A deep exon cryptic splice site promotes aberrant intron retention in a von Willebrand disease patient

A translationally silent single nucleotide mutation, in exon 44 of the von Willebrand factor (VWF) gene, is associated with inefficient removal of intron 44 in a von Willebrand disease (VWD) patient. This intron retention (IR) event was previously attributed to altered secondary structure that sequesters the normal splice donor site. We propose an alternative mechanism: that the mutation introduces a cryptic splice donor site that interferes with function of the annotated site to favor IR. We evaluated both models using minigene splicing reporters engineered to vary in secondary structure and/or cryptic splice site content. Analysis of reporter splicing efficiency in transfected K562 cells suggested that the mutation-generated internal splice site was sufficient to induce substantial IR. Mutations predicted to vary secondary structure at the annotated site had modest effects on IR, and also shifted the balance of residual splicing between the cryptic site and annotated site, supporting competition between the sites. Further studies demonstrated that introduction of cryptic splice donor motifs at other positions in E44 did not promote IR, indicating that interference with the annotated site is context-dependent. We conclude that mutant deep exon splice sites can interfere with proper splicing by inducing IR.