Preprint Charting the Circulating Proteome in ME/CFS: Cross System Profiling and Mechanistic insights, 2025, Hoel, Fluge, Mella+

[SNT Gatchaman]

Charting the Circulating Proteome in ME/CFS: Cross System Profiling and Mechanistic insights
August Hoel; Fredrik Hoel; Sissel Elisabeth Furesund Dyrstad; Henrique Chapola; Ingrid Gurvin Rekeland; Kristin Risa; Kine Alme; Kari Sorland; Karl Albert Brokstad; Hans-Peter Marti; Olav Mella; Oystein Fluge; Karl Johan Tronstad

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a debilitating condition often triggered by infections. The underlying mechanism remains poorly understood, and diagnostic markers and effective treatments are presently lacking.

We performed aptamer-based serum proteomics in 54 ME/CFS patients and 27 healthy controls and identified 1823 of 7326 aptamers reporting differences between the groups (845 after false discovery rate (FDR) correction).

Distinct patterns of tissue- and process-specific changes were seen. There was a broad increase in secreted proteins, while intracellular proteins, e.g. from skeletal muscle, particularly showed reduction. Immune cell-specific signatures indicated immune reprogramming, including a distinct reduction in neutrophil-associated proteins. Focused secretome analysis supported intensified regulatory interactions related to immune activity, inflammation, vasculature, and metabolism. Validation of measurements using antibody-based methods confirmed findings for a selection of proteins.

The uncovered serum proteome patterns in ME/CFS patients help clarify a multifaceted pathophysiology and offer a foundation for future therapy and biomarker discovery efforts.

Link | PDF (Preprint: MedRxiv) [Open Access]

 

[Utsikt]

The patients are from RituxME and CycloME. Age and gender matched controls.

 

[Hutan]

The introduction is really nicely written, I think.

Aptamer microarray proteomics is a discovery platform capable of simultaneously measuring a high number of proteins from small sample volumes, which utilize the protein-specific binding affinity of small single-stranded DNA or RNA molecules (aptamers) 26. This technology has been used successfully to study circulatory factors involved in aging 27, kidney disease 26, diabetes 28, and other diseases. To our knowledge, it has been employed in only two previous ME/CFS studies, both using relatively small study cohorts 29,30

ref 29 Germain, A., Levine, S.M., and Hanson, M.R. (2021). In-Depth Analysis of the Plasma Proteome in ME/CFS Exposes Disrupted Ephrin-Eph and Immune System Signaling. S4ME thread

ref 30Walitt, B., Singh, K., Lamunion, S.R., Hallett, M., Jacobson, S., Chen, K., Enose-Akahata, Y., Apps, R., Barb, J.J., Bedard, P., et al. (2024). Deep phenotyping of post-infectious myalgic encephalomyelitis/chronic fatigue syndrome. S4ME thread

 

[Hutan]

That data looks to be all in the Supplementary Tables, which is great. The data is anonymised and just uses age bands.

The serum samples were analyzed using the SomaScan aptamer-based detection platform, yielding a dataset for 7326 aptamers (6494 protein targets, 6409 identified proteins) (SupplData1). In total, as many as 1823 of the aptamers showed statistically significant differences (p < 0.05) between theME/CFS group and the HC group, with 845 showing q-significant changes (SupplData2). This corresponds to 1610 different proteins (751 q-significant) demonstrating altered serum concentrations.

Very good to see that the PCA was done using all of the aptamers, unlike the study we were looking at recently that only used the features that were significantly different between the groups and then miraculously found amazing separation of the groups.

The PCA (Principal Component Analysis) plot with group overlay showed partial separation between the ME/CFS and HC subjects (Figure 1B-C; SupplData3), primarily along the PC2 dimension (y-axis, 9.6% explained variance). The groups largely overlapped across the PC1 dimension (x-axis, 14.5% explained variance), indicating that certain elements of intragroup variation are shared between the groups. This makes sense, as PC1 was more influenced by the covariate factors, age, sex, BMI, and fasting state, as indicated by the respective loadings, compared to PC2 (Figure 1C; variable, PC1, PC2, p;BMI, 0.943 -0.333, 0.106; age, -0.981, -0.195, 0.381; sex, -0.925, -0.380, 0.091; fasting, -0.830, -0.557, 0.046).

Since group separation along PC2 did not seem to be driven by these covariate factors, this may point to more disease-specific changes. Accordingly, the major PC2-driving proteins (i.e. with high loading) expressed more statistically significant differences between the two groups, compared to major PC1-driving proteins.

 

[Hutan]

Proteins that were the most different

The list of the 25 protein targets showing the sharpest increase or decrease in ME/CFS patients
indicated specific changes of key interest (Figure 1E). The proteins that showed the most decline were predominantly intracellular proteins, such as histones from the nucleus and enzymes involved in metabolic pathways. In contrast, many proteins involved in secretory and systemic regulation of the immune system and metabolism showed significant increase. Additionally, several factors related to vascular function were among the most affected proteins, either increased or decreased. Individual data are shown for a selection of the most significantly affected proteins (Figure 1F).

So we can search on them, I've written them here

Downregulated
MMP17, DPYSL3, PPM1F, WWP1, LDHB, H2AZ1, ID2, CXADR, COMP, ATP5IF1, ALDOA, CELF2, TAGLN3, ANTXR2, IFI16, VIM, YY1, GAPDH, H2BC21 (Seq 2), H2AC1, H2BC21 (Seq 1), H2BC12, H2BU1, H1-2, H1-10

Upregulated
MCTS1, FABP3, FABP4 (Seq1), FAB4 (Seq2), CRABP2, None, ERP29, CFD, LIPG, CMPK1, TMED9, C6, RARRES2, FZD8, PGRMC1, GRIA4, PCSKIN, LMAN2, IL26, CANT1, TRAPPC3, FAM20A, PSMB3, OCRL, CHST12

 

[SNT Gatchaman]

The widespread decrease of intracellular proteins released from skeletal muscle to blood may have different explanations. Possible systematic variation, such as dilution effects due to possible blood volume changes in ME/CFS, seems unlikely based on the specificity of the findings related to different tissues and processes. It could be speculated that reduced leakage of muscle proteins into the bloodstream could be associated with low physical activity and less muscle mass, but this is not convincingly supported since the data were adjusted for age, BMI, and sex, which indirectly integrates variation in muscle mass and activity. Yet, we did observe a reduction in COMP, a component of cartilage matrix that temporarily increases in serum upon physical exercise.

Alternative explanations may involve aspects of skeletal muscle tissue maintenance and protein washout, and potentially, impaired tissue perfusion causing reduced protein flux from the interstitial fluid to blood. Given that different muscle proteins have different release pathways and clearance dynamics, it seems plausible that a pathomechanism involving vascular dysfunction and microvascular shunting could lead to reduced drainage of cellular protein remains from the tissue in ME/CFS patients.

Based on the overall pattern of lower serum concentrations of many muscle-related proteins, the selective increase in myoglobin is of a certain interest, as it suggests muscle tissue oxygenation as a potential contributing factor. Nevertheless, as the increase in myoglobin was not reproduced by the antibody-based method, concern needs to be taken for the interpretation, and further investigations are required.

Myoglobin in blood is a sensitive marker for muscle injury, but since the increase in ME/CFS patients was not accompanied by increases of other markers such as CKM or LDH, it does not seem to reflect tissue damage or differences in muscle mass in this case. In support, others have found reduced blood levels of creatine kinase in ME/CFS patients. Instead, it may be speculated that the possible specific increase in myoglobin could be mechanistically associated with tissue hypoxia adaptation, serving as a compensation that contributes to improved muscle oxygenation.

The primary function of myoglobin is to store and facilitate the transfer of oxygen from the cell membrane to the mitochondria inside muscle cells. This may align with our finding that serum from ME/CFS patients contain factors that seem to induce compensatory increases in mitochondrial respiration in muscle cells. Furthermore, this may involve elements of physiological mitigation that could be beneficial in resting ME/CFS patients, when blood lactate levels usually are within the normal range. However, upon exertion, the limitations become apparent, as indicated by the abnormal rise in lactate concentration at low workloads.

Oxygen-dependent mitochondrial ATP production is also a determinant of endothelial cell control of vascular tone, and red blood cell function and deformability. Capillary alterations in skeletal muscle and changes in hypoxia-related blood factors were recently reported in long COVID patients, and roles of immune dysfunction, inefficient oxygen transport and inflammatory disequilibrium have been proposed as drivers of persisting symptoms, which may be relevant for ME/CFS. This notion is further supported by the activation of the inherent hypoxia response in muscle tissue, as the expression of WASF3, a target gene for the HIF1A transcription factor, was increased in ME/CFS patients.

 

[forestglip]

54 ME/CFS and 27 HC.

We analyzed blood serum from 54 ME/CFS patient biobank samples collected at baseline in two previous clinical intervention trials: RituxME (ClinicalTrials.gov NCT02229942, 2014–2017) 31 and CycloME (ClinicalTrials.gov NCT02444091, 2015–2020) 32 (Table 1, Figure 1A). The healthy control (HC) group consisted of 27 sex- and age-matched individuals.

6494 proteins tested. 751 with significant q values.

The serum samples were analyzed using the SomaScan aptamer-based detection platform, yielding a dataset for 7326 aptamers (6494 protein targets, 6409 identified proteins) (SupplData1). In total, as many as 1823 of the aptamers showed statistically significant differences (p < 0.05) between the ME/CFS group and the HC group, with 845 showing q-significant changes (SupplData2). This corresponds to 1610 different proteins (751 q-significant) demonstrating altered serum concentrations.

Down: intracellular proteins, including histones, metabolic enzymes | Up: immune system, metabolism | Up or down: vascular function

The proteins that showed the most decline were predominantly intracellular proteins, such as histones from the nucleus and enzymes involved in metabolic pathways. In contrast, many proteins involved in secretory and systemic regulation of the immune system and metabolism showed significant increase. Additionally, several factors related to vascular function were among the most affected proteins, either increased or decreased.

Controlling for metabotype (what is metabotype?) and SF-36 physical function score to try to control for physical activity.

775 [aptamers] were only associated with the ME/CFS diagnosis and not influenced by the metabotype nor the SF-36PF score. Enrichment analysis indicated an impact on annotated functions such as protein localization to the plasma membrane, Golgi transport, and endosomal transport.

Reduction in granulocyte associated proteins. 40% of neutrophil associated proteins were decreased. (Does this mean significantly decreased? How many were significantly increased?)

For bone marrow proteins, proteins such as PADI4, BPI, and MPO, had a sharp reduction, which may indicate altered granulocyte/neutrophil cell function. [...] The reduced amount of granulocyte proteins was not associated with abnormally low neutrophil counts (the most abundant type of granulocytes) or other leukocyte types in the patients. [...] Furthermore, comparing a list of proteins associated with neutrophil granules and stimulated neutrophil protein release, we found that about 40% or more of the proteins reported to be released by activated neutrophils showed lower serum concentrations in the ME/CFS group compared to the HC group, suggesting a suppressive effect on overall neutrophil activity.

I don't know what Ephrin is, but they said it's interesting so maybe it's interesting.

Interestingly, four members of the Ephrin subfamily A receptors (EPHA) and their ligands (EFNA) displayed concordance, supporting the involvement of altered EPHA-EFNA signaling in ME/CFS, as previously suggested (29).

29 is Germain, A., Levine, S.M., and Hanson, M.R. (2021). In-Depth Analysis of the Plasma Proteome in ME/CFS Exposes Disrupted Ephrin-Eph and Immune System Signaling. Proteomes 9, 6. https://10.3390/proteomes9010006 S4ME

They followed up this aptamer-based protein measurement method on a smaller set of proteins using an antibody-based method. It appears to be on different participants as well.

To validate and expand on findings from the aptamer-based analyses, we used antibody-based methods (ELISA and Luminex) to measure a panel of serum proteins related to immunity, inflammation, coagulation, and stressed energy metabolism. Of the 77 proteins measured on the Luminex platform (ME/CFS, n = 83; HC, n = 48), 49 proteins expressed quantifiable serum concentrations at the group level (SupplData9).

To investigate serum proteome changes in ME/CFS, we utilized 54 baseline ME/CFS patient samples from the two clinical intervention studies RituxME and CycloME, and 27 sex- and age-matched healthy controls, randomly selected from the ME/CFS biobank at Haukeland University Hospital. [...] In the validation measurements towards the end, samples were randomly picked from the ME/CFS biobank, including up to 212 ME/CFS patients and 66 healthy individuals.

Is the 212 and 66 the total number of people in the ME/CFS biobank, while 83 and 48 are the number they chose for the validation portion? It says they randomly selected participants for both stages of the study. Did they try to avoid overlap?

Looking at SupplData9, I am confused about the numbers. I see 83 ME/CFS and 29 HC, not 83 and 48. And I see data for 57 proteins, not 77.

But anyway, the validation stage seemed to mostly agree with the first stage findings:

There was a reasonably good correlation between the antibody-based and aptamer-based measurements, and importantly, significant effects in ME/CFS patients versus HC subjects were generally reproduced (Figures 6A-B). Thus, the selected antibody-based measurements validated key findings from the aptamer-based analyses. However, the increase in myoglobin was not reproduced by the antibody- based method.

I think there's been talk of GDF15 on the forum. No difference here:

For GDF15, the ELISA analysis showed no difference between the ME/CFS and HC groups, which supported the finding of the aptamer-based analysis.

MCTS1, gene involved in T-cell lymphoma, most increased overall.

The most increased protein was malignant T cell-amplified sequence 1 (MCTS1), an oncogene originally found in human T-cell lymphoma that recently was identified as a potential biomarker for COVID-19-related thrombosis (39).

39 is Qi, P., Huang, M., and Li, T. (2022). Screening the Potential Biomarkers of COVID-19-Related Thrombosis Through Bioinformatics Analysis. Front Genet 13, 889348. https://10.3389/fgene.2022.889348.

 

[hotblack]

So we can search on them, I've written them here

And here’s links to the genecards (edit: put in a spoiler tag to not clog up the discussion)

Spoiler: Genecard links

Downregulated
MMP17
DPYSL3
PPM1F
WWP1
LDHB
H2AZ1
ID2
CXADR
COMP
ATP5IF1
ALDOA
CELF2
TAGLN3
ANTXR2
IFI16
VIM
YY1
GAPDH
H2BC21
H2AC1
H2BC21 (second hit)
H2BC12
H2BU1
H1-2
H1-10

Upregulated
MCTS1
FABP3
FABP4
FAB4 (this one is invalid, I think a typo and it was a second hit for FABP4)
CRABP2
ERP29
CFD
LIPG
CMPK1
TMED9
C6
RARRES2
FZD8
PGRMC1
GRIA4
PCSKIN
LMAN2
IL26
CANT1
TRAPPC3
FAM20A
PSMB3
OCRL
CHST12

 

[SNT Gatchaman]

Controlling for metabotype (what is metabotype?)

The three metabotypes identified in A map of metabolic phenotypes in patients with myalgic encephalomyelitis/chronic fatigue syndrome (2021, JCI Insight)

I don't know what Ephrin is, but they said it's interesting so maybe it's interesting.

See In-Depth Analysis of the Plasma Proteome in ME/CFS Exposes Disrupted Ephrin-Eph and Immune System Signaling (2021, Proteomes)

 

[Hutan]

There's lots that is interesting in this paper, not least that we are seeing some replications of findings.

Myoglobin in blood is a sensitive marker for muscle injury, but since the increase in ME/CFS patients was not accompanied by increases of other markers such as CKM or LDH, it does not seem to reflect tissue damage or differences in muscle mass in this case. In support, others have found reduced blood levels of creatine kinase in ME/CFS patients. Instead, it may be speculated that the possible specific increase in myoglobin could be mechanistically associated with tissue hypoxia adaptation, serving as a compensation that contributes to improved muscle oxygenation.

It will be good to hear from the researchers what they have found since the paper was submitted for publication regarding the lack of replication from the antibody-based method.

I'm interested in this as I reliably got 3-day PEM from massages. I get brown urine from time to time, some symptoms seem to be like rhabdomyolysis.

On the question of high myoglobin and low creatine, AI says:
Low serum creatine kinase (CK) levels in the context of connective tissue diseases, even with high myoglobin, can be a complex clinical scenario. While CK is a marker for muscle damage, it doesn't always accurately reflect the severity or extent of inflammation, especially in connective tissue diseases. Myoglobin is released in larger quantities than CK during muscle damage, so high myoglobin with low CK could indicate a more subtle chronic inflammatory process or muscle injury rather than severe rhabdomyolysis.
one of the references

I wonder if some of the cohorts actually have a (mixed) connective tissue disease? It seems to me that there is a significant overlap of symptoms with ME/CFS.

 

[Jonathan Edwards]

What I see here is

1. A lot of evidence for reduced activity lowering levels of proteins.
2. The study is yet another powerful indicator that there is no inflammation or damage going on.
3. Hidden in plain sight is one increased protein of interest (the top one): MCTS1. Gene card says: "Notably, it positively regulates interferon gamma immunity to mycobacteria by enhancing the translation of JAK2 (PubMed:37875108)."

 

[hotblack]

Hidden in plain sight is one increased protein of interest (the top one): MCTS1. Gene card says: "Notably, it positively regulates interferon gamma immunity to mycobacteria by enhancing the translation of JAK2

So there could be an MCTS1 variation that rather than impairing functionality here enhances it? Another one of the various contributory factors?

 

[Jonathan Edwards]

So there could be an MCTS1 variation that rather than impairing functionality here enhances it? Another one of the various contributory factors?

It only just struck me in the last few days that finding raised levels of a protein (or RNA) in patients may be more of a sign of a genetic predisposition than a 'driving force' in a process. That came up with CD24. I could never see why an acquired disease should push up CD24 across a whole cell population. But of course if it is a disease process that is facilitated by a genetic tendency to express more CD24 it is not a problem.

Here we are looking at proteomics so a raised MCTS1 protein could again either be just that process has set up that uses MCTS1 or it could be that people who tend to express a lot of MCTS1 are at risk of flipping in to this process. Either way the MCTS1 protein is presumably part of the process (unless there is linkage disequilibrium which I doubt) but in the second case you will not find the increased protein just restricted to the cells involved in the process. Moreover, you do not need to invoke some further upstream event that drives more MCTS1 as part of the process. The more MCTS1 comes with your genes.

 

[hotblack]

It only just struck me in the last few days that finding raised levels of a protein (or RNA) in patients may be more of a sign of a genetic predisposition than a 'driving force' in a process.

That’s what I was wondering, cause or effect. Useful to know it could be either. I guess the only way we’ll know for sure is from genetic studies?

 

[Sasha]

I wonder if the lack of highly sedentary controls could be a problem here. I haven't read the paper but from a quote above:

It could be speculated that reduced leakage of muscle proteins into the bloodstream could be associated with low physical activity and less muscle mass, but this is not convincingly supported since the data were adjusted for age, BMI, and sex, which indirectly integrates variation in muscle mass and activity.

I don't think that matching for BMI is necessarily enough. I have a normal BMI but have lost muscle and gained fat, due to my extreme inactivity. One of those fancy electrical checks for body composition would have helped (the ones with proper kit where you get electrodes stuck all over you).

 

[Simon M]

It only just struck me in the last few days that finding raised levels of a protein (or RNA) in patients may be more of a sign of a genetic predisposition than a 'driving force' in a process.

Perhaps we should be cautious in relying on genetic explanations for a disease with only weak heritability.Almost everything that will come up in any GWAS will be common (so widespread in the population) and with a weak effect (though the effect is most likely to be on gene expression). Many people with the illness won’t have the risk genotype. Should we expect such a small effect to show in a study like this, particularly given the level of correction for multiple comparisons?

 

[Hutan]

The paper linked above talks about an X-linked recessive MCTS1 deficiency in males - MCTS1 is on the X chromosome.

I'm not sure how the gene works, if just one version works or if both contribute to the functionality. But, if there is an allele that increases interferon gamma responsiveness to a signal, perhaps that could be a reason for the female predominance in ME/CFS? I did see some papers that suggested that females produce more interferon gamma than men.

 

[Jonathan Edwards]

I don't think that matching for BMI is necessarily enough. I have a normal BMI but have lost muscle and gained fat, due to my extreme inactivity.

I think that is important and it may be relevant to the Beentjes proteomics findings on lipid metabolism and insulin resistance. It may be hard to factor all these things out completely though.

 

[Jonathan Edwards]

Perhaps we should be cautious in relying on genetic explanations for a disease with only weak heritability.

Maybe, but I am just making the point that if we see proteins coming up on proteomics or cell labelling studies we should not just assume that the disease is switching them on. It may be that the disease occurs in people who always switch that protein on more. The protein is still likely to be involved in the disease but we don't have look for a mysterious signal in the disease mechanism that is pushing it up. If a disease occurs just because of normal processes overstepping a threshold then there may be no other extra mysterious cause.

Common polymorphisms can have quite big effects on disease. B27 has a huge predisposing effect for Reiter's but the heritability of Reiter's is probably very low - much lower than ME/CFS. The intriguing thing is that we still have no idea why B27 does this, since it seems to function the same way as all other HLA-B alleles. But we know it has major effects on expression of a whole range of infectious disease - so it is n't to do with one magic peptide fitting somewhere.

 

[Jonathan Edwards]

I'm not sure how the gene works, if just one version works or if both contribute to the functionality. But, if there is an allele that increases interferon gamma responsively, perhaps that could be a reason for female predominance? I did see some papers that suggested that females produce more interferon gamma than men.

Interesting thought. I included in our paper an Indian paper that showed more gamma interferon responsiveness in women with TB. (And we did actually have a paragraph in brackets that suggested that women being more sensitive to gamma interferon signalling might obviate the need to invoke antibody!)