Involvement of tryptophan hydroxylase 2 gene polymorphisms in susceptibility to tic disorder in Chinese Han population

Tic disorder (TD) is a neuropsychiatric disorder characterized by involuntary tics (multiple motor and/or vocal tics). Tics wax and wane over time and are more intense in prepubertal boys. A large proportion of affected children reach a quiescent stage around early adolescence [1‐3]. Recent epidemiological studies have changed the traditional perception that TD is rare, and demonstrate a prevalence rates >1000 per 100000 children [4‐7]. Co morbidity of TD with attention deficit hyperactivity disorder (ADHD) and obsessive-compulsive disorder (OCD) is common [8].

The exact cause of TD is unknown. A heritable component is proposed to play a major role in the pathophysiology of TD, as shown by family studies, evaluations of monozygotic and dizygotic twins, and by follow-up investigations of at-risk children [9‐12]. However, the susceptible genes or relevant DNA sequence variations that might be involved in the pathogenesis of TD have not been identified [13, 14]. Abnormalities of neurotransmitter systems such as dopamine and serotonin (5-hydroxytryptamine, 5-HT) are also proposed to contribute to the symptoms of the disorder. Serotonergic projections from the dorsal raphe area have been shown to inhibit dopamine function in the striatum and cortex, where they inhibit the synaptic release of dopamine, and probably the synthesis of dopamine. Data from neurochemical studies have shown that generally decreased concentrations of 5-hydroxyindolacetic acid (5-HIAA) in cerebrospinal fluid (CSF) in TD patients could reflect the inadequate modulation of dopaminergic activity by serotonergic neurons [15]. Data from pharmacological studies also indicate the use of serotonin reuptake inhibitors (SRIs) is effective in tic reduction in some TD children [16, 17]. Tryptophan hydroxylase (TPH) is a rate-limiting enzyme that catalyses the synthesis of 5-HT from tryptophan [18]. Until recently, it was thought that there was only one gene encoding TPH, referred to as tryptophan hydroxylase isoform (TPH1). However, TPH knockout studies in mice (mice TPH−/−) showed normal amounts of 5-HT in certain brain regions compared to their wild-type siblings (mice TPH+/+) [19]. Furthermore, TPH knockout mice exhibited no significant differences in 5-HT-related behaviors. This observation confirmed the hypothesis of the existence of a second TPH isoform (TPH2) [19]. The expression of both isoforms of TPH is partially exclusive. Several studies demonstrate that in humans TPH2 is exclusively expressed in serotonergic neurons of the brain, with the predominant expression in the Raphe nuclei of the midbrain, whereas TPH1 is mostly expressed in peripheral tissues [20]. TPH2 is located on chromosome 12q21.1; it spans a region of 93.5 kb and harbors 11 exons [20].

TPH2 is strategically placed to control 5-HT synthesis in the brain. For this reason, there has been great interest in identifying genetic variants that affect TPH2 enzymatic activity or the expression levels of the TPH2 gene. However, extensive DNA sequencing of the TPH2 gene has shown that the polymorphisms change the amino-acid sequence of the TPH2 protein were rare [21, 22]. The research has, therefore, focused on identifying genetic variants that influence the TPH2 gene expression [23]. Some studies suggest that the human TPH2 gene includes several single nucleotide polymorphisms (SNPs) which include G to T base substitution in the transcriptional control region at position −703 (rs4570625). Recently, the functional relevance of rs4570625 in the transcriptional control region of the TPH2 gene was investigated by many groups [23‐26]. In addition, the haplotype analysis of rs4570625 and rs4565946 by Mossner et al. found an association between TD and the G-C haplotype (and to a lesser extent, the T-C haplotype). This came from the association of the CC genotype and the C allele of the intronic SNP rs4565946 [27].

The current study was undertaken to investigate whether the TPH2 polymorphisms (SNP rs4570625 and rs4565945) act as susceptibility loci for TD in the Chinese Han population. We also investigated the possible association between these polymorphisms and severity scored with the Yale Global Tic Severity Scale (YGTSS) [28].

TD is thought to be caused by a complex interaction of environmental and biological factors, with multiple genes acting at distinct neural circuits, such as the brain serotonin system. Several studies have investigated an association between the TPH2 gene and other psychiatric disorders, but only one study to date has demonstrated an association between TPH2 and Tourette’s syndrome (TS) [27]. Mossner et al. investigated an association between two SNPs (rs4570625 and rs4565946) of the TPH2 gene and TS in German samples and showed that the C allele and CC genotype of rs4565946 was associated with TS [27]. This finding is not in line with our results, which suggest that the C allele is the major allele for TPH2 rs4565946 in Chinese subjects, indicating that there are ethnic differences in comparison with European, Indian, and other populations (Table 7). Our results do, however, suggest that the T allele of rs4565946 may be the risk allele in the Chinese Han population, and that individuals with T allele may be more susceptible to TD than those with C allele. We, therefore, propose that the TT genotype might produce different effects on TD in subjects in different ethnic groups.

It has been suggested that alteration in TPH2 gene expression might result in central serotonergic system dysfunction, which may in turn lead to abnormality of behavior and increased susceptibility to psychiatric disorders such as TS [27]. Chen et al. demonstrated that the expression of human TPH2 is regulated by both the 5’-UTR and common polymorphisms in the 5’ regulatory region of TPH2 [26]. These researchers also suggest that promoter rs4570625 (−703 G/T) is likely to influence the transcription rate of TPH2, a proposal supported by transcription factor binding sites for function prediction (http://​www.​cbrc.​jp/​research/​db/​TFSEARCH.​html). In our study, TD children had significantly higher frequencies of the rs4570625 G allele than the normal controls in males, suggesting that the G allele might increase the risk of TD in the Chinese Han population.

Additional evidence has shown that the T allele of rs4570625 is related to the low expression of TPH2 and increased activity in the prefrontal brain [31, 32]. James F et al. demonstrated that levels of tryptophan in cerebrospinal fluid were inversely associated with tic severity in TS subjects [33]. In our studies, we found an increase in G-allele in high score subgroup of male TD children. For rs4570625, the allele G type might affect the transcription factor binding ability (CdxA and HSF); therefore, the allele G type can change the transcription regulation of the TPH2 gene. The results indicated that the allele G of rs4570625 might have relationship with the severity of tic symptoms among male TS children in the Chinese Han population.

A recent study provided evidence that promoter rs4570625 polymorphism was not associated with down-regulated expression of TPH2 [30]. Whether SNP rs4570625 is a direct disease-causing polymorphism, therefore, remains a subject of further study.

To the best of our knowledge, no previous studies have investigated whether the intronic variant (SNP rs4565946) has any functional effects. In our study, the intronic polymorphism rs4565946 resulted in strong linkage disequilibrium (D’ = 0.933, r² = 0.429) with a functional polymorphism of rs4570625. Considering the association of TD with the T-G and C-G haplotypes, and given results for each SNP and the observation that the C allele located in intron 2 is in linkage disequilibrium with the functional T allele of the promoter SNP, it is possible that the intronic variant may also be functionally important in the development of TD.

There are a number of limitations to the present study. Firstly, the relatively small sample size and only two SNPs were studied, may have reduced the power to detect the main effects of (serotonin-related) TPH2 gene polymorphisms and limit the generalization of our results to the population as a whole. Secondly, nearly 23.5% of TD children in our study had ADHD, therefore, our results could be confounded by co-existing ADHD. Thirdly, no information was available about the medications and behavioral therapeutic interventions of the TD patients at the recruitment. For these reasons our data should be interpreted cautiously.

This is the first study to assess whether polymorphic variants of TPH2 are associated with TD in the Chinese Han population. Our results clearly link TPH2 variations to the pathogenesis of TD and further support the etiological relevance of 5-HT signaling in TD.