RESUMEN
Millions of patients, with suspected complex neurogenetic disorders, living in resource limited regions around the world have no access to genetic testing despite the rapidly expanding availability and decreasing costs of genetic testing in first world nations. The barriers to increasing availability of genetic testing in resource limited nations are multifactorial but can be attributed, in large part, to a lack of awareness of the power of genetic testing to lead to a rapid, cost-effective, diagnosis that potentially will have profound clinical implications on treatment and patient outcomes. We report our experience with whole exome sequencing (WES) done for the first time in 5 patients of African descent with a suspected neurogenetic disorder living in a resource limited setting on the Eastern Caribbean island of Barbados. A diagnostic pathogenic mutation was found in 3 patients in the SCN1A, STXBP1 and SCN4A, who clinically were diagnosed with Dravet syndrome, Lennox-Gastaut syndrome, paramytonia and seizures respectively. A variant of undetermined significance was found in a patient with global developmental delays, hypotonia, with abnormal eye movements. In one patient WES was non-diagnostic. This result highlights the high yield of WES in carefully selected patients with a neurologic disease and the need for increase access to genetic testing in resource limited settings globally.
Asunto(s)
Secuenciación del Exoma/métodos , Pruebas Genéticas/métodos , Enfermedades del Sistema Nervioso/diagnóstico , Adulto , Barbados , Niño , Análisis Costo-Beneficio , Pruebas Genéticas/economía , Humanos , Lactante , Proteínas Munc18/genética , Mutación , Canal de Sodio Activado por Voltaje NAV1.1/genética , Canal de Sodio Activado por Voltaje NAV1.4/genética , Enfermedades del Sistema Nervioso/genética , Enfermedades del Sistema Nervioso/fisiopatología , Secuenciación del Exoma/economía , Adulto JovenRESUMEN
The spermatozoon is a very specialized cell capable of carrying out a limited set of functions with high efficiency. Sperm are then excellent model cells to dissect fundamental processes such as regulated exocytosis. The secretion of the single dense-core granule of mammalian spermatozoa relies on the same highly conserved molecules and goes through the same stages as exocytosis in other types of cells. In this study, we describe the presence of Munc18-1 in human sperm and show that this protein has an essential role in acrosomal exocytosis. We observed that inactivation of endogenous Munc18-1 with a specific antibody precluded the stabilization of trans-SNARE complexes and inhibited acrosomal exocytosis. Addition of recombinant Munc18-1 blocked secretion by sequestering monomeric syntaxin, an effect that was rescued by α-soluble NSF attachment protein. By electron microscopy, we observed that both the anti-Munc18-1 antibody and recombinant Munc18-1 inhibited the docking of the acrosome to the plasma membrane. In conclusion, our results indicate that Munc18-1 plays a key role in the dynamics of trans-SNARE complex assembly and/or stabilization, a process that is necessary for the docking of the outer acrosomal membrane to the plasma membrane and subsequent fusion pore opening.
Asunto(s)
Acrosoma/metabolismo , Membrana Celular/metabolismo , Exocitosis/fisiología , Proteínas Munc18/metabolismo , Proteínas SNARE/metabolismo , Acrosoma/ultraestructura , Reacción Acrosómica/fisiología , Anticuerpos/química , Membrana Celular/genética , Humanos , Masculino , Proteínas Munc18/genética , Estabilidad Proteica , Proteínas SNARE/genéticaRESUMEN
The structure and evolution of the SEC1 gene were examined for the first time in New World primates of the genera Alouatta, Aotus, Ateles, Brachyteles, Callicebus, Callithrix, Cebus, Chiropotes, Lagothrix, Leontopithecus, Pithecia, Saguinus, and Saimiri. This gene has a high CG content (63.8%) and an estimated heterogeneous size ranging from 795 (Callithrix) to 1041 bp (Pithecia), due to numerous indel events. Similar to other fucosyltransferases, three conserved regions are shared by these primates, except for the callitrichines, Aotus and Pithecia, in which indel events resulted in premature stop codons that are related to the production of a supposedly non-functional protein. Phylogenetic analysis of the SEC1 gene, transition/transversion rates, and nucleotide sequence alignment support the hypothesis that primate SEC1 evolved by divergent evolution, and that the lack of activity in some lineages occurred independently at least twice in New World primates, once in the Aotus-Cebus-Callitrichinae group and again in Pithecia. Likelihood-based inference of ancestral states for the activity of SEC1 leads us to suppose that inactivation of SEC1 in the Callitrichinae was a result of a more complex series of events than in Pithecia.
Asunto(s)
Cebidae/genética , Evolución Molecular , Proteínas Munc18/genética , Secuencia de Aminoácidos , Animales , Cebidae/clasificación , Humanos , Datos de Secuencia Molecular , Filogenia , Alineación de SecuenciaRESUMEN
Prolactin induces maturation of insulin secretion in cultured neonatal rat islets. In this study, we investigated whether the improved secretory response to glucose caused by prolactin involves alteration in the expression, association and phosphorylation of several proteins that participate in these processes. Messenger RNA was extracted from neonatal rat islets cultured for 5 days in the presence of prolactin and reverse transcribed. Gene expression was analyzed by semi-quantitative RT-PCR and by Western blotting for proteins. The gene transcription and protein expression of kinesin and MAP-2 were increased in prolactin-treated islets compared to the controls. The association and phosphorylation of proteins was analyzed by immunoprecipitation followed by Western blotting, after acute exposure to prolactin. Prolactin increased the association between SNARE proteins and kinesin/MAP-2 while the association of munc-18/syntaxin 1A was decreased. Serine phosphorylation of SNAP-25, syntaxin 1A, munc-18, MAP-2 was significantly higher whereas kinesin phosphorylation was decreased in prolactin-treated islets. There was an increase in SNARE complex formation in islets stimulated with prolactin, 22 mM glucose, 40 mM K(+), 200 microM carbachol and 1 microM PMA. The prolactin-induced increase in the formation of SNARE complex and syntaxin 1A phosphorylation was inhibited by PD098059 and U0126, inhibitors of the MAPK pathway. These findings indicate that prolactin primes pancreatic beta-cells to release insulin by increasing the expression and phosphorylation/association of proteins implicated in the secretory machinery and the MAPK/PKC pathway is important for this effect.