DEPRESSION

AND

ANXIETY 0 : 16 (2011)

Research Article
ROLE OF COMT, 5-HT1A, AND SERT GENETIC POLYMORPHISMS ON ANTIDEPRESSANT RESPONSE TO TRANSCRANIAL MAGNETIC STIMULATION
Malaguti Alessia, M.D., Rossini David, M.D., Lucca Adelio, M.D., Magri Lorenzo, PSy.D., Lorenzi Cristina, Ph.D., Pirovano Adele, Ph.D., Colombo Cristina, Smeraldi Enrico, M.D., and Zanardi Raffaella, M.D.

Background: Transcranial Magnetic Stimulation (TMS) is an effective technique in the treatment of depression, specifically in drug-resistant patients. However, there is little data available on the influence of genetic variables on TMS response. Methods: We analyzed the role of three genetic polymorphisms that affected the antidepressant response: serotonin transporter promoter region (SERTPR) polymorphism, 5-HT1A serotonergic receptor promoter region polymorphism (rs6295), and the coding region of COMT gene polymorphism (rs4680). Ninety patients with a major depressive drug-resistant episode due to a Major Depressive Disorder or to a Bipolar Disorder were included in our study. Patients underwent high frequency TMS, focused on the left prefrontal cortex, for 2 weeks. At study completion, the response rate was 45.5%. Effects of gene polymorphisms on clinical improvement were analyzed with an analysis of variance with each gene (SERTPR, 5-HT1A, and COMT) as factors and the Hamilton Rating Scale for Depression variation from baseline to the end of the treatment as a dependent variable. Results: We found a significant model in which three factors were not significant (diagnosis, COMT, and SERTPR), whereas factor 5-HT1A showed a significant influence on the outcome, with patients with C/C genotype showing a greater improvement than G/G and C/G and no difference between G/G and C/G. Conclusion: According to our data, 5-HT1A polymorphism may play a role in influencing TMS response. The effect of COMT and SERTPR did not reach statistical significance. The analysis of these and other candidate genes in larger samples could help explain genetic influence on TMS response. Depression and r 2011 Wiley-Liss, Inc. Anxiety 0:16, 2011. Key words: TMS; genetic; depression; COMT, 5-HT1A; serotonin transporter

ranscranial Magnetic Stimulation (TMS) is a noninvasive method to stimulate brain cortex studied for the treatment of neurological and psychiatric diseases. In particular, treatment-resistant major depression is the disorder in which TMS has most extensively been applied, mainly as an add-on therapy.[1,2] The vast majority of research applied trains of high frequency over the left dorsolateral prefrontal cortex (DLPFC), in an attempt to enhance neuronal activity of a cortical area that may be involved in the pathophysiology of depression.[3,4] In order to explain the variability of response in depressed patients, different clinical and demographic variables have been evaluated.[5,6] However, few data are available so far concerning
r 2011 Wiley-Liss, Inc.

INTRODUCTION

the influence of genetic variables on TMS clinical response.[79]

San Raffaele Scientific Institute, Vita-Salute San Raffaele University, Clinical Neurosciences Department, Milan, Italy
Correspondence to: Zanardi Raffaella, Clinical Neurosciences

Department, San Raffaele Scientific Institute, Vita-Salute San Raffaele University, Via Stamira dAncona, 20 Milan 20127 Italy. E-mail: zanardi.raffaella@hsr.it The authors report they have no financial relationships within the past 3 years to disclose. Received for publication 15 November 2010; Revised 23 February 2011; Accepted 26 February 2011 DOI 10.1002/da.20815 Published online in Wiley Online Library (wileyonlinelibrary.com).

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Many studies have investigated the mechanism underlying the therapeutic effects of TMS, focusing on serotonergic and dopaminergic systems. With respect to the serotonergic system, animal studies have shown a modulatory effect of prefrontal TMS on serotonergic transmission. After TMS, it has been observed that 5-HT increased in the hippocampus and amygdala,[10] 5-HT1A binding sites increased in amygdala and frontal cortex,[11] 5-HT2A receptors were downregulated in striatum and frontal cortex,[12] 5-HT1A and 5-HT1B presynaptic receptors decreased,[13] and mRNA for the serotonin transports lowered in the brain stem, cerebellum, and cerebrum.[14] Moreover, a study on healthy volunteers conducted with the (11)C-Mtrp/PET method found the acute TMS of the left DLPFC modulates aspects of tryptophan/5-HT metabolism in limbic areas.[15] For these reasons, in a previous article, we analyzed the polymorphism of serotonergic genes as possible predictors of TMS response.[7] In particular, we investigated the polymorphism of the serotonin transporter promoter region (SERTPR)[16] and of the 5-HT1A serotonergic receptor promoter region (rs6295).[17] Considering SERTPR, we observed an influence of the genotype on the response independently from the type of stimulation (active versus sham). Specifically, patients with the l allele showed a better amelioration than s/s patients. Considering 5-HT1A, we found that the subjects genotype differentially affects response depending on the type of stimulation, active and sham. In particular, C/C patients showed a significantly larger difference between active and sham stimulation. With respect to the dopaminergic system, animal studies have shown TMS influence on dopamine transmission. In rats, an increased extracellular concentration of dopamine in the dorsolateral hippocampus, nucleus accumbens septi, and dorsal striatum after acute TMS (20 Hz) of frontal brain regions has been shown.[18] Similar effects in the mesostriatal and mesolimbic dopaminergic system have been reported by other groups.[19] In humans, prefrontal cortex TMS has been demonstrated to induce the release of endogenous dopamine in the ipsilateral caudate nucleus.[20] In depressed patients, an acute 10 Hz TMS over the left DLPFC has been observed to induce a release of endogenous striatal dopamine.[21] More recently, Cho and Strafella have observed dopaminergic changes in the ipsilateral anterior cingulated cortex and medial orbitofrontal cortex following acute left 10 Hz TMS of the DLPFC.[22] Therefore, another site of interest is the functional polymorphism of the coding gene of the catechol-Omethyltransferase (COMT) enzyme (rs4680),[23] that inactivates catecholamines, such as dopamine. Many studies have focused on the influence of COMT polymorphic gene on antidepressant effect.[2428] In this study, we analyzed the influence of genetic variables on the TMS clinical response. In particular, we analyzed the coding region of COMT gene
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polymorphism (rs4680). Moreover, we reanalyzed SERTPR polymorphism and 5-HT1A serotonergic receptor promoter region polymorphism (rs6295)[7] in patients that underwent active TMS treatment in this completely new sample.

MATERIALS AND METHODS

PATIENTS
In this study, we considered 90 inpatients consequently admitted to our Research Centre for Mood Disorders (San Raffaele Hospital, Milan, Italy) that gave consent for genetic analysis. All subjects suffered from a major depressive episode, due to a Major Depressive Disorder (N 5 66) or Bipolar Disorder (N 5 24) without psychotic features and not due to a medical condition or induced by a substance. Lifetime diagnoses were assigned by trained psychiatrists on the basis of unstructured clinical interviews and medical records, according to DSM-IV criteria and following the best estimate procedure.[29] All patients were considered drug resistant because they showed a lack of improvement to at least two different treatments with antidepressant at adequate dosage and duration, administered during the current episode and selected on the basis of clinical experience.[30] During the trial, they were on selective serotonin reuptake inhibitor (SSRI) (n 5 47), SNRI (n 5 27), or TCA (n 5 16). All currently administered drugs were maintained at a stable dosage throughout the trial. The exclusion criteria were age (younger than 18 years or older than 65), the presence of any concomitant Axis I diagnosis, psychotic features, personality disorders, somatic or neurological illnesses impairing psychiatric evaluation, pregnancy, and a 21-item Hamilton Rating Scale for Depression (HAM-D) less than 21. A physical examination, laboratory analysis, and electrocardiogram were routinely performed before inclusion. Patients bearing pacemakers, mobile metal implants, implanted medical pumps, or metal clips placed inside the skull were also excluded in accordance with the safety criteria for TMS.[31] When the procedure was completely explained to the subjects, their written informed consent was obtained. The study was approved by the Ethical Committee of San Raffaele Hospital and was conducted in accordance with the Declaration of Helsinki and the Good Clinical Practice Guidelines.

CLINICAL ASSESSMENT
Assessment was performed using the 21-item HAM-D,[32] administered at baseline and weekly thereafter for 2 weeks. The assessment was performed by two trained psychiatrists, blind to treatment conditions, with a good interrater reliability (interclass correlation coefficient on HAM-D 5 .95). Response was defined as Z50% decrease in the HAM-D total score from baseline.

TMS METHODS
All patients underwent active treatment. We used a Magstim Rapid Stimulator for biphasic pulses (Magstim Company Ltd., Wales, UK, Withland LJK) with a focal 70 mm eight-shaped coil. Before the first treatment, the resting motor threshold (MT) was determined as the lowest intensity able to induce 5 out of 10 times an involuntary movement of the right abductor pollicis brevis muscle. The site of stimulation was defined by the neuronavigational method (SofTaxic Optic, EMS company, Bologna, Italy).[33] The left DLPFC was stimulated with the following parameters: 20 Hz, 30 trains of 30 pulses (2 sec each, with 28 sec intertrain interval), for 900 pulses per day. Stimulation was applied at 100% of the MT with the coil held flat on the scalp (tangentially) and the

Research Article: Genetic Influence on Antidepressant Response to TMS

handle creating a 451 angle with the midline, in the left posterior direction. Stimulation was performed for 10 consecutive working days (from Monday to Friday for 2 weeks).

DNA ANALYSIS
All patients underwent a venous blood sample for genotypic analysis. PCR-based techniques were used to determine the three studied polymorphism. Genomic DNA was extracted from whole blood by manual extraction, using the Illustra blood genomicPrep Midi Flow kit (GE Healthcare, Milan, Italy). Genotyping of COMT rs4680 was performed with the following primers: 50 -ACT GTG GCT ACT CAG CTG TG-30 , 50 -CCT TTT TCC AGG TCT GAC AA-30 . PCR product (169 bp) was digested using NlaIII (New England Biolabs, England, UK); fragments were separated in 3% Seakem agarose gels (BMA, BioWhittaker Molecular Applications. Rockland, ME). The cleaved bands were visualized by ultraviolet light. Depending on the presence of two or three restriction NlaIII sites, either three fragments 114, 29, and 26 bp (allele G or Val) or four fragments 96, 29, 26, and 18 bp (allele A or Met) were produced. Determination of SERTPR and 5-HT1A variants was carried out as described.[34,35] Given the low number of patients enrolled in this study, SERTPR polymorphism PCR products were analyzed by agarose gel which allowed the differentiation of the long (528 bp) from the short (484 bp) alleles. When we reach an adequate number of patients, we will be able to analyze the genotype according to Nakamura.[36] To analyze 5-HT1A polymorphisms, the PCR product was genotyped by means of a Genetic Analyzer (MegaBace 500, GE Healthcare) after having purified DNA by SEPHADEX (SIGMA, Italy).

cervical pain. None of the patients had to take concomitant medications for the above-mentioned side effects. At the end of the 2-week treatment, 41 out of 90 pharmaco-resistant depressive patients (45.55% of the whole sample) were responders. Clinical and demographic characteristics of enrolled patients are summarized in Table 1. There are no statistical significant baseline differences either in demographic or clinical variables between the groups, according to the SERTPR, 5-HT1A, and COMT genes polymorphisms. Genotype frequencies of the SERTPR and COMT polymorphisms were in the HardyWeinberg equilibrium (w2 5 0.41, df 5 1, P 5.523; w2 5 0.24, df 5 1, P 5.623), whereas frequencies of 5-HT1A were not (w2 5 9.43, df 5 1, P 5.002). Results of the main effects of the ANOVA showed a significant model (F 5 2.693; P 5.015). In this model, three factors were not significant: diagnosis (F 5 2.591; P 5.112), COMT (F 5 0.291; P 5.748), and SERTPR (F 5 1.313; P 5.275), whereas factor 5-HT1A showed a significant influence on the outcome (F 5 5.089; P 5.008). Scheffe post hoc multiple comparison showed that patients with C/C genotype had a greater improvement than G/G (P 5.015) and C/G (P 5.019), whereas there was no difference between G/G and C/G (P 5.912).

STATISTICAL ANALYSIS
One-way analysis of variance (ANOVA) and the chi-square test were used to investigate the differences among genotypes for demographic and baseline clinical variables. Effects of gene polymorphisms on clinical improvement were analyzed with an ANOVA with each gene (SERTPR, 5-HT1A, and COMT) as factors (with three levels, one for each genotype) and HAM-D variation from the baseline to the end of treatment as a dependent variable.

DISCUSSION
Our results confirmed the antidepressant efficacy of TMS in drug-resistant depressives. Almost 50% of our patients showed a response to treatment. Considering that we treated patients resistant to pharmacological treatment, these results are very encouraging and promising. Our data are in line with previous findings.[37,38] The sample was not in the HardyWeinberg equilibrium for the 5-HT1A, probably because of an excess of patients with G allele; we hypothesized that this finding could be due to the high amount of drugresistant depressed subjects.[17] We evaluated the rTMS response with respect to genetic polymorphism of the promoter region of the 5-HT1A receptor gene. Some studies reported that C/C

RESULTS
All patients completed the study and no one dropped out. The TMS was generally well tolerated. We did not observe major adverse events, except for headache and

TABLE 1. Clinical and demographic characteristics of enrolled patients

Mean Age, years Onset of illness, years Episode duration, weeks HAM-D baseline Sex M/F Diagnosis UP/BP Drugs (SSRI/SNRI/TCA) 53.96 39.21 17.13 25.81 25/65 67/23 47/27/16 SD 10.69 15.06 8.45 3.05 P (SERTPR)a .971 .954 .668 .622 .508 .258 .712 P (5-HT1A)b .224 .137 .219 .568 .441 .901 .253 P (COMT)c .177 .463 .580 .213 .145 .239 .442

t-test and w2() for continuous or categorical variables. a Differences among serotonin transporter genotype groups (l/l versus l/s versus s/s). b Differences among 5-HT1A genotype groups (G/G versus G/C versus C/C). c Differences among COMT genotype groups (met/met versus val/met versus val/val). Depression and Anxiety

Malaguti et al.

Figure 1. Time course of HAM-D scores between 5-HT1a genotypes (C/C versus C/G and G/G) during the 2 weeks of TMS stimulation.

subjects have a better response to serotonergic antidepressant drugs if compared to other genotypes.[17,3942] Our results are in line with the following observation: C/C subjects showed a better outcome after TMS. In more detail, we found that C/C subjects have a better clinical outcome than G carrier subjects. In agreement with previous findings,[7] it seems that (Fig. 1) 5-HT1A polymorphism may influence TMS response. Similar to antidepressant drugs, the 5-HT1A presinaptic receptor desensitization has been demonstrated in an animal study with TMS.[43] Moreover, an influence on SSRIs antidepressant action by drugs acting on 5-HT1A receptors has been previously observed; for example, using pindolol, a mixed b-adrenoreceptor, and 5-HT1A antagonist.[4446] TMS action on 5-HT1A receptors may occur through a direct pharmacological action (as an increase of 5-HT release or a decrease of 5-HT uptake) or through an electromagnetic action. Regarding this, Espinosa et al.[47] found that the binding properties of another G-protein coupled receptor, the 5-HT1B, can be changed by the exposure to alternated current and direct current magnetic fields. It is not clear yet which one between these mechanisms plays a predominant role, and it also seems difficult to explain in which way patients with C/C genotype showed a more pronounced improvement of their psychopathological symptoms after TMS. It is possible that the serotonergic neurotransmission of those patients with a lower 5-HT1A function (C/C patients) is more affected by the downregulation of such function, as observed after TMS treatment. We also evaluated the rTMS response with respect to genetic polymorphism of the promoter region of serotonin transporter gene. Even if some studies, including Asian patients, are discordant,[4850] many
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research groups found that homozygotes for the long variant had a better response to different SSRIs when compared to heterozygotes and homozygotes for the short variant.[34,5156] In our previous article,[7] we observed an influence of the genotype on the response independent of the type of stimulation (active versus sham), with patients with the l allele showing a better amelioration than s/s patients. In a recent article that evaluated SERTPR polymorphism as a predictor of TMS response in drug-resistant depressed patients, a significantly better clinical response in homozygote subjects for l allele compared to s allele carriers has been found.[8] In this study, we did not find any significant influence of SERTPR polymorphism on TMS response. Other studies regarding SERTPRs influence on TMS response are aimed at better clarifying this topic. Finally, we evaluated TMS response with respect to COMT polymorphism, finding no significant influence on treatment response. Published studies on the influence of COMT genotype on antidepressants response gave contrasting results. A negative effect of met COMT variant was reported on mirtazapine, paroxetine,[24] and citalopram response.[53] Different studies evaluating COMT polymorphism influence on electroconvulsive therapy also sustained a better influence of val allele.[5759] On the other hand, a recent study suggested a COMT gene polymorphism influence on antidepressant response to sleep deprivation combined with light therapy in bipolar depressed patients, with a better response in carriers of the met variant.[27] These results are similar to those observed in a previous study, in which met COMT variant showed a significantly better antidepressant response to paroxetine.[26] Finally, a recent trial did not observe any difference in the distribution of COMT gene polymorphisms in the treatment response to SSRIs.[28] No studies are available yet on COMT influence on TMS response. Some limits of this trial should be considered. Because the majority of available literature data reported the efficacy of TMS in combination with antidepressant drugs, our ethical committee approved for this specific study the use of TMS plus antidepressants in drug-resistant patients. Anyway, in a few literature data, an antidepressant efficacy of TMS alone was observed. The possibility to perform a TMS study in depressed drugs free patients could be useful to assess the influence of 5-HT1a polymorphism on the response to TMS alone. Moreover, in our study, the rather small number of subjects in some subgroups, different antidepressants taken by patients, and the fact that the sample was not in HardyWeinberg equilibrium for the 5-HT1A should be taken into account.

CONCLUSION
In conclusion, our results represent one of the first TMS genetic studies assessing the influence of functional 5-HT1A, SERTPR, and COMT gene polymorphisms on response to trascranial magnetic

Research Article: Genetic Influence on Antidepressant Response to TMS

stimulation in patients with a major depressive drugresistant episode. The analysis of these and other candidate genes in larger samples, homogeneous in terms of antidepressants assumed, could help to better explain the genetic influence on TMS response.

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