ABSTRACT
Malattia Leventinese/Doyne honeycomb retinal dystrophy (ML/DHRD) is an age-related macular degeneration-like (AMD-like) retinal dystrophy caused by an autosomal dominant R345W mutation in the secreted glycoprotein, fibulin-3 (F3). To identify new small molecules that reduce F3 production in retinal pigmented epithelium (RPE) cells, we knocked-in a luminescent peptide tag (HiBiT) into the endogenous F3 locus that enabled simple, sensitive, and high-throughput detection of the protein. The GSK3 inhibitor, CHIR99021 (CHIR), significantly reduced F3 burden (expression, secretion, and intracellular levels) in immortalized RPE and non-RPE cells. Low-level, long-term CHIR treatment promoted remodeling of the RPE extracellular matrix, reducing sub-RPE deposit-associated proteins (e.g., amelotin, complement component 3, collagen IV, and fibronectin), while increasing RPE differentiation factors (e.g., tyrosinase, and pigment epithelium-derived factor). In vivo, treatment of 8-month-old R345W+/+ knockin mice with CHIR (25 mg/kg i.p., 1 mo) was well tolerated and significantly reduced R345W F3-associated AMD-like basal laminar deposit number and size, thereby preventing the main pathological feature in these mice. This is an important demonstration of small molecule-based prevention of AMD-like pathology in ML/DHRD mice and may herald a rejuvenation of interest in GSK3 inhibition for the treatment of retinal degenerative diseases, including potentially AMD itself.
Subject(s)
Extracellular Matrix Proteins , Extracellular Matrix , Macular Degeneration , Retinal Pigment Epithelium , Animals , Mice , Retinal Pigment Epithelium/pathology , Retinal Pigment Epithelium/metabolism , Retinal Pigment Epithelium/drug effects , Macular Degeneration/pathology , Macular Degeneration/genetics , Macular Degeneration/drug therapy , Macular Degeneration/metabolism , Humans , Extracellular Matrix Proteins/metabolism , Extracellular Matrix Proteins/genetics , Extracellular Matrix/metabolism , Extracellular Matrix/drug effects , Pyridines/pharmacology , Pyrimidines/pharmacology , Glycogen Synthase Kinase 3/metabolism , Glycogen Synthase Kinase 3/antagonists & inhibitors , Glycogen Synthase Kinase 3/genetics , Disease Models, Animal , Retinal Dystrophies/metabolism , Retinal Dystrophies/pathology , Retinal Dystrophies/genetics , Optic Disk Drusen/congenitalABSTRACT
Purpose: Biallelic pathogenic variants in the gene encoding the ATP-binding cassette transporter ABCA4 are the leading cause of irreversible vision loss in inherited retinal dystrophies (IRDs). Interpretation of ABCA4 variants is challenging, due to cis-modifying and hypomorphic variants. We have previously detected 10 missense variants of unknown significance (VUS) in patients with suspected ABCA4-retinal dystrophies (ABCA4-RDs) in Norway. In this study, we functionally characterized the VUS to aid interpretation of the variants and to determine if they are associated with the disease. Methods: The ABCA4 VUS were expressed in HEK293T cells and the ABCA4 expression level and ATPase activity were determined and correlated with the patients' phenotype. The functional data further used for reclassification of the VUS following the American College of Medical Genetics and Genomics (ACMG) guidelines. Results: Of the 10 VUSs, 2 variants, Cys205Phe and Asn415Thr, were categorized as functionally severe. The age at presentation in the 2 patients carrying these variants was divergent and seemed to be driven by the patients' second pathogenic variants Gly1961Glu and c.5461-10T>C, respectively. Three variants, Val643Gly, Pro799Leu, and Val1433Ile were categorized as functionally moderate, and were found in patients with intermediate/late age at presentation. The remaining five variants were categorized as functionally normal/mild. Based on our data, c.614G>T p.(Cys205Phe), c.1244A>C p.(Asn415Thr), and c.2396C>T p.(Pro799Leu) were reclassified to (likely) pathogenic, while 4 of the functionally normal/mild variants could be reclassified to likely benign. Conclusions: Functional analyses of ABCA4 variants are a helpful tool in variant classification and enable us to better predict the disease severity in patients with ABCA4-RDs.
Subject(s)
ATP-Binding Cassette Transporters , Mutation, Missense , Phenotype , Retinal Dystrophies , Humans , Retinal Dystrophies/genetics , Retinal Dystrophies/metabolism , Retinal Dystrophies/diagnosis , ATP-Binding Cassette Transporters/genetics , Female , Male , HEK293 Cells , DNA Mutational Analysis , Pedigree , AdultABSTRACT
BACKGROUND: Inherited retinal dystrophies (IRDs) are a group of debilitating visual disorders characterized by the progressive degeneration of photoreceptors, which ultimately lead to blindness. Among the causes of this condition, mutations in the PCYT1A gene, which encodes the rate-limiting enzyme responsible for phosphatidylcholine (PC) de novo synthesis via the Kennedy pathway, have been identified. However, the precise mechanisms underlying the association between PCYT1A mutations and IRDs remain unclear. To address this knowledge gap, we focused on elucidating the functions of PCYT1A in the retina. RESULTS: We found that PCYT1A is highly expressed in Müller glial (MG) cells in the inner nuclear layer (INL) of the retina. Subsequently, we generated a retina-specific knockout mouse model in which the Pcyt1a gene was targeted (Pcyt1a-RKO or RKO mice) to investigate the molecular mechanisms underlying IRDs caused by PCYT1A mutations. Our findings revealed that the deletion of Pcyt1a resulted in retinal degenerative phenotypes, including reduced scotopic electroretinogram (ERG) responses and progressive degeneration of photoreceptor cells, accompanied by loss of cells in the INL. Furthermore, through proteomic and bioinformatic analyses, we identified dysregulated retinal fatty acid metabolism and activation of the ferroptosis signalling pathway in RKO mice. Importantly, we found that PCYT1A deficiency did not lead to an overall reduction in PC synthesis within the retina. Instead, this deficiency appeared to disrupt free fatty acid metabolism and ultimately trigger ferroptosis. CONCLUSIONS: This study reveals a novel mechanism by which mutations in PCYT1A contribute to the development of IRDs, shedding light on the interplay between fatty acid metabolism and retinal degenerative diseases, and provides new insights into the treatment of IRDs.
Subject(s)
Fatty Acids , Ferroptosis , Mice, Knockout , Retina , Animals , Mice , Choline-Phosphate Cytidylyltransferase/genetics , Choline-Phosphate Cytidylyltransferase/metabolism , Fatty Acids/metabolism , Ferroptosis/physiology , Ferroptosis/genetics , Retina/metabolism , Retinal Dystrophies/genetics , Retinal Dystrophies/metabolismABSTRACT
Inherited retinal dystrophies (IRDs) are progressive diseases leading to vision loss. Mutation in the eyes shut homolog (EYS) gene is one of the most frequent causes of IRD. However, the mechanism of photoreceptor cell degeneration by mutant EYS has not been fully elucidated. Here, we generated retinal organoids from induced pluripotent stem cells (iPSCs) derived from patients with EYS-associated retinal dystrophy (EYS-RD). In photoreceptor cells of RD organoids, both EYS and G protein-coupled receptor kinase 7 (GRK7), one of the proteins handling phototoxicity, were not in the outer segment, where they are physiologically present. Furthermore, photoreceptor cells in RD organoids were vulnerable to light stimuli, and especially to blue light. Mislocalization of GRK7, which was also observed in eys-knockout zebrafish, was reversed by delivering control EYS into photoreceptor cells of RD organoids. These findings suggest that avoiding phototoxicity would be a potential therapeutic approach for EYS-RD.
Subject(s)
Induced Pluripotent Stem Cells , Organoids , Retinal Dystrophies , Zebrafish , Animals , Humans , Eye Proteins/genetics , Eye Proteins/metabolism , Induced Pluripotent Stem Cells/metabolism , Light/adverse effects , Mutation , Organoids/metabolism , Retina/metabolism , Retina/pathology , Retinal Dystrophies/therapy , Retinal Dystrophies/genetics , Retinal Dystrophies/metabolismABSTRACT
Acyl-CoA-binding domain-containing protein 5-related retinal dystrophy with leukodystrophy (ACBD5) is a peroxisomal disorder due to deficiency of ACBD5. Presenting features include retinal dystrophy, progressive leukodystrophy, and ataxia. Only seven cases of ACBD5-related retinal dystrophy have been reported in the literature to date, including one other case diagnosed in adulthood. Here we report a case with novel compound heterozygous ACBD5 mutations, presenting with the common features of rod monochromatism and progressive leukodystrophy with spasticity and ataxia. Additional novel clinical features included head and neck tremor and ovarian insufficiency. The patient's symptoms were present since infancy, but a diagnosis was only reached in adulthood when whole exome sequencing was performed. This case, which reports two novel mutations and additional clinical manifestations, contributes to the emerging phenotype of ACBD5-related retinal dystrophy with leukodystrophy, and delineation of the natural history and disease progression.
Subject(s)
Primary Ovarian Insufficiency , Retinal Dystrophies , Female , Humans , Mutation , Pedigree , Retinal Dystrophies/diagnosis , Retinal Dystrophies/genetics , Retinal Dystrophies/metabolism , Phenotype , Primary Ovarian Insufficiency/diagnosis , Primary Ovarian Insufficiency/genetics , Ataxia , Membrane Proteins/genetics , Adaptor Proteins, Signal Transducing/geneticsABSTRACT
Choroideremia (CHM) is an X-linked chorioretinal dystrophy leading to progressive retinal degeneration that results in blindness by late adulthood. It is caused by mutations in the CHM gene encoding the Rab Escort Protein 1 (REP1), which plays a crucial role in the prenylation of Rab proteins ensuring correct intracellular trafficking. Gene augmentation is a promising therapeutic strategy, and there are several completed and ongoing clinical trials for treating CHM using adeno-associated virus (AAV) vectors. However, late-phase trials have failed to show significant functional improvements and have raised safety concerns about inflammatory events potentially caused by the use of viruses. Therefore, alternative non-viral therapies are desirable. Episomal scaffold/matrix attachment region (S/MAR)-based plasmid vectors were generated containing the human CHM coding sequence, a GFP reporter gene, and ubiquitous promoters (pS/MAR-CHM). The vectors were assessed in two choroideremia disease model systems: (1) CHM patient-derived fibroblasts and (2) chmru848 zebrafish, using Western blotting to detect REP1 protein expression and in vitro prenylation assays to assess the rescue of prenylation function. Retinal immunohistochemistry was used to investigate vector expression and photoreceptor morphology in injected zebrafish retinas. The pS/MAR-CHM vectors generated persistent REP1 expression in CHM patient fibroblasts and showed a significant rescue of prenylation function by 75%, indicating correction of the underlying biochemical defect associated with CHM. In addition, GFP and human REP1 expression were detected in zebrafish microinjected with the pS/MAR-CHM at the one-cell stage. Injected chmru848 zebrafish showed increased survival, prenylation function, and improved retinal photoreceptor morphology. Non-viral S/MAR vectors show promise as a potential gene-augmentation strategy without the use of immunogenic viral components, which could be applicable to many inherited retinal disease genes.
Subject(s)
Choroideremia , Retinal Dystrophies , Animals , Humans , Adult , Choroideremia/genetics , Choroideremia/therapy , Choroideremia/metabolism , Zebrafish/genetics , Zebrafish/metabolism , Retina/metabolism , Mutation , Retinal Dystrophies/metabolism , Plasmids , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolismABSTRACT
The ITM2B-related retinal dystrophy (ITM2B-RD) was identified within patients carrying the autosomal dominant variant [c.782A > C, p.(Glu261Ala)] in ITM2B from whom induced pluripotent stem cell (IPSC) lines were previously generated. Here, we report the generation of three isogenic control iPSC lines from the derived affected subject cell line (ITM2B-5286-3) using CRISPR/Cas9 engineering. The three generated lines express pluripotency markers, can be differentiated into the three germ layers and present a normal karyotype. The generated iPSC lines can be used to study the implications of ITM2B-RD variant in vitro.
Subject(s)
Induced Pluripotent Stem Cells , Retinal Dystrophies , Humans , Induced Pluripotent Stem Cells/metabolism , CRISPR-Cas Systems/genetics , Retinal Dystrophies/genetics , Retinal Dystrophies/metabolism , Cell Differentiation , Mutation , Adaptor Proteins, Signal Transducing/geneticsABSTRACT
CLCN2 encodes a two-pore homodimeric chloride channel protein (CLC-2) that is widely expressed in human tissues. The association between Clcn2 and the retina is well-established in mice, as loss-of-function of CLC-2 can cause retinopathy in mice; however, the ocular phenotypes caused by CLCN2 mutations in humans and the underlying mechanisms remain unclear. The present study aimed to define the ocular features and reveal the pathogenic mechanisms of CLCN2 variants associated with retinal degeneration in humans using an in vitro overexpression system, as well as patient-induced pluripotent stem cell (iPSC)-derived retinal pigment epithelium (RPE) cells and retinal organoids (ROs). A patient carrying the homozygous c.2257C > T (p.R753X) nonsense CLCN2 mutation was followed up for > 6 years. Ocular features were comprehensively characterized with multimodality imaging and functional examination. The patient presented with severe bilateral retinal degeneration with loss of photoreceptor and RPE. In vitro, mutant CLC-2 maintained the correct subcellular localization, but with reduced channel function compared to wild-type CLC-2 in HEK293T cells. Additionally, patient iPSC-derived RPE cells carrying the CLCN2 mutation exhibited dysfunctional ClC-2 chloride channels and outer segment phagocytosis. Notably, these functions were rescued following the repair of the CLCN2 mutation using the CRISPR-Cas9 system. However, this variant did not cause significant photoreceptor degeneration in patient-derived ROs, indicating that dysfunctional RPE is likely the primary cause of biallelic CLCN2 variant-mediated retinopathy. This study is the first to establish the confirmatory ocular features of human CLCN2-related retinal degeneration, and reveal a pathogenic mechanism associated with biallelic CLCN2 variants, providing new insights into the cause of inherited retinal dystrophies.
Subject(s)
Induced Pluripotent Stem Cells , Retinal Dystrophies , Animals , Humans , Mice , Chloride Channels/genetics , Codon, Nonsense , HEK293 Cells , Mutation , Phagocytosis/genetics , Reactive Oxygen Species/metabolism , Retinal Dystrophies/metabolism , Retinal Pigment Epithelium/metabolism , Retinal Pigment Epithelium/pathologyABSTRACT
Inherited retinal dystrophies (IRDs) are congenital retinal degenerative diseases that have various inheritance patterns, including dominant, recessive, X-linked, and mitochondrial. These diseases are most often the result of defects in rod and/or cone photoreceptor and retinal pigment epithelium function, development, or both. The genes associated with these diseases, when mutated, produce altered protein products that have downstream effects in pathways critical to vision, including phototransduction, the visual cycle, photoreceptor development, cellular respiration, and retinal homeostasis. The aim of this manuscript is to provide a comprehensive review of the underlying molecular mechanisms of pathogenesis of IRDs by delving into many of the genes associated with IRD development, their protein products, and the pathways interrupted by genetic mutation.
Subject(s)
Retinal Dystrophies , Humans , Retinal Dystrophies/genetics , Retinal Dystrophies/metabolism , Retina/metabolism , Retinal Cone Photoreceptor Cells , Mutation , Vision, OcularABSTRACT
Pathogenic variants in the Retinitis pigmentosa GTPase regulator (RPGR) gene lead to a clinically severe form of X-linked retinal dystrophy. However, it remains unclear why some variants cause a predominant rod, while others result in a cone-dominated phenotype. Post-translational glutamylation of the photoreceptor-specific RPGRORF15 isoform by the TTLL5 enzyme is essential for its optimal function in photoreceptors, and loss of TTLL5 leads to retinal dystrophy with a cone phenotype. Here we show that RPGR retinal disease, studied in a single cohort of 116 male patients, leads to a clear progressive shift from rod- to cone-dominating phenotype as the RPGRORF15 variant location approaches the distal part of the Open Reading Frame 15 (ORF15) region. The rod photoreceptor involvement on the contrary diminishes along the RGPR sequence, and the variants associated with the cone only phenotype are located predominantly in the very distal part, including the C-terminal basic domain. Moreover, these distal truncating RPGRORF15 variants disrupt the interaction with TTLL5 and lead to a significant impairment of RPGR glutamylation. Thus, consistent with the phenotype of TTLL5 pathogenic variants, our study shows that RPGRORF15 variants, which disrupt its basic domain and the interaction with TTLL5, also impair RPGR glutamylation and lead to the cone phenotype. This has implications for ongoing gene therapy clinical trials where the application of RPGR with impaired glutamylation may be less effective in treating RGPR dystrophies and may even convert a rod-cone dystrophy into a cone dystrophy phenotype.
Subject(s)
Cone-Rod Dystrophies , Retinal Dystrophies , Humans , Male , Cone-Rod Dystrophies/genetics , Cone-Rod Dystrophies/metabolism , Eye Proteins/genetics , Eye Proteins/metabolism , Open Reading Frames/genetics , Open Reading Frames/physiology , Phenotype , Retinal Cone Photoreceptor Cells/metabolism , Retinal Dystrophies/genetics , Retinal Dystrophies/metabolism , Glutamic Acid/metabolismABSTRACT
PROM1-related retinal dystrophy (PROM1-RD) is a group of hereditary retinal disorder characterized by the progressive damage of the photoreceptors. We generated and identified two induced pluripotent stem cell (iPSC) lines carrying homozygous or heterozygous nonsense mutation c.619G > T (p.E207X) in PROM1 gene from a patient with PROM1-RD and his healthy mother, respectively. Both iPSC lines maintained the typical stem cell morphology, genomic stability and pluripotency. These iPSC lines have great potential to elucidate the disease mechanisms and develop the feasible treatments of PROM1-RD.
Subject(s)
AC133 Antigen , Induced Pluripotent Stem Cells , Retinal Dystrophies , Humans , AC133 Antigen/genetics , AC133 Antigen/metabolism , Codon, Nonsense/genetics , Heterozygote , Homozygote , Induced Pluripotent Stem Cells/metabolism , Mutation , Retinal Dystrophies/genetics , Retinal Dystrophies/metabolismABSTRACT
Photoreceptor degeneration leads to irreversible vision loss in humans with retinal dystrophies such as retinitis pigmentosa. Whereas photoreceptor loss is permanent in mammals, zebrafish possesses the ability to regenerate retinal neurons and restore visual function. Following acute damage, Müller glia (MG) re-enter the cell cycle and produce multipotent progenitors whose progeny differentiate into mature neurons. Both MG reprogramming and proliferation of retinal progenitor cells require reactive microglia and associated inflammatory signaling. Paradoxically, in zebrafish models of retinal degeneration, photoreceptor death does not induce the MG to reprogram and regenerate lost cells. Here, we used male and female zebrafish cep290 mutants to demonstrate that progressive cone degeneration generates an immune response but does not stimulate MG proliferation. Acute light damage triggered photoreceptor regeneration in cep290 mutants but cones were only restored to prelesion densities. Using irf8 mutant zebrafish, we found that the chronic absence of microglia reduced inflammation and rescued cone degeneration in cep290 mutants. Finally, single-cell RNA-sequencing revealed sustained expression of notch3 in MG of cep290 mutants and inhibition of Notch signaling induced MG to re-enter the cell cycle. Our findings provide new insights on the requirements for MG to proliferate and the potential for immunosuppression to prolong photoreceptor survival.SIGNIFICANCE STATEMENT Inherited retinal degenerations (IRDs) are genetic diseases that lead to the progressive loss of photoreceptors and the permanent loss of vision. Zebrafish can regenerate photoreceptors after acute injury by reprogramming Müller glia (MG) into stem-like cells that produce retinal progenitors, but this regenerative process fails to occur in zebrafish models of IRDs. Here, we show that Notch pathway inhibition can promote photoreceptor regeneration in models of progressive degeneration and that immunosuppression can prevent photoreceptor loss. These results offer insight into the pathways that promote MG-dependent regeneration and the role of inflammation in photoreceptor degeneration.
Subject(s)
Retinal Degeneration , Retinal Dystrophies , Animals , Animals, Genetically Modified , Cell Proliferation , Female , Immunosuppression Therapy , Inflammation/metabolism , Male , Mammals , Regeneration/physiology , Retina/physiology , Retinal Cone Photoreceptor Cells/physiology , Retinal Degeneration/pathology , Retinal Dystrophies/metabolism , Zebrafish , Zebrafish Proteins/metabolismABSTRACT
Inherited retinal dystrophies are a group of disorders characterized by the progressive degeneration of photoreceptors leading to loss of the visual function and eventually to legal blindness. Although next generation sequencing (NGS) has revolutionized the molecular diagnosis of these diseases, the pathogenicity of some mutations casts doubts. After the screening of 208 patients with a panel of 117 genes, we obtained 383 variants that were analysed in silico with bioinformatic prediction programs. Based on the results of these tools, we selected 15 variants for their functional assessment. Therefore, we carried out minigene assays to unveil whether they could affect the splicing of the corresponding gene. As a whole, seven variants were found to induce aberrant splicing in the following genes: BEST1, CACNA2D4, PRCD, RIMS1, FSCN2, MERTK and MAK. This study shows the efficacy of a workflow, based on the association of the Minimum Allele Frequency, family co-segregation, in silico predictions and in vitro assays to determine the effect of potential splice site variants identified by DNA-based NGS. These findings improve the molecular diagnosis of inherited retinal dystrophies and will allow some patients to benefit from the upcoming gene-based therapeutic strategies.
Subject(s)
Mutation , RNA Splicing , Retinal Dystrophies/genetics , Computational Biology , DNA Mutational Analysis , Gene Frequency , Genetic Predisposition to Disease , Heredity , High-Throughput Nucleotide Sequencing , Humans , Models, Genetic , Retinal Dystrophies/diagnosis , Retinal Dystrophies/metabolismABSTRACT
Pathogenic variants in CRB1 lead to diverse recessive retinal disorders from severe Leber congenital amaurosis to isolated macular dystrophy. Until recently, no clear phenotype-genotype correlation and no appropriate mouse models existed. Herein, we reappraise the phenotype-genotype correlation of 50 patients with regards to the recently identified CRB1 isoforms: a canonical long isoform A localized in Müller cells (12 exons) and a short isoform B predominant in photoreceptors (7 exons). Twenty-eight patients with early onset retinal dystrophy (EORD) consistently had a severe Müller impairment, with variable impact on the photoreceptors, regardless of isoform B expression. Among them, two patients expressing wild type isoform B carried one variant in exon 12, which specifically damaged intracellular protein interactions in Müller cells. Thirteen retinitis pigmentosa patients had mainly missense variants in laminin G-like domains and expressed at least 50% of isoform A. Eight patients with the c.498_506del variant had macular dystrophy. In one family homozygous for the c.1562C>T variant, the brother had EORD and the sister macular dystrophy. In contrast with the mouse model, these data highlight the key role of Müller cells in the severity of CRB1-related dystrophies in humans, which should be taken into consideration for future clinical trials.
Subject(s)
Ependymoglial Cells/pathology , Eye Proteins/genetics , Eye Proteins/metabolism , Macular Degeneration/pathology , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mutation , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Retinal Dystrophies/pathology , Retinitis Pigmentosa/pathology , Adolescent , Age of Onset , Alternative Splicing , Child , Child, Preschool , Ependymoglial Cells/metabolism , Eye Proteins/chemistry , Female , Genetic Association Studies , Humans , Infant , Macular Degeneration/genetics , Macular Degeneration/metabolism , Male , Membrane Proteins/chemistry , Models, Molecular , Mutation, Missense , Nerve Tissue Proteins/chemistry , Point Mutation , Retinal Dystrophies/genetics , Retinal Dystrophies/metabolism , Retinitis Pigmentosa/genetics , Retinitis Pigmentosa/metabolism , Retrospective Studies , Sequence Deletion , Young AdultABSTRACT
INTRODUCTION: Biallelic pathogenic RPE65 variants are related to a spectrum of clinically overlapping inherited retinal dystrophies (IRD). Most affected individuals progress to severe disease, with 50% of patients becoming legally blind by 20 years of age. Deeper knowledge of the mutational spectrum and the phenotype-genotype correlation in RPE65-related IRD is needed. PATIENTS AND METHODS: Forty-five affected subjects from 27 unrelated families with a clinical diagnosis of RPE65-related IRD were included. Clinical evaluation consisted of self-reported ophthalmological history and objective ophthalmological examination. Patients' genotype was classified according to variant class (truncating or missense) or to variant location at different protein domains. The main phenotypic outcome measure was age at onset (AAO) of symptomatic disease and a Kaplan-Meier analysis of disease symptom event-free survival was performed. RESULTS: Twenty-nine different RPE65 variants were identified in our cohort, 7 of them novel. Patients carrying two missense alleles showed a later disease onset than those with 1 or 2 truncating variants (log-rank test p <0.05). While 60% of patients carrying a missense/missense genotype presented symptoms before or during the first year of life, almost all patients with at least 1 truncating allele (91%) had an AAO ≤1 year (p <0.05). CONCLUSION: Our findings suggest an association between the type of RPE65 variant carried and AAO. These findings provide useful data on RPE65-associated IRD phenotypes and may help improve clinical and therapeutic management of these patients.
Subject(s)
DNA/genetics , Genetic Association Studies/methods , Mutation , Retinal Dystrophies/genetics , cis-trans-Isomerases/genetics , Adolescent , Alleles , Child , Child, Preschool , DNA Mutational Analysis , Electroretinography , Female , Genotype , Humans , Infant , Male , Pedigree , Phenotype , Retinal Dystrophies/diagnosis , Retinal Dystrophies/metabolism , Young Adult , cis-trans-Isomerases/metabolismABSTRACT
Purpose: The purpose of this study was to perform a detailed longitudinal phenotyping and genetic characterization of 32 Italian patients with a nonsyndromic retinal dystrophy and mutations in the CEP290 gene. Methods: We reviewed the clinical history and examinations of 32 patients with a nonsyndromic retinal dystrophy due to mutations in the CEP290 gene, followed up (mean follow-up: 5.9 years) at 3 Italian centers. The clinical examinations included: best corrected visual acuity (BCVA), optical coherence tomography (OCT), and full-field electroretinogram (ERG). Results: Patients (mean age = 19.0 ± 3.4 years) had a mean BCVA of 1.73 ± 0.20 logMAR. Longitudinal analysis of BCVA showed a nonsignificant decline. Central retinal thickness (CRT) declined significantly with age at an exponential rate of 1.0%/year (P = 0.001). At disease onset, most patients (19/32; 49.4%) had nystagmus. The absence of nystagmus was significantly associated with better BCVA and more preserved CRT (P < 0.05). ERG showed undetectable responses in most patients (64.0%), whereas reduced scotopic and photopic responses were observed in four patients (16.0%) who had no nystagmus. We identified 35 different variants, among which 12 were novel. Our genotype-phenotype correlation analysis shows a significantly worse BCVA in patients harboring a loss-of-function mutation and the deep-intronic variant c.2991+1655A>G. Conclusions: Our study highlights a mild phenotype of the disease, characterized by absence of nystagmus, good visual acuity, considerably preserved retinal morphology, and recordable ERG, confirming the wide spectrum of CEP290-related retinal dystrophies. Finally, in our cohort, the deep intronic variant c.2991+1655A>G was associated with a more severe phenotype.
Subject(s)
Antigens, Neoplasm/genetics , Cell Cycle Proteins/genetics , Cytoskeletal Proteins/genetics , DNA/genetics , Mutation , Retina/diagnostic imaging , Retinal Dystrophies/genetics , Tomography, Optical Coherence/methods , Adolescent , Antigens, Neoplasm/metabolism , Cell Cycle Proteins/metabolism , Child , Child, Preschool , Cytoskeletal Proteins/metabolism , DNA Mutational Analysis , Electroretinography , Female , Follow-Up Studies , Humans , Male , Pedigree , Phenotype , Retinal Dystrophies/diagnosis , Retinal Dystrophies/metabolism , Severity of Illness Index , Time Factors , Young AdultABSTRACT
Biallelic STX3 variants were previously reported in five individuals with the severe congenital enteropathy, microvillus inclusion disease (MVID). Here, we provide a significant extension of the phenotypic spectrum caused by STX3 variants. We report ten individuals of diverse geographic origin with biallelic STX3 loss-of-function variants, identified through exome sequencing, single-nucleotide polymorphism array-based homozygosity mapping, and international collaboration. The evaluated individuals all presented with MVID. Eight individuals also displayed early-onset severe retinal dystrophy, i.e., syndromic-intestinal and retinal-disease. These individuals harbored STX3 variants that affected both the retinal and intestinal STX3 transcripts, whereas STX3 variants affected only the intestinal transcript in individuals with solitary MVID. That STX3 is essential for retinal photoreceptor survival was confirmed by the creation of a rod photoreceptor-specific STX3 knockout mouse model which revealed a time-dependent reduction in the number of rod photoreceptors, thinning of the outer nuclear layer, and the eventual loss of both rod and cone photoreceptors. Together, our results provide a link between STX3 loss-of-function variants and a human retinal dystrophy. Depending on the genomic site of a human loss-of-function STX3 variant, it can cause MVID, the novel intestinal-retinal syndrome reported here or, hypothetically, an isolated retinal dystrophy.
Subject(s)
Eye Diseases, Hereditary/genetics , Intestinal Mucosa/metabolism , Malabsorption Syndromes/genetics , Microvilli/pathology , Mucolipidoses/genetics , Polymorphism, Single Nucleotide , Qa-SNARE Proteins/genetics , Retinal Cone Photoreceptor Cells/metabolism , Retinal Dystrophies/genetics , Aged , Aged, 80 and over , Alcohol Oxidoreductases/genetics , Alcohol Oxidoreductases/metabolism , Animals , Autopsy , Co-Repressor Proteins/genetics , Co-Repressor Proteins/metabolism , Eye Diseases, Hereditary/metabolism , Eye Diseases, Hereditary/pathology , Female , Gene Expression Regulation , Homozygote , Humans , Intestinal Mucosa/pathology , Malabsorption Syndromes/metabolism , Malabsorption Syndromes/pathology , Mice , Mice, Knockout , Microvilli/genetics , Microvilli/metabolism , Mucolipidoses/metabolism , Mucolipidoses/pathology , Phenotype , Qa-SNARE Proteins/deficiency , RNA, Messenger/genetics , RNA, Messenger/metabolism , Retinal Cone Photoreceptor Cells/pathology , Retinal Dystrophies/metabolism , Retinal Dystrophies/pathology , Sensory Rhodopsins/genetics , Sensory Rhodopsins/metabolism , Exome SequencingABSTRACT
The retina is a highly active metabolic organ that displays a particular vulnerability to genetic and environmental factors causing stress and homeostatic imbalance. Mitochondria constitute a bioenergetic hub that coordinates stress response and cellular homeostasis, therefore structural and functional regulation of the mitochondrial dynamic network is essential for the mammalian retina. CERKL (ceramide kinase like) is a retinal degeneration gene whose mutations cause Retinitis Pigmentosa in humans, a visual disorder characterized by photoreceptors neurodegeneration and progressive vision loss. CERKL produces multiple isoforms with a dynamic subcellular localization. Here we show that a pool of CERKL isoforms localizes at mitochondria in mouse retinal ganglion cells. The depletion of CERKL levels in CerklKD/KO(knockdown/knockout) mouse retinas cause increase of autophagy, mitochondrial fragmentation, alteration of mitochondrial distribution, and dysfunction of mitochondrial-dependent bioenergetics and metabolism. Our results support CERKL as a regulator of autophagy and mitochondrial biology in the mammalian retina.
Subject(s)
Mitochondria/metabolism , Phosphotransferases (Alcohol Group Acceptor)/deficiency , Retina/metabolism , Retinal Dystrophies/metabolism , Retinal Ganglion Cells/metabolism , Animals , Autophagy/physiology , Cells, Cultured , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Mitochondria/genetics , Mitochondria/ultrastructure , Phosphotransferases (Alcohol Group Acceptor)/genetics , Retina/ultrastructure , Retinal Dystrophies/genetics , Retinal Dystrophies/pathology , Retinal Ganglion Cells/ultrastructure , Retinitis Pigmentosa/genetics , Retinitis Pigmentosa/metabolism , Retinitis Pigmentosa/pathologyABSTRACT
PURPOSE: To determine whether functional vision and visual function improvements after voretigene neparvovec (VN; Luxturna [Spark Therapeutics, Inc]) administration in patients with biallelic RPE65 mutation-associated inherited retinal disease are maintained at 3 to 4 years and to review safety outcomes. DESIGN: Open-label, randomized, controlled phase 3 trial. PARTICIPANTS: Thirty-one individuals were enrolled and randomized 2:1 to intervention (n = 21) or control (n = 10). One participant from each group withdrew before, or at, randomization. METHODS: Patients in the original intervention (OI) group received bilateral subretinal VN injections. Delayed intervention (DI) patients served as control participants for 1 year then received VN. MAIN OUTCOME MEASURES: Change from injection baseline in bilateral performance on the multiluminance mobility test (MLMT), a measure of ambulatory navigation, and change from injection baseline in full-field light sensitivity threshold white light, visual field (VF), and visual acuity (VA). RESULTS: Mean bilateral MLMT change scores at year 4 for OI patients and year 3 for DI patients were 1.7 and 2.4, respectively, with 71% of patients with a year 3 visit able to pass MLMT at the lowest light level. Mean change in full-field light sensitivity threshold white light, averaged over both eyes at year 4 for OI patients and year 3 for DI patients, was -1.90 log10(cd.s/m2) and -2.91 log10(cd.s/m2), respectively. Mean change in Goldmann kinetic VF III4e sum total degrees, averaged across both eyes, was 197.7 at year 4 for OI patients and 157.9 at year 3 for DI patients. Mean change in VA (Holladay scale), averaged across both eyes, was -0.003 logarithm of the minimum angle of resolution (logMAR) at year 4 for OI patients and -0.06 logMAR at year 3 for DI patients. One OI patient experienced retinal detachment at approximately year 4 that impacted VA for the OI group. No product-related serious adverse events (AEs) occurred, nor did any deleterious immune responses. CONCLUSIONS: Improvements in ambulatory navigation, light sensitivity, and VF were consistent in both intervention groups. Overall, improvements were maintained up to 3 to 4 years, with ongoing observation. The safety profile of VN was consistent with vitrectomy and the subretinal injection procedure and was similar between intervention groups, with no product-related serious AEs reported.
Subject(s)
Genetic Therapy/methods , Genetic Vectors/administration & dosage , Mutation , Retinal Dystrophies/drug therapy , Visual Acuity , cis-trans-Isomerases/administration & dosage , Adolescent , Adult , Child , Child, Preschool , Female , Follow-Up Studies , Humans , Injections, Intraocular , Male , Retina , Retinal Dystrophies/genetics , Retinal Dystrophies/metabolism , Time Factors , Treatment Outcome , Visual Fields , Young Adult , cis-trans-Isomerases/genetics , cis-trans-Isomerases/metabolismABSTRACT
Peroxisomes play an essential role in lipid metabolism via interaction with other intracellular organelles. The information about the role of the Acyl-CoA-binding domain containing-protein 5 (ACBD5) in these interactions in human cells is emerging. Moreover, a few patients with retinal dystrophy and leukodystrophy caused by pathogenic variants in ACBD5 have been recently introduced. Here, we present a 36-year-old female with retinal dystrophy, leukodystrophy, and psychomotor regression due to a novel homozygous variant in ACBD5. Our study adds to the growing knowledge of this peroxisomal disorder by providing phenotypic details of the first adult patient.