Le persone affette da malattia di Lyme, o coloro i quali sospettano di averla, si trovano spesso a navigare in un mare di informazione e disinformazione. Nel seguito riporto documenti riassuntivi relativi alla variante europea della malattia.
In Europa abbiamo delle linee guida del 2010 sia per la Lyme (link) che per la neuroborreliosi (link). In Europa abbiamo anche un analogo del centers for disease control and prevention (CDC) americano, che ha la pagina sulla malattia di Lyme dove c’è una dettagliata revisione della letteratura del 2016 (link).
Una ridotta capacità delle NK di uccidere cellule invase da virus (vedi figura) è stata dimostrata in più studi sulla ME/CFS. In particolare, ben 16 studi hanno dimostrato una ridotta citotossicità delle NK quando le cellule K562 siano usate come bersaglio (IOM, 2015). Uno dei primi studi in merito credo sia quello di Caligiuri e colleghi del 1987 (Caligiuri et al., 1987). In seguito si poté chiarire che questa difettosa citotossicità risulta legata a una ridotta concentrazione intracellulare (nelle NK) di perforina (Maher et al., 2005), l’enzima che tanto le NK che le T CD8+ usano per indurre l’apopotosi delle cellule infette. In studi ancora posteriori è stato accertato sorprendentemente che, a fronte di una concentrazione ridotta di perforina, si ha un aumento della espressione di RNA messaggero relativo a questa molecola, sempre nelle NK (Brenu EW, 2011). E’ come se le NK tentassero di aumentare la concentrazione intracellulare di perforina, senza riuscirci. Ebbene, è stato dimostrata in vitro la capacità dell’Ampligen di migliorare il funzionamento delle NK provenienti da pazienti CFS (Strayer et al., 2015). Sfortunatamente non esistono dati in vivo, tuttavia anche la misura della citotossicità delle NK sembra avere il potenziale di identificare un gruppo di pazienti che potrebbero beneficiare del farmaco. Si consideri inoltre che la citotossicità delle NK è ridotta anche in altre patologie, come il lupus eritematoso sistemico e la sindrome di Sjögren (Struyf NG et al. 1990).
In Italia la misurazione della “citotossicità spontanea delle NK” (utilizzando cellule K562 come bersaglio) è un esame previsto in molte regioni (se non tutte) e tabulato con codice 90.59.3 e costa una ventina di euro (vedi link per Lombardia). Può essere eseguito presso il Policlinico Umberto Primo di Roma (UOC Immunologia e Immunopatologia). In figura 1 sono riportati i livelli di citotossicità di tre pazienti, confrontati con due controlli sani. La citotossicità è calcolata come percentuale di cellule bersagio uccise e viene riportata per diversi valori del rapporto seguente:
numero di cellule effettrici/numero di cellule bersaglio
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In the chart you find below I collected the single nucleotide polymorphisms (SNPs) that have been associated with ME/CFS byGriffith University’s researchers, to the extent that they are included in the SNPs read by the common23ndME genetic test. Between 2015 and 2016 the research team published at least four genetics studies, identifying a total of 65 SNPs associated with ME/CFS (Johnston S, Staines D et al. 2016), (Marshall-Gradisnik S, Huth T et al. 2016), (Marshall-Gradisnik S, Johnston S et al. 2016), (Marshall-Gradisnik S, Smith P et al. 2015). Of these, only 23 match the data provided by the 23andME test. In the chart I report these 23 SNPs, their frequency in healthy controls (HC) and patients (CFS), Odd Ratios (OR) and p-values; in addition, I report the respective base pairs for three patients (Pt. 1, 2, 3). In yellow are the genotypes mainly associated with the pathology, in green those that seem to have a protective role against the pathology. For studies no. 1, 2 and 3 I reported the ORs, whereas for study no. 4 I reported the p-values. ORs greater than 1 indicate the genotype’s association with the pathology; ORs less than 1 indicate its protective role against the pathology. P-values less than 0.05 indicate genotypes associated with the pathology.
Alpha-adrenergic receptor 1
AA genotype for rs2322333 in adrenergic receptor α1 (ADRA1A) has been demonstrated to have a protective role against ME/CFS (Johnston S, Staines D et al. 2016). As shown in the chart, none of the three patients is a carrier for this genotype. ADRA1A receptors are involved in vasoconstriction of blood vessels throughout the body, including the skin, gastrointestinal system, genitourinary system, kidney, and brain. In the brain, these receptors exert effects on the hypothalamic-pituitary-adrenocortical (HPA) axis and in motor functions. In ME/CFS orthostatic intolerance (POTS and/or orthostatic hypotension) is frequent to be found: in fact, this symptom is featured in the diagnostic criteria (IOM, 2015). Midodrine, an agonist of he ADRA1A receptor, has been suggested as a treatment for ME/CFS in a case study (Naschitz J et al 2004). Bearing all the above in mind, position rs2322333 may be linked to some important mechanism at the core of ME/CFS.
Figure. TRPM3 gene expression in different human tissues.
Nicotinic cholinergic receptors ACHRN
As shown in the chart, patient no. 1 presents two polymorphisms in the gene for nicotinic acetylcholine receptor alpha-2 subunit (ACHRN2), which are associated with ME/CFS. Nicotinic acetylcholine receptors are found both in Peripheral Nervous System (sympathetic and parasympathetic) and in the neuromuscular junction. They are present in the Central Nervous System too. Moreover, the alpha-2 subunit is expressed also by various lymphocytes (B cells, T cells, monocytes), and the same applies to the beta-4 subunit, which presents potentially pathogenic polymorphisms in patient no. 2 and 3. Eventual vulnerabilities in these receptors may lead to countless effects: they may, for example, constitute a predisposing risk factor for orthostatic intolerance, a main feature of ME/CFS. Besides, pyridostigmine, a cholinesterase inhibitor, the enzyme involved in degradation of acetylcholine, has been successfully used in at least one study on ME/CFS patients (Kawamura Y et al. 2003).
TRPM3 ionic channels
Griffith University has published two studies arguing for a connection between ME/CFS and impaired transient receptor potential melastatin 3 (TRPM3) calcium channels. After demonstrating a significant reduction in TRPM3 cell surface expression in NK in patients, compared to healthy controls (Nguyen T et al. 2016), researchers have proved this abnormality to impact calcium influx in NK and postulated this mechanism as the very cause of the reduced NK cytotoxicity observed in numerous studies. Being TRPM3 expressed in many tissues (sensory neurons, kidneys, brain, hypophysis, pancreas: see figure), researchers have suggested its malfunction to be the physiological basis of ME/CFS (Nguyen T et al. 2016). In addition, given previous data (by the same research team) regarding the statistic association between ME/CFS and TRPM3 polymorphisms (Johnston S, Staines D et al. 2016), (Marshall-Gradisnik S, Huth T et al. 2016), (Marshall-Gradisnik S, Johnston S et al. 2016), the authors have suggested this dysfunction to be possibly due to a genetic predisposition. Looking at our three patients, each one of them is a carrier for at least one genetic variant of TRPM3 associated with ME/CFS. Particularly, genotype CT in rs1328153 position is present in all of them.
Some days ago I wrote a letter to a researcher who is currently involved in the study of ME/CFS. I sent him some relatively rare genetic variants that I had found analyzing my own exome and the one of another patient (see this post). He was so kind to reply to my mail. He answered with a simple and – at the same time – very interesting note. If there was a genetic predisposition to ME/CFS – he observed – it would be common, very prevalent in the general population. Otherwise, we could not explain the epidemic episodes of the disease, like the one that happened in Lake Tahoe (Nevada), or in Lyndonville (New York), or in Bergen (Norway), and so forth. He left me with this problem “to think at night”, as he wrote.
Well, I did my homework. A genetic predisposition to ME/CFS has been suggested by a study on familial clustering of ME/CFS from a data bank of Utah health care system. They have found a significant increase in ME/CFS relative risk among first, second, and third degree relatives, compared with the general population (Albright F. et al. 2011). The problem is: acknowledged a genetic predisposition, how prevalent is it?
In 2004, a large outbreak of Giardia duodenalis struck the city of Bergen, in Norway. Of 1252 laboratory-confirmed cases, 347 reported chronic fatigue three years later. 53 of them were selected for a study and 41.5% of them were found to fit the criteria for ME/CFS (Mørch K et al. 2013). If we assume the same percentage for all the 347 patients who were symptomatic three years after the outbreak, we find that 144 of the original cases of laboratory-confirmed infection developed ME/CFS. This points to a prevalence of 11,5% in the general population for the genetic predisposition to ME/CFS.
In order to confirm this result, I then considered a very well known study on the prevalence of ME/CFS among Australian patients who went to their doctor for infections due to Epstein-Barr virus, Coxiella burnetii, and Ross River virus. They found that after six months from the infection, 28/253 participants (11%) met the diagnostic criteria for ME/CFS (Hickie I. et al. 2006).
The conclusion of this very short and poor analysis is that if there was a genetic predisposition, it would be present in 11% of the general population. And yet, ME/CFS is much less prevalent. But if we consider the two studies mentioned, we could argue that we need a major infection (one that requires medical care and blood tests) in order to trigger this predisposition. So we would have a genetic predisposition highly prevalent (1 in 10 individuals!) but with low penetrance (only a small percentage of those who carry the genetic predisposition ends up developing the disease).
Now, if we assume that the genes involved in this predisposition are n and that these genes are transmitted independently one from another, then we have:
p_1 × p_2 × … × p_n = 0.11
where p_i is the prevalence of the variation on the i-th gene involved. This means that if we assume that the genetic predisposition is due to two or more genes, then each of these variants has a prevalence higher than 0.33.
We present an attempt at exome analysis in two ME/CFS patients. Pt. 1 presents a mild form of carboxypeptidase N (CPN1) deficiency (a missense in exon 3) while Pt. 2 revealed two rare intronic variants in the same gene. CPN1 is an enzyme that inactivates kinins and complement proteins split products (such as C4a, a known anaphylatoxin). Therefore, CPN1 deficiency could explain C4a increase after exercise and mast cell abnormalities previously reported in ME/CFS. It could also explain the high prevalence of POTS in ME/CFS since kinins are vasodilators.
Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a debilitating disease characterized by cognitive deficits, fatigue, orthostatic intolerance with symptoms exacerbated after exertion (IOM, 2015). This disease has no known cause but several abnormalities have been observed in energy metabolism (Tomas C. and Newton J. 2018), immune system and gut flora (Blomberg J. et al. 2018), brain (Zeineh MM. et al. 2014). In this population of patients, several abnormalities have been found to be triggered by exercise, such as abnormal aerobic performance (Snell C. et al. 2013), enhanced gene expression of specific receptors (White AT. et al. 2012), abnormal gut flora translocation (Shukla SK et al. 2015) and failure in blood clearance of complement protein 4 split product A (C4a) (Sorensen B et al. 2003). An increase in C4a is part of the human physiologic response to physical exercise, but these levels return to baseline within 30 minutes to 2 hours (Dufaux B et al. 1991) while in ME/CFS there is a peak in serum C4a six hours after exertion. A possible explanation for slow C4a inactivation could be a problem in carboxypeptidase N (CPN1), an enzyme involved in the inactivation of C3a, C4a, C5a. CPN1 is required for kinins inactivation too, such as bradykinin, kalladin (Hugli T. 1978), (Plummer TH et Hurwitz MY 1978), that are vasodilators. We report on the case of a ME/CFS patient (Pt. 1) with a missense variant in CPN1 gene that is linked to reduced function of the enzyme and of another ME/CFS patient (Pt. 2) with rare variants in introns 1 and 6 of the same gene with uncertain significance (table 1, figure 1).
Materials and Methods
Whole exome sequencing (WES) has been performed on cells from the saliva of two ME/CFS patients, with an average 100X coverage (Dante Labs). The first search for pathogenic variants and insertions/deletions was performed with the software EVE, provided by Sequencing.com. A further refinement of the search was conducted by manual insertion of these SNPs in VarSome. The search for possible unknown pathogenic variants within the gene for CPN1 has been performed using Integrative Genome Viewer (IGV), an opensource tool for genetic data analysis.
Results from the analysis of the two exomes performed with EVE and refined with VarSome are collected in table 2 (Pt. 1) and table 3 (Pt. 2).
Pt. 2 is carrier of a mitochondrial disease (table 3, first line): a missense in gene for medium-chain acyl-CoA dehydrogenase (MCAD) which leads to mild functional impairment of the enzyme involved in the oxidation of fatty acids (44% residual activity) (Koster KL. et al. 2014).
Pt. 2 is also homozygous for a variation in gene arylsulfatase A (ARSA) that is linked to a residual activity of only 10% of normal (Gomez-Ospina N. 2010). Arylsulfatase A deficiency (also known as metachromatic leukodystrophy or MLD) is a disorder of impaired breakdown of sulfatides (cerebroside sulfate or 3-0-sulfo-galactosylceramide), sulfate-containing lipids that occur throughout the body and are found in greatest abundance in nervous tissue, kidneys, and testes. Sulfatides are critical constituents in the nervous system, where they comprise approximately 5% of the myelin lipids. Sulfatide accumulation in the nervous system eventually leads to myelin breakdown (leukodystrophy) and a progressive neurologic disorder (Von Figura et al 2001). Nevertheless, this genotype does not cause MLD, and this benign condition of reduced ARSA activity is called ARSA pseudodeficiency. There are about 4 homozygotes in 1000 persons among non-Finnish Europeans (VarSome)
Pt. 1 is a carrier of a missense in gene CPN1 (table 2, first line) which leads to a loss of more than 60% of activity, according to a study on a single patient (Mathews KP. et al. 1980), (Cao H. et Hegele RA. 2003). The study of gene CPN1 in both patients (using IGV) has led to the identification of two rare variants (frequency less than 0.002) in intron 1 and 6 of one allele from Pt. 2 (table 1, figure 1). In MCAD no other damaging variations have been identified in these two patients by direct inspection with IGV (data not shown).
Whole exome sequencing (WES) is a technique that aims at the sequencing of the fraction of our genome that encodes for proteins: about 30 million base pairs (1% of the all the human DNA) divided into about 20 thousand genes (Ng SB et al. 2009). It has become increasingly clear that the use of WES can positively improve the rate of diagnosis and decrease the time needed for a definitive diagnosis in patients with rare genetic diseases (Sawyer SL et al. 2016). WES also positively impacts the ability to discover new pathogenic variants in known disease genes (Polychronakos C. et Seng KC. 2011) and the discovery of completely new disease genes (Boycott KM 2013). ME/CFS seems to have a genetic component: a US study found clear evidence of familial clustering and elevated risk for the disease among relatives of ME/CFS cases (Albright F et al. 2011) and several SNPs in various genes have been reported as more prevalent in ME/CFS patients versus healthy controls (Wang T et al. 2017). And yet, no studies that analyzed whole exomes of ME/CFS patients have been published, to my knowledge.
In this study, we searched for known genetic diseases in the exomes of two ME/CFS patients who fit the IOM criteria for SEID (IOM, 2015), with postural orthostatic tachycardia syndrome (POTS) identified by positive tilt table test. We detected a missense variant in CPN1 (rs61751507) in Pt. 1 (heterozygosis) that has been associated to a loss of activity of the enzyme of at least 60% in a previous study (Mathews KP. et al. 1980), (Cao H. et Hegele RA. 2003). We then found that, although Pt. 2 was not a carrier of this SNP, she had two rare SNPs in intron 1 (rs188667294) and 6 (rs113386068) of gene CPN1 (both present in less than 1/500 alleles, table 1, figure 1). These intronic variations have not been studied, to our knowledge, so their pathogenicity can’t be excluded at present. Variations in introns can be damaging just as missense and nonsense mutations in exons; suffice to say that the main known pathogenic SNP of gene CPN1 is a substitution in intron 1 (Cao H. et Hegele RA. 2003).
Carboxypeptidase N (CPN1) is an enzyme involved in the inactivation of C3a, C4a, C5a, and of kinins (bradykinin, kalladin) (Hugli T. 1978), (Plummer TH et Hurwitz MY 1978). In ME/CFS the physiologic increase in blood of C4a (the split product of the complement protein C4) after exercise is significantly more pronounced than in healthy controls as if there was a defect in C4a inactivation (Sorensen B et al. 2003). Such a defect could very well be a loss of function in CPN1, as found in Pt 1. Moreover, CPN1 is involved in inactivation of bradykinin, which is known to induce vasodilatation (Siltari A. et al. 2016), therefore CPN1 deficiency could play a role in POTS and in orthostatic intolerance in general. Both patients have a tilt table test positive for POTS. C4a has been recently considered to play a causal role in the cognitive deficit of schizophrenia, because of its role in synapsis pruning (Sekar, A et al, 2016); therefore a failure in its inactivation could be implicated in the incapacitating cognitive defects lamented by ME/CFS patients.
Only two patients with CPN1 deficiency have been reported so far in medical literature (Mathews KP. et al. 1980), (Willemse Jl et al. 2008), and the enzymatic defect has been associated to angioedema that most often involved the face and tongue, urticaria, and hay fever and asthma precipitated by exercise. This clinical presentation could be due, at least in part, to mast cell activation: in fact, C4a is a known anaphylatoxin that induces mast cells degranulation and release of histamine (Erdei A. et al. 2004). That said, we can observe that even if the clinical presentation of the only two known cases of CPN1 deficiency doesn’t fit the clinical picture of ME/CFS, mast cell activation syndrome (MCAS) has some commonalities with ME/CFS (Theoharides, TC et al. 2005), and mast cell abnormalities have been reported among ME/CFS patients (Nguyen T. et al. 2016). So we can’t exclude that activation of mast cells by a failure in C4a inactivation may lead to ME/CFS symptoms. The role of exercise as a trigger for symptoms in CPN1 deficiency is also highly suggestive because this is a pathognomonic feature of ME/CFS.
CPN1 deficiency is present (even if in a mild form) in Pt. 1, while Pt. 2 presents two rare intronic variants whose pathogenic role can’t be excluded. CPN1 deficiency could explain the abnormal increase of C4a after exercise and might be a contributing factor to post-exertional malaise and cognitive symptoms in ME/CFS. A search for pathogenetic SNPs in gene CPN1 among ME/CFS patients would clarify the role (if any) of this gene.
Acknowledgments. I would like to thank Chiara Scarpellini for her careful collection of annotations for each of the 2 hundred or so variants found by EVE within the exomes of Pt. 1 and Pt. 2 (table 2 and table 3).