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Title
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Functional characterization of the new 8q21 Asthma risk locus
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Author
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Abstract
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| Genome wide association studies (GWAS) provide a powerful tool to identify genetic variants associated with asthma risk. However, the target genes for many allergy risk variants discovered to date are unknown. In a recent GWAS, Ferreira et al. identified a new association between asthma risk and common variants located on chromosome 8q21. The overarching aim of this thesis was to elucidate the biological mechanisms underlying this association. Specifically, the goals of this study were to identify the gene(s) underlying the observed association and to study their contribution to asthma pathophysiology. Using genetic data from the 1000 Genomes Project, we first identified 118 variants in linkage disequilibrium (LD; r2>0.6) with the sentinel allergy risk SNP (rs7009110) on chromosome 8q21. Of these, 35 were found to overlap one of four Putative Regulatory Elements (PREs) identified in this region in a lymphoblastoid cell line (LCL), based on epigenetic marks measured by the ENCODE project. Results from analysis of gene expression data generated for LCLs (n=373) by the Geuvadis consortium indicated that rs7009110 is associated with the expression of only one nearby gene: PAG1 - located 732 kb away. PAG1 encodes a transmembrane adaptor protein localized to lipid rafts, which is highly expressed in immune cells. Results from chromosome conformation capture (3C) experiments showed that PREs in the region of association physically interacted with the promoter of PAG1. Furthermore, results from luciferase reporter assays demonstrated that one of these PREs (PRE 3) acted as a transcriptional enhancer on PAG1 exclusively when it carried the rs2370615:C asthma predisposing allele. This variant, which is in complete LD with rs7009110, was found to disrupt the binding of the Foxo3a transcription factor to PRE3. As such, rs2370615 represents a putative functional variant underlying the association between rs7009110 and asthma. In addition to PAG1, other genes in the 8q21 region could be targets of asthma risk variants, despite the lack of evidence for an association between rs7009110 and gene expression. The closest gene is ZBTB10 (75 kb away), a repressor of the specificity proteins (Sp1, Sp3 and SP4), which are transcription factors known to regulate several immune-related genes. Therefore, based on distance to rs7009110 and its known function, we prioritised ZBTB10 to investigate if it could represent an additional target gene of 8q21 asthma risk variants. Results from 3C assays showed that the same PREs that interacted with the PAG1 promoter also interacted with the ZBTB10 promoter. Results from luciferase assays for ZBTB10 were inconclusive: PRE 2 enhanced the activity of the ZBTB10 promoter, and this effect was inhibited by an asthma risk allele but these differences were not statistically significant. Together, these results indicate that ZBTB10 is also likely to be a target of 8q21 asthma risk variants. To study the contribution of PAG1 and ZBTB10 to asthma pathophysiology, we performed in vivo experiments using wild type (WT), Pag1-/- and Zbtb10het mice, which included challenging mice with an allergen or virus, both able to induce airway inflammation. Results from these experiments indicate that Pag1 expression might regulate granulocyte, lymphocyte and/or airway epithelial cell function. On the other hand, the expression of Zbtb10 was critical for embryonic development and could play a role in immune cell development as well as activation during the allergen sensitization phase. In conclusion we have identified PAG1 and ZBTB10 as target genes of 8q21 asthma risk variants and showed that both genes play important roles in the immune system and development of airway inflammation. |
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Language
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English
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Publication
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Queensland
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University of Queensland
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2018
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DOI
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10.14264/UQL.2018.547
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Volume/pages
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358 p.
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