Three new possible autoepitopes in ME/CFS

Paolo Maccallini

Abstract

I have performed a set of analysis on experimental data already published about autoimmunity to muscarinic receptors in ME/CFS. My predictions are that extracellular loop 2 and 3, and also transmembrane helix 5 of both muscarinic cholinergic receptors 4 and 3, are main autoantigens in a subset of ME/CFS patients. Moreover, I have found that autoimmunity to M4 and M3 ChR is independent of autoimmunity to beta 2 adrenergic receptor, also reported in ME/CFS patients.  

Introduction

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a debilitating disease characterized by cognitive deficits, fatigue, orthostatic intolerance with symptoms exacerbated after exertion (post-exertional malaise, PEM) (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, gut flora (Blomberg J. et al. 2018), brain (Zeineh MM. et al. 2014). A possible role for autoantibodies in the pathogenesis of the disease has been suggested by the finding of reactivity of patient sera to two nuclear antigens (Nishikai, et al., 1997), (Nishikai, et al., 2001), to cardiolipin (Hokama, et al., 2009), to HSP60 (Elfaitouri A. et al. 2013), and to muscarinic cholinergic (M ChR) and beta adrenergic receptors (ß AdR) (Tanaka S et al. 2003), (Loebel M et al. 2016); reactivity that was significantly elevated when compared to healthy contols. Reactivity to adrenergic and muscarinic Ch receptors has been confirmed by two independent groups, but these results have not been published yet (R). A role for autoantibodies in at least a subgroup of patients has also been suggested by a response to rituximab, a CD20 B cells depleting agent (Fluge Ø. et al. 2011), (Fluge Ø. et al. 20115), and to immunoadsorption (Scheibenbogen C. et al 2018). Sera response to muscarinic cholinergic receptors is confirmed in two studies but both of them used an immune assay with proteins coated on a plate. This kind of test does not allow to identify the exact autoepitope on the receptor and – even more importantly – it is subjected to false positive results because it exposes to sera surfaces of receptors that are hidden when they are in their physiological position (Ramanathan S et al 2016). Nevertheless, the amount of data provided in the study by Loebel et al. where reactivity of sera to 5 subtypes of muscarinic cholinergic receptors have been measured simultaneously, has – in our opinion – the potential to unveil the exact autoepitope(s). Thus, we performed a bioinformatical analysis on experimental data from this study in order to extract hidden information. We used a software for the in silico study of B cell epitope cross-reactivity (Maccallini P. et al. 2018) and a software for amino acid protrusion index calculation (Ponomarenko J. et al., 2008).  Our prediction is that patients sera mainly react to three epitopes that belong to the second and third extracellular loop of M3 and M4 ChR, but also to a hidden epitope of the same two receptors, leading to possible false positive results of this test. We have also found that the reactivity to beta 2 adrenergic receptor (ß2 AdR) found in the study by Loebel et al. is not due to the same antibody that reacts to muscarinic cholinergic receptors.

Methods

Search for cross-reactive epitopes. Cross-reactivity between muscarinic cholinergic receptors M4 and M3, and between M4 and M1 has been studied in silico using EPITOPE, a software already described (Maccallini P. et al. 2018). Briefly, EPITOPE searches for cross-reactive epitopes shared between two proteins (let’s say protein A and protein B) by comparing each possible 7-mer peptide of A with each possible 7-mer peptide of B. The comparison is made using the algorithm by Needleman and Wunsch (Needleman SB. and Wunsch CD. 1970)  with a gap model a + b·x, where a is the opening gap penalty, b is the extending one, and x is the extension of the gap. A penalty for gaps at the end of the alignment was also assumed. The choice for gap penalties and substitution matrix were done according to the theory already developed for peptide alignments (Altschul SF. 1991), (Karlin S. and Altschul SF. 1990). Available experimental data on cross-reactivity between γ enolase and α enolase (McAleese SM. et al. 1988)  have been used for EPITOPE calibration: a score >60 was considered the cut-off for cross-reactivity, a score below 50 indicates non-cross-reactive epitopes; a score between 50 and 60 defines a borderline result. A simpler version of EPITOPE has been used for single local alignments. The main program used for M4-M3 comparison, its subroutine NeWalign and the substitution matrix employed are available for download. Primary structures used in this work have been downloaded from UniProt and are the following ones: M1 ChR (P11229), M3 ChR (P20309), M4 ChR (P08173), B2 AdR (P07550).

Surface exposure. In order to select only those 7-mer peptides that are on the surface of proteins, we have considered their mean protrusion indexes. A protrusion index of at least 0.5 has been considered the cut-off for surface exposure. Protrusion indexes of single amino acids have been calculated with ElliPro. A protrusion index of 0.5 means that the amino acid is outside the ellipsoid of inertia which includes 50% of the centers of mass of all the amino acids of the protein (Ponomarenko J. et al., 2008). For M4 ChR we have used the crystal structure 5DSG (Thal DM. et al. 2016). The 3D structure of human M3 ChR has not been experimentally determined yet, so we have used a theoretical model built using murine M3 ChR (PDB ID: 4DAJA) as a template, provided by ModBase.

M ChR plot
Figure 1. The position of the first amino acid of each possible 7-mer peptide of M4 ChR is reported on the abscissa, the score for the comparison of each of these peptides with M1 ChR (blue line) and M3 ChR (orange line) is reported on the ordinate. N terminus, extracellular loop 1, 2 and 3 are also indicated. Scores above the yellow line indicate cross-reactivity, scores below the blue line indicate a lack of cross-reactivity.

Selection criteria. Our purpose is to predict to what epitopes of M3 and M4 ChRs sera from ME/CFS patients react. So we search for M4 ChRs 7-mer peptides that are cross-reactive to M3 ChR, but non-cross-reactive to M1 ChR. Moreover, they have to present surface exposure both on M4 and on M3 ChR (otherwise antibodies can’t reach them). So, selection criteria for M4 ChR epitopes are as follows:

  1. they have to be cross-reactive to M3 ChR;
  2. they have to be non-cross reactive to M1 ChR or borderline;
  3. they have to present a mean protrusion index ≥0.5;
  4. M3 ChR peptides to which thy cross-react have to present a mean protrusion index ≥0.5.

We will refer to strict criteria when we assume only non-cross-reactivity in 2, while weak selection criteria are fulfilled when M4 ChR epitopes have borderline reactivity to M3 Chr peptides.

M4 vs M1, M3
Figure 2. Distribution of the scores from the comparison of M4 ChR with M1 ChR (left) and with M3 ChR (right). M3 ChR presents a slightly higher mean score.

Results

The search for 7-mer peptides of M4 ChR that are cross-reactive to M3 ChR found 108 sequences. We then studied cross-reactivity to M1 ChR for each of these peptides and we found that 11 of them are non-cross-reactive and that other 9  peptides have borderline reactivity. None of these 20 peptides presented a cross-reactivity to B2 AdR (Table 1S, column 1). Scores between peptides of M4 ChR and the other two muscarinic cholinergic receptors are plotted in Figure 1. The distribution of scores from the comparison of M3 ChR with M1 ChR and with M3 ChR are reported in Figure 2. For the M4 ChR 20 epitopes mentioned above, we calculated the mean protrusion indexes and we did the same calculation for their cross-reactive peptides on M3 ChR. We also indicated their position with respect to the plasma membrane. All these data are collected in Table 2S. Once we apply selection criteria on these 20 peptides, we obtain 9 epitopes (Table 1). Of these selected epitopes, one belongs to a transmembrane helix: peptide 186-192 of M4 ChR, which cross-reacts to peptide 231-237 of M3 ChR. Peptide 418-431 of M4 ChR is partially immersed in the plasmatic membrane, even though its cross-reactive peptide of M3 ChR is entirely exposed to the extracellular space, and the same applies to the other two epitopes found (figure 1). Peptide 175-181 of M4 ChR cross-reacts to peptide 211-217 of M3 ChR; peptide 186-192 of M4 Chr cross-reacts to peptide 222-228 of M3 ChR; peptide 418-431 of M4 Chr cross-reacts to peptide 513-522 of M3 ChR. Sequences that fulfill selection criteria and their respective inverted sequences are collected in  Table 2.

Table 1
Table 1. This is the collection of M4 Chr 7-mer peptides that are cross-reactive to M3 ChR; are not cross-reactive or borderline with M1 ChR; have a mean protrusion index higher than 0.5; are cross-reactive with epitopes of M3 ChR with a protrusion index higher than 0.5.

Discussion

B cells autoimmunity to muscarinic cholinergic receptors in ME/CFS has been reported in two studies (Tanaka S et al. 2003), (Loebel M et al. 2016) and this finding has been recently confirmed by two other independent groups who have not published yet (R). The two studies mentioned used full-length proteins coated on a plate in order to perform the immune assay. With this kind of technique we may have both false positives (due to the fact that sera react with peptides that are not in the extracellular domain) and false negatives (due to protein denaturation, which leads to the formation of epitopes that would not be present if the protein were correctly folded) as has been reported in the case of anti-MOG antibodies (Ramanathan S et al 2016). A way to solve the possible inaccuracy of these data would thus be to measure sera reactivity with a cell-based assay (CBA) which is a test where receptors are expressed by eukaryotic cells and thus they are held in their physiological position.

M4_M3_ChR_weaker
Figure 1. Peptides of table 1 that belong to the extracellular domain of M3 and M4 ChR are here highlighted directly on the 3D structures of their respective receptors.

Nevertheless, we can still try to extract hidden information from experimental data and predict the position of the epitope(s) ME/CFS patients sera react to. Knowing that sera from patients react to M4, M3 ChRs and that there is a low correlation between reactivity to M4 ChR and reactivity to M1 ChR (Loebel M et al. 2016) we selected 7-mer peptides of M4 ChR that cross-react (in silico) to M3 ChR but not to M1 ChR (Table 2S). We then selected, among them, only those peptides that have surface exposure on their respective proteins (Table 1). The result is that patient sera react to extracellular loops 2 and 3 of both M3 and M4 ChRs (Figure 1), but also to a hidden antigen, a peptide of transmembrane helix 5 of both M3 and M4 ChR.

Our results are of interest because extracellular loops 2 and 3 of M3 ChR are known autoepitopes in Sjögren’s syndrome (Ss) (Deng C. et al. 2915). Moreover, sera from patients with orthostatic hypotension (OH) react to extracellular loop 2 of M3 ChR, where they show an agonistic effect, thus acting as vasodilators (Li H. et al. 2012). OH, a form of orthostatic intolerance has been reported in ME/CFS patients (Bou-Holaigah et al. 1995) while fatigue similar to post-exertional malaise have been described in Ss (Segal B. et al. 2008). A pathogenic role of these antibodies in fatigue for both ME/CFS and Sjögren syndrome could perhaps be due to their vasodilatory effect.

Our analysis unveiled reactivity to a hidden autoepitope, which belongs to transmembrane helix 5 of M3 and of M4 ChR. This epitope is buried inside the plasma membrane when these two receptors are in their physiological position, so this reactivity can’t contribute to the pathogenesis of ME/CFS.

None of the 7-mer peptides of M4 ChR that cross-react to M3 ChR and at the same time don’t cross-react to M1 ChR presents in silico reactivity to B2 AdR. This means that in those patients whose sera present reactivity to both M4-M3 ChR and B2 AdR, there are two distinct autoantibodies. This prediction of our model is consistent with the low correlation found by Loebel and colleagues between anti-M4 ChR and anti-B2 AdR antibodies (Loebel M et al. 2016).

Most B cells epitopes on non-denaturated proteins (i.e. proteins that conserve their tertiary structure) are believed to be conformational (Morris, 2007), so a significant limitation of this study is due to the fact that our analysis considers only linear epitopes. Nevertheless, the main limitation of this study remains by far my encephalopathy.

Conclusion

This analysis of previously published data suggests a role for the second and the third extracellular loop of M4 and M3 ChR as autoantigens in ME/CFS. It also predicts the presence of a hidden autoantigen and thus a risk of false-positive results with standard ELISA.  The eight peptides found by this analysis and their inverse sequences (Table 2) should be employed as query sequences for the search for possible triggering pathogens and for other autoantigens. These predictions should be tested using both cell-based assays and ELISA tests with these 8 peptides coated on the plate.

Table 2.PNG
Table 2. Peptides belonging to M4 and M4 ChR that fulfill our selection criteria are collected on the left. On the right, their reverse sequences. These 16 peptides can be used in BLAST in order to serach for triggering pathogens and for other possible autoepitopes.

 

Supplementary material. The following two tables represent the first two steps of the analysis presented in this paper. M4 ChR 7-mer peptides that are cross-reactive to M3 ChR are collected in Table 1S, while those of them that are non-cross-reactive (or borderline) to M1 ChR are collected in Table 2S.

TableS.png
Table 1S. Peptides of M4 ChR that are cross-reactive to M3 ChR are collected in the first column. In the second column are collected the scores of these 7-mer peptides obtained from the comparison with M1 ChR. For those that obtained a score below 60, the score from the comparison with B2 AdR is reported in column 5. Positions of peptides of interest that belong to M3ChR and B2 AdR are collected in columns 4 and 6 respectively.
Table 2S.PNG
Table 2S. These 20 peptides are those M4 ChR peptides that cross-react to M3 ChR and at the same time are non-cross-reactive or borderline when compared to M1 ChR. Reactivity to B2 AdR is also indicated, as well as positions with respect to the plasma membrane and mean protrusion indexes. On the left are indicated those peptides of M4 ChR that pass the selection according to our criteria. Both a strict selection and a selection with more weak criteria are reported.

 

 

 

 

 

Annunci

Cellule NK nella ME/CFS

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).

 

cytotoxicitye
Figura 1. Citotossicità delle NK in tre pazienti ME/CFS e in due controlli sani. La citotossicità è calcolata come percentuale di cellule bersaglio che sopravvivono dopo 4 ore di incubazione con concentrazioni crescenti di NK. Grafico di Paolo Maccallini.

 

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

NK
Figura 2. Attivazione e funzione citotossica delle cellule NK. Disegno di Paolo Maccallini.

 

Australian genes

Australian genes

This is the translation into English of an article originally written in Italian. I thank Chiara Scarpellini for the translation.

Four studies, 23 polymorphisms, and one chart

In the chart you find below I collected the single nucleotide polymorphisms (SNPs) that have been associated with ME/CFS by Griffith University’s researchers, to the extent that they are included in the SNPs read by the common 23ndME 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.

genome study

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.

TRPM3 expression
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.

 

The prevalence of the predisposition to ME/CFS (if any)

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 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). So we find a slightly lower prevalence in this case, but we should consider that in the second study the diagnosis was made after six months from the infection, while in the first paper the clinical picture was evaluated after three years. The difference could be due to a certain degree of spontaneous recovery (4% of patients?) that has been reported (anectodal) during the first years from the beginning of the disease.

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.

Is Carboxypeptidase N deficiency a contributing factor in ME/CFS and POTS?

Abstract

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.

Introduction

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).

Table 1.PNG
Table 1. This is a collection of the variants within gene CPN1 found in Pt. 1 and 2. The verdict is the one given by VarSome. A damaging SNP present in exon 3 of CPN1 from Pt. 1 and two rare SNPs found in intron 1 and intron 6 of CPN1 from Pt. 2 are highlighted in orange.
Cattura.PNG
Figure 1. SNPs and InDels along gene CPN1 found in Pt. 1 (first row) and Pt. 2 (second row) as reported by IGV.

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

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).

 

exome 1
Table 2. Possible pathogenic variants found in exome from Pt. 1.
exome 2.PNG
Table 3. Possible pathogenic variants found in exome from Pt. 2.

Discussion

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.

Conclusion

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).

Tempo di convegni

Con l’arrivo della bella stagione, inizia la migrazione dei professionisti del settore sanitario alla volta dei convegni medici. Questa’anno vale la pena ricordare due appuntamenti nostrani, a ridosso l’uno dell’atro:

Nel primo congresso sarò relatore anche io, nel secondo si parlerà di una ricerca a cui ho contribuito.

Errata corrigenda!

This paper is a review on anti-G protein-coupled receptors antibodies in rheumatic diseases. Beta-adrenergic receptors and muscarinic receptors belong to this important class of membrane proteins. I was eager to check if they had included ME/CFS among those diseases associated with this kind of autoantibodies, but I was skeptical. I was surprised to read that, in fact, they mentioned ME/CFS (they call it CFS) in table I, along with Sjögren, Systemic Sclerosis and other autoimmune diseases. But there are some mistakes: they correctly cited the paper by Loebel and colleagues, but they associated CFS with the wrong type of antibodies. Loebel et al. found that the only association that was statistically significant was with anti-beta 2 adrenergic receptors, and anti-M3/M4 muscarinic receptors (you can read that even from the abstract) while in this paper CFS has been associated with anti-beta 1, but not with anti-beta 2, and with antibodies to all the first 4 subtypes of muscarinic receptors. Moreover, they forgot to mention both the paper by Tanaka S et al. and the work by Yamamoto S et al., where an association with anti-M1 was reported. I wrote a mail to one of the authors, but she never answered. The journal is Nature Reviews, Rheumatology.