ABSTRACT
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.
Subject(s)
Central Serous Chorioretinopathy , Macular Degeneration , Humans , Genome-Wide Association Study , Genetic Predisposition to Disease , Central Serous Chorioretinopathy/diagnosis , Central Serous Chorioretinopathy/genetics , Macular Degeneration/genetics , Genotype , Polymorphism, Single Nucleotide , Genetic LociABSTRACT
Long interspersed nuclear element-1 (L1)mediated reverse transcription (RT) of Alu RNA into cytoplasmic Alu complementary DNA (cDNA) has been implicated in retinal pigmented epithelium (RPE) degeneration. The mechanism of Alu cDNAinduced cytotoxicity and its relevance to human disease are unknown. Here we report that Alu cDNA is highly enriched in the RPE of human eyes with geographic atrophy, an untreatable form of age-related macular degeneration. We demonstrate that the DNA sensor cGAS engages Alu cDNA to induce cytosolic mitochondrial DNA escape, which amplifies cGAS activation, triggering RPE degeneration via the inflammasome. The L1-extinct rice rat was resistant to Alu RNAinduced Alu cDNA synthesis and RPE degeneration, which were enabled upon L1-RT overexpression. Nucleoside RT inhibitors (NRTIs), which inhibit both L1-RT and inflammasome activity, and NRTI derivatives (Kamuvudines) that inhibit inflammasome, but not RT, both block Alu cDNA toxicity, identifying inflammasome activation as the terminal effector of RPE degeneration.
ABSTRACT
Alu retroelements propagate via retrotransposition by hijacking long interspersed nuclear element-1 (L1) reverse transcriptase (RT) and endonuclease activities. Reverse transcription of Alu RNA into complementary DNA (cDNA) is presumed to occur exclusively in the nucleus at the genomic integration site. Whether Alu cDNA is synthesized independently of genomic integration is unknown. Alu RNA promotes retinal pigmented epithelium (RPE) death in geographic atrophy, an untreatable type of age-related macular degeneration. We report that Alu RNA-induced RPE degeneration is mediated via cytoplasmic L1-reverse-transcribed Alu cDNA independently of retrotransposition. Alu RNA did not induce cDNA production or RPE degeneration in L1-inhibited animals or human cells. Alu reverse transcription can be initiated in the cytoplasm via self-priming of Alu RNA. In four health insurance databases, use of nucleoside RT inhibitors was associated with reduced risk of developing atrophic macular degeneration (pooled adjusted hazard ratio, 0.616; 95% confidence interval, 0.493-0.770), thus identifying inhibitors of this Alu replication cycle shunt as potential therapies for a major cause of blindness.
Subject(s)
Alu Elements/genetics , Long Interspersed Nucleotide Elements/genetics , Macular Degeneration/genetics , Retinal Pigments/metabolism , Animals , Cytoplasm/genetics , DNA, Complementary/genetics , Epithelium/metabolism , Epithelium/pathology , Humans , Macular Degeneration/pathology , Retinal Pigments/biosynthesis , Retroelements/genetics , Reverse Transcription/geneticsABSTRACT
Degeneration of the retinal pigmented epithelium (RPE) and aberrant blood vessel growth in the eye are advanced-stage processes in blinding diseases such as age-related macular degeneration (AMD), which affect hundreds of millions of people worldwide. Loss of the RNase DICER1, an essential factor in micro-RNA biogenesis, is implicated in RPE atrophy. However, the functional implications of DICER1 loss in choroidal and retinal neovascularization are unknown. Here, we report that two independent hypomorphic mouse strains, as well as a separate model of postnatal RPE-specific DICER1 ablation, all presented with spontaneous RPE degeneration and choroidal and retinal neovascularization. DICER1 hypomorphic mice lacking critical inflammasome components or the innate immune adaptor MyD88 developed less severe RPE atrophy and pathological neovascularization. DICER1 abundance was also reduced in retinas of the JR5558 mouse model of spontaneous choroidal neovascularization. Finally, adenoassociated vector-mediated gene delivery of a truncated DICER1 variant (OptiDicer) reduced spontaneous choroidal neovascularization in JR5558 mice. Collectively, these findings significantly expand the repertoire of DICER1 in preserving retinal homeostasis by preventing both RPE degeneration and pathological neovascularization.
Subject(s)
DEAD-box RNA Helicases/metabolism , Macular Degeneration/metabolism , Retinal Pigment Epithelium/blood supply , Ribonuclease III/metabolism , Animals , Choroidal Neovascularization/genetics , Choroidal Neovascularization/metabolism , Choroidal Neovascularization/pathology , Choroidal Neovascularization/physiopathology , DEAD-box RNA Helicases/genetics , Humans , Macular Degeneration/genetics , Macular Degeneration/pathology , Macular Degeneration/physiopathology , Mice , Mice, Inbred C57BL , Mice, Knockout , Retinal Degeneration/genetics , Retinal Degeneration/metabolism , Retinal Degeneration/pathology , Retinal Degeneration/physiopathology , Retinal Neovascularization/genetics , Retinal Neovascularization/metabolism , Retinal Neovascularization/parasitology , Retinal Neovascularization/physiopathology , Retinal Pigment Epithelium/metabolism , Retinal Pigment Epithelium/pathology , Ribonuclease III/geneticsABSTRACT
The traditional classification of hypertensive retinopathy was based on the Keith-Wagener-Barker (KWB) grading, which is a subjective scaling system, and it is difficult to distinguish between the first and second grades. Retinal and choroidal vasculatures are affected by systemic hypertension, although retinal vasculature changes with age, axial length, intraocular pressure, and retinal diseases. It is necessary to establish a new objective method to assess hypertensive vascular changes. In the present study, we have examined the vasculature of the macular choriocapillaris in order to establish a new objective method to assess hypertensive vascular changes using optical coherence tomography angiography (OCTA). Choriocapillaris vessel density (VD), vessel length, and vessel diameter index in a 3 × 3 mm macular area were measured by OTCA in a total of 567 volunteers (361 healthy subjects and 206 subjects with systemic hypertension) who attended a basic health check-up. Ocular factors, systemic factors, and medications were evaluated. We detected significant differences in normative choriocapillaris vasculature between the left and right eyes in 53 healthy subjects and revealed correlations between age, intraocular pressure, axial length, and choriocapillaris vasculature in 308 healthy subjects. Normative foveal VD was correlated with age only and the efficiency was weak. The analysis of 206 right eyes (KWB grade 0, 159 eyes; grade 1, 35 eyes; and grade 2, 12 eyes) revealed that foveal VD was strongly correlated with KWB grade only (P < 0.001). This is the first report suggesting that OCTA for foveal choriocapillaris measurement by OCTA would might provide the advantage of evaluating be objective method for evaluating the progression of systemic hypertension.
Subject(s)
Choroid/blood supply , Hypertension/pathology , Macula Lutea/blood supply , Macula Lutea/pathology , Retinal Vessels/pathology , Case-Control Studies , Female , Fluorescein Angiography/methods , Healthy Volunteers , Humans , Intraocular Pressure/physiology , Male , Middle Aged , Retinal Diseases/pathology , Tomography, Optical Coherence/methodsABSTRACT
PURPOSE: To investigate the microvascular changes in choroidal neovascularization (CNV) using optical coherence tomography angiography (OCTA) during anti-vascular endothelial growth factor (VEGF) therapies. METHODS: We retrospectively collected data on consecutive treatment-naïve eyes with typical age-related macular degeneration that initially received 3 aflibercept injections. OCTA was performed at baseline and at 1, 2, and 4 months of follow-up. The CNV images were analyzed using open-source software to assess vessel area and junction density. RESULTS: Fifteen eyes of 15 patients were included. The mean vessel area at baseline was 0.50 ± 0.33 mm2; at 1, 2, and 4 months, the ratios of change in vessel area from baseline were 66.6 ± 38.8%, 80.5 ± 25.5%, and 94.0 ± 29.3%, respectively. The vessel area was significantly reduced at 1 month from that at baseline (p = 0.0015) but significantly increased at 4 months from that at 1 month (p = 0.011). The mean junction density was also significantly reduced from 4.70 ± 1.30/mm at baseline to 3.82 ± 1.06/mm at 1 month (p = 0.00084). However, junction density did not continue to decrease at 2 and 4 months. CONCLUSION: OCTA quantification revealed that CNV rebounded after repeat aflibercept injections despite shrinking in response to the first injection.
Subject(s)
Choroid/blood supply , Choroidal Neovascularization/drug therapy , Fluorescein Angiography/methods , Receptors, Vascular Endothelial Growth Factor/administration & dosage , Recombinant Fusion Proteins/administration & dosage , Tomography, Optical Coherence/methods , Aged , Aged, 80 and over , Choroidal Neovascularization/diagnosis , Female , Follow-Up Studies , Fundus Oculi , Humans , Intravitreal Injections , Male , Middle Aged , Receptors, Vascular Endothelial Growth Factor/antagonists & inhibitors , Retrospective StudiesABSTRACT
Geographic atrophy is a blinding form of age-related macular degeneration characterized by retinal pigmented epithelium (RPE) death; the RPE also exhibits DICER1 deficiency, resultant accumulation of endogenous Alu-retroelement RNA, and NLRP3-inflammasome activation. How the inflammasome is activated in this untreatable disease is largely unknown. Here we demonstrate that RPE degeneration in human-cell-culture and mouse models is driven by a noncanonical-inflammasome pathway that activates caspase-4 (caspase-11 in mice) and caspase-1, and requires cyclic GMP-AMP synthase (cGAS)-dependent interferon-ß production and gasdermin D-dependent interleukin-18 secretion. Decreased DICER1 levels or Alu-RNA accumulation triggers cytosolic escape of mitochondrial DNA, which engages cGAS. Moreover, caspase-4, gasdermin D, interferon-ß, and cGAS levels were elevated in the RPE in human eyes with geographic atrophy. Collectively, these data highlight an unexpected role of cGAS in responding to mobile-element transcripts, reveal cGAS-driven interferon signaling as a conduit for mitochondrial-damage-induced inflammasome activation, expand the immune-sensing repertoire of cGAS and caspase-4 to noninfectious human disease, and identify new potential targets for treatment of a major cause of blindness.
Subject(s)
Geographic Atrophy/enzymology , Inflammasomes/metabolism , Nucleotidyltransferases/metabolism , Animals , DEAD-box RNA Helicases/genetics , Humans , Interferon Type I/metabolism , Mice , Retinal Pigment Epithelium/metabolism , Ribonuclease III/genetics , Signal TransductionABSTRACT
Human intravenous immune globulin (IVIg), a purified IgG fraction composed of ~ 60% IgG1 and obtained from the pooled plasma of thousands of donors, is clinically used for a wide range of diseases. The biological actions of IVIg are incompletely understood and have been attributed both to the polyclonal antibodies therein and also to their IgG (IgG) Fc regions. Recently, we demonstrated that multiple therapeutic human IgG1 antibodies suppress angiogenesis in a target-independent manner via FcγRI, a high-affinity receptor for IgG1. Here we show that IVIg possesses similar anti-angiogenic activity and inhibited blood vessel growth in five different mouse models of prevalent human diseases, namely, neovascular age-related macular degeneration, corneal neovascularization, colorectal cancer, fibrosarcoma and peripheral arterial ischemic disease. Angioinhibition was mediated by the Fc region of IVIg, required FcγRI and had similar potency in transgenic mice expressing human FcγRs. Finally, IVIg therapy administered to humans for the treatment of inflammatory or autoimmune diseases reduced kidney and muscle blood vessel densities. These data place IVIg, an agent approved by the US Food and Drug Administration, as a novel angioinhibitory drug in doses that are currently administered in the clinical setting. In addition, they raise the possibility of an unintended effect of IVIg on blood vessels.
ABSTRACT
Aberrant angiogenesis is implicated in diseases affecting nearly 10% of the world's population. The most widely used anti-angiogenic drug is bevacizumab, a humanized IgG1 monoclonal antibody that targets human VEGFA. Although bevacizumab does not recognize mouse Vegfa, it inhibits angiogenesis in mice. Here we show bevacizumab suppressed angiogenesis in three mouse models not via Vegfa blockade but rather Fc-mediated signaling through FcγRI (CD64) and c-Cbl, impairing macrophage migration. Other approved humanized or human IgG1 antibodies without mouse targets (adalimumab, alemtuzumab, ofatumumab, omalizumab, palivizumab and tocilizumab), mouse IgG2a, and overexpression of human IgG1-Fc or mouse IgG2a-Fc, also inhibited angiogenesis in wild-type and FcγR humanized mice. This anti-angiogenic effect was abolished by Fcgr1 ablation or knockdown, Fc cleavage, IgG-Fc inhibition, disruption of Fc-FcγR interaction, or elimination of FcRγ-initated signaling. Furthermore, bevacizumab's Fc region potentiated its anti-angiogenic activity in humanized VEGFA mice. Finally, mice deficient in FcγRI exhibited increased developmental and pathological angiogenesis. These findings reveal an unexpected anti-angiogenic function for FcγRI and a potentially concerning off-target effect of hIgG1 therapies.
ABSTRACT
Age-related macular degeneration (AMD) is a major cause of blindness in the elderly. Previous sequencing studies of AMD susceptibility genes have revealed the association of rare coding variants in CFH, CFI, C3 and C9 in European population; however, the impact of rare or low-frequency coding variants on AMD susceptibility in other populations is largely unknown. To identify the role of low-frequency coding variants on exudative AMD susceptibility in a Japanese population, we analysed the association of coding variants of 34 AMD candidate genes in the two-stage design by a multiplex PCR-based target sequencing method. We used a total of 2,886 (1st: 827, 2nd: 2,059) exudative AMD cases including typical AMD, polypoidal choroidal vasculopathy, and retinal angiomatous proliferation and 9,337 (1st: 3,247 2nd: 6,090) controls. Gene-based analysis found a significant association of low-frequency variants (minor allele frequency (MAF) < 0.05) in CETP, C2 and CFB. The association of CETP remained after conditioned with all known genome-wide association study (GWAS) associated variants. In addition, when we included only disruptive variants, enrichment of rare variants (MAF < 0.01) was also observed after conditioned with all GWAS associated variants (P = 1.03 × 10−6, odds ratio (OR) = 2.48). Haplotype and conditional analysis of the C2-CFB-SKIV2L locus showed a low-frequency variant (R74H) in CFB would be individually associated with AMD susceptibility independent of the GWAS associated SNP. These findings highlight the importance of target sequencing to reveal the impact of rare or low-frequency coding variants on disease susceptibility in different ethnic populations.
Subject(s)
Cholesterol Ester Transfer Proteins/genetics , Complement Factor B/genetics , Macular Degeneration/genetics , Aged , Base Sequence , Case-Control Studies , Cholesterol Ester Transfer Proteins/metabolism , Complement C2/genetics , Complement Factor B/metabolism , Complement Factor H/genetics , Female , Gene Frequency , Genetic Predisposition to Disease , Genome-Wide Association Study/methods , Genotype , Haplotypes , Humans , Japan , Macular Degeneration/epidemiology , Macular Degeneration/metabolism , Male , Polymorphism, Single NucleotideABSTRACT
Excess iron induces tissue damage and is implicated in age-related macular degeneration (AMD). Iron toxicity is widely attributed to hydroxyl radical formation through Fenton's 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.
Subject(s)
Alu Elements , Carrier Proteins/metabolism , Iron/toxicity , Retinal Pigment Epithelium/metabolism , Animals , Carrier Proteins/genetics , Caspase 1/genetics , Caspase 1/metabolism , DEAD-box RNA Helicases/genetics , DEAD-box RNA Helicases/metabolism , Inflammasomes/metabolism , Iron/pharmacology , Mice , Mice, Inbred C57BL , NLR Family, Pyrin Domain-Containing 3 Protein , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Retinal Pigment Epithelium/drug effects , Ribonuclease III/genetics , Ribonuclease III/metabolismABSTRACT
Nucleoside reverse transcriptase inhibitors (NRTIs) are mainstay therapeutics for HIV that block retrovirus replication. Alu (an endogenous retroelement that also requires reverse transcriptase for its life cycle)-derived RNAs activate P2X7 and the NLRP3 inflammasome to cause cell death of the retinal pigment epithelium in geographic atrophy, a type of age-related macular degeneration. We found that NRTIs inhibit P2X7-mediated NLRP3 inflammasome activation independent of reverse transcriptase inhibition. Multiple approved and clinically relevant NRTIs prevented caspase-1 activation, the effector of the NLRP3 inflammasome, induced by Alu RNA. NRTIs were efficacious in mouse models of geographic atrophy, choroidal neovascularization, graft-versus-host disease, and sterile liver inflammation. Our findings suggest that NRTIs are ripe for drug repurposing in P2X7-driven diseases.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Inflammasomes/drug effects , Reverse Transcriptase Inhibitors/pharmacology , Alu Elements , Animals , Anti-Inflammatory Agents, Non-Steroidal/therapeutic use , Apoptosis/drug effects , Carrier Proteins/metabolism , Caspase 1/metabolism , Choroidal Neovascularization/drug therapy , Disease Models, Animal , Geographic Atrophy/drug therapy , Graft vs Host Disease/drug therapy , Hepatitis/drug therapy , Liver/drug effects , Mice , NLR Family, Pyrin Domain-Containing 3 Protein , Receptors, Purinergic P2X7/metabolism , Retinal Pigment Epithelium/drug effects , Retinal Pigment Epithelium/metabolism , Retinal Pigment Epithelium/physiology , Reverse Transcriptase Inhibitors/therapeutic useABSTRACT
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.
Subject(s)
Alu Elements , Apoptosis , Caspase 8/metabolism , DEAD-box RNA Helicases/metabolism , Eye Proteins/metabolism , Macular Degeneration/metabolism , RNA/metabolism , Ribonuclease III/metabolism , Animals , Caspase 8/genetics , DEAD-box RNA Helicases/genetics , Eye Proteins/genetics , Humans , Interleukin-18/genetics , Interleukin-18/metabolism , Macular Degeneration/pathology , Mice , Mice, Knockout , RNA/genetics , Ribonuclease III/genetics , Up-Regulation/geneticsABSTRACT
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.
Subject(s)
Alu Elements , Geographic Atrophy/physiopathology , Inflammasomes/physiology , Receptors, Purinergic P2X7/physiology , Signal Transduction/physiology , Toll-Like Receptors/physiology , Animals , Carrier Proteins/antagonists & inhibitors , Carrier Proteins/metabolism , Caspase 1/metabolism , Disease Models, Animal , Glyburide/pharmacology , Humans , Interleukin-18/metabolism , Mice , NF-kappa B/antagonists & inhibitors , NF-kappa B/metabolism , Nitriles/pharmacology , Purinergic P2X Receptor Antagonists/pharmacology , RNA, Messenger/metabolism , Retinal Pigment Epithelium/cytology , Sulfones/pharmacologyABSTRACT
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.
Subject(s)
Gene Rearrangement/genetics , Genome, Human/genetics , Protein Serine-Threonine Kinases/genetics , Retinitis Pigmentosa/genetics , Animals , Base Sequence , Frameshift Mutation/genetics , Genetics, Medical , Genome-Wide Association Study , Humans , Japan , Molecular Sequence Data , NIMA-Related Kinases , Sequence Analysis, DNA , United States , ZebrafishABSTRACT
Vascular endothelial growth factor (VEGF) is recognized as a principal mediator of vessel growth. VEGF regulates various endothelial cellular processes, including cell migration, proliferation, and survival, through the serine threonine protein kinase Akt. The Akt substrate girdin, an actin-binding protein, is known to regulate VEGF-mediated postnatal angiogenesis. However, the role of girdin and its phosphorylation in neonatal retinal vascular development and ocular pathological neovascularization in vivo has not been elucidated. In the present study, therefore, we investigated these processes using Girdin(+/-) mice lacking one copy of the girdin gene and girdin S1416A knockin (Girdin-KI(SA/SA)) mice in which the phosphorylation site of girdin is completely disrupted. We used three mouse models of pathological ocular neovascularization: oxygen-induced retinopathy (a mouse model of ischemic retinopathies), laser-induced choroidal neovascularization, and a human VEGF transgenic mouse that overexpresses human VEGF specifically in photoreceptor cells and generates pathological neovascularization in the retina. Neonatal vascular development was delayed and pathological neovascularization was decreased in both Girdin(+/-) mice and Girdin-KI(SA/SA) mice. These results demonstrate that girdin and its phosphorylation play an important role in neonatal vascular development and in pathological neovascularization in the retina.
Subject(s)
Blood Vessels/growth & development , Microfilament Proteins/metabolism , Neovascularization, Physiologic , Retinal Neovascularization/pathology , Vesicular Transport Proteins/metabolism , Animals , Animals, Newborn , Blood Vessels/drug effects , Blood Vessels/metabolism , Cell Movement/drug effects , Cell Proliferation/drug effects , Chromones/pharmacology , Disease Models, Animal , Female , Gene Knockdown Techniques , Human Umbilical Vein Endothelial Cells/drug effects , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Mice , Microfilament Proteins/genetics , Morpholines/pharmacology , Neovascularization, Physiologic/drug effects , Phosphatidylinositol 3-Kinases/metabolism , Phosphorylation/drug effects , Protein Transport/drug effects , Proto-Oncogene Proteins c-akt/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Small Interfering/metabolism , Retina/drug effects , Retina/metabolism , Retina/pathology , Retinal Neovascularization/metabolism , Signal Transduction/drug effects , Vascular Endothelial Growth Factor A/metabolism , Vesicular Transport Proteins/geneticsABSTRACT
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 Ménière's 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 Ménière's 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 Ménière's 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 Ménière's 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 Ménière's disease as a group may deepen our understanding of each disease.
Subject(s)
Aqueous Humor/physiology , Cerebrospinal Fluid/physiology , Endolymphatic Hydrops/pathology , Meniere Disease/pathology , Contrast Media , Ear, Inner/anatomy & histology , Endolymph/physiology , Endolymphatic Hydrops/physiopathology , Eye/anatomy & histology , Gadolinium , Humans , Magnetic Resonance Imaging , Meniere Disease/physiopathology , Vitreous Body/physiologyABSTRACT
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.
Subject(s)
Axial Length, Eye/physiopathology , Myopia, Degenerative/physiopathology , Aged , Aged, 80 and over , Case-Control Studies , Choroid/pathology , Female , Humans , Intraocular Pressure/physiology , Male , Middle Aged , Myopia, Degenerative/pathology , Retina/pathology , Retinal Pigment Epithelium/pathology , Retrospective Studies , Tomography, Optical CoherenceABSTRACT
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.