Computational protocol: An interaction between Nrf2 polymorphisms and smoking status affects annual decline in FEV1: a longitudinal retrospective cohort study

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Protocol publication

[…] Using JPT (Japanese in Tokyo, Japan) genotype data (PhaseIII/Rel#2, Feb09, on NCBI B36 assembly, dbSNP b126) from the International HapMap project, four tag SNPs (rs2001350, rs6726395, rs1962142, rs2364722) were identified in the 34.38 kb Nrf2 gene region (chromosome 2, position 177,803,285-177,837,663). We used the multi-marker predictor method implemented in the Tagger program []. Tag set was generated using a threshold r2 of 0.8 and a minor allele frequency of > 0.1. Genomic DNA was extracted from samples of whole blood from each of the 915 participants by an automated DNA extraction system (QuickGene-610L, FUJIFILM, Tokyo, Japan). Genotyping of these 4 bi-allelic SNPs were attempted for each participant by the pre-designed TaqMan allele-specific polymerase chain reaction (PCR) assays according to the manufacturer's instruction (Applied Biosystems, Foster City, CA).It has been reported that 3 SNPs (rs6721961, rs6706649, and rs35652124) located in the promoter region of the Nrf2 gene affect the transcriptional activity of Nrf2 []. Genotyping for rs6721961 was carried out for each participant by the TaqMan technique using a pair of primers and a pair of oligonucleotide probes designed and synthesized by Applied Biosystems. The sequences of the primers were as follows: forward, 5'-CAGTGGGCCCTGCCTAG-3'; reverse, 5'-TCAGGGTGACTGCGAACAC-3'. The TaqMan fluorescence-labeled oligonucleotide probes were 5'-[VIC]-TGGACAGCGCCGGCAG-3' and 5'-[FAM]-TGTGGACAGCTCCGGCAG-3'. Because rs6706649 and rs35652124 are only 2 base pairs apart, the allele-specific probe technique was not appropriate for genotyping. Instead, for these 2 SNPs, direct DNA sequencing analysis was performed for 50 subjects (25 major allele homozygotes and 25 minor allele homozygotes for rs6726395 SNP). PCR amplification was carried out with 50 ng genomic DNA and a pair of primers flanking the 2 SNPs by a GeneAmp PCR System (Applied Biosystems). The primer sequences were as follows: forward, 5'-AGAGGTTCTCTTGGGGTTCC-3'; reverse, 5'-AGAACCTTGCCCTGCTTTTA-3'. The amplified 343-bp PCR DNA products were sequenced using the same primers and the dideoxynucleotide chain termination method available as a fluorescent sequencing kit (DNA Sequencing Kit; Applied Biosystems) and an automated sequencer (ABI PRISM 3130; Applied Biosystems) according to the manufacturers' instruction. [...] Data are expressed as mean ± SD, unless otherwise stated. Statistical analysis was performed using SYSTAT software, version 13 (Systat Software, Inc., Chicago, IL). Statistical tests with a p value < 0.05 were considered significant.Values of annual FEV1 decline were computed for each individual across the repeated measurements using a linear mixed-effect model. We used a random intercept to take into account the heterogeneity across subjects and the correlation induced by having repeated observations on the same subjects.We performed univariate analysis to evaluate association of annual decline in FEV1 with clinical characteristics. For categorical variables such as gender and smoking status, Student's t tests and one-way analyses of variance with Bonferroni post hoc correction were used for comparisons of 2 and 3 group means, respectively. For continuous variables such as age, body mass index (BMI), PFT measurements, and total serum IgE levels, the correlation with annual decline in FEV1 was assessed by Pearson correlation coefficient analysis.All polymorphisms were tested for Hardy-Weinberg equilibrium using Haploview 4.2 software[]. Estimates of pairwise linkage disequilibrium (LD) between the loci were calculated using r2 []. The associations of genotypes with annual decline in FEV1 were analyzed by multivariate linear regressions adjusted for potential confounding factors such as sex, age, BMI, FEV1/FVC ratio, total serum IgE levels, smoking status (never, ex, or current), smoking index (0, 0-200 or > 200), and affection of bronchial asthma. Correction for multiple comparisons was done by the Bonferroni's method. The interaction effect of genotypes and smoking status on the annual decline in FEV1 was analyzed using general linear models adjusted for the same confounding factors except for smoking behavior.Association of the rs6726395 genotypes with the mRNA expression levels of Nrf2 was analyzed using GENEVAR database[], which shows mRNA expression profiles of 3 cell types (fibroblast, lymphoblastoid cell line and T-cell) derived from umbilical cords of 75 Geneva GenCord individuals [].For analyses of association between haplotypes and annual FEV1 decline, we used the Haplo. score program, which adjusts for the same covariates and calculates simulation p values for each haplotype []. […]

Pipeline specifications

Software tools Tagger, Haploview, Genevar
Databases dbSNP International HapMap Project
Application GWAS