Original Paper Received: March 19, 2014 Accepted after revision: August 24, 2014 Published online: January 9, 2015
Neuropsychobiology 2014;70:220–227 DOI: 10.1159/000368120
Longitudinal Monitoring of the Serotonin Transporter Gene Expression to Assess Major Depressive Episode Evolution Raoul Belzeaux a, c, d Anderson Loundou b Jean-Michel Azorin c, d Jean Naudin c El Chérif Ibrahim a, d
a
Aix-Marseille Université, CNRS, CRN2M UMR 7286, and b Faculté de Médecine Timone, Unité d’Aide Méthodologique, Aix-Marseille Université, and c AP-HM, Hôpital Sainte Marguerite, Pôle de Psychiatrie Universitaire Solaris, Marseille, and d FondaMental, Fondation de Recherche et de Soins en Santé Mentale, Créteil, France
Key Words Biomarker · mRNA · SLC6A4 · Antidepressant · Major depression · Blood
Abstract Background: Mood disorders are frequently characterized by uncertain prognosis and studying mRNA expression variations in blood cells represents a promising avenue of identifying biomarkers for mood disorders. State-dependent gene expression variations have been described during a major depressive episode (MDE), in particular for SLC6A4 mRNA, but how this transcript varies in relation to MDE evolution remains unclear. In this study, we prospectively assessed time trends of SCL6A4 mRNA expression in responder and nonresponder patients. Methods: We examined SLC6A4 mRNA expression in blood samples from 13 patients treated for severe MDE and their matched controls by reverse transcription and quantitative PCR. All subjects were followed for 30 weeks. Patients were classified as either responders or nonresponders based on improvement of depression according to the 17-item Hamilton Depression Rating Scale. Using a longitudinal design, we ascertained mRNA expression at baseline, 2, 8, and 30 weeks and compared mRNA
© 2015 S. Karger AG, Basel 0302–282X/15/0704–0220$39.50/0 E-Mail [email protected] www.karger.com/nps
expression between responder and nonresponder patients, and matched controls. Results: We observed a decrease of SLC6A4 mRNA expression in responder patients across a 30week follow-up, while nonresponder patients exhibited upregulated SLC6A4 mRNA. Conclusion: Peripheral SLC6A4 mRNA expression could serve as a biomarker for monitoring and follow-up during an MDE and may help to more appropriately select individualized treatments. © 2015 S. Karger AG, Basel
Introduction
Major depressive disorder (MDD) is a frequent and severe psychiatric condition [1]. Unfortunately, MDD prognosis and treatment efficacy remain unpredictable and more than 30% of patients suffering from a major depressive episode (MDE) fail to improve after a first-line treatment in both naturalistic and phase III efficacy trials [2, 3]. Although important efforts have been made to rationalize treatment guidelines concerning affective disorders [4], the primary care physician or the psychiatrist generally faces the ‘trial and error’ paradigm when selecting the most suitable antidepressant medication for each El Chérif Ibrahim CNRS, CRN2M-UMR 7286, Faculté de Médecine Nord de Marseille Aix-Marseille Université, 51 Bd Pierre-Dramard FR–13344 Marseille Cedex 15 (France) E-Mail el-cherif.ibrahim @ univ-amu.fr
patient [5, 6]. In this context, biomarker identification and validation are important goals of research, opening the door to personalized medicine in psychiatry [7]. However, the development of biomarkers is difficult due to the complexity of clinical presentation and pathophysiology of MDD. First of all, MDD evolves in a highly unstable and unpredictable fashion, with a random succession of remission, relapse, recovery, recurrence, and chronic episodes [8]. Secondly, the pathophysiology of MDD remains unclear, even though genetic vulnerability and gene-to-environment interactions have been described [9–11]. Pharmacogenetic studies aiming to personalize the treatment of depression are based on genetic variations associated with treatment response phenotype. Indeed, previous studies have described significant associations between certain polymorphisms and antidepressant response, suggesting that the clinical use of pharmacogenomics might improve the cost/effectiveness ratio of MDE treatment [12, 13]. To date, however, pharmacogenomics have not been incorporated into genetic tests for prediction of antidepressant response in clinical practice [2, 7, 14]. In this context, gene expression can bridge the gap between genetic variation and treatment response as an intermediate phenotype [15]. Gene expression studies provide a window on gene and environment interactions, due to the regulation of gene expression implicating both genetic background as well as epigenetic imprinting. Moreover, gene expression studies offer the possibility to define quantitative biomarkers that could be followed across time and clinical evolution in prospective studies and define state-dependent biological markers. Indeed, although the correlation of gene expression between blood and brain is not obvious, several studies have suggested that peripheral gene expression, particularly in blood cells, could be relevant to study the pathophysiology of psychiatric disorders and to define such biomarkers [16–22]. In this context, several previous studies proposed using mRNA expression as potential diagnostic biomarkers of MDD, based on gene expression differences between patients and healthy controls [18, 19, 23–25]. More precisely, some studies proposed gene expression in peripheral blood as biomarkers for treatment response [26]. In a recent study, Cattaneo et al. [27] distinguished two kinds of gene expression biomarkers, i.e. ‘predictors’ which predict treatment response before treatment and ‘targets’ which change longitudinally only in patients who respond to treatment. In light of this definition, we and others have described ‘predictor’ biomarkers [18, 28, 29],
while only Cattaneo et al. [27] described well-defined ‘target’ biomarkers, i.e. IL-6 and FKBP-5 within a selection of candidate genes. We and others have described state-dependent gene expression variation during MDE, in particular for SLC6A4 mRNA. Two previous studies described a decrease of gene expression in peripheral blood of patients between baseline (patients were recruited with DSM-IV MDE criteria) and at 8–12 weeks when clinical improvement was demonstrated [30, 31]. In contrast, we observed an increase of SLC6A4 mRNA expression in a similar clinical design [19]. To the best of our knowledge, no previous study described gene expression variation in a larger time window and little is known about SLC6A4 mRNA expression in healthy subject across time. Serotonin transporter plays a key role in MDD pathophysiology, as it has been linked to depression in candidate gene studies [9] and in gene-to-environment interaction studies [10]. The serotonin transporter protein (5HTT) is the main target of many antidepressants, although links between pathophysiology and therapeutic effects of antidepressants are still a matter of debate [32, 33]. It remains unclear whether the role of 5HTT involves transcriptional and/or a posttranscriptional regulation [34]. Therefore, we still do not know how SLC6A4 mRNA should vary in relation to the MDE treatment response. Based on previous studies on SLC6A4 mRNA gene expression variation in peripheral tissues, we aimed to explore if SLC6A4 mRNA, measured in peripheral blood mononuclear cells (PBMCs), could be a target biomarker during MDE that varies longitudinally in responder patients.
SLC6A4 mRNA during Major Depressive Episode
Neuropsychobiology 2014;70:220–227 DOI: 10.1159/000368120
Methods Design Setting and Subjects The study design was a naturalistic, prospective, longitudinal and comparative study with assessments of MDE patients and healthy controls at baseline (week 0), and at 2, 8 and 30 weeks after inclusion. Patients who met the Diagnostic and Statistical Manual of Mental Disorders, fourth edition, text revision (DSM-IV-TR) criteria for MDD were recruited for the study [35]. Inclusion criteria were: (i) treated or untreated MDE and (ii) 17-item Hamilton Rating Scale for Depression (HDRS-17) score ≥20 corresponding to severe or very severe MDE [36]. Patients with bipolar disorder, schizophrenia, personality disorder, substance use disorder, medical comorbidities, abnormal body temperature or laboratory tests (especially blood cell count), and neurological disorders were excluded. Patients were clinically evaluated using an adapted standardized procedure (SCID-I/P) [37]. Sociodemographic information, including age and gender, was collected. The severity of MDE was evaluated with a French version of HDRS-17 at each evalua-
221
tion [38]. Episode duration, number of previous failed antidepressant treatments before inclusion, melancholic features during the MDE, family history of mood disorder and other severe psychiatric disorders, history of suicide attempts, and tobacco smocking were evaluated. Patients were classified as responders and nonresponders based on the consensus definition of clinical response corresponding to a minimal reduction of 50% of the HDRS-17 score at baseline evaluation and at 8 weeks. Responder status was defined by the persistence of observed clinical improvement at 8 weeks to the end of the 30-week follow-up [8]. This ensures that patients who either relapsed or exhibit an oscillating response pattern can be excluded, as previously described [39]. To standardize antidepressant exposure between responders and nonresponders at baseline, antidepressant treatment was converted in equivalent doses of imipramine, as suggested by Bollini et al. [40]. For the control group, age- and sex-matched subjects were evaluated to exclude patients with a history of psychiatric disorder using the French version of a standardized interview validated for health control subjects (SCID-NP). Moreover, all participants were carefully interviewed to exclude the presence of psychiatric disorders and they were clinically examined to eliminate any medical conditions. Tobacco use was also evaluated. All experiments on human subjects were conducted in accordance with the latest version of the Declaration of Helsinki. The project was approved by the local ethics committee (Comité de Protection des Personnes, CPP Sud Méditerranée II, Marseille, France, study registered under number 09.025) and written informed consent was obtained after a complete description of the study to the subjects. Blood mRNA Extraction At each evaluation, 8–10 ml of venous blood was collected from fasting patients and matched controls in EDTA tubes between 7:00 and 9:00 a.m. and processed within 2 h. PBMCs were isolated from the blood by Ficoll density centrifugation. Total RNA was extracted from the PBMCs with the mirVana miRNA isolation kit (Ambion, Austin, Tex., USA) according to the manufacturer’s protocol. RNA concentration was determined using a NanoDrop ND1000 spectrophotometer (NanoDrop Technologies, Wilmington, Del., USA). RNA integrity was assessed on an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, Calif., USA), and all samples exhibited an RNA integrity number superior to 8.
(i.e. difference between the target and the reference mRNA
Neuropsychobiology 2014;70:220–227 DOI: 10.1159/000368120
Longitudinal Monitoring of the Serotonin Transporter Gene Expression to Assess Major Depressive Episode Evolution Raoul Belzeaux a, c, d Anderson Loundou b Jean-Michel Azorin c, d Jean Naudin c El Chérif Ibrahim a, d
a
Aix-Marseille Université, CNRS, CRN2M UMR 7286, and b Faculté de Médecine Timone, Unité d’Aide Méthodologique, Aix-Marseille Université, and c AP-HM, Hôpital Sainte Marguerite, Pôle de Psychiatrie Universitaire Solaris, Marseille, and d FondaMental, Fondation de Recherche et de Soins en Santé Mentale, Créteil, France
Key Words Biomarker · mRNA · SLC6A4 · Antidepressant · Major depression · Blood
Abstract Background: Mood disorders are frequently characterized by uncertain prognosis and studying mRNA expression variations in blood cells represents a promising avenue of identifying biomarkers for mood disorders. State-dependent gene expression variations have been described during a major depressive episode (MDE), in particular for SLC6A4 mRNA, but how this transcript varies in relation to MDE evolution remains unclear. In this study, we prospectively assessed time trends of SCL6A4 mRNA expression in responder and nonresponder patients. Methods: We examined SLC6A4 mRNA expression in blood samples from 13 patients treated for severe MDE and their matched controls by reverse transcription and quantitative PCR. All subjects were followed for 30 weeks. Patients were classified as either responders or nonresponders based on improvement of depression according to the 17-item Hamilton Depression Rating Scale. Using a longitudinal design, we ascertained mRNA expression at baseline, 2, 8, and 30 weeks and compared mRNA
© 2015 S. Karger AG, Basel 0302–282X/15/0704–0220$39.50/0 E-Mail [email protected] www.karger.com/nps
expression between responder and nonresponder patients, and matched controls. Results: We observed a decrease of SLC6A4 mRNA expression in responder patients across a 30week follow-up, while nonresponder patients exhibited upregulated SLC6A4 mRNA. Conclusion: Peripheral SLC6A4 mRNA expression could serve as a biomarker for monitoring and follow-up during an MDE and may help to more appropriately select individualized treatments. © 2015 S. Karger AG, Basel
Introduction
Major depressive disorder (MDD) is a frequent and severe psychiatric condition [1]. Unfortunately, MDD prognosis and treatment efficacy remain unpredictable and more than 30% of patients suffering from a major depressive episode (MDE) fail to improve after a first-line treatment in both naturalistic and phase III efficacy trials [2, 3]. Although important efforts have been made to rationalize treatment guidelines concerning affective disorders [4], the primary care physician or the psychiatrist generally faces the ‘trial and error’ paradigm when selecting the most suitable antidepressant medication for each El Chérif Ibrahim CNRS, CRN2M-UMR 7286, Faculté de Médecine Nord de Marseille Aix-Marseille Université, 51 Bd Pierre-Dramard FR–13344 Marseille Cedex 15 (France) E-Mail el-cherif.ibrahim @ univ-amu.fr
patient [5, 6]. In this context, biomarker identification and validation are important goals of research, opening the door to personalized medicine in psychiatry [7]. However, the development of biomarkers is difficult due to the complexity of clinical presentation and pathophysiology of MDD. First of all, MDD evolves in a highly unstable and unpredictable fashion, with a random succession of remission, relapse, recovery, recurrence, and chronic episodes [8]. Secondly, the pathophysiology of MDD remains unclear, even though genetic vulnerability and gene-to-environment interactions have been described [9–11]. Pharmacogenetic studies aiming to personalize the treatment of depression are based on genetic variations associated with treatment response phenotype. Indeed, previous studies have described significant associations between certain polymorphisms and antidepressant response, suggesting that the clinical use of pharmacogenomics might improve the cost/effectiveness ratio of MDE treatment [12, 13]. To date, however, pharmacogenomics have not been incorporated into genetic tests for prediction of antidepressant response in clinical practice [2, 7, 14]. In this context, gene expression can bridge the gap between genetic variation and treatment response as an intermediate phenotype [15]. Gene expression studies provide a window on gene and environment interactions, due to the regulation of gene expression implicating both genetic background as well as epigenetic imprinting. Moreover, gene expression studies offer the possibility to define quantitative biomarkers that could be followed across time and clinical evolution in prospective studies and define state-dependent biological markers. Indeed, although the correlation of gene expression between blood and brain is not obvious, several studies have suggested that peripheral gene expression, particularly in blood cells, could be relevant to study the pathophysiology of psychiatric disorders and to define such biomarkers [16–22]. In this context, several previous studies proposed using mRNA expression as potential diagnostic biomarkers of MDD, based on gene expression differences between patients and healthy controls [18, 19, 23–25]. More precisely, some studies proposed gene expression in peripheral blood as biomarkers for treatment response [26]. In a recent study, Cattaneo et al. [27] distinguished two kinds of gene expression biomarkers, i.e. ‘predictors’ which predict treatment response before treatment and ‘targets’ which change longitudinally only in patients who respond to treatment. In light of this definition, we and others have described ‘predictor’ biomarkers [18, 28, 29],
while only Cattaneo et al. [27] described well-defined ‘target’ biomarkers, i.e. IL-6 and FKBP-5 within a selection of candidate genes. We and others have described state-dependent gene expression variation during MDE, in particular for SLC6A4 mRNA. Two previous studies described a decrease of gene expression in peripheral blood of patients between baseline (patients were recruited with DSM-IV MDE criteria) and at 8–12 weeks when clinical improvement was demonstrated [30, 31]. In contrast, we observed an increase of SLC6A4 mRNA expression in a similar clinical design [19]. To the best of our knowledge, no previous study described gene expression variation in a larger time window and little is known about SLC6A4 mRNA expression in healthy subject across time. Serotonin transporter plays a key role in MDD pathophysiology, as it has been linked to depression in candidate gene studies [9] and in gene-to-environment interaction studies [10]. The serotonin transporter protein (5HTT) is the main target of many antidepressants, although links between pathophysiology and therapeutic effects of antidepressants are still a matter of debate [32, 33]. It remains unclear whether the role of 5HTT involves transcriptional and/or a posttranscriptional regulation [34]. Therefore, we still do not know how SLC6A4 mRNA should vary in relation to the MDE treatment response. Based on previous studies on SLC6A4 mRNA gene expression variation in peripheral tissues, we aimed to explore if SLC6A4 mRNA, measured in peripheral blood mononuclear cells (PBMCs), could be a target biomarker during MDE that varies longitudinally in responder patients.
SLC6A4 mRNA during Major Depressive Episode
Neuropsychobiology 2014;70:220–227 DOI: 10.1159/000368120
Methods Design Setting and Subjects The study design was a naturalistic, prospective, longitudinal and comparative study with assessments of MDE patients and healthy controls at baseline (week 0), and at 2, 8 and 30 weeks after inclusion. Patients who met the Diagnostic and Statistical Manual of Mental Disorders, fourth edition, text revision (DSM-IV-TR) criteria for MDD were recruited for the study [35]. Inclusion criteria were: (i) treated or untreated MDE and (ii) 17-item Hamilton Rating Scale for Depression (HDRS-17) score ≥20 corresponding to severe or very severe MDE [36]. Patients with bipolar disorder, schizophrenia, personality disorder, substance use disorder, medical comorbidities, abnormal body temperature or laboratory tests (especially blood cell count), and neurological disorders were excluded. Patients were clinically evaluated using an adapted standardized procedure (SCID-I/P) [37]. Sociodemographic information, including age and gender, was collected. The severity of MDE was evaluated with a French version of HDRS-17 at each evalua-
221
tion [38]. Episode duration, number of previous failed antidepressant treatments before inclusion, melancholic features during the MDE, family history of mood disorder and other severe psychiatric disorders, history of suicide attempts, and tobacco smocking were evaluated. Patients were classified as responders and nonresponders based on the consensus definition of clinical response corresponding to a minimal reduction of 50% of the HDRS-17 score at baseline evaluation and at 8 weeks. Responder status was defined by the persistence of observed clinical improvement at 8 weeks to the end of the 30-week follow-up [8]. This ensures that patients who either relapsed or exhibit an oscillating response pattern can be excluded, as previously described [39]. To standardize antidepressant exposure between responders and nonresponders at baseline, antidepressant treatment was converted in equivalent doses of imipramine, as suggested by Bollini et al. [40]. For the control group, age- and sex-matched subjects were evaluated to exclude patients with a history of psychiatric disorder using the French version of a standardized interview validated for health control subjects (SCID-NP). Moreover, all participants were carefully interviewed to exclude the presence of psychiatric disorders and they were clinically examined to eliminate any medical conditions. Tobacco use was also evaluated. All experiments on human subjects were conducted in accordance with the latest version of the Declaration of Helsinki. The project was approved by the local ethics committee (Comité de Protection des Personnes, CPP Sud Méditerranée II, Marseille, France, study registered under number 09.025) and written informed consent was obtained after a complete description of the study to the subjects. Blood mRNA Extraction At each evaluation, 8–10 ml of venous blood was collected from fasting patients and matched controls in EDTA tubes between 7:00 and 9:00 a.m. and processed within 2 h. PBMCs were isolated from the blood by Ficoll density centrifugation. Total RNA was extracted from the PBMCs with the mirVana miRNA isolation kit (Ambion, Austin, Tex., USA) according to the manufacturer’s protocol. RNA concentration was determined using a NanoDrop ND1000 spectrophotometer (NanoDrop Technologies, Wilmington, Del., USA). RNA integrity was assessed on an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, Calif., USA), and all samples exhibited an RNA integrity number superior to 8.
(i.e. difference between the target and the reference mRNA
