Limited Evidence for Parent-of-Origin Effects in Inflammatory Bowel Disease Associated Loci

Background

Genome-wide association studies of two main forms of inflammatory bowel diseases (IBD), Crohn’s disease (CD) and ulcerative colitis (UC), have identified 99 susceptibility loci, but these explain only ∼23% of the genetic risk. Part of the ‘hidden heritability’ could be in transmissible genetic effects in which mRNA expression in the offspring depends on the parental origin of the allele (genomic imprinting), since children whose mothers have CD are more often affected than children with affected fathers. We analyzed parent-of-origin (POO) effects in Dutch and Indian cohorts of IBD patients.

Methods

We selected 28 genetic loci associated with both CD and UC, and tested them for POO effects in 181 Dutch IBD case-parent trios. Three susceptibility variants in NOD2 were tested in 111 CD trios and a significant finding was re-evaluated in 598 German trios. The UC-associated gene, BTNL2, reportedly imprinted, was tested in 70 Dutch UC trios. Finally, we used 62 independent Indian UC trios to test POO effects of five established Indian UC risk loci.

Results

We identified POO effects for NOD2 (L1007fs; OR = 21.0, P-value = 0.013) for CD; these results could not be replicated in an independent cohort (OR = 0.97, P-value = 0.95). A POO effect in IBD was observed for IL12B (OR = 3.2, P-value = 0.019) and PRDM1 (OR = 5.6, P-value = 0.04). In the Indian trios the IL10 locus showed a POO effect (OR = 0.2, P-value = 0.03).

Conclusions

Little is known about the effect of genomic imprinting in complex diseases such as IBD. We present limited evidence for POO effects for the tested IBD loci. POO effects explain part of the hidden heritability for complex genetic diseases but need to be investigated further.     

Exon-skipping Splice Variants of Excitatory Amino Acid Transporter-2 (EAAT2) Form Heteromeric Complexes with Full-length EAAT2

The glial transporter excitatory amino acid transporter-2 (EAAT2) is the main mediator of glutamate clearance in brain. The wild-type transporter (EAAT2wt) forms trimeric membrane complexes in which each protomer functions autonomously. Several EAAT2 variants are found in control and Alzheimer-diseased human brains; their expression increases with pathological severity. These variants might alter EAAT2wt-mediated transport by abrogating membrane trafficking, or by changing the configuration or functionality of the assembled transporter complex. HEK293 cells were transfected with EAAT2wt; EAAT2b, a C-terminal variant; or either of two exon-skipping variants: alone or in combination. Surface biotinylation studies showed that only the exon-7 deletion variant was not trafficked to the membrane when transfected alone, and that all variants could reach the membrane when co-transfected with EAAT2wt. Fluorescence resonance energy transfer (FRET) studies showed that co-transfected EAAT2wt and EAAT2 splice variants were expressed in close proximity. Glutamate transporter function was measured using a whole cell patch clamp technique, or by changes in membrane potential indexed by a voltage-sensitive fluorescent dye (FMP assay): the two methods gave comparable results. Cells transfected with EAAT2wt or EAAT2b showed glutamate-dependent membrane potential changes consistent with functional expression. Cells transfected with EAAT2 exon-skipping variants alone gave no response to glutamate. Co-transfection of EAAT2wt (or EAAT2b) and splice variants in various ratios significantly raised glutamate EC₅₀ and decreased Hill coefficients. We conclude that exon-skipping variants form heteromeric complexes with EAAT2wt or EAAT2b that traffic to the membrane but show reduced glutamate-dependent activity. This could allow glutamate to accumulate extracellularly and promote excitotoxicity.     

Virus progeny of murine cytomegalovirus bacterial artificial chromosome pSM3fr show reduced growth in salivary Glands due to a fixed mutation of MCK-2

Murine cytomegalovirus (MCMV) Smith strain has been cloned as a bacterial artificial chromosome (BAC) named pSM3fr and used for analysis of virus gene functions in vitro and in vivo. When sequencing the complete BAC genome, we identified a frameshift mutation within the open reading frame (ORF) encoding MCMV chemokine homologue MCK-2. This mutation would result in a truncated MCK-2 protein. When mice were infected with pSM3fr-derived virus, we observed reduced virus production in salivary glands, which could be reverted by repair of the frameshift mutation. When looking for the source of the mutation, we consistently found that virus stocks of cell culture-passaged MCMV Smith strain are mixtures of viruses with or without the MCK-2 mutation. We conclude that the MCK-2 mutation in the pSM3fr BAC is the result of clonal selection during the BAC cloning procedure.     

Limitation of lowland riverine bacterioplankton by dissolved organic carbon and inorganic nutrients

Flow regulation in lowland rivers has reduced the amount of allochthonous dissolved organic carbon (DOC) entering main channels through less frequent wetting of benches, flood runners and floodplains. The hypothesis tested was that lowland riverine bacterioplankton are DOC limited when flow events are absent and simulating an increase in assimilable DOC similar to that expected during an environmental flow will lead to heterotrophic dominance. Experiments took place in the Namoi River, a highly regulated lowland river in Australia. Specifically, in situ microcosms were used to examine the responses of bacterioplankton and phytoplankton to various additions of DOC as glucose or leaf leachate, with and without additions of inorganic nutrients. The results indicated that ambient DOC availability limited the bacterioplankton for the three seasons over which we conducted the experiments. When DOC was added alone, dissolved oxygen concentrations decreased primarily because of increased bacterial respiration and bacterioplankton growth generally increased relative to controls. Additions of DOC alone led to a pattern of decreased chlorophyll a concentration relative to controls, except for willow leachate. Additions of inorganic nutrients alone increased chlorophyll a concentrations above controls, indicating limitation of phytoplankton. These findings support our hypothesis. Based on the present results, environmental flows should increase the duration of allochthonously driven heterotrophic dominance, thus shifting regulated lowland rivers to more natural (pre-regulation) conditions for greater periods.     

The chloride permeation pathway of a glutamate transporter and its proximity to the glutamate translocation pathway

Excitatory amino acid transporters (EAATs) regulate glutamate concentrations in the brain to maintain normal excitatory synaptic transmission. A widely accepted view of transporters is that they consist of a pore with alternating access to the intracellular and extracellular solutions, which serves to couple ion movement to the movement of substrate. However, recent observations that EAATs, and also a number of other neurotransmitter transporters, can also function as ligand-gated chloride channels have blurred the distinctions between transporters and ion channels. Here we show that mutations in the second transmembrane domain (TM2) of EAAT1 alter anion permeation properties without affecting glutamate transport and that a number of TM2 residues are accessible to the external aqueous solution. Furthermore, we demonstrate that the extracellular edge of TM2 is in close proximity to a membrane-associated domain that influences glutamate transport. This study will provide the foundation for beginning to understand how transporters can function as both transporters and ion channels.     

Reduced connectivity in the self-processing network of schizophrenia patients with poor insight

Lack of insight (unawareness of illness) is a common and clinically relevant feature of schizophrenia. Reduced levels of self-referential processing have been proposed as a mechanism underlying poor insight. The default mode network (DMN) has been implicated as a key node in the circuit for self-referential processing. We hypothesized that during resting state the DMN network would show decreased connectivity in schizophrenia patients with poor insight compared to patients with good insight. Patients with schizophrenia were recruited from mental health care centers in the north of the Netherlands and categorized in groups having good insight (n = 25) or poor insight (n = 19). All subjects underwent a resting state fMRI scan. A healthy control group (n = 30) was used as a reference. Functional connectivity of the anterior and posterior part of the DMN, identified using Independent Component Analysis, was compared between groups. Patients with poor insight showed lower connectivity of the ACC within the anterior DMN component and precuneus within the posterior DMN component compared to patients with good insight. Connectivity between the anterior and posterior part of the DMN was lower in patients than controls, and qualitatively different between the good and poor insight patient groups. As predicted, subjects with poor insight in psychosis showed decreased connectivity in DMN regions implicated in self-referential processing, although this concerned only part of the network. This finding is compatible with theories implying a role of reduced self-referential processing as a mechanism contributing to poor insight.     

Comparative study of 137Cs distribution in broilers and pheasants and possibilities for protection

The aim of the present study was to investigate distribution of (137)Cs in leg and breast meat of broilers and pheasants following single alimentary contamination and administration of two protectors (AFCF and clinoptilolite). The birds were administered a single dose of (137)CsCl, with an activity of 750?Bq. Protectors were given via gastric tube or mixed in the forage pellets. AFCF given via gastric tube decreased the (137)Cs concentration by a factor of 7.8 in broilers leg meat and 7.4 in broilers breast meat. When AFCF was mixed in pellets, the (137)Cs concentration was 19.5 times lower in broilers leg meat and 22.1 times lower in broilers breast meat, than in the control group. In pheasants, AFCF administered via gastric tube decreased the (137)Cs concentration by a factor of 12.4 in leg meat and by a factor of 13.7 in breast meat, respectively. In group 4, where pheasants were administered AFCF mixed in pellets, the (137)Cs concentration was 3.7 times lower in leg and breast meat, than in the control group. For comparison, clinoptilolite administered via gastric tube decreased the (137)Cs concentration 1.8 times in broilers leg meat and 2.0 times in breast meat, compared to the control group. In pheasants, (137)Cs concentration was 2.9 times lower in leg meat and 2.6 times lower in breast meat. Clinoptilolite mixed in the feed had relatively low efficiency of protection in broilers ((137)Cs concentration was 1.4 times lower in leg meat and 1.6 lower in breast meat). A similar trend was observed in pheasants ((137)Cs concentration was 1.6 lower in leg and breast meat).     

Effects of Tyrosine Kinase Inhibitors and CXCR4 Antagonist on Tumor Growth and Angiogenesis in Rat Glioma Model: MRI and Protein Analysis Study

The aim of the study was to determine the antiangiogenic efficacy of vatalanib, sunitinib, and AMD3100 in an animal model of human glioblastoma (GBM) by using dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) and tumor protein expression analysis. Orthotopic GBM-bearing animals were randomly assigned either to control group or vatalanib, sunitinib, and AMD3100 treatment groups. Following 2 weeks of drug treatment, tumor growth and vascular parameters were measured using DCE-MRI. Expression of different angiogenic factors in tumor extracts was measured using a membrane-based human antibody array kit. Tumor angiogenesis and invasion were determined by immunohistochemistry. DCE-MRI showed a significant increase in tumor size after vatalanib treatment. AMD3100-treated group showed a significant decrease in a number of vascular parameters determined by DCE-MRI. AMD3100 significantly decreased the expression of different angiogenic factors compared to sunitinib or vatalanib; however, there were no significant changes in vascular density among the groups. Sunitinib-treated animals showed significantly higher migration of the invasive cells, whereas in both vatalanib- and AMD3100-treated animals the invasive cell migration distance was significantly lower compared to that of control. Vatalanib and sunitinib resulted in suboptimal therapeutic effect, but AMD3100 treatment resulted in a significant reduction in tumor growth, permeability, interstitial space volume, and invasion of tumor cells in an animal model of GBM.