A Novel Mutation Glyl672→Arg in Type 2A and a Homozygous Mutation in Type 2B von Willebrand Disease

1996 ◽  
Vol 76 (02) ◽  
pp. 253-257 ◽  
Author(s):  
Takeshi Hagiwara ◽  
Hiroshi Inaba ◽  
Shinichi Yoshida ◽  
Keiko Nagaizumi ◽  
Morio Arai ◽  
...  
Keyword(s):  
Missense Mutation ◽  
Novel Mutation ◽  
Point Mutations ◽  
Japanese Patients ◽  
Von Willebrand Disease ◽  
Homozygous Mutation ◽  
Missense Mutations ◽  
Reaction Products ◽  
Type 3 ◽  
Von Willebrand

SummaryGenetic materials from 16 unrelated Japanese patients with von Willebrand disease (vWD) were analyzed for mutations. Exon 28 of the von Willebrand factor (vWF) gene, where point mutations have been found most frequent, was screened by various restriction-enzyme analyses. Six patients were observed to have abnormal restriction patterns. By sequence analyses of the polymerase chain-reaction products, we identified a homozygous R1308C missense mutation in a patient with type 2B vWD; R1597W, R1597Q, G1609R and G1672R missense mutations in five patients with type 2A; and a G1659ter nonsense mutation in a patient with type 3 vWD. The G1672R was a novel missense mutation of the carboxyl-terminal end of the A2 domain. In addition, we detected an A/C polymorphism at nucleotide 4915 with HaeIII. There was no particular linkage disequilibrium of the A/C polymorphism, either with the G/A polymorphism at nucleotide 4391 detected with Hphl or with the C/T at 4891 detected with BstEll.

Three Distinct Candidate Point Mutations of the von Willebrand Factor Gene in Four Patients with Type IIA von Willebrand Disease

1992 ◽  
Vol 67 (06) ◽  
pp. 612-617 ◽  
Author(s):  
Isamu Sugiura ◽  
Tadashi Matsushita ◽  
Mitsune Tanimoto ◽  
Isao Takahashi ◽  
Tomio Yamazaki ◽  
...  
Keyword(s):  
Amino Acid ◽  
Von Willebrand Factor ◽  
Cleavage Site ◽  
Point Mutations ◽  
Pcr Amplification ◽  
Von Willebrand Disease ◽  
Missense Mutations ◽  
Allele Specific ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryType IIA von Willebrand disease (vWD) is the most common type II vWD and is characterized by the selective loss of large and intermediate sized multimers. One explanation for this disorder has been postulated to be a qualitative defect in von Willebrand factor (vWF) which results in increased susceptibility to proteolysis at the bond between residues Tyr842 and Met843. Four missense mutations that may cause type IIA vWD have recently been identified near the cleavage site. We analyzed the molecular basis for type IIA vWD in six patients. A 512 bp DNA sequence spanning the proteolytic cleavage site was targeted for PCR amplification and sequencing. We exploited a difference in restriction sites between the vWF gene and the pseudogene and have designed allele-specific oligomer used with PCR to distinguish these two genes. Three candidate missense mutations; Ser743 (TCG) → Leu (TTG), Leu799 (CTG) → Pro (CCG), and Arg834 (CGG) → Trp (TGG) were identified in 4 out of 6 patients. The amino acid substitution at Arg834 has been reported previously, but the other substitutions at Ser743 and Leu799 are novel candidate mutations locating 99 and 43 amino acids to the N-terminal side of the cleavage site, respectively. Our results indicate that amino acid substitutions located relatively distant from the cleavage site may also be involved in type IIA vWD.

Mutation distribution in the von Willebrand factor gene related to the different von Willebrand disease (VWD) types in a cohort of VWD patients

2012 ◽  
Vol 108 (10) ◽  
pp. 662-671 ◽  
Author(s):  
Hamideh Yadegari ◽  
Julia Driesen ◽  
Anna Pavlova ◽  
Arijit Biswas ◽  
Hans-Jörg Hertfelder ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Splice Site ◽  
Von Willebrand Disease ◽  
Missense Mutations ◽  
Large Deletions ◽  
Type 3 ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryVon Willebrand disease (VWD) is the most common inherited bleeding disorder caused by quantitative or qualitative defects of the von Willebrand factor (VWF). VWD is classified into three types – type 1 (partial quantitative deficiencies), type 2 (qualitative defects) and type 3 (complete deficiency of VWF). In this study we explored genotype and phenotype characteristics of patients with VWD with the aim of dissecting the distribution of mutations in different types of VWD. One hundred fourteen patients belonging to 78 families diagnosed to have VWD were studied. Mutation analysis was performed by direct sequencing of the VWF. Large deletions were investigated by multiplex ligation-dependent probe amplification (MLPA) analysis. The impact of novel candidate missense mutations and potential splice site mutations was predicted by in silico assessments. We identified mutations in 66 index patients (IPs) (84.6%). Mutation detection rate was 68%, 94% and 94% for VWD type 1, 2 and 3, respectively. In total, 68 different putative mutations were detected comprising 37 missense mutations (54.4%), 10 small deletions (14.7%), two small insertions (2.9%), seven nonsense mutations (10.3%), five splice-site mutations (7.4%), six large deletions (8.8%) and one silent mutation (1.5%). Twenty-six of these mutations were novel. Furthermore, in type 1 and type 2 VWD, the majority of identified mutations (74% vs. 88.1%) were missense substitutions while mutations in type 3 VWD mostly caused null alleles (82%). Genotyping in VWD is a helpful tool to further elucidate the pathogenesis of VWD and to establish the relationship between genotype and phenotype.

Molecular Characterization of a Multiethnic Group of 21 Patients with Type 3 von Willebrand Disease

2000 ◽  
Vol 84 (10) ◽  
pp. 536-540 ◽  
Author(s):  
Giovanna Cozzi ◽  
Maria Canciani ◽  
Flora Peyvandi ◽  
Alok Srivastava ◽  
Augusto Federici ◽  
...  
Keyword(s):  
Ethnic Origin ◽  
Von Willebrand Disease ◽  
Null Alleles ◽  
Missense Mutations ◽  
Nonsense Mutations ◽  
Molecular Defects ◽  
Large Gene ◽  
Type 3 ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryType 3 von Willebrand disease is a rare autosomal disorder characterized by unmeasurable levels of von Willebrand factor and severe hemorrhagic symptoms. We studied a multiethnic group of 37 patients, from Italy (n = 14), Iran (n = 10) and India (n = 13) to identify the molecular defects and to evaluate genetic heterogeneity among these populations. Twenty-one patients (6 Italians, 9 Iranians and 6 Indians) were fully characterized at the molecular level. Twenty-four different gene alterations were identified, 20 of which have not been described previously. The majority of the mutations caused null alleles, 11 being nonsense mutations (Q218*, W222*, R365*, R373*, E644*, Q706*, S1338*, Q1346*, Y1542*, R1659*, E2129*), 4 small deletions (437delG, 2680delC, 6431delT, del 8491-8499), 3 possible splice site mutations [IVS9(-1)g→a, IVS29(+10)c→t, IVS40(-1)g → c], 3 candidate missense mutations (C275S, C2174G, C2804Y), 2 small insertions (7375insC, 7921insC) and 1 large gene deletion. The latter mutation was associated with the development of alloantibodies to VWF, but this complication was also found in a patient homozygous for a nonsense mutation (Q1346*). Due to the ethnic origin of the patients most of them were the offspring of consanguineous marriages and so were homozygous for the mutations found (18/21). Our results indicate that molecular defects responsible for type 3 VWD are scattered throughout the entire VWF gene (from exon 3 to 52), and that there is no prevalent and common gene defect in the three populations studied by us.

Characterization of the Genetic Defects in Recessive Type 1 and Type 3 von Willebrand Disease Patients of Italian Origin

1998 ◽  
Vol 79 (04) ◽  
pp. 709-717 ◽  
Author(s):  
Giancarlo Castaman ◽  
Hans Vos ◽  
Rogier Bertina ◽  
Francesco Rodeghiero ◽  
Jeroen Eikenboom
Keyword(s):  
Stop Codon ◽  
Von Willebrand Disease ◽  
Genetic Defects ◽  
Missense Mutations ◽  
Molecular Defects ◽  
Recessive Type ◽  
Italian Origin ◽  
Type 3 ◽  
Von Willebrand

SummaryThe genetic defects causing recessive type 1 and type 3 von Wille-brand disease (VWD) in eight families from the northern part of Italy have been investigated. Mutations were identified in 14 of the 16 disease-associated von Willebrand factor (VWF) genes. Only one mutation, a stop codon in exon 45, was previously reported. Several new mutations were identified: one cytosine insertion in exon 42, one guanine deletion in exon 28, one probably complete VWF gene deletion, one substitution in the 3’ splice site of intron 13, one possible gene conversion, and three candidate missense mutations. One missense mutation, the substitution of a cysteine in exon 42, was identified in all type 3 VWD patients that were previously characterized as a subgroup with significant increase of factor VIII procoagulant activity after desmopressin infusion. This paper demonstrates again that the molecular defects of quantitative VWD are diverse and located throughout the entire VWF gene.

Phenotypic and molecular characterisation of type 3 von Willebrand disease in a cohort of Indian patients

2013 ◽  
Vol 109 (04) ◽  
pp. 652-660 ◽  
Author(s):  
Ulrich Budde ◽  
Rifat Jan ◽  
Florian Oyen ◽  
Meganathan Kannan ◽  
Reinhard Schneppenheim ◽  
...  
Keyword(s):  
Direct Sequencing ◽  
Mutation Screening ◽  
Strand Break ◽  
Von Willebrand Disease ◽  
Missense Mutations ◽  
Large Deletions ◽  
High Propensity ◽  
Type 3 ◽  
Von Willebrand ◽  
Willebrand Factor

SummarySevere type 3 VWD (VWD3) is characterised by complete absence or presence of trace amounts of non-functional von Willebrand factor (VWF). The study was designed to evaluate the VWF mutations in VWD3 patients and characterise the breakpoints of two identified homozygous novel large deletions. Patients were diagnosed by conventional tests and VWF multimer analysis. Mutation screening was performed in 19 VWD3 patients by direct sequencing of VWF including flanking intronic sequence and multiplex ligation-dependent probe amplification (MLPA) analysis. Breakpoint characterisation of two identified novel large deletions was done using walking primers and long spanning PCR. A total of 21 different mutations including 15 (71.4%) novel ones were identified in 17 (89.5%) patients. Of these mutations, five (23.8%) were nonsense (p.R1659*, p.R1779*, p.R1853*, p.Q2470*, p.Q2520*), one was a putative splice site (p.M814I) and seven (33.3%) were deletions (p.L254fs*48, p.C849fs*60, p.L1871fs*6, p.E2720fs*24) including three novel large deletions of exon 14–15, 80,830bp (−41510_657+7928A*del) and 2,231bp [1534–2072T_c.1692G*del(p.512fs*terminus)] respectively. A patient carried gene conversion comprising of pseudogene harbouring mutations. The missense mutations (p.G19R, p.K355R, p.D437Y, p.C633R, p.M771V, p.G2044D, p.C2491R) appear to play a major role and were identified in seven (36.8%) patients. In conclusion, a high frequency of novel mutations suggests the high propensity of VWF for new mutations. Missense and deletion mutations found to be a common cause of VWD3 in cohort of Indian VWD3 patients. Breakpoints characterisation of two large deletions reveals the double strand break and non-homologous recombination as deletions mechanism.

Novel Mutations in Types 2 & 3 von Willebrand Disease and Correlation with von Willebrand Factor Multimer Patterns.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2136-2136 ◽  
Author(s):  
Ruetima Titapiwatanakun ◽  
Jennifer C. Guenther ◽  
Yan W. Asmann ◽  
Todd M. Daniels ◽  
John A. Heit ◽  
...  
Keyword(s):  
Genetic Testing ◽  
Banding Pattern ◽  
Infrared Imaging ◽  
Imaging System ◽  
Novel Mutation ◽  
Von Willebrand Disease ◽  
Compound Heterozygous ◽  
Novel Mutations ◽  
Type 3 ◽  
Von Willebrand

Abstract Background & Objectives: Diagnosis and classification of VWD are currently based on integration of personal and family bleeding histories and results of protein-based diagnostic tests (VWF:Ag, VWF:RCo, FVIII:C, VWF:CB and VWF multimers) which have performance limitations. Genetic testing is emerging as a complementary diagnostic tool. We have identified mutations and correlated VWF multimer patterns in our patients (pt) with types 2 & 3 VWD, and report novel candidate mutations. Patients & Methods: Unrelated pt with type 3 (n=6) and subtypes 2 (n=22) VWD, from Mayo Comprehensive Hemophilia Center, consented to this IRB approved study. PCR amplification, from genomic DNA, of VWF gene (splice junctions, coding and promoter regions), avoiding pseudogene amplification, followed by ABI® sequencing and analysis (Mutation Surveyor: SoftGenetic®) were performed. Mutations were compared to VWD (ISTH), NCBI NR nucleotide and DV SNP databases. Comparison with available VWF sequences from other vertebrate species was performed. Selected regions not known to contain mutations remain to be sequenced. VWF multimer analysis was performed using a novel in-gel immunostaining and infrared imaging system. Results: Type 2A VWD (n=10), a novel mutation in exon 28 (E28) (V1524G: 2 pt) with characteristic VWF multimer abnormalities (decreased HMWM, increased satellite banding, suggesting enhanced VWF proteolysis). Type 2B VWD (n=6), 2 pt were compound heterozygous for 2 previously reported mutations, the first in E28 (R1306W)/E48 (T2647M) and the second in E28 (V1316M)/E20 (R854Q). Type 2M VWD (n=4): an 84 yr female with personal (spontaneous and post-surgical) and family bleeding histories, VWF:RCo 72%, VWF:Ag 118% (RCo:Ag ratio 0.61), normal FVIII and platelets, and aberrant VWF multimer banding pattern without substantive reduction of HMWM, had a novel mutation in E52 (S2775C; conserved in mouse, rat, chicken and dog); a 14 yr female with a history of bleeding, VWF:RCo 34%, VWF:Ag 68% (RCo:Ag ratio 0.5) and normal FVIII and platelets, was compound heterozygous for a known mutation (R1399C subtype not classified in VWD database) and two novel mutations: E30 (P1725S) (conserved in mouse, rat, dog, cow and chimp) & E49 (T2666M) (conserved in mouse, dog, rat not in chicken). VWF multimers demonstrated abnormal banding pattern without substantive reduction of HMWM; 2 unrelated pt and families with ultra-large and smeary multimers, previously classified as Vicenza variant, had R1374C mutations and were reclassified as Type 2M VWD. Type 2N VWD (n=1) previously reported mutation E20 (R854Q) was confirmed. Type 3 VWD, 2 pt had novel mutations, the first in E22 (E950X) and the second was a compound heterozygote for E43 (R2478Q)/E40 (E2322V) mutations. In general, patients with similar mutations had similar multimer patterns. Conclusions: We report novel candidate mutations in types 2 & 3 VWD and demonstrate novel VWF multimer patterns with our in-gel staining system. Multimer patterns correlated well with the underlying genotype. The novel mutations are likely causative, given their occurrence at highly conserved residues, but this needs to be confirmed with expression studies. Integration of genetic testing into the diagnostic workup of VWD will potentially lead to more accurate diagnosis and subtyping of VWD, and may further refine protein based testing and provide additional biological insight into VWD.

The Use of High Resolution Melting (HRM) Analysis for Molecular Gene Defects of Type 3 Von Willebrand Disease: Studies of An Italian Cohort of 10 Patients.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3498-3498
Author(s):  
Maria Solimando ◽  
Augusto B. Federici ◽  
Luciano Baronciani ◽  
Alberto Mussetti ◽  
Margherita Punzo ◽  
...  
Keyword(s):  
Direct Sequencing ◽  
Large Deletion ◽  
Von Willebrand Disease ◽  
Missense Mutations ◽  
Duplex Pcr ◽  
Hrm Analysis ◽  
Large Deletions ◽  
Large Gene ◽  
Type 3 ◽  
Von Willebrand

Abstract Abstract 3498 Poster Board III-435 Introduction Type 3 von Willebrand disease (VWD3) is a severe autosomal recessive inherited bleeding disorder caused by a virtually complete absence of von Willebrand Factor (VWF). Classically, patients are homozygous or compound heterozygous for null alleles due to nonsense mutations, small insertions/deletions, splice site defects or, more rarely, large gene deletions spread throughout the VWF gene. Nevertheless, several missense mutations have also been reported. Aims of the study, patients and methods The aim of this study was to investigate the molecular basis of VWD3 in 10 Italian patients using DNA direct sequencing, High Resolution Melting (HRM) analysis and duplex PCR. HRM is a simple, low-cost, and rapid PCR-based method for detecting sequence variation by measuring changes in the melting temperature of double stranded DNA. Duplex PCR was used to screen for the presence of some known large deletions causing VWD3: the 61-kb deletion encompassing exons 6-16 (Xie et al. Blood Cells Mol Dis. 2006; 36: 385), the 253-kb deletion involving the whole VWF gene (Schneppenheim et al. J Thromb Haemost. 2007; 5: 722), the exons 1-3 deletion (Mohl et al. J Thromb Haemost. 2008; 6: 1729), and the exons 4-5 deletion (Sutherland et al. Blood. 2009; 114: 1091). Results and discussion Twenty-four exons were analyzed by direct sequencing, 21 exons by HRM and, so far, 6 exons using both methods. The following mutations were identified in 8 of the 10 patients investigated: 2157delA/7729+7C>T; C2184S*/undetermined; Q1526X*/C2325S*; del ex1-3/3940delG*; 8155+1G>T*/8155+1G>T*; E1549X*/undetermined; 658-2A>G*/658-2A>G*; del ex 1-3/undetermined. Direct sequencing revealed 7 mutations, HRM analysis could detect 2 defects (2157delA, C2325S) and duplex PCR identified one large deletion. Seven of these 11 mutations were novel (indicated with *). Two patients were found to carry mutations in the homozygous state. To confirm these findings, their parents will have to be investigated in order to exclude the presence of a large gene deletion in one of the alleles. Interestingly, the large deletion involving exons 1-3, which was previously reported in the Hungarian population, was also found in 2 unrelated patients. Two missense mutations were identified, both involving a cysteine residue, further suggesting the importance of these residues in the correct folding/processing/secretion of the neo-synthesized VWF. In those patients who still remain uncharacterized further analysis should be performed to search for intronic mutations or heterozygous large deletions responsible for aberrant splicing/post-transcriptional events. Conclusion Based on these preliminary data, HRM analysis, to our knowledge used for the first time in the molecular diagnosis of VWD3, in our hands seems to be an accurate and rapid method for mutational screening of VWF gene. However, so far, the presence of many polymorphic sites in the VWF coding region has strongly limited the use of this technique to 21 exons of the gene. Disclosures: Baronciani: Bayer Awards: Research Funding.

scholarly journals Re-establishment of VWF-dependent Weibel-Palade bodies in VWD endothelial cells

Blood ◽  
2005 ◽  
Vol 105 (1) ◽  
pp. 145-152 ◽  
Author(s):  
Sandra L. Haberichter ◽  
Elizabeth P. Merricks ◽  
Scot A. Fahs ◽  
Pamela A. Christopherson ◽  
Timothy C. Nichols ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Canine Model ◽  
Von Willebrand Disease ◽  
Homozygous Mutation ◽  
Wild Type ◽  
Aortic Endothelial Cells ◽  
Chapel Hill ◽  
Type 3 ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Type 3 von Willebrand disease (VWD) is a severe hemorrhagic defect in humans. We now identify the homozygous mutation in the Chapel Hill strain of canine type 3 VWD that results in premature termination of von Willebrand factor (VWF) protein synthesis. We cultured endothelium from VWD and normal dogs to study intracellular VWF trafficking and Weibel-Palade body formation. Weibel-Palade bodies could not be identified in the canine VWD aortic endothelial cells (VWD-AECs) by P-selectin, VWFpp, or VWF immunostaining and confocal microscopy. We demonstrate the reestablishment of Weibel-Palade bodies that recruit endogenous P-selectin by expressing wild-type VWF in VWD-AECs. Expression of mutant VWF proteins confirmed that VWF multimerization is not necessary for Weibel-Palade body creation. Although the VWF propeptide is required for the formation of Weibel-Palade bodies, it cannot independently induce the formation of the granule. These VWF-null endothelial cells provide a unique opportunity to examine the biogenesis of Weibel-Palade bodies in endothelium from a canine model of type 3 VWD.

scholarly journals Identification and characterisation of mutations associated with von Willebrand disease in a Turkish patient cohort

2013 ◽  
Vol 110 (08) ◽  
pp. 264-274 ◽  
Author(s):  
Adel Abuzenadah ◽  
Ashley Cartwright ◽  
Nawal Al-Shammari ◽  
Rachael Coyle ◽  
Michaela Eckert ◽  
...  
Keyword(s):  
Von Willebrand Disease ◽  
Compound Heterozygous ◽  
Missense Mutations ◽  
End Joining ◽  
Turkish Patient ◽  
Non Homologous End Joining ◽  
Type 3 ◽  
Von Willebrand ◽  
High Degree

SummarySeveral cohort studies have investigated the molecular basis of von Willebrand disease (VWD); however, these have mostly focused on European and North American populations. This study aimed to investigate mutation spectrum in 26 index cases (IC) from Turkey diagnosed with all three VWD types, the majority (73%) with parents who were knowingly related. IC were screened for mutations using multiplex ligation-dependent probe amplification and analysis of all von Willebrand factor gene (VWF) exons and exon/intron boundaries. Selected missense mutations were expressed in vitro. Candidate VWF mutations were identified in 25 of 26 IC and included propeptide missense mutations in four IC (two resulting in type 1 and two in recessive 2A), all influencing VWF expression in vitro. Four missense mutations, a nonsense mutation and a small in-frame insertion resulting in type 2A were also identified. Of 15 type 3 VWD IC, 13 were homozygous and two compound heterozygous for 14 candidate mutations predicted to result in lack of expression and two propeptide missense changes. Identification of intronic breakpoints of an exon 17–18 deletion suggested that the mutation resulted from non-homologous end joining. This study provides further insight into the pathogenesis of VWD in a population with a high degree of consanguineous partnerships.

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