Progress in Neuro-Psychopharmacology & Biological Psychiatry 30 (2006) 953 – 957 www.elsevier.com/locate/pnpbp
Serotonin transporter polymorphisms and clinical response to sertraline across ethnicities Chee Hong Ng a,⁎, Simon Easteal b , Susan Tan b , Isaac Schweitzer a , Brian Kong Wai Ho c , Salina Aziz c b
a Professorial Unit, The Melbourne Clinic, University of Melbourne, Australia John Curtin School of Medicine, Australian National University, Canberra, Australia c Department of Psychiatry, Universiti Putra Malaysia, Kuala Lumpur, Malaysia
Available online 3 April 2006
Abstract The aim of this pilot study was to examine the relationship between clinical response, adverse effects, sertraline (SERT) plasma concentrations and the genetic polymorphism of the serotonin transporter gene-linked polymorphic region (5HTTLPR) in 2 ethnic patient groups. The study involved 45 patients in a clinical trial who received a fixed dose regimen of 50 mg SERT for one week, then a variable-dose regimen for a further 6 weeks for major depressive disorder. At weeks 1 and 6, the following assessments were completed: Hamilton Depression Rating Scale (HDRS), Clinical Global Impression (CGI), drug adverse reaction scale and measurement of plasma SERT levels. Genomic analysis for the long and short allele variants of the 5HTTLPR polymorphism was also carried out. Caucasian subjects had a higher rate of l/l genotype while Chinese subjects had higher frequencies of l/s and s/s genotypes. Comparison of the subjects with the 5HTTLPR s/s genotype and those with the l/l and l/s genotypes found no significant differences in the HDRS scores, CGI scores, response rates, adverse effects and SERT plasma concentrations at week 6. © 2006 Elsevier Inc. All rights reserved. Keywords: Adverse effects; 5-HTT polymorphisms; Plasma concentrations; Sertraline
1. Introduction In spite of current advances in the pharmacotherapy of mood disorders, there remains substantial inter-individual and interethnic variability in antidepressant response. Although the determinants of such variations are not completely understood, genetic factors are thought to play a large role. The serotonin transporter (5HTT) is a prime target of action of selective serotonin reuptake inhibitors (SSRIs). Drug transporter systems Abbreviations: CGI, Clinical Global Impression; HDRS, Hamilton Depression Rating Scale; 5HTT, serotonin transporter; 5HTTLPR, serotonin transporter-linked polymorphic region; l/l and l/s, long allele genotypes; LUNSERS, Liverpool University Neuroleptic Side Effect Rating Scale; MAOIs, monoamine oxidase inhibitors; s, short allelic variant; SERT, sertraline; s/s, short allele genotype; SSRIs, selective serotonin reuptake inhibitors; l, long allelic variant; STin2, polymorphism in the 5HTT gene occurring in intron 2. ⁎ Corresponding author. Department of Psychiatry, University of Melbourne, The Melbourne Clinic, 130 Church St, Richmond 3121, Victoria, Australia. Tel.: +61 3 9420 9350; fax: +61 3 9428 5990. E-mail address:
[email protected] (C.H. Ng). 0278-5846/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.pnpbp.2006.02.015
are subject to functional polymorphisms, which may affect both drug availability and response to medication. The rate of polymorphism of the long and short allele of the serotonin transporter gene (5HTTLPR) located on chromosome 17, varies between ethnic groups. Long (l) and short (s) allele frequencies in Caucasians are 57% and 43%, respectively (Lesch et al., 1996), while in Koreans the frequencies are 14% and 86% (Lee et al., 2004). A number of studies have shown a relationship between the functional polymorphisms of the 5HTTLPR and treatment response to SSRIs (Smeraldi et al., 1998; Pollock et al., 2000; Zanardi et al., 2000, 2001; Arias et al., 2003). These studies in general found that depressed patients with the long allele genotypes (l/l and l/s) showed a greater response to SSRIs than those with the short allele (s/s) (Smits et al., 2004). However, divergent results have been found in recent studies in Korean and Japanese depressed subjects (Kim et al., 2000; Yoshida et al., 2002). This may be due to ethnic differences in the 5HTTLPR polymorphism and/or pharmacogenetics of drug targets (Ng et al., 2004).
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Cross-ethnic studies of the 5HTT genotype and response are needed to further elucidate the impact of genotype on antidepressant response patterns, and to examine if these are applicable to diverse population subgroups. This prospective pilot study aimed to test if differences exist in the response to sertraline (SERT) in relation to 5HTTLPR polymorphism when conducted across two ethnic groups where distinct inter-ethnic profiles of the allelic variations have previously been found. It is hypothesized that the functional polymorphisms of the 5HTTLPR gene influence the response to SERT but may be subject to the genotype–ethnicity interaction in Chinese and Caucasian patients. 2. Methods
HDRS at the Australian and Malaysian study sites received the same training module for investigators. The inter-rater reliability coefficient for all raters was consistently good (alpha value N 0.9). A validated translated Chinese version of the HDRS (Zheng et al., 1988) was used when necessary. Responders were defined as those subjects with a decrease in HDRS by ≥ 50% from the baseline to week 6. Side effects were assessed using a modified version of the Liverpool University Neuroleptic Side Effect Rating Scale (LUNSERS) (Day et al., 1995) which included the addition of three items: feeling anxious, increased appetite and loss of appetite. A translated Chinese version of the LUNSERS was also available for use. In addition, a subscale of serotonergic side effects was separated out from the main scale to include SSRI-specific adverse effects only.
2.1. Patient population 2.4. Plasma concentration measurements Patients with major depressive disorder were recruited from The Melbourne Clinic in Australia and Hospital Kuala Lumpur in Malaysia. Two groups comprising of 15 Caucasian patients (all Australian) and 30 Chinese patients (17 living in Australia and 13 from Malaysia) were matched according to age, sex and severity of depression. All patients were at least 18 years of age and had a diagnosis of major depressive disorder according to the DSM-IV criteria (American Psychiatric Association, 1994) with a minimum Hamilton Depression Rating Scale 17-items (HDRS) score at a baseline of 18. They were not previously refractory or intolerant to SERT, and had completed appropriate washout period for any previous antidepressant treatment (3 days for all SSRIs and venlafaxine except fluoxetine, 7 days for mirtazepine and tricyclics, 14 days for irreversible MAOIs, 28 days for fluoxetine) and had no electroconvulsive therapy treatment (ECT) in the previous 6 months. Patients with primary or comorbid diagnosis of schizophrenia, schizoaffective disorder, rapid cycling bipolar disorder, alcohol or substance dependence, dementia or significant medical condition were excluded from the study. The ethnicity of each group was specifically defined as either Caucasian or Chinese (for at least 3 generations). 2.2. Drug administration SERT was administered initially at a fixed-dose regimen of 50 mg for one week, followed by dosages ranging from 25– 200 mg/day according to clinical response over the 6 weeks treatment duration. Psychotropic drugs were not allowed except for hypnotics (e.g. zopiclone 7.5–15 mg) or anxiolytics (e.g. lorazepam 1–3 mg) for severe anxiety. Prior approval from the relevant institutional research and ethics committees and written consent were obtained before the commencement of the study.
Blood samples (12 to 20 h after the last dose) were collected by venepuncture at baseline and weeks 1 and 6. Plasma was stored at − 20 °C then sent to the same biochemical laboratory to standardise the assay methodology for plasma SERT. The sampling time and dosage of medication were documented. SERT concentrations in the plasma samples were analysed by Gas Chromatography–Mass Spectrometry using a standard method (details available from the authors or reference). 2.5. DNA genotyping for 5HTTLPR polymorphism Plasma samples originally collected for SERT assay were sent to a common genetics laboratory. DNA was extracted from 400 μl plasma from each sample using a QIAamp DNA Mini Kit (QIAGEN Inc.). The genomic DNA was amplified with a Genomiphi DNA Amplification Kit (Amersham) according to the Amersham protocol with 30 to 100 ng of DNA used for each polymerase chain reaction (PCR). The primers employed were stpr5 (5′ GGC GTT GCC GCT CTG AAT GC 3′) and stpr3 (5′ GAG GGA CTG AGC TGG ACA ACC AC 3′). PCR reactions mixture consisted of 20 μl volume, using 2 μl of DNA, 2 μl of 10× PCR buffer, 2 μl of 2 mM dNTPs, 0.8 μl of 25 mM MgCl2, 0.5 μl of 10 μM of each primer, Taq DNA polymerase and ddH2O to make a total volume of 20 μl. The PCR condition was a cycle of pre-denaturation at 95 °C for 3 min, 35 cycles of denaturation at 95 °C for 30 s denaturing, annealing at 61 °C for 30 s and extension at 72 °C for 1 min. The final cycles PCR reaction were at 72 °C for 7 min and 25 °C for 5 min. With electrophoresis, the amplified products were run at 130 V for 2.5 h, resolved on 2.5% agarose gel and visualized with ethidium bromide. Alleles were designated as short (484 bp) and long (528 bp) against a DNA marker in genotyping for the 5HTTLPR polymorphism.
2.3. Clinical measures 2.6. Statistical data analysis Efficacy was assessed using the 17-item HDRS and Clinical Global Impression for severity of illness and global improvement (CGI) (Guy, 1976). These evaluations were done at baseline and at weeks 1 and 6 of treatment. All raters for the
In the analysis of the 5HTTLPR genotype, the rates of the long (l) and short alleles (s) were compared between the ethnic groups. Those with homozygous 5HTTLPR s/s genotype were
C.H. Ng et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 30 (2006) 953–957 Table 1 Frequencies of the serotonin transporter promoter (5HTTLPR) gene variants 5HTTLPR
l allele
s allele
l/l genotype
Caucasian subjects Chinese subjects
12 (55%)
10 (45%)
5 (46%)
l/s genotype 2 (18%)
s/s genotype 4 (36%)
15 (31%)
33 (69%)
1 (4%)
13 (54%)
10 (42%)
compared with those with l/s and l/l genotypes in relation to the efficacy, side-effects and plasma SERT variables as reported in previous studies. Descriptive statistics and χ2 tests were included where applicable. All statistical tests were performed at a significant level of ∝ = 0.05. Between group and within group comparisons over time for differential outcome measures were assessed by repeated measures of ANOVA (with time as the within subject factor and genotype as the between subjects factor). The ANOVA for HDRS, CGI and LUNSERS were also done using also gender, baseline weight and dietary factors (alcohol, nicotine and caffeine) as covariates. 3. Results The sample comprised of 23 females and 22 males of mean (± SD) age 43.5 ± 13.2 years with a baseline HDRS of 23.6 ± 4.3. There were no significant differences in the baseline demographics between the Australian Chinese and Malaysian Chinese, hence they were combined for further analyses. All in the intention to treat groups were analysed with 45 subjects followed up at week 1 and 39 subjects at week 6. The functional polymorphisms of the 5HTTLPR gene were analysed for all subjects but genotype results were unavailable for 10 subjects due to sample quality. The allelic frequencies for these subjects are displayed in Table 1. The frequencies of l and s alleles in Caucasians were 55% and 45%, respectively, whereas in Chinese subjects, they were 31% and 69%. In terms of the frequencies of 5HTTLPR homozygous and heterozygous genotypes, Caucasian subjects had a higher proportion of l/ l genotype while Chinese subjects had higher frequencies of l/s and s/s genotypes (χ2 = 9.83, p = 0.007). The homozygous 5HTTLPR s/s genotype group was compared with the combined l/l and l/s genotypes (Table 2). One-way ANOVA did not reveal any significant differences between the two groups for age, gender, ethnicity, baseline weight, duration of illness, HDRS score, CGI score, and LUNSERS score at baseline. Table 2 Demographics and baseline ratings (mean ± SD) for the genotype groups
Age (years) Female:male Caucasians:Chinese Baseline weight (kg) Duration of illness (years) Baseline HDRS Baseline CGI-S Baseline LUNSERS
5HTTLPR l/l and l/s (n = 21)
5HTTLPR s/s (n = 14)
42.2 ± 11.4 13:8 7:14 71.2 ± 23.7 1.52 ± 0.68
40.6 ± 13.9 4:10 4:10 69.5 ± 17.6 1.57 ± 0.76
23.5 ± 4.2 4.14 ± 0.66 67.4 ± 24.5
22.6 ± 3.4 4.07 ± 0.48 74.4 ± 26.6
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Table 3 Rating scores and sertraline data (mean ± SD) at weeks 1 and 6 for the genotype groups Week 1
HDRS CGI-S LUNSERS Dose (mg) Plasma SERT (ng/ml) Plasma SERT/ dose a b
Week 6
5HTTLPR l/l and l/s
5HTTLPR s/s
5HTTLPR l/l and l/s
5HTTLPR s/s
18.3 ± 5.1 2.91 ± 1.18 56.8 ± 30.0 61.9 ± 20.3
17.0 ± 5.5 2.78 ± 0.80 63.4 ± 23.8 50 ± 17.0
7.7 ± 5.8
9.0 ± 6.4
0.15 ± 0.11
0.18 ± 0.12
10.4 ± 5.1 2.0 ± 0.77 41.3 ± 20.7 83.3 ± 33.2 (n = 18) 12.8 ± 6.8 (n = 17) 0.16 ± 0.097 (n = 17)
8.6 ± 4.0 1.73 ± 0.65 44.0 ± 16.3 56.8 ± 16.2 (n = 11)a 14.2 ± 9.7 (n = 13) 0.23 ± 0.11 (n = 11)b
F = 6.07, df 1,27, p = 0.020. F = 3.67, df 1,27, p = 0.066.
The mean HDRS score decreased significantly for both groups during treatment (Table 3; F(3,84)= 153.8, p b 0.001). However, no significant difference was seen in the mean HDRS scores between the genotype groups (F(1,28) = 1.53, p = 0.23) and no significant genotype by time interaction was found (F(1,28) = 0.01, p = 0.95). The proportion of responders in the s/s genotype group was 66.6%, which was higher than the l/l and l/s groups (55.7%) but the difference was not statistically significant (χ2 = 0.37, p = 0.54). The CGI scores decreased significantly for both groups across the study period (Table 3; F(3,81)= 35.9, p b 0.001) but no significant difference was observed between the genotype groups (F(1,27) = 0.72, p = 0.40). The interaction between group and time was also not significant (F(1,28) = 0.19, p = 0.67). To explore the effect of 5HTTLPR genotype on drug side effects and tolerance, the LUNSERS scores were compared between the genotype groups. Although the LUNSERS scores decreased significantly over time for both groups (F(3,81) = 19.8, p b 0.001), no significant difference was observed between the genotype groups (F(1,27) = 0.24, p = 0.63). Similarly, the serotonergic side effects subscale scores were not significantly different between groups (F(1,27) = 0.25, p = 0.62). The mean SERT dose, plasma concentrations and dose/plasma concentration ratio at weeks 1 and 6 are also presented in Table 3. Plasma SERT concentrations were not different between the two genotypes at either week. The dose of SERT was lower for the s/s group, although only significantly so for week 6 (F(1,27) = 6.07, p = 0.020), and the ratio of plasma SERT/dose was almost significantly higher for the s/s group at week 6 (F(1,27) = 3.67, p = 0.066). 4. Discussion 4.1. SERT efficacy The 5HTTLPR polymorphisms have been shown to affect serotonin transporter mRNA synthesis leading to changes in the 5HTT expression and 5HT cellular uptake (Lesch et al., 1996). The short variant of 5HTTLPR has been associated with a 50% reduction in the transcriptional activity and serotonin uptake
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(Heils et al., 1996). Furthermore, the functional polymorphism of the 5HTTLPR gene has been implicated in the antidepressant response to the SSRIs. Recent studies have found a greater response in depressed subjects with l alleles compared to those with s alleles (Smits et al., 2004). The results from this pilot study however do not support an association between the 5HTTLPR polymorphisms and SERT efficacy in a mixed ethnic sample. 4.2. Ethnic differences in 5HTTLPR allele The distribution of the 5HTTLPR allele frequencies in the Caucasian and Chinese group was consistent with those observed in the studies of Caucasians and Korean populations, respectively (Lesch et al., 1996; Lee et al., 2004). The rates of l/ l, l/s and s/s variants of the 5HTTLPR in the Chinese subjects in this study (4%, 54% and 42%) were also similar to the frequencies found in the Korean study by Kim et al. (2000) (4%, 39% and 57%), respectively. Consequently, variations in the ethnic frequencies of the 5HTTLPR polymorphisms may affect the genotype influence on SSRI antidepressant response within a particular population. Furthermore, if there are ethnic differences in the effect of the l and s alleles on treatment efficacy, as suggested by the inconsistent findings in Asian samples (Kim et al., 2000), genotype–ethnicity interaction on SSRI treatment outcome must also be considered. Further prospective cross-ethnic studies using comparable SSRI levels are needed to determine the effect of both 5HTTLPR genotypes and ethnicity on SSRI response. The lack of any significant findings may arise from the inadequate power of this study due to the mixed ethnicity, which could increase the genotype variability in the sample. To ensure satisfactory effect size, adequate subject numbers should be included to allow for sample stratification into the different ethnic groups in future studies. A second polymorphism in the 5HTT gene occurring in intron 2 (STin2) may also be linked to the 5HTT gene expression and antidepressant response (Kim et al., 2000). Further studies measuring both 5HTTLPR and STin2 variant alleles could clarify the relative contributing effects of both polymorphisms on the SSRI efficacy. 4.3. Effects of other genetic polymorphisms In addition, polymorphisms of enzymes responsible for the metabolism of the SSRIs may also affect clinical response. In this study, there was no difference between the groups in plasma SERT concentrations however, the dose of SERT was significantly lower in the short allele group leading to a higher plasma SERT/dose ratio in that group. This is possibly accounted for by the higher number of Chinese subjects in the short allele group. Slower rates of metabolism of psychotropic drugs and increased drug sensitivity have been previously shown in Asian populations including Chinese ethnicity (Lin et al., 1996). Both lower drug clearance or lower dosage requirement may explain the differences found in the s/s group. Hence, future studies should also include the analysis of the relevant polymorphisms of cytochrome P450 enzymes to determine the metabolic genotype of the study subjects.
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