Serotonin receptor 2A, 2C, 1A genes and response to lithium prophylaxis in mood disorders

Journal of Psychiatric Research 34 (2000) 89±98

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Serotonin receptor 2A, 2C, 1A genes and response to lithium prophylaxis in mood disorders Alessandro Serretti*, Cristina Lorenzi, Roberta Lilli, Enrico Smeraldi Istituto Scienti®co Ospedale San Ra€aele, Department of Neuropsychiatric Sciences, University of Milan School of Medicine, Italy Received 31 July 1999; received in revised form 10 November 1999; accepted 10 January 2000

Abstract The aim of this study was to investigate the in¯uence of serotonin receptors 2A, 2C and 1A gene variants on lithium prophylactic ecacy in mood disorders. One hundred and twenty-four subjects a€ected by bipolar (n = 102) and major depressive (n = 22) disorder were followed prospectively for an average of 52 months and were typed for 5-HT2A (T102C: n = 111, HTP: n = 104), 5-HT2C (n = 110) and 5-HT1A (n = 61) variants. Both 5-HT2A and 5-HT2C variants were not associated with lithium outcome. Consideration of possible strati®cation e€ects like gender, polarity, family history, age at onset and duration of lithium treatment did not in¯uence results. No 5-HT1A gene variant was identi®ed. 5-HT2A and 2C variants are not, therefore, associated with lithium prophylactic ecacy in mood disorders. 7 2000 Elsevier Science Ltd. All rights reserved. Keywords: Lithium; Bipolar disorder; Follow-up studies; Treatment outcome; Serotonin receptors

1. Introduction Lithium is the ®rst line therapy for the prophylaxis of mood disorders recurrencies, but its ecacy is highly variable (Anonymous, 1994). Clinical predictors account for less than half of lithium prophylactic ecacy (Abou-Saleh, 1993; Goodwin and Jamison, 1990; Maj et al., 1985, 1989; O'Connell et al., 1991; Schou, 1989), the remaining variance could be due to clinical or environmental variables not considered or to genetic factors. Despite the fact that formal genetic studies of lithium response heritability have not been performed, there is evidence suggesting a strong genetic component for lithium ecacy. A positive family history of bipolar illness has been associated with good outcome (Grof et al., 1994; Maj et al., 1984; Mendlewicz et al., 1972) and, conversely, lithium responder probands proved to have a higher genetic loading when * Corresponding author. Tel.: +39-02-2643-3264; fax: +39-022643-3265. E-mail address: [email protected] (A. Serretti).

compared to non-responders (Morabito et al., 1982; Smeraldi et al., 1984a,b). In light of this evidence, lithium response has been used as a tool to select homogeneous samples for genetic studies (Cavazzoni et al., 1996; Turecki et al., 1996). Possible genetic predictors of lithium response have not, however, yet been evaluated. Lithium activity is supposed to be mediated by second-messenger or non-speci®c ionic ¯ux e€ects (Belmaker et al., 1996). But other complementary mechanisms may in¯uence this activity (ElMallakh, 1996). Disturbances of the serotoninergic neurotransmitter system have been implicated in the pathogenesis of mood disorders (Benkelfat, 1993; Jacobs and Fornal, 1995) and serotonin (5-HT) has also been repeatedly implicated in the mechanism of action of lithium. Short- and long-term lithium treatment enhanced 5HT e‚ux in rat limbic regions (Baptista et al., 1990; Pei et al., 1995; Sharp et al., 1991; Treiser et al., 1981), increased the turnover of 5-HT in either frontal cortex or hippocampus, caused the down-regulation of postsynaptic 5-HT1 and 5-HT2 receptors (Hotta et al., 1986), enhanced both electrophysiological and beha-

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vioral responses mediated by postsynaptic 5-HT1A receptors (Goodwin, 1989), and signi®cantly decreased 5-HT1 binding sites in the hippocampus (Odagaki et al., 1990). Moreover, the chronic administration of lithium in rats increased the density of 5-HT transporters in frontal, temporal, and entorhinal-piriform cortices. Interestingly, the only modi®cations observed were located in regions containing nerve terminals of 5-HT neurons, while regions with cell bodies remained una€ected. These e€ects were completely reversed following a recovery of 48 h without lithium (Carli et al., 1997; Carli and Reader, 1997). Other ®ndings support the view that lithium treatment acts through an enhancement of 5-HT function: lithium salts induced signi®cant increases in plasma free 5-HT (Artigas et al., 1989), and the prolactin response to tryptophan was signi®cantly enhanced after short-term lithium treatment (Price et al., 1989). Finally, studies in humans generally suggest that lithium has a net enhancing e€ect on 5-HT function (El-Mallakh, 1996). However, this view is not univocally accepted because others suggested that the e€ects of lithium on neuronal excitability appeared independent of changes in endogenous 5-HT (Cassidy et al., 1998; Chaoulo€ et al., 1992; Lacaille et al., 1992; Manji et al., 1991). Taken together, the previous studies evidence lithium's e€ects on 5-HT function at the levels of precursor uptake, synthesis, storage, catabolism, release, receptors, and receptor±e€ector interactions. The weight of this evidence suggests that lithium's primary actions on 5-HT may be presynaptic, with many secondary postsynaptic e€ects. These actions of lithium may serve to correct 5-HT function abnormalities involved in the pathogenesis of mood disorders (Price et al., 1990). When investigating possible genetic susceptibility factors for lithium response, a number of genes related to the serotoninergic system should therefore be analyzed. The human serotonin receptor 2A (5-HT2A) gene is located on chromosome 13 (13q14-21) and it consists of three exons spanning more than 20 kb. Polymorphisms of the 5-HT2A gene have been extensively investigated in psychiatric disorders. A silent T/ C mutation in amino acid 102 (T102C) has been reported to be associated with schizophrenia (Inayama et al., 1996). This ®nding has been either con®rmed (Erdmann et al., 1996; Joober et al., 1999; Spurlock et al., 1998; Tay et al., 1997; Williams et al., 1996) and not con®rmed (Arranz et al., 1996; Chen et al., 1997; Hallmayer et al., 1994; Hawi et al., 1997; Ishigaki et al., 1996; Jonsson et al., 1996; Kouzmenko et al., 1999; Lin et al., 1999; Malhotra et al., 1996; Ohara et al., 1999; Sasaki et al., 1996; Shinkai et al., 1998; Verga et al., 1997). Mood disorders have been investigated to a lesser extent, one study reported an excess of the T102 variant in major depressive disorder

(Zhang et al., 1997), but no association with bipolar disorder has been reported (Arranz et al., 1997; Gutierrez et al., 1997; Mahieu et al., 1997). Finally, homozygosity for the wild allele of another polymorphism located in the promoter region of the gene (C-1420 T) was higher among non-responders to clozapine treatment than in responders (Arranz et al., 1998) and in alcohol dependence (Nakamura et al., 1999). The function of 5-HT2C receptors has been suggested to be abnormal in individuals with neuropsychiatric disorders (Cowen, 1993; Heath and Hen, 1995). The complete DNA encoding the 5-HT2C has been cloned; the gene contains six exons and ®ve introns spanning at least 230 kb of DNA (Xq24, Xie et al., 1996). Using SSCP analysis it has been identi®ed a Cys23-Ser23 substitution (designated 5-HT2Ccys and 5-HT2Cser) in the ®rst hydrophobic region. The 5HT2Ccys and 5-HT2Cser receptor proteins seem to not di€er in their responses to serotonin under baseline conditions (in vitro experiments) (Lappalainen et al., 1995). Instead, this substitution alters the pharmacological properties of the protein suggesting an in¯uence in case of system abnormalities such as drug treatment or illness episodes (Martin et al., 1998; Moreau et al., 1996). In humans a small excess of the Ser23 allele in female bipolars was reported (Gutierrez et al., 1996; Oruc et al., 1997). Considering others mental disorders, no (Cavallini et al., 1998; Lentes et al., 1997) or marginal (Goldman, 1995; Holmes et al., 1998; Oliveira and Zatz, 1999; Pandey et al., 1996) associations were reported. Finally, schizophrenia does not seem associated with 5HT2C gene variants (Ozaki, 1996) but it resulted associated with time spent in hospital since the ®rst admission (Segman et al., 1997). The 5-HT1A receptor is of relevant interest because it is an important locus for the action of antidepressant treatments (Goodwin, 1989). Microdialysis studies have evidenced that the combination of a 5-HT-uptake inhibitor and 5-HT1A-autoreceptor antagonist produce much greater availability of 5-HT in the synaptic cleft in terms of much faster induction of subsensitivity of 5-HT1A autoreceptors (De Montigny et al., 1993). These changes may be relevant to the therapeutic e€ect of repeated ECT in depression (Burnet et al., 1995). Clinical trials based on this hypothesis have revealed that combination therapy with a 5-HT-uptake inhibitor and 5-HT1A-autoreceptor antagonist ameliorated the therapeutic ecacy in depressive patients (Artigas et al., 1994, 1996; Zanardi et al., 1997). The 5HT1A receptor gene is located at 5.q11.2-q13 (Fargin et al., 1988; Kobilka et al., 1987), the same region has been correlated with schizophrenia (Gilliam et al., 1989). However negative replications followed (Curtis et al., 1993; Inayama et al., 1996) also when considering neuroleptic malignant syndrome (Kawanishi et al., 1998a). A number of polymorphisms were identi®ed in

A. Serretti et al. / Journal of Psychiatric Research 34 (2000) 89±98

the human 5-HT1A receptor gene, two of this alter receptor's amino acid composition encoding amino acid substitutions of a glycine to serine at amino acid 22 and an isoleucine to valine at amino acid 28, respectively (Nakhai et al., 1995). A number of studies suggested that this mutation is unlikely to play a signi®cant role in the genetic predisposition to psychiatric disorders (Brett et al., 1995; Erdmann et al., 1995; Kawanishi et al., 1998a; Lam et al., 1996; Xie et al., 1995; Zhang et al., 1997). Finally, other rare variants have been reported (Erdmann et al., 1995; Kawanishi et al., 1998b; Lam et al., 1996; Xie et al., 1995). We previously investigated possible gene variants associated with lithium ecacy (Serretti et al., 1999a; Serretti et al., 1998) and we evidenced a possible association with tryptophan hydroxylase polymorphism (Serretti et al., 1999b). In the present paper, we hypothesized that gene variants at 5-HT1A, 2A and 2C could be involved in individual susceptibility to lithium prophylactic ecacy in mood disorders. 2. Materials and methods 2.1. Subjects One hundred and twenty four subjects consecutively admitted to the Lithium Clinic for Mood Disorders of S. Ra€aele Hospital in Milan were included in this study (male/female = 49(39.8%)/75(60.2%); bipolar/ major depressives = 102(82.2%)/22(17.8%)) (Franchini et al., 1994, 1996). Patients considered for this study were part of the sample collected in the context of the European Collaborative Project on A€ective Disorders (Souery et al., 1998), a preliminary analysis of the pooled sample has been presented for dopamine receptor D2, D3, D4 and tyrosine hydroxylase genes (Lipp et al., 1997) and further papers about case-control studies are in preparation. Previous analyses on the same sample have been reported (Serretti et al., 1998; Serretti et al., 1999b). All patients were evaluated using the Schedule for A€ective Disorders and Schizophrenia (SADS, Endicott and Spitzer, 1978) and/or the Operational Criteria for Psychotic Illness checklist (OPCRIT, McGun et al., 1991). Lifetime diagnoses were assigned according to DSMIV criteria on the basis of interviews and all available sources of information. Information about the illness before contact with our center were collected by a psychiatrist experienced in mood disorders following the best estimate procedure, by interviewing subjects, family members, previous health professionals and obtaining records when possible (Leckman et al., 1982). The course of illness was therefore retrospectively evaluated by a ®rst psychiatrist who produced a life chart with a method similar to the LIFE technique, di€ering for a longer

91

duration of the period investigated (Keller et al., 1987); a second experienced psychiatrist reviewed the chart and, if no consensus was obtained, a third senior psychiatrist was involved. If disagreement continued, the patient was excluded. However, no subject was excluded because of disagreement. The presence of concomitant diagnoses of mental retardation, drug dependence, or other Axis I disorders, together with severe somatic or neurological illnesses represented exclusion criteria. Lithium was initiated if a diagnosis of bipolar disorder of major depressive disorder recurrent was formulated and in absence of counterindications (Anonymous, 1994). All enrolled patients received lithium as the only maintenance therapy with doses adjusted to obtain 12-h plasma levels within standard therapeutic range, benzodiazepines were at times administered mostly at bedtime. The mean lithium plasma levels ranged between 0.4 and 0.7 mEq/l for plasma levels and between 0.2 and 0.4 mEq/ l for red blood cell levels. Determination of lithium levels in each patient was performed every 3 months and, at the same time, the clinical condition of patients was evaluated using the Hamilton Rating Scale for Depression (21-HAMD, Hamilton, 1967) and Manic Rating Scale for mania (Young et al., 1978). Evaluations were performed by the psychiatrist who follows the patients during both in- and outpatient therapy, our psychiatrist sta€ is constantly trained for reliability on those scales (Gasperini et al., 1993b; Gatti et al., 1996; Zanardi et al., 1996; Zanardi et al., 2000). If patients presented a major depressive (HAMD >18) or a manic (Young rating scale >20) episode after an euthymic period of at least 6 months, they were recognized as having a new recurrence (Frank et al., 1991) and received additional care (unstandardized treatment and hospitalization when needed) and treatment according to the judgment of their clinician. Informed consent was obtained after the procedure had been fully explained from all probands, who were unrelated and of Italian descent with antecedents from all parts of the country. Ecacy of lithium treatment is dicult to establish (Goodwin and Jamison, 1990) and the more straightforward method is to evaluate a complete absence of illness episodes after lithium treatment, but this happens only in a limited number of subjects. The large majority experiences a decrease in number of illness episodes or in their severity (Goodwin and Jamison, 1990). However, the raw number of episodes is dependent from the time of follow-up, and, on the other hand, measures weighted on the time of follow-up may vary considerably for very short observations. Therefore we reported both raw and weighted measurements and we excluded cases followed for less than 18 months. The recurrence rates before and during prophylaxis were evaluated by taking into account the

92

A. Serretti et al. / Journal of Psychiatric Research 34 (2000) 89±98

occurring episodes of illness over the months from the onset to the beginning of lithium prophylaxis (pretreatment frequency = number of episodes/month duration of illness before lithium treatment 100) and the occurring episodes from the beginning of prophylaxis to the moment of assessment (on-lithium treatment frequency = number of recurrences/month duration of lithium treatment 100). The ecacy of prophylactic treatment was evaluated by calculating the di€erence between the pre-treatment and the onlithium treatment frequency (Franchini et al., 1994; Gasperini et al., 1993a). 2.2. DNA analysis DNA was extracted from leucocytes by NaCl precipitation (Lahiri and Nurnberger, 1991). Polymerase chain reaction (PCR) was performed with the following primers: for 5-HT2A T102C: 5 ' CGC CCG CCG CGC CCC GCG CCC GTC CCG CCG TCT GCT ACA AGT TCT GGC TT 3 ', and 5 ' CTG CAG CTT TTT CTC TAG GG 3 '; for C-1420 T: 5 ' AAG CTG CAA GGT AGC AAC AGC 3 ' and 5 ' AAC CAA CTT ATT TCC TAC CAC 3 '; for 5-HT2C 5 ' CTG CCA TGA TCA CAA GGA TG 3 ' and 5' GGC CTA TTG GTT TGG CCA T 3' and, ®nally, for 5-HT1A 5 ' TGC TTG GGT CTC TGC ATT C 3 ' and 5 ' CAC TTG GTA GCT GAC GGT CA 3'. 100 ng of genomic DNA was diluted to 5 ml and heated to 998C for 3 min. Then a reaction mix containing 0.025 Uml Taq Polymerase (Perkin±Elmer, Monza, Italy) 1X PCR Bu€er (Perkin±Elmer, Monza, Italy), 0.2 mM of each primer, 200 mM of dATP, 200 mM of dCTP, 200 mM of dTTP, 200 mM of dGTP was added in a total volume of 10 ml. PCR was performed with the following annealing temperature: 608C for 5-HT2A, 588C for 5-HT2C and 578C for 5-HT1A. The PCR products were digested by the HpaII or BsaJI or NlaIII or BsmAI restriction enzymes (New England BioLabs) respectivelly for the four studied polimorphisms: 5HT2A (T102C, C-1420 T), 5-HT2C and 5-HT1A; then the digested products were separated on a high resolution agarose gel and visualized by ethidium bromide. 2.3. Statistical analysis Di€erences were assessed using Analysis of Variance (ANOVA), with Newmann±Keuls test evaluating posthoc comparisons. Analysis of Covariance (ANCOVA) was used to include possible confounders. Alpha levels were considered signi®cant when less than 0.05. The power of our sample for detecting di€erences among variants was calculated considering an alpha value of 0.05 two tailed. With these parameters in our sample we had a high power (0.80) to detect a medium e€ect size ( f = 0.33) that corresponded to a di€erence of ap-

proximately nine points between the two major genotypes on the di€erence between pre-treatment index and the ongoing-lithium treatment index for 5-HT2A T102C (Cohen, 1988). For HTP and 5-HT2C the power was slightly lower, minimum e€ect size 0.32 and 0.39/0.50 (Females/Males) respectively. 5-HT1A variants were not present in our sample. The EH package was used for the analysis of disequilibrium between 5HT2A T102C and C-1420 T (Terwilliger and Ott, 1994). 3. Results Of the 124 patients, 32 (26.4%) had no recurrences during follow up (28 bipolars and four major depressives), 77 (62.1%) showed a decrease in episode frequency and the remaining 15 (11.5%) worsened. The whole sample showed a signi®cant reduction in episode frequency after lithium treatment (pre-lithium versus on-lithium: 7.84 2 8.13 vs 4.65 2 5.96; t = 3.53; d.f.=246; p < 0.0005). Haplotype analysis (EH) revealed a marginal disequilibrium between the two 5-HT2A markers (chisquare = 2.79, d.f.=1, p = 0.09). Tables 1 and 2 show the sample divided according to 5-HT2A variants. No association is reported when considering both raw and weighted outcome measures. Consideration of possible strati®cation e€ects like gender, family history, age at onset and duration of lithium treatment did not reveal any association either (data not shown). Bipolar subjects were also analyzed separately (last line in tables) but they did not di€er from the total sample. 5-HT2C marker is located on the X chromosome, therefore we analyzed separately males and females (Table 3). No association is reported when considering both raw and weighted outcome measures, and consideration of possible strati®cation e€ects did not signi®cantly in¯uence results (data not shown). 5-HT1A could not be analyzed because all subjects resulted wild type, with no variant. Subjects were in Hardy± Weinberg equilibrium (5-HT2A T102C: chi-square = 0.0, d.f.=1, p = n.s.; 5-HT2A HTP: chi-square = 2.9, d.f.=1, p = n.s.; 5-HT2C: chi-square = 1.9, d.f.=1, p = n.s.). 4. Discussion Serotonin receptor 2A, 2C and 1A gene variants were not associated with lithium outcome in our sample of mood disorder subjects. This was also true when known clinical and demographic risk factors, such as polarity, gender, onset, family history, and length of lithium administration, were controlled for. To our knowledge, genetic liability fac-

A. Serretti et al. / Journal of Psychiatric Research 34 (2000) 89±98

93

Table 1 Clinical variables and lithium outcome measures across 5-HT2A T102C variants. Genotypes were not associated with both raw and weighted outcome measures 5-HT2A T102C

102 T/102 T

102 T/102C

102C/102C

All groups 114

ANOVA (d.f.=111,2) F

Number of subjects

29

57

28

p

Current age (yr) Onset of illness (yr) Duration of illness (months) Duration of illness before lithium treatment (months) Duration of lithium treatment (months) Pre-lithium manic and depressive episodes On-lithium manic and depressive episodes Pre-lithium treatment frequency On-lithium treatment frequency Pre-on lithium treatment. Frequency di€erence Pre-on lithium treatment. Frequency di€erence Bipolar subjects (n = 93)

44.59213.25 32.52210.53 145.242103.34 96.28293.61 48.97242.22 3.9722.31 1.3922.41 8.429.21 4.3927.27 4.01210.53

45.07213.61 31.93211.55 157.682106.57 109299.6 48.68231.13 3.9823.62 2.0422.04 8.0927.97 4.6225.98 3.4628.99

49.14213.08 32.3629.91 201.432154.66 152.462147.07 48.96221 5.523.45 2.4422.52 7.2926.52 4.6825.32 2.6129.28

45.95213.4 32.18210.82 165.262120.19 116.442112.74 48.82232.05 4.3523.33 1.9722.27 7.9727.93 4.5826.13 3.3929.40

1.07 0.03 1.81 2.06 0.00 2.26 1.54 0.15 0.02 0.16

0.35 0.97 0.17 0.13 1.00 0.11 0.22 0.86 0.98 0.85

3.5029.10

2.8929.09

3.0629.71

3.0829.15

0.03

0.97

ence the overall activity of the system (Bradley and Blakely, 1997). Factors associated with drug response may erroneously be considered predictors if they are associated with a mood disorder's outcome independent of therapy. We are currently collecting longitudinal data to investigate the role of clinical and genetic factors on the time course of the illness. Most studies performed in tertiary care settings, like the present one, may be biased because the use of selection criteria limits the extent to which the sample is representative (Maj et al., 1998). In fact, lithium prophylaxis may be less e€ective in routine clinical practice than in specialized treatment centers (Goldberg et al., 1996). But the purpose of this study was to investigate predictive factors, which is more likely the less confounding variables, like Axis I comorbidity or substance abuse, are present. The power of our sample was enough to detect a

tors for lithium response have not yet been studied. Our center is currently testing a number of possible candidate genes within both the dopaminergic and serotoninergic systems. We previously reported a possible liability factor analyzing the tryptophan hydroxylase polymorphism (Serretti et al., 1999b) and we excluded the Dopamine receptor D2 (DRD2), D3 (DRD3), D4 exon 3 (DRD4), and g-aminobutyric acid type A (GABAA) receptor alpha-1 subunit (GABRA1) gene variants (Serretti et al., 1999a; Serretti et al., 1998). Even though our results do not support the hypothesis of an involvement of 5-HT2A, 2C and 1A in individual susceptibility to lithium response, they do not exclude an in¯uence of the serotoninergic system. We did not scan the whole pathway, and further regulation of receptor protein expression (i.e. promoters, enhancers or post-trancriptional splicing) may in¯u-

Table 2 Clinical variables and lithium outcome measures across 5-HT2A C-1420 T variants. Genotypes were not associated with both raw and weighted outcome measures 5-HT2A HTP (C-1420 T) Number of subjects

-1420C/-1420C

-1420C/-1420 T -1420 T/-1420 T All groups

89

13

Current age (yr) 46.43213.02 51.69213.02 Onset of illness (yr) 32.24210.77 39.23211.86 Duration of illness (months) 170.432116.85 149.542111.67 Duration of illness before lithium treatment (months) 119.042110.57 99.852107.25 Duration of lithium treatment (months) 51.38231.93 49.69221.49 Pre-lithium manic and depressive episodes 4.4423.37 4.1523.39 On-lithium manic and depressive episodes 2.1622.35 1.9222.14 Pre-lithium treatment frequency 7.7327.75 10.33210.86 On-lithium treatment frequency 4.926.41 3.6322.91 Pre-on lithium treatment. Frequency di€erence 2.8329.27 6.7211.44 Pre-on lithium treatment. Frequency di€erence Bipolar subjects (n = 84) 2.2629.07 5.55211.97

2

104

ANOVA (d.f.=101,2) F p

40.5216.26 29.523.54 1322152.74 1062121.62 26231.11 2.520.71 121.41 5.7825.97 2.0922.95 3.728.91

46.97213.08 33.06211.01 167.082115.83 116.392109.42 50.68230.77 4.3723.34 2.1122.31 8.0228.14 4.6926.04 3.3329.54

1.17 2.46 0.27 0.18 0.67 0.36 0.29 0.65 0.43 0.93

0.31 0.09 0.76 0.83 0.51 0.70 0.75 0.52 0.65 0.40

2.7129.38

0.79

0.46

10.020.0

94

A. Serretti et al. / Journal of Psychiatric Research 34 (2000) 89±98

Table 3 Clinical variables and lithium outcome measures across 5-HT2C. Males and females are displayed separately. In both cases genotypes were not associated with both raw and weighted outcome measures 5-HT2C Cys23Ser Males

Cys23

Ser23

All groups 41

ANOVA (d.f.=39,1) F

Number of subjects

29

12

p

Current age (yr) Onset of illness (yr) Duration of illness (months) Duration of illness before lithium treatment (months) Duration of lithium treatment (months) Pre-lithium manic and depressive episodes On-lithium manic and depressive episodes Pre-lithium treatment frequency On-lithium treatment frequency Pre-on lithium treatment. Frequency di€erence Pre-on lithium treatment. Frequency di€erence Bipolar subjects (n = 37)

44.38214.59 32.38211.54 1442102.93 101.83290.86 42.17224.1 3.6922.98 1.2121.32 9.1728.56 4.1126.78 5.06210.42

39.33212.76 30.0829.56 111264.34 71.25258.18 39.75217.21 3.1721.47 121.1 8.3628.53 2.7923.44 5.5827.36

42.9214.11 31.71210.93 134.34293.73 92.88283.12 41.46222.12 3.5422.62 1.1521.25 8.9328.45 3.7225.99 5.2129.54

1.09 0.37 1.05 1.15 0.10 0.33 0.21 0.08 0.41 0.02

0.30 0.55 0.31 0.29 0.75 0.57 0.65 0.78 0.53 0.88

4.98210.94

5.5727.36

5.1729.82

0.03

0.87

5-HT2C Cys23Ser Females

Cys23-Cys23

Cys23-Ser23

Ser23-Ser23

All groups

Number of subjects

48

17

4

Current age (yr) Onset of illness (yr) Duration of illness (months) Duration of illness before lithium treatment (months) Duration of lithium treatment (months) Pre-lithium manic and depressive episodes On-lithium manic and depressive episodes Pre-lithium treatment frequency On-lithium treatment frequency Pre-on lithium treatment. Frequency di€erence Pre-on lithium treatment. Frequency di€erence Bipolar subjects (n = 52)

48.38212.7 33.27212.15 181.52124.65 127.672121.71 53.83234.07 5.1924 2.4522.56 7.4427.59 4.6724.71 2.7729.04

45.41210.44 33.8229.1 139.06289.3 79.24259.93 59.82243.76 3.5321.7 2.1322.53 7.1225.9 4.9729.49 2.1527.94

51.5218.08 27.2523.77 2912208.16 241.252211.09 49.75213.43 5.524.12 4.7522.99 10.61215.35 9.0923.95 1.53216.99

2.0228.28

1.1629.73

standardized di€erence (e€ect size) up to d = 0.33 (for T102C, depending on the frequency of the risk genotype, considering a power of 0.8 and alpha 0.05 twotailed), that corresponds to a di€erence of nine points on the lithium ecacy index and to an explained variance of 10%. Thus it is possible that smaller di€erences were missed. We previously reported that administration of lithium late in the illness is less e€ective (Franchini et al., 2000), this could bias our results. However we never observed a signi®cant di€erence in our samples, only in the female sample for 5-HT2C we observed a borderline di€erence that, in fact, caused a poorer response in the Ser23-Ser23 group, but this did not reach the statistical signi®cance. A limitation of the present study is that a number of clinical variables were not considered, such as number of days of hospitalization, the episodes' sequence type (depression/ mania) (Maj et al., 1989), life events or the time-course of plasma lithium levels. With regard to this last point, lithium levels were maintained within range values in our setting and, though high-range serum levels have been associated with better outcome, there have been

ÿ6.6025.98

69

ANOVA (d.f.=66,2) F

p

47.83212.41 33.06211.15 177.392125.16 122.322119.85 55.07235.63 4.823.61 2.5122.6 7.5427.68 526.19 2.5529.17

0.54 0.58 2.59 3.33 0.22 1.42 1.71 0.34 0.94 0.05

0.59 0.56 0.08 0.04 0.80 0.25 0.19 0.71 0.40 0.95

1.3528.56

1.44

0.25

doubts raised as to their relevance (Gelenberg et al., 1989; Page et al., 1987). Ethnic origin is frequently a cause of strati®cation bias but our sample was composed of subjects with Italian antecedents for at least two generations and, though genetic heterogeneity have been evidenced for some isolate populations (Barbujani and Sokal, 1991), Italy is characterized by a substantial genetic homogeneity (Gasparini et al., 1997). In conclusion, 5-HT2A and 2C variants were not associated with lithium prophylactic ecacy in mood disorders. 5-HT1A was not informative in our sample.

Acknowledgements We acknowledge Linda Franchini MD for her help in data collection. This work was partially supported by the BIOMED 2 grant BMH4-CT97-2307.

A. Serretti et al. / Journal of Psychiatric Research 34 (2000) 89±98

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