Gaetano R. Barile

Variation in factor B (BF) and complement component 2 (C2) genes is associated with age-related macular degeneration

Age-related macular degeneration (AMD) is the most common form of irreversible blindness in developed countries. Variants in the factor H gene (CFH, also known as HF1), which encodes a major inhibitor of the alternative complement pathway, are associated with the risk for developing AMD. Here we test the hypothesis that variation in genes encoding other regulatory proteins of the same pathway is associated with AMD. We screened factor B (BF) and complement component 2 (C2) genes, located in the major histocompatibility complex class III region, for genetic variation in two independent cohorts comprising ∼900 individuals with AMD and ∼400 matched controls. Haplotype analyses identify a statistically significant common risk haplotype (H1) and two protective haplotypes. The L9H variant of BF and the E318D variant of C2 (H10), as well as a variant in intron 10 of C2 and the R32Q variant of BF (H7), confer a significantly reduced risk of AMD (odds ratio = 0.45 and 0.36, respectively). Combined analysis of the C2 and BF haplotypes and CFH variants shows that variation in the two loci can predict the clinical outcome in 74% of the affected individuals and 56% of the controls. These data expand and refine our understanding of the genetic risk for AMD.

Genome-wide association study of advanced age-related macular degeneration identifies a role of the hepatic lipase gene (LIPC)

Advanced age-related macular degeneration (AMD) is the leading cause of late onset blindness. We present results of a genome-wide association study of 979 advanced AMD cases and 1,709 controls using the Affymetrix 6.0 platform with replication in seven additional cohorts (totaling 5,789 unrelated cases and 4,234 unrelated controls). We also present a comprehensive analysis of copy-number variations and polymorphisms for AMD. Our discovery data implicated the association between AMD and a variant in the hepatic lipase gene (LIPC) in the high-density lipoprotein cholesterol (HDL) pathway (discovery P = 4.53e-05 for rs493258). Our LIPC association was strongest for a functional promoter variant, rs10468017, (P = 1.34e-08), that influences LIPC expression and serum HDL levels with a protective effect of the minor T allele (HDL increasing) for advanced wet and dry AMD. The association we found with LIPC was corroborated by the Michigan/Penn/Mayo genome-wide association study; the locus near the tissue inhibitor of metalloproteinase 3 was corroborated by our replication cohort for rs9621532 with P = 3.71e-09. We observed weaker associations with other HDL loci (ABCA1, P = 9.73e-04; cholesterylester transfer protein, P = 1.41e-03; FADS1-3, P = 2.69e-02). Based on a lack of consistent association between HDL increasing alleles and AMD risk, the LIPC association may not be the result of an effect on HDL levels, but it could represent a pleiotropic effect of the same functional component. Results implicate different biologic pathways than previously reported and provide new avenues for prevention and treatment of AMD.

Common Variants near FRK/COL10A1 and VEGFA are Associated with Advanced Age-related Macular Degeneration

Despite significant progress in the identification of genetic loci for age-related macular degeneration (AMD), not all of the heritability has been explained. To identify variants which contribute to the remaining genetic susceptibility, we performed the largest meta-analysis of genome-wide association studies to date for advanced AMD. We imputed 6 036 699 single-nucleotide polymorphisms with the 1000 Genomes Project reference genotypes on 2594 cases and 4134 controls with follow-up replication of top signals in 5640 cases and 52 174 controls. We identified two new common susceptibility alleles, rs1999930 on 6q21-q22.3 near FRK/COL10A1 [odds ratio (OR) 0.87; P = 1.1 × 10−8] and rs4711751 on 6p12 near VEGFA (OR 1.15; P = 8.7 × 10−9). In addition to the two novel loci, 10 previously reported loci in ARMS2/HTRA1 (rs10490924), CFH (rs1061170, and rs1410996), CFB (rs641153), C3 (rs2230199), C2 (rs9332739), CFI (rs10033900), LIPC (rs10468017), TIMP3 (rs9621532) and CETP (rs3764261) were confirmed with genome-wide significant signals in this large study. Loci in the recently reported genes ABCA1 and COL8A1 were also detected with suggestive evidence of association with advanced AMD. The novel variants identified in this study suggest that angiogenesis (VEGFA) and extracellular collagen matrix (FRK/COL10A1) pathways contribute to the development of advanced AMD.

Reticular Macular Disease

PURPOSE To present a unified description of reticular macular disease (RMD), a common clinical entity that includes reticular pseudodrusen (RPD) and confers high-risk of progression to advanced age-related macular degeneration. DESIGN Population-based, retrospective, cross-sectional study. Forty-two patients with reticular findings in at least one imaging method, of whom 21 were followed up. METHODS RMD was defined as RPD in color or red-free photography, in a reticular pattern on scanning laser ophthalmoscope imaging (autofluorescence scans, infrared photographs, or indocyanine green angiography), or both. Color and red-free images were contrast-enhanced, and color photographs were examined in green and blue channels. Image registration in different methods allowed comparison of areas involved and assessment of lesion colocalization. RESULTS RMD generally was present in both photography and scanning laser ophthalmoscope imaging. When present in two image methods, areas of RMD either largely overlapped or one fell within the other. Individual lesions had high spatial correspondence. Serial imaging showed faded to absent findings in eyes in which choroidal neovascularization developed. CONCLUSIONS RMD is a single disease entity with stereotypical presentations in multiple imaging methods, of which RPD is one. Autofluorescence, infrared imaging, and indocyanine green angiography suggest that it involves the retinal pigment epithelium and choriocapillaris, whereas photographic patterns implicate the inner choroid. Infrared imaging, unlike other methods, can demonstrate RMD in the central macula. RMD is associated with progression to advanced age-related macular degeneration, perhaps on an inflammatory basis. RMD deserves wider recognition among clinicians caring for elderly patients.

Intravitreal triamcinolone for the treatment of refractory diabetic macular oedema with hard exudates: an optical coherence tomography study.

Aim: To investigate the use of intravitreal triamcinolone acetonide (IVTA) for the treatment of diabetic macular oedema (DMO) unresponsive to previous laser photocoagulation. Method: A retrospective, interventional, non-comparative case series. There were 30 eyes of 22 consecutive patients with refractory DMO. An intravitreal injection of triamcinolone acetonide at the dose of 4 mg in 0.1 ml was administered. Best corrected visual acuity was measured at each examination. In addition the central macular thickness was quantitatively measured by optical coherence tomography (OCT) examination at each visit. The amount of hard exudates deposition in the macula was subjectively evaluated using colour fundus photographs. Results: 30 eyes of 22 patients completed 6 months or more of follow up and were included in the study. Mean (SD) visual acuity improved from 0.17 (0.12) at baseline to 0.34 (0.18), 0.36 (0.16), and 0.31 (0.17) at the 1, 3, and 6 month follow up respectively. Mean (SD) OCT macular thickness decreased from 476 (98.32) μm at baseline to 277.46 (96.77) μm, 255.33 (95.73) μm, and 331.25 (146.76) μm at the 1, 3, and 6 month follow up period respectively. 18 and seven eyes completed 12 months and 18 months of follow up, respectively. Mean (SD) visual acuity was 0.36 (0.15) and 0.35 (0.16) at the 12 and 18 month follow up period respectively. 12 eyes received two, seven eyes received three, and two eyes received four IVTA injections. The mean (SD) interval between the first and second IVTA injection was 5.7 (2.67) months and between the second and third was 5.7 (3.25) months. Hard exudates were present in the macula at baseline in all eyes. Progressive reduction in the number and size of the hard exudates was noted after IVTA in all cases. Intraocular pressure was raised above 21 mm Hg in 12 (40%) of 30 eyes. Two eyes developed posterior subcapsular cataract and two developed vitreous haemorrhage. Conclusions: IVTA is a promising treatment for patients with DMO refractory to laser treatment. IVTA is effective in improving vision, reducing macular thickness, and inducing reabsorption of hard exudates. Further investigation is warranted to assess the safety of IVTA for the treatment of DMO.

Persistent indocyanine green fluorescence after vitrectomy for macular hole

PURPOSE To evaluate the persistence of indocyanine green (ICG) autofluorescence after ICG-assisted internal limiting membrane peeling for macular hole surgery. DESIGN Interventional case series. METHODS Retrospective institutional study. Four eyes of four patients who underwent pars plana vitrectomy with ICG-assisted internal limiting membrane peeling for macular hole repair were imaged for ICG autofluorescence at 795 nm with a scanning laser ophthalmoscope. The main outcome measure was persistence of ICG autofluorescence. RESULTS All four patients demonstrated persistent ICG fluorescence in the central macula up to 8 months postsurgery. CONCLUSIONS Persistent ICG signal was noted in the macula months after vitrectomy for macular hole surgery. The persistence of ICG autofluorescence could be responsible for delayed photochemical damage to the retinal pigment epithelium. Further studies must quantify the risk of retinal pigment epithelium injury when ICG-assisted internal limiting membrane peeling is used in macular hole surgery.

Remote image based retinopathy of prematurity diagnosis: a receiver operating characteristic analysis of accuracy

Background/aims: Telemedicine offers potential to improve the accessibility and quality of diagnosis of retinopathy of prematurity (ROP). The aim of this study was to measure accuracy of remote image based ROP diagnosis by three readers using receiver operating characteristic (ROC) analysis. Methods: 64 hospitalised infants who met ROP examination criteria underwent two consecutive bedside procedures: dilated examination by an experienced paediatric ophthalmologist and digital retinal imaging with a commercially available wide angle camera. 410 images from 163 eyes were reviewed independently by three trained ophthalmologist readers, who classified each eye into one of four categories: no ROP, mild ROP, type 2 prethreshold ROP, or ROP requiring treatment. Sensitivity and specificity for detection of mild or worse ROP, type 2 prethreshold or worse ROP, and ROP requiring treatment were determined, compared to a reference standard of dilated ophthalmoscopy. ROC curves were generated by calculating values for each reader at three diagnostic cut-off levels: mild or worse ROP (that is, reader was asked whether image sets represented mild or worse ROP), type 2 prethreshold or worse ROP (that is, reader was asked whether image sets represented type 2 prethreshold or worse ROP), and ROP requiring treatment. Results: Areas under ROC curves ranged from 0.747–0.896 for detection of mild or worse ROP, 0.905–0.946 for detection of type 2 prethreshold or worse ROP, and 0.941–0.968 for detection of ROP requiring treatment. Conclusions: Remote interpretation is highly accurate among multiple readers for the detection of ROP requiring treatment, but less so for detection of mild or worse ROP.

Heritability and Genome-Wide Association Study to Assess Genetic Differences between Advanced Age-Related Macular Degeneration Subtypes

PURPOSE To investigate whether the 2 subtypes of advanced age-related macular degeneration (AMD), choroidal neovascularization (CNV), and geographic atrophy (GA) segregate separately in families and to identify which genetic variants are associated with these 2 subtypes. DESIGN Sibling correlation study and genome-wide association study (GWAS). PARTICIPANTS For the sibling correlation study, 209 sibling pairs with advanced AMD were included. For the GWAS, 2594 participants with advanced AMD subtypes and 4134 controls were included. Replication cohorts included 5383 advanced AMD participants and 15 240 controls. METHODS Participants had the AMD grade assigned based on fundus photography, examination, or both. To determine heritability of advanced AMD subtypes, a sibling correlation study was performed. For the GWAS, genome-wide genotyping was conducted and 6 036 699 single nucleotide polymorphisms (SNPs) were imputed. Then, the SNPs were analyzed with a generalized linear model controlling for genotyping platform and genetic ancestry. The most significant associations were evaluated in independent cohorts. MAIN OUTCOME MEASURES Concordance of advanced AMD subtypes in sibling pairs and associations between SNPs with GA and CNV advanced AMD subtypes. RESULTS The difference between the observed and expected proportion of siblings concordant for the same subtype of advanced AMD was different to a statistically significant degree (P = 4.2 × 10(-5)), meaning that in siblings of probands with CNV or GA, the same advanced subtype is more likely to develop. In the analysis comparing participants with CNV to those with GA, a statistically significant association was observed at the ARMS2/HTRA1 locus (rs10490924; odds ratio [OR], 1.47; P = 4.3 × 10(-9)), which was confirmed in the replication samples (OR, 1.38; P = 7.4 × 10(-14) for combined discovery and replication analysis). CONCLUSIONS Whether CNV versus GA develops in a patient with AMD is determined in part by genetic variation. In this large GWAS meta-analysis and replication analysis, the ARMS2/HTRA1 locus confers increased risk for both advanced AMD subtypes, but imparts greater risk for CNV than for GA. This locus explains a small proportion of the excess sibling correlation for advanced AMD subtype. Other loci were detected with suggestive associations that differ for advanced AMD subtypes and deserve follow-up in additional studies.

BEVACIZUMAB (AVASTIN) AND RANIBIZUMAB (LUCENTIS) FOR CHOROIDAL NEOVASCULARIZATION IN MULTIFOCAL CHOROIDITIS

Background: Multifocal choroiditis (MFC) is an inflammatory condition, occasionally associated with choroidal neovascularization (CNV). Bevacizumab (Avastin) and ranibizumab (Lucentis) are therapies that target vascular endothelial growth factor. Bevacizumab and ranibizumab have been used successfully to treat CNV in age-related and myopic macular degeneration. Purpose: To describe the treatment of MFC-associated CNV with intravitreal bevacizumab and/or ranibizumab. Design: Retrospective interventional case series. Participants: Six eyes of five patients with MFC-associated CNV were treated with intravitreal bevacizumab and/or ranibizumab. Main Outcome Measures: Visual acuity at 1, 3, and 6 months after the initial injection. Results: Previous therapies (number of eyes treated) included sub-Tenons corticosteroids (2), intravitreal corticosteroids (1), photodynamic therapy (1), and thermal laser (1). The mean number (range) of antivascular endothelial growth factor injections per eye was 2.3 (1–6). The mean duration (range) of follow-up per patient was 41.5 (25–69) weeks. Five of six eyes improved to 20/30 acuity or better at 6 months. One eye suffered a subfoveal rip of the retinal pigment epithelium with 20/400 acuity. There was a qualitative decrease in clinical and angiographic evidence of CNV. Conclusions: Bevacizumab and ranibizumab were effective at improving visual acuity over 6 months in a small series of patients with MFC-associated CNV. Tears of the retinal pigment epithelium may occur after intravitreal antivascular endothelial growth factor therapy in MFC-associated CNV.

G1961E mutant allele in the Stargardt disease gene ABCA4 causes bull's eye maculopathy

The aim of this study was to characterize the pathological and functional consequences of the G1961E mutant allele in the Stargardt disease gene ABCA4. Data from 15 patients were retrospectively reviewed and all the patients had at least one G1961E mutation. Comprehensive ophthalmic examination, full-field and pattern electroretinograms, and fundus autofluorescence (FAF) imaging were performed on all patients. Microperimetry, spectral-domain optical coherence tomography (OCT), and fluorescein angiography were performed in selected cases. Genetic screening was performed using the ABCR400 micro-array that currently detects 496 distinct ABCA4 variants. All patients had normal full-field scotopic and photopic electroretinograms (ERGs) and abnormal pattern electroretinograms (PERGs) performed on both eyes, and all the fundi had bulls eye maculopathy without retinal flecks on FAF. On OCT, 1 patient had disorganization of photoreceptor outer segment, 2 had outer nuclear layer (ONL) thinning likely due to photoreceptor atrophy proximal to the foveal center, and 3 had additional retinal pigment epithelium (RPE) atrophy. On microperimetry, 6 patients had eccentric superior fixation and amongst this group, 5 had an absolute scotoma in the foveal area. DNA analysis revealed that 3 patients were homozygous G1961E/G1961E and the rest were compound heterozygotes for G1961E and other ABCA4 mutations. The G1961E allele in either homozygosity or heterozygosity is associated with anatomical and functional pathologies limited to the parafoveal region and a trend to delayed onset of symptoms, relative to other manifestations of ABCA4 mutations. Our observations support the hypothesis that the G1961E allele contributes to localized macular changes rather than generalized retinal dysfunction, and is a cause of bulls eye maculopathy in either the homozygosity or heterozygosity state. In addition, genetic testing provides precise diagnosis of the underlying maculopathy, and current non-invasive imaging techniques could be used to detect photoreceptor damage at the earliest clinical onset of the disease.