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      Deep-intronic ABCA4 variants explain missing heritability in Stargardt disease and allow correction of splice defects by antisense oligonucleotides

      research-article
      , PhD 1 , 2 , , PhD 1 , 3 , , MSc 1 , 3 , , MD 3 , 4 , , PhD 5 , , MD 3 , 4 , , MD, PhD 6 , , PhD 1 , 2 , , MSc 1 , 3 , , MD 7 , , MD, PhD 8 , , MD, PhD 9 , , MD, PhD 4 , 9 , , MD 10 , , PhD 11 , 12 , , MSc 5 , , PhD 13 , 14 , , PhD 13 , 14 , , PhD 15 , 16 , , MD, PhD 16 , 17 , , MD, PhD 13 , 14 , , MD, PhD 18 , , PhD 19 , , PhD 19 , 20 , , PhD 19 , , MD, PhD 3 , 4 , , MD, PhD 5 , , PhD 1 , 3 , , PhD 1 , 3 , , , PhD 1 , 3 ,
      Genetics in Medicine
      Nature Publishing Group US
      ABCA4, antisense oligonucleotide, deep-intronic variant, missing heritability, Stargardt disease

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          Abstract

          Purpose

          Using 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.

          Methods

          Sequencing 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.

          Results

          In 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.

          Conclusion

          Our 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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          Most cited references26

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          Deep intronic mutations and human disease.

          Next-generation sequencing has revolutionized clinical diagnostic testing. Yet, for a substantial proportion of patients, sequence information restricted to exons and exon-intron boundaries fails to identify the genetic cause of the disease. Here we review evidence from mRNA analysis and entire genomic sequencing indicating that pathogenic mutations can occur deep within the introns of over 75 disease-associated genes. Deleterious DNA variants located more than 100 base pairs away from exon-intron junctions most commonly lead to pseudo-exon inclusion due to activation of non-canonical splice sites or changes in splicing regulatory elements. Additionally, deep intronic mutations can disrupt transcription regulatory motifs and non-coding RNA genes. This review aims to highlight the importance of studying variation in deep intronic sequence as a cause of monogenic disorders as well as hereditary cancer syndromes.
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            Next-generation genetic testing for retinitis pigmentosa

            Molecular diagnostics for patients with retinitis pigmentosa (RP) has been hampered by extreme genetic and clinical heterogeneity, with 52 causative genes known to date. Here, we developed a comprehensive next-generation sequencing (NGS) approach for the clinical molecular diagnostics of RP. All known inherited retinal disease genes (n = 111) were captured and simultaneously analyzed using NGS in 100 RP patients without a molecular diagnosis. A systematic data analysis pipeline was developed and validated to prioritize and predict the pathogenicity of all genetic variants identified in each patient, which enabled us to reduce the number of potential pathogenic variants from approximately 1,200 to zero to nine per patient. Subsequent segregation analysis and in silico predictions of pathogenicity resulted in a molecular diagnosis in 36 RP patients, comprising 27 recessive, six dominant, and three X-linked cases. Intriguingly, De novo mutations were present in at least three out of 28 isolated cases with causative mutations. This study demonstrates the enormous potential and clinical utility of NGS in molecular diagnosis of genetically heterogeneous diseases such as RP. De novo dominant mutations appear to play a significant role in patients with isolated RP, having major implications for genetic counselling.
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              Autosomal recessive retinitis pigmentosa and cone-rod dystrophy caused by splice site mutations in the Stargardt's disease gene ABCR.

              Ophthalmological and molecular genetic studies were performed in a consanguineous family with individuals showing either retinitis pigmentosa (RP) or cone-rod dystrophy (CRD). Assuming pseudodominant (recessive) inheritance of allelic defects, linkage analysis positioned the causal gene at 1p21-p13 (lod score 4.22), a genomic segment known to harbor the ABCR gene involved in Stargardt's disease (STGD) and age-related macular degeneration (AMD). We completed the exon-intron structure of the ABCR gene and detected a severe homozygous 5[prime] splice site mutation, IVS30+1G->T, in the four RP patients. The five CRD patients in this family are compound heterozygotes for the IVS30+1G->T mutation and a 5[prime] splice site mutation in intron 40 (IVS40+5G->A). Both splice site mutations were found heterozygously in two unrelated STGD patients, but not in 100 control individuals. In these patients the second mutation was either a missense mutation or unknown. Since thus far no STGD patients have been reported to carry two ABCR null alleles and taking into account that the RP phenotype is more severe than the STGD phenotype, we hypothesize that the intron 30 splice site mutation represents a true null allele. Since the intron 30 mutation is found heterozygously in the CRD patients, the IVS40+5G->A mutation probably renders the exon 40 5[prime] splice site partially functional. These results show that mutations in the ABCR gene not only result in STGD and AMD, but can also cause autosomal recessive RP and CRD. Since the heterozygote frequency for ABCR mutations is estimated at 0.02, mutations in ABCR might be an important cause of autosomal recessive and sporadic forms of RP and CRD.
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                Author and article information

                Contributors
                rob.collin@radboudumc.nl
                frans.cremers@radboudumc.nl
                Journal
                Genet Med
                Genet. Med
                Genetics in Medicine
                Nature Publishing Group US (New York )
                1098-3600
                1530-0366
                15 January 2019
                15 January 2019
                2019
                : 21
                : 8
                : 1751-1760
                Affiliations
                [1 ]ISNI 0000 0004 0444 9382, GRID grid.10417.33, Department of Human Genetics, , Radboud University Medical Center, ; Nijmegen, The Netherlands
                [2 ]ISNI 0000 0004 0444 9382, GRID grid.10417.33, 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 ]ISNI 0000 0004 0444 9382, GRID grid.10417.33, Department of Ophthalmology, , Radboud University Medical Center, ; Nijmegen, The Netherlands
                [5 ]ISNI 0000 0004 0626 3303, GRID grid.410566.0, Center for Medical Genetics, Ghent University and Ghent University Hospital, ; Ghent, Belgium
                [6 ]ISNI 0000 0001 0009 7699, GRID grid.414699.7, 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 ]ISNI 000000040459992X, GRID grid.5645.2, Department of Ophthalmology, , Erasmus Medical Center, ; Rotterdam, The Netherlands
                [10 ]Service d′Exploration de la Vision CHU, Lille, France
                [11 ]ISNI 0000 0001 2097 0141, GRID grid.121334.6, Institute for Neurosciences of Montpellier INSERM U1051, University of Montpellier, ; Montpellier, France
                [12 ]ISNI 0000 0004 0639 125X, GRID grid.417836.f, Centre d′Etude du Polymorphisme Humain, Fondation Jean Dausset, ; Paris, France
                [13 ]ISNI 0000000121901201, GRID grid.83440.3b, UCL Institute of Ophthalmology, ; London, UK
                [14 ]ISNI 0000 0000 8726 5837, GRID grid.439257.e, Moorfields Eye Hospital, ; London, UK
                [15 ]ISNI 0000000121885934, GRID grid.5335.0, Department of Haematology, , University of Cambridge, ; Cambridge, UK
                [16 ]ISNI 0000 0004 0383 8386, GRID grid.24029.3d, NIHR BioResource, Cambridge University Hospitals NHS Foundation Trust, Cambridge Biomedical Campus, ; Cambridge, UK
                [17 ]ISNI 0000000121885934, GRID grid.5335.0, Department of Medical Genetics, , Cambridge Institute for Medical Research, University of Cambridge, ; Cambridge, UK
                [18 ]ISNI 0000 0004 0471 8845, GRID grid.410463.4, Univ. Lille, Inserm UMR-S 1172, CHU Lille, Biochemistry and Molecular Biology Department - UF Génopathies, ; Lille, France
                [19 ]ISNI 0000 0001 2190 5763, GRID grid.7727.5, Institut für Humangenetik, Universität Regensburg, ; Regensburg, Germany
                [20 ]ISNI 0000 0004 1937 0626, GRID grid.4714.6, Department of Medical Epidemiology and Biostatistics, , Karolinska Institutet, ; Stockholm, Sweden
                Article
                414
                10.1038/s41436-018-0414-9
                6752325
                30643219
                16302b3b-e061-4f67-b173-a9e98fe1bc0c
                © The Author(s) 2019

                Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

                History
                : 19 April 2018
                : 7 December 2018
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                © American College of Medical Genetics and Genomics 2019

                Genetics
                abca4,antisense oligonucleotide,deep-intronic variant,missing heritability,stargardt disease

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