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The purpose of this paper is to identify likely pathogenic non-coding variants in inherited retinal dystrophy (IRD) genes, using genome sequencing (GS). Patients with IRD were recruited to the study and underwent comprehensive ophthalmological evaluation and GS. The results of GS were investigated through virtual gene panel analysis, and plausible pathogenic variants and clinical phenotype evaluated by the multidisciplinary team (MDT) discussion. For unsolved patients in whom a specific gene was suspected to harbor a missed pathogenic variant, targeted re-analysis of non-coding regions was performed on GS data. Candidate variants were functionally tested by messenger RNA analysis, minigene or luciferase reporter assays. Previously unreported, likely pathogenic, non-coding variants in 7 genes (PRPF31, NDP, IFT140, CRB1, USH2A, BBS10 and GUCY2D), were identified in 11 patients. These were shown to lead to mis-splicing (PRPF31, IFT140, CRB1 and USH2A) or altered transcription levels (BBS10 and GUCY2D). MDT-led, phenotype-driven, non-coding variant re-analysis of GS is effective in identifying the missing causative alleles.

PURPOSE: A key property to consider in all genetic tests is clinical utility, the ability of the test to influence patient management and health outcomes. Here we assess the current clinical utility of genetic testing in diverse pediatric inherited eye disorders (IEDs).METHODS: Two hundred one unrelated children (0-5 years old) with IEDs were ascertained through the database of the North West Genomic Laboratory Hub, Manchester, UK. The cohort was collected over a 7-year period (2011-2018) and included 74 children with bilateral cataracts, 8 with bilateral ectopia lentis, 28 with bilateral anterior segment dysgenesis, 32 with albinism, and 59 with inherited retinal disorders. All participants underwent panel-based genetic testing.RESULTS: The diagnostic yield of genetic testing for the cohort was 64% (ranging from 39% to 91% depending on the condition). The test result led to altered management (including preventing additional investigations or resulting in the introduction of personalized surveillance measures) in 33% of probands (75% for ectopia lentis, 50% for cataracts, 33% for inherited retinal disorders, 7% for anterior segment dysgenesis, 3% for albinism).CONCLUSION: Genetic testing helped identify an etiological diagnosis in the majority of preschool children with IEDs. This prevented additional unnecessary testing and provided the opportunity for anticipatory guidance in significant subsets of patients.

PurposeThe widespread adoption of genomic testing for individuals with ophthalmic disorders has increased demand on diagnostic genomic services for these conditions. Moreover, the clinical utility of a molecular diagnosis for individuals with inherited ophthalmic disorders is increasingly placing pressure on the speed and accuracy of genomic testing.MethodsWe created EyeG2P, a publically available resource to assist diagnostic filtering of genomic datasets for ophthalmic conditions, utilising the Ensembl Variant Effect Predictor. We assessed the sensitivity of EyeG2P for 1234 individuals with a broad range of conditions, who had previously received a confirmed molecular diagnosis through routine genomic diagnostic approaches. For a prospective cohort of 83 individuals, we also assessed the precision of EyeG2P in comparision to routine genomic diagnostic approaches.ResultsWe observed that EyeG2P had a 99.5% sensitivity for genomic variants previously identified as a molecular diagnosis for 1234 individuals. EyeG2P enabled a significant increase in precision in comparison to routine testing strategies (pConclusionAutomated filtering of genomic variants through EyeG2P can increase the efficiency of diagnostic testing for individuals with a broad range of inherited ophthalmic disorders.

Purpose North Carolina macular dystrophy (NCMD) is an autosomal dominant, congenital disorder affecting the central retina. Here, we report clinical and genetic findings in three families segregating NCMD and use epigenomic datasets from human tissues to gain insights into the effect of NCMD-implicated variants. Methods Clinical assessment and genetic testing were performed. Publicly available transcriptomic and epigenomic datasets were analyzed and the activity-by-contact method for scoring enhancer elements and linking them to target genes was used. Results A previously described, heterozygous, noncoding variant upstream of the PRDM13 gene was detected in all six affected study participants (chr6:100,040,987G>C [GRCh37/hg19]). Interfamilial and intrafamilial variability were observed; the visual acuity ranged from 0.0 to 1.6 LogMAR and fundoscopic findings ranged from visually insignificant, confluent, drusen-like macular deposits to coloboma-like macular lesions. Variable degrees of peripheral retinal spots (which were easily detected on widefield retinal imaging) were observed in all study subjects. Notably, a 6-year-old patient developed choroidal neovascularization and required treatment with intravitreal bevacizumab injections. Computational analysis of the five single nucleotide variants that have been implicated in NCMD revealed that these noncoding changes lie within two putative enhancer elements; these elements are predicted to interact with PRDM13 in the developing human retina. PRDM13 was found to be expressed in the fetal retina, with greatest expression in the amacrine precursor cell population. Conclusions We provide further evidence supporting the role of PRDM13 dysregulation in the pathogenesis of NCMD and highlight the usefulness of widefield retinal imaging in individuals suspected to have this condition.

PurposeNorth Carolina macular dystrophy (NCMD) is an autosomal dominant, congenital disorder affecting the central retina. Here, we report clinical and genetic findings in three families segregating NCMD and use epigenomic datasets from human tissues to gain insights into the effect of NCMD-implicated variants.MethodsClinical assessment and genetic testing were performed. Publicly-available transcriptomic and epigenomic datasets were analyzed and the ‘Activity-by-Contact’ (ABC) method for scoring enhancer elements and linking them to target genes was used.ResultsA previously-described, heterozygous, non-coding variant upstream of the PRDM13 gene was detected in all six affected study participants (chr6:100,040,987G>C [GRCh37/hg19]). Inter- and intra-familial variability were observed; the visual acuity ranged from 0.0 to 1.6 LogMAR and fundoscopic findings ranged from visually insignificant, confluent, drusen-like macular deposits to coloboma-like macular lesions. Variable degrees of peripheral retinal spots (which were easily detected on widefield retinal imaging) were observed in all study subjects. Notably, a 6-year-old patient developed choroidal neovascularization and required treatment with intravitreal bevacizumab injections. Computational analysis of the five single nucleotide variants that have been implicated in NCMD revealed that these non-coding changes lie within two putative enhancer elements; these elements are predicted to interact with PRDM13 in the developing human retina. PRDM13 was found to be expressed in the fetal retina, with highest expression in the amacrine precursor cell population.ConclusionsWe provide further evidence supporting the role of PRDM13 dysregulation in the pathogenesis of NCMD and highlight the utility of widefield retinal imaging in individuals suspected to have this condition.

Thirty percent of all inherited retinal disease (IRD) is accounted for by conditions with extra-ocular features. This study aimed to establish the genetic diagnostic pick-up rate for IRD patients with one or more extra-ocular features undergoing panel-based screening in a clinical setting. One hundred and six participants, tested on a gene panel which contained both isolated and syndromic IRD genes, were retrospectively ascertained from the Manchester Genomic Diagnostics Laboratory database spanning 6 years (2012–2017). Phenotypic features were extracted from the clinical notes and classified according to Human Phenotype Ontology; all identified genetic variants were interpreted in accordance to the American College of Medical Genetics and Genomics guidelines. Overall, 49% (n = 52) of patients received a probable genetic diagnosis. A further 6% (n = 6) had a single disease-associated variant in an autosomal recessive disease-relevant gene. Fifty-two percent (n = 55) of patients had a clinical diagnosis at the time of testing. Of these, 71% (n = 39) received a probable genetic diagnosis. By contrast, for those without a provisional clinical diagnosis (n = 51), only 25% (n = 13) received a probable genetic diagnosis. The clinical diagnosis of Usher (n = 33) and Bardet–Biedl syndrome (n = 10) was confirmed in 67% (n = 22) and 80% (n = 8), respectively. The testing diagnostic rate in patients with clinically diagnosed multisystemic IRD conditions was significantly higher than those without one (71% versus 25%;pvalue

Next-generation sequencing has revolutionized rare disease diagnostics, but many patients remain without a molecular diagnosis, particularly because many candidate variants usually survive despite strict filtering. Exomiser was launched in 2014 as a Java tool that performs an integrative analysis of patients&rsquo, sequencing data and their phenotypes encoded with Human Phenotype Ontology (HPO) terms. It prioritizes variants by leveraging information on variant frequency, predicted pathogenicity, and gene-phenotype associations derived from human diseases, model organisms, and protein&ndash, protein interactions. Early published releases of Exomiser were able to prioritize disease-causative variants as top candidates in up to 97% of simulated whole-exomes. The size of the tested real patient datasets published so far are very limited. Here, we present the latest Exomiser version 12.0.1 with many new features. We assessed the performance using a set of 134 whole-exomes from patients with a range of rare retinal diseases and known molecular diagnosis. Using default settings, Exomiser ranked the correct diagnosed variants as the top candidate in 74% of the dataset and top 5 in 94%, not using the patients&rsquo, HPO profiles (i.e., variant-only analysis) decreased the performance to 3% and 27%, respectively. In conclusion, Exomiser is an effective support tool for rare Mendelian phenotype-driven variant prioritization.

Objective: USH2A -related disorders are characterised by genetic and phenotypic heterogeneity, and are associated with an spectrum of sensory deficits, ranging from deaf blindness to blindness with normal hearing. It has been previously proposed that the presence of specific USH2A alleles can be predictive of unaffected hearing . This study reports the clinical and genetic findings in a group of patients with USH2A - related disease and evaluates the validity of the allelic hierarchy model. Patients and intervention: USH2A variants from 27 adults with syndromic and nonsyndromic USH2A -related disease were analysed according to a previously reported model of allelic hierarchy. The analysis was replicated on genotype-phenotype correlation information from 197 individuals previously reported in 2 external datasets.Main Outcome Measure: Genotype-phenotype correlations in USH2A -related disease.Results: A valid allelic hierarchy model was observed in 93% of individuals with nonsyndromic USH2A -retinopathy (n=14/15) and in 100% of patients with classic Usher syndrome type IIa (n=8/8). Furthermore, when two large external cohorts of cases were combined, the allelic hierarchy model was valid across 85.7% (n=78/91) of individuals with non-syndromic USH2A -retinopathy and 95% (n=123/129) of individuals with classic Usher syndrome type II (p=0.012, Chi-square test). Notably, analysis of all three patient datasets revealed that USH2A protein truncating variants were reported most frequently in individuals with hearing loss.Conclusion: Genetic testing results in individuals suspected to have an USH2A - related disorder have the potential to facilitate personalised audiological surveillance and rehabilitation pathways.

Linear scleroderma is a characteristic form of scleroderma that typically affects children. Ocular manifestations may be present, especially when the frontoparietal area of the head is affected. We present the case of a 5-year-old boy with craniofacial linear scleroderma ("en coup de sabre") who developed exudative retinal detachment. Angiographic and neuroimaging findings are presented, and the importance of regular fundus examination is highlighted.

To gain insights into microgravity-induced ophthalmic changes (microgravity ocular syndrome), and as part of a project investigating effects of future planetary habitats, we investigated the effect of acute hypercapnia following 10-day bed rest and hypoxia on posterior eye structures. Female subjects ( N = 7) completed three 10-day experimental interventions: 1) normoxic bed rest [NBR; partial pressure of inspired O2 (PiO2) = 132.9 ± 0.3 Torr]; 2) hypoxic ambulatory confinement (HAMB; PiO2 = 90.4 ± 0.3 Torr); and 3) hypoxic bed rest (HBR; n = 12; PiO2 = 90.4 ± 0.3 Torr). Before and on the last day of each intervention, optical coherence tomography (OCT) of the optic disk was performed, and the thicknesses of the retinal nerve fiber layer (RNFL), retina, and choroid were measured. OCT examinations were conducted with the subjects breathing the prevailing normocapnic breathing mixture (either normoxic or hypoxic) and then following a 10-min period of breathing the same gas mixture, but with the addition of 1% CO2. Choroidal thickness was greater during both bed-rest conditions (NBR and HBR) compared with the ambulatory (HAMB) condition (ANOVA, P < 0.001). Increases in RNFL thickness compared with baseline were observed in the hypoxic trials (HBR, P < 0.001; and HAMB, P = 0.021), but not the normoxic trial (NBR). A further increase in RNFL thickness ( P = 0.019) was observed after the 10-min hypercapnic trial in the NBR condition only. The fact that choroidal thickness was not affected by Po2 or Pco2, but increased by bed rest, suggests a hydrostatic rather than a vasoactive effect. The increments in RNFL thickness were most likely associated with local hypoxia and hypercapnia-induced dilatation of the retinal blood vessels.

Persistent placoid maculopathy (PPM) is a bilateral inflammatory chorioretinopathy characterized by long-standing plaque-like macular lesions. No systemic manifestations have been reported to date. We describe a case of PPM complicated by cerebral vasculitis, suggesting that neurological symptoms, including headache, should be enquired about in all PPM subjects.

Purpose To evaluate the phenotypic variability and natural history of ocular disease in a cohort of 28 individuals with MYO7A -related disease. Mutations in the MYO7A gene are the most common cause of Usher syndrome type 1, characterized by profound congenital deafness, vestibular arreflexia, and progressive retinal degeneration. Design Retrospective case series. Participants Twenty-eight patients from 26 families (age range, 3–65 years; median, 32) with 2 likely disease-causing variants in MYO7A . Methods Clinical investigations included fundus photography, optical coherence tomography, fundus autofluorescence (FAF) imaging, and audiologic and vestibular assessments. Longitudinal visual acuity and FAF data (over a 3-year period) were available for 20 and 10 study subjects, respectively. Main Outcome Measures Clinical, structural, and functional characteristics. Results All patients with MYO7A mutations presented with features consistent with Usher type 1. The median visual acuity for the cohort was 0.39 logarithm of the minimum angle of resolution (logMAR; range, 0.0–2.7) and visual acuity in logMAR correlated with age (Spearman's rank correlation coefficient, r = 0.71; P 0.0001). Survival analysis revealed that acuity ≤0.22 logMAR was maintained in 50% of studied subjects until age 33.9; legal blindness based on loss of acuity (≥1.00 logMAR) or loss of field (≤20°) was reached at a median age of 40.6 years. Three distinct patterns were observed on FAF imaging: 13 of 22 patients tested had relatively preserved foveal autofluorescence surrounded by a ring of high density, 4 of 22 had increased signal in the fovea with no obvious hyperautofluorescent ring, and 5 of 22 had widespread hypoautofluorescence corresponding to retinal pigment epithelial atrophy. Despite a number of cases presenting with a milder phenotype, there seemed to be no obvious genotype–phenotype correlation. Conclusions MYO7A -related ocular disease is variable. Central vision typically remains preserved at least until the third decade of life, with 50% of affected individuals reaching legal blindness by 40 years of age. Distinct phenotypic subsets were identified on FAF imaging. A specific allele, previously reported in nonsyndromic deafness, may be associated with a mild retinopathy.

Complement component C3 is the central complement component and a key inflammatory protein activated in age-related macular degeneration (AMD). AMD is associated with genetic variation in complement proteins that results in enhanced activation of C3 through the complement alternative pathway. These include complement factor H (CFH), a negative regulator of C3 activation. Both C3 inhibition and/or CFH augmentation are potential therapeutic strategies in AMD. Herein, we examined retinal integrity in aged (12 months) mice deficient in both factors H and C3 (CFH(-/-).C3(-/-)), CFH alone (CFH(-/-)), or C3 alone (C3(-/-)), and wild-type mice (C57BL/6). Retinal function was assessed by electroretinography, and retinal morphological features were analyzed at light and electron microscope levels. Retinas were also stained for amyloid β (Aβ) deposition, inflammation, and macrophage accumulation. Contrary to expectation, electroretinograms of CFH(-/-).C3(-/-) mice displayed more severely reduced responses than those of other mice. All mutant strains showed significant photoreceptor loss and thickening of Bruch's membrane compared with wild-type C57BL/6, but these changes were greater in CFH(-/-).C3(-/-) mice. CFH(-/-).C3(-/-) mice had significantly more Aβ on Bruch's membrane, fewer macrophages, and high levels of retinal inflammation than the other groups. Our data show that both uncontrolled C3 activation (CFH(-/-)) and complete absence of C3 (CFH(-/-).C3(-/-) and C3(-/-)) negatively affect aged retinas. These findings suggest that strategies that inhibit C3 in AMD may be deleterious.

To assess whether laser treatment to the retinal pigment epithelium anterior to drusen in eyes of patients with EFEMP1-related maculopathy affects visual acuity, deposit volume, and retinal sensitivity.Prospective, interventional case series.In 11 patients with autosomal dominant drusen and confirmed disease-causing EFEMP1 mutation, the worse-seeing eye was treated with Argon green laser (10 to 15 laser spots; 200-μm spot size, 0.1-second duration, 80 to 120 mW). Patients were examined before treatment as well as 1, 3, 6, and 12 months after the procedure. Clinical assessment included visual acuity, fundus-controlled perimetry, spectral-domain optical coherence tomography, and autofluorescence imaging. Custom-made software allowed for coregistration of fundus-controlled perimetry and spectral-domain optical coherence tomography data sets. The main outcome measures were change in visual acuity, retinal sensitivity, and drusen volume.The untreated eyes lost an average of 0.8 letters, whereas the treated eyes gained an average of 4.9 letters. For fundus-controlled perimetry, locus-by-locus differences in sensitivity were calculated between pretreatment and posttreatment assessments; subsequently, the overall difference in the treated and untreated eye was compared. Five patients showed significant improvement in retinal sensitivity, 5 patients showed no change, and 1 patient showed significant deterioration. An increase in mean drusen thickness was observed in the untreated eyes, but not in the treated eyes (P = .0322). The thickness of the drusen correlated with retinal sensitivity (ρ = -0.49; P.0001). Safety was demonstrated and no adverse events were observed.Low-energy laser treatment is safe and may be effective in the treatment of autosomal dominant drusen. Further evaluation with long-term assessment is required to confirm the benefits.

Purpose To describe phenotypic variability and to report novel mutational data in patients with gyrate atrophy. Design Retrospective case series. Participants Seven unrelated patients (10 to 52 years of age) with clinical and biochemical evidence of gyrate atrophy. Methods Detailed ophthalmologic examination, fundus photography, fundus autofluorescence (FAF) imaging, spectral-domain optical coherence tomography, and microperimetry testing were performed. The coding region and intron–exon boundaries of ornithine aminotransferase (OAT) were analyzed. OAT mRNA was isolated from peripheral blood leucocytes of 1 patient and analyzed. Main Outcome Measures OAT mutation status and resultant clinical, structural, and functional characteristics. Results Funduscopy revealed circular areas of chorioretinal atrophy, and FAF imaging showed sharply demarcated areas of increased or preserved signal in all 7 patients. Spectral-domain optical coherence tomography revealed multiple intraretinal cystic spaces and hyperreflective deposit in the ganglion cell layer of all study subjects. Round tubular, rosette-like structures located in the outer nuclear layer of the retinae of the 4 older patients were observed (termed outer retinal tubulation ). Thickening was evident in the foveolae of younger patients, despite the posterior pole appearing relatively preserved. Macular function, assessed by microperimetry, was preserved over areas of normal or increased autofluorescence. However, sensitivity was reduced even in structurally intact parts of the retina. The molecular pathologic features were determined in all study subjects: 9 mutations, 4 novel, were detected in the OAT gene. OAT mRNA was isolated from blood leukocytes, and monoallelic expression of a mutated allele was demonstrated in 1 patient. Conclusions Fundus autofluorescence imaging can reveal the extent of neurosensory dysfunction in gyrate atrophy patients. Macular edema is a uniform finding; the fovea is relatively thick in early stages of disease and retinal tubulation is present in advanced disease. Analysis of leukocyte RNA complements the high sensitivity of conventional sequencing of genomic DNA for mutation detection in this gene. Financial Disclosure(s) The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Purpose: To report the results of intravitreal treatment with bevacizumab in neovascular age-related macular degeneration (AMD) after a loading dose (LD) of three monthly injections followed by an optical coherence tomography (OCT)-guided strategy, based on best-corrected visual acuity (VA) and number of injections required over 1 year. Methods: A series of consecutive cases of 149 eyes of 147 patients received three or more intravitreal injections of bevacizumab (1.25 mg) for neovascular AMD over a 1-year period. The patients underwent ophthalmological examinations: measurement of the VA, fluorescein angiography, dilated fundus examination at baseline; VA, OCT and dilated fundus examination at monthly follow-up visits. Repeated injections were given each month for the first 3 months (LD); thereafter, injections were only administered if leakage or macular oedema were present. Results: Mean baseline VA was 51 ± 14 letters, which improved to 58 ± 15 letters (p

Autosomal dominant drusen is a dominantly inherited maculopathy first described by Doyne in 1899 [1]. The condition is characterised by macular drusen and is associated with slowly progressive loss of central visual acuity. A single heterozygous missense mutation (p.Arg345Trp) in the EFEMP1 (EGF-containing fibulin-like extracellular matrix protein 1) gene encoding fibulin-3 is responsible for the autosomal dominant drusen phenotype in all patients and families so far reported [2].

In a subset of inherited retinal degenerations (including cone, cone-rod, and macular dystrophies), cone photoreceptors are more severely affected than rods; ABCA4 mutations are the most common cause of this heterogeneous class of disorders. To identify retinal-disease-associated genes, we performed exome sequencing in 28 individuals with "cone-first" retinal disease and clinical features atypical for ABCA4 retinopathy. We then conducted a gene-based case-control association study with an internal exome data set as the control group. TTLL5, encoding a tubulin glutamylase, was highlighted as the most likely disease-associated gene; 2 of 28 affected subjects harbored presumed loss-of-function variants: c.[1586_1589delAGAG];[1586_1589delAGAG], p.[Glu529Valfs(∗)2];[Glu529Valfs(∗)2], and c.[401delT(;)3354GA], p.[Leu134Argfs(∗)45(;)Trp1118(∗)]. We then inspected previously collected exome sequence data from individuals with related phenotypes and found two siblings with homozygous nonsense variant c.1627GT (p.Glu543(∗)) in TTLL5. Subsequently, we tested a panel of 55 probands with retinal dystrophy for TTLL5 mutations; one proband had a homozygous missense change (c.1627GA [p.Glu543Lys]). The retinal phenotype was highly similar in three of four families; the sibling pair had a more severe, early-onset disease. In human and murine retinae, TTLL5 localized to the centrioles at the base of the connecting cilium. TTLL5 has been previously reported to be essential for the correct function of sperm flagella in mice and play a role in polyglutamylation of primary cilia in vitro. Notably, genes involved in the polyglutamylation and deglutamylation of tubulin have been associated with photoreceptor degeneration in mice. The electrophysiological and fundus autofluorescence imaging presented here should facilitate the molecular diagnosis in further families.

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.

Purpose To further investigate functional aspects of hyperautofluorescent ring, seen in retinal dystrophies. Methods Full ophthalmologic examination was performed, autofluorescence imaging was done by Heidelberg Engineering Spectralis/OCT, microperimetry with MP1 and electrophysiology. Results Hzperautofluorescent ring exhibited different characteristics in RP-related diseases and very heterogenous characteristics in cone dystrophies, cone/rod dystrophies and macular dystrophies such as Stargardt disease, Best's disease and other dystrophies affecting central retina. Conclusion Hyperautofluorescent ring is a specific feature that represents boundary between functioning and disfunctional retina and its origin is at present unknown. Different forms of ring are seen in different diseases and its functional correlations are important to understand the underlying mechanisms.

To investigate the clinical use of the large-field pattern electroretinogram (PERG) as an adjunct test to the International-standard PERG in an unselected sequential cohort of patients referred for routine electrophysiologic assessment.Pattern electroretinograms to both 15° × 11° (International Society for Clinical Electrophysiology of Vision Standard) and 30° × 22° (large field) checkerboard field sizes were recorded in 277 consecutive electrophysiology patients, aged 10-79 years. Most patients had additional tests including full-field electroretinogram, electrooculogram, multifocal electroretinograms, or cortical visual evoked potential. Patient data were compared with data from 27 control subjects.Satisfactory 2-field PERG data were obtained in 91% (N = 253) of patients; data from 24 patients (9%) were excluded because of poor compliance (n = 17) or nystagmus (n = 7). Standard PERGs were consistent with macular dysfunction in 44% of cases; large-field PERG revealed macular dysfunction in an additional 8% of eyes and helped to distinguish between localized central, predominantly paracentral, and widespread macular dysfunction. The results were consistent with multifocal electroretinogram and/or imaging studies on the same patients. In some patients with optic nerve disease, the large-field PERG provided clearer evidence of normal macular function than the standard PERG.Routine use of the large-field PERG is a valuable complement to standard-field PERG testing in the evaluation and management of patients with different forms of macular or generalized retinal dysfunction and can be useful in patients with optic nerve disease.

Background Amyloid beta (Aβ) accumulates in the ageing central nervous system and is associated with a number of age-related diseases, including age-related macular degeneration (AMD) in the eye. AMD is characterised by accumulation of extracellular deposits called drusen in which Aβ is a key constituent. Aβ activates the complement cascade and its deposition is associated with activated macrophages. So far, little is known about the quantitative measurements of Aβ accumulation and definitions of its relative sites of ocular deposition in the normal ageing mouse. Methodology/Principal Findings We have traced Aβ accumulation quantitatively in the ageing mouse retina using immunohistochemistry and Western blot analysis. We reveal that it is not only deposited at Bruch's membrane and along blood vessels, but unexpectedly, it also coats photoreceptor outer segments. While Aβ is present at all sites of deposition from 3 months of age, it increases markedly from 6 months onward. Progressive accumulation of deposits on outer segments was confirmed with scanning electron microscopy, revealing age-related changes in their morphology. Such progress of accumulation of Aβ on photoreceptor outer segments with age was also confirmed in human retinae using immunohistochemistry. We also chart the macrophage response to increases in Aβ showing up-regulation in their numbers using both confocal laser imaging of the eye in vivo followed by in vitro immunostaining. With age macrophages become bloated with cellular debris including Aβ, however, their increasing numbers fail to stop Aβ accumulation. Conclusions Increasing Aβ deposition in blood vessels and Bruch's membrane will impact upon retinal perfusion and clearance of cellular waste products from the outer retina, a region of very high metabolic activity. This accumulation of Aβ may contribute to the 30% reduction of photoreceptors found throughout life and the shortening of those that remain. The coating of Aβ on outer segments may also have an impact upon visual function with age.

Purpose To evaluate the morphology and function of the hyperautofluorescent parafoveal ring in patients with retinitis pigmentosa (RP) using optical coherence tomography (OCT) and microperimetry (MP). Methods Twelve patients with RP who had hyperautofluorescent parafoveal ring were examined. In all patients, full ophthalmological examination, MP (Nidek Technologies MP1 Microperimeter), OCT (Topcon 3D OCT-1000) and fundus autofluoresence (AF) imaging (Heidelberg Engineering HRA) were performed. The results of OCT imaging and MP testing were superimposed on the AF image. We investigated whether the AF diameter correlated with photoreceptor inner segment/outer segment junction (IS/OS line). Results On the site of hyperautofluorescent ring, the integrity of the photoreceptor layer was affected and the IS/OS line was absent. In the encircled area of normal AF, a preserved photoreceptor layer with an IS/OS line was present. A strong correlation (r = 0.83, P < 0.0001) between the horizontal diameter of the encircled area of normal AF and the length of the IS/OS line was seen. The mean retinal sensitivity was significantly lower (P < 0.0001) over the area of hyperautofluorescent ring (2.0 dB, CI 1.1 to 2.8) compared to the mean retinal sensitivity of the encircled area of normal AF (9.2 dB, CI 6.6 to 11.9). Outside the ring, the majority of patients seemed to have an absence of photoreceptor layer with no light perception, even if AF pattern appeared well preserved. Conclusion The hyperautofluorescent ring demonstrates border of abnormal retinal morphology and function, which are only preserved within the area it encircles. It appears that the ring represents an area of intensive photoreceptor degradation.

Aims: To investigate the importance of a larger stimulus field for pattern electroretinography (PERG) in evaluating macular function in Stargardt disease, and to determine the relationship between PERG and spectral-domain optical coherence tomography (SD-OCT). Methods: In this prospective cross-sectional study, PERG from standard (12°×16°) and larger (24°×32°) stimulus fields and SD-OCT were recorded in 18 patients with genetically confirmed Stargardt disease, and in 18 control subjects. Results: A PERG P50 response to the larger stimulus field was detectable in 86% of eyes, with a mean P50 amplitude of 2.3 μV, compared with 22% and 1.0 μV for the standard stimulus field. The specificity and sensitivity of PERG to the standard stimulus field were greater than for the larger field. For both PERG P50 and N95, the differences in their amplitudes between the standard and larger stimulus fields correlated significantly with visual acuity and SD-OCT parameters. Conclusion: The higher sensitivity and specificity of PERG to the standard stimulus field provide detection of early maculopathy in Stargardt disease, while PERG with the larger stimulus field allows for longer follow-up. The PERG amplitude for the larger stimulus field correlated with severity of transverse photoreceptor loss in SD-OCT. These methods are complementary for evaluation of progression of photoreceptor damage in patients with Stargardt disease.

Purpose The aim of our study was to evaluate retinal function in patients with genetically determined mutation in the ABCR gene by correlating retinal morphology with functional and electrophysiological tests. Methods 12 patients (10F, 2M, VA: 0,2±0,2) were included in the study. The central 10o visual fields were tested with Octopus M2 TOP and microperimetry (MP1, Nidek technologies), that enables one to compare central retinal sensitivity and fixation patterns in relation to the fundus image. PERG and mfERG were recorded in all the patients according to the ISCEV standards. Results mfERG showed reduced responses mostly in the inner three rings (ring 1: 33,9% of mean normal value, ring 2: 35,4%, ring 3: 57,5%, ring 4: 75,5%, ring 5: 84% of mean normal value). There was good correlation between mfERG and pattern P50 (r=0,7, p=0,0001) and N95 responses (r= 0,6, p=0,004). mfERG appears more sensitive for central retinal testing than pattern ERG. There was a high correlation found between microperimetry (MP) and static perimetry (MD, r = 0.6, p=0.008). Shift of fixation to the preferred retinal locus(seen on MP) was found in 8 out of 16 eyes tested with VA 0,2 and less. In patients with absolute central scotoma and shifting of fixation, scotoma may erroneously be interpreted as eccentric when seen only with static perimetry. Conclusion In patients with visual acuity 0,2 and lower, the fixation shift to the preferred retinal locus was observed. Cautious interpretation of the static perimetry and mfERG as well as PERG is needed in patients with eccentric and nonstable fixation.

Purpose To evaluate the effects of intravitreal treatment with bevacizumab on vision and anatomic outcome in patients with neovascular AMD. Methods Retrospective review of 153 eyes that received three or more intravitreal injections of bevacizumab (1.25 mg) for neovascular AMD over a one-year period. Patients underwent ophthalmological examinations, measurements of the best-corrected visual acuity (VA), fluorescein angiography and optical coherence tomography, at baseline and at monthly follow-up visits. Repeated injections were given in the presence of persistent leakage or retinal oedema. We analyzed the data obtained at three time intervals: 1 month (first evaluation) after the third injection, 6 months (second evaluation), and one year (third evaluation) after the onset of treatment. Changes from baseline in VA, central retinal thickness (CRT), and total macular volume (TMV) were analyzed using paired t-tests. Results Mean baseline VA improved from 50.5 to 57.6 letters (P < 0.0001, N = 153) at first evaluation, 58.3 letters (P < 0.0001, N = 119) at second evaluation, and 59.5 letters (P = 0.002, N = 48) at third evaluation. Baseline mean CRT (344.6 μm) and baseline mean TMV (8.6 mm3) decreased at the first evaluation, to 219.0 μm (P < 0.0001) and 7.2 mm3 (P < 0.0001), respectively. No systemic or serious ocular side effects were noted. Conclusion Intravitreal bevacizumab is an effective treatment for neovascular AMD, resulting in significant functional and anatomical improvement seen up to one year. After one year, VA in 27% of patients was improved by 15 letters or more, was maintained stable in 67% of patients, and was worsened by 15 letters or more in 6% of patients.

Visual processes continue to mature well into childhood, due to the development of the retina, optic nerve, visual pathway and visual cortex. Normal development of visual acuity and maturation of visual evoked potentials (VEPs) have been well studied, although rarely for their correlation. The purpose of this study was to investigate the same population of infants and preschool children for their VEP maturation to flash, pattern-reversal (reversal) and pattern-onset (onset) stimulation, which are normally used in our everyday clinical protocol, and to determine the relationships between the VEP parameters obtained and the development of visual acuity.Forty-one healthy children from 1.5 months to 7.5 years old were included. Their visual acuity for distance was tested with Teller Acuity Cards (2 years of age) and Cambridge Visual Acuity Crowding Cards (2 years of age). VEP latencies and amplitudes were evaluated to flash (P2 wave), reversal (P100 wave) and onset (C1 wave) binocular stimulation. For reversal and onset stimulation, checkerboard pattern (check) sizes of 25', 50' and 100' were used.Age-dependent exponential decreases in latencies to flash, reversal and onset stimulation were seen. For amplitudes, there was an age-dependent increase only to onset stimulation. There was a significant correlation between VEPs and visual acuity (P0.05) for latencies to flash, reversal and onset stimulation and amplitudes to onset stimulation.These findings indicate the expected maturation of flash, reversal and onset VEPs, and demonstrate their correlation to normal development of visual acuity. Maturation of VEP latencies is associated with development of visual acuity.

Flecked-retina syndromes, including fundus flavimaculatus, fundus albipunctatus, and benign fleck retina, comprise a group of disorders with widespread or limited distribution of yellow-white retinal lesions of various sizes and configurations. Three siblings who have benign fleck retina and were born to consanguineous parents are the basis of this report. A combination of homozygosity mapping and exome sequencing helped to identify a homozygous missense mutation, c.133GT (p.Gly45Cys), in PLA2G5, a gene encoding a secreted phospholipase (group V phospholipase A(2)). A screen of a further four unrelated individuals with benign fleck retina detected biallelic variants in the same gene in three patients. In contrast, no loss of function or common (minor-allele frequency0.05%) nonsynonymous PLA2G5 variants have been previously reported (EVS, dbSNP, 1000 Genomes Project) or were detected in an internal database of 224 exomes (from subjects with adult onset neurodegenerative disease and without a diagnosis of ophthalmic disease). All seven affected individuals had fundoscopic features compatible with those previously described in benign fleck retina and no visual or electrophysiological deficits. No medical history of major illness was reported. Levels of low-density lipoprotein were mildly elevated in two patients. Optical coherence tomography and fundus autofluorescence findings suggest that group V phospholipase A(2) plays a role in the phagocytosis of photoreceptor outer-segment discs by the retinal pigment epithelium. Surprisingly, immunohistochemical staining of human retinal tissue revealed localization of the protein predominantly in the inner and outer plexiform layers.

Background Usher Syndrome is the leading cause of inherited deaf-blindness. It is divided into three subtypes, of which the most common is Usher type 2, and the USH2A gene accounts for 75-80% of cases. Despite recent sequencing strategies, in our cohort a significant proportion of individuals with Usher type 2 have just one heterozygous disease-causing mutation in USH2A, or no convincing disease-causing mutations across nine Usher genes. The purpose of this study was to improve the molecular diagnosis in these families by screening USH2A for duplications, heterozygous deletions and a common pathogenic deep intronic variant USH2A: c.7595-2144A>G. Methods Forty-nine Usher type 2 or atypical Usher families who had missing mutations (mono-allelic USH2A or no mutations following Sanger sequencing of nine Usher genes) were screened for duplications/deletions using the USH2A SALSA MLPA reagent kit (MRC-Holland). Identification of USH2A: c.7595-2144A>G was achieved by Sanger sequencing. Mutations were confirmed by a combination of reverse transcription PCR using RNA extracted from nasal epithelial cells or fibroblasts, and by array comparative genomic hybridisation with sequencing across the genomic breakpoints. Results Eight mutations were identified in 23 Usher type 2 families (35%) with one previously identified heterozygous disease-causing mutation in USH2A. These consisted of five heterozygous deletions, one duplication, and two heterozygous instances of the pathogenic variant USH2A: c.7595-2144A>G. No variants were found in the 15 Usher type 2 families with no previously identified disease-causing mutations. In 11 atypical families, none of whom had any previously identified convincing disease-causing mutations, the mutation USH2A: c.7595-2144A>G was identified in a heterozygous state in one family. All five deletions and the heterozygous duplication we report here are novel. This is the first time that a duplication in USH2A has been reported as a cause of Usher syndrome. Conclusions We found that 8 of 23 (35%) of ‘missing’ mutations in Usher type 2 probands with only a single heterozygous USH2A mutation detected with Sanger sequencing could be attributed to deletions, duplications or a pathogenic deep intronic variant. Future mutation detection strategies and genetic counselling will need to take into account the prevalence of these types of mutations in order to provide a more comprehensive diagnostic service.