Tetsuhiro Yasuma

Whole genome sequencing in patients with retinitis pigmentosa reveals pathogenic DNA structural changes and NEK2 as a new disease gene

Significance Retinitis pigmentosa (RP) is a genetic disease that causes progressive blindness and that is caused by mutations in more than 50 genes. Conventional methods for identification of both RP mutations and novel RP genes involve the screening of DNA sequences spanning coding exons. In our work, we conversely test the use of whole genome sequencing, a technique that takes into account all variants from both the coding and noncoding regions of the human genome. In our approach, we identify a number of unique RP mutations, a previously undescribed disease gene, as well as pathogenic structural DNA rearrangements originating in introns. We performed whole genome sequencing in 16 unrelated patients with autosomal recessive retinitis pigmentosa (ARRP), a disease characterized by progressive retinal degeneration and caused by mutations in over 50 genes, in search of pathogenic DNA variants. Eight patients were from North America, whereas eight were Japanese, a population for which ARRP seems to have different genetic drivers. Using a specific workflow, we assessed both the coding and noncoding regions of the human genome, including the evaluation of highly polymorphic SNPs, structural and copy number variations, as well as 69 control genomes sequenced by the same procedures. We detected homozygous or compound heterozygous mutations in 7 genes associated with ARRP (USH2A, RDH12, CNGB1, EYS, PDE6B, DFNB31, and CERKL) in eight patients, three Japanese and five Americans. Fourteen of the 16 mutant alleles identified were previously unknown. Among these, there was a 2.3-kb deletion in USH2A and an inverted duplication of ∼446 kb in EYS, which would have likely escaped conventional screening techniques or exome sequencing. Moreover, in another Japanese patient, we identified a homozygous frameshift (p.L206fs), absent in more than 2,500 chromosomes from ethnically matched controls, in the ciliary gene NEK2, encoding a serine/threonine-protein kinase. Inactivation of this gene in zebrafish induced retinal photoreceptor defects that were rescued by human NEK2 mRNA. In addition to identifying a previously undescribed ARRP gene, our study highlights the importance of rare structural DNA variations in Mendelian diseases and advocates the need for screening approaches that transcend the analysis of the coding sequences of the human genome.

Intravitreal injection of bevacizumab for macular edema secondary to branch retinal vein occlusion:results after 12 months and multiple regression analysis.

Purpose: To evaluate the 12-month follow-up results of intravitreal bevacizumab therapy for macular edema secondary to branch retinal vein occlusion and to identify the pretreatment factors that were associated with an improvement of the final visual outcome. Methods: Fifty eyes of 50 patients with macular edema secondary to branch retinal vein occlusion received an injection of 1.25 mg/0.05 mL bevacizumab. Additional injections were done when recurrence of macular edema occurred or the treatment was not effective. The best-corrected visual acuity and foveal thickness were measured. Stepwise multiple regression analyses were also performed. Results: The mean logarithm of the minimum angle of resolution visual acuity improved significantly from 0.53 to 0.26, and the mean foveal thickness decreased significantly from 523 to 305 &mgr;m during the 12-month follow-up period. The mean number of injections was 2.0 (range, 1–4). Stepwise multiple regression analyses showed that younger patients had both better visual acuity at 12 months and greater improvement of visual acuity during 12 months. In addition, better pretreatment visual acuity was associated with better visual acuity at 12 months but with less improvement of the visual acuity. Conclusion: Intravitreal bevacizumab therapy can be a long-term effective treatment for macular edema secondary to branch retinal vein occlusion.

TLR-Independent and P2X7-Dependent Signaling Mediate Alu RNA-Induced NLRP3 Inflammasome Activation in Geographic Atrophy

PURPOSE Accumulation of Alu RNA transcripts due to DICER1 deficiency in the retinal pigmented epithelium (RPE) promotes geographic atrophy. Recently we showed that Alu RNA activated the NLRP3 inflammasome, leading to RPE cell death via interleukin-18 (IL-18)-mediated MyD88 signaling. However, the molecular basis for NLRP3 inflammasome activation by Alu RNA is not well understood. We sought to decipher the key signaling events triggered by Alu RNA that lead to priming and activation of the NLRP3 inflammasome and, ultimately, to RPE degeneration by investigating the roles of the purinoreceptor P2X7, the transcription factor NF-κB, and the Toll-like receptors (TLRs) in these processes. METHODS Human and mouse RPE cells were transfected with a plasmid encoding an Alu element (pAlu) or an in vitro-transcribed Alu RNA. Inflammasome priming was assessed by measuring NLRP3 and IL18 mRNA levels by real-time quantitative PCR. Using immunoblotting, we assessed NF-κB activation by monitoring phosphorylation of its p65 subunit, and inflammasome activation by monitoring caspase-1 cleavage into its active form. RPE degeneration was induced in mice by subretinal transfection of pAlu or Alu RNA. The NF-κB inhibitor BAY 11-7082, the P2X7 receptor antagonist A-740003, and the NLRP3 inflammasome inhibitor glyburide were delivered by intravitreous injections. We studied wild-type (WT) C57Bl/6J, P2rx7(-/-), Nfkb1(-/-), and Tlr23479(-/-) mice. RPE degeneration was assessed by fundus photography and zonula occludens-1 (ZO-1) staining of mouse RPE. RESULTS Alu RNA-induced NF-κB activation, independent of TLR-1, -2, -3, -4, -6, -7, and -9 signaling, was required for priming the NLRP3 inflammasome. Nfkb1(-/-) and P2rx7(-/-) mice and WT mice treated with the pharmacological inhibitors of NF-κB, P2X7, or NLRP3, were protected against Alu RNA-induced RPE degeneration. CONCLUSIONS NF-κB and P2X7 are critical signaling intermediates in Alu RNA-induced inflammasome priming and RPE degeneration. These molecules are novel targets for rational drug development for geographic atrophy.

DICER1/Alu RNA dysmetabolism induces Caspase-8–mediated cell death in age-related macular degeneration

Significance Geographic atrophy is a late stage of age-related macular degeneration (AMD) that causes blindness in millions worldwide characterized by death of the retinal pigmented epithelium (RPE). We previously reported that RPE death is due to a deficiency in the enzyme DICER1, which leads to accumulation of toxic Alu RNA. We also demonstrated that Alu RNA causes RPE death by activating an immune platform called the NLRP3 inflammasome. However, the precise mechanisms of RPE death in this disease remained unresolved. The present study indicates that Alu RNA induces RPE death by activating the enzyme Caspase-8 downstream of inflammasome activation and that blocking Caspase-8 rescues RPE degeneration. This implicates apoptosis as the cell death pathway responsible for Alu RNA cytotoxicity, and these findings provide new potential therapeutic targets for this disease. Geographic atrophy, an advanced form of age-related macular degeneration (AMD) characterized by death of the retinal pigmented epithelium (RPE), causes untreatable blindness in millions worldwide. The RPE of human eyes with geographic atrophy accumulates toxic Alu RNA in response to a deficit in the enzyme DICER1, which in turn leads to activation of the NLRP3 inflammasome and elaboration of IL-18. Despite these recent insights, it is still unclear how RPE cells die during the course of the disease. In this study, we implicate the involvement of Caspase-8 as a critical mediator of RPE degeneration. Here we show that DICER1 deficiency, Alu RNA accumulation, and IL-18 up-regulation lead to RPE cell death via activation of Caspase-8 through a Fas ligand-dependent mechanism. Coupled with our observation of increased Caspase-8 expression in the RPE of human eyes with geographic atrophy, our findings provide a rationale for targeting this apoptotic pathway in this disease.

Iron Toxicity in the Retina Requires Alu RNA and the NLRP3 Inflammasome

Excess iron induces tissue damage and is implicated in age-related macular degeneration (AMD). Iron toxicity is widely attributed to hydroxyl radical formation through Fentons reaction. We report that excess iron, but not other Fenton catalytic metals, induces activation of the NLRP3 inflammasome, a pathway also implicated in AMD. Additionally, iron-induced degeneration of the retinal pigmented epithelium (RPE) is suppressed in mice lacking inflammasome components caspase-1/11 or Nlrp3 or by inhibition of caspase-1. Iron overload increases abundance of RNAs transcribed from short interspersed nuclear elements (SINEs): Alu RNAs and the rodent equivalent B1 and B2 RNAs, which are inflammasome agonists. Targeting Alu or B2 RNA prevents iron-induced inflammasome activation and RPE degeneration. Iron-induced SINE RNA accumulation is due to suppression of DICER1 via sequestration of the co-factor poly(C)-binding protein 2 (PCBP2). These findings reveal an unexpected mechanism of iron toxicity, with implications for AMD and neurodegenerative diseases associated with excess iron.

C9-R95X Polymorphism in Patients with Neovascular Age-Related Macular Degeneration

PURPOSE A non-sense mutation at codon 95 in the gene encoding complement factor C9 (C9-R95X) is found most frequently among Japanese. The authors investigated the association between C9-R95X and Japanese patients with neovascular age-related macular degeneration (AMD) and polypoidal choroidal vasculopathy (PCV). METHODS The presence of the C9-R95X polymorphism was assessed by direct sequencing in Japanese patients with either PCV (n = 105) or neovascular AMD (n = 198) and 396 control subjects. Multivariate regression analyses were conducted. Photocoagulation was applied in the eyes of mice with a heterozygous defect in the C3 gene and control wild-type mice. Photocoagulation was also applied to wild-type mice before either anti-C9 antibody or isotype IgG was injected into the eyes. The eyes were collected later for measurement of vascular endothelial growth factor (VEGF) and histological evaluation of choroidal neovascularization (CNV). RESULTS The frequency of those with one or two C9-R95X variants was lower in neovascular AMD (2.02%) than in PCV (5.71%) and controls (6.05%). The presence of C9-R95X conferred a 4.7-fold reduction (95% confidence interval, 1.2-18.1; P = 0.021) in the risk for neovascular AMD after adjusting for the major AMD risk factors. A heterozygous defect in the C3 gene was associated with the reduced growth of laser-induced CNV, as was intraocular injection of anti-C9 antibody. This reduced CNV growth was accompanied by a decreased level of secreted VEGF in the intraocular fluid. CONCLUSIONS These findings support the notion that the haploinsufficiency of C9, a terminal complement complex component, engenders reduced intraocular secretion of VEGF and decreased risk for CNV development.

IL-18 is not therapeutic for neovascular age-related macular degeneration

Up to 50 million people worldwide are afflicted with the devastating blinding disease age-related macular degeneration (AMD)1–3. The vast majority of patients have the currently untreatable “dry” or atrophic form of AMD, characterized by NLRP3 inflammasome-driven degeneration of the retinal pigment epithelium (RPE) supportive cell layer4,5. Blockade of the NLRP3 inflammasome is a next-generation therapeutic target in dry AMD; however, it was recently reported that inflammasome-mediated production of IL18 potentially safeguards the retina against the other, often more visually devastating form of AMD, for which dry AMD patients are at greatly increased risk of developing, known as choroidal neovascularization (CNV)6. Therefore, it is essential, prior to initiating inflammasome-targeting clinical trials, to directly and rigorously assess whether modulating IL18 or the NLRP3 inflammasome affects CNV and RPE cell health.

Axial length increases and related changes in highly myopic normal eyes with myopic complications in fellow eyes.

Purpose: To determine whether the axial length (AL) in highly myopic normal adult eyes with myopic complications in the fellow eyes increases significantly during a 1-year interval and to investigate the relationships between the changes in the AL and different ocular parameters. Methods: The medical records of 20 highly myopic normal eyes whose fellow eyes had myopic complications were reviewed. The AL, subfoveal choroidal thickness, height of a posterior staphyloma, and length of the retinal pigment epithelium from the fovea to 3-mm superior, inferior, nasal, and temporal retina were measured twice at an interval of approximately 1 year. The changes in these ocular parameters and their correlations were investigated. Results: The AL increased, the choroid became thinner (both P < 0.001), the superior (P < 0.05) and temporal (P < 0.01) staphyloma height increased, and the superior and temporal retinal pigment epithelial length increased (both P < 0.01). All the changes were significant. Stepwise analyses indicated that the factor most associated with the increase in the AL was the increase in the superior retinal pigment epithelial length (P < 0.001). Conclusion: Our results indicate that the AL can increase significantly in highly myopic normal adult eyes during a 1-year interval, and the increase in the posterior staphyloma height is the most likely cause for the increased AL.

A perspective from magnetic resonance imaging findings of the inner ear: Relationships among cerebrospinal, ocular and inner ear fluids.

Visualization of endolymphatic hydrops has been performed using magnetic resonance imaging (MRI) after intratympanic or intravenous gadolinium (Gd) injection. Our recent findings indicate that just as the prevalence of asymptomatic glaucoma is greater than that of symptomatic glaucoma, there are also many cases of asymptomatic endolymphatic hydrops. It is assumed that the asymptomatic endolymphatic hydrops that precedes Ménières disease is found more frequently using MRI than with other techniques. Gd in the inner ear moves into the cerebrospinal fluid (CSF) via the internal auditory meatus. Gd enhancement is also recognized in the ocular fluid after the intravenous Gd administration. In this paper, the relationships between CSF, ocular fluid and inner ear fluid are reviewed. The central nervous system, eye and inner ear contain specialized extracellular fluids that are essential for maintaining their function: CSF, ocular fluid consisting of vitreous humor and aqueous humor, and inner ear fluid consisting of perilymph and endolymph. Abnormal accumulation of or pressure elevation in these fluids is associated with hydrocephalus, glaucoma and Ménières disease, respectively. The dura mater and the arachnoid membrane of the optic nerve canal and inner ear meatus are very close to the eye and the inner ear, respectively. It has been reported that low CSF pressure is associated with glaucoma and endolymphatic hydrops. In glaucoma and Ménières disease, nerve damage to ganglion cells rather than damage of the sensory cells is directly associated with progression of the disease. Retinal ganglion cells in glaucoma and spiral ganglion cells in Ménières disease are targets of the abnormal accumulation of, or increased pressure in, the extracellular fluid, just as neurons are damaged in hydrocephalus. Studies on hydrocephalus, glaucoma and Ménières disease as a group may deepen our understanding of each disease.

Suppression of Choroidal Neovascularization and Quantitative and Qualitative Inhibition of VEGF and CCL2 by Heparin

PURPOSE To study the effect of heparin on the development of laser-induced choroidal neovascularization (CNV) and to assess the underlying molecular mechanisms. METHODS Bone marrow transplantation (BMT) was conducted by intravenous injection of green fluorescence protein (GFP)-labeled bone marrow cells (1 × 10(7) cells) into irradiated (9 Gy) C57BL/6J mice. Laser photocoagulation was applied to induce CNV; subsequently, unfractionated heparin or phosphate-buffered saline was injected into mice that did or did not undergo BMT. The area of CNV, distribution of injected heparin, and quantities of infiltrating cells positive for Griffonia simplicifolia (GS) and GFP inside and outside the CNV were evaluated. Effects of heparin on the secretion of VEGF, CCL2, and TNF-α by ARPE19 cells and on the binding of VEGF, CCL2, TNF-α, and their receptors were analyzed in vitro. RESULTS Intravitreal injection of heparin at higher doses reduced the size of the CNV. Heparin localized at the vascular structures and photoreceptor layers adjacent to the laser scar. Only GS-positive cells infiltrating outside the CNV were reduced significantly, but not those inside the CNV or those expressing GFP. Relative decreases in VEGF and CCL2 levels were observed in media of ARPE19 cells at higher heparin concentrations. In vitro binding assays revealed that heparin and porcine ocular fluid, respectively, suppressed the binding of VEGF to VEGFR2 and CCL2 to CCR2. CONCLUSIONS Intravitreal heparin injection inhibited CNV development. Reduced VEGF and CCL2 secretion by RPE cells and suppression of VEGF-VEGFR2 and CCL2-CCR2 interactions at the laser site mediated by heparin may contribute to the pharmacologic effect.