Computational protocol: Genetic Variants of TSLP and Asthma in an Admixed Urban Population

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Protocol publication

[…] Contiguous single nucleotide polymorphisms (SNPs) in the TSLP region on chromosome 5q were identified in the International Haplotype (HapMap) project (International Haplotype Consortium 2003) using data from the European Americans (CEU) and the West African population (YRI). The program Tagger was used to select representative SNPs from the TSLP gene with high linkage disequilibrium (LD) (minor allele frequency >5% and r2≥0.8). Genotyping was performed at the Robert S. Boas Center for Genomics and Human genetics on an Illumina BeadStation 500G Golden Gate custom panel using unamplified DNA extracted from blood. Genotyping reproducibility was verified with duplicates. Ten SNPs in TSLP were successfully genotyped with call rate greater than 99% and minor allele frequency (MAF) greater than 1%. The genotypic information and the Hardy-Weinberg analysis results are summarized in .Genome-wide genotyping, quality control measures, and imputation in the replication cohorts (APCAT) has been described previously . Briefly, each cohort was genotyped on a platform containing >500,000 SNPs, and, after quality control the genotypes at >2.2 million genotypes were imputed using the HapMap CEU panel as a reference.Ancestral informative markers (AIMs) with the maximal absolute difference in allele frequency between ancestral populations were used to differentiate continental origins most likely to be represented in the NYUBAR population including diverse Hispanic ancestry . We genotyped 213 AIMs to adjust for population admixture in associate tests , . [...] Genotype frequencies of each SNP were tested for the concordance with Hardy-Weinberg equilibrium (HWE) using Pearson's chi-square test in the overall population and then in the control population using R package HardyWeinberg (http://cran.r-project.org/web/packages/HardyWeinberg/index.html). Two approaches, the principal component analysis (PCA) method and the Bayesian STRUCTURE method (version 2.2.3) were implemented using 213 AIMs to adjust for population stratification. Either the first five principal component scores from the PCA approach or the posterior probabilities from the STRUCTURE approach were included in the association analysis as ancestry covariates to adjust for population stratification.Single SNP association tests with asthma susceptibility assuming an additive allelic effect were performed using the logistic regression, including covariates of age, BMI, income, education and ancestry covariates. Subgroup analyses stratified by smoking status into ex-smokers and never-smokers were also conducted. To test whether multiple genetic variants from the TSLP gene were associated with asthma, haplotypes were reconstructed using the EM algorithm and the haplotype-specific association were tested using the score test approach based on the generalized linear model using R package haplo.stats (http://cran.r-project.org/web/packages/haplo.stats/index.html). All analyses were performed using R 2.9.1.For the APCAT cohorts, each study separately performed a single marker association analysis assuming an additive allelic model for the imputed data set consisting of ∼2.2 million SNPs. SNPs with low imputation quality (r2<0.3 for MACH) were removed from all studies. Each cohort was stratified into current-smokers, ex-smokers and never-smokers, and the association analysis was further performed within each stratum. The analysis was performed using the logistic regression, adjusted for age, gender and the first ten principal components, and accounting for uncertainty at imputed genotypes. The GEE logistic regression test of the GWAF package in R (http://cran.r-project.org/web/packages/GWAF/) was used to correct for familial relatedness in FHS. Test statistics from individual studies were corrected for inflation using genomic control, and then combined using meta-analysis by combining the regression coefficients and standard errors from each study, implemented in METAL (http://www.sph.umich.edu/csg/abecasis/Metal/index.html). […]

Pipeline specifications

Software tools Tagger, GATK, haplo.stats
Application GWAS
Organisms Nicotiana tabacum
Diseases Asthma
Chemicals Smoke