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.
| Original language | English |
|---|---|
| Pages (from-to) | 1751-1760 |
| Number of pages | 10 |
| Journal | Genetics in Medicine |
| Volume | 21 |
| Issue number | 8 |
| Early online date | 15 Jan 2019 |
| DOIs | |
| Publication status | Published - Aug 2019 |
Bibliographical note
We thank Ellen Blokland, Lonneke Duijkers, Duaa Elmelik, Anita Hoogendoorn, Marlie Jacobs-Camps, Saskia van der Velde-Visser, and Marijke Zonneveld-Vrieling for technical assistance. We thank Sabine Defoort, Hélène Dollfus, Isabelle Drumare, Christian P. Hamel, Karsten Hufendiek, Cord Huchzermeyer, Herbert Jägle, Ulrich Kellner, Philipp Rating, Klaus Rüther, Eric Souied, Georg Spital, and Xavier Zanlonghi for their cooperation and ascertaining STGD1 cases. This work was supported by the FP7-PEOPLE-2012-ITN programme EyeTN, agreement 317472 (to F.P.M.C.); the Macula Vision Research Foundation (to F.P.M.C.); the Foundation Fighting Blindness USA, grant no. PPA-0517-0717-RAD (to A.G., C.B.H., F.P.M.C., R.W.J.C., and S.A.); the RP Fighting Blindness UK (RetinaUK), grant no. GR591 (to F.P.M.C. and S.A.); the Rotterdamse Stichting Blindenbelangen, the Stichting Blindenhulp, and the Stichting tot Verbetering van het Lot der Blinden (to F.P.M.C. and S.A.); and by the Landelijke Stichting voor Blinden en Slechtzienden, Macula Degeneratie fonds, and the Stichting Blinden-Penning, which contributed through Uitzicht 2016-12 (to F.P.M.C. and S.A.). This work was also supported by the Algemene Nederlandse Vereniging ter Voorkoming van Blindheid, Stichting Blinden-Penning, Landelijke Stichting voor Blinden en Slechtzienden, Stichting Oogfonds Nederland, Stichting Macula Degeneratie Fonds, and Stichting Retina Nederland Fonds, which contributed through UitZicht 2015-31, together with the Rotterdamse Stichting Blindenbelangen, Stichting Blindenhulp, Stichting tot Verbetering van het Lot der Blinden, Stichting voor Ooglijders, and Stichting Dowilvo (to A.G. and R.W.J.C.); the Stichting Macula Degeneratie Fonds; and the Stichting A.F. Deutman Researchfonds Oogheelkunde (to C.B.H.). This work was also supported by the Algemene Nederlandse Vereniging ter Voorkoming van Blindheid and Landelijke Stichting voor Blinden en Slechtzienden, which contributed through UitZicht 2014-13, together with the Rotterdamse Stichting Blindenbelangen, Stichting Blindenhulp, and the Stichting tot Verbetering van het Lot der Blinden (to F.P.M.C.), the Ghent University Research Fund (BOF15/GOA/011), the Research Foundation Flanders (FVO) G0C6715N, and the Hercules Foundation AUGE/13/023 and JED Foundation to E.D.B. M.B. was PhD fellow of the FWO and recipient of a grant of the funds for Research in Ophthalmology (FRO). E.D.B. is Senior Clinical Investigator of the FWO (1802215N). This work was also supported by the National Institute for Health Research (NIHR) Biomedical Centre at Moorfields and UCL Institute of Ophthalmology (to A.W.), UK NIHR Rare Disease Translational Research Consortium (to G.A. and A.W.), NIHR for the NIHR BioResource (RG65966) (to F.L.R.) by grants from the Federal Ministry of Education and Research (BMBF) (ref. IDs 01GM0851 and 01GM1108B) (to B.H.F.W.). G.A. is supported by a Fight for Sight UK Early Career Investigator Award. The funding organizations had no role in the design or conduct of this research, and provided unrestricted grants. This study made use of data generated by the Genome of the Netherlands Project. Funding for the project was provided by the Netherlands Organization for Scientific Research under award number 184021007, dated 9 July 2009 and made available as a Rainbow Project of the Biobanking and Biomolecular Research Infrastructure Netherlands (BBMRI-NL). Samples where contributed by LifeLines (http://lifelines.nl/lifelines-research/general), the Leiden Longevity Study (http://www.healthy-ageing.nl; http://www.langleven.net), the Netherlands Twin Registry (NTR: http://www.tweelingenregister.org), the Rotterdam studies (http://www.erasmus-epidemiology.nl/rotterdamstudy), and the Genetic Research in Isolated Populations program (http://www.epib.nl/research/geneticepi/research.html#gip). The sequencing was carried out in collaboration with the Beijing Institute for Genomics (BGI).Data Availability Statement
The online version of this article (https://doi.org/10.1038/s41436-018-0414-9) contains supplementary material, which is available
to authorized users.
Keywords
- ABCA4
- antisense oligonucleotide
- deep-intronic variant
- missing heritability
- Stargardt disease