Alterations of mRNA processing and stability as a pathogenic mechanism in von Willebrand factor quantitative deficiencies

Introduction: von Willebrand disease (VWD) is an inherited bleeding disorder due to a deficiency or abnormality of von Willebrand factor (VWF), associated with heterogeneous phenotypes. While VWD mutations acting at the protein level have been deeply investigated, fewer data are available on genetic defects affecting VWF mRNA. Aim: The aim of this study was to characterize the molecular mechanism underlying VWD in three patients. Methods: Mutational screening of the patients (P1–3) was accomplished by DNA sequencing of all VWF exons and splicing junctions. Platelet mRNA was analyzed by reverse‐transcription (RT)‐PCR and real‐time RT‐PCR. Results: P1 is a compound heterozygote for a c.1534‐3C>A transversion in intron 13 and for a nonsense mutation (p.Q77X) in exon 4. P2 is heterozygous for a splicing mutation in intron 9 (c.1109+2T>C). RT‐PCR assays on the patient’s platelet RNA revealed three mRNA populations: (i) wild type; (ii) lacking exon 9; and (iii) lacking exons 8 and 9. P3 showed a novel homozygous splicing mutation in intron 46 (c.7770+1G>T), producing three different mRNA species: (i) retaining the first 25 bp of intron 46; (ii) skipping exon 46; and (iii) skipping exon 46 while retaining 5 bp of intron 45. Whenever possible, the effect of mutations on the levels of VWF transcripts was analyzed, showing that mRNA variants containing a premature termination codon are downregulated, probably by the nonsense‐mediated mRNA decay pathway. Conclusions: The identification of the genetic basis of VWD in three patients confirmed that mutations leading to null alleles in the VWF gene are associated with allele‐specific mRNA degradation.