Patient Sampling and Phenotyping
Saudi MI Study 1: Over a period spanning 2019–2020, samples and data from consecutive subjects with myocardial infarction (MI) attending the Cardiology Clinics, King Fahd Hospital of the University, Al-Khobar, and King Fahd Hospital, Alhafof, Saudi Arabia, were collected for inclusion in this study. Participants ranged in age from 25–66 and were clinically diagnosed with myocardial infarction at the point of recruitment. Clinical diagnosis of MI was derived according to the fourth universal definition of MI [19]. The phenotype data of all subjects were reviewed by a cardiologist consultant to verify uniformity among sites and eligibility according to study criteria. Eligibility for each of the individual cases were reviewed by the consultant committee and assessed for inclusion. For secondary analyses: T2D and hypertension were defined using WHO criteria; LDL, HDL, total cholesterol and Troponin I were determined using: Direct LDL-, Ultra HDL-, Cholesterol- and STAT High Sensitive Troponin I- Alinity c Reagent kits (Abbott, Wiesbaden, Germany) [20–21].
Saudi MI Study 2
Details of the MI cases and controls in this Saudi study are described in detail in a 2016 GWAS of CAD/MI by Wakil et al. [22]. MI cases were evaluated for suspected CAD/MI cases who underwent coronary angiography and echocardiography (ECG) abnormalities at the Catheterization Centre of King Faisal Heart Institute, King Faisal Specialist Hospital and Research Centre, Riyadh (KFSHRC), Saudi Arabia, and represented all five regions of the country. Changes in the biomarkers, myoglobin, cardiac troponin T, pro-brain natriuretic peptide and pro-calcitonin were also assessed. Two experienced interventional cardiologists independently reviewed patient records for the presence of ischemia as per recommendations of the Joint ESC/ACCF/AHA/WHF Task Force for the Redefinition of MI [23]. Exclusion criteria for the cases included major cardiac rhythm disturbances, history of cerebral vascular disease, neurological disorder, psychiatric illness, substance abuse. Controls consisted of individuals in KFSHRC undergoing heart valvular disease surgery, and subjects with chest pain, but were established to have no significant coronary stenosis by angiography. There were 3,481 MI cases available after delineating MI from CAD-alone cases, with 2,299 controls.
MI cases, controls, phenotype ascertainment, ancestry information for the UK Biobank and CARDIoGRAMplusC4D Consortium MI case and controls GWAS datasets comprising 56,278 MI cases and 577,716 non-MI controls are described in detail elsewhere [9].
For the Saudi MI Study 1, ethical approval was obtained from the Imam Abdulrahman Bin Faisal University Institutional Review Board (IRB) committee (IRB- 2019-01-104) and the study was conducted according to the ethical principles of the Declaration of Helsinki and Good Clinical Practice guidelines. Informed written consent in English, with a verified translation in Arabic, was obtained from all participants in accordance with the IRB rules. The Saudi MI Study 2 protocol was approved by the Institutional Review Board (IRB) at the King Faisal Specialist Hospital and Research Centre. Summary level GWAS datasets for the UK BioBank and CardioGRAMplusC4D were downloaded through a resource database outlined in Hartiala et al. [9].
Generation of genotype data and imputation
Saudi MI Study 1
Peripheral blood samples were collected in EDTA tubes and stored at 4oC before extraction of genomic DNA using Gentra, Puregene Blood kits (Qiagen, Maryland, USA) according to the manufacturer’s protocol. DNA concentrations and purity were estimated by fluorometry using a NanoDrop 2000 Spectrophotometer (Thermo Fisher, MA, USA) and were diluted to 20 ng/µl. GWG was then performed using the Infinium Global Screening Array v3.0 (Illumina, CA, USA) which captures 654,027 SNPs or monomorphic/rare variants. Genotype data was clustered using the Illumina GenomeStudio software and standard quality control was performed using PLINK [24]. Normalized intensities for all samples were generated using optiCall clustering [25]. Raw genotypes were imputed using the 1000 Genomes Project (1KGP) v3 multi-ethnic reference panel through the Michigan Imputation Server [26]. The genotype data was subjected to quality control with variants with < 90% missingness and consistency against Haplotype Reference Consortium (HRC) reference panel for strand, reference/alternative alleles, SNP names and genome build positions. Furthermore, the imputed data were subjected to quality control to retain variants with imputation INFO scores of R2 > 0.3 using Minimac, a 99% genotyping and sample call rate, and minor allele frequency (MAF) > 0.01 [27]. Variants with Hardy-Weinberg equilibrium (HWE) p-value < 1x10-8 were excluded from the analyses. Principal Component Analyses (PCA) was computed using fastPCA module in eigensoft package [28]. The data points were then projected on the 1KGP populations [29].
Saudi MI Study 2
DNA, GWG and QC is described in detail in by Wakil et al. [22]. In brief though GWG was performed using Affymetrix Axiom Genome-Wide “ASI Array” (Asian population) with ~ 537,800 directly genotyped SNPs passing quality control filtering. CARDIoGRAMplusC4D and UKBioBank GWAS data and imputation are fully described in outlined in Hartiala et al. [9].
Statistical analyses
Genome-wide Association Studies Meta-analyses: The variants passing quality controlled for imputed dosage data was used to perform genome-wide association analyses for MI cases and controls. To account for the relatedness in the dataset, the analyses for Saudi study 1 and 2 were performed using REGENIE[30]. The associations were adjusted by age, sex, and the first 4 principal components. Two GWAS meta-analyses were performed for discovery of MI loci. Firstly, the Saudi MI study 1 and 2 were meta-analyzed using PLINK 2.0. Secondly the Saudi MI study 1 and 2 were further meta-analyzed with the CARDIoGRAMplusC4D and UK Biobank MI datasets using PLINK 2.0 [31].