Association study of tryptophan hydroxylase 2 gene polymorphisms in panic disorder

Experimental studies on serotonin (5-HT) availability suggest a role for 5-HT synthesis rate in panicogenesis. Recently, it has been discovered that the tryptophan hydroxylase gene isoform 2 (TPH2), rather than TPH1, is preferentially expressed in the neuronal tissue and, therefore, is primarily responsible for the regulation of brain 5-HT synthesis. In the present case-control genetic association study we investigated whether panic disorder (PD) phenotypes are related to two single nucleotide polymorphisms (SNP) of TPH2, rs1386494 A/G and rs1386483 C/T. The study sample consisted of 213 (163 females and 50 males) PD patients with or without affective comorbidity and 303 (212 females and 91 males) matched healthy control subjects. The allelic and genotypic analyses in the total sample did not demonstrate significant association of PD with the studied SNPs, suggesting that these polymorphisms may not play a robust role in predisposition to PD. However, an association with rs1386494 SNP was observed in the subgroup of female patients with pure PD phenotype, indicating a possible gender-specific effect of TPH2 gene variants in PD.     

A functional variant of the tryptophan hydroxylase 2 gene impacts working memory: a genetic imaging study

Imaging studies have demonstrated that prefrontal and parietal regions are activated during working memory (WM) tasks. Recently some molecular genetic studies reported associations between a functional promoter polymorphism of the tryptophan hydroxylase 2 gene (TPH2), that regulates the synthesis of serotonin, and attention. In 49 healthy Caucasian subjects the role of the TPH2 -703 G/T polymorphism for WM was tested by means of an imaging genomics approach in an n-back task. fMRI data showed an increased activation for the 2-back as compared to the 0-back condition for a large network in prefrontal and parietal areas. Although behavioural data showed no performance differences between the genotype groups of the -703 G/T a significantly stronger activation of the TT genotype carriers in BA 6, BA 46, and BA 40 was visible in contrast to the GT and GG groups. Present findings in congruence with previous findings support the hypothesis that TT carriers compensate deficits in executive control functions by increased brain activity.     

Tryptophan hydroxylase 2 gene is associated with major depressive disorder in a female Chinese population

Background

Previous candidate gene studies of major depressive disorder (MDD) have provided inconclusive evidence of association for genes with strong biological rationale for MDD. In this study, we aimed to investigate the association of tryptophan hydroxylase 2 gene with MDD and its treatment response in the Chinese Han population.

Methods

Three hundred and sixty eight depressed patients who met DSM-IV criteria for major depressive disorder were recruited for the study. 371 normal controls were recruited from local community. Patients and normal controls were genotyped for TPH2 (rs4290270 and rs7305115) variants by polymerase chain reaction. Male and female subjects were analyzed separately.

Results

No differences were found in the frequencies of the single alleles and genotypes of the tested polymorphisms between MDD patients and normal group. The frequency of the A-A haplotype was significantly higher in female MDD compared to healthy female controls (P < 0.05). No significant association with treatment response was discovered in haplotype and single-marker analysis.

Limitations

This study lacks a placebo control and we cannot definitively exclude the possibility that some patients in the responder group responded to the placebo effect alone.

Conclusion

The result suggests that TPH2 gene may have a gender dependent effect on susceptibility to MDD but not with its treatment response in Chinese Han population. Further studies are needed to replicate the association that we observed.     

Common genetic variations in TPH1/TPH2 genes are not associated with schizophrenia in Japanese population

Alteration of serotonin transmission in the brain of patients with schizophrenia has been reported in postmortem brain studies, cerebrospinal fluid studies, and pharmacological challenges. Although a genetic association of tryptophan hydroxylase isoform 1 (TPH1), the rate-limiting enzyme in serotonin synthesis, with schizophrenia has been suggested by recent systematic meta-analyses, the newly identified neuronal isoform TPH2 is more relevant to the central nervous system and the association of TPH2 gene with schizophrenia has been much less explored. We, therefore, explored the association of TPH2 gene with schizophrenia using a case–control study of 720 Japanese populations and also tried to replicate the association of the TPH1 rs1800532 (A218C) single nucleotide polymorphism (SNP) with schizophrenia. We selected 15 tagging SNPs in the TPH2 gene. We found no significant differences in genotypic distributions (uncorrected P = 0.18–0.98) or allelic frequencies (uncorrected P = 0.18–0.98) of the 15 SNPs between the schizophrenia and control groups. Haplotypes constructed with these SNPs were also not associated with schizophrenia (uncorrected P = 0.12–0.97). The genotypic and allelic distribution of the TPH1 rs1800532 SNP was also not different between the case and control groups in our samples. In addition, a subsequent meta-analysis including our results did not showed a significant association with schizophrenia in Asian populations. Our findings suggest that neither common genetic variations of TPH1 nor TPH2 are likely to contribute to the genetic susceptibility to schizophrenia in Japanese population.     

A new gain-of-function allele in chimpanzee tryptophan hydroxylase 2 and the comparison of its enzyme activity with that in humans and rats

Tryptophan hydroxylase 2 (TPH2) is a rate-limiting enzyme of neuronal serotonin biosynthesis. Recently, two single nucleotide polymorphisms (SNPs) at the exon 11 coding region that resulted in amino acid substitutions in the C-terminal domain have been reported to affect enzyme activity in humans and mice. We determined 175 base-pair sequences of the exon 11 region in nine primate species from all recognized lineages. All nucleotide sequence substitutions were synonymous, with the exception of one adenine (A) to guanine (G) substitution at the 1404th position in the open reading frame (ORF). This substitution leads to a glutamine (Q) to arginine (R) amino acid substitution at the 468th position within chimpanzee sequences. The frequency of the G allele was 0.24 among 66 chimpanzees. Therefore, it is a novel SNP observed in chimpanzees, and we have named these two alleles as ch468Q and ch468R, respectively. When expressed in HeLa cells, ch468R caused an approximate 20% increase in enzyme function during L-5-hydroxytryptophan (5HTP) production (P<0.001). We also surveyed the interspecies difference in enzyme activity among human, chimpanzee, and rat. Although the rat showed an identical amino acid sequence at the C-terminal region as those of human and ch468Q, the rat enzyme was more active than those of human or chimpanzee (P<0.001), indicating the importance of substitutions in other regions. Our findings on the chimpanzee SNP will be a useful genetic marker in understanding the individual difference in the serotonin-related behavior.     

Rhesus monkey tryptophan hydroxylase-2 coding region haplotypes affect mRNA stability

Tryptophan hydroxylase-2 (TPH2) synthesizes neuronal 5-HT and its genetic variance is associated with numerous behavioral traits and psychiatric disorders. This study characterized the functional significance of two nonsynonymous single nucleotide polymorphisms (SNPs) (C74A and G223A) in rhesus monkey TPH2 (mTPH2). Four haplotypes of mTPH2 were cloned into pcDNA3.1 and stably transfected into PC12 cells. The levels of mTPH2 mRNA and protein were assessed by quantitative real-time PCR and Western blot, respectively, while the intracellular 5-HT was measured by enzyme-linked immunosorbent assay (ELISA). The variant A-A haplotype showed significantly higher levels of mTPH2 mRNA and protein, as well as significantly higher 5-HT production than the wild-type C-G haplotype, while the other two variant haplotypes (C-A and A-G) also tended to produce more 5-HT than C-G haplotype when stably expressed in PC12 cells. Both C74A and G223A were predicted to change mRNA secondary structure, and analysis of the mRNA stability showed that the wild-type C-G haplotype mRNA degrades more quickly than mRNAs of the mutant mTPH2 haplotypes in both stable PC12 and transient HEK-293 cells. This study demonstrates that nonsynonymous SNPs in mTPH2 can affect mRNA stability. Our findings provide an additional mechanism by which nonsynonymous SNPs affect TPH2 function, and further our understanding of TPH2 gene expression regulation.     

Effect of metals and phenylalanine on the activity of human tryptophan hydroxylase-2: comparison with that on tyrosine hydroxylase activity

We cloned the human tryptophan hydroxylase-2 (hTPH2) gene by RT-PCR, and expressed and purified its product as a maltose-binding protein (MBP)-fusion protein. We investigated the effects of essential divalent cations and L-phenylalanine (L-Phe) on the hTPH2 activity for the first time, and compared them with those on human tyrosine hydroxylase (hTH1) activity. We found that cobaltous and manganous ions inhibited the activities of both enzymes but that hTH1 was affected at lower concentrations than hTPH2. From kinetic analyses, we found that phenylalanine acted as an inhibitor more strongly against hTPH2 than against hTH1. These data are important for elucidating the molecular mechanism underlying the alterations in the contents of serotonin and catecholamines in the brain under pathological and physiological conditions, such as hyperphenylalaninemia and chronic manganese toxicity.     

Additive effects of serotonin transporter and tryptophan hydroxylase-2 gene variation on neural correlates of affective processing

Individual differences in brain response to emotional stimuli have previously been associated with gene variations within the serotonin transporter (5-HTT) and tryptophan hydroxylase-2 (TPH2) genes. We recently reported that these two genes exhibit an additive effect, based on recordings of event-related potentials (ERPs) from individuals viewing emotional scenes. The current study was designed to replicate and extent this initial report in an independent study sample, and use functional magnetic resonance imaging (fMRI) to identify specific neural loci that may mediate the 5-HTT-TPH2 additive effect. Furthermore, we sought to obtain convergent evidence for a gene-gene additive effect by collecting fMRI data from the same individuals engaged in two different cognitive-affective tasks, using emotional and neutral facial expressions and word stimuli. We found evidence for an additive effect of 5-HTT-TPH2 genotype, which was most robust in the putamen, a region rich in both 5-HTT and TPH2 protein, but was also observed in the amygdala at a less stringent threshold, and in other cortical regions. The additive effect was more robust effect for visuospatial than for verbal stimuli, and more robust for negatively than for positively valenced stimuli. These findings confirm and extend the additive effect of two critical genes in the serotonergic regulation of neural processing of affective stimuli, and identify the striatum as a critical site where is gene-gene regulation takes place.     

Lack of association between the tryptophan hydroxylase gene A218C polymorphism and attention‐deficit hyperactivity disorder in Chinese Han population

Previous studies have suggested that the serotonergic (5‐HT) system might be involved in the development of Attention‐deficit hyperactivity disorder (ADHD). ADHD is frequently characterized by aggressive and impulsive behavior, a major symptom associated with reduction in serotonergic function. The tryptophan hydroxylase (TPH) gene is a reasonable candidate for ADHD because it encodes the rate‐limiting enzyme in the process of 5‐HT biosynthesis. In this study, we examined the relationship between the A218C polymorphism in TPH gene and ADHD. Sixty‐nine ADHD patients and their biological parents were investigated. The A218C polymorphism in intron 7 of TPH gene was detected by PCR‐RFLP method. No allele or genotype concerned with this A218C polymorphism was found to be associated with ADHD when analyzed with the haplotype relative risk method. Therefore, our data indicate that the TPH gene A218C polymorphism may not be a susceptibility factor of ADHD in the Chinese Han population. © 2001 Wiley‐Liss, Inc.     

TPH2 polymorphisms and alcohol-related suicide

Substantial evidence from family, twin, and adoption studies corroborates implication of genetic and environmental factors, as well as their interactions, on suicidal behavior and alcoholism risk. Serotonergic disfunction seems to be involved in the pathophysiology of substance abuse, and has also an important role in suicidal behavior. Recent studies of the tryptophan hydroxylase 2 showed mild or no association with suicide and alcohol-related suicide. We performed SNP and alcohol analysis on 388 suicide victims and 227 controls. The results showed association between suicide (Pχ2=0.043) and alcohol-related suicide (Pχ2=0.021) for SNP Rs1843809. A tendency for association was determined also for polymorphism Rs1386493 (Pχ2=0.055) and alcohol-related suicide. Data acquired from psychological autopsies in a subsample of suicide victims (n=79) determined more impulsive behavior (Pχ2=0.016) and verbal aggressive behavior (Pχ2=0.025) in the subgroup with alcohol misuse or dependency. In conclusion, our results suggest implication of polymorphisms in suicide and alcohol-related suicide, but further studies are needed to clarify the interplay among serotonergic system disfunction, suicide, alcohol dependence, impulsivity and the role of TPH2 enzyme.     

Hypotensive but not normotensive haemorrhage increases tryptophan hydroxylase-2 mRNA in caudal midline medulla

Severe blood loss triggers shock, a precipitous hypotension and bradycardia. The integrity of (i) neurons in the vasodepressor region of the caudal midline medulla and (ii) central 5-HT neurotransmission are critical for the expression of haemorrhagic shock. This study investigated whether progressive blood loss triggers altered synthesis of 5-HT in the vasodepressor region of the caudal midline medulla by measuring changes in relative expression levels of tryptophan hydroxylase 2 (TpH 2) mRNA, the rate-limiting enzyme in the synthesis of neuronal 5-HT. Hypotensive but not normotensive haemorrhage triggered a significant increase in TpH 2 mRNA in the vasodepressor region of the caudal midline medulla, identifying an important role for 5-HT-containing caudal midline medullary neurons in haemorrhagic shock.     

Functional properties of missense variants of human tryptophan hydroxylase 2

Tryptophan hydroxylase 2 (TPH2) catalyzes the rate‐limiting step in serotonin biosynthesis in the nervous system. Several variants of human TPH2 have been reported to be associated with a spectrum of neuropsychiatric disorders such as unipolar major depression, bipolar disorder, suicidality, and attention‐deficit/hyperactivity disorder (ADHD). We used three different expression systems: rabbit reticulocyte lysate, Escherichia coli, and human embryonic kidney cells, to identify functional effects of all human TPH2 missense variants reported to date. The properties of mutants affecting the regulatory domain, that is, p.Leu36Val, p.Leu36Pro, p.Ser41Tyr, and p.Arg55Cys, were indistinguishable from the wild‐type (WT). Moderate loss‐of‐function effects were observed for mutants in the catalytic and oligomerization domains, that is, p.Pro206Ser, p.Ala328Val, p.Arg441His, and p.Asp479Glu, which were manifested via stability and solubility effects, whereas p.Arg303Trp had severely reduced solubility and was completely inactive. All variants were tested as substrates for protein kinase A and were found to have similar phosphorylation stoichiometries. A standardized assay protocol as described here for activity and solubility screening should also be useful for determining properties of other TPH2 variants that will be discovered in the future. Hum Mutat 30:1–8, 2009. © 2009 Wiley‐Liss, Inc.     

A dimensional impulsive‐aggressive phenotype is associated with the A218C polymorphism of the tryptophan hydroxylase gene: A pilot study in well‐characterized impulsive inpatients

Tryptophan hydroxylase (TPH) is the rate‐limiting enzyme in the biosynthesis of serotonin, and association and linkage studies of its variants in suicidal and impulsive‐aggressive behavior have brought conflicting results. This pilot study was designed to investigate whether TPH A218C genotypes could be associated with impulsive behavioral tendencies (IBTs) in consecutively admitted nonpsychotic nonorganic inpatients. Patients (20 females and 34 males; age, 38.8 ± 11.8) did not differ from healthy nonimpulsive controls (16 females and 11 males; age, 35.2 ± 10.2) regarding TPH genotypes, but in the patients, the number of IBT was related to the presence of the 218C allele. It was concluded that impulsive‐aggressive behavior may be associated with the TPH genotype in well‐characterized impulsive patients and that the present results stress the importance of considering impulsiveness‐aggressiveness in studies investigating the relationship between suicidal behavior and TPH genotypes. © 2002 Wiley‐Liss, Inc.     

Acoustic stimulation in vivo and corticotropin-releasing factor in vitro increase tryptophan hydroxylase activity in the rat caudal dorsal raphe nucleus

Exposure of rats to unpredictable loud sound pulses increases activity of the rate-limiting enzyme for serotonin synthesis, tryptophan hydroxylase (TPH), in the median raphe nucleus (MnR) and a mesolimbocortical serotonergic system. Corticotropin-releasing factor (CRF)-induced activation of a subset of serotonergic neurons in the caudal dorsal raphe nucleus (DR) may underlie stress-related increases in TPH activity in the MnR and a mesolimbocortical serotonergic system. An in vivo acoustic stimulation paradigm and an in vitro brain slice preparation were designed to test the hypothesis that stress-related stimuli and CRF receptor activation have convergent actions on TPH activity in the caudal DR (DRC). We measured 5-hydroxytryptophan (5-HTP) accumulation as an index of TPH activity following inhibition of aromatic amino acid decarboxylase (using NSD-1015). To examine effects of acoustic stimulation on TPH activity, male Wistar rats, pretreated with NSD-1015, were exposed to a 30 min sham, predictable or unpredictable acoustic stimulation paradigm; brains were frozen and microdissected for analyses of tissue 5-HTP concentrations in subregions of the DR. To examine the effect of CRF receptor activation on TPH activity, freshly prepared brain slices were exposed to CRF (0–2000 nM) for 10 min in the presence of NSD-1015, then frozen and microdissected for analysis of tissue 5-HTP concentrations. Increases in TPH activity in the DRC, but not other subregions, were observed in both paradigms. These findings are consistent with the hypothesis that stress-related increases in TPH activity are mediated via effects of CRF or CRF-related neuropeptides on a mesolimbocortical serotonergic system originating in the DRC.     

The mTPH2 C1473G single nucleotide polymorphism is not responsible for behavioural differences between mouse strains

Tryptophan hydroxylase 2 (TPH2) is the rate limiting enzyme of serotonin synthesis in the brain. A recently described functional (C1473G) single nucleotide polymorphism in mouse TPH2 resulting in vitro in a strongly decreased enzymatic activity was suspected to be responsible for the observed differences in 5-HT levels and behaviour between mice strains. We bred two substrains of C57BL/6 mice carrying the two isoforms and could show that both exhibit equal TPH activity, brain 5-HT content and behaviour. These data indicate that the distinct behavioural characteristics of mouse strains are not due to differences in TPH2 activity, but to other variations in the genetic background.     

Tryptophan hydroxylase 2 genotype determines brain serotonin synthesis but not tissue content in C57Bl/6 and BALB/c congenic mice

Tryptophan hydroxylase 2 (TPH2) catalyzes the rate-limiting step in the synthesis of brain serotonin (5-HT). In a previous report, a single nucleotide polymorphism in mTph2 (C1473G) reduced 5-HT synthesis by 55%. Mouse strains expressing the 1473C allele, such as C57Bl/6, have higher 5-HT synthesis rates than strains expressing the 1473G allele, such as BALB/c. Many studies have attributed strain differences to Tph2 genotype without ruling out the potential role of alterations in other genes. To test the role of the C1473G polymorphism in strain differences, we generated C57Bl/6 and BALB/c mice congenic for the Tph2 locus. We found that the 1473G allele reduced 5-HT synthesis in C57Bl/6 mice but had no effect on 5-HT tissue content except for a slight reduction (15%) in the frontal cortex. In BALB/c mice, the 1473C allele increased 5-HT synthesis but again did not affect 5-HT tissue content. At the same time, 5-hydroxyindoleacetic acid (5-HIAA) was significantly elevated in BALB/c congenic mice. In C57Bl/6 mice, there was no effect of genotype on 5-HIAA levels. BALB/c mice had lower expression of monoamine oxidase A and B than C57Bl/6 mice, but there was no effect of Tph2 genotype. On the tail suspension test, escitalopram treatment reduced immobility regardless of genotype. These data demonstrate that the C1473G polymorphism determines differences in 5-HT synthesis rates among strains but only minimally affects 5-HT tissue levels.     

A Tryptophan Hydroxylase 2 Gene Polymorphism is Associated with Panic Disorder

Panic disorder (PD) is a complex and heterogeneous psychiatric condition. Dysfunction within the serotonergic system has been hypothesized to play an important role in PD. The novel brain-specific serotonin synthesizing enzyme, tryptophan hydroxylase 2 (TPH2), which represents the rate-limiting enzyme of serotonin production in the brain, may therefore be of particular importance in PD. We investigated the TPH2 703G/T SNP for association with PD. Patients with PD (n = 108), and control subjects (n = 247), were genotyped for rs4570625 (TPH2 703G/T). Male and female subjects were analyzed separately. The severity of their symptoms was measured using the Spielberger state-trait anxiety inventory (STAI), panic disorder severity scale (PDSS), anxiety sensitivity index (ASI), acute panic inventory (API), and Hamilton’s rating scale for depression (HAMD). The genotype and allele frequencies of the PD patients and controls were analyzed using χ ² statistics. There was a significant difference in the allele frequency in rs4570625 between the PD patients and normal controls. The T allele was significantly less frequent in the PD patients. We also found a significant association with rs4570625 in the female subgroup. There was no difference in symptom severity among the genotypes of this polymorphism. This result suggests that rs4570625 polymorphism may play a significant role in the pathogenesis of PD. Moreover, rs4570625 may have a gender-dependent effect on susceptibility to PD. Further studies are needed to replicate the association that we observed. Edited by Tatiana Foroud.     

Tryptophan hydroxylase 1 gene (TPH1) moderates the influence of social support on depressive symptoms in adults

BACKGROUND: Tryptophan hydroxylases (TPHs) are involved in the biosynthesis of serotonin and are therefore candidate genes for psychiatric disorders, including depression. We examined whether the common 218 A > C and 779 A > C polymorphisms in the tryptophan hydroxylase 1 gene (TPH1) moderated the association between perceived social support and sub-clinical depressive symptoms in adults. METHODS: The subjects were a randomly selected subsample (n=341) of individuals participating in the Cardiovascular Risk in Young Finns study, who had data on social support on one assessment time and depressive symptoms on two assessment times. Social support was assessed on the Perceived Social Support Scale Revised (PSSS-R) and depressive symptoms on a modified version of the Beck's Depression Inventory (BDI). RESULTS: We found that low social support predicted depressive symptoms more strongly in individuals carrying A alleles of the TPH1 than in others. The interaction effect was observed in a cross-sectional analysis and when predicting depressive symptoms over a four-year period. LIMITATIONS: We did not have data on TPH2, which has recently been identified as the primary TPH isomorphism affecting serotonin synthesis in the brain. CONCLUSIONS: TPH1 gene may be involved in the development of depressive symptoms by moderating the impact of depressogenic social influences.     

Arylamine N-acetyltransferase 2 gene polymorphism in an Algerian population

Background: The arylamine N-acetyltransferase 2 (NAT2) is a key enzyme in the biotransformation of xenobiotics. NAT2 gene polymorphisms have been associated with the risk of isoniazid hepatotoxicity and these polymorphisms change among different populations.

Aim: The objective of this study is to investigate NAT2 polymorphisms in order to predict the prevalence of NAT2 phenotype in an Algerian population.

Subjects and methods: Genotyping of NAT2 was done using a PCR-RFLP method. Haplotype was analysed using the software package PHASE, version 2.0.

Results: The major haplotypes were NAT2*5B (23.72%), NAT2*6?A (18.61%), NAT2*4 (14.60%) and NAT2*5?F (10%). The average of the expected slow acetylator phenotype was 53%.

Conclusion: Our results suggest that the high frequency of slow acetylator phenotype requires investigation into its possible association with ATDH.     

INSIG2 gene rs7566605 polymorphism is associated with severe obesity in Japanese

The single nucleotide polymorphism (SNP) rs7566605 in the upstream region of the insulin-induced gene 2 (INSIG2) is associated with the obesity phenotype in many Caucasian populations. In Japanese, this association with the obesity phenotype is not clear. To investigate the relationship between rs7566605 and obesity in Japanese, we genotyped rs7566605 from severely obese subjects [n = 908, body mass index (BMI) >/= 30 kg/m(2)] and normal-weight control subjects (n = 1495, BMI < 25 kg/m(2)). A case-control association analysis revealed that rs7566605 was significantly associated with obesity in Japanese. The P value in the minor allele recessive mode was 0.00020, and the odds ratio (OR) adjusted for gender and age was 1.61 [95% confidential interval (CI) = 1.24-2.09]. Obesity-associated phenotypes, which included the level of BMI, plasma glucose, hemoglobin A1c, total cholesterol, triglycerides, high-density lipoprotein (HDL) cholesterol, and blood pressure, were not associated with the rs7566605 genotype. Thus, rs7566605 in the upstream region of the INSIG2 gene was found to be associated with obesity, i.e., severe obesity, in Japanese.