Original Article Effects of UGT1A3, UGT1A6, and …

1 Int J Clin Exp Pathol 2016;9(4) /ISSN:1936-2625/IJCEP0021911 Original ArticleEffects of UGT1A3, UGT1A6, and UGT2B7 genetic polymorphisms on plasma concentration of valproic acid in south Chinese epilepsy patientsXiongrong Shen1*, Jingbo Bi3*, Quankun Liu2, Zhihong Ma4, Lishan Min4, Limin Xu4, Shuixin Yang1, Yingrong Chen4 Departments of 1 Clinical Pharmacology, 2 Neurology, Huzhou Central Hospital, Huzhou, Zhejiang Province, China; 3 Department of Central Analysis, Huzhou Environmental Monitoring Center, Huzhou, Zhejiang Province, China; 4 Huzhou Key Laboratory of Molecular Medicine, Huzhou Central Hospital, Huzhou, Zhejiang Province, China. *Equal December 15, 2015; Accepted February 25, 2016; Epub April 1, 2016; Published April 15, 2016 Abstract: Valproic acid (VPA) is one of the most commonly used anti-epileptic drugs in epilepsy patients. The large in-terindividual variability in plasma VPA concentration may reflect functional consequence of genetic polymorphisms in genes encoding drug-metabolizing enzymes, especially uridine 5 -diphospho (UDP)-glucuronosyltransferase (UGT).

2 This study was aimed to determine the relationship between plasma VPA concentration and the polymor-phisms of UGT1A3, UGT1A6 and UGT2B7 in south Chinese patients with epilepsy. UGT1A3, UGT1A6 and UGT2B7 polymorphisms were identified by polymerase chain reaction-ligase detection reaction (PCR-LDR) in 97 epileptic treated with VPA monotherapy. The steady-state plasma concentrations of VPA were measured by High Performance Liquid Chromatography (HPLC) and associated with UGT1A3, UGT1A6 and UGT2B7 polymorphisms. The UGT1A3 A17G polymorphism showed a significant influence, with higher VAP Cs in carrier of AA genotype than in AG genotype (P = ). For UGT1A3 C133T, significant difference in VAP Cs were detected between carriers of CC and CT geno-types, with higher VAP Cs in carrier of CC genotype than in CT genotype (P = ). For UGT1A6 A552C, patients who were carriers of AA and AC genotypes were characterized with a significant higher VAP Cs than carrier of CC genotype. Therefore, these results suggest that the polymorphisms of UGT1A3 A17G, UGT1A3 C133T and UGT1A6 A552C in epilepsy patients may affect VPA : UGT1A3, UGT1A6, UGT2B7, epilepsy, valproic acid, polymorphismsIntroductionValproic acid (VPA) is one of the most common-ly used anti-epileptic drugs in epilepsy patients [1, 2].

3 VPA has a narrow therapeutic range (50-150 g/mL) in the treatment of epilepsy and shows large individual variability in both treat-ment dose and plasma concentration [3-5]. In order to control seizures, reduce and prevent the incidence of adverse reactions, monitoring VPA concentrations is necessary for clinic use. The large interindividual variability may reflect functional consequence of genetic polymor-phisms in genes encoding drug-metabolizing enzymes [6, 7].There are three main metabolic pathways in the metabolism of VPA, mitochondrial beta oxida-tion, glucuronidation conjugation and CYP-catalyzed terminal desaturation and hydroxyl-ation [8]. In particular, UGTs, such as UGT1A3, UGT1A6 and UGT2B7, play a pivotal role because glucuronide metabolites account for an up to 50% of VPA dose [9]. UGT polymor-phisms were found to be most important for explaining the considerable variation observed in valproate levels in epilepsy patients.

4 However, several previous studies on the influ-ence of UGT polymorphisms on VPA plasma concentration were reported, the results were not consistent [10-13]. In addition, most previ-ous researches used polymerase chain reac-tion-restriction fragment length polymorphism (PCR-RFLP) to detect the influence of UGT poly-UGT1A3, UGT1A6, and UGT2B7 polymorphisms and valproic acid concentration in epilepsy4514 Int J Clin Exp Pathol 2016;9(4):4513-4522 Table 1. Patient characteristicsCharacteristicsAge, mean SD (year) (male/female)60/37 Body weight, mean SD (kg) (U/L) (U/L) ( mol/L) doses, mean SD (mg/kg/d) VPA concentration, mean SD ( g/mL) plasma VPA concentration (VPA Cs), [( g/mL)/(mg/kg)] : alanine transaminase; AST: aspartate aminotransferase; Cr: creatinine; Reference range: ALT: 7-40 U/L; AST: 13-35 U/L; Cr: on VPA concentration in epilepsy patients. But PCR-RFLP method has great limi-tations because it is only applicable to the frag-ments that affected the restriction endonucle-ase.

5 It also has a trouble experimental opera-tion and a long experimental period. As a meth-od of high accuracy, strong versatility, high throughput, and simple operation, polymerase chain reaction-ligase detection reaction (PCR-LDR) had widely used to identify the single nucleotide polymorphisms (SNPs), but it was not used to detect UGT polymorphisms [14]. Therefore, the present study was designed to use PCR-LDR to determine UGT1A3, UGT1A6 and UGT2B7 polymorphisms in south Chinese patients with epilepsy on VPA monotherapy and how these polymorphisms affect VPA and methodsPatients and blood samplingA total 97 epileptic patients were enrolled between January 2014 and July 2015 at the department of Neurology at Huzhou Central Hospital (Huzhou, China). Patients diagnosed with partial seizures or generalized seizures with normal liver and kidney function, were treated with VPA as monotherapy.

6 All patients were informed of the purpose of this study and signed a written informed consent form. After a minimum of one month or continuous VPA (Deparkin, Sanofi-Synthelabo Minsheng Pharmaceutical Hangzhou, China) treatments, each whole blood sample was collected in a 5-mL sodium citrate tube (BD Vacutainer, New Jersey, USA) from each patient. The blood sam-ples were separated into two tubes, one of which was immediately centrifuged at 700 g for 10 min to obtain plasma and then stored at -80 C until used for drug analysis, and the other was immediately stored at -20 C until used for DNA quantitationSteady-state trough plasma concentrations of VPA were determined by an Agilent 1260 High-Performance Liquid Chromatography (Agilent Technologies, CA, USA) analysis. The method developed in this study was validated for biosa-mple analysis in a linear range of 4-160 g/ml, within- and between-batch variations were <10%, and the lower limit was 4 g/ml.

7 Genotyping proceduresGenomic DNA was extracted from periphe- ral blood using AxyPrep DNA Mini Kits accord-ing to the manufacture s recommendations (Axygen, USA) and stored at -20 C. DNA con-centrations were determined by spectropho-tometry at 260 nm. The UGT1A3 (A17G, T31C, G81A, C133T, T140C and A477G), UGT1A6 (A552C and A541G) and UGT2B7 (G211T and A268G) polymorphisms were studied by poly-merase chain reaction-ligase detection reac-tion (PCR-LDR). Primers were synthesized by Shanghai HANYU Biological Engineering Ltd. Each set of ligase detection reaction probes comprised one common probe and two discrim-inating probes for the two types. The target DNA sequences were amplified using the multi-plex PCR method. PCR was carried out for each subject at a final volume of 20 l containing 2 l of 1 PCR buffer, l of mM MgCl2, 2 l of UGT1A3, UGT1A6, and UGT2B7 polymorphisms and valproic acid concentration in epilepsy4515 Int J Clin Exp Pathol 2016;9(4):4513-4522 UGT1A3, UGT1A6, and UGT2B7 polymorphisms and valproic acid concentration in epilepsy4516 Int J Clin Exp Pathol 2016;9(4):4513-4522 Figure 1.

8 UGT1A3 genotyping analysis by PCR-LDR. A. UGT1A3 A17G. B. UGT1A3 T31C. C. UGT1A3 G81A. D. UGT1A3 C133T. E. UGT1A3 T140C. F. UGT1A3 2. UGT1A6 and UGT2B7 genotyping analysis by PCR-LDR. A. UGT1A6 A552C. B. UGT1A6 A541G. C. UGT2B7 G211T. D. UGT2B7 , UGT1A6, and UGT2B7 polymorphisms and valproic acid concentration in epilepsy4517 Int J Clin Exp Pathol 2016;9(4):4513-4522 Table 3. Genotype frequencies of the UGT1A6 and UGT2B7 polymorphismsGenotypeNumber of patientsFrequency (%)X2 PUGT1A6 GG00 Table 2. Genotype frequencies of the UGT1A3 polymorphismsGenotypeNumber of patientsFrequency (%)X2 PUGT1A3 AG77. 2 CC00 UGT1A3 TT00 UGT1A3 mM deoxynucleotide triphosphate, 2 l of primers, l of Taq Polymerase, l of ddH2O and 50 ng of genomic DNA. Thermal cycling was performed using the Gene Amp PCR system 9600 (Perkin Elmer, USA) with ini-tial denaturation for 2 minutes at 95 C, fol-lowed by 35 cycles of denaturation at 94 C for 90 sec, annealing at 65 C for 1 min and exten-sion at 72 C for 1 min with final extension at 72 C for 7 min.

9 The ligation reaction for each subject was carried out in a final volume of 10 l, containing 1 l of NEB Taq DNA ligase buffer, 1 l of 2 pmol/ l probe mix, l of 2 U Taq DNA ligase (NEB, USA), 4 l of ddH2O and 4 l of the multi-PCR product. A total of 35 cycles for the ligase detection reaction were performed at 95 C for 2 min, followed by 94 C for 30 sec and 60 C for 2 min. The fluorescent products of the ligase detection reaction were differenti-ated using the PRISM 3730 (Applied Bio- systems, USA).Statistical analysisStatistical analysis was performed using SPSS version (SPSS Inc., Armonk, NY, USA). Hardy-Weinberg equilibrium (HWH) was deter-mined in each group using the chi-square test. Steady-state trough plasma concentrations of VPA were standardized by adjusting with patients weight and dose and expressed as Cs [Cs = trough plasma concentration/(dairy dose/weight)]. The patients demographic character-istics including age and body weight, VAP con-centrations and adjusted plasma VPA concen-tration (Cs) among different UGT1A3, UGT1A6 and UGT2B7 functional genotypes were com-pared using analysis of variance (ANOVA) or Student s t-test.

10 P value less than was considered as statistically significant. ResultsPatients characteristicsNinety-seven patients characteristics includ-ing sex, age, body weight, alanine transamina- se (ALT), aspartate aminotransferase (AST), creatinine (Cr), VPA daily dose, plasma VPA con-centration, and adjusted plasma VPA concen-tration are presented in Table 1. To explore the Effects of genetic factors on interindividual variabilities in VPA plasma concentrations in this study of epilepsy patients, the UGTs poly-morphisms were analyzed by PCR-LDR (Figures UGT1A3, UGT1A6, and UGT2B7 polymorphisms and valproic acid concentration in epilepsy4518 Int J Clin Exp Pathol 2016;9(4):4513-4522 Table 4. Effect of UGT1A3 genotypes on VPA doses, VPA concentrations and adjusted VPA concentra-tions (Cs) in all studied epileptic (years)Weihgt (kg)VPA dose (mg/kg/day)VPA conccntration ( g/mL)VPA Cs [( g/mL)/(mg/kg)]UGT1A3 * #TT0-----P : *UGT1A3 A17G AA vs AG, P< ; #UGT1A3 C133T CC vs CT, P< 5.)