An RNA Based Approach to vWD Type 1 Mutation Analysis.

Von Willebrand disease (vWD) (MIM 193400) is a heterogeneous bleeding disorder caused by both quantitative (type 1 and 3) and qualitative (type 2) defects of von Willebrand factor. Whilst type 2 vWD has been reasonably well studied, due to the localisation of mutations within specific domains of the vWF protein, the large size (~178 kb spread over 52 exons) and highly polymorphic nature of the gene, together with the heterogeneity of mutations has made the study of the molecular basis of type 1 vWD difficult using conventional approaches to mutation analysis such as dHPLC or CSGE.

We have developed an RNA based approach which allows the amplification of the entire vWF mRNA (~9 kb) in 13 overlapping PCR fragments, ranging in size from 590 bp to 1040 bp. Following reverse transcription, the 13 fragments are PCR amplified and subjected to direct sequence analysis on an ABI-3100. ‘Pre-screening’ using a technique such as dHPLC or CSGE was not considered to be advantageous due to the highly polymorphic nature of the gene. The use of sophisticated sequence alignment and comparison software (SeqScape^®, Applied Biosystems), which allows known polymorphisms to be highlighted on the reference sequence, enables a very rapid screening of the reverse transcribed vWF mRNA. Analysis at the RNA level has the additional advantage of eliminating interference from the vWF pseudogene which is not transcribed. One potential disadvantage of an RNA based approach to mutation screening is that transcript from the mutant allele may not be present, as may result from nonsense-mediated decay of mRNAs containing Stop codons, unstable transcripts resulting from aberrant splicing or large deletions. The highly polymorphic nature of the vWF gene may help to demonstrate the presence of two alleles by showing heterozygosity at various loci. However, we routinely performed a quantitative RT-PCR assay on an ABI-7700 to prove that RNA was being expressed from both alleles.

Early studies have identified causative mutations in 6 patients. Three patients were found to be heterozygous for missense mutations, one previously reported (Pro2063Ser) and two novel (Cys1225Trp and Ile2177Val). One patient was heterozygous for a previously reported nonsense mutation, Arg2434Term. A severely deficient type 3 patient was homozygous for the absence of exon 50 from her vWF mRNA, the result of a T→ A change in the intron 50 donor splice site.

The final patient was heterozygous for a previously reported missense mutation associated with a type 2A phenotype (Ile1628Thr). A further two patients have been studied with no causative mutation being identified, providing further support for the fact that not all cases of phenotypic type 1 vWD are due to a defect in the vWF gene.

We believe that an RNA based approach makes the study of the molecular basis of type 1 vWD more feasible, allowing confirmation of the diagnosis and subsequent family studies in many cases. This could potentially provide an additional diagnostic tool in what is a difficult area clinically. The identification of novel type 1 vWD mutations will also help enhance our understanding of the molecular basis of vWD.