Asthma is a chronic inflammatory disorder mainly characterized by reversible airflow obstruction, bronchial hyperresponsiveness, and dyspnea. The functional alterations in airway smooth muscle usually observed in asthmatic patients have been associated with increased expression of the smooth muscle myosin light chain kinase isoform (smMLCK).1Benayoun L. Druilhe A. Dombret M.C. Aubier M. Pretolani M. Airway structural alterations selectively associated with severe asthma.Am J Respir Crit Care Med. 2003; 167: 1360-1368Crossref PubMed Scopus (680) Google Scholar This constitutes a key cytoskeleton effector of the smooth muscle contractile machinery and is encoded by the myosin light chain kinase (MYLK) gene. Two MYLK single nucleotide polymorphisms (SNPs), which are common in Africans (>30%) but rare in Europeans (≤1%), have been associated with asthma in African American and Afro-Caribbean subjects.2Flores C. Ma S.F. Maresso K. Ober C. Garcia J.G. A variant of the myosin light chain kinase gene is associated with severe asthma in African Americans.Genet Epidemiol. 2007; 31: 296-305Crossref PubMed Scopus (54) Google Scholar, 3Gao L. Grant A.V. Rafaels N. Stockton-Porter M. Watkins T. Gao P. et al.Polymorphisms in the myosin light chain kinase gene that confer risk of severe sepsis are associated with a lower risk of asthma.J Allergy Clin Immunol. 2007; 119: 1111-1118Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar However, none of the MYLK variants have attained the strict Bonferroni-corrected level of genome-wide significance in a genome-wide association study (GWAS) of asthma. This discrepancy might arise from the low coverage of this region on commercial arrays, the genetic specificities of the populations studied, or both because risk factors in one population might not generalize to another.4Galanter J.M. Gignoux C.R. Torgerson D.G. Roth L.A. Eng C. Oh S.S. et al.Genome-wide association study and admixture mapping identify different asthma-associated loci in Latinos: the Genes-environments & Admixture in Latino Americans study.J Allergy Clin Immunol. 2014; 134: 295-305Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar Here we aimed to fine map the association of SNPs from the MYLK gene with asthma in case-control studies from Spanish (n = 3219) and Latino (n = 4650) populations. In the discovery stage DNA samples from 606 patients with physician-diagnosed asthma from the Genetics of Asthma (GOA) study in the Spanish population (GOA I) were compared with 1258 nonasthmatic subjects. Top associated SNPs were then replicated in 2 independent Spanish studies: GOA II and the Genetics of Asthma study in the Spanish population from Malaga (GOAM). GOA II included 248 asthmatic cases and 537 control subjects genotyped with the Axiom Genome-Wide CEU 1 Array (Affymetrix, Santa Clara, Calif), whereas GOAM comprised 320 asthma cases and 250 control subjects genotyped by using TaqMan allelic discrimination assays (Life Technologies, Carlsbad, Calif). Further replication was assessed in 2 Latino studies using existing genome-wide genotyping data: the Genetics of Asthma in Latino Americans (GALA I) study,4Galanter J.M. Gignoux C.R. Torgerson D.G. Roth L.A. Eng C. Oh S.S. et al.Genome-wide association study and admixture mapping identify different asthma-associated loci in Latinos: the Genes-environments & Admixture in Latino Americans study.J Allergy Clin Immunol. 2014; 134: 295-305Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar consisting of 529 cases and 347 control subjects, and the Genes-Environments & Admixture in Latino Americans (GALA II) study,5Torgerson D.G. Gignoux C.R. Galanter J.M. Drake K.A. Roth L.A. Eng C. et al.Case-control admixture mapping in Latino populations enriches for known asthma-associated genes.J Allergy Clin Immunol. 2012; 130: 76-82.e12Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar including 1893 cases and 1881 control subjects. Detailed descriptions of the study design and sample characteristics can be found in Fig E1, Tables E1 and E2, and the Methods section in this article's Online Repository at www.jacionline.org. In the discovery stage a total of 29 tagging SNPs (tSNPs) were selected from HapMap II to analyze variants with a minor allele frequency (MAF) of 5% or greater from the MYLK gene in European subjects. Genotyping was performed with the iPLEX Gold assay on the MassARRAY system (Sequenom, San Diego, Calif). A total of 26 SNPs passed quality control (after removing monomorphic SNPs or with a P value of 1.7 × 10−3 or less for Hardy-Weinberg equilibrium [HWE] in control subjects, see Table E3 in this article's Online Repository at www.jacionline.org). After imputation, 272 SNPs with MAFs of 5% or greater and a squared correlation between imputed and true genotypes (Rsq) of 0.3 or greater were kept for association testing by using logistic regression models (88% of the total variants with MAFs of ≥5% in Europeans). Principal components were used as covariates to adjust for population stratification, as previously described.6Pino-Yanes M. Corrales A. Acosta-Herrera M. Perez-Rodriguez E. Cumplido J. Campo P. et al.HLA-DRB1*15:01 allele protects from asthma susceptibility.J Allergy Clin Immunol. 2014; 134: 1201-1203Abstract Full Text Full Text PDF PubMed Google Scholar Three significantly associated SNPs were observed after Bonferroni correction (P ≤ 1.8 × 10−4). Two intronic SNPs, rs77820417 and rs78442149, in perfect linkage disequilibrium (LD; r2 = 1) and with MAFs of 9% showed the most significant associations (for both SNPs: odds ratio [OR] of 2.71 [95% CI, 1.79-4.11] for the minor allele, P = 2.78 × 10−6; Fig 1 and see Table E4 in this article's Online Repository at www.jacionline.org). We followed up SNP rs77820417 for replication in 4 independent studies. Association was significant in GOAM (OR, 2.32; 95% CI, 1.23-4.36; P = 4.17 × 10−3), GALA I (OR, 1.73; 95% CI, 1.03-2.93; P = .040), and GALA II (OR, 1.31; 95% CI, 1.05-1.64; P = .019) but not GOA II (OR, 0.88; 95% CI, 0.48-1.61; P = .669). Because of the heterogeneity of effects among studies (P = .007, Cochran Q test), a random-effects meta-analysis of the 7869 subjects was performed, confirming the strong association of rs77820417 with asthma susceptibility (OR, 1.66; 95% CI, 1.14-2.42; P = 1.57 × 10−7; Table I). We further explored the association of rs77820417 with asthma exacerbations in the GALA II study, as defined by the presence of 1 or more asthma-related events (hospitalizations, emergency department visits, and oral steroid use) over the 12 months before recruitment and adjusting for the use of medication during the same period. The A allele, which is associated with asthma risk, was also associated with increased risk of asthma exacerbations (OR, 1.80; 95% CI, 1.08-2.99; P = .023). The associated SNP is located within the MYLK gene region encoding the smMLCK isoform, which participates in smooth muscle cell contractility. smMLCK activation is a critical step in cytoskeletal rearrangement, providing dynamic regulation of cell shape, cell motility, and adhesion, which are involved in the remodeling processes underlying asthma.7Garcia J.G. Lazar V. Gilbert-McClain L.I. Gallagher P.J. Verin A.D. Myosin light chain kinase in endothelium: molecular cloning and regulation.Am J Respir Cell Mol Biol. 1997; 16: 489-494Crossref PubMed Scopus (174) Google ScholarTable ISummary of association testing of rs77820417 with asthma susceptibilityStudySample size (cases/control subjects)MAFOR (95% CI)P valueGOA I1864 (606/1258)0.0902.71 (1.79-4.11)2.78 × 10−6GOA II785 (248/537)0.0840.88 (0.48-1.61).669GOAM570 (320/250)0.0522.32 (1.23-4.36)4.17 × 10−3GALA I876 (529/347)0.0731.73 (1.03-2.93).040GALA II3774 (1893/1881)0.0441.31 (1.05-1.64).019Meta-analysis7869 (3596/4273)—1.66 (1.14-2.42)1.57 × 10−7P values of .05 or less, indicating statistical significance, are shown in boldface. Open table in a new tab P values of .05 or less, indicating statistical significance, are shown in boldface. To date, GWASs have firmly identified susceptibility genes underlying asthma risk, although most of the studies were performed by using HapMap-based inferences, where the coverage for genetic variation is limited compared with the information provided by the 1000 Genomes Project (1KGP). In fact, neither the top hit observed in the current study nor its proxy (rs78442149) were tested for association in the GABRIEL (http://www.cng.fr/gabriel/results.html) or EVE consortia, the largest GWAS meta-analyses in asthma performed in Europeans8Moffatt M.F. Gut I.G. Demenais F. Strachan D.P. Bouzigon E. Heath S. et al.A large-scale, consortium-based genomewide association study of asthma.N Engl J Med. 2010; 363: 1211-1221Crossref PubMed Scopus (1508) Google Scholar and multiethnic groups.9Torgerson D.G. Ampleford E.J. Chiu G.Y. Gauderman W.J. Gignoux C.R. Graves P.E. et al.Meta-analysis of genome-wide association studies of asthma in ethnically diverse North American populations.Nat Genet. 2011; 43: 887-892Crossref PubMed Scopus (628) Google Scholar No other variants from 1KGP are in moderate LD with those 2 SNPs (highest r2 = 0.25). However, 1 MYLK variant was associated with asthma in the GABRIEL consortium (rs7633133, P = 1.01 × 10−7; Fig 1). This SNP shows an MAF of 1% in 1KGP European subjects and therefore was not tested in our study. In the EVE consortium, although no MYLK SNP showed an outstanding significance (minimum P = .01, Fig 1), a 4-fold enrichment of significant associations was observed in Latino subjects (P = 7.03 × 10−5, Fisher exact test) but not in European or African American subjects (P = .720 and P = 1.0, respectively; see Table E5 and the Methods section in this article's Online Repository at www.jacionline.org). One striking aspect of our study is the large effect sizes found for the association of rs77820417 with asthma susceptibility and exacerbations, which is only comparable with the effect reported for an SNP in GSDMB with early childhood asthma with severe exacerbations.10Bonnelykke K. Sleiman P. Nielsen K. Kreiner-Moller E. Mercader J.M. Belgrave D. et al.A genome-wide association study identifies CDHR3 as a susceptibility locus for early childhood asthma with severe exacerbations.Nat Genet. 2014; 46: 51-55Crossref PubMed Scopus (409) Google Scholar However, this effect size might be confounded by the large sex and age differences among cases and control subjects in the discovery sample. Despite this, the validation across multiple studies of both children and adults with different sex balance suggests that the result from the discovery study is not a false-positive result. The catalog of asthma susceptibility genes could be more comprehensive if imputation based on 1KGP data were exploited to meta-analyze existing GWAS data. In addition, analysis of diverse populations also contributes with new susceptibility loci because many GWAS hits are not transferable to all populations.4Galanter J.M. Gignoux C.R. Torgerson D.G. Roth L.A. Eng C. Oh S.S. et al.Genome-wide association study and admixture mapping identify different asthma-associated loci in Latinos: the Genes-environments & Admixture in Latino Americans study.J Allergy Clin Immunol. 2014; 134: 295-305Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar In fact, higher North African ancestry is detected in southwestern Europe and is decreased in northern latitudes.11Botigue L.R. Henn B.M. Gravel S. Maples B.K. Gignoux C.R. Corona E. et al.Gene flow from North Africa contributes to differential human genetic diversity in southern Europe.Proc Natl Acad Sci U S A. 2013; 110: 11791-11796Crossref PubMed Scopus (131) Google Scholar Therefore novel susceptibility loci for asthma could be revealed in populations of Spanish descent. In summary, we identified an MYLK SNP association with asthma in subjects of Spanish descent, showing suggestive genome-wide significance. Future studies will be needed to confirm its importance in other populations. We thank Servicio de Apoyo Informático a la Investigación, ULL (SAII) for the HPC support; the GALA investigators, recruiters, participants, and study coordinators; and the EVE consortium for granting access to summary data. This study was conducted by using 5 case-control studies with unrelated subjects reporting at least 4 grandparents of Spanish or Latino origin. Demographic and clinical data are summarized in Tables E1 and E2 for the Spanish and Latino studies, respectively. All local institutional review boards and ethics committees approved the studies, and participants/parents provided written assent/consent, respectively. This study included patients with 2 generations of Spanish descent. The group of cases included participants who fulfilled the Global Initiative for Asthma guidelines for diagnosis and classification of asthma (http://www.ginasthma.com) and were collected from respiratory medicine and allergy departments in hospitals from Spain as part of GOA I.E1Pino-Yanes M. Sanchez-Machin I. Cumplido J. Figueroa J. Torres-Galvan M.J. Gonzalez R. et al.IL-1 receptor-associated kinase 3 gene (IRAK3) variants associate with asthma in a replication study in the Spanish population.J Allergy Clin Immunol. 2012; 129 (e1-10): 573-575Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar This study included 607 patients with a physician's diagnosis of asthma who were using specific medication for asthma symptoms and were older than 5 years. We recorded basic demographic data (age, sex, and smoking habits), age at diagnosis of the disease, asthma severity, family history of asthma and allergic diseases, allergic symptoms (rhinitis, atopic dermatitis, and food and drug allergy), basal pulmonary function measurements, and medication use for symptomatic treatment. Atopy was confirmed by the presence of a positive skin prick test response (a wheal with a diameter 3 mm greater than that elicited by the saline control) to one of 7 common allergens, including dust mite, epithelium, pollen, fungi, food, latex, and others or by specific serum IgE levels of greater than 0.35 IU/mL. Allergens evaluated for specific IgE were dust mite (Dermatophagoides pteronyssinus, Dermatophagoides farinae, Glycyphagus species, Blomia tropicalis, Acarus siro, Tyrophagus putrescentiae, Lepidoglyphus destructor, and Euroglyphus maynei), epithelia (Felis domesticus, Canis familiaris, Equus caballus, and Oryctolagus cuniculus), pollen (Olea europaea, Salsola species, Lolium perenne, Artemisia vulgaris, Parietaria species, Platanus species, Chenopodium species, Plantago species, Rumex species, and Cupressus species), fungi (Penicillium notatum, Alternaria alternata, Aspergillus fumigatus, and Cladosporium herbarum), food (cow's milk, hen's egg, peanut, soybean, wheat, fish, shrimp, crabs, lobster, clams, oysters, mussels, banana, chestnut, and kiwi), latex, and cockroach (Blattella germanica). Control samples were obtained from the Spanish National DNA Biobank (http://www.bancoadn.org), a member of the Public Population Project in Genomics Consortium (http://www.p3gobservatory.org). After signing an informed consent form, donors self-declared general health status, physical activity, transportation and nutrition habits, employment and qualification, demographics, tobacco smoking history, alcohol consumption, genealogic information, residence, and mother's language of preference. In addition, personal and family histories of diseases, including infectious, cancerous, blood and circulatory, endocrine, mental and behavioral, respiratory (including asthma symptoms), immunologic (including allergies), bone, congenital, skin and digestive, and eye and hearing disorders, were documented. No information from medical records was incorporated or revised, and no medical testing was performed on donors. We minimized the presence of atopy among control subjects by excluding those participants self-reporting personal or family history of allergic or pulmonary diseases. We also minimized the differences in ancestry composition against cases by only including samples from patients with at least 4 grandparents born in Spain. A total of 1271 DNA samples from unrelated subjects were finally selected as controls for the discovery study. However, because the number of samples fulfilling these requirements was limited in the Spanish National DNA Biobank, the final design resulted in large differences in age and sex composition, with older age and higher proportion of male subjects among control subjects than among cases (see Table E1). Genotype data for SNPs with MAFs of 5% or greater for subjects with ancestry from northern and western Europe (CEU) were downloaded from the HapMap II project.E2Frazer K.A. Ballinger D.G. Cox D.R. Hinds D.A. Stuve L.L. Gibbs R.A. et al.A second generation human haplotype map of over 3.1 million SNPs.Nature. 2007; 449: 851-861Crossref PubMed Scopus (3621) Google Scholar We selected the region in chromosome 3 comprising hg19 positions 123329143 to 123605149 to get the full transcript of MYLK (NM_053025) and to include 2 kb of its flanking regions. Haplotypes of MYLK were reconstructed for the CEU data by using PHASE v2.1E3Stephens M. Smith N.J. Donnelly P. A new statistical method for haplotype reconstruction from population data.Am J Hum Genet. 2001; 68: 978-989Abstract Full Text Full Text PDF PubMed Scopus (6472) Google Scholar under default parameters. The tSNP selection was performed on haplotypes showing a frequency of 1% or greater to provide a haplotype r2 value of 0.85 or greater using the multiple-marker selection algorithm implemented in TagIT 3.03 software.E4Weale M.E. Depondt C. Macdonald S.J. Smith A. Lai P.S. Shorvon S.D. et al.Selection and evaluation of tagging SNPs in the neuronal-sodium-channel gene SCN1A: implications for linkage-disequilibrium gene mapping.Am J Hum Genet. 2003; 73: 551-565Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar The final set of tSNPs consisted of 29 SNPs. DNA extracted from blood (GFX kit; GE Healthcare, Little Chalfont, United Kingdom) or saliva (Oragene DNA; DNA GenotekInc, Ottawa, Ontario, Canada) was whole genome amplified with the Illustra GenomiPhiV2 DNA Amplification Kit (GE Healthcare, Pittsburgh, Pa). The concentration was quantified by using a SYBR Green I–based DNA quantification method (Molecular Probes, Eugene, Ore). Genotyping was performed with the iPLEX Gold assay on MassARRAY at the University of Chicago Sequencing Facility and was blind to disease status. We included duplicated samples (approximately 6%) to monitor genotyping quality. Genotype calls were performed automatically by using TYPER 3.4 software (Sequenom, San Diego, Calif). Summary quality control measures were generated by using SNPing,E5Sun X. Ma S.F. Wade M.S. Flores C. Pino-Yanes M. Moitra J. et al.Functional variants of the sphingosine-1-phosphate receptor 1 gene associate with asthma susceptibility.J Allergy Clin Immunol. 2010; 126 (e1-3): 241-249Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar and monomorphic SNPs and those that deviated from HWE within control subjects after multiple comparison adjustment (P ≤ 1.7 × 10−3 [α-corrected = .05/29]) were excluded from the analyses (see Table E2). After removing samples with genotype call rates of less than 80%, 606 cases and 1258 control subjects were retained for association testing. Ninety-three ancestry informative markers of population structure within Europe were also genotyped with the iPLEX Gold assay to reduce the risk for false-positive results, allowing correcting for major population stratification effects among European and Spanish populations.E6Pino-Yanes M. Corrales A. Basaldua S. Hernandez A. Guerra L. Villar J. et al.North African influences and potential bias in case-control association studies in the Spanish population.PLoS One. 2011; 6: e18389Crossref PubMed Scopus (24) Google Scholar, E7Price A.L. Butler J. Patterson N. Capelli C. Pascali V.L. Scarnicci F. et al.Discerning the ancestry of European Americans in genetic association studies.PLoS Genet. 2008; 4: e236Crossref PubMed Scopus (264) Google Scholar Principal component analysis with EIGENSOFT 4.2E8Price A.L. Patterson N.J. Plenge R.M. Weinblatt M.E. Shadick N.A. Reich D. Principal components analysis corrects for stratification in genome-wide association studies.Nat Genet. 2006; 38: 904-909Crossref PubMed Scopus (6963) Google Scholar was used to derive the scores for the first principal component to be used as ancestry estimates in cases and control subjects. MaCH 1.0E9Li Y. Willer C.J. Ding J. Scheet P. Abecasis G.R. MaCH: using sequence and genotype data to estimate haplotypes and unobserved genotypes.Genet Epidemiol. 2010; 34: 816-834Crossref PubMed Scopus (1504) Google Scholar was used for SNP imputation by using as reference the Phase 1 data for European samples (May 2011) deposited in 1KGP.E10Abecasis G.R. Auton A. Brooks L.D. DePristo M.A. Durbin R.M. Handsaker R.E. et al.An integrated map of genetic variation from 1,092 human genomes.Nature. 2012; 491: 56-65Crossref PubMed Scopus (5751) Google Scholar Association testing was performed by using Mach2datE9Li Y. Willer C.J. Ding J. Scheet P. Abecasis G.R. MaCH: using sequence and genotype data to estimate haplotypes and unobserved genotypes.Genet Epidemiol. 2010; 34: 816-834Crossref PubMed Scopus (1504) Google Scholar for SNPs showing MAFs of 5% or greater, ensuring that tested SNPs had good-quality imputation scores (Rsq > 0.3). Imputed variants with MAFs of less than 5% were filtered out for several reasons: (1) the selection of tSNPs was based on HapMap II information to cover variants with frequencies of 5% or greater but not rarer variants; (2) observations in diverse populations suggest that genotype imputation errors produce the greatest power reduction for rarer variantsE11Huang L. Wang C. Rosenberg N.A. The relationship between imputation error and statistical power in genetic association studies in diverse populations.Am J Hum Genet. 2009; 85: 692-698Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar; and (3) the discovery sample has low statistical power to detect associations of low-frequency variants (<5%). For example, by using Quanto (http://biostats.usc.edu/Quanto.html) and assuming an OR of 2 and a Bonferroni-corrected P value of 1.8 × 10−4, the statistical power of the discovery study was 55% for MAFs of 3%, whereas it was 87% for MAFs of 5%. Representation of association results was then performed with LocusZoom 1.1E12Pruim R.J. Welch R.P. Sanna S. Teslovich T.M. Chines P.S. Gliedt T.P. et al.LocusZoom: regional visualization of genome-wide association scan results.Bioinformatics. 2010; 26: 2336-2337Crossref PubMed Scopus (1697) Google Scholar based on LD data from hg19 deposited in 1KGP. Functional annotation on the plots was performed with UCSC Genome Browser data for dbSNP build 142. Regulatory variants were assessed with the Variant Annotation Integrator tool based on ChIP-seq experimentsE13Rosenbloom K.R. Sloan C.A. Malladi V.S. Dreszer T.R. Learned K. Kirkup V.M. et al.ENCODE data in the UCSC Genome Browser: year 5 update.Nucleic Acids Res. 2013; 41: D56-D63Crossref PubMed Scopus (581) Google Scholar for 161 factors from the ENCODE Project. Multiple comparison adjustment was performed by using Bonferroni correction for the number of SNPs tested (P < 1.8 × 10−4 [α-corrected = .05/272]). SNPs with the most significant association after multiple comparisons adjustment were followed up in replication studies. GOA II included a total of 248 asthma samples collected during 2011 and 2012 from the same centers as in GOA I. Recruitment protocols of asthma cases in GOA II and GOA I were the same (including that the diagnosis of asthma was made by a physician, patients had an active use of specific medication for asthma symptoms according to severity, and their age was >5 years), and the clinical characterization of asthma and atopy was similar.E14Pino-Yanes M. Corrales A. Acosta-Herrera M. Perez-Rodriguez E. Cumplido J. Campo P. et al.HLA-DRB1*15:01 allele protects from asthma susceptibility.J Allergy Clin Immunol. 2014; 134: 1201-1203Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar Control subjects included a total of 537 DNA samples from subjects who self-reported having at least 2 generations of ancestors born in Spain and without personal or family history of chronic diseases. These subjects were obtained from the Spanish National Genotyping Center, Santiago de Compostela Node (CeGen, http://www.cegen.org), consisting of unrelated healthy adult subjects collected from hospitals across Spain by Fundacion Publica Galega de Medicina Xenomica (FPGMX, http://fundacion.xenomica.org). After signing an informed consent form, donors self-declared general health status, demographics, residence, and personal and family history of infectious, cancerous, mental, cardiovascular, or immunologic diseases were documented. No information from medical records was incorporated or revised, and no medical testing was performed on these control subjects. Samples were genome-wide genotyped on the Axiom Genome-Wide Human CEU Array (Affymetrix, Santa Clara, Calif) by using the services provided by the National Genotyping Center (CeGen), Universidad de Santiago de Compostela Node, as described elsewhere.E14Pino-Yanes M. Corrales A. Acosta-Herrera M. Perez-Rodriguez E. Cumplido J. Campo P. et al.HLA-DRB1*15:01 allele protects from asthma susceptibility.J Allergy Clin Immunol. 2014; 134: 1201-1203Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar Genotype calls were obtained simultaneously for all cases and control subjects by using the Axiom GT1 algorithm on the Affymetrix Genotyping Console (GTC), according to the manufacturer's recommendations. Quality of data was assessed for dish quality control and the metrics recommended by the manufacturer, call rate (≥95%), MAF of 1% or greater, genotype missing rate differences between cases and control subjects (P > 1 × 10−5), HWE P values of greater than 1 × 10−6 in control subjects, concordance between genetically predicted and reported sex, and showing no relatedness (PI_HAT < 0.2). A total of 484,597 variants passed all quality control filters and had 99.3% of mean genotype concordance among duplicated samples. The genomic inflation factor of this study was 1.038 adjusting for the 2 first principal components EIGENSOFT.E8Price A.L. Patterson N.J. Plenge R.M. Weinblatt M.E. Shadick N.A. Reich D. Principal components analysis corrects for stratification in genome-wide association studies.Nat Genet. 2006; 38: 904-909Crossref PubMed Scopus (6963) Google Scholar GIANT v.3 haplotypes from the integrated Phase I release of 1KGPE10Abecasis G.R. Auton A. Brooks L.D. DePristo M.A. Durbin R.M. Handsaker R.E. et al.An integrated map of genetic variation from 1,092 human genomes.Nature. 2012; 491: 56-65Crossref PubMed Scopus (5751) Google Scholar were used as references to impute untyped SNPs by using MinimacE15Howie B. Fuchsberger C. Stephens M. Marchini J. Abecasis G.R. Fast and accurate genotype imputation in genome-wide association studies through pre-phasing.Nat Genet. 2012; 44: 955-959Crossref PubMed Scopus (1223) Google Scholar for the entirety of chromosome 3, which was previously phased with SHAPEIT version 2.E16Delaneau O. Coulonges C. Zagury J.F. Shape-IT: new rapid and accurate algorithm for haplotype inference.BMC Bioinformatics. 2008; 9: 540Crossref PubMed Scopus (107) Google Scholar The SNP rs77820417 showed an imputation quality score (Rsq) of 0.39. Association testing was performed by means of logistic regression models using Mach2dat.E9Li Y. Willer C.J. Ding J. Scheet P. Abecasis G.R. MaCH: using sequence and genotype data to estimate haplotypes and unobserved genotypes.Genet Epidemiol. 2010; 34: 816-834Crossref PubMed Scopus (1504) Google Scholar GOAM consisted of 320 asthmatic cases collected from allergy departments in 2 Spanish centers from the Canary Islands and Malaga fulfilling the Global Initiative for Asthma guidelines for diagnosis and classification of asthma and 250 control subjects collected from the Department of Allergy in the Carlos Haya Hospital (Malaga, Spain).E14Pino-Yanes M. Corrales A. Acosta-Herrera M. Perez-Rodriguez E. Cumplido J. Campo P. et al.HLA-DRB1*15:01 allele protects from asthma susceptibility.J Allergy Clin Immunol. 2014; 134: 1201-1203Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar Asthmatic patients had physician-diagnosed disease with active specific medication for asthmatic symptoms according to severity and were 5 years or older. Patients were demographically and clinically characterized as in GOA I and GOA II. Atopy was confirmed by evaluating allergens that are common in Malaga and included dust mites (D pteronyssinus and L destructor), epithelia (F domesticus and C familiaris), pollen (O europaea, Salsola species, L perenne, Phleum pratense, A vulgaris, Parietaria species, Platanus species, Chenopodium species, Plantago species, and Cupressus species), fungi (A alternata and A fumigatus), and latex. Control subjects were ascertained for asthma and atopy while accompanying the patients at routine visits to the allergy department. These subjects were unrelated to cases and were demographically and clinically characterized for the presence of a personal or family history of asthma and atopy. Although none of these control subjects were given a diagnosis of asthma at the time of enrollment, 130 of them reported having a personal or family history of atopy. Genotyping of rs77820417 was assessed by using TaqMan allelic discrimination assays (Applied Biosystems, Foster City, Calif), with automated calls generated by using a 7500 Fast Real-Time
