Massarray platform

As described in our previous studies [7 (link), 17 (link)], we employed a two-step approach to select tagSNPs for the genes of interest. Briefly, Haploview software (http://www.broadinstitute.org/mpg/haploview) was used to minimize the number of SNPs that needs to be genotyped and FastSNP search (http://FastSNP.ibms.sinica.edu.tw/) was performed to predict their functional effects. The predicted function of each tagSNP selected in this study were summarized in Supplementary table 1. Genomic DNA was isolated from peripheral blood lymphocytes by the routine phenol–chloroform method. The genotypes of 13 tagSNPs (rs4711690, rs6458238, rs9471643, rs6941539, rs6912200, rs3789210, and rs6939861 of PGC; rs10983755 and rs11536878 of TLR4; rs12229892 of PTPN11; and rs1143623, rs1143627, and rs1143643 of IL1B) were selected and assessed by the Sequenom MassARRAY platform (Sequenom, San Diego, CA, USA) according to the manufacturer's instructions [7 (link), 17 (link)]. Each DNA sample was diluted to a working concentration of 50 ng/μL for genotyping. All samples were randomly placed on the 384-well plates and the operator confirming the SNP genotyping calls was blinded to disease status. Randomly selected samples had repeat genotypes performed and 100% concordance was confirmed.

We genotyped the rs11591147 (R46L) genetic variant of PCSK9, which was selected from the International HapMap database (http://hapmap.ncbi.nlm.nih.gov/). The LOF PCSK9 genetic variant rs11591147 (R46L) is associated with lower levels of plasma PCSK9 [24 (link)]. Genomic DNA was extracted from peripheral leukocytes that had been isolated from anticoagulated venous blood using a QIAamp DNA Blood Kit (Qiagen Iberia SL, Madrid, Spain) according to the manufacturer’s instructions. The DNA was genotyped for the genetic variant on the Sequenom MassARRAY platform using the iPLEX Gold protocol as specified by the manufacturer (Sequenom Inc., San Diego, CA) [25 (link)]. The genotypes of 5 of the samples were confirmed using duplicate Sequenom runs, and they showed 100 % consistency. Genotyping was performed at the Spanish National Genotyping Centre.

DNA extraction was performed with a DNA extraction kit (Qiagen, Hilden, Germany). The genotyping for the replication cohort was conducted by the Sequenom MassARRAY platform (Sequenom, Inc., San Diego, CA, USA), and the genotyping yield was higher than 99.5%.

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.

All SNPs with minor allele frequencies (MAF) ≥ 0.01 were searched for between 15 kb upstream and 15 kb downstream of the IBSP and PTHLH genes in the HapMap HCB database by Haploview v4.225 (link). A total of 27 SNPs in the IBSP gene and 24 SNPs in the PTHLH gene were identified. Three additional SNPs in the IBSP gene were included from previous reports17 (link)24 (link). Based on the above criteria, 54 SNPs were included in further analyses (Supplementary Table S1). Peripheral blood was drawn from a vein into a sterile tube containing ethylenediamine tetraacetic acid (EDTA). Genomic DNA was extracted from peripheral blood leukocytes according to the manufacturer’s protocol (Genomic DNA kit, Axygen Scientific Inc., California, USA). DNA was stored at −20 °C for SNP analysis. Genotyping was performed for all SNPs using the MassARRAY platform (Sequenom, San Diego, California, USA). Briefly, SNPs were genotyped using high-throughput, matrix-assisted laser desorption ionization–time-of-flight (MALDI–TOF) mass spectrometry. The resulting spectra were processed using Typer Analyzer software (Sequenom), and genotype data were generated from the samples. As the final genotype call rate of each SNP was greater than 98% and the overall genotyping call rate was 99.6%, the reliability of further statistical analysis was ensured.

Screening of EGFR and KRAS mutations was performed by Sequenom MassARRAY platform with OncoFOCUS Panel v1.0 (Supplementary Table S1, Sequenom, San Diego, CA). Mutiplex PCR reaction for tumor DNA (20 ng) was admixed with Taq polymerase (0.2 units), PCR primer (2.5 pmol) and dNTP (25 mM, Sequenom, PCR accessory and Enzyme kit) in 5 μl volume. Thermocycling was programmed as 94  °C for 4-minutes followed by 45 cycles of 94°C for 20-sec, 56 °C for 30-sec and 72 °C for 1-minute, then 72 °C for 3-minutes, respectively. After deactivation of unincorporated dNTPs by using shrimp alkaline phosphatase (0.3 units), single base extension reactions was performed by iPLEX Pro single base extension reactions by using Sequenom, iPLEX Pro kit according to manufacturer’s instructions. After removing residual salt from the reactions by cation-exchange resins, 7 nl of the purified primer extension reaction was loaded onto a matrix pad of a SpectroCHIP (Sequenom, San Diego, CA), analyzed using a MassARRAY Analyzer 4, and the calling by clustering analysis with TYPER 4.0 software.

Six SNPs, five prothrombotic SNPs [rs1799963 (F2), rs6025 (F5, Leiden), rs2066865 (FGG), rs2036914 (F11), and rs8176719 (ABO)] from our previous study (47 (link)), due to their confirmed and large effect size, and the rs121909567 SNP in the SERPINC1 gene, from the so-called antithrombin Budapest3 (ATBp3 mutation) study (36 (link)), were included in the present study. ATBp3 is the common cause of antithrombin (AT) deficiency in Hungary, and the founder effect of this mutation was previously considered in the Roma population (36 (link), 56 (link)).
The assay design and genotyping were performed by Karolinska University Hospital, Stockholm, Sweden Mutation Analysis Core Facility (MAF). A MassARRAY platform (Sequenom, CA, USA) with iPLEX Gold chemistry was used for genotyping. Quality control, validation, and concordance analysis were conducted by MAF.