Oocyte-Specific Deletion of Slc6a9 Encoding the GLYT1 Glycine Transporter Eliminates Glycine Transport in Mouse Preimplantation Embryos and Their Ability to Counter Hypertonic Stress.

Early preimplantation mouse embryos are sensitive to increased osmolarity, which can block their development. To overcome this, they accumulate organic osmolytes to maintain cell volume. The main organic osmolyte used by early mouse embryos is glycine. Glycine is transported during the mature egg and 1-cell to 4-cell embryo stages by a transporter identified as GLYT1, encoded by the Slc6a9 gene. Here, we have produced an oocyte-specific knockout of Slc6a9 by crossing mice that have a segment of the gene flanked by LoxP elements with transgenic mice expressing iCre driven by the oocyte-specific Gdf9 promoter. Slc6a9 null oocytes failed to develop glycine transport activity during meiotic maturation. However, females with these oocytes were fertile. When enclosed in their cumulus-oocyte complex, Slc6a9 null oocytes could accumulate glycine via GLYT1 transport in their coupled cumulus cells, which may support female fertility in vivo. In vitro, embryos derived from Slc6a9 null oocytes displayed a clear phenotype. While glycine rescued complete preimplantation development of wild type embryos from increased osmolarity, embryos derived from null oocytes failed to develop past the 2-cell stage even with glycine. Thus, Slc6a9 is required for glycine transport and protection against increased osmolarity in mouse eggs and early embryos.

Dual Role of Dysfunctional Asc-1 Transporter in Distinct Human Pathologies, Human Startle Disease, and Developmental Delay.

Human startle disease is associated with mutations in distinct genes encoding glycine receptors, transporters or interacting proteins at glycinergic synapses in spinal cord and brainstem. However, a significant number of diagnosed patients does not carry a mutation in the common genes GLRA1, GLRB, and SLC6A5 Recently, studies on solute carrier 7 subfamily 10 (SLC7A10; Asc-1, alanine-serine-cysteine transporter) knock-out (KO) mice displaying a startle disease-like phenotype hypothesized that this transporter might represent a novel candidate for human startle disease. Here, we screened 51 patients from our patient cohort negative for the common genes and found three exonic (one missense, two synonymous), seven intronic, and single nucleotide changes in the 5' and 3' untranslated regions (UTRs) in Asc-1. The identified missense mutation Asc-1G307R from a patient with startle disease and developmental delay was investigated in functional studies. At the molecular level, the mutation Asc-1G307R did not interfere with cell-surface expression, but disrupted glycine uptake. Substitution of glycine at position 307 to other amino acids, e.g., to alanine or tryptophan did not affect trafficking or glycine transport. By contrast, G307K disrupted glycine transport similar to the G307R mutation found in the patient. Structurally, the disrupted function in variants carrying positively charged residues can be explained by local structural rearrangements because of the large positively charged side chain. Thus, our data suggest that SLC7A10 may represent a rare but novel gene associated with human startle disease and developmental delay.

Startle Disease: New Molecular Insights into an Old Neurological Disorder.

Startle disease (SD) is characterized by enhanced startle responses, generalized muscle stiffness, unexpected falling, and fatal apnea episodes due to disturbed feedback inhibition in the spinal cord and brainstem of affected individuals. Mutations within the glycine receptor (GlyR) subunit and glycine transporter 2 (GlyT2) genes have been identified in individuals with SD. Impaired inhibitory neurotransmission in SD is due to pre- and/or postsynaptic GlyR or presynaptic GlyT2 dysfunctions. Previous research has focused on mutated GlyRs and GlyT2 that impair ion channel/transporter function or trafficking. With insights provided by recently solved cryo-electron microscopy and X-ray structures of GlyRs, a detailed picture of structural transitions important for receptor gating has emerged, allowing a deeper understanding of SD at the molecular level. Moreover, studies on novel SD mutations have demonstrated a higher complexity of SD, with identification of additional clinical signs and symptoms and interaction partners representing key players for fine-tuning synaptic processes. Although our knowledge has steadily improved during the last years, changes in synaptic localization and GlyR or GlyT2 homeostasis under disease conditions are not yet completely understood. Combined proteomics, interactomics, and high-resolution microscopy techniques are required to reveal alterations in receptor dynamics at the synaptic level under disease conditions.

Cross-Linking and Functional Analyses for Dimerization of a Cysteine Mutant of Glycine Transporter 1.

Glycine transporter 1 (GlyT1) is responsible for the reuptake of glycine, which regulates glutamate signaling as a co-agonist with N-methyl-D-aspartic acid (NMDA) receptors in the excitatory synapse and has been proposed to be a potential target in the development of therapies for a broad range of disorders of the central nervous system. Despite significant progress in characterizing structure and transport mechanism of the transporter, the regulation of transport function through oligomerization remains to be understood. In the present work, association of two forms of GlyT1 into dimers and higher order oligomers was detected by coimmunoprecipitation. To investigate functional properties of dimers of a GlyT1 cysteine mutant L288C, we performed oxidative cross-linking of the positioned cysteine residues in extracellular loop 3 (EL3) near the extracellular end of TM6. By analyzing the effect of copper phenanthroline (CuP)-induced dimerization on transport function, cross-linking of L288C was found to inhibit transport activity. In addition, an intramolecular ion pair Lys286-Glu289 was revealed to be critical for stabilizing EL3 in a conformation that modulates CuP-induced dimerization and transport function of the GlyT1 L288C mutant. Furthermore, the influence of transporter conformation on GlyT1 L288C dimerization was investigated. The substrate glycine, in the presence of both Na+ and Cl-, significantly reduced oxidative cross-linking, suggesting a large-scale rotation of the bundle domain during substrate transport impairs interfacial interactions between L288C protomers. The present study provides new insights into structural and functional elements regulating GlyT1 transport activity through its dimerization or oligomerization.

Genome-wide methylation analysis of post-mortem cerebellum samples supports the role of peroxisomes in autism spectrum disorder.

Aim: We tested the hypothesis that a subset of patients with autism spectrum disorder (ASD) contains candidate genes with high DNA methylation differences (effective values) that potentially affect one of the two alleles. Materials & methods: Genome-wide DNA methylation comparisons were made on cerebellum samples from 30 patients and 45 controls. Results: 12 genes with high effective values, including GSDMD, MMACHC, SLC6A5 and NKX6-2, implicated in ASD and other neuropsychiatric disorders were identified. Monoallelic promoter methylation and downregulation were observed for SERHL (serine hydrolase-like) and CAT (catalase) genes associated with peroxisome function. Conclusion: These data are consistent with the hypothesis implicating impaired peroxisome function/biogenesis for ASD. A similar approach holds promise for identifying rare epimutations in ASD and other complex disorders.

Genetic susceptibility of autism spectrum disorder (ASD) is well recognized but found only in one among 30­50% of identical twins. Epigenetic modifications such as DNA methylation underlie differences in gene function without alteration in the sequence. In this study, we identified large DNA methylation changes in nine gene promoters in cerebellum tissues of patients with ASD. Several were already implicated in ASD and other neuropsychiatric disorders. Promoters of two genes (SERHL and CAT) involved in peroxisome function showed increased methylation and low-level expression. The two abnormalities identified support the hypothesis associating ASD with defects in peroxisomes that are involved in detoxification of reactive oxygen species.

Hereditary Hyperekplexia in Saudi Arabia.

BACKGROUND: Hyperekplexia is a rare disorder characterized by exaggerated startle responses to unexpected sensory stimuli, recurrent apneas, and stiffness. Only few studies have been published on this disorder in populations with high rates of consanguinity. METHODS: We retrospectively reviewed Saudi patients with genetically confirmed hereditary hyperekplexia using a standard questionnaire that was sent to nine major referral hospitals in Saudi Arabia. RESULTS: A total of 22 Saudi patients (11 males, 11 females) from 20 unrelated families who had hereditary hyperekplexia were included. Based on molecular studies, they were classified into different subtypes: SLC6A5 variant (12 patients, 54.5%), GLRB variant (seven patients, 31.8%), and GLRA1 variant (three patients, 13.7%). All patients were homozygous for the respective causal variant. The combined carrier frequency of hereditary hyperekplexia for the encountered founder mutations in the Saudi population is 10.9 per 10,000, which translates to a minimum disease burden of 13 patients per 1,000,000. CONCLUSION: Our study provides comprehensive epidemiologic information, prevalence figures, and clinical characteristics of a large cohort of patients with hereditary hyperekplexia.

Regulation of the Glycine Transporter GLYT1 by microRNAs.

The glycine transporter GLYT1 participates in inhibitory and excitatory neurotransmission by controlling the reuptake of this neuroactive substance from synapses. Over the past few years, microRNAs have emerged as potent negative regulators of gene expression. In this report, we investigate the possible regulation of GLYT1 by microRNAs. TargetScan software predicted the existence of multiple targets for microRNAs within the 3' UTR of the human GLYT1 (miR-7, miR-30, miR-96, miR-137 and miR-141), and as they are all conserved among mammalian orthologues, their effects on GLYT1 expression were determined experimentally. Dual reporter bioluminescent assays showed that only miR-96 and miR-137 down-regulated expression of the Renilla reporter fused to the 3' UTR of GLYT1. Mutations introduced into the target sequences blocked this inhibitory effect. Consistently, these two microRNAs downregulated the uptake of [3H]glycine into glial C6 cells, a cell line where GLYT1 is the main carrier for glycine. Moreover, the expression of endogenous GLYT1 in primary mixed cultures from rat spinal cord was decreased upon lentiviral expression of miR-96 and miR-137. Although the bulk of GLYT1 is glial, it is abundantly expressed in glycinergic neurons of the retina and in smaller amounts in glutamatergic neurons though the brain. Since miR-96 in the retina is strongly downregulated by light exposure, when rats were maintained in darkness for a few hours we observed a concomitant increase of GLYT1 expression, suggesting that at least miR-96 might be an important negative regulator of GLYT1 under physiological conditions.

Functional Conservation and Genetic Divergence of Chordate Glycinergic Neurotransmission: Insights from Amphioxus Glycine Transporters.

Glycine is an important neurotransmitter in vertebrates, performing both excitatory and inhibitory actions. Synaptic levels of glycine are tightly controlled by the action of two glycine transporters, GlyT1 and GlyT2, located on the surface of glial cells and neurons, respectively. Only limited information is available on glycinergic neurotransmission in invertebrates, and the evolution of glycinergic neurotransmission is poorly understood. Here, by combining phylogenetic and gene expression analyses, we characterized the glycine transporter complement of amphioxus, an important invertebrate model for studying the evolution of chordates. We show that amphioxus possess three glycine transporter genes. Two of these (GlyT2.1 and GlyT2.2) are closely related to GlyT2 of vertebrates, whereas the third (GlyT) is a member of an ancestral clade of deuterostome glycine transporters. GlyT2.2 expression is predominantly non-neural, whereas GlyT and GlyT2.1 are widely expressed in the amphioxus nervous system and are differentially expressed, respectively, in neurons and glia. Vertebrate glycinergic neurons express GlyT2 and glia GlyT1, suggesting that the evolution of the chordate glycinergic system was accompanied by a paralog-specific inversion of gene expression. Despite this genetic divergence between amphioxus and vertebrates, we found strong evidence for conservation in the role glycinergic neurotransmission plays during larval swimming, the implication being that the neural networks controlling the rhythmic movement of chordate bodies may be homologous.

Glycine Transporters and Receptors as Targets for Analgesics.

The suitability of modulating glycinergic neurotransmission for the treatment of inflammatory and chronic pain has gained widespread recognition, with glycine receptors (GlyRs) and glycine transporters (GlyT1 and GlyT2) now considered key therapeutic targets [...].

The presynaptic glycine transporter GlyT2 is regulated by the Hedgehog pathway in vitro and in vivo.

The identity of a glycinergic synapse is maintained presynaptically by the activity of a surface glycine transporter, GlyT2, which recaptures glycine back to presynaptic terminals to preserve vesicular glycine content. GlyT2 loss-of-function mutations cause Hyperekplexia, a rare neurological disease in which loss of glycinergic neurotransmission causes generalized stiffness and strong motor alterations. However, the molecular underpinnings controlling GlyT2 activity remain poorly understood. In this work, we identify the Hedgehog pathway as a robust controller of GlyT2 expression and transport activity. Modulating the activation state of the Hedgehog pathway in vitro in rodent primary spinal cord neurons or in vivo in zebrafish embryos induced a selective control in GlyT2 expression, regulating GlyT2 transport activity. Our results indicate that activation of Hedgehog reduces GlyT2 expression by increasing its ubiquitination and degradation. This work describes a new molecular link between the Hedgehog signaling pathway and presynaptic glycine availability.

Structural Determinants of the Neuronal Glycine Transporter 2 for the Selective Inhibitors ALX1393 and ORG25543.

The neuronal glycine transporter GlyT2 modulates inhibitory glycinergic neurotransmission by controlling the extracellular concentration of synaptic glycine and the supply of neurotransmitter to the presynaptic terminal. Spinal cord glycinergic neurons present in the dorsal horn diminish their activity in pathological pain conditions and behave as gate keepers of the touch-pain circuitry. The pharmacological blockade of GlyT2 reduces the progression of the painful signal to rostral areas of the central nervous system by increasing glycine extracellular levels, so it has analgesic action. O-[(2-benzyloxyphenyl-3-fluorophenyl)methyl]-l-serine (ALX1393) and N-[[1-(dimethylamino)cyclopentyl]methyl]-3,5-dimethoxy-4-(phenylmethoxy)benzamide (ORG25543) are two selective GlyT2 inhibitors with nanomolar affinity for the transporter and analgesic effects in pain animal models, although with deficiencies which preclude further clinical development. In this report, we performed a comparative ligand docking of ALX1393 and ORG25543 on a validated GlyT2 structural model including all ligand sites constructed by homology with the crystallized dopamine transporter from Drosophila melanogaster. Molecular dynamics simulations and energy analysis of the complex and functional analysis of a series of point mutants permitted to determine the structural determinants of ALX1393 and ORG25543 discrimination by GlyT2. The ligands establish simultaneous contacts with residues present in transmembrane domains 1, 3, 6, and 8 and block the transporter in outward-facing conformation and hence inhibit glycine transport. In addition, differential interactions of ALX1393 with the cation bound at Na1 site and ORG25543 with TM10 define the differential sites of the inhibitors and explain some of their individual features. Structural information about the interactions with GlyT2 may provide useful tools for new drug discovery.

Rescue of two trafficking-defective variants of the neuronal glycine transporter GlyT2 associated to hyperekplexia.

Hyperekplexia is a rare sensorimotor syndrome characterized by pathological startle reflex in response to unexpected trivial stimuli for which there is no specific treatment. Neonates suffer from hypertonia and are at high risk of sudden death due to apnea episodes. Mutations in the human SLC6A5 gene encoding the neuronal glycine transporter GlyT2 may disrupt the inhibitory glycinergic neurotransmission and cause a presynaptic form of the disease. The phenotype of missense mutations giving rise to protein misfolding but maintaining residual activity could be rescued by facilitating folding or intracellular trafficking. In this report, we characterized the trafficking properties of two mutants associated with hyperekplexia (A277T and Y707C, rat numbering). Transporter molecules were partially retained in the endoplasmic reticulum showing increased interaction with the endoplasmic reticulum chaperone calnexin. One transporter variant had export difficulties and increased ubiquitination levels, suggestive of enhanced endoplasmic reticulum-associated degradation. However, the two mutant transporters were amenable to correction by calnexin overexpression. Within the search for compounds capable of rescuing mutant phenotypes, we found that the arachidonic acid derivative N-arachidonoyl glycine can rescue the trafficking defects of the two variants in heterologous cells and rat brain cortical neurons. N-arachidonoyl glycine improves the endoplasmic reticulum output by reducing the interaction transporter/calnexin, increasing membrane expression and improving transport activity in a comparable way as the well-established chemical chaperone 4-phenyl-butyrate. This work identifies N-arachidonoyl glycine as a promising compound with potential for hyperekplexia therapy.

Modulation of Glycinergic Neurotransmission may Contribute to the Analgesic Effects of Propacetamol.

Treating neuropathic pain remains challenging, and therefore new pharmacological strategies are urgently required. Here, the enhancement of glycinergic neurotransmission by either facilitating glycine receptors (GlyR) or inhibiting glycine transporter (GlyT) function to increase extracellular glycine concentration appears promising. Propacetamol is a N,N-diethylester of acetaminophen, a non-opioid analgesic used to treat mild pain conditions. In vivo, it is hydrolysed into N,N-diethylglycine (DEG) and acetaminophen. DEG has structural similarities to known alternative GlyT1 substrates. In this study, we analyzed possible effects of propacetamol, or its metabolite N,N-diethylglycine (DEG), on GlyRs or GlyTs function by using a two-electrode voltage clamp approach in Xenopus laevis oocytes. Our data demonstrate that, although propacetamol or acetaminophen had no effect on the function of the analysed glycine-responsive proteins, the propacetamol metabolite DEG acted as a low-affine substrate for both GlyT1 (EC50 > 7.6 mM) and GlyT2 (EC50 > 5.2 mM). It also acted as a mild positive allosteric modulator of GlyRα1 function at intermediate concentrations. Taken together, our data show that DEG influences both glycine transporter and receptor function, and therefore could facilitate glycinergic neurotransmission in a multimodal manner.

Elevated preoptic brain activity in zebrafish glial glycine transporter mutants is linked to lethargy-like behaviors and delayed emergence from anesthesia.

Delayed emergence from anesthesia was previously reported in a case study of a child with Glycine Encephalopathy. To investigate the neural basis of this delayed emergence, we developed a zebrafish glial glycine transporter (glyt1 - / -) mutant model. We compared locomotor behaviors; dose-response curves for tricaine, ketamine, and 2,6-diisopropylphenol (propofol); time to emergence from these anesthetics; and time to emergence from propofol after craniotomy in glyt1-/- mutants and their siblings. To identify differentially active brain regions in glyt1-/- mutants, we used pERK immunohistochemistry as a proxy for brain-wide neuronal activity. We show that glyt1-/- mutants initiated normal bouts of movement less frequently indicating lethargy-like behaviors. Despite similar anesthesia dose-response curves, glyt1-/- mutants took over twice as long as their siblings to emerge from ketamine or propofol, mimicking findings from the human case study. Reducing glycine levels rescued timely emergence in glyt1-/- mutants, pointing to a causal role for elevated glycine. Brain-wide pERK staining showed elevated activity in hypnotic brain regions in glyt1-/- mutants under baseline conditions and a delay in sensorimotor integration during emergence from anesthesia. Our study links elevated activity in preoptic brain regions and reduced sensorimotor integration to lethargy-like behaviors and delayed emergence from propofol in glyt1-/- mutants.

The allosteric inhibition of glycine transporter 2 by bioactive lipid analgesics is controlled by penetration into a deep lipid cavity.

The role of lipids in modulating membrane protein function is an emerging and rapidly growing area of research. The rational design of lipids that target membrane proteins for the treatment of pathological conditions is a novel extension in this field and provides a step forward in our understanding of membrane transporters. Bioactive lipids show considerable promise as analgesics for the treatment of chronic pain and bind to a high-affinity allosteric-binding site on the human glycine transporter 2 (GlyT2 or SLC6A5). Here, we use a combination of medicinal chemistry, electrophysiology, and computational modeling to develop a rational structure-activity relationship for lipid inhibitors and demonstrate the key role of the lipid tail interactions for GlyT2 inhibition. Specifically, we examine how lipid inhibitor head group stereochemistry, tail length, and double-bond position promote enhanced inhibition. Overall, the l-stereoisomer is generally a better inhibitor than the d-stereoisomer, longer tail length correlates with greater potency, and the position of the double bond influences the activity of the inhibitor. We propose that the binding of the lipid inhibitor deep into the allosteric-binding pocket is critical for inhibition. Furthermore, this provides insight into the mechanism of inhibition of GlyT2 and highlights how lipids can modulate the activity of membrane proteins by binding to cavities between helices. The principles identified in this work have broader implications for the development of a larger class of compounds that could target SLC6 transporters for disease treatment.

SLC6A20 transporter: a novel regulator of brain glycine homeostasis and NMDAR function.

Glycine transporters (GlyT1 and GlyT2) that regulate levels of brain glycine, an inhibitory neurotransmitter with co-agonist activity for NMDA receptors (NMDARs), have been considered to be important targets for the treatment of brain disorders with suppressed NMDAR function such as schizophrenia. However, it remains unclear whether other amino acid transporters expressed in the brain can also regulate brain glycine levels and NMDAR function. Here, we report that SLC6A20A, an amino acid transporter known to transport proline based on in vitro data but is understudied in the brain, regulates proline and glycine levels and NMDAR function in the mouse brain. SLC6A20A transcript and protein levels were abnormally increased in mice carrying a mutant PTEN protein lacking the C terminus through enhanced ß-catenin binding to the Slc6a20a gene. These mice displayed reduced extracellular levels of brain proline and glycine and decreased NMDAR currents. Elevating glycine levels back to normal ranges by antisense oligonucleotide-induced SLC6A20 knockdown, or the competitive GlyT1 antagonist sarcosine, normalized NMDAR currents and repetitive climbing behavior observed in these mice. Conversely, mice lacking SLC6A20A displayed increased extracellular glycine levels and NMDAR currents. Lastly, both mouse and human SLC6A20 proteins mediated proline and glycine transports, and SLC6A20 proteins could be detected in human neurons. These results suggest that SLC6A20 regulates proline and glycine homeostasis in the brain and that SLC6A20 inhibition has therapeutic potential for brain disorders involving NMDAR hypofunction.

GLYT1 encephalopathy: Further delineation of disease phenotype and discussion of pathophysiological mechanisms.

GLYT1 encephalopathy is a form of glycine encephalopathy caused by disturbance of glycine transport. The phenotypic spectrum of the disease has not yet been completely described, as only four unrelated families with the disorder have been reported to date. Common features of affected patients include neonatal hypotonia, respiratory failure, encephalopathy, myoclonic jerks, dysmorphic features, and musculoeskeletal anomalies. All reported affected patients harbor biallelic genetic variants in SLC6A9. SNP array together with Sanger sequencing were performed in a newborn with arthrogryposis and severe neurological impairment. The novel genetic variant c.997delC in SLC6A9 was detected in homozygous state in the patient. At protein level, the predicted change is p.(Arg333Alafs*3), which most probably results in a loss of protein function. The variant cosegregated with the disease in the family. A subsequent pregnancy with ultrasound anomalies was also affected. The proband presented the core phenotypic features of GLYT1 encephalopathy, but also a burst suppression pattern on the electroencephalogram, a clinical feature not previously associated with the disorder. Our results suggest that the appearance of this pattern correlates with higher cerebrospinal fluid glycine levels and cerebrospinal fluid/plasma glycine ratios. A detailed discussion on the possible pathophysiological mechanisms of the disorder is also provided.

A System for Assessing Dual Action Modulators of Glycine Transporters and Glycine Receptors.

Reduced inhibitory glycinergic neurotransmission is implicated in a number of neurological conditions such as neuropathic pain, schizophrenia, epilepsy and hyperekplexia. Restoring glycinergic signalling may be an effective method of treating these pathologies. Glycine transporters (GlyTs) control synaptic and extra-synaptic glycine concentrations and slowing the reuptake of glycine using specific GlyT inhibitors will increase glycine extracellular concentrations and increase glycine receptor (GlyR) activation. Glycinergic neurotransmission can also be improved through positive allosteric modulation (PAM) of GlyRs. Despite efforts to manipulate this synapse, no therapeutics currently target it. We propose that dual action modulators of both GlyTs and GlyRs may show greater therapeutic potential than those targeting individual proteins. To show this, we have characterized a co-expression system in Xenopus laevis oocytes consisting of GlyT1 or GlyT2 co-expressed with GlyRα1. We use two electrode voltage clamp recording techniques to measure the impact of GlyTs on GlyRs and the effects of modulators of these proteins. We show that increases in GlyT density in close proximity to GlyRs diminish receptor currents. Reductions in GlyR mediated currents are not observed when non-transportable GlyR agonists are applied or when Na+ is not available. GlyTs reduce glycine concentrations across different concentration ranges, corresponding with their ion-coupling stoichiometry, and full receptor currents can be restored when GlyTs are blocked with selective inhibitors. We show that partial inhibition of GlyT2 and modest GlyRα1 potentiation using a dual action compound, is as useful in restoring GlyR currents as a full and potent single target GlyT2 inhibitor or single target GlyRα1 PAM. The co-expression system developed in this study will provide a robust means for assessing the likely impact of GlyR PAMs and GlyT inhibitors on glycine neurotransmission.

Identification of "regulation of RhoA activity panel" as a prognostic and predictive biomarker for gastric cancer.

RhoA is a member of the RHO family GTPases and is associated with essential functions in gastric cancer. In this study, we identified a gastric cancer biomarker, termed the "regulation of RhoA activity panel" (RRAP). Patients with gastric cancer from The Cancer Genome Atlas database were divided into training (N=160) and validation (N=155) cohorts. A cohort of 109 Chinese gastric cancer patients was utilized as an independent validation. Patients with mutated RRAP showed significantly better overall survival than patients with wild type RRAP. We also analyzed the association between RRAP and the migration capacity, immune-related signatures, and the tumor microenvironment. RRAP-mutant tumors had a significantly lower degree of lymph node metastasis and lower activities of migration-related pathways. These tumors also showed significantly increased immune cell infiltration and cytotoxic activity. Furthermore, two independent patient cohorts who received immune checkpoint blockade therapy were assessed for RRAP mutant status. As expected, for both immunotherapy cohorts, higher response rates to immune checkpoint blockade therapy were observed in patients with RRAP-mutant tumors than in patients with wild type RRAP tumors. Overall, this study indicates that the RRAP gene set is a potential biomarker for gastric cancer prognosis and therapeutic selection.

Do damaging variants of SLC6A9, the gene for the glycine transporter 1 (GlyT-1), protect against schizophrenia?

The aim of the investigation was to test whether genetic variants predicted to impair the functionality of SLC6A9, which codes for the GlyT-1 glycine transporter, are protective against schizophrenia. In an exome sequenced sample of 4225 schizophrenia cases and 5834 controls, variants occurring in SLC6A9 were annotated and weights were assigned using GENEVARASSOC. Genotype counts were compared using SCOREASSOC. Variants predicted to be deleterious by SIFT and damaging by PolyPhen were examined. Genotypes at 1:44466494-G/A seemed likely to be erroneous. If these were ignored then there were 15 damaging variants in controls and five in cases. The results are consistent with the hypothesis that variants which damage SLC6A9 are protective against schizophrenia but a larger sample would be required to confirm this.