Search

Purpose In a cohort of eight families (11 patients) with autosomal recessive retinitis pigmentosa (arRP), we clinically characterized disease associated with mutations in CNGB1. Methods Visual function was determined by measuring the patients' visual acuity, dark- and light-adapted perimetry, and by full-field electroretinography. Retinal structure was evaluated with spectral-domain optical coherence tomography, fundus imaging, and autofluorescence imaging. Results Age of onset ranged from 4 to 49 years (mean [SD] 26 [17], median 27 years). The age at visit was 27-54 years, mean 37 (17). The range of visual acuity was logMAR -0.1 to 1.3 (Snellen 20/16 to 20/400) in the right eye and -0.1 to 0.9 (Snellen 20/16 to 20/160) in the left eye. Electrophysiological testing in five patients showed an absence of the rod response. Cone responses ranged from normal to severely reduced. The patients exhibited loss of rod vision more severe than cone vision. Funduscopic images showed widespread retinal degeneration with pigment clumping, optic disk pallor, arteriole attenuation, and a peri-foveal ring of hyper autofluorescence. Three families were tested for olfactory dysfunction and results indicated mild to complete anosmia in individuals with mutations in CNGB1. Genetic analysis revealed 6 novel variants, c.2127 C > G, p.Phe709Leu; c.1431 C > A, p.Cys477*; c.2034 G > A, p.Trp678*; c.2092 T > C, p.Cys698Arg; and c.583 + 2 T > C, c.2305-34 G > A and 3 variants that have been previously described, c.2957A>T, p.Asn986Ile; c.2544dup, p.Leu849Alafs*3; and c.2492 + 1 G > A. Discussion This is the first report for six novel CNGB1 variants associated with arRP. Two families had olfactory dysfunction in patients with arRP and family members who were heterozygous for a CNGB1 mutation. Additionally, findings demonstrated variable penetrance and expressivity of disease in these patients.

Human induced pluripotent stem cells (iPSCs) are differentiated into three-dimensional (3D) retinal organoids to study retinogenesis and diseases that would otherwise be impossible. The complexity and low yield in current protocols remain a technical challenge, particularly for inexperienced personnel. Differentiation protocols require labor-intensive and time-consuming dissection of optic vesicles (OVs). Here we compare this method with a suspension method of developing retinal organoids. iPSCs were differentiated with standard protocols but the suspension-grown method omitted the re-plating of embryoid bodies and dissection of OVs. All other media and treatments were identical between developmental methods. Developmental maturation was evaluated with RT-qPCR and immunocytochemistry. Dissection- and suspension-derived retinal organoids displayed temporal biogenesis of retinal cell types. Differences in retinal organoids generated by the two methods of differentiation included temporal developmental and the organization of neural retina layers. Retinal organoids grown in suspension showed delayed development and disorganized retinal layers compared to the dissected retinal organoids. We found that omitting the re-plating of EBs to form OVs resulted in numerous OVs that were easy to identify and matured along a retinal lineage. While more efficient, the suspension method led to retinal organoids with disorganized retinal layers compared to those obtained using conventional dissection protocols.

More than 200 different mutations in peripherin-2 (PRPH2) are associated with multiple subtypes of inherited retinal diseases (IRDs), including retinitis pigmentosa and cone or macular diseases. Our goal was to understand how the poorly characterized PRPH2 mutation p.Pro210Arg (P210R) affects visual function and retinal structure as well as gain insight into the mechanism driving the clinical pathology.Eleven patients had clinical assessments including best-corrected visual acuity (BCVA), full field and multifocal electroretinography (ERG), static (spot size V) and kinetic perimetry (Octopus 900), and dark-adapted chromatic (DAC; Medmont; spot size V) perimetry. Images were acquired with the Optos ultra-wide field camera and spectral-domain optical coherence tomography (SD-OCT). Molecular characteristics of the P210R mutant protein were evaluated in vitro.Patients with the P210R mutation had BCVA (Snellen) ranging from 20/15 to 20/80. Perimetry showed a reduction in sensitivity, while ERG findings suggested that cone function was more impaired than rod function. Scotomas were identified corresponding to atrophic retinal lesions. Imaging revealed heterogeneous outer retinal changes such as hyperfluorescent flecks, hypo-autofluorescence (AF) regions of atrophy, and thinning of the photoreceptor layer on SD-OCT. In vitro findings suggested that P210R-Prph2 retains the ability to interact with binding partner Rom1 but abnormally accumulates in the endoplasmic reticulum (ER), suggesting the protein does not fold properly.Rod and cone sensitivities were decreased in subjects with the P210R mutation in PRPH2. There was scotomatous vision loss that occurred within the macula, likely due to atrophy that occurs after drusen have formed and have begun to resolve. This suggests that although rod and cone photoreceptors are dependent on PRPH2, preventing blindness in this specific subgroup of patients could involve therapeutics that impede the formation or lifecycle of drusen.

Inherited retinal diseases (IRD) comprise a heterogeneous set of clinical and genetic disorders that lead to blindness. Given the emerging opportunities in precision medicine and gene thera-py, it has become increasingly important to determine whether DNA variants with uncertain significance (VUS) are responsible for the patients’ IRD. This research was performed to assess the functional consequence of six VUS identified in patients with IRD. Clinical assessments in-cluded an ophthalmic examination, best corrected visual acuity, and kinetic perimetry. Imaging was acquired with the Optos ultra-widefield camera and spectral-domain optical coherence to-mography (SD-OCT). Genetic testing was performed by Molecular Vision Laboratories. VUS that were predicted to alter splicing were analyzed with a minigene assay which revealed that VUS in the genes OPA1, CNGB1, and CLUAP1 altered spicing mechanisms. Due to the emerging gene and cell therapies, these results expand the genotype-phenotype correlations for patients diag-nosed with an IRD.

Purpose Dark-adapted visual fields were obtained from patients with inherited retinal degeneration (IRD) and controls to evaluate the effect that age, retinal region, and disease had on scotopic sensitivity. Intra- and intersession test-retest repeatabilities for patients and controls were measured to establish significant change for longitudinal studies. Methods A total of 41 patients with IRD and 30 controls had one eye dilated and dark-adapted for 40 minutes. Scotopic sensitivity was measured with a Medmont dark-adapted chromatic (DAC) perimeter (size V stimulus, 200-ms duration, background luminance < 0.0001 cd/m2, dynamic range 0-75 decibel [dB]). Mixed effects analysis was performed to analyze age, retinal eccentricity, and sensitivity. The intra-/intersession coefficients of repeatability (CR) were calculated for controls and patients with IRD. Results Each additional year was associated with lower sensitivity (-0.22 dB) per year in normal controls over age 50 compared to younger controls (12-49 years). The superior field had lower sensitivity than the inferior, but the nasal field was not different compared to the temporal field in normal controls. The CR for intra- and intersession testing on mean sensitivity (MS)/pointwise sensitivity (PWS) were ±1.5/±8.5 and ±3.3/±9.8 dB, respectively, for patients with IRD. Control MS/PWS CR were ±1.5/±6.1 dB for intrasession and ±1.7/±6.8 dB for intersession DAC perimetry. Conclusions The DAC perimeter is an important asset because it tests a wide field of scotopic vision. The CR are comparable to those of other perimetry devices. Effects of age and retinal region should be considered when assessing scotopic sensitivity measured with the DAC perimeter.

PURPOSE: In a cohort of 8 families (11 patients) with autosomal recessive retinitis pigmentosa (arRP), we clinically characterized disease associated with mutations in CNGB1. METHODS: Visual function was determined by measuring the patients’ visual acuity, dark- and light-adapted perimetry, and by full field electroretinography. Retinal structure was evaluated with spectral-domain optical coherence tomography, fundus imaging, and autofluorescence imaging. RESULTS: Age of onset ranged from 4 to 49 years (mean [SD] 26 [17], median 27 years). The age at visit was 27 – 54 years, mean 37 (17). The range of visual acuity was logMAR −0.1 to 1.3 (Snellen 20/16 to 20/400) in the right eye and −0.1 to 0.9 (Snellen 20/16 to 20/160) in the left eye. Electrophysiological testing in 5 patients showed an absence of the rod response. Cone responses ranged from normal to severely reduced. The patients exhibited loss of rod vision more severe than cone vision. Funduscopic images showed widespread retinal degeneration with pigment clumping, optic disk pallor, arteriole attenuation, and a peri-foveal ring of hyper autofluorescence. Three families were tested for olfactory dysfunction and results indicated mild to complete anosmia in individuals with mutations in CNGB1. Genetic analysis revealed 6 novel variants, c.2127C>G, p.Phe709Leu; c.1431C>A, p.Cys477*; c.2034G>A, p.Trp678*; c.2092T>C, p.Cys698Arg; and c.583+2T>C, c.2305-34G>A and 3 variants that have been previously described, c.2957A>T, p.Asn986Ile; c.2544dup, p.Leu849Alafs*3; and c.2492+1G>A. DISCUSSION: This is the first report for 6 novel CNGB1 variants associated with arRP. Two families had olfactory dysfunction in patients with arRP and family members who were heterozygous for a CNGB1 mutation. Additionally, findings demonstrated variable penetrance and expressivity of disease in these patients.

Retinal organoids derived from inducible pluripotent stem cells were used to gain insight into the role of l-DOPA during human retinal development. Dopaminergic gene expression was indicated by assessing two dopamine receptors (DRD1 and DRD2), DOPA decarboxylase (DDC), and tyrosine hydroxylase (TH) via quantitative reverse transcription-polymerase chain reaction at various developmental stages. TH transcript levels started to express around day (D) 42, reached maximal expression ∼D63 and then decreased thereafter. At D29, proliferating retinal progenitors expressed DRD1, DRD2, and DDC at various levels of mRNA throughout the day. In the presence of l-DOPA, D29 retinal organoids expressed DRD1 but DRD2 mRNA expression was suppressed. Additionally, l-DOPA upregulated TH mRNA prior to dopaminergic amacrine cell (DAC) development. After the appearance of DACs, l-DOPA phase shifted expression of DRD2 and synchronized mRNA expression of DDC, DRD2, and TH. The present results suggest unique mechanisms for DA signaling at different stages of development in the human retina.

PurposeTo evaluate the long-term efficacy of ciliary neurotrophic factor delivered via an intraocular encapsulated cell implant for the treatment of retinitis pigmentosa.DesignLong-term follow-up of a multicenter, sham-controlled study.MethodsThirty-six patients at 3 CNTF4 sites were randomly assigned to receive a high- or low-dose implant in 1 eye and sham surgery in the fellow eye. The primary endpoint (change in visual field sensitivity at 12months) had been reported previously. Here we measure long-term visual acuity, visual field, and optical coherence tomography (OCT) outcomes in 24 patients either retaining or explanting the device at 24months relative to sham-treated eyes.ResultsEyes retaining the implant showed significantly greater visual field loss from baseline than either explanted eyes or sham eyes through 42months. By 60months and continuing through 96months, visual field loss was comparable among sham-treated eyes, eyes retaining the implant, and explanted eyes, as was visual acuity and OCT macular volume.ConclusionsOver the short term, ciliary neurotrophic factor released continuously from an intravitreal implant led to loss of total visual field sensitivity that was greater than the natural progression in the sham-treated eye. This additional loss of sensitivity related to the active implant was reversible when the implant was removed. Over the long term (60-96months), there was no evidence of efficacy for visual acuity, visual field sensitivity, or OCT measures of retinal structure.

Purpose Mutations in the inosine monophosphate dehydrogenase 1 (IMPDH1) gene are a common cause of inherited retinal degeneration (IRD). Due to species- and tissue-dependent expression of IMPDH1, there are no appropriate models of human IMPDH1 disease. Therefore, a limited understanding remains of disease expression and rates of progression for IMPDH1-related IRD. Methods We evaluated semiautomated kinetic and chromatic static perimetry, spectral-domain optical coherence tomography (SD-OCT), and ultra-wide field fundus images with autofluorescence in a cohort of 12 patients (ages 11-58 at first visit). Ten patients had longitudinal data for which rates of progression were estimated. Results Visual acuities were relatively stable over time and the photoreceptors within the central retina remained intact. Perifoveal photoreceptor loss measured over a period of years coincided with visual fields, which were constricted and progressed over time in all patients. Rod sensitivity showed a similar pattern of defect to that of the kinetic perimetry and the autofluorescence ultra-wide field imaging. Full-field electroretinograms were severely reduced and the dark-adapted rod and mixed responses were extinguished at earlier visits than the light-adapted cone responses. Conclusions There was variability in disease severity at the first visit, but results show that the peripheral retina is more susceptible to the deleterious consequences of an IMPDH1 mutation. Given the pattern of degeneration and the alternatively spliced isoforms of IMPDH1, potential interventions may consider targeting the periphery early in disease, modulating transcript expression, and/or preserving central vision at late stages of the disease. Translational relevance These results inform clinical prognosis and offer evidence strategies toward therapeutic intervention.

PURPOSE Although rod photoreceptors are initially affected in retinitis pigmentosa (RP), the full-field of rod vision is not routinely characterized due to the unavailability of commercial devices detecting rod sensitivity. The purpose of this study was to quantify rod-mediated vision in the peripheral field from patients with RP using a new commercially available perimeter. METHODS Participants had one eye dilated and dark-adapted for 45 minutes. A dark-adapted chromatic (DAC) perimeter tested 80 loci 144° horizontally and 72° vertically with cyan stimuli. The number of rod-mediated loci (RML) were analyzed based on normal cone sensitivity (method 1) and associated with full-field electroretinography (ERG) responses by Pearson's r correlation and linear regression. In a second cohort of patients with RP, RML were identified by two-color perimetry (cyan and red; method 2). The two methods for ascribing rod function were compared by Bland-Altman analysis. RESULTS Method 1 RML were correlated with responses to the 0.01 cd.s/m2 flash (P < 0.001), while total sensitivity to the cyan stimulus showed correlation with responses to the 3.0 cd.s/m2 flash (P < 0.0001). Method 2 detected a mean of 10 additional RML compared to method 1. CONCLUSIONS Scotopic fields measured with the DAC detected rod sensitivity across the full visual field, even in some patients who had nondetectable rod ERGs. Two-color perimetry is warranted when sensitivity to the cyan stimulus is reduced to ≤20 dB to get a true estimation of rod function. TRANSLATIONAL RELEVANCE Many genetic forms of retinitis pigmentosa (RP) are caused by mutations in rod-specific genes. However, treatment trials for patients with RP have relied primarily on photopic (cone-mediated) tests as outcome measures because there are a limited number of available testing methods designed to evaluate rod function. Thus, efficient methods for quantifying rod-mediated vision are needed for the rapidly increasing numbers of clinical trials.

Background The purpose of this study is to determine the maximum tolerated dose of a single intravitreal injection of aminoguanidine and 1400W, 2 inhibitors of inducible nitric oxide synthase, in rabbit eyes. Inhibition of inducible nitric oxide synthase has already been shown to be beneficial in various animal models of diabetic eye disease. Methods Groups of 4 New Zealand white rabbits were injected with balanced salt solution in the right eye and a single dose of either aminoguanidine (5, 1, 0.25 mg) or 1400W (2 mg and 0.4 mg) in the left eye. Toxicity was assessed by slit-lamp and fundus examination, intraocular pressure and pachymetric measurements, and electrophysiologic and histologic analysis. Results Eyes injected with high doses of aminoguanidine (5 mg) or 1400W (2 mg) demonstrated severe retinal vascular attenuation and infarction. Lower doses of intravitreal aminoguanidine (1 mg) and 1400W (0.4 mg) caused no significant toxic ocular effects in rabbit eyes. Conclusion If the difference in vitreal volume between rabbit eyes and human eyes is taken into account, aminoguanidine (2.7 mg) and 1400W (1 mg) would be reasonable intravitreal doses to test for safety and efficacy in early clinical trials.

Juvenile X-linked retinoschisis (XLRS) is a congenital macular degeneration affecting 1/5000 to 1/25,000 worldwide.1,–3 The gene associated with XLRS, Retinoschisin (RS1),4 translates to a retinoschisin protein (RS1), which assists in maintaining retinal structure by binding to the photoreceptors and bipolar cells. Patients are diagnosed in their primary school years with clinical characteristics of bilateral retinal splitting5–8 and an electronegative electroretinography (ERG) response with preserved a-wave.9 Best corrected visual acuity (BCVA) typically ranges from 20/50 to 20/120 (0.5–0.8 log minimum angle of resolution [logMAR]) and remains stable until the fifth or sixth decade of life when the cavities resolve and visual acuity decreases.10 Shape discrimination hyperacuity (SDH) and contour integration perimetry (CIP) in patients with intermediate AMD show significant deficits, with macular edema exacerbating the loss of the ability to detect distortions in circular shapes.11,12 These tests assess the global integration of visual stimuli over a large retinal area. The SDH tests parafoveal acuity, whereas the CIP determines retinal acuity outside of the central 3°. Due to the foveal edema in XLRS we hypothesize that the global integration measured by SDH/CIP may be affected, although some patients retain a relatively good BCVA. Spectral-domain optical coherence tomography (SDOCT) studies in XLRS have been reported,5,7,13,14 but rarely correlated with fundus-guided perimetry15 or shape discrimination. Clinical attributes of XLRS have been characterized, but concise relationships with structure and psychophysical function need further exploration. A multicenter natural history study of XLRS was designed to understand disease progression and determine suitable outcome measures for future gene therapy trials. The results reported here were obtained from a single visit. A subset of patients were tested with additional measures so that we could determine whether photoreceptor outer segment (OS) and/or total retina (TR) thickness could predict performance on visual tasks such as BCVA, fundus-guided perimetry, SDH, and CIP in patients with XLRS.

Stargardt-like macular dystrophy-3 (STGD3) is a juvenile-onset disease caused by mutations in ELOVL4 (elongation of very long fatty acids-4). This gene product catalyzes the elongation of long chain saturated and polyunsaturated fatty acids (LC-FAs and LC-PUFAs) into very long chain FAs and PUFAs (VLC-FAs and VLC-PUFAs). These mutations cause a frame shift in the ELOVL4 transcript, introducing a premature stop codon that results in the translation of a truncated protein that has lost a C-terminus endoplasmic reticulum (ER) retention/retrieval signal. The truncated protein is not targeted to the ER, the site of very long-chain PUFA (VLC-PUFA; 28-40 carbons) synthesis. Expression of the ELOVL4 gene is limited mainly to the brain, testis, skin, and photoreceptor cells of the retina. While the skin and brain contain very long chain saturated fatty acids (VLC-FAs), the other tissues expressing ELOVL4 contain VLC-PUFAs, with sperm and the retina having the highest levels. This review focuses on the current information available concerning the role of VLC-PUFAs in the retina.

Stargardt-like macular dystrophy-3 (STGD3) is a juvenile-onset disease caused by mutations in ELOVL4 (elongation of very long fatty acids-4). This gene product catalyzes the elongation of long chain saturated and polyunsaturated fatty acids (LC-FAs and LC-PUFAs) into very long chain FAs and PUFAs (VLC-FAs and VLC-PUFAs). These mutations cause a frame shift in the ELOVL4 transcript, introducing a premature stop codon that results in the translation of a truncated protein that has lost a C-terminus endoplasmic reticulum (ER) retention/retrieval signal. The truncated protein is not targeted to the ER, the site of very long-chain PUFA (VLC-PUFA; 28-40 carbons) synthesis. Expression of the ELOVL4 gene is limited mainly to the brain, testis, skin, and photoreceptor cells of the retina. While the skin and brain contain very long chain saturated fatty acids (VLC-FAs), the other tissues expressing ELOVL4 contain VLC-PUFAs, with sperm and the retina having the highest levels. This review focuses on the current information available concerning the role of VLC-PUFAs in the retina.

Purpose Juvenile-onset autosomal dominant Stargardt-like macular dystrophy (STGD3) is caused by mutations in ELOVL4 (elongation of very long fatty acids-4), an elongase necessary for the biosynthesis of very long chain fatty acids (VLC-FAs ≥ C26). Photoreceptors are enriched with VLC polyunsaturated fatty acids (VLC-PUFAs), which are necessary for long-term survival of rod photoreceptors. The purpose of these studies was to determine the effect of deletion of VLC-PUFAs on rod synaptic function in retinas of mice conditionally depleted (KO) of Elovl4. Methods Retina function was assessed in wild-type (WT) and KO by electroretinography. Outer plexiform structure was evaluated by immunofluorescence and transmission electron microscopy. Single-cell recordings measured rod ion channel operation and rod bipolar glutamate signaling. Sucrose gradient centrifugation was used to isolate synaptosomes from bovine retina. Proteins and lipids were analyzed by Western blotting and tandem mass spectroscopy, respectively. Results Inner retinal responses (b-wave, oscillatory potentials, and scotopic threshold responses) of the ERG were decreased in the KO mice compared to controls. However the rod ion channel operation and bipolar glutamate responses were comparable between groups. Biochemical analysis revealed that conventional and ribbon synapses have VLC-PUFAs. Ultrastructural analysis showed that the outer plexiform layer was disorganized and the diameter of vesicles in rod terminals was smaller in the KO mice. Conclusions Very long chain PUFAs affect rod function by contributing to synaptic vesicle size, which may alter the dynamics of synaptic transmission, ultimately resulting in a loss of neuronal connectivity and death of rod photoreceptors.

Autosomal dominant Stargardt-like macular dystrophy (STGD3) is a juvenile-onset disease that is caused by mutations in Elovl4 (elongation of very long fatty acids-4). The Elovl4 catalyzes the first step in the conversion of C24 and longer fatty acids (FAs) to very long-chain FAs (VLC-FAs, ≥C26). Photoreceptors are particularly rich in VLC polyunsaturated FAs (VLC-PUFA). To explore the role of VLC-PUFAs in photoreceptors, we conditionally deleted Elovl4 in the mouse retina.Proteins were analyzed by Western blotting and lipids by gas chromatography (GC)-mass spectrometry, GC-flame ionization detection, and tandem mass spectrometry. Retina function was assessed by electroretinography (ERG), and structure was evaluated by bright field, immunofluorescence, and transmission electron microscopy.Conditional deletion (KO) of retinal Elovl4 reduced RNA and protein levels by 91% and 96%, respectively. Total retina VLC-PUFAs were reduced by 88% compared to the wild type (WT) levels. Retinal VLC-PUFAs incorporated in phosphatidylcholine were less abundant at 12 months compared to 8-week-old levels. Amplitudes of the ERG a-wave were reduced by 22%, consistent with photoreceptor degeneration (11% loss of photoreceptors). Analysis of the rod a-wave responses gave no evidence of a role for VLC-PUFA in visual transduction. However, there were significant reductions in rod b-wave amplitudes (30%) that could not be explained by loss of rod photoreceptors. There was no effect of VLC-PUFA reduction on cone ERG responses, and cone density was not different between the WT and KO mice at 12 months of age.The VLC-PUFAs are important for rod, but not cone, function and for rod photoreceptor longevity.

Elongation of Very Long chain fatty acids-like 4 (ELOVL4) is a fatty acid elongase responsible for the biosynthesis of very long chain (VLC; ≥ C26) fatty acids in the retina, brain, skin, Meibomian gland, and testes. Heterozygous inheritance of mutant ELOVL4 causes juvenile macular degeneration in autosomal dominant Stargardt-like macular dystrophy (STGD3). Retinal photoreceptors are enriched with VLC polyunsaturated fatty acids (VLC-PUFAs), which have been shown by our group and others to be necessary for the survival of rod photoreceptors. Our group performed a series of studies using mice conditionally depleted of retinal Elovl4 (KO) aimed at understanding the role of VLC-PUFAs in long-term retinal health and function, focusing on the role of these fatty acids in mediating synaptic function between the photoreceptors and the rest of the neural retina. The absence of VLC-PUFA from the retina of KO mice resulted in a marked decrease in retinal b-wave responses of the electroretinogram as well as a decrease in the amplitude of the oscillatory potentials mediated by the neural retina. Although there were no measureable differences between KO and wild type (WT) mice in either pre-synaptic rod calcium channel function or post-synaptic bipolar cell glutamate receptor responses, ultrastructural analysis revealed a marked decrease in the diameter of synaptic vesicles in rod terminals. Recent quantification suggests that this decrease in synaptic vesicle size due to the absence of VLC-PUFAs in KO mice, and the consequent decrease in glutamate content, could account for the decrease in b-wave response amplitudes that were previously measured in these animals.