Changes in DNA methylation profiles of myalgic encephalomyelitis/chronic fatigue syndrome patients reflect systemic dysfunctions, Helliwell et al 2020

[John Mac]

Background
Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a lifelong debilitating disease with a complex pathology not yet clearly defined.
Susceptibility to ME/CFS involves genetic predisposition and exposure to environmental factors, suggesting an epigenetic association.
Epigenetic studies with other ME/CFS cohorts have used array-based technology to identify differentially methylated individual sites.
Changes in RNA quantities and protein abundance have been documented in our previous investigations with the same ME/CFS cohort used for this study.

Results
DNA from a well-characterised New Zealand cohort of 10 ME/CFS patients and 10 age-/sex-matched healthy controls was isolated from peripheral blood mononuclear (PBMC) cells, and used to generate reduced genome-scale DNA methylation maps using reduced representation bisulphite sequencing (RRBS).
The sequencing data were analysed utilising the DMAP analysis pipeline to identify differentially methylated fragments, and the MethylKit pipeline was used to quantify methylation differences at individual CpG sites.
DMAP identified 76 differentially methylated fragments and Methylkit identified 394 differentially methylated cytosines that included both hyper- and hypo-methylation.
Four clusters were identified where differentially methylated DNA fragments overlapped with or were within close proximity to multiple differentially methylated individual cytosines.
These clusters identified regulatory regions for 17 protein encoding genes related to metabolic and immune activity.
Analysis of differentially methylated gene bodies (exons/introns) identified 122 unique genes. Comparison with other studies on PBMCs from ME/CFS patients and controls with array technology showed 59% of the genes identified in this study were also found in one or more of these studies.
Functional pathway enrichment analysis identified 30 associated pathways.
These included immune, metabolic and neurological-related functions differentially regulated in ME/CFS patients compared to the matched healthy controls.

Conclusions
Major differences were identified in the DNA methylation patterns of ME/CFS patients that clearly distinguished them from the healthy controls.
Over half found in gene bodies with RRBS in this study had been identified in other ME/CFS studies using the same cells but with array technology.
Within the enriched functional immune, metabolic and neurological pathways, a number of enriched neurotransmitter and neuropeptide reactome pathways highlighted a disturbed neurological pathophysiology within the patient group.

https://clinicalepigeneticsjournal.biomedcentral.com/articles/10.1186/s13148-020-00960-z

 

[Simon M]

Like all Warren Tate studies, this seems to be done meticulously well, but on a very small sample. The finding that there was a 59% overlap of the specific genes identified in this study with other studies using (inferior) array-based technology, together with the clustering around types of pathways, suggest the findings might be relevant.



Hopefully this small study will help generate funding for a larger study that also looks at controls with relevant diseases.

 

[Sasha]

Like all Warren Tate studies, this seems to be done meticulously well, but on a very small sample. The finding that there was a 59% overlap of the specific genes identified in this study with other studies using (inferior) array-based technology, together with the clustering around types of pathways, suggest the findings might be relevant.



Hopefully this small study will help generate funding for a larger study that also looks at controls with relevant diseases.

Is this the sort of difference that the GWAS study would identify, @Simon M?

 

[Jonathan Edwards]

Is this the sort of difference that the GWAS study would identify, @Simon M?

This is unrelated to the GWAS study. Tate is looking at how much genes are turned on or off, not what variants of genes there are to start with.

I agree with Simon that the study seems careful. The problem for me is that it is hard to know what to make of information about control of gene expression in white blood cells (many of which are doing nothing much or going nowhere) in the absence of something weight expect that to explain, like a raised CRP or fever or cytokine production changes in metabolic rate or whatever. The key thing about the GWAS is that it looks for things that were there before the ME started and which, if different in PWME, more or less have to be implicated, even if indirectly, in mechanism.

 

[ME/CFS Skeptic]

Summary with quotes:

This is the first study of its kind to explore the reduced DNA methylome of ME/CFS patient PBMCs utilising reduced representation bisulphite sequencing (RRBS).

...

ME/CFS patients and controls were recruited from Dunedin NZ. Diagnosis of ME/CFS was made by Dr Rosamund Vallings of the Howick Health and Medical Centre, Auckland, NZ, using the Canadian consensus diagnostic criteria

...

Using DMAP analysis, 76 DNA fragments were identified as differentially methylated out of 146,575 analysed RRBS fragments (with statistical thresholds set at a raw P-value <0.05 and a minimum methylation difference of 15%).

...

We have used a stringent P-value threshold without false discovery rate correction in order to not lose true positives from this analysis, considering our sample size is low.

...

We compared the available gene lists produced by the array-based analyses [6–10] performed in these five previous investigations with those derived from our New Zealand study using RRBS. This revealed that 59% (72/122) of the genes identified in the New Zealand study had been observed in one or more of the previous studies, with 34% (42/122) observed in two comparable studies using PBMCs [8–9].

 

[Hoopoe]

The KEGG pathway ‘Circadian entrainment’ was identified in this analysis through genes associated with hypo-methylation within internal introns. This is potentially indicative of an irregular circadian rhythm being established in ME/CFS patients vs. healthy controls, especially with the hypo-methylated ryanodine receptor RYR1 having an important role setting the clock to the evening phase in response to light [46]. Our previous transcriptome study performed on the same NZ cohort of patients observed changes in the expression of genes encoding circadian rhythm-related proteins, indicating a disruption to circadian regulation [38]. The correlations across these biological investigations with patient symptoms are providing a significant crossover with the overall clinical presentation of ME/CFS, with symptoms such as fatigue, sleep and cognitive dysfunctions, flu-like symptoms and metabolic and immune disruptions all having links to disrupted circadian activity.

Severely unrefreshing sleep on some days with no apparent pattern or cause was the first symptom I had. Only later did I develop ME/CFS.

Later on the unrefreshing sleep (of lesser severity) became part of postexertional malaise.

Although I can see how for example this methylation pattern might be due to the lifestyle of patients who spend a lot of time in front of blue light emitting screens because they can't do much else.

 

[Hoopoe]

Also this summer I made sure to get a lot of sun and it seemed to be quite helpful (but I first had to get used to hot summer weather and felt worse for the first few weeks). When autumn arrived, I had a relapse.

 

[Simon M]

Is this the sort of difference that the GWAS study would identify, @Simon M?

No. These are differences in methylation of DNA - think of methylation as a tag that alters the activity of genes near the tagged DNA. So as @Jonathan Edwards says, it's about gene control (through methylation), not differences in DNA sequences that GWAS detect.

Using DMAP analysis, 76 DNA fragments were identified as differentially methylated out of 146,575 analysed RRBS fragments (with statistical thresholds set at a raw P-value <0.05 and a minimum methylation difference of 15%).

We have used a stringent P-value threshold without false discovery rate correction in order to not lose true positives from this analysis, considering our sample size is low.

Oh, that is disappointing. Though I'm surprised only 76 fragments were more than 15% different between patients and controls out of 146,575 fragments, and that they clustered in what seem to be relevant areas to mecfs.

I would still like to see a much bigger study and one with disease controls.

 

[Hoopoe]

@Simon M what do you think about the comparison to other studies? The authors say that a substantial portion of genes identified are the same across all the studies performed so far, with these studies utilizing different diagnostic criteria, recruiting patients from across a wide variety of ages and countries of residence. It seems encouraging that in all this heterogeneity there is a lot in common in terms of gene expression.

 

[Ravn]

Would be grateful for a plain English translation. Anyone?

Hopefully this small study will help generate funding for a larger study that also looks at controls with relevant diseases.

Our study demonstrates how DNA methylation has provided an imprint of multiple systemic changes in ME/CFS with links to disease pathophysiology. Comparisons with previous relevant publications have provided compelling support that the genes identified in this work are reflecting changes specific to an ME/CFS state. Many of the specific targets highlighted can now become the focus of validation and stimulation of further work to ameliorate the devastating effects of ME/CFS on those affected by the disease.

Looks like your wish is mirrored by the authors @Simon M. The bit I bolded reads to me as a plea for others elsewhere with better funding streams to follow up and expand on the results. Because funding in NZ goes like this:

The project was funded by a grant from the Healthcare Otago Charitable Trust, with support from the Associated New Zealand Myalgic Encephalomyelitis Society (ANZMES), and by generous private donations.

So, a small local charity, an even smaller patient organisation plus private donations (from what I've heard they come in dribs and drabs leaving the team to live from week to week).

@DMissa - you get a few mentions in the paper. What's your take on it?

 

[DMissa]

Hey Ravn, thanks for the tag. I hadn't seen this yet.

I've only had a quick read but the statistical approach seems to be commendable as others have already flagged. Good to see a new technical approach too.

So obviously, to discuss function/expression of specific gene products based on epigenetics, generally speaking, is inferential, so I won't go too deep. The authors have already done a nice job!

Just one or two points after first read:

It's interesting that Complex 1 overexpression is implicated again from a different angle.

The UCP2 hypermethylation is interesting. Uncoupling protein can be a major contributor to "proton leak" - pmf depletion not by ATP synthesis. In ME lymphoblasts this is elevated. If UCP is reduced in expression as is possible with the hypermethylation, it could indicate that something else is increasingly contributing towards proton leak. Could also still be a significant UCP contribution - remains to be seen.