Disease-specific variant interpretation highlighted the genetic findings in 2325 Japanese patients with retinitis pigmentosa and allied diseases.

BACKGROUND: As gene-specific therapy for inherited retinal dystrophy (IRD) advances, unified variant interpretation across institutes is becoming increasingly important. This study aims to update the genetic findings of 86 retinitis pigmentosa (RP)-related genes in a large number of Japanese patients with RP by applying the standardised variant interpretation guidelines for Japanese patients with IRD (J-IRD-VI guidelines) built upon the American College of Medical Genetics and Genomics and the Association for Molecular Pathology rules, and assess the contribution of these genes in RP-allied diseases. METHODS: We assessed 2325 probands with RP (n=2155, including n=1204 sequenced previously with the same sequencing panel) and allied diseases (n=170, newly analysed), including Usher syndrome, Leber congenital amaurosis and cone-rod dystrophy (CRD). Target sequencing using a panel of 86 genes was performed. The variants were interpreted according to the J-IRD-VI guidelines. RESULTS: A total of 3564 variants were detected, of which 524 variants were interpreted as pathogenic or likely pathogenic. Among these 524 variants, 280 (53.4%) had been either undetected or interpreted as variants of unknown significance or benign variants in our earlier study of 1204 patients with RP. This led to a genetic diagnostic rate in 38.6% of patients with RP, with EYS accounting for 46.7% of the genetically solved patients, showing a 9% increase in diagnostic rate from our earlier study. The genetic diagnostic rate for patients with CRD was 28.2%, with RP-related genes significantly contributing over other allied diseases. CONCLUSION: A large-scale genetic analysis using the J-IRD-VI guidelines highlighted the population-specific genetic findings for Japanese patients with IRD; these findings serve as a foundation for the clinical application of gene-specific therapies.

Association Between Torque Teno Virus and Systemic Immunodeficiency in Patients With Uveitis With a Suspected Infectious Etiology.

PURPOSE: To determine the correlation between the presence of torque teno virus (TTV) in the aqueous humor of patients with uveitis and clinical information, including immunodeficiency history. DESIGN: Multicenter, retrospective, cross-sectional study. METHODS: Fifty-eight patients with uveitis with a suspected infectious etiology and 24 controls with cataract or age-related macular degeneration were included. We used quantitative polymerase chain reaction to test all subjects for TTV and multiplex polymerase chain reaction to test uveitis subjects for common ocular pathogens. When possible, both serum and aqueous humor samples were tested. Ocular TTV positivity was compared with age, sex, and a history of systemic immunodeficiency with logistic analysis. RESULTS: Ocular TTV positivity was found in 23%, 11%, and 0% of patients with herpetic uveitis, nonherpetic uveitis, and controls, respectively. Among patients with herpes infection, positivity for ocular TTV was found in 43%, 8%, 14%, and 50% of patients with cytomegalovirus retinitis, iridocyclitis, acute retinal necrosis, and Epstein-Barr virus-positive uveitis, respectively. Patients with cytomegalovirus retinitis showed a significantly higher rate of ocular TTV infection than controls (P = .008). Serum analysis revealed TTV positivity in 90% of patients with uveitis and in 100% of controls. Age- and gender-adjusted logistic analysis revealed a correlation between ocular TTV positivity and systemic immunodeficiency (P = .01), but no correlations between ocular TTV and age, gender, or viral pathogenic type. CONCLUSIONS: These findings suggest that positivity for ocular TTV was correlated with a clinical history of systemic immunodeficiency.

Cost-effective sequence analysis of 113 genes in 1,192 probands with retinitis pigmentosa and Leber congenital amaurosis.

Introduction: Retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA) are two groups of inherited retinal diseases (IRDs) where the rod photoreceptors degenerate followed by the cone photoreceptors of the retina. A genetic diagnosis for IRDs is challenging since >280 genes are associated with these conditions. While whole exome sequencing (WES) is commonly used by diagnostic facilities, the costs and required infrastructure prevent its global applicability. Previous studies have shown the cost-effectiveness of sequence analysis using single molecule Molecular Inversion Probes (smMIPs) in a cohort of patients diagnosed with Stargardt disease and other maculopathies. Methods: Here, we introduce a smMIPs panel that targets the exons and splice sites of all currently known genes associated with RP and LCA, the entire RPE65 gene, known causative deep-intronic variants leading to pseudo-exons, and part of the RP17 region associated with autosomal dominant RP, by using a total of 16,812 smMIPs. The RP-LCA smMIPs panel was used to screen 1,192 probands from an international cohort of predominantly RP and LCA cases. Results and discussion: After genetic analysis, a diagnostic yield of 56% was obtained which is on par with results from WES analysis. The effectiveness and the reduced costs compared to WES renders the RP-LCA smMIPs panel a competitive approach to provide IRD patients with a genetic diagnosis, especially in countries with restricted access to genetic testing.

Genome-Wide Association Study of Age-Related Macular Degeneration Reveals 2 New Loci Implying Shared Genetic Components with Central Serous Chorioretinopathy.

PURPOSE: To investigate the genetic architecture of age-related macular degeneration (AMD) in a Japanese population. DESIGN: Genome-wide association study (GWAS). PARTICIPANTS: Three thousand seven hundred seventy-two patients with AMD and 16 770 control participants from the Japanese population were enrolled in the association analyses. METHODS: We conducted a meta-analysis of 2 independent GWASs that included a total of 2663 patients with AMD and 9471 control participants using the imputation reference panel for genotype imputation specified for the Japanese population (n = 3541). A replication study was performed using an independent set of 1109 patients with AMD and 7299 control participants. MAIN OUTCOME MEASURES: Associations of genetic variants with AMD. RESULTS: A meta-analysis of the 2 GWASs identified 6 loci significantly associated with AMD (P < 5.0 × 10-8). Of these loci, 4 were known to be associated with AMD (CFH, C2/FB, TNFRSF10A, and ARMS2), and 2 were novel (rs4147157 near WBP1L and rs76228488 near GATA5). The newly identified associations were confirmed in a replication study (P < 0.01). After the meta-analysis of all datasets, we observed strong associations in these loci (P = 1.88 × 10-12 and P = 1.35 × 10-9 for meta-analysis for rs4147157 and rs76228488, respectively). When we looked up the associations in the reported central serous chorioretinopathy (CSC) GWAS conducted in the Japanese population, both loci were associated significantly with CSC (P = 4.86 × 10-3 and P = 4.28 × 10-3 for rs4147157 and rs76228488, respectively). We performed a genetic colocalization analysis for these loci and estimated that the posterior probabilities of shared causal variants between AMD and CSC were 0.39 and 0.60 for WBP1L and GATA5, respectively. Genetic correlation analysis focusing on the epidemiologically suggested clinical risk factors implicated shared polygenic architecture between AMD and smoking cessation (rg [the measure of genetic correlation] = -0.33; P = 0.01; false discovery rate, 0.099). CONCLUSIONS: Our findings imply shared genetic components conferring the risk of both AMD and CSC. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found after the references.

Dimethyl Fumarate Protects Retinal Pigment Epithelium from Blue Light-Induced Oxidative Damage via the Nrf2 Pathway.

The purpose of this study is to investigate the protective effect of dimethyl fumarate (DMF), the methyl-ester of fumaric acid, against blue-light (BL) exposure in retinal pigment epithelial (RPE) cells. ARPE-19 cells, a human RPE cell line, were cultured with DMF followed by exposure to BL. Reactive oxygen species (ROS) generation, cell viability, and cell death rate were determined. Real-time polymerase chain reaction and Western blotting were performed to determine the change in nuclear factor (erythroid-derived)-like 2 (NRF2) expression. Twenty-seven inflammatory cytokines in the supernatant of culture medium were measured. BL exposure induced ROS generation in ARPE-19 cells, which DMF alleviated in a concentration-dependent manner. BL exposure increased the ARPE-19 cell death rate, which DMF alleviated. BL exposure induced ARPE-19 cell apoptosis, again alleviated by DMF. Under BL exposure, DMF increased the NRF2 mRNA level and promoted NRF2 expression in the nucleus. BL also strongly increased interleukin (IL)-1ß and fibroblast growth factor (FGF) expression. BL strongly induced RPE cell damage with apoptotic change while DMF mainly reduced inflammation in BL-induced RPE damage, resulting in blockade of cell death. DMF has a protective effect in RPE cells against BL exposure via activation of the NRF2 pathway.

Three cases of brolucizumab-associated retinal vasculitis treated with systemic and local steroid therapy.

PURPOSE: To describe three Japanese cases of retinal vasculitis that occurred following intravitreal brolucizumab injections and the systemic and local steroid treatment administered. CASES: Three patients developed intraocular inflammation (IOI) and retinal vasculitis following the first injection of brolucizumab for age-related macular degeneration. For two eyes, monthly aflibercept injections did not control exudation, and therapy was changed to brolucizumab; one eye was treatment-naïve. All three patients noticed blurry vision and floaters 11-18 days after brolucizumab injections, and the treated eyes exhibited anterior chamber cells, fine keratic precipitates, vitreous cells, and vitreous haze. Ultra-widefield color images of the fundus showed retinal hemorrhage in the peripheral retina and, in two cases vascular sheathing. Ultra-widefield fluorescein angiography (FA) showed segmental vascular leakage in all eyes and leakage from the optic disc in two eyes. Vascular filling defects were noted in the peripheral retinae of two eyes. Brolucizumab-associated retinal vasculitis was diagnosed, and treated with 30 mg/day of oral prednisolone, subtenon triamcinolone acetonide injection (20 mg/0.5 ml), and 0.1% betamethasone sodium phosphate solution. After 1 week, color fundus images and FA showed improvements in vascular sheathing, leakage from retinal vessels, and optic disc leakage, but the vascular filling defects remained. Visual acuity was restored in all three eyes 6 weeks after the onset. CONCLUSION: Brolucizumab-associated IOI, including retinal vasculitis and retinal occlusion, is a rare but important adverse event that can cause severe vision loss. Prompt diagnosis with FA and treatment with systemic or local steroids should be considered.

INTRAOPERATIVE AND POSTOPERATIVE MONITORING OF AUTOLOGOUS NEUROSENSORY RETINAL FLAP TRANSPLANTATION FOR A REFRACTORY MACULAR HOLE ASSOCIATED WITH HIGH MYOPIA.

PURPOSE: To describe the intraoperative and postoperative morphological and functional outcomes after autologous neurosensory retinal flap transplantation (ART) for a high myopia-related refractory macular hole (MH). METHODS: This prospective interventional study enrolled five eyes of five patients (age range 54-84 years) with highly myopic refractory MHs who underwent ART. All cases were evaluated with intraoperative optical coherence tomography and postoperative optical coherence tomography, optical coherence tomography angiography, and microperimetry for at least 6 months postoperatively. RESULTS: Intraoperatively, the MH was covered by an ART flap with a persistent small subretinal space that was filled with the ART flap after 4 days to 6 days. Optical coherence tomography discriminated the original from the transplanted retina. The mean basal diameter of the original MH decreased from 1,504 ± 684 µm preoperatively to 1,111 ± 356 µm postoperatively. The best-corrected visual acuity improved in two cases, was stable in two cases, and deteriorated in one case. Microperimetry demonstrated no obvious postoperative changes in the fixation points and the absolute scotoma corresponding to the base of MHs with chorioretinal atrophy. In two eyes, choroidal neovascularization developed beneath the transplanted retinas. CONCLUSION: Transplanted tissue was in a fixed position by 1 week postoperatively with a decreased diameter of the original MH. Postoperative fixation points were on the original retina at the MH edge. Because choroidal neovascularization may develop, detailed monitoring is required.

Changes in retinochoroidal thickness after vitrectomy for proliferative diabetic retinopathy.

PURPOSE: To evaluate the changes in the peripheral retinochoroidal thickness after pars plana vitrectomy (PPV) with scatter photocoagulation for proliferative diabetic retinopathy (PDR). METHODS: Small gauge PPV was performed on 22 eyes with PDR with scatter photocoagulation, and on 32 eyes with an epiretinal membrane (ERM) without photocoagulation as control. The peripheral retinochoroidal thickness was measured at 5 mm from the limbus in the four quadrants using anterior segment optical coherence tomography preoperatively, and 3 days and 1 and 2 weeks after the surgery. In eyes with a peripheral choroidal detachment, the retinochoroidal thickness and the height of choroidal detachment were measured separately. The total peripheral thickness was defined as the sum of retinochoroidal thickness and the height of choroidal detachment. RESULTS: A significant larger number of eyes developed a choroidal detachment in the PDR group than in the ERM group 3 days after surgery (P < 0.001). The total peripheral choroidal thickness 3 days after surgery was significantly thicker than that before surgery in the PDR group (P = 0.009). The increase in the total peripheral thickness in the PDR group was significantly greater than that in the ERM group at 3 days after surgery (P = 0.007). The number of photocoagulation burns was significantly and positively correlated with the total peripheral thickness (r = 0.57, P = 0.006). CONCLUSIONS: We conclude that the transient thickening of the total peripheral thickness in early postoperative stage for PDR was due to the intraoperative scatter photocoagulation.

Electroretinograms and level of aqueous vascular endothelial growth factor in eyes with hemicentral retinal vein occlusion or branch retinal vein occlusion.

PURPOSE: Hemicentral retinal vein occlusion (hCRVO) is a disease related to CRVO but not to branch retinal vein occlusion (BRVO). We reported a significant correlation between aqueous vascular endothelial growth factor (VEGF) levels and the implicit time of 30-Hz flicker electroretinogram (ERG) in CRVO eyes. The purpose of this study was to compare aqueous VEGF levels and ERG components between hCRVO and BRVO eyes. METHODS: The medical records of patients with macular edema secondary to hCRVO (12 eyes) or BRVO (16 eyes) and received an intravitreal injection of bevacizumab (IVB) at the Nagoya University Hospital from July 2009 to May 2013 were reviewed. Full-field ERGs were recorded before the IVB. Aqueous humor was collected just before the IVB to measure VEGF concentration. Differences in aqueous VEGF level and ERG components between hCRVO and BRVO eyes were determined. RESULTS: Mean aqueous VEGF concentration in hCRVO eyes was significantly higher than that in BRVO eyes (504 vs. 148 pg/ml, P < 0.05). The implicit time of 30-Hz flicker ERG was significantly longer in hCRVO than in BRVO eyes (33.5 vs. 29.8 ms, P < 0.01). CONCLUSION: The significant difference in VEGF levels in aqueous and implicit times of 30-Hz flicker ERG suggest that retinal ischemia is more manifest in hCRVO than in BRVO eyes.

Changes in vitreous temperature during intravitreal surgery.

PURPOSE: To determine how different intraoperative surgical procedures affect the midvitreous temperature. METHODS: The vitreous temperatures were monitored continuously with an intravitreal thermocouple in 87 eyes of 81 cases undergoing vitrectomy. Thirty-three eyes had diabetic retinopathy (DR), 35 eyes had an epiretinal membrane, and 19 eyes had an idiopathic macular hole. In eyes with DR, the correlation between the number of photocoagulations (PCs) and the change in temperature was analyzed. The temperature was also recorded before and after combined phacoemulsification and aspiration (PEA) and vitrectomy in 10 eyes. RESULTS: The average midvitreal temperature before the vitrectomy was 33.0 ± 1.3°C, 30.7 ± 1.7°C after core vitrectomy, 32.9 ± 1.3°C after membrane peeling, and 29.2 ± 1.4°C after peripheral vitrectomy. The temperature before PC was 29.8 ± 1.3°C, and it increased to 31.5 ± 1.9°C post-PC. The differences in the temperatures between consecutive procedures were significant (P < 0.01, respectively, Wilcoxon signed-rank test). The difference in the temperatures of the same procedures among the different diseases was not significant except after membrane peeling. A significant correlation was detected between the number of PCs and the duration of the PCs, and between the duration of PCs and the change in vitreous temperature after PC (r = 0.719, P = 0.0010, and r = 0.800, P = 0.0002, respectively, Spearman's rank correlation coefficient test). The temperature after PEA decreased significantly by 2.3°C. CONCLUSIONS: Our results showed that vitreous temperatures vary during different vitrectomy procedures.

Age-dependent alteration of intraocular soluble heparan sulfate levels and its implications for proliferative diabetic retinopathy.

PURPOSE: To assess the relationship between intraocular soluble heparan sulfate (HS) concentration and age in subjects with and without diabetic retinopathy. METHODS: Vitreous from subjects with idiopathic maculopathies (n=17), i.e., macula hole or epiretinal membrane, or nonproliferative diabetic retinopathy (non-PDR; n=5) and aqueous humor from subjects with PDR (n=16), non-PDR (n=7), or cataracts (n=15) was collected. The levels of HS and vascular endothelial growth factor (VEGF) were measured using enzyme-linked immunosorbent assay. Concentrations of sulfated glycosaminoglycan were determined through dimethylmethylene blue-based assay. The effect of the vitreal HS level on the binding of exogenous VEGF to surface-bound heparin was determined in vitro. RESULTS: The level of HS in vitreous samples from subjects with idiopathic maculopathies increased concomitantly with age (p=0.020, R²=0.327). Meanwhile, HS levels in aqueous humor were lower in PDR subjects than in non-PDR (p=0.003) and cataract subjects (p=0.007). However, the PDR subjects were significantly younger than the non-PDR subjects (p<0.001) or cataract subjects (p<0.001). When the three groups were controlled for age, the levels of HS glycosaminoglycans were no longer different between the three (p=0.247). The increasing level of HS or sulfated glycosaminoglycan in the vitreous was associated with its increased inhibitory effect on interaction between VEGF and surface heparin in vitro (p=0.014, R²=0.377). CONCLUSIONS: The HS level of the intraocular fluid increased with age. The possible link between low HS in intraocular fluid and increased localization of VEGF at the retinal surface may provide one explanation for the higher susceptibility of younger subjects with diabetes mellitus to developing PDR.

Influence of preoperative intravitreal bevacizumab on visual function in eyes with proliferative diabetic retinopathy.

PURPOSE: Although bevacizumab, a humanized monoclonal antibody against vascular endothelial growth factor (VEGF), is effective in treating ocular neovascularization, there are some concerns about whether blocking VEGF might be harmful to retinal neurons. The purpose of this study was to evaluate the effects of preoperative intravitreal bevacizumab (IVB) on the visual function of eyes with proliferative diabetic retinopathy (PDR). METHODS: Thirty eyes of 23 patients (13 men and 10 women) with PDR who were treated at the Nagoya University Hospital from November 2006 to October 2009 were studied. All of the eyes were treated with 1.25 mg/0.05 ml of IVB 2-8 days before the vitrectomy. The protocol was approved by the Institutional Review Board of Nagoya University, and a written informed consent was obtained from each patient. All of the eyes had an active proliferative membrane with vitreous hemorrhage, but the fundus was visible. The mean age of the patients was 41.6 ± 10 years (range, 27-59), and the mean follow-up period was 9.7 ± 8.9 months (range, 1-24) after the vitrectomy. The visual acuity (VA) was measured, the visual fields were determined by Goldmann perimetery, and full-field electroretinograms (ERGs) were recorded before IVB, and before and after the vitrectomy. Fluorescein angiography was also performed before and after IVB. The area of the visual field was measured using a computer software (Scion Image). RESULTS: All eyes showed a regression of the new vessels and a reduction of fluorescent leakage from the new vessels after IVB. In addition, there was less bleeding during the removal of the proliferative membrane. The average VA was improved postoperatively from 20/250 to 20/70. However, there was no significant change in the amplitudes of the a- (from 261.4 to 259.2 µV) and b-waves (from 256.9 to 253.3 µV) of the ERGs, and there was no significant change in the visual field area after the surgery (from 8,322.5 to 7,496.3 degrees(2)). No significant ocular or systemic adverse events were observed. CONCLUSION: IVB-assisted vitrectomy led to an improvement of the VA in eyes with PDR without significant adverse events. There was no change in the visual fields and ERGs. Although only a small number of patients were studied, we conclude that IVB is most likely not harmful to retinal neurons if bevacizumab is washed out in less than 1 week. In addition, preoperative IVB made the surgery much easier by decreasing the activity of new vessels.

Significant correlation between electroretinogram parameters and ocular vascular endothelial growth factor concentration in central retinal vein occlusion eyes.

PURPOSE: Central retinal vein occlusion (CRVO) leads to retinal ischemia, which then induces an upregulation of vascular endothelial growth factor (VEGF). The aim of this study was to determine whether a significant correlation exists between the ocular VEGF levels and the amplitudes and implicit times of different components of the electroretinogram (ERG) in eyes with a CRVO. METHODS: The medical records of the 20 consecutive patients who had macular edema secondary to CRVO and were examined at the Nagoya University Hospital from November 2008 to February 2010 were reviewed. Because all the patients were scheduled to receive an intravitreal injection of bevacizumab (IVB), it was possible to collect samples of the aqueous humor before the IVB. The correlation between the different components of the ERGs and the VEGF concentration in the aqueous was determined. RESULTS: The mean VEGF concentration of the aqueous humor was 416 pg/mL with a range of 100-1260 pg/mL. The b/a ratio of the single flash ERGs (P = 0.049; ρ = -0.45), implicit times of the cone a-wave (P = 0.028; ρ = 0.50), cone b-wave (P = 0.0059; ρ = 0.63), and 30 Hz flicker ERGs (P = 0.0058; ρ = 0.63) were significantly correlated with the VEGF concentration in the aqueous. CONCLUSIONS: The significant correlations between the different components of the ERGs and the aqueous VEGF concentration indicate that full-field ERGs can be used to detect the CRVO patients at a high risk of developing neovascularization of the iris.

Regression of macular edema secondary to branch retinal vein occlusion during anti-TNF-alpha therapy for rheumatoid arthritis.

A patient with macular edema secondary to a branch retinal vein occlusion (BRVO) was treated with intravenous injections of infliximab, an antitumor necrosis factor (TNF)-alpha antibody, for her rheumatoid arthritis (RA). Before the injection, the thickness of the right fovea, determined by optical coherent tomography, was 629 mum and the best-corrected visual acuity (BCVA) was 20/50. After eight injections of infliximab and 10 months after the first injection, her foveal thickness was decreased to 293 mum and the visual acuity improved to 20/20. There was no recurrence of macular edema during the infliximab injections. However, the infliximab injection was stopped because the patient developed pneumonia. Eight months after stopping the infliximab injection, her foveal thickness increased to 494 mum. To treat the RA, her orthopedists began weekly subcutaneous injections of etanercept, a fusion protein of a section of the TNF receptor and immunoglobulin. Five months later, the foveal thickness had decreased to 260 mum, and the visual acuity remained at 20/25(+). Because TNF-alpha is known to break down the blood-retinal barrier, the improvements in our case suggest that TNF-alpha plays a role in the pathogenesis of macular edema in some patients with BRVO.