The Prevalence of Reduced VWF Survival Phenotype (Type 1C VWD) in Type 1 Index Cases Recruited into the TS Zimmerman Program for the Molecular and Clinical Biology of VWD (ZPMCB - VWD).

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2133-2133 ◽  
Author(s):  
Sandra L. Haberichter ◽  
P.A. Christopherson ◽  
C.L. Perry ◽  
A. Lee ◽  
P.A. Morateck ◽  
...  
Keyword(s):  
Normal Distribution ◽  
Half Life ◽  
Treatment Strategies ◽  
Essentially Normal ◽  
Normal Ranges ◽  
Bleeding Score ◽  
Clinical Biology ◽  
Index Cases ◽  
Normal Controls

Abstract The decreased survival of von Willebrand factor (VWF) in plasma has recently been identified as a novel mechanism causing type 1 VWD. Patients with this phenotype are characterized by a substantially reduced plasma VWF:Ag (< 30 IU/dL), a robust response to DDAVP, and a reduced half-life of VWF:Ag after DDAVP administration. Although a normal distribution of plasma VWF multimers appears to be present, the multimer satellite bands are decreased in intensity. A few mutations have been identified in type 1C VWD patients including C1130F/G/R, W1144G, R1205H, and S2179F. Identification of type 1 VWD patients with this phenotype is clinically important because DDAVP may not be the treatment of choice in this subset of patients due to the reduced half-life of the VWF released into plasma. Although previous studies have facilitated the characterization of this phenotype, the prevalence of the reduced survival, type 1C VWD has not yet been determined. We have previously reported that assay of plasma VWF:Ag and VWF propeptide (VWFpp) can be used to identify type 1C VWD patients having reduced VWF survival. These individuals have a substantially increased ratio of steady-state plasma VWFpp/VWF:Ag in contrast to normal individuals where the ratio is equal to one. We subsequently established blood group-specific normal ranges for VWFpp/VWF:Ag by assay of 279 healthy blood donors. Here we report the assay of VWF:Ag and VWFpp in 192 normal controls and 61 type 1 VWD index cases recruited from eight US centers within the TS Zimmerman Program for the Molecular and Clinical Biology of VWD (ZPMCB - VWD) study. VWD type 1C individuals previously reported from our center were excluded from the current study. Using VWF:Ag and VWFpp levels determined for the 192 normal controls, we established a 2-standard deviation normal range for VWFpp/VWF:Ag ratio of 0.5 to 1.6, consistent with our previous studies. Of the 61 type 1 VWD index cases, three individuals were identified with a substantially increased VWFpp/VWF:Ag ratio (mean = 5.0). One individual was found to have the well-characterized R1205H VWF gene mutation that has been associated with reduced VWF survival. These individuals demonstrated laboratory parameters consistent with a reduced VWF survival phenotype including moderate to severely decreased plasma VWF:Ag (mean = 16.6 IU/dL), a diminished intensity of multimer satellite bands amongst an essentially normal distribution of plasma multimers, and an elevated bleeding score (mean = 4.7). Together these data indicate a reduced survival phenotype (type 1C) in 4.9% of type 1 VWD index cases in the ZPMCB - VWD study. Identification of type 1 VWD individuals with a reduced plasma VWF survival phenotype is clinically essential as these individuals may require alternative treatment strategies.

The Complete Type I VWD Cohort Of The Zimmerman Program For The Molecular and Clinical Biology Of VWD - Phenotypic Assignment, Mutation Frequency, and Bleeding Assessment

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 332-332 ◽  
Author(s):  
Robert R. Montgomery ◽  
Pamela Christopherson ◽  
Daniel B. Bellissimo ◽  
Joan Cox Gill ◽  
Sandra L. Haberichter ◽  
...  
Keyword(s):  
Family Members ◽  
Normal Range ◽  
The Usa ◽  
Abnormal Bleeding ◽  
Bleeding Score ◽  
Clinical Biology ◽  
Severe Type ◽  
Index Cases ◽  
Normal Controls

Abstract The Zimmerman Program for the Molecular and Clinical Biology of VWD (ZPMCBVWD) is an NIH Program Project for the study of VWD in the USA and collaboration with ongoing studies in Canada and the University of Sheffield. Study subjects were recruited from 9 Primary Clinical Centers and 21 Secondary Clinical Centers across the USA and had to have the prior diagnosis of VWD on an intention to treat basis. We recruited 651 index cases, 2017 family members, and 247 normal controls. Of the index cases 152 had type 2 or 3 VWD or other variants and not part of this report. The remaining 499 represents the now completed Type 1 VWD cohort. All 2915 study subjects underwent extensive laboratory testing including VWF:Ag, VWF:RCo, VWF multimeric analysis, FVIII activity, VWFpp, and VWF:CB (collagen binding), blood typing, and VWF linkage analysis. A detailed quantitative bleeding score (BS) was also performed on all 2915 individuals. Index cases had full length VWF sequencing with mutation confirmation and exclusion in family members. When indicated VWF:F8B (FVIII binding), VWF:PB (platelet binding to GPIb), VWF:IbCo (spontaneous binding to modified GPIb in absence of ristocetin), and repeat VWF testing in a second laboratory by different methods. When possible, historical laboratory results obtained at the time of initial diagnosis were obtained and entered into our Investig-8 Database. Based upon clinical laboratory studies and phenotypic assignment, this cohort was found to include 232 type 1 VWD (VWF:Ag or VWF:RCo <40 IU/dL including 66 type 1C or severe type 1 VWD); 93 low-VWF (LVWF with lab studies between 40 IU/dL and the lower end of the normal range); 119 type 1H (historical levels below the normal range but not substantiated in current testing); and 55 individuals that tested within the normal range and did not have historic levels that were low. Full-length VWF sequencing was performed on all index cases and sequence variations (SVs) were identified, and where possible, compared to clinical phenotypes reported in the Sheffield Database. SVs were identified in 53% of the Type 1 VWD cohort (excluding those that were normal on testing without abnormal historic results). Of the 232 Type 1 VWD with VWF levels <40, 74% had SVs. In further analysis of this group, 100% of severe type 1, and 85% of type 1C had SVs. Looking at this in the type 1 with VWF:Ag <40 IU/dL by level of VWF:Ag, 87% of those 1 with VWF:Ag of 2-10, 93% with VWF:Ag 11-20, 71% with VWF:Ag of 21-30, 67% with VWF:Ag of 31-40, and 52% with VWF:Ag of >40 had SVs. The milder phenotypes demonstrate SVs in 39% of the LVWF subjects and 30% of the Type 1H subjects. In the individuals entered into the study as VWD subjects that on central testing had normal levels of VWF on entry into the study and did not have an abnormal historic VWF determination, only 22% had SVs. A similar approach was undertaken to compare the quantitative BS using the ISTH Bleeding Assessment Tool. For this analysis, a score of 5 or greater was considered abnormal without regard to age or sex. Abnormal BS were identified in 63% of those with VWF:Ag of 2-10, 66% of those 11-20, 64% of those 21-30, 48% of those 31-40, and 58% of those >40. In LVWF subjects, 52% had abnormal BS and in Type 1H subjects 57% were abnormal. Based upon a similar laboratory analysis, all 2017 family members underwent phenotypic assignment and were found to either be affected or unaffected family members. 713 were phenotyped as being an affected family member and 1296 as unaffected family members. Abnormal bleeding scores were identified in 38% of affected family members and 19% of unaffected family members. This report summarizes some of the results on the completed Type 1 VWD cohort that was part of the ZPMCBVWD. All 2915 subjects (including index cases, family members, affected family members and normal controls) underwent extensive laboratory testing and phenotype assignment. Of the 499 subjects in the Type 1 VWD cohort, 325 had low VWF and 232 had VWF levels <40 IU/dL. Based on the drop of SV frequency, the level of VWF <40 may provide a possible demarcation to define VWD. In this study the frequency of abnormal bleeding score was not helpful in further defining this critical level for diagnosis - possibly because the bleeding score was ascertained at the entry into the study rather than at the time of diagnosis as done in earlier studies. Further analysis of this cohort can be expected to shed further understanding of the molecular and clinical biology of VWD. Disclosures: No relevant conflicts of interest to declare.

Novel VWF Sequence Variations Identified in Normal Controls and Index Cases Enrolled in the TS Zimmerman Program for the Molecular and Clinical Biology of VWD (ZPMCB-VWD).

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 709-709 ◽  
Author(s):  
Daniel B. Bellissimo ◽  
P.A. Christopherson ◽  
S.L. Haberichter ◽  
V.H. Flood ◽  
J.C. Gill ◽  
...  
Keyword(s):  
Normal Control ◽  
Normal Control Group ◽  
Control Group ◽  
Normal Individuals ◽  
Sequence Variations ◽  
Clinical Biology ◽  
Von Willebrand ◽  
Index Cases ◽  
Normal Controls

Abstract Von Willebrand disease (VWD) is caused by quantitative (types 1 and 3) and qualitative (type 2) defects in von Willebrand factor (VWF). The TS Zimmerman Program for the Molecular and Clinical Biology of VWD is a multinational Program Project established to further the study of VWD in the United States and to contrast these studies with the studies initiated previously in the EU and Canada. As one of the components of this study we sought further insight into the clinical expression and penetrance of established types of VWD by performing full gene DNA sequence analysis in VWD patients and normal controls. This report is an interim report of the first 50 index cases and 113 normal individuals recruited into this study. Twenty four of these index cases were found to have known mutations, four of which had a second new mutation, and 11 cases had 1 or 2 new mutations. In cases where mutations were identified, 46% of the identified mutations were new mutations that have not been reported in the Sheffield VWF Mutation Database. In 15 patients, no mutations were identified in the coding region, although analysis of the non-coding regions is still in progress. Five of the mutations were deletions, insertions, or nonsense mutations that have clear functional consequences. The other 12 mutations were missense mutations. Since VWF polymorphisms are not well characterized in all exons, we have also completed studies of the first 113 normal control individuals in our study. These are individuals without a bleeding history and in whom full VWF laboratory testing and VWF sequencing was also undertaken. Since some estimates in the EU and Canadian studies have determined the prevalence of VWF mutations varies by the severity of type 1 VWD patients, we wanted to determine the frequency of VWF variation in a normal population and determine if sequence variations correlate with VWF levels. There were three linked common polymorphims identified in normal African Americans that are discussed elsewhere and are not included in this present analysis. We found 19 new sequence variations in the normal control group of which three (2900G>A, 6554G>A, 7997C>T) were found individually in 4–6% of the normal control samples. In addition, in 12 normal control samples we identified 6 sequence variations that were previously reported as VWF mutations. Four were reported as type 1 mutations (2220 G>A, 3686T>G, 3692A>C, 6859C>T) and two as type 2N mutations (2451T>A, 2771G>A). The 2220G>A and 2451T>A mutations were seen in 6 normal controls (5%) and 5 of these 6 normal controls had both mutations. In another normal control, both 3686T>G and 3692A>C were identified. Although the reported prevalence of VWD is 1% or greater, the frequency of these mutations in our normal controls is higher than expected (as high as 5%). In our normal control group, the mean VWF:Ag concentration in the patients with polymorphisms/mutations did not differ from the normal control group as a whole and did not cluster on the lower end of the normal range. Thus, the data on our normal individuals suggest that VWF gene variation is considerable and that many mutations and polymorphisms remain to be identified. Differentiation of those that affect the diagnosis of VWD and/or hemorrhagic risk continues to be difficult.

Novel VWF Sequence Variations Identified in a Second Cohort of Index Cases Enrolled in the TS Zimmerman Program for the Molecular and Clinical Biology of VWD (ZPMCB-VWD).

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3379-3379
Author(s):  
Daniel B. Bellissimo ◽  
Pamela A. Christopherson ◽  
Sandra L. Haberichter ◽  
Veronica H. Flood ◽  
Joan Cox Gill ◽  
...  
Keyword(s):  
African Americans ◽  
Coding Region ◽  
New Mutation ◽  
Sequence Variations ◽  
Clinical Biology ◽  
Type 3 ◽  
Index Cases ◽  
Normal Controls ◽  
New Mutations

Abstract The TS Zimmerman Program for the Molecular and Clinical Biology of von Willenbrand disease (VWD) is a multinational Program Project established to further the study of VWD in the United States and to contrast these studies with the studies initiated previously in the EU and Canada. In order to gain further insight into the clinical expression and penetrance of established types of VWD, we performed full gene DNA sequence analysis on VWD patients and normal controls. Previously, we reported new sequence variations identified from 50 VWD index cases and 113 normal controls in our study. This is an updated report of the sequence variations identified in a second cohort of 44 index cases with type 1, 2 and 3 VWD and 48 normal controls. Fourteen of these index cases have known mutations, 3 of which also have a second new mutation. Seven additional index cases had 1 or 2 new mutations. Three cases had new polymorphisms identified in our first cohort. Thirteen new mutations were identified in type 1 and type 3 patients including 3 nonsense mutations, 2 insertions, and 8 missense mutations. In cases where mutations were identified, 48% of the identified mutations were new mutations that have not been reported in the Sheffield VWF Mutation Database. A similar frequency of new mutation was observed in our first cohort (46%). In 20 of 44 (45%) patients with either type 1 or type 3 VWD, no sequence variations were identified in the VWF coding region. In our previous cohort, sequence variations were not detected in 30% of patients. Mutations in the non-coding region of the gene or mutations not detectable by DNA sequencing have not been ruled out in this group of patients. Since VWF polymorphisms are not well characterized in all exons and in different ethnic groups, full VWF laboratory testing and gene sequencing of over 160 normal controls was completed in our study. In our first report of the 113 normal controls, we identified 19 new sequence variations that were found mainly in African Americans. In the second cohort of 48 normal controls, we identified 2 new sequence variations (1087C>T and 1463C>G). The decreasing number of new sequence variations in the normal controls is in contrast to the index cases where a similar percent of new mutations were detected in both cohorts and may indicate that the majority of polymorphisms have been identified. Seven of 19 new sequence variations seen in the first cohort of normal controls were also found in the second cohort further supporting that they are polymorphisms. Previously, we identified six sequence variations that were previously reported as VWF mutations. In this study, the same six sequence variations (2220G>A, 2451T>A, 2771G>A, 3686T>G, 3692A>C and 6859C>T) were detected in the normal controls providing further evidence that these sequence variations are most likely polymorphisms. In addition, we detected two other reported mutations (6187C>T; P2063S and 2561G>A; R854Q) each in two normal controls. Our data indicate that sequencing of large numbers of normal controls is important to aid in differentiating mutations from polymorphisms. This study emphasizes the importance of understanding the ethnic-specific sequence variations in African Americans such that polymorphisms are not misidentified as mutations. Our data also suggest that the genetic variation in the VWF gene is extensive and that many low frequency mutations and polymorphisms remain to be identified. Differentiating polymorphisms from disease-causing sequence variations that affect the diagnosis of VWD and/or hemorrhagic risk is important but continues to be challenging in this bleeding disorder.

Quantitative Measurement of VWF Binding to the Platelet Integrin αIIbβ3 - Abnormalities Identified in Individuals with VWD

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1107-1107
Author(s):  
Robert R. Montgomery ◽  
Veronica H Flood ◽  
Jieqing Zhu ◽  
Pamela A Christopherson ◽  
Peter J. Newman ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Conflicts Of Interest ◽  
Functional Screening ◽  
Integrin Αiibβ3 ◽  
Binding Ratio ◽  
Clinical Biology ◽  
The One ◽  
Index Cases ◽  
Normal Controls

Abstract Most studies of VWF function have focused on VWF interactions with platelet GPIb, FVIII, and specific sites and types of collagen. Early studies carried out by Ruggeri and Montgomery demonstrated that in addition to binding GPIb on platelets, VWF also bound to the GPIIb-IIIa receptor (also known as integrin αIIbβ3), but only in the absence of fibrinogen. Ruggeri and other groups have demonstrated that under flow, VWF interaction with GPIIb-IIIa is also critical to VWF-platelet biology. A large systematic study of VWF binding to GPIIb-IIIa (αIIbβ3) has not been undertaken previously in a cohort of type 1 and 2 VWD subjects. Studies were carried out on 619 VWD index cases (both type 1 and type 2 VWD) and 84 normal controls enrolled in the Zimmerman Program for the Molecular and Clinical Biology of VWD (ZPMCB-VWD) using a novel assay of αIIbβ3 binding of VWF. Zhu and coworkers expressed the soluble αIIbβ3 headpiece and previously characterized this in the open position when bound to ligand. This αIIbβ3 headpiece was used for our studies. An antigen capture ELISA was developed using the αIIbβ3 headpiece to capture VWF and to compare binding to the VWF:Ag as a ratio of VWF-αIIbβ3/VWF:Ag. ELISA plates were coated with the GPIIIa-specific monoclonal antibody (MAb), AP3 and used to capture the αIIbβ3 headpiece. Since fibrinogen blocks VWF binding to αIIbβ3, plasma samples were heated at 56°C to precipitate fibrinogen, and then centrifuged to remove the precipitate. The resulting heat defibrinated plasma was utilized in the assay. Bound VWF was quantified using biotin-labeled AVW15, a MAb to VWF. The reference was the ISTH SSC Plasma Standard. As specific positive and negative controls, we used wild-type recombinant VWF containing the normal RGDS sequence at position 2507-2510, and mutant VWF containing the RGDE sequence at the same position. As an additional control, the monoclonal antibody 7E3, known to block aggregation by binding to β3 was used to block αIIbβ3/VWF interactions. While binding of wt-VWF to αIIbβ3 was robust, no binding of the RGDE mutant was observed (<1% of wt). 7E3 completely inhibited binding of VWF to αIIbβ3. Interestingly, two other MAbs known to block the binding of Fg to αIIbβ3, AP2 and 10E5, did not inhibit VWF binding. The 84 normal controls had a mean VWF-αIIbβ3/VWF:Ag binding ratio of 0.87 with a 5% and 95% confidence limit of 0.53-1.39. 152 type 1 VWD (VWF <30IU/dL) samples had a modestly reduced binding ratio of 0.62 (0.36-0.85) with a significance of p<0.0001. The binding ratios of 55 type 2A and 41 2B VWD samples were markedly reduced with a ratio of 0.293 (0.12-0.67) and 0.40 (0.23-0.81) both with a significance of 0.0001. Since both of these latter VWF variants have abnormal VWF multimers, we tested recombinant D-pro VWF (no VWF propeptide present) and Y87S mutant VWF (absent VWF multimerization). No binding to αIIbβ3 was observed with VWF from either of these constructs. Among our ZPMCB-VWD index cases, we also had individuals with low VWF (VWF:Ag 30-50 IU/dL) that had an intermediate reduction between the normal controls and type 1 subjects. Type 2N VWD samples demonstrated no abnormal binding to αIIbβ3 and type 2M subjects had minimal differences. While VWF binding to αIIbβ3 has been recognized previously, it has not been systematically studied in subjects with VWD. We identified a modest decrease in αIIbβ3 binding of VWF in type 1 VWD and in clinical subjects with low VWF. Furthermore, type 2A and 2B subjects have a much more profound reduction in αIIbβ3 binding and suggest an importance of normal multimeric VWF to the functional binding of VWF to αIIbβ3. Mutating the RGDS sequence in VWF, or adding 7E3 to block αIIbβ3, abrogate this VWF binding. To date we have not identified any of our enrolled ZPMCB-VWD subjects with mutation of the RGDS sequence or in the C4 domain of VWF, although mutations in this region recently have been reported by Legendre et al. at the ISTH2015 meeting. Such mutations are not identified with current VWF functional screening assays, but specific assays of αIIbβ3-VWF interaction, such as the one described here, can be used and suggest there are specific potential mutations and variants with abnormal multimers that have abnormal αIIbβ3 binding. The qualitative and quantitative assessment of VWF function continues to be complicated. Disclosures No relevant conflicts of interest to declare.

Critical Importance Of VWF Propeptide (VWFpp) In The Diagnosis Of Type 1 Von Willebrand Disease (VWD)

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 331-331 ◽  
Author(s):  
Sandra L. Haberichter ◽  
Pamela A. Christopherson ◽  
Veronica H. Flood ◽  
Joan Cox Gill ◽  
Daniel B. Bellissimo ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Von Willebrand Disease ◽  
Promoter Polymorphisms ◽  
Normal Platelet ◽  
Bleeding Score ◽  
Clinical Biology ◽  
Type 3 ◽  
Severe Type ◽  
Index Cases

Abstract Type 1 VWD is the most common form of VWD and is characterized by quantitative deficiency of VWF. Mechanisms causing type 1 VWD include decreased VWF synthesis due to promoter polymorphisms, decreased VWF secretion with intracellular retention/degradation, and increased clearance of VWF from plasma (type 1C VWD). VWF and its propeptide (VWFpp) are released into plasma in equimolar amounts but have very different half-lives (8 – 12 hours and 2-3 hours, respectively). The assay of VWFpp can be used to assess VWF synthesis, secretion, and clearance. Reduced VWFpp level indicates reduced VWF synthesis or secretion. An increased VWFpp/VWF:Ag ratio is expected when VWF is cleared rapidly from plasma (type 1C VWD), while decreased VWF secretion/intracellular retention results in a normal ratio. We enrolled 502 type 1 VWD index cases with a pre-existing diagnosis of type 1 VWD through the Zimmerman Program for the Molecular and Clinical Biology of VWD. We confirmed 262 index cases as type 1 VWD (VWF:Ag or VWF:RCo ≤ 40 IU/dL). Of the confirmed type 1 VWD cases, 58 met the criteria for type 1C VWD with VWFpp/VWF:Ag ≥ 3 and VWF:Ag ≤ 30 IU/dL, and 12 met the criteria for “Type 1 – Severe” with VWF:Ag of 1 – 5 IU/dL and VWFpp/VWF:Ag < 3. Type 1C VWD comprised 22% of all type 1 VWD cases. The type 1C cohort included several individuals previously diagnosed as type 2A (11), type 2M (2), and “Unclassified” (3). The most significant reclassification involved 7 cases previously diagnosed as type 3 VWD – these patient had detectable VWF:Ag (2 – 6 IU/dL) and VWFpp (14 – 66 IU/dL), and elevated VWFpp/VWF:Ag (4.2 – 33.0). Although plasma VWF:Ag is very low in these patients, they might be expected to have normal platelet stores of VWF:Ag, unlike type 3 VWD patients. Type 3 VWD patients in our study had undetectable (<1 IU/dL) VWFpp and VWF:Ag. In fact, type 1C VWD comprised the majority of severe type 1 VWD cases. At very low VWF:Ag levels (2 – 10 IU/dL), 76% of index cases had a type 1C phenotype, while the remainder had normal VWFpp/VWF:Ag consistent with a decreased secretion phenotype. In the VWF:Ag of 11 – 20 IU/dL group, 38% were type 1C. At still higher VWF:Ag levels (21 – 30 IU/dL), only 7% had a type 1C phenotype and the majority had a decreased secretion phenotype. Additionally, 43% of the type 1C cohort had VWFpp levels below the normal range, indicating reduced synthesis/secretion is also involved in the mechanism causing type 1C VWD. Mutations associated with type 1C were located in the VWF D3, A1, and D4 domains while decreased secretion variants were located primarily in the D1, D2, and D3 domains. Somewhat surprisingly, no difference in bleeding score was identified between type 1C and other type 1 patients. We can conclude that a reduced VWF survival phenotype (type 1C) is very frequent in severe type 1 VWD patients with VWF:Ag < 20 IU/dL. Identification of type 1C VWD is important because the rapid clearance of VWF in these patients may preclude the use of desmopressin, which is commonly given in type 1 VWD, but may be insufficient to achieve hemostasis in type 1C patients. Disclosures: No relevant conflicts of interest to declare.

Upstream Mechanisms Causing Type 1C Von Willebrand Disease (VWD): Contribution Of Defective Von Willebrand Factor (VWF) Multimerization, Regulated Storage, and Secretion

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3571-3571
Author(s):  
Sandra L. Haberichter ◽  
David A Jakab ◽  
Paula M. Jacobi
Keyword(s):  
Conflicts Of Interest ◽  
Von Willebrand Disease ◽  
Hek293 Cells ◽  
Essentially Normal ◽  
Intracellular Processing ◽  
Clinical Biology ◽  
And Function ◽  
Von Willebrand ◽  
Index Cases

Abstract One mechanism causing type 1 VWD is the reduced survival of VWF in plasma (type 1C VWD), characterized by markedly decreased VWF:Ag and VWF half-life, essentially normal multimers, increased ratio of VWF propeptide (VWFpp) to VWF:Ag, robust response to DDAVP, and normal ratios of VWF:CB, FVIII, or VWF:RCo to VWF:Ag. We enrolled 502 index cases with a pre-existing diagnosis of type 1 VWD through the Zimmerman Program for the Molecular and Clinical Biology of VWD. We confirmed 262 of the index cases as type 1 VWD (VWF:Ag or VWF:RCo ≤ 40 IU/dL). Of these, 58 met the criteria for type 1C VWD with VWFpp/VWF:Ag ≥ 3 and VWF:Ag ≤ 30 IU/dL. Sequence variations were identified in the VWF D3, A1, A2, and D4 domains. Little is known regarding the mechanisms causing type 1C VWD, but it has been assumed that VWF undergoes normal intracellular processing and secretion with rapid clearance upon release into plasma. We hypothesized that defective intracellular processing may contribute to the type 1C phenotype. We studied 10 type 1C variants including C1130Y, W1144G, R1205H, N1231S, R1315C, V1411E, R1527W, N2041S, Y2160C, and S2179F. Variants were expressed alone (homozygously) or with wild-type (WT) VWF (heterozygously) in HEK293T cells and VWF secretion, multimer structure, and binding to collagen (types III and VI), GPIb-alpha, and FVIII was analyzed. To assess regulated storage, variants were expressed homozygously in HEK293 cells where WT VWF forms elongated pseudo-Weibel-Palade bodies (pWPB). Five variants (C1130Y, R1315C, V1411E, N2041S, Y2160C) had severely decreased secretion and defective multimerization when homozygously expressed. These variants did not form pWPB, but appeared to co-localize with the endoplasmic reticulum, consistent with the severely impaired secretion. One variant, W1144G, had mildly reduced secretion, formed only dimeric VWF, and unexpectedly did not form pWPB. These multimer defective variants demonstrated decreased collagen binding and GPIb-alpha binding as would be predicted. The remaining variants (R1205H, N1231S, R1527W, S2179F) were normally secreted, multimerized, stored in pWPB, and had normal binding to FVIII, collagen, and GPIb-alpha. Interestingly, FVIII binding to homozygous VWF D3 variants C1130Y and W1144G was substantially reduced. This result is not entirely unexpected as the FVIII binding region in VWF has been mapped to the D’-D3 region. Co-expression with WT VWF essentially corrected defective secretion, although some variants still had moderately reduced secretion. Multimer structure appeared normal for all heterozygous variants, although staining which discriminates between variant and WT alleles revealed that for some variants, little variant VWF was actually expressed when transfected at a 1:1 ratio with WT. In sum, when variants were homozygously expressed, we observed a constellation of processing and functional defects. Only R1205H, N1231S, R1527W, and S2179F variants demonstrated normal processing and function. Heterozygous expression (consistent with patients) corrected most of the observed defects, although reduced secretion persisted for a subset of variants. We can conclude that while reduced plasma survival of VWF is a major determinant of the type 1C phenotype, additional upstream processing defects may contribute to the severity of the overall VWD phenotype. Disclosures: No relevant conflicts of interest to declare.

Spectrum of Type 2 Von Willebrand Disease in the Zimmerman Program

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 472-472 ◽  
Author(s):  
Veronica H Flood ◽  
Pamela A Christopherson ◽  
Daniel B Bellissimo ◽  
Joan Cox Gill ◽  
Sandra L Haberichter ◽  
...  
Keyword(s):  
Sequence Variation ◽  
Von Willebrand Disease ◽  
Sequence Variations ◽  
Platelet Binding ◽  
The Mean ◽  
Bleeding Score ◽  
Von Willebrand ◽  
Index Cases

Abstract While von Willebrand disease (VWD) is the most common inherited bleeding disorder, most patients have quantitative defects in von Willebrand factor (VWF). The qualitative variants, collectively termed type 2 VWD, are less common, but also in general more severe than type 1 VWD. However, despite a common laboratory phenotype of decreased VWF:RCo/VWF:Ag ratio for types 2A, 2B, and 2M VWD, the clinical phenotype is highly variable. We examined index cases and affected family members enrolled in the Zimmerman Program with a phenotypic diagnosis of type 2 VWD. All subjects had factor VIII (FVIII), VWF antigen (VWF:Ag), VWF ristocetin cofactor activity (VWF:RCo), and multimer distribution analyzed in a central laboratory. For calculation of mean VWF:RCo values, a level of 5 was assigned to subjects with VWF:RCo below the laboratory lower limit of detection of 10 IU/dL. A platelet binding assay was also performed using a gain of function GPIb containing 2 mutations that enable spontaneous binding to VWF in the absence of ristocetin (VWF:GPIbM). Full length VWF gene sequencing was performed for all index cases. Targeted sequencing was performed for family members to ascertain the presence or absence of sequence variations found in the index case. Bleeding symptoms were quantified using the ISTH bleeding assessment tool and reported as bleeding scores (BS). Mean FVIII, VWF:Ag, VWF:RCo, and BS are listed in the table below for each type 2 variant. For type 2A VWD, 113 subjects have been enrolled to date. All had an abnormal multimer distribution with loss of high molecular weight multimers. 6 type 2A subjects had a VWF:RCo/VWF:Ag ratio of ≤0.7. The lowest VWF:RCo levels were seen in the type 2A cohort with 60% <10. 98% of type 2A subjects had an identified sequence variation on full length sequencing. 25% had the p.R1597W sequence variation and an additional 4 subjects had p.R1597Q. The mean bleeding score for the subjects with sequence variations at 1597 was 10.6. 11% of subjects had p.R1374H, which correlated with a higher mean bleeding score of 12.4. Mean bleeding score for the remainder of the type 2A subjects was lower, at 6.6, suggesting that differences in VWF genetics may account for differences in phenotype, despite the common type 2A laboratory presentation of reduced VWF:RCo and loss of high molecular weight multimers. 44 type 2B subjects have been enrolled to date, all with abnormal multimer distribution and either documented abnormal VWF-platelet binding or a presence of a known type 2B sequence variation. Sequence variations were found in 100% of subjects. The most common sequence variations were p.V1316M (20%), p.R1306W (18%), p.R1341Q (11%), and p.H1268Y (9%). Mean VWF:RCo/VWF:Ag ratios ranged from 0.32-1.12, suggesting that a normal VWF:RCo/VWF:Ag ratio cannot completely exclude the possibility of type 2B VWD. Most (94%) had increased VWF:GPIbM. Subjects with p.V1316M and p.R1306W/Q sequence variations had lower VWF:RCo compared to subjects with p.R1341Q/W but mean bleeding scores did not differ. 59 type 2M subjects have been enrolled to date. Mean VWF:RCo/VWF:Ag ratio was 0.46 (range 0.14-0.7). Sequence variations were found in 93% of subjects. R1374C was found in 13 members from one family. While mean VWF levels were similar to the entire 2M group, a wide range in VWF:Ag and VWF:RCo/VWF:Ag ratio was observed, accompanied by a corresponding range in BS from 0-8. This suggests that other modifiers of phenotype may be present aside from the VWF sequence variation. 11 type 2N subjects have been enrolled to date, all with low VWF binding to FVIII. Sequence variations were found in 100% of this cohort. R854Q was present in 89% of subjects. Bleeding scores were highest for homozygous 2N sequence variations. Overall, the mean BS for type 1 VWD subjects was 6.3, the mean BS for type 2 VWD subjects was 7.5, and the mean BS for type 3 VWD subjects was 16.8. Types 2A and 2N had higher bleeding scores on average than type 2B, and type 2M subjects had on average the lowest bleeding scores. Although heterogeneity was seen across all the type 2 variants, both laboratory testing and genetic testing are useful in categorizing and phenotyping type 2 VWD. Table. FVIII (mean) VWF:Ag (mean) VWF:RCo (mean) BS (mean) Type 2A 47 34 12 8.7 Type 2B 45 36 23 7.1 Type 2M 62 54 21 5.4 Type 2N 30 69 76 8.3 Disclosures Montgomery: Immucor: Patents & Royalties.

scholarly journals No Association Between Normalization of VWF Levels and Bleeding Phenotype in Patients with Type 1 VWD - from the Win Study

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2577-2577 ◽  
Author(s):  
Johan Boender ◽  
Jeroen Eikenboom ◽  
Karin Fijnvandraat ◽  
Waander van Heerde ◽  
Karina Meijer ◽  
...  
Keyword(s):  
Family History ◽  
Blood Group ◽  
Research Funding ◽  
Von Willebrand Disease ◽  
Younger Adults ◽  
Bleeding Score ◽  
Von Willebrand ◽  
Index Cases ◽  
Over Time

Abstract Von Willebrand factor (VWF) levels vary over time and increase throughout life in both healthy individuals and patients with von Willebrand disease (VWD). Especially in type 1 VWD patients, this increase may result in normalization of VWF levels. It is not yet known if normalization of VWF levels ameliorates bleeding symptoms in VWD patients. We have recently shown that elderly type 1 VWD patients had similar bleeding tendency as younger adults.1 However, many elderly patients in this study had relatively low VWF levels and many younger adults had relatively high VWF levels.1 The aim of the current study was to investigate the association between normalization of VWF levels and the bleeding phenotype in type 1 VWD patients. We included patients from the nationwide cross-sectional "Willebrand in the Netherlands" Study, with lowest historical VWF antigen (VWF:Ag) and/or VWF activity (VWF:Act) ≤30 U/dL. At inclusion, blood was sampled for central measurement of VWF:Ag and VWF:Act and VWF to collagen binding (VWF:CB). Central measurements were available in 367 type 1 VWD patients. Based on these central measurements, patients were divided into three groups: definite VWD (central VWF:Ag and/or VWF:Act and/or VWF:CB ≤30 U/dL, n=152); low VWF (central VWF:Ag and/or VWF:Act and/or VWF:CB 31-50 U/dL , n=120) and historical VWD with presently normalized levels (central VWF:Ag and VWF:Act and VWF:CB ≥51 U/dL, n=95). Age differed between groups: median age was 43 years in definite VWD patients, 45 years in low VWF and 50 years in historical VWD patients (p<0.01). No difference in sex distribution was found, see table 1. Of definite VWD patients, 59% had blood group O compared with 77% of low VWF and 73% historical VWD patients (p<0.01). A variant in the VWF gene was most common in definite VWD patients: 55/76 (72%) patients in whom mutation analysis was performed had a variant compared with 22/41 (54%) low VWF and 13/41 (32%) historical VWD patients (p<0.001). Of definite VWD patients, 72% had a positive family history, compared with 39% of low VWF and 27% of historical VWD patients (p<0.001). In contrast, 69% of historical VWD patients were index cases, compared with 67% of "low VWF" and 39% of definite VWD patients (p<0.001). Median Tosetto Bleeding Score (BS) did not differ between the three groups as it was respectively 9, 8 and 9 in definite VWD patients , low VWF patients and historical VWD patients (p=NS), see table 1. The incidence of bleeding episodes requiring treatment with desmopressin or clotting factor concentrate in the year prior to inclusion also did not differ between groups as it was 18% in definite VWD, 22% in "low VWF" and 27% in historical VWD patients (p=NS). In many countries VWF levels 60 IU/dL are used as the cut-off value for abnormal VWF levels. Using this value as cut-off between low VWF and historical VWD, 151 (42%) patients had low VWF and 64 (17%) patients had historical VWD. Using this cut-off value had no major effect on bleeding phenotype or patient characteristics (data not shown). In conclusion, patients with higher VWF levels at the time of study were older and less often had a variant in the VWF gene. More historical VWD patients were index cases, underlining their negative family history. In contrast, blood group O was more common in patients with normalized VWF levels, suggesting that factors outside the VWF gene have a more important effect on VWF levels in this group. Importantly, the bleeding score was similar in all groups. The bleeding score is a reflection of bleeding symptoms that have occurred throughout one's life and may therefore not detect changes in bleeding phenotype over time. Importantly, the bleeding incidence requiring treatment in the year prior to inclusion was also similar in all groups, regardless of VWF levels. Our study suggests that normalization of VWF levels is not associated with the bleeding phenotype in type 1 VWD patients. However, this study is limited by its retrospective design and prospective studies are required to assess the bleeding phenotype and bleeding rate in more detail and to identify patients at increased or decreased risk of bleeding. 1Sanders YV, Giezenaar MA, Laros-van Gorkom BA, et al. von Willebrand disease and aging: an evolving phenotype. J Thromb Haemost. 2014;12(7):1066-1075. Disclosures Boender: CSL Behring: Research Funding. Eikenboom:CSL Behring: Research Funding. Fijnvandraat:CSL Behring: Research Funding; Bayer: Research Funding. Meijer:Bayer: Honoraria, Research Funding; Baxter: Research Funding; Pfizer: Research Funding; Sanquin: Honoraria, Research Funding; Boehringer Ingelheim: Honoraria; Bristol-Myers Squibb: Honoraria. Mauser-Bunschoten:Baxter: Research Funding; Bayer: Research Funding; CSL Behring: Research Funding; Novo Nordisk: Research Funding; Griffols: Research Funding; Sanquin: Research Funding. Cnossen:Novo Nordisk: Research Funding; CSL Behring: Other: Travel Funding, Research Funding; Pfizer: Research Funding; Novartis: Research Funding; Baxalta: Research Funding; Bayer: Research Funding. Laros-van Gorkom:CSL Behring: Research Funding. van der Bom:CSL Behring: Research Funding; Novo Nordisk: Research Funding; Pfizer: Research Funding; Bayer: Research Funding; Baxalta: Research Funding. Leebeek:UniQure: Consultancy; Netherlands Hemophilia Foundation: Research Funding; Baxter: Research Funding; CSL Behring: Research Funding.

Phenotypic Variability in Carriers of Von Willebrand Factor Truncating Sequence Variants in the Zimmerman Program

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2833-2833
Author(s):  
Daniel B Bellissimo ◽  
Rupa A Udani ◽  
Pamela A. Christopherson ◽  
Kenneth D. Friedman ◽  
Joan Cox Gill ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Family Members ◽  
The Other ◽  
Normal Range ◽  
Bleeding Score ◽  
Type 3 ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Index Cases

Abstract Von Willebrand disease (VWD) is caused by quantitative (types 1 and 3) and qualitative (type 2) defects in von Willebrand factor (VWF). VWD type 1 and 2 are autosomal dominant with variable expression while type 3 is autosomal recessive. The majority of patients have quantitative defects in VWF. The clinical phenotype is highly variable. This variability is evident in the normal population as the VWF antigen levels range from 50-240 IU/dl. In order to investigate variability in clinical expression of VWD, we examined type 1 or type 3 VWD index cases and their family members enrolled in the Zimmerman program where a VWF truncating variant wasfound. These variants included nonsense, splice site, deletions and duplications. All subjects had factor VIII (FVIII), VWF antigen (VWF:Ag), VWF ristocetin cofactor activity (VWF:RCo), and multimer distribution analyzed in a central laboratory. VWF gene sequencing was performed for all index cases. Targeted sequencing was performed for family members to ascertain the presence or absence of sequence variations found in the index case. Bleeding symptoms were quantified using the ISTH bleeding assessment tool and reported as bleeding scores. TableNumber of Truncating Variants210Number of Subjects257515VWF:Ag (IU/dl): Avg ±SD2.0±2.545±2099±32VWF:Ag Range0-106-11657-182VWF:Ag 25th-75th Percentile0-3.032-5671-112ISTH Bleeding Score: Avg±SD17.6±8.14.4±4.32.1±2.5ISTH Bleeding Score: 25th-75th Percentile10.0-23.51-60-4.0 The VWF:Ag level was significantly different between all three groups (P<0.0001). As expected, type 3 subjects with two truncating variants had the significantly lower VWF:Ag and higher bleeding scores. There was less variability in VWF:Ag and bleeding score compared to the subjects in the other two groups. In subjects with no truncating variants, the unaffected family members, the average VWF:Ag and bleeding score were similar to values observed in healthy controls. The subjects with one variant were the largest group and the most variable in VWD phenotype. The average VWF:Ag was 45±20, slightly below the cutoff of the normal range but the antigen level in this group ranged from 6-116. Thirty-five percent of subjects had VWF:Ag greater than 50 and in the normal range. Approximately 65% had VWF:Ag below 50; about one half were blood type O. 17% had VWF:Ag levels below 30. Although the bleeding score was only slightly higher than the subjects with no variants (P=0.038), the bleeding score ranged from 0-17. Even within a family, highly variable Ag levels were observed. Although numbers were small, when only families with type 3 VWD were considered, mean antigens were higher and mean bleeding scores lower for subjects heterozygous for a truncating allele compared to a non-truncating allele. For a series of subjects with identical truncating alleles, those heterozygous subjects from families with a diagnosis of type 3 VWD had lower bleeding scores than heterozygous subjects from families with only type 1 VWD, despite similar antigen levels, suggesting less concern for mild bleeding symptoms in families where some members have severe bleeding. In summary, carriers of a single VWF truncating allele have a variable phenotype. A wide range of Ag levels and bleeding scores were observed. In 35% of subjects, the Ag level is normal (>50) suggesting the lack of expression from one VWF allele can be compensated by the other VWF allele. The majority of subjects with a single VWF truncating allele had antigen levels typical for type 1 VWD. This suggests that additional environmental or genetic factors are required for symptomaticVWD. Disclosures No relevant conflicts of interest to declare.

scholarly journals Variability in Bleeding Phenotype in Type 3 VWD Families

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2466-2466
Author(s):  
Pamela A Christopherson ◽  
Veronica H Flood ◽  
Sandra L Haberichter ◽  
Joan Cox Gill ◽  
Robert R Montgomery ◽  
...  
Keyword(s):  
Oral Cavity ◽  
Family Members ◽  
Study Entry ◽  
Null Alleles ◽  
Healthy Controls ◽  
Bleeding Score ◽  
Type 3 ◽  
Von Willebrand ◽  
Index Cases

Abstract Type 3 von Willebrand Disease (VWD) is a rare and severe form of VWD characterized by complete absence of von Willebrand factor (VWF). Patients with type 3 VWD typically present with moderate to severe mucocutaneous bleeding as well as muscle hematomas and hemarthroses. While inheritance has classically been considered autosomal recessive, there is increasing evidence for co-dominant inheritance, with heterozygous carriers affected with low VWF levels. We sought to explore the bleeding phenotype in type 3 index cases (IC) and family members (FM) enrolled in the Zimmerman Program study using the ISTH bleeding assessment tool (ISTH-BAT), and to correlate baseline bleeding score (ISTH BS) and sequence variant (SV) location as well as analyze prospective bleeding diatheses over time using an interim bleeding score (iBAT). Analysis included type 3 VWD index cases (IC), type 3 FM, type 1 affected family members (AFM) and healthy controls (HC). Clinical laboratory phenotyping was performed at BloodCenter of Wisconsin and included FVIII, VWF:Ag, VWF:RCo, VWFpp, VWF:CB3 and multimer analysis. Genotyping involved full-length VWF sequencing, array comparative genomic hybridization to detect deletions and Fabric Genomics Opal software to determine pathological variants. Bleeding symptoms were quantified using the ISTH-BAT bleeding score where baseline scores (ISTH BS) included entire history at time of enrollment and interim bleeding scores (iBAT) that represented bleeding occurring since the initial ISTH BS was obtained. Type 3 obligate carriers (OC) were defined as either having offspring with type 3 VWD or being an offspring of an individual with type 3 VWD. Type 1 AFM cohort included subjects with type 1/low VWF who were related to a type 1 IC. The type 3 families consisted of 45 unrelated type 3 IC, 56 OC with type 1/low VWF (Type 3 OC), 23 normal unaffected OC, and 8 unaffected family members (UFM). Clinical phenotyping and study entry ISTH BS are detailed in Table 1. Type 3 OC with type 1/low VWF (n=56) had mean VWF:Ag and VWF:RCo lab values which were not different from type 1/low VWF AFM (n=483) however both FVIII (68 vs 56, p<0.001) and ISTH BS (1 vs 3, p<0.0001)were both significantly different. OC without evidence of VWD (n=23) had mean VWF:Ag (79) and FVIII (122) that was significantly different (p<0.01) from healthy controls, however there was no difference in ISTH BS. The median ISTH BS in the type 3 IC was 15, with no difference between males and females. Adults had a median ISTH BS of 18 that was significantly increased (p<0.0005) compared to a score of 11 in pediatric subjects (<18). Only 1 type 3 IC did not have an abnormal BS which was an 8 year old female. No difference in ISTH BS was observed according to number of SV or between those with or without null alleles. There was also no difference in ISTH BS (13) of IC with SV in the propeptide region compared to SV in other regions of VWF (15). The highest median baseline subscores reported were hemarthorsis (3), followed by nose bleeds (2), bruising (2), and oral cavity bleeding (2) and bleeding from minor wounds (1). 17 type 3 IC had at least one follow-up visit where median total iBAT score was 11 with most bleeding due to nosebleeds, bruising, bleeding from minor wounds and oral cavity bleeding (iBAT subscores=1). Prospective bleeding was analyzed in 12 adult type 3 IC using a baseline ISTH BS cutoff of 10 to evaluate if bleeding score would predict future bleeding. 10 subjects had ISTH BS >10 (mean age 33, BS 22) and 2 had BS<10 (mean age 33, BS 10). Those with a baseline ISTH BS >10 had a median iBAT of 13 compared to those with ISTH BS<10 with an iBAT of 6, however this was not significantly different (p=0.06) most likely due to the small number of subjects. While type 3 carriers may have low VWF levels, their bleeding is mild compared to type 1 affected family members, and not significantly different from OC with normal levels. There is a difference in type 3 index case bleeding subscores from baseline study entry compared to interim bleeding most likely reflecting the effect of treatment. In this small cohort of prospective bleeding data, the baseline bleeding score did not seem to predict future bleeding as measured by the interim bleeding score. Continued prospective studies of these patients should provide valuable insight into the variability and changes in bleeding in type 3 VWD families. Table 1. Table 1. Disclosures Montgomery: BCW: Patents & Royalties: GPIbM assay patent to the BloodCenter of Wisconsin.

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