Adaptive optics: principles and applications in ophthalmology

This is a comprehensive review of the principles and applications of adaptive optics (AO) in ophthalmology. It has been combined with flood illumination ophthalmoscopy, scanning laser ophthalmoscopy, as well as optical coherence tomography to image photoreceptors, retinal pigment epithelium (RPE), retinal ganglion cells, lamina cribrosa and the retinal vasculature. In this review, we highlight the clinical studies that have utilised AO to understand disease mechanisms. However, there are some limitations to using AO in a clinical setting including the cost of running an AO imaging service, the time needed to scan patients, the lack of normative databases and the very small size of area imaged. However, it is undoubtedly an exceptional research tool that enables visualisation of the retina at a cellular level

Serum Vascular Endothelial Growth Factor Levels in the IVAN Trial; Relationships with Drug, Dosing, and Systemic Serious Adverse Events

Purpose: To describe serum vascular endothelial growth factor (sVEGF) in patients with neovascular age-related macular degeneration (nAMD) receiving anti-VEGF agents and associations between sVEGF and systemic serious adverse events (SSAEs). Design: Exploratory analyses of a randomized controlled trial that enrolled 610 participants with nAMD and compared 2 anti-VEGF antibodies, ranibizumab and bevacizumab, and 2 treatment regimens, monthly vs. discontinuous, with 2 years' follow-up. Participants: Adults aged 50+ years with treatment-naïve nAMD and a visual acuity of ≥25 letters (Snellen equivalent 20/320) in the affected eye. Methods: Intravitreal injection of anti-VEGF antibodies. Main Outcome Measures: sVEGF and occurrence of SSAE, with particular interest in arteriothromboembolic events (ATE) and immunologically mediated events (IME). Results: On average, sVEGF (measured at months 0, 1, 11, 12, 23, and 24) decreased from a geometric mean of 168 pg/mL at baseline to 64 pg/mL at month 24. The decrease was greater with bevacizumab than with ranibizumab and was dependent on time since last treatment; at month 24 sVEGF was 11% lower with bevacizumab if treated ≥3 months previously, 51% lower if treated 2 months previously, and 76% lower if treated the previous month, compared with ranibizumab. The hazard of experiencing an ATE increased with age (hazard ratio [HR] = 2.01; 95% confidence interval [CI] = 1.32-3.05; P = 0.001) and higher sVEGF (HR = 1.16; 95% CI = 1.03-1.30, per 100 unit rise in sVEGF; P = 0.013). There was no association between sVEGF and the hazard of an IME (HR = 1.01; 95% CI = 0.76-1.33; P = 0.942); however, the hazard of an IME was significantly increased by treatment with bevacizumab compared with ranibizumab (HR = 3.53; 95% CI = 1.35-9.22; P = 0.010). The hazard of an "other SSAE" (not categorized as ATE or IME) increased with age (HR 1.51, 95% CI 1.14-2.01, P = 0.005) and decreased if an injection had been administered within the previous month (HR = 0.68; 95% CI = 0.45-1.03; P = 0.069). Conclusions: The decrease in sVEGF is greater with bevacizumab than with ranibizumab, but this difference is eliminated when treatment is withheld for 3 months. Higher sVEGF increased the hazard of an ATE and bevacizumab increases the hazard of an IME compared with ranibizumab

Associations with Retinal Pigment Epithelium Thickness Measures in a Large Cohort: Results from the UK Biobank

PURPOSE: To describe associations of ocular and systemic factors with retinal pigment epithelium (RPE)-Bruch's membrane (BM) complex thickness as measured by spectral-domain (SD) optical coherence tomography (OCT). DESIGN: Multisite community-based study. This research has been conducted using the UK Biobank Resource. PARTICIPANTS: Sixty-seven thousand three hundred eighteen people 40 to 69 years old received questionnaires, physical examination, and eye examination, including macular SD OCT. Systematic selection process identified 34 652 eyes with high-quality SD OCT images from normal individuals for analysis. METHODS: We included people with no self-reported ocular disease, diabetes, or neurologic disorders; visual acuity of ≥20/25; refraction between -6 diopters (D) to 6 D, and IOP of 6 to 21 mmHg. Only high-quality, well-centered SD OCT images with central, stable fixation were included. Descriptive statistics, t tests, and regression analyses were performed. Multivariate regression modeling was used to adjust for covariates and to identify relationships between RPE-BM thickness and ocular and systemic features. MAIN OUTCOME MEASURES: Retinal pigment epithelium-BM thickness, as measured by SD OCT segmentation using Topcon Advanced Boundary Segmentation at 9 Early Treatment of Diabetic Retinopathy Study subfields. RESULTS: Mean RPE-BM thickness was 26.3 μm (standard deviation, 4.8 μm) at central subfield. Multivariate regression with age stratification showed that RPE thinning became apparent after age 45. Among those aged ≤45, RPE-BM was significantly thicker among those of black or mixed/other race (+3.61 and +1.77 μm vs. white, respectively; P 45, RPE-BM was significantly thinner with older age (-0.10 μm/year; P < 0.001), Asian ethnicity (-0.45 μm vs. white; P = 0.02), taller height (-0.02 μm/cm; P < 0.001), higher IOP (-0.03 μm/mmHg; P < 0.001), and regular smoking (-0.27 μm vs. nonsmokers; P = 0.02). In contrast, RPE-BM was significantly thicker among black or mixed/other race (+3.29 μm and +0.81 μm vs. white, respectively; P < 0.001) and higher hyperopia (+0.28 μm/D; P < 0.001). There was no significant association with sex or Chinese ethnicity. CONCLUSIONS: We describe novel findings of RPE-BM thickness in normal individuals, a structure that varies with age, ethnicity, refraction, IOP, and smoking. The significant association with IOP is especially interesting and may have relevance for the etiology of glaucoma, while the association between age and smoking may have relevance for the etiology of age-related macular degeneration

Association of ambient air pollution with age-related macular degeneration and retinal thickness in UK Biobank

AIM: To examine the associations of air pollution with both self-reported age-related macular degeneration (AMD), and in vivo measures of retinal sublayer thicknesses. METHODS: We included 115 954 UK Biobank participants aged 40-69 years old in this cross-sectional study. Ambient air pollution measures included particulate matter, nitrogen dioxide (NO2) and nitrogen oxides (NOx). Participants with self-reported ocular conditions, high refractive error ( +6 diopters) and poor spectral-domain optical coherence tomography (SD-OCT) image were excluded. Self-reported AMD was used to identify overt disease. SD-OCT imaging derived photoreceptor sublayer thickness and retinal pigment epithelium (RPE) layer thickness were used as structural biomarkers of AMD for 52 602 participants. We examined the associations of ambient air pollution with self-reported AMD and both photoreceptor sublayers and RPE layer thicknesses. RESULTS: After adjusting for covariates, people who were exposed to higher fine ambient particulate matter with an aerodynamic diameter <2.5 µm (PM2.5, per IQR increase) had higher odds of self-reported AMD (OR=1.08, p=0.036), thinner photoreceptor synaptic region (β=-0.16 µm, p=2.0 × 10-5), thicker photoreceptor inner segment layer (β=0.04 µm, p=0.001) and thinner RPE (β=-0.13 µm, p=0.002). Higher levels of PM2.5 absorbance and NO2 were associated with thicker photoreceptor inner and outer segment layers, and a thinner RPE layer. Higher levels of PM10 (PM with an aerodynamic diameter <10 µm) was associated with thicker photoreceptor outer segment and thinner RPE, while higher exposure to NOx was associated with thinner photoreceptor synaptic region. CONCLUSION: Greater exposure to PM2.5 was associated with self-reported AMD, while PM2.5, PM2.5 absorbance, PM10, NO2 and NOx were all associated with differences in retinal layer thickness

Modeling the Structural Consequences of \u3cem\u3eBEST1\u3c/em\u3e Missense Mutations

Mutations in the bestrophin-1 gene (BEST1) are an important cause of inherited retinal disorders. Hitherto, over 100 unique allelic variants have been linked to the human BEST1 (hBEST1), and associated with disease phenotypes, broadly termed as bestrophinopathies. A spontaneous animal model recapitulating BEST1-related phenotypes, canine multifocal retinopathy (cmr), is caused by mutations in the canine gene ortholog (cBEST1). We have recently characterized molecular consequences of cmr, demonstrating defective protein trafficking as a result of G161D (cmr2) mutation. To further investigate the pathological effects of BEST1 missense mutations, canine and human peptide fragments derived from the protein sequence have been studied in silico as models for early events in the protein folding. The results showed that G161D as well as I201T substitutions cause severe conformational changes in the structure of bestrophin-1, suggesting protein misfolding as an underlying disease mechanism. The comparative modeling studies expand our insights into BEST1 pathogenesis

A clinical and molecular characterisation of CRB1-associated maculopathy

To date, over 150 disease-associated variants in CRB1 have been described, resulting in a range of retinal disease phenotypes including Leber congenital amaurosis and retinitis pigmentosa. Despite this, no genotype–phenotype correlations are currently recognised. We performed a retrospective review of electronic patient records to identify patients with macular dystrophy due to bi-allelic variants in CRB1. In total, seven unrelated individuals were identified. The median age at presentation was 21 years, with a median acuity of 0.55 decimalised Snellen units (IQR = 0.43). The follow-up period ranged from 0 to 19 years (median = 2.0 years), with a median final decimalised Snellen acuity of 0.65 (IQR = 0.70). Fundoscopy revealed only a subtly altered foveal reflex, which evolved into a bull’s-eye pattern of outer retinal atrophy. Optical coherence tomography identified structural changes—intraretinal cysts in the early stages of disease, and later outer retinal atrophy. Genetic testing revealed that one rare allele (c.498_506del, p.(Ile167_Gly169del)) was present in all patients, with one patient being homozygous for the variant and six being heterozygous. In trans with this, one variant recurred twice (p.(Cys896Ter)), while the four remaining alleles were each observed once (p.(Pro1381Thr), p.(Ser478ProfsTer24), p.(Cys195Phe) and p.(Arg764Cys)). These findings show that the rare CRB1 variant, c.498_506del, is strongly associated with localised retinal dysfunction. The clinical findings are much milder than those observed with bi-allelic, loss-of-function variants in CRB1, suggesting this in-frame deletion acts as a hypomorphic allele. This is the most prevalent disease-causing CRB1 variant identified in the non-Asian population to date

Cohort profile: design and methods in the eye and vision consortium of UK Biobank

PURPOSE: To describe the rationale, methods and research potential of eye and vision measures available in UK Biobank. PARTICIPANTS: UK Biobank is a large, multisite, prospective cohort study. Extensive lifestyle and health questionnaires, a range of physical measures and collection of biological specimens are collected. The scope of UK Biobank was extended midway through data collection to include assessments of other measures of health, including eyes and vision. The eye assessment at baseline included questionnaires detailing past ophthalmic and family history, measurement of visual acuity, refractive error and keratometry, intraocular pressure (IOP), corneal biomechanics, spectral domain optical coherence tomography (OCT) of the macula and a disc-macula fundus photograph. Since recruitment, UK Biobank has collected accelerometer data and begun multimodal imaging data (including brain, heart and abdominal MRI) in 100 000 participants. Dense genotypic data and a panel of 20 biochemistry measures are available, and linkage to medical health records for the full cohort has begun. FINDINGS TO DATE: A total of 502 665 people aged between 40 and 69 were recruited to participate in UK Biobank. Of these, 117 175 took part in baseline assessment of vision, IOP, refraction and keratometry. A subgroup of 67 321 underwent OCT and retinal photography. The introduction of eye and vision measures in UK Biobank was accompanied by intensive training, support and a data monitoring quality control process. FUTURE PLANS: UK Biobank is one of the largest prospective cohorts worldwide with extensive data on ophthalmic diseases and conditions. Data collection is an ongoing process and a repeat of the baseline assessment including the questionnaires, measurements and sample collection will be performed in subsets of 25 000 participants every 2-3 years. The depth and breadth of this dataset, coupled with its open-access policy, will create a powerful resource for all researchers to investigate the eye diseases in later life

Multiple domains in the Crumbs Homolog 2a (Crb2a) protein are required for regulating rod photoreceptor size

Background Vertebrate retinal photoreceptors are morphologically complex cells that have two apical regions, the inner segment and the outer segment. The outer segment is a modified cilium and is continuously regenerated throughout life. The molecular and cellular mechanisms that underlie vertebrate photoreceptor morphogenesis and the maintenance of the outer segment are largely unknown. The Crumbs (Crb) complex is a key regulator of apical membrane identity and size in epithelia and in Drosophila photoreceptors. Mutations in the human gene CRUMBS HOMOLOG 1 (CRB1) are associated with early and severe vision loss. Drosophila Crumbs and vertebrate Crb1 and Crumbs homolog 2 (Crb2) proteins are structurally similar, all are single pass transmembrane proteins with a large extracellular domain containing multiple laminin- and EGF-like repeats and a small intracellular domain containing a FERM-binding domain and a PDZ-binding domain. In order to begin to understand the role of the Crb family of proteins in vertebrate photoreceptors we generated stable transgenic zebrafish in which rod photoreceptors overexpress full-length Crb2a protein and several other Crb2a constructs engineered to lack specific domains. Results We examined the localization of Crb2a constructs and their effects on rod morphology. We found that only the full-length Crb2a protein approximated the normal localization of Crb2a protein apical to adherens junctions in the photoreceptor inner segment. Several Crb2a construct proteins localized abnormally to the outer segment and one construct localized abnormally to the cell body. Overexpression of full-length Crb2a greatly increased inner segment size while expression of several other constructs increased outer segment size. Conclusions Our observations suggest that particular domains in Crb2a regulate its localization and thus may regulate its regionalized function. Our results also suggest that the PDZ-binding domain in Crb2a might bring a protein(s) into the Crb complex that alters the function of the FERM-binding domain

Mutation screening of retinal dystrophy patients by targeted capture from tagged pooled DNAs and next generation sequencing.

Purpose: Retinal dystrophies are genetically heterogeneous, resulting from mutations in over 200 genes. Prior to the development of massively parallel sequencing, comprehensive genetic screening was unobtainable for most patients. Identifying the causative genetic mutation facilitates genetic counselling, carrier testing and prenatal/pre-implantation diagnosis, and often leads to a clearer prognosis. In addition, in a proportion of cases, when the mutation is known treatment can be optimised and patients are eligible for enrolment into clinical trials for gene-specific therapies. Methods: Patient genomic DNA was sheared, tagged and pooled in batches of four samples, prior to targeted capture and next generation sequencing. The enrichment reagent was designed against genes listed on the RetNet database (July 2010). Sequence data were aligned to the human genome and variants were filtered to identify potential pathogenic mutations. These were confirmed by Sanger sequencing. Results: Molecular analysis of 20 DNAs from retinal dystrophy patients identified likely pathogenic mutations in 12 cases, many of them known and/or confirmed by segregation. These included previously described mutations in ABCA4 (c.6088C>T,p.R2030*; c.5882G>A,p.G1961E), BBS2 (c.1895G>C,p.R632P), GUCY2D (c.2512C>T,p.R838C), PROM1 (c.1117C>T,p.R373C), RDH12 (c.601T>C,p.C201R; c.506G>A,p.R169Q), RPGRIP1 (c.3565C>T,p.R1189*) and SPATA7 (c.253C>T,p.R85*) and new mutations in ABCA4 (c.3328+1G>C), CRB1 (c.2832_2842+23del), RP2 (c.884-1G>T) and USH2A (c.12874A>G,p.N4292D). Conclusions: Tagging and pooling DNA prior to targeted capture of known retinal dystrophy genes identified mutations in 60% of cases. This relatively high success rate may reflect enrichment for consanguineous cases in the local Yorkshire population, and the use of multiplex families. Nevertheless this is a promising high throughput approach to retinal dystrophy diagnostics

Mutation Screening of Multiple Genes in Spanish Patients with Autosomal Recessive Retinitis Pigmentosa by Targeted Resequencing

Retinitis Pigmentosa (RP) is a heterogeneous group of inherited retinal dystrophies characterised ultimately by the loss of photoreceptor cells. RP is the leading cause of visual loss in individuals younger than 60 years, with a prevalence of about 1 in 4000. The molecular genetic diagnosis of autosomal recessive RP (arRP) is challenging due to the large genetic and clinical heterogeneity. Traditional methods for sequencing arRP genes are often laborious and not easily available and a screening technique that enables the rapid detection of the genetic cause would be very helpful in the clinical practice. The goal of this study was to develop and apply microarray-based resequencing technology capable of detecting both known and novel mutations on a single high-throughput platform. Hence, the coding regions and exon/intron boundaries of 16 arRP genes were resequenced using microarrays in 102 Spanish patients with clinical diagnosis of arRP. All the detected variations were confirmed by direct sequencing and potential pathogenicity was assessed by functional predictions and frequency in controls. For validation purposes 4 positive controls for variants consisting of previously identified changes were hybridized on the array. As a result of the screening, we detected 44 variants, of which 15 are very likely pathogenic detected in 14 arRP families (14%). Finally, the design of this array can easily be transformed in an equivalent diagnostic system based on targeted enrichment followed by next generation sequencing