"V体育安卓版" GRIN2B encephalopathy: novel findings on phenotype, variant clustering, functional consequences and treatment aspects

"VSports最新版本" Patients

We reviewed the clinical and genetic data of patients in whom GRIN2B de novo variants were detected within diagnostic or research settings focusing on neurodevelopmental disorders. Referring physicians provided detailed clinical information via a standardised clinical questionnaire VSports app下载. Molecular diagnostics were performed by targeted panel sequencing,14 whole exome sequencing (WES) or array comparative genomic hybridisation. All legal guardians provided informed written consent for genetic testing in accordance with the respective national ethics guidelines and with approval of the local ethics committees in the participating study centres.

Variant classification

Variants in GRIN2B (NM_000834. 3) were determined to be pathogenic, likely pathogenic or of unknown significance according to established guidelines of the American College of Medical Genetics (ACMG) (tables 1 and 2). 15 All variants classified as (likely) pathogenic constitute de novo variants. The database of the Exome Aggregation Consortium (ExAC) served as the control population V体育官网. 16.

GRIN2B physiology

We used cDNA for wild-type (WT) human NMDA GluN1-1a (hereafter GluN1; NP_015566) and GluN2B subunits in pCI-neo (NP_000827. 1). 17 The mutant GluN2B constructs were generated by site-directed mutagenesis using the Quik-Change protocol (Stratagene). Synthesis and injection of cRNA into Xenopus laevis oocytes (Ecocyte Inc. ) and two-electrode voltage-clamp current (TEVC) recordings were performed at −40 mV and 23°C (unless otherwise stated), as previously described. 18 Recording electrodes were filled with 0. 3 M KCl for voltage electrode and 3 M KCl for the current electrode. The recording solution contained (in mM) 90 NaCl, 1 KCl, 10 HEPES, 0 VSports注册入口. 5 BaCl2 and 0. 01 EDTA (pH 7. 4). The concentration-response curves were fitted with the following equations: .

where nH is the Hill slope, EC₅₀ is the concentration of the agonist that produces a half-maximal effect, IC₅₀ is the concentration of the antagonist that produces a half-maximal effect, and minimum is the degree of residual inhibition at a saturating concentration of the antagonist. The effects of co-application of increasing concentrations of memantine and maximally effective concentrations of glutamate (100 μM) and glycine (30 μM) on the response of NMDAR were determined using TEVC recordings from oocytes co-expressing GluN1 with the WT or the mutant GluN2B. The concentration-effect curves were recorded at a holding potential of −40 mV and fitted with equation 2.

Patient treatment

Patients were offered memantine treatment after functional confirmation of a gain-of-function variant retaining memantine sensitivity in vitro. In four patients, oral memantine treatment was initiated aiming for doses of 0.5–0.6 mg/kg body weight per day referring to the dosage of a treatment trial in GRIN2A encephalopathy.¹⁹ Patients were neurologically assessed prior and during memantine treatment. Individual treatment trials were approved by the local ethics boards of the participating centres.

V体育平台登录 - Phenotypic spectrum

All patients carrying a (likely) pathogenic GRIN2B variant presented with DD, ID and/or ASD (86/86; 100%, table 1, table 2, detailed phenotypic data in online supplementary table 3). The level of ID could be specified in 52 of 58 patients, with a majority displaying severe ID (31/52; 60%), whereas smaller proportions had moderate (13/52; 25%) or mild ID (8/52; 15%). Autistic features were seen in 28% of patients (16/58). At the time of data ascertainment, about half of the patients have had seizures (30/58; 52%), with a variable age of onset ranging from birth to 9 years and the frequency of seizures varying from multiple per day to a few seizures per year. Patients presented with generalised seizures (18/30; 60%, mostly tonic or tonic-clonic), focal seizures (14/30; 47%) and/or epileptic spasms (11/30; 37%). EEG patterns comprised hypsarrhythmia, focal, multifocal and/or generalised epileptiform activity. Follow-up data concerning seizure outcome were available for 22 patients, with one half becoming seizure-free (11/22; 50%) and the other half remaining refractory to therapy. No obvious treatment strategy using conventional antiepileptic drugs (AED) was associated with a higher likelihood of seizure freedom. At least 31 patients displayed hypotonia (31/58; 53%), which required tube feeding in five patients (5/31; 16%). Spasticity was documented in 14 patients (14/58; 24%). In addition, six patients presented with dystonic, dyskinetic or choreiform movement disorders (6/58; 10%). Four patients (4/58; 7%) showed signs of developmental regression, two of whom were temporary and one case had recurrent regression. Microcephaly was seen in multiple patients (11/58; 19%) and cortical visual impairment (CVI) was reported in four (4/58; 7%), three of whom also had a malformation of cortical development (MCD).

Neuroimaging data

Neuroimaging was performed in 44 of 58 patients: six patients (6/44, 14%) showed a consistent MCD intermediate between typical polymicrogyria (PMG) and the cortical appearance of tubulinopathies, consisting of mixed large and small gyri separated by shallow sulci, a smooth grey-white border and little infolding (figure 1). These patients also had hypoplastic corpus callosum of varying degrees, enlarged and mildly dysplastic basal ganglia, hippocampal dysplasia with thick leaves and open hilus as well as enlarged tecta (figure 1A, E and M). One patient had no septum pellucidum (figure 1C). Generalised cerebral volume loss, compatible with cerebral atrophy, was described in four additional patients (4/44; 9%).

VSports - Genetic spectrum

The 86 (likely) pathogenic variants in GRIN2B comprise 52 distinct missense variants. Variants cluster within or in very close proximity to the ligand-binding domains S1 and S2, as well as transmembrane domains M1–M4 (figure 2A, table 1). The only variant escaping this clustering pattern is p.(Ile150Val) in the amino-terminal domain (ATD). Although most variants were unique, five variants occurred multiple times: p.(Arg540His), p.(Gly689Ser), p.(Arg696His), p.(Ile751Thr) and p.(Gly820Ala). The 21 distinct pathogenic variants presumably leading to truncation/haploinsufficiency comprised nonsense and frameshift variants (n=11), splice site variants (n=3), chromosomal rearrangements (n=3) as well as gross deletions encompassing only GRIN2B (n=4) (figure 2B, table 2). According to the ACMG criteria, five de novo variants were classified as variants of unknown significance (see online supplement 2).¹⁵

V体育安卓版 - Frequency of GRIN2B encephalopathy

Several of our collaborators (RSM, DS, CS and SB) performed different diagnostic panel sequencing approaches in 3136 independent epilepsy patients revealing seven (likely) pathogenic GRIN2B variants and a diagnostic frequency of 0.22%. Three diagnostic WES trio cohorts reported 8051 patients with neurodevelopmental disorders (defined as human phenotype ontology terms (1) abnormality of the nervous system, (2) multiple congenital anomalies, (3) seizures or (4) ASD.^20–22 Overall, 15 pathogenic/likely pathogenic GRIN2B variants (14 missense, 1 frameshift) were identified, equalling a similar frequency of 0.19%. Expanding the data of the Deciphering Developmental Disorders Study,²³ 14 de novo missense variants in GRIN2B are significantly enriched (p value 2×10⁻¹⁷) in a combined cohort of WES trio data (n=8051) of patients with neurodevelopmental disorders (see online supplement 3). Among 209 independent individuals with PMG, we identified two patients with de novo GRIN2B variants.

"VSports最新版本" Functional investigation

Seven of 17 variants evaluated showed small apparent currents (less than 15 nA for 1000 μM glutamate and 100 μM glycine) (see online supplementary table 1). Because it was unclear whether these small currents were agonist evoked, the properties of these variants could not be studied further. We assessed the pharmacological properties of NMDAR containing the remaining 10 GluN2B mutants. Three variants (p.(Ser541Arg), p.(Val558Ile), and p.(Ile655Phe)) increased glutamate EC₅₀ values (ie, decreased glutamate potency) by 6.7-fold, 2.8-fold and 3.7-fold (table 3), indicating that higher concentrations of glutamate are needed to activate these receptors. The variant p.(Ser541Arg) increased glycine EC₅₀ values by 2.9-fold compared with WT receptors. Three variants (p.(Ser810Arg), p.(Met818Thr), and p.(Al-a819Thr)) decreased both the glutamate and glycine EC₅₀ values (ie, increased glutamate and glycine potency) (table 3), allowing these mutant receptors to be activated by lower concentration of agonists, suggesting a potential gain-of-function, provided trafficking and other features of receptor function are unchanged.

Voltage-dependent inhibition by extracellular Mg²⁺ and negative modulation by extracellular protons are two important features of NMDAR function. The voltage-dependent potency of Mg²⁺ inhibition showed that p.(Gly611Val) and p.(Ile655Phe) increased the Mg²⁺ IC₅₀ value from 25 μM for WT NMDAR to over 1000 μM and 220 μM for NMDAR containing GluN2B-Gly611Val and GluN2B-Ile655Phe, respectively (−60 mV holding potential; p<0.05, one-way analysis of variance) (table 3). Proton sensitivity was evaluated by comparing the current amplitude at two different extracellular pH values (6.8 vs. 7.6). Four mutant GluN2B subunits (p.(Ile655Phe), p.(Ser810Arg), p.(Met818Thr) and p.(Ala819Thr)) show significantly larger current response at pH 6.8 compared with pH 7.6 than WT receptors (table 3), suggesting that these variants significantly reduce tonic proton inhibition. This could result in more current flowing through the channel when the receptor is bound by agonists. Taken together, these data suggest that three variants (p.(Ser810Arg), p.(Met818Thr) and p.(Ala819Thr)) are likely overactive under resting conditions as a result of both the increased activation at low concentrations of agonists, reduced voltage-dependent Mg²⁺ block and reduced proton inhibition. The combination of these effects could potentially contribute to hyperexcitability and thus to patients’ epileptic phenotype. Further data are needed to determine how receptors with mixed functional changes (decreased agonist potency, decreased regulation by Mg²⁺ or protons) will alter, for example, charge transfer during synaptic transmission.

Evaluation of NMDAR antagonist memantine

The Food and Drug Administration-approved NMDAR antagonist memantine²⁴ has been suggested to have anticonvulsant effects in some epilepsy animal models²⁵ and was previously used off-label to effectively treat one patient with a gain-of-function GluN2A variant (p.(Leu812Met)).¹⁹ Thus, we evaluated its ability to inhibit six potential gain-of-function GluN2B variants (p.(Gly611Val), p.(Asn615Ile), p.(Val618Gly), p.(Ser810Arg), p.(Met818Thr) and p.(Ala819Thr))⁹ (see online supplementary figure 1 and supplementary table 2). The data indicated GluN2B-Gly611Val and GluN2B-Asn615Ile decreased memantine IC₅₀ by 1.7-fold and 3-fold, respectively (ie, increased potency; 1.0 μM for p.(Gly611Val) and 0.44 μM for p.(Asn615Il)e compared with 1.7 μM of WT). The other four variants (p.(Val618Gly), p.(Ser810Arg), p.(Met818Thr) and p.(Ala819Thr)) increased the IC₅₀ values (ie, decreased potency) compared with WT (see online supplementary figure 1 and supplementary table 2), suggesting that memantine can reduce NMDAR hyperactivity caused by these variants, although several mutants showed a reduced potency compared with WT receptors.

Targeted treatment with memantine

In four out of these six patients, we added memantine to the AED regimen (p.(Gly611Val), p.(Asn615Ile), p.(Val618Gly) and p.(Met818Thr)) (see online supplement 1). Parents and physicians initially observed beneficial effects such as improvements in awareness, behaviour and sleep. However, there were no changes in seizure frequencies and none of the potential benefits could be sufficiently objectivised. Long-term follow-up data were available in only one patient (p.(Val618Gly)) and did not suggest significant improvement.

Phenotypic spectrum

In agreement with the known GRIN2B-associated features, our data confirm that all patients had DD and a majority developed severe ID, with autistic features in a quarter of patients. Half of the patients developed seizures with a broad heterogeneity with respect to age of onset, seizure semiology, EEG features and outcome. Expanding our previous observation, 37% of patients presented with epileptic spasms.⁹ Less frequent findings of GRIN2B encephalopathy include generalised cerebral volume loss, CVI, hyperkinetic movement disorders (dystonia, dyskinesia, chorea) and developmental regression. These features have been repeatedly observed in other GRIN-associated encephalopathies,^{4,5,10,12,19,26,27} suggesting a shared phenotypic spectrum, with differences likely reflecting variant class, location and effect in the various NMDAR subunits. However, we did not observe EEG patterns of continuous spikes and waves during sleep or centrotemporal spikes in our GRIN2B cohort, which appears to reflect a milder end of the GRIN spectrum that is so far predominantly associated with GRIN2A.²⁸ As systematic genetic testing is more likely performed in patients with severe early-onset disorders, the mentioned frequencies of, for example, severe ID and epilepsy in GRIN2B encephalopathy are probably overestimated due to ascertainment bias.

V体育官网 - Malformation of cortical development

Six patients presented with a consistent MCD intermediate between typical PMG and tubulinopathies. This has also been referred to as tubulinopathy-related dysgyria.²⁹ Whereas tubulinopathies are classified as disorders of neuronal migration leading to cortical dysgenesis,³⁰ PMG is considered to be due to postmigrational disruption of cortical development with fusion of cortical laminae.³¹ Knockdown of Grin2b in rat has been shown to disturb proper neuronal migration³² and glutamate has been implemented in its regulation.³³ As GluN2B is predominantly expressed prenatally, disruption of neuronal migration seems to be the possible cause of GRIN2B-associated MCD. All six individuals had a very similar degree of severity and we did not observe less pronounced patterns of dysgyria in others. We can largely exclude the co-occurrence of, for example, known genetic tubulinopathies in all six cases, as individuals were screened by (trio)WES (n=4) or a panel targeting MCD genes (n=2). Other than in GRIN2B, we did not identify any other putatively causative variants in all six cases. Deficient ion channels are known to cause a plethora of human neurodevelopmental disorders, with the exception of abnormal cell migration. Despite the observation of six patients with pathogenic SCN1A variants who also had focal cortical dysplasia or bilateral periventricular nodular heterotopia,³⁴ the identification of six unrelated individuals with (likely) pathogenic variants in GRIN2B and a consistent MCD expands the phenotypic spectrum and reveals novel phenotypic consequences of channelopathies.

"V体育平台登录" Genetic spectrum

GRIN2B is a gene with a significantly reduced number of missense variants in controls, indicating a selective constraint.¹⁶ All (likely) pathogenic missense variants cluster within or in direct proximity to ligand-binding sites and transmembrane domains. These regions are largely spared by (likely) benign single nucleotide variants (SNV) in ExAC controls, suggesting that missense variants within these functionally important and conserved domains are only scarcely tolerated.^35,36 In contrast, the ATD and carboxy-terminal domain (CTD), as well as two small segments within S1 and M1, are markedly enriched for (likely) benign SNV in controls. The likely pathogenic missense variant p.(I-le150Val) escapes this clustering pattern, as it is located within a stretch of ~300 bp that contains very few missense variants within the ATD. It remains unclear whether or not this variant alters subunit assembly, ligand-binding or allosteric regulation by extracellular Zn^2+.1,2

The ratio of expected versus observed numbers of truncating variants of 33.9:0 in controls and the probability of loss-of-function intolerant score of 1.00 suggest that GRIN2B haploinsufficiency is most likely not tolerated.¹⁶ We observed seven truncating variants in the CTD establishing a premature termination codon in the last exon and therefore likely escaping nonsense-mediated mRNA decay and possibly not significantly altering protein function^15,37: two de novo truncating variants in patients with ID,²⁰ one de novo frameshift variant in a healthy control individual³⁸ and four frameshift variants reported in ExAC. The ExAC-annotated frameshift variant c.99dupC, p.(Ser34Glnfs*25) occurring in 27 controls conflicts with the hypothesis of haploinsufficiency. However, this variant is part of a homopolymer stretch of seven recurrent C nucleotides, raising the suspicion of a technical artefact despite having been described as pathogenic de novo variant via different sequencing approaches in an individual with ASD.³⁹

Genotype-phenotype correlations

MCD-associated variants are located in the transmembrane domain M3 (p.(Ala636Val), p.(Ala639Val) and p.(Ile655Phe)), in the ligand-binding domain S2 (p.(Arg693Ser)) or within the linker domain connecting S2 and M4 (p.(Ser810Arg) and p.(Ser810Asn)). Both missense variants at position 810 (p.(Ser810Arg) and p. (Ser810Asn)) were associated with MCD, whereas position 636 also showed two different (likely) pathogenic missense variants, with only p.(Ala636Val) being seen in MCD.⁴⁰ Thus, the specific functional mechanisms mediating MCD still remain to be elucidated. In contrast to our previous hypothesis,⁹ we did not find a significant correlation between variant class (missense vs truncation) and occurrence of seizures (Fisher’s exact test, p=0.1187). However, there was a significant correlation between variant class and intellectual outcome (mild-moderate vs severe ID) (Fisher’s exact test, p=0.0079), with truncation carriers tending to present with mild or moderate instead of severe ID.

Functional investigation

We evaluated several different aspects of receptor function, as it is possible that variants could have opposing effects on receptor activity (eg, Ile655Phe). We found that several other variants (Ser810Arg, Met818Thr, Ala819Thr) showed multiple differences in their properties that each enhanced NMDAR function, suggesting a potential gain-of-function of NMDAR assuming there is no change in the fraction of receptors that reach the cell surface or synapse. Such functional changes could lead to excito-toxic cell death, circuit rewiring, changes in neuronal migration or persistent increases in excitatory synaptic and non-synaptic drive through surface receptors. Thus there are multiple ways these variant receptors could contribute to clinical symptoms. Extensive functional analyses of rare variants in the GluN2B agonist binding domain recently showed the multifaceted and sometimes conflicting consequences that these variants can have on NMDAR activity.³⁶

"VSports" Targeted treatment with memantine

Initial subjective improvements in awareness, behaviour and sleep could not be sufficiently objectivised, and seizure frequencies showed no significant changes. In three cases, the missense variant led to significant loss of Mg²⁺ block engaging a different mechanism compared with the multiple means by which channel function was enhanced in a patient with p.(Leu812Met) in GRIN2A, which responded favourably to memantine treatment.¹⁹ In comparison, the variant of the fourth individual (p.(Met818Thr)) displayed similarities with respect to, for example, the increased glycine and glutamate potency of the NMDAR; however this does not hold true for patients’ treatment response to memantine. At present, it remains entirely unclear whether memantine is effective at all in this patient population and what factors (eg, variant location, mechanism of gain-of-function, patient age, memantine dose, differences in blood and brain concentrations) might influence memantine treatment response. Although some variants retain sensitivity to channel blockers like memantine in vitro, translating these potential benefits into patient care still remains elusive. Options for personalised therapy in GRIN2B encephalopathy still require more systematic and thorough evidence best through double-blinded prospective trials with a homogeneous patient population in terms of variant class.

Summary

Given the frequency of about 0.2% among individuals with neurodevelopmental disorders and/or childhood-onset epilepsy, GRIN2B encephalopathy appears to be a recurrent and distinct diagnosis. Our observations of novel features expand the phenotypic spectrum and suggest novel consequences of channelopathies resulting in disturbed neuronal migration. We found phenotypic similarities and a marked clustering of missense variants in ligand-binding and transmembrane domains paralleling other GRIN-associated disorders. Despite the limited conclusions regarding treatment with memantine in four patients with potential gain-of-function variants, the diverse functional consequences of (likely) pathogenic variants possibly enable future personalised therapeutic approaches in patients with GRIN2B encephalopathy.