Analysis of gene variants in the GASH/Sal model of epilepsy

Elena Díaz-Casado,María Jara-Acevedo,Ricardo Gómez-Nieto,Luis J. Muñoz,José M. de Pereda,Dolores E. López,Giuseppe Biagini

doi:10.1371/journal.pone.0229953

Abstract

Epilepsy is a complex neurological disorder characterized by sudden and recurrent seizures, which are caused by various factors, including genetic abnormalities. Several animal models of epilepsy mimic the different symptoms of this disorder. In particular, the genetic audiogenic seizure hamster from Salamanca (GASH/Sal) animals exhibit sound-induced seizures similar to the generalized tonic seizures observed in epileptic patients. However, the genetic alterations underlying the audiogenic seizure susceptibility of the GASH/Sal model remain unknown. In addition, gene variations in the GASH/Sal might have a close resemblance with those described in humans with epilepsy, which is a prerequisite for any new preclinical studies that target genetic abnormalities. Here, we performed whole exome sequencing (WES) in GASH/Sal animals and their corresponding controls to identify and characterize the mutational landscape of the GASH/Sal strain. After filtering the results, moderate- and high-impact variants were validated by Sanger sequencing, assessing the possible impact of the mutations by “in silico” reconstruction of the encoded proteins and analyzing their corresponding biological pathways. Lastly, we quantified gene expression levels by RT-qPCR. In the GASH/Sal model, WES showed the presence of 342 variations, in which 21 were classified as high-impact mutations. After a full bioinformatics analysis to highlight the high quality and reliable variants, the presence of 3 high-impact and 15 moderate-impact variants were identified. Gene expression analysis of the high-impact variants of Asb14 (ankyrin repeat and SOCS Box Containing 14), Msh3 (MutS Homolog 3) and Arhgef38 (Rho Guanine Nucleotide Exchange Factor 38) genes showed a higher expression in the GASH/Sal than in control hamsters. In silico analysis of the functional consequences indicated that those mutations in the three encoded proteins would have severe functional alterations. By functional analysis of the variants, we detected 44 significantly enriched pathways, including the glutamatergic synapse pathway. The data show three high-impact mutations with a major impact on the function of the proteins encoded by these genes, although no mutation in these three genes has been associated with some type of epilepsy until now. Furthermore, GASH/Sal animals also showed gene variants associated with different types of epilepsy that has been extensively documented, as well as mutations in other genes that encode proteins with functions related to neuronal excitability, which could be implied in the phenotype of the GASH/Sal. Our findings provide valuable genetic and biological pathway data associated to the genetic burden of the audiogenic seizure susceptibility and reinforce the need to validate the role of each key mutation in the phenotype of the GASH/Sal model.

Highlights

Epilepsy is a complex neurological disorder, defined by sudden and recurrent seizures, which involves various underlying conditions associated with its etiology
13 GASH/Sal animals from the inbred strain maintained at the vivarium of the University of Salamanca (USAL, Spain) and 12 wild-type Syrian golden hamsters (RjHan: AURA) from Janvier Labs (Le Genest-Saint-Isle, France), that were used as a control group
We focused our study on these mutations because they were the most likely to have severe effects on the encoded proteins and on their function

Summary

Introduction

Epilepsy is a complex neurological disorder, defined by sudden and recurrent seizures, which involves various underlying conditions associated with its etiology. Epilepsy is one of the most common neurological diseases, affecting approximately 50 million people worldwide [1,2]. The causes of epilepsy are diverse and heterogeneous, some epilepsy syndromes are genetic and most often heritable conditions resulting from a pathogenic variant (mutation) with significant functional effects [3]. With the advent of whole genome sequencing (WGS), more complex genetic mechanisms underlying many forms of epilepsy are being identified [6,7]. Whole exome sequencing (WES) makes it possible to identify alleles with direct functional consequences on proteins; most known human disease-causing variants reside within the exome [8,9]

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Results

Conclusion