IT202100012572A1 - DIAGNOSTIC BIOMARKER OF INFLAMMATORY MYOPATHY - Google Patents
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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Description
Descrizione del brevetto per invenzione industriale avente per titolo: Description of the patent for an industrial invention entitled:
?BIOMARCATORE DIAGNOSTICO DI MIOPATIA INFIAMMATORIA? ?DIAGNOSTIC BIOMARKER OF INFLAMMATORY MYOPATHY?
La presente invenzione ha per oggetto un metodo diagnostico per determinare se un soggetto ? affetto da una miopatia infiammatoria e un kit per l?attuazione di detto metodo. The present invention relates to a diagnostic method for determining whether a subject is suffering from an inflammatory myopathy and a kit for implementing this method.
Sato della tecnica Knowledge of the technique
Le miopatie infiammatorie idiopatiche (IIM), dette anche miositi, sono un gruppo di malattie rare (incidenza di 5-10 casi/anno/milione di abitanti e prevalenza di 50-100 casi/milione di abitanti) caratterizzate da mialgie e debolezza muscolare associate a infiltrazione linfocitaria nel tessuto muscolare. La causa di queste malattie ? tuttora non completamente definita: numerosi dati suggeriscono una componente autoimmune e una probabile predisposizione genetica a cui ? possibile si sovrappongano fattori ambientali, infettivi o tossici. Idiopathic inflammatory myopathies (IIM), also called myositis, are a group of rare diseases (incidence of 5-10 cases/year/million inhabitants and prevalence of 50-100 cases/million inhabitants) characterized by associated myalgias and muscle weakness lymphocytic infiltration into muscle tissue. The cause of these diseases? still not completely defined: numerous data suggest an autoimmune component and a probable genetic predisposition to which? possible overlapping environmental, infectious or toxic factors.
Le IMM comprendono la dermatomiosite (DM), la polimiosite (PM), la miopatia necrotizzante (NM) e la miosite a corpi inclusi (IBM). Queste condizioni sono accomunate dalla presenza di debolezza muscolare e mialgie, dall?aumento della Creatinchinasi, dalla presenza di segni elettromiografici di sofferenza muscolare con attivit? spontanea e di alterazioni del segnale muscolare alla risonanza magnetica e vengono distinte tra di loro sulla base di elementi clinici (entit? e distribuzione della debolezza, interessamento di altri organi/tessuti) e laboratoristici (presenza di autoanticorpi), della risposta alla terapia e di specifici aspetti istopatologici [1, 2, 3]. IMMs include dermatomyositis (DM), polymyositis (PM), necrotizing myopathy (NM), and inclusion body myositis (IBM). These conditions are united by the presence of muscle weakness and myalgias, by the increase of Creatine Kinase, by the presence of electromyographic signs of muscular pain with activity? and muscle signal alterations on magnetic resonance imaging and are distinguished from each other on the basis of clinical elements (extent and distribution of weakness, involvement of other organs/tissues) and laboratory (presence of autoantibodies), response to therapy and specific histopathological aspects [1, 2, 3].
La diagnosi ? basata sui criteri di Bohan e Peter (1975) [4] rivisti nella nuova classificazione EULAR/ACR del 2017 [5, 6] che prevedono la biopsia muscolare come metodica cruciale nell?iter diagnostico, in quanto non solo in grado di definire la diagnosi, distinguendo le miositi dalle miopatie ereditarie, ma anche capace di differenziare fra loro le diverse entit? clinico-patologiche. Nella DM le alterazioni sono prevalenti a livello delle strutture capillari endo e perimisiali con conseguente deposizione di immunocomplessi a carico dell?endotelio che porta ad attivazione del complemento e quindi lisi cellulare [7] e atrofia perifascicolare. Nella PM l?infiltrato cellulare ? costituito da macrofagi e linfociti citotossici CD8+ che possono circondare ed invadere fibre muscolari non necrotiche. Un ruolo fondamentale nell?interazione tra fibra muscolare e cellule immunitarie pare ricoperto dal fattore maggiore di istocompatibilit? (MHC-I) che viene up-regolato dalla fibra muscolare in condizioni pro-infiammatorie [8]. L?IBM, considerata la miopatia acquisita pi? frequente dopo i 50 anni, viene anch'essa diagnosticata secondo criteri clinici e istopatologici [2, 9]. La biopsia muscolare mostra aspetti infiammatori sovrapponibili a quelli della PM associati ad aspetti degenerativi. La componente degenerativa ? rappresentata dalla presenza di rimmed vacuoles e dalla deposizione intracellulare di ?-amiloide e di diverse proteine quali la p-tau, presenilina 1, apolipoproteina, ?-tubulina, clusterina, ?-synucleina e gelsolina [10]. Questa forma in particolare rappresenta una sfida per la complessit? del suo meccanismo patogenetico e per l?assenza di un trattamento specifico. The diagnosis ? based on the criteria of Bohan and Peter (1975) [4] revised in the new EULAR/ACR classification of 2017 [5, 6] which envisage muscle biopsy as a crucial method in the diagnostic process, as it is not only able to define the diagnosis , distinguishing myositis from hereditary myopathies, but also capable of differentiating between them the different entities? clinical-pathological. In DM the alterations are prevalent at the level of the endo and perimysial capillary structures with consequent deposition of immune complexes in the endothelium which leads to complement activation and therefore cell lysis [7] and perifascicular atrophy. In PM the cellular infiltrate ? made up of macrophages and CD8+ cytotoxic lymphocytes that can surround and invade non-necrotic muscle fibers. A fundamental role in the interaction between muscle fiber and immune cells seems to be covered by the major histocompatibility factor? (MHC-I) which is up-regulated by muscle fiber in pro-inflammatory conditions [8]. The IBM, considered the most acquired myopathy? frequent after the age of 50, it is also diagnosed according to clinical and histopathological criteria [2, 9]. The muscle biopsy shows inflammatory aspects superimposable on those of PM associated with degenerative aspects. The degenerative component? represented by the presence of rimmed vacuoles and by the intracellular deposition of ?-amyloid and of various proteins such as p-tau, presenilin 1, apolipoprotein, ?-tubulin, clusterin, ?-synuclein and gelsolin [10]. This shape in particular poses a challenge to the complexity of the design. of its pathogenetic mechanism and for the absence of a specific treatment.
L'individuazione di nuovi marcatori biologici-molecolari potrebbe migliorare sia l'efficienza diagnostica sia lo sviluppo di nuovi approcci terapeutici. Lo stato dell?arte in tema di patogenesi delle miositi conferma l?idea che esista una componente infiammatoria cronica antigene-stimolata comune che porta al progressivo peggioramento della debolezza muscolare. L?approfondimento sui pathways e sulle molecole critiche nel rapporto tra infiammazione e degenerazione potrebbe essere la chiave di lettura per la comprensione della progressione della malattia. In particolare, i microRNAs (miRs) [11] sono regolatori critici dell?infiammazione mediata da citochine e della differenziazione dei mioblasti (cellule satelliti) (MiRs-1, 133a, 133b, e 206) [12]. Alcuni recenti dati della letteratura evidenziano alterazioni nell?espressione dei miRs nelle biopsie di pazienti con miosite con conseguente inibizione alla differenziazione in senso miogenico e quindi deficit rigenerativo, substrato biologico della debolezza muscolare. Inoltre, un'alterata espressione dei miRs circolanti nei soggetti miositici [13, 14, 15] ? gi? stata indagata per approfondire le conoscenze sul meccanismo patogenetico, suggerendo che la loro identificazione possa essere utile per la diagnosi. The identification of new biological-molecular markers could improve both the diagnostic efficiency and the development of new therapeutic approaches. The state of the art in the field of myositis pathogenesis confirms the idea that there is a common antigen-stimulated chronic inflammatory component that leads to the progressive worsening of muscle weakness. Insight into the critical pathways and molecules in the relationship between inflammation and degeneration could be the key to understanding the progression of the disease. In particular, microRNAs (miRs) [11] are critical regulators of cytokine-mediated inflammation and myoblast (satellite cell) differentiation (MiRs-1, 133a, 133b, and 206) [12]. Some recent literature data show alterations in miRs expression in biopsies of patients with myositis with consequent inhibition of differentiation in the myogenic sense and therefore regenerative deficit, biological substrate of muscle weakness. Furthermore, an altered expression of circulating miRs in myositis subjects [13, 14, 15] ? already been investigated to deepen the knowledge on the pathogenetic mechanism, suggesting that their identification could be useful for the diagnosis.
Descrizione dell?invenzione Description of the invention
Si ? ora trovato un metodo diagnostico basato sui miRs circolanti in grado di fornire un metodo rapido e di facile analisi a supporto del clinico. Si ? identificato in particolare un indice dato dal rapporto tra miR-206 e miR-409-3p misurati nel plasma in grado di discriminare i pazienti miositici da quelli non miositici, cio? quei pazienti affetti da altre patologie muscolari che si presentano con manifestazioni cliniche simili. Yes ? now found a diagnostic method based on circulating miRs able to provide a quick and easy method of analysis to support the clinician. Yes ? identified in particular an index given by the ratio between miR-206 and miR-409-3p measured in plasma capable of discriminating myositic patients from non-myositic ones, that is? those patients affected by other muscle pathologies presenting with similar clinical manifestations.
In un suo primo aspetto, l?invenzione ha pertanto per oggetto un metodo in vitro per la diagnosi di miopatie infiammatorie in un soggetto che comprende: In a first aspect thereof, the invention therefore relates to an in vitro method for the diagnosis of inflammatory myopathies in a subject comprising:
a) la determinazione del rapporto tra le quantit? di miR-206 e di miR-409-3p misurate in un campione di plasma del soggetto; a) the determination of the relationship between the quantities? of miR-206 and miR-409-3p measured in a plasma sample of the subject;
b) il confronto del rapporto miR-206/miR-409-3p determinato nello stadio a) con un valore soglia discriminante fra soggetti miositici e soggetti nonmiositici. b) the comparison of the miR-206/miR-409-3p ratio determined in step a) with a threshold value discriminating between myositic and nonmyositic subjects.
Il valore soglia discriminante pu? essere ottenuto da un?analisi statistica di una popolazione di soggetti con diagnosi positiva o negativa di miosite, ad esempio mediante una curva ROC. The discriminating threshold value can? be obtained from a statistical analysis of a population of subjects with a positive or negative diagnosis of myositis, for example by means of a ROC curve.
La quantit? di miR-206 e di miR-409-3p pu? essere determinata con metodi noti ad esempio, mediante RT-PCR quantitativa sull?RNA totale estratto dal plasma utilizzando uno spike-in esogeno come normalizzatore. The quantity? of miR-206 and miR-409-3p pu? be determined by known methods, for example, by quantitative RT-PCR on total RNA extracted from plasma using an exogenous spike-in as a normalizer.
Un secondo aspetto dell?invenzione riguarda un kit per la diagnosi di miopatie infiammatorie che comprende reagenti per l?estrazione di RNA da plasma, oligonucleotidi complementari a miR-206 e miR-409-3p e reagenti per RT-PCR quantitativa. A second aspect of the invention relates to a kit for the diagnosis of inflammatory myopathies comprising reagents for the extraction of RNA from plasma, oligonucleotides complementary to miR-206 and miR-409-3p and reagents for quantitative RT-PCR.
Un ulteriore aspetto dell?invenzione riguarda l?uso del rapporto miR-206/miR-409-3p come biomarcatore di miopatie infiammatorie. A further aspect of the invention concerns the use of the miR-206/miR-409-3p ratio as a biomarker of inflammatory myopathies.
Il metodo oggetto dell?invenzione ? vantaggioso rispetto alle metodiche esistenti, complesse e non specifiche: dopo la formulazione del sospetto diagnostico vengono infatti generalmente avviati numerosi esami, tra cui elettromiografia, analisi ematobiochimiche, risonanza magnetica e biopsia muscolare, metodica invasiva da considerare tuttora il gold standard. L?iter comporta quindi alti costi e lunghi tempi di diagnosi che non permettono l?applicazione di un protocollo terapeutico specifico ed efficace in tempi rapidi, con un vantaggio quindi importante per il paziente. The method object of the invention ? advantageous compared to existing, complex and non-specific methods: in fact, after the formulation of the diagnostic suspicion, numerous tests are generally started, including electromyography, haematobiochemical analyses, magnetic resonance imaging and muscle biopsy, an invasive method that is still considered the gold standard. The process therefore involves high costs and long diagnostic times which do not allow the application of a specific and effective therapeutic protocol quickly, with an important advantage for the patient.
Il metodo dell?invenzione, attuabile all?inizio dell?iter diagnostico, consente di velocizzare l?identificazione o l?esclusione della miosite per accelerare la diagnosi e quindi anche l?inizio del trattamento terapeutico. The method of the invention, which can be implemented at the beginning of the diagnostic procedure, allows to speed up the identification or exclusion of the myositis in order to speed up the diagnosis and therefore also the beginning of the therapeutic treatment.
Il metodo dell?invenzione consente inoltre un notevole risparmio economico considerando che l?attuale iter diagnostico risulta costoso a causa delle molteplici analisi da effettuare. The method of the invention also allows a considerable economic saving considering that the current diagnostic process is expensive due to the multiple analyzes to be performed.
Descrizione dettagliata dell?invenzione Detailed description of the invention
Sono stati arruolati pazienti affetti da sospetta miopatia infiammatoria, successivamente ad approvazione dello studio da parte del comitato etico locale. Patients with suspected inflammatory myopathy were enrolled, following approval of the study by the local ethics committee.
Per ogni paziente ? stato effettuato un prelievo ematico in provetta con anticoagulante EDTA per la raccolta di sangue intero e successiva separazione del plasma. I campioni sono stati processati entro le 2 ore dal prelievo, centrifugando a 2000xg per 20 minuti a 4?C. Il plasma raccolto ? stato aliquotato e conservato a -80?C.? Sui campioni di plasma ? stata effettuata l?analisi dei miRs, con una prima fase di estrazione dell?RNA. Da 100 ?l di plasma, l?RNA totale ? stato estratto con il kit Total RNA Purification (NorgenBiotek Corp.). L?RNA ? stato poi utilizzato per effettuare l?analisi dei miRs con applicazione della tecnologia Taqman (Thermo Fisher Scientific) che prevede una retrotrascrizione miR-specifica e una successiva amplificazione in retro-transcription quantitative or real time PCR (RT-qPCR). For each patient? a blood sample was taken in a test tube with EDTA anticoagulant for the collection of whole blood and subsequent plasma separation. The samples were processed within 2 hours of collection by centrifuging at 2000xg for 20 minutes at 4?C. The collected plasma ? been aliquoted and stored at -80?C.? On the plasma samples ? the miRs analysis was carried out, with a first phase of RNA extraction. From 100 ?l of plasma, the total RNA ? was extracted with the Total RNA Purification kit (NorgenBiotek Corp.). The RNA ? It was then used to carry out the analysis of the miRs with the application of Taqman technology (Thermo Fisher Scientific) which involves miR-specific reverse transcription and subsequent amplification in quantitative or real time PCR retro-transcription (RT-qPCR).
L?espressione relativa ? stata calcolata utilizzando come miR di riferimento il cel-miR-39 (spike in sintetico introdotto durante la fase di estrazione dell?RNA dal campione di plasma), calcolando il delta Ct (?Ct = CtmiR X- Ctcel-miR-39) e successivamente il 2<-?Ct >seguendo le procedure standard. Le sequenze di miR-409-3p e miR-206 sono riportate di seguito: The relative expression ? was calculated using the cel-miR-39 (synthetic spike introduced during the extraction of RNA from the plasma sample) as reference miR, calculating the delta Ct (?Ct = CtmiR X- Ctcel-miR-39) and then the 2<-?Ct > following the standard procedures. The sequences of miR-409-3p and miR-206 are given below:
hsa-miR-409-3p: GAAUGUUGCUCGGUGAACCCCU (SEQ ID 1) hsa-miR-409-3p: GAAUGUUGCUCGGUGAACCCCU (SEQ ID 1)
hsa-miR-206: UGGAAUGUAAGGAAGUGUGUGG (SEQ ID 2). hsa-miR-206: UGGAAUGUAAGGAAGUGUGUGG (SEQ ID 2).
Dall?espressione relativa dei due miRs ? stato ricavato un indice dato dal rapporto dei due miRs con al numeratore il miR-206 e al denominatore il miR-409-3p (mir-206/miR-409-3p) in grado di identificare in maniera significativa i pazienti miositici da quelli non miositici e dai controlli. From the relative expression of the two miRs ? an index was obtained given by the ratio of the two miRs with the miR-206 in the numerator and the miR-409-3p in the denominator (mir-206/miR-409-3p) able to significantly identify myositis patients from those not myositis and controls.
Descrizione delle Figure Description of the Figures
Figura 1. Indice analizzato nei tre gruppi di studio. Figure 1. Index analyzed in the three study groups.
Il valore del rapporto miR-206/miR-409-3p ? stato misurato nel plasma dei soggetti nei diversi gruppi di studio: 10 pazienti miositici, 5 pazienti non miositici (affetti da altre patologie muscolari) e 30 soggetti di controllo. I dati sono riportati come boxplot (con valore della mediana). La differenza tra i gruppi ? stata valutata con il test non parametrico Kruskal Wallis, e le differenze significative sono considerate con valore p < 0,05 (* p < 0,05; * p < 0,01). The value of the miR-206/miR-409-3p ratio? was measured in the plasma of subjects in the different study groups: 10 myositic patients, 5 non-myositic patients (affected by other muscle pathologies) and 30 control subjects. Data are reported as boxplot (with median value). The difference between the groups ? was evaluated with the Kruskal Wallis nonparametric test, and significant differences are considered with p-value < 0.05 (* p < 0.05; * p < 0.01).
Figura 2. Analisi della curva di ROC Figure 2. ROC curve analysis
L?analisi ? stata effettuata per definire la qualit? dell?indice diagnostico proposto nel discriminare i pazienti miositici da quelli non miositici. L?analisi ? stata realizzata sui valori del rapporto tra i due miRs (miR-206/miR-409-3p) misurati sui 10 pazienti miositici e 5 pazienti non miositici. L?area sotto la curva ? risultata uguale a 1, valore massimo, identificando un valore soglia di 1,94 con 100% di sensibilit? e di specificit? nel discriminare i due gruppi. The analysis? was carried out to define the quality? of the proposed diagnostic index in discriminating myositic patients from non-myositic ones. The analysis? was performed on the values of the ratio between the two miRs (miR-206/miR-409-3p) measured on 10 myositic patients and 5 non-myositic patients. The area under the curve? resulted equal to 1, the maximum value, identifying a threshold value of 1.94 with 100% of sensitivity? and specificity? in discriminating between the two groups.
Figura 3. Analisi bootstrap dei dati Figure 3. Bootstrap analysis of data
Il bootstrap dei dati ? stato effettuato per definire i valori al di fuori del quale c?? un?alta confidenza nel valore discriminante, mentre per i valori attorno a quello soglia (1,95) ? richiesta una pi? cauta valutazione della discriminazione, in particolare nel range di valori compresi tra 0,96 e 4,11. Data bootstrapping ? been made to define the values out of which c?? a? high confidence in the discriminating value, while for the values around the threshold (1.95) ? request a pi? cautious evaluation of discrimination, especially in the range of values between 0.96 and 4.11.
Parte sperimentale Experimental part
Nello studio sono stati analizzati 10 soggetti miositici e 5 non miositici, utilizzando inoltre un gruppo di controllo di 30 soggetti sani di et? compresa tra i 50 e gli 80 anni. Per ognuno di questi soggetti sono stati misurati entrambi i miRs riportati come espressione relativa da cui poi ? stato ottenuto il valore del rapporto tra questi due miRs, che identifica l?indice diagnostico da noi proposto come riportato in Tabella 1. In the study, 10 myositic and 5 non-myositic subjects were analysed, also using a control group of 30 healthy subjects aged between 50 and 80 years old. For each of these subjects both miRs were measured and reported as a relative expression from which then ? the value of the ratio between these two miRs was obtained, which identifies the diagnostic index proposed by us as reported in Table 1.
Il rapporto dei due miRs risulta significativamente diverso tra i gruppi di studio, e in particolare tra i pazienti miositici rispetto ai non miositici, cos? come rispetto al gruppo di controllo, come riportato in Figura 1. The ratio of the two miRs is significantly different between the study groups, and in particular between myositis patients compared to non-myositis patients, so as compared to the control group, as reported in Figure 1.
Con i dati ? stata effettuata l?analisi della curva ROC per identificare la specificit? e sensibilit? dell?indice individuato (rapporto tra i due miRs, cio? miR-206/miR-409-3p) come biomarcatore diagnostico per distinguere i pazienti miositici da quelli non miositici. I risultati dell?analisi sono riportati in Figura 2. L?area sotto la curva (AUC) risulta uguale a 1, che ? il valore massimo ottenibile, ed ? stato identificato il valore soglia dell?indice uguale a 1,94 con 100% di sensibilit? e 100% di specificit? del marcatore in grado di discriminare i pazienti miositici dai non miositici. With the data ? was carried out? analysis of the curve ROC to identify the specificity? and sensitivity? of the index identified (ratio between the two miRs, i.e. miR-206/miR-409-3p) as a diagnostic biomarker to distinguish myositic from non-myositic patients. The results of the analysis are shown in Figure 2. The area under the curve (AUC) is equal to 1, which is? the maximum obtainable value, and ? been identified the threshold value of? index equal to 1.94 with 100% of sensibility? and 100% specificity? of the marker capable of discriminating myositic from non-myositic patients.
? stata effettuata un?ulteriore analisi di bootstrapping dei dati (per valutare il possibile effetto del caso con il resampling dei campioni) come riportato in Figura 3. ? stato confermato il valore soglia discriminante uguale a 1,95 definendo un intervallo di valori al di fuori del quale c?? un?alta confidenza nel valore discriminante, delimitando attorno al valore soglia una zona ?grigia? compresa tra 4,11 e 0,96. In pratica, per un indice con valore al di sopra di 4,11 il paziente sar? definito come miositico, al di sotto del valore 0,96 sar? definito come non miositico. Mentre ottenendo un valore compreso tra 1,95 e 4,11 il paziente potrebbe essere miositico, ma sar? richiesta una verifica ulteriore per effettuare correttamente la diagnosi. Similmente, i valori compresi tra 0,96 e 1,95 potrebbero indicare un paziente non miositico, ma con necessit? di ulteriore conferma del dato. ? A further bootstrapping analysis of the data was carried out (to evaluate the possible effect of the case with the resampling of the samples) as shown in Figure 3. ? the discriminating threshold value equal to 1.95 was confirmed by defining a range of values outside which c?? a?high confidence in the discriminant value, delimiting a ?grey? area around the threshold value? between 4.11 and 0.96. In practice, for an index with a value above 4.11 the patient will be? defined as myositic, below the value of 0.96 will be? defined as non-myositic. While getting a value between 1.95 and 4.11 the patient could be myositic, but will? further verification is required to make the correct diagnosis. Similarly, values between 0.96 and 1.95 could indicate a non-myositic patient, but with the need for for further confirmation of the data.
Tabella 1. Pazienti arruolati nello studio con relativi valori dei miRs analizzati riportati come espressione relativa e indice diagnostico ricavato come rapporto tra i due miRs Table 1. Patients enrolled in the study with relative values of the miRs analyzed reported as relative expression and diagnostic index obtained as the ratio between the two miRs
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