A nonhuman primate model of inherited retinal disease.

Inherited retinal degenerations are a common cause of untreatable blindness worldwide, with retinitis pigmentosa and cone dystrophy affecting approximately 1 in 3500 and 1 in 10,000 individuals, respectively. A major limitation to the development of effective therapies is the lack of availability of animal models that fully replicate the human condition. Particularly for cone disorders, rodent, canine, and feline models with no true macula have substantive limitations. By contrast, the cone-rich macula of a nonhuman primate (NHP) closely mirrors that of the human retina. Consequently, well-defined NHP models of heritable retinal diseases, particularly cone disorders that are predictive of human conditions, are necessary to more efficiently advance new therapies for patients. We have identified 4 related NHPs at the California National Primate Research Center with visual impairment and findings from clinical ophthalmic examination, advanced retinal imaging, and electrophysiology consistent with achromatopsia. Genetic sequencing confirmed a homozygous R565Q missense mutation in the catalytic domain of PDE6C, a cone-specific phototransduction enzyme associated with achromatopsia in humans. Biochemical studies demonstrate that the mutant mRNA is translated into a stable protein that displays normal cellular localization but is unable to hydrolyze cyclic GMP (cGMP). This NHP model of a cone disorder will not only serve as a therapeutic testing ground for achromatopsia gene replacement, but also for optimization of gene editing in the macula and of cone cell replacement in general.

Do cGMP Levels Drive the Speed of Photoreceptor Degeneration?

Humans with mutations in the phototransduction pathway develop forms of retinal degeneration, such as retinitis pigmentosa, cone dystrophy, or Leber congenital amaurosis. Similarly, numerous phototransduction mutant animal models resemble retinal degeneration. In our lab, using a zebrafish model, we study cone-specific phototransduction mutants. cGMP is the second messenger in the phototransduction pathway, and abnormal cGMP levels are associated with photoreceptor death. Rd1, a rod-specific phosphodiesterase 6 (Pde6) subunit mutant in mice, is one of the most widely used animal models for retinal degeneration. Rd1 mutant mice accumulate cGMP, causing rapid photoreceptor degeneration. However, much less is known about photoreceptor mutants producing abnormally low levels of cGMP. Here, focusing on Pde6 mutants in zebrafish and mice, we propose a correlation between cGMP levels and speed of photoreceptor degeneration.

Decreased catalytic activity and altered activation properties of PDE6C mutants associated with autosomal recessive achromatopsia.

Mutations in the gene encoding the catalytic subunit of the cone photoreceptor phosphodiesterase (PDE6C) have been recently reported in patients with autosomal recessive inherited achromatopsia (ACHM) and early-onset cone photoreceptor dysfunction. Here we present the results of a comprehensive study on PDE6C mutations including the mutation spectrum, its prevalence in a large cohort of ACHM/cone dysfunction patients, the clinical phenotype and the functional characterization of mutant PDE6C proteins. Twelve affected patients from seven independent families segregating PDE6C mutations were identified in our total patient cohort of 492 independent families. Eleven different PDE6C mutations were found including two nonsense mutations, three mutations affecting transcript splicing as shown by minigene assays, one 1 bp-insertion and five missense mutations. We also performed a detailed functional characterization of six missense mutations applying the baculovirus system to express recombinant mutant and wildtype chimeric PDE6C/PDE5 proteins in Sf9 insect cells. Purified proteins were analyzed using Western blotting, phosphodiesterase (PDE) activity measurements as well as inhibition assays by zaprinast and Pγ. Four of the six PDE6C missense mutations led to baseline PDE activities and most likely represent functional null alleles. For two mutations, p.E790K and p.Y323N, we observed reduction in PDE activity of approximately 60% and 80%, respectively. We also observed differences for Pγ inhibition. The p.E790K mutant, with an IC50 value of 2.7 nm is 20.7-fold more sensitive for Pγ inhibition, whereas the p.Y323N mutant with an IC50 of 158 nm is 3-fold less sensitive when compared with the wildtype control.

Associations between platelet monoamine oxidase-B activity and acquired colour vision loss in a fish-eating population.

Platelet monoamine oxidase-B (MAO-B) has been considered a surrogate biochemical marker of neurotoxicity, as it may reflect changes in the monoaminergic system in the brain. Colour vision discrimination, in part a dopamine dependent process, has been used to identify early neurological effects of some environmental and industrial neurotoxicants. The objective of this cross-sectional study was to explore the relationship between platelet MAO-B activity and acquired colour discrimination capacity in fish-consumers from the St. Lawrence River region of Canada. Assessment of acquired dyschromatopsia was determined using the Lanthony D-15 desaturated panel test. Participants classified with dyschromatopsia (n=81) had significantly lower MAO-B activity when compared to those with normal colour vision (n=32) (26.5+/-9.6 versus 31.0+/-9.9 nmol/min/20 microg, P=0.030)). Similarly, Bowman's Colour Confusion Index (CCI) was inversely correlated with MAO-B activity when the vision test was performed with the worst eye only (r=-0.245, P=0.009), the best eye only (r=-0.188, P=0.048) and with both eyes together (r=-0.309, P=0.001). Associations remained significant after adjustment for age and gender when both eyes (P=0.003) and the worst eye (P=0.045) were tested. Adjustment for heavy smoking weakened the association between MAO-B and CCI in the worst eye (P=0.140), but did not alter this association for both eyes (P=0.006). Adjustment for blood-mercury concentrations did not change the association. This study suggests a relationship between reduced MAO-B activity and acquired colour vision loss and both are associated with tobacco smoking. Therefore, results show that platelet MAO-B may be used as a surrogate biochemical marker of acquired colour vision loss.

Identification, genomic structure, and screening of the vacuolar proton-ATPase membrane sector-associated protein M8-9 gene within the COD1 critical region (Xp11.4).

PURPOSE: Our goal is to identify the gene responsible for X-linked cone-rod dystrophy (COD1) that has been localized to a limited region of Xp11.4. METHODS: A complete physical contig of the COD1 region was partially sequenced and subjected to BLAST searches to identify homologies with GenBank ESTs. ESTs were analyzed for overlapping or related cDNA sequences and retinal expression by PCR screening of multiple human retina cDNA libraries. RACE was performed to complete the missing 5' end of the transcripts. Transcripts were compared with genomic sequences to specify intron-exon boundaries. Genomic DNAs from COD1-affected males from 3 families were screened for mutations using direct PCR sequencing of the exons. RESULTS: The vacuolar proton-ATPase membrane sector-associated protein M8-9 (APT6M8-9) gene was identified within our critical region. We confirmed its retinal expression and its genomic location in our physical contig. Eight exons (with flanking intronic sequences) were characterized from partial cDNA sequence and genomic sequence data. An additional 5' end exon was identified using RACE. No mutations were found in the COD1-affected males. CONCLUSIONS: The combination of disease mapping and information from the Human Genome project has enabled us to identify candidate genes within the COD1 region, including APT6M8-9 gene. We found no evidence that this gene is responsible for COD1 in our families, but it may be an important candidate for other diseases that have been mapped to this region of the X chromosome.

Selective loss of S-cones in diabetic retinopathy.

OBJECTIVE: To determine whether selective cone loss could explain the acquired tritan-like color confusion found in diabetic retinopathy. METHODS: Terminal deoxynucleotidyl transferase-mediated biotin-deoxyuridine triphosphate nick end labeling (TUNEL) was employed on paraffin sections of retinas from 5 donors with diabetic retinopathy. For quantitative analysis, postmortem retinas were obtained from 13 human donors; 7 from patients with various durations and stages of diabetic retinopathy (4 background, 3 proliferative) and 6 controls. Enzyme histochemical analysis for carbonic anhydrase (CA) was used to distinguish L/M-cones (positive for CA) from S-cones (negative for CA). Cone topography was determined by sampling 360 degrees from 0.1 to 1.5 mm of foveal eccentricity and along the horizontal meridians from 1.5 to 15.0 mm. RESULTS: Rare cells in both the inner and outer nuclear layers of the diabetic eyes were positively labeled with the TUNEL method. The CA staining revealed incomplete and patchy losses of S-cones that were limited to the diabetic retinas. Statistically significant reduction in the density of S-cones was found at nearly all foveal eccentricities from 0.1 mm to 15.0 mm. This was not the case for the L/M-cones. On average, for all locations, the percentage of S-cones compared with L/M-cones was decreased by 21.0% +/- 3.4% with respect to the controls. CONCLUSION: The S-cones selectively die in diabetic retinopathy. CLINICAL RELEVANCE: Selective loss of S-cones may contribute to the tritan-like color vision deficit seen in patients with diabetic retinopathy.

Impairment of colour vision in workers exposed to organic solvents.

OBJECTIVES: To investigate loss of colour vision related to exposure to solvents and the role of three enzyme polymorphisms in modifying the risk in exposed workers. METHODS: A sample was studied of 68 male dockyard workers and 42 male community controls with and without neuropsychological symptoms from a previous cross sectional study. Indices of cumulative and intensity based exposure to solvents were calculated for all subjects. Alcohol, drug, and smoking histories were obtained. Colour vision was tested by Lanthony D15d colour vision test. Genotype of glutathione S-transferase M1 and T1 and N-acetyltransferase 2 polymorphisms were determined. RESULTS: The relation between impairment of colour vision and exposure to solvents was investigated with multiple regression techniques. Increasing annual exposure to solvents was significantly associated with reduced colour vision (p=0.029). Impairment of colour vision was not associated with neuropsychological symptoms as measured by the Q16 solvent symptom questionnaire. No significant association was found between acquired impairment of colour vision and genetic polymorphisms when GSTM1, GSTT1 or NAT2 phenotypes were included in the analyses. CONCLUSIONS: Exposure to mixed solvents is associated with impairment in colour vision, the risk increases with increasing exposure. The risk of impairment of colour vision was not altered in this study by the presence of different GSTM1, GSTT1 or NAT2 polymorphisms.

Family in which Duchenne's muscular dystrophy and protan colour blindness are segregating.

A family is recorded in which Duchenne's muscular dystrophy and protan colour blindness are segregating. Of 4 members of the second generation at least one is a recombinant. The lod scores have been calculated and added to those already published.