Computational protocol: Assessing the Validity of Asthma Associations for Eight Candidate Genes and Age at Diagnosis Effects

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Protocol publication

[…] To reduce the risk for false positives due to major population stratification effects, a total of 83 European ancestry informative markers (termed EuroAIMs) were determined in case and control samples. These EuroAIMs allowed to correct for major differences in Spanish populations due to the North African genetic influences observed in this population, with a mean value of 5–9% for mainland populations and 16–20% for Canary Islanders . A principal component analysis (PCA) based on these genetic markers was used to derive the ancestry estimates in cases and controls as scores of the first principal component (PC1), by means of EIGENSOFT . A full list of EuroAIMs used and the genotyping procedures have been detailed elsewhere , . [...] Clinical and demographical data were analyzed by means of the χ2-test and the Mann-Whitney U-test using R version 2.15 . Departures from Hardy-Weinberg equilibrium (HWE) were evaluated separately for cases and controls using an exact test , by means of a custom script for STATISTICA (StatSoft Inc., Tulsa, OK) . However, as deviations in cases have been considered a symptom of disease association –, only those tSNPs deviating significantly from HWE in the control group were filtered out from further analyses (threshold p-value  = 7.0E-04 after considering the multiple comparisons performed). Individual tSNP associations were tested under an additive model by means of regression analysis with SNPassoc . For that, PC1 scores were included as a covariate in regression models to adjust associations for population stratification, and allele effects were estimated as odds ratios (ORs) with 95% confidence intervals (CIs).Additionally, MaCH 1.0 was used to impute untyped SNPs with data from 380 European individuals deposited in The 1000 Genomes Project (1KGP), May 2011 version . Association testing was performed using Mach2dat adjusting for the PC1 scores. This analysis was conducted using allele dosages for those SNPs showing MAF≥10% and Rsq>0.3, ensuring that all SNPs considered for association testing were accurately imputed (with >90% of SNPs having Rsq>0.8, and with a mean Rsq across all imputed SNPs of 0.95 [IQR: 0.91–0.97]) ().For each gene by separate, a conditional regression-based analysis was used to point out the independent association signals of each locus by including all SNPs associated at nominal significance. We then tested if association tests of the SNPs that represented nominal independent associations within each gene improved considering age-at-onset-varying effects, by implementing a sequential addition (SA) of cases . For that, the age at diagnosis was utilized as a proxy for the age-at-onset of the disease, which was not recorded for most patients, and cases were grouped in categories of quartiles of age (14 [n = 155], 26 [n = 291], 39 [n = 427], and 82 years [n = 606]). The age at diagnosis cutoff obtained was next used to select a sub-sample of cases for which associations were tested again, both for tSNPs and imputed SNPs. LD patterns and regional association results were represented using LocusZoom 1.1 based on LD data from hg18 deposited by 1KGP .To judge the significance of SNP associations in the context of the multiple comparisons performed, a false discovery rate (FDR) was calculated using QVALUE . A FDR threshold of 5% (p-value ≤0.0012) was established to declare study-wise significance to limit the expected proportion of false positives incurred in the study when a particular individual SNP test was called significant. This was assessed considering altogether the p-values from all SNPs analyzed, both genotyped and imputed, the tests from the SA of cases to obtain the age cutoff at which the allele effects were largest, and all the comparisons performed (i.e. associations with asthma, atopic asthma, and age-of-onset before the cutoffs). Functional annotation of associated SNPs was carried out using the software HaploReg . […]

Pipeline specifications

Software tools EIGENSOFT, Statistica, SNPassoc, LocusZoom, HaploReg
Applications Miscellaneous, Population genetic analysis, GWAS
Diseases Asthma