Tetrahydrobiopterin

[mariovitali]

I am starting a new thread for this topic as it may be important. Apparently Tetrahydrobiopterin (aka BH4) has some very interesting associations to Kynurenine pathway, Nitric Oxide production, EBV and many more.

I am tagging @wigglethemouse for the SNPs that i provide :


GCH1

s4411417 (Risk C)
rs752688 (Risk T)
rs10483639 (Risk C)
rs3783641 (Risk A)
rs8007267 (Risk T)
rs10137071 (Risk T)
rs841 (Risk A)

SPR

rs6730083 (Risk G)
rs1876487 (Risk A)




More on BH4 / Tetrahydrobioptrin :


Connection with ME/CFS (?)

The existence of chronic ONS in the CNS following the activation of microglia and astrocytes subsequent to the existence of chronic peripheral inflammation may also explain the development of cerebral hypoperfusion in CFS patients. Briefly, high levels of NO and ROS result in oxidative damage to lipids, proteins and DNA in the endothelial cells of the BBB resulting in a pattern of escalating damage to such cells and a concomitant loss of the cytoprotective effects of NO normally derived from endothelial nitric oxide synthase (eNOS) (Lucas et al. 2015; Morris and Maes 2014). This is the result of oxidative inactivation of tetrahydrobiopterin (BH4), which is one of the enzyme’s essential cofactors, and changes in levels of arginine and calcium ions (Burghardt et al. 2013; Mitchell et al. 2007; Montezano and Touyz 2012). It should also be noted at this juncture that chronic peripheral inflammation can also impair endothelial eNOS function (Burghardt et al. 2013).

https://link.springer.com/article/10.1007/s11011-019-0388-6


Connection with Kynurenine :


http://www.jbc.org/content/291/2/652.full



Connection with NRF2

https://www.sciencedirect.com/science/article/pii/S0891584914010764



BH4 Connection with IDO, Quinolinic acid, Glutamate


https://www.frontiersin.org/articles/10.3389/fnins.2018.00499/full


EDIT : With DRP1

https://www.sciencedirect.com/science/article/pii/S0891584917300126

 

[Lisa108]

rs4411417: CC
rs752688: TT
rs8007267: TT
rs10483639: CC
rs3783641: AA
rs8007267: TT
rs10137071: TT


rs6730083: AA
rs1876487: CC

So that is 8/9 for me?

 

[mariovitali]

rs4411417: CC
rs752688: TT
rs8007267: TT
rs10483639: CC
rs3783641: AA
rs8007267: TT
rs10137071: TT


rs6730083: AA
rs1876487: CC

So that is 8/9 for me?

If the risk alleles are correct , then yes ! Hopefully someone who is far more knowledgeable than me can confirm the alternative (risk) alleles that i found.

 

[mariovitali]

I made a correction for rs6730083. the Risk allele is G, not A. Apparently on Sequencing.com they had this one wrong :



So they show 'A' as the alternative and Refernce is 'G' when actually it is the other way around after looking at dbSNP. Lesson learned.

I went through all of the rest variants and they are correct. @Lisa108 despite this, i believe that it is interesting that you have many homozygous mutations.

For the record, me and a severely ill patient have heterozygous mutations on all of these variants , apart from rs6730083

 

[wigglethemouse]

GCH1

s4411417 (Risk C)
rs752688 (Risk T)
rs8007267 (Risk T)
rs10483639 (Risk C)
rs3783641 (Risk A)
rs8007267 (Risk T)
rs10137071 (Risk T)

SPR

rs6730083 (Risk G)
rs1876487 (Risk A)

These are all very common

Gene GCH1

rs4411417
Alleles T>C 22% (Reference>Variant)
https://www.ncbi.nlm.nih.gov/snp/rs4411417

rs752688
Alleles C>T 22%
https://www.ncbi.nlm.nih.gov/snp/rs752688

rs8007267
Alleles C>T 30%
https://www.ncbi.nlm.nih.gov/snp/rs8007267

rs10483639
Alleles G>C 26%
https://www.ncbi.nlm.nih.gov/snp/rs10483639

rs3783641
Alleles T>A 24% / T>C
https://www.ncbi.nlm.nih.gov/snp/rs3783641


rs8007267
Alleles C>T 30%
https://www.ncbi.nlm.nih.gov/snp/rs8007267

rs10137071
Alleles C>G / C>T 41%
https://www.ncbi.nlm.nih.gov/snp/rs10137071


Gene SPR

rs6730083
Alleles G>A 17%
https://www.ncbi.nlm.nih.gov/snp/rs6730083

rs1876487
Alleles A>C 46%
https://www.ncbi.nlm.nih.gov/snp/rs1876487

 

[wigglethemouse]

@mariovitali
I don't see much excitement on gnomAD Broad Institute database. No protein coding genes. Some variants have no transcripts...... Here are the links.

Gene GCH1

rs4411417
https://gnomad.broadinstitute.org/variant/14-54853845-T-C?dataset=gnomad_r3

rs752688
https://gnomad.broadinstitute.org/variant/14-54844851-C-T?dataset=gnomad_r3

rs8007267
https://gnomad.broadinstitute.org/variant/14-54912273-C-T?dataset=gnomad_r3

rs10483639
https://gnomad.broadinstitute.org/variant/14-54839739-G-C?dataset=gnomad_r3

rs3783641
https://gnomad.broadinstitute.org/variant/14-54893421-T-A?dataset=gnomad_r3

rs8007267
https://gnomad.broadinstitute.org/variant/14-54912273-C-T?dataset=gnomad_r3

rs10137071
https://gnomad.broadinstitute.org/variant/14-54903622-C-T?dataset=gnomad_r3

Gene SPR

rs6730083
https://gnomad.broadinstitute.org/variant/2-72888868-G-A?dataset=gnomad_r3

rs1876487
https://gnomad.broadinstitute.org/variant/2-72887223-A-C?dataset=gnomad_r3

 

[mariovitali]

@wigglethemouse Thank you.

Unfortunately i do not understand -since these are all indeed common mutations- why they are shown in Disgenet as having some effect. Example for rs3783641 :

http://www.disgenet.org/browser/2/1/1/rs3783641/

Protein measurement
0.700 GeneticVariation GWASDB A genome-wide association study of circulating galectin-3. 23056639 2012
Pain
0.020 GeneticVariation BEFREE All subjects were genotyped at rs8007267 and rs3783641 to determine the frequency of the GCH1 pain-protective haplotype. 19014702 2009
Pain
0.020 GeneticVariation BEFREE Diagnosis of the pain-protective GCH1 haplotype was possible with 100% sensitivity and specificity by screening for just 3 GCH1 genetic variants that span the entire DNA range of the haplotype: c.-9610G>A (dbSNP rs8007267G>A) in the 5' untranslated region, c.343 + 8900A>T (dbSNP rs3783641A>T) in intron 1, and c.*4279 (dbSNP rs10483639C>G) in the 3' untranslated region. 17363416 2007
Agnosia for Pain
0.010 GeneticVariation BEFREE The number of days until analgesia was not required during the first postoperative week was associated with GCH1 SNPs (rs8007267, P=0.05; rs3783641, P=0.01; rs10483636, P=0.002). 20842020 2011
Widespread Chronic Pain
0.010 GeneticVariation BEFREE Three SNPs forming a proposed "pain-protective" haplotype in GCH1 (rs10483639, rs3783641 and rs8007267) and two SNPs in OPRM1 (rs1777971 (A118G) and rs563649) were genotyped in cases with persistent CWP (CWP present at >or=2 time-points) and controls who were pain-free at all time-points. 19775452 2010

and another

Protein measurement
0.700 GeneticVariation GWASDB A genome-wide association study of circulating galectin-3. 23056639 2012
Pain
0.040 GeneticVariation BEFREE The presence of a GCH1 haplotype with high BH4 in populations of African ancestry could explain the association of rs8007267 with sickle cell anemia pain crises. 24136375 2014
Pain
0.040 GeneticVariation BEFREE Three SNPs forming a proposed "pain-protective" haplotype in GCH1 (rs10483639, rs3783641 and rs8007267) and two SNPs in OPRM1 (rs1777971 (A118G) and rs563649) were genotyped in cases with persistent CWP (CWP present at >or=2 time-points) and controls who were pain-free at all time-points. 19775452 2010
Pain
0.040 GeneticVariation BEFREE All subjects were genotyped at rs8007267 and rs3783641 to determine the frequency of the GCH1 pain-protective haplotype. 19014702 2009
Pain
0.040 GeneticVariation BEFREE Diagnosis of the pain-protective GCH1 haplotype was possible with 100% sensitivity and specificity by screening for just 3 GCH1 genetic variants that span the entire DNA range of the haplotype: c.-9610G>A (dbSNP rs8007267G>A) in the 5' untranslated region, c.343 + 8900A>T (dbSNP rs3783641A>T) in intron 1, and c.*4279 (dbSNP rs10483639C>G) in the 3' untranslated region. 17363416 2007
Anemia, Sickle Cell
0.010 GeneticVariation BEFREE The presence of a GCH1 haplotype with high BH4 in populations of African ancestry could explain the association of rs8007267 with sickle cell anemia pain crises. 24136375 2014
Mood Disorders
0.010 GeneticVariation BEFREE Thus, we considered the GTP cyclohydrolase gene (GCH1) to be a good candidate gene in the pathophysiology of MDs and of the serotonin selective reuptake inhibitors (SSRIs) response in MDD, and conducted a case-control study utilizing three SNPs (rs8007267, rs3783641 and rs841) and moderate sample sizes (405 MDD patients, including 262 patients treated by SSRIs, 1022 BP patients and 1805 controls). 22770721 2013
Agnosia for Pain
0.010 GeneticVariation BEFREE The number of days until analgesia was not required during the first postoperative week was associated with GCH1 SNPs (rs8007267, P=0.05; rs3783641, P=0.01; rs10483636, P=0.002). 20842020 2011
Widespread Chronic Pain
0.010 GeneticVariation BEFREE Three SNPs forming a proposed "pain-protective" haplotype in GCH1 (rs10483639, rs3783641 and rs8007267) and two SNPs in OPRM1 (rs1777971 (A118G) and rs563649) were genotyped in cases with persistent CWP (CWP present at >or=2 time-points) and controls who were pain-free at all time-points.

http://www.disgenet.org/browser/2/1/1/rs8007267/

 

[mariovitali]

@wigglethemouse Another example from dbSNP for rs8007267 and rs10483639 (both on the list i provided). Lower levels of BH4 is what we are looking for !

GCH1 haplotype determines vascular and plasma biopterin availability in coronary artery disease effects on vascular superoxide production and endothelial function. Part of a haplotype, rs10483639(C)-rs8007267(T), associated with lower levels of tetrahydrobiopterin.[PMID 18598896]

 

[wigglethemouse]

@wigglethemouse Another example from dbSNP for rs8007267 and rs10483639 (both on the list i provided). Lower levels of BH4 is what we are looking for !

Okay, from GCH1 Haplotype Determines Vascular and Plasma Biopterin Availability in Coronary Artery Disease: Effects on Vascular Superoxide Production and Endothelial Function that you reference above

The GCH1 haplotypes were defined by 3 polymorphisms: rs8007267G<A, rs3783641A<T, and rs10483639C<G (X haplotype: A, T, G; O haplotype: any other combination).

Results

Haplotype frequencies were OO 70.6%, XO 27.4%, and XX 2.0%. The X haplotype was associated with significantly lower vascular GCH1 messenger ribonucleic acid expression and substantial reductions in both plasma and vascular BH4 levels. In X haplotype carriers both vascular superoxide and L-NAME–inhibitable superoxide were significantly increased, and were associated with reduced vasorelaxations to acetylcholine.

We first examined the effect of GCH1 haplotype on the levels of plasma biopterins. Patients with the X haplotype had significantly lower plasma levels of both BH4 and total biopterins than patients homozygous for the common O haplotype; median plasma BH4 levels in patients with XX genotype were reduced by approximately 80% compared with OO patients.

I interpret this as 2% of study patients with all of the following has BH4 reduced by 80%
rs8007267 AA
rs3783641 TT
rs10483639 GG

These (i.e. AA, TT, GG) are the reference alleles not the variants that you are referencing? The OO haplotype frequency is 70.6% in this study.

Hope I got that right @mariovitali

 

[Amw66]

BH4 is the short cut to methionine synthesis if the normal.pathway is compromised ( mthfr mutations being common)
Is there an" order" for BH4 function/ use which would compromise other actions if it was prioritised?

It strikes me that it's not potentially the I.ia t a couple of mutations for conditions but the cumulative effect of many.

 

[mariovitali]

@wigglethemouse

Yes that is correct. However i am finding information regarding the haplotype mentioned in that paper as gs224

https://www.snpedia.com/index.php/Gs224

I do not know why there is a discrepancy but the text in the link i provided writes :

GCH1 haplotype determines vascular and plasma biopterin availability in coronary artery disease effects on vascular superoxide production and endothelial function. The haplotype rs10483639(C)-rs3783641(A)-rs8007267(T) is associated with lower levels of tetrahydrobiopterin.[PMID 18598896]

On my first post i provide the variants along with their alt. alleles which match completely with the ones quoted above. This haplotype is referred as haplotype gs226. In my -limited- understanding there appears to be a discrepancy between the text quoted above and the study. Moreover user nandixon discusses about GCH1 here, quoteing the same alt. alleles :

https://forums.phoenixrising.me/thr...-bh4-relation-to-nos.19570/page-2#post-592334

In that post , angiotensin is being mentioned (something that comes up over and over to my machine learning runs). Also norepinephrine and epinephrine are closely related to BH4. Vitamin C (which replenishes BH4) appears to be useful in POTS :


https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3191072/


From all the above i will definitely be looking at this more closely, something i didn't do in the past.

 

[mariovitali]

@wigglethemouse @Lisa108 I added rs841 (Risk A) to the list.

It is very interessting that in the following link on rs841, lower HRV (Heart rate variability) is mentioned and also decreased baroreflex.

https://www.snpedia.com/index.php/Rs841


FWIW I am heterozygous on rs841 and the severely ill patient is as well.

 

[Lisa108]

rs 841: AA

Dang, I just wanted to change my avatar...

 

[wigglethemouse]

https://www.ncbi.nlm.nih.gov/clinvar/variation/313386/
rs841 is very common - 22%.
https://www.ncbi.nlm.nih.gov/snp/rs841

Clinvar has 3 citations stating benign.
https://www.ncbi.nlm.nih.gov/clinvar/variation/313386/

It is very interesting that in the following link on rs841, lower HRV (Heart rate variability) is mentioned and also decreased baroreflex.

https://www.snpedia.com/index.php/Rs841

I followed the link but don't see the scientific source for lower HRV and decreased baroreflex. I did find this though linking rs841 with C+273T
Tetrahydrobiopterin, Superoxide and Vascular Dysfunction
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2852262/

The discovery of frequent GTPCH mRNA variants in the 3-untranslated regions (3’-UTR) C+273T (rs841) was recently reported [26].

Reference 26

26. Zhang L, Rao F, Zhang K, Khandrika S, Das M, Vaingankar SM, Bao X, Rana BK, Smith DW, Wessel J, Salem RM, Rodriguez-Flore JL, Mahata SK, Schork NJ, Ziegler MG, O’Connor DT. Discovery of common human genetic variants of GTP cyclohydrolase 1 (GCH1) governing nitric oxide, autonomic activity and cardiovascular risk. J. Clin. Invest. 2007;117:2658–2671. [PMC free article] [PubMed] [Google Scholar]

where the text states

GCH1 C59038T (3′-UTRs C+243T).
The 3′-UTRs variant predicted NO excretion (P = 0.0086), though not neopterin or catecholamine secretion (Table (Table4).4). Significant C+243T associations were also found for autonomic traits: baroreceptor coupling (P = 0.0414), maximum pulse interval (i.e., minimum heart rate; P = 0.0075), and pulse interval variability (heart rate variability; P = 0.0498). The 3′-UTRs polymorphism did not associate with basal (resting, average) BP or heart rate in these predominantly normotensive subjects (Table (Table4). 4).

So, that paper is all about C+243T, not C+273T. A typo. I guess the first paper meant C+243T.
Just to double check
rs841 is Chr 14 position 54843774 G->A. Lets look it up
https://gnomad.broadinstitute.org/region/14-54843754-54843794?dataset=gnomad_r3
the table at the bottom shows c.*12+8C>T

I've not been able to see what the variant position on the chromosome is for C+243T. Do you know?



When I first started looking at my 23andMe data I got excited about each mutation found. Then it was explained to me that
* the body has many genes affecting the same protein and so backup mechanisms exist in many situations.
* if you do find a gene that is damaging both in studies, and SIFT, that has a high CADD score it doesn't mean it is disease causing in your case.
* Ideally a genetic study is tied with a proteomics study. If in proteomics you see proteins very high or low you can then look at the genetic data to see if there is a link. Unfortunately to date I don't believe we have seen a large proteomics study in ME. And on an individual basis you would want to run a medical test to confirm the disease mechanism explained by the mutation if possible.
* You probably also want to look at your siblings data and parents data to see if any of them have the same mutation/symptom.

I guess what I'm trying to say is that it is all very complicated, way above my head, and easy to jump to assumptions. Sorry to be Debby Downer. We need to follow-up multiple citations where possible.

 

[wigglethemouse]

I do not know why there is a discrepancy but the text in the link i provided writes :

I can only think that the original paper used < rather than > and that confused people reading it. e.g. they wrote G<A rather than the more usual A>G

The GCH1 haplotypes were defined by 3 polymorphisms: rs8007267G<A, rs3783641A<T, and rs10483639C<G (X haplotype: A, T, G; O haplotype: any other combination).

 

[mariovitali]

I guess what I'm trying to say is that it is all very complicated, way above my head, and easy to jump to assumptions. Sorry to be Debby Downer. We need to follow-up multiple citations where possible.

I agree, we should be very careful in not accepting but also not dismissing things easily and Thank you once again for this detective work.

Here is a post i made on PR :

https://forums.phoenixrising.me/thr...e-treatment-for-cfs.37244/page-29#post-634381

So back then, i did notice some issues with protein but believed that it was attributed to "protein misfolding". I discuss how by not eating protein for 20 days and then introducing it, i got Sensorineural Hearing Loss (= lost my hearing on one ear).

Also, i felt the worst when i would switch to a low fat, low carb diet (which means that i had to go high-protein). Given the re-visit to GCH1 and Phenylalanine metabolism, i stopped eating meat for the past 9 days and dropped every supplement i take apart from Metafolin and Vitamin C (to replenish BH4). No symptoms so far but i have to pass the 15-days mark. Numerous times in the past i would try to stop the supplements i was taking and within 7-10 days i would get symptoms with the first one being tinnitus.

If this does not happen and when i re-introduce protein i get tinnitus then that will confirm the origin of problems for myself. I will report back.

More potential interesting "signals" are coming up that require further investigation. This i found on the ME association regarding a machine learning approach that was able to differentiate ME/CFS vs Controls :

https://www.meassociation.org.uk/wp...n-MECFS-using-Raman-spectroscopy-06.09.18.pdf

@Andy Dr Morten is being mentioned. Could we ask what is his opinion with Phenylalanine on ME/CFS patients by any chance?


@wigglethemouse I did not have the time to look in the paper discussing the Machine Learning approach extensively. The algorithm they use is Ok but they could have used more advanced algorithms. I will have to elaborate more on the paper :

https://pubs.rsc.org/en/Content/ArticleLanding/2018/AN/C8AN01437J#!divAbstract

 

[wigglethemouse]

https://www.meassociation.org.uk/wp...n-MECFS-using-Raman-spectroscopy-06.09.18.pdf

@Andy Dr Morten is being mentioned. Could we ask what is his opinion with Phenylalanine on ME/CFS patients by any chance?

phenylalanine is definitely on my radar. Chris Armstrong was the first to highlight it.

Metabolic profiling reveals anomalous energy metabolism and oxidative stress pathways in Chronic Fatigue Syndrome patients.
https://www.researchgate.net/public...pathways_in_chronic_fatigue_syndrome_patients

3.2 Altered blood metabolites in patients with ME/CFS
The absolute concentrations of six blood metabolites from the 1H NMR analysis were significantly altered. Glucose levels were increased whereas acetate, glutamate, hypoxanthine, lactate, and phenylalanine were decreased in ME/CFS patients (Fig. 3A). When blood metabolites were analyzed as a function of total metabolite concentrations (relative abundance data) there were again, six metabolites that were significantly different (Fig. 3A). For this analysis aspartate and glucose were increased whereas acetate, glutamate, hypoxanthine, lactate, and phenylalanine decreased in ME/CFS patients.

Karl Morten then identified phenylalanine in a model cell and in patients in the work you mentioned. He didn't talk about it in the Q&A here on s4me............

And soon we should expect more data from the Jarred Younger good day bad day paper where samples sent to Chris Armstrong measured metabolites............

 

[mariovitali]

@wigglethemouse i believe at one point we had a discussion about mast cell activation syndrome (MCAS). Yet one more possible signal regarding BH4 and its role in NO :

Mast cells are allergy cells responsible for immediate allergic reactions. They cause allergic symptoms by releasing products called “mediators” stored inside them or made by them. In allergic reactions, this release occurs when the allergy antibody IgE, which is present on the mast cell surfaces, binds to proteins that cause allergies, called allergens. This triggering is called activation, and the release of these mediators is called degranulation.

So, mast cell degranulation. Then i find this :

Tetrahydrobiopterin, a critical factor in the production and role of nitric oxide in mast cells.
Gilchrist M1, Hesslinger C, Befus AD.
Author information
1
Glaxo-Heritage Asthma Research Laboratory, Pulmonary Research Group, Department of Medicine, University of Alberta, Edmonton, Alberta T6G 2S2, Canada. mark.gilchrist@ualberta.ca
Abstract
Mast cells (MC) are biologically potent, ubiquitously distributed immune cells with fundamental roles in host integrity and disease. MC diversity and function is regulated by exogenous nitric oxide; however, the production and function of endogenously produced NO in MC is enigmatic. We used rat peritoneal MC (PMC) as an in vivo model to examine intracellular NO production. Live cell confocal analysis of PMC using the NO-sensitive probe diaminofluorescein showed distinct patterns of intracellular NO formation with either antigen (Ag)/IgE (short term) or interferon-gamma (IFN-gamma) (long term). Ag/IgE-induced NO production is preceded by increased intracellular Ca2+, implying constitutive nitric-oxide synthase (NOS) activity. NO formation inhibits MC degranulation. NOS has obligate requirements for tetrahydrobiopterin (BH4), a product of GTP-cyclohydrolase I (CHI), IFN-gamma-stimulated PMC increased CHI mRNA, protein, and enzymatic activity, while decreasing CHI feedback regulatory protein mRNA, causing sustained NO production. Treatment with the CHI inhibitor, 2,4-diamino-6-hydroxypyrimidine, inhibited NO in both IFN-gamma and Ag/IgE systems, increasing MC degranulation. Reconstitution with the exogenous BH4 substrate, sepiapterin, restored NO formation and inhibited exocytosis. Thus, Ag/IgE and IFN-gamma induced intracellular NO plays a key role in MC mediator release, and alterations in NOS activity via BH4 availability may be critical to the heterogeneous responsiveness of MC.

 

[wigglethemouse]

Very interesting link @mariovitali

There are also papers stating mast cells produce NO as well as react to NO
http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0074-02762005000900003

ABSTRACT

Mast cells (MC) are important in the numerous physiological processes of homeostasis and disease. Most notably, MC are critical effectors in the development and exacerbation of allergic disorders. Nitric oxide (NO) is a diatomic radical produced by nitric oxide synthase (NOS), and has pluripotent cell signaling and cytotoxic properties. NO can influence many MC functions. Recent evidence shows the source of this NO can be from the mast cell itself. Governing the production of this endogenous NO, through alterations in the expression of tetrahydrobiopterin (BH4), a NOS cofactor, has stabilizing effects on MC degranulation. Furthermore, NO regulates the synthesis and secretion of de novo generated mediators, including leukotrienes and chemokines. These novel observations add to the growing body of knowledge surrounding the role of NO in the MC.

I wish I knew what Chris Armstrong was working on regarding Nitrogen

 

[mariovitali]

@wigglethemouse

Of course i cannot be sure that this is what he is looking at but many interesting topics we've been discussing. From a Nitrogen metabolism page (link below) :

and (see NMDA, AMPA, Kainate below, related to excitotoxicity)

https://themedicalbiochemistrypage.org/nitrogen-metabolism.php