Introduction: Sexual assault and a history of childhood sexual abuse (CSA) are related to posttraumatic stress disorder (PTSD) development. Long interspersed nuclear elements (LINE-1) are transposable elements, and their methylation is used to infer DNA global methylation. DNA methylation can be affected by trauma exposition which in turn would be associated with PTSD. Thus, we investigated if the LINE-1 methylation pattern is related to PTSD symptoms in females with a history of CSA. Methods: This is a case-control study that examined, at baseline (W1), 64 women victims of sexual assault diagnosed with PTSD and 31 patients with PTSD who completed the 1-year follow-up (W2). Participants were categorized into two groups according to the presence of CSA (PTSDCSA+: NW1 = 19, NW2 = 10; PTSDCSA-: NW1 = 45, NW2 = 21). PTSD symptoms (re-experiencing, avoidance, hyperarousal, alterations in cognition/mood) were assessed using the Clinician-Administered PTSD Scale, and the history of CSA was assessed by the Childhood Trauma Questionnaire. LINE-1 methylation was measured in three sites (CpG1, CpG2, CpG3) located in the 5’UTR region using bisulfite conversion followed by pyrosequencing. Linear regression models were performed to test the relation between LINE-1 CpG sites methylation and PTSD symptoms. Results: We found a negative association between CpG2 methylation and hyperarousal symptoms among those in the PTSDCSA+ group in W1 (adjusted p = 0.003) compared to the PTSDCSA- group (p > 0.05). Still, no association was observed between other PTSD symptoms and other CpG sites. Further, in the longitudinal analysis, LINE-1 hypomethylation was no longer observed in PTSD participants exposed to CSA. Conclusion: Our findings suggest that LINE-1 methylation may help understand the relationship between trauma and PTSD. However, more studies are needed to investigate LINE-1 as an epigenetic marker of psychiatric disorders.

Sexual assault is a significant trigger of posttraumatic stress disorder (PTSD), a severe and prevalent psychiatric disorder characterized by symptoms of re-experiencing, avoidance, hyperarousal, and alterations in cognition and mood. In Brazil, it is estimated that about 45% of sexually assaulted women develop PTSD, suggesting a high incidence of PTSD after sexual assault [1]. Furthermore, childhood sexual abuse (CSA) is a common type of trauma, mainly in girls, and one in five women reports a history of CSA [2, 3]. CSA has been associated with an increased risk for trauma symptoms like PTSD [4]. Previous studies found that women who have experienced child sexual abuse are more likely to develop PTSD compared to women survivors of trauma but with no history of child sexual abuse [5, 6].

Early trauma, multiple exposures to traumatic events, genetic vulnerability, and environmental factors may contribute to the liability of developing PTSD [7‒9]. Epigenetics studies arise as one way to understand the interaction of genetic factors with environmental exposure to trauma in PTSD [10]. DNA methylation at cytosine-guanine dinucleotides (CpG sites) is one of the most studied epigenetic mechanisms related to the etiology of PTSD [11]. Other epigenetic mechanisms such as noncoding RNA and histone modification are poorly explored in the literature [12, 13].

Epigenome-wide studies and candidate gene studies found that methylation of genes involved in the hypothalamic-pituitary-adrenal (HPA) axis and immune system are related to epigenetic regulation in PTSD [10]. Specifically, the methylation in gene promoters related to stress, neuronal plasticity, neurotransmission, and immune response may be linked to PTSD [14‒17]. Further, there is a growing body of research linking childhood maltreatment with DNA methylation, showing differential methylation of genes associated with the stress response, such as the HPA axis and glucocorticoid receptors in children and adults with a history of childhood trauma [18‒22]. Furthermore, evidence shows that methylation of repetitive elements, such as long interspersed nuclear element 1 (LINE-1), might be associated with the development of psychiatric disorders, such as depression [23, 24], PTSD [25], and first-episode schizophrenia [26], including those with a history of childhood trauma [27].

LINE-1 is a retrotransposon corresponding to approximately 17% of the human genome [28, 29]. These sequences are highly methylated in somatic tissues; previous studies suggest that more than one-third of DNA methylation occurs in repetitive elements, like LINE-1 [30]. As LINE-1 has large genome dissemination, LINE-1 methylation status may be considered a proxy for the global DNA methylation level [31]. Previous studies suggest that alterations in the methylation of LINE-1 sequences can influence the gene expression status, induce genetic variation through recombination or rearrangements, contribute to the differentiation of neurons during brain development, and lead to genomic instability in cells [32, 33].

Methylation in the CpG regions of the 5'UTR portion in several cell types is generally described in the literature as suppressors of LINE-1 expression; however, it is not clear how LINE-1 may be associated with psychiatric diseases. Although DNA methylation is widely investigated, few studies have investigated methylation in repeated regions of DNA, such as the LINE-1 region, associated with psychiatric disorders. Thus, the role of LINE-1 methylation in the etiology of PTSD remains unclear/unexplored.

We hypothesized that exposure to early trauma, such as CSA, could influence the changes in a DNA methylation pattern, such as methylation of LINE-1 sequences, which could increase the severity of PTSD symptoms. Thus, we investigated whether PTSD symptoms were associated with LINE-1 methylation in a cohort of sexually assaulted women with and without a history of CSA, which was assessed using multiple assessments (baseline and 1-year follow-up). To the best of our knowledge, there have been no studies to date that have examined associations between PTSD and LINE-1 methylation in sexually assaulted women.

Study Design

This study was part of a project investigating the genetic basis of PTSD in a Brazilian sample of civilian women aged 18–45 years old who had been recently sexually assaulted (1 to 6 months before the study inclusion). More information about this cohort is provided in the study by Coimbra et al. [34]. The Research Ethics Committee of Universidade Federal de São Paulo (UNIFESP) approved the study protocol, and all participants provided written informed consent.

This study presents clinical and genetics measurements from two time-point assessments: baseline (wave-1) and 1-year follow-up (wave-2) (shown in Fig. 1). We investigated both time points’ LINE-1 methylation levels in a group of women with PTSD and CSA histories versus a group of women with PTSD who reported no CSA according to the Childhood Trauma Questionnaire (CTQ).

Fig. 1.

Overview of the study workflow.

Fig. 1.

Overview of the study workflow.

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Sample and Eligibility Criteria

Participants were recruited at Hospital Pérola Byington (HPB) in São Paulo, Brazil. HPB is the largest public health service offering gynecological care for victims of sexual assault in São Paulo. At wave-1, 64 recently sexually assaulted participants were assessed for PTSD symptoms using the Clinician-Administered PTSD Scale for DSM-5 (CAPS-5). At wave-2, 31 patients returned for follow-up assessments. According to the CAPS-5 assessment, of the 31 patients, 18 PTSD cases continued to meet a PTSD diagnosis (persistent PTSD – CAPS-5 score =20), and 13 participants remitted from the disorder (PTSD remitters – CAPS-5 score <20). Further, we separated the participants into groups based on their history of CSA: PTSDCSA+ and PTSDCSA- (shown in Fig. 1).

Eligible participants were women aged 18–45 who suffered a sexual assault between one and 6 months before study inclusion. At wave-1, the exclusion criteria were: (a) a diagnosis of sexually transmissible disease, (b) a diagnosis of schizophrenia or bipolar disorder, (c) any uncontrolled clinical disease, (d) current use of corticosteroid medications, (e) menopausal symptoms, and (f) pregnancy. Moreover, we excluded women who were undergoing any kind of psychiatric or psychotherapeutic treatment at wave-1. A small number of participants (n = 8) had a history of previous antidepressant use. Women who used any psychiatric medication up to 6 months before study enrollment were excluded.

Assessments

PTSD symptoms were assessed at waves 1 and 2 using the CAPS-5, which is a 30-item structured interview that provides frequency and symptom severity based on PTSD symptom clusters (avoidance, intrusion symptoms, negative alterations in cognition and mood, alterations in arousal and reactivity) [35, 36]. Responses are measured on a 5-point scale from 0 to 4, with 0 = never/not at all severe and 4 = most of the time/very intense. The CAPS-5 symptom severity score was calculated by summing severity scores for all PTSD symptoms (CAPS-5 total) or by summing the individual item severity scores for symptom clusters, being that the higher the score, the more severe the PTSD symptoms. Further, the CAPS-5 was used to make a categorical PTSD diagnosis according to the total score: persistent PTSD – CAPS-5 score =20 and PTSD remitters – CAPS-5 score <20.

The CTQ was applied to all participants in wave-1. The CTQ is a questionnaire widely used by psychologists and psychiatrists to measure the prevalence of retrospective childhood trauma. We used a short-form version of the CTQ (CTQ-SF) validity in Portuguese. CTQ-SF is a self-report retrospective instrument of 28 items to assess early traumatic experiences related to emotional abuse, physical abuse, sexual abuse, emotional neglect, and physical neglect [37]. Responses are measured on a 5-point Likert scale (1 = never true, 2 = true, 3 = sometimes true, 4 = often true, 5 = very often true). All five abuse and neglect subscales are sums of the scoring of five questions with a score range from 5 to 25; these scores fall into four categories: none to low trauma exposure, low to moderate trauma exposure, moderate to severe trauma exposure, and severe to extreme trauma exposure. The same items can also be used as a summative scale for each type of trauma evaluated (emotional abuse, physical abuse, sexual abuse, emotional neglect, and physical neglect). For the sexual abuse subscale, we categorize the score into two categories according to the CTQ specification: the presence of any trauma related to sexual contact or trying to make sexual touch in an adult (score more than 5) and the absence of CSA (score equal to 5).

The Alcohol, Smoking and Substance Involvement Screening Test (ASSIST) aims for the early identification of substance use related to health risks and substance use disorders. It assesses the use of nine classes of psychoactive substances (tobacco, alcohol, marijuana, cocaine, stimulants, sedatives, inhalants, hallucinogens, and opiates) [38]. ASSIST is a structured questionnaire containing eight questions. The maximum possible score is 39 for each type of evaluated substance, except in the case of tobacco, in which the maximum score is 31. Scores above 27 indicate a high risk for dependence.

LINE-1 DNA Methylation

Peripheral blood was collected from participants in each wave, and DNA was isolated using the Gentra Puregene Kit (QIAGEN, USA) following the manufacturer’s standard protocol. LINE-1 DNA methylation was analyzed using pyrosequencing as described previously by Florea, 2013 [39]. Briefly, bisulfite treatment of DNA was performed using an EpiTect Fast Bisulfite Conversion Kit (QIAGEN, Germany). Initially, a conventional polymerase chain reaction (PCR) was performed for amplification of the region of interest (i.e., TTYGTGGTGYGTYGTTT – where Y is the region subject to methylation) of the bisulfite-converted DNA samples using the PyroMark Q24 CPG LINE1 kit primers (QIAGEN, USA) that use the reverse primer biotinylated as a starter sequence for the pyrosequencing reaction. Then, the biotinylated PCR product was isolated by binding to Streptavidin Sepharose® HP beads (GE Healthcare Life Sciences, Sweden) using the Immobilization of PCR Products to Streptavidin Sepharose® HP Beads protocol. Subsequently, the biotinylated single-stranded amplicon from these samples were submitted to pyrosequencing on the PyroMark Q24 Sequencer (QIAGEN, USA). PyroMark Q24 CpG methylation software (QIAGEN, USA) was used to quantify mean percentage methylation across three CpG sites in 5'UTR: CpG1 at position 328, CpG2 at position 321, and CpG3 at position 318, on the report of GenBank accession number X58075.

Statistics

A descriptive analysis of variables evaluated in this study (PTSD symptoms, LINE-1 methylation, age, tobacco use) was conducted for all participants stratified by CSA exposure (PTSDCSA+ vs. PTSDCSA-) using the independent sample T-test. Correlations between CSA and PTSD symptoms (total, re-experiencing, avoidance, hyperarousal, and alterations in cognition/mood) were evaluated using Pearson’s correlation. Relationships between LINE-1 methylation (%) and CSA results were tested by linear regression.

Further, 15 cross-sectional linear regression models were conducted to investigate the relationships between LINE-1 methylation levels (CpG1, CpG2, and CpG3) and PTSD symptoms according to the CSA exposure (PTSDCSA+ vs. PTSDCSA-), totalizing 30 models (15 models for wave-1 and 15 models for wave-2). We included PTSD CAPS-5 clusters as independent variables (i.e., CAPS-5 scores – total, re-experiencing, avoidance, hyperarousal, and alterations in cognition/mood) and LINE-1 methylation levels (CpG1, CpG2, and CpG3) as dependent variables. As age and smoking status have been related to alteration in methylation status, we included age and the ASSIST score for tobacco use as covariates for these linear models. Thus, our models were made by following this equation: percentage of LINE-1 methylation ˜ (CAPS-5 cluster + age + tobacco score use+ random error), being that we analyzed singly each CpG as a dependent variable per model and singly each CAPS-5 cluster as an independent variable per model. In the linear regression analyses, Bonferroni correction for multiple comparisons was used to adjust the p value. We multiplied the uncorrected p value by the number of comparisons made (n = 30). The alpha level was set at 0.05 (significance level of p value or adjusted p value <0.05).

All statistical analyses were conducted using the software Statistical Package for the Social Sciences (SPSS version 24). We used a tool online to draft the figures (https://chart-studio.plotly.com/).

Descriptive Analysis

For the descriptive analysis of the sample, we performed the independent T-tests to assess whether the CSA groups (PTSDCSA+ and PTSDCSA-, respectively, exposed and unexposed CSA) differed in terms of the following continuous variables: age (wave-1 and wave-2), PTSD symptoms (wave-1 and wave-2), and tobacco use score (Table 1). In Table 2, we can observe the means of these variables.

Table 1.

Descriptive analysis

Variablestdfp value95% confidence interval
Age (wave-1) 0.89 58 0.37 -2.05 -5.41 
Age (wave-2) 0.36 32 0.71 -4.51 -6.47 
Total CAPS-5 (wave-1) -0.49 58 0.62 -6.42 -3.88 
PTSD re-experiencing (wave-1) -0.69 58 0.48 -2.54 -1.23 
PTSD hyperarousal (wave-1) -0.19 58 0.84 -2.02 -1.66 
PTSD avoidance (wave-1) 0.25 58 0.81 -0.49 -0.63 
PTSD alterations in cognition and mood (wave-1) -1.83 58 0.07 -4.61 -0.21 
Total CAPS-5 (wave-2) -1.52 27 0.13 -22.46 -3.30 
PTSD re-experiencing (wave-2) -2.23 27 0.03 -5.16 -0.21 
PTSD hyperarousal (wave-2) -0.67 27 0.51 -5.29 -2.67 
PTSD avoidance (wave-2) -1.72 27 0.09 -3.56 -0.31 
PTSD alterations in cognition and mood (wave-2) -1.39 27 0.17 -9.74 -1.85 
ASSIST score for tobacco (wave-1) -1.07 21.72 0.29 -6.44 -2.03 
Variablestdfp value95% confidence interval
Age (wave-1) 0.89 58 0.37 -2.05 -5.41 
Age (wave-2) 0.36 32 0.71 -4.51 -6.47 
Total CAPS-5 (wave-1) -0.49 58 0.62 -6.42 -3.88 
PTSD re-experiencing (wave-1) -0.69 58 0.48 -2.54 -1.23 
PTSD hyperarousal (wave-1) -0.19 58 0.84 -2.02 -1.66 
PTSD avoidance (wave-1) 0.25 58 0.81 -0.49 -0.63 
PTSD alterations in cognition and mood (wave-1) -1.83 58 0.07 -4.61 -0.21 
Total CAPS-5 (wave-2) -1.52 27 0.13 -22.46 -3.30 
PTSD re-experiencing (wave-2) -2.23 27 0.03 -5.16 -0.21 
PTSD hyperarousal (wave-2) -0.67 27 0.51 -5.29 -2.67 
PTSD avoidance (wave-2) -1.72 27 0.09 -3.56 -0.31 
PTSD alterations in cognition and mood (wave-2) -1.39 27 0.17 -9.74 -1.85 
ASSIST score for tobacco (wave-1) -1.07 21.72 0.29 -6.44 -2.03 
Table 2.

Description of the mean of PTSD symptom clusters, age, and smoking score between PTSD patients with and without a history of CSA

VariableCSANMeanStandard deviation
Wave-1 
Total No 46 42.63 9.64 
Yes 18 44.38 7.81 
Re-experiencing No 46 10.78 3.59 
Yes 18 11.44 2.79 
Hyperarousal No 46 11.23 3.21 
Yes 18 11.61 3.27 
Avoidance No 46 5.34 0.947 
Yes 18 5.33 1.28 
Alterations in cognition and mood No 46 14.82 4.40 
Yes 18 17.22 3.70 
Age No 42 24.61 7.17 
Yes 18 22.94 5.04 
Tobacco use No 42 1.74 4.56 
Yes 18 3.94 8.14 
Wave-2 
Total No 20 16.95 14.73 
Yes 10 27.00 18.05 
Re-experiencing No 20 2.95 2.72 
Yes 10 5.80 3.73 
Hyperarousal No 20 5.70 4.23 
Yes 10 7.10 6.06 
Avoidance No 20 2.35 2.32 
Yes 10 4.10 2.60 
Alterations in cognition and mood No 20 5.95 6.95 
Yes 10 10.00 7.43 
Age No 23 27.34 8.07 
Yes 11 26.36 5.44 
VariableCSANMeanStandard deviation
Wave-1 
Total No 46 42.63 9.64 
Yes 18 44.38 7.81 
Re-experiencing No 46 10.78 3.59 
Yes 18 11.44 2.79 
Hyperarousal No 46 11.23 3.21 
Yes 18 11.61 3.27 
Avoidance No 46 5.34 0.947 
Yes 18 5.33 1.28 
Alterations in cognition and mood No 46 14.82 4.40 
Yes 18 17.22 3.70 
Age No 42 24.61 7.17 
Yes 18 22.94 5.04 
Tobacco use No 42 1.74 4.56 
Yes 18 3.94 8.14 
Wave-2 
Total No 20 16.95 14.73 
Yes 10 27.00 18.05 
Re-experiencing No 20 2.95 2.72 
Yes 10 5.80 3.73 
Hyperarousal No 20 5.70 4.23 
Yes 10 7.10 6.06 
Avoidance No 20 2.35 2.32 
Yes 10 4.10 2.60 
Alterations in cognition and mood No 20 5.95 6.95 
Yes 10 10.00 7.43 
Age No 23 27.34 8.07 
Yes 11 26.36 5.44 

No significant difference in PTSD symptoms was found between CSA groups (PTSDCSA+ and PTSDCSA-), except for PTSD re-experiencing symptoms measured at wave-2. The mean of PTSD re-experiencing symptoms was statistically significant between groups with and without a history of CSA (t = -2.23, DF = 27, p = 0.034), showing that the mean of this symptom was major in the PTSDCSA+ group compared to the group unexposed to CSA (shown in Fig. 2). No significant between-group differences were observed in demographic characteristics such as age and tobacco use status (Table 1).

Fig. 2.

Distribution of PTSD symptom scores based on CAPS-5 clusters between PTSD patients with and without a history of CSA.

Fig. 2.

Distribution of PTSD symptom scores based on CAPS-5 clusters between PTSD patients with and without a history of CSA.

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Correlation between CSA and PTSD Symptoms and LINE-1 Methylation

CSA exposure and increase PTSD symptoms were not significantly correlated; however, the direction of all correlations tested was positive. Table 3 shows all correlations conducted for multiple assessments of PTSD symptoms and CSA.

Table 3.

Pearson’s correlations results

VariablesCAPS-5 totalPTSD re-experiencingPTSD hyperarousalPTSD avoidancePTSD alterations in cognition and mood
Waves wave 1/wave 2 wave 1/wave 2 wave 1/wave 2 wave 1/wave 2 wave 1/wave 2 
CSA Pearson correlation 0.05/0.26 0.18/0.34 0.14/0.18 0.20/0.27 0.13/0.21 
p 0.66/0.15 0.17/0.05 0.27/0.32 0.12/0.13 0.31/0.23 
N 60/31 60/31 60/31 60/31 60/31 
VariablesCAPS-5 totalPTSD re-experiencingPTSD hyperarousalPTSD avoidancePTSD alterations in cognition and mood
Waves wave 1/wave 2 wave 1/wave 2 wave 1/wave 2 wave 1/wave 2 wave 1/wave 2 
CSA Pearson correlation 0.05/0.26 0.18/0.34 0.14/0.18 0.20/0.27 0.13/0.21 
p 0.66/0.15 0.17/0.05 0.27/0.32 0.12/0.13 0.31/0.23 
N 60/31 60/31 60/31 60/31 60/31 

CSA, childhood sexual abuse.

The methylation level was analyzed separately for 3 sites in the LINE-1 promoter. No significant relationships were observed between LINE-1 methylation levels for each of the 3 analyzed CpG sites (CpG1, CpG2, CpG3) and scores of CSA independent of the evaluated wave (p > 0.05; Table 4). LINE-1 DNA methylation measurements were available for 64 participants in wave-1 and 31 participants in wave-2, with no significant difference of mean methylation values between exposed and unexposed to CSA (CpG1wave-1 = 0.73; CpG2wave-1 = 0.77; CpG3wave-1 = 0.91; CpG1wave-2 = 0.99; CpG2wave-2 = 0.17; CpG3wave-2 = 0.72) (shown in Fig. 3).

Table 4.

Relationships between LINE-1 methylation (%) and score of CSA results

Testsp valueB95% confidence interval
Wave-1 ? PTSD patients 
CpG1~Score CSA 0.53 -0.05 -0.23 to 0.12 
CpG2~Score CSA 0.48 -0.07 -0.28 to 0.13 
CpG3~Score CSA 0.41 -0.08 -0.29 to 0.12 
Wave-2 ? persistent PTSD 
CpG1~Score CSA 0.42 0.07 -0.12 to 0.28 
CpG2~Score CSA 0.96 0.01 -0.29 to 0.31 
CpG3~Score CSA 0.81 -0.02 -0.27 to 0.21 
Wave-2 ? PTSD remitters 
CpG1~Score CSA 0.92 0.01 -0.36 to 0.23 
CpG2~Score CSA 0.71 0.06 -0.31to 0.42 
CpG3~Score CSA 0.55 -0.11 -0.49 to 0.28 
Testsp valueB95% confidence interval
Wave-1 ? PTSD patients 
CpG1~Score CSA 0.53 -0.05 -0.23 to 0.12 
CpG2~Score CSA 0.48 -0.07 -0.28 to 0.13 
CpG3~Score CSA 0.41 -0.08 -0.29 to 0.12 
Wave-2 ? persistent PTSD 
CpG1~Score CSA 0.42 0.07 -0.12 to 0.28 
CpG2~Score CSA 0.96 0.01 -0.29 to 0.31 
CpG3~Score CSA 0.81 -0.02 -0.27 to 0.21 
Wave-2 ? PTSD remitters 
CpG1~Score CSA 0.92 0.01 -0.36 to 0.23 
CpG2~Score CSA 0.71 0.06 -0.31to 0.42 
CpG3~Score CSA 0.55 -0.11 -0.49 to 0.28 
Fig. 3.

Distribution of LINE-1 methylation (%) in patients with PTSD with and without a history of CSA.

Fig. 3.

Distribution of LINE-1 methylation (%) in patients with PTSD with and without a history of CSA.

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LINE-1 Methylation and PTSD Symptoms Findings

We identified that the severity of hyperarousal symptoms measured at wave-1 was nominally related to low LINE-1 methylation levels in PTSD patients who suffered CSA (CpG1wave-1: p = 0.004, B = -0.55, 95% CI = -0.89 to -0.21; CpG2 wave-1: p = 0.001, B = -0.63, 95% CI = -0.96 to -0.31; and CpG3 wave-1: p = 0.003, B = -0.65, 95% CI = -1.04 to -0.26) but not in the PTSD patients without a history of CSA (CpG1wave-1: p > 0.05, CpG2wave-1: p > 0.05, and CpG3wave-1: p > 0.05) (Table 5). However, after Bonferroni correction for multiple comparisons (n = 30), only the negative association between methylation levels of the CpG2 site and hyperarousal symptoms remains significant (adjusted p value = 0.03). At wave-2, decreasing of hyperarousal symptoms was not associated with LINE-1 methylation pattern independent of the CpG site evaluated (CpG1, CpG2, and CpG3) (p > 0.05) in patients with or without a history of CSA. We identified no significant associations between the other PTSD cluster symptoms (CAPS-5 total, avoidance, re-experiencing, and negative alterations in cognitions and mood) and the mean percentage of LINE-1 methylation levels of 3 CpG sites (CpG1, CpG2, and CpG3) measured at waves 1 or 2 (p > 0.05) independent of a history of CSA.

Table 5.

Main results of relationship of PTSD symptoms on LINE-1 methylation levels

Modelp valueB95% confidence interval
Wave-1 - PTSDCSA+    
CpG1 ˜ CAPS-5 hyperarousal 0.004 -0.55 -0.89 to -0.21 
CpG1 ˜ CAPS-5 re-experiencing 0.18 -0.32 -0.83 to 0.17 
CpG1 ˜ CAPS-5 avoidance 0.46 -0.43 -1.65 to 0.79 
CpG1 ˜ CAPS-5 mood/cognition 0.48 0.13 -0.26 to 0.53 
CpG1 ˜ total CAPS-5 0.17 -0.12 -0.31 to 0.06 
CpG2 ˜ CAPS-5 hyperarousal 0.001 -0.63 -0.96 to -0.31 
CpG2 ˜ CAPS-5 re-experiencing 0.034 -0.53  
CpG2 ˜ CAPS-5 avoidance 0.11 -0.95 -2.15 to 0.24 
CpG2 ˜ CAPS-5 mood/cognition 0.48 0.14 -0.28 to 0.56 
CpG2 ˜ total CAPS-5 0.053 -0.053 -1,08 to 0.008 
CpG3 ˜ CAPS-5 hyperarousal 0.003 -0.65' -1.04 to -0.26 
CpG3 ˜ CAPS-5 re-experiencing 0.053 -0.53 -1.08 to 0.008 
CpG3 ˜ CAPS-5 avoidance 0.21 -0.82 2.19 to 0.53 
CpG3 ˜ CAPS-5 mood/cognition 0.376 1,19 -0.26 to 0.65 
CpG3 ˜ total CAPS-5 0.56 -0.06 -0.28 to 0.16 
Wave-1 - PTSDCSA-    
CpG1 ˜ CAPS-5 hyperarousal 0.81 -0.03 -0.31 to 0.24 
CpG1 ˜ CAPS-5 re-experiencing 0.96 -0.006 -0.26 to 0.25 
CpG1 ˜ CAPS-5 avoidance 0.33 0.51 -0.54 to 1.55 
CpG1 ˜ CAPS-5 mood/cognition 0.42 0.8 -0.12 to 0.28 
CpG1 ˜ total CAPS-5 0.2 0.05 -0.03 to 0.15 
CpG2 ˜ CAPS-5 hyperarousal 0.57 0.09 -0.24 to 0.44 
CpG2 ˜ CAPS-5 re-experiencing 0.46 0.11 -0.21 to 0.43 
CpG2 ˜ CAPS-5 avoidance 0.04 1.27 0.038 to 2.51 
CpG2 ˜ CAPS-5 mood/cognition 0.67 0.05 -0.19 to 0.29 
CpG2 ˜ total CAPS-5 0.08 0.09 -0.13 to 0 
CpG3 ˜ CAPS-5 hyperarousal 0.52 0.1 -0.23 to 0.44 
CpG3 ˜ CAPS-5 re-experiencing 0.61 0.07 -0.23 to 0.39 
CpG3 ˜ CAPS-5 avoidance 0.11 1.01 -0.23 to 2.25 
CpG3 ˜ CAPS-5 mood/cognition 0.67 0.05 -0.19 to 0.29 
CpG3 ˜ total CAPS-5 0.11 0.08 -0.02 to 0.19 
Wave-2 - PTSDCSA+    
CpG1 ˜ CAPS-5 hyperarousal 0.49 -0.11 -0.52 to 0.28 
CpG1 ˜ CAPS-5 re-experiencing 0.49 -0.19 -0.85 to 0.46 
CpG1 ˜ CAPS-5 avoidance 0.48 -0.25 -1.08 to 0.57 
CpG1 ˜ CAPS-5 mood/cognition 0.76 -0.04 -0.35 to 0.27 
CpG1 ˜ total CAPS-5 0.53 -0.03 -0.17 to 0.11 
CpG2 ˜ CAPS-5 hyperarousal 0.61 0.14 -0.51 to 0.81 
CpG2 ˜ CAPS-5 re-experiencing 0.75 -0.14 -1.22 to 0.94 
CpG2 ˜ CAPS-5 avoidance 0.96 0.02 -1.35 to 1,41 
CpG2 ˜ CAPS-5 mood/cognition 0.41 0.17 -0.31 to 0.65 
CpG2 ˜ total CAPS-5 0.63 0.04 -0.17 to 0.27 
CpG3 ˜ CAPS-5 hyperarousal 0.46 0.17 -0.37 to 0.72 
CpG3 ˜ CAPS-5 re-experiencing 0.91 0.04 -0.89 to 0.98 
CpG3 ˜ CAPS-5 avoidance 0.69 0.19 -0.97 to 1.37 
CpG3 ˜ CAPS-5 mood/cognition 0.23 0.21 0.17 to 0.59 
CpG3 ˜ total CAPS-5 0.39 0.07 -0.11 to 0.25 
Wave-2 - PTSDCSA-    
CpG1 ˜ CAPS-5 hyperarousal 0.68 0.04 -0.16 to 0.24 
CpG1 ˜ CAPS-5 re-experiencing 0.39 -0.13 -0.47 to 0.19 
CpG1 ˜ CAPS-5 avoidance 0.35 -0.17 -0.55 to 0.21 
CpG1 ˜ CAPS-5 mood/cognition 0.38 -0.05 -0.19 to 0.07 
CpG1 ˜ total CAPS-5 0.56 -0.17 -0.08 to 0.04 
CpG2 ˜ CAPS-5 hyperarousal 0.71 0.04 -0.18 to 0.26 
CpG2 ˜ CAPS-5 re-experiencing 0.71 -0.06 -0.44 to 0.31 
CpG2 ˜ CAPS-5 avoidance 0.75 -0.06 -0.48 to 036 
CpG2 ˜ CAPS-5 mood/cognition 0.85 -0.01 -0.16 to 0.13 
CpG2 ˜ total CAPS-5 0.93 -0.003 -0.07 to 0.06 
CpG3 ˜ CAPS-5 hyperarousal 0.75 0.03 -0.19 to 0.25 
CpG3 ˜ CAPS-5 re-experiencing 0.45 -0.13 -0.49 to 0.23 
CpG3 ˜ CAPS-5 avoidance 0.57 -0.11 -0.52 to 0.31 
CpG3 ˜ CAPS-5 mood/cognition 0.78 -0.02 -0.16 to 0.13 
CpG3 ˜ total CAPS-5 0.8 -0.008 -0.07 to 0.05 
Modelp valueB95% confidence interval
Wave-1 - PTSDCSA+    
CpG1 ˜ CAPS-5 hyperarousal 0.004 -0.55 -0.89 to -0.21 
CpG1 ˜ CAPS-5 re-experiencing 0.18 -0.32 -0.83 to 0.17 
CpG1 ˜ CAPS-5 avoidance 0.46 -0.43 -1.65 to 0.79 
CpG1 ˜ CAPS-5 mood/cognition 0.48 0.13 -0.26 to 0.53 
CpG1 ˜ total CAPS-5 0.17 -0.12 -0.31 to 0.06 
CpG2 ˜ CAPS-5 hyperarousal 0.001 -0.63 -0.96 to -0.31 
CpG2 ˜ CAPS-5 re-experiencing 0.034 -0.53  
CpG2 ˜ CAPS-5 avoidance 0.11 -0.95 -2.15 to 0.24 
CpG2 ˜ CAPS-5 mood/cognition 0.48 0.14 -0.28 to 0.56 
CpG2 ˜ total CAPS-5 0.053 -0.053 -1,08 to 0.008 
CpG3 ˜ CAPS-5 hyperarousal 0.003 -0.65' -1.04 to -0.26 
CpG3 ˜ CAPS-5 re-experiencing 0.053 -0.53 -1.08 to 0.008 
CpG3 ˜ CAPS-5 avoidance 0.21 -0.82 2.19 to 0.53 
CpG3 ˜ CAPS-5 mood/cognition 0.376 1,19 -0.26 to 0.65 
CpG3 ˜ total CAPS-5 0.56 -0.06 -0.28 to 0.16 
Wave-1 - PTSDCSA-    
CpG1 ˜ CAPS-5 hyperarousal 0.81 -0.03 -0.31 to 0.24 
CpG1 ˜ CAPS-5 re-experiencing 0.96 -0.006 -0.26 to 0.25 
CpG1 ˜ CAPS-5 avoidance 0.33 0.51 -0.54 to 1.55 
CpG1 ˜ CAPS-5 mood/cognition 0.42 0.8 -0.12 to 0.28 
CpG1 ˜ total CAPS-5 0.2 0.05 -0.03 to 0.15 
CpG2 ˜ CAPS-5 hyperarousal 0.57 0.09 -0.24 to 0.44 
CpG2 ˜ CAPS-5 re-experiencing 0.46 0.11 -0.21 to 0.43 
CpG2 ˜ CAPS-5 avoidance 0.04 1.27 0.038 to 2.51 
CpG2 ˜ CAPS-5 mood/cognition 0.67 0.05 -0.19 to 0.29 
CpG2 ˜ total CAPS-5 0.08 0.09 -0.13 to 0 
CpG3 ˜ CAPS-5 hyperarousal 0.52 0.1 -0.23 to 0.44 
CpG3 ˜ CAPS-5 re-experiencing 0.61 0.07 -0.23 to 0.39 
CpG3 ˜ CAPS-5 avoidance 0.11 1.01 -0.23 to 2.25 
CpG3 ˜ CAPS-5 mood/cognition 0.67 0.05 -0.19 to 0.29 
CpG3 ˜ total CAPS-5 0.11 0.08 -0.02 to 0.19 
Wave-2 - PTSDCSA+    
CpG1 ˜ CAPS-5 hyperarousal 0.49 -0.11 -0.52 to 0.28 
CpG1 ˜ CAPS-5 re-experiencing 0.49 -0.19 -0.85 to 0.46 
CpG1 ˜ CAPS-5 avoidance 0.48 -0.25 -1.08 to 0.57 
CpG1 ˜ CAPS-5 mood/cognition 0.76 -0.04 -0.35 to 0.27 
CpG1 ˜ total CAPS-5 0.53 -0.03 -0.17 to 0.11 
CpG2 ˜ CAPS-5 hyperarousal 0.61 0.14 -0.51 to 0.81 
CpG2 ˜ CAPS-5 re-experiencing 0.75 -0.14 -1.22 to 0.94 
CpG2 ˜ CAPS-5 avoidance 0.96 0.02 -1.35 to 1,41 
CpG2 ˜ CAPS-5 mood/cognition 0.41 0.17 -0.31 to 0.65 
CpG2 ˜ total CAPS-5 0.63 0.04 -0.17 to 0.27 
CpG3 ˜ CAPS-5 hyperarousal 0.46 0.17 -0.37 to 0.72 
CpG3 ˜ CAPS-5 re-experiencing 0.91 0.04 -0.89 to 0.98 
CpG3 ˜ CAPS-5 avoidance 0.69 0.19 -0.97 to 1.37 
CpG3 ˜ CAPS-5 mood/cognition 0.23 0.21 0.17 to 0.59 
CpG3 ˜ total CAPS-5 0.39 0.07 -0.11 to 0.25 
Wave-2 - PTSDCSA-    
CpG1 ˜ CAPS-5 hyperarousal 0.68 0.04 -0.16 to 0.24 
CpG1 ˜ CAPS-5 re-experiencing 0.39 -0.13 -0.47 to 0.19 
CpG1 ˜ CAPS-5 avoidance 0.35 -0.17 -0.55 to 0.21 
CpG1 ˜ CAPS-5 mood/cognition 0.38 -0.05 -0.19 to 0.07 
CpG1 ˜ total CAPS-5 0.56 -0.17 -0.08 to 0.04 
CpG2 ˜ CAPS-5 hyperarousal 0.71 0.04 -0.18 to 0.26 
CpG2 ˜ CAPS-5 re-experiencing 0.71 -0.06 -0.44 to 0.31 
CpG2 ˜ CAPS-5 avoidance 0.75 -0.06 -0.48 to 036 
CpG2 ˜ CAPS-5 mood/cognition 0.85 -0.01 -0.16 to 0.13 
CpG2 ˜ total CAPS-5 0.93 -0.003 -0.07 to 0.06 
CpG3 ˜ CAPS-5 hyperarousal 0.75 0.03 -0.19 to 0.25 
CpG3 ˜ CAPS-5 re-experiencing 0.45 -0.13 -0.49 to 0.23 
CpG3 ˜ CAPS-5 avoidance 0.57 -0.11 -0.52 to 0.31 
CpG3 ˜ CAPS-5 mood/cognition 0.78 -0.02 -0.16 to 0.13 
CpG3 ˜ total CAPS-5 0.8 -0.008 -0.07 to 0.05 

In this longitudinal study, we investigated whether LINE-1 methylation levels differ between sexually assaulted women with a history of CSA compared to sexually assaulted women without a history of CSA according to PTSD symptom severity. At wave-1, we observed an association between the LINE-1 methylation level of CpG2 and PTSD hyperarousal symptoms assessed in women sexually abused as a child. Interestingly, after follow-up analysis, we did not find an association between any PTSD symptom and the LINE-1 methylation level of the three CpG sites investigated.

Most epigenetic studies on PTSD have examined the methylation status of CpG sites near or within genes [10, 40‒42]. The methylation status of repetitive DNA elements, such as LINE-1, is little investigated despite being a significant contributor to global DNA methylation patterns, given the large presence of these sequences in the genome [43]. Previous studies hypothesized that hypomethylation of the LINE-1 region is associated with behavioral dysfunction and psychiatric disorders possibly due to genomic instability associated with the pathogenic cascade of neurodegeneration, possibly interrupting gene expression [44, 45]. The biological mechanisms are still not very clear in the literature. LINE-1 can influence gene expression status, which may contribute to transcription interruption, insertion mutations, or DNA breaks, leading to genomic instability in cells. Further, LINE-1 can induce genetic variation through recombination or rearrangements, or mutations [32, 33]. Furthermore, somatic retrotransposition in the brain may also contribute to the etiology of some psychiatric disorders. Finally, LINE-1 has also been linked to inflammatory processes [33, 43, 46‒48]. All these mechanisms can somehow influence the development of psychiatric disorders, such as PTSD.

We found a single study in the literature about the effects of LINE-1 methylation on PTSD. This study reported hypomethylation of LINE-1 in US military service members with a PTSD diagnosis [25], suggesting that LINE-1 methylation may be involved in the stress response regulatory system and the immune system, which may represent an adaptive response to stress-fighting mechanisms that may decrease the risk for development of PTSD. However, we did not find any study investigating the association of LINE-1 methylation with PTSD symptoms.

Furthermore, Misiak et al. [27] found that a history of childhood trauma (emotional abuse and trauma) was able to predict a lower mean LINE-1 methylation level in patients with first-episode schizophrenia. However, there is not enough evidence to state whether hypomethylation at different CpG sites of LINE-1 occurs due to disease course, trauma experience, or whether some regions of the genome are naturally more prone to undergo methylation.

Our study focused on the investigation of PTSD symptoms and not PTSD diagnosis due to the complexity of this phenotype. This proposal to investigate symptomatology is currently gaining prominence in the literature, such as the Research Domain Criteria (RDoC) project, which aims to understand the etiology of psychiatric disorders [49], based on the domain of symptoms. Further, PTSD symptoms are characterized by alterations in biological systems such as overactivity of the HPA axis, which could reveal specific factors associated with PTSD states [50, 51].

Hyperarousal is the predominant PTSD symptom cluster investigated in genetic studies [8, 52‒54]. Severe PTSD symptoms related to brain hyperactivity or increased arousal like hyperarousal symptoms may expose the individual to psychological stress by alterations in the HPA axis, which could influence DNA methylation [55‒58]. A previous gene association study found that FK506-binding protein 5 (FKBP5) polymorphisms may predict the severity of PTSD symptoms such as hyperarousal [59] in veterans diagnosed with PTSD and with a history of CSA.

The relationship between DNA methylation and PTSD symptoms is complex and not fully understood. Windy McNerney et al. [60] found that interaction between NR3C1 (glucocorticoid receptor gene) methylation and hippocampal size is linked to lower hyperarousal scores in veterans with PTSD. Meanwhile, Vukojevic et al. [61] did not find an association between hyperarousal symptoms and NR3C1 methylation in genocide survivors with PTSD. Wolf et al. [62] found that PTSD hyperarousal symptoms were associated with accelerated DNA methylation age in veterans with PTSD. Nonetheless, the authors observed a significant association between DNA methylation and PTSD hyperarousal symptoms. Ziegler et al. [63] found an association between monoamine oxidase A (MAOA) gene hypermethylation and the severity of PTSD hyperarousal symptoms in male civilians of the Southeastern Europe population. Although some studies focus on the methylation of candidate genes related to PTSD symptoms, like NR3C1 [64], MAOA [65], spermatogenesis and centriole-associated 1-like gene (SPATC1L) [42], and the longevity gene klotho KL[41], our study is the first to find an association between the global DNA methylation and PTSD hyperarousal symptoms in a sample of women exposed to a recent sexual assault and with a history of CSA.

LINE-1 methylation may be an important factor related to the development of psychiatric disorders; however, the effects of LINE-1 methylation on psychiatric diagnosis are controversial. More studies are needed to be able to investigate LINE-1 as an epigenetic marker of psychiatric disorders and to better comprehend the biological mechanisms underlying the role of LINE-1 in their pathophysiology. Nonetheless, understanding the relationship between LINE-1 methylation and the risk to develop psychiatric disorders may help in the development of new treatments and methodologies for clinical purposes.

The present results must be interpreted considering some limitations. First, this study focused on a sample of young sexually assaulted women. Future studies should elucidate if our findings can be reproduced in both men and women survivors of other types of traumatic experiences. Second, our study has a limited number of participants, reducing the statistical power to identify the minor effects of LINE-1 methylation on PTSD. Third, we used peripheral blood for DNA methylation analysis because it is a less invasive method and an easily accessible tissue, which allows the analysis of a more significant number of patients. However, some recent studies have found a high correlation between brain (tissue-specific) and blood (peripheral) methylation, suggesting that blood may be a good target tissue for methylation studies.

In conclusion, the results indicate that the LINE-1 hypomethylation pattern was associated with higher PTSD hyperarousal scores in women who were recently sexually assaulted and who had a history of CSA. Conversely, we did not observe an association between PTSD symptoms and alterations in LINE-1 methylation levels in participants with no history of CSA. Future research could investigate the relationship between global DNA methylation, history of CSA, and PTSD cluster symptoms in larger sample sizes, including female and male subjects.

We wish to thank the staff at the Hospital Pérola Byington for recruitment of case patients for participation in this study.

This study protocol was reviewed and approved by Research Ethics Committee of Universidade Federal de São Paulo (UNIFESP), approval number (CAAE: 30332214.8.0000.5505). Patients and participants provided their written informed consent to participate in this study.

The authors have no conflicts of interest to declare.

This work was supported by a research grant from the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP2014/12559-5). CMC was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP 2015/26473-8). BMC was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES/Brasil) – Finance-Code-001. Additional grant support came from CNPq 303389/2016-8 and CNPq 312464/2018-5.

C.M.C. and S.I.B. participated in the conception and design of this study. C.M.C. drafted the article. C.M.C., B.M.C., A.B., V.K.O., A.M.F., M.F.M., and S.I.B. participated in data collection, data analysis, and data interpretation. D.F.M. aided in the data analysis and interpretation. B.M.C., A.B., D.F.M., V.K.O., A.M.F., M.F.M., and S.I.B. made critical revisions to this article and agreed on the final article before submission. All the authors contributed to the article and approved the submitted version.

The original contributions presented in the study are included in the article, and further inquiries can be directed to the corresponding author.

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