Integration of molecular and clinical data of 40 unrelated von Willebrand Disease families in a Spanish locus-specific mutation database: first release including 58 mutations

The VWF gene (VWF) extends along 178 kb in the genome and comprises a total of 52 exons, whose sizes range from 40 to 1400 bp.1 Systematic identification of the responsible mutations has been hampered by the large size and complex genomic organization of VWF, which is highly polymorphic and has a highly homologous (>96%) partial pseudogene in chromosome 22.2,3 For several years, our group has been involved in the genetic study of the VWF, and this effort led to the design and optimization of a technique for complete sequencing of all exons and the intronic flanking regions of the gene, with very favorable results.4 Herein, we present the VWF Mutation Registry at Hemobase (VWFdb@Hemobase), a freely accessible online database (http://www.vwf.hemo-base.com) developed to facilitate rapid publication, access to, and interpretation of the VWF mutations identified in our laboratory.

All the records in the database contain data from genetic testing carried out at the Molecular Diagnosis and Therapy Unit of the Blood and Tissue Bank in patients clinically diagnosed, mainly at the Hemophilia Unit of Vall d'Hebron Hospital (Barcelona), but also at other Spanish hospitals. All patients were diagnosed as having VWD and met the ISTH criteria.5,6 Genetic testing of patients and relatives was approved by the Ethics Committee of Vall d’Hebron Hospital, and all participants provided their informed consent. Genomic DNA was extracted and the complete VWF of the index case of unrelated families was amplified and sequenced with a previously described method.4 The numbering and nomenclature used for the mutations identified follow the recommendations of the International Society on Thrombosis and Haemostasis (ISTH) Scientific Subcommittee on von Willebrand Disease.7 It is the same as that applied in the ISTH SSC VWF Database (http://www.vwf.group.shef.ac.uk/), thus facilitating comparison between the two databases and with previous publications.

The VWFdb@Hemobase is published online (Figure 1) as a subdomain of Hemobase.com, a website that hosts the Spanish hemophilia A and B mutation databases. The content and structure follow several recommendations from the Human Genome Variation Society (HGVS)8,9 for locus-specific mutation database (LSDB) design and curation: covers many aspects of the variants, provides quality assessment for each entry, and contains information about the disease and resulting clinical phenotype. In this first release of the database, 93 individuals are included: 40 index cases and 53 relatives. A total of 58 mutations identified are distributed over the entire gene (Online Supplementary Figure S1): 11 type 1, 15 type 2A, 2 type 2M, 3 type 2N, one type 2 unclassified and 26 type 3. There were 44 substitutions, including 5 nonsense mutations and 6 mutations involved in a single gene conversion event, one indel, 2 insertions, and 11 potential splice site mutations (PSSM). The database contains 19 mutations first described by our group, 12 of which were reported in a previous publication;4 the remaining 7 are presented herein including molecular and phenotypic data (Table 1). These mutations (4 missense, 2 PSSM, and one insertion) were found in 3 type 1, 3 type 3, and one type 2 patient.

Table 1.Summary of molecular and clinical data associated to new mutations and their location in the VWF gene. Results of the in silico analysis of missense and potential splice site mutations.

Without the help of functional studies, it is often difficult to decide whether a particular sequence variant is associated with disease or represents a rare polymorphism with no deleterious effect. In this regard, molecular studies are being conducted at the mRNA level to determine the true impact of PSSM (I Corrales, L Ramírez, R Parra and F Vidal, manuscript in preparation). However, software tools can provide useful information before in-depth mutation analysis is initiated. The results of in silico analysis of new mutations are shown in Table 1. The predicted impact of the novel missense mutations described was evaluated with PolyPhen (http://genetics.bwh.harvard.edu/pph/) and the PSSM with NetGene2 (http://www.cbs.dtu.dk/services/NetGene2/). Because of the limitations of in silico tools10 a second program was used to perform the analysis: Sort Intolerant from Tolerant (SIFT; http://blocks.fhcrc.org/sift/SIFT.html) for missense mutations and Human Splicing Finder (HSF; http://www.umd.be/SSF) for PSSM.

Figure 1.Screenshot of the web page for clinical data (http://www.vwf.hemobase.com), showing the patient-specific clinical and coagulation variables (blood group, laboratory coagulation parameters, symptoms, sex, geographical origin, etc.). The related molecular data (A) and family tree with inheritance information (B) can be easily consulted from this page by direct access with pop-up windows. The emergent molecular data table summarizes the significant information about the mutation(s) found in the patient. In addition, there is a link to PubMed entries of the articles reporting this variant. The website was built using the template-based website design tool RapidWeaver 4.1 (Realmac Software). The VWF database was developed using MS Access software (Microsoft Corporation), and ASPRunnerPro (Xlinesoft), a database management tool, was used to create a set of Active Server Pages to access, modify, display, and navigate the data on the web.

An excellent International database of VWF mutations has been assembled by the University of Sheffield, UK.11 All the mutations contained in the database have been sent (and will be submitted in the future) to the ISTH VWF database. There had been only 18 entries from Spanish laboratories before our participation with 41 different (58 total) mutations, which made us a significant single-laboratory contributor to the database. The ISTH VWF database provides users with a rich repository of mutations from worldwide contributing researchers, but its aim differs somewhat from ours. The VWFdb@Hemobase provides additional supplementary data that is not compiled by the international database, such as tabulated laboratory studies, STR gene tracking and pedigree representation of mutation and disease inheritance in the families recorded (Figure 1).

A major feature in designing the database user interface was the capability to browse and search for mutations in an intuitive and straightforward manner. Users of the VWFdb@Hemobase can view the information via 4 different main web pages: the advanced search page, a fast route to find mutations; the clinical data page, showing relevant patient-specific clinical and laboratory parameters; the molecular data page, which includes all mutation information in an individualized manner; and the international VWF database page, which corresponds to an up-to-date copy of the ISTH SSC VWF Database.11 An advanced search system allows easy retrieval of any mutation in the database. The VWFdb@Hemobase also includes general facts about VWD, the classification system into subtypes, clinical features of the disease, diagnostic difficulties involved, and the biochemical and molecular characteristics of VWF.

In conclusion, we present an online VWD LSDB containing the putative mutations responsible for VWD identified in several families after complete VWF sequencing of the index case. This database endeavors to assure that the relevant data obtained from molecular study of VWD patients will contribute to a better understanding of the mechanisms involved in the pathophysiology of this disease, providing a dynamic view of the molecular epidemiology of VWD in our population. Furthermore, it is a practical instrument to maintain updated information for clinicians and researchers,8 and to offer fast and simple access to new data that will grow rapidly with the introduction of the next generation sequencing platforms.12

Footnotes

• Funding: this work was supported in part with a grant (FIS, PI080385) from the Fondo de Investigaciones Sanitarias, Spanish Ministry of Science and Innovation, and by the Fundació Privada Catalana de l’Hemofília.