RESUMEN
Hereditary angioedema (HAE) is a disease caused by defects in the C1 inhibitor gene (SERPING1/C1NH). We screened the entire C1NH gene for mutations in a large series of 87 Spanish families (77 with type I, and 10 with type II HAE) by SSCP, sequencing, Southern blotting, and quantitative multiplex PCR of short fluorescent fragments (QMPSF), and we characterized several defects at the mRNA level. We found large rearrangements in 13 families, and point mutations or microdeletions/insertions in 74 families. The 13 large rearrangements included nine exon deletions, of which at least eight were distinct, two were distinct exon duplications, and two were rearrangements whose precise nature could not be determined. We confirmed that exon 4 is particularly prone to rearrangements. Thirty-six mutations were unreported, and included 10 microdeletions/insertions, 10 missense, five nonsense, eight splicing, and three splicing or missense mutations. Moreover, we detected six novel uncharacterized sequence variants (USV). RT-PCR studies showed that in addition to several intronic splice site mutations tested, the exonic mutations c.882C>G and c.884T>G, located near the 3' end of exon 5, also produced exon skipping. This is the first evidence of SERPING1/C1NH mutations in coding regions that differ from the canonical splice sites that affect splicing, which suggests the presence of an exonic splicing enhancer (ESE) in exon 5.
Asunto(s)
Proteínas Inactivadoras del Complemento 1/genética , Predisposición Genética a la Enfermedad , Mutación , Serpinas/genética , Empalme Alternativo , Angioedema/genética , Proteína Inhibidora del Complemento C1 , Elementos de Facilitación Genéticos , Exones , Salud de la Familia , Eliminación de Gen , Humanos , Mutación Puntual , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , EspañaRESUMEN
Hereditary angioedema (HAE) is caused by mutations in the C1 inhibitor gene (SERPING1, C1NH) and the result is C1 inhibitor deficiency, either in levels or function. We have searched exon 8 for mutations by direct sequencing and analyzed the rest of the exons by SSCP in 87 Spanish families affected by HAE. Out of 87 screened families, we have detected exon 8 mutations in 26. Among these, 17 different mutations were identified: 14 point mutations and 3 frameshift. Seven of the point mutations and the three frameshift were not previously reported. Mutations were: S438P; R444P; V451G; W460X; V468D; G471E; X479R; S417fsX427; I440fsX450; E429fsX450. The rest of the families presented previously reported mutations, 5 missense and two nonsense. In none of the 26 families was an additional change identified in the rest of the exons by SSCP, and, in 20 out of the 22 families with point mutation, we verified that the mutation did not affect a healthy relative. Seven of these families had no history of the disease, and in five of them we were able to verify that the progenitors did not have the mutation. Therefore, they were de novo mutations.
Asunto(s)
Angioedema/genética , Proteínas Inactivadoras del Complemento 1/genética , Exones , Mutación , Serpinas/genética , Proteína Inhibidora del Complemento C1 , Mutación del Sistema de Lectura , Humanos , Mutación Puntual , Polimorfismo Conformacional Retorcido-SimpleRESUMEN
Neuroblastoma (NB) is a pediatric tumor of the sympathetic nervous system, which is often associated with elevated catecholamines. More than half of patients with metastatic NB relapse and survival is extremely poor with current therapies. In a high-throughput screen of FDA-approved drugs we identified anti-NB activity for the nonselective ß-adrenergic receptor antagonist propranolol hydrochloride. Propranolol inhibited growth of a panel of fifteen NB cell lines irrespective of MYCN status, and treatment induced apoptosis and decreased proliferation. Activity was dependent on inhibition of the ß2, and not ß1, adrenergic receptor, and treatment resulted in activation of p53 and p73 signaling in vitro. The majority of NB cell lines and primary tumors express ß2 adrenergic receptor and higher mRNA levels correlate with improved patient survival, but expression levels did not correlate with in vitro sensitivity to propranolol. Furthermore, propranolol is synergistic with the topoisomerase I inhibitor SN-38 and propranolol inhibits growth of NB xenografts in vivo at doses similar to those used to treat infants with hemangiomas and hypertension. Taken together, our results suggest that propranolol has activity against NB and thus should be considered in combination treatments for patients with relapsed and refractory NB.
Asunto(s)
Antagonistas Adrenérgicos beta/farmacología , Neoplasias del Sistema Nervioso/tratamiento farmacológico , Neuroblastoma/tratamiento farmacológico , Propranolol/farmacología , Animales , Apoptosis/efectos de los fármacos , Enfermedades del Sistema Nervioso Autónomo/tratamiento farmacológico , Enfermedades del Sistema Nervioso Autónomo/metabolismo , Enfermedades del Sistema Nervioso Autónomo/patología , Procesos de Crecimiento Celular/efectos de los fármacos , Proteínas de Unión al ADN , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Neoplasias del Sistema Nervioso/metabolismo , Neoplasias del Sistema Nervioso/patología , Neuroblastoma/metabolismo , Neuroblastoma/patología , Proteínas Nucleares , Receptores Adrenérgicos beta/metabolismo , Proteína Tumoral p73 , Proteínas Supresoras de Tumor , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The p53 family of transcription factors is a key regulator of cell proliferation and death. In this report we identify the eukaryotic translation elongation factor 1-alpha 1 (eEF1A1) to be a novel p53 and p73 interacting protein. Previous studies have demonstrated that eEF1A1 has translation-independent roles in cancer. We report that overexpression of eEF1A1 specifically inhibits p53-, p73- and chemotherapy-induced apoptosis resulting in chemoresistance. Short-interfering RNA-mediated silencing of eEF1A1 increases chemosensitivity in cell lines bearing wild type p53, but not in p53 null cells. Furthermore, silencing of eEF1A1 partially rescues the chemoresistance observed in response to p53 or p73 knockdown, suggesting that eEF1A1 is a negative regulator of the pro-apoptotic function of p53 and p73. Thus, in the context of p53-family signaling, eEF1A1 has anti-apoptotic properties. These findings identify a novel mechanism of regulation of the p53 family of proteins by eEF1A1 providing additional insight into potential targets to sensitize tumors to chemotherapy.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis , Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/metabolismo , Factor 1 de Elongación Peptídica/fisiología , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Camptotecina/farmacología , Cisplatino/farmacología , Doxorrubicina/farmacología , Resistencia a Antineoplásicos , Etopósido/farmacología , Expresión Génica , Técnicas de Silenciamiento del Gen , Células HEK293 , Células HeLa , Humanos , ARN Interferente Pequeño/genética , Proteína Tumoral p73RESUMEN
BACKGROUND: C1 Inhibitor (C1-Inh) deficiency causes angioedema and can be hereditary (HAE), caused by mutations in the C1-Inh gene (C1NH), or acquired (AAE). Patients with HAE show a complement profile different from that of patients with AAE with normal levels of C1 (C1q, C1r, and C1s). OBJECTIVE: We sought to characterize the complement profile of a patient with HAE and a mutation in homozygosis in the C1NH gene (c.1576T>G, Ile462Ser) and study his family. METHODS: Biochemical diagnosis of HAE was confirmed by analyzing the C1NH gene. Further studies on the levels and activation states of the C1q, C1r, C1s, and C1-Inh components of the classical pathway of complement activation were also performed. RESULTS: Another 7 members of the family were given diagnoses of HAE: 1 was homozygous and 6 were heterozygous for the C1NH mutation c.1576T>G. The homozygous patients showed undetectable C1q levels, reduced C1s levels, the circulating active form of C1r, and a C1-Inh mostly in its cleaved inactive form in plasma. CONCLUSION: This is the first report of patients homozygous for a mutation affecting the coding region of C1NH. These patients showed a unique activation and consumption profile of the classical complement activation pathway different from that commonly observed in patients with HAE but similar to that of patients with AAE. CLINICAL IMPLICATIONS: The most common HAE treatment is attenuated androgens, which increase the C1NH gene transcription levels. Because the homozygous patients lack a wild-type allele, long-term prophylactic treatment with attenuated androgens might not be advisable.
Asunto(s)
Angioedema/genética , Proteína Inhibidora del Complemento C1/genética , Homocigoto , Adolescente , Adulto , Anciano , Angioedema/metabolismo , Western Blotting , Complemento C1/metabolismo , Proteína Inhibidora del Complemento C1/metabolismo , Femenino , Genes/genética , Humanos , Masculino , Persona de Mediana Edad , Mutación , Sistemas de Lectura Abierta/genética , LinajeRESUMEN
Mutations in p73 are rare in cancer. Emerging evidence suggests that the relative expression of various p73 isoforms may contribute to tumorigenesis. Alternative promoters and N-terminal splicing result in the transcription and processing of either full-length (TA) or N-terminally truncated (deltaN) p73 isoforms. TAp73 possesses pro-apoptotic functions, while deltaNp73 has anti-apoptotic properties via functional inhibition of TAp73 and p53. Here, we report that TAp73, but not deltaNp73, is covalently modified by NEDD8 under physiologic conditions in an Mdm2-dependent manner. Co-expression of NEDP1, a cysteine protease that specifically cleaves NEDD8 conjugates, was shown to deneddylate TAp73. In addition, blockage of the endogenous NEDD8 pathway increased TAp73-mediated transactivation of p53- and p73-responsive promoter-driven reporter activity, and in conjunction, neddylated TAp73 species were found preferentially in the cytoplasm. These results suggest that Mdm2 attenuates TAp73 transactivation function, at least in part, by promoting NEDD8-dependent TAp73 cytoplasmic localization and provide the first evidence of a covalent post-translational modification exclusively targeting the TA isoforms of p73.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Activación Transcripcional , Proteínas Supresoras de Tumor/metabolismo , Ubiquitinas/metabolismo , Células Cultivadas , Citoplasma/metabolismo , Proteínas de Unión al ADN/genética , Endopeptidasas/metabolismo , Genes Dominantes , Humanos , Immunoblotting , Inmunoprecipitación , Riñón/metabolismo , Riñón/patología , Luciferasas/metabolismo , Proteína NEDD8 , Proteínas Nucleares/genética , Osteosarcoma/genética , Osteosarcoma/metabolismo , Osteosarcoma/patología , Plásmidos , Regiones Promotoras Genéticas/genética , Procesamiento Proteico-Postraduccional , Proteínas Proto-Oncogénicas c-mdm2/genética , Fracciones Subcelulares , Transcripción Genética , Proteína Tumoral p73 , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor/genética , Ubiquitinas/genéticaRESUMEN
BACKGROUND: Hereditary angioedema (HAE) is a rare disease caused by C1 inhibitor mutations. Although more than 100 mutations have been described, epidemiologic data are lacking; therefore, we developed a Spanish HAE patient registry. OBJECTIVE: To study the prevalence of HAE and the current state of diagnosis and treatment of this disease in Spain. METHODS: Epidemiologic data were obtained by direct contact with physicians who treat patients with HAE and with patients themselves. Diagnosis was evaluated by measuring C1 inhibitor levels and function, and most families also underwent genetic studies. RESULTS: We registered 444 patients (minimal prevalence, 1.09 per 100,000 inhabitants), many of whom are asymptomatic (never having symptoms) (n = 61, 13.7%). Most symptomatic patients (62.9%) receive long-term prophylaxis with attenuated androgens (80.9%) and antifibrinolytic agents (22.8%), alone or in combination, but no patients are receiving long-term prophylaxis with C1 inhibitor. There is a long delay in diagnosis (mean, 13.1 years). Nine patients underwent a tracheotomy as a consequence of a laryngeal attack, and 30 families recalled a total of 38 relatives who died of HAE, which underlines the severity of the illness. CONCLUSIONS: The detected minimal prevalence of HAE in Spain is 1.09 per 100,000 inhabitants. Because this is a rare disease and some patients may be misdiagnosed, this prevalence could be higher.
Asunto(s)
Angioedema/genética , Proteínas Inactivadoras del Complemento 1/deficiencia , Adulto , Angioedema/epidemiología , Proteínas Inactivadoras del Complemento 1/genética , Análisis Mutacional de ADN , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mutación , Prevalencia , Sistema de Registros , España/epidemiologíaRESUMEN
Hereditary angioedema (HAE), a rare but life-threatening condition, manifests as acute attacks of facial, laryngeal, genital, or peripheral swelling or abdominal pain secondary to intra-abdominal edema. Resulting from mutations affecting C1 esterase inhibitor (C1-INH), inhibitor of the first complement system component, attacks are not histamine-mediated and do not respond to antihistamines or corticosteroids. Low awareness and resemblance to other disorders often delay diagnosis; despite availability of C1-INH replacement in some countries, no approved, safe acute attack therapy exists in the United States. The biennial C1 Esterase Inhibitor Deficiency Workshops resulted from a European initiative for better knowledge and treatment of HAE and related diseases. This supplement contains work presented at the third workshop and expanded content toward a definitive picture of angioedema in the absence of allergy. Most notably, it includes cumulative genetic investigations; multinational laboratory diagnosis recommendations; current pathogenesis hypotheses; suggested prophylaxis and acute attack treatment, including home treatment; future treatment options; and analysis of patient subpopulations, including pediatric patients and patients whose angioedema worsened during pregnancy or hormone administration. Causes and management of acquired angioedema and a new type of angioedema with normal C1-INH are also discussed. Collaborative patient and physician efforts, crucial in rare diseases, are emphasized. This supplement seeks to raise awareness and aid diagnosis of HAE, optimize treatment for all patients, and provide a platform for further research in this rare, partially understood disorder.