Hamostaseologie ; 24(1): 71-6, 2004 Feb.
Article in German | MEDLINE | ID: mdl-15029276

ABSTRACT

Thrombotic thrombocytopenic purpura (TTP) is a micro-angiopathic disease due to deficiency of the specific VWF cleaving protease (VWF-CP) ADAMTS13. The acquired form is caused by autoantibodies against VWF-CP, whereas mutations of the ADAMTS13 gene are responsible for inherited TTP. In childhood both forms exist with predominance of inherited TTP. The phenotype of TTP in childhood can be rather variable. Besides the classical clinical picture, abortive forms may occur that can delay the identification of patients at risk. The patients are frequently assumed to suffer from idiopathic thrombocytopenia (ITP) or Evans syndrome. Further efforts are necessary to accelerate correct diagnosis and to establish a risk-adapted prophylactic therapy.


Subject(s)
Purpura, Thrombotic Thrombocytopenic/genetics , ADAM Proteins , ADAMTS13 Protein , Child , Child, Preschool , Diagnosis, Differential , Hemolysis , Humans , Infant , Infant, Newborn , Metalloendopeptidases/genetics , Phenotype , Prognosis , Purpura, Thrombotic Thrombocytopenic/diagnosis
16.
Blood ; 97(7): 2059-66, 2001 Apr 01.
Article in English | MEDLINE | ID: mdl-11264172

ABSTRACT

Dimerization defects of von Willebrand factor (vWF) protomers underlie von Willebrand disease (vWD) type 2A, subtype IID (vWD 2A/IID), and corresponding mutations have been identified at the 3' end of the vWF gene in exon 52. This study identified and expressed 2 additional mutations in this region, a homozygous defect in a patient with vWD type 3 (C2754W) and a heterozygous frameshift mutation (8566delC) in a patient with vWD type 2A, subtype IIE. Both mutations involve cysteine residues that we propose are possibly essential for dimerization. To prove this hypothesis, transient recombinant expression of each of the 2 mutations introduced in the carboxy-terminal vWF fragment II and in the complete vWF complementary DNA, respectively, were carried out in COS-7 cells and compared with expression of vWD 2A/IID mutation C2773R and the wild-type (WT) sequence in COS-7 cells. Recombinant WT vWF fragment II assembled correctly into a dimer, whereas recombinant mutant fragments were monomeric. Homozygous expression of recombinant mutant full-length vWF resulted in additional dimers, probably through disulfide bonding at the amino-terminal multimerization site, whereas recombinant WT vWF correctly assembled into multimers. Coexpression of recombinant mutant and recombinant WT vWF reproduced the multimer patterns observed in heterozygous individuals. Our results suggest that a common defect of vWF biosynthesis--lack of vWF dimerization--may cause diverse types and subtypes of vWD. We also confirmed previous studies that found that disulfide bonding at the vWF amino-terminal is independent of dimerization at the vWF carboxy-terminal. (Blood. 2001;97:2059-2066)


Subject(s)
von Willebrand Diseases/metabolism , von Willebrand Factor/chemistry , Adult , Amino Acid Substitution , Animals , COS Cells , Chlorocebus aethiops , Cystine/chemistry , DNA Mutational Analysis , Dimerization , Female , Frameshift Mutation , Gene Expression , Heterozygote , Humans , Male , Mutation, Missense , Pedigree , Phenotype , Protein Conformation , Recombinant Fusion Proteins/chemistry , Sequence Deletion , Structure-Activity Relationship , Transfection , von Willebrand Diseases/classification , von Willebrand Diseases/genetics , von Willebrand Factor/genetics
18.
Arch Dis Child ; 81(1): 57-9, 1999 Jul.
Article in English | MEDLINE | ID: mdl-10373137

ABSTRACT

The case of an 8 year old boy with both nocturnal enuresis and nephrogenic diabetes insipidus is presented. Diagnosis of nephrogenic diabetes insipidus was based on a typical medical history, the characteristic result of a fluid restriction test, the lack of an effect of 1-desamino-8-D-arginine (DDAVP) on both urine osmolality and plasma coagulation factors and, finally, the detection of a hemizygous missense mutation within the arginine vasopressin (AVP) receptor gene. Hydrochlorothiazide treatment and dietary measures reduced the patient's urine volume to one third of its original volume. However, this had no effect on enuresis. The daily intranasal application of DDAVP did not further reduce urine output but dramatically decreased the frequency of bed wetting. This observation contradicts the common notion that the therapeutic effect of DDAVP in nocturnal enuresis is the result of compensation for a nocturnal AVP deficit. Rather, it points to a different mode of action of DDAVP in patients with enuresis. It is hypothesised that central AVP receptors are a target of DDAVP and that they might play an important role in the pathogenesis of nocturnal enuresis.


Subject(s)
Circadian Rhythm , Deamino Arginine Vasopressin/therapeutic use , Diabetes Insipidus, Nephrogenic/complications , Enuresis/drug therapy , Renal Agents/therapeutic use , Child , Enuresis/complications , Humans , Male
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