Haptoglobin phenotypes and structural variants associate with post-exertional malaise and cognitive dysfunction in [ME], 2025, Moezzi, Moreau et al

[Nightsong]

Abstract:

Background​

Myalgic encephalomyelitis (ME) is a chronic, multisystem illness characterized by post-exertional malaise (PEM) and cognitive dysfunction, yet the molecular mechanisms driving these hallmark symptoms remain unclear. This study investigated haptoglobin (Hp) as a potential biomarker of PEM severity and cognitive impairment in ME, with a focus on Hp phenotypes and structural proteoforms.

Methods​

A longitudinal case–control study was conducted in 140 ME patients and 44 matched sedentary healthy controls. In the discovery phase, global plasma proteomic profiling was performed in 61 ME patients and 20 controls before and after a standardized, non-invasive stress protocol in order to induce PEM. Associations between Hp levels, phenotype, and cognitive performance were assessed. In the validation phase, plasma Hp concentrations and proteoform composition were analyzed in an independent cohort of 89 ME patients and 24 controls using high-performance liquid chromatography (HPLC).

Results​

ME patients demonstrated a significant reduction in Hp levels following post-exertional stress. Lower baseline Hp concentrations were associated with impaired cognitive performance. Hp phenotypes were differentially associated with symptom burden, with the Hp2-1 phenotype enriched in ME and linked to greater PEM severity and cognitive deficits compared to Hp1-1 and Hp2-2. HPLC analysis revealed altered Hp proteoform profiles in the Hp2-1 subgroup, including increased high-mass tetrameric and pentameric forms and shorter retention times indicative of structural changes. In contrast, the Hp1-1 phenotype was associated with milder symptoms and greater cognitive resilience.

Conclusions​

These findings suggest that Hp phenotype and proteoform structure modulate the physiological response to post-exertion in ME, offering insight into the molecular basis of PEM and its clinical heterogeneity. Hp may serve as a translational biomarker for patient stratification and a potential therapeutic target to mitigate oxidative stress and cognitive dysfunction in ME.

Haptoglobin phenotypes and structural variants associate with post-exertional malaise and cognitive dysfunction in myalgic encephalomyelitis - Journal of Translational Medicine

Background Myalgic encephalomyelitis (ME) is a chronic, multisystem illness characterized by post-exertional malaise (PEM) and cognitive dysfunction, yet the molecular mechanisms driving these hallmark symptoms remain unclear. This study investigated haptoglobin (Hp) as a potential biomarker of...

translational-medicine.biomedcentral.com

 

[forestglip]

Proteomic analysis revealed that Hp levels were significantly higher at baseline compared to 90 min post-stress in the ME group (1.84-fold change, p = 0.007). This stress-induced decrease in Hp level was specific to ME patients and was not observed in healthy controls, in whom Hp levels remained undetectable at both time points.

The pronounced post-exertional reduction in Hp, a key scavenger of free hemoglobin, in ME patients suggests a diminished capacity to buffer hemolysis-associated oxidative and inflammatory stress.

Supporting this, the non-invasive cerebral monitoring using MASIMO's NIRS revealed a significant post-PEM increase in cerebral total hemoglobin in ME patients, a pattern absent in healthy controls (Supplementary Figure S4).

Importantly, our cohort was composed primarily of severely affected, housebound individuals, patients who are rarely included in clinical studies due to the demands of protocols such as cardiopulmonary exercise testing (CPET), which typically excludes this population.

The standardized 90-min provocation maneuver used here was carefully tailored to this severity level and, based on extensive clinical experience, is sufficient to trigger PEM in this group.

 

[Utsikt]

Myalgic encephalomyelitis (ME) is a chronic, disabling disease characterized by profound fatigue, unrefreshing sleep, and cognitive impairment. The hallmark symptom, post-exertional malaise (PEM), involves exacerbation of symptoms following minimal exertion (physical or mental) and lacks a clear mechanistic explanation [1, 2].

PEM severity was further evaluated using a modified version of the DePaul Post-Exertional Malaise Questionnaire (DPEMQ), which was administered by a clinical research nurse five days after the stress test [18]. This version focused on the frequency and severity of symptoms during the immediate post-exercise period rather than over the previous six months. Thirteen items contributed to the total DPEMQ score, with higher scores indicating greater PEM severity.

I think 18 is this:

https://www.s4me.info/threads/the-development-of-an-instrument-to-assess-post-exertional-malaise-in-patients-with-myalgic-encephalomyelitis-and-cfs-2018-jason-et-al.6370/

I don’t fully understand how they measured PEM or what they mean by it. I wish they spent more effort on explaining their understanding of PEM and showing a timeline.

They say the controls were sedentary, but I can’t find any data on activity levels. Can we exclude the possibility that the observed differences are due to very inactive muscles being stimulated compared to somewhat active muscles being stimulated?

 

[Kitty]

Can we exclude the possibility that the observed differences are due to very inactive muscles being stimulated compared to somewhat active muscles being stimulated?

I wondered that.

It would have been good to include some moderately ill people too; in terms of how much they use the arm muscles being stimulated, they'd probably match sedentary controls quite well. But if a project can only include one cohort due to funding limitations, it is important to prioritise severely ill people.

 

[Utsikt]

I wondered that.

It would have been good to include some moderately ill people too; in terms of how much they use the arm muscles being stimulated, they'd probably match sedentary controls quite well. But if a project can only include one cohort due to funding limitations, it is important to prioritise severely ill people.

In principle I agree that prioritising the severe is important, but only if it doesn’t reduce the value of the information you get from the study.

What they were trying to figure out is if the body of pwME/CFS reacts abnormally to external stimuli of the muscles in the arm. If you are unable to control the large confounder of baseline activity for that muscles group when studying the severe, wouldn’t it be better to study moderate or even mild patients first, and then move on to the severe to see if the findings replicate there?

Unless you’ve got a reason to think that the severe are somehow different from the moderate/mild, in other ways than just being more sick with the same thing?

 

[forestglip]

If you are unable to control the large confounder of baseline activity for that muscles group when studying the severe, wouldn’t it be better to study moderate or even mild patients first, and then move on to the severe to see if the findings replicate there?

On the other hand, we're talking about a disease where proteomics and exercise tests have already been tried several times in less severe people. If there actually are differentially expressed proteins, we expect them to probably have tiny effect sizes, and when you're correcting for a hundred tests, you might miss them.

So it might be better to compare to people who are severe, to get as large of effect sizes as possible, so they aren't missed. Then try to replicate just the proteins of interest in groups that better match.

 

[Utsikt]

On the other hand, we're talking about a disease where proteomics and exercise tests have already been tried several times in less severe people. If there actually are differentially expressed proteins, we expect them to probably have tiny effect sizes, and when you're correcting for a hundred tests, you might miss them.

So it might be better to compare to people who are severe, to get as large of effect sizes as possible, so they aren't missed. Then try to replicate just the proteins of interest in groups that better match.

That’s a good point.

My main worry is that you’ll just end up finding noise all the time due confounders and chasing red herrings. Maybe a three group setup would be better - HC, severe and moderate/mild, or HC, severe and another disease?

 

[Simon M]

That’s a good point.

My main worry is that you’ll just end up finding noise all the time due confounders and chasing red herrings. Maybe a three group setup would be better - HC, severe and moderate/mild, or HC, severe and another disease?

I'm sure that's been done a few times. And quite a few of those severe ended up halfway between health controls and mild/moderate, sometimes with generous explanations of how this makes sense. Which makes me wonder if it's a case of more red herring is.

 

[ChronicallyOverIt]

Is there any connection here to the HFE gene found in the DecodeME?

 

[Mij]

September 16, 2025

​

OMF’s Collaborative Center at Montreal, in collaboration with our Collaborative Center at Uppsala and the Computational Research Center for Complex Diseases, has released a new paper: Haptoglobin phenotypes and structural variants associate with post-exertional malaise and cognitive dysfunction in myalgic encephalomyelitis.

From the Desk of Dr. Alain Moreau, Director of the ME/CFS Collaborative Research Center at Montreal:​

What is this study about?

This study investigated a blood protein called haptoglobin (Hp), which normally protects the body from damage when red blood cells break down. OMF researchers from Montreal, Uppsala, and the OMF computation team looked at how Hp levels and genetic variants behave in people with ME/CFS, especially during post-exertional malaise (PEM)—the worsening of symptoms after even minor activity.

Why does it matter?

They found that after a mild stress challenge, people with ME/CFS showed a sharp drop in Hp levels, while healthy controls did not. Low Hp was linked to worse PEM symptoms and poorer cognitive performance (“brain fog”). Importantly, the study also revealed that certain genetic forms of Hp influence how severe PEM becomes: patients with the Hp2-1 genotype had stronger PEM and more cognitive problems, while those with Hp1-1 were more resilient. This shows that genetics directly contribute to vulnerability in ME/CFS.

What is the potential impact for people with ME/CFS?

This is the first strong evidence of a genetic contribution to PEM in ME/CFS. Hp could serve as a biomarker to better diagnose ME/CFS, identify patient subgroups, and guide clinical management. In the future, therapies that restore or replace Hp function (e.g., intravenous infusion of recombinant Hp-1-1 protein) might help protect genetically at-risk patients from post-exertional crashes and cognitive decline, offering a new pathway toward targeted treatment.

Read the full publication in the Journal of Translational Medicine.

 

[Hutan]

Among the proteins altered in response to stress, Hp emerged as the most significantly dysregulated in ME patients (Table 2, Fig. 2 and Supplementary Figure S6). Proteomic analysis revealed that Hp levels were significantly higher at baseline compared to 90 min post-stress in the ME group (1.84-fold change, p = 0.007). This stress-induced decrease in Hp level was specific to ME patients and was not observed in healthy controls, in whom Hp levels remained undetectable at both time points. This distinct post-stress pattern positions Hp as a potential disease-specific marker of PEM. The pronounced post-exertional reduction in Hp, a key scavenger of free hemoglobin, in ME patients suggests a diminished capacity to buffer hemolysis-associated oxidative and inflammatory stress.

The paper says that Hp levels were undetectable before and after the stressor in the healthy controls. But for the ME group, HP was present at baseline, but decreased after the stressor.

To me, the situation as described there for the ME/CFS group is normal. It is not normal to have no Hp.

Normal haptoglobin (Hp) levels in adults generally range from about 40 to 200 mg/dL, but this can vary slightly depending on the laboratory and individual factors like age and gender.

Reading on, there is an explanation.

The identification of Hp as a top candidate biomarker warrants careful consideration in light of the technical protocol employed. The plasma samples used in the global proteomic assay were processed using a MAR-14 immunodepletion column, which targets and removes high-abundance plasma proteins, including acute-phase reactants such as Hp. In theory, this step should have substantially reduced or eliminated detectable Hp levels across all samples. The fact that Hp remained quantifiable among the ME group, and showed consistent, biologically plausible differences post-stress, raises important questions about the molecular form and binding context of the Hp detected.

So, actually the processing was supposed to remove "high-abundance plasma proteins" including Hp. So, the processing explains why there was no Hp in the healthy controls. It doesn't explain why Hp was found in the ME/CFS group. It seems possible that there was a lab error. They are suggesting that the difference might be related to some difference in ME/CFS Hp.

To validate and extend the discovery phase results obtained using MAR-14-depleted plasma proteomics, a high-sensitivity method for direct Hp quantification in total plasma (without prior depletion) was applied to the same cohort. This orthogonal approach confirmed a significant post-stress reduction in Hp levels in ME patients, with the medium effect size (Fig. 4). In contrast, no significant change in Hp levels was found in HC group, confirming the disease specificity of the stress-induced Hp depletion (Fig. 5). This replication strengthens the evidence that Hp is a stress-responsive protein in ME and may be a biomarker of PEM.

So, there they are saying that they used another method, this time directly quantifying Hp levels in the plasma. Figure 5 gives those actual levels.


Chart A is for the ME/CFS group; Chart B is for the control group. Note the different y axis scale. Really, there's nothing much to the claimed differences between the ME/CFS and control groups, and even from before and after the stressor. Maybe the stressor just isn't enough to make a difference?

It is normal for levels to decrease a bit after exercise, so that very small change found in the ME/CFS group is not remarkable.

Perhaps there is something about ME/CFS Hp that resulted in it still being present after that first processing approach that should have removed it. It's worth looking into more. But, it could easily just be some sort of an error, some difference in the sample processing.

 

[Hutan]

Is that y axis scale a Log base 2 scale, as in
Log2 (6) = 2.585 mg/dL
Log2 (9) = 3.17 mg/dL
?

If so, then the mean value in the ME/CFS group dropped from about 3.0 to 2.9; while the mean value of the controls also dropped from 3.0 to 2.9, as best I can work out, and rounding to one decimal place.

Why would you use a log scale rather than just the actual levels? Also, the numbers don't seem to line up with the mg/dL levels that are widely reported as being normal (40-200). So, was the assay unusual?

It's looking a bit underwhelming in terms of the headline finding from an initial assay of over a hundred proteins.

 

[forestglip]

Is that y axis scale a Log base 2 scale, as in
Log2 (6) = 2.585 mg/dL
Log2 (9) = 3.17 mg/dL
?

If so, then the mean value in the ME/CFS group dropped from about 3.0 to 2.9; while the mean value of the controls also dropped from 3.0 to 2.9, as best I can work out, and rounding to one decimal place.

No, it'd be log2(x mg/dL) = [number on y axis]

So reverse the calculation. To go from the y axis to the real measurement, you do 2^number. So a value of 8 on the chart equals 2^8=256 mg/dL.

Why would you use a log scale rather than just the actual levels?

I think it's normally done when the data is skewed. Taking the log makes it more normally distributed, which helps make certain statistical tests and visualization work better.

 

[Hutan]

Oh, yes, whoops.
So for the ME/CFS chart, approx from 256 to 181.
For the healthy control chart, approx 216 to 210
That's rough eyeballing.

The top value there is something like 675, so I can sort of see why they used the log scale, but using the actual values would have made it clearer that the within-cohort variation massively swamps the between-cohort and between-timepoint variation. And that the decrease in the mean value for the ME/CFS cohort after the application of the stressor is pretty small.

 

[forestglip]

The top value there is something like 675, so I can sort of see why they used the log scale, but using the actual values would have made it clearer that the within-cohort variation massively swamps the between-cohort and between-timepoint variation. And that the decrease in the mean value for the ME/CFS cohort after the application of the stressor is pretty small.

I don't know. It'd be great to have large effect sizes, but the next best thing is at least being significant. Until we have a reason to forget about it, like a study that finds the opposite, I think it's fine to speculate about. Small but significant can still be biologically meaningful.

You mentioned that it's underwhelming in a study of over a hundred proteins, which I assume is implying that you're likely to get a false positive if you're testing so many things. But they did multiple test correction, so that's not the case.

Edit: On second thought, I don't really like the method of saying that if a protein significantly decreased in one group but didn't significantly decrease in another, that it means the groups are significantly different. I think there should be a direct comparison between groups to make it convincing.

They also talk about several other haptoglobin related findings. But notably, even though the first effect they talk about is change in haptoglobin after stress, the symptom correlation they found (fig. 4) is with baseline haptoglobin. So between groups, the important metric is change between timepoints, but within the ME/CFS group the important metric is a single timepoint.

I didn't really try to dig into the later Hp structural phenotype findings.

 

[Hutan]

I don't know. It'd be great to have large effect sizes, but the next best thing is at least being significant. Until we have a reason to forget about it, like a study that finds the opposite, I think it's fine to speculate about. Small but significant can still be biologically meaningful.

You mentioned that it's underwhelming in a study of over a hundred proteins, which I assume is implying that you're likely to get a false positive if you're testing so many things. But they did multiple test correction, so that's not the case.

I'm not sure if the assumption about the multiple test correction being applied to haptoglobin is right.

I noted upthread

So, actually the processing was supposed to remove "high-abundance plasma proteins" including Hp. So, the processing explains why there was no Hp in the healthy controls. It doesn't explain why Hp was found in the ME/CFS group. It seems possible that there was a lab error. They are suggesting that the difference might be related to some difference in ME/CFS Hp.

The Methods section notes that haptoglobin was one of the proteins that was purposefully depleted in the proteomics analysis.

In order to enrich the plasma samples with low abundant proteins for the subsequent LC–MS/MS analysis, fourteen of the most highly abundant proteins (human albumin, IgG, α1-antitrypsin, IgA, transferrin, haptoglobin, fibrinogen, α2-macroglobulin, α1-acid glycoprotein, apolipoprotein A-I, apolipoprotein A-II, IgM, transthyretin, and complement C3) were depleted using a Multi Affinity Removal Spin (MARS) Cartridge Human-14, 0.45 mL, (Agilent Technologies) according to the manufacturer’s instructions.

So, the quantification of the haptoglobin levels comes from a separate analysis done when they were trying to work out why, when they had tried to remove haptoglobin in the proteomic analysis, they had still found some in the ME/CFS samples. I don't think the FDR adjustment applies to the haptoglobin finding.

They think the reason why they still found haptoglobin in the ME/CFS samples after trying to deplete it is because the ME/CFS haptoglobin has some special properties. There was a lot of processing steps going on there. Unless the samples were processed in random order, I think it is at least possible, and probably likely that some processing difference accounted for the remaining haptoglobin in the ME/CFS samples.

So, they looked at all those low abundance proteins, and then I think they chose to chase up a finding that actually was for something they weren't planning to look at. And, if you look at that chart (Fig 5) of the levels of haptoglobin they measured using another method, the levels don't appear to be significantly different between the controls and ME/CFS samples.

Kudos to them for looking out for serendipitous findings and then following through with further investigations. I haven't looked yet at their analysis of the haptoglobin variations, maybe there is something there. I just think it's worth keeping in mind that the difference in actual levels in Fig 5 is very minimal, and that the differences in the first stage of the analysis between controls and ME/CFS samples arose from an attempt to deplete haptoglobin from samples.

 

[forestglip]

Kudos to them for looking out for serendipitous findings and then following through with further investigations. I haven't looked yet at their analysis of the haptoglobin variations, maybe there is something there. I just think it's worth keeping in mind that the difference in actual levels is very minimal, and that the differences in the first stage of the analysis between controls and ME/CFS samples arose from an attempt to deplete haptoglobin from samples.

Ah yes, you read it more carefully than I did. Though I think it's possible it would have passed multiple test correction if they had considered all the other original proteins for the second analysis as well. In the first depleted protein analysis, haptoglobin was significantly decreased after the stress test with a p-value of 0.007, which was still significant after correction. In the second validation test, it had p<0.01 (I didn't see an exact p-value). So it's possible it was just as significantly decreased, but since they didn't retest everything else, they didn't explicitly do multiple test correction.

Though again, what the above can show is whether haptoglobin decreased in ME/CFS. It doesn't show that haptoglobin decreased significantly more than it did in healthy controls.

I just saw that there are more than three times as many people in the ME group compared to the control group. So controls not being significant may have more to do with the small sample size than because of a smaller effect. And even if they had identical sample sizes, one being significant and one not isn't enough for showing a difference between groups.