RESUMEN
Granular corneal dystrophy (GCD) is featured by corneal deposits of transforming growth factor beta-induced gene (TGFBI) mediated by the TGF-ß (transforming growth factor-ß)/Smad signaling. However, the roles of c-Jun amino-terminal kinase (JNK) pathway in GCD pathogenesis remains unexplored, which was investigated in this study. JNK signaling activation and inhibition in primary corneal fibroblasts were obtained by treatments with anisomycin and SP600125, respectively. Protein abundance and phosphorylation were detected by immunoblotting. Cell viability and apoptosis were analyzed by CCK-8 and flow cytometry respectively. TGFBI deposit and autophagy progression were assessed by immunofluorescence. The results found that JNK1 expression and phosphorylation were greatly increased in corneal tissues from GCD2 patients. JNK signaling activation impaired the viability and promoted apoptosis and autophagy processes in primary corneal fibroblasts, along with Smad2/3 phosphorylation, TGFBI accumulation and Bcl-2 suppression. Autophagy related proteins, such as ATG5 (autophagy related 5), ATG12 (autophagy related 12) and LC3B (microtubule-associated protein 1 light chain 3 beta), were also increased in anisomycin or TGF-ß1 treated corneal fibroblasts. However, SP600125 effectively reversed the above effect induced by TGF-ß1 treatment in corneal fibroblasts, including the TGF-ß-induced autophagy progression. The results suggested that JNK signaling was activated in GCD2 corneal tissues, and it mediated the TGF-ß-induced TGFBI protein accumulation and apoptosis of corneal fibroblasts during GCD2 pathogenesis.
Asunto(s)
Apoptosis , Córnea/patología , Distrofias Hereditarias de la Córnea/enzimología , Distrofias Hereditarias de la Córnea/patología , Proteínas de la Matriz Extracelular/metabolismo , Fibroblastos/patología , Sistema de Señalización de MAP Quinasas , Factor de Crecimiento Transformador beta/farmacología , Apoptosis/efectos de los fármacos , Autofagia , Fibroblastos/metabolismo , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteínas Smad/metabolismo , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Autosomal-dominant Schnyder corneal dystrophy (SCD) is characterized by corneal opacification owing to overaccumulation of cholesterol. SCD is caused by mutations in UBIAD1, which utilizes geranylgeranyl pyrophosphate (GGpp) to synthesize vitamin K2. Using cultured cells, we previously showed that sterols trigger binding of UBIAD1 to the cholesterol biosynthetic enzyme HMG CoA reductase (HMGCR), thereby inhibiting its endoplasmic reticulum (ER)-associated degradation (ERAD) (Schumacher et al. 2015). GGpp triggers release of UBIAD1 from HMGCR, allowing maximal ERAD and ER-to-Golgi transport of UBIAD1. SCD-associated UBIAD1 resists GGpp-induced release and is sequestered in ER to inhibit ERAD. We now report knockin mice expressing SCD-associated UBIAD1 accumulate HMGCR in several tissues resulting from ER sequestration of mutant UBIAD1 and inhibition of HMGCR ERAD. Corneas from aged knockin mice exhibit signs of opacification and sterol overaccumulation. These results establish the physiological significance of UBIAD1 in cholesterol homeostasis and indicate inhibition of HMGCR ERAD contributes to SCD pathogenesis.
Asunto(s)
Distrofias Hereditarias de la Córnea/metabolismo , Dimetilaliltranstransferasa/metabolismo , Retículo Endoplásmico/metabolismo , Hidroximetilglutaril-CoA Reductasas/metabolismo , Animales , Distrofias Hereditarias de la Córnea/enzimología , Dimetilaliltranstransferasa/genética , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , ProteolisisRESUMEN
Bietti's crystalline dystrophy (BCD) is an intractable and progressive chorioretinal degenerative disease caused by mutations in the CYP4V2 gene, resulting in blindness in most patients. Although we and others have shown that retinal pigment epithelium (RPE) cells are primarily impaired in patients with BCD, the underlying mechanisms of RPE cell damage are still unclear because we lack access to appropriate disease models and to lesion-affected cells from patients with BCD. Here, we generated human RPE cells from induced pluripotent stem cells (iPSCs) derived from patients with BCD carrying a CYP4V2 mutation and successfully established an in vitro model of BCD, i.e., BCD patient-specific iPSC-RPE cells. In this model, RPE cells showed degenerative changes of vacuolated cytoplasm similar to those in postmortem specimens from patients with BCD. BCD iPSC-RPE cells exhibited lysosomal dysfunction and impairment of autophagy flux, followed by cell death. Lipidomic analyses revealed the accumulation of glucosylceramide and free cholesterol in BCD-affected cells. Notably, we found that reducing free cholesterol by cyclodextrins or δ-tocopherol in RPE cells rescued BCD phenotypes, whereas glucosylceramide reduction did not affect the BCD phenotype. Our data provide evidence that reducing intracellular free cholesterol may have therapeutic efficacy in patients with BCD.
Asunto(s)
Colesterol/metabolismo , Distrofias Hereditarias de la Córnea/metabolismo , Enfermedades de la Retina/metabolismo , Animales , Colesterol/análisis , Distrofias Hereditarias de la Córnea/dietoterapia , Distrofias Hereditarias de la Córnea/enzimología , Distrofias Hereditarias de la Córnea/genética , Familia 4 del Citocromo P450/genética , Familia 4 del Citocromo P450/metabolismo , Humanos , Ratones , Mutación , Fenotipo , Enfermedades de la Retina/dietoterapia , Enfermedades de la Retina/enzimología , Enfermedades de la Retina/genética , Epitelio Pigmentado de la Retina/enzimología , Epitelio Pigmentado de la Retina/metabolismoRESUMEN
Granular corneal dystrophy type 2 (GCD2) is an autosomal dominant disorder that is caused by a point mutation in transforming growth factor-ß-induced gene-h3 (TGFBI), which encodes transforming growth factor-ß-induced protein (TGFBIp). Recently, we found that the autophagic clearance of mutant-TGFBIp is delayed in GCD2 corneal fibroblasts; however, any potential correlation between mutant-TGFBIp turnover and autophagy-lysosome pathway remains unknown. Here, we report that mutant-TGFBIp is accumulated and that autophagy, a key clearance pathway for mutant-TGFBIp, is induced in primary cultured GCD2 homozygous (HO) and wild-type (WT) corneal fibroblasts that express exogenously introduced mutant-TGFBIp. Mutant-TGFBI colocalized with LC3-enriched cytosolic vesicles and cathepsin D in primary cultured GCD2 corneal fibroblasts. We also observed reduced levels of raptor (regulatory-associated protein of the mammalian target of rapamycin [mTOR]) in GCD2 corneal fibroblasts and WT corneal fibroblasts expressing mutant-TGFBIp. Strikingly, treatment with MG132, a ubiquitin/proteasome system inhibitor, significantly increased the levels of both total and ubiquitinated raptor in GCD2 corneal fibroblasts. The levels of the autophagy marker LC3-II were also increased in WT corneal fibroblasts that were treated with shRNA against raptor. However, mutant-TGFBIp accumulated in autophagosomes or/and lysosomes in spite of the significant activation of basal autophagy in GCD2 corneal fibroblasts. These results suggest that an insufficient autophagy-lysosome pathway might be responsible for the intracellular accumulation of mutant-TGFBIp during the pathogenesis of GCD2.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Autofagia , Distrofias Hereditarias de la Córnea/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Ubiquitina/metabolismo , Distrofias Hereditarias de la Córnea/enzimología , Humanos , Proteolisis , Proteína Reguladora Asociada a mTORRESUMEN
PURPOSE: We investigated the effect of an aldose reductase inhibitor (ARI) and the role of matrix metalloproteinase (MMP)-10 on recovery after corneal epithelium removal in a rat diabetic keratopathy model. METHODS: Three-week-old Sprague-Dawley rats were fed the following diets for 6 weeks: normal MF chow (MF), 50% galactose (Gal), and 50% Gal containing 0.01% ARI (Gal +ARI). The corneal epithelium was removed using n-heptanol, and the area of epithelial defects was photographed and measured every 24 h. Real-time reverse transcriptase PCR, western blotting, and immunohistochemistry were used to determine the expression profile of MMP-10 and integrin α3. RESULTS: Compared to the MF control group, the amount of galactitol in the Gal group increased approximately 200-fold, which was reduced to sevenfold by ARI treatment. The area of corneal erosion in the Gal group was significantly larger than in the MF group at 72 h and thereafter (p<0.01, unpaired t test). The expression level of MMP-10 was enhanced at both the protein and mRNA levels by exposure to a high concentration of Gal, while integrin α3 expression decreased at the protein level but remained unchanged at the mRNA level. Delayed epithelial wound healing and alterations in the expression levels of MMP-10 and integrin α3 were normalized by ARI. The corneal erosion closure rate was significantly decreased with topical recombinant MMP-10. CONCLUSIONS: These studies confirm that the increased expression of MMP-10 induced by Gal feeding is counteracted by ARI treatment and suggest a role of MMP-10 in modulating corneal epithelial wound healing.
Asunto(s)
Distrofias Hereditarias de la Córnea/enzimología , Diabetes Mellitus Experimental/enzimología , Inhibidores Enzimáticos/farmacología , Epitelio Corneal/efectos de los fármacos , Galactosa/administración & dosificación , Metaloproteinasa 10 de la Matriz/genética , Cicatrización de Heridas/efectos de los fármacos , Administración Oral , Administración Tópica , Aldehído Reductasa/antagonistas & inhibidores , Aldehído Reductasa/genética , Aldehído Reductasa/metabolismo , Animales , Distrofias Hereditarias de la Córnea/complicaciones , Distrofias Hereditarias de la Córnea/patología , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/patología , Dieta , Epitelio Corneal/enzimología , Epitelio Corneal/lesiones , Epitelio Corneal/patología , Galactitol/metabolismo , Galactosa/metabolismo , Regulación de la Expresión Génica , Integrina alfa3/genética , Integrina alfa3/metabolismo , Masculino , Metaloproteinasa 10 de la Matriz/metabolismo , Metaloproteinasa 10 de la Matriz/farmacología , Ratas , Ratas Sprague-Dawley , Transducción de Señal , Cicatrización de Heridas/genéticaRESUMEN
Bietti's crystalline corneoretinal dystrophy (BCD) is a recessive degenerative eye disease caused by germline mutations in the CYP4V2 gene. More than 80% of mutant alleles consist of three mutations, that is, two splice-site alterations and one missense mutation, c.992C>A, which translates to p.H331P. In the present study, we analyzed the expression of CYP4 family members in human tissues and conducted functional studies with the wild-type and p.H331P enzymes, to elucidate the link between CYP4V2 activity and BCD. Expression analysis of 17 CYP1 to CYP4 genes showed CYP4V2 to be a major cytochrome P450 in ARPE-19 cells (a human cell line spontaneously generated from normal human retinal pigmented epithelium) and the only detectable CYP4 transcript. Immunohistochemical analyses demonstrated that CYP4V2 protein was present in epithelial cells of the retina and cornea and the enzyme was localized to endoplasmic reticulum. Recombinant reconstituted CYP4V2 protein metabolized eicosapentaenoic acid and docosahexaenoic acid (an important constituent of the retina) to their respective ω-hydroxylated products at rates similar to those observed with purified CYP4F2, which is an established hepatic polyunsaturated fatty acid (PUFA) hydroxylase. The disease-associated p.H331P variant was undetectable in Western blot analyses of HepG2 cells stably transduced with lentiviral expression vectors. Finally, overexpression of functional CYP4V2 in HepG2 cells altered lipid homeostasis. We demonstrated that CYP4V2 protein is expressed at high levels in ocular target tissues of BCD, that the enzyme is metabolically active toward PUFAs, and that the functional deficit among patients with BCD who carry the H331P variant is most likely a consequence of the instability of the mutant protein.
Asunto(s)
Córnea/metabolismo , Distrofias Hereditarias de la Córnea/enzimología , Sistema Enzimático del Citocromo P-450/metabolismo , Ácidos Docosahexaenoicos/metabolismo , Ácido Eicosapentaenoico/metabolismo , Retina/metabolismo , Enfermedades de la Retina/enzimología , Línea Celular , Sistema Enzimático del Citocromo P-450/genética , Familia 4 del Citocromo P450 , Células Hep G2 , Humanos , Metabolismo de los Lípidos , Mutación Missense , Especificidad de Órganos , ARN Mensajero/metabolismo , Proteínas Recombinantes/metabolismo , Epitelio Pigmentado de la Retina/citologíaRESUMEN
PURPOSE: Bietti crystalline dystrophy (BCD) is a rare autosomal recessive disorder caused by mutation of the cytochrome P450, family 4, subfamily V, polypeptide 2 (CYP4V2) gene and characterized by retinal pigmentary abnormalities and scattered deposits of crystals in the retina and the marginal cornea. The aim of this study was to investigate the spectrum of mutations in CYP4V2 in Lebanese families, and to characterize the phenotype of patients affected with BCD. METHODS: Nine patients from three unrelated Lebanese families were clinically and molecularly investigated. Detailed characterization of the patients' phenotype was performed with comprehensive ophthalmic examination, color vision study, fundus photography, visual field testing, retinal fluorescein angiography, electroretinography, and electrooculography. One family was followed for 12 years. The 11 exons of the CYP4V2 gene were sequenced. RESULTS: Symptoms consisting of night blindness, loss of paracentral visual field, and disturbed color vision were apparent during the third decade of life. Ophthalmoscopy revealed posterior pole crystalline deposits and areas of retinal pigment epithelium atrophy. Fluorescein angiography disclosed geographic areas of the pigment epithelium layer and choriocapillaris atrophy in the posterior pole and fundus periphery. The most striking findings were those of normal electroretinographic responses in some patients and clinical heterogeneity. Two mutations in CYP4V2 were found: p.I111T (c.332T>C) in exon 3 in two families and the novel p.V458M (c.1372G>A) mutation in exon 9 in one family. CONCLUSIONS: These patients are affected with Bietti crystalline dystrophy without corneal involvement. Variation in disease severity and electroretinographic responses suggests that environmental or additional genetic factors influence the course of the retinal disease. The CYP4V2 p.I111T (c.332T>C) mutant allele may be especially prevalent among patients with BCD in Lebanon, resulting from a single founder.
Asunto(s)
Distrofias Hereditarias de la Córnea/genética , Sistema Enzimático del Citocromo P-450/genética , Mutación , Retina/patología , Enfermedades de la Retina/genética , Adulto , Anciano , Alelos , Secuencia de Bases , Distrofias Hereditarias de la Córnea/enzimología , Distrofias Hereditarias de la Córnea/patología , Familia 4 del Citocromo P450 , Electrooculografía , Electrorretinografía , Exones , Femenino , Angiografía con Fluoresceína , Genes Recesivos , Humanos , Líbano , Masculino , Datos de Secuencia Molecular , Linaje , Fenotipo , Retina/enzimología , Enfermedades de la Retina/enzimología , Enfermedades de la Retina/patología , Análisis de Secuencia de ADNAsunto(s)
Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/fisiología , Distrofias Hereditarias de la Córnea/enzimología , Familia 4 del Citocromo P450 , Humanos , Metabolismo de los Lípidos , Estructura Molecular , Enfermedades de la Retina/enzimología , Distrofias Retinianas/enzimologíaRESUMEN
The cytochrome P450 (CYP) 4 family of enzymes contains several recently identified membersthat are referred to as "orphan P450s" because their endogenous substrates are unknown.Human CYP4V2 and CYP4F22 are two such orphan P450s that are strongly linked to ocular andskin disease, respectively. Genetic analyses have identified a wide spectrum of mutations in the CYP4V2gene from patients suffering from Bietti's crystalline corneoretinal dystrophy, and mutations in theCYP4F22 gene have been linked to lamellar ichthyosis. The strong genedisease associations provideunique opportunities for elucidating the substrate specificity of these orphan P450s and unraveling thebiochemical pathways that may be impacted in patients with CYP4V2 and CYP4F22 functional deficits.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Ictiosis Lamelar/enzimología , Animales , Distrofias Hereditarias de la Córnea/enzimología , Distrofias Hereditarias de la Córnea/etiología , Distrofias Hereditarias de la Córnea/genética , Distrofias Hereditarias de la Córnea/metabolismo , Sistema Enzimático del Citocromo P-450/genética , Familia 4 del Citocromo P450 , Ácidos Grasos/metabolismo , Humanos , Ictiosis Lamelar/genética , Ictiosis Lamelar/metabolismo , Enfermedades de la Retina/enzimología , Enfermedades de la Retina/etiología , Enfermedades de la Retina/genética , Enfermedades de la Retina/metabolismo , Especificidad por SustratoAsunto(s)
Distrofias Hereditarias de la Córnea/etiología , Peroxidación de Lípido/fisiología , Estrés Oxidativo/fisiología , Estudios de Casos y Controles , Distrofias Hereditarias de la Córnea/enzimología , Femenino , Humanos , Masculino , Óxido Nítrico Sintasa de Tipo II/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismoRESUMEN
BACKGROUND: Mutations in a novel gene, UBIAD1, were recently found to cause the autosomal dominant eye disease Schnyder corneal dystrophy (SCD). SCD is characterized by an abnormal deposition of cholesterol and phospholipids in the cornea resulting in progressive corneal opacification and visual loss. We characterized lesions in the UBIAD1 gene in new SCD families and examined protein homology, localization, and structure. METHODOLOGY/PRINCIPAL FINDINGS: We characterized five novel mutations in the UBIAD1 gene in ten SCD families, including a first SCD family of Native American ethnicity. Examination of protein homology revealed that SCD altered amino acids which were highly conserved across species. Cell lines were established from patients including keratocytes obtained after corneal transplant surgery and lymphoblastoid cell lines from Epstein-Barr virus immortalized peripheral blood mononuclear cells. These were used to determine the subcellular localization of mutant and wild type protein, and to examine cholesterol metabolite ratios. Immunohistochemistry using antibodies specific for UBIAD1 protein in keratocytes revealed that both wild type and N102S protein were localized sub-cellularly to mitochondria. Analysis of cholesterol metabolites in patient cell line extracts showed no significant alteration in the presence of mutant protein indicating a potentially novel function of the UBIAD1 protein in cholesterol biochemistry. Molecular modeling was used to develop a model of human UBIAD1 protein in a membrane and revealed potentially critical roles for amino acids mutated in SCD. Potential primary and secondary substrate binding sites were identified and docking simulations indicated likely substrates including prenyl and phenolic molecules. CONCLUSIONS/SIGNIFICANCE: Accumulating evidence from the SCD familial mutation spectrum, protein homology across species, and molecular modeling suggest that protein function is likely down-regulated by SCD mutations. Mitochondrial UBIAD1 protein appears to have a highly conserved function that, at least in humans, is involved in cholesterol metabolism in a novel manner.
Asunto(s)
Distrofias Hereditarias de la Córnea/enzimología , Distrofias Hereditarias de la Córnea/genética , Dimetilaliltranstransferasa/genética , Mitocondrias/enzimología , Mitocondrias/genética , Mutación/genética , Proteínas/genética , Secuencia de Aminoácidos , Aminoácidos , Secuencia de Bases , Colesterol/metabolismo , Secuencia Conservada , Córnea/enzimología , Córnea/patología , Distrofias Hereditarias de la Córnea/patología , Análisis Mutacional de ADN , Demografía , Familia , Humanos , Modelos Lineales , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Transporte de Proteínas , Proteínas/químicaRESUMEN
Granular corneal dystrophy type II (GCD II) is an autosomal dominant disorder characterized by age-dependent progressive accumulation of transforming growth factor-beta-induced protein (TGFBIp) deposits in the corneal stroma. Several studies have suggested that corneal fibroblasts may decline with age in response to oxidative stress. To investigate whether oxidative stress is involved in the pathogenesis of GCD II, we assayed antioxidant enzymes, oxidative damage, and susceptibility to reactive oxygen species-induced cell death in primary cultured corneal fibroblasts (PCFs) from GCD II patients and healthy subjects. We found elevated protein levels of Mn-superoxide dismutase, Cu/Zn-superoxide dismutase, glutathione peroxidase, and glutathione reductase, as well as increased CAT mRNA and decreased catalase protein in GCD II PCFs. Furthermore, catalase is down-regulated in normal PCFs transfected with transforming growth factor-beta-induced gene-h3. We also observed an increase in not only intracellular reactive oxygen species and H(2)O(2) levels, but also malondialdehyde, 4-hydroxynonenal, and protein carbonyls levels in GCD II PCFs. Greater immunoreactivity for malondialdehyde was observed in the corneal tissue of GCD II patients. In addition, we observed a decrease in Bcl-2 and Bcl-xL levels and an increase in Bax and Bok levels in GCD II PCFs. Finally, GCD II PCFs are more susceptible to H(2)O(2)-induced cell death. Together, these results suggest that oxidative damage induced by decreased catalase is involved in GCD II pathogenesis, and antioxidant agents represent a possible treatment strategy.
Asunto(s)
Catalasa/biosíntesis , Córnea/fisiopatología , Distrofias Hereditarias de la Córnea/fisiopatología , Fibroblastos/patología , Estrés Oxidativo/fisiología , Adolescente , Adulto , Western Blotting , Catalasa/genética , Células Cultivadas , Niño , Córnea/enzimología , Distrofias Hereditarias de la Córnea/enzimología , Distrofias Hereditarias de la Córnea/genética , Proteínas de la Matriz Extracelular/biosíntesis , Proteínas de la Matriz Extracelular/genética , Femenino , Fibroblastos/enzimología , Citometría de Flujo , Humanos , Peróxido de Hidrógeno/metabolismo , Inmunohistoquímica , Masculino , Persona de Mediana Edad , ARN Interferente Pequeño , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transfección , Factor de Crecimiento Transformador beta/biosíntesis , Factor de Crecimiento Transformador beta/genética , Adulto JovenRESUMEN
PURPOSE: To identify mutations in the carbohydrate sulfotransferase gene (CHST6) for a Chinese family with macular corneal dystrophy (MCD) and to investigate the histopathological changes in the affected cornea. METHODS: A corneal button of the proband was obtained by penetrating keratoplasty. The half button and ultrathin sections from the other half button were examined with special stains under a light microscope (LM) and an electron microscope (EM) separately. Genomic DNA was extracted from peripheral blood of 11 family members, and the coding region of CHST6 was amplified by the polymerase chain reaction (PCR) method. The PCR products were analyzed by direct sequencing and restriction enzyme digestion. RESULTS: The positive reaction to colloidal iron stain (extracellular blue accumulations in the stroma) was detected under light microscopy. Transmission electron microscopy revealed the enlargement of smooth endoplasmic reticulum and the presence of intracytoplasmic vacuoles. The compound heterozygous mutations, c.892C>T and c.1072T>C, were identified in exon 3 of CHST6 in three patients. The two transversions resulted in the substitution of a stop codon for glutamine at codon 298 (p.Q298X) and a missense mutation at codon 358, tyrosine to histidine (p.Y358H). The six unaffected family individuals carried alternative heterozygous mutations. These two mutations were not detected in any of the 100 control subjects. CONCLUSIONS: Those novel compound heterozygous mutations were thought to contribute to the loss of CHST6 function, which induced the abnormal metabolism of keratan sulfate (KS) that deposited in the corneal stroma. It could be proved by the observation of a positive stain reaction and the enlarged collagen fibers as well as hyperplastic fibroblasts under microscopes.
Asunto(s)
Pueblo Asiatico/genética , Distrofias Hereditarias de la Córnea/enzimología , Distrofias Hereditarias de la Córnea/genética , Mutación/genética , Sulfotransferasas/genética , Secuencia de Bases , China , Córnea/patología , Córnea/ultraestructura , Distrofias Hereditarias de la Córnea/patología , Análisis Mutacional de ADN , Familia , Femenino , Humanos , Masculino , Datos de Secuencia Molecular , Linaje , Reacción en Cadena de la Polimerasa , Mapeo Restrictivo , Carbohidrato SulfotransferasasRESUMEN
PURPOSE: To define the histopathology of Salzmann nodular degeneration and suggest potential mechanisms involved in its pathogenesis. METHODS: Archived corneal biopsy specimens from 5 patients with Salzmann nodular degeneration were evaluated by chemical and immunohistochemical staining to describe the structure of the Salzmann nodules and phenotypes of nodule epithelium and stromal cells. RESULTS: Each Salzmann nodule appeared as a hypercellular mound of extracellular matrix located between a thinned corneal epithelium and a fragmented Bowman layer. Stromal cells within each nodule stained positively for vimentin, consistent with a fibroblast phenotype, whereas the epithelial cells overlying each nodule were positive for matrix metalloproteinase-2 and negative for matrix metalloproteinase-9. CONCLUSIONS: The observed epithelial expression of matrix metalloproteinase-2 overlying Salzmann nodules is consistent with chronic epithelial wounding in the disorder but does not identify a cause-effect relationship. Salzmann nodules might develop because of enzymatic disruption of the Bowman layer, anterior migration and proliferation of keratocytes, and secondary deposition of extracellular matrix. Alternatively, desiccation secondary to the elevation of the nodule might induce increased epithelial metalloproteinase expression.
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Lámina Limitante Anterior/patología , Distrofias Hereditarias de la Córnea/patología , Epitelio Corneal/patología , Adulto , Anciano , Anciano de 80 o más Años , Lámina Limitante Anterior/enzimología , Distrofias Hereditarias de la Córnea/enzimología , Epitelio Corneal/enzimología , Femenino , Humanos , Masculino , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Persona de Mediana Edad , FenotipoRESUMEN
Schnyder crystalline corneal dystrophy (SCCD, MIM 121800) is a rare autosomal dominant disease characterized by progressive opacification of the cornea resulting from the local accumulation of lipids, and associated in some cases with systemic dyslipidemia. Although previous studies of the genetics of SCCD have localized the defective gene to a 1.58 Mbp interval on chromosome 1p, exhaustive sequencing of positional candidate genes has thus far failed to reveal causal mutations. We have ascertained a large multigenerational family in Nova Scotia affected with SCCD in which we have confirmed linkage to the same general area of chromosome 1. Intensive fine mapping in our family revealed a 1.3 Mbp candidate interval overlapping that previously reported. Sequencing of genes in our interval led to the identification of five putative causal mutations in gene UBIAD1, in our family as well as in four other small families of various geographic origins. UBIAD1 encodes a potential prenyltransferase, and is reported to interact physically with apolipoprotein E. UBIAD1 may play a direct role in intracellular cholesterol biochemistry, or may prenylate other proteins regulating cholesterol transport and storage.
Asunto(s)
Distrofias Hereditarias de la Córnea , Dimetilaliltranstransferasa/genética , Mutación , Proteínas/genética , Secuencia de Aminoácidos , Animales , Colesterol/metabolismo , Mapeo Cromosómico , Biología Computacional , Córnea/patología , Distrofias Hereditarias de la Córnea/enzimología , Distrofias Hereditarias de la Córnea/genética , Análisis Mutacional de ADN , Dimetilaliltranstransferasa/química , Dimetilaliltranstransferasa/metabolismo , Femenino , Ligamiento Genético , Genotipo , Haplotipos , Humanos , Masculino , Persona de Mediana Edad , Modelos Moleculares , Datos de Secuencia Molecular , Nueva Escocia , Linaje , Conformación Proteica , Proteínas/química , Proteínas/metabolismo , Alineación de SecuenciaRESUMEN
OBJECTIVE: To assess the main clinical, genetic, histopathological and ultrastructural features of Mexican patients with macular corneal dystrophy, and to compare the results with those previously reported. METHOD: We analyzed six cases where a histopathologic diagnosis of macular corneal dystrophy had been made between 1957 and 2004. RESULTS: Clinically, all corneas showed focal grayish-white stromal opacities with diffuse edges. Histopathologically, intrastromal granules stained strongly positive with Alcian blue and colloidal iron. Transmission electron microscopy showed enlargement of smooth endoplasmic reticulum and the presence of intracytoplasmic vacuoles that corresponded to glycosaminoglycans. Genetic analysis showed novel mutations in the CHST6 gene in 2 of the patients. CONCLUSIONS: Females were more affected than males and the mean age at the time of diagnosis was older than that reported previously, however the clinical, histopathological and ultrastructural features were similar to those of previous reports. As described in other cases in the literature, in some instances a disorder is found in CHST6 gene as a basis for this condition.
Asunto(s)
Distrofias Hereditarias de la Córnea , Adulto , Distrofias Hereditarias de la Córnea/diagnóstico , Distrofias Hereditarias de la Córnea/enzimología , Distrofias Hereditarias de la Córnea/genética , Distrofias Hereditarias de la Córnea/patología , Distrofias Hereditarias de la Córnea/ultraestructura , Sustancia Propia/patología , Femenino , Humanos , Masculino , Microscopía Electrónica de Transmisión , Mutación , Factores Sexuales , Sulfotransferasas/genéticaAsunto(s)
Sulfotransferasas/fisiología , Animales , Distrofias Hereditarias de la Córnea/enzimología , Distrofias Hereditarias de la Córnea/genética , Distrofias Hereditarias de la Córnea/metabolismo , Humanos , Sulfato de Queratano/biosíntesis , Ratones , Mutación , Proteoglicanos/biosíntesis , Sulfotransferasas/genética , Carbohidrato SulfotransferasasRESUMEN
PURPOSE: The aim of this study was to examine the carbohydrate sulfotransferase 6 (CHST6) gene for mutations in a sibship with both macular corneal dystrophy (MCD) types I and II. DESIGN: Clinically relevant laboratory investigation. METHODS: The coding region of the CHST6 gene was examined for mutations. RESULTS: In one sibling, MCD type I was due to a homozygous C1110T (Arg140end) mutation in CHST6. Two MCD type II individuals exhibited three heterozygous nucleotide changes: C1110T, G1360A (Gly223Asp), and G1685T (Gln331His). Analysis of the upstream region was performed on one individual with MCD type II, and no upstream deletion or substitution was found. CONCLUSIONS: These findings fit the haplotype analysis that we reported previously and indicate that the predicted protein that is encoded by CHST6 is more severely affected in the individual with MCD type I than in the siblings with MCD type II.
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Codón sin Sentido , Distrofias Hereditarias de la Córnea/genética , Mutación Missense , Sulfotransferasas/genética , Distrofias Hereditarias de la Córnea/clasificación , Distrofias Hereditarias de la Córnea/enzimología , Femenino , Haplotipos , Humanos , Masculino , Linaje , Hermanos , Carbohidrato SulfotransferasasRESUMEN
PURPOSE: To further characterize the mutations within the CHST6 gene responsible for causing macular corneal dystrophy in a cohort of affected patients from the United States. DESIGN: Experimental study. METHODS: Genomic DNA was extracted from buccal epithelium of 16 affected patients (14 families), 17 unaffected relatives, and 127 controls, followed by polymerase chain reaction amplification and direct sequencing of the CHST6 coding region. Subtyping of affected patients into type I and II macular corneal dystrophy was performed by measuring antigenic keratan sulfate (AgKS) serum levels. Haplotype analysis was performed in families that demonstrated common mutations. RESULTS: CHST6 coding region analysis in 10 patients identified as having type I macular corneal dystrophy revealed 10 sequence changes: eight missense mutations, four of which are novel (Met104Val, Tyr110Cys, Gln122Pro, and Leu276Pro) and four of which have been reported previously (Ser51Leu, Pro72Ser, Cys102Gly, and Leu200Arg); one novel homozygous nonsense mutation in two patients from a single family (c. 1683C>T, Gln331X); and one frameshift mutation in a heterozygous state in a single patient (c.1744_1751dupGTGCGCTG). Mutation analysis in the four patients identified as having type II macular corneal dystrophy (serum samples were not obtained from two affected patients) revealed three patients heterozygous for either the c.923G>C, c.969C>A, or c.1519T>C sequence changes. The fourth patient was compound heterozygous for c.969C>A and c.1291T>G. None of these changes was observed in 127 control individuals. Haplotype analysis using microsatellite markers flanking the CHST6 gene did not reveal a common founder for the Leu200Arg (1291T>G) missense mutation, present in five families, identifying this position as a mutation hot-spot. CONCLUSIONS: A variety of previously unreported mutations in the coding region of the CHST6 gene are associated with type I macular corneal dystrophy in a cohort of patients from the United States.
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Distrofias Hereditarias de la Córnea/genética , Mutación , Sulfotransferasas/genética , Adulto , Secuencia de Aminoácidos , Distrofias Hereditarias de la Córnea/enzimología , Análisis Mutacional de ADN , Amplificación de Genes , Genotipo , Haplotipos , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADN , Estados Unidos , Carbohidrato SulfotransferasasRESUMEN
OBJECTIVE: To further characterize the role of the carbohydrate sulfotransferase (CHST6) gene in macular corneal dystrophy (MCD) through identification of causative mutations in a cohort of affected patients from southern India. METHODS: Genomic DNA was extracted from buccal epithelium of 75 patients (51 families) with MCD, 33 unaffected relatives, and 48 healthy volunteers. The coding region of the CHST6 gene was evaluated by means of polymerase chain reaction amplification and direct sequencing. Subtyping of MCD into types I and II was performed by measuring serum levels of antigenic keratan sulfate. RESULTS: Seventy patients were classified as having type I MCD, and 5 patients as having type II MCD. Analysis of the CHST6 coding region in patients with type I MCD identified 11 homozygous missense mutations (Leu22Arg, His42Tyr, Arg50Cys, Arg50Leu, Ser53Leu, Arg97Pro, Cys102Tyr, Arg127Cys, Arg205Gln, His249Pro, and Glu274Lys), 2 compound heterozygous missense mutations (Arg93His and Ala206Thr), 5 homozygous deletion mutations (delCG707-708, delC890, delA1237, del1748-1770, and delORF), and 2 homozygous replacement mutations (ACCTAC 1273 GGT, and GCG 1304 AT). One patient with type II MCD was heterozygous for the C890 deletion mutation, whereas 4 possessed no CHST6 coding region mutations. CONCLUSION: A variety of previously unreported mutations in the coding region of the CHST6 gene are associated with type I MCD in a cohort of patients in southern India. CLINICAL RELEVANCE: An improved understanding of the genetic basis of MCD allows for earlier, more accurate diagnosis of affected individuals, and may provide the foundation for the development of novel disease treatments.