Serotonin transporter gene polymorphisms and treatment-resistant depression

Progress in Neuro-Psychopharmacology & Biological Psychiatry 34 (2010) 934–939

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Progress in Neuro-Psychopharmacology & Biological Psychiatry j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / p n p

Serotonin transporter gene polymorphisms and treatment-resistant depression Cristian Bonvicini a, Alessandra Minelli a,b, Catia Scassellati a, Marco Bortolomasi b, Matilde Segala b, Riccardo Sartori c, Mario Giacopuzzi b, Massimo Gennarelli a,d,⁎ a

Genetic Unit, I.R.C.C.S. “San Giovanni di Dio”, Fatebenefratelli, Brescia, Italy Psychiatric Hospital “Villa Santa Chiara”, Verona, Italy Department of Psychology and Cultural Anthropology, University of Verona, Verona, Italy d Department of Biomedical Sciences and Biotechnologies, Biology and Genetic Division, Brescia University School of Medicine, Brescia, Italy b c

a r t i c l e

i n f o

Article history: Received 15 March 2010 Received in revised form 16 April 2010 Accepted 21 April 2010 Available online 5 May 2010 Keywords: rs25531 SLC6A4 Treatment-resistant depression 5-HTTLPR

a b s t r a c t Major Depression Disorder (MDD) is a serious mental illness that is one of the most disabling diseases worldwide. In addition, approximately 15% of depression patients are defined treatment-resistant (TRD). Preclinical and genetic studies show that serotonin modulation dysfunction exists in patients with TRD. Some polymorphisms in the promoter region of the serotonin transporter gene (SLC6A4) are likely to be involved in the pathogenesis/treatment of MDD; however, no data are available concerning TRD. Therefore, in order to investigate the possible influence of SLC6A4 polymorphisms on the risk of TRD, we genotyped 310 DSM-IV MDD treatment-resistant patients and 284 healthy volunteers. We analysed the most studied polymorphism 5-HTTLPR (L/S) and a single nucleotide substitution, rs25531 (A/G), in relation to different functional haplotype combinations. However the correct mapping of rs25531 is still debated whether it is within or outside the insertion. Our sequencing analysis showed that rs25531 is immediately outside of the 5-HTTLPR segment. Differences in 5-HTTLPR allele (p = 0.04) and in L allele carriers (p b 0.05) were observed between the two groups. Concerning the estimated haplotype analyses, LALA homozygote haplotype was more represented among the control subjects (p = 0.01, OR = 0.64 95%CI: 0.45–0.91). In conclusion, this study reports a protective effect of the LALA haplotype on TRD, supporting the hypothesis that lower serotonin transporter transcription alleles are correlated to a common resistant depression mechanism. © 2010 Elsevier Inc. All rights reserved.

1. Introduction Major Depressive Disorder (MDD) is a disabling psychiatric condition that is among the top five leading causes of disability and disease burden throughout the world. The pharmacological therapy is substantially effective, although approximately 15% of patients are classified as resistant or refractory (Berlim and Turecki, 2007). Abbreviations: 5-HTTLPR, serotonin-transporter-linked promoter region, (43bp Ins=L allele/Del=S allele); ANOVA, analysis of variance; ATD, acute tryptophan depletion; DSM-IV, Diagnostic and Statistical Manual of Mental Disorders; ECT, Electroconvulsive therapy; HWE, Hardy-Weinberg Equilibrium; M.I.N.I., Mini International Neuropsychiatric Interview; M.M.S.E., Mini Mental State Examination; MAF, minor Allele Frequency; MDD, major depressive disorder; PCR, polymerase chain reaction; rMDD, remitted major depressive disorder; rTMS, repetitive transcranial magnetic stimulation; SLC6A4, serotonin transporter gene; SNP, single nucleotide polymorphism; SSRI, Selective Serotonin Reuptake Inhibitor; STin2, 17-bp variable tandem repeat intron 2; TCA, trcyclic antidepressant; TD, tryptophan depletion; TRD, treatment-resistant depression. ⁎ Corresponding author. Massimo Gennarelli, Genetic Unit, I.R.C.C.S. “San Giovanni di Dio”, Fatebenefratelli, Via Pilastroni, 4-25123 Brescia, Italy. Tel.: +39 030 3501453; fax: +39 030 3533513. E-mail address: [email protected] (M. Gennarelli). 0278-5846/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.pnpbp.2010.04.020

The underlying mechanism of response to treatment is multifactorial and involves environmental and genetic factors as well as their interactions (Uher, 2008). In particular it has been observed that antidepressant response clusters in families supporting a role for genetic variation in the response, although the degree of heritability is still unknown (Franchini et al., 1998; O'Reilly et al., 1994). Differences in clinical response may be attributable to 5-HTTLPR polymorphism of the serotonin transporter gene SLC6A4. The S allele of 5-HTTLPR has been reported to be associated with increased risk for non-response to treatment to SSRIs (Horstmann and Binder, 2009; Huezo-Diaz et al., 2009; Serretti et al., 2007). Specifically, functional studies of the activity of the SLC6A4 promoter in transfected cell lines, human post-mortem brain, and lymphoblasts confirmed that the L allele of the functional polymorphism 5-HTTLPR is associated with higher levels of transcriptional activity and influences the rate of serotonin uptake more than the S variant (Hu et al., 2006; Lipsky et al., 2009; Ruhe et al., 2009). As regard to the response to other antidepressants not SSRIs (Joyce et al., 2003; Lee et al., 2004; Minov et al., 2001; Murphy et al., 2004; Yoshida et al., 2002) as well as to non-medication treatments such as sleep deprivation (Baghai et al., 2003; Benedetti et al., 2003) and repetitive transcranial magnetic stimulation (rTMS) (Baghai et al., 2003;

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Benedetti et al., 2003; Bocchio-Chiavetto et al., 2008), associations have been reported. This suggests that SLC6A4 could be a common genetic modulator of response to treatment regardless of modality, due to its involvement in downstream mechanisms of action of several antidepressant drugs. Recently, it has been critically discussed that the analysis of 5-HTTLPR is incomplete because other polymorphisms have been found in the proximity of the Ins/Del locus, such as rs25531, rs25532, rs2020933, and a 17-bp variable tandem repeat in the second intron (STin2) (Damberg, 2005; Hu et al., 2006; Lipsky et al., 2009; Wendland et al., 2008). In particular, rs25531, the polymorphism nearest 5-HTTLPR, results in an A to G substitution and has been shown to modulate the effect of 5-HTTLPR on transcriptional efficacy. However, the exact location of this SNP is uncertain; while some authors have indicated that rs25531 resides within of 5-HTTLPR (Hu et al., 2005; Wray et al., 2009), others have shown that is located immediately outside (Kraft et al., 2005; Nakamura et al., 2000; Wendland et al., 2006). Nonetheless, rs25531 has been associated with the response to fluoxetine (Kraft et al., 2005) and fluvoxamine (Smeraldi et al., 2006), while other studies have reported negative results with escitalopram (Maron et al., 2009) and citalopram (Hu et al., 2007; Kraft et al., 2007; Lekman et al., 2008; Mrazek et al., 2009). However, patients carrying the SA, SG or LG haplotypes were more likely to experience adverse effects of SSRIs (Hu et al., 2007; Lekman et al., 2008; Maron et al., 2009). To date, only one study has explored the potential role of 5-HTTLPR in treatment-resistant depression (TRD) (Kishida et al., 2007), showing that this polymorphism is not involved in the mechanism of resistance to treatment in mood disorder patients. Furthermore, no evidence is available concerning the influence of the rs25531 polymorphism on resistance to antidepressant treatment. Therefore, we studied the role of the 5-HTTLPR and rs25531 polymorphisms in a representative sample of Italian TRD patients and controls. 2. Methods 2.1. Sample A total of 310 DSM-IV severe MDD patients were enrolled voluntarily in the study, which was approved by the Local Ethics Committee, and written informed consent was obtained. All of the patients had been referred to the Psychiatric Hospital ‘Villa S. Chiara’ in Verona and were judged to be treatment-resistant depressed patients. Treatment resistance to antidepressant drugs was defined as the failure of the patient to respond to two or more adequate trials of two or more different classes of antidepressant drugs and as a failure to respond to an adequate trial of a tricyclic (TCA) referred to Stage III of Thase and Rush Staging Method (Thase and Rush, 1997). In particular, an adequate therapy typically consists of one or more trials with antidepressants that have established efficacy in MDD and that are prescribed in at least standard dosages (that is, dosages that have shown efficacy in randomized trials) for a duration long enough to produce significant therapeutic effects (Berlim and Turecki, 2007). Sixty-two patients showed comorbidity in Axis I (Generalised Anxiety Disorder or Panic Attacks) and 64 showed symptoms of Axis II disorders (Dependent and Obsessive–Compulsive personality disorders) as a secondary diagnosis (the total number exceeds the number of subjects due to the presence of comorbidity). Exclusion criteria included the following: a) mental retardation and cognitive disorders; b) a lifetime history of schizophrenic, schizoaffective, or bipolar disorders; c) personality disorders, substance abuse, alcohol abuse or dependency, obsessive–compulsive disorder, post-traumatic stress disorder, as primary diagnosis; d) comorbidity with eating disorders. The control sample consisted of 284 healthy volunteers screened for lifetime DSM-IV Axis I disorders diagnoses and Axis I diagnosis of firstdegree relatives through the Mini-International Neuropsychiatric Interview (M.I.N.I.) (Sheehan et al., 1998). Subjects who obtained a score lower than 27/30 at the Mini Mental State Examination (M.M.S.E.)

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Table 1 Sociodemographic characteristics of the TRD and control samples. Characteristic

Age strata 18–30 31–50 51–70 Over 70 Mean age (± SD) Sex Female Male Education University High school Professional school Elementary school Mean years (± SD) Marital status Single Married or cohabiting Divorced Widowed Work status Student Employed Housewife Unemployed Pension

TRD patients (310) %

Healthy subjects (284) %

Statistic; p

4% 27% 56% 13% 56.3 (± 13.2)

15% 39% 35% 11% 49.0 (± 16.4)

F = 35.76; b 0.0001

67% 33%

52% 48%

χ2 = 13.86; b0.001

4% 26% 33% 37% 9 (± 4.0)

32% 31% 27% 10% 13 (± 5.0)

F = 150.34; b0.0001

19% 62% 8% 11%

23% 60% 10% 6%

χ2 = 7.12; 0.07

1% 29% 33% 4% 33%

13% 44% 19% 1% 23%

χ2 = 48.02; b0.0001

(Folstein et al., 1975) was excluded from the study. Both patients and controls were origin of Italian descent. Sociodemographic characteristics of the samples are shown in Table 1. 2.2. 5-HTTLPR L/S and rs25531 A/G genotyping analyses The Puregene kit (Gentra System) was used to isolate genomic DNA from peripheral white blood cell samples, according to the manufacturer's protocol. Genotyping of the 5-HTTLPR polymorphism was performed according to Gelernter et al. (1997). PCR products were directly used for 5HTTLPR genotyping. PCR products (15 μl) were digested with the MspI enzyme (Fermentas) to confirm the 5HTTLPR/rs25531 genotype. The MspI enzyme recognises and cuts a C|CGG sequence, leading to the following fragments: LA = (325 + 62 + 33) bp, LG = (174 + 150 + 62 + 33) bp, SA = (281 + 62 + 33) bp and SG = (150 + 131 + 62 + 33) bp. The digestion was performed overnight at 37 °C and analysed via agarose gel electrophoresis with an agarose concentration of 3.5%. Both strands of the PCR fragments of 30 subjects (10 for each genotype: LALA, LALG and SASA) were directly sequenced using the ABI 3130xl DNA analyser (Applied Biosystems, Foster City, CA, USA) and analysed using SeqScape Software version 2.6 (Applied Biosystems, Foster City, CA, USA). 2.3. Statistical analyses Using PLINK software we performed Hardy–Weinberg Equilibrium (HWE) and Chi-square test to compare allele, genotype and estimated haplotype frequency distributions between cases and controls. Phased haplotypes were performed by PLINK and their distributions were compared between two groups using the χ2 test. ORs with their corresponding 95% CIs were used to quantify the association between genotype and TRD. Chi-square (χ2) test was used to evaluate the association between groups and categorical variables, while analysis of variance (ANOVA)

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was used for computing possible differences between Groups for Age and Education. In all these analyses, a nominal level of significance p = 0.05 was accepted and corrected according to the Bonferroni procedure (the corrected alpha is .05 / N = with N number of contrasts). SPSS statistical software, edition 12.0 for Windows (SPSS Inc. Chicago, IL) was used. The association study showed a power ≥80% by using a Quanto program version 1.2.4 with the following parameters: 5-HTTLPR S and rs25531 G minor allele frequency (MAF) in the European origin population of 40% and 6%, respectively, p = 0.05, OR = 2.0, mode of inheritance: dominant; population risk of 10%.

Table 2 Allele and genotype distributions of the 5-HTTLPR and rs25531 polymorphisms in SLC6A4 gene in TRD patients and in controls. Polymorphisms 5-HTTLPR Allele frequencies L S Genotype frequencies LL LS SS Carriers L

TRD patients (n = 310)

Healthy subjects (n = 284)

n (freq. %) 356 (0.57) 264 (0.43) n (freq. %) 105 (0.34) 146 (0.47) 59 (0.19) n (freq. %) 251 (0.81)

n (freq. %) 359 (0.63) 209 (0.37) n (freq. %) 112 (0.39) 135 (0.48) 37 (0.13) n (freq. %) 247 (0.87)

205 (0.66)

172 (0.61)

n (freq. %) 579 (0.93) 41 (0.07) n (freq. %) 269 (0.87) 41 (0.13) 0 (0.0) n (freq. %) 310 (1.00) 41 (0.13)

n (freq. %) 537 (0.95) 31 (0.05) n (freq. %) 253 (0.89) 31 (0.11) 0 (0.0) n (freq. %) 284 (1.00) 31 (0.11)

3. Results S

We sequenced 30 individuals (10 for each genotype: LALA, LALG, and SASA) and randomly between patients and controls in order to determine the position of rs25531. Data have shown that this SNP is located in the sixth repetitive element (ζ) of 5-HTTLPR, according to the repeat architecture described by Nakamura et al. (2000) (Fig. 1). In the genotyping analyses, we did not detect an SG haplotype both in the patients or controls. This is consistent with previous reports (Kraft et al., 2005; Mrazek et al., 2009), observing that the G allele of rs25531 occurs very rarely on haplotypes with the S version of the Ins/ Del promoter variation.

rs25531 Allele frequencies A G Genotype frequencies AA AG GG Carriers A G

χ2 = 4.14, p = 0.04

χ2 = 4.57, p = 0.10

χ2 = 3.94, p b 0.05 OR = 0.64 95%CI: 0.41–1.00 χ2 = 1.98, p = 0.16

χ2 = 0.69, p = 0.40

χ2 = 074, p = 0.39

Not applicable χ2 = 074, p = 0.39

Fig. 1. Schematic overview and sequence of the 16 repetitive region containing 5-HTTLPR (43 bp Ins/Del start in the VII(ο) to finish IX(η) repetitive elements (marked by brackets and underlined sequence)) and the rs25531 functional SNP (located in the VI (ζ) repetitive element (designated by bracket “[A/G]”)). Individual elements of the repetitive region are depicted by Romanic numbers and Greek letters, according to the nomenclature introduced by Nakamura et al. (2000). Note that, as we reported, the definition of the exact beginning and end of 5-HTTLPR is contrasted between authors (Hu et al., 2005; Kraft et al., 2005; Nakamura et al., 2000; Wendland et al., 2006; Wray et al., 2009). Two chromatograms from the amplified genomic DNA of SASA and LALG genotypes are shown in the left-hand side of the figure. The rs25531 polymorphism is located 18 bp upstream of 5-HTTLPR.

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The distribution of genotypes was in Hardy–Weinberg equilibrium (5-HTTLPR, patients p = 0.56 and controls, p = 0.80; rs25531, patients p = 0.38 and controls, p = 1.00). The allele and genotype distributions of the 5-HTTLPR and rs25531 polymorphisms are presented in Table 2. The results showed, for 5-HTTLPR polymorphism, differences between the two populations in allele (p = 0.04) and in L allele carrier frequencies (χ2 = 3.94, p b 0.05, OR = 0.64 95%CI: 0.41–1.00). No evidence of association was observed for the rs25531 allele and genotype frequencies. In addition, we analysed the estimated and phased haplotypes distributions transforming the classification into a triallelic/biallelic model according to SLC6A4 allele expression levels: SASA, LGSA, LGLG as S′S′; LASA and LALG as L′S′; LALA as L′L′ (Hu et al., 2006; Parsey et al., 2006) (Table 3). Concerning the estimated haplotypes analyses, the frequencies of clustered haplotypes were different between patients and controls (p = 0.02). As regard the estimated phased haplotypes, the associations were found for haplotype frequencies and for those clustered (p = 0.02 and p = 0.04 respectively) but they were lost after Bonferroni correction (p = 0.1 and p = 0.12, respectively). A difference was found in the analysis of L′L′ vs. L′S′ + S′S′ (χ2 = 6.21, p = 0.01, OR = 0.64 95%CI: 0.45–0.91). The two groups were not homogeneous for the following features: Gender (67% and 52% females in patients and controls, respectively; χ2 = 13.86, p b 0.001), Age (healthy subjects 49.0 ± 16.4 (mean ± SD) years; patients 56.3 ± 13.2; F = 35.76; p b 0.0001) and Education (controls 13 ± 5 (mean ± SD) years; patients 9 ± 4; F = 150.34; p b 0.0001). Thus, to estimate the possible effects of these variables on the significant results, we carried out an ANOVA including Groups, Education and Gender as Independent Variables, Age as a Covariate and L allele carriers/LALA as Dependent Variables. In the analysis on L allele carriers only Gender, apart from Groups, showed effects (F = 4,435 p = 0.036), nevertheless, Partial Eta Squared Indices estimated the effect size to be very low (0.009). Moreover, the interaction “Groups × Gender” had no effect on the dependent variable. For the LALA ANOVA analysis, no effect of Gender was found (p = 0.08).

Table 3 Estimated and phased haplotypes distributions of the 5-HTTLPR and rs25531 polymorphisms in SLC6A4 gene in TRD patients and in controls.

Haplotype frequencies LA LG SA Clustered haplotype frequencies LA LG + SA Phased haplotype frequencies LALA LALG LASA LG S A SASA Clustered phased haplotype frequencies L′L′ L′S′ S′S′ Carriers of phased haplotypes (S′S′) vs. (L′L′ + L′S′) L′L′ vs. (L′S′ + S′S′)

TRD patients (n = 310)

Healthy subjects (n = 284)

n (freq. %) 315 (0.50) 41 (0.07) 264 (0.43) n (freq. %)

n (freq. %) 328 (0.57) 31 (0.06) 209 (0.37) n (freq. %)

315 (0.50) 305 (0.50) n (freq. %)

328 (0.57) 240 (0.43) n (freq. %)

χ2 = 5.75, p = 0.02

78 (0.25) 27 (0.09) 132 (0.43) 14 (0.04) 59 (0.19) n (freq. %)

98 (0.35) 14 (0.05) 118 (0.41) 17 (0.06) 37 (0.13) n (freq. %)

χ2 = 11.39, p = 0.02

78 (0.25) 159 (0.51) 73 (0.24) n (freq. %)

98 (0.35) 132 (0.46) 54 (0.19) n (freq. %)

73 (0.24) 78 (0.25)

54 (0.19) 98 (0.35)

χ2 = 5.78, p = 0.06

χ2 = 6.50, p = 0.04

χ2 = 1.81, p = 0.18 χ2 = 6.21, p = 0.01, OR = 0.64 95%CI: 0.45–0.91

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Two separate Independent-Samples T-test, one with Groups (patients and controls) and the other with Gender (Male and Female) as Grouping Variables, showed that only Groups affects the dependent variables (no statistical effect of Gender on the dependent variables occurred, p N 0.05). We tested their possible influence by carrying out post-hoc analyses comparing the patient sub-samples due to the presence of comorbidity in our sample. No differences in the frequency of genotypes between each group were detected (data not shown). 4. Discussion Primarily, our sequence data showed that the location of rs25531 is immediately outside of the 5-HTTLPR segment, which confirms the original polymorphism site suggested by Nakamura et al. (2000). On this basis, this is the first study on the 5-HTTLPR and rs25531 as two independent polymorphisms, and on the relative estimated/phased haplotypes in a sample of TRD patients and in control subjects. The minor G allele of rs25531 is continually in phase with the 5-HTTLPR long allele and attenuates the transcriptional efficacy compared to the 5-HTTLPR short allele. Therefore, the modulation of 5-HTTLPR by rs25531 results in haplotypes with a high (LA) or low (LG, SA or SG) transcriptional efficacy (Hu et al., 2006; Martin et al., 2007). On this basis, we clustered the phased haplotypes and the results evidenced that the carriers of LALA (alias L′L′) resulted more represented in the control group (p = 0.01), suggesting that this haplotype may have a protective effect associated to TRD (OR = 0.64 95%CI: 0.45–0.91). To date, only one study has investigated the association between 5-HTTLPR and TRD in a European origin population (Kishida et al., 2007), and negative results were reported. The authors found a deviation from Hardy–Weinberg equilibrium in their treatmentresistant patients because the 5-HTTLPR SS genotype was less frequent than expected in patients. In our analysis, this HWE deviation was not observed; in contrast, we found that the 5-HTTLPR SS genotype was more frequent in our patients. This result supports the literature reporting an excess of the S allele in depressive patients (Holsboer, 2008; Kiyohara and Yoshimasu, 2009). Therefore, Kishida et al. (2007) hypothesised that the observed discrepancy may have been due to a selection bias in their TRD patients because the 5-HTTLPR SS frequency observed in their patients (10%) was lower than those previously reported (21%–28%) in European descendent depressive patients (Smits et al., 2004). The 5-HTTLPR SS frequency observed in our study (19%) closely resembles the results obtained in research conducted by Smits et al. (2004). Other studies have reported findings contradictory to those of the present study regarding the association between the “triallelic 5HTTLPR/rs25531” polymorphism and the patient response to antidepressant drugs (Hu et al., 2007; Kraft et al., 2007; Kraft et al., 2005; Maron et al., 2009; Mrazek et al., 2009; Ruhe et al., 2009; Smeraldi et al., 2006). Our data support the hypothesis that the LALA haplotype may represent a protective marker for TRD in depressive patients, as supported by previous studies (Kraft et al., 2005; Ruhe et al., 2009; Smeraldi et al., 2006). Furthermore, lower expressing haplotypes (LG, SA or SG) increased the adverse effects of antidepressant drugs (Hu et al., 2007; Maron et al., 2009), predicted greater severity of MDD with moderate to severe life events (Zalsman et al., 2006), and increased amygdala reactivity to masked emotional faces that in turn correlate with lifetime psychiatric hospitalisation as an index of chronicity (Dannlowski et al., 2008). Recent studies (Firk and Markus, 2009; Roiser et al., 2006) have demonstrated that the S allele at 5-HTTLPR affects responses to mild stress and/or acute tryptophan depletion (ATD). These data suggest that S allele carriers may be more vulnerable to serotoninergic manipulations, resulting in an increased susceptibility to depression. Furthermore, the results highlight the importance of genetic variation

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in determining individual responses to pharmacological treatments. Neumeister's study (Neumeister et al., 2006) showed differential effects of the rs25531 polymorphism on behavioural and neural responses to tryptophan depletion (TD) in controls and in patients with remitted MDD (rMDD). The authors found that rMDD patients carrying the LA allele display a transient return of depressive symptoms during TD. In addition, the haplotypes and their putative functional equivalents (LG, SA or SG) were found to be associated with increased levels of depressogenic attributions for negative events, supporting the role of these haplotypes in cognitive vulnerability to depression (Sheikh et al., 2008). This work needs additional comments. 1) One limitation could be the absence of definitive, consensual, and standardised operational criteria for TRD. Depression is considered to be resistant when at least two trials with antidepressants from different pharmacologic classes (adequate in terms of dosage, duration, and compliance) fail to produce a significant clinical improvement (Berlim and Turecki, 2007). We utilised the Thase and Rush's (1997) five levels of resistance model, a more stringent method that is a very useful tool in the classification of TRD (Berlim and Turecki, 2007). The whole sample of our patients was resistant to SSRIs and TCAs; however it was more heterogeneous because some patients were resistant also to other antidepressant drugs (i.e. SNRIs) and/or to augmentation (i.e. ECT, rTMS). Unfortunately the Thase and Rush method does not consider the role of the other drugs and augmentation. Another important step toward the assessment of TRD concerns the level of treatment non-adherence because it is estimated at about 20% of cases considered resistant. The plasma dosage might solve the nonadherence problem, furthermore it permits the identification of rapid metabolizers (Serretti et al, 2008). Nevertheless the use of antidepressant plasma levels has provided contrasting results (Serretti et al., 2008), and regarding to metabolizer status, the heterogeneous pharmacokinetics profiles of different antidepressant classes reduces the effect of this confounding factor. 2) Heterogeneity of antidepressant drug treatments. However Mathew et al. (2008) and Wong and Licinio (2004) support that the primary mode of action of all antidepressant agents is related to an enhancement of monoaminergic neurotransmission and despite differences in the profile of receptor occupancy, antidepressant drugs show comparable efficacy across drug classes. 3) Absence of comparison with MDD responder patients group. In our study we compared TRD patients versus controls; however the involvement of L allele in MDD patient responders to SSRIs monotherapy was largely supported by numerous studies (see for review Horstmann and Binder, 2009). 4) Comorbidity. Many clinical features, such as anxiety comorbidity (mainly panic disorder and social phobia) and personality disorders have been associated with TRD (Souery et al., 2007). However, studies related to the involvement of personality disorders in resistance to treatment showed contrasting results (Petersen et al., 2002; Souery et al., 2007). Nevertheless, in order to control for this possible confounding variable, we computed post-hoc analyses between patient subsamples, and no effect was observed. Nonetheless, further investigations need to be conducted using larger comorbidity sub-samples of depressed subjects. 5) Stressful life events. Although somewhat controversial, there is still support in the literature that the association between 5-HTTLPR and both depression and response to antidepressant medication is moderated by either current or past life events (Goldman et al., 2010; Keers et al., 2010; Munafo et al., 2009). 6) Sample size. Recently, to achieve sufficient statistical power and replicable results, a frequent request is to have large samples from a minimum of 500 cases and 500 controls. However our analysis showed a sample power ≥80% and in relation to the other studies (Anttila et al., 2007a,b; Garriock et al., 2005; Kishida et al., 2007; Zhang et al., 2005), we employed the larger sample of patients and also healthy subjects.

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