Multiple splicing defects caused by hERG splice site mutation 2592+1G>A associated with long QT syndrome

Matthew R. Stump, Qiuming Gong, Zhengfeng Zhou

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    6 Citations (Scopus)

    Abstract

    Long QT syndrome type 2 (LQT2) is caused by mutations in the human ether-a-go-go-related gene (hERG). Cryptic splice site activation in hERG has recently been identified as a novel pathogenic mechanism of LQT2. In this report, we characterize a hERG splice site mutation, 2592+1G>A, which occurs at the 5= splice site of intron 10. Reverse transcription-PCR analyses using hERG minigenes transfected into human embryonic kidney-293 cells and HL-1 cardiomyocytes revealed that the 2592+1G>A mutation disrupted normal splicing and caused multiple splicing defects: the activation of cryptic splice sites within exon 10 and intron 10 and complete intron 10 retention. We performed functional and biochemical analyses of the major splice product, hERGΔ24, in which 24 amino acids within the cyclic nucleotide binding domain of the hERG channel COOH-terminus is deleted. Patch-clamp experiments revealed that the splice mutant did not generate hERG current. Western blot and immunostaining studies showed that mutant channels did not traffic to the cell surface. Coexpression of wild-type hERG and hERGΔ24 resulted in signifi-cant dominant-negative suppression of hERG current via the intracellular retention of the wild-type channels. Our results demonstrate that 2592+1G>A causes multiple splicing defects, consistent with the pathogenic mechanisms of long QT syndrome.

    Original languageEnglish (US)
    JournalAmerican Journal of Physiology - Heart and Circulatory Physiology
    Volume300
    Issue number1
    DOIs
    StatePublished - Jan 2011

    Fingerprint

    Long QT Syndrome
    Ether
    Mutation
    Genes
    RNA Splice Sites
    Introns
    Cyclic Nucleotides
    Cardiac Myocytes
    Reverse Transcription
    Exons
    Western Blotting
    Kidney
    Amino Acids
    Polymerase Chain Reaction

    Keywords

    • Ion channels
    • Protein trafficking
    • RNA splicing

    ASJC Scopus subject areas

    • Physiology
    • Physiology (medical)
    • Cardiology and Cardiovascular Medicine

    Cite this

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    abstract = "Long QT syndrome type 2 (LQT2) is caused by mutations in the human ether-a-go-go-related gene (hERG). Cryptic splice site activation in hERG has recently been identified as a novel pathogenic mechanism of LQT2. In this report, we characterize a hERG splice site mutation, 2592+1G>A, which occurs at the 5= splice site of intron 10. Reverse transcription-PCR analyses using hERG minigenes transfected into human embryonic kidney-293 cells and HL-1 cardiomyocytes revealed that the 2592+1G>A mutation disrupted normal splicing and caused multiple splicing defects: the activation of cryptic splice sites within exon 10 and intron 10 and complete intron 10 retention. We performed functional and biochemical analyses of the major splice product, hERGΔ24, in which 24 amino acids within the cyclic nucleotide binding domain of the hERG channel COOH-terminus is deleted. Patch-clamp experiments revealed that the splice mutant did not generate hERG current. Western blot and immunostaining studies showed that mutant channels did not traffic to the cell surface. Coexpression of wild-type hERG and hERGΔ24 resulted in signifi-cant dominant-negative suppression of hERG current via the intracellular retention of the wild-type channels. Our results demonstrate that 2592+1G>A causes multiple splicing defects, consistent with the pathogenic mechanisms of long QT syndrome.",
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    N2 - Long QT syndrome type 2 (LQT2) is caused by mutations in the human ether-a-go-go-related gene (hERG). Cryptic splice site activation in hERG has recently been identified as a novel pathogenic mechanism of LQT2. In this report, we characterize a hERG splice site mutation, 2592+1G>A, which occurs at the 5= splice site of intron 10. Reverse transcription-PCR analyses using hERG minigenes transfected into human embryonic kidney-293 cells and HL-1 cardiomyocytes revealed that the 2592+1G>A mutation disrupted normal splicing and caused multiple splicing defects: the activation of cryptic splice sites within exon 10 and intron 10 and complete intron 10 retention. We performed functional and biochemical analyses of the major splice product, hERGΔ24, in which 24 amino acids within the cyclic nucleotide binding domain of the hERG channel COOH-terminus is deleted. Patch-clamp experiments revealed that the splice mutant did not generate hERG current. Western blot and immunostaining studies showed that mutant channels did not traffic to the cell surface. Coexpression of wild-type hERG and hERGΔ24 resulted in signifi-cant dominant-negative suppression of hERG current via the intracellular retention of the wild-type channels. Our results demonstrate that 2592+1G>A causes multiple splicing defects, consistent with the pathogenic mechanisms of long QT syndrome.

    AB - Long QT syndrome type 2 (LQT2) is caused by mutations in the human ether-a-go-go-related gene (hERG). Cryptic splice site activation in hERG has recently been identified as a novel pathogenic mechanism of LQT2. In this report, we characterize a hERG splice site mutation, 2592+1G>A, which occurs at the 5= splice site of intron 10. Reverse transcription-PCR analyses using hERG minigenes transfected into human embryonic kidney-293 cells and HL-1 cardiomyocytes revealed that the 2592+1G>A mutation disrupted normal splicing and caused multiple splicing defects: the activation of cryptic splice sites within exon 10 and intron 10 and complete intron 10 retention. We performed functional and biochemical analyses of the major splice product, hERGΔ24, in which 24 amino acids within the cyclic nucleotide binding domain of the hERG channel COOH-terminus is deleted. Patch-clamp experiments revealed that the splice mutant did not generate hERG current. Western blot and immunostaining studies showed that mutant channels did not traffic to the cell surface. Coexpression of wild-type hERG and hERGΔ24 resulted in signifi-cant dominant-negative suppression of hERG current via the intracellular retention of the wild-type channels. Our results demonstrate that 2592+1G>A causes multiple splicing defects, consistent with the pathogenic mechanisms of long QT syndrome.

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