Research Article

Association between the polymorphisms in the ATP-binding cassette genes ABCB1 and ABCC2 and the risk of drug-resistant epilepsy in a Chinese Han population

Published: November 25, 2016
Genet. Mol. Res. 15(4): gmr15048752 DOI: https://doi.org/10.4238/gmr15048752
Cite this Article:
(2016). Association between the polymorphisms in the ATP-binding cassette genes ABCB1 and ABCC2 and the risk of drug-resistant epilepsy in a Chinese Han population. Genet. Mol. Res. 15(4): gmr15048752. https://doi.org/10.4238/gmr15048752
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Abstract

Epilepsy is a common disease of the nervous system; approximately 20-30% of all patients with epilepsy are reported resistant to antiepileptic drugs. ABCB1 and ABCC2 are members of ATP-binding cassette transporter (ABC) family that is involved in the excretion of antiepileptic drugs. In this case-control study, we have investigated the role of ABCB1 rs1045642 and rs2032582 and ABCC2 rs2273697 and rs717620 single nucleotide polymorphisms in antiepileptic drug-resistance in patients with epilepsy. A total of 254 patients with epilepsy (104 drug-resistant and 150 drug-responsive) were recruited from the People’s Hospital of Wuhan University between March 2013 and April 2014. The correlation between the demographic, clinical, and genotypic characteristics of the patients and risk of drug resistance was statistically analyzed. Patients with drug-resistant epilepsy were more likely to present symptomatic epilepsy (χ2 = 22.29, P < 0.001) compared to those with drug-responsive epilepsy. The TT genotype of the ABCB1 rs717620 polymorphism was associated with a higher risk of drug-resistant epilepsy compared to the CC genotype [odds ratio (OR) = 2.97, 95% confidence interval (CI) = 1.11-8.29]. The TT genotype of ABCB1 rs717620 was also related with an increased risk of drug-resistant epilepsy (OR = 2.64, 95%CI = 1.03-7.13) compared to the CC+CT genotype in the recessive model. Thus, our study suggests that the ABCC2 rs717620 polymorphism is associated with resistance to antiepileptic drugs in Chinese patients with epilepsy.

 

INTRODUCTION

Epilepsy is a common disease affecting the nervous system; the estimated prevalence and morbidity rates of epilepsy are approximately 2.1-7.8% and 28.8/100,000 in China (Pi et al., 2014; Tang et al., 2014; Zhou et al., 2014). Epilepsy is caused by super-synchronous discharges from the brain neurons, resulting in sudden and repetitive short bursts of central nervous system dysfunction. Epilepsy is treated mainly by pharmaceutical drugs; while these drugs are effective in controlling the symptoms of ~60% epileptic patients, approximately 20-30% of these patients have been shown to be resistant to antiepileptic drugs (Giussani et al., 2016; Huang et al., 2016). Pharmacogenetic factors may play a key role in individualized sensitivity to antiepileptic drugs. Previous studies have identified several genetic factors that are involved in drug resistance in patients with epilepsy, such as ABCC2, SCN1A, SCN2A, KCNJ10, ATP1A2, and ATP1A3 (Escalante-Santiago et al., 2014; Ma et al., 2014; Guo et al., 2015; Qu et al., 2015; Wang et al., 2015).

Multidrug transporters play a major role in the drug resistance mechanism. Lazarowski and Czornyj (2011) reported that overexpression of multidrug transporters could result in excessive transport (and hence, elimination) of antiepileptic drugs from the lesions, thereby influencing the on-site concentration and effect of the drug. The ATP-binding cassette (ABC) family is a multidrug transporter family; ABCB1 (P-glycoprotein, P-gp) is an important member of the ABC family that was among the first to be identified (Begley, 2004). High P-gp expression could result in the transfer of lipid-soluble antiepileptic drugs out of the brain tissue, reducing the concentration of antiepileptic drugs in the blood and decreasing their efficacy (Akamine et al., 2012). ABCC2 is another member of the ABC family that is involved in the excretion of antiepileptic drugs (Urry et al., 2009; Subenthiran et al., 2013b). Single nucleotide polymorphisms (SNPs) in ABCB1 and ABCC2 could influence the expression and functions of the resultant proteins, and may be associated with drug resistance in patients with epilepsy. Previous studies have reported a correlation between the ABCB1 rs1045642 and rs2032582 and ABCC2 rs2273697 and rs717620 polymorphisms and drug resistance in patients with epilepsy; however, the results of these studies are inconsistent (Keangpraphun et al., 2015; Li et al., 2015; Yu et al., 2015; Zhou et al., 2015). In this case-control study, the role of ABCB1 rs1045642 and rs2032582 and ABCC2 rs2273697 and rs717620 SNPs in the resistance to antiepileptic drugs was investigated in patients with epilepsy.

MATERIAL AND METHODS

Subjects

A hospital-based case-control design was employed in this study. A total of 254 patients with epilepsy were recruited from the People’s Hospital of Wuhan University between March 2013 and April 2014. Patients conforming to the following criteria were included in this study: Han Chinese, aged between 5 and 80 years, and confirmed to be epileptic based on the diagnostic criteria provided by the International League against Epilepsy in 1981. Patients without serious adverse reactions, poor drug-use adherence, and end-stage liver or kidney diseases were excluded from the study.

All included patients were administered antiepileptic drugs within one year prior to enrollment. Response to antiepileptic drugs was characterized by a lack of epileptic seizures after drug administration. Resistance to antiepileptic drugs was characterized by at least four episodes of epileptic seizure despite receiving at least three types of antiepileptic drugs over a one-year period prior to the study. Based on the effective treatment criteria, 104 patients showed resistance to antiepileptic drugs, whereas 150 patients showed a positive response to antiepileptic drugs.

The clinical variables, including gender, age, use of antiepileptic drugs, body mass index, type of seizures, and etiology of the disease, were collected from the patient medical records. Written informed consent was obtained from all subjects prior to enrollment. The procedures employed in this study were approved by the Ethics Committee of the People’s Hospital of Wuhan University.

DNA extraction and genotyping

Peripheral blood (5 mL) was collected from each subject in vacuum tubes containing 5% ethylenediaminetetraacetic acid. DNA was extracted from these samples using the TIANamp Blood DNA kit (Tiangen, Beijing, China) according to the manufacturer instructions. The ABCB1 rs1045642 and rs2032582 and ABCC2 rs2273697 and rs717620 polymorphisms were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The forward and reverse primer sequences were designed as for PCR analysis as follows: ABCB1 rs1045642: 5'-TGTTTTCAGCTGCTTFATGG-3' and 5'-AAGGCATGTATGTTGGCCTC-3'; ABCB1 rs2032582: 5'-CTGGACAAGCACTGAAAGATAAGA-3' and 5'-TGGCTTTGCTACTTTCTGTAAGTT-3'; ABCC2 rs2273697: 5'-GGGCAAAGAAGTGTGTGGAT-3' and 5'-ACATCAGGTTCACTGTTTCTCCCA-3'; and ABCC2 rs717620: 5'-TAAATGGTTGGGATGAAAGG-3' and 5'-GCTTTAGACCAATTGCACATC-3'.

The amplification was performed in a 20-μL reaction mixture comprising 2 μL 10X PCR Buffer, 1 μL dNTP (2.5 mM), 0.4 μL forward primer (10 μM), 0.4 μL reverse primer (10 μM), 0.2 μL rTaq enzyme (5 U/μL), 1 μL gDNA, and 15 μL ddH2O. The ABCB1 rs1045642 and rs2032582 and ABCC2 rs2273697 and rs717620 polymorphisms were amplified as summarized in Table 1. Restriction digestion was performed using a 20-μL digestion mixture comprising 2 μL 10X PCR buffer, 0.5 μL restriction enzyme, and 10 μL PCR amplified mixture, and 7.5 μL ddH2O. The enzyme-digested products were electrophoresed on an agarose gel.

Restriction enzymes and PCR conditions for the ABCB1 rs1045642 and rs2032582 and ABCC2 rs2273697 and rs717620 polymorphisms.

Gene polymorphism Restriction enzyme Amplification reaction
ABCB1 rs1045642 BpiI Initial denaturation at 98°C for 5 min; 36 cycles at 98°C for 30 s, 58°C for 30 s, and 72°C for 30 s; and a final extension at 72°C for 5 min
ABCB1 rs2032582 NcoI Initial denaturation at 98°C for 5 min; 36 cycles at 98°C for 30 s, 54°C for 30 s, and 72°C for 30 s; and a final extension at 72°C for 5 min
ABCC2 rs2273697 BsiEI Initial denaturation at 98°C for 5 min; 36 cycles at 98°C for 30 s, 56°C for 30 s, and 72°C for 30 s; and a final extension at 72°C for 5 min
ABCC2 rs717620 Bspl43I Initial denaturation at 98°C for 5 min; 36 cycles at 98°C for 30 s, 56°C for 30 s, and 72°C for 30 s; and a final extension at 72°C for 5 min

Statistical analysis

The differences in demographic and clinical variables between patients and controls were analyzed by the Student t-test and chi-square test. Departure of the ABCB1 rs1045642 and rs2032582 and ABCC2 rs2273697 and rs71762 polymorphism genotype frequencies from the Hardy-Weinberg equilibrium (HWE) was calculated using the Pearson χ2 test. The relationships between the ABCB1 rs1045642 and rs2032582 and ABCC2 rs2273697 and rs71762 polymorphisms and drug-resistant epilepsy were determined by multiple logistic regression analyses and estimated as the odds ratios (ORs) and 95% confidence intervals (CIs). The data was statistically analyzed using SPSS v.16.0 (SPSS Inc., Chicago, IL, USA). Differences with P values <0.05 were considered statistically significant.

RESULTS

The demographic and clinical variables of the investigated subjects are summarized in Table 2. The mean age of patients with drug-resistant and drug-responsive epilepsy was 7.60 ± 15.82 and 7.2 ± 16.14, respectively. Patients with drug-resistant and drug-responsive epilepsy included 61 (58.65%) and 84 (56.00%) males and 43 (41.35%) and 66 (44.00%) females, respectively. Patients with drug-resistant epilepsy were more likely to present symptomatic epilepsy (χ2 = 22.29, P < 0.001) compared to those with drug-responsive epilepsy. We found no significant differences between the age (t = 0.20, P = 0.42), age at first onset (t = 1.49, P = 0.07), gender (χ2 = 0.18, P = 0.67), and type of seizures (χ2 = 3.54, P = 0.17) in the two groups.

Demographic and clinical variables of epileptic patients.

Variables Drug-resistant (N = 104) % Drug-responsive (N = 150) % Chi-square test P value
Mean age, years 7.60 ± 15.82 7.2 ± 16.14 0.20 0.42
Age at first onset, years 2.15 ± 2.70 2.65 ± 2.58 1.49 0.07
Gender
Male 61 58.65 84 56.00
Female 43 41.35 66 44.00 0.18 0.67
Type of seizures
Simple partial 24 23.08 29 19.33
Complex partial 26 25.00 26 17.33
Generalized 54 51.92 95 63.33 3.54 0.17
Etiology
Idiopathic 49 47.12 114 76.00
Symptomatic 55 52.88 36 24.00 22.29 < 0.001
The genotypic distributions of the ABCB1 rs1045642 and rs2032582 and ABCC2 rs2273697 and rs717620 polymorphisms are summarized in Table 3. The CC, CT, and TT genotypes of ABCC2 rs717620 were significantly different between the drug-responsive and drug-resistant epilepsy groups (χ2 = 6.01, P = 0.04). However, no significant differences were found in the genotypic frequencies of ABCB1 rs1045642 (χ2 = 1.61, P = 0.45) and rs2032582 (χ2 = 0.22, P = 0.90) and ABCC2 rs2273697 (χ2 = 0.24, P = 0.89) between the two study groups.

Genotype frequencies of ABCB1 rs1045642 and rs2032582 and ABCC2 rs2273697 and rs717620 polymorphisms.

Genotypes Drug-resistant (N = 104) % Drug-responsive (N = 150) % χ2 P value χ2 for HWE P value
Drug-resistant Drug-responsive Drug-resistant Drug-responsive
rs1045642
CC 43 41.35 61 40.67
CT 48 46.15 77 51.33
TT 13 12.50 12 8.00 1.61 0.45 0.005 0.94 3.34 0.07
rs2032582
GG 35 33.65 54 36.00
GT/GA 53 50.96 72 48.00
TT/TA 16 15.38 24 16.00 0.22 0.90 0.31 0.58 < 0.001 1.00
rs2273697
GG 79 75.96 112 74.67
GA 23 22.12 36 24.00
AA 2 1.92 2 1.33 0.24 0.89 0.05 0.83 0.22 0.64
rs717620
CC 46 44.23 82 54.67
CT 43 41.35 59 39.33
TT 15 14.42 9 6.00 6.01 0.04 0.89 0.35 0.14 0.71

HWE, Hardy-Weinberg equilibrium.

The associations between the ABCB1 rs1045642 and rs2032582 and ABCC2 rs2273697 and rs717620 polymorphisms and risk of drug-resistant epilepsy are summarized in Table 4. Logistic regression analysis indicated that the TT genotype of ABCB1 rs717620 was associated with a higher risk of drug-resistant epilepsy compared to the CC genotype (OR = 2.97, 95%CI = 1.11-8.29). The TT genotype of ABCB1 rs717620 was correlated with an increased risk of drug-resistant epilepsy (OR = 2.64, 95%CI = 1.03-7.13) compared to the CC+CT genotype in the recessive model. However, no significant correlation was found between the ABCB1 rs1045642 and rs2032582 and ABCC2 rs2273697 polymorphisms and risk of drug-resistant epilepsy.

Association between ABCB1 rs1045642 and rs2032582 and ABCC2 rs2273697 and rs717620 polymorphisms and risk of drug-resistant epilepsy.

Genotypes Drug-resistant (N = 104) % Drug-responsive (N = 150) % OR (95% CI)1 P value
rs1045642
Codominant
CC 43 41.35 61 40.67 1.0 (Ref.) -
CT 48 46.15 77 51.33 0.88 (0.50-1.56) 0.65
TT 13 12.50 12 8.00 1.54 (0.58-4.07) 0.33
Dominant
CC 43 41.35 61 40.67 1.0 (Ref.) -
CT+TT 61 58.65 89 59.33 0.97 (0.57-1.67) 0.91
Recessive
CC+CT 91 87.5 138 92 1.0 (Ref.) -
TT 13 12.5 12 8 1.64 (0.66-4.12) 0.24
rs2032582
Codominant
GG 35 33.65 54 36.00 1.0 (Ref.) -
GT/GA 53 50.96 72 48.00 1.14 (0.63-2.06) 0.65
TT/TA 16 15.39 24 16.00 1.03 (0.44-2.35) 0.90
Dominant
GG 35 33.65 54 36 1.0 (Ref.) -
GT/GA + TT/TA 69 66.35 96 64 1.11 (0.63-1.95) 0.70
Recessive
GG + GT/GA 88 84.61 126 84 1.0 (Ref.) -
TT/TA 69 15.39 96 64 1.03 (0.67-1.59) 0.89
rs2273697
Codominant
GG 79 75.96 112 74.67 1.0 (Ref.) -
GA 23 22.12 36 24.00 0.91 (0.47-1.71) 0.75
AA 2 1.92 2 1.33 1.42 (0.10-19.90) 0.89
Dominant
GG 79 75.96 112 74.67 1.0 (Ref.) -
GA + AA 25 24.04 38 25.33 0.93 (0.50-1.73) 0.81
Recessive
GG + GA 102 98.08 148 98.67 1.0 (Ref.) -
AA 2 1.92 2 1.33 1.45 (0.10-20.29) 0.71
rs717620
Codominant
CC 46 44.23 82 54.67 1.0 (Ref.) -
CT 43 41.35 59 39.33 1.30 (0.74-2.29) 0.34
TT 15 14.42 9 6.00 2.97 (1.11-8.29) 0.04
Dominant
CC 46 44.23 82 54.67 1.0 (Ref.) -
CT + TT 58 55.77 68 45.33 1.52 (0.89-2.60) 0.10
Recessive
CC + CT 89 85.58 141 94 1.0 (Ref.) -
TT 15 14.42 9 6 2.64 (1.03-7.13) 0.02

1Adjusted for age, sex and etiology. OR = odds ratio; CI = confidence interval.

An analysis of the interactions between the demographic and clinical variables (environment) and the ABCB1 rs1045642 and rs2032582 and ABCC2 rs2273697 and rs717620 polymorphisms revealed that this interaction did not influence the risk of drug-resistant epilepsy.

DISCUSSION

In this study, we attempted to evaluate the relationship between the ABCB1 rs1045642 and rs2032582 and ABCC2 rs2273697 and rs717620 polymorphisms and risk of drug-resistant epilepsy; the results revealed that the TT genotype of the ABCC2 rs717620 polymorphism is significantly associated with the risk of drug-resistant epilepsy compared to the wild-type genotype.

Multidrug resistant protein 2, encoded by ABCC2, is a multidrug transporter distributed in the blood-brain barrier, endothelial cells, astrocytes, and the surfaces of some neurons (König et al., 2013). Multidrug transporters regulate the two-way transfer balance of drugs in the blood and brain tissues to influence the drug concentration in the brain tissue, thereby influencing its resistance to antiepileptic drugs (Glauser and Pippenger, 2000). Previous studies have indicated that multidrug transporters could influence the efficacy of antiepileptic drugs (Zimprich et al., 2004; Ufer et al., 2009). Therefore, the ABCC2 expression could influence the mechanism of transport of antiepileptic drugs in the brain tissues (Nies et al., 2004). A SNP is an insertion, deletion, or substitution of nucleic acid bases leading to a polymorphism in the gene sequence (Friedberg, 2003). Genetic polymorphisms can change the structure and quantity of the gene product, ultimately affecting the function of the product. Polymorphisms in ABCC2 could influence the expression of this protein, therein affecting its response or resistance to antiepileptic drugs.

Several previous studies have reported an association between the ABCC2 rs2273697 and rs717620 polymorphisms and risk of drug-resistant epilepsy, but with conflicting results (Seo et al., 2008; Kwan et al., 2011; Hilger et al., 2012; Qu et al., 2012; Sporis et al., 2013; Subenthiran et al., 2013a; Escalante-Santiago et al., 2014; Ma et al., 2014). Qu et al. (2012) revealed a significant association between the ABCC2 rs2273697 and rs717620 polymorphisms and resistance to antiepileptic drugs in Chinese epileptic patients. Subenthiran et al. (2013a) indicated a correlation between the ABCC2 rs2273697 and rs717620 polymorphisms and resistance to antiepileptic drugs in a study comprising 152 and 162 anti-epileptic drug-responsive and -resistant patients, respectively (Subenthiran et al., 2013a). Ma et al. (2014) reported a possible correlation between the ABCC2 rs2273697 polymorphism and resistance to antiepileptic drugs in 453 Chinese Han patients with epilepsy. However, other studies have reported that the ABCC2 rs2273697 and rs717620 polymorphisms were not associated with resistance to antiepileptic drugs (Seo et al., 2008; Kwan et al., 2011; Hilger et al., 2012; Sporis et al., 2013). In this study, we reported a significant correlation between the ABCC2 rs717620 polymorphism and resistance to antiepileptic drugs. The discrepancies among these results may be attributed to differences in the sample populations and sizes, etiology of epilepsy, and patient selection criteria.

The results of this study are subject to two limitations. First, the patients were selected from a single region in China; therefore, the sample may not be representative of the general population. However, the genotype frequencies of the ABCB1 and ABCC2 polymorphisms were in accordance with the HWE, indicating that the samples could be representative of the general population. Second, the sample size was quite small, resulting in low statistical power to compare the differences between groups.

In conclusion, our study suggests that the ABCC2 rs717620 polymorphism is associated with antiepileptic drug resistance in Chinese patients with epilepsy. Further studies with larger sample sizes and subjects with diverse ethnic backgrounds are required to confirm the mechanism by which the ABCB1 and ABCC2 polymorphisms resist antiepileptic drugs.

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