Massarray platform

Tumour DNA was tested for the BRAF codon 597 and 600 mutations and KRAS codons 12 and 13 mutations in 98 adenomas and 401 CRC samples. Mutations of BRAF (nucleotides 1790 and 1799) and KRAS (nucleotides 35 and 38) were analysed by genotyping assay on the MassARRAY platform (Sequenom, San Diego, CA, USA). PCR and extension primers for these mutations were designed using MassARRAY Assay Design 3.0 software (Sequenom) and applying default single-base extension settings and default parameters (Additional file 1: Table S1). DNA was amplified by PCR, and a single-base extension reaction was performed using a custom mixture of nucleotides and extension primers that hybridised immediately adjacent to the mutations. Reaction products were transferred to a SpectroCHIP (Sequenom), and mass differences were analysed using MALDI-TOF mass spectrometry to identify the extended base at the possible mutation site. Repeat Sanger sequencing on an ABI PRISM 3730 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) was used to reconfirm the results of MassARRAY and rule out the possibility that any mutations were missed due to the sensitivity of the MassARRAY platform. Primers used to amplify and sequence exon 15 of BRAF and exon 1 of KRAS are shown in Additional file 1: Table S1.

DNA was extracted from whole blood using the MaxiPrep Kit (QIAGEN, Sollentuna, Sweden). SNPs were genotyped using a Sequenom massARRAY platform or TaqMan allelic discrimination assay with an ABI Prism 7900 sequence detection system (Applied Biosystems, Foster City, CA, USA). The success rate of genotyping was > 90%. Replication genotyping of 6% of the samples showed > 98% concordance. All SNPs were in Hardy–Weinberg equilibrium (HWE), except for rs11920090 and rs6467136, which significantly deviated in women who did not develop diabetes postpartum (p < 0.01), and were eventually excluded from the analysis. We analysed 12 SNPs previously shown to be associated with measures of insulin secretion [11 (link), 12 (link), 14 (link)], and 4 SNPs previously shown to be associated with measures of insulin resistance in GWAS [10 (link), 11 (link), 13 ], for association with disposition index and HOMA-IR, respectively, in women with previous GDM. We also analysed 70 (2 out of 72 were excluded for not being in HWE) SNPs, previously associated with diabetes in GWAS [11 (link), 15 (link), 18 (link)], for association with diabetes postpartum.