Abstract: Results from pharmacogenetic investigations of methylphenidate (MPH) response in patients with ADHD are still inconsistent, especially among adults. This study investigates the role of genetic variants (SLC6A4, HTR1B, TPH2, DBH, DRD4, COMT, and SNAP25) in the response to MPH in a sample of 164 adults. Genes were chosen owing to previous evidence for an influence in ADHD susceptibility. No significant differences in allele or genotype frequencies between MPH responders and nonresponders were detected. In conclusion, our findings do not support an effect of these genes in the pharmacogenetics of MPH among adults with ADHD.
From the *Department of Genetics, Instituto de Biociências, Universidade Federal do Rio Grande do Sul; †Adult ADHD Outpatient Clinic, Hospital de Clínicas de Porto Alegre; and ‡Department of Psychiatry, Faculdade de Medicina, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil.
Received August 25, 2011; accepted after revision April 4, 2012.
Reprints: Claiton H.D. Bau, MD, PhD, Departamento de Genética, Instituto de Biociências, UFRGS, Caixa Postal 15053, CEP 91501-970, Porto Alegre, RS, Brazil (e-mail: firstname.lastname@example.org).
CNPq–Instituto do Milênio, FIPE-HCPA, FAPERGS, DECIT/SCTIE/MS/PPSUS, and PRONEX funded this study.
Attention-deficit/hyperactivity disorder (ADHD) is a complex and highly heritable neuropsychiatric disorder that affects children and adults worldwide. It is characterized by symptoms of inattention, impulsivity, and hyperactivity to a degree that is inconsistent with normal developmental level.1
There is abundant evidence that the treatment with the psychostimulant methylphenidate (MPH) is frequently efficacious in attenuating ADHD symptoms in children and adult patients. Nevertheless, approximately 30% of the patients do not show a satisfactory clinical response to the treatment.2,3
Pharmacogenetic investigations of MPH response in patients with ADHD have focused mainly in genes of the catecholamine pathway.4–6 In children samples, there are more than 30 studies; and some genetic variants have been significantly associated with the response to treatment.6 In adults, however, there are only 4 published studies that have investigated the dopamine transporter (DAT1/SLC6A3),7–9 the dopamine D4 receptor (DRD4)8, the norepinephrine transporter (NET1/SLC6A2),8 and the α-2A-adrenergic receptor (ADRA2A) genes.10 So far, only DAT1 was associated with the response to MPH in a sample of 42 adults.8
Results from pharmacogenetic studies are still not conclusive, especially for adults. Therefore, additional investigations are required. In this context, the objective of our study was to investigate the role of genetic variants in response to MPH in a sample of adults with ADHD. We selected 11 polymorphisms in 7 genes that have already been investigated in children samples: serotonin transporter (5HTT/SLC6A4) and receptor 1B (HTR1B), tryptophan hydroxylase-2 (TPH2), dopamine β-hydroxylase (DBH), catechol-O-methyltransferase (COMT), synaptosomal-associated protein 25 (SNAP25), and DRD4. All these genes present significant evidence for association with ADHD susceptibility or significant heterogeneity in effect size across studies in a meta-analytic review. Therefore, they are strong candidate genes for a pharmacogenetic study.11
MATERIALS AND METHODS
The sample is composed of 164 adults with ADHD from the ADHD Outpatient Program at the Hospital de Clinicas de Porto Alegre, RS, Brazil. The inclusion criteria were the following: (a) Native-Brazilian of European descent; (b) age 18 years or older; (c) fulfillment of Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition diagnostic criteria for ADHD (American Psychiatric Association, 1994), both currently and during childhood; and (d) eligibility to immediate-release methylphenidate (IR-MPH) treatment. Exclusion criteria were the presence of (a) clinical contraindication to IR-MPH; (b) any significant neurological disease (eg, delirium, dementia, epilepsy, head trauma, and multiple sclerosis); (c) current or history of psychosis; (d) intelligence quotient (IQ) of less than 70; and (e) current unstabilized comorbid disorders. Less than 20% of the patients had used MPH in their lifetime, but none of them was currently using this medication when included in the study.
The project was carried out in accordance with the Declaration of Helsinki and was approved by the institutional review board (IRB) of the hospital (IRB No. 00000921). All patients signed an informed consent.
The diagnostic procedures for ADHD and comorbidities in our unit have been described elsewhere.12–15
Pharmacological Intervention and Drug Response
This protocol is part of a larger study on predictors of MPH treatment response, including phenotypic characteristics16 and pharmacogenetic studies.7,10 A detailed description of this pharmacogenetic protocol is in Contini et al.10 Briefly, patients were treated with weekly increases in IR-MPH dose until symptom control or occurrence of limiting adverse effects. All patients took at least the minimum MPH dose of 0.3 mg/kg per day. The outcome measures of MPH treatment were the Portuguese version of the Swanson, Nolan and Pelham Rating Scale version IV (SNAP-IV)17 and the Clinical Global Impression—Severity scale,18 applied at the beginning of the treatment (baseline levels) and after the 30th day of treatment (end point).
Drug response was assessed by both categorical and dimensional approaches. The a priori categorical definition of response was a 30% or greater symptom reduction in SNAP-IV and a Clinical Global Impression—Severity score of 2 points or less. The dimensional evaluation of drug-response was measured by the variation in SNAP-IV scores (SNAP-IV baseline to SNAP-IV end-point scores).
MATERIALS AND METHODS
DNA was extracted from whole blood by an adaptation of Lahiri and Nurnberger.19 The polymorphisms in DRD4 (variable number of tandem repeats, exon 3), SLC6A4 (5-HTTLPR), DBH (rs1611115), and HTR1B (rs6296) genes were amplified using the polymerase chain reaction (PCR) conditions adapted from Roman et al,20 Grevet et al,21 Kohnke et al,22 and Guimarães et al,23 respectively. The remaining polymorphisms (HTR1B-rs11568817, HTR1B-rs13212041, TPH2-rs1843809, TPH2-rs4570625, COMT-rs4680, SNAP25-rs3746544, and SNAP25-rs363020) were genotyped using the Taqman SNP genotyping assays (Applied Biosystems), according to the manufacturer’s recommended protocol.
The association between specific alleles or genotypes with the categorical response to MPH treatment was analyzed by logistic regression analyses. Genetic effects in the dimensional variation in SNAP-IV scores after the MPH treatment were analyzed by analysis of covariance, considering baseline scores as covariates. Potential confounders (demographic characteristics, IQ, ADHD subtype, comorbidities, use of concomitant medication, and MPH dose) were included as covariates using a statistical definition (association with both the study factor and outcome for a P ≤ 0.20).24 All analyses were conducted using SPSS version 18.0 software (SPSS Inc, Chicago, Ill).
Subjects were 164 patients comprising 89 men and 75 women. The mean (SD) estimated full IQ of the sample was 101.6 (9.5). Mean (SD) baseline scores for the overall symptoms of ADHD according to SNAP-IV was 1.71 (0.52). The mean (SD) MPH dose was 0.15 (0.06) mg/kg per day at baseline and 0.52 (0.20) mg/kg per day at end point.
According to our a priori categorical definition of MPH treatment response, 136 patients (83%) were classified as responders. Twenty-eight participants (17%) failed to show a clinical response to the treatment.
The clinical characteristics that differ between the responders and the nonresponders at P ≤ 0.20 are given in Table 1. In the remaining characteristics, IQ scores, ADHD subtype, lifetime comorbidities (bipolar disorders, generalized anxiety disorder, oppositional defiant disorder, and nicotine use), SNAP-IV baseline scores, concomitant use of medication, and MPH dose, the groups did not differ (all P > 0.20).
The estimated allele frequencies for the polymorphisms in our sample are in Table 2. The genotype distribution in all polymorphisms are in Hardy-Weinberg equilibrium (all P > 0.10). Considering the fact that some cell sizes were very small after stratification according to genotype and treatment response, the statistical analysis for MPH response was performed between carriers versus noncarriers of the risk allele. The specific allele or genotype tested was defined based on previous ADHD pharmacogenetic studies or by the frequency of the rare allele. The risk allele for the polymorphisms in the DRD4 (variable number of tandem repeats, exon 3), SL6A4 (5-HTTLPR), and HTR1B (rs6296) genes were based on the study conducted by Zeni et al25 in Brazilian children with ADHD. For the COMT (rs4680) and SNAP25 (rs3746544) genes, the risk alleles were defined as in Salatino-Oliveira et al26 and McGough et al,27 respectively. In the remaining polymorphisms (rs363020, rs1845809, rs4570625, rs1611115, rs11568817, and rs13212041), carriers ofthe rare alleles were pooled with the heterozygous subjects.
There were no significant differences in allele or genotype frequencies between MPH responders and nonresponders in any polymorphisms (Table 2). Likewise, there are no significant effects of the polymorphisms on the response to MPH evaluated through the variation between pretreatment and posttreatment SNAP-IV scores (data not shown, detailed results for all polymorphisms are available upon request).
This study suggests that a group of genes with strong evidence for a role in childhood ADHD is not associated with response to MPH in adults with ADHD. Except for DBH, all genes that we studied had already been investigated in ADHD pharmacogenetics. However, it is important to note that almost all studies were conducted in children samples.
Considering the genes included in this study, the overall results from previous MPH pharmacogenetic studies in children are not conclusive. Even for the genes for which there are reports of both effects and noneffects on the MPH response (DRD4, COMT, and SNAP25), more studies are needed to dissect and confirm findings. On the other hand, the results regarding the serotonergic genes are more consistent, excluding an important role of this neurotransmitter system in the response to MPH treatment in children.4–6 Considering the noradrenergic system, some investigations have pointed for a significant role of the SLC6A2 gene in the response to MPH in children.6,28 In adults, however, this particular gene seems to be not relevant.8
The interpretation of our findings should consider the naturalistic design of this study and absence of a placebo group. The rate of improvement of ADHD symptoms, however, was similar to those generally found in placebo-controlled studies. A second point to ponder is the average final MPH dose of 0.52 mg/kg per day, which is sometimes considered low. Although there is evidence that it may be effective,29 and our own data show a sharp symptom decrease, we cannot rule out the possibility that some patients could require more robust dosing of MPH to attain an adequate clinical response. The lack of a strictly standardized medication titration should also be considered. However, the fact that all patients were treated in a comparable manner by the same experienced psychiatrist trained in our protocol minimizes, to some extent, the limitations of the approach. Another issue is the risk of a type II error. Recoding genotypes in only 2 categories was necessary considering the limited sample size available. However, this approach may reduce the power of the analysis, when it introduces the risk or assuming an incorrect mode of transmission. The fact that our sample size is one of the largest presented in pharmacogenetic investigations of ADHD, especially among adults, shows the need for further research with bigger sample sizes. Finally, sex differences in the sample composition of children and adults might influence pharmacogenetic studies and turn inconsistent the results from different age groups.
In conclusion, our findings fail to support a significant role of 7 relevant genes in the clinical response to MPH treatment in ADHD, at least among Brazilian adults. Together with our previous findings for the DAT17 and ADRA2A10 genes, we suggest that the classical candidate genes in ADHD susceptibility do not show relevant effects on MPH response in this Brazilian sample.
The authors thank Rafael G. Karam, Katiane Silva, Paula O. G. da Silva, and Eduardo Vitola for help in the sample collection of ADHD patients.
AUTHOR DISCLOSURE INFORMATION
The ADHD Program received unrestricted educational and research support from the following pharmaceutical companies in the last 3 years: Abbott, Bristol-Myers Squibb, Eli Lilly, Janssen-Cilag, Novartis, and Shire. Dr Belmonte-de-Abreu is on the speakers’ bureau or is a consultant for Janssen-Cilag and Bristol-Myers Squibb. Dr Rohde was on the speakers’ bureau and/or acted as consultant for Eli Lilly, Janssen-Cilag, Novartis, and Shire in the last 3 years (less than U$10,000 per year and reflecting less than 5% of his gross income per year). He also receives travel awards (air tickets + hotel) for taking part in psychiatric meetings from Novartis and Janssen-Cilag.
1. Purper-Ouakil D, Ramoz N, Lepagnol-Bestel AM, et al.. Neurobiology of attention deficit/hyperactivity disorder. Pediatr Res
. 2011; 69: 69R–76R.
2. Heal DJ, Cheetham SC, Smith SL. The neuropharmacology of ADHD drugs in vivo: insights on efficacy and safety. Neuropharmacology
. 2009; 57: 608–618.
3. Wilens TE. Effects of methylphenidate on the catecholaminergic system in attention-deficit/hyperactivity disorder. J Clin Psychopharmacol
. 2008; 28: S46–S53.
4. Froehlich TE, McGough JJ, Stein MA. Progress and promise of attention-deficit hyperactivity disorder pharmacogenetics. CNS Drugs
. 2010; 24: 99–117.
5. Kieling C, Genro JP, Hutz MH, et al.. A current update on ADHD pharmacogenomics. Pharmacogenomics
. 2010; 11: 407–419.
6. Polanczyk G, Bigarella MP, Hutz MH, et al.. Pharmacogenetic approach for a better drug treatment in children. Curr Pharm Des
. 2010; 16: 2462–2473.
7. Contini V, Victor MM, Marques FZ, et al.. Response to methylphenidate is not influenced by DAT1 polymorphisms in a sample of Brazilian adult patients with ADHD. J Neural Transm
. 2010; 117: 269–276.
8. Kooij JS, Boonstra AM, Vermeulen SH, et al.. Response to methylphenidate in adults with ADHD is associated with a polymorphism in SLC6A3 (DAT1). Am J Med Genet B Neuropsychiatr Genet
. 2008; 147B: 201–208.
9. Mick E, Biederman J, Spencer T, et al.. Absence of association with DAT1 polymorphism and response to methylphenidate in a sample of adults with ADHD. Am J Med Genet B Neuropsychiatr Genet
. 2006; 141B: 890–894.
10. Contini V, Victor MM, Cerqueira CC, et al.. Adrenergic alpha2A receptor gene is not associated with methylphenidate response in adults with ADHD. Eur Arch Psychiatry Clin Neurosci
. 2011; 261: 205–211.
11. Gizer IR, Ficks C, Waldman ID. Candidate gene studies of ADHD: a meta-analytic review. Hum Genet
. 2009; 126: 51–90.
12. Fischer AG, Bau CH, Grevet EH, et al.. The role of comorbid major depressive disorder in the clinical presentation of adult ADHD. J Psychiatr Res
. 2007; 41: 991–996.
13. Grevet EH, Bau CH, Salgado CA, et al.. [Interrater reliability for diagnosis in adults of attention deficit hyperactivity disorder and oppositional defiant disorder using K-SADS-E]. Arq Neuropsiquiatr
. 2005; 63: 307–310.
14. Kalil KL, Bau CH, Grevet EH, et al.. Smoking is associated with lower performance in WAIS-R Block Design scores in adults with ADHD. Nicotine Tob Res
. 2008; 10: 683–688.
15. Karam RG, Bau CH, Salgado CA, et al.. Late-onset ADHD in adults: milder, but still dysfunctional. J Psychiatr Res
. 2009; 43: 697–701.
16. Victor MM, Grevet EH, Salgado CA, et al.. Reasons for pretreatment attrition and dropout from methylphenidate in adults with attention-deficit/hyperactivity disorder: the role of comorbidities. J Clin Psychopharmacol
. 2009; 29: 614–616.
17. Swanson JM. School-based Assessment and Interventions for ADD Students
. Irvine, CA: KC Publishing; 1992.
18. Guy W. ECDEU Assessment Manual for Psychopharmacology, Revised ed
. Washington, DC: US Department of Health Education and Welfare; 1976.
19. Lahiri DK, Nurnberger JI Jr. A rapid non-enzymatic method for the preparation of HMW DNA from blood for RFLP studies. Nucleic Acids Res
. 1991; 19: 5444.
20. Roman T, Bau CH, Almeida S, et al.. Lack of association of the dopamine D4 receptor gene polymorphism with alcoholism in a Brazilian population. Addict Biol
. 1999; 4: 203–207.
21. Grevet EH, Marques FZ, Salgado CA, et al.. Serotonin transporter gene polymorphism and the phenotypic heterogeneity of adult ADHD. J Neural Transm
. 2007; 114: 1631–1636.
22. Kohnke MD, Zabetian CP, Anderson GM, et al.. A genotype-controlled analysis of plasma dopamine beta-hydroxylase in healthy and alcoholic subjects: evidence for alcohol-related differences in noradrenergic function. Biol Psychiatry
. 2002; 52: 1151–1158.
23. Guimaraes AP, Schmitz M, Polanczyk GV, et al.. Further evidence for the association between attention deficit/hyperactivity disorder and the serotonin receptor 1B gene. J Neural Transm
. 2009; 116: 1675–1680.
24. Maldonado G, Greenland S. Simulation study of confounder-selection strategies. Am J Epidemiol
. 1993; 138: 923–936.
25. Zeni CP, Guimaraes AP, Polanczyk GV, et al.. No significant association between response to methylphenidate and genes of the dopaminergic and serotonergic systems in a sample of Brazilian children with attention-deficit/hyperactivity disorder. Am J Med Genet B Neuropsychiatr Genet
. 2007; 144B: 391–394.
26. Salatino-Oliveira A, Genro JP, Zeni C, et al.. Catechol-O
-methyltransferase valine158methionine polymorphism moderates methylphenidate effects on oppositional symptoms in boys with attention-deficit/hyperactivity disorder. Biol Psychiatry
. 2011; 70: 216–221.
27. McGough J, McCracken J, Swanson J, et al.. Pharmacogenetics of methylphenidate response in preschoolers with ADHD. J Am Acad Child Adolesc Psychiatry
. 2006; 45: 1314–1322.
28. Song J, Song DH, Jhung K, et al.. Norepinephrine transporter gene (SLC6A2) is involved with methylphenidate response in Korean children with attention deficit hyperactivity disorder. Int Clin Psychopharmacol
. 2011; 26: 107–113.
29. Rosler M, Fischer R, Ammer R, et al.. A randomised, placebo-controlled, 24-week, study of low-dose extended-release methylphenidate in adults with attention-deficit/hyperactivity disorder. Eur Arch Psychiatry Clin Neurosci
. 2009; 259: 120–129.