Construction and initial electrophysiological characterization of an in vitro system expressing the wild-type SCN1A gene and mutations associated with Dravet Syndrome

Author(s)

Demetrio Saul Lindo Samanamud

• Document type: Doctoral Thesis
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Faculdade de Ciências Médicas
• Defense date: 2022-12-12

Examining board members

Íscia Teresinha Lopes Cendes; Simoni Helena Avansini; Fábio Rogério; José Eduardo Peixoto Santos

• Advisor: Íscia Teresinha Lopes Cendes; Andre Schwambach Vieira

Abstract

Epilepsies are a group of diseases with specific signs and symptoms, with typical characteristics on the electroencephalogram, and which often have their etiological aspects, such as genetic, structural, metabolic, and infectious. Currently, it is recognized that many types of epilepsy have a genetic etiological factor, of a monogenic nature. In this context, the SCN1A gene has recognized importance, having been linked to several epilepsy phenotypes. This gene encodes the ?-1 subunit of the voltage-gated sodium channel (Nav1.1). This channel is a multiprotein complex, whose function is to regulate the transport of sodium ions between the extracellular and intracellular environment. Genetic variants in the SCN1A gene can cause changes in the biophysical properties of the sodium channel, which may cause changes in the flow of sodium ions, leading to neuronal hyperexcitability. This way, most patients with Dravet syndrome (DS), an epileptic and developmental encephalopathy characterized by difficult-to-control seizures, associated with varying degrees of intellectual disability and neuropsychomotor developmental delay, have mutations that occur, in general, "de novo" in the SCN1A gene. Indeed, previous studies by our group determined that 81% of patients with DS present variants classified as pathogenic or possibly pathogenic in the SCN1A gene, most of which are of the "missense" type and some of the "nonsense" and "frameshift" types. ", which produce truncated proteins. In general, these different types of disease-linked variants lead to loss of sodium channel function, which ultimately determines the pathophysiology of DS. However, despite this common mechanism, there is great diversity in the phenotypes of epilepsies associated with mutations in the SCN1A gene. This aspect, associated with the ongoing difficulty that still exists in determining the link between genetic variants and the phenotype of patients, makes molecular diagnosis complex. This happens because, currently, the determination of the association between the genetic variation and the phenotypic manifestation of the disease is determined by a combination of criteria that include: the use of predictive tools of the impact of the variants in the protein function, frequency of the variants in the general population, and, mainly, functional studies of alterations in the protein. This last criterion is crucial in elucidating more conclusive evidence on determining the pathogenicity of the variants found in patients. However, functional studies are rare; in most cases, there is no functional criterion to establish the genetic diagnosis. In this scenario, our work aimed to carry out biophysical studies of three mutations that were found in patients with the DS phenotype in our casuistry, they are c.5177G>A, c.5329delG, c.5434T>C that produces the mutant proteins W1726X, V1777fsX1778, and W1812R, respectively. For these mutations, it was not possible to use "in silico" prediction tools in the pathogenicity analysis. Thus, we express these mutant proteins in Hek-293T cells because they do not show endogenous expression of the sodium channel and we use the Patch Clamp technique to characterize the biophysical properties of these mutant channels. Our results, for the three cases studied, show that the current density decreases markedly about the sodium channel without mutation, indicating loss of function in varying degrees. Thus, we conclude that in the three cases, the genetic variation present in the SCN1A gene may be related to the DS phenotype presented by the patients. Our work, in addition to leading to a resolution of the molecular diagnosis of the patients under study and understanding the functional effect of each mutation, establishes the bases for the implementation of this type of study in the inference of the function of the sodium channel in our setting (AU)

• FAPESP's process: 16/03896-3 - Functional characterization of sodium channel mutations associated with epilepsy
• Grantee: Demetrio Saul Lindo Samanamud
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)