ABSTRACT
Inherited retinal diseases (IRDs) are a heterogeneous group of blinding disorders, which result in dysfunction or death of the light-sensing cone and rod photoreceptors. Despite individual IRDs (Inherited retinal disease) being rare, collectively, they affect up to 1:2000 people worldwide, causing a significant socioeconomic burden, especially when cone-mediated central vision is affected. This study uses the Pde6ccpfl1 mouse model of achromatopsia, a cone-specific vision loss IRD (Inherited retinal disease), to investigate the potential gene-independent therapeutic benefits of a histone demethylase inhibitor GSK-J4 on cone cell survival. We investigated the effects of GSK-J4 treatment on cone cell survival in vivo and ex vivo and changes in cone-specific gene expression via single-cell RNA sequencing. A single intravitreal GSK-J4 injection led to transcriptional changes in pathways involved in mitochondrial dysfunction, endoplasmic reticulum stress, among other key epigenetic pathways, highlighting the complex interplay between methylation and acetylation in healthy and diseased cones. Furthermore, continuous administration of GSK-J4 in retinal explants increased cone survival. Our results suggest that IRD (Inherited retinal disease)-affected cones respond positively to epigenetic modulation of histones, indicating the potential of this approach in developing a broad class of novel therapies to slow cone degeneration.
Subject(s)
Color Vision Defects , Cone Dystrophy , Animals , Color Vision Defects/metabolism , Cone Dystrophy/metabolism , Disease Models, Animal , Histones/metabolism , Humans , Mice , Retinal Cone Photoreceptor Cells/metabolismABSTRACT
Certain combinations of common variants in exon 3 of OPN1LW and OPN1MW, the genes encoding the apo-protein of the long- and middle-wavelength sensitive cone photoreceptor visual pigments in humans, induce splicing defects and have been associated with dyschromatopsia and cone dysfunction syndromes. Here we report the identification of a novel exon 3 haplotype, G-C-G-A-T-T-G-G (referring to nucleotide variants at cDNA positions c.453, c.457, c.465, c.511, c.513, c.521, c.532, and c.538) deduced to encode a pigment with the amino acid residues L-I-V-V-A at positions p.153, p.171, p.174, p.178, and p.180, in OPN1LW or OPN1MW or both in a series of seven patients from four families with cone dysfunction. Applying minigene assays for all observed exon 3 haplotypes in the patients, we demonstrated that the novel exon 3 haplotype L-I-V-V-A induces a strong but incomplete splicing defect with 3-5% of residual correctly spliced transcripts. Minigene splicing outcomes were similar in HEK293 cells and the human retinoblastoma cell line WERI-Rb1, the latter retaining a cone photoreceptor expression profile including endogenous OPN1LW and OPN1MW gene expression. Patients carrying the novel L-I-V-V-A haplotype presented with a mild form of Blue Cone Monochromacy or Bornholm Eye Disease-like phenotype with reduced visual acuity, reduced cone electroretinography responses, red-green color vision defects, and frequently with severe myopia.
Subject(s)
Color Vision Defects , Rod Opsins/genetics , Color Vision Defects/genetics , Color Vision Defects/metabolism , Exons/genetics , HEK293 Cells , Haplotypes , Humans , Retinal Cone Photoreceptor Cells/metabolism , Rod Opsins/metabolismABSTRACT
Cone photoreceptor dysfunction represents a clinically heterogenous group of disorders characterized by nystagmus, photophobia, reduced central or color vision, and macular dystrophy. Here, we described the molecular findings and clinical manifestations of achromatopsia, a partial or total absence of color vision, co-segregating with three known missense variants of CNGA3 in three large consanguineous Pakistani families. Fundus examination and optical coherence tomography (OCT) imaging revealed myopia, thin retina, retinal pigment epithelial cells loss at fovea/perifovea, and macular atrophy. Combination of Sanger and whole exome sequencing revealed three known homozygous missense variants (c.827A>G, p.(Asn276Ser); c.847C>T, p.(Arg283Trp); c.1279C>T, p.(Arg427Cys)) in CNGA3, the α-subunit of the cyclic nucleotide-gated cation channel in cone photoreceptor cells. All three variants are predicted to replace evolutionary conserved amino acids, and to be pathogenic by specific in silico programs, consistent with the observed altered membrane targeting of CNGA3 in heterologous cells. Insights from our study will facilitate counseling regarding the molecular and phenotypic landscape of CNGA3-related cone dystrophies.
Subject(s)
Color Vision Defects , Retinal Cone Photoreceptor Cells , Color Vision Defects/diagnosis , Color Vision Defects/genetics , Color Vision Defects/metabolism , Cyclic Nucleotide-Gated Cation Channels/genetics , Humans , Mutation , PakistanABSTRACT
Numerous missense mutations in cyclic nucleotide-gated (CNG) channels cause achromatopsia and retinitis pigmentosa, but the underlying pathogenic mechanisms are often unclear. We investigated the structural basis and molecular/cellular effects of R410W, an achromatopsia-associated, presumed loss-of-function mutation in human CNGA3. Cryo-EM structures of the Caenorhabditis elegans TAX-4 CNG channel carrying the analogous mutation, R421W, show that most apo channels are open. R421, located in the gating ring, interacts with the S4 segment in the closed state. R421W disrupts this interaction, destabilizes the closed state, and stabilizes the open state. CNGA3_R410W/CNGB3 and TAX4_R421W channels are spontaneously active without cGMP and induce cell death, suggesting cone degeneration triggered by spontaneous CNG channel activity as a possible cause of achromatopsia. Our study sheds new light on CNG channel allosteric gating, provides an impetus for a reevaluation of reported loss-of-function CNG channel missense disease mutations, and has implications for mutation-specific treatment of retinopathy.
Subject(s)
Color Vision Defects , Cyclic Nucleotide-Gated Cation Channels , Color Vision Defects/genetics , Color Vision Defects/metabolism , Color Vision Defects/pathology , Cyclic Nucleotide-Gated Cation Channels/chemistry , Cyclic Nucleotide-Gated Cation Channels/genetics , Cyclic Nucleotide-Gated Cation Channels/metabolism , Humans , Light Signal Transduction , Mutation, Missense , Retinal Cone Photoreceptor CellsABSTRACT
PURPOSE: To characterize red-green and tritan color discrimination in eyes with macular telangiectasia Type II (MacTel). METHODS: Color discrimination was assessed by metameric matching methods using an Oculus MR Anomaloscope. Red-green color discrimination was assessed using the Rayleigh equation, and tritan color discrimination was assessed using the Moreland equation. Results were expressed as anomalquotient (AQ) and tritanomalquotient (TAQ) units, respectively. RESULTS: Seventeen eyes with MacTel were compared with 16 control eyes with normal vision. Twelve eyes with MacTel demonstrated abnormal color matches; except for two eyes with red-shifted Rayleigh matches, the primary abnormality evident was reduced color discrimination. On average, Rayleigh matching ranges were significantly widened in MacTel (0.518 ± 0.066 AQ units) compared with normal (0.14 ± 0.03 AQ units; P < 0.0001). Similarly, Moreland matching ranges were significantly wider (0.794 ± 0.109 TAQ units) than normal control subjects (0.204 ± 0.070 TAQ units; P < 0.0001). Losses in color discrimination did not correlate significantly with the best-corrected visual acuity, although Moreland matching ranges were significantly correlated to Rayleigh matching ranges. CONCLUSION: MacTel results in a combined acquired red-green and tritan color vision deficiency. A minority of eyes demonstrated red-shifted Rayleigh matches, consistent with decreases in cone photopigment optical density.
Subject(s)
Color Vision Defects/etiology , Cone Opsins/metabolism , Retinal Cone Photoreceptor Cells/metabolism , Retinal Telangiectasis/complications , Aged , Aged, 80 and over , Color Perception Tests , Color Vision Defects/metabolism , Cross-Sectional Studies , Female , Humans , Male , Middle Aged , Prospective StudiesABSTRACT
Sporadic occurrence of inherited eye disorders has been reported in cattle but so far pathogenic variants were found only for rare forms of cataract but not for retinopathies. The aim of this study was to characterize the phenotype and the genetic aetiology of a recessive form of congenital day-blindness observed in several cases of purebred Original Braunvieh cattle. Electroretinography in an affected calf revealed absent cone-mediated function, whereas the rods continue to function normally. Brain areas involved in vision were morphologically normal. When targeting cones by immunofluorescence, a decrease in cone number and an accumulation of beta subunits of cone cyclic-nucleotide gated channel (CNGB3) in the outer plexiform layer of affected animals was obvious. Achromatopsia is a monogenic Mendelian disease characterized by the loss of cone photoreceptor function resulting in day-blindness, total color-blindness, and decreased central visual acuity. After SNP genotyping and subsequent homozygosity mapping with twelve affected cattle, we performed whole-genome sequencing and variant calling of three cases. We identified a single missense variant in the bovine CNGB3 gene situated in a ~2.5 Mb homozygous genome region on chromosome 14 shared between all cases. All affected cattle were homozygous carriers of the p.Asp251Asn mutation that was predicted to be deleterious, affecting an evolutionary conserved residue. In conclusion, we have evidence for the occurrence of a breed-specific novel CNGB3-related form of recessively inherited achromatopsia in Original Braunvieh cattle which we have designated OH1 showing an allele frequency of the deleterious allele of ~8%. The identification of carriers will enable selection against this inherited disorder. The studied cattle might serve as an animal model to further elucidate the function of CNGB3 in mammals.
Subject(s)
Alleles , Color Vision Defects/genetics , Cyclic Nucleotide-Gated Cation Channels/genetics , Mutation, Missense , Protein Subunits/genetics , Retinal Cone Photoreceptor Cells/metabolism , Amino Acid Substitution , Animals , Asparagine/metabolism , Aspartic Acid/metabolism , Cattle , Color Vision Defects/diagnostic imaging , Color Vision Defects/metabolism , Color Vision Defects/pathology , Cyclic Nucleotide-Gated Cation Channels/deficiency , Electroretinography , Female , Gene Expression , Gene Frequency , Homozygote , Male , Phenotype , Protein Subunits/deficiency , Retinal Cone Photoreceptor Cells/pathology , Retinal Rod Photoreceptor Cells/cytology , Retinal Rod Photoreceptor Cells/metabolism , Whole Genome SequencingABSTRACT
Achromatopsia is characterized by amblyopia, photophobia, nystagmus, and color blindness. Previous animal models of achromatopsia have shown promising results using gene augmentation to restore cone function. However, the optimal therapeutic window to elicit recovery remains unknown. Here, we attempted two rounds of gene augmentation to generate recoverable mouse models of achromatopsia including a Cnga3 model with a knock-in stop cassette in intron 5 using Easi-CRISPR (Efficient additions with ssDNA inserts-CRISPR) and targeted embryonic stem (ES) cells. This model demonstrated that only 20% of CNGA3 levels in homozygotes derived from target ES cells remained, as compared to normal CNGA3 levels. Despite the low percentage of remaining protein, the knock-in mouse model continued to generate normal cone phototransduction. Our results showed that a small amount of normal CNGA3 protein is sufficient to form "functional" CNG channels and achieve physiological demand for proper cone phototransduction. Thus, it can be concluded that mutating the Cnga3 locus to disrupt the functional tetrameric CNG channels may ultimately require more potent STOP cassettes to generate a reversible achromatopsia mouse model. Our data also possess implications for future CNGA3-associated achromatopsia clinical trials, whereby restoration of only 20% functional CNGA3 protein may be sufficient to form functional CNG channels and thus rescue cone response.
Subject(s)
Color Vision Defects/genetics , Cyclic Nucleotide-Gated Cation Channels/genetics , Disease Models, Animal , Gene Editing , Mutation , Animals , Clustered Regularly Interspaced Short Palindromic Repeats , Color Vision Defects/metabolism , Gene Knock-In Techniques , Mice , Retinal Cone Photoreceptor Cells/metabolism , Retinal Cone Photoreceptor Cells/physiologyABSTRACT
Purpose: Emmetropization is the process of adjusting ocular growth to the focal plane in order to achieve a clear image. Chromatic light may be involved as a cue to guide this process. Achromats are color blind and lack normal cone function; they are often described as being hyperopic, indicating a failure to emmetropize. We aim to describe the refraction and refractive development in a population of genetically characterized achromats. Methods: Refractive error data were collected retrospectively from 28 medical records of CNGB3 c.1148delC homozygous achromats. The distribution of spherical equivalent refractive error (SER) and spherical error was analyzed in adults. The refractive development in children was analyzed by documenting astigmatic refractive error and calculating median SER in 1-year age groups and by analyzing the individual development when possible. Results: The distribution of SER and spherical error resembled a Gaussian distribution, indicating that emmetropization was disturbed in achromats, but we found indication of some decrease in SER during the first years of childhood. The prevalence of refractive errors was high and broadly distributed. Astigmatic refractive errors were frequent but did not seem to increase with age. Conclusions: Refractive development in achromats is more complicated than a complete failure to emmetropize. The spread of refractive errors is larger than previously documented. Results presented here support the theory that chromatic cues and cone photoreceptors may play a role in emmetropization in humans but that it is not essential.
Subject(s)
Accommodation, Ocular/physiology , Color Vision Defects/genetics , Cyclic Nucleotide-Gated Cation Channels/genetics , Refraction, Ocular/physiology , Refractive Errors/etiology , Adolescent , Adult , Aged , Color Vision Defects/complications , Color Vision Defects/metabolism , Cyclic Nucleotide-Gated Cation Channels/metabolism , Female , Follow-Up Studies , Humans , Male , Middle Aged , Refractive Errors/metabolism , Refractive Errors/physiopathology , Retrospective Studies , Time Factors , Young AdultABSTRACT
Purpose: Psychophysical and genetic testing provide substantial information about color vision phenotype and genotype. However, neither reveals how color vision phenotypes and genotypes manifest themselves in individual cones, where color vision and its anomalies are thought to originate. Here, we use adaptive-optics phase-sensitive optical coherence tomography (AO-PSOCT) to investigate these relationships. Methods: We used AO-PSOCT to measure cone function-optical response to light stimulation-in each of 16 human subjects with different phenotypes and genotypes of color vision (five color-normal, three deuteranopic, two protanopic, and six deuteranomalous trichromatic subjects). We classified three spectral types of cones (S, M, and L), and we measured cone structure-namely cone density, cone mosaic arrangement, and spatial arrangement of cone types. Results: For the different phenotypes, our cone function results show that (1) color normals possess S, M, and L cones; (2) deuteranopes are missing M cones but are normal otherwise; (3) protanopes are missing L cones but are normal otherwise; and (4) deuteranomalous trichromats are missing M cones but contain evidence of at least two subtypes of L cones. Cone function was consistent with the subjects' genotype in which only the first two M and L genes in the gene array are expressed and was correlated with the estimated spectral separation between photopigments, including in the deuteranomalous trichromats. The L/M cone ratio was highly variable in the color normals. No association was found between cone density and the genotypes and phenotypes investigated, and the cone mosaic arrangement was altered in the dichromats. Conclusions: AO-PSOCT is a novel method for assessing color vision phenotype and genotype in single cone cells.
Subject(s)
Color Vision Defects/genetics , Color Vision/genetics , Retinal Cone Photoreceptor Cells/metabolism , Retinal Pigments/metabolism , Adult , Color Perception/physiology , Color Vision Defects/metabolism , Color Vision Defects/pathology , Female , Genotype , Humans , Male , Middle Aged , Phenotype , Retinal Cone Photoreceptor Cells/pathology , Tomography, Optical Coherence/methods , Young AdultABSTRACT
Mutations in the CNGA3 gene, which encodes the A subunit of the cyclic guanosine monophosphate (cGMP)-gated cation channel in cone photoreceptor outer segments, cause total colour blindness, also referred to as achromatopsia. Cones lacking this channel protein are non-functional, accumulate high levels of the second messenger cGMP and degenerate over time after induction of ER stress. The cell death mechanisms that lead to loss of affected cones are only partially understood. Here, we explored the disease mechanisms in the Cnga3 knockout (KO) mouse model of achromatopsia. We found that another important effector of cGMP, the cGMP-dependent protein kinase 2 (Prkg2) is crucially involved in cGMP cytotoxicity of cones in Cnga3 KO mice. Virus-mediated knockdown or genetic ablation of Prkg2 in Cnga3 KO mice counteracted degeneration and preserved the number of cones. Analysis of markers of endoplasmic reticulum stress and unfolded protein response confirmed that induction of these processes in Cnga3 KO cones also depends on Prkg2. In conclusion, we identified Prkg2 as a novel key mediator of cone photoreceptor degeneration in achromatopsia. Our data suggest that this cGMP mediator could be a novel pharmacological target for future neuroprotective therapies.
Subject(s)
Color Vision Defects/etiology , Color Vision Defects/metabolism , Cyclic GMP-Dependent Protein Kinase Type II/metabolism , Cyclic Nucleotide-Gated Cation Channels/deficiency , Retinal Cone Photoreceptor Cells/metabolism , Animals , Biomarkers , Color Vision Defects/pathology , Cyclic GMP/metabolism , Cyclic GMP-Dependent Protein Kinase Type II/genetics , Disease Models, Animal , Disease Susceptibility , Endoplasmic Reticulum Stress , Fluorescent Antibody Technique , Gene Expression , Mice , Mice, Knockout , Microscopy, Confocal , Models, Biological , Retinal Degeneration/etiology , Retinal Degeneration/metabolism , Retinal Degeneration/pathology , Unfolded Protein ResponseABSTRACT
Purpose: To perform deep phenotyping of subjects with PDE6C achromatopsia and examine disease natural history. Methods: Eight subjects with disease-causing variants in PDE6C were assessed in detail, including clinical phenotype, best-corrected visual acuity, fundus autofluorescence, and optical coherence tomography. Six subjects also had confocal and nonconfocal adaptive optics scanning light ophthalmoscopy, axial length, international standard pattern and full-field electroretinography (ERG), short-wavelength flash (S-cone) ERGs, and color vision testing. Results: All subjects presented with early-onset nystagmus, decreased best-corrected visual acuity, light sensitivity, and severe color vision loss, and five of them had high myopia. We identified three novel disease-causing variants and provide phenotype data associated with nine variants for the first time. No subjects had foveal hypoplasia or residual ellipsoid zone (EZ) at the foveal center; one had an absent EZ, three had a hyporeflective zone, and four had outer retinal atrophy. The mean width of the central EZ lesion on optical coherence tomography at baseline was 1923 µm. The mean annual increase in EZ lesion size was 48.3 µm. Fundus autofluorescence revealed a central hypoautofluorescence with a surrounding ring of increased signal (n = 5). The mean hypoautofluorescent area at baseline was 3.33 mm2 and increased in size by a mean of 0.13 mm2/year. Nonconfocal adaptive optics scanning light ophthalmoscopy revealed residual foveal cones in only one of two cases. Full-field ERGs were consistent with severe generalized cone system dysfunction but with relative preservation of S-cone sensitivity. Conclusions: PDE6C retinopathy is a severe cone dysfunction syndrome often presenting as typical achromatopsia but without foveal hypoplasia. Myopia and slowly progressive maculopathy are common features. There are few (if any) residual foveal cones for intervention in older adults.
Subject(s)
Color Vision Defects/genetics , Color Vision/physiology , Cyclic Nucleotide Phosphodiesterases, Type 6/genetics , Eye Proteins/genetics , Visual Acuity , Adolescent , Adult , Child , Color Vision Defects/diagnosis , Color Vision Defects/metabolism , Cyclic Nucleotide Phosphodiesterases, Type 6/metabolism , Electroretinography , Eye Proteins/metabolism , Female , Follow-Up Studies , Forecasting , Humans , Male , Middle Aged , Ophthalmoscopy , Phenotype , Tomography, Optical Coherence/methods , Young AdultABSTRACT
BACKGROUND: Color vision impairment (CVI) has been reported in dementia with Lewy bodies (DLB) and prodromal Lewy body disease (pro-LBD). OBJECTIVE: In order to better characterize the diagnostic value of CVI testing, we compared the prevalence of CVI in patients with with Lewy body disease compared to Alzheimer's disease (AD), and we examined clinical and imaging characteristics associated with CVI in patients with DLB and suspected pro-LBD. METHODS: We retrospectively reviewed medical records, dopamine transporter (DaT-SPECT) imaging, and volumetric MRI from patients with AD, DLB, and suspected pro-LBD who underwent an online Farnsworth D-15 color vision test. RESULTS: 111 patients (62 DLB, 25 pro-LBD, and 24 AD) were included with a median age of 75 years. Newly diagnosed CVI was present in 67% of patients with DLB, 44% of patients with pro-LBD, and 18% of patients with AD. In patients with DLB, CVI was associated with lower Montreal Cognitive Assessment (MoCA) scores and lower sub-scores in visuospatial/executive function, naming, and language. In a multivariable logistic regression model, a diagnosis of DLB or pro-LBD compared to AD, and a lower composite MoCA score in visuospatial/executive function, naming, and language were associated with CVI controlling for age and gender. Among 17 DLB patients who underwent volumetric MRI, patients with CVI (nâ=â9) demonstrated lower normative volumetric percentiles in the right transverse superior temporal lobe. CONCLUSION: We provide further evidence that CVI can help differentiate DLB from AD, and we suggest that CVI may be an indicator of cognitive decline and disease progression in DLB.
Subject(s)
Brain/diagnostic imaging , Color Vision Defects/diagnostic imaging , Color Vision/physiology , Lewy Body Disease/diagnostic imaging , Aged , Aged, 80 and over , Brain/metabolism , Brain/physiopathology , Cognition/physiology , Color Vision Defects/complications , Color Vision Defects/metabolism , Color Vision Defects/physiopathology , Dopamine Plasma Membrane Transport Proteins/metabolism , Female , Humans , Lewy Body Disease/complications , Lewy Body Disease/metabolism , Lewy Body Disease/physiopathology , Magnetic Resonance Imaging , Male , Middle Aged , Neuropsychological Tests , Retrospective StudiesABSTRACT
Incomplete achromatopsia (ACHM) is a disorder in which there is function defect of cone photoreceptors in the retina and individuals with such disease retain residual color vision. Here, we have generated an induced pluripotent stem cell (iPSC) line carrying a homozygous mutation in the PDE6C gene, already related with this vision disorder. Skin fibroblasts from a patient with incomplete ACHM were reprogrammed to iPSCs by the non-integrative Sendai-virus method. Finally, the iPSC line has been characterized expressing the pluripotency markers and being capable to differentiate to endoderm, mesoderm and ectoderm in vitro.
Subject(s)
Cell Line/cytology , Color Vision Defects/genetics , Cyclic Nucleotide Phosphodiesterases, Type 6/genetics , Eye Proteins/genetics , Genetic Diseases, X-Linked/genetics , Induced Pluripotent Stem Cells/metabolism , Adult , Cell Differentiation , Cell Line/metabolism , Cells, Cultured , Color Vision Defects/metabolism , Color Vision Defects/physiopathology , Cyclic Nucleotide Phosphodiesterases, Type 6/metabolism , Eye Proteins/metabolism , Fibroblasts/cytology , Fibroblasts/metabolism , Genetic Diseases, X-Linked/metabolism , Genetic Diseases, X-Linked/physiopathology , Homozygote , Humans , Induced Pluripotent Stem Cells/cytology , Male , MutationABSTRACT
Cone photoreceptor cyclic-nucleotide gated channels (CNG) are tetrameric proteins composed of subunits from CNGA3 and CNGB3. These channels transduce light information into electrical signals carried by both Na+ and Ca2+ ions. More than 100 mutations in the CNGA3 gene are associated with the inherited retinal disorder, achromatopsia 2 (ACHM2), which results in attenuation or loss of color vision, daylight blindness, and reduced visual acuity. Classical techniques to measure CNG channel function utilize patch clamp electrophysiology measuring Na currents in the absence of divalent cations, yet intracellular Ca2+ regulates both light and dark adaptation in photoreceptors. We developed a fluorescence-based, high-throughput Ca2+ flux assay using yellow fluorescent protein (YFP) tagged CNGA3 channels expressed in HEK293 cells which allow monitoring for folding defects in mutant channels. The cell permeant cGMP analog, 8-(4-chlorophenylthio)-cGMP (CPT-cGMP), was used to activate Ca2+ flux. The assay was validated using wild-type CNGA3 homomeric and heteromeric channels and ACHM2-associated homomeric mutant CNG channels, CNGA3-R427C, CNGA3-E590K, and CNGA3-L633P. Additionally, we examined two naturally occurring canine mutations causing day-blindness previously studied by patch clamp. We compared the CPT-cGMP K0.5 values of the channels with patch clamp values from previous studies. The assay provides a screen for modulation of gating and/or rescue of trafficking and/or misfolding defects in ACHM2-associated CNG channels. Importantly, the calcium flux assay is advantageous compared to patch clamp as it allows the ability to monitor CNG channel activity in the presence of calcium.
Subject(s)
Calcium/metabolism , Color Vision Defects/metabolism , Cyclic Nucleotide-Gated Cation Channels/metabolism , Retinal Cone Photoreceptor Cells/metabolism , HEK293 Cells , Humans , Patch-Clamp Techniques , Protein Transport , Retina/metabolismABSTRACT
Mutations in CNGA3 and CNGB3, the genes encoding the subunits of the tetrameric cone photoreceptor cyclic nucleotide-gated ion channel, cause achromatopsia, a congenital retinal disorder characterized by loss of cone function. However, a small number of patients carrying the CNGB3/c.1208G>A;p.R403Q mutation present with a variable retinal phenotype ranging from complete and incomplete achromatopsia to moderate cone dysfunction or progressive cone dystrophy. By exploring a large patient cohort and published cases, we identified 16 unrelated individuals who were homozygous or (compound-)heterozygous for the CNGB3/c.1208G>A;p.R403Q mutation. In-depth genetic and clinical analysis revealed a co-occurrence of a mutant CNGA3 allele in a high proportion of these patients (10 of 16), likely contributing to the disease phenotype. To verify these findings, we generated a Cngb3R403Q/R403Q mouse model, which was crossbred with Cnga3-deficient (Cnga3-/-) mice to obtain triallelic Cnga3+/- Cngb3R403Q/R403Q mutants. As in human subjects, there was a striking genotype-phenotype correlation, since the presence of 1 Cnga3-null allele exacerbated the cone dystrophy phenotype in Cngb3R403Q/R403Q mice. These findings strongly suggest a digenic and triallelic inheritance pattern in a subset of patients with achromatopsia/severe cone dystrophy linked to the CNGB3/p.R403Q mutation, with important implications for diagnosis, prognosis, and genetic counseling.
Subject(s)
Color Vision Defects , Cyclic Nucleotide-Gated Cation Channels , Heterozygote , Ion Channel Gating , Mutation, Missense , Retinal Cone Photoreceptor Cells , Retinal Diseases , Amino Acid Substitution , Animals , Color Vision Defects/genetics , Color Vision Defects/metabolism , Color Vision Defects/pathology , Cyclic Nucleotide-Gated Cation Channels/genetics , Cyclic Nucleotide-Gated Cation Channels/metabolism , Disease Models, Animal , HEK293 Cells , Humans , Mice , Mice, Transgenic , Mutation , Retinal Cone Photoreceptor Cells/metabolism , Retinal Cone Photoreceptor Cells/pathology , Retinal Diseases/genetics , Retinal Diseases/metabolism , Retinal Diseases/pathologyABSTRACT
Color vision deficiency (color blindness) is an inherited genetic ocular disorder. While no cure for this disorder currently exists, several methods can be used to increase the color perception of those affected. One such method is the use of color filtering glasses which are based on Bragg filters. While these glasses are effective, they are high cost, bulky, and incompatible with other vision correction eyeglasses. In this work, a rhodamine derivative is incorporated in commercial contact lenses to filter out the specific wavelength bands (≈545-575 nm) to correct color vision blindness. The biocompatibility assessment of the dyed contact lenses in human corneal fibroblasts and human corneal epithelial cells shows no toxicity and cell viability remains at 99% after 72 h. This study demonstrates the potential of the dyed contact lenses in wavelength filtering and color vision deficiency management.
Subject(s)
Color Vision Defects/therapy , Contact Lenses, Hydrophilic , Color Vision Defects/metabolism , Cornea/cytology , Cornea/metabolism , Epithelial Cells/cytology , Epithelial Cells/metabolism , Female , Humans , Male , Materials Testing , Rhodamines/chemistryABSTRACT
The cyclic nucleotide-gated (CNG) channel - composed of CNGA3 and CNGB3 subunits - mediates the influx of cations in cone photoreceptors after light stimulation and thus is a key element in cone phototransduction. Mutations in CNGA3 and CNGB3 are associated with achromatopsia, a rare autosomal recessive retinal disorder. Here, we demonstrate that the presence of an early nonsense mutation in CNGA3 induces the usage of a downstream alternative translation initiation site giving rise to a short CNGA3 isoform. The expression of this short isoform was verified by Western blot analysis and DAB staining of HEK293â¯cells and cone photoreceptor-like 661W cells expressing CNGA3-GST fusion constructs. Functionality of the short isoform was confirmed by a cellular calcium influx assay. Furthermore, patients carrying an early nonsense mutation were analyzed for residual cone photoreceptor function in order to identify a potential role of the short isoform to modify the clinical outcome in achromatopsia patients. Yet the results suggest that the short isoform is not able to compensate for the loss of the long isoform leaving the biological role of this variant unclear.
Subject(s)
Codon, Nonsense/genetics , Color Vision Defects/genetics , Cyclic Nucleotide-Gated Cation Channels/genetics , Gene Expression Regulation/physiology , Peptide Chain Initiation, Translational/genetics , Protein Isoforms/genetics , Animals , Blotting, Western , Cell Line , Color Vision Defects/metabolism , Electrophoresis, Polyacrylamide Gel , HEK293 Cells/metabolism , Humans , Immunohistochemistry , Mice , Polymerase Chain Reaction , Retinal Cone Photoreceptor Cells/metabolism , TransfectionABSTRACT
Purpose: Blue cone monochromacy (BCM) is an X-linked congenital vision disorder characterized by complete loss or severely reduced L- and M-cone function. Patients with BCM display poor visual acuity, severely impaired color discrimination, myopia, nystagmus, and minimally detectable cone-mediated electroretinogram. Recent studies of patients with BCM with adaptive optics scanning laser ophthalmoscopy (AOSLO) showed that they have a disrupted cone mosaic with reduced numbers of cones in the fovea that is normally dominated by L- and M-cones. The remaining cones in the fovea have significantly shortened outer segments but retain sufficient structural integrity to serve as potential gene therapy targets. In this study, we tested whether exogenously expressed human L- and M-opsins can rescue M-cone function in an M-opsin knockout (Opn1mw-/- ) mouse model for BCM. Methods: Adeno-associated virus type 5 (AAV5) vectors expressing OPN1LW, OPN1MW, or C-terminal tagged OPN1LW-Myc, or OPN1MW-HA driven by a cone-specific promoter were injected subretinally into one eye of Opn1mw-/- mice, while the contralateral eye served as the uninjected control. Expression of cone pigments was determined with western blotting and their cellular localization identified with immunohistochemistry. M-cone function was analyzed with electroretinogram (ERG). Antibodies against cone phototransduction proteins were used to study cone outer segment (OS) morphology in untreated and treated Opn1mw-/- eyes. Results: We showed that cones in the dorsal retina of the Opn1mw-/- mouse do not form outer segments, resembling cones that lack outer segments in the human BCM fovea. We further showed that AAV5-mediated expression of either human M- or L-opsin individually or combined promotes regrowth of cone outer segments and rescues M-cone function in the treated Opn1mw-/- dorsal retina. Conclusions: Exogenously expressed human opsins can regenerate cone outer segments and rescue M-cone function in Opn1mw-/- mice, thus providing a proof-of-concept gene therapy in an animal model of BCM.
Subject(s)
Color Vision Defects/therapy , Fovea Centralis/metabolism , Genetic Therapy/methods , Retinal Photoreceptor Cell Outer Segment/metabolism , Rod Opsins/genetics , Animals , Color Vision Defects/genetics , Color Vision Defects/metabolism , Color Vision Defects/pathology , Dependovirus/genetics , Dependovirus/metabolism , Disease Models, Animal , Fovea Centralis/pathology , Gene Expression , Genetic Complementation Test , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Humans , Mice , Mice, Knockout , Ophthalmoscopy , Promoter Regions, Genetic , Retinal Photoreceptor Cell Outer Segment/pathology , Rod Opsins/metabolism , TransgenesABSTRACT
Mutations in PDE6 genes encoding the effector enzymes in rods and cones underlie severe retinal diseases including retinitis pigmentosa (RP), autosomal dominant congenital stationary night blindness (adCSNB), and achromatopsia (ACHM). Here we examined a spectrum of pathogenic missense mutations in PDE6 using the system based on co-expression of cone PDE6C with its specialized chaperone AIPL1 and the regulatory Pγ subunit as a potent co-chaperone. We uncovered two mechanisms of PDE6C mutations underlying ACHM: (a) folding defects leading to expression of catalytically inactive proteins and (b) markedly diminished ability of Pγ to co-chaperone mutant PDE6C proteins thereby dramatically reducing the levels of functional enzyme. The mechanism of the Rambusch adCSNB associated with the H258N substitution in PDE6B was probed through the analysis of the model mutant PDE6C-H262N. We identified two interrelated deficits of PDE6C-H262N: disruption of the inhibitory interaction of Pγ with mutant PDE6C that markedly reduced the ability of Pγ to augment the enzyme folding. Thus, we conclude that the Rambusch adCSNB is triggered by low levels of the constitutively active PDE6. Finally, we examined PDE6C-L858V, which models PDE6B-L854V, an RP-linked mutation that alters the protein isoprenyl modification. This analysis suggests that the type of prenyl modifications does not impact the folding of PDE6, but it modulates the enzyme affinity for its trafficking partner PDE6D. Hence, the pathogenicity of PDE6B-L854V likely arises from its trafficking deficiency. Taken together, our results demonstrate the effectiveness of the PDE6C expression system to evaluate pathogenicity and elucidate the mechanisms of PDE6 mutations in retinal diseases.
Subject(s)
Cyclic Nucleotide Phosphodiesterases, Type 6/genetics , Eye Proteins/genetics , Mutation, Missense , Retinal Diseases/genetics , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Animals , Carrier Proteins/genetics , Carrier Proteins/metabolism , Color Vision Defects/genetics , Color Vision Defects/metabolism , Cyclic Nucleotide Phosphodiesterases, Type 6/analysis , Cyclic Nucleotide Phosphodiesterases, Type 6/metabolism , Eye Diseases, Hereditary/genetics , Eye Diseases, Hereditary/metabolism , Eye Proteins/analysis , Eye Proteins/metabolism , Gene Expression , Genetic Diseases, X-Linked/genetics , Genetic Diseases, X-Linked/metabolism , HEK293 Cells , Humans , Mice , Models, Molecular , Myopia/genetics , Myopia/metabolism , Night Blindness/genetics , Night Blindness/metabolism , Protein Folding , Protein Prenylation , Retinal Diseases/metabolismABSTRACT
Endoplasmic reticulum (ER) stress and mislocalization of improperly folded proteins have been shown to contribute to photoreceptor death in models of inherited retinal degenerative diseases. In particular, mice with cone cyclic nucleotide-gated (CNG) channel deficiency, a model for achromatopsia, display both early-onset ER stress and opsin mistrafficking. By 2 weeks of age, these mice show elevated signaling from all three arms of the ER-stress pathway, and by 1 month, cone opsin is improperly distributed away from its normal outer segment location to other retinal layers. This work investigated the role of Ca2+-release channels in ER stress, protein mislocalization, and cone death in a mouse model of CNG-channel deficiency. We examined whether preservation of luminal Ca2+ stores through pharmacological and genetic suppression of ER Ca2+ efflux protects cones by attenuating ER stress. We demonstrated that the inhibition of ER Ca2+-efflux channels reduced all three arms of ER-stress signaling while improving opsin trafficking to cone outer segments and decreasing cone death by 20-35%. Cone-specific gene deletion of the inositol-1,4,5-trisphosphate receptor type I (IP3R1) also significantly increased cone density in the CNG-channel-deficient mice, suggesting that IP3R1 signaling contributes to Ca2+ homeostasis and cone survival. Consistent with the important contribution of organellar Ca2+ signaling in this achromatopsia mouse model, significant differences in dynamic intraorganellar Ca2+ levels were detected in CNG-channel-deficient cones. These results thus identify a novel molecular link between Ca2+ homeostasis and cone degeneration, thereby revealing novel therapeutic targets to preserve cones in inherited retinal degenerative diseases.