Kaspar

Genomic DNA was extracted from whole-blood frozen samples collected at the inclusion in any of the abovementioned studies using the Qiagen FlexiGene Kit (Qiagen, Hilden, Germany) in accordance with the manufacturer's instructions.
Putatively functional SNPs with minor allele frequency of at least 5% in European population were selected for analysis: all nonsynonymous SNPs and SNPs in 3′ and 5′ untranslated regions. No SNPs in intronic regions were selected. Additionally, some SNPs were selected based on previously published literature. Ten different polymorphisms in three MMP genes fulfilling these criteria were genotyped: MMP2 rs243865, rs243849, and rs7201; MMP9 rs17576, rs17577, rs2250889, and rs20544; and MMP14 rs1042703, rs1042704, and rs743257. Predicted function of these polymorphisms was assessed using SNP function prediction [22 (link)]. For SNPs in 5′ or 3′ untranslated regions, HaploReg v4.1 [23 (link)] and GTEx [24 (link)] were also used.
The genotyping of all the SNPs was carried out using a fluorescence-based competitive allele-specific assay (KASPar), according to the manufacturer's instructions (LGC Genomics, UK). For all investigated polymorphisms, 15% of samples were genotyped in duplicates. Genotyping quality control criteria included 100% duplicate call rate and 95% SNP-wise call rate.

Genotyping of NPHSII samples was performed on DNA extracted from blood and carried out using Taqman (Applied Biosystems, Life Technologies, Carlsbad, CA, USA) and KASPar (LGC, Teddington, UK) genotyping assays as well as restriction fragment length polymorphism (RFLP) analysis. For the CoRDia SMI + RR arm, saliva was collected using the Oragene-DNA OG-250 and DNA manually purified using DNA Genotek’s PrepIt-L2P DNA extraction kit (DNA Genotek Inc., Ontario, Canada). Individuals in the CoRDia SMI + RR arm and selected NPHSII samples (n = 185) were genotyped using the Cardiac Risk Prediction Array (Randox Laboratories Ltd, Crumlin, Co Antrim, UK) according to the manufacturer’s instructions. The Cardiac Risk Prediction Array is a multiplex SNP genotyping system which uses Randox’s Biochip Array Technology [13 (link)] to genotype 19 CHD risk SNPs. The protocol involves using multiplex PCR to amplify target DNA in an allele-specific manner. Amplicons are detected by hybridisation to spatially tethered probes on the biochip array surface. Each position on the biochip array corresponds to a specific allele and genotypes are determined using the Evidence Investigator Analyser. In some instances (e.g. for rare genotypes) Sanger sequencing was used to confirm the result.