Deep-intronic ABCA4 variants explain missing heritability in Stargardt disease and allow correction of splice defects by antisense oligonucleotides |
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| Institution: | 1. Department of Human Genetics, Radboud University Medical Center, Nijmegen, The Netherlands.;2. Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, The Netherlands.;3. Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Center, Nijmegen, The Netherlands.;4. Department of Ophthalmology, Radboud University Medical Center, Nijmegen, The Netherlands.;5. Center for Medical Genetics, Ghent University and Ghent University Hospital, Ghent, Belgium.;6. The Rotterdam Eye Hospital and the Rotterdam Ophthalmic Institute, Rotterdam, The Netherlands.;7. Department of Medical Genetics, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands.;8. Department of Ophthalmology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands.;9. Department of Ophthalmology, Erasmus Medical Center, Rotterdam, The Netherlands.;10. Service d′Exploration de la Vision CHU, Lille, France.;11. Institute for Neurosciences of Montpellier INSERM U1051, University of Montpellier, Montpellier, France.;12. Centre d′ Etude du Polymorphisme Humain, Fondation Jean Dausset, Paris, France.;13. UCL Institute of Ophthalmology, London, UK.;14. Moorfields Eye Hospital, London, UK.;15. Department of Haematology, University of Cambridge, Cambridge, UK.;16. NIHR BioResource, Cambridge University Hospitals NHS Foundation Trust, Cambridge Biomedical Campus, Cambridge, UK.;17. Department of Medical Genetics, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK.;18. Univ. Lille, Inserm UMR-S 1172, CHU Lille, Biochemistry and Molecular Biology Department - UF Génopathies, Lille, France.;19. Institut für Humangenetik, Universität Regensburg, Regensburg, Germany.;20. Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, Stockholm, Sweden;1. Department of Human Genetics, Radboud University Medical Center, Nijmegen, The Netherlands.;2. Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, The Netherlands.;3. Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Center, Nijmegen, The Netherlands.;4. Department of Ophthalmology, Radboud University Medical Center, Nijmegen, The Netherlands.;5. Center for Medical Genetics, Ghent University and Ghent University Hospital, Ghent, Belgium.;6. The Rotterdam Eye Hospital and the Rotterdam Ophthalmic Institute, Rotterdam, The Netherlands.;7. Department of Medical Genetics, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands.;8. Department of Ophthalmology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands.;9. Department of Ophthalmology, Erasmus Medical Center, Rotterdam, The Netherlands.;10. Service d′Exploration de la Vision CHU, Lille, France.;11. Institute for Neurosciences of Montpellier INSERM U1051, University of Montpellier, Montpellier, France.;12. Centre d′ Etude du Polymorphisme Humain, Fondation Jean Dausset, Paris, France.;13. UCL Institute of Ophthalmology, London, UK.;14. Moorfields Eye Hospital, London, UK.;15. Department of Haematology, University of Cambridge, Cambridge, UK.;16. NIHR BioResource, Cambridge University Hospitals NHS Foundation Trust, Cambridge Biomedical Campus, Cambridge, UK.;17. Department of Medical Genetics, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK.;18. Univ. Lille, Inserm UMR-S 1172, CHU Lille, Biochemistry and Molecular Biology Department - UF Génopathies, Lille, France.;19. Institut für Humangenetik, Universität Regensburg, Regensburg, Germany.;20. Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, Stockholm, Sweden |
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| Abstract: | PurposeUsing exome sequencing, the underlying variants in many persons with autosomal recessive diseases remain undetected. We explored autosomal recessive Stargardt disease (STGD1) as a model to identify the missing heritability.MethodsSequencing of ABCA4 was performed in 8 STGD1 cases with one variant and p.Asn1868Ile in trans, 25 cases with one variant, and 3 cases with no ABCA4 variant. The effect of intronic variants was analyzed using in vitro splice assays in HEK293T cells and patient-derived fibroblasts. Antisense oligonucleotides were used to correct splice defects.ResultsIn 24 of the probands (67%), one known and five novel deep-intronic variants were found. The five novel variants resulted in messenger RNA pseudoexon inclusions, due to strengthening of cryptic splice sites or by disrupting a splicing silencer motif. Variant c.769-784C>T showed partial insertion of a pseudoexon and was found in cis with c.5603A>T (p.Asn1868Ile), so its causal role could not be fully established. Variant c.4253+43G>A resulted in partial skipping of exon 28. Remarkably, antisense oligonucleotides targeting the aberrant splice processes resulted in (partial) correction of all splicing defects.ConclusionOur data demonstrate the importance of assessing noncoding variants in genetic diseases, and show the great potential of splice modulation therapy for deep-intronic variants. |
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