Introduction

Statins hold a central place in the therapy of coronary heart disease (CHD), exerting a multifaceted effect on key pathogenetic mechanisms of atherothrombosis. The primary therapeutic goals for patients with CHD, who are categorized as having very high, and in some cases extreme, cardiovascular risk, are achieving target levels of low-density lipoprotein cholesterol (LDL-C) — below 1.4 mmol/L or below 1.0 mmol/L, respectively — or reducing its concentration by 50% or more from baseline. This is critically important, as a 1 mmol/L reduction in LDL-C lowers the risk of cardiovascular events by 20% [1, 2]. Besides achieving target LDL-C levels, normalizing high-density lipoprotein cholesterol (HDL-C) levels is also an important factor. Many studies have demonstrated that low HDL-C levels increase the risk of developing cardiovascular diseases [3]. A number of studies confirm that statins are effective not only in reducing LDL-C levels but also in increasing HDL-C levels [4].

The clinical efficacy of statins in CHD has been confirmed by large-scale studies. For instance, the landmark 4S study (Scandinavian Simvastatin Survival Study), which included 4,444 CHD patients aged 35–70 years with total cholesterol levels of 5.5–8.0 mmol/L, was a key work confirming the effectiveness of statins [5]. The particular role of statins is evident in the treatment of acute coronary syndrome (ACS) as a form of destabilization of chronic CHD, where initiating optimal therapy early during hospitalization is especially crucial. The benefits of early intensive statin therapy in patients with ACS were studied in the large MIRACL trial (Myocardial Ischemia Reduction with Aggressive Cholesterol Lowering) [6], which showed that atorvastatin therapy was associated with an absolute risk reduction in the rate of rehospitalization. The efficacy of statins compared to placebo in significantly reducing mortality, myocardial infarction, unstable angina, the need for percutaneous and surgical coronary revascularization, and stroke in patients with stable CHD has also been demonstrated in another randomized placebo-controlled trial [7].

However, despite the proven efficacy of statins, not all patients achieve target LDL-C and total cholesterol levels. Treatment inefficacy may be due to a combination of factors, including poor adherence to treatment, non-compliance with a lipid-lowering diet, and prescription of an incorrect drug dosage, which can lead to insufficient effect or adverse drug reactions. Another common reason is the patient's voluntary withdrawal from therapy. One study investigating reasons for withdrawal found that the most common causes were unwillingness to change lifestyle, general rejection of statins, and fear of side effects [8]. Currently, an important aspect of statin therapy inefficacy may be the presence of mutant alleles of the CYP3A4 gene, responsible for statin metabolism. Personalized medicine is currently a leading direction, and pharmacogenetics is one of its key tools, allowing for an individualized approach to patient treatment based on their genetic characteristics [9].

Statin metabolism is primarily carried out by the CYP450 system. Studying CYP450 genes when prescribing statins plays a key role in ensuring the safety and efficacy of therapy. Most statins (with the exception of pravastatin) are metabolized in the liver by CYP450 enzymes, as are approximately 75% of drugs [5, 10]. Molecular studies have identified several CYP3A4 variants, with the most important variant allele of this gene being CYP3A4*1B (rs2740574). Data on the influence of this polymorphism on treatment efficacy and the metabolic activity of the CYP3A4 enzyme are contradictory [11]. A study by Rosales A et al. showed that carriage of the polymorphism is associated with high lipid-lowering efficacy of atorvastatin and reduced CYP3A4 enzyme activity in the liver, which could lead to a slower drug metabolism rate and an increased risk of adverse effects [12]. Meanwhile, a large randomized comparative study, GEOSTAT-1, examined the CYP3A5*1 allelic variant. It was shown that the target LDL-C level was observed significantly more often in patients with variant CYP3A5 genotypes [13]. These results underscore the importance of CHD prevention and the need for active detection and correction of hypercholesterolemia in practically healthy individuals from risk groups.

Objective

To evaluate the influence of CYP3A4 gene polymorphisms on the lipid-lowering efficacy of atorvastatin in patients with coronary heart disease in real-world clinical practice.

Materials and methods

The study included 96 patients with coronary heart disease who were inpatients at the Regional Vascular Center of the E.E. Volosevich First City Clinical Hospital in Arkhangelsk between February 2024 and July 2024.

Inclusion criteria: voluntary informed consent to participate in the study; confirmed coronary heart disease (ICD codes I20.0-I25); hospitalization in the emergency cardiology department of the E.E. Volosevich First City Clinical Hospital of the Arkhangelsk Region; atorvastatin therapy; age over 18 years.

Exclusion criteria: withdrawal of consent at any stage of the study.

To determine the allelic variants A/G (rs2740574), Leu293Pro (rs28371759), Phe189Ser (rs4987161) of the CYP3A4 gene, real-time polymerase chain reaction (PCR) was performed on a Bio-Rad CFX96 Touch amplifier. The "DNA-Extran-1" reagent kit manufactured by "Syntol" was used for DNA extraction from whole blood. The study design was approved by the local ethics committee of Northern State Medical University (protocol No. 11/12-24).

Statistical processing of the data obtained during the study was performed using descriptive and analytical statistics with the STATA 2014 program.

Results

The patient sample consisted of 58 men (60.4%) and 38 women (39.6%). Patient ages ranged from 39 to 100 years (Me=70 [59; 75]), among women from 52 to 100 years (Me=75 [72; 83]), and among men from 39 to 86 years (Me=64 [55; 71]). The mean total cholesterol level upon hospital admission was 5.19±1.43 mmol/L, LDL-C was 3.23±1.19 mmol/L, and HDL-C was 1.1 (0.9; 1.34) mmol/L. All patients received statins as part of their baseline CHD therapy during the hospital stay; based on clinical guidelines, 92.7% of patients received atorvastatin, and 7.3% received rosuvastatin. Furthermore, 93.1% of patients were taking atorvastatin before hospitalization. The clinical and anamnestic characteristics of the patients are presented in Table 1.

Table 1

Characteristics of patients included in the study

ParameterNumber (total number of study subjects — 96)Age70 years (from 59 to 75 years)Sex (n (%))Male — 58 (60.4%) Female — 38 (39.6%)Arterial hypertension (n (%)): None6 (6.25%)Stage 10 (0%)Stage 23 (3.13%)Stage 387 (90.63%)Diabetes mellitus (n (%)): None71 (73.96%)Type 13 (3.13%)Type 222 (22.92%)Pre-obesity (n (%))38 (39.58%)Obesity (n (%)): Class 121 (21.88%)Class 25 (5.2%)Class 31 (1.04%)Smoking (n (%))32 (33.33%)Took statins before hospitalization (n (%))29 (30.2%)Atorvastatin27 (93.1% of those on prior statins)Rosuvastatin1 (3.45% of those on prior statins)Rosuvastatin + ezetimibe1 (3.45% of those on prior statins)SBP (mm Hg)140 (123; 160)DBP (mm Hg)80.64±13.22Total Cholesterol (mmol/L)5.3 (4.08; 6.19)LDL-C (mmol/L)3.23±1.19HDL-C (mmol/L)1.1 (0.9; 1.34)

In patients with CHD hospitalized in the emergency cardiology department of the E.E. Volosevich First City Clinical Hospital in Arkhangelsk, allelic variants of the CYP3A4 gene responsible for statin metabolism were analyzed using molecular genetic analysis. The distribution of allelic polymorphisms in the studied genes is presented in Table 2.

Table 2

Frequency of CYP3A4 gene genotypes and alleles in patients, % (n --- number of alleles)

Genotype/AlleleFrequencyCYP3A4_3 A/G (rs2740574) A/A84.4% (81)A/G14.6% (14)G/G1.0% (1)A91.7% (172)G8.3% (16)CYP3A4 Phe189Ser (rs4987161) T/T100% (96)T/C0%C/C0%T100% (192)C0%CYP3A4_2 Leu293Pro (rs28371759) T/T99.0% (95)T/C1.0% (1)C/C0%T99.5% (191)C0.5% (1)

According to the results of molecular genetic testing for the A/G (rs2740574) polymorphism of the CYP3A4 gene, the AA genotype was detected in 84.4% (n=81) of cases, AG in 14.6% (n=14), and the GG genotype in 1.0% (n=1). The frequency of the A allele was 91.7%, and the G allele was 8.3%. Genotyping for carriage of the CYP3A4 (Leu293Pro, rs28371759) allelic variants showed the following results: the TT genotype was detected in 98.96% (n=95), the heterozygous TC allele occurred in 1.04% (n=1), and the homozygous CC polymorphism was not detected. The frequency of the T allele was 99.48%, and the C allele was 0.52%. In the study of the CYP3A4 (Phe189Ser, rs4987161) polymorphism, TT was detected in 100% (n=96); no heterozygotes or homozygotes for the minor allele were found.

The prevalence of mutant alleles of this gene in the study sample was compared with other populations. Analysis of CYP3A4_3 A/G (rs2740574) allele frequencies in patients with CHD compared to the global sample showed no statistically significant differences (p=0.2373). However, statistically significant differences were found with the European (p=0.0005) and Russian (p=0.0095) samples (Table 3).

Table 3

Comparative analysis of CYP3A4_3 A/G (rs2740574) gene allele frequencies in patients with CHD and a healthy population [14, 15]

PopulationSample (N)/total alleles (n)Frequency of G allele CYP3A4_3 A/G (n (%))ResultCHD patients in Arkhangelsk96/19216 (8.33%)-Global [14]69736/139,47215,347 (11%)χ²=1.40 p=0.2373Europe [14]54420/108,8403,954 (3.63%)χ²=12.07 p=0.0005Russia [15]443/88635 (4%)χ²=6.73 p=0.0095

Analysis of CYP3A4_2 Leu293Pro (rs28371759) gene allele frequencies in patients with CHD revealed significant, statistically significant differences compared to global (p=0) and European (p=0) populations (Table 4), which may be explained by the small sample size.

Table 4

Comparative analysis of CYP3A4_2 Leu293Pro (rs28371759) gene allele frequencies in patients with coronary heart disease and in a healthy population [14, 15]

PopulationSample (N)/total alleles (n)Frequency of C allele CYP3A4_2 Leu293Pro (n (%))CHD patients in ArkhangelskCHD patients in Arkhangelsk96/1921 (0.52%)-Global [48]309888/619,776324 (0.052%)χ²~(1)~=17.93; p=0Europe [48]262030/524,0608 (0.0015%)χ²~(1)~=604.78; p=0

When comparing allele frequencies of the CYP3A4 Phe189Ser (rs4987161) gene, no statistically significant differences were found with the global (p=0.9439) or European (p=0.9609) populations (Table 5). The mutant allele was not detected, most likely due to the small sample size.

Table 5

Comparative analysis of CYP3A4 Phe189Ser (rs4987161) gene allele frequencies in healthy and diseased individuals

PopulationSample (N)/total alleles (n)Frequency of C allele CYP3A4 Phe189Ser (n (%))CHD patients in ArkhangelskCHD patients in Arkhangelsk96/1920 (0)-Global [52]48410/96,8205 (0.005)χ²~(1)~=0; p=0.9439Europe [52]39938/79,8762 (0.0025)χ²~(1)~ = 0; p=0.9609

To assess the impact of allelic polymorphism on the therapeutic response to treatment with atorvastatin at a dose of 80 mg per day, the dynamics of lipid profile parameters were analyzed in 15 patients with A/G and G/G genotypes (Table 6), in whom the mutant G allele was identified. Since the occurrence frequencies of the minor alleles for the CYP3A4_2 Leu293Pro (rs28371759) and CYP3A4 (Phe189Ser, rs4987161) genes were 0–0.5%, lipid profile dynamics were not assessed in these cases.

Table 6

Dynamics of lipid profile in carriers of the CYP3A4 A/G (rs2740574) polymorphism G allele during atorvastatin therapy (n=15)

VariableGenotype A/G and G/GGenotype A/At, p At hospitalizationAfter 3 months of treatmentAt hospitalizationAfter 3 months of treatment Total Cholesterol (mmol/L)5.38±1.493.23±0.964.8±1.213.9±1.41t= -3.5973 p=0.0019LDL-C (mmol/L)3.54±1.171.58±0.623.67±0.892.1±0.87t= -4.2939 p=0.0004HDL-C (mmol/L)1.1±0.31.3±0.10.91±0.121.01±0.17t= -3.8712 p=0.0025

Atorvastatin therapy in carriers of the A/G (rs2740574) polymorphism of the CYP3A4 gene demonstrated a significant reduction in total cholesterol (p=0.0019) and LDL-C (p=0.0004) after 8±4 weeks of treatment. At the same time, changes in HDL-C levels were not statistically significant. These results are consistent with data indicating a more pronounced lipid-lowering response to atorvastatin therapy in carriers of the G allele. Despite the pronounced effect, the study's limitations — small sample size (n=15), predominance of patients on a high dose of atorvastatin (80 mg per day — 75% of the group), and lack of control for external factors — do not allow for a definitive conclusion about the polymorphism's impact on the efficacy of atorvastatin therapy.

Discussion

The obtained results demonstrate significant differences in the epidemiology of CYP3A4 A/G (rs2740574) gene allelic variants among patients with CHD in Arkhangelsk compared to European and Russian populations. The predominance of the wild-type homozygous genotype and the relatively high frequency of the mutant G allele (8.3%) may be related to the genetic characteristics of the northern Russian population, where a founder effect or genetic drift, due to historically lower population migration, is possible. Similar regional differences in CYP3A4 allele frequencies have been previously described in studies on pharmacogenetics in isolated populations [16, 17].

Regarding the other studied polymorphisms, an extremely low frequency of minor alleles was detected in this sample. For the CYP3A4_2 Leu293Pro (rs28371759) polymorphism, the frequency of the minor C allele was 0.5% (heterozygous genotype T/C — 1.0%), which was statistically significantly different from frequencies in global (p=0.0000) and European (p=0.0000) populations. For the CYP3A4 Phe189Ser (rs4987161) polymorphism, the minor C allele was not detected, which is consistent with its known low frequency in European and global populations and was likely exacerbated by the limited sample size. The complete absence of variant alleles at the Phe189Ser locus and their minimal presence at the Leu293Pro locus did not allow for an analysis of their impact on treatment efficacy. However, the very fact of their detection, even in isolated cases, indicates the presence of a specific genetic background in the studied population, requiring further investigation in larger samples.

The observed significant reduction in total cholesterol and LDL-C levels in carriers of the mutant G allele of CYP3A4 A/G (rs2740574) may be explained by altered metabolic activity of the enzyme. The rs2740574 (A>G) polymorphism in the promoter region of the CYP3A4 gene is associated with altered enzyme induction, which may lead to variability in statin clearance [18]. Carriers of the G allele may exhibit reduced CYP3A4 activity, leading to slower metabolism of atorvastatin and, consequently, a more pronounced lipid-lowering effect [19].

Data on the influence of the CYP3A4 A/G (rs2740574) polymorphism on treatment efficacy and the metabolic activity of the CYP3A4 enzyme are quite contradictory. On one hand, carriers of the mutant G allele show high lipid-lowering efficacy of atorvastatin, as well as higher drug concentrations in the blood and reduced CYP3A4 enzyme activity in the liver, which could lead to a slower drug metabolism rate and an increased risk of adverse effects. On the other hand, some studies demonstrate an association of this same allele with higher LDL-C levels after treatment and, presumably, increased CYP3A4 enzyme activity [1, 20]. Similar results were obtained in a study by Maslub MG et al., where in Egyptians, the mutant CYP3A4 A/G (rs2740574) alleles were associated with higher blood concentrations of atorvastatin and lower triglyceride levels after treatment [21]. However, another study demonstrated that the AG or GG genotype of CYP3A4*1B was associated with higher LDL-C levels in patients with hypercholesterolemia taking 10 mg of atorvastatin compared to patients with the AA genotype [22]. Furthermore, mutant CYP3A4*1B may be associated with a lower risk of elevated plasma drug levels in patients taking atorvastatin and simvastatin, and with increased CYP3A4 enzyme activity [11, 23].

Thus, the identified differences in the lipid-lowering efficacy of atorvastatin in carriers of the CYP3A4 G (rs2740574) allele are due to altered drug pharmacokinetics, underscoring the importance of a personalized approach to prescribing statins, considering patients' pharmacogenetic characteristics. Although the impact of the CYP3A4_2 (Leu293Pro) and CYP3A4 (Phe189Ser) polymorphisms could not be assessed in our study due to their low frequency, their potential functional significance, related to amino acid substitutions in the enzyme structure, requires further investigation within larger-scale pharmacogenetic studies in northern Russian populations.

Study limitations

Despite the significant results obtained, this study has several limitations that should be considered when interpreting the data:

Recommendations for future research

To overcome these limitations, it is advisable to: increase the sample size, especially for rare genotypes; ensure a standardized follow-up protocol with mandatory lipid profile monitoring; consider additional genetic and non-genetic factors influencing treatment response; and conduct multicenter studies to enhance data representativeness.

Conclusion

This study, conducted in real-world clinical practice in Arkhangelsk, demonstrated a predominance of wild-type homozygous genotypes for the studied CYP3A4 gene polymorphisms. Among the mutant variants, the G allele (8.3%) of the CYP3A4 A/G (rs2740574) polymorphism was the most frequent. For the CYP3A4_2 Leu293Pro (rs28371759) polymorphism, the minor C allele was detected with a frequency of 0.5%, while for the CYP3A4 Phe189Ser (rs4987161) polymorphism, minor alleles were not found in the study sample.

Significant differences were found in the prevalence of CYP3A4 A/G (rs2740574) gene allelic variants in the studied sample of CHD patients from Arkhangelsk compared to the European (p=0.0005) and Russian (p=0.0095) populations. Statistically significant differences were also identified in the distribution of CYP3A4_2 Leu293Pro (rs28371759) alleles compared to global and European data, which, along with the unique frequency profile of CYP3A4 A/G, may reflect the genetic specificity of the northern population.

Analysis of the impact of the CYP3A4 A/G (rs2740574) polymorphism on treatment efficacy showed that in patients with CHD — carriers of the mutant G allele — there is a significant reduction in total cholesterol (p=0.0019) and low-density lipoprotein cholesterol (p=0.0004) after 2–4 months of atorvastatin therapy. Due to the extremely low occurrence frequency, it was not possible to assess the impact of the CYP3A4_2 (Leu293Pro) and CYP3A4 (Phe189Ser) polymorphisms on lipid metabolism.

This study demonstrates the importance of implementing pharmacogenetic testing in patients with coronary heart disease receiving statin therapy. The obtained data underscore the need for further population pharmacogenetic studies with larger sample sizes to assess the contribution of rare, but potentially significant, polymorphisms such as CYP3A4_2 Leu293Pro (rs28371759) to the efficacy and safety of lipid-lowering therapy.