Specification of field: molecular psychiatry and psychobiology
*Tadafumi Kato, MD, PhD, Laboratory for Molecular Dynamics of Mental Disorders, RIKEN Brain Science Institute, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan. Email: firstname.lastname@example.org
Aim: Serotonin receptor 2C (HTR2C) has been postulated as being involved in the etiology or pathophysiology of mental disorders such as bipolar disorder, major depression and schizophrenia. We previously revealed the altered mRNA expression and RNA editing of HTR2C in the postmortem brains of patients with mental disorders. Here we examined the relationship between genetic variations and expression level or RNA editing level of HTR2C in the human brain.
Methods: We performed mutation screening of the HTR2C gene by sequencing all exons, exon–intron boundaries, and promoter region in the same cohort used for expression and RNA editing studies (n = 58). Using the detected genetic variations, we examined the relationship between genetic variations and expression or RNA editing level.
Results and conclusion: We did not find novel mutations or single nucleotide polymorphisms that were specific to patients. Genotype and haplotype-based analyses revealed that genetic variations of HTR2C did not account for observed altered expression or RNA editing level of HTR2C in the brain.
SEROTONIN IS A monoamine neurotransmitter that plays important roles in regulation of mood, appetite, sexual behavior and anxiety. Serotonin receptor 2C (HTR2C) encodes a G protein-coupled receptor that activates phospholipase C. Knock-out mice for this gene showed various phenotypic and behavioral alterations including seizure, enhanced exploration of a novel environment, and dysregulation of anxiety-related behaviors,1–3 implicating the involvement of this gene in the pathophysiology of mental disorders. Several studies also reported the significant associations between genetic variations of HTR2C and major depression (MD), bipolar disorder (BD),4,5 increased risk for lifetime hospitalization in schizophrenia (SZ),6 and psychotic symptoms of Alzheimer' s disease.7,8 Among the known 14 serotonin receptors, HTR2C has a unique feature in that its pre-mRNA undergoes adenosine-to-inosine (A-to-I) RNA editing by adenosine deaminases.9 In A-to-I RNA editing, the translation machinery recognizes inosine as guanosine, resulting in changes in amino acid sequences. In cases of HTR2C, there are five editable positions (termed sites A to E) in exon 5 (Fig. 1), which theoretically result in a generation of up to 24 different HTR2C, in terms of amino acid sequences.9,10
In the studies using postmortem brains, decreased expression of HTR2C was reported in patients with SZ,11,12 and altered RNA editing in patients with SZ,13 MD,14 and BD15 as well as an animal model of depression.16 Consistent with these results, we have also revealed decreased expression of HTR2C in brains of patients with SZ and BD,17 and altered RNA editing status in major depression and suicide victims.18 To examine the possible cause of the altered status of expression and RNA editing, we here performed mutation screening of the HTR2C gene in the same cohort used in previous studies. We then examined the effect of genetic variations on expression and RNA editing level of HTR2C in the brain.
Postmortem liver samples corresponding to prefrontal cortex samples, using previous expression17 and RNA editing18 studies, were donated by the Stanley Medical Research Institute (SMRI). This sample set, called Consortium Collection, consisted of patients with BD, MD and SZ, as well as control subjects. Each diagnostic group consisted of 15 subjects. Diagnosis had been made according to the DSM-IV criteria. Detailed information about subjects are found elsewhere.19 Of the 60 liver samples, we successfully extracted genomic DNA from 58 samples (15 BD, 15 MD, 14 SZ, and 14 controls) by standard phenol-chloroform extraction method.
Polymerase chain reaction and DNA sequencing
To screen the mutations in the HTR2C genomic region, we sequenced all exons, exon–intron boundaries, and the promoter region (Fig. 1). The polymerase chain reaction (PCR)-amplified regions, primer pairs and PCR conditions are available upon request. PCR products were sequenced with a BigDye Terminator v3.1 Cycle Sequencing Kit and a 3730xl DNA analyzer (Applied Biosystems, Foster City, CA). The number of dinucleotide repeats at a microsatellite (rs3834996) was determined by 3130xl Genetic Analyzer (Applied Biosystems) using primers FAM-labeled 5′-TGCTGTTTGTTGAAATGAAATG-3′ and 5′-GTTTCTTGAGAGGGAAGGCTTTCTCAA-3′.
In the genotype- and haplotype-based analyses, one-way anova was conducted using the genotype or haplotype as the factor variable, and expression level or RNA editing efficiency as the dependent variable using spss 11.0J software (spss Japan). In cases where the number of informative genotypes or haplotypes was too small (≤2), we omitted that group from the anova. We employed the Student's t-test for two-group comparison. P < 0.05 was considered to be significant. Previously determined HTR2C expression level17 and RNA editing efficiency18 in postmortem prefrontal cortex (Brodmann's Area 10) were used for data analysis. Of the five edited sites, editing efficiency of two sites (sites A and D) were available.20 Power analysis was performed using G*power software.21
The genomic structure of the HTR2C gene (Xq24) is shown in Fig. 1. We sequenced all six exons, exon–intron boundaries and the promoter region (about 500 bp upstream region from exon 1) in a total of 58 subjects (15 BD, 15 MD, 14 SZ, and 14 controls). We detected seven SNP and a dinucleotide (GT) repeat polymorphism in these subjects (Fig. 1). All polymorphisms are known genetic variations and could be found in the SNP database. Among them, the rs3813929 (−795C/T) was reported to alter transcriptional activities by in vitro reporter assays, and the rs6318 (Cys23Ser) was shown to alter receptor activity.22 First, we examined whether each of the eight polymorphisms affected the mRNA expression or RNA editing level of HTR2C in the brain. We did not find significant effects of polymorphisms on the expression level of HTR2C, including −795C/T (Fig. 2) and Cys23Ser functional polymorphisms (data not shown). Similarly, we did not find significant effects of genetic variations on the RNA editing efficiency at site A or D (data not shown). We also confirmed that there were no significant effects of genetic variations on expression or RNA editing level using only control subjects (data not shown).
We then performed haplotype-based analysis using five polymorphisms in the promoter region (SNP #1–5 in Fig. 1). We conducted anova using the three representative haplotype combinations (diplotypes; A(GT)16GCG/A(GT)16GCG or A(GT)16GCG/-, A(GT)16GCG/G(GT)13ATC, and G(GT)13ATC/G(GT)13ATC or G(GT)13ATC/-) as factor variables. However, we could not detect significant differences of expression or RNA editing efficiency across three diplotypes (Fig. 3). We then performed haplotype-based analysis using all detected polymorphisms (SNP #1–8 in Fig. 1) in the HTR2C. Although there were 12 diplotypes within 58 subjects, six diplotypes were omitted from analysis because of the small number of subjects (n ≤ 2). Using the remaining six diplotypes as factor variables, we performed anova again. However, we could not detect significant differences of expression or RNA editing efficiency across haplotypes (data not shown). We also examined the relationship between expression level and RNA editing efficiency of HTR2C in the brain, which was not addressed in the previous studies, but we found no significant correlations between them (R = 0.118, P = 0.450 for site A, and R = 0.163, P = 0.297 for site D). Because HTR2C is located on chromosome X, we then performed the same analyses in male and female groups, respectively. However, we did not find significant relationship between these variables.
In the sample set used in this study, HTR2C expression showed a 1.55-fold decrease in BD and a 1.46-fold decrease in SZ.17 We also reported a tendency toward an increase of RNA editing efficiency at site D in MD subjects and at site A in suicide victims.18 Sequence analysis of HTR2C in the same cohort revealed that genetic variations of HTR2C did not affect the expression or RNA editing status in the brain. A similar result with regard to HTR2C expression in the brain was also reported by Castensson et al.11 They compared the effect of haplotypes, which were similar to this study on the expression level of HTR2C in the brain, and they did not find significant differences either.11
In contrast, in vitro reporter assays revealed that promoter genotype or haplotype of HTR2C affected the transcriptional activity.23–26 Among the studies, McCarthy et al. and Hill et al. used the haplotype constructs that were comparable to our study, for reporter assays.25,26 They reported that haplotype (GT)16GCG showed higher promoter activity than (GT)13ATC. The cause of discrepancy might be due to the relatively small sample size (n = 58) in this study, which lacks the statistical power to detect the small effects. Indeed, power analysis revealed that, in our sample size, we could detect a ‘large’ effect size (f = 0.4)27 at the power of 0.55 in anova. However, it is also clear that the observed decreased expression of HTR2C in brains of patients cannot be fully explained by the genetic variations of HTR2C.
Therefore, other factors may be involved in the observed altered expression and RNA editing statuses of HTR2C in patients with mental disorders. Because we did not detect correlations between expression and the RNA editing level of HTR2C, independent factors may contribute to their altered statuses in the brain.
Postmortem samples were donated by the SMRI, courtesy of Drs Michael B. Knable, E. Fuller Torrey, Maree J. Webster, and Robert H. Yolken. We are indebted to the Research Resource Center of our institute for DNA sequencing analysis.