scholarly journals Genotypes of European and Iranian patients with type 3 von Willebrand disease enrolled in 3WINTERS-IPS

2021 ◽  
Vol 5 (15) ◽  
pp. 2987-3001
Author(s):  
Luciano Baronciani ◽  
Ian Peake ◽  
Reinhard Schneppenheim ◽  
Anne Goodeve ◽  
Minoo Ahmadinejad ◽  
...  
Keyword(s):  
Missense Variant ◽  
European Population ◽  
Von Willebrand Disease ◽  
Null Alleles ◽  
Severe Bleeding ◽  
Compound Heterozygous ◽  
Large Deletions ◽  
Type 3 ◽  
Von Willebrand ◽  
Iranian Patients

Abstract Type 3 von Willebrand disease (VWD3) is a rare and severe bleeding disorder characterized by often undetectable von Willebrand factor (VWF) plasma levels, a recessive inheritance pattern, and heterogeneous genotype. The objective of this study was to identify the VWF defects in 265 European and Iranian patients with VWD3 enrolled in 3WINTERS-IPS (Type 3 Von Willebrand International Registries Inhibitor Prospective Study). All analyses were performed in centralized laboratories. The VWF genotype was studied in 231 patients with available DNA (121 [115 families] from Europe [EU], and 110 [91 families] from Iran [IR]). Among 206 unrelated patients, 134 were homozygous (EU/IR = 57/77) and 50 were compound heterozygous (EU/IR = 43/7) for VWF variants. In 22 patients, no or only one variant was found. A total of 154 different VWF variants (EU/IR = 101/58 [5 shared]) were identified among the 379 affected alleles (EU/IR = 210/169), of which 48 (EU/IR = 18/30) were novel. The variants p.Arg1659*, p.Arg1853*, p.Arg2535*, p.Cys275Ser, and delEx1_Ex5 were found in both European and Iranian VWD3 patients. Sixty variants were identified only in a single allele (EU/IR = 50/10), whereas 18 were recurrent (≥3 patients) within 144 affected alleles. Nine large deletions and one large insertion were found. Although most variants predicted null alleles, 21% of patients carried at least 1 missense variant. VWD3 genotype was more heterogeneous in the European population than in the Iranian population, with nearly twice as many different variants. A higher number of novel variants were found in the Iranian VWD3 patients.

scholarly journals Profile of Mutations Identified in the 3WINTERS-IPS Project on European & Iranian Patients with Previously Diagnosed Type 3 Von Willebrand Disease.

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1184-1184
Author(s):  
Luciano Baronciani ◽  
Flora Peyvandi ◽  
Anne Goodeve ◽  
Reinhard Schneppenheim ◽  
Zahra Badiee ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Research Funding ◽  
Advisory Board ◽  
Von Willebrand Disease ◽  
Compound Heterozygous ◽  
Advisory Committees ◽  
Type 3 ◽  
Von Willebrand ◽  
Two Populations ◽  
Iranian Patients

Abstract Background: The type 3 Von Willebrand International RegistrieSInhibitor Prospective Study (3WINTERS-IPS) is a no-profit, investigator initiated, multicenter, European-Iranian observational, retrospective and prospective study on patients with diagnosis of type 3 VWD. Patients with type 3 von Willebrand Disease (VWD3) have markedly reduced levels of von Willebrand factor (VWF) and very severe bleeding phenotype. Due to the recessive inheritance pattern, VWD3 is by definition a rare bleeding disorder (1:Million) but its prevalence may increase in countries like Iran with consanguineous marriages. Aim: To identify the VWF genetic defects in a cohort of European and Iranian patients with previously diagnosed VWD3 enrolled into the 3WINTERS-IPS project. Methods: Patients classified locally as VWD3 were enrolled in the study following informed consent. 141 patients were from 9 different European countries and 119 patients were from the Islamic Republic of Iran. Plasma/buffy-coat samples were sent to expert labs to confirm patient's laboratory phenotype and to perform molecular analysis. PCR and Sanger sequencing/ next generation sequencing and multiplex-ligation dependent probe amplification were used in Hamburg, Sheffield and Milan to confirm previously identified variants or to seek previously unidentified variants. Results: DNA samples from 122 patients from Europe and 114 patients from Iran were analyzed at the molecular level. Of the 236 VWD3 patients under evaluation 24 are still in progress. Of the 212 fully evaluated patients 139 were homozygous (EU/IR=46/93) and 43 were compound heterozygous (EU/IR=36/7). In the remaining 30 patients no variants were identified in 19 samples (EU/IR=6/13) and only one variant was found in the remaining 11 cases (EU/IR=10/1). 135 (EU/IR=82/53) different gene defects were identified among the 375 (EU/IR=174/201) alleles found in this study. Of these 135 variants identified 51(EU/IR=22/29) were not reported on the www.ensembl.org database. The distribution of the different type of variants identified in the two populations is shown in the Figure. The two charts are showing quite similar percentages of the variants identified, with a main exception for the Small deletions and Small insertions. Only five variants are shared among the two populations. Three of these are the "hotspot" variants at the Arg codon, p.Arg1659* (EU/IR=9/8), p.Arg1853* (EU/IR=2/3) and p.Arg2535* (EU/IR=1/2). However, a missense variant , p.Cys275Ser (EU/IR=1/2) and a large deletion, delEx1_Ex5 (EU/IR=1/2) were also found in both populations. Fifteen variants were recurrent and were found in 154 alleles, whereas 49 variants were found only once in the heterozygous state (EU/IR=40/9) and 50 variants were found only twice, mainly in the homozygous state (EU/IR=25/25). Six large deletions were identified (delEx1_Ex3, delEx1_Ex5, delEx14_Ex15, delEx17, delEx35_Ex52 and delEx1_Ex52) and a duplication (dupEx1_Ex28), nevertheless 52 alleles with missense variants were identified (EU/IR=20/32). Discussion: As expected, the majority of the Iranian patients were found to be homozygous (Homozygous/Compound Heterozygous=93/7) reflecting a high rate of consanguinity, nevertheless half of the European patients were found to be homozygous (Homozygous/Compound Heterozygous=46/36). The European populations demonstrated a higher heterogeneity of variants with 82 different variants among the 175 mutated alleles vs 53 different variants among the 201 mutated alleles identified in the Iranian population. Nevertheless, a higher number of previously unreported variants was found in the Iranian population (29) vs the European one (22), probably due to bias of previous investigations performed in European patients. Figure Figure. Disclosures Peyvandi: Ablynx: Other: Member of Advisory Board, Speakers Bureau; Shire: Speakers Bureau; Roche: Speakers Bureau; Grifols: Speakers Bureau; Grifols: Speakers Bureau; Novo Nordisk: Speakers Bureau; Sobi: Speakers Bureau; Sobi: Speakers Bureau; Novo Nordisk: Speakers Bureau; Kedrion: Consultancy; Novo Nordisk: Speakers Bureau; Octapharma US: Honoraria; Novo Nordisk: Speakers Bureau; Sobi: Speakers Bureau; Ablynx: Other: Member of Advisory Board, Speakers Bureau; Kedrion: Consultancy; Novo Nordisk: Speakers Bureau; Kedrion: Consultancy; Ablynx: Other: Member of Advisory Board, Speakers Bureau; Octapharma US: Honoraria; Shire: Speakers Bureau; Roche: Speakers Bureau; Kedrion: Consultancy; Kedrion: Consultancy; Ablynx: Other: Member of Advisory Board, Speakers Bureau; Octapharma US: Honoraria; Octapharma US: Honoraria; Sobi: Speakers Bureau; Roche: Speakers Bureau; Octapharma US: Honoraria; Shire: Speakers Bureau; Sobi: Speakers Bureau; Roche: Speakers Bureau; Roche: Speakers Bureau; Shire: Speakers Bureau; Ablynx: Other: Member of Advisory Board, Speakers Bureau; Grifols: Speakers Bureau; Grifols: Speakers Bureau; Grifols: Speakers Bureau; Shire: Speakers Bureau. Schneppenheim:CSL Behring: Consultancy; SHIRE: Consultancy. Berntorp:Octapharma: Consultancy; CSL Behring: Consultancy; Shire: Consultancy, Other: honoraria for lecturing . Eikenboom:CSL: Research Funding. Mannucci:Bayer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Kedrion: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Grifols: Speakers Bureau; Alexion: Speakers Bureau; Baxalta/Shire: Speakers Bureau; Novo Nordisk: Speakers Bureau. Mazzucconi:Baxalta-Shire: Consultancy, Speakers Bureau; Bayer: Consultancy, Speakers Bureau; Novartis,: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Novo Nordisk: Consultancy, Speakers Bureau; CSL Behring: Consultancy, Speakers Bureau. Oldenburg:Swedish Orphan Biovitrum: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Shire: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees; Grifols: Honoraria, Membership on an entity's Board of Directors or advisory committees; Biogen Idec: Honoraria, Membership on an entity's Board of Directors or advisory committees; Chugai: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Biotest: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; CSL Behring: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novo Nordisk: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Octapharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Molecular characterization of Iranian patients with type 3 von Willebrand disease

Haemophilia ◽  
2009 ◽  
Vol 15 (5) ◽  
pp. 1058-1064 ◽  
Author(s):  
S. SHAHBAZI ◽  
R. MAHDIAN ◽  
F. A. ALA ◽  
J.-M. LAVERGNE ◽  
C. V. DENIS ◽  
...  
Keyword(s):  
Molecular Characterization ◽  
Von Willebrand Disease ◽  
Type 3 ◽  
Von Willebrand ◽  
Iranian Patients

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.

scholarly journals Social participation is reduced in type 3 Von Willebrand disease patients and in patients with a severe bleeding phenotype

Haemophilia ◽  
2021 ◽  
Author(s):  
Eva K. Kempers ◽  
Calvin B. Kwawegen ◽  
Joke Meris ◽  
Saskia E. M. Schols ◽  
Karin P. M. Galen ◽  
...  
Keyword(s):  
Social Participation ◽  
Von Willebrand Disease ◽  
Severe Bleeding ◽  
Type 3 ◽  
Von Willebrand

scholarly journals Clustering of Bleeding Symptoms in Patients Previously Diagnosed As Type 3 Von Willebrand Disease: Results from a Large Cohort of Type 3 Von Willebrand Disease (the 3Winters-Ips Project)

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2465-2465
Author(s):  
Alberto Tosetto ◽  
Zahra Badiee ◽  
Mohammad-Reza Baghaipour ◽  
Luciano Baronciani ◽  
Javier Battle ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Research Funding ◽  
Advisory Board ◽  
Von Willebrand Disease ◽  
Severe Bleeding ◽  
Advisory Committees ◽  
Bleeding Score ◽  
Type 3 ◽  
Von Willebrand

Abstract Patients with type 3 von Willebrand Disease (VWD) usually have markedly reduced FVIII/VWF levels and very severe bleeding manifestations but, because of their rarity, their bleeding phenotype is poorly described. We aimed at evaluating the distribution of bleeding symptoms in patients with type 3 VWD, comparing them with previously available data from a cohort of type 1 patients, and describing site-specific clustering of bleeding symptoms in these patients. We analyzed clinical data from the type 3 Von Willebrand International RegistrieS Inhibitor Prospective Study (3WINTERS-IPS),a no-profit, investigators initiated, multicenter, European-Iranian observational, retrospective and prospective study on patients with diagnosis of type 3 VWD. Aims of the 3WINTERS-IPS is 3-fold: a) to identify the main phenotypic and molecular characteristics of a large cohort of VWD patients; b) to evaluate the risk factors responsible for the severe bleeding phenotype; c) to assess the efficacy and safety of the treatment with VWF concentrates with or without FVIII including the risk of anti-VWF antibodies. Retrospective information on bleeding symptoms at presentation was collected using the MCMDM-1 VWD bleeding questionnaire, and bleeding severity summarized as bleeding score. Individual bleeding symptoms were considered as relevant when having a score >1 (hence requiring medical attention). Data was compared with that retrieved from the MCMDM-1 VWD study database on patients affected by type 1 VWD (index cases and affected family members). The study enrolled a total of 260 patients, of which we analysed 243 patients with available bleeding score at recruitment. The median age at study inclusion was 29 years (interquantile range, 26.5 years); 140 were females (53.8%). There were 108 patients of Iranian descent, while the remaining of patients were from Europe. The median number of bleeding symptoms was 5, and the median bleeding score was 15 (interquantile range, 13). Only 7/243 patients (2.8%) had a single bleeding symptom. Epistaxis was the most frequent relevant symptom, being present in 195 patients (80.2%), followed by menorrhagia in 99 females (70.7%). Males had a higher frequency of hemarthroses and hematomas than females (53.4% vs 42.1% and 40.8% vs 27.1%, respectively). When comparing the clinical presentation of type 3 vs. type 1 VWD, clearly increased bleeding scores were evident for all age-classes and even in paediatric cases. The association between symptoms having a relative frequency >20% is presented in the circle diagram, showing that some symptoms appeared to cluster with others in a variable degree (e.g., menorrhagia with epistaxis, hemarthrosis or oral cavity bleeding; post-extraction bleeding again with epistaxis, hemarthrosis or oral cavity bleeding; surgical bleeding or gastrointestinal bleeding with epistaxis alone). These findings confirm the severity of type 3 VWD and extend the knowledge of symptoms distribution in the widest available cohort of type 3 VWD patients. Disclosures Tosetto: Stago, Novo-Nordisk, BMS: Speakers Bureau; Werfen: Other: Member of Advisory Board, Speakers Bureau. Berntorp:Octapharma: Consultancy; CSL Behring: Consultancy; Shire: Consultancy, Other: honoraria for lecturing . Eikenboom:CSL: Research Funding. Mazzucconi:Baxalta-Shire: Consultancy, Speakers Bureau; Bayer: Consultancy, Speakers Bureau; Novartis,: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Novo Nordisk: Consultancy, Speakers Bureau; CSL Behring: Consultancy, Speakers Bureau. Oldenburg:Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Biogen Idec: Honoraria, Membership on an entity's Board of Directors or advisory committees; Chugai: Honoraria, Membership on an entity's Board of Directors or advisory committees; Grifols: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees; Shire: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Octapharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novo Nordisk: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; CSL Behring: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Biotest: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Swedish Orphan Biovitrum: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Peyvandi:Kedrion: Consultancy; Ablynx: Other: Member of Advisory Board, Speakers Bureau; Shire: Speakers Bureau; Roche: Speakers Bureau; Shire: Speakers Bureau; Kedrion: Consultancy; Kedrion: Consultancy; Ablynx: Other: Member of Advisory Board, Speakers Bureau; Grifols: Speakers Bureau; Roche: Speakers Bureau; Octapharma US: Honoraria; Octapharma US: Honoraria; Sobi: Speakers Bureau; Ablynx: Other: Member of Advisory Board, Speakers Bureau; Kedrion: Consultancy; Shire: Speakers Bureau; Roche: Speakers Bureau; Roche: Speakers Bureau; Novo Nordisk: Speakers Bureau; Shire: Speakers Bureau; Ablynx: Other: Member of Advisory Board, Speakers Bureau; Grifols: Speakers Bureau; Shire: Speakers Bureau; Ablynx: Other: Member of Advisory Board, Speakers Bureau; Grifols: Speakers Bureau; Novo Nordisk: Speakers Bureau; Octapharma US: Honoraria; Octapharma US: Honoraria; Sobi: Speakers Bureau; Grifols: Speakers Bureau; Grifols: Speakers Bureau; Kedrion: Consultancy; Sobi: Speakers Bureau; Roche: Speakers Bureau; Novo Nordisk: Speakers Bureau; Novo Nordisk: Speakers Bureau; Novo Nordisk: Speakers Bureau; Sobi: Speakers Bureau; Octapharma US: Honoraria; Sobi: Speakers Bureau. Schneppenheim:SHIRE: Consultancy; CSL Behring: Consultancy. Tiede:Alnylam, Bayer, Biogen Idec, Biotest, Bristol-Myers-Squibb, Boehringer Ingelheim, CSL Behring, Leo Pharma, Novo Nordisk, Octapharma, Pfizer, Roche, Shire, and SOBI: Consultancy; Alnylam, Bayer, Biogen Idec, Biotest, Bristol-Myers-Squibb, Boehringer Ingelheim, CSL Behring, Leo Pharma, Novo Nordisk, Octapharma, Pfizer, Roche, Shire, and SOBI: Honoraria; Alnylam, Bayer, Biogen Idec, Biotest, Bristol-Myers-Squibb, Boehringer Ingelheim, CSL Behring, Leo Pharma, Novo Nordisk, Octapharma, Pfizer, Roche, Shire, and SOBI: Research Funding.

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.

Coinheritance of Severe von Willebrand Disease With Glanzmann Thrombasthenia

2010 ◽  
Vol 16 (5) ◽  
pp. 529-532 ◽  
Author(s):  
Firdos Ahmad ◽  
Meganathan Kannan ◽  
Kamal Kishor ◽  
Renu Saxena
Keyword(s):  
Replacement Therapy ◽  
Dna Analysis ◽  
Von Willebrand Disease ◽  
Severe Bleeding ◽  
Glanzmann Thrombasthenia ◽  
Truncating Mutation ◽  
Coagulation Tests ◽  
Hemostatic Disorders ◽  
Type 3 ◽  
Von Willebrand

A 35-years old male patient presented severe bleeding was diagnosed to have type 3 von Willebrand disease (VWD) and carrier for Glanzmann thrombasthenia (GT). Propositus and family members were studied through basic coagulation tests and genomic DNA analysis. Two offspring of the family were diagnosed to have GT through platelet aggregation along with VWD carrier. The patient with VWD was found positive for homozygous truncating mutation R1659X in VWF gene, and all offspring were heterozygous carriers of null allele. Hence, propositus was a carrier of GT with severe type 3 VWD and wife was a carrier of GT. Thus, it is concluded that there is importance of careful studies of patients even from nonconsanguineous families to exclude unusual coinheritance of congenital hemostatic disorders. If single replacement therapy in patient not responding well then probably co-expression of coagulopathies required and multiple replacement therapy should be given according to clinical and laboratory features.

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.

scholarly journals Establishment of Blood Outgrowth Endothelial Cells from a Type 3 Von Willebrand Disease (VWD) Patient with a Neutralizing Anti-VWF Inhibitors

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1508-1508 ◽  
Author(s):  
Naomi Sanda ◽  
Nobuaki Suzuki ◽  
Mayuko Kishimoto ◽  
Natsumi Kameyama ◽  
Yoko Kajiura ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Dna Sequences ◽  
Mononuclear Cells ◽  
Flow Cytometric Analysis ◽  
Von Willebrand Disease ◽  
Compound Heterozygous ◽  
Nonsense Mutations ◽  
Flow Cytometric ◽  
Type 3 ◽  
Von Willebrand

Abstract Background: Type 3 von Willebrand disease (VWD) is characterized by a complete loss type of von Willebrand Factor (VWF) with the rarest disease frequency and hemorrhagic symptom is the most severe among other VWD types. The development of alloantibodies directed against VWF occurs in approximately 10% of patients with type 3 VWD. In this study, we found the VWF gene alterations and established blood outgrowth endothelial cells (BOECs) a from a Japanese type 3 VWD patient with an anti-VWF inhibitors. Case: A 5-year-old woman suffered from epistaxis, purpura and easy bruising and has been diagnosed as VWD. Her plasma level of FVIII:C was 1.8% and VWF:Ag and VWF:RCo was <5% and <10%, respectively. Her plasma lacked VWF multimers, indicating that she had type 3 VWD. To treat her frequent muco-cutaneous bleeding, purified plasma-derived FVIII/VWF concentrates were administrated followed by inhibitor development. After about 20ED of FVIII/VWF concentrates, an anaphylactic symptom comprised of cough, dyspnea, and wheezing developed at the time of dosing. An high titer inhibitory antibody against VWF was confirmed by a Bethesda assay based on a assay of VWF:RCo. Her younger sister also had type 3 VWD. Methods: Patients samples were collected after the written informed consent has bee obtained. We performed MLPA for genetic deletion or insertion. Then we amplified the all exons including the exon/intron boundaries of the VWF gene by PCR using allele-specific primers, and analyzed DNA sequences of the patient. Peripheral blood mononuclear cells were obtained from the patient and BOECs were then established. Briefly, buffy coat mononuclear cells were isolated and seeded onto a 6-well tissue culture plate precoated with type 1 rat tail collagen at 37°C, 5% CO2, in a humidified incubator. Medium was changed daily for 7 days and then every other day until the first passage. The endothelial identity of the BOECs was confirmed by flow cytometric and immunofluorescence analyses using endothelial markers with antibodies to CD31, VE-Cadherin (CD144) and VEGFR-2 (CD309). Results: The large deletion and insertion of VWF gene were not found. Direct sequencing showed the propositus and her sister were compound heterozygous for an E2341X (c.7021G>T) mutation in exon41 and a Y2631X (c.7892-7893insA) mutation in exon48. These were therefore two novel nonsense mutations and normal VWF polypeptides could not be translated. Flow cytometric analysis indicated that established BOECs expressed cell surface CD31 and CD309, whereas CD34 was not detected. By immunofluorescence analysis of fixed BOECs, VWF signal was remarkably reduced from the patient, in spite of normal VE-Cadherin expression. VWF specific ELISA was performed for both cell supernatant and lysates of patient’s BOECs but immunodetectable VWF was not secreted. Discussion: We identified two novel nonsense mutations causing type3 VWD. BOECs established from the patient reproduced the phenotype of the disease, suggesting that BOEC analysis is useful for studying the molecular pathogenesis of von Willebrand disease. Disclosures No relevant conflicts of interest to declare.

Sign in / Sign up

Export Citation Format

Share Document