Name of Student:
Loubna Akhabir
Research Supervisor: Dr. Andrew Sandford
Title of Presentation: Functional follow-up of a TSLP SNP associated with asthma
Abstract
Introduction: The aim of this work is to perform functional assays to uncover the mechanism underlying their involvement of TSLP SNP rs1837253 in asthma pathogenesis. TSLP has been shown to be
sufficient to initiate experimental allergic airway inflammation using mouse models and its gene expression has been shown to increase in murine and human asthmatic lungs. TSLP is a cytokine secreted
by airway epithelial cells in response to different stimuli such as TLR ligands or viruses. The signaling
cascade resulting from the binding of TSLP to its receptor on mast cells, dendritic cells and activated T
cells result in the promotion of a Th2-type inflammation.
The SNP rs1837253 was identified as a putative causal SNP based on consistent previous association
data both from candidate gene and genome-wide association studies; as well as the absence of significant linkage disequilibrium with other polymorphisms in the region. Previous data from our lab showed
the presence of this SNP in a (formaldehyde-assisted isolation of regulatory elements) FAIRE-enriched
DNA region.
Experimental design: DNA samples from lung of asthmatics and controls were genotyped for our
candidate SNP using Taqman technology and gene expression assays (qPCR) were performed to compare levels of expression between the different genotypes. These qPCR experiments were conducted
for both isoforms of TSLP (short and long isoforms). ANOVA tests were used to compare gene expression levels between groups.
In silico analysis was performed to predict binding of regulatory nuclear proteins on the SNP site. Electrophoretic mobility shift assays (EMSA) are underway in order to test for a potential differential binding of a regulatory protein depending on the SNP allele.
Results: Taqman genotyping was successful with 100% call rate; the data were in Hardy Weinberg
equilibrium. No homozygotes for the minor allele were present.
qPCR did not result in differential gene expression between phenotypes (fatal asthma or non asthma)
nor between genotypes. In all likelihood, this is due to a low N: 6/8 for the phenotypes and 7/7/0 for the
3 genotypes; in addition, these data are solely for the short isoform as we were unable to detect higher
enough levels of the long isoforms. Efforts are underway to increase production of the TSLP long
isoforms by stimulation of airway epithelial cells from asthmatics and controls, increase the number of
samples as well as pursue DNA and RNA samples from a different source (blood).
Preliminary EMSA data show differential binding of a nuclear protein derived from A549 cells to the T
allele and little or no binding of the C allele. In silico analysis resulted in the identification of potential
candidates for the binding as members of the FOX family, GR receptor alpha and members of the
HMG box group of proteins. Cold completion experiments using consensus sequences of candidate
factors are in progress.
Conclusion: The objective of this research is to explain the asthma association of a singleton SNP 2.5
Kb from the gene of interest, TSLP. The ultimate goal of these functional studies is to reach a greater
understanding of the molecular pathogenesis of asthma and eventually pave the way for novel therapies
targeting the source of inflammation rather than life-long therapies aimed at dampening inflammation
and easing symptoms.