IDO2 gene mutations - SNPs

[daftasabrush]

Background
The metabolic trap hypothesis being investigated by the OMF is currently investigating IDO2, with the Severely Ill Big Data Project finding IDO2 mutations in the 20 patients ill with M.E.
This question is about how someone can check if they have the mutations found based on genetic data analyzed by popular saliva tests.

Gene data
Many people have had their genetic data analyzed from saliva tests, eg by 23andMe or Ancestry DNA. This results in a large file that can be searched for different SNPs. Various projects have collected this data eg Nova Southeastern's ME/CFS Gene Study run by Nancy Klimas.

Question 1
I believe 5 or 6 IDO2 mutations were identified, that meant IDO2 was either not working at all or not working properly.
How can you find a list of these to compare with you own genetic data?
What seems to be needed is the rsid and the corresponding allele values.

Source 1:
The diagram on p5 may contain part of the answer, but what exactly to look for in the file?
https://www.omf.ngo/wp-content/uploads/2018/11/Edited-Robert-Phair-Metabolic-Trap.pdf

Source 2:
Diagnostics 2019, 9(3), 82; https://doi.org/10.3390/diagnostics9030082
The IDO Metabolic Trap Hypothesis for the Etiology of ME/CFS
Table 1. Common and rare mutations in IDO2 identified as damaging 3.

eg for mutation R248W -
snp rs10109853
Allele ref > alt C > T
exon 9
Min pop AF 0.418 (minimum alternate allele frequency (expressed as a fraction) reported [28] for any sampled population)
Max pop AF 0.487 (maximum alternate allele frequency reported for any sampled population)
SIFT damaging
PROVEAN deleterious
POLYPHEN probably damaging

so with your genetic data, suppose it says:
rsid rs10109853
chromosome 1
allele 1 G
allele 2 G
* how does this compare to the damaging mutation?
*Do alleles 1 and 2 need to be any letter between C and T?
*Does the exon need to match the chromosome number to have that mutation?
*Are the min / max values relevant, or is that missing from the genetic data analysis?
*Where to look for the R248W? Is this the name of a gene or something else?

Question 2
Are there any particular values that should be there, that would cause a problem in IDO2 if they are missing?

Please forgive me for any basic misunderstandings or confusion and the many wrong words. I am extremely brain fogged and it's taken me so very long just to get this far.

 

[Kitty]

The rsids are given at the top of the table you mention. They appear to be:

rs10109853
rs4503083
rs4736794
rs35212142
rs774492001

Not all of them will necessarily be reported in direct-to-consumer genetic testing.

*Do alleles 1 and 2 need to be any letter between C and T?

This means that the reference (more frequent) allele is C and the alternative (less frequent) is T. However, the so-called 'alternative' allele is still very common, appearing in between 41% and 48% of all sampled populations.

Where to look for the R248W? Is this the name of a gene or something else?

This is one way of referring to a specific variant within a particular gene. It isn't always described this way in
direct-to-consumer test reports – as you say, the rsid is easier to look for.

so with your genetic data, suppose it says:
rsid rs10109853
chromosome 1
allele 1 G
allele 2 G
* how does this compare to the damaging mutation?

This is about how the gene is 'read', or the orientation. Here's an article that might make this a bit easier to understand:

https://www.snpedia.com/index.php/Orientation


It's worth remembering that the metabolic trap is still only a theory at the moment. These mutation are described as 'damaging', but we don't know whether they actually have meaningful consequences. For instance, a variant might mean the body is only able to produce small amounts of a given enzyme, which can look important at first sight – but there could be compensating mechanisms elsewhere, which mean it has little or no effect on the body's ability to function normally.

 

[Barry]

@RDP said:

Okay, so on the left hand side is a plot of tryptophan, that's the molecule that's the substrate for this enzyme. The molecule that's converted to other molecules. On the vertical axis is the flux through that enzyme, that means how many molecules are converted to product in a unit time, so maybe a thousand molecules per second, something like that.

This intrigues me, because the flux described here relates to molecule conversions per unit time. The biological pathways between this and pwME's energy utilisation is way above my head I'm afraid, but ultimately the energy limitation pwME primarily suffer from (I'm pretty sure) is energy availability per unit time - i.e. power.

Does this IDO2 fault ultimately translate into a reduction in available power for pwME?

 

[daftasabrush]

I think I'm getting there, but a few more questions:

1. are alleles T > C equivalent to C > T (ie does positive or negative matter)?

2. can chromosome number be ignored, because the chromosome number either isn't important or would always be the same?
(I noticed the gene mutations didn't state what chromosome each was found in)

3. What is the exon, and does it further restrict whether a gene is matched? (ie if exon isn't reported in DNA results, does that mean only some of them will match)?

4. What are the min / max values about?

5. Finally, the research paper says Table 1. Common and rare mutations in IDO2 identified as damaging 3.

So is a match to that table (potentially) damaging? Or is it saying that if you have those rsid numbers, and if your alleles are different to those in the table then only that is (potentially) damaging?

 

[Kitty]

I have to go out, but I'll do this one because it's fairly straightforward:

1. are alleles T > C equivalent to C > T (ie does positive or negative matter)?

The symbol '>' is used to indicate 'greater than'. So 9>8 just means 9 is more than 8.

'<' means 'lesser than' in the same way.

T>C usually indicates that more people in the sampled population have the T allele than the C.

C>T usually indicates that more people in the sampled population have the C allele than the T.

 

[Barry]

1. are alleles T > C equivalent to C > T (ie does positive or negative matter)?

The symbol '>' is used to indicate 'greater than'. So 9>8 just means 9 is more than 8.

'<' means 'lesser than' in the same way.

T>C usually indicates that more people in the sampled population have the T allele than the C.

C>T usually indicates that more people in the sampled population have the C allele than the T.

I don't know if the values in question can go negative or not, but if so then to be purist:

8>-9 would be true of course. Unless for any negative quantities it were only their magnitudes that mattered, and not their sign.

 

[Barry]

A few very elementary questions, but I'm interested.

I emphasise this is purely hypothetical, as I imagine it would be extremely foolish and dangerous to try this in practice! So please don't!

Given that humans cannot manufacture tryptophan and can only obtain it from foods, what would happen if someone were to eat a tryptophan-free diet? (i.e. Identical diet to normal except without the tryptophan - impossible in practice I imagine). Seeing as once in the trap the high tryptophan density is actually preventing its conversion to kynurenine, would there be much difference? Is @RDP saying that once the trap is fully established, then although the extra-cellular tryptophan density would drop, the intra-cellular tryptophan density would remain high? Is it the intra-cellular tryptophan density that is the key interest here?

 

[sb4]

A few very elementary questions, but I'm interested.

I emphasise this is purely hypothetical, as I imagine it would be extremely foolish and dangerous to try this in practice! So please don't!

Given that humans cannot manufacture tryptophan and can only obtain it from foods, what would happen if someone were to eat a tryptophan-free diet? (i.e. Identical diet to normal except without the tryptophan - impossible in practice I imagine). Seeing as once in the trap the high tryptophan density is actually preventing its conversion to kynurenine, would there be much difference? Is @RDP saying that once the trap is fully established, then although the extra-cellular tryptophan density would drop, the intra-cellular tryptophan density would remain high? Is it the intra-cellular tryptophan density that is the key interest here?

Somebody over on PR did a very low / zero tryp diet and if I remember correctly didn't notice any improvements however I think he said he did notice worsening with high tryp foods.

 

[Trish]

Trouble is tryptophan is an essential amino acid present in protein and I assume needed for most protein based cell structures and functions in the body, so deficiency sounds to me like a bad idea.

 

[Barry]

Trouble is tryptophan is an essential amino acid present in protein and I assume needed for most protein based cell structures and functions in the body, so deficiency sounds to me like a bad idea.

But from @RDP's talk tryptophan's primary benefit is it being converted to kynurenine, which is depleted anyway once in the IDO trap; the tryptophan in itself is of no use other than being converted to something else. Or am I missing something crucial here?

But I do also see it gets converted to seratonin, which would suffer if no tryptophan were available.

It was really a thought experiment, to try and understand if dropping the tryptophan concentration would pull the molecular conversion back into the IDO1 operating range, where conversion could still work within those limits, and out of the IDO2 operating range which is broken. But I think (but not at all sure) if this is where the 10 day / 8 week issue comes into play).

 

[Trish]

But from @RDP's talk tryptophan's primary benefit is it being converted to kynurenine, which is depleted anyway once in the IDO trap; the tryptophan in itself is of no use other than being converted to something else. Or am I missing something crucial here?

I may be the blind leading the blind here. I'm sure our biochemists could answer this better than I can, but as I understand it there are 20 essential amino acids, of which tryptophan is one.

Amino acids bind together into complex structures to create proteins. Many of the structures in the body are proteins, for example actin and myosin, the two large molecules in muscles that make them contract, are proteins made from thousands of amino acids, including tryptophan. For example, google told me this:

Actin contains four tryptophan residues, W79, W86, W340, and W356, all located in subdomain 1 of the protein.

And this paper tells me myosin includes tryptophan.
http://www.jbc.org/content/275/33/25481.full.html

So my point was that tryptophan's role in the serotonin and kynurenine pathway is not its only role in the body. So we shouldn't try to deprive ourselves of it completely.

 

[Barry]

So my point was that tryptophan's role in the serotonin and kynurenine pathway is not its only role in the body. So we shouldn't try to deprive ourselves of it completely.

Which is why I say it was a thought experiment. I acknowledge I did not fully appreciate tryptophan had other important roles*, but in the context of this thought experiment it's of no consequence anyway, because no one is suggesting to do it for real at all ... I emphasised that right at the beginning.

If this were an engineering system I could potentially just isolate the bits I was interested in testing, and not fuss with the other bits. But with real biology and real living people you clearly cannot do that, hence trying it as a thought experiment. Just pretending you could look at the IDO trap aspect in isolation, and saying if tryptophan concentration could be dropped to zero for just that bit of the biology, what would happen? I suspect that is what Robert Phair has already covered anyway, but I got lost along the way. Especially not understanding if it is only the intra-cellular tryptophan concentrations that are significant, and if after 8 weeks those stay stuck at high levels no matter what else you do.

*Based on Robert Phair's comments: "There are other small or minor fates but big ones are the kynurenine pathway that goes from east to west in this diagram. ... just to make the system complete, there's a pathway going from north to south here that also is important for the fate of tryptophan and it is the production of serotonin")

 

[Barry]

Somebody over on PR did a very low / zero tryp diet and if I remember correctly didn't notice any improvements however I think he said he did notice worsening with high tryp foods.

As I said in my original post, I think it would be extremely silly to try such an experiment for real, for all sorts of reasons: Not properly trialled; probably cannot have a tryptophan-free diet without losing all sorts of other essential dietary needs; as @Trish points out, tryptophan needed elsewhere; etc, etc.

 

[daftasabrush]

I don't know if the values in question can go negative or not, but if so then to be purist:

8>-9 would be true of course. Unless for any negative quantities it were only their magnitudes that mattered, and not their sign.

The meaning is not the same as the "greater than" mathematical one, it's about forward and backward direction and which alleles are equivalent to others. If you check the orientation link on SNPedia above it explains positive and negative and how the letters are paired, eg:

In terms of the nucleotides for that SNP, the pairing of A with T,and C with G, in the DNA double helix means that an A on the plus strand by definition is a T on the minus strand, and vice versa, and a C on the plus strand means a G on the minus strand (and vice versa).

Each person's DNA analysis result will include the two alleles eg T > A.

 

[daftasabrush]

Ron Davis has explicitly said: do not experiment with this. Doing so can put the body into a much worse state which it may not be possible to get out of.

Tryptophan - Kynurenine is not the only tryptophan pathway, and there have already been huge safety concerns leading to L-typtophan supplements being withdrawn from use. Clearly we haven't developed the same condition as those taking L-typtophan so it is not simply a matter of lowering tryptophan. Tryptophan also impacts serotonin, and melatonin. Too much serotonin is also extremely dangerous (life threatening), yet too little is dangerous also.

The point of my post was: how you can tell if you have one of those IDO2 gene mutations based on data analyzed from saliva?
(But also, how to do comparisons in general - the ME/CFS Gene Study by Nancy Klimas has reported it's first results based on saliva sample consumer testing).

 

[Barry]

Ron Davis has explicitly said: do not experiment with this. Doing so can put the body into a much worse state which it may not be possible to get out of.
...
The point of my post was: how you can tell if you have one of those IDO2 gene mutations based on data analyzed from saliva?

And the point for my post was to:

• Make it very clear from the outset that to try anything for real would be very stupid.
• Trying to understand something from a purely hypothetical, thought experiment perspective, which is a valid investigative approach.

 

[wigglethemouse]

For clarity here are the five IDO2 variants of interest from table 1 of Robert Phair's paper. Thought I would repost here as posts on other threads are asking as well.

According to Robert Phair's paper the first two variants in the table have been shown experimentally to affect IDO2. The other 4 are predicted to be somewhat damaging by 3 different simulation tools.

The two most common damaging mutations, R248W and Y359STOP, are known to abolish enzyme
activity in an in vitro cell kynurenine production assay [34]. While the corresponding experiments for
I140V, S252T, and N257K have not been reported, the SIFT, PROVEAN, and POLYPHEN predictions are
suggestive and could contribute compound heterozygosity for individuals who are merely heterozygous
for R248W or Y359STOP. There are no such common mutations in IDO1 or in the remainder of the
kynurenine pathway.

I am homozygous (both gene copies) for rs10109853, the first variant, so it is seems from this information that my IDO2 is non-functional.

 

[Hoopoe]

What does minimum alternate allele frequency (expressed as fraction) mean?

Does a value of 0.418 mean that 418 out of 1000 people have the mutation? Or that in 1000 people, there are 418 mutations (some of them homozygous?)

 

[wigglethemouse]

Does a value of 0.418 mean that 418 out of 1000 people have the mutation? Or that in 1000 people, there are 418 mutations (some of them homozygous?)

I find it confusing too. Hope this will make a bit of sense.........

Looking at this variant on openSNP helps visualize the answer with real world data and not calculated data.
https://opensnp.org/snps/rs10109853


The T variant of rs10109853 is showing as having an allele frequency of 48%. This is the probability of having it on one allele. You need to look at genotype frequency to understand the occurrence in the population as humans have two alleles in most chromosomes. Here it shows CT as 48%, and TT as 24% meaning that 72% of the population has the variant.

There is an explanation of allele frequency vs population (genotype) frequency here.
https://biologydictionary.net/allele-frequency/

If p is the frequency of the normal dominant allele and q is the variant then
1 = p*p + 2pq + q*q

You have to start with q the variant. In the case above q = T is 0.48 or 48%. p = C is calculated as 1-q = 0.52 or 52%.
The TT genotype (population frequency) is q*q = 23%
The CC genotype = p*p = 27%
The CT genotype = 2pq = 50%

According to the paper referenced in Robert Phairs paper the IDO2 level will be reduced to <10% in the TT genotype. I'm not sure about the CT genotype. What Phair says is if the person with the CT genotype has another variant listed in the table then those two variants might be compounded such that the combination severely affects IDO2 output.

The math for multiple variants (e.g. combining any variants found of the 5 listed in the table) gets very confusing as some allele frequencies are different in different populations. The paper referenced in Robert Phairs paper shows the first variant is more prevalent in the European population and the second variant in the table is more prevalent in the Asian population. Here is the link where this is shown in the supplementary materials of the referenced paper. See figure S5 on page 5 and S6 on page 6.
https://cancerres.aacrjournals.org/...7/08/02/67.15.7082.DC1/IDO2_Supp_Figs_1-7.pdf

 

[dreampop]

Sorry if I'm not too bright, but the process for checking these would be going to 23andme, going to id02 and just searchign for the snp#s? Because I got no results. I have a WGA whose data I put into promethease and the SNPS didn't come up there either (though it only shows 1 snp on IDO2 which is weird)