Genetic and epigenetic regulation of Catechol-O-methyltransferase in relation to inflammation in chronic fatigue syndrome and Fibromyalgia

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. Supplementary information The online version contains supplementary material available at 10.1186/s12967-022-03662-7.

. Pain thresholds are supposed to increase while the contralateral hand is receiving tonic painful stimuli (hot water bath). Therefore, ceiling effect would mask the effect of the conditioning stimulus, as the highest possible value is reached before the CPM paradigm. In fact, CPM effect was found statistically significant only in the patient group (p<.05 at within-group paired test, when comparing thresholds before and after CPM). Data inspection revealed that this was clearly due to the ceiling effect seen in the healthy control group.
As correlation analyses showed among pain thresholds were moderately correlated in both the healthy control and the CFS/FM group (all r>.400), we used predictive values, rather than actual values, for further analyses. Briefly, we created two separated regression models (one for cold thresholds and one for heat thresholds) with pain threshold at the hand as a dependent variable, and pain threshold at the leg and neck as independent variable. The hand site was chosen as a dependent variable because it was the most reliable of the three measures, according to the interclass correlation coefficient (hand cold threshold: a=.935, p<.001; hand heat threshold: a=.915, p<.001). The models calculated a regression coefficient and returned a predicted value which was based on the pain threshold at the hand site, but considered the other two thresholds as well. In this way, we were able to create two new variables that we considered a measure for cold sensitivity and heat sensitivity, respectively. Predicted values indeed showed less ceiling effect and were less skewed towards the limit values (0°C for cold thresholds and 50°C for heat threshold). See table S1-2 below. Between-group difference were still significant at both timepoints on both independent samples t-tests, and in repeated-measure mixed linear model (Table S1-2). Standard deviation was reduced and did not overlapped with the device limit values anymore. In addition, within-group paired t-tests showed that CPM had a significant effect on both patients with CFS/FM and controls (see Table S1-2).
We believe that this statistical approach to pain thresholds is able to retrieve the most useful information without distorting the original data, and should be considered when ceiling effect is negatively influencing pain thresholds assessment.

PCR and Sequencing Primer design
For COMT DNA methylation analyses, we used PyroMark Assay Design Software 2.0 to design forward, reverse, and sequencing primers in order to assess DNA methylation in three regions of interest -MB-COMT, S-COMT, and Exon IV (where two out of three polymorphisms are located -rs4818 and rs4680). Regions of interest were selected based on previous literature, which showed these regions to influence gene expression. 2,3 Using a gradient PCR Device (Veriti 96-Well Thermal Cycler, Applied Biosystems, ThermoFisher Scientific, Belgium), we undertook the validation process to detect which annealing temperature worked best. PCR cycling protocol was set as follows: •   Figure S2-1 below report details on the primer validation procedure. We reported only two examples, in order to clarify the procedure. The same procedure applies to all primers.

Randomisation and positive controls for pyrosequencing analyses
Pyrosequencing was performed on 24-well plates using a Q24 Pyrosequencer device (Qiagen, Hilde, Germany). Sample randomisation was performed in order to reduce the bias that might have accumulated during the lab procedures, and to ensure that each plate would include both patients and controls in random order. DNA positive (highly methylated) controls used to validate the pyrosequencing. We included two wells per analysis, in different plates (see Table S2-2 below). One of the two controls in the MB-COMTa plate did not work and variability could not be calculated. Mean methylation for each positive control in the two wells, and the intersample variability. Positive controls were expected to be highly methylated (>80%) and with low variability (up to 5%). Only MB-COMTa and Exon IV did not reach 80% methylation. However, variation was small so this will unlikely impact on the analyses. Plus, even in the case of higher variation, all samples are randomised, and the repeated measure design allowed us to test each subject twice. Frequency and haplotypes for the genetic polymorphisms rs4818, rs4633, and rs4680 There was a strong linkage disequilibrium between the genetic polymorphisms rs4818, rs4680 and rs4633 (Table S2- (Table S2-4).
Haplotypes were not associated with CFS/FM as was shown by the similarities in individual haplotype frequencies and overall distribution of the haplotype frequencies (analysis via SPSS) between healthy controls and patients with CFS/FM (Table S2-5)