vWF Gene Research Articles

Characterization and Expression of an In-Frame Exon 33-34 Deletion Causing Type 1 VWD

Introduction: In recent years, the role of copy number variation (CNV) in the pathogenesis of type 1 von Willebrand disease (VWD) has been extensively explored. Large cohort studies including the MCMDM-1VWD and French studies have identified a total of eight partial VWF gene deletions, involving single and multiple exons, as well as one duplication. Furthermore, breakpoints have been identified within the MCMDM-1VWD cohort study for deletions involving exons 3, 32-34 and 33-34.Aim: To characterize and determine heterozygous deletion breakpoints of a deletion within the Zimmerman Program for the Molecular and Clinical Biology of VWD (ZPMCB-VWD) cohort, previously identified through array CGH analysis and to determine the pathogenesis of the deletion through in vitro analysis.Methods: Identification of deletion breakpoints utilized a deletion-specific multiplex PCR approach, whereby two forward primers and one reverse primer were designed to determine the heterozygous exon deletion, based on previous optimization within the MCMDM-1VWD cohort. VWF antigen (VWF:Ag), VWF propeptide (VWFpp), and multimer distribution were performed in a central laboratory. Recombinant exon 33-34 deletion mutant (rVWFdel33-34) was created using site directed mutagenesis. Recombinant vectors were transiently transfected into HEK293T cells and Renilla used as a transfection control. Conditioned media and cellular lysates were collected 48hr post-transfection and analysed by VWF:Ag ELISA, comparing to wild-type VWF (rVWFwt) expression.Results: Three families enrolled in the ZPMCB-VWD displayed a deletion of VWF exon 33-34. The deletion was determined to be 3.4kb in size and led to an in-frame loss of exons 33-34, with breakpoints being identical to those identified in the MCMDM-1VWD study (c.5620+872_5842+2440delinsGCAGCATAAGCATAAAGC). VWFpp/VWF:Ag ratios were elevated in all affected family members with the exon 33-34 deletion (mean 4.3) as opposed to unaffected family members (mean 1.3). These results are consistent with findings in the MCMDM-1VWD family (mean VWFpp/VWF:Ag of 4.8), suggesting increased clearance of VWF. In silico analysis of the breakpoint region suggested no significant repetitive elements and microhomology mediated end-joining was hypothesised as the potential mechanism leading to the deletion. Expression of recombinant mutant rVWFdel33-34 revealed that secretion reduced by 52% in the homozygous state (p<0.0001) when compared to rVWFwt expression. The deletion also resulted in a significant 35% reduction in secretion in the heterozygous state (p<0.0001). However, no intracellular retention was observed in either mutant state.Conclusion: Analysis of the exon 33-34 deletion breakpoint revealed no highly homologous repetitive sequences but there was a DNA insertion of 18 bp at the deletion breakpoint. In vitro expression of the exon 33-34 deletion suggests that there is a moderate effect on VWF secretion into conditioned media, without increased intracellular retention. The exon 33-34 deletion leads to the loss of nine cysteine residues. Inter- and intra- disulphide bonding between cysteine residues within VWF monomers and multimers is known to be functionally important in facilitating correct VWF structural orientation and folding within D4-CK domains. Loss of these cysteines may explain the moderate reduction in VWF secretion and potentially reduced plasma survival of the variant VWF. DisclosuresMontgomery:Immucor: Patents & Royalties.

Molecular Genetic Analysis in Patients with a Phenotype-Based Putative Diagnosis of Von Willebrand’s Disease Type 1

The conclusive diagnosis of von Willebrand’s disease (VWD) type 1 represents a challenging task. In addition to repeatedly performed phenotypic assessment, genetic analysis of the VWF gene may help establish the diagnosis. The aim of the present study was to assess the contribution of molecular genetic analysis in confirming the diagnosis in cases of suspected VWD type 1. Twenty-one patients (18 females, 3 males) with bleeding tendency were preliminary classified as likely or possible VWD type 1 based on phenotypic assays (VWF:Ag, VWF:RCoF < 50 % or 51-59 %, respectively) and further investigated by direct sequencing and MLPA analysis of the VWF gene. Ten different heterozygous mutations were identified in eleven patients (52 % confirmation rate). The mutation Y1584C (exon 28) was found in two unrelated patients. Most patients (9 of 11) demonstrated missense mutations in exons 20, 21, 28, 45 and 49. Newly identified mutations were K2566N (exon 45) and a 15 bp insertion predicting L168insX (exon 5). Large deletions and duplications were not detected. The results confirm the varying molecular pathology underlying VWD type 1. Molecular genetic analysis represents a useful approach to conclusively establish the diagnosis of VWD type 1 in approximately 50 % of patients with a phenotype-based preliminary diagnosis. DisclosuresNo relevant conflicts of interest to declare.

Phenotypic and Genotypic Correlations in 218 Patients with VWD Including Platelet Function

▪Objectives: In the past, progress in the diagnosis of von Willebrand Disease (VWD) due to more advanced molecular techniques allowed the first phenotype-genotype correlations. In contrast, improvement of methods for phenotypic description has not developed comparably. Therefore, diagnosis of VWD remains difficult because the relationship between laboratory assays and clinical presentation remains uncertain. Here, we present genetic results combined with laboratory and clinical data of patients with various types of VWD.Methods: Long-range PCRs combined with sequencing were used to completely sequence VWF exons and exon-intron flanking regions. Laboratory investigations included Factor VIII:C, VWF antigen, VWF GP Ib-binding, PFA-200 closure time, VWF collagen binding, multimeric analysis, a standardized bleeding score and blood group. In addition, CRP and BMI were analyzed to assess environmental confounders. Closure time measured by PFA-200 was correlated with the clinical type of VWD, VWF domains carrying mutations, e.g. A1 domain which is known to play a role in VWF-collagen-interaction (YEE, A. & KRETZ, C., 2014) and those missense mutations which occurred in our cohort, respectively.Results: Molecular analysis of the VWF gene in 218 patients with VWD has led to the identification of 1350 exonic missense sequence variations, 936 were heterozygous and 414 homozygous. Furthermore, we found 1114 silent sequence variations (761 heterozygous and 353 homozygous). Numerous intronic variants were detected, in addition. High allele frequencies of sequence variation were detected in exon 13, 18, 20, 28, 35 and 42, respectively. We identified a Thr1381Ala mutation in 162 patients, Ser1378Phe mutation (n=15), Arg1379Lys mutation (n=12) and in 6 patients we detected Arg1399His mutation. These residues are located on VWF A1 domain coding for a collagen binding region.In the total group, mean PFA-Coll/ADP was 111.9 ± 43.3 sec and mean PFA-Coll/Epi was 156.8 ± 53.0 sec. In VWD type 1-patients mean PFA-Coll/ADP was 115.6 ± 45.8 sec and mean PFA-Coll/Epi was 165.4 ± 56.8 sec and in patients with type 2N 99.9 ± 19.3 sec and 130.8 ± 22.9 sec. In patients with VWD type 3 mean PFA-Coll/ADP was 269.0 ± 43.8 sec and mean PFA-Coll/Epi was 292.5 ± 10.6 sec. The differences between these groups were highly significant (p≤ .001). By analysing the closure time according to the mutations in the collagen binding region the mean PFA-Coll/ADP in patients with Thr1381Ala was 112.3 ± 43.8 sec and the mean PFA-Coll/Epi was 163.5 ± 52.1 sec, in Ser1378Phe or Arg1379Lys mean PFA-Col/ADP was 114.1 ± 42.3 and mean PFA-Coll/Epi was 164.8 ± 46.8 sec. In Arg1399His mean PFA-Coll/ADP was 104.8 ± 25.7 sec and the mean PFA-Coll/Epi was 183.4 ± 63.1 sec showing the strongest prolongation. Further analyses with other frequent mutations will be presented, too.Conclusion: Here, we present a large number of newly described mutations in various regions of the VWF gene including mutations in the collagen binding region of domain A1. Although some mutations are located in the collagen binding region which is believed to promote binding to collagens type IV and VI patients showed prolonged closure time using the PFA-200 Collagen/Epinephrine cartridge which works with collagen type I but not cross reacting to collagens type IV or VI. Furthermore, even when a comprehensive panel of assays including genetic analysis is used both diagnosis as well as classification of VWD remain uncertain in some cases. Thus, further investigation of phenotypic genotypic correlations in VWD is warranted to better understand the VWF molecule. DisclosuresNo relevant conflicts of interest to declare.

New insights into genotype and phenotype of VWD

AbstractRecent advances in VWD research have improved our understanding of the genotype and phenotype of VWD. The VWF gene is highly polymorphic, with a large number of sequence variations reported in healthy individuals. This can lead to some difficulty when attempting to discern genotype–phenotype correlations because sequence variations may not represent disease. In type 1 VWD, mutations can be found throughout the VWF gene, but likely pathogenic sequence variations are found in only ∼2/3 of type 1 VWD patients. Sequence variations in type 2 VWD are located in the region corresponding to the defect in the VWF protein found in each type 2 variant. In type 3 VWD, sequence variations are not confined to a specific region of the VWF gene and also include large deletions that may not be picked up using conventional sequencing techniques. Use of genetic testing may be most helpful in diagnosis of type 2 VWD, in which a larger number of known, well characterized mutations are present and demonstration of one of these may help to confirm the diagnosis. Bleeding symptoms in general are more severe with decreasing VWF levels and more severe in type 2 and type 3 VWD compared with type 1 VWD. Prediction of phenotype for an individual patient, however, is still difficult, and the addition of genetic data will be most helpful in ascertaining the correct diagnosis for VWD patients.

New insights into genotype and phenotype of VWD.

Recent advances in VWD research have improved our understanding of the genotype and phenotype of VWD. The VWF gene is highly polymorphic, with a large number of sequence variations reported in healthy individuals. This can lead to some difficulty when attempting to discern genotype-phenotype correlations because sequence variations may not represent disease. In type 1 VWD, mutations can be found throughout the VWF gene, but likely pathogenic sequence variations are found in only ∼2/3 of type 1 VWD patients. Sequence variations in type 2 VWD are located in the region corresponding to the defect in the VWF protein found in each type 2 variant. In type 3 VWD, sequence variations are not confined to a specific region of the VWF gene and also include large deletions that may not be picked up using conventional sequencing techniques. Use of genetic testing may be most helpful in diagnosis of type 2 VWD, in which a larger number of known, well characterized mutations are present and demonstration of one of these may help to confirm the diagnosis. Bleeding symptoms in general are more severe with decreasing VWF levels and more severe in type 2 and type 3 VWD compared with type 1 VWD. Prediction of phenotype for an individual patient, however, is still difficult, and the addition of genetic data will be most helpful in ascertaining the correct diagnosis for VWD patients.

Clinical and laboratory diagnosis of VWD.

VWD is the most common inherited bleeding disorder and is due to a deficiency and/or abnormality of VWF. VWD is inherited in an autosomal-dominant or autosomal-recessive pattern, but women are apparently more symptomatic. Three main criteria are required for correct diagnoses of VWD: (1) positive bleeding history since childhood, (2) reduced VWF activity in plasma, and (3) history of bleeding in the family. The bleeding score, together with baseline VWF levels and family history, have been proposed as more evidence-based criteria for VWD. Measurements of a reduced VWF activity in plasma are essential for the diagnosis of VWD; assays for the evaluation of the interactions between VWF and platelet glycoprotein Ib receptor with or without ristocetin, as well as VWF collagen binding, are currently in use. However, other tests such as VWF antigen, factor VIII, ristocetin-induced platelet agglutination, multimeric analysis, VWF propeptide, VWF/FVIII binding assay, and assessment of biological response to desmopressin are necessary to characterize VWD types. Levels of VWF activities <30 U/dL have been associated with a bleeding phenotype and the presence of mutations in the VWF gene.

Effect of Purification of Human Adipose-derived Mesenchymal Stem Cells on the Expression of vWF Cell Factor Under Chemical and Mechanical Conditions

Introduction: Human adipose-derived mesenchymal stem cells (hADSCs) are easily accessible in the body, and under appropriate conditions, they can be directed toward various phenotypes. Therefore, hADSCs have been considered as a potential cell source for tissue engineering applications. hADSCs are able to differentiate into endothelial cells which covers the interior surface of vessels, in vivo. CD271 is a surface marker for hADSCs and hADSCs purified based on this marker (ADSCs-CD271+) are more capable of differentiating into various cells such as endothelial cells. In the present work, ADSCs-CD271+ were exposed to endothelial growth factor (VEGF) in vitro, and using a perfusion bioreactor, the effect of shear stress (a mechanical signal that is naturally produced by blood flowing ), on their differentiation into endothelial cells was investigated. Materials and Method: hADSCs were isolated from fat tissue and cultured in DMEM containing 10% FBS. Then they were characterized in passage 2, using flow cytometery technique. CD271expression level was examined and flow cytometry using antibodies was used to separate ADSCs-CD271+ from (p2) ADSCs. ADSCs-CD271+ were cultured and characterized and their expression level was examined. ADSCs and ADSCs-CD271+ in passage 3 were infused into 2 mm-diameter-silicone tubes coated with collagen I. The four experimental groups included control group (cells exposed to DMEM + 10% FBS, for 7 days), chemical group (cells exposed to differentiation medium containing DMEM, 3% FBS and 50 ng/ml of VEGF, for 7 days), mechanical group (cells were in a designed perfusion bioreactor, under application of 4 dyn/cm2 of shear stress for 24 hours) as well as chemical- mechanical group. Real-Time PCR was used to quantify the expression level of vWF (the factor specifying endothelial cells) in the experimental groups. Results: in the current work, ADSCs showed 1% of CD271 marker, which was increased to 52% after purification. Evaluation of surface markers using flow cytometry confirmed the mesenchymal characteristics of both cell groups. According to the results from Real-time PCR, the chemical-mechanical group, the mechanical group and then the chemical group (in both purified and unpurified cells) showed the highest expression of vWF, respectively. The expression of vWF was generally higher in ADSCs-CD271+ groups compared ADSCs ones. Conclusion: The findings of this study imply that MSCs purification as well as combining the chemical and mechanical factors can improve vWF expression. Keywords: Adipose-derived mesenchymal stem cell, CD271,Differentiation, VEGF, shear stress, vWF gene.

Abstract 37: Secretion of von Willebrand Factor by Endothelial Cells Links Salt to Hypercoagulability and Thrombosis

Hypercoagulability increases the risk of thrombi that cause cardiovascular events. Dehydration and hypernatremia are often accompanied by thrombosis, but the mechanisms are not clear. Von Willebrand Factor is secreted by endothelium, affecting aggregation of platelets and promoting activation of the coagulation cascade and formation of thrombi. Here we show that in culture of primary Human Umbilical Vein Endothelia Cells, elevating medium osmolality to 320-380 mosmol/kg by adding NaCl reversibly increases both vWF mRNA and vWF secretion. The high NaCl increases expression of tonicity regulated transcription factor NFAT5 and its binding to promoter of vWF gene, suggesting that vWF upregulation is caused by hypertonic signaling. To elevate NaCl in vivo, we modeled mild dehydration, subjecting mice to water restriction (WR) for 9 days by feeding them with gel food containing 30% of water. Such WR elevates blood sodium from 145.1±0.5 to 150.2±1.3 mmol/l and activates hypertonic signaling as evidenced from increased expression of NFAT5 in tissues. WR increased vWF mRNA in liver and lung and raised vWF protein in blood. Immunostaining of liver revealed increased production of vWF protein by endothelium and increased number of microthrombi inside capillaries. WR also increased blood level of D-dimer, a fibrinogen degradation product indicative of ongoing coagulation and thrombolysis. We conclude that elevation of extracellular sodium within the physiological range raises expression and secretion of von Willebrand Factor sufficiently to increase coagulability of blood and risk of thrombosis. The results suggest that hydration and salt intake are modifiable factors that affect coagulability and thrombosis through high salt-dependent secretion of vWF from endothelial cells.

Abstract 195: Characterization of DNA Modules in Regulating VWF Vascular Bed--Specific Expression

Expression of von Willebrand Factor (vWF) in the endothelium is regulated by vascular bed-specific pathways. A region of the human vWF gene between -843 and -620 was previously shown to direct expression in the endothelium of capillaries and a subset of larger blood vessels in the heart and skeletal muscle. Here, our goal was to further delineate the responsible DNA sequences. A series of constructs containing 5’-deletions, internal deletions or point mutations of the vWF promoter coupled to LacZ were targeted to the Hprt locus of mice using homologous recombination, and the resulting animals were analyzed for reporter gene expression. The findings demonstrate that a three-base-pair fragment within vWF promoter is necessary for expression in capillary but not large vessel endothelium in heart and skeletal muscle. Although the sequence does not conform to established DNA binding sites, it bound to nuclear protein as determined by elecrophoretic mobility shift assay. Mass spectrometry of the DNA-protein complex revealed several potential binding transcription factors. The heart- and skeletal muscle-specific promoter region did not response to shear stress or mechanical stress. Bisulfite sequencing revealed differential methylation of the human vWF promoter in endothelial cells from expressing and non-expressing vascular beds. These differences were altered by the mutation at the short cis-element. Together, these data support a model of modular gene regulation in which distinct DNA sequences in the vWF promoter direct expression in different vascular beds.

Each flying fox on its own branch: A phylogenetic tree for Pteropus and related genera (Chiroptera: Pteropodidae)

Pteropodidae is a diverse Old World family of non-echolocating, frugivorous and nectarivorous bats that includes the flying foxes (genus Pteropus) and allied genera. The subfamily Pteropodinae includes the largest living bats and is distributed across an immense geographic range from islands in East Africa to the Cook Islands of Polynesia. These bats are keystone species in their ecosystems and some carry zoonotic diseases that are increasingly a focus of interest in biomedical research. Here we present a comprehensive phylogeny for pteropodines focused on Pteropus. The analyses included 50 of the ∼63 species of Pteropus and 11 species from 7 related genera. We obtained sequences of the cytochrome b and the 12S rRNA mitochondrial genes for all species and sequences of the nuclear RAG1, vWF, and BRCA1 genes for a subsample of taxa. Some of the sequences of Pteropus were obtained from skin biopsies of museum specimens including that of an extinct species, P. tokudae. The resulting trees recovered Pteropus as monophyletic, although further work is needed to determine whether P. personatus belongs in the genus. Monophyly of the majority of traditionally-recognized Pteropus species groups was rejected, but statistical support was strong for several clades on which we based a new classification of the Pteropus species into 13 species groups. Other noteworthy results emerged regarding species status of several problematic taxa, including recognition of P. capistratus and P. ennisae as distinct species, paraphyly of the P. hypomelanus complex, and conspecific status of P. pelewensis pelewensis and P. p. yapensis. Relationships among the pteropodine genera were not completely resolved with the current dataset. Divergence time analysis suggests that Pteropus originated in the Miocene and that two independent bursts of diversification occurred in the Pleistocene in different regions of the Indo-Pacific realm.

The UK National External Quality Assessment Scheme for Heritable Bleeding Disorders

Molecular genetic analysis of families with hemophilia and other heritable bleeding disorders is a frequently requested laboratory investigation. In the United Kingdom, laboratories undertaking genetic testing must participate in a recognized external quality assessment scheme for formal accreditation. The UK National External Quality Assessment Scheme (UK NEQAS) for heritable bleeding disorders was established in its current format in 2003, and currently has 27 registered participants in the United Kingdom, the European Union (EU), and the non-EU countries. Two exercises per annum are circulated to participants comprising either whole blood or DNA isolated from cell lines, and laboratories are allowed 6 weeks to analyze the samples and generate a report. Reports are assessed by a panel comprising clinicians and scientists with expertise in this area. Samples to date have involved analysis of the F8 gene (10 exercises), the F9 gene (4 exercises), and the VWF gene (3 exercises) and have comprised a wide spectrum of mutations representing the routine workload encountered in the molecular genetics laboratory. The majority of laboratories in each exercise passed, but a small number did not and reasons for failing included clerical errors, genotyping inaccuracies, and a failure to correctly interpret data. Overall we have seen an improvement in quality of reports submitted for assessment, with a more concise format that will be of value to referring clinicians and counsellors. Informal feedback from participants has been very positive.

Left atrial endocardial dysfunction and platelet activation in patients with atrial fibrillation and mitral stenosis

This study demonstrated left atrial endocardial dysfunction and platelet activation in patients with atrial fibrillation and mitral stenosis. Study included 80 patients with mitral stenosis and atrial fibrillation (40 each with and without left atrial thrombosis), 15 healthy volunteers, and 10 left atrial appendage (LAA) specimens from donor hearts. Blood samples were collected through peripheral vein and left atrium, with peripheral blood samples of volunteers as controls. LAA specimens were collected during operations. LAA expressions of von Willebrand factor (vWF) and P-selectin were determined immunohistochemically; plasma concentrations were measured by enzyme-linked immunosorbent assay. LAA expressions of vWF and P-selectin genes in were quantitated with real-time fluorescent quantitative polymerase chain reaction. The difference in vWF and P-selectin plasma levels between left atrial and peripheral venous blood was not significant; however, peripheral plasma levels of vWF and P-selectin were significantly higher in those with thrombosis than without thrombosis, which in turn were higher than in healthy subjects. Both vWF and P-selectin proteins were stained in both left atrial endocardium and cardiomyocytes. The normalized vWF gene expression relative to control was 3.04 in patients with thrombosis and 2.16 in those without thrombosis (P<.01). The difference in P-selectin gene expressions among the groups was not significant. No differences were observed in plasma levels of vWF and P-selectin between left atrial and peripheral venous blood. Over expression of vWF gene in LAA may contribute to increased plasma vWF levels. P-selectin and vWF together may play a role in thrombosis.

SNP Analysis Of The VWF GENE Identifies Multiple Common Variants THAT Affect VWF Levels and OCCUR At Different Frequencies In Patients With TYPE 1 VWD

von Willebrand disease (VWD) type 1 is characterized by incomplete penetrance and variable expressivity. Individuals with very low levels of VWF (< 20 IU/dl) typically have mutations in the VWF gene. However, no such mutations can be identified in a significant subset of type 1 VWD patients, particularly those with VWF levels > 30 IU/dL. ABO is the best-characterized genetic modifier of VWF levels, accounting for up to 30% of the genetic variability and blood type O is overrepresented in patients with VWD type 1. VWF is highly polymorphic with over 1,100 reported single nucleotide variants (SNVs). The impact of many of these changes remains unknown. We hypothesize that common variants in VWF affect VWF levels and may play a role in the diagnosis of VWD type 1. We genotyped 94 tagging SNPs through VWF in a large Amish family (n=445) and study their association with VWF levels. We performed single SNP association tests using age, gender and ABO status as covariates and multiple testing corrections were implemented. We observed significant associations (p value ≤ 0.0005) between VWF levels and twelve tagging SNPs: rs2362479 (telomeric to VWF), rs10849363 and rs7964554 (intron 4); rs216308 (intron 24); rs216310 and rs216311 (exon 28); rs11612370 and rs11612384 (intron 33); rs216330, rs11064003, and rs2239162 (intron 37); and rs216293 (intron 38). To replicate the association of these SNPs with VWF levels in a larger Caucasian population, we conducted association analyses in three population-based cohorts of healthy individuals: 1) the Zimmerman Program for Molecular and Clinical Biology of VWD (ZPMCB-VWD) control cohort (n=150), 2) the Genes and Blood-Clotting Study (GABC) cohort (n=934) and 3) the Trinity Student Study (TSS) cohort (n=2,145). All association analyses were modeled including age, gender, rs687289 (a proxy marker of blood group O) and adjusted for multiple testing. Three SNP associations were replicated in the ZPMCB-VWD control cohort. An additional four SNP associations were replicated in the ZPMCB-VWD control and TSS cohorts. Associations of the intron 37 variants rs11064003 and rs2239162 with VWF levels were replicated in all cohorts and reached genome wide significance with p values of 1.34 x 10 -11 and 7.35 x 10 -15 respectively in the TSS cohort. We then genotyped the 12 candidate SNPs that showed significance in the original family in a cohort of 310 Caucasian VWD type 1 index cases (ZPMCB-VWD). We compared the minor allele frequency (MAF) of the tagging SNPs between the ZPMCB-VWD control cohort (n=150) and the ZPMCB-VWD type 1 index case cohort (n=310).The minor allele of rs7964554 (associated with lower VWF levels) was more common (46.2%) in patients with VWD type 1 than in controls (34.0%), and that difference was statistically significant (p=0.0005). The minor alleles of rs11612370, rs11612384, rs11064003, and rs2239162 (all associated with higher VWF levels) were less common (p=0.018, 0.009, 0.012, and 0.0004, respectively) in VWD type 1 index cases compared to controls. In this population if the subject has VWD type 1, then the odds of being heterozygous for the minor allele of rs7964554 were 1.47 (95% CI: 1.08, 2.00; p=0.014). Similarly, the odds of being heterozygous for the minor allele of rs2239162 were 0.64 (95% CI: 0.45, 0.91; p=0.014). In summary we have identified several common variants in VWF that have a strong effect on VWF levels and more importantly are either under or over represented in patients with VWD type 1 indicating that may influence its diagnosis. Disclosures:Shapiro:Baxter: Consultancy, Global steering committees Other, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novo Nordisk: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bayer: Global steering committees, Global steering committees Other, Membership on an entity's Board of Directors or advisory committees, Research Funding; Inspiration: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; CSL Behring: Research Funding; Biogen Idec: Research Funding. Di Paola:CSL Behring: Consultancy; Pfizer: DSMB, DSMB Other.

Next‐generation sequencing study finds an excess of rare, coding single‐nucleotide variants of ADAMTS13 in patients with deep vein thrombosis

The considerable genetic predisposition to deep vein thrombosis (DVT) is only partially accounted for by known genetic risk variants. Rare single-nucleotide variants (SNVs) of the coding areas of hemostatic genes may explain part of this missing heritability. The ADAMTS13 and VWF genes encode two interconnected proteins with fundamental hemostatic functions, the disruption of which may result in thrombosis. To study the distribution and burden of rare coding SNVs of ADAMTS13 and VWF found by sequencing in cases and controls of DVT. The protein-coding areas of 186 hemostatic/proinflammatory genes were sequenced by next-generation technology in 94 thrombophilia-negative patients with DVT and 98 controls. Gene-specific information on ADAMTS13 and VWF was used to study the association between DVT and rare coding SNVs of the two genes. More than 70billion base pairs of raw sequence data were produced to sequence the 700-kb target area with a median redundancy of ×45 in 192 individuals. Most of the 4366 SNVs identified were rare and non-synonymous, indicating pathogenetic potential. Rare (frequency of <1%) and low-frequency (<5%) coding SNVs of ADAMTS13 were associated with DVT (prevalence 17% vs. 4%; odds ratio [OR]4.8 and 95% confidence interval [CI]1.6-15.0 for rare coding; prevalence 36% vs. 23%, OR1.9 and 95% CI1.0-3.5 for low-frequency coding). Patients with rare coding SNVs of ADAMTS13 had lower plasma levels of ADAMTS-13 activity than patients without them. SNVs of VWF were not associated with DVT. We found an excess of rare coding SNVs of the ADAMTS13 gene in patients with DVT.

Cytokine-induced Chromatin Modifications of the Type I Collagen Alpha 2 Gene during Intestinal Endothelial-to-Mesenchymal Transition

Fibrosis of the intestine is currently an irreversible complication of inflammatory bowel disease; yet, little is understood of the underlying pathogenesis and antifibrotic strategies remain elusive. To develop effective therapies, knowledge of the mechanism of transcription and excessive deposition of type I collagen, a hallmark of fibrosis, is needed. We have shown previously that endothelial-to-mesenchymal transition (EndoMT) contributes to the pool of intestinal fibrotic cells and that a cytokine cocktail (interleukin 1-β, tumor necrosis factor α, and transforming growth factor β) induces collagen I alpha 2 (COL1A2) mRNA and protein. Chromatin immunoprecipitation assays on pure cultures of human intestinal mucosal endothelial cells undergoing EndoMT were performed with antibodies to specific histone modifications and RNA polymerase II. Reverse transcriptase-PCR was used to quantify the levels of Col1A2 and endothelial-specific von Willebrand factor (vWF) mRNA. We showed that cytokines induce selective chromatin modifications (histone 4 hyperacetylation, and hypermethylation of histone 3) and phosphorylated RNA polymerase II at the COL1A2 promoter. Hypoacetylated and hypomethylated histone 3 was detected on the repressed vWF gene. Prolonged exposure to cytokines (16 days) retained hyperacetylation of select lysines in H4 on the COL1A2 promoter. Removal of cytokines after 16 days and continued culture for 10 days showed persistent hyperacetylation at lysine 16 in histone H4. This is the first study to show that COL1A2 gene expression is associated with cytokine-induced, temporally ordered, and persistent chromatin modifications and suggests that these are important determinants of gene expression in EndoMT and intestinal fibrosis.

The 1000 Genomes Project: paving the way for personalized genomic medicine.

The 1000 Genomes Project: paving the way for personalized genomic medicine.

Spatial and Temporal Aspects of Occurrence ofMogeraSpecies in the Japanese Islands Inferred from Mitochondrial and Nuclear Gene Sequences

We assessed dispersal and vicariant events in four species of Japanese moles in the genera Mogera and Euroscaptor to better understand the factors shaping intra- and interspecific differentiation in Japanese moles. We used the combined viewpoints of molecular phylogeny and historical geology using nucleotide sequences of mitochondrial (cytochrome b; Cytb) and nuclear (A2ab, Bmp4, Tcf25, vWf) genes. The divergence times estimated from the molecular data were verified with available geological data on the chronology of fluctuations in sea level in the Korea Strait, assuming sequential migration and speciation events. This produced possible migration times of 5.6, 3.5, 2.4, and 1.3 million years ago for four species of Japanese moles, Euroscaptor mizura, Mogera tokudae, M. imaizumii, and M. wogura, respectively. For the western Japanese mole M. wogura, Cytb sequences revealed four major phylogroups with strong geographic affinities in southwestern Central Honshu (I), western Honshu/Shikoku (II), Kyushu/westernmost Honshu (III), and Korea/Russian Primorye (IV). The nuclear gene sequences supported the distinctiveness of phylogroups I and IV, indicating long, independent evolutionary histories. In contrast, phylogroups II and III were merged into a single geographic group based on the nuclear gene data. Intraspecific divergences in M. imaizumii and M. tokudae were rather apparent in Cytb but not in nuclear gene sequences. The results suggest that repeated dispersal events have occurred between the Asian continent and the Japanese Islands, and intensive vicariant events associated with abiotic and biotic factors have created higher levels of species and genetic diversities in moles occurring on the Japanese Islands.

Changes of plasma vWF level in response to the improvement of air quality: an observation of 114 healthy young adults

Plasma von Willebrand factor (vWF) is an important factor involving in hemostasis and various cardiovascular diseases. Air pollution is related to many respiratory and cardiovascular diseases. During the Olympic Games Beijing 2008 period (August 8 to September 17, 2008) when air quality in Beijing improved greatly, we studied the relationship between plasma vWF level and the factors of air pollution index (API), ABO blood group, and polymorphisms in vWF gene in healthy young adults. We recruited 114 healthy medical students. In a period of more than 4 months around the period of Olympic Games Beijing 2008, six blood samples at stages 1 and 2 (before Olympic Games), stages 3 and 4 (during Olympic Games), and stages 5 and 6 (after Olympic Games) were taken from every participant for the measurement of plasma vWF level and genotyping of three SNPs (rs7954855, rs7965413, and rs216311) in vWF gene. Daily air pollution index near their living places was obtained from the officially published data. The average API began to decrease from stage 2, reached to nadir in stages 3 and 4, and increased but was still lower in stages 5 and 6. Plasma vWF decreased during the experimental period in all participants. The average plasma vWF decreased from stage 2 and remained lower in stages 3-6. vWF level varied greatly among the participants (from 30 to 170 %) but decreased proportionately when we analyzed their levels individually. Participants with O blood type had lower plasma vWF level than those with A, B, and AB blood types. Those with the SNP in vWF gene causing homozygous threonine at codon 1381 had lower plasma vWF level than those with homozygous alanine or heterozygous alanine/threonine. In the 114 normal individuals, the average plasma vWF level decreased during the period of Olympic Games Beijing 2008 when air quality improved greatly. This suggests that control of air pollution may be useful to prevent some diseases such as cardiovascular diseases.

Rare Coding Single Nucleotide Variants of ADAMTS13 Are Associated with Deep Vein Thrombosis in a Next-Generation Sequencing Association Study

AbstractAbstract 107The considerable genetic predisposition to deep vein thrombosis (DVT) is only partially accounted for by known genetic risk variants. Genome-wide association studies investigating hundreds of thousands of common single nucleotide variants (SNVs) identified a number of associations, but missing heritability remains. Rare (i.e. minor allele frequency below 1%) and low-frequency (i.e. minor allele frequency below 5%) SNVs of the coding area may be responsible for at least part of this missing heritability. In order to investigate this, we sequenced 700,000 base pairs of genomic DNA including the protein coding exons and intron-exon boundaries of 186 hemostatic/pro-inflammatory genes. Indexed genomic DNA libraries were co-captured on NimbleGen HD2 2.1M-probe chips and capture products were sequenced on SOLiD 4 platforms. More than 70 billion base-pairs of raw sequence data were produced to sequence the target area with a median redundancy of 45X in 94 thrombophilia-negative patients with DVT and 98 controls. Most of the 4366 SNVs identified were rare, novel and nonsynonymous indicating pathogenetic potential. We tested the association of coding SNVs in the ADAMTS13 and VWF genes, encoding two interconnected proteins with fundamental roles in hemostasis. Sequencing of the two genes yielded 109 variants, 108 SNVs and a c.8241_8442del frameshift deletion in exon 51 of VWF. Being a carrier of rare coding (prevalence in DVT: 17% [n=16]; prevalence in controls 4% [n=4]; odds ratio [OR]: 4.8; 95% confidence interval [CI]: 1.6–15.0), rare nonsynonymous (prevalence in DVT: 11% [n=10]; prevalence in controls 3% [n=3]; OR: 3.8; 95% CI: 1.0–14.2) or low-frequency coding (prevalence in DVT: 36% [n=34]; prevalence in controls 23% [n=23]; OR: 1.9; 95% CI: 1.0–3.5) SNVs of ADAMTS13 was associated with DVT. Carrying rare or low-frequency SNVs of VWF was not associated with DVT. The 11 different rare missense variants of ADAMTS13 found in DVT patients had never been described in association with congenital thrombotic thrombocytopenic purpura. Patients carrying at least one ADAMTS13 mutation of the categories associated with DVT had lower plasmatic levels of ADAMTS13 activity compared to patients without mutations. The change in ADAMTS13 activity was −7% (95% CI: −24 to 10%) for patients with rare coding ADAMTS13 mutations, −12% (95% CI: −30 to 6%) for patients with rare nonsynonymous mutations and −29% (95% CI: −52 to −6%) for patients with missense mutations predicted to be damaging for protein function by SIFT. Our results uncover for the first time a link between ADAMTS13, an important regulator of hemostasis implicated in microvascular thrombosis, and DVT. Disclosures:No relevant conflicts of interest to declare.

Intracellular Retention, Enhanced Clearance, and Defective FVIII Binding Are Common Features of Von Willebrand Factor D'-D3 Domain Mutations in Patients with Von Willebrand Disease Type 1 From the European Mcmdm-1VWD Study

AbstractAbstract 99 Background:Von Willebrand disease (VWD) type 1 is characterized by a partial reduction of structurally and functionally normal VWF with normal VWF multimers. As part of a large European study (Molecular and Clinical Markers for the Diagnosis and Management of Type 1 von Willebrand Disease (MCMDM-1VWD) patients previously diagnosed with VWD type 1 were studied systematically to assess the phenotypic and genotypic spectrum. Objective:To confirm the pathogenicity of VWF gene mutations and to elucidate the molecular mechanisms of VWD type 1. Patients and methods:VWD type 1 patients were recruited by twelve expert centers in nine European countries. VWF genotyping was performed in all index cases (IC). The eight mutations studied here are located in the VWF D'-D3 domain and corresponded to 57 patients from 19 families. They were reproduced by recombinant expression with subsequent phenotypic characterization, two of them in cis and one in trans with a second mutation. Results and Discussion:Intracellular VWF:Ag of all mutants was normal or near normal suggesting normal expression levels. However, seven mutations (p.M771I, p.I1094T, p.C1130R, p.C1130G, p.C1130F, p.W1144G and p.Y1146C) caused intracellular retention and impaired VWF secretion. In addition, we observed a major loss of high molecular weight multimers as in type 2A and a novel finding of a severe VWF:FVIII binding defect in most of the homozygously expressed mutants. Additional mutations either in cis or in trans had no modifying effect. The recombinant VWD type 1 Vicenza mutation p.R1205H with or without the allelic variant p.M740I seen in three Italian IC was secreted normally and had normal function leaving enhanced clearance of mutant VWF as the only pathomechanism. In conclusion, the majority of mutations in the D3 domain impair VWF multimerization, cause intracellular retention and correlate with defective FVIII binding. An elevated ratio of VWF propeptide to VWF:Ag suggests enhanced VWF clearance as an important pathomechanism of most mutations and particularly of p.R1205H. Disclosures:No relevant conflicts of interest to declare.