1. Introduction
Trait anxiety is defined as an individual’s disposition to experience frequent and intense anxiety and worry in response to various stress situations. Individuals with high trait-anxious (HTA) are considered more susceptible to clinical anxiety and depression [
1,
2], which refers to individuals with HTA as pre-existing forms of anxiety. A large number of behavioral studies have revealed that individuals with HTA exhibit cognitive and emotional disorders, which are similar to anxiety disorders and behavioral biases, such as excessive uneasiness and concern about uncertain events on critical symptoms, threatening information, attention bias, persistent attention, low tolerance for uncertain information, and rejection or dislike of negative results or information [
3,
4,
5]. The reasons for those results may be related to the information processing efficiency caused by anxious emotion [
6]. Evidence from these studies indicates that individuals with high trait anxiety have similar behavioral performance compared to anxiety disorders. Most importantly, there is consistency that excessive concerns about uncertainty point to the developmental links between high trait anxiety and anxiety disorders.
Serotonin (5-HT) is an important signaling molecule and neurotransmitter, which is widely distributed in the central nervous system and surrounding tissues. In recent years, molecular genetic studies have shown that dysfunction of the 5-HT is closely related to anxiety, depression, loss of appetite, sleeping nap, decreased activity, sexual dysfunction, endocrine function disorder, etc. 5-HT is involved in the regulation of various mental activities and is closely related to psychiatric diseases. Almost all serotonin receptor subtypes are involved in antidepressant or anxiolytic effects [
7]. 5-hydroxytryptamine receptor 1B(5-HT1B), an inhibitory G-protein coupled metabotropic receptor that decreases cAMP, is highly expressed in the striatum, pallidum, accumbens nucleus, substantia nigra, and ventral tegmental area [
8]. 5-HT1B plays an important role in regulating serotonergic neurotransmission. It has been reported that the function of 5-HT1Bacts both presynaptically, as an inhibitory autoreceptor located on terminals of serotonin neurons and postsynaptically, as an inhibitory heteroreceptorcontrolling the release of other neurotransmitters [
9].
Various animal studies have also demonstrated that 5-HT1B plays a role in anxiety-like and anxiolytic-like effects. 5-HT1B gene knockout mice exhibited reduced anxiety and hyperactivity [
10]. Nautiyal and colleagues showed that the forebrain 5-HT1B heteroreceptors expressed during an early postnatal period might contribute to the development of the neural systems underlying adult aggression and proved that distinct heteroreceptors acting during adulthood were involved in mediating impulsivity [
11]. Interestingly, mice lacking 5-HT1B autoreceptors presented decreased anxiety in the open field test [
9]. Similarly, studies byLin and Parsons [
12] indicated that stimulation of 5-HT1B receptor increased anxiety-like behavior in the elevated plus-maze test in rats, suggesting the role of this receptor subtype in the pathology and treatment of anxiety. Non-selective 5-HT1B/1D receptors agonist GR127935 also showed anxiolytic-like properties [
13]. The observed antianxiety-like effect might be linked to the postsynaptic 5-HT1B receptors or/and 5-HT1B heteroreceptors [
14]. These studies suggested that the 5-HT1B receptor might play an important role in trait anxiety. However, its exact role is yet unclear.
Previous human studies reported the associations between the different polymorphisms in the gene coding for 5-HT1B and alcohol dependence [
15,
16], alcohol abuse [
17], aggressive behaviour [
18,
19], anger and hostility [
20], attention-deficit/hyperactivity disorder (ADHD) [
21], substance abuse [
22], and schizophrenia [
23,
24]. Although the current genome-wide literature could not find an association of 5-HT1B with anxiety or related phenotypes, including depression and neuroticism in some European populations [
25,
26,
27], a few studies have reported the associations between the different polymorphisms in 5-HT1B and some phenotypes, including anxiety and depression in Chinese and Americans [
28,
29,
30,
31]. The most frequently studied 5-HT1B gene variants are rs6296, rs13212041, rs6297, rs11568817, and rs130058. Both rs11568817 and rs130058 were significantly associated with substance use disorders, while rs11568817 was associated with nicotine dependence in men with ADHD. Alcohol-dependent individuals with rs13212041 CC genotype were more frequent compared to the carriers of the T allele in the group with early onset of alcohol abuse [
17]. The 3′-untranslated region (3′-UTR) variant rs13212041 potentially enables the microRNA-mediated regulation of 5-HT1B expression [
20]. The variant of rs6296 has been associated with attention-deficit hyperactivity disorder [
32], aggressive behavior in children [
33], substance misuse disorder, and major depression [
31]. These allelic associations with trait anxiety are not consistently found. The etiology and pathogenesis of trait anxiety are still largely unclear. It is commonly believed that trait anxiety results from different underlying neurobiological mechanisms, such as genetic and environmental influences. Thus, we hypothesized that 5-HT1B might be involved in the development of trait anxiety in Chinese Han college students.
To our knowledge, this is the first study to examine the association between 5-HT1B gene polymorphism and trait anxiety in Chinese Han subjects. The results might provide novel insights into the serotonergic regulation mechanisms underlying trait anxiety; it could also help further differentiation of trait anxiety and potential improvement of the prediction for anxiety disorders.
4. Discussion
College students are increasingly reporting common mental health problems, such as depression and anxiety [
35]. It is very important to explore the prevalence and genetic causes of trait anxiety of Chinese college students. 5-HT1B receptor plays important roles in multiple behavioral traits, such as locomotion, feeding, and thermoregulation, and also in arterial contractile regulation mechanisms. The 5-HT1B receptor has been the focus of much neuropsychiatric and neuropharmacological research [
38]. The intronless human 5-HT1B, located at 6q14.3–q16.3 (GDB 132312), encodes a 390-amino-acid polypeptide. Many polymorphisms in the coding sequence and UTRs were screened, and multiple correlation studies were carried out in 5-HT1B [
39]. To the best of our knowledge, this is the first study reporting the association of HTR1B rs13212041 with trait anxiety.
In the present study, we investigated four SNPs in 5-HT1B among 851 individuals of Han Chinese students, including 463 HTA individuals and 388 LTA individuals. According to previously reported observations, there is no functional study available for these SNPs, including rs11568817, rs130058, rs6297, and rs13212041 with trait anxiety, but few studies have investigated their roles in other mental disorders. Evidence of association was found between the functional SNP (rs130058) and alcohol, cocaine, and heroin dependence [
40]. The rs130058 SNP within 5-HT1B was demonstrated to have a differential association with increasing suicidal ideation depending on the antidepressant type [
41]. Some contribution of the functional promoter combination (rs11568817, rs130058)was found with self-reported anger and hostility among young men [
20]. The association between three SNPs (rs11568817, rs130058, rs6297) and the susceptibility to schizophrenia and anxiety disorders has not been previously reported [
24]. According to previous studies, only a few studies have investigated the association of rs13212041 polymorphism in 5-HT1B with alcohol dependence [
17] and schizophrenia [
23]. We demonstrated that rs13212041 in 5-HT1B was significantly associated with the personality development of trait anxiety in the Chinese Han population. The frequency of the TC genotype in LTA individuals was significantly higher than in HTA individuals. Both the C-allele, TC genotype, and TC + CC genotype were significantly associated with LTA. FPRP and statistical power were calculated for the positive findings for the samples [
42]. At the prior probability level of 0.1, the significant association for rs13212041 remained noteworthy. Our study provides evidence that rs13212041 may be involved in the protective effect of trait anxiety in China Han college subjects.In future studies, we will explore the potential function of rs13212041 to discover the mechanism of treatment, prevention (through nutritional or environmental changes), and diagnosis.
From the results of bioinformatics analysis, we also found the rs13212041 might affect the proteins bound of 5-HT1B with the FOS gene. FOS genes encode leucine zipper proteins that can dimerise with proteins of the JUN family, thereby forming the transcription factor complex AP-1, which can regulate the expression of 5-HT1B [
43]. Based on these, we proposed a hypothesis that rs13212041 could influence the proteins bound of 5-HT1B with the FOS gene, indirectly regulate the downstream gene expression, and affect the expression of 5-HT1B through the gene expression network. The specific mechanism needs to be demonstrated further. Moreover, Jensen et al. characterized the SNP (rs13212041; T1997C) in the distal 3’-UTR of 5-HT1B messenger RNA that disrupts a binding site for miR-96 [
44].
MicroRNAs are 20–21 nucleotide ribonucleic acids that regulate gene expression by binding to complementary sites on messenger RNA, triggering mRNA degradation and/or inhibition of translation [
45,
46]. Jensen et al. showed that the rs13212041 polymorphism modulates the gene expression by binding to miR-96, and the C-allele of rs13212041 may attenuate the regulatory function of miR-96 [
44]. In our study, the “TC” genotype and “C” allele of 5-HT1B rs13212041 were significantly associated with low trait anxiety. The C-allele of rs13212041 appeared to drive the dominant protective effect of trait anxiety. We presumed the “TC” genotype and “C” allele could disrupt 5-HT1B receptor expression by miR-96. Maybe this pattern suggests that the microRNA-binding site polymorphism has great behavioral effects. Further functional assay is necessary to explore the function and the underlying mechanism of rs13212041 polymorphism.
Given that trait anxiety is a complex disease affected by the interaction of genetic and environmental factors, polygenic or SNP-SNP interaction studies may help to discover the risk factors of trait anxiety. Of note, MDR is a powerful method to detect gene-gene interactions without main gene effects in case-control studies of complex diseases [
47]. Further, the MDR was used to analyze the interactions of these four SNPs, the result of the present study suggested that rs13212041 was the best single-locus model to predict trait anxiety and a combination of rs13212041 and rs6297 was the best multi-loci model. The interaction Fruchterman–Reingoldgraph also further confirmed that rs13212041 and rs6297 had a strong correlation, suggesting that HTR1B polymorphisms had an additive effect on the risk of trait anxiety development.
As a fundamental form of genetic variation and inheritance unit, haplotype may affect the phenotypes either directly by affecting promoter activity and protein structure, or indirectly through untyped causal variation near the marker [
48]. Therefore, haplotype association is of great significance for revealing the etiology of complex phenotypes. In the study, haplotype analysis implied that haplotype C
rs13212041T
rs130058T
rs6297A
rs11568817 may be a protective haplotype in trait anxiety. Previously,
HTR1B haplotypes were associated with
HTR1B gene expression [
20]. These hinted to us that HTR1B haplotypes could be a potential trait anxiety risk factor.
Limitations
One of the study limitations is a lack of careful determination of trait anxiety phenotypes, using nuclear magnetic resonance (NMR), electroencephalogram (EEG), and other effective anxiety laboratory indicators. In the future, we would like to enlarge sample size and complete the phenotype information to evaluate the association between 5-HT1B SNPs and trait anxiety phenotypes. In addition, although 5-HT1BSNPs might be associated with trait anxiety, the results were not significant after multiple testing correction (p < 0.05/4). Thus, the present findings need to be confirmed in future studies with a large sample size. Besides, a replication experiment in a different cohort will strengthen our findings. However, our association analysis firstly displayed that rs13212041 was a susceptible site for anxiety, which might lay a good working foundation for subsequent in-depth experimental molecular research.