Volume 7 Issue 7 - February 20, 2009
Association of Functional Polymorphisms of the Human Tryptophan Hydroxylase 2 Gene with Risk for Bipolar Disorder in Han Chinese
Yi-Mei J. Lin1, Shin-Chih Chao2, Tsung-Ming Chen1, Te-Jen Lai3, Jia-Shing Chen1, H. Sunny Sun1,2,*

1Institute of Basic Medical Sciences and 2Institute of Molecular Medicine, National Cheng Kung University Medical College, Tainan
3Department of Psychiatry, Chung Shan Medical University Hospital, Taichung, Taiwan
hssun@mail.ncku.edu.tw

Arch Gen Psychiatry. 2007; 64(9):1015-1024

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Background- Serotonin (5-Hydroxytryptamine) is a neurotransmitter that controls a wide range of physiological events in the central nervous system. Accordingly, disruption of serotonergic function has been implicated in the pathogenesis of many psychiatric disorders, including bipolar disorder (BPD). The previous inconclusive studies suggest the earlier discovered TPH1 may not relate to the development of various neuron-system related diseases or only have a minor effect. The novel neuronal form (TPH2) was identified recently and represents 71% amino acid sequence identity with TPH1. And previous studies suggest a duality hypothesis that TPH1 response for the peripheral serotonergic effects and the major CNS effects regulated by TPH2. Despite possible associations between polymorphisms in human TPH2 and many psychiatric disorders including bipolar disorder (BPD) being reported, the functional effect and susceptibility loci of such polymorphisms for BPD have not yet been identified.
       
Identify sequence variants of TPH2 gene in Han Chinese - To illustrate the role of the TPH2 gene in etiology of BPD, we have undertaken a systematic approach to screen polymorphisms among all exons and promoter of the human TPH2 gene in Han Chinese population. Ten single-nucleotide variants have been identified, including two unique SNPs have not been reported in any public databases or literature, thus they are new and probably are population-specific polymorphisms in our population. The detail information of these 10 variants is summarized in Table 1.

Genetic association analysis and linkage disequilibrium (LD) mapping suggest the TPH2 gene involved in the etiology of BPD - Our study, using single-locus analysis, LD-mapping, and haplotype analysis, provided genetic support for the association of TPH2 with BPD. Significant haplotype association of TPH2 polymorphisms and BPD was identified (P<0.001). And as demonstrated in Figure 1, the distinctly different patterns of LD between patients and controls also suggest the possible involvement of TPH2 gene in etiology of BPD.
Table 1 Sequence variants of the human TPH2 gene identified in this study

a.Genomic localizations of SNPs are given in bp relative to the transcription start site (position 0).
b.Information is from the dbSNP database (http://www.ncbi.nlm.nih.gov/SNP/; Build 124, 2005), NA: not available.
c.The G33775A was observed only in a heterozygous individual. The minor allele frequency was less than 1%; thus, it is considered a mutation

Figure 1. LD plot between case and control groups has shown different pattern in the 5’ region of TPH2 loci. Absolute standardized linkage disequilibrium coefficient (D’) was plotted using the Graphical Overview of Linkage Disequilibrium (GOLD) program. Red indicates complete linkage (D’ =±1), and blue indicates no linkage (D’ = 0). The six loci are numbered in the 5’- > 3’- orientation. (Selected from Arch Gen Psych 2007; 64(9):1015-1024)

Evidences of altered TPH2 protein function involved in the development of BPD - Beside the genetic association, we ask whether the sequence variants of associated genes are functional thus have biological effect on gene’s function. Data from our study indicated the allelic alteration of polymorphisms in the promoter and exon 2 of TPH2 caused noteworthy functional losses in promoter and enzyme activities, respectively, indicating the potential susceptibility loci for BPD. The two promoter SNPs, T-703G and T-473A, are commonly present in many populations and are predicted that base changes at both positions can affect POU3F2 binding. Using an in vitro luciferase reporter assay, we demonstrated that significant loss in promoter activity is indeed associated with the GA haplotype (Figure 2A). Gel shift assays indicate a reduction in POU3F2 binding affinity of probes containing the -703G and -473A alleles when compared with the -703T- and -473T-conatining probes, respectively. These data provide evidence that the two promoter SNPs can affect POU3F2 binding and modulate TPH2 gene expression (Figure 2B). The other functional C2755A (S41Y) polymorphism is located on the diverse N-terminal region of human TPH2 proteins and sequence alignment indicated the position is highly conserved in the TPH2 proteins across species. Our data using recombinant constructs demonstrated that TPH2-41Y fusion protein exhibits lower enzyme activity than TPH2-41S fusion protein (Figure 2C). While SNPs on the promoter and exon 2 show significant functional alterations in gene expression and TPH2 activity, respectively; their associations with BPD at single-locus levels are not significant. These results indicate that an association of partial functional loss in each SNP with BPD may be weak, but that the combination of several common variants may have a dramatic impact on the etiology of BPD.
Figure 2. Allelic alteration of TPH2 polymorphisms caused noteworthy functional losses in promoter and enzyme activities.  (A) Promoter activity of the T-703G and T-473A polymorphisms. The levels of promoter activity were represented by relative luciferase activities and expressed as mean ± standard deviation (SD). (B) Electrophoretic mobility shift analysis (EMSA) of T-703G and T-473A polymorphisms. These data indicated the -473 T to A and -703 T to G alterations indeed reduce POU3F2 protein binding affinity in IMR-32 cells. (C)  Serotonin levels in SH-SY5Y cells expressing TPH2_41Y was ~40% lower than TPH2_41S transfected cells (P = 0.002). Serotonin levels were measured in medium of transfected cells using serotonin ELISA. (Selected from Arch Gen Psych 2007; 64(9):1015-1024)

Evidences of interaction between two tryptophan hydroxylase genes co-contribute to the development of BPD - The current conventional wisdom is that genes in the same pathway or involve in the same regulatory network tend to have risk interaction. TPH1 and TPH2 are paralogous proteins that have distinct structural and kinetic properties but share common resources for serotonin production, and the question of whether these two proteins are regulated independently, and thus do not interact, remains unanswered. We propose that the TPH1 gene alone has little or no effect on the etiology of BPD, but it may interact with the TPH2 to influence BPD development. Our data indeed support this hypothesis via examining the co-expressing of TPH1 and TPH2 in various parts of the human brain (Figure 3A), and further provide statistical evidence, for the first time, that the interaction between these two TPH genes might confer the risk for BPD. We found the odds ratio for BPD changed from 3.73 of TAG haplotype to either 4.81 or 1.68, depending on the combined effect of the two TPH genotypes (Figure 3B). While the interaction between two TPH genes merits further investigation, our findings suggest that the interactive effect be considered in the future studies of serotonin-related disorders.
Figure 3. Interactive effect exists between two tryptophan hydroxylase genes (TPH1 and TPH2). (A) Both TPH1 and TPH2 are expressed in various regions of the human brain. (B) Interaction between TPH1 and TPH2 genes increases the risk of BPD. The odds ratio associated with BPD changed from original 3.73 to either 4.81 or 1.68, depending on the combined effect of the two TPH genotypes. (Selected from Arch Gen Psych 2007; 64(9):1015-1024)

Concluding Remarks- The discovery of TPH2 has changed ideas about linking the TPH1 polymorphism with a variety of psychiatric diseases including BPD. However, the polymorphisms that represent susceptibility loci of the TPH2 have not yet been identified before our current publication. Our study, which used LD mapping and haplotype analysis, provided significant support for the association of TPH2 with BPD. In addition, we demonstrated the function of 3 TPH2 polymorphisms, 2 common variants in the promoter region and 1 novel variant in the exon 2, the combination of which may underlie the risk for BPD. Results from our study support the common disease/common variant (CDCV) hypothesis, that the effect of each SNP just can lead partially functional lost, and combination of several common variants can make the BPD occurs. This is also the first study to propose the interaction between the two TPH genes and the interactive effect between the two TPHs in the etiology of BPD. And we believe that the present study redefines the role of TPHs in the development of BPD.
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