6 XAV-939 Methods Explained

Genotyping of the independent smoker cohort (276 additional smokers) was performed at the CeGen genotyping facilities, in the CNIO Node (Centro Nacional de Genotipado, Genoma Espa?a). As a quality control, 5% of the genotyped samples had their genotypes confirmed by Sanger sequencing. Quality control and case�Ccontrol association analyses for both Veracode and Kaspar assays were performed using PLINK software, version 1.07 (http://pngu.mgh.harvard.edu/purcell/plink/) (Purcell et al. 2007). SNPs with a low genotyping rate (<95%), not fulfilling the Hardy�CWeinberg equilibrium (HWE: P < 0.05), or with a minimum allele frequency below 5%, as well as samples with a low genotyping rate (<95%), were <a href="http://www.selleckchem.com/products/AC-220.html">Quizartinib datasheet excluded from the association analyses. We tested for association for two phenotypes: smoking behavior, and level of addiction based on the Fagerstr?m score. For this, we considered smoking behavior as a dichotomic phenotype (smoker vs. non-smoker), and we used the total Fagerstr?m score, either as a quantitative or a nominal variable: minimally (<4), moderately (4�C6) and highly (7�C10) dependent. All <a href="http://www.selleckchem.com/products/XAV-939.html">XAV-939 chemical structure association tests included sex and age as covariates, and they were corrected for multiple testing based on the Bonferroni correction (35 markers: P < 0.0014). Linkage disequilibrium (LD) between polymorphisms and haplotype block structures were evaluated using Haploview software (version 4.1). Regions of strong LD were defined according to the confidence intervals algorithm (Gabriel et al. 2002). We studied haplotypes present at a frequency of at least 5%, and estimated the significance of the best result via a permutation procedure (2000 permutations). The SNPassoc R package was used for all analyses (Gonzalez et al. tuclazepam 2007). We used WGAviewer (Ge et al. 2008) and Pupasuite 3.1 (http://pupasuite.bioinfo.cipf.es/) to assess the possible functional consequences of the SNPs associated with smoking behavior. The GENEVAR tool of WGAviewer was used to evaluate the effects of SNPs on gene expression, based on Hapmap genotype and expression data (Stranger et al. 2005, 2007). Pupasuite was used to predict the pathogenicity of an SNP based on the disruption of potential transcription factor binding sites, the creation or disruption of splice sites, its location in miRNA sequences or targets, or in promoter or conserved regions. Ten SNPs (4 with a low genotyping rate, 1 not fulfilling HWE and 5 with a low MAF) and 20 samples (with a low genotyping rate) did not fulfill the aforementioned quality control criteria and, thus, a total of 38 SNPs were analyzed in 153 cases (TAB_Discovery) and 583 controls (CVH1) (Fig. 1 and Table 2).