SYNGAP1 encephalopathy:A distinctive generalized developmental and epileptic encephalopathy

Objective To delineate the epileptology, a key part of the SYNGAP1 phenotypic spectrum, in a large patient cohort. Methods Patients were recruited via investigators' practices or social media. We included patients with (likely) pathogenic SYNGAP1 variants or chromosome 6p21.32 microdeletions incorporating SYNGAP1. We analyzed patients' phenotypes using a standardized epilepsy questionnaire, medical records, EEG, MRI, and seizure videos. Results We included 57 patients (53% male, median age 8 years) with SYNGAP1 mutations (n = 53) or microdeletions (n = 4). Of the 57 patients, 56 had epilepsy: generalized in 55, with focal seizures in 7 and infantile spasms in 1. Median seizure onset age was 2 years. A novel type of drop attack was identified comprising eyelid myoclonia evolving to a myoclonic-atonic (n = 5) or atonic (n = 8) seizure. Seizure types included eyelid myoclonia with absences (65%), myoclonic seizures (34%), atypical (20%) and typical (18%) absences, and atonic seizures (14%), triggered by eating in 25%. Developmental delay preceded seizure onset in 54 of 56 (96%) patients for whom early developmental history was available. Developmental plateauing or regression occurred with seizures in 56 in the context of a developmental and epileptic encephalopathy (DEE). Fifty-five of 57 patients had intellectual disability, which was moderate to severe in 50. Other common features included behavioral problems (73%); high pain threshold (72%); eating problems, including oral aversion (68%); hypotonia (67%); sleeping problems (62%); autism spectrum disorder (54%); and ataxia or gait abnormalities (51%). Conclusions SYNGAP1 mutations cause a generalized DEE with a distinctive syndrome combining epilepsy with eyelid myoclonia with absences and myoclonic-atonic seizures, as well as a predilection to seizures triggered by eating.</p

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Supplementary Table 3: Anti-epileptic drug use in patients with SYNGAP1 mutations or deletion

A de novo loss-of-function GRIN2A mutation associated with childhood focal epilepsy and acquired epileptic aphasia.

OBJECTIVE:N-methyl-D-aspartate receptors (NMDAR) subunit GRIN2A/GluN2A mutations have been identified in patients with various neurological diseases, such as epilepsy and intellectual disability / developmental delay (ID/DD). In this study, we investigated the phenotype and underlying molecular mechanism of a GRIN2A missense mutation identified by next generation sequencing on idiopathic focal epilepsy using in vitro electrophysiology. METHODS:Genomic DNA of patients with epilepsy and ID/DD were sequenced by targeted next-generation sequencing within 300 genes related to epilepsy and ID/DD. The effects of one missense GRIN2A mutation on NMDAR function were evaluated by two-electrode voltage clamp current recordings and whole cell voltage clamp current recordings. RESULTS:We identified one de novo missense GRIN2A mutation (Asp731Asn, GluN2A(D731N)) in a child with unexplained epilepsy and DD. The D731N mutation is located in a portion of the agonist-binding domain (ABD) in the GluN2A subunit, which is the binding pocket for agonist glutamate. This residue in the ABD is conserved among vertebrate species and all other NMDAR subunits, suggesting an important role in receptor function. The proband shows developmental delay as well as EEG-confirmed seizure activity. Functional analyses reveal that the GluN2A(D731N) mutation decreases glutamate potency by over 3,000-fold, reduces amplitude of current response, shortens synaptic-like response time course, and decreases channel open probability, while enhancing sensitivity to negative allosteric modulators, including extracellular proton and zinc inhibition. The combined effects reduce NMDAR function. SIGNIFICANCE:We identified a de novo missense mutation in the GRIN2A gene in a patient with childhood focal epilepsy and acquired epileptic aphasia. The mutant decreases NMDAR activation suggesting NMDAR hypofunction may contribute to the epilepsy pathogenesis

Summary of pharmacological data for GluN2A(D731N).

<p>Summary of pharmacological data for GluN2A(D731N).</p

Summary of channel open probability for GluN2A(D731N).

<p>Summary of channel open probability for GluN2A(D731N).</p

GluN2A(D731N) enhances sensitivity to endogenous proton and zinc ions.

<p>(<b>A</b>) The composite proton concentration-response curves show an enhanced sensitivity of the GluN2A(D731N)-containing receptors to proton compared to the WT NMDA receptors; the abscissa shows hydrogen ion activity. (<b>B</b>) Summary of proton sensitivity of WT GluN2A and mutant receptors, evaluated by current ratio at pH 6.8 to pH 7.6 (in the presence of 30 mM glutamate and 100 μM glycine). Di-heteromeric (h2A-D731N), one-copy and two-copy mutant tri-heteromeric (D731N/2A and D731N/D731N) receptors show a decreased current ratio, indicating enhanced proton sensitivity. (<b>C</b>) The composite zinc concentration-response curves show an enhanced sensitivity of the GluN2A(D731N)-containing receptors to zinc compared to the WT NMDA receptors. (<b>D</b>) Composite Mg²⁺ concentration-response curves for di-heteromeric receptors indicate a similar Mg²⁺ inhibition of GluN2A(D731N)-containing receptors. The data were generated by TEVC recordings on <i>Xenopus</i> oocyte at holding potential of -40 mV for proton concentration-response curves, and -20 mV for zinc, and -60 mV for Mg²⁺ concentration-response curves. Fitted parameters are in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170818#pone.0170818.t002" target="_blank">Table 2</a>.</p

GluN2A(D731N) reduces the agonist potency.

<p>(<b>A,B</b>) Representative TEVC recordings obtained from oocytes expressing WT GluN1/GluN2A (WT 2A) receptors (<b>A</b>) and GluN1/GluN2A(D731N) (2A-D731N) receptors (<b>B</b>) in which the currents were evoked by increasing concentrations (μM) of glutamate (in the presence of 100 μM glycine) at the holding potential of -40 mV. (<b>C,D</b>) Composite concentration-response curves of glutamate and glycine for di-heteromeric receptors GluN1/GluN2A (WT 2A) and GluN1/GluN2A-D731N (2A-D731N). (<b>E,F</b>) Composite concentration-response curves of glutamate and glycine for tri-heteromeric receptors GluN1/GluN2A/GluN2A (2A/2A), GluN1/GluN2A(D731N)/GluN2A (D731N/2A) and GluN1/GluN2A(D731N)/GluN2A(D731N) (D731N/D731N). (<b>C,E</b>) The composite glutamate (in the presence of 100 μM glycine) concentration-response curves reveal a significant decrease in glutamate potency in both di-heteromeric (<b>C</b>) and tri-heteromeric (<b>E</b>) GluN2A(D731N)-containing NMDARs compared to wild type receptors. A single copy D731N-containing receptor (D731N/2A) (<b>E</b>) showed an intermediate but a dominantly negative effect on glutamate potency. The traces for D731N-contianing receptors (dash lines in panels <b>C</b> and <b>E</b>) were fitted by predicted glutamate concentrations of maximal responses. (<b>D,F</b>) The composite glycine (in the presence of 3–30 mM glutamate) concentration-response curves indicate a mild, but significantly reduced glycine potency in both di-heteromeric (<b>D</b>) and tri-heteromeric (<b>F</b>) GluN2A(D731N) receptors. Error bars are SEM, and are shown when larger than symbol size.</p

GluN2A(D731N) decreases current amplitudes and shortens synaptic-like response time course.

<p>The representative current response time course was generated by the whole cell voltage clamp recording (V_HOLD -60 mV) of GluN1/GluN2A (WT 2A, in BLACK) and GluN1/GluN2A-D731N (2A-D731N, in RED) receptor responses to rapid application of long (1.5 sec) (<b>A,B</b>) and brief (5 ms) (<b>C,D</b>) application of 30 mM glutamate. Panels <b>B</b> and <b>D</b> showed normalized responses to the WT response at the moment glutamate were removed. The mutant D731N-containing receptors showed an accelerated deactivation time course (<i>right panel</i> in <b>B,D</b>). Saturating glycine (100 μM) was present in all of solutions. Fitted parameters describing the response time course are given in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170818#pone.0170818.t003" target="_blank">Table 3</a>.</b></p

Summary of patients' information.

<p>Summary of patients' information.</p