A splice site mutation in hERG leads to cryptic splicing in human long QT syndrome

Qiuming Gong, Li Zhang, Arthur J. Moss, G. Michael Vincent, Michael J. Ackerman, Jeffrey C. Robinson, Melanie A. Jones, David J. Tester, Zhengfeng Zhou

    Research output: Contribution to journalArticle

    18 Citations (Scopus)

    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.

    Original languageEnglish (US)
    Pages (from-to)502-509
    Number of pages8
    JournalJournal of Molecular and Cellular Cardiology
    Volume44
    Issue number3
    DOIs
    StatePublished - Mar 2008

    Fingerprint

    Long QT Syndrome
    Ether
    RNA Splice Sites
    Mutation
    Introns
    Genes
    Amino Acids
    Messenger RNA
    HEK293 Cells
    Cyclic Nucleotides
    Muscle Cells
    Proteins
    Western Blotting
    Genotype
    Cell Membrane
    Lymphocytes
    RNA
    Phenotype
    Polymerase Chain Reaction
    human KCNH2 protein

    Keywords

    • Arrhythmia
    • Long QT syndrome
    • Myocytes
    • Splicing mutation
    • Sudden death

    ASJC Scopus subject areas

    • Molecular Biology
    • Cardiology and Cardiovascular Medicine

    Cite this

    A splice site mutation in hERG leads to cryptic splicing in human long QT syndrome. / Gong, Qiuming; Zhang, Li; Moss, Arthur J.; Vincent, G. Michael; Ackerman, Michael J.; Robinson, Jeffrey C.; Jones, Melanie A.; Tester, David J.; Zhou, Zhengfeng.

    In: Journal of Molecular and Cellular Cardiology, Vol. 44, No. 3, 03.2008, p. 502-509.

    Research output: Contribution to journalArticle

    Gong, Q, Zhang, L, Moss, AJ, Vincent, GM, Ackerman, MJ, Robinson, JC, Jones, MA, Tester, DJ & Zhou, Z 2008, 'A splice site mutation in hERG leads to cryptic splicing in human long QT syndrome', Journal of Molecular and Cellular Cardiology, vol. 44, no. 3, pp. 502-509. https://doi.org/10.1016/j.yjmcc.2008.01.002
    Gong, Qiuming ; Zhang, Li ; Moss, Arthur J. ; Vincent, G. Michael ; Ackerman, Michael J. ; Robinson, Jeffrey C. ; Jones, Melanie A. ; Tester, David J. ; Zhou, Zhengfeng. / A splice site mutation in hERG leads to cryptic splicing in human long QT syndrome. In: Journal of Molecular and Cellular Cardiology. 2008 ; Vol. 44, No. 3. pp. 502-509.
    @article{77b4aece54a647e0bfcdd171511aa392,
    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.",
    keywords = "Arrhythmia, Long QT syndrome, Myocytes, Splicing mutation, Sudden death",
    author = "Qiuming Gong and Li Zhang and Moss, {Arthur J.} and Vincent, {G. Michael} and Ackerman, {Michael J.} and Robinson, {Jeffrey C.} and Jones, {Melanie A.} and Tester, {David J.} and Zhengfeng Zhou",
    year = "2008",
    month = "3",
    doi = "10.1016/j.yjmcc.2008.01.002",
    language = "English (US)",
    volume = "44",
    pages = "502--509",
    journal = "Journal of Molecular and Cellular Cardiology",
    issn = "0022-2828",
    publisher = "Academic Press Inc.",
    number = "3",

    }

    TY - JOUR

    T1 - A splice site mutation in hERG leads to cryptic splicing in human long QT syndrome

    AU - Gong, Qiuming

    AU - Zhang, Li

    AU - Moss, Arthur J.

    AU - Vincent, G. Michael

    AU - Ackerman, Michael J.

    AU - Robinson, Jeffrey C.

    AU - Jones, Melanie A.

    AU - Tester, David J.

    AU - Zhou, Zhengfeng

    PY - 2008/3

    Y1 - 2008/3

    N2 - 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.

    AB - 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.

    KW - Arrhythmia

    KW - Long QT syndrome

    KW - Myocytes

    KW - Splicing mutation

    KW - Sudden death

    UR - http://www.scopus.com/inward/record.url?scp=40649127397&partnerID=8YFLogxK

    UR - http://www.scopus.com/inward/citedby.url?scp=40649127397&partnerID=8YFLogxK

    U2 - 10.1016/j.yjmcc.2008.01.002

    DO - 10.1016/j.yjmcc.2008.01.002

    M3 - Article

    C2 - 18272172

    AN - SCOPUS:40649127397

    VL - 44

    SP - 502

    EP - 509

    JO - Journal of Molecular and Cellular Cardiology

    JF - Journal of Molecular and Cellular Cardiology

    SN - 0022-2828

    IS - 3

    ER -