Long-term effect of gene therapy on Leber's congenital amaurosis.

BACKGROUND: Mutations in RPE65 cause Leber's congenital amaurosis, a progressive retinal degenerative disease that severely impairs sight in children. Gene therapy can result in modest improvements in night vision, but knowledge of its efficacy in humans is limited. METHODS: We performed a phase 1-2 open-label trial involving 12 participants to evaluate the safety and efficacy of gene therapy with a recombinant adeno-associated virus 2/2 (rAAV2/2) vector carrying the RPE65 complementary DNA, and measured visual function over the course of 3 years. Four participants were administered a lower dose of the vector, and 8 were administered a higher dose. In a parallel study in dogs, we investigated the relationship among vector dose, visual function, and electroretinography (ERG) findings. RESULTS: Improvements in retinal sensitivity were evident, to varying extents, in six participants for up to 3 years, peaking at 6 to 12 months after treatment and then declining. No associated improvement in retinal function was detected by means of ERG. Three participants had intraocular inflammation, and two had clinically significant deterioration of visual acuity. The reduction in central retinal thickness varied among participants. In dogs, RPE65 gene therapy with the same vector at lower doses improved vision-guided behavior, but only higher doses resulted in improvements in retinal function that were detectable with the use of ERG. CONCLUSIONS: Gene therapy with rAAV2/2 RPE65 vector improved retinal sensitivity, albeit modestly and temporarily. Comparison with the results obtained in the dog model indicates that there is a species difference in the amount of RPE65 required to drive the visual cycle and that the demand for RPE65 in affected persons was not met to the extent required for a durable, robust effect. (Funded by the National Institute for Health Research and others; ClinicalTrials.gov number, NCT00643747.).

Scotopic and Photopic Microperimetry in Patients With Reticular Drusen and Age-Related Macular Degeneration.

IMPORTANCE: Clinical observations suggest that patients with age-related macular degeneration (AMD) have vision problems, particularly in dim light conditions. Previous studies on structural-functional analysis in patients with AMD with reticular drusen (RDR) have focused on photopic sensitivity testing but have not specifically assessed scotopic function. OBJECTIVE: To evaluate retinal function by scotopic and photopic microperimetry in patients with AMD and a well-demarcated area of RDR. DESIGN, SETTING, AND PARTICIPANTS: Prospective case series in a referral center of 22 eyes from 18 patients (mean age, 74.7 years; range, 62-87 years). The study was conducted from June 1, 2014, to October 31, 2014. INTERVENTIONS: With the use of combined confocal scanning laser ophthalmoscopy and spectral-domain optical coherence tomography imaging, retinal areas with RDR (category 1) and no visible pathologic alterations (category 2) were identified in each eye. Scotopic and photopic microperimetry (MP-1S; Nidek Technologies) was performed using a grid with 56 stimulus points. MAIN OUTCOMES AND MEASURES: Comparison of mean threshold sensitivities for each category for scotopic and photopic microperimetry. RESULTS: In all eyes, areas of category 1 showed a relative and sharply demarcated reduction of scotopic threshold values compared with areas of category 2, but only less-pronounced differences were seen for photopic testing. Statistical analysis in the 18 eyes in which the 1.0-log unit neutral density filter was applied revealed a difference of scotopic threshold values in areas of category 1 (mean, 13.5 dB [95% CI, 12.1-15.0]) vs category 2 (mean, 18.3 dB; [95% CI, 17.4-19.3] (P ≤ .001). For photopic testing, the mean threshold values were 16.8 dB (95% CI, 15.5-18.2) in category 1 and 18.4 dB (95% CI, 17.1-19.6) in category 2 (P = .03). CONCLUSIONS AND RELEVANCE: The results of this study suggest that rod function is more severely affected than cone function in retinal areas with RDR. This differential structural-functional correlation underscores the functional relevance of RDR in patients with AMD.

A longitudinal study of Stargardt disease: quantitative assessment of fundus autofluorescence, progression, and genotype correlations.

PURPOSE: We characterized subtypes of fundus autofluorescence (AF) and the progression of retinal atrophy, and correlated these findings with genotype in Stargardt disease. METHODS: Full clinical examination and AF imaging was undertaken in 68 patients with Stargardt disease. The baseline data were compared to those at follow-up. Patients were classified into three AF subtypes: type 1 had a localized low signal at the fovea surrounded by a homogeneous background, type 2 had a localized low signal at the macula surrounded by a heterogeneous background with numerous foci of abnormal signal, and type 3 had multiple low signal areas at the posterior pole with a heterogeneous background. At baseline, there were 19 patients with type 1, 41 with type 2, and 8 with type 3 disease. The areas of reduced AF signal were measured and rate of atrophy enlargement (RAE) was calculated as the difference of the atrophy size over time (mm²) divided by the follow-up interval (years). Molecular screening of ABCA4 was undertaken. RESULTS: The mean follow-up interval was 9.1 years. A total of 42% cases with type 1 disease progressed to type 2, and 12% with type 2 progressed to type 3. The RAE (mm²/y) based upon baseline AF subtypes was significantly different; 0.06 in type 1, 0.67 in type 2, and 4.37 in type 3. ABCA4 variants were identified in 57 patients. There was a significant association between AF subtype and genotype. CONCLUSIONS: The AF pattern at baseline influences the enlargement of atrophy over time and has genetic correlates. These data are likely to assist in the provision of counseling on prognosis in Stargardt disease and be valuable for future clinical trials.

Comparison of fundus autofluorescence with photopic and scotopic fine matrix mapping in patients with retinitis pigmentosa: 4- to 8-year follow-up.

PURPOSE: To assess the significance and evolution of parafoveal rings of high-density fundus autofluorescence (AF) in 12 patients with retinitis pigmentosa (RP). METHODS: Twelve patients with autosomal recessive RP or Usher syndrome type 2 were ascertained who had a parafoveal ring of high-density AF and a visual acuity of 20/30 or better at baseline. Photopic and scotopic fine matrix mapping (FMM) were performed to test sensitivity across the macula. AF imaging and FMM were repeated after 4 to 8 years and optical coherence tomography (OCT) performed. RESULTS: The size of the AF ring reduced over time and disappeared in one subject. Photopic thresholds were normal over the fovea; thresholds were elevated by 0.6 log units over the ring and by 1.2 log units external to the ring at baseline and differed by less than 0.1 log unit at follow-up. Mild photopic losses close to the internal edge of the ring were detected at baseline or follow-up in all. Mean scotopic thresholds over parafoveal areas within the ring were markedly elevated in 8 of 10 at baseline and were severely elevated in 9 of 11 at follow-up. The eccentricity of the inner edge of the AF ring corresponded closely with the lateral extent of the inner segment ellipsoid band in the OCT image. CONCLUSIONS: Ring constriction was largely coincident with progressive centripetal photopic threshold elevation led by worsening of rod photoreceptor function. The rate of constriction differed across patients, and a ring may reach a critical minimum before disappearing, at which stage central visual loss occurs. The structural and functional changes associated with rings of increased autofluorescence confirm that they provide an objective index of macular involvement and may aid the management of RP patients and the monitoring of future treatment efficacy.

Early onset retinal dystrophy due to mutations in LRAT: molecular analysis and detailed phenotypic study.

PURPOSE: To report novel variants and characterize the phenotype associated with the autosomal recessive retinal dystrophy caused by mutations in the lecithin retinol acyltransferase (LRAT) gene. METHODS: A total of 149 patients with Leber's congenital amaurosis (LCA) or early onset retinal dystrophy were screened for mutations in LCA-associated genes using an arrayed-primer extension (APEX) genotyping microarray (Asper Ophthalmics). LRAT sequencing was subsequently performed in this 148-patient panel. Patients identified with mutations underwent further detailed phenotyping. RESULTS: APEX analysis identified one patient with a previously reported homozygous LRAT mutation. Sequencing of the panel identified three additional patients with novel homozygous LRAT mutations in exon 2. All four patients had severe progressive nyctalopia, visual field constriction, and photophilia in childhood. Visual acuity ranged from 0.22 logMAR to hand motion. Funduscopy revealed severe retinal pigment epithelial atrophy and minimal retinal pigmentation. Asteroid hyalosis and macular epiretinal fibrosis were frequent. All demonstrated reduced fundus autofluorescence. Optical coherence tomography identified disrupted retinal lamination, outer-retinal debris, and an unidentifiable photoreceptor layer in two cases. Full-field electroretinograms were undetectable or showed severe rod-cone dysfunction. Photopic perimetry revealed severe visual field constriction. Dark-adapted perimetry demonstrated markedly reduced photoreceptor sensitivity. Dark-adapted spectral sensitivity measurements identified functioning rods in two of three patients. All three had severely reduced L- and M-cone sensitivity and poor color discrimination. CONCLUSIONS: LRAT mutations cause a severe, early childhood onset, progressive retinal dystrophy. Phenotypic similarities to the retinal dysfunction associated with RPE-specific protein 65 kDa mutations, another visual cycle gene, suggest that LRAT deficiency may show a good response to novel therapies.

Serial imaging and structure-function correlates of high-density rings of fundus autofluorescence in retinitis pigmentosa.

PURPOSE: To document the evolution and functional and structural significance of parafoveal rings of high-density fundus autofluorescence (AF) in patients with retinitis pigmentosa and preserved visual acuity. METHODS: Fifty-two patients with nonsyndromic retinitis pigmentosa or Usher syndrome, who had a parafoveal ring of high-density AF and a visual acuity of 20/30 or better, were ascertained. All had international standard full-field electroretinography and pattern electroretinography. Autofluorescence imaging was repeated in 30 patients after periods of up to 9.3 years. Of the 52 patients, 35 underwent optical coherence tomography. RESULTS: Progressive constriction of the ring was detected in 17 patients. Ring radius reduced by up to 40% at a mean rate of between 0.8% and 15.8% per year. In 1 patient, a small ring was replaced by irregular AF; visual acuity deteriorated over the same period. There was a high correspondence between the lateral extent of the preserved optical coherence tomography inner segment/outer segment band and the diameter of the ring along the same optical coherence tomographic scan plane (slope, 0.9; r = 0.97; P < 0.005; n = 35) and between preserved inner segment/outer segment lamina and the pattern electroretinography P50 measure of macular function (R = 0.72; P < 0.005; n = 34). CONCLUSION: Rings of increased AF surround areas of preserved outer retina and preserved photopic function. Serial fundus AF may provide prognostic indicators for preservation of central acuity and potentially assist in the identification and evaluation of patients suitable for treatment aimed at preservation of remaining function.

Retinal specific measurement of dark-adapted visual function: validation of a modified microperimeter.

BACKGROUND: Scotopic function is an important marker of many retinal diseases and is increasingly used as an outcome measure in clinical trials, such as those investigating gene therapy for Lebers congenital amaurosis. Scotopic visual function has traditionally been measured using an adapted perimetry system such as the Humphrey field analyser (HFA). However this system does not control for fixation errors or poor fixation stability. Here we evaluate the use of an adapted microperimeter to measure visual function at defined retinal regions under scotopic conditions. METHODS: A MP-1 microperimeter (Nidek Technologies, Italy) was modified by adding a 1 log unit Neutral Density filter and a 530 nm shortpass filter within the optical path of the instrument. Stray light was shielded. Fine matrix mapping perimetry was performed on five younger (< 35 years) and five older (> 65 years) subjects with no eye disease and good vision. All subjects were fully dark adapted before testing and pupils were dilated with 1% tropicamide. Tests was performed once on the modified MP-1 microperimeter and once using a modified HFA, in a counterbalanced order. RESULTS: A foveal scotopic scotoma with a sensitivity reduction of >1 log unit was found using each instrument. In addition, the MP-1 system showed the retinal location of the foveal scotoma. Mean test time was 25 minutes for the MP-1 and 32 minutes for the HFA. DISCUSSION: A modified MP-1 microperimeter can be used to measure scotopic retinal function, creating results which are comparable to the modified Humphrey field analyser. Advantages of the MP-1 system include the ability to track the retina through testing, retinal localisation of the scotoma and a faster test time.

Localisation and significance of in vivo near-infrared autofluorescent signal in retinal imaging.

AIM: To evaluate near-infrared (NIR) autofluorescence (AF) in patients with geographic atrophy (GA) secondary to age-related macular degeneration and to investigate the origin of the signal by in vivo and histological analysis in rats and in a human donor eye. METHODS: Confocal scanning laser ophthalmoscopy in vivo imaging, including blue (excitation: 488 nm, emission 500-700 nm) and NIR (excitation: 790 nm, emission >810 nm) AF was performed in 21 eyes of 18 GA patients. Pigmented and albino rats underwent with the same device both in vivo and post-mortem imaging. For the latter, cryostat prepared retinal cross-sections were imaged using an additional customised magnification lens. Finally, cross-sections of a 49-year old human donor eye were recorded. RESULTS: Atrophic areas in GA were characterised by low NIR AF intensities. In the junctional zone of atrophy, focal areas of increased intensity were seen which appeared to seldom correlate to blue AF findings. Confocal live scanning in pigmented rats identified the maximum of the NIR AF signal in the outer retina, with histological confirmation of the signal origin localised to the retinal pigment epithelium and sclera in both animals and human donor eye. No NIR AF was found in the retina of young non-pigmented rats. DISCUSSION: This study further underscores the assumption that melanin is the main source of NIR AF in the healthy retina. Increased NIR AF intensities in the junctional zone in GA may represent accumulation of melanolipofuscin, which may reflect disease activity and thus may allow for early identification of patients at high-risk of GA enlargement.

Structural and functional changes over time in MacTel patients.

PURPOSE: To investigate functional and morphologic alterations over a 1-year review analysis in patients with type 2 idiopathic juxtafoveal retinal telangiectasia (MacTel). METHODS: Nine eyes of 9 patients with MacTel underwent repeated scotopic and photopic fine matrix mapping (FMM), 10-2 photopic microperimetry, and imaging studies. RESULTS: Early Treatment Diabetic Retinopathy Study visual acuity assessment showed a median difference between examinations of 1.0 letter (range, -3 to 4 letters). The difference of sensitivity values of all test points was 4.5 dB (range, 0.4 -5.5 dB) for microperimetry 1, 0.4 dB (range, -0.8 to 1.7 dB) for photopic, and -1.7 dB (range, -6.1 to 1.0 dB) for scotopic fine matrix mapping, respectively. The difference in test points of more than a 10-dB loss compared with age-matched controls was higher for scotopic than for photopic testing (P= 0.03, Wilcoxon signed ranks test). Small progression of scotoma correlated with a slight increase in retinal blood vessel dilatation and hyperfluorescence and subtle enlargement of pigmented plaques. CONCLUSION: Changes in central visual acuity and microperimetry testing after 1 year most likely do not extend beyond test-retest variability. The deterioration of scotopic sensitivity confirms our previous results of more severe rod compared with cone dysfunction in MacTel. Changes in fine detail visual function over a 1-year period may be useful parameters for interventional trials.

Post-capsulotomy dysphotopsia in monofocal versus multifocal lenses.

PURPOSE: To compare the photic symptoms experienced by patients with the monofocal SI30 intraocular lens (IOL) with the refractive multifocal SA40 Array IOL after capsulotomy. METHODS: In this prospective cohort study, 49 eyes of 49 patients (20 multifocal, 29 monofocal IOLs) were assessed following uncomplicated cataract extraction and Nd:YAG capsulotomy equal to the scotopic pupillary diameter. Subjects with post-operative refraction of +/-1.00 or more DS, +/-1.00 or more DC, concurrent ocular pathology, LogMAR acuity of worse than 0.3 for distance or 1.0 for near were excluded. Glare and halo were assessed objectively with computer-generated psychophysical tests (Glare and Halo) and subjective dysphotopic symptoms were evaluated with Tester, Javitt, Winther-Neilsen and Sedgewick questionnaires. RESULTS: No significant difference was found for mean halo size (square degrees) between monofocals (121.33) and multifocals (97.32, p = 0.207) or for mean glare (percentage contrast), monofocals (7.881) and multifocals (7.353, p = 0.812). No significant differences in the subjective appreciation of dysphotopsia were found: Tester (p = 0.358), Javitt (p = 0.29), Winther-Neilson (p = 0.54) and Sedgewick questionnaires (p = 0.134). CONCLUSION: The posterior capsule is an important optical medium, which has not been fully considered in other comparative studies and its complete removal eliminates any confounding contribution. The results suggest that after capsulotomy, there is no significant difference in objective or subjective photic phenomena between monofocal and multifocal silicone lenses. Dysphotopic symptoms in patients with refractive multifocal IOLs were comparable to monofocal IOL patients after capsulotomy.

Effect of gene therapy on visual function in Leber's congenital amaurosis.

Early-onset, severe retinal dystrophy caused by mutations in the gene encoding retinal pigment epithelium-specific 65-kD protein (RPE65) is associated with poor vision at birth and complete loss of vision in early adulthood. We administered to three young adult patients subretinal injections of recombinant adeno-associated virus vector 2/2 expressing RPE65 complementary DNA (cDNA) under the control of a human RPE65 promoter. There were no serious adverse events. There was no clinically significant change in visual acuity or in peripheral visual fields on Goldmann perimetry in any of the three patients. We detected no change in retinal responses on electroretinography. One patient had significant improvement in visual function on microperimetry and on dark-adapted perimetry. This patient also showed improvement in a subjective test of visual mobility. These findings provide support for further clinical studies of this experimental approach in other patients with mutant RPE65. (ClinicalTrials.gov number, NCT00643747 [ClinicalTrials.gov].).

Correlation of functional impairment and morphological alterations in patients with group 2A idiopathic juxtafoveal retinal telangiectasia.

OBJECTIVE: To correlate functional impairment with morphological alterations in patients with group 2A idiopathic juxtafoveal retinal telangiectasia. METHODS: As part of the Macular Telangiectasia Project, a cohort of 10 patients underwent additional functional testing and imaging studies including photopic and scotopic fine matrix mapping, microperimetry, reflectance, and autofluorescence imaging with scanning laser ophthalmoscopy. RESULTS: From clinical stage 2 to 5, scotopic central function was reduced, which corresponded to depletion of macular pigment density. From clinical stage 3 onward, severe photopic and scotopic scotomata with up to 30 dB of loss were found next to fixation and were not totally confined to abnormalities seen with standard imaging modalities. The number of test points with loss of 10 dB or more was significantly greater for scotopic testing than for photopic testing (P = .007, Wilcoxon signed rank test). CONCLUSIONS: Rod function may be more severely affected than cone function in patients with group 2A idiopathic juxtafoveal retinal telangiectasia, and this may occur early in the disease progression. Severe reduction in retinal sensitivity is spatially confined to morphological alterations seen with scanning laser ophthalmoscopy imaging. The findings imply that idiopathic juxtafoveal retinal telangiectasia is not solely a vascular disease and that early neuronal involvement may be implicated in the pathogenesis of the disease.

Macular and retinal dysfunction of unknown origin in adults with normal fundi: evidence for an autoimmune pathophysiology.

Here we report the discovery of and phenotypic characterization of a retinal disorder of unknown origin in adults using clinical, electrophysiological and psychophysical techniques, and to seek the presence of circulating retinal autoantibodies in the sera of these patients. Sixteen patients were identified with progressive bilateral visual loss over a period of months. Ten of the patients were male, and the average age was 55.3 years (range from 43 to 76 years). Known causes such as carcinoma-associated retinopathy, acute zonal occult outer retinopathy and hereditary cone dystrophy appeared unlikely. Investigations included electrophysiology, fundus autofluorescence imaging and psychophysical tests. The sera of these patients were analyzed with indirect immunocytochemistry and Western immunoblot analysis on murine (BALB/c) retinal tissue for the presence of retinal autoantibodies. Bilateral visual loss and photophobia progressed over a period of months to years (average 28.7 months, range 3-67) and subsequently stabilized. No abnormality was observed by biomicroscopy, angiography or autofluorescence imaging. Electrophysiology indicated predominant cone-system dysfunction, either macular or generalized, and post-phototransduction involvement in 9 patients (56%). Photopic and scotopic visual fields and dark adaptation kinetics showed both cone and rod system involvement in all cases. Heterogeneous immunohistochemical staining patterns were seen with the sera of these patients as compared with controls. A majority of the affected patients (9/15) stained with an antinuclear pattern. The retinal autoantibodies from the sera of most patients reacted with the retinal proteins of molecular weight between 34 and 40 kDa. The aetiology of this distinctive retinal disorder therefore appears to be mediated through an autoimmune mechanism.

Functional characteristics of patients with retinal dystrophy that manifest abnormal parafoveal annuli of high density fundus autofluorescence; a review and update.

PURPOSE: To examine the presence and functional significance of annular fundus autofluorescence abnormalities in patients with different retinal dystrophies. METHODS: Eighty one patients were ascertained who had a parafoveal ring of high density on fundus autofluorescence imaging. Sixty two had had a clinical diagnosis of retinitis pigmentosa (RP) or Usher syndrome with normal visual acuity. Others included a case of Leber congenital amaurosis and genetically confirmed cases of cone or cone-rod dystrophy (GUCA1A, RPGR, RIMS1), "cone dystrophy with supernormal rod ERG" (KCNV2) and X-linked retinoschisis (RS1). International-standard full-field and pattern electroretinography (ERG; PERG) were performed. Some patients with rod-cone or cone-rod dystrophy underwent multifocal ERG (mfERG) testing and photopic and scotopic fine matrix mapping (FMM). RESULTS: In patients with RP, the radius of the parafoveal ring of high density correlated with PERG P50 (R = 0.83, P < 0.0005, N = 62) and encircled areas of preserved photopic function. In the other patients, AF rings either resembled those seen in RP or encircled an area of central atrophy. Ring radius was inversely related to the PERG P50 component in 4 of 18 cases with a detectable response. FMM showed that arcs of high density were associated with a gradient of sensitivity change. CONCLUSIONS: Parafoveal rings of high density autofluorescence are a non-specific manifestation of retinal dysfunction that can occur in different retinal dystrophies. Electrophysiology remains essential for accurate diagnosis. The high correlation of autofluorescence with PERG, mfERG and FMM demonstrates that AF abnormalities have functional significance and may help identify suitable patients and retinal areas amenable to future therapeutic intervention.

Complement factor H deficiency in aged mice causes retinal abnormalities and visual dysfunction.

Age-related macular degeneration is the most common form of legal blindness in westernized societies, and polymorphisms in the gene encoding complement factor H (CFH) are associated with susceptibility to age-related macular degeneration in more than half of affected individuals. To investigate the relationship between complement factor H (CFH) and retinal disease, we performed functional and anatomical analysis in 2-year-old CFH-deficient (cfh(-/-)) mice. cfh(-/-) animals exhibited significantly reduced visual acuity and rod response amplitudes on electroretinography compared with age-matched controls. Retinal imaging by confocal scanning laser ophthalmoscopy revealed an increase in autofluorescent subretinal deposits in the cfh(-/-) mice, whereas the fundus and vasculature appeared normal. Examination of tissue sections showed an accumulation of complement C3 in the neural retina of the cfh(-/-) mice, together with a decrease in electron-dense material, thinning of Bruch's membrane, changes in the cellular distribution of retinal pigment epithelial cell organelles, and disorganization of rod photoreceptor outer segments. Collectively, these data show that, in the absence of any specific exogenous challenge to the innate immune system, CFH is critically required for the long-term functional health of the retina.

Xanthophyll accumulation in the human retina during supplementation with lutein or zeaxanthin - the LUXEA (LUtein Xanthophyll Eye Accumulation) study.

The xanthophylls lutein (L) and zeaxanthin (Z) form the macular pigment with the highest density in the macula lutea. We investigated Macular Pigment Optical Density (MPOD) responses to supplementation with identically formulated (Actilease) L or Z (OPTISHARP) or L+Z over 6-12 months using doses of 10 or 20mg/day. MPOD as well as blue light sensitivity in fovea and parafovea were measured monthly by heterochromatic flicker photometry. Average xanthophyll plasma concentrations, analysed monthly by HPLC, increased up to 27-fold. MPOD increased by 15% upon L or L+Z supplementation. Supplementation of Z alone produced similar pigment accumulation in fovea and parafovea, which confounded MPOD measurements. After correction for this, a 14% MPOD increase resulted for Z. Thus, during supplementation with xanthophylls, L is predominantly deposited in the fovea while Z deposition appears to cover a wider retinal area. This may be relevant to health and disease of the retina.

Maculopathy due to the R345W substitution in fibulin-3: distinct clinical features, disease variability, and extent of retinal dysfunction.

PURPOSE: To determine (1) clinical features that distinguish maculopathy due to the R345W substitution in fibulin-3 from other forms of inherited or early-onset drusen, (2) the phenotypic variability, and (3) the extent of retinal disease in those with a positive molecular diagnosis. METHODS: Affected individuals underwent ophthalmic examination, digital color fundus photography, fundus autofluorescence (AF) imaging, and psychophysical testing with automated photopic and dark-adapted perimetry and fine matrix mapping. Blood samples were taken for DNA extraction and screening for the R345W mutation in fibulin-3. Patients were subsequently divided into mutation-positive and -negative groups, to compare the identified phenotypic findings in these two sets of subjects. RESULTS: Twenty-nine subjects from 19 families were ascertained with inherited or early-onset drusen. Twenty-four (83%) subjects from 15 families were found to harbor the R345W fibulin-3 mutation. Peripapillary deposition and a radial distribution of macular drusen were consistent, distinguishing signs in the mutation-positive group. Subretinal neovascular membrane (SRNVM) was a rare occurrence, affecting only 1 of 48 eyes, whereas hyperpigmentation and atrophy of the retinal pigment epithelium (RPE) were common in older mutation-positive patients. Increased AF corresponding to the drusen was detected in both the mutation-positive and -negative groups. The phenotype in the group of patients positive for the R345W mutation was extremely variable, with evidence of interocular, intrafamilial, and interfamilial variability in visual loss, natural history, ophthalmoscopic findings, autofluorescence imaging, and psychophysical data. The novel finding of nonpenetrance was observed in a 62-year-old asymptomatic, mutation-positive man. The findings from detailed perimetry performed on a subset of subjects were consistent with the presence of widespread retinal dysfunction not isolated to the macula. CONCLUSIONS: Marked inter- and intrafamilial variation associated with the fibulin-3 R345W mutation in terms of retinal appearance, severity, progression, and nonpenetrance were identified. It was noted that SRNVM is a rare occurrence in R345W fibulin-3 maculopathy. These findings are helpful for advice regarding prognosis and for genetic counseling. The findings established that the presence of peripapillary deposit is highly likely to indicate that a patient carries the R345W mutation.

Pars plana vitrectomy with and without peeling of the inner limiting membrane for diabetic macular edema.

PURPOSE: A prospective, comparative, nonrandomized study to evaluate the efficacy of pars plana vitrectomy (PPV) with and without inner limiting membrane (ILM) peeling for persistent diffuse clinically significant macular edema. METHODS: Eighteen patients with persistent diffuse clinically significant macular edema despite laser photocoagulation were recruited for the study. Clinical assessment included determination of best-corrected visual acuity, fundus fluorescein angiography, optical coherence tomography, and perifoveal cone function testing. Eight patients underwent PPV with elevation and removal of the posterior hyaloid alone, and 10 patients underwent vitrectomy and ILM peeling. The follow-up was 12 months. RESULTS: Patients with ILM peeling had improvement in foveal thickness (P = 0.07) and significant improvement in the macular volume (P = 0.039) 12 months after surgery but did not have significant improvement in Early Treatment Diabetic Retinopathy Study vision or perifoveal cone function. There was no significant difference in outcome parameters between the no peeling group and the ILM peeling group. CONCLUSIONS: In this prospective, comparative study of PPV with and without ILM peeling for diffuse clinically significant macular edema, structural improvement was seen but with limited visual improvement after ILM peeling.

Assessment of neuroprotective effects of glutamate modulation on glaucoma-related retinal ganglion cell apoptosis in vivo.

PURPOSE: To assess the neuroprotective effects of different glutamate modulation strategies, with a nonselective (MK801) and a selective (ifenprodil) NMDA receptor antagonist and a metabotropic glutamate receptor agonist (mGluR Group II, LY354740), in glaucoma-related in vivo rat models of retinal ganglion cell (RGC) apoptosis. METHODS: RGC apoptosis was induced in Dark Agouti (DA) rats by staurosporine (SSP) treatment. Single agents MK801, ifenprodil, or LY354740, or MK801 and LY354740 combined, were administrated intravitreally at different doses. Eyes were imaged in vivo using a recently established technique and the results confirmed histologically. The most effective combined therapy regimen of MK801 and LY354740 was then assessed in a chronic ocular hypertension (OHT) rat model with application at 0, 1, and 2 weeks after OHT surgery and the effects assessed as described before. RESULTS: All strategies of glutamate modulation reduced SSP-induced-RGC apoptosis compared with the control, in a dose-dependent manner: MK801 (R2= 0.8863), ifenprodil (R2= 0.4587), and LY354740 (R2= 0.9094), with EC50s of 0.074, 0.0138, and 19 nanomoles, respectively. The most effective combination dose of MK801 and LY354740 was 0.06 and 20 nanomoles (P < 0.05), respectively, and the optimal timing of the therapy was 0 weeks after OHT surgery (P < 0.05). CONCLUSIONS: This novel SSP model was validated as a useful tool for screening neuroprotective strategies in vivo. Group II mGluR modulation may be a useful treatment for RGC death. Combination therapy optimized to limit neurotoxic effects of MK801 may be an effective neuroprotective approach in retinal degenerative disease. Furthermore, treatments that minimize secondary RGC degeneration may be most useful in glaucoma.

Central retinal thickness is positively correlated with macular pigment optical density.

Macular pigment (MP) has been suggested to have a protective role in age-related macular degeneration by reducing the amount of oxidative stress on the retina. MP levels peak at the foveal center, where it is found predominantly in the receptor axon and inner plexiform layers of the retina. The purpose of this study was to investigate the relationship between central retinal thickness and macular pigment optical density in a group of healthy subjects. We report that macular pigment optical density (MPOD) has a significant and positive relationship with central retinal thickness as measured by optical coherence tomography. The strength of the observed relationship (r approximately 0.30) was independent of the technique used to measure MPOD, whether heterochromatic flicker photometry (HFP) or 2-wavelength autofluorescence (AF). Of note, there was no statistically demonstrable relationship between MPOD at an eccentricity of 1- or 2-degrees and central retinal thickness. This finding has important implications for future studies investigating MPOD, and its response to dietary modification/supplementation.