A Novel Frameshift Mutation Results in the Absence of VWF mRNA: The Utility of Quantitative Analysis in the Diagnosis of Type 1 VWD.
Authors: Hadonou Medard , Cutler Jacky A. , Savidge Geoffrey F. , Madan Bella , Mitchell Mike J. 
Published: April 2020

Abstract
Von Willebrand disease (VWD) is an autosomal bleeding disorder caused by qualitative (type 2 VWD) or quantitative (type 1 and type 3 VWD) defects of plasma VWF: complete absence of VWF from plasma distinguishes type 3 from the partial deficiency type 1 VWD. It is often difficult to assign a definitive diagnosis of type 1 VWD due to variable penetrance and phenotype and the influence of other factors, such as ABO blood group. Mutation analysis, identifying a causative mutation, can provide confirmation of a diagnosis of type 1 VWD, which may help with patient management and family studies. The large size of the VWF gene (~180 kb, 52 exons) makes mutation analysis difficult and time consuming at the DNA level, as type 1 mutations are heterogeneous and spread throughout the gene. We have developed an RNA based approach, which allows the amplification of the entire VWF mRNA (~9 kb) in 13 overlapping PCR fragments. However, this approach is dependent on the expression of a stable mRNA containing the mutation.
A mother and son with suspected type 1 VWD were referred for mutation detection, and confirmation of diagnosis. Analysis of the mother by RT-PCR of ectopic transcript from leukocyte RNA, and sequencing of the entire coding region of the VWF gene failed to identify a causative mutation. She was apparently homozygous for all known SNPs throughout the gene. Quantitative RNA analysis was performed on an ABI 7500 and demonstrated a significant reduction in the amount of VWF mRNA in the patient compared to normal controls when normalised against a second autosomal ectopic transcript. This suggested that VWF mRNA may be only present from one allele. Sequence analysis, following RT-PCR, of the son showed him to be homozygous for the alternate allele for two SNPs located within exon 18 of the VWF gene, indicating no maternal contribution. Subsequent genomic analysis of exon 18 showed the son to be heterozygous for both SNPs and also for a single nucleotide insertion (c.2438dupG). Sequence analysis at the genomic level confirmed heterozygosity for the insertion in the mother. The identified insertion results in the production of a premature termination codon (PTC) at codon 818 in exon 19. However as the mutation is located very close to the exon 18 donor splice junction the possibility of it affecting splicing was also considered. Aberrant splicing had not been detected in the original RNA analysis, and splice site prediction software did not suggest any reduction in splicing efficiency. The presence of a PTC suggests nonsense-mediated decay (NMD) as the probable mechanism for the absence of mRNA from this allele.
Subsequent mutation analysis of unrelated patients identified the presence of c.2438dupG in one other patient, suggesting that this mutation is not uncommon. Mutations resulting in a null allele have been shown to be highly prevalent in VWD and it is likely that many are associated with the absence of expressed mRNA. This has been demonstrated in at least one other nonsense mutation, R2535X (Eikenboom et al). RNA analysis has been shown to have many advantages over genomic sequencing, but is reliant on the presence of stably expressed transcripts from both alleles. Quantitative RNA analysis should therefore be considered as a front line approach in the molecular diagnosis of type 1 VWD.