Apolipoprotein E (APOE) Gene Polymorphisms in Libyan Patients with Coronary Heart Disease: A Pilot Descriptive Study with Methodological Considerations for Future Research

Technical Summary: APOE Gene Polymorphisms in Libyan CHD Patients

Problem Statement
Coronary heart disease (CHD) remains a leading cause of global mortality, driven by complex interactions between lifestyle, environment, and genetics. While the Apolipoprotein E (APOE) gene polymorphisms (specifically alleles ε2, ε3, and ε4) are well-documented regulators of lipoprotein metabolism and CHD risk in various populations, there is a complete absence of genetic data regarding these polymorphisms in North African populations, including Libya. This study addresses the gap in understanding the genetic architecture of CHD in Libya and investigates the feasibility of conducting such genetic research in the region.

Methodology
This was a cross-sectional pilot descriptive study conducted between January and October 2025 involving 60 clinically confirmed CHD patients recruited from two cardiac centers in Tripoli and Zliten, Libya.

• Participants: Adults (>18 years) with confirmed CHD and at least one additional risk factor (hypertension, diabetes, dyslipidemia, or smoking). Six of 66 initially enrolled patients were excluded due to invalid genotyping results.
• Genotyping: APOE polymorphisms (rs429358 and rs7412) were primarily identified using the CVD Strip Assay (Vienna Lab Diagnostics GmbH), a reverse-hybridization assay.
• Validation: A separate validation subset of 28 individuals (14 CHD patients, 14 healthy controls) was genotyped in parallel using the Strip Assay and an independent real-time PCR kit (YZYMED Biotechnology Co., Ltd.) to assess methodological concordance. No orthogonal sequencing reference (e.g., Sanger sequencing) was used as a gold standard.
• Analysis: Data were analyzed using SPSS v27. Associations were tested using chi-square or Fisher's exact tests. Cohen's κ was used to measure concordance between genotyping platforms.
• Design Constraints: The study was a pilot with no formal sample size calculation. Crucially, a matched control group could not be included in the final analysis due to exclusions, limiting the study to descriptive frequency analysis rather than risk estimation.

Key Results

• Genotype Distribution: Among the 60 CHD patients, the ε2/ε4 genotype was the most prevalent (45.0%), followed by ε3/ε4 (30.0%), ε2/ε3 (16.7%), and ε3/ε3 (8.3%). Genotypes containing the ε4 allele accounted for 75.0% of the cohort. The ε4 allele frequency was 37.5%.
• Clinical Associations: A significant association was observed between smoking status and APOE genotype among male patients (p = 0.001), though this was based on a small subgroup (n = 24). No significant associations were found between genotype and residence, gender, or comorbidities.
• Lipid Profiles: 60.0% of patients presented with normal lipid profiles at the time of assessment, attributed to ongoing lipid-lowering therapy (71.7% on statins), which obscured potential genotype-phenotype relationships.
• Methodological Concordance: The validation subset revealed a critical discrepancy between genotyping methods. Concordance between the Strip Assay and real-time PCR was only 34.8% (κ = 0.19), indicating "slight agreement." The real-time PCR method disproportionately assigned ε3/ε3 genotypes, suggesting an under-calling of heterozygosity.

Key Contributions

1. First Genetic Data for Libya: This study provides the initial documentation of APOE genotype frequencies among Libyan patients with CHD, filling a significant void in North African genetic epidemiology.
2. Methodological Warning: The study identifies severe reliability issues with the specific genotyping platforms used. The low concordance rate (34.8%) between the Strip Assay and real-time PCR, without sequencing validation, highlights a critical need for orthogonal validation (e.g., Sanger sequencing) in future genetic studies within this population.
3. Feasibility Assessment: The paper serves as a feasibility study, outlining the logistical and technical challenges of conducting genetic research in the Libyan healthcare setting, including the impact of pharmacological masking on lipid phenotypes.

Significance and Claims
The authors explicitly frame this work as a pilot descriptive study and a hypothesis-generating effort rather than a definitive clinical association study.

• Preliminary Nature: The authors caution that the high frequency of ε4-containing genotypes (75.0%) cannot be interpreted as a confirmed risk factor for CHD in Libyans due to the absence of a matched control group. The observed frequencies may reflect population-specific founder effects, selection bias, or background genetics rather than disease association.
• Call for Rigor: The primary significance of the paper lies in its methodological lessons. It asserts that future research must prioritize larger, multicenter case-control studies with matched controls, standardized genotyping protocols, and mandatory orthogonal validation (such as Sanger sequencing) to ensure data accuracy.
• Future Directions: The study concludes that while the findings suggest APOE polymorphisms may contribute to CHD susceptibility in this population, these results are preliminary and require confirmation in adequately powered studies that account for confounders like medication use and gene-environment interactions.