title: A splice site mutation in hERG leads to cryptic splicing in human long QT syndrome.
abstract: Mutations in the human ether-a-go-go-related gene (hERG) cause type 2 long QT syndrome. In this study, we investigated the pathogenic mechanism of the hERG splice site mutation 2398+1G>C and the genotype-phenotype relationship of mutation carriers in three unrelated kindreds with long QT syndrome. The effect of 2398+1G>C on mRNA splicing was studied by analysis of RNA isolated from lymphocytes of index patients and using minigenes expressed in HEK293 cells and neonatal rat ventricular myocytes. RT-PCR analysis revealed that the 2398+1G>C mutation disrupted the normal splicing and activated a cryptic splice donor site in intron 9, leading to the inclusion of 54 nt of the intron 9 sequence in hERG mRNA. The cryptic splicing resulted in an in-frame insertion of 18 amino acids in the middle of the cyclic nucleotide binding domain. In patch clamp experiments the splice mutant did not generate hERG current. Western blot and immunostaining studies showed that the mutant expressed an immature form of hERG protein that failed to reach the plasma membrane. Coexpression of the mutant and wild-type channels led to a dominant negative suppression of wild-type channel function by intracellular retention of heteromeric channels. Our results demonstrate that 2398+1G>C activates a cryptic site and generates a full-length hERG protein with an insertion of 18 amino acids, which leads to a trafficking defect of the mutant channel.
authors.affiliation: Division of Cardiovascular Medicine, Oregon Health and Science University, Portland, OR , USA.
authors.forename: Qiuming
authors.initials: Q
authors.lastname: Gong
authors.forename: Li
authors.initials: L
authors.lastname: Zhang
authors.forename: Arthur J
authors.initials: AJ
authors.lastname: Moss
authors.forename: G Michael
authors.initials: GM
authors.lastname: Vincent
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Ackerman
authors.forename: Jeffrey C
authors.initials: JC
authors.lastname: Robinson
authors.forename: Melanie A
authors.initials: MA
authors.lastname: Jones
authors.forename: David J
authors.initials: DJ
authors.lastname: Tester
authors.forename: Zhengfeng
authors.initials: Z
authors.lastname: Zhou
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D000831:Animals, Newborn; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D002478:Cells, Cultured; D000072237:ERG1 Potassium Channel; D051638:Ether-A-Go-Go Potassium Channels / Q000235:genetics* / Q000502:physiology; D006801:Humans; D047468:Immunoprecipitation; D008133:Long QT Syndrome / Q000235:genetics* / Q000473:pathology / Q000503:physiopathology; D008214:Lymphocytes / Q000378:metabolism; D008564:Membrane Potentials; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D032342:Muscle Cells / Q000166:cytology / Q000378:metabolism / Q000502:physiology; D009154:Mutation; D012326:RNA Splicing / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
chemical_list: D000072237:ERG1 Potassium Channel; D051638:Ether-A-Go-Go Potassium Channels; C000606913:KCNH2 protein, human; D012333:RNA, Messenger