Neuropharmacology 57 (2009) 731733

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Neuropharmacology
journal homepage: www.elsevier.com/locate/neuropharm

Carboxylesterase 1 gene polymorphism and methylphenidate response in ADHD

Zsoa Nemoda a, *, Nora Angyal a, Zsanett Tarnok b, Julia Gadoros b, Maria Sasvari-Szekely a
a
Institute of Medical Chemistry, Molecular Biology and Pathobiochemistry, Semmelweis University, Budapest, Hungary
b
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: Methylphenidate (MPH) is the most frequently prescribed drug in the treatment of attention decit
Received 7 May 2009 hyperactivity disorder (ADHD). Several pharmacogenetic studies suggested that catecholamine candidate
Received in revised form genes inuence individual MPH-responses, but these results are mostly contradictory. Genetic analyses
18 August 2009
of MPH metabolizing carboxylesterase 1 enzyme (CES1) have not been carried out, whereas, meta-
Accepted 25 August 2009
analysis of CYP2D6 genetic variants has been already indicated signicant pharmacogenetic differences
in atomoxetine treatment. Here we present an association analysis of the CES1 Gly143Glu functional
Keywords:
polymorphism in a Hungarian ADHD group (n 173). The genotype frequencies were similar to that of
Attention decit hyperactivity
disorder (ADHD) the general population (5.8% vs 4.1% of Gly/Glu heterozygote). Pharmacogenetic analysis was conducted
Methylphenidate (MPH) among 122 ADHD children treated with MPH. Neither the categorical analysis comparing 90 responders
Pharmacogenetics vs 32 non-responders, nor the dimensional analysis of Inattention and HyperactivityImpulsivity score
Carboxylesterase 1 (CES1) reduction showed a signicant 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) 2.33, p 0.022).
This result warrants for further investigations of the CES1 gene in larger ADHD samples.
2009 Elsevier Ltd. All rights reserved.

1. Introduction effects). Most of the studies investigating MPH-response in ADHD

Attention decit 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 rst-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 inuence the pharmacokinetic and pharmacodynamic
properties of the medicine (drug metabolism, efciency, side
* Corresponding author at: Institute of Medical Chemistry, Molecular Biology and
Pathobiochemistry, Semmelweis University, Budapest, POB 260, H-1444, Hungary.
Tel.: 36 1 4591500x60134; fax: 36 1 2662615.
E-mail address: zsoa.nemoda@eok.sote.hu (Z. Nemoda).
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 rst-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) identied 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).

0028-3908/$ see front matter 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.neuropharm.2009.08.014
732 Z. Nemoda et al. / Neuropharmacology 57 (2009) 731733
2. Methods and materials
Previously published ADHD patient population (n 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 1030 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 32) was done during the rst
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 HyperactivityImpulsivity severity scores (03 points for 99
items). Analysis of variance investigating the effect of MPH on ADHD-RS scores after
the rst 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 conrmed 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 -CCCAGGTGATGGTGTG-
GAT-30 forward and 50 -GCCTTACTGTGGAACCTAGCTAAGC-30 reverse primers to
include a control restriction site (51 bp fragment is cleaved from the 251 bp PCR-
fragment at every sample). The Glu-allele specic 168 bp fragment was separated
from the Gly-specic 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 signicant deviation from the
HardyWeinberg equilibrium was detected (p 0.695 in the ADHD group, p 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 signicant difference in the
genotype distribution (Pearson c2 0.651, df 1, p 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 0.021,
df 1, Fishers exact test p 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 signicantly reduced ADHD-RS scores after one
month of treatment (F(2,119) 27.63, p < 0.001, h2 0.317, power 1),
for both Inattention (F(1,120) 40.17, p < 0.001, h2 0.251) and
HyperactivityImpulsivity (F(1,120) 55.33, p < 0.001, h2 0.316).
Although signicant multivariate interaction of MPH-effect and CES1
genotype was observed (F(2,119) 5.02, p 0.008, h2 0.078,
power 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 groups score decreased from
15.00  4.32 to 12.14  5.27, F(1,120) 1.22, p 0.272, h2 0.01. At the
HyperactivityImpulsivity scale, the Gly/Gly groups 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) 0.81, p 0.37, h2 0.007.
Finally, we compared the average doses required to achieve the
therapeutic effect among the responders. The Gly/Glu hetero-
zygotes (n 5) required less MPH compared to the Gly/Gly
homozygotes (n 85): the average daily dose was 0.410  0.127 vs
0.572  0.153 mg/kg, t(1,88) 2.33, p 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 rst 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
efciently norepinephrine transporter as well (Han and Gu, 2006).
In contrast to selective norepinephrine reuptake inhibitor atom-
oxetine, 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 metabo-
lizers, 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 Glu-
variant 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 515 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 effec-
tiveness of MPH was assessed every month in the rst six months
when the children and their parents came back for control exam-
inations 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.
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