Genetic and transcriptional analysis of inflammatory bowel disease-associated pathways in patients with GUCY2C-linked familial diarrhea.

OBJECTIVE: Activating mutations in the GUCY2C gene, which encodes the epithelial receptor guanylate cyclase C, cause diarrhea due to increased loss of sodium chloride to the intestinal lumen. Patients with familial GUCY2C diarrhea syndrome (FGDS) are predisposed to inflammatory bowel disease (IBD). We investigated whether genes in the guanylate cyclase C pathway are enriched for association with IBD and reversely whether genetic or transcriptional changes associated with IBD are found in FGDS patients. METHODS: (1) A set of 27 genes from the guanylate cyclase C pathway was tested for enrichment of association with IBD by Gene Set Enrichment Analysis, using genome-wide association summary statistics from 12,882 IBD patients and 21,770 controls. (2) We genotyped 163 known IBD risk loci and sequenced NOD2 in 22 patients with FGDS. Eight of them had concomitant Crohn's disease. (3) Global gene expression analysis was performed in ileal tissue from patients with FGDS, Crohn's disease and healthy individuals. RESULTS: The guanylate cyclase C gene set showed a significant enrichment of association in IBD genome-wide association data. Risk variants in NOD2 were found in 7/8 FGDS patients with concomitant Crohn's disease and in 2/14 FDGS patients without Crohn's disease. In ileal tissue, downregulation of metallothioneins characterized FGDS patients compared to healthy controls. CONCLUSIONS: Our results support a role of guanylate cyclase C signaling and disturbed electrolyte homeostasis in development of IBD. Furthermore, downregulation of metallothioneins in the ileal mucosa of FGDS patients may contribute to IBD development, possibly alongside effects from NOD2 risk variants.

LOC689986, a unique gene showing specific expression in restricted areas of the rodent neocortex.

BACKGROUND: The neocortex is a highly specialised and complex brain structure, involved in numerous tasks, ranging from processing and interpretation of somatosensory information, to control of motor functions. The normal function linked to distinct neocortical areas might involve control of highly specific gene expression, and in order to identify such regionally enriched genes, we previously analysed the global gene expression in three different cortical regions (frontomedial, temporal and occipital cortex) from the adult rat brain. We identified distinct sets of differentially expressed genes. One of these genes, namely the hypothetical protein LOC689986 (LOC689986), was of particular interest, due to an almost exclusive expression in the temporal cortex. RESULTS: Detailed analysis of LOC689986 in the adult rat brain confirmed the expression in confined areas of parieto-temporal cortex, and revealed highly specific expression in layer 4 of the somatosensory cortex, with sharp borders towards the neighbouring motor cortex. In addition, LOC689986 was found to be translated in vivo, and was detected in the somatosensory cortex and in the Purkinje cells of the cerebellar cortex. The protein was present in neuronal dendrites and also in astrocyte cells. Finally, this unique gene is apparently specific for, and highly conserved in, the vertebrate lineage. CONCLUSIONS: In this study, we have partially characterised the highly conserved LOC689986 gene, which is specific to the vertebrate linage. The gene displays a distinct pattern of expression in layer 4 of the somatosensory cortex, and areas of the parieto-temporal cortex in rodents.

Patients with schizophrenia and bipolar disorder display a similar global gene expression signature in whole blood that reflects elevated proportion of immature neutrophil cells with association to lipid changes.

Schizophrenia (SCZ) and bipolar disorder (BD) share clinical characteristics, genetic susceptibility, and immune alterations. We aimed to identify differential transcriptional patterns in peripheral blood cells of patients with SCZ or BD versus healthy controls (HC). We analyzed microarray-based global gene expression data in whole blood from a cohort of SCZ (N = 329), BD (N = 203) and HC (N = 189). In total, 65 genes were significantly differentially expressed in SCZ and 125 in BD, as compared to HC, with similar ratio of up- and downregulated genes in both disorders. Among the top differentially expressed genes, we found an innate immunity signature that was shared between SCZ and BD, consisting of a cluster of upregulated genes (e.g., OLFM4, ELANE, BPI and MPO) that indicate an increased fraction of immature neutrophils. Several of these genes displayed sex differences in the expression pattern, and post-hoc analysis demonstrated a positive correlation with triglyceride and a negative correlation with HDL cholesterol. We found that many of the downregulated genes in SCZ and BD were associated with smoking. These findings of neutrophil granulocyte-associated transcriptome signatures in both SCZ and BD point at altered innate immunity pathways with association to lipid changes and potential for clinical translation.

Gene expression in the rat brain: high similarity but unique differences between frontomedial-, temporal- and occipital cortex.

BACKGROUND: The six-layered neocortex of the mammalian brain may appear largely homologous, but is in reality a modular structure of anatomically and functionally distinct areas. However, global gene expression seems to be almost identical across the cerebral cortex and only a few genes have so far been reported to show regional enrichment in specific cortical areas. RESULTS: In the present study on adult rat brain, we have corroborated the strikingly similar gene expression among cortical areas. However, differential expression analysis has allowed for the identification of 30, 24 and 11 genes enriched in frontomedial -, temporal- or occipital cortex, respectively. A large proportion of these 65 genes appear to be involved in signal transduction, including the ion channel Fxyd6, the neuropeptide Grp and the nuclear receptor Rorb. We also find that the majority of these genes display increased expression levels around birth and show distinct preferences for certain cortical layers and cell types in rodents. CONCLUSIONS: Since specific patterns of expression often are linked to equally specialised biological functions, we propose that these cortex sub-region enriched genes are important for proper functioning of the cortical regions in question.

Gene expression profiles in rat brain disclose CNS signature genes and regional patterns of functional specialisation.

BACKGROUND: The mammalian brain is divided into distinct regions with structural and neurophysiological differences. As a result, gene expression is likely to vary between regions in relation to their cellular composition and neuronal function. In order to improve our knowledge and understanding of regional patterns of gene expression in the CNS, we have generated a global map of gene expression in selected regions of the adult rat brain (frontomedial-, temporal- and occipital cortex, hippocampus, striatum and cerebellum; both right and left sides) as well as in three major non-neural tissues (spleen, liver and kidney) using the Applied Biosystems Rat Genome Survey Microarray. RESULTS: By unsupervised hierarchical clustering, we found that the transcriptome within a region was highly conserved among individual rats and that there were no systematic differences between the two hemispheres (right versus left side). Further, we identified distinct sets of genes showing significant regional enrichment. Functional annotation of each of these gene sets clearly reflected several important physiological features of the region in question, including synaptic transmission within the cortex, neurogenesis in hippocampus and G-protein-mediated signalling in striatum. In addition, we were able to reveal potentially new regional features, such as mRNA transcription- and neurogenesis-annotated activities in cerebellum and differential use of glutamate signalling between regions. Finally, we determined a set of 'CNS-signature' genes that uncover characteristics of several common neuronal processes in the CNS, with marked over-representation of specific features of synaptic transmission, ion transport and cell communication, as well as numerous novel unclassified genes. CONCLUSION: We have generated a global map of gene expression in the rat brain and used this to determine functional processes and pathways that have a regional preference or ubiquitous distribution within the CNS, respectively. The existence of shared specialised neuronal activities in CNS is interesting in a context of potential functional redundancy, and future studies should further explore the overall characteristics of CNS-specific versus region-specific gene profiles in the brain.

Hyperoxia retards growth and induces apoptosis and loss of glands and blood vessels in DMBA-induced rat mammary tumors.

BACKGROUND: This study investigated the effects of hyperoxic treatment on growth, angiogenesis, apoptosis, general morphology and gene expression in DMBA-induced rat mammary tumors. METHODS: One group of animals was exposed to normobaric hyperoxia (1 bar, pO2 = 1.0 bar) and another group was exposed to hyperbaric hyperoxia (1.5 bar, pO2 = 1.5 bar). A third group was treated with the commonly used chemotherapeutic drug 5- Fluorouracil (5-FU), whereas animals housed under normal atmosphere (1 bar, pO2 = 0.2 bar) served as controls. All treatments were performed on day 1, 4, 7 and 10 for 90 min. Tumor growth was calculated from caliper measurements. Biological effects of the treatment, was determined by assessment of vascular morphology (immunostaining for von Willebrandt factor) and apoptosis (TUNEL staining). Detailed gene expression profiles were obtained and verified by quantitative rtPCR. RESULTS: Tumor growth was significantly reduced (~57-66 %) after hyperoxic treatment compared to control and even more than 5-FU (~36 %). Light microscopic observations of the tumor tissue showed large empty spaces within the tissue after hyperoxic treatment, probably due to loss of glands as indicated by a strong down-regulation of glandular secretory proteins. A significant reduction in mean vascular density (30-50%) was found after hyperoxic treatment. Furthermore, increased apoptosis (18-21%) was found after hyperoxic treatment. CONCLUSION: Thus, by increasing the pO2 in mammary tumor tissue using normobaric and moderate hyperbaric oxygen therapy, a significant retardation in tumor growth is achieved, by loss of glands, reduction in vascular density and enhanced cell death. Hyperbaric oxygen should therefore be further evaluated as a tumor treatment.

Subchronic olanzapine exposure leads to increased expression of myelination-related genes in rat fronto-medial cortex.

Schizophrenia is a psychotic disorder with severe and disabling symptoms, such as hallucinations, delusions, blunted affect and social withdrawal. The neuropathology remains elusive, but disturbances in immunity-related processes, neuronal connectivity and myelination have consistently been linked to schizophrenia. Antipsychotic drugs can be efficient in reducing symptoms, acting primarily on the dopamine system, but additional biological targets are likely to exist. Here we have screened for novel mechanisms of action in an animal model, using adult rats exposed to long-acting olanzapine, achieving stable and clinically relevant antipsychotic drug concentrations. By microarray-based examination of global gene expression in the fronto-medial cortex, at the single gene- and gene-set level, we observed downregulation of two neuropeptide-encoding genes, Vgf and Cort (fold change -1,25 and -1,48, respectively) in response to olanzapine exposure. Furthermore, we demonstrated significant upregulation of five out of ~2000 GO predefined gene sets after olanzapine exposure. Strikingly, all were linked to myelination and oligodendrocyte development; "Ensheathment of neurons", "Axon ensheathment", "Myelination", "Myelin sheath" and "Oligodendrocyte development" (FDR-values < 25). Sixteen of the leading edge genes in these gene sets were analysed independently by qPCR, of which 11 genes displayed significant upregulation, including Plp1, Mal, Mag and Cnp (fold change: 1,30, 1,50, 1,30 and 1,15, respectively). Several of the upregulated genes (e.g. MAG, MAL and CNP) have previously been reported as downregulated in post-mortem brain samples from schizophrenia patients. Although caution needs to be taken when extrapolating results from animal studies to humans, the data suggest a role for olanzapine in alleviating myelination-related dysfunction in schizophrenia.

Antidepressant drugs activate SREBP and up-regulate cholesterol and fatty acid biosynthesis in human glial cells.

Dysfunction of glial lipid metabolism and abnormal myelination has recently been reported in both schizophrenia and bipolar disorder. Cholesterol is a major component of myelin, and glia-produced cholesterol serves as a glial growth factor in synaptogenesis. We have recently demonstrated that antipsychotic drugs activate the sterol regulatory element-binding protein (SREBP) transcription factors in human and rat glial cells, with subsequent up-regulation of numerous downstream genes involved in cholesterol and fatty acid biosynthesis. Since this stimulation of cellular lipogenesis could represent a new mechanism of action of psychotropic drugs, we investigated whether antidepressants and mood-stabilizers were able to induce a similar activation of SREBP-controlled lipid biosynthesis. Cultured human glioma cells (GaMg) were exposed to the antidepressant drugs imipramine, amitriptyline, clomipramine, citalopram, fluoxetine, mirtazapine and bupropion and the mood-stabilizers/antiepileptics lithium, valproate and carbamazepine. All antidepressant drugs activated the SREBP system with subsequent up-regulation of the downstream lipogenesis-related genes, although to a markedly different extent. The mood-stabilizers did not affect the SREBPs or the downstream genes. These results link antidepressant drugs, but not mood-stabilizers, to SREBP-mediated activation of cellular lipogenesis, and demonstrate a functional similarity between antipsychotic and antidepressant molecular drug action.

Syndromic X-linked intellectual disability segregating with a missense variant in RLIM.

We describe a three-generation Norwegian family with a novel X-linked intellectual disability (XLID) syndrome characterized by subtle facial dysmorphism, autism and severe feeding problems. By exome sequencing we detected a rare missense variant (c.1067A>G, p.(Tyr356Cys)) in the RLIM gene, in two affected male second cousins. Sanger sequencing confirmed the presence of the variant in the four affected males (none of whom were siblings) and in three mothers available for testing. The variant was not present in 100 normal Norwegian controls, has not been reported in variant databases and is deleterious according to in silico prediction tools. The clinical phenotype and the variant co-segregate, yielding a LOD score of 3.0 for linkage to the shared region (36.09 Mb), which contains 242 genes. No other shared rare variants on the X chromosome were detected in the two affected exome-sequenced individuals, and all female carriers had an extremely skewed X-chromosome inactivation pattern. RLIM encodes RING zinc finger protein 12 (RNF12), an ubiquitin ligase that is essential for X inactivation in mice and that acts as a co-regulator of a range of transcription factors, particularly those containing a LIM homeodomain. Tyrosine in position 356 in RNF12 is located within a highly conserved domain essential for binding such transcription factors. Expression of RNF12 is widespread during embryogenesis, and is particularly high in the outer layers of the cerebral cortex. Functional studies are needed to prove a definite causal relationship between the variant and the phenotype. Subsequent reports may confirm a role for RLIM variants in patients with XLID.

The interleukin 1 receptor family.

Interleukin-1 is a key inflammatory cytokine that mediates its effects through a type I receptor and a receptor accessory protein. These two molecules are members of a wider family of proteins that have in common the presence of immunoglobulin domains in the extracellular region of the protein and a TIR domain in the cytoplasmic region. The nature of this family of proteins and their signal transduction pathway is discussed in this review.

Cloning of a Salmo salar interleukin-1 receptor-like cDNA.

The interleukin-1 receptor/toll-like receptor (IL-1R/TLR) superfamily, defined by a cytosolic Toll/IL-1R (TIR) signalling domain, participates in host responses to injury and infection. We describe in this study the cloning of a cDNA encoding a Salmo salar interleukin-1 receptor-like protein (SalIL-1RLP). SalIL-1RLP comprises a potential signal peptide, three extracellular immunoglobulin domains, a short transmembrane region and an intracellular region that contains the TIR domain. The predicted amino acid sequence of SalIL-1RLP displays 43-44% similarities and 31% identities to chicken and human IL-1RI sequences. Within the intracellular region, SalIL-1RLP displays highest similarity (59%) and identity (46%) to the chicken IL-1RI sequence. Two different 5' distal UTRs were identified among six salmon IL-1RLP clones. The six clones, however, displayed identical 5' proximal UTRs, coding regions and 3' UTRs. SalIL-1RLP expression is induced in liver, head kidney, spleen and gills upon injection of salmon with bacterial lipopolysaccharide. Sequence comparisons, protein domain structures, expression patterns and phylogenetic analyses indicate that SalIL-1RLP is most closely related to type I interleukin-1 receptors and interleukin-1 receptor related proteins.

Peli1 promotes microglia-mediated CNS inflammation by regulating Traf3 degradation.

Microglia are crucial for the pathogenesis of multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE). Here we show that the E3 ubiquitin ligase Peli1 is abundantly expressed in microglia and promotes microglial activation during the course of EAE induction. Peli1 mediates the induction of chemokines and proinflammatory cytokines in microglia and thereby promotes recruitment of T cells into the central nervous system. The severity of EAE is reduced in Peli1-deficient mice despite their competent induction of inflammatory T cells in the peripheral lymphoid organs. Notably, Peli1 regulates Toll-like receptor (TLR) pathway signaling by promoting degradation of TNF receptor-associated factor 3 (Traf3), a potent inhibitor of mitogen-activated protein kinase (MAPK) activation and gene induction. Ablation of Traf3 restores microglial activation and CNS inflammation after the induction of EAE in Peli1-deficient mice. These findings establish Peli1 as a microglia-specific mediator of autoimmune neuroinflammation and suggest a previously unknown signaling mechanism of Peli1 function.

Gene-based analysis of regionally enriched cortical genes in GWAS data sets of cognitive traits and psychiatric disorders.

BACKGROUND: Despite its estimated high heritability, the genetic architecture leading to differences in cognitive performance remains poorly understood. Different cortical regions play important roles in normal cognitive functioning and impairment. Recently, we reported on sets of regionally enriched genes in three different cortical areas (frontomedial, temporal and occipital cortices) of the adult rat brain. It has been suggested that genes preferentially, or specifically, expressed in one region or organ reflect functional specialisation. Employing a gene-based approach to the analysis, we used the regionally enriched cortical genes to mine a genome-wide association study (GWAS) of the Norwegian Cognitive NeuroGenetics (NCNG) sample of healthy adults for association to nine psychometric tests measures. In addition, we explored GWAS data sets for the serious psychiatric disorders schizophrenia (SCZ) (n = 3 samples) and bipolar affective disorder (BP) (n = 3 samples), to which cognitive impairment is linked. PRINCIPAL FINDINGS: At the single gene level, the temporal cortex enriched gene RAR-related orphan receptor B (RORB) showed the strongest overall association, namely to a test of verbal intelligence (Vocabulary, P = 7.7E-04). We also applied gene set enrichment analysis (GSEA) to test the candidate genes, as gene sets, for enrichment of association signal in the NCNG GWAS and in GWASs of BP and of SCZ. We found that genes differentially expressed in the temporal cortex showed a significant enrichment of association signal in a test measure of non-verbal intelligence (Reasoning) in the NCNG sample. CONCLUSION: Our gene-based approach suggests that RORB could be involved in verbal intelligence differences, while the genes enriched in the temporal cortex might be important to intellectual functions as measured by a test of reasoning in the healthy population. These findings warrant further replication in independent samples on cognitive traits.

Upregulation of immunoglobulin-related genes in cortical sections from multiple sclerosis patients.

Multiple sclerosis (MS) is a demyelinating disease of the central nervous system (CNS). Microarray-based global gene expression profiling is a promising method, used to study potential genes involved in the pathogenesis of the disease. In the present study, we have examined global gene expression in normal-appearing gray matter and gray matter lesions from the cortex of MS patients, and compared them with cortical gray matter samples from controls. We observed a massive upregulation of immunoglobulin (Ig)-related genes in cortical sections of MS patients. Using immunohistochemistry, the activation of Ig genes seems to occur within plasma cells in the meninges. As synthesis of oligoclonal IgGs has been hypothesized to be caused by the activation of Epstein-Barr virus (EBV)-infected B-cells, we screened the brain samples for the presence of EBV by real-time quantitative polymerase chain reaction (qPCR) and immunohistochemistry, but no evidence of active or latent EBV infection was detected. This study demonstrates that genes involved in the synthesis of Igs are upregulated in MS patients and that this activation is caused by a small number of meningeal plasma cells that are not infected by EBV. The findings indicate that the Ig-producing B-cells found in the cerebrospinal fluid (CSF) of MS patients could have meningeal origin.

Switch from stress response to homeobox transcription factors in adipose tissue after profound fat loss.

BACKGROUND: In obesity, impaired adipose tissue function may promote secondary disease through ectopic lipid accumulation and excess release of adipokines, resulting in systemic low-grade inflammation, insulin resistance and organ dysfunction. However, several of the genes regulating adipose tissue function in obesity are yet to be identified. METHODOLOGY/PRINCIPAL FINDINGS: In order to identify novel candidate genes that may regulate adipose tissue function, we analyzed global gene expression in abdominal subcutaneous adipose tissue before and one year after bariatric surgery (biliopancreatic diversion with duodenal switch, BPD/DS) (n = 16). Adipose tissue from lean healthy individuals was also analyzed (n = 13). Two different microarray platforms (AB 1700 and Illumina) were used to measure the differential gene expression, and the results were further validated by qPCR. Surgery reduced BMI from 53.3 to 33.1 kg/m(2). The majority of differentially expressed genes were down-regulated after profound fat loss, including transcription factors involved in stress response, inflammation, and immune cell function (e.g., FOS, JUN, ETS, C/EBPB, C/EBPD). Interestingly, a distinct set of genes was up-regulated after fat loss, including homeobox transcription factors (IRX3, IRX5, HOXA5, HOXA9, HOXB5, HOXC6, EMX2, PRRX1) and extracellular matrix structural proteins (COL1A1, COL1A2, COL3A1, COL5A1, COL6A3). CONCLUSIONS/SIGNIFICANCE: The data demonstrate a marked switch of transcription factors in adipose tissue after profound fat loss, providing new molecular insight into a dichotomy between stress response and metabolically favorable tissue development. Our findings implicate homeobox transcription factors as important regulators of adipose tissue function.

An 8.9 Mb 19p13 duplication associated with precocious puberty and a sporadic 3.9 Mb 2q23.3q24.1 deletion containing NR4A2 in mentally retarded members of a family with an intrachromosomal 19p-into-19q between-arm insertion.

In a 2 and a half-year-old girl with onset of puberty before the age of 5 months, short stature, hand anomalies and severe mental retardation, an 8.9 Mb interstitial 19p13 duplication containing 215 predicted genes was detected. It was initially assumed that the duplication involved the kisspeptin receptor gene, GPR54, known to stimulate induction of puberty, but more refined duplication mapping excluded this possibility. In an attempt to further understand the genotype-phenotype correlation, global gene expression was measured in skin fibroblasts. The overall expression pattern was quite similar to controls, and only about 25% of the duplicated genes had an expression level that was increased by more than 1.3-fold, with no obvious changes that could explain the precocious puberty. The proband's mother carried a balanced between-arm insertion of the duplicated segment that resembled a pericentric inversion. The same insertion was found in several other family members, including one who had lost a daughter with severe mental retardation and menarche at the age of 10 years. Another close relative was severely mentally retarded, but neither dysmorphic nor microcephalic. His phenotype was initially ascribed to a presumed cryptic chromosome 19 imbalance caused by the 19p-into19q insertion, but subsequent array-CGH detected a 3.9-Mb deletion of 2q23.3q24.1. This novel microdeletion involves seven genes, of which FMNL2, a suggested regulator of Rho-GTPases, and NR4A2, an essential gene for differentiation of dopaminergic neurons, may be critical genes for the proposed 2q23q24 microdeletion syndrome.

Cloning of the Atlantic salmon (Salmo salar) IL-1 receptor associated protein.

In mammals, the pro-inflammatory cytokine interleukin-1 signals through a receptor complex containing a type I interleukin-1 receptor (IL-1RI) and a receptor associated protein (IL-1RAcP). Previously, we have described a cDNA from Atlantic salmon encoding a molecule with homology to the mammalian IL-RI. This molecule was named IL-1 receptor like protein (IL-1RLP) in the absence of functional data to support its proposed role as the salmon IL-1RI. Here, we describe the cloning and characterisation of a cDNA encoding salmon IL-1RAcP. Like other members of the IL-1R family, the salmon IL-1RAcP encodes three extracellular immunoglobulin-like domains and a cytoplasmic Toll/Interleukin-1 receptor (TIR) domain involved in signalling. Specific binding of salmon IL-1RAcP to IL-1RLP was shown by co-immunoprecipitation studies.

Cloning and characterisation of a putative ST2L homologue from Atlantic salmon (Salmo salar).

The ST2L receptor is a member of the interleukin-1 (IL-1) receptor family and has previously been cloned from human, mouse, rat and chicken. This orphan receptor has no known physiological role but has been implicated in T helper cell type 2 effector function. We describe in this report the cloning and characterisation of a cDNA encoding a homologue of ST2L in Atlantic salmon (Salmo salar). The salmon ST2L cDNA is 2364bp in length and has an open reading frame encoding a polypeptide of 582 amino acids. Similar to other members of the IL-1 receptor (IL-1R) family, the predicted protein has a potential signal peptide, extracellular immunoglobulin-like domains, a short transmembrane region and a characteristic cytoplasmic Toll-IL-1R domain. The predicted protein shows 33% identity and 44% similarity to the chicken ST2L homologue. Phylogenetic analyses cluster the putative salmon ST2L with the chicken and the mammalian ST2L homologues, away from the other members of the IL-1R family. Salmon ST2L is constitutively expressed in brain, white and red blood cells, head kidney, liver, gills and muscle, with highest level of expression in spleen. In vivo stimulation of salmon with lipopolysaccaride does not appear to have a significant effect on expression of the ST2L homologue.