Parkinsonism and Related Disorders 20 (2014) 170e175
Contents lists available at ScienceDirect
Parkinsonism and Related Disorders journal homepage: www.elsevier.com/locate/parkreldis
Dopa-decarboxylase gene polymorphisms affect the motor response to L-dopa in Parkinson’s disease David Devos a, b, *, Stéphanie Lejeune a, c, Florence Cormier-Dequaire a, c, Khadija Tahiri c, Fanny Charbonnier-Beaupel c, d, e, Nathalie Rouaix f, Alain Duhamel f, Bernard Sablonnière f, Anne-Marie Bonnet g, Cecilia Bonnet a, Noel Zahr e, Jean Costentin h, Marie Vidailhet a, c, g, Jean-Christophe Corvol a, c, g a
INSERM (French National Institute of Medical Research and Health), APHP (Assistance Publique Hopitaux de Paris), Clinical Investigation Center (CIC9503), Pitié-Salpêtrière Hospital, Paris, France b Lille Nord de France University, Department of Medical Pharmacology, Lille University Medical Center, Faculty of Medicine of Lille 2, EA 4610, France c INSERM, UMRS_975 unit, UPMC (Pierre and Marie Curie University), CNRS UMR7525 CR-ICM, Paris, France d APHP (Assistance Publique Hopitaux de Paris), Pitié-Salpêtrière Hospital, Department of Pharmacy, France e APHP, Pitié-Salpêtrière Hospital, Department of Pharmacology, Paris, France f Lille Nord de France University, Department of Molecular Biology, Lille University Medical Center, France g APHP, Pitié-Salpêtrière Hospital, Department of Neurology, France h University of Rouen, Neuropharmacology Laboratory, Rouen, France
a r t i c l e i n f o
a b s t r a c t
Article history: Received 31 July 2013 Received in revised form 5 October 2013 Accepted 15 October 2013
Background: In Parkinson’s disease (PD), the response to L-dopa is highly variable and unpredictable. The major pathway for dopamine synthesis from L-dopa is decarboxylation by aromatic L-amino acid decarboxylase (AAAD, encoded by the DDC gene). Objective: To determine the motor response to L-dopa in PD patients as a function of the DDC gene promoter polymorphisms (rs921451 T > C polymorphism (DDCT/C) and rs3837091 AGAG del (DDCAGAG/ )). Methods: Thirty-three Caucasian PD patients underwent an acute L-dopa challenge together with the peripheral AAAD inhibitor benserazide and were genotyped for rs921451 and rs3837091. The primary efficacy criterion was the motor response to L-dopa, as estimated by the area under the curve for the change in the Unified Parkinson’s Disease Rating Scale part III (UPDRS) score relative to baseline (AUCDUPDRS) in the 4 h following L-dopa administration. Secondary endpoints were pharmacokinetic parameters for plasma levels of L-dopa and dopamine. Investigators and patients were blinded to genotypes data throughout the study. Results: When adjusted for the L-dopa dose, the AUCDUPDRS was significantly lower in DDCCC/CT patients (n ¼ 14) than in DDCTT patients (n ¼ 19) and significantly lower in DDC/ or AGAG/ patients (n ¼ 8) than in DDCAGAG/AGAG patients (n ¼ 25). There were no significant intergroup differences in plasma pharmacokinetic parameters for L-dopa and dopamine. Discussion: The rs921451 and rs3837091 polymorphisms of the DDC gene promoter influence the motor response to L-dopa but do not significantly change peripheral pharmacokinetic parameters for L-dopa and dopamine. Our results suggest that DDC may be a genetic modifier of the L-dopa response in Parkinson’s disease. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Parkinson’s disease Dopamine L-amino acid decarboxylase Genetic L-dopa
1. Introduction
* Corresponding author. Département de Pharmacologie Médicale, Université Lille Nord de France, CHRU de Lille, F-59037 Lille, France. E-mail address: [email protected] (D. Devos). 1353-8020/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.parkreldis.2013.10.017
Parkinson’s disease (PD) is the second most frequent neurodegenerative disorder worldwide and affects more than 1% of the population over the age of 60 [1]. The loss of dopamine production in the striatum (as a result of progressive neuronal degeneration in the substantia nigra pars compacta) is the primary chemical disease
D. Devos et al. / Parkinsonism and Related Disorders 20 (2014) 170e175
marker for PD. Given that dopamine deficiency is the main cause of symptoms in PD, pharmacological treatment of this disease has focused on the restoration of dopaminergic transmission within the nigrostriatal pathway [2]. Since dopamine does not cross the digestive mucosa or the blood brain barrier, its precursor L-dopa has been developed as an orally administered medication. It is known that a significant proportion of the L-dopa dose is metabolized to dopamine in the gut by the enzyme aromatic L-amino acid decarboxylase (AAAD), with only 30% reaching the bloodstream. Hence, L-dopa is generally combined with AAAD inhibitors (AAADIs) such as carbidopa (at an L-dopa/inhibitor dose ratio of 10/1 or 4/1) and benserazide (at an L-dopa/inhibitor dose ratio of 4/1). The use of AAADIs nearly triples the oral bioavailability of L-dopa and markedly reduces both the therapeutic dose required and the severity of dopamine-mediated gastrointestinal and cardiovascular sideeffects [3e5]. Since its introduction in 1960, L-dopa treatment has remained the most effective therapeutic strategy in PD [3]. However, the practical value of chronic oral L-dopa is hampered by interindividual variations in the treatment response (i.e. the absolute level of improvement on L-dopa) [6] and intra-individual variations (i.e. motor fluctuations over time). Given that the major pathway for the synthesis of dopamine from L-dopa in vivo is decarboxylation by AAAD (encoded by the DDC gene) [7], we hypothesized that variations in the response to this drug in PD could be due (at least in part) to polymorphisms in the DDC gene. The possible association between DDC polymorphisms and the response to L-dopa in PD has never previously been investigated. Genetic polymorphisms within DDC have been analyzed for their potential association with diseases in which the mesolimbic dopamine system is altered, such as smoking and bipolar affective disorder [8e11]. We reasoned that DDC polymorphisms associated with diseases of the mesolimbic dopamine system may have also a functional interaction with the nigrostriatal dopamine system and thus the motor response to Ldopa in PD. We chose two fairly frequent sequence variations (rs921451 and rs3837091) from the same region of the DDC gene promoter. In genome-wide linkage studies, the single nucleotide polymorphism (SNP) rs921451 (an intronic nucleotide substitution, T > C) reportedly has a “suggestive linkage” with nicotine dependence [8,9]. The rs3837091 polymorphism (a four-base-pair deletion in the non-translated exon 1 (AGAG del (DDCAGAG/))) is significantly associated with bipolar affective disorders [10,11]. This deletion comprises a GAGA sequence located at the start of a (GA)5 repeat array. In Drosophila, it is known that this type of GA-rich sequence can act as a binding site for the important GAGA transcription factor (GAF). It has further been shown that a reduction in repeat length (from (GA)4 to (GA)2) reduces the relative affinity for GAF [12]. These observations suggest that this 4-bp deletion may have a functional impact on the expression of DDC [11,12]. To the best of our knowledge, there are no published reports of pharmacogenetic modulation of the response to L-dopa. The objective of the present study was to determine the impact of these DDC polymorphisms on the response to an acute L-dopa challenge in PD patients. 2. Patients and methods 2.1. Patients and procedure In the present study, we took advantage of the genotyping work performed as part of a previously described double-blind, placebocontrolled, randomized crossover trial assessing the impact of the COMT polymorphism (rs4608) on the entacapone response [13]. In the latter study, consecutive patients, homozygous for Val158 or Met158 COMT, received two standardized, acute challenges with L-
171
dopa and placebo or entacapone (with a 1-week washout interval). We selected data from patients undergoing a standardized acute challenge with L-dopa and placebo and excluded data from patients receiving entacapone. Male and female PD patients were recruited consecutively in the Department of Neurology at Pitié-Salpetrière Hospital (Paris, France) between September 2006 and June 2009. All patients were over the age of 30 and suffered from motor fluctuations. The exclusion criteria included atypical PD, dementia and treatment with clozapine or monoamine oxidase inhibitors. Investigators and patients were blinded to the genotype data throughout the study. The study (ClinicalTrials #NCT00373087, http://www. clinicaltrials.gov) was carried out in accordance with the Declaration of Helsinki, Good Clinical Practice and French legislation at the Neuroscience Clinical Investigation Center (CIC-9503) at Pitié-Salpetrière Hospital. The study was approved by the French drug agency (Agence nationale de sécurité du médicament et des produits de santé) and the investigational review board at Pitié-Salpetrière Hospital. All patients provided written, informed consent prior to involvement in any other study procedures. L-dopa challenges were performed in the morning in fasting patients, after the withdrawal of L-dopa, dopaminergic agonists and entacapone for at least 18 h, as previously described [13]. The Ldopa dose administered for the challenge was the equivalent morning dose of L-dopa (i.e. first morning dose of L-dopa plus the Ldopa equivalent dose of the first morning dose of dopamine agonist) plus an additional 50 mg of L-dopa. The L-dopa used was immediate-release L-dopa þ 25% benserazide (Roche, Neuilly-SurSeine, France). Patients were allowed to have a light breakfast 20e30 min after dosing. Motor disability on the Unified Parkinson Disease Rating Scale [UPDRS] part III was scored by the same investigator at baseline, 15, 30, 45 and 60 min and every 30 min thereafter up to 4 h after L-dopa administration. In order to determine the plasma pharmacokinetics of L-dopa, blood samples were collected at 0, 20, 40, and 60 min and then every 30 min up to 4 h after L-dopa administration. The samples taken at baseline and 1, 2 and 4 h were also used to determine the plasma pharmacokinetics of dopamine. Our primary objective was to assess the effect of the polymorphisms on the motor response to L-dopa. The pre-specified primary efficacy criterion was the area under the curve (AUC) for the improvement in the motor UPDRS part III (motor) score (DUPDRS) after acute L-dopa administration, relative to baseline (AUCDUPDRS, calculated using a linear trapezoidal method). The pre-specified secondary end-points were (i) the maximum improvement in the UPDRS part III (motor) score, relative to baseline (maximal response: RmaxDUPDRS), and (ii) the time at which RmaxDUPDRS was achieved (TmaxDUPDRS). We also analyzed pharmacokinetic parameters for L-dopa (the AUC for the plasma concentration during the challenge (AUCDOPA), the peak concentration (CmaxDOPA) and the peak time (TmaxDOPA)) and dopamine (the AUC for the plasma concentration, i.e. AUCdopamine). 2.2. Genotyping Genomic DNA was extracted from venous blood samples using standard procedures. The DDC SNP rs921451 was analyzed in an allelic discrimination TaqManÒ, assay running on an ABI PRISM 7900 sequence detection system (Life Technologies Corporation, Carlsbad, USA). Primers were chosen by using OligoExplorer 1.4 software (genelink.com) to take account of the hybridization temperature, the GC content and the primer’s size and location relative to the SNP. The upper and lower primers were 50 -GGGCACCACACACACAC-30 and 50 -CTCTTTGCCTGGTTCTCG-30 , respectively. The rs3837091 polymorphism is characterized by a 4-bp deletion
172
D. Devos et al. / Parkinsonism and Related Disorders 20 (2014) 170e175
Table 1 Baseline characteristics of the patients in each group.
Age (years) Gender ratio Disease duration (years) Hoehn and Yahr scale score (off L-dopa) Baseline UPDRS part III motor score (off L-dopa) Usual daily dose of dopaminergic agonist (mg) Usual L-dopa daily dose (mg/day) Usual total levodopa daily dose equivalent (mg/day) L-dopa dose in the acute challenge (mg) Frequency of COMT val/val (H/H)
DDCCC/CT N ¼ 14
DDCTT N ¼ 19
DDCAGAG/; N¼8
63 13 8/6 11.8 5 2.6 0.7 33 10 201 101 688 252 889 262 228 61 0.5
62 9 15/4 10 4 2.2 0.4 35.6 10 203 133 708 304 911 366 241 56 0.52
63 17 5/3 10 4 2.5 0.6 35.6 5 201 94 717 262 918 260 231 70 0.5
/
DDCAGAG/AGAG N ¼ 25
p values
62 8 18/7 11.6 5 2.4 0.6 33.8 11 217 120 694 289 910 338 234 61 0.5
0.5 (0.6) Ficher exact test: 0.3 (0.6) 0.5 (0.1) 0.2 (0.5) 0.6 (0.9) 0.52 (0.3) 0.87 (0.7) 0.7 (0.5) 0.2 (0.8) Ficher exact test : 1 (1)
Values are quoted as the mean SD. LEDD: L-dopa equivalent daily dose (dopaminergic agonist þ L-dopa). The p values indicate the level of significance of the differences between DDCCC/CT and DDCTT and in brackets between DDCAGAG/ or / and DDCAGAG/AGAG.
(AGAG) and so we sequenced a fragment of the DDC gene (using the Sanger method and after PCR amplification) in a 3730 DNA Analyser (AB Applied Biosystems). We then used polyacrylamide gel electrophoresis to size-select fragments and thus identify DDC/, DDCAGAG/, DDCAGAG/AGAG genotypes. For each patient, two controls for each genotype were analyzed concomitantly. 2.3. Plasma concentrations of L-dopa and dopamine Plasma levels of L-dopa were measured as previously described [14]. Briefly, after addition of dihydroxybenzylamine as an internal standard and protein precipitation with perchloric acid, L-dopa was measured in the supernatant by High-Performance Liquid Chromatography (HPLC) coupled with electrochemical detection at 0.82 V relative to an Ag/AgCl reference electrode (Coulochem III, ThermoFisher, Thermo Scientific, Sunnyvale, USA). The lower limit of quantitation was 50 ng/ml, with a relative standard deviation of
Contents lists available at ScienceDirect
Parkinsonism and Related Disorders journal homepage: www.elsevier.com/locate/parkreldis
Dopa-decarboxylase gene polymorphisms affect the motor response to L-dopa in Parkinson’s disease David Devos a, b, *, Stéphanie Lejeune a, c, Florence Cormier-Dequaire a, c, Khadija Tahiri c, Fanny Charbonnier-Beaupel c, d, e, Nathalie Rouaix f, Alain Duhamel f, Bernard Sablonnière f, Anne-Marie Bonnet g, Cecilia Bonnet a, Noel Zahr e, Jean Costentin h, Marie Vidailhet a, c, g, Jean-Christophe Corvol a, c, g a
INSERM (French National Institute of Medical Research and Health), APHP (Assistance Publique Hopitaux de Paris), Clinical Investigation Center (CIC9503), Pitié-Salpêtrière Hospital, Paris, France b Lille Nord de France University, Department of Medical Pharmacology, Lille University Medical Center, Faculty of Medicine of Lille 2, EA 4610, France c INSERM, UMRS_975 unit, UPMC (Pierre and Marie Curie University), CNRS UMR7525 CR-ICM, Paris, France d APHP (Assistance Publique Hopitaux de Paris), Pitié-Salpêtrière Hospital, Department of Pharmacy, France e APHP, Pitié-Salpêtrière Hospital, Department of Pharmacology, Paris, France f Lille Nord de France University, Department of Molecular Biology, Lille University Medical Center, France g APHP, Pitié-Salpêtrière Hospital, Department of Neurology, France h University of Rouen, Neuropharmacology Laboratory, Rouen, France
a r t i c l e i n f o
a b s t r a c t
Article history: Received 31 July 2013 Received in revised form 5 October 2013 Accepted 15 October 2013
Background: In Parkinson’s disease (PD), the response to L-dopa is highly variable and unpredictable. The major pathway for dopamine synthesis from L-dopa is decarboxylation by aromatic L-amino acid decarboxylase (AAAD, encoded by the DDC gene). Objective: To determine the motor response to L-dopa in PD patients as a function of the DDC gene promoter polymorphisms (rs921451 T > C polymorphism (DDCT/C) and rs3837091 AGAG del (DDCAGAG/ )). Methods: Thirty-three Caucasian PD patients underwent an acute L-dopa challenge together with the peripheral AAAD inhibitor benserazide and were genotyped for rs921451 and rs3837091. The primary efficacy criterion was the motor response to L-dopa, as estimated by the area under the curve for the change in the Unified Parkinson’s Disease Rating Scale part III (UPDRS) score relative to baseline (AUCDUPDRS) in the 4 h following L-dopa administration. Secondary endpoints were pharmacokinetic parameters for plasma levels of L-dopa and dopamine. Investigators and patients were blinded to genotypes data throughout the study. Results: When adjusted for the L-dopa dose, the AUCDUPDRS was significantly lower in DDCCC/CT patients (n ¼ 14) than in DDCTT patients (n ¼ 19) and significantly lower in DDC/ or AGAG/ patients (n ¼ 8) than in DDCAGAG/AGAG patients (n ¼ 25). There were no significant intergroup differences in plasma pharmacokinetic parameters for L-dopa and dopamine. Discussion: The rs921451 and rs3837091 polymorphisms of the DDC gene promoter influence the motor response to L-dopa but do not significantly change peripheral pharmacokinetic parameters for L-dopa and dopamine. Our results suggest that DDC may be a genetic modifier of the L-dopa response in Parkinson’s disease. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Parkinson’s disease Dopamine L-amino acid decarboxylase Genetic L-dopa
1. Introduction
* Corresponding author. Département de Pharmacologie Médicale, Université Lille Nord de France, CHRU de Lille, F-59037 Lille, France. E-mail address: [email protected] (D. Devos). 1353-8020/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.parkreldis.2013.10.017
Parkinson’s disease (PD) is the second most frequent neurodegenerative disorder worldwide and affects more than 1% of the population over the age of 60 [1]. The loss of dopamine production in the striatum (as a result of progressive neuronal degeneration in the substantia nigra pars compacta) is the primary chemical disease
D. Devos et al. / Parkinsonism and Related Disorders 20 (2014) 170e175
marker for PD. Given that dopamine deficiency is the main cause of symptoms in PD, pharmacological treatment of this disease has focused on the restoration of dopaminergic transmission within the nigrostriatal pathway [2]. Since dopamine does not cross the digestive mucosa or the blood brain barrier, its precursor L-dopa has been developed as an orally administered medication. It is known that a significant proportion of the L-dopa dose is metabolized to dopamine in the gut by the enzyme aromatic L-amino acid decarboxylase (AAAD), with only 30% reaching the bloodstream. Hence, L-dopa is generally combined with AAAD inhibitors (AAADIs) such as carbidopa (at an L-dopa/inhibitor dose ratio of 10/1 or 4/1) and benserazide (at an L-dopa/inhibitor dose ratio of 4/1). The use of AAADIs nearly triples the oral bioavailability of L-dopa and markedly reduces both the therapeutic dose required and the severity of dopamine-mediated gastrointestinal and cardiovascular sideeffects [3e5]. Since its introduction in 1960, L-dopa treatment has remained the most effective therapeutic strategy in PD [3]. However, the practical value of chronic oral L-dopa is hampered by interindividual variations in the treatment response (i.e. the absolute level of improvement on L-dopa) [6] and intra-individual variations (i.e. motor fluctuations over time). Given that the major pathway for the synthesis of dopamine from L-dopa in vivo is decarboxylation by AAAD (encoded by the DDC gene) [7], we hypothesized that variations in the response to this drug in PD could be due (at least in part) to polymorphisms in the DDC gene. The possible association between DDC polymorphisms and the response to L-dopa in PD has never previously been investigated. Genetic polymorphisms within DDC have been analyzed for their potential association with diseases in which the mesolimbic dopamine system is altered, such as smoking and bipolar affective disorder [8e11]. We reasoned that DDC polymorphisms associated with diseases of the mesolimbic dopamine system may have also a functional interaction with the nigrostriatal dopamine system and thus the motor response to Ldopa in PD. We chose two fairly frequent sequence variations (rs921451 and rs3837091) from the same region of the DDC gene promoter. In genome-wide linkage studies, the single nucleotide polymorphism (SNP) rs921451 (an intronic nucleotide substitution, T > C) reportedly has a “suggestive linkage” with nicotine dependence [8,9]. The rs3837091 polymorphism (a four-base-pair deletion in the non-translated exon 1 (AGAG del (DDCAGAG/))) is significantly associated with bipolar affective disorders [10,11]. This deletion comprises a GAGA sequence located at the start of a (GA)5 repeat array. In Drosophila, it is known that this type of GA-rich sequence can act as a binding site for the important GAGA transcription factor (GAF). It has further been shown that a reduction in repeat length (from (GA)4 to (GA)2) reduces the relative affinity for GAF [12]. These observations suggest that this 4-bp deletion may have a functional impact on the expression of DDC [11,12]. To the best of our knowledge, there are no published reports of pharmacogenetic modulation of the response to L-dopa. The objective of the present study was to determine the impact of these DDC polymorphisms on the response to an acute L-dopa challenge in PD patients. 2. Patients and methods 2.1. Patients and procedure In the present study, we took advantage of the genotyping work performed as part of a previously described double-blind, placebocontrolled, randomized crossover trial assessing the impact of the COMT polymorphism (rs4608) on the entacapone response [13]. In the latter study, consecutive patients, homozygous for Val158 or Met158 COMT, received two standardized, acute challenges with L-
171
dopa and placebo or entacapone (with a 1-week washout interval). We selected data from patients undergoing a standardized acute challenge with L-dopa and placebo and excluded data from patients receiving entacapone. Male and female PD patients were recruited consecutively in the Department of Neurology at Pitié-Salpetrière Hospital (Paris, France) between September 2006 and June 2009. All patients were over the age of 30 and suffered from motor fluctuations. The exclusion criteria included atypical PD, dementia and treatment with clozapine or monoamine oxidase inhibitors. Investigators and patients were blinded to the genotype data throughout the study. The study (ClinicalTrials #NCT00373087, http://www. clinicaltrials.gov) was carried out in accordance with the Declaration of Helsinki, Good Clinical Practice and French legislation at the Neuroscience Clinical Investigation Center (CIC-9503) at Pitié-Salpetrière Hospital. The study was approved by the French drug agency (Agence nationale de sécurité du médicament et des produits de santé) and the investigational review board at Pitié-Salpetrière Hospital. All patients provided written, informed consent prior to involvement in any other study procedures. L-dopa challenges were performed in the morning in fasting patients, after the withdrawal of L-dopa, dopaminergic agonists and entacapone for at least 18 h, as previously described [13]. The Ldopa dose administered for the challenge was the equivalent morning dose of L-dopa (i.e. first morning dose of L-dopa plus the Ldopa equivalent dose of the first morning dose of dopamine agonist) plus an additional 50 mg of L-dopa. The L-dopa used was immediate-release L-dopa þ 25% benserazide (Roche, Neuilly-SurSeine, France). Patients were allowed to have a light breakfast 20e30 min after dosing. Motor disability on the Unified Parkinson Disease Rating Scale [UPDRS] part III was scored by the same investigator at baseline, 15, 30, 45 and 60 min and every 30 min thereafter up to 4 h after L-dopa administration. In order to determine the plasma pharmacokinetics of L-dopa, blood samples were collected at 0, 20, 40, and 60 min and then every 30 min up to 4 h after L-dopa administration. The samples taken at baseline and 1, 2 and 4 h were also used to determine the plasma pharmacokinetics of dopamine. Our primary objective was to assess the effect of the polymorphisms on the motor response to L-dopa. The pre-specified primary efficacy criterion was the area under the curve (AUC) for the improvement in the motor UPDRS part III (motor) score (DUPDRS) after acute L-dopa administration, relative to baseline (AUCDUPDRS, calculated using a linear trapezoidal method). The pre-specified secondary end-points were (i) the maximum improvement in the UPDRS part III (motor) score, relative to baseline (maximal response: RmaxDUPDRS), and (ii) the time at which RmaxDUPDRS was achieved (TmaxDUPDRS). We also analyzed pharmacokinetic parameters for L-dopa (the AUC for the plasma concentration during the challenge (AUCDOPA), the peak concentration (CmaxDOPA) and the peak time (TmaxDOPA)) and dopamine (the AUC for the plasma concentration, i.e. AUCdopamine). 2.2. Genotyping Genomic DNA was extracted from venous blood samples using standard procedures. The DDC SNP rs921451 was analyzed in an allelic discrimination TaqManÒ, assay running on an ABI PRISM 7900 sequence detection system (Life Technologies Corporation, Carlsbad, USA). Primers were chosen by using OligoExplorer 1.4 software (genelink.com) to take account of the hybridization temperature, the GC content and the primer’s size and location relative to the SNP. The upper and lower primers were 50 -GGGCACCACACACACAC-30 and 50 -CTCTTTGCCTGGTTCTCG-30 , respectively. The rs3837091 polymorphism is characterized by a 4-bp deletion
172
D. Devos et al. / Parkinsonism and Related Disorders 20 (2014) 170e175
Table 1 Baseline characteristics of the patients in each group.
Age (years) Gender ratio Disease duration (years) Hoehn and Yahr scale score (off L-dopa) Baseline UPDRS part III motor score (off L-dopa) Usual daily dose of dopaminergic agonist (mg) Usual L-dopa daily dose (mg/day) Usual total levodopa daily dose equivalent (mg/day) L-dopa dose in the acute challenge (mg) Frequency of COMT val/val (H/H)
DDCCC/CT N ¼ 14
DDCTT N ¼ 19
DDCAGAG/; N¼8
63 13 8/6 11.8 5 2.6 0.7 33 10 201 101 688 252 889 262 228 61 0.5
62 9 15/4 10 4 2.2 0.4 35.6 10 203 133 708 304 911 366 241 56 0.52
63 17 5/3 10 4 2.5 0.6 35.6 5 201 94 717 262 918 260 231 70 0.5
/
DDCAGAG/AGAG N ¼ 25
p values
62 8 18/7 11.6 5 2.4 0.6 33.8 11 217 120 694 289 910 338 234 61 0.5
0.5 (0.6) Ficher exact test: 0.3 (0.6) 0.5 (0.1) 0.2 (0.5) 0.6 (0.9) 0.52 (0.3) 0.87 (0.7) 0.7 (0.5) 0.2 (0.8) Ficher exact test : 1 (1)
Values are quoted as the mean SD. LEDD: L-dopa equivalent daily dose (dopaminergic agonist þ L-dopa). The p values indicate the level of significance of the differences between DDCCC/CT and DDCTT and in brackets between DDCAGAG/ or / and DDCAGAG/AGAG.
(AGAG) and so we sequenced a fragment of the DDC gene (using the Sanger method and after PCR amplification) in a 3730 DNA Analyser (AB Applied Biosystems). We then used polyacrylamide gel electrophoresis to size-select fragments and thus identify DDC/, DDCAGAG/, DDCAGAG/AGAG genotypes. For each patient, two controls for each genotype were analyzed concomitantly. 2.3. Plasma concentrations of L-dopa and dopamine Plasma levels of L-dopa were measured as previously described [14]. Briefly, after addition of dihydroxybenzylamine as an internal standard and protein precipitation with perchloric acid, L-dopa was measured in the supernatant by High-Performance Liquid Chromatography (HPLC) coupled with electrochemical detection at 0.82 V relative to an Ag/AgCl reference electrode (Coulochem III, ThermoFisher, Thermo Scientific, Sunnyvale, USA). The lower limit of quantitation was 50 ng/ml, with a relative standard deviation of
