**Background:** Cardiovascular diseases (CVDs) are the leading cause of mortality globally, accounting for approximately 74.4% of all fatalities in 2019. Myocardial infarction (MI) is a major CVD subtype with high mortality and disability rates, and is among the top 10 causes of death in Pakistan. Both lifestyle and genetic factors contribute to CVD development, with heritability estimates of 40–80%. The APOE gene (chromosome 19) encodes a serum glycoprotein critical for lipid transport and metabolism; its two common SNPs (rs429358 and rs7412) form three alleles (ε2, ε3, ε4) and six genotypes. The PON1 gene (chromosome 7q21.3-q22.1) encodes a membrane-bound glycoprotein with anti-inflammatory and anti-oxidative properties; its common polymorphisms L55M and Q192R have been linked to lipid disorders. Despite increasing CAD incidence in Pakistan, no prior study had examined these variants in the Pashtun ethnic population of Khyber Pakhtunkhwa (KP).
**Methods:** This case-control study enrolled 300 age- and gender-matched individuals of Pashtun ethnicity (200 MI cases, 100 healthy controls) from July 2018 to July 2019. Cases were recruited from three tertiary care hospitals in Peshawar (Lady Reading Hospital, Hayatabad Medical Complex, Khyber Teaching Hospital), while controls were collected from various districts of KP. MI diagnosis followed ACC/AHA classification, confirmed by a senior cardiologist based on medical records, cardiac enzymes, ECG abnormalities, and angiography/echocardiography. CAD was defined as stenosis >50% in at least one significant coronary artery segment. Controls had no lumen stenosis (<50%) or physical indications of CVD. Inclusion criteria for cases: confirmed MI, Pakistani Pashtun origin, age ≥30 years. Exclusion criteria: age >80 or <30 years, mental illness, severe liver disease, malignant tumor, renal dysfunction. Blood samples (2.5 mL in EDTA and plain tubes) were collected after overnight fasting. Genomic DNA was extracted using the WizPrep DNA extraction kit. DNA pools were created from all 200 MI patients and 100 controls. Whole Exome Sequencing (WES) identified 12 variants in APOE and 16 in PON1. Selected SNPs were genotyped using the Sequenom MassARRAY platform. Statistical analysis was performed using SPSS; categorical data were analyzed with Chi-square test, continuous variables expressed as mean±SD. Odds ratios were estimated using binary logistic regression with 95% confidence intervals, adjusted for age, gender, smoking, family history of MI, TC, and LDL-C.
**Key Results:** Co-morbidities were more prevalent in cases vs. controls: hypertension (55% vs. 36%, p=0.001) and diabetes mellitus (47.5% vs. 32%, p=0.007). Family history of MI was present in 55.5% of cases vs. 21% of controls (p<0.001). Most cases had a sedentary lifestyle (70% vs. 30%, p=0.029). Male smoking was 58.5% in cases vs. 26% in controls (p<0.001). Lipid profiles showed significantly higher TC (265±15 vs. 230±12 mg/dL, p<0.001), LDL-C (125±14 vs. 100±8 mg/dL, p<0.001), and lower HDL-C (58±7 vs. 60±5 mg/dL, p<0.001) in cases. WES identified 33,329 exonic SNPs including 3,600 homozygous, 29,729 heterozygous, 31,488 synonymous, 1,086 deletions, 68 pathogenic, 3,456 missense, and 460 probably damaging variants. For APOE, the ε3/ε4 genotype showed significant association with MI [OR=2.13 (95% CI: 1.32–2.65), p=0.031], and the ε4 allele was significantly associated [OR=2.11 (95% CI: 1.25–2.43), p=0.030]. The ε3 allele was most common (73% cases, 81% controls). For PON1 Q192R, the RR genotype was significantly associated with MI [OR=1.523 (95% CI: 1.087–2.132), p=0.048], and the R allele showed significant association [OR=1.353 (95% CI: 0.959–1.910), p=0.048]. The R allele frequency was 40% in cases vs. 30% in controls. No significant association was found for PON1 L55M genotypes or alleles (all p>0.05). Multivariable logistic regression confirmed that the ε4 allele [adjusted OR=1.98 (95% CI: 1.16–2.36), p=0.024] and R allele [adjusted OR=1.25 (95% CI: 1.22–2.43), p=0.037] remained significant predictors of MI after adjustment for conventional risk factors.
**Clinical Implications:** This study provides the first evidence that APOE rs429358 (ε4 allele) and PON1 Q192R (R allele) are significantly associated with MI risk in the Pashtun population of KP, Pakistan. These variants may serve as potential susceptibility biomarkers for MI risk assessment in this ethnic group. The findings align with studies in Chinese, Russian, Colombian, South Indian Tamil, and Asian Indian populations, but differ from studies in African Caribbean and Turkish populations, highlighting ethnic variability. The study's limitations include a small sample size (n=300), lack of corresponding protein level measurements, and restriction to a single ethnic group, limiting generalizability. Larger, multi-ethnic studies incorporating protein expression analysis are needed to validate these findings.