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Human Molecular Genetics Advance Access originally published online on September 27, 2007
Human Molecular Genetics 2008 17(1):87-97; doi:10.1093/hmg/ddm286
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Brain-specific tryptophan hydroxylase 2 (TPH2): a functional Pro206Ser substitution and variation in the 5'-region are associated with bipolar affective disorder

Sven Cichon1,2,*, Ingeborg Winge5, Manuel Mattheisen1,3, Alexander Georgi6, Anna Karpushova1, Jan Freudenberg7, Yun Freudenberg-Hua2, Gulia Babadjanova8, Ann Van Den Bogaert9, Lilia I. Abramova10, Sofia Kapiletti11, Per M. Knappskog12, Jeffrey McKinney5, Wolfgang Maier4, Rami Abou Jamra2, Thomas G. Schulze6, Johannes Schumacher2, Peter Propping2, Marcella Rietschel6, Jan Haavik5 and Markus M. Nöthen1,2

1 Department of Genomics, Life and Brain Center 2 Institute of Human Genetics 3 Institute for Medical Biometry, Informatics and Epidemiology and 4 Department of Psychiatry, University of Bonn, Bonn, Germany 5 Department of Biomedicine, University of Bergen, Bergen, Norway 6 Central Institute for Mental Health, Division Genetic Epidemiology in Psychiatry, Mannheim, Germany 7 Department of Neurology, Laboratories of Neurogenetics, University of California, San Francisco, USA 8 Russian State Medical University, Moscow, Russia 9 Department of Molecular Genetics, Flanders Interuniversity Institute for Biotechnology, University of Antwerp, Antwerp, Belgium 10 Mental Health Research Center, Moscow, Russia 11 Moscow Research Institute of Psychiatry, Moscow, Russia 12 Center for Medical Genetics and Molecular Medicine, Haukeland University Hospital, Bergen, Norway

* To whom correspondence should be addressed at: Department of Genomics Life and Brain Center University of Bonn Sigmund-Freud Street 25, D-53127 Bonn, Germany. Tel: +49 2286885405; Fax: +49 2286885401; Email: sven.cichon{at}uni-bonn.de

Received June 25, 2007; Accepted September 24, 2007


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
The neurotransmitter serotonin [5-hydroxytryptamine (5-HT)] controls a broad range of biological functions that are disturbed in affective disorder. In the brain, 5-HT production is controlled by tryptophan hydroxylase 2 (TPH2). In order to assess the possible contribution of TPH2 genetic variability to the aetiology of bipolar affective disorder (BPAD), we systematically investigated common and rare genetic variation in the TPH2 gene through a sequential sequencing and SNP-based genotyping approach. Our study sample comprised two cohorts of BPAD from Germany and Russia, totalling 883 patients and 1300 controls. SNPs located in a haplotype block covering the 5' region of the gene as well as a rare, non-synonymous SNP, resulting in a Pro206Ser substitution, showed significant association with bipolar disorder. The odds ratio for the minor allele in the pooled sample was 1.5 (95% CI 1.2–1.9) for rs11178997 (in the 5'-associated haplotype block) and 4.8 (95% CI 1.6–14.8) for rs17110563 encoding the Pro206Ser substitution. Examination of the functional effects of TPH2 Pro206Ser provided evidence for a reduced thermal stability and solubility of the mutated enzyme, suggesting reduced 5-HT production in the brain as a pathophysiological mechanism in BPAD.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
Bipolar affective disorder (BPAD) is a severe psychiatric disorder characterized by episodes of mania and depression with a lifetime prevalence of 1.3–1.6% (1) in all human populations. The occurrence of at least one manic episode distinguishes BPAD from major depressive disorder (MDD), another common affective disorder. Both BPAD and MDD share depressive symptoms as a common liability factor and a substantial percentage of patients commit suicide. The neurotransmitter serotonin [5-hydroxytryptamine (5-HT)] has been shown to influence a broad range of behavioural functions that are disturbed in affective disorder, including the control of mood, sleep-wake cycle, appetite, sexual behaviour and cognition (2,3). Decreased concentrations of the 5-HT metabolite 5-hydroxyindole acetic acid in cerebrospinal fluid and brain tissues of depressed individuals and suicide victims/attempters strongly suggest a dysregulation of serotonergic neurotransmission in affective disorder (2,4,5). Further, most antidepressant drugs (e.g. serotonin-selective reuptake inhibitors and tricyclic antidepressants) exert their effect by increasing the levels of extracellular 5-HT by inhibiting its reuptake or blocking its metabolism. It is therefore likely that naturally occurring variants of genes involved in the regulation of 5-HT levels could play a role in the development of affective disorder. A very strong candidate in this respect is the gene for the recently identified brain-specific isoform of tryptophan hydroxylase (TPH), designated TPH2, which is the rate-limiting enzyme in the biosynthesis of 5-HT in neural tissue (6). In fact, recent studies have provided preliminary evidence for an involvement of TPH2 variants in MDD and BPAD (7–13) as well as in suicide (10,13–15). With the exception of Zhang et al. (9), who reported the Arg441His missense mutation in MDD, no genetic variants of obvious functional relevance could be identified up to now.

The aim of our study is to investigate the possible contribution of genetic variants of TPH2 to the development of BPAD. On the one hand, we made use of haplotype tagging SNPs as provided by the International HapMap Project, to test for the contribution of common variants. Since it is not clear to what extent rare variation (i.e. ≤1% minor allele frequency)—which is under-represented in common databases—might have an impact on BPAD, we also re-sequenced the coding region of the TPH2 gene in 182 chromosomes. One rare, non-synonymous SNP in exon 6 (resulting in Pro206Ser) was identified and also included in the study.

We show that a common haplotype block covering the 5' region of the gene and the rare, non-synonymous change are associated with BPAD in two samples of German and Russian origin. Further, we present comprehensive functional investigations of mutated TPH2 (Pro206Ser) that strongly suggest that this is one of the first genetic variants of pathophysiological relevance identified in BPAD so far.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
In the present study, we systematically investigated TPH2 variability in two cohorts with BPAD. We selected 12 haplotype tagging (ht)SNPs that captured all haplotypes at a population frequency >5%. In addition, we included two SNPs (rs11178997 and rs11178998) from the 5'-regulatory and 5'-untranslated region. Apart from these frequent SNPs that are best suited to detect common susceptibility variants, we aimed to include rare, coding variants that tend to be under-represented in the common databases and that are not adequately tested for by using HapMap data alone. Such infrequent non-synonymous variants may well be disease-relevant and highly penetrant, as demonstrated by the Arg441His variant described by Zhang et al. (9). We therefore re-sequenced the complete coding region (11 exons) in a total of 45 BPAD patients of German descent and 46 population-based control individuals, originating from various European countries proportional to their relative population size (16). The only non-synonymous change was found in exon 6 (c.757 C->T), giving rise to a substitution of a proline in position 206 of the TPH2 protein with a serine (after submission to dbSNP now known as rs17110563). We included rs17110563 in our study and eventually genotyped a set of 15 SNPs in two patient–control samples with BPAD.

In the first sample, comprising 636 BPAD patients and 1070 population-based controls of German descent, we found significant association with three SNPs that are located in haplotype block 1, which covers part of the 5' regulatory region and exons 1 and 2 (Table 1). The minor alleles of SNPs rs11178997, rs11178998 and rs7954758 were significantly over-represented in BPAD patients when compared with controls (P = 0.0039–0.0051, OR 1.5). These three SNPs are in almost perfect LD, with pairwise r2 values of 0.992–1.0 and D' values of 1.0 (Supplementary Material, Table S1). For one of these SNPs, rs11178997, Scheuch et al. (17) recently demonstrated that the A-allele reduces TPH2 transcriptional activity in serotonergic neurons from rat raphe nuclei and in human small cell lung carcinoma cells. In our German BPAD sample, genotype distributions for the associated SNP rs11178997 (P = 0.0047) showed an OR of 1.36 for TA-heterozygous individuals and 4.00 for homozygous risk-allele carriers (Table 2).


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  		Table 1. Results of association analysis of SNPs in the TPH2 gene with BPAD

 


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  		Table 2. Genotype distributions of associated TPH2 SNPs rs11178997 and rs17110563

 
The rare, non-synonymous SNP rs17110563 also showed association: nine out of 636 BPAD patients were heterozygous for Pro206Ser, compared with only three out of 1070 controls (P = 0.0066, OR 5.1). We noticed that eight out of nine (88.9%) Pro206Ser heterozygous BPAD patients had a strong family history for affective disorder with multiple affected family members. The homozygous wild-type individuals, in contrast, comprised only 66.5% family history positive patients. Strikingly, when we looked up information for the three heterozygous individuals in our population-based control sample, we found that one was affected with MDD and had a strong family history of affective disorder (mother and one uncle Bipolar I, grandfather MDD). When we excluded this individual and re-analysed the sample, the OR for the rs17110563 CT genotype increased from 5.1 to 7.7 and the P-value decreased from 0.0066 to 0.0021 (Table 2). For three of the heterozygous patients, we had DNA of additional family members available that allowed us to study the segregation of the Ser206 variant in these families (Fig. 1). Our results are suggestive of non-random co-segregation of the Ser206 (T-allele) variant with affective disorder. In each family, the parent who would be expected to carry the mutation, in fact, is a carrier (Fig. 1, a/002, b/003, c/003). Moreover, in eight situations, where the transmission of the C/T genotype to affected offspring can be traced (from a/002 to a/004, a/005, a/006, and a/008; from b/003 to b/001 and b/004; from c/003 to c/001 and c/002), the T-allele is always transmitted (100% transmissions compared with 50% assuming random segregation). At the same time, Pro206Ser is not fully penetrant as indicated by individuals a/007 and b/003, who are carriers of the T-allele, but do not have a psychiatric diagnosis. Another exception from perfect co-segregation is individual c/005 who is not a carrier of the T-allele, but is affected with BPAD (bipolar I disorder according to DSM-IV).


Figure 1
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  		Figure 1. Three BPAD families (A–C), of which the index patient (indicated by an arrow) was included in the German BPAD sample and was identified to be heterozygous for SNP rs17110563 (C/T), encoding the Pro206Ser substitution. Heterozygous individuals (C/T) are carriers of the ‘mutated’ Ser206 (T) allele, homozygous (C/C) individuals are homozygous for the wild-type Pro206 (C) allele. Squares denote males, circles females and crossed-out symbols represent deceased individuals. Unfilled symbols represent individuals without a psychiatric diagnosis. Individuals with colour-coding (half black, half colour) have a DSMIV diagnosis that we used to define as ‘affected’ in our previous disease model used for affecteds-only linkage analyses of BPAD families (32). Individuals marked in red are bipolar I (BPI) individuals, blue individuals have a diagnosis of recurrent major depression (RUP or MDD), green denotes a diagnosis of schizoaffective disorder (SA). Checkered symbols respresent individuals with a minor psychiatric diagnosis that was not included in our disease model of BPAD.

 
Since TPH2 had been reported to be associated with suicidality, we subdivided the German BPAD sample (for which information on this symptom was available) into a group that had experienced suicidal ideation and a group without suicidal thoughts and tested for association with the four BPAD-associated SNPs (Table 3). The comparison of the group of patients with suicidal ideation against controls produced almost identical OR for the three SNPs in the 5' region (rs11178997, rs11178998, rs7954758) as the group of patients without suicidal ideation against controls (Table 3). The frequency of the non-synonymous SNP appears to be higher in the group with suicidal ideation. We also compared the suicidal ideation versus no suicidal ideation groups directly and found no significant difference between these two groups. These results suggest that both subgroups contribute similarly to the association finding and that there is no specific effect of the associated TPH2 SNPs on suicidal ideations.


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  		Table 3. Results of association analysis of four associated TPH2 SNPs in BPAD with and without suicidal ideation

 
In an attempt to replicate our results from the German sample, we tested the 15 SNPs in an independent, but smaller sample from Russia, consisting of 247 BPAD patients and 230 controls. We observed very similar overrepresentations of risk alleles in both samples (Table 1). The three SNPs in the 5' region showed a non-significant trend (P = 0.067–0.085) and heterozygosity for the rare, non-synonymous SNP occurred four times in the patients and once in the controls (P = 0.21). Since there were no significant differences between SNP allele frequencies among German and Russian controls, we decided to combine the two samples to increase statistical power. As shown in Tables 1 and 2, the significance levels for the associated SNPs increased in the combined sample.

Pro206 is part of a highly conserved domain in all the aromatic amino acid hydroxylases (18). We have recently generated a homology-based model of human TPH2 based on the crystal structure of the related enzyme tyrosine hydroxylase (TH) (19). Inspection of the wild type (WT) and mutant enzymes (Fig. 2A) revealed that proline 206 is found at the surface of TPH2, close to the neighbouring subunit and far from the active site (24.6 Å from the active site iron atom to the C{alpha} atom of Pro206). The strong genetic association of Pro206Ser with BPAD, its location in a conserved region of the protein and the substitution of the hydrophobic imino acid proline with the polar serine residue prompted us to assume that it might be a functionally relevant mutation that in itself confers a disease risk by changing the functional properties of TPH2. We therefore produced WT (TPH2 Pro206) and the TPH2 Pro206Ser mutant in Escherichia coli and human embryonic kidney (HEK 293) cells and compared their expression levels, solubilities, thermal stabilities, secondary structure and catalytic properties (Fig. 2, Table 4). We also compared the properties of the Pro206Ser TPH2 mutant with the Arg441His variant, which has been reported to be strongly associated with MDD in an American sample, has an 80% loss of function in serotonin production in PC12 cells (9) and recently has been shown to have strongly reduced thermal stability and solubility in vitro (19). Although Arg441His was not observed in any of the 2183 individuals studied here (and therefore supposedly represents a locally confined founder-mutation), we tested the effect of this mutation using the same methods as for Pro206Ser and compared the relative effects of the mutations. TPH2 Pro206Ser had similar catalytic activity (Vmax) compared with the WT (Table 4), but had decreased solubility (Fig. 2B) and thermal stability. At 37, 50 and 55°C, the rate of TPH2 Pro206Ser inactivation was 3–6-fold higher than that for the WT enzyme (Fig. 2C, data not shown for 50°C). In comparison, the TPH2 Arg441His had reduced catalytic activity (Vmax) compared with the WT, and its solubility and thermal stability were even lower than those for the Pro206Ser mutant (Table 4) (19).


Figure 2
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  		Figure 2. Secondary structure, solubilities and thermal stabilities of WT and mutant TPH2. (A) Model of the tetrameric TPH2 with the four subunits marked in different colours. The positions of the two known amino acid substitutions are indicated by a circle (Pro206Ser) and a square (Arg441His). (B) Relative solubilities of WT and mutant TPH2 were measured by comparing the band intensities of Coomassie stained SDS–PAGE gels of the soluble fraction and the pellet (30 µg of total protein in each lane) (33). The arrow indicates the position of the TPH2-MBP fusion protein. (C) Thermal stability of WT and mutant TPH2. Enzyme samples were incubated at 37 or 55°C and residual TPH2 activity (in % of initial activity at t0) was assayed every 2–5 min. See Table 4 for a summary of enzyme properties.

 


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  		Table 4. Biochemical properties of wild-type and mutant human TPH2

 
Given that all Pro206Ser mutation carriers observed in the course of our study were heterozygous, we also wanted to mimic this situation by co-expressing equimolar amounts of WT and mutant protein in HEK 293 cells. As shown in Figure 3A, the TPH activity in cell lysates containing a 50/50 mixture of the WT and Pro206Ser mutant was significantly lower than that for cells transfected with identical amounts of pure WT plasmid. Likewise, the amount of TPH2, as detected by western blotting, was also significantly lower for the mutant protein and for the mixture of mutant and WT protein. However, the level of precision in these experiments was not adequate to allow firm conclusions about possible dominant interallelic effects of these enzyme forms.


Figure 3
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  		Figure 3. The effect of heteroallelic expression of TPH2 in HEK 293 cells and using in vitro transcription and translation. (A) Enzyme activity and amount of TPH2 protein detected in HEK 293 cells transfected with pure TPH2 WT plasmid, pure mutant plasmid or a 50/50 mixture of the WT and mutant plasmids. (B) TPH activity in reticulocyte lysates containing pure TPH2 WT plasmid, pure Pro206Ser plasmid, pure Arg441His plasmid or a 50/50 mixture of the WT and mutant plasmids. The values in (A) represent the mean±SEM of three to nine measurements. All values for the mutants and the mixture of mutant and WT enzyme were significantly different from the pure WT enzyme (*P < 0.05, ANOVA).

 
In order to examine this possibility in further detail, we also employed an alternative experimental system where the WT and mutated proteins were in vitro transcribed and translated either alone or in combination (Table 4, Fig. 3B). Using [35S]methionine labelling and SDS–PAGE with autoradiography, one major band of 57 kDa molecular mass was observed for all the different proteins, corresponding to the subunit molecular mass of TPH2 (20). The intensity of this protein band was identical for the WT and mutant enzymes, indicating similar expression levels of the proteins. As for HEK 293 cells, the specific activity of the TPH2 Arg441His and Pro206Ser mutants were significantly lower than that for the WT enzyme. The activity of the 50/50 mixed enzymes was lower than that for the pure WT, and slightly lower than the average of the pure enzymes, i.e. 2.2 pmol/min/mg for the Pro206Ser/WT mixture, compared with an expected value of 2.4 pmol/min/mg, and 1.4 pmol/min/mg for the Arg441His/WT mixture, compared with an average value of 2.0 pmol/min/mg. These values are consistent with a mild dominant negative effect of the Pro206Ser mutation and a moderate negative effect of the Arg441His mutation.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
Our overall genetic and biochemical data suggest that genetic variation of TPH2 is involved in the aetiology of BPAD. At least two independent risk-conferring variants are present in our samples: a rare variant, rs17110563 (MAF <<1% in controls), encoding a Pro206Ser substitution, and a more frequent haplotype block of at least 3 kb size (including rs11178997, MAF ~6%) spanning a genomic region of putative regulatory relevance. The strength of our study is that it independently shows the same effect of both risk-conferring variants in two different BPAD samples. Although these results are encouraging, replication studies in other independent samples will be necessary to confirm our finding.

We are not aware of any other published study that has tested rs17110563 in BPAD or other neuropsychiatric disorders. On the other hand, rs11178997 had previously been tested for association with BPAD in the NIMH wave three pedigrees, but failed to show evidence there (13). In this study, however, only 33 families were informative for marker rs11178997, with the consequence of low power to detect an association of the magnitude observed in our study. Another recent study (11) investigated this SNP in Swedish MDD and BPAD samples. Rs11178997 was significantly associated with MDD (135 patients/364 controls), however, showing an under-representation of allele A (which is a risk allele for BPAD in our study). The BPAD sample (182 patients/364 controls) was not significantly associated with rs11178997 (11). Interestingly, however, the A-allele was over-represented in the Swedish BPAD patients (7% in patients, 5% in controls; P = 0.16) which is a distribution comparable with the one seen in our German and Russian BPAD samples (8.6% in the German and Russian BPAD patients, 5.9% in controls; P = 0.00046; Table 1).

Overlapping with the submission of our study for publication, Scheuch et al. (17) reported results of functional studies investigating the effect of TPH2 promoter polymorphisms on transcriptional activity in serotonergic neurons from rat raphe nuclei and human small lung carcinoma cells. Interestingly, they found that the A-allele of rs11178997, which is associated with BPAD in our study, significantly reduces TPH2 transcriptional activity by 22% in serotonergic raphe neurons and 7% in lung carcinoma cells. These data suggest that A/rs11178997 may be the functionally relevant variant underlying the association of a haplotype block in the 5' region of the TPH2 gene with BPAD in our study.

Other groups have previously tested whether polymorphisms in the TPH2 promoter region exert an effect on TPH2 expression. DeLuca et al. (15) found a significantly greater amount of TPH2 mRNA in 35 postmortem dorsolateral prefrontal cortex samples from BPAD patients versus 35 control brain samples, but failed to show a correlation between mRNA amount and two promoter SNPs, among them rs11178997. Another recent TPH2 expression study on 48 postmortem median raphe nuclei samples found evidence for a correlation of haplotypes spanning introns 5 through 8 with TPH2 mRNA levels. Unfortunately, the total of 22 SNPs tested in their study did not include rs11178997, nor any other highly correlated (r2) SNP (21).

Our functional data show that the Pro206Ser missense mutant is also one of the first identified genetic variants of likely pathophysiological relevance in BPAD. The most striking property of TPH2 Ser206 is its decreased stability and solubility, possibly leading to an increased rate of cellular turnover, as demonstrated by its decreased protein levels in mammalian cells. This could lead to the decreased cerebral serotonin levels that appear to be a risk factor for affective disorder (3).

The pathogenic role of mutations affecting the closely related enzymes phenylalanine hydroxylase (PAH) and TH is well established. Destabilizing missense mutations in PAH and TH can be found in all exons of the genes and are the most common causes of phenylketonuria and recessive DOPA responsive dystonia/juvenile Parkinsonism, respectively (see PAHdb knowledge database at http://www.pahdb.mcgill.ca/) (22). However, as shown for PAH, it is difficult to predict the exact residual activity of the enzymes in vivo solely based on studies of the isolated proteins.

As we observed only heterozygous carriers for the Pro206Ser mutation, it was important to examine the effect of simultaneous expression of equimolar amounts of WT and mutant proteins. Using two different expression systems, we demonstrated that the final TPH activity is significantly reduced if WT TPH2 and either Pro206Ser or Arg441His are expressed together. Using the in vitro transcription and translation system, we could also show that the final activity of the ‘hybrid’ proteins is slightly lower than the average of the two pure mutant proteins, consistent with a moderate dominant gain of dysfunction effect of the mutants. For human PAH and TPH, it has been shown that subunits of the WT enzyme interact with subunits of the mutants (23,24), and if different mutant subunits of PAH are expressed together, the specific activity of the hybrid protein is 21–88% lower than that anticipated from their average values (negative interallelic complementation) (25). One mechanism that can explain the decreased activity of PAH and TPH is a destabilization of the heterotetrameric enzymes caused by an unstable subunit. The Pro206Ser and Arg441His variants are both unstable forms of TPH2 (Table 4 and Fig. 2). More detailed studies are required to determine the exact molecular mechanisms of such a destabilization of heterotetramers and whether this also occurs in the intact human brain. However, as 5-HT is essential for brain development, even a moderate transient deficiency of this transmitter could lead to permanent alterations of brain function (26).

In conclusion, our results provide evidence that both common and rare variants in TPH2 contribute to BPAD within the same population. Pro206Ser appears to be an infrequent susceptibility variant with a relatively high penetrance, as indicated by its large genotypic OR (6.5 in the combined German/Russian BPAD sample), its presence in patients with a positive family history of affective disorder and by its co-segregation with affective disorder in three families tested by us. Our study supports the notion that re-sequencing studies to detect such low-frequency/high-penetrance variants are still needed in genetically complex diseases such as BPAD and that detection of such variants should complement HapMap-based investigation of common variation to identify the full spectrum of susceptibility variants present at a given locus.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
German and Russian patient and control samples, recruitment and phenotpye characterization
For all patient and control individuals, written informed consent was obtained prior to study participation. Protocols and procedures were approved by the Ethics Committees of the Faculties of Medicine at the Universities of Bonn and Mannheim/Heidelberg and by the Ethics Committee of the Scientific Center of Mental Health of the Russian Academy of Medical Science in Moscow.

The German BPAD sample was recruited from consecutive admissions to the inpatient unit of the Department of Psychiatry and Psychotherapy of the University of Bonn and of the Central Institute of Mental Health in Mannheim. The sample comprised 636 patients with a DSM-IV lifetime diagnosis of bipolar I disorder, made by a consensus best-estimate procedure (27) based on all available information, including a structured interview (SCID-I) (28), medical records and the family history method. We also used the OPCRIT (29) system to obtain detailed polydiagnostic documentation of symptoms.

Fifty-three percent of the patients were female, 47% male. The mean age was 43.7±13.1 years (median age 43.0), and the mean age of onset was 27.5±10.7 years (median age of onset 25.0). All individuals and their parents were of German descent.

The German control sample consisted of 1070 population-based individuals, 51.5% were females, 48.5% were males. The mean age was 48.4±15.5 years (median age 48.0). The control individuals were questioned for a history of psychiatric disorder. All individuals were German. Seventy-four (6.8%) patients had one or more non-German grandparent originating from Europe, the Americas, former Soviet Union or the Middle East.

The Russian BPAD sample consisted of 247 DSM-IV diagnosed patients with bipolar I disorder, following the same diagnostic procedure as described for the German patient sample, 55.9% females and 44.1% males. Mean age of onset was 26.8±11.3 years (median age of onset 23.0). One hundred and seventy (68.3%) were of Russian origin, the remaining patients originated from other parts of the former Soviet Union. Of the 230 controls, 54.3% were female and 45.7% were male. Their mean age was 38.1±15.4 years, 183 (79.2%) were of Russian origin, the remaining controls originated from other parts of the former Soviet Union.

Genotyping procedure
Genotyping was performed using the MassARRAY system on a Sequenom Compact MALDI-TOF device (Sequenom Inc., San Diego, CA, USA). Primer sequences and PCR/assay conditions can be obtained from the authors upon request. Genotype call rates were >98.0% for all SNPs tested, 2% of individuals were genotyped in duplicate and there were no replication errors. Samples (cases and controls) with a call rate <85% (i.e. allowing only ≤2 missing genotypes per individual) were excluded from the analysis.

Statistical analysis
Hardy–Weinberg equilibrium and intermarker LD as expressed by r2 and D' was calculated and visualized using the HAPLOVIEW v.3.32 software (http://www.broad.mit.edu/mpg/haploview/). LD measures for all 15 markers tested in all three control populations are given in Supplementary Material, Table S1. Patient–control single-marker association analysis was performed using the program FAMHAP (30). For comparison, all calculations were also performed using the UNPHASED software package version 3.0.6 (31) (http://www.mrc-bsu.cam.ac.uk/personal/frank/software/unphased/). In addition, the non-synonymous SNP (rs17110563) was analysed using Fisher's exact test. The results were almost identical to the results obtained with the Cochrane–Armitage trend test used in FAMHAP (data not shown). ANOVA and pairwise t-tests were used to compare the properties of the mutant forms of TPH2 with the WT enzyme.

Expression and purification of P206S
The Pro206Ser mutation was introduced in TPH2 cDNA using the Quik Change site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA) using primers from Sigma (forward [5'-AATTATGGTCAGTCCATTCCCAGGGTG], reverse [5'-CACCCTGGGAATGGACTGACCATATTT]). The sequences of all TPH expression clones were verified by DNA sequencing. Expression and purification of WT, Pro206Ser and Arg441His TPH2 as N-terminal 6xHis-maltose-binding protein (MBP) fusion proteins in E. coli, followed by cleavage with tobacco etch virus protease was performed as described (19). The relative solubility of the proteins was determined after the cells had been harvested by centrifugation at 4000g for 20 min at 4°C (18).

Immunostaining
Extracts of bacteria and mammalian cells and purified proteins were analysed by SDS–PAGE, transferred to a nitrocellulose membrane and immunostained. The primary goat polyclonal TPH2 antibody ab 40846 from Abcam (Cambridge, UK) was applied at a 1:2000 dilution and the secondary donkey anti-goat antibody ab 6566 from Abcam was applied at a 1:5000 dilution. Detection was made by enhanced chemiluminescence and band intensities was measured using a camera and Quantity One software provided by Bio-Rad, (Hercules, CA, USA).

Enzyme assay
TPH activity was assayed at 30°C in a standard reaction mixture (100 µl final volume) containing 40 mM NaHEPES, pH 7.0, 0.05 mg/ml catalase, 10 µM ferrous ammonium sulphate, 2.5 mM dithiothreitol, 100 µM L-tryptophan (L-Trp) and 250 µM tetrahydrobiopterin, essentially as described previously (20). Kinetic parameters were calculated by non-linear regression curve fitting using the equation vo = Vmax[S]/(Km+[S]+[S]2/Ksi), where Ksi is the substrate inhibition constant.

Measurement of temperature stability
Enzyme samples (3 µM) in 400 mM NaHEPES, pH 7.0, were incubated at 37, 50 or 55°C for up to 1 h and then assayed for TPH activity. The data were fitted with exponential decay curves and rate constants were calculated using the equation Et = E0e–kt+a, where E0 is the initial enzyme activity, Et the activity at time t, k the decay rate constant, and a an apparent plateau value. Thermal stability of the Pro206Ser mutant was also determined using circular dichroism spectroscopy as described (19).

Expression of TPH2 in mammalian cells
Human embryonic kidney (HED 293) cells were transfected with the pcDNA5-TPH2 expression vector (WT, Pro206Ser and Arg441His) using lipofectamine (Gibco) as described by the manufacturer, and the proteins were expressed as described. The cells were harvested after 48 h, the lysed cells were centrifuged (14 000 r.p.m. at 4°C for 10 min in an Eppendorf microcentrifuge) and the crude cell extracts were kept in liquid nitrogen until used for SDS/PAGE/immunoblotting and assay of enzyme activity.

In vitro transcription and translation of TPH2
Using a commercial rabbit reticulocyte-coupled transcription and translation system (Promega) WT and mutant TPH2 were expressed either alone or in a 50/50 mixture of WT and mutant protein, using 1 µg of total plasmid DNA per reaction and 1 h reaction time. Using [35S]methionine labelling, the conditions were optimized to achieve identical production of the proteins which were subjected to gel filtration before activity assays were performed for 18 min at 30°C in the presence of 100 µM tryptophan and 50 µM tetrahydrobiopterin (20).

Molecular modeling/prediction analysis
A homology model of TPH2 was prepared using the crystal structure of tetrameric TH (45% sequence identity) and the SWISS-MODEL automated protein homology-modelling server. The optimal conformation of the Pro206Ser mutant was determined as described (19).


   SUPPLEMENTARY MATERIAL
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
FUNDING
 REFERENCES
 
Supplementary Material is available at HMG Online.


   FUNDING
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
REFERENCES
 
This work was supported by the National Genomic Network of the ‘Bundesministerium für Bildung und Forschung’ (BMBF). M.M.N. received support from the Alfried Krupp von Bohlen und Halbach-Stiftung and J.H. from the Research Council of Norway.


   ACKNOWLEDGEMENTS
 
The authors are most grateful to the patients for their co-operation. We thank Dr. G. Chuchalin (Russian State Medical University, Moscow, Russia), Drs. A. Tiganov, G. Panteleyeva, V. Artyuch (Mental Health Research Center, Moscow), and Drs. V. Krasnov and S. Mosolov (Moscow Research Institute of Psychiatry, Moscow) for valuable help with recruiting patients and controls for this study. We are grateful to Dr. C. Schmäl for carefully reading the manuscript.

Conflict of Interest statement. The authors declare that they have no conflict of interest.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 

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