Mutation distribution in the von Willebrand factor gene related to the different von Willebrand disease (VWD) types in a cohort of VWD patients

Abstract

Von Willebrand disease (VWD) is the most common inherited bleeding disorder caused by quantitative or qualitative defects of the von Willebrand factor (VWF). VWD is classified into three types--type 1 (partial quantitative deficiencies), type 2 (qualitative defects) and type 3 (complete deficiency of VWF). In this study we explored genotype and phenotype characteristics of patients with VWD with the aim of dissecting the distribution of mutations in different types of VWD. One hundred fourteen patients belonging to 78 families diagnosed to have VWD were studied. Mutation analysis was performed by direct sequencing of the VWF . Large deletions were investigated by multiplex ligation-dependent probe amplification (MLPA) analysis. The impact of novel candidate missense mutations and potential splice site mutations was predicted by in silico assessments. We identified mutations in 66 index patients (IPs) (84.6%). Mutation detection rate was 68%, 94% and 94% for VWD type 1, 2 and 3, respectively. In total, 68 different putative mutations were detected comprising 37 missense mutations (54.4%), 10 small deletions (14.7%), two small insertions (2.9%), seven nonsense mutations (10.3%), five splice-site mutations (7.4%), six large deletions (8.8%) and one silent mutation (1.5%). Twenty-six of these mutations were novel. Furthermore, in type 1 and type 2 VWD, the majority of identified mutations (74% vs. 88.1%) were missense substitutions while mutations in type 3 VWD mostly caused null alleles (82%). Genotyping in VWD is a helpful tool to further elucidate the pathogenesis of VWD and to establish the relationship between genotype and phenotype.

Intron retention resulting from a silent mutation in the VWF gene that structurally influences the 5′ splice site

“…[19][20][21] We had previously reported a translationally silent mutation c.7464C.T (p.Gly2488) in exon 44 of VWF, 25 bp after the 39ss and 85 bp downstream of the 59ss, in a patient with type 1 VWD. 22 In the present study, in vivo and ex vivo transcript analysis unexpectedly revealed that it inhibited splicing at the 59 donor splice site, despite its 85-bp distance to the 59ss, and led to an aberrant transcript with a retained intronic region in mature mRNA. The previously described mechanisms for exonic substitutions, such as contribution to cisregulatory elements, could not provide a plausible explanation for this event.…”

Clinical and laboratory variability in a cohort of patients diagnosed with type 1 VWD in the United States

“…38 However, our reported frequency of VWF sequence variations of 62% in all subjects with type 1 VWD is similar to that reported in several other studies, including the United Kingdom, Canadian, European Union, and German studies. [12][13][14]39 Four subjects with VWF sequence variations had large deletions that would not have been picked up on conventional sequencing but were picked up via comparative genomic hybridization.…”

Insights into pathological mechanisms of missense mutations in C-terminal domains of von Willebrand factor causing qualitative or quantitative von Willebrand disease

“…16 Interestingly, these gene alterations were associated with either quantitative (types 1 and 3) or qualitative VWD (type 2A). We transiently expressed the mutations in vitro, and characterized their effect on multimer assembly, biogenesis of WPB and secretion.…”

“…17 Laboratory investigations of VWF antigen (VWF:Ag), VWF ristocetin cofactor activity (VWF:RCo), factor VIII coagulant activity (FVIII:C) and VWF multimers [1.2% (w/v) and 1.6% (w/v) agarose gels] were performed as previously described. 16,18 All mutations were identified by direct sequencing of the VWF coding region as previously described. 16 This study was approved by the local ethics committee and informed consent was obtained from all patients.…”

“…16,18 All mutations were identified by direct sequencing of the VWF coding region as previously described. 16 This study was approved by the local ethics committee and informed consent was obtained from all patients. …”