Department of Central Analysis, Huzhou Environmental Monitoring Center, Huzhou, Zhejiang Province, China; 4

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1 Int J Clin Exp Pathol 2016;9(4): /ISSN: /IJCEP Original Article Effects of UGT1A3, UGT1A6, and UGT2B7 genetic polymorphisms on plasma concentration of valproic acid in south Chinese epilepsy patients Xiongrong Shen 1*, Jingbo Bi 3*, Quankun Liu 2, Zhihong Ma 4, Lishan Min 4, Limin Xu 4, Shuixin Yang 1, Yingrong Chen 4 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 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 Valproic acid (VPA) is one of the most commonly used anti-epileptic drugs in epilepsy patients [1, 2]. VPA has a narrow therapeutic range ( μg/ml) in the treatment of epilepsy and shows large individual variability in both treatment 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 polymorphisms in genes encoding drug-metabolizing enzymes [6, 7]. There are three main metabolic pathways in the metabolism of VPA, mitochondrial beta oxidation, glucuronidation conjugation and CYPcatalyzed terminal desaturation and hydroxylation [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 polymorphisms were found to be most important for explaining the considerable variation observed in valproate levels in epilepsy patients. However, several previous studies on the influence of UGT polymorphisms on VPA plasma concentration were reported, the results were not consistent [10-13]. In addition, most previous researches used polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) to detect the influence of UGT poly-

2 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/ 3.63±1.52 ALT: alanine transaminase; AST: aspartate aminotransferase; Cr: creatinine; Reference range: ALT: 7-40 U/L; AST: U/L; Cr: μmol/l. morphisms on VPA concentration in epilepsy patients. But PCR-RFLP method has great limitations because it is only applicable to the fragments that affected the restriction endonuclease. It also has a trouble experimental operation and a long experimental period. As a method 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 levels. Materials and methods Patients and blood sampling A 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. 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 samples 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 isolation. VPA quantitation Steady-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 biosample analysis in a linear range of μg/ml, within- and between-batch variations were <10%, and the lower limit was 4 μg/ml. Genotyping procedures Genomic DNA was extracted from peripheral blood using AxyPrep DNA Mini Kits according to the manufacture s recommendations (Axygen, USA) and stored at -20 C. DNA concentrations were determined by spectrophotometry 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 polymerase chain reaction-ligase detection reaction (PCR-LDR). Primers were synthesized by Shanghai HANYU Biological Engineering Ltd. Each set of ligase detection reaction probes comprised one common probe and two discriminating probes for the two types. The target DNA sequences were amplified using the multiplex PCR method. PCR was carried out for each subject at a final volume of 20 μl containing 2 μl of 1 PCR buffer, 0.6 μl of 3.0 mm MgCl 2, 2 μl of 4514 Int J Clin Exp Pathol 2016;9(4):

3 4515 Int J Clin Exp Pathol 2016;9(4):

4 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 Int J Clin Exp Pathol 2016;9(4):

5 Table 2. Genotype frequencies of the UGT1A3 polymorphisms Genotype Table 3. Genotype frequencies of the UGT1A6 and UGT2B7 polymorphisms Genotype Number of patients Number of patients Frequency (%) UGT1A3 A17G AA AG GG UGT1A3 T32C TT TC CC 0 0 UGT1A3 G81A GG GA AA UGT1A3 C133T CC CT TT 0 0 UGT1A3 T140C TT TC CC UGT1A3 A477G AA AG GG Frequency (%) UGT1A6 A552C AA AC CC UGT1A6 A541G AA AG GG UGT2B7 G211T GG GT TT UGT2B7 A268G AA AG GG 0 0 X 2 X 2 P P 2.0 mm deoxynucleotide triphosphate, 2 μl of primers, 0.2 μl of Taq Polymerase, 12.2 μl of ddh 2 O and 50 ng of genomic DNA. Thermal cycling was performed using the Gene Amp PCR system 9600 (Perkin Elmer, USA) with initial denaturation for 2 minutes at 95 C, followed by 35 cycles of denaturation at 94 C for 90 sec, annealing at 65 C for 1 min and extension at 72 C for 1 min with final extension at 72 C for 7 min. The ligation reaction for each subject was carried out in a final volume of 10 μl, containing 1 μl of NEB Taq DNA ligase buffer, 1 μl of 2 pmol/μl probe mix, 0.05 μl of 2 U Taq DNA ligase (NEB, USA), 4 μl of ddh 2 O and 4 μl 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 differentiated using the PRISM 3730 (Applied Biosystems, USA). Statistical analysis Statistical analysis was performed using SPSS version 19.0 (SPSS Inc., Armonk, NY, USA). Hardy-Weinberg equilibrium (HWH) was determined 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 characteristics including age and body weight, VAP concentrations and adjusted plasma VPA concentration (Cs) among different UGT1A3, UGT1A6 and UGT2B7 functional genotypes were compared using analysis of variance (ANOVA) or Student s t-test. P value less than 0.05 was considered as statistically significant. Results Patients characteristics Ninety-seven patients characteristics including sex, age, body weight, alanine transaminase (ALT), aspartate aminotransferase (AST), creatinine (Cr), VPA daily dose, plasma VPA concentration, and adjusted plasma VPA concentration 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 polymorphisms were analyzed by PCR-LDR (Figures 4517 Int J Clin Exp Pathol 2016;9(4):

6 Table 4. Effect of UGT1A3 genotypes on VPA doses, VPA concentrations and adjusted VPA concentrations (Cs) in all studied epileptic patients Gene Genotype No. Age (years) Weihgt (kg) VPA dose (mg/kg/day) VPA conccntration (μg/ml) VPA Cs [(μg/ UGT1A3 A17G AA ± ± ± ± ±1.54 AG ± ± ± ± ±0.35 * GG P value UGT1A3 T31C TT ± ± ± ± ±1.54 TC ± ± ± ± ±1.29 CC P value UGT1A3 G81A GG ± ± ± ± ±1.07 GA ± ± ± ± ±2.01 AA ± ± ± ± ±1.80 P value UGT1A3 C133T CC ± ± ± ± ±1.54 CT ± ± ± ± ±1.09 # TT P value UGT1A3 T140C TT ± ± ± ± ±1.30 TC ± ± ± ± ±1.68 CC ± ± ± ± ±2.53 P value UGT1A3 A477G AA ± ± ± ± ±1.02 AG ± ± ± ± ±2.04 GG ± ± ± ± ±1.74 P value 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 Gene Genotype N Age (years) Weihgt (kg) VPA dose (mg/kg/day) VPA conccntration (μg/ml) VPA Cs [(μg/ UGT1A6 A552C AA ± ± ± ± ±1.40 * AC ± ± ± ± ±1.24 * CC ± ± ± ± ±1.06 P value UGT1A6 A541G AA ± ± ± ± ±1.43 AG ± ± ± ± ±1.67 GG ± ± ± ± ±1.06 P value UGT2B7 G211T GG ± ± ± ± ±1.49 GT ± ± ± ± ±1.73 TT ± ± ± ± ±0.45 P value UGT2B7 A268G AA ± ± ± ± ±1.56 AG ± ± ± ± ±0.98 GG P value Cs: * UGT1A6 A552C AA vs CC, AC vs CC, P< Int J Clin Exp Pathol 2016;9(4):

7 Table 6. Frequencies of UGT1A3 polymorphisms and adjusted VPA concentration in various populations Genotype Chinese patients with epilepsy Chu et al. (n = 136) * Chu et al. (n = 242) # Current study (n = 97) N (%) VAP Cs [(μg/ N (%) N (%) VAP Cs [(μg/ A17G AA 116 (85.3) 4.76± (88.8) 89 (91.8) 3.75±1.54 AG 18 (13.2) 3.49±1.43 a 27 (11.2) 7 (7.2) 2.47±0.35 b GG 2 (1.5) 3.01~ (1.0) 1.38 P value T31C TT 65 (47.8) 4.70± (47.1) 0 - TC 59 (43.4) 4.45± (45.5) 4 (4.1) 3.68±1.29 CC 12 (8.8) 4.52± (7.4) 93 (95.9) 3.63±1.54 P value 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 C133T CC 122 (89.7) 4.54± (91.3) 84 (86.6) 3.78±1.54 CT 14 (10.3) 4.90± (8.7) 13 (13.4) 2.69±1.09 c TT P value T140C TT 109 (80.1) 4.56± (75.6) 55 (56.7) 3.42±1.30 TC 24 (17.7) 4.64± (22.7) 37 (38.1) 3.94±1.68 CC 3 (2.2) 5.21± (1.7) 5 (5.2) 3.67±2.53 P value 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 * 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: a UGT1A3 A17G AA vs AG, P<0.05 (Chu et al.); VAP Cs: b UGT1A3 A17G AA vs AG, P<0.05; c UGT1A3 C133T CC vs CT, P<0.05 (Current study). 1 and 2). The frequencies of each UGT1A3, UGT1A6 and UGT2B7 genotype are shown in Tables 2 and 3. The frequencies were all consistent with the Hardy-Weinberg equilibrium. Influence of UGT1A3, UGT1A6, and UGT2B7 polymorphism on plasma VPA concentrations To determine the influence of polymorphisms of the identified UGTs on VPA concentration, association analysis of UGT1A3, UGT1A6 and UGT2B7 SNPs and VPA Cs were observed in all studied epileptic patients (Tables 4 and 5). Among all of the UGT1A3 SNPs detected, 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). There were no significant differences in VAP Cs among the genotypes of UGT1A3 T32C, G81A, T140C and A477G. For UGT1A6 A552C, patients who were carriers of AA and AC genotypes were characterized with a significant higher VAP Cs 4519 Int J Clin Exp Pathol 2016;9(4):

8 Table 7. Frequencies of UGT1A6 polymorphisms and adjusted VPA concentration in various populations Genotype Chinese patients with epilepsy Current study (n = 97) Sun et al. (n = 67) Tan et al. (n = 40) Chu et al. (n = 136) VAP Cs [(μg/ VAP Cs [(μg/ VAP Cs [(μg/ VAP Cs [(μg/ N (%) N (%) N (%) N (%) UGT1A6 A552C AA 40 (59.7) 4.32± (57.5) 6.95± (61.0) 4.55± (54.6) 3.88±1.40 c AC 24 (35.8) 3.43±0.30 a 13 (32.5) 3.59±1.11 b 50 (36.8) 4.61± (40.2) 3.10±1.24 c CC 3 (4.5) 3.16±0.84 a 4 (10.0) 3 (2.2) 4.82± (5.2) 2.76±1.06 P value <0.05 < UGT1A6 A541G AA NS NS NS NS 88 (64.7) 4.52± (56.7) 3.86±1.43 AG NS NS NS NS 45 (33.1) 4.60± (38.1) 3.44±1.67 GG NS NS NS NS 3 (2.2) 5.92± (5.2) 2.76±1.06 P value NS: Not studied. VAP Cs: a UGT1A6 A552C AA vs AC, AA vs CC, P<0.05 (Sun et al.); b UGT1A6 A552C AA vs AC+CC, P<0.05 (Tan et al.); VAP Cs: c UGT1A6 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 Genotype Ma et al. (n = 248) Sun et al. (n = 40) Current study (n = 97) N (%) VAP Cs [(μg/ N (%) VAP Cs [(μg/ N (%) VAP Cs [(μg/ 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 UGT2B7 A268G AA 147 (59.3) 3.80± (76.5) 2.28± (94.8) 3.63±1.56 AG 90 (36.3) 3.61± (22.5) 2.30± (5.2) 3.59±0.98 GG 11 (4.4) 1.76±0.31 a 1 (1.0) P value NS: Not studied. VAP Cs: a UGT2B7 A268G AA vs GG, P<0.05 (Ma et al.). than carrier of CC genotype. The polymorphisms of UGT1A6 A541G, UGT2B7 A268G and UGT 2B7 G211T had no significant effect on VPA Cs. Discussion Large interindividual variability is detected in VPA pharmacokinetics and pharmacodynamics, which may be contributed by genetic polymorphisms. Previous reports showed that UGT contributed to around 50% of VPA metabolism in human. UGT1A3, UGT1A6, and UGT2B7 are three major UGT isoforms involved in VPA metabolism [15-18]. In this study, we evaluated the effects of UGT1A3, UGT1A6, and UGT2B7 genetic polymorphisms on plasma concentration of VPA in south Chinese epilepsy patients. The genotype frequencies and the relationship between UGT1A3, UGT1A6, and UGT2B7 and adjusted VPA concentration in the current study compared to previous studies [11, 17-19] in Chinese patients with epilepsyare shown in Tables 6-8. The genotype frequencies of UGT1A3 A17G, T31C, G81A, C133T, T140C and A477G in previous study [9], no matter the epilepsy patients treated concomitantly with VPA and other antiepileptic drugs or VPA monotherapy were similar to our research. They also showed a significant influence of UGT1A3 A17G polymorphism on plasma concentration of VPA in Chinese epilepsy patients, with higher VAP Cs in carrier of AA genotype than in AG genotype. For UGT1A6 A552C, VAP Cs of patients who were carriers of AA genotype was significant higher than carrier of CC genotype. We deduced that it would be important to determine UGT1A3 A17G and UGT1A6 A552C polymorphisms of epilepsy patients in clinic use of VPA Int J Clin Exp Pathol 2016;9(4):

9 Furthermore, as a high accuracy, strong versatility, high throughput, and simple operation, PCR-LDR was used to detect UGT polymorphisms in this research. We produced a multiplex RCR-LDR method, which could detect the polymorphisms of ten target gene nucleotide positions simultaneously. It could save time and cost of patients, so we believe that this method could be a suitable method to use in the clinic of UGT polymorphism detection. However, a small sample size was the limitation of this study. Otherwise, it is known that VPA biotransformation involve several enzymatic processes, such as UGTs, CYPs, and mitochondrial beta oxidation [20]. Our finding regarding the role of UGT1A3, UGT1A6, and UGT2B7 on the overall elimination of VPA were limited, so it is necessary to elucidate the association between VPA concentration and polymorphisms of other enzymes involved in VPA biotransformation. In conclusion, our study demonstrate that the UGT1A3 A17G, C133T and UGT1A6 A552C polymorphism are important factors influencing VPA concentration, whereas UGT2B7 G211T and A268G had no obvious impacts in South Chinese epilepsy patients. This might provide useful genetic information for personalized VPA therapy in epileptic patients. Acknowledgements The study was supported by grants from General Public Technology and Application Project Fund of Huzhou City (No. 2014GYB03). Disclosure of conflict of interest None. Address correspondence to: Dr. Yingrong Chen, Huzhou Key Laboratory of Molecular Medicine, Huzhou Central Hospital, 198 Hongqi Road, Huzhou , Zhejiang Province, China. Tel: ; Fax: ; chenyingrong2006@163.com References [1] Schatzberg AF and Nemeroff CB. Textbook of psychopharmacology. 2nd edition. Washington DC: The American Psychiatric Press; pp [2] Nakashima H, Oniki K, Nishimura M, Ogusu N, Shimomasuda M, Ono T, Matsuda K, Yasui- Furukori N, Nakagawa K, Ishitsu T, Saruwatari J. Determination of the optimal concentration of valproicacid in patients with epilepsy: a population pharmacokinetic-pharmacodynamic analysis. PLoS One 2015; 10: e [3] Tan L, Yu JT, Sun YP, Ou JR, Song JH, Yu Y. The influence of cytochrome oxidase CYP2A6, CYP2B6, and CYP2C9 polymorphisms on the plasma concentrations of valproic acid in epileptic patients. Clin Neurol Neurosurg 2010; 112: [4] Barker-Haliski ML, Dahle EJ, Heck TD, Pruess TH, Vanegas F, Wilcox KS and White HS. Evaluating an etiologically relevant platform for therapy development for temporal lobe epilepsy: effects of carbamazepine and valproic acid on acute seizures and chronic behavioral comorbidities in the Theiler s murine encephalomyelitis virus mouse model. J Pharmacol Exp Ther 2015; 353: [5] Ding J, Wang Y, Lin W, Wang C, Zhao L, Li X, Zhao Z, Miao L and Jiao Z. A population pharmacokinetic model of valproic acid in pediatric patients with epilepsy: a non-linear pharmacokinetic model based on protein-binding saturation. Clin Pharmacokinet 2015; 54: [6] Perucca E. Clinically relevant drug interactions with antiepileptic drugs. Br J Clin Pharmacol 2006; 61: [7] Ogungbenro K, Aarons L; CRESim & Epi-CRE- Sim Project Groups. A physiologically based pharmacokinetic model for Valproic acid in adults and children. Eur J Pharm Sci 2014; 63: [8] Fang J, Chen S, Tong N, Chen L, An D, Mu J and Zhou D. Metabolic syndrome among Chinese obese patients with epilepsy on sodium valproate. Seizure 2012; 21: [9] Argikar UA and Remmel RP. Effect of aging on glucuronidation of valproic acid in human liver microsomes and the role of UDP-glucuronosyl transferase UGT1A4, UGT1A8, and UGT1A10. Drug Metab Dispos 2009; 37: [10] Guo Y, Hu C, He X, Qiu F and Zhao L. Effects of UGT1A6, UGT2B7, and CYP2C9 genotypes on plasma concentrations of valproic acid in Chinese children with epilepsy. Drug Metab Pharmacokinet 2012; 27: [11] Chu XM, Zhang LF, Wang GJ, Zhang SN, Zhou JH and Hao HP. Influence of UDP-glucuronosyltransferase polymorphisms on valproic acid pharmacokinetics in Chinese epilepsy patients. Eur J Clin Pharmacol 2012; 68: [12] Munisamy M, Tripathi M, Behari M, Raghavan S, Jain DC, Ramanujam B, Arumugam K, Rajakannan T, Mallayasamy SR and Subbiah V Int J Clin Exp Pathol 2016;9(4):

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