The identification and isolation of sperm cells from biological samples remain a pivotal component of forensic analysis in sexual offense investigations. Although differential lysis (DL) is the standard method for sperm cell separation, it is time-consuming and frequently inadequate for generating informative male DNA profiles, particularly when the epithelial-to-sperm cell ratio is unfavorable. These limitations have driven interest in alternative separation techniques, such as fluorescence-activated cell sorting (FACS), which enables the detection and isolation of individual cells from mixed populations based on phenotypic markers. This study therefore aimed to establish and systematically validate a FACS-based methodology, referred to as spermFACS, for the separation of sperm cells from vaginal swab samples for routine implementation, following ISO/IEC 17025 guidelines. Notably, spermFACS enabled the recovery of informative male autosomal DNA profiles from mixtures with sperm/epithelial cell ratios as low as 1:7500 and from postcoital samples collected up to 120 h after intercourse. Supported by the calculation of likelihood ratios, a superior separation efficiency and purity of the sperm fraction was thus demonstrated in a comparative analysis with DL. These findings underscore the utility of spermFACS in delayed-report sexual assault cases and highlight its potential in the deconvolution of complex mixtures, such as those encountered in multiple offender rapes. By overcoming key limitations of traditional methods, the integration of spermFACS into forensic workflows may enhance the ability to identify offenders and contribute meaningfully to the resolution of sexual assault cases.

There is a broad consensus that forensic tests for the prediction of externally visible characteristics (EVC) and analysis of biogeographic ancestry (BGA) of an individual are technically reliable. However, interpretation of the results and population-specific genotype distribution patterns remains challenging. EVC and BGA analyses provide valuable information for population genetics studies and as investigative leads for criminal cases, as well as for historical and contemporary identification tests. However, inaccurate or incorrect predictions, for example, from subjective bias in the interpretations made, have the potential to misdirect police investigations. The legal situation regarding EVC and BGA testing varies by country: ranging from countries where it is explicitly prohibited, to those without specific regulations on biogeographic ancestry prediction, and others that have already enacted laws governing its use. The reluctance to utilize these analyses is not only due to legal restrictions and data protection concerns, but also to initial limited sets of sufficiently comprehensive forensic DNA assays. Forensic BGA marker panels typically contain up to ∼300 SNPs. This relatively small number of genetic markers, along with limited reference population data, complicates the interpretation of results from donors of unknown origin. This paper presents the results of a collaborative EDNAP study, which, for the first time, evaluated the approach to reporting EVC and BGA data between international laboratories. For the study, DNA from nine individuals with self-reported ancestry was collected and analysed using various forensic panels differing in the number and composition of ancestry-informative markers genotyped, comprising: the Precision ID mtDNA Whole Genome Panel, the VISAGE Basic Tool and the VISAGE Enhanced Tool for Appearance and Ancestry Prediction, and the Ion AmpliSeq™ PhenoTrivium Panel. To ensure full data protection, all SNP genotypes and uniparental marker haplotypes obtained were not shared with third parties. Instead, the genetic data were analysed using a range of commonly used population analysis software packages. These analysis outcomes were then distributed to twelve European forensic laboratories (both academic and law enforcement institutions), who were asked to prepare reports based on their interpretation of the phenotypes and ancestry they inferred from the analysis data. A questionnaire sent alongside the genetic information, aimed to evaluate which difficulties were encountered by the participants in processing the BGA analysis data they were given.

Numerous forensic age prediction models based on DNA methylation markers have been developed, each differing in the number of predictive markers, statistical method, biomatrix, and sequencing platform used. This variability highlighted the need for more uniformity in the development of epigenetic clocks. To partially address this need, the VISAGE Consortium introduced the VISAGE enhanced tool assay, a multi-tissue assay that targets eight age-associated genes (ELOVL2, EDARADD, ASPA, FHL2, MIR29B2CHG, KLF14, TRIM59, and PDE4C). So far, three models were built using this assay for age prediction in blood, buccal cells, and bones, based on Illumina MiSeq sequencing data with the v3 reagent kit (2 × 300 bp). Unfortunately, the existing models are neither publicly accessible nor permitted for use in forensic casework. To address this limitation, we developed our own age estimation model utilising the VISAGE enhanced tool assay in combination with the Illumina NovaSeq 6000 platform and the v1.5 reagent kit (2 × 150 bp). By employing the same assay, we streamlined the workflow and enhanced uniformity, as there was no need to identify additional age-associated genes. By adjusting the assay’s primer concentrations, we achieved sufficient read depths to accurately determine methylation levels, even for longer amplicons with partial sequencing strand coverage. This modified assay was used to develop an age estimation model in blood (n = 98) with a mean absolute error (MAE) of 3.22 years and root mean squared error (RMSE) of 3.77 years in the test set (n = 30). Overall, this study demonstrated that by adjusting primer concentrations, equal age estimation performances can be achieved with the added benefit of drastically reduced costs and turn-around-time by using a 2 × 150 bp sequencing strategy. Additionally, this study was the first to independently validate the VISAGE enhanced tool assay on a different sequencing platform, exploring its potential for broader applications and partially answering the need for more uniformity.

The increasing numbers of sexual violence and unresolved rape cases require alternative approaches with higher evidential value to complement existing forensic tools. Predicting recent intercourse is crucial in forensic casework on sexual assaults. In this work, we assessed whether sexual intercourse can be predicted based on the vaginal microbiome and compared it to the gold standard method of semen detection. Using a prediction model based on microbiome of 3043 women, intercourse was predicted with 71

Predicting recent intercourse is crucial in forensic casework on sexual assaults. In this work, we assessed whether sexual intercourse can be predicted based on the vaginal microbiome and compared it to the gold standard method of semen detection. Using a prediction model based on microbiome of 3,043 women, intercourse was predicted with 71% accuracy in a balanced cross-validation machine learning setting. Next, this prediction model was validated in a longitudinal intervention study and tested on forensic sexual assault cases. The developed predictor could accurately establish intercourse in 82% cases. Yet, underwear was found to hold an even greater evidential value and replace the more invasive vaginal sampling for semen detection in some cases, with an accuracy of 95%. This was confirmed through a retrospective analysis of 207 forensic sexual assault cases. Taken together, this study revealed the potential of both microbiome profiling on vaginal swabs and semen detection on underwear for forensic casework.

Forensic autopsies remain indispensable for accurately determining the cause and manner of death. However, pathologists face significant challenges inherent to the complex process of postmortem diagnostics (PMD), including the potential for diagnostic errors. The implementation of quality assurance (QA) mechanisms is crucial for minimizing these errors. Nonetheless, the lack of QA programs, specifically tailored for forensic pathology, continues to pose a significant obstacle. This article explores the enhancement of PMD after the adoption of ISO 17020 accreditation at a Belgian forensic institute in 2010. Drawing on over a decade of experience, it details on the development and implementation of label-specific standard operating procedures (SOPs) designed to address the various types of deaths in forensic practice. Additionally, it underscores the necessity of adapting international directives from leading organizations into effective local protocols, aiming to standardize practices and improve efficacy. Through a comparative review of existing international guidelines, this study provides forensic pathologists and institutions worldwide with practical strategies for qualitative standardization and improving their PMD.

In a previous study, we have provided the first proof that chronological age can be estimated through DNA methylation (DNAm) patterns in fingernails and toenails. DNAm data of 15 CpGs located in 4 genetic markers (ASPA, EDARADD, ELOVL2 and PDE4C) were evaluated, of which variable selection yielded age prediction models with a mean absolute deviation (MAD) ranging from 7.68 to 9.36 years, depending on the sampling location. Three additional age-associated markers (KLF14, MIR29B2CHG and TRIM59) were assessed in the current study with the goal of increasing the prediction accuracy of the model initially constructed for toenails. This new and improved age estimation assay yielded an MAD of 4.82 and 5.61 years for the training and test set, respectively. The feasibility of the application for post-mortem cases was also demonstrated through testing a limited set of samples collected from deceased individuals.

Over the past decade, age prediction based on DNA methylation has become a vastly investigated topic; many age prediction models have been developed based on different DNAm markers and using various tissues. However, the potential of using nails to this end has not yet been explored. Their inherent resistance to decay and ease of sampling would offer an advantage in cases where post-mortem degradation poses challenges concerning sample collection and DNA-extraction. In the current study, clippings from both fingernails and toenails were collected from 108 living test subjects (age range: 0-96 years). The methylation status of 15 CpGs located in 4 previously established age-related markers (ASPA, EDARADD, PDE4C, ELOVL2) was investigated through pyrosequencing of bisulphite converted DNA. Significant dissimilarities in methylation levels were observed between all four limbs, hence both limb-specific age prediction models and prediction models combining multiple sampling locations were developed. When applied to their respective test sets, these models yielded a mean absolute deviation between predicted and chronological age ranging from 5.48 to 9.36 years when using ordinary least squares regression. In addition, the assay was tested on methylation data derived from 5 nail samples collected from deceased individuals, demonstrating its feasibility for application in post-mortem cases. In conclusion, this study provides the first proof that chronological age can be assessed through DNA methylation patterns in nails.

BACKGROUND:Daily Hassles (DH) or daily stress - is a mild type of stressor with unique contributions to psychological distress. Yet, most prior studies that investigate the effects of stressful life experiences focus on childhood trauma or on early life stress and little is known about the effects of DH on epigenetic changes in stress system related genes and on the physiological response to social stressors.METHODS:In the present study, conducted among 101 early adolescents (mean age = 11.61; SD = 0.64), we investigated whether Autonomic Nervous System (ANS) (namely heart rate and heart rate variability) and Hypothalamic-Pituitary-Adrenal (HPA) axis functioning (measured as cortisol stress reactivity and recovery) are associated with DNA methylation (DNAm) in the glucocorticoid receptor gene (NR3C1), the level of DH and their interaction. To assess the stress system functioning the TSST protocol was used.RESULTS:Our findings show that higher NR3C1 DNAm in interaction with higher levels of daily hassles, is associated with blunted HPA axis reactivity to psychosocial stress. In addition, higher levels of DH are associated with extended HPA axis stress recovery. In addition, participants with higher NR3C1 DNAm had lower ANS adaptability to stress, specifically lower parasympathetic withdrawal; for heart rate variability this effect was strongest for participants with higher level of DH.CONCLUSIONS:The observation that interaction effects between NR3C1 DNAm levels and daily stress on the functioning of the stress-systems, are already detectable in young adolescents, highlights the importance of early interventions, not only in the case of trauma, but also daily stress. This might help to prevent stress-induced mental and physical disorders later in life.

The localization of latent blood traces at crime scenes is generally performed using fluorescent stains although infrared light has previously been recognized as an effective localization test for bloodstains as it is a non-destructive and non-contact technique. The goal of this study was to evaluate infrared photography for the detection of latent bloodstains on a large number of objects with different compositions frequently encountered at crime scenes. In this study we show that infrared light photography was able to detect bloodstains deposited on 71.7% of materials while bloodstains on 29.2% of materials could only be detected using infrared photography and not through visual photography. Bloodstain could be detected on most fabrics composed of 100% polyester, 100% cotton and 100% wool or a combination of these fibers with other types of fiber such as nylon or viscose. For other materials such as leather, tiles, wood, bricks, parquet, infrared did not improve the visibility of the bloodstains. Finally, the influence of the time of bloodstain deposition was tested over the period of 1 week and 1 month and did not reveal major differences compared to stains after 24 h drying time suggesting that time has little influence on the ability of infrared light and visual light to detect latent bloodstains.

Background Catechol-O-methyltransferase (COMT) has been shown to influence clinical pain, descending modulation, and exercise-induced symptom worsening. COMT regulates nociceptive processing and inflammation, key pathophysiological features of Chronic Fatigue Syndrome and Fibromyalgia (CFS/FM). We aimed to determine the interactions between genetic and epigenetic mechanisms regulating COMT and its influence on inflammatory markers and symptoms in patients with CFS/FM. Methods. A case-control study with repeated-measures design was used to reduce the chance of false positive and increase the power of our findings. Fifty-four participants (28 patients with CFS/FM and 26 controls) were assessed twice within 4 days. The assessment included clinical questionnaires, neurophysiological assessment (pain thresholds, temporal summation, and conditioned pain modulation), and blood withdrawal in order to assess rs4818, rs4633, and rs4680 COMT polymorphisms and perform haplotype estimation, DNA methylation in the COMT gene (both MB-COMT and S-COMT promoters), and cytokine expression (TNF-α, IFN-γ, IL-6, and TGF-β). Results. COMT haplotypes were associated with DNA methylation in the S-COMT promoter, TGF-β expression, and symptoms. However, this was not specific for one condition. Significant between-group differences were found for increased DNA methylation in the MB-COMT promoter and decreased IFN-γ expression in patients. Discussion Our results are consistent with basic and clinical research, providing interesting insights into genetic-epigenetic regulatory mechanisms. MB-COMT DNA methylation might be an independent factor contributing to the pathophysiology of CFS/FM. Further research on DNA methylation in complex conditions such as CFS/FM is warranted. We recommend future research to employ a repeated-measure design to control for biomarkers variability and within-subject changes.

Forensic DNA analysis of cartridges and fired cartridge casings remains challenging, possibly due to the heat and pressure generated during firing of the weapon as well as metal ions from the casings that have been suggested to initiate DNA degradation and inhibit PCR during the DNA profiling process. Even though recently developed DNA recovery protocols have shown to significantly improve DNA yields and DNA profile success rates no information is available on whether the time interval between contact and the DNA recovery process has an influence on these outcomes. In the current study 40 cartridges and 40 fired cartridge casings were left untreated for 24 h or 1 week after which the rinse-and-swab technique was used to collect DNA. Higher DNA yields and higher DNA profile success rates were obtained from cartridges compared to fired cartridge casings. The same general observation was made when cartridges and fired cartridge casings were processed after 24 h compared to after 1 week. In addition, DNA profiles suitable for comparison could still be generated from samples when real-time PCR quantification indicated DNA concentrations < 0.001 ng/μl, suggesting that quantification results may not be reliable when assessing the presence of DNA on such items. In conclusion, the results indicate that cartridges and fired cartridge casings should be processed for DNA profiling as soon as possible and that DNA quantification results should be interpreted with caution as DNA profiles suitable for comparison could be missed.

The detection and separation of spermatozoa is crucial in the forensic investigation of alleged sexual assault cases. Differential lysis and microscopy-based techniques are conventional methods for the isolation of spermatozoa, though are time-consuming and frequently fail with samples containing an unfavourable sperm/epithelial cell ratio. Successful separation by means of fluorescence-activated cell sorting (FACS) has previously been reported, yet little efforts have been dedicated towards the further improvement or routine implementation of this technique. With this ongoing research, a methodology is being developed to sort sperm from epithelial cells by combining the Sperm Hy-Liter™ staining kit and FACS. Sorted sperm cells are then subjected to a direct lysis and low volume PCR (LV-PCR) protocol. Preliminary results demonstrate the successful separation of both cell types at a sperm/epithelial cell ratio of up to 1:500. The direct lysis and LV-PCR protocol allows to produce full haploid profiles from single sperm cells and mostly full diploid profiles from 10 spermatozoa. These data suggest that the proposed methods are potentially viable for forensic casework, yet additional testing is required for validation purposes.

RNA analysis of post-mortem tissues, or thanatotranscriptomics, has become a topic of interest in forensic science due to the essential information it can provide in forensic investigations. Several studies have previously investigated the effect of death on gene transcription, but it has never been conducted with samples of the same individual. For the first time, a longitudinal mRNA expression analysis study was performed with post-mortem human blood samples from individuals with a known time of death. The results reveal that, after death, two clearly differentiated groups of up- and down-regulated genes can be detected. Pathway analysis suggests active processes that promote cell survival and DNA damage repair, rather than passive degradation, are the source of early post-mortem changes of gene expression in blood. In addition, a generalized linear model with an elastic net restriction predicted post-mortem interval with a root mean square error of 4.75 h. In conclusion, we demonstrate that post-mortem gene expression data can be used as biomarkers to estimate the post-mortem interval though further validation using independent sample sets is required before use in forensic casework.

Automated Teller Machine bombings are an increasing societal problem that are often committed using Improvised Explosive Devices. The evolution in IEDs and the negative consequences for society require new security measures to prevent these crimes. Ink staining and security smoke devices are added to cash cassettes, in order to protect ATMs and prevent ATM bombings. Traces found at crime scenes, such as fingerprints and DNA, can contribute to the identification of perpetrators. However, the effect of ink staining and security smoke devices on dactyloscopy and DNA profiling is still unknown. In the current study, we demonstrate that procedures using Citrus Cleaner or sulfosalicylic acid were successful in removing ink and security smoke deposited on plastic plates but did result in the massive loss of fingerprint information as only a low number (4%) of good quality fingerprints were recovered after smoke contamination. Secondly, security ink Sun Blue ES2, but not SICPA Green and Sun Blue ES1, had a significant impact on DNA profiling success. DNA concentrations obtained from blood spiked swabs decreased with increasing ink concentration resulting in a complete loss of genotype information with the addition of ≥10 μl Sun Blue ES2. No noticeable PCR inhibition or DNA degradation was detected during quantification, but a decreased efficiency of DNA extraction could not be excluded. Security smoke, on the other hand, does not seem to have a significant influence on DNA analysis. Precautions must therefore be taken in order to avoid contaminating DNA swabs with ink during sampling. Thirdly, only a single negative impression of a glove in ink and a single glove print were able to be visualized with white fingerprint powder on detonated cash cassettes. In conclusion, the detection of glove prints and fingerprints is limited and security ink, contrary to smoke, after detonation can have a negative influence on downstream DNA analysis procedures.

Background There is a growing interest in the role of brain-derived neurotrophic factor (BDNF) in major depressive disorder (MDD). BDNF potentially exhibits opposite effects in the pathways linked to anhedonia and reward learning on the one hand and cognitive performance, on the other hand. However, the epigenetic mechanisms behind this remain unknown. In the present study, we aimed to investigate the interplay of DNA methylation of different BDNF exons and the common Val66Met polymorphism on anhedonia, reward learning and cognitive performance in MDD. Methods We recruited 80 depressed patients and 58 age- and gender-matched healthy controls. Participants underwent clinical assessment including neuropsychological testing and a probabilistic reward task to assess reward learning. Val66Met polymorphism and DNA methylation of BDNF promoters I, IV and exon IX were assessed from whole blood derived DNA, using pyrosequencing. Results BDNF promoter I methylation was lower in MDD patients ( p = 0.042) and was negatively associated with self-reported anhedonia. In depressed patients, both Val66Met polymorphism and DNA methylation of promoter I were significantly associated with reward bias ( p < 0.050 and p = 0.040, respectively), without an interaction effect. On the other hand, methylation of exon IX had a negative impact on executive functioning ( p = 0.002) and mediated the effect of Val66Met on this outcome in patients with MDD. Conclusions Our results provide the first evidence of Val66Met susceptibility to differential epigenetic regulation of BDNF exons in reward learning and executive functioning in MDD, which needs to be further explored.

BACKGROUND:Work-related stress and burnout have become major occupational health concerns. Dysregulation of HPA axis is considered one of the central mechanisms and is potentially moderated through epigenetics. In the present study, we aim to investigate epigenetic regulation of the HPA axis in burnout, by focusing on salivary cortisol and cortisone and DNA methylation of the glucocorticoid receptor gene (NR3C1) and the serotonin transporter gene (SLC6A4).METHODS:We conducted a cross-sectional study with 59 subjects with burnout and 70 healthy controls recruited from the general population. All participants underwent a clinical interview and psychological assessment. Saliva samples were collected at 0, 30 and 60 min after awakening and were used to quantify cortisol and cortisone. Pyrosequencing was performed on whole blood-derived DNA to assess DNA methylation.RESULTS:There were no between-group differences in cortisol levels, whereas burnout participants had higher levels of cortisone. Job stress was associated with increased cortisol and cortisone. We observed both increased and decreased NR3C1 and SLC6A4 methylation in the burnout group compared to the control group. Some of these methylation changes correlated with burnout symptoms dimensionally. Increased methylation in a specific CpG in the SLC6A4 promoter region moderated the association between job stress and burnout. DNA methylation in this CpG was also associated with increased cortisol. In addition, average methylation of NR3C1 was negatively associated with cortisone levels.LIMITATIONS:This is a cross-sectional study and therefore no conclusions on causality could be made.CONCLUSIONS:We provide first evidence of changes in DNA methylation of NR3C1 and SLC6A4 in burnout, which were further associated with cortisol and cortisone. Further, increased cortisol and cortisone seemed to reflect job stress rather than burnout itself.

Background: Epigenetic changes are considered the main mechanisms behind the interplay of environment and genetic susceptibility in major depressive disorder (MDD). However, studies focusing on epigenetic dysregulation of the HPA axis stress response in MDD are lacking. Our objective was to simultaneously asses DNA methylation of the glucocorticoid receptor gene (NR3C1) and serotonin transporter gene (SLC6A4) and HPA axis response to stress in MDD. Methods: We recruited 80 depressed inpatients and 58 gender and age matched healthy controls. All participants underwent the Trier Social Stress Test (TSST) and salivary cortisol was repeatedly measured to assess HPA axis reactivity. DNA methylation of the NR3C1 (exon 1 F) and SLC6A4 CpG islands was quantified from whole blood DNA. In the MDD group, clinical assessment was repeated at 8-week follow-up to test the predictive potential of DNA methylation for symptom improvement. Results: Depressed patients had blunted cortisol reactivity to TSST compared to healthy controls (p = 0.01). In addition, they presented with increased average SLC6A4 (p = 0.003) and NR3C1 methylation (p = 0.03), as well as methylation of two individual NR3C1 CpG loci overlapping with the NGFI-A-binding sites (CpG12 and CpG20). Methylation of one of these two loci (CpG20) predicted lower symptom improvement at the follow-up (p = 0.007). Both, average NR3C1 and SLC6A4 methylation were associated with lower cortisol reactivity in the MDD group and explained about 16% of variability in cortisol response to TSST. Conclusions: We provide evidence of the role of NR3C1 and SLC6A4 DNA methylation in HPA axis dysregulation in MDD, which needs to be further explored.

We aimed to generate equation to predict arterial lactate (a-Lac) using venous lactate (v-Lac) and other lab data.A prospective cross-sectional study was conducted on emergency patients in the emergency department for 6 months at a general hospital in Tokyo, Japan. We collected arterial and venous gas analysis data. Patients were eligible for entry into the study if an arterial blood gas analysis was required for appropriate diagnostic care by the treating physician. Univariate linear regression analysis was conducted to generate an equation to calculate a-Lac incorporating only v-Lac. A multivariate forward stepwise logistic regression model (p-value of 0.05 for entry, 0.1 for removal) was used to generate an equation including v-Lac and other potentially relevant variables. Bland-Altman plot was drawn and the two equations were compared for model fitting using R-squares.Seventy-two arterial samples from 72 participants (61% male; mean age, 58.2 years) were included in the study. An initial regression equation was derived from univariate linear regression analysis:“(a-Lac) = − 0.259 + (v-Lac) × 0.996”. Subsequent multivariate forward stepwise logistic regression analysis, incorporating v-Lac and Po2, generated the following equation:“(a-Lac) = − 0.469+(venous Po2) × 0.005 + (v-Lac) × 0.997”. Calculated R-squares by single and multiple regression were 0.94 and 0.96, respectively.v-Lac estimates showed a high correlation with arterial values and our data provide two clinically useful equations to calculate a-Lac from v-Lac data. Considering clinical flexibility, “Lac = − 0.259 + v-Lac × 0.996” might be more useful while avoiding a time-consuming and invasive procedure.

ObjectiveThe epigenetics of neurotrophic factors holds the potential to unravel the mechanisms underlying the pathophysiology of complex conditions such as chronic fatigue syndrome (CFS). This study was undertaken to explore the role of brain‐derived neurotrophic factor (BDNF) genetics, epigenetics, and protein expression in patients with both CFS and comorbid fibromyalgia (CFS/FM).MethodsA repeated‐measures study was conducted in 54 participants (28 patients with CFS/FM and 26 matched healthy controls). Participants underwent a comprehensive assessment, including questionnaires, sensory testing, and blood withdrawal. Serum BDNF (sBDNF) protein levels were measured using enzyme‐linked immunosorbent assay, while polymorphism and DNA methylation were measured in blood using pyrosequencing technology. To assess the temporal stability of the measures, participants underwent the same assessment twice within 4 days.ResultsRepeated‐measures mixed linear models were used for between‐group analysis, with mean differences and 95% confidence intervals (95% CIs) shown. Compared to controls, serum BNDF was higher in patients with CFS/FM (F = 15.703; mean difference 3.31 ng/ml [95% CI 1.65, 4.96]; P = 0.001), whereas BDNF DNA methylation in exon 9 was lower (F = 7.543; mean difference −2.16% [95% CI −3.93, −0.83]; P = 0.007). BDNF DNA methylation was mediated by the Val66Met (rs6265) polymorphism. Lower methylation in the same region predicted higher sBDNF levels (F = 7.137, β = −0.408 [95% CI −0.711, −0.105]; P = 0.009), which in turn predicted participants’ symptoms (F = 14.410, β = 3.747 [95% CI 1.79, 5.71]; P = 0.001) and widespread hyperalgesia (F = 4.147, β = 0.04 [95% CI 0.01, 0.08]; P = 0.044).ConclusionOur findings indicate that sBDNF levels are elevated in patients with CFS/FM and that BDNF methylation in exon 9 accounts for the regulation of protein expression. Altered BDNF levels might represent a key mechanism explaining CFS/FM pathophysiology.