233 patients with PD were recruited from inpatient and outpatient populations in the Department of Psychosomatic, Sichuan Provincial People’s Hospital between May 2015 and December 2018(recruitment flow chart in Figure S1). Diagnosis of PD was conducted according to the Structured Clinical Interview for the Diagnostic and Statistical Manual of Mental Disorders fourth edition (DSM-IV) (SCID-1), which was administered by trained clinical psychiatrists. Patients with neurological diseases, and/or past or current episodes of MDD, generalized anxiety disorder(GAD), manic disorder, bipolar disorder, schizophrenia or any other psychiatric disorders were excluded. In addition, 231 healthy controls were recruited from the Center of Health Examination, Sichuan Provincial People’s Hospital. The SCID-1 was also performed to exclude a lifetime or current diagnosis of PD, MDD, GAD, among others.
All subjects in this study were Han Chinese, and all subjects were free of acute or chronic somatic disorders. All patients were free of antidepressants or other psychotropic medications intake within 2 weeks before their examination. Demographic data and clinical presentations were obtained from medical records or qualified interviews. A 3-mL EDTA-anticoagulated peripheral blood sample was collected from every individual.
Panic Disorder Severity Scale(PDSS)
The Panic Disorder Severity Scale is a valid and reliable inventory. 7 items are included in this scale, and each item is scored 0–4 on a Likert scale ranging from “none” to “extremely severe” . Also, the scale was translated into Chinese, and the PDSS-Chinese Version(PDSS-CV) has good internal consistency (Cronbach’s alpha) with the overall score (0.83).
Setraline (100-200mg qd) was administered to all patients for 4-week period. Other psychotropic medications were not permitted during the study except for benzodiazepine, which was prescribed occasionally for insomnia with a minimal dosage at bedtime. PDSS was assessed to all patients at baseline and after 4 weeks of treatment. Response was defined as a 40% or greater reduction in scores on the pre-treatment PDSS.
SNP Selection and Genotyping
The SNP selected was based on literature search and minor allele frequency(MAF) >0.05 of east Asian population from public databases. The references were listed in the Table S1. SNP genotyping was performed using an improved multiplex ligation detection reaction (iMLDR) technique which developed by Genesky Biotechnologies Inc. (Shanghai, China). The primer information of the reaction mixtures is described in Tables S2 and S3. The 20 μl multiplex polymerase chain reaction (PCR) included 1 x GC-I buffer (Takara), 0.3 mM dNTP, 3.0 mM Mg2+ , 1 U HotStar Taq Polymerase (Qiagen Inc), 1 μl genomic DNA(5-10ng/μl), and 1μl Multiplex-PCR primermix. In addition, The 5-HTT of PCR reaction volume(10μl) included 10 x buffer I (Qiagen Inc), Q Solution(Qiagen Inc.), 0.2 mM Mg 2+, 1 U HotStarTaq polymerase (Qiagen Inc.), 1 μl genomic DNA, and 1μl PCR primermix. The PCR cycling program was as follows: denaturation at 95 °C for 2 min, followed by 11 cycles of 94 °C for 20s, annealing at 65 °C for 40 s and 72 °C for 1.5 min, and each cycle decreased 0.5°C. The third step was set as 24 cycles of 94°C for 20s, 59°C for 30 s, finally, 72 °C for 2 min and 4 °C for hold. In addition, the 5-HTT of cycling program for PCR was 95 °C for 2 min, 35 cycles of amplification consisted of 94 °C for 20 s and 72 °C for 1.5 min, and final extension at 68 °C for 60 min and a hold at 4°C. 5U SAP and 2 ExonucleaseⅠwere used to purify the PCR product at 37 °C and then 15 min of 75 °C for inactivation. 2 μl purified multiplex PCR product, together with 6 μl ddH2O, were added into the ligation reaction containing 1 μl of 10× ligation buffer, 0.25 μl Tag DNA ligase, 5′ ligationprimer (1μM) 0.4μl, 3′ ligationprimer (2μM) 0.4μl. The ligation cycling program was set as 38 cycles× (94 °C for 1.5 min and 56 °C for 4 min), and a hold at 4°C. After PCR, 0.5 μl ligation product was taken for Sequencing with 0.5 μl Liz500 Size Standard, and 9 μl HiDi mixture (ABI3730XL). The raw data were analyzed by Gene Mapper v4.1 software (AppliedBiosystems, USA). All primers, probes and labeling oligos were designed by and ordered from Genesky Biotechnologies Inc.
The data were analyzed using SPSS version 18.0 software (SPSS Inc., Chicago, IL, USA). Student’s t-tests were used for inter group comparisons of continuous variables, with Pearson’s chi-square tests utilized for categorical variables. P values for the Hardy-Weinberg equilibrium (HWE) were tested by Pearson’s chi-square test, and P > 0.05 indicated no significant deviation in allele and genotype frequencies among subjects. Associations between SNPs and disease status were determined based on the distributions of allelic frequencies and genetic models (additive, dominant, and recessive model), and odds ratios (ORs) and 95% confidence intervals (CIs) were performed in unconditional logistic regression analysis using PLINK v1.07, 20 adjusting for age, gender, educational level and resident location. To protect from Type I error, a Bonferroni correction was conducted. For all analyses, statistical tests were two-tailed and an alpha level of 0.05 was used to define statistical significance. Power calculation (α = 0.05) was performed using Power and Sample Size Calculation Software (3.6.1).