Computational protocol: PADI2 Is Significantly Associated with Rheumatoid Arthritis

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Protocol publication

[…] Genomic DNA was extracted from whole blood samples using the Omega E-Z 96 Blood DNA kit (Omega, USA) according to the manufacturer’s protocol. After extraction, the genomic DNA was diluted to a final concentration of 15–20 ng/µl for use in the genotyping assays.Tag single nucleotide polymorphisms (tag SNPs) across the PAD locus were identified by searching the HapMap database , . Only SNPs with a minor allele frequency (MAF) greater than 5% and a pair-wise r2≥0.8 were considered. Candidate SNPs were submitted to Illumina for a design score. Finally, seventeen SNPs were selected, as described in .The seventeen SNPs in were genotyped using a custom-designed Illumina 96-SNP VeraCode microarray. Peripheral blood samples were collected from patients with RA (n = 267, 183 females) and AS (n = 51, 10 females). RA patients had a mean age of 51.7 years, while AS patients had a mean age of 35.9 years. A total of 160 (58 females) healthy individuals with a mean age of 48.0 years were blood donors. The work was completed at Beijing Yimei Tongde, a China-based company, which provided technical services for the genotyping.The microarray results were verified with an allele-specific MALDI-TOF mass spectrometry assay (Sequenom MassARRAY). Five SNPs, including rs2235926, rs2057094, rs2076616, rs1204898 and rs10788668, were genotyped in a cohort of 307 patients with RA (252 females), 324 patients with AS (45 females) and 509 healthy controls (194 females), none of whom were included in the experiments utilizing the Illumina microarray. The RA patients had a mean age of 51.43 years, the AS patients had a mean age of 28.88 years, and the healthy individuals had a mean age of 62.88 years. The polymorphism-spanning fragments were amplified by the polymerase chain reaction (PCR), and genotyping was performed using the Sequenom MassARRAY iPLEX platform. Primers for the amplification and extension reactions were designed using MassARRAY Assay Design version 3.1 software (Sequenom, San Diego, CA). The SNP genotypes were obtained according to the protocol provided by the manufacturer. The work was completed at Bioyoung Tech, a China-based company, which also provided technical services for the genotyping.The genotyping quality was evaluated by a detailed quality control procedure consisting of a >95% success identification rate, duplicate identification of the genotypes, internal positive control samples and testing for the Hardy-Weinberg Equilibrium (HWE). The SNPs were analyzed for association by comparing the MAF between the cases and controls. Dominant and recessive models were considered with respect to the minor allele. Associations between the SNPs and the diseases were evaluated using odds ratios (ORs) with 95% confidence intervals (CIs). Fisher’s exact test was used for comparisons between categorical variables. P values less than 0.05 were considered statistically significant. Genotypic associations were assessed using Plink v1.07 (http://pngu.mgh.harvard.edu/purcell/plink/) and SHEsis (http://analysis.bio-x.cn/myAnalysis.php) software . A Bonferroni single-step correction was performed using the Plink v1.07. Linkage disequilibrium (LD), coefficient (D′ and r2) and haplotype was estimated by Haploview 4.2 (http://www.broad.mit.edu/mpg/haploview/) . […]

Pipeline specifications

Software tools PLINK, Haploview
Application GWAS
Organisms Homo sapiens
Diseases Arthritis, Rheumatoid, Osteoarthritis, Spondylitis, Ankylosing