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Foundation Fighting Blindness USA Project Program Award Splice Modulation to Treat Inherited Retinal Diseases
Through a 5-year program award from FFB USA, we are searching for deep-intronic variants in genes associated with autosomal recessive retinal dystrophies in patients that carry one causal coding variant. Non-coding variants can create new splice sites or activate cryptic splice sites and thereby result in the inclusion of a pseudoexon into the mRNA. In addition, we will be searching for cis-regulatory variants that have an effect on transcription. Candidate disease variants will be tested in vitro using sophisticated splicing vectors and HEK293T transfections. For selected cases, somatic cells of patients will be reprogrammed into induced pluripotent stem cells which will be differentiated to photoreceptor precursor cells (PPCs). Mutation- or gene-specific RT-PCR will be performed to validate RNA splicing defects.
Researchers: Kim Rodenburg, MSc, Frans Cremers, PhD; Susanne Roosing, PhD
Former researcher: Zeinab Fadaie, PhD
Identifying elusive variants in autosomal dominant RP and cystoid Macular Dystrophy
Through a RadboudUMC grant, we search for non-coding sequence variants and structural variants in autosomal dominant RD families. We reprogram fibroblasts or blood cells of patients into induced pluripotent stem cells (iPSCs) in our Stem Cell Technology Center. iPSCs are being differentiated into photoreceptor progenitor cells or retinal organoids. RNA extracted from these cells is analyzed by RT-PCR. In parallel, whole genome sequencing is performed to identify intronic variants and structural variants.
Researchers: Suzanne de Bruijn, PhD; Silvia Albert, PhD; Frans Cremers, PhD; Susanne Roosing, PhD
In parallel, through a UitZicht grant, we investigate these long-studied large IRD families with an unresolved disease locus as well as other IRD families that remain unresolved after WGS analysis. By applying Bionano optical mapping and long-read sequencing through Pacbio, we aim to identify the underlying genetic defect in these families.
Researchers: Kim Rodenburg, MSc; Suzanne de Bruijn, PhD; Susanne Roosing, PhD
Former researchers: Zeinab Fadaie, PhD
Shedding light on unexplained inherited retinal diseases in Ireland and the Netherlands
Through a grant from Fighting Blindness Ireland the Dublin group consisting of Prof. Jane Farrar and Dr. Mathew Carrigan, and the Nijmegen group consisting of Dr. Susanne Roosing and Prof. dr. Frans Cremers, will try to identify the underlying mutations in persons with Stargardt disease and (after sequencing coding exons) one or no ABCA4 mutation from Ireland, as well as, after targeted next-generation sequencing, mono-allelic persons with other inherited retinal diseases from Ireland, as well as (after whole exome sequencing), persons with inherited retinal diseases from the Netherlands and no causal variant(s). For Stargardt disease, we will employ an smMIPs-approach; for other inherited retinal diseases we will use whole genome sequencing.
Researchers: Laura Whelan, MSc; Frans Cremers, PhD; Jane Farrar, PhD (Dublin); Adrian Dockery (Dublin); Susanne Roosing, PhD
Former researchers: Victor de Jager, MSc; Stephanie Cornelis, MSc
Identification and treatment of non-coding USH2A variants underlying Usher syndrome and retinitis pigmentosa
Through a grant from the Velux Stiftung, we will search for non-coding variants in the USH2A gene in persons with Usher syndrome type II or autosomal recessive retinitis pigmentosa and one proven coding variant. We will test their causality using a midigene splice assay in HEK293T cells. Subsequently, we will design a therapy based on antisense oligonucleotide targeting of the USH2A pre-mRNA.
Researchers: Janine Reurink, MSc; Hannie Kremer, PhD; Erwin van Wyk, PhD; Frans Cremers, PhD; Susanne Roosing, PhD
Former researcher: Marco Aben, MSc
Prediction of tissue-specific splicing to improve diagnosis of inherited retinal diseases
Through an Radboudumc grant, we will assess deep learning based splice prediction tools like SpliceAI and DARTS that show an improvement in the identification of splice altering variants. However, these fail to identify deep intronic variants affecting tissue-specific splice events in the retina as prediction tools are not trained on retina RNA data. Sequencing of the human retina revealed hundreds of retina-specific exons that are missing in the training data of those tools. The goal of this project is to increase the diagnostic yield of IRDs by creating a tissue specific deep learning splice prediction tool for the retina.
Researchers: Tabea Riepe, MSc; Peter-Bram 't Hoen, PhD; Susanne Roosing, PhD; Frans Cremers, PhD.
Deciphering the mechanisms underlying variable expression and non-penetrance of Stargardt disease
Through an Individual Investigator Award from the Foundation Fighting Blindness Inc. USA, we will search for genetic and non-genetic modifiers of Stargardt disease. In a recent study, we identified non-penetrance or late-onset of Stargardt disease due to the combination of the frequent coding ABCA4 variant p.Asn1868Ile on one allele and a severe mutation on the other allele. To search for genetic and non-genetic modifiers of disease, we will collect bi-allelic sibpairs in whom there is a major difference in onset of Stargardt disease. We will perform whole genome sequencing in these sibpairs and search for major factors.
Researchers: Victor de Jager, Msc; Stephanie Cornelis, MSc; Femke Bults, Bsc; Esmee Runhart, MD, MSc; Susanne Roosing, PhD; Frans Cremers, PhD
CONSORTIA AND DATABASES
European Retinal Disease Consortium: ERDC
While aiming to uncover novel strategies to finding new causative genes for IRD, this cannot be pursued without extensive collaboration with other research groups in Europe and the world. The urgency for collaborations has become more clear as individual research groups have interesting candidate genes which may explain IRD in single families. The European Retinal Disease Consortium (ERDC; www.erdc.info) has a long-standing and ongoing collaboration in sharing patient resources, data and candidate genes as well as technologies. Susanne Roosing is the chair and coordinator where today, the ERDC contains 22 research groups of which most are European based, three are from Israel and two are American and Canadian. This collaboration has resulted in over 160 joint publications and many joint projects, where the aim is to provide all IRD patients with a genetic diagnosis.
Leiden Open (source) Variation Databases (LOVDs)
LOVDs have been established for all inherited retinal diseases and we aim to complete all LOVDs for ~150 non-syndromic IRD-associated genes and Usher syndrome, to be completed with published variants and (anonymous) patient IDs in the 2020-2023 period. For several genes, the LOVD is already complete. Susanne Roosing is curator for the cone-dystrophy and cone-rod dystrophy genes.
An overview of relevant last-author and first-author publications. For a list of all publications see PubMed
Fadaie Z, Neveling K, Mantere T, Derks R, Haer-Wigman L, den Ouden A, Kwint M, O'Gorman L, Valkenburg D, Hoyng CB, Gilissen C, Vissers LELM, Nelen M, Cremers FPM, Hoischen A, Roosing S. Long-read technologies identify a hidden inverted duplication in a family with choroideremia. HGG Adv. 2021 Jul 20;2(4):100046.
Fadaie Z, Whelan L, Ben-Yosef T, Dockery A, Corradi Z, Gilissen C, Haer-Wigman L, Corominas J, Astuti GDN, de Rooij L, van den Born LI, Klaver CCW, Hoyng CB, Wynne N, Duignan ES, Kenna PF, Cremers FPM, Farrar GJ, Roosing S. Whole genome sequencing and in vitro splice assays reveal genetic causes for inherited retinal diseases.NPJ Genom Med. 2021 Nov 18;6(1):97.
Reurink J, Dockery A, Oziębło D, Farrar GJ, Ołdak M, Ten Brink JB, Bergen AA, Rinne T, Yntema HG, Pennings RJE, van den Born LI, Aben M, Oostrik J, Venselaar H, Plomp AS, Khan MI, van Wijk E, Cremers FPM, Roosing S, Kremer H. Molecular Inversion Probe-Based Sequencing of USH2A Exons and Splice Sites as a Cost-Effective Screening Tool in USH2 and arRP Cases. Int J Mol Sci. 2021 Jun 15;22(12):6419
Riepe TV, Khan M, Roosing S, Cremers FPM, 't Hoen PAC. Benchmarking deep learning splice prediction tools using functional splice assays.
Hum Mutat. 2021 Jul;42(7):799-810.
Fadaie Z, Whelan L, Dockery A, Li CHZ, van den Born LI, Hoyng CB, Gilissen C, Corominas J, Rowlands C, Megaw R, Lampe AK, Cremers FPM, Farrar GJ, Ellingford JM, Kenna PF, Roosing S. BBS1 branchpoint variant is associated with non-syndromic retinitis pigmentosa. J Med Genet. 2022 May;59(5):438-444.
de Bruijn SE, Fadaie Z, Cremers FPM, Kremer H, Roosing S. The Impact of Modern Technologies on Molecular Diagnostic Success Rates, with a Focus on Inherited Retinal Dystrophy and Hearing Loss. Int J Mol Sci. 2021 Mar 14;22(6):2943.
de Bruijn SE, Fiorentino A, Ottaviani D, Fanucchi S, Melo US, Corral-Serrano JC, Mulders T, Georgiou M, Rivolta C, Pontikos N, Arno G, Roberts L, Greenberg J, Albert S, Gilissen C, Aben M, Rebello G, Mead S, Raymond FL, Corominas J, Smith CEL, Kremer H, Downes S, Black GC, Webster AR, Inglehearn CF, van den Born LI, Koenekoop RK, Michaelides M, Ramesar RS, Hoyng CB, Mundlos S, Mhlanga MM, Cremers FPM, Cheetham ME, Roosing S, Hardcastle AJ. Structural Variants Create New Topological-Associated Domains and Ectopic Retinal Enhancer-Gene Contact in Dominant Retinitis Pigmentosa. Am J Hum Genet. 2020 Nov 5;107(5):802-814.
Fadaie, Z, Khan, M, del Pozo-Valero, M, Cornelis, S.S., Ayuso, C, ABCA4 Studygroup, Cremers, F.P.M., Roosing, S. Identification of splice defects due to non-canonical splice site or deep-intronic variants in ABCA4, Hum Mutat. 2019 Dec;40(12):2365-2376.
Verbakel, SK, Fadaie, Z, Klevering, BJ, van Genderen, MM, Feenstra, I, Cremers, FPM, Hoyng, CB, Roosing, S. The identification of a RNA splice variant in TULP1 in two siblings with early-onset photoreceptor dystrophy, Mol Genet Genomic Med. 2019 Apr 4:e660.
Khan M, Fadaie Z, Cornelis SS, Cremers FPM, Roosing S. Identification and Analysis of Genes Associated with Inherited Retinal Diseases. Methods Mol Biol. 2019;1834:3-27.
de Bruijn, SE, Verbakel SK, de Vrieze E, Kremer H, Cremers FPM, Hoyng CB, van den Born LI, Roosing S. Homozygous variants in gene KIAA1549, encoding a ciliary protein, are associated with autosomal recessive retinitis pigmentosa. J Med Genet 2018;0:1–8.
Astuti, GDN, van den Born LI, Khan MI, Hamel CP, Bocquet B, Manes G, Quinodoz M, Ali M, Toomes C, McKibbin M, El-Asrag ME, Haer-Wigman L, Inglehearn CF, Black GCM, Hoyng CB, Cremers FPM, Roosing S, Identification of inherited retinal disease-associated genetic variants in 11 possible candidate genes, Genes (Basel). 2018 Jan 10;9(1).
Roosing S, Cremers FPM, Riemslag FCC, Zonneveld-Vrieling MN, Talsma H, Klessens-Godfroy FJM, den Hollander AI, van den Born LI A rare form of retinal dystrophy caused by hypomorphic nonsense mutations in CEP290, Genes (Basel). 2017 Aug 22;8(8).
Roosing S, Hofree M, Kim S, Scott E, Copeland B, Romani M, Silhavy JL, Rosti RO, Schroth J, Mazza T, Miccinilli E, Zaki MS, Swoboda KJ, Milisa-Drautz J, Dobyns WB, Mikati M, İncecik F, Azam M, Borgatti R, Romaniello R, Boustany RM, Clericuzio CL, D'Arrigo S, Strømme P, Boltshauser E, Stanzial F, Mirabelli-Badenier M, Moroni I, Bertini E, Emma F, Steinlin M, Hildebrandt F, Johnson CA, Freilinger M, Vaux KK, Gabriel SB, Aza-Blanc P, Heynen-Genel S, Ideker T, Dynlacht BD, Lee JE, Valente EM, Kim J, Gleeson JG. Functional genome-wide siRNA screen identifies KIAA0586 as mutated in Joubert syndrome. Elife. 2015 May 30;4.
Roosing S, van den Born LI, Sangermano R, Banfi S, Koenekoop RK, Zonneveld-Vrieling MN, Klaver CC, van Lith-Verhoeven JJ, Cremers FPM, den Hollander AI, Hoyng CB. Mutations in MFSD8, encoding a lysosomal membrane protein, are associated with nonsyndromic autosomal recessive macular dystrophy. Ophthalmology. 2015 Jan;122(1):170-9.
Roosing S*, Lamers IJ*, de Vrieze E*, van den Born LI*, Lambertus S, Arts HH; POC1B Study Group, Peters TA, Hoyng CB, Kremer H, Hetterschijt L, Letteboer SJ, van Wijk E#, Roepman R#, den Hollander AI#, Cremers FPM#. Disruption of the basal body protein POC1B results in autosomal-recessive cone-rod dystrophy. Am J Hum Genet. 2014 Aug 7;95(2):131-42.
Roosing S, Thiadens AAHJ, Hoyng CB, Klaver CCW, den Hollander AI, Cremers FPM. Causes and consequences of inherited cone disorders. Prog Retin Eye Res. 2014 Sep;42:1-26.