title: Functional assessment of potential splice site variants in arrhythmogenic right ventricular dysplasia/cardiomyopathy.
abstract: BACKGROUND
  Interpretation of genetic screening results in arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) often is difficult. Pathogenicity of variants with uncertain clinical significance may be predicted by software algorithms. However, functional assessment can unambiguously demonstrate the effect of such variants.
  
  
  OBJECTIVE
  The purpose of this study was to perform functional analysis of potential splice site variants in ARVD/C patients.
  
  
  METHODS
  Nine variants in desmosomal (PKP2, JUP, DSG2, DSC2) genes with potential RNA splicing effect were analyzed. The variants were found in patients who fulfilled 2010 ARVD/C Task Force Criteria (n = 7) or had suspected ARVD/C (n = 2). Total RNA was isolated from fresh blood samples and subjected to reverse transcriptase polymerase chain reaction.
  
  
  RESULTS
  An effect on splicing was predicted by software algorithms for all variants. Of the 9 variants, 5 were intronic and 4 exonic. RNA analysis showed a functional effect on mRNA splicing by exon skipping, generation of new splice sites, or activation of cryptic sites in 6 variants. All 5 intronic variants tested severely impaired splicing. Only 1 of 4 exonic potential splice site variants was shown to have a deleterious effect on splicing. The remaining 3 exonic variants had no detectable effect on splicing, and heterozygous presence in mRNA confirmed biallelic expression.
  
  
  CONCLUSIONS
  Six variants of uncertain clinical significance in the PKP2, JUP, and DSG2 genes showed a deleterious effect on mRNA splicing, indicating these are ARVD/C-related pathogenic splice site mutations. These results highlight the importance of functional assessment of potential splice site variants to improve patient care and facilitate cascade screening.
authors.affiliation: Department of Cardiology, University Medical Center Utrecht and ICIN-Netherlands Heart Institute, Utrecht, The Netherlands.
authors.forename: Judith A
authors.initials: JA
authors.lastname: Groeneweg
authors.affiliation: Department of Medical Genetics, University Medical Center Utrecht, Utrecht, The Netherlands.
authors.forename: Amber
authors.initials: A
authors.lastname: Ummels
authors.affiliation: Department of Medical Genetics, University Medical Center Utrecht, Utrecht, The Netherlands.
authors.forename: Marcel
authors.initials: M
authors.lastname: Mulder
authors.affiliation: Department of Medical Genetics, Academic Medical Center, Amsterdam, The Netherlands.
authors.forename: Hennie
authors.initials: H
authors.lastname: Bikker
authors.affiliation: Department of Medical Genetics, University Medical Center Utrecht, Utrecht, The Netherlands.
authors.forename: Jasper J
authors.initials: JJ
authors.lastname: van der Smagt
authors.affiliation: Department of Medical Genetics, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Anneke M
authors.initials: AM
authors.lastname: van Mil
authors.affiliation: Department of Clinical Genetics, St. George's Hospital Medical School, London, United Kingdom.
authors.forename: Tessa
authors.initials: T
authors.lastname: Homfray
authors.affiliation: Department of Medical Genetics, University Medical Center Utrecht, Utrecht, The Netherlands.
authors.forename: Jan G
authors.initials: JG
authors.lastname: Post
authors.affiliation: Department of Cardiology, Isala Klinieken, Zwolle, The Netherlands.
authors.forename: Arif
authors.initials: A
authors.lastname: Elvan
authors.affiliation: Department of Cardiology, University Medical Center Utrecht, Utrecht, The Netherlands.
authors.forename: Jeroen F
authors.initials: JF
authors.lastname: van der Heijden
authors.affiliation: Department of Medical Genetics, VU Medical Center, Amsterdam, The Netherlands.
authors.forename: Arjan C
authors.initials: AC
authors.lastname: Houweling
authors.affiliation: Department of Genetics, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands.
authors.forename: Jan D H
authors.initials: JD
authors.lastname: Jongbloed
authors.affiliation: Department of Cardiology, Academic Medical Center, Amsterdam, The Netherlands.
authors.forename: Arthur A M
authors.initials: AA
authors.lastname: Wilde
authors.affiliation: Department of Genetics, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands; Durrer Center for Cardiogenetic Research, Utrecht, The Netherlands.
authors.forename: J Peter
authors.initials: JP
authors.lastname: van Tintelen
authors.affiliation: Department of Cardiology, University Medical Center Utrecht and ICIN-Netherlands Heart Institute, Utrecht, The Netherlands.
authors.forename: Richard N
authors.initials: RN
authors.lastname: Hauer
authors.affiliation: Department of Medical Genetics, University Medical Center Utrecht, Utrecht, The Netherlands. Electronic address: D.Dooijes@umcutrecht.nl.
authors.forename: Dennis
authors.initials: D
authors.lastname: Dooijes
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000465:Algorithms; D000483:Alleles; D019571:Arrhythmogenic Right Ventricular Dysplasia / Q000235:genetics* / Q000503:physiopathology*; D002135:Calcium-Binding Proteins / Q000235:genetics; D009202:Cardiomyopathies / Q000235:genetics* / Q000503:physiopathology*; D051187:Desmocollins / Q000235:genetics; D051301:Desmoglein 2 / Q000235:genetics; D051180:Desmoplakins / Q000235:genetics; D004562:Electrocardiography; D005091:Exons; D005260:Female; D014644:Genetic Variation; D006801:Humans; D007438:Introns; D008297:Male; D008565:Membrane Proteins / Q000235:genetics; D008875:Middle Aged; D009154:Mutation; D010375:Pedigree; D051191:Plakophilins / Q000235:genetics; D022821:RNA Splice Sites; D012333:RNA, Messenger / Q000097:blood; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012984:Software; D051185:gamma Catenin; D000099211:Phospholamban
chemical_list: D002135:Calcium-Binding Proteins; D051187:Desmocollins; D051301:Desmoglein 2; D051180:Desmoplakins; D008565:Membrane Proteins; D051191:Plakophilins; D022821:RNA Splice Sites; D012333:RNA, Messenger; D051185:gamma Catenin; D000099211:Phospholamban; C081561:DSC2 protein, human; C495314:DSG2 protein, human; C495294:DSP protein, human; C495292:JUP protein, human; C495337:PKP2 protein, human; C528696:TMEM43 protein, human
keywords: Arrhythmogenic right ventricular dysplasia/cardiomyopathy; Desmosomes; Genetics; RNA analysis; Splice site mutations; Ventricular arrhythmias