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PURPOSE: Inherited retinal diseases (IRDs) are a group of monogenic conditions that can lead to progressive blindness. Their missing heritability is still considerable, due in part to the presence of disease genes that await molecular identification. The purpose of this work was to identify novel genetic associations with IRDs. METHODS: Patients underwent a comprehensive ophthalmological evaluation using standard-of-care tests, such as detailed retinal imaging (macular optical coherence tomography and short-wavelength fundus autofluorescence) and electrophysiological testing. Exome and genome sequencing, as well as computer-assisted data analysis were used for genotyping and detection of DNA variants. A minigene-driven splicing assay was performed to validate the deleterious effects of 1 of such variants. RESULTS: We identified 8 unrelated families from Hungary, the United States, Israel, and The Netherlands with members presenting with a form of autosomal recessive and nonsyndromic retinal degeneration, predominantly described as rod-cone dystrophy but also including cases of cone/cone-rod dystrophy. Age of disease onset was very variable, with some patients experiencing first symptoms during their fourth decade of life or later. Myopia greater than 5 diopters was present in 5 of 7 cases with available refractive data, and retinal detachment was reported in 2 cases. All ascertained patients carried biallelic loss-of-function variants in UBAP1L (HGNC: 40028), a gene with unknown function and with homologies to UBAP1, encoding a protein involved in ubiquitin metabolism. One of these pathogenic variants, the intronic NM_001163692.2:c.910-7G>A substitution, was identified in 5 unrelated families. Minigene-driven splicing assays in HEK293T cells confirmed that this DNA change is responsible for the creation of a new acceptor splice site, resulting in aberrant splicing. CONCLUSION: We identified UBAP1L as a novel IRD gene. Although its function is currently unknown, UBAP1L is almost exclusively expressed in photoreceptors and the retinal pigment epithelium, hence possibly explaining the link between pathogenic variants in this gene and an ocular phenotype.
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Linaje , Degeneración Retiniana , Humanos , Masculino , Femenino , Adulto , Degeneración Retiniana/genética , Persona de Mediana Edad , Mutación con Pérdida de Función , Genes Recesivos , Niño , Adolescente , Distrofias de Conos y Bastones/genética , Hungría , Adulto Joven , Predisposición Genética a la EnfermedadRESUMEN
PURPOSE: Gyrate atrophy of the choroid and retina (GACR) is an autosomal recessive inherited metabolic disorder (IMD) characterised by progressive retinal degeneration, leading to severe visual impairment. The rapid developments in ophthalmic genetic therapies warrant knowledge on clinical phenotype of eligible diseases such as GACR to define future therapeutic parameters in clinical trials. METHODS: Retrospective chart analysis was performed in nineteen patients. Data were analysed using IBM SPSS Statistics version 28.0.1.1. RESULTS: Nineteen patients were included with a mean age of 32.6 years (range 8-58). Mean age at onset of ophthalmic symptoms was 7.9 years (range 3-16). Median logMAR of visual acuity at inclusion was 0.26 (range -0.18-3.00). Mean age at cataract surgery was 28.8 years (n = 11 patients). Mean spherical equivalent of the refractive error was -8.96 (range -20.87 to -2.25). Cystoid maculopathy was present in 68% of patients, with a loss of integrity of the foveal ellipsoid zone (EZ) in 24/38 eyes. Of the 14 patients treated with dietary protein restriction, the four patients who started the diet before age 10 showed most benefit. CONCLUSION: This study demonstrates the severe ophthalmic disease course associated with GACR, as well as possible benefit of early dietary treatment. In addition to visual loss, patients experience severe myopia, early-onset cataract, and CME. There is a loss of foveal EZ integrity at a young age, emphasising the need for early diagnosis enabling current and future therapeutic interventions.
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The cause of autosomal-dominant retinitis pigmentosa (adRP), which leads to loss of vision and blindness, was investigated in families lacking a molecular diagnosis. A refined locus for adRP on Chr17q22 (RP17) was delineated through genotyping and genome sequencing, leading to the identification of structural variants (SVs) that segregate with disease. Eight different complex SVs were characterized in 22 adRP-affected families with >300 affected individuals. All RP17 SVs had breakpoints within a genomic region spanning YPEL2 to LINC01476. To investigate the mechanism of disease, we reprogrammed fibroblasts from affected individuals and controls into induced pluripotent stem cells (iPSCs) and differentiated them into photoreceptor precursor cells (PPCs) or retinal organoids (ROs). Hi-C was performed on ROs, and differential expression of regional genes and a retinal enhancer RNA at this locus was assessed by qPCR. The epigenetic landscape of the region, and Hi-C RO data, showed that YPEL2 sits within its own topologically associating domain (TAD), rich in enhancers with binding sites for retinal transcription factors. The Hi-C map of RP17 ROs revealed creation of a neo-TAD with ectopic contacts between GDPD1 and retinal enhancers, and modeling of all RP17 SVs was consistent with neo-TADs leading to ectopic retinal-specific enhancer-GDPD1 accessibility. qPCR confirmed increased expression of GDPD1 and increased expression of the retinal enhancer that enters the neo-TAD. Altered TAD structure resulting in increased retinal expression of GDPD1 is the likely convergent mechanism of disease, consistent with a dominant gain of function. Our study highlights the importance of SVs as a genomic mechanism in unsolved Mendelian diseases.
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Cromosomas Humanos Par 17/química , Proteínas Nucleares/genética , Hidrolasas Diéster Fosfóricas/genética , Células Fotorreceptoras Retinianas Conos/metabolismo , Retinitis Pigmentosa/genética , Factores de Transcripción/genética , Adulto , Secuencia de Aminoácidos , Diferenciación Celular , Reprogramación Celular , Niño , Mapeo Cromosómico , Estudios de Cohortes , Elementos de Facilitación Genéticos , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Expresión Génica , Genes Dominantes , Genoma Humano , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/patología , Masculino , Proteínas Nucleares/metabolismo , Organoides/metabolismo , Organoides/patología , Hidrolasas Diéster Fosfóricas/metabolismo , Polimorfismo Genético , Cultivo Primario de Células , Células Fotorreceptoras Retinianas Conos/patología , Retinitis Pigmentosa/diagnóstico , Retinitis Pigmentosa/metabolismo , Retinitis Pigmentosa/patología , Factores de Transcripción/metabolismo , Secuenciación Completa del GenomaRESUMEN
PURPOSE: Structural variants (SVs) play an important role in inherited retinal diseases (IRD). Although the identification of SVs significantly improved upon the availability of genome sequencing, it is expected that involvement of SVs in IRDs is higher than anticipated. We revisited short-read genome sequencing data to enhance the identification of gene-disruptive SVs. METHODS: Optical genome mapping was performed to improve SV detection in short-read genome sequencing-negative cases. In addition, reanalysis of short-read genome sequencing data was performed to improve the interpretation of SVs and to re-establish SV prioritization criteria. RESULTS: In a monoallelic USH2A case, optical genome mapping identified a pericentric inversion (173 megabase), with 1 breakpoint disrupting USH2A. Retrospectively, the variant could be observed in genome sequencing data but was previously deemed false positive. Reanalysis of short-read genome sequencing data (427 IRD cases) was performed which yielded 30 pathogenic SVs affecting, among other genes, USH2A (n = 15), PRPF31 (n = 3), and EYS (n = 2). Eight of these (>25%) were overlooked during previous analyses. CONCLUSION: Critical evaluation of our findings allowed us to re-establish and improve our SV prioritization and interpretation guidelines, which will prevent missing pathogenic events in future analyses. Our data suggest that more attention should be paid to SV interpretation and the current contribution of SVs in IRDs is still underestimated.
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Genoma Humano , Enfermedades de la Retina , Humanos , Estudios Retrospectivos , Genoma Humano/genética , Mapeo Cromosómico , Análisis de Secuencia , Enfermedades de la Retina/genética , Variación Estructural del Genoma , Proteínas del Ojo/genéticaRESUMEN
BACKGROUND: Inherited retinal diseases (IRDs) can be caused by variants in >270 genes. The Bardet-Biedl syndrome 1 (BBS1) gene is one of these genes and may be associated with syndromic and non-syndromic autosomal recessive retinitis pigmentosa (RP). Here, we identified a branchpoint variant in BBS1 and assessed its pathogenicity by in vitro functional analysis. METHODS: Whole genome sequencing was performed for three unrelated monoallelic BBS1 cases with non-syndromic RP. A fourth case received MGCM 105 gene panel analysis. Functional analysis using a midigene splice assay was performed for the putative pathogenic branchpoint variant in BBS1. After confirmation of its pathogenicity, patients were clinically re-evaluated, including assessment of non-ocular features of Bardet-Biedl syndrome. RESULTS: Clinical assessments of probands showed that all individuals displayed non-syndromic RP with macular involvement. Through detailed variant analysis and prioritisation, two pathogenic variants in BBS1, the most common missense variant, c.1169T>G (p.(Met390Arg)), and a branchpoint variant, c.592-21A>T, were identified. Segregation analysis confirmed that in all families, probands were compound heterozygous for c.1169T>G and c.592-21A>T. Functional analysis of the branchpoint variant revealed a complex splicing defect including exon 8 and exon 7/8 skipping, and partial in-frame deletion of exon 8. CONCLUSION: A putative severe branchpoint variant in BBS1, together with a mild missense variant, underlies non-syndromic RP in four unrelated individuals. To our knowledge, this is the first report of a pathogenic branchpoint variant in IRDs that results in a complex splice defect. In addition, this research highlights the importance of the analysis of non-coding regions in order to provide a conclusive molecular diagnosis.
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Síndrome de Bardet-Biedl , Retinitis Pigmentosa , Síndrome de Bardet-Biedl/diagnóstico , Síndrome de Bardet-Biedl/genética , Síndrome de Bardet-Biedl/patología , Análisis Mutacional de ADN , Humanos , Proteínas Asociadas a Microtúbulos/genética , Mutación/genética , Linaje , Retina/patología , Retinitis Pigmentosa/diagnóstico , Retinitis Pigmentosa/genética , Retinitis Pigmentosa/patologíaRESUMEN
INTRODUCTION: An increasing number of gene-specific therapies are being developed for inherited retinal degenerations (IRDs). Identification of well-characterized patients is an emerging need. We conducted the second multinational survey among the
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Degeneración Retiniana , Adulto , Humanos , Degeneración Retiniana/diagnóstico , Degeneración Retiniana/genética , Degeneración Retiniana/terapia , Estudios de Seguimiento , Pruebas de Visión , Proyectos de Investigación , Europa (Continente)RESUMEN
INTRODUCTION: The aim of this study was to evaluate the current management of RPE65 biallelic mutation-associated inherited retinal degeneration (RPE65-IRD) in Europe since market authorization of voretigene neparvovec (VN, LuxturnaTM) in 2018. By July 2022, over 200 patients have been treated outside the USA, of whom about 90% in Europe. We conducted among all centers of the European Vision Institute Clinical Research Network (
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Calidad de Vida , Degeneración Retiniana , Humanos , Preescolar , Niño , Adolescente , Adulto Joven , Adulto , Persona de Mediana Edad , Estudios de Seguimiento , Degeneración Retiniana/genética , Degeneración Retiniana/terapia , Proyectos de Investigación , Europa (Continente) , MutaciónRESUMEN
PURPOSE: To describe the natural course, phenotype, and genotype of patients with X-linked retinoschisis (XLRS). DESIGN: Retrospective cohort study. PARTICIPANTS: Three hundred forty patients with XLRS from 178 presumably unrelated families. METHODS: This multicenter, retrospective cohort study reviewed medical records of patients with XLRS for medical history, symptoms, visual acuity (VA), ophthalmoscopy, full-field electroretinography, and retinal imaging (fundus photography, spectral-domain [SD] OCT, fundus autofluorescence). MAIN OUTCOME MEASURES: Age at onset, age at diagnosis, severity of visual impairment, annual visual decline, and electroretinography and imaging findings. RESULTS: Three hundred forty patients were included with a mean follow-up time of 13.2 years (range, 0.1-50.1 years). The median ages to reach mild visual impairment and low vision were 12 and 25 years, respectively. Severe visual impairment and blindness were observed predominantly in patients older than 40 years, with a predicted prevalence of 35% and 25%, respectively, at 60 years of age. The VA increased slightly during the first 2 decades of life and subsequently transitioned into an average annual decline of 0.44% (P < 0.001). No significant difference was found in decline of VA between variants that were predicted to be severe and mild (P = 0.239). The integrity of the ellipsoid zone (EZ) as well as the photoreceptor outer segment (PROS) length in the fovea on SD OCT correlated significantly with VA (Spearman's ρ = -0.759 [P < 0.001] and -0.592 [P = 0.012], respectively). Fifty-three different RS1 variants were found. The most common variants were the founder variant c.214GâA (p.(Glu72Lys)) (101 patients [38.7%]) and a deletion of exon 3 (38 patients [14.6%]). CONCLUSIONS: Large variabilities in phenotype and natural course of XLRS were seen in this study. In most patients, XLRS showed a slow deterioration starting in the second decade of life, suggesting an optimal window of opportunity for treatment within the first 3 decades of life. The integrity of EZ as well as the PROS length on SD OCT may be important in choosing optimal candidates for treatment and as potential structural end points in future therapeutic studies. No clear genotype-phenotype correlation was found.
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Proteínas del Ojo/genética , Retinosquisis/diagnóstico , Retinosquisis/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Ceguera/diagnóstico , Ceguera/fisiopatología , Niño , Preescolar , Electrorretinografía , Femenino , Estudios de Seguimiento , Estudios de Asociación Genética , Humanos , Lactante , Masculino , Persona de Mediana Edad , Oftalmoscopía , Imagen Óptica , Retina/diagnóstico por imagen , Retina/fisiopatología , Segmento Externo de las Células Fotorreceptoras Retinianas/patología , Retinosquisis/fisiopatología , Estudios Retrospectivos , Tomografía de Coherencia Óptica , Baja Visión/diagnóstico , Baja Visión/fisiopatología , Agudeza Visual/fisiologíaRESUMEN
Mutations in PRPH2, encoding peripherin-2, are associated with the development of a wide variety of inherited retinal diseases (IRDs). To determine the causality of the many PRPH2 variants that have been discovered over the last decades, we surveyed all published PRPH2 variants up to July 2020, describing 720 index patients that in total carried 245 unique variants. In addition, we identified seven novel PRPH2 variants in eight additional index patients. The pathogenicity of all variants was determined using the ACMG guidelines. With this, 107 variants were classified as pathogenic, 92 as likely pathogenic, one as benign, and two as likely benign. The remaining 50 variants were classified as variants of uncertain significance. Interestingly, of the total 252 PRPH2 variants, more than half (n = 137) were missense variants. All variants were uploaded into the Leiden Open source Variation and ClinVar databases. Our study underscores the need for experimental assays for variants of unknown significance to improve pathogenicity classification, which would allow us to better understand genotype-phenotype correlations, and in the long-term, hopefully also support the development of therapeutic strategies for patients with PRPH2-associated IRD.
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Periferinas/genética , Enfermedades de la Retina , Estudios de Asociación Genética , Humanos , Mutación , Mutación Missense , Enfermedades de la Retina/genéticaRESUMEN
Stargardt disease is caused by variants in the ABCA4 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 ABCA4 gene. These midigenes were used to analyze the effect of all 44 reported and three novel NCSS variants on ABCA4 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 ABCA4 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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Transportadoras de Casetes de Unión a ATP/genética , Degeneración Macular/congénito , Precursores del ARN/genética , Sitios de Empalme de ARN , Empalme del ARN , Transportadoras de Casetes de Unión a ATP/biosíntesis , Adulto , Femenino , Humanos , Degeneración Macular/genética , Degeneración Macular/metabolismo , Masculino , Precursores del ARN/metabolismo , Enfermedad de StargardtRESUMEN
PURPOSE: To investigate the natural history of RHO-associated retinitis pigmentosa (RP). METHODS: A multicenter, medical chart review of 100 patients with autosomal dominant RHO-associated RP. RESULTS: Based on visual fields, time-to-event analysis revealed median ages of 52 and 79 years to reach low vision (central visual field <20°) and blindness (central visual field <10°), respectively. For the best-corrected visual acuity (BCVA), the median age to reach mild impairment (20/67 ≤ BCVA < 20/40) was 72 years, whereas this could not be computed for lower acuities. Disease progression was significantly faster in patients with a generalized RP phenotype (n = 75; 75%) than that in patients with a sector RP phenotype (n = 25; 25%), in terms of decline rates of the BCVA (P < 0.001) and V4e retinal seeing areas (P < 0.005). The foveal thickness of the photoreceptor-retinal pigment epithelium (PR + RPE) complex correlated significantly with BCVA (Spearman's ρ = 0.733; P < 0.001). CONCLUSION: Based on central visual fields, the optimal window of intervention for RHO-associated RP is before the 5th decade of life. Significant differences in disease progression are present between generalized and sector RP phenotypes. Our findings suggest that the PR + RPE complex is a potential surrogate endpoint for the BCVA in future studies.
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Proteínas de Fase Aguda/genética , Predicción , Epitelio Pigmentado de la Retina/patología , Retinitis Pigmentosa/diagnóstico , Agudeza Visual , Campos Visuales/fisiología , Proteínas de Fase Aguda/metabolismo , Anciano , Electrorretinografía , Femenino , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Fenotipo , Retinitis Pigmentosa/sangre , Retinitis Pigmentosa/genética , Estudios Retrospectivos , Tomografía de Coherencia Óptica/métodosRESUMEN
PURPOSE: An increasing number of gene therapies are developed for Inherited Retinal Degenerations (IRD). To date, 1 treatment has been approved for clinical use (FDA USA 2017, EMA Europe 2018, MoHAP UAE 2019, SFDA Saudi Arabia 2019, Swiss Medic Switzerland 2020, TGA Australia 2020, and BFR Brazil 2020). While such therapies do not provide complete cure, they may halt degeneration or partially restore function. Identification of well-characterized patients is an emerging need. We conducted the first multinational survey to understand the management of IRDs in Europe. METHODS: An electronic survey questionnaire containing 112 questions was developed and sent to the 101 EVICR.net clinical centers (14 European countries and Israel). RESULTS: The overall response rate was 49%. Only 14% of responding centers do not see IRD patients; 52% that manage IRD patients follow ≥200 patients, 16% > 1,000. Databases exist in 86% of the centers; of these, 75% are local files, 28% local Web-based database, and 19% national Web-based. IRD patients are referred to EVICR.net centers mainly by general ophthalmologists, patient self-referrals, and medical retina specialists. Most IRD patients are first seen in adulthood. Most prominent signs and symptoms depend on the age of onset, for example, nystagmus in infancy, or night blindness, and reduced visual acuity at older age. The time from inquiring for first appointment and clinical diagnosis varies among countries: in 29% of centers, the mean time is <4 weeks, although can be up to 35 months in others. The time to genetic diagnosis is ≥4 weeks, the maximum 10 years, likely depending on access to genetic testing, and the improvement of the tests available. Comprehensive eye examination always includes autofluorescence imaging and perimetry (86% static, 76% kinetic, and 21% microperimetry), and frequently optical coherence tomography (OCT) (95%), electroretinography (93%), and fundus photography (93%). Identified genotypes were reported in 40-80% patients by 69% of centers, and in 80-100% by 5%. Genetic testing is provided by public health insurance in 77% of centers, private health insurance in 38%, center budget in 13%, research funds in 18%; and 15% of centers do not have access to genetic testing. CONCLUSION: At the start of this era of ocular gene therapy for IRD patients, this first international survey on management of IRDs in Europe highlights significant heterogeneity between centers and across countries and provides important baseline data for researchers, clinicians, pharmaceutical companies, and investors.
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Degeneración Retiniana , Electrorretinografía , Humanos , Retina , Degeneración Retiniana/genética , Degeneración Retiniana/terapia , Encuestas y Cuestionarios , Pruebas del Campo VisualRESUMEN
INTRODUCTION: Treatment of exudative age-related macular degeneration (ARMD) has shifted to pro re nata and treat-extend-stop strategies. However, a rational discontinuation strategy is lacking. To develop such a strategy, it is important to determine choroidal neovascularization (CNV) recurrence rates after anti-VEGF treatment is discontinued. Here we report prospective data on persistent and recurrent CNV activity after discontinuation of bevacizumab treatment. METHODS: This prospective, single-center clinical trial enrolled 191 patients with exudative ARMD. Patients were randomly assigned to receiving intravitreal bevacizumab injections every 4, 6, or 8 weeks for 1 year. CNV activity was determined in the 157 patients who completed the 1-year treatment regimen. Patients with inactive CNV were then followed for signs of CNV reactivation. RESULTS: After 1 year of treatment, 66 (42%) of the 157 patients still had signs of persistent active CNV. Of the remaining 91 (58%) patients, 61 (67%) needed retreatment for active CNV within the first year after discontinuation of treatment (mean 4.28 ± 0.29 months). CNV was reactivated in 50 (80%) of the 61 patients within 6 months after their final treatment for CNV. CONCLUSION: Based on quiescent disease, anti-VEGF therapy was discontinued in 58% of the patients after they received bevacizu-mab injections every 4, 6, or 8 weeks for 1 year; 67% showed reactivated CNV within a year after discontinuation. The high reactivation rate of CNV shown in this study should help clinicians to develop rational discontinuation protocols. TRIAL REGISTRATION: This study is registered as NTR1174 at http://www.trialregister.nl.
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Neovascularización Coroidal , Degeneración Macular , Inhibidores de la Angiogénesis/uso terapéutico , Bevacizumab/uso terapéutico , Neovascularización Coroidal/diagnóstico , Neovascularización Coroidal/tratamiento farmacológico , Humanos , Degeneración Macular/diagnóstico , Degeneración Macular/tratamiento farmacológico , Estudios Prospectivos , Factor A de Crecimiento Endotelial Vascular , Agudeza VisualRESUMEN
PURPOSE: The first ocular gene augmentation therapy, voretigene neparvovec (VN) (Luxturna®), has been approved for clinical use in an increasing number of countries (FDA USA 2017, EMA Europe 2018, MoHAP United Arab Emirates 2019, SFDA Saudi Arabia 2019, Swiss Medic Switzerland 2020, TGA Australia 2020, BFR Brazil 2020). Among the EVICR.net clinical centers, we conducted the first multinational survey to understand distribution, diagnostic work-up, and management of inherited retinal degeneration (IRD) cases in Europe with a special focus on RPE65 mutation-associated IRDs. METHODS: An electronic survey questionnaire including 35 questions specifically addressing RPE65 mutation-associated IRDs was developed and sent to the 101 EVICR.net clinical centers. RESULTS: The overall response rate was 49%. Forty-two centers see IRD patients, and 22/42 follow patients with confirmed biallelic RPE65 mutations. Fifteen of the 22 centers (68%) and 3/22 (14%) follow 1-5 and 6-10 patients with homozygous RPE65 mutations, respectively. Additionally, 15/22 (68%) and 3/22 (14%) follow 1-5 and >20 patients with compound heterozygous RPE65 mutations, respectively. Fifty-nine percent of mutations were ACMG Class 4 and 5 (at least 1 allele), 82.8% reported previously and 17.2% novel. Referral diagnoses (the mean per center) were Leber congenital amaurosis (38.2%), early-onset severe retinal degeneration (16.8%), rod-cone-dystrophy/retinitis pigmentosa (RP) (28.1%), and unclassified visual impairment (17.0%). Twenty-five percent of the centers changed the referral diagnosis in >47.5% of cases; 32% follow a specific referral process for RPE65 mutation-associated IRD patients. Annual follow-up visits are done in 55% of the centers and biannual visits in 23%. In 32%, other centers also follow the patients. Kinetic perimetry is done in 82%, static perimetry in 45%, and microperimetry in 18% of the centers. Full-field light stimulus threshold testing with blue and red stimuli to quantify the rod and cone function is used in 6/22 centers (27%). A mobility course is available in one center (5%). CONCLUSION: This first multinational survey on management of patients with RPE65 mutation-associated IRDs in Europe shows that about half of the responding EVICR.net centers have such patients under care. There is heterogeneity in diagnoses and management practices. At the start of clinical practice experience with VN, these data provide a useful baseline and highlight the need for consensus/guidelines to inform standard of care in this new era of gene therapy.
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cis-trans-Isomerasas/genética , Europa (Continente) , Humanos , Amaurosis Congénita de Leber/diagnóstico , Amaurosis Congénita de Leber/genética , Amaurosis Congénita de Leber/terapia , Mutación , Distrofias Retinianas , Encuestas y CuestionariosRESUMEN
A substantial proportion of subjects with autosomal recessive retinitis pigmentosa (arRP) or Usher syndrome type II (USH2) lacks a genetic diagnosis due to incomplete USH2A screening in the early days of genetic testing. These cases lack eligibility for optimal genetic counseling and future therapy. USH2A defects are the most frequent cause of USH2 and are also causative in individuals with arRP. Therefore, USH2A is an important target for genetic screening. The aim of this study was to assess unscreened or incompletely screened and unexplained USH2 and arRP cases for (likely) pathogenic USH2A variants. Molecular inversion probe (MIP)-based sequencing was performed for the USH2A exons and their flanking regions, as well as published deep-intronic variants. This was done to identify single nucleotide variants (SNVs) and copy number variants (CNVs) in 29 unscreened or partially pre-screened USH2 and 11 partially pre-screened arRP subjects. In 29 out of these 40 cases, two (likely) pathogenic variants were successfully identified. Four of the identified SNVs and one CNV were novel. One previously identified synonymous variant was demonstrated to affect pre-mRNA splicing. In conclusion, genetic diagnoses were obtained for a majority of cases, which confirms that MIP-based sequencing is an effective screening tool for USH2A. Seven unexplained cases were selected for future analysis with whole genome sequencing.
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Análisis Costo-Beneficio , Exones/genética , Proteínas de la Matriz Extracelular/genética , Sondas Moleculares/metabolismo , Sitios de Empalme de ARN/genética , Retinitis Pigmentosa/genética , Análisis de Secuencia de ADN , Síndromes de Usher/genética , Secuencia de Bases , Variaciones en el Número de Copia de ADN/genética , Eliminación de Gen , Humanos , Polimorfismo de Nucleótido Simple/genética , Retinitis Pigmentosa/economía , Síndromes de Usher/economíaRESUMEN
PURPOSE: Missing heritability in human diseases represents a major challenge, and this is particularly true for ABCA4-associated Stargardt disease (STGD1). We aimed to elucidate the genomic and transcriptomic variation in 1054 unsolved STGD and STGD-like probands. METHODS: Sequencing of the complete 128-kb ABCA4 gene was performed using single-molecule molecular inversion probes (smMIPs), based on a semiautomated and cost-effective method. Structural variants (SVs) were identified using relative read coverage analyses and putative splice defects were studied using in vitro assays. RESULTS: In 448 biallelic probands 14 known and 13 novel deep-intronic variants were found, resulting in pseudoexon (PE) insertions or exon elongations in 105 alleles. Intriguingly, intron 13 variants c.1938-621G>A and c.1938-514G>A resulted in dual PE insertions consisting of the same upstream, but different downstream PEs. The intron 44 variant c.6148-84A>T resulted in two PE insertions and flanking exon deletions. Eleven distinct large deletions were found, two of which contained small inverted segments. Uniparental isodisomy of chromosome 1 was identified in one proband. CONCLUSION: Deep sequencing of ABCA4 and midigene-based splice assays allowed the identification of SVs and causal deep-intronic variants in 25% of biallelic STGD1 cases, which represents a model study that can be applied to other inherited diseases.
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Degeneración Macular , Transcriptoma , Transportadoras de Casetes de Unión a ATP/genética , Genómica , Humanos , Intrones , Degeneración Macular/genética , Mutación , Linaje , Enfermedad de StargardtRESUMEN
PURPOSE: To study the disease course of RPE65-associated inherited retinal degenerations (IRDs) as a function of the genotype, define a critical age for blindness, and identify potential modifiers. METHODS: Forty-five patients with IRD from 33 families with biallelic RPE65 mutations, 28 stemming from a genetic isolate. We collected retrospective data from medical charts. Coexisting variants in 108 IRD-associated genes were identified with Molecular Inversion Probe analysis. RESULTS: Most patients were diagnosed within the first years of life. Daytime visual function ranged from near-normal to blindness in the first four decades and met WHO criteria for blindness for visual acuity and visual field in the fifth decade. p.(Thr368His) was the most common variant (54%). Intrafamilial variability and interfamilial variability in disease severity and progression were observed. Molecular Inversion Probe analysis confirmed all RPE65 variants and identified one additional variant in LRAT and one in EYS in two separate patients. CONCLUSION: All patients with RPE65-associated IRDs developed symptoms within the first year of life. Visual function in childhood and adolescence varied but deteriorated inevitably toward blindness after age 40. In this study, genotype was not predictive of clinical course. The variance in severity of disease could not be explained by double hits in other IRD genes.
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Mutación , Degeneración Retiniana/genética , cis-trans-Isomerasas/genética , Adolescente , Adulto , Niño , Preescolar , Electrorretinografía , Femenino , Estudios de Asociación Genética , Genotipo , Humanos , Lactante , Recién Nacido , Estudios Longitudinales , Masculino , Persona de Mediana Edad , Retina/fisiopatología , Degeneración Retiniana/diagnóstico por imagen , Degeneración Retiniana/fisiopatología , Estudios Retrospectivos , Tomografía de Coherencia Óptica , Agudeza Visual/fisiología , Campos Visuales/fisiología , Adulto JovenRESUMEN
BACKGROUND: Transcorneal electrical stimulation (TES) has been suggested as a possible treatment for retinitis pigmentosa (RP). OBJECTIVE: To expand the safety assessment of repeated applications of an electrical current from a DTL-like electrode in patients with RP. METHODS: This single-arm open label interventional safety trial included a total of 105 RP patients from 11 European centers, who received weekly TES for 6 months on 1 eye followed by observation for another 6 months without stimulation. The primary outcome measure was safety, indicated by the frequency and severity of adverse events. Secondary measures included intraocular pressure and central retinal thickness. Visual field and visual acuity were examined using the methods available at each site. RESULTS: Dry eye sensation was the most common adverse event recorded (37.5%). Serious adverse events secondary to TES were not observed. Most adverse events were mild and all resolved without sequelae. The secondary outcome measures revealed no significant or clinically relevant changes. CONCLUSION: The present results confirm the excellent safety profile of TES. Transient dry eye symptoms were the most common adverse event.
Asunto(s)
Terapia por Estimulación Eléctrica/instrumentación , Retinitis Pigmentosa/terapia , Agudeza Visual , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Electrorretinografía , Diseño de Equipo , Femenino , Estudios de Seguimiento , Humanos , Presión Intraocular/fisiología , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Retinitis Pigmentosa/diagnóstico , Resultado del Tratamiento , Adulto JovenRESUMEN
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.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Oligonucleótidos Antisentido/genética , Isoformas de Proteínas/genética , Enfermedad de Stargardt/genética , Adolescente , Adulto , Anciano , Niño , Exones/genética , Células HEK293 , Humanos , Intrones/genética , Persona de Mediana Edad , Mutación/genética , Oligonucleótidos Antisentido/farmacología , Linaje , Polimorfismo de Nucleótido Simple/genética , Empalme del ARN/genética , Enfermedad de Stargardt/patología , Adulto JovenRESUMEN
BACKGROUND: Retinitis pigmentosa (RP) shows substantial genetic heterogeneity. It has been estimated that in approximately 60%-80% of RP cases, the genetic diagnosis can be found using whole exome sequencing (WES). In this study, the purpose was to identify causative variants in individuals with genetically unexplained retinal disease, which included one consanguineous family with two affected siblings and one case with RP. METHODS: To identify the genetic defect, WES was performed in both probands, and clinical analysis was performed. To obtain insight into the function of KIAA1549 in photoreceptors, mRNA expression, knockdown and protein localisation studies were performed. RESULTS: Through analysis of WES data, based on population allele frequencies, and in silico prediction tools, we identified a homozygous missense variant and a homozygous frameshift variant in KIAA1549 that segregate in two unrelated families. Kiaa1549 was found to localise at the connecting cilium of the photoreceptor cells and the synapses of the mouse retina. Both variants affect the long transcript of KIAA1549, which encodes a 1950 amino acid protein and shows prominent brain expression. The shorter transcript encodes a 734 amino acid protein with a high retinal expression and is affected by the identified missense variant. Strikingly, knockdown of the long transcript also leads to decreased expression of the short transcript likely explaining the non-syndromic retinal phenotype caused by the two variants targeting different transcripts. CONCLUSION: In conclusion, our results underscore the causality of segregating variants in KIAA1549 for autosomal recessive RP. Moreover, our data indicate that KIAA1549 plays a role in photoreceptor function.