233 patients with PD were recruited from inpatient and outpatient populations in the Department of Psychosomatic, Sichuan Provincial Peoples Hospital between May 2015 and December 2018(see 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 performed by trained psychiatrists. Patients with neurological diseases, and/or past or current episodes of major depression disorder, generalized anxiety disorder, 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 Peoples Hospital. The SCID was also administered by a trained clinical psychiatrist to exclude a lifetime or current diagnosis of PD, major depression, generalized anxiety disorder, among others.
All subjects in this study were Han Chinese in China, 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 comprises 7 items, and participants are instructed to rate each item from 0-4 based on the severity of each symptom, with possible responses ranging from none to extremely severe . The scale was translated into Chinese by Xiong HF , and the Panic Disorder Severity Scale-Chinese Version has good internal consistency (Cronbachs alpha) with the overall score (0.83).
All patients received treatment for a 4-week period with sertraline(100-200 mg qd)), other psychotropic medications were not permitted during the study except for benzodiazepine, which was prescribed occasionally for insomnia with a minimal dosage at bedtime. PD severity was assessed using the PDSS at baseline and after 4 weeks of treatment. The response of PD was defined as a reduction of the pre-treatment PDSS score of at least 40%.
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 multiplex polymerase chain reaction(PCR) reaction volume(20μl) included 1 x GC-I buffer (Takara), 3.0 mM Mg 2+ , 0.3 mM dNTP, 1 U HotStar Taq Polymerase (Qiagen Inc., Hilden, Germany), 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 cycling program for PCR was 95 °C for 2 min, followed by 11 Cycles of 94 °C for 20 s, 65 °C for 40 s and 72 °C for 1.5 min, and each cycle decreased 0.5°C. The third step, 24 cycles of 94°C for 20s, 59°C for 30 s, finally, 72 °C for 2 min and 4 °C and a hold at 4°C. 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. The PCR product was purified with 5 U SAP and 2 U Exonuclease I at 37 °C for 1 h and then 15 min of 75 °C inactivation. The ligation reaction contained 1 μl of 10× ligation buffer, 0.25 μl Tag DNA ligase, 5′ ligationprimer(1μM) 0.4μl, 3′ ligationprimer (2μM) 0.4μl, 2μl purified multiplex PCR product, and 6 μl ddH 2 O mixture. The ligation cycling program was 38 cycles× ( 94 °C for 1.5 min and 56 °C for 4 min), and a hold at 4°C. Sequencing was conducted on 0.5 μl ligation product, 0.5 μl Liz500 Size Standard, and 9 μl Hi-Di mixture (ABI3730XL). The raw data were analyzed by Gene Mapper v4.1 software (AppliedBiosystems, Foster City,CA 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). Students t-tests were used for inter group comparisons of continuous variables, with Pearsons chi-square tests utilized for categorical variables. P values for the Hardy-Weinberg equilibrium (HWE) were tested by Pearsons 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).