Received: 17 March 2022 | Revised: 1 May 2022 | Accepted: 4 May 2022 DOI: 10.1111/pai.13802 ORIGINAL ARTICLE Multi-­ancestry genome-­wide association study of asthma exacerbations Esther Herrera-­Luis1 | Victor E. Ortega2 | Elizabeth J. Ampleford3 | Yang Yie Sio4 | Raquel Granell5 | Emmely de Roos6,7 | Natalie Terzikhan6,7 | Ernesto Elorduy Vergara8 | Natalia Hernandez-­Pacheco9,10 | Javier Perez-­Garcia1 | Elena Martin-­Gonzalez1 | Fabian Lorenzo-­Diaz1,11 | Simone Hashimoto12 | Paul Brinkman12 | U-­BIOPRED Study Group* | Andrea L. Jorgensen13 | Qi Yan14 | Erick Forno15 | 16,17,18 5 1 Susanne J. Vijverberg | Ryan Lethem | Antonio Espuela-­Ortiz | Mario Gorenjak19 | Celeste Eng20 | Ruperto González-­Pérez21,22 | José M. Hernández-­Pérez23,24 | Paloma Poza-­Guedes21,22 | Olaia Sardón25,26 | Paula Corcuera25 | Greg A. Hawkins27 | Annalisa Marsico28 | Thomas Bahmer29,30 | Klaus F. Rabe29,30 | Gesine Hansen31 | Matthias Volkmar Kopp32,33,34 | Raimon Rios35 | Maria Jesus Cruz10,36 | Francisco-­Javier González-­Barcala37 | José María Olaguibel10,38 | 10,39 10,40 41 42 Vicente Plaza | Santiago Quirce | Glorisa Canino | Michelle Cloutier | 10,43 44 26 Victoria del Pozo | Jose R. Rodriguez-­Santana | Javier Korta-­Murua | 10,45 46 47 48 Jesús Villar | Uroš Potočnik | Camila Figueiredo | Michael Kabesch | 49,50 51 52,53,54 Somnath Mukhopadhyay | Munir Pirmohamed | Daniel B. Hawcutt | 9,55 50 56 Erik Melén | Colin N. Palmer | Steve Turner | 16,17,18 57,58,59 Anke H. Maitland-­van der Zee | Erika von Mutius | Juan C. Celedón15 | 6,7,60 4 61 61 Guy Brusselle | Fook Tim Chew | Eugene Bleecker | Deborah Meyers | 22,62 1,10,63 Esteban G. Burchard | Maria Pino-­Yanes 1 Genomics and Health Group, Department of Biochemistry, Microbiology, Cell Biology and Genetics, Universidad de La Laguna (ULL), San Cristóbal de La Laguna, Tenerife, Spain 2 Division of Respiratory Medicine, Department of Internal Medicine, Mayo Clinic, Scottsdale, Arizona, USA 3 Department of Internal Medicine, Center for Precision Medicine, Wake Forest School of Medicine, Winston-­Salem, North Carolina, USA 4 Department of Biological Sciences, National University of Singapore, Singapore City, Singapore 5 MRC Integrative Epidemiology Unit (IEU), Population Health Sciences, Bristol Medical School, University of Bristol, Bristol, UK 6 Department of Epidemiology, Erasmus University Medical Center, Rotterdam, The Netherlands 7 Department of Respiratory Medicine, Ghent University Hospital, Ghent, Belgium 8 Institute of Computation Biology, Helmholtz Zentrum München, German Research Center for Environmental Health, Munich, Germany 9 Department of Clinical Sciences and Education, Södersjukhuset, Karolinska Institutet, Stockholm, Sweden Abbreviations: 1KGP, 1000 Genomes Project; CDK6, cyclin-­dependent kinase 6; CI, confidence interval; GAG, glycosaminoglycan; GTEx, genotype-­tissue expression; GWAS, genome-­wide association study; LPS, lipopolysaccharide; MAF, minor allele frequency; meQTL, methylation quantitative trait loci; OR, odds ratio; PPAR-­α , peroxisome proliferator-­ activating receptor α; RR, relative risk; SNP, single-­nucleotide polymorphism; TLR4, Toll-­like receptor 4; TNFα, tumor necrosis factor α. *The members of the U-­BIOPRED Study Group are provided in the Appendix S1. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2022 The Authors. Pediatric Allergy and Immunology published by European Academy of Allergy and Clinical Immunology and John Wiley & Sons Ltd. Pediatr Allergy Immunol. 2022;33:e13802.  wileyonlinelibrary.com/journal/pai | 1 of 17 https://doi.org/10.1111/pai.13802 | 13993038, 2022, 6, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/pai.13802 by Readcube (Labtiva Inc.), Wiley Online Library on [02/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 2 of 17 HERRERA-­LUIS et al. 10 CIBER de Enfermedades Respiratorias (CIBERES), Madrid, Spain 11 Instituto Universitario de Enfermedades Tropicales y Salud Pública de Canarias (IUETSPC), Universidad de La Laguna (ULL), San Cristóbal de La Laguna, Tenerife, Spain 12 Department of Respiratory Medicine, Amsterdam University Medical Center, University of Amsterdam, Amsterdam, The Netherlands 13 Department of Health Data Science, Institute of Population Health, University of Liverpool, Liverpool, UK 14 Department of Obstetrics and Gynecology, Columbia University Irving Medical Center, New York, New York, USA 15 Division of Pediatric Pulmonary Medicine, UPMC Children's Hospital of Pittsburgh, University of Pittsburgh, Pittsburgh, Pennsylvania, USA 16 Department of Respiratory Medicine, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands 17 Division of Pharmacoepidemiology and Clinical Pharmacology, Faculty of Science, Utrecht University, Utrecht, The Netherlands 18 Department of Paediatric Respiratory Medicine and Allergy, Emma's Children Hospital, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands 19 Center for Human Molecular Genetics and Pharmacogenomics, Faculty of Medicine, University of Maribor, Maribor, Slovenia 20 Department of Medicine, University of California San Francisco, San Francisco, California, USA 21 Allergy Department, Hospital Universitario de Canarias, Santa Cruz de Tenerife, Tenerife, Spain 22 Severe Asthma Unit, Allergy Department, Hospital Universitario de Canarias, Santa Cruz de Tenerife, Tenerife, Spain 23 Pulmonary Medicine, Hospital Universitario de N.S de Candelaria, Santa Cruz de Tenerife, Spain 24 Pulmonary Medicine, Hospital General de La Palma, La Palma, Santa Cruz de Tenerife, Spain 25 Division of Pediatric Respiratory Medicine, Hospital Universitario Donostia, San Sebastián, Spain 26 Department of Pediatrics, University of the Basque Country (UPV/EHU), San Sebastián, Spain 27 Department of Biochemistry, Wake Forest School of Medicine, Winston-­Salem, North Carolina, USA 28 Computational Health Center, Helmholtz Zentrum München, German Research Center for Environmental Health, Munich, Germany 29 LungenClinic Grosshansdorf, Pneumology, Grosshansdorf, Germany 30 Airway Research Center North (ARCN), Members of the Germany Center for Lung Research (DZL), Grosshansdorf, Germany 31 Department of Pediatric Pneumology, Allergology and Neonatology, Hannover Medical School, Hannover, Germany 32 Division of Pediatric Pneumology & Allergology, University Medical Center Schleswig-­Holstein, Lübeck, Germany 33 Airway Research Center North (ARCN), Members of the Germany Center for Lung Research (DZL), Lübeck, Germany 34 Department of Paediatric Respiratory Medicine, Inselspital, University Children's Hospital of Bern, University of Bern, Bern, Switzerland 35 Programa de Pós Graduação em Imunologia (PPGIm), Instituto de Ciências da Saúde, Universidade Federal da Bahia (UFBA), Salvador, Brazil 36 Servicio de Neumología, Hospital Vall d’Hebron, Barcelona, Spain 37 Servicio de Neumología, Complejo Hospitalario Universitario de Santiago, Santiago de Compostela, Spain 38 Servicio de Alergología, Complejo Hospitalario de Navarra, Pamplona, Navarra, Spain 39 Departamento de Medicina Respiratoria, Hospital de la Santa Creu i Sant Pau, Instituto de Investigación Biomédica Sant Pau (IIB Sant Pau), Barcelona, Spain 40 Department of Allergy, La Paz University Hospital, IdiPAZ, Madrid, Spain 41 Behavioral Sciences Research Institute, University of Puerto Rico, San Juan, Puerto Rico 42 Department of Pediatrics, University of Connecticut, Farmington, Connecticut, USA 43 Immunology Department, Instituto de Investigación Sanitaria Hospital Universitario Fundación Jiménez Díaz, Madrid, Spain 44 Centro de Neumología Pediátrica, San Juan, Puerto Rico 45 Multidisciplinary Organ Dysfunction Evaluation Research Network, Research Unit, Hospital Universitario Dr. Negrín, Las Palmas de Gran Canaria, Spain 46 Laboratory for Biochemistry, Molecular Biology and Genomics, Faculty for Chemistry and Chemical Engineering, University of Maribor, Maribor, Slovenia 47 Instituto de Ciências da Saúde, Universidade Federal da Bahia, Salvador, Brazil 48 Department of Paediatric Pneumology and Allergy, University Children's Hospital Regensburg (KUNO), Regensburg, Germany 49 Academic Department of Paediatrics, Brighton and Sussex Medical School, Royal Alexandra Children's Hospital, Brighton, UK 50 Population Pharmacogenetics Group, Biomedical Research Institute, Ninewells Hospital and Medical School, University of Dundee, Dundee, UK 51 Department of Pharmacology and Therapeutics, Institute of Systems, Molecular and Integrative Biology, University of Liverpool, Liverpool, UK 52 Department of Women's and Children's Health, University of Liverpool, Liverpool, UK 53 Alder Hey Children's Hospital, Liverpool, UK 54 NIHR Alder Hey Clinical Research Facility, Alder Hey Children's Hospital, Liverpool, UK 55 Sachs’ Children’s Hospital, South General Hospital, Stockholm, Sweden 56 Child Health, University of Aberdeen, Aberdeen, UK 57 Institute for Asthma and Allergy Prevention, Helmholtz Zentrum München, German Research Center for Environmental Health, Munich, Germany 58 Dr von Hauner Children's Hospital, Ludwig-­Maximilians-­Universität, Munich, Germany 59 Comprehensive Pneumology Center Munich (CPC-­M), Member of the German Center for Lung Research, Munich, Germany 60 Department of Respiratory Medicine, Erasmus University Medical Center, Rotterdam, The Netherlands 61 Division of Genetics, Genomics, and Precision Medicine, Department of Internal Medicine, University of Arizona College of Medicine, Tucson, Arizona, USA 62 Department of Bioengineering and Therapeutic Sciences, University of California San Francisco, San Francisco, California, USA 63 Instituto de Tecnologías Biomédicas (ITB), Universidad de La Laguna (ULL), San Cristóbal de La Laguna, Tenerife, Spain | 13993038, 2022, 6, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/pai.13802 by Readcube (Labtiva Inc.), Wiley Online Library on [02/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License HERRERA-­LUIS et al. 3 of 17 Correspondence Maria Pino-­Yanes, Genomics and Health Abstract Group, Department of Biochemistry, Background: Asthma exacerbations are a serious public health concern due to high Microbiology, Cell Biology and Genetics, Universidad de La Laguna (ULL), Apartado healthcare resource utilization, work/school productivity loss, impact on quality of 456, 38200 San Cristóbal de La Laguna, life, and risk of mortality. The genetic basis of asthma exacerbations has been stud- Tenerife, Spain. Email: mdelpino@ull.edu.es ied in several populations, but no prior study has performed a multi-­ancestry meta-­ analysis of genome-­wide association studies (meta-­GWAS) for this trait. We aimed to Funding information This work was funded by the Spanish identify common genetic loci associated with asthma exacerbations across diverse Ministry of Science and Innovation MCIN/ populations and to assess their functional role in regulating DNA methylation and AEI/10.13039/501100011033, and the European Regional Development Fund gene expression. “ERDF A way of making Europe” by the Methods: A meta-­GWAS of asthma exacerbations in 4989 Europeans, 2181 European Union (SAF2017-­83417R), by MCIN/AEI/10.13039/501100011033 Hispanics/Latinos, 1250 Singaporean Chinese, and 972 African Americans analyzed (PID2020-­116274RB-­I 00) and by the 9.6 million genetic variants. Suggestively associated variants (p ≤ 5 × 10−5) were as- Allergopharma-­E AACI award 2021. This study was also supported by sessed for replication in 36,477 European and 1078 non-­European asthma patients. the SysPharmPedia grant from the Functional effects on DNA methylation were assessed in 595 Hispanic/Latino and ERACoSysMed 1st Joint Transnational Call from the European Union under African American asthma patients and in publicly available databases. The effect on the Horizon 2020. GALA II and SAGE gene expression was evaluated in silico. studies were supported by the Sandler Family Foundation, the American Asthma Results: One hundred and twenty-­six independent variants were suggestively associated Foundation, the RWJF Amos Medical with asthma exacerbations in the discovery phase. Two variants independently repli- Faculty Development Program, Harry Wm. and Diana V. Hind Distinguished cated: rs12091010 located at vascular cell adhesion molecule-­1/exostosin like glycosyl- Professor in Pharmaceutical Sciences transferase-­2 (VCAM1/EXTL2) (discovery: odds ratio (ORT allele) = 0.82, p = 9.05 × 10−6 and II, the National Heart, Lung, and Blood Institute of the National Institutes of replication: ORT allele = 0.89, p = 5.35 × 10−3) and rs943126 from pantothenate kinase 1 Health (R01HL117004, R01HL128439, (PANK1) (discovery: ORC allele = 0.85, p = 3.10 × 10−5 and replication: ORC allele = 0.89, R01HL135156, X01HL134589, R01HL141992, and R01HL141845), p = 1.30 × 10−2). Both variants regulate gene expression of genes where they locate and National Institute of Health and DNA methylation levels of nearby genes in whole blood. Environmental Health Sciences (R01ES015794 and R21ES24844); Conclusions: This multi-­ancestry study revealed novel suggestive regulatory loci for the National Institute on Minority asthma exacerbations located in genomic regions participating in inflammation and host Health and Health Disparities (NIMHD) (P60MD006902, R01MD010443, and defense. R56MD013312); the National Institute of General Medical Sciences (NIGMS) KEYWORDS (RL5GM118984); the Tobacco-­Related asthma exacerbations, EXTL2, GWAS, PANK1, single-­nucleotide polymorphism Disease Research Program (24RT-­0 025 and 27IR-­0 030); and the National Human Genome Research Institute (NHGRI) (U01HG009080) to EGB. The PACMAN study was funded by a strategic alliance between GlaxoSmithKline and Utrecht Institute for Pharmaceutical Sciences. The Slovenia study was financially supported by the Slovenian Research Agency (research core funding No. P3-­0 067) and from SysPharmPediA grant, co-­financed by the Ministry of Education, Science and Sport Slovenia (MIZS) (contract number C3330-­16-­500106). The SHARE Bioresource (GoSHARE) and SHARE have ongoing funding from NHS Research Scotland and were established by funding from The Wellcome Trust Biomedical Resource [Grant No. 099177/Z/12/Z]. Genotyping of samples from BREATHE, PAGES, and GoSHARE was funded by AC15/00015 and conducted at the Genotyping National Centre (CeGEN) CeGen-­PRB3-­ISCIII; supported by ISCIII and European Regional Development | 13993038, 2022, 6, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/pai.13802 by Readcube (Labtiva Inc.), Wiley Online Library on [02/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 4 of 17 HERRERA-­LUIS et al. Fund (ERDF) (PT17/0019). ALSPAC was supported by the UK Medical Research Council and Wellcome (102215/2/13/2) and the University of Bristol. The Swedish Heart-­Lung Foundation, the Swedish Research Council, and Region Stockholm (ALF project and database maintenance) funded the BAMSE study. The PASS study was funded by the NHS Chair of Pharmacogenetics via the UK Department of Health. U-­BIOPRED was funded by the Innovative Medicines Initiative (IMI) Joint Undertaking, under grant agreement no. 115010, resources for which are composed of financial contribution from the European Union's Seventh Framework Programme (FP7/2007-­2013) and kind contributions from companies in the European Federation of Pharmaceutical Industries and Associations (EFPIA). Genotyping of samples from GEMAS and MEGA studies was funded by the Spanish Ministry of Science and Innovation (SAF2017-­87417R) at the Spanish National Cancer Research Centre, in the Human Genotyping lab, a member of CeGen, PRB3, and was supported by grant PT17/0019, of the PE I+D+i 2013-­ 2016, funded by ISCIII and ERDF. The genotyping of GEMAS was also partially funded by Fundación Canaria Instituto de Investigación Sanitaria de Canarias (PIFIISC19/17). The Rotterdam Study was funded by Erasmus Medical Center and Erasmus University Rotterdam; Netherlands Organization for the Health Research and Development (ZonMw); the Research Institute for Diseases in the Elderly (RIDE); the Ministry of Education, Culture and Science, the Ministry for Health, Welfare and Sports, the European Commission (DG XII), and the Municipality of Rotterdam. ALLIANCE Cohort was funded by grants from the German Federal Ministry of Education and Research (Bundesministerium für Bildung und Forschung, BMBF) as part of the German Centre for Lung Research (DZL) funding. The Hartford-­Puerto Rico study was funded by the U.S. National Institutes of Health (grant HL07966 to JCC). MP-­Y was funded by the Ramón y Cajal Program (RYC-­2015-­17205) by MCIN/ AEI/10.13039/501100011033 and by the European Social Fund “ESF Investing in your future”. MP-­Y and JV were supported by CIBER de Enfermedades Respiratorias, Instituto de Salud Carlos III, Spain (CB/06/06/1088). EH-­L was supported by a fellowship awarded by MCIN/AEI/10.13039/501100011033 and by “ESF Investing in your future” (PRE2018-­0 83837). JP-­G was supported by a fellowship awarded by Spanish Ministry of Universities (FPU19/02175). AE-­O reports funding from the Spanish Ministry of Science, Innovation, and Universities (MICIU) and Universidad de La Laguna (ULL). NH-­P was supported | 13993038, 2022, 6, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/pai.13802 by Readcube (Labtiva Inc.), Wiley Online Library on [02/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License HERRERA-­LUIS et al. 5 of 17 by a Medium-­Term Research Fellowship by the European Academy of Allergy and Clinical Immunology (EAACI) and a Long-­Term Research Fellowship by the European Respiratory Society (ERS) (LTRF202101-­0 0861). UP and MG were supported by the Ministry of Education, Science and Sport of the Republic of Slovenia, grant PERMEABLE (contract number C3330-­19-­252012). SCSGES results were contributed by authors FTC and YYS. FTC has received research support from the Singapore Ministry of Education Academic Research Fund, Singapore Immunology Network (SIgN), National Medical Research Council (NMRC) (Singapore), Biomedical Research Council (BMRC) (Singapore), and the Agency for Science Technology and Research (A*STAR) (Singapore); Grant Numbers: N-­154-­0 00-­038-­0 01, R-­154-­ 000-­191-­112, R-­154-­0 00-­4 04-­112, R-­154-­ 000-­553-­112, R-­154-­0 00-­565-­112, R-­ 154-­0 00-­630-­112, R-­154-­0 00-­A08-­592, R-­154-­0 00-­A 27-­597, R-­154-­0 00-­A91-­592, R-­154-­0 00-­A95-­592, R-­154-­0 00-­ B99-­114, BMRC/01/1/21/18/077, BMRC/04/1/21/19/315, SIgN-­06-­0 06, SIgN-­0 8-­020, NMRC/1150/2008, and H17/01/a0/008. F.T.C. has received consulting fees from Sime Darby Technology Centre; First Resources Ltd; Genting Plantation, and Olam International, outside the submitted work. YYS has received research support from the NUS Resilience & Growth Postdoctoral Fellowships with grant number: R-­141-­0 00-­036-­281. QY conducted the analysis from Hartford-­ Puerto Rico and United Kingdom Biobank studies. QY was funded by the U.S. National Institutes of Health (HL138098). All funding agencies had no role in the study design, data collection, and analysis, decision to publish, or preparation of the manuscript. The views expressed are those of the authors and not necessarily those of the any funder, National Health Service (NHS), the National Institute for Health Research (NIHR) or the United Kingdom Department of Health. Editor: Ömer Kalayci 1 | I NTRO D U C TI O N Asthma exacerbations are defined as worsening of respira- tory symptoms requiring hospitalization, unscheduled/emergency Asthma is a common chronic inflammatory airway disorder affect- asthma care, and/or use of systemic corticosteroids.3 Prevention of ing over 300 million people worldwide. The disparities in asthma asthma exacerbations is a major public health priority due to their prevalence across populations reflect a complex interplay between associated consequences on health (i.e., decreased quality of life, ac- environmental exposures (i.e., air pollution and viral infections), celerated decline in lung function, or mortality), school attendance, behavioral and socioeconomic factors (i.e., treatment adherence work productivity, and healthcare costs.1,4,5 To date, the best predic- and healthcare access), and genetic ancestry, which is inferred tor of future exacerbations is the occurrence of one in the previous from whole-­genome variation and tracks geographic and histori- year.6 Thus, identifying potential biomarkers to guide the reduction cal factors and the aforementioned factors influencing asthma and prevention of exacerbations is a priority for therapeutics devel- 1,2 prevalence. opment and for precision medicine of asthma. | 13993038, 2022, 6, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/pai.13802 by Readcube (Labtiva Inc.), Wiley Online Library on [02/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 6 of 17 HERRERA-­LUIS et al. With the advent of high-­throughput sequencing and genotyping technologies, the study of the genetic contributions to asthma ex- Key Message acerbations has shifted from hypothesis-­driven, limited candidate-­ A large multi-­ancestry meta-­analysis of GWAS of asthma gene strategies to genome-­wide association studies (GWAS).7–­14 exacerbations revealed two novel susceptibility loci lo- Pharmacogenomics studies of asthma exacerbations as an out- cated close to PANK1 and at the intergenic region of come of treatment response have identified five suggestive asso- VCAM1 and EXTL2. These loci decreased PANK1 and ciations for asthma exacerbations despite inhaled corticosteroids 9 8 11 EXTL2 gene expression in whole blood, respectively. Both (CMTR1, APOBEC3B-­APOBEC3C, and CACNA2D3-­WNT5A ), or genetic variants were associated with DNA methylation long-­acting beta2-­agonists (TBX3 and EPHA7).10 Beyond pharma- levels at CpG sites nearby. Our results identified two gene cogenomics, other studies have focused on asthma exacerbations targets for asthma exacerbations that should be further independently of treatment. In European-­ descent populations, explored to assess their specific role in asthma. CDHR3, CTNNA3, and HLA-­DQB1 have been associated with se- 7,13 vere asthma exacerbations. More recently, the representation of ethnically diverse populations has increased in GWAS of asthma exacerbations. A meta-­analysis of GWAS in Hispanic/Latino chil- dren identified a single-­nucleotide polymorphism (SNP) at FLJ22447 two studies, and 201 Filipinos from one study (Table S2). A detailed that modulated KCNJ2-­AS1 expression in nasal epithelium through description of each study is available in the Appendix S1. All stud- 12 DNA methylation. In Hispanic/Latinos and African Americans, a ies included were approved by their respective Institutional review genome-­wide significant locus for asthma with exacerbations regu- boards, and written informed consent was provided by participants lated LINC01913 lung gene expression and DNA methylation levels or their parents/caregivers. All methods followed the Declaration of of the PKDCC gene in whole blood.14 However, none of those stud- Helsinki guidelines. ies has approached the search for genetic determinants of asthma Assessment of genetic ancestry was performed using principal exacerbations independently of treatment from a multi-­ancestry component analysis. The Haplotype Reference Consortium (r1.1 framework. 2016)15 was used as the reference imputation panel for most stud- To improve our understanding on genetic and biological mecha- ies, except for Avon Longitudinal Study of Parents and Children nisms of asthma exacerbations across multiple populations, we con- (ALSPAC) and Singapore Cross Sectional Genetic Epidemiology ducted the first multi-­ancestry meta-­analysis of GWAS of asthma Study (SCSGES), which used the phase 3 of the 1000 Genomes exacerbations independently of treatment and attempted to vali- Project (1KGP).16 Genotyping and imputation procedures for the dis- date previous associations. Then, we conducted in silico and in vivo covery and replication studies are detailed in the Appendix S1 and downstream analyses to assess the potential functional effects of Tables S1 and S2. the associated SNPs over DNA methylation and gene expression. 2.2 | Association analysis 2 | M E TH O D S Association between genetic variants and asthma exacerbations was 2.1 | Study design and study populations tested using logistic regression models including age, sex, and principal components from the genotype matrix (if needed to correct for popu- We performed a two-­stage study to identify genetic variants associ- lation stratification) (Table S1). Analyses were conducted separately ated with asthma exacerbations, defined as a binary variable based on for each study using PLINK 2.0,17 EPACTS 3.2.618 or rvtests 2.1.0.19 the presence of emergency care, hospitalizations, or administration Results were filtered with the EasyQC software20 to retain variants of systemic corticosteroids because of asthma. We also considered with a minor allele frequency (MAF) ≥ 1% and imputation quality 3 a definition of moderate exacerbations, comprising unscheduled R2 ≥ .3, absolute value of the beta coefficient <10, standard error of general practitioner or pulmonary specialist visits and school ab- the beta included in the interval [0,10], and minor allele cut-­off ≥6. sence, as no information on the former variables was available for In the discovery phase, genetic variants that were available some studies. A period of 6–­24 months or ever was considered de- in at least two ethnic-­specific studies were meta-­analyzed with pending on the data available for each study (Tables S1 and S2). In METASOFT, 21 using fixed-­effects or random-­effects models based the discovery phase, we performed a multi-­ancestry meta-­analysis on the heterogeneity among studies (measured by the Cochran’s Q of GWAS of asthma exacerbations in 9392 patients with asthma test p-­value). Ethnic-­specific results were then combined in a multi-­ from 12 studies, including 4989 European-­descents from nine stud- ancestry meta-­analysis. Independent variants (r2 ≤ .8) with sugges- ies, 2181 Hispanics/Latinos, 1250 Singaporean Chinese, and 972 tive association22 at p ≤ 5 × 10−5 within 1 Megabase were identified African Americans. We attempted to replicate the findings from with GCTA-­COJO v1.93.223 using the 1KGP reference.16 These the discovery phase in a total of 37,555 participants with asthma, variants were evaluated in the replication stage, following the same including 36,477 Europeans from seven studies, 877 Latinos from procedures as in the discovery phase. Evidence of replication was | 13993038, 2022, 6, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/pai.13802 by Readcube (Labtiva Inc.), Wiley Online Library on [02/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License HERRERA-­LUIS et al. 7 of 17 considered if the variants showed consistent direction of effects Americans and 241 Puerto Ricans from Genes-­Environments & with the discovery stage at p ≤ .05. Admixture in Latino Americans (GALA II) and 215 African Americans from the Study of African Americans, Asthma, Genes & Environments (SAGE) studies. Linear regression models were corrected for asthma 2.3 | Assessment of shared genetic basis of asthma exacerbations status, age, sex, genetic ancestry, ReFACTor compo- exacerbations with other traits nents as a proxy of cell heterogeneity, and methylation batch (when appropriate). The results from Mexican Americans and Puerto To identify groups of genes previously associated with other traits, Ricans assayed with different methylation arrays were then meta-­ we used a Gene-­Set Enrichment Analysis (GSEA), as implemented analyzed for each sub-­ethnic group with METASOFT. 21 SNP-­CpG in FUMA GWAS24 via the GENE2FUNC algorithm, and queried the pairs were considered significant at Storey q-­value <.05. In silico GWAS catalog. 25 SNPs with p ≤ 1 × 10−4 in the discovery phase of evidence of functional effects of variants on gene expression and the meta-­analysis of GWAS were mapped to the closest gene using DNA methylation was assessed using QTLbase, 29 Genotype-­Tissue 26 the UCSC Table Browser tool. A false discovery rate (FDR) of 5% Expression (GTEx) v8 Portal,30 PhenoScanner v231 and eFORGE- was used to declare significance. ­TF.32 Long-­distance chromatin interactions were determined using To estimate the pairwise genome-­wide genetic correlations (R g) the ChiCP tool.33 between asthma exacerbations and other traits, we compared our findings with publicly available GWAS summary statistics via LD score regression using LDHub. 27 As most of the GWAS have been 2.7 | Validation of previous associations conducted in European populations, the analysis was restricted to predominantly European-­descent individuals to maximize the sta- A literature search for all studies reporting genetic loci significantly tistical power. A Bonferroni-­corrected significance threshold of associated with asthma exacerbations was conducted, as described p < .05/711 traits = 6.48 × 10−5 was applied. in the Appendix S1. Association results in the discovery stage were extracted and significance threshold was defined as p = .05/ number of tested SNPs to adjust for multiple testing. 2.4 | Sensitivity analysis To assess the robustness of the genetic associations, we conducted 3 | R E S U LT S sensitivity analyses for the time-­dependent probability occurrence of exacerbations, the effect of Body Mass Index (BMI), obesity, 3.1 | Characteristics of the patients asthma severity, and age group. Moreover, we evaluated the asso- ciation of the variants with asthma susceptibility, as detailed in the In the discovery phase, we analyzed 2781 exacerbators and Appendix S1. Studies from the discovery stage that had covariate 6611 non-­exacerbators; 53.1% were predominantly Europeans, data available were considered. 23.2% Hispanics/Latinos, 13.3% Singaporean Chinese, and 10.3% African Americans. The percentage of exacerbators ranged from 9.1% to 65.2% in Europeans, and reached 58.8% in Hispanics/ 2.5 | Methylation profiling and quality control Latinos, 46.1% in African ­Americans, and 3.4% in Singaporeans. The replication phase included 37,555 individuals with asthma (3030 ex- Whole blood DNA methylation from Hispanics/Latinos and African acerbators and 34,525 non-­exacerbators) where most participants Americans was profiled using the Infinium HumanMethylation450 were of European ­descent (97.1%), followed by Latinos (2.3%) and BeadChip or the Infinium Methylation EPIC BeadChip arrays. Briefly, Filipinos (0.5%). The percentage of exacerbators ranged from 4.8% low-­quality probes and samples, outliers of DNA methylation, and sam- to 65.2% in Europeans, reached approximately 43% in Latinos, and ples with sex mismatch or mixed genotype distributions on the control 1.3% in Filipinos (Tables S1 and S2). Regarding sex, 51.7% and 42.9% SNP probes were excluded. Standard background correction, dye-­bias were male participants in the discovery and replication phases, correction, inter-­array normalization, and probe-­type bias adjustment respectively. were performed, and beta values were transformed to M-­values for bet- ter statistical performance. Quality control is detailed in the Appendix S1. 3.2 | Discovery phase 2.6 | Functional assessment of associated SNPs The quantile–­quantile plots did not show major genomic inflation due to population stratification in each individual study (Figure S1), the DNA methylation quantitative trait loci (meQTL) analyses were combined results from individuals of European-­descent (Figure S2), conducted using fastQTL 28 for CpG sites within 1 Mb of SNPs or the multi-­ancestry meta-­analysis (Figure S3). In the multi-­ancestry with MAF ≥ 0.01 in at least 10 samples, separately in 139 Mexican meta-­analysis of 9,634,748 variants, 447 SNPs exhibited suggestive | 13993038, 2022, 6, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/pai.13802 by Readcube (Labtiva Inc.), Wiley Online Library on [02/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 8 of 17 HERRERA-­LUIS et al. association (Table S3). The most significant association was the in- (R g = 0.55, p = 3.89 × 10−2), asthma (R g = 0.32, p = 3.99 × 10−2), and tronic SNP rs6888198 within the cadherin-­12 (CDH12) gene at chro- BMI (R g = 0.19, p = 4.76 × 10−2). However, the associations did not mosome 5p14.3 (odds ratio [OR] for C allele: 1.37, 95% confidence remain significant after Bonferroni correction. interval [CI]: 1.23–­1.54, p = 1.95 × 10−8) (Figure 1, Figure S4). 3.5 | Sensitivity analysis 3.3 | Replication phase To assess the robustness of associations that replicated across Fifteen of the 126 independent variants identified in the discovery stages to the time-­dependent probability of occurrence of exacer- phase were not available for replication as they were mostly present bations, stratified analyses were performed in European-­descents in African ­Americans and Hispanics/Latinos (Table S3). Two of the from the discovery stage that reported exacerbations for 6 vs. 106 variants present in more than one ethnic group were consist- 12 months. Consistent effects per period were observed across pe- ently associated with asthma exacerbations (Table 1): rs12091010 riods (Table 2). −3 [VCAM1/EXTL2, OR for T allele: 0.89 (0.82–­0.97), p = 5.35 × 10 ] As the post-­GWAS analyses revealed significant enrichment/ (Figure 2) and rs943126 [PANK1, OR for C allele: 0.92 (0.86–­0.98), correlation at p < .05 with fat mass/distribution, the association p = 1.30 × 10−2] (Figure 3). In the meta-­analysis across both phases, of rs12091010 and rs943126 after additional adjustment by BMI/ these variants reached an association p-­value of 4.23 × 10−7 and obesity was examined in individuals from the discovery phase with 4.93 × 10−6, respectively. From five variants that were present only BMI data available. Moreover, the effect of asthma severity alone in non-­Europeans in the replication stage, none exhibited p < .05 or combined with BMI/obesity on the genetic association exacer- in any other population group (Table S4). Even though rs6888198 bations was evaluated. The effects sizes of the genetic association reached genome-­ wide significance in the discovery and showed after additional adjustment by these variables remained consistent consistent effects among Europeans in the replication phase, this with the effects reported in the discovery stage (Table S7). SNP had opposite effects in Latinos and Filipinos, which resulted We next investigated if the observed effects could differ across in the lack of replication in the multi-­ancestry replication phase age groups in those studies that analyzed exclusively children or (Table 1, Figure S5). adults, but the effect sizes remained consistent across age groups (Table S8). Moreover, we assessed if the effects could be driven by the underlying asthma syndrome, rather than asthma exacerbations, 3.4 | Gene-­set enrichment and genome-­wide and no significant association with asthma was found in results from genetic correlation analysis the UK Biobank or the Michigan Genomics Initiative (Table S9). Enrichment analysis of associations from the multi-­ancestry discov- ery GWAS including 959 SNPs associated with asthma exacerba- 3.6 | Functional exploration of variants associated tions at p ≤ 1 × 10−4 revealed significant enrichment in several traits, with asthma exacerbations including treatment response (min p = 2.77 × 10−6), neurological conditions (min p = 4.62 × 10−5), obesity (min p = 6.52 × 10−5), or We next assessed the association between DNA methylation lev- waist-­to-­hip ratio (min p = 1.88 × 10−7) (Table S5). els in whole blood at 525 and 538 CpG sites with rs12091010 A total of 16 traits exhibited genetic correlation with asthma ex- and rs943126, respectively. A total of 7 and 1 SNP-­CpG pairs for acerbations at p < .05 (Table S6), including wheeze or whistling in the rs943126 and rs12091010 exhibited Storey q < .05, respectively last year (R g = 0.47, p = 1.01 × 10−2), emphysema/chronic bronchitis (Table 3 and Table S10). Two of these replicated consistently in F I G U R E 1 Manhattan plot of the results of the discovery stage of the multi-­ancestry meta-­analysis of GWAS of asthma exacerbations (represented as -­log10 p-­value on the y-­axis) along the chromosome position of the variants analyzed (x-­axis). The suggestive (p = 5 × 10−5) and genome-­wide (p = 5 × 10−8) significance thresholds are indicated by the black line and dark gray lines | 13993038, 2022, 6, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/pai.13802 by Readcube (Labtiva Inc.), Wiley Online Library on [02/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License HERRERA-­LUIS et al. 9 of 17 Europeans for rs943126 (cg25770176 and cg00475140). In silico TA B L E 1 Association results for the top hit in the discovery stage and sentinel variants with significant and consistent effects in the discovery and replication phases, and the meta-­analysis Cochran's 1.89E-­02 4.47E-­01 7.91E-­02 analyses revealed 10 SNP-­CpGs pairs, 3 of which showed consistent Meta-­analysis (discovery and replication) effects in Hispanics/Latinos and African Americans at Storey q < .05 Qp (Tables S11 and S12) including the previous two pairs and rs943126- ­cg03948048. The eight significant CpG sites in minority children 4.23E-­07 4.93E-­06 2.41E-­06 showed significant enrichment (q < .001) in transcription factor (TF) motifs in lung (Table S13). Besides, the T allele of rs12091010 was p associated with decreased EXTL2 expression in whole blood from 0.89 (0.85–­0.94) 0.86 (0.81–­0.91) 1.24 (1.05–­1.45) OR (95% CI) Europeans, according to PhenoScanner.31 The C allele of rs943126 was associated with increased expression of PANK1 in whole blood from Europeans (Table S14). Both variants showed evidence of long-­ range chromatin interaction with several genes in lymphoblastoid cells, including VCAM1 and EXTL2 for rs12091010 and PANK1 for Cochran's rs943126 (Table S15). 3.87E-­01 4.92E-­01 7.18E-­01 Qp 3.7 | Validation of previous associations 1.30E-­02 5.35E-­03 7.72E-­01 We next examined 47 previous genetic loci for asthma exacerb p ations7,8,12,13,34–­36 and moderate-­to-­s evere asthma37 for associa- tion with asthma exacerbations in the discovery phase. A total 0.92 (0.86–­0.98) 0.89 (0.82–­0.97) 1.02 (0.90–­1.15) of 5 variants had p < .05 in Europeans, 2 in Hispanics/Latinos, 5 OR (95% CI) Replication in African Americans, and 1 in Singaporean Chinese (Table S16). These were in loci previously associated with asthma exacerba- The variant identifier corresponds to chromosomal position (hg19) followed by non-­tested allele and tested allele. tions (GSDMB, RAD50, HLA-­D QB1, ADAM33, VDR, and CDHR3) or moderate-­to-­s evere asthma (IKZF3, TSLP, MUC5AC, C11orf30, Cochran's 4.83E-­01 5.82E- ­01 8.01E-­02 SMAD3, and WDR36). However, none of the SNPs exceeded the stringent Bonferroni-­ corrected threshold for significance Qp (p = .05/47 = 1.06 × 10 −3). 9.05E-­06 1.95E-­0 8 3.10E-­05 4 | DISCUSSION p To our knowledge, this is the first multi-­ancestry meta-­analysis of 0.82 (0.75–­0.90) 0.85 (0.78–­0.92) 1.37 (1.23–­1.54) Abbreviations: 95% CI, 95% confidence interval; OR, odds ratio; p, p-­value. GWAS of asthma exacerbations independently of treatment includ- OR (95% CI) Discovery ing European, Hispanic/Latino, Asian, and African ­American patients with asthma. In our combined analysis of 46,947 individuals with asthma, two regulatory SNPs were significantly and consistently as- sociated with asthma exacerbations in most of the studies included Closest gene in the discovery and replication phases, independently of the type of exacerbation and the time period for which the exacerbation status CDH12 PANK1 EXTL2 was assessed. The SNP rs120910109 was located in the intergenic region of the VCAM1/EXTL2 genes, whereas rs943126 was harbored within intron 1 of PANK1. rs12091010 rs6888198 rs943126 VCAM1 encodes a surface protein predominantly expressed in endothelial cells that modulates leukocyte adhesion and trans-­ rsID endothelial migration in response to pro-­inflammatory cytokines, and lipopolysaccharide (LPS) among other factors.38,39 VCAM1 is involved across both phases 1:101210560:C:T 10:91376299:T:C in cancer progression and several immunological disorders, including 5:22659406:T:C asthma.38 In the ovalbumin mice model, anti-­VCAM1 reduced airway hyperresponsiveness and eosinophilic inflammation.40 On the other hand, EXTL2 encodes an enzyme that controls glycosaminoglycan IDa (GAG) biosynthesis via transference of N-­acetylgalactosamine and a | 13993038, 2022, 6, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/pai.13802 by Readcube (Labtiva Inc.), Wiley Online Library on [02/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 10 of 17 HERRERA-­LUIS et al. F I G U R E 2 Forest plot of the association results for rs12091010 (VCAM1/EXTL2) in the meta-­analysis of GWAS of asthma exacerbations. ALSPAC (discovery), SCSGES (discovery), and the subset of samples from BREATHE genotyped with the Illumina Infinium CoreExome-­24 BeadChip (replication) had no genotyped or imputed data for rs12091010 F I G U R E 3 Forest plot of the association results for rs943126 (PANK1) in the meta-­analysis of GWAS of asthma exacerbations. The subset of samples from BREATHE genotyped with the Illumina Infinium CoreExome-­24 BeadChip (replication) had no available genotyped or imputed data for rs943126 HERRERA-­LUIS et al. TA B L E 2 Sensitivity analysis for rs12091010 and rs943126 in individuals from the discovery stage Exacerbations in the last 6 months Exacerbations in the last 12 months European-­descent populations European-­descent populations Multi-­ancestry meta-­analysis rsID OR (95% CI) p Cochran's Q p OR (95% CI) p Cochran's Q p OR (95% CI) p Cochran's Q p −1 −1 −2 −1 −5 rs12091010 0.84 (0.67–­1.04) 1.08 × 10 5.14 × 10 0.86 (0.72–­1.03) 9.13 × 10 6.25 × 10 0.82 (0.74–­0.90) 3.45 × 10 4.84 × 10 −1 rs943126 0.78 (0.64–­0.96) 2.13 × 10 −2 8.61 × 10 −1 0.88 (0.74–­1.04) 1.26 × 10 −1 8.28 × 10 −1 0.85 (0.78–­0.93) 3.29 × 10 −4 7.50 × 10 −2 Abbreviations: 95% CI, 95% confidence interval; OR, odds ratio; p, p-­value. TA B L E 3 Results from the meQTL analysis in whole blood in the GALA II and SAGE studies for genome-­wide significant hit in the discovery and two SNPs that were replicated Mexican Americans Puerto Ricans African Americans Meta-­analysis SNP-­CpG pair Position (hg19) Closest genes Coef SE p Coef SE p Coef SE p Coef SE p Cochran's Q p Storey q rs943126-­cg26800131 91574784 KIF20B −0.21 0.11 2.99E-­0 4 −0.07 0.04 6.99E-­02 −0.07 0.04 8.87E-­02 −0.18 0.10 1.85E-­06 4.45E−03 9.95E-­0 4 rs943126-­cg14920044 91296311 SLC16A12 0.09 0.05 9.77E-­02 0.24 0.06 1.68E-­05 0.12 0.06 4.68E-­02 0.15 0.03 4.61E-­06 1.24E-­01 1.24E-­03 rs943126-­cg20654695 91444521 KIF20B/PANK1 −0.10 0.08 2.12E-­01 −0.06 0.04 9.04E-­02 −0.15 0.03 2.04E-­05 −0.10 0.02 9.96E-­06 1.98E−01 1.79E-­03 rs943126-­cg25770176 91405685 PANK1 −0.09 0.03 1.43E-­03 0.00 0.09 2.78E-­01 −0.07 0.02 3.00E-­03 −0.07 0.02 1.86E-­05 1.49E−01 2.50E-­03 rs12091010-­cg05612904 101491636 DPH5 −0.07 0.04 6.99E-­02 −0.21 0.11 2.99E-­0 4 −0.09 0.04 2.72E-­02 −0.10 0.02 2.31E-­05 9.52E−02 1.20E-­02 rs943126-­cg00475140 91404454 PANK1 −0.21 0.07 1.39E-­03 −0.13 0.08 1.05E-­01 −0.19 0.09 3.19E-­02 −0.18 0.04 4.28E-­05 5.25E−01 4.60E-­03 rs943126-­cg15620114 91296457 SLC16A12 0.09 0.08 2.64E-­01 0.27 0.09 3.87E-­03 0.20 0.08 7.76E-­03 0.18 0.05 1.53E-­0 4 5.70E−01 1.32E-­02 rs943126-­cg04957662 91411382 KIF20B/PANK1 −0.34 0.79 6.69E-­01 −1.16 0.29 1.33E-­0 4 −0.77 0.26 2.89E-­03 −1.02 0.27 1.72E-­0 4 3.14E−01 1.32E-­02 Abbreviations: Coef, Coefficient of the regression; p, p-­value; SE, standard error; Storey q, Storey q-­value. Bold numbers corresponds to values less than .05 for both Storey q and p. | 11 of 17 13993038, 2022, 6, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/pai.13802 by Readcube (Labtiva Inc.), Wiley Online Library on [02/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License | 13993038, 2022, 6, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/pai.13802 by Readcube (Labtiva Inc.), Wiley Online Library on [02/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 12 of 17 HERRERA-­LUIS et al. N-­acetylglucosamine to the glycosaminoglycan-­protein linkage re- promoting epithelial-­mesenchymal transition via activation of the 41 42 gion. Decreased EXTL2 causes an over-­accumulation of GAGs Snail transcription factor pathway. CDH12 expression is positively that can promote inflammation in injured areas.43,44 Moreover, modulated by the chemotactic factor CCL2,53,54 whose levels in- −/− in bone marrow-­derived macrophages from EXTL2 mice, there creases in blood and airway smooth muscle from asthma patients is overproduction of key molecules involved in inflammation and compared to healthy controls.55 extracellular matrix remodeling, including tumor necrosis factor We also attempted to assess previously associated loci for asthma α (TNFα) and several matrix metalloproteinases.43 In a scenario of exacerbations or moderate-­to-­severe asthma for association with overaccumulation of GAGs under the loss of EXTL2 in macrophages, asthma exacerbations in multiple ethnic groups. Although several GAGs act as inflammatory mediators with strong Toll-­like receptor variants showed association at p < .05, none surpassed the stringent 44 4 (TLR4) agonist capacity. Interestingly, genetic variation in both Bonferroni correction, which could be due to differences in study VCAM1 and EXTL2 is associated with blood cell counts and multiple design, phenotype definition, ethnicity, and clinical characteristics, sclerosis, according to the GWAS catalog. 25 among others. Of note, none of the previous findings was initially de- PANK1 catalyzes coenzyme A biosynthesis, regulated by the scribed in Asian or African populations, which highlights the need to transcription factor peroxisome proliferator-­activating receptor α increase ethnic diversity in genomic studies of asthma exacerbations. (PPAR-­α),45 a key anti-­inflammatory factor in asthma.46 A decrease Our study has several limitations. First, the VCAM1/EXTL2 and in PPAR-­α expression is accompanied by a decrease in the expres- PANK1 loci did not surpass a stringent Bonferroni threshold of sion of PANK1 and miR-­107, which is encoded within the intron 5 4.7 × 10−4 (p = .05/106 variants) in the replication stage nor the of PANK1. TLR4 can also downregulate miR-­107. In turn, this leads genome-­wide significance in the combined analysis from all studies. to a higher cyclin-­dependent kinase 6 (CDK6) expression and sub- Second, these loci exhibited modest effects sizes, which could im- sequently increases the adhesion of macrophages in response to pact the clinical relevance of these loci. Third, the history of asthma LPS.45 Bioproducts from bacterial infections, such as LPS, can trig- exacerbations was based on retrospective questionnaires in all co- ger an inflammatory response and increase airway hyperrespon- horts but COMPASS, a randomized, prospective clinical trial. Fourth, siveness and risk of asthma exacerbations.47,48 Moreover, p53 can to bring together large sample sizes necessary to map susceptibility regulate cell cycle progression via upregulation of PANK1 after DNA variants, we considered studies where asthma exacerbations were damage 49 and metabolism.50 reported for the previous 6 to 24 months or ever, which may have in- To prioritize gene targets, we assessed the functional capacity troduced some heterogeneity in the phenotype. Moreover, the repli- of relevant SNPs.51 Both rs12091010 and rs943126 exhibited an cation stage comprised mostly European individuals, which hindered association with DNA methylation at several nearby CpG sites in our capability to replicate associations driven in the discovery stage whole blood from African Americans and Hispanics/Latinos with by non-­Europeans. Despite these limitations, our findings exhibited asthma. Additionally, the SNPs rs12091010 and rs943126 were consistent effects for the VCAM1/EXTL2 and PANK1 loci indepen- associated with EXTL2 and PANK1 gene expression in whole blood dent of the time period assessed. Future studies in adequately pow- from Europeans. Specifically, the T allele of rs12091010, located at ered and phenotypically harmonized cohorts should untangle the 6 kb downstream of the 3′ UTR of VCAM1 and 150 kb upstream of role of these loci in the time-­to-­first exacerbation, the annual num- the transcription start site of EXTL2, was associated with lower odds ber of exacerbations, or the temporal distance among events, ex- of having asthma exacerbations and decreased EXTL2 expression31 plore other epigenetic mechanisms known to be involved in asthma The T allele is more common among Latinos/Admixed Americans, (e.g., histone modifications or miRNAs), 56 and the biological func- followed by Europeans, Africans, and East Asians (Figure S6). tion of these genes. Moreover, although asthma exacerbation risk is The T allele of rs943126 at PANK1, which is less common among influenced by sex in an age-­dependent manner, 57 and our analyses Europeans than the rest of populations (Figure S7), was associated were corrected for sex, future genome-­wide gene-­by-­sex interaction with a higher risk of asthma exacerbations in the combined analy- scans may reveal the influence of sex on the genetic susceptibility to sis of the discovery and replication phases and with decreased gene exacerbations. On the other hand, we acknowledge several study expression of PANK1 in whole blood from Europeans according to strengths. First, we leveraged clinical and genetic data from 46,947 PhenoScanner.31 However, these eQTL effects were not validated asthma patients from different ethnicities from 18 independent in the GTEx data.30 studies. Our study had statistical power ≥80% to detect associations In the discovery phase, the most significant association was with MAF > 17% and relative risk (RR) >1.20 in the discovery stage located at the intronic SNP rs6888198 (CDH12), but no evidence and for variants with MAF ≥ 1%, and was powered at 80% to detect of replication was found in the second stage (p > .05) despite the associations with larger effect sizes (RR ≥ 1.85). Second, we identi- consistency of the direction of the effect across study phases. fied novel, biologically plausible genetic factors of asthma exacerba- Interestingly, rs6888198 showed variable MAF among popula- tions demonstrated by transcriptomics and epigenomics studies and tions, with the largest MAF among Africans and Latinos (Figure S8). evidence for prior literature. Moreover, we accounted for blood cell-­ CDH12 has been associated with angiogenesis and progression of type heterogeneity to overcome the limitations of analyzing mixed several types of cancers.52–­54 Specifically, in colorectal cancer, it cell types tissues. 56,58 Third, we evaluated previous genetic signals has been suggested that CDH12 increases cancer cell migration by from asthma exacerbations in populations from several ancestries. | 13993038, 2022, 6, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/pai.13802 by Readcube (Labtiva Inc.), Wiley Online Library on [02/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License HERRERA-­LUIS et al. 13 of 17 We identified suggestive loci for asthma exacerbations with and editing (supporting). Paloma Poza-­Guedes: Resources (equal); consistent genetic effects across individuals from varying ances- investigation (supporting); writing—­review and editing (supporting). tral backgrounds using a multi-­ancestry approach. We also demon- Olaia Sardón: Resources (equal); investigation (supporting); writing—­ strated that these loci are biologically functional and regulate RNA review and editing (supporting). Paula Corcuera: Resources (equal); expression and adjacent CpG site DNA methylation as meQTL in investigation (supporting); writing—­review and editing (supporting). whole blood cells. Our findings highlight VCAM1, EXTL2, and PANK1 Greg A. Hawkins: Investigation (supporting); writing—­review and as functional loci for asthma exacerbations applicable to people editing (supporting). Annalisa Marsico: Data curation (support- across different ancestral backgrounds, warranting future inves- review and editing (supporting). Thomas Bahmer: ing); writing—­ tigation of these novel genomic mechanisms underlying asthma Investigation (supporting); writing—­ review and editing (support- exacerbations. ing). Klaus F. Rabe: Investigation (supporting); writing—­review and editing (supporting). Gesine Hansen: Investigation (supporting); AU T H O R C O N T R I B U T I O N S writing—­review and editing (supporting). Matthias Volkmar Kopp: Esther Herrera-­Luis: Conceptualization (supporting); data curation Investigation (supporting); writing—­ review and editing (support- (equal); formal analysis (lead); methodology (equal); writing original ing). Raimon Rios: Formal analysis (supporting); writing—­ review draft (lead); writing—­review and editing (equal). Victor E. Ortega: and editing (supporting). Maria Jesus Cruz: Investigation (support- Resources (equal); writing—­review and editing (supporting); fund- review and editing (supporting). Francisco-­Javier ing); writing—­ ing acquisition (lead). Elizabeth J. 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Paul Brinkman: Resources (equal); data curation (equal); (equal); writing—­review and editing (supporting). Daniel B. Hawcutt: writing—­review and editing (supporting). Andrea L. Jorgensen: Data Resources (equal); data curation (equal), writing—­review and edit- curation (equal); formal analysis (supporting); writing—­review and ed- ing (supporting). Erik Melén: Resources (equal), writing—­review and iting (supporting). Qi Yan: Formal analysis (supporting). Erick Forno: editing (supporting). Colin N. Palmer: Resources (equal), writing—­ Data curation (equal); formal analysis (supporting); writing—­review review and editing (supporting). Steve Turner: Resources (equal), and editing (supporting). Susanne J. Vijverberg: Resources (equal); review and editing (supporting). Anke H. Maitland:-­ writing—­ van data curation (equal); writing—­review and editing (supporting). Ryan der Zee: Resources (supporting), writing—­review and editing (sup- Lethem: Data curation (equal), writing—­review and editing (support- porting). Erika von Mutius: Resources (equal); writing—­review and ing). Antonio Espuela-­Ortiz: Formal analysis (supporting), writing—­ editing (supporting). Juan C. Celedón: Resources (equal); writing—­ review and editing (supporting). Mario Gorenjak: Resources (equal); review and editing (supporting). Guy Brusselle: Resources (equal); investigation (supporting); data curation (equal); formal analysis writing—­review and editing (supporting). Fook Tim Chew: Resources (supporting); writing—­review and editing (supporting). Celeste Eng: (equal); writing—­review and editing (supporting). Eugene Bleecker: Investigation (supporting); writing—­review and editing (supporting). Resources (equal); writing—­review and editing (supporting). Deborah Ruperto González-Pérez: Resources (equal); investigation (support- Meyers: Resources (equal); writing—­review and editing (supporting). ing); ​writing—­review and editing (supporting). José M. Hernández-­ Esteban G. Burchard: Resources (equal); funding acquisition (lead); Pérez: Resources (equal); investigation (supporting); writing—­review supervision (supporting); writing—­review and editing (supporting). | 13993038, 2022, 6, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/pai.13802 by Readcube (Labtiva Inc.), Wiley Online Library on [02/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 14 of 17 HERRERA-­LUIS et al. Maria Pino-­Yanes: Resources (equal); conceptualization (lead); su- de Salud Carlos III. DH received grant support from by NIHR for pervision (lead); funding acquisition (lead); methodology (equal); work on NIHR Alder Hey Clinical Research Facility, received pay- writing original draft (supporting); writing—­review and editing (lead). ment for medicolegal report writing not related to asthma or phar- macogenomics for UK family court as an expert in pediatric clinical AC K N OW L E D G M E N T S pharmacology. FJ-­B received fees from ALK, Astra-­Zeneca (AZ), Bial, The authors thank the patients, families, recruiters, healthcare pro- Chiesi, Gebro Pharma, GlaxoSmithKline (GSK), Menarini, Rovi, Roxall, viders, and community clinics for their participation in the studies Sanofi, Stallergenes-­Greer and Teva. G-­B received fees from AZ, analyzed in this manuscript. The authors also acknowledge the con- GSK, Boehringer-­Ingelheim, Novartis, Chiesi and Sanofi. JC received tribution of the high-­performance compute cluster Wynton HPC research materials from Pharmavite and GSK and Merck in order underlying UCSF’s Research Computing Capability to the results to provide medications free of cost to participants in NIH-­funded of this research and Sandra Salazar for her support as GALA II and studies, unrelated to the current work. VO received grants from the SAGE study coordinator. The authors thank the Centro Nacional de National Heart, Lung, and Blood Institute, has participated in Data Genotipado-­ Plataforma de Recursos Biomoleculares-­ Instituto de Safety Monitoring Boards for Regeneron and Sanofi, and participated Salud Carlos III (CeGen-­PRB3-­ISCIII; www.cegen.org) for providing as a Chair of the section on Genetics and Genomics of the American the genotyping services. The authors also acknowledge all the fami- Thoracic Society. MVK has received grants from the German Federal lies who took part in ALSPAC, the midwives for their help in recruit- Ministry of Education and Research, fees from Allergopharma GmbH, ing them, and the whole ALSPAC team, which includes interviewers, Sanofi Aventis GmbH, Infectopharm GmbH, Vertex GmbH, and Leti computer and laboratory technicians, clerical workers, research sci- GmbH, has participated in Data Safety Monitoring Boards for Sanofi entists, volunteers, managers, receptionists, and nurses. Aventis GmbH, and is the president of the German-­Swiss-­Austrian Society of Pediatric Pulmonology (GPP). NHP received support from C O N FL I C T O F I N T E R E S T the Instituto de Salud Carlos III, the European Social Funds from the AE-­O received grants from the Spanish Ministry of Science, European Union “ESF invests in your future,” the European Academy Innovation, and Universities (MICIU) and Universidad de La Laguna of Allergy and Clinical Immunology, and the European Respiratory (ULL). EH-­L , and MP-­Y report funding from the Spanish Ministry Society. MP has received grants from NHS Chair of Pharmacogenetic of Science and Innovation (MCIN/AEI/10.13039/501100011033) grant from UK Department of Health, has received partnership fund- and by the European Social Fund “ESF Investing in your future” ing for the following: MRC Clinical Pharmacology Training Scheme by the European Union. JP-­G reports funding from the Spanish (co-­funded by MRC and Roche, UCB, Eli Lilly and Novartis); Joint Ministry of Universities. MP-­Y and FLD report grants from MCIN/ PhD funding from EPSRC and AZ, and grant funding from Vistagen AEI/10.13039/501100011033 and the European Regional Therapeutics. He has also unrestricted educational grant support Development Fund “ERDF A way of making Europe” by the European for the UK Pharmacogenetics and Stratified Medicine Network from Union. MP-­Y reports grant support from GlaxoSmithKline, Spain Bristol-­Myers Squibb and UCB. He has developed an HLA genotyp- paid to Fundación Canaria Instituto de Investigación Sanitaria de ing panel with MC Diagnostics, but does not benefit financially from Canarias (FIISC) for a project outside the submitted work. MP-­Y this. MP is part of the IMI Consortium ARDAT (www.ardat.org). SQ and JV reports grants from Instituto de Salud Carlos III, Madrid, has received fees from GSK, AZ, Sanofi, Teva, Novartis, and Chiesi. Spain. JV also reports funding by ISCIII and the European Regional SJ-­HV has received grants from SysPharmPediA EraNet. VdP has Development Fund “ERDF A way of making Europe”. JMH-­P has received fees from AZ and GSK. VP has received fees from Sanofi, received fees from CSL Behring, GSK, Astra-­Zeneca, laborato- AZ, Chiesi, MSD, and Boehringer Ingelheim, grant support from rios Menarini, Boehringer Ingelheim, FAES, laboratorios Esteve, MSD, Chiesi Institutional, and Menarini. EvM has received grants Laboratorios Ferrer, Mundipharma, Laboratorios Rovi, Roche, from the German Federal Ministry of Education and Research and Novartis, GRIFOLS, Pfizer, Acthelion-­Jansen, Chiesi y Laboratorios the Bavarian State Ministry of Health and Care, royalties/licenses Bial for the realization of courses, talks, consultancies, and other from Elsevier GmbH, Georg Thieme Verlag, Springer-­Verlag GmbH activities related to his professional activity. FTC has received re- and Elsevier Ltd. EvM has recieved fees from the Chinese University search support from the Singapore Ministry of Education Academic of Hongkong, European Commission, HiPP GmbH & Co KG, AZ, Research Fund, Singapore Immunology Network (SIgN), National Imperial College London, Massachusetts Medical Society, Springer-­ Medical Research Council (NMRC) (Singapore), Biomedical Research Verlag GmbH, Elsevier Ltd., Böhringer Ingelheim International Council (BMRC) (Singapore), and the Agency for Science Technology GmbH, European Respiratory Society (ERS), Universiteit Utrecht, and Research (A*STAR) (Singapore). FTC has received consult- Faculteit Diergeneeskunde, Universität Salzburg, Springer Medizin ing fees from Sime Darby Technology Centre; First Resources Ltd; Verlag GmbH, Japanese Society of Pediatric Allergy and Clinical Genting Plantation, and Olam International, outside the submitted Immunology (JSPACI), Klinikum Rechts der Isar, University of work. YYS has received research support from the NUS Resilience Colorado, Paul-­Martini-­Stiftung, Astra Zeneca, Imperial College & Growth Postdoctoral Fellowships. UP and MG received grants London, Children´s Hospital Research Institute of Manitoba, from the Ministry of Education, Science and Sport from Slovenia, the Kompetenzzentrum für Ernährung (Kern), OM Pharma S.A., Swedish Slovenian Research Agency. M-­JC received grants from the Instituto Pediatric Society for Allergy and Lung Medicine, Chinese College of | 13993038, 2022, 6, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/pai.13802 by Readcube (Labtiva Inc.), Wiley Online Library on [02/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License HERRERA-­LUIS et al. 15 of 17 Allergy and Asthma (CCAA), Verein zur Förderung der Pneumologie Simone Hashimoto https://orcid.org/0000-0001-8995-3817 am Krankenhaus Großhansdorf e.V., Pneumologie Developpement, Qi Yan https://orcid.org/0000-0002-5236-9673 Mondial Congress & Events GmbH & Co. KG, American Academy of Erick Forno https://orcid.org/0000-0001-6497-9885 Allergy, Asthma & Immunology, Imperial College London, Margaux Susanne J. Vijverberg https://orcid.org/0000-0002-4579-4081 Orange, Volkswagen Stiftung, Böhringer Ingelheim International Antonio Espuela-­Ortiz https://orcid.org/0000-0003-0572-9549 GmbH, European Respiratory Society (ERS), Universiteit Utrecht, Mario Gorenjak https://orcid.org/0000-0003-4208-9683 Faculteit Diergeneeskunde, Österreichische Gesellschaft f. José M. Hernández-­Pérez https://orcid. Allergologie u. Immunologie, Massachusetts Medical Society, org/0000-0002-2920-212X OM Pharma S. A., Hanson Wade Ltd., iKOMM GmbH, DSI Dansk Paloma Poza-­Guedes https://orcid.org/0000-0001-8516-6648 Borneastma Center, American Thoracic Society, HiPP GmbH & Co Paula Corcuera https://orcid.org/0000-0001-8661-7033 KG, Universiteit Utrecht, Faculteit Bètawetenschappen. EvM has José María Olaguibel https://orcid.org/0000-0002-9126-4559 patents No. PCT/EP2019/085016, EP21189353.2. 2021. and PCT/ Vicente Plaza https://orcid.org/0000-0003-2567-5496 US2021/016918. 2021. pending, royalties paid to ProtectImmun Santiago Quirce https://orcid.org/0000-0001-8086-0921 for patent EP2361632 and patents EP1411977, EP1637147, and EP Michelle Cloutier https://orcid.org/0000-0001-8316-6409 1964570 licensed to ProtectImmun. EvM is a member of the EXPANSE Victoria del Pozo https://orcid.org/0000-0001-6228-1969 Scientific Advisory Board, Member of the BEAMS External Scientific Camila Figueiredo https://orcid.org/0000-0003-1356-6188 Advisory Board (ESAB), Member of the Editorial Board of “The Munir Pirmohamed https://orcid.org/0000-0002-7534-7266 Journal of Allergy and Clinical Immunology: In Practice”, Member Daniel B. Hawcutt https://orcid.org/0000-0002-8120-6507 of the Scientific Advisory Board of the Children's Respiratory and Erik Melén https://orcid.org/0000-0002-8248-0663 Environmental Workgroup (CREW), Member of the International Colin N. Palmer https://orcid.org/0000-0002-6415-6560 Scientific & Societal Advisory Board (ISSAB) of Utrecht Life Sciences Steve Turner https://orcid.org/0000-0001-8393-5060 (ULS), University of Utrecht, Member of External Review Panel of the Anke H. Maitland-­van der Zee https://orcid. Faculty of Veterinary Science, University of Utrecht, Member of the org/0000-0002-0414-3442 Selection Committee for the Gottfried Wilhelm Leibniz Programme Erika von Mutius https://orcid.org/0000-0002-8893-4515 (DFG), Member of the International Advisory Board of Asthma UK Juan C. Celedón https://orcid.org/0000-0002-6139-5320 Centre for Applied Research (AUKCAR), Member of the International Guy Brusselle https://orcid.org/0000-0001-7021-8505 Advisory Board of “The Lancet Respiratory Medicine”, Member of Fook Tim Chew https://orcid.org/0000-0003-1337-5146 the Scientific Advisory Board of the CHILD (Canadian Healthy Infant Esteban G. Burchard https://orcid.org/0000-0001-7475-2035 Longitudinal Development) study, McMaster University, Hamilton, Maria Pino-­Yanes https://orcid.org/0000-0003-0332-437X Canada, Asthma UK Centre for Applied Research and the Pediatric Scientific Advisory Board Iceland. The other authors declare no con- REFERENCES flict of interest. 1. 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