Keywords von Willebrand factor - von Willebrand disease - type 2M - collagen-binding domain
Introduction
Von Willebrand disease (VWD) is caused by genetic variants in the von Willebrand factor
(VWF ) gene resulting in quantitative (types 1 and 3) and qualitative (type 2) deficiencies
of VWF. Main characteristics are prolonged bleeding time, tendency for hematomas,
and mucocutaneous bleedings such as epistaxis and menorrhagia. Type 2M VWD is due
to autosomal dominant variants either in A1 or A3 domain of VWF. Variants in A1 domain
lead to defective binding of VWF to platelet GpIb without loss of high-molecular-weight
multimers. Defects in A3 domain are characterized by reduced binding activity of VWF
to subendothelial collagen type I and type III.[1 ] Patients with a collagen-binding defect show low values for VWF:CB (VWF:collagen-binding
activity) and VWF:CB/VWF:antigen (Ag) ratio, whereas other VWF parameters and multimeric
patterns are normal.[2 ] Genetic analysis using next-generation sequencing (NGS) for the large VWF has replaced former sequential Sanger sequencing.
Case Report
We investigated two siblings (one girl and one boy) with a bleeding diathesis. The
boy (13 years old) suffered from several bleedings after tonsillectomy and recurrent
epistaxis. The older sister (16 years old) presented with menorrhagia, intermittent
gum bleeding, and easy bruising. Both siblings showed severely decreased values for
VWF:CB (≤0.11 U/mL) and VWF:CB/VWF:Ag ratio (0.06 and 0.16, respectively). VWF:CB
measured in the patients corresponds to collagen type I (Takeda, Austria GmbH). VWF
activity (VWF:GPIbM) was analyzed using VWF:Ac INNOVANCE VWF Ac (Siemens Healthcare
Diagnostics). Values for VWF:Ag, VWF:Ac, factor VIII activity ([Table 1 ]), and multimer analysis ([Fig. 1 ]) were normal. For genetic analysis, we performed NGS using enriched gene panel diagnostics
(Nextera Rapid Custom Enrichment Kit followed by sequencing on a MiSeq; both Illumina,
San Diego, California, United States). The coverage for the enriched sequence of the
VWF reached 99% for 20× and 97% for 100 × . In exon 30 (NM_000552.3), we identified a
heterozygous nsSNV (c.5192C > T) ([Fig. 2 ]). The nsSNV is listed in dbSNP (rs764077750) and gnomAD (genome aggregation database)
with rare minor allele frequency (ALL: 0.0012%). The variant is leading to an amino
acid substitution from serine to leucine at position 1731 (p.Ser1731Leu) within the
A3 domain of VWF. The A3 domain is considered to be the major binding site for collagen
types I and III. In silico pathogenicity prediction is concordant pathogenic (SIFT,
MutationTaster, PolyPhen2, CADD; [Table 2 ]). The variant is absent in the locus-specific database EAHAD and the public version
of HGMD (Human Gene Mutation Database) (accessed July 10, 2021). However, the amino
acid position p.1731 has been described with threonine instead of wild-type serine
(p.Ser1731Thr) to be present in two patients with reduced binding of VWF to collagen.
Expression of the VWF mutant p.Ser1731Thr in COS7 cells confirmed the functional defect.[3 ] Based on these findings, we classified p.Ser1731Leu as novel likely pathogenic variant within the A3 domain of VWF causing VWD type 2M CBD.
Table 1
VWF diagnostic for the two siblings
Patient
VWF:Ag [U/mL]
normal: 0.6–1.5
VWF:CB [U/mL]
normal: 0.6–1.5
VWF:CB/VWF:Ag ratio
normal: 0.8–1.5
VWF:Ac [%]
normal: 46–179
Multimer analysis
FVIII activity [%]
normal: 60–150
Brother
0.70
0.09
0.13
60
Normal
100
0.83
0.05
0.06
68
n.d.
n.d.
Sister
0.69
0.11
0.16
57
Normal
90
0.74
0.05
0.07
67
n.d.
n.d.
Abbreviations: n.d., not done; VWF, von Willebrand factor.
Fig. 1 SDS agarose gel electrophoresis of von Willebrand factor multimers, visualized by
enzyme immunostaining after capillary transfer onto PVDF (polyvinylidene difluoride)
membranes. Multimeric analysis was performed by SDS-agarose gel electrophoresis in
1.0% (A ) and in 2.2% (B ) SDS-agarose gels (demonstrated for the boy).
Fig. 2 Molecular genetic analysis; NM_000552.3(VWF):c.5192[C > T];[=] (p.S1731L).
Table 2
In silico pathogenicity prediction for VWF variant p.Ser1731Leu
Program
Output
SIFT
Deleterious (score: 0.01, median: 3.34)
MutationTaster
Disease causing (prob: 1)
PolyPhen2 HumDiv
Probably damaging (score: 0.983)
CADD score
26.5
Abbreviation: VWF, von Willebrand factor.
