title: Photoreceptor Progenitor mRNA Analysis Reveals Exon Skipping Resulting from the ABCA4 c.5461-10T→C Mutation in Stargardt Disease.
abstract: To elucidate the functional effect of the ABCA4 variant c.5461-10T→C, one of the most frequent variants associated with Stargardt disease (STGD1).
  
  
  
  Case series.
  
  
  
  Seventeen persons with STGD1 carrying ABCA4 variants and 1 control participant.
  
  
  
  Haplotype analysis of 4 homozygotes and 11 heterozygotes for c.5461-10T→C and sequence analysis of the ABCA4 gene for a homozygous proband. Fibroblasts were reprogrammed from 3 persons with STGD1 into induced pluripotent stem cells, which were differentiated into photoreceptor progenitor cells (PPCs). The effect of the c.5461-10T→C variant on RNA splicing by reverse-transcription polymerase chain reaction was analyzed using PPC mRNA. In vitro assays were performed with minigene constructs containing ABCA4 exon 39. We analyzed the natural history and ophthalmologic characteristics of 4 persons homozygous for c.5461-10T→C.
  
  
  
  Haplotype and rare variant data for ABCA4, RNA splice defects, age at diagnosis, visual acuity, fundus appearance, visual field, electroretinography (ERG) results, fluorescein angiography results, and fundus autofluorescence findings.
  
  
  
  The frequent ABCA4 variant c.5461-10T→C has a subtle effect on splicing based on prediction programs. A founder haplotype containing c.5461-10T→C was found to span approximately 96 kb of ABCA4 and did not contain other rare sequence variants. Patient-derived PPCs showed skipping of exon 39 or exons 39 and 40 in the mRNA. HEK293T cell transduction with minigenes carrying exon 39 showed that the splice defects were the result of the c.5461-10T→C variant. All 4 subjects carrying the c.5461-10T→C variant in a homozygous state showed a young age of STGD1 onset, with low visual acuity at presentation and abnormal cone ERG results. All 4 demonstrated severe cone-rod dystrophy before 20 years of age and were legally blind by 25 years of age.
  
  
  
  The ABCA4 variant c.5461-10T→C is located on a founder haplotype lacking other disease-causing rare sequence variants. In vitro studies revealed that it leads to mRNA exon skipping and ABCA4 protein truncation. Given the severe phenotype in persons homozygous for this variant, we conclude that this variant results in the absence of ABCA4 activity.
authors.affiliation: Department of Human Genetics, Radboud University Medical Center, Nijmegen, The Netherlands; Radboud Institute of Molecular Life Sciences, Nijmegen, The Netherlands.
authors.forename: Riccardo
authors.initials: R
authors.lastname: Sangermano
authors.affiliation: Department of Ophthalmology, Radboud University Medical Center, Nijmegen, The Netherlands.
authors.forename: Nathalie M
authors.initials: NM
authors.lastname: Bax
authors.affiliation: Center for Medical Genetics, Ghent University and Ghent University Hospital, Ghent, Belgium.
authors.forename: Miriam
authors.initials: M
authors.lastname: Bauwens
authors.affiliation: Rotterdam Eye Hospital, Rotterdam, The Netherlands.
authors.forename: L Ingeborgh
authors.initials: LI
authors.lastname: van den Born
authors.affiliation: Center for Medical Genetics, Ghent University and Ghent University Hospital, Ghent, Belgium.
authors.forename: Elfride
authors.initials: E
authors.lastname: De Baere
authors.affiliation: Department of Human Genetics, Radboud University Medical Center, Nijmegen, The Netherlands; Radboud Institute of Molecular Life Sciences, Nijmegen, The Netherlands.
authors.forename: Alejandro
authors.initials: A
authors.lastname: Garanto
authors.affiliation: Department of Human Genetics, Radboud University Medical Center, Nijmegen, The Netherlands; Radboud Institute of Molecular Life Sciences, Nijmegen, The Netherlands.
authors.forename: Rob W J
authors.initials: RW
authors.lastname: Collin
authors.affiliation: Department of Human Genetics, Radboud University Medical Center, Nijmegen, The Netherlands.
authors.forename: Angelique S A
authors.initials: AS
authors.lastname: Goercharn-Ramlal
authors.affiliation: Department of Human Genetics, Radboud University Medical Center, Nijmegen, The Netherlands.
authors.forename: Anke H A
authors.initials: AH
authors.lastname: den Engelsman-van Dijk
authors.affiliation: Universitätsaugenklinik, Ruprecht-Karls-Universität, Heidelberg, Heidelberg, Germany.
authors.forename: Klaus
authors.initials: K
authors.lastname: Rohrschneider
authors.affiliation: Department of Ophthalmology, Radboud University Medical Center, Nijmegen, The Netherlands.
authors.forename: Carel B
authors.initials: CB
authors.lastname: Hoyng
authors.affiliation: Department of Human Genetics, Radboud University Medical Center, Nijmegen, The Netherlands; Radboud Institute of Molecular Life Sciences, Nijmegen, The Netherlands. Electronic address: Frans.Cremers@radboudumc.nl.
authors.forename: Frans P M
authors.initials: FP
authors.lastname: Cremers
authors.affiliation: Department of Human Genetics, Radboud University Medical Center, Nijmegen, The Netherlands; Radboud Institute of Molecular Life Sciences, Nijmegen, The Netherlands. Electronic address: Silvia.Albert@radboudumc.nl.
authors.forename: Silvia
authors.initials: S
authors.lastname: Albert
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000235:genetics*; D000328:Adult; D017398:Alternative Splicing; D004252:DNA Mutational Analysis; D004596:Electroretinography; D005091:Exons / Q000235:genetics*; D005260:Female; D005347:Fibroblasts / Q000378:metabolism; D005451:Fluorescein Angiography; D057809:HEK293 Cells; D006239:Haplotypes; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000378:metabolism; D008268:Macular Degeneration / Q000151:congenital* / Q000175:diagnosis / Q000235:genetics / Q000503:physiopathology; D008297:Male; D008875:Middle Aged; D020419:Photoreceptor Cells, Vertebrate / Q000502:physiology*; D020641:Polymorphism, Single Nucleotide; D022821:RNA Splice Sites / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000080362:Stargardt Disease; D013234:Stem Cells / Q000502:physiology*; D014162:Transfection; D014792:Visual Acuity / Q000502:physiology; D014794:Visual Fields / Q000502:physiology; D055815:Young Adult
chemical_list: C056475:ABCA4 protein, human; D018528:ATP-Binding Cassette Transporters; D022821:RNA Splice Sites; D012333:RNA, Messenger