Massarray iplex

From the total number of patients recruited, 515 self-reported white European patients with endoscopically confirmed PUD (206 cases) and 309 controls (124 control group A, 185 control group B) were included in the replication cohort. Cases and controls included in the replication cohort were recruited later than those included in the discovery cohort. Lead variants with a p-value below 5*10⁻⁶ in a clear delineated LD block in the association with aspirin-induced PUD in the discovery cohort were subsequently typed in the replication cohort using the Agena MassArray iPLEX platform (Agena Bioscience Inc, San Diego, CA, USA) according to the manufacturer's protocols and subsequently tested in using the same logistic regression model and methodology in SNPTEST as previously described. SNPs were excluded when MAF<0·01, call rate <95% and HWE p>0·0001 [exact test]. Samples were excluded if genotyping call rate <90%. Meta-analysis combining the association summary data of both cohorts was undertaken under an inverse-variance weighted fixed-effects model using GWAMA [32] (link).

Genomic DNA was extracted from peripheral blood using GoldMag‐Mini Whole Blood Genomic DNA Purification Kits (GoldMag Co. Ltd., Xi'an City, China), and quantified with a spectrophotometer (NanoDrop 2000; Thermo Fisher Scientific, Waltham, MA, United States). To have sufficient DNA for further reactions, polymerase chain reaction (PCR) was applied to each sample. Then SAP purification was performed to remove the remaining dNTP and amplified primers in PCR products. Using a MassARRAY Nanodispenser (Agena Bioscience, San Diego, CA), standardized genotyping reactions were dispensed onto a 384‐well spectroCHIP. The repeated control samples were set in every genotyping plate and the concordance was more than 99%. The genotyping of these SNPs was carried out on the MassARRAY iPLEX (Agena Bioscience, San Diego, CA) platform using the allele‐specific matrix‐assisted laser desorption ionization‐time of flight mass spectrometry (MALDI‐TOF‐MS). Genotyping results were outputted by Agena Bioscience TYPER 4.0. Haploview software package (version 4.2) was used to analyze the linkage disequilibrium (LD), haplotype construction, genetic association at polymorphism loci and haplotype blocks were defined according to the criteria laid out by Gabriel and others.25

Using the database of 1,000 Genomes Project (http://www.1000genomes.org/) and dbSNP (https://www.ncbi.nlm.nih.gov/SNP/), candidate SNPs in the ITPR2 gene with minor allele frequencies (MAFs)> 5% in the global population were selected. A total of eight SNPs (rs1049376, rs11048526, rs11048556, rs11048585, rs16931011, rs10842759, rs2230372, and rs7134213) were selected for further genotyping.
Peripheral blood samples (5 ml) were obtained from each participant. Genomic DNA was extracted from peripheral blood of cases and controls using the GoldMag‐Mini whole blood Genomic DNA Purification Kit (GoldMag Co. Ltd., Xi'an city, China), as recommended by the manufacturer's instructions (Geng et al., 2015). DNA concentration was determined by the NanoDrop Lite spectrophotometer (Thermo Fisher Scientific, Waltham, MA) (Wang et al., 2015). MassARRAY Nanodispenser (Agena Bioscience, San Diego, CA) was used to design primers for amplification process and single base extension reactions (Jin et al., 2015). SNP genotyping was carried out on the MassARRAY iPLEX (Agena Bioscience, SanDiego, CA) platform. Agena Bioscience Typer 4.0 software was used to manage and analyze SNP genotypic data.