In-Depth Analysis of the Plasma Proteome in ME/CFS Exposes Disrupted Ephrin-Eph and Immune System Signaling, 2021, Hanson et al

[John Mac]

Abstract:
Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a disabling disease
with worldwide prevalence and limited therapies exclusively aimed at treating symptoms.
To gain insights into the molecular disruptions in ME/CFS, we utilized an aptamer-based technology that quantified 4790 unique human proteins, allowing us to obtain the largest proteomics dataset yet available for this disease, detecting highly abundant proteins as well as rare proteins over a ninelog dynamic range.

We report a pilot study of 20 ME/CFS patients and 20 controls, all females.

Significant differences in the levels of 19 proteins between cohorts implicate pathways related to the extracellular matrix, the immune system and cell–cell communication.

Outputs of pathway and cluster analyses robustly highlight the ephrin pathway, which is involved in cell–cell signaling and regulation of an expansive variety of biological processes, including axon guidance, angiogenesis, epithelial cell migration, and immune response.

Receiver Operating Characteristic (ROC) curve analyses distinguish the plasma proteomes of ME/CFS patients from controls with a high degree of accuracy (Area Under the Curve (AUC) > 0.85), and even higher when using protein ratios (AUC up to 0.95), that include some protein pairs with established biological relevance.

Our results illustrate the promise of plasma proteomics for diagnosing and deciphering the molecular basis of ME/CFS

https://www.mdpi.com/2227-7382/9/1/6

 

[Jonathan Edwards]

Ephrin sounds an interesting new signal.

 

[Jonathan Edwards]

https://www.frontiersin.org/articles/10.3389/fimmu.2019.01473/full

Gives some background to Ephrins/Ephs.
They seem to do a lot.

 

[Hoopoe]

Going through every interaction found to be disrupted in ME/CFS patients compared to controls would distract from the purpose of this analysis. Instead, the principal conclusion is the number of significantly altered interactions that disproportionately affect a few proteins (Table 6). Indeed, CILP2 and CGA FSHB have over 7% of their total interactions inverted in our ME/CFS cohort compared to the control cohort.

CGA and FSHB constitute follicle stimulating hormone (FSH). I had FSH tested and it was very low, near zero if I recall correctly.

 

[ME/CFS Skeptic]

So if I understand correctly, this preliminary study tested 4790 unique human proteins in a small sample of 20 ME/CFS patients and 20 controls. After controlling for multiple testing, 9 proteins showed statistically significant differences between the two groups.

These are summarized as follows:

two proteins (PXDN and MXRA7) are involved in the extracellular matrix, while AIF1L plays a role in the cytoskeleton. Four of the proteins (Ephrin-A4, TIMD3, TNF SR-I, and IL-18 BPa) can be linked to the immune response, with TIMD3 and IL-18 BPa having an inhibitory role, while only the soluble form, but not the membranebound form, of TNF sR-I has an inhibitory role

The authors did some additional analyses and mention that their data points to disrupted Ephrin-Eph signalling.

The Eph proteins from the superfamily of transmembrane tyrosine kinase receptors as well as their membrane-tethered ephrin ligands appear, directly or indirectly, in many of our figures and tables

It's difficult to say what the disrupted Ephrin-Eph signalling means exactly because it is involved in so many different bodily processes. Will be interesting to see if the results pointing towards Ephrin can be confirmed in larger studies.

 

[wigglethemouse]

This group has been very good at sharing data in supplementary files. For anyone interested there are two Excel files and one doc file in a zip archive here
https://www.mdpi.com/2227-7382/9/1/6/s1

I sorted "Supplementary File 1.xlsx" by fold change, filtered p<=0.01, and magnitude of log2(fold change)>0.5 (FC>1.42) and this is what I get. Some of these findings are discussed in the paper but I could not see GUCA2B discussed. This protein may be related to intestinal salts and water (and/or kidneys) according to gene cards, and there is a 5 fold change reduction between healthy control and ME/CFS with p=0.01. This paper although using the sample methods of sample collection as the previous paper does not list gut issues as symptom so it is not possible to tell if there is a relationship in the current data.



Here is what Gene Cards says which seems to impy possible digestion, salt, and water balance in the intestines. Not sure if I'm reading this right.
https://www.genecards.org/cgi-bin/carddisp.pl?gene=GUCA2B

Entrez Gene Summary for GUCA2B Gene
This gene encodes a preproprotein that is proteolytically processed to generate multiple protein products, including uroguanylin, a member of the guanylin family of peptides and an endogenous ligand of the guanylate cyclase-C receptor. Binding of this peptide to its cognate receptor stimulates an increase in cyclic GMP and may regulate salt and water homeostasis in the intestine and kidneys. [provided by RefSeq, Nov 2015]

GeneCards Summary for GUCA2B Gene
GUCA2B (Guanylate Cyclase Activator 2B) is a Protein Coding gene. Diseases associated with GUCA2B include Secretory Diarrhea and Colorectal Cancer. Among its related pathways are Myometrial Relaxation and Contraction Pathways and Metabolism. Gene Ontology (GO) annotations related to this gene include calcium sensitive guanylate cyclase activator activity and guanylate cyclase activator activity. An important paralog of this gene is GUCA2A.

UniProtKB/Swiss-Prot Summary for GUCA2B Gene
Endogenous activator of intestinal guanylate cyclase. It stimulates this enzyme through the same receptor binding region as the heat-stable enterotoxins. May be a potent physiological regulator of intestinal fluid and electrolyte transport. May be an autocrine/paracrine regulator of intestinal salt and water transport.

I used the string-db tool described in the paper to look at the protein relationships for GUCA2B but there is no data in the Excel spreadsheet to quantify those other proteins related to GUCA2B

 

[DMissa]

. Some of these findings are discussed in the paper but I could not see GUCA2B discussed

Q value is 0.42 which excludes it when correcting for multiple comparisons by BH method, would explain its absence from a global-level analysis using tighter Q thresholds.

 

[Hutan]

Our results illustrate the promise of plasma proteomics for diagnosing and deciphering the molecular basis of ME/CFS

I haven't read the paper yet but it looks like this group are well aware that the small sample in this study means that this is just a start. I hope they get more funding for this.

With the potential for possibly two or more illnesses being included under the ME/CFS label, it would be interesting to look at the scatter plots of some of the proteins.

 

[wigglethemouse]

I haven't read the paper yet but it looks like this group are well aware that the small sample in this study means that this is just a start. I hope they get more funding for this.

They are aware.

Our paper on proteins present in plasma from ME/CFS cases vs controls appeared today. Despite a small sample size (20/20) due to the cost of the assays, important new information was obtained about biological disruptions in ME/CFS.

According to the paper this was funded by Cornell and NIH. The NIH grant was a 5 year funded grant 2013-2017 totalling $2,956,287
Immune Cell Gene Expression And Predictive Models In CFS
https://projectreporter.nih.gov/project_info_description.cfm?aid=9042926&icde=0

As an aside, at Stanford collection of ~1000 patient samples of ME and comparison illness is complete but lacks funding to process. This has been collected for the Stanford multi-omics project led by Fereshteh Jahanbani. Proteomics is one of the omics waiting to be performed. I hope results from pilot studies like this Hanson one will open up funding paths.

 

[Hutan]

One issue is that some protein biomarkers might only be different when the body is stressed. For example, one team reported finding that, in healthy people, plasma Cys34 changed to its oxidised form when muscles were worked, but it didn't in people with ME/CFS. Surely there will be proteins that are permanently different, but it's something to keep in mind I guess, if one group finds a difference and another doesn't.

 

[Milo]

Did anyone notice that dr Levine picked patients meeting the Fukuda criteria? This is unusual unless they used a cohort of patients whose blood was sitting in the freezer for a while

 

[Ravn]

Did anyone notice that dr Levine picked patients meeting the Fukuda criteria?

Fukuda would be fine if they made sure it was Fukuda with PEM. Which I'm hoping Dr Levine would have done. But unfortunately not made clear in the paper.

Can anyone explain what the significance of protein ratios is and how they relate to "biological relevance"?

 

[Snow Leopard]

This is my confirmation bias talking, but I am very excited about this study. I have been trying to explain links as to why certain neurological syndromes (GBS for example) often lead to long term CFS-like symptoms, despite apparent neurological recovery. This is why I've been interested in axonal guidance pathways and cellular adhesion pathways (along with the fact that viruses like to bind to certain receptors in these pathways...)

I'm going to be poring over this one for a while...

Oh and by the way,
Ephrin receptor A2 is an epithelial cell receptor for Epstein-Barr virus entry
https://pubmed.ncbi.nlm.nih.gov/29292383/

side note, here is a table of EPH - Ephrin receptor interactions:
https://www.frontiersin.org/files/A...10-01473-HTML/image_m/fimmu-10-01473-t001.jpg

https://www.frontiersin.org/articles/10.3389/fimmu.2019.01473/full

Gives some background to Ephrins/Ephs.
They seem to do a lot.

Guess how Ephrins regulate cellular adhesions?
(A certain class of receptors like to co-locate with RTKs...)

Also interesting is that Ephrins are involved in "bidirectional" signalling and the fact that they play key roles in the development and function of proprioceptive and other sensory afferents.

edit - there is some good stuff and some bad stuff in the paper. The AUC for the protein pairs look a bit spurious. The small sample size is still a problem, with both false positives and false negatives. From the supplementary data, there are a few other proteins that have low P values, but borderline high Q values that are either previously of interest (HSP70), or other ephrins, and semaphorins and netrins which are also associated in the same processes as ephrins.

I also note that activation of EphB receptors leads to muscle hyperalgesia in a rat model:
https://journals.sagepub.com/doi/full/10.1177/1744806920984079

 

[Hoopoe]

I am wondering: how do you go from disruption of these proteins to an illness like ME/CFS?

Ephrins play a role in the neuronal dendrites and synapses and that seems like it might be a way.

Ephrin regulation of synapse formation, function and plasticity

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3631567/

 

[Snow Leopard]

I am wondering: how do you go from disruption of these proteins to an illness like ME/CFS?

That's the mystery isn't it? The hypothesis needs to be quite specific and tick a lot of boxes (including many things that have been ruled out).

 

[ME/CFS Skeptic]

The hypothesis needs to be quite specific and tick a lot of boxes (including many things that have been ruled out).

IMHO, the most important thing a hypothesis needs to explain is how a mechanism could cause the severe and chronic disability seen in ME/CFS while not showing any major disturbances in normal medical tests and various markers that have been tested repeatedly in ME/CFS patients. That's one of the reasons I'm not very enthusiastic about hypotheses like the one discussed here

There must be some mechanism or bodily function that we can't see or measure that is disturbed. But the interesting thing is that it doesn't leave many clues behind in the many biomarkers that modern medicine can already measure.

When we'll finally find a disruption of proteins in ME/CFS I suspect it will be more like the tip of the iceberg rather than the pathology that is causing ME/CFS. So I wouldn't think too much about how the disrupted proteins (if we find something that is robust) might cause ME/CFS and think more about what it might signal.

 

[Snow Leopard]

There must be some mechanism or bodily function that we can't see or measure that is disturbed. But the interesting thing is that it doesn't leave many clues behind in the many biomarkers that modern medicine can already measure.

This may seem surprising, but highly sensitive and specific blood based biomarkers are rare. Many diseases do not have them.

Most blood tests are quite superficial and test very little of what goes on in the body. The first is that there are tens of thousands of things to test, the second is that for a biomarker to be found in blood, it either has to be due to cells in circulation, or somehow spill over into blood (or be entirely missing from blood in the case of deficiencies). This leaves plenty of signalling problems that won't show up in blood tests.

I agree we should think about what it might signal - specifically, consider what it means on each of the biological scales - from biochemistry, to cellular chemistry, to localised tissue and so on. And most importantly, place it in context of all the other findings so far - both positive findings and everything that has been ruled out.

Nonetheless, I am reminded of the hypotheses that we were discussing a few weeks ago - which I suggested could involve dysregulated cellular adhesions (sadly I didn't mention Ephrins!). But the key mystery was still: how this led to other symptoms.
https://www.s4me.info/threads/the-possibility-of-autoimmunity-or-auto-reactivity-in-me-cfs.18640/

 

[ME/CFS Skeptic]

This may seem surprising, but highly sensitive and specific blood based biomarkers are rare. Many diseases do not have them.

Yes but in ME/CFS there aren't even non-specific biomarkers.

A person can be incredibly sick yet his main test come back pretty much normal. I'm not sure but I suspect in most other illnesses it will be easier to find abnormalities, even with low specificity, if a person has gotten so ill.

 

[Snow Leopard]

Yes but in ME/CFS there aren't even non-specific biomarkers.

I presume you are referring to sensitive blood based biomarker tests.

We do have a good candidate for a reliable biomarker (in over 10 studies): workrate at ventilatory threshold on the 2 Day CPET. The only problem is that it isn't practical for many patients to undergo.

All of this still begs the question though, is it just that we haven't found the right test(s) yet?

 

[Kitty]

There must be some mechanism or bodily function that we can't see or measure that is disturbed. But the interesting thing is that it doesn't leave many clues behind in the many biomarkers that modern medicine can already measure.

There are lots of other clues, though. I guess when doctors were figuring out what causes Type 1 diabetes, they had to rely on these to diagnose it until someone realised that blood sugar might be important and worked out how to test it. The lack of a test didn't mean that it was impossible to diagnose it, and it's possible that other indications of health vs disease used at the time looked pretty normal.

It's still possible that, when we do unpick the mechanism, it will come to look quite quickly as if it should have been much more obvious from the clues.