Disturbances in glutamate signaling caused by disruption of N-methyl-D-aspartate-type glutamate receptor (NMDAR) have been implicated in schizophrenia. Findings suggested that miR-219, miR-132 and miR-107 could involve in NMDAR signaling by influencing the expression of pathway genes or the signaling transmission and single nucleotide polymorphisms (SNPs) within miRNA genes or miRNA target sites could result in their functional changes. Therefore, we hypothesized that SNPs in miRNAs and/or their target sites were associated with schizophrenia. 3 SNPs in hsa-pri-miR-219/132/107 and 6 SNPs in 3'UTRs of GRIN2A/2B/3A and CAMK2G were selected and genotyped in a case-control study of 1041 schizophrenia cases and 953 healthy controls in Chinese Han population. In the present study, GRIN2B rs890 showed significant associations with schizophrenia. Further functional analyses showed that the rs890 variant C allele led to significantly lower luciferase activity, compared with the A allele. MDR analysis showed that a 4-locus model including rs107822, rs2306327, rs890 and rs12342026 was the best model. These findings suggest that GRIN2B may be associated with schizophrenia and interaction effects of the polymorphisms in hsa-miR-219, CAKM2G, GRIN2B and GRIN3A may confer susceptibility to schizophrenia in the Chinese Han population.

Expression of miR-137 is downregulated in brain tissue from patients with depression and suicidal behavior, and is also downregulated in peripheral blood from stroke patients. However, it is not yet known if miR-137 acts as a bridge between stroke and depression. To test this, we used middle cerebral artery occlusion and chronic mild stress to establish a post-stroke depression model in rats. Compared with controls, we found significantly lower miR-137 levels in the brain and peripheral blood from post-stroke depression rats. Injection of a miR-137 antagonist into the brain ventricles upregulated miR-137 levels, and improved behavioral changes in post-stroke depression rats. Luciferase assays showed miR-137 bound to the 3'UTR of Grin2A, regulating Grin2A expression in a neuronal cell line. Grin2A gene overexpression in the brain of post-stroke depression rats, noticeably suppressed the inhibitory effect of miR-137 on post-stroke depression. Overall, our results show that miR-137 suppresses Grin2A protein expression through binding to Grin2A mRNA, thereby exerting an inhibitory effect on post-stroke depression. Our results offer a new therapeutic direction for post-stroke depression.

OBJECTIVE

The epilepsy-aphasia spectrum (EAS) is a heterogeneous group of age-dependent childhood disorders characterized by sleep-activated discharges associated with infrequent seizures and language, cognitive, and behavioral deficits. Defects in the GRIN2A gene, encoding a subunit of glutamate-gated N-methyl-d-aspartate (NMDA) receptors, represent the most important cause of EAS identified so far. Neocortical or thalamic lesions were detected in a subset of severe EAS disorders, and more subtle anomalies were reported in patients with so-called "benign" phenotypes. However, whether brain structural alterations exist in the context of GRIN2A defects is unknown.

METHODS

Magnetic resonance diffusion tensor imaging (MR-DTI) was used to perform longitudinal analysis of the brain at 3 developmental timepoints in living mice genetically knocked out (KO) for Grin2a. In addition, electroencephalography (EEG) was recorded using multisite extracellular electrodes to characterize the neocortical activity in vivo.

RESULTS

Microstructural alterations were detected in the neocortex, the corpus callosum, the hippocampus, and the thalamus of Grin2a KO mice. Most MR-DTI alterations were detected at a specific developmental stage when mice were aged 30 days, but not at earlier (15 days) or later (2 months) ages. EEG analysis detected epileptiform discharges in Grin2a KO mice in the third postnatal week.

CONCLUSIONS

Grin2a KO mice replicated several anomalies found in patients with EAS disorders. Transient structural alterations detected by MR-DTI recalled the age-dependent course of EAS disorders, which in humans start during childhood and show variable outcome at the onset of adolescence. Together with the epileptiform discharges detected in young Grin2a KO mice, our data suggested the existence of early anomalies in the maturation of the neocortical and thalamocortical systems. Whereas the possible relationship of those anomalies with sleep warrants further investigations, our data suggest that Grin2a KO mice may serve as an animal model to study the neuronal mechanisms of EAS disorders and to design new therapeutic strategies.

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.

CONCLUSIONS

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.

Recently, mutations and deletions in the GRIN2A gene have been identified to predispose to benign and severe idiopathic focal epilepsies (IFE), revealing a higher incidence of GRIN2A alterations among the more severe phenotypes. This study aimed to explore the phenotypic boundaries of GRIN2A mutations by investigating patients with the two most common epilepsy syndromes: (i) idiopathic generalized epilepsy (IGE) and (ii) temporal lobe epilepsy (TLE). Whole exome sequencing data of 238 patients with IGE as well as Sanger sequencing of 84 patients with TLE were evaluated for GRIN2A sequence alterations. Two additional independent cohorts comprising 1469 IGE and 330 TLE patients were screened for structural deletions (>40kb) involving GRIN2A. Apart from a presumably benign, non-segregating variant in a patient with juvenile absence epilepsy, neither mutations nor deletions were detected in either cohort. These findings suggest that mutations in GRIN2A preferentially are involved in genetic variance of pediatric IFE and do not contribute significantly to either adult focal epilepsies as TLE or generalized epilepsies.

Schizophrenia is a severe, complex mental disorder. Abnormal glutamate neurotransmission mediated by decreased expression of N-methyl-d-aspartic acid receptors (NMDArs) and its endogenous co-agonist d-serine (d-Ser) has been proposed as one of the hypotheses of the pathogenesis of schizophrenia. GRIN2A gene promoter polymorphism causes changes in the regulation of the expression of NMDAr subunit genes. Our study is aimed at evaluating a possible association between GRIN2A promoter GT polymorphisms and schizophrenia in the Han Chinese population in Shaanxi and the relationship between serum d-Ser levels and GRIN2A (GT)n in schizophrenia. Four hundred and twenty patients with schizophrenia and 410 healthy individuals were recruited in this study and GRIN2A (GT)n repeats as well as serum d-Ser levels were measured in all of the subjects. Nineteen alleles were found in (GT)n locus. The allele frequency of (GT)21, (GT)22 and (GT)23 in schizophrenic subjects was significantly lower compared with the mentally healthy controls, while the allele (GT)26 was significantly more frequent than in normal persons. Transcriptional activity of GRIN2A promoter was gradually suppressed with the increase in the length of the (GT)n repeats. d-Ser levels in the serum of the GRIN2A (GT)21 schizophrenic patients were significantly lower than those of the GRIN2A (GT)21 healthy control. A significant correlation between serum d-Ser levels and GRIN2A (GT)21 in schizophrenia was detected. GRIN2A (GT)21 may play a significant role in the etiology of schizophrenia among the Chinese Han population of Shaanxi.

We have recently described a hemi-deletion on chromosome 9p24.2 at the SLC1A1 gene locus and its co-segregation with schizophrenia in an extended Palauan pedigree. This finding represents a point of convergence for several pathophysiological models of schizophrenia. The present report sought to characterize the biological consequences of this hemi-deletion. Dual luciferase assays demonstrated that the partially deleted allele (lacking exon 1 and the native promoter) can drive expression of a 5'-truncated SLC1A1 using sequence upstream of exon 2 as a surrogate promoter. However, confocal microscopy and electrophysiological recordings demonstrate that the 5'-truncated SLC1A1 lacks normal membrane localization and glutamate transport ability. To identify downstream consequences of the hemi-deletion, we first used a themed qRT-PCR array to compare expression of 84 GABA and glutamate genes in RNA from peripheral blood leukocytes in deletion carriers (n = 11) versus noncarriers (n = 8) as well as deletion carriers with psychosis (n = 5) versus those without (n = 3). Then, targeted RNA-Seq (TREx) was used to quantify expression of 375 genes associated with neuropsychiatric disorders in HEK293 cells subjected to either knockdown of SLC1A1 or overexpression of full-length or 5'-truncated SLC1A1. Expression changes of several genes strongly implicated in schizophrenia pathophysiology were detected (e.g. SLC1A2, SLC1A3, SLC1A6, SLC7A11, GRIN2A, GRIA1 and DLX1).

Glioblastoma is the most frequent and aggressive primary brain tumor, characterized by extensive brain invasion and rarely, systemic metastases. The pathogenesis of metastatic glioblastoma is largely unknown. We present the first integrated clinical/histologic/genetic analysis of 5 distinct brain and lung foci from a unique case of recurrent, multifocal, multicentric and metastatic glioblastoma. The initial right frontotemporal gliosarcoma received standard surgical/chemoradiation therapy and recurred 1.5 years later, co-occurring with three additional masses localized to the ipsilateral temporal lobe, cerebellum and lung. Synchronous metastatic lung carcinoma was suspected in this long-term smoker patient with family history of cancer. However, glioblastoma was confirmed in all tumors, although with different morphologic patterns, including ependymomatous and epithelioid. Genomic profiling revealed a germline FANCD2 variant of unknown significance, and a 4-gene somatic mutation signature shared by all tumors, consisting of TERT promoter and PTEN, RB1 and TP53 tumor suppressor mutations. Additional GRIN2A and ATM heterozygous mutations were selected in the cerebellar and lung foci, but were variably present in the supratentorial foci, indicating reduced post-therapeutic genetic evolution in brain foci despite morphologic variability. Significant genetic drift characterized the lung metastasis, likely explaining the known resistance of circulating glioblastoma cells to systemic seeding. MET overexpression was detected in the initial gliosarcoma and lung metastasis, possibly contributing to invasiveness. This comprehensive analysis sheds light on the temporospatial evolution of glioblastoma and underscores the importance of genetic testing for diagnosis and personalized therapy.

Fractionated whole-brain irradiation (fWBI) is a mainstay of treatment for patients with intracranial neoplasia; however late-delayed radiation-induced normal tissue injury remains a major adverse consequence of treatment, with deleterious effects on quality of life for affected patients. We hypothesize that cerebrovascular injury and remodeling after fWBI results in ischemic injury to dependent white matter, which contributes to the observed cognitive dysfunction. To evaluate molecular effectors of radiation-induced brain injury (RIBI), real-time quantitative polymerase chain reaction (RT-qPCR) was performed on the dorsolateral prefrontal cortex (DLPFC, Brodmann area 46), hippocampus and temporal white matter of 4 male Rhesus macaques (age 6-11 years), which had received 40 Gray (Gy) fWBI (8 fractions of 5 Gy each, twice per week), and 3 control comparators. All fWBI animals developed neurologic impairment; humane euthanasia was elected at a median of 6 months. Radiation-induced brain injury was confirmed histopathologically in all animals, characterized by white matter degeneration and necrosis, and multifocal cerebrovascular injury consisting of perivascular edema, abnormal angiogenesis and perivascular extracellular matrix deposition. Herein we demonstrate that RIBI is associated with white matter-specific up-regulation of hypoxia-associated lactate dehydrogenase A (LDHA) and that increased gene expression of fibronectin 1 (FN1), SERPINE1 and matrix metalloprotease 2 (MMP2) may contribute to cerebrovascular remodeling in late-delayed RIBI. Additionally, vascular stability and maturation associated tumor necrosis super family member 15 (TNFSF15) and vascular endothelial growth factor beta (VEGFB) mRNAs were increased within temporal white matter. We also demonstrate that radiation-induced brain injury is associated with decreases in white matter-specific expression of neurotransmitter receptors SYP, GRIN2A and GRIA4. We additionally provide evidence that macrophage/microglial mediated neuroinflammation may contribute to RIBI through increased gene expression of the macrophage chemoattractant CCL2 and macrophage/microglia associated CD68. Global patterns in cerebral gene expression varied significantly between regions examined (P < 0.0001, Friedman's test), with effects most prominent within cerebral white matter.

Lithium has been the first-line treatment for bipolar disorder (BD) for more than six decades. Although the molecular effects of lithium have been studied extensively and gene expression changes are generally believed to be involved, the specific mechanisms of action that mediate mood regulation are still not known. In this study, a multi-step approach was used to explore the transcriptional changes that may underlie lithium's therapeutic efficacy. First, we identified genes that are associated both with lithium exposure and with BD, and second, we performed differential expression analysis of these genes in brain tissue samples from BD patients (n = 42) and healthy controls (n = 42). To identify genes that are regulated by lithium exposure, we used high-sensitivity RNA-sequencing of corpus callosum (CC) tissue samples from lithium-treated (n = 8) and non-treated (n = 9) rats. We found that lithium exposure significantly affected 1108 genes (FDR < 0.05), 702 upregulated and 406 downregulated. These genes were mostly enriched for molecular functions related to signal transduction, including well-established lithium-related pathways such as mTOR and Wnt signaling. To identify genes with differential expression in BD, we performed expression quantitative trait loci (eQTL) analysis on BD-associated genetic variants from the most recent genome-wide association study (GWAS) using three different gene expression databases. We found 307 unique eQTL genes regulated by BD-associated variants, of which 12 were also significantly modulated by lithium treatment in rats. Two of these showed differential expression in the CC of BD cases: RPS23 was significantly downregulated (p = 0.0036, fc = 0.80), while GRIN2A showed suggestive evidence of downregulation in BD (p = 0.056, fc = 0.65). Crucially, GRIN2A was also significantly upregulated by lithium in the rat brains (p = 2.2e-5, fc = 1.6), which suggests that modulation of GRIN2A expression may be a part of the therapeutic effect of the drug. These results indicate that the recent upsurge in research on this central component of the glutamatergic system, as a target of novel therapeutic agents for affective disorders, is warranted and should be intensified.

Schizophrenia is considered a neurodevelopmental disorder. Recent reports relate synaptic alterations with disease etiology. The inbred Roman High- (RHA-I) and Low- (RLA-I) Avoidance rat strains are a congenital neurobehavioral model, with the RHA-I displaying schizophrenia-related behaviors and serotonin 2A (5-HT2A) and metabotropic glutamate 2 (mGlu2) receptor alterations in the prefrontal cortex (PFC). We performed a comprehensive characterization of the RHA-I/RLA-I rats by real-time qPCR and Western blotting for 5-HT1A, 5-HT2A, mGlu2, dopamine 1 and dopamine 2 receptors (DRD1 and DRD2), AMPA receptor subunits Gria1, Gria2 and NMDA receptor subunits Grin1, Grin2a and Grin2b, as well as pre- and post-synaptic components in PFC and hippocampus (HIP). Besides corroborating decreased mGlu2 (Grm2) expression, we found increased mRNA levels for Snap25, Synaptophysin (Syp), Homer1 and Neuregulin-1 (Nrg1) in the PFC of the RHA-I and decreased expression of Vamp1, and Snapin in the HIP. We also showed alterations in Vamp1, Grin2b, Syp, Snap25 and Nrg1 at protein levels. mRNA levels of Brain Derived Neurotrophic Factor (BDNF) were increased in the PFC of the RHA-I rats, with no differences in the HIP, while BDNF protein levels were decreased in PFC and increased in HIP. To investigate the temporal dynamics of these synaptic markers during neurodevelopment, we made use of the open source BrainCloud™ dataset, and found that SYP, GRIN2B, NRG1, HOMER1, DRD1 and BDNF expression is upregulated in PFC during childhood and adolescence, suggesting a more immature neurobiological endophenotype in the RHA-I strain.

Regulation of N-methyl-d-aspartate (NMDA) subunits NR2A and NR2B expression during status epilepticus (SE) remains incompletely understood. Here we explored the role of brain-enriched microRNA (miR)-139-5p in this process.

miRNA microarray was performed to examine changes in miRNA expression in the rat pilocarpine model following NMDA-receptor blockade. The dynamic expression patterns of miR-139-5p, NR2A, and NR2B levels were measured in rats during the three phases of temporal lobe epilepsy (TLE) development using quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot. Similar expression methods were applied to hippocampi obtained from patients with TLE and from normal controls. Moreover, miR-139-5p agomir and antagomir were utilized to explore the role of miR-139-5p in determining NMDA-receptor subunit expression patterns.

We identified 18 miRNAs that were significantly altered in the rat pilocarpine model following NMDA-receptor blockade. Of these, miR-139-5p was significantly up-regulated and Grin2A was predicted as its potential putative target. In patients with TLE, miR-139-5p expression was significantly down-regulated, whereas NR2A and NR2B levels were significantly up-regulated. In the rat model of SE, miR-139-5p expression was down-regulated while NR2A was up-regulated in the acute and chronic phases, but not in the latent phase. NR2B expression was up-regulated during the three phases of TLE development. Overexpression of miR-139-5p decreased, whereas depletion of miR-139-5p enhanced the expression levels of NR2A, but not NR2B, induced by pilocarpine treatment. Of interest, NMDA nonselective antagonist and NR2A selective antagonist enhanced miR-139-5p levels suppressed by pilocarpine treatment, whereas the NR2B selective antagonist was ineffective.

These findings elucidate the potential role of miR-139-5p in NMDA-receptor involvement in TLE development and may provide novel therapeutic targets for the future treatment of TLE.

Epilepsy is a common and genetically heterogeneous disorder among children. Advances in next-generation sequencing have revealed that numerous epilepsy genes, helped us improve the understanding of mechanisms underlying epileptogenesis, and guided the development of treatments. We identified 39 candidate variants in 21 genes, including 37 that were pathogenic or likely pathogenic variants according to the American College of Medical Genetics and Genomics scoring system and two variants of uncertain significance that were considered causative after they were associated with clinical characteristics. Thirty were de novo variants (76.9%), and 20 variants had not previously been reported (51.3%). We obtained a diagnosis in 39 of the 141 probands (27.7%). The most frequently mutated gene was SCN1A; KCNQ2, KCNT1, PCDH19, STXBP1, SCN2A, TSC2, and PRRT2 were mutated in more than one individual; ANKRD11, CDKL5, DCX, DEPDC5, GABRB3, GRIN2A, IQSEC2, KCNA2, KCNB1, KCNJ6, TSC1, SCN9A, and SCN1B were mutated in a single individual. In addition, we detected a nonsense variant in a candidate gene KCND1 and considered it as a new candidate epilepsy gene, which needed further functional study. Consequently, large number of unreported variants were detected, diverse phenotypes were associated with known epilepsy genes. Changes in clinical management beyond genetic counseling were suggested.

Sudden unexpected death in epilepsy is an unpredicted condition in patients with a diagnosis of epilepsy, and autopsy does not conclusively identify cause of death. Although the pathophysiological mechanisms that underlie this entity remain unknown, the fact that epilepsy can affect cardiac function is not surprising. The genetic factors involving ion channels co-expressed in the heart and brain and other candidate genes have been previously described. In the present study, 20 epilepsy patients with personal or family history of heart rhythm disturbance/cardiac arrhythmias/sudden death were sequenced using a custom re-sequencing panel. Twenty-six relatives were genetically analysed to ascertain the family segregation in ten individuals. Four subjects revealed variants with positive genotype-phenotype segregation: four missense variants in the CDKL5, CNTNAP2, GRIN2A and ADGRV1 genes and one copy number variant in KCNQ1. The potential pathogenic role of variants in new candidate genes will need further studies in larger cohorts, and the evaluation of the potential pathogenic role in the cardio-cerebral mechanisms requires in vivo/in vitro studies. In addition to family segregation, evaluation of the potential pathogenic roles of these variants in cardio-cerebral mechanisms by in vivo/in vitro studies should also be performed. The potential pathogenic role of variants in new candidate genes will need further studies in larger cohorts.

Evidence indicates that experiencing early-life stress (ELS) is a risk factor for the development of mental disorders such as depression. Maternal separation stress (MS) is a valid animal model of ELS that caused to induce long-lasting effects on the brain and behaviors of animals. It hypothesized that adolescence is a critical stage in which the brain is still developing, and applying (non)pharmacological therapies in this period may attenuate the effects of ELS on the brain and behavior. Male rats were subjected to MS from postnatal day (PND) 2-14, and the stressed animals were then treated with (1) chronic fluoxetine (FLX) (5 mg/kg) and (2) voluntary running wheel exercise (RW) from PND 30, for 30 days. Then, we subjected the animals to behavioral and molecular assessments at PND 60. Our data showed that MS provoked depressive-like behaviors in rats, tested by the forced swimming test, splash test, and sucrose preference test. Additionally, we found that MS increased the gene expression of the NR2A (and not NR2B) subunit of N-methyl-D-aspartate (NMDA) receptors in the hippocampus of adult rats. Both FLX and RW treatments during adolescence were able to mitigate the negative effects of ELS on stressed animals. These results highlighted the importance of adolescence in treating stressed animals with FLX/voluntary RW exercise to alleviate the depressive effects of ELS. In addition, we found that ELS altered the transcriptional level of Grin2a (and not Grin2b) in the hippocampus. Finally, our results showed that FLX/voluntary RW exercise during adolescence could normalize altered expression of Grin2a in the hippocampus of adult rats.

Tumor mutation burden (TMB) serves as an effective biomarker predicting efficacy of mono-immunotherapy for non-small cell lung cancer (NSCLC). Establishing a precise TMB predicting model is essential to select which populations are likely to respond to immunotherapy or prognosis and to maximize the benefits of treatment. In this study, available Formalin-fixed paraffin embedded tumor tissues were collected from 499 patients with NSCLC. Targeted sequencing of 636 cancer related genes was performed, and TMB was calculated. Distribution of TMB was significantly (p < 0.001) correlated with sex, clinical features (pathological/histological subtype, pathological stage, lymph node metastasis, and lympho-vascular invasion). It was also significantly (p < 0.001) associated with mutations in genes like TP53, EGFR, PIK3CA, KRAS, EPHA3, TSHZ3, FAT3, NAV3, KEAP1, NFE2L2, PTPRD, LRRK2, STK11, NF1, KMT2D, and GRIN2A. No significant correlations were found between TMB and age, neuro-invasion (p = 0.125), and tumor location (p = 0.696). Patients with KRAS p.G12 mutations and FAT3 missense mutations were associated (p < 0.001) with TMB. TP53 mutations also influence TMB distribution (P < 0.001). TMB was reversely related to EGFR mutations (P < 0.001) but did not differ by mutation types. According to multivariate logistic regression model, genomic parameters could effectively construct model predicting TMB, which may be improved by introducing clinical information. Our study demonstrates that genomic together with clinical features yielded a better reliable model predicting TMB-high status. A simplified model consisting of less than 20 genes and couples of clinical parameters were sought to be useful to provide TMB status with less cost and waiting time.

Keywords: early-stage non-small-cell lung cancer; genomics; histology; model; tumor mutation burden (TMB).

Five N-methyl-D-aspartate receptor subunit genes and six metabotropic glutamate receptor subtype genes have been assigned to particular rat chromosomes by using a rat x mouse somatic cell hybrid clone panel. N-Methyl-D-aspartate receptor subunit genes (gene symbol, GRIN) GRIN1, GRIN2A, GRIN2B, GRIN2C, and GRIN2D have been assigned to chromosomes (Chr) 3, 10, 4, 10, and 1, respectively. Metabotropic glutamate receptor subtype genes (gene symbol, GRM) GRM1, GRM2, GRM3, GRM4, GRM5, and GRM6 have been assigned to Chr 1, 8, 4, 20, 1, and 10, respectively. In addition, GRIN2A and GRM6 loci were successfully localized on Chr 10 linkage maps by linkage analyses. The genetic distances between loci in cM (+/- SD) are as follows: GRIN2A-28.6(+/- 7.0)-RR24-23.3(+/- 6.4)-MYHSE, from a linkage analysis using the (SHR x WTC)F1 x WTC cross, and RR24-4.2(+/- 2.9)-GRM6-4.2(+/- 2.9)-MMYHSE-2.1(+/- 2.1)-ASGR, SHBG-27.1(+/- 6.4)-PPY, from a linkage analysis using the (ZI x TM)F1 x ZI cross.

Multiple espilon subunits are major determinants of the diversity of the N-methyl-D-aspartate (NMDA) receptor channel. The four epsilon subunit mRNAs exhibit distinct expression patterns in the brain. In an attempt to elucidate the molecular basis of selective and characteristic expression of the NMDA receptor channel subunits, we have isolated the gene encoding the mouse NMDA receptor epsilon 3 subunit and have determined its structural organization. The epsilon 3 subunit gene spans 17.5 kb and consists of 14 exons. The major transcription start site is 439 bp upstream of the ATG initiation codon as determined by primer extension and S1 nucleas protection analyses. Two polyadenylation sites are 397 (or 398) and 402 bp downstream of the termination codon. The 5'-flanking region of the epsilon 3 subunit gene contains GC-rich segments including consensus sequences for binding of the transcription factors Spl and EGR-1. The murine chromosomal locations of the five NMDA receptor channel subunits, the epsilon 1 (Grin2a), epsilon 2 (Grin2b), epsilon 3 (Grin2c), epsilon 4 (Grin2d) and zeta 1 (Grinl) subunits, were determined using an interspecific backcross mapping panel derived from crosses of [(C57BL/6JxM. spretus) F1xC57BL/6J] mice. Each of these genes mapped to a single chromosome location. The mapping results assigned the five loci to five different mouse autosomes, indicating that they have become well dispersed among mouse chromosomes.

OBJECTIVE

To determine genetic variability within the N-methyl-D-aspartate receptor 2A sub-unit (GRIN2A) gene promoter and its association with concussion recovery time. The hypothesis tested was that there would be a difference in allele and/or genotype distribution between two groups of athletes with normal and prolonged recovery.

METHODS

DNA was extracted from saliva collected from a total of 87 athletes with a physician-diagnosed concussion. The (GT) variable number tandem repeats (VNTR) within the promoter region of GRIN2A was genotyped. The long (L) allele was an allele with ≥25 repeats and the short (S) allele was an allele with <25 repeats in the GT tract. Participants' recovery time was followed prospectively and was categorized as normal (≤60 days) or prolonged (>60 days).

RESULTS

LL carriers were 6-times more likely to recover longer than 60 days following the concussive event (p = 0.0433) when compared to SS carriers. Additionally, L allele carriers were found more frequently in the prolonged recovery group (p = 0.048).

CONCLUSIONS

Determining genetic influence on concussion recovery will aid in future development of genetic counselling. The clinical relevance of genotyping athletes could improve management of athletes who experience concussion injuries.

In the developing nervous system, ordered neuronal activity patterns can occur even in the absence of sensory input and to investigate how these arise, we have used the model system of the embryonic chicken spinal motor circuit, focusing on motor neurons of the lateral motor column (LMC). At the earliest stages of their molecular differentiation, we can detect differences between medial and lateral LMC neurons in terms of expression of neurotransmitter receptor subunits, including CHRNA5, CHRNA7, GRIN2A, GRIK1, HTR1A and HTR1B, as well as the KCC2 transporter. Using patch-clamp recordings we also demonstrate that medial and lateral LMC motor neurons have subtly different activity patterns that reflect the differential expression of neurotransmitter receptor subunits. Using a combination of patch-clamp recordings in single neurons and calcium-imaging of motor neuron populations, we demonstrate that inhibition of nicotinic, muscarinic or GABA-ergic activity, has profound effects of motor circuit activity during the initial stages of neuromuscular junction formation. Finally, by analysing the activity of large populations of motor neurons at different developmental stages, we show that the asynchronous, disordered neuronal activity that occurs at early stages of circuit formation develops into organised, synchronous activity evident at the stage of LMC neuron muscle innervation. In light of the considerable diversity of neurotransmitter receptor expression, activity patterns in the LMC are surprisingly similar between neuronal types, however the emergence of patterned activity, in conjunction with the differential expression of transmitter systems likely leads to the development of near-mature patterns of locomotor activity by perinatal ages.

Heroin dependence is a debilitating psychiatric disorder with a complex inheritance mechanism. Genetic polymorphisms in functional regions of the glutamate receptor, N-methyl D-aspartate 2A (GRIN2A) gene, which encodes the 2A subunit of the N-methyl D-aspartate (NMDA) receptor, may modulate the risk of heroin addiction. We investigated the potential association between 8 single nucleotide polymorphisms (SNPs) of the GRIN2A gene (SNPs rs3219790, rs1014531, rs8044472, rs8045712, rs9933624, rs9940680, rs1420040, and rs767749) and heroin addiction using the MassARRAY system and GeneScan. A total of 405 heroin-addicted patients and 397 healthy control subjects were recruited for this study. Statistically significant differences were observed for rs3219790 in the promoter region of the GRIN2A gene. The frequency of the (GT)26 repeats in the heroin addiction group was significantly higher than that in the control group [X(2) = 5.475, P = 0.019, odds ratio (OR) = 1.367, 95% confidence interval (CI) = 1.051-1.776]. Strong linkage disequilibrium was observed in block 1 (D'>> 0.9). However, significant evidence of linkage disequilibrium was not observed between the 7 SNPs in our sample population. These data suggest that GRIN2A gene polymorphisms confer susceptibility to heroin addiction and support the hypothesis that dysfunction of GRIN2A is involved in the pathophysiological process of heroin addiction.