ORIGINAL INVESTIGATION

Association study of the glycogen synthase kinase-3b genepolymorphism with prophylactic lithium response in bipolar patients

ALEKSANDRA SZCZEPANKIEWICZ1, JANUSZ K. RYBAKOWSKI2, ALEKSANDRA

SUWALSKA2, MARIA SKIBINSKA1, ANNA LESZCZYNSKA-RODZIEWICZ2,

MONIKA DMITRZAK-WEGLARZ1, PIOTR M. CZERSKI1 & JOANNA HAUSER1,2

1Laboratory of Psychiatric Genetics, Department of Psychiatry, Poznan University of Medical Sciences, Poznan, Poland, and2Department of Adult Psychiatry, Poznan University of Medical Sciences, Poznan, Poland

AbstractA relationship between response to lithium prophylaxis and T-50C polymorphism of glycogen synthase kinase-3b (GSK-3b) gene was investigated in 89 bipolar patients (41 male and 48 female) who have been taking lithium for at least 5 years.The patients were delineated as excellent responders, partial responders and non-responders to lithium. The resultsobtained suggest that this polymorphism may not be related to the degree of prophylactic lithium response.

Key words: Association, bipolar disorder, glycogen synthase kinase-3b gene, lithium response

Introduction

The enzyme, glycogen synthase kinase-3b (GSK-

3b), is essential player in a number of intracellular

signaling pathways and regulates several transcrip-

tion factors and cytoskeletal elements. GSK-3b is

pro-apoptotic factor and plays a significant role in

neuroprotection processes. The pathogenic impor-

tance of GSK-3b has been recently implicated in

such illnesses as bipolar mood disorder and Alzhei-

mer’s disease (Gould et al. 2004b).

The gene for GSK-3b was mapped to 3q21.1

region and several polymorphisms of this gene have

been described. The �50 T/C polymorphism is

localized in an untranscribed region of the promoter

that is not putative transcriptional binding site (Russ

et al. 2001). However, it is located in an effective

promoter region (nucleotide �171 to �/29) of the

gene encoding GSK-3b and decreased expression of

the gene was found after deletion of this promoter

region (Lau et al. 1999).

Lithium is one of the most commonly used drugs

in the prophylaxis and treatment of bipolar disorder.

The mechanisms of mood-stabilizing effect of

lithium incorporate its effect on the processes of

intracellular signalling and neuronal plasticity. Many

possible targets of mood-normalizing action of

lithium have been delineated such as phosphatidyli-

nositol system, brain-derived neurotrophic factor

(BDNF) and also GSK-3b (Manji and Zarate

2002). In our recent study, an association was

demonstrated between lithium prophylactic re-

sponse and BDNF gene polymorphisms (Ryba-

kowski et al. 2005).

Lithium exerts inhibitory effect on the GSK-3

activity at concentrations relevant for bipolar dis-

order treatment, thus promoting impaired cellular

resilience and synaptic plasticity in this illness

(Gould et al. 2004a; Li et al. 2002). In addition,

lithium also affects circadian clocks which distur-

bances may play pathogenic role in bipolar disorder

(Padiath et al. 2004). GSK3-b is the mammalian

orthologue of an enzyme which regulates molecular

clock in Drosophila (Martinek et al. 2001) and it was

observed that long-term lithium administration, via

reduction of GSK-3 activity, lengthens the circadian

clock (Iwahana et al. 2004).

Recently, Benedetti et al. (2005) reported that the

efficacy of long-term lithium prophylaxis is influ-

enced by the T-50C polymorphism in GSK-3b gene.

In their sample, a better efficacy of lithium was

observed in the carriers of mutant C allele of this

Correspondence: Aleksandra Szczepankiewicz, Laboratory of Psychiatric Genetics, Poznan University of Medical Sciences, ul. Szpitalna

27/33, 60-572 Poznan, Poland. Tel: �/48 61 8491311. Fax: �/48 61 8480-392. E-mail: alszczep@amp.edu.pl

The World Journal of Biological Psychiatry, 2006; 7(3): 158�161

(Received 20 July 2005; accepted 22 December 2005)

ISSN 1562-2975 print/ISSN 1814-1412 online # 2006 Taylor & Francis

DOI: 10.1080/15622970600554711

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polymorphism. In the present study we make an

attempt to verify their findings in thoroughly char-

acterized group of bipolar patients receiving long-

term lithium prophylaxis.

Experimental procedures

Patients

The study was performed on 89 patients with

bipolar disorder (n�/82 of bipolar I and n�/7 of

bipolar II) according to DSM-IV and ICD-10

criteria using SCID, recruited from Wielkopolska

region, attending the Outpatient Lithium Clinic at

the Department of Psychiatry, Poznan University of

Medical Sciences. All patients involved in the study

(41 males with a mean age of 49 years, SD�/12; 48

females with a mean age of 47 years, SD�/15) have

been treated with lithium carbonate for at least 5

years (5�27 years, mean 15 years). Serum concen-

tration of lithium has been maintained in the range

between 0.5�0.8 mmol/l.

The course of illness was assessed retrospectively,

based on the analysis of medical outpatient charts,

inpatient records and semi-structured reviews. The

efficacy of lithium treatment was assessed according

to the following criteria: excellent lithium responders

(ER) had no affective episodes on lithium; partial

lithium responders (PR) showed 50% reduction in

the episode index (number of episodes per year to

pre-lithium period); lithium non-responders (NR)

showedB/50% reduction, no change or worsening in

the episode index.

All patients gave written consent to the study.

The study was approved by the Local Bioethics

Committee.

Genotyping

The DNA was extracted from 10 ml of EDTA

anticoagulated whole blood using the salting out

method (Miller et al. 1988). A 344-basepair frag-

ment of the GSK-3b gene was amplified by PCR

reaction with set of primers described by Russ et al.

(2001) using PTC-200 (MJ Research) thermal

cycler. PCR product (4.0 ml) was then digested

with AluI restriction endonuclease (MBI Fermen-

tas). The uncut PCR product size was 344 bp. After

RFLP analysis, the following alleles were observed:

uncut C allele (127 bp), for Tallele the bands of 220

and 124 bp. Restriction analysis for uncut C allele

was repeated to confirm the results.

Statistical analysis

The Pearson’s chi-square (x2)-test and Fisher’s exact

test were applied to test differences in the genotypic

and allelic (respectively) distribution between groups

of lithium responders. Additionally, stepwise logistic

regression analysis was performed including the

GSK-3b polymorphism and lithium response as

covariates. Calculations were performed using the

computer programme SPSS version 11.5. For small

numbers of patients in cells (see Table I, below 5) we

performed Fisher�Freeman�Halton test with Stat-

Xact-4 v 4.0.1 programme.

Results

Genotype distribution of analysed population was in

concordance with Hardy�Weinberg equilibrium

(P�/0.18). Among this group, 23 patients (25.8%)

were classified as excellent responders (ER), 47

(52.8%) as partial responders (PR) and 19

(21.3%) as non-responders (NR) to lithium prophy-

laxis. Clinical data of these patients are presented in

Table I.

No significant differences in genotype distribu-

tions and allele frequencies between GSK-3b T-50C

polymorphism and the degree of lithium response

was found (P�/0.646 and P�/0.675, respectively).

Data are shown in Table II. Analysis by gender did

not reveal any significant differences in genotype and

allele frequencies, either (P�/0.788 for males, P�/

0.649 for females).

Due to very small number in the case of

homozygous patients, we performed the Fisher�Freeman�Halton test considering small amount in

some cells in Table I (below 5). However, no

Table I. Clinical characteristics of the bipolar patients on lithium prophylaxis.

Total (n�/89) ER (n�/23) PR (n�/47) NR (n�/19)

Age, years [mean9/SD] 54.89/12.3 57.89/14.2 53.29/12.2 54.19/8.4

Gender [M:F] 38:51 11:12 16:31 10:9

Family history of psychiatric illness, N (%) 41 (46.6%) 11 (40.7%) 24 (53.3%) 6 (37.5%)

Age at onset, years [mean9/SD] 31.59/10.7 33.09/11.6 31.19/10.9 30.19/8.5

Duration of illness before lithium, years [mean9/SD] 7.49/7.4 9.79/9.6 5.69/5.9 8.79/6.0

Duration of lithium treatment, years [mean9/SD] 14.69/7.3 14.09/7.1 15.39/7.9 13.89/5.8

Affective episodes before lithium, N [mean�/SD] 6.29/4.1 7.09/3.6 6.09/4.5 5.89/3.9

Affective episodes on lithium, N [mean�/SD] 3.39/3.9 0 3.59/2.7 8.29/4.8

Association study of GSK-3b T-50C polymorphism and lithium response 159

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statistically significant differences were found be-

tween the analysed groups (P�/0.6977).

Discussion

The main finding of our study is lack of significant

association between T-50C polymorphism in the

GSK-3b gene and the degree of prophylactic re-

sponse to lithium carbonate in carefully character-

ized group of bipolar patients. Thus, the results

obtained in our analysis are not consistent with the

previous study performed by Benedetti et al. (2005).

In our study, in comparison to that of Benedetti’s

group, a nearly identical number of bipolar patients

was involved (89 vs. 88, respectively). On the other

hand, the duration of lithium prophylaxis in our

study was much longer (at least 5�27 years on

lithium) what enabled more precise assessment of

quality of lithium prophylactic effect. Our division of

bipolar patients on lithium into three groups (ex-

cellent, partial and non-responders) makes it possi-

ble to analyse an association between GSK-3b and

prophylactic lithium response in the groups of

bipolar patients with distinct clinical characteristics.

Excellent lithium responders are characterised by

episodic course of disease and low rates of comor-

bidity (MacQueen et al. 2005). On the other hand,

family studies showed that lithium non-responders

had 10 times higher prevalence of schizophrenia in

first-degree relatives compared to lithium responders

(Grof et al. 1994). Therefore, our analysis may be

complementary to that of Benedetti et al. (2005) and

extend it.

A closer inspection of Table II reveals that the

ratio of ER/NR in carriers of C allele (patients with

T/C�/C/C genotypes) was higher than ER/NR ratio

in patients with T/T genotype (1.5 and 0.9, respec-

tively). Therefore, this may be a slight hint to

suppose that ER may be more likely to have C allele

than NR. Although it may resemble a relationship

found by Italian investigators, this is not supported

by any statistical significance for neither genotypes

nor alleles.

In conclusion, the role of GSK-3b in the patho-

genesis of bipolar illness and in the prophylactic

action of lithium remains to be further elucidated.

Some association has been found between GSK-3bgene polymorphism with some features of illness

such as later onset or antidepressant response to

total sleep deprivation (Benedetti et al. 2004a,b). On

the other hand, studies of GSK-3b in postmortem

brains of bipolar patients were negative (Beasley et

al. 2002; Lesort et al. 1999). Although the study of

Benedetti et al. (2005) suggested an association

between GSK-3b gene polymorphism and lithium

prophylactic effect, we were not able to confirm their

results with our population of patients.

Acknowledgements/Statement of interest

This study was supported by the Polish Committee

of Scientific research (KBN), grants no. 2P05B

01 226 and 2P05B 00 226. Dr P.M.C. is the

recipient of a 2004 Annual Stipend for Young

Scientists from the Foundation for Polish Science

(FNP).

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