Quantitative and qualitative characterization of retinal dystrophies in canine models of inherited retinal diseases using spectral domain optical coherence tomography (SD-OCT).

The purpose of this study was to establish spectral domain optical coherence tomography (SD-OCT) assessment data in well-established canine models of inherited retinal dystrophies: PDE6B-rod-cone dysplasia 1 (RCD1: early onset retinitis pigmentosa), PRCD-progressive rod-cone degeneration (PRCD: late onset retinitis pigmentosa), CNGB3-achromatopsia, and RPE65-Leber congenital amaurosis (LCA). High resolution SD-OCT images of the retina were acquired from both eyes in 5 planes: temporal; superotemporal; superior; nasal; and inferior in adult dogs with: RCD1 (n = 4 dogs, median age: 1.5 yrs); PRCD (n = 2, 4.3 yrs); LCA (n = 3, 5.2 yrs); achromatopsia (n = 3, 4.2 yrs); and wild types (wt, n = 6, 5.5 yrs). Total, inner and outer retinal thicknesses and ellipsoid zone were analyzed. In selected animals, histomorphometric evaluations were performed. In dogs with RCD1, PRCD, and LCA, the thickness of the outer retina was, compared to wt, significantly decreased (p ≤ 0.02) in all OCT imaging planes, and in superotemporal and inferior imaging planes in dogs with achromatopsia. No significant thinning was observed in inner retina thickness in any disease model except in the inferior imaging plane in dogs with RCD1. Dogs with RCD1, PRCD, and LCA had significantly more areas with disrupted ellipsoid zone in the presumed area centralis than wt (p ≤ 0.001). OCT findings provide baseline information for research of retinal dystrophies using these canine models.

Disease Progression in CNGA3 and CNGB3 Retinopathy; Characteristics of Slovenian Cohort and Proposed OCT Staging Based on Pooled Data from 126 Patients from 7 Studies.

Achromatopsia has been proposed to be a morphologically predominately stable retinopathy with rare reports of progression of structural changes in the macula. A five-grade system of optical coherence tomography (OCT) features has been used for the classification of structural macular changes. However, their association with age remains questionable. We characterized the Slovenian cohort of 12 patients with pathogenic variants in CNGA3 or CNGB3 who had been followed up with OCT for up to 9 years. Based on observed structural changes in association with age, the following four-stage classification of retinal morphological changes was proposed: (I) preserved inner segment ellipsoid band (Ise), (II) disrupted ISe, (III) ISe loss and (IV) ISe and RPE loss. Data from six previously published studies reporting OCT morphology in CNGA3 and CNGB3 patients were additionally collected, forming the largest CNGA3/CNGB3 cohort to date, comprising 126 patients aged 1-71 years. Multiple regression analysis showed a significant correlation of OCT stage with age (p < 0.001) and no correlation with gene (p > 0.05). The median ages of patients with stages I-IV were 12 years, 23 years, 27 years and 48 years, respectively, and no patient older than 50 years had continuous ISe. Our findings suggest that achromatopsia presents with slowly but steadily progressive structural changes of the macular outer retinal layers. However, whether morphological changes in time follow the proposed four-stage linear pattern needs to be confirmed in a long-term study.

Clinical and Molecular Characterization of Achromatopsia Patients: A Longitudinal Study.

Achromatopsia (ACHM) is a rare genetic disorder of infantile onset affecting cone photoreceptors. To determine the extent of progressive retinal changes in achromatopsia, we performed a detailed longitudinal phenotyping and genetic characterization of an Italian cohort comprising 21 ACHM patients (17 unrelated families). Molecular genetic testing identified biallelic pathogenic mutations in known ACHM genes, including four novel variants. At baseline, the patients presented a reduced best corrected visual acuity (BCVA), reduced macular sensitivity (MS), normal dark-adapted electroretinogram (ERG) responses and undetectable or severely reduced light-adapted ERG. The longitudinal analysis of 16 patients (mean follow-up: 5.4 ± 1.0 years) showed a significant decline of BCVA (0.012 logMAR/year) and MS (-0.16 dB/year). Light-adapted and flicker ERG responses decreased below noise level in three and two patients, respectively. Only two patients (12.5%) progressed to a worst OCT grading during the follow-up. Our findings corroborate the notion that ACHM is a progressive disease in terms of BCVA, MS and ERG responses, and affects slowly the structural integrity of the retina. These observations can serve towards the development of guidelines for patient selection and intervention timing in forthcoming gene replacement therapies.

Molecular and clinical characterization of Thai patients with achromatopsia: identification of three novel disease-associated variants in the CNGA3 and CNGB3 genes.

PURPOSE: Achromatopsia (ACHM) is an autosomal recessive cone disorder characterized by pendular nystagmus, photophobia, reduced visual acuity, and partial or total absence of color vision. Mutations in six genes (CNGA3, CNGB3, GNAT2, PDE6C, PDE6H, and ATF6) have been reported in ACHM. There is no information on these disease-associated genes in Thai population. This study aimed to investigate the molecular and clinical characteristics in Thai patients with ACHM. METHODS: Seven unrelated Thai patients with ACHM were recruited. Detailed ophthalmologic examination was performed. Polymerase chain reaction (PCR)-coupled single-strand conformation polymorphism (SSCP) screening followed by Sanger sequencing was used to identify sequence variants in all exons and splice junctions of three genes (CNGA3, CNGB3, and GNAT2). The pathogenicity of the detected variants was interpreted. Segregation analysis was performed to determine variant sharing in available family members. RESULTS: Four patients displayed different SSCP migration patterns. Sequence analysis revealed a reported pathogenic and a novel disease-associated variant in the CNGA3 gene. For the CNGB3 gene, we found two novel disease-associated variants and a reported variant of uncertain significance (VUS). Segregation analysis confirmed that the variants identified in each patient were present in the heterozygous state in their corresponding family members, which was consistent with an autosomal recessive mode of inheritance. CONCLUSIONS: This study demonstrated the first molecular and clinical characterization of ACHM in Thai patients. The identification of disease-associated genes in a specific population leads to a personalized gene therapy benefiting those affected patients.

Deep-intronic variants in CNGB3 cause achromatopsia by pseudoexon activation.

Our comprehensive cohort of 1100 unrelated achromatopsia (ACHM) patients comprises a considerable number of cases (~5%) harboring only a single pathogenic variant in the major ACHM gene CNGB3. We sequenced the entire CNGB3 locus in 33 of these patients to find a second variant which eventually explained the patients' phenotype. Forty-seven intronic CNGB3 variants were identified in 28 subjects after a filtering step based on frequency and the exclusion of variants found in cis with pathogenic alleles. In a second step, in silico prediction tools were used to filter out those variants with little odds of being deleterious. This left three variants that were analyzed using heterologous splicing assays. Variant c.1663-1205G>A, found in 14 subjects, and variant c.1663-2137C>T, found in two subjects, were indeed shown to exert a splicing defect by causing pseudoexon insertion into the transcript. Subsequent screening of further unsolved CNGB3 subjects identified four additional cases harboring the c.1663-1205G>A variant which makes it the eighth most frequent CNGB3 variant in our cohort. Compound heterozygosity could be validated in ten cases. Our study demonstrates that whole gene sequencing can be a powerful approach to identify the second pathogenic allele in patients apparently harboring only one disease-causing variant.

CNGB3 mutation spectrum including copy number variations in 552 achromatopsia patients.

Achromatopsia is a rare autosomal recessive cone disorder characterized by color vision defects, photophobia, nystagmus, and severely reduced visual acuity. The disease is caused by mutations in genes encoding crucial components of the cone phototransduction cascade (CNGA3, CNGB3, GNAT2, PDE6C, and PDE6H) or in ATF6, involved in the unfolded protein response. CNGB3 encoding the beta subunit of the cyclic nucleotide-gated ion channel in cone photoreceptors is the major achromatopsia gene. Here, we present a comprehensive spectrum of CNGB3 mutations and their prevalence in a cohort of 1074 independent families clinically diagnosed with achromatopsia. Of these, 485 (45.2%) carried mutations in CNGB3. We identified a total of 98 different potentially disease-causing CNGB3 variants, 58 of which are novel. About 10% of patients with CNGB3 mutations only harbored a single heterozygous variant. Therefore, we performed quantitative real-time PCR in 43 of such single heterozygotes in search of the missing allele, followed by microarray-based comparative genomic hybridization and breakpoint mapping. We discovered nine different heterozygous copy number variations encompassing one to 10 consecutive exons in 16 unrelated patients. Moreover, one additional patient with a homozygous CNGB3 deletion encompassing exons 4-18 was identified, highlighting the importance of CNV analysis for this gene.

Hereditary Retinal Dystrophy.

As our understanding of the genetic basis for inherited retinal disease has expanded, gene therapy has advanced into clinical development. When the gene mutations associated with inherited retinal dystrophies were identified, it became possible to create animal models in which individual gene were altered to match the human mutations. The retina of these animals were then characterized to assess whether the mutated genes produced retinal phenotypes characteristic of disease-affected patients. Following the identification of a subpopulation of patients with the affected gene and the development of techniques for the viral gene transduction of retinal cells, it has become possible to deliver a copy of the normal gene into the retinal sites of the mutated genes. When this was performed in animal models of monogenic diseases, at an early stage of retinal degeneration when the affected cells remained viable, successful gene augmentation corrected the structural and functional lesions characteristic of the specific diseases in the areas of the retina that were successfully transduced. These studies provided the essential proof-of-concept needed to advance monogenic gene therapies into clinic development; these therapies include treatments for: Leber's congenital amaurosis type 2, caused by mutations to RPE65, retinoid isomerohydrolase; choroideremia, caused by mutations to REP1, Rab escort protein 1; autosomal recessive Stargardt disease, caused by mutations to ABCA4, the photoreceptor-specific ATP-binding transporter; Usher 1B disease caused by mutations to MYO7A, myosin heavy chain 7; X-linked juvenile retinoschisis caused by mutations to RS1, retinoschisin; autosomal recessive retinitis pigmentosa caused by mutations to MERTK, the proto-oncogene tyrosine-protein kinase MER; Leber's hereditary optic neuropathy caused by mutations to ND4, mitochondrial nicotinamide adenine dinucleotide ubiquinone oxidoreductase (complex I) subunit 4 and achromatopsia, caused by mutations to CNGA3, cyclic nucleotide-gated channel alpha 3 and CNGB3, cyclic nucleotide-gated channel beta 3. This review includes a tabulated summary of treatments for these monogenic retinal dystrophies that have entered into clinical development, as well as a brief summary of the preclinical data that supported their advancement into clinical development.

[Molecular genetic diagnosis of Stargardt disease]. / Molekuliarno-geneticheskaia diagnostika bolezni Shtargardta.

AIM: To comparatively evaluate the efficacy of genetic screening in patients with Stargardt disease (SD) by using an express panel of 5 most common ABCA4 mutations and performing massive parallel sequencing of all coding regions of the ABCA4, ELOVL4, PROM1, and CNGB3 genes. MATERIAL AND METHODS: MLPA analysis for 5 ABCA4 mutations, namely p.G863A, p.L541P, p.A1038V, p.G1961E, and p.P1380L, was done in 54 patients with SD. In 25 patients, massive parallel sequencing of coding regions (exons) and neighboring introns of the ABCA4, ELOVL4, PROM1, and CNGB3 genes was also performed. RESULTS: Gene testing for 5 ABCA4 mutations showed that 50% of patients (27 patients) harbored one mutation and 13% - two mutations. At massive parallel sequencing (25 patients), two pathogenic alleles were found in 21 patients (84%), one mutation - in 23 patients (91.7%). The majority of mutations was accounted for by the ABCA4 gene (83% of all mutation-positive patients). CONCLUSION: Sequencing of exons and neighboring introns of the ABCA4, ELOVL4, PROM1, and CNGB3 genes with the new molecular genetic diagnostic system enabled confirmation of the diagnosis of SD in 84% of patients. High prevalence of p.L541P, p.A1038V, and p.G1961E mutations of the ABCA4 gene has been established.

Achromatopsia in three sibling Labrador Retrievers in the UK.

Achromatopsia was identified in three Labrador Retriever littermates. The dogs demonstrated day blindness, negotiating obstacles under low-light conditions, but apparently blind when outdoors. One of the dogs presented with immature bilateral diffuse posterior cortical cataracts and clinical signs of day blindness became apparent following cataract extraction surgery. Electroretinography demonstrated an absence of a cone photoreceptor response to a bright stimulus and a flicker response of 30 Hz in all three dogs. No fundic lesions have been apparent ophthalmoscopically in any of the dogs as the initial presentation of each case. No abnormalities were detected with DNA screening for known mutations of the CNGB3 gene in any of the dogs.

Intact high-level visual functions in congenital rod-monochromacy.

High-level visual functions such as reading and face recognition rely on global processes, which are often insensitive to high spatial frequencies. However, it is unknown whether a sharp cone signal is necessary for the development of these skills or whether a blurry rod signal is sufficient. CNGA3/B3-achromatopsia is a congenital disease stemming from cone dysfunction, leading to rod-only vision characterized by nystagmus, impaired acuity, and complete color blindness. We tested reading and face recognition in CNGA3/B3-achromatopsia patients (ACHM) to determine whether a rod signal is sufficient for these skills to develop. We tested 10 ACHM and 10 controls in three experiments under dark and light conditions. Initially, we evaluated acuity along the eccentricity axis. Later, we tested reading speed and upright/inverted face matching accuracy while tracking participants' eye movements. Given that ACHM patients' acuity under light conditions resembled that of controls under dark conditions, we selected these conditions for comparison. Remarkably, ACHM reading speed, face recognition abilities, and susceptibility to face inversion were not inferior to those of controls. Additionally, ACHM patients exhibited similar eye movements to controls, focusing their attention on specific areas of words and faces that indicate expertise. Despite the evident low-level limitations, ACHM patients demonstrated notable high-level visual skills, suggesting that rod-only vision is sufficient for the development of proficient reading and face recognition. These findings not only corroborate empirical evidence for high-level vision models but also enrich the discussion regarding the reasons for high-level deficits observed in individuals who have gained sight late in life.

Absent meibomian glands and cone dystrophy in ADULT syndrome: identification by whole exome sequencing of pathogenic variants in two causal genes TP63 and CNGB3.

BACKGROUND: Ectrodactyly is a rare congenital limb malformation characterized by a deep median cleft of the hand and/or foot due to the absence of central rays. It could be isolated or depicts a part of diverse syndromic forms. Heterozygous pathogenic variants in the TP63 gene are responsible for at least four rare syndromic human disorders associated with ectrodactyly. Among them, ADULT (Acro-Dermato-Ungual-Lacrimal-Tooth) syndrome is characterized by ectodermal dysplasia, excessive freckling, nail dysplasia, and lacrimal duct obstruction, in addition to ectrodactyly and/or syndactyly. Ophthalmic findings are very common in TP63-related disorders, consisting mainly of lacrimal duct hypoplasia. Absent meibomian glands have also been well documented in EEC3 (Ectrodactyly Ectodermal dysplasia Cleft lip/palate) syndrome but not in ADULT syndrome. METHODS: We report a case of syndromic ectrodactyly consistent with ADULT syndrome, with an additional ophthalmic manifestation of agenesis of meibomian glands. The proband, as well as her elder sister, presented with congenital cone dystrophy.The molecular investigation was performed in the proband using Whole Exome Sequencing. Family segregation of the identified variants was confirmed by Sanger sequencing. RESULTS: Two clinically relevant variants were found in the proband: the novel de novo heterozygous missense c.931A > G (p.Ser311Gly) in the TP63 gene classified as pathogenic, and the homozygous nonsense pathogenic c.1810C > T (p.Arg604Ter) in the CNGB3 gene. The same homozygous CNGB3 variation was also found in the sister, explaining the cone dystrophy in both cases. CONCLUSIONS: Whole Exome Sequencing allowed dual molecular diagnoses: de novo TP63-related syndromic ectrodactyly and familial CNGB3-related congenital cone dystrophy.

Phenotypic characteristics of Danish patients with achromatopsia.

PURPOSE: To describe the phenotype of Danish patients with genetically verified achromatopsia (ACHM) with special focus on signs of progression on structural or functional parameters, and possible genotype-phenotype correlations. METHODS: Forty-eight patients were identified, with disease-causing variants in five different genes: CNGA3, CNGB3, GNAT2, PDE6C and PDE6H. Longitudinal evaluation was possible for 11 patients and 27 patients participated in a renewed in-depth phenotyping consisting of visual acuity assessment, optical coherence tomography (OCT), fundus autofluorescence, colour vision evaluation, contrast sensitivity, mesopic microperimetry and full-field electroretinography. Foveal morphology was evaluated based on OCT images for all 48 patients using a grading system based on the integrity of the hyperreflective photoreceptor band, the inner segment ellipsoid zone (ISe). Signs of progression were evaluated based on longitudinal data and correlation with age. RESULTS: We found a statistically significant positive correlation between OCT grade and age (Spearman ρ = 0.62, p < 0.0001) and we observed changes in the foveal morphology in 2 of 11 patients with ≥5 years of follow-up. We did not find any convincing correlation between age and functional parameters (visual acuity, retinal sensitivity and contrast sensitivity) nor did we find correlation between structural and functional parameters, or any clear genotype-phenotype correlation. CONCLUSIONS: Some patients with ACHM demonstrate signs of progressive foveal changes in OCT characteristics with increasing age. This is relevant in terms of possible new treatments. However, functional characteristics, such as visual acuity, remained stable despite changing foveal structure. Thus, seen from a patient perspective, ACHM can still be considered a non-progressive condition.

Phenotyping and genotyping inherited retinal diseases: Molecular genetics, clinical and imaging features, and therapeutics of macular dystrophies, cone and cone-rod dystrophies, rod-cone dystrophies, Leber congenital amaurosis, and cone dysfunction syndromes.

Inherited retinal diseases (IRD) are a leading cause of blindness in the working age population and in children. The scope of this review is to familiarise clinicians and scientists with the current landscape of molecular genetics, clinical phenotype, retinal imaging and therapeutic prospects/completed trials in IRD. Herein we present in a comprehensive and concise manner: (i) macular dystrophies (Stargardt disease (ABCA4), X-linked retinoschisis (RS1), Best disease (BEST1), PRPH2-associated pattern dystrophy, Sorsby fundus dystrophy (TIMP3), and autosomal dominant drusen (EFEMP1)), (ii) cone and cone-rod dystrophies (GUCA1A, PRPH2, ABCA4, KCNV2 and RPGR), (iii) predominant rod or rod-cone dystrophies (retinitis pigmentosa, enhanced S-Cone syndrome (NR2E3), Bietti crystalline corneoretinal dystrophy (CYP4V2)), (iv) Leber congenital amaurosis/early-onset severe retinal dystrophy (GUCY2D, CEP290, CRB1, RDH12, RPE65, TULP1, AIPL1 and NMNAT1), (v) cone dysfunction syndromes (achromatopsia (CNGA3, CNGB3, PDE6C, PDE6H, GNAT2, ATF6), X-linked cone dysfunction with myopia and dichromacy (Bornholm Eye disease; OPN1LW/OPN1MW array), oligocone trichromacy, and blue-cone monochromatism (OPN1LW/OPN1MW array)). Whilst we use the aforementioned classical phenotypic groupings, a key feature of IRD is that it is characterised by tremendous heterogeneity and variable expressivity, with several of the above genes associated with a range of phenotypes.

Molecular and Clinical Characterization of CNGA3 and CNGB3 Genes in Brazilian Patients Affected with Achromatopsia.

Achromatopsia (ACHM) is a congenital cone photoreceptor disorder characterized by reduced visual acuity, nystagmus, photophobia, and very poor or absent color vision. Pathogenic variants in six genes encoding proteins composing the cone phototransduction cascade (CNGA3, CNGB3, PDE6C, PDE6H, GNAT2) and of the unfolded protein response (ATF6) have been related to ACHM cases, while CNGA3 and CNGB3 alone are responsible for most cases. Herein, we provide a clinical and molecular overview of 42 Brazilian patients from 38 families affected with ACHM related to biallelic pathogenic variants in the CNGA3 and CNGB3 genes. Patients' genotype and phenotype were retrospectively evaluated. The majority of CNGA3 variants were missense, and the most prevalent CNGB3 variant was c.1148delC (p.Thr383Ilefs*13), resulting in a frameshift and premature stop codon, which is compatible with previous publications in the literature. A novel variant c.1893T>A (p.Tyr631*) in the CNGB3 gene is reported for the first time in this study. A great variability in morphologic findings was observed in our patients, although no consistent correlation with age and disease stage in OCT foveal morphology was found. The better understanding of the genetic variants landscape in the Brazilian population will help in the diagnosis of this disease.

Clinical and Genetic Features of Korean Patients with Achromatopsia.

This multicenter study aimed to characterize Korean patients with achromatopsia. The patients' genotypes and phenotypes were retrospectively evaluated. Twenty-one patients (with a mean age at the baseline of 10.9 years) were enrolled and followed up for a mean of 7.3 years. A targeted gene panel or exome sequencing was performed. The pathogenic variants of the four genes and their frequencies were identified. CNGA3 and PDE6C were equally the most prevalent genes: CNGA3 (N = 8, 38.1%), PDE6C (N = 8, 38.1%), CNGB3 (N = 3, 14.3%), and GNAT2 (N = 2, 9.5%). The degree of functional and structural defects varied among the patients. The patients' age exhibited no significant correlation with structural defects. During the follow-up, the visual acuity and retinal thickness did not change significantly. In CNGA3-achromatopsia patients, a proportion of patients with a normal foveal ellipsoid zone on the OCT was significantly higher than that of patients with other causative genes (62.5% vs. 16.7%; p = 0.023). In PDE6C-achromatopsia patients, the same proportion was significantly lower than that of patients with other causative genes (0% vs. 58.3%; p = 0.003). Korean patients with achromatopsia showed similar clinical features but a higher prevalence of PDE6C variants than those of other ethnic groups. The retinal phenotypes of the PDE6C variants were more likely to be worse than those of other genes.

Genetic and Clinical Characterization of Danish Achromatopsia Patients.

Achromatopsia is a rare congenital condition with cone photoreceptor dysfunction causing color blindness, reduced vision, nystagmus and photophobia. New treatments are being developed, but the current evidence is still conflicting regarding possible progression over time, and there is no clear genotype-phenotype correlation. This natural history study aimed to further explore the course of disease and potential clinical differences between various genotypes. The retrospective design allowed for the study of a large cohort with a long follow-up. Patients were identified from the Danish national registries. If not already available, genetic analysis was offered to the patient. Clinical data from 1945-2022 were retrieved from medical records and included best-corrected visual acuity (BCVA), color vision, refractive error, nystagmus, visual fields and fundoscopic findings. We identified variants believed to be disease causing in five of the known achromatopsia genes: CNGA3; CNGB3; GNAT2; PDE6C and PDE6H; and novel variants were identified in CNGB3 and PDE6C. Progressive deterioration of BCVA only attributable to achromatopsia was found in three of 58 patients. Progressive phenotype was seen with variants in CNGB3 and PDE6C. The results indicate that myopia could be more frequently occurring with variants in GNAT2, PDE6C and PDE6H and support the evidence that achromatopsia is a predominantly stationary condition with respect to BCVA. Although a clear genotype-phenotype correlation can still not be concluded, there may be differences in phenotypical characteristics with variants in different genes.

Progress in treating inherited retinal diseases: Early subretinal gene therapy clinical trials and candidates for future initiatives.

Due to improved phenotyping and genetic characterization, the field of 'incurable' and 'blinding' inherited retinal diseases (IRDs) has moved substantially forward. Decades of ascertainment of IRD patient data from Philadelphia and Toronto centers illustrate the progress from Mendelian genetic types to molecular diagnoses. Molecular genetics have been used not only to clarify diagnoses and to direct counseling but also to enable the first clinical trials of gene-based treatment in these diseases. An overview of the recent reports of gene augmentation clinical trials by subretinal injections is used to reflect on the reasons why there has been limited success in this early venture into therapy. These first-in human experiences have taught that there is a need for advancing the techniques of delivery of the gene products - not only for refining further subretinal trials, but also for evaluating intravitreal delivery. Candidate IRDs for intravitreal gene delivery are then suggested to illustrate some of the disorders that may be amenable to improvement of remaining central vision with the least photoreceptor trauma. A more detailed understanding of the human IRDs to be considered for therapy and the calculated potential for efficacy should be among the routine prerequisites for initiating a clinical trial.

Molecular genetic cause of achromatopsia in two patients of Czech origin.

INTRODUCTION: Achromatopsia is an autosomal recessive retinal disorder with an estimated prevalence ranging from 1 in 30.000 to 50.000. The disease is caused by mutations in six different genes. The aim of the study was to perform molecular genetic analysis in 11 unrelated probands with a clinical diagnosis of achromatopsia and to describe clinical findings in those that were found to carry biallelic pathogenic mutations. METHODS: All probands and their parents underwent ophthalmic examination. Mutation detection was performed using Sanger sequencing of CNGB3 exons 6, 7, 9-13, which have been found to harbour most disease-causing mutations in patients with achromatopsia of European origin. RESULTS: Three known pathogenic variants in CNGB3 were identified in 2 probands. Proband 1 was a compound heterozygote for the c.819_826del; p.(Arg274Valfs*13) and c.1006G>T; p.(Glu336*). Proband 2 carried the c.1148del; p.(Thr383Ilefs*13) in a homozygous state. The best corrected visual acuity in proband 1 (aged 19 years) was 0.1 in both eyes, in proband 2 (aged 8 years) 0.05 in the right eye and 0.1 in the left eye. Both individuals had nystagmus, photophobia, and absence of colour discrimination. Fundus examination appeared normal however spectral-domain optical coherence tomography revealed subtle bilaterally symmetrical structural changes in the fovea. CONCLUSION: Molecular genetic analysis of Czech patients with achromatopsia was performed for the first time. Identification of disease-causing mutations in achromatopsia is important for establishing an early diagnosis, participation in clinical trials assessing gene therapies and may be also used for preimplantation genetic diagnosis.

AAV-mediated human CNGB3 restores cone function in an all-cone mouse model of CNGB3 achromatopsia.

Complete congenital achromatopsia is a devastating hereditary visual disorder. Mutations in the CNGB3 gene account for more than 50% of all known cases of achromatopsia. This work investigated the efficiency of subretinal (SR) delivered AAV8 (Y447, 733F) vector containing a human PR2.1 promoter and a human CNGB3 cDNA in Cngb3 -/-/ Nrl -/- mice. The Cngb3 -/-/ Nrl -/- mouse was a cone-dominant model with Cngb3 channel deficiency, which partially mimicked the all-cone foveal structure of human achromatopsia with CNGB3 mutations. Following SR delivery of the vector, AAV-mediated CNGB3 expression restored cone function which was assessed by the restoration of the cone-mediated electroretinogram (ERG) and immunohistochemistry. This therapeutic rescue resulted in long-term improvement of retinal function with the restoration of cone ERG amplitude. This study demonstrated an AAV-mediated gene therapy in a cone-dominant mouse model using a human gene construct and provided the potential to be utilized in clinical trials.

Inherited macular degeneration-associated mutations in CNGB3 increase the ligand sensitivity and spontaneous open probability of cone cyclic nucleotide-gated channels.

Cyclic nucleotide gated (CNG) channels are a critical component of the visual transduction cascade in the vertebrate retina. Mutations in the genes encoding these channels have been associated with a spectrum of inherited retinal disorders. To gain insight into their pathophysiological mechanisms, we have investigated the functional consequences of several CNGB3 mutations, previously associated with macular degeneration (Y469D and L595F) or complete achromatopsia (S156F, P309L, and G558C), by expressing these subunits in combination with wild-type CNGA3 in Xenopus oocytes and characterizing them using patch-clamp recordings in the inside-out configuration. These mutations did not prevent the formation of functional heteromeric channels, as indicated by sensitivity to block by L-cis-diltiazem. With the exception of S156F, each of the mutant channels displayed electrophysiological properties reflecting enhanced channel activity at physiological concentrations of cGMP (i.e., a gain-of-function phenotype). The increased channel activity produced by these mutations resulted from either increased functional expression levels, or increased sensitivity to cyclic nucleotides. Furthermore, L595F increased the spontaneous open probability in the absence of activating ligand, signifying a ligand independent gain-of-function change. In addition to the CNGB3 disease-associate mutations, we characterized the effects of several common CNGB3 and CNGA3 single-nucleotide polymorphisms (SNPs) on heteromeric CNGA3+CNGB3 channel function. Two of the SNPs examined (A3-T153M, and B3-W234C) produced decreased ligand sensitivity for heteromeric CNG channels. These changes may contribute to background disease susceptibility when combined with other genetic or non-genetic factors. Together, these studies help to define the underlying molecular phenotype for mutations relating to CNG channel disease pathogenesis.