A Deep Exon Cryptic Splice Site Promotes Aberrant Intron Retention in a Von Willebrand Disease Patient

Abstract

A translationally silent single nucleotide mutation in exon 44 (E44) 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 reordered E44 secondary structure that sequesters the normal splice donor site. We propose an alternative mechanism: the mutation introduces a cryptic splice donor site that interferes with the 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 splicing efficiency in transfected K562 cells suggested that the mutation-generated cryptic splice site in E44 was sufficient to induce substantial IR. Mutations predicted to vary secondary structure at the annotated site also had modest effects on IR and shifted the balance of residual splicing between the cryptic site and annotated site, supporting competition among 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.

Case Report: A Novel Intronic Mutation in AIFM1 Associated With Fatal Encephalomyopathy and Mitochondrial Disease in Infant

“…Most of these splicing mutations occur within or close to conserved consensus donor (+1/+2) or acceptor (−1/−2) splice sites ( 17 ). However, mutations remote from the splice donor or acceptor sites have also been shown to lead to aberrant splicing in a number of reports ( 18 – 20 ). Thus, intronic mutations particularly within the splice sites (∼20 bp) must be considered more readily when evaluating sequence variants, especially when tributary mutation in coding regions or canonical splice sites cannot be found.…”

A Homozygous Deep Intronic Variant Causes Von Willebrand Factor Deficiency and Lack of Endothelial-Specific Secretory Organelles, Weibel–Palade Bodies

“…However, this number might be undervalued due to the lack of transcript assays. The previously reported VWD-associated splicing mutations occur within or close to the conserved consensus 5′ donor or 3′ ss (e.g., c.323 + 1 G > T, or c.2547−13 T > A), as well as exonic modulatory splicing elements, which cause either exon skipping or intron retention [ 30 , 31 , 32 , 33 , 34 ]. All these VWD-causing splicing mutations were detected by Sanger sequencing or targeted NGS, which cover exons and exon–intron junctions (usually up to 50 bp of introns).…”