ME/CFS Research Review

Simon McGrath explores the big biomedical stories

Tag: Ponting

Significant association of DNA variants with self-reported ME/CFS

Guest blog by Professor Chris Ponting and colleagues.


A new analysis using data from UK Biobank indicates that one version of a particular gene increases the risk of ME/CFS in women. The gene codes for a transporter protein in the mitochondrial membrane and plays a critical role in the urea cycle, which is important for removing ammonia from the body. Reduced levels of the transporter protein, which are expected for the gene variant associated with ME/CFS, are likely to impair mitochondrial function. If replicated later, this finding would provide the first evidence that a person’s risk for ME/CFS is caused by changes to mitochondrial function.


On June 11 2018 we posted a blog describing an analysis of the UK Biobank’s data, drawn from half-a-million individuals from around the UK. The data implied that there is a genetic contribution to an individual’s risk of ME/CFS but it did not provide strong evidence that change in any one section of DNA explained this risk.

This analysis is called a genome-wide association study, or GWAS for short. When there is an association between a biological factor, such as cytokine changes, and an illness, it is not normally possible to say whether the factor is a cause of the illness or simply a consequence of being ill.  But because the genetic variation comes first it must contribute to a cause rather than being an effect of the illness.


A GWAS asks whether the frequency of a DNA letter difference predicts whether a person has, or has not, a disease;

This prediction is not perfect and almost always reflects only a slight preference for people with this DNA difference to part of the disease cohort;

Importantly, GWAS predictions indicate genetic cause. This is because inherited disease (except cancer) does not predictably cause DNA mutation.

We are blogging again because a new analysis has revealed a promising finding.

This new analysis is again of the UK Biobank data and has been posted by Ben Neale’s lab. After discarding data that they considered lower quality, they were left with data from 194,174 females and 167,020 males and kindly made their results freely available to all.

Female-only GWAS

Considering males and females together they identified no specific region of the human genome whose DNA variants were significantly associated with self-reported CFS/ME. (One variant, rs148723539, is possibly indicated [p = 2.3×10-9] but this is not supported by adjacent variants.)

However, the female-only analysis revealed a single region, on chromosome 13. Ten DNA variants (single nucleotide variants, SNVs) were significantly associated [SNVs with minor allele frequency <0.001 or that were “low confidence” were filtered out] using a probability threshold of p < 5×10-8.

These 10 SNVs are inherited down the generations together (they are in “linkage disequilibrium” [LD]) and so this looks like just one association, rather than ten different ones. The 10 SNVs all lie in a 51,000 base region that surrounds the SLC25A15 gene (Figure 1 below).


Figure 1

Their conclusion is that DNA variation in this part of the genome slightly changes a woman’s risk of having a ME/CFS diagnosis. This must mean that one or more DNA differences in this part of the genome cause this risk change. But because all 10 differences are inherited together, it is not clear which one or ones are causing the increase in disease risk. Pinning down the causal DNA changes will require detailed experimental research.

We can talk about a DNA letter representing increased risk (this is the “risk allele”). For example, the 41,353,297th DNA letter on chromosome 13 is either G or A (the site is given the code rs7337312). You have two copies of chromosome 13, so also two versions of this DNA letter: you can have G twice (“GG”), A twice (“AA”) or G and A (“GA”). People across the world vary in the frequency of G or A but in the UK Biobank population it turns out that the frequency of G is about the same as that of A (~50%).

The Ben Neale lab result implies that having a DNA letter G at this position on chromosome 13 predicts a slight increase in risk of having a ME/CFS diagnosis; having two such letters G increases the risk further over having just one G. The frequency of G of ~50% means that roughly three-quarters of these UK Biobank females have at least one risk G.

Another of the Neale results is that women that have G at this position tend to have very slightly lower lymphocyte count (rs7337312; p = 4.6×10-7; other variants in LD have p < 5×10-8).  Lymphocytes are white blood cells, mostly B cells and T cells. They also show that G at this position significantly and slightly increases two biomechanical properties of the cornea (corneal hysteresis and corneal resistance factor).

ornt biofactors

Figure 2. Genetic effects on ME/CFS risk & blood cell counts, and additional predictions

Ornithine Transporter type 1

Figure 1 shows that the GWAS genetic associations are for DNA differences that lie in-or-around a particular gene called SLC25A15. There is another gene in this Figure which is a pseudogene (TPTE2P5) which does not make protein and is less likely to alter physiological function when mutated. This does not necessarily immediately implicate SLC25A15 as the gene through which this genetic effect is transacted.

However, other data indicates that people that have G at this position tend to produce slightly lower amounts of SLC25A15 RNA (in aorta, colon, hippocampus, transformed lymphocytes and other samples, but not in whole blood, liver, muscle, cerebellum etc; Figure 2). So SLC25A15 is an excellent candidate for the gene whose activity alters between GG, GA and AA individuals thereby changing ME/CFS disease risk.

ornt tp t1

3D model of ORNT1

So what do we know about SLC25A15? Interestingly, it encodes a protein called Ornithine Transporter type 1 (ORNT1). This transports ornithine (as well as lysine and arginine) across the inner membrane of mitochondria to the mitochondrial matrix. Ornithine is an amino acid (but not incorporated into proteins) that plays a role in the urea cycle. This cycle plays an essential part in removing ammonia from the body (see point iv below).

This analysis predicts that if you have a letter G at this position then (Figure 2):

(i) if you are female, then you have a greater risk of ME/CFS;

(ii) many of your cells (for example in the heart or hippocampus, but not in muscle or liver) would tend to produce less ORNT1 RNA and less ORNT1 protein; and,

(iii) if so, then ornithine would build up in these cells and mitochondrial function overall would be impaired; and,

(iv) ammonia would accumulate in the blood.

Some of these predictions are drawn from what is known about individuals whose SLC25A15 genes are both defective, causing ornithine translocase deficiency (also known as HHH syndrome). This is a severe disease often leading to coma due to hyperammonemia, learning difficulties and also lethargy. A good review of HHH syndrome can be found here.

So do these predictions agree with what others have found when comparing people with ME/CFS with control individuals? In large part yes, but not always:

(a) Yamano et al. (2016) showed that there is an increased ornithine/citrulline ratio in ME/CFS individuals

(b) Naviaux et al. PNAS (2016) found that ornithine is moderately high for males and for females

(c) Nevertheless, Armstrong et al. (2012) found the opposite, a significant reduction of ornithine (P<0.05) in the blood of CFS samples.

What are this study’s limitations?

  1. Only one genomic region was identified, so a much larger study is required with about 20,000 ME/CFS cases to find more.
  2. This association would need replication in an independent study.
  3. We do not know whether males with ME/CFS show this genetic association. Males in UK Biobank with self-reported ME/CFS are 2.4-fold fewer than females, so a male-only GWAS is substantially less well powered to find true results.
  4. Even if true, then this association would alter risk for ME/CFS by only a small amount and certainly not for most people.
  5. The urea cycle occurs mostly in mitochondria in liver cells, yet SLC25A15 expression is found not to be different between GG, GA and AA in this organ. SLC25A15 is expressed also at moderate levels in pancreas and small intestine, yet these are not major sites of the urea cycle.
  6. We do not know whether slightly lower amounts of SLC25A15 RNA (in aorta, colon, hippocampus, transformed lymphocytes and other samples) result in lower amounts of SLC25A15 protein. This would need to be true for the hypothesis to be correct.
  7. We do not know whether the various observations on ME/CFS, lymphocyte percentage and cornea biomechanics are relevant to one another.
  8. Its findings should not be used to alter clinical treatment without consulting with a GP.

What are its strengths?

  1. If replicated, this would be the first piece of genetic evidence that mitochondrial dysfunction (specifically ornithine transport) is causal of ME/CFS susceptibility (as opposed to being a consequence of ME/CFS disease).
  2. It would focus clinicians’ attention on these cellular processes for diagnosing and stratifying ME/CFS cases.

With thanks to Ben Neale and his lab for making their results freely available, and to Simon McGrath for comments and hosting this blog.

Chris P. Ponting1, Neil Clark1, Mark Jones2.

1 MRC Institute of Genetics & Molecular Medicine, MRC Human Genetics Unit. 2 UCB Pharma.

Image credits: ORNT1 3D computational model, Wang & Chou, 2012.

Analysis of data from 500,000 individuals in UK Biobank demonstrates an inherited component to ME/CFS

Guest blog by Professor Chris Ponting and colleagues.

UK Biobank – a national biobank different from the ME/CFS biobank – has data from around 500,000 individuals, including both healthy people and those with one or more of the many different diseases in the UK population. About 2,000 people in the sample reported that they had been given a diagnosis of CFS.​

Analysis of data from this biobank indicates an inherited biological component for ME/CFS. The results show only one statistically significant change in a particular section of DNA and even this is problematic. This analysis indicates that a much bigger study, with many more ME/CFS cases, will be needed to indicate which genes and biological pathways are altered in people with ME/CFS.​



Chris Ponting

Myalgic encephalomyelitis (ME, also described as chronic fatigue syndrome, CFS) is a devastating long-term condition affecting 250,000 UK individuals. People with ME experience severe, disabling fatigue associated with post-exertional malaise. A few make good progress and may recover, while most others remain ill for years and may never recover. There is no known cause, or effective treatment for most. Consequently, it is vital to try new approaches to understand the reasons for the development of the condition.

This blog sets out what we can glean from the release, last summer, of data from about 500,000 individuals who make up the UK Biobank. (This biobank is not to be confused with the UK ME/CFS Biobank, UKMEB.) The data were acquired from individuals between 40 and 69 years of age in 2006-2010 who live across the UK. These people provided samples (e.g. blood, urine and saliva) and answered questionnaires. In addition, for some of these people their electronic health record data are being linked in. Importantly for this blog, the DNA variation (‘genotype’) of all the volunteer participants has been determined.

Genetic variation can provide insights into the causes of disease when these have a heritable component (i.e. are inherited down through the generations). DNA sequence is not altered by disease (except in cancer) and so variants can reveal the causes, rather than consequences, of disease.


Here we draw heavily from an analysis of the UK Biobank data by Oriol Canela-Xandri, Konrad Rawlik and Albert Tenesa which is described in a preprint available from bioRxiv. (The authors have kindly shared their results in this way in order to share results with others before the findings have been peer reviewed.)

From this (specifically, Supplemental Table 1) we see that data were analysed from 1,829 people among the UK Biobank cohort who self-reported as having been diagnosed with ME/CFS. The table also provides five pieces of information:

(1) The prevalence of ME/CFS among UK Biobank individuals was 0.448%. In other words, picking any person randomly in the UK then there is an even chance that they know someone with ME/CFS if they know about 200 people.

(2) There is a reasonably strong female bias: the prevalence rates are female = 0.611%; male = 0.255%; so there are 2.4-fold more females than males with ME/CFS in the UK Biobank cohort.

(3) Extrapolating these numbers to the UK as a whole, here are the full population prevalence predictions (using 2016 estimates for UK census populations).

Female Male Total
ENGLAND 171,630 69,339 240,969
SCOTLAND 16,784 6,781 23,565
WALES 9,668 3,906 13,574
N IRELAND 5,783 2,336 8,119
UK (total) 203,865 82,362 286,227

There is one caveat that should be mentioned with respect to these numbers. This is that the 500,000 people assessed in the Biobank, despite being recruited for assessment at 22 centres in Scotland, Wales and England, are not fully representative of the general population. There appears to be a “healthy volunteer” selection bias which would imply that the prevalence estimates are lower-bound values. Furthermore, if ME/CFS prevalence is different in other groups then this is not accounted for in the numbers above.

(4) ME/CFS has a biological component because the heritability of ME/CFS is not zero. Canela-Xandri et al. estimate that the genetic heritability (liability scale) is 0.080. This is slightly lower than the median heritability of heritable binary traits (0.11; see Figure 1). So among all such things measured, it’s in the lower half of the heritability, but not zero. Note that this doesn’t rule out non-heritable biological causes.

(5) The analysis identifies one, and only one, DNA position whose genetic variation associates with (in part) ME/CFS susceptibility. (The plot below is called a Manhattan plot and any point above the dashed line is predicted to be a significant “hit”. Each dot represents a position (X axis) along a chromosome – shown alternatively in red and blue – and its position on the Y-axis indicates the statistical significance of the association: the higher the better.)

Manhattan plot biobank GWAS

Statistical significance for the association between each DNA position and ME/CFS across 22 chromosomes. The arrow highlights the one “significant hit”.

This proposed “significant hit” is on chromosome 10 (position 74828696; rs150954845). The calculated p-value is 2.5×10-12. This DNA change (A-to-T) is predicted to alter a protein called P4HA1, changing an aspartic acid (“D”; GAT) for a valine (“V”; GTT) at its 124th amino acid position.  P4HA1 is prolyl 4-hydroxylase subunit alpha 1: in other words, one part of prolyl 4-hydroxylase, a key enzyme in collagen synthesis. We know what this molecule looks like and where the aspartic acid (D124) occurs within it (below; courtesy of Luis Sanchez-Pulido).


We can even see at a resolution of 10-10 of a metre what effect such a change would have on the protein (below; courtesy of Luis Sanchez-Pulido).



So, should we believe that this amino acid change alters someone’s risk of developing ME? For five reasons we need to be cautious:

(a) ME is a complex condition, likely to be caused by many DNA changes each of small effect acting together with the environment, so the fact that only one association was found indicates that the study is under-powered. This means that it doesn’t have the number of patients sufficient to provide the statistical power needed to detect the major DNA changes associated with the illness: more individuals means greater statistical power.

(b) Second, this part of the protein is not conserved across evolution. There is even a nematode worm known that has a valine at exactly the position (124) that would be predicted to alter risk for ME in humans. This isn’t conclusive, but an amino acid change at a position that is shared across different species would have given us greater confidence in the prediction.

(c) Third, very few people have this amino acid change. Only 0.01% of the population have this alteration, and at such low levels it is difficult to calculate levels of significance accurately particularly when the numbers of people self-reporting with ME (here, n=1,829) are so much lower than the entire cohort (500,000).

(d) Fourth, this association was not reported to be significant in a separate study.

(e) The study relies of self-report of receiving diagnosis of chronic fatigue syndrome, so these cases have not been diagnosed by researchers as meeting any particular definition of ME/CFS.


  1. If the UK Biobank prevalence of ME/CFS is repeated across different populations, then 34 million people worldwide will have this disorder, 2.4-fold more women than men.
  1. ME/CFS has a biological component, as shown by its non-zero heritability in UK Biobank.
  1. To obtain robust indications of which genes and which biological pathways are altered in which cells or tissues in people living with ME/CFS, then a much larger study is required. A GWAS with ten- or twenty-thousand cases, is likely to be necessary. Results will then need to be replicated in a separate cohort.

Chris Ponting, Luis Sanchez-Pulido, Katie Nicoll-Baines, Thibaud Boutin and Shona Kerr.

With thanks to Cathie Sudlow, Veronique Vitart, Oriol Canela-Xandri and Albert Tenesa for helpful comments.

MRC Human Genetics Unit at the MRC Institute of Genetics and Molecular Medicine, University of Edinburgh, Western General Hospital, Crewe Road South, Edinburgh, EH4 2XU, UK

A plan to replicate Mark Davis’s remarkable findings of immune activation in ME/CFS


Chris Ponting

A team led by Edinburgh University’s Professor Chris Ponting has won funding for a PhD student who would follow up and expand on remarkable recent findings made at Stanford University, where Dr Mark Davis may have pinpointed a major issue in the immune system in ME/CFS.

Last year, Davis produced strong evidence of T cell clonal expansion, similar to that seen in illnesses including multiple sclerosis and acute Lyme disease. His discovery came from a new and sophisticated way of looking at the immune system in ME/CFS, which revealed the strongest evidence yet for immune activation in this disease. These results could indicate autoimmunity or a response to infection in ME/CFS, and could point to the core problem in the disease.


Human T cell (photo: NIAID)

So if the results of the PhD work are as hoped then they could narrow down the cause of ME/CFS for some patients. The ultimate aim is for this research to form part of a concerted effort that provides affordable diagnostic tests and targets for treatment.

The PhD will be based at Ponting’s Computational and Disease Genomics lab in Edinburgh. The £90,000 cost of the PhD will be jointly funded by Action for ME and the Scottish Government’s Chief Scientist’s Office. Samples from patients will be provided by the UK ME/CFS Biobank, subject to a successful full application to the Biobank.

Ponting has drawn in impressive collaborators for this project: senior immunologist Professor Georg Höllander from Oxford University and molecular biologist Dr Lia Chappell from the prestigious Wellcome Sanger Institute. I am pleased to say that I have also been involved with this study from the outset and the successful PhD candidate will engage with more patients as the project gets underway.

The immune-system biology behind the study: T cells and their receptors

Many people have heard of B cells and the antibodies they produce; T cells are their cousins. T cells come in many forms, but the relevant ones here are called killer (CD8) T cells. These act like a security force, checking cells and killing off any that look sick because they are infected or cancerous. Much like with B cells and their antibodies, T cells are produced in countless varieties, each version specifically binding to just one molecule (called an antigen). It’s this specific binding that allows a T cell to pick off a cell infected with a particular pathogen (or affected by another problem) while avoiding collateral damage.

Because these varieties are produced randomly, most turn out to be completely useless. But all the T cells hang around, hoping to find a pathogen molecule (antigen) or tumour protein they happen to recognise, like a lock searching for the key that will open it. If a T cell finds its “key”, the T cell is activated and starts dividing to produce identical copies of itself – clones – in a process called clonal expansion. This new army of clones then heads off to target the infected or cancerous cells. And when things go awry with the immune system, T cells can end up targeting a person’s own molecules – “self” antigens – leading to autoimmunity.

Mark Davis’s study was comparing T cell clonal expansion in healthy people with that in several diseases, including multiple sclerosis (an autoimmune disease), and acute Lyme disease (an infection). At a late stage in the project, he won a grant to include samples from ME/CFS patients as well and discovered that clonal expansion in these patients was similar to that seen in these other diseases.

Measuring clonal expansion

But how do you measure clonal expansion? Every cell in a clone shares an identical T cell receptor (TCR) – the lock responsible for recognising the key of a specific molecule on the pathogen or tumour cell. So the process is to laboriously sequence the T cell receptor of every T cell and then count the matches. When a lot of T cells have matching TCRs then this means that there has been clonal expansion.

Mark Davis did this for six ME/CFS patients, and the results are shown in the figure below.

TCR Davis just mecfs

Image taken from a video of Davis’s presentation

The figure takes a little understanding, so here’s what the first pie chart shows. It’s for Patient L3-07. The white area of the chart shows that just over half of the tested cells were unique (no other cells had identical TCRs), indicating no clonal expansion for those cells. The coloured pie slices indicate the scale of clonal expansion. For example, there were two clones with more than 20 copies of the same TCR, as indicated by the two pie slices marked in red. The other coloured slices show the number of copies for clones with fewer copies.

The number underneath the chart shows that this patient’s sample contained 323 T cells.

After examining clonal expansion in ME/CFS, Davis then compared the results for ME/CFS with those for other illnesses. Examples from four patients with multiple sclerosis, ME/CFS, or a recent Lyme infection and from four healthy controls are shown below.

TCR Davis all diseases clearer

Image taken from a video of Davis’s presentation

The contrast between the disease patients – including the CFS patients – and the healthy group is striking. In the healthy individuals, about 90% of T cells are unique, with only a few expanded into clones. The ME/CFS patients look far more like patients with multiple sclerosis or Lyme. This is an astonishing result and, if replicated, could point firmly to the underlying disease process for at least some patients. It could also yield biomarkers.

Mark Davis is already trying to identify the antigen(s) that might be triggering clonal expansion, which could identify self-antigens (autoimmunity) or specific pathogens that have triggered the problem. That could pave the way to developing new treatments.

The new PhD project will use similar but improved technology. Because every cell must be sequenced separately, T cell sequencing is currently very expensive work, which effectively limits how many samples can be processed.

Dr Lia Chappell at Sanger will develop the method further in order to dramatically lower costs, allowing much bigger samples to be analysed, and the technique will be implemented by the PhD student. The goal is to sequence TCRs for 10,000 cells per patient and for many more patients. The initial target is 50 patients and 50 controls but the ultimate ambition is to eventually sequence the T cells for all samples in the UK ME/CFS Biobank, and possibly for other cohorts as well.

How the study came about

Chris Ponting is an old friend and I told him about Mark Davis’s remarkable study when I saw the video about it from last year’s Open Medicine Foundation symposium at Stanford. Chris also thought this work was exciting and important, though I hadn’t expected him to follow up on it with a research project. However, by a stroke of luck, Chris was part of a consortium at the Wellcome Sanger Institute that was looking to find a cheaper way to sequence T cell receptors. The consortium is headed by Höllander with Chappell developing the core technology and both agreed to collaborate with Chris on this new project. Chappell and Höllander are now adapting their technology to suit this new study.

Clonal expansion could be the smoking gun for what’s going wrong in ME/CFS. So if the findings of clonal expansion check out then this work could help to track down the cause of ME/CFS for at least some patients. The new technology being developed could also ultimately lead to an affordable way for doctors to measure clonal expansion, helping with diagnosis.

It’s impressive that Action for ME and the Scottish Government’s Chief Scientist’s Office are funding this important work. Replication is central to establishing robust results, and I can’t think of a more important finding to try to replicate than that of T cell clonal expansion.

Powered by WordPress & Theme by Anders Norén