Publications by authors named "Rob W J Collin"

116 Publications

Antisense Oligonucleotide-Based Rescue of Aberrant Splicing Defects Caused by 15 Pathogenic Variants in .

Int J Mol Sci 2021 Apr 28;22(9). Epub 2021 Apr 28.

Departments of Pediatrics and Human Genetics, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, 6525GA Nijmegen, The Netherlands.

The discovery of novel intronic variants in the locus has contributed significantly to solving the missing heritability in Stargardt disease (STGD1). The increasing number of variants affecting pre-mRNA splicing makes a suitable candidate for antisense oligonucleotide (AON)-based splicing modulation therapies. In this study, AON-based splicing modulation was assessed for 15 recently described intronic variants (three near-exon and 12 deep-intronic variants). In total, 26 AONs were designed and tested in vitro using a midigene-based splice system. Overall, partial or complete splicing correction was observed for two variants causing exon elongation and all variants causing pseudoexon inclusion. Together, our results confirm the high potential of AONs for the development of future RNA therapies to correct splicing defects causing STGD1.
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http://dx.doi.org/10.3390/ijms22094621DOI Listing
April 2021

Delivery of oligonucleotide-based therapeutics: challenges and opportunities.

EMBO Mol Med 2021 Apr 6;13(4):e13243. Epub 2021 Apr 6.

Neuromuscular Disorders Group, Biocruces Bizkaia Health Research Institute, Barakaldo, Spain.

Nucleic acid-based therapeutics that regulate gene expression have been developed towards clinical use at a steady pace for several decades, but in recent years the field has been accelerating. To date, there are 11 marketed products based on antisense oligonucleotides, aptamers and small interfering RNAs, and many others are in the pipeline for both academia and industry. A major technology trigger for this development has been progress in oligonucleotide chemistry to improve the drug properties and reduce cost of goods, but the main hurdle for the application to a wider range of disorders is delivery to target tissues. The adoption of delivery technologies, such as conjugates or nanoparticles, has been a game changer for many therapeutic indications, but many others are still awaiting their eureka moment. Here, we cover the variety of methods developed to deliver nucleic acid-based therapeutics across biological barriers and the model systems used to test them. We discuss important safety considerations and regulatory requirements for synthetic oligonucleotide chemistries and the hurdles for translating laboratory breakthroughs to the clinic. Recent advances in the delivery of nucleic acid-based therapeutics and in the development of model systems, as well as safety considerations and regulatory requirements for synthetic oligonucleotide chemistries are discussed in this review on oligonucleotide-based therapeutics.
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http://dx.doi.org/10.15252/emmm.202013243DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8033518PMC
April 2021

Detailed Phenotyping and Therapeutic Strategies for Intronic ABCA4 Variants in Stargardt Disease.

Mol Ther Nucleic Acids 2020 Sep 12;21:412-427. Epub 2020 Jun 12.

Department of Human Genetics, Radboud University Medical Center, Nijmegen, the Netherlands; Donders Institute for Brain, Cognition and Behavior, Radboud University Medical Center, Nijmegen, the Netherlands. Electronic address:

Stargardt disease is a progressive retinal disorder caused by bi-allelic mutations in the ABCA4 gene that encodes the ATP-binding cassette, subfamily A, member 4 transporter protein. Over the past few years, we and others have identified several pathogenic variants that reside within the introns of ABCA4, including a recurrent variant in intron 36 (c.5196+1137G>A) of which the pathogenicity so far remained controversial. Detailed clinical characterization of this variant confirmed its pathogenic nature, and classified it as an allele of intermediate severity. Moreover, we discovered several additional ABCA4 variants clustering in intron 36. Several of these variants resulted in aberrant splicing of ABCA4, i.e., the inclusion of pseudoexons, while the splicing defects caused by the recurrent c.5196+1137G>A variant strongly increased upon differentiation of patient-derived induced pluripotent stem cells into retina-like cells. Finally, all splicing defects could be rescued by the administration of antisense oligonucleotides that were designed to specifically block the pseudoexon insertion, including rescue in 3D retinal organoids harboring the c.5196+1137G>A variant. Our data illustrate the importance of intronic variants in ABCA4 and expand the therapeutic possibilities for overcoming splicing defects in Stargardt disease.
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http://dx.doi.org/10.1016/j.omtn.2020.06.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7352060PMC
September 2020

PCARE and WASF3 regulate ciliary F-actin assembly that is required for the initiation of photoreceptor outer segment disk formation.

Proc Natl Acad Sci U S A 2020 05 20;117(18):9922-9931. Epub 2020 Apr 20.

Department of Human Genetics, Radboud University Medical Center, 6525 GA, Nijmegen, The Netherlands.

The outer segments (OS) of rod and cone photoreceptor cells are specialized sensory cilia that contain hundreds of opsin-loaded stacked membrane disks that enable phototransduction. The biogenesis of these disks is initiated at the OS base, but the driving force has been debated. Here, we studied the function of the protein encoded by the photoreceptor-specific gene , which is mutated in inherited retinal dystrophy (RP54). We demonstrate that C2orf71/PCARE (photoreceptor cilium actin regulator) can interact with the Arp2/3 complex activator WASF3, and efficiently recruits it to the primary cilium. Ectopic coexpression of PCARE and WASF3 in ciliated cells results in the remarkable expansion of the ciliary tip. This process was disrupted by small interfering RNA (siRNA)-based down-regulation of an actin regulator, by pharmacological inhibition of actin polymerization, and by the expression of PCARE harboring a retinal dystrophy-associated missense mutation. Using human retinal organoids and mouse retina, we observed that a similar actin dynamics-driven process is operational at the base of the photoreceptor OS where the PCARE module and actin colocalize, but which is abrogated in mice. The observation that several proteins involved in retinal ciliopathies are translocated to these expansions renders it a potential common denominator in the pathomechanisms of these hereditary disorders. Together, our work suggests that PCARE is an actin-associated protein that interacts with WASF3 to regulate the actin-driven expansion of the ciliary membrane at the initiation of new outer segment disk formation.
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http://dx.doi.org/10.1073/pnas.1903125117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7211956PMC
May 2020

Clinical spectrum, genetic complexity and therapeutic approaches for retinal disease caused by ABCA4 mutations.

Prog Retin Eye Res 2020 11 9;79:100861. Epub 2020 Apr 9.

Department of Ophthalmology, Columbia University, New York, NY, 10032, USA; Department of Pathology & Cell Biology, Columbia University, New York, NY, 10032, USA. Electronic address:

The ABCA4 protein (then called a "rim protein") was first identified in 1978 in the rims and incisures of rod photoreceptors. The corresponding gene, ABCA4, was cloned in 1997, and variants were identified as the cause of autosomal recessive Stargardt disease (STGD1). Over the next two decades, variation in ABCA4 has been attributed to phenotypes other than the classically defined STGD1 or fundus flavimaculatus, ranging from early onset and fast progressing cone-rod dystrophy and retinitis pigmentosa-like phenotypes to very late onset cases of mostly mild disease sometimes resembling, and confused with, age-related macular degeneration. Similarly, analysis of the ABCA4 locus uncovered a trove of genetic information, including >1200 disease-causing mutations of varying severity, and of all types - missense, nonsense, small deletions/insertions, and splicing affecting variants, of which many are located deep-intronic. Altogether, this has greatly expanded our understanding of complexity not only of the diseases caused by ABCA4 mutations, but of all Mendelian diseases in general. This review provides an in depth assessment of the cumulative knowledge of ABCA4-associated retinopathy - clinical manifestations, genetic complexity, pathophysiology as well as current and proposed therapeutic approaches.
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http://dx.doi.org/10.1016/j.preteyeres.2020.100861DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7544654PMC
November 2020

In or Out? New Insights on Exon Recognition through Splice-Site Interdependency.

Int J Mol Sci 2020 03 26;21(7). Epub 2020 Mar 26.

Department of Human Genetics, Radboud University Medical Center, 6525 GA Nijmegen, The Netherlands.

Noncanonical splice-site mutations are an important cause of inherited diseases. Based on in vitro and stem-cell-based studies, some splice-site variants show a stronger splice defect than expected based on their predicted effects, suggesting that other sequence motifs influence the outcome. We investigated whether splice defects due to human-inherited-disease-associated variants in noncanonical splice-site sequences in , , and could be rescued by strengthening the splice site on the other side of the exon. Noncanonical 5'- and 3'-splice-site variants were selected. Rescue variants were introduced based on an increase in predicted splice-site strength, and the effects of these variants were analyzed using in vitro splice assays in HEK293T cells. Exon skipping due to five variants in noncanonical splice sites of exons in , , and could be partially or completely rescued by increasing the predicted strengths of the other splice site of the same exon. We named this mechanism "splicing interdependency", and it is likely based on exon recognition by splicing machinery. Awareness of this interdependency is of importance in the classification of noncanonical splice-site variants associated with disease and may open new opportunities for treatments.
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http://dx.doi.org/10.3390/ijms21072300DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7177576PMC
March 2020

Modeling ZNF408-Associated FEVR in Zebrafish Results in Abnormal Retinal Vasculature.

Invest Ophthalmol Vis Sci 2020 02;61(2):39

,.

Purpose: Familial exudative vitreoretinopathy (FEVR) is an inherited retinal disease in which the retinal vasculature is affected. Patients with FEVR typically lack or have abnormal vasculature in the peripheral retina, the outcome of which can range from mild visual impairment to complete blindness. A missense mutation (p.His455Tyr) in ZNF408 was identified in an autosomal dominant FEVR family. Little, however, is known about the molecular role of ZNF408 and how its defect leads to the clinical features of FEVR.

Methods: Using CRISPR/Cas9 technology, two homozygous mutant zebrafish models with truncated znf408 were generated, as well as one heterozygous and one homozygous missense znf408 model in which the human p.His455Tyr mutation is mimicked.

Results: Intriguingly, all three znf408-mutant zebrafish strains demonstrated progressive retinal vascular pathology, initially characterized by a deficient hyaloid vessel development at 5 days postfertilization (dpf) leading to vascular insufficiency in the retina. The generation of stable mutant lines allowed long-term follow up studies, which showed ectopic retinal vascular hyper-sprouting at 90 dpf and extensive vascular leakage at 180 dpf.

Conclusions: Together, our data demonstrate an important role for znf408 in the development and maintenance of the vascular system within the retina.
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http://dx.doi.org/10.1167/iovs.61.2.39DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7329629PMC
February 2020

Correction: P-cadherin mutations are associated with high basal Wnt activity and stemness in canine mammary tumor cell lines.

Oncotarget 2020 Feb 4;11(5):573. Epub 2020 Feb 4.

Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.

[This corrects the article DOI: 10.18632/oncotarget.26873.].
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http://dx.doi.org/10.18632/oncotarget.27475DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7007293PMC
February 2020

Preface of Special Issue "Molecular Therapies for Inherited Retinal Diseases".

Genes (Basel) 2020 02 5;11(2). Epub 2020 Feb 5.

Department of Human Genetics and Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Center, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands.

Inherited retinal diseases (IRDs) are a group of progressive disorders that lead to severe visual impairment or even complete blindness. IRDs display a vast heterogeneity, clinically as well as genetically, with over 250 genes identified in which mutations can cause one or more clinical subtypes of IRD. Long considered incurable diseases, intense research over the last two decades, combined with major technological advancements, have enabled the development of the first therapeutic approaches for these diseases. The approval of LuxturnaTM (voretigene neparvovec), a gene augmentation therapy vector for RPE65-associated IRD, by the US Food and Drug Administration and the European Medicines Agency, is considered a true milestone in the field, and has led to the development of similar, or different therapeutic strategies for many other subtypes of IRD. Despite these major achievements, there are still many aspects that can-and need to-be improved, including more insights into the relationship between genetic variation and cellular dysfunction, optimization of the vectors and sequences used, improving delivery methods, as well as understanding and modulating the (local) immune response. In addition, the extreme rarity of some genetic subtypes of IRDs poses an enormous challenge on the development of novel therapies, in terms of e.g., costs and regulatory affairs.[...].
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http://dx.doi.org/10.3390/genes11020169DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7074026PMC
February 2020

Molecular Therapies for Inherited Retinal Diseases-Current Standing, Opportunities and Challenges.

Genes (Basel) 2019 08 28;10(9). Epub 2019 Aug 28.

Department of Human Genetics and Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Center, 6525GA Nijmegen, The Netherlands.

Inherited retinal diseases (IRDs) are both genetically and clinically highly heterogeneous and have long been considered incurable. Following the successful development of a gene augmentation therapy for biallelic -associated IRD, this view has changed. As a result, many different therapeutic approaches are currently being developed, in particular a large variety of molecular therapies. These are depending on the severity of the retinal degeneration, knowledge of the pathophysiological mechanism underlying each subtype of IRD, and the therapeutic target molecule. DNA therapies include approaches such as gene augmentation therapy, genome editing and optogenetics. For some genetic subtypes of IRD, RNA therapies and compound therapies have also shown considerable therapeutic potential. In this review, we summarize the current state-of-the-art of various therapeutic approaches, including the pros and cons of each strategy, and outline the future challenges that lie ahead in the combat against IRDs.
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http://dx.doi.org/10.3390/genes10090654DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6770110PMC
August 2019

Antisense Oligonucleotide Screening to Optimize the Rescue of the Splicing Defect Caused by the Recurrent Deep-Intronic Variant c.4539+2001G>A in Stargardt Disease.

Genes (Basel) 2019 06 14;10(6). Epub 2019 Jun 14.

Department of Human Genetics and Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Center, 6525GA Nijmegen, The Netherlands.

Deep-sequencing of the locus has revealed that ~10% of autosomal recessive Stargardt disease (STGD1) cases are caused by deep-intronic mutations. One of the most recurrent deep-intronic variants in the Belgian and Dutch STGD1 population is the c.4539+2001G>A mutation. This variant introduces a 345-nt pseudoexon to the mRNA transcript in a retina-specific manner. Antisense oligonucleotides (AONs) are short sequences of RNA that can modulate splicing. In this work, we designed 26 different AONs to perform a thorough screening to identify the most effective AONs to correct splicing defects associated with c.4539+2001G>A. All AONs were tested in patient-derived induced pluripotent stem cells (iPSCs) that were differentiated to photoreceptor precursor cells (PPCs). AON efficacy was assessed through RNA analysis and was based on correction efficacy, and AONs were grouped and their properties assessed. We (a) identified nine AONs with significant correction efficacies (>50%), (b) confirmed that a single nucleotide mismatch was sufficient to significantly decrease AON efficacy, and (c) found potential correlations between efficacy and some of the parameters analyzed. Overall, our results show that AON-based splicing modulation holds great potential for treating Stargardt disease caused by splicing defects in .
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http://dx.doi.org/10.3390/genes10060452DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6628380PMC
June 2019

Extending the Spectrum of EYS-Associated Retinal Disease to Macular Dystrophy.

Invest Ophthalmol Vis Sci 2019 05;60(6):2049-2063

The Rotterdam Eye Hospital, Rotterdam, The Netherlands.

Purpose: To assess the phenotypic variability and natural course of inherited retinal diseases (IRDs) caused by EYS mutations.

Methods: Multiethnic cohort study (N = 30) with biallelic EYS variants from a clinical IRD database (retinitis pigmentosa [RP], N = 27; cone-rod dystrophy [CRD], N = 1; and macular dystrophy, N = 2). In vitro minigene splice assay was performed to determine the effect on EYS pre-mRNA splicing of the c.1299+5_1299+8del variant in macular dystrophy patients.

Results: We found 27 different EYS variants in RP patients and 7 were novel. The rate of visual field loss of the V4e isopter area was -0.84 ± 0.44 ln(deg2) per year, and the rate of visual acuity loss was 0.75 Early Treatment Diabetic Retinopathy Study letters per year. Ellipsoid zone width was correlated with area of the hyperautofluorescent ring, with rs = 0.78 and P < 0.001. Rate of decline in ellipsoid zone width was -57 ± 17 μm per year (P < 0.01) (n = 14) or -3.69% ± 0.51% from baseline per year (P < 0.001). An isolated CRD patient carried a homozygous EYS variant (c.9405T>A), previously identified in RP patients. Two siblings with macular dystrophy carried compound heterozygous EYS variants: c.1299+5_1299+8del and c.6050G>T. The former was novel and shown to result in skipping of exon 8, and the latter was a known RP variant.

Conclusions: We report on EYS-associated macular dystrophy, extending the spectrum of EYS-associated IRDs. We observed heterogeneity between RP patients in age of onset and disease progression. Identical EYS variants were found in cases with RP, CRD, and macular dystrophy. Screening for EYS variants in CRD and macular dystrophy patients might increase the diagnostic yield in previously unsolved cases.
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http://dx.doi.org/10.1167/iovs.18-25531DOI Listing
May 2019

ABCA4-associated disease as a model for missing heritability in autosomal recessive disorders: novel noncoding splice, cis-regulatory, structural, and recurrent hypomorphic variants.

Genet Med 2019 08 23;21(8):1761-1771. Epub 2019 Jan 23.

Center for Medical Genetics Ghent, Ghent University and Ghent University Hospital, Ghent, Belgium.

Purpose: ABCA4-associated disease, a recessive retinal dystrophy, is hallmarked by a large proportion of patients with only one pathogenic ABCA4 variant, suggestive for missing heritability.

Methods: By locus-specific analysis of ABCA4, combined with extensive functional studies, we aimed to unravel the missing alleles in a cohort of 67 patients (p), with one (p = 64) or no (p = 3) identified coding pathogenic variants of ABCA4.

Results: We identified eight pathogenic (deep-)intronic ABCA4 splice variants, of which five are novel and six structural variants, four of which are novel, including two duplications. Together, these variants account for the missing alleles in 40.3% of patients. Furthermore, two novel variants with a putative cis-regulatory effect were identified. The common hypomorphic variant c.5603A>T p.(Asn1868Ile) was found as a candidate second allele in 43.3% of patients. Overall, we have elucidated the missing heritability in 83.6% of our cohort. In addition, we successfully rescued three deep-intronic variants using antisense oligonucleotide (AON)-mediated treatment in HEK 293-T cells and in patient-derived fibroblast cells.

Conclusion: Noncoding pathogenic variants, novel structural variants, and a common hypomorphic allele of the ABCA4 gene explain the majority of unsolved cases with ABCA4-associated disease, rendering this retinopathy a model for missing heritability in autosomal recessive disorders.
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http://dx.doi.org/10.1038/s41436-018-0420-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6752479PMC
August 2019

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

Genet Med 2019 08 15;21(8):1751-1760. Epub 2019 Jan 15.

Department of Human Genetics, Radboud University Medical Center, Nijmegen, The Netherlands.

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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http://dx.doi.org/10.1038/s41436-018-0414-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6752325PMC
August 2019

Clinical Characterization of 66 Patients With Congenital Retinal Disease Due to the Deep-Intronic c.2991+1655A>G Mutation in CEP290.

Invest Ophthalmol Vis Sci 2018 09;59(11):4384-4391

Radboud University Medical Center, Donders Institute for Brain, Cognition and Behavior, Cognitive Neuroscience Department, Nijmegen, The Netherlands.

Purpose: To describe the phenotypic spectrum of retinal disease caused by the c.2991+1655A>G mutation in CEP290 and to compare disease severity between homozygous and compound heterozygous patients.

Methods: Medical records were reviewed for best-corrected visual acuity (BCVA), age of onset, fundoscopy descriptions. Foveal outer nuclear layer (ONL) and ellipsoid zone (EZ) presence was assessed using spectral-domain optical coherence tomography (SD-OCT). Differences between compound heterozygous and homozygous patients were analyzed based on visual performance and visual development.

Results: A total of 66 patients were included. The majority of patients had either light perception or no light perception. In the remaining group of 14 patients, median BCVA was 20/195 Snellen (0.99 LogMAR; range 0.12-1.90) for the right eye, and 20/148 Snellen (0.87 LogMAR; range 0.22-1.90) for the left. Homozygous patients tended to be more likely to develop light perception compared to more severely affected compound heterozygous patients (P = 0.080) and are more likely to improve from no light perception to light perception (P = 0.022) before the age of 6 years. OCT data were available in 12 patients, 11 of whom had retained foveal ONL and EZ integrity up to 48 years (median 23 years) of age.

Conclusions: Homozygous patients seem less severely affected compared to their compound-heterozygous peers. Improvement of visual function may occur in the early years of life, suggesting a time window for therapeutic intervention up to the approximate age of 17 years. This period may be extended by an intact foveal ONL and EZ on OCT.
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http://dx.doi.org/10.1167/iovs.18-24817DOI Listing
September 2018

Detection and quantification of a KIF11 mosaicism in a subject presenting familial exudative vitreoretinopathy with microcephaly.

Eur J Hum Genet 2018 12 4;26(12):1819-1823. Epub 2018 Sep 4.

Department of Human Genetics, Radboud University Medical Center, Nijmegen, The Netherlands.

Familial exudative vitreoretinopathy (FEVR) is an inherited retinal disorder, which is primarily characterized by abnormal development of retinal vasculature. In this study, we reported a subject presenting the clinical features of FEVR as well as microcephaly. Screening of the KIF11 gene in this patient revealed a novel heterozygous protein-truncating variant (c.2717del, p.(L906*), NM_004523.3). Segregation analysis in the unaffected parents using Sanger sequencing suggested the variant to be present in a mosaic state in the unaffected mother. KIF11 exon 19 which harbors the variant was amplified from the proband and her father, as well as three different tissues of the mother, followed by amplicon-based deep sequencing. This analysis revealed that the variant is present in different tissues of the mother at various rates, i.e. in blood (16.9%), saliva (20.7%), or skin biopsy-derived fibroblast cells (6.6%). These data demonstrate the importance of deep sequencing in unaffected parents upon detection of a genetic defect in isolated cases to detect possible mosaicisms, enabling a  more reliable recurrence risk assessment and thereby improve genetic counseling.
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http://dx.doi.org/10.1038/s41431-018-0243-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6244075PMC
December 2018

Splice-Modulating Oligonucleotide QR-110 Restores CEP290 mRNA and Function in Human c.2991+1655A>G LCA10 Models.

Mol Ther Nucleic Acids 2018 Sep 23;12:730-740. Epub 2018 Jul 23.

UCL Institute of Ophthalmology, London, UK. Electronic address:

Leber congenital amaurosis type 10 (LCA10) is a severe inherited retinal dystrophy associated with mutations in CEP290. The deep intronic c.2991+1655A>G mutation in CEP290 is the most common mutation in LCA10 individuals and represents an ideal target for oligonucleotide therapeutics. Here, a panel of antisense oligonucleotides was designed to correct the splicing defect associated with the mutation and screened for efficacy and safety. This identified QR-110 as the best-performing molecule. QR-110 restored wild-type CEP290 mRNA and protein expression levels in CEP290 c.2991+1655A>G homozygous and compound heterozygous LCA10 primary fibroblasts. Furthermore, in homozygous three-dimensional iPSC-derived retinal organoids, QR-110 showed a dose-dependent restoration of mRNA and protein function, as measured by percentage and length of photoreceptor cilia, without off-target effects. Localization studies in wild-type mice and rabbits showed that QR-110 readily reached all retinal layers, with an estimated half-life of 58 days. It was well tolerated following intravitreal injection in monkeys. In conclusion, the pharmacodynamic, pharmacokinetic, and safety properties make QR-110 a promising candidate for treating LCA10, and clinical development is currently ongoing.
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http://dx.doi.org/10.1016/j.omtn.2018.07.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6092551PMC
September 2018

Eyes shut homolog is important for the maintenance of photoreceptor morphology and visual function in zebrafish.

PLoS One 2018 27;13(7):e0200789. Epub 2018 Jul 27.

Department of Human Genetics, Radboud University Medical Center, Nijmegen, The Netherlands.

Mutations in eyes shut homolog (EYS), a gene predominantly expressed in the photoreceptor cells of the retina, are among the most frequent causes of autosomal recessive (ar) retinitis pigmentosa (RP), a progressive retinal disorder. Due to the absence of EYS in several rodent species and its retina-specific expression, still little is known about the exact function of EYS and the pathogenic mechanism underlying EYS-associated RP. We characterized eys in zebrafish, by RT-PCR analysis on zebrafish eye-derived RNA, which led to the identification of a 8,715 nucleotide coding sequence that is divided over 46 exons. The transcript is predicted to encode a 2,905-aa protein that contains 39 EGF-like domains and five laminin A G-like domains, which overall shows 33% identity with human EYS. To study the function of EYS, we generated a stable eysrmc101/rmc101 mutant zebrafish model using CRISPR/Cas9 technology. The introduced lesion is predicted to result in premature termination of protein synthesis and lead to loss of Eys function. Immunohistochemistry on retinal sections revealed that Eys localizes at the region of the connecting cilium and that both rhodopsin and cone transducin are mislocalized in the absence of Eys. Electroretinogram recordings showed diminished b-wave amplitudes in eysrmc101/rmc101 zebrafish (5 dpf) compared to age- and strain-matched wild-type larvae. In addition, decreased locomotor activity in response to light stimuli was observed in eys mutant larvae. Altogether, our study shows that absence of Eys leads to a disorganized retinal architecture and causes visual dysfunction in zebrafish.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0200789PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6063403PMC
January 2019

An FEVR-associated mutation in ZNF408 alters the expression of genes involved in the development of vasculature.

Hum Mol Genet 2018 10;27(20):3519-3527

Department of Human Genetics, Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Center, Nijmegen, Netherlands.

Familial exudative vitreoretinopathy (FEVR) is an inherited retinal disorder hallmarked by an abnormal development of retinal vasculature. A missense mutation in ZNF408 (p.H455Y) was reported to underlie autosomal dominant FEVR in a large Dutch family, and ZNF408 was shown to play a role in the development of vasculature. Nonetheless, little is known about the molecular mechanism of ZNF408-associated FEVR. To investigate this, an in vitro model of ZNF408-associated FEVR was generated by overexpressing wild-type and p.H455Y ZNF408 in human umbilical vein endothelial cells. Cells overexpressing mutant ZNF408 were unable to form a capillary-like network in an in vitro tube formation assay, thereby mimicking the clinical feature observed in patients with FEVR. Intriguingly, transcriptome analysis revealed that genes involved in the development of vasculature were deregulated by the p.H455Y mutation. Chromatin immunoprecipitation showed that p.H455Y ZNF408 has reduced DNA-binding ability, as compared to the wild-type protein. The fact that the p.H455Y mutation disrupts the expression of genes important for the development of vasculature sheds further light on the molecular mechanisms underlying ZNF408-associated FEVR.
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http://dx.doi.org/10.1093/hmg/ddy244DOI Listing
October 2018

C2orf71a/pcare1 is important for photoreceptor outer segment morphogenesis and visual function in zebrafish.

Sci Rep 2018 06 26;8(1):9675. Epub 2018 Jun 26.

Department of Human Genetics, Radboud University Medical Center, Nijmegen, The Netherlands.

Mutations in C2orf71 are causative for autosomal recessive retinitis pigmentosa and occasionally cone-rod dystrophy. We have recently discovered that the protein encoded by this gene is important for modulation of the ciliary membrane through the recruitment of an actin assembly module, and have therefore renamed the gene to PCARE (photoreceptor cilium actin regulator). Here, we report on the identification of two copies of the c2orf71/pcare gene in zebrafish, pcare1 and pcare2. To study the role of the gene most similar to human PCARE, pcare1, we have generated a stable pcare1 mutant zebrafish model (designated pcare1 ) in which the coding sequence was disrupted using CRISPR/Cas9 technology. Retinas of both embryonic (5 dpf) and adult (6 mpf) pcare1 zebrafish display a clear disorganization of photoreceptor outer segments, resembling the phenotype observed in Pcare mice. Optokinetic response and visual motor response measurements indicated visual impairment in pcare1 zebrafish larvae at 5 dpf. In addition, electroretinogram measurements showed decreased b-wave amplitudes in pcare1 zebrafish as compared to age- and strain-matched wild-type larvae, indicating a defect in the transretinal current. Altogether, our data show that lack of pcare1 causes a retinal phenotype in zebrafish and indicate that the function of the PCARE gene is conserved across species.
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http://dx.doi.org/10.1038/s41598-018-27928-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6018674PMC
June 2018

Antisense Oligonucleotide-Based Splice Correction of a Deep-Intronic Mutation in CHM Underlying Choroideremia.

Adv Exp Med Biol 2018 ;1074:83-89

Department of Human Genetics, Radboud University Medical Center, Nijmegen, The Netherlands.

Choroideremia is a progressive genetic eye disorder caused by mutations in the CHM gene that encodes the Rab escort protein-1 (REP-1). One of the many CHM mutations described so far is a deep-intronic variant, c.315-4587T>A, that creates a novel splice acceptor site resulting in the insertion of a 98-bp pseudoexon in the CHM transcript. Antisense oligonucleotides (AONs) are a potential therapeutic tool for correcting splice defects, as they have the properties to bind to the pre-mRNA and redirect the splicing process. Previously, we used AONs to correct aberrant splicing events caused by a recurrent intronic mutation in CEP290 underlying Leber congenital amaurosis. Here, we expand the use of these therapeutic molecules for the c.315-4587T>A deep-intronic mutation in CHM by demonstrating splice correction in patient-derived lymphoblast cells.
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http://dx.doi.org/10.1007/978-3-319-75402-4_11DOI Listing
May 2019

Non-syndromic retinitis pigmentosa.

Prog Retin Eye Res 2018 09 27;66:157-186. Epub 2018 Mar 27.

Department of Ophthalmology, Donders Institute for Brain, Cognition and Behavior, Radboud University Medical Center, Nijmegen, The Netherlands. Electronic address:

Retinitis pigmentosa (RP) encompasses a group of inherited retinal dystrophies characterized by the primary degeneration of rod and cone photoreceptors. RP is a leading cause of visual disability, with a worldwide prevalence of 1:4000. Although the majority of RP cases are non-syndromic, 20-30% of patients with RP also have an associated non-ocular condition. RP typically manifests with night blindness in adolescence, followed by concentric visual field loss, reflecting the principal dysfunction of rod photoreceptors; central vision loss occurs later in life due to cone dysfunction. Photoreceptor function measured with an electroretinogram is markedly reduced or even absent. Optical coherence tomography (OCT) and fundus autofluorescence (FAF) imaging show a progressive loss of outer retinal layers and altered lipofuscin distribution in a characteristic pattern. Over the past three decades, a vast number of disease-causing variants in more than 80 genes have been associated with non-syndromic RP. The wide heterogeneity of RP makes it challenging to describe the clinical findings and pathogenesis. In this review, we provide a comprehensive overview of the clinical characteristics of RP specific to genetically defined patient subsets. We supply a unique atlas with color fundus photographs of most RP subtypes, and we discuss the relevant considerations with respect to differential diagnoses. In addition, we discuss the genes involved in the pathogenesis of RP, as well as the retinal processes that are affected by pathogenic mutations in these genes. Finally, we review management strategies for patients with RP, including counseling, visual rehabilitation, and current and emerging therapeutic options.
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http://dx.doi.org/10.1016/j.preteyeres.2018.03.005DOI Listing
September 2018

Identification and Rescue of Splice Defects Caused by Two Neighboring Deep-Intronic ABCA4 Mutations Underlying Stargardt Disease.

Am J Hum Genet 2018 04 8;102(4):517-527. Epub 2018 Mar 8.

Department of Human Genetics, Radboud University Medical Center, 6525 GA Nijmegen, the Netherlands; Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Center, 6525 EN Nijmegen, the Netherlands. Electronic address:

Sequence analysis of the coding regions and splice site sequences in inherited retinal diseases is not able to uncover ∼40% of the causal variants. Whole-genome sequencing can identify most of the non-coding variants, but their interpretation is still very challenging, in particular when the relevant gene is expressed in a tissue-specific manner. Deep-intronic variants in ABCA4 have been associated with autosomal-recessive Stargardt disease (STGD1), but the exact pathogenic mechanism is unknown. By generating photoreceptor precursor cells (PPCs) from fibroblasts obtained from individuals with STGD1, we demonstrated that two neighboring deep-intronic ABCA4 variants (c.4539+2001G>A and c.4539+2028C>T) result in a retina-specific 345-nt pseudoexon insertion (predicted protein change: p.Arg1514Leufs36), likely due to the creation of exonic enhancers. Administration of antisense oligonucleotides (AONs) targeting the 345-nt pseudoexon can significantly rescue the splicing defect observed in PPCs of two individuals with these mutations. Intriguingly, an AON that is complementary to c.4539+2001G>A rescued the splicing defect only in PPCs derived from an individual with STGD1 with this but not the other mutation, demonstrating the high specificity of AONs. In addition, a single AON molecule rescued splicing defects associated with different neighboring mutations, thereby providing new strategies for the treatment of persons with STGD1. As many genes associated with human genetic conditions are expressed in specific tissues and pre-mRNA splicing may also rely on organ-specific factors, our approach to investigate and treat splicing variants using differentiated cells derived from individuals with STGD1 can be applied to any tissue of interest.
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http://dx.doi.org/10.1016/j.ajhg.2018.02.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5985352PMC
April 2018

Antisense Oligonucleotide-Based Splicing Correction in Individuals with Leber Congenital Amaurosis due to Compound Heterozygosity for the c.2991+1655A>G Mutation in CEP290.

Int J Mol Sci 2018 Mar 7;19(3). Epub 2018 Mar 7.

Department of Human Genetics, Radboud University Medical Center, 6525 GA Nijmegen, The Netherlands.

Leber congenital amaurosis (LCA) is a rare inherited retinal disorder affecting approximately 1:50,000 people worldwide. So far, mutations in 25 genes have been associated with LCA, with (encoding the Centrosomal protein of 290 kDa) being the most frequently mutated gene. The most recurrent LCA-causing mutation, c.2991+1655A>G, causes the insertion of a pseudoexon into a variable proportion of transcripts. We previously demonstrated that antisense oligonucleotides (AONs) have a high therapeutic potential for patients homozygously harbouring this mutation, although to date, it is unclear whether rescuing one single allele is enough to restore CEP290 function. Here, we assessed the AON efficacy at RNA, protein and cellular levels in samples that are compound heterozygous for this mutation, together with a protein-truncating mutation in . We demonstrate that AONs can efficiently restore splicing and increase protein levels. However, due to a high variability in ciliation among the patient-derived cell lines, the efficacy of the AONs was more difficult to assess at the cellular level. This observation points towards the importance of the severity of the second allele and possibly other genetic variants present in each individual. Overall, AONs seem to be a promising tool to treat -associated LCA, not only in homozygous but also in compound heterozygous carriers of the c.2991+1655A>G variant.
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http://dx.doi.org/10.3390/ijms19030753DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5877614PMC
March 2018

Autosomal Recessive NRL Mutations in Patients with Enhanced S-Cone Syndrome.

Genes (Basel) 2018 Jan 30;9(2). Epub 2018 Jan 30.

The Rotterdam Eye Hospital, 3011 BH Rotterdam, The Netherlands.

Enhanced S-cone syndrome (ESCS) is mainly associated with mutations in the gene. However, rare mutations in the gene have been reported in patients with ESCS. We report on an ESCS phenotype in additional patients with autosomal recessive (ar) mutations. Three Moroccan patients of two different families with ar mutations were enrolled in this study. The mutation in the DNA of one patient, from a consanguineous marriage, was detected by homozygosity mapping. The mutation in the DNA of two siblings from a second family was detected in a targeted next-generation sequencing project. Full ophthalmic examination was performed, including best-corrected visual acuity, slit-lamp biomicroscopy, funduscopy, Goldmann kinetic perimetry, optical coherence tomography, fundus autofluorescence, and extended electroretinography including an amber stimulus on a blue background and a blue stimulus on an amber background. One patient carried a homozygous missense mutation (c.508C>A; p.Arg170Ser) in the gene, whereas the same mutation was identified heterozygously in the two siblings of a second family, in combination with a one base-pair deletion (c.654del; p.Cys219Valfs*4) on the other allele. All patients had reduced visual acuity and showed a typical clumped pigmentary retinal degeneration (CPRD). Foveal schisis-like changes were observed in the oldest patient. An electroretinogram (ERG) under dark-adapted conditions showed absent responses for low stimulus strengths and reduced responses for high stimulus strengths, with constant b-wave latencies despite increasing stimulus strength. A relatively high amplitude was detected with a blue stimulus on an amber background, while an amber stimulus on a blue background showed reduced responses. The ar mutations cause a phenotype with typical CPRD. This phenotype has previously been described in patients with ESCS caused by mutations, and rarely by mutations. Based on our findings in ERG testing, we conclude that S-cone function is enhanced in our patients in a similar manner as in patients with -associated ESCS, confirming previous reports of as a second gene to cause ESCS.
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http://dx.doi.org/10.3390/genes9020068DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5852564PMC
January 2018

Design and In Vitro Use of Antisense Oligonucleotides to Correct Pre-mRNA Splicing Defects in Inherited Retinal Dystrophies.

Methods Mol Biol 2018 ;1715:61-78

Department of Human Genetics, Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Center, Nijmegen, The Netherlands.

Antisense oligonucleotides (AONs) are small molecules able to bind to the pre-mRNA and modulate splicing. The increasing amount of intronic mutations leading to pseudoexon insertion in genes underlying inherited retinal dystrophies (IRDs) has highlighted the potential of AONs as a therapeutic tool for these disorders. Here we describe how to design and test AON molecules in vitro in order to correct pre-mRNA splicing defects involved in IRDs.
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http://dx.doi.org/10.1007/978-1-4939-7522-8_5DOI Listing
July 2018

midigenes reveal the full splice spectrum of all reported noncanonical splice site variants in Stargardt disease.

Genome Res 2018 01 21;28(1):100-110. Epub 2017 Nov 21.

Department of Human Genetics and Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Center, 6525 GA Nijmegen, The Netherlands.

Stargardt disease is caused by variants in the gene, a significant part of which are noncanonical splice site (NCSS) variants. In case a gene of interest is not expressed in available somatic cells, small genomic fragments carrying potential disease-associated variants are tested for splice abnormalities using in vitro splice assays. We recently discovered that when using small minigenes lacking the proper genomic context, in vitro results do not correlate with splice defects observed in patient cells. We therefore devised a novel strategy in which a bacterial artificial chromosome was employed to generate midigenes, splice vectors of varying lengths (up to 11.7 kb) covering almost the entire gene. These midigenes were used to analyze the effect of all 44 reported and three novel NCSS variants on pre-mRNA splicing. Intriguingly, multi-exon skipping events were observed, as well as exon elongation and intron retention. The analysis of all reported NCSS variants in allowed us to reveal the nature of aberrant splicing events and to classify the severity of these mutations based on the residual fraction of wild-type mRNA. Our strategy to generate large overlapping splice vectors carrying multiple exons, creating a toolbox for robust and high-throughput analysis of splice variants, can be applied to all human genes.
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http://dx.doi.org/10.1101/gr.226621.117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5749174PMC
January 2018

EYS mutation update: In silico assessment of 271 reported and 26 novel variants in patients with retinitis pigmentosa.

Hum Mutat 2018 02 26;39(2):177-186. Epub 2017 Dec 26.

Department of Human Genetics, Radboud University Medical Center, Nijmegen, The Netherlands.

Mutations in Eyes shut homolog (EYS) are one of the most common causes of autosomal recessive (ar) retinitis pigmentosa (RP), a progressive blinding disorder. The exact function of the EYS protein and the pathogenic mechanisms underlying EYS-associated RP are still poorly understood, which hampers the interpretation of the causality of many EYS variants discovered to date. We collected all reported EYS variants present in 377 arRP index cases published before June 2017, and uploaded them in the Leiden Open Variation Database (www.LOVD.nl/EYS). We also describe 36 additional index cases, carrying 26 novel variants. Of the 297 unique EYS variants identified, almost half (n = 130) are predicted to result in premature truncation of the EYS protein. Classification of all variants using the American College of Medical Genetics and Genomics guidelines revealed that the predicted pathogenicity of these variants cover the complete spectrum ranging from likely benign to pathogenic, although especially missense variants largely fall in the category of uncertain significance. Besides the identification of likely benign alleles previously reported as being probably pathogenic, our comprehensive analysis underscores the need of functional assays to assess the causality of EYS variants, in order to improve molecular diagnostics and counseling of patients with EYS-associated RP.
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http://dx.doi.org/10.1002/humu.23371DOI Listing
February 2018

Therapeutic effects of the mitochondrial ROS-redox modulator KH176 in a mammalian model of Leigh Disease.

Sci Rep 2017 09 15;7(1):11733. Epub 2017 Sep 15.

Department of Pediatrics, Radboud Center for Mitochondrial Medicine, Radboud University Medical Center, Nijmegen, The Netherlands.

Leigh Disease is a progressive neurometabolic disorder for which a clinical effective treatment is currently still lacking. Here, we report on the therapeutic efficacy of KH176, a new chemical entity derivative of Trolox, in Ndufs4 mice, a mammalian model for Leigh Disease. Using in vivo brain diffusion tensor imaging, we show a loss of brain microstructural coherence in Ndufs4 mice in the cerebral cortex, external capsule and cerebral peduncle. These findings are in line with the white matter diffusivity changes described in mitochondrial disease patients. Long-term KH176 treatment retained brain microstructural coherence in the external capsule in Ndufs4 mice and normalized the increased lipid peroxidation in this area and the cerebral cortex. Furthermore, KH176 treatment was able to significantly improve rotarod and gait performance and reduced the degeneration of retinal ganglion cells in Ndufs4 mice. These in vivo findings show that further development of KH176 as a potential treatment for mitochondrial disorders is worthwhile to pursue. Clinical trial studies to explore the potency, safety and efficacy of KH176 are ongoing.
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http://dx.doi.org/10.1038/s41598-017-09417-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5601915PMC
September 2017