Asunción García-Sánchez

Genome-wide association studies (GWAS) and their importance in asthma

Asthma is a complex disease determined by the interaction of different genes and environmental factors. The first genetic investigations in asthma were candidate gene association studies and linkage studies. In recent years research has focused on association studies that scan the entire genome without any prior conditioning hypothesis: the so-called genome-wide association studies (GWAS). The first GWAS was published in 2007, and described a new locus associated to asthma in chromosome 17q12-q21, involving the ORMDL3, GSDMB and ZPBP2 genes (a description of the genes named in the manuscript are listed in Table 1). None of these genes would have been selected in a classical genetic association study since it was not known they could be implicated in asthma. To date, a number of GWAS studies in asthma have been made, with the identification of about 1000 candidate genes. Coordination of the different research groups in international consortiums and the application of new technologies such as new generation sequencing will help discover new implicated genes and improve our understanding of the molecular mechanisms underlying the disease.

Analysis of FOXP3 gene in children with allergy and autoimmune diseases.

BACKGROUND Allergy and autoimmunity are important immunological entities underlying chronic diseases in children. In some cases both entities develop simultaneously in the same patient. FOXP3 gene codes for a transcription factor involved in regulation of the immune system. Considering that regulatory T cells are involved in controlling immunological disease development, and the relevant role of FOXP3 in this kind of T cells, the objective of this study was to analyse the FOXP3 gene in the most prevalent autoimmune diseases and/or allergies in childhood in a European population. METHODS A total of 255 Caucasian individuals, 95 controls and 160 patients diagnosed with allergic, autoimmune or both diseases were included in this study. The molecular analysis of FOXP3 was performed by DNA sequencing following the recommendations for quality of the European Molecular Genetics Quality Network. Genomic DNA was extracted from peripheral blood of all participants and was amplified using the polymerase chain reaction. After the visualisation of the amplified fragments by agarose gel-electrophoresis, they were sequenced. RESULTS Thirteen different polymorphisms in FOXP3 gene were found, seven of which had not been previously described. The mutated allele of SNP 7340C>T was observed more frequently in the group of male children suffering from both allergic and autoimmune diseases simultaneously (p=0.004, OR=16.2 [1.34-195.15]). CONCLUSIONS In this study we identified for first time genetic variants of FOXP3 that are significantly more frequent in children who share allergic and autoimmune diseases. These variants mainly affect regulatory sequences that could alter the expression levels of FOXP3 modifying its function including its role in Treg cells.

Review on Pharmacogenetics and Pharmacogenomics Applied to the Study of Asthma

Nearly one-half of asthmatic patients do not respond to the most common therapies. Evidence suggests that genetic factors may be involved in the heterogeneity in therapeutic response and adverse events to asthma therapies. We focus on the three major classes of asthma medication: β-adrenergic receptor agonist, inhaled corticosteroids, and leukotriene modifiers. Pharmacogenetics and pharmacogenomics studies have identified several candidate genes associated with drug response.In this chapter, the main pharmacogenetic and pharmacogenomic studies in addition to the future perspectives in personalized medicine will be reviewed. The ideal treatment of asthma would be a tailored approach to health care in which adverse effects are minimized and the therapeutic benefit for an individual asthmatic is maximized leading to a more cost-effective care.

Pharmacogenetics and the treatment of asthma

Heterogeneity defines both the natural history of asthma as well as patients response to treatment. Pharmacogenomics contribute to understand the genetic basis of drug response and thus to define new therapeutic targets or molecular biomarkers to evaluate treatment effectiveness. This review is initially focused on different genes so far involved in the pharmacological response to asthma treatment. Specific considerations regarding allergic asthma, the pharmacogenetics aspects of polypharmacy and the application of pharmacogenomics in new drugs in asthma will also be addressed. Finally, future perspectives related to epigenetic regulatory elements and the potential impact of systems biology in pharmacogenetics of asthma will be considered.

Protocol for Lipid-Mediated Transient Transfection in A549 Epithelial Lung Cell Line

Trials of transfection in eukaryotic cells are essential tools for the study of gene and protein function. They have been used in a wide range of research fields. In this chapter, a method of transient transfection of the A549 cell line, human lung cells of alveolar epithelium, with an expression plasmid is described. In addition, the fundamental characteristics of this experimental procedure are addressed.

Cell Culture Techniques: Corticosteroid Treatment in A549 Human Lung Epithelial Cell.

Experimentation with cell cultures is widespread in different areas of basic science as well as in the development of biotechnology applied to medicine. Cellular models are applied to the study of respiratory diseases. In this chapter, we present a protocol of basic cell culture using A549 Human lung adenocarcinoma epithelial cells as model. Corticosteroid therapy is used to treat respiratory diseases such asthma. Thus, we also describe a protocol of lung epithelial cell culture treated with dexamethasone to illustrate an example of monitoring the effects of a drug in a lung cell culture assay.

Chromatin Immunoprecipitation: Application to the Study of Asthma.

Chromatin immunoprecipitation (ChIP) is a technique for studying interactions between proteins and DNA in living cells. A protein of interest is selectively immunoprecipitated from a chromatin preparation, to analyze the DNA sequences involved. ChIP can be used to determine whether a transcription factor interacts with a candidate target gene and to map the localization of histones with posttranslational modifications on the genome.The protein-DNA interactions are captured in vivo by chemical cross-linking. Cell lysis, DNA fragmentation, and immunoaffinity purification of the protein of interest allow to co-purify DNA fragments that are associated with that protein. The enriched protein-DNA population is ready to be quantified by PCR to detect precipitated DNA fragments. The combination of ChIP with DNA microarray analysis (ChIP-on-chip) and high-throughput sequencing (ChIP-seq) has enabled to obtain profiles of transcription factor occupancy sites and histone modifications throughout the genome.

Cluster Analysis Identifies 3 Phenotypes within Allergic Asthma

BACKGROUND Asthma is a heterogeneous chronic disease with different clinical expressions and responses to treatment. In recent years, several unbiased approaches based on clinical, physiological, and molecular features have described several phenotypes of asthma. Some phenotypes are allergic, but little is known about whether these phenotypes can be further subdivided. OBJECTIVE We aimed to phenotype patients with allergic asthma using an unbiased approach based on multivariate classification techniques (unsupervised hierarchical cluster analysis). METHODS From a total of 54 variables of 225 patients with well-characterized allergic asthma diagnosed following American Thoracic Society (ATS) recommendation, positive skin prick test to aeroallergens, and concordant symptoms, we finally selected 19 variables by multiple correspondence analyses. Then a cluster analysis was performed. RESULTS Three groups were identified. Cluster 1 was constituted by patients with intermittent or mild persistent asthma, without family antecedents of atopy, asthma, or rhinitis. This group showed the lowest total IgE levels. Cluster 2 was constituted by patients with mild asthma with a family history of atopy, asthma, or rhinitis. Total IgE levels were intermediate. Cluster 3 included patients with moderate or severe persistent asthma that needed treatment with corticosteroids and long-acting β-agonists. This group showed the highest total IgE levels. CONCLUSIONS We identified 3 phenotypes of allergic asthma in our population. Furthermore, we described 2 phenotypes of mild atopic asthma mainly differentiated by a family history of allergy.

PTGDR gene expression and response to dexamethasone treatment in an in vitro model

Asthma is a multifactorial pathology influenced by environmental and genetic factors. Glucocorticoid treatment decreases symptoms by regulating genes involved in the inflammatory process through binding to specific DNA sequences. Polymorphisms located in the promoter region of the Prostaglandin D Receptor (PTGDR) gene have been related to asthma. We aimed to analyze the effect of PTGDR promoter haplotypes on gene expression and response to corticosteroid therapy. A549 lung epithelial cells were transfected with vectors carrying four different PTGDR haplotypes (CTCT, CCCC, CCCT and TCCT), and treated with dexamethasone. Different approaches to study the promoter activity (Dual Luciferase Reporter System), gene expression levels (qPCR) and cytokine secretion (Multiplexed Bead-based Flow Cytometric) were used. In addition, in silico analysis was also performed. Cells carrying the TCCT haplotype showed the lowest promoter activity (p-value<0.05) and mRNA expression levels in basal conditions. After dexamethasone treatment, cells carrying the wild-type variant CTCT showed the highest response, and those carrying the TCCT variant the lowest (p-value<0.05) in luciferase assays. Different transcription factor binding patterns were identified in silico. Moreover, differences in cytokine secretion were also found among different promoter haplotypes. Polymorphisms of PTGDR gene influence basal promoter activity and gene expression, as well as the cytokine secretory pattern. Furthermore, an association between these positions and response to corticoid treatment was observed.

Real-Time PCR for Gene Expression Quantification in Asthma.

The quantitative real-time PCR (qPCR) has become the reference technique for studying gene expression in recent years. The application of qPCR to the study of asthma provides very useful information regarding the gene expression mechanisms. The quantification of RNA from cDNA can be performed by using fluorescent dyes or specific sequence probes. Here, we describe the protocol to quantify gene expression levels using SYBR Green as fluorescent dye. The protocol starts with the RNA extraction, followed by reverse transcription to obtain cDNA, quantification and finally data analysis.