GSTCD and INTS12 Regulation and Expression in the Human Lung Ma’en Obeidat1,4☯, Suzanne Miller1☯, Kelly Probert1, Charlotte K. Billington1, Amanda P. Henry1, Emily Hodge1, Carl P. Nelson1, Ceri E. Stewart1, Caroline Swan1, Louise V. Wain2, María Soler Artigas2, Erik Melén3, Kevin Ushey4, Ke Hao5, Maxime Lamontagne7, Yohan Bossé6,7, Dirkje S. Postma8, Martin D. Tobin2,9, Ian Sayers1, Ian P. Hall1* 1 Division of Respiratory Medicine, University of Nottingham, Queen’s Medical Center, Nottingham, United Kingdom, 2 Genetic Epidemiology Group, Department of Health Sciences, University of Leicester, Leicester, United Kingdom, 3 Institute of Environmental Medicine, Karolinska Institutet and Sachs’ Children’s Hospital, Stockholm, Sweden, 4 James Hogg Research Centre, Institute for Heart and Lung Health, University of British Columbia, Vancouver, British Columbia, Canada, 5 Department of Genetics and Genomic Sciences, Icahn Institute of Genomics and Multiscale Biology, Icahn School of Medicine at Mount Sinai, New York, United States of America, 6 Department of Molecular Medicine, Laval University, Québec City, Canada, 7 Institut Universitaire de Cardiologie et de Pneumologie de Québec, Laval University, Québec City, Canada, 8 Department of Pulmonology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands, 9 National Institute for Health Research (NIHR) Leicester Respiratory Biomedical Research Unit, Glenfield Hospital, Leicester, United Kingdom Abstract Genome-Wide Association Study (GWAS) meta-analyses have identified a strong association signal for lung function, which maps to a region on 4q24 containing two oppositely transcribed genes: glutathione S-transferase, C- terminal domain containing (GSTCD) and integrator complex subunit 12 (INTS12). Both genes were found to be expressed in a range of human airway cell types. The promoter regions and transcription start sites were determined in mRNA from human lung and a novel splice variant was identified for each gene. We obtained the following evidence for GSTCD and INTS12 co-regulation and expression: (i) correlated mRNA expression was observed both via Q-PCR and in a lung expression quantitative trait loci (eQTL) study, (ii) induction of both GSTCD and INTS12 mRNA expression in human airway smooth muscle cells was seen in response to TGFβ1, (iii) a lung eQTL study revealed that both GSTCD and INTS12 mRNA levels positively correlate with percent predicted FEV1, and (iv) FEV1 GWAS associated SNPs in 4q24 were found to act as an eQTL for INTS12 in a number of tissues. In fixed sections of human lung tissue, GSTCD protein expression was ubiquitous, whereas INTS12 expression was predominantly in epithelial cells and pneumocytes. During human fetal lung development, GSTCD protein expression was observed to be highest at the earlier pseudoglandular stage (10-12 weeks) compared with the later canalicular stage (17-19 weeks), whereas INTS12 expression levels did not alter throughout these stages. Knowledge of the transcriptional and translational regulation and expression of GSTCD and INTS12 provides important insights into the potential role of these genes in determining lung function. Future work is warranted to fully define the functions of INTS12 and GSTCD. Citation: Obeidat M, Miller S, Probert K, Billington CK, Henry AP, et al. (2013) GSTCD and INTS12 Regulation and Expression in the Human Lung. PLoS ONE 8(9): e74630. doi:10.1371/journal.pone.0074630 Editor: Rory Edward Morty, University of Giessen Lung Center, Germany Received May 2, 2013; Accepted August 5, 2013; Published September 18, 2013 Copyright: © 2013 Obeidat et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The work performed at the University of Nottingham and included in this manuscript was funded by the Medical Research Council (G1000861). Martin D. Tobin holds a Medical Research Council Senior Clinical Fellowship (G0902313) (www.MRC.ac.uk). The resequencing study performed at the Univserity of Leicester was funded by Pfizer. Yohan Bossé is a research scholar from the Heart and Stroke Foundation of Canada. Erik Melen has received funding from The Swedish Research Council, The Swedish Heart-Lung Foundation and Stockholm County Council (ALF). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript Competing interests: The resequencing study performed at the University of Leicester was funded by Pfizer. There are no patents, products in development or marketed products to declare. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors. * E-mail: [email protected] ☯ These authors contributed equally to this work. PLOS ONE | www.plosone.org 1 September 2013 | Volume 8 | Issue 9 | e74630 GSTCD and INTS12 Regulation in the Human Lung Introduction correlation=0.48, P=0.0002) [15]. This, and the fact that the 4q24 locus has the strongest novel association with lung Forced expiratory volume in one second (FEV1), forced vital function in the multiple GWAS meta-analyses conducted so far, capacity (FVC), and the ratio of FEV1 to FVC (FEV 1/FVC) are have provided the rationale to further investigate this locus to commonly used to assess pulmonary function and these unravel the mechanisms underlying this association. measurements are integral to the diagnosis of chronic Here we report the genetic architecture of GSTCD and obstructive pulmonary disease (COPD). Reduced FEV 1/FVC INTS12, and show that both genes are expressed in a range of defines airway obstruction, whereas reduced FEV1 grades the cell types present in the lung. We report one novel splice severity of airway obstruction [1]. These measures also predict variant for each gene. Furthermore we show that the genome- population morbidity and mortality [2]. wide significant SNPs at 4q24 act as expression quantitative Pulmonary function is determined both by environmental and trait loci (eQTL) for INTS12, and provide evidence that the genetic factors. Tobacco smoking is the major environmental peak association signal maps to a region rich in elements likely risk factor for the development of COPD in the developed to be important for transcriptional regulation of these genes. world. A genetic contribution to pulmonary function is well Interestingly, these appear to be coordinately regulated in the established with heritability estimates reaching as high as 77% lung. Expression levels were also shown to correlate with lung for FEV1 [3]. Three large scale meta-analyses of genome-wide function measures. Finally, we describe the expression of association studies (GWAS) of lung function measures have GSTCD and INTS12 proteins in human lung tissue and assess recently been published [4-6]: these studies identified a total of how expression is altered during human fetal development. 26 novel loci associated with either FEV1 or FEV 1/FVC. Genome-wide interaction analyses with smoking later identified Results three additional regions of potential importance for lung function [7]. One of the strongest association signals identified Gene arrangement via PCR and RACE was with intronic Single Nucleotide Polymorphisms (SNPs) in a Using cDNA synthesised from RNA extracted from total lung, region at 4q24 containing two oppositely transcribed genes: human airway smooth muscle (HASM) cells and normal human glutathione S-transferase, C-terminal domain containing bronchial epithelial cells (HBEC), Reverse Transcription (RT)- (GSTCD) and integrator complex subunit 12 (INTS12). In the PCR was initially performed to investigate whether gene SpiroMeta study [5], SNP rs10516526 in intron 5 of GSTCD arrangements are consistent with those reported by NCBI (the -23 was associated with FEV1 (P=2.18x10 in the joint meta- National Center for Biotechnology Information). For both analysis of discovery and replication cohorts (n=53,309)). In the GSTCD and INTS12 a novel splice variant was discovered via combined SpiroMeta CHARGE meta-analysis with larger this method (named here “variant 3”). Figure 1 outlines sample size (discovery n=48,201), SNP rs10516526 also schematically the two published variants in addition to the showed the strongest association in the 4q24 locus for FEV1 novel variant 3 in each case. The SpiroMeta genome-wide (P=4.75x10-14) [6]. Subsequent reports by SpiroMeta significant SNPs for association with FEV1 in this locus are investigators and others have also implicated this locus for annotated on the schematic in Figure 1 along with the relevant association with COPD [8,9]. association statistical P values [5]. At present, little is known about the potential function of To confirm published or reveal novel transcription start site GSTCD – all four entries on PubMed are related to recent (TSS) s for both GSTCD and INTS12, Rapid Amplification of GWAS studies [4,5,8,9]. GSTCD is so named because of cDNA Ends (RACE) was performed. For GSTCD, a total of 30 homology with the glutathione S-transferase (GST) super clones were sequenced and analysed. 5’ RACE confirmed the family of enzymes [10]. GSTs are also involved in the presence of variant 2 (NM_024751.2) in 13 of these (43%), detoxification of products of oxidative stress [11] and synthesis albeit with multiple TSSs spanning a region of 50bp;