Von Willebrand Factor Gene Research Articles

Assessing the Safety of Various VWF Regimens in Patients with Clinically Severe VWD: A Natural History Study

Background: Von Willebrand disease (VWD) is the most common inherited bleeding disorder in humans affecting up to 1% of the population, while symptomatic prevalence is likely closer to 0.1%. A deficiency of von Willebrand factor (VWF) can be quantitative (type 1 or type 3) or qualitative (type 2) and lead to a bleeding diathesis of variable intensity roughly correlating with functional activity. Diagnosis can be challenging due to variable penetrance and large influence of multiple pre-analytic variables and a wide testing coefficient of variation. Treatment for VWD is focused on replacement of defective or deficient VWF with a plasma-derived or recombinant VWF-containing product, release and elevation of endogenous stores of VWF with Desmopressin (DDAVP), or prevention of premature fibrinolysis with an antifibrinolytic, such as aminocaproic acid. Although there is relative consensus on the management of mild VWD, there is scarce literature about the optimal treatment of patients with severe disease, especially in regard to factor replacement. Real World evidence for the use of primary (prior to significant bleeding) or secondary (following development of significant bleeding) prophylaxis is lacking with the majority of studies relying heavily on retrospective data. Additionally, ongoing VWD prophylaxis studies typically only allow participants to enroll if they previously have not been on prophylaxis, limiting our ability to learn about this growing population of patients. Study Design and Methods: Approximately 1,900 VWD patients were identified in the ATHNdataset with a VWF:Ag or VWF:RCo of ≤ 30%, with ~170 of these on prophylaxis. This group, in addition to those VWD patients with clinically significant bleeding and ≤ 40% of normal VWF:Ag or VWF:RCo, provide a potential unmet opportunity to examine prophylaxis and treatment patterns. Furthermore, a standardized laboratory assessment (including a standardized diagnostic battery, genetic evaluation of VWF gene, and inhibitor testing) will provide significant enrichment of the ATHNdataset by fully characterizing patients that are highly likely to utilize factor concentrates. Inclusion criteria are patients with severe VWD defined as type 3 VWD, or VWF:RCo, VWF:GP1bM or VWF:Ag≤ 30%, patients with clinically severe VWD as defined by VWF:Rco, VWF:GP1bM or VWF:Ag ≤ 40% with severe bleeding phenotype requiring recurrent use of factor concentrates, and co-enrollment in the ATHNdataset. Patients with platelet-type or acquired VWD are excluded. The primary objective is to assess the safety of various VWF regimens for different indications (on-demand, surgery, and prophylaxis) in adult and pediatric patients with clinically severe VWD. Safety is measured by the number of reported events as defined by the European Haemophilia Safety Surveillance (EUHASS) program. Secondary objectives are to enrich and analyze data from clinically severe congenital VWD patients by collecting laboratory data; to establish sub-studies for patients who are treated with VWF products on demand or who have started on or switched to a particular VWF containing product; to evaluate the use of factor replacement as prophylaxis in a cohort of severe VWD participants over 6 month time periods; to describe bleeding events, changes in overall bleeding, and annualized bleed rate as measured by the International Society on Thrombosis and Haemostasis (ISTH) Bleeding Assessment Tool (BAT) and if applicable the Pictorial Bleed Assessment Chart (PBAC); and to describe real-world effectiveness of VWD treatment as measured by health care utilization and quality of life measures (PROMIS® and V-WIQ questionnaires). Descriptive statistics will be calculated to analyze the primary and secondary outcomes. For each categorical variable, its frequency and percentage will be reported. In terms of a continuous measurement, its mean, median, standard deviation, interquartile range, minimum, and maximum values will be disclosed. The study will attempt to enroll a target number of at least 50 participants who are receiving VONVENDI but will not mandate the use of VONVENDI. More study design details are outlined in Table 1. Disclosures Sidonio: Genetech: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda-Shire: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bioverativ: Membership on an entity's Board of Directors or advisory committees, Research Funding; Octapharma: Membership on an entity's Board of Directors or advisory committees, Research Funding; Grifols: Membership on an entity's Board of Directors or advisory committees, Research Funding; Biomarin: Membership on an entity's Board of Directors or advisory committees; Uniqure: Membership on an entity's Board of Directors or advisory committees; Novo Nordisk: Membership on an entity's Board of Directors or advisory committees; Kedrion: Research Funding.

Characterization of the mutation spectrum in a Pakistani cohort of type 3 von Willebrand disease.

Type 3 von Willebrand disease (VWD), a severe autosomal recessive hereditary bleeding disorder, is described by the virtual absence of von Willebrand factor (VWF). In consanguineous populations, for example Pakistan, the disease is reported with a higher incidence rate than the worldwide prevalence. This study aims to characterize molecular pathology and clinical profile of type 3 VWD cohort of Pakistani origin. In total, 48 patients were enrolled in the current study. Initially, the index patients (IPs) were evaluated by a standardized questionnaire for recording bleeding manifestations and by performing conventional coagulation tests. The diagnosis of VWD type 3 was confirmed by VWF antigens less than 5IU/dL. Direct sequencing of VWF gene (VWF) was carried out to identify causative gene variations. We evaluated the potential consequence of novel splice site and missense variations by predictive computational programs and in silico structural analysis. VWF mutations were detected in 46 out of 48 IPs (95.8%), predominantly as homozygous variants. In total, twenty-nine different gene defects were characterized in this cohort from which 10 (34.5%) are novel. The majority of the mutations were null alleles (66%; including gene conversions, nonsense, splice site variations, small deletions and insertions), and 34% of them were missense substitutions. Herein, we reported for the first time, the pattern of gene defects in Pakistani type 3 VWD cohort. We identified a wide heterogeneous mutation spectrum along with variability in the type of bleeding episodes.

Characterization of a Porcine Model for Von Willebrand Disease Type 1 and 3 Regarding Expression of Angiogenic Mediators in the Nonpregnant Female Reproductive Tract.

Von Willebrand disease (VWD), a blood coagulation disorder, is also known to cause angiodysplasia. Hitherto, no animal model has been found with angiodysplasia that can be studied in vivo. In addition, VWD patients tend to have a higher incidence of miscarriages for reasons unknown. Thus, we aimed to examine the influence of von Willebrand factor (VWF) on the female reproductive tract histology and the expression and distribution of angiogenic factors in a porcine model for VWD types 1 and 3. The disease-causing tandem duplication within the VWF gene occurred naturally in these pigs, making them a rare and valuable model. Reproductive organs of 6 animals (2 of each mutant genotype and 2 wildtype (WT) animals) were harvested. Genotype plus phenotype were confirmed. Several angiogenic factors were chosen for possible connections to VWF and analyzed alongside VWF by immunohistochemistry and quantitative gene expression studies. VWD type 3 animals showed angiodysplasia in the uterus and shifting of integrin αVβ₃ from the apical membrane of uterine epithelium to the cytoplasm accompanied by increased vascular endothelial growth factor (VEGF) expression. Varying staining patterns for angiopoietin (Ang)-2 were observed among the genotypes. As compared with WT, the ovaries of the VWD type 3 animals showed decreased gene expression of ANG2 and increased gene expression of TIE (tyrosine kinase with immunoglobulin and epidermal growth factor homology domains) 2, with some differences in the ANG/TIE-system among the mutant genotypes. In conclusion, severely reduced VWF seems to evoke angiodysplasia in the porcine uterus. Varying distribution and expression of angiogenic factors suggest that this large animal model is promising for investigation of influence of VWF on angiogenesis in larger groups.

The role of genetics in the pathogenesis and diagnosis of type 1 Von Willebrand disease.

Von Willebrand disease (VWD) is a common bleeding disorder, but diagnosis of VWD is challenging, particularly with type 1 VWD. Although most clinicians use specific tests of von Willebrand factor (VWF) activity to classify patients with VWD, genetic testing for VWF defects is another potential method of diagnosis. Studies of patients with type 1 VWD report consistently that many, but not all, study participants have VWF gene defects. Certain populations, including those with VWF levels less than 30 IU/dl and those with clearance defects, are more likely to have a VWF sequence variant. In addition, a number of loci outside the VWF gene have been shown to affect VWF levels, including ABO, CLEC4M, STXBP5, and STAB2. Genetic defects in VWF are common, but not all defects lead to disease. Type 1 VWD in particular does not always have an associated VWF sequence variant. New data stemming from genome-wide association studies on modifier genes suggest that the etiology of type 1 VWD is multifactorial.

Cryptic non-canonical splice site activation is part of the mechanism that abolishes multimer organization in the c.2269_2270del von Willebrand factor

We report a new pathogenic mechanism in von Willebrand disease involving the use of a non-canonical splicing site. The proband, carrying the homozygous c.2269_2270del mutation previously classified as a type 3 mutation, showed severely reduced plasma and platelet von Willebrand factor antigen levels and functions, and no factor VIII binding capacity. A particular von Willebrand factor multimer pattern emerged in plasma, characterized by the presence of only two oligomers: the dimer and an unusually large band, with no intermediate components. There were von Willebrand factor multimers in platelets, but each band ran more slowly than the normal counterpart. No anti-von Willebrand factor antibodies were detectable. The proband was classified as having severe type 1 von Willebrand disease. Seeking the relationship between phenotype and genotype, we found the c.2269_2270del mutation associated with three different RNA: r.2269_2270del (RNAI), giving a truncated von Willebrand factor protein; r.[2269_2270del;2282_2288del] (RNAII), resulting from activation of a cryptic “AG” splicing site; and r.[2269_2270del;2281_2282insAG] (RNAIII), where the wild-type “AG” acceptor of exon 18 was retained due to the non-canonical 2279-2280 “CG” acceptor splicing site being used. The aberrant RNAII and RNAIII caused the alteration of the furin cleavage and binding sites, respectively, both resulting in a von Willebrand factor protein characterized by the persistence of von Willebrand factor propeptide, as confirmed by western blot analysis of the recombinant mutated von Willebrand factor molecules produced in vitro. Taken together, these findings explain the residual von Willebrand factor synthesis, slower-running multimers, and absent factor VIII binding capacity. The apparently pure gene null mutation c.2269_2270del profoundly alters von Willebrand factor gene splicing, inducing a complex RNA expression pattern.

Update on Molecular Testing in von Willebrand Disease

Diagnosis of von Willebrand disease (VWD) depends on personal and family history of bleeding and confirmatory laboratory testing. Currently available phenotypic tests for VWD contain potential sources for error that may distort results. Despite an exponential growth of information about the von Willebrand factor gene (VWF), the role of molecular diagnosis in VWD is still controversial. Due to the complexity and high cost of conventional molecular analyses, some investigators have recommended limiting this approach to distinguish suspected type 2N VWD from hemophilia A, type 2B from platelet-type VWD, and the exploration of type 3 VWD. New genetic methodologies and approaches are becoming available, but there is still some reluctance for their implementation in VWD diagnosis. This article discusses the pros and cons of molecular testing in VWD considering the experience obtained through the multicenter project "Molecular and Clinical Profile of VWD in Spain (PCM-EVW-ES)."

Treatment of cancer stem cells from human colon adenocarcinoma cell line HT-29 with resveratrol and sulindac induced mesenchymal-endothelial transition rate.

In the current experiment, the combined regime of resveratrol and a Wnt-3a inhibitor, sulindac, were examined on the angiogenic potential of cancer stem cells from human colon adenocarcinoma cell line HT-29 during 7days. Cancer stem cells were enriched via a magnetic-activated cell sorter technique and cultured in endothelial induction medium containing sulindac and resveratrol. Expression of endothelial markers such as the von Willebrand factor (vWF) and vascular endothelial cadherin (VE-cadherin) and genes participating in mesenchymal-to-epithelial transition was studied by real-time PCR assay. Protein levels of Wnt-3a and angiogenic factor YKL-40 were examined by western blotting. ELISA was used to determine the level of N-acetylgalactosaminyltransferase 11 (GALNT11) during mesenchymal-endothelial transition. Autophagy status was monitored by PCR array under treatment with the resveratrol plus sulindac. Results showed that resveratrol and sulindac had the potential to decrease the cell survival of HT-29 cancer cells and the clonogenic capacity of cancer stem cells compared with the control (p < 0.05). The expression of VE-cadherin and vWF was induced in cancer stem cells incubated with endothelial differentiation medium enriched with resveratrol (p < 0.05). Interestingly, the Wnt-3a level was increased in the presence of resveratrol and sulindac (p < 0.05). YKL-40 was reduced after cell exposure to sulindac and resveratrol. The intracellular content of resistance factor GALNT11 was diminished after treatment with resveratrol (p < 0.05). Resveratrol had the potential to induce the transcription of autophagy signaling genes in cancer stem cells during endothelial differentiation (p < 0.05). These data show that resveratrol could increase cancer stem cell trans-differentiation toward endothelial lineage while decrease cell resistance by modulation of autophagy signaling and GALNT11 synthesis.

Regulation of von Willebrand Factor Gene in Endothelial Cells That Are Programmed to Pluripotency and Differentiated Back to Endothelial Cells.

Endothelial cells play a central role in physiological function and pathophysiology of blood vessels in health and disease. However, the molecular mechanism that establishes the endothelial phenotype, and contributes to its signature cell type-specific gene expression, is not yet understood. We studied the regulation of a highly endothelial-specific gene, von Willebrand factor (VWF), in induced pluripotent stem cells generated from primary endothelial cells (human umbilical vein endothelial cells [HUVEC] into a pluripotent state [HiPS]) and subsequently differentiated back into endothelial cells. This allowed us to explore how VWF expression is regulated when the endothelial phenotype is revoked (endothelial cells to HiPS), and re-established (HiPS back to endothelial cells [EC-Diff]). HiPS were generated from HUVECs, their pluripotency established, and then differentiated back to endothelial cells. We established phenotypic characteristics and robust angiogenic function of EC-Diff. Gene array analyses, VWF chromatin modifications, and transacting factors binding assays were performed on the three cell types (HUVEC, HiPS, and EC-Diff). The results demonstrated that generally cohorts of transacting factors that function as transcriptional activators, and those that contribute to histone acetylation and DNA demethylation, were significantly decreased in HiPS compared with HUVECs and EC-Diff. In contrast, there were significant increases in the gene expression levels of epigenetic modifiers that function as methyl transferases in HiPS compared with endothelial cells. The results demonstrated that alterations in chromatin modifications of the VWF gene, in addition to expression and binding of transacting factors that specifically function as activators, are responsible for establishing endothelial specific regulation of the VWF gene. Stem Cells 2019;37:542-554.

Von Willebrand Factor Gene Expression in Primary Lower Grade Glioma: Mutually Co-Occurring Mutations in von Willebrand Factor, ATRX, and TP53.

BackgroundVenous thromboembolism is a common complication in patients with glioma. The clotting factor von Willebrand factor (VWF) is a highly adhesive procoagulant molecule that mediates platelet adhesion to endothelial and subendothelial surfaces. In the current analysis, we examined The Cancer Genome Atlas (TCGA) data to assess the VWF gene in patients with lower grade gliomas.MethodsFor newly diagnosed gliomas, we evaluated the association between VWF and overall survival in the Genomic Data Commons TCGA Lower Grade Glioma (LGG) dataset in TCGA. Simple statistics were calculated to identify patterns of mutual exclusivity or co-occurrence of VWF mutations. For each pair of query genes an odds ratio was calculated that indicates the likelihood that the mutations in the two genes are mutually exclusive or co-occurrent across the selected cases. To determine whether the identified relationship was significant for a gene pair, Fisher's exact test was performed.ResultsLower grade gliomas with less VWF gene expression had significantly better survival than those with more VWF gene expression (hazard ratio 0.64, 95% confidence interval 0.44 to 0.92, p=0.015 log rank test). When we analyzed the data with Cox regression, VWF expression had a significant effect on survival (p=0.02) that was unrelated to the effect of IDH1 expression (p=0.062), TP53 expression (p=0.135), independent of ATRX expression (p=0.021) and histology (astrocytoma versus oligoastrocytoma and oligodendroglioma, p=0.002). VWF mutations significantly co-occur with mutations in TP53 and ATRX (p<0.001).ConclusionThe deleterious prognostic effect of VWF expression and its co-occurrent mutations with TP53 and ATRX in lower grade gliomas are not surprising, given VWF's role in other cancers. Therefore, VWF gene expression may be a clinically important risk marker in lower grade glioma.

Increased RNA Expression of von Willebrand Factor Gene Is Associated With Infiltrating Lobular Breast Cancer and Normal PAM50 Subtype.

Infiltrating lobular carcinoma (ILC) is the second most common histologicaI subtype of breast cancer, accounting for 10% of all cases. ILC has a characteristic genomic profile. ILC shows a high frequency of cadherin 1 (CDH1) mutations, along with loss of phosphatase and tensin homolog (PTEN), activation of alpha serine/threonine kinase (AKT), and mutations in T-box transcription factor (TBX3) and forkhead box protein A1 (FOXA1). We suspected that another gene, von Willebrand factor (VWF), might also be part of the profile, since coagulation tests reveal significant differences in patients with breast cancer. For newly-diagnosed breast cancer, the association between VWF and histology in the GDC Breast Cancer dataset in The Cancer Genome Atlas (TCGA) was evaluated. The following were used to access and analyze the data: Genomic Data Commons Data Portal (https://portal.gdc.cancer.gov/); Xena browser (https://xenabrowser.net); cBioportal (http://cbioportal.org); Oncomine (https://oncomine.org); and Prediction Analysis of Microarray 50 (PAM50). Patients with ILC had higher VWF RNA expression than patients with infiltrating ductal carcinoma and other histology. The difference of expression was present to the same degree in both pre-menopausal and post-menopausal patients. Nine alterations in VWF and PTEN were significantly co-occurrent. Considering all histologies in 843 samples, Tukey's honest significant difference post hoc test showed that VWF RNA expression of the normal subtype was significantly greater than that of the other subtypes (p<0.001). Our finding of significantly higher VWF RNA expression in the PAM50 normal (non-basal-like) breast cancer subtype suggests that VWF protein measurement might complement or corroborate PAM50 results. VWF and PAM50 results both suggesting a low risk of recurrence might make the decision whether to give chemotherapy easier, especially if VWF protein were an independent predictor.

Genotypic and Phenotypic Analysis of Adolescents with Heavy Menstrual Bleeding and Low Von Willebrand Activity - Interim Report of a Multi-Center Study

Genotypic and Phenotypic Analysis of Adolescents with Heavy Menstrual Bleeding and Low Von Willebrand Activity - Interim Report of a Multi-Center Study

Distribution of Pathogenic Variants in Von Willebrand Disease Type 2A in the American Population: Results from a National Clinical Reference Laboratory

Introductionvon Willebrand disease (VWD) is a common inherited bleeding disorder with a reported incidence ranging from 0.01% to 1%. VWD is classified as quantitative (types 1 and 3) and qualitative (type 2) defects in von Willebrand factor (VWF). Classical VWD type 2A, the most common qualitative defect of VWD, is characterized by decreased VWF-dependent platelet adhesion and a selective deficiency of high molecular weight VWF multimers. The majority of type 2A VWD is inherited in an autosomal dominant fashion. This subtype is caused by missense variants acting through a variety of mechanisms including defects in multimer assembly or an intrinsically increased sensitivity to cleavage by ADAMTS-13. The majority of pathogenic mutations are located in exon 28, predominantly affecting the A2 domain. Missense mutations have also been reported in the D3 and A1 domains (exons 22 and 25-28). Less common missense mutations include those responsible for dimerization (CK domain, exon 52) and multimerization defects (D2 domain, exons 11-17). Our objective was to determine the genomic distribution of mutations underlying type 2A VWD in patients referred to a USA-based clinical reference laboratory.MethodsIn a clinical reference laboratory, BloodCenter of Wisconsin, VWF gene sequence analysis results from January 2009 to July 2018 were reviewed. Molecular testing was performed by polymerase chain reaction (PCR) amplification and bi-directional Sanger sequencing or capture-based enrichment and next-generation sequencing (NGS). Complete coding region and splice junction of VWF gene was compared to the reference sequence (NM_00552.3). Variant interpretation was performed according to American College of Medical Genetics (ACMG) standards and guidelines by utilizing genome databases, population frequency data, variant specific literature search and predictive computational tools. Variant interpretation was reviewed by a team comprising of genetic counselors, molecular experts and experts in coagulation disorders correlating these findings with the patient's clinical phenotype provided.ResultsPathogenic variants were identified in 46 cases received. Of these, 56.5 % (26 of 46) were in exon 28 of the VWF gene.(Figure 1) 21.7% of mutations (10 out of 46) were identified in exon 26, 4.3% were identified in exon 25 (2 of 46) and 6.5% (3 out of 46) were identified in exon 52 of VWF gene. The remaining 10.7% were identified in exons 6,11,12,22 and 27 (1 each). The distribution of these mutations according to VWF domains was also studied. 39% of these mutations were found in the A2 domain, 30.4% were identified in the D3 domain, and 13% were found in the A1 domain. A small number of these mutations were identified in the D1 domain (1/46), D2 domain (2/46) and CK domain (2/46). Phenotypic information was available for most patients evaluated.ConclusionsIn a predominantly Caucasian population with others of mixed ethnicities, the distribution of VWD type 2A mutations was studied in patients evaluated in a clinical reference laboratory. Most mutations were identified in VWF Exon 28 and in the A2 domain. Next generation sequencing platform enables sequencing of large amounts of DNA in parallel thereby allowing all VWF exons to be evaluated at one time. However, in smaller laboratories, without access to a NGS platform, initial evaluation of the VWF Exon 28 for evaluation of VWD type 2A variants is a cost effective measure. Subsequently, sequencing of other less implicated exons may be performed utilizing a reflexive algorithm. Genetic testing can be very useful in diagnosis of type 2 variants, where mutations in specific regions correspond to the defect in VWF function. [Display omitted] DisclosuresFriedman:Shire: Membership on an entity's Board of Directors or advisory committees; CSL Behring: Membership on an entity's Board of Directors or advisory committees.

Elevated Von Willebrand Factor May Abrogate Bleeding Phenotype in a Male with a Non-Null F8 Mutation

Introduction: Although FVIII activity (FVIII:C) correlates with phenotype in most Hemophilia A males, some have bleeding phenotypes milder than predicted by FVIII:C. Analysis of these individuals may provide additional insight into the relationship between F8 genotype and bleeding phenotype, identify additional factors that modulate FVIII, refine the classification of Hemophilia A and improve clinical management.We present a male subject who carries a c.5999G>C mutation in F8. This mutation has been reported in an individual with a severe bleeding phenotype in the EAHAD F8 Variant Database (www.factorviii-db.org). Our subject was serendipitously diagnosed at age 80, and has never had abnormal bleeding despite numerous challenges, including multiple dental extractions, adult circumcision, internal hemorrhoid ligation and cardiac catheterization. In an effort to resolve this genotype-phenotype discrepancy, we performed FVIII and von Willebrand Factor (VWF) assays, quantitated the extent of alternative splicing in F8, and screened for mutations in additional genes known to impact hemostasis. We validated a partial splicing defect that is consistent with mild Hemophilia A, but surprisingly cannot explain the lack of a bleeding phenotype.Methods: Peripheral blood was tested by one-stage FVIII:C and chromogenic FVIII assays, FVIII antigen ELISA, VWF antigen and activity assays, and von Willebrand Disease (VWD) Type 2 Normandy binding assay.To evaluate F8 splice variants, quantitative RT-PCR (qRT-PCR) was performed on RNA isolated from peripheral blood using primers that distinguish FVIII isoforms. Samples were tested in quadruplicate, with sample input normalized relative to GAPDH. Screening for exonic mutations in F8, F5, F2 and VWF genes was undertaken by Next Generation Sequencing (NGS).Results and Conclusions: In contrast to the previously reported severe c.5999G>C patient, this subject has reduced FVIII activity consistent with mild Hemophilia A measured by both FVIII activity assays. FVIII antigen assessment by ELISA gave similar results (0.35 IU/mL).To better understand FVIII:C levels in this individual, we sought to characterize F8 transcript isoforms. The c.5999G>C resides within the splice acceptor site of exon 19 and may result in two FVIII isoforms. One isoform produces a full-length FVIII containing a missense mutation (p.Gly2000Ala). The second isoform skips the 117 bp exon 19, but is predicted to retain an open reading frame; however, its location in the A3 domain likely disrupts secretion or function (Donadon et al., Haematologica 2018). The relative abundance of F8 variants evaluated by qRT-PCR demonstrated that despite a modest overall increase in F8 transcript relative to a normal control (1.6 fold higher expression of F8 at exon 18), the predominant isoform in this subject lacks exon 19, resulting in substantial depletion of the full-length isoform (0.3 relative to a normal control). These data indicate the full-length protein likely retains function, with transcript levels that explain residual FVIII activity.In an effort to explain absent bleeding phenotype, NGS was performed. The c.5999G>C F8 mutation was confirmed, while the thrombophilic Factor V Leiden and Prothrombin G20210A mutations were excluded. Sequencing also excluded mutations implicated in Type 2 Normandy VWD. Additional clinical testing may provide an explanation. VWF was elevated as indicated by VWF antigen and Ristocetin cofactor activity (>400%, >100%, respectively). Furthermore, VWF:FVIII binding activity by ELISA showed a higher than normal binding ratio (>1.42). VWF multimer analysis indicated normal distribution. Therefore, we propose elevated VWF abrogates this subject's bleeding phenotype.Altogether, our study expands our understanding of the functional consequences of this mutation by demonstrating that this mutation can result in FVIII levels predicting mild Hemophilia A. This case also underscores the complexities of predicting phenotype for mutations that result in partial splicing defects. Perhaps more intriguing is the lack of bleeding phenotype in this elderly individual with numerous hemostatic challenges, that might be related to increased VWF. These data support additional studies investigating the role of VWF in ameliorating bleeding risk in Hemophilia A. DisclosuresNo relevant conflicts of interest to declare.

Characterization of VWF gene conversions causing von Willebrand disease.

We previously reported that von Willebrand Factor gene (VWF) conversions are a relatively frequent cause of von Willebrand disease (VWD), however, their molecular pathomechanisms resulting in variant phenotypes is largely unknown. Here, we characterized VWF conversions harbouring missense and synonymous mutations, through generating a series of mutant constructs followed by transient expression in 293 cells, and qualitative and quantitative analysis of recombinant VWF (rVWF). The characterization of mutant rVWF showed the critical roles of synonymous variants in the pathogenicity of VWF conversions. The gene conversion variants p.Val1229Gly, p.Asn1231Thr, p.Asn1231Ser and p.Ala1464Pro in the absence of synonymous p.Ser1263= and p.Gln1449= showed minimal effect on rVWF synthesis and activity. Interestingly, a construct including the synonymous variants displayed significantly low rVWF expression and activity. The variant p.Pro1266Leu showed gain of rVWF function toward glycoprotein Ibα; surprisingly, this function was significantly abolished in the presence of gene conversion variants p.Val1229Gly-p.Asn1231Thr. Taken together, our expression studies suggest that synonymous variants in the combination of other gene conversion variants suppress the protein expression, possibly due to defective primary mRNA structure or processing. The variants p.Val1229Gly-p.Asn1231Thr affected the VWF gain of function caused by variant p.Pro1266Leu, probably due to conformational changes in VWF.

3015 - Hierarchically Related Lineage-Restricted Fates of Multipotent Hematopoietic Stem Cells

Self-renewing bone marrow hematopoietic stem cells (HSCs) secure blood cell replenishment in homeostasis and upon stress stimuli. Single cell transplantations uncovered considerable heterogeneity among HSCs (Müller-Sieburg et al. Blood 2002; Dykstra et al. Cell Stem Cell 2007; Challen et al. Cell Stem Cell 2010), also supported by findings in unperturbed hematopoiesis (Busch et al. Nature 2015; Pei et al. Nature 2017; Rodriguez-Fraticelli et al. Nature 2018). Expression of the megakaryocytic (Mk) von Willebrand factor gene (Vwf) enriches for platelet-biased HSCs (Sanjuan-Pla et al. Nature 2013). Some studies also suggested direct generation of unipotent self-renewing Mk progenitors within the phenotypic HSC compartment, implicating uncoupling between self-renewal and multipotency (Yamamoto et al. Cell 2013; Haas et al. Cell Stem Cell 2015). In this study (Carrelha et al. Nature 2018), sensitive tracking of progenitors and mature cells of the Mk/platelet (P), erythroid (E), myeloid (M), B and T cell lineages upon single HSC transplantation revealed a stable hierarchical framework for lineage-restricted fates of HSCs. The Vwf- HSC compartment includes long-term (LT) and short-term (ST) multilineage HSCs, with lymphoid-biased output resulting from ST-HSC exhaustion. In addition to multilineage HSCs, the Vwf+ compartment includes distinct subsets adopting PEMB-restricted, PEM-restricted, PE-restricted and P-restricted fates. Sustained self-renewal potential and unfulfilled multipotency of these fate-restricted HSCs is revealed through serial transplantation and in vitro assays, respectively. The only unilineage-fate subset of serially-transplantable HSCs replenish exclusively the Mk lineage branch of the hematopoietic tree, and no LT-HSC subset was found contributing exclusively to another single lineage. Genetic lineage tracing with Vwf-Cre inducible mouse model also supports an important role for platelet-biased HSCs in unperturbed hematopoiesis. Our findings uncover a limited repertoire of LT-HSC subsets, defined by hierarchically organised propensities for replenishment of a restricted set of blood lineages, prior to loss of self-renewal and multipotency.

Von Willebrand disease in the elderly: clinical perspectives.

Von Willebrand disease (VWD) is an inherited bleeding disorder that affects up to 1% of the population. In most cases, VWD results from a mutation in the von Willebrand Factor (VWF) gene, which alters the amount and function of VWF, a key glycoprotein in both primary and secondary hemostasis. A comprehensive analysis of patients with VWD should include VWF activity, antigen levels, platelet function, and a careful bleeding history. Treatment options include antifibrinolytics, desmopressin, and VWF replacement therapy. VWF levels fluctuate due to age, stress, environmental exposures, and pharmacologic treatment. Treatment guidelines exist to treat and prevent bleeding for patients undergoing surgery and medical procedures, but often these must be reevaluated in the setting of age-related comorbidities including cardiovascular events, venous thrombosis, and malignancy. In addition, many age-related complications are associated with a secondary acquired von Willebrand syndrome (AVWS), including malignancies, hypothyroidism, cardiovascular diseases, and cardiac replacement devices. The current literature is limited by a lack of older patients in clinical trials. Larger studies are needed to determine if age-related comorbidities affect VWD patients at different frequencies than the general elderly population. There is also a significant need for registry-based studies to evaluate many age-related comorbidities in VWD patients.

Type-3 von Willebrand disease in India-Clinical spectrum and molecular profile.

Type 3 von Willebrand disease (VWD) is the rare and most severe form of VWD which results from a near-complete deficiency of the von Willebrand factor (VWF). This study evaluates in detail the molecular pathology of type-3 VWD in India. One hundred and two patients from 90 families were evaluated. Phenotypic data, including bleeding scores (BS), were documented using structured questionnaires. Diagnosis of type 3 VWD was based on undetectable VWF antigen levels in the plasma. Genomic DNA from these patients was screened for mutations in VWF gene. Structural modeling and expression studies were carried out for missense mutations. Out of 102 patients, mutations could be identified in 91% (n=93). Fifty-five different gene variants were identified. Thirty-four (61.8%) were novel. Mutations could be identified in both the alleles in 90 patients, while no causative mutation could be identified in 9 patients; twenty-four (23.5%) patients had mutations clustered in the propeptide region of VWF. Interestingly, five mutations accounted for the defects in 37/93 (39.8%) patients. Structural analysis and in vitro studies on missense mutations imply impaired processes associated with secretion of VWF. This study is one of the largest series to define the molecular basis of type-3 VWD.

Role of multimeric analysis of von Willebrand factor (VWF) in von Willebrand disease (VWD) diagnosis: Lessons from the PCM-EVW-ES Spanish project.

The multimeric analysis (MA) of plasma von Willebrand factor (VWF) evaluates structural integrity and helps in the diagnosis of von Willebrand disease (VWD). This assay is a matter of controversy, being considered by some investigators cumbersome and only slightly informative. The centralised study ‘Molecular and Clinical Profile of von Willebrand Disease in Spain (PCM-EVW-ES)’ has been carried out by including the phenotypic assessment and the genetic analysis by next generation sequencing (NGS) of the VWF gene (VWF). The aim of the present study was to evaluate the role of MA to the diagnosis of these patients and their potential discrepancies. Two hundred and seventy out of 480 patients centrally diagnosed with VWD had normal multimers, 168 had abnormal multimers and 42 a total absence of multimers. VWF MA was of great significance in the diagnosis of 83 patients (17.3%), it was also of help in the diagnosis achieved in 365 additional patients (76%) and was not informative in 32 cases (6.7%). With regard to discrepancies, 110 out of 480 (23%) patients centrally diagnosed with VWD presented some kind of discordance between VWF:RCo/VWF:Ag and/or VWF:CB/VWF:Ag ratios, multimeric study and/or genetic results. The VWF MA was key in the presence of novel mutations as well as in cases with phenotypic discrepancies. A comparison between the contribution of MA and VWF:CB showed a clearly higher contribution of the former in the diagnostic process. These data seem to reinforce the relevance of the VWF MA in VWD diagnosis, despite all its limitations.

Novel Von Willebrand factor gene polymorphism presenting as purpura

Novel Von Willebrand factor gene polymorphism presenting as purpura

Common VWF sequence variants associated with higher VWF and FVIII are less frequent in subjects diagnosed with type 1 VWD

Unlabelled Box BackgroundGenetic variation in the VWF gene is associated with von Willebrand factor (VWF) and factor VIII (FVIII) levels in healthy individuals. ObjectivesWe hypothesized that VWF sequence variants associated with higher VWF or FVIII could impact the diagnosis of type 1 von Willebrand disease (VWD). MethodsWe examined VWF antigen (VWF:Ag), VWF ristocetin cofactor activity (VWF:RCo), VWF propeptide (VWFpp), and FVIII levels along with VWF gene sequencing in 256 healthy control and 97 type 1 VWD subjects as part of a cross‐sectional study. ResultsWe found several VWF sequence variants (VWF c.2880G>A and VWF c.2365A>G(;)c.2385T>C, found in linkage disequilibrium) associated with higher VWF and FVIII levels in healthy controls (P<.001 for both variants). In addition, these variants were significantly more common in controls than in subjects diagnosed with type 1 VWD and VWF:Ag <30 (P<.005). The decreased variant frequencies in type 1 VWD was not seen in other VWD types. VWF:Ag, VWF:RCo, and FVIII were not statistically different in type 1 VWD subjects who had these VWF variants compared to type 1 VWD patients without them. There was no difference in ABO blood group, VWF propeptide levels (excluding subjects with known VWF clearance defects), or bleeding score using the ISTH bleeding assessment tool. ConclusionsThese data suggest that certain VWF sequence variants associated with elevated FVIII and VWF levels may protect against reduced VWF levels. These findings were independent of other pathogenic sequence variants in VWF, suggesting a possible independent effect of c.2880G>A and c.2365A>G(;)c.2385T>C on VWF levels.