Intron retention resulting from a silent mutation in the VWF gene that structurally influences the 5′ splice site

Abstract

Key Points• This study demonstrates allosteric RNA structure alteration resulting from an exonic variation, thereby interfering with splicing.• This study details a novel mechanism by which silent mutation distant to the 59 splice site could still result in intron retention.Disease-associated silent mutations are considered to affect the accurate premessenger RNA (mRNA) splicing either by influencing regulatory elements, leading to exon skipping, or by creating a new cryptic splice site. This study describes a new molecular pathological mechanism by which a silent mutation inhibits splicing and leads to intron retention. We identified a heterozygous silent mutation, c.7464C>T, in exon 44 of the von Willebrand factor (VWF) gene in a family with type 1 von Willebrand disease. In vivo and ex vivo transcript analysis revealed an aberrantly spliced transcript, with intron 44 retained in the mRNA, implying disruption of the first catalytic step of splicing at the 59 splice site (59ss). The abnormal transcript with the retained intronic region coded a truncated protein that lacked the carboxy-terminal end of the VWF protein. Confocal immunofluorescence characterizations of blood outgrowth endothelial cells derived from the patient confirmed the presence of the truncated protein by demonstrating accumulation of VWF in the endoplasmic reticulum. In silico pre-mRNA secondary and tertiary structure analysis revealed that this substitution, despite its distal position from the 59ss (85 bp downstream), induces cis alterations in pre-mRNA structure that result in the formation of a stable hairpin at the 59ss. This hairpin sequesters the 59ss residues involved in U1 small nuclear RNA interactions, thereby inhibiting excision of the pre-mRNA intronic region. This study is the first to show the allosteric-like/far-reaching effect of an exonic variation on pre-mRNA splicing that is mediated by structural changes in the pre-mRNA. (Blood. 2016;128(17):2144-2152

Intron retention as a component of regulated gene expression programs

“…AONs can also be employed to modulate IR in different contexts and channel decay or protection mechanisms that enable control over mRNA and consequently protein expression. Careful design of AONs to block splice sites or mutations that induce IR (Yadegari et al 2016 ) would be feasible to target specific introns (Kralovicova et al 2014 , 2016 ). Analysis of the mechanisms of regulation of cassette exons benefitted hugely from the ability to curate sets of tightly co-regulated events from mRNA-Seq data, followed by computational deciphering of the key features of these events, and detailed molecular dissection of individual events that exemplify the co-regulated program (Barash et al 2010 ; Chen and Manley 2009 ; Fu and Ares 2014 ; Matlin et al 2005 ).…”

The genetic background of antibiotic resistance among clinical uropathogenic Escherichia coli strains

“…Also, the antibiotics, especially fluoroquinolones can induce the response of SOS systems, which can be responsible for DNA changes in bacteria genome [ 78 , 79 ]. There is some evidence that silence mutations might cause a phenotypic effect, they can especially have an influence on the regulation of transcription [ 80 – 82 ]; possibly they can also change the affinity of the hot spot to fluoroquinolones. Nevertheless, we can conclude that the hot spot of parC is more specific but less sensitive to fluoroquinolones (more silent mutations), whereas gyrA conversely—a lot of missense mutations give the phenotypic effect but not in the hot spot of gyrA .…”

Update on Molecular Testing in von Willebrand Disease

“…In addition to splice site consensus sequence mutations, deep intronic, nonsense, missense, and synonymous mutations in VWF can also lead to a disturbing effect in splicing. [64][65][66] It has long been suspected, but not shown systematically, that the pathogenic effect of these variants is related to alterations on splicing. Historically, missense mutations have been interpreted as protein-altering variants and the synonymous variants as "benign," thereby potentially leading to an underappreciation of the frequency of the aberrant splicing as a pathologic mechanism in these types of mutations.…”