Participants and procedure
According to the DSM-IV-TR diagnostic criteria [1] for PTSD, 128 subjects with PTSD and 128 (non-PTSD) controls were recruited from a psychiatric outpatient clinic at the Veterans Health Service (VHS) Medical Center. All subjects were of Korean ethnicity and male veterans who had served on active duty during the Vietnam War. Individuals with a history of head trauma, organic brain syndrome including cerebrovascular accidents or dementia, major psychiatric disorders including psychosis or bipolar disorder, or substance dependence other than alcohol and nicotine were excluded. The study was approved by the institutional review board of the VHS Medical Center, South Korea(BOHUN 2016-02-007). All subjects gave their written informed consent before participating in this study.
Measures
For assessing PTSD, we used the Clinician-Administered PTSD Scale (CAPS), a structured clinical interview, which is considered the gold standard for diagnosing PTSD [27, 28]. The diagnosis of PTSD was determined by symptom frequency and intensity based on the liberal scoring rule of the CAPS [29]. In addition, the Combat Exposure Scale (CES), a self-reporting scale, was administered for measuring the level of wartime traumatic stressors experienced by the combatants [30]. The total CES scores were divided into five categories of combat exposure: light (1–8), light–moderate (9–16), moderate (17–24), moderate–heavy (25–32), and heavy (33–41). The Alcohol Use Disorders Identification Test (AUDIT) was also used to assess hazardous and harmful alcohol use [31].
Genotyping
Seven tagging single nucleotide polymorphisms (SNPs) in the USP46 (rs346005, rs10034164, rs2244291, rs12646800, rs6554557, rs17675844, and rs10517263) were selected based on a prior genetic association study in a Japanese population [26]. Subjects donated a blood sample through venipuncture, and the DNA of each subject was isolated using standard techniques. The genotyping procedures were carried out using single base primer extension assay using the ABI PRISM SNaPShot multiplex kit (ABI, Foster City, CA, USA) according to the manufacturer’s recommendations. The forward and reverse primer pairs used for the SNaPshot assay and genetic information for all tested SNPs are presented in Table S1 in Additional file 1 (Supplementary information). Analysis was performed using Genemapper software (version 3.0; Applied Biosystems) in the DNA Link, Inc. (Seoul, South Korea).
Data Analyses
Demographic and clinical characteristics between subjects with and without PTSD were compared using χ2-test or Student’s t-test on the Statistical Package for the Social Sciences version 25.0 (SPSS Inc., Chicago, IL, USA). The Hardy–Weinberg equilibrium for each SNP in the control group was calculated by χ2-test.
Single-marker analyses were performed using the R package SNPassoc [32]. Between-group comparisons of genotype frequency differences for diagnostic status were performed by logistic regression analysis considering different genetic inheritance models. The outcome variable was analyzed yielding odds ratios (ORs) with 95% confidence intervals (CIs) and p-values. Associations between haplotype distributions and PTSD status were examined using the ‘haplo.score’ function of R package haplo.stats [33]. Haploblock structure and linkage disequilibrium (LD) patterns obtained from the seven SNPs were constructed using the Haploview ver. 4.2 (http://www.broad.mit.edu/mpg/haploview). In further analysis, we conducted linear regression analyses for three clusters (re-experiencing, avoidance, and hyperarousal) of PTSD symptoms considering PTSD as continuous phenotypes. All analyses were adjusted for demographic factors including age, education year, socio-economic status, and marital status; the 5 levels of CES, and AUDIT scores (harmful alcohol drinking). In all analyses, p-value of less than 0.05 was considered as nominally significant (uncorrected p < 0.05). The statistical threshold was corrected using the Bonferroni method for the total number of SNPs (α = 0.05/7 = 0.0071).