Cloning of canine galactokinase (GALK1) and evaluation as a candidate gene for hereditary cataracts in Labrador retrievers.
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Animals↗
Biomedical subjects
G D Aguirre
Publications and source records attributed to G D Aguirre.
Cloning and mapping of canine KIAA1753.
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Animals↗
Cloning of the canine delta tubulin cDNA (TUBD) and mapping to CFA9.
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Animals↗
Calcium channel blocker D-cis-diltiazem does not slow retinal degeneration in the PDE6B mutant rcd1 canine model of retinitis pigmentosa.
PURPOSE: D-cis-diltiazem, a calcium channel blocker, has been reported to enhance photoreceptor survival in the rd mouse, a model of retinitis pigmentosa (RP) resulting from mutation of the PDE6B gene. We tested the hypothesis that diltiazem treatment would similarly rescue the canine rcd1 model of RP, which is also caused by a null mutation in the PDE6B gene. METHODS: D-cis-diltiazem was delivered orally twice daily to rcd1 affected dogs beginning at 4 weeks of age; untreated age-matched rcd1 dogs served as controls. At 14 weeks, electroretinograms (ERG) were performed on all animals; 14 dogs were euthanized at this age, and 2 dogs at 25 weeks of age. Eyes were enucleated, fixed, and processed for routine histological examination. RESULTS: No significant differences were found in ERG or histopathologic parameters between diltiazem-treated and untreated rcd1 dogs. Neither rcd1 group showed a rod b-wave; ERGs evoked by single white flashes (dark- or light-adapted) and flicker were also identical between groups. Similarly, treated and untreated animals did not differ in the degree of preservation of the photoreceptor layer, confirmed in cell counts within the outer nuclear layer. CONCLUSIONS: Treatment of rcd1 affected dogs with D-cis-diltiazem did not modify the photoreceptor disease when results were assessed using either ERG or histopathologic criteria. The positive photoreceptor-rescue effect of calcium channel blockers reported in the rd mouse was thus not generalizable to another species with retinal degeneration due to mutation in the PDE6B gene. Caution needs to be exerted in extrapolation to the comparable human forms of RP.
3',5'-Cyclic-GMP Phosphodiesterases↗
Concentric retinitis pigmentosa: clinicopathologic correlations.
Progressive concentric (centripetal) loss of vision is one pattern of visual field loss in retinitis pigmentosa. This study provides the first clinicopathologic correlations for this form of retinitis pigmentosa. A family with autosomal dominant concentric retinitis pigmentosa was examined clinically and with visual function tests. A post-mortem eye of an affected 94 year old family member was processed for histopathology and immunocytochemistry with retinal cell specific antibodies. Unrelated simplex/multiplex patients with concentric retinitis pigmentosa were also examined. Affected family members of the eye donor and patients from the other families had prominent peripheral pigmentary retinopathy with more normal appearing central retina, good visual acuity, concentric field loss, normal or near normal rod and cone sensitivity within the preserved visual field, and reduced rod and cone electroretinograms. The eye donor, at age 90, had good acuity and function in a central island. Grossly, the central region of the donor retina appeared thinned but otherwise normal, while the far periphery contained heavy bone spicule pigment. Microscopically the central retina showed photoreceptor outer segment shortening and some photoreceptor cell loss. The mid periphery had a sharp line of demarcation where more central photoreceptors were near normal except for very short outer segments and peripheral photoreceptors were absent. Rods and cones showed abrupt loss of outer segments and cell death at this interface. It is concluded that concentric retinitis pigmentosa is a rare but recognizable phenotype with slowly progressive photoreceptor death from the far periphery toward the central retina. The disease is retina-wide but shows regional variation in severity of degeneration; photoreceptor death is severe in the peripheral retina with an abrupt edge between viable and degenerate photoreceptors. Peripheral to central gradients of unknown retinal molecule(s) may be defective or modify photoreceptor degeneration in concentric retinitis pigmentosa.
Adolescent↗
Evaluation of retinal photoreceptors and pigment epithelium in a female carrier of choroideremia.
PURPOSE: To clarify the pathogenesis of choroideremia. STUDY DESIGN: Human tissue study. TISSUES: Eyes of an 88-year-old symptomatic female carrier of choroideremia (CHM) and six normal, age-matched donors. METHODS: The eyes were processed for histopathologic examination, including immunocytochemistry with an antibody against the CHM gene product, REP-1, and retinal cell-specific markers. RESULTS: The CHM carrier retina showed patchy degeneration, but the photoreceptor and retinal pigment epithelium (RPE) loss appeared to be independent. The choriocapillaris was normal except where retinal areas were severely degenerate. The CHM gene product, REP-1, was localized to the cytoplasm of rods but not cones. CONCLUSIONS: It has generally been considered that photoreceptor degeneration in CHM is secondary to loss of the choriocapillaris or RPE. This study suggests that the rod photoreceptors are a primary site of disease in CHM.
Adaptor Proteins, Signal Transducing↗
Gene therapy restores vision in a canine model of childhood blindness.
The relationship between the neurosensory photoreceptors and the adjacent retinal pigment epithelium (RPE) controls not only normal retinal function, but also the pathogenesis of hereditary retinal degenerations. The molecular bases for both primary photoreceptor and RPE diseases that cause blindness have been identified. Gene therapy has been used successfully to slow degeneration in rodent models of primary photoreceptor diseases, but efficacy of gene therapy directed at photoreceptors and RPE in a large-animal model of human disease has not been reported. Here we study one of the most clinically severe retinal degenerations, Leber congenital amaurosis (LCA). LCA causes near total blindness in infancy and can result from mutations in RPE65 (LCA, type II; MIM 180069 and 204100). A naturally occurring animal model, the RPE65-/- dog, suffers from early and severe visual impairment similar to that seen in human LCA. We used a recombinant adeno-associated virus (AAV) carrying wild-type RPE65 (AAV-RPE65) to test the efficacy of gene therapy in this model. Our results indicate that visual function was restored in this large animal model of childhood blindness.
Animals↗
Cloning of canine gamma-tubulin (TUBG1) cDNA and mapping to CFA9.
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Animals↗
Adenoviral vector-mediated beta-glucuronidase cDNA transfer to treat MPS VII RPE in vitro.
PURPOSE: To develop an effective therapy for treating glycosaminoglycan (GAG) storage in mucopolysaccharidosis VII (MPS VII) retinal pigment epithelium (RPE) in vitro using adenoviral vector mediated human beta-glucuronidase cDNA (Ad-GUSB) transfer. METHODS: Ad-GUSB was used to infect RPE at confluency. The transduction condition was optimized varying time of infection and number of infectious particles. The beta-glucuronidase (GUSB) activity was measured in transduced cells and media using a fluorogenic substrate. The GAG profiles were examined by metabolically labeling RPE with (35)Na(2)SO(4). RESULTS: Transduced RPE, irrespective of species or disease status, expressed a high level of beta-glucuronidase. The expressed enzyme restored normal levels of GAGs in the RPE cells of homozygous affected MPS VII dogs by metabolizing stored GAGs. The over-expressed enzyme (>10 000 nmoles/hr/mg) failed to restore normal level of GAGs. A high level of GUSB expression was maintained in vitro at least nine weeks. CONCLUSIONS: Adenoviral vector could mediate transfer of GUSB in MPS VII affected RPE and RPE of various species, and the expression was observed to be stable in vitro. However, controlled expression of GUSB was essential for the metabolism of stored GAGs to achieve normal levels.
Adenoviridae↗
Fine mapping of canine XLPRA establishes homology of the human and canine RP3 intervals.
PURPOSE: Canine X-linked progressive retinal atrophy (XLPRA) is a hereditary, progressive retinal degeneration that has been mapped previously to the canine X chromosome in a region flanked by the dystrophin (DMD) and tissue inhibitor of metalloproteinase 1 (TIMP1) genes, and is tightly linked to the gene RPGR. The comparable region of the human X chromosome includes the disease locus for RP3, an X-linked form of retinitis pigmentosa, although the current canine disease interval is much larger. METHODS: To refine the map of the canine XLPRA disease interval, 11 X-linked markers were mapped, both meiotically, in two extensive canine pedigrees informative for XLPRA, and on a 3000-rad canine-hamster radiation hybrid (RH) panel. A 12th marker was mapped on the RH panel alone. RESULTS: The integrated map of this region of CFAX now covers approximately 47.3 centimorgans (cM) and 194 centirays (cR)(3000), and demonstrates strong conservation of synteny between humans and dogs. Genes defining the human RP3 zero-recombination interval (human homologue of mouse t complex [TCTE1L], sushi repeat-containing protein, X chromosome [SRPX], and retinitis pigmentosa guanosine triphosphatase [GTPase] regulator [RPGR]) are tightly linked to each other, to the XLPRA locus, and to the gene ornithine transcarbamylase (OTC) in dogs. CONCLUSIONS: Strong conservation of gene order was demonstrated in the short arm of the X chromosome between dogs and humans as was homology of the canine XLPRA and human RP3 intervals. These results create a valuable tool for investigating canine XLPRA and other X-linked eye diseases in dogs.
Animals↗
Loss of cone molecular markers in rhodopsin-mutant human retinas with retinitis pigmentosa.
PURPOSE: To examine the effect of rhodopsin mutations on cone photoreceptors in human retinas with retinitis pigmentosa (RP). METHODS: Four RP retinas with rhodopsin mutations and four normal retinas were examined by immunofluorescence with a battery of cell-specific antibodies against cone and rod cytoplasmic and outer segment membrane proteins. Areas of the retinas were studied that showed maximal preservation of photoreceptor structure. RESULTS: All four RP retinas showed loss of rods, ranging from mild (T-17-M), to more severe (P-23-H), to advanced degeneration (Q-64-ter and G-106-R). The majority of cones in the T-17-M and P-23-H retinas were cytologically normal but showed loss of immunoreactivity for the cytoplasmic proteins 7G6, calbindin, and X-arrestin. The cone outer segments (OS) remained positive for cone opsins and peripherin-2 (rds/peripherin). All remaining cones in the Q-64-ter and G-106-R retinas were degenerate, with short to absent OS, but had strong reactivity for these cytoplasmic and OS membrane markers. Cones in the maculas of the RP retinas were degenerate, with short to absent OS, but retained strong labeling for the cytoplasmic and OS proteins. CONCLUSIONS: Even before cones show cytologic changes in response to rod cell degeneration, they lose immunoreactivity for certain cytoplasmic proteins. These cones later show shortening and loss of OS, although their OS membrane proteins remain well labeled. Cones may down regulate expression of both cytoplasmic and outer segment membrane proteins in response to mutant rod cell dysfunction and/or cell death in human RP retinas. Such cytologic and immunocytochemical changes in the cones may presage death of these critical cells in the later stages of RP.
Aged↗
Mapping of X-linked progressive retinal atrophy (XLPRA), the canine homolog of retinitis pigmentosa 3 (RP3).
X-linked progressive retinal atrophy (XLPRA) in the Siberian husky dog is a naturally occurring X-linked retinopathy closely resembling X-linked retinitis pigmentosa (XLRP) in humans. In affected males, initial degeneration of rods is followed by cone degeneration and complete retinal atrophy; carrier females have random patches of rod degeneration consistent with random X chromosome inactivation. By typing the XLPRA pedigree with five intragenic markers [dystrophin, retinitis pigmentosa GTPase regulator ( RPGR ), tissue inhibitor of metalloproteinases 1, androgen receptor and factor IX], we established a linkage map of the canine X chromosome, and confirmed that the order of these five genes is identical to that on the human X. XLPRA was tightly linked to an intragenic RPGR polymorphism (LOD 11.7, zero recombination), thus confirming locus homology with RP3. We cloned the full-length canine RPGR cDNA and three additional splice variants. No disease-causing mutation was found in the RPGR-coding sequence of the four splice variants characterized, a finding similar to approximately 80% of human XLRP patients whose disease maps to the RP3 locus. In addition, there were no significant differences in the proportional expression of each splice variant in normal and pre-degenerate XLPRA-affected retina. Expression of all RPGR splice variants increased later in the disease, when retinas were undergoing active degeneration. The results provide further evidence of cross-species retention of a complex splicing pattern in the 3' portion of RPGR, the functional significance of which is unknown. In addition, the possibility of another disease locus in the RP3 region is supported.
Alternative Splicing↗
Molecular cloning, characterization and expression of a novel retinal clusterin-like protein cDNA.
A novel gene expressed predominantly in retina, but detected at a conspicuously lower level in retina of canine progressive rod cone degeneration (prcd), has been identified by suppression subtractive hybridization and retinal cDNA library screening. The characterized region of cDNA of the novel gene includes 1017 nucleotides of coding sequence predicted to encode a protein of 338 amino acids (M(r) 39389), 791 nucleotides of 5'-untranslated region (UTR), and 300 nucleotides of 3'-UTR including the poly(A)(+) tail. Multiple transcripts were detected in retina by Northern blot analysis, and a lower level of expression was observed in brain and liver by RT-PCR. The transcript appears to be developmentally regulated with a burst in gene expression at a time period (34 postnatal days) that coincides with the photoreceptor differentiation phase of retinal development. The deduced amino acid sequence from the cDNA of the novel gene has 24% identity and 48% similarity with the multifunctional glycoprotein clusterin. Hence, the putative gene product from the novel transcript has been named clusterin-like protein 1 (CLUL1). The human homologue of CLUL1 cDNA has 84 and 70% identity at the level of nucleotides and amino acids, respectively, with the characterized canine cDNA. The presence of a stretch of 128 amino acids in the putative human CLUL1, not detected in canine CLUL1, suggests alternate splicing events. An STS database search revealed that the human homologue of CLUL1 maps to chromosome 18p, a location not yet reported to harbor an RP locus. Tissue-specific expression of CLUL1 in retina, and its lower abundance in different forms of PRA suggest that this novel gene may represent an as-yet unidentified locus for a retinal disorder.
Amino Acid Sequence↗
Molecular characterization and mapping of canine cGMP-phosphodiesterase delta subunit (PDE6D).
cGMP-phosphodiesterase (PDE) is composed of two catalytic (alpha and beta) and two identical inhibitory (gamma) subunits. The human gene (PDE6D) encoding a new subunit (delta) has been characterized and mapped to the long arm of chromosome 2 (HSA2q35-q36) where a new autosomal recessive retinitis pigmentosa (arRP) locus (RP26) has been localized. Characterization of the canine PDE6D shows the gene is about 4.2kb containing four exons interrupted by three introns; the size of the cDNA is 1059bp with an open reading frame (ORF) of 453bp. A single transcript of identical size (1.43kb) was detected in all tissues examined (liver, lung, spleen, kidney, heart, brain and retina), with the highest abundance in the retina. Canine PDE6D has been localized to canine radiation hybrid group 14-a, which extends conserved synteny between the dog, human chromosome 2q and mouse chromosome 1. The characterization of the canine PDE6D gene and its mapping provide important information for testing causal association of the gene with canine retinal degenerations, in particular rod-cone dysplasia 2 (rcd2) in collie dogs. This disease is characterized by abnormal retinal cGMP metabolism due to a deficiency in cGMP-PDE activity, yet the alpha, beta and gamma subunits of PDE have been excluded as candidate gene loci.
3',5'-Cyclic-GMP Phosphodiesterases↗
A novel retinal degeneration locus identified by linkage and comparative mapping of canine early retinal degeneration.
Early retinal degeneration (erd) is an early onset progressive retinal atrophy, a hereditary canine retinal disease phenotypically similar to human retinitis pigmentosa (RP). In previous efforts to identify the erd locus, canine homologs of genes causally associated with RP in humans, such as opsin (RHO), the beta-subunit gene for cyclic GMP phosphodiesterase (PDE6B), and RDS/peripherin, were excluded. A genome-wide screen was undertaken on canine families segregating the erd disease. Analysis of over 150 canine-specific markers has localized erd to a single linkage group comprising two previously identified canine linkage groups, 20 and 26, corresponding to canine radiation hybrid groups RH.34-a and RH.40-a. Multipoint analysis places erd in the interval between marker FH2289 (distance 23.6 cM) and FH2407 (5.9 cM) with a lod score of 12.23. Although the erd linkage group has not been assigned to an identified canine chromosome, conserved synteny of this linkage group with human 12p13-q13 suggests several candidates for erd and identifies a novel retinal degeneration locus. The rapid progress now occurring in canine genetics will expedite identification of the genes and molecular mechanisms underlying the inherited traits and diseases that make the dog a unique asset for study of mammalian traits.
Animals↗
Evaluation of cGMP-phosphodiesterase (PDE) subunits for causal association with rod-cone dysplasia 2 (rcd2), a canine model of abnormal retinal cGMP metabolism.
Rod-cone dysplasia types 1 (rcd1; Irish setter) and 2 (rcd2; collie) in dogs are early onset forms of progressive retinal atrophy (PRA) which serve as models of retinitis pigmentosa (RP) in humans. As both rcd1 and rcd2 result from abnormal retinal cGMP metabolism associated with a deficiency in cGMP-phosphodiesterase (PDE) activity, and a nonsense mutation in the PDE6B subunit gene has been shown to cause rcd1, the genes encoding the four subunits of the PDE complex (PDE6A, PDE6B, PDE6G and PDE6D) make compelling candidates for the rcd2 locus. We adopted diverse strategies to evaluate causal association of the four PDE subunit genes with the rcd2 phenotype. Identification in an informative pedigree of obligate recombinations between intragenic polymorphisms within PDE6A and PDE6D and the rcd2 locus unequivocally excludes these two genes. PDE6B was excluded by a breeding strategy demonstrating nonallelism of rcd1 and rcd2. Direct sequencing of PDE6G from an rcd2 -homozygous collie dog revealed no abnormality in the entire genomic sequence. To evaluate cosegregation between PDE6G and rcd2, advantage was taken of prior knowledge that PDE6G and Galactokinase 1 (GALK1) localize to the same canine-rodent somatic hybrid cell line. Linkage analysis using a single nucleotide polymorphism (SNP) in the PDE6G gene, and a (CA)n repeat polymorphism in the GALK1 gene, which were both segregating in an unrelated pedigree, established close linkage of these two genes (theta = 0; Z = 4.21). Identification of obligate recombinations between GALK1 and the rcd2 locus in an informative rcd2 pedigree thus excluded PDE6G as a candidate gene for rcd2; the exclusion distance between GALK1 and rcd2 is at least 0.35 cM. These results therefore exclude the entire set of genes coding for the rod PDE complex as candidates for rcd2.
3',5'-Cyclic-GMP Phosphodiesterases↗
A hypothesis to explain the reduced blood levels of docosahexaenoic acid in inherited retinal degenerations caused by mutations in genes encoding retina-specific proteins.
Some humans and animals with inherited retinal degenerations (RD) have lower blood levels of docosahexaenoic acid (22:6n-3) than controls. As a result of recent studies, clearly the low blood 22:6n-3 phenotype is found in multiple RD phenotypes and no mutation thus far identified in humans or animals is involved in lipid metabolism. Therefore, it seems reasonable to suggest that the primary defect is not in 22:6n-3 metabolism, but rather in some common convergent pathway that ultimately leads to the reduction of blood and tissue 22:6n-3 levels. One possibility is that the different mutations produce a metabolic stress that provokes structural and biochemical adaptive changes in photoreceptor cells and their rod outer segments. If the stress is oxidant, the retina could downregulate 22:6n-3 and upregulate antioxidant defenses. How such a stress could lead to changes in blood levels of 22:6n-3 is not obvious. However, the consistent finding of the 22:6n-3 phenotype in many different retinal degeneration genotypes suggests that some form of communication exists between the retina and other tissues that serves to reduce blood levels of 22:6n-3.
Animals↗
Anchoring of canine linkage groups with chromosome-specific markers.
A high-resolution genetic map with polymorphic markers spaced frequently throughout the genome is a key resource for identifying genes that control specific traits or diseases. The lack of rigorous selection against genetic disorders has resulted in many breeds of dog suffering from a very high frequency of genetic diseases, which tend to be breed-specific and usually inherited as autosomal recessive or apparently complex genetic traits. Many of these closely resemble human genetic disorders in their clinical and pathologic features and are likely to be caused by mutations in homologous genes. To identify loci important in canine disease genes, as well as traits associated with morphological and behavioral variation, we are developing a genetic map of the canine genome. Here we report on an updated version of the canine linkage map, which includes 341 mapped markers distributed over the X and 37 autosomal linkage groups. The average distance between markers on the map is 9.0 cM, and the linkage groups provide estimated coverage of over 95% of the genome. Fourteen linkage groups contain either gene-associated or anonymous markers localized to cosmids that have been assigned to specific canine chromosomes by FISH. These 14 linkage groups contain 150 microsatellite markers and allow us to assign 40% of the linkage groups to specific canine chromosomes. This new version of the map is of sufficient density and characterization to initiate mapping of traits of interest.
Animals↗