May 2024
Dr Valerio Vittone
Dr Graham Exelby
Genetics is the study of heritable changes in gene activity or function due to the direct alteration of the DNA sequence. Epigenetics is the study of heritable changes in gene activity or function that is not associated with any change of the DNA sequence itself.
Although virtually all cells in an organism contain the same genetic information, not all genes are expressed simultaneously by all cell types. In a broader sense, epigenetic mechanisms mediate the diversified gene expression profiles in a variety of cells and tissues in multicellular organisms.(1)
Gene expression in the brain is dramatically regulated by a variety of stimuli. While the role of neural activity has been extensively studied, less is known about the effects of metabolism and nutrition on transcriptional control mechanisms in the brain. In the last twenty years, it has become clear that epigenetics plays a crucial role in modulating central nervous system functions and finally behaviour. (2)
Polygenic risk scores
A polygenic risk score (PRS) estimates the genetic risk of an individual for some disease or trait, calculated by aggregating the effect of many common variants (usually defined as minor allele frequency 1%) in the genome, each of which can have a small effect on a person’s genetic risk. associated with the condition. A PRS is the weighted sum of a collection of genetic variants, usually single nucleotide polymorphisms (SNPs) or single base-pair variations from the reference genome. (3)
Our reports are generated using cutting edge AI and machine-learning technology. Robust methodologies in AI including Deep Learning, Bayesian Machine learning, and Hyperdimensional Computing for imputation are the foundations for our genetic models. Using a technique called genetic imputation, it is now possible to use the ~750,000 SNPs that our world-class Illumina GSA sequencing machine in Germany provides on a chip and use the gene imputation data to compare to into up to 83 million additional SNP variants with an accuracy rate of 98%.
Critical DNA mutations in POTS and Long-Covid
From the DNA studies by Dr Valerio Vittone, the mutations in the TLRs, mast cell membrane and in Dao enzyme function are major contributors to the pathogenesis of Long COVID, and when combined with his other findings, eg COMT, PEMT mutations, clear paths are emerging to reduce the severity of COVID infections and in the management of Long COVID. Problems such as persisting thromboinflammation as may be seen in persistently elevated D-Dimer may be identified with DNA as there are no available biomarkers for many of these mutations.
Using a technique called genetic imputation, it is now possible to use the ~750,000 SNPs on a chip into up to 83 million additional SNP variants with an accuracy rate of 98%.
More than 400 genes are differentially expressed in long covid patients. Ongoing DNA assays in Long COVID that confirm similar findings warrant further investigation in a broader cohort of Long COVID subtypes.
There is increasing DNA evidence that multiple mutations in the Toll-Like Receptors (especially “first responders” TLR2 and TLR4) play a large role in the individual immune response, and associated with “downstream” mutations can create a domino effect responsible for the individual problems being caused by Covid. These are common and look to provide directions for management in patients not responding to the first line mast cell blockade.
The TLR4 mutations appear to be potentially one of the critical “molecular connections” associated with the abnormally low “biomarkers” used to measure the inflammatory responses seen in many Covid patients. These patients have often “fallen below the radar” while in fact have an impaired immune response. – see Long COVID Immune Dysfunction
When looking at recovering Long Covid patients, there are mutations in mast cell function, with one on the mast cell membrane and two critical enzymes (namely DAO & HNMT) involved in clearing histamines in different tissues in the body.
The Dao enzyme (Diamine oxidase) is involved in the metabolism, oxidation, and inactivation of histamine and other polyamines such as putrescine or spermidine. DAO is produced in kidneys, thymus and intestinal lining that breaks down excess histamine in the body, particularly in the gut.
HNMT Histamine N-methyltransferase) catalyzes the methylation of histamine in the presence of S-adenosylmethionine (SAM-e) forming N-methylhistamine. HNMT is involved in metabolism of intracellular histamine, mainly in kidneys and liver, but also in bronchi, large intestine, ovary, prostate, spinal cord, spleen, trachea. and peripheral tissue.
Vascular complications in COVID-patients have been linked phosphatidylserine to thrombo-inflammation. Thrombo-inflammation plays a critical role through complement activation and cytokine release, platelet overactivity, apoptosis (thrombocytopathy), as well as coagulation abnormalities (coagulopathy).((46)(47) Phosphatidylserine is associated with increased thrombo-inflammation and vascular complications, and can trigger the blood coagulation cascade, the complement system, inflammation and reside on activated immune cells. Phosphatidylserine is synthesized from phosphatidylcholine through a Ca2+ -dependent reaction in the endoplasmic reticulum. PEMT mutations, as well as other thrombophilia-related genes eg Factor V Leidon are likely to be associated with persisting elevated D-Dimer levels in Long COVID.
The Critical Mutations in POTS/ Long Covid:
A single mutation, by itself, may be insufficient to cause the symptoms of Long Covid. However, we have been recognising patterns that allow clinicians to treat POTS (and increasingly, long COVID). As the sensitisation from the microglial activation and small fibre neuropathy is controlled, we can work with management protocols.
The mechanical and other activators and drivers do not necessarily cause symptoms in other people. DNA polymorphisms and sensitisation make the difference. This list is incomplete, but we are normally able to show the impact of mutations in POTS, Long Covid and comorbidities, eg Hashimoto’s and rheumatoid arthritis.
Attempts to reduce POTS to one or 2 causes or depend on management of dysfunctional mast cells and for example is usually fraught with failure as it is a summation of multiple metabolic, psychological, genetic, infective, traumatic, mechanical, dietary and inflammatory factors.
Even when looking at DNA many practitioners might attribute everything to “methylation,” but this for example would discount “glycation” and similarly attempts to reduce the impact of DNA mutations in vital areas eg PEMT, Apo E4 and STAT3 can in reality be potentially harmful if one is singled out without a clear knowledge of underlying metabolic pathways and how they interact. Dr Vittone, when describing the importance of glycation in POTS “One hypothesis could be that AGEs may contribute to endothelial dysfunction or vascular stiffness, exacerbating the orthostatic intolerance seen in POTS patients. Moreover, AGEs could interact with the receptor for AGEs (RAGE) on immune cells, potentially causing inflammation that may alter autonomic function."
More than 400 genes differentially expressed in long covid patients. There is seldom only 1 mutation involved. The major mutations are as follows, with more detailed information in TLR4, mast cell, TRP, COMT, STAT3, APOE4 and NFkB and CCL2.
Toll-Like Receptors (especially “first responders” TLR2 and TLR4) play a large role in the individual immune response, and associated with “downstream” mutations can create a domino effect responsible for the individual problems being caused by Covid. TLR4 receptors are on Natural Killer Cells (NK cells)
TLR2 senses the SARS-CoV-2 envelope protein (E), resulting in production of inflammatory cytokines and chemokines, contributing to the hyperinflammatory state and tissue damage seen in severe Covid. The severity of the Covid infection is largely determined by the E Protein /TLR2 activation rather than the S protein.(42)
TLR4 signalling is activated by the Spike protein (S). This can lead to a pro-thrombotic and pro-inflammatory state contributing to severe complications eg myocardial infarction and acute lung injury.(43)(44)
The endosomal TLR3 senses intracellular viral dsRNA. Activated TLR regulates the production of proinflammatory factors through a series of signalling in the NF‐κB pathway and activates IRF3/7 to produce I IFN. (42)(44) A DNA variant in TLR3 has also been identified as increasing susceptibility and mortality to acute COVID infections by decreasing TLR3 expression and impairing recognition of SARS-Co-V dsRNA. (42) These results suggest perivascular inflammation may be a critical factor in Long COVID, but the role of these receptors in Long COVID associated POTS has not been established.
Mast cells- membrane, HNMT, Dao enzyme function. Activation triggers include
TLR activation
Oxidative stress (Reactive Oxygen Species)
Extracellular mitochondrial components from damaged mitochondria
Glutathione depletion (compromised clearance of free radicals)
COMT (impaired processing of catecholamines, 2 types, fast and slow COMT- clue is family history breast cancer, autism, ADHD, POTS/dysautonomia, fibromyalgia, Parkinson’s disease)- see below.
Methylation mutations eg MTHFR- the 677 MTHFR mutation typically is associated with increased homocysteine, and affects collagen function via SAMe and other molecules as well as increased thrombotic risk, and plaque formation in different tissues.
Elevated plasma levels of homocysteine are a metabolic risk factor for atherosclerotic vascular disease, as shown in numerous clinical studies that linked elevated homocysteine levels to de novo and recurrent cardiovascular events. High levels of homocysteine promote oxidative stress in vascular cells and tissue because of the formation of reactive oxygen species (ROS), which have been strongly implicated in the development of atherosclerosis.
BHMT, PEMT and CHKA are connected to MTHFR and the folate and methylation cycle so mutations in genes like MTR and MTFHR indirectly affect them too, eg in the BHMT-catalyzed reaction, betaine, also known as trimethylglycine, acts as a methyl donor, transferring one of its methyl groups to homocysteine. This transfer results in the conversion of homocysteine to methionine and betaine to dimethylglycine.
PEMT mutations- Phosphatidylethanolamine N-methyltransferase (PEMT) catalyses phosphatidylcholine synthesis. PEMT and similar mutations are involved in vascular complications, neurodegeneration and thrombo-inflammation. It is thought that PEMT gene polymorphisms are associated with non-alcoholic fatty liver disease (NAFLD).(48) Fatigue is a common symptom of PEMT mutations and its associated mitochondrial dysfunction, and is thought to be involved in neurodegenerative disease. PEMT is involved in the biosynthesis of phosphatidylcholine (PC) from phosphatidylethanolamine (PE), and likely to be the underlying culprit in persistent D-Dimer tests in Long Covid. At present, only DNA can reveal whether this is present as there are no biomarkers
The CHKA gene codes for the choline kinase alpha enzyme, which is involved in the pathway of reactions that coverts choline into phosphatidylcholine needed for cell membranes.
NLRP3 mutations look to play a major role in this neurodegenerative/inflammatory process as University of Qld researchers Woodruff et al demonstrated that SARS drives NLRP3 inflammasome activation in human microglia through spike protein.
TRP mutations- TRPM3 appears critical in NK (Natural Killer) immune cell function, with implications for Ca2+ signalling, cell function,(4) and glymphatic function
The transient receptor potential melastatin subfamily 3 (TRPM3) is one of the most primitive receptors in the body, activated by a wide variety of agents, from bacteria and viruses to temperature and environmental factors such as perfumes. This diversity made it a logical suspect for a condition like CFS that has so many different triggers in different people.
Prof Sonya Marshall-Gradisnik and the Griffith University Chronic Fatigue team working with TRPM3 function in the research into chronic fatigue at Griffith University (4)(5)(6) have linked mutations in this pathway with “glymphatic” function with consequent reduced clearance of waste solutes from the brain with production of fatigue and brain fog and the therapeutic benefit of Low Dose Naltrexone
TRPA1 is a key ion channel that detects oxidative stress and a range of endogenous and exogenous chemicals (smoke, solvents, cold air).
o TRPM3 activity is impaired in CFS/ME patients suggesting changes in intracellular Ca2+ concentration, which may impact NK cellular functions. This investigation further helps to understand the intracellular-mediated roles in NK cells and confirm the potential role of TRPM3 ion channels in the aetiology and pathomechanism of CFS/ME.(4)(5)
Excessive or dysregulated TRPM3 expression can result in neurological consequences.(52)
Excessive TRPM7 expression, particularly in PV GABAergic neurons, appears to exacerbate neuronal damage and dysfunction following brain ischaemia. Reducing TRPM7 levels in these neurons has stronger neuroprotective effects compared to reducing it in glutamatergic neuron o
Current CFS research encompasses how to correct the over-expression of TRPM3a7 variant that is over-expressed in ME CFS and long COVID. This then allows normal function of several cellular ion channels. We found over expression of TRPM3 and 4 o
Prof Pete Smith describes that low dose naltrexone (LDN) is both a TLR4 modulator, is an NLRP3 inhibitor and well as restoring TRPM3 function
STAT 3 : This plays an important role in inflammation and tumorigenesis by regulating cell proliferation, differentiation and metabolism. It is a critical factor in IL-6 regulation. There are no statistics published as yet on Covid-related malignancy. Its pathways can however be seen through the DNA pathways already uncovered- eg TLR4/ RAGE/STAT3. The direct effect of Covid on natural Killer cells (NK cells) looks also to be critical as this cell’s function is vital in controlling malignancy.-see Long COVID Immune Dysfunction
IL-6 and other Interleukins
Oxidative stress and mitochondrial mutations eg eNOS, SOD2. NO metabolism- associated with the development of FMS and pain sensitization, and a likely problem in Long Covid.
Lipoprotein a (LPa): High levels of LPa, found in 20% of European descent, increases the inflammatory response and thrombotic risk in COVID-19.(7)
APO E4 - The Apolipoprotein E allele 4 is a major genetic risk factor for Alzheimer's disease, as this lipid carrier is important for maintaining homeostasis necessary for a healthy environment of the brain. This mutation is seen in around 15 to 20% of the general population, with 2-3% being homozygous with the increased risks that are associated. This is emerging as a significant mutation in resistant cognitive impairment. Of note there has been research into the use of ARB medication candesartan and telmisartan in reducing risk- this is the subject under close scrutiny, particularly with increasing evidence of neuroprotection afforded by candesartan and telmisartan.
APO E is particularly concentrated in astrocytic processes at the pial surface and around the blood vessels. In addition, the choroid plexus and tanycytes in the wall of the third ventricle also produce Apolipoprotein E. Thus, Apolipoprotein E production is co-localized with CSF production sites and transport pathways suggesting that lipids are transported by the glymphatic system.
The glymphatic system is thought to play a central role in macroscopic distribution of lipids in the brain and that medium to large lipid soluble molecules might require carrier particles in order to be delivered via the CSF. Astrocytes thus play a key role in lipid synthesis and lipid distribution by releasing lipid carrier proteins, such as Apolipoprotein E, and in maintaining the highway for distribution, the glymphatic system.
APO E4 mutation also affects arteries, significantly increasing coronary artery disease risk ,decreased mitochondrial function, decreased insulin sensitivity, increased insulin resistance, fatty liver and progression to cirrhosis (APO E4 contributing to altered VLDL metabolism and increased atherosclerosis).
Core inflammatory genes: Inflammasone activation, oxidative stress and mitochondrial damage
· NF-KB
· IL-6
· CRP
· CCL2
· IL-1B
· TNF
Malignancy risk:
· TLR4/RAGE/STAT3
· COMT
· NFKB
· CCL2
Clotting: simplified. Interesting as stress alters D Dimer, IL6 is one of these
· TLR4/IL6/PEMT
· MTHFR, Factor V Leidon, prothrombin gene, Protein C and S deficiencies
· TLR3
TLR4 Mutations:
Toll-like Receptor 4 (TLR4) is a protein receptor found on the surface of immune cells, including Natural Killer Cells (NK cells), which play a critical role in the innate immune response to viral infections and cancer. When TLR4 on NK cells is activated, it triggers a signalling cascade that leads to the production of cytokines and chemokines, which help activate other immune cells to fight the threat.
There is increasing DNA evidence that multiple mutations in the Toll-Like Receptors (especially “first responders” TLR2 and TLR4) play a large role in the individual immune response, and associated with “downstream” mutations can create a domino effect responsible for the individual problems being caused by Covid. These are common and look to provide directions for management in patients not responding to the first line mast cell blockade. The TLR4 mutations may explain the effectiveness of Low Dose Naltrexone in chronic fatigue and cognitive impairment.
Diseases driven by TLR4 signalling due to its involvement in inflammation and immune responses include:
Sepsis
Inflammatory bowel disease including Crohn’s disease and Ulcerative Colitis by promoting production of inflammatory cytokines and chemokines
Arteriosclerosis, with promotion of inflammation in the arterial wall, leading to plaque formation. Toll-like receptor-4 (TLR4) signalling pathway has the role of hub in preserving aorta homeostasis, but also in contributing to the onset of degenerative aorta diseases, such as aneurysms. As the result of a sustained/excessive activation of TLR4 signalling pathway, expressed on both endothelial and vascular smooth muscle cells, followed by its cross-talk with other pathways, and also by TGF-β, NO, MMP, NF-Kβ pathways.(8)
Rheumatoid arthritis, by inducing inflammatory cytokines and promoting synovial inflammation
Asthma
Chronic obstructive airways disease
Neurodegenerative disease including Alzheimers disease, Parkinson’s Disease and Multiple Sclerosis. TLR4 contributes to age-related cognitive decline due to neuroinflammation and apoptosis through TLR4/MyD88/NF-κB signalling pathway via a modulation of hippocampal neurogenesis and synaptic function.(9)
Non-alcoholic fatty liver disease (NAFLD) with TLR4 signalling involved in development of inflammation and fibrosis which can progress to non-alcoholic steatohepatitis (NASH) and eventually cirrhosis or liver cancer
Chronic kidney disease with the inflammation and fibrosis leading to progressive kidney damage.
It is important to recognize that TLR4 signalling alone may not be the sole cause of these diseases but acts in concert with other factors including genetic predisposition, environmental factors and additional signalling pathways.
TLR4 Modulators
Low dose naltrexone. There is evidence suggesting that naltrexone, and potentially LDN, modulates Toll-like receptor 4 (TLR4) signalling. A study by Wang et al. (10) demonstrated that naltrexone could attenuate TLR4 signalling in glial cells, leading to reduced neuroinflammation, complementing the work at Griffith University in LDN modulation of TRPM3 Ion channels in NK cells (11).
Nigella sativa commonly known as black seed or black cumin has anti-inflammatory, anti-oxidant, anti-clotting and immune-modulatory effects. It modulates cytokine production and it is suggested it a TLR4 modulator (12).
There is considerable research on its active ingredient Thymoquinone downregulating TLR2, TLR4, TNF-α, IL-1, and NFκB expression in diseases such as rheumatoid arthritis (13) as well as Alzheimers disease and Parkinsons disease(14)(15)(16)(17).
Badary et al (18) described how TQ could potentially inhibit COVID-19 by binding to the receptor-binding domain on the spike protein of SARS-CoV-2, thus hindering hinder virus entry into the host cell.
Additionally, they felt it may bind to the SARS-CoV-2 envelope protein and inhibit its ion channel and pore formation activity as well as other studies showing that TQ might display inhibitory action against the SARS CoV2 protease, which would halt viral replication.(18)
Curcumin has been shown in various studies to modulate TLR4 signalling in various cell types. It has also been shown to inhibit the nuclear factor kappa B (NF-kB) signalling pathway.
PPARγ agonists: Peroxisome proliferator-activated receptor-gamma (PPARγ) agonists, such as pioglitazone and rosiglitazone, have been shown to inhibit TLR4 expression and signaling in various cell types. Telmisartan is a partial agonist
Glutathione is a tripeptide antioxidant composed of three amino acids: cysteine, glutamic acid, and glycine. It is a crucial molecule in maintaining cellular redox balance and detoxification processes. Although glutathione is not a direct modulator of Toll-like receptor 4 (TLR4), it has been shown to have indirect effects on TLR4 signaling and related inflammation. A study by Vlahopoulos et al. (19) showed that glutathione depletion resulted in increased activation of TLR4 and NF-κB signaling pathways in macrophages, leading to enhanced production of pro-inflammatory cytokines. This suggests that maintaining adequate glutathione levels is important for modulating TLR4 signaling and regulating inflammation.
Sirtuin activators: Molecules like resveratrol and nicotinamide riboside, which activate sirtuins, might indirectly influence TLR4 signaling through their effects on sirtuins like SIRT1 and SIRT6. Nicotinamide riboside is structurally similar to the easily available nicotinamide, as it incorporates NAD+ into its structure.(49)
Nicotinamide mononucleotide (NMN) is a precursor of nicotinamide adenine dinucleotide (NAD+), a crucial coenzyme involved in various cellular processes, including energy metabolism, DNA repair, and regulation of cellular aging. While there is no direct evidence that NMN specifically modulates Toll-like receptor 4 (TLR4), it may have indirect effects on TLR4 signalling and inflammation through its role in enhancing NAD+ levels and sirtuin activation. A study by Schug et al. (20) found that SIRT6 could inhibit NF-κB signalling, a downstream target of TLR4 activation, and thus reduce inflammation.
Amitriptyline is a tricyclic antidepressant (TCA) commonly used to treat depression, neuropathic pain, and other chronic pain conditions. While the primary mechanism of action of amitriptyline is the inhibition of serotonin and norepinephrine reuptake, it has also been shown to have other effects, including anti-inflammatory and immunomodulatory properties. There is limited research on the direct interaction between amitriptyline and TLR4. However, some studies have reported that amitriptyline may have anti-inflammatory effects that could be indirectly related to TLR4 signalling.(21)(22)
TLR4 and TRP
TLR4 signalling and TRP are 2 separate biological pathways that can intersect in certain contexts. TLR4 signalling can activate TRP channels, such as TRPA1 and TRPV4, which are expressed on immune cells. The activation of these channels can lead to an influx of calcium ions into the cell, which can modulate downstream signalling events, including the activation of the NF-κB pathway.
TRP refers to a family of ion channels found on a cell membrane of a variety of cells, including immune cells. These channels are involved in a wide range of intracellular processes, eg pain sensation, temperature sensing and regulation of intracellular calcium levels.
Researchers, notably Prof Sonya Marshall-Gradisnik and the Griffith University Chronic Fatigue team working with TRPM3 function in chronic fatigue at Griffith University have linked mutations in this pathway with “glymphatic” function and fatigue and the therapeutic benefit of Low Dose Naltrexone, while Drs Grubb and Gunning(50) have demonstrated chronic inflammatory changes in POTS patients. Adding these to Barnden’s (51) work in chronic fatigue syndrome (ME/CFS), demonstrating abnormal MRI correlations with symptom severity and autonomic measures have suggested impaired nerve signal conduction within the brainstem while seemingly unrelated, provide a tantalising explanation for much of the fatigue, autonomic instability and cognitive impairment that link all of these.
TRP mutations- TRPM3 appears critical in NK (Natural Killer) immune cell function, with implications for Ca2+ signalling and cell function.(4) The transient receptor potential melastatin subfamily 3 (TRPM3) is one of the most primitive receptors in the body, activated by a wide variety of agents, from bacteria and viruses to temperature and environmental factors such as perfumes.
This diversity made it a logical suspect for a condition like CFS that has so many different triggers in different people.
Prof Sonya Marshall-Gradisnik and the Griffith University Chronic Fatigue team (4)(5)(6) working with TRPM3 function in the research into chronic fatigue at Griffith University have linked mutations in this pathway with “glymphatic” function with consequent reduced clearance of waste solutes from the brain with production of fatigue and brain fog and the therapeutic benefit of Low Dose Naltrexone
TRPA1 is a key ion channel that detects oxidative stress and a range of endogenous and exogenous chemicals (smoke, solvents, cold air).
TRPM3 activity is impaired in CFS/ME patients suggesting changes in intracellular Ca2+ concentration, which may impact NK cellular functions. This investigation further helps to understand the intracellular-mediated roles in NK cells and confirm the potential role of TRPM3 ion channels in the aetiology and pathomechanism of CFS/ME.(4)(5)
Excessive or dysregulated TRPM3 expression can result in neurological consequences.(52)
Excessive TRPM7 expression, particularly in PV GABAergic neurons, appears to exacerbate neuronal damage and dysfunction following brain ischaemia. Reducing TRPM7 levels in these neurons has stronger neuroprotective effects compared to reducing it in glutamatergic neurons.(53)
o Current CFS research encompasses how to correct the over-expression of TRPM3a7 variant that is over-expressed in ME CFS and long COVID. This then allows normal function of several cellular ion channels. We found over expression of TRPM3 and 4
o Allergist/Immunologist researcher Prof Pete Smith describes that low dose naltrexone (LDN) is both a TLR4 modulator, is an NLRP3 inhibitor and well as restoring TRPM3 function.
Ion channels and mutations in Natural Killer cells
Ion channels open and close in response to a signal from receptors and allow cations or anions in or out of the glial cell membrane, thus changing the electrical potential. Research from Griffith University has demonstrated mutations in transient receptor potential ion channels in chronic fatigue syndrome- notably the TRPM3 ion channels in NK cells. (6)
Prof Marshall-Gradisnik and Eaton-Fitch (23) report: “An overlapping feature of these disruptions to the nervous, GI, and immune systems is calcium (Ca2+) signalling. Ca2+ is a universal second messenger that affects numerous biological processes in all cell types. Transient receptor potential (TRP) channels are a superfamily of nonselective ion channels with high permeability to Ca2+.”
“TRP channels are widely expressed by cells composing multiple organ systems, including the nervous, musculoskeletal, cardiovascular, and GI systems, all of which are associated with symptoms reported by ME/ CFS and Long COVID patients. Single-nucleotide polymorphisms have been identified to impair TRPM3 (also called melastatin) ion channel function and Ca2+ mobilization in NK cells of ME/CFS patients. Impaired TRP channel function and Ca2+ signalling is likely to impede cell and mitochondrial function, resulting in cognitive, immune, and GI manifestations.”(23)
Mast Cell Mutations:
Mast Cell Activation is a characteristic in Long Covid and POTS. Dr Vittone has identified a number of mutations affecting mast cell function - one on the membrane and additional mutations of two critical enzymes (namely DAO & HNMT) involved in clearing histamines in different tissues in the body. Mast cell mutations that affect body’s ability to respond to mast cell activation and threats mediated through mast cells. These are major mutations in POTS and thus far in Long Covid. The primary ones are on the mast cell membranes and in the function of Dao and HNMT enzyme.
The mast cell is a potent immune cell known for its functions in host defence responses and diseases, such as asthma and allergies. “Mast cells play a key role in homeostatic mechanisms and surveillance, recognizing and responding to different pathogens, and tissue injury. An abundance of mast cells reside in connective tissue that borders with the external world (the skin as well as gastrointestinal, respiratory, and urogenital tracts.)
The Dao enzyme (Diamine oxidase) is involved in the metabolism, oxidation, and inactivation of histamine and other polyamines such as putrescine or spermidine. DAO is produced in kidneys, thymus and intestinal lining that breaks down excess histamine in the body, particularly in the gut.
HNMT Histamine N-methyltransferase) catalyzes the methylation of histamine in the presence of S-adenosylmethionine (SAM-e) forming N-methylhistamine. HNMT is involved in metabolism of intracellular histamine, mainly in kidneys and liver, but also in bronchi, large intestine, ovary, prostate, spinal cord, spleen, trachea. and peripheral tissue.
COMT mutations
Catechol-O-Methyltransferase (COMT) is one of several enzymes that degrade catecholamines eg dopamine, adrenaline, nor-adrenaline, catecholestrogens and various drugs. COMT introduces a methyl group to the catecholamines which is donated by S-adenosylmethionine (SAM).(26) Therefore you need adequate SAM for COMT to work. Having too little SAM and too much SAH (s-adenosylhomocysteine) from undermethylation results in COMT inhibition as well.(27) For this reason, MTHFR SNPs that cause undermethylation and COMT SNPs that lower COMT levels are a bad combination.(24)
COMT gene production is itself influenced by methylation. (25) Usually, methylation shuts down gene production.
Oestrogen also signals mast cells to release histamines via its ER receptor on Mast cells. This is a critical mutation in POTS. COMT gene production is itself influenced by methylation (in the presence of SAMe a product of the methylation cycle), which in turn is affected by multiple mutations, some very common such as the MTHFR genes.
There is an association between COMT mutations with malignancy especially breast cancer, and also endometriosis, pain perception,(39) Parkinson’s disease,(38) and auto-immune disease. Then there are various oxidative stress and inflammatory mutations. These combinations are amenable to epigenetic modification Dr Vittone can provide with a personalized protocol based on individual genetics.
The COMT gene is found on chromosome 22, which is the second smallest human chromosome, spanning more than 51 million DNA building blocks (base pairs) and representing between 1.5 and 2 percent of the total DNA in cells. This is sometimes called the “warrior gene,”as those with the G/G:Val/Val with fast COMT activity have higher pain threshholds. The COMT gene has many variants, but one of the most well-studied is a single nucleotide polymorphism (SNP) called rs4680. The COMT gene codes for the COMT enzyme, which breaks down dopamine in the brain's prefrontal cortex.(40)
The wild-type allele is a (G), coding for a valine amino acid; the (A) substitution polymorphism changes the amino acid to a methionine. This alters the structure of the resultant enzyme such that its activity is only 25% of the wild type. As a result, A allele carriers have more dopamine in their prefrontal cortex, which may be responsible for many neuropsychological associations.(40) Studies in Parkinson’s disease have shown that genetic variations in COMT gene are associated with levodopa response variability.(38)
The most common genotype in Caucasians with intermediate pain sensitivity is the A/G:Val/Met, while the slow COMT with 40% lower COMT activity has lower pain threshold, higher dopamine, and they experience more pain, and this is more common in fibromyalgia patients.
rs4680(A) = Worrier. Met, more exploratory, lower COMT enzymatic activity, therefore higher dopamine levels; lower pain threshold, enhanced vulnerability to stress, yet also more efficient at processing information under most conditions
rs4680(G) = Warrior. Val, less exploratory, higher COMT enzymatic activity, therefore lower dopamine levels; higher pain threshold, better stress resiliency, albeit with a modest reduction in executive cognition performance under most conditions (40)
STAT3
The basic DNA mutations are complicated by multiple others, especially STAT3 which increases risks of a variety of malignancy especially lymphoma as well as rheumatoid arthritis, food allergy, autoimmune diseases eg SLE, MS, inflammatory bowel disease including Crohn’s disease and ulcerative colitis and immunodeficiency disorders as well as malignancies- leukaemia, lymphoma and solid tumours breast, lung and liver.
STAT3 gene on chromosome 17 encodes a transcription factor that plays a critical role in mediating cytokine-induced changes in gene expression. Gain-of-function mutations in the STAT3 gene have been reported to cause multi-organ early onset auto-immune diseases; such as thyroid disease, diabetes, intestinal inflammation, and low blood counts, while constitutive STAT3 activation is associated with various human cancers and commonly suggests poor prognosis.(41)
Both Valerio and Prof Pete Smith have been looking at the pathway from TLR4 , RAGE receptor then STAT3 activation. Other important mutations include CLL2, NFKb that are best identified through personal DNA assessment.
NFkB (Nuclear factor kappa-light-chain-enhancer of activated B cells)
NFkB is a protein complex that plays a crucial role in regulating the immune response, inflammation, and cell survival. The primary function of NFkB is to control gene expression in response to various signals, such as pro-inflammatory cytokines, bacterial or viral products, stress, and oxidative damage. In its inactive state, NFkB is located in the cytoplasm, where it is bound to its inhibitory proteins (IκBs). Upon activation by a stimulus, IκBs are phosphorylated and degraded, releasing NFkB to translocate to the nucleus. In the nucleus, NFKB binds to specific DNA sequences called κB sites and regulates the transcription of target genes involved in inflammation, immune response, cell survival, and apoptosis.
NFkB activation is tightly regulated to prevent excessive or prolonged inflammation, which can lead to tissue damage and chronic diseases. Dysregulation of NFkB signalling has been implicated in various health conditions, including autoimmune disorders, inflammatory diseases, cancer, and neurodegenerative diseases.
CCL2 (monocyte chemoattractant protein-1)
CCL2, also known as monocyte chemoattractant protein-1 (MCP-1), is a chemokine that is involved in the recruitment and activation of monocytes and other immune cells.
It helps guide immune cells towards tissue injury and inflammation
Stimulates superoxide and further expression of pro-inflammatory genes
CCL2 signalling moves aluminium to the brain where it accumulates- Neurons and microglia are especially vulnerable to aluminium toxicity
Increased CCL2 is associated with:
Greater deficits in cognitive function
Aberrant behaviour
Impaired development
Genetic variations in the CCL2 gene are associated with an increased risk of developing diseases such as rheumatoid arthritis, multiple sclerosis, and HIV-associated dementia. Dysregulation of CCL2 expression has been implicated in the pathogenesis of various other health conditions, including atherosclerosis, asthma, and cancer. For example, high levels of CCL2 expression in tumour cells have been shown to promote tumour growth and metastasis by attracting immune cells to the tumour microenvironment.(293)
Disease associated with CCL2
IBS: There is some evidence that CCL2 may be involved in the inflammation and pain associated with irritable bowel syndrome (IBS). Elevated levels of CCL2 have been found in the intestinal mucosa of patients with IBS.
Mast cell activation syndrome (MCAS): CCL2 has been identified as a mediator that can be released by activated mast cells, and elevated levels of CCL2 have been found in the serum of patients with MCAS.
Breast cancer: CCL2 has been implicated in the growth and spread of breast cancer cells, and high levels of CCL2 have been associated with a poor prognosis.
Fibromyalgia: Some studies have suggested that CCL2 may play a role in the pathogenesis of fibromyalgia, as elevated levels of CCL2 have been found in the cerebrospinal fluid of patients with fibromyalgia.
Migraine with aura: CCL2 has been implicated in the inflammation and pain associated with migraine headaches, including those with aura.
Chronic fatigue: Elevated levels of CCL2 have been found in the serum of patients with chronic fatigue syndrome (CFS), suggesting a role for CCL2 in the pathogenesis of this condition.
Chronic pain: CCL2 has been implicated in the pathogenesis of chronic pain, and elevated levels of CCL2 have been found in the serum and cerebrospinal fluid of patients with chronic pain conditions.
Postural orthostatic hypertension (POTS): CCL2 has been found to be elevated in the serum of patients with POTS, suggesting a role for CCL2 in the autonomic dysfunction associated with this condition.
Connective tissue disease: CCL2 has been implicated in the pathogenesis of connective tissue diseases, including systemic sclerosis and rheumatoid arthritis.
Hypertension: CCL2 has been found to be elevated in the serum of patients with hypertension, and has been implicated in the pathogenesis of hypertension.
Raynaud’s: CCL2 has been implicated in the pathogenesis of Raynaud's phenomenon, a condition characterized by constriction of the blood vessels in the fingers and toes in response to cold or stress.
Autonomic dysfunction: CCL2 has been found to be elevated in the serum of patients with autonomic dysfunction, suggesting a role for CCL2 in the pathogenesis of this condition.
Pelvic congestion: CCL2 has been implicated in the pathogenesis of pelvic congestion syndrome, a condition characterized by chronic pelvic pain.
ADHD: CCL2 has been found to be elevated in the serum of children with attention deficit hyperactivity disorder (ADHD), suggesting a role for CCL2 in the pathogenesis of this condition.
Autism: Increased levels of CC2 are found in the brain of children with autism. (291)(292)
TOC (transverse ovarian vein compression syndrome): CCL2 has been found to be elevated in the serum of patients with TOC, a condition in which the ovarian vein is compressed, leading to pelvic pain.
MALs (median arcuate ligament syndrome): CCL2 has been found to be elevated in the serum of patients with MALs, a condition characterized by chronic abdominal pain.
Retrograde ovarian flow: CCL2 has been implicated in the pathogenesis of retrograde ovarian flow, a condition in which the menstrual blood flows backward into the abdomen instead of out of the body.
CCL2 modulators:
Several CCL2 modulators are currently being utilized. These include:
· Monoclonal antibodies
· Antisense oligonucleotides
· RNA interference therapeutics
· Small molecule inhibitors
Supplements thought to be of benefit include:
· Omega-3 fatty acids
· Curcumin
· Green tea
· Vitamin D
· Nigella sativa
· NAC
Methylation
A major epigenetic mechanism involving direct chemical modification to the DNA is called DNA methylation. It is now well recognized that DNA methylation, in concert with other regulators, is a major epigenetic factor influencing gene activities. DNA methylation involves the transfer of a methyl group onto the C5 position of the cytosine to form 5-methylcytosine.(28)
DNA methylation regulates gene expression. Methyl groups can be added directly to the DNA molecule and this process can determine the way DNA is transcribed to RNA, a fundamental first step in the process of gene expression by recruiting proteins involved in gene repression or by inhibiting the binding of transcription factor(s) to DNA. This area of biology is extraordinarily complex because regulation of any gene, or modification of any particular protein or amino acid, is intricately related to a myriad of tightly and exquisitely regulated biological processes. Accordingly, there are many unknowns about the effects of methylation on a given human disease.(28)
Methylation is catalyzed by enzymes; such methylation can be involved in modification of heavy metals, regulation of gene expression, regulation of protein function and RNA processing. (257) Methylation is responsible for cellular repair, detoxification and neurotransmitter production and health immune system function. Genetic mutations in the methylation pathways can have serious consequences in disease processes. Most people are aware of the MTHFR mutations but in reality this is the tip of the iceberg.
The methylation cycle is essential for mental and physical health. It is critical to the metabolism of catecholamines in the synapse via an enzyme (COMT) as well as the synthesis of ‘depression-relevant' compounds such as melatonin, myelin basic protein, carnitine, CoQ10, etc. Methylation is required to inactivate histamine. Basic nutrients necessary for normal function of this cycle include B12, glycine, serine, activated B6, selenium, cysteine, methionine and folic acid. (29)
S-Adenosyl methionine (SAM or SAMe) a common co-substrate involved in methyl group transfers, transsulfuration, and aminopropylation. Although these anabolic reactions occur throughout the body, most SAM is produced and consumed in the liver. More than 40 methyl transfers from SAM are known, to various substrates such as nucleic acids, proteins, lipids and secondary metabolities. It is made from adenosine triphosphate (ATP) and methionine by methionine acetyltransferase. It facilitates DNA enzymatic actions required for conversion of neurotransmitters eg nor epinephrine, dopamine and serotonin. It helps maintain acetylcholine levels necessary for cognitive function and increases hepatic glutathione levels.(30) Glutathione is a critical antioxidant capable of preventing damage to important cellular components caused for example by reactive oxygen species, free radicals, peroxides, lipid peroxides and heavy metals.
There are several potential uses of SAMe, and a substantial number of studies on depression, osteoarthritis and liver diseases have been completed but the evidence is not conclusive. There are no satisfactory studies in pregnancy, may not be safe for people with bipolar disorder. SAMe may interact with some medicines eg levodopa (Parkinson’s disease) or other dietary supplements, eg St John’s Wort and there is a theoretical risk in the immunocompromised (eg in HIV) that it may provoke Pneumocystis infections.(31) Serotonin syndrome is a potential risk when used with other medication eg tramadol.
Metabolomic background
SARS-CoV-2 acts directly or indirectly on multiple “molecular fronts” causing immune dysregulation and disease progression. It causes major disturbances in serum metabolite levels, associated with the severity of the immune response.
AMP-Activated Protein Kinase (AMPK) is a key physiological energy sensor. Its activity is mostly regulated by glucose availability. Transmission of signals through AMPK modulates pathways of cellular metabolism, growth, and proliferation to maintain cellular energy homeostasis. Dr Vittone describes how the critical elements are AMPK activations, as AMPK down-regulates the inflammasome ,specifically NFKB, and also activates autophagy and mitophagy, the 2 cellular mechanisms to clear malignant cells and damaged mitochondria.
Metabolomic profiling in Covid patients revealed a major urinary increase of tryptophan metabolites kynurenine, 3-hydroxykynurenine and 3-hydroxyanthranilate. Urine levels of kynurenines (KYN) were associated with disease severity and systemic inflammation. Increased urinary levels of neutral amino acids and amino acid proline were also common in COVID-19, suggesting specific transport defects. Urine metabolomics identified major alterations in the tryptophan-kynurenine pathway, consistent with changes in host metabolism during SARS-CoV-2 infection.(32) Urine KYN is also significantly associated with Parkinson’s Disease (PD) severity and mild cognitive impairment.(33)
Researchers have been looking closely at dietary nutrients in slowing the progression of those very important neurodegenerative diseases PD and Alzheimers disease (AD). For example, choline is a B-like vitamin nutrient found in common foods that is important in various cell functions. It serves as a methyl donor and as a precursor for production of cell membranes. Choline is also the precursor for acetylcholine, a neurotransmitter which activates the alpha7 nicotinic acetylcholine receptor (α7nAchR), and also acts as an agonist for the Sigma-1 R (σ1R). These receptors regulate CNS immune response, and their dysregulation contributes to AD pathogenesis.(34)
Laboratory studies demonstrated lifelong choline supplementation significantly reduced amyloid-β plaque load and improved spatial memory, changes linked to a decrease of the amyloidogenic processing of APP, reductions in disease-associated microglial activation, and a downregulation of the α7nAch and σ1 receptors. (34)
Phosphatidylcholine is used in the synthesis of acetylcholine which is an important neurotransmitter in the brain. The liver is able to produce phosphatidylcholine on its own, although it may not produce enough to optimally support peak mental performance and overall brain health.
Phosphatidylserine facilitates the activation of signalling proteins and receptors that are critical for neuronal survival, differentiation and synaptic neurotransmission and is required for healthy nerve cell membranes and myelin. Aging of the human brain is associated with biochemical alterations and structural deterioration that impair neurotransmission.
Phosphatidylserine is absorbed efficiently from the gut in humans, crosses the blood-brain barrier, and safely slows, halts, or reverses biochemical alterations and structural deterioration in nerve cells. It supports human cognitive functions, including the formation of short-term memory, the consolidation of long-term memory, the ability to create new memories, the ability to retrieve memories, the ability to learn and recall information, the ability to focus attention and concentrate, the ability to reason and solve problems, language skills, and the ability to communicate. It also supports locomotor functions, especially rapid reactions and reflexes.(35)
PEMT (Phosphatidylethanolamine N-methyltransferase) mutations
PEMT (Phosphatidylethanolamine N-methyltransferase) mutations are associated with fatigue, fatty liver, and increased neurodegenerative risk. Disrupted PEMT and other lipoproteins (eg APO E4, LPa, APOB) may create problems which also affect the transport of fats in the body. Elevated homocysteine promotes atherosclerosis through increased oxidant stress, impaired endothelial function, and induction of thrombosis. The homocysteine and abnormal lipoproteins may interact to increase the inflammatory and oxidative stress response. This linking may provide the missing link to the non-resolving elevated D-Dimer measurements for microemboli, as well as persisting cognitive impairment despite mast cell blockade.
Phosphatidylserine (PS) is a phospholipid present on the inner leaflet of the plasma membrane in eukaryotic cells. Under normal conditions, PS is maintained within the inner leaflet by flippase enzymes. However, during cellular stress or apoptosis, PS becomes externalized on the outer leaflet of the plasma membrane. This externalization is a signal for macrophages to recognize and engulf apoptotic cells in a process called efferocytosis. This mechanism usually maintains a balance between pro- and anti-inflammatory responses, preventing excessive inflammation.
So the balance is the key . This balance can be disrupted by PEMT with deleterious variants, as when there is an increased exposure of PS on the cell surface, it can lead to thrombo-inflammation through the following molecular pathways:
Activation of blood coagulation: Externalized PS provides a negatively charged surface that facilitates the assembly of coagulation factors, promoting the formation of prothrombinase and tenase complexes, ultimately leading to the generation of thrombin and fibrin clot formation.
Platelet activation and aggregation: PS exposure on the activated platelets also promotes platelet activation and aggregation, further amplifying the coagulation cascade.
Inflammation: Externalized PS can interact with various immune cells like monocytes and neutrophils, activating them and leading to the release of pro-inflammatory cytokines and chemokines. This can lead to a vicious cycle of inflammation and coagulation, collectively termed as thrombo-inflammation.
Phosphatidylserine and Covid-associated thrombo-inflammation
Mutations in the PEMT gene could potentially lead to an imbalance in the levels of different phospholipids, including PS, in the plasma membrane. This could result in increased PS exposure, contributing to the thrombo-inflammatory state and vascular complications
Ramya Dwivedi (36) describes that “vascular complications in COVID-patients links phosphatidylserine (PS) to thrombo-inflammation. Thrombo-inflammation plays a critical role through complement activation and cytokine release, platelet overactivity, apoptosis (thrombocytopathy), as well as coagulation abnormalities (coagulopathy).”(36) Rauchet al (37) found phosphatidylserine was associated with increased thrombo-inflammation and vascular complications, and can trigger the blood coagulation cascade, the complement system, inflammation and reside on activated immune cells.
She continues: “To understand the related aspects of COVID-19 in detail, Rauchet et al (37) compared peripheral blood mononuclear cells (PBMC) between COVID-19 patients, healthy individuals, and those recovered from COVID-19. They found that the PS+ platelet-derived microparticles (PMP)-associated PBMC correlated with lymphopenia and disease severity, showing a higher correlation than the commonly used laboratory diagnostic markers such as IL-6 (Interleukin-6), D-Dimer (a fibrin degradation product used to assess clot formation) and C-reactive protein (CRP is a protein found in plasma that rises in response to inflammation. CRP is an “acute phase protein” of hepatic origin that increases following IL-6 secretion by macrophages and T- cells. Its physiological role is to bind to lysophosphatidylcholine expressed on the surface of dead or dying cells in order to activate the complement system via Complement Component 1q or C1q).”(36) Activation of the C1 complex initiates the “classical complement pathway,” part of the innate immune system.
“It is known that vascular complications may occur in COVID-19 patients where autoantibodies may target phosphatidylserine (PS)/prothrombin complexes. PS is a plasma membrane component that is actively retained by an ATP-requiring process at the inner membrane surface in living cells; it participates in the immune response. PS-enveloped foreign particles (such as microparticles or viruses) are released from cells to interact with extracellular proteins, including coagulation and complement systems.”(36)
“Once activated, the platelet-derived microparticles (PMPs) are released, which cause thrombin formation, coagulation, activation of the complement system, and inflammation in a PS-dependent manner. COVID-19 severity is associated with immune abnormalities such as increased inflammatory cytokines, immune cell exhaustion, and general lymphopenia. Hyperactivated platelets, elevated levels of circulating PMPs, and an increased risk of thromboembolic complications - related to PS-associated immune mechanisms - contribute to COVID-19 disease severity and death.” (36)
“The study significantly identified the severity of COVID-19 disease correlating with high significance with the frequencies of PS+ PBMC of COVID-19 patients. Moreover, they also reported that the elevated frequencies of PS+PBMC over time, for up to 10 weeks in some patients - indicating that the presence of PS+PBMC in COVID-19 patients might be long-lasting. Also, they observed that in recovering patients, the PS+PBMC returned to the levels of healthy controls.” (36)
Summary
This document is intended to summarise our current research on the aetiology and management of POTS and Long-COVID from the discoveries of the DNA mutations that underpin many of the symptoms of these conditions. It helps to understand why the inflammatory response is so severe, and provides a springboard for the investigation and management of the exaggerated inflammatory and microembolic responses to the SARS-CoV-2 virus and increasingly the development of a recovery program based on individual DNA assessment matched to clinical assessment.
The complexity of the inflammatory and metabolic mutations that underpin Long Covid in many people appears to be best managed by incorporating individualized DNA studies and using the information to vary management programs.
Our findings may enable physicians to help their Long-COVID and POTS patients now, often with conservative measures such as physical therapy, dietary change, and medications that have a well-established safety profile and are often available without a prescription. It is though, like opening Pandora’s Box. There are various sub-types we are seeing that will require a larger cohort to fully assess. The complex nature of the viral assault and management programs systems are discussed in the other Long Covid discussion documents in the website. References are provided in separate documentation.
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