CNGB3 Research Articles

Genetic testing for Stargardt macular dystrophy

Abstract We studied the scientific literature and disease guidelines in order to summarize the clinical utility of genetic testing for Stargardt macular dystrophy (STGD). STGD is mostly inherited in an autosomal recessive manner and rarely in an autosomal dominant manner, with an overall prevalence of 1-5 per 10 000 live births. It is caused by variations in the ABCA4, CNGB3, ELOVL4, PRPH2 and PROM1 genes. Clinical diagnosis is based on ophthalmological examination, fluorescein angiography, electroretinography, visual field testing, optical coherence tomography and color testing. The genetic test is useful for confirming diagnosis, and for differential diagnosis, couple risk assessment and access to clinical trials.

Genetic testing for achromatopsia

Abstract We studied the scientific literature and disease guidelines in order to summarize the clinical utility of genetic testing for achromatopsia. The disease has autosomal recessive inheritance, a prevalence of 1/30000-1/50000, and is caused by mutations in the CNGB3, CNGA3, GNAT2, PDE6C, ATF6 and PDE6H genes. Clinical diagnosis is by ophthalmological examination, color vision testing and electrophysiological testing. Genetic testing is useful for confirming diagnosis and for differential diagnosis, couple risk assessment and access to clinical trials.

Clinical and Genetic Evaluation of a Cohort of Pediatric Patients with Severe Inherited Retinal Dystrophies.

We performed a clinical and genetic characterization of a pediatric cohort of patients with inherited retinal dystrophy (IRD) to identify the most suitable cases for gene therapy. The cohort comprised 43 patients, aged between 2 and 18 years, with severe isolated IRD at the time of presentation. The ophthalmological characterization also included assessment of the photoreceptor layer integrity in the macular region (ellipsoid zone (EZ) band). In parallel, we carried out a targeted, next-generation sequencing (NGS)-based analysis using a panel that covers over 150 genes with either an established or a candidate role in IRD pathogenesis. Based on the ophthalmological assessment, the cohort was composed of 24 Leber congenital amaurosis, 14 early onset retinitis pigmentosa, and 5 achromatopsia patients. We identified causative mutations in 58.1% of the cases. We also found novel genotype-phenotype correlations in patients harboring mutations in the CEP290 and CNGB3 genes. The EZ band was detectable in 40% of the analyzed cases, also in patients with genotypes usually associated with severe clinical manifestations. This study provides the first detailed clinical-genetic assessment of severe IRDs with infantile onset and lays the foundation of a standardized protocol for the selection of patients that are more likely to benefit from gene replacement therapeutic approaches.

Next generation sequencing identified novel heterozygous nonsense mutation in CNGB1 gene associated with retinitis pigmentosa in a Chinese patient.

Retinitis pigmentosa (RP) is a severe hereditary eye disease characterized by progressive degeneration of photoreceptors and subsequent loss of vision. Retinitis pigmentosa (RP) is a clinically and genetically heterogeneous group of retinal diseases. Germline mutations of CNGB1 is associated with retinitis pigmentosa. We have identified and investigated a 34-year-old Chinese man with markedly have night vision blindness and loss of midperipheral visual field. The proband also lose his far peripheral visual field and also central vision. Proband’s retinal pigment deposits visible on fundus examination and primary loss of rod photoreceptor cells followed by secondary loss of cone photoreceptors. Target exome capture based next generation sequencing and Sanger sequencing identified novel nonsense mutation, c.1917G>A and a reported mutation, c.2361C>A, in the CNGB1 gene. Both the nonsense mutations are predicted to lead to the formation of a premature stop codon which finally results into formation of truncated CNGB1 protein product which finally predicted to be disease causing. According to the variant classification guidelines of ACMG, these two variants are categorized as “likely pathogenic” variants. Our findings expand the mutational spectra of CNGB1 and are valuable in the mutation-based pre- and post-natal screening and genetic diagnosis for retinitis pigmentosa.

Myotis rufoniger genome sequence and analyses: M. rufoniger's genomic feature and the decreasing effective population size of Myotis bats.

Myotis rufoniger is a vesper bat in the genus Myotis. Here we report the whole genome sequence and analyses of the M. rufoniger. We generated 124 Gb of short-read DNA sequences with an estimated genome size of 1.88 Gb at a sequencing depth of 66× fold. The sequences were aligned to M. brandtii bat reference genome at a mapping rate of 96.50% covering 95.71% coding sequence region at 10× coverage. The divergence time of Myotis bat family is estimated to be 11.5 million years, and the divergence time between M. rufoniger and its closest species M. davidii is estimated to be 10.4 million years. We found 1,239 function-altering M. rufoniger specific amino acid sequences from 929 genes compared to other Myotis bat and mammalian genomes. The functional enrichment test of the 929 genes detected amino acid changes in melanin associated DCT, SLC45A2, TYRP1, and OCA2 genes possibly responsible for the M. rufoniger’s red fur color and a general coloration in Myotis. N6AMT1 gene, associated with arsenic resistance, showed a high degree of function alteration in M. rufoniger. We further confirmed that the M. rufoniger also has bat-specific sequences within FSHB, GHR, IGF1R, TP53, MDM2, SLC45A2, RGS7BP, RHO, OPN1SW, and CNGB3 genes that have already been published to be related to bat’s reproduction, lifespan, flight, low vision, and echolocation. Additionally, our demographic history analysis found that the effective population size of Myotis clade has been consistently decreasing since ~30k years ago. M. rufoniger’s effective population size was the lowest in Myotis bats, confirming its relatively low genetic diversity.

Molecular genetic diagnosis of Stargardt disease

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. 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. 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). 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.

Cav1.4 L-Type Calcium Channels Contribute to Calpain Activation in Degenerating Photoreceptors of rd1 Mice.

Retinitis pigmentosa is an inherited blinding disorder characterized by progressive degeneration and loss of photoreceptors. The exact mechanism of degeneration and cell death of photoreceptors is not known, but is thought to involve disturbed Ca2+—signaling. Ca2+ can enter the photoreceptor cell via outer segment cyclic nucleotide-gated (CNG) channels or synaptic Cav1.4 L-type voltage-gated calcium channels (VGCC). Previously, we have shown that genetic ablation of the Cngb1 gene encoding the B subunit of the rod CNG channel delays the fast progressing degeneration in the rd1 mutant mouse model of retinitis pigmentosa. In this study, we crossbred rd1 mice with the Cacna1f-deficient mouse lacking the Cav1.4 α1 subunit of the L-type VGCC. Longitudinal in vivo examinations of photoreceptor layer thickness by optical coherence tomography revealed a significant, but not sustained delay of retinal degeneration in Cacna1f x rd1 double mutant mice compared to rd1 mice. This was accompanied by a reduction of TUNEL positive cells in the early phase of rod degeneration. Remarkably, Cacna1f x rd1 double mutant mice displayed a strong decrease in the activation of the Ca2+-dependent protease calpain during photoreceptor loss. Our results show that genetic deletion of the synaptic Cav1.4 L-type VGCCs impairs calpain activation and leads to a short-term preservation of photoreceptors in the rd1 mouse.

88. Safety and Biodistribution Study of rAAV2tYF-PR1.7-hCNGB3 in Nonhuman Primates

Background: AGTC is developing a recombinant AAV vector expressing the human CNGB3 gene for treatment of achromatopsia, an inherited retinal disorder characterized by markedly reduced visual acuity, extreme light sensitivity and absence of color discrimination. Here we report results of a toxicology and biodistribution study of this vector administered by subretinal injection in cynomolgus macaques. Methods: Three groups of animals (n=2 males and 2 females per group) received a subretinal injection in one eye of 300 µL containing either vehicle or rAAV2tYF-PR1.7-hCNGB3 at one of two concentrations (4 × 1011 or 4 × 1012 vg/mL) and were evaluated for safety and biodistribution over a 3-month period prior to euthanasia. Toxicity assessment was based on mortality, clinical observations, body weights, ophthalmic examinations, intraocular pressure (IOP) measurements, electroretinography (ERG), visual evoked potentials (VEP), and clinical and anatomic pathology. Vector shedding and biodistribution was assessed by qPCR analyses. Immune responses to AAV and hCNGB3 were measured by ELISA, Elispot, or neutralization antibody assay for AAV2tYF. Results: There was no evidence of local or systemic toxicity and no changes in IOP, VEP responses, or hematology, coagulation or clinical chemistry parameters and no clinically important changes in ERG responses. Aqueous cells, sometimes with aqueous flare, were observed at the Day 3 evaluation in all groups and generally resolved or were at the mild (1+) levels by Week 4 and absent on Week 8 and thereafter except in one high dose animal. Posterior segment findings consisted of varying degrees of dose-related white vitreous cell, vitreous haze, white retinal perivascular sheathing, and white to grey-white subretinal infiltrates within and outside of the injection site. Vitreous haze resolved by Day 8 in eyes given vehicle control, by Week 4 in the low dose group and by Week 13 in the high dose group. Vitreous cells were observed at the mild (trace or 1+) level in the vehicle control group and resolved by Study Weeks 9 or 13 but persisted through Week 13 in a dose-related fashion in the low and high dose groups. Serum neutralizing antibodies against AAV2tYF were detected in all animals given vector. There were no T cell responses to AAV capsid peptides and no antibody or T cell responses to hCNGB3. Mononuclear cell infiltrates in the vitreous body/optic disc, of minimal intensity, in the vector-injected eye of all animals at both dose levels. All other tissues collected for histopathological examination showed no abnormalities. Results of biodistribution studies demonstrated that the vector did not spread widely or consistently outside the injected eye. High levels of vector DNA were found in vector-injected eyes but minimal or no vector DNA was found in any other tissue. Conclusions: Subretinal injection of rAAV2tYF-PR1.7-hCNGB3 at concentrations of 4 × 1011 or 4 × 1012 vg/mL was associated with a dose-related anterior and posterior segment inflammatory response that improved over time. There was no evidence of systemic toxicity and no changes in IOP, VEP responses, or hematology, coagulation or clinical chemistry parameters and no clinically important changes in ERG responses. These results support the use of rAAV2tYF-PR1.7-hCNGB3 in clinical studies in patients with achromatopsia. A Phase 1/2 clinical trial evaluating rAAV2tYF-PR1.7-hCNGB3 in patients with achromatopsia is scheduled to begin in 2016.

299. Safety and Biodistribution Study of rAAV2tYF-PR1.7-hCNGB3 in CNGB3-Deficient Mice

Background: AGTC is developing a recombinant adeno-associated virus (rAAV) vector expressing the human CNGB3 gene, for treatment of achromatopsia, an inherited retinal disorder characterized by markedly reduced visual acuity, extreme light sensitivity and absence of color discrimination. Here we report results of a toxicology and biodistribution study of this vector administered by subretinal injection in CNGB3-deficient mice. Methods: Three groups of CNGB3-deficient mice (n= 35 per sex per group) received a subretinal injection in one eye of 1 µL of vehicle (balanced salt solution with 0.014% Tween 20) or rAAV2tYF-PR1.7-hCNGB3 vector at a concentration of 1 × 1012 vg/mL (1 × 109 vg/eye) or 4 × 1012 vg/mL (4 × 109 vg/eye). The other eye was untreated. Ten animals/sex/group were used for toxicology evaluation with ophthalmic examinations and pathological evaluations, 10 animals/sex/group were used for biodistribution evaluation, and 15 animals/sex/group were used for efficacy evaluation. Half the animals in the biodistribution and toxicology groups were euthanized 4 weeks after vector administration and the remaining animals were euthanized 12 weeks after vector administration. For animals in the biodistribution groups, blood for qPCR analysis was obtained on Study Days 3, 8 and at euthanasia. At necropsy, samples of eyes, brain, heart, liver, gall bladder, kidneys, spleen, thymus, lungs, adrenals, ovaries, epididymides and testes were obtained for histopathology (for animals in the toxicology groups) or DNA PCR (for animals in the biodistribution groups). For animals scheduled for efficacy evaluations, electroretinography (ERG) testing included scotopic and photopic tests performed at Week 4, 8, and 12 on each eye and serum was collected at euthanasia for measurement of antibodies to AAV and hCNGB3. Results: There were no test article-related changes in clinical observations, body weights, food consumption, ocular examinations, clinical pathology parameters, organ weights, or macroscopic observations at necropsy. Cone-mediated ERG responses were detected after vector administration in the treated eyes in 90% of animals in the higher dose group, 31% of animals in the lower dose group, and none of the untreated or vehicle-treated eyes. Microscopic pathology results demonstrated minimal mononuclear cell infiltrates in the retina and vitreous of some animals at the interim euthanasia, and in the vitreous of some animals at the terminal euthanasia. Serum anti-AAV antibodies developed in most vector-injected animals. No animals developed antibodies to hCNGB3. Biodistribution studies demonstrated high levels of vector DNA in vector-injected eyes but little or no vector DNA in non-ocular tissue. Conclusions: Subretinal injection of rAAV2tYF-PR1.7-hCNGB3 in CNGB3-deficient mice was associated with no clinically important toxicology findings, rescue of cone-mediated ERG responses in vector-treated eyes, and vector DNA detection limited primarily to vector-injected eyes. These results support the use of rAAV2tYF-PR1.7-hCNGB3 in clinical studies in patients with achromatopsia caused by CNGB3 mutations. A Phase 1/2 clinical trial evaluating rAAV2tYF-PR1.7-hCNGB3 is scheduled to begin in 2016.

Safety and Biodistribution Evaluation in Cynomolgus Macaques of rAAV2tYF-PR1.7-hCNGB3, a Recombinant AAV Vector for Treatment of Achromatopsia.

Applied Genetic Technologies Corporation (AGTC) is developing rAAV2tYF-PR1.7-hCNGB3, a recombinant adeno-associated viral (rAAV) vector expressing the human CNGB3 gene, for treatment of achromatopsia, an inherited retinal disorder characterized by markedly reduced visual acuity, extreme light sensitivity, and absence of color discrimination. We report here results of a study evaluating the safety and biodistribution of rAAV2tYF-PR1.7-hCNGB3 in cynomolgus macaques. Three groups of animals (n = 2 males and 2 females per group) received a subretinal injection in one eye of 300 μl containing either vehicle or rAAV2tYF-PR1.7-hCNGB3 at one of two concentrations (4 × 10(11) or 4 × 10(12) vector genomes/ml) and were evaluated over a 3-month period before being euthanized. Administration of rAAV2tYF-PR1.7-hCNGB3 was associated with a dose-related anterior and posterior segment inflammatory response that was greater than that observed in eyes injected with the vehicle control. Most manifestations of inflammation improved over time except that vitreous cells persisted in vector-treated eyes until the end of the study. One animal in the lower vector dose group was euthanized on study day 5, based on a clinical diagnosis of endophthalmitis. There were no test article-related effects on intraocular pressure, visual evoked potential responses, hematology or clinical chemistry parameters, or gross necropsy observations. Histopathological examination demonstrated minimal mononuclear infiltrates in all vector-injected eyes. Serum anti-AAV antibodies developed in all vector-injected animals. No animals developed antibodies to CNGB3. Biodistribution studies demonstrated high levels of vector DNA in the injected eye but minimal or no vector DNA in any other tissue. These results support the use of rAAV2tYF-PR1.7-hCNGB3 in clinical studies in patients with achromatopsia caused by CNGB3 mutations.

Safety and Biodistribution Evaluation in CNGB3-Deficient Mice of rAAV2tYF-PR1.7-hCNGB3, a Recombinant AAV Vector for Treatment of Achromatopsia.

Applied Genetic Technologies Corporation (AGTC) is developing rAAV2tYF-PR1.7-hCNGB3, a recombinant adeno-associated virus (rAAV) vector expressing the human CNGB3 gene, for treatment of achromatopsia, an inherited retinal disorder characterized by markedly reduced visual acuity, extreme light sensitivity, and absence of color discrimination. We report here results of a study evaluating safety and biodistribution of rAAV2tYF-PR1.7-hCNGB3 in CNGB3-deficient mice. Three groups of animals (n = 35 males and 35 females per group) received a subretinal injection in one eye of 1 μl containing either vehicle or rAAV2tYF-PR1.7-hCNGB3 at one of two dose concentrations (1 × 10(12) or 4.2 × 10(12) vg/ml) and were euthanized 4 or 13 weeks later. There were no test-article-related changes in clinical observations, body weights, food consumption, ocular examinations, clinical pathology parameters, organ weights, or macroscopic observations at necropsy. Cone-mediated electroretinography (ERG) responses were detected after vector administration in the treated eyes in 90% of animals in the higher dose group and 31% of animals in the lower dose group. Rod-mediated ERG responses were reduced in the treated eye for all groups, with the greatest reduction in males given the higher dose of vector, but returned to normal by the end of the study. Microscopic pathology results demonstrated minimal mononuclear cell infiltrates in the retina and vitreous of some animals at the interim euthanasia and in the vitreous of some animals at the terminal euthanasia. Serum anti-AAV antibodies developed in most vector-injected animals. No animals developed antibodies to hCNGB3. Biodistribution studies demonstrated high levels of vector DNA in vector-injected eyes but little or no vector DNA in nonocular tissue. These results support the use of rAAV2tYF-PR1.7-hCNGB3 in clinical studies in patients with achromatopsia caused by CNGB3 mutations.

Cone-Specific Promoters for Gene Therapy of Achromatopsia and Other Retinal Diseases.

Adeno-associated viral (AAV) vectors containing cone-specific promoters have rescued cone photoreceptor function in mouse and dog models of achromatopsia, but cone-specific promoters have not been optimized for use in primates. Using AAV vectors administered by subretinal injection, we evaluated a series of promoters based on the human L-opsin promoter, or a chimeric human cone transducin promoter, for their ability to drive gene expression of green fluorescent protein (GFP) in mice and nonhuman primates. Each of these promoters directed high-level GFP expression in mouse photoreceptors. In primates, subretinal injection of an AAV-GFP vector containing a 1.7-kb L-opsin promoter (PR1.7) achieved strong and specific GFP expression in all cone photoreceptors and was more efficient than a vector containing the 2.1-kb L-opsin promoter that was used in AAV vectors that rescued cone function in mouse and dog models of achromatopsia. A chimeric cone transducin promoter that directed strong GFP expression in mouse and dog cone photoreceptors was unable to drive GFP expression in primate cones. An AAV vector expressing a human CNGB3 gene driven by the PR1.7 promoter rescued cone function in the mouse model of achromatopsia. These results have informed the design of an AAV vector for treatment of patients with achromatopsia.

Advances in genetic study of achromatopsia

Achromatopsia is a kind of autosomal recessive cone disorder. It occurs with nystagmus, photophobia, inability of color discrimination and severely reduced visual acuity. Five pathogenic genes had been reported to be associated with achromatopsia: cyclic nucleotide-gated(CNG)A3, CNGB3, guanine nucleotide binding protein alpha transduction active pepitide 2(GNAT2), phosphodiesterase (PDE)6C and PDE6H. They are crucial for cone phototransduction. Mutations of these genes can induce achromatopsia. Gene therapy, which can recover partial visual function, has been successfully used for the treatment of achromatopsia in animal model. Clinical features, pathogenic genes functions and mutations, the animal models and gene therapy of achromatopsia were reviewed. Key words: Achromatopsia; Mutation; Disease models, animal; Gene therapy

Achromatopsia: a review.

The purposes of this article are to examine the literature published on achromatopsia and provide a comprehensive review of the clinical disease, genetic characteristics, and potential for therapy. Specifically, this article will describe recent advances in gene therapy in animal models, clinical features in human, and barriers to human translation. Building on prior success with adeno-associated virus (AAV) therapy in mice models for achromatopsia with mutations in the CNGB3, CNGA3, or GNAT2 genes, multiple cone-specific promoters have recently been developed and shown success in mice and nonhuman primates. A sheep CNGA3 model has also been characterized. Two clinical trials are under way: one to better characterize humans with achromatopsia and another to study a ciliary neurotrophic factor (CNTF) implant as a treatment for patients with the CNGB3 mutation. Genetic understanding and disease characterization of achromatopsia continues to evolve, as do gene therapy tools and animal models. The potential for the treatment of achromatopsia in humans with gene therapy shows great promise.

Cyclic nucleotide-gated channels and retinal cone photoreceptor cells function

Cyclic nucleotide-gated (CNG) channels are ion channels which are activated by the binding of cyclic guanosine monophosphate (cGMP) or cyclic adenosine monophosphate (cAMP), they play a central role in the signal transduction pathways of vision and olfaction.Six different genes encode CNG protein, containing four A subunits (A1-A4) and two B subunits (B1 and B3). CNGA3 and CNGB3 have been found to be implicated in achromatopsia-associated mutations.Recently, a huge amount of researches showed the good responses to gene therapy in achromatopsia animal models.This article briefly reviewed the physiological roles of CNG channel in retinal cone photoreceptor cells and the recent research achievements of gene therapy in CNG channel-deficient mouse models with achromatopsia. Key words: Cyclic nucleotide-gated channel; Retinal cone photoreceptor cells; Gene, knockout

Spectral-Domain Optical Coherence Tomography Staging and Autofluorescence Imaging in Achromatopsia

IMPORTANCE Evidence is mounting that achromatopsia is a progressive retinal degeneration, and treatments for this condition are on the horizon. OBJECTIVES To categorize achromatopsia into clinically identifiable stages using spectral-domain optical coherence tomography and to describe fundus autofluorescence imaging in this condition. DESIGN, SETTING, AND PARTICIPANTS A prospective observational study was performed between 2010 and 2012 at the Edward S. Harkness Eye Institute, New York-Presbyterian Hospital. Participants included 17 patients (aged 10-62 years) with full-field electroretinography-confirmed achromatopsia. MAIN OUTCOMES AND MEASURES Spectral-domain optical coherence tomography features and staging system, fundus autofluorescence and near-infrared reflectance features and their correlation to optical coherence tomography, and genetic mutations served as the outcomes and measures. RESULTS Achromatopsia was categorized into 5 stages on spectral-domain optical coherence tomography: stage 1 (2 patients [12%]), intact outer retina; stage 2 (2 patients [12%]), inner segment ellipsoid line disruption; stage 3 (5 patients [29%]), presence of an optically empty space; stage 4 (5 patients [29%]), optically empty space with partial retinal pigment epithelium disruption; and stage 5 (3 patients [18%]), complete retinal pigment epithelium disruption and/or loss of the outer nuclear layer. Stage 1 patients showed isolated hyperreflectivity of the external limiting membrane in the fovea, and the external limiting membrane was hyperreflective above each optically empty space. On near infrared reflectance imaging, the fovea was normal, hyporeflective, or showed both hyporeflective and hyperreflective features. All patients demonstrated autofluorescence abnormalities in the fovea and/or parafovea: 9 participants (53%) had reduced or absent autofluorescence surrounded by increased autofluorescence, 4 individuals (24%) showed only reduced or absent autofluorescence, 3 patients (18%) displayed only increased autofluorescence, and 1 individual (6%) exhibited decreased macular pigment contrast. Inner segment ellipsoid line loss generally correlated with the area of reduced autofluorescence, but hyperautofluorescence extended into this region in 2 patients (12%). Bilateral coloboma-like atrophic macular lesions were observed in 1 patient (6%). Five novel mutations were identified (4 in the CNGA3 gene and 1 in the CNGB3 gene). CONCLUSIONS AND RELEVANCE Achromatopsia often demonstrates hyperautofluorescence suggestive of progressive retinal degeneration. The proposed staging system facilitates classification of the disease into different phases of progression and may have therapeutic implications.

Rip3 knockdown rescues photoreceptor cell death in blind pde6c zebrafish

Achromatopsia is a progressive autosomal recessive retinal disease characterized by early loss of cone photoreceptors and later rod photoreceptor loss. In most cases, mutations have been identified in CNGA3, CNGB3, GNAT2, PDE6C or PDE6H genes. Owing to this genetic heterogeneity, mutation-independent therapeutic schemes aimed at preventing cone cell death are very attractive treatment strategies. In pde6c(w59) mutant zebrafish, cone photoreceptors expressed high levels of receptor-interacting protein kinase 1 (RIP1) and receptor-interacting protein kinase 3 (RIP3) kinases, key regulators of necroptotic cell death. In contrast, rod photoreceptor cells were alternatively immunopositive for caspase-3 indicating activation of caspase-dependent apoptosis in these cells. Morpholino gene knockdown of rip3 in pde6c(w59) embryos rescued the dying cone photoreceptors by inhibiting the formation of reactive oxygen species and by inhibiting second-order neuron remodelling in the inner retina. In rip3 morphant larvae, visual function was restored in the cones by upregulation of the rod phosphodiesterase genes (pde6a and pde6b), compensating for the lack of cone pde6c suggesting that cones are able to adapt to their local environment. Furthermore, we demonstrated through pharmacological inhibition of RIP1 and RIP3 activity that cone cell death was also delayed. Collectively, these results demonstrate that the underlying mechanism of cone cell death in the pde6c(w59) mutant retina is through necroptosis, whereas rod photoreceptor bystander death occurs through a caspase-dependent mechanism. This suggests that targeting the RIP kinase signalling pathway could be an effective therapeutic intervention in retinal degeneration patients. As bystander cell death is an important feature of many retinal diseases, combinatorial approaches targeting different cell death pathways may evolve as an important general principle in treatment.

A CNGB1 Frameshift Mutation in Papillon and Phalène Dogs with Progressive Retinal Atrophy

Progressive retinal degenerations are the most common causes of complete blindness both in human and in dogs. Canine progressive retinal atrophy (PRA) or degeneration resembles human retinitis pigmentosa (RP) and is characterized by a progressive loss of rod photoreceptor cells followed by a loss of cone function. The primary clinical signs are detected as vision impairment in a dim light. Although several genes have been associated with PRAs, there are still PRAs of unknown genetic cause in many breeds, including Papillons and Phalènes. We have performed a genome wide association and linkage studies in cohort of 6 affected Papillons and Phalènes and 14 healthy control dogs to map a novel PRA locus on canine chromosome 2, with a 1.9 Mb shared homozygous region in the affected dogs. Parallel exome sequencing of a trio identified an indel mutation, including a 1-bp deletion, followed by a 6-bp insertion in the CNGB1 gene. This mutation causes a frameshift and premature stop codon leading to probable nonsense mediated decay (NMD) of the CNGB1 mRNA. The mutation segregated with the disease and was confirmed in a larger cohort of 145 Papillons and Phalènes (PFisher = 1.4×10−8) with a carrier frequency of 17.2 %. This breed specific mutation was not present in 334 healthy dogs from 10 other breeds or 121 PRA affected dogs from 44 other breeds. CNGB1 is important for the photoreceptor cell function its defects have been previously associated with retinal degeneration in both human and mouse. Our study indicates that a frameshift mutation in CNGB1 is a cause of PRA in Papillons and Phalènes and establishes the breed as a large functional animal model for further characterization of retinal CNGB1 biology and possible retinal gene therapy trials. This study enables also the development of a genetic test for breeding purposes.

Genomic deletion of CNGB3 is identical by descent in multiple canine breeds and causes achromatopsia

BackgroundAchromatopsia is an autosomal recessive disease characterized by the loss of cone photoreceptor function that results in day-blindness, total colorblindness, and decreased central visual acuity. The most common causes for the disease are mutations in the CNGB3 gene, coding for the beta subunit of the cyclic nucleotide-gated channels in cones. CNGB3-achromatopsia, or cone degeneration (cd), is also known to occur in two canine breeds, the Alaskan malamute (AM) and the German shorthaired pointer.ResultsHere we report an in-depth characterization of the achromatopsia phenotype in a new canine breed, the miniature Australian shepherd (MAS). Genotyping revealed that the dog was homozygous for a complete genomic deletion of the CNGB3 gene, as has been previously observed in the AM. Identical breakpoints on chromosome 29 were identified in both the affected AM and MAS with a resulting deletion of 404,820 bp. Pooled DNA samples of unrelated purebred Australian shepherd, MAS, Siberian husky, Samoyed and Alaskan sled dogs were screened for the presence of the affected allele; one Siberian husky and three Alaskan sled dogs were identified as carriers. The affected chromosomes from the AM, MAS, and Siberian husky were genotyped for 147 SNPs in a 3.93 Mb interval within the cd locus. An identical shared affected haplotype, 0.5 Mb long, was observed in all three breeds and defined the minimal linkage disequilibrium (LD) across breeds. This supports the idea that the mutated allele was identical by descent (IBD).ConclusionWe report the occurrence of CNGB3-achromatopsia in a new canine breed, the MAS. The CNGB3-deletion allele previously described in the AM was also observed in a homozygous state in the affected MAS, as well as in a heterozygous carrier state in a Siberian husky and Alaskan sled dogs. All affected alleles were shown to be IBD, strongly suggesting an affected founder effect. Since the MAS is not known to be genetically related to the AM, other breeds may potentially carry the same cd-allele and be affected by achromatopsia.

Monitoring CNGA3 and CNGB3 Subunit Expression in Retinas of Day-Blind Canines with Inherited Deletion or Missense CNGB3 Asp262Asn Mutations Show Progressive Loss of Both CNGB3 and CNGA3 Expression

Cone cyclic nucleotide-gated (CNG) channels are heteromeric, composed of CNGA3 and CNGB3 subunits. Inherited canine day blindness is similar to human achromatopsia and results in loss of cone function. Affected dogs have inherited deletion (-/-) or missense (m/m) Asp262Asn mutations in the CNGB3 gene. Cone ERG studies from m/m dogs show early, progressive loss of cone function with complete loss by ∼6-weeks. Immunohistochemical analysis of m/m and -/- retinal tissues using an anti-CNGA3 antibody show outer segment expression until ∼6 weeks; no immunoreactivity is observed at 1 year. A polyclonal antibody was generated against the C-terminus of canine CNGB3 to investigate expression in the m/m dogs. Immunohistochemistry on 6-week, 8-week and one-year old m/m retinas showed no expression of CNGB3. Immunoblots of retinal homogenates from m/m and -/- mutant dogs showed no reactivity although the antibody recognizes the mutant protein as demonstrated with heterologously-expressed human B3-D262N (canine numbering). Previous qRT-PCR studies examined expression of both CNGA3 and CNGB3 mRNA. Levels of CNGA3 were similar for unaffected, m/m and -/- retinas; CNGB3 mRNA levels were similar for unaffected and m/m dogs but, as expected, levels in -/- retinas were non-detectable. The affected m/m and -/- dogs provide models for inherited human channelopathies including achromatopsia with the potential for direct retinal examination of affected dogs during development and following gene therapy. Results with m/m dogs show that the CNGB3 subunits are degraded; importantly, both the m/m and -/- dogs loose expression of CNGA3 in outer segments. This surprising result implies that an intact CNGB3 is a requirement for CNGA3 expression, a result not predicted or replicated in heterologous expression of CNGA3 channels.