Blindness Genetics

**Integrated genomics and transcriptomics for ABCA4-associated Stargardt disease**

Through a grant from the Marie Curie Innovative Training Network entitled ‘StarT’, European Training Network to Diagnose, Understand and Treat Stargardt Disease, a Frequent Inherited Blinding Disorder, we focus on the identification of deep-intronic and non-canonical splice site defects in ABCA4, the most frequently mutated gene in IRDs. In collaboration with Dr. E. de Baere and Dr. M. Bauwens in Ghent, we performed ABCA4 locus sequencing in 65 STGD1 probands. The effect of rare variants is validated using in vitro splice assays in HEK393T cells and ‘in vivo’ by RT-PCR of retinal-like cells generated from patients’ somatic cells. Through many collaborations 3,000 genetically unsolved STGD1 cases are being will be analysed for non-coding and structural variants in ABCA4. In case variants result in pseudoexons, we employ antisense oligonucleotides to block erroneous splicing in collaboration with Dr. Rob Collin.
Researchers: Frans Cremers, PhD; Stephanie Cornelis, MSc; Zelia Corradi, MSc.

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 variants p.Asn1868Ile or p.Gly1961Glu 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: Susanne Roosing, PhD; Stephanie Cornelis, MSc; Catherina Li, MSc; Victor de Jager, MSc

Identifying elusive variants in autosomal dominant RP and cystoid MD

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, MSc; Silvia Albert, PhD; Frans Cremers, PhD; Susanne Roosing, PhD;

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. 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: Susanne Roosing, PhD; Frans Cremers, PhD; Zeinab Fadaie, MSc

Natural exon skipping in ABCA4 mRNA and its modulation as a novel genetic therapy for Stargardt disease

By using a grant from the Retina UK (formerly Fighting Blindness UK), we will investigate the underlying basis of natural exon skipping in the ABCA4 gene. By identifying the involved sequence motifs, we will try to modulate the natural exon skipping to design a novel genetic therapy for Stargardt disease.
Researchers: Frans Cremers PhD; Rob Collin, PhD; Alex Garanto, PhD; Tomasz Tomkiewisc, MSc

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. Adrian Dockery, and the Nijmegen group consisting of Dr. Susanne Roosing and Prof. Dr. Frans Cremers, 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 employ an smMIPs-approach; for other inherited retinal diseases we will use whole genome sequencing.
Researchers: Frans Cremers, PhD; Jane Farrar, PhD (Dublin); Adrian Dockery (Dublin); Susanne Roosing, PhD; Victor de Jager, MSc; Stephanie Cornelis, MSc; Laura Whelan, 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; Susanne Roosing, PhD; Frans Cremers, PhD

High-throughput sequencing of inherited and multifactorial macula disease-associated genes and risk factors allows identification of genetic interactions and modifiers

Through funds from Health Research Charities Ireland – Health Research Board, Stichting Blindenhulp, Stichting tot Verbetering van het Lot der Blinden, Stichting Oogfonds, Stichting voor Ooglijders, and ProRetina, we aim to perform cost-effective smMIPs-based sequence analysis of 110 genes implicated in inherited and age-related macular diseases in 1,000 probands with inherited macular diseases, 1,000 probands with age-related macular degeneration, and 1,000 control persons. We consider that many genetically unsolved inherited maculopathy cases may in fact be age-related maculopathy cases and vice versa.
Researchers: Frans Cremers, PhD; Claire-Marie Dhaenens, PhD (Lille); Jane Farrar, PhD; Rebekkah Hitti-Malin, PhD; Anneke den Hollander, PhD; Susanne Roosing, PhD

Cost-effective identification of causal variants in 110 genes underlying Leber congenital amaurosis and retinitis pigmentosa in 4,000 probands, with a special focus on RPE65

In collaboration with Novartis, we plan to perform cost-effective sequence analysis using a new smMIPs platform targeting ~110 genes implicated in retinitis pigmentosa (RP) and/or Leber congenital amaurosis (LCA). We will sequence 4,000 probands with RP and LCA ascertained by international collaborators. We will pay special attention to the RPE65 gene by sequencing all exons and introns.
Researchers: Frans Cremers, PhD; Susanne Roosing, PhD, Alexander Hoischen, PhD; Rebekkah Hitti-Malin, PhD; Anna Tracewska-Siemiatkowska, PhD; postdoc and technician vacancies.

Comprehensive registry and in silico assessment of variants associated with non-syndromic inherited retinal diseases, Bardet-Biedl syndrome and Usher syndrome

Through a grant from the Foundation Fighting Blindness USA, we established an international network consisting of 20 colleagues in 10 teams, each of which will focus on one of the clinical subtypes of inherited retinal diseases. They aim to collect all published genetic variants and the cases in which these were found, in the 2020-2022 period. After data curation and variant pathogenicity classification according to ACMG guidelines, all data will be uploaded in gene-specific Leiden Open (source) Variation databases (see: www.lovd.nl/gene symbol] and ClinVar. Manuscripts are collected that contain genetic defects in a total of 190 different genes.
Researchers: Nijmegen: Frans Cremers, PhD; Susanne Roosing, PhD; Leiden: Ivo Fokkema, PhD, Johan den Dunnen, PhD; Julia Lopez-Hernandez

CONSORTIA AND DATABASES

National clinical database

It is estimated that there are approximately 5000 individuals with inherited retinal diseases in the Netherlands. In order to perform the abovementioned studies, I initiated a national program entitled RD5000 in which all major clinical centers in the Netherlands participate. One of the major goals is to create a multicenter, webbased opthalmogenetic database (rd5000.db) that contains detailed opthalmologic data and basic genetic data of persons with IRDs.Currently, clinical and genetic data have been registered for ~ 3,000 cases.

My main national collaborators are:
• Dr. C.J.F. Boon, Leiden University Medical Center, Leiden
• Dr. F.N. Boonstra, Bartimeus Zeist
• Dr. L.I. van den Born, Rotterdam Eye Hospital, Rotterdam
• Prof. dr. C.C.W. Klaver, Erasmus Medical Center, Rotterdam

European Retinal Disease Consortium: ERDC

The most frequently mutated retinal disease genes have already been identified. As we are now searching for less frequently mutated genes, we initiated a European collaboration coined European Retinal Disease Consortium which consists of 20 research groups in 11 countries.

Contact
Name: Telephone: Email:	F.P.M. Cremers 024-3613750 Frans.Cremers@radboudumc.nl
Name: Telephone: Email:	S. Roosing 024-3668901 susanne.roosing@radboudumc.nl

Visiting and shipping address:	Department of Human Genetics Route 855 Geert Grooteplein Zuid 10 6525 GA Nijmegen The Netherlands
Postal address:	Department of Human Genetics (855) P.O. Box 9101 6500 HB Nijmegen The Netherlands

Main grants

Health Research Charities Ireland – Health Research Board

High-throughput sequencing of inherited and multifactorial macula disease-associated genes and risk factors allows identification of genetic interactions and modifiers; co-applicants: F.P.M. Cremers, G.J. Farrar (Dublin), A.I. den Hollander, S. Roosing, C.-M. Dhaenens (Lille); € 300.000,-; 06/2021 – 05/2024.

Horizon 2020, Marie Sklodowska-Curie Innovative Training Network

European Training Network to Diagnose, Understand and Treat Stargardt Disease, a Frequent Inherited Blinding Disorder -‘StarT’ (813490). Eight academic centers, 2 companies and 5 partner organisations participate. Coordinator: E. de Baere, Ghent. Other academic members: K. Vleminckx, F. Coppieters, Ghent; F.P.M. Cremers, R.W.J. Collin, Nijmegen; S. Banfi, A. Auricchio, Naples; S. Kohl, Tuebingen; C. Toomes, Leeds; J-L. Gomez-Skarmeta, Barcelona; G.J. Farrar, Dublin; M.E. Cheetham, London. Commercial partners: M. Lako, NewCells, Newcastle and P. Adamson, ProQR, Leiden. Partner organizations: Retina International, Fighting Blindness Ireland, 20Med Therapeutics, F. Hofmann – La Roche Ltd. and the European Vision Institute. Total budget: € 3.776.714; Nijmegen part: € 531.240; 11/2018 – 10/2022.

European Joint Programme on Rare Diseases (EJPRD) - ZonMw

Solving missing heritability in inherited retinal diseases using integrated omics and gene editing in human cellular and animal models (Solve-RET). PI: E. de Baere; co-PIs: S. Banfi, F.P.M. Cremers, H. Dollfus, J.L. Gomez-Skarmeta, P. Liskova, M. Spielmann, C. Rivolta. Total budget: € 1,654,600; Nijmegen part: € 250,000,-; 06/2020 – 05/2023.

Foundation Fighting Blindness Inc USA

Project Program Award entitled ‘Splice Modulation to Treat Inherited Retinal Diseases’; coordinator: F.P.M. Cremers; co-investigators: R.W.J. Collin, H.A.R. van Wijk, A. Garanto, C.B. Hoyng, S. Roosing, S. Albert, R. van Huet, module 1 budget $ 542.800,-; total budget: $ 2.500.000; 05/2017 – 04/2022

Velux Foundation

Identification and treatment of non-coding USH2A variants underlying Usher syndrome and retinitis pigmentosa; PIs, H. Kremer, F.P.M. Cremers, E. van Wyk, S. Roosing; € 397,000; 05/2018 – 04/2022.

Foundation Fighting Blindness Inc USA

Individual Investigator Award entitled ‘Deciphering the mechanisms underlying variable expression and non-penetrance of Stargardt disease’; PI: F.P.M. Cremers; co-PI: S. Roosing; budget: $ 299.147,- ; 06/2018 – 05/2021.

Fighting Blindness Ireland

Shedding light on unexplained inherited retinal diseases in Ireland and the Netherlands; co-applicants: J. Farrar, F.P.M. Cremers, S. Roosing, M. Carrigan; € 340.000; 03/2018 – 03/2021.

RetinaUK

Identification and functional characterization of the missing ABCA4 variants in Stargardt disease; F. Cremers (co-applicants: S. Albert, M.I. Khan); £ 216.686; 7/2017 – 6/2020.

RetinaUK - Macular Society

Natural exon skipping in ABCA4 mRNA and its modulation as a novel genetic therapy for Stargardt disease; R.W.J. Collin, F.P.M. Cremers; £ 120.000; 11/2018 – 10/2021.

Awards

F.P.M. Cremers: Ed Golllob Board of Directors Award 2020, for the best manuscript in the field of Ophthalmology in 2019

F.P.M. Cremers: Candle In The Dark - Childvision Research Award (2013)

F.P.M. Cremers: Internationalisations Award from the Radboud University Nijmegen (2010).

F.P.M. Cremers: First European Vision Award October 2007, Portoroz, European Vision Institute.

F.P.M. Cremers: Royal Netherlands Academy of Arts and Sciences (KNAW) fellowship (1991 - 1996). Identification of novel genes for inherited retinal disorders.

F.P.M. Cremers: Retinitis Pigmentosa Award for the Prevention of Blindness 1990, February 1991, Essen, Germany.

F.P.M. Cremers: ‘Student award’ in the category ‘predoctoral basic’ at the 41st Annual Meeting of the American Society of Human Genetics’, October 1990, Cincinnati, USA.

F.P.M. Cremers: Individual Investigator Award of the Foundation Fighting Blindness Inc USA entitled ‘Deciphering the mechanisms underlying variable expression and non-penetrance of Stargardt disease’, May 2018.

Recent Publications Frans Cremers

Cornelis, S. S., Runhart, E. H., Bauwens, M., Corradi, Z., de Baere, E., Roosing, S., Haer-Wigman, L., Dhaenens, C-M., Vulto-van Silfhout, A.T., Cremers, F. P. M. Personalized genetic counseling for Stargardt disease: offspring risk estimates based on variant severity (2022) Am. J. Hum. Genet. S0002-9297(22)00008-8. doi: 10.1016/j.ajhg.2022.01.008. Online ahead of print.

Runhart EH, Khan M, Cornelis SS, Roosing S, Del Pozo-Valero M, Lamey TM, Liskova P, Roberts L, Stöhr H, Klaver CCW, Hoyng CB, Cremers FPM, Dhaenens CM; ABCA4 Disease Consortium Study Group (2020) Association of Sex With Frequent and Mild ABCA4 Alleles in Stargardt Disease. JAMA Ophthalmol. 138, 1035-1042

Cremers FPM, Lee W, Collin RWJ, Allikmets R. Clinical spectrum, genetic complexity and therapeutic approaches for retinal disease caused by ABCA4 mutations. (2020) Prog. Retin. Eye Res. Apr 9:100861. doi: 10.1016/j.preteyeres. 100861. [Epub ahead of print] Review.

Khan M, Cornelis SS, Pozo-Valero MD, Whelan L, Runhart EH, Mishra K, Bults F, AlSwaiti Y, AlTalbishi A, De Baere E, Banfi S, Banin E, Bauwens M, Ben-Yosef T, Boon CJF, van den Born LI, Defoort S, Devos A, Dockery A, Dudakova L, Fakin A, Farrar GJ, Sallum JMF, Fujinami K, Gilissen C, Glavač D, Gorin MB, Greenberg J, Hayashi T, Hettinga YM, Hoischen A, Hoyng CB, Hufendiek K, Jägle H, Kamakari S, Karali M, Kellner U, Klaver CCW, Kousal B, Lamey TM, MacDonald IM, Matynia A, McLaren TL, Mena MD, Meunier I, Miller R, Newman H, Ntozini B, Oldak M, Pieterse M, Podhajcer OL, Puech B, Ramesar R, Rüther K, Salameh M, Salles MV, Sharon D, Simonelli F, Spital G, Steehouwer M, Szaflik JP, Thompson JA, Thuillier C, Tracewska AM, van Zweeden M, Vincent AL, Zanlonghi X, Liskova P, Stöhr H, Roach JN, Ayuso C, Roberts L, Weber BHF, Dhaenens CM, Cremers FPM (2020) Resolving the dark matter of ABCA4 for 1054 Stargardt disease probands through integrated genomics and transcriptomics. Genet. Med. 22:1235-1246

*Sangermano R, *Garanto A, *Khan M, Runhart EH, Bauwens M, Bax NM, van den Born LI, Khan MI, Cornelis SS, Verheij JBGM, Pott JWR, Thiadens AAHJ, Klaver CCW, Puech B, Meunier I, Naessens S, Blokland EAW, Elmelik DM, Arno G, Fakin A, Carss KJ, Raymond FL, Webster AR, Dhaenens C-M, Stöhr H, Grassmann F, Weber BHF, Hoyng CB, de Baere E, Albert S, **Collin RWJ, **Cremers FPM. Identification and rescue of splice defects due to deep-intronic ABCA4 variants in Stargardt disease (2019) Genet. Med., 21:1751-1760.

Sangermano R, Khan M, Cornelis SS, Richelle V, Albert S, Garanto A, Elmelik D, Qamar R, Lugtenberg D, van den Born LI, Collin RWJ, Cremers FPM. ABCA4 midigenes reveal the full splice spectrum of all reported noncanonical splice site variants in Stargardt disease (2018) Genome Res. 28,100-110.

*Albert S, *Garanto A, Sangermano R, Khan M, Bax N, Hoyng CB, Zernant J, Lee W, Allikmets R, **Collin RWJ, **Cremers FPM. Identification and rescue of splice defects caused by two neighboring deep-intronic ABCA4 mutations underlying Stargardt disease. (2018) Am. J. Hum. Genet., 102, 517-527. *Shared first authors; **shared senior authors.

For a list of all publications see PubMed

Recent Publications Susanne Roosing

*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 (2020) Structural Variants Create New Topological-Associated Domains and Ectopic Retinal Enhancer-Gene Contact in Dominant Retinitis Pigmentosa. Am. J. Hum. Genet. 107, 802-814. *Shared first authors; **shared senior authors.

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; 40:2365-2237

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.

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. doi: 10.7554/eLife.06602.

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 AA, Hoyng CB, Klaver CC, den Hollander AI, Cremers FP. Causes and consequences of inherited cone disorders. Prog Retin Eye Res. 2014 Sep;42:1-26.

For a list of all publications see PubMed