Genetic Methods

4.2.1 Deoxyribonucleic Acid (DNA) Extraction, Genotyping and Quality Control (I-V)

In YFS, genomic DNA was extracted from peripheral blood leukocytes using a commercially available kit and the Qiagen BioRobot M48 Workstation according to the manufacturer’s instructions (Qiagen, Hilden, Germany). Genotyping was performed on 2,556 samples using a custom-built Illumina Human 670k BeadChip at the Welcome Trust Sanger Institute. Genotypes were called using the Illuminus clustering algorithm. Fifty-six samples failed to meet the Sanger genotyping pipeline QC criteria (i.e. duplicated samples, heterozygosity, low call rate, or Sequenom fingerprint discrepancy). Out of the remaining 2,500 samples, one failed the gender check, three were removed due to low genotyping call rate (< 0.95), and 54 were excluded for possible relatedness (pi-hat > 0.2). Based on the Hardy–

Weinberg equilibrium (HWE) test, 11,766 SNPs were excluded (p ≤ 10-6), and 7,746 SNPs failed the missingness test (call rate < 0.95) and another 34,596 failed the frequency test (MAF < 0.01). After quality control, 2,442 samples and 546,677 genotyped SNPs were available for further analysis (Smith, Chen et al. 2010).

Imputation of SNPs means using the LD structure of the genome in prediction of the SNPs that have not been genotyped (Marchini, Howie 2010). Genotype imputation was performed using MACH 1.0 (Li, Willer et al. 2009, Li, Willer et al.

2010) and HapMap II CEU (release 22, NCBI build 36, dbSNP 126) samples as a reference. Palindromic A/T and C/G SNPs were removed before imputation.

After imputation, 2,543,887 SNPs were available. SNPs with a squared correlation (r2) of ≥ 0.30 between imputed and true genotypes were considered well imputed.

In FINCAVAS and ANGES, for the DNA extraction, 9.0 ml ethylenediaminetetraacetic acid (EDTA) whole blood was drawn from the participants and stored at -20 °C. Genomic DNA was extracted from peripheral blood leukocytes by using the QIAamp DNA Blood Midi Kit and automated biorobot M48 extraction (QIAGEN GmbH, Hilden, Germany). Genotyping was completed for 2,824 samples using the Illumina Cardio-Metabo Chip (Illumina

Inc., San Diego, CA, USA) at the Helmholtz Zentrum, München, Germany. The chips were scanned with the Illumina iScan system and genotypes called with Illumina GenomeStudio software. The following quality control filters were applied: GenCall score < 0.2, sample and SNP call rate < 0.95, HWE p value < 10-6, MAF < 0.01, cryptic relatedness (pi-hat > 0.2), and gender check. After quality control, 2,620 samples and 120,721 SNPs were available. Both genotype and clinical data were available for 2,390 samples in FINCAVAS and for 808 in ANGES.

In LURIC, genomic DNA was prepared from EDTA anticoagulated peripheral blood using a common salting-out procedure. Genotyping was accomplished for 2,966 samples using a custom-built Illumina 200k BeadChip (Cardio-Metabo Chip) at the Institute of Human Genetics at the Department of Genomics, Life & Brain Center, University of Bonn, Germany. Forty-seven samples failed to meet the QC criteria (i.e. duplicated samples, possible relatedness, low call rate [< 0.95], or gender discrepancy). Out of the SNPs, 5,384 failed to meet the QC criteria (HWE test [p ≤ 10–6], call rate < 0.95). After quality control, 2,919 samples and 191,341 genotyped SNPs were available for further analysis.

Both genotype and clinical data were available for 2,912 samples in this study.

In KORA, genotyping was accomplished by using the IBC 50K array, which is an Illumina iSelect genotyping array designed to test ~50,000 SNPs identified through genome-wide meta-analyses associated with a range of cardiovascular, metabolic, and inflammatory syndromes (Keating, Tischfield et al. 2008).

The studied top SNP (rs676210) passed QC (call rate > 0.95, MAF > 0.01, HWE p > 10-6) in all cohorts.

For the WTCCC2 studies and METASTROKE, 2,858 cases and 5,716 matched controls genotyped using the Immunochip platform; and 3,940 cases genotyped using either the Illumina 610k or 660k platforms matched with 6,379 controls genotyped on the Illumina Human 1.2M Duo (UK), Illumina Human 550k (German) and Illumina 610k platforms (Italian). Bead intensity data were processed and normalized in BeadStudio (Illumina); data for successfully genotyped samples were extracted and genotypes called within the collections using Illuminus. German controls were genotyped on the Illumina Human550k platform, and intensity data were processed and normalised for each sample in GenomeStudio (Illumina) using the Illumina cluster file HumanHap550v3.

Standard quality control procedures were undertaken on all centres, before centre-wise imputation to the 1000 Genomes phase 1 integrated variant set (March 2012),

using IMPUTE v2.2.0. SNPs with poor imputation quality (info<0.3) or low minor allele frequency (MAF<0.01) were discarded.

In CHARGE cohorts, the nine studies used commercial genotyping platforms available from Illumina and Affymetrix. Each study performed genotyping quality control checks and imputed the approximately 2.5 million polymorphic autosomal SNPs described in the HapMap CEU population for each participant using available imputation methods.

4.2.2 Messenger Ribonucleic Acid (mRNA) Isolation, Microarrays and Quantitative Real-Time PCR of mRNAs (III-V)

In TVS, fresh tissue samples were immediately soaked in RNALater solution (Ambion Inc.) and homogenized using an Ultra-Turrax® T80 homogenizer (IKA) (Oksala, Pärssinen et al. 2013). RNA was extracted with the Trizol reagent (Invitrogen) and miRNEasy® Mini-Kit (Qiagen) with the RNase-Free DNase Set (Qiagen) according to the manufacturers’ instructions. The RNA isolation protocol was validated by analysing the integrity of the RNA with the RNA 6000 Nano Chip Kit (Agilent).

The quality of the RNA samples was evaluated spectrophotometrically, and the samples were stored in –80°C. The expression levels were analyzed with an Illumina HumanHT-12 v3 Expression BeadChip (Illumina) analyzing 47 000 transcripts of all known genes, gene candidates, and splice variants. 300–500 ng of RNA was reverse transcribed into cRNA and biotin-UTP labelled using the Illumina TotalPrep RNA Amplification Kit (Ambion), and 1500 ng of cRNA was then hybridized to the Illumina HumanHT-12 v3 Expression BeadChip.

The BeadChips were scanned with the Illumina iScan system. After background subtraction, raw intensity data was exported using the Illumina GenomeStudio software. Further data processing was conducted by means of R language and appropriate Bioconductor modules. Data was log2-transformed and robust multichip average and robust spline normalization (rma_rsn) was used.

After background subtraction, raw intensity data were exported using the Illumina GenomeStudio software. Raw expression data were imported into R version 3.1.1 (http://www.r-project.org/), log2 transformed and normalized by the locally estimated scatterplot smoothing normalization method implemented in the R/Bioconductor package Lumi (www.bioconductor.org) (Oksala, Pärssinen et al.

2013). Locally estimated scatterplot smoothing normalization was selected for the

data because it gave the best accuracy in comparison with quantitative reverse transcription polymerase chain reaction data for artery samples (Raitoharju, Seppälä et al. 2013). Data quality control criteria included detection of outlier arrays based on the low number of robustly expressed genes and hierarchical clustering. Artery samples (n=92: 68 plaque, 24 LITA) fulfilled all data quality control criteria. Probes were considered robustly expressed if the detection was P<0.05 for at least half of the samples in the data set. Both HDAC9 and MMP12 were robustly expressed in human atherosclerotic plaques and LITAs.

The high MMP12 expression in the samples was also confirmed using reverse transcription polymerase chain reaction (RT-PCR). The accuracy, sensitivity and specificity between the HumanHT-12 v3 Expression BeadChip and TagMan qRT-PCR low-density array (LDA) in TVS have been validated earlier (Raitoharju, Seppälä et al. 2013).