RESUMEN
We sought to identify the impacts of Friedreich's ataxia (FRDA) on cardiomyocytes. FRDA is an autosomal recessive degenerative condition with neuronal and non-neuronal manifestations, the latter including progressive cardiomyopathy of the left ventricle, the leading cause of death in FRDA. Little is known about the cellular pathogenesis of FRDA in cardiomyocytes. Induced pluripotent stem cells (iPSCs) were derived from three FRDA individuals with characterized GAA repeats. The cells were differentiated into cardiomyocytes to assess phenotypes. FRDA iPSC- cardiomyocytes retained low levels of FRATAXIN (FXN) mRNA and protein. Electrophysiology revealed an increased variation of FRDA- cardiomyocyte beating rates which was prevented by addition of nifedipine, suggestive of a calcium handling deficiency. Finally, calcium imaging was performed and we identified small amplitude, diastolic and systolic calcium transients confirming a deficiency in calcium handling. We defined a robust FRDA cardiac-specific electrophysiological profile in patient-derived iPSCs which could be used for high throughput compound screening. This cell-specific signature will contribute to the identification and screening of novel treatments for this life-threatening disease.
Asunto(s)
Señalización del Calcio , Calcio/metabolismo , Diferenciación Celular , Linaje de la Célula , Ataxia de Friedreich/metabolismo , Frecuencia Cardíaca , Células Madre Pluripotentes Inducidas/metabolismo , Miocitos Cardíacos/metabolismo , Potenciales de Acción , Línea Celular , Separación Celular/métodos , Femenino , Ataxia de Friedreich/genética , Ataxia de Friedreich/patología , Regulación de la Expresión Génica , Humanos , Células Madre Pluripotentes Inducidas/patología , Proteínas de Unión a Hierro/genética , Proteínas de Unión a Hierro/metabolismo , Masculino , Miocitos Cardíacos/patología , Fenotipo , ARN Mensajero/genética , ARN Mensajero/metabolismo , FrataxinaRESUMEN
OBJECTIVE: Friedreich ataxia (FRDA) is an inherited neurodegenerative disease characterized by ataxia and cardiomyopathy. Homozygous GAA trinucleotide repeat expansions in the first intron of FXN occur in 96% of affected individuals and reduce frataxin expression. Remaining individuals are compound heterozygous for a GAA expansion and a FXN point/insertion/deletion mutation. We examined disease-causing mutations and the impact on frataxin structure/function and clinical outcome in FRDA. METHODS: We compared clinical information from 111 compound heterozygotes and 131 individuals with homozygous expansions. Frataxin mutations were examined using structural modeling, stability analyses and systematic literature review, and categorized into four groups: (1) homozygous expansions, and three compound heterozygote groups; (2) null (no frataxin produced); (3) moderate/strong impact; and (4) minimal impact. Mean age of onset and the presence of cardiomyopathy and diabetes mellitus were compared using regression analyses. RESULTS: Mutations in the hydrophobic core of frataxin affected stability whereas surface residue mutations affected interactions with iron sulfur cluster assembly and heme biosynthetic proteins. The null group of compound heterozygotes had significantly earlier age of onset and increased diabetes mellitus, compared to the homozygous expansion group. There were no significant differences in mean age of onset between homozygotes and the minimal and moderate/strong impact groups. INTERPRETATION: In compound heterozygotes, expression of partially functional mutant frataxin delays age of onset and reduces diabetes mellitus, compared to those with no frataxin expression from the non-expanded allele. This integrated analysis of categorized frataxin mutations and their correlation with clinical outcome provide a definitive resource for investigating disease pathogenesis in FRDA.
Asunto(s)
Diabetes Mellitus/epidemiología , Diabetes Mellitus/genética , Ataxia de Friedreich/epidemiología , Ataxia de Friedreich/genética , Proteínas de Unión a Hierro/genética , Pérdida de Heterocigocidad/genética , Adolescente , Adulto , Distribución por Edad , Causalidad , Niño , Preescolar , Comorbilidad , Femenino , Marcadores Genéticos/genética , Predisposición Genética a la Enfermedad/epidemiología , Predisposición Genética a la Enfermedad/genética , Humanos , Incidencia , Lactante , Masculino , Pronóstico , Factores de Riesgo , Distribución por Sexo , Victoria/epidemiología , Adulto Joven , FrataxinaRESUMEN
Friedreich ataxia (FRDA) is due to a triplet repeat expansion in FXN, resulting in deficiency of the mitochondrial protein frataxin. Resveratrol is a naturally occurring polyphenol, identified to increase frataxin expression in cellular and mouse models of FRDA and has anti-oxidant properties. This open-label, non-randomized trial evaluated the effect of two different doses of resveratrol on peripheral blood mononuclear cell (PBMC) frataxin levels over a 12-week period in individuals with FRDA. Secondary outcome measures included PMBC FXN mRNA, oxidative stress markers, and clinical measures of disease severity. Safety and tolerability were studied. Twenty-four participants completed the study; 12 received low-dose resveratrol (1 g daily) and 12 high-dose resveratrol (5 g daily). PBMC frataxin levels did not change in either dosage group [low-dose group change: 0.08 pg/µg protein (95% CI -0.05, 0.21, p = 0.21); high-dose group change: 0.03 pg/µg protein (95% CI -0.10, 0.15, p = 0.62)]. Improvement in neurologic function was evident in the high-dose group [change in Friedreich Ataxia Rating Scale -3.4 points, 95% CI (-6.6, -0.3), p = 0.036], but not the low-dose group. Significant improvements in audiologic and speech measures, and in the oxidative stress marker plasma F2-isoprostane were demonstrated in the high-dose group only. There were no improvements in cardiac measures or patient-reported outcome measures. No serious adverse events were recorded. Gastrointestinal side-effects were a common, dose-related adverse event. This open-label study shows no effect of resveratrol on frataxin levels in FRDA, but suggests that independent positive clinical and biologic effects of high-dose resveratrol may exist. Further assessment of efficacy is warranted in a randomized placebo-controlled trial.
Asunto(s)
Antioxidantes/uso terapéutico , Ataxia de Friedreich/tratamiento farmacológico , Ataxia de Friedreich/metabolismo , Proteínas de Unión a Hierro/metabolismo , Estilbenos/uso terapéutico , 8-Hidroxi-2'-Desoxicoguanosina , Adulto , Desoxiguanosina/análogos & derivados , Desoxiguanosina/metabolismo , F2-Isoprostanos/sangre , Femenino , Análisis de Fourier , Humanos , Proteínas de Unión a Hierro/genética , Leucocitos Mononucleares/metabolismo , Masculino , Persona de Mediana Edad , ARN Mensajero/metabolismo , Resveratrol , Resultado del Tratamiento , Adulto Joven , FrataxinaRESUMEN
Neurodegenerative disorders such as Friedreich ataxia (FRDA) present significant challenges in developing effective therapeutic intervention. Current treatments aim to manage symptoms and thus improve quality of life, but none can cure, nor are proven to slow, the neurodegeneration inherent to this disease. The primary clinical features of FRDA include progressive ataxia and shortened life span, with complications of cardiomyopathy being the major cause of death. FRDA is most commonly caused by an expanded GAA trinucleotide repeat in the first intron of FXN that leads to reduced levels of frataxin, a mitochondrial protein important for iron metabolism. The GAA expansion in FRDA does not alter the coding sequence of FXN. It results in reduced production of structurally normal frataxin, and hence any increase in protein level is expected to be therapeutically beneficial. Recently, there has been increased interest in developing novel therapeutic applications like cell and/or gene therapies, and these cutting-edge applications could provide effective treatment options for FRDA. Importantly, since individuals with FRDA produce frataxin at low levels, increased expression should not elicit an immune response. Here we review the advances to date and highlight the future potential for cell and gene therapy to treat this debilitating disease.
Asunto(s)
Ataxia de Friedreich/terapia , Terapia Genética , Trasplante de Células Madre , Animales , Células de la Médula Ósea/fisiología , Vectores Genéticos , Humanos , Transducción GenéticaRESUMEN
Friedreich ataxia (FRDA) is a devastating neurodegenerative disease caused by mutations in the frataxin gene (FXN). Frataxin is an essential protein which localizes to the mitochondria and is required for the synthesis of iron-sulfur clusters and heme. Most individuals with FRDA are homozygous for trinucleotide GAA.TTC repeat expansions in intron 1 of FXN. The instability of these GAA.TTC repeats, the formation of non-B DNA GAA.TTC structures, and accompanying epigenetic changes lead to reduced FXN transcript and frataxin protein. This 'loss of frataxin' is considered the main driver of disease pathology with mitochondria-rich tissues such as the heart and the brain most affected. While our understanding of FRDA etiology has advanced in recent years, exactly how reduced frataxin leads to disease remains largely unknown. Most therapeutic strategies aim to increase frataxin, yet there are other underlying aspects of the molecular pathology that could impact disease progression and severity. These include RNA toxicity due to antisense RNAs, dysregulated splicing and microRNAs, and repeat-associated protein toxicity via RAN translation. Here we review the diverse array of molecular events that have been shown to influence clinical outcome in FRDA. We also examine additional pathogenic factors from other trinucleotide repeat diseases which could be potentially important in FRDA.
Asunto(s)
Ataxia de Friedreich/genética , Ataxia de Friedreich/patología , Proteínas de Unión a Hierro/genética , Expansión de Repetición de Trinucleótido/genética , Alelos , Humanos , FrataxinaRESUMEN
BACKGROUND: Friedreich ataxia (FRDA) generally results from reduced frataxin, a mitochondrial protein involved in iron metabolism. We assessed whether HFE p.C282Y and/or p.H63D heterozygosity modifies age at disease onset or disease severity in individuals with FRDA. METHODS: One hundred seventy individuals with FRDA were assessed for the association of HFE p.C282Y and p.H63D with (1) age at disease onset and (2) Friedreich Ataxia Rating Scale (FARS) score. RESULTS: After adjusting for the smaller FXN GAA repeat size and sex, individuals with FRDA and heterozygous for p.C282Y had disease onset on average 3.72 years earlier than those homozygous for the wild-type amino acid (P = 0.02). Neither mutation affected disease severity as measured by FARS. CONCLUSIONS: It is hypothesized that the association between p.C282Y heterozygosity and an earlier age at FRDA onset relates to exacerbation of the already dysregulated iron metabolism that plays a major role in the pathogenesis of FRDA.
Asunto(s)
Ataxia de Friedreich/genética , Antígenos de Histocompatibilidad Clase I/genética , Proteínas de la Membrana/genética , Mutación Puntual/genética , Adolescente , Adulto , Edad de Inicio , Anciano , Niño , Femenino , Genotipo , Proteína de la Hemocromatosis , Heterocigoto , Humanos , Proteínas de Unión a Hierro/genética , Masculino , Persona de Mediana Edad , Proteínas Mitocondriales/metabolismo , Adulto Joven , FrataxinaRESUMEN
Accumulating evidence supports the important role for epigenetic changes in modulating clinical parameters of complex disorders, including neurodegenerative disease. Several conditions, including fragile X syndrome and Huntington's disease are caused by trinucleotide repeat (TNR) expansions in or near specific genes. Highlighting the link between epigenetic disruption and disease phenotype, recent studies have established significant correlations between clinical features, expansion size, gene expression, the chromatin profile, and DNA methylation in regions surrounding the TNR. Given the debilitating and sometimes fatal consequences of TNR disorders, understanding how an altered epigenetic profile impacts clinical outcome warrants further attention, and could provide key insights for developing novel epigenetic therapies and biomarkers. This review presents the current evidence of epigenetic changes in several TNR diseases.
Asunto(s)
Enfermedad/genética , Epigénesis Genética , Expansión de Repetición de Trinucleótido , Animales , HumanosRESUMEN
OBJECTIVE: Friedreich ataxia (FA) is the most common ataxia and results from an expanded GAA repeat in the first intron of FXN. This leads to epigenetic modifications and reduced frataxin. We investigated the relationships between genetic, epigenetic, and clinical parameters in a large case-control study of FA. METHODS: Clinical data and samples were obtained from individuals with FA during annual visits to our dedicated FA clinic. GAA expansions were evaluated by polymerase chain reaction (PCR) and restriction endonuclease digest. DNA methylation was measured using bisulfite-based EpiTYPER MassARRAY (Sequenom, San Diego, CA). FXN expression was determined using real-time reverse transcriptase PCR. Significant correlations between the different parameters were examined using the nonparametric Spearman rank correlation coefficient, as well as univariate and multivariate regression modeling. RESULTS: Characteristic DNA methylation was identified upstream and downstream of the expansion, and validated in an independent FA cohort. Univariate and multivariate analyses showed significant inverse correlations between upstream methylation and FXN expression, and variation in downstream methylation and age of onset. FXN expression also inversely correlated with the Friedreich Ataxia Rating Scale score, an indicator of disease severity. INTERPRETATION: These novel findings provide compelling evidence for the link between the GAA expansion, the DNA methylation profile, FXN expression, and clinical outcome in FA. Epigenetic profiling of FXN could be used to gain greater insight into disease onset and progression, but also as a biomarker to learn more about specific treatment responses and pharmacological mechanism(s). This work also highlights the potential for developing therapies aimed at increasing frataxin levels to treat this debilitating disease.
Asunto(s)
Metilación de ADN/genética , Ataxia de Friedreich/genética , Marcadores Genéticos/genética , Proteínas de Unión a Hierro/genética , Expansión de Repetición de Trinucleótido/genética , Adolescente , Adulto , Anciano , Estudios de Casos y Controles , Niño , Progresión de la Enfermedad , Epigénesis Genética , Femenino , Humanos , Masculino , Persona de Mediana Edad , Secuencias Repetitivas de Ácidos Nucleicos , Adulto Joven , FrataxinaRESUMEN
Friedreich ataxia (FRDA), the most common of the hereditary ataxias, is an autosomal recessive, multisystem disorder characterised by progressive ataxia, sensory symptoms, weakness, scoliosis and cardiomyopathy. FRDA is caused by a GAA expansion in intron one of the FXN gene, leading to reduced levels of the encoded protein frataxin, which is thought to regulate cellular iron homeostasis. The cerebellar and spinocerebellar dysfunction seen in FRDA has known effects on motor function; however until recently slowed information processing has been the main feature consistently reported by the limited studies addressing cognitive function in FRDA. This chapter will systematically review the current literature regarding the neuropathological and neurobehavioural phenotype associated with FRDA. It will evaluate more recent evidence adopting systematic experimental methodologies that postulate that the neurobehavioural phenotype associated with FRDA is likely to involve impairment in cerebello-cortico connectivity.
Asunto(s)
Cerebelo/fisiopatología , Corteza Cerebral/fisiopatología , Ataxia de Friedreich/fisiopatología , Proteínas de Unión a Hierro/genética , Médula Espinal/fisiopatología , Expansión de Repetición de Trinucleótido , Cerebelo/metabolismo , Cerebelo/patología , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Ataxia de Friedreich/genética , Ataxia de Friedreich/metabolismo , Ataxia de Friedreich/patología , Estudios de Asociación Genética , Humanos , Intrones , Hierro/metabolismo , Fenotipo , Médula Espinal/metabolismo , Médula Espinal/patología , FrataxinaRESUMEN
Friedreich ataxia (FRDA) is an autosomal recessive neurodegenerative disease most commonly caused by a GAA trinucleotide repeat expansion in the first intron of FXN, which reduces expression of the mitochondrial protein frataxin. Approximately 98% of individuals with FRDA are homozygous for GAA expansions, with the remaining 2% compound heterozygotes for a GAA expansion and a point mutation within FXN. Two siblings with early onset of symptoms experienced rapid loss of ambulation by 8 and 10 years. Diagnostic testing for FRDA demonstrated one GAA repeat expansion of 1010 repeats and one non-expanded allele. Sequencing all five exons of FXN identified a novel deletion-insertion mutation in exon 3 (c.371_376del6ins15), which results in a modified frataxin protein sequence at amino acid positions 124-127. Specifically, the amino acid sequence changes from DVSF to VHLEDT, increasing frataxin from 211 residues to 214. Using the known structure of human frataxin, a theoretical 3D model of the mutant protein was developed. In the event that the modified protein is expressed and stable, it is predicted that the acidic interface of frataxin, known to be involved in iron binding and interactions with the iron-sulphur cluster assembly factor IscU, would be impaired.
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Exones , Ataxia de Friedreich/genética , Ataxia de Friedreich/fisiopatología , Mutación INDEL , Proteínas de Unión a Hierro/genética , Hermanos , Adolescente , Niño , Preescolar , Análisis Mutacional de ADN , Femenino , Humanos , Proteínas de Unión a Hierro/química , Masculino , Modelos Moleculares , Fenotipo , Expansión de Repetición de Trinucleótido , Adulto Joven , FrataxinaRESUMEN
The Zap1 transcription factor of Saccharomyces cerevisiae plays a central role in zinc homeostasis by controlling the expression of genes involved in zinc metabolism. Zap1 is active in zinc-limited cells and repressed in replete cells. At the transcriptional level, Zap1 controls its own expression via positive autoregulation. In addition, Zap1's two activation domains are regulated independently of each other by zinc binding directly to those regions and repressing activation function. In this report, we show that Zap1 DNA binding is also inhibited by zinc. DMS footprinting showed that Zap1 target gene promoter occupancy is regulated with or without transcriptional autoregulation. These results were confirmed using chromatin immunoprecipitation. Zinc regulation of DNA binding activity mapped to the DNA binding domain indicating other parts of Zap1 are unnecessary for this control. Overexpression of Zap1 overrode DNA binding regulation and resulted in constitutive promoter occupancy. Under these conditions of constitutive binding, both the zinc dose response of Zap1 activity and cellular zinc accumulation were altered suggesting the importance of DNA binding control to zinc homeostasis. Thus, our results indicated that zinc regulates Zap1 activity post-translationally via three independent mechanisms, all of which contribute to the overall zinc responsiveness of Zap1.
Asunto(s)
ADN/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismo , Zinc/metabolismo , Transporte Activo de Núcleo Celular , Secuencia de Bases , Núcleo Celular/metabolismo , ADN/genética , Homeostasis , Datos de Secuencia Molecular , Unión Proteica , Estructura Terciaria de Proteína , Elementos de Respuesta/genética , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Factores de Transcripción/química , Factores de Transcripción/genética , Transcripción GenéticaRESUMEN
Friedreich ataxia (FRDA) is an autosomal recessive disorder characterised by neurodegeneration and cardiomyopathy. It is caused by a trinucleotide (GAA) repeat expansion in the first intron of the FXN gene that results in reduced synthesis of FXN mRNA and its protein product, frataxin. We report the generation of induced pluripotent stem (iPS) cell lines derived from skin fibroblasts from two FRDA patients. Each of the patient-derived iPS (FA-iPS) cell lines maintain the GAA repeat expansion and the reduced FXN mRNA expression that are characteristic of the patient. The FA-iPS cells are pluripotent and form teratomas when injected into nude mice. We demonstrate that following in vitro differentiation the FA-iPS cells give rise to the two cell types primarily affected in FRDA, peripheral neurons and cardiomyocytes. The FA-iPS cell lines have the potential to provide valuable models to study the cellular pathology of FRDA and to develop high-throughput drug screening assays. We have previously demonstrated that stable insertion of a functional human BAC containing the intact FXN gene into stem cells results in the expression of frataxin protein in differentiated neurons. As such, iPS cell lines derived from FRDA patients, following correction of the mutated gene, could provide a useful source of immunocompatible cells for transplantation therapy.
Asunto(s)
Línea Celular , Ataxia de Friedreich/patología , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/fisiología , Animales , Biomarcadores/metabolismo , Diferenciación Celular , Reprogramación Celular , Ataxia de Friedreich/genética , Humanos , Proteínas de Unión a Hierro/genética , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Teratoma/patología , Expansión de Repetición de Trinucleótido , FrataxinaRESUMEN
Pathogenic activation of the LMO2 proto-oncogene by an oncoretroviral vector insertion in a clinical trial for X-linked severe combined immunodeficiency (X-SCID) has prompted safety concerns. We used an adeno-associated virus vector to achieve targeted insertion of a gamma-retroviral long terminal repeat (LTR) driving a GFP expression cassette with flanking loxP sites in a human T-cell line at the precise location of vector integration in one of the patients with X-SCID. The LTR-GFP cassette was inserted into the first intron of the LMO2 gene, resulting in strong activation of LMO2. Cre-mediated cassette exchange was used to replace the original LTR-GFP cassette with one flanked by insulator elements leading to a several fold reduction in LMO2 expression. The LTR-GFP cassette was also replaced with a globin gene regulatory cassette that failed to activate the LMO2 gene in lymphoid cells. A gamma-retroviral vector with 2 intact LTRs resulted in activation of the LMO2 gene when inserted into the first intron, but a self-inactivating lentiviral vector with an internal cellular promoter and flanking insulator elements did not activate the LMO2 gene. Thus, this system is useful for comparing the safety profiles of vector cassettes with various regulatory elements for their potential for proto-oncogene activation.
Asunto(s)
Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica , Vectores Genéticos , Metaloproteínas/genética , Proto-Oncogenes , Retroviridae/genética , Proteínas Adaptadoras Transductoras de Señales , Cromosomas Humanos X , ADN de Neoplasias/genética , Genes Reporteros , Humanos , Intrones , Células Jurkat , Proteínas con Dominio LIM , Plásmidos , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas/genética , Inmunodeficiencia Combinada Grave/genéticaRESUMEN
Retroviral vectors have been developed for gene therapy of blood disorders because they achieve long-term expression of the transgene. However, interactions between the regulatory elements contained in such vectors and cellular genes may result in pathogenic proto-oncogene activation. We designed a cassette consisting of a splice acceptor followed by the coding sequences for Green Fluorescent Protein (GFP) and a polyadenylation site which was inserted in a reverse orientation in self-inactivating lentiviral vectors. Retroviral vectors integrate preferentially in genes and in one-half of the integrations, the expression cassette will be in a tandem orientation with respect to the gene's promoter and potentially expressed when the trapped promoter is intrinsically active or activated by regulatory elements within the vector. Approximately 10% of the integrations of the control vector with only the GFP cassette resulted in expression of the GFP marker. The trapping cassette in vectors containing globin regulatory elements was expressed more frequently in erythroleukemia (K562) cells than the control cassette only vector but there was no increase in promoter trapping efficiency in HeLa cells. In contrast, we found that addition of a gammaretroviral long terminal repeat increased the frequency of GFP expression both in K562 and HeLa cells. Promoter activation in K562 cells by vectors containing globin regulatory elements was significantly reduced by addition of flanking insulator elements.
Asunto(s)
Células Eritroides/metabolismo , Globinas/genética , Elementos Aisladores/genética , Regiones Promotoras Genéticas/genética , Secuencias Reguladoras de Ácidos Nucleicos/genética , Cromatina/genética , Técnicas de Transferencia de Gen , Terapia Genética , Vectores Genéticos , Células HeLa , Humanos , Células K562 , Proto-Oncogenes MasRESUMEN
Lentiviral vectors efficiently transduce quiescent stem cells and are being evaluated for gene therapy of blood dis-orders. The risk of genotoxicity as a result of insertional mutagenesis is an important safety consideration. The hy-persensitive site 4 insulator from the chicken beta-globin locus (cHS4) possesses chromatin bar-rier and enhancer-blocking functions. A control lentiviral vector encoding green fluorescent protein was compared with a vector in which the cHS4 insulator element flanked the green fluorescent protein expression cassette in single cell isolates of transduced human T cells (Jurkat) after 9 days in culture. The insulator had minimal effect on mean fluorescent intensity and only modestly reduced the variability of green fluorescent protein expression among indi-vidual single cell isolates. Most unique integration sites were within genes, but the insulator-containing vector had a moderate predilection to integrate near the transcriptional start site compared with the control vector. Clonal domi-nance developed in cultures of cells containing the integrated vector genomes, as reflected by the recovery of mul-tiple single cell isolates containing the same integration site. We infer that certain integrations conferred a prolifera-tive or survival advantage by affecting gene expression through insertional mutagenesis, leading to this clonal dominance. This effect was diminished by including the insulator element in the vector genome.