Association studies of 5-HT 2A and 5-HT 2C serotonin receptor gene polymorphisms with prophylactic lithium response in bipolar patients
Monika Dmitrzak-Wêglarz1, Janusz K. Rybakowski2, Aleksandra Suwalska2, Agnieszka S³opieñ3, Piotr M. Czerski1, Anna Leszczyñska-Rodziewicz2, Joanna Hauser1,2
Laboratory of Psychiatric Genetics at Department of Psychiatry, Department of Adult Psychiatry,!Department of Child and Adolescent Psychiatry, Poznañ University of Medical Sciences, Szpitalna 27/33, PL 60-572 Poznañ, Poland Correspondence: Monika Dmitrzak-Wêglarz, e-mail: mweglarz2003@yahoo.com
Abstract:
Lithium is one of the most commonly used drugs in the prophylaxis and treatment of bipolar disorder. The mechanisms of mood stabilization by lithium incorporate its effect on serotonergic neurotransmission. This paper investigates a relationship between response to lithium prophylaxis and polymorphisms in two genes: T102C of 5-HT )receptor and G68C (Cys23Ser) of 5-HT +
serotonin receptor gene. Genotypes were estimated in 92 bipolar patients (39 males and 53 females) who have been taking lithium for at least 5 years. The patients were classified as excellent responders, partial responders and non-responders to lithium. The obtained results suggest that these polymorphisms may not be related to the degree of prophylactic lithium response.
Key words:
lithium prophylaxis, bipolar disorder, 5-HT )– serotonin receptor 2A gene, 5-HT +– serotonin receptor 2C gene, pharmacogenetics
Abbreviations: DNA – deoxyribonucleic acid, DSM-IV – Di- agnostic and Statistical Manual, 4th edn., EDTA – ethylenedia- minetetraacetic acid, ER – excellent lithium responders, 5-HT – serotonin, 5-HT )– serotonin receptor 2A, 5-HT + – sero- tonin receptor 2C, 5-HTT – serotonin transporter gene, ICD-10 – International Classification of Disease, 10th edn., NR – lith- ium non-responders, PCR – polymerase chain reaction, PR – partial lithium responders, RFLP – restriction fragment length polymorphism, TPH – tryptophan hydroxylase
Introduction
Lithium salts have been used for the prevention of re- currences in bipolar affective illness since the 1960s.
Such a procedure is in some countries still considered
a first-choice treatment. The response to lithium pro- phylaxis is variable, with about 30% of subjects being excellent responders, and 25% of non-responders. Al- though recently the claims were made that lithium prophylaxis has been less effective nowadays than it used to be, our recent study did not show the decline in the efficacy of lithium in patients entering treat- ment in the 1970s compared to those starting in the 1980s [9]. The high degree of response to lithium (ex- cellent lithium responders) was regarded as clinical endophenotypic marker of bipolar affective illness [15].
As early as in the 1980s, it has been proposed that serotonergic system plays an important role in the mechanism of prophylactic action of lithium [7]. This has been supported by recent pharmacogenetic re-
Pharmacological Reports 2005, 57, 761765 ISSN 1734-1140
Copyright © 2005 by Institute of Pharmacology Polish Academy of Sciences
search which showed an association between lithium prophylactic efficacy and polymorphism of some genes of serotonergic system such as tryptophan hy- droxylase gene [11] and serotonin transporter gene, although the relationship was not identical in all stud- ies [2, 12, 14]. In our recent study with carefully se- lected patients on long-term lithium prophylaxis we found that the s/s genotype and s allele of serotonin transporter promoter length polymorphism were more frequent in lithium non-responders than in excellent and partial responders [10].
Neurobiological studies have demonstrated lithium influence on serotonin receptor2 (5-HT2) receptors and an association of this effect with pharmacological ac- tion of the drug in bipolar illness. Kitamura et al. [4]
reported an inhibition of 5-HT2Ahyperfunction (wet- dog shakes) in rats by co-administration of lithium and imipramine but not by imipramine alone. Basse- lin et al. [1] showed that chronic lithium administra- tion to rats selectively modified 5-HT2A/2C receptor signalingvia arachidonic acid. Pandey et al. [8] found an up-regulation of platelet 5-HT2Areceptors in pa- tients with bipolar disorder after lithium administra- tion. These data suggest that 5-HT2receptors may be involved in the response to lithium treatment in bipo- lar illness. However, in the only one genetic study of an association between serotonin receptor 5-HT2A, 5-HT2Cgene polymorphism and lithium response in bipolar affective disorder, Serretti et al. [13] reported that either 5-HT2Aor 5-HT2Cgene variants were not associated with lithium outcome.
In the present paper, we addressed the issue of a possible association between 5-HT2A and 5-HT2C gene variants and response to lithium prophylaxis in Polish population in our group of highly selected pa- tients with bipolar affective disorder receiving lithium for a long time.
Materials and Methods
Subjects
The subjects of the study were 92 patients with bipo- lar affective disorders (39 males, 53 females), aged 30–77 (mean age 54 years). Diagnosis was made ac- cording to International Classification of Disease, 10th edn. (ICD-10) and Diagnostic and Statistical
Manual, 4th edn. (DSM-IV) criteria, by two research- ers. All patients have been treated with lithium car- bonate for at least five years (5–27 years, mean 15 years). Serum concentration of lithium has been maintained in the range between 0.5–0.8 mmol/l.
The group of patients was ethnically homogenous and was recruited only from Wielkopolska region of Poland. The patients gave written consent for taking part in genetic association studies. The study was ap- proved by the Local Ethical Committee, University of Medical Sciences in Poznañ.
The efficacy of lithium treatment was assessed ac- cording to the criteria described in our previous paper [10]:
1. Excellent lithium responders (ER) had no affective episodes on lithium.
2. Partial lithium responders (PR) showed 50% reduc- tion in the episode index, defined as number of epi- sodes per year compared to pre-lithium period.
3. Lithium non-responders (NR) showed < 50% re- duction, no change or worsening in the episode index, defined as number of episodes per year compared to pre-lithium period.
The course of illness was assessed retrospectively, based on the analysis of medical outpatient charts, in- patient records (if applicable) and semi-structured in- terviews.
Genotyping
Genomic deoxyribonucleic acid (DNA) was extracted from 10 ml of ethylenediaminetetraacetic acid (EDTA) supplemented whole blood using salting out method [6].
For the assessment of T102C polymorphism, a 410-basepair fragment of the 5-HT2Agene was am- plified by polymerase chain reaction (PCR) with primer pair described by Du et al. [3]. PCR products were digested by Eco72I restriction endonuclease.
The uncut product size was 410 bp (allele T), for al- lele C comprised the cut bands of 248 and 162 bp.
For the assessment of Cys23Ser polymorphism of 5-HT2Cgene, PCR primers described by Lappalainen et al. [5] were used. PCR product was digested by HinfI restriction endonuclease. After RFLP analysis, the following alleles were observed: for allele G (Cys23) the uncut PCR product of size 182 bp, for al- lele C (Ser23) the cut bands of 164 and 18 bp.
The genotyping was performed without knowledge of the subject’s clinical status.
Results
In our group, 24 patients (26%) were classified as ER, 48 (52%) as PR, and 20 (21%) as NR to lithium pro- phylaxis. Clinical data of these patients are presented in Table 1.
Age at onset of the illness, duration of illness be- fore treatment introduction and on lithium as well as number of affective episodes before lithium treatment did not differ between the studied subgroups of patients.
Genotype distributions in the studied patients were in agreement with Hardy-Weinberg equilibrium for 5-HT2Apolymorphism (p = 0.589) but not for 5-HT2C polymorphism (p < 0.001).
The distribution of genotypes and alleles for 5-HT2Aand 5-HT2C gene polymorphism in patients with various response to lithium prophylaxis is pre- sented in Table 2.
No significant differences in genotype distributions and allele frequencies between T102C 5-HT2Apoly- morphism and the degree of lithium response was
Response to lithium prophylaxis and serotonin 5-HT receptors
Monika Dmitrzak-Wêglarz et al.
Tab 1. Clinical characteristics of the patient groups
Total n = 92
ER n = 24
PR n = 48
NR n = 20
Age years [mean ± SD] 54.8 ± 12.3 57.8 ± 14.2 53.2 ± 12.2 54.1 ± 8.4
Gender [M:F] 39:53 11:13 17:31 11:9
Family history of psychiatric illness N (%) 41 (46.6%) 11 (40.7%) 24 (53.3%) 6 (37.5%) Age at onset – years [mean ± SD] 31.5 ± 10.7 33.0 ± 11.6 31.1 ± 10.9 30.1 ± 8.5 Duration of illness before lithium – years [mean ± SD] 7.4 ± 7.4 9.7 ± 9.6 5.6 ± 5.9 8.7 ± 6.0 Duration of lithium treatment – years [mean ± SD] 14.6 ± 7.3 14.0 ± 7.1 15.3 ± 7.9 13.8 ± 5.8 Affective episodes before lithium N [mean ± SD] 6.2 ± 4.1 7.0 ± 3.6 6.0 ± 4.5 5.8 ± 3.9
Affective episodes on lithium N [mean ± SD] 3.3 ± 3.9 0 3.5 ± 2.7 8.2 ± 4.8
ER (excellent lithium responders): no affective episodes on lithium. PR (partial lithium responders): 50% reduction in the episode index, defined as number of episodes per year compared to pre-lithium period. NR (non-responders): < 50% reduction, no change or worsening in the epi- sode index, defined as number of episodes per year compared to pre-lithium period
Tab 2. Comparison of genotypes and alleles in 5-HT )and 5-HT +gene polymorphisms in patients with various responses to lithium prophy- laxis
Lithium response Genotypes
5-HT ) n = 92
Alleles 5-HT )
Genotypes 5-HT +
n = 88
Alleles 5-HT +
T/T T/C C/C T C G/G G/C C/C G C
ER 9
37.5%
11 45.8%
4 16.7%
29 60.4%
19 39.6%
19 82.6%
1 4.3%
3 13.1%
39 84.8%
7 15.2%
PR 20
41.7%
25 52.0%
3 6.3%
65 67.7%
31 32.3%
34 75.5%
6 13.3%
5 11.2%
74 82.2%
16 17.8%
ER + PR 29
40.2%
36 50.0%
7 9.8%
91 64.5%
50 35.5%
53 77.9%
7 10.3%
8 11.8%
113 83.0%
23 17.0%
NR 7
35.0%
9 45%
4 20.0%
23 57.5%
17 42.5%
17 85.0%
1 5.0%
2 10.0%
35 87.5%
5 12.5%
Difference, ERvs. PR vs. NR for 5-HT )–c2 = 3.225, df = 4, p = 0.521 for genotypes, p = 0.460 for alleles. Difference, ER + PR vs. NR for 5-HT ) –c2 = 1.574, df = 2, p = 0.455 for genotypes, p = 0.359 for alleles. Difference, ER vs. PR vs. NR for 5-HT +–c2 = 2.113, df = 4, p = 0.715 for genotypes, p = 0.741 for alleles. Difference, ER + PRvs. NR for 5-HT +–c2 = 0.616, df = 2, p = 0.735 for genotypes, p = 0.627 for alleles. ER – excellent lithum responders, PR – partial lithum responders, NR – non-responders
found (p = 0.521 and p = 0.460, respectively). There was no association between the analyzed Cys23Ser 5-HT2Cpolymorphism and lithium response (p = 0.715 for genotypes; p = 0.741 for alleles). Analysis by re- sponse or lack of response (ER + PR versus NR) did not reveal any significant differences in genotype and allele frequencies, both for 5-HT2A(p = 0.455, p = 0.359) and 5-HT2C(p = 0.735, p = 0.627) polymorphisms.
Discussion
The main finding of our study is showing the lack of association between either T102C 5-HT2Aor G68C 5-HT2Cpolymorphisms and a quality of lithium pro- phylaxis in our highly selected population of patients with bipolar mood disorder receiving lithium for the average of 15 years. These results corroborate those of the previous study performed by Italian investiga- tors [13]. Pharmacogenetic data obtained so far show a possible connection of lithium response with genes related to synthesis and reuptake of serotonin (trypto- phan hydroxylase, serotonin transporter) but not for serotonin receptors. Although the results of pharma- cological and neurobiological studies are highly sug- gestive, the association of lithium prophylaxis with a polymorphism of any serotonin receptor gene has not yet been demonstrated.
However, the results obtained in this paper as well as those of Serretti et al. [13] do not exclude an influ- ence of serotonin receptor genes on lithium response.
Firstly, the polymorphisms investigated here may be involved in gene expression independent of lithium response. Secondly, a deviation from Hardy-Weinberg equilibrium for 5-HT2Cgene may be a compounding factor in the analysis. Thirdly, although the number of patients in the present study was higher than in the previous one (92 versus 54) [10], it may still be con- sidered rather small for this kind of genetic associa- tion research. On the other hand, the group of patients studied was ethnically homogenous and thoroughly characterized clinically. The duration of lithium ad- ministration (minimum 5 years, average 15 years) made it possible to precisely assess the quality of lith- ium prophylactic effect in the patients.
In conclusion, the results of our study confirmed the lack of effect of polymorphisms of two serotonin
receptor genes on lithium prophylactic efficacy in Polish bipolar patients.
Acknowledgments:
This research was supported by the State Committee for Scientific Research (KBN), grant No 2PO5B 012 26. Dr P.M.C is the recipient of a 2004 Annual Fellowship for Young Scientists from the Foundation for Polish Science (FNP).
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Received:
May 17, 2005; in revised form: October 5, 2005.
Response to lithium prophylaxis and serotonin 5-HT receptors
Monika Dmitrzak-Wêglarz et al.
