title: A KCNH2 branch point mutation causing aberrant splicing contributes to an explanation of genotype-negative long QT syndrome.
abstract: BACKGROUND
  Genetic screening of long QT syndrome (LQTS) fails to identify disease-causing mutations in about 30% of patients. So far, molecular screening has focused mainly on coding sequence mutations or on substitutions at canonical splice sites.
  
  
  OBJECTIVE
  The purpose of this study was to explore the possibility that intronic variants not at canonical splice sites might affect splicing regulatory elements, lead to aberrant transcripts, and cause LQTS.
  
  
  METHODS
  Molecular screening was performed through DHPLC and sequence analysis. The role of the intronic mutation identified was assessed with a hybrid minigene splicing assay.
  
  
  RESULTS
  A three-generation LQTS family was investigated. Molecular screening failed to identify an obvious disease-causing mutation in the coding sequences of the major LQTS genes but revealed an intronic A-to-G substitution in KCNH2 (IVS9-28A/G) cosegregating with the clinical phenotype in family members. In vitro analysis proved that the mutation disrupts the acceptor splice site definition by affecting the branch point (BP) sequence and promoting intron retention. We further demonstrated a tight functional relationship between the BP and the polypyrimidine tract, whose weakness is responsible for the pathological effect of the IVS9-28A/G mutation.
  
  
  CONCLUSIONS
  We identified a novel BP mutation in KCNH2 that disrupts the intron 9 acceptor splice site definition and causes LQT2. The present finding demonstrates that intronic mutations affecting pre-mRNA processing may contribute to the failure of traditional molecular screening in identifying disease-causing mutations in LQTS subjects and offers a rationale strategy for the reduction of genotype-negative cases.
authors.affiliation: Department of Cardiology, University of Pavia, and IRCCS Fondazione Policlinico S. Matteo, Pavia, Italy. l.crotti@smatteo.pv.it
authors.forename: Lia
authors.initials: L
authors.lastname: Crotti
authors.forename: Marzena A
authors.initials: MA
authors.lastname: Lewandowska
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Schwartz
authors.forename: Roberto
authors.initials: R
authors.lastname: Insolia
authors.forename: Matteo
authors.initials: M
authors.lastname: Pedrazzini
authors.forename: Erica
authors.initials: E
authors.lastname: Bussani
authors.forename: Federica
authors.initials: F
authors.lastname: Dagradi
authors.forename: Alfred L
authors.initials: AL
authors.lastname: George
authors.forename: Franco
authors.initials: F
authors.lastname: Pagani
mesh_terms: D000328:Adult; D016757:Death, Sudden, Cardiac; D000072237:ERG1 Potassium Channel; D051638:Ether-A-Go-Go Potassium Channels / Q000235:genetics*; D005260:Female; D005820:Genetic Testing; D005838:Genotype; D006801:Humans; D007438:Introns; D008126:Lod Score; D008133:Long QT Syndrome / Q000235:genetics*; D008297:Male; D010375:Pedigree; D010641:Phenotype; D017354:Point Mutation; D022821:RNA Splice Sites; D012326:RNA Splicing; D014158:Transcription, Genetic
chemical_list: D000072237:ERG1 Potassium Channel; D051638:Ether-A-Go-Go Potassium Channels; C000606913:KCNH2 protein, human; D022821:RNA Splice Sites