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Andras M Komaromy - One of the best experts on this subject based on the ideXlab platform.
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Novel retinopathy in related Gordon setters: a clinical, behavioral, electrophysiological, and genetic investigation
Veterinary ophthalmology, 2015Co-Authors: Kathryn L. Good, Andras M Komaromy, Philip H. Kass, Ron OfriAbstract:Purpose To conduct ophthalmic, behavioral, electrophysiological, and genetic testing on two related Gordon setters presented for Day Blindness and compare findings with those of nine related and unrelated Gordon setters. Methods All dogs underwent comprehensive ophthalmic examination. Maze testing was conducted under different light intensities. Rod and cone function was assessed electroretinographically. DNA samples were screened for five canine retinal disease gene mutations. Results Ophthalmic examination was unremarkable in all dogs. There was no notable difference between Day blind dogs and the reference population in scotopic and mesopic maze tests. Day blind dogs performed worse in the photopic maze with slower course completion time and more obstacle collisions. Electroretinography revealed extinguished cone function in Day blind dogs and depressed rod responses in all but two reference dogs. One reference population dog presented with Day Blindness 1 year after initial examination. Mutations that cause achromatopsia (in CNGB3) and cone-rod dystrophies (in ADAM9 and IQCB1) were not detected in any dog tested, although five reference dogs were carriers of the mutation in C2orf71 that causes rod–cone degeneration 4 (rcd4) in Gordon setters and in polski owczarek nizinny dogs. Conclusions This report describes a novel retinopathy in related Gordon setters that has clinical signs and vision testing results consistent with achromatopsia but electroretinographic results suggestive of cone-rod dystrophy. The majority of Gordon setters in this study had low rod responses on electroretinography but it is unclear whether this was indicative of rod dysfunction or normal for the breed. Longer-term observation of affected individuals is warranted.
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A cyclic nucleotide-gated channel mutation associated with canine Daylight Blindness provides insight into a role for the S2 segment Tri-Asp motif in channel biogenesis
PloS one, 2014Co-Authors: Naoto Tanaka, Andras M Komaromy, Lucie Delemotte, Michael L. Klein, Jacqueline C. TanakaAbstract:Cone cyclic nucleotide-gated channels are tetramers formed by CNGA3 and CNGB3 subunits; CNGA3 subunits function as homotetrameric channels but CNGB3 exhibits channel function only when co-expressed with CNGA3. An aspartatic acid (Asp) to asparagine (Asn) missense mutation at position 262 in the canine CNGB3 (D262N) subunit results in loss of cone function (Daylight Blindness), suggesting an important role for this aspartic acid residue in channel biogenesis and/or function. Asp 262 is located in a conserved region of the second transmembrane segment containing three Asp residues designated the Tri-Asp motif. This motif is conserved in all CNG channels. Here we examine mutations in canine CNGA3 homomeric channels using a combination of experimental and computational approaches. Mutations of these conserved Asp residues result in the absence of nucleotide-activated currents in heterologous expression. A fluorescent tag on CNGA3 shows mislocalization of mutant channels. Co-expressing CNGB3 Tri-Asp mutants with wild type CNGA3 results in some functional channels, however, their electrophysiological characterization matches the properties of homomeric CNGA3 channels. This failure to record heteromeric currents suggests that Asp/Asn mutations affect heteromeric subunit assembly. A homology model of S1–S6 of the CNGA3 channel was generated and relaxed in a membrane using molecular dynamics simulations. The model predicts that the Tri-Asp motif is involved in non-specific salt bridge pairings with positive residues of S3/S4. We propose that the D262N mutation in dogs with CNGB3-Day Blindness results in the loss of these inter-helical interactions altering the electrostatic equilibrium within in the S1–S4 bundle. Because residues analogous to Tri-Asp in the voltage-gated Shaker potassium channel family were implicated in monomer folding, we hypothesize that destabilizing these electrostatic interactions impairs the monomer folding state in D262N mutant CNG channels during biogenesis.
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Do Acidic Residues in the Tri-Asp Motif of the CNGA3 S2 Domain Form Required Pairings with Positive Residues of the S1-S4 Bundle? Evidence from Day-Blind Dogs and Insights from a Molecular Model of CNGA3 S1-S6 with Md Simulations
Biophysical Journal, 2014Co-Authors: Naoto Tanaka, Andras M Komaromy, Lucie Delemotte, Michael L. Klein, Jacqueline C. TanakaAbstract:Cone CNGA3 homo-tetramers were expressed in tSA201 cells. Missense mutations were generated in a highly conserved region of S2 containing three Asp residues denoted as the Tri-Asp motif. This motif is conserved in all CNG channel subunits. We substituted Asp 231 with Asn which resulted in complete loss of channel function. This conserved missense mutation is similar to Asp267Asn in canine CNGB3 which is associated with the loss of cone function resulting in Day-Blindness in the dogs. Further substitutions of all acidic residues in the Tri-Asp motif were generated with Ile, Asn, Cys, or Glu. Cyclic GMP-activated current data from excised patches of cells expressing CNGA3 channels showed that any substitution of an Asp residue resulted in the loss of channel function. Further, Asp substitutions resulted in mislocalization of a fluorescent tagged subunit suggestive of improper folding and ER retention. Our results highlight a crucial role for the Tri-Asp motif in channel biogenesis as well as function. A homology model of the S1-S6 domains of the cCNGA3 channel was created, using Kv1.2/2.1 as a template, and relaxed in a membrane with molecular dynamics simulations. The model shows that the Tri-Asp residues are involved in salt bridge pairings with positive residues of S2-S4. We hypothesize that the canine Asp/Asn mutation alters the electrostatic equilibrium in the S1-S4 bundle. In previous Shaker channel studies, conserved acidic residues in the Tri-Asp motif have been implicated in monomer folding. We, therefore, suggest that loss of the side chain cross bridges in the missense dog mutation destabilizes electrostatic interactions impairing the monomer folding state during biogenesis.
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Genomic deletion of CNGB3 is identical by descent in multiple canine breeds and causes achromatopsia
BMC Genetics, 2013Co-Authors: Orly Goldstein, Susan E. Pearce-kelling, Amy M Knollinger, Debbie Holley, Heather J Huson, Anna V Kukekova, Gregory M. Acland, Andras M KomaromyAbstract:Background Achromatopsia 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. Results Here 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). Conclusion We 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.
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Genomic deletion of CNGB3 is identical by descent in multiple canine breeds and causes achromatopsia
BMC genetics, 2013Co-Authors: Connie Y. Yeh, Orly Goldstein, Susan E. Pearce-kelling, Amy M Knollinger, Debbie Holley, Heather J Huson, Anna V Kukekova, Gregory M. Acland, Andras M KomaromyAbstract:Background Achromatopsia 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.
Jacqueline C. Tanaka - One of the best experts on this subject based on the ideXlab platform.
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A cyclic nucleotide-gated channel mutation associated with canine Daylight Blindness provides insight into a role for the S2 segment Tri-Asp motif in channel biogenesis
PloS one, 2014Co-Authors: Naoto Tanaka, Andras M Komaromy, Lucie Delemotte, Michael L. Klein, Jacqueline C. TanakaAbstract:Cone cyclic nucleotide-gated channels are tetramers formed by CNGA3 and CNGB3 subunits; CNGA3 subunits function as homotetrameric channels but CNGB3 exhibits channel function only when co-expressed with CNGA3. An aspartatic acid (Asp) to asparagine (Asn) missense mutation at position 262 in the canine CNGB3 (D262N) subunit results in loss of cone function (Daylight Blindness), suggesting an important role for this aspartic acid residue in channel biogenesis and/or function. Asp 262 is located in a conserved region of the second transmembrane segment containing three Asp residues designated the Tri-Asp motif. This motif is conserved in all CNG channels. Here we examine mutations in canine CNGA3 homomeric channels using a combination of experimental and computational approaches. Mutations of these conserved Asp residues result in the absence of nucleotide-activated currents in heterologous expression. A fluorescent tag on CNGA3 shows mislocalization of mutant channels. Co-expressing CNGB3 Tri-Asp mutants with wild type CNGA3 results in some functional channels, however, their electrophysiological characterization matches the properties of homomeric CNGA3 channels. This failure to record heteromeric currents suggests that Asp/Asn mutations affect heteromeric subunit assembly. A homology model of S1–S6 of the CNGA3 channel was generated and relaxed in a membrane using molecular dynamics simulations. The model predicts that the Tri-Asp motif is involved in non-specific salt bridge pairings with positive residues of S3/S4. We propose that the D262N mutation in dogs with CNGB3-Day Blindness results in the loss of these inter-helical interactions altering the electrostatic equilibrium within in the S1–S4 bundle. Because residues analogous to Tri-Asp in the voltage-gated Shaker potassium channel family were implicated in monomer folding, we hypothesize that destabilizing these electrostatic interactions impairs the monomer folding state in D262N mutant CNG channels during biogenesis.
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Do Acidic Residues in the Tri-Asp Motif of the CNGA3 S2 Domain Form Required Pairings with Positive Residues of the S1-S4 Bundle? Evidence from Day-Blind Dogs and Insights from a Molecular Model of CNGA3 S1-S6 with Md Simulations
Biophysical Journal, 2014Co-Authors: Naoto Tanaka, Andras M Komaromy, Lucie Delemotte, Michael L. Klein, Jacqueline C. TanakaAbstract:Cone CNGA3 homo-tetramers were expressed in tSA201 cells. Missense mutations were generated in a highly conserved region of S2 containing three Asp residues denoted as the Tri-Asp motif. This motif is conserved in all CNG channel subunits. We substituted Asp 231 with Asn which resulted in complete loss of channel function. This conserved missense mutation is similar to Asp267Asn in canine CNGB3 which is associated with the loss of cone function resulting in Day-Blindness in the dogs. Further substitutions of all acidic residues in the Tri-Asp motif were generated with Ile, Asn, Cys, or Glu. Cyclic GMP-activated current data from excised patches of cells expressing CNGA3 channels showed that any substitution of an Asp residue resulted in the loss of channel function. Further, Asp substitutions resulted in mislocalization of a fluorescent tagged subunit suggestive of improper folding and ER retention. Our results highlight a crucial role for the Tri-Asp motif in channel biogenesis as well as function. A homology model of the S1-S6 domains of the cCNGA3 channel was created, using Kv1.2/2.1 as a template, and relaxed in a membrane with molecular dynamics simulations. The model shows that the Tri-Asp residues are involved in salt bridge pairings with positive residues of S2-S4. We hypothesize that the canine Asp/Asn mutation alters the electrostatic equilibrium in the S1-S4 bundle. In previous Shaker channel studies, conserved acidic residues in the Tri-Asp motif have been implicated in monomer folding. We, therefore, suggest that loss of the side chain cross bridges in the missense dog mutation destabilizes electrostatic interactions impairing the monomer folding state during biogenesis.
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A Canine CNGB3 Channelopathy Suggests that Changes in Calcium Homeostasis Result in Progressive Loss of Cone Function
Biophysical Journal, 2013Co-Authors: Naoto Tanaka, Andras M Komaromy, Amalaris Gonzalez, Jacqueline C. TanakaAbstract:Canine Day-Blindness, a model for human achromatopsia, is associated with loss of cone function due either to the deletion or a missense Asp (D) 262 to Asn (N) mutation in CNGB3. Asp 262 resides in an acidic motif in the S2 transmembrane helix conserved in all CNG channel subunits and members of the Shaker K+ superfamily. Tetrameric cyclic nucleotide-gated (CNG) channels are formed from CNGA3 and CNGB3 subunits and transduce light information in cone photoreceptor outer segments. In canine Day-Blindness, the CNGB3-D262N mutation leads to loss of cone function between 4 and ∼10 weeks suggesting progressive physiological changes. We investigated the missense mutation using the human CNGB3, previously used in gene therapy to restore cone function in young dogs (Hum Mol Genet 2010 19: 2581). Canine CNGA3 was co-expressed with hCNGB3; the most significant functional difference between homomeric and heteromeric currents was an ∼10 fold increase in PCa/PNa in heteromeric channels. Co-expression of cCNGA3 with hCNGB3-D262N (canine numbering) result in the absence of functional heteromeric channels with evidence of some homomeric CNGA3 channels. We suggest that alterations in calcium homeostasis associated with the missense mutation in CNGB3 contribute to the loss of cone function. We generated mutations in the Asp residues in CNGA3 channels. We investigated substitutions in the three Asp residues in S2 and all mutations examined resulted in the loss of channel function underscoring the essential role for these residues in channel function. Studies in voltage-gated channels show electrostatic interactions between the acidic residues in S2 and residues in S3 and S4 transmembrane domains. Our future experiments will explore the role of these acidic residues in intra-subunit helical interactions using mutagenesis and molecular modeling.
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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
Biophysical Journal, 2013Co-Authors: Amaliris Gonzalez, Jacqueline C. Tanaka, Andras M KomaromyAbstract: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.
Naoto Tanaka - One of the best experts on this subject based on the ideXlab platform.
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A cyclic nucleotide-gated channel mutation associated with canine Daylight Blindness provides insight into a role for the S2 segment Tri-Asp motif in channel biogenesis
PloS one, 2014Co-Authors: Naoto Tanaka, Andras M Komaromy, Lucie Delemotte, Michael L. Klein, Jacqueline C. TanakaAbstract:Cone cyclic nucleotide-gated channels are tetramers formed by CNGA3 and CNGB3 subunits; CNGA3 subunits function as homotetrameric channels but CNGB3 exhibits channel function only when co-expressed with CNGA3. An aspartatic acid (Asp) to asparagine (Asn) missense mutation at position 262 in the canine CNGB3 (D262N) subunit results in loss of cone function (Daylight Blindness), suggesting an important role for this aspartic acid residue in channel biogenesis and/or function. Asp 262 is located in a conserved region of the second transmembrane segment containing three Asp residues designated the Tri-Asp motif. This motif is conserved in all CNG channels. Here we examine mutations in canine CNGA3 homomeric channels using a combination of experimental and computational approaches. Mutations of these conserved Asp residues result in the absence of nucleotide-activated currents in heterologous expression. A fluorescent tag on CNGA3 shows mislocalization of mutant channels. Co-expressing CNGB3 Tri-Asp mutants with wild type CNGA3 results in some functional channels, however, their electrophysiological characterization matches the properties of homomeric CNGA3 channels. This failure to record heteromeric currents suggests that Asp/Asn mutations affect heteromeric subunit assembly. A homology model of S1–S6 of the CNGA3 channel was generated and relaxed in a membrane using molecular dynamics simulations. The model predicts that the Tri-Asp motif is involved in non-specific salt bridge pairings with positive residues of S3/S4. We propose that the D262N mutation in dogs with CNGB3-Day Blindness results in the loss of these inter-helical interactions altering the electrostatic equilibrium within in the S1–S4 bundle. Because residues analogous to Tri-Asp in the voltage-gated Shaker potassium channel family were implicated in monomer folding, we hypothesize that destabilizing these electrostatic interactions impairs the monomer folding state in D262N mutant CNG channels during biogenesis.
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Do Acidic Residues in the Tri-Asp Motif of the CNGA3 S2 Domain Form Required Pairings with Positive Residues of the S1-S4 Bundle? Evidence from Day-Blind Dogs and Insights from a Molecular Model of CNGA3 S1-S6 with Md Simulations
Biophysical Journal, 2014Co-Authors: Naoto Tanaka, Andras M Komaromy, Lucie Delemotte, Michael L. Klein, Jacqueline C. TanakaAbstract:Cone CNGA3 homo-tetramers were expressed in tSA201 cells. Missense mutations were generated in a highly conserved region of S2 containing three Asp residues denoted as the Tri-Asp motif. This motif is conserved in all CNG channel subunits. We substituted Asp 231 with Asn which resulted in complete loss of channel function. This conserved missense mutation is similar to Asp267Asn in canine CNGB3 which is associated with the loss of cone function resulting in Day-Blindness in the dogs. Further substitutions of all acidic residues in the Tri-Asp motif were generated with Ile, Asn, Cys, or Glu. Cyclic GMP-activated current data from excised patches of cells expressing CNGA3 channels showed that any substitution of an Asp residue resulted in the loss of channel function. Further, Asp substitutions resulted in mislocalization of a fluorescent tagged subunit suggestive of improper folding and ER retention. Our results highlight a crucial role for the Tri-Asp motif in channel biogenesis as well as function. A homology model of the S1-S6 domains of the cCNGA3 channel was created, using Kv1.2/2.1 as a template, and relaxed in a membrane with molecular dynamics simulations. The model shows that the Tri-Asp residues are involved in salt bridge pairings with positive residues of S2-S4. We hypothesize that the canine Asp/Asn mutation alters the electrostatic equilibrium in the S1-S4 bundle. In previous Shaker channel studies, conserved acidic residues in the Tri-Asp motif have been implicated in monomer folding. We, therefore, suggest that loss of the side chain cross bridges in the missense dog mutation destabilizes electrostatic interactions impairing the monomer folding state during biogenesis.
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A Canine CNGB3 Channelopathy Suggests that Changes in Calcium Homeostasis Result in Progressive Loss of Cone Function
Biophysical Journal, 2013Co-Authors: Naoto Tanaka, Andras M Komaromy, Amalaris Gonzalez, Jacqueline C. TanakaAbstract:Canine Day-Blindness, a model for human achromatopsia, is associated with loss of cone function due either to the deletion or a missense Asp (D) 262 to Asn (N) mutation in CNGB3. Asp 262 resides in an acidic motif in the S2 transmembrane helix conserved in all CNG channel subunits and members of the Shaker K+ superfamily. Tetrameric cyclic nucleotide-gated (CNG) channels are formed from CNGA3 and CNGB3 subunits and transduce light information in cone photoreceptor outer segments. In canine Day-Blindness, the CNGB3-D262N mutation leads to loss of cone function between 4 and ∼10 weeks suggesting progressive physiological changes. We investigated the missense mutation using the human CNGB3, previously used in gene therapy to restore cone function in young dogs (Hum Mol Genet 2010 19: 2581). Canine CNGA3 was co-expressed with hCNGB3; the most significant functional difference between homomeric and heteromeric currents was an ∼10 fold increase in PCa/PNa in heteromeric channels. Co-expression of cCNGA3 with hCNGB3-D262N (canine numbering) result in the absence of functional heteromeric channels with evidence of some homomeric CNGA3 channels. We suggest that alterations in calcium homeostasis associated with the missense mutation in CNGB3 contribute to the loss of cone function. We generated mutations in the Asp residues in CNGA3 channels. We investigated substitutions in the three Asp residues in S2 and all mutations examined resulted in the loss of channel function underscoring the essential role for these residues in channel function. Studies in voltage-gated channels show electrostatic interactions between the acidic residues in S2 and residues in S3 and S4 transmembrane domains. Our future experiments will explore the role of these acidic residues in intra-subunit helical interactions using mutagenesis and molecular modeling.
Kristina Narfström - One of the best experts on this subject based on the ideXlab platform.
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Functional and structural changes in the retina of wire-haired dachshunds with early-onset cone-rod dystrophy.
Investigative Opthalmology & Visual Science, 2008Co-Authors: Ernst Otto Ropstad, Kristina Narfström, Frode Lingaas, Caroline Wiik, Anitha Bruun, Ellen BjerkåsAbstract:PURPOSE. To describe and classify the morphologic changes in a naturally occurring dog model of early-onset cone-rod dystrophy (CRD) and to correlate these with earlier described clinical characteristics of the disease in dogs. METHODS. Purpose-bred Standard Wire-Haired Dachshunds (SWHDs) derived from a large pedigree of dogs with earlyonset CRD were euthanatized at defined ages to characterize morphologic changes in the disease process. Specimens were examined by light microscopy, including morphometric studies, electron microscopy, and immunohistochemistry. Peanut agglutinin (PNA), protein kinase C (PKC), synaptophysin (Syn), rhodopsin (Rho)-63, glial fibrillary acidic protein (GFAP), and short-wavelength cone opsin (OS) were used for immunohistochemical characterization. RESULTS. The photopic cone-system-derived ERG amplitudes were already significantly reduced or nonrecordable in CRD-affected dogs at 5 weeks, the earliest age studied. The outer retina was morphologically most severely affected initially, with a subsequent degeneration of the inner retina. Cone degeneration was more pronounced than rod degeneration in young CRD-affected dogs. There was a marked phenotypic variation based on morphologic findings in the affected dogs. At the earliest time point studied (5-8 weeks) cone photoreceptor and glial cell abnormalities were observed, in accordance with earlier studies based on electrophysiological and clinical findings in which Day Blindness and abnormal cone ERGs were observed in young affected SWHD puppies. Preliminary genetic studies have indicated an autosomal recessive mode of inheritance for the defect. CONCLUSIONS. Through functional and structural characterization, early-onset cone abnormalities were found, consistent with a cone dysplasia at an age when rod structure was normal. Further studies are in progress to identify the gene(s) involved in this retinal disease process. The presently described natural animal model of primary cone dysplasia followed by rod degeneration may provide further insight into the human counterpart. Further studies are needed to ascertain an autosomal recessive mode of inheritance for CRD in the SWHD. (Less)
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congenital stationary night Blindness in the dog common mutation in the rpe65 gene indicates founder effect
Molecular Vision, 1998Co-Authors: Gustavo D. Aguirre, Kristina Narfström, Victoria J Baldwin, Sue Pearcekelling, Gregory M. AclandAbstract:Purpose: To clone and characterize the canine RPE65 cDNA from normal dog, examine for mutations, and establish if the mutation identified in Swedish briard dogs with retinal dystrophy is present in dogs of the same breed that originated from the United States and other countries, and are affected with congenital stationary night Blindness. Methods: Fifteen briard dogs were studied, of which 10 were affected with csnb, and five were clinically normal. In addition, we tested samples from four Swedish dogs, and samples from a briard affected with progressive retinal atrophy. RPE65 cDNA was cloned a from retinal cDNA library by PCR, and from canine retina by RT-PCR. ERG and morphology were used to characterize csnb. Results: The normal RPE65 cDNA spans 1724 nucleotides (GenBank accession number AF084537), and includes 1602 nucleotides of coding sequence; the deduced amino acid sequence shares 98%, 97%, and 93% identity with homologous human, bovine, and rat sequences, respectively. A homozygous four nucleotide (AAGA) deletion, representing nucleotides 487-490 of wildtype RPE65 sequence, was found only in csnb and retinal dystrophy affected dogs; heterozygous animals had normal and mutant alleles. The mutation produces a frameshift, causing a deduced mistranslation with a premature stop codon. The mutation causes retinal dysfunction and RPE accumulation of lipid vacuoles. Conclusions: Identification of the same mutation in csnb and retinal dystrophy confirms the molecular identity of the two disorders. A common mutation in dogs derived from different countries suggests a founder effect causing the propagation of a common mutant allele in the population at risk. Disciplines Comparative and Laboratory Animal Medicine | Genetic Phenomena | Medicine and Health Sciences | Veterinary Medicine Comments PMID: 9808841 http://www.molvis.org/molvis/v4/p13 At the time of publication, author Gustavo Aguirre was affiliated with the James A. Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University. Currently, he is a faculty member at the Vet Med school at the University of Pennsylvania. Author(s) Gustavo D. Aguirre, Victoria Baldwin, Sue Pearce-Kelling, Kristina Narfstrom, Kunal Ray, and Gregory M. Acland This journal article is available at ScholarlyCommons: http://repository.upenn.edu/vet_papers/39 Molecular Vision 1998; 4:23 Received 5 October 1998 | Accepted 29 October 1998 | Published 30 October 1998 The briard dog is affected with a recessively inherited retinal disorder characterized by congenital night Blindness with various degrees of visual impairment under photopic illumination. Vision in affected dogs ranges from normal Day vision to profound Day Blindness [1]. The disease was initially described in Swedish dogs as a stationary disorder analogous to human congenital stationary night Blindness (CSNB [2]). More recently, the disease has been described as having a progressive component, and has been termed hereditary retinal dystrophy [3,4]. Along with the visual impairment, affected dogs have an abnormal electroretinogram (ERG); in general, the recorded responses are normal in waveform, but show a marked diminution of response amplitudes, similar to a “Riggs type” ERG in man. The ERG recorded under DC conditions shows complete absence of the a-, b-, and c-waves, with the latter waveform being replaced by a very slow negative potential which develops when the stimulus intensity is greater than 3 log units above the normal b-wave threshold. The authors interpret the abnormalities in the aand b-waves as representing a delay in rod phototransduction [5]. A similar disease is also recognized in other countries, including France, Canada, and the United States. In the US, the disease is termed congenital stationary night Blindness, and csnb has been designated as the gene symbol for the disease locus. Apart from the above studies in Swedish briard dogs, no other systematic investigation of the disease has been reported, nor has there been definitive proof that csnb and retinal dystrophy represent the same disorder. During a presentation on the mutation spectrum of the RPE65 gene in childhood onset retinal dystrophies at the 1998 ARVO meeting, Andreas Gal and associates stated that a 4nucleotide (AAGA) deletion in the RPE65 gene was responsible for hereditary retinal dystrophy in the Swedish briard dog [6]. This presentation prompted us to clone and characterize the canine RPE65 cDNA from normal dog, examine for mutations in the coding sequence of this gene, and establish if the mutation described in Swedish briards is present in dogs of the same breed that originated from the United States and other countries. Our results indicate that the same four nucleotide deletion in the RPE65 gene is the mutation causing csnb in the briard dog. METHODS Animals: Briard dogs affected with csnb and related and unrelated phenotypically normal dogs have been examined to characterize the disease phenotype, and examine for mutations in the RPE65 gene. Overall, we have studied 15 briard dogs, of which 10 were affected with csnb, and five were clinically normal. These dogs came primarily from the US and Canada, of breeding stock that originated from the US and © Molecular Vision Correspondence to: Gustavo D. Aguirre, James A. Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, NY, 14853-6401, USA; Phone: (607) 256-5620; FAX: (607) 256-5689; email: gda1@cornell.edu Dr. Ray is now at the Indian Institute of Chemical Biology, Calcutta, India. Congenital stationary night Blindness in the dog: common mutation in the RPE65 gene indicates founder effect Gustavo D. Aguirre, Victoria Baldwin, Sue Pearce-Kelling, Kristina Narfstrom,Kunal Ray, Gregory M. Acland James A. Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, NY, USA; Department of Medicine & Surgery, Faculty of Veterinary Medicine, Swedish University of Agricultural Sciences, Uppsala,
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Ultrastructural changes of the retina and the retinal pigment epithelium in briard dogs with hereditary congenital night Blindness and partial Day Blindness
Experimental eye research, 1992Co-Authors: Anders Wrigstad, Sven Erik G. Nilsson, Kristina NarfströmAbstract:Abstract The offspring of two Briard dogs (brother and sister) with congenital, clinically stationary night Blindness showed an aggravation of the disease with severe impairment of Day vision in addition to night Blindness. This ultrastructural study was performed on four such second generation puppies at the age of 4 months. The neuroretina and retinal pigment epithelium (RPE) from four locations were studied: the central area (immediately temporal to the optic disc); the centre of the tapetal area; the upper periphery (border of tapetal area); and the lower periphery (non-tapetal area). The RPE showed large inclusions, seemingly lipid in nature, mainly in the central and tapetal areas of the retina. Small, membrane bound, electrondense inclusions were scattered in the RPE cytoplasm in all areas examined. The small inclusions were found to be less numerous in normal than in affected dogs and may be lysosomal in nature. Forty to fifty percent of the rod outer segments in the tapetal area showed disorientation of the disc membranes, whereas the corresponding figures were 20–40% in the central and lower peripheral areas and 6–25% in the upper peripheral area. No structural abnormalities were found in the rod inner segments or synaptic bodies. The cones were better preserved. The inner retina appeared normal. These electron microscopic findings seem to correspond to a previously published electrophysiologic evaluation, indicating a defective and delayed rod function (virtually no scotopic a- and b-waves), a better preserved cone function (photopic flicker responses present, although reduced) and impaired RPE activity (a prominent, slow negative potential of long latency at the site of the c-wave). It appears that these Briard dogs, showing structural changes of the rod outer segments in addition to pigment epithelial inclusions, mainly located in the posterior pole, comprise a pigment epitheliopathy and retinopathy morphologically different from other hereditary canine retinopathies that have been described earlier in the literature and different from animal models of congenital night Blindness.
Eyal Banin - One of the best experts on this subject based on the ideXlab platform.
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Six Years and Counting: Restoration of Photopic Retinal Function and Visual Behavior Following Gene Augmentation Therapy in a Sheep Model of CNGA3 Achromatopsia
Human gene therapy, 2018Co-Authors: Ron Ofri, Alexey Obolensky, Edward Averbukh, Elisha Gootwine, Raaya Ezra-elia, Maya Ross, Hen Honig, Alexander Rosov, William W. Hauswirth, Eyal BaninAbstract:Achromatopsia causes severely reduced visual acuity, photoaversion, and inability to discern colors due to cone photoreceptor dysfunction. In 2010, we reported on Day-Blindness in sheep caused by a...
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gene augmentation therapy for a missense substitution in the cgmp binding domain of ovine cnga3 gene restores vision in Day blind sheep
Investigative Ophthalmology & Visual Science, 2017Co-Authors: Elisha Gootwine, Alexey Obolensky, Hen Honig, Esther Yamin, Mazen Abusiam, Alex Rosov, Tali Nitzan, Andrey Shirak, Raaya Ezraelia, Eyal BaninAbstract:Purpose Applying CNGA3 gene augmentation therapy to cure a novel causative mutation underlying achromatopsia (ACHM) in sheep. Methods Impaired vision that spontaneously appeared in newborn lambs was characterized by behavioral, electroretinographic (ERG), and histologic techniques. Deep-sequencing reads of an affected lamb and an unaffected lamb were compared within conserved genomic regions orthologous to human genes involved in similar visual impairment. Observed nonsynonymous amino acid substitutions were classified by their deleteriousness score. The putative causative mutation was assessed by producing compound CNGA3 heterozygotes and applying gene augmentation therapy using the orthologous human cDNA. Results Behavioral assessment revealed Day Blindness, and subsequent ERG examination showed attenuated photopic responses. Histologic and immunohistochemical examination of affected sheep eyes did not reveal degeneration, and cone photoreceptors expressing CNGA3 were present. Bioinformatics and sequencing analyses suggested a c.1618G>A, p.Gly540Ser substitution in the GMP-binding domain of CNGA3 as the causative mutation. This was confirmed by genetic concordance test and by genetic complementation experiment: All five compound CNGA3 heterozygotes, carrying both p.Arg236* and p.Gly540Ser mutations in CNGA3, were Day-blind. Furthermore, subretinal delivery of the intact human CNGA3 gene using an adeno-associated viral vector (AAV) restored photopic vision in two affected p.Gly540Ser homozygous rams. Conclusions The c.1618G>A, p.Gly540Ser substitution in CNGA3 was identified as the causative mutation for a novel form of ACHM in Awassi sheep. Gene augmentation therapy restored vision in the affected sheep. This novel mutation provides a large-animal model that is valid for most human CNGA3 ACHM patients; the majority of them carry missense rather than premature-termination mutations.
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Gene Augmentation Therapy Restores Retinal Function and Visual Behavior in a Sheep Model of CNGA3 Achromatopsia.
Molecular therapy : the journal of the American Society of Gene Therapy, 2015Co-Authors: Eyal Banin, Alexey Obolensky, Elisha Gootwine, Raaya Ezra-elia, Hen Honig, Alexander Rosov, Ayala Ejzenberg, Lina Zelinger, Esther Yamin, Dror SharonAbstract:Achromatopsia is a hereditary form of Day Blindness caused by cone photoreceptor dysfunction. Affected patients suffer from congenital color Blindness, photosensitivity, and low visual acuity. Mutations in the CNGA3 gene are a major cause of achromatopsia, and a sheep model of this disease was recently characterized by our group. Here, we report that unilateral subretinal delivery of an adeno-associated virus serotype 5 (AAV5) vector carrying either the mouse or the human intact CNGA3 gene under the control of the red/green opsin promoter results in long-term recovery of visual function in CNGA3-mutant sheep. Treated animals demonstrated shorter maze passage times and a reduced number of collisions with obstacles compared with their pretreatment status, with values close to those of unaffected sheep. This effect was abolished when the treated eye was patched. Electroretinography (ERG) showed marked improvement in cone function. Retinal expression of the transfected human and mouse CNGA3 genes at the mRNA level was shown by polymerase chain reaction (PCR), and cone-specific expression of CNGA3 protein was demonstrated by immunohistochemisrty. The rescue effect has so far been maintained for over 3 years in the first-treated animals, with no obvious ocular or systemic side effects. The results support future application of subretinal AAV5-mediated gene-augmentation therapy in CNGA3 achromatopsia patients.
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Flicker cone function in normal and Day blind sheep: a large animal model for human achromatopsia caused by CNGA3 mutation
Documenta Ophthalmologica, 2014Co-Authors: Raaya Ezra-elia, Alexey Obolensky, Edward Averbukh, Eyal Banin, Elisha Gootwine, Hen Honig, Alexander Rosov, William W. Hauswirth, Ron OfriAbstract:Purpose Recently we reported on Day Blindness in sheep caused by a mutation in the CNGA3 gene, thus making affected sheep a naturally occurring large animal model for therapeutic intervention in CNGA3 achromatopsia patients. The purpose of this study was to characterize flicker cone function in normal and Day blind sheep, with the aim of generating a normative data base for ongoing gene therapy studies. Methods Electoretinographic (ERG) cone responses were evoked with full-field conditions in 10 normal, 6 heterozygous carriers and 36 Day blind sheep. Following light adaptation (10 min, 30 cd/m^2), responses were recorded at four increasing light intensities (1, 2.5, 5 and 10 cd s/m^2). At each of these intensities, a single photopic flash response followed by 8 cone flicker responses (10–80 Hz) was recorded. Results were used to generate a normative data base for the three groups. Differences between Day blind and normal control animals were tested in two age-matched groups ( n = 10 per group). Results The normal sheep cone ERG wave is bipartite in nature, with critical flicker fusion frequency (CFF) >80 Hz. In all four flash intensities, the single photopic flash a-wave and b-wave amplitudes were significantly lower ( p
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Flicker cone function in normal and Day blind sheep: a large animal model for human achromatopsia caused by CNGA3 mutation
Documenta ophthalmologica. Advances in ophthalmology, 2014Co-Authors: Raaya Ezra-elia, Alexey Obolensky, Edward Averbukh, Eyal Banin, Elisha Gootwine, Hen Honig, Alexander Rosov, William W. Hauswirth, Ron OfriAbstract:Purpose Recently we reported on Day Blindness in sheep caused by a mutation in the CNGA3 gene, thus making affected sheep a naturally occurring large animal model for therapeutic intervention in CNGA3 achromatopsia patients. The purpose of this study was to characterize flicker cone function in normal and Day blind sheep, with the aim of generating a normative data base for ongoing gene therapy studies.
