Microarray Profiling of Lymphocytes in Internal Diseases With an Altered Immune Response: Potential and Methodology

Recently it has become possible to investigate expression of all human genes with microarray technique. The authors provide arguments to consider peripheral white blood cells and in particular lymphocytes as a model for the investigation of pathophysiology of asthma, RA, and SLE diseases in which inflammation is a major component. Lymphocytes are an alternative to tissue biopsies that are most often difficult to collect systematically. Lymphocytes express more than 75% of the human genome, and, being an important part of the immune system, they play a central role in the pathogenesis of asthma, RA, and SLE. Here we review alterations of gene expression in lymphocytes and methodological aspects of the microarray technique in these diseases. Lymphocytic genes may become activated because of a general nonspecific versus disease-specific mechanism. The authors suppose that in these diseases microarray profiles of gene expression in lymphocytes can be disease specific, rather than inflammation specific. Some potentials and pitfalls of the array technologies are discussed. Optimal clinical designs aimed to identify disease-specific genes are proposed. Lymphocytes can be explored for research, diagnostic, and possible treatment purposes in these diseases, but their precise value should be clarified in future investigation

Microarrays

Microarrays are revolutionizing genetics by making it possible to genotype hundreds of thousands of DNA markers and to assess the expression (RNA transcripts) of all of the genes in the genome. Microarrays are slides the size of a postage stamp that contain millions of DNA sequences to which single-stranded DNA or RNA can hybridize. This miniaturization requires little DNA or RNA and makes the method fast and inexpensive; multiple assays of each target make the method highly accurate. DNA microarrays with hundreds of thousands of DNA markers have made it possible to conduct systematic scans of the entire genome to identify genetic associations with complex disorders or dimensions likely to be influenced by many genes of small effect size. RNA microarrays can provide snapshots of gene expression across all of the genes in the genome at any time in any tissue, which has far-reaching applications such as structural and functional ‘genetic neuroimaging’ and providing a biological basis for understanding environmental influence

Gamma-aminobutyric acid, a potential tumor suppressor for small airway-derived lung adenocarcinoma

Pulmonary adenocarcinoma (PAC) is the leading type of lung cancer in smokers and non-smokers that arises in most cases from small airway epithelial cells. PAC has a high mortality due to its aggressive behavior and resistance to cancer therapeutics. We have shown previously that the proliferation of human PAC cells NCI-H322 and immortalized human small airway epithelial cells HPL1D is stimulated by cyclic adenosine monophosphate (cAMP)/protein kinase A-dependent phosphorylation of cyclic adenosine monophosphate response element-binding (CREB) protein and transactivation of the epidermal growth factor receptor and that this pathway is activated by beta-1-adrenoreceptors (β1-ARs) and the non-genomic estrogen receptor beta. Our current in vitro studies with HPL1D and NCI-H322 cells showed that signaling via the gamma-amino butyric acid receptor (GABABR) strongly inhibited base level and isoproterenol-induced cAMP, p-CREB, cyclic adenosine monophosphate response element-luciferase activity and p-extracellular regulated kinase-1 (ERK1)/2 and effectively blocked DNA synthesis and cell migration. The inhibitory effects of gamma-amino butyric acid (GABA) were disinhibited by the GABABR antagonist CGP-35348 or GABABR knockdown. Immunohistochemical investigation of hamster lungs showed significant underexpression of GABA in animals with small airway-derived PACs induced by the nicotine-derived carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). These findings suggest that GABA may have tumor suppressor function in small airway epithelia and the PACs derived from them and that downregulation of GABA by NNK may contribute to the development of this cancer in smokers. Our findings suggest that marker-guided treatment with GABA or a GABABR agonist of individuals with downregulated pulmonary GABA may provide a novel targeted approach for the prevention of PAC in smokers

Identification of Phosphoglycerate Kinase 1 (PGK1) as a reference gene for quantitative gene expression measurements in human blood RNA

<p>Abstract</p> <p>Background</p> <p>Blood is a convenient sample and increasingly used for quantitative gene expression measurements with a variety of diseases including chronic fatigue syndrome (CFS). Quantitative gene expression measurements require normalization of target genes to reference genes that are stable and independent from variables being tested in the experiment. Because there are no genes that are useful for all situations, reference gene selection is an essential step to any quantitative reverse transcription-PCR protocol. Many publications have described appropriate genes for a wide variety of tissues and experimental conditions, however, reference genes that may be suitable for the analysis of CFS, or human blood RNA derived from whole blood as well as isolated peripheral blood mononuclear cells (PBMCs), have not been described.</p> <p>Findings</p> <p>Literature review and analyses of our unpublished microarray data were used to narrow down the pool of candidate reference genes to six. We assayed whole blood RNA from Tempus tubes and cell preparation tube (CPT)-collected PBMC RNA from 46 subjects, and used the geNorm and NormFinder algorithms to select the most stable reference genes. <it>Phosphoglycerate kinase 1 (PGK1) </it>was one of the optimal normalization genes for both whole blood and PBMC RNA, however, additional genes differed for the two sample types; <it>Ribosomal protein large, P0 (RPLP0</it>) for PBMC RNA and <it>Peptidylprolyl isomerase B </it>(<it>PPIB) </it>for whole blood RNA. We also show that the use of a single reference gene is sufficient for normalization when the most stable candidates are used.</p> <p>Conclusions</p> <p>We have identified <it>PGK1 </it>as a stable reference gene for use with whole blood RNA and RNA derived from PBMC. When stable genes are selected it is possible to use a single gene for normalization rather than two or three. Optimal normalization will improve the ability of results from PBMC RNA to be compared with those from whole blood RNA and potentially allows comparison of gene expression results from blood RNA collected and processed by different methods with the intention of biomarker discovery. Results of this study should facilitate large-scale molecular epidemiologic studies using blood RNA as the target of quantitative gene expression measurements.</p

No relationship between 2',3'-cyclic nucleotide 3'-phosphodiesterase and schizophrenia in the Chinese Han population: an expression study and meta-analysis

<p>Abstract</p> <p>Background</p> <p>2',3'-Cyclic nucleotide 3'-phosphodiesterase (<it>CNP</it>), one of the promising candidate genes for schizophrenia, plays a key part in the oligodendrocyte function and in myelination. The present study aims to investigate the relationship between <it>CNP </it>and schizophrenia in the Chinese population and the effect of different factors on the expression level of <it>CNP </it>in schizophrenia.</p> <p>Methods</p> <p>Five <it>CNP </it>single nucleotide polymorphisms (SNPs) were investigated in a Chinese Han schizophrenia case-control sample set (n = 180) using direct sequencing. The results were included in the following meta-analysis. Quantitative real-time polymerase chain reaction (PCR) was conducted to examine <it>CNP </it>expression levels in peripheral blood lymphocytes.</p> <p>Results</p> <p>Factors including gender, genotype, sub-diagnosis and antipsychotics-treatment were found not to contribute to the expression regulation of the <it>CNP </it>gene in schizophrenia. Our meta-analysis produced similar negative results.</p> <p>Conclusion</p> <p>The results suggest that the <it>CNP </it>gene may not be involved in the etiology and pathology of schizophrenia in the Chinese population.</p

Changes in Mood States and Biomarkers During Peginterferon and Ribavirin Treatment of Chronic Hepatitis C

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/74953/1/j.1572-0241.2008.02106.x.pd

Is human blood a good surrogate for brain tissue in transcriptional studies?

Abstract Background Since human brain tissue is often unavailable for transcriptional profiling studies, blood expression data is frequently used as a substitute. The underlying hypothesis in such studies is that genes expressed in brain tissue leave a transcriptional footprint in blood. We tested this hypothesis by relating three human brain expression data sets (from cortex, cerebellum and caudate nucleus) to two large human blood expression data sets (comprised of 1463 individuals). Results We found mean expression levels were weakly correlated between the brain and blood data (r range: [0.24,0.32]). Further, we tested whether co-expression relationships were preserved between the three brain regions and blood. Only a handful of brain co-expression modules showed strong evidence of preservation and these modules could be combined into a single large blood module. We also identified highly connected intramodular "hub" genes inside preserved modules. These preserved intramodular hub genes had the following properties: first, their expression levels tended to be significantly more heritable than those from non-preserved intramodular hub genes (p &lt; 10-90); second, they had highly significant positive correlations with the following cluster of differentiation genes: CD58, CD47, CD48, CD53 and CD164; third, a significant number of them were known to be involved in infection mechanisms, post-transcriptional and post-translational modification and other basic processes. Conclusions Overall, we find transcriptome organization is poorly preserved between brain and blood. However, the subset of preserved co-expression relationships characterized here may aid future efforts to identify blood biomarkers for neurological and neuropsychiatric diseases when brain tissue samples are unavailable

Gene expression analyses in breast cancer epidemiology: the Norwegian Women and Cancer postgenome cohort study

Introduction The introduction of high-throughput technologies, also called -omics technologies, into epidemiology has raised the need for high-quality observational studies to reduce several sources of error and bias. Methods The Norwegian Women and Cancer (NOWAC) postgenome cohort study consists of approximately 50,000 women born between 1943 and 1957 who gave blood samples between 2003 and 2006 and filled out a two-page questionnaire. Blood was collected in such a way that RNA is preserved and can be used for gene expression analyses. The women are part of the NOWAC study consisting of 172,471 women 30 to 70 years of age at recruitment from 1991 to 2006 who answered one to three questionnaires on diet, medication use, and lifestyle. In collaboration with the Norwegian Breast Cancer Group, every NOWAC participant born between 1943 and 1957 who is admitted to a collaborating hospital for a diagnostic biopsy or for surgery of breast cancer will be asked to donate a tumor biopsy and two blood samples. In parallel, at least three controls are approached for each breast cancer case in order to obtain blood samples from at least two controls per case. The controls are drawn at random from NOWAC matched by time of follow-up and age. In addition, 400 normal breast tissues as well as blood samples will be collected among healthy women participating at the Norwegian Mammography Screening program at the Breast Imaging Center at the University Hospital of North-Norway, Tromsø. Results The NOWAC postgenome cohort offers a unique opportunity (a) to study blood-derived gene expression profiles as a diagnostic test for breast cancer in a nested case-control design with adjustment for confounding factors related to different exposures, (b) to improve the reliability and accuracy of this approach by adjusting for an individual's genotype (for example, variants in genes coding for hormone and drug-metabolizing and detoxifying enzymes), (c) to study gene expression profiles from peripheral blood as surrogate tissue to biomonitor defined exposure (for example, hormone) and its association with disease risk (that is, breast cancer), and (d) to study gene variants (single nucleotide polymorphisms and copy number variations) and environmental exposure (endogenous and exogenous hormones) and their influence on the incidence of different molecular subtypes of breast cancer. Conclusion The NOWAC postgenome cohort combining a valid epidemiological approach with richness of biological samples should make an important contribution to the study of the etiology and system biology of breast cancer

Genome-wide DNA methylation levels and altered cortisol stress reactivity following childhood trauma in humans

DNA methylation likely plays a role in the regulation of human stress reactivity. Here we show that in a genome-wide analysis of blood DNA methylation in 85 healthy individuals, a locus in the Kit ligand gene (KITLG; cg27512205) showed the strongest association with cortisol stress reactivity (P=5.8 � 10?6). Replication was obtained in two independent samples using either blood (N=45, P=0.001) or buccal cells (N=255, P=0.004). KITLG methylation strongly mediates the relationship between childhood trauma and cortisol stress reactivity in the discovery sample (32% mediation). Its genomic location, a CpG island shore within an H3K27ac enhancer mark, and the correlation between methylation in the blood and prefrontal cortex provide further evidence that KITLG methylation is functionally relevant for the programming of stress reactivity in the human brain. Our results extend preclinical evidence for epigenetic regulation of stress reactivity to humans and provide leads to enhance our understanding of the neurobiological pathways underlying stress vulnerability

Gene Expression Studies in Major Depression

The dramatic technical advances in methods to measure gene expression on a genome-wide level thus far have not been paralleled by breakthrough discoveries in psychiatric disorders—including major depression (MD)—using these hypothesis-free approaches. In this review, we first describe the methodologic advances made in gene expression analysis, from quantitative polymerase chain reaction to next-generation sequencing. We then discuss issues in gene expression experiments specific to MD, ranging from the choice of target tissues to the characterization of the case group. We provide a synopsis of the gene expression studies published thus far for MD, with a focus on studies using mRNA microarray methods. Finally, we discuss possible new strategies for the gene expression studies in MD that circumvent some of the addressed issues