RESUMO
How genetic defects trigger the molecular changes that cause late-onset disease is important for understanding disease progression and therapeutic development. Fuchs' endothelial corneal dystrophy (FECD) is an RNA-mediated disease caused by a trinucleotide CTG expansion in an intron within the TCF4 gene. The mutant intronic CUG RNA is present at one-two copies per cell, posing a challenge to understand how a rare RNA can cause disease. Late-onset FECD is a uniquely advantageous model for studying how RNA triggers disease because: (i) Affected tissue is routinely removed during surgery; (ii) The expanded CTG mutation is one of the most prevalent disease-causing mutations, making it possible to obtain pre-symptomatic tissue from eye bank donors to probe how gene expression changes precede disease; and (iii) The affected tissue is a homogeneous single cell monolayer, facilitating accurate transcriptome analysis. Here, we use RNA sequencing (RNAseq) to compare tissue from individuals who are pre-symptomatic (Pre_S) to tissue from patients with late stage FECD (FECD_REP). The abundance of mutant repeat intronic RNA in Pre_S and FECD_REP tissue is elevated due to increased half-life in a corneal cells. In Pre_S tissue, changes in splicing and extracellular matrix gene expression foreshadow the changes observed in advanced disease and predict the activation of the fibrosis pathway and immune system seen in late-stage patients. The absolute magnitude of splicing changes is similar in pre-symptomatic and late stage tissue. Our data identify gene candidates for early drivers of disease and biomarkers that may represent diagnostic and therapeutic targets for FECD. We conclude that changes in alternative splicing and gene expression are observable decades prior to the diagnosis of late-onset trinucleotide repeat disease.
Assuntos
Distrofia Endotelial de Fuchs/genética , Fator de Transcrição 4/genética , Expansão das Repetições de Trinucleotídeos/genética , Repetições de Trinucleotídeos/genética , Adulto , Idoso , Biomarcadores/metabolismo , Córnea/metabolismo , Córnea/patologia , Feminino , Distrofia Endotelial de Fuchs/patologia , Distrofia Endotelial de Fuchs/terapia , Regulação da Expressão Gênica/genética , Predisposição Genética para Doença , Humanos , Íntrons/genética , Masculino , Pessoa de Meia-Idade , Mutação/genética , Especificidade de Órgãos/genética , Análise de Sequência de RNARESUMO
Purpose: Retinitis pigmentosa (RP) is an inherited retinal disorder that results in the degeneration of photoreceptor cells, ultimately leading to severe visual impairment. We characterized a consanguineous family from Southern India wherein a 25 year old individual presented with night blindness since childhood. The purpose of this study was to identify the causative mutation for RP in this individual as well as characterize how the mutation may ultimately affect protein function. Methods: We performed a complete ophthalmologic examination of the proband followed by exome sequencing. The likely causative mutation was identified and modeled in cultured cells, evaluating its expression, solubility (both with western blotting), subcellular distribution, (confocal microscopy), and testing whether this variant induced endoplasmic reticulum (ER) stress (quantitative PCR [qPCR] and western blotting). Results: The proband presented with generalized and parafoveal retinal pigmented epithelium (RPE) atrophy with bone spicule-like pigmentation in the midperiphery and arteriolar attenuation. Optical coherence tomography scans through the macula of both eyes showed atrophy of the outer retinal layers with loss of the ellipsoid zone, whereas the systemic examination of this individual was normal. The proband's parents and sibling were asymptomatic and had normal funduscopic examinations. We discovered a novel homozygous p.Pro388Ser mutation in the tubby-like protein 1 (TULP1) gene in the individual with RP. In cultured cells, the P388S mutation does not alter the subcellular distribution of TULP1 or induce ER stress when compared to wild-type TULP1, but instead significantly lowers protein stability as indicated with steady-state and cycloheximide-chase experiments. Conclusions: These results add to the list of known mutations in TULP1 identified in individuals with RP and suggest a possible unique pathogenic mechanism in TULP1-induced RP, which may be shared among select mutations in TULP1.
Assuntos
Proteínas do Olho/genética , Mutação de Sentido Incorreto/genética , Retinose Pigmentar/genética , Adulto , Western Blotting , Consanguinidade , Eletrorretinografia , Homozigoto , Humanos , Índia , Masculino , Microscopia Confocal , Linhagem , Reação em Cadeia da Polimerase em Tempo Real , Retinose Pigmentar/diagnóstico , Retinose Pigmentar/fisiopatologia , Tomografia de Coerência Óptica , Acuidade Visual/fisiologia , Sequenciamento do ExomaRESUMO
Fuchs' endothelial corneal dystrophy (FECD) is the most common repeat expansion disorder. FECD impacts 4% of U.S. population and is the leading indication for corneal transplantation. Most cases are caused by an expanded intronic CUG tract in the TCF4 gene that forms nuclear foci, sequesters splicing factors and impairs splicing. We investigated the sense and antisense RNA landscape at the FECD gene and find that the sense-expanded repeat transcript is the predominant species in patient corneas. In patient tissue, sense foci number were negatively correlated with age and showed no correlation with sex. Each endothelial cell has â¼2 sense foci and each foci is single RNA molecule. We designed antisense oligonucleotides (ASOs) to target the mutant-repetitive RNA and demonstrated potent inhibition of foci in patient-derived cells. Ex vivo treatment of FECD human corneas effectively inhibits foci and reverses pathological changes in splicing. FECD has the potential to be a model for treating many trinucleotide repeat diseases and targeting the TCF4 expansion with ASOs represents a promising therapeutic strategy to prevent and treat FECD.
Assuntos
Distrofia Endotelial de Fuchs/genética , Distrofia Endotelial de Fuchs/metabolismo , Fator de Transcrição 4/genética , Idoso , Idoso de 80 Anos ou mais , Alelos , Endotélio Corneano/metabolismo , Feminino , Predisposição Genética para Doença , Humanos , Íntrons , Masculino , Pessoa de Meia-Idade , Oligorribonucleotídeos Antissenso/genética , Oligorribonucleotídeos Antissenso/uso terapêutico , RNA/metabolismo , Splicing de RNA , Fator de Transcrição 4/metabolismo , Fatores de Transcrição/genética , Expansão das Repetições de TrinucleotídeosRESUMO
PURPOSE: To identify the causative mutation in two siblings from a consanguineous family in India with retinitis pigmentosa (RP) and polydactyly without other findings of Bardet-Biedl syndrome (BBS). We also performed functional characterization of the mutant protein to explore its role in this limited form of BBS. METHODS: The siblings underwent a thorough ophthalmological examination, including retinal optical coherence tomography (OCT) imaging, and an extensive physical examination with abdominal ultrasonography to characterize the disease phenotype. Next-generation sequencing (NGS) using a panel targeting retinal degeneration genes was performed on genomic DNA samples from the siblings and parents. Upon identification of the causative mutation, functional characterization was accomplished by performing protein-protein interaction studies in human embryonic kidney (HEK-293T) and human adult retinal pigmented epithelium (ARPE-19) cells. RESULTS: The two siblings showed signs of RP and polydactyly. The patients did not have truncal obesity, renal anomalies, hydrometrocolpos, congenital heart disease, or overt cognitive defects. NGS identified a homozygous c.1184A>G mutation in the MKKS/BBS6 gene in both patients resulting in a p.H395R substitution in the MKKS/BBS6 protein. This mutant protein decreased the interaction of MKKS/BBS6 with BBS12 but did so to a different extent in the HEK-293T versus ARPE-19 cells. Nonetheless, the effect of the H395R variant on disrupting interactions with BBS12 was not as profound as other reported MKKS/BBS6 mutations associated with syndromic RP. CONCLUSIONS: We identified a novel H395R substitution in MKKS/BBS6 that results in a unique phenotype of only RP and polydactyly. Our observations reaffirm the notion that mutations in MKKS/BBS6 cause phenotypic heterogeneity and do not always result in classic MKKS or BBS findings.
Assuntos
Anormalidades Múltiplas/genética , Síndrome de Bardet-Biedl/genética , Chaperoninas do Grupo II/genética , Cardiopatias Congênitas/genética , Hidrocolpos/genética , Mutação de Sentido Incorreto , Polidactilia/genética , Retinose Pigmentar/genética , Doenças Uterinas/genética , Adolescente , Western Blotting , Consanguinidade , Análise Mutacional de DNA , Feminino , Células HEK293 , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Linhagem , Plasmídeos , Epitélio Pigmentado da Retina/citologia , Irmãos , Tomografia de Coerência ÓpticaRESUMO
OBJECTIVE: To correlate corneal endothelium-Descemet membrane (EDM) layer parameters of scroll tightness with donor age, endothelial cell density (ECD), and history of diabetes. METHODS: Endothelium-Descemet membrane layer scrolls were harvested from 26 corneoscleral buttons using the SCUBA technique by a cornea-fellowship trained ophthalmologist masked to donor age. Two independent outcome parameters were used to characterize the scrolling severity of successfully harvested tissue: scroll width and tendency for EDM scroll formation (referred to as scroll rating on a 1-4 scale: incomplete scroll formation to tightly scrolled). RESULTS: Mean donor age was 59 ± 17 (15-69) years. Mean ECD of EDM scroll was 2,451 ± 626 (range: 1,307-3,195) cells per square millimeter. Using stepwise linear regression, a significant correlation was found between scroll width and donor age (R=0.497, P<0.05). Additionally, a significant inverse correlation was found between scroll width and ECD (R=-0.605, P<0.05). There was no statistically significant correlation between a donor history of diabetes and the parameters of scrolling tendency. CONCLUSIONS: Our data suggest that using older donors reduces EDM scroll tightness.
Assuntos
Perda de Células Endoteliais da Córnea/patologia , Lâmina Limitante Posterior/fisiologia , Ceratoplastia Endotelial com Remoção da Lâmina Limitante Posterior , Endotélio Corneano/fisiologia , Doadores de Tecidos , Adolescente , Adulto , Fatores Etários , Idoso , Fenômenos Biomecânicos , Contagem de Células , Lâmina Limitante Posterior/cirurgia , Diabetes Mellitus , Endotélio Corneano/citologia , Endotélio Corneano/transplante , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Análise de Regressão , Adulto JovemRESUMO
PURPOSE: To describe the phenotypic characteristics and clinical course of a sporadic case of congenital fibrosis of the extraocular muscles (CFEOM) and Möbius syndrome with a de novo mutation in the KIF21A gene encoding a kinesin motor protein. METHODS: An individual with the rare combination of CFEOM and Möbius syndrome underwent comprehensive ophthalmologic and neurological evaluations. Magnetic resonance imaging (MRI) including diffusion tensor imaging (DTI) tractigraphy at 3T field strength was used to evaluate orbital, encephalic, and intracranial nerve integrity. The proband and her healthy parents underwent screening for mutations in the KIF21A, PHOX2A, and TUBB3 genes. RESULTS: The patient exhibited congenital, nonprogressive, bilateral external ophthalmoplegia, bilateral ptosis, bilateral facial palsy, and developmental delay. Her inability to blink resulted in severe exposure keratopathy and subsequent corneal perforation requiring a penetrating keratoplasty. MRI revealed an unremarkable configuration of the axial central nervous system and preservation of the intracranial portion of cranial nerves I, II, III, V, VI, VII, and VIII (cranial nerve IV is not normally visualized by MRI). A novel and de novo heterozygous KIF21A mutation (c.1056C>G, p.Asp352Glu) in a highly conserved region of the gene was present in the proband. CONCLUSIONS: The reported KIF21A D352E mutation and associated phenotype further expand the clinical and mutational spectrum of CFEOM and Möbius syndrome.
Assuntos
Oftalmopatias Hereditárias/genética , Fibrose/genética , Cinesinas/genética , Síndrome de Möbius/genética , Mutação/genética , Transtornos da Motilidade Ocular/genética , Sequência de Aminoácidos , Sequência de Bases , Criança , Sequência Conservada , Análise Mutacional de DNA , Oftalmopatias Hereditárias/fisiopatologia , Feminino , Fibrose/fisiopatologia , Fixação Ocular , Humanos , Cinesinas/química , Imageamento por Ressonância Magnética , Síndrome de Möbius/fisiopatologia , Dados de Sequência Molecular , Transtornos da Motilidade Ocular/fisiopatologiaRESUMO
BACKGROUND: Late-onset Fuchs' endothelial corneal dystrophy (FECD) is a degenerative disease of cornea and the leading indication for corneal transplantation. Genetically, FECD patients can be categorized as with (RE+) or without (RE-) the CTG trinucleotide repeat expansion in the transcription factor 4 gene. The molecular mechanisms underlying FECD remain unclear, though there are plausible pathogenic models proposed for RE+ FECD. METHOD: In this study, we performed a meta-analysis on RNA sequencing datasets of FECD corneal endothelium including 3 RE+ datasets and 2 RE- datasets, aiming to compare the transcriptomic profiles of RE+ and RE- FECD. Gene differential expression analysis, co-expression networks analysis, and pathway analysis were conducted. RESULTS: There was a striking similarity between RE+ and RE- transcriptomes. There were 1,184 genes significantly upregulated and 1,018 genes significantly downregulated in both RE+ and RE- cases. Pathway analysis identified multiple biological processes significantly enriched in both-mitochondrial functions, energy-related processes, ER-nucleus signaling pathway, demethylation, and RNA splicing were negatively enriched, whereas small GTPase mediated signaling, actin-filament processes, extracellular matrix organization, stem cell differentiation, and neutrophil mediated immunity were positively enriched. The translational initiation process was downregulated in the RE+ transcriptomes. Gene co-expression analysis identified modules with relatively distinct biological processes enriched including downregulation of mitochondrial respiratory chain complex assembly. The majority of oxidative phosphorylation (OXPHOS) subunit genes, as well as their upstream regulator gene estrogen-related receptor alpha (ESRRA), encoding ERRα, were downregulated in both RE+ and RE- cases, and the expression level of ESRRA was correlated with that of OXPHOS subunit genes. CONCLUSION: Meta-analysis increased the power of detecting differentially expressed genes. Integrating differential expression analysis with co-expression analysis helped understand the underlying molecular mechanisms. FECD RE+ and RE- transcriptomic profiles are much alike with the hallmark of downregulation of genes in pathways related to ERRα-mediated OXPHOS.
Assuntos
Endotélio Corneano , Distrofia Endotelial de Fuchs , Humanos , Endotélio Corneano/metabolismo , Fosforilação Oxidativa , Fator de Transcrição 4/genética , Distrofia Endotelial de Fuchs/genética , Distrofia Endotelial de Fuchs/patologia , Perfilação da Expressão GênicaRESUMO
Purpose: In the United States, 70% of Fuchs' endothelial corneal dystrophy (FECD) cases are caused by an intronic trinucleotide repeat expansion in the TCF4 gene. CUG repeat RNA transcripts from this expansion accumulate as nuclear foci in the corneal endothelium. In this study, we sought to detect foci in other anterior segment cell types and assess their molecular impact. Methods: We examined CUG repeat RNA foci appearance, expression of downstream affected genes, gene splicing, and TCF4 RNA expression in corneal endothelium, corneal stromal keratocytes, corneal epithelium, trabecular meshwork cells, and lens epithelium. Results: CUG repeat RNA foci, the hallmark of FECD in corneal endothelium (found in 84% of endothelial cells), are less detectable in trabecular meshwork cells (41%), much less prevalent in stromal keratocytes (11%) or corneal epithelium (4%), and absent in lens epithelium. With few exceptions including mis-splicing in the trabecular meshwork, differential gene expression and splicing changes associated with the expanded repeat in corneal endothelial cells are not observed in other cell types. Expression of the TCF4 transcripts including full-length isoforms containing the repeat sequence at the 5' end is much higher in the corneal endothelium or trabecular meshwork than in the corneal stroma or corneal epithelium. Conclusions: Expression of the CUG repeat containing TCF4 transcripts is higher in the corneal endothelium, likely contributing to foci formation and the large molecular and pathologic impact on those cells. Further studies are warranted to examine any glaucoma risk and impact of the observed foci in the trabecular meshwork of these patients.
Assuntos
Distrofia Endotelial de Fuchs , Expansão das Repetições de Trinucleotídeos , Humanos , Expansão das Repetições de Trinucleotídeos/genética , Células Endoteliais/metabolismo , Fator de Transcrição 4/genética , Distrofia Endotelial de Fuchs/metabolismo , Endotélio Corneano/metabolismo , RNA/genética , RNA/metabolismoRESUMO
Objective: Seventy percent of Fuchs' endothelial corneal dystrophy (FECD) cases are caused by an intronic trinucleotide repeat expansion in the transcription factor 4 gene (TCF4). The objective of this study was to characterize the corneal subbasal nerve plexus and corneal haze in patients with FECD with (RE+) and without the trinucleotide repeat expansion (RE-) and to assess the correlation of these parameters with disease severity. Design: Cross-sectional, single-center study. Participants: Fifty-two eyes of 29 subjects with a modified Krachmer grade of FECD severity from 1 to 6 were included in the study. Fifteen of the 29 subjects carried an expanded TCF4 allele length of ≥ 40 cytosine-thymine-guanine repeats (RE+). Main Outcomes Measures: In vivo confocal microscopy assessments of corneal nerve fiber length (CNFL), corneal nerve branch density, corneal nerve fiber density (CNFD), and anterior corneal stromal backscatter (haze); Scheimpflug tomography densitometry measurements of haze in anterior, central, and posterior corneal layers. Results: Using confocal microscopy, we detected a negative correlation between FECD severity and both CNFL and CNFD in the eyes of RE+ subjects (Spearman ρ = -0.45, P = 0.029 and ρ = -0.62, P = 0.0015, respectively) but not in the eyes of RE- subjects. Additionally, CNFD negatively correlated with the repeat length of the expanded allele in the RE+ subjects (Spearman ρ = -0.42, P = 0.038). We found a positive correlation between anterior stromal backscatter and severity in both the RE+ and RE- groups (ρ = 0.60, P = 0.0023 and ρ = 0.44, P = 0.024, respectively). The anterior, central, and posterior Scheimpflug densitometry measurements also positively correlated with severity in both the RE+ and RE- groups (P = 5.5 × 10-5, 2.5 × 10-4, and 2.9 × 10-4, respectively, after adjusting for the expansion status in a pooled analysis. However, for patients with severe FECD (Krachmer grades 5 and 6), the posterior densitometry measurements were higher in the RE+ group than in the RE- group (P < 0.05). Conclusions: Loss of corneal nerves in FECD supports the classification of the TCF4 trinucleotide repeat expansion disorder as a neurodegenerative disease. Haze in the anterior, central, and posterior cornea correlate with severity, irrespective of the genotype. Quantitative assessments of corneal nerves and corneal haze may be useful to gauge and monitor FECD disease severity in RE+ patients.
RESUMO
One advantage of antisense oligonucleotides (ASOs) for drug development is their long-lasting gene knockdown after administration in vivo. In this study, we examine the effect on gene expression after intraocular injection in target tissues in the eye. We examined expression levels of the Malat1 gene after intracameral or intravitreal (IV) injection of an anti-Malat1 ASO in corneal epithelium/stroma, corneal endothelium, lens capsule epithelium, neurosensory retina, and retinal pigment epithelium/choroid of the mouse eye. We assessed potency of the compound at 7 days as well as duration of the gene knockdown at 14, 28, 60, 90, and 120 days. The ASO was more potent when delivered by IV injection relative to intracameral injection, regardless of whether the tissues analyzed were at the front or back of the eye. For corneal endothelium, inhibition was >50% after 120 days for ASO at 50 µg. At IV dosages of 6 µg, we observed >75% inhibition of gene expression in the retina and lens epithelium for up to 120 days. ASOs have potential as long-lasting gene knockdown agents in the mouse eye, but efficacy varies depending on the specific ocular target tissue and injection protocol.
Assuntos
Oligonucleotídeos Antissenso , Retina , Camundongos , Animais , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/farmacologia , Endotélio Corneano , Expressão GênicaRESUMO
Fuchs' corneal endothelial dystrophy (FECD) is a major cause of vision loss. Corneal transplantation is the only effective curative treatment, but this surgery has limitations. A pharmacological intervention would complement surgery and be beneficial for many patients. FECD is caused by an expanded CUG repeat within intron 2 of the TCF4 RNA. Agents that recognize the expanded repeat can reverse the splicing defects associated with the disease. Successful drug development will require diverse strategies for optimizing the efficacy of anti-CUG oligomers. In this study, we evaluate anti-CUG morpholinos conjugated to cyclic cell penetrating peptides. The morpholino domain of the conjugate is complementary to the repeat, while the peptide has been optimized for import across cell membranes. We show that morpholino conjugates can enter corneal endothelial cells and block the CUG RNA foci associated with the disease. These experiments support morpholino peptide conjugates as an approach for developing anti-CUG therapies for FECD.
RESUMO
Purpose: We sought to define the role of Wwtr1 in murine ocular structure and function and determine the role of mechanotransduction in Fuchs' endothelial corneal dystrophy (FECD), with emphasis on interactions between corneal endothelial cells (CEnCs) and Descemet's membrane (DM). Methods: A Wwtr1 deficient mouse colony was established, and advanced ocular imaging, atomic force microscope (AFM), and histology/immunofluorescence were performed. Corneal endothelial wound healing was assessed using cryoinjury and phototherapeutic keratectomy in Wwtr1 deficient mice. Expression of WWTR1/TAZ was determined in the corneal endothelium from normal and FECD-affected patients; WWTR1 was screened for coding sequence variants in this FECD cohort. Results: Mice deficient in Wwtr1 had reduced CEnC density, abnormal CEnC morphology, softer DM, and thinner corneas versus wildtype controls by 2 months of age. Additionally, CEnCs had altered expression and localization of Na/K-ATPase and ZO-1. Further, Wwtr1 deficient mice had impaired CEnC wound healing. The WWTR1 transcript was highly expressed in healthy human CEnCs comparable to other genes implicated in FECD pathogenesis. Although WWTR1 mRNA expression was comparable between healthy and FECD-affected patients, WWTR1/TAZ protein concentrations were higher and localized to the nucleus surrounding guttae. No genetic associations were found in WWTR1 and FECD in a patient cohort compared to controls. Conclusions: There are common phenotypic abnormalities seen between Wwtr1 deficient and FECD-affected patients, suggesting that Wwtr1 deficient mice could function as a murine model of late-onset FECD. Despite the lack of a genetic association between FECD and WWTR1, aberrant WWTR1/TAZ protein subcellular localization and degradation may play critical roles in the pathogenesis of FECD.
Assuntos
Células Endoteliais , Distrofia Endotelial de Fuchs , Humanos , Camundongos , Animais , Células Endoteliais/metabolismo , Mecanotransdução Celular , Distrofia Endotelial de Fuchs/patologia , Endotélio Corneano/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional , Proteínas Adaptadoras de Transdução de Sinal/metabolismoRESUMO
OBJECTIVES: The prevalence of primary open-angle glaucoma (POAG) in patients with corneal endothelial dystrophy has not been previously studied. Prevalence of POAG in patients with endothelial dystrophy was compared with that in the general population to determine the presence of a relationship between the diseases. DESIGN: Retrospective case-control study. METHODS: A study of the prevalence of POAG in 430 eyes of 215 patients with endothelial dystrophy was conducted. Patients followed for less than 6 months were excluded. Relative risk of POAG was calculated using age- and race-matched control data from the Baltimore Eye Survey and the Los Angeles Latino Eye Survey for comparison. Ocular hypertension (OHT) and secondary glaucoma (SG) rates after penetrating keratoplasty (PK) and Descemet stripping endothelial keratoplasty (DSEK) were separately analyzed. RESULTS: Relative risk of POAG in white, African American, and Hispanic patients with endothelial dystrophy was 0.94, 2.59, and 3.7, respectively (P = 0.89, 95% confidence interval [CI], -0.028 to 0.0289; P = 0.13; 95% CI, 0.011-0.274; P = 0.055; 95% CI, 0.0423-0.356). Relative risk of SG and combined OHT/SG in PK versus DSEK was 4.15 and 1.95 (P < 0.001; 95% CI, 0.0654-0.322; P = 0.005; 95% CI, 0.116-0.332), respectively. No differences in OHT/SG rates were found comparing PK-triple with PK, DSEK-triple with DSEK, and repeat with primary PK or DSEK (P = 0.98; P = 0.62; P = 0.95; P = 0.87), respectively. CONCLUSIONS: No increased risk of POAG was found in patients with endothelial dystrophy. Increased prevalence of OHT/SG was shown with PK versus DSEK; possible mechanisms include mechanical closure of Schlemm's canal by running suture and prolonged steroid use.
Assuntos
Distrofia Endotelial de Fuchs/complicações , Glaucoma de Ângulo Aberto/epidemiologia , Negro ou Afro-Americano , Estudos de Casos e Controles , Feminino , Distrofia Endotelial de Fuchs/etnologia , Distrofia Endotelial de Fuchs/cirurgia , Glaucoma de Ângulo Aberto/etnologia , Hispânico ou Latino , Humanos , Masculino , Hipertensão Ocular/epidemiologia , Hipertensão Ocular/etnologia , Prevalência , Estudos Retrospectivos , Fatores de Risco , Estados Unidos/epidemiologiaRESUMO
PURPOSE: To compare the corneal higher-order aberration (HOA) after Descemet's stripping automated endothelial keratoplasty (DSAEK) and penetrating keratoplasty (PKP), and in age-matched controls. DESIGN: Cross-sectional, non-comparative interventional case series. PARTICIPANTS: Thirty-one eyes of 28 patients who underwent DSAEK, 20 eyes of 16 patients who underwent PKP, and 31 eyes of 31 control patients. INTERVENTION: The corneal topography and HOAs of the central 4- and 6-mm zones from anterior and posterior corneal surfaces were evaluated postoperatively with the Scheimpflug rotating imaging system (Oculus Gmbh, Wetzlar, Germany). MAIN OUTCOME MEASURES: Anterior and posterior corneal HOAs. RESULTS: The mean anterior corneal total HOAs of the central 4 and 6 mm were 0.599+/-0.288 microm and 1.215+/-0.496 microm, respectively, in eyes that underwent DSAEK; 1.730+/-0.826 microm and 3.349+/-1.490 microm, respectively, in eyes that underwent PKP; and 0.439+/-0.163 microm and 0.921+/-0.300 microm, respectively, in controls. Although the mean anterior corneal total HOAs of the central 4 and 6 mm were significantly higher in eyes that underwent PKP than in eyes that underwent DSAEK and in controls (P<0.01), there was no significant difference in anterior corneal total HOAs of the central 4 and 6 mm between eyes that underwent DSAEK and controls. The mean posterior corneal total HOAs of the central 4 and 6 mm were 3.680+/-1.586 microm and 7.142+/-3.011 microm, respectively, in eyes that underwent DSAEK; 2.957+/-1.238 microm and 5.314+/-2.095 microm, respectively, in eyes that underwent PKP; and 0.818+/-0.193 microm and 1.609+/-0.344 microm, respectively, in controls. Although there was no significant difference in posterior corneal total HOAs of the central 4 mm between the DSAEK group and the PKP group, the posterior corneal HOAs of the central 6 mm were significantly higher in the DSAEK group than in the PKP group (P<0.01). CONCLUSIONS: Although posterior corneal HOAs are significantly higher in eyes that underwent DSAEK, anterior corneal HOAs are not significantly different in eyes that underwent DSAEK than those of age-matched controls.
Assuntos
Aberrações de Frente de Onda da Córnea/etiologia , Ceratoplastia Endotelial com Remoção da Lâmina Limitante Posterior , Ceratoplastia Penetrante , Complicações Pós-Operatórias , Adulto , Idoso , Idoso de 80 Anos ou mais , Doenças da Córnea/cirurgia , Topografia da Córnea , Estudos Transversais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Refração Ocular/fisiologia , Acuidade Visual/fisiologiaRESUMO
OBJECTIVE: To compare the effect on aqueous tear (AT) evaporation rate of Systane and Optive at 30 min postinstillation in patients with dry eye. METHODS: In a crossover study of 20 patients with keratoconjunctivitis sicca, the evaporation rate of AT was measured. Evaporometry was used at two relative humidity (RH) ranges of 25% to 35% and 35% to 45%. The measurements were made at baseline (before the instillation of the study agent) and at 30 min after the instillation of 40 µL of either Systane or Optive per randomization assignment per visit with a 1-week interval between visits. RESULTS: No significant effects on AT evaporation rates at both RHs were found between study agents. CONCLUSIONS: In our study, neither Systane nor Optive has a significant impact on AT evaporation at 30 min postinstillation in patients with dry eye.
Assuntos
Ceratoconjuntivite Seca/tratamento farmacológico , Soluções Oftálmicas/química , Soluções Oftálmicas/uso terapêutico , Adulto , Idoso , Estudos Cross-Over , Feminino , Humanos , Umidade , Masculino , Pessoa de Meia-Idade , Volatilização , Adulto JovemRESUMO
PURPOSE: To identify the incidence of and risk factors for intraocular pressure (IOP) elevation after Descemet stripping automated endothelial keratoplasty (DSAEK). METHODS: Retrospective review was conducted of 68 consecutive DSAEK procedures alone, or in combination with phacoemulsification with intraocular lens implantation or exchange, performed by two surgeons at the University of Texas Southwestern Medical Center between 2005 and 2009. Eyes that developed IOP elevation above 21 mm Hg after DSAEK and requiring initiation or escalation of glaucoma therapy were evaluated. RESULTS: Thirty-seven (54%) eyes showed IOP elevation responsive to medical treatment by a mean follow-up of 11.38 +/- 7.81 months. Six (8.8%) eyes required glaucoma surgery. In the eyes, which developed elevated IOP, gonioscopy did not reveal any new peripheral anterior synechiae formation. Prolonged topical steroid usage, rebubbling, combined DSAEK/cataract surgery, or repeat DSAEK were not significant factors (P>0.05) for development of elevated IOP, but history of previous glaucoma or ocular hypertension (OHTN) was significant (P=0.007). CONCLUSIONS: Intraocular pressure elevation is not uncommon in eyes after DSAEK, but most cases can be controlled with conservative management. Intraocular pressure elevation post-DSAEK occurred by mechanisms other than peripheral anterior synechial angle closure. The only significant risk factor for development of elevated IOP in our series was a previous history of glaucoma or OHTN.
Assuntos
Doenças da Córnea/cirurgia , Transplante de Córnea/efeitos adversos , Lâmina Limitante Posterior/cirurgia , Endotélio Corneano/transplante , Pressão Intraocular , Hipertensão Ocular/etiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Doenças da Córnea/fisiopatologia , Transplante de Córnea/métodos , Lâmina Limitante Posterior/patologia , Endotélio Corneano/patologia , Feminino , Seguimentos , Humanos , Incidência , Masculino , Pessoa de Meia-Idade , Hipertensão Ocular/epidemiologia , Hipertensão Ocular/fisiopatologia , Complicações Pós-Operatórias , Estudos Retrospectivos , Fatores de Risco , Texas/epidemiologia , Tomografia de Coerência ÓpticaRESUMO
Purpose: Fuchs' endothelial corneal dystrophy (FECD) is the leading indication for corneal transplantation. Seventy percent of cases are caused by an intronic CTG triplet repeat expansion in the TCF4 gene that results in accumulation of pathogenic expanded CUG repeat RNA (CUGexp) as nuclear foci in corneal endothelium. A catalytically dead Cas9 (dCas9) can serve as an effective guide to target genomic DNA or RNA transcripts. Here, we examined the utility of the clustered regularly interspaced short palindromic repeats (CRISPR)-dCas9 system to effectively target and reduce CUGexp. Methods: We delivered dCas9 and repeat-targeting single guide RNA (sgRNA) expression plasmids to patient-derived endothelial cells using lipofection or lentiviral transduction. We used fluorescence in situ hybridization (FISH) and RNA dot-blot hybridization to quantify CUGexp foci and repeat RNA levels, respectively. TCF4 expression levels were assessed using quantitative PCR (qPCR). Results: Using FISH, we found that expression of both dCas9 and a (CAG)n sgRNA complementary to CUGexp are necessary to reduce foci. We observed a reduction in percentage of cells with foci from 59% to 5.6% and number of foci per 100 cells from 73.4 to 7.45 (P < 0.001) in cells stably expressing dCas9-(CAG)n sgRNA but saw no decrease in cells expressing dCas9-(CUG)n sgRNA or nontargeting control sgRNA. In cells with dCas9-(CAG)n sgRNA, we detected a reduction in CUGexp RNA by dot-blot without any reduction in TCF4 mRNA levels using qPCR. Conclusions: Using CRISPR-dCas9 to target the trinucleotide repeat is a promising treatment for FECD contingent on effective in vivo delivery. Translational Relevance: This work advances a gene therapy for a common age-related degenerative disorder.
Assuntos
Células Endoteliais , Distrofia Endotelial de Fuchs , Distrofia Endotelial de Fuchs/genética , Humanos , Hibridização in Situ Fluorescente , Fator de Transcrição 4/genética , Expansão das Repetições de Trinucleotídeos/genéticaRESUMO
PURPOSE: To investigate the long-term corneal changes in patients with Fuchs endothelial corneal dystrophy contributing to superior postoperative visual outcomes after Descemet membrane endothelial keratoplasty (DMEK) compared with Descemet stripping automated endothelial keratoplasty (DSAEK). METHODS: Using retrospective analysis, we evaluated 9 patients with Fuchs endothelial corneal dystrophy who underwent DSAEK in 1 eye and DMEK in the fellow eye. Patients were genotyped for the triplet repeat expansion in the TCF4 gene and imaged using optical coherence tomography, Scheimpflug imaging, and in vivo confocal microscopy through focusing. RESULTS: Eight of 9 subjects were genotyped, and all were found to harbor the triplet repeat expansion. The average time between endothelial keratoplasty and imaging was 76 ± 22 and 37 ± 9 months after DSAEK and DMEK, respectively. The mean best spectacle-corrected visual acuity (logMAR) was 0.04 ± 0.05 and 0.11 ± 0.03 in the DMEK eyes versus DSAEK eyes (P = 0.02), respectively. Posterior corneal higher order aberrations were less in the DMEK eyes compared with fellow DSAEK eyes (0.25 ± 0.06 and 0.66 ± 0.25, respectively, P ≤ 0.01). Using confocal microscopy through focusing, we found that the persistent anterior stromal haze was correlated between the right and left eyes (R = 0.73, P ≤ 0.05), but total stromal backscattering was higher for the DSAEK eyes (P ≤ 0.05). CONCLUSIONS: DSAEK inherently results in higher total stromal backscattering (haze) compared with DMEK because of the addition of stromal tissue. Lower higher order aberrations of the posterior cornea and lower total stromal backscattering (haze) may both contribute to superior visual outcomes after DMEK compared with DSAEK.
Assuntos
Opacidade da Córnea/fisiopatologia , Substância Própria/fisiopatologia , Aberrações de Frente de Onda da Córnea/fisiopatologia , Ceratoplastia Endotelial com Remoção da Lâmina Limitante Posterior/métodos , Distrofia Endotelial de Fuchs/cirurgia , Aberrometria , Idoso , Idoso de 80 Anos ou mais , Feminino , Distrofia Endotelial de Fuchs/genética , Distrofia Endotelial de Fuchs/fisiopatologia , Técnicas de Genotipagem , Humanos , Masculino , Microscopia Confocal , Estudos Retrospectivos , Tomografia de Coerência Óptica , Fator de Transcrição 4/genética , Expansão das Repetições de Trinucleotídeos/genética , Acuidade Visual/fisiologiaRESUMO
Antisense oligonucleotides (ASOs) are synthetic nucleic acids that recognize complementary RNA sequences inside cells and modulate gene expression. In this study, we explore the feasibility of ASO delivery to the cornea. We used quantitative polymerase chain reaction to test the efficacy of a benchmark ASO targeting a noncoding nuclear RNA, Metastasis-Associated Lung Adenocarcinoma Transcript 1 (MALAT1), in a human corneal endothelial cell line, ex vivo human corneas, and in vivo in mice. In vivo delivery was via intravitreal or intracameral injections as well as topical administration. The anti-MALAT1 ASO significantly reduced expression of MALAT1 in a corneal endothelial cell line. We achieved a dose-dependent reduction of target gene expression in endothelial tissue from ex vivo human donor corneas. In vivo mouse experiments confirmed MALAT1 reduction in whole corneal tissue via intravitreal and intracameral routes, 82% and 71% knockdown, respectively (P < 0.001). Effects persisted up to at least 21 days, 32% (P < 0.05) and 43% (P < 0.05) knockdown, respectively. We developed protocols for the isolation and analysis of mouse corneal endothelium and observed reduction in MALAT1 expression upon both intravitreal and intracameral administrations, 64% (P < 0.05) and 63% (P < 0.05) knockdown, respectively. These data open the possibility of using ASOs to treat corneal disease.
Assuntos
Doenças da Córnea/terapia , Distrofia Endotelial de Fuchs/terapia , Oligonucleotídeos Antissenso/farmacologia , RNA Longo não Codificante/genética , Animais , Córnea/efeitos dos fármacos , Córnea/patologia , Doenças da Córnea/genética , Doenças da Córnea/patologia , Modelos Animais de Doenças , Endotélio Corneano/efeitos dos fármacos , Endotélio Corneano/patologia , Distrofia Endotelial de Fuchs/genética , Distrofia Endotelial de Fuchs/patologia , Humanos , Camundongos , RNA Longo não Codificante/antagonistas & inibidoresRESUMO
OBJECTIVES: To report the efficacy and safety profile of sutureless and adhesiveless amniotic membrane device (ProKera, Bio-Tissue, Inc., Miami, FL) in the management of various ocular surface and orbital disorders. METHODS: Twenty eyes of 20 patients underwent placement of 21 ProKera implants between March 2006 and July 2007 at the University of Texas Southwestern Medical Center. Patient demographics, indications for placement, and duration of ProKera retention were recorded. Outcomes measured included corneal epithelial healing, visual acuity, patient tolerance, and adverse events. RESULTS: ProKera was most commonly used in patients with corneal neovascularization with or without limbal stem-cell deficiency (10 eyes). Other indications included recurrent pterygium or pseudopterygium (three eyes), anophthalmic orbit contraction (two eyes), persistent epithelial defects (two eyes), severe thinning in a corneal ulcer (one eye), benign hereditary intraepithelial dyskeratosis (one eye), and band keratopathy (one eye). The mean duration of ProKera retention was 25.3 days (range, 0-125) visual acuity improved in 12 eyes (60%). Immediate adverse events included residual epithelial defects after removal (five eyes) and spontaneous extrusion of the implant (four eyes). Six patients (30%) reported eye pain or headache and four eyes (20%) had recurrence of the primary pathology. CONCLUSIONS: Sutureless and adhesiveless amniotic membrane transplantation is a safe and effective method to promote healing and reconstruction of the ocular surface and orbit with minimal side effects. Recurrence of the underlying primary pathology remains a concern. The advent of a newer, softer conformer ring may improve patient tolerability and limit discomfort.