All participants provided written informed consent. The study protocol was approved by the ethics committee of Shaanxi Normal University. Participants in the study were China Han subjects, recruited from freshman or senior. Subjects were asked to provide venous blood samples and fill out State-Trait Anxiety Inventory (STAI) (Wenli Li,1995；Spielberger et al.,1983). The number of participants that filled out State-Trait Anxiety Inventory (STAI) was 2645, then 2529 valid questionnaires were received. We filtered the top 25% of the participants as the HTA group (case) and last 25% of the participants as the LTA group (control) followed by the score of STAI by SPSS quartile method. At last, 851 participants with valid DNA and valid data were enrolled for our study, including 388 individuals with HTA and 463 individuals with LTA with age and gender matched. The classic case-control research paradigm to conduct in our research. Questionnaire test (post-test) was performed on the subjects before genotyping, according to the self-report of the participants, all the participants had no history of long-term medication, no symptoms of mental or neurological disorders.
Blood collection and DNA isolation
Peripheral blood samples (2ml) were obtained from each participant. Genomic DNA was extracted from peripheral blood of cases and controls using the GoldMag-Mini whole blood Genomic DNA Purification Kit (GoldMag Co.Ltd., Xi'an city, China), as recommended by the manufacturer's instructions. DNA concentration was determined by the NanoDrop Lite spectrophotometer (ThermoFisher Scientfic, Waltham,MA). The concentration of all DNA samples was normalized to 20 ng/ul.
SNP inclusion and screening criteria are as follows: 1) according to the HTR1B polymorphism distribution, we selected SNPs with favorable polymorphisms (MAF≥0.1); 2) as the function of the HTR1B coding region has been studied extensively, SNPs located within HTR1B regulatory regions were selected 3) based on prior reports, polymorphisms in HTR1B gene which have been well studied in other mental disorders including alchol abuse, ADHD cormorbidities, anger and hostility, schizophrenia, but current knowledge of the association between trait anxiety and HTR1B polymorphisms in the Chinese Han population have not been well studied.. At last, four polymorphisms of HTR1B gene including rs11568817, rs130058, rs6297 and rs13212041 were selected in this study.
These four HTR1B gene polymorphisms were genotyped according to the procedure of iPLEX single base extension amplification technology. MassARRAY Nanodispenser (Agena Bioscience, San Diego, CA) was used to design primers for amplification process and single base extension reactions. SNP genotyping was carried out on the MassARRAY iPLEX (Agena Biosience, SanDiego, CA) platform. Agena Bionsience Typer 4.0 software was used to manage and analyze SNP genotypic data. iPLEX primer for HTR1B genotyping in this work as listed in the Supplementary file S1.
Quantitative data were shown as median ± standard deviation (SD). Student’s t-test was used to compare the differences of quantitative data, and χ2 test was applied for qualitative data comparison. Deviation from Hardy–Weinberg equilibrium (HWE) of genotypic distribution of each SNP in controls was analyzed using Fisher’s exact test. In addition, the Pearson’s χ2 and Fisher’s exact tests were used to calculate the allele frequencies of case and control, and MAF in controls was defined as baseline. After adjusting for age and gender, odds ratios (ORs) and 95% confidence interval (95% CI) were calculated using unconditional logistic regression analysis. The relationship between the selected SNPs and trait anxiety was calculated using genotypic model analysis (codominant, dominant, recessive, over-dominant, and log additive) by (SPSS 21, Chicago, IL). The analyses, which included linkage disequilibrium (LD), haplotype construction, and genetic association at polymorphism loci, were performed using the Haploview software package (Haploview version 4.2). In the LD analysis, pairwise distance among SNPs 500 kb was ignored. D′ value was used to evaluate the LD for each pair of SNPs, and variants with the red square indicated that the two related sites were in complete LD (D′=1). D′ value equaling to 0.8 indicated that the related SNPs formed one block[34-35]. Haplotypes were constructed using SNPs in the same LD block, and haplotype frequency of > 0.05 was selected. Finally, four SNPs in HTR1B gene from each LD block was selected in the current study. In addition to statistical analysis, we also conducted a number of bioinformatics mining for the identified SNPs of HTR1B gene, the SNPs information of HTR1B gene was retrieved from the National Center for Biotechnology Information (NCBI) database of SNPs (dbSNP (http://www.ncbi.nlm.nih.gov/snp/). The bioinformatics tools including SIFT, PolyPhen, FATHMM, Mutation Accessor, UTRscan Server, MirSNP, PolymiRTS, miRNASNP were used to identify the potential functional SNPs in human HTR1B gene. Statistical analyses were performed using Microsoft Excel (Microsoft Corporation, Redmond, WA, USA) and SPSS (SPSS 21, Chicago, IL)) statistical package. In the study, all the P-value were two-sided, and P < 0.05 was defined as statistically significant.