Introduction

Several epidemiological studies have indicated that genetic susceptibility constitutes a major predisposing factor for bipolar (manic-depressive) illness. The pattern of inheritance is probably complex, involving multiple genes of weak to moderate effects in interaction with undefined environmental factors. Several chromosomal regions and candidate genes have been proposed, but there is still no conclusive evidence for specific genetic factor(s) involved in the etiology of manic-depressive illness^1,2.

Similarly, there is an incomplete knowledge about the molecular mechanisms underlying the therapeutic efficacy of lithium, which is the most commonly used mood-stabilizing drug in the treatment of bipolar disorder. Lithium has, however, been shown to affect directly and indirectly a large number of (neural) signaling factors.^3,4,5 Among the possible drug targets, the phosphoinositide second messenger system has been intensively studied with respect to its potential role in explaining disease susceptibility or therapeutic action of lithium. The integrity and sustainability of phosphoinositide signaling, which produces the second messengers 1,4,5-trisphosphate and diacylglycerol, depend on a continuous supply of free myo-inositol for the resynthesis of precursor phospholipids. The recycling of inositol (poly)phosphates to free myo-inositol involves several different enzymes, including myo-inositol monophosphatase (IMPase) and inositol polyphosphate 1-phosphatase, which are both uncompetitively inhibited by lithium at therapeutically relevant concentrations.^6,7,8,9 Myo-inositol can also be synthesized de novo from glucose 6-phosphate (involving the IMPase) or transported into the cell by the high-affinity sodium/myo-inositol cotransporter (SMIT).

More than a decade ago, Berridge et al^10,11 proposed that the mood-stabilizing effects of lithium could be explained by the lithium-mediated inhibition of the IMPase enzyme, known as the inositol depletion hypothesis. The IMPase catalyzes the final step in the inositol recycling, by dephosphorylating inositol monophosphates to free myo-inositol. The IMPase-encoding myo-inositol monophosphatase 1 (IMPA1) gene is localized to chromosome 8q21.13–21.3.¹² Recently, a gene on chromosome 18p11.2, named IMPA2, was predicted to encode an IMPase of high similarity to the IMPA1-encoded protein, especially in the active site and the ion-binding regions.^12,13 However, the IMPA2-encoded protein has not yet been isolated or functionally characterized.

Whereas no linkage or association studies so far have provided evidence for a role of IMPA1 in bipolar disorder, there have been many reports that may suggest that IMPA2 could be a genetic susceptibility factor for this disease. Several independent linkage studies have indicated the presence of a susceptibility locus for bipolar disorder (as well as schizophrenia) on chromosome 18p11.2.^{14,15,16,17,18,19} Recently, Yoshikawa et al²⁰ reported an association between various IMPA2 polymorphisms and schizophrenia (but not bipolar disorder) in a large Japanese sample. Other studies have found some evidence for altered IMPase enzyme activity and changes in IMPA1 and myo-inositol monophosphatase 1 (IMPA2) expression levels in bipolar patients.^21,22,23 Moreover, lithium treatment of bipolar patients leads to a reduction in myo-inositol levels in certain brain regions.^24,25 It was recently published that the three mood-stabilizing drugs lithium, valproate and carbamazepine inhibited the collapse of growth cones and increased the growth cone area in sensory neurons.²⁶ These effects were reversed by inositol supplementation, thus implicating inositol depletion as part of a common mechanism of action for the mood stabilizers.

The genomic structure and polymorphisms of the human IMPA1 and IMPA2 genes have been reported previously.^{12,20,27,28,29} In this study, we aimed to further investigate the possible role of the two IMPase-encoding genes in bipolar disorder. An initial scanning for additional variations in IMPA1 and IMPA2 was performed in a limited number of Norwegian manic-depressive patients and normal controls, followed by a family-based association study using a sample of bipolar Palestinian Arab families.

Materials and methods

DNA specimens from a Norwegian pilot sample

Blood specimens for the pilot study were obtained from 44 Norwegian lithium-treated patients with a firm diagnosis of bipolar disorder according to DSM-III-R criteria (SCID interview). The lithium response of the patients was classified retrospectively according to the clinical history, with comparison of the frequency, duration and severity of episodes before and after treatment. Of the patients included, 16 were classified as lithium responders and 16 were classified as lithium nonresponders. Written informed consent was obtained from the participants and the study was carried out with the approval of the regional ethical committee. In all, 48 healthy, anonymous blood donors, recruited from the local blood bank, were used as control subjects. DNA was isolated manually from leukocytes by phenol–chloroform extraction of EDTA-anticoagulated blood or automatically using a DNA extractor (ABI model 341; Applied Biosystems, Foster City, CA, USA).

DNA specimens from a Palestinian Arab trio sample

All patients were of Arab ethnicity and recruited from the Bethlehem and Jerusalem areas. The patients were interviewed by an experienced psychiatrist using the SCID interview. The diagnosis of bipolar disorder was assigned on the basis of the interview and medical records according to DSM-IV criteria. Probands were diagnosed as bipolar-I disorder. All patients gave informed consent and the protocol was approved by the local IRB committee. Genomic DNA was extracted from peripheral blood or transformed lymphocytes using phenol–chloroform or commercial kits. The sample consisted of 75 nuclear families, where 74 families included both parents and the total number of affected offspring was 92.

Polymorphism scanning

An initial polymorphism scanning was performed by direct DNA sequencing of promoters and exons with flanking regions in IMPA1 and IMPA2, using DNA from 25 Norwegian subjects; 23 bipolar patients, randomly chosen from the pilot sample and two control individuals. The marked over-representation of patients compared to control subjects was intended to increase the probability of identifying possible disease-associated DNA variants.

PCR products of exons 2–9 with flanking regions of IMPA1 were obtained as reported previously.¹² The GC-rich exon 1 and promoter region was amplified as a 1142 bp PCR fragment with the following primers: A101, forward (5'-GAGCCTCAGTTTCCTCATCTGT-CT-3') and A102, reverse (5'-I -3').

Polymorphism scanning of exons 2–8 with flanking regions of IMPA2 has been reported previously.²⁸ The GC-rich exon 1 and promoter region in IMPA2 was amplified as a 1555 bp fragment, using the following PCR primers: B103, forward (5'-CATGGGCCAGCAAGAGAATC-3') and B102, reverse (5'-CAGCACAAAGGCTCCTTTCAC-3').

For PCR amplification, we used the Taq DNA polymerase kit (Qiagen, San Francisco, CA, USA) according to the manufacturer's recommendations. For GC-rich regions, the Q-solution buffer (Qiagen) was added to a final 1 concentration. PCR conditions were as follows: an initial denaturing step at 94°C for 1 min, then 35 cycles of 94°C for 20 s, 55–60°C for 15 s and 72°C for 1–2 min.

The PCR products were purified using the Exo-SAP It kit (USB Corporation, Cleveland, OH, USA). All PCR products were sequenced bidirectionally with the corresponding PCR primers, using the ABI Prism Big Dye Terminator Cycle Sequencing Kit (Applied Biosystems). For the sequencing of the GC-rich PCR products, betaine was added to a final 1 M concentration. The reactions were analyzed on a 3100 Genetic Analyzer (Applied Biosystems).

Genotyping

Three IMPA1 polymorphisms (written with bold letters in Figure 1) and 10 IMPA2 polymorphisms (written with bold letters in Figure 2) were selected for further association studies. Allele and genotype frequencies of selected IMPA1 and IMPA2 single-nucleotide polymorphisms (SNPs) were pretested in a Norwegian case/control pilot sample, followed by a family-based association study in samples from Palestinian Arabs.

Figure 1.

Overview of DNA variants localized in the human IMPA1 gene on chromosome 8q21.13–q21.3. Numbered boxes represent the corresponding exons that encode the mRNA transcript. Positions of start and stop codons are marked with arrows. The localization and nature of the detected IMPA1 polymorphisms are indicated. Polymorphisms selected for further genotyping and association studies in this study are written with bold letters. SNPs registered in the database of SNPs are noted with SNP ID numbers in parenthesis.

Full figure and legend (39K)

Figure 2.

Overview of DNA variants localized in the human IMPA2 gene on chromosome 18p11.2. Numbered boxes represent the corresponding exons that encode the mRNA transcript. Positions of start and stop codons are marked with arrows. Localization and nature of the detected IMPA2 polymorphisms are indicated. As an example, -241_-237dup denotes that the five nucleotides in positions -241 to -237 were duplicated. Polymorphisms selected in this study for further genotyping and association studies are written with bold letters. IMPA2 polymorphisms, also reported by Yoshikawa et al, are described with the corresponding nomenclature in parenthesis and marked with^*. IMPA2 polymorphisms found to be associated with schizophrenia in Japanese samples are marked with ^**. SNPs registered in the database of SNPs are noted with SNP ID numbers in parenthesis.

Full figure and legend (44K)

Six genomic regions (three from IMPA1 and three from IMPA2, respectively) were amplified by PCR (primer sequences are available upon request). For genotyping in the association studies, DNA sequencing was performed in one direction, with one of the corresponding PCR primers. The procedures for PCRs, product purification and DNA sequencing are described above.

All sequence chromatograms for the genotyping procedure were analyzed using the Staden Package available at http://www.mrc-lmb.cam.ac.uk/pubseq/index.html. Pregap4 was used for processing and assembling the chromatogram files, and Gap4 for viewing the resulting sequence assemblages and for the identification of DNA differences.

Calculating linkage disequilibrium (LD) measures between DNA polymorphisms

To calculate LD measures between polymorphisms, we used the two-locus linkage disequilibrium (2LD) calculator, which is freely available at http://www.iop.kcl.ac.uk/IoP/Departments/PsychMed/GEpiBSt/software.2ld.shtml.³⁰

Case/control association analysis of the Norwegian pilot sample

Allele frequencies were calculated for each genotype, and the expected genotype frequencies were calculated from the allele frequencies under the assumption of Hardy–Weinberg equilibrium. The differences in allele and genotype frequencies between cases and controls were determined using Pearson's ² test.

The descriptive statistics for observed differences in allele or genotype distribution with the corresponding P-values were analyzed using the program package, SPSS, version 10.0 (SPSS Inc., Chicago, IL, USA) in combination with StatXact for windows, version 4.0.1 (Cytel Software Corp., Cambridge, MA, USA).

Transmission disequilibrium test (TDT) of the Palestinian Arab trio sample

A TDT³¹ was accomplished as a family-based model for association studies in the Palestinian Arab trios. The TDT considers different alleles at particular DNA markers and tests for an excess of transmission to affected offspring, generating a composite ² statistic. We used the TDT/STDT software that is freely available at http://genomics.med.upenn.edu/spielman/TDT.htm. The corresponding P-values were calculated using the McNemar's test with the StatXact software.

To examine the possible transmission disequilibrium between SNP-based haplotypes and disease, we used the program TRANSMIT (version 2.5.4) freely available at http://www-gene.cimr.cam.ac.uk/clayton/software/. This program performs a TDT-like test of excess transmission of each haplotype formed by the available SNP's as well as a global test of differential transmission.³² Systematic combinations ('sliding window' approach) of two-, three- and four-marker haplotype transmission analysis were performed as described by Straub et al.³³ The corresponding P-values were calculated on http://www.graphpad.com/quickcalcs/PValue1.cfm.

Nucleotide sequences and polymorphism nomenclature

The complete genomic sequence of IMPA1, including SNPs registered in the database of SNPs (dbSNP), is reported in genomic contig NT_008183 by the NCBI Reference Sequence project (RefSeq; http://www.ncbi.nlm.nih.gov/RefSeq). Sequence data of the IMPA1 exons with flanking regions are deposited in the EMBL/GenBank Data Libraries under accession numbers Y11360-Y11366.

The complete genomic sequence of IMPA2, including SNPs registered in the dbSNP, is reported in genomic contig NT_010859 by NCBI RefSeq. Sequence data of IMPA2 exons with flanking regions and its corresponding promoter are deposited in the EMBL/GenBank Data Libraries under accession numbers AF157096-AF157102.

The positions of the polymorphisms have been described relative to the open-reading frames of IMPA1 and IMPA2, respectively, defining the A of the ATG start codon as position +1, as suggested by the Nomenclature Working Group.^34,35

Results

IMPA1

In the initial DNA scanning of 23 bipolar patients and two control individuals from Norway, a total of nine IMPA1 polymorphisms were detected (see Figure 1) of which only one has been published previously.¹² Three polymorphisms (-800C>T, -742C>T and -446T>C) were localized in the promoter region, two polymorphisms (197+29ins16bp and 197+54G>A) in intron 3, one polymorphism (718+101C>T) in intron 8 and three polymorphisms (986G>T, 1789G>A and 2030G>A) in the 3'-UTR of exon 9.

-446T>C, 986G>T, 1789G>A and 2030G>A were the most frequent IMPA1 polymorphisms in the Norwegian pilot sample (0.42, 0.15, 0.42 and 0.42, respectively), and these markers were all in LD with each other (D'>0.98; results not shown). The remaining polymorphisms detected in IMPA1 had allele frequencies below 0.13. Owing to their higher frequency, the SNPs -446T>C, 986G>T and 2030G>A were selected for further association studies.

In the extended Norwegian pilot sample (44 patients and 48 controls), no associations were found between the selected IMPA1 polymorphisms and bipolar disorder, neither with respect to disease susceptibility nor with variation in lithium response (data not shown).

In the sample of Palestinian Arabs (92 trios), none of the parental IMPA1 SNP alleles were unequally transmitted to affected offspring (data not shown). The most frequent estimated IMPA1 haplotypes were [-446T; 986G; 2030G] (0.55), [-446C; 986G; 2030A] (0.31) and [-446C; 986T; 2030A] (0.10). None of the estimated IMPA1 haplotypes were unequally transmitted to affected offspring (data not shown).

IMPA2

In all, 16 polymorphisms in IMPA2 have been published previously.^20,28,29 In the present study, we detected five new DNA variants in the GC-rich promoter region (-1209T>G, -1051T>G, -1031C>A, -241_-237dup and -207T>C) (Figure 2).

Three regions were selected for further genotyping in association studies of bipolar disorder (see Figure 2). The first site, localized in the promoter region, was selected due to its putative functional importance. Also, the -461C>T, -241_-237dup, -207T>C and -185A>G polymorphisms were relatively frequent in the Norwegian pilot sample (0.26, 0.11, 0.25 and 0.23, respectively). For the association analysis of complex disorders, it has been recommended that SNP frequencies should range between 0.1 and 0.4.³⁶ The second region around exon 2 contained the 97-15G>A, 159T>C and 230+141G>A DNA variants (with frequencies of 0.13, 0.02 and 0.12, respectively), of which 159T>C despite its low frequency, was considered interesting due to a recently reported association to bipolar disorder.²⁹ The third selected region surrounding exon 5 contained the infrequent polymorphisms 382-44G>A, 443G>A (R148Q) and 490+13_14insA (with the frequencies of 0.08, 0.01 and 0.02, respectively). The 443G>A variant has been predicted to give an amino-acid change from a basic arginine to a polar glutamine residue in codon 148, which was of special interest as a putative functional IMPA2 polymorphism.²⁸ The LD coefficients were calculated for the different combinations of the 10 IMPA2 SNPs, using the parental genotypes of the Palestinian trios (Table 1). As could be expected, markers located in the same region (ie in the 5'-end, exon 2 and exon 5 regions of IMPA2) were linked to each other. Especially, it should be noted that the frequent -461C and -207T alleles in the promoter region were in strong LD to each other.

Table 1 - LD coefficients for IMPA2 polymorphisms in Palestinian Arab parents.

Full table

In the extended Norwegian pilot sample, an apparent association was found between the IMPA2 159C allele and bipolar disorder (44 patients, frequency of 159C allele=9/88; 47 controls, frequency of 159C allele=2/94; ²=5.25, d.f.=1, P=0.03, no Bonferroni correction for multiple testing) (complete data are available upon request). No association was found between the IMPA2 variants and varying lithium response in the Norwegian bipolar patients (results not shown).

More intriguing data were obtained when testing the transmission of selected IMPA2 polymorphisms in the sample of Palestinian Arab bipolar families, as two SNPs located in the IMPA2 promoter were apparently strongly associated with bipolar disorder (Table 2). The -461C and -207 T alleles were significantly more often transmitted than nontransmitted to bipolar offspring (P=0.006 and 0.002, respectively; no Bonferroni correction for multiple testing). These polymorphisms were strongly linked to each other (Table 1; D'=0.97) and in most families, the -461C and -207 T variants seemed to be transmitted together. Two nearby polymorphisms in the IMPA2 promoter, -241_-237dup and -185A>G, demonstrated a similar trend of association to bipolar disorder (Table 2). No differences were found between paternal and maternal inheritance of the IMPA2 polymorphisms.

Table 2 - Transmitted IMPA2 polymorphisms in families of bipolar Palestinian Arabs.

Full table

Based on the IMPA2 genotypes in the trio samples of Palestinian Arabs, consecutive and systematic two-, three- and four-locus haplotype transmission analyses were performed. Table 3 shows the sliding windows of the IMPA2 three-locus haplotype transmission analysis, demonstrating that certain combinations of the DNA variants in the promoter and 5'-end were significantly more often transmitted to the affected offspring than expected. The effect was most pronounced in the ultimate 5'-end of the investigated region of the gene, with a gradual decrease in preferential transmission towards the 3'-end of IMPA2. Similar results were obtained in the two- and four-locus haplotype transmission analyses (data not shown).

Table 3 - Transmission of overlapping IMPA2 three-marker haplotypes.

Full table

The most probable promoter haplotypes were then estimated (Table 4). The four promoter polymorphisms, -461C>T, -241_-237dup, -207T>C and -185A>G, formed nine of 16 possible haplotypes. Four of these estimated major haplotypes had a frequency above 0.05 (Table 4). The remaining infrequent haplotypes (frequencies <0.05) were flagged and aggregated, and subsequently, the transmission of the most common haplotypes was tested. The most frequent haplotype, [-461C; -241_-237dup-negative; -207T; -185A] was transmitted significantly more often than expected to the bipolar offspring. Accordingly, the second most frequent haplotype, [-461T; -241_-237dup-negative; -207C; -185G] was transmitted significantly less often than expected to the cases.

Table 4 - Transmission of IMPA2 promoter haplotypes in Palestinian Arab trios.

Full table

Discussion

IMPA1

The lithium-inhibited IMPA1-encoded IMPase was the biological background for the inositol depletion hypothesis on the mechanism of action of lithium in bipolar disorder. ^9,10,37 However, there have so far been no firm reports on the linkage of manic-depressive illness to chromosome 8q21. In the present study, neither of the IMPA1 polymorphisms examined was associated with bipolar disorder. Consequently, the present data do not support the human IMPA1 gene as a genetic risk factor in manic-depressive illness.

IMPA2

We here report an apparently significant association between two IMPA2 promoter SNPs (-461C>T and -207T>C) and bipolar disorder in a trio sample of Palestinian Arabs. Two nearby promoter polymorphisms demonstrated a trend of association, indicating the importance of certain combinations of DNA variants in the 5'-end of IMPA2. This assumption was indeed supported by systematic three-locus and estimated haplotype transmission analyses, which demonstrated that the most frequent IMPA2 5'-end haplotype was transmitted significantly more often than expected to the bipolar offspring. This apparent association between IMPA2 promoter variants and haplotypes and manic-depressive illness supports the possible role of IMPA2 or a nearby gene as a susceptibility factor in bipolar disorder.

Interestingly, the human IMPA2 gene is localized on chromosome 18p11.2 where several independent studies have reported linkage to bipolar disorder.^{14,15,16,18,19} A susceptibility locus on chromosome 18p11.2 has also been demonstrated in a linkage study on schizophrenia, in which the statistical significance was increased by including unipolar affective disorder in a wide definition of the affected phenotype category.¹⁷ These data are further strengthened by a recent report on a significant association between schizophrenia (but not bipolar disorder) and three different IMPA2 polymorphisms (-185A>G, 97-15G>A and 800C>T) in a Japanese sample.²⁰ It is therefore plausible that DNA variants in one or more loci on chromosome 18p11.2, such as the IMPA2 gene, may contribute as genetic susceptibility factor(s) for bipolar disorder, in particular, and functional psychoses in general. However, it should be noted that the apparent importance of the IMPA2 promoter polymorphisms was not confirmed in the small Norwegian pilot sample, although an association between bipolar disorder and the 159C variant in exon 2 of IMPA2 was indicated. Interestingly, the same 159C allele showed a trend of association to bipolar disorder in a sample from the NIMH Genetics Initiative.²⁹

The data are, however, not corrected for multiple testing, and should therefore be considered with some caution. In short, we have tested a total of 13 DNA variants in two genes in two separate samples of limited size. Multiple testing like this raises the risk of producing false-positive results by chance. The Bonferroni method can be used to correct for multiple testing. However, this method assumes independency between different tests, whereas DNA variants in the same gene are indeed linked on the same chromosomal strand and not transmitted independently of each other, as was clearly demonstrated by the calculation of LD coefficients. Therefore, the Bonferroni correction was considered as too conservative for calculating corrected P-values in this study. Despite the risk of false-positive results (type I errors), we consider the apparent association between IMPA2 promoter polymorphisms and manic-depressive illness as highly interesting, and we recommend further studies of these polymorphisms in additional and larger samples.

Since the polymorphisms associated with bipolar disorder in Palestinian Arabs are located in the promoter region, they may have a functional role in the regulation of the IMPA2 expression. Three of the promoter variants (-241_-237dup, -207T>C and -185A>G) are present in a region of alternative transcription start sites,^12,13 where -241_-237dup represents the five first nucleotides of the transcript reported by Yoshikawa et al.²⁹ The -207T>C polymorphism is localized in a putative MZF1 regulatory site.²⁸ There is some evidence for altered IMPase enzyme activity²¹ and changes in the IMPA2 expression levels²³ in bipolar patients compared to control individuals. These differences could possibly be explained by DNA variants in the regulative regions of the IMPA2 promoter and/or the 5'-UTR of the mRNA transcript. Indeed, the degrees of transmission disequilibrium observed for the (-461C>T and -207T>C SNPs in the Palestinian Arab sample is rather large in the context of complex disorders. It is therefore a possibility that these polymorphisms could constitute the susceptibility-causing variants. Further studies are necessary to determine any functional effects of the IMPA2 promoter polymorphisms in relation to disease susceptibility in bipolar patients.

Moreover, it has been reported that the cellular level of myo-inositol is decreased in post-mortem brain tissue³⁸ and lymphocytes³⁹ from bipolar patients, as compared to healthy controls. These data are somewhat unexpected in light of the demonstration that the mood-stabilizers lithium, valproate and carbamazepine decrease the cellular content of myo-inositol in neuronal cell lines,^40,41,42 and inositol depletion has been implicated as part of a common mechanism of action for these drugs. This apparent discrepancy might be explained by recent results showing that these mood stabilizers increase inositol uptake at low cellular inositol levels and, on the other hand, reduce the inositol uptake at high cellular inositol levels.⁴² The possible influence on IMPA2 expression levels by different mood stabilizers at varying cellular inositol levels should be further studied for the different IMPA2 promoter haplotypes.

The proposed IMPase2 protein has not been isolated and characterized for its biochemical properties. Indeed, it has not even been experimentally demonstrated that IMPase2 displays IMPase activity. It is therefore not known whether IMPase1 and IMPase2 will favor different substrates and whether the lithium inhibition differs between these proteins. Consequently, it is essential to isolate and separate the IMPase1 and IMPase2 enzymes in future studies, to measure and compare the enzyme activities (K_m and V_max) for different substrates and the efficiency of lithium inhibition under different conditions. As a first step, we have cloned and expressed the human IMPA2 cDNA in Escherichia coli cells (unpublished data).

It is a possibility that the different IMPA2 alleles and haplotypes are in LD with DNA variants in other nearby candidate loci. One of these genes, G-olf, is located less than 100 kb from the IMPA2 gene in distal direction on region 18p11.2 (see contig sequence NT_010859.10). Certain intragenic G-olf DNA alleles have been reported to be preferentially transmitted to schizophrenic patients.¹⁷ In addition, a novel gene, MPPE1, is localized between IMPA2 and G-olf. The proposed MPPE1 protein, a metallophosphoesterase, shows similarity to the exonuclease family involved in DNA repair and telomere length maintenance.⁴³ It should be noted that some conflicting data have been reported for the arrangements of genes on region 18p11.2 (NT_010859 vs Reyes et al⁴⁴).

In conclusion, we have demonstrated an apparently significant association of IMPA2 promoter alleles with bipolar disorder in Palestinian Arab trios, although the data were not corrected for multiple testing. The association was not replicated in Norwegian cases, although a weaker association was observed for a different polymorphism in the same gene. The positive association of IMPA2 to bipolar disorder should be confirmed in additional samples. Our results, however, support the earlier linkage reports of bipolar disorder to this locus and sustain the candidature of IMPA2 as a possible susceptibility gene in manic-depressive illness.

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Acknowledgements

We would like to thank the anonymous reviewers for very valuable comments on this manuscript. We also thank Anne-Karin Gulbrandsen for assisting in the genotyping of the DNA polymorphisms. This work was supported by Dr Einar Martens' Research Fund and the Research Council of Norway (Mental Health Programme).