title: Intron retention resulting from a silent mutation in the VWF gene that structurally influences the 5' splice site.
abstract: Disease-associated silent mutations are considered to affect the accurate pre-messenger 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 5' splice site (5'ss). 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 5'ss (85 bp downstream), induces cis alterations in pre-mRNA structure that result in the formation of a stable hairpin at the 5'ss. This hairpin sequesters the 5'ss 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.
authors.affiliation: Institute of Experimental Haematology and Transfusion Medicine, University Clinic Bonn, Bonn, Germany.
authors.forename: Hamideh
authors.identifier: 0000-0002-2006-8497
authors.initials: H
authors.lastname: Yadegari
authors.affiliation: Institute of Experimental Haematology and Transfusion Medicine, University Clinic Bonn, Bonn, Germany.
authors.forename: Arijit
authors.identifier: 0000-0002-4103-5854
authors.initials: A
authors.lastname: Biswas
authors.affiliation: Institute of Experimental Haematology and Transfusion Medicine, University Clinic Bonn, Bonn, Germany.
authors.forename: Mohammad Suhail
authors.initials: MS
authors.lastname: Akhter
authors.affiliation: Institute of Experimental Haematology and Transfusion Medicine, University Clinic Bonn, Bonn, Germany.
authors.forename: Julia
authors.initials: J
authors.lastname: Driesen
authors.affiliation: Institute of Experimental Haematology and Transfusion Medicine, University Clinic Bonn, Bonn, Germany.
authors.forename: Vytautas
authors.initials: V
authors.lastname: Ivaskevicius
authors.affiliation: Institute of Experimental Haematology and Transfusion Medicine, University Clinic Bonn, Bonn, Germany.
authors.forename: Natascha
authors.initials: N
authors.lastname: Marquardt
authors.affiliation: Institute of Experimental Haematology and Transfusion Medicine, University Clinic Bonn, Bonn, Germany.
authors.forename: Johannes
authors.identifier: 0000-0003-2655-3503
authors.initials: J
authors.lastname: Oldenburg
mesh_terms: D002648:Child; D005260:Female; D006801:Humans; D007438:Introns / Q000235:genetics*; D018613:Microscopy, Confocal; D062105:Molecular Docking Simulation; D060885:Multiplex Polymerase Chain Reaction; D016133:Polymerase Chain Reaction; D022821:RNA Splice Sites / Q000235:genetics*; D012326:RNA Splicing / Q000235:genetics; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics; D000069456:Silent Mutation / Q000235:genetics; D056725:von Willebrand Disease, Type 1 / Q000235:genetics*; D014841:von Willebrand Factor / Q000235:genetics*
chemical_list: D022821:RNA Splice Sites; D012333:RNA, Messenger; D014841:von Willebrand Factor