Original Article Effects of UGT1A3, UGT1A6, and UGT2B7 Genetic Polymorphisms on Plasma Concentration of Valproic Acid in South Chinese Epilepsy Patients

Int J Clin Exp Pathol 2016;9(4):4513-4522 www.ijcep.com /ISSN:1936-2625/IJCEP0021911

Original Article Effects of UGT1A3, UGT1A6, and UGT2B7 genetic polymorphisms on plasma concentration of valproic acid in south Chinese epilepsy patients

Xiongrong Shen1*, Jingbo Bi3*, Quankun Liu2, Zhihong Ma4, Lishan Min4, Limin Xu4, Shuixin Yang1, Yingrong Chen4

Departments of 1Clinical Pharmacology, 2Neurology, Huzhou Central Hospital, Huzhou, Zhejiang Province, China; 3Department of Central Analysis, Huzhou Environmental Monitoring Center, Huzhou, Zhejiang Province, China; 4Huzhou Key Laboratory of Molecular Medicine, Huzhou Central Hospital, Huzhou, Zhejiang Province, China. *Equal contributors. Received 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 interindividual 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). This study was aimed to determine the relationship between plasma VPA concentration and the polymorphisms 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 = 0.034). For UGT1A3 C133T, significant difference in VAP Cs were detected between carriers of CC and CT genotypes, with higher VAP Cs in carrier of CC genotype than in CT genotype (P = 0.016). 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 concentration.

Keywords: UGT1A3, UGT1A6, UGT2B7, epilepsy, valproic acid, polymorphisms

Introduction tion, glucuronidation conjugation and CYP- catalyzed terminal desaturation and hydroxyl- Valproic acid (VPA) is one of the most common- ation [8]. In particular, UGTs, such as UGT1A3, ly used anti-epileptic drugs in epilepsy patients UGT1A6 and UGT2B7, play a pivotal role [1, 2]. VPA has a narrow therapeutic range (50- because glucuronide metabolites account for 150 μg/mL) in the treatment of epilepsy and an up to 50% of VPA dose [9]. UGT polymor- shows large individual variability in both treat- phisms were found to be most important for ment dose and plasma concentration [3-5]. In explaining the considerable variation observed order to control seizures, reduce and prevent in valproate levels in epilepsy patients. the incidence of adverse reactions, monitoring VPA concentrations is necessary for clinic use. However, several previous studies on the influ- The large interindividual variability may reflect ence of UGT polymorphisms on VPA plasma functional consequence of genetic polymor- concentration were reported, the results were phisms in genes encoding drug-metabolizing enzymes [6, 7]. not consistent [10-13]. In addition, most previous researches used polymerase chain reac- There are three main metabolic pathways in the tion-restriction fragment length polymorphism metabolism of VPA, mitochondrial beta oxida- (PCR-RFLP) to detect the influence of UGT poly- UGT1A3, UGT1A6, and UGT2B7 polymorphisms and valproic acid concentration in epilepsy

Table 1. Patient characteristics Characteristics Age, mean ± SD (year) 31.5±18.5 Sex (male/female) 60/37 Body weight, mean ± SD (kg) 53.5±16.3 ALT (U/L) 20.7±14.8 AST (U/L) 23.8±8.4 Cr (μmol/L) 80.1±18.9 VPA doses, mean ± SD (mg/kg/d) 16.3±4.6 Plasma VPA concentration, mean ± SD (μg/mL) 53.4±16.5 Adjusted plasma VPA concentration (VPA Cs), [(μg/mL)/(mg/kg)] 3.63±1.52 ALT: alanine transaminase; AST: aspartate aminotransferase; Cr: creatinine; Reference range: ALT: 7-40 U/L; AST: 13-35 U/L; Cr: 44-132.6 μmol/L. morphisms on VPA concentration in epilepsy which was immediately centrifuged at 700 g for patients. But PCR-RFLP method has great limi- 10 min to obtain plasma and then stored at tations because it is only applicable to the frag- -80°C until used for drug analysis, and the ments that affected the restriction endonucle- other was immediately stored at -20°C until ase. It also has a trouble experimental opera- used for DNA isolation. tion and a long experimental period. As a method of high accuracy, strong versatility, high VPA quantitation throughput, and simple operation, polymerase chain reaction-ligase detection reaction (PCR- Steady-state trough plasma concentrations of LDR) had widely used to identify the single VPA were determined by an Agilent 1260 High- nucleotide polymorphisms (SNPs), but it was Performance Liquid Chromatography (Agilent not used to detect UGT polymorphisms [14]. Technologies, CA, USA) analysis. The method Therefore, the present study was designed to developed in this study was validated for biosa- use PCR-LDR to determine UGT1A3, UGT1A6 mple analysis in a linear range of 4-160 μg/ml, and UGT2B7 polymorphisms in south Chinese within- and between-batch variations were patients with epilepsy on VPA monotherapy and <10%, and the lower limit was 4 μg/ml. how these polymorphisms affect VPA levels. Genotyping procedures Materials and methods Genomic DNA was extracted from periphe- Patients and blood sampling ral blood using AxyPrep DNA Mini Kits accord- ing to the manufacture’s recommendations A total 97 epileptic patients were enrolled (Axygen, USA) and stored at -20°C. DNA con- between January 2014 and July 2015 at the centrations were determined by spectropho- department of Neurology at Huzhou Central tometry at 260 nm. The UGT1A3 (A17G, T31C, Hospital (Huzhou, China). Patients diagnosed G81A, C133T, T140C and A477G), UGT1A6 with partial seizures or generalized seizures with normal liver and kidney function, were (A552C and A541G) and UGT2B7 (G211T and treated with VPA as monotherapy. All patients A268G) polymorphisms were studied by poly- were informed of the purpose of this study and merase chain reaction-ligase detection reac- signed a written informed consent form. tion (PCR-LDR). Primers were synthesized by Shanghai HANYU Biological Engineering Ltd. After a minimum of one month or continuous Each set of ligase detection reaction probes VPA (Deparkin, Sanofi-Synthelabo Minsheng comprised one common probe and two discrim- Pharmaceutical Hangzhou, China) treatments, inating probes for the two types. The target each whole blood sample was collected in a DNA sequences were amplified using the multi- 5-mL sodium citrate tube (BD Vacutainer, New plex PCR method. PCR was carried out for each Jersey, USA) from each patient. The blood sam- subject at a final volume of 20 μl containing 2 μl ples were separated into two tubes, one of of 1×PCR buffer, 0.6 μl of 3.0 mM MgCl2, 2 μl of

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Figure 1. UGT1A3 genotyping analysis by PCR-LDR. A. UGT1A3 A17G. B. UGT1A3 T31C. C. UGT1A3 G81A. D. UGT1A3 C133T. E. UGT1A3 T140C. F. UGT1A3 A477G.

Figure 2. UGT1A6 and UGT2B7 genotyping analysis by PCR-LDR. A. UGT1A6 A552C. B. UGT1A6 A541G. C. UGT2B7 G211T. D. UGT2B7 A268G.

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Table 2. Genotype frequencies of the UGT1A3 2.0 mM deoxynucleotide triphosphate, 2 μl of polymorphisms primers, 0.2 μl of Taq Polymerase, 12.2 μl of Number of Frequency ddH2O and 50 ng of genomic DNA. Thermal Genotype X2 P patients (%) cycling was performed using the Gene Amp UGT1A3 A17G 3.30 0.07 PCR system 9600 (Perkin Elmer, USA) with ini- AA 89 91.8 tial denaturation for 2 minutes at 95°C, followed by 35 cycles of denaturation at 94°C for AG 7 7.2 90 sec, annealing at 65°C for 1 min and exten- GG 1 1.0 sion at 72°C for 1 min with final extension at UGT1A3 T32C 0.04 0.84 72°C for 7 min. The ligation reaction for each TT 93 95.9 subject was carried out in a final volume of 10 TC 4 4.1 μl, containing 1 μl of NEB Taq DNA ligase buffer, CC 0 0 1 μl of 2 pmol/μl probe mix, 0.05 μl of 2 U Taq

UGT1A3 G81A 0.81 0.37 DNA ligase (NEB, USA), 4 μl of ddH2O and 4 μl of GG 53 54.6 the multi-PCR product. A total of 35 cycles for GA 35 36.1 the ligase detection reaction were performed AA 9 9.3 at 95°C for 2 min, followed by 94°C for 30 sec UGT1A3 C133T 0.50 0.48 and 60°C for 2 min. The fluorescent products of the ligase detection reaction were differenti- CC 84 86.6 ated using the PRISM 3730 (Applied Bio- CT 13 13.4 systems, USA). TT 0 0 UGT1A3 T140C 0.15 0.70 Statistical analysis TT 55 56.7 Statistical analysis was performed using SPSS TC 37 38.1 version 19.0 (SPSS Inc., Armonk, NY, USA). CC 5 5.2 Hardy-Weinberg equilibrium (HWH) was deter- UGT1A3 A477G 0.15 0.70 mined in each group using the chi-square test. AA 52 53.6 Steady-state trough plasma concentrations of AG 37 38.1 VPA were standardized by adjusting with GG 8 8.2 patients’ weight and dose and expressed as Cs [Cs = trough plasma concentration/(dairy dose/ weight)]. The patients’ demographic character- Table 3. Genotype frequencies of the UGT1A6 istics including age and body weight, VAP con- and UGT2B7 polymorphisms centrations and adjusted plasma VPA concen- Number of Frequency Genotype X2 P tration (Cs) among different UGT1A3, UGT1A6 patients (%) and UGT2B7 functional genotypes were com- UGT1A6 A552C 0.41 0.52 pared using analysis of variance (ANOVA) or AA 53 54.6 Student’s t-test. P value less than 0.05 was AC 39 40.2 considered as statistically significant. CC 5 5.2 Results UGT1A6 A541G 0.15 0.70 AA 55 56.7 Patients’ characteristics AG 37 38.1 GG 5 5.2 Ninety-seven patients’ characteristics includ- UGT2B7 G211T 1.33 0.25 ing sex, age, body weight, alanine transaminase (ALT), aspartate aminotransferase (AST), GG 70 72.2 creatinine (Cr), VPA daily dose, plasma VPA con- GT 23 23.7 centration, and adjusted plasma VPA concen- TT 4 4.1 tration are presented in Table 1. To explore the UGT2B7 A268G 0.07 0.79 effects of genetic factors on interindividual AA 92 94.8 variabilities in VPA plasma concentrations in AG 5 5.2 this study of epilepsy patients, the UGTs poly- GG 0 0 morphisms were analyzed by PCR-LDR (Figures

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Table 4. Effect of UGT1A3 genotypes on VPA doses, VPA concentrations and adjusted VPA concentrations (Cs) in all studied epileptic patients VPA dose VPA conccntration VPA Cs [(μg/ Gene Genotype No. Age (years) Weihgt (kg) (mg/kg/day) (μg/mL) mL)/(mg/kg)] UGT1A3 A17G AA 89 30.82±17.95 53.07±16.43 16.23±4.81 57.53±21.79 3.75±1.54 AG 7 43.71±26.14 60.43±17.41 15.87±4.47 38.44±8.82 2.47±0.35* GG 1 9.00 31.00 16.10 22.30 1.38 P value 0.105 0.212 0.982 0.024 0.034 UGT1A3 T31C TT 93 31.43±19.15 53.20±16.89 16.23±4.75 55.95±22.25 3.63±1.54 TC 4 33.75±9.78 57.50±6.03 15.48±5.14 52.08±6.85 3.68±1.29 CC 0 - - - - - P value 0.811 0.614 0.757 0.730 0.947 UGT1A3 G81A GG 53 33.74±16.76 56.04±13.63 15.96±3.63 55.41±18.36 3.53±1.07 GA 35 26.37±19.88 48.06±19.56 17.23±5.88 59.96±26.85 3.83±2.01 AA 9 38.56±23.36 58.33±16.50 13.60±4.94 41.81±12.78 3.45±1.80 P value 0.099 0.054 0.105 0.081 0.618 UGT1A3 C133T CC 84 32.95±19.01 54.40±15.54 15.88±4.57 57.04±21.63 3.78±1.54 CT 13 22.31±15.18 46.69±21.85 18.25±5.47 47.72±22.19 2.69±1.09# TT 0 - - - - - P value 0.057 0.119 0.094 0.153 0.016 UGT1A3 T140C TT 55 29.22±18.50 52.33±17.51 16.21±4.85 52.16±19.18 3.42±1.30 TC 37 34.22±19.22 54.29±14.79 16.47±4.56 62.61±24.64 3.94±1.68 CC 5 37.00±19.65 58.00±21.27 15.04±5.58 45.18±15.84 3.67±2.53 P value 0.371 0.701 0.806 0.052 0.277 UGT1A3 A477G AA 52 33.25±17.20 55.13±15.11 16.13±3.43 56.75±17.33 3.60±1.02 AG 37 26.16±19.10 48.81±18.50 17.08±6.00 57.34±27.78 3.67±2.04 GG 8 45.12±21.06 63.00±10.65 12.53±4.14 52.38±12.28 3.66±1.74 P value 0.021 0.046 0.046 0.192 0.974 Cs: *UGT1A3 A17G AA vs AG, P<0.05; #UGT1A3 C133T CC vs CT, P<0.05.

Table 5. Effect of UGT1A6 and UGT2B7 genotypes on VPA doses, VPA concentrations and adjusted VPA concentrations (Cs) in all studied epileptic patients VPA dose VPA conccntration VPA Cs [(μg/ Gene Genotype N Age (years) Weihgt (kg) (mg/kg/day) (μg/mL) mL)/(mg/kg)] UGT1A6 A552C AA 53 32.21±18.68 55.04±15.26 15.45±4.47 57.79±20.90 3.88±1.40* AC 39 31.54±19.80 50.85±18.53 17.20±5.23 50.86±21.72 3.10±1.24* CC 5 24.20±13.26 55.40±17.47 16.32±1.50 44.94±16.76 2.76±1.06 P value 0.666 0.474 0.214 0.181 0.011 UGT1A6 A541G AA 55 32.21±18.68 55.04±15.26 15.45±4.47 57.42±21.26 3.86±1.43 AG 37 31.54±19.80 50.85±18.52 17.20±5.23 54.97±23.15 3.44±1.67 GG 5 24.20±13.26 55.40±14.74 16.32±1.50 44.94±16.76 2.76±1.06 P value 0.666 0.474 0.214 0.457 0.182 UGT2B7 G211T GG 70 31.66±18.44 54.00±17.05 16.09±4.44 57.40±22.73 3.73±1.49 GT 23 37.39±15.74 51.96±15.28 16.18±5.82 52.24±20.23 3.50±1.73 TT 4 53.00±31.07 50.50±19.33 18.25±2.24 47.92±11.05 2.63±0.45 P value 0.041 0.827 0.679 0.475 0.340 UGT2B7 A268G AA 92 30.99±18.79 52.89±16.85 16.20±4.85 55.60±21.94 3.63±1.56 AG 5 41.40±18.92 62.20±6.61 16.24±1.94 59.20±21.53 3.59±0.98 GG 0 - - - - - P value 0.231 0.224 0.984 0.722 0.947 Cs: *UGT1A6 A552C AA vs CC, AC vs CC, P<0.05.

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Table 6. Frequencies of UGT1A3 polymorphisms and adjusted VPA concentration in various populations Chinese patients with epilepsy Current study (n = 97) Genotype Chu et al. (n = 136)* Chu et al. (n = 242)# N (%) VAP Cs [(μg/mL)/(mg/kg)] N (%) N (%) VAP Cs [(μg/mL)/(mg/kg)] A17G AA 116 (85.3) 4.76±1.41 215 (88.8) 89 (91.8) 3.75±1.54 AG 18 (13.2) 3.49±1.43a 27 (11.2) 7 (7.2) 2.47±0.35b GG 2 (1.5) 3.01~1.84 0 1 (1.0) 1.38 P value 0.001 0.034 T31C TT 65 (47.8) 4.70±1.37 114 (47.1) 0 - TC 59 (43.4) 4.45±1.47 110 (45.5) 4 (4.1) 3.68±1.29 CC 12 (8.8) 4.52±2.13 18 (7.4) 93 (95.9) 3.63±1.54 P value 0.653 0.947 G81A GG NA NA 113 (46.7) 53 (54.6) 3.53±1.07 GA NA NA 112 (46.3) 35 (36.1) 3.83±2.01 AA NA NA 17 (7.0) 9 (9.3) 3.45±1.80 P value 0.618 C133T CC 122 (89.7) 4.54±1.38 221 (91.3) 84 (86.6) 3.78±1.54 CT 14 (10.3) 4.90±2.24 21 (8.7) 13 (13.4) 2.69±1.09c TT 0 - 0 0 - P value 0.662 0.016 T140C TT 109 (80.1) 4.56±1.54 183 (75.6) 55 (56.7) 3.42±1.30 TC 24 (17.7) 4.64±1.26 55 (22.7) 37 (38.1) 3.94±1.68 CC 3 (2.2) 5.21±0.73 4 (1.7) 5 (5.2) 3.67±2.53 P value 0.509 0.277 A477G AA NA NA 116 (47.9) 52 (53.6) 3.60±1.02 AG NA NA 110 (45.5) 37 (38.1) 3.67±2.04 GG NA NA 16 (6.6) 8 (8.2) 3.66±1.74 P value 0.974 *Chinese patients with epilepsy treated with VPA monotherapy. #Chinese patients with epilepsy treated concomitantly with VPA and other antiepileptic drugs. NA: Not analyzed; VAP Cs: aUGT1A3 A17G AA vs AG, P<0.05 (Chu et al.); VAP Cs: bUGT1A3 A17G AA vs AG, P<0.05; cUGT1A3 C133T CC vs CT, P<0.05 (Current study).

1 and 2). The frequencies of each UGT1A3, Among all of the UGT1A3 SNPs detected, the UGT1A6 and UGT2B7 genotype are shown in UGT1A3 A17G polymorphism showed a signifi- Tables 2 and 3. The frequencies were all con- cant influence, with higher VAP Cs in carrier of sistent with the Hardy-Weinberg equilibrium. AA genotype than in AG genotype (P = 0.034). For UGT1A3 C133T, Significant difference in Influence of UGT1A3, UGT1A6, and UGT2B7 VAP Cs were detected between carriers of CC polymorphism on plasma VPA concentrations and CT genotypes, with higher VAP Cs in carrier of CC genotype than in CT genotype (P = 0.016). To determine the influence of polymorphisms of There were no significant differences in VAP Cs the identified UGTs on VPA concentration, asso- among the genotypes of UGT1A3 T32C, G81A, ciation analysis of UGT1A3, UGT1A6 and T140C and A477G. For UGT1A6 A552C, patients UGT2B7 SNPs and VPA Cs were observed in all who were carriers of AA and AC genotypes were studied epileptic patients (Tables 4 and 5). characterized with a significant higher VAP Cs

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Table 7. Frequencies of UGT1A6 polymorphisms and adjusted VPA concentration in various populations Chinese patients with epilepsy Current study (n = 97) Sun et al. (n = 67) Tan et al. (n = 40) Chu et al. (n = 136) Genotype VAP Cs [(μg/ VAP Cs [(μg/ VAP Cs [(μg/ VAP Cs [(μg/ N (%) N (%) N (%) N (%) mL)/(mg/kg)] mL)/(mg/kg)] mL)/(mg/kg)] mL)/(mg/kg)] UGT1A6 A552C AA 40 (59.7) 4.32±0.21 23 (57.5) 6.95±2.98 83 (61.0) 4.55±1.45 53 (54.6) 3.88±1.40c AC 24 (35.8) 3.43±0.30a 13 (32.5) 3.59±1.11b 50 (36.8) 4.61±1.55 39 (40.2) 3.10±1.24c CC 3 (4.5) 3.16±0.84a 4 (10.0) 3 (2.2) 4.82±1.75 5 (5.2) 2.76±1.06 P value <0.05 <0.01 0.691 0.011 UGT1A6 A541G AA NS NS NS NS 88 (64.7) 4.52±1.38 55 (56.7) 3.86±1.43 AG NS NS NS NS 45 (33.1) 4.60±1.58 37 (38.1) 3.44±1.67 GG NS NS NS NS 3 (2.2) 5.92±2.72 5 (5.2) 2.76±1.06 P value 0.619 0.182 NS: Not studied. VAP Cs: aUGT1A6 A552C AA vs AC, AA vs CC, P<0.05 (Sun et al.); bUGT1A6 A552C AA vs AC+CC, P<0.05 (Tan et al.); VAP Cs: cUGT1A6 A552C AA vs CC, AC vs CC, P<0.05, P<0.05 (Current study).

Table 8. Frequencies of UGT2B7 polymorphisms and adjusted VPA concentration in various populations Chinese patients with epilepsy Current study (n = 97) Genotype Ma et al. (n = 248) Sun et al. (n = 40) N (%) VAP Cs [(μg/mL)/(mg/kg)] N (%) VAP Cs [(μg/mL)/(mg/kg)] N (%) VAP Cs [(μg/mL)/(mg/kg)] UGT2B7 G211T GG 14 (5.6) 5.99±0.98 NS NS 70 (72.2) 3.73±1.49 GT 121 (48.8) 3.70±0.29 NS NS 23 (23.7) 3.50±1.73 TT 113 (45.6) 3.26±0.23 NS NS 4 (4.1) 2.63±0.45 P value 0.23 0.340 UGT2B7 A268G AA 147 (59.3) 3.80±0.25 78 (76.5) 2.28±1.32 92 (94.8) 3.63±1.56 AG 90 (36.3) 3.61±0.30 23 (22.5) 2.30±1.38 5 (5.2) 3.59±0.98 GG 11 (4.4) 1.76±0.31a 1 (1.0) 1.55 0 - P value 0.005 0.197 0.947 NS: Not studied. VAP Cs: aUGT2B7 A268G AA vs GG, P<0.05 (Ma et al.). than carrier of CC genotype. The polymor- adjusted VPA concentration in the current study phisms of UGT1A6 A541G, UGT2B7 A268G and compared to previous studies [11, 17-19] in UGT 2B7 G211T had no significant effect on Chinese patients with epilepsyare shown in VPA Cs. Tables 6-8. The genotype frequencies of UGT1A3 A17G, T31C, G81A, C133T, T140C and Discussion A477G in previous study [9], no matter the epi- Large interindividual variability is detected in lepsy patients treated concomitantly with VPA VPA pharmacokinetics and pharmacodynam- and other antiepileptic drugs or VPA monother- ics, which may be contributed by genetic poly- apy were similar to our research. They also morphisms. Previous reports showed that UGT showed a significant influence of UGT1A3 A17G contributed to around 50% of VPA metabolism polymorphism on plasma concentration of VPA in human. UGT1A3, UGT1A6, and UGT2B7 are in Chinese epilepsy patients, with higher VAP three major UGT isoforms involved in VPA Cs in carrier of AA genotype than in AG geno- metabolism [15-18]. In this study, we evaluated type. For UGT1A6 A552C, VAP Cs of patients the effects of UGT1A3, UGT1A6, and UGT2B7 who were carriers of AA genotype was signifi- genetic polymorphisms on plasma concentra- cant higher than carrier of CC genotype. We tion of VPA in south Chinese epilepsy patients. deduced that it would be important to determine UGT1A3 A17G and UGT1A6 A552C poly- The genotype frequencies and the relationship morphisms of epilepsy patients in clinic use of between UGT1A3, UGT1A6, and UGT2B7 and VPA.

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Furthermore, as a high accuracy, strong versa- [2] Nakashima H, Oniki K, Nishimura M, Ogusu N, tility, high throughput, and simple operation, Shimomasuda M, Ono T, Matsuda K, Yasui- PCR-LDR was used to detect UGT polymor- Furukori N, Nakagawa K, Ishitsu T, Saruwatari phisms in this research. We produced a multi- J. Determination of the optimal concentration of valproicacid in patients with epilepsy: a plex RCR-LDR method, which could detect the population pharmacokinetic-pharmacodynam- polymorphisms of ten target gene nucleotide ic analysis. PLoS One 2015; 10: e0141266. positions simultaneously. It could save time [3] Tan L, Yu JT, Sun YP, Ou JR, Song JH, Yu Y. The and cost of patients, so we believe that this influence of cytochrome oxidase CYP2A6, method could be a suitable method to use in CYP2B6, and CYP2C9 polymorphisms on the the clinic of UGT polymorphism detection. plasma concentrations of valproic acid in epileptic patients. Clin Neurol Neurosurg 2010; However, a small sample size was the limitation 112: 320-323. of this study. Otherwise, it is known that VPA [4] Barker-Haliski ML, Dahle EJ, Heck TD, Pruess TH, Vanegas F, Wilcox KS and White HS. biotransformation involve several enzymatic Evaluating an etiologically relevant platform for processes, such as UGTs, CYPs, and mitochon- therapy development for temporal lobe epilep- drial beta oxidation [20]. Our finding regarding sy: effects of carbamazepine and valproic acid the role of UGT1A3, UGT1A6, and UGT2B7 on on acute seizures and chronic behavioral co- the overall elimination of VPA were limited, so it morbidities in the Theiler’s murine encephalo- is necessary to elucidate the association be- myelitis virus mouse model. J Pharmacol Exp tween VPA concentration and polymorphisms Ther 2015; 353: 318-329. of other enzymes involved in VPA biotrans- [5] Ding J, Wang Y, Lin W, Wang C, Zhao L, Li X, formation. Zhao Z, Miao L and Jiao Z. A population pharmacokinetic model of valproic acid in pediatric patients with epilepsy: a non-linear pharmaco- In conclusion, our study demonstrate that the kinetic model based on protein-binding satura- UGT1A3 A17G, C133T and UGT1A6 A552C tion. Clin Pharmacokinet 2015; 54: 305-317. polymorphism are important factors influenc- [6] Perucca E. Clinically relevant drug interactions ing VPA concentration, whereas UGT2B7 G211T with antiepileptic drugs. Br J Clin Pharmacol and A268G had no obvious impacts in South 2006; 61: 246-255. Chinese epilepsy patients. This might provide [7] Ogungbenro K, Aarons L; CRESim & Epi-CRE- useful genetic information for personalized VPA Sim Project Groups. A physiologically based therapy in epileptic patients. pharmacokinetic model for Valproic acid in adults and children. Eur J Pharm Sci 2014; 63: Acknowledgements 45-52. [8] Fang J, Chen S, Tong N, Chen L, An D, Mu J and Zhou D. Metabolic syndrome among Chinese The study was supported by grants from Ge- obese patients with epilepsy on sodium valpro- neral Public Technology and Application Project ate. Seizure 2012; 21: 578-582. Fund of Huzhou City (No. 2014GYB03). [9] Argikar UA and Remmel RP. Effect of aging on glucuronidation of valproic acid in human liver Disclosure of conflict of interest microsomes and the role of UDP-glucuronosyl transferase UGT1A4, UGT1A8, and UGT1A10. None. Drug Metab Dispos 2009; 37: 229-236. [10] Guo Y, Hu C, He X, Qiu F and Zhao L. Effects of Address correspondence to: Dr. Yingrong Chen, UGT1A6, UGT2B7, and CYP2C9 genotypes on Huzhou Key Laboratory of Molecular Medicine, plasma concentrations of valproic acid in Huzhou Central Hospital, 198 Hongqi Road, Huzhou Chinese children with epilepsy. Drug Metab 313000, Zhejiang Province, China. Tel: 86-572- Pharmacokinet 2012; 27: 536-542. 2555802; Fax: 86-572-2555802; E-mail: chenyin- [11] Chu XM, Zhang LF, Wang GJ, Zhang SN, Zhou [email protected] JH and Hao HP. Influence of UDP-glucuronosyltransferase polymorphisms on valproic acid References pharmacokinetics in Chinese epilepsy patients. Eur J Clin Pharmacol 2012; 68: 1395- [1] Schatzberg AF and Nemeroff CB. Textbook of 1401. psychopharmacology. 2nd edition. Washington [12] Munisamy M, Tripathi M, Behari M, Raghavan DC: The American Psychiatric Press; 1998. pp. S, Jain DC, Ramanujam B, Arumugam K, 514-546. Rajakannan T, Mallayasamy SR and Subbiah V.

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The effect of uridine diphosphate glucuronos- [16] Hung CC, Ho JL, Chang WL, Tai JJ, Hsieh TJ, yltransferase (UGT)1A6 genetic polymorphism Hsieh YW, Liou HH. Association of genetic vari- on valproic acidpharmacokinetics in Indian pa- ants in six candidate genes with valproic ac- tients with epilepsy: a pharmacogenetic ap- id therapy optimization, Pharmacogenomics proach. Mol Diagn Ther 2013; 17: 319-326. 2011; 12: 1107-1117. [13] Inoue K, Suzuki E, Yazawa R, Yamamoto Y, [17] Sun YP, Tan L, Wang Y and Song JH. Effect of Takahashi T, Takahashi Y, Imai K, Koyama S, UGT1A6 genetic polymorphisms on the metab- Inoue Y, Tsuji D, Hayashi H and Itoh K. Influence olism of sodium valproate. Natl Med J China of uridine diphosphate glucuronosyltransfer- 2007; 87: 2033-2035. ase 2B7 -161C>T polymorphism on the con- [18] Tan XY, Zhang XP and Qiu ZJ. Association be- centration ofvalproic acid in pediatric epilepsy tween genetic polymorphisms of UGT1A6 and patients. Ther Drug Monit 2014; 36: 406-409. serum Valproate concentration. Pharmaceuti- [14] Xie F, Qian Q, Chen Z, Ma G and Feng Y. cal and Clinical Research 2014; 22: 299-301. Chitinase 3-like 1 gene-329G/A polymor- [19] Ma H, Zhang T, Gong Z, Zhou B, Zou M, Xiao S phism, plasma concentration and risk of coro- and Zhu W. Effect of UGT2B7 genetic variants nary heart disease in a Chinese population. on serum valproic acid concentration. Zhong Gene 2012; 499: 135-138. Nan Da Xue Xue Bao Yi Xue Ban 2013; 38: [15] Liu L, Zhao L, Wang Q, Qiu F, Wu X and Ma Y. 766-772. Influence of valproic acid concentration and [20] Argikar UA and Remmel RP. Effect of aging on polymorphism of UGT1A4*3, UGT2B7 -161C>T glucuronidation of valproic acid in human liver and UGT2B7*2 on serum concentration of la- microsomes and the role of UDP-glucuronosyl motrigine in Chinese epileptic children. Eur J transferase UGT1A4, UGT1A8, and UGT1A10. Clin Pharmacol 2015; 71: 1341-1347. Drug Metab Dispos 2009; 37: 229-236.

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