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Keywords:

  • bipolar disorder;
  • circadian clock genes;
  • lithium

Abstract

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. Disclosures
  8. References

Objectives

The therapeutic action of lithium in bipolar mood disorder may be connected with its effect on biological rhythms. In the present study, an attempt was made to investigate an association between multiple single nucleotide polymorphisms (SNPs) and their haplotypes pertaining to four genes involved in regulation of biological rhythms [circadian locomotor output cycle kaput (CLOCK), aryl hydrocarbon receptor nuclear translocator-like (ARNTL), timeless circadian clock (TIMELESS), period circadian clock 3 (PER 3)], and the efficacy of lithium prophylaxis.

Methods

The study was performed on 115 patients with bipolar mood disorder (45 males, 70 females) with a mean age of 52 ± 12 years, with lithium prophylaxis for 22 ± 8 years, recruited from the outpatients in the Department of Psychiatry, Poznan University of Medical Sciences. The assessment of the lithium prophylactic response was made retrospectively using the Alda scale. Genotyping was done for nine SNPs of the CLOCK gene, 18 SNPs of the ARNTL gene, six SNPs of the timeless circadian clock (TIM) gene, and nine SNPs of the PER3 gene.

Results

An association with the degree of lithium prophylaxis was found for six SNPs and three haplotype blocks of the ARNTL gene, and two SNPs and one haplotype block of the TIM gene. No association with SNPs or haplotypes of the CLOCK and PER3 genes was observed.

Conclusions

The results suggest that the ARNTL and TIM genes may be associated with the lithium prophylactic response in bipolar illness. This association may be related to the role of these genes in the predisposition to bipolar mood disorder. Of special interest may be polymorphisms of these genes involved both in the predisposition to bipolar mood disorder and the lithium response.

Disturbances in circadian and circannual rhythms in bipolar disorder have been documented both in clinical and neurobiological studies. Subjects with bipolar disorder have abnormal sleep/wake cycles and abnormal circadian patterns of hormone activity. Moreover, the symptoms of bipolar disorder are often seasonal, with periods of depression in winter and mania in the summer [1].

Biological rhythms have been regulated with a number of genes, termed clock genes. At the molecular–genetic level, candidate gene studies showed that some clock genes, such as aryl hydrocarbon receptor nuclear translocator-like (ARNTL), circadian locomotor output cycle kaput (CLOCK), endothelial growth factor regulated-3 (EGR 3), period circadian clock 3 (PER3), retinoid-related orphan nuclear receptor-beta (RORB), timeless circadian clock (TIM), and vasoactive intestinal peptide (VIP), have been associated with a predisposition to bipolar illness [2-7]. Although such an association has not been obtained in genome-wide association studies (GWAS), McCarthy et al. [8], while reanalyzing the GWAS data, suggested a possible association between circadian clock genes and bipolar disorder and also with the lithium response.

Lithium has been a cornerstone for the long-term treatment of bipolar disorder [9]. A number of neurobiological hypotheses have been put forward concerning the mechanism of therapeutic action of lithium in bipolar mood disorder. There is some evidence that such an effect of lithium may be also connected with its action on biological rhythms. It has been shown that lithium produces a lengthening of the circadian period in a variety of organisms, including humans [10], and is a strong inhibitor of glycogen synthase kinase 3β (GSK-3β) [11], implicated in synaptic plasticity, apoptosis, and the circadian cycle [12]. Also, a mouse model of bipolar disorder has been developed in which a mutation in the CLOCK gene, a component of the cellular circadian clock, causes mania-like behavioral abnormalities which are lithium sensitive [13].

In view of the above, it is surprising that the studies on the association between lithium efficacy and the polymorphism of genes connected with circadian rhythms have not been numerous. In regard to GSK-3β, Italian investigators demonstrated an association between the functional −50 T/C polymorphism of the GSK-3β gene and the lithium response [14] but this was not confirmed in two other studies, including our own [15, 16]. However, in a network coordinating circadian rhythms, GSK-3β interacts with a number of proteins, including Rev-Erba, and, indeed, in two recent studies a variant of the Rev-Erba gene has been shown to be associated with the prophylactic lithium response [17, 18].

Eleven years ago, Canadian researchers (Martin Alda and colleagues) introduced a scale allowing the quantitative retrospective assessment of the prophylactic lithium response [19]. This scale, which will be further referred to as the Alda scale, can be effectively used in molecular–genetic studies investigating the association between gene polymorphism and the magnitude of the lithium response.

In the present study, an attempt has been made to investigate the association between multiple single nucleotide polymorphisms (SNPs) of four clock genes (CLOCK, ARNTL, TIM, PER3) and the efficacy of lithium prophylaxis, as estimated by the Alda scale.

Patients and methods

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. Disclosures
  8. References

Patients

The study was performed on 115 patients with bipolar mood disorder [45 males, 70 females, with an age range of 31–80 years; mean ± standard deviation (SD) = 52 ± 12 years (males 50 ± 13; females 54 ± 11 years)], recruited from the outpatient clinic, Department of Psychiatry, University of Medical Sciences, Poznan, Poland. Consensus diagnosis by at least two psychiatrists was made for each patient, according to DSM-IV criteria (Structured Clinical Interview for DSM-IV Axis I Disorders) [20]. Bipolar I disorder was diagnosed in 95 patients and bipolar II disorder in 20 patients. The duration of illness was 9–55 years (32 ± 11 years: males = 30 ± 12; females = 32 ±10 years). Patients had been treated with lithium carbonate for at least five years (6–39 years). The mean duration of lithium therapy in the whole group was 22 ± 8 years, with 22 ± 8 years in males and 22 ± 9 years in females. The patients were attending the same outpatient clinic for the entire period of lithium administration. The serum concentration of lithium was maintained in the range of 0.5–0.8 mmol/L. The course of illness was assessed retrospectively, based on the results of the Alda scale.

The study was approved by the Bioethics Committee (Poznan University of Medical Sciences) and all patients gave their informed consent after the nature of the procedures had been fully explained to them.

The assessment of the lithium prophylactic response

The assessment of the magnitude of the lithium prophylactic response was made using the Alda scale [19]. In this scale, retrospective criteria of the lithium response have been established: Criterion A rates the degree of response (activity of the illness while on adequate lithium treatment) on the ten-point scale; and Criteria B1–B5 establish whether there is a causal relationship between the improvement and the treatment. Criteria B include: B1: the number of episodes experienced while off treatment; B2: the frequency of episodes experienced while off treatment; B3: the duration of the treatment; B4: compliance during period(s) of stability; and B5: use of additional medications during the periods of stability. The total score is obtained by subtracting Criteria B from Criterion A and may be in the range of 0–10 points. Each patient was assessed by two clinicians (JKR and SK) and the mean of the two assessments was recorded. The inter-rater reliability value was 0.91. A first study of the reliability of the scale has been previously published [21].

Method of genotyping

The DNA was extracted from 10 mL of EDTA-anticoagulated whole blood using the salting-out method [22]. The SNP selection included the following criteria: functionality (in experimental studies), high frequency [minor allele frequency (MAF) > 0.1], indication as a tag SNP in Haploview v4.1 according to the HapMap database [Genome Browser release#24 (Phase 1 and 2: full dataset)] for the Caucasian population or previously reported associations for psychiatric disorders (both positive and negative findings). The SNPs chosen included the coding regions of known functionality as well as the non-coding regions (introns, untranslated regions) which may possibly affect gene regulation. The polymorphisms of CLOCK, ARNTL, TIM, and PER3 genes were genotyped using TaqMan SNP Genotyping assays (Applied Biosystems, Foster City, CA, USA) and TaqMan Genotyping Master Mix (Applied Biosystems). The list of SNPs analyzed in the ID numbers of TaqMan assays are presented in Table 1. All the assays were validated and pre-designed except for six polymorphisms (rs1481892, rs10864315, rs4908694, rs2172563, rs2640909, rs10462021), for which custom assays were designed. The amplification for TaqMan SNP genotyping assay plates was carried out using ABI PRISM® 7900HT Sequence Detection System (Applied Biosystems). Data acquisition and analysis were performed using the allelic discrimination analysis module in SDS v2.1 software (Applied Biosystems). For genotyping quality control, genomic control DNA samples and negative control samples (water) for each reaction plate were included. To check for genotyping accuracy, 10% of randomly chosen samples for each SNP were repeatedly analyzed. This enabled identification of identical genotypes in all repeated samples. Due to the low number of samples and the lack of a control group of healthy subjects, the systematic genotyping errors could not be detected based on departure from the Hardy–Weinberg equilibrium. The genotyping was performed without knowing the clinical status of the subject. The polymorphisms of four genes studied are listed in Table 1.

Table 1. Description of polymorphisms analyzed
GeneSNP IDChromosomal positionCustom nameMAFAllelesTaqMan assay IDFunction
  1. ARNTL = aryl hydrocarbon receptor nuclear translocator-like; CLOCK = circadian locomotor output cycle kaput; ID = identification; MAF = minor allele frequency; PER 3 = period circadian clock 3; SNP = single nucleotide polymorphism; TIMELESS = timeless circadian clock; UTR = untranslated region.

CLOCK rs1801260559961263111C/T0.275A:GC___8746719_203′UTR
 rs380514856001567 0.367A:CC__27519005_10Intron
 rs684947456013219 0.325G:AC__11821338_10Intron
 rs1193259556018354 0.433A:GC____296556_10Intron
 rs1264827156062879 0.292G:CC____251897_10Intron
 rs685052456076754 0.450G:CC__11821294_10Intron
 rs1264950756380484 0.331A:GC__1836992_10Intron
 rs434084456023613 0.375A:CC__31137420_10Intron
 rs53465455984977 0.140A:GC____769781_10Intron
ARNTL rs148189213258497 0.258G:CCustom assayIntron
 rs414638813263181 0.233C:TC___1870648_10Intron
 rs1076607513275163 0.233C:TC___1870671_10Intron
 rs475714213282271 0.317A:GC___1870681_10Intron
 rs739694313285555 0.292G:CC___1870682_10Intron
 rs1182409213302870 0.283C:TC___2160476_10Intron
 rs794795113312606 0.275G:AC___2160488_10Intron
 rs793706013319391 0.392T:CC__29136982_10Intron
 rs156243813320776 0.305C:TC___2160492_10Intron
 rs381636013324326 0.333C:TC__25813227_10Intron
 rs712630313327111 0.408T:CC___2160497_10Intron
 rs378932713341892 0.467A:GC___2160503_20Intron
 rs1102277813347436 0.358T:GC__31248681_10Intron
 rs1160099613352742 0.483T:CC___2160507_10Intron
 rs1102277913353386 0.208G:AC___2160509_10Intron
 rs1102278013353485 0.492T:CC___2160510_10Intron
 rs710728713269545 0.233G:TC___1870658_10Intron
 rs198235013306707 0.430A:GC___2160480_10Intron
TIMELESS rs229173955100920Pro1018Leu0.467A:GC__15966257_10Exon 25
 rs229173855101548 0.396T:CC___3134217_1_Intron
 rs730206055115359 0.439T:CC___2690225_10Intron
 rs1087689055120018 0.492A:TC___2690213_10Intron
 rs1117185655128086 0.457C:TC__31820742_10Intron
 rs227966555113961Leu38Leu0.483C:GC__15968332_10Exon 3
PER3 rs8367557769114 0.425A:CC___2510236_20Intron
 rs2287277770423 0.450C:TC__11673507_10Intron
 rs108643157772668 0.317C:TCustom assayIntron
 rs49086947773485 0.200C:TCustom assayIntron
 rs2286827778933 0.392T:CC___8881633_20Intron
 rs2286427785880 0.475T:CC___2510264_10Intron
 rs21725637796630 0.242G:ACustom assayIntron
 rs26409097812704Met1028Thr0.186T:CCustom assayExon 18
 rs104620217819720His1149Arg0.151A:GCustom assayExon 20

Statistics

The degree of response to lithium, according to the Alda scale, was divided into four quartiles. Pearson's χ2 test and Fisher's exact test were used for calculating differences in genotypic and allelic distribution between the high and low quartiles, and between the high- and low-quartile subjects versus the remaining subjects. Identification of haplotypes composed of three or more contiguous SNPs from each gene studied was performed. Differences in haplotype associations were calculated by comparing the high and low quartiles of the response to lithium. Calculations were performed using the Statistica 10 version statistical package (Statsoft-Polska, Poland). The level of statistical significance was determined at p < 0.05. The Bonferroni correction was performed for verifying differences obtained in genotypes, and the test of 1,000 permutations was performed for verifying differences obtained in haplotypes.

Results

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. Disclosures
  8. References

The mean ± SD value for the Alda scale of the whole group was 6.40 ± 2.72. The values 0–5 were placed in the low quartile, while the values 8–10 were placed in the high quartile.

The differences in genotypic and allelic distribution between high and low quartiles and between high- and low-quartile subjects versus remaining subjects in the polymorphisms of the CLOCK, ARNTL, TIM, and PER3 genes are shown in Tables 2–5.

Table 2. p-values for differences in genotypic and allelic distribution between the high quartile (HQ) and low quartile (LQ) and between high- and low-quartile subjects versus remaining subjects in polymorphisms of the CLOCK gene
GeneSNP IDHQ (#4) versus LQ (#1)HQ (#4) versus RQ (#1, 2, 3)LQ (#1) versus RQ (#2, 3, 4)
GenotypesAllelesGenotypesAllelesGenotypesAlleles
  1. CLOCK = circadian locomotor output cycle kaput; ID = identification; RQ = remaining quartiles; SNP = single nucleotide polymorphism.

  2. a

    Fisher's exact test, two-sided.

CLOCK rs18012600.85970.60000.92750.87400.72580.4341
 rs38051480.67720.86570.94821.00000.50180.7649
 rs6849474 0.8147a0.8600 0.6722a0.7476 0.8314a0.8721
 rs119325950.60040.86600.51150.64320.74100.8796
 rs12648271 0.6549a0.7158 0.6820a0.6197 0.8384a0.8678
 rs68505240.80450.62630.90970.76840.05240.4607
 rs12649507 0.8181a0.8608 0.5404a0.63311.000a1.0000
 rs43408440.53691.00000.83441.00000.43020.8767
 rs534654 0.2580a0.3348 0.0937a0.1445 0.6771a0.7170
Table 3. p-Values for differences in genotypic and allelic distribution between the high quartile (HQ) and low quartile (LQ) and between high- and low-quartile subjects versus remaining subjects in polymorphisms of the ARNTL gene
GeneSNP IDHQ (#4) versus LQ (#1)HQ (#4) versus RQ (#1, 2, 3)LQ (#1) versus RQ (#2, 3, 4)
GenotypesAllelesGenotypesAllelesGenotypesAlleles
  1. Significant differences are marked in bold. ARNTL = aryl hydrocarbon receptor nuclear translocator-like; ID = identification; RQ = remaining quartiles; SNP = single nucleotide polymorphism.

  2. a

    Fisher's exact test, two-sided.

ARNTL rs1481892 0.1122a0.1451 0.1008a0.1814 0.1462a0.2366
 rs41463880.0568 0.0425 0.10730.0665 0.0263 0.0667
 rs10766075 0.0355 0.16700.09190.6280 0.0063 0.0364
 rs4757142 0.8195a0.8465 0.4155a0.49581.000a1.0000
 rs73969430.07200.26300.11470.7683 0.0304 0.0759
 rs11824092 0.0439 0.2541 0.0413 0.1950 0.0210 0.0487
 rs7947951 0.6500a0.7287 0.5362a0.6382 0.6809a0.7536
 rs79370600.48860.86950.67250.65680.37961.0000
 rs15624380.35750.30730.54670.43400.17230.2806
 rs38163600.14040.30870.25630.28280.09530.3567
 rs71263030.25781.00000.63800.77110.08470.5605
 rs37893270.18900.14130.23100.14030.28170.1841
 rs11022778 0.6549a0.7130 0.6844a0.7377 0.5471a0.6174
 rs116009960.1078 0.0363 0.14060.05510.17810.0766
 rs110227791.000a1.0000 0.8187a0.83211.000a1.0000
 rs110227800.1078 0.0363 0.14060.05510.17810.0766
 rs7107287 0.0500 0.0394 0.08950.0613 0.0254 0.0432
 rs1982350 0.0415 0.5233 0.0293 0.8832 0.0281 0.1058
Table 4. p-values for differences in genotypic and allelic distribution between the high quartile (HQ) and low quartile (LQ) and between high- and low-quartile subjects versus remaining subjects in polymorphisms of the TIM gene
GeneSNP IDHQ (#4) versus LQ (#1)HQ (#4) versus RQ (#1, 2, 3)LQ (#1) versus RQ (#2, 3, 4)
GenotypesAllelesGenotypesAllelesGenotypesAlleles
  1. Significant differences are marked in bold. ID = identification; RQ = remaining quartiles; SNP = single nucleotide polymorphism; TIM = timeless circadian clock.

TIM rs22917390.23260.49390.07980.26330.48401.0000
 rs22917380.20450.20570.09920.08510.46960.4725
 rs73020600.38360.32560.32130.22940.54480.5482
 rs10876890 0.0408 0.0552 0.0386 0.05700.09870.1413
 rs111718560.21850.19410.17250.13690.38120.8791
 rs2279665 0.0395 0.0834 0.0129 0.11120.19310.2458
Table 5. p-values for differences in genotypic and allelic distribution between the high quartile (HQ) and low quartile (LQ) and between high- and low-quartile subjects versus remaining subjects in polymorphisms of the PER3 gene
GeneSNP IDHQ (#4) versus LQ (#1)HQ (#4) versus RQ (#1, 2, 3)LQ (#1) versus RQ (#2, 3, 4)
GenotypesAllelesGenotypesAllelesGenotypesAlleles
  1. ID = identification; PER3 = period circadian clock 3; RQ = remaining quartiles; SNP = single nucleotide polymorphism.

  2. a

    Fisher's exact test, two-sided.

PER3 rs8367550.55181.00000.39311.00000.83501.0000
 rs2287270.40030.51070.27630.54600.73160.7669
 rs108643150.19490.24330.25690.21680.22950.4391
 rs49086940.82690.84310.61500.85440.95950.8569
 rs2286820.13120.74970.08340.56100.33800.8836
 rs2286420.84011.00000.66910.66050.94890.7713
 rs2172563 0.4938a0.5543 0.5342a0.5908 0.5294a0.5878
 rs2640909 0.4959a0.5665 0.4071a0.4898 0.6785a0.7289
 rs104620210.61780.81950.47021.00000.71590.8340

Eight polymorphisms of the ARNTL gene (rs 4146388, rs10766075, rs7396943, rs11824092, rs11600996, rs11022780, rs7107287, and rs1982350) and two SNPs of the TIM gene (rs10976890 and rs2279665) showed some association with the degree of lithium prophylactic response measured using the Alda scale. However, the significance of this association disappeared after a Bonferroni correction was performed. No association was found with any of the polymorphisms of the CLOCK and PER3 genes.

The linkage disequilibrium map and haplotype blocks of four genes in which a comparison of quartiles 1 and 4 of the lithium response was carried out are shown in Figure 1, where the relative position and linkage disequilibrium estimates between all analyzed polymorphism are presented.

image

Figure 1. Linkage disequilibrium map and haplotype blocks of four genes in which a comparison of quartiles #1 and #4 of the lithium response was carried out. Colored squares correspond to D′ values, with numerical estimates given within the squares.

Download figure to PowerPoint

Two blocks of haplotypes were identified for the CLOCK gene, four for the ARNTL gene, two for the TIM gene, and three for the PER3 gene. Association analysis showed a significant difference in the distribution between the first and fourth quartiles of the lithium response for three haplotype blocks of the ARNTL gene and for one block of the TIM gene, namely:

  1. Haplotype block, seven SNPs of the ARNTL gene:

    image

    Haplotype variant CGCGGCG was significantly less frequent in quartile 1 (0.239), compared with quartile 4 (0.399), p = 0.031. Haplotype variant TTGCTACGCGGCG was significantly more frequent in quartile 1 (0.303) compared with quartile 4 (0.160), p = 0.031.

  2. Haplotype block, four SNPs of the ARNTL gene:

    image

    Haplotype variant GCCC was significantly less frequent in quartile 1 (0.002) compared with quartile 4 (0.075), p = 0.016.

  3. Haplotype block, three SNPs of the ARNTL gene:

    image

    Haplotype variant CGT was significantly more frequent in quartile 1 (0.680) compared with quartile 4 (0.512), p = 0.031.

  4. Haplotype block, three SNPs of the TIM gene:

    image

    Haplotype variant CTA was significantly less frequent in quartile 1 (0.013) compared with quartile 4 (0.680), p = 0.031.

However, none of these differences in the four haplotype blocks above remained significant after a test of 1,000 permutations was performed. No association was found with any of the haplotype blocks of the CLOCK and PER3 genes.

Discussion

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. Disclosures
  8. References

The main finding of the present study is that the polymorphisms of two clock genes (ARNTL and TIM) show an association with lithium prophylactic activity. This adds to the findings of other genetic studies performed by candidate gene methodology in which an association has been found between the lithium response and polymorphisms of other circadian clock genes such as Rev-Erba and GSK-3β.

The findings of associations with lithium prophylactic efficacy can be discussed in the context of the possible importance of the ARNTL and TIM genes in the pathogenesis of bipolar disorder. The most robust association in this respect has been obtained with ARNTL, also known as BMAL1, located on chromosome 11p15. The absence of this gene in the mouse results in a rapid and complete loss of the circadian rhythm [23]. In addition, the ARNTL gene is involved in the integration of the mammalian clock and energy metabolism, and its expression, as well as that of Rev-Erba, another gene associated with the lithium response, is stimulated by the transcriptional coactivator PGC-1α [24]. ARNTL and PER3 were the only clock genes out of the ten genes investigated which were found to be associated with bipolar disorder in the studies by Nievergelt et al. [2] and Mansour et al. [3]. Interestingly, two of the significant ARNTL gene polymorphisms that were associated with bipolar disorder in the Mansour et al. study (rs7107287 and rs1982350) were connected in our study with the degree of lithium response both in genotypic and allelic frequency calculations as well as a part of the largest haplotype block of seven SNPs. These polymorphisms are both located in the intronic region. Although several studies have found important regulatory sequences in the intronic region that regulate the expression patterns, splicing, and the amount of gene transcription [25], to our knowledge, a functionality of this SNP in gene expression has not been demonstrated. Nevertheless, a concordance of our results with those of the Mansour et al. study may suggest such a possibility.

Some associations of TIM with a predisposition to bipolar disorder and also with circadian phenotypes of this disorder were found in the studies by Mansour et al. [3] and Shi et al. [4]. The rs2279665 polymorphism identified by Mansour et al. as being connected with bipolar disorder was also associated in our study with the degree of lithium response both in genotypic and allelic calculation as well as a part of the haplotype block containing three SNPs. This SNP, located in exon3, is a synonymous nucleotide change which does not code for a protein change. However, a concordance of our results with those obtained by Mansour et al. may suggest its functionality.

We did not find an association between the lithium prophylactic response and polymorphism of the CLOCK and PER3 genes either in the genotypic or haplotype analysis. This lack of an association with polymorphism of the CLOCK gene was surprising. The disruption of the CLOCK gene in the mouse results in hyperactivity, decreased sleep, and an increase in reward behavior, which is analogous to the situation in human mania, where the chronic administration of lithium restores many of these behavioral responses to wild-type levels [13]. Some studies have shown an association between the T3111C SNP (rs 1801260) of the CLOCK gene and clinical and circadian phenotypes of bipolar disorder [26, 27]. In our study, no association was found between this SNP and the lithium response. However, given the fact that ARNTL binds to CLOCK and regulates its activity [28], it may be speculated that the changes in ARNTL may also impact CLOCK function. Finally, with regard to lithium efficacy and the PER3 gene, we were unable to confirm the results of studies showing an association between several SNPs of this gene and both a predisposition to bipolar disorder and clinical phenotypes pertaining to this disorder [2, 29, 30].

The sample size of our group was similar to that in other publications analyzing the association between gene polymorphism and the lithium response. The power of association tests was estimated at the level of about 5%, which is characteristic for analysis of complex disorders.

The limitation of our study may have been that significant associations obtained between SNPs and the degree of lithium response mostly disappeared after Bonferroni correction, and those between haplotypes and the lithium response disappeared after a test of 1,000 permutations was performed. However, the strength of our study was the long period of administration of lithium to our patients, which enabled a precise retrospective analysis to be made of the degree of the prophylactic effect of lithium.

Acknowledgement

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. Disclosures
  8. References

This research was supported by the Polish National Science Centre, grant NN-402-4671-40.

Disclosures

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. Disclosures
  8. References

The authors of this paper do not have any commercial associations that might pose a conflict of interest in connection with this manuscript.

References

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. Disclosures
  8. References