Computational protocol: Evidence for genetic association of RORB with bipolar disorder

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Protocol publication

[…] SNP genotype information for the CEPH population (Utah residents of northern and western European ancestry) was downloaded from the Phase II HapMap data (release #20) for regions surrounding each gene (750.7 kb for RORA and 208.1 kb for RORB). We used the Tagger program as implemented in Haploview[] to select pair-wise tag-SNPs with minor allele frequencies (MAF) = 0.05 and an r2 threshold of 0.8. In total, 332 tag-SNPs from RORA (99% of alleles captured; mean r2 = 0.959) and 44 tag-SNPs from RORB (98% of alleles captured; mean r2 = 0.959) were chosen for genotyping. Primers were designed using MassARRAY's Assay Design software (Bruker-Sequenom, USA) and were purchased from Integrated DNA Technologies (USA). Genotyping of samples was performed as single-base extension reactions (iPLEX) using the MassARRAY mass spectrometry system as previously described []. A list of the genotyped SNPs and the assay primers used can be found in Additional file . [...] A number of quality control measures were implemented to ensure accuracy of the data collected. Genotypes from intra- and inter-plate controls were compared for identity, and negative test controls were confirmed to have no genotypes called. In addition, assays that failed in over 10% of the samples (14 SNPs) were excluded and samples that failed in over 10% of the assays (33 samples) were excluded. The genotyping rate in the remaining individuals was 99.38%. Families with greater than 5% Mendelian errors (4 families) and SNPs with greater than 10% Mendelian errors (1 SNP) were excluded, and genotypes causing remaining Mendelian errors were set to missing. SNPs out of Hardy-Weinberg Equilibrium (P < 0.001; 6 SNPs) were excluded from analysis. These measures resulted in a final set of 312 RORA SNPs (93% of alleles captured; mean r2 = 0.959), and 43 RORB SNPs (98% of alleles captured; mean r2 = 0.959).Family-based transmission disequilibrium tests (TDT) and case-control association tests were conducted independently on the two sample sets using the program PLINK[]. Bonferroni correction for multiple testing was implemented based on the number of SNPs analyzed per gene. The critical P-value for a positive association was therefore 1.6 × 10-4 for SNPs from RORA and 1.2 × 10-3 for SNPs from RORB. In addition to these separate analyses for the case-control and the family-based samples, a combined odds ratio for the two sample sets was determined via the method described in Kazeem and Farrall []. Haplotype analyses were performed for both sample sets using the Confidence Intervals algorithm in Haploview [,]. Haplotype block associations were considered significant with a two-tailed permutation-based P-value < 0.05 after 1000 permutations.Association with genotyped SNPs and age-at-onset (AAO) of bipolar disorder was analyzed by performing a quantitative trait analysis using the option qfam in PLINK. This option takes into account family structure information, so we were able to include data from the family-based sample and the case-control sample in the same analysis. The AAO phenotype was set to missing for all controls. Results were considered significant with a permutation-based P-value < 0.05 after 1000 permutations. […]

Pipeline specifications

Software tools Haploview, PLINK
Application GWAS
Organisms Mus musculus, Homo sapiens