ABCB1 polymorphism: prevalence and association with clinical,laboratory and demographic factors in patients with ischemic stroke

Introduction

Active transport of xenobiotics across biological cell membranes, consuming energy in the form of adenosine triphosphate (ATP), is mediated by transport systems belonging to the superfamily of ATP-binding cassette transporters (ABC transporters). One representative of this superfamily is P-glycoprotein (P-gp), whose name derives from the term "permeability glycoprotein" and which was previously referred to as multidrug resistance protein 1 (MDR1). P-gp is encoded by the ABCB1 gene, located on chromosome 7 in the p21-21.1 region and comprising 28 exons (coding sequences) [1, 2]. Over twenty single nucleotide substitution variants, termed single nucleotide polymorphisms (SNPs), have been identified; some lead to increased P-gp ATPase activity without elevating ABCB1 RNA or protein levels. The most extensively studied genotypes are CT and TT of the ABCB1 (C3435T) polymorphism, which result in altered functional activity of P-gp [3].

P-gp has been identified on the biliary surface of hepatocytes, on epithelial cells of the small and large intestine, in the membrane of proximal renal tubule cells, in endothelial cells of histohematic barriers—including the blood-brain, blood-ovarian, blood-testis, and blood-placental barriers—as well as in cells of the immune system (mature macrophages, killer cells, T- and B-lymphocytes, monocytes) and in epithelial cells of the adrenal cortex. Thus, P-gp contributes to the pharmacokinetics of drugs by impeding their absorption in the intestine, kidneys, and liver, as well as their passage across histohematic barriers [4, 5].

Since P-gp is encoded in capillary endothelial cells, its expression level, regulated by the ABCB1 gene, may also be associated with organ and system dysfunction [6]. Consequently, ABCB1 (C3435T), which is associated with the development of adverse drug reactions and a predisposition to several diseases, represents a promising pharmacogenetic marker [7].

Objective

To determine the frequency of the ABCB1 (C3435T, rs1045642) polymorphism in patients with ischemic stroke and to assess its association with clinical, laboratory, and demographic parameters.

Materials and Methods

The study was approved by the Local Ethics Committee of the Irkutsk Order of the Badge of Honour Regional Clinical Hospital (Protocol No. 73 dated November 29, 2016) and was conducted in 2017–2018. The study included 120 patients with non-cardiogenic ischemic stroke (IS) hospitalized in the neurological department for patients with acute cerebrovascular accident (Irkutsk Regional Clinical Hospital). The mean age of patients was 61.6 ± 7.7 years. The diagnosis of IS was established according to the classification of cerebrovascular disorders (Schmidt E.V., 1985). Ethnic characteristics of the patients were not considered.

Inclusion criteria: non-cardiogenic ischemic stroke; age from 18 to 74 years; signed informed consent from the patient.
Exclusion criteria: cardiogenic stroke; hemorrhagic stroke.

The clinical and demographic characteristics of the study group are presented in Table 1.

Table 1. Clinical and demographic characteristics of patients, n=120

Assessment of comparability of laboratory parameters between groups stratified by the ABCB1 (C3435T, rs1045642) allelic variant in patients with IS is presented in Table 2.

Table 2. Comparability of laboratory parameters between groups stratified by the ABCB1 allelic variant (C3435T) in patients with ischemic stroke, n=120

Molecular genetic analysis was performed at the Research Institute of Molecular and Personalized Medicine, Russian Medical Academy of Continuing Professional Education, Moscow, Russia. Deoxyribonucleic acid was isolated from peripheral venous blood leukocytes using the DNA-Extran-1 reagent kit (Sintol CJSC, Moscow, Russia). The presence of polymorphic markers of the ABCB1 (C3435T) gene was determined by real-time polymerase chain reaction (Real-Time PCR) using SNP-Screen reagent kits (Sintol CJSC, Moscow, Russia) according to the manufacturer's instructions on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad Laboratories, Inc., USA).

Clinical interpretation of ABCB1 (C3435T) polymorphisms: The presence of the CC genotype is considered indicative of normal P-gp activity. The presence of the T allele is associated with altered pharmacokinetic characteristics of drugs [8].

Statistical analysis

Statistical analysis of the study results was performed using STATISTICA 6.1 (serial No. AXXR010E749701FA) and BIOSTAT (S. Glantz, 1999) software packages. To confirm the validity of using the obtained statistical results for describing quantitative data, conformity to the normal distribution law was assessed; a p-value < 0.05 was considered statistically significant. Independent distribution of alleles in the studied polymorphism was assessed for conformity to the Hardy-Weinberg equilibrium using a calculator (Michael H. Court, 2005–2008); the results of the population study conformed to the Hardy-Weinberg equilibrium at p > 0.05. Comparability of clinical and demographic characteristics and genotyping results was assessed using Pearson's χ² test. Differences in mean quantitative laboratory parameters were evaluated using the Mann-Whitney U test.

Results

The ABCB1 (C3435T, rs1045642) polymorphism was determined in 120 patients with IS. The CC genotype was identified in 22 (18.0%) patients, while CT and TT genotypes were significantly more frequent: CT in 67 (56.0%) and TT in 31 (26.0%) patients. The distribution of genotypes among IS patients for ABCB1 (C3435T, rs1045642) conformed to the Hardy-Weinberg equilibrium (χ² = 1.81; p = 0.18).

To assess the comparability of clinical features of IS, laboratory parameters, and demographic indicators between patients with different ABCB1 allelic variants (C3435T, rs1045642), all patients were divided into two groups: carriers of the CC genotype (n = 22, 18.0%) and the combined group of carriers of CT+TT genotypes (n = 98, 82.0%).

When assessing the comparability of clinical and demographic characteristics with ABCB1 (C3435T, rs1045642) genotyping results, a statistically significant difference was observed in the frequency of diabetes mellitus and overweight/obesity among IS patients. Diabetes mellitus and obesity were more frequently identified in the group of patients carrying CT+TT genotypes: for diabetes mellitus, 31.6% vs. 18.2% (p = 0.023); for overweight/obesity, 36.7% vs. 18.2% (p = 0.003).

In both genotype groups (CC and CT+TT), analysis using Pearson's χ² test revealed no statistically significant association with the clinical features of IS (history of transient ischemic attack, risk level for recurrent stroke, pathogenetic subtype). Furthermore, no statistically significant difference was found between these groups regarding the number or proportion of IS patients who responded to clopidogrel antiplatelet therapy based on light-transmission aggregometry results.

When assessing the comparability of mean quantitative laboratory parameters between carriers of the CC and CT+TT genotypes of the ABCB1 (C3435T, rs1045642) polymorphism using the Mann-Whitney U test, it was found that in the complete blood count, the mean leukocyte and neutrophil levels were significantly higher in the patient group with the CC genotype. The leukocyte level was 9.2 ± 2.3 × 10⁹ cells/L vs. 8.3 ± 5.6 × 10⁹ cells/L (p = 0.036), and the mean neutrophil level was 6.2 ± 2.9 × 10⁹ cells/L vs. 5.0 ± 2.1 × 10⁹ cells/L (p = 0.045). Regarding blood biochemical markers, including blood creatinine and creatinine clearance calculated by the Cockcroft-Gault formula, no significant differences were found between the analyzed genotype groups (CC vs. CT+TT) for the ABCB1 (C3435T, rs1045642) allelic variants.

Discussion

The study of the ABCB1 (C3435T) polymorphism in cerebrovascular pathology is of considerable relevance. To date, data exist on the ABCB1 polymorphism in patients with IS and transient ischemic attacks. It is known that in patients with IS, the prevalence of the "altered" CT and TT genotypes for the ABCB1 (C3435T) allelic variant was higher than the CC genotype (12.30%): 48.36% and 39.34%, respectively [9]. Pan Y et al. determined a high cumulative frequency of the CT/TT polymorphism in ABCB1 (C3435T) among patients with cerebral stroke, amounting to 65.3% [10]. In our study, we also found that CT and TT genotypes were more frequent in IS patients: CC was identified in 18.0%, CT in 56.0%, and TT in 26.0% of patients (82% in total).

The study by Surendiran A et al. did not reveal statistically significant differences in body mass index or glycemic status parameters in patients with diabetes mellitus carrying the variant CT and TT genotypes of ABCB1 (C3435T) [11]. Results from a study of the ABCB1 (C3435T) allelic variant polymorphism in Moroccan patients with Alzheimer's disease showed that the CT genotype and the T allele were statistically more frequent in healthy control individuals than in Alzheimer's patients (p = 0.015 and p = 0.04, respectively). Furthermore, in these patients, carriage of the ABCB1 (C3435T) allelic variant was associated with diabetes (p = 0.015) and age (p = 0.054) [12].

Some pharmacogenetic studies have focused on the influence of the ABCB1 (C3435T) allelic variant polymorphism in IS patients on the effect of antiplatelet therapy. A meta-analysis by Junjie L et al. demonstrated a potential association between the ABCB1 (C3435T) polymorphism and resistance to clopidogrel and acetylsalicylic acid in patients with IS. Resistance to acetylsalicylic acid in IS patients correlated significantly with the ABCB1 (C3435T, rs1045642) polymorphism (allelic model: p = 0.010; homozygous model: p = 0.047; heterozygous model: p = 0.132; dominant model: p = 0.021; recessive model: p = 0.045). Meanwhile, the authors found that the ABCB1 (C3435T, rs1045642) polymorphism may be significantly associated with clopidogrel resistance in IS (homozygous model: p = 0.000; heterozygous model: p = 0.895; dominant model: p = 0.435; recessive model: p = 0.000) [13]. The study by Yurek E et al. also showed that among patients with acetylsalicylic acid resistance, a higher number of heterozygous (CT) and homozygous (TT) genotypes for ABCB1 (C3435T) were identified (p = 0.001) [14]. In a Moroccan population of patients with acute coronary syndrome taking clopidogrel, no effect of genetic variations or demographic factors on platelet activity was detected. The frequency of the ABCB1 (C3435T) T allele among non-responders to clopidogrel therapy was higher (78.9%) compared to responders (52.8%), but this difference was not significant (p = 0.057). Demographic characteristics, comorbidities, and concomitant treatments were also not associated with clopidogrel response [15].

In 2022, results from a study including 691 patients were published, evaluating the association between the neutrophil-to-lymphocyte ratio and clinical outcomes in IS and transient ischemic attack. A higher neutrophil-to-lymphocyte ratio indicated a worse clinical outcome at 90 days (p < 0.001). Multivariate logistic regression suggested that a high neutrophil-to-lymphocyte ratio was an adverse predictor of 90-day outcome (p < 0.001) [16].

The study by Chen X et al. investigated the association of the ABCB1 (C3435T, rs1045642) gene with renal function impairment in the context of arterial hypertension. The final analysis included 306 patients: 170 hypertensive cases and 136 controls. Compared to the control group, the case group had higher rates of alcohol consumption (65.3% vs. 52.9%, p = 0.029), body mass index (p = 0.032), systolic blood pressure (p < 0.001), total cholesterol (p = 0.004), blood urea nitrogen (p = 0.029), creatinine (p = 0.024), uric acid (p = 0.011), estimated glomerular filtration rate (p < 0.001), and platelet count (p = 0.003). No significant differences were observed for other parameters. The distribution of ABCB1 (C3435T, rs1045642) genotype frequencies was statistically significant between the study groups (p < 0.001). Among hypertensive patients, carriers of the TT genotype exhibited a higher risk of renal function impairment compared to patients with the CC genotype [17].

Conclusions

We determined the frequency of the ABCB1 (C3435T, rs1045642) polymorphism in patients with ischemic stroke. Our results correlate with findings from other authors involving similar patient populations. In our study, we established that diabetes mellitus and obesity were more frequently identified in the group of patients carrying CT+TT genotypes. These data may influence the prioritization of pharmacogenetic test implementation, both for cerebrovascular pathology and for other diseases.

Analysis of the presented data underscores the need for further investigation of the ABCB1 (C3435T) allelic variant in cerebral stroke and assessment of its influence on combination pharmacotherapy (acetylsalicylic acid, clopidogrel, apixaban, rivaroxaban, dabigatran), which would enhance both the efficacy and safety of such treatment.