A synonymous (c.3390 C > T ) or a splice‐site (c.3380‐2 A > G ) mutation causes exon 26 skipping in four patients with von W illebrand disease (2 A / IIE )

Summary Introduction We characterized four unrelated patients with von W illebrand disease type 2 A / IIE , sharing the same von W illebrand factor ( VWF ) in‐frame deletion (p.[ P 1127_ G 1180delins R ];[=]) resulting from exon 26 skipping (Δ26). Objectives To identify the VWF mutations and how they caused the m RNA splicing alteration, to evaluate the deletion by in vitro expression studies, and to assess whether or not the heterogeneity of the patients’ phenotype might be related to a different degree of expression of the deleted subunit in patient plasma VWF . Methods Sequence analysis was performed with patient genomic DNA and platelet m RNA . Semiquantitative RT ‐ PCR was also carried out to compare the expression of the wild‐type ( WT ) and Δ26 alleles in the four patients. In silico analysis was performed with prediction splicing programs. Expression studies were performed to evaluate mutant recombinant VWF (r VWF ) (Δ26 and Δ26/ WT ) as compared with WT r VWF . Results Three patients shared the synonymous single‐nucleotide substitution ( SSS ) c.[3390 C > T ];[=], whereas the novel mutation c.[3380‐2 A > G ];[=] was present in the fourth patient. Semiquantitative RT ‐ PCR of platelet m RNA revealed a different ratio of the WT and Δ26 alleles in the patients, consistent with the different VWF : FVIIIB values present in patient plasma. Expression studies confirmed reduced VWF – FVIII binding of r VWF ‐Δ26/ WT . Conclusions SSS can induce alternative splicing, and those like c.3390 C > T , which impact on the poorly conserved splicing regulatory elements, are difficult to predict, so that their role can be evaluated only by m RNA analysis. Moreover, these mutations seem to have different effects on the efficiency of alternative splicing, producing heterogeneous VWF variants among the four patients.