RESUMEN
Changes in the anterior segment of the eye due to type 2 diabetes mellitus (T2DM) are not well-characterized, in part due to the lack of a reliable animal model. This study evaluated changes in the anterior segment, including crystalline lens health, corneal endothelial cell density, aqueous humor metabolites, and ciliary body vasculature, in a rat model of T2DM compared with human eyes. Male Sprague-Dawley rats were fed a high-fat diet (45% fat) or normal diet, and rats fed the high-fat diet were injected with streptozotocin intraperitoneally to generate a model of T2DM. Cataract formation and corneal endothelial cell density were assessed using microscopic analysis. Diabetes-related rat aqueous humor alterations were assessed using metabolomics screening. Transmission electron microscopy was used to assess qualitative ultrastructural changes ciliary process microvessels at the site of aqueous formation in the eyes of diabetic rats and humans. Eyes from the diabetic rats demonstrated cataracts, lower corneal endothelial cell densities, altered aqueous metabolites, and ciliary body ultrastructural changes, including vascular endothelial cell activation, pericyte degeneration, perivascular edema, and basement membrane reduplication. These findings recapitulated diabetic changes in human eyes. These results support the use of this model for studying ocular manifestations of T2DM and support a hypothesis postulating blood-aqueous barrier breakdown and vascular leakage at the ciliary body as a mechanism for diabetic anterior segment pathology.
Asunto(s)
Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 2 , Ratas Sprague-Dawley , Animales , Diabetes Mellitus Tipo 2/patología , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/complicaciones , Masculino , Ratas , Humanos , Diabetes Mellitus Experimental/patología , Diabetes Mellitus Experimental/complicaciones , Modelos Animales de Enfermedad , Segmento Anterior del Ojo/patología , Humor Acuoso/metabolismo , Catarata/patología , Catarata/metabolismo , Cristalino/patología , Cristalino/metabolismo , Cristalino/ultraestructura , Cuerpo Ciliar/patología , Cuerpo Ciliar/metabolismo , Dieta Alta en Grasa/efectos adversosRESUMEN
Bardet-Beidl syndrome (BBS) manifests from genetic mutations encoding for one or more BBS proteins. BBS4 loss impacts olfactory ciliation and odor detection, yet the cellular mechanisms remain unclear. Here, we report that Bbs4-/- mice exhibit shorter and fewer olfactory sensory neuron (OSN) cilia despite retaining odorant receptor localization. Within Bbs4-/- OSN cilia, we observed asynchronous rates of IFT-A/B particle movements, indicating miscoordination in IFT complex trafficking. Within the OSN dendritic knob, the basal bodies are dynamic, with incorporation of ectopically expressed centrin-2 and γ-tubulin occurring after nascent ciliogenesis. Importantly, BBS4 loss results in the reduction of basal body numbers separate from cilia loss. Adenoviral expression of BBS4 restored OSN cilia lengths and was sufficient to re-establish odor detection, but failed to rescue ciliary and basal body numbers. Our results yield a model for the plurality of BBS4 functions in OSNs that includes intraciliary and periciliary roles that can explain the loss of cilia and penetrance of ciliopathy phenotypes in olfactory neurons.
Asunto(s)
Síndrome de Bardet-Biedl/metabolismo , Cilios/fisiología , Flagelos/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Neuronas Receptoras Olfatorias/fisiología , Animales , Cuerpos Basales/patología , Células Cultivadas , Modelos Animales de Enfermedad , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Asociadas a Microtúbulos/genética , Fenotipo , Transporte de Proteínas , Olfato , Combinación Trimetoprim y Sulfametoxazol/metabolismo , Tubulina (Proteína)/metabolismoRESUMEN
Genetic mutations disrupting the structure and function of primary cilia cause various inherited retinal diseases in humans. Bardet-Biedl syndrome (BBS) is a genetically heterogeneous, pleiotropic ciliopathy characterized by retinal degeneration, obesity, postaxial polydactyly, intellectual disability, and genital and renal abnormalities. To gain insight into the mechanisms of retinal degeneration in BBS, we developed a congenital knockout mouse of Bbs8, as well as conditional mouse models in which function of the BBSome (a protein complex that mediates ciliary trafficking) can be temporally inactivated or restored. We demonstrate that BBS mutant mice have defects in retinal outer segment morphogenesis. We further demonstrate that removal of Bbs8 in adult mice affects photoreceptor function and disrupts the structural integrity of the outer segment. Notably, using a mouse model in which a gene trap inhibiting Bbs8 gene expression can be removed by an inducible FLP recombinase, we show that when BBS8 is restored in immature retinas with malformed outer segments, outer segment extension can resume normally and malformed outer segment discs are displaced distally by normal outer segment structures. Over time, the retinas of the rescued mice become morphologically and functionally normal, indicating that there is a window of plasticity when initial retinal outer segment morphogenesis defects can be ameliorated.
Asunto(s)
Morfogénesis/fisiología , Células Fotorreceptoras/metabolismo , Transporte de Proteínas/fisiología , Animales , Síndrome de Bardet-Biedl/genética , Síndrome de Bardet-Biedl/metabolismo , Síndrome de Bardet-Biedl/patología , Cilios/metabolismo , Ratones , Ratones Noqueados , Modelos Animales , Morfogénesis/genética , Mutación/genética , Transporte de Proteínas/genética , Retina/metabolismo , Retina/fisiologíaRESUMEN
Olfactory dysfunction is a pervasive but underappreciated health concern that affects personal safety and quality of life. Patients with olfactory dysfunctions have limited therapeutic options, particularly those involving congenital diseases. Bardet-Biedl syndrome (BBS) is one such disorder, where olfactory loss and other symptoms manifest from defective cilium morphology and/or function in various cell types/tissues. Olfactory sensory neurons (OSNs) of BBS mutant mice lack the capacity to build/maintain cilia, rendering the cells incapable of odor detection. Here we examined OSN cilium defects in Bbs1 mutant mice and assessed the utility of gene therapy to restore ciliation and function in young and adult mice. Bbs1 mutant mice possessed short residual OSN cilia in which BBSome protein trafficking and odorant detection were defective. Gene therapy with an adenovirus-delivered wild-type Bbs1 gene restored OSN ciliation, corrected BBSome cilium trafficking defects, and returned acute odor responses. Finally, using clinically approved AAV serotypes, we demonstrate, for the first time, the capacity of AAVs to restore ciliation and odor detection in OSNs of Bbs1 mutants. Together, our data demonstrate that OSN ciliogenesis can be promoted in differentiated cells of young and adult Bbs1 mutants and highlight the potential of gene therapy as a viable restorative treatment for congenital olfactory disorders.
Asunto(s)
Síndrome de Bardet-Biedl/genética , Síndrome de Bardet-Biedl/fisiopatología , Terapia Genética , Neuronas Receptoras Olfatorias/metabolismo , Alelos , Animales , Síndrome de Bardet-Biedl/terapia , Cilios/metabolismo , Cilios/patología , Dependovirus/genética , Modelos Animales de Enfermedad , Expresión Génica Ectópica , Expresión Génica , Técnicas de Transferencia de Gen , Vectores Genéticos/administración & dosificación , Vectores Genéticos/genética , Humanos , Ratones , Ratones Noqueados , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Mutación , Percepción Olfatoria/genética , Fenotipo , Transporte de Proteínas , Transducción GenéticaRESUMEN
Retinitis pigmentosa is a genetically heterogeneous group of inherited ocular disorders characterized by progressive photoreceptor cell loss, night blindness, constriction of the visual field, and progressive visual disability. Homozygosity mapping and gene expression studies identified a 2 exon gene, C2ORF71. The encoded protein has no homologs and is highly expressed in the eye, where it is specifically expressed in photoreceptor cells. Two mutations were found in C2ORF71 in human RP patients: A nonsense mutation (p.W253X) in the first exon is likely to be a null allele; the second, a missense mutation (p.I201F) within a highly conserved region of the protein, leads to proteosomal degradation. Bioinformatic and functional studies identified and validated sites of lipid modification within the first three amino acids of the C2ORF71 protein. Using morpholino oligonucleotides to knockdown c2orf71 expression in zebrafish results in visual defects, confirming that C2ORF71 plays an important role in the development of normal vision. Finally, localization of C2ORF71 to primary cilia in cultured cells suggests that the protein is likely to localize to the connecting cilium or outer segment of photoreceptor cells.
Asunto(s)
Ojo/metabolismo , Mutación , Células Fotorreceptoras de Vertebrados/metabolismo , Proteínas/genética , Retinitis Pigmentosa/genética , Ceguera/genética , Cilios/genética , Cilios/metabolismo , Exones , Proteínas del Ojo/genética , Homocigoto , Humanos , Mutación Missense , Retinitis Pigmentosa/metabolismoRESUMEN
Bardet-Biedl Syndrome (BBS) is a heterogeneous syndromic form of retinal degeneration. We have identified a novel transcript of a known BBS gene, BBS3 (ARL6), which includes an additional exon. This transcript, BBS3L, is evolutionally conserved and is expressed predominantly in the eye, suggesting a specialized role in vision. Using antisense oligonucleotide knockdown in zebrafish, we previously demonstrated that bbs3 knockdown results in the cardinal features of BBS in zebrafish, including defects to the ciliated Kupffer's Vesicle and delayed retrograde melanosome transport. Unlike bbs3, knockdown of bbs3L does not result in Kupffer's Vesicle or melanosome transport defects, rather its knockdown leads to impaired visual function and mislocalization of the photopigment green cone opsin. Moreover, BBS3L RNA, but not BBS3 RNA, is sufficient to rescue both the vision defect as well as green opsin localization in the zebrafish retina. In order to demonstrate a role for Bbs3L function in the mammalian eye, we generated a Bbs3L-null mouse that presents with disruption of the normal photoreceptor architecture. Bbs3L-null mice lack key features of previously published Bbs-null mice, including obesity. These data demonstrate that the BBS3L transcript is required for proper retinal function and organization.
Asunto(s)
Factores de Ribosilacion-ADP/metabolismo , Visión Ocular , Proteínas de Pez Cebra/metabolismo , Factores de Ribosilacion-ADP/química , Factores de Ribosilacion-ADP/deficiencia , Factores de Ribosilacion-ADP/genética , Secuencia de Aminoácidos , Animales , Síndrome de Bardet-Biedl/complicaciones , Síndrome de Bardet-Biedl/genética , Síndrome de Bardet-Biedl/patología , Síndrome de Bardet-Biedl/fisiopatología , Anomalías del Ojo/complicaciones , Anomalías del Ojo/patología , Anomalías del Ojo/fisiopatología , Ganglios/efectos de los fármacos , Ganglios/metabolismo , Ganglios/patología , Regulación de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Humanos , Ratones , Ratones Mutantes , Datos de Secuencia Molecular , Oligonucleótidos Antisentido/farmacología , Especificidad de Órganos/efectos de los fármacos , Fenotipo , Células Fotorreceptoras de Vertebrados/efectos de los fármacos , Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/patología , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transporte de Proteínas/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reflejo de Sobresalto/efectos de los fármacos , Opsinas de Bastones/metabolismo , Visión Ocular/efectos de los fármacos , Pez Cebra , Proteínas de Pez Cebra/química , Proteínas de Pez Cebra/genéticaRESUMEN
Bardet-Biedl syndrome (BBS, OMIM 209900) is a genetic disorder with the primary features of obesity, pigmentary retinopathy, polydactyly, renal malformations, mental retardation and hypogenitalism. Individuals with BBS are also at increased risk for diabetes mellitus, hypertension and congenital heart disease. What was once thought to be a homogeneous autosomal recessive disorder is now known to map to at least six loci: 11q13 (BBS1), 16q21 (BBS2), 3p13 p12 (BBS3), 15q22.3 q23 (BBS4), 2q31 (BBS5) and 20p12 (BBS6). There has been considerable interest in identifying the genes that underlie BBS, because some components of the phenotype are common. Cases of BBS mapping ro BBS6 are caused by mutations in MKKS; mutations in this gene also cause McKusick-Kaufman syndrome (hydrometrocolpos, post-axial polydactyly and congenital heart defects). In addition, we recently used positional cloning to identify the genes underlying BBS2 (ref. 16) and BBS4 (ref. 17). The BBS6 protein has similarity to a Thermoplasma acidophilum chaperonin, whereas BBS2 and BBS4 have no significant similarity to chaperonins. It has recently been suggested that three mutated alleles (two at one locus, and a third at a second locus) may be required for manifestation of BBS (triallelic inheritance). Here we report the identification of the gene BBS1 and show that a missense mutation of this gene is a frequent cause of BBS. In addition, we provide data showing that this common mutation is not involved in triallelic inheritance.
Asunto(s)
Síndrome de Bardet-Biedl/genética , Proteínas/genética , Regulación del Desarrollo de la Expresión Génica , Homocigoto , Humanos , Proteínas Asociadas a Microtúbulos , Datos de Secuencia Molecular , Mutación , Mutación Missense , Linaje , Polimorfismo Conformacional Retorcido-Simple , Proteínas/metabolismoRESUMEN
Diabetes mellitus is a multiorgan systemic disease impacting numerous ocular structures that results in significant ocular morbidity and often results in more frequent corneal and glaucoma surgeries for affected individuals. We hypothesize that the systemic metabolic and proteomic derangement observed in the progression of diabetes influences the composition of the aqueous humor (AH), which ultimately impacts the anterior segment health of the eye. To identify changes associated with diabetes progression, we mapped the metabolite profile and proteome of AH samples from patients with varying severities of type II diabetes (T2DM). Patients were classified as nondiabetic (ND or control), non-insulin-dependent diabetic without advanced features of disease (NAD-ni), insulin-dependent diabetic without advanced features (NAD-i), or diabetic with advanced features (AD). AH samples collected from the anterior chamber during elective ophthalmic surgery were evaluated for metabolite and protein expression changes associated with diabetic severity via gas chromatography/mass spectrometry and ultra-high performance liquid chromatography tandem mass spectrometry, respectively. Metabolic and proteomic pathway analyses were conducted utilizing MetaboAnalyst 4.0 and Ingenuity Pathway Analysis. A total of 14 control, 12 NAD-ni, 4 NAD-I, and 14 AD samples were included for analysis. Elevated levels of several branched amino acids (e.g., valine, leucine, isoleucine), and lipid metabolites (e.g., palmitate) were found only with increasing diabetic severity (i.e., the AD group). Similar proteomic trends were noted in amino acid and fatty acid metabolism and the unfolded protein/stress response. These results represent the first report of both metabolomic and proteomic evaluation of aqueous humor. Diabetes results in metabolic and proteomic perturbations detectable in the AH, and unique changes become manifest as T2DM severity worsens. Changes in AH composition may serve as an indicator of disease severity, risk assessment of anterior segment cells and structures, and potential future therapies.
Asunto(s)
Humor Acuoso , Diabetes Mellitus Tipo 2 , Humanos , Humor Acuoso/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Proteómica , NAD/metabolismo , Cromatografía LiquidaRESUMEN
Bardet-Biedl syndrome (BBS) is a rare hereditary autosomal recessive disease associated with several features including obesity, hypertension, and renal abnormalities. The underlying mechanisms of renal defects associated with BBS remain poorly defined. We examined the histological, molecular, and functional renal changes in BBS mouse models that have features of the human disorder. Interestingly, obese hypertensive Bbs4(-/-) mice exhibited inflammatory infiltration and renal cysts, whereas the obese normotensive Bbs2(-/-) mice had only minor inflammatory infiltration. Accordingly, the expression level of inducible nitric oxide synthase was elevated in the kidney of both BBS mice with a more marked increase in Bbs4(-/-) mice. In contrast, endothelial nitric oxide synthase expression was decreased in Bbs4(-/-), but not Bbs2(-/-), mice. Similarly, the expression levels of transient receptor potential vanilloid 1 and 4 channels as well as ß- and γ-subunits of epithelial Na channel were significantly reduced only in the kidney of Bbs4(-/-) mice. Metabolic studies revealed changes in urine output and urinary concentrations of creatinine, blood urea nitrogen, sodium, and potassium with a more pronounced effect in Bbs4(-/-) mice. Finally, we found that calorie restriction which prevented obesity in BBS mice reversed the morphological and molecular changes found in Bbs2(-/-) and Bbs4(-/-) mice, indicating the kidney abnormalities associated with BBS are obesity related. These findings extend our understanding of the function of BBS proteins and emphasize the importance of these proteins in renal physiology.
Asunto(s)
Síndrome de Bardet-Biedl/genética , Riñón/anomalías , Proteínas Asociadas a Microtúbulos/genética , Proteínas/genética , Animales , Nitrógeno de la Urea Sanguínea , Restricción Calórica , Creatinina/orina , Canales Epiteliales de Sodio/análisis , Femenino , Hipertensión/genética , Masculino , Ratones , Óxido Nítrico Sintasa de Tipo III/análisis , Obesidad/genética , Oliguria/fisiopatología , Potasio/orina , Sodio/orina , Canales Catiónicos TRPV/análisisRESUMEN
Bardet-Biedl syndrome (BBS) is a heterogeneous genetic disorder characterized by many features, including obesity and cardiovascular disease. We previously developed knockout mouse models of 3 BBS genes: BBS2, BBS4, and BBS6. To dissect the mechanisms involved in the metabolic disorders associated with BBS, we assessed the development of obesity in these mouse models and found that BBS-null mice were hyperphagic, had low locomotor activity, and had elevated circulating levels of the hormone leptin. The effect of exogenous leptin on body weight and food intake was attenuated in BBS mice, which suggests that leptin resistance may contribute to hyperleptinemia. In other mouse models of obesity, leptin resistance may be selective rather than systemic; although mice became resistant to leptin's anorectic effects, the ability to increase renal sympathetic nerve activity (SNA) was preserved. Although all 3 of the BBS mouse models were similarly resistant to leptin, the sensitivity of renal SNA to leptin was maintained in Bbs4 -/- and Bbs6 -/- mice, but not in Bbs2 -/- mice. Consequently, Bbs4 -/- and Bbs6 -/- mice had higher baseline renal SNA and arterial pressure and a greater reduction in arterial pressure in response to ganglionic blockade. Furthermore, we found that BBS mice had a decreased hypothalamic expression of proopiomelanocortin, which suggests that BBS genes play an important role in maintaining leptin sensitivity in proopiomelanocortin neurons.
Asunto(s)
Síndrome de Bardet-Biedl/metabolismo , Hipertensión/metabolismo , Leptina/farmacología , Proteínas Asociadas a Microtúbulos/metabolismo , Chaperonas Moleculares/metabolismo , Obesidad/metabolismo , Proteínas/metabolismo , Animales , Síndrome de Bardet-Biedl/genética , Síndrome de Bardet-Biedl/patología , Barrera Hematoencefálica/efectos de los fármacos , Peso Corporal/efectos de los fármacos , Modelos Animales de Enfermedad , Electrocardiografía , Regulación de la Expresión Génica/efectos de los fármacos , Chaperoninas del Grupo II , Hipertensión/genética , Imagen por Resonancia Magnética , Ratones , Ratones Noqueados , Proteínas Asociadas a Microtúbulos/deficiencia , Proteínas Asociadas a Microtúbulos/genética , Chaperonas Moleculares/genética , Obesidad/genética , Tamaño de los Órganos , Proteínas/genéticaRESUMEN
Mitochondrial function is essential for the viability of aerobic eukaryotic cells, as mitochondria provide energy through the generation of adenosine triphosphate (ATP), regulate cellular metabolism, provide redox balancing, participate in immune signaling, and can initiate apoptosis. Mitochondria are dynamic organelles that participate in a cyclical and ongoing process of regeneration and autophagy (clearance), termed mitophagy specifically for mitochondrial (macro)autophagy. An imbalance in mitochondrial function toward mitochondrial dysfunction can be catastrophic for cells and has been characterized in several common ophthalmic diseases. In this article, we review mitochondrial homeostasis in detail, focusing on the balance of mitochondrial dynamics including the processes of fission and fusion, and provide a description of the mechanisms involved in mitophagy. Furthermore, this article reviews investigations of ocular diseases with impaired mitophagy, including Fuchs endothelial corneal dystrophy, primary open-angle glaucoma, diabetic retinopathy, and age-related macular degeneration, as well as several primary mitochondrial diseases with ocular phenotypes that display impaired mitophagy, including mitochondrial encephalopathy lactic acidosis stroke, Leber hereditary optic neuropathy, and chronic progressive external ophthalmoplegia. The results of various studies using cell culture, animal, and human tissue models are presented and reflect a growing awareness of mitophagy impairment as an important feature of ophthalmic disease pathology. As this review indicates, it is imperative that mitophagy be investigated as a targetable mechanism in developing therapies for ocular diseases characterized by oxidative stress and mitochondrial dysfunction.
Asunto(s)
Retinopatía Diabética/fisiopatología , Distrofia Endotelial de Fuchs/fisiopatología , Glaucoma de Ángulo Abierto/fisiopatología , Degeneración Macular/fisiopatología , Mitocondrias/fisiología , Enfermedades Mitocondriales/fisiopatología , Mitofagia/fisiología , Animales , Humanos , Terapia Molecular DirigidaRESUMEN
Defective cellular metabolism, impaired mitochondrial function, and increased cell death are major problems that adversely affect donor tissues during hypothermic preservation prior to transplantation. These problems are thought to arise from accumulated reactive oxygen species (ROS) inside cells. Oxidative stress acting on the cells of organs and tissues preserved in hypothermic conditions before surgery, as is the case for cornea transplantation, is thought to be a major reason behind cell death prior to surgery and decreased graft survival after transplantation. We have recently discovered that ubiquinol - the reduced and active form of coenzyme Q10 and a powerful antioxidant - significantly enhances mitochondrial function and reduces apoptosis in human donor corneal endothelial cells. However, ubiquinol is highly lipophilic, underscoring the need for an aqueous-based formulation of this molecule. Herein, we report a highly dispersible and stable formulation comprising a complex of ubiquinol and gamma cyclodextrin (γ-CD) for use in aqueous-phase ophthalmic products. Docking studies showed that γ-CD has the strongest binding affinity with ubiquinol compared to α- or ß-CD. Complexed ubiquinol showed significantly higher stability compared to free ubiquinol in different aqueous ophthalmic products including Optisol-GS® corneal storage medium, balanced salt solution for intraocular irrigation, and topical Refresh® artificial tear eye drops. Greater ROS scavenging activity was noted in a cell model with high basal metabolism and ROS generation (A549) and in HCEC-B4G12 human corneal endothelial cells after treatment with ubiquinol/γ-CD compared to free ubiquinol. Furthermore, complexed ubiquinol was more effective at lowering ROS, and at far lower concentrations, compared to free ubiquinol. Complexed ubiquinol inhibited lipid peroxidation and protected HCEC-B4G12 cells against erastin-induced ferroptosis. No evidence of cellular toxicity was detected in HCEC-B4G12 cells after treatment with complexed ubiquinol. Using a vertical diffusion system, a topically applied inclusion complex of γ-CD and a lipophilic dye (coumarin-6) demonstrated transcorneal penetrance in porcine corneas and the capacity for the γ-CD vehicle to deliver drug to the corneal endothelium. Using the same model, topically applied ubiquinol/γ-CD complex penetrated the entire thickness of human donor corneas with markedly greater ubiquinol retention in the endothelium compared to free ubiquinol. Lastly, the penetrance of ubiquinol/γ-CD complex was assayed using human donor corneas preserved for 7 days in Optisol-GS® per standard industry practices, and demonstrated higher amounts of ubiquinol retained in the corneal endothelium compared to free ubiquinol. In summary, ubiquinol complexed with γ-CD is a highly stable composition that can be incorporated into a variety of aqueous-phase products for ophthalmic use including donor corneal storage media and topical eye drops to scavenge ROS and protect corneal endothelial cells against oxidative damage.
Asunto(s)
Trasplante de Córnea , Células Endoteliales , Animales , Córnea , Medio de Cultivo Libre de Suero , Dextranos , Endotelio Corneal , Gentamicinas , Humanos , Preservación de Órganos , Porcinos , Ubiquinona/análogos & derivadosRESUMEN
Bardet-Biedl syndrome (BBS) is an autosomal recessive disease characterized by retinal dystrophy, polydactyly, obesity, learning disabilities, renal involvement, and male hypogenitalism. BBS is genetically heterogeneous with mutations of 14 genes, accounting for approximately 70% of cases. Triallelic inheritance has been suggested in about 5% of cases. Forty-nine unrelated BBS patients were screened for mutations by DHPLC analysis in BBS1, BBS2, BBS4, BBS6/MKKS, BBS10, and BBS12. The selected genes either account for more than 5% of the mutational load or are commonly reported in triallelic inheritance. Eight patients with only one or no BBS mutation were further investigated by single nucleotide polymorphism (SNP) analysis. In total, mutations were detected in 44 patients. Twenty percent had two mutations in BBS1, 18% in BBS2, 4% in BBS9, 43% in BBS10, and 2% in BBS12. Five patients were heterozygous for a sequence variation in BBS6/MKKS. We found eight patients with three sequence variations in two genes, which could be explained by triallelic inheritance, by the prevalence of heterozygous carriers or the third sequence variations representing rare polymorphisms. All changes found in a second BBS gene were amino acid substitutions. Genotype-phenotype correlations suggest a milder phenotype for BBS1 compared to BBS2 and BBS10, which we ascribe to the hypomorphic p.Met390Arg-mutation.
Asunto(s)
Síndrome de Bardet-Biedl/genética , Mutación/genética , Alelos , Secuencia de Aminoácidos , Secuencia de Bases , Secuencia Conservada , Análisis Mutacional de ADN , Dinamarca , Evolución Molecular , Femenino , Estudios de Asociación Genética , Genotipo , Chaperoninas del Grupo II/química , Chaperoninas del Grupo II/genética , Humanos , Patrón de Herencia/genética , Masculino , Datos de Secuencia Molecular , Polimorfismo de Nucleótido Simple/genéticaRESUMEN
Bardet-Biedl syndrome (BBS) is a genetically heterogeneous disorder that results in retinal degeneration, obesity, cognitive impairment, polydactyly, renal abnormalities, and hypogenitalism. Of the 12 known BBS genes, BBS1 is the most commonly mutated, and a single missense mutation (M390R) accounts for approximately 80% of BBS1 cases. To gain insight into the function of BBS1, we generated a Bbs1(M390R/M390R) knockin mouse model. Mice homozygous for the M390R mutation recapitulated aspects of the human phenotype, including retinal degeneration, male infertility, and obesity. The obese mutant mice were hyperphagic and hyperleptinemic and exhibited reduced locomotor activity but no elevation in mean arterial blood pressure. Morphological evaluation of Bbs1 mutant brain neuroanatomy revealed ventriculomegaly of the lateral and third ventricles, thinning of the cerebral cortex, and reduced volume of the corpus striatum and hippocampus. Similar abnormalities were also observed in the brains of Bbs2(-/-), Bbs4(-/-), and Bbs6(-/-) mice, establishing these neuroanatomical defects as a previously undescribed BBS mouse model phenotype. Ultrastructural examination of the ependymal cell cilia that line the enlarged third ventricle of the Bbs1 mutant brains showed that, whereas the 9 + 2 arrangement of axonemal microtubules was intact, elongated cilia and cilia with abnormally swollen distal ends were present. Together with data from transmission electron microscopy analysis of photoreceptor cell connecting cilia, the Bbs1 M390R mutation does not affect axonemal structure, but it may play a role in the regulation of cilia assembly and/or function.
Asunto(s)
Síndrome de Bardet-Biedl/genética , Síndrome de Bardet-Biedl/patología , Modelos Animales de Enfermedad , Ratones/genética , Proteínas Asociadas a Microtúbulos/genética , Animales , Ventrículos Cerebrales/patología , Cilios/ultraestructura , Masculino , Mutación , Obesidad/genética , Enfermedades de la Retina/genética , Cola del Espermatozoide/patologíaRESUMEN
PURPOSE: To determine whether ubiquinol improves mitochondrial function and cell viability in human donor corneal endothelial cells during hypothermic corneal tissue storage. METHODS: Endothelial cell Descemet membrane tissues were treated with 10 µM ubiquinol, the reduced form of the antioxidant coenzyme Q10, for 5 days in Optisol-GS storage media before assaying for mitochondrial activity using extracellular flux analysis of oxygen consumption. In addition, endothelial cell Descemet membrane tissues were analyzed for cell viability using apoptosis and necrosis assays. Control tissues from mate corneas were treated with diluent only, and comparisons were analyzed for differences. RESULTS: A total of 13 donor corneal tissues with a mean (SEM) preservation time of 11.8 days (0.4) were included for the analysis. Treatment with 10 µM ubiquinol increased spare respiratory capacity by 174% (P = 0.001), maximal respiration by 93% (P = 0.003), and proton leak by 80% (P = 0.047) compared with controls. Cells treated with ubiquinol had no significant change in cell necrosis or apoptosis. CONCLUSIONS: Preliminary testing in donor corneal tissue at specified doses indicates that ubiquinol may be a useful biocompatible additive to hypothermic corneal storage media that increases corneal endothelial cell mitochondrial function. Additional investigations are indicated to further study and optimize the dose and formulation of ubiquinol for use in preserving donor corneal tissue function during hypothermic storage.
Asunto(s)
Respiración de la Célula/fisiología , Endotelio Corneal/efectos de los fármacos , Micronutrientes/farmacología , Mitocondrias/metabolismo , Ubiquinona/análogos & derivados , Anciano , Recuento de Células , Supervivencia Celular/efectos de los fármacos , Sulfatos de Condroitina , Mezclas Complejas , Criopreservación , Lámina Limitante Posterior/efectos de los fármacos , Dextranos , Femenino , Gentamicinas , Humanos , Masculino , Persona de Mediana Edad , Preservación de Órganos , Soluciones Preservantes de Órganos , Donantes de Tejidos , Ubiquinona/farmacologíaRESUMEN
PURPOSE: To identify and characterize gene expression changes associated with photoreceptor cell loss in a Bbs4-knockout mouse model of retinal degeneration. METHODS: Differential gene expression in the eyes of 5-month-old Bbs4(-/-) mice undergoing retinal degeneration were analyzed using gene microarrays (Affymetrix, Santa Clara, CA). Elevated ocular transcripts were confirmed by Northern blotting of RNA from Bbs4(-/-) and three additional mouse models of Bardet-Biedl Syndrome (BBS). TUNEL assays and transmission electron microscopy were used to study cell death and photoreceptor morphology in these mice. RESULTS: Three hundred fifty-four probes were differentially expressed in Bbs4(-/-) eyes compared with controls using a twofold cutoff. Numerous vision-related transcripts decreased because of photoreceptor cell loss. Increased expression of the stress response genes Edn2, Lcn2, Serpina3n, and Socs3 was noted at 5 months of age and as early as postnatal week 4 in the eyes of four BBS mouse model strains. A burst of apoptotic activity in the photoreceptor outer nuclear layer at postnatal week 2 and highly disorganized outer segments by postnatal weeks 4 to 6 was observed in all four strains. CONCLUSIONS: The specific loss of photoreceptors in Bbs4(-)(/)(-) mice allows us to identify a set of genes that are preferentially expressed in photoreceptors compared with other cell types found in the eye and is a valuable resource in the continuing search for genes involved in retinal disease. The molecular and morphologic changes observed in young BBS animal model eyes implies that BBS proteins play a critical, early role in establishing the correct structure and function of photoreceptors.
Asunto(s)
Proteínas de Fase Aguda/genética , Síndrome de Bardet-Biedl/genética , Regulación de la Expresión Génica , Proteínas Asociadas a Microtúbulos/genética , Células Fotorreceptoras de Vertebrados/metabolismo , Degeneración Retiniana/genética , Animales , Apoptosis , Síndrome de Bardet-Biedl/metabolismo , Síndrome de Bardet-Biedl/patología , Northern Blotting , Endotelina-2/genética , Perfilación de la Expresión Génica , Etiquetado Corte-Fin in Situ , Lipocalina 2 , Lipocalinas , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas Oncogénicas/genética , Células Fotorreceptoras de Vertebrados/patología , Degeneración Retiniana/metabolismo , Degeneración Retiniana/patología , Serpinas/genética , Proteína 3 Supresora de la Señalización de Citocinas , Proteínas Supresoras de la Señalización de Citocinas/genéticaRESUMEN
We report a 22-year-old female with a variant of the Axenfeld-Rieger Syndrome (ARS) and discuss its relation with the subtelomeric 6p deletion. An ARS variant has been described in two familial cases of Axenfeld-Rieger Anomaly (ARA) featuring specific extra ocular manifestations-hypertelorism, midface hypoplasia, mild sensorial deafness, hydrocephaly, psychomotor delay and flattened femoral epiphyses. We proposed that this set of characteristics represents a separate syndrome within the ARS. On the other hand, there have been reported four cases with cryptic de novo pure 6pter microdeletions detected by specific subtelomeric probes in patients with ARS characteristics. We describe a 6pter deletion detected by SNP genotyping and confirmed by FISH and MLPA involving the FOXC1 gene in a patient with ocular and systemic findings that fit perfectly with the variant mentioned above. We conclude that the ARS variant belongs to the ARS phenotypic spectrum, which includes flattened femoral epiphyses as a feature.
Asunto(s)
Anomalías Múltiples/genética , Deleción Cromosómica , Cromosomas Humanos Par 6 , Anomalías del Ojo/genética , Variación Genética , Anomalías Múltiples/diagnóstico por imagen , Adulto , Mapeo Cromosómico , Sondas de ADN , Femenino , Factores de Transcripción Forkhead/genética , Genotipo , Cadera/diagnóstico por imagen , Humanos , Hipertelorismo/genética , Hibridación Fluorescente in Situ , Discapacidad Intelectual/genética , Cariotipificación , Polimorfismo de Nucleótido Simple , Radiografía , SíndromeRESUMEN
PURPOSE: Mutations of the forkhead transcription factor gene FOXC1 result in anterior segment anomalies. No description of the spectrum of defects resulting from a single point mutation of this gene exists in the ophthalmology literature. We have screened all available patients with Axenfeld-Rieger genes (PITX2 and FOXC1). In this report, we clinically characterize the spectrum of ocular and systemic manifestations in one family resulting from a previously reported point mutation (Phe112Ser) in FOXC1. DESIGN: Observational case series. METHODS: Ten members of a multigenerational family were examined for signs of glaucoma, anterior segment abnormalities, and systemic features of Axenfeld-Rieger syndrome. The examinations were performed in an ophthalmology examination room or in the patients' homes. Blood was obtained from 10 members and screened for mutations in FOXC1 using direct DNA sequencing. RESULTS: A single mutation causing a T to C change in codon 112 (Phe112Ser) of FOXC1 was present in six members of the family. Five of these six patients were examined and all demonstrated anterior segment anomalies. One patient had Axenfeld anomaly, one had Rieger syndrome, and one had both Axenfeld anomaly and Peters anomaly. Additionally, some members demonstrated cardiac abnormalities, which may be secondary to their FOXC1 mutation. CONCLUSIONS: A wide spectrum of clinical phenotypes can result from a single point mutation of FOXC1. This report confirms that Rieger syndrome (with dental and facial abnormalities) can be caused by a mutation in FOXC1. It is also the first report of Peters anomaly being caused by a FOXC1 mutation.
Asunto(s)
Anomalías Múltiples/genética , Segmento Anterior del Ojo/anomalías , Proteínas de Unión al ADN , Anomalías del Ojo/genética , Iris/anomalías , Mutación Puntual , Factores de Transcripción/genética , Adolescente , Adulto , Anciano , Niño , Opacidad de la Córnea/genética , Análisis Mutacional de ADN , Huesos Faciales/anomalías , Femenino , Factores de Transcripción Forkhead , Glaucoma/genética , Humanos , Masculino , Linaje , Síndrome , Anomalías Dentarias/genéticaRESUMEN
PURPOSE: Axenfeld-Rieger syndrome is a genetically heterogeneous, autosomal dominant disorder that is characterized by anterior segment defects, glaucoma, and extraocular anomalies. This study examined the two genes known to cause Rieger syndrome, PITX2 and FOXC1, for mutations in five Brazilian families with Axenfeld-Rieger syndrome. METHODS: Five families with a total of 23 persons affected by Axenfeld-Rieger syndrome were recruited for this study. A sequencing-based mutation screen was undertaken for the PITX2 and FOXC1 genes. Linkage analysis was used to study one large family for which no mutations were detected in the PITX2 or FOXC1 genes. RESULTS: Two of the five families harbored mutations in the PITX2 gene, but none of the families had a detectable FOXC1 mutation. Haplotypic analysis of three Rieger syndrome regions in a large family with Axenfeld-Rieger syndrome excluded linkage to the 4q25 (PITX2), 6p25 (FOXC1), and 13q14 (RIEG2) regions. CONCLUSIONS: It appears that the PITX2 gene is responsible for a significant portion of Axenfeld-Rieger syndrome in the Brazilian population. Furthermore, there is also evidence for the presence of genetic heterogeneity of the disorder within the Brazilian population. Finally, a large family with Axenfeld-Rieger syndrome has been identified that does not appear to harbor any of the three known loci. Axenfeld-Rieger syndrome gene segregation in this family likely represents a novel locus.