Mutational analysis of the ARX gene and whole exome sequencing in epileptic encephalopathy patients

Abstract

The term epileptic encephalopathy (EE) is used to describe epileptic syndromes with generally early onset, within the first year of life, refractory to drug treatment and poor developmental outcome. The genetics of epileptic encephalopathy disorders can be monogenic or complex. ARX gene mutations were observed in epileptic encephalopathy phenotypes such as Ohtahara and West Syndrome. The most common mutation seen in ARX gene is the polyalanine tract expansion, which is due to duplication of a 24 base-pair polyalanine tract that increases the alanine number to 20. ARX is a homeobox-containing gene encoding for Homeobox protein ARX which has roles in normal brain development. The polyalanine expansion in the ARX gene results in intellectual disability. Epileptic encephalopathy diagnosis cannot be solely made based on clinical criteria, and ARX gene mutations and repeat expansions are considered as an important parameter for both diagnosis and treatment. The aim of this study was to explore the genetic basis of epileptic encephalopathy. Genetic causes of EE are heterogeneous; hence, whole exome sequencing (WES) is necessary to reveal the causative variants. However, repeat expansions cannot be detected by WES technique. Therefore, initially ARX gene mutations and repeat expansions in epilepsy patients with EE were analysed by Agilent and Sanger sequencing in patients with ARX-related phenotypes. In addition, WES was performed in 3 patients with EE and without ARX mutations to reveal pathological variants, and candidate variants were determined. For ARX repeat analysis, exon 2.1 part with the repeat region of 16 patients that present ARX-related phenotype was screened using Agilent system, revealing no significant repeat number increase in the patients. Other exon regions were analysed by PCR and Sanger sequencing for the 16 patients. Analyses of Sanger sequencing data using FinchTV software revealed no mutations in the exons of ARX gene. WES was then performed in 3 out of 16 EE patients with ARX-related disease phenotype. After family segregation analysis, CUL4B and MAP2 variants were selected as the strongest candidates responsible for EE phenotype in two patients.