Von Willebrand Factor Gene Research Articles

A novel platelet-type von Willebrand disease mutation (GP1BA p.Met255Ile) associated with type 2B “Malmö/New York” von Willebrand disease

Interaction between von Willebrand factor (VWF) and platelet GPIbα is required for primary haemostasis. Lack or loss-of-function in the ligand-receptor pair results in bleeding complications. Paradoxically, gain-of-function mutations in VWF or GPIbα also result in bleeding complications as observed in type 2B von Willebrand disease (VWD) and platelet-type- (PT-) VWD, respectively. A similar phenotype is observed with increased ristocetin-induced platelet agglutination and disappearance of the highest molecular weight multimers of VWF. We evaluated a patient with a bleeding disorder and a biological presentation compatible with type 2B VWD. VWF and platelet functional assays, sequencing of the VWF and GP1BA genes, and expression studies in HEK cells were performed. Sequencing of the VWF gene in the propositus revealed a heterozygous p.Pro1266Leu mutation previously found in type 2B VWD Malmö/New York. These variants are characterised by a mild phenotype and a normal VWF multimer composition suggesting the presence of a second mutation in our propositus. Sequencing of the GP1BA gene revealed a heterozygous c.765G>A substitution changing Met at position 255 of GPIbα to Ile. This new mutation is located in the β-switch domain where five other gain-of-function mutations have been reported in PT-VWD. Expression of GPIbα Ile255 in HEK GPIb-IX cells resulted in enhanced VWF binding compared to wild-type, similar to known PT-VWD mutations (p.Val249, p.Ser249 and p.Val255) indicating that it contributes to the propositus defects. This first report associating PT- with type 2B VWD illustrates the importance of combining biological assays with genetic testing to better understand the clinical phenotype.

Intron retention resulting from a silent mutation in the VWF gene that structurally influences the 5′ splice site

AbstractDisease-associated silent mutations are considered to affect the accurate pre–messenger RNA (mRNA) splicing either by influencing regulatory elements, leading to exon skipping, or by creating a new cryptic splice site. This study describes a new molecular pathological mechanism by which a silent mutation inhibits splicing and leads to intron retention. We identified a heterozygous silent mutation, c.7464C>T, in exon 44 of the von Willebrand factor (VWF) gene in a family with type 1 von Willebrand disease. In vivo and ex vivo transcript analysis revealed an aberrantly spliced transcript, with intron 44 retained in the mRNA, implying disruption of the first catalytic step of splicing at the 5′ splice site (5′ss). The abnormal transcript with the retained intronic region coded a truncated protein that lacked the carboxy-terminal end of the VWF protein. Confocal immunofluorescence characterizations of blood outgrowth endothelial cells derived from the patient confirmed the presence of the truncated protein by demonstrating accumulation of VWF in the endoplasmic reticulum. In silico pre-mRNA secondary and tertiary structure analysis revealed that this substitution, despite its distal position from the 5′ss (85 bp downstream), induces cis alterations in pre-mRNA structure that result in the formation of a stable hairpin at the 5′ss. This hairpin sequesters the 5′ss residues involved in U1 small nuclear RNA interactions, thereby inhibiting excision of the pre-mRNA intronic region. This study is the first to show the allosteric-like/far-reaching effect of an exonic variation on pre-mRNA splicing that is mediated by structural changes in the pre-mRNA.

How much do we really know about von Willebrand disease?

In the last nine decades, large advances have been made toward the characterization of the pathogenic basis and clinical management of von Willebrand disease (VWD), the most prevalent inherited bleeding disorder. Pathological variations at the von Willebrand factor (VWF) locus present as a range of both quantitative and qualitative abnormalities that make up the complex clinical spectrum of VWD. This review describes the current understanding of the pathobiological basis of VWD. The molecular basis of type 2 (qualitative abnormalities) and type 3 VWD (total quantitative deficiency) have been well characterized in recent decades. However, knowledge of type 1 VWD (partial quantitative deficiency) remains incomplete because of the allelic and locus heterogeneity of this trait, and is complicated by genetic variability at the VWF gene, interactions between the VWF gene and the environment, and the involvement of external modifying loci. Recent genome wide association studies and linkage analyses have sought to identify additional genes that modify the type 1 VWD phenotype. Understanding the pathogenic basis of VWD will facilitate the development of novel treatment regimens for this disorder, and improve the ability to provide complementary molecular diagnostics for type 1 VWD.

Genome‐wide studies of von Willebrand factor propeptide identify loci contributing to variation in propeptide levels and von Willebrand factor clearance

Essentials Variants at ABO, von Willebrand Factor (VWF) and 2q12 contribute to the variation in plasma in VWF. We performed a genome-wide association study of plasma VWF propeptide in 3,238 individuals. ABO, VWF and 2q12 loci had weak or no association or linkage with plasma VWFpp levels. VWF associated variants at ABO, VWF and 2q12 loci primarily affect VWF clearance rates. Background Previous studies identified common variants at the ABO and VWF loci and unknown variants in a chromosome 2q12 linkage interval that contributed to the variation in plasma von Willebrand factor (VWF) levels. Whereas the association with ABO haplotypes can be explained by differential VWF clearance, little is known about the mechanisms underlying the association with VWF single-nucleotide polymorphisms (SNPs) or with variants in the chromosome 2 linkage interval. VWF propeptide (VWFpp) and mature VWF are encoded by the VWF gene and secreted at the same rate, but have different plasma half-lives. Therefore, comparison of VWFpp and VWF association signals can be used to assess whether the variants are primarily affecting synthesis/secretion or clearance. Methods We measured plasma VWFpp levels and performed genome-wide linkage and association studies in 3238 young and healthy individuals for whom VWF levels had been analyzed previously. Results and conclusions Common variants in an intergenic region on chromosome 7q11 were associated with VWFpp levels. We found that ABO serotype-specific SNPs were associated with VWFpp levels in the same direction as for VWF, but with a much lower effect size. Neither the association at VWF nor the linkage on chromosome 2 previously reported for VWF was observed for VWFpp. Taken together, these results suggest that the major genetic factors affecting plasma VWF levels, i.e. variants at ABO, VWF and a locus on chromosome 2, operate primarily through their effects on VWF clearance.

Characterization of aberrant splicing of von Willebrand factor in von Willebrand disease: an underrecognized mechanism

AbstractApproximately 10% of von Willebrand factor (VWF) gene mutations are thought to alter messenger RNA (mRNA) splicing through disruption of consensus splice sites. This mechanism is likely underrecognized and affected by mutations outside consensus splice sites. During VWF synthesis, splicing abnormalities lead to qualitative defects or quantitative deficiencies in VWF. This study investigated the pathologic mechanism acting in 3 von Willebrand disease (VWD) families with putative splicing mutations using patient-derived blood outgrowth endothelial cells (BOECs) and a heterologous human embryonic kidney (HEK 293(T)) cell model. The exonic mutation c.3538G>A causes 3 in-frame splicing variants (23del, 26del, and 23/26del) which cannot bind platelets, blood coagulation factor VIII, or collagen, causing VWD through dominant-negative intracellular retention of coexpressed wild-type (WT) VWF, and increased trafficking to lysosomes. Individuals heterozygous for the c.5842+1G>C mutation produce exon 33 skipping, exons 33-34 skipping, and WT VWF transcripts. Pathogenic intracellular retention of VWF lacking exons 33-34 causes their VWD. The branch site mutation c.6599-20A>T causes type 1 VWD through mRNA degradation of exon 38 skipping transcripts. Splicing ratios of aberrant transcripts and coexpressed WT were altered in the BOECs with exposure to shear stress. This study provides evidence of mutations outside consensus splice sites disrupting splicing and introduces the concept that VWF splicing is affected by shear stress on endothelial cells.

Functional characterisation of the type 1 von Willebrand disease candidate VWF gene variants: p.M771I, p.L881R and p.P1413L.

Abnormalities in the biosynthetic pathway or increased clearance of plasma von Willebrand factor (VWF) are likely to contribute to decreased plasma VWF levels in inherited type 1 von Willebrand disease (VWD). Recent studies demonstrated that 65% of type 1 VWD patients have candidate VWF mutations, the majority of which are missense variants. The purpose of this study was to explore the effects of three VWF missense mutations (p.M771I, p.L881R and p.P1413L) located in different functional domains of VWF, reported as candidate mutations in type 1 VWD patients in the course of the MCMDM-1VWD study. The focus of these studies was on the intracellular biosynthetic processing and localisation of VWF in a heterologous cell system. Molecular dynamic simulation for p.M771I and p.P1413L was also performed to analyse the conformational effects of the changes. As determined by immunofluorescence antibody staining and confocal microscopy of HEK293 cells, the intracellular localisation of recombinant VWF with the p.M771I variation was impaired. Transient transfection studies and phorbol myristate acetate stimulation in COS-7 cells revealed significant intracellular retention. In addition, major loss of VWF multimers was observed for only the p.M771I mutation. Molecular dynamic simulations on p.M771I mutant VWF revealed distinct structural rearrangements including a large deviation in the E' domain, and significant loss of β-sheet secondary structure. The pathogenic effects of candidate VWF gene mutations were explored in this study. In vitro expression studies in heterologous cell systems revealed impaired secretion of VWF and a dominant negative effect on the processing of the wild-type protein for only the p.M771I mutation and none of the mutations affected the regulated secretion.

Controversies in Inherited Bleeding Disorders.

Recent years are witnessing key developments in treatment approaches for congenital bleeding disorders (CBD), in particular the hemophilias, but also other factor deficiencies.1,2 Moreover, cooperative multinational efforts are providing advances in our knowledge of pathophysiological, clinical, and management aspects of these disorders, including the more rare abnormalities.3–6 However, despite being in an era of novel insights and paradigms of treatment, many issues remain controversial or need further research and/or confirmation in clinical practice. Along these lines, the 16 chapters presented in this latest bumper issue of Seminars in Thrombosis and Hemostasis deal with some of these debated or evolving concepts, presenting and discussing the most recent literature information and the available expert recommendations. The assessment of patients with suspected or known CBD is based on accurate and appropriate data from clinical history and laboratory testing. Therefore, two papers at the beginning of the issue focus on these crucial aspects.7,8 Starting from the clinical validation in type 1 von Willebrand disease (VWD), Tosetto reports on the potential clinical usefulness of bleeding assessment tools (BAT), these being first developed essentially as research tools for the quantification of bleeding symptoms and the study of phenotype/ genotype correlations.7 Indeed, although disease-specific implementations should be widely validated in most cases, these tools can provide amain advantage of standardizing the diagnostic process, allowing a rational approach to the laboratory diagnosis, in particular for mild bleeding disorders that may require a complex laboratory work-up. Moreover bleeding severity assessed by these tools has been shown to correlate with the long-term probability of bleeding, thus representing an interesting predictor of disease severity.7 As regards laboratory issues, in viewof the general lack of agreed approaches for the diagnostics of bleeding disorders, Lippi and colleagues provide pragmatic guidance to improve the identification and diagnosis of CBD due to abnormalities of secondary hemostasis, even in nonspecialized laboratories.8 A strategy based on the collection of personal and family history and the results of first-line tests (i.e., prothrombin time, activated partial thromboplastin time, and fibrinogen) is proposed, followed by secondor third-line tests that will definitely establish the specific nature and the severity of the bleeding defect. Indeed, second-line tests (specific factor assays, on the basis of the detected abnormalities in firstline assays) will provide the basis for a preliminary diagnosis, which will then be supported by third-line tests (namely, immunological assays of clotting factors and molecular biology).8 VWD is the most common CBD; however, its identification and management still provides many challenges.9 Three articles in this issue deal with these problems, reflecting a complex and not completely elucidated pathophysiological background. Castaman and Federici review recent evidence regarding one of the most intriguing VWD subtypes, type 2B.10 This rare autosomally dominant inherited variant is due to mutations clustered in exon 28 of the von Willebrand Factor (VWF) gene encoding for the A1 domain, involved in VWF binding to platelet glycoprotein Ib-α. The mutant VWF, normally synthesized and assembled by endothelial cells, shows heightened affinity binding to its platelet receptor. This results in in vivo platelet clumping that is correlatedwith a variable degree of thrombocytopenia, in particular under specific clinical circumstances. However, recent studies show that a true platelet defect is also present in VWD 2B, since morphological and functional defects are detected in these patients, possibly contributing to the clinical and laboratory heterogeneity.10 In the frame of the phenotypical variability in type 2 VWD, the often misdiagnosed subtypes 2A and 2M are being increasingly studied, showing both similarities and differences in various aspects of their presentation to clinicians, researchers, and laboratories. Favaloro and colleagues

Von Willebrand Factor Gene Variants Associate with Herpes simplex Encephalitis.

Herpes simplex encephalitis (HSE) is a rare complication of Herpes simplex virus type-1 infection. It results in severe parenchymal damage in the brain. Although viral latency in neurons is very common in the population, it remains unclear why certain individuals develop HSE. Here we explore potential host genetic variants predisposing to HSE. In order to investigate this we used a rat HSE model comparing the HSE susceptible SHR (Spontaneously Hypertensive Rats) with the asymptomatic infection of BN (Brown Norway). Notably, both strains have HSV-1 spread to the CNS at four days after infection. A genome wide linkage analysis of 29 infected HXB/BXH RILs (recombinant inbred lines—generated from the prior two strains), displayed variable susceptibility to HSE enabling the definition of a significant QTL (quantitative trait locus) named Hse6 towards the end of chromosome 4 (160.89–174Mb) containing the Vwf (von Willebrand factor) gene. This was the only gene in the QTL with both cis-regulation in the brain and included several non-synonymous SNPs (single nucleotide polymorphism). Intriguingly, in human chromosome 12 several SNPs within the intronic region between exon 43 and 44 of the VWF gene were associated with human HSE pathogenesis. In particular, rs917859 is nominally associated with an odds ratio of 1.5 (95% CI 1.11–2.02; p-value = 0.008) after genotyping in 115 HSE cases and 428 controls. Although there are possibly several genetic and environmental factors involved in development of HSE, our study identifies variants of the VWF gene as candidates for susceptibility in experimental and human HSE.

Clinical and laboratory variability in a cohort of patients diagnosed with type 1 VWD in the United States

Abstractvon Willebrand disease (VWD) is the most common inherited bleeding disorder, and type 1 VWD is the most common VWD variant. Despite its frequency, diagnosis of type 1 VWD remains the subject of debate. In order to study the spectrum of type 1 VWD in the United States, the Zimmerman Program enrolled 482 subjects with a previous diagnosis of type 1 VWD without stringent laboratory diagnostic criteria. von Willebrand factor (VWF) laboratory testing and full-length VWF gene sequencing was performed for all index cases and healthy control subjects in a central laboratory. Bleeding phenotype was characterized using the International Society on Thrombosis and Haemostasis bleeding assessment tool. At study entry, 64% of subjects had VWF antigen (VWF:Ag) or VWF ristocetin cofactor activity below the lower limit of normal, whereas 36% had normal VWF levels. VWF sequence variations were most frequent in subjects with VWF:Ag <30 IU/dL (82%), whereas subjects with type 1 VWD and VWF:Ag ≥30 IU/dL had an intermediate frequency of variants (44%). Subjects whose VWF testing was normal at study entry had a similar rate of sequence variations as the healthy controls (14%). All subjects with severe type 1 VWD and VWF:Ag ≤5 IU/dL had an abnormal bleeding score (BS), but otherwise BS did not correlate with VWF:Ag. Subjects with a historical diagnosis of type 1 VWD had similar rates of abnormal BS compared with subjects with low VWF levels at study entry. Type 1 VWD in the United States is highly variable, and bleeding symptoms are frequent in this population.

Type 2B von Willebrand Disease: A Matter of Plasma Plus Platelet Abnormality.

Type 2B von Willebrand disease (VWD2B) is a rare, autosomal-dominant inherited bleeding disorder, characterized by an enhanced ristocetin-induced platelet aggregation in platelet-rich plasma and often with variable degree of thrombocytopenia and loss of high-molecular-weight multimers von Willebrand factor (VWF). All these phenomena are caused by a mutant VWF, normally synthesized and assembled by endothelial cells, but with heightened affinity binding to the platelet receptor glycoprotein Ib-α (GpIb-α). When this abnormal VWF is released into the circulation and under specific clinical circumstances, in vivo platelet clumping is observed. Mutations, invariably clustered in exon 28 of the VWF gene encoding for the VWF A1 domain involved in VWF binding to GpIb-α, are responsible for VWD2B phenotype. Clinical and laboratory phenotype appears strongly related to the type of VWF-causative mutations. However, recent evidences suggest that a true platelet defect is also present in this type, with several morphological and functional abnormalities being detected in a subset of VWD2B patients.

Regulation of VWF expression, and secretion in health and disease.

Von Willebrand factor (VWF) is a large multidomain, multimeric glycoprotein that plays an essential role in regulating the balance between blood clotting and bleeding. Aberrant VWF regulation can lead to a spectrum of diseases extending from bleeding disorders [Von Willebrand disease (VWD)] to aberrant thrombotic thrombocytopenic purpura (TTP). Understanding the biology of VWF expression and secretion is essential for developing novel targeted therapies for VWF-related hemostasis disorders. A number of recent elegant in-vitro and in-vivo studies will be highlighted, including the discovery of intronic splicing in the VWF gene, microRNA-regulated VWF gene expression, and syntaxin binding protein and autophagy mediated VWF secretion. Compared with the already established critical role of VWF in VWD and TTP pathophysiology, additional clinical studies have clarified and reinforced the association of elevated plasma levels of VWF with an increased risk of stroke, myocardial infarction, venous thrombosis, and diabetic thrombotic complications. Moreover, experimental mouse models of ischemic stroke and myocardial infarction have further supported VWF as a potential therapeutic target. VWF biosynthesis, maturation, and secretion is a complex process, which mandates tight regulation. Significant progress has been made in our understandings of VWF expression and secretion and its association with thrombotic diseases, contributing to the development of novel targeting VWF drugs for prevention and treatment of deficient and enhanced hemostasis.

Current and emerging approaches for assessing von Willebrand disease in 2016.

C), ristocetin-induced platelet agglutination (RIPA), multimeric analysis (VWF:MA), VWF propeptide (VWFpp), VWF:FVIII binding assay (VWF:FVIIIB), and the assessment of biological response to desmopressin (DDAVP) are necessary to characterize VWD types. Levels of VWF activities <30U/dL have been associated with a bleeding phenotype and the presence of mutations in the VWF gene.

Association of von Willebrand factor gene polymorphisms with coronary artery disease among ethnic southern Han Chinese

To assess the association of rs216293 T/G and rs1063857 T/C polymorphisms of von Willebrand factor (vWF) gene with the morbidity of coronary artery disease (CAD) and the number of involved vessels among an ethnic Han Chinese population from Zhejiang province. A case-control study was conducted. For 246 patients and 156 unaffected controls, the frequencies of genotypes and alleles of the rs216293T/G and rs1063857T/C polymorphisms were determined, and their association with CAD and the numbers of involved vessels were assessed. The frequencies of G allele of rs216293 and C allele of rs1063857 were higher in the CAD patients compared with those of the controls (30.3% vs.23.7%, chi-square=4.107, P=0.043; 7.7% vs. 4.2%, chi-square=4.066, P=0.044). The G allele of the rs216293 polymorphism and C allele of the rs1063857 polymorphism were both higher in the CAD patients compared with the controls (53.7% vs.41.0%, chi-square=6.098, P=0.014; 15.4% vs. 8.3%, chi-square=4.361, P=0.037). After adjusting the influence factors by logistic regression analysis, the G allele carriers of rs216293 and the C allele carriers of rs1063857 showed an increased risk for CAD (OR=1.625, 95%CI: 1.060-2.492, P=0.026; OR=2.305, 95% CI: 1.142-4.654, P=0.040). No significant difference was detected in the frequency of both rs216293 and rs1063857 among patients with single or multiple vessels (P>0.05). The rs216293T/G and rs1063857T/C polymorphisms of the vWF gene are both associated with the risk for CAD among the selected population. The G allele of the rs216293 polymorphism and C allele of the rs1063857 polymorphism may be the genetic determinants for CAD.

Human endothelial and foetal femur-derived stem cell co-cultures modulate osteogenesis and angiogenesis.

BackgroundA dynamic vasculature is a prerequisite for bone formation where the interaction of bone cells and endothelial cells is essential for both the development and the healing process of bone. Enhanced understanding of the specific mediators involved in bone cell and endothelial cell interactions offers new avenues for skeletal regenerative applications. This study has investigated the osteogenic and angiogenic potential of co-cultures of human foetal diaphyseal or epiphyseal cells with human umbilical vein endothelial cells (HUVEC) in the presence and absence of vascular endothelial growth factor (VEGF) supplementation.MethodsEarly osteogenic activities of the co-cultures (±VEGF) were assessed by alkaline phosphatase (ALP) activity. Osteogenic and angiogenic gene expression was measured using quantitative polymerase chain reaction. An ex vivo organotypic embryonic chick (E11) femur culture model was used to determine the osteogenic effects of VEGF as determined using micro-computed tomography (μCT) and Alcian blue/Sirius red histochemistry and immunocytochemistry for expression of CD31.ResultsALP activity and gene expression of ALP and Type-1 collagen was enhanced in foetal skeletal/HUVECs co-cultures. In foetal diaphyseal/HUVECs co-cultures, VEGF reduced the levels of ALP activity and displayed a negligible effect on von Willebrand factor (vWF) and VEGF gene expression. In contrast, VEGF supplementation was observed to significantly increase FLT-1 and KDR gene expression in co-cultures with modulation of expression enhanced, compared to VEGF skeletal monocultures. In the organotypic chick model, addition of VEGF significantly enhanced bone formation, which coincided with elevated levels of CD31-positive cells in the mid-diaphyseal region of the femurs.ConclusionThese studies demonstrate a differential skeletal response of early foetal skeletal cells, when co-cultured with endothelial cells and the potential of co-culture models for bone repair. The differential effect of VEGF supplementation on markers of angiogenesis and osteogenesis in co-cultures and organ cultures, demonstrate the importance of the intricate temporal coordination of osteogenic and angiogenic processes during bone formation and implications therein for effective approaches to bone regenerative therapies.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-015-0270-3) contains supplementary material, which is available to authorized users.

Sulodexide Reduces the Proinflammatory Effect of Serum from Patients with Peripheral Artery Disease in Human Arterial Endothelial Cells

Background/Aims: Dysfunction of the arterial endothelial cells promotes the progression of atherosclerosis. We studied how exposure of human arterial endothelial cells to atherosclerotic serum from patients with peripheral artery disease changes the secretory activity of these cells, and whether that reaction is modified by sulodexide. Methods: Endothelial cells in in vitro culture were exposed to standard culture medium ± 100pg/mL Interleukin-1(IL-1) or to medium supplemented with 20% atherosclerotic serum. Afterwards, the expression of genes responsible for the synthesis of Interleukin-6 (IL-6), Vascular Cell Adhesion Protein-1 (VCAM-1) and Von Willebrand Factor (VWF) was evaluated, together with the secretion of these compounds. Additionally, the effect of sulodexide on these processes was studied. Results: Atherosclerotic serum stimulated the expression of IL6, VCAM-1 and VWF genes in endothelial cells, which was followed by increased secretion of these compounds by 179%, 121% and 116%, respectively. Sulodexide (0.5 LRU/mL) reduced atherosclerotic serum-induced increased expression of genes for IL-6 (-32%), VCAM-1 (-20%) and VWF (-42%), and lowered secretion of these molecules: IL-6 (-27%), VCAM-1(-27%), VWF (-25%). Sulodexide also reduced, in a dose- dependent manner, secretion of IL6 from unstimulated and stimulated with IL-1 endothelial cells. Conclusions: Atherosclerotic serum induces proinflammatory and prothrombotic phenotype in arterial endothelium, which is partially reduced by sulodexide, via inhibition of genes expression, and in consequence lower secretory activity.

Management of an uncommon form of type 2M VWD: a single centre experience

Abstract We report a single centre’s experience of the diagnosis and management of an uncommon form of type 2 von Willebrand disease (VWD) in members of two unrelated families. The affected patients presented with mild to moderate bleeding phenotypes and accompanying MCMDM-1 VWD bleeding assessment tool scores of 5 or less. Genetic analysis in both families confirmed a missense mutation in exon 30 of the von Willebrand factor (VWF) gene, a single base substitution T&gt;A at nucleotide 5282 which led to change at codon 1761 from methionine to lysine (M1761K). This mutation lies within the A3 domain of the VWF protein, a region that is important for collagen binding. All affected patients were found to have normal coagulation profiles, normal VWF multimers and normal VWF assays except the VWF collagen-binding (VWF: CB) assay levels, which were significantly reduced. Desmopressin effected a good response in all treated patients, with a 3- to 5-fold rise of VWF:CB levels. However, there was variability in the degree to which VWF:CB levels remained elevated. Surgical procedures, including the delivery of one patient, were able to be managed with either desmopressin and/or tranexamic acid alone, with little need for recourse to VWF factor concentrate therapy.

Genetic Analysis of Von Willebrand Disease By Using the Exome Sequencing Approach

Abstract Background: Von Willebrand disease (VWD) is the most common inherited bleeding disorder characterized by quantitative and/or qualitative von Willebrand factor (VWF) abnormalities. VWF gene is a large gene consists of 52 exons, wheras its pseudogene corresponds to exons 23-34 that makes the analysis much complicated. In this study, we performed genetic analyses of the VWF gene from 20 Japanese VWD patients, aimed to find the causative genetic mutations by using various techniques including the exome sequencing approach, and to explore the diversity of yet unknown VWD pathogenetic mechanisms. Methods: Patients' samples were collected after the written informed consent has been obtained. First, we analyzed exon 28 of VWF genes from peripheral blood genomic DNA of the patients by PCR using allele-specific primers, and analyzed DNA sequences of the patients by Sanger sequencing method. If patients show the type 3 phenotype, the possibility of deletion or insertion of the gene was taken into consideration, then MLPA method was selected. In cases the gene mutation was not detected by exon 28 sequencing or MLPA, we analyzed all exons of VWF by Sanger method. In selected patients, the exome sequencing was employed by using the next-generation sequencer Hi-Seq 2500 (Illumina) and exomes were captured using SureSelect XT Human All Exon V5+UTRs kit (Agilent Technologies). Results: Twenty VWD cases were studied. As a result of the VWF exon 28 sequencing, we identified causative gene mutation in 12 cases (10 cases were type 2, 2 cases were type 1). Gene mutations found in exon 28 were p.R1308C, p.R1315C, p.V1316M, p.R1334W, p.A1461D, p.L1503R, p.S1506L, p.R1597Q, p.G1609R. In one case and her sister of type 3, MLPA could not detect the large deletions or insertions. Sanger sequencing showed the they were compound heterozygous for an p.E2341X (c.7021G&gt;T) mutation in exon 41 and a p.Y2631X (c.7892-7893insA) mutation in exon 48. In another case of type 3, we used exome sequencing and found a novel homozygous gene mutation p.G2752S (c.8254G&gt;A) in exon 52. The mutation was confirmed by Sanger sequencing and the propositus was homozygote for the mutation, while the parents and two sons had the same mutation in the heterozygote. In the multimer analysis of plasma, multimers larger than the dimer in size were hardly visible, but there was a faint monomer band was present in the plasma. Multimeric patterns of his family were normal. Discussion: We analyzed the VWF genes of 20 VWD cases and identified causative gene mutations in 14 cases including three novel mutations (p.E2341X, p.Y2631X, p.G2752S). We previously demonstrated BOECs established from the type 3 patient (p.E2341X and p.Y2631X) reproduced the phenotype of the disease (ASH 2014). Meanwhile, G2752S mutation is located in CTCK domain, therefore possibly it affects the VWF dimer formation. However, the causative gene mutations were not detected in 6 cases (5 cases were type 1, 1 case was type 2) by exome sequencing. Although previous observations have pointed out that causative VWF gene mutations are not detectable in about 30 % of entire type 1 cases, a newer approach would be needed to elucidate molecular pathogenesis of VWD. Disclosures Suzuki: Baxalta: Honoraria; Novo Nordisk Pharma: Honoraria; Bayer Healthcare: Honoraria. Matsushita:Biogen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Baxalta: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bayer Healthcare: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Asahi Kasei Pharma: Honoraria, Research Funding, Speakers Bureau; Kaketsuken: Honoraria, Research Funding, Speakers Bureau; Seamens: Speakers Bureau; Japan Blood Products Organization: Honoraria, Research Funding; Kyowa-Kirin: Honoraria, Research Funding; Novartis Pharma: Honoraria, Speakers Bureau; Sysmex: Speakers Bureau; Chugai Pharmaceutical Co., Ltd.: Research Funding; Novo Nordisk Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Eisai: Research Funding; Nihon Pharmaceutical: Honoraria, Research Funding, Speakers Bureau; Octapharma AG: Honoraria; CLS-Behling: Research Funding.

Domain Distribution of Type 1 VWD Sequence Variants and Impact on Clinical and Laboratory Phenotype in the Zimmerman Program

Type 1 von Willebrand disease (VWD) is a common inherited bleeding disorder, characterized by a quantitative deficiency of von Willebrand factor (VWF). The molecular basis continues to merit exploration, especially since the genetic cause has not been determined in 30-40% of cases in previous studies. As part of the Zimmerman Program for the Molecular and Biology of VWD (ZPMCB-VWD) study, we sought to identify the sequence variants (SV) within the VWF gene that result in type 1 VWD and correlate these to clinical laboratory phenotype and clinical severity and determine the frequency of type 1 subjects who do not have a gene defect. We also wanted to examine the types of sequence variants that cause type 1, type 1-Severe (1S), type 1C and low VWF (LVWF), and determine the influence of SV location on VWF levels and risk of bleeding. 310 type 1 Index Cases (IC) enrolled in the ZPMCB-VWD were analyzed, including 69 type 1 (VWF:Ag&lt;30), 9 type 1S (VWF:Ag&lt;5), 57 type 1C (VWF:Ag&lt;30; VWFpp/VWF:Ag&gt;3), and 175 LVWF (VWF:Ag or VWF:RCo 30-50). Bleeding symptoms were quantified using the ISTH bleeding score (ISTH BS). Clinical VWF testing was done in a central laboratory at BloodCenter of Wisconsin and included FVIII, VWF:Ag, VWF:RCo, VWFpp, and VWF:CB. Full-length VWF sequencing was performed, including 5' and 3' regions and intron/exon boundaries. SV found in &gt;1% of the healthy control population were excluded from this analysis. Mutation negative cases were further investigated by comparative genomic hybridization (CGH) to identify any large deletions or duplications in the VWF gene. SV or large deletions were identified in 62% (n=192) of this cohort with quantitative VWF deficiency, whereas 38% (n=118) had no variants found. Of those with no sequence variant identified, 10% were type 1, 5% type 1C and 85% LVWF. There were 142 SV found in this cohort. 75% of the subjects had missense mutations, 8% small insertions/deletions, 9% splice site mutations, 3% nonsense mutations and 1% large deletions. The median number of SV found in individuals was 1, however 23% had more than 1 SV present (range 1 to 4). Mean VWF:Ag (IU/dl) was examined to determine the influence of SV location on levels and median ISTH BS was assessed as a measure of bleeding severity by domain: Table.VWF DomainD1D2D'D3A1A2A3D4C1-C6CKNone# Subjects24343460251324304118VWF:Ag (IU/dl)18211614311221253839ISTH BS5665495515 Type 1S subjects had SV that were enriched in D1, D2 and A3 domains. 52% of type 1C variants were found in the A1 domain followed by 19% in D'D3 and 17% in D4. Both type 1 and LVWF subjects had SV that were evenly distributed throughout the gene, but LVWF had the majority of SV found in the A2, D2 and C1-C6 domains. Type 1S subjects showed a difference in mutation type compared to rest of the type 1 cohort with 31% intronic/splice site mutations, 19% nonsense mutations, 19% insertions/deletions and only 25% missense mutations. In contrast, missense mutations accounted for 68% of the mutations in type 1, 91% in type 1C and 80% in LVWF. The presence of predominantly truncating or loss of function variants in type 1S correlates with lower VWF levels and increased bleeding. In conclusion, we found that 62% of our type 1 cohort did have potentially causative SVs identified, with the majority caused by missense mutations (75%). Type 1C and type 1S tend to have SV isolated in certain domains whereas SV in type 1 and LVWF tend to be spread throughout the gene. SVs in the A3 domain demonstrated the lowest VWF:Ag (12 IU/dl) and highest ISTH BS (9) while changes in the CK domain had the highest VWF:Ag (38 IU/dl) and lowest ISTH BS (1). SVs or large deletions were not found in 38% of this cohort, indicating that other molecular factors either intronic or outside of the VWF gene may be the cause of low VWF and bleeding in these subjects. Disclosures Friedman: CSL Behring: Consultancy; Instrumentation Laboratories: Consultancy; Novo Nordisk: Consultancy; Alexion: Speakers Bureau. Gill:Baxalta, Bayer, and CSL-Behring: Membership on an entity's Board of Directors or advisory committees.

Molecular Genetics Analysis of Von Williebrand Disease: Studies in Cohort Pakistani Patients

Introduction: Von Willebrand disease (VWD) type III is the second most common inherited bleeding disorder in Pakistan. It is caused by severe quantitative deficiency of plasma Von Willebrand factor (VWF). Absence of VWF increases the severity of disease and is caused by homozygous/ compound heterozygous mutations in Von Willebrand factor gene.Objective: The aim of study is to characterize molecular genetics of Von Willebrand type III in Pakistani population.Setting: NIBD & BMT Karachi, Chughtais lab, Children's Hospital Lahore, PAEC Islamabad and HMC Peshawar.Material And Method: In this cohort study of Von Willebrand disease (VWD) type III, Blood samples of 48 unrelated patients of type III VWD were collected in National Institute Of Blood Disease & Bone Marrow Transplant (NIBD) Karachi, Chughtais lab, Children's Hospital Lahore, PAEC Islamabad and HMC Peshawar. Genomic DNA was extracted from peripheral blood by QIAamp DNA Blood mini kit (Qiagen) and Exon specific PCR was done for VWF gene and Direct gene sequenced on automated ABI-3130 Genetic Analyzer (Applied Biosystems). Sequence Variations in VWF were checked on ISTH-SSC VWD homepage (http://www.vwf.group.shef.ac.uk/), Ensemble genome browser (http://www.ensembl.org/), VWFdb hemobase and biobase biological database (http://www.hgmd.cf.ac.uk/ac/index.php). We have adopted the nomenclature for numbering amino acids of Human genome variation society (HGVS). Statistics analysis was done SPSSv17.Results: Sequence analysis detected mutations in 46 (95.83%) out of 48 samples. VWD type III has led to identification of 28 cases (60.8%) homozygous and 18 (39.1%) were compound heterozygous. We have indentified total 31 Mutations distributed as 17 missense mutations (54%), 7 nonsense mutations (22%) 2 small deletions (6%) , 2 insertion mutations (6%) and 3 splice site mutations (9%).19 of these were newly mutations in this cohort study.. Further method multiplex ligation dependent probe amplification (MLPA) is needed for detection of large deletions in two patients. Nonsense c.3931C>T, p.Q1311*was founder mutation in Pakistani patients.Conclusion: In cohort study, missense mutations are detected as common in among patients and most of the mutations identified in this cohort were homozygous due to Consanguinity in the family of the patients. DisclosuresOldenburg:SOBI: Consultancy.

Role of Von Willebrand Factor in Female Carriers from an Extended Family with Mild Hemophilia A

Background: Hemophilia A (HA) is an X-linked disorder that primarily affects males, although female carriers can exhibit bleeding phenotypes. Factor VIII activity levels (FVIII:C) in XX females are influenced by X chromosome inactivation (XCI), a process that silences each parental X in a proportion of cells. XCI skewing can decrease FVIII expression by preferentially inactivating the normal X chromosome. FVIII:C is further modulated by factors such as ABO blood groups and von Willebrand factor (VWF). The D'/D3 domain of VWF binds circulating FVIII protein, preventing proteolytic degradation. In type 2 Normandy von Willebrand disease (2N VWD), D'/D3 mutations decrease affinity to FVIII and result in bleeding events similar to mild/moderate HA. Variants in VWF also affect the pharmacokinetics of recombinant FVIII. Current clinical screening tests detect the 3 VWF mutations responsible for >90% of 2N, but report variants not directly responsible for 2N as clinically benign. However, common polymorphisms are known to affect FVIII:C in normal individuals. Therefore, a better understanding of how specific alterations in VWF modulate HA phenotype is necessary to interpret clinical presentation and refine management with factor concentrates. This is particularly important in HA individuals carrying mild/moderate mutations. To evaluate VWF variants in HA carriers, we focused on an extended pedigree that includes four obligate carriers from a family with mild/moderate HA (FVIII: p.Ala723Thr). FVIII:C varied and largely correlated with XCI skewing. Nevertheless, the FVIII:VWF interaction prompted us to identify VWF gene variants that could further modulate FVIII:C and contribute to bleeding in this family.Methods: To identify D'/D3 VWF variants that impact FVIII binding, primers were designed to amplify exons 17-20 and 24-27 on chromosome 12. A chromosome 22 pseudogene, with 97% identity to VWF exons 24-34, complicated primer design. Primers specific to VWF were selected by targeting regions that differed from the pseudogene and were verified by digestion with a restriction enzyme unique to each chromosome 12 exon. The PCR products were amplified and sequenced from the four female carriers and a control male relative.Results: After excluding the three most common mutations responsible for 2N, seven other variants were identified. Four of these were intronic polymorphisms and a synonymous variant at p.Asn1138 not associated with VWD and presumed to be clinically benign. All but one of these have been described in normal individuals. Two females were heterozygous for missense variant rs1063856 (p.Thr789Ala) and synonymous polymorphism rs1063857 (p.Tyr795=) that are in linkage disequilibrium and are likely to impact FVIII:C and VWF antigen (VWF:Ag) levels. These common variants, found in up to 36% of Caucasians and 58% of African Americans, are reported to increase VWF:Ag and FVIII:C jn heterozygotes (9 IU/dL and 7 IU/dL respectively). Neither ABO blood groups nor XCI skewing could fully explain the differences in FVIII:C activity observed with this variant.Conclusions: We propose that VWF variants rs1063856/rs1063857 may contribute to FVIII:C differences between two females in the pedigree with similar XCI skewing. We conclude that consideration of VWF variants is important for fully understanding bleeding phenotype and treatment responses in female carriers and males in families with mild/moderate HA. These findings support the need for expanded studies into the role of FVIII and VWF variant interactions in additional unrelated HA individuals. DisclosuresNo relevant conflicts of interest to declare.