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Jean Bennett - One of the best experts on this subject based on the ideXlab platform.
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spliceosome mediated pre mrna trans splicing can repair CEP290 mrna
Molecular therapy. Nucleic acids, 2018Co-Authors: Scott J. Dooley, Devin S. Mcdougald, Krishna J. Fisher, Jeanette L. Bennicelli, Lloyd G. Mitchell, Jean BennettAbstract:Ocular gene therapy with recombinant adeno-associated virus (AAV) has shown vector-mediated gene augmentation to be safe and efficacious in the retina in one set of diseases (retinitis pigmentosa and Leber congenital amaurosis (LCA) caused by RPE65 deficiency), with excellent safety profiles to date and potential for efficacy in several additional diseases. However, size constraints imposed by the packaging capacity of the AAV genome restrict application to diseases with coding sequence lengths that are less than 5,000 nt. The most prevalent retinal diseases with monogenic inheritance are caused by mutations in genes that exceed this capacity. Here, we designed a spliceosome mediated pre-mRNA trans-splicing strategy to rescue expression of CEP290, which is associated with Leber congenital amaurosis type 10 (LCA10) and several syndromic diseases including Joubert syndrome. We used this reagent to demonstrate editing of CEP290 in cell lines in vitro and in vivo in a mini-gene mouse model. This study is the first to show broad editing of CEP290 transcripts and in vivo proof of concept for editing of CEP290 transcripts in photoreceptors and paves the way for future studies evaluating therapeutic effects.
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Spliceosome-Mediated Pre-mRNA trans-Splicing Can Repair CEP290 mRNA
'Elsevier BV', 2018Co-Authors: Scott J. Dooley, Devin S. Mcdougald, Krishna J. Fisher, Jeanette L. Bennicelli, Lloyd G. Mitchell, Jean BennettAbstract:Ocular gene therapy with recombinant adeno-associated virus (AAV) has shown vector-mediated gene augmentation to be safe and efficacious in the retina in one set of diseases (retinitis pigmentosa and Leber congenital amaurosis (LCA) caused by RPE65 deficiency), with excellent safety profiles to date and potential for efficacy in several additional diseases. However, size constraints imposed by the packaging capacity of the AAV genome restrict application to diseases with coding sequence lengths that are less than 5,000 nt. The most prevalent retinal diseases with monogenic inheritance are caused by mutations in genes that exceed this capacity. Here, we designed a spliceosome mediated pre-mRNA trans-splicing strategy to rescue expression of CEP290, which is associated with Leber congenital amaurosis type 10 (LCA10) and several syndromic diseases including Joubert syndrome. We used this reagent to demonstrate editing of CEP290 in cell lines in vitro and in vivo in a mini-gene mouse model. This study is the first to show broad editing of CEP290 transcripts and in vivo proof of concept for editing of CEP290 transcripts in photoreceptors and paves the way for future studies evaluating therapeutic effects. Keywords: gene therapy, molecular genetics, RNA editing, cell models, animal models, LCA10, CEP290, trans-splicin
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in vitro and in vivo rescue of aberrant splicing in CEP290 associated lca by antisense oligonucleotide delivery
Human Molecular Genetics, 2016Co-Authors: Alejandro Garanto, Lonneke Duijkers, Jean Bennett, Daniel C Chung, Julio C Corralserrano, Muriel Messchaert, Ru Xiao, Luk H Vandenberghe, Rob W J CollinAbstract:Leber congenital amaurosis (LCA) is a severe disorder resulting in visual impairment usually starting in the first year of life. The most frequent genetic cause of LCA is an intronic mutation in CEP290 (c.2991 + 1655A > G) that creates a cryptic splice donor site resulting in the insertion of a pseudoexon (exon X) into CEP290 mRNA. Previously, we showed that naked antisense oligonucleotides (AONs) effectively restored normal CEP290 splicing in patient-derived lymphoblastoid cells. We here explore the therapeutic potential of naked and adeno-associated virus (AAV)-packaged AONs in vitro and in vivo In both cases, AON delivery fully restored CEP290 pre-mRNA splicing, significantly increased CEP290 protein levels and rescued a ciliary phenotype present in patient-derived fibroblast cells. Moreover, administration of naked and AAV-packaged AONs to the retina of a humanized mutant CEP290 mouse model, carrying the intronic mutation, showed a statistically significant reduction of exon X-containing CEP290 transcripts, without compromising the retinal structure. Together, our data highlight the tremendous therapeutic prospective of AONs for the treatment of not only CEP290-associated LCA but potentially many other subtypes of retinal dystrophy caused by splicing mutations.
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basal exon skipping and genetic pleiotropy a predictive model of disease pathogenesis
Science Translational Medicine, 2015Co-Authors: Theodore G Drivas, Edwin M. Stone, Adam Wojno, Budd A Tucker, Jean BennettAbstract:Genetic pleiotropy, the phenomenon by which mutations in the same gene result in markedly different disease phenotypes, has proven difficult to explain with traditional models of disease pathogenesis. We have developed a model of pleiotropic disease that explains, through the process of basal exon skipping, how different mutations in the same gene can differentially affect protein production, with the total amount of protein produced correlating with disease severity. Mutations in the centrosomal protein of 290 kDa (CEP290) gene are associated with a spectrum of phenotypically distinct human diseases (the ciliopathies). Molecular biologic examination of CEP290 transcript and protein expression in cells from patients carrying CEP290 mutations, measured by quantitative polymerase chain reaction and Western blotting, correlated with disease severity and corroborated our model. We show that basal exon skipping may be the mechanism underlying the disease pleiotropy caused by CEP290 mutations. Applying our model to a different disease gene, CC2D2A (coiled-coil and C2 domains–containing protein 2A), we found that the same correlations held true. Our model explains the phenotypic diversity of two different inherited ciliopathies and may establish a new model for the pathogenesis of other pleiotropic human diseases.
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CEP290 and the primary cilium.
Advances in Experimental Medicine and Biology, 2014Co-Authors: Theodore G Drivas, Jean BennettAbstract:The protein CEP290 has recently emerged as a major player in the biology of the cilium and as a causative protein in a number of human syndromic diseases, most of which are associated with the devastating blinding disease Leber congenital amaurosis. (Coppieters et al., Hum Mutat 31, 2010, 1097–1108) CEP290 is known to be an important component of the primary cilium, localizing to the Y-links of the ciliary transition zone and having a role in the regulation of transport in and out of the ciliary compartment (Craige et al., J Cell Biol 190, 2010, 927–940). While many mutations in CEP290 have been identified in human patients, how these mutations result in the spectrum of human disease attributed to the protein remain unknown. As we begin to learn more about the normal role of CEP290, it is likely that we will begin to shed light on how these mutations result in the various CEP290 disease phenotypes. Here we discuss many of these diverse aspects of CEP290 biology and pathology in an attempt to link what we know about the molecular mechanisms of CEP290 function with what we know about CEP290-asociated disease.
Rob W J Collin - One of the best experts on this subject based on the ideXlab platform.
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antisense oligonucleotide based splicing correction in individuals with leber congenital amaurosis due to compound heterozygosity for the c 2991 1655a g mutation in CEP290
International Journal of Molecular Sciences, 2018Co-Authors: Lonneke Duijkers, Rob W J Collin, Ingeborgh L Van Den Born, John Neidhardt, Nathalie M Bax, Laurence Pierrache, Jeroen B Klevering, Alejandro GarantoAbstract: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 CEP290 (encoding the Centrosomal protein of 290 kDa) being the most frequently mutated gene. The most recurrent LCA-causing CEP290 mutation, c.2991+1655A>G, causes the insertion of a pseudoexon into a variable proportion of CEP290 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 CEP290. 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 CEP290-associated LCA, not only in homozygous but also in compound heterozygous carriers of the c.2991+1655A>G variant.
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in vitro and in vivo rescue of aberrant splicing in CEP290 associated lca by antisense oligonucleotide delivery
Human Molecular Genetics, 2016Co-Authors: Alejandro Garanto, Lonneke Duijkers, Jean Bennett, Daniel C Chung, Julio C Corralserrano, Muriel Messchaert, Ru Xiao, Luk H Vandenberghe, Rob W J CollinAbstract:Leber congenital amaurosis (LCA) is a severe disorder resulting in visual impairment usually starting in the first year of life. The most frequent genetic cause of LCA is an intronic mutation in CEP290 (c.2991 + 1655A > G) that creates a cryptic splice donor site resulting in the insertion of a pseudoexon (exon X) into CEP290 mRNA. Previously, we showed that naked antisense oligonucleotides (AONs) effectively restored normal CEP290 splicing in patient-derived lymphoblastoid cells. We here explore the therapeutic potential of naked and adeno-associated virus (AAV)-packaged AONs in vitro and in vivo In both cases, AON delivery fully restored CEP290 pre-mRNA splicing, significantly increased CEP290 protein levels and rescued a ciliary phenotype present in patient-derived fibroblast cells. Moreover, administration of naked and AAV-packaged AONs to the retina of a humanized mutant CEP290 mouse model, carrying the intronic mutation, showed a statistically significant reduction of exon X-containing CEP290 transcripts, without compromising the retinal structure. Together, our data highlight the tremendous therapeutic prospective of AONs for the treatment of not only CEP290-associated LCA but potentially many other subtypes of retinal dystrophy caused by splicing mutations.
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species dependent splice recognition of a cryptic exon resulting from a recurrent intronic CEP290 mutation that causes congenital blindness
International Journal of Molecular Sciences, 2015Co-Authors: Alejandro Garanto, Lonneke Duijkers, Rob W J CollinAbstract:A mutation in intron 26 of CEP290 (c.2991+1655A>G) is the most common genetic cause of Leber congenital amaurosis (LCA), a severe type of inherited retinal degeneration. This mutation creates a cryptic splice donor site, resulting in the insertion of an aberrant exon (exon X) into ~50% of all CEP290 transcripts. A humanized mouse model with this mutation did not recapitulate the aberrant CEP290 splicing observed in LCA patients, suggesting differential recognition of cryptic splice sites between species. To further assess this phenomenon, we generated two CEP290 minigene constructs, with and without the intronic mutation, and transfected these in cell lines of various species. RT-PCR analysis revealed that exon X is well recognized by the splicing machinery in human and non-human primate cell lines. Intriguingly, this recognition decreases in cell lines derived from species such as dog and rodents, and it is completely absent in Drosophila. In addition, other cryptic splicing events corresponding to sequences in intron 26 of CEP290 were observed to varying degrees in the different cell lines. Together, these results highlight the complexity of splice site recognition among different species, and show that care is warranted when generating animal models to mimic splice site mutations in vivo.
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unexpected CEP290 mrna splicing in a humanized knock in mouse model for leber congenital amaurosis
PLOS ONE, 2013Co-Authors: Alejandro Garanto, Sylvia E. C. Van Beersum, Frans P M Cremers, Theo A Peters, Ronald Roepman, Rob W J CollinAbstract:Leber congenital amaurosis (LCA) is the most severe form of retinal dystrophy with an onset in the first year of life. The most frequent genetic cause of LCA, accounting for up to 15% of all LCA cases in Europe and North-America, is a mutation (c.2991+1655AG) in intron 26 of CEP290. This mutation generates a cryptic splice donor site resulting in the insertion of an aberrant exon (exon X) containing a premature stop codon to CEP290 mRNA. In order to study the pathophysiology of the intronic CEP290 mutation, we generated two humanized knock-in mouse models each carrying ~6.3 kb of the human CEP290 gene, either with or without the intronic mutation. Transcriptional characterization of these mouse models revealed an unexpected splice pattern of CEP290 mRNA, especially in the retina. In both models, a new cryptic exon (coined exon Y) was identified in ~5 to 12% of all CEP290 transcripts. This exon Y was expressed in all murine tissues analyzed but not detected in human retina or fibroblasts of LCA patients. In addition, exon x that is characteristic of LCA in humans, was expressed at only very low levels in the retina of the LCA mouse model. Western blot and immunohistochemical analyses did not reveal any differences between the two transgenic models and wild-type mice. Together, our results show clear differences in the recognition of splice sites between mice and humans, and emphasize that care is warranted when generating animal models for human genetic diseases caused by splice mutations.
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antisense oligonucleotide aon based therapy for leber congenital amaurosis caused by a frequent mutation in CEP290
Molecular therapy. Nucleic acids, 2012Co-Authors: Rob W J Collin, Jean Bennett, Anneke Den I Hollander, Saskia D Van Der Veldevisser, Jeannette L Bennicelli, Frans P M CremersAbstract:Leber congenital amaurosis (LCA) is the most severe form of inherited retinal degeneration, with an onset in the first year of life. The most frequent mutation that causes LCA, present in at least 10% of individuals with LCA from North-American and Northern-European descent, is an intronic mutation in CEP290 that results in the inclusion of an aberrant exon in the CEP290 mRNA. Here, we describe a genetic therapy approach that is based on antisense oligonucleotides (AONs), small RNA molecules that are able to redirect normal splicing of aberrantly processed pre-mRNA. Immortalized lymphoblastoid cells of individuals with LCA homozygously carrying the intronic CEP290 mutation were transfected with several AONs that target the aberrant exon that is incorporated in the mutant CEP290 mRNA. Subsequent RNA isolation and reverse transcription-PCR analysis revealed that a number of AONs were capable of almost fully redirecting normal CEP290 splicing, in a dose-dependent manner. Other AONs however, displayed no effect on CEP290 splicing at all, indicating that the rescue of aberrant CEP290 splicing shows a high degree of sequence specificity. Together, our data show that AON-based therapy is a promising therapeutic approach for CEP290-associated LCA that warrants future research in animal models to develop a cure for this blinding disease.
Hemant Khanna - One of the best experts on this subject based on the ideXlab platform.
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Ciliopathy-associated protein CEP290 modifies the severity of retinal degeneration due to loss of RPGR
Human molecular genetics, 2016Co-Authors: Kollu N. Rao, Wolfgang Baehr, Cecinio C Ronquillo, Wei Zhang, Hemant KhannaAbstract:Mutations in RPGR (retinitis pigmentosa GTPase regulator) are the most common cause of X-linked RP, a severe blindness disorder. RPGR mutations result in clinically variable disease with early- to late-onset phenotypic presentation. Molecular mechanisms underlying such heterogeneity are unclear. Here we show that phenotypic expression of Rpgr-loss in mice is influenced genetically by the loss of CEP290, a human ciliopathy gene. We found that Rpgrko/Y mice with a heterozygous hypomorphic allele of CEP290 (CEP290rd16/+) but not of a heterozygous null allele of CEP290 (CEP290null/+) or of other ciliopathy genes, Rpgrip1, Nphp1, Nphp4 and Nphp5, exhibit relatively early onset (by 3 months of age) retinal degeneration and dysfunction when compared with the onset at ∼7 months of age in the Rpgrko/Y mice. We also observed disorganized photoreceptor outer-segment morphology and defective trafficking of opsins in the Rpgrko/Y::CEP290rd16/+ mice. Together with a physical interaction between RPGR and the C-terminal domain of CEP290, our data suggest that RPGR and CEP290 genetically interact and highlight the involvement of hypomorphic alleles of genes as potential modifiers of heterogeneous retinal ciliopathies.
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loss of raf 1 kinase inhibitory protein delays early onset severe retinal ciliopathy in CEP290rd16 mouse
Investigative Ophthalmology & Visual Science, 2014Co-Authors: Balajikarthick Subramanian, Manisha Anand, Naheed W Khan, Hemant KhannaAbstract:Purpose Mutations in the cilia-centrosomal protein of centrosomal protein of 290 kDa (CEP290) result in severe ciliopathies, including autosomal recessive early onset childhood blindness disorder Leber congenital amaurosis (LCA). The CEP290(rd16) (retinal degeneration 16) mouse model of CEP290-LCA exhibits accumulation of CEP290-interacting protein Raf-1 kinase inhibitory protein (RKIP) prior to onset of retinal degeneration (by postnatal day P14). We hypothesized that reducing RKIP levels in the CEP290(rd16) mouse will delay or improve retinal phenotype. Methods We generated double mutant mice by combining the CEP290(rd16) and Rkip(ko) alleles (CEP290(rd16):Rkip(+/ko) and CEP290(rd16):Rkip(ko/ko)). Retinal function was assessed by ERG and retinal morphology and protein trafficking were assessed by histology, transmission electron microscopy (TEM), and immunofluorescence analysis. Cell death was examined by apoptosis. Results Prior to testing our hypothesis, we examined ERG and retinal morphology of Rkip(ko/ko) mice and did not find any detectable differences compared with wild-type mice. The CEP290(rd16):Rkip(+/ko) mice exhibited similar retinopathy as CEP290(rd16); however, CEP290(rd16): Rkip(ko/ko) double knockout mice demonstrated a substantial improvement (>9-fold) in photoreceptor function and structure at P18 as of CEP290(rd16) mice. We consistently detected transient preservation of photoreceptors at P18 and polarized trafficking of opsins to sensory cilia in the double mutant mice; however, retinal degeneration ensued by P30. Conclusions Our studies implicate CEP290-RKIP pathway in CEP290-retinal degeneration and suggest that targeting RKIP levels can delay photoreceptor degeneration, assisting in extending the time-window for treating such rapidly progressing blindness disorder.
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ciliary transition zone tz proteins rpgr and CEP290 role in photoreceptor cilia and degenerative diseases
Expert Opinion on Therapeutic Targets, 2012Co-Authors: Manisha Anand, Hemant KhannaAbstract:Introduction: Primary cilia are microtubule-based extensions of the plasma membrane in nearly all cell types. In vertebrate photoreceptors, the sensory cilium develops as outer segment (OS) that contains the photopigment rhodopsin and other proteins necessary for phototransduction. The distinct composition of proteins and lipids in the OS membrane is maintained by the selective barrier located at the border between the basal body and the ciliary compartment, called the transition zone (TZ). Areas covered: In this review, we will discuss the identification and function of two ciliary TZ proteins, RPGR (retinitis pigmentosa GTPase regulator) and CEP290. Mutations in these proteins account for a majority of retinopathies due to ciliary dysfunction. We will also discuss the potential of such information in designing therapeutic approaches to treat cilia-dependent photoreceptor degenerative diseases. Expert opinion: RPGR and CEP290 perform overlapping yet distinct functions in regulating trafficking of cargo v...
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cp110 suppresses primary cilia formation through its interaction with CEP290 a protein deficient in human ciliary disease
Developmental Cell, 2008Co-Authors: William Y Tsang, Hemant Khanna, Anand Swaroop, Carine Bossard, Johan Peranen, Vivek Malhotra, Brian David DynlachtAbstract:Summary Primary cilia are nonmotile organelles implicated in signaling and sensory functions. Understanding how primary cilia assemble could shed light on the many human diseases caused by mutations in ciliary proteins. The centrosomal protein CP110 is known to suppress ciliogenesis through an unknown mechanism. Here, we report that CP110 interacts with CEP290— a protein whose deficiency is implicated in human ciliary disease—in a discrete complex separable from other CP110 complexes involved in regulating the centrosome cycle. Ablation of CEP290 prevents ciliogenesis without affecting centrosome function or cell-cycle progression. Interaction with CEP290 is absolutely required for the ability of CP110 to suppress primary cilia formation. Furthermore, CEP290 and CP110 interact with Rab8a, a small GTPase required for cilia assembly. Depletion of CEP290 interferes with localization of Rab8a to centrosomes and cilia. Our results suggest that CEP290 cooperates with Rab8a to promote ciliogenesis and that this function is antagonized by CP110.
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centrosomal ciliary gene CEP290 nphp6 mutations result in blindness with unexpected sparing of photoreceptors and visual brain implications for therapy of leber congenital amaurosis
Human Mutation, 2007Co-Authors: Hemant Khanna, Artur V Cideciyan, Alexander Sumaroka, Tomas S Aleman, Samuel G Jacobson, Geoffrey K Aguirre, Sharon B Schwartz, Elizabeth A M Windsor, Shirley HeAbstract:Howard Hughes Medical Instituteand Department of Ophthalmology, University of Iowa Carver College of Medicine, Iowa City, IowaCommunicated by Andrew WilkieMutations in the centrosomal-ciliary gene CEP290/NPHP6 are associated with Joubert syndrome and are themost common cause of the childhood recessive blindness known as Leber congenital amaurosis (LCA). Anin-frame deletion in CEP290 shows rapid degeneration in the rod-rich mouse retina. To explore the mechanismsof the human retinal disease, we studied CEP290-LCA in patients of different ages (7–48 years) and comparedresults to CEP290-mutant mice. Unexpectedly, blind CEP290-mutant human retinas retained photoreceptorand inner laminar architecture in the cone-rich central retina, independent of severity of visual loss.Surrounding the cone-rich island was photoreceptor loss and distorted retina, suggesting neural-glialremodeling. The mutant mouse retina at 4–6 weeks of age showed similar features of retinal remodeling,with altered neural and synaptic laminae and Muller glial activation. The visual brain pathways in CEP290-LCAwere anatomically intact. Our findings of preserved foveal cones and visual brain anatomy in LCAwith CEP290mutations, despite severe blindness and rapid rod cell death, suggest an opportunity for visual restorationof central vision in this common form of inherited blindness. Hum Mutat 28(11), 1074–1083, 2007.Published 2007 Wiley-Liss, Inc.
Edwin M. Stone - One of the best experts on this subject based on the ideXlab platform.
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Mutations Result in Blindness With Unexpected Sparing of Photoreceptors and Visual Brain:
2016Co-Authors: Centrosomal-ciliary Gene Cep/nphp, Edwin M. StoneAbstract:Mutations in the centrosomal-ciliary gene CEP290/NPHP6 are associated with Joubert syndrome and are the most common cause of the childhood recessive blindness known as Leber congenital amaurosis (LCA). An in-frame deletion in CEP290 shows rapid degeneration in the rod-rich mouse retina. To explore the mechanisms of the human retinal disease, we studied CEP290-LCA in patients of different ages (7–48 years) and compared results to CEP290-mutant mice. Unexpectedly, blind CEP290-mutant human retinas retained photoreceptor and inner laminar architecture in the cone-rich central retina, independent of severity of visual loss. Surrounding the cone-rich island was photoreceptor loss and distorted retina, suggesting neural-glial remodeling. The mutant mouse retina at 4–6 weeks of age showed similar features of retinal remodeling, with altered neural and synaptic laminae and Muller glial activation. The visual brain pathways in CEP290-LCA were anatomically intact. Our findings of preserved foveal cones and visual brain anatomy in LCAwith CEP290 mutations, despite severe blindness and rapid rod cell death, suggest an opportunity for visual restoration of central vision in this common form of inherited blindness. Hum Mutat 28(11), 1074–1083, 2007
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full field pupillary light responses luminance thresholds and light discomfort thresholds in CEP290 leber congenital amaurosis patients
Investigative Ophthalmology & Visual Science, 2015Co-Authors: Frederick T Collison, Jason C Park, Gerald A Fishman, Jason J Mcanany, Edwin M. StoneAbstract:Leber congenital amaurosis (LCA) refers to a group of severe inherited retinal disorders characterized by profound congenital vision impairment, nystagmus, and most often autosomal recessive inheritance. Mutations in the gene CEP290 (also known as NPHP6)1 have been found in at least 21% of LCA patients descended from various parts of Europe,1–3 making it the most common cause of nonsyndromic LCA in those populations. The CEP290 protein localizes to the base of the connecting cilium of photoreceptors,4 and loss of function of this protein in the human retina leads to an early loss of rod photoreceptor function and structure, as well as poor cone-mediated central vision. Both photopic and scotopic full-field electroretinograms (ERGs) tend to be nondetectable, even at an early age.1,3,5 Visual acuity in patients with CEP290 mutations is typically poor, with most falling in the range of hand motion to no light perception.6–8 In spite of poor central vision, optical coherence tomography has shown that foveal cone structure can be relatively preserved decades longer than rod structure,5,9,10 a finding that suggests the potential for therapeutic intervention. Owing to very poor visual acuity and mild photoaversion, LCA-CEP290 has been referred to as a “cone–rod dystrophy.”1–3 On the other hand, LCA-CEP290 has more recently been referred to as a “rod–cone dystrophy”7 owing to relative sparing of the macula and early loss of rod structure.5 Given the poor fixation and large visual sensitivity losses in LCA-CEP290 patients, typical clinical measures of visual function are not ideally suited either to assess functional losses or to track changes in visual function over time. This is important because substantial progress has been made in the development of gene therapies for application to patients with LCA-CEP290, but techniques for assessing therapeutic efficacy in these patients are relatively limited. The primary purpose of this study was to address this limitation by investigating the use of three full-field tests of visual function in patients with nonsyndromic LCA-CEP290: (1) pupillary light reflexes (PLRs); (2) luminance thresholds; and (3) light discomfort (photoaversion). A secondary purpose of this study was to investigate the disagreement in classification of LCA-CEP290 as either a “cone–rod” or “rod–cone” dystrophy through the use of chromatic stimuli to study the relative contributions of different photoreceptor types (rods versus cones) to the PLR and luminance thresholds, as well as through the inclusion of light discomfort threshold (LDT) testing. For the PLR, we followed a published method that uses 1-second chromatic stimuli at various luminance levels to target rod, cone and melanopsin responses.11 For the luminance thresholds, we tested patients with the full-field stimulus threshold test (FST),12,13 also using chromatic stimuli to determine rod- versus cone-mediation of dark adapted thresholds. As a measure of photoaversion, we adapted a published measure of LDT14,15 by using a full-field stimulus, and we added chromatic stimuli to evaluate the effect of wavelength on LDTs in LCA-CEP290 patients.
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basal exon skipping and genetic pleiotropy a predictive model of disease pathogenesis
Science Translational Medicine, 2015Co-Authors: Theodore G Drivas, Edwin M. Stone, Adam Wojno, Budd A Tucker, Jean BennettAbstract:Genetic pleiotropy, the phenomenon by which mutations in the same gene result in markedly different disease phenotypes, has proven difficult to explain with traditional models of disease pathogenesis. We have developed a model of pleiotropic disease that explains, through the process of basal exon skipping, how different mutations in the same gene can differentially affect protein production, with the total amount of protein produced correlating with disease severity. Mutations in the centrosomal protein of 290 kDa (CEP290) gene are associated with a spectrum of phenotypically distinct human diseases (the ciliopathies). Molecular biologic examination of CEP290 transcript and protein expression in cells from patients carrying CEP290 mutations, measured by quantitative polymerase chain reaction and Western blotting, correlated with disease severity and corroborated our model. We show that basal exon skipping may be the mechanism underlying the disease pleiotropy caused by CEP290 mutations. Applying our model to a different disease gene, CC2D2A (coiled-coil and C2 domains–containing protein 2A), we found that the same correlations held true. Our model explains the phenotypic diversity of two different inherited ciliopathies and may establish a new model for the pathogenesis of other pleiotropic human diseases.
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natural history of cone disease in the murine model of leber congenital amaurosis due to CEP290 mutation determining the timing and expectation of therapy
PLOS ONE, 2014Co-Authors: Shannon E. Boye, Budd A Tucker, Alexander Sumaroka, Wei Chieh Huang, Alejandro J. Roman, Sanford L. Boye, Renee C. Ryals, Melani B. Olivares, Qing Ruan, Edwin M. StoneAbstract:Background Mutations in the CEP290 (cilia-centrosomal protein 290 kDa) gene in Leber congenital amaurosis (LCA) cause early onset visual loss but retained cone photoreceptors in the fovea, which is the potential therapeutic target. A cone-only mouse model carrying a CEP290 gene mutation, rd16;Nrl−/−, was engineered to mimic the human disease. In the current study, we determined the natural history of retinal structure and function in this murine model to permit design of pre-clinical proof-of-concept studies and allow progress to be made toward human therapy. Analyses of retinal structure and visual function in CEP290-LCA patients were also performed for comparison with the results in the model.
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Structure and function in the central retina of CEP290-LCA patients.
2014Co-Authors: Shannon E. Boye, Budd A Tucker, Alexander Sumaroka, Wei Chieh Huang, Alejandro J. Roman, Sanford L. Boye, Renee C. Ryals, Melani B. Olivares, Qing Ruan, Edwin M. StoneAbstract:(A) Cross-sectional OCT scans along the horizontal meridian through the fovea in a normal subject, a CEP290-LCA patient, and an RP patient. ONL is highlighted in blue. Inset shows location of scan. (B) Relationship of foveal ONL thickness and visual acuity in CEP290-LCA patients. Bar graph represents the average ±1SD foveal ONL thickness of eyes in the different visual acuity ranges (n = 3, for 0.1–1 LogMAR; n = 5, for 1–2 LogMAR; and n = 11, for 2–NLP). Dashed line is lower limit of normal and emphasizes that despite low acuities, foveal ONL is within normal limits. Inset, data from a series of RP patients plotted similarly to show the more expected relationship between structure and visual acuity in retinal degenerations (n = 3, for 0–0.2 LogMAR; n = 20, for 0.2–1 LogMAR; and n = 3 for 1–2 LogMAR). Dashed line is also lower limit of normal. (C) Relationship in CEP290-LCA patients of width of the ONL in the central retina and patient age at time of examination. ONL width was unable to be defined in a 32-year-old CEP290-LCA patient with maculopathy. Solid line is linear regression. Inset, traced central ONL peaks in representative patients of different ages.
Isabelle Perrault - One of the best experts on this subject based on the ideXlab platform.
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description of two siblings with apparently severe CEP290 mutations and unusually mild retinal disease unrelated to basal exon skipping or nonsense associated altered splicing
Advances in Experimental Medicine and Biology, 2019Co-Authors: Iris Barny, Isabelle Perrault, Sabine Defoortdhellemmes, Helene Dollfus, Josseline Kaplan, Marlene Rio, Jeanmichel Rozet, Xavier GerardAbstract:CEP290 mutations cause a spectrum of ciliopathies, including Leber congenital amaurosis. Milder retinal diseases have been ascribed to exclusion of CEP290 mutant exons through basal exon skipping (BES) and/or nonsense-associated altered splicing (NAS). Here, we report two siblings with some preserved vision despite biallelism for presumably severe CEP290 mutations: a maternal splice site change in intron 18 (c.1824 + 3A > G) and a paternal c.6869dup (p.Asn2290Lysfs∗6) in exon 50 that introduces a premature termination codon (PTC) within the same exon. Analyzing mRNAs from fibroblasts of the two siblings, we detected no BES or NAS which could have enabled the production of PTC-free CEP290 isoforms from the paternal allele. In contrast, we reveal partial alteration of exon 18 donor splice site, allowing the transcription of some correctly spliced CEP290 mRNAs from the maternal allele which likely account for the mild retinal disease. This observation adds further variability to the mechanisms underlying CEP290 pleiotropy.
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basal exon skipping and nonsense associated altered splicing allows bypassing complete CEP290 loss of function in individuals with unusually mild retinal disease
Human Molecular Genetics, 2018Co-Authors: Iris Barny, Isabelle Perrault, Tania Attiebitach, Marlene Rio, Christel Michel, Mickael Soussan, Nicolas Goudin, Sophie Thomas, Christian P Hamel, Helene DollfusAbstract:CEP290 mutations cause a spectrum of ciliopathies from Leber congenital amaurosis type 10 (LCA10) to embryo-lethal Meckel syndrome (MKS). Using panel-based molecular diagnosis testing for inherited retinal diseases, we identified two individuals with some preserved vision despite biallelism for presumably truncating CEP290 mutations. The first one carried a homozygous 1 base pair deletion in Exon 17, introducing a premature termination codon (PTC) in Exon 18 (c.1666del; p.Ile556Phefs*17). mRNA analysis revealed a basal exon skipping (BES) of Exon 18, providing mutant cells with the ability to escape protein truncation, while disrupting the reading frame in controls. The second individual harbored compound heterozygous nonsense mutations in Exon 8 (c.508A>T, p.Lys170*) and Exon 32 (c.4090G>T, p.Glu1364*), respectively. Some CEP290 lacking Exon 8 were detected in mutant fibroblasts but not in controls whereas some skipping of Exon 32 occurred in both lines, but with higher amplitude in the mutant. Considering that the deletion of either exon maintains the reading frame in either line, skipping in mutant cells likely involves nonsense-associated altered splicing alone (Exon 8), or with BES (Exon 32). Skipping of PTC-containing exons in mutant cells allowed production of CEP290 isoforms with preserved ability to assemble into a high molecular weight complex and to interact efficiently with proteins important for cilia formation and intraflagellar trafficking. In contrast, studying LCA10 and MKS fibroblasts we show moderate to severe cilia alterations, providing support for a correlation between disease severity and the ability of cells to express shortened, yet functional, CEP290 isoforms.
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Abnormal respiratory cilia in non-syndromic Leber congenital amaurosis with CEP290 mutations
Journal of Medical Genetics, 2010Co-Authors: Jean Francois Papon, Isabelle Perrault, Sylvain Hanein, Sylvie Gerber, Xavier Gerard, Andre Coste, Bruno Louis, Lucas Fares-taie, Sabine Defoort-dhellemmes, Anne Marie VojtekAbstract:Background : Leber congenital amaurosis (LCA) is the earliest and most severe inherited retinal degeneration. Isolated forms of LCA frequently result from mutation of the CEP290 gene which is expressed in various ciliated tissues. Methods : Seven LCA patients with CEP290 mutations were investigated to study otorhinolaryngologic phenotype and respiratory cilia. Nasal biopsies and brushing were performed to study cilia ultrastructure using transmission electron microscopy and ciliary beating using high-speed videomicroscopy, respectively. CEP290 expression in normal nasal epithelium was studied using real time RT-PCR. Results : When electron microscopy was feasible (5/7), high levels of respiratory cilia defects were detected. The main defects concerned dynein arms, central complex and/or peripheral microtubules. All patients had a rarefaction of ciliated cells and a variable proportion of short cilia. Frequent but moderate and heterogeneous clinical and ciliary beating abnormalities were found. CEP290 was highly expressed in the neural retina and nasal epithelial cells compared to other tissues. Discussion : These data provide the first clear demonstration of respiratory cilia ultrastructural defects in LCA patients with CEP290 mutations. The frequency of these findings in LCA patients along with the high expression of CEP290 in nasal epithelium suggest that CEP290 has an important role in the proper development of both the respiratory ciliary structures and the connecting cilia of photoreceptors. The presence of respiratory symptoms in patients could represent additional clinical criteria to direct CEP290 genotyping of patients affected with the genetically heterogeneous cone-rod dystrophy subtype of LCA.
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expanding CEP290 mutational spectrum in ciliopathies
American Journal of Medical Genetics Part A, 2009Co-Authors: Francesco Brancati, Sophie Audollent, Isabelle Perrault, Lorena Travaglini, Tania Attiebitach, Enrico Bertini, Josseline Kaplan, Miriam IannicelliAbstract:Ciliopathies are an expanding group of rare conditions characterized by multiorgan involvement, that are caused by mutations in genes encoding for proteins of the primary cilium or its apparatus. Among these genes, CEP290 bears an intriguing allelic spectrum, being commonly mutated in Joubert syndrome and related disorders (JSRD), Meckel syndrome (MKS), Senior-Loken syndrome and isolated Leber congenital amaurosis (LCA). Although these conditions are recessively inherited, in a subset of patients only one CEP290 mutation could be detected. To assess whether genomic rearrangements involving the CEP290 gene could represent a possible mutational mechanism in these cases, exon dosage analysis on genomic DNA was performed in two groups of CEP290 heterozygous patients, including five JSRD/MKS cases and four LCA, respectively. In one JSRD patient, we identified a large heterozygous deletion encompassing CEP290 C-terminus that resulted in marked reduction of mRNA expression. No copy number alterations were identified in the remaining probands. The present work expands the CEP290 genotypic spectrum to include multiexon deletions. Although this mechanism does not appear to be frequent, screening for genomic rearrangements should be considered in patients in whom a single CEP290 mutated allele was identified.
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spectrum of nphp6 CEP290 mutations in leber congenital amaurosis and delineation of the associated phenotype
Human Mutation, 2007Co-Authors: Isabelle Perrault, Elisa Fazzi, Nathalie Delphin, Sylvain Hanein, Sylvie Gerber, Jeanlouis Dufier, O Roche, Sabine Defoortdhellemmes, Helene Dollfus, Arnold MunnichAbstract:Leber congenital amaurosis (LCA) is the earliest and most severe retinal degeneration responsible for congenital blindness. Hitherto, 13 LCA genes have been mapped, nine of which have been identified. Recently, mutations in the NPHP6/CEP290 gene were shown to account for Joubert and Senior-Loken syndromes and to represent a frequent cause of isolated LCA. All LCA patients shared an intronic mutation resulting in an aberrantly spliced transcript and low levels of wild-type transcript that was believed to explain the absence of cerebellar and renal involvement in these patients. Here, we confirm the high frequency of NPHP6/CEP290 mutations in our series of LCA families hailing worldwide (22%). However, we show that conversely to other LCA genes, NPHP6 is involved in families of European descent only (38/38). A total of 24 different mutations were found, 23 of which are novel (one founder mutation in the North region of France). All mutations but two were either nonsense, frameshift, or splice-site changes. The common NPHP6/CEP290 intronic mutation accounted for 43% (33/76) of all disease alleles. Twelve families did not carry this common intronic mutation. At least 10 out of them harboured two mutations expected to truncate the protein questioning the relevance of the assumption according to which the retinal-restricted phenotype in LCA patient could be due to a residual NPHP6/CEP290 activity. Finally, we show that all patients were affected with the cone-rod subtype of the disease whatever their NPHP6/CEP290 genotype.