Stanford Community Symposium 2018: Phair, Metabolic traps, Tryptophan trap

[Chris Ponting]

I'm not a scientist so apologies if these questions are naive.

As I read this, the protein is not precisely repaired back to its original form, but more 'patched' so its functionality is restored, albeit with with two additional components that cancel out each others' effects. From this, can we be certain that the protein's behaviour is fully restored? And can we be confident it will not exhibit some additional behaviour, interactions, that the original would not? Is there a risk in presuming such a refactored protein will behave the same, but might in fact have some additional side effects up its sleeve that may be hard to preempt?

In practice is that as simple as it sounds? If you don't know up front what it is you are trying to tease apart, how hard is it to know what questions to ask in order to do so?

Great questions.
(1) Until the experiment is done we will not know for sure. But because natural selection is good at weeding out "bad" mutations over 100s/1000s of generations, we must have a strong expectation that the combined variants (cancelling each other) produce a "good", functioning protein. The alternative hypothesis - that large numbers of people carry one version that produces a nonfunctioning protein - must be much less likely.
(2) "how hard is it to know what questions to ask in order to do so?" Hard - because you need to guess ahead of time what questions are most relevant without knowing what the genetics will uncover. In the project that is being planned we will use questionnaires developed and in common use by the LSHTM CureME Biobank. But this is why recruiting a cohort who are happy to be recontacted with new questions every year or so is a good way to go.

 

[lunarainbows]

Hi, please bear with me as I have severe M.E and struggle a lot cognitively (despite having a maths & physics PhD background.. makes me very sad), although have tried to follow parts of this. Is it the case that @Chris Ponting is a member of the UK biobank? And his data does not support the paper that Dr Phair published? Why is this? And why do the OMF think this is @RDP ? and why does Chris ponting think this is?
I had been very excited and heartened by the paper that was published.

 

[wigglethemouse]

I needed some time to look into this. By reading the Broad investigators' manuscript on MNVs (here: https://www.biorxiv.org/content/biorxiv/early/2019/03/10/573378.1.full.pdf) and looking at the Supplemental Data for this paper this is the conclusion: the annotation of Tyr359Ter/Stop appears to be wrong, and for an interesting reason (see Figure 1 of their paper).

When Single Nucleotide Variants (SNVs) are interpreted they are mostly considered one-by-one and, indeed, if this one is considered alone then the annotation is Tyr359 to a Stop Codon implying that the protein is prematurely truncated making it effectively dead. What the Broad Investigators have done differently is to consider *multiple* variants together, and when they do this they declare that the annotation is dubious. Biologically, there is at least another variant nearby that 'repairs' the annotation to a Stop Codon.

@RDP @Chris Ponting . I get lost following the explanation but this is what I found. Let me know if this makes sense?

Here is the coding MVV file that I think Chris is describing. I have extracted the variant described from the file
https://storage.googleapis.com/gnomad-public/release/2.1/mnv/gnomad_mnv_coding.tsv

39872934   8   ENSG00000188676   IDO2   ENST00000502986   Rescued nonsense   8-39872934-A-T   8-39872935-T-A   8-39872934-AT-TA   missense_variant   stop_gained   missense_variant   tAt/tTt   taT/taA   tAT/tTA   Y/F   Y/*   Y/L   None   HC   None   2.0   2.0   7.138981695650932e-06   0   2   280326.0   62658   280152.0   1.0   1.0   4.019163370952702e-06   0   1   248936.0   []   56546   248808.0   []   1   1   3.1904032669729455e-05   0   1   31390.0   []   6112   31344.0   []

The Multi-Nucleotide Variant in the file is described on this web page
https://gnomad.broadinstitute.org/variant/8-39872934-AT-TA

If you go back to the original variant page you will see that this MNV seems to apply to only two individuals in the database
https://gnomad.broadinstitute.org/variant/8-39872935-T-A

So if it is only two individuals that have this MNV I think we can say that for most people the STOP is valid. Am I right?

 

[Chris Ponting]

RE: "So if it is only two individuals that have this MNV I think we can say that for most people the STOP is valid. Am I right?"

Yes, you're correct: only two individuals, I hadn't spotted this. Thank you.
So for nearly all people that have the variant it does, indeed, truncate the protein. Thanks for putting me right on this.

Nevertheless, people who carry this variant are not more likely to be ME/CFS cases than controls, according to UK Biobank data.

 

[wigglethemouse]

If anyone is interested you can search by variant with the "Phewas" search option and you will get a table you can sort by p-value for that variant. I couldn't see any phenotype that was relevant for all four of the 4 "damaging" IDO2 variants the database supports. Might be of interest to @RDP

e.g. here is the phenotype table for IDO2 Y359STOP variant rs4503083. You will have to sort by p-value yourself by clicking on the down arrow next to "p-value".
http://geneatlas.roslin.ed.ac.uk/phewas/?variant=rs4503083&representation=table

 

[Andy]

I'm obviously not either of the two tagged people in your post but thought I'd try and help out.

Is it the case that @Chris Ponting is a member of the UK biobank?

No, to the best of my knowledge he isn't a member of the UK Biobank (not to be confused with the UK ME/CFS Biobank). He does, however, have access to the data from the UK Biobank and has experience in this area.

For background, from https://www.ed.ac.uk/profile/chris-ponting, "Professor Chris Ponting is Chair of Medical Bioinformatics and a Principal Investigator at the MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine."

And his data does not support the paper that Dr Phair published?

Correct. The data from the UK Biobank shows that, for those people who self-reported a diagnosis of CFS, they were no more likely to have the IDO mutation than anyone else in the Biobank cohort.

And why do the OMF think this is @RDP ?

Well, first we need to bear in mind that the paper is a proposed theory that needs to be validated. But I believe I'm right in saying that the authors report that, of 20 people tested by the OMF, all 20 are positive for the IDO mutation.

I won't try to answer the question why there is this seeming difference between the Biobank data and the metabolic trap theory, I'll leave that to the experts to figure out.

Hope this helps.

 

[Trish]

Well, first we need to bear in mind that the paper is a proposed theory that needs to be validated. But I believe I'm right in saying that the authors report that, of 20 people tested by the OMF, all 20 are positive for the IDO mutation.

I think that particular group of patients all have very severe ME. I guess it's possible that having that particular mutation could contribute to making ME worse maybe? Perhaps a series of metabolic traps with more severely affected people having more than one? There may be other mutations that affect other people with less severe ME. All speculation on my part.
Edit: RDP has explained I got this wrong - they now have a larger sample of over 60 with all levels of severity with the mutation, which means my speculation is wrong!

Even if this particular metabolic trap doesn't pan out, I'm loving learning about the hypothesis, and reading this discussion. Thank you @Chris Ponting and Robert Phair (@RDP).

 

[Sunshine3]

Well, first we need to bear in mind that the paper is a proposed theory that needs to be validated. But I believe I'm right in saying that the authors report that, of 20 people tested by the OMF, all 20 are positive for the IDO mutation.

As far as I am aware OMF have at this point tested 65 patients and all test positive for IDO mutation.

 

[wigglethemouse]

For the fun of it I took a look at the high p value phenotypes for IDO1 and IDO2. Not sure it means much for the metabolic trap theory, but it was fun to play.

@RDP This IDO2 variant looks interesting in the UK Biobank data. It is not a variant you mentioned before, maybe because of low MAF of 0.01%
https://gnomad.broadinstitute.org/variant/8-39847306-C-T

This is the data in the UK Biobank for phenotype "B98 Other specified infectious agents as the cause of diseases classified to other chapters" for variant rs199869245. There were 918 cases in the database.

Variant              Chr  Position    beta              pv                MAF
rs199869245     8     39847306   -0.025354   6.3966e-09  0.0001173

Source : http://geneatlas.roslin.ed.ac.uk/search/?traits=82&variants=rs199869245

Given the low MAF I don't know how reliable this is but it does meet the -log10(pv)>8 criteria and infectious agent is interesting


Also I took a look at IDO1 variants in case that is of interest to you. This is the data for phenotype "O00-O08 Pregnancy with abortive outcome". There were 3984 cases in the database.
https://gnomad.broadinstitute.org/variant/8-39778730-C-T

Variant         Chr    Position       beta            pv                MAF
rs11995570   8       39778730    0.027008    9.1014e-09  0.00152519
rs10113525   8       39775845    0.025189    9.5611e-08  0.00148379
rs7840765     8       39776922    0.022857    6.6065e-08  0.00184858

http://geneatlas.roslin.ed.ac.uk/search/?traits=254&variants=rs10113525+rs11995570+rs7840765

 

[Barry]

Perhaps a series of metabolic traps with more severely affected people having more than one?

Or maybe a series of adverse contributing factors, only one or some of which might be metabolic traps. Possibly a metabolic trap mechanism could be the final straw pushing people into the severe state. This makes me ponder the possibility that severity might not simply be a smooth continuum of severities, but maybe discontinuous stages, depending on whether a given mechanism kicks in or not.

 

[Robert 1973]

I felt we had to say there is a low probability because even if I'm correct that ME/CFS epidemics/outbreaks require common damaging mutations, there are still 207 other possibilities.

I must say, I found this rather refreshing to read in the paper. Having become so tired of people trying to convince us that completely implausible and unhelpful ideas about ME/CFS are true, the suggestion that the theory has a low probability of being right actually increased my confidence in the authors and therefore, paradoxically, my perception of the probability of the theory being right.

A few more questions:

– Am I correct in thinking that a metabolic trap could also exist with no predisposing damaging mutations in enzymes or transporters? After all, even if this theory is correct, most people with a broken IDO2 will never develop ME/CFS, so presumably the same trap could theoretically have existed if everybody had a damaging IDO2 mutation – or even if IDO2 had never evolved.

– I understand that the authors looked for common mutations in human enzymes because of the existence of epidemics but, if it is assumed that there are predisposing genetic factors in ME/CFS, is it not at least as likely that the predisposing mutations would be in the genes which affect the probability of cytosolic tryptophan concentrations rising above the critical threshold in the first place rather than genes which determine whether or not someone falls into the trap when the critical level is reached? After all, if the theory is correct, it would to be the concentration of cytosolic tryptophan which is the most significant factor in determining whether or not someone develops ME/CFS.

– This makes me wonder, how could a metabolic trap be identified if there were no predisposing mutations in any of the enzymes or transporters?

– @RDP Can you tell us whether the testing of the theory based on metabolic tracer kinetics (as described in your paper) is happening, or likely to happen?

– If this type of metabolic trap is shown to be theoretically possible, would that mean that it is also probable that it exists in at least some people? Are there currently any good reasons to think that the trap does not occur in anyone?

– If it transpired that this (or any other) metabolic trap only applied to a small sub-group of people with ME/CFS, is there a danger that it could be wrongly rejected based on genetic analysis without getting to the stage of testing with tracer kinetics analysis?

– If IDO2 is broken in most people without it causing illness, is there any suggestion as to why the enzyme evolved, and why it appears to have subsequently been abandoned by evolution? Could it be that elevated cytosolic tryptophan concentration levels were once much more common for some reason?


Thanks again to Chris Ponting and Robert Phair for taking the time to respond to questions. Great to have you both involved with research and engaged with patients.

 

[Amw66]

Or maybe a series of adverse contributing factors, only one or some of which might be metabolic traps. Possibly a metabolic trap mechanism could be the final straw pushing people into the severe state. This makes me ponder the possibility that severity might not simply be a smooth continuum of severities, but maybe discontinuous stages, depending on whether a given mechanism kicks in or not.

Check out Craig Robinson's maths catastrophe theory as applied to ME

 

[fossil]

What kind of triggers can induce an increase of tryptophan in the cytosol?

I wonder if a trigger in susceptible individuals could be a drug or even supplement that people commonly use when they get an acute illness like the flu to alleviate symptoms.

I just googled, acetaminophen and tryptophan, just for fun, and this article came up. Disclaimer - I have no idea how factual it is, or if it's relevant to this discussion, but the authors say they have found that acetaminophen can cause falsely low serotonin levels in the test they perform, and that patients should therefore be advised to discontinue use before testing.

Interference of paracetamol (acetaminophen) with a commercially available high-performance liquid chromatography analysis of serotonin leading to falsely low serotonin levels

Albrecht Pfäfflin*, Karsten Müssig*, Erwin Schleicher

First Published January 22, 2009 Research Article

https://journals.sagepub.com/doi/full/10.1258/acb.2008.008116


If I'm remembering correctly that tryptophan is the precursor to serotonin, I wonder if acetaminophen could inhibit the breakdown of tryptophan to serotonin somehow?

And if someone had the IDO mutations and they were also a slow metabolizer of acetaminophen, could that make them more susceptible?

Please note, this is just a random thought, happy for someone to point out that it's nonsense. I was wondering if maybe even something simple like vitamin c could affect tryptophan levels in some way since so many people take it when they have the flu, I just haven't got around to googling that one yet.

 

[lunarainbows]

I think that particular group of patients all have very severe ME. I guess it's possible that having that particular mutation could contribute to making ME worse maybe? Perhaps a series of metabolic traps with more severely affected people having more than one? There may be other mutations that affect other people with less severe ME. All speculation on my part.

Even if this particular metabolic trap doesn't pan out, I'm loving learning about the hypothesis, and reading this discussion. Thank you @Chris Ponting and Robert Phair (@RDP).

Thank you Trish. I do wonder then (and I’m sure this has been talked about already, sorry I just can’t read/understand all the replies),1) could it be the case that some of the people reporting M.E/CFS in the UK biobank may be misdiagnosed or not reporting as accurately as could be done if specifically looked at by an ME physician. And in the UK, where I am, I do think of a few people who I believe have been misdiagnosed..I think it’s also in the way CFS & Medically Unexplained Illnesses (MUS)/Persistent Physical Symptoms (PPS) is diagnosed here and the criteria isn’t very strict. Although for that to affect the biobank data, I’m sure that number of misdiagnosis would have to be quite big. So what’s you said about severely ill patients and having extra metabolic traps could be something too.


2) I suspect that those diagnosed with severe M.E will be far more likely to actually have M.E (as in the OMF study). which is why I’m happy the OMF is using severely ill patients predominantly in many of these studies and hypotheses. Is there a way for the biobank to do the same? Severe meaning predominantly bedbound and very severe meaning unable to leave bed at all and requiring 24 hour care I think are the (loose) definitions.

I'm obviously not either of the two tagged people in your post but thought I'd try and help out.


No, to the best of my knowledge he isn't a member of the UK Biobank (not to be confused with the UK ME/CFS Biobank). He does, however, have access to the data from the UK Biobank and has experience in this area.

For background, from https://www.ed.ac.uk/profile/chris-ponting, "Professor Chris Ponting is Chair of Medical Bioinformatics and a Principal Investigator at the MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine."


Correct. The data from the UK Biobank shows that, for those people who self-reported a diagnosis of CFS, they were no more likely to have the IDO mutation than anyone else in the Biobank cohort.


Well, first we need to bear in mind that the paper is a proposed theory that needs to be validated. But I believe I'm right in saying that the authors report that, of 20 people tested by the OMF, all 20 are positive for the IDO mutation.

I won't try to answer the question why there is this seeming difference between the Biobank data and the metabolic trap theory, I'll leave that to the experts to figure out.

Hope this helps.

Thank you very much Andy. Very sweet of you

 

[Andy]

Is there a way for the biobank to do the same?

No, I don't believe they can, but the proposed GWAS study that Chris would be part of would be looking, in part, at samples stored at the UK ME/CFS Biobank which, as they visit patients in their home, have many samples from severely affected patients. Should it actually go ahead, this then would provide data to support, or not, the metabolic trap theory.

 

[lunarainbows]

No, I don't believe they can, but the proposed GWAS study that Chris would be part of would be looking, in part, at samples stored at the UK ME/CFS Biobank which, as they visit patients in their home, have many samples from severely affected patients. Should it actually go ahead, this then would provide data to support, or not, the metabolic trap theory.

Ah! What does GWAS stand for? That definitely sounds like a better way of providing data - visiting severely ill people in their homes.

Edit: brain fog so I got confused. Just editing the part below.

I used to think the UK biobank and the ME/CFS biobank were part of the same thing. Does the ME/CFS biobank not have data on severely ill patients and could they not use that data? (You’ve just answered that in the above reply so please ignore this part).

I’m still a little confused though as to why we have two biobanks!

 

[Trish]

Ah! What does GWAS stand for? That definitely sounds like a better way of providing data - visiting severely ill people in their homes.

Edit: brain fog so I got confused. Just editing the part below.

I used to think the UK biobank and the ME/CFS biobank were part of the same thing. Does the ME/CFS biobank not have data on severely ill patients and could they not use that data? (You’ve just answered that in the above reply so please ignore this part).

I’m still a little confused though as to why we have two biobanks!

There are people much better qualified to answer this than me, but here's my lay person's attempt to sort out the confusion:

The UK biobank is a huge study of hundreds of thousands of people sampled from the UK population funded by the government. They filled in what conditions they had been diagnosed and their whole genome was analysed. A few hundred of them put ME or CFS on their forms, and Chris Ponting has studied the genetic results from these people. Thread with more detail here

The UK ME/CFS biobank is a disease specific biobank set up and funded by the ME charities. It is much smaller. (about 200 ME cases. Edit: plus controls). The samples and questionnaire data collected are being used to study many different aspects of ME. As far as I know they have not had funding to do a whole genome study on these cases.

There are other disease specific biobanks for studying all aspects of those diseases.

Chris Ponting and others are hoping to set up a large (about 20,000) study of ME patients genetics. It would be too expensive to do the whole genome (about £1000 per person) so they plan to do a GWAS (genome wide association study, about £100 per person). This collects information on regions of genes so is less specific, but can indicate where there are mutations that may be important. (a bit like looking for variations in selected whole paragraphs in a book, rather than every letter in a book). Thread with more detail here

The metabolic trap hypothesis discussed on this thread came at the genetics from a completely different angle, looking for common genetic variations that might lead to specific types of metabolic problems, and making a hypothesis about one of these that occurred more commonly in the Ron Davis study group than in the general population.

Edited to add links.

 

[Andy]

It is much smaller. (about 200 ME cases).

Actually over 600.

 

[Trish]

Actually over 600.

Thanks Andy. I thought the 600 included the healthy controls and MS cases.

 

[Andy]

Thanks Andy. I thought the 600 included the healthy controls and MS cases.

Ah, yes, looks like it's less than the 600.

Over 600 participants have been included in this study.

Samples are available from four groups of participants:

• Mild and moderate ME/CFS
• Severe (home/bed-bound) ME/CFS
• Multiple sclerosis
• Healthy controls

https://cureme.lshtm.ac.uk/researchers/accessing-the-biobank/

200 still seems a bit low but it's not the full 600 obviously. I can get it confirmed if we need to know exactly.