De Novo Mutations in YWHAG Cause Early-Onset Epilepsy

Abstract

Massively parallel sequencing has revealed many de novo mutations in the etiology of developmental and epileptic encephalopathies (EEs), highlighting their genetic heterogeneity. Additional candidate genes have been prioritized in silico by their co-expression in the brain. Here, we evaluate rare coding variability in 20 candidates nominated with the use of a reference gene set of 51 established EE-associated genes. Variants within the 20 candidate genes were extracted from exome-sequencing data of 42 subjects with EE and no previous genetic diagnosis. We identified 7 rare non-synonymous variants in 7 of 20 genes and performed Sanger sequence validation in affected probands and parental samples. De novo variants were found only in SLC1A2 (aka EAAT2 or GLT1) (c.244G>A [p.Gly82Arg]) and YWHAG (aka 14-3-3γ) (c.394C>T [p.Arg132Cys]), highlighting the potential cause of EE in 5% (2/42) of subjects. Seven additional subjects with de novo variants in SLC1A2 (n = 1) and YWHAG (n = 6) were subsequently identified through online tools. We identified a highly significant enrichment of de novo variants in YWHAG, establishing their role in early-onset epilepsy, and we provide additional support for the prior assignment of SLC1A2. Hence, in silico modeling of brain co-expression is an efficient method for nominating EE-associated genes to further elucidate the disorder's etiology and genotype-phenotype correlations.

Keywords: SLC1A2; YWHAG; de novo variants; epileptic encephalopathy; whole-exome sequencing.

Figure 1

De Novo SLC1A2 and YWHAG Mutations in Individuals with Early-Onset Epilepsy (A) Top: transmembrane topology of EAAT2 (adapted from Yernool et al.³⁰) and localization of the variants identified in this study (green) or previously reported (black) (the asterisk indicates a recurrent variant). Bottom: partial sequence alignment of EAAT2 orthologs and different human EAAT proteins surrounding p.Pro289Arg (indicated by an arrow). Identical residues across all proteins are shown in black, and residues identical to the human EAAT2 are in gray. GenBank accession numbers are as follows: Homo sapiens, NP_004162.2; Macaca mulatta, NP_001248598.1; Mus musculus, NP_001070982.1; Gallus gallus, NP_001012917.1; Xenopus tropicalis, XP_002937340.1; Dario rerio, NP_956273.1; Caenorhabditis elegans, NP_001024393.1; human EAAT1, NP_004163.3; human EAAT3, NP_004161.4; human EAAT4, NP_005062.1; and human EAAT5, NP_006662.3. (B) Top: crystal structure of 14-3-3γ (PDB: 5D3E). Left: dimeric 14-3-3γ is shown as green ribbons, and the phosphopeptide ligand is shown as an orange stick. Right: close-up view of the binding groove, side chains of the residues crucial for the phosphopeptide binding, and hydrogen bonds (green dashed lines). Images were generated with Swiss-Pdb Viewer v.4.10. Bottom: partial sequence alignment of 14-3-3γ orthologs and different human 14-3-3 proteins surrounding the mutated residues identified in this study (green) or previously reported (black). Identical residues across all proteins are shown in black, and residues identical to the human 14-3-3γ are in gray. GenBank accession numbers are as follows: Homo sapiens, NP_036611.2; Macaca mulatta, NP_001181365.1; Mus musculus, NP_061359.2; Gallus gallus, NP_001026648.1; Xenopus tropicalis, NP_001072309.1; Dario rerio, NP_998187.1; Caenorhabditis elegans, AAA61872.1; human 14-3-3β, NP_003395.1; human 14-3-3ε, NP_006752.1; human 14-3-3 ζ, NP_001129174.1; human 14-3-3 η, NP_003396.1; human 14-3-3 θ, NP_006817.1; and human 14-3-3 NP_006133.1. Sequences were aligned with CLUSTAL Omega. Asterisks indicate positions with a single fully conserved residue, colons indicate conservation between groups with strongly similar properties, and periods indicate conservation between groups with weakly similar properties.

Figure 2

MRI Findings in Individuals with SLC1A2 Mutations (A–G) Subject A. Sagittal T1 (A) and axial T2 (B) at 6 weeks revealed a thin corpus callosum but within normal limits for age. Axial T2 (C and D) and sagittal T1 (E) at 2 years revealed volume loss and a thin corpus callosum, increased T2 white-matter signal intensity, delayed myelination, decreased T2 signal of thalami, and increased T2 signal of putamen and caudate. At 4 years, axial T2 (F) and sagittal T1 (G) showed progressive volume loss; persistent abnormal signal in thalami, putamen, and caudate; a focal area of increased T2 signal in the left lentiform nucleus; and a thin corpus callosum. (H–J) Subject C. Axial T2 (H) and sagittal T1 (I) at 2 months were normal. At 2.5 years, axial T2 (J) revealed atrophy, delayed myelination, and bilateral T2 prolongation in caudate heads and putamina.