Polymorphisms in the SLC6A4 and HTR2A Genes Influence Treatment Outcome Following Antidepressant Therapy

Polymorphisms in the SLC6A4 and HTR2A genes influence treatment outcome following antidepressant therapy

MJV Wilkie1, G Smith1, RK Day2, The majority of antidepressant drugs act by increasing synaptic serotonin 2 3 levels in the brain. Genetic variation in serotonin-related genes may therefore K Matthews , D Smith , influence antidepressant efficacy. In this study, nine polymorphisms in four 3 4 D Blackwood , IC Reid serotonin receptor genes (HTR1B, HTR2A, HTR5A and HTR6) and the and CR Wolf1 serotonin transporter gene (SLC6A4) were analysed to investigate their influence on antidepressant response in a well-characterized unipolar 1Biomedical Research Centre, Ninewells Hospital depressive population (n ¼ 166) following a protocolized treatment regimen. and Medical School, University of Dundee, 5-HTTLPR short-allele homozygotes were significantly associated with Dundee, UK; 2Department of Psychiatry, Ninewells Hospital and Medical School, Dundee, both remission (odds ratios (OR) ¼ 4.00, P ¼ 0.04) and response UK; 3Division of Psychiatry, Royal Edinburgh (OR ¼ 5.06, P ¼ 0.02) following second switch treatment, with a similar Hospital, University of Edinburgh, Edinburgh, UK trend observed following initial treatment and paroxetine therapy. Following 4 and Department of Mental Health, Institute of initial treatment, unipolar patients homozygous for the SLC6A4 intron 2 Medical Sciences, Forresterhill Hospital, Aberdeen, UK repeat polymorphism were significantly associated with lack of remission (OR ¼ 0.38, P ¼ 0.02) and lack of response (OR ¼ 0.42, P ¼ 0.01). Addition- Correspondence: ally, the HTR2A C1354T polymorphism showed an association with remission Professor CR Wolf, Biomedical Research Centre, (OR ¼ 7.50, P ¼ 0.002) and response (OR ¼ 5.25, P ¼ 0.01) following Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, paroxetine therapy. These results suggest that genetically determined Scotland, UK. variation in serotonin receptor genes makes a significant contribution to E-mail: [email protected] the efficacy of commonly prescribed antidepressant drugs. The Pharmacogenomics Journal (2009) 9, 61–70; doi:10.1038/sj.tpj.6500491; published online 5 February 2008

Keywords: serotonin; depression; antidepressants; treatment response; polymorphism; pharmacogenetics

Introduction

Serotonin receptors can be directly targeted by antidepressants, for example imipramine directly antagonizes the serotonin receptor 2A (HTR2A), whereas clomipramine and mianserin have been shown to target serotonin receptor 6 (HTR6).1 The majority of serotonin receptors are G-protein-coupled receptors that modulate intra-cellular levels of cAMP. The majority of studies investigating the association between genetic variations in the serotonin genes and antidepressant response have analysed polymorphisms in the serotonin transporter gene (SLC6A4), which is targeted by selective serotonin reuptake inhibitors (SSRIs) and the majority of tricyclic antidepressants (TCAs).2 The SLC6A4 gene is located on chromosome 17q11.1–q12, spans 31 kb, Received 25 April 2006; revised 18 January 3 2007; accepted 26 November 2007; published consists of 14 exons and encodes a 630 amino-acid protein. Two common online 5 February 2008 polymorphisms have been shown to affect expression of SLC6A4: a 44 bp Polymorphisms and antidepressant response MJV Wilkie et al 62

deletion/insertion polymorphism (5-HTTLPR) in the 50 remitters (w2 ¼ 5.23, P ¼ 0.07) and responders and non- flanking regulatory region of the SLC6A4 gene and a second responders (w2 ¼ 5.74, P ¼ 0.06), following initial drug variable number of tandem repeats (VNTR) polymorphism treatment (Table 1B). In both cases, the frequency of in intron 2. This VNTR polymorphism consists of 9, 10 or 12 individuals carrying two copies of the 12-copy repeat was copies of a 16–17 bp repeat element. significantly associated with lack of remission (OR ¼ 0.38, The serotonin system plays a central role in the action of P ¼ 0.02) and lack of response (OR ¼ 0.42, P ¼ 0.01) (Table 2A). antidepressant drugs. Genetic variation that may affect the No significant associations were found between ADR integrity of this system may therefore influence treatment incidence and this genotype following initial therapy response. In this study, we have investigated a number of (w2 ¼ 3.08, P ¼ 0.55). polymorphisms in a variety of serotonin receptor genes to determine whether inheritance of specific polymorphisms Second switch therapy. No significant associations were is associated with response to a variety of commonly found following second switch treatment for the SLC6A4 prescribed antidepressants. intron 2 polymorphism, although 12-copy repeat homozygote carriers were more frequent in non-remitter Results (45 vs 36%, w2 ¼ 1.67, P ¼ 0.80) and non-responder (40 vs 37%, w2 ¼ 1.49, P ¼ 0.83) groups. 5-HTTLPR Initial therapy. Following initial drug treatment, a higher frequency of 5-HTTLPR short-allele homozygotes was Paroxetine therapy. Higher frequencies of the 12 allele observed in responders compared to non-responders (29 vs homozygote genotype were also observed in non-remitters 2 19%, w2 ¼ 2.77, P ¼ 0.25), although this failed to reach (45 vs 28%, w ¼ 2.10, P ¼ 0.36) and non-responders (46 vs 2 significance. Similarly, no significant differences in 33%, w ¼ 2.94, P ¼ 0.23) to paroxetine treatment (Table 1B), genotype distribution were associated with remission although this did not reach statistical significance. (w2 ¼ 0.25, P ¼ 0.88) or adverse drug reaction (ADR) 2 incidence (w ¼ 2.52, P ¼ 0.28) (Table 1A). HTR2A T102C and C1354T Initial therapy. Two polymorphisms were analysed in the

Second switch therapy. Significant differences in 5-HTTLPR HTR2A gene. HTR2A T102C genotype did not influence genotype frequencies were observed following second response to initial drug therapy, assessed in terms of switch treatment (Table 1A). Inheritance of two copies of remission (w2 ¼ 0.42, P ¼ 0.81), response (w2 ¼ 0.10, P ¼ 0.95) the 5-HTTLPR short allele was associated with both or ADR incidence (w2 ¼ 1.23, P ¼ 0.54). In addition, no remission (w2 ¼ 5.98, P ¼ 0.05) and response (w2 ¼ 7.40, associations were observed in response to initial therapy P ¼ 0.025) following longer-term treatment, while a higher correlating HTR2A C1354T genotype and remission frequency of heterozygotes was found in the non-remission (w2 ¼ 2.20, P ¼ 0.33), response (w2 ¼ 2.02, P ¼ 0.36) and ADR and non-response groups (Table 1A). These results suggest incidence (w2 ¼ 0.03, P ¼ 0.86). a recessive mode of action for the short allele, with homozygotes associated with remission (odds ratios Second switch therapy. Following second switch treatment, (OR) ¼ 4.00, P ¼ 0.04) and response (OR ¼ 5.06, P ¼ 0.02) the HTR2A T C genotype frequencies did not significantly (Table 2b). 5-HTTLPR genotype did not influence ADR 102 differ between remitters and non-remitters (w2 ¼ 1.40, incidence (w2 ¼ 0.39, P ¼ 0.82). P ¼ 0.50), responders and non-responders (w2 ¼ 0.85, P ¼ 0.65), and between ADR and non-ADR groups Paroxetine therapy. Following paroxetine treatment, the 2 (w ¼ 0.45, P ¼ 0.80). The HTR2A C1354T genotype did not 5-HTTLPR short/short genotype was more frequent in influence second switch therapy in terms of remission responders compared to non-responders (40 vs 19%, (w2 ¼ 0.03, P ¼ 0.98), response (w2 ¼ 0.69, P ¼ 0.71) and ADR w2 P ¼ 4.20, ¼ 0.12) and in remitters compared to non- incidence (w2 ¼ 3.01, P ¼ 0.22). remitters (38 vs 24%, w2 ¼ 2.24, P ¼ 0.33). However, due to lower number of patients in this group, these figures failed to reach significance. ADRs following paroxetine therapy Paroxetine therapy. HTR2A C1354T heterozygotes were were not influenced by 5-HTTLPR genotype (w2 ¼ 2.94, significantly associated with improved response to P ¼ 0.23). paroxetine therapy. There was a significantly higher frequency of heterozygotes in the remitter and response Early onset depressive population. The 5-HTTLPR short-allele groups in comparison to the non-remitter (OR ¼ 7.50, homozygotes genotype was more frequent in patients P ¼ 0.002) and non-response groups (OR ¼ 5.25, P ¼ 0.01) responding to antidepressant treatment (28 vs 14%, (Table 2C). The HTR2A C102T genotype did not influence 2 2 w2 ¼ 5.88, P ¼ 0.05) (Table 3). remission (w ¼ 0.65, P ¼ 0.72) or response (w ¼ 1.36, P ¼ 0.51) but was associated with the incidence of ADRs SLC6A4 intron 2 (w2 ¼ 9.73, P ¼ 0.008) following paroxetine treatment with Initial therapy. Genotype frequencies for the SLC6A4 intron patients inheriting the HTR2A C102C genotype associated 2 polymorphism differed between remitters and non- with the incidence of ADRs (OR ¼ 24.69, P ¼ 0.007).

The Pharmacogenomics Journal Table 1 Genotype frequency distribution in response groups following initial, second switch and paroxetine therapy

Response following initial therapy Response following second switch therapy Response following paroxetine therapy

Remitter Non-remitter Responder Non-responder ADR Non-ADR Remitter Non-remitter Responder Non-responder ADR Non-ADR Remitter Non-remitter Responder Non-responder ADR Non-ADR (n ¼ 39) (n ¼ 124) (n ¼ 58) (n ¼ 105) (n ¼ 15) (n ¼ 148) (n ¼ 70) (n ¼ 40) (n ¼ 98) (n ¼ 30) (n ¼ 36) (n ¼ 105) (n ¼ 21) (n ¼ 55) (n ¼ 30) (n ¼ 46) (n ¼ 4) (n ¼ 72)

5-HTTLPR A Long/long 12 (30%) 40 (32%) 15 (26%) 37 (35%) 7 (46%) 45 (31%) 26 (37%) 14 (35%) 34 (35%) 9 (30%) 10 (28%) 35 (33%) 7 (33%) 17 (31%) 9 (30%) 15 (33%) 2 (50%) 22 (31%) Long/short 17 (44%) 57 (46%) 26 (45%) 48 (46%) 4 (27%) 70 (47%) 27 (39%) 23 (58%) 38 (39%) 19 (63%) 17 (47%) 45 (43%) 6 (29%) 25 (45%) 9 (30%) 22 (48%) 0 31 (43%) Short/short 10 (26%) 27 (22%) 17 (29%) 20 (19%) 4 (27%) 33 (22%) 17 (24%) 3 (7%) 26 (26%) 2 (7%) 9 (25%) 25 (24%) 8 (38%) 13 (24%) 12 (40%) 9 (19%) 2 (50%) 19 (26%) w2 0.25 2.77 2.52 5.98 7.40 0.39 2.24 4.20 2.94 P-value 0.88 0.25 0.28 0.05 0.025 0.82 0.33 0.12 0.23 SLC6A4 intron 2 B 12/12 10 (26%) 59 (47%) 17 (29%) 52 (49%) 8 (53%) 61 (41%) 25 (36%) 18 (45%) 36 (37%) 12 (40%) 21 (58%) 35 (33%) 6 (28%) 25 (45%) 10 (33%) 21 (46%) 3 (75%) 28 (39%) 12/10 22 (56%) 47 (38%) 30 (52%) 39 (37%) 7 (47%) 62 (42%) 32 (46%) 16 (40%) 46 (47%) 12 (40%) 10 (28%) 52 (49%) 12 (57%) 22 (40%) 17 (57%) 17 (37%) 1 (25%) 33 (46%) 10/10 5 (13%) 17 (14%) 9 (16%) 13 (12%) 0 22 (15%) 11 (16%) 5 (13%) 14 (14%) 5 (17%) 4 (11%) 16 (15%) 1 (5%) 7 (13%) 1 (3.3%) 7 (15%) 0 8 (11%) 12/9 1 (2.5%) 1 (1%) 1 (1.5%) 1 (2%) 0 2 (1.5%) 1 (1%) 1 (2%) 1 (1%) 1 (3%) 1 (3%) 1 (1.5%) 1 (5%) 1 (2%) 1 (3.3%) 1 (2%) 0 2 (3%) 10/9 1 (2.5%) 0 1 (1.5%) 0 0 1 (0.5%) 1 (1%) 0 1 (1%) 0 0 1 (1.5%) 1 (5%) 0 1 (3.3%) 0 0 1 (1%) w2 5.23 5.74 3.08 1.67 1.49 8.40 2.10 2.94 2.20 P-value 0.07 0.06 0.55 0.80 0.83 0.08 0.36 0.23 0.33

HTR6 C CC 33 (85%) 96 (77%) 50 (86%) 79 (75%) 10 (67%) 119 (80%) 57 (81%) 28 (70%) 77 (79%) 24 (80%) 30 (83%) 82 (78%) 18 (86%) 43 (78%) 27 (90%) 34 (74%) 3 (75%) 58 (81%) CT 6 (15%) 25 (20%) 8 (14%) 23 (23%) 5 (33%) 26 (18%) 13 (19%) 10 (25%) 20 (20%) 5 (17%) 6 (17%) 21 (20%) 3 (14%) 10 (18%) 3 (10%) 10 (22%) 1 (25%) 12 (17%) TT 0 3 (3%) 0 3 (2%) 0 3 (2%) 0 2 (5%) 1 (1%) 1 (3%) 0 2 (2%) 0 2 (4%) 0 2 (4%) 0 2 (2%) w2 0.54 2.10 0.84 4.43 0.96 0.93 0.17 2.04 0.07 P-value 0.46 0.15 0.36 0.11 0.62 0.63 0.68 0.15 0.79

HTR1B AÀ161 T D AA 19 (49%) 67 (54%) 29 (50%) 57 (54%) 8 (53%) 78 (53%) 36 (52%) 24 (60%) 47 (48%) 20 (67%) 15 (42%) 56 (53%) 8 (38%) 31 (56%) 14 (46.7%) 25 (54%) 1 (25%) 38 (53%) AT 18 (46%) 48 (39%) 25 (43%) 41 (39%) 6 (40%) 60 (40%) 31 (44%) 11 (28%) 45 (46%) 7 (23%) 19 (53%) 42 (40%) 13 (62%) 20 (36%) 15 (50%) 18 (39%) 2 (50%) 31 (43%) TT 2 (5%) 9 (7%) 4 (7%) 7 (7%) 1 (7%) 10 (7%) 3 (4%) 5 (12%) 6 (6%) 3 (10%) 2 (5%) 7 (7%) 0 4 (8%) 1 (3.3%) 3 (7%) 1 (25%) 3 (4%) w2 0.76 0.29 0.002 4.58 4.91 1.79 1.37 1.05 3.74 P-value 0.68 0.87 1.00 0.10 0.09 0.41 0.24 0.59 0.15 J Wilkie MJV response antidepressant and Polymorphisms

HTR1B G816 C E GG 23 (59%) 66 (53%) 34 (59%) 55 (52%) 10 (67%) 79 (53%) 41 (59%) 23 (58%) 57 (58%) 16 (53%) 21 (58%) 55 (52%) 15 (71%) 30 (54%) 20 (67%) 25 (54%) 2 (50%) 43 (60%) GC 14 (36%) 48 (39%) 19 (33%) 43 (41%) 4 (27%) 58 (39%) 22 (31%) 16 (40%) 31 (32%) 13 (43%) 14 (39%) 39 (37%) 6 (29%) 22 (40%) 8 (26%) 20 (43%) 2 (50%) 26 (36%) CC 2 (5%) 10 (8%) 5 (8%) 7 (7%) 1 (6%) 11 (8%) 7 (10%) 1 (2%) 10 (10%) 1 (4%) 1 (3%) 11 (11%) 0 3 (6%) 2 (7%) 1 (3%) 0 3 (4%) al et w2 0.59 1.12 1.01 2.52 2.27 2.07 1.16 2.79 0.15 P-value 0.75 0.57 0.60 0.28 0.32 0.36 0.28 0.25 0.70

HTR2A T102 C F CC 15 (38%) 41 (33%) 20 (34%) 36 (34%) 7 (47%) 49 (33%) 26 (37%) 14 (35%) 34 (35%) 9 (30%) 13 (36%) 35 (33%) 5 (24%) 18 (32%) 7 (23%) 16 (35%) 4 (100%) 19 (26%) CT 17 (44%) 57 (46%) 27 (47%) 47 (45%) 6 (40%) 68 (46%) 28 (40%) 20 (50%) 43 (44%) 16 (53%) 17 (47%) 47 (45%) 11 (52%) 24 (44%) 16 (54%) 19 (41%) 0 35 (49%) TT 7 (18%) 26 (21%) 11 (19%) 22 (21%) 2 (13%) 31 (21%) 16 (23%) 6 (15%) 21 (21%) 5 (17%) 6 (17%) 23 (22%) 5 (24%) 13 (24%) 7 (23%) 11 (24%) 0 18 (25%) w2 0.42 0.10 1.23 1.40 0.85 0.45 0.65 1.36 9.73 P-value 0.81 0.95 0.54 0.50 0.65 0.80 0.72 0.51 0.008

HTR2A C1354 T G CC 29 (74%) 104 (84%) 44 (76%) 89 (85%) 13 (87%) 120 (81%) 56 (80%) 32 (80%) 76 (78%) 25 (83%) 30 (83%) 83 (79%) 12 (57%) 46 (84%) 19 (63%) 39 (85%) 4 (100%) 54 (75%) CT 8 (21%) 14 (11%) 10 (17%) 12 (11%) 2 (13%) 20 (14%) 10 (14%) 6 (15%) 15 (15%) 4 (13%) 6 (17%) 14 (13%) 9 (43%) 5 (9%) 10 (33%) 4 (9%) 0 14 (19%) TT 2 (5%) 6 (5%) 4 (7%) 4 (4%) 0 8 (5%) 4 (6%) 2 (5%) 7 (7%) 1 (4%) 0 8 (8%) 0 4 (7%) 1 (4%) 3 (6%) 0 4 (6%) w2 2.20 2.02 0.03 0.03 0.69 3.01 4.53 7.43 1.31 P-value 0.33 0.36 0.86 0.98 0.71 0.22 0.03 0.02 0.52 h hraoeoisJournal Pharmacogenomics The HTR5A GÀ19 C H GG 14 (36%) 66 (53%) 22 (38%) 58 (55%) 8 (53%) 72 (49%) 33 (47%) 22 (55%) 46 (47%) 17 (57%) 20 (56%) 50 (48%) 9 (43%) 27 (49%) 13 (43%) 23 (50%) 3 (75%) 33 (46%) GC 20 (51%) 48 (39%) 29 (50%) 39 (37%) 6 (40%) 52 (42%) 29 (41%) 15 (38%) 41 (42%) 11 (37%) 11 (30%) 44 (42%) 11 (52%) 21 (38%) 14 (47%) 18 (39%) 0 32 (44%) CC 5 (13%) 10 (8%) 7 (12%) 8 (8%) 1 (7%) 14 (9%) 8 (12%) 3 (7%) 11 (11%) 2 (6%) 5 (14%) 11 (10%) 1 (5%) 7 (13%) 3 (10%) 5 (11%) 1 (25%) 7 (10%) w2 3.67 4.57 0.19 0.81 1.06 1.50 1.77 0.43 3.30 P-value 0.16 0.10 0.91 0.67 0.59 0.47 0.41 0.81 0.19

HTR5A A12 T I AA 0 7 (6%) 1 (2%) 6 (6%) 2 (13%) 5 (3%) 3 (4%) 1 (2%) 3 (3%) 1 (3%) 1 (3%) 4 (4%) 1 (5%) 2 (4%) 1 (3%) 2 (4%) 1 (25%) 2 (3%) AT 16 (41%) 52 (42%) 24 (41%) 44 (42%) 5 (33%) 63 (43%) 29 (41%) 14 (35%) 41 (42%) 8 (27%) 14 (39%) 44 (42%) 11 (52%) 25 (45%) 14 (47%) 22 (48%) 2 (50%) 34 (47%) TT 23 (59%) 65 (52%) 33 (57%) 55 (52%) 8 (54%) 80 (54%) 38 (55%) 25 (63%) 54 (55%) 21 (70%) 21 (58%) 57 (54%) 9 (43%) 28 (51%) 15 (50%) 22 (48%) 1 (25%) 36 (50%) w2 2.44 1.53 3.42 0.79 2.26 1.13 0.41 0.07 5.23 P-value 0.30 0.47 0.18 0.67 0.32 0.57 0.82 0.97 0.07 63 Groups frequencies highlighted in bold text indicate where genotype frequencies produced w2 values greater than 5 or P-values o0.05. Polymorphisms and antidepressant response MJV Wilkie et al 64

Table 2 Further analysis of genotype effect on response

Polymorphism Response group Genotype association Odds ratio 95% Confidence P-value interval

A. Initial response SLC6A4 intron 2 Remitters vs non-remitters 12/12 vs remaining genotypes 0.38 0.17, 0.85 0.02

SLC6A4 intron 2 Responders vs non-responders 12/12 vs remaining genotypes 0.42 0.21, 0.84 0.01

B. Second switch response SLC6A4 intron 2 ADR vs no ADR 12/12 vs remaining genotypes 2.68 1.24, 5.83 0.02 5-HTTLPR Remitters vs non-remitters Short/short vs long/short+long/long 4.00 1.10, 14.50 0.04 5-HTTLPR Responders vs non-responders Short/short vs long/short+long/long 5.06 1.10, 22.70 0.02

C. Paroxetine response HTR2A T102C ADR vs no ADR CC vs CT+TT 24.69 1.23, 480.40 0.007 HTR2A C1354T Remitters vs non-remitters CT vs CC+TT 7.50 2.12, 26.50 0.002 HTR2A C1354T Responders vs non-responders CT vs CC+TT 5.25 1.47, 18.81 0.01

Table 3 5-HTTLPR genotype in Edinburgh early onset population They also observed an increase in heterozygotes in non- responders (58 vs 32%), suggesting that the short allele was 5-HTTLPR Responder Non-responder acting in a recessive manner. A second study of 66 depressed Japanese patients also found that short-allele homozygotes Long/long 24% 53% were more frequent in responders to fluvoxamine (69 vs Long/short 48% 33% 32%, P ¼ 0.01). Again, heterozygote frequencies were higher Short/short 28% 14% w2 5.88 in non-responders (47 vs 31%) which further suggests a 5 P-value 0.05 recessive allele effect. Both these studies were, however, carried out in Oriental populations where the frequencies of Bold value signifies Pp0.05. the 5-HTTLPR genotypes differ from Caucasian populations, with the short/short genotype frequency approximately 25% in Caucasian populations but 57% in Asian populations.2 However, a recent study using a mixed American

HTR1B AÀ161T, HTR1B C816G, HTR5A GÀ19C, HTR5A A12T and population (78% Caucasians, 6% African American, 7% HTR6 C267T Hispanic, 5% Asian and 3% other) also found the short allele Table 1 illustrates that no significant difference in genotype to be associated with response to fluoxetine.6 frequency between response groups was observed for any In contrast to these findings, the majority of previous polymorphisms in the remaining 5-HT receptor genes. studies investigating the inheritance of 5-HTTLPR genotype on antidepressant efficacy have found the short allele to be associated with reduced antidepressant response.7–16 This Discussion may be explained by a number of variables that differ between the studies. First, variation in the criteria used to The serotonin system is targeted by antidepressants. Our measure response has differed between studies, with a hypothesis was that genetic variation in genes influencing reduction of 50% or more from baseline Hamilton serotonin signalling cascades may therefore be associated Depression Rating Scale (HAMD) scores used in certain with antidepressant response. studies,4,5,7,13,15–17 but an HAMD score of 8 or less was used We demonstrated a significant association between 5- to determine response in other studies.8,12 In addition, a HTTLPR short-allele homozygotes and both remission and further criterion for response was used in certain studies response following second switch drug therapy. In support where differences in HAMD score were analysed before and of these findings, a similar association was observed in a after treatment.11,14,18 Such differences in determining second distinct and pathologically different depressive response may potentially lead to situations where one population. individual may be classified as a responder in one study This observation has previously been reported in three but as a non-responder in another. Our study assessed both studies. Kim et al.4 reported that the 5-HTTLPR short/short response and remission to eliminate this variability and genotype was more frequent in responders to either found the 5-HTTLPR short-allele homozygotes to be more fluoxetine or paroxetine treatment (64 vs 36%, P ¼ 0.007). frequent in both groups.

The Pharmacogenomics Journal Polymorphisms and antidepressant response MJV Wilkie et al 65

Second, inclusion criteria have also differed between In comparison to the 5-HTTLPR polymorphism, only two studies, for example some studies included bipolar studies have previously investigated the effect of the SLC6A4 patients,10 others unipolar12,13,15 and some both.11 These intron 2 polymorphism on antidepressant response. Kim differences may result in variation in response, for example et al.4,19 found the 12-copy repeat to be associated with bipolar patients going through a manic phase may be paroxetine and fluoxetine response in 120 depressed observed as antidepressant responders. By analysing early Koreans, although a Japanese study found no association onset and unipolar depressive populations, we have con- with this polymorphism and fluvoxamine response. Our firmed that the 5-HTTLPR genotype has the same effect in findings suggest that individuals homozygous for the two separate depressive populations. 12-copy repeat are more likely to have a poor therapeutic A third variable differing between studies was the time point outcome following antidepressant treatment. at which HAMD scores were evaluated. A number of studies The difference in results between our study and Kim et al.’s determined response after 4 weeks,11,17,18 6 weeks4,5,8,12 or had findings may be due to the differences in allele frequencies multiple time points when the HAMD score was measured.13–16 between Oriental and Caucasians populations. The Response in our study was assessed following initial therapy frequency for the 12-copy allele homozygotes was 42% in that gave an indication of how genotype influenced initial our study, but 81% in the Korean population4 and 71% in a drug treatment, while the effect on longer-term treatment Japanese population.19 was assessed following second switch therapy. The 12-copy allele of the SLC6A4 intron 2 polymorphism To assess whether there were inherent differences in has been associated with increased gene transcription20,21 depression scores in our responsive and non-responsive and the 5-HTTLPR short allele with reduced transcrip- patients, baseline HAMD scores were compared in responders/ tion.22,23 We have shown that 5-HTTLPR short-allele homo- non-responders, remitters/non-remitters and in patients zygotes are associated with favourable response, while suffering/not suffering ADRs following both initial and homozygotes for the SLC6A4 intron 2 12-copy allele are second switch therapy (Table 4). Although significant associated with poorer response, suggesting that increased differences in HAMD scores were observed between remitters/ transcription of the SLC6A4 gene results in a higher non-remitters and responders/non-responders, we do not probability of poor antidepressant response. believe that these represent true clinical differences between The polymorphisms in the SLC6A4 gene showed the same response groups. In addition, as we did not observe similar trend independent of the population analysed, suggesting associations for all genotypes tested, we are confident that that these polymorphisms influence response irrespective of our results do not simply reflect inherent differences in time and drug type. However, when response to paroxetine depression severity. was analysed, two further polymorphisms in the HTR2A

Table 4 Type of antidepressants prescribed and number of remitters, responders and adverse drug reactions following initial and second switch therapy and in an early onset depressive population

Initial therapy Second switch therapya Early onset depressives b,c

A. Number and type of antidepressants prescribed SSRI 104 131 57 TCA 30 100 12 MAOI 1 20 3 SNRI 2 5 24 Other* 26 51 10 Total 163 307 106

B. Response and remission following initial and second switch drug therapy Remitters 39 (24%) 70 (64%) — Non-remitters 124 (76%) 40 (36%) — Responders 59 (36%) 98 (77%) 58 (83%) Non-responders 104 (64%) 30 (33%) 12 (17%) ADR 15 (9%) 36 (26%) — No ADR 148 (91%) 105 (74%) —

Abbreviations: ADR, adverse drug reaction; MAOI, monoamine oxidase inhibitor; SNRI, seletive norepinephrine reuptake inhibitor; SSRI, selective serotonin reuptake inhibitor; TCA, tricyclic antidepressant. *Other refers to mirtazapine, nefazodone and trazadone. aFewer patients were included in second switch analysis due to previous remission and/or reduced patient compliance. bEarly onset depressive patients were often prescribed more than one medication before clinical response was measured. cRemission and ADR were not measured in this population.

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gene showed significant associations to therapeutic out- in pharmacogenetic studies analysing SLC6A4 polymor- come suggesting a paroxetine-specific action. phisms. A number of possible confounding factors (for A number of studies have provided evidence that the example, axis II comorbidity, drug plasma levels) were not HTR2A receptor is involved in susceptibility to depression recorded in this and previous similar studies, while other and in achieving antidepressant response. The first of these potential confounding factors (specific psychotherapy inter- showed the HTR2A receptor to be overexpressed in ventions, antipsychotic medications) were not used in depressed patients.24 A further study showed that down- patients in this study, all of which may go some way to regulation of this receptor was observed after paroxetine rationalize differences in reported results. However, the therapy.25 Stress activates HTR2A receptors and this has patient group and treatment regimen used in this study is been shown to down-regulate the downstream brain-derived representative of routine clinical practice in the United neurotrophic factor (BDNF) in rat models.26–28 BDNF has Kingdom. It has been suggested that many patients recruited been shown to be upregulated following antidepressant to previous antidepressant trials represent only a minority of treatment and downregulated in depressed subjects.29,30 patients treated for depression in routine clinical prac- Levels of BDNF were decreased in the rat hippocampus tice.34,35 For pharmacogenetics to be used as a prognostic following HTR2A agonist treatment, an effect which was tool to facilitate the development of personalized medicine, blocked when an HTR2A antagonist was administered.31 we must be able to demonstrate that genetic factors can Finally, imipramine, nefazodone and mirtazapine directly influence therapeutic response under ‘real-world’ condi- antagonize HTR2A in vitro and antidepressants may therefore tions. It is also of obvious importance that our observations exert their function by antagonizing HTR2A receptors.28,32 are replicated in larger, independently recruited patient

We found a significant association between HTR2A C1354T groups, particularly as the present study involved the genotype and both remission and response to paroxetine. simultaneous analysis of multiple SNPs. This polymorphism results in a histidine to tyrosine amino- acid change at position 452. It was expected that the HTR2A

T1354T homozygotes would have been associated with better paroxetine response, although no differences in this geno- Materials and methods type were seen between responders and non-responders to paroxetine or SSRIs as a whole, suggesting that the Subjects heterozygote phenotype differs in a non-additive-dependent Unipolar depressive population. Patients diagnosed with manner. unipolar depression (n ¼ 166) were recruited from the

The HTR2A C1354T single nucleotide polymorphism (SNP) Affective Disorder Clinic (ADC) in the Department of has only been studied as a modifier of antidepressant Psychiatry at Ninewells Hospital, Dundee, UK. Entry response in two previous reports. The first one found no criteria was an age of between 18 and 65 years and association in depressed patients (n ¼ 173) given a variety of diagnosis of unipolar depression by ICD-10 criteria, with a antidepressant treatments,18 however the second report minimum Hamilton score of 18. All patients were of found the HTR2A T1354 allele to be associated with response Caucasian descent (52 men, 114 women, mean age 6 to fluoxetine. This suggests that the HTR2A C1354T 43.42±11.34 years) and were treated according to a polymorphism influences the efficacy of only SSRIs, an protocolized regimen. The aim of the research clinic was observation that was also made in this study. Further studies to prospectively identify a cohort of patients with investigating this allele and response to SSRIs are warranted. treatment-resistant depression (defined as failure to

The HTR2A C102T polymorphism showed a possible respond to adequate dose and duration of two association with susceptibility to ADRs following paroxetine antidepressants). Exclusion criteria were contraindications treatment, however the small number of patients suffering to drug therapy including pregnancy, lactation and severe an ADR means this finding must be treated with caution. A physical disorder. Patients were treated following a previous study investigating the HTR2A C102Tpolymorphism protocolized regimen. Prior to referral to the ADC, the and antidepressant efficacy found the same association, majority of patients had been prescribed antidepressants where individuals carrying the HTR2A C102C genotype were by their GP or community mental health team but at an more likely to suffer an ADR following paroxetine, but not inadequate dose. The first step of the ADC protocol was 32 mirtazapine therapy. The HTR2A C102T polymorphism did therefore to optimize any ongoing antidepressant therapy, not, however, influence response or remission to anti- that is prescribe the maximum tolerated dose for a depressant therapy in this patient group, a finding also minimum of 6 weeks. Lack of response to this initial described by Cusin et al.33 when investigating the influence therapy resulted in a trial of an SSRI antidepressant of this polymorphism on the efficacy of fluvoxamine and (paroxetine up to 50 mg per day or citalopram up to 60 mg paroxetine. per day if paroxetine was not tolerated). Lack of response to In conclusion, we found significant associations between paroxetine resulted in the TCA imipramine being prescribed the 5-HTTLPR and SLC6A4 intron 2 genotypes with both (up to 300 mg per day) or lofepramine (up to 280 mg per remission and response following initial and second switch day) if imipramine was not tolerated. If the initial therapy therapy. These findings conflict with a number of previously was already with an SSRI then the second treatment trial was published studies and add to the heterogeneity of observations with a TCA. Lack of response to imipramine or lofepramine

The Pharmacogenomics Journal Table 5 PCR/RFLP conditions

Name of polymorphism PCR reaction mix (Promega Taq PCR amplification Primers Banding patterns to determine polymerase system) 25 ml total conditions genotype volume

0 0 Serotonin transporter 1 Â PCR buffer, 1.25 mM MgCl2, 94 1C 5 min 34 Â (94 1C Forward ¼ 5 -GGCGTTGCCGCTCTGAATGC-3 Run out for 1 h on 2% (SLC6A4) promoter 0.77 mM DMSO, 0.2 mM dNTPs, 30 s 60 1C35s721C Reverse ¼ 50-GAGGGACTGAGCTGGACAA agarose gel polymorphism (5-HTTLPR) 0.45 mM each primer, 0.75 units 1 min) 72 1C 5 min CCAC-30 InsIns ¼ 528 bp enzyme mix (Promega), InsDel ¼ 528, 484 bp DelDel ¼ 484 bp 0 0 Serotonin transporter 1 Â PCR buffer, 1.25 mM MgCl2, 94 1C 5 min 34 Â (94 1C Forward ¼ 5 -GTCAGTATCACAGGCTGCGAG-3 Run out for 1 h on 2% (SLC6A4) intron 2 0.2 mM dNTPs, 0.45 mM each 30 s 55 1C35s721C Reverse ¼ 50-TGTTCCTAGTCTTACGCCAGTG-30 agarose gel polymorphism primer, 0.75 units enzyme mix 1 min) 72 1C 5 min 12/12 ¼ 204 bp (Promega), 12/10 ¼ 170, 204 bp 10/10 ¼ 170 bp 12/9 ¼ 204, 153 bp 10/9 ¼ 170, 153 bp 9/9 ¼ 153 bp 0 0 HTR2A T102 C polymorphism 1 Â PCR buffer, 1.25 mM MgCl2, 95 1C 5 min 34 Â (95 1C Forward ¼ 5 -TTGAGCTCAACTACGAACTCC-3 PCR gives product of 385 bp 0.77 mM DMSO, 0.2 mM dNTPs, 30 s 55 1C30s721C30s) Reverse ¼ 50-ACCCATACAGGATGGTTAACAT-3’ digested with HpaII at 37 1C 0.45 mM each primer, 0.75 units 72 1C 5 min for 3 h : enzyme mix (Promega), CC ¼ 314, 71 bp CT ¼ 385, 314, 71 bp TT ¼ 385 bp 0 Wilkie MJV response antidepressant and Polymorphisms HTR2A C1354 T 1 Â PCR Buffer, 1.25 mM MgCl2, 95 1C 5 min 34 Â (95 1C Forward ¼ 5 -GGTTATCTCTCTTCAGCAGT PCR gives product of 440 bp polymorphism 0.77 mM DMSO, 0.2 mM dNTPs, 30 s 58 1C30s721C30s) CAACC-3’ digested with BsaMI at 65 1C 0.4 mM each primer, 0.75 units 72 1C 5 min Reverse ¼ 50-GAGGCATACAGATATGAT for 3 h: tal et enzyme mix (Promega), CGTTGG-30 HisHis ¼ 250, 190 bp HisTyr ¼ 440, 250, 190 bp TyrTyr ¼ 440 bp 0 0 HTR5A G19 C polymorphism 1 Â PCR buffer, 1.25 mM MgCl2, 95 1C 5 min 34 Â (95 1C Forward ¼ 5 -AGAGGTTGCAAACATCCGGA-3 PCR gives product of 299 bp 0.77 mM DMSO, 0.2 mM dNTPs, 30 s 59 1C30s721C30s) Reverse ¼ 50-AGGATGGTCGCCAGCACC-30 digested with HpaII at 37 1C 0.35 mM each Primer, 0.75 units 72 1C 5 min for 3 h: enzyme mix (Promega), CC ¼ 83, 216 bp CG ¼ 83, 216, 299 bp GG ¼ 299 bp 0 0 HTR5A A12 T polymorphism 1 Â PCR buffer, 1.25 mM MgCl2, 95 1C 5 min 34 Â (95 1C Forward ¼ 5 -AGAGGTTGCAAACATCCGGA-3 PCR gives product of 299 bp 0.2 mM dNTPs, 0.35 mM each 30 s 59 1C30s721C30s) Reverse ¼ 50-AGGATGGTCGCCAGCACC-30 digested with BseNI at 65 1C h hraoeoisJournal Pharmacogenomics The primer, 0.75 units enzyme mix 72 1C 5 min for 3 h: (Promega), AA ¼ 114, 185 bp AT ¼ 299, 114, 185 bp TT ¼ 299 bp 0 0 HTR1B A-161 T polymorphism 1 Â PCR buffer, 1.5 mM MgCl2, 95 1C 5 min 34 Â (95 1C Forward ¼ 5 -AGGAACAACCACAGACGC-3 PCR gives product of 255 bp 0.77 mM DMSO, 0.2 mM dNTPs, 30 s 55 1C30s721C30s) Reverse ¼ 50-TTCCTCCATGGCTCTCCT-30 digested with Hsp92II at 0.37 mM each primer, 0.6 units 72 1C 5 min 37 1C for 3 h: enzyme mix (Promega), AA ¼ 120, 83, 50 bp AT ¼ 205, 120, 83, 50 bp TT ¼ 205, 50 bp 67 Polymorphisms and antidepressant response MJV Wilkie et al 68 C

1 resulted in a final treatment with the monoamine oxidase Iat inhibitor (MAOI) phenelzine (up to 90 mg per day). Rsa Each treatment was prescribed for a minimum of 6 weeks

cII at 37 before clinical assessment. The HAMD was used to deter-

Hin mine mental status and therapeutic response following initial therapy at least 6 weeks following commencement of treatment. Second switch therapy was assessed at a mini- 520 bp 510, 310, 210 bp 310, 210 bp 116, 84 bp

200, 116, 84 bp mum of 18 weeks. Response was defined as a 50% or greater 200 bp C for 3 h: ¼ ¼ ¼ ¼ ¼ 1 ¼ reduction from baseline HAMD score with remission defined GG PCR gives product ofdigested 520 bp with for 3 h: CC CG PCR gives product of 200 bps digested with 37 CC CT TT genotype as an HAMD score of 7 or less. Occurrence of ADRs was also

0 recorded. Any patient who reached second switch treatment had been exposed, prospectively, to an adequate dose and duration of at least two antidepressants and was deemed to be treatment resistant.

Early onset depressive population. Patients with early onset depression (n ¼ 102) were recruited from the Department of Psychiatry at the Royal Edinburgh Hospital. Patients were diagnosed as suffering from early onset unipolar or bipolar depression according to Diagnostic and Statistical Manual of Mental Disorders, fourth edition criteria36–39 with a

-CTTCTTCTGGCGTCAGGCTAA-3 -AACTTCTTCCTGGTGTCGCTC maximum age of 22. Twenty-one subjects of the early 0 0 -GCCATGTGAAGAAGTCAAAGA -ATGAGCAGGTAGCGGTCCAG 0 0 5 5

5 5 onset depressed population were diagnosed with bipolar ¼ ¼ ¼ ¼ depression with the remainder (81 patients) diagnosed with 0 0 0 unipolar depression. Antidepressant response data were collected for 70 early TG-3 GC-3 Reverse Reverse TTC-3 Forward Primers Banding patterns to determine Forward onset unipolar patients. Criteria for response included a C C formal HAMD rating score at 12 weeks, as well as an 1 1 C 1

C30s) assessment of previous history of treatment response 1 (95 (95 obtained from patient notes. Â Â Following written informed consent, whole blood was C 5 min

1 collected from all study participants and stored in EDTA C 1 min 72 C30s72 1 1 anti-coagulated blood tubes at À20 1C prior to further C 5 min 34 C 5 min C 5 min 34 analysis. 1 1 1 95 30 s 59 72 PCR amplification conditions 95 30 s 55 1 min) 72 Ethics

, The study was approved by the Tayside Committee on 2 ,

2 Medical Research Ethics and the Lothian Research Ethics Committee. Written informed consent was obtained from dNTPs, dNTPs, MgCl MgCl l total M M

M all study participants. m M Clinical data Clinical response data were obtained for 163 unipolar patients from Dundee with data for 3 patients unobtainable. DMSO, 0.2 m DMSO, 0.2 m

each primer, 0.75 units The number and type of antidepressant drugs prescribed each primer, 0.6 units M M M m

M following initial and second switch therapy are summarized PCR buffer, 1.5 m PCR buffer, 1.25 m m in Table 4A, while Table 4B details the number of patients Â Â enzyme mix (Promega), 0.77 m volume 0.3 0.28 enzyme mix (Promega), 0.77 m polymerase system) 25 remitting, responding or suffering an ADR following initial and second switch drug therapy. The number of patients receiving second switch treatment was lower than following initial therapy due to patients responding to previous drug treatments and/or reduced patient compliance. In total, 19 patients did not respond to initial therapy and failed to

G polymorphism 1 return to the ADC. A further 16 patients failed to respond to T polymorphism 1 Continued

816 initial and first switch therapy and did not return to the 267 ADC for second switch treatment. Analysis of clinical outcome following treatment with the HTR6 C Table 5 Name of polymorphism PCR reaction mixHTR1B (Promega C Taq Abbreviations: DMSO, dimethyl sulfoxide; dNTP, deoxyribonucleotide triphosphate; PCR, polymerase chain reaction. most commonly prescribed drug, paroxetine, was also

The Pharmacogenomics Journal Polymorphisms and antidepressant response MJV Wilkie et al 69

carried out to determine if polymorphisms in serotonin References transporter and receptor genes influenced response specifi- cally to paroxetine therapy. 1 Monsma Jr FJ, Shen Y, Ward RP, Hamblin MW, Sibley DR. Cloning and expression of a novel serotonin receptor with high affinity for tricyclic A total of 70 clinical data sets were collected from early psychotropic drugs. Mol Pharmacol 1993; 43: 320–327. onset depressive patients. Table 4A and B detail the number 2 Smits KM, Smits LJ, Schouten JS, Stelma FF, Nelemans P, Prins MH. and type of antidepressant drugs prescribed to these Influence of SERTPR and STin2 in the serotonin transporter gene on the individuals and the number of responders and non- effect of selective serotonin reuptake inhibitors in depression: a systematic review. Mol Psychiatry 2004; 9: 433–441. responders to drug treatment. It was not possible to collect 3 Lesch KP, Balling U, Gross J, Strauss K, Wolozin BL, Murphy DL et al. remission or ADR data for this population due to differences Organization of the human serotonin transporter gene. J Neural Transm in study parameters between the collection centres. Gen Sect 1994; 95: 157–162. 4 Kim DK, Lim SW, Lee S, Sohn SE, Kim S, Hahn CG et al. Serotonin Molecular analysis transporter gene polymorphism and antidepressant response. Neuro- DNA was extracted from 200 ml of whole blood using a report 2000; 11: 215–219. 5 Yoshida K, Ito K, Sato K, Takahasti H, Kamata M, Higuchi H et al. QIAamp DNA Blood Kit (Qiagen, Crawley, West Sussex, UK) Influence of the serotonin transporter gene-linked polymorphic region according to the manufacturer’s instructions. on the antidepressant response to fluvoxamine in Japanese depressed patients. Prog Neuropsychopharmacol Biol Psychiatry 2002; 26: 383–386. Genotyping for HTR1B AÀ161T(rs130058), HTR1B C G(rs6296), HTR6 C T(rs1805054), 5-HTTLPR and 6 Peters EJ, Slager SL, McGrath PJ, Knowles JA, Hamilton SP. Investigation 816 267 of serotonin-related genes in antidepressant response. Mol Psychiatry the SLC6A4 intron 2 polymorphism was carried out as 2004; 9: 879–889. previously described.4,40,41 Novel PCR-restriction fragment 7 Hong CJ, Chen TJ, Yu YW, Tsai SJ. Response to fluoxetine and serotonin length polymorphism (RFLP) assays were designed for 1A receptor (C-1019G) polymorphism in Taiwan Chinese major the polymorphisms HTR2A C T(rs6314), HTR2A depressive disorder. Pharmacogenomics J 2006; 6: 27–33. 1354 8 Smeraldi E, Zanardi R, Benedetti F, Di Bella D, Perez J, Catalano M. T102C(rs6313), HTR5A GÀ19C and HTR5A A12T(rs6320). Polymorphism within the promoter of the serotonin transporter gene Table 5 summarizes the PCR protocols and amplification and antidepressant efficacy of fluvoxamine. 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The Pharmacogenomics Journal