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

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.

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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 ◽

10.1182/blood.v110.11.2133.2133 ◽

2007 ◽

Vol 110 (11) ◽

pp. 2133-2133 ◽

Cited By ~ 1

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.

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

Blood ◽

10.1182/blood.v124.21.2833.2833 ◽

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.

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

Blood ◽

10.1182/blood.v122.21.331.331 ◽

2013 ◽

Vol 122 (21) ◽

pp. 331-331 ◽

Cited By ~ 1

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.

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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 ◽

10.1182/blood.v110.11.709.709 ◽

2007 ◽

Vol 110 (11) ◽

pp. 709-709 ◽

Cited By ~ 1

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.

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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 ◽

10.1182/blood.v112.11.3379.3379 ◽

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.

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

Blood ◽

10.1182/blood.v126.23.1107.1107 ◽

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.

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Variability in Bleeding Phenotype in Type 3 VWD Families

Blood ◽

10.1182/blood-2018-99-112961 ◽

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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Bleeding Scores in Von Willebrand Disease (VWD) Re-Visited: Analysis of the TS Zimmerman Program for the Molecular and Clinical Biology of VWD

Blood ◽

10.1182/blood.v112.11.425.425 ◽

2008 ◽

Vol 112 (11) ◽

pp. 425-425 ◽

Cited By ~ 4

Author(s):

Joan Cox Gill ◽

Pamela A Christopherson ◽

Veronica H Flood ◽

Kenneth D Friedman ◽

Robert R. Montgomery ◽

...

Keyword(s):

Dna Sequence ◽

Sequence Variation ◽

Family Members ◽

Dna Sequence Variation ◽

Bleeding Score ◽

Clinical Biology ◽

Type 3 ◽

Bleeding Severity

Abstract Quantitation of bleeding severity in VWD has recently been investigated utilizing a standardized bleeding score developed through a European study (MCMDM-1) of a large cohort of VWD type 1 families but has not been evaluated in other types of VWD. We analyzed data from the TS Zimmerman Program for the Molecular and Clinical Biology of VWD in order to determine the relative impact of various VWD molecular variants on the clinical bleeding phenotype of individuals with VWF abnormalities. After obtaining informed consent, demographic data and bleeding histories were obtained from subjects through administration of a standardized questionnaire by trained personnel; bleeding score (BS) was calculated by the method of the MCMDM-1. VWF assays were performed in the central laboratory, including quantitation of VWF by VWF:Ag; functional analysis by ristocetin cofactor (VWF:RCo) and collagen binding (VWF:CB); multimer analysis; and DNA sequencing of the VWF gene including coding sequences, intron-exon boundaries and promoter regions. A total of 212 Index Cases (IC) including 149 with type 1 VWD, 17 type 2A, 8 2B, 4 2M, 6 2N and 28 type 3), 346 affected family members and 417 unaffected family members of the IC were identified by their primary hematologist and compared to 234 Normal Control subjects (NC). Subjects with VWD ranged in age from 1–76 years, median 17; NC ages ranged from 2–74 years, median 40. Male to female ratios were similar in type 1 VWD (0.44) and NC (0.34) but equivalent in type 2 (0.94) and type 3 VWD (0.87). Subjects with each type of VWD had significantly higher BS (median, 25–75% interquartile range) than NC (p<0.001); type 1 IC (6, 3–10); type 2 IC (7, 3–13); type 3 IC (15, 10.5–23); and NC (0, −1–1). Age was significantly correlated with BS in type 1 IC (p<0.0001). Males vs. females within each group did not have significantly different BS, nor did those with blood group O vs. non-O; there were significant differences in comparisons of BS in IC vs. NC (p<.001) in each of these subgroups. BS (median, 25–75% interquartile range) in families differed between the IC (6, 3–10), affected family members (1, −1–4), unaffected family members (0, −1–2) and NC (0, −1–1). VWF DNA sequencing revealed known mutations or new SNPs in 21/57 (37%) type 1 IC and 17/48 (35%) NC. In contrast to results from the MCMDM-1, there was no difference in BS in those with or without a DNA sequence variation within the type 1 IC group. In addition, there were no differences in BS within the NC group in the presence or absence of a sequence variation but significant BS differences were found in IC compared with NC (p<.001) regardless of the presence of a DNA sequence variation. Comparisons of BS (mean ± SD) in IC with type 1 VWD and VWF:Ag< 30 (N=20) compared to IC with type 2 VWD and VWF:Ag<30 (N=31) revealed higher BS in type 2 IC (8.5 ± 6.3) than type 1 IC (6.9 ± 5); p<0.05). This study of the quantitation of bleeding severity in subjects with both quantitative defects in VWD (type 1 and 3) and qualitative defects (type 2) revealed that the BS is significantly higher compared with NC in those with type 2 and 3 VWD as well as has been previously shown in type 1 VWD. The presence of a DNA sequence variation was not associated with higher bleeding scores in type1 VWD. A comparison of BS in type 1 IC vs. type 2 IC with similar concentrations of VWF in plasma (VWF:Ag), suggests that the molecular abnormalities present in type 2 subjects results in a more severe bleeding phenotype in spite of the same concentration of antigen. The strong correlation of age with bleeding score warrants additional investigation of bleeding history instruments to identify pediatric patients at risk. We conclude that improved understanding of the clinical consequences of VWF defects will be enhanced by investigation of their impact on clinical bleeding phenotypes in well characterized patients with VWD.

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Correlation of a Condensed Bleeding Score with New Diagnosis of a Congenital Bleeding Disorder in Patients Referred to a Tertiary Centre

Blood ◽

10.1182/blood.v118.21.1226.1226 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1226-1226

Author(s):

Deepa Ranjani Jayakody Arachchillage ◽

Tina Biss ◽

John Hanley ◽

Kate Talks

Keyword(s):

Laboratory Tests ◽

Conflicts Of Interest ◽

Bleeding Disorder ◽

Bleeding Tendency ◽

Factor Xi ◽

Factor Xi Deficiency ◽

Number Of Patients ◽

Abnormal Bleeding ◽

Bleeding Score

Abstract Abstract 1226 The performance and utility of a condensed bleeding score (Bowman et al, J Thromb Haemost., 2008;6:2062) in relation to the diagnosis of a congenital bleeding disorder in new referrals to a regional haemostasis clinic over an 8 month period is presented. Between November 2010 and June 2011, 50 patients over the age of 16 (median age, 31 years; range, 16–79), including 32 females, were referred for investigation of a possible congenital bleeding disorder following detection of abnormal coagulation results and/or presentation with a bleeding history. A bleeding score was performed as part of their initial assessment. 12(24%) patients were from local referral and 38(76%) patients were referred from other hospitals in the region for further investigation of a suspected bleeding disorder. Basic coagulation tests (activated partial thromboplastin time (APTT), prothrombin time Clauss fibrinogen and platelet count) were normal in the referred patients from other centres. 50% (6/12) of the local referrals were for investigation of a prolonged APTT detected on routine coagulation screening prior to major surgery. The median bleeding score was 6 with a range of −1 to 14 (Table 1). The presence of a congenital bleeding disorder was confirmed in 31 of the 50 patients (62%), including 19/31 (61%) of the female patients and 12/31(39%) of the males. Correlation of an abnormal bleeding score (score ≥ 4) with diagnosis of a congenital bleeding disorder was only seen for diagnosis of type 1 Von Willebrand Disease (VWD) (Table 2). Analysis of the cases with low scores and abnormal results identified two groups of patients; firstly, those who had not yet had a significant haemostatic challenge, and secondly, those in whom the abnormal coagulation results were explained by a non-haemostatically significant reduction in a coagulation factor level (e.g. FVII, 15%; dysfibrinogenaemia; F XII deficiency). These clinically insignificant laboratory abnormalities explain the discrepancy between the number of patients with abnormal laboratory tests (35) and the number of patients diagnosed with a congenital bleeding disorder (31).Table 1Bleeding score (range)Number of patients with normal lab resultsNumber of patients with abnormal lab results−1 to +1382–44105–74128–102311–1422Total1535Table 2DiagnosisNumber of patientsMedian bleeding scoreAge rangeType 1 VWD116 (4–10)17–51Type 2 VWD48 (5–13)17–36Factor XI123 (1–8)17–76Platelet function defect46 (2–9)17–57 Compared to previous reports the range of scores found with this assessment tool was narrow and could not exclude patients from further laboratory assessment. However the condensed bleeding score has only been validated prospectively for the diagnosis of type 1 VWD and all patients in this cohort who were diagnosed with type 1 VWD had an abnormal bleeding score (≥ 4). This observation supports the role of this scoring system in the assessment of patients for type 1 VWD. The use of the condensed bleeding score in assessing patients with suspected factor XI deficiency is difficult due to the lack of a phenotypic relationship between residual factor XI activity and a bleeding tendency. Furthermore, although factor XI deficiency is a rare congenital bleeding disorder in our cohort of patients 12/31(39%) were diagnosed with factor XI deficiency. This may explain the overall lack of correlation between bleeding score and diagnosis of a congenital bleeding disorder. Patients who have an abnormal bleeding score but normal laboratory tests need consideration of further investigations before concluding they are normal. The possibility of an acquired bleeding disorder should be considered. A thorough drug history is also important as one of the patients with a bleeding score of 14 was taking a non-steroidal anti-inflammatory drug. The use of the condensed bleeding score in the detection of congenital bleeding disorders other than type 1 VWD requires further validation in a larger number of patients. Disclosures: No relevant conflicts of interest to declare.

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Bleeding Patterns in Type I VWD in Effect of VWF Levels: An Individual Participant Data Meta-Analysis of Three Cohorts

Blood ◽

10.1182/blood-2018-99-115728 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1180-1180

Author(s):

Alberto Tosetto ◽

Pamela A Christopherson ◽

Jeroen C.J. Eikenboom ◽

Julie Grabell ◽

Paula D. James ◽

...

Keyword(s):

Cluster Analysis ◽

Blood Group ◽

Research Funding ◽

Family Members ◽

Type I ◽

Unsupervised Cluster ◽

Pooled Data ◽

Index Cases ◽

Unsupervised Cluster Analysis

Abstract Type I von Willebrand disease (VWD) is the most frequent bleeding disorder, with a prevalence of 10-50 cases per 10,000 persons. VWD usually shows an autosomal dominant inheritance pattern, at least in families having VWF:Ag levels below 30 IU/dL. There is considerable uncertainty whether patients having only mildly reduced VWF levels (in the range 30-50 IU/dL) should be diagnosed as having VWD or if they should be considered as a separate clinical entity, broadly referred to as "reduced VWF patients." We hypothesize that these patients may have a clinically distinct bleeding pattern, in terms of presenting symptoms at diagnosis, bleeding severity, and possibly even clinical. We pooled data from three cohorts of patients. The MCMDM-1 VWD Study is a multicenter survey on type 1 VWD that recruited 154 type 1 VWD families from nine European countries. The Kingston cohort recruited consecutive patients with type 1 VWD diagnosed in Kingston, Canada and includes patients screened because of bleeding symptoms or family history of VWD over a 10 year period. The Zimmerman Program for the Molecular and Clinical Biology of VWD is a multicenter study that enrolled VWD patients mostly from US clinical centres, primarily carrying a diagnosis of type 1 VWD (315 families) but also including type 2 and type 3 subjects. For the present study, only patients with a confirmed diagnosis of type 1 VWD were retained. From the three cohorts, demographic, laboratory and clinical data were abstracted, including severity of bleeding symptoms classified according to the MCMDM-1 study criteria. Only data from index cases and affected family members were retained for the present analysis. Bleeding symptoms receiving a score >=2 (and therefore requiring some type of medical intervention) were classified as "clinically relevant". VWF:Ag and VWF:RCo were measured centrally by the reference core lab of each of the three studies, against the WHO International Standard. For this purpose of the present study, Type 1 VWD is defined as VWF:Ag levels (or VWF:RCo for the Zimmerman Program) below 30 IU/dL; affected family members and index cases with VWF:Ag levels equal or above 30 IU/dL were classified as "low-VWF" patients. Pooled data from the three studies resulted in 1411 patients. At multivariate analysis, blood group O females with lower bleeding scores were the characteristics associated with the "low-VWF" patients; interestingly, the number of clinically relevant bleeding symptoms was associated with the phenotype independently from the total bleeding score. For all considered bleeding symptoms, VWD patients had a higher prevalence of relevant bleeding, with the notable exception of menorrhagia. Particularly in index cases, menorrhagia but also post-surgical bleeding was increased in patients having the "low-VWF" phenotype, although only for menorrhagia the difference was statistically significant (p=0.022). Unsupervised cluster analysis of symptom distribution disclosed three subgroups of patients. The first two were composed by males (first group) or females (second group), both having few or none bleeding symptoms. The third identified by unsupervised cluster analysis group was composed of highly symptomatic patients, predominantly women. In these patients, mucous ("wet") bleeding (epistaxis and menorrhagia) was common together with post-surgical or extraction bleeding. This pattern was not correlated with mean VWF:Ag level or "low-VWF" or Type 1 VWD phenotype. We conclude that "low-VWF" patients are more frequently blood group O, older females, with lower average bleeding scores and number of symptoms and possibly selected because of menorrhagia. A group of "severe bleeders" may be identified (n=270, 19%), having a similar distribution of "low VWF" and "VWD" phenotypes. Disclosures Tosetto: Stago, Novo-Nordisk, BMS: Speakers Bureau; Werfen: Other: Member of Advisory Board, Speakers Bureau. Eikenboom:CSL: Research Funding. James:CSL Behring: Research Funding; Shire: Research Funding; Bayer: Research Funding. Montgomery:BCW: Patents & Royalties: GPIbM assay patent to the BloodCenter of Wisconsin.

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