Exon array data analysis using Affymetrix power tools and R statistical software

The use of microarray technology to measure gene expression on a genome-wide scale has been well established for more than a decade. Methods to process and analyse the vast quantity of expression data generated by a typical microarray experiment are similarly well-established. The Affymetrix Exon 1.0 ST array is a relatively new type of array, which has the capability to assess expression at the individual exon level. This allows a more comprehensive analysis of the transcriptome, and in particular enables the study of alternative splicing, a gene regulation mechanism important in both normal conditions and in diseases. Some aspects of exon array data analysis are shared with those for standard gene expression data but others present new challenges that have required development of novel tools. Here, I will introduce the exon array and present a detailed example tutorial for analysis of data generated using this platform

Variability of gene expression profiles in human blood and lymphoblastoid cell lines

BACKGROUND: Readily accessible samples such as peripheral blood or cell lines are increasingly being used in large cohorts to characterise gene expression differences between a patient group and healthy controls. However, cell and RNA isolation procedures and the variety of cell types that make up whole blood can affect gene expression measurements. We therefore systematically investigated global gene expression profiles in peripheral blood from six individuals collected during two visits by comparing five of the following cell and RNA isolation methods: whole blood (PAXgene), peripheral blood mononuclear cells (PBMCs), lymphoblastoid cell lines (LCLs), CD19 and CD20 specific B-cell subsets. RESULTS: Gene expression measurements were clearly discriminated by isolation method although the reproducibility was high for all methods (range rho = 0.90-1.00). The PAXgene samples showed a decrease in the number of expressed genes (P < 1*10(-16)) with higher variability (P < 1*10(-16)) compared to the other methods. Differentially expressed probes between PAXgene and PBMCs were correlated with the number of monocytes, lymphocytes, neutrophils or erythrocytes. The correlations (rho = 0.83; rho = 0.79) of the expression levels of detected probes between LCLs and B-cell subsets were much lower compared to the two B-cell isolation methods (rho = 0.98). Gene ontology analysis of detected genes showed that genes involved in inflammatory responses are enriched in B-cells CD19 and CD20 whereas genes involved in alcohol metabolic process and the cell cycle were enriched in LCLs. CONCLUSION: Gene expression profiles in blood-based samples are strongly dependent on the predominant constituent cell type(s) and RNA isolation method. It is crucial to understand the differences and variability of gene expression measurements between cell and RNA isolation procedures, and their relevance to disease processes, before application in large clinical studies

Metabolic changes in schizophrenia and human brain evolution.

BACKGROUND: Despite decades of research, the molecular changes responsible for the evolution of human cognitive abilities remain unknown. Comparative evolutionary studies provide detailed information about DNA sequence and mRNA expression differences between humans and other primates but, in the absence of other information, it has proved very difficult to identify molecular pathways relevant to human cognition. RESULTS: Here, we compare changes in gene expression and metabolite concentrations in the human brain and compare them to the changes seen in a disorder known to affect human cognitive abilities, schizophrenia. We find that both genes and metabolites relating to energy metabolism and energy-expensive brain functions are altered in schizophrenia and, at the same time, appear to have changed rapidly during recent human evolution, probably as a result of positive selection. CONCLUSION: Our findings, along with several previous studies, suggest that the evolution of human cognitive abilities was accompanied by adaptive changes in brain metabolism, potentially pushing the human brain to the limit of its metabolic capabilities.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Short and long-read genome sequencing methodologies for somatic variant detection; genomic analysis of a patient with diffuse large B-cell lymphoma

Recent advances in throughput and accuracy mean that the Oxford Nanopore Technologies PromethiON platform is a now a viable solution for genome sequencing. Much of the validation of bioinformatic tools for this long-read data has focussed on calling germline variants (including structural variants). Somatic variants are outnumbered many-fold by germline variants and their detection is further complicated by the effects of tumour purity/subclonality. Here, we evaluate the extent to which Nanopore sequencing enables detection and analysis of somatic variation. We do this through sequencing tumour and germline genomes for a patient with diffuse B-cell lymphoma and comparing results with 150 bp short-read sequencing of the same samples. Calling germline single nucleotide variants (SNVs) from specific chromosomes of the long-read data achieved good specificity and sensitivity. However, results of somatic SNV calling highlight the need for the development of specialised joint calling algorithms. We find the comparative genome-wide performance of different tools varies significantly between structural variant types, and suggest long reads are especially advantageous for calling large somatic deletions and duplications. Finally, we highlight the utility of long reads for phasing clinically relevant variants, confirming that a somatic 1.6 Mb deletion and a p.(Arg249Met) mutation involving TP53 are oriented in trans

Distinct regulation of hippocampal neuroplasticity and ciliary genes by corticosteroid receptors

Glucocorticoid hormones (GCs) are of critical importance for maintaining brain health, but their involvement in mental disorders is poorly understood. Here the authors show how GCs act through hippocampal mineralocorticoid and glucocorticoid receptors to impact the gene regulatory programs underpinning neuronal plasticity, ciliogenesis and behavioral adaptation

Maternal high-fat diet interacts with embryonic Cited2 genotype to reduce Pitx2c expression and enhance penetrance of left–right patterning defects

Deficiency of the transcription factor Cited2 in mice results in cardiac malformation, adrenal agenesis, neural tube, placental defects and partially penetrant cardiopulmonary laterality defects resulting from an abnormal Nodal->Pitx2c pathway. Here we show that a maternal high-fat diet more than doubles the penetrance of laterality defects and, surprisingly, induces palatal clefting in Cited2-deficient embryos. Both maternal diet and Cited2 deletion reduce embryo weight and kidney and thymus volume. Expression profiling identified 40 embryonic transcripts including Pitx2 that were significantly affected by embryonic genotype-maternal diet interaction. We show that a high-fat diet reduces Pitx2c levels >2-fold in Cited2-deficient embryos. Taken together, these results define a novel interaction between maternal high-fat diet and embryonic Cited2 deficiency that affects Pitx2c expression and results in abnormal laterality. They suggest that appropriate modifications of maternal diet may prevent such defects in humans

Cytokines and inflammatory mediators: 25. Certolizumab Pegol has a Different Profile from the other Anti-TNFS, Including Golimumab, in a Variety of in Vitro Assays

Background: Activities of the anti-TNFs, certolizumab pegol (CZP), etanercept (ETA), infliximab (IFX) and adalimumab (ADA), have been compared in a range of in vitro assays. CZP is the only licensed PEGylated Fab' anti-TNF; ETA is a fusion protein with an IgG1 Fc, and IFX and ADA are both antibodies with an IgG1 Fc. Golimumab (GLM) is a monoclonal IgG1 TNF inhibitor recently approved for a number of indications; it is thus of interest to assess the in vitro activity of GLM. In vitro assays previously used were neutralisation of TNF in the L929 bioassay, inhibition of LPS-driven cytokine production by monocytes, induction of apoptosis in activated lymphocytes and monocytes, and induction of neutrophil necrosis. Methods: Neutralisation of human TNF was assessed in the L929 bioassay using a range of concentrations of the anti-TNFs and a fixed concentration of TNF (100 pg/mL). Activity of the anti-TNFs at inhibiting LPS-driven IL-1β secretion by monocytes was assessed by incubating peripheral blood monocytes with various concentrations of the anti-TNF for 1 hour (hr) and then washing the cells. LPS was added for 4 hrs, the supernatants collected and the IL-1β level measured by ELISA. To assess induction of apoptosis, peripheral blood lymphocytes were activated for 2 days with 2 μg/mL CD3/CD28 and monocytes with 300 U/mL IL-4 and GMCSF for 3 days. The effect of the anti-TNFs on apoptosis was assessed by Annexin V staining using flow cytometry 24 hrs later. The effect of the anti-TNFs on neutrophil necrosis was determined by measuring myeloperoxidase release after 12 hrs. An isotype-matched control was used in all assays except the L929 bioassay. Results: IC90 neutralisation activity of the anti-TNFs in the L929 bioassay was 0.3 ng/mL for ETA, 4 ng/mL for GLM, 15 ng/mL for ADA, and 20 ng/mL for IFX, compared with 2.5 ng/mL for CZP. CZP was the most potent inhibitor of LPS-driven IL-1β secretion (IC50 ∼0.1 ng/mL), followed by GLM (20 ng/mL) and IFX (50 ng/mL). GLM, ADA, IFX and ETA induced apoptosis of monocytes and lymphocytes to a similar degree reaching a level of 23% and ∼40% at 100 μg/mL, respectively. CZP caused no increase in apoptosis above the levels seen with the isotype-matched control. In the neutrophil necrosis assay, ADA,IFX and GLM caused ∼70% necrosis at 100 μg/mL, and ETA 48%. CZP did not increase the level of necrosis above the level of the control. Conclusions: Bioactivity of the IgG1 molecules GLM, IFX and ADA in neutralising human TNF was inferior to that of CZP and ETA. CZP, the only PEGylated anti-TNF, had a different profile to the other anti-TNFs as it was the most potent at inhibiting LPS-driven IL-1β production by monocytes, did not induce apoptosis of activated monocytes and lymphocytes, and did not cause neutrophil necrosis. The clinical relevance of these in vitro effects is unknown. Nevertheless, these assays show interesting in vitro differences between the anti-TNFs. Disclosure statement: G.F. and A.N. are employees of UC

The Cerebral Microvasculature in Schizophrenia: A Laser Capture Microdissection Study

BACKGROUND: Previous studies of brain and peripheral tissues in schizophrenia patients have indicated impaired energy supply to the brain. A number of studies have also demonstrated dysfunction of the microvasculature in schizophrenia patients. Together these findings are consistent with a hypothesis of blood-brain barrier dysfunction in schizophrenia. In this study, we have investigated the cerebral vascular endothelium of schizophrenia patients at the level of transcriptomics. METHODOLOGY/PRINCIPAL FINDINGS: We used laser capture microdissection to isolate both microvascular endothelial cells and neurons from post mortem brain tissue from schizophrenia patients and healthy controls. RNA was isolated from these cell populations, amplified, and analysed using two independent microarray platforms, Affymetrix HG133plus2.0 GeneChips and CodeLink Whole Human Genome arrays. In the first instance, we used the dataset to compare the neuronal and endothelial data, in order to demonstrate that the predicted differences between cell types could be detected using this methodology. We then compared neuronal and endothelial data separately between schizophrenic subjects and controls. Analysis of the endothelial samples showed differences in gene expression between schizophrenics and controls which were reproducible in a second microarray platform. Functional profiling revealed that these changes were primarily found in genes relating to inflammatory processes. CONCLUSIONS/SIGNIFICANCE: This study provides preliminary evidence of molecular alterations of the cerebral microvasculature in schizophrenia patients, suggestive of a hypo-inflammatory state in this tissue type. Further investigation of the blood-brain barrier in schizophrenia is warranted

Foxp2 Regulates Gene Networks Implicated in Neurite Outgrowth in the Developing Brain

Forkhead-box protein P2 is a transcription factor that has been associated with intriguing aspects of cognitive function in humans, non-human mammals, and song-learning birds. Heterozygous mutations of the human FOXP2 gene cause a monogenic speech and language disorder. Reduced functional dosage of the mouse version (Foxp2) causes deficient cortico-striatal synaptic plasticity and impairs motor-skill learning. Moreover, the songbird orthologue appears critically important for vocal learning. Across diverse vertebrate species, this well-conserved transcription factor is highly expressed in the developing and adult central nervous system. Very little is known about the mechanisms regulated by Foxp2 during brain development. We used an integrated functional genomics strategy to robustly define Foxp2-dependent pathways, both direct and indirect targets, in the embryonic brain. Specifically, we performed genome-wide in vivo ChIP–chip screens for Foxp2-binding and thereby identified a set of 264 high-confidence neural targets under strict, empirically derived significance thresholds. The findings, coupled to expression profiling and in situ hybridization of brain tissue from wild-type and mutant mouse embryos, strongly highlighted gene networks linked to neurite development. We followed up our genomics data with functional experiments, showing that Foxp2 impacts on neurite outgrowth in primary neurons and in neuronal cell models. Our data indicate that Foxp2 modulates neuronal network formation, by directly and indirectly regulating mRNAs involved in the development and plasticity of neuronal connections

A mouse model with a frameshift mutation in the nuclear factor I/X (NFIX) gene has phenotypic features of Marshall-Smith Syndrome

The nuclear factor I/X (NFIX) gene encodes a ubiquitously expressed transcription factor whose mutations lead to two allelic disorders characterized by developmental, skeletal, and neural abnormalities, namely, Malan syndrome (MAL) and Marshall–Smith syndrome (MSS). NFIX mutations associated with MAL mainly cluster in exon 2 and are cleared by nonsense-mediated decay (NMD) leading to NFIX haploinsufficiency, whereas NFIX mutations associated with MSS are clustered in exons 6–10 and escape NMD and result in the production of dominant-negative mutant NFIX proteins. Thus, different NFIX mutations have distinct consequences on NFIX expression. To elucidate the in vivo effects of MSS-associated NFIX exon 7 mutations, we used CRISPR-Cas9 to generate mouse models with exon 7 deletions that comprised: a frameshift deletion of two nucleotides (Nfix Del2); in-frame deletion of 24 nucleotides (Nfix Del24); and deletion of 140 nucleotides (Nfix Del140). Nfix+/Del2, Nfix+/Del24, Nfix+/Del140, NfixDel24/Del24, and NfixDel140/Del140 mice were viable, normal, and fertile, with no skeletal abnormalities, but NfixDel2/Del2 mice had significantly reduced viability (p < 0.002) and died at 2–3 weeks of age. Nfix Del2 was not cleared by NMD, and NfixDel2/Del2 mice, when compared to Nfix+/+ and Nfix+/Del2 mice, had: growth retardation; short stature with kyphosis; reduced skull length; marked porosity of the vertebrae with decreased vertebral and femoral bone mineral content; and reduced caudal vertebrae height and femur length. Plasma biochemistry analysis revealed NfixDel2/Del2 mice to have increased total alkaline phosphatase activity but decreased C-terminal telopeptide and procollagen-type-1-N-terminal propeptide concentrations compared to Nfix+/+ and Nfix+/Del2 mice. NfixDel2/Del2 mice were also found to have enlarged cerebral cortices and ventricular areas but smaller dentate gyrus compared to Nfix+/+ mice. Thus, NfixDel2/Del2 mice provide a model for studying the in vivo effects of NFIX mutants that escape NMD and result in developmental abnormalities of the skeletal and neural tissues that are associated with MSS