RESUMEN
Epilepsy will affect nearly 3% of people at some point during their lifetime. Previous copy number variants (CNVs) studies of epilepsy have used array-based technology and were restricted to the detection of large or exonic events. In contrast, whole-genome sequencing (WGS) has the potential to more comprehensively profile CNVs but existing analytic methods suffer from limited accuracy. We show that this is in part due to the non-uniformity of read coverage, even after intra-sample normalization. To improve on this, we developed PopSV, an algorithm that uses multiple samples to control for technical variation and enables the robust detection of CNVs. Using WGS and PopSV, we performed a comprehensive characterization of CNVs in 198 individuals affected with epilepsy and 301 controls. For both large and small variants, we found an enrichment of rare exonic events in epilepsy patients, especially in genes with predicted loss-of-function intolerance. Notably, this genome-wide survey also revealed an enrichment of rare non-coding CNVs near previously known epilepsy genes. This enrichment was strongest for non-coding CNVs located within 100 Kbp of an epilepsy gene and in regions associated with changes in the gene expression, such as expression QTLs or DNase I hypersensitive sites. Finally, we report on 21 potentially damaging events that could be associated with known or new candidate epilepsy genes. Our results suggest that comprehensive sequence-based profiling of CNVs could help explain a larger fraction of epilepsy cases.
Asunto(s)
Variaciones en el Número de Copia de ADN , Epilepsia/genética , Estudios de Casos y Controles , Estudios de Cohortes , Humanos , Sitios de Carácter Cuantitativo , Secuenciación Completa del GenomaRESUMEN
Scn1b-null mice have a severe neurological and cardiac phenotype. Human mutations in SCN1B result in epilepsy and cardiac arrhythmia. SCN1B is expressed as two developmentally regulated splice variants, ß1 and ß1B, that are each expressed in brain and heart in rodents and humans. Here, we studied the structure and function of ß1B and investigated a novel human SCN1B epilepsy-related mutation (p.G257R) unique to ß1B. We show that wild-type ß1B is not a transmembrane protein, but a soluble protein expressed predominantly during embryonic development that promotes neurite outgrowth. Association of ß1B with voltage-gated Na+ channels Na(v)1.1 or Na(v)1.3 is not detectable by immunoprecipitation and ß1B does not affect Na(v)1.3 cell surface expression as measured by [(3)H]saxitoxin binding. However, ß1B coexpression results in subtle alteration of Na(v)1.3 currents in transfected cells, suggesting that ß1B may modulate Na+ current in brain. Similar to the previously characterized p.R125C mutation, p.G257R results in intracellular retention of ß1B, generating a functional null allele. In contrast, two other SCN1B mutations associated with epilepsy, p.C121W and p.R85H, are expressed at the cell surface. We propose that ß1B p.G257R may contribute to epilepsy through a mechanism that includes intracellular retention resulting in aberrant neuronal pathfinding.
Asunto(s)
Epilepsia/genética , Mutación/genética , Canales de Sodio/genética , Canales de Sodio/metabolismo , Secuencia de Aminoácidos , Animales , Animales Recién Nacidos , Arginina/genética , Biotinilación/métodos , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Células Cultivadas , Cerebelo , Cricetinae , Cricetulus , Femenino , Regulación del Desarrollo de la Expresión Génica/genética , Genotipo , Glicina/genética , Humanos , Inmunoprecipitación/métodos , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Canal de Sodio Activado por Voltaje NAV1.3 , Neuritas/metabolismo , Neuronas/citología , Neuronas/fisiología , Técnicas de Placa-Clamp , Isoformas de Proteínas/genética , ARN Mensajero/metabolismo , Saxitoxina/farmacocinética , Canales de Sodio/deficiencia , Estadísticas no Paramétricas , Transfección/métodos , Tritio/farmacocinética , Subunidad beta-1 de Canal de Sodio Activado por VoltajeRESUMEN
The With No lysine (K) family of serine/threonine kinase (WNK) defines a small family of kinases with significant roles in ion homeostasis. WNK1 has been shown to have different isoforms due to what seems to be largely tissue specific splicing. Here, we used two distinct in situ hybridization riboprobes on developing and adult mouse tissues to make a comparative analysis of Wnk1 and its sensory associated splice isoform, Wnk1/Hsn2. The hybridization signals in developing mouse tissues, which were prepared at embryonic day e10.5 and e12.5, revealed a homogenous expression profile with both probes. At e15.5 and in the newborn mouse, the two probes revealed different expression profiles with prominent signals in nervous system tissues and also other tissues such as kidney, thymus and testis. In adult mouse tissues, the two expression profiles appeared even more restricted to the nervous tissues, kidney, thymus and testis, with no detectable signal in the other tissues. Throughout the nervous system, sensory tissues, as well as in Cornu Ammonis 1 (CA1), CA2 and CA3 areas of the hippocampus, were strongly labeled with both probes. Hybridization signals were also strongly detected in Schwann and supporting satellite cells. Our results show that the expression profiles of Wnk1 isoforms change during the development, and that the expression of the Wnk1 splice variant containing the Hsn2 exon is prominent during developing and in adult mouse tissues, suggesting its important role in the development and maintenance of the nervous system.