SEARCH

SEARCH BY CITATION

Keywords:

  • multimer analysis;
  • mutation;
  • type 1;
  • von Willebrand disease;
  • von Willebrand factor

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. Addendum
  8. Acknowledgements
  9. Disclosure of Conflict of Interests
  10. References

Summary.  Background: Type 1 von Willebrand disease (VWD) is a congenital bleeding disorder characterized by a partial quantitative deficiency of plasma von Willebrand factor (VWF) in the absence of structural and/or functional VWF defects. Accurate assessment of the quantity and quality of plasma VWF is difficult but is a prerequisite for correct classification. Objective: To evaluate the proportion of misclassification of patients historically diagnosed with type 1 VWD using detailed analysis of the VWF multimer structure. Patients and methods: Previously diagnosed type 1 VWD families and healthy controls were recruited by 12 expert centers in nine European countries. Phenotypic characterization comprised plasma VWF parameters and multimer analysis using low- and intermediate-resolution gels combined with an optimized visualization system. VWF genotyping was performed in all index cases (ICs). Results: Abnormal multimers were present in 57 out of 150 ICs; however, only 29 out of these 57 (51%) had VWF ristocetin cofactor to antigen ratio below 0.7. In most cases multimer abnormalities were subtle, and only two cases had a significant loss of the largest multimers. Conclusions: Of the cases previously diagnosed as type 1 VWD, 38% showed abnormal multimers. Depending on the classification criteria used, 22 out of these 57 cases (15% of the total cohort) may be reclassified as type 2, emphasizing the requirement for multimer analysis compared with a mere ratio of VWF functional parameters and VWF:Ag. This is further supported by the finding that even slightly aberrant multimers are highly predictive for the presence of VWF mutations.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. Addendum
  8. Acknowledgements
  9. Disclosure of Conflict of Interests
  10. References

von Willebrand disease (VWD) is the most common congenital bleeding disorder and demonstrates autosomal inheritance of mucocutaneous bleeding symptoms and reduced plasma von Willebrand factor (VWF) levels and is usually caused by genetic defects in the VWF gene (VWF) [1–3]. Type 1 VWD is characterized by a partial quantitative deficiency of plasma VWF, whereas qualitatively abnormal variants of VWF are classified as type 2 VWD [4,5]. Provisional diagnostic criteria for type 1 VWD were published recently on behalf of the Subcommittee on VWF of the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis (ISTH-SSC on VWF) [6].

As type 1 VWD is characterized by a partial reduction of structurally and functionally normal VWF, it is often difficult to distinguish type 1 VWD patients from healthy individuals with VWF levels at the lower end of the normal distribution curve. VWF multimer analysis is used to determine the structural integrity of plasma VWF. Many diagnostic laboratories use multimer analysis to determine whether there is a loss of high molecular weight (HMW) multimers to help classify patients with type 2 VWD. However, using an optimized system, more minor abnormalities such as a relative rather than absolute lack of HMW multimers plus abnormalities in the oligomer structure can be determined [7].

To systematically study the value of clinical, phenotypic and molecular characteristics in the diagnosis of type 1 VWD, a multicenter European study was initiated: ‘Molecular and Clinical Markers for the Diagnosis and Management of Type 1 von Willebrand disease (MCMDM-1VWD)’ (http://www.euvwd.group.shef.ac.uk/). Previously reported data from this study has focused on bleeding symptoms, prediction of VWD based on low plasma VWF levels, co-segregation of VWD with VWF and mutation analysis [8–11]. The main aim of the present study was to evaluate the proportion of misclassification, even in centers experienced in VWD diagnosis, of patients historically diagnosed with type 1 VWD using detailed analysis of VWF multimer structure. Specific multimer profiles were evaluated in relation to patient phenotypic characteristics, genotypes, and the current VWD classification. We compared the value of multimer analysis with the VWF ristocetin cofactor activity (VWF:RCo) to the VWF antigen (VWF:Ag) ratio in the classification of VWD. Finally, the predictive value of multimer analysis with respect to genotype was evaluated.

Patients and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. Addendum
  8. Acknowledgements
  9. Disclosure of Conflict of Interests
  10. References

Study design

The MCMDM-1VWD study is an EU-funded survey on type 1 VWD. For further details of study design and recruitment see http://www.euvwd.group.shef.ac.uk/ and references [8–11]. Twelve VWD expert centers (P1–P12) in nine European countries recruited 154 families historically diagnosed with type 1 VWD. Informed consent was obtained from all subjects. Each family comprised an index case (IC) with VWD, at least one further affected family member (AFM), plus unaffected family members (UFM). One hundred and fifty ICs could be completely analyzed. Each center recruited about 100 healthy controls (HC).

Phenotype and genotype

Plasma samples were assessed for VWF:Ag, VWF:RCo and factor VIII activity (FVIII:C) as described [9,11]. Ristocetin-induced platelet agglutination (RIPA) using two final ristocetin concentrations (0.5 mg mL−1 and 1.25 mg mL−1) was determined in platelet-rich plasma obtained at enrollment from each IC to exclude type 2B VWD. The VWF gene of each IC was analyzed for candidate mutations as previously described [8].

VWF multimer analysis

Electrophoresis and blotting  SDS-agarose gel electrophoresis was carried out essentially as described [12–14]. Briefly, medium- (1.6%) and low-resolution (1.2%) gels (LGT agarose type VII; Sigma, Munich, Germany) were prepared. Electrophoresis was performed for 16 h at 55 volts. VWF multimers were transferred to nitrocellulose filters by electroblotting using transfer buffer (0.05 m phosphate, pH7.4 with 0.04 m SDS, without methanol). All incubation and washing steps were performed in low-fat milk. Filters were incubated overnight at room temperature (RT) in a 1:2000 dilution of polyclonal rabbit anti-human VWF antibody (DakoCytomation, Glostrup, Denmark). After three washing steps they were incubated for 2 h at RT in the second antibody diluted 1:3000 (Goat anti-rabbit IgG HRP conjugate; BioRad Laboratories, Hercules, CA, USA). Thereafter, filters were washed three times, incubated twice for 30 seconds in buffer (20 mm Tris–HCl, 500 mm NaCl, pH7.5) to remove the milk and placed into the video-detection system (Fluorchem™; Alpha Innotech Corp., San Leandro, CA, USA). Filters were overlaid with 5 mL solution containing 0.4 mg mL−1 luminol, 0.01 mg mL−1 4-iodophenol (both from Sigma-Aldrich Chemie, Steinheim, Germany) and 2.5 μL mL−1 30% H2O2 (Perhydrol, Merck, Darmstadt, Germany) in Tris buffer (20 mm Tris–HCl, 500 mm NaCl, pH7.5). Typical exposure times were between 30 s and 5 min.

Qualitative and quantitative evaluation  Multimer analysis was performed in all samples from the families. By having IC, AFM and UFM on one gel together, qualitative defects were detected more readily. VWF multimers of patients’ plasma were classified as either abnormal multimers (AbM) or normal multimers (NM) by comparison with the reference plasma (pool of 30 HC). AbM were defined as a deviation from a normal distribution; either loss of HMW multimers or presence of supranormal multimers on low-resolution gels or as abnormal migration of individual oligomers or abnormal separation into triplets/quintuplets on medium-resolution gels. In some samples there was no clear separation between individual oligomer triplets, leading to a smeary multimer pattern, these samples were also designated abnormal. Further classification of samples into type 1 and type 2 VWD and sub-classification was performed according to ISTH guidelines [4,5].

Quantitative, densitometric gel analysis of all samples was performed using software provided with the video-detection system (AlphaEaseFC Stand Aalone software; Alpha Innotech Corp., San Leandro, CA, USA). Whenever possible, samples with the same quantity of VWF:Ag were applied to the gels. For densitometric evaluation, small, intermediate and large multimers were defined as oligomers 1–5, 6–10 and > 10, respectively. In low-resolution gels, the smallest multimers (1 and 2) migrated together and the corresponding first peak (protomer) was counted as two oligomers, while in higher-resolutions gels, where the smallest oligomers were better separated, the protomer was counted as one oligomer. Evaluation of multimer patterns was performed by the first author, blinded for the results of mutation status. Percentage figures in Tables 1–5 are relative amounts of large multimers compared with normal pooled plasma on each gel. This is a different figure than the distribution per sample over large, intermediate and small multimers. For 70 HC, we found the relative amount of large multimers to be 100 ± 19.9 [mean ± SD% (observed range 76–169%)]. Thus a decrease of the large multimers was defined as < 70%.

Table 1.  von Willebrand disease (VWD) type 2 A (IIE) ranked according to expression of abnormal multimers
ICVWF:Ag IU dL−1VWF:RCo IU dL−1VWF:RCo/ VWF:AgLarge multimers low resolution %*Multimer pattern, medium resolutionMutation typeExon/ intronAmino acid change(Sub) type
  1. *% of the corresponding normal plasma (pool of 30 HC = 100%). +++, typical pattern; ++, typical pattern but less pronounced; +, minor abnormality; (sub) type according to the 2006 classification [5]. nd, no mutation detected.

P9F2 II217100.5949.3+++Missense26C1130R2A(IIE)
P9F4 II11270.5848.7+++Missense26C1130R2A(IIE)
P9F7 II122130.5967.4+++Missense26C1130R2A(IIE)
P9F5 II210101.0057.0+++Missense26C1130G2A(IIE)
P2F9 II4840.5073.2+++Missense26C1130F2A(IIE)
P2F18 I2830.3833.2+++Missense26C1130F2A(IIE)
P2F6 II113110.8577.2+++Missense Missense26 49C1130F R2263P2A(IIE)
P10F10 II114130.9394.4+++Missense Missense26 28Y1146C S1378F2A(IIE)
P3F13 I142130.3145.5+++Missense Missense21 28R924Q R1315L2A(IIE)
P9F1 I231120.3973.2+++Missense26W1144G2A(IIE)
P5F4 II12030.1596.1++Missense28R1342C1
P2F1 II1930.33139.7++Deletion Missense28 28V1485fs Y1584C1
P2F22 I219261.37148.6++Missense32V1822G1
P3F11 II119271.4270.8++Missense45Q2520P1
P6F14 III136280.7875.2++nd1
P9F3 I212110.9274.5++nd1
P9F6 II11430.2175.5+Missense28G1415D1
P9F13 III114141.0068.9+Missense28G1415D1
P7F9 I244360.82114.9+SpliceInt 20Exon 21 skip1
P7F13 II12170.33129.5+SpliceInt 21Exon 21 skip1
Table 2.  von Willebrand disease (VWD) type 2A(sm) ranked according to the expression of abnormal multimers
ICVWF:Ag IU dL−1VWF:RCo IU dL−1VWF:RCo/ VWF:AgLarge multimers low resolution %*Multimer pattern, medium resolutionMutation typeExon/ intronAmino acid change(Sub) type
  1. *, +++, see Table 1. This mutation is probably not the mutation responsible for the phenotype.

P4F1 II12030.1594.2+++Missense28R1374C2A
P4F3 II11070.7082.6+++Missense28R1374C2A
F4F9 II11540.2785.8+++Missense28R1374C2A
P4F4 I11030.3071.3+++Missense Missense28 43R1374C P2145S2A
P9F8 I125100.4052.0+++Missense28R1315C2A
P4F8 II1730.4358.2+++Missense28R1315C2A
P3F1 II11830.1770.4+++Deletion28D1277_L1278delinsE2A
P6F1 II132230.7264.9+++Missense21R854Q2A
Table 3.  von Willebrand disease (VWD) type 2A miscellaneous
ICSubtypeVWF:Ag IU dL−1VWF:RCo IU dL−1VWF:RCo/ VWF:AgLarge multimers low resolution %*Mutation typeExon/ intronAmino acid change(Sub) type
  1. *, see Table 1. Type 2B mutation, occurring on same allele as R1315H. §Index case and affected family member shown, mutation only in AFM.

P6F3II1IB1630.1974.5Missense28S1285P1
P12F10 II1IB1930.1667.2Missense28I1416N2A
P5F3 II2IB27110.4191.0Missense Missense28 28P1266L R1315H1
P6F13II1IB1670.4456.4Missense28L1307P2A
P3F4 III1IIA29100.3437.1Missense28R1374H2A
P3F5 II1IIA74160.2228.7Missense28R1374H2A
P3F6 II1Unspecified830.3845.1Missense27R1205H1
P3F7II1Unspecified331.0064.1Missense Missense31 43R1779X S2469P1
P11F2 III10Unspecified7101.4369.7Missense27R1205H1
P7F5F II4§Unspecified72771.0769.1nd1
P7F5F I1§Unspecified531.2541.8Frameshift18L812fs2A
P12F3 II1Unspecified1040.40131.4Missense27R1205H1
Table 4.  von Willebrand disease (VWD) type 2M (smf, sm)
ICSubtypeVWF:Ag IU dL−1VWF:RCo IU dL−1VWF:RCo/ VWF:AgLarge multimers low resolution %*Mutation typeExon/ IntronAmino acid change(Sub) type
  1. *, see Table 1. sm, smeary pattern; f, faster moving oligomers.

P2F5 II22M(smf) 25251.00104.0Missense43R2464C1
P2F14 II22M(smf)33300.91122.3Missense43R2464C1
P1F4II12M(smf)19251.32133.1Missense Missense28 43Y1584C R2464C1
P2F16 II22M(smf)35340.97116.5Missense42C2362F1
P5F2 II22M(smf)20190.9594.7Missense40C2304Y1
P5F10 II12M(smf)22200.91108.2Missense43G2441C1
P7F22 I22M(smf)38330.87123.7Missense43C2477Y1
P10F2 I12M(smf)28401.4385.0Missense45C2477S1
P12F12 II22M(sm)29421.4596.6Missense38C2257S1
P3F15 III22M(sm)2090.4570.7Missense28R1315C1
P9F15 I12M(sm)10ndnd81.1Missense28R1315C1
P5F9 II22M26190.7397.3Missense38L2207P1
P5F8 I32M32381.19156.5SpliceInt 21Exon 21skip1
Table 5.  von Willebrand disease (VWD) type 2M (Vicenza)
ICSubtypeVWF:Ag IU dL−1VWF:RCo IU dL−1VWF:RCo/ VWF:AgLarge multimers low resolution %*Mutation typeExon/ intronAmino acid change(Sub) type
  1. *, see Table 1. V, Vicenza. §The only sample with supranormal multimers.

P1F1 II12M(V) 530.60138.1Missense27R1205H1 Vic)
P3F3 III12M(V) §801081.35149.8Missense27R1205H1(Vic)
P2F2II12M(V)1460.43150.9Missense Missense17 27M740I R1205H1(Vic)
P2F8 II12M(V)860.75144.3Missense17 27M740I R1205H1(Vic)
P2 F17 II12M(V)630.50129.0Missense17 27M740I R1205H1(Vic)

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. Addendum
  8. Acknowledgements
  9. Disclosure of Conflict of Interests
  10. References

Normal vs. abnormal multimers

VWF multimers of 150 IC, 278 AFM, 312 UFM and 70 HC randomly chosen from the 1166 HC included in the study were evaluated on low- and intermediate-resolution gels. Fifty-seven of the 150 ICs (38%) showed abnormalities in their multimer distribution or oligomer composition (further referred to as group 1) and in nearly all of those ICs (54 of 57, 95%) mutations were identified. Ninety-three of the 150 ICs (62%) showed completely normal multimers (NM) using both low- and medium-resolution gels. Among the 93 with NM, mutations were identified in 51 (55%) (group 2). No mutations were identified in the remaining 42 (45%) with NM (group 3). No multimer abnormalities were detected among the 70 HC. Although abnormal, according to our stringent criteria, the abnormalities observed in group 1 patients were much more subtle than usually seen in type 2A VWD, especially the ‘classic’ previous type IIA. A significant loss of largest multimers and prominent outer sub-bands was only seen in two families (P3F4 and P3F5); with the same mutation in the A1-domain (R1374H) and even in these families the grade of abnormality was relatively small.

Specific multimer abnormalities

Among the 57 ICs with multimer abnormalities, several patterns could be identified. Initial subdivision was into those with a selective deficiency of HMW multimers (type 2A) and those without a selective deficiency of HMW multimers (type 2M) [4]. Further subdivision was based on specific characteristics of the migration pattern.

VWD type 2A  Thirty-nine of the 57 ICs with AbM had some degree of loss of HMW multimers and were allocated to type 2A (Tables 1–3). In 20 families, the multimer pattern was characterized by a relative decrease of the large multimers and oligomers showed barely visible outer sub-bands with some smear around the central bands (Fig. 1) or in two families (P3F11 & P6F14) clearly visible inner sub-bands. This pattern is indicative of a multimerization defect and of reduced proteolytic processing by ADAMTS13 and can be designated as type 2A(IIE) according to the first description by Zimmerman et al. [14]. This pattern was pronounced in ten of the 20 families; typical but less pronounced in six and only mildly abnormal in four. Phenotypes, genotypes, and multimer patterns are summarized in Table 1.

image

Figure 1.  Family P2F9 with the C1130F mutation classified as von Willebrand disease (VWD) type 2A(IIE) in medium-A) and low- (B) resolution gels. Unaffected family member (FM) II1 (lane 1) and the normal-pooled plasma (NP) (lane 3) show a normal pattern, while index case (IC) II4 (lane 5) and affected family member (AFMs) (II2 and II3, lanes 2 and 4, respectively) show besides a small loss of the largest multimers, a significant relative decrease of large multimers together with a paucity of subbands.

Download figure to PowerPoint

In eight families, we observed a significant relative decrease of the large multimers in the presence of supranormal multimers in low-resolution gels. Quintuplets had a paucity of outer sub-bands and a smear was clearly visible around the central bands and in the space between the individual oligomers (Fig. 2). This pattern was designated type 2A (sm = smeary) (Table 2).

image

Figure 2.  Family P4F1 with the R1374C mutation classified as von Willebrand disease (VWD) type 2 A(sm) in medium-(A) and low- (B) resolution gels. UFM II2 (lane 1) and normal-pooled plasma (NP) (lane 4) show a normal pattern, while the index case (IC) II1 (lane 6) and affected family members (AFMs) (I1, III1 and III2, lanes 5, 2, 3, respectively) show a significant relative decrease of large multimers together with a smeary pattern. In the low-resolution gel supranormal multimers are clearly visible.

Download figure to PowerPoint

In four families, all multimers were present with a significant relative decrease of the large multimers, but in contrast to type 2A(IIE), with a normal structure of triplets/quintuplets (Table 3). This subtype can be designated 2A(IB) according to the description by Hoyer et al. [15]. Only two ICs were identified that fitted the classic pattern of type 2A(IIA), as indicated above (Table 3). Finally, five families could not be allocated to a specific VWD subtype (Table 3). One of these samples (P3F7II1) could not be evaluated properly as a result of a very low VWF:Ag (3 IU dL−1). The cryoprecipitate showed an abnormal pattern with loss of the large multimers, faster running central bands and a smeary appearance. In another sample (P3F6II1) with very low VWF:Ag, the pattern of the un-concentrated sample was indicative of type 2A. However, the cryoprecipitate showed normal multimers (Fig. 3). In another family (P11F2), the signal was low but strong enough to show the presence of all multimers with a relative loss of the large multimers and enhanced outer sub-bands which differed from those seen in type 2A(IIA). This pattern possibly resulted from in vitro proteolysis during sample storage or transportation. In P7F5, the IC’s grandfather had clearly abnormal VWF. Large multimers were relatively decreased with a unique, largely abnormal triplet structure. Although visible in un-concentrated plasma, this pattern was more pronounced in cryoprecipitate. The IC showed normal VWF properties and multimers were normal with the exception of a double band, particularly in oligomer 4 (Fig. 4). This peculiar multimer pattern was confirmed at a second visit of the family 3 years later.

image

Figure 3.  P3F6 with the R1205H mutation classified as von Willebrand disease (VWD) type 2A (unspecified) in a low-resolution gel with normal (A) and long (B) exposure times. UFM I3 (lane 1) and normal-pooled plasma (NP) (lane 3) show a normal pattern, while the index case (IC) II1 (lane 5) and affected family members (AFMs) (I1 and II2, lanes 2 and 4, respectively) show barely visible multimers suggestive of a reduction of large multimers. In contrast, the cryoprecipitates (lanes 6–8 samples) show a normal pattern. In the longer exposure these patterns become more evident. Supranormal multimers are absent.

Download figure to PowerPoint

image

Figure 4.  Family P7F5 with no detectable mutation classified as von Willebrand disease (VWD) type 2A (unspecified) (AFM I1) and VWD type 1 (IC II4) in medium- (A) and low- (B) resolution gels. UFM I2 (lane 3) and normal-pooled plasma (NP) (lane 4) show a normal pattern, while the index case (IC) II4 (lane 1) and affected family members (AFMs) (III1 and III2, lanes 5 and 6, respectively) show a double band best seen in oligomer 4 (arrows). While the neat plasma in the grandfather I1 of the IC (lane 2) with an exon 18 frameshift mutation (L812fs) shows barely visible faint bands (B), the cryoprecipitate (A) shows a grossly abnormal multimer pattern together with a loss of the largest multimers.

Download figure to PowerPoint

VWD type 2M  Eighteen of 57 ICs with AbM were allocated to type 2M (Tables 4 and 5). In eleven ICs, the presence of all multimers was observed, with supranormal multimers in some, and quintuplets with paucity of outer sub-bands and a smear clearly visible around the central bands and in the space between the individual oligomers (Table 4). This type was designated 2M (sm = smeary). In eight of these families, faster running oligomer bands were also detected (Table 4, Fig. 5), designated type 2M (smf = smeary pattern and faster running oligomers). In two other families, a type 2M phenotype without a smeary pattern was diagnosed, but the quintuplet/triplet structure was slightly abnormal with a paucity of outer sub-bands.

image

Figure 5.  P2F5 with the R2464C mutation classified as von Willebrand disease (VWD) type 2M(smf) in medium- (A) and low- (B) resolution gels. UFMs I2 and II1 (lanes 4 and 5) and normal-pooled plasma (NP) (lane 2) show a normal pattern, while the index case (IC) II2 (lane 1) and affected family member (AFM) (I1, lane 3) show the presence of all multimers together with a smeary pattern. The individual oligomers are running faster (arrows).

Download figure to PowerPoint

In five families, all multimers were present and supranormal multimers were visible in one family (P3F3). The quintuplet/triplet structure was abnormal with a paucity of all sub-bands. This group was designated 2M (V = Vicenza) (Table 5).

Correlations between multimer phenotype and VWF genotype

The multimer patterns were categorized according to the patterns listed in Tables 1–5. An overview of the different patterns observed is given in Fig. 6. The characteristic multimer pattern of VWD type 2A(IIE) (Table 1) is in the majority of ICs associated with mutations in exons 26 or 28 (14 out of 20 ICs). Strikingly, three different mutations at C1130 were associated with the 2A(IIE) pattern. In six out of the eight ICs with the type 2A(sm) pattern (Table 2) a mutation was identified in exon 28 that introduces a new cysteine. In all families with the type 2M(smf) or 2M (sm) pattern (Table 4), the responsible mutations involved cysteines. These cysteine mutations may be responsible for additional structural alterations of VWF that explains the presence of the smeary material between the multimer bands. The multimer pattern of type 2M(V), sometimes referred to as type 1 (Vicenza), was diagnostic for patients with the R1205H mutation (Table 5). The hallmark of the mutation R1205H, which may be associated with a second mutation (M740I) [16], is the presence of supranormal multimers [17]. However, only 5 out of10 ICs with R1205H demonstrated a 2M(V) pattern, two of the remaining five had normal multimers, whereas three had an unspecified profile (Table 3) suggesting a poor correlation between phenotype and genotype.

image

Figure 6.  Mature von Willebrand factor (VWF) protein showing the domain structure and main associated functions. Multimer gels indicate the multimer profile of normal-pooled plasma (NP) and those of individuals with various different von Willebrand disease (VWD) subtypes together with mutations detected. *bdg, binding; **coll, collagen; ***dim, dimerization.

Download figure to PowerPoint

Based on identified multimer patterns, the responsible mutated exon was predicted in a blinded fashion in 26 out of the 57 ICs with AbM (46%) and this prediction was correct in 22 out of 26 ICs (85%).

VWF:RCo/VWF:Ag ratio as a predictor of multimer abnormality

According to the 1994 VWD classification [4], 38% of all 150 ICs in the current study had multimer abnormalities and 26% could be reclassified as having 2A and 12% as 2M VWD. A VWF:RCo/VWF:Ag ratio of < 0.7 is suggestive of a functional VWF defect; however, of ICs with AbM only 29 out of 57 (51%) had a low VWF:RCo/VWF:Ag ratio below 0.7. This indicates that the VWF:RCo/VWF:Ag ratio is not always reliable for classification. The prediction of multimer abnormalities by a reduced VWF:RCo/VWF:Ag ratio < 0.7 was higher in the combined type 2A IC (25 out of 39 = 64%, Tables 1–3) than in the combined 2M IC (4 out of 18 = 22%, Tables 4 and 5). Abnormal ratios (< 0.7) were consistently detected in IC and AFM only of families with the 2A(sm) mutations, where all affected family members with R1374C, R1315C and D1277_L1278delinsE had such ratios (Table 2).When we consider the updated 2006 classification [5], that allows more subtle multimer abnormalities to be classified as type 1 VWD, 15% of 150 ICs in this study could still be reclassified as type 2 VWD. Of these 22 type 2 VWD ICs, 77% had a VWF:RCo/VWF:Ag ratio < 0.7 and of those with minimal multimer abnormalities fitting type 1 VWD under the 2006 VWD classification, 34% had a ratio < 0.7 (Table 6).

Table 6.  VWF:RCo/VWF:Ag ratio and multimer abnormalities
Multimer patternNo. of IC of total cohortNo. of IC per group with VWF:RCo/ VWF:Ag <0.7
n % n %
  1. *Subtyping according to the 2006 VWD classification [5]. Of 150 IC analysed.

Abnormal57382951
Type 2*22151777
Type 1*35231234
Normal93621112

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. Addendum
  8. Acknowledgements
  9. Disclosure of Conflict of Interests
  10. References

The aim of the present analysis of the MCMDM-1VWD study was to evaluate the extent of misclassification of patients historically diagnosed with type 1 VWD. Thirty-eight percent (57) of the ICs historically diagnosed with type 1 VWD showed some multimer abnormalities. These could be divided into 2A and 2M dependant on relative loss of HMW multimers, and then further subdivided based on other multimer characteristics (Tables 1–5). These subdivisions may not always be of clinical relevance and therefore we do not suggest that the VWD subtypes should be split into these ever-smaller groupings for diagnostic purposes. However, the subdivisions facilitate understanding of pathophysiological processes perturbed by abnormal VWF and in many cases enable predictions of mutation type and location to be made.

The multimer abnormalities identified in this study are very subtle. According to the majority of publications, type 2 patients show clearly abnormal multimers (type 2A) or largely diminished VWF:RCo when compared with VWF:Ag (type 2M). Patients with more subtle abnormalities have been described, but mostly in single families (e.g. type IIE [14]). All other published type 2 patients have a clear IIA phenotype (loss of large and intermediate multimers and enhanced proteolysis) or belong to the rare phenotypes IIC and IID with gross multimer abnormalities, in which false classification as type 1 is impossible because of the clear-cut phenotype. Multimer patterns observed in this study are often so subtle that they may have been interpreted in the past as normal and may be beyond the resolution of multimer analysis in many laboratories today. However, according to the 1994 VWD classification [4], which was strict in that no qualitative abnormalities should be present in type 1 patients, all abnormal multimer patterns were grouped (57 ICs) and subsequent analyses of the cohort were performed by this stratification. The updated 2006 VWD classification [5], which was published after completion of the study, uses a more flexible type 1 definition and allows qualitative abnormalities as long as they do not hamper normal functionality and allow successful desmopressin treatment. Using this more flexible definition, 22 out of 57 ICs with abnormal multimers would still fit the criteria for type 2 VWD (Table 6). Owing to insensitive multimer methods, not allowing detection of relative losses of large multimers, even experienced laboratories will classify these 22 patients as type 1. In some publications, authors were aware of missing multimers, but found the multimer abnormality insufficient to classify these patients as type 2, for example, patients with C1130F [18]. The 2M(smf, sm) phenotype with non-proteolyzed multimers may not be readily detectable with low-resolution gels. Because of the presence of all multimers, these patients are not classified as type 2M as long as the ratio between VWF:RCo and VWF:Ag is normal (> 0.7).

Discrimination between the different multimer profiles of 2A and 2M patterns is sometimes equivocal. In three families (P3F6, P11F2 and P12F3) affected members with R1205H were allocated to type 2A instead of 2M(V). In these families the VWF:Ag was between 3 and 10 IU dL−1. Additionally in two families, the large multimers were decreased to 45% and 70% of the normal control. A third family showed evidence of supranormal multimers, but had a clearly abnormal triplet pattern indicative of enhanced proteolysis. Four families with R1315C in exon 28 were allocated to the subtypes 2A(sm) (P4F8 and P9F8) and 2M(sm) (P3F15 and P9F15). Those in the 2A subtype showed a significant (relative) decrease of the large multimers (52% and 58% of the normal control) compared with 71% and 81% in subtype 2M. Different sample handling could explain the phenotypic difference but this is unlikely as samples from two families with different phenotypes were prepared by the same center (P9). Phenotypic difference remained after reevaluation when we were aware of this difference in families with the same mutation. Previous studies for R1315C were interpreted either as type 2M or type 2A by two groups [19,20], supporting our findings.

Even although subtyping of VWD based on multimers may not always be of clinical relevance, identification of even subtle multimer abnormalities has pathophysiological significance. Despite the sometimes minor multimer alterations, the AbM group is clearly separable from the NM groups. The median VWF:Ag is much lower in AbM (19 IU dL−1) than in NM (47 IU dL−1) [8]. Linkage to VWF is much stronger in AbM than in NM (Lod score 20.4 and 5.69, linkage proportion 1.0 and 0.46, respectively) [11]. The chance of finding a mutation is much higher in the group with AbM (95%) than NM (55%) [8]. Accurate multimer analysis therefore adds valuable information to the phenotype, especially with respect to inheritance. Furthermore, the multimer pattern predicted the mutation site in 26 out of 57 (46%) families with AbM. With a new understanding about the 2M(smf) mutations (all cases involve cysteine residues located between the D4 and the CK-domains), mutation site may be predictable in about 70% of patients with AbM, and this may facilitate mutation analysis.

Assessment of qualitative variants can be attempted by the use of ratios between functional (VWF:RCo) and quantitative parameters (VWF:Ag), and by evaluation of multimerization using low- and intermediate-resolution gels. The use of ratios has been proposed but not validated [21]. Using a ratio of < 0.7, only 29 out of 57 families (51%) with AbM would have been defined as qualitatively abnormal, indicating that this ratio is not reliable for classification. The 2006 VWD classification [5] appears to correlate better with the VWF:RCo/VWF:Ag ratio [4] as 77% of the 22 ICs that would still be classified as type 2 VWD according to the 2006 classification have a VWF:RCo/VWF:Ag ratio < 0.7 (Table 6). When comparing low- and intermediate-resolution gels, all families with AbM were detected with intermediate gels. Low-resolution gels missed 14 out of 57 families and thus have a power of 75% to detect families with qualitative defects. Genotyping detects many more families with mutations, as in the 93 families with normal multimers, 51 families had a mutation. However, in three families with qualitative abnormalities no mutation was detectable. We have good reasons to assume that in cases, VWF is indeed mutated and thus none of our methods reached 100% sensitivity. In the MCMDM-1VWD cohort, the VWF:RCo/VWF:Ag ratio was clearly inferior to low-resolution multimer gels, while this gel resolution was inferior to intermediate-resolution multimer gels.

Results from the study reinforce the role and importance of multimer analysis in VWD analysis. This role is complementary to other phenotypic tests. It may provide insight into molecular abnormalities which other analyses cannot, for example through satellite bands alterations. Although this methodology remains complex, its simplification and better standardization should be pursued. We conclude that even centers experienced in diagnosis and management of VWD often misclassify patients as type 1 VWD that should be classified as type 2. The extent of this misclassification is smaller when the 2006 classification [5] is considered. Subtle multimer abnormalities may be of limited clinical significance, but they identify a subgroup of patients with a very high chance of having a VWF mutation and indicate pathophysiological mechanisms perturbed by those mutations.

Addendum

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. Addendum
  8. Acknowledgements
  9. Disclosure of Conflict of Interests
  10. References

Contribution of authors

Study initiation and coordination: A. Goodeve, F. Rodeghiero and I. Peake; study design, data collection and performing laboratory analyses: U. Budde, R. Schneppenheim, J. Eikenboom, G. Castaman, A. B. Federici, J. Batlle, A. Pérez, D. Meyer, C. Mazurier, J. Goudemand, J. Ingerslev, D. Habart, Z. Vorlova, L. Holmberg, S. Lethagen, J. Pasi and F. Hill; analysis and interpretation of results: U. Budde, R. Schneppenheim, J. Eikenboom, A. Goodeve, K. Will, E. Drewke, G. Castaman, F. Rodeghiero, A. B. Federici, J. Goudemand, S. Lethagen and I. Peake; lead authors of initial manuscript: U. Budde, R. Schneppenheim, J. Eikenboom and A. Goodeve; revisions of draft manuscripts: U. Budde, R. Schneppenheim, J. Eikenboom, A. Goodeve and I. Peake; review and approval of final manuscript: U. Budde, R. Schneppenheim, J. Eikenboom, A. Goodeve, G. Castaman, F. Rodeghiero, A. B. Federici, J. Batlle, D. Meyer, C. Mazurier, J. Goudemand, J. Ingerslev, D. Habart, Z. Vorlova, L. Holmberg, S. Lethagen, J. Pasi, F. Hill and I. Peake.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. Addendum
  8. Acknowledgements
  9. Disclosure of Conflict of Interests
  10. References

The European Community under the Fifth Framework Programme (QLG1-CT-2000-00387) financially supported this study.

We would like to thank E. Jennings, M. Makris, H. Powell, M. Walker, L. Marsden, A. Al-Buhairan, S. Joyce, A. Bowyer, J. Anson, A. Tosetto, A. Cappelletti, M. Bernardi, K. Bertoncello, R. Bader, L. Baronciani, M. Canciani, F. Gianniello, E. Fressinaud, A. S. Ribba, A. Stephanian, L. Hilbert, C. Caron, E. Gomez, V. van Marion, J. Lambert, F. Oyen, T. Obser, J. Suttnar, J. Dudlova, C. Hallden, C. Watson, J. Warren, S. Mughal, W. Lester, A. Guilliatt, S. Enayat, G. Surdhar, and P. Short for their contributions to this study.

Disclosure of Conflict of Interests

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. Addendum
  8. Acknowledgements
  9. Disclosure of Conflict of Interests
  10. References

The authors state that they have no conflict of interest.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. Addendum
  8. Acknowledgements
  9. Disclosure of Conflict of Interests
  10. References
  • 1
    Rodeghiero F , Castaman GC , Dini E . Epidemiological investigation of the prevalence of von Willebrand’s disease . Blood 1987 ; 69 : 4549 .
  • 2
    Werner EJ , Emmett H , Tucker E , Giroux D , Schultz J , Abshire T . Prevalence of von Willebrand disease in children. A multiethnic study . J Pediatr 1993 ; 123 : 938 .
  • 3
    Sadler JE . Biochemistry and genetics of von Willebrand factor . Annu Rev Biochem 1998 ; 67 : 395424 .
  • 4
    Sadler JE . A revised classification of von Willebrand disease. For the Subcommittee on von Willebrand Factor of the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis . Thromb Haemost 1994 ; 71 : 5205 .
  • 5
    Sadler JE , Budde U , Eikenboom JCJ , Favaloro EJ , Hill FG , Holmberg L , Ingerslev J , Lee CA , Lillicrap D , Mannucci PM , Mazurier C , Meyer D , Nichols WL , Nishino M , Peake IR , Rodeghiero F , Schneppenheim R , Ruggeri ZM , Srivastava A , Montgomery RR , et al. ; Working Party on von Willebrand Disease Classification . Update on the pathophysiology and classification of von Willebrand disease: a report of the Subcommittee on von Willebrand Factor . J Thromb Haemost 2006 ; 4 : 210314 .
  • 6
    Sadler JE , Rodeghiero F . Provisional criteria for the diagnosis of VWD type 1 . J Thromb Haemost 2005 ; 3 : 7757 .
  • 7
    Budde U , Pieconka A , Will K , Schneppenheim R . Laboratory testing for von Willebrand disease: contribution of multimer analysis to diagnosis and classification . Semin Thromb Hemost 2006 ; 32 : 51421 .
  • 8
    Goodeve A , Eikenboom J , Castaman G , Rodeghiero F , Federici AB , Battle J , Meyer D , Mazurier C , Goudemand J , Schneppenheim R , Budde U , Ingerslev J , Habart D , Vorlova Z , Holmberg L , Lethagen S , Pasi J , Hill F , Hashemi Soteh M , Baronciani L et al. Phenotype and genotype of a cohort of families historically diagnosed with type 1 von Willebrand disease in the European study, molecular and clinical markers for the diagnosis and management of type 1 von Willebrand disease (MCMDM-1VWD) . Blood 2007 ; 109 : 11221 .
  • 9
    Tosetto A , Rodeghiero F , Castaman G , Goodeve A , Federici AB , Battle J , Meyer D , Fressinaud E , Mazurier C , Goudemand J , Eikenboom J , Schneppenheim R , Budde U , Ingerslev J , Vorlova Z , Habart D , Holmberg L , Lethagen S , Pasi J , Hill F et al. A quantitative analysis of bleeding symptoms in type 1 von Willebrand disease: results from a multicenter European study (MCMDM-1 VWD) . J Thromb Haemost 2006 ; 4 : 76673 .
  • 10
    Tosetto A , Rodeghiero F , Castaman G , Bernardi M , Bertoncello K , Goodeve A , Federici AB , Battle J , Meyer D , Mazurier C , Goudemand J , Eikenboom J , Schneppenheim R , Budde U , Ingerslev J , Vorlova Z , Habart D , Holmberg L , Lethagen S , Pasi J et al. Impact of plasma von Willebrand factor levels in the diagnosis of type 1 von Willebrand disease: results from a multicenter European study (MCMDM-1VWD) . J Thromb Haemost 2007 ; 5 : 71521 .
  • 11
    Eikenboom J , van Marion V , Putter H , Goodeve A , Rodeghiero F , Castaman G , Federici AB , Battle J , Meyer D , Mazurier C , Goudemand J , Schneppenheim R , Budde U , Ingerslev J , Vorlova Z , Habart D , Holmberg L , Lethagen S , Pasi J , Hill F et al. Linkage Analysis in Families Diagnosed with Type 1 von Willebrand Disease in the European Study, Molecular and Clinical Markers for the Diagnosis and Management of Type 1 VWD (MCMDM-1VWD) . J Thromb Haemost 2006 ; 4 : 77482 .
  • 12
    Ruggeri ZM , Zimmerman TS . Variant von Willebrand’s disease. Characterization of two subtypes by analysis of multimeric composition of factor VIII/von Willebrand factor in plasma and platelets . J Clin Invest 1980 ; 65 : 131825 .
  • 13
    Schneppenheim R , Plendl H , Budde U . Luminography – an alternative assay for detection of von Willebrand factor multimers . Thromb Haemost 1988 ; 60 : 1336 .
  • 14
    Zimmerman TS , Dent JA , Ruggeri ZM , Nannini LH . Subunit composition of plasma von Willebrand factor. Cleavage is present in normal individuals, increased in IIA and IIB von Willebrand disease, but minimal in variants with aberrant structure of individual oligomers (types IIC, IID and IIE) . J Clin Invest 1986 ; 77 : 94751 .
  • 15
    Hoyer LW , Rizza CR , Tuddenham EG , Carta CA , Armitage H , Rotblat F . von Willebrand factor multimer patterns in von Willebrand’s disease . Br J Haematol 1983 ; 55 : 493507 .
  • 16
    Castaman G , Missiglia E , Federici AB , Schneppenheim R , Rodeghiero F . An additional unique candidate mutation (G2470A; M740I) in the original families with von Willebrand disease type 2M Vicenza and the G3864A (R1205H) mutation . Thromb Haemost 2000 ; 84 : 83501 .
  • 17
    Schneppenheim R , Federici AB , Budde U , Castaman G , Drewke E , Krey S , Mannucci PM , Riesen G , Rodeghiero F , Zieger B , Zimmermann R . Von Willebrand disease type 2M “Vicenza” in Italian and German patients: identification of the first candidate mutation(G3864A; R1205H) in 8 families . Thromb Haemost 2000 ; 83 : 13640 .
  • 18
    Eikenboom JCJ , Matushita T , Reitsma PH , Tuley EA , Castaman G , Briet E , Sadler JE . Dominant type I von Willebrand disease caused by mutated Cysteine residues in the D3 domain of von Willebrand factor . Blood 1996 ; 88 : 243341 .
  • 19
    Casana P , Martinez F , Espinos C , Haya S , Lorenzo JI , Aznar JA . Search for mutations in a segment of exon 28 of the human von Willebrand factor gene: new mutations, R1315C and R1341W, associated with type 2M and 2B variants . Am J Hematol 1998 ; 59 : 5763 .
  • 20
    Ribba AS , Hilbert L , Lavergne J-M , Fressinaud E , Boyer-Neumann C , Temisien C , Juhan-Vague I , Goudemand J , Girma J-P , Mazurier C , Meyer D . The arginine-522-cysteine (R1315C) mutation within the A1 loop of von Willebrand factor induces abnormal folding with a loss of function resulting in type 2A-like phenotype of von Willebrand disease: study of 10 patients and mutated recombinant von Willebrand factor . Blood 2001 ; 97 : 9529 .
  • 21
    Federici AB . Diagnosis of von Willebrand disease . Haemophilia 1998 ; 4 : 65460 .