Reconstitution of the phosphodiesterase 6 maturation process important for photoreceptor cell function.

The sixth family phosphodiesterases (PDE6) are principal effector enzymes of the phototransduction cascade in rods and cones. Maturation of nascent PDE6 protein into a functional enzyme relies on a coordinated action of ubiquitous chaperone HSP90, its specialized cochaperone aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1), and the regulatory Pγ-subunit of PDE6. Deficits in PDE6 maturation and function underlie severe visual disorders and blindness. Here, to elucidate the roles of HSP90, AIPL1, and Pγ in the maturation process, we developed the heterologous expression system of human cone PDE6C in insect cells allowing characterization of the purified enzyme. We demonstrate that in the absence of Pγ, HSP90, and AIPL1 convert the inactive and aggregating PDE6C species into dimeric PDE6C that is predominantly misassembled. Nonetheless, a small fraction of PDE6C is properly assembled and fully functional. From the analysis of mutant mice that lack both rod Pγ and PDE6C, we conclude that, in contrast to the cone enzyme, no maturation of rod PDE6AB occurs in the absence of Pγ. Co-expression of PDE6C with AIPL1 and Pγ in insect cells leads to a fully mature enzyme that is equivalent to retinal PDE6. Lastly, using immature PDE6C and purified chaperone components, we reconstituted the process of the client maturation in vitro. Based on this analysis we propose a scheme for the PDE6 maturation process.

Knockout of CaV1.3 L-type calcium channels in a mouse model of retinitis pigmentosa.

Retinitis Pigmentosa is a genetically heterogeneous, degenerative retinal disorder characterized by gradual dysfunction and death of photoreceptors, first rods and later cones, and progressive blindness. Studies suggested that application of L-type calcium channel blockers rescues photoreceptors in paradigms related to Ca2+ overflow. To investigate whether Cav1.3 L-type channels have protective effects in the retina, we established a new mouse model by crossing rd10, modeling autosomal-recessive RP, with Cav1.3 deficient mice (rd10/Cav1.3KO). Our immunohistochemical analyses revealed an influence of Cav1.3 channels on the degenerative process of photoreceptors. The absence of Cav1.3 delayed the centre-to-periphery degeneration of rods indicated by a significantly higher number of photoreceptor rows and, consequently, of cones. In accordance with a preserved number of cones we observed a regular row of cone somas in rd10/Cav1.3-KO retinas. Surviving rod photoreceptors maintained synaptic contacts with rod bipolar cells. However, the delay in degeneration was only observed up to postnatal day 45. Although we observed a reduction in the spontaneous oscillatory retinal activity during multielectrode array analyses, measurable functional preservation was lacking in behavioural tests. In conclusion, Cav1.3 channels contribute to photoreceptor degeneration in rd10 retinas but photoreceptor temporary rescue might rather be achieved indirectly through other retinal cell layers.

Long-term effects of human induced pluripotent stem cell-derived retinal cell transplantation in Pde6b knockout rats.

Retinal degenerative disorders, including age-related macular degeneration and retinitis pigmentosa (RP), are characterized by the irreversible loss of photoreceptor cells and retinal pigment epithelial (RPE) cells; however, the long-term effect of implanting both human induced pluripotent stem cell (hiPSC)-derived RPE and photoreceptor for retinal regeneration has not yet been investigated. In this study, we evaluated the long-term effects of hiPSC-derived RPE and photoreceptor cell transplantation in Pde6b knockout rats to study RP; cells were injected into the subretinal space of the right eyes of rats before the appearance of signs of retinal degeneration at 2-3 weeks of age. Ten months after transplantation, we evaluated the cells using fundus photography, optical coherence tomography, and histological evaluation, and no abnormal cell proliferation was observed. A relatively large number of transplanted cells persisted during the first 4 months; subsequently, the number of these cells decreased gradually. Notably, immunohistochemical analysis revealed that the hiPSC-derived retinal cells showed characteristics of both RPE cells and photoreceptors of human origin after transplantation. Functional analysis of vision by scotopic electroretinogram revealed significant preservation of vision after transplantation. Our study suggests that the transplantation of hiPSC-derived retinal cells, including RPE cells and photoreceptors, has a potential therapeutic effect against irreversible retinal degenerative diseases.

Metabolite therapy guided by liquid biopsy proteomics delays retinal neurodegeneration.

BACKGROUND: Neurodegenerative diseases are incurable disorders caused by progressive neuronal cell death. Retinitis pigmentosa (RP) is a blinding neurodegenerative disease that results in photoreceptor death and progresses to the loss of the entire retinal network. We previously found that proteomic analysis of the adjacent vitreous served as way to indirectly biopsy the retina and identify changes in the retinal proteome. METHODS: We analyzed protein expression in liquid vitreous biopsies from autosomal recessive (ar)RP patients with PDE6A mutations and arRP mice with Pde6ɑ mutations. Proteomic analysis of retina and vitreous samples identified molecular pathways affected at the onset of photoreceptor death. Based on affected molecular pathways, arRP mice were treated with a ketogenic diet or metabolites involved in fatty-acid synthesis, oxidative phosphorylation, and the tricarboxylic acid (TCA) cycle. FINDINGS: Dietary supplementation of a single metabolite, ɑ-ketoglutarate, increased docosahexaeonic acid levels, provided neuroprotection, and enhanced visual function in arRP mice. A ketogenic diet delayed photoreceptor cell loss, while vitamin B supplementation had a limited effect. Finally, desorption electrospray ionization mass spectrometry imaging (DESI-MSI) on ɑ-ketoglutarate-treated mice revealed restoration of metabolites that correlated with our proteomic findings: uridine, dihydrouridine, and thymidine (pyrimidine and purine metabolism), glutamine and glutamate (glutamine/glutamate conversion), and succinic and aconitic acid (TCA cycle). INTERPRETATION: This study demonstrates that replenishing TCA cycle metabolites via oral supplementation prolongs retinal function and provides a neuroprotective effect on the photoreceptor cells and inner retinal network. FUNDING: NIH grants [R01EY026682, R01EY024665, R01EY025225, R01EY024698, R21AG050437, P30EY026877, 5P30EY019007, R01EY018213, F30EYE027986, T32GM007337, 5P30CA013696], NSF grant CHE-1734082.

Single AAV-mediated mutation replacement genome editing in limited number of photoreceptors restores vision in mice.

Supplementing wildtype copies of functionally defective genes with adeno-associated virus (AAV) is a strategy being explored clinically for various retinal dystrophies. However, the low cargo limit of this vector allows its use in only a fraction of patients with mutations in relatively small pathogenic genes. To overcome this issue, we developed a single AAV platform that allows local replacement of a mutated sequence with its wildtype counterpart, based on combined CRISPR-Cas9 and micro-homology-mediated end-joining (MMEJ). In blind mice, the mutation replacement rescued approximately 10% of photoreceptors, resulting in an improvement in light sensitivity and an increase in visual acuity. These effects were comparable to restoration mediated by gene supplementation, which targets a greater number of photoreceptors. This strategy may be applied for the treatment of inherited disorders caused by mutations in larger genes, for which conventional gene supplementation therapy is not currently feasible.

On the pivotal role of Elovl3/ELOVL3 in meibogenesis and ocular physiology of mice.

The purpose of this study was to examine the role of Elovl3 gene in meibogenesis and the impact of ELOVL3 protein ablation on the physiology of the mouse ocular surface and Meibomian glands (MGs). Elovl3 knockout, ELOVL3-ablated (E3hom) mice and their wild type littermates (E3wt) were studied side by side. E3hom mice had abnormal ocular phenotypes such as delayed eye opening, weeping eyes, crusty eyelids, eyelid edema, highly vascularized cornea and tarsal plates (TPs), slit eye, and increased tearing that resemble symptoms observed in human subjects with various forms of dry eye, MG dysfunction and blepharitis. Lipid profiling of E3hom TPs was conducted using chromatography and mass spectrometry. The analyses revealed that the lipid composition of E3hom TPs was strikingly different from that of their E3wt littermates. The mutation affected major classes of meibomian lipids - cholesteryl esters, wax esters, and cholesteryl esters of (O)-acylated w-hydroxy fatty acids. The studies illuminated the central role of ELOVL3 in producing C21:0-C29:0 fatty acids, including odd-chain and branched ones. Ablation of ELOVL3 leads to selective changes in the lipid composition of meibum, making E3hom mice instrumental in studying the mechanisms of the biosynthesis of meibum and modeling various pathologies of human ocular surface and adnexa.-Butovich, I. A., Wilkerson, A., Bhat, N., McMahon, A., Yuksel, S. On the pivotal role of Elovl3/ELOVL3 in meibogenesis and ocular physiology of mice.

Rescuing cones and daylight vision in retinitis pigmentosa mice.

Hallmark of retinitis pigmentosa (RP) is the primary, genetic degeneration of rods followed by secondary loss of cones, caused by still elusive biologic mechanisms. We previously shown that exposure of rd10 mutant mice, modeling autosomal recessive RP, to environmental enrichment (EE), with enhanced motor, sensorial and social stimuli, results into a sensible delay of retinal degeneration and vision loss. Searching for effectors of EE-mediated retinal protection, we performed transcriptome analysis of the retina of rd10 enriched and control mice and found that gene expression at the peaks of rod and cone degeneration is characterized by a strong inflammatory/immune response, which is however measurably lower in enrichment conditions. Treating rd10 mice with dexamethasone during the period of maximum photoreceptors death lowered retinal inflammation and caused a preservation of cones and cone-mediated vision. Our findings indicate a link between retinal inflammation and bystander cone degeneration, reinforcing the notion that cone vision in RP can be preserved using anti-inflammatory approaches.-Guadagni, V., Biagioni, M., Novelli, E., Aretini, P., Mazzanti, C. M., Strettoi, E. Rescuing cones and daylight vision in retinitis pigmentosa mice.

A novel intronic mutation of PDE6B is a major cause of autosomal recessive retinitis pigmentosa among Caucasus Jews.

Purpose: To identify the genetic basis for retinitis pigmentosa (RP) in a cohort of Jewish patients from Caucasia. Methods: Patients underwent a detailed ophthalmic evaluation, including funduscopic examination, visual field testing, optical coherence tomography (OCT), and electrophysiological tests, electroretinography (ERG) and visual evoked potentials (VEP). Genetic analysis was performed with a combination of whole exome sequencing (WES) and Sanger sequencing. Bioinformatic analysis of the WES results was performed via a customized pipeline. Pathogenicity of the identified intronic variant was evaluated in silico using the web tool Human Splicing Finder, and in vitro, using a minigene-based splicing assay. Linkage disequilibrium (LD) analysis was used to demonstrate a founder effect, and the decay of LD over generations around the mutation in Caucasus Jewish chromosomes was modeled to estimate the age of the most recent common ancestor. Results: In eight patients with RP from six unrelated families, all of Caucasus Jewish ancestry, we identified a novel homozygous intronic variant, located at position -9 of PDE6B intron 15. The c.1921-9C>G variant was predicted to generate a novel acceptor splice site, nine bases upstream of the original splice site of intron 15. In vitro splicing assay demonstrated that this novel acceptor splice site is used instead of the wild-type site, leading to an 8-bp insertion into exon 16, which is predicted to cause a frameshift. The presence of a common ancestral haplotype in mutation-bearing chromosomes was compatible with a founder effect. Conclusions: The PDE6B c.1921-9C>G intronic mutation is a founder mutation that accounts for at least 40% (6/15 families) of autosomal recessive RP among Caucasus Jews. This result is highly important for molecular diagnosis, carrier screening, and genetic counseling in this population.

The PDE6 mutation in the rd10 retinal degeneration mouse model causes protein mislocalization and instability and promotes cell death through increased ion influx.

The retinal degeneration model rd10 contains a missense mutation of the catalytic PDE6 ß subunit, which hydrolyzes cGMP in response to light. This model produces cell death more slowly than others caused by PDE6 loss of function, making it of particular interest for studying potential therapeutics. We used morphology, biochemistry, and single-cell physiology to examine the mechanism of rd10 degeneration. Our results show that the mutation produces no alteration of Pde6b RNA but does dramatically decrease maximal and basal PDE6 activity, apparently caused by a decrease in protein stability and transport. The enzymatic properties of the remaining mutant PDE6 appear to be nearly normal. We demonstrate that an increase in free cGMP, which would result from decreased PDE6 activity and serve to increase opening of the cGMP-gated channels and calcium influx, is an underlying cause of cell death: degeneration of rd10/Cngb1-/- double mutants is slower than the parent rd10 line. Paradoxically, degeneration in rd10/Cngb1-/- is also slower than in Cngb1-/- This rescue is correlated with a lowering of cGMP content in Cngb1-/- retinas and suggests that it may be caused by mislocalization of active PDE6. Single-cell recordings from rd10 rods show that the rates of rise and decay of the response are significantly slower; simulations indicate that these changes are primarily the result of the decrease in PDE6 concentration and rod collecting area. Together, these results provide insights into the complex mechanisms that underlie rd10-mediated retinal degeneration and a cautionary note for analysis of therapeutic interventions.

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.

PKG-Dependent Cell Death in 661W Cone Photoreceptor-like Cell Cultures (Experimental Study).

In humans cone photoreceptors are responsible for high-resolution colour vision. A variety of retinal diseases can compromise cone viability, and, at present, no satisfactory treatment options are available. Here, we present data towards establishing a reliable, high-throughput assay system that will facilitate the search for cone neuroprotective compounds using the murine-photoreceptor cell line 661 W. To further characterize 661 W cells, a retinal marker study was performed, followed by the induction of cell death using paradigms over-activating cGMP-dependent protein kinase G (PKG). We found that 661 W cells may be used to mimic specific aspects of cone degeneration and may thus be valuable for future compound screening studies.

Electrophysiology Alterations in Primary Visual Cortex Neurons of Retinal Degeneration (S334ter-line-3) Rats.

The dynamic nature of the brain is critical for the success of treatments aimed at restoring vision at the retinal level. The success of these treatments relies highly on the functionality of the surviving neurons along the entire visual pathway. Electrophysiological properties at the retina level have been investigated during the progression of retinal degeneration; however, little is known about the changes in electrophysiological properties that occur in the primary visual cortex (V1) during the course of retinal degeneration. By conducting extracellular recording, we examined the electrophysiological properties of V1 in S334ter-line-3 rats (a transgenic model of retinal degeneration developed to express a rhodopsin mutation similar to that found in human retinitis pigmentosa patients). We measured the orientation tuning, spatial and temporal frequency tunings and the receptive field (RF) size for 127 V1 neurons from 11 S334ter-3 rats and 10 Long-Evans (LE) rats. V1 neurons in the S334ter-3 rats showed weaker orientation selectivity, lower optimal spatial and temporal frequency values and a smaller receptive field size compared to the LE rats. These results suggest that the visual cognitive ability significantly changes during retinal degeneration.

AAV-mediated Gene Therapy Halts Retinal Degeneration in PDE6ß-deficient Dogs.

We previously reported that subretinal injection of AAV2/5 RK.cpde6ß allowed long-term preservation of photoreceptor function and vision in the rod-cone dysplasia type 1 (rcd1) dog, a large animal model of naturally occurring PDE6ß deficiency. The present study builds on these earlier findings to provide a detailed assessment of the long-term effects of gene therapy on the spatiotemporal pattern of retinal degeneration in rcd1 dogs treated at 20 days of age. We analyzed the density distribution of the retinal layers and of particular photoreceptor cells in 3.5-year-old treated and untreated rcd1 dogs. Whereas no rods were observed outside the bleb or in untreated eyes, gene transfer halted rod degeneration in all vector-exposed regions. Moreover, while gene therapy resulted in the preservation of cones, glial cells and both the inner nuclear and ganglion cell layers, no cells remained in vector-unexposed retinas, except in the visual streak. Finally, the retinal structure of treated 3.5-year-old rcd1 dogs was identical to that of unaffected 4-month-old rcd1 dogs, indicating near complete preservation. Our findings indicate that gene therapy arrests the degenerative process even if intervention is initiated after the onset of photoreceptor degeneration, and point to significant potential of this therapeutic approach in future clinical trials.

Circadian clock control of connexin36 phosphorylation in retinal photoreceptors of the CBA/CaJ mouse strain.

The gap-junction-forming protein connexin36 (Cx36) represents the anatomical substrate of photoreceptor electrical coupling in mammals. The strength of coupling is directly correlated to the phosphorylation of Cx36 at two regulatory sites: Ser110 and Ser293. Our previous work demonstrated that the extent of biotinylated tracer coupling between photoreceptor cells, which provides an index of the extent of electrical coupling, depends on the mouse strain. In the C57Bl/6J strain, light or dopamine reduces tracer coupling and Cx36 phosphorylation in photoreceptors. Conversely, darkness or a dopaminergic antagonist increases tracer coupling and Cx36 phosphorylation, regardless of the daytime. In the CBA/CaJ strain, photoreceptor tracer coupling is not only regulated by light and dopamine, but also by a circadian clock, a type of oscillator with a period close to 24 h and intrinsic to the retina, so that under prolonged dark-adapted conditions tracer coupling is broader at night compared to daytime. In the current study, we examined whether the modulation of photoreceptor coupling by a circadian clock in the CBA/CaJ mouse photoreceptors reflected a change in Cx36 protein expression and/or phosphorylation. We found no significant change in Cx36 expression or in the number of Cx36 gap junction among the conditions examined. However, we found that Cx36 phosphorylation is higher under dark-adapted conditions at night than in the daytime, and is the lowest under prolonged illumination at any time of the day/night cycle. Our observations are consistent with the view that the circadian clock regulation of photoreceptor electrical coupling is mouse strain-dependent and highlights the critical position of Cx36 phosphorylation in the control of photoreceptor coupling.

The effect of photoreceptor degeneration on ganglion cell morphology.

Retinitis pigmentosa refers to a family of inherited photoreceptor degenerations resulting in blindness. During and after photoreceptor loss, neurons of the inner retina are known to undergo plastic changes. Here, we have investigated in detail whether ganglion cells are altered at late stages of degeneration, well after the total loss of photoreceptors. We used mice, rd1-Thy1, that carry a mutation in the ß-subunit of phosphodiesterase 6 and a fluorescent protein that labels a subset of ganglion cells and B6-Thy1 control mice. Retinal wholemounts from mice aged 3-11 months were processed for immunohistochemistry and analyzed. Ganglion cells were classified based on soma area, dendritic field size, and branching of dendrites. The dendritic fields of some ganglion cells were further analyzed for their length, area and quantity of branching points. There was a decrease in size and level of branching of A2, B1, and D type ganglion cells in the degenerated retina at 11 months of age. In contrast, C1 ganglion cells remained unchanged. In addition, there was a shift in the proportion of ganglion cells ramifying in the different layers of the inner plexiform layer. Careful analysis of the dendrites of ganglion cells revealed some projecting to new, more distal regions of the inner plexiform layer. We propose that these changes in ganglion cell morphology could impact the function of individual cells as well as the retinal circuitry in the degenerated retina.

Loss of Pde6 reduces cell body Ca(2+) transients within photoreceptors.

Modulation of Ca(2+) within cells is tightly regulated through complex and dynamic interactions between the plasma membrane and internal compartments. In this study, we exploit in vivo imaging strategies based on genetically encoded Ca(2+) indicators to define changes in perikaryal Ca(2+) concentration of intact photoreceptors. We developed double-transgenic zebrafish larvae expressing GCaMP3 in all cones and tdTomato in long-wavelength cones to test the hypothesis that photoreceptor degeneration induced by mutations in the phosphodiesterase-6 (Pde6) gene is driven by excessive [Ca(2+)]i levels within the cell body. Arguing against Ca(2+) overload in Pde6 mutant photoreceptors, simultaneous analysis of cone photoreceptor morphology and Ca(2+) fluxes revealed that degeneration of pde6c(w59) mutant cones, which lack the cone-specific cGMP phosphodiesterase, is not associated with sustained increases in perikaryal [Ca(2+)]i. Analysis of [Ca(2+)]i in dissociated Pde6ß(rd1)mouse rods shows conservation of this finding across vertebrates. In vivo, transient and Pde6-independent Ca(2+) elevations ('flashes') were detected throughout the inner segment and the synapse. As the mutant cells proceeded to degenerate, these Ca(2+) fluxes diminished. This study thus provides insight into Ca(2+) dynamics in a common form of inherited blindness and uncovers a dramatic, light-independent modulation of [Ca(2+)]i that occurs in normal cones.

Restoration of vision in the pde6ß-deficient dog, a large animal model of rod-cone dystrophy.

Defects in the ß subunit of rod cGMP phosphodiesterase 6 (PDE6ß) are associated with autosomal recessive retinitis pigmentosa (RP), a childhood blinding disease with early retinal degeneration and vision loss. To date, there is no treatment for this pathology. The aim of this preclinical study was to test recombinant adeno-associated virus (AAV)-mediated gene addition therapy in the rod-cone dysplasia type 1 (rcd1) dog, a large animal model of naturally occurring PDE6ß deficiency that strongly resembles the human pathology. A total of eight rcd1 dogs were injected subretinally with AAV2/5RK.cpde6ß (n = 4) or AAV2/8RK.cpde6ß (n = 4). In vivo and post-mortem morphological analysis showed a significant preservation of the retinal structure in transduced areas of both AAV2/5RK.cpde6ß- and AAV2/8RK.cpde6ß-treated retinas. Moreover, substantial rod-derived electroretinography (ERG) signals were recorded as soon as 1 month postinjection (35% of normal eyes) and remained stable for at least 18 months (the duration of the study) in treated eyes. Rod-responses were undetectable in untreated contralateral eyes. Most importantly, dim-light vision was restored in all treated rcd1 dogs. These results demonstrate for the first time that gene therapy effectively restores long-term retinal function and vision in a large animal model of autosomal recessive rod-cone dystrophy, and provide great promise for human treatment.

Do calcium channel blockers rescue dying photoreceptors in the Pde6b ( rd1 ) mouse?

Retinitis pigmentosa (RP) is a genetically heterogeneous set of blinding diseases that affects more than a million people worldwide. In humans, ~5-8% of recessive and dominant RP cases are caused by nonsense mutations in the Pde6b gene coding for the ss-subunit of the rod photoreceptor cGMP phosphodiesterase 6 (PDE6-ss). The study of the disease has been greatly aided by the Pde6b ( rd1 ) (rd1) mouse model of RP carrying a null PDE6ss allele. Degenerating rd1 rods were found to experience a pathological increase in intracellular calcium concentration ('Ca overload') when they enter the apoptotic process at postnatal day 10. A 1999 study suggested that the Ca(2+) channel antagonist D-cis diltiazem delays the kinetics of rd1 rod degeneration, conferring partial rescue of scotopic vision. Subsequent reports were mixed: whereas several studies failed to replicate the original results, others appeared to confirm the neuroprotective effects of Ca(2+) channel antagonists such as diltiazem, nilvadipine and verapamil. We discuss the discrepancies between the results of different groups and suggest plausible causes for the discordant results. We also discuss potential involvement of recently identified Ca(2+)-dependent mechanisms that include protective calcium ATPase mechanisms, ryanodine and IP3 calcium stores, and store operated channels in Pde6b ( rd1 ) neurodegeneration.

Stimulation of the insulin/mTOR pathway delays cone death in a mouse model of retinitis pigmentosa.

Retinitis pigmentosa is an incurable retinal disease that leads to blindness. One puzzling aspect concerns the progression of the disease. Although most mutations that cause retinitis pigmentosa are in rod photoreceptor-specific genes, cone photoreceptors also die as a result of such mutations. To understand the mechanism of non-autonomous cone death, we analyzed four mouse models harboring mutations in rod-specific genes. We found changes in the insulin/mammalian target of rapamycin pathway that coincided with the activation of autophagy during the period of cone death. We increased or decreased the insulin level and measured the survival of cones in one of the models. Mice that were treated systemically with insulin had prolonged cone survival, whereas depletion of endogenous insulin had the opposite effect. These data suggest that the non-autonomous cone death in retinitis pigmentosa could, at least in part, be a result of the starvation of cones.

A novel mutation and phenotypes in phosphodiesterase 6 deficiency.

PURPOSE: To develop a systematic approach for the molecular diagnosis of retinitis pigmentosa (RP) and to report new genotype-phenotype correlations for phosphodiesterase 6 (PDE6)-based RP mutations. DESIGN: Clinical and molecular studies on a retrospective case series. METHODS: We screened 40 unrelated RP patients with an autosomal recessive RP microarray. Individuals with RP caused by PDE6 deficiency underwent genetic segregation and phenotype analysis. RESULTS: A disease-associated allele was identified in 32% of patients. Two probands (5%) had PDE6 mutations. The first proband was a compound heterozygote for known R102C and N216S alleles in PDE6A (MIM#180071). Pedigree analysis determined that the N216S variant was benign and direct sequencing discovered a novel, S303C allele. The second proband had a homozygous D600N mutation in the PDE6B gene (MIM#180072). Visual acuities of PDE6-deficient patients ranged from 20/40 to 20/200. Clinical studies showed unusual vitreomacular traction, cystoid macular edema, macular atrophy, and ring hyperfluorescence in PDE6-deficient patients. Such extensive vitreoretinal degeneration is not characteristic of photoreceptor-specific enzyme deficiencies. CONCLUSION: High-throughput deoxyribonucleic acid microarray chips can be used in combination with clinical imaging to precisely characterize patients with RP. Identifying the precise mutation in RP may become the standard of care as gene therapy emerges.