Asthma is a chronic inflammatory disease associated with genetic and environmental factors. The HLA locus is the most polymorphic and gene-dense region of the human genome and has been associated with a large number of infectious and autoimmune diseases.1Gregersen P.K. Behrens T.W. Genetics of autoimmune diseases–disorders of immune homeostasis.Nat Rev Genet. 2006; 7: 917-928Crossref PubMed Scopus (154) Google Scholar Before the advent of genome-wide association studies (GWAS), HLA-DRB1 and HLA-DQB1 genes were independently associated with asthma and related traits in several candidate gene association studies.2Ober C. Hoffjan S. Asthma genetics 2006: the long and winding road to gene discovery.Genes Immun. 2006; 7: 95-100Crossref PubMed Scopus (530) Google Scholar The importance of these genes in the pathogenesis of asthma has recently been corroborated by both individual and meta-analyzed GWAS.3Hirota T. Takahashi A. Kubo M. Tsunoda T. Tomita K. Doi S. et al.Genome-wide association study identifies three new susceptibility loci for adult asthma in the Japanese population.Nat Genet. 2011; 43: 893-896Crossref PubMed Scopus (267) Google Scholar, 4Li X. Howard T.D. Zheng S.L. Haselkorn T. Peters S.P. Meyers D.A. et al.Genome-wide association study of asthma identifies RAD50-IL13 and HLA-DR/DQ regions.J Allergy Clin Immunol. 2010; 125: 328-335.e11Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar, 5Moffatt 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 (1543) Google Scholar In spite of the evidence, the interpretation of these associations can be problematic because of the complex relationship between the allele at single-nucleotide polymorphisms (SNPs) and the variation at classical HLA alleles. Interestingly, classic alleles associate with stronger effects than individual SNPs and constitute the most likely functional variants.6Dilthey A. Leslie S. Moutsianas L. Shen J. Cox C. Nelson M.R. et al.Multi-population classical HLA type imputation.PLoS Comput Biol. 2013; 9: e1002877Crossref PubMed Scopus (129) Google Scholar Here, we aimed to test the association of SNPs from HLA-DRB1 and HLA-DQB1 with asthma in Spanish samples, and to uncover the classic alleles that are involved in the susceptibility to the disease. In the discovery stage, DNA samples from 574 physician-diagnosed asthmatic patients from the Genetics of Asthma (GOA) study in the Spanish population were compared with samples of 1186 nonasthmatic subjects obtained from the Spanish National DNA Biobank (www.bancoadn.org).7Pino-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: 573-575.e10Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar An independent sample of 568 asthma cases and 787 controls was used for replication. For further description of the study design, see Fig E1, Online Repository text, and Table E1 in this article's Online Repository at www.jacionline.org. A total of 22 SNPs capable of predicting classic alleles from HLA-DRB1 and HLA-DQB1 in European populations were genotyped in the discovery sample using a combination of different methods (see Table E2 in this article's Online Repository at www.jacionline.org).8Leslie S. Donnelly P. McVean G. A statistical method for predicting classical HLA alleles from SNP data.Am J Hum Genet. 2008; 82: 48-56Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar Their performance on predicting HLA-DRB1 and HLA-DQB1 classic alleles was first assessed by genotyping 313 DNA samples from healthy Spanish individuals with paired data for classic alleles at a 4-digit resolution (Luminex, Austin, Tex). To impute classic alleles, we used a reference data set with more than 2500 samples of European ancestry with dense SNP data and classical HLA allele typing.6Dilthey A. Leslie S. Moutsianas L. Shen J. Cox C. Nelson M.R. et al.Multi-population classical HLA type imputation.PLoS Comput Biol. 2013; 9: e1002877Crossref PubMed Scopus (129) Google Scholar We used a new probabilistic approach (HLP*IMP:02), which delivers increased accuracy on European samples, even under conditions of reduced SNP coverage.6Dilthey A. Leslie S. Moutsianas L. Shen J. Cox C. Nelson M.R. et al.Multi-population classical HLA type imputation.PLoS Comput Biol. 2013; 9: e1002877Crossref PubMed Scopus (129) Google Scholar The imputed alleles for the training sample were compared with the observed classic alleles at 2-digit and 4-digit resolution. Only those classic alleles that attained 80% or more sensitivity, specificity, and positive and negative predictive values and that were common in the training sample (frequency ≥5%) were tested for association. The association of SNPs with asthma in the discovery sample was tested using logistic regression models and included previously obtained ancestry scores as covariates to adjust for population stratification.7Pino-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: 573-575.e10Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar In this stage, we performed multiple testing adjustments for SNPs and classic alleles by means of 105 permutations in which case-control labels were swapped while maintaining the haplotype structure. Seventeen SNPs were successfully genotyped and passed quality control checks (see Online Repository text and Table E2). Four of them were associated with asthma in the discovery sample after multiple comparison adjustments (1.24 ≤ odds ratio [OR] ≤ 1.94, 2.8 × 10−7 ≤ P ≤ .002) (see Table E3 and Fig E2 in this article's Online Repository at www.jacionline.org) and 2 of them were nominally significant in replication samples (P ≤ .008) (Table I). A meta-analysis confirmed the consistency of the effects for the association of rs3135388 and rs6457617 with asthma (P = 7.8 × 10−5 and 3.0 × 10−5, respectively) (Table I), and conditional regression analysis in the overall sample revealed their independent association (P = 1.3 × 10−4 for rs3135388 and P = 1.5 × 10−4 for rs6457617), consistent with their weak linkage disequilibrium (r2 = 0.06).Table ISummary of association testing of HLA-DQB1 and HLA-DRB1 SNPs and classic alleles with asthma susceptibilityrs#/geneEffect alleleDiscovery sample (n = 1760)Replication sample (n = 1355)Meta-analysis (n = 3115)OR (95% CI)P valueOR (95% CI)P valueQ testP value∗Cochran's Q test for heterogeneity.OR (95% CI)FEP valueREP valuers2395175A1.94 (1.51-2.49)2.8 × 10−7†Significant P values in individual samples and those that revealed a consistent effect in the meta-analysis.0.99 (0.72-1.35).931.0011.39 (0.72-2.70)‡Effect and interval derived from the RE model as suggested by the Q test.6.6 × 10−54.5 × 10−6rs3135388A0.66 (0.50-0.87).002†Significant P values in individual samples and those that revealed a consistent effect in the meta-analysis.0.63 (0.41-0.97).008†Significant P values in individual samples and those that revealed a consistent effect in the meta-analysis..9760.66 (0.54-0.81)§Effect and interval derived from the FE model as suggested by the Q test.7.8 × 10−5†Significant P values in individual samples and those that revealed a consistent effect in the meta-analysis.9.4 × 10−5rs6457617T1.25 (1.08-1.44).003†Significant P values in individual samples and those that revealed a consistent effect in the meta-analysis.1.26 (1.07-1.48).005†Significant P values in individual samples and those that revealed a consistent effect in the meta-analysis..9471.25 (1.13-1.39)§Effect and interval derived from the FE model as suggested by the Q test.3.0 × 10−5†Significant P values in individual samples and those that revealed a consistent effect in the meta-analysis.3.6 × 10−5rs7764856A1.24 (1.07-1.45).005†Significant P values in individual samples and those that revealed a consistent effect in the meta-analysis.1.00 (0.84-1.19).973.0631.13 (1.01-1.27)§Effect and interval derived from the FE model as suggested by the Q test..029.028HLA-DQB1∗Cochran's Q test for heterogeneity.060.67 (0.56-0.80)1.0 × 10−5†Significant P values in individual samples and those that revealed a consistent effect in the meta-analysis.0.97 (0.91-1.02).249‖Classical allele data were available only for a subset of 785 samples.1.3 × 10−40.81 (0.57-1.16)‡Effect and interval derived from the RE model as suggested by the Q test.4.5 × 10−31.2 × 10−4HLA-DRB1∗Cochran's Q test for heterogeneity.150.64 (0.49-0.84).001†Significant P values in individual samples and those that revealed a consistent effect in the meta-analysis.0.87 (0.80-0.95).001†Significant P values in individual samples and those that revealed a consistent effect in the meta-analysis.‖Classical allele data were available only for a subset of 785 samples..0260.77 (0.57-1.03)‡Effect and interval derived from the RE model as suggested by the Q test.1.2 × 10−41.3 × 10−4†Significant P values in individual samples and those that revealed a consistent effect in the meta-analysis.HLA-DRB1∗Cochran's Q test for heterogeneity.15:010.65 (0.49-0.86).002†Significant P values in individual samples and those that revealed a consistent effect in the meta-analysis.0.66 (0.49-0.90).008†Significant P values in individual samples and those that revealed a consistent effect in the meta-analysis..9180.66 (0.53-0.81)§Effect and interval derived from the FE model as suggested by the Q test.6.7 × 10−5†Significant P values in individual samples and those that revealed a consistent effect in the meta-analysis.8.0 × 10−5FE, Fixed-effects; RE, random-effects.∗ Cochran's Q test for heterogeneity.† Significant P values in individual samples and those that revealed a consistent effect in the meta-analysis.‡ Effect and interval derived from the RE model as suggested by the Q test.§ Effect and interval derived from the FE model as suggested by the Q test.‖ Classical allele data were available only for a subset of 785 samples. Open table in a new tab FE, Fixed-effects; RE, random-effects. In silico analysis of expression quantitative trait loci (eQTLs) revealed the role of associated SNPs as eQTLs for HLA-DRB1 and/or HLA-DRB5 in lymphoblastoid cells derived from European individuals (see Table E4 in this article's Online Repository at www.jacionline.org). The SNP rs3135388 was also located on an enhancer histone mark site in B-lymphocyte cells and a transcription factor binding site as demonstrated by empirical data from the Encyclopedia of DNA Elements (ENCODE) (www.genome.gov). In addition, our results for rs6457617 constitute a replication of a GWAS hit for asthma in Japanese.3Hirota T. Takahashi A. Kubo M. Tsunoda T. Tomita K. Doi S. et al.Genome-wide association study identifies three new susceptibility loci for adult asthma in the Japanese population.Nat Genet. 2011; 43: 893-896Crossref PubMed Scopus (267) Google Scholar Interestingly, the 2 associated SNPs showed consistent effects with those reported for lipid traits (see Table E5 in this article's Online Repository at www.jacionline.org). Quality assessment of the imputation of the 76 classic alleles detected in the training sample (see Table E6 in this article's Online Repository at www.jacionline.org) revealed that 14 of the 25 common alleles passed our quality criteria and were followed-up for association analysis using logistic regression models. Three classic alleles were associated with asthma in the discovery sample after multiple testing adjustments (see Table E7 in this article's Online Repository at www.jacionline.org): HLA-DQB1*06, HLA-DRB1*15, and HLA-DRB1*15:01 (.001 ≤ P ≤ 1.0 × 10−5). However, only 2 of them replicated at nominal significance: HLA-DRB1*15 and HLA-DRB1*15:01 (P = .001 and .008, respectively). A meta-analysis confirmed a consistent protective effect of HLA-DRB1*15:01 for asthma across all the samples (OR, 0.66; 95% CI, 0.53-0.81; P = 6.7 × 10−5). Interestingly, HLA-DRB1*15:01 has previously been associated with an increased risk for multiple sclerosis and narcolepsy. This result is consistent with other susceptibility genes that have been shown to have opposite effects in asthma and autoimmune diseases (Table E5).9Li X. Ampleford E.J. Howard T.D. Moore W.C. Torgerson D.G. Li H. et al.Genome-wide association studies of asthma indicate opposite immunopathogenesis direction from autoimmune diseases.J Allergy Clin Immunol. 2012; 130: 861-868.e7Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 10Noguchi E. Sakamoto H. Hirota T. Ochiai K. Imoto Y. Sakashita M. et al.Genome-wide association study identifies HLA-DP as a susceptibility gene for pediatric asthma in Asian populations.PLoS Genet. 2011; 7: e1002170Crossref PubMed Scopus (104) Google Scholar However, this study is the first revealing such an effect for a classic HLA allele. Because a previous study has found an association between SNPs from the HLA region and sensitization to specific allergens,11Hinds D.A. McMahon G. Kiefer A.K. Do C.B. Eriksson N. Evans D.M. et al.A genome-wide association meta-analysis of self-reported allergy identifies shared and allergy-specific susceptibility loci.Nat Genet. 2013; 45: 907-911Crossref PubMed Scopus (189) Google Scholar we performed a post hoc analysis for sensitization to the 3 most common specific allergens within the asthma cases (see Online Repository text), and identified SNP rs2395175 as associated with the presence of sensitization to dust mites, pollens, and animal epithelia in the discovery sample (P ≤ .004), with both harmful and protective associations, depending on the allergen (see Table E8 in this article's Online Repository at www.jacionline.org). However, only the association with animal epithelia sensitization was replicated in independent samples (P ≤ .003), showing a large effect size in the meta-analysis (OR, 2.00; 95% CI; 1.43-2.81; P = 5.6 × 10−5) (Table II). This SNP was also an eQTL for HLA-DRB1 and/or HLA-DRB5 in lymphoblastoid cells (Table E4).Table IISummary of association testing of the SNP rs2395175 with allergic sensitization against dust mites, pollens, and animal epitheliaAllergenDiscovery sampleReplication sampleMeta-analysisSample size∗Number of individuals with positive and negative sensitization (sensitized: nonsensitized).OR (95% CI)P valueSample size∗Number of individuals with positive and negative sensitization (sensitized: nonsensitized).OR (95% CI)P valueSample size∗Number of individuals with positive and negative sensitization (sensitized: nonsensitized).Q testP value†Cochran's Q test for heterogeneity.OR (95% CI)FEP valueREP valueDust mites253:1732.69 (1.58-4.59)2.7 × 10−4‡Significant P values in individual samples and those that revealed a consistent effect in the meta-analysis.437:1101.18 (0.64-2.20).597690:283.0481.81 (0.81-4.06)§Effect and interval derived from the RE model as suggested by the Q test..002.002Pollens163:2620.46 (0.27-0.79).004‡Significant P values in individual samples and those that revealed a consistent effect in the meta-analysis.224:3441.06 (0.64-1.76).830387:606.0290.70 (0.31-1.57)§Effect and interval derived from the RE model as suggested by the Q test..069.042Animal epithelia128:2822.07 (1.28-3.35).003‡Significant P values in individual samples and those that revealed a consistent effect in the meta-analysis.219:3491.94 (1.20-3.12).007‡Significant P values in individual samples and those that revealed a consistent effect in the meta-analysis.347:631.8422.00 (1.43-2.81)‖Effect and interval derived from the FE model as suggested by the Q test.5.6 × 10−5‡Significant P values in individual samples and those that revealed a consistent effect in the meta-analysis.6.7 × 10−5‡Significant P values in individual samples and those that revealed a consistent effect in the meta-analysis.FE, Fixed-effects; RE, random-effects.∗ Number of individuals with positive and negative sensitization (sensitized: nonsensitized).† Cochran's Q test for heterogeneity.‡ Significant P values in individual samples and those that revealed a consistent effect in the meta-analysis.§ Effect and interval derived from the RE model as suggested by the Q test.‖ Effect and interval derived from the FE model as suggested by the Q test. Open table in a new tab FE, Fixed-effects; RE, random-effects. Our study has 2 main limitations. First, it had more than 80% statistical power for variants with an OR of more than 1.3, but not for smaller effect sizes (see Fig E3 in this article's Online Repository at www.jacionline.org). Second, association testing of the full spectrum of classic alleles at these 2 genes was impracticable, both because of sample size limitations and because of the imperfect predictive ability of the SNPs retained for classic allele imputation. Consequently, we analyzed only 56% of common classic alleles in this population (see Online Repository text). It is possible that we missed other interesting associations, as suggested by the fact that a weighted score including the 2 SNPs associated with asthma explained slightly higher phenotypic variance than did a model including the classic alleles (Nagelkerke's R2 = 0.14 vs 0.10, respectively). In summary, a deeper examination of HLA genes has revealed a classic allele that shows pleiotropic effects for asthma and other immune-related diseases and likely constitutes a putative causal variant. Analysis of SNPs also revealed shared genetic risk factors between asthma and lipid levels. Finally, we provided evidence supporting the role of HLA polymorphisms in specific allergic sensitization. We thank Alexander Dilthey, Gonçalo Abecasis, and Christian Fuchsberger for their help with software tools, Servicio de Apoyo Informático a la Investigación (ULL) for the HPC support, and Katherine K. Nishimura for proofreading the manuscript. Download .doc (.33 MB) Help with doc files Online Repository DataFig E2View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E3View Large Image Figure ViewerDownload Hi-res image Download (PPT)

HLA-DRB1*15:01 allele protects from asthma susceptibility