RESUMEN
Myopia is the most common eye disorder, caused by heterogeneous genetic and environmental factors. Rare progressive and stationary inherited retinal disorders are often associated with high myopia. Genes implicated in myopia encode proteins involved in a variety of biological processes including eye morphogenesis, extracellular matrix organization, visual perception, circadian rhythms, and retinal signaling. Differentially expressed genes (DEGs) identified in animal models mimicking myopia are helpful in suggesting candidate genes implicated in human myopia. Complete congenital stationary night blindness (cCSNB) in humans and animal models represents an ON-bipolar cell signal transmission defect and is also associated with high myopia. Thus, it represents also an interesting model to identify myopia-related genes, as well as disease mechanisms. While the origin of night blindness is molecularly well established, further research is needed to elucidate the mechanisms of myopia development in subjects with cCSNB. Using whole transcriptome analysis on three different mouse models of cCSNB (in Gpr179-/-, Lrit3-/- and Grm6-/-), we identified novel actors of the retinal signaling cascade, which are also novel candidate genes for myopia. Meta-analysis of our transcriptomic data with published transcriptomic databases and genome-wide association studies from myopia cases led us to propose new biological/cellular processes/mechanisms potentially at the origin of myopia in cCSNB subjects. The results provide a foundation to guide the development of pharmacological myopia therapies.
Asunto(s)
Enfermedades Hereditarias del Ojo , Enfermedades Genéticas Ligadas al Cromosoma X , Miopía , Ceguera Nocturna , Animales , Ratones , Humanos , Ceguera Nocturna/genética , Estudio de Asociación del Genoma Completo , Electrorretinografía/métodos , Mutación , Enfermedades Hereditarias del Ojo/genética , Enfermedades Hereditarias del Ojo/metabolismo , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Enfermedades Genéticas Ligadas al Cromosoma X/metabolismo , Miopía/genética , Proteínas de la Membrana/genéticaRESUMEN
Mutations in GPR179 are one of the most common causes of autosomal recessive complete congenital stationary night blindness (cCSNB). This retinal disease is characterized in patients by impaired dim and night vision, associated with other ocular symptoms, including high myopia. cCSNB is caused by a complete loss of signal transmission from photoreceptors to ON-bipolar cells. In this study, we hypothesized that the lack of Gpr179 and the subsequent impaired ON-pathway could lead to myopic features in a mouse model of cCSNB. Using ultra performance liquid chromatography, we show that adult Gpr179-/- mice have a significant decrease in both retinal dopamine and 3,4-dihydroxyphenylacetic acid, compared to Gpr179+/+ mice. This alteration of the dopaminergic system is thought to be correlated with an increased susceptibility to lens-induced myopia but does not affect the natural refractive development. Altogether, our data added a novel myopia model, which could be used to identify therapeutic interventions.
Asunto(s)
Enfermedades Genéticas Ligadas al Cromosoma X , Miopía , Ceguera Nocturna , Ratones , Animales , Electrorretinografía/métodos , Ceguera Nocturna/genética , Retina , Miopía/genética , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Receptores Acoplados a Proteínas G/genéticaRESUMEN
The purpose of this work was to identify the gene defect underlying a relatively mild rod-cone dystrophy (RCD), lacking disease-causing variants in known genes implicated in inherited retinal disorders (IRD), and provide transcriptomic and immunolocalization data to highlight the best candidate. The DNA of the female patient originating from a consanguineous family revealed no large duplication or deletion, but several large homozygous regions. In one of these, a homozygous frameshift variant, c.244_246delins17 p.(Trp82Valfs*4); predicted to lead to a nonfunctional protein, was identified in CCDC51. CCDC51 encodes the mitochondrial coiled-coil domain containing 51 protein, also called MITOK. MITOK ablation causes mitochondrial dysfunction. Here we show for the first time that CCDC51/MITOK localizes in the retina and more specifically in the inner segments of the photoreceptors, well known to contain mitochondria. Mitochondrial proteins have previously been implicated in IRD, although usually in association with syndromic disease, unlike our present case. Together, our findings add another ultra-rare mutation implicated in non-syndromic IRD, whose pathogenic mechanism in the retina needs to be further elucidated.
Asunto(s)
Distrofias de Conos y Bastones/patología , Genes Recesivos , Proteínas Mitocondriales/genética , Mutación , Canales de Potasio/genética , Adulto , Distrofias de Conos y Bastones/etiología , Distrofias de Conos y Bastones/metabolismo , Femenino , Humanos , Masculino , Linaje , FenotipoRESUMEN
Complete congenital stationary night blindness (cCSNB) due to mutations in TRPM1, GRM6, GPR179, NYX, or leucine-rich repeat immunoglobulin-like transmembrane domain 3 (LRIT3) is an incurable inherited retinal disorder characterized by an ON-bipolar cell (ON-BC) defect. Since the disease is non-degenerative and stable, treatment could theoretically be administrated at any time in life, making it a promising target for gene therapy. Until now, adeno-associated virus (AAV)-mediated therapies lead to significant functional improvements only in newborn cCSNB mice. Here we aimed to restore protein localization and function in adult Lrit3 -/ - mice. LRIT3 localizes in the outer plexiform layer and is crucial for TRPM1 localization at the dendritic tips of ON-BCs and the electroretinogram (ERG)-b-wave. AAV2-7m8-Lrit3 intravitreal injections were performed targeting either ON-BCs, photoreceptors (PRs), or both. Protein localization of LRIT3 and TRPM1 at the rod-to-rod BC synapse, functional rescue of scotopic responses, and ON-responses detection at the ganglion cell level were achieved in a few mice when ON-BCs alone or both PRs and ON-BCs, were targeted. More importantly, a significant number of treated adult Lrit3 -/- mice revealed an ERG b-wave recovery under scotopic conditions, improved optomotor responses, and on-time ON-responses at the ganglion cell level when PRs were targeted. Functional rescue was maintained for at least 4 months after treatment.
RESUMEN
Mutations in GPR179 lead to autosomal recessive complete congenital stationary night blindness (cCSNB). This condition represents a signal transmission defect from the photoreceptors to the ON-bipolar cells. To confirm the phenotype, better understand the pathogenic mechanism in vivo, and provide a model for therapeutic approaches, a Gpr179 knock-out mouse model was genetically and functionally characterized. We confirmed that the insertion of a neo/lac Z cassette in intron 1 of Gpr179 disrupts the same gene. Spectral domain optical coherence tomography reveals no obvious retinal structure abnormalities. Gpr179 knock-out mice exhibit a so-called no-b-wave (nob) phenotype with severely reduced b-wave amplitudes in the electroretinogram. Optomotor tests reveal decreased optomotor responses under scotopic conditions. Consistent with the genetic disruption of Gpr179, GPR179 is absent at the dendritic tips of ON-bipolar cells. While proteins of the same signal transmission cascade (GRM6, LRIT3, and TRPM1) are correctly localized, other proteins (RGS7, RGS11, and GNB5) known to regulate GRM6 are absent at the dendritic tips of ON-bipolar cells. These results add a new model of cCSNB, which is important to better understand the role of GPR179, its implication in patients with cCSNB, and its use for the development of therapies.
Asunto(s)
Modelos Animales de Enfermedad , Enfermedades Hereditarias del Ojo/genética , Enfermedades Hereditarias del Ojo/patología , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Enfermedades Genéticas Ligadas al Cromosoma X/patología , Miopía/genética , Miopía/patología , Ceguera Nocturna/genética , Ceguera Nocturna/patología , Receptores Acoplados a Proteínas G/fisiología , Retina/patología , Animales , Femenino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Fenotipo , Retina/metabolismo , Transducción de SeñalRESUMEN
Purpose: Complete congenital stationary night blindness (cCSNB) is an incurable inherited retinal disorder characterized by an ON-bipolar cell (ON-BC) defect. GRM6 mutations are the third most prevalent cause of cCSNB. The Grm6-/- mouse model mimics the human phenotype, showing no b-wave in the electroretinogram (ERG) and a loss of mGluR6 and other proteins of the same cascade at the outer plexiform layer (OPL). Our aim was to restore protein localization and function in Grm6-/- adult mice targeting specifically ON-BCs or the whole retina. Methods: Adeno-associated virus-encoding Grm6 under two different promoters (GRM6-Grm6 and CAG-Grm6) were injected intravitreally in P15 Grm6-/- mice. ERG recordings at 2 and 4 months were performed in Grm6+/+, untreated and treated Grm6-/- mice. Similarly, immunolocalization studies were performed on retinal slices before or after treatment using antibodies against mGluR6, TRPM1, GPR179, RGS7, RGS11, Gß5, and dystrophin. Results: Following treatment, mGluR6 was localized to the dendritic tips of ON-BCs when expressed with either promoter. The relocalization efficiency in mGluR6-transduced retinas at the OPL was 2.5% versus 11% when the GRM6-Grm6 and CAG-Grm6 were used, respectively. Albeit no functional rescue was seen in ERGs, relocalization of TRPM1, GPR179, and Gß5 was also noted using both constructs. The restoration of the localization of RGS7, RGS11, and dystrophin was more obvious in retinas treated with GRM6-Grm6 than in retinas treated with CAG-Grm6. Conclusions: Our findings show the potential of treating cCSNB with GRM6 mutations; however, it appears that the transduction rate must be improved to restore visual function.
Asunto(s)
Dependovirus/genética , Modelos Animales de Enfermedad , Enfermedades Hereditarias del Ojo/metabolismo , Técnicas de Transferencia de Gen , Enfermedades Genéticas Ligadas al Cromosoma X/metabolismo , Miopía/metabolismo , Ceguera Nocturna/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Células Bipolares de la Retina/metabolismo , Animales , Electrorretinografía , Enfermedades Hereditarias del Ojo/fisiopatología , Enfermedades Genéticas Ligadas al Cromosoma X/fisiopatología , Vectores Genéticos , Inyecciones Intravítreas , Ratones , Ratones Endogámicos C57BL , Miopía/fisiopatología , Ceguera Nocturna/fisiopatología , Regiones Promotoras Genéticas , Receptores de Glutamato Metabotrópico/genética , Retina/fisiopatología , TransfecciónRESUMEN
PURPOSE: To reappraise the presentation and the course of ITM2B-related retinal dystrophy and give further insights into ITM2B expression in the retina. METHODS: The clinical data of nine subjects with ITM2B-related retinal dystrophy were retrospectively reviewed. The genetic mutation was assessed for its influence on splicing in cultured fibroblasts. The cellular expression of ITM2B within the inner retina was investigated in wild-type mice through mRNA in situ hybridization. RESULTS: All patients complained of decreased vision and mild photophobia around their twenties-thirties. The peculiar feature was the hyperreflective material on optical coherence tomography within the inner retina and the central outer nuclear layer with thinning of the retinal nerve fiber layer. Although retinal imaging revealed very mild or no changes over the years, the visual acuity slowly decreased with about one Early Treatment Diabetic Retinopathy Study letter per year. Finally, full-field electroretinography showed a mildly progressive inner retinal and cone dysfunction. ITM2B mRNA is expressed in all cellular types of the inner retina. Disease mechanism most likely involves mutant protein misfolding and/or modified protein interaction rather than misplicing. CONCLUSION: ITM2B-related retinal dystrophy is a peculiar, rare, slowly progressive retinal degeneration. Functional examinations (full-field electroretinography and visual acuity) seem more accurate in monitoring the progression in these patients because imaging tends to be stable over the years.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Distrofias Retinianas/genética , Anciano , Animales , Modelos Animales de Enfermedad , Electrorretinografía , Femenino , Regulación de la Expresión Génica/fisiología , Humanos , Hibridación in Situ , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Imagen Óptica , Fenotipo , ARN Mensajero/genética , Retina/fisiopatología , Distrofias Retinianas/diagnóstico por imagen , Distrofias Retinianas/fisiopatología , Estudios Retrospectivos , Tomografía de Coherencia Óptica , Agudeza Visual/fisiologíaRESUMEN
Rod-cone dystrophy (RCD), also called retinitis pigmentosa, is characterized by rod followed by cone photoreceptor degeneration, leading to gradual visual loss. Mutations in over 65 genes have been associated with non-syndromic RCD explaining 60% to 70% of cases, with novel gene defects possibly accounting for the unsolved cases. Homozygosity mapping and whole-exome sequencing applied to a case of autosomal recessive non-syndromic RCD from a consanguineous union identified a homozygous variant in WDR34. Mutations in WDR34 have been previously associated with severe ciliopathy syndromes possibly associated with a retinal dystrophy. This is the first report of a homozygous mutation in WDR34 associated with non-syndromic RCD.
Asunto(s)
Proteínas Portadoras/genética , Distrofias de Conos y Bastones/genética , Adulto , Estudios de Asociación Genética , Humanos , Masculino , Linaje , Repeticiones WD40RESUMEN
Melanoma-associated retinopathy (MAR) is a rare paraneoplastic retinal disorder usually occurring in the context of metastatic melanoma. Patients present with night blindness, photopsias and a constriction of the visual field. MAR is an auto-immune disorder characterized by the production of autoantibodies targeting retinal proteins, especially autoantibodies reacting to the cation channel TRPM1 produced in melanocytes and ON-bipolar cells. TRPM1 has at least three different isoforms which vary in the N-terminal region of the protein. In this study, we report the case of three new MAR patients presenting different anti-TRPM1 autoantibodies reacting to the three isoforms of TRPM1 with variable binding affinity. Two sera recognized all isoforms of TRPM1, while one recognized only the two longest isoforms upon immunolocalization studies on overexpressing cells. Similarly, the former two sera reacted with all TRPM1 isoforms on western blot, but an immunoprecipitation enrichment step was necessary to detect all isoforms with the latter serum. In contrast, all sera labelled ON-bipolar cells on Tprm1+/+ but not on Trpm1-/- mouse retina as shown by co-immunolocalization. This confirms that the MAR sera specifically detect TRPM1. Most likely, the anti-TRPM1 autoantibodies of different patients vary in affinity and concentration. In addition, the binding of autoantibodies to TRPM1 may be conformation-dependent, with epitopes being inaccessible in some constructs (truncated polypeptides versus full-length TRPM1) or applications (western blotting versus immunohistochemistry). Therefore, we propose that a combination of different methods should be used to test for the presence of anti-TRPM1 autoantibodies in the sera of MAR patients.
Asunto(s)
Autoanticuerpos/sangre , Melanoma/inmunología , Síndromes Paraneoplásicos Oculares/inmunología , Retina/inmunología , Enfermedades de la Retina/inmunología , Canales Catiónicos TRPM/inmunología , Anciano , Animales , Células COS , Chlorocebus aethiops , Femenino , Humanos , Masculino , Melanoma/patología , Persona de Mediana Edad , Retina/patologíaRESUMEN
Human induced pluripotent stem cell (iPSC) lines were generated from fibroblasts of a patient affected with an autosomal dominant retinal dystrophy carrying the mutation c.782A>C, p.Glu261Ala in ITM2B and from an unaffected brother. Three different iPSC lines were generated and characterized from primary dermal fibroblasts of the affected subject and two from the unaffected brother. All iPSC lines expressed the pluripotency markers, were able to differentiate into the three germ layers and presented normal karyotypes. This cellular model will provide a powerful tool to study this retinal dystrophy and better understand the role of ITM2B.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Técnicas de Cultivo de Célula/métodos , Línea Celular/patología , Células Madre Pluripotentes Inducidas/patología , Mutación/genética , Distrofias Retinianas/genética , Distrofias Retinianas/patología , Hermanos , Secuencia de Bases , Humanos , Masculino , Persona de Mediana Edad , Reproducibilidad de los ResultadosRESUMEN
Phenotypes observed in a large cohort of patients with cone and cone-rod dystrophies (COD/CORDs) are described based on multimodal retinal imaging features in order to help in analyzing massive next-generation sequencing data. Structural abnormalities of 58 subjects with molecular diagnosis of COD/CORDs were analyzed through specific retinal imaging including spectral-domain optical coherence tomography (SD-OCT) and fundus autofluorescence (BAF/IRAF). Findings were analyzed with the underlying genetic defects. A ring of increased autofluorescence was mainly observed in patients with CRX and GUCY2D mutations (33% and 22% of cases respectively). "Speckled" autofluorescence was observed with mutations in three different genes (ABCA4 64%; C2Orf71 and PRPH2, 18% each). Peripapillary sparing was only found in association with mutations in ABCA4, although only present in 40% of such genotypes. Regarding SD-OCT, specific outer retinal abnormalities were more commonly observed in particular genotypes: focal retrofoveal interruption and GUCY2D mutations (50%), foveal sparing and CRX mutations (50%), and outer retinal atrophy associated with hyperreflective dots and ABCA4 mutations (69%). This study outlines the phenotypic heterogeneity of COD/CORDs hampering statistical correlations. A larger study correlating retinal imaging with genetic results is necessary to identify specific clinical features that may help in selecting pathogenic variants generated by high-throughput sequencing.
Asunto(s)
Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Fenotipo , Distrofias Retinianas/diagnóstico , Distrofias Retinianas/genética , Adolescente , Adulto , Alelos , Biomarcadores , Niño , Preescolar , Distrofias de Conos y Bastones/diagnóstico , Distrofias de Conos y Bastones/genética , Electrorretinografía , Femenino , Fondo de Ojo , Estudios de Asociación Genética/métodos , Genotipo , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Lactante , Masculino , Persona de Mediana Edad , Mutación , Células Fotorreceptoras Retinianas Conos/metabolismo , Tomografía de Coherencia Óptica , Adulto JovenAsunto(s)
Enfermedades Hereditarias del Ojo/genética , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Mutación Missense , Miopía/genética , Ceguera Nocturna/genética , Receptores de Glutamato/genética , Adulto , Consanguinidad , Electrorretinografía , Exones/genética , Enfermedades Hereditarias del Ojo/diagnóstico , Enfermedades Hereditarias del Ojo/fisiopatología , Femenino , Estudios de Seguimiento , Enfermedades Genéticas Ligadas al Cromosoma X/diagnóstico , Enfermedades Genéticas Ligadas al Cromosoma X/fisiopatología , Humanos , Masculino , Miopía/diagnóstico , Miopía/fisiopatología , Ceguera Nocturna/diagnóstico , Ceguera Nocturna/fisiopatología , Linaje , Retina/fisiopatologíaRESUMEN
Inherited retinal disorders (IRD) represent clinically and genetically heterogeneous diseases. To date, pathogenic variants have been identified in ~260 genes. Albeit that many genes are implicated in IRD, for 30-50% of the cases, the gene defect is unknown. These cases may be explained by novel gene defects, by overlooked structural variants, by variants in intronic, promoter or more distant regulatory regions, and represent synonymous variants of known genes contributing to the dysfunction of the respective proteins. Patients with one subgroup of IRD, namely incomplete congenital stationary night blindness (icCSNB), show a very specific phenotype. The major cause of this condition is the presence of a hemizygous pathogenic variant in CACNA1F. A comprehensive study applying direct Sanger sequencing of the gene-coding regions, exome and genome sequencing applied to a large cohort of patients with a clinical diagnosis of icCSNB revealed indeed that seven of the 189 CACNA1F-related cases have intronic and synonymous disease-causing variants leading to missplicing as validated by minigene approaches. These findings highlight that gene-locus sequencing may be a very efficient method in detecting disease-causing variants in clinically well-characterized patients with a diagnosis of IRD, like icCSNB.
Asunto(s)
Canales de Calcio Tipo L/genética , Enfermedades Hereditarias del Ojo/genética , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Mutación , Miopía/genética , Ceguera Nocturna/genética , Análisis de Secuencia de ADN/métodos , Predisposición Genética a la Enfermedad , Hemicigoto , Humanos , Intrones , Masculino , Linaje , Empalme del ARN , Mutación SilenciosaRESUMEN
Autosomal dominant congenital stationary night blindness (adCSNB) is rare and results from altered phototransduction giving a Riggs type of electroretinogram (ERG) with loss of the rod a-wave and small b-waves. These patients usually have normal vision in light. Only few mutations in genes coding for proteins of the phototransduction cascade lead to this condition; most of these gene defects cause progressive rod-cone dystrophy. Mutation analysis of an adCSNB family with a Riggs-type ERG revealed a novel variant (c.155T>A p.Ile52Asn) in GNAT1 coding for the α-subunit of transducin, cosegregating with the phenotype. Domain predictions and 3D-modelling suggest that the variant does not affect the GTP-binding site as other GNAT1 adCSNB mutations do. It affects a predicted nuclear localization signal and a part of the first α-helix, which is distant from the GTP-binding site. The subcellular protein localization of this and other mutant GNAT1 proteins implicated in CSNB are unaltered in mammalian GNAT1 overexpressing cells. Our findings add a third GNAT1 mutation causing adCSNB and suggest that different pathogenic mechanisms may cause this condition.
Asunto(s)
Enfermedades Hereditarias del Ojo/genética , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Proteínas de Unión al GTP Heterotriméricas/genética , Mutación Missense/genética , Miopía/genética , Ceguera Nocturna/genética , Adulto , Femenino , Proteínas de Unión al GTP Heterotriméricas/química , Heterocigoto , Humanos , Masculino , Transducina , Adulto JovenRESUMEN
We identified herein additional patients with rod-cone dystrophy (RCD) displaying mutations in KIZ, encoding the ciliary centrosomal protein kizuna and performed functional characterization of the respective protein in human fibroblasts and of its mouse ortholog PLK1S1 in the retina. Mutation screening was done by targeted next generation sequencing and subsequent Sanger sequencing validation. KIZ mRNA levels were assessed on blood and serum-deprived human fibroblasts from a control individual and a patient, compound heterozygous for the c.52G>T (p.Glu18*) and c.119_122del (p.Lys40Ilefs*14) mutations in KIZ. KIZ localization, documentation of cilium length and immunoblotting were performed in these two fibroblast cell lines. In addition, PLK1S1 immunolocalization was conducted in mouse retinal cryosections and isolated rod photoreceptors. Analyses of additional RCD patients enabled the identification of two homozygous mutations in KIZ, the known c.226C>T (p.Arg76*) mutation and a novel variant, the c.3G>A (p.Met1?) mutation. Albeit the expression levels of KIZ were three-times lower in the patient than controls in whole blood cells, further analyses in control- and mutant KIZ patient-derived fibroblasts unexpectedly revealed no significant difference between the two genotypes. Furthermore, the averaged monocilia length in the two fibroblast cell lines was similar, consistent with the preserved immunolocalization of KIZ at the basal body of the primary cilia. Analyses in mouse retina and isolated rod photoreceptors showed PLK1S1 localization at the base of the photoreceptor connecting cilium. In conclusion, two additional patients with mutations in KIZ were identified, further supporting that defects in KIZ/PLK1S1, detected at the basal body of the primary cilia in fibroblasts, and the photoreceptor connecting cilium in mouse, respectively, are involved in RCD. However, albeit the mutations were predicted to lead to nonsense mediated mRNA decay, we could not detect changes upon expression levels, protein localization or cilia length in KIZ-mutated fibroblast cells. Together, our findings unveil the limitations of fibroblasts as a cellular model for RCD and call for other models such as induced pluripotent stem cells to shed light on retinal pathogenic mechanisms of KIZ mutations.
RESUMEN
GNAT1, encoding the transducin subunit Gα, is an important element of the phototransduction cascade. Mutations in this gene have been associated with autosomal dominant and autosomal recessive congenital stationary night blindness. Recently, a homozygous truncating GNAT1 mutation was identified in a patient with late-onset rod-cone dystrophy. After exclusion of mutations in genes underlying progressive inherited retinal disorders, by targeted next generation sequencing, a 32 year-old male sporadic case with severe rod-cone dystrophy and his unaffected parents were investigated by whole exome sequencing. This led to the identification of a homozygous nonsense variant, c.963C>A p.(Cys321*) in GNAT1, which was confirmed by Sanger sequencing. The mother was heterozygous for this variant whereas the variant was absent in the father. c.963C>A p.(Cys321*) is predicted to produce a shorter protein that lacks critical sites for the phototransduction cascade. Our work confirms that the phenotype and the mode of inheritance associated with GNAT1 variants can vary from autosomal dominant, autosomal recessive congenital stationary night blindness to autosomal recessive rod-cone dystrophy.
Asunto(s)
Codón sin Sentido , Distrofias de Conos y Bastones/genética , Proteínas de Unión al GTP Heterotriméricas/genética , Adulto , Análisis Mutacional de ADN , Secuenciación de Nucleótidos de Alto Rendimiento , Homocigoto , Humanos , Masculino , Retinitis Pigmentosa/genética , TransducinaRESUMEN
Congenital stationary night blindness (CSNB) is a heterogeneous group of non-progressive inherited retinal disorders with characteristic electroretinogram (ERG) abnormalities. Riggs and Schubert-Bornschein are subtypes of CSNB and demonstrate distinct ERG features. Riggs CSNB demonstrates selective rod photoreceptor dysfunction and occurs due to mutations in genes encoding proteins involved in rod phototransduction cascade; night blindness is the only symptom and eye examination is otherwise normal. Schubert-Bornschein CSNB is a consequence of impaired signal transmission between the photoreceptors and bipolar cells. Schubert-Bornschein CSNB is subdivided into complete CSNB with an ON bipolar signaling defect and incomplete CSNB with both ON and OFF pathway involvement. Both subtypes are associated with variable degrees of night blindness or photophobia, reduced visual acuity, high myopia, and nystagmus. Whole-exome sequencing of a family screened negative for mutations in genes associated with CSNB identified biallelic mutations in the guanine nucleotide-binding protein subunit beta-3 gene (GNB3). Two siblings were compound heterozygous for a deletion (c.170_172delAGA [p.Lys57del]) and a nonsense mutation (c.1017G>A [p.Trp339(∗)]). The maternal aunt was homozygous for the nonsense mutation (c.1017G>A [p.Trp339(∗)]). Mutational analysis of GNB3 in a cohort of 58 subjects with CSNB identified a sporadic case individual with a homozygous GNB3 mutation (c.200C>T [p.Ser67Phe]). GNB3 encodes the ß subunit of G protein heterotrimer (Gαßγ) and is known to modulate ON bipolar cell signaling and cone transducin function in mice. Affected human subjects showed an unusual CSNB phenotype with variable degrees of ON bipolar dysfunction and reduced cone sensitivity. This unique retinal disorder with dual anomaly in visual processing expands our knowledge about retinal signaling.
Asunto(s)
Enfermedades Hereditarias del Ojo/etiología , Genes Recesivos/genética , Enfermedades Genéticas Ligadas al Cromosoma X/etiología , Proteínas de Unión al GTP Heterotriméricas/genética , Mutación/genética , Miopía/etiología , Ceguera Nocturna/etiología , Alelos , Secuencia de Aminoácidos , Animales , Estudios de Casos y Controles , Electrorretinografía , Enfermedades Hereditarias del Ojo/patología , Femenino , Enfermedades Genéticas Ligadas al Cromosoma X/patología , Genotipo , Proteínas de Unión al GTP Heterotriméricas/química , Homocigoto , Humanos , Masculino , Ratones , Persona de Mediana Edad , Miopía/patología , Ceguera Nocturna/patología , Linaje , Fenotipo , Conformación Proteica , Homología de Secuencia de Aminoácido , Agudeza Visual/genéticaRESUMEN
BACKGROUND: Cone and cone-rod dystrophies are clinically and genetically heterogeneous inherited retinal disorders with predominant cone impairment. They should be distinguished from the more common group of rod-cone dystrophies (retinitis pigmentosa) due to their more severe visual prognosis with early central vision loss. The purpose of our study was to document mutation spectrum of a large French cohort of cone and cone-rod dystrophies. METHODS: We applied Next-Generation Sequencing targeting a panel of 123 genes implicated in retinal diseases to 96 patients. A systematic filtering approach was used to identify likely disease causing variants, subsequently confirmed by Sanger sequencing and co-segregation analysis when possible. RESULTS: Overall, the likely causative mutations were detected in 62.1 % of cases, revealing 33 known and 35 novel mutations. This rate was higher for autosomal dominant (100 %) than autosomal recessive cases (53.8 %). Mutations in ABCA4 and GUCY2D were responsible for 19.2 % and 29.4 % of resolved cases with recessive and dominant inheritance, respectively. Furthermore, unexpected genotype-phenotype correlations were identified, confirming the complexity of inherited retinal disorders with phenotypic overlap between cone-rod dystrophies and other retinal diseases. CONCLUSIONS: In summary, this time-efficient approach allowed mutation detection in the most important cohort of cone-rod dystrophies investigated so far covering the largest number of genes. Association of known gene defects with novel phenotypes and mode of inheritance were established.
Asunto(s)
Genotipo , Secuenciación de Nucleótidos de Alto Rendimiento/estadística & datos numéricos , Mutación , Fenotipo , Retinitis Pigmentosa/genética , Estudios de Cohortes , Variaciones en el Número de Copia de ADN , Femenino , Francia , Humanos , Masculino , LinajeRESUMEN
Rod-cone dystrophy, also known as retinitis pigmentosa (RP), is the most common inherited degenerative photoreceptor disease, for which no therapy is currently available. The P23H rat is one of the most commonly used autosomal dominant RP models. It has been created by incorporation of a mutated mouse rhodopsin (Rho) transgene in the wild-type (WT) Sprague Dawley rat. Detailed genetic characterization of this transgenic animal has however never been fully reported. Here we filled this knowledge gap on P23H Line 1 rat (P23H-1) and provide additional phenotypic information applying non-invasive and state-of-the-art in vivo techniques that are relevant for preclinical therapeutic evaluations. Transgene sequence was analyzed by Sanger sequencing. Using quantitative PCR, transgene copy number was calculated and its expression measured in retinal tissue. Full field electroretinography (ERG) and spectral domain optical coherence tomography (SD-OCT) were performed at 1-, 2-, 3- and 6-months of age. Sanger sequencing revealed that P23H-1 rat carries the mutated mouse genomic Rho sequence from the promoter to the 3' UTR. Transgene copy numbers were estimated at 9 and 18 copies in the hemizygous and homozygous rats respectively. In 1-month-old hemizygous P23H-1 rats, transgene expression represented 43% of all Rho expressed alleles. ERG showed a progressive rod-cone dysfunction peaking at 6 months-of-age. SD-OCT confirmed a progressive thinning of the photoreceptor cell layer leading to the disappearance of the outer retina by 6 months with additional morphological changes in the inner retinal cell layers in hemizygous P23H-1 rats. These results provide precise genotypic information of the P23H-1 rat with additional phenotypic characterization that will serve basis for therapeutic interventions, especially for those aiming at gene editing.
Asunto(s)
Retinitis Pigmentosa/genética , Rodopsina/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Visión de Colores , Modelos Animales de Enfermedad , Electrorretinografía , Dosificación de Gen , Hemicigoto , Datos de Secuencia Molecular , Mutación/genética , Fenotipo , Ratas Sprague-Dawley , Ratas Transgénicas , Retina/patología , Rodopsina/química , Análisis de Secuencia de ADN , Tomografía de Coherencia Óptica , TransgenesRESUMEN
Mutations in LRIT3 lead to complete congenital stationary night blindness (cCSNB). The exact role of LRIT3 in ON-bipolar cell signaling cascade remains to be elucidated. Recently, we have characterized a novel mouse model lacking Lrit3 [no b-wave 6, (Lrit3(nob6/nob6) )], which displays similar abnormalities to patients with cCSNB with LRIT3 mutations. Here we compare the localization of components of the ON-bipolar cell signaling cascade in wild-type and Lrit3(nob6/nob6) retinal sections by immunofluorescence confocal microscopy. An anti-LRIT3 antibody was generated. Immunofluorescent staining of LRIT3 in wild-type mice revealed a specific punctate labeling in the outer plexiform layer (OPL), which was absent in Lrit3(nob6/nob6) mice. LRIT3 did not co-localize with ribeye or calbindin but co-localized with mGluR6. TRPM1 staining was severely decreased at the dendritic tips of all depolarizing bipolar cells in Lrit3(nob6/nob6) mice. mGluR6, GPR179, RGS7, RGS11 and Gß5 immunofluorescence was absent at the dendritic tips of cone ON-bipolar cells in Lrit3(nob6/nob6) mice, while it was present at the dendritic tips of rod bipolar cells. Furthermore, peanut agglutinin (PNA) labeling was severely reduced in the OPL in Lrit3(nob6/nob6) mice. This study confirmed the localization of LRIT3 at the dendritic tips of depolarizing bipolar cells in mouse retina and demonstrated the dependence of TRPM1 localization on the presence of LRIT3. As tested components of the ON-bipolar cell signaling cascade and PNA revealed disrupted localization, an additional function of LRIT3 in cone synapse formation is suggested. These results point to a possibly different regulation of the mGluR6 signaling cascade between rod and cone ON-bipolar cells.