Mechanisms of allergy and clinical immunology
A genome-wide survey of CD4+ lymphocyte regulatory genetic variants identifies novel asthma genes

https://doi.org/10.1016/j.jaci.2014.04.011Get rights and content

Background

Genome-wide association studies have yet to identify the majority of genetic variants involved in asthma. We hypothesized that expression quantitative trait locus (eQTL) mapping can identify novel asthma genes by enabling prioritization of putative functional variants for association testing.

Objective

We evaluated 6706 cis-acting expression-associated variants (eSNPs) identified through a genome-wide eQTL survey of CD4+ lymphocytes for association with asthma.

Methods

eSNPs were tested for association with asthma in 359 asthmatic patients and 846 control subjects from the Childhood Asthma Management Program, with verification by using family-based testing. Significant associations were tested for replication in 579 parent-child trios with asthma from Costa Rica. Further functional validation was performed by using formaldehyde-assisted isolation of regulatory elements (FAIRE) quantitative PCR and chromatin immunoprecipitation PCR in lung-derived epithelial cell lines (Beas-2B and A549) and Jurkat cells, a leukemia cell line derived from T lymphocytes.

Results

Cis-acting eSNPs demonstrated associations with asthma in both cohorts. We confirmed the previously reported association of ORMDL3/GSDMB variants with asthma (combined P = 2.9 × 10−8). Reproducible associations were also observed for eSNPs in 3 additional genes: fatty acid desaturase 2 (FADS2; P = .002), N-acetyl-α-D-galactosaminidase (NAGA; P = .0002), and Factor XIII, A1 (F13A1; P = .0001). Subsequently, we demonstrated that FADS2 mRNA is increased in CD4+ lymphocytes in asthmatic patients and that the associated eSNPs reside within DNA segments with histone modifications that denote open chromatin status and confer enhancer activity.

Conclusions

Our results demonstrate the utility of eQTL mapping in the identification of novel asthma genes and provide evidence for the importance of FADS2, NAGA, and F13A1 in the pathogenesis of asthma.

Section snippets

Methods

We provide a brief description of the cohorts below, with details shown in the supplementary text in this article's Online Repository at www.jacionline.org. The Institutional Review Board of Brigham and Women's Hospital approved these studies. Subject recruitment and procedures for the cohorts have been previously described.7, 11, 12

Asthma association testing of eSNPs

The baseline characteristics of the CAMP and Costa Rican index cases included in this study are presented in Table I. Baseline characteristics were similar between the CAMP and Costa Rican cohorts, including measures of asthma severity and lung function.

Of the 6706 SNPs identified as CD4+ lymphocyte cis-acting expression-associated eSNPs, 143 were associated with asthma under an additive genetic model in the CAMP cohort in either the case-control or family-based association analyses (see Table

Discussion

Although GWASs of complex traits have offered insight into the genetic underpinnings of complex diseases, such as asthma, the discovered variants explain only a fraction of the genetic contribution to such diseases.4 In part this is likely due to the stringent multiple-comparison burden imposed when testing millions of (mostly functionless) variants. Strategies that guide prioritization of functional genetic markers for testing can address this issue. We and others have demonstrated that eSNPs

The Asthma BRIDGE Consortium

Asthma BRIDGE Data Coordinating Center

CAMP Genetics Ancillary Study

Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts

Benjamin A. Raby, MD, MPH (PI); Scott T. Weiss, MD, MSc (PI); Vincent Carey, PhD; Weiliang Qiu, PhD; Roxanne Kelly, BSc; Jody Sylvia Senter, MS; John Ziniti, BSc; Diana Tubbs; Brooke Schumann; Damien Croteau-Chonka, PhD.

Childhood Asthma Research and Education (CARE) Network

Arizona Respiratory Center, University of Arizona (Coordinating Center), Tucson,

References (44)

  • D.G. Torgerson et al.

    Meta-analysis of genome-wide association studies of asthma in ethnically diverse North American populations

    Nat Genet

    (2011)
  • M.F. Moffatt et al.

    A large-scale, consortium-based genomewide association study of asthma

    N Engl J Med

    (2010)
  • T.A. Manolio et al.

    Finding the missing heritability of complex diseases

    Nature

    (2009)
  • E.E. Schadt et al.

    Genetics of gene expression surveyed in maize, mouse and man

    Nature

    (2003)
  • D.L. Nicolae et al.

    Trait-associated SNPs are more likely to be eQTLs: annotation to enhance discovery from GWAS

    PLoS Genet

    (2010)
  • A. Murphy et al.

    Mapping of numerous disease-associated expression polymorphisms in primary peripheral blood CD4+ lymphocytes

    Hum Mol Genet

    (2010)
  • W. Gu et al.

    Fringe controls naive CD4(+)T cells differentiation through modulating notch signaling in asthmatic rat models

    PLoS One

    (2012)
  • M. Busse et al.

    ICOS mediates the generation and function of CD4+CD25+Foxp3+ regulatory T cells conveying respiratory tolerance

    J Immunol

    (2012)
  • S.M.A. Sharma et al.

    eQTL mapping in peripheral blood CD4+ lymphocytes identifies replicable novel asthma susceptibility loci, augmenting standard genome-wide association testing [abstract]

    (2010)
  • The Childhood Asthma Management Program (CAMP): design, rationale, and methods. Childhood Asthma Management Program Research Group

    Control Clin Trials

    (1999)
  • A.L. Price et al.

    Principal components analysis corrects for stratification in genome-wide association studies

    Nat Genet

    (2006)
  • R. Fisher

    Combining independent tests of significance

    Am Stat

    (1948)
  • Cited by (48)

    • Genetic variants and risk of asthma in an American Indian population

      2017, Annals of Allergy, Asthma and Immunology
      Citation Excerpt :

      There are several, well-replicated genetic variants that have been previously associated with asthma, notably variants at 17q21.7,13–17 Although associated single-nucleotide polymorphisms (SNPs) in this region encompass at least a 380-Kb span,7 including 5 annotated genes,13,18 initial attention has centered primarily on ORMDL3 and GSDMB. Calcium homeostasis, sphingolipid metabolism, and lymphocyte function are affected by variants that influence expression of ORMDL3,19–22 whereas GSDMB is highly expressed in T lymphocytes and thus potentially affects the inflammatory response, particularly to viral infection.16,23

    • Interleukins (from IL-1 to IL-38), interferons, transforming growth factor β, and TNF-α: Receptors, functions, and roles in diseases

      2016, Journal of Allergy and Clinical Immunology
      Citation Excerpt :

      CD4+ T cells are divided into distinct subsets according to their cytokine profile.2 They differentiate from naive T cells, and their cytokine expression profile depends on the types of antigen-presenting cells (APCs), the type of the initial innate immune response, the adjuvanticity of the molecules presented with the antigen, and the existence and dose of many small molecules and other cytokines in the microenvironment.7 CD4+ naive T cells can differentiate into TH1, TH2, TH9, TH17, TH22, and follicular effector T cells, as well as different subsets of regulatory T (Treg) cells.4,8,9

    View all citing articles on Scopus

    Supported by R01 HL086601, RC2 HL101543, R37 HL066289, and K08 HL096833. All work on data collected from the CAMP Genetic Ancillary Study was conducted at the Channing Laboratory of the Brigham and Women's Hospital under appropriate CAMP policies and human subject protections. This work is supported by grants R01 HL086601 and RC2 HL101543 from the National Heart, Lung, and Blood Institute/National Institutes of Health (NIH/NHLBI). The CAMP Genetics Ancillary Study is supported by U01 HL075419, U01 HL65899, and P01 HL083069 and through the Colorado CTSA grant 1 UL1 RR025780 from the NIH and National Center for Research Resources. B.E.H. was supported by NIH K99 HL105663. S.S. receives additional support from K08 HL096833 from NIH/NHLBI. Additional support was provided by NIH P01ES011627 (PI: F. Gilliland). Supported in part by the Division of Intramural Research, National Institute of Environmental Health Sciences, NIH.

    Disclosure of potential conflict of interest: S. Sharma, R. Strunk, A. E. Berger, J. Fan, M. P. Boorgula, C. Ober, and V. J. Carey have received research support from the National Institutes of Health. S. J. Szefler has received research support from and has received travel support from the National Heart, Lung, and Blood Institute; has received consultancy fees from Merck, Genentech, Boehringer Ingelheim, and GlaxoSmithKline; has received research support from GlaxoSmithKline; has received lecture fees from Merck; has received payment for manuscript preparation from Genentech; and has a patent with the NHLBI CARE Network. M. Castro has received research support from the National Institutes of Health; has received support for travel from National Institutes of Health (for AsthmaNet); has consultancy arrangements with Asthmatx/Boston Scientific, Genentech, IPS, Pulmagen, and Sanofi Aventis; has received 1 or more grants from or has 1 or more grants pending with Asthmatx/Boston Scientific, Amgen, Ception/Cephalon/Teva, Genentech, MedImmune, Merck, Novartis, GlaxoSmithKline, Sanofi Aventis, Vectura, Next Bio, and KaloBios; has received 1 or more payments for lecturing from or is on the speakers' bureau for Merck, GlaxoSmithKline, Genentech, Asthmatx/Boston Scientific, and Boehringer Ingelheim; and has received royalties from Elsevier. G. B. Diette has received research support from the Baurenschmidt Foundation. B. M. Vonakis has received research support from the NIH; has received consultancy fees from Sanofi-US; is employed by Johns Hopkins University; has received research support from the NHLBI, Sanofi-US, Novaremed, Los Alamos National Laboratory, and Luna Innovations; has received lecture fees from the NIH; and has received travel support from the AAI. L. Avila has received research support from the Costa Rica Genetics of Asthma Study (R37 HL066289). R. F. Lemanske, Jr, has received research support from, has received consultancy fees from, and has received participation fees from the NIH; is a board member for the American Academy of Allergy, Asthma & Immunology (AAAAI); has received consultancy fees from Merck, Sepracor, SA Boney and Associates, GlaxoSmithKline, the American Institute of Research, Genentech, and Double Helix Development; and Boerhinger Ingelheim; is employed by the University of Wisconsin School of Medicine and Public Health; has received research support from the National Heart, Lung, and Blood Institute and Pharmaxis; has received lecture fees from the Michigan Public Health Institute, Allegheny General Hospital, the American Academy of Pediatrics, West Allegheny Health Systems, California Chapter 4, the AAP, the Colorado Allergy Society, the Pennsylvania Allergy and Asthma Association, Harvard Pilgrim Health, the California Society of Allergy, the NYC Allergy Society, the World Allergy Organization, the American College of Chest Physicians, APAPARI, and the Western Society of Allergy, Asthma, and Immunology; has received payment for manuscript preparation from the AAAAI; and has received royalties from Elsevier and UpToDate. J. Solway has received research support from NIH grant U54 TR000016 and NIH grant K12 HL090003; is a board member for PulmOne Advanced Medical Devices; has received consultancy fees from Hollister, Cytokinetics, and the Novartis Institute for Biomedical Research; has received research support from the NIH (grants P50 HL107171, R01 HL097805, P01 HL090554, T32 HL07605, U10 HL098096, R01 HL107361, U19 AI095230, K12 HL119995, and R01 HL113395) and AstraZeneca; has a patent with and has received royalties from Boston Scientific; is a Principal Investigator on the Chicago Metropolitan AsthmaNet Consortium site funded by the NHLBI and is participating in an AsthmaNet clinical trial by using 2 medications donated by Sunovion Pharmaceuticals. S. R. White has provided expert testimony for Shook, Hardy & Bacon, LLC; has received research support from the National Institute of Allergy and Infectious Diseases, the NHLBI, and the University of Chicago CTSA; and has received lecture fees from Pfizer. D. Nicolae has received research support from the NIH/NHLBI. K. Barnes has received research support from the NIH; is a board member for Genentech; has received consultancy fees from Sanofi-Aventis, Sirius Genomics, and Merck; is employed by the JHU; has received research support from the NIH and the NHLBI; and has received royalties from UpToDate. F. Martinez has received research support from the NIH and has received travel support from Abbott and Merck. J. C. Celedón has received consultancy fees from Genentech; has received research support from the NIH; and has received royalties from UpToDate. S. T. Weiss has received consultancy fees from Novartis. B. A. Raby has received research support from the NIH, has received consultancy fees from Merck, and has received royalties from UpToDate.

    These authors contributed equally to this work.

    View full text