Epigenetic modification of the glucocorticoid receptor gene is linked to traumatic memory and post-traumatic stress disorder risk in genocide survivors

The quote only partly states this claim.

In healthy men, NR3C1 CpG3 methylation was positively correlated with brain activity during successful recognition of previously seen pictures in the pars triangularis and pars orbitalis of the inferior frontal gyrus, cuneus, and vicinity of superior temporal and superior frontal cortices, at a whole-brain FWE-corrected threshold.

“In men, we found a whole-brain, family-wise error (FWE) multiple-comparison corrected ( p FWE corrected Ͻ 0.05, p uncorrected Ͻ 0.001) positive correlation between methylation levels at the CpG3 site of the NR3C1 promoter and brain activity related to the successful recognition of previously seen pictures in the pars triangularis and pars orbitalis of the inferior frontal gyrus, in the cuneus, and”

• AssumptionRests on an assumption: DNA methylation measured in saliva reflects the epigenetic state relevant to memory functions and PTSD risk.

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

In healthy subjects, individual NR3C1 CpG3 methylation levels were not significantly correlated with encoding-related brain activity.

“Moreover, we did not find significant correlations of individual methylation levels with encoding-related brain activity.”

• AssumptionRests on an assumption: DNA methylation measured in saliva reflects the epigenetic state relevant to memory functions and PTSD risk.

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

In the Rwandan sample, age was not significantly associated with NR3C1 DNA methylation in either men or women.

“Furthermore, age was not significantly associated with DNA methylation in either gender ( p ϭ 0.10 and 0.14 for men and women, respectively).”

• AssumptionRests on an assumption: DNA methylation measured in saliva reflects the epigenetic state relevant to memory functions and PTSD risk.

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

In healthy subjects, free-recall performance was not significantly related to NR3C1 CpG3 methylation in either men or women.

“Free-recall performance was not related to methylation levels at NR3C1 CpG3 (men: ␤ ϭ Ϫ0.291, p ϭ 0.16; women: ␤ ϭ Ϫ0.048, p ϭ 0.74).”

• AssumptionRests on an assumption: DNA methylation measured in saliva reflects the epigenetic state relevant to memory functions and PTSD risk.

May combine two claims.

In healthy men, NR3C1 CpG3 methylation was significantly associated with arousal during encoding of negative pictures, but not neutral or positive pictures.

“Furthermore, DNA methylation at this site in men was significantly associated with arousal during encoding of negative pictures, but not of neutral or positive pictures (Table 3).”

• AssumptionRests on an assumption: DNA methylation measured in saliva reflects the epigenetic state relevant to memory functions and PTSD risk.

The quote only partly states this claim.

In healthy women, NR3C1 CpG3 methylation was not significantly associated with recognition-related brain activity.

“We did not observe significant associations between methylation levels at the CpG3 site and brain activity in females.”

• AssumptionRests on an assumption: DNA methylation measured in saliva reflects the epigenetic state relevant to memory functions and PTSD risk.

May combine two claims.

DNA methylation at the NGFI-A binding site of NR3C1 negatively correlates with NR3C1 expression in saliva, for both exon 1F and total NR3C1, in males and females.

“exon 1F and total NR3C1 expression (␤ ϭ Ϫ0.812, p ϭ 0.001 and ␤ ϭ Ϫ0.759, p ϭ 0.003, respectively; in both males and females, p Յ 0.05).Thus, DNA methylation at the NGFI-A transcriptional factor-binding site of NR3C1 correlated negatively with NR3C1 expression in saliva.”

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

In healthy Swiss women, NR3C1 CpG3 methylation is not negatively associated with recognition memory performance.

“Recognition of all pictures ␤ 0.065 Ϫ0.455 p value 0.667 0.012*”

• Also stated in: § Discussion · hedged

  “Recognition of negative pictures corrected for arousal ␤ 0.289 Ϫ0.481 p value 0.050* 0.010**”

May combine two claims.

In Rwandan men, NR3C1 CpG3 methylation is negatively associated with lifetime PTSD risk (nominal p=0.008, β=−0.519), while in women the association is not significant (p=0.243, β=−0.170).

“Lifetime PTSD risk was assessed against NR3C1 CpG3 DNA methylation and the number of lifetime traumatic event types via binary logistic regression (n males ϭ 83, p nominal_males ϭ 0.008, ␤ males ϭ Ϫ0.519; n females ϭ 69, p nominal_females ϭ 0.243, ␤ females ϭ Ϫ0.170).”

The quote only partly states this claim.

In healthy men, NR3C1 CpG3 methylation is positively correlated with brain activity in the right ventrolateral prefrontal cortex (pars triangularis and pars orbitalis of the inferior frontal gyrus) and the cuneus during successful recognition of previously seen pictures.

“Displayed are voxels with a positive correlation between methylation values (at NR3C1_CpG3) and activity, using color-coded t values.The blue circles show the activation in the pars triangularis and pars orbitalis of the inferior frontal gyrus (centered at 44, 25, Ϫ4; peak p FWE corrected Ͻ 0.05; displayed at p uncorrected Ͻ 0.001; a randomeffects, linear-regression model).”

May combine two claims.

In Rwandan male genocide survivors, NR3C1 CpG3 methylation is negatively correlated with intrusion symptoms, and this association survives Bonferroni correction for eight CpGs.

“Spearman's Ϫ0.235 Ϫ0.039 Ϫ0.355 Ϫ0.128 Ϫ0.203 Ϫ0.059 Ϫ0.118 Ϫ0.18 p value Nominal 0.037* 0.733 0.001*** 0.261 0.073 0.606 0.3 0.112 Bonferroni correction for 8 CpGs 0.296 1 0.008 1 0.584 1 1 0.896”

• Also stated in: § Discussion · plain

  “The association between CpG3 and intrusions reached Bonferronicorrected statistical significance (after correcting for 8 tests; P Bonferroni ϭ 0.008).”

May combine two claims.

In Rwandan male genocide survivors, NR3C1 CpG3 methylation is negatively correlated with hyperarousal symptoms at nominal significance, but this does not survive Bonferroni correction for eight CpGs.

“Spearman's Ϫ0.187 Ϫ0.097 Ϫ0.346 Ϫ0.038 Ϫ0.165 Ϫ0.074 Ϫ0.08 Ϫ0.053 p value Nominal 0.099 0.396 0.009** 0.74 0.145 0.514 0.483 0.645 Bonferroni correction for 8 CpGs 0.792 1 0.072 1 1 1 1 1”

May combine two claims. The quote only partly states this claim.

In healthy Swiss men, higher NR3C1 CpG3 methylation is associated with worse recognition memory performance, including for all pictures and for positive, negative, and neutral pictures corrected for arousal.

“Recognition of all pictures ␤ 0.065 Ϫ0.455 p value 0.667 0.012*”

• Also stated in: § Discussion · plain

  “Recognition of negative pictures corrected for arousal ␤ 0.289 Ϫ0.481 p value 0.050* 0.010**”

• Also stated in: § Discussion · plain

  “Recognition of neutral pictures corrected for arousal ␤ Ϫ0.014 Ϫ0.643 p value 0.927 0.002**”

May combine two claims.

Increased NR3C1-1F CpG3 methylation is associated with less intrusive memory in genocide survivors and less picture recognition memory in healthy population, but only in males.

“Indeed, we found that increased NR3C1-1 F CpG3 methylation was associated with less intrusive memory (genocide survivors) and less picturerecognition memory (healthy population), but only in males.”

May combine two claims.

Methylation levels at the NR3C1-1F correlated negatively with recognition memory in men, but not in women.

“Specifically, we found that methylation levels at the NR3C1-1 F correlated negatively with recognition memory in men, but not in women.”

Classified as probably not a substantial claim.

DNA methylation of the NR3C1 promoter is inversely correlated with lifetime PTSD risk.

“Two recent studies suggested that DNA methylation of the GR gene promoter are inversely correlated with lifetime PTSD risk (Labonte ´et al., 2014;Yehuda et al., 2014).”

• cites Labonte ´et al., 2014Not checked yet

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• cites Yehuda et al., 2014Not checked yet

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May combine two claims.

NR3C1 CpG3 methylation is a stable trait that likely preexisted the traumatic event in the Rwandan population.

“These findings suggest that methylation at this specific site is a stable trait and likely preexisted the traumatic event in the Rwandan population.”

• AssumptionRests on an assumption: The healthy Swiss sample provides an appropriate reference for inferring that NR3C1 CpG3 methylation is stable and preexisted trauma in Rwandan survivors.

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

The BclI polymorphism of NR3C1 is associated with glucocorticoid sensitivity, emotional memory in healthy individuals, and traumatic memories and PTSD symptoms in critically ill patients.

“Accordingly, it has been shown that the BclI polymorphism of NR3C1 (nuclear receptor subfamily 3, group C, member 1), the gene encoding the GR, is associated with sensitivity to glucocorticoids (van Rossum et al., 2003), emotional memory in healthy individuals (Ackermann et al., 2013), traumatic memories and PTSD symptoms in critically ill patients (Hauer et al., 2011).”

• cites van Rossum et al., 2003Not checked yet

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• cites Ackermann et al., 2013Not checked yet

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• cites Hauer et al., 2011Not checked yet

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

All samples were analyzed in quadruplicates from two independent bisulfite conversions.

“All samples were analyzed in quadruplicates from two independent bisulfite conversions.”

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

All laboratory procedures were conducted in a blind, randomized order.

“All laboratory procedures were conducted in a blind, randomized order, including DNA and RNA isolations, bisulfite conversion, PCR, pyrosequencing, and expression analysis.”

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

The translated instruments' psychometric properties were investigated in a validation study with a two-week retest and cross-validation with expert rating.

“Following the translations, the psychometric properties of the translated scales were investigated in a validation study that included a retest spanning a two-week period and a cross-validation with expert rating (Neuner et al., 2008).”

• cites Neuner et al., 2008Not checked yet

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

Correlation between NR3C1 CpG3 methylation and NR3C1 expression was corrected for age independently in two genders.

“Correlation between NR3C1 CpG3 DNA methylation and NR3C1 expression was examined by linear regression model, corrected for age independently in two genders.”

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

Bonferroni correction was used to account for multiple testing.

“Bonferroni correction was implemented to account for multiple testing procedures.”

Classified as probably not a substantial claim.

PTSD symptom cluster scores were divided by the sum of lifetime traumatic event types to account for trauma load and ceiling effects.

“To account for trauma load and ceiling effects (Kolassa et al., 2010), PTSD symptom cluster scores were divided by the sum of lifetime traumatic event types (i.e., symptoms scores per traumatic event type; de Quervain et al., 2007).”

• cites Kolassa et al., 2010Not checked yet

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• cites de Quervain et al., 2007Not checked yet

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

Allele-specific DNA demethylation of FKBP5 mediates gene-childhood trauma interactions.

“Moreover, allele-specific DNA demethylation of FKBP5, a regulator of the GR complex, has been found to mediate gene-childhood trauma interactions (Klengel et al., 2013).”

• cites Klengel et al., 2013Not checked yet

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

Traumatic load measured by the number of different traumatic event types is considered more reliable than measuring the frequency of traumatic events.

“This measure is considered more reliable than assessing the frequency of traumatic events (Neuner et al., 2008).”

• cites Neuner et al., 2008Not checked yet

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Classified as probably not a substantial claim.

Lifetime PTSD status was assessed using binary logistic regression with NR3C1 CpG3 methylation as predictor and total lifetime traumatic event types as covariate.

“The relationship between DNA methylation at NR3C1 promoter and lifetime PTSD status was assessed using binary logistic regression, with NR3C1 CpG3 DNA methylation as quantitative predictor and the sum of life traumatic event types as a covariate.”

May combine two claims.

Altered HPA axis regulation and a high number of glucocorticoid receptors are pretrauma risk factors for PTSD.

“there is evidence suggesting that altered HPA axis regulation and a high number of glucocorticoid receptors (GRs) represent a pretrauma risk factor for the disorder (Yehuda, 2009;van Zuiden et al., 2011van Zuiden et al., , 2013)).”

• cites Yehuda, 2009Not checked yet

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• cites van Zuiden et al., 2011Not checked yet

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• cites van Zuiden et al., 2013Not checked yet

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PCR temperature bias and calibration were tested using calibrator samples with known methylation levels.

“Testing for PCR temperature bias and calibration was done by introducing a series of calibrator samples with known methylation levels.”

May combine two claims.

PTSD is not accompanied by increased glucocorticoid levels but by enhanced HPA axis feedback.

“In contrast to a state of chronic stress, PTSD is not paralleled by increased glucocorticoid levels (Meewisse et al., 2007), but rather by enhanced hypothalamus-pituitary-adrenal (HPA) axis feedback (Yehuda, 2002;Rohleder et al., 2004;Pitman et al., 2012).”

• cites Meewisse et al., 2007Not checked yet

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• cites Yehuda, 2002Not checked yet

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• cites Rohleder et al., 2004Not checked yet

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The NGFI-A binding site in the NR3C1 exon 1F promoter was previously reported as important for epigenetic regulation of GR gene expression.

“including the exon 1F promoter (NR3C1-1 F ) with the NGFI-A binding site, previously reported as important for epigenetic regulation of GR gene expression (Weaver et al., 2004)”

• cites Weaver et al., 2004Not checked yet

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

Only one person per household was interviewed to exclude genetic relatives from the samples.

“Additionally, to exclude genetic relatives in the samples, only one person per household was interviewed.”

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

fMRI data from one male and two female subjects were corrupted and excluded from the study.

“fMRI data from one male and two female subjects were corrupted and therefore were not included in the study.”

May combine two claims.

The present study indicates that DNA methylation at the NR3C1 exon 1F promoter is linked to traumatic memories and PTSD risk in male, but not female, genocide survivors.

“The present study indicates that DNA methylation at the NR3C1 exon 1 F promoter is linked to traumatic memories and PTSD risk in male, but not in female genocide survivors.”

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

The exon 1F region is a human orthologue of the rat exon 1-7 and was previously reported as differentially methylated.

“It spans the exon 1F region, a human orthologue of the rat exon 1-7 (Turner and Muller, 2005), previously reported as differentially methylated (Oberlander et al., 2008;McGowan et al., 2009).”

• cites Turner and Muller, 2005Not checked yet

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• cites Oberlander et al., 2008Not checked yet

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• cites McGowan et al., 2009Not checked yet

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

In male combat veterans, DNA methylation at NR3C1-1F in peripheral blood mononuclear cells is inversely correlated with PTSD risk, NR3C1 expression, cortisol decline to dexamethasone, and clinical PTSD measures.

“A recent study in male combat veterans reported that DNA methylation at NR3C1-1 F in peripheral blood mononuclear cells was inversely correlated with PTSD risk, NR3C1 gene expression, cortisol decline in response to dexamethasone, and clinical measures of PTSD, including poorer sleep (Yehuda et al., 2014).”

• cites Yehuda et al., 2014Not checked yet

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

DNA methylation of NR3C1-1B and NR3C1-1C promoters in sorted T-lymphocytes is inversely correlated with PTSD risk and morning cortisol levels in individuals with different trauma backgrounds.

“Another study reported that DNA methylation of NR3C1-1 B and NR3C1-1 C promoters in sorted T-lymphocytes is also inversely correlated with PTSD risk and morning cortisol levels in individuals with different trauma background (Labonte ´et al., 2014).”

• cites Labonte et al., 2014Not checked yet

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

Hypermethylation at the human NR3C1-1F promoter NGFI-A binding site is associated with perinatal stress and maternal depression.

“Hypermethylation at the human ortholog NR3C1-1 F promoter NGFI-A binding site was likewise associated to perinatal stress and maternal depression (Oberlander et al., 2008).”

• cites Oberlander et al., 2008Not checked yet

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May combine two claims.

DNA methylation at the NR3C1-1F promoter is preferentially related to the memory aspects of PTSD symptomatology and PTSD risk in men.

“In conclusion, we provide evidence that DNA methylation at the NR3C1-1 F promoter is preferentially related to the memory aspects of PTSD symptomatology and PTSD risk in men.”

May combine two claims.

Epigenetic marks at the NR3C1 locus are set during a sensitive period around birth and seem to remain stable throughout life in rodents and humans.

“Epigenetic marks at the NR3C1 locus are set during a sensitive period around birth and seem to remain stable throughout life in rodents (Weaver et al., 2004;Mueller and Bale, 2008;Suderman et al., 2012) and humans (Oberlander et al., 2008;McGowan et al., 2009;Radtke et al., 2011;Suderman et al., 2012).”

• cites Weaver et al., 2004Not checked yet

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• cites Mueller and Bale, 2008Not checked yet

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• cites Suderman et al., 2012Not checked yet

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Classified as probably not a substantial claim.

The study applied Bonferroni correction for multiple comparisons across the eight CpG sites and for sex and three PTSD symptom clusters.

“Bonferroni correction for 8 CpGs, sex, and 3 PTSD symptom clusters”

May combine two claims.

In rodents, specific 5' NR3C1-1 7 promoter sites are targets of DNA methylation, leading to suppressed NGFI-A binding and decreased NR3C1 gene expression.

“Studies in rodents have shown that specific 5Ј NR3C1-1 7 promoter sites are targets of DNA methylation, leading to suppressed NGFI-A binding and consequently decreased NR3C1 gene expression (Weaver et al., 2007).”

• cites Weaver et al., 2007Not checked yet

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High numbers of glucocorticoid receptors before trauma are a risk factor for developing PTSD.

“Whereas high numbers of GRs before trauma have been found to be a risk factor for developing PTSD (van Zuiden et al., 2011)”

• cites van Zuiden et al., 2011Not checked yet

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Pharmacological elevation of glucocorticoid levels during or after a traumatic event seems to prevent and reduce PTSD symptoms.

“a pharmacological elevation of glucocorticoid levels during or after a traumatic event seems to prevent and reduce PTSD symptoms (Schelling et al., 2001(Schelling et al., , 2004;;Aerni et al., 2004).”

• cites Schelling et al., 2001Not checked yet

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• cites Schelling et al., 2004Not checked yet

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• cites Aerni et al., 2004Not checked yet

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The right ventrolateral prefrontal cortex is involved in retrieval attempt and effort rather than retrieval success.

“Interestingly, the right VLPFC has been discussed as a brain region involved in retrieval attempt and effort rather than retrieval success (Taylor et al., 2004;Badre and Wagner, 2007)”

• cites Taylor et al., 2004Not checked yet

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• cites Badre and Wagner, 2007Not checked yet

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

Glucocorticoid effects on memory are more pronounced in men than in women, or even restricted to men.

“Indeed, the finding that NR3C1-1 F methylation was related to memory only in men is in line with reports that found glucocorticoid effects on memory to be more pronounced in men than in women, or even restricted to men (Andreano and Cahill, 2006;Preuss and Wolf, 2009;Cornelisse et al., 2011).”

• cites Andreano and Cahill, 2006Not checked yet

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• cites Preuss and Wolf, 2009Not checked yet

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• cites Cornelisse et al., 2011Not checked yet

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The ventrolateral prefrontal cortex is involved in the neurocircuitry of PTSD.

“Moreover, a recent meta-analysis has pointed to an involvement of the VLPFC in the neurocircuitry of PTSD (Hayes et al., 2012).”

• cites Hayes et al., 2012Not checked yet

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