Frequency Of The Polymorphism In MDR1 And MRP2 Genes And Association Of These Polymorphisms With Multidrug-resistant Epilepsy

Epilepsy is one of the most common neurological disorders characterized by sudden, recurrent and transient disturbance of cerebral function caused by an abnormal neuronal discharge. It is estimated that 20-30% of epilepsy patients do not respond adequately to currently available medication. Many researches suggest that this phenomenon is associated with pharmacoresistance genes. This project is consisted of three parts: With genetic-epidemiology methods, the main aim is to find the frequency of the polymorphim in MDR1 and MRP2, and association of these polymorphisms with multidrug-resistant epilepsy, and the association of these polymorphisms with carbamazepine and sodium valproate amount and blood concentration.Part 1 Frequency of the polymorphim in MDR1 and MRP2 and association of these polymorphisms with multidrug-resistant epilepsyObjective: Determine the frequency of polymorphism at exon 26 (C3435T) in Multidrug Resistance 1 gene (MDR1) and exon 28 (C3972T) in Multidrug Resestance-associated protein 2 gene (MRP2) in epileptic patients and to study the association of these polymorphisms with pharmacoresi stance.Materials and methods: This is a retrospective case-control study design. DNA was extracted from 5ml of whole blood using standard phenol-chloroform extraction method. A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used for the detection of C3435T single-nucleotide polymorphism (SNP), and real-time quantitative PCR was used for the detection of C3972T SNP. With retrospective case-control study methods, we analysised the frequency of these two polymorphims and association of them with multidrug-resistant epilepsy. Rusults:Three genotypes (CC, CT and TT) were detected in MDR1 C3435T, and of all persons, 24．5% were CC genotype, 59．5% were CT genotype, and 16．0% were TT genotype. No difference was found in the C3435T polymorphism between 258 epilepsy patients and 371 healthy persons(p=0．124) . Two genotypes (GG and GA) were detected in MRP2 C3972T, and of all persons, 64．8% were GG genotype and 35．2% were GA genotype. No difference was found in the C3972T polymorphism between 246 epilepsy patients and 399 healthy persons(p=0．437) .In MDR1 polymorphism, the frequency of CC genotype was significantly higher in 141 pharmacoresistant patients than that in 117 responsive patients, and the frequency of TT genotype was significantly higher in responsive group than that in pharmacoresistant group, and the frequency of CT genotype was a little higher in responsive group than that in pharmacoresistant group. (p=0．000) . In MRP2 polymorphism, the frequency of GG genotype was a little higher in 135 pharmacoresistant patients than that in 111 responsive patients, and the frequency of GA genotype was a little higer in responsive group than that in pharmacoresistant group, but no significant difference was found (p=0．587) .With multiple logistic regression analysis, the CC genotype in MDR1 gene was an independent risk factor for pharmacoresistance. Independent of other risks such as "age", "sex", "partial seizure" ,"generalized seizure with partial seizure" , "onset age" and "the length between onset and treatment" , the CC genotype was associated with a 3．761-fold increased risk for pharmacoresistance than TT genotype(OR =3．761, p=0．002, 95%CI: 1．632-8．667) , and the CT genotype was associated with a 1．488-fold increased risk for pharmacoresistance than TT genotype (OR=1．488, p=0．271, 95%CI: 0．734-3．016) . Independent of the same factors, the GG genotype in MRP2 gene was associated with a 1．174-fold increased risk for pharmacoresistance than GA genotype(OR=1．174, p=0．589, 95% CI : 0．655-2．104) . Besides, "partial seizure" was an independent risk factor for pharmacoresistance.Conclusions: No difference was found between the frequency of CC, CT and TT genotype in MDR1 gene , the frequency of GG and GA genotype in epilepsy patients with that in normal controls. The CC genotype in MDR1 gene was associated with resistance to the antiepileptic treatment. No association was found between the GG genotype in MRP2 gene and pharmacoresistance.Part2 Follow-up of curative effect in epilepsy patients with different genotypes in MDR1 and MRP2 genesObjective: Find the influence on curative effect of different genotypes MDR1 and MRP2 genes and validate the results in Part 1 study.Materials and methods: After having followed the curative effect for patients with different genotypes and observed the pharmacoresistant rates among different genotypes, and analyze the relative risk for pharmacoresistance of different genotypes.Rusults: Significant difference was found about pharmacoresistant and responsive rates among CC, CT and TT genotype groups in MDR1 gene(p=0．01) . The pharmacoresistant rate in CC genotype group was 40% , and was higher than that in CT group(20．2%) and TT group(16．0%). Though pharmacoresistant rate of GG genotype group was 26．3%, a little higher than that of GA group, no significant difference was found there. Cox regression analysis suggest that independent of other factors such as "follow period" , "age" , "sex" , "partial seizure" and "generalized seizure with partial seizure" , the CC genotype in MDR1 gene was associated with a 1．735-fold increased relative risk for pharmacoresistance than CT genotype (RR=1．735, p=0．077, 95%CI: 0．943-3．194 ) and with a 2．879-fold increased relative risk for pharmacoresistance than TT genotype ( RR=2．879 , p=0．031 , 95% CI: 1．101-7．530) . No influence was found of the GG genotype in MRP2 on pharmacoresi stance.Conclusions: Patients with CC genotype in MDR1 gene may tend to resist to antiepileptic drugs treatment, and the TT genotype may tend to be responsive to treatment.Part 3 Association of the polymorphism in MDR1 and MRP2 genes and drug amount and concentration of carbamazepine(CBZ) and sodium valproate(VPA)Objective: Compare the difference of CBZ and VPA drug amount and concentration in different genotype groups. Materials and methods: Detect the CBZ blood concentration and CBZE with high-performance liquid chromatography, and VPA blood concentration with Fluorescent polarization immunoassay. Compare the CBZ and VPA drug amount and blood concentration in different genotype groups.Rusults: The CBZ amount/kg, CBZE blood concentration/kg and VPA amount/kg in pharmacoresistant group were much higher than that in responsive group. The CBZ blood concentration/kg was a little higher in pharmacoresistant group than that in the other group, and the VPA blood concentration/kg was a little lower in pharmacoresistant group than that in the other. In MDR1 genotype group, the CBZ amount/kg, CBZ and CBZE blood concentration/kg in CC genotype group were higher than that in CT genotype group and were the lowest in TT enotype group. And the VPA amount/kg was the highest in in TT genotype group, the lowest in CT group, and blood concentration/kg was the highest in CC genotype group and the lowest in TT genotype group. No significant difference was found in the above.In MRP2 genotype group, the CBZ amount/kg was higher in GG group, and CBZ and CBZE blood concentration/kg in GG genotype group were lower than that in GA genotype group, while the VPA amount/kg and blood concentration/kg in GG genotype group were higher than that in the other. No significant difference was found in the above.Conclusions: The genotypes in MDR1 was associated with the CBZ amount and blood concentration. In order to control seizure, the patients with CC genotype may need higher amount and blood concentration of CBZ than patients with TT genotype. No association was found among the genotypes in MDR1 with VPA amount and blood concentration. Also no association was found between the genotypes in MRP2 with CBZ and VPA amount and blood concentration.