Genes Known to Escape X Chromosome Inactivation Predict Co

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Genes known to escape X chromosome inactivation predict co-morbid chronic musculoskeletal pain and posttraumatic stress symptom development in women following trauma exposure

Abstract

Co-morbid chronic musculoskeletal pain (CMSP) and posttraumatic stress symptoms (PTSS) are frequent sequelae of motor vehicle collision (MVC), are associated with greater disability than either outcome alone, and are more prevalent in women vs men. In the current study we assessed for evidence that gene transcripts originating from the X chromosome (XCh) contribute to sex differences in vulnerability to CMSP/PTSS after MVC. Nested samples were drawn from a longitudinal study of

African American individuals, and CMSP (0-10 Numeric Rating Scale) and PTSS (Impact of Events

Scale, Revised) outcomes were assessed six months following MVC. Blood RNA were sequenced

(n=101) and the relationship between XCh mRNA expression levels and co-morbid CMSP/PTSS outcomes was evaluated using logistic regression analyses. A disproportionate number of peritraumatic

XCh mRNA predicting CMSP/PTSS in women were genes previously found to escape XCh inactivation

(XCI) (11/40, z=-2.9, p=0.004). Secondary analyses assessing gene ontology relationships between these genes identified an enrichment in genes known to influence neuronal plasticity. Further, the relationship of expression of two critical regulators of XCI, XIST and YY1, was different in women developing CMSP/PTSS. Together, these data suggest that XCh genes that escape inactivation may contribute to sex differences in vulnerability to CMSP/PTSS after MVC.

Key Words sex differences; XIST; anxiety; hyperalgesia; RNA

Introduction

Exposure to traumatic events is common in life.1 In industrialized nations, motor vehicle collisions

(MVCs) are one of the most common types of trauma1, with over 50 million MVCs occurring worldwide each year2. The great majority of individuals experiencing MVC do not have serious injury; in the US, more than 90% of individuals seen in the emergency department after MVC are discharged home after evaluation3. A substantial proportion of such individuals develop chronic musculoskeletal pain (CMSP)4-

6 and/or posttraumatic stress symptoms (PTSS)7-9. Some individuals develop both CMSP and PTSS

(CMSP/PTSS); such individuals experience greater mental and physical disability than those with either outcome alone10-14, are are an important outcome group for further study15.

Most individuals who develop CMSP/PTSS following MVC are women.16-18 This fact is consistent with the marked increase in CMSP and PTSS burden experienced by women vs. men in other settings.19-25 Additionally, data from animal models suggest that there are sex differences in response to stress/priming events that lead to increased hyperalgesia and deficits in fear extinction26-30. Despite substantial sex differences, genetic/molecular mechanisms responsible for increased CMSP/PTSS vulnerability in women remain poorly understood.

One biological mechanism that may contribute to increased CMSP/PTSS vulnerability in women is altered expression of messenger RNA (mRNA) originating from the X chromosome (XCh). Women have two copies of the XCh (XX), while men have only one copy (XY). In women one copy of each XCh gene is silenced via a process termed XCh inactivation (XCI). Mechanisms important to XCh silencing include epigenetic/chromatin modification and the coating of one XCh by the long non-coding RNA

(lncRNA), XIST31-34. The latter mechanism, XIST RNA coating, requires the YY1 protein to activate the

XIST RNA and tether it to the XCh35. Interestingly, despite such inactivation mechanisms, 10-15% of

XCh genes escape XCh inactivation (XCI) and are bi-allelically expressed36. XCh genes that escape

XCI have been shown to play a pathogenic role in many diseases37-47.

Increasing evidence suggests that altered expression of mRNA originating from the XCh in general, and escape from XCI in particular, may be an important mechanism contributing to sex differences in CMSP/PTSS susceptibility. XIST RNA has been found to be dysregulated in women with

CMSP/PTSS-related major affective disorders37,38, sex choromosome abnormalities contribute to a range of brain-health disorders48, XCI mechanisms have been implicated in nervous system development and function49, and in mice, YY1 expression is associated with inflammatory pain and stress-induced analgesia50.

In this study, we assessed for evidence that expression differences in mRNA originating from the XCh contribute to the increased incidence of CMSP/PTSS in women experiencing MVC. We hypothesized that altered expression of mRNA originating from the XCh would predict the development of co-morbid CMSP/PTSS in such women. In addition, we hypothesized that XCh genes known to escape from XCI would predict CMSP/PTSS development, and play an important role. In addition, we evaluated whether the peritraumatic expression of critical regulators of XCI, XIST and YY1, also predict

CMSP/PTSS outcomes.

Materials/Subjects and Methods

Parent longitudinal cohort study of women and men experiencing MVC

This prospective longitudinal study enrolled African American individuals ≥ 18 and ≤ 65 years of age who presented within 24 hours of MVC to one of eleven emergency departments (EDs) in six states/districts (Michigan, Pennsylvania, Florida, Alabama, Massachusetts, and Washington D.C.) between July 2012 and July 2015. The study only enrolled African Americans because of the pressing need for pain studies that focus on such understudied, high risk groups.51-53 This study has been described in detail previously.54 In brief, individuals who did not have a fracture or other injury requiring hospital admission were screened for eligibility. Patients who were not alert and oriented were excluded, as were patients who did not self-identify as African American, pregnant patients, prisoners, patients unable to read and understand English, or patients taking opioids above a total daily dose of

30 mg of oral morphine or equivalent. The study was approved by the institutional review boards of all participating hospitals. Each participant provided written informed consent before enrollment.

MVC-related pain intensity and distribution in the past week was assessed six months following

MVC using the modified Regional Pain Scale.55 Overall pain intensity was evaluated via a 0 (no pain) to 10 (maximum possible pain) numeric rating scale (NRS).56 MVC-related PTSS was assessed six months following MVC using the Impact of Event Scale: Revised (IESR).57 This 22-item questionnaire assesses symptoms such as physical reactions, dreams, and reminders of the stressful event on a scale of 0-4. The questionnaire also includes assessments for avoidance, intrusion and hyperarousal subscales; total scores can range from 0-88.

Sample selection and outcome definitions

To increase power, samples were not selected from the parent cohort study for analyses at random. Rather, individuals with relatively high levels and relatively low levels of pain and PTSS over time after MVC were selected from the overall cohort for gene expression analyses (more extreme phenotypes). In addition, only parent cohort participants who at the time of the emergency department visit reported little or no pain in the weeks prior to MVC were selected, and only participant samples with an RNA integrity score of 7 or greater were selected for sequncing. This selection occurred intermittently throughout the parent study by a single investigator (SDL), when samples were selected for gene expression analyses, and well prior to planning of these specific analyses.

Individuals with co-morbid CMSP/PTSS were defined based on previously validated cut-offs for moderate-severe pain (NRS ≥ 4) and PTSS (IES-R score ≥ 33).58 Individuals with co-morbid

CMSP/PTSS were compared to controls (individuals reporting neither CMSP nor PTSS). The average

NRS and IES-R scores of women who developed co-morbid CMSP/PTSS was 7.98 (SD 1.71) and

59.33 (SD 15.79), respectively, while the NRS and IES-R scores of women who recovered was 0.71

(SD 1.10) and 8.51 (SD 8.51), respectively.

RNA collection and isolation from MVC study participants

Research assistants collected blood samples in the ED at the time of enrollment using PAXgene

RNA tubes. Total RNA was isolated using the PAXgene blood miRNA kit (QIAGEN) and RNA was stored at -80˚C until use. RNA concentration and purity were measured using a NanoDrop One

(Nanodrop Technologies, Wilmington, DE), and RNA integrity was measured using an Agilent 2100

Bioanalyzer (Agilent Technologies, Santa Clara, CA).

Next Generation Sequencing and RNA read count normalization

Template libraries for total RNA sequencing were produced from 600ng total RNA using Ovation

Human Blood RNA-Seq Library Systems kit (NuGen, San Carlos, CA) according to manufacturer’s specifications. Libraries were multiplexed in groups of six and sequenced on a HiSeq 2500 at the

University of North Carolina at Chapel Hill High Throughput Sequencing Facility. Raw sequencing reads were aligned to the human hg19 genome assembly using STAR (version 2.4.2a).59 Expression levels of each transcript (n=20,353) were estimated via RSEM60 using University of California Santa Cruz

(UCSC) known gene transcript and gene definitions. Raw RSEM read counts for all samples were normalized to the overall upper quartile61 before comparison and visualization. Consistent with study goals, only non-pseudoautosomal region (non-PAR) messenger RNA (mRNA) originating from the X chromosome (n=783) with mean read counts ≥ 10 (n=724) were included in analyses. As a quality control measure and verification of appropriate male/female categorization based on genetic attributes

(vs. self-reported gender), median XIST RNA read counts were compared between women and men in this cohort: median XIST RNA read counts were 99,806 in women and 339 in men.

Statistical analyses

Sample sociodemographic characteristics were summarized using standard descriptive statistics. Logistic regression analyses adjusted for ED study site, education level, and participant age were used to assess the relationship between XCh gene expression levels and co-morbid CMSP/PTSS

6 months following MVC, and to derive β coefficients, Z-score, standard error and p-value significance.

RNA expression levels were scaled using the ‘Scale’ function in R. For this exploratory study, a p-value significance threshold for each of the different hypotheses assessed was set at p < 0.05. Gene expression values meeting this threshold were used to generate a heatmap comparing relative expression levels of significant genes in women who develop CMSP/PTSS vs recovery following MVC.

To determine whether there was a statistically significant difference in proportions of genes from two different groups, a z score and p value were calculated by comparing the proportions between the two groups using a two-tailed significance test.

Competitive gene set analyses (CGSA) were performed using the limma package in

R/Bioconductor, version 3.34.962. The Camera function was used to test whether specified sets of genes (e.g. EscapeSet) were more highly ranked in an ordered list of genes than would be expected by chance. While some CGSA methods (e.g., WilcoxGST) assume that each gene is conditionally independent of other genes, Camera adjusts for inter-gene correlation and is thus considered a more rigorous analysis for large sets of related genes63.

The relationship between XIST RNA expression levels and YY1 mRNA expression levels with

CMSP or PTSS was assessed using bivariate analyses (Pearson Correlation) in stratified outcome groups (women who developed co-morbid CMSP/PTSS vs those who recovered). All statistical analyses were carried out using SPSS software v24.0, SAS software v9.4, or R Studio v3.3.3.

Pathway analyses

The Ingenuity Pathway Analysis (IPA, Qiagen, 2018) software was used to assess functional relationships between XCh genes that predicted CMSP/PTSS, XCh genes known to escape XCI that predicted CMSP/PTSS (“EscapeSet”, n=9) and XChSet (n=31) separately. A core expression anlysis was done to assess the similar functionalities between genes in the context of curated Canonical pathways in the IPA library. Functional relationships identified using IPA were validated across three other freely-available pathway analysis tools available online: DAVID64,65, PANTHER66, and

GATHER67.

Results

Study participants / Cohort

Study analyses were performed on a subsample of participants from a parent prospective observational study of African American (AA) women and men who presented to the ED for evaluation after MVC. This subsample was comprised of participants who were selected to have gene expression analyses performed on RNA blood samples obtained from them in the ED. Participants who reported little or no pain in the weeks prior to MVC and who had more extreme outcome phenotypes (little or no symptoms, or more substantial persistent symptoms) were selected for gene expression analyses.

Samples with an RNA integrity score of 7 or greater were used in the present analyses. This method removed those with prior pain and enriched the study subsample for the outcome of interest (i.e., 25% of parent cohort (n=930) had co-morbid CMSP/PTSS six months following MVC, vs. 51% of the study subsample (n = 101)). The majority of these study subsample participants were between twenty and forty years of age and had not completed college; a little more than 60% were female (Table 1). The majority were drivers, and more than half reported severe damage to their vehicle (Table 1).

Peritraumatic pain and distress were common in the cohort, and consistent with selection critieria, pain symptoms in the weeks prior to MVC were mild (Table 1).

Associations between altered peritraumatic expression of XCh mRNA and CMSP/PTSS outcomes in women vs. men

In logistic regression analyses adjusted for participant age, education level, and enrollment study site, forty mRNA originating from the XCh were differentially expressed in the early aftermath of MVC among women who developed CMSP/PTSS (n=30) vs those who recovered (n=35) (p < 0.05; Figure

1 and Table 2). Differences in expression of these RNA transcripts ranged from -4 to 4 fold at the individual level (Figure 1) and 0.64 to 1.58 fold at the group level (Table 2). These mRNAs clustered into two main groups, one group showing predominately increased expression of differentially expressed XCh mRNA in women who developed CMSP/PTSS (n=23 mRNA) and another group with predominately decreased expression in women who developed CMSP/PTSS (n=17 mRNA) (Figure 1). These RNA transcripts have been identified as having a variety of broad ranging functions such as regulation of transcription/translation (e.g. EIF1AX, EIF2S3), immune cell function (e.g. CD40LG, TLR8,

SH2D1A), and cell communication/signaling (e.g. GPR174, GPRASP1) (Supplementary Table 1). In parallel logistic regression analyses conducted in men who developed CMSP/PTSS (n=21) vs those who recovered (n=15), twenty-five mRNA were differentially expressed; these mRNAs did not cluster into well-defined groups as they did in women (Supplementary Figure 1). Differences in expression of these RNA transcripts ranged from 0.58 to 15.71 fold at the group level (Supplementary Table 2).

Similar analyses of CMSP/PTSS-associated XCh genes in men indicated that differentially expressed

XCh mRNA were nearly entirely distinct from those identified in women (only RBBP7 and SEPT6 that were associated with CMSP/PTSS in both sexes).

In women, but not in men, XCh gene transcripts that predict post-MVC co-morbid CMSP/PTSS are enriched in gene transcripts that have been shown previously to escape XCI

In women, 11/40 (28%) of XCh mRNA differentially expressed in those who subsequently developed CMSP/PTSS have been shown in previous literature to escape XCI68-71 (Figure 2). When compared to the total number of XCh genes shown in previous literature to escape XCI71 (93/783, 12%,

Supplementary Table 3), the percentage of escape genes predicting CMSP/PTSS in women was much higher than would be expected due to chance (z = -2.9, p = 0.004, Figure 2A). In contrast, in men the twenty-five XCh mRNA differentially expressed in those who did vs. did not develop

CMSP/PTSS were not enriched for genes that escape XCI (3/25, 12%; z = -0.0187, p = 0.984).

The great majority of XCh gene transcripts found in previous literature to escape XCI and that were differentially expressed in the early aftermath of MVC among women developing CMSP/PTSS were differentially expressed in a direction consistent with escape from XCI

Consistent with the direction of effect that would be expected of escape gene transcripts, nine out of eleven XCh gene transcripts found in previous literature to escape XCI and that were differentially expressed in the early aftermath of MVC among women developing CMSP/PTSS were expressed at higher levels in women who developed CMSP/PTSS vs those who recovered. This set of nine genes was the focus of the following analyses and are herein referred to as “EscapeSet” genes. Consistent with the chromosomal location of most genes that escape XCI72, the majority of EscapeSet transcripts originate from the distal end of the short arm of the XCh (Figure 2B).

EscapeSet gene transcripts demonstrated greater differential expression in women who developed CMSP/PTSS than both other differentially expressed XCh genes and other sets of genes

In competitive gene set analyses (CGSA) that further account for inter-gene correlation (Camera,

Bioconductor/R), EscapeSet genes were more differentially expressed (in the direction of increased expression) in women who developed CMSP/PTSS vs those who recovered than the other 31 differentially expressed XCh genes (the “XChSet”, p=0.004, Figure 2C). The EscapeSet was also more differentially expressed in women who developed CMSP/PTSS vs recovered in comparison to other randomly selected sets of nine genes from the XCh (Direction: up-regulated, p= 8.81x10-15, Figure 2C) and when compared to any other set of nine genes from the full transcriptome (Directoin: up-regulated, p =4.75x10-12, Figure 2C).

EscapeSet pathway analyses indicate an enrichment for genes involved in protein translation

Shared relationships between EscapeSet genes were evaluated via pathway analysis (IPA software). An enrichment of the Eukaryotic Initiation Factor 2 (EIF2) Signaling/Protein Biosynthesis

Pathway was identified (Canonical pathway enrichment function, p=9.9x10-5), a pathway which regulates translation in response to a range of stress-related signals73. Enrichment of this pathway was mainly driven by differential expression of escape genes EIF1AX, EIF2S3, and RPS4X, which have been shown to play a role in the regulation of transcription and translation. Additionally, using the

Network analysis function in IPA, we found that EscapeSet genes are all connected to IL2 regulation/function/signaling (Figure 2D). The association between EscapeSet genes and translational regulation was confirmed with additional pathway analysis software including GATHER (enrichment in protein biosynthesis (GO:0006412, Bayes Factor = 3, p value = 0.002)), PANTHER (translation initiation” (GO:0003743, >100 fold enrichment, p = 1.65x10-4)), and DAVID (enrichment in translational initiation (GO:0006413, p value = 1.8x10-3)).

Expression levels of the non-coding RNA XIST and the transcription factor YY1 were differentially associated with each other and with CMSP/PTSS outcomes when comparing women who developed CMSP/PTSS vs women who recovered following MVC

Based on our results showing that women who develop CMSP/PTSS following MVC over- express more genes that escape XCI than expected based on chance, we hypothesized that these at- risk women would also exhibit further evidence of altered XCI. Therefore, we examined the relationship between the expression of two major regulators of XCI, XIST and YY1, and CMSP or PTSS outcomes in women who recovered and in women who developed co-morbid CMSP/PTSS following MVC. We first assessed a relationship between XIST RNA expression and PTSS severity and found that in women who recovered there was no statistically significant relationship (r = -0.113, p = 0.520, Figure

3A). However, in women who developed co-morbid CMSP/PTSS, we found a negative relationship between XIST RNA and PTSS severity, with trend level statistical significance (r = -0.294, p = 0.114,

Figure 3A). Similar (though non-significant) relationships were observed when assessing the correlation between XIST RNA expression levels and CMSP severity in women (Supplementary

Figure 2) but no such relationships were observed in men (Supplementary Figure 3A).

We next examined whether there was a relationship between YY1 mRNA expression and PTSS and found that in women who recovered, there was no statistically significant relationship (r = 0.187, p

= 0.281, Figure 3A). However, in women who developed co-morbid CMSP/PTSS, we found a positive relationship between YY1 mRNA expression and PTSS severity, with trend level statistical significance

(r = 0.351, p = 0.057, Figure 3A). The relationship between YY1 mRNA expression and CMSP severity in women was not as pronounced as was observed for PTSS severity (Supplementary Figure 2) and no statistically significant relationships were observed in men (Supplementary Figure 4).

To further extend these analyses, we examined whether there were different relationships between XIST RNA and YY1 RNA expression in women who recovered vs women who developed co- morbid CMSP/PTSS following MVC. In women who recovered, we detected a negative correlation with trend level statistical significance (r = -0.315, p = 0.065, Figure 3B). In comparison, in women who developed co-morbid CMSP/PTSS, we detected a strong and highly significant negative correlation between these RNA molecules (r = -0.582, p = 0.0007, Figure 3B). In contrast, in men, we detected positive correlations between XIST and YY1 RNA expression levels in both outcome groups

(Supplementary Figure 3B).

Discussion

In the current study, we found that forty genes originating from the XCh were differentially expressed in the early aftermath of MVC among women who subsequently developed CMSP/PTSS vs those who recovered, and twenty five XCh genes were differentially expressed in men. Differentially expressed XCh genes in women demonstrated greater fold change than men, were nearly entirely distinct from men, clustered into two well-defined groups, and unlike men were greatly enriched for genes previously shown to escape XCI. In addition, the expression of XCh genes known to escape XCI appeared to be particularly dysregulated, as this subset of nine genes exhibited greater differential expression than any other nine gene set from the XCh or the full transcriptome. Importantly, consistent with potential escape from XCI, in women who developed CMSP/PTSS these genes exhibited increased expression. These results, together with data indicating that women who developed

CMSP/PTSS following MVC exhibited altered relationships between XIST and YY1 RNA expression levels, provide evidence that support the hypothesis that genes that escape XCI might contribute to the increased vulnerability to CMSP/PTSS in women experiencing MVC.

Pathway analyses to look for clues regarding mechanisms through which differentially expressed

XCh genes that escape XCI might contribute to increased CMSP/PTSS vulnerability in women implicated EIF2 signaling. The EIF2 pathway has been shown to be involved in the cellular response to diverse stressors73-75. More recently, the EIF2 pathway in neurons has also been shown to be involved in processes related to learning and neuroplasticity76-78; processes central to the alterations in central and peripheral nervous system function that mediate the development of CMSP and PTSS.

Pathway analyses also indicated that differentially expressed XCh genes that escape XCI influence IL-

2 signaling/function. IL-2 has been implicated in the pathogenesis of both chronic pain79,80 and PTSS81-

84. Further studies evaluating the potential contribution of EIF2 and IL-2 signaling to sex differences in pain and PTSS outcomes after MVC and other stressors are warrented.

Our results demonstrating a negative correlation between XIST RNA expression levels and

PTSS or CMSP severity in women who developed co-morbid CMSP/PTSS was interesting in light of our consistent results showing that genes known to escape from XCI predict CMSP/PTSS development in women following MVC. These data suggest that low levels of XIST RNA expression (and thus potentially less coating of the inactive XCh) may allow certain XCh RNA to escape XCI in women who go on to develop CMSP/PTSS. Of note, in addition to the amount of XIST RNA available to silence the

XCh, available evidence suggest that regulation of intracellular location of XIST RNA may also be important. For instance, in an elegant study by Wang et al, women with systemic lupus erythematosus

(SLE) exhibited dispersed localization of XIST in mature naïve lymphocytes, leading to gene escape from XCI in these cells85. This finding is of interest because our data our potentially consistent with the mechanism, and like CMSP/PTSS, SLE is a disorder that disproportionately affects women. Wang et al. also examined expression of the YY1 protein and its ability to recruit XIST to the inactive XCh, and showed that YY1 was important for XIST localization. Interestingly, we found that the relationship between YY1 and PTSS severity (but not CMSP severity) and the relationship between YY1 and XIST is more pronounced in women who developed CMSP/PTSS, indicating that the amount and the relationship between these RNA molecules might contribute to gene escape from XCI in these women.

However, these results should be interpreted with caution as they are only correlative, and further mechanistic studies are needed.

The strengths of this study included our ability to obtain nested samples from a prospective study of trauma survivors with high follow-up rates, our ability to increase power via selection of more extreme phenotypes (reduced type II error), the quality of the RNA data used in the study, and our ability to look for evidence that genes that escape XCI contribute to sex differences in vulnerability to post-MVC

CMSP/PTSS using multiple complementary approaches in both women and men (i.e., gene expression analyses, pathway analyses, XIST and YY1 RNA association analyses). However, several limitations should also be considered when interpreting our results. First, the main findings of the study have not been replicated in a second cohort of post-trauma survivors. This is an essential next step for future studies. Second, this study did not adjust for multiple hypothesis testing. This has the disadvantage of producing false positive associations (i.e. Type I error), but is optimal given the exploratory nature of the study and our relatively small sample size. Third, this work was performed in only African American individuals. Therefore, whether there are also ethnic differences in the association between XCh genes and CMSP/PTSS is not known. Fourth, we relied on previous studies for the identification of escape genes and did not assess escape status in our cohort. However, most of the escape genes identified as predictors of CMSP/PTSS have been shown across a number of studies to escape XCI71. Finally, we have not performed mechanistic studies to more fully explicate relationships between XIST and YY1

RNA expression levels in women who develop CMSP/PTSS vs recover. Further studies are needed to replicate these findings and, if present, to better understand the underlying mechanisms driving these differences.

In conclusion, we report the first evidence suggesting that differential expression of genes originating from the X chromosome, including genes that escape X chromosome inactivation, may contribute to the increased vulnerability to co-morbid CMSP/PTSS in women vs. men experiencing

MVC. The expression of expression of a set of XCh genes known to escape XCI appeared to be particularly dysregulated/influential; pathway analyses indicate that the affect of these genes may include influencing the EIF2 pathwway, which is involved in neuronal neuroplasticity. Further suggesting a role in CMSP/PTSS pathogenesis of X chromosome genes that escape inactivation, women who developed CMSP/PTSS following MVC exhibited altered relationships between XIST and

YY1 RNA expression levels. Future studies are needed to further understand the potential role of X chromosome gene expression in general, and X chromosome genes that escape X chromosome inactivation in particular, in mediating sex differences in vulnerability to CMSP/PTSS after MVC.

Acknowledgements

First and foremost, I would like to thank Dr. Sarah Linnstaedt, who has supported me throughout my thesis with her unconditional patience and knowledge. I am especially grateful for the other members of the Linnstadt Lab: Connie Chen and Yue Pan, without them, this thesis would not have been possible.

Funding for this study was provided by the National Institute of Arthritis, Musculoskeletal, and

Skin Diseases (R01AR060852, PI: McLean), by the Mayday Fund (PIs: McLean and Linnstaedt), by a

Future Leaders in Pain Grant from The American Pain Society (PI: Linnstaedt), and by the Tom and

Elizabeth Long Excellence Fund for Honors administered by Honors Carolina.

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Table 1. Participant characteristics Participants, n 101 Female, n (%) 65 (64.4) Age, years, mean (SD) 31.4 (10.8) Education, n (%) HS or less 44 (43.6) Post-HS not college 4 (4) Some college 34 (33.7) College 15 (14.9) Post-college 4 (4) Collision characteristics Driver, n (%) 72 (71.3) Severe vehicle damage, n (%) 58 (57.4) Seatbelt worn, n (%) 86 (85.1) Airbag deployed, n (%) 38 (37.6) Pain level prior to MVC (0-10 NRS), mean (SD) 1.0 (2.2) Overall pain in the ED (0-10 NRS), mean (SD) 6.9 (2.3) Peritraumatic distress level*, mean (SD) 24.8 (12.2) *distress measured with the peritraumatic distress inventory

Figure 1. Heatmap illustrating X chromosome genes that are differentially expressed in women who recover vs develop co-morbid CMSP/PTSS six months following motor vehicle collision (MVC) (n=65). Each column represents a female participant from the study, with individuals who recovered on the left (light gray bar) and individuals who developed CMSP/PTSS on the right (dark gray bar). Each row represents an X chromosome mRNA (with the corresponding official gene symbol to the right) that significantly predicts CMSP/PTSS development following MVC. Bolded mRNA represent genes that have been shown previously to escape X chromosome inactivation. The dendrogram on the left of the heatmap indicates the hierarchical clustering of X chromosome genes associated with CMSP/PTSS. CMSP = chronic musculoskeletal pain, PTSS = posttraumatic stress symptoms.

Table 2. X Chromosome mRNA that predict co-morbid CMSP/PTSS in women six months following motor vehicle collision (n=65).

a β Std. Z- p RNA Fold Difference b Value Error score Value SH2D1A 1.58 (655.1/413.7) 1.55 0.49 3.15 0.002

RPS4X 1.29 (17512.3/13585.3) 1.28 0.41 3.15 0.002 ELF4 0.82 (4319.1/5236.8) -1.39 0.46 -3.04 0.002 TCEAL8 1.47 (217.9/147.7) 1.06 0.38 2.75 0.006 SEPT6 1.18 (12224.9/10333.6) 1.02 0.38 2.72 0.007 GPR174 1.35 (533.6/394.3) 1.13 0.44 2.59 0.010

SLC38A5 0.64 (3491.9/5445) -1.03 0.40 -2.57 0.010 PRPS1 1.18 (554.8/468.2) 0.91 0.35 2.57 0.010 PRKX 1.14 (2237.1/1954.2) 0.99 0.39 2.56 0.010 TFE3 0.85 (3400.6/4004.5) -0.91 0.36 -2.52 0.012 MSN 0.83 (24891.8/30062.4) -0.99 0.40 -2.52 0.012 WAS 0.8 (9341.2/11624.8) -1.02 0.41 -2.50 0.012 RPL10 1.27 (22451.2/17620) 0.99 0.40 2.49 0.013 BEX4 1.33 (304.2/228.9) 0.83 0.34 2.46 0.014

TAF9B 1.19 (615.4/518.2) 0.85 0.35 2.45 0.014 RBMX 1.16 (4245.2/3666) 1.05 0.43 2.44 0.015 CA5B 1.18 (1412.8/1196.6) 0.95 0.39 2.42 0.016 GPRASP1 1.25 (943.2/755.2) 0.83 0.35 2.35 0.019 REPS2 0.76 (1989.7/2607.2) -0.75 0.33 -2.31 0.021 CD40LG 1.15 (1303.5/1132) 0.80 0.35 2.26 0.024 SPIN2A 0.69 (44.2/64.4) -0.78 0.35 -2.22 0.027 BEX2 1.49 (120.5/80.6) 0.82 0.37 2.21 0.027

MPP1 0.79 (7647.5/9736.2) -0.83 0.38 -2.20 0.028 EIF1AX 1.3 (1458.3/1119.9) 0.88 0.40 2.20 0.028 TLR8 0.78 (6849.1/8826.4) -0.74 0.34 -2.19 0.028 VSIG1 1.18 (477.8/403.9) 0.71 0.32 2.19 0.029 VMA21 1.19 (823.4/694.8) 0.74 0.34 2.18 0.029

OTUD5 0.91 (2165.2/2378.1) -0.72 0.33 -2.15 0.032 GPR34 1.28 (240.1/187.2) 0.69 0.32 2.14 0.033 G6PD 0.85 (3445.5/4054.8) -0.80 0.38 -2.13 0.033 WDR13 0.89 (803.1/899.7) -0.64 0.30 -2.12 0.034 SAT1 0.79 (2970.5/3756.3) -0.73 0.35 -2.11 0.035 RBBP7 1.18 (882.8/745.3) 0.74 0.36 2.09 0.036 SCML1 1.2 (273.1/227) 0.69 0.33 2.09 0.037 ITM2A 1.32 (1274/966) 0.90 0.44 2.06 0.039 UBA1 0.91 (5976.2/6559.7) -0.78 0.38 -2.03 0.042 WWC3 0.87 (3304.1/3805.4) -0.64 0.32 -2.00 0.045 EIF2S3 1.11 (5000.1/4511.8) 0.69 0.34 2.00 0.045 CFP 0.84 (4061.4/4819.9) -0.69 0.34 -1.99 0.046 NKRF 1.17 (554.5/472.7) 0.64 0.33 1.98 0.048 aFold difference was calculated by dividing the average sequencing read counts for women who developed co-morbid CMSP/PTSS by the average sequencing read counts for women who recovered following MVC. bp values were calculated using logistic regression analyses adjusted for participant age, enrollment study site, and education level. Bolded mRNA have been defined previously as genes that escape X chromosome inactivation.

A Escape Genes B Escape Genes

X chromosome Genes CMSP/PTSS Associated Genes

Difference in proportions, z-score = -2.9, p = 0.004

C Test group: CMSP/PTSS associated “EscapeSet” Comparison group: Directiond P value CMSP/PTSS XCISeta Up 0.0038 Other XCh genesb Up 8.81E-15 Other genesc Up 4.75E-12

D EIF2 Signaling

Figure 2. In women, gene transcripts previously shown to escape X chromosome inactivation (XCI) are important predictors of co-morbid CMSP/PTSS. A) Escape genes are over-represented in the list of 40 X chromosome (XCh) genes that significantly predict co-morbid CMSP/PTSS in women following motor vehicle collision. The pie chart on the left represents the proportion of all XCh genes that have been shown to escape XCI (n=93, yellow wedge) out of the total number of non-PAR XCh genes (n=783); the pie chart on the right represents the proportion of genes that have been shown to escape XCI (n=11) out of the total number of XCh genes that predict CMSP/PTSS in women following MVC. B) Schematic representation of the location of “EscapeSet” genes on the X chromosome. C) Results of competitive gene set analyses indicate that EscapeSet genes are more differentially expressed in women who develop co-morbid CMSP/PTSS vs recover following MVC than aXChSet genes, bany other set of nine XCh genes, and cany other set of nine randomly selected genes from the full transcriptome. dDirection of expression of genes in women who developed co-morbid CMSP/PTSS relative to expression of the same genes in women who recover following MVC (i.e. “Up” indicates the genes are higher expressed in women with co-morbid CMSP/PTSS). D) Network analysis results based on EscapeSet genes using Ingenuity design software (IPA, Qiagen).

4 0 1 0 0

r = - 0 . 1 1 3 8 0 3 0 p = 0 . 5 2 0

6 0

2 0 4 0 1 0 2 0 r = - 0 . 2 9 4 p = 0 . 1 1 4

0 0 5 0 0 0 0 1 0 0 0 0 0 1 5 0 0 0 0 2 0 0 0 0 0 1 0 0 0 0 0 1 5 0 0 0 0 XIST RNA expression levels

1 0 0 3 0 r = 0 . 1 8 7 p = 0 . 2 8 1 8 0 2 0 6 0 1 0 Posttraumatic StressSeverity Symptom Posttraumatic 4 0 r = 0 . 3 5 1 p = 0 . 0 5 7 0 2 0 1 0 0 0 2 0 0 0 3 0 0 0 1 0 0 0 2 0 0 0 3 0 0 0 YY1 RNA expression levels

2 0 0 0 0 0

2 0 0 0 0 0 r = -0 .3 1 5 1 5 0 0 0 0 p = 0 .0 6 5 1 5 0 0 0 0

1 0 0 0 0 0 1 0 0 0 0 0

RNA expressionRNA 5 0 0 0 0 5 0 0 0 0 r = - 0 . 5 8 2 p = 0 . 0 0 0 7

XIST 0 0 1 0 0 0 2 0 0 0 3 0 0 0 1 5 0 0 2 0 0 0 2 5 0 0 3 0 0 0 3 5 0 0 YY1 RNA expression levels

Figure 3. Relationship between RNA expression levels of key regulators of X Chromosome inactivation, XIST and YY1, and posttraumatic stress symptom (PTSS) severity scores in women following MVC trauma. A) XIST RNA expression levels were examined in relation to PTSS severity scores in women who recovered following MVC (top left, purple triangles, n=35), and in women who developed co-morbid CMSP/PTSS following MVC (top right, teal circles, n=30). YY1 RNA expression levels were also analyzed in relation to PTSS severity scores in women who recovered following MVC (bottom left, purple triangles, n=35), and in women who developed co- morbid CMSP/PTSS following MVC (bottom right, teal circles, n=30). B) The relationship between XIST RNA expression levels and YY1 RNA expression levels in women who recovered following MVC (left, green squares, n=35) and in women who developed co-morbid CMSP/PTSS following MVC (right, yellow diamonds, n=30). RNA expression levels were measured via RNA sequencing of blood collected in the early aftermath of MVC. Bivariate analyses were used to derive Pearson correlation coefficients and corresponding p values.

Supplementary Table 1. Putative gene functions for XCh mRNA identified as predictors of co-morbid CMSP/PTSS in women following motor vehicle collision trauma. mRNA Official Gene Name Gene Function SH2D1A SH2 domain containing 1A stimulation of the immune system (T and B cells) RPS4X ribosomal protein S4, X-linked transcriptional regulation ELF4 E74 like ETS transcription factor 4 transcriptional activator, Immune system TCEAL8 transcription elongation factor A like 8 transcriptional regulation SEPT6 septin 6 GTPase required for cytokinesis

GPR174 GPCR with unidentified ligand (similar family members G protein-coupled receptor 174 activate neurotransmitters, hormones) SLC38A5 solute carrier family 38 member 5 amino acid transporter PRPS1 phosphoribosyl pyrophosphate synthetase 1 enzyme involved in nucleotide synthesis serine threonine protein kinase involved in immune cell PRKX maturation. Also serotonin, adrenergic, dopaminergic, protein kinase, X-linked GABA, and endogenous opioid pathways. TFE3 transcription factor binding to IGHM enhancer 3 transcription factor for genes downstream of TGF-beta MSN moesin functions in cell-cell recognition and cell movement transduction of signals from cell surface to actin WAS cytoskeleton. Involved in immune regulation and Wiskott-Aldrich syndrome microthrombocytopenia RPL10 ribosomal protein L10 transcriptional regulation BEX4 brain expressed X-linked 4 regulation of transcription TAF9B TATA-box binding protein associated factor 9 transcriptional regulation RNA binding protein involved in the regulation of RBMX RNA binding motif protein, X-linked transcription and translation mitochondrial carbonic anhydrase involved in wide range CA5B carbonic anhydrase 5B of biological activities GPCR that interacts with DRD2, OPRD1, ADRB2, DRD4, GPRASP1 G protein-coupled receptor associated sorting protein 1 CB1R regulates endocytosis and down regulates growth factor REPS2 RALBP1 associated Eps domain containing 2 receptors CD40LG CD40 ligand ligand on T cells that regulates B cell function involved in regulation of cell cycle progression and exhibits SPIN2A spindlin family member 2A H3K4me3-binding activity BEX2 brain expressed X-linked 2 transcriptional regulation regulates cell proliferation, signaling pathways, intercellular MPP1 membrane palmitoylated protein 1 junctions EIF1AX eukaryotic translation initiation factor 1A translation initiation TLR8 toll like receptor 8 immune system function VSIG1 V-set and immunoglobulin domain containing 1 member of the junctional adhesion molecule VMA21 vacuolar ATPase assembly factor required for assembly of lysosomal vacuolar ATPase suppressor of type I interferon-dependent innate immune OTUD5 OTU deubiquitinase 5 response GPR34 G protein-coupled receptor 34 GPCR involved in the immune response G6PD glucose-6-phosphate dehydrogenase enzyme involved in NADPH production involved in signal transduction, apoptosis, cell cycle WDR13 WD repeat domain 13 progression, gene regulation SAT1 spermidine/spermine N1-acetyltransferase 1 enzyme involved in polyamine metabolism RBBP7 RB binding protein 7, chromatin remodeling factor cell proliferation and differentiation helps maintain transcriptionally repressive state of SCML1 Scm polycomb group protein like 1 homeotic genes throughout development ITM2A integral membrane protein 2A potential role in osteo- and chondrogenic differentiation UBA1 ubiquitin like modifier activating enzyme 1 DNA repair WWC3 WWC family member 3 kinase binding and protein complex scaffold

EIF2S3 eukaryotic translation initiation factor 2 subunit gamma translation initiation positively regulates the alternative complement pathway of CFP complement factor properdin the innate immune system NKRF NFKB repressing factor transcriptional repression of NFKB responsive genes

Supplementary Figure 1. Heatmap illustrating X chromosome genes that are differentially expressed in men who recover vs develop co-morbid CMSP/PTSS six months following motor vehicle collision (MVC) (n=36). Each column represents a male participant from the study, with individuals who recovered on the left (light gray bar) and individuals who developed CMSP/PTSS on the right (dark gray bar). Each row represents an X chromosome mRNA (with the corresponding official gene symbol to the right) that significantly predicts CMSP/PTSS development following MVC. Bolded mRNA represents genes that have been shown previously to escape X chromosome inactivation. The dendrogram on the left of the heatmap indicates the hierarchical clustering of X chromosome genes associated with CMSP/PTSS. CMSP = chronic musculoskeletal pain, PTSS = posttraumatic stress symptoms.

Supplementary Table 2. XCh mRNA that predict co-morbid CMSP/PTSS in men six months following motor vehicle collision (n=36).

a β Std. Z- p RNA Fold Difference Value Error score Valueb TSR2 1.5 (349.9/233.8) 1.59 0.59 2.70 0.007 USP11 1.25 (870.1/694.1) 1.92 0.71 2.69 0.007 RNF113A 1.32(408.4/308.7) 1.28 0.50 2.55 0.011 SMC1A 1.32 (3157.6/2386.2) 1.83 0.74 2.48 0.013 NONO 1.18 (5181.3/4384.3) 1.56 0.65 2.40 0.017 FLNA 1.33 (31586.2/23673.7) 1.13 0.49 2.30 0.021 UPF3B 1.54 (409.5/266.3) 1.33 0.58 2.29 0.022 SEPT6 1.22 (8523.2/7007.8) 1.30 0.61 2.15 0.032 TNMD 3.16 (46.2/14.6) 1.81 0.85 2.13 0.033 IL2RG 1.29 (9020.7/6979.2) 1.79 0.84 2.12 0.034 RBMX2 1.32 (267.1/202.3) 1.07 0.50 2.12 0.034 ZRSR2 1.38 (518.9/374.7) 0.99 0.47 2.09 0.036 FMR1 1.29 (1418.9/1098.2) 0.92 0.44 2.09 0.037 HTATSF1 1.43 (589.5/413.3) 1.00 0.48 2.08 0.038 ARHGEF6 1.2 (4523.6/3757.8) 1.05 0.51 2.07 0.038 TREX2 0.59 (29.6/50.3) -0.99 0.48 -2.07 0.038 P2RY4 0.58 (33/56.8) -0.91 0.44 -2.06 0.04 CXorf58 1.86 (33.9/18.2) 1.50 0.73 2.06 0.04 BCOR 1.22 (2273/1857.9) 1.08 0.53 2.05 0.04 DIAPH2 1.23 (1392.9/1128.9) 0.92 0.45 2.04 0.041 HUWE1 1.23 (8522.9/6922.1) 1.07 0.53 2.04 0.042 RBBP7 1.28 (774.3/603.8) 1.00 0.50 2.02 0.043 HDAC6 1.2 (1060.4/886.2) 1.01 0.50 2.02 0.044 ARMCX3 1.23 (401.8/325.4) 0.90 0.45 1.99 0.047 GAGE1 15.71 (11/0.7) 3.02 1.52 1.98 0.048 aFold difference was calculated by dividing the average sequencing read counts for men who developed co-morbid CMSP/PTSS by the average sequencing read counts for men who recovered following MVC. bp values were calculated using logistic regression analyses adjusted for participant age, enrollment study site, and education level. Bolded mRNA have been defined previously as genes that escape X chromosome inactivation.

Supplementary Table 3. Genes known to escape X chromosome inactivation as described by Carolyn Brown and colleagues (see manuscript for associated reference). Mostly Mostly Variable Escapea Escapeb Variable Escapec Escape d ARSH GYG2 ASB11 KIAA1210 ARSE PRKX ARSD BEND2 SH2D1A SHROOM2 STS MXRA5 PPEF1 FRMD7 GRPR

PNPLA4 NLGN4X PHEX MGC1612184848 NHS FAM9C KAL13730 CXorf21 PLAC1 PABPC5 ACE2 TCEANC MED14 BRS3 COL4A6 ZRSR2 RAB9A TIMP1 CD40LG PLS3 S100G TRAPPC2 WAS MAGEA11 NAA10 SYAP1 OFD1 DGAT2L6 MAGEA8 HCFC1 TXLNG GPM6B FOXO4 TMEM187 EIF1AX GEMIN8 CXorf65 EIF2S3 CA5B MED149282 ZFX AP1S2 TSIX VENTXP1 CTPS2 COX7B

CXorf38 RBBP7 P2RY10 DDX3X USP9X GPR174 FUNDC1 KDM6A POF1B UBA1 CDK16 SRPX2 INE1 KDM5C NXF3 TAF7L IQSEC2 WBP551186 NXF5 SMC1A GLRA4 ZCCHC16340595 RPS4X GUCY2F GPR112139378 NAP1L3 RGAG157529

VGLL1 HTR2C ALG13

L1CAM TRPC5 agene has been shown to escape from X Chromosome inactivation (XCI) as identified in a wide range of studies. bgene has been shown to escape from XCI in the majority of studies (2 of 3 or 3 of 4 reviewed studies). cgene has been shown in a wide range of studies to variably escape from XCI in some individuals or tissues. dgene has been shown in the majority of studies to variably escape from XCI in some individuals or tissues.

4 1 5 r = 0 . 0 7 5 r = - 0 . 1 0 5 p = 0 . 6 6 9 p = 0 . 5 8 0 3 1 0

5 1

0 0 5 0 0 0 0 1 0 0 0 0 0 1 5 0 0 0 0 2 0 0 0 0 0 5 0 0 0 0 1 0 0 0 0 0 1 5 0 0 0 0 2 0 0 0 0 0 XIST RNA expression levels

4 1 5 r = 0 . 1 4 3 r = 0 . 2 3 4 p = 0 . 4 5 3 3 p = 0 . 1 7 7

1 0

Chronic Musculoskeletal PainSeverity Musculoskeletal Chronic 5 1

0 0 1 0 0 0 2 0 0 0 3 0 0 0 1 5 0 0 2 0 0 0 2 5 0 0 3 0 0 0 3 5 0 0

YY1 RNA expression levels

Supplementary Figure 2. Relationship between RNA expression levels of key regulators of X Chromosome inactivation, XIST and YY1, and chronic musculoskeletal pain (CMSP) severity scores in women following MVC trauma. A) XIST RNA expression levels were examined in relation to CMSP severity scores in women who recovered following MVC (top left, purple triangles, n=35), and in women who developed co-morbid CMSP/PTSS following MVC (top right, teal circles, n=30). YY1 RNA expression levels were also analyzed in relation to CMSP severity scores in women who recovered following MVC (bottom left, purple triangles, n=35), and in women who developed co-morbid CMSP/PTSS following MVC (bottom right, teal circles, n=30).

2 0 1 0 0

1 5 8 0

r = 0 . 0 3 2 6 0 p = 0 . 9 1 0 1 0 4 0 5 2 0 r = 0 . 2 5 0 p = 0 . 2 7 5 0 0 0 5 0 0 1 0 0 0 1 5 0 0 0 5 0 0 1 0 0 0 1 5 0 0 2 0 0 0 XIST RNA expression levels

2 0 1 0 0

8 0 1 5 6 0 1 0

StressSeverity Symptom Posttraumatic 4 0 r = 0 . 2 9 6 5 p = 0 . 2 8 4 2 0 r = 0 . 1 2 3 p = 0 . 5 9 6

0 0 1 5 0 0 2 0 0 0 2 5 0 0 3 0 0 0 3 5 0 0 1 0 0 0 2 0 0 0 3 0 0 0 YY1 RNA expression levels

2 0 0 0 1 5 0 0 r = 0 . 6 2 5 p = 0 . 0 1 3 r = 0 . 3 5 3 1 5 0 0 p = 0 . 1 1 6 1 0 0 0

1 0 0 0

5 0 0 RNA expressionRNA

5 0 0

XIST 0 0 1 5 0 0 2 0 0 0 2 5 0 0 3 0 0 0 1 0 0 0 2 0 0 0 3 0 0 0 4 0 0 0

YY1 RNA expression levels

Supplementary Figure 3. Relationship between RNA expression levels of key regulators of X Chromosome inactivation, XIST and YY1, and posttraumatic stress symptom (PTSS) severity scores in men following MVC trauma. A) XIST RNA expression levels were examined in relation to PTSS severity scores in men who recovered following MVC (top left, purple triangles, n=15), and in men who developed co-morbid CMSP/PTSS following MVC (top right, teal circles, n=21). YY1 RNA expression levels were also analyzed in relation to PTSS severity scores in men who recovered following MVC (bottom left, purple triangles, n=15), and in men who developed co-morbid CMSP/PTSS following MVC (bottom right, teal circles, n=21). B) The relationship between XIST RNA expression levels and YY1 RNA expression levels in men who recovered following MVC (left, green squares, n=15) and in men who developed co-morbid CMSP/PTSS following MVC (right, yellow diamonds, n=21). RNA expression levels were measured via RNA sequencing of blood collected in the early aftermath of MVC. Bivariate analyses were used to derive Pearson correlation coefficients and corresponding p values.

4 1 5

r = 0 . 3 5 8 1 0 p = 0 . 1 9 0 2

5 r = 0 . 4 4 6 1 p = 0 . 0 4 3

0 0 0 5 0 0 1 0 0 0 1 5 0 0 0 5 0 0 1 0 0 0 1 5 0 0 2 0 0 0 XIST RNA expression levels

1 5 r = - 0 . 1 5 1 3 p = 0 . 5 1 2

1 0

2 r = 0 . 1 4 6 p = 0 . 6 0 4

Chronic Musculoskeletal PainSeverity Musculoskeletal Chronic 5 1

0 0 1 5 0 0 2 0 0 0 2 5 0 0 3 0 0 0 1 0 0 0 2 0 0 0 3 0 0 0 4 0 0 0 YY1 RNA expression levels

Supplementary Figure 4. Relationship between RNA expression levels of key regulators of X Chromosome inactivation, XIST and YY1, and chronic musculoskeletal pain (CMSP) severity scores in men following MVC trauma. A) XIST RNA expression levels were examined in relation to CMSP severity scores in men who recovered following MVC (top left, purple triangles, n=15), and in men who developed co-morbid CMSP/PTSS following MVC (top right, teal circles, n=21). YY1 RNA expression levels were also analyzed in relation to CMSP severity scores in men who recovered following MVC (bottom left, purple triangles, n=15), and in men who developed co-morbid CMSP/PTSS following MVC (bottom right, teal circles, n=21).