Bead express platform

Patients included in the study were genotyped using the Illumina Immunochip-v1 on the Illumina Bead Express platform at the Broad Institute (Cambridge, MA). The Immunochip is an Illumina genotyping platform containing 196,524 polymorphisms (718 small insertion deletions, 195,806 SNPs), with dense coverage of known major immune and inflammatory diseases loci.

DNA samples were derived from whole blood collected from study subjects at the time of VARA enrolment. Quantitative polymerase chain reaction was used to genotype patients’ DNA. SNP selection for FPGS and GGH utilised a tagSNP approach to detect associations with SNP blocks defined by linkage disequilibrium (LD), utilising the following parameters: 1) LD blocks were defined using a Caucasian LD map TagSNPs with an r²=0.8; 2) minor allele frequency (MAF) >0.1; 3) range of −1,500 basepairs (bps) from the initiation codon to +1,500 bps from the termination codon; and 4) 1 SNP/LD bin. MTHFR SNPs were chosen a priori based upon previously published reports (16 (link), 18 (link)).The patients were genotyped for the following SNPs: FPGS (rs7033913, rs10760503, rs10106); GGH (rs12548933, rs7010484, rs4617146, rs719235, rs11988534) and MTHFR (rs1801131, rs1801133). Genotyping was performed using either a BeadExpress platform (Illumina, San Diego, CA) (SNPs for FPGS, GGH, and MTHFR 1801133) or by TaqMan assay (MTHFR 1801131) using a Gene Amp 9700 PCR machine (Applied Biosystems, Foster City, CA) with endpoint analysis on a PRISM 7900HT Sequence Detection System (Applied Biosystems). Additionally, the participants were genotyped for the human leukocyte antigen shared epitope (HLA-DRB1-SE) containing alleles as previously described (24 (link)).