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

Severe polyposis in Apc1322T mice is associated with submaximal Wnt signalling and increased expression of the stem cell marker Lgr5

Background and aims: Adenomatous polyposis coli (APC) is a tumour suppressor gene mutated in the germline of patients with familial adenomatous polyposis (FAP) and somatically in most colorectal cancers. APC mutations impair β-catenin degradation, resulting in increased Wnt signalling. The most frequent APC mutation is a codon 1309 truncation that is associated with severe FAP. A previous study compared two mouse models of intestinal tumorigenesis, ApcR850X (Min) and. Apc1322T (1322T), the latter a model of human codon 1309 changes. 1322T mice had more severe polyposis but, surprisingly, these tumours had lower levels of nuclear β-catenin than Min tumours. The consequences of these different β-catenin levels were investigated. Methods: Enterocytes were isolated from 1322T and Min tumours by microdissection and gene expression profiling was performed. Differentially expressed Wnt targets and other stem cell markers were validated using quantitative PCR, in situ hybridisation and immunohistochemistry. Results: As expected, lower nuclear β-catenin levels in 1322T lesions were associated with generally lower levels of Wnt target expression. However, expression of the Wnt target and stem cell marker Lgr5 was significantly higher in 1322T tumours than in Min tumours. Other stem cell markers (Musashi1, Bmi1 and the Wnt target Cd44) were also at higher levels in 1322T tumours. In addition, expression of the Bmp antagonist Gremlin1 was higher in 1322T tumours, together with lower Bmp2 and Bmp4 expression. Conclusions: The severe phenotype caused by truncation of Apc at codon 1322 is associated with an increased number of stem cells. Thus, a submaximal level of Wnt signalling favours the stem cell phenotype and this may promote tumorigenesis. A level of Wnt signalling exists that is too high for optimal tumour growth

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

Metabolic profiling of aortic stenosis and hypertrophic cardiomyopathy identifies mechanistic contrasts in substrate utilization

Aortic stenosis (AS) and hypertrophic cardiomyopathy (HCM) are distinct disorders leading to left ventricular hypertrophy (LVH), but whether cardiac metabolism substantially differs between these in humans remains to be elucidated. We undertook an invasive (aortic root, coronary sinus) metabolic profiling in patients with severe AS and HCM in comparison with non‐LVH controls to investigate cardiac fuel selection and metabolic remodeling. These patients were assessed under different physiological states (at rest, during stress induced by pacing). The identified changes in the metabolome were further validated by metabolomic and orthogonal transcriptomic analysis, in separately recruited patient cohorts. We identified a highly discriminant metabolomic signature in severe AS in all samples, regardless of sampling site, characterized by striking accumulation of long‐chain acylcarnitines, intermediates of fatty acid transport across the inner mitochondrial membrane, and validated this in a separate cohort. Mechanistically, we identify a downregulation in the PPAR‐α transcriptional network, including expression of genes regulating fatty acid oxidation (FAO). In silico modeling of β‐oxidation demonstrated that flux could be inhibited by both the accumulation of fatty acids as a substrate for mitochondria and the accumulation of medium‐chain carnitines which induce competitive inhibition of the acyl‐CoA dehydrogenases. We present a comprehensive analysis of changes in the metabolic pathways (transcriptome to metabolome) in severe AS, and its comparison to HCM. Our results demonstrate a progressive impairment of β‐oxidation from HCM to AS, particularly for FAO of long‐chain fatty acids, and that the PPAR‐α signaling network may be a specific metabolic therapeutic target in AS

Metabolic profiling of aortic stenosis and hypertrophic cardiomyopathy identifies mechanistic contrasts in substrate utilization

Open Access via the Wiley Agreement UKRI | Medical Research Council (MRC), Grant/Award Number: MR/ P011705/1, MC_UP_A090_1006 and MR/S010483; British Heart Foundation (BHF); NIHR | NIHR Oxford Biomedical Research Centre (OxBRC)Peer reviewe

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

Impact of spliceosome mutations on RNA splicing in myelodysplasia: dysregulated genes/pathways and clinical associations.

SF3B1, SRSF2, and U2AF1 are the most frequently mutated splicing factor genes in the myelodysplastic syndromes (MDS). We have performed a comprehensive and systematic analysis to determine the effect of these commonly mutated splicing factors on pre-mRNA splicing in the bone marrow stem/progenitor cells and in the erythroid and myeloid precursors in splicing factor mutant MDS. Using RNA-seq, we determined the aberrantly spliced genes and dysregulated pathways in CD34+ cells of 84 patients with MDS. Splicing factor mutations result in different alterations in splicing and largely affect different genes, but these converge in common dysregulated pathways and cellular processes, focused on RNA splicing, protein synthesis, and mitochondrial dysfunction, suggesting common mechanisms of action in MDS. Many of these dysregulated pathways and cellular processes can be linked to the known disease pathophysiology associated with splicing factor mutations in MDS, whereas several others have not been previously associated with MDS, such as sirtuin signaling. We identified aberrantly spliced events associated with clinical variables, and isoforms that independently predict survival in MDS and implicate dysregulation of focal adhesion and extracellular exosomes as drivers of poor survival. Aberrantly spliced genes and dysregulated pathways were identified in the MDS-affected lineages in splicing factor mutant MDS. Functional studies demonstrated that knockdown of the mitosis regulators SEPT2 and AKAP8, aberrantly spliced target genes of SF3B1 and SRSF2 mutations, respectively, led to impaired erythroid cell growth and differentiation. This study illuminates the effect of the common spliceosome mutations on the MDS phenotype and provides novel insights into disease pathophysiology