DLG3 variants caused X-linked epilepsy with/without neurodevelopmental disorders and the genotype-phenotype correlation

Abstract

Background: The DLG3 gene encodes disks large membrane-associated guanylate kinase scaffold protein 3, which plays essential roles in the clustering of N-methyl-D-aspartate receptors (NMDARs) at excitatory synapses. Previously, DLG3 has been identified as the causative gene of X-linked intellectual developmental disorder-90 (XLID-90; OMIM# 300850). This study aims to explore the phenotypic spectrum of DLG3 and the genotype-phenotype correlation.

Methods: Trios-based whole-exome sequencing was performed in patients with epilepsy of unknown causes. To analyze the genotype-phenotype correlations, previously reported DLG3 variants were systematically reviewed.

Results: DLG3 variants were identified in seven unrelated cases with epilepsy. These variants had no hemizygous frequencies in controls. All variants were predicted to be damaging by silico tools and alter the hydrogen bonds with surrounding residues and/or protein stability. Four cases mainly presented with generalized seizures, including generalized tonic-clonic and myoclonic seizures, and the other three cases exhibited secondary generalized tonic-clonic seizures and focal seizures. Multifocal discharges were recorded in all cases during electroencephalography monitoring, including the four cases with generalized discharges initially but multifocal discharges after drug treating. Protein-protein interaction network analysis revealed that DLG3 interacts with 52 genes with high confidence, in which the majority of disease-causing genes were associated with a wide spectrum of neurodevelopmental disorder (NDD) and epilepsy. Three patients with variants locating outside functional domains all achieved seizure-free, while the four patients with variants locating in functional domains presented poor control of seizures. Analysis of previously reported cases revealed that patients with non-null variants presented higher percentages of epilepsy than those with null variants, suggesting a genotype-phenotype correlation.

Significance: This study suggested that DLG3 variants were associated with epilepsy with/without NDD, expanding the phenotypic spectrum of DLG3. The observed genotype-phenotype correlation potentially contributes to the understanding of the underlying mechanisms driving phenotypic variation.

Keywords: DLG3 gene; Genotype-phenotype correlation; epilepsy; neurodevelopmental disorder; variants.

FIGURE 1

Genetic data of cases with DLG3 variants. (A) Pedigrees of the cases with DLG3 mutations and their corresponding phenotypes. (B) DNA sequencing chromatograms of the cases with DLG3 variants. Red arrows indicate the positions of the mutations. (C) The amino acid sequence alignment showed that the six identified missense variants are residues with high conservation in mammals.

FIGURE 2

Schematic illustration of hydrogen bond changes, hydrophobicity changes and protein stability prediction. (A) Three-dimensional structure of DLG3 protein and localization of DLG3 variants of this study. (B) Fauchère and Pliska hydrophobicity scale exhibited the hydrophobicity of 20 amino acids. Abscissa: from left to right, hydrophobicity gradually decreased. Blue amino acids are hydrophobic, green amino acids are neutral, and yellow amino acids are hydrophilic. Amino acids with high positive values are more hydrophobic, whereas amino acids with low negative values are more hydrophilic. (C) Hydrogen bond changes and Gibbs free energy of folding (DDG) values of DLG3 variants. The residues where the mutations occurred are shown as green rods. The red dotted line represents hydrogen bonds. Arrows indicate the positions with hydrogen bond changes. Three of six variants were predicted to alter hydrogen bonds with surrounding residues. All variants were predicted to decrease protein stability.

FIGURE 3

Representative EEG and magnetic resonance imaging (MRI) of the cases with DLG3 variants. (A) Interictal EEG of case 1 at 2 years of age showed bilateral and multifocal spike-slow waves. (B) Interictal EEG of case 6 at 7 years of age showed bilateral multifocal spikes and spikes-slow waves (obtained). (C,D) Interictal EEG of case 7 at 9 years of age showed spike-slow waves predominant at the left posterior head (C) and eye closure sensitivity (D). (E) The MRI of case 4 at the age of 16 years showed gray matter heterotopia.

FIGURE 4

Schematic illustration of variant location. Schematic diagram of the DLG3 protein and the localization of the DLG3 variants identified in this study. Variants identified in patients with seizure-free are shown in black. Variants identified in patients with poor control of seizures are shown in red.

FIGURE 5

Genotype–phenotype correlation analysis of DLG3 variants. (A) Schematic diagram of the DLG3 protein and the localization of the DLG3 variants identified in this study and reported previously. Variants associated with intellectual disability (ID) are shown in black, variants associated with epilepsy are shown in green, and variants associated with epilepsy and neurodevelopmental disorders (NDDs) are shown in red. * Means termination codon. (B) Pie chart of the genotype distribution of DLG3 variants. (C) The stacked bar chart of inherited patterns and origination of the DLG3 variants (n = 27). (D) Venn diagram of the phenotypes of patients with DLG3 variants. A total of 27 variants were identified, including sixteen variants associated with intellectual disability, nine variants associated with both intellectual disability and epilepsy, and two variants associated with epilepsy. (E) The stacked bar chart of phenotypes of the variants with different genotypes. Variants with non-null variants presented higher percentages in patients with epilepsy with/without NDD than null variants (10/14 vs. 1/13; p = 0.001).

FIGURE 6

Analysis of genes interacting with DLG3. The DLG3 protein interacted with 52 proteins with high confidence [(left), minimum required interaction score ≥ 0.7, STRING database], including five genes identified to be associated with both epilepsy and neurodevelopmental disorder (NDD), eleven genes identified to be associated with NDD with seizures, four genes identified to be associated with NDD without seizures, four genes identified to be associated with other phenotypes, and 28 genes not identified to be associated with phenotypes (right).

FIGURE 7

Schematic diagram of the possible association between DLG3 variants on synaptic damage and related phenotypes. (A) Possible synaptic damage and postsynaptic current alteration of various DLG3 variants. cLOF, complete loss of function; DNE, dominant-negative effects; GOF, gain of function; NMDARE, N-methyl-D-aspartate receptors; pLOF, partial loss of function. (B) The possible association between phenotypes and synaptic damage. Variants of mild or moderate damage would cause subtle functional alteration of synapse with abnormal electrophysiological activity and subsequently epilepsies and/or intellectual disability; while variants of complete loss of function would lead to decreased synaptic conduction, subsequently intellectual disability.