GENETIC ETIOLOGY AND CLINICAL FEATURES OF ACHROMATOPSIA IN JAPAN.

PURPOSE: To ascertain the characteristics of achromatopsia (ACHM) in Japan by analyzing the genetic and phenotypic features of patients with ACHM. METHODS: The medical records of 52 patients from 47 Japanese families who were clinically diagnosed with ACHM were reviewed in this retrospective observational study. RESULTS: Thirty-six causative variants of ACHM were identified in 26 families via whole-exome sequencing: PDE6C (12 families), CNGA3 (10 families), CNGB3 (two families), and GNAT2 (two families). However, none of the 6 causative variants that are known to cause ACHM, or the 275 other genes listed in RetNet, were observed in 19 families. A significant trend toward older age and worsening of ellipsoid zone disruption on optical coherence tomography images was observed (P < 0.01). Progressive ellipsoid zone disruptions were observed in 13 eyes of seven patients during the follow-up visits. These patients harbored one or more variants in PDE6C. CONCLUSION: The ACHM phenotype observed in this study was similar to those observed in previous reports; however, the causative gene variants differed from those in Europe. The low identification ratio of causative genes in whole-exome sequencing suggests the presence of unique hotspots in Japanese patients with ACHM that were not detectable via ordinal whole-exome sequencing.

Genetic characterization of 1210 Japanese pedigrees with inherited retinal diseases by whole-exome sequencing.

Inherited retinal diseases (IRDs) comprise a phenotypically and genetically heterogeneous group of ocular disorders that cause visual loss via progressive retinal degeneration. Here, we report the genetic characterization of 1210 IRD pedigrees enrolled through the Japan Eye Genetic Consortium and analyzed by whole exome sequencing. The most common phenotype was retinitis pigmentosa (RP, 43%), followed by macular dystrophy/cone- or cone-rod dystrophy (MD/CORD, 13%). In total, 67 causal genes were identified in 37% (448/1210) of the pedigrees. The first and second most frequently mutated genes were EYS and RP1, associated primarily with autosomal recessive (ar) RP, and RP and arMD/CORD, respectively. Examinations of variant frequency in total and by phenotype showed high accountability of a frequent EYS missense variant (c.2528G>A). In addition to the two known EYS founder mutations (c.4957dupA and c.8805C>G) of arRP, we observed a frequent RP1 variant (c.5797C>T) in patients with arMD/CORD.

Binding of Gtf2i-ß/δ transcription factors to the ARMS2 gene leads to increased circulating HTRA1 in AMD patients and in vitro.

The disease-initiating molecular events for age-related macular degeneration (AMD), a multifactorial retinal disease affecting many millions of elderly individuals worldwide, are still unknown. Of the over 30 risk and protective loci so far associated with AMD through whole genome-wide association studies (GWAS), the Age-Related Maculopathy Susceptibility 2 (ARMS2) gene locus represents one of the most highly associated risk regions for AMD. A unique insertion/deletion (in/del) sequence located immediately upstream of the High Temperature Requirement A1 (HTRA1) gene in this region confers high risk for AMD. Using electrophoretic mobility shift assay (EMSA), we identified that two Gtf2i-ß/δ transcription factor isoforms bind to the cis-element 5'- ATTAATAACC-3' contained in this in/del sequence. The binding of these transcription factors leads to enhanced upregulation of transcription of the secretory serine protease HTRA1 in transfected cells and AMD patient-derived induced pluripotent stem cells (iPSCs). Overexpression of Htra1 in mice using a CAG-promoter demonstrated increased blood concentration of Htra1 protein, caused upregulation of vascular endothelial growth factor (VEGF), and produced a choroidal neovascularization (CNV)-like phenotype. Finally, a comparison of 478 AMD patients to 481 healthy, age-matched controls from Japan, India, Australia, and the USA showed a statistically increased level of secreted HTRA1 blood concentration in AMD patients compared with age-matched controls. Taken together, these results suggest a common mechanism across ethnicities whereby increased systemic blood circulation of secreted serine protease HTRA1 leads to subsequent degradation of Bruch's membrane and eventual CNV in AMD.

Novel mutations in malonyl-CoA-acyl carrier protein transacylase provoke autosomal recessive optic neuropathy.

Inherited optic neuropathies are rare eye diseases of optic nerve dysfunction that present in various genetic forms. Previously, mutation in three genes encoding mitochondrial proteins has been implicated in autosomal recessive forms of optic atrophy that involve progressive degeneration of optic nerve and retinal ganglion cells (RGC). Using whole exome analysis, a novel double homozygous mutation p.L81R and pR212W in malonyl CoA-acyl carrier protein transacylase (MCAT), a mitochondrial protein involved in fatty acid biosynthesis, has now been identified as responsible for an autosomal recessive optic neuropathy from a Chinese consanguineous family. MCAT is expressed in RGC that are rich in mitochondria. The disease variants lead to structurally unstable MCAT protein with significantly reduced intracellular expression. RGC-specific knockdown of Mcat in mice, lead to an attenuated retinal neurofiber layer, that resembles the phenotype of optic neuropathy. These results indicated that MCAT plays an essential role in mitochondrial function and maintenance of RGC axons, while novel MCAT p.L81R and p.R212W mutations can lead to optic neuropathy.

LRRTM4-C538Y novel gene mutation is associated with hereditary macular degeneration with novel dysfunction of ON-type bipolar cells.

The macula is a unique structure in higher primates, where cone and rod photoreceptors show highest density in the fovea and the surrounding area, respectively. The hereditary macular dystrophies represent a heterozygous group of rare disorders characterized by central visual loss and atrophy of the macula and surrounding retina. Here we report an atypical absence of ON-type bipolar cell response in a Japanese patient with autosomal dominant macular dystrophy (adMD). To identify a causal genetic mutation for the adMD, we performed whole-exome sequencing (WES) on four affected and four-non affected members of the family for three generations, and identified a novel p.C538Y mutation in a post-synaptic gene, LRRTM4. WES analysis revealed seven rare genetic variations in patients. We further referred to our in-house WES data from 1360 families with inherited retinal diseases, and found that only p.C538Y mutation in LRRTM4 was associated with adMD-affected patients. Combinatorial filtration using public database of single-nucleotide polymorphism frequency and genotype-phenotype annotated database identified novel mutation in atypical adMD.

Compound heterozygous mutations in a mouse model of Leber congenital amaurosis reveal the role of CCT2 in photoreceptor maintenance.

TRiC/CCT is a chaperonin complex required for the folding of cytoplasmic proteins. Although mutations in each subunit of TRiC/CCT are associated with various human neurodegenerative diseases, their impact in mammalian models has not yet been examined. A compound heterozygous mutation in CCT2 (p.[Thr400Pro]; p.[Arg516His]) is causal for Leber congenital amaurosis. Here, we generate mice carrying each mutation and show that Arg516His (R516H) homozygosity causes photoreceptor degeneration accompanied by a significant depletion of TRiC/CCT substrate proteins in the retina. In contrast, Thr400Pro (T400P) homozygosity results in embryonic lethality, and the compound heterozygous mutant (T400P/R516H) mouse showed aberrant cone cell lamination and died 2 weeks after birth. Finally, CCDC181 is identified as a interacting protein for CCTß protein, and its localization to photoreceptor connecting cilia is compromised in the mutant mouse. Our results demonstrate the distinct impact of each mutation in vivo and suggest a requirement for CCTß in ciliary maintenance.

Retinal pigment epithelium-specific ablation of GPx4 in adult mice recapitulates key features of geographic atrophy in age-related macular degeneration.

Age-related macular degeneration (AMD) is a leading cause of irreversible vision loss in the elderly population, particularly the late-stage of dry AMD known as geographic atrophy (GA), lacks effective treatment options. Genetic mouse models of AMD have revealed the significance of impaired lipid metabolism and anti-oxidative capacity in early/intermediate stage of AMD, but remains unclear in GA that severely damages visual function. Here, to investigate the potential relevance of peroxidized lipids in RPE for late-stage dry AMD, GPx4fl/fl mice underwent subretinal injections of RPE-specific AAV-Cre vector or control AAV vector. RPE-specific GPx4 deficiency led to rapid RPE degeneration resembling key features of late-stage dry AMD, including preceding loss of RPE cell polarity, accumulation of acrolein, malondialdehyde, and 4-hydroxynonenal, photoreceptor loss, lipofuscin-laden subretinal melanophage infiltration, and complement activation. Treatment with α-tocopherol and ferrostatin-1 mitigated RPE degeneration, and shrunk mitochondria were observed in GPx4 deficient mice, suggesting involvement of ferroptosis. Unexpectedly, necrostatin-1s, an inhibitor of necroptosis, also ameliorated RPE degeneration, and activation of RIP3 and MLKL along with inactivation of caspase-8 was observed, indicating crosstalk between ferroptosis and necroptosis pathways. Our findings shed light on the intricate mechanisms underlying RPE degeneration in AMD and highlight GPx4/lipid peroxidation as potential therapeutic targets. RPE-specific ablation of GPx4 in mice provides a valuable tool for further elucidating the interplay between lipid peroxidation, cell death pathways, and AMD pathogenesis, offering new insights for preclinical research and therapeutic development targeting GA.

Optical coherence tomography findings of the peripheral retina in patients with congenital X-linked retinoschisis.

Introduction: Congenital X-linked retinoschisis (XLRS) presents as macular retinoschisis/degeneration in almost all patients and as peripheral retinoschisis in half the patients. Although the optical coherence tomography (OCT) findings of macular retinoschisis have been well investigated, those of peripheral retinoschisis have rarely been reported. This study aimed to report the ultra-widefield OCT findings of the peripheral retina in patients with XLRS. Methods: Medical records of 10 Japanese patients (19 eyes) with clinically and/or genetically diagnosed XLRS were retrospectively reviewed. Funduscopic, electroretinographic, and OCT findings were reviewed and evaluated. Some were also genetically evaluated for the RS1 gene. Results: OCT of the macula revealed schises and/or cystoid changes in the inner nuclear layer (INL) and outer nuclear layer. In contrast, OCT of the peripheral retina revealed schises and/or cystoid changes in the INL in eight eyes (44%), and/or splitting in the ganglion cell layer (GCL) in 10 (56%) of the 18 eyes with clear OCT images. No schisis or cystoid changes were found in the peripheral OCT images of eight eyes (44%). A 16-year-old boy presented with retinal splitting of the GCL and INL of the inferior retina, although he had no ophthalmoscopic peripheral retinoschisis. Genetic examinations were performed on three patients, all of whom had reported missense mutations in the RS1 gene. Conclusion: In XLRS, peripheral bullous retinoschisis results from GCL splitting in the retina. One of the 10 patients with XLRS showed intraretinal retinoschisis in the GCL in the inferior periphery, which was unremarkable on ophthalmoscopy (occult retinoschisis). Although both peripheral bullous retinoschisis and occult retinoschisis showed splitting/cystic changes in the GCL, further studies are needed to determine whether occult retinoschisis progresses to bullous retinoschisis.

METTL23 mutation alters histone H3R17 methylation in normal-tension glaucoma.

Normal-tension glaucoma (NTG) is a heterogeneous disease characterized by retinal ganglion cell (RGC) death leading to cupping of the optic nerve head and visual field loss at normal intraocular pressure (IOP). The pathogenesis of NTG remains unclear. Here, we describe a single nucleotide mutation in exon 2 of the methyltransferase-like 23 (METTL23) gene identified in 3 generations of a Japanese family with NTG. This mutation caused METTL23 mRNA aberrant splicing, which abolished normal protein production and altered subcellular localization. Mettl23-knock-in (Mettl23+/G and Mettl23G/G) and -knockout (Mettl23+/- and Mettl23-/-) mice developed a glaucoma phenotype without elevated IOP. METTL23 is a histone arginine methyltransferase expressed in murine and macaque RGCs. However, the novel mutation reduced METTL23 expression in RGCs of Mettl23G/G mice, which recapitulated both clinical and biological phenotypes. Moreover, our findings demonstrated that METTL23 catalyzed the dimethylation of H3R17 in the retina and was required for the transcription of pS2, an estrogen receptor α target gene that was critical for RGC homeostasis through the negative regulation of NF-κB-mediated TNF-α and IL-1ß feedback. These findings suggest an etiologic role of METTL23 in NTG with tissue-specific pathology.

LIM family transcription factors regulate the subtype-specific morphogenesis of retinal horizontal cells at post-migratory stages.

In the nervous system, transcription factor expression in progenitor and/or nascent neurons regulates cell type specification. Although the functions of these transcription factors at early stages are well established, whether or not they are required during late developmental periods remains an open question. To address this issue, we conditionally manipulated gene expression using a recently developed transposon-mediated gene transfer system combined with in ovo electroporation. In chicken retinas, horizontal cells are classified into three subtypes according to their characteristic neuronal morphology. Two LIM family transcription factors, Lim1 and Isl1, begin to be expressed in a distinct subset of nascent retinal neurons, which results in complementary expression of these genes in mature retinas in type I and type II/III horizontal cells, respectively. Overexpression of Isl1 in post-migratory horizontal cells represses endogenous Lim1 expression and increases the number of neurons with a dendritic morphology characteristic of type II horizontal cells, which normally express Isl1. Inhibition of Lim1 function by expression of a dominant negative form Lim1 perturbs axonal morphogenesis of type I horizontal cells. Therefore, we propose that LIM family transcription factors are required for subtype-specific morphogenesis of horizontal cells at later stages of retinal development.

Cellular FLIP inhibits beta-catenin ubiquitylation and enhances Wnt signaling.

Cellular FLIP (cFLIP) is a close homologue of caspase 8 without caspase activity that inhibits Fas signaling. The cFLIP protein is often expressed in human tumors and is believed to suppress antitumor immune responses involving the Fas system. Here, we report that a long form of cFLIP (cFLIP-L) inhibits beta-catenin ubiquitylation and increases endogenous cytosolic beta-catenin, which results in translocation of beta-catenin into nuclei and induction of beta-catenin-dependent gene expression in cFLIP-L-expressing cells. When cells stably expressing cFLIP-L were stimulated with Wnt3a, enhanced Wnt signaling was observed compared with the control cells. Conversely, depletion of endogenous cFLIP results in reduced Wnt signaling. Furthermore, cFLIP-L increases secondary-body axis formation when coinjected with suboptimal doses of beta-catenin into early Xenopus embryos. Down-regulation of FADD by RNA-mediated interference abolishes the beta-catenin-dependent gene expression induced by cFLIP-L. These results indicate that cFLIP-L, in cooperation with FADD, enhances canonical Wnt signaling by inhibiting proteasomal degradation of beta-catenin, thus suggesting an additional mechanism involved with tumorgenesis, in addition to inhibiting Fas signaling.

Identification of Novel Mutations in the LRR-Cap Domain of C21orf2 in Japanese Patients With Retinitis Pigmentosa and Cone-Rod Dystrophy.

PURPOSE: C21orf2 encodes a ciliary protein related to syndromic and nonsyndromic retinal degeneration. The purpose of this study was to identify novel mutations of C21orf2 associated with syndromic autosomal recessive retinitis pigmentosa (arRP) and autosomal recessive cone-rod dystrophy (arCRD) by using whole exome sequencing of a Japanese cohort. METHODS: Whole exome sequencing was performed on DNA from affected and healthy members from 147 families with retinal degenerations. Identified nonsense and missense mutations were further restricted by using the reported single nucleotide variation frequencies and inherited patterns. The effect of the mutations was examined by in vitro assays. RESULTS: Novel mutations in C21orf2 were found in Japanese patients with arRP with skeletal defects or arCRD. Compound heterozygous mutations, from one family (p.V111M and p.Y107H), and a homozygous mutation, from another family (p.Y107C), were all located in the leucine-rich repeat C-terminal domain required for protein stabilization. C21orf2 was expressed in the retina through the developing to the mature stage, and the protein localized to the photoreceptor cilia in the adult retina. In vitro expression showed reduced levels and affected localizations of mutated protein products compared to the wild type. CONCLUSIONS: The identified C21orf2 mutations decreased protein stability and affected cytoplasmic localization of C21orf2. Since C21orf2 was required for ciliogenesis, our data suggested that reduced levels of functional C21orf2 induced photoreceptor degradation through abnormal cilia formation, leading to arRP or arCRD in the retina.

Proliferation potential of Müller glia after retinal damage varies between mouse strains.

Retinal Müller glia can serve as a source for regeneration of damaged retinal neurons in fish, birds and mammals. However, the proliferation rate of Müller glia has been reported to be low in the mammalian retina. To overcome this problem, growth factors and morphogens have been studied as potent promoters of Müller glial proliferation, but the molecular mechanisms that limit the proliferation of Müller glia in the mammalian retina remain unknown. In the present study, we found that the degree of damage-induced Müller glia proliferation varies across mouse strains. In mouse line 129×1/SvJ (129), there was a significantly larger proliferative response compared with that observed in C57BL/6 (B6) after photoreceptor cell death. Treatment with a Glycogen synthase kinase 3 (GSK3) inhibitor enhanced the proliferation of Müller glia in 129 but not in B6 mouse retinas. We therefore focused on the different gene expression patterns during retinal degeneration between B6 and 129. Expression levels of Cyclin D1 and Nestin correlated with the degree of Müller glial proliferation. A comparison of genome-wide gene expression between B6 and 129 showed that distinct sets of genes were upregulated in the retinas after damage, including immune response genes and chromatin remodeling factors.

Characterization of human induced pluripotent stem cell-derived retinal pigment epithelium cell sheets aiming for clinical application.

Age-related macular degeneration (AMD) causes severe visual impairment due in part to age-dependent impairment of retinal pigment epithelium (RPE). It has been suggested that autologous human induced pluripotent stem cells (hiPSCs) may represent a useful cell source for the generation of graft RPE. We generated hiPSC-derived RPE (hiPSC-RPE) cell sheets optimized to meet clinical use requirements, including quality, quantity, consistency, and safety. These cell sheets are generated as a monolayer of cells without any artificial scaffolds, express typical RPE markers, form tight junctions that exhibit polarized secretion of growth factors, and show phagocytotic ability and gene-expression patterns similar to those of native RPE. Additionally, upon transplantation, autologous nonhuman primate iPSC-RPE cell sheets showed no immune rejection or tumor formation. These results suggest that autologous hiPSC-RPE cell sheets may serve as a useful form of graft for use in tissue replacement therapy for AMD.

Xenopus ADAMTS1 negatively modulates FGF signaling independent of its metalloprotease activity.

We have isolated the Xenopus ortholog of ADAMTS1 (a disintegrin and metalloprotease with thrombospondin motifs), XADAMTS1, which is expressed in the presumptive ectoderm, then the Spemann organizer, and later in the trunk organizer region and posterior ectoderm in the Xenopus embryo. We show that, when overexpressed in the dorsal marginal zone or in the anterior ectoderm by mRNA injection, XADAMTS1 inhibits gastrulation or generates embryos with an enlarged cement gland, respectively. XADAMTS1 also reduces the expression of Xbra in both whole embryos and FGF-treated animal caps. These effects of XADAMTS1 are likely to be due to its inhibition of the Ras-MAPK cascade because XADAMTS1 inhibits the phosphorylation of ERK by FGF4 in animal caps. Deletion analysis of XADAMTS1 revealed that a combination of the signal peptide and the C-terminal region containing the thrombospondin type 1 repeats is necessary and sufficient for this function, whereas the metalloprotease domain is dispensable. In addition, loss-of-function analysis with antisense morpholino oligos showed that knockdown of XADAMTS1 sensitizes animal caps to Xbra induction by FGF2. These data suggest that secreted XADAMTS1 negatively modulates FGF signaling in the Xenopus embryo.