Exome sequencing reveals new causal mutations in children with epileptic encephalopathies

Abstract

Purpose: The management of epilepsy in children is particularly challenging when seizures are resistant to antiepileptic medications, or undergo many changes in seizure type over time, or have comorbid cognitive, behavioral, or motor deficits. Despite efforts to classify such epilepsies based on clinical and electroencephalographic criteria, many children never receive a definitive etiologic diagnosis. Whole exome sequencing (WES) is proving to be a highly effective method for identifying de novo variants that cause neurologic disorders, especially those associated with abnormal brain development. Herein we explore the utility of WES for identifying candidate causal de novo variants in a cohort of children with heterogeneous sporadic epilepsies without etiologic diagnoses.

Methods: We performed WES (mean coverage approximately 40×) on 10 trios comprised of unaffected parents and a child with sporadic epilepsy characterized by difficult-to-control seizures and some combination of developmental delay, epileptic encephalopathy, autistic features, cognitive impairment, or motor deficits. Sequence processing and variant calling were performed using standard bioinformatics tools. A custom filtering system was used to prioritize de novo variants of possible functional significance for validation by Sanger sequencing.

Key findings: In 9 of 10 probands, we identified one or more de novo variants predicted to alter protein function, for a total of 15. Four probands had de novo mutations in genes previously shown to harbor heterozygous mutations in patients with severe, early onset epilepsies (two in SCN1A, and one each in CDKL5 and EEF1A2). In three children, the de novo variants were in genes with functional roles that are plausibly relevant to epilepsy (KCNH5, CLCN4, and ARHGEF15). The variant in KCNH5 alters one of the highly conserved arginine residues of the voltage sensor of the encoded voltage-gated potassium channel. In vitro analyses using cell-based assays revealed that the CLCN4 mutation greatly impaired ion transport by the ClC-4 2Cl(-) /H(+) -exchanger and that the mutation in ARHGEF15 reduced GEF exchange activity of the gene product, Ephexin5, by about 50%. Of interest, these seven probands all presented with seizures within the first 6 months of life, and six of these have intractable seizures.

Significance: The finding that 7 of 10 children carried de novo mutations in genes of known or plausible clinical significance to neuronal excitability suggests that WES will be of use for the molecular genetic diagnosis of sporadic epilepsies in children, especially when seizures are of early onset and difficult to control.

Keywords: ARHGEF15; CDKL5; CLCN4; Epileptic encephalopathy; SCN1A.

Figure 1. Automated hierarchical filtering system to identify real de novo mutations

All variants that fit a de novo inheritance pattern (variant allele present in the proband but neither parent) are first examined for evidence of reads with the variant allele in either parent despite the actual genotype called (aa/aa/ab or aa/aa/bb). When <30% reads containing the alternate allele were present in the parent, variants are placed into Level 7 (Kong et al. (Kong et al., 2012)) because of a likely false heterozygote call in the proband. When >10% of reads with the variant allele were in one or both of the parents, variants are placed into Level 6 (because of a likely false homozygote reference call in the parent). Remaining variants within 5bp of an indel are assigned to Level 5 due to possible misalignment. Remaining variants in proximity to other (at least one) Mendelian Inheritance Errors (MIEs) (i.e. in the same gene) are assigned to Level 4 because of either possible misalignment or poor region capture/sequencing. Remaining variants within a coverage in the proband > 2 standard deviations from the mean are placed into Level 3 due to likely lying in repetitive sequence. Remaining variants found in large population genetic variation screens (i.e. 1000 Genomes and the 5400 exomes of ESP) are assigned to Level 2 because of likely non-pathogenicity or systematic sequencing artifacts. The remaining variants are assigned to Level 1 and represent those putative de novo variants that are most likely to be real and are Sanger validated. Variants with a frequency < 0.001 in Level 2 are also Sanger validated.

Figure 2. Characterization of putative epilepsy-causing mutation in human CLC-4

(A) Overexpression of human ClC-4 wildtype and mutant protein (green) in HeLa cells show that the G544R mutation does not alter the subcellular localization of the protein. Nuclei are stained with DAPI in blue. (B) Current voltage-relationships of ClC-4 WT and ClC-4^G544R expressed in Xenopus oocytes. The results were obtained with at least five oocytes from four batches of oocytes. Control measurements were done on uninjected oocytes. Error bars indicate standard error of the mean (some error bars are smaller than symbol size).

Figure 3. De novo mutation at Arg604Cys disrupts Ephexin5’s guanine nucleotide exchange activity

A) Lysates from HEK 293T cells transfected with control plasmid, human WT-Ephexin5, or R604C were subjected to the G-LISA activation assay. Error bars indicate SEM (n=4) *p < .01, Student’s T-test. B) Mouse WT-Ephexin5, or R612C were assayed for RhoA activation similar to A). Error bars indicate SEM (n=3) *p < .05, Student’s T-test. C) Representative quantitative Western Blot using LiCor Odyssey IR software from lysates used in A) and B). Protein samples were run on an SDS-PAGE gel and Ephexin5 protein was detected using a rabbit polyclonal antibody raised against the N-terminus. D) Western Blot quantification for all samples. Error bars indicate SEM (n=4 per human conditions; n=3 per mouse conditions), p > .05, Student’s T-test.