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Cited by 19Epigenetics and DNA MethylationStress Responses and CortisolTranscranial Magnetic Stimulation Studies

Dynamic DNA methylation changes in the COMT gene promoter region in response to mental stress and its modulation by transcranial direct current stimulation

Open access
Wiegand, Ariane, Blickle, Arne, Brückmann, Christof, Weller, Simone, Nieratschker, Vanessa, Plewnia, Christian·2021·Biomolecules
DOI 10.3390/biom11111726PMC PMC8615564

Published version of a preprint

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Abstract

Changes in epigenetic modifications present a mechanism how environmental factors, such as the experience of stress, can alter gene regulation. While stress-related disorders have consistently been associated with differential DNA methylation, little is known about the time scale in which these alterations emerge. We investigated dynamic DNA methylation changes in whole blood of 42 healthy male individuals in response to a stressful cognitive task, its association with concentration changes in cortisol, and its modulation by transcranial direct current stimulation (tDCS). We observed a continuous increase in COMT promotor DNA methylation which correlated with higher saliva cortisol levels and was still detectable one week later. However, this lasting effect was suppressed by concurrent activity-enhancing anodal tDCS to the dorsolateral prefrontal cortex. Our findings support the significance of gene-specific DNA methylation in whole blood as potential biomarkers for stress-related effects. Moreover, they suggest alternative molecular mechanisms possibly involved in lasting behavioral effects of tDCS.

AI snapshot

Written from the paper’s full text.

TL;DR

In 42 healthy male participants, a stressful adaptive 2-back PASAT induced a continuous increase in COMT promoter DNA methylation in whole blood that correlated with salivary cortisol and was still detectable one week later. Concurrent anodal tDCS to the left dorsolateral prefrontal cortex suppressed this lasting methylation increase.

Key findings

  • During session 1, COMT promoter DNA methylation increased from 57.70% pre-task to 59.33% at 90 min post-task (t(41) = −4.30, p < 0.001, |d| = 0.66), with significant increases from 20 min post-task onward (58.69% methylated; t(41) < −2.48, p < 0.017, |d| > 0.38).
  • Changes in DNA methylation correlated with changes in salivary cortisol during session 1 (r = 0.359, p = 0.019); cortisol increased in session 1 (t(41) = −2.50, p = 0.017, |d| = 0.39) but not session 2 (t(41) = −0.26, p = 0.80).
  • The session-1 methylation increase remained detectable at the start of session 2 in sham-treated participants (t(20) = −2.95, p = 0.008, |d| = 0.64) but not in anodal-treated participants (t(20) = −0.13, p = 0.90); the interaction of first-session stimulation and session predicted DNA methylation (B = 1.54, SE = 0.68, β = 0.21, t(40) = 2.26, p = 0.030).
  • tDCS did not significantly affect task performance (stimulation: B = −0.39, SE = 0.97, β = −0.02, t(202) = −0.40, p = 0.69) or cortisol levels (stimulation: B = 0.02, SE = 0.03, β = 0.06, t(121) = 0.76, p = 0.45).

Methods

  • Single-blind, sham-controlled cross-over study in 42 healthy male non-smoking participants of European descent aged 18–30 years, recruited as a pilot cohort of 22 and a replication cohort of 20; each participant completed two sessions 7 days apart with anodal or sham tDCS to the left dorsolateral prefrontal cortex (F3) during an adaptive 2-back PASAT.
  • Blood was sampled before stimulation, immediately after task completion, and 20, 40, 60, and 90 min after task completion; saliva cortisol was sampled at session start and 30 min after task onset; affect was assessed with the PANAS pre, post, and 90 min follow-up.
  • DNA methylation was quantified by pyrosequencing at two CpG sites in the S-COMT promoter region (chr22:19,950,055 and chr22:19,950,064) and expressed as the mean of the two sites; cortisol was measured by ELISA. Multilevel models in R included fixed effects of stimulation, session, and time/task block, random intercepts and slopes, and an AR(1) error term; Pearson correlations tested DNA methylation–cortisol and affect–cortisol associations.

Limitations

  • The paper states that this was an explorative first human study with no prior effect sizes for power estimation, and that the sample size, while adequate for tDCS effects, requires replication in randomized clinical trials using a parallel design.
  • The paper notes that the observed DNA methylation differences were relatively small, functional relevance needs clarification with gene expression data, and whole-blood DNA methylation may not directly reflect neuronal tissue; cell-type composition could confound whole-blood methylation, though the authors argue this is unlikely within 20 min.
  • The paper states that the PASAT is a relatively mild stressor and that no cortisol response occurred in session 2, likely due to adaptation; venous catheter placement shortly before baseline sampling could have affected baseline measurements. The text also makes evident that the sample was restricted to young, healthy, right-handed, non-smoking male participants of European descent, limiting generalizability.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.3390/biom11111726

Cites

67 of 67 references verified0 citation errors · 0 not checkable

Statistics

40 tests recomputed · 0 with the reported and recomputed p on opposite sides of .05

Claims the paper makes

0 of 54 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    The interaction of stimulation and time did not significantly predict COMT DNA methylation levels.

    “While the interaction of stimulation and time (B = −0.09, SE = 0.23, β = −0.01, t(457) = −0.37, p = 0.71) did not predict the outcome variable significantly”
    Quote in the paperSubstantial claim
  • Findingown data

    During session 2, there was no change in COMT DNA methylation from pre to any post time point.

    “For session 2, no change in DNAm from time point pre to any post time point was observed (|t(41)| < 1.8, p > 0.08).”
    Quote in the paperSubstantial claim
  • Findingown data

    The increase in COMT DNA methylation during session 1 was not detectable at the beginning of session 2 in anodal-treated participants.

    “As depicted in Figure 3B, the increase in DNAm during the first session could still be detected in the beginning of session 2 in sham-treated participants (t(20) = −2.95, p = 0.008, |d| = 0.64), but not in anodal-treated participants (t(20) = −0.13, p = 0.90).”
    Quote in the paperSubstantial claim
  • Findingown data

    The interaction of stimulation in first session and session significantly predicted COMT DNA methylation levels at the beginning of each session.

    “The interaction of stimulation in first session and session significantly predicted DNAm levels at the beginning of each session (B = 1.54, SE = 0.68, β = 0.21, t(40) = 2.26, p = 0.030).”
    Quote in the paperSubstantial claim
  • Findingown data

    Time significantly predicted COMT DNA methylation levels in the linear mixed model.

    “In a linear mixed model with the predictors stimulation (anodal, sham), session (1, 2) and time (hours), stimulation (B = 0.98, SE = 0.42, β = 0.13, t(457) = 2.34, p = 0.020) and time (B = 0.65, SE = 0.17, β = 0.06, t(457) = 3.75, p < 0.001) predicted DNAm levels significantly”
    Quote in the paperSubstantial claim
  • May combine two claims. 20
  • Classified as probably not a substantial claim. 34
  • The quote only partly states this claim. 1

  • May combine two claims.

    Findingown data

    During session 2, the number of correct trials increased significantly from task block 1 to 3 and from 2 to 3, but not from 1 to 2.

    “During session 2, there was a significant increase from task block 1 to task block 3 (t(41) = −2.63 p = 0.012, |d| = 0.41) and from task block 2 to task block 3 (t(41) = −2.98 p = 0.005, |d| = 0.46), but not from task block 1 to task block 2 (t(41) = −0.33 p = 0.74).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Session significantly predicted task performance, with increasing number of correct trials from the first to the second session.

    “Task block (B = 3.80, SE = 0.99, β = 0.24, t(202) = 3.84, p < 0.001) and session (B = 9.53, SE = 0.79, β = 0.60, t(202) = 12.07, p < 0.001) significantly predicted task performance due to an increasing number of correct trials over the course of the three task blocks within each session and from the first to the second session, respectively.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    During session 1, the number of correct trials increased significantly from task block 1 to 2, from 2 to 3, and from 1 to 3.

    “Follow-up t-tests showed significant increases in the number of correct trials from task block 1 to task block 2 (t(41) = −2.86, p = 0.007, |d| = 0.44), from task block 2 to task block 3 (t(41) = −3.20, p = 0.003, |d| = 0.49) and from task block 1 to task block 3 (t(41) = −5.94, p < 0.001, |d| = 0.92) during session 1.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    There was an increase in negative affect by trend during the first, but not during the second experimental session.

    “Similar to the observed changes in cortisol and DNAm levels, there was also an increase in negative affect by trend during the first, but not during the second experimental session.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    These data support the notion of dynamic DNA methylation in response to mental stress that is associated with changes in cortisol levels and can be modulated by tDCS.

    “These data support the notion of dynamic DNAm in response to mental stress that is associated with changes in cortisol levels and can be modulated by tDCS.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Previous studies have associated differential DNAm patterns with diseases, such as post-traumatic stress disorder, which can be triggered by the experience of a single stressful life event.

    “However, previous studies have associated differential DNAm patterns with diseases, such as post-traumatic stress disorder, which can be triggered by the experience of a single stressful life event [42].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [42] Yehuda, 2009, The relevance of epigenetics to PTSD: Implications for the DSM-V, J. Trauma. Stress source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    COMT is involved in the degradation of dopamine and plays a critical role in cognitive processes and executive functioning.

    “COMT is involved in the degradation of dopamine and, therefore, plays a critical role in cognitive processes and executive functioning [19,20].”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [19] Schacht, 2016, COMT val158met moderation of dopaminergic drug effects on cognitive function: A critical review, Pharm. J.

      Not checked in the free run.

    • cites [20] Geller, 2017, Associations of the COMT Val158Met polymorphism with working memory and intelligence–A review and meta-analysis, Intelligence source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    A titration assay using standardized bisulfite-converted control DNA samples with established DNA methylation levels was performed to detect disparate amplification of unmethylated DNA fragments.

    “To detect disparate amplification of unmethylated DNA fragments, a titration assay using standardized bisulfite-converted control DNA samples (EpiTect Control DNA, Qiagen, Hilden, Germany) with established DNAm levels of 0%, 25%, 50%, 75%, and 100% DNAm was performed.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    The study used a single-blind, sham-controlled cross-over design.

    “The study followed a single-blind, sham-controlled cross-over design.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Study design and conductcitation

    The severity of multicollinearity was assessed by VIF, and to assure VIF <10, the interaction of stimulation and session and the three-way interaction were eliminated from models for task performance, DNA methylation and cortisol levels.

    “The severity of multicollinearity was assessed by the variance inflation factor (VIF). To assure a VIF < 10, the interaction of stimulation and session and, hence, the three-way interaction of stimulation, session, and time (or task block) was eliminated from the models for task performance, DNAm and cortisol levels [38,39].”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [38] Menard, S. (1995). An introduction to logistic regression diagnostics. Applied Logistic Regression Analysis, Sage.

      Not checked in the free run.

    • cites [39] Alin, 2010, Multicollinearity, Wiley Interdiscip. Rev. Comput. Stat. source

      Not checked in the free run.

  • May combine two claims.

    Study design and conductown data

    The two randomized stimulation-order groups showed no significant differences with respect to age, years of education, math performance, body mass index, global severity index, or COMT Val108/158Met genotype.

    “The two resulting groups showed no significant differences with respect to age (t(40) = 1.52, p = 0.14), years of formal education (t(40) = 1.06, p = 0.30), math performance at school (t(36) = −0.54, p = 0.59), body mass index (t(40) = −0.24, p = 0.81), global severity index (SCL-90-R) (t(40) = 0.59, p = 0.56), or COMT Val108/158Met genotype (x2 = 0.15, p = 0.93).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductown data

    Cortisol concentrations were determined in duplicates, and the mean coefficient of variation was below 10%.

    “Cortisol concentrations were determined in duplicates, and the mean coefficient of variation was below 10%.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    The order of stimulation was randomized and counterbalanced across participants.

    “The order of stimulation was randomized and counterbalanced across participants.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    The effect of PASAT performance on COMT DNA methylation over time differs between the first and second session, and the tDCS effect occurs between and not within the interventions.

    “This indicates that the effect of the PASAT performance on COMT DNAm over time differs between the first and the second session and that the tDCS effect occurs between and not within the interventions.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    An increase in OXTR DNAm was observed already 10 min after exposure to the TSST.

    “An increase in OXTR DNAm was observed already 10 min after the exposure to the TSST [11]”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [11] Unternaehrer, 2012, Dynamic changes in DNA methylation of stress-associated genes (OXTR, BDNF) after acute psychosocial stress, Transl. Psychiatry source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    DNA methylation plays an important role in gene regulation.

    “Currently, one of the best studied epigenetic modifications is DNA methylation (DNAm), which plays an important role in gene regulation [1].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [1] Klose, 2006, Genomic DNA methylation: The mark and its mediators, Trends Biochem. Sci. source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    DNAm levels have been regarded as rather stable, long-term epigenetic marks in somatic cells, which might even be maintained over numerous cell divisions.

    “In fact, DNAm levels have been regarded as rather stable, long-term epigenetic marks in somatic cells, which might even be maintained over numerous cell divisions [41].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [41] Reik, 2007, Stability and flexibility of epigenetic gene regulation in mammalian development, Nature source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given

    Each DNA methylation sample was analyzed twice, and samples with a deviation ≥3% between duplicates were repeated.

    “Each sample was analyzed twice, and the mean percentage was used for further analysis. Samples with a deviation ≥ 3% between duplicates were repeated.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    tDCS has been shown to modulate neuroplasticity.

    “transcranial direct current stimulation (tDCS), a non-invasive brain stimulation technique, which has been shown to modulate neuroplasticity [12].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [12] Nitsche, 2008, Transcranial direct current stimulation: State of the art 2008, Brain Stimul. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    To ensure blinding effectiveness, participants were asked for tDCS adverse effects at the end of each session.

    “To ensure blinding effectiveness, participants were asked for tDCS adverse effects at the end of each session (see Supplementary Table S1).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductown data

    The study was approved by the University of Tübingen local ethics committee and conducted in accordance with the Declaration of Helsinki.

    “Prior to study inclusion, all participants gave written informed consent to the experimental procedure approved by the University of Tübingen local ethics committee. The study was conducted in accordance with the Declaration of Helsinki in its latest version.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Venous catheter placement was done with a relatively short interval before the first blood sample was collected, which could have affected baseline measurements.

    “Nevertheless, venous catheter placement was done with a relatively short interval before the first blood sample was collected, which could have affected baseline measurements.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisereasoning

    Alterations in blood cell composition might affect DNAm.

    “One confounding factor which might affect DNAm are alterations in blood cell composition.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    Taken together, these data support the hypothesis that dynamic epigenetic modifications are invoked immediately by exposure to stress and associated with the released stress hormones.

    “Taken together, these data support the hypothesis that dynamic epigenetic modifications are invoked immediately by exposure to stress and associated with the released stress hormones.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    In the brain, the primarily expressed mRNA encodes the membrane-bound isoform (MB-COMT); however, there is evidence that the S-COMT form is also expressed to a lower extent.

    “Furthermore, in the brain, the primarily expressed mRNA is encoding the membrane-bound isoform (MB-COMT); however, there is evidence that, to a lower extent, the S-COMT form is also expressed [52].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [52] Tenhunen, 1994, Genomic organization of the human catechol O-methyltransferase gene and its expression from two distinct promoters, Eur. J. Biochem. source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Interpretationreasoning

    Being already mentally prepared to encounter a difficult task that comes along with frequent negative feedback might attenuate the stress experience.

    “Being already mentally prepared to encounter a difficult task that comes along with frequent negative feedback might attenuate the stress experience.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationreasoning

    Changes in COMT expression levels could consequently affect catecholamine concentrations.

    “and changes in its expression levels could consequently affect catecholamine concentrations.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: The COMT enzyme is involved in the degradation of catecholamines, such as dopamine.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Changes in FKBP5 gene expression occur within 70 min.

    “and changes in FKBP5 gene expression within 70 min [43].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [43] Poidinger, 2015, FKBP5 genotype-dependent DNA methylation and mRNA regulation after psychosocial stress in remitted depression and healthy controls, Int. J. Neuropsychopharmacol. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). The quote only partly states this claim.

    Study design and conductreasoning

    This potential difference in cumulated cortisol excretion was not captured by the experimental design.

    “This was not captured by our experimental design”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationreasoning

    It is possible that active and sham stimulated subjects differed in the cumulated amount of cortisol excretion over the time of the experiment.

    “it is possible that these differed in the cumulated amount of cortisol excretion over the time of the experiment.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    In a mouse model, activity enhancing tDCS can lead to alterations in histone modifications, chromatin remodeling and changes in gene expression.

    “It has been previously shown in a mouse model that activity enhancing tDCS can lead to alterations in histone modifications, chromatin remodeling and changes in gene expression [60].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [60] Podda, 2016, Anodal transcranial direct current stimulation boosts synaptic plasticity and memory in mice via epigenetic regulation of Bdnf expression, Sci. Rep. source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    Participants are adapting to the task, which is why a stress response is only elicited when the task is unfamiliar.

    “Probably, participants are adapting to the task, which is why a stress response is only elicited when the task is unfamiliar.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Previous findings report DNAm differences after glucocorticoid exposure.

    “This is also in line with previous findings reporting DNAm differences after glucocorticoid exposure [44,45].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [44] Klengel, 2013, Allele-specific FKBP5 DNA demethylation mediates gene–childhood trauma interactions, Nat. Neurosci. source

      Not checked in the free run.

    • cites [45] Braun, 2019, Genome-wide DNA methylation investigation of glucocorticoid exposure within buccal samples, Psychiatry Clin. Neurosci. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationreasoning

    Since cortisol can cross the blood brain barrier, it might elicit similar effects on DNAm in neuronal tissue.

    “Since cortisol can cross the blood brain barrier, it might elicit similar effects on DNAm in neuronal tissue.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: Cortisol can cross the blood brain barrier.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    A modulation of the stress response by brain stimulation has been reported previously.

    “but a modulation of the stress response by brain stimulation has been reported previously [58,59]”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [58] Brunoni, 2013, Polarity-and valence-dependent effects of prefrontal transcranial direct current stimulation on heart rate variability and salivary cortisol, Psychoneuroendocrinology source

      Not checked in the free run.

    • cites [59] Sarkar, 2014, Cognitive enhancement or cognitive cost: Trait-specific outcomes of brain stimulation in the case of mathematics anxiety, J. Neurosci. source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    The COMT gene interacts with cognitive performance and tDCS effects.

    “Furthermore, the COMT gene interacts with cognitive performance and tDCS effects, supporting its eligibility as candidate gene [16,49].”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [16] Wiegand, 2016, Genetic Modulation of Transcranial Direct Current Stimulation Effects on Cognition, Front. Hum Neurosci. source

      Not checked in the free run.

    • cites [49] Hosak, 2007, Role of the COMT gene Val158Met polymorphism in mental disorders: A review, Eur. Psychiatry source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationcitation

    If COMT DNAm is affected to a similar extent in neuronal tissue, effects on COMT expression levels in the brain might be diverse.

    “Therefore, if COMT DNAm is affected to a similar extent in neuronal tissue, effects on COMT expression levels in the brain might be diverse [53].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [53] Jjingo, 2012, On the presence and role of human gene-body DNA methylation, Oncotarget source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    There are two isoforms of COMT regulated by two different promoters.

    “There are two isoforms of COMT regulated by two different promotors.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [52] Tenhunen, 1994, Genomic organization of the human catechol O-methyltransferase gene and its expression from two distinct promoters, Eur. J. Biochem. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The PASAT task is originally designed as a measure of information processing ability.

    “Of note, the PASAT task is originally designed as a measure of information processing ability [54].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [54] Gronwall, 1977, Paced auditory serial-addition task: A measure of recovery from concussion, Percept. Mot. Ski. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Previous research suggests that activity-enhancing stimulation applied over the left dorsolateral prefrontal cortex increases cognitive control over emotion and, thereby, leads to stabilization in affect.

    “Previous research suggests that activity-enhancing stimulation applied over the left dorsolateral prefrontal cortex increases cognitive control over emotion and, thereby, leads to stabilization in affect [56,57].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [56] Plewnia, C., Schroeder, P.A., Kunze, R., Faehling, F., and Wolkenstein, L. (2015). Keep calm and carry on: Improved frustration tolerance and processing speed by transcranial direct current stimulation (tDCS). PLoS ONE, 10. source

      Not checked in the free run.

    • cites [57] Wolkenstein, 2013, Amelioration of cognitive control in depression by transcranial direct current stimulation, Biol. Psychiatry source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Interpretationreasoning

    DNAm measured in whole blood might be influenced by cell type composition.

    “DNAm measured in whole blood might be influenced by cell type composition.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Epigenetic patterns are cell type specific.

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    Studies investigating stress-induced immunological reactions report changes in leukocyte counts after severe physical or acute psychological stress, which is often accompanied by increased glucocorticoid levels.

    “Studies investigating stress-induced immunological reactions report changes in leukocyte counts after severe physical or acute psychological stress, which is often accompanied by increased glucocorticoid levels [64].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [64] Koch, 2010, Immune response to exercise, Braz. J. Biomotricity

      Not checked in the free run.

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    Whether these effects can already occur within a narrow time frame of two hours is disputed and potentially species specific.

    “Whether these effects can already occur within a narrow time frame of two hours is disputed and potentially species specific [65].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [65] Davis, 2008, The use of leukocyte profiles to measure stress in vertebrates: A review for ecologists, Funct. Ecol. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    A polymorphism within BDNF has been shown to interact with the effects of tDCS.

    “In particular, a polymorphism within BDNF, the gene encoding the brain-derived neurotrophic factor, has been shown to interact with the effects of tDCS [16].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [16] Wiegand, 2016, Genetic Modulation of Transcranial Direct Current Stimulation Effects on Cognition, Front. Hum Neurosci. source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    According to the iMETHYL database, the methylation level of the investigated CpG sites in the COMT gene promoter region is very similar between neutrophils and monocytes but substantially lower for CD4-positive T-lymphocytes.

    “According to the iMETHYL database, which provides cell type specific DNAm patterns based on a Japanese population, the methylation level of the investigated CpG sites in the COMT gene promoter region is very similar between neutrophils and monocytes but substantially lower for CD4-positve T-lymphocytes [61,62,63].”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [61] Komaki, 2018, iMETHYL: An integrative database of human DNA methylation, gene expression, and genomic variation, Hum. Genome Var. source

      Not checked in the free run.

    • cites [62] Hachiya, 2017, Genome-wide identification of inter-individually variable DNA methylation sites improves the efficacy of epigenetic association studies, NPJ Genom. Med. source

      Not checked in the free run.

    • cites [63] Tohoku, I. (2019, March 21). Medical Megabank Organization, iMETHYL Database; over 100 Japanese whole Genome DNA Methylation Database from Monocytes, CD4+ T Cells, and Neutrophils. Available online: http://imethyl.iwate-megabank.org/.

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Previous studies have shown that DNAm might not only change in association with stress exposure but also in response to different psychological treatments, such as cognitive-behavioral therapy (CBT).

    “Previous studies have shown that DNAm might not only change in association with stress exposure but also in response to different psychological treatments, such as cognitive-behavioral therapy (CBT) [68].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [68] Schiele, 2020, The applied implications of epigenetics in anxiety, affective and stress-related disorders-A review and synthesis on psychosocial stress, psychotherapy and prevention, Clin. Psychol. Rev. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    In a mouse model, previous research has observed differences in epigenetic patterns of the BDNF gene in response to tDCS.

    “Moreover, in a mouse model, previous research has observed differences in epigenetic patterns of the BDNF gene in response to tDCS [60].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [60] Podda, 2016, Anodal transcranial direct current stimulation boosts synaptic plasticity and memory in mice via epigenetic regulation of Bdnf expression, Sci. Rep. source

      Not checked in the free run.

47 of 47

  • The paper relies on this without support.

    Few studies have reported dynamic changes in DNAm in response to a mental stress paradigm.

    “To date, few studies have reported dynamic changes in DNAm in response to a mental stress paradigm.”

  • The paper relies on this without support.

    Little is known about the time frame of the formation of these methylation changes and the amount of stress load required to induce these changes.

    “Yet, little is known about the time frame of the formation of these methylation changes and the amount of stress load required to induce these changes.”

  • The paper relies on this without support.

    Cortisol can cross the blood brain barrier.

    “Since cortisol can cross the blood brain barrier”

  • The paper relies on this without support.

    The COMT enzyme is involved in the degradation of catecholamines, such as dopamine.

    “The COMT enzyme is involved in the degradation of catecholamines, such as dopamine”

  • The paper relies on this without support.

    The PASAT presents a relatively mild stressor.

    “Considering that the PASAT only presents a relatively mild stressor”

  • The paper relies on this without support.

    Epigenetic patterns are cell type specific.

    “Since epigenetic patterns are cell type specific”

  • The authors state this as an assumption.

    tDCS effects have small to intermediate effect sizes.

    “we can assume small to intermediate effect sizes [69]”

  • The paper relies on this without support.

    This is the first human study investigating the effects of tDCS on DNAm changes.

    “this is the first human study investigating the effects of tDCS on DNAm changes”
8 of 8

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • In depression, hypermethylation of COMT is associated with MDD, and often correlated with stress exposure and treatment response.

    “Studies in depression show that hypermethylation of COMT is associated with MDD, and often correlated with stress exposure and treatment response”

    From The clinical use of epigenetics in psychiatry: a narrative review of epigenetic mechanisms, key candidate genes, and precision psychiatry (2025) · cites this paper as [78]

    The Discussion reports increased COMT promoter methylation after a stressful task, correlated with increased salivary cortisol. But the Participants section says people with a history of mental illness were excluded; the paper does not establish an association between COMT hypermethylation and MDD or examine depression treatment response. (LLM, conf=0.88)

    Experimental — accuracy not yet measured

Statistics that don’t add up

Each reported p-value recomputed from its test statistic and degrees of freedom.

  • Inconsistent p-value: t(40) = −0.39, p = .97

    Minor

    The paper reports p = .97 here; recomputing from the test statistic and its degrees of freedom gives p = .699 (two-tailed). The check cannot tell whether the statistic or the p-value is the misprint.

    Reported p = .97 · recomputed p = .699

1 of 1

Statistics recomputed

Every reported test result, recomputed from its test statistic and degrees of freedom. A result shows as not adding up only once an independent check has confirmed it.

  • Doesn’t add upt(40) = -0.39reported p = .97 · recomputed p = .699

    “t(40) = −0.39, p = .97”

  • Not confirmedt(41) < -2.48reported p < .017 · recomputed p = .017

    “t(41) < −2.48, p < .017”

  • Consistentt(40) = 1.13reported p = .26 · recomputed p = .265

    “t(40) = 1.13, p = .26”

  • Consistentt(40) = 1.52reported p = .14 · recomputed p = .136

    “t(40) = 1.52, p = .14”

  • Consistentt(40) = 1.06reported p = .3 · recomputed p = .296

    “t(40) = 1.06, p = .30”

  • Consistentt(36) = -0.54reported p = .59 · recomputed p = .593

    “t(36) = −0.54, p = .59”

  • Consistentt(40) = -0.24reported p = .81 · recomputed p = .812

    “t(40) = −0.24, p = .81”

  • Consistentt(40) = 0.59reported p = .56 · recomputed p = .559

    “t(40) = 0.59, p = .56”

  • Consistentt(202) = 3.84reported p < .001 · recomputed p < .001

    “t(202) = 3.84, p < .001”

  • Consistentt(202) = 12.07reported p < .001 · recomputed p < .001

    “t(202) = 12.07, p < .001”

  • Consistentt(202) = -2.11reported p = .036 · recomputed p = .036

    “t(202) = −2.11, p = .036”

  • Consistentt(202) = -0.4reported p = .69 · recomputed p = .690

    “t(202) = −0.40, p = .69”

  • Consistentt(202) = 1.19reported p = .23 · recomputed p = .235

    “t(202) = 1.19, p = .23”

  • Consistentt(41) = -2.86reported p = .007 · recomputed p = .007

    “t(41) = −2.86, p = .007”

  • Consistentt(41) = -3.2reported p = .003 · recomputed p = .003

    “t(41) = −3.20, p = .003”

  • Consistentt(41) = -5.94reported p < .001 · recomputed p < .001

    “t(41) = −5.94, p < .001”

  • Consistentt(199) = -2.45reported p = .015 · recomputed p = .015

    “t(199) = −2.45, p = .015”

  • Consistentt(199) = -3.84reported p < .001 · recomputed p < .001

    “t(199) = −3.84, p < .001”

  • Consistentt(199) = 1.59reported p = .11 · recomputed p = .113

    “t(199) = 1.59, p = .11”

  • Consistentt(199) = 3.05reported p = .003 · recomputed p = .003

    “t(199) = 3.05, p = .003”

  • Consistentt(199) = -1.85reported p = .065 · recomputed p = .066

    “t(199) = −1.85, p = .065”

  • Consistentt(20) = -1.95reported p = .066 · recomputed p = .065

    “t(20) = −1.95, p = .066”

  • Consistentt(20) = -0.38reported p = .71 · recomputed p = .708

    “t(20) = −0.38, p = .71”

  • Consistentt(20) = -1.16reported p = .26 · recomputed p = .260

    “t(20) = −1.16, p = .26”

  • Consistentt(20) = 0.13reported p = .9 · recomputed p = .898

    “t(20) = 0.13, p = .90”

  • Consistentt(457) = 2.34reported p = .02 · recomputed p = .020

    “t(457) = 2.34, p = .020”

  • Consistentt(457) = 3.75reported p < .001 · recomputed p < .001

    “t(457) = 3.75, p < .001”

  • Consistentt(457) = -0.37reported p = .71 · recomputed p = .712

    “t(457) = −0.37, p = .71”

  • Consistentt(457) = -3.77reported p < .001 · recomputed p < .001

    “t(457) = −3.77, p < .001”

  • Consistentt(41) = -4.3reported p < .001 · recomputed p < .001

    “t(41) = −4.30, p < .001”

  • Consistentt(40) = 2.26reported p = .03 · recomputed p = .029

    “t(40) = 2.26, p = .030”

  • Consistentt(20) = -2.95reported p = .008 · recomputed p = .008

    “t(20) = −2.95, p = .008”

  • Consistentt(20) = -0.13reported p = .9 · recomputed p = .898

    “t(20) = −0.13, p = .90”

  • Consistentt(121) = -3.4reported p < .001 · recomputed p < .001

    “t(121) = −3.40, p < .001”

  • Consistentt(121) = 1.9reported p = .06 · recomputed p = .060

    “t(121) = 1.90, p = .060”

  • Consistentt(121) = 0.76reported p = .45 · recomputed p = .449

    “t(121) = 0.76, p = .45”

  • Consistentt(121) = -0.54reported p = .59 · recomputed p = .590

    “t(121) = −0.54, p = .59”

  • Consistentt(121) = -2.6reported p = .011 · recomputed p = .010

    “t(121) = −2.60, p = .011”

  • Consistentt(41) = -2.5reported p = .017 · recomputed p = .017

    “t(41) = −2.50, p = .017”

  • Consistentt(41) = -0.26reported p = .8 · recomputed p = .796

    “t(41) = −0.26, p = .80”

40 of 40

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Age-comparison t statistic and p-value disagree

    Minor

    For 40 degrees of freedom, a two-sided t-test with t = −0.39 gives p ≈ 0.70, not 0.97.

  • Task-block interaction has opposite coefficient signs

    Minor

    The unstandardized and standardized estimates for the same session-by-task-block interaction have opposite signs. Standardizing a coefficient cannot reverse its direction.

2 of 2

Cites

Flagged first, then by citations

6 of the 73 references couldn’t be matched to a known paper.

Research works printed but not linked: 2

  1. Pinheiro, J., and Bates, D. (2006). Mixed-Effects Models in S and S-PLUS, Springer Science & Business Media.
  2. Lejuez, 2003, A modified computer version of the Paced Auditory Serial Addition Task (PASAT) as a laboratory-based stressor, Behav. Ther., 26, 290

Cited by

Sorted by citations

Related

Authors
Wiegand, A., Blickle, A., Brückmann, C., Weller, S., Nieratschker, V., & Plewnia, C.
Affiliations
Universitätsklinikum Tübingen (DE)
Corresponding author
Wiegand, Ariane
ORCID
Venue
Biomolecules
Publisher
MDPI AG
Published
2021-11-19
Volume
11
Issue
11
Pages
1726
ISSN
2218-273X (ISSN-L)
DOI
10.3390/biom11111726
PMC
PMC8615564
OpenAlex
W3216145433
Semantic Scholar
f362865d42df8a4241c23f248970f81238f0d235
Cited by
19
References
73
Citations by year
2026: 3 · 2025: 4 · 2024: 5 · 2023: 5 · 2022: 2
FWCI
0.94
Type
journal article
Language
en
Open access
Licence
Locations
  • Biomolecules (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
Federal Ministry of Education and Research (FKZ 01EE1403D)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Epigenetics and DNA Methylation
  • Life Sciences › Neuroscience › Behavioral Neuroscience › Stress Responses and Cortisol
  • Life Sciences › Neuroscience › Neurology › Transcranial Magnetic Stimulation Studies
Keywords
DNA methylation, psychological stress, transcranial direct current stimulation, dorsolateral prefrontal cortex, cortisol, epigenetic biomarkers, stress-related disorders, anodal tDCS
MeSH terms
Adult, Catechol O-Methyltransferase, Humans, Hydrocortisone, Male, Promoter Regions, Genetic, Saliva, Stress, Psychological, DNA Methylation, Epigenesis, Genetic, Young Adult, Transcranial Direct Current Stimulation

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-08.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-08.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

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