title: Identification and functional characterization of KCNQ1 mutations around the exon 7-intron 7 junction affecting the splicing process.
abstract: BACKGROUND
  KCNQ1 gene encodes the delayed rectifier K(+) channel in cardiac muscle, and its mutations cause long QT syndrome type 1 (LQT1). Especially exercise-related cardiac events predominate in LQT1. We previously reported that a KCNQ1 splicing mutation displays LQT1 phenotypes.
  
  
  RESULTS
  We identified novel mutation at the third base of intron 7 (IVS7 +3A>G) in exercise-induced LQT1 patients. Minigene assay in COS7 cells and RT-PCR analysis of patients' lymphocytes demonstrated the presence of exon 7-deficient mRNA in IVS7 +3A>G, as well as c.1032G>A, but not in c.1022C>T. Real-time RT-PCR demonstrated that both IVS7 +3A>G and c.1032G>A carrier expressed significant amounts of exon-skipping mRNAs (18.8% and 44.8% of total KCNQ1 mRNA). Current recordings from Xenopus oocytes injected cRNA by simulating its ratios of exon skipping displayed a significant reduction in currents to 64.8 ± 4.5% for IVS7 +3A>G and to 41.4 ± 9.5% for c.1032G>A carrier, respectively, compared to the condition without splicing error. Computer simulation incorporating these quantitative results revealed the pronounced QT prolongation under beta-adrenergic stimulation in IVS7 +3A>G carrier model.
  
  
  CONCLUSIONS
  Here we report a novel splicing mutation IVS7 +3A>G, identified in a family with mild form LQT1 phenotypes, and examined functional outcome in comparison with three other variants around the exon 7-intron 7 junction. In addition to c.1032G>A mutation, IVS7 +3A>G generates exon-skipping mRNAs, and thereby causing LQT1 phenotype. The severity of clinical phenotypes appeared to differ between the two splicing-related mutations and to result from the amount of resultant mRNAs and their functional consequences.
authors.affiliation: Department of Cardiovascular and Respiratory Medicine, Shiga University of Medical Science, Otsu, Japan.
authors.forename: Keiko
authors.initials: K
authors.lastname: Tsuji-Wakisaka
authors.forename: Masaharu
authors.initials: M
authors.lastname: Akao
authors.forename: Takahiro M
authors.initials: TM
authors.lastname: Ishii
authors.forename: Takashi
authors.initials: T
authors.lastname: Ashihara
authors.forename: Takeru
authors.initials: T
authors.lastname: Makiyama
authors.forename: Seiko
authors.initials: S
authors.lastname: Ohno
authors.forename: Futoshi
authors.initials: F
authors.lastname: Toyoda
authors.forename: Kenichi
authors.initials: K
authors.lastname: Dochi
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Matsuura
authors.forename: Minoru
authors.initials: M
authors.lastname: Horie
mesh_terms: D000293:Adolescent; D000328:Adult; D000818:Animals; D001483:Base Sequence; D003198:Computer Simulation; D005091:Exons / Q000235:genetics*; D005260:Female; D006579:Heterozygote; D006801:Humans; D007438:Introns / Q000235:genetics*; D051657:KCNQ1 Potassium Channel / Q000235:genetics*; D008133:Long QT Syndrome / Q000235:genetics*; D008297:Male; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D009865:Oocytes / Q000378:metabolism; D010375:Pedigree; D010641:Phenotype; D012326:RNA Splicing / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014982:Xenopus laevis / Q000378:metabolism
chemical_list: D051657:KCNQ1 Potassium Channel; C496909:KCNQ1 protein, human; D012333:RNA, Messenger