Sequensing of exon 28 of Von Willebrand factor in five patients with type 2 Von Willebrand disease

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Date
2009-05
Authors
Mothabeng, Maliengoane Sylvia
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Publisher
University of the Free State
Abstract
English: Von Willebrand disease (VWD) is a common bleeding disorder caused by either quantitative (type1 and 3) or qualitative (type 2) defects of von Willebrand factor (VWF). The diagnosis of VWD usually requires a panel of tests. Several analyses therefore are required to diagnose VWD. These tests are also subjected to pitfalls and it is important to take the pitfalls in to consideration when diagnosing VWD. Despite all these tests, the diagnosis and classification of VWD often remains a challenge. Identification of mutations that cause functional defects of VWF (type 2 VWD) is needed to improve the diagnosis of the disease. Mutations that cause functional abnormalities of VWF occur mostly in exon 28 of the VWF gene. Exon 28 primarily encodes the platelet GPIb and collagen binding domains of VWF (A1 domains) and the ADAMTS13 cleavage domain (A2 domains). Recently, studies in industrialised countries have been conducted on finding mutations on exon 28 but none have been done on South African populations. In this study we searched for mutations in exon 28 of the VWF gene in 5 patients with functional defects of VWF in order to set up the method for genetic analysis of VWD. We used two patients with type 2M, two with type 2B and one with type 2A VWD in this study. The whole exon 28 was analysed in four specific fragments, using PCR with primers that mismatch the pseudogene. The mutations were identified by automatic sequencing of the different fragments. The following polymorphisms were detected. A silent SNP 4641T/C in all five patients, the SNP 4141A/G in three patients, a silent SNP 3795G/A in one patient and a novel silent SNP 4923G/A in another patient. It is important to note that we found a novel SNP in an African patient with type 2B VWD, since no polymorphisms reported in exon 28 were from African populations. Several studies have proven the importance of mutational analysis is solving laboratory diagnosis paradox. The mutations found in the patients with type 2 VWD confirm the diagnosis and validates the importance of molecular diagnosis in VWD. With this study, we have successfully implemented a method to detect mutations in exon 28 of the VWF gene.
Afrikaans: Von Willebrand siekte (VWS) is die mees algemeenste bloedingsiekte wat veroorsaak word deur kwalitatiewe (tipe 1 en 3) and kwantitatiewe (tipe 2) afwykings van Von Willebrand factor (VWF). Die diagnose van VWS benodig ’n paneel van toetse en dus is verskeie toetse nodig om die siekte te diagnoseer. Hierdie toetse het ook tekortkominge en dit is nodig om hierdie tekortkominge in aanmerking te neem wanneer VWS gediagnoseer word. Die diagnose en klassifikasie van VWS by steeds ’n uitdaging, nieteenstaande dat al hierdie toetse beskikbaar is. Dit is nodig om mutasies wat funksionele defekte van VWF veroorsaak, te identifiseer om sodoende die diagnose van VWS te verbeter. Sulke mutasies kom meestal in ekson 28 van die VWF geen voor. Ekson 28 kodeer vir die plaatjie- en kollageen bindingsdomeine van VWF, asook die ADAMTS13 snydingsdomein. Studies waar daar na mutasies in ekson 28 van die VWF geen gesoek is, is slegs in ontwikkelende lande gedoen en geen so ’n studie is nog op Suid Afrikaanse of Afrika populasies gedoen nie. Ons het in hierdie studie na mutasies in ekson 28 van die VWF geen gesoek in 5 pasiënte met funksionele defekte van VWF. Dit is uitgevoer om die metode daar te stel wat gebruik kan word om genetiese analise in VWS te bepaal. Ons het twee pasiënte met tipe 2M, twee met tipe 2B an een met tipe 2A VWS in die studie ingesluit. Die hele ekson 28 is in 4 spesifieke fragmente ge-analiseer deur polimerase-kettingreaksies te doen met pylstukke wat van die pseudogeen verskil. Ons het die volgende enkel-nukleotied-polimorfismes (ENP) gevind. Die 4641T/C ENP in al vyf pasiënte, die 4141A/G ENP in 3 pasiënte, die 3795G/A ENP in een pasiënt en ‘n nuwe ENP 4923G/A in ‘n ander pasiënt. Dis is van belang dat hierdie nuwe ENP in ’n swart pasiënt met tipe 2B VWS gevind is, aangesien nog geen ENP’s in VWF ekson 28 van swart pasiënte beskryf is nie. Verskeie studies het al die belang van mutasie-analise aangetoon in die oorkoming van die diagnostiese uitdagings van VWS. Die mutasies wat in ons type 2 VWS pasiënte gevind is, bevestig die belang van molekulere diagnose in VWS. Ons het die metode om mutasies in ekson28 van die VWF geen aan te toon suksesvol met hierdie studie geimplementeer.
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Dissertation (M.Med.Sc. (Haematology and Cell Biology))--University of the Free State, 2009, Von Willebrand disease, Blood coagulation disorders -- Genetic aspects, Von Willebrand factor, Diagnosis, Laboratory, Molecular pathogenesis, Inherited bleeding disorders, Mutations, Polymorphisms, Genetics
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