RESUMEN
TGFBIp, also known as keratoepithelin and ßig-h3, is among the most abundant proteins in the human cornea, and approximately 60% is associated with the insoluble fraction following extraction in sodium dodecyl sulfate (SDS) sample buffer. TGFBIp is of particular interest because a wide range of mutations causes amyloid or fuchsinophilic crystalloid deposits in the cornea leading to visual impairment. We show that the SDS-insoluble fraction of TGFBIp from porcine and human corneas is covalently linked via a reducible bond to the NC3 domain of type XII collagen in a TGFBIp:type XII collagen stoichiometric ratio of 2:1. Because type XII collagen is anchored to striated collagen fibers of the extracellular matrix, its interaction with TGFBIp is likely to provide anchoring for cells to the extracellular matrix through the integrin binding capability of TGFBIp. Furthermore, the TGFBIp-type XII collagen molecule will affect our understanding of the molecular pathogenesis of the TGFBI-linked corneal dystrophies.
Asunto(s)
Colágeno Tipo XII/química , Colágeno Tipo XII/metabolismo , Córnea/química , Proteínas de la Matriz Extracelular/química , Factor de Crecimiento Transformador beta/química , Animales , Disulfuros/química , Disulfuros/metabolismo , Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Humanos , Peso Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Solubilidad , Porcinos , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Fuchs corneal dystrophy (FCD) is a degenerative genetic disorder of the corneal endothelium that represents one of the most common causes of corneal transplantation in the United States. Despite its high prevalence (4% over the age of 40), the underlying genetic basis of FCD is largely unknown. Here we report missense mutations in TCF8, a transcription factor whose haploinsufficiency causes posterior polymorphous corneal dystrophy (PPCD), in a cohort of late-onset FCD patients. In contrast to PPCD-causing mutations, all of which are null, FCD-associated mutations encode rare missense changes suggested to cause loss of function by an in vivo complementation assay. Importantly, segregation of a recurring p.Q840P mutation in a large, multigenerational FCD pedigree showed this allele to be sufficient but not necessary for pathogenesis. Execution of a genome-wide scan conditioned for the presence of the 840P allele identified an additional late-onset FCD locus on chromosome 9p, whereas haplotype analysis indicated that the presence of the TCF8 allele and the disease haplotype on 9p leads to a severe FCD manifestation with poor prognosis. Our data suggest that PPCD and FCD are allelic variants of the same disease continuum and that genetic interaction between genes that cause corneal dystrophies can modulate the expressivity of the phenotype.
Asunto(s)
Cromosomas Humanos Par 9/genética , Distrofias Hereditarias de la Córnea/genética , Distrofia Endotelial de Fuchs/genética , Proteínas de Homeodominio/genética , Mutación Missense , Factores de Transcripción/genética , Adulto , Edad de Inicio , Anciano , Alelos , Análisis Mutacional de ADN , Femenino , Predisposición Genética a la Enfermedad , Humanos , Masculino , Persona de Mediana Edad , Homeobox 1 de Unión a la E-Box con Dedos de ZincRESUMEN
PURPOSE: Specific mutations in the transforming growth factor beta induced (TGFBI) gene are associated with lattice corneal dystrophy (LCD) type 1 and its variants. In this study, we performed an in-depth proteomic analysis of human corneal amyloid deposits associated with the heterozygous A546D mutation in TGFBI. METHODS: Corneal amyloid deposits and the surrounding corneal stroma were procured by laser capture microdissection from a patient with an A546D mutation in TGFBI. Proteins in the captured corneal samples and healthy corneal stroma were identified with liquid chromatography-tandem mass spectrometry and quantified by calculating exponentially modified Protein Abundance Index values. Mass spectrometry data were further compared for identifying enriched regions of transforming growth factor beta induced protein (TGFBIp/keratoepithelin/ßig-h3) and detecting proteolytic cleavage sites in TGFBIp. RESULTS: A C-terminal fragment of TGFBIp containing residues Y571-R588 derived from the fourth fasciclin 1 domain (FAS1-4), serum amyloid P-component, apolipoprotein A-IV, clusterin, and serine protease HtrA1 were significantly enriched in the amyloid deposits compared to the healthy cornea. The proteolytic cleavage sites in TGFBIp from the diseased cornea are in accordance with the activity of serine protease HtrA1. We also identified small amounts of the serine protease kallikrein-14 in the amyloid deposits. CONCLUSIONS: Corneal amyloid caused by the A546D mutation in TGFBI involves several proteins associated with other varieties of amyloidosis. The proteomic data suggest that the sequence 571-YHIGDEILVSGGIGALVR-588 contains the amyloid core of the FAS1-4 domain of TGFBIp and point at serine protease HtrA1 as the most likely candidate responsible for the proteolytic processing of amyloidogenic and aggregated TGFBIp, which explains the accumulation of HtrA1 in the amyloid deposits. With relevance to identifying serine proteases, we also found glia-derived nexin (protease-nexin 1) in the amyloid deposits, making this serine protease inhibitor a good candidate for the physiologically relevant inhibitor of one of the amyloid-associated serine proteases in the cornea and probably in other tissues. Noteworthy, the present results are in accordance with our findings from a previous study of corneal amyloid deposits caused by the V624M mutation in TGFBI, suggesting a common mechanism for lattice corneal dystrophies (LCDs) associated with mutations in the TGFBIp FAS1-4 domain.
Asunto(s)
Córnea/metabolismo , Córnea/patología , Proteínas de la Matriz Extracelular/metabolismo , Placa Amiloide/metabolismo , Serina Endopeptidasas/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Anciano , Secuencia de Aminoácidos , Análisis por Conglomerados , Distrofias Hereditarias de la Córnea/metabolismo , Distrofias Hereditarias de la Córnea/patología , Sustancia Propia/metabolismo , Sustancia Propia/patología , Proteínas de la Matriz Extracelular/química , Femenino , Serina Peptidasa A1 que Requiere Temperaturas Altas , Humanos , Microdisección , Datos de Secuencia Molecular , Inhibidores de Proteasas/metabolismo , Estructura Terciaria de Proteína , Proteolisis , Proteómica , Alineación de Secuencia , Espectrometría de Masas en Tándem , Factor de Crecimiento Transformador beta/química , Tripsina/metabolismoRESUMEN
PURPOSE: Fuchs endothelial corneal dystrophy (FECD) is a genetically heterogeneous disorder that has been primarily studied in patients of European or Asian ancestry. Given the sparse literature on African Americans with FECD, we sought to characterize the genetic variation in three known FECD candidate genes in African American patients with FECD. METHODS: Over an 8-year period, we enrolled 47 African American probands with FECD. All participants were clinically examined with slit-lamp biomicroscopy, and when corneal tissue specimens were available, histopathologic confirmation of the clinical diagnosis was obtained. The coding regions of known FECD susceptibility genes collagen, type VIII, alpha 2 (COL8A2); solute carrier family 4, sodium borate transporter, member 11 (SLC4A11); and zinc finger E-box binding homeobox 1 (ZEB1 [also known as TCF8]) were Sanger sequenced in the 47 probands using DNA isolated from blood samples. RESULTS: Twenty-two coding variants were detected across the COL8A2, SLC4A11, and ZEB1 genes; six were nonsynonymous variants. Three novel coding variants were detected: a synonymous variant each in COL8A2 and SLC4A11 and one nonsynonymous variant in ZEB1 (p.P559S), which is predicted to be benign and tolerated, thus making its physiologic consequence uncertain. CONCLUSIONS: Variation in the COL8A2, SLC4A11, and ZEB1 genes is present in only a small fraction of our African American cases and as such does not appear to significantly contribute to the genetic risk of FECD in African Americans. This observation is on par with findings from previous sequencing studies involving European or Asian ancestry patients with FECD.
Asunto(s)
Proteínas de Transporte de Anión/genética , Antiportadores/genética , Negro o Afroamericano , Colágeno Tipo VIII/genética , Distrofia Endotelial de Fuchs/genética , Proteínas de Homeodominio/genética , Mutación , Factores de Transcripción/genética , Adulto , Anciano , Anciano de 80 o más Años , Córnea/metabolismo , Córnea/patología , Femenino , Distrofia Endotelial de Fuchs/etnología , Distrofia Endotelial de Fuchs/patología , Expresión Génica , Pruebas Genéticas , Variación Genética , Genotipo , Heterocigoto , Homocigoto , Humanos , Masculino , Persona de Mediana Edad , Riesgo , Análisis de Secuencia de ADN , Homeobox 1 de Unión a la E-Box con Dedos de ZincRESUMEN
Mutations in the human TGFBI gene encoding TGFBIp have been linked to protein deposits in the cornea leading to visual impairment. The protein consists of an N-terminal Cys-rich EMI domain and four consecutive fasciclin 1 (FAS1) domains. We have compared the stabilities of wild-type (WT) human TGFBIp and six mutants known to produce phenotypically distinct deposits in the cornea. Amino acid substitutions in the first FAS1 (FAS1-1) domain (R124H, R124L, and R124C) did not alter the stability. However, substitutions within the fourth FAS1 (FAS1-4) domain (A546T, R555Q, and R555W) affected the overall stability of intact TGFBIp revealing the following stability ranking R555W>WT>R555Q>A546T. Significantly, the stability ranking of the isolated FAS1-4 domains mirrored the behavior of the intact protein. In addition, it was linked to the aggregation propensity as the least stable mutant (A546T) forms amyloid fibrils while the more stable variants generate non-amyloid amorphous deposits in vivo. Significantly, the data suggested that both an increase and a decrease in the stability of FAS1-4 may unleash a disease mechanism. In contrast, amino acid substitutions in FAS1-1 did not affect the stability of the intact TGFBIp suggesting that molecular the mechanism of disease differs depending on the FAS1 domain carrying the mutation.
Asunto(s)
Sustitución de Aminoácidos , Amiloide/metabolismo , Córnea/metabolismo , Distrofias Hereditarias de la Córnea/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Mutación Missense , Factor de Crecimiento Transformador beta/metabolismo , Amiloide/genética , Distrofias Hereditarias de la Córnea/genética , Proteínas de la Matriz Extracelular/genética , Células HEK293 , Humanos , Estabilidad Proteica , Estructura Terciaria de Proteína , Factor de Crecimiento Transformador beta/genéticaRESUMEN
Different types of granular corneal dystrophy (GCD) and lattice corneal dystrophy (LCD) are associated with mutations in the transforming growth factor beta induced gene (TGFBI). These dystrophies are characterized by the formation of non-amyloid granular deposits (GCDs) and amyloid (LCD type 1 and its variants) in the cornea. Typical corneal non-amyloid deposits from GCD type 2 (R124H), amyloid from a variant of LCD type 1 (V624M) and disease-free tissue controls were procured by laser capture microdissection and analyzed by tandem mass spectrometry. Label-free quantitative comparisons of deposits and controls suggested that the non-amyloid sample (R124H) specifically accumulated transforming growth factor beta induced protein (TGFBIp/keratoepithelin/ßig-h3), serum amyloid P-component, clusterin, type III collagen, keratin 3, and histone H3-like protein. The amyloid (V624M) similarly accumulated serum amyloid P-component and clusterin but also a C-terminal fragment of TGFBIp containing residues Y571-R588 derived from the fourth fasciclin-1 domain (FAS1-4), apolipoprotein E and apolipoprotein A-IV. Significantly, analyses of the amyloid sample also revealed the presence of the serine protease Htr (High-temperature requirement) A1 and a number of proteolytic cleavage sites in the FAS1-4 domain of TGFBIp. These cleavage sites were consistent with the ligand binding and proteolytic activity of HtrA1 suggesting that it plays a role in the proteolytic processing of the amyloidogenic FAS1-4 domain. Taken together, the data suggest that the amyloidogenic-prone region of the fourth FAS1 domain of TGFBIp encompasses the Y571-R588 peptide and that HtrA1 is involved in the proteolytic processing of TGFBIp-derived amyloid in vivo.
Asunto(s)
Amiloidosis Familiar/metabolismo , Distrofias Hereditarias de la Córnea/metabolismo , Sustancia Propia/metabolismo , Proteínas de la Matriz Extracelular/genética , Mutación , Placa Amiloide/metabolismo , Factor de Crecimiento Transformador beta/genética , Amiloidosis Familiar/genética , Apolipoproteínas/metabolismo , Cromatografía Liquida , Clusterina/metabolismo , Colágeno Tipo III/metabolismo , Distrofias Hereditarias de la Córnea/genética , Humanos , Queratina-3/metabolismo , Captura por Microdisección con Láser , Proteolisis , Proteómica , Componente Amiloide P Sérico/metabolismo , Espectrometría de Masas en TándemRESUMEN
Transforming growth factor beta induced protein (TGFBIp, also named keratoepithelin) is an extracellular matrix protein abundant in the cornea. The purpose of this study was to determine the expression and processing of TGFBIp in the normal human cornea during postnatal development and aging. TGFBIp in corneas from individuals ranging from six months to 86 years of age was detected and quantified by immunoblotting. The level of TGFBIp in the cornea increases about 30% between 6 and 14 years of age, and adult corneas contain 0.7-0.8 microg TGFBIp per mg wet tissue. Two-dimensional (2-D) immunoblots of the corneal extracts showed a characteristic "zig-zag" pattern formed by different lower-molecular mass TGFBIp isoforms (30-60 kDa). However, the relative abundance of the different isoforms was different between infant corneas (<1 year) and the child/adult corneas (>6 years). Matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) data of TGFBIp isoforms separated on large 2-D gels show that TGFBIp is proteolytically processed from the N-terminus. This observation was supported by in silico 2-D gel electrophoresis showing that sequential proteolytical trimming events from the N-terminus of mature TGFBIp generate TGFBIp isoforms which form a similar "zig-zag" pattern when separated by 2-D polyacrylamide gel electrophoresis (PAGE). This study shows that in humans TGFBIp is more abundant in mature corneas than in the developing cornea and that the processing of TGFBIp changes during postnatal development of the cornea. In addition, TGFBIp appears to be degraded in a highly orchestrated manner in the normal human cornea with the resulting C-terminal fragments being retained in the cornea. The age-related changes in the expression and processing of corneal TGFBIp suggests that TGFBIp may play a role in the postnatal development and maturation of the cornea. Furthermore, these observations may be relevant to the age at which mutant TGFBIp deposits in the cornea in those dystrophies caused by mutations in the transforming growth factor beta induced gene (TGFBI) as well as the mechanisms of corneal protein deposition.
Asunto(s)
Envejecimiento/fisiología , Córnea/crecimiento & desarrollo , Córnea/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Adolescente , Anciano de 80 o más Años , Niño , Electroforesis en Gel Bidimensional , Femenino , Humanos , Immunoblotting , Lactante , Masculino , Persona de Mediana Edad , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Adulto JovenRESUMEN
PURPOSE: To determine the immunophenotypes of macular corneal dystrophy (MCD) in Indian patients and to correlate them with mutations in the carbohydrate 6-sulfotransferase (CHST6) gene. METHODS: Sixty-four patients from 53 families with MCD that were previously screened for mutations in CHST6 were included in an immunophenotype analysis. Antigenic keratan sulfate (AgKS) in serum as well as corneal tissue was evaluated in 31 families. Only cornea was evaluated in 11 families, and only serum was evaluated in 11 families. AgKS was detected in formalin-fixed, paraffin-embedded corneal sections by immunohistochemistry and in serum by ELISA using a monoclonal antibody against sulfated forms of KS in patients with MCD as well as normal controls. RESULTS: Analysis of corneal and/or serum AgKS disclosed MCD type I (27 families), MCD type IA (5 families), and MCD type II (3 families) in the cases studied. An additional 10 families were either MCD type I or MCD type IA since only serum AgKS data were available. Seven families manifested atypical immunophenotypes since the corneal AgKS expression was either of MCD type I or MCD type IA, but serum AgKS levels ranged from 19 ng/ml to 388 ng/ml. More than one immunophenotype was detected amongst siblings in two families. Each immunophenotype was associated with mutational heterogeneity in CHST6. CONCLUSIONS: MCD type I was the predominant immunophenotype in the Indian population studied followed by MCD type IA and then MCD type II. We detected further immunophenotypic heterogeneity by finding atypical patterns of AgKS reactivity in a subset of families. There were no simple correlations between immunophenotypes and specific mutations in CHST6, suggesting that factors other than CHST6 mutations may be contributing to the immunophenotypes in MCD.
Asunto(s)
Distrofias Hereditarias de la Córnea/genética , Sulfato de Queratano/inmunología , Mutación , Sulfotransferasas/genética , Córnea/inmunología , Córnea/patología , Distrofias Hereditarias de la Córnea/inmunología , Distrofias Hereditarias de la Córnea/metabolismo , Distrofias Hereditarias de la Córnea/patología , Familia , Humanos , Inmunohistoquímica , India , Sulfato de Queratano/análisis , Sulfato de Queratano/sangre , Fenotipo , Estadística como Asunto , Sulfotransferasas/metabolismo , Carbohidrato SulfotransferasasRESUMEN
Transforming growth factor beta-induced protein (TGFBIp) has been linked to several corneal dystrophies as certain point mutations in the protein may give rise to a progressive accumulation of insoluble protein material in the human cornea. Little is known about the biological functions of this extracellular protein, which is expressed in various tissues throughout the human body. However, it has been found to interact with a number of extracellular matrix macromolecules such as collagens and proteoglycans. Structural information about TGFBIp might prove to be a valuable tool in the elucidation of its function and its role in corneal dystrophies caused by mutations in the TGFBI gene. A simple method for the purification of wild-type and mutant forms of recombinant human TGFBIp from human cells under native conditions is presented here. Moreover, the crystallization and preliminary X-ray analysis of TGFBIp are reported.
Asunto(s)
Proteínas de la Matriz Extracelular/química , Proteínas de la Matriz Extracelular/aislamiento & purificación , Proteínas Mutantes/química , Proteínas Mutantes/aislamiento & purificación , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Factor de Crecimiento Transformador beta/química , Factor de Crecimiento Transformador beta/aislamiento & purificación , Cromatografía de Afinidad , Cristalización , Cristalografía por Rayos X , Electroforesis en Gel de Poliacrilamida , Humanos , Mutación/genéticaRESUMEN
PURPOSE: To evaluate the TGFBI gene and the encoded transforming growth factor beta-induced protein (TGFBIp) in a 47-year-old African-American patient with an unusual atypical asymmetric lattice corneal dystrophy (LCD). METHODS: The eyes of the proband and his brother were examined by slit-lamp biomicroscopy and their clinical records were reviewed. All 17 exons of TGFBI were evaluated in genomic DNA extracted from blood or buccal epithelial cell samples from the proband and his family members. The corneal tissue of the proband was examined histopathologically, and TGFBIp was analyzed in half of an excised corneal button. RESULTS: The proband (who had an unusual atypical asymmetric LCD) and his brother (who had mild bilateral deep stromal opacities) were found to have homozygous Val624Met mutations in TFGBI. The proband's daughter who was heterozygous for the Val624Met mutation had no reported ophthalmic abnormalities. The corneal tissue from the proband contained TGFBIp with the Val624Met mutation. Patients with LCD have different clinical phenotypes based on their genotype. Molecular genetic analyses are becoming increasingly important in making precise diagnoses and prognostic predictions about inherited corneal disorders. CONCLUSIONS: A novel Val624Met homozygous mutation in TGFBI was associated with atypical LCD in two family members. Symptomatic corneal disease was absent at the age of 24 years in the offspring of the proband who was heterozygous for this mutation. This is an apparent example of a TGFBI mutation that becomes evident when it is homozygous. The finding of Val624Met mutated TGFBIp in a approximately 65 kDa protein band in a reduced sodium dodecyl sulfate (SDS) gel suggests that the accumulated protein was intact TGFBIp and not a fragment of TGFBIp.
Asunto(s)
Distrofias Hereditarias de la Córnea/genética , Proteínas de la Matriz Extracelular/genética , Homocigoto , Mutación , Factor de Crecimiento Transformador beta/genética , Secuencia de Bases , Distrofias Hereditarias de la Córnea/patología , Humanos , Masculino , Metionina , Persona de Mediana Edad , ValinaRESUMEN
AIMS: To evaluate mutations in the transforming-growth-factor-beta-induced (TGFBI) gene in patients of Czech origin with autosomal dominant corneal dystrophies. METHODS: The coding sequence of the TGFBI gene was analysed in 22 affected Czech individuals from 7 apparently unrelated families. Comparison of phenotype to genotype was performed. RESULTS: A H626P mutation, previously only described in a family with a variant of lattice corneal dystrophy (LCD), was detected in one family with superficial geographic corneal opacities. Light microscopy of 2 samples obtained following either a prior superficial keratectomy or keratoplasty showed amyloid but no fuchsinophilic deposits. In a family with LCD type I, an R124C mutation was identified. The R124L mutation was shown to be causative of Reis-Bucklers corneal dystrophy in 2 families. A family with Thiel-Behnke corneal dystrophy exhibited an R555Q mutation. In 2 families with granular corneal dystrophy type I, the typical R555W change was identified. CONCLUSION: The phenotype of the family with the H626P mutation differed from the phenotype previously reported for this change.
Asunto(s)
Catarata/genética , Distrofias Hereditarias de la Córnea/genética , Proteínas de la Matriz Extracelular/genética , Mutación , Factor de Crecimiento Transformador beta/genética , Adulto , Amiloide/metabolismo , Catarata/metabolismo , Catarata/patología , Córnea/metabolismo , Córnea/patología , Distrofias Hereditarias de la Córnea/clasificación , Distrofias Hereditarias de la Córnea/metabolismo , República Checa , Femenino , Histidina , Humanos , Masculino , Persona de Mediana Edad , Fenotipo , ProlinaRESUMEN
PURPOSE: Several inherited corneal disorders in humans result from mutations in the transforming growth factor beta induced gene (TGFBI), which encodes for the extracellular transforming growth factor beta induced protein (TGFBIp) that is one of the most abundant proteins in the cornea. We previously reported a significant amount of TGFBIp in plasma by immunoblotting using the only TGFBIp antiserum (anti-p68(beta ig-h3)) available at that time (anti-p68(beta ig-h3) was generated against residues Val210-His683 of TGFBIp). This observation raised the possibility that a fraction of corneal TGFBIp may originate from the plasma. However, recent experiments in our laboratory indicated that the anti-p68(beta ig-h3) antiserum cross-reacts with an environmental protein contaminant. Therefore, we investigated the specificity of the originally utilized anti-p68(beta ig-h3) antiserum and re-evaluated the amount of TGFBIp in human plasma by immunoblotting using a new specific antiserum. METHODS: The observed cross-reactivity of the previously utilized anti-p68(beta ig-h3) antiserum was tested by immunoblotting and the antigen identity was determined by mass spectrometry. A part of human TGFBI encoding an NH2-terminal 11.4 kDa fragment of TGFBIp (residues Gly134-Ile236) was amplified by polymerase chain reaction (PCR) and cloned in E. coli. The TGFBIp fragment was expressed in E. coli, purified by Ni2+-affinity chromatography, and used to immunize rabbits to produce a specific antiserum (anti-TGFBIp(134-236)). To enhance the detection of possible TGFBIp in plasma by allowing a higher sample load, albumin and immunoglobulin G (IgG) were specifically depleted from normal human plasma by affinity chromatography. The presence of TGFBIp in plasma was investigated by immunoblotting using the anti-TGFBIp(134-236) antiserum. Purified TGFBIp from porcine corneas was used for estimation of the TGFBIp detection limit. RESULTS: The previously utilized TGFBIp antiserum, anti-p68(beta ig-h3), cross-reacted with human keratin-1, a common environmental protein contaminant. Thus, the anti-p68(beta ig-h3) antiserum recognizes both TGFBIp and keratin-1. In contrast, the anti-TGFBIp(134-236) antiserum reacted with TGFBIp but showed no indication of reactivity with other proteins in plasma. Using this antiserum, TGFBIp was not detected in crude or albumin/IgG-depleted human plasma and the detection limit of TGFBIp using immunoblotting was estimated to be 10 ng. CONCLUSIONS: Our failure to detect TGFBIp in human plasma using a highly specific antiserum suggests that TGFBIp is not present in a physiologically relevant concentration in human plasma. The previous impression that normal human plasma contains a significant amount of TGFBIp by immunoblotting was due to the utilization of a less specific antiserum that recognizes both TGFBIp and human keratin-1. Together with other results, our observation makes it unlikely that TGFBIp is imported into the cornea from the circulation as reported for other abundant extracellular corneal proteins and suggests corneal origin of TGFBIp deposits in individuals with inherited corneal diseases caused by mutations in the TGFBI gene.
Asunto(s)
Córnea/metabolismo , Enfermedades de la Córnea/genética , Enfermedades de la Córnea/metabolismo , Proteínas de la Matriz Extracelular/sangre , Proteínas de la Matriz Extracelular/genética , Mutación , Factor de Crecimiento Transformador beta/sangre , Factor de Crecimiento Transformador beta/genética , Animales , Artefactos , Enfermedades de la Córnea/sangre , Epítopos , Proteínas de la Matriz Extracelular/metabolismo , Humanos , Sueros Inmunes/inmunología , Immunoblotting , Queratina-1/inmunología , Porcinos , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
PURPOSE: To report the histopathologic features in an eye with Sveinsson chorioretinal atrophy (SCRA), also termed helicoid peripapillary chorioretinal degeneration, for the first time. PARTICIPANT: An 82-year-old woman clinically and genetically confirmed to have SCRA. DESIGN: Examination of an eye obtained after death. METHOD: Light microscopic examination of an eye of an 82-year-old woman documented to have SCRA since the age of 10 years. MAIN OUTCOME MEASURE: The findings in ocular tissues were determined by light microscopy. RESULTS: In the most advanced areas of chorioretinal atrophy, the sensory retina, retinal pigment epithelium (RPE), choriocapillaris, and choroid were absent. In the transition between affected and unaffected areas, the RPE and the outer segments of the photoreceptors only were affected. The optic nerve was smaller than normal, but well myelinated. Other ocular tissues retained a relatively normal appearance for a patient who had died at this age. CONCLUSIONS: The mildest and presumably earliest morphologic changes involved the photoreceptor outer segments, the RPE, and choriocapillaris in this progressive degenerative disease of the retina and choroid.
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Enfermedades de la Coroides/patología , Disco Óptico/patología , Células Fotorreceptoras de Vertebrados/patología , Epitelio Pigmentado Ocular/patología , Degeneración Retiniana/patología , Anciano de 80 o más Años , Atrofia , Enfermedades de la Coroides/genética , Femenino , Humanos , Nervio Óptico/patología , Retina/patología , Degeneración Retiniana/genéticaRESUMEN
PURPOSE: To locate the mildest and/or earliest changes in the retina and/or choroid in Sveinsson chorioretinal atrophy (SCRA), using more advanced techniques than previous studies. METHODS: We used fundus photography, intravenous fluorescein angiography (IVFA) enhanced ocular coherence tomography (OCT) scans, microperimetry and multifocal electroretinography (mfERG) in an attempt to locate the mildest changes in SCRA. Eight patients with SCRA were examined. To improve the resolution of OCT scans, several consecutive recorded B-scans were retrieved for each location of interest. The scans were processed off-line with an averaging algorithm developed to maximally reduce laser speckle (noise). Static microperimetry was performed using the Rodenstock scanning laser ophthalmoscope (SLO). RESULTS: Biomicroscopy and fundus photographs disclosed an apparent thinning of the retinal pigment epithelium (RPE) in the areas minimally affected, where possible changes in the transparent sensory retina were not visible. In minimally affected areas a choriocapillaris filling defect was evident on IVFA, but some choroidal blood vessels remained open. High-resolution OCT scans in normal eyes showed three highly reflective outer layers, probably representing the junction of the inner and outer photoreceptor segments in the case of the innermost layer, the interdigitizing outer photoreceptors and RPE villi in the case of the middle layer, and the outer RPE in the case of the outermost layer. The middle layer was absent in the transition between affected and unaffected areas in all eyes with SCRA. In the more severely affected areas, the innermost layer was discontinuous and associated with increasing thinning of the outermost layer. Microperimetry of the transition areas sometimes showed clearly defined lesions that were non-responsive to stimuli. It also revealed elevated thresholds (10-15 dB) at the margins and normal thresholds in apparently unaffected areas. CONCLUSIONS: The mildest changes seen in SCRA on OCT are found at the outer photoreceptor/RPE junction; the changes in the outer RPE, choriocapillaris and inner photoreceptor segments may be secondary. Corresponding functional deficits are confirmed on microperimetry and mfERG.
Asunto(s)
Enfermedades de la Coroides/diagnóstico , Epitelio Pigmentado Ocular , Enfermedades de la Retina/diagnóstico , Segmento Externo de la Célula en Bastón , Adulto , Atrofia , Electrorretinografía/métodos , Femenino , Angiografía con Fluoresceína , Fondo de Ojo , Humanos , Rayos Láser , Masculino , Persona de Mediana Edad , Oftalmoscopía , Epitelio Pigmentado Ocular/patología , Segmento Externo de la Célula en Bastón/patología , Tomografía de Coherencia Óptica , Pruebas del Campo Visual/métodosRESUMEN
The structure of the cornea is vital to its transparency, and dystrophies that disrupt corneal organization are highly heritable. To understand the genetic aetiology of Fuchs endothelial corneal dystrophy (FECD), the most prevalent corneal disorder requiring transplantation, we conducted a genome-wide association study (GWAS) on 1,404 FECD cases and 2,564 controls of European ancestry, followed by replication and meta-analysis, for a total of 2,075 cases and 3,342 controls. We identify three novel loci meeting genome-wide significance (P<5 × 10-8): KANK4 rs79742895, LAMC1 rs3768617 and LINC00970/ATP1B1 rs1200114. We also observe an overwhelming effect of the established TCF4 locus. Interestingly, we detect differential sex-specific association at LAMC1, with greater risk in women, and TCF4, with greater risk in men. Combining GWAS results with biological evidence we expand the knowledge of common FECD loci from one to four, and provide a deeper understanding of the underlying pathogenic basis of FECD.
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Distrofia Endotelial de Fuchs/genética , Sitios Genéticos , Estudio de Asociación del Genoma Completo , Humanos , Curva ROC , Reproducibilidad de los Resultados , Factores de RiesgoRESUMEN
The lattice corneal dystrophies (LCD) and granular corneal dystrophies (GCD) are autosomal dominant disorders of the corneal stroma. They are bilateral, progressive conditions characterized by the formation of opacities arising due to the deposition of insoluble material in the corneal stroma leading to visual impairment. The LCDs and GCDs are distinguished from each other and are divided into subtypes on the basis of the clinical appearance of the opacities, clinical features of the disease, and on histopathological staining properties of the deposits. The GCDs and most types of LCD arise from mutations in the transforming growth factor beta-induced (TGFBI) gene on chromosome 5q31. Over 30 mutations causing LCD and GCD have been identified so far in the TGFBI. There are two mutation hotspots corresponding to arginine residues at positions 124 and 555 of the transforming growth factor beta induced protein (TGFBIp) and they are the most frequent sites of mutation in various populations. Mutations at either of these two hotspots result in specific types of LCD or GCD. The majority of identified mutations involve residues in the fourth fasciclin-like domain of TGFBIp.
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Distrofias Hereditarias de la Córnea/diagnóstico , Distrofias Hereditarias de la Córnea/genética , Proteínas de la Matriz Extracelular/genética , Mutación Missense , Factor de Crecimiento Transformador beta/genética , Distrofias Hereditarias de la Córnea/clasificación , Humanos , FenotipoRESUMEN
PURPOSE: To evaluate mutations in the carbohydrate sulfotransferase-6 (CHST6) gene in American subjects with macular corneal dystrophy (MCD). METHODS: We analyzed CHST6 in 57 patients from 31 families with MCD from the United States, 57 carriers (parents or children), and 27 unaffected blood relatives of affected subjects. We compared the observed nucleotide sequences with those found by numerous investigators in other populations with MCD and in controls. RESULTS: In 24 families, the corneal disorder could be explained by mutations in the coding region of CHST6 or in the region upstream of this gene in both the maternal and paternal chromosome. In most instances of MCD a homozygous or heterozygous missense mutation in exon 3 of CHST6 was found. Six cases resulted from a deletion upstream of CHST6. CONCLUSIONS: Nucleotide changes within the coding region of CHST6 are predicted to alter the encoded protein significantly within evolutionary conserved parts of the encoded sulfotransferase. Our findings support the hypothesis that CHST6 mutations are cardinal to the pathogenesis of MCD. Moreover, the observation that some cases of MCD cannot be explained by mutations in CHST6 suggests that MCD may result from other subtle changes in CHST6 or from genetic heterogeneity.
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Distrofias Hereditarias de la Córnea/genética , Sulfotransferasas/genética , Regiones no Traducidas 5'/genética , Cromosomas , Elementos Transponibles de ADN , Padre , Mutación del Sistema de Lectura , Eliminación de Gen , Humanos , Biología Molecular/métodos , Madres , Mutación , América del Norte , Polimorfismo de Nucleótido Simple , Carbohidrato SulfotransferasasRESUMEN
Increased biochemical knowledge of normal and diseased corneas is essential for the understanding of corneal homeostasis and pathophysiology. In a recent study, we characterized the proteome of the normal human cornea and identified 141 distinct proteins. This dataset represents the most comprehensive protein study of the cornea to date and provides a useful reference for further studies of normal and diseased human corneas. The list of identified proteins is available at the Cornea Protein Database. In the present paper, we review the utilized procedures for extraction and fractionation of corneal proteins and discuss the potential roles of the identified proteins in relation to homeostasis, diseases, and wound-healing of the cornea. In addition, we compare the list of identified proteins with high quality gene expression libraries (cDNA libraries) and Serial Analysis of Gene Expression (SAGE) data. Of the 141 proteins, 86 (61%) were recognized in cDNA libraries from the corneas of dogs and rabbits, or humans with keratoconus, and 98 (69.5%) were recognized in SAGE data of mouse and human corneas. However, the percentages of identified genes in each of the protein functional groups differed markedly. Thus, exceptionally few of the traditional blood/plasma proteins and immune defense proteins that were identified in the human cornea were recognized in the gene expression libraries of the cornea. This observation strongly indicates that these abundant corneal proteins are not expressed in the cornea but originate from the surrounding pericorneal tissue.
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Proteínas Sanguíneas/metabolismo , Biología Computacional/métodos , Córnea/metabolismo , Proteoma/metabolismo , Animales , HumanosRESUMEN
PURPOSE: To identify CHST6 mutations in five additional Icelandic cases of macular corneal dystrophy (MCD) type I and in four families with MCD type II from Iceland. METHODS: Genomic DNA was extracted from blood leukocytes of patients with MCD, their healthy family members, and from control individuals. CHST6 mutations were determined by PCR-sequencing. Immunophenotypes of MCD were determined by measuring antigenic keratan sulfate (AgKS) levels in serum and by an immunohistochemical study on corneal tissue. RESULTS: Five additional cases of MCD type I and four families with MCD type II from Iceland were studied. A homozygous p.A128V mutation in the coding region of the CHST6 gene was identified in four of the five MCD type I cases. The other person with MCD type I was a compound heterozygote for p.A128V and a frameshift p.V6fs resulting from a 10-base pair insertion (c.15_16insATGCTGTGCG). Four of five individuals with MCD type II were compound heterozygotes for p.A128V and p.V329L, thus sharing the same p.A128V mutation as MCD type I. One patient with MCD type II was homozygous for p.V329L. The p.V329L mutation was only found in MCD type II patients. An analysis of the upstream region of CHST6 disclosed no upstream deletion or replacements in Icelandic patients with MCD type II. CONCLUSIONS: The findings fit the haplotype analysis that we reported previously in Icelandic MCD families and indicate that different mutations in CHST6 cause MCD type I and type II in Iceland.
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Distrofias Hereditarias de la Córnea/clasificación , Distrofias Hereditarias de la Córnea/genética , Mutación , Sulfotransferasas/genética , Alanina , Elementos Transponibles de ADN , Femenino , Mutación del Sistema de Lectura , Heterocigoto , Homocigoto , Humanos , Islandia , Inmunohistoquímica , Inmunofenotipificación , Leucina , Masculino , Linaje , Reacción en Cadena de la Polimerasa , Valina , Carbohidrato SulfotransferasasRESUMEN
OBJECTIVE: To review 30 years' clinical experience with Fuchs corneal endothelial dystrophy leading to penetrating keratoplasty (PK). METHODS: We reviewed the clinical records of patients diagnosed histopathologically as having Fuchs corneal endothelial dystrophy who underwent PK at Duke University Medical Center between January 1, 1972, and December 31, 2001. This observational case series included 424 patients (546 eyes). RESULTS: Women represented 77.6% of patients. Mean age at the time of PK for all patients was 69.2 years. Bilateral PK was required in 28.8% of patients, and the mean interval between the 2 PKs was 3.2 years. There was no difference with regard to sex in the likelihood of requiring bilateral PK (P=.59). Among 376 documented family histories, 13.6% were known to be positive for Fuchs dystrophy. Patients with a positive family history underwent PK a mean of 5 years earlier (P<.002) and were more likely to require bilateral PK (P<.003). Patients who underwent bilateral PK were twice as likely to have a positive family history compared with those undergoing unilateral PK (P<.001). Mean visual acuities at corneal thicknesses of 539 to 650 microm, 651 to 750 microm, and greater than 750 microm were 20/60, 20/60, and 20/80, respectively. Patients who underwent bilateral PK and had a preceding cataract extraction on 1 eye required PK of the pseudophakic eye on average 3.2 years earlier than the fellow eye. The mean time from cataract extraction to PK was 2.2 years. CONCLUSIONS: This large study affirms that Fuchs dystrophy is a disorder of aging that predominantly affects women (3.5:1) and is often familial. Pachymetry-determined corneal thickness was a poor predictor of visual acuity until extreme levels of corneal edema were reached. Cataract extraction in an eye with Fuchs dystrophy leads to earlier PK.