A Laboratory Phenotype/Genotype Correlation of 1167 French Patients From 670 Families With von Willebrand Disease: A New Epidemiologic Picture

Abstract

von Willebrand disease (VWD) is a genetic bleeding disease due to a defect of von Willebrand factor (VWF), a glycoprotein crucial for platelet adhesion to the subendothelium after vascular injury. VWD include quantitative defects of VWF, either partial (type 1 with VWF levels <50 IU/dL) or virtually total (type 3 with undetectable VWF levels) and also qualitative defects of VWF (type 2 variants with discrepant antigenic and functional VWF levels). The most bleeding forms of VWD usually do not concern type 1 patients with the mildest VWF defects (VWF levels between 30 and 50 IU/dL). The French reference center for VWD performed a laboratory phenotypic and genotypic analysis in 1167 VWD patients (670 families) selected by their basic biologic phenotype: type 3, type 2, and type 1 with VWF levels <30 IU/dL. In these patients indeed, to achieve an accurate diagnosis of VWD type and subtype is crucial for the management (treatment and genetic counseling). A phenotype/genotype correlation was present in 99.3% of cases; 323 distinct VWF sequence variations (58% of novel) were identified (missense 67% versus truncating 33%). The distribution of VWD types was: 25% of type 1, 8% of type 3, 66% of type 2 (2A: 18%, 2B: 17%, 2M: 19%, 2N: 12%), and 1% of undetermined type. Type 1 VWD was related either to a defective synthesis/secretion or to an accelerated clearance of VWF. In type 3 VWD, bi-allelic mutations of VWF were found in almost all patients. In type 2A, the most frequent mechanism was a hyper-proteolysis of VWF. Type 2B showed 85% of patients with deleterious mutations (distinct from type 2B New York). Type 2M was linked to a defective binding of VWF to platelet glycoprotein Ib or to collagen. Type 2N VWD included almost half type 2N/3. This biologic study emphasizes the complex mechanisms for both quantitative and qualitative VWF defects in VWD. In addition, this study provides a new epidemiologic picture of the most bleeding forms of VWD in which qualitative defects are predominant.

FIGURE 1

Distribution of von Willebrand disease (VWD) types in the global cohort (n = 1167 patients) and in the index cases (n = 670 cases). The proportion of each VWD type (1, 2A, 2B, 2M, 2N, and 3) is represented with blue histograms for the global cohort of 1167 patients and with red histograms for the 670 index cases (IC). These proportions are similar for both groups of patients within each VWD type. IC = index cases, VWD = von Willebrand disease.

FIGURE 2

Phenotype/genotype correlation in 670 index cases (IC) patients with von Willebrand disease (VWD). Among 670 IC, after second level phenotypic assays and direct sequencing of VWF gene, 648 patients exhibited a genotype supporting their phenotype: 151 patients had type 1 VWD, 121 patients type 2A, 112 patients type 2B, 127 patients type 2M, 81 patients type 2N, 49 patients type 3, and 7 patients undetermined (U) VWD. In contrast, after direct sequencing, the genotype was not supporting the phenotype in 22 patients (17 patients with no mutation and 5 patients with only 1 mutation although recessive inheritance) who thus underwent Multiplex Ligation-dependent Probe Amplification (MLPA) analysis. MLPA helped identifying large deletions, insertions, and duplications in 14 patients consisting in 10 type 1, 1 type 2A (IIC), and 3 type 3. Finally, 8 patients remained with a genotype either not explaining their phenotype (6 type 1 patients with no mutation found) or only partially supporting their phenotype (2 type 3 patients with a single mutation found).IC = index cases, MLPA = multiplex ligation-dependent probe amplification, VWD = von Willebrand disease.

FIGURE 3

Distribution of von Willebrand disease (VWD) types and subtypes in 670 index cases patients. The central ring represents the proportion of VWD types in the 670 IC patients: 25% of type 1, 18% of type 2A, 17% of type 2B, 19% of type 2M, 12% of type 2N, 8% of type 3, and 1% of undetermined (U) VWD. The peripheral disks indicate the proportion of the miscellaneous mechanisms responsible for some VWD type. For type 1 VWD, the main mechanism was classical dominant negative mutations of VWF gene leading to synthesis/secretion defects (53%), whereas mutations responsible for an accelerated VWF clearance represented 20% of cases; heterozygous mutations usually described in type 3 VWD accounted for 20% of patients (type 3 carriers); mutations within VWF propeptide cleavage site were rare (3.5%) and the mechanism remained unknown in 3.5% of cases (no mutation found). In patients with type 2A VWD, the most common subtype was 2A(IIA) (62%) including a large majority of mutations within the A2 VWF domain inducing an increased VWF proteolysis by ADAMTS13 (59%), whereas mutations within the A1 VWF domain were rare (4%); subtype 2A(IIE) was frequent, accounting for 34.5% of type 2A, whereas subtypes 2A(IIC) and 2A(IID) were rare, representing 3% and 1%, respectively. Type 2B VWD included either a majority of classical 2B mutations located within the A1 VWF domain (85%) or some New York mutations within the D3 VWF domain (15%). In patients with type 2M VWD, the most common mechanism was mutations within the A1 VWF domain inducing a 2M/2A-like phenotype (63%) related to a defective binding of VWF to GPIb; the 2M phenotype consisted in a decreased binding of VWF to collagen (37%) related either to mutations within the A1 VWF domain or the A3 VWF domain. Type 2N VWD included patients with either bi-allelic 2N mutations (2N/2N, 54%) or a combination of a mono-allelic 2N mutation (expressed allele) with a mutation inducing a silent allele (2N/3, 46%).IC = index cases, VWD = von Willebrand disease, VWF = von Willebrand factor.

FIGURE 4

Sequences variations of VWF gene in 161 patients with type 1 von Willebrand disease (VWD). In our 161 patients with type 1 VWD, 105 distinct sequence variations spread all over VWF gene were identified. Novel mutations are indicated in blue. Missense mutations (51%) are presented on the top and truncating sequence variations (49%) are indicated on the bottom. In type 1 “IC” (clearance), the most frequent mutations were either the Vicenza mutation p.Arg1205His or others like p.Arg1205Cys, p.Arg1205Leu, p.Cys1165Phe, and dup.exon35–37. Six patients had mutations localized within the propeptide-cleavage site (p.Arg763Gly or p.Arg763Met). Interestingly, almost half of the truncating mutations found at the heterozygous state in our patients with type 1 VWD were also found in association with another mutation in our patients with type 3 VWD. IC = index cases, VWD = von Willebrand disease.

FIGURE 5

Sequences variations of VWF gene in 54 patients with type 3 von Willebrand disease (VWD). In our 54 patients with type 3 VWD, 61 distinct sequence variations were identified. Novel mutations are indicated in blue. They were spread all over VWF gene with a hot spot on the N-terminal part of VWF (D domains). Truncating mutations (82%) are indicated at the bottom of the figure; missense mutations (18%) are indicated at the top of the figure.VWD = von Willebrand disease, VWF = von Willebrand factor.

FIGURE 6

Mutations of VWF gene in 442 patients with type 2 von Willebrand disease (VWD). In our 442 patients with type 2 VWD, 118 distinct mutations were identified. Novel mutations are indicated in blue. In type 2A VWD (56 distinct mutations), most mutations (59%) were located in the A2 domain consisting in subtype 2A(IIA) leading to an increased VWF proteolysis (mainly mutations p.Arg1597Trp, p.Gly1609Arg, and p. Ile1626Thr); 30.5% of mutations were located in the D3 domain consisting in subtype 2A(IIE) (mainly mutations p.Cys1101Arg, p.Tyr1146Cys, and p.Cys1157Phe). Mutations located in either the D2 domain consisting in subtype 2A(IIC), or the A1 domain consisting in subtype 2A(IIA) leading to a decreased VWF secretion (mainly mutations p.Cys1272Arg, p.Cys1272Tyr, and p.Cys1458Tyr) or the CK domain (subtype 2A[IID]) were few (5%, 3.5%, and 2%, respectively). In type 2B VWD, 13 distinct mutations were described, most of them (85%) being located in the A1 domain (most frequently p.Arg1306Trp, p.Arg1308Cys, p.Pro1337Leu, and p.Arg1341Gln); some other mutations responsible for the specific type 2B “New York” (15%) were located in the D3-A1 junction (p.Pro1266Leu or p.Pro1266Gln). In type 2M VWD (40 distinct mutations), most mutations (78%) were located in the A1 domain (mainly p.Arg1315Cys and p.Arg1374Cys), whereas the other mutations (22%) were located within the A3 domain (p.Leu1696Arg was the most frequent mutation). In type 2N VWD, 8 distinct 2N mutations located in the D′-D3 domains were found (the p.Arg854Gln being the most frequent mutation as present in 90% of patients). VWD = von Willebrand disease, VWF = von Willebrand factor.