Carboxylesterase 1 gene polymorphism and methylphenidate response in ADHD

Neuropharmacology 57 (2009) 731–733

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Carboxylesterase 1 gene polymorphism and methylphenidate response in ADHD Zsofia Nemoda a, *, Nora Angyal a, Zsanett Tarnok b, Julia Gadoros b, Maria Sasvari-Szekely a a b

Institute of Medical Chemistry, Molecular Biology and Pathobiochemistry, Semmelweis University, Budapest, Hungary Vadaskert Child and Adolescent Psychiatric Clinic, Budapest, Hungary

a r t i c l e i n f o

a b s t r a c t

Article history: Received 7 May 2009 Received in revised form 18 August 2009 Accepted 25 August 2009

Methylphenidate (MPH) is the most frequently prescribed drug in the treatment of attention deficit hyperactivity disorder (ADHD). Several pharmacogenetic studies suggested that catecholamine candidate genes influence individual MPH-responses, but these results are mostly contradictory. Genetic analyses of MPH metabolizing carboxylesterase 1 enzyme (CES1) have not been carried out, whereas, metaanalysis of CYP2D6 genetic variants has been already indicated significant pharmacogenetic differences in atomoxetine treatment. Here we present an association analysis of the CES1 Gly143Glu functional polymorphism in a Hungarian ADHD group (n ¼ 173). The genotype frequencies were similar to that of the general population (5.8% vs 4.1% of Gly/Glu heterozygote). Pharmacogenetic analysis was conducted among 122 ADHD children treated with MPH. Neither the categorical analysis comparing 90 responders vs 32 non-responders, nor the dimensional analysis of Inattention and Hyperactivity–Impulsivity score reduction showed a significant main genotype effect. However, analyzing the daily dose, we observed an association with the rare 143Glu-variant: 5 patients in the responder group carrying the Glu-allele required lower doses of MPH for symptom reduction (0.410  0.127 vs 0.572  0.153 mg/kg, t(1,88) ¼ 2.33, p ¼ 0.022). This result warrants for further investigations of the CES1 gene in larger ADHD samples. Ó 2009 Elsevier Ltd. All rights reserved.

Keywords: Attention deficit hyperactivity disorder (ADHD) Methylphenidate (MPH) Pharmacogenetics Carboxylesterase 1 (CES1)

1. Introduction Attention deficit hyperactivity disorder (ADHD) is one of the most prevalent childhood-onset psychiatric disorders, affecting 5% of school-age children worldwide (Polanczyk et al., 2007). Psychostimulant drugs such as methylphenidate (MPH) and amphetamines are first-line treatments for this disorder, but they are effective only 70% of the cases (Biederman and Spencer, 2008). With global rise in ADHD diagnosis, the international drug consumption of stimulants has been increasing in the past decade (Singh, 2008), therefore, identifying genetic and/or biological markers predicting drug response has turned into a public health concern. Pharmacogenetic studies would hopefully lead to individualized treatment protocols in the near future by providing a panel of informative genetic markers to check before starting a pharmacotherapy. Pharmacogenetic studies try to explore how individual genetic variations influence the pharmacokinetic and pharmacodynamic properties of the medicine (drug metabolism, efficiency, side

* Corresponding author at: Institute of Medical Chemistry, Molecular Biology and Pathobiochemistry, Semmelweis University, Budapest, POB 260, H-1444, Hungary. Tel.: þ36 1 4591500x60134; fax: þ36 1 2662615. E-mail address: zsofi[email protected] (Z. Nemoda). 0028-3908/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2009.08.014

effects). Most of the studies investigating MPH-response in ADHD focused on genetic variants of drug targets, such as transporters and receptors (reviewed by Stein and McGough, 2008), and little attention has been paid to genetic variability of drug metabolism. In contrast to amphetamine and atomoxetine, which are metabolized by hepatic cytochrome P450 (CYP) enzymes, MPH is converted to inactive ritalinic acid by carboxylesterase. Liver carboxylesterase 1 (CES1) is responsible for the hydrolysis of both D- and L-isomer of MPH (the two components of Ritalin, one of the most frequently prescribed drug in ADHD), and for the resulting first-pass metabolism of the drug (Sun et al., 2004). Therefore, we selected a recently reported functional variant of the CES1 gene for pharmacogenetic analysis. Zhu et al. (2008) identified two CES1 variants with reduced enzyme activity in a compound heterozygote subject participating in an MPH pharmacokinetics study. The 428G-A transition in exon 4 of the CES1 gene results in glycine-to-glutamate substitution (Gly143Glu), whereas a 1-bp deletion (780delT) in exon 6 produces a frameshift and premature truncation of the protein (Asp260fs). Since the Asp260fs is extremely rare variant (no other person out of the reported 925 subjects carried this variant), we chose the Gly143Glu polymorphism which had 3.7% minor allele frequency in Caucasian population. None of the previously described single nucleotide polymorphisms (SNPs) in the CES1 gene was regarded as functional, because they were not associated with altered mRNA levels in either normal or tumor tissue (Marsh et al., 2004).

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2. Methods and materials Previously published ADHD patient population (n ¼ 173, mean age: 9.14  2.6; 87.3% male and 12.7% female) was analyzed in this study, 122 children (mean age: 9.6  2.6; 88.5% male and 11.5% female) participated in a prospective MPH-response analysis (for detailed demographic and clinical characteristics see Supplementary Table 1). The study was approved by the Local Ethics Committee (TUKEB), patients and their parents provided written informed consent for their participation. Patients participating in the drug-response study were given 10–30 mg methylphenidate according to their body weight, in two doses (morning and noon). The daily dose thereby ranged from 0.22 to 0.95 mg/kg/day, in average 0.55  0.15 mg/kg/day. Categorization of the non-responder children (n ¼ 32) was done during the first three months (see Kereszturi et al., 2008 for details). Drug response was also assessed by dimensional approach using the ADHD Rating Scale (ADHD-RS; DuPaul, 1998) Inattention and Hyperactivity–Impulsivity severity scores (0–3 points for 9–9 items). Analysis of variance investigating the effect of MPH on ADHD-RS scores after the first month was carried out in a repeated measures design including genotype as a between-subject factor. Genotyping of the CES1 Gly143Glu SNP (rs71647871) was done using published primers and TaqMan probes (Zhu et al., 2008) on 7300 Real-Time PCR System (Applied BioSystem, Foster City, USA). The accuracy of the method was confirmed by sequencing 6 selected samples (4 heterozygotes and 2 homozygotes). In addition, the ADHD samples were checked by AluI digestion (Glu-allele creates an AGCT restriction site). The PCR reaction was carried out with 50 -CCCAGGTGATGGTGTGGAT-30 forward and 50 -GCCTTACTGTGGAACCTAGCTAAGC-30 reverse primers to include a control restriction site (51 bp fragment is cleaved from the 251 bp PCRfragment at every sample). The Glu-allele specific 168 bp fragment was separated from the Gly-specific 200 bp fragment by 2.5% agarose gel electrophoresis. For comparison reasons, allele and genotype frequencies of the general Hungarian population were assessed by genotyping 268 control subjects (university students, mean age: 20.51  1.83, 50.4% male, 49.6% female). Both control and patient samples consisted of unrelated individuals of Caucasian origin, hence creating ethnically homogenous populations. No significant deviation from the Hardy–Weinberg equilibrium was detected (p ¼ 0.695 in the ADHD group, p ¼ 0.732 in the control group). Since there was no Glu/Glu homozygote genotype, the chi-square analyses were computed for a 2  2 table.

3. Results A control Hungarian sample was genotyped in addition to the ADHD sample to compare allele and genotype frequencies of the CES1 Gly143Glu polymorphism. No Glu/Glu homozygote subject was found in either groups. The Gly/Glu heterozygote frequency was 4.1% in the control group (11 out of 268) and 5.8% in the ADHD group (10 out of 173), indicating no significant difference in the genotype distribution (Pearson c2 ¼ 0.651, df ¼ 1, p ¼ 0.420). The minor allele frequency (MAF: 2.1% and 2.9%) was comparable to earlier report (3.7%, Zhu et al., 2008). Methylphenidate response was assessed in 122 ADHD children. Using the categorical grouping system, 90 patients (73.8%) were described as responder, while 32 (26.2%) were non-responder. Genotype frequencies did not differ between the two patient groups: there were 5 Gly/Glu heterozygotes among the responders, and 2 heterozygotes in the non-responder group (5.6% vs 6.3%, c2 ¼ 0.021, df ¼ 1, Fisher’s exact test p ¼ 0.591). In the dimensional approach, repeated measures analyses of variance were carried out to test the effect of MPH on ADHD-RS scores, and its potential interaction with CES1 genotype. MPH significantly reduced ADHD-RS scores after one month of treatment (F(2,119) ¼ 27.63, p < 0.001, h2 ¼ 0.317, power ¼ 1), for both Inattention (F(1,120) ¼ 40.17, p < 0.001, h2 ¼ 0.251) and Hyperactivity–Impulsivity (F(1,120) ¼ 55.33, p < 0.001, h2 ¼ 0.316). Although significant multivariate interaction of MPH-effect and CES1 genotype was observed (F(2,119) ¼ 5.02, p ¼ 0.008, h2 ¼ 0.078, power ¼ 0.807), it was not explained by either subscales separately: Inattention scores of the Gly/Gly group decreased from 16.31  5.00 to 12.25  5.30, whereas the Gly/Glu group’s score decreased from 15.00  4.32 to 12.14  5.27, F(1,120) ¼ 1.22, p ¼ 0.272, h2 ¼ 0.01. At the Hyperactivity–Impulsivity scale, the Gly/Gly group’s score decreased from 16.37  5.55 to 12.56  5.60, while the Gly/Glu group had 18.57  6.40 at baseline and 13.71  5.41 after one month of MPH-treatment, F(1,120) ¼ 0.81, p ¼ 0.37, h2 ¼ 0.007.

Finally, we compared the average doses required to achieve the therapeutic effect among the responders. The Gly/Glu heterozygotes (n ¼ 5) required less MPH compared to the Gly/Gly homozygotes (n ¼ 85): the average daily dose was 0.410  0.127 vs 0.572  0.153 mg/kg, t(1,88) ¼ 2.33, p ¼ 0.022. 4. Discussion Pharmacogenetic analysis of the CES1 Gly143Glu polymorphism and MPH-response was carried out among 122 Hungarian ADHD children. To our best knowledge, this is the first report about CES1 genetic variant in an ADHD sample. Hitherto, only genetic variants of drug targets have been studied in connection to MPH-response. In the therapeutic effects of MPH-treatment dopamine transporter blockade is thought to have crucial role, although MPH blocks efficiently norepinephrine transporter as well (Han and Gu, 2006). In contrast to selective norepinephrine reuptake inhibitor atomoxetine, genetic analyses of drug metabolizing enzymes have not been carried out in relation to MPH, probably because lack of knowledge of potentially interesting candidate polymorphisms. Atomoxetine is metabolized by CYP2D6, which genetic variants are well known in humans. A recent meta-analysis of several clinical trials reported pharmacogenetic differences between poor and extensive metabolizers (Michelson et al., 2007). Poor metabolizers displayed greater symptom improvement than extensive metabolizers, but several side effects, such as decreased appetite or tremor, were also more frequent in poor metabolizers, most likely due to higher plasma drug concentration. Our genetic analyses of MPH-response indicated an association with the rare CES1 143Glu-variant: patients carrying the Glu-allele required lower doses of MPH for symptom reduction. Since the Gluvariant has reduced enzyme activity, patient with this genetic variant might have higher plasma drug level, similarly to that of the CYP2D6 poor metabolizers treated with atomoxetine. Our study was not a placebo-controlled clinical trial, the starting MPH dose was 5–15 mg (depending on the body weight) in the morning for every patient and supplemented with another dose at noon for those who needed it for afternoon activities at school. The effectiveness of MPH was assessed every month in the first six months when the children and their parents came back for control examinations and drug-prescription. Unfortunately, no detailed data was available about side effects in our sample. The main limitation of the present study is the small group size, there were only 7 patients carrying the 143 Glu-allele, and the difference in daily doses was relatively small (0.410 vs 0.572 mg/kg) and was based on 5 patients with Gly/Glu genotype responding to MPH. Therefore, our results should be regarded as preliminary and require further analyses in larger patient samples. Acknowledgement This work was supported by Hungarian OTKA fund F67784. Appendix. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.neuropharm.2009.08.014. References Biederman, J., Spencer, T.J., 2008. Psychopharmacological interventions. Child Adolesc. Psychiatr. Clin. N. Am. 17 (2), 439–458 (xi). DuPaul, G.J., 1998. ADHD Rating Scale-IV: Checklists, Norms and Clinical Interpretations. Guilford Press, New York. Han, D.D., Gu, H.H., 2006. Comparison of the monoamine transporters from human and mouse in their sensitivities to psychostimulant drugs. BMC Pharmacol. 3 (6), 6.

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