Ethics, approvals, and study registration

The study protocol was approved by The Committees on Health Research Ethics for the Capital Region of Denmark (approval number: H-19010072), hence, the study complied with all relevant ethical regulations including the Declaration of Helsinki. The handling of data complied with the rules and regulations set forward by the data responsible institution in the Capital Region of Denmark (approval numbers: VD-2019-161 / P-2021-126). The study was registered on ClinicalTrials.gov (registration number: NCT03974048). Informed consent for participation was obtained as described below.

Study design

Study setup and reporting were conducted in accordance with the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guidelines31. This was a prospective, observational epigenome-wide association study (EWAS) investigating a possible association between severe traumatic injury and alterations in the whole blood DNA methylation profile. Ideally, the study would have been set up to compare the pre-trauma DNA methylation profile with the post-trauma DNA methylation profile of a cohort of patients exposed to severe traumatic injury. However, as traumatic injury is acute and unpredictable, we assumed it would only be possible to obtain pre-trauma samples from a small proportion of trauma patients. Therefore, a cohort of patients admitted for elective surgery was included as a control group. The surgical procedure was considered a “controlled” form of traumatic injury, and for this group we were able to obtain both pre-surgery and post-surgery samples. Pre-trauma blood/DNA samples were obtained from the Danish National Biobank (DNB), when inclusion of all patients was complete.

Study population and informed consent

Trauma patients: We included trauma patients 18–65 years of age who were admitted to the Trauma Center at RH following a trauma team activation from June 2019 to April 2021. Exclusion criteria were secondary transfers, prehospital cardiac arrest, more than four hours between the time of trauma and the first study related blood sample, and transfusion with PRBC before the first study related blood sample was obtained. Prehospital transfusion with freeze-dried or liquid plasma or in-hospital transfusion with fresh frozen plasma were not exclusion criteria. Trauma patients were included regardless of the severity of the trauma. After the initial examination and treatment phase (tertiary survey) all patients had an ISS determined by review of their medical records performed by a certified Abbreviated Injury Scale (AIS) coding specialist usually within seven days32. Patients with ISS > 15 (considered severe trauma) were included for DNA methylation profiling and compared with the surgical control group. Blood samples from trauma patients with ISS < 15 were stored, but not handled further in this study. It is not possible to obtain an ISS of 15.

All trauma patients were considered temporarily unable to provide informed consent at the time of inclusion in the study. Informed consent was sought from the patient or his/her next-of-kin as soon as possible after the acute clinical examination and treatment phase.

Surgical patients: In the control group, we included patients 18–65 years of age who were scheduled for major, elective orthopaedic or spine surgery at either RH or RH/Glostrup Hospital between September 2019 and January 2021. Surgical procedures included Ganz osteotomy, lower extremity surgery (total hip arthroplasty [THA], total knee arthroplasty [TKA]), or spinal fusion with an expected duration of at least 60 min and anticipated minor to moderate blood loss. We excluded patients who had surgery due to fractures or cancer and patients who had previously been exposed to severe trauma (assessed by asking the patient about prior traumatic exposure). Patients who were re-operated within 30 days after the surgery were excluded from the follow-up blood sample.

All surgical patients provided informed consent before inclusion in the study.

Study procedures

Trauma patients: All trauma patients admitted to the Trauma Center at RH who were initially considered eligible for inclusion had a study blood sample (“post-trauma” blood sample, see below) taken in the trauma bay by a laboratory technician. Hereafter, a member of the research staff assessed the medical record in relation to the in- and exclusion criteria and sought informed consent if the patient was still eligible for inclusion. Post-trauma blood samples from patients deemed ineligible or where informed consent was not obtained, were destroyed.

Pre-trauma blood samples: Upon completion of trauma patient inclusion, we queried the DNB for previously collected blood- or DNA samples from the included trauma patients with ISS > 15. The DNB contains millions of biological samples from the Danish population, and these samples were either excess material from other research biobanks or national screenings. We used the included trauma patients’ unique Central Person Register (CPR) number (which is assigned to all Danish citizens) to identify biological samples containing DNA, which had been collected for another purpose before the patients’ trauma date.

Surgical patients: The surgical patients had a peripheral venipuncture (“pre-surgery” blood sample) performed immediately prior to incision. Again, immediately after surgery and no later than four hours after wound closure another peripheral venipuncture (“post-surgery” blood sample) was performed.

Follow-up at 30–45 days: We contacted trauma patients with an ISS > 15 and the surgical patients in whom both the pre-surgery and post-surgery blood sample had been obtained. After consent, a follow-up peripheral venipuncture was performed 30–45 days after trauma/surgery (“30d-trauma” and “30d-surgery” blood sample, respectively). Thus, all included trauma patients had a set of two or three blood samples ((pre-trauma), post-trauma, 30d-trauma), and all included surgical patients had a set of three blood samples (pre-surgery, post-surgery, 30d-surgery).

Data collection: Demographic, trauma- and surgery-related, and in-hospital data were obtained by the research staff from the patients’ electronic medical records.

Outcome measures

Outcome measures included differential DNA methylation of either individual CpG sites or regions in the genome of whole blood DNA. We compared findings within the trauma group (analysis 1, 2, and 3; paired analyses), within the surgical group (analyses 4 and 5; paired analyses), and between the two study groups (analyses 6 and 7; unpaired analyses) (Fig. 2). In addition, the DNA methylation profiles of the post-trauma blood samples were compared for trauma patients with an uncomplicated vs. complicated recovery (analysis 8; unpaired). A complicated recovery was defined as a stay exceeding 48 h in the intensive care unit (ICU) during the trauma-related hospital admission.

Blood sampling and circulating leucocyte DNA methylation profiling

Blood sampling was performed by trained laboratory technicians by peripheral venipuncture in the antecubital fossa using the SAFETY Blood Collection Set + Holder (Greiner Bio-One, Kremsmünster, Austria). A total volume of 21 millilitres (9 + 9 + 3 mililiters) was drawn in VACUETTE® EDTA coated tubes (Greiner Bio-One, Kremsmünster, Austria). All blood samples were taken to the biobank within five hours upon obtainment and stored at 4 degrees Celsius until further handling. Samples delivered to the biobank on weekdays (Monday-Friday) from 8 AM to 3 PM were handled on the same day, and samples delivered at other times were handled on the first coming weekday. Samples were separated into full blood samples and plasma samples. Full blood samples were pipetted into 850 µL matrix tubes (Thermo Fisher Scientific, Waltham, Massachusetts, USA), and samples for plasma obtainment were centrifuged at 1800 Relative Centrifugal Force (RFG) at 4 degrees Celsius for 10 minutes and stored in 850 µL matrix tubes (Thermo Fisher Scientific, Waltham, Massachusetts, USA). All samples were subsequently stored at -80 degrees Celsius.

Only samples from patients with a complete set of blood samples were further processed. DNA was extracted from whole blood using the Chemagen technology (ChemagicStar, Perkin Elmer) following the instructions given by the manufacturer. In brief, DNA was isolated with magnetic beads after addition of lysis buffer and proteases to the samples, and finally eluated by adding an elution buffer.

The extracted DNA was bisulfite treated to convert unmethylated cytosines to uracil. Then, the DNA was hybridized to Infinium MethylationEPIC BeadChips (Illumina, San Diego, CA, USA) and further underwent single base extension, fluorescent staining, signal amplification, and scanning using the iScan System (all procedures were performed by Eurofins Genomics, Galten, Denmark). Finally, the intensities of the probes were extracted and analysed using the GenomeStudio Software V2001.1 (Illumina, San Diego, CA, USA). Each MethylationEPIC BeadChip held eight samples, and samples were randomly distributed on the BeadChips to minimize systematic bias. Each BeadChip interrogated 863,904 CpG sites distributed throughout the genome33.

Data analysis

IDAT files were extracted from the GenomeStudio Software V2001.1 (Illumina, San Diego, CA, USA) for each sample and imported into Rstudio version 4.1.0 (2021-05-08) – “Camp Pontanezen” using the minfi package (version 1.38.0)34,35. The level of methylation at each CpG site was measured as a beta(β)-value ranging from 0 (no methylation) to 1 (complete methylation). The ShinyMethyl package (version 1.28.0) was used to assess the quality of the data36. Individual samples that did not pass quality control in order to be sufficiently analysed were excluded (trauma patients: n = 0, surgical patients, n = 1) from further analysis.

Samples passing quality control (trauma patients: n = 60, surgical patients, n = 57) were normalized using a Subset-quantile Within Array Normalization (SWAN)35 method to reduce the technical variability within and between arrays. CpGs and single nucleotide extensions containing single nucleotide polymorphisms (SNPs) were dropped. To control for batch effects, including changes in cell type composition, we calculated surrogate variables using the SmartSVA package (version 0.1.3)37 and included the surrogate variables as covariates in subsequent analyses.

For each sample group comparison (see paragraph on outcome measures and Fig. 2) differential methylation at single CpG sites were determined using the CpGAssoc package (version 2.6.0)38 after inclusion of the surrogate variables as covariates. To correct for multiple testing a False Discovery Rate (FDR) using the Benjamini-Hochberg method was applied resulting in a q-value (adjusted p-value). A q-value below 0.05 was considered statistically significant. All analyses were, in addition to surrogate variables, adjusted for age and sex. Paired analyses within the trauma group were adjusted for ISS, and paired analyses within the surgical group were adjusted for type of surgical procedure.

DMRs were identified and ranked using the DMRcate package (version 2.6.0)39. We used a bandwidth of 1000 nucleotides (lambda = 1000), a scaling factor of 2 (C = 2), and corrected the results for multiple testing using the Benjamini-Hochberg method as recommended by the authors of the DMRcate package39. Annotation was done according to hg19.

Descriptive statistics were reported with median and interquartile range (IQR) and frequencies and percentages for continuous and categorical variables, respectively.

Sample size

As there was limited knowledge on DNA methylation changes following severe trauma, we were not able to properly estimate the effect size of trauma on the DNA methylation profile. Based on studies of non-trauma populations that have found effect sizes of 0–25% difference in genome-wide DNA methylation levels between cases and controls40,41,42, we assumed that a 15% change in the mean methylation difference between our comparison groups was relevant to detect following exposure to severe trauma. A simulation study set up to estimate power and adequate sample size for EWAS43 found that to detect a 15% mean methylation difference between two groups with 80% power and a genome-wide significance level of 1 × 10−6, 54 patients were needed in each group. Thus, we needed to include 54 trauma patients and 54 surgical patients to obtain 80% power at a genome-wide significance level of 1 × 10−6. To account for potential DNA samples of low quality we decided to continue inclusion of patients until we had a full set of blood samples from 60 trauma patients and 60 surgical patients.

Enrichment analyses

To identify perturbated pathways or biological functions, we performed enrichment analyses of the genes related to the significant DMRs. We used EnrichR44 and included the following pathway databases: Reactome 202245, BioPlanet 201946, and BioCarta 2016.