Computational protocol: Analysis of positional candidate genes in the AAA1 susceptibility locus for abdominal aortic aneurysms on chromosome 19

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Protocol publication

[…] Genes in the linkage interval on chromosome 19 were prioritized functionally using Gene Ontology (GO) [] and Kyoto Encyclopedia of Genes and Genomes (KEGG) [] annotations. mRNA expression of each gene in AAA and control abdominal aorta was also assessed using data from a previously described microarray-based mRNA expression profiling study []. Altogether 55 SNPs with a minor allele frequency (MAF) of 0.10 or greater that were representative of major haplotype blocks in the Caucasian population were identified in and around the nine positional functional candidate genes selected for the study. Each SNP was assessed for its SNP quality score from Illumina and a custom Illumina BeadChip was designed using those SNPs considered to have sufficient designability. The SNPs are listed in Additional file , Table S1.Power calculations were performed using the Genetic Power Calculator [] (http://pngu.mgh.harvard.edu/~purcell/gpc/). We assumed that the polymorphism and the disease locus were in complete linkage disequilibrium (LD) and that they had the same allele frequencies, i.e., the polymorphism was the disease locus. Assuming a disease locus with an additive effect and a disease prevalence of 0.02, our sample size of 394 cases and 419 controls had an 80% power to detect a susceptibility locus with a genotypic relative risk (GRR) ≥ 1.2 (2.4 for two copies of risk alleles) at a significance level of 0.05 for a SNP with a high risk allele frequency (HAF) ≥ 0.2.SNPs were genotyped using the Illumina GoldenGate assay [] and the call rate of each SNP was evaluated using GenCall Software (version 6.1.3.28, Illumina, San Diego, CA). Deviation from Hardy-Weinberg equilibrium (HWE) was evaluated using an exact test [] as implemented in Haploview [] and nominal p-values without correction for multiple testing are reported in Additional File . Allelic association was tested using a Pearson χ2 test with one degree of freedom (DF). To assess the potential for population stratification, HWE and allelic association were also tested separately in the Belgian and Canadian subpopulations. An additional test of association was performed using the general linear mixed model approach implemented in the Statistical Analysis for Genetic Epidemiology (S.A.G.E.) [] program ASSOC, which yields both Wald and likelihood ratio test (LRT) statistics. Both tests gave very similar results and therefore, we report only results from the LRT which is considered more robust in small sample sizes. False discovery rates (FDR) are reported for the LRT. Haplotype association was analyzed using a score test [] implemented in the haplo.score function in the package HaploStats for the R statistical language and environment []. For genes with evidence of association, LD plots were generated from the case and control genotypes separately using HaploView to examine LD structure. [...] Genomic DNA and RNA samples used in DNA sequencing are listed in Additional files (Table S2) and (Table S3). Primer pairs to amplify the coding sequence of CEBPG, cDNA sequence of PEPD, and exonic sequences of CD22 were designed using PrimerQuestSM, a primer design tool based on Primer3 software [] available on the Integrated DNA Technologies website (http://www.idtdna.com/Scitools/Applications/Primerquest/; IDT, Coralville, IA). Primers were selected using predicted melting points, primer hairpins and primer-dimer interactions calculated using the SciTools programs in PrimerQuestSM. DNA amplification by PCR was performed under the following conditions. Final concentrations were 50 mM KCl, 1.5 or 2 mM MgCl2 (primer-pair specific; see Additional file , Table S4), 2 μM dNTPs, 0.167 μM of each primer, 0.025 U/μl AmpliTaq Gold enzyme (Applied Biosystems) in 10 mM Tris-HCl, pH 8.3. Template DNA constituted 1/10th of the final volume for WGA-genomic DNA or 1/20th for cDNA. Template was denatured by heating to 94°C for 10 minutes, which was followed by 40 cycles of denaturation (94°C), annealing (variable) and elongation (72°C), and a final three-minute elongation incubation at 72°C. Detailed cycle times and annealing temperatures are found in Additional file , Table S4.PCR products were separated by agarose gel electrophoresis to assess the sizes of bands and the specificity of the reaction. PCR products with single clean bands of the correct molecular weight were purified for DNA sequencing using Montage™ PCR purification columns (Millipore, Billerica, MA).DNA sequencing was performed using the Cycle Sequencing (Applied Biosystems, Foster City, CA) method at the Applied Genomics Technology Center (AGTC) at Wayne State University. Primers used for DNA sequencing can be found in Additional file , Table S5. Sequence variants were identified by alignment of sequences using BioX software (available at https://www.lagercrantz.name/projects/biox) and manual examination of the sequence plots. [...] To assess the potential function of identified sequence variants, in silico functional analyses were carried out. The SIFT (Sorting Intolerant from Tolerant) algorithm [] was used to predict the structural effects of non-synonymous substitutions. NetPhos [] was used to predict the creation or destruction of phosphorylation sites. To assess the potential effect of 3'-untranslated region (UTR) sequence variants on microRNA (miRNA) binding, predicted binding sites were obtained from the MicroCosm Targets Database [,] (http://www.ebi.ac.uk/enright-srv/microcosm/htdocs/targets/v5/). […]

Pipeline specifications

Software tools SIFT, NetPhos
Application PTM analysis
Organisms Homo sapiens
Diseases Aneurysm, Aortic Aneurysm, Aortic Aneurysm, Abdominal