Background: GRIN-related disorders (GRD), the so-called grinpathies, is a group of rare encephalopathies caused by mutations affecting GRIN genes (mostly GRIN1, GRIN2A and GRIN2B genes), which encode for the GluN subunit of the N-methyl D-aspartate (NMDA) type ionotropic glutamate receptors. A growing number of functional studies indicate that GRIN-encoded GluN1 subunit disturbances can be dichotomically classified into gain- and loss-of-function, although intermediate complex scenarios are often present.

Methods: In this study, we aimed to delineate the structural and functional alterations of GRIN1 disease-associated variants, and their correlations with clinical symptoms in a Spanish cohort of 15 paediatric encephalopathy patients harbouring these variants.

Results: Patients harbouring GRIN1 disease-associated variants have been clinically deeply-phenotyped. Further, using computational and in vitro approaches, we identified different critical checkpoints affecting GluN1 biogenesis (protein stability, subunit assembly and surface trafficking) and/or NMDAR biophysical properties, and their association with GRD clinical symptoms.

Conclusions: Our findings show a strong correlation between GRIN1 variants-associated structural and functional outcomes. This structural-functional stratification provides relevant insights of genotype-phenotype association, contributing to future precision medicine of GRIN1-related encephalopathies.

Keywords: GRIN-related disorders; NMDA receptors; glutamatergic neurotransmission; neurodevelopmental disorders.

Lower N-methyl-d-aspartate receptor (NMDAR) GluN1 subunit levels and heightened neuroinflammation are found in the cortex in schizophrenia. Since neuroinflammation can lead to changes in NMDAR function, it is possible that these observations are linked in schizophrenia. We aimed to extend our previous studies by measuring molecular indices of NMDARs that define key functional properties of this receptor - particularly the ratio of GluN2A and GluN2B subunits - in dorsolateral prefrontal cortex (DLPFC) from schizophrenia and control cases (37/37). We sought to test whether changes in these measures are specific to the subset of schizophrenia cases with high levels of inflammation-related mRNAs, defined as a high inflammatory subgroup. Quantitative autoradiography was used to detect 'functional' NMDARs ([³H]MK-801), GluN1-coupled-GluN2A subunits ([³H]CGP-39653), and GluN1-coupled-GluN2B subunits ([³H]Ifenprodil). Quantitative RT-PCR was used to measure NMDAR subunit transcripts (GRIN1, GRIN2A and GRIN2B). The ratios of GluN2A:GluN2B binding and GRIN2A:GRIN2B mRNAs were calculated as an index of putative NMDAR composition. We found: 1) GluN2A binding, and 2) the ratios of GluN2A:GluN2B binding and GRIN2A:GRIN2B mRNAs were lower in schizophrenia cases versus controls (p < 0.05), and 3) lower GluN2A:GluN2B binding and GRIN2A:GRIN2B mRNA ratios were exaggerated in the high inflammation/schizophrenia subgroup compared to the low inflammation/control subgroup (p < 0.05). No other NMDAR-related indices were significantly changed in the high inflammation/schizophrenia subgroup. This suggests that neuroinflammation may alter NMDAR stoichiometry rather than targeting total NMDAR levels overall, and future studies could aim to determine if anti-inflammatory treatment can alleviate this aspect of NMDAR-related pathology.

The heritable component of schizophrenia (SCH) as a polygenic trait is represented by numerous variants from a heterogeneous group of genes each contributing a relatively small effect. Various SNPs have already been found and analyzed in genes encoding the NMDAR subunits. However, less is known about genetic variations of genes encoding the AMPA and kainate receptor subunits. We analyzed sixteen iGluR genes in full length to determine the sequence variability of iGluR genes. Our aim was to describe the rate of genetic variability, its distribution, and the co-occurrence of variants and to identify new candidate risk variants or haplotypes. The cumulative effect of genetic risk was then estimated using a simple scoring model. GRIN2A-B, GRIN3A-B, and GRIK4 genes showed significantly increased genetic variation in SCH patients. The fixation index statistic revealed eight intronic haplotypes and an additional four intronic SNPs within the sequences of iGluR genes associated with SCH (p < 0.05). The haplotypes were used in the proposed simple scoring model and moreover as a test for genetic predisposition to schizophrenia. The positive likelihood ratio for the scoring model test reached 7.11. We also observed 41 protein-altering variants (38 missense variants, four frameshifts, and one nonsense variant) that were not significantly associated with SCH. Our data suggest that some intronic regulatory regions of iGluR genes and their common variability are among the components from which the genetic predisposition to SCH is composed.

Keywords: BrainAGE; SNP; genetic variations; haplotypes; intron; ionotropic glutamate receptor; schizophrenia; scoring model.

Schizophrenia is a disabling neuropsychiatric disorder of adulthood onset with high heritability. Worldwide collaborations have identified an association of ~270 common loci, with small individual effects and hence weak clinical implications. The recent technological feasibility of exome sequencing enables the identification of rare variants of high penetrance that refine previous findings and improve risk assessment and prognosis. We recruited two multiplex Pakistani families, having 11 patients and 19 unaffected individuals in three generations. We performed genome-wide SNP genotyping, next-generation mate pairing and whole-exome sequencing of selected members to unveil genetic components. Candidate variants were screened in unrelated cohorts of 508 cases, 300 controls and fifteen families (with 51 affected and 47 unaffected individuals) of Pakistani origin. The structural impact of substituted residues was assessed through in silico modeling using iTASSER. In one family, we identified a rare novel microduplication (5q14.1_q14.2) encompassing critical genes involved in glutamate signaling, such as CMYA5, HOMER and RasGRF2. The second family segregates two ultra-rare, predicted pathogenic variants in the GRIN2A (NM_001134407.3: c.3505C>T, (p.R1169W) and in the NRG3 NM_001010848.4: c.1951G>A, (p.E651K). These genes encode for parts of AMPA and NMDA receptors of glutamatergic neurotransmission, respectively, and the variants are predicted to compromise protein function by destabilizing their structures. The variants were absent in the aforementioned cohorts. Our findings suggest that rare, highly penetrant variants of genes involved in glutamatergic neurotransmission are contributing to the etiology of schizophrenia in these families. It also highlights that genetic investigations of multiplex, multigenerational families could be a powerful approach to identify rare genetic variants involved in complex disorders.

Keywords: duplication; familial schizophrenia; glutamatergic neurotransmission; rare variants.

Immune escape and low response to immunotherapy are crucial challenges in present lung cancer treatment. In this study, we constructed a new immune-related classifier based on CXCL13/CXCR5, an important tumor microenvironment component and strongly related with the formation of tertiary lymphoid structures (TLSs) in tumor microenvironment. With the classifier, we divided patients into two main clusters and each cluster was further divided into subcluster (A1, A2, B1, B2, B3). In the later analysis, we noticed that patients in subcluster B3 had a distinct advantage over patients in A1 in survival time and immune infiltration, suggesting a more favorable response to immunotherapy. Moreover, we demonstrated the genetic and epigenetic regulation related to the subclusters and recovered four key differentially expressed genes (ERBB4, GRIN2A, IL2RA, CCND2). With several experiments, we verified the unique role of CCND2 in tumor metastasis and T cell apoptosis. Overexpressing CCND2 could significantly impair cancer cell abilities of migration and invasion and downregulate PD-1/PD-L1 signaling, which may be the cause of T cell apoptosis reduction. In the end, we constructed a regression risk model that could successfully predict ICI response. To sum up, our study established new stratification models that can successfully predict patient survival and response to ICI. And using integrative analysis of multi-omics data, four key DEGs were noticed, and CCND2, one of the four genes, was identified as a potential treatment target because of its effect in tumor metastasis and T cell apoptosis.

Keywords: CCND2; CXCL13; Immunotherapy; Lung adenocarcinoma; Tertiary lymphoid structures; Tumor immune microenvironment.

Glutamate, the major excitatory neurotransmitter, plays a ubiquitous role in most aspects of normal brain functioning. Its indispensable position is paradoxically doubled by a high excitotoxic potential following disruption of its dynamic equilibrium. Several lines of evidence have suggested the involvement of the glutamatergic N-methyl-D-aspartate receptor (NMDAR) in learning, memory formation, and human cognition. Furthermore, NMDARs play a pivotal role in various neuropsychiatric disorders, recently being identified as an important locus for disease-associated genomic variation. The GRIN2A gene encodes the NMDAR's GluN2A subunit. Genetic alterations of GRIN2A result in phenotypic pleiotropy, predisposing to a broad range of epilepsy syndromes, with an elusive and unpredictable evolution and response to treatment. The archetypal GRIN2A-related phenotype comprises the idiopathic focal epilepsies (IFEs), with a higher incidence of GRIN2A mutants among entities at the more severe end of the spectrum. We report the case of a patient heterozygous for GRIN2A, c.1081C>T, presenting with febrile convulsions and later superimposed atonic seizures, expressive language delay, and macrocephaly. As the number of reported GRIN2A variants is continuously increasing, the phenotypic boundaries gradually grow faint. Therefore, it is fundamental to maintain an acute critical awareness of the possible genetic etiology of different epilepsy syndromes. So far, therapeutic strategies rely on empirical observations relating genotypes to specific drugs, but the overall success of treatment remains unpredictable. Deciphering the functional consequences of individual GRIN2A variants could lead to the development of precision therapeutic approaches for patients carrying NMDAR mutations.

Keywords: GRIN2A gene; N-methyl-D-aspartate receptor (NMDAR); child; genetic epilepsy.

Aim: We investigated GRIN1, GRIN2A, GRIN2B and LINE-1 DNA methylation in first-episode schizophrenia patients, their nonaffected siblings and age- and sex-matched controls testing for associations between DNA methylation and exposition to childhood trauma. Materials & methods: The Childhood Trauma Questionnaire evaluated the history of childhood trauma. Genomic DNA was bisulfite converted and pyrosequencing was employed to quantify DNA methylation. Results: GRIN2A, GRIN2B and LINE-1 DNA methylation was not associated with childhood trauma in patients, siblings and controls. Siblings with childhood trauma had hypermethylation at CpG1 of GRIN1 compared with siblings without trauma. Conclusion: Childhood trauma may influence GRIN1 methylation in subjects with liability to psychosis, but not in frank schizophrenia or controls.

Keywords: DNA methylation; GRIN1; GRIN2A; GRIN2B; NMDAR genes; childhood trauma; first-episode schizophrenia; siblings.

Different neuronal alterations within glutamatergic system seem to be crucial for developing of cocaine-seeking behavior. Cocaine exposure provokes a modulation of the NMDA receptor subunit expression in rodents, which probably contributes to cocaine-induced behavioral alterations. The aim of this study was to examine the composition of the NMDA receptor subunits in the brain structures in rats with the history of cocaine self-administration after cocaine abstinence (i) in an enriched environment, (ii) in an isolated condition, (iii) with extinction training, or (iv) without instrumental task, as well as the Grin1 (encoding GluN1) and Grin2A (encoding GluN2A) gene expression were evaluated after 10-day extinction training in rat brain structures. In the present study, we observed changes only following cocaine abstinence with extinction training, when the increased GluN2A subunit levels were seen in the postsynaptic density fraction but not in the whole homogenate of the prelimbic cortex (PLC) and dorsal hippocampus (dHIP) in rats previously self-administered cocaine. At the same time, extinction training did not change the Grin1 and Grin2A gene expression in these structures. In conclusion, NMDA receptor subunit modulation observed following cocaine abstinence with extinction training may represent a potential target in cocaine-seeking behavior.

Keywords: Cocaine abstinence; Cocaine self-administration; NMDA receptor subunit.

There is a hypothesis of the involvement of the glutamatergic system in the development of autism. It has been shown that the chronic experience in daily intermale confrontations leads to disturbances in social behavior: a decrease in communicativeness, disturbances of socialization, emergence of stereotypical behaviors that can be considered as symptoms of the autistic spectrum disorders. So, the aim of this study was to investigate changes in the expression of glutamatergic (GG) and autism-related (GA) genes in the hippocampus of animals with impaired social behavior caused by repeated experience of social defeat or aggression in daily agonistic confrontations. To form groups of animals with contrasting behaviors, a model of sensory contact (chronic social stress) was used. The collected brain samples were sequenced at JSC Genoanalytica (http://genoanalytica.ru/ , Moscow, Russia). Transcriptomic analysis revealed a down-regulation of autism-related (Shank3, Auts2, Ctnnd2, Nrxn2) and glutamatergic (Grm4) genes in aggressive mice. At the same time, the expression of GA-related genes (Shank2, Nlgn2, Ptcdh10, Reln, Arx) and GG genes (Grik3, Grm2, Grm4, Slc17a7, Slc1a4, Slc25a22) excluding Grin2a was increased in defeated mice. Correlative analysis revealed a statistically significant association between GG and GA expression. These results can serve as a confirmation of the participation of the glutamatergic system in the pathophysiology of the autistic spectrum disorder.

В настоящее время существует представление о вовлеченности глутаматергической системы (ГГ) в механизмы развития аутизма. В предыдущих исследованиях нами было показано, что негативный социальный опыт, приобретенный в ежедневных межсамцовых конфронтациях, приводит к нарушениям в социальном поведении: снижению коммуникативности, нарушению социализации, появлению стереотипных форм поведения, которые могут рассматриваться как симптомы аутистического спектра. В связи с этим целью нашей работы было изучение с помощью транскриптомного анализа изменений экспрессии генов, кодирующих белки, вовлеченные в функционирование глутаматергической системы, и генов, связанных с патологией аутизма (ГА), в гиппокампе. В эксперименте использовали животных с нарушениями социального поведения, вызванными повторным опытом социальных побед или поражений в ежедневных агонистических взаимодействиях. Для формирования групп животных с контрастными типами поведения использовали модель сенсорного контакта (хронического социального стресса). Полученные образцы мозга были секвенированы в ЗАО «Геноаналитика» (http://genoanalytica.ru/, Россия, Москва). Транскриптомный анализ показал, что у агрессивных животных снижается экспрессия генов Shank3, Auts2, Ctnnd2, Nrxn2, для которых показано участие в развитии аутизма, а также глутаматергического гена Grm4. В то же время у животных с негативным социальным опытом экспрессия ГА Shank2, Nlgn2, Ptcdh10, Reln, Arx возрастает. При этом ГГ (Grik3, Grm2, Grm4, Slc17a7, Slc1a4, Slc25a22), за исключением гена Grin2a, повышают свою экспрессию. Корреляционный анализ выявил статистически значимую взаимосвязь из- мененной экспрессии ГГ и ГА. Полученные результаты, с одной стороны, могут служить подтверждением участия ГГ в патофизиологии развития симптомов аутистического спектра, с другой – свидетельствовать о коэкспрессии ГГ и ГА в гиппокампе, развивающейся под влиянием социальной среды. Так как большинство ГА, изменивших экспрессию в настоящем исследовании, являются генами, связанными с клеточным скелетом и внеклеточным матриксом, в частности участвующими в формировании синапсов, а ГГ, изменившие свою экспрессию, – генами, кодирующими субъединицы рецепторов, то можно предположить, что вовлечение ГГ в патофизиологию аутизма происходит на уровне рецепторов.

Keywords: RNA-seq; autism; autism-related genes; glutamatergic genes; hippocampus; social experience.

The activation of the endocannabinoid system controls the release of many neurotransmitters involved in the brain reward pathways, including glutamate. Both endocannabinoid and glutamate systems are crucial for alcohol relapse. In the present study, we hypothesize that N-methyl-D-aspartate (NMDA) glutamate receptors regulate the ability of a priming dose of WIN 55,212-2 to cross-reinstate ethanol-induced conditioned place preference (CPP). To test this hypothesis, ethanol-induced (1.0 g/kg, 10% w/v, i.p.) CPP (unbiased method) was established using male adult Wistar rats. After CPP extinction, one group of animals received WIN 55,212-2 (1.0 and 2.0 mg/kg, i.p.), the cannabinoid receptor 1 (CB1) agonist, or ethanol, and the other group received memantine (3.0 or 10 mg/kg, i.p.), the NMDA antagonist and WIN 55,212-2 on the reinstatement day. Our results showed that a priming injection of WIN 55,212-2 (2.0 mg/kg, i.p.) reinstated (cross-reinstated) ethanol-induced CPP with similar efficacy to ethanol. Memantine (3.0 or 10 mg/kg, i.p.) pretreatment blocked this WIN 55,212-2 effect. Furthermore, our experiments indicated that ethanol withdrawal (7 days withdrawal after 10 days ethanol administration) down-regulated the CNR1 (encoding CB1), GRIN1/2A (encoding GluN1 and GluN2A subunit of the NMDA receptor) genes expression in the prefrontal cortex and dorsal striatum, but up-regulated these in the hippocampus, confirming the involvement of these receptors in ethanol rewarding effects. Thus, our results show that the endocannabinoid system is involved in the motivational properties of ethanol, and glutamate may control cannabinoid induced relapse into ethanol seeking behavior.

Keywords: CNR1; GRIN1; GRIN2A; cannabinoid; ethanol relapse; glutamate; rats.

Excessive prenatal opioid exposure may lead to the development of Neonatal Opioid Withdrawal Syndrome (NOWS). RNA-seq was done on 64 formalin-fixed paraffin-embedded placental tissue samples from 32 mothers with opioid use disorder, with newborns with NOWS that required treatment, and 32 prenatally unexposed controls. We identified 93 differentially expressed genes in the placentas of infants with NOWS compared to unexposed controls. There were 4 up- and 89 downregulated genes. Among these, 7 genes CYP1A1, APOB, RPH3A, NRXN1, LINC01206, AL157396.1, UNC80 achieved an FDR p-value of <0.01. The remaining 87 genes were significant with FDR p-value <0.05. The 4 upregulated, CYP1A1, FP671120.3, RAD1, RN7SL856P, and the 10 most significantly downregulated genes were RNA5SP364, GRIN2A, UNC5D, DMBT1P1, MIR3976HG, LINC02199, LINC02822, PANTR1, AC012178.1, CTNNA2. Ingenuity Pathway Analysis identified the 7 most likely to play an important role in the etiology of NOWS. Our study expands insights into the genetic mechanisms of NOWS development.

Keywords: Differentially expressed genes; In utero drug exposure; Neonatal abstinence syndrome; Transcription factors; Transcriptional regulatory element.

N-methyl-D-aspartate (NMDA) receptors mediate excitatory neurotransmission in the nervous system and are preferentially inhibited by general anesthetics such as sevoflurane. Spontaneous movement is a common complication during sevoflurane anesthesia induction and seriously affects operations. In this study, we investigated the relationship between NMDA polymorphisms and spontaneous movement during sevoflurane induction. This prospective clinical study enrolled 393 patients undergoing sevoflurane anesthesia as part of their surgical routine. In the GRIN1, GRIN2A, and GRIN2B genes, 13 polymorphisms that form a heteromeric complex as part of the NMDA receptor were selected using Haploview and genotyped using matrix-assisted laser desorption ionization-time of flight mass spectrometry MassARRAY. Both RNAfold and Genotype-Tissue Expression portals were used to identify gene expression profiles. Our data showed that 35.8% of subjects exhibited spontaneous movement. The GRIN2A rs12918566 polymorphism was associated with spontaneous movement during sevoflurane induction. A logistic regression analysis of additive, dominant, and recessive models indicated a significant association (odds ratio [OR] (95% confidence limit [CI]): 0.58 (0.42-0.80), p = .00086; OR (95% CI): 0.51 (0.31-0.84), p = .0075, and OR (95% CI): 0.47 (0.27-0.81), p = .0060, respectively). After false discovery rate (FDR) correction, the additive model was still significant with a P_FDR =0.010. Bioinformatics demonstrated that the rs12918566 genomic variation affected GRIN2A expression in brain tissue. We also revealed that GRIN2A rs12918566 was significantly associated with spontaneous movement during sevoflurane induction. We believe the NMDA receptor plays an important role in regulating the anesthetic effects of sevoflurane.

Keywords: N-methyl-D-aspartate; expression; polymorphism; sevoflurane; spontaneous movement.

Epilepsy is characterized by highly abnormal synchronous discharge of brain neurons, and ion channels are fundamental in the generation and modulation of neural excitability. Considering that abnormal methylation can either activate or repress genes, this study was designed to explore the DNA methylation signature of pathogenic genes encoding ion channels in temporal lobe epilepsy (TLE). In total, 38 TLE patients and 38 healthy controls were enrolled in the study, and genomic DNA and total protein of the lymphocytes were extracted from peripheral blood samples to assess methylation and protein levels. The DNA methylation levels of all 12 genes examined were significantly lower in the TLE group than in the control group. After false-positive correction, 83.3% (10/12) of these genes, namely, gamma-aminobutyric acid type A receptor subunit beta1 (GABRB1), gamma-aminobutyric acid type A receptor subunit beta2 (GABRB2), gamma-aminobutyric acid type A receptor subunit beta1 (GABRB3), glutamate ionotropic receptor NMDA type subunit 1 (GRIN1), glutamate ionotropic receptor NMDA type subunit 2A (GRIN2A), glutamate ionotropic receptor NMDA type subunit 2B (GRIN2B), hyperpolarization activated cyclic nucleotide gated potassium channel 1 (HCN1), potassium voltage-gated channel subfamily A member 2 (KCNA2), potassium voltage-gated channel subfamily B member 1 (KCNB1), and potassium sodium-activated channel subfamily T member 1 (KCNT1), were still differentially expressed. Among these ion channels, HCN1 and KCNA2 were selected to evaluate the effects of DNA methylation, and the levels of these proteins were inversely upregulated in the TLE group compared to the control group. As the genes identified as having differential methylation levels are involved in both excitatory and inhibitory ion channels, this study observed by binary logistic regression that hypermethylated GARAB1 was an independent risk factor for TLE, indicating that the overwhelming effect of ion channels on TLE is probably inhibitory from the perspective of DNA methylation. All these findings support the involvement of DNA methylation in TLE pathologies, but the mechanisms need to be further investigated.

Keywords: DNA methylation; epigenetic; genetic susceptibility; ion channels; temporal lobe epilepsy.

Purpose: The aim of this study was to increase knowledge of genes associated with anorexia nervosa (AN) and their diagnostic offer, using a next generation sequencing (NGS) panel for the identification of genetic variants. The rationale underlying this test is that we first analyze the genes associated with syndromic forms of AN, then genes that were found to carry rare variants in AN patients who had undergone segregation analysis, and finally candidate genes intervening in the same molecular pathways or identified by GWAS or in mouse models.

Methods: We developed an NGS gene panel and used it to screen 68 Italian AN patients (63 females, 5 males). The panel included 162 genes. Family segregation study was conducted on available relatives of probands who reported significant genetic variants.

Results: In our analysis, we found potentially deleterious variants in 2 genes (PDE11A and SLC25A13) associated with syndromic forms of anorexia and predicted deleterious variants in the following 12 genes: CD36, CACNA1C, DRD4, EPHX2, ESR1, GRIN2A, GRIN3B, LRP2, NPY4R, PTGS2, PTPN22 and SGPP2. Furthermore, by Sanger sequencing of the promoter region of NNAT, we confirmed the involvement of this gene in the pathogenesis of AN. Family segregation studies further strengthened the possible causative role of CACNA1C, DRD4, GRIN2A, PTGS2, SGPP2, SLC25A13 and NNAT genes in AN etiology.

Conclusion: The major finding of our study is the confirmation of the involvement of the NNAT gene in the pathogenesis of AN; furthermore, this study suggests that NGS-based testing can play an important role in the diagnostic evaluation of AN, excluding syndromic forms and increasing knowledge of the genetic etiology of AN.

Level of evidence: Level I, experimental study.

Keywords: Anorexia nervosa; Eating disorders; NGS analysis; Rare variants.

Mutations in N-methyl-d-aspartate receptors (NMDAR) subunits have been implicated in a growing number of human neurodevelopmental disorders. Previously, a de novo mutation in GRIN2A, encoding the GluN2A subunit, was identified in a patient with severe epilepsy and developmental delay. This missense mutation, which leads to GluN2A-P552R, produces significant dendrotoxicity in transfected rodent cortical neurons, as evidenced by pronounced dendritic blebbing. This injurious process can be prevented by treatment with the NMDA antagonist memantine. Given the increasing use of FDA approved NMDA antagonists to treat patients with GRIN mutations, who may have seizures refractory to traditional anti-epileptic drugs, we investigated whether additional NMDA antagonists were effective in attenuating neurotoxicity associated with GluN2A-P552R expression. Intriguingly, we found that while treatment with memantine can effectively block GluN2A-P552R-mediated dendrotoxicity, treatment with ketamine does not, despite the fact that both drugs work as open NMDAR channel blockers. Interestingly, we found that neurons expressing GluN2A-P552R were more vulnerable to an excitotoxic insult-an effect that, in this case, could be equally rescued by both memantine and ketamine. These findings suggest that GluN2A-P552R induced dendrotoxicity and increased vulnerability to excitotoxic stress are mediated through two distinct mechanisms. The differences between memantine and ketamine in halting GluN2A-P552R dendrotoxicity could not be explained by NMDA antagonist induced changes in MAP or Src kinase activation, previously shown to participate in NMDA-induced excitotoxicity. Our findings strongly suggest that not all NMDA antagonists may be of equal clinical utility in treating GRIN2A-mediated neurological disorders, despite a shared mechanism of action.

Keywords: GRIN2A gene; GluN2A subunit; N-methyl-d-aspartate receptor; channelopathy; ketamine; memantine; precision medicine.

Patients with monoallelic bromodomain and PHD finger-containing protein 1 (BRPF1) mutations showed intellectual disability. The hippocampus has essential roles in learning and memory. Our previous work indicated that Brpf1 was specifically and strongly expressed in the hippocampus from the perinatal period to adulthood. We hypothesized that mouse Brpf1 plays critical roles in the morphology and function of hippocampal neurons, and its deficiency leads to learning and memory deficits. To test this, we performed immunofluorescence, whole-cell patch clamp, and mRNA-Seq on shBrpf1-infected primary cultured hippocampal neurons to study the effect of Brpf1 knockdown on neuronal morphology, electrophysiological characteristics, and gene regulation. In addition, we performed stereotactic injection into adult mouse hippocampus to knock down Brpf1 in vivo and examined the learning and memory ability by Morris water maze. We found that mild knockdown of Brpf1 reduced mEPSC frequency of cultured hippocampal neurons, before any significant changes of dendritic morphology showed. We also found that Brpf1 mild knockdown in the hippocampus showed a decreasing trend on the spatial learning and memory ability of mice. Finally, mRNA-Seq analyses showed that genes related to learning, memory, and synaptic transmission (such as C1ql1, Gpr17, Htr1d, Glra1, Cxcl10, and Grin2a) were dysregulated upon Brpf1 knockdown. Our results showed that Brpf1 mild knockdown attenuated hippocampal excitatory synaptic transmission and reduced spatial learning and memory ability, which helps explain the symptoms of patients with BRPF1 mutations.

Keywords: BRPF1; Morris water maze; dendritic morphology; electrophysiology; intellectual disability; mRNA-seq; primary cultured hippocampal neurons; stereotactic hippocampal injection.

Background: Alzheimer's disease (AD) is the most prevalent cause of dementia. Large RNA-Sequencing data sets have provided detailed molecular profiles of AD, but they cannot differentiate causal vs. non-causal gene expression changes.

Method: We combined bioinformatic prioritization of human data with genetic screens in Drosophila melanogaster to identify human brain transcriptional changes that exacerbate or ameliorate neurodegeneration induced by Tau or amyloid-β (Aβ), the proteins which form the hallmark pathologic aggregates that define AD. First, we used large, AD-associated coexpression networks derived from Accelerating Medicines Partnership (AMP)-AD human brain RNA-Seq data to nominate candidate AD drivers based on multiple bioinformatic criteria. We then tested nominated drivers for modification of Tau- or Aβ-induced neurodegeneration in vivo using a locomotor assay in Drosophila. For one module of our interest, PHGbrown, we analyzed its overlap with published differentially expressed gene (DEG) sets from mouse models to explore its potential functional roles in AD. Finally, we used the L1000 Connectivity Map database of perturbation-associated gene expression changes to select small molecules which either reverse or promote the implicated AD-associated gene expression signatures that are causally linked with disease progression.

Result: Through our screen, we identified a module, PHGbrown, with 14 of its 27 prioritized genes (51.9%) significantly modifying Tau/Aβ neurotoxicity in our screen. We integrated our screen data with PHGbrown's module structure to identify a subnetwork containing genes involved in synaptic function including GRIN2A, GRIN2B, KCNJ6, GABRB2, and GABRB3. This synaptic subnetwork is downregulated in human AD and contains multiple loss-of-function suppressors of Tau/Aβ neurotoxicity. Interestingly, PHGbrown shows overlap and concordant down-regulation with published mouse models of excitotoxicity. Our results suggest that PHGbrown down-regulation may compensate for excitotoxic injury in AD. Based on in silico analysis of L1000 Connectivity Map data, the protein kinase C inhibitor, Chelerythrine, was identified as a promising "network therapy" capable of promoting PHGbrown down-regulation and resilience to AD pathophysiology.

Conclusion: By integrating human coexpression networks and Drosophila genetic screen data, our results establish a causal chain connecting Tau/Aβ with neurodegeneration through network-level gene expression changes. We additionally identify candidate drugs targeting causal subnetworks for development of network modulating therapies.

Monoamine neurotransmitter abundance affects motor control, emotion, and cognitive function and is regulated by monoamine oxidases. Among these, Monoamine oxidase A (MAOA) catalyzes the degradation of dopamine, norepinephrine, and serotonin into their inactive metabolites. Loss-of-function mutations in the X-linked MAOA gene have been associated with Brunner syndrome, which is characterized by various forms of impulsivity, maladaptive externalizing behavior, and mild intellectual disability. Impaired MAOA activity in individuals with Brunner syndrome results in bioamine aberration, but it is currently unknown how this affects neuronal function, specifically in dopaminergic (DA) neurons. Here we generated human induced pluripotent stem cell (hiPSC)-derived DA neurons from three individuals with Brunner syndrome carrying different mutations and characterized neuronal properties at the single cell and neuronal network level in vitro. DA neurons of Brunner syndrome patients showed reduced synaptic density but exhibited hyperactive network activity. Intrinsic functional properties and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)-mediated synaptic transmission were not affected in DA neurons of individuals with Brunner syndrome. Instead, we show that the neuronal network hyperactivity is mediated by upregulation of the GRIN2A and GRIN2B subunits of the N-methyl-d-aspartate receptor (NMDAR), resulting in increased NMDAR-mediated currents. By correcting a MAOA missense mutation with CRISPR/Cas9 genome editing we normalized GRIN2A and GRIN2B expression, NMDAR function and neuronal population activity to control levels. Our data suggest that MAOA mutations in Brunner syndrome increase the activity of dopaminergic neurons through upregulation of NMDAR function, which may contribute to the etiology of Brunner syndrome associated phenotypes.

Keywords: Brunner syndrome; Dopaminergic neuron; Human iPSC; MAOA; Microelectrode array; NMDA receptor.

Pathogenic missense variants in GRIN2A and GRIN2B may result in gain or loss of function (GoF/LoF) of the N-methyl-D-aspartate receptor (NMDAR). This observation gave rise to the hypothesis of successfully treating GRIN-related disorders due to LoF variants with co-agonists of the NMDAR. In this respect, we describe a retrospectively collected series of ten individuals with GRIN2A- or GRIN2B-related disorders who were treated with L-serine, each within an independent n-of-1 trial. Our cohort comprises one individual with a LoF missense variant with clinical improvements confirming the above hypothesis and replicating a previous n-of-1 trial. A second individual with a GoF missense variant was erroneously treated with L-serine and experienced immediate temporary behavioral deterioration further supporting the supposed functional pathomechanism. Eight additional individuals with null variants (that had been interpreted as loss-of-function variants despite not being missense) again showed clinical improvements. Among all nine individuals with LoF missense or null variants, L-serine treatment was associated with improvements in behavior in eight (89%), in development in four (44%), and/or in EEG or seizure frequency in four (44%). None of these nine individuals experienced side effects or adverse findings in the context of L-serine treatment. In summary, we describe the first evidence that L-serine treatment may not only be associated with clinical improvements in GRIN-related disorders due to LoF missense but particularly also null variants.

Keywords: Developmental and epileptic encephalopathy; Epilepsy; Precision medicine; Retrospective observational case series; Targeted treatment.

This study analysed genomic mutations in basal cell carcinoma using whole exome sequencing of DNA specimens obtained from 20 Korean patients. Histological evaluation determined that 15 (75%) were low-risk basal cell carcinomas, and 5 (25%) were high-risk basal cell carcinomas. Seventy-five percent of the basal cell carcinomas harboured somatic mutations in hedge-hog pathway genes (PTCH1, 40% and SMO, 50%) and 45% harboured mutations in TP53. LRP1B was the most frequently mutated gene in high-risk basal cell carcinomas, SMO was the most frequently mutated gene in low-risk basal cell carcinomas. Specifically, LRP1B, ROS1, PTCH1, KMT2C, NSD1 and ARID1A mutations were more frequent in high-risk basal cell carcinomas than in low-risk basal cell carcinomas. However, copy number gains of the ROS1 gene were observed only in low-risk basal cell carcinomas. Other basal cell carcinoma related genes found in this study include: KDR, KMT2D, FAT1, FAT4, GRIN2A, ERBB4, NOTCH2, PDE4DIP, TET1, ZFHX3 and PREX2. These results provide insight into basal cell carcinoma in non-Caucasians.

Keywords: genetics; histopathology; mutation; skin cancer; whole exome sequencing; basal cell carcinoma.

Introduction: Prenatal stress (PS) can cause depression in offspring. However, the underlying biological mechanism of these influences is still unclear. This work was implemented to investigate the molecular mechanisms of depressive-like behavior of offspring rats insulted with PS.

Methods: Relative quantitative phosphoproteomics of the hippocampus of PS susceptibility (PS-S) and control (CON) rat offspring was performed using liquid chromatography-tandem mass spectrometry to confirm known pathways and to identify new mechanisms involved in depression.

Results: A total of 6790 phosphopeptides, 9817 phosphorylation sites, and 2978 phosphoproteins were detected. Among the 2978 phosphoproteins, 1760 (59.09%) had more than two phosphorylated sites, the ENSRNOP00000023460 protein had more than 117 phosphorylated sites, and the average distribution of modification sites per 100 amino acids was 2.97. There were 197 different phosphopeptides, including 140 increased phosphopeptides and 57 decreased phosphopeptides in the PS-S offspring rats, compared to the CON offspring rats. These differential phosphopeptides corresponded to 100 upregulated and 44 downregulated phosphoproteins, respectively. Gene ontology enrichment analysis revealed that these different phosphoproteins in the top five enriched terms in the cellular component, molecular function, and biological proces categories were involved in a total of 35 different phosphoproteins, and these phosphoproteins were mainly related to myelin-, microtubule- and synapse-associated proteins. The enrichment of Kyoto Encyclopedia of Genes and Genome pathways was found to be involved in many essential biological pathways, and the top five pathways included amphetamine addiction, insulin secretion, Cushing syndrome, and the circadian entrainment signaling pathway. These first five pathways were related to nine phosphoproteins, including Adcy9, Apc, Cacna1c, Camk2a, Camk2b, Camk2g, Ctnnd2, Grin2a, and Stx1a. The full data are available via ProteomeXchange with identifier PXD019117.

Conclusion: We preliminarily identified 144 different phosphoproteins involved in myelin, microtubule, and synapse formation and plasticity in the hippocampus of susceptible offspring rats exposed to PS.

Keywords: depressive-like behaviors; hippocampus; offspring; phosphoproteomics; prenatal stress.

Large international consortia examining the genomic architecture of the epilepsies focus on large diagnostic subgroupings such as "all focal epilepsy" and "all genetic generalized epilepsy". In addition, phenotypic data are generally entered into these large discovery databases in a unidirectional manner at one point in time only. However, there are many smaller phenotypic subgroupings in epilepsy, many of which may have unique genomic risk factors. Such a subgrouping or "microphenotype" may be defined as an uncommon or rare phenotype that is well recognized by epileptologists and the epilepsy community, and which may or may not be formally recognized within the International League Against Epilepsy classification system. Here we examine the genetic structure of a number of such microphenotypes and report in particular on two interesting clinical phenotypes, Jeavons syndrome and pediatric status epilepticus. Although no single gene reached exome-wide statistical significance to be associated with any of the diagnostic categories, we observe enrichment of rare damaging variants in established epilepsy genes among Landau-Kleffner patients (GRIN2A) and pediatric status epilepticus patients (MECP2, SCN1A, SCN2A, SCN8A).

Keywords: Jeavons syndrome; genomics; microphenotype; pediatric status epilepticus.

Background: Schizophrenia is a complex mental disorder with a high heritability. Dysfunction of the N-methyl-D-aspartate (NMDA)-type glutamate receptors may be involved in the pathogenesis of schizophrenia. In this study, we examined the contribution of GRIN2A and GRIN2B (Glutamate Ionotropic Receptor NMDA Type Subunit 2A/2B) polymorphisms to the clinical features of schizophrenia, such as the leading symptoms, the type of course, and the age of onset.

Methods: A population of 402 Russian patients with schizophrenia from the Siberian region was investigated. Genotyping of seventeen single-nucleotide polymorphisms (SNPs) in GRIN2A and GRIN2B was performed using QuantStudio™ 3D Digital PCR System Life Technologies amplifier using TaqMan Validated SNP Genotyping Assay kits (Applied Biosystems). The results were analyzed using Chi-square and the Fisher's exact tests.

Results: We found an association of GRIN2A rs7206256 and rs11644461 and GRIN2B rs7313149 with the early onset (before the age of 18 years old) schizophrenia. We did not reveal any associations of GRIN2A and GRIN2B polymorphisms with leading (positive vs. negative) symptoms or type of course (continuous vs. episodic) of schizophrenia.

Conclusions: In the study, we confirmed the involvement of the GRIN2A and GRIN2B genes in the early onset of schizophrenia in a Russian population of the Siberian region.

Keywords: GRIN2A; GRIN2B; early onset; genes; genetic polymorphism; schizophrenia.

Human genetic sequencing has implicated epigenetic and synaptic aberrations as the most prominent risk factors for autism. Here we show that autistic patients exhibit the significantly lower histone acetylation and elevated HDAC2 expression in prefrontal cortex (PFC). The diminished histone acetylation is also recaptured in an autism mouse model with the deficiency of the Shank3 gene encoding a synaptic scaffolding protein. Treating young (5-week-old) Shank3-deficient mice with a 4-week ketogenic diet, which can act as an endogenous inhibitor of class I HDACs via the major product β-hydroxybutyrate, elevates the level of histone acetylation in PFC neurons. Behavioral assays indicate that ketogenic diet treatment leads to the prolonged rescue of social preference deficits in Shank3-deficient mice. The HDAC downstream target genes encoding NMDA receptor subunits, GRIN2A and GRIN2B, are significantly reduced in PFC of autistic humans. Ketogenic diet treatment of Shank3-deficient mice elevates the transcription and histone acetylation of Grin2a and Grin2b, and restores the diminished NMDAR synaptic function in PFC neurons. These results suggest that the ketogenic diet provides a promising therapeutic strategy for social deficits in autism via the restoration of histone acetylation and gene expression in the brain.