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WO2012004276A2 - Marqueurs biologiques multiprotéines de la sclérose latérale amyotrophique dans les cellules mononucléaires du sang périphérique, procédés et kits de diagnostic - Google Patents

Marqueurs biologiques multiprotéines de la sclérose latérale amyotrophique dans les cellules mononucléaires du sang périphérique, procédés et kits de diagnostic Download PDF

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WO2012004276A2
WO2012004276A2 PCT/EP2011/061344 EP2011061344W WO2012004276A2 WO 2012004276 A2 WO2012004276 A2 WO 2012004276A2 EP 2011061344 W EP2011061344 W EP 2011061344W WO 2012004276 A2 WO2012004276 A2 WO 2012004276A2
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als
seq
protein
biomarkers
proteins
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WO2012004276A3 (fr
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Valentina Bonetto
Caterina Bendotti
Gabriele Mora
Massimo Corbo
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Fondazione Telethon
Fondazione Salvatore Maugeri Clinica Del Lavoro E Della Riabilitazione
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2835—Movement disorders, e.g. Parkinson, Huntington, Tourette
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/50—Determining the risk of developing a disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/56—Staging of a disease; Further complications associated with the disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/60—Complex ways of combining multiple protein biomarkers for diagnosis

Definitions

  • ALS Amyotrophic lateral sclerosis
  • PBMC peripheral blood mononuclear cells
  • PBMC protein profile changes detected in PBMC of ALS patients are suggestive of possible pathogenic mechanisms such as endoplasmic reticulum stress, nitrative stress, disturbances in redox regulation and R A processing, previously detected in spinal cord of patients and animal models.
  • ALS Amyotrophic lateral sclerosis
  • diagnosis (1) Its incidence is between 1.5 and 2.5 per 100.000 per year (2): approximately 90% of cases are sporadic and the remaining 10% are familial.
  • the diagnosis is mostly based on clinical assessment with a history of progression of symptoms and is thus made with a delay of about a year from symptom onset (3), quite likely beyond the therapeutic window of a disease-modifying drug (4).
  • the clinical course varies widely. No ALS biomarkers are currently in clinical use except for the genetic forms (10% of ALS cases).
  • ALS bio markers are needed not only to support early diagnosis, but also to monitor disease progression, and to accelerate (by decreasing the length and the cost of clinical trials) the identification of effective treatments, that are not available at the moment.
  • PBMC peripheral blood mononuclear cells
  • a proteomic approach such as 2D difference gel electrophoresis (2D DIGE) offers the possibility to reveal multiple protein alterations and is a robust system for identification of potential protein biomarkers (15).
  • PBMC are readily accessible clinical samples and offer a series of advantages over serum/plasma and cerebrospinal fluid (CSF).
  • CSF cerebrospinal fluid
  • Bio-fluids have wide inter- individual variability and a broad range of protein abundance, which make them difficult to analyze by proteomic approaches (16, 17).
  • the cellular proteome is relatively stable, less complex to analyze and gives direct information on alterations of cellular pathways, hence insights into possible pathogenic mechanisms.
  • Massignan et al. (22) refers to PDI and CypA as potential pharmacological targets.
  • WO2010148323 differs from the present invention as it is directed to detecting presence, absence, or quantity of an Xenotropic Murine Leukemia Virus-Related Virus (XMRV) antigen, immunopeptide or po!ynucleic acid in PBMCs.
  • XMRV Xenotropic Murine Leukemia Virus-Related Virus
  • WO2011005628 discloses methods for diagnosing ALS and determining drug efficacy based on the characterization of TDP-43 in a tissue, specifically in cerebrospinal fluid.
  • PBMCs are not mentioned.
  • HSC70 was present in hyaline inclusions (Watanabe M et al. Histological evidence of protein aggregation in mutant SOD1 transgenic mice and in amyotrophic lateral sclerosis neural tissues. Neurobiol Dis 2001, 8:933-941), and TDP-43 was identified as the major component of the ubiquitinated inclusions (Neumann M et al. Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Science 2006, 314: 130-133).
  • US2008/0289964 discloses an assay for the diagnosis of patients with ALS or ALS-like disorders that manifest ALS-like symptoms based on the use of 2D gel electrophoresis to separate the complex mixture of proteins found in blood serum.
  • the other proteins identified in this invention are not nitrotyrosine modified.
  • PBMC peripheral blood mononuclear cells
  • PBMCs peripheral blood mononuclear cells
  • Object of the invention is therefore a method to diagnose and/or to make prognostic predictions and/or to monitor the efficacy of a therapy of Amyotrophic lateral sclerosis (ALS) in a subject comprising the steps of:
  • PBMCs peripheral blood mononuclear cells
  • the method to diagnose and/or to make prognostic predictions and/or to monitor the efficacy of a therapy of Amyotrophic lateral sclerosis (ALS) in a subject comprises the steps of:
  • FUBP1 Far upstream element-binding protein 1
  • Glutathione S-transferase omega-1 Glutathione S-transferase omega-1 (GSTOl)
  • HSC70 Heat shock cognate 71 kDa protein
  • CypA peptidyl-prolyl cis-trans isomerase A
  • Actin NT tyrosine nitrated actin
  • SEQ ID No. 24 heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2/Bl) (SEQ ID No.
  • PBMCs peripheral blood mononuclear cells
  • the method is performed by detecting or measuring the amount of at least: chloride intracellular channel protein 1 (CLIC1) (SEQ ID No. 15); tyrosine nitrated actin (actin NT ) (SEQ ID No. 24); heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2/Bl) (SEQ ID No. 40), or allelic variants, or protein precursors, or functional fragments, or post-trans lationally modified iso forms or gene transcripts thereof. More preferably the method should further detect or measure the amount of Inter leukin-1 receptor-associated kinase 4 (IRAK4) (SEQ ID No.
  • IRAK4 Inter leukin-1 receptor-associated kinase 4
  • step a) is performed by further detecting or measuring the amount of at least: TDP-43 (SEQ ID No. 42); protein disulfide-isomerase A3 (ERp57) (SEQ ID No. 20); peptidyl-prolyl cis-trans isomerase A (CypA) (SEQ ID No. 18), or allelic variants, or protein precursor, or functional fragments, or post-translationally modified isoforms or gene transcripts thereof.
  • TDP-43 SEQ ID No. 42
  • protein disulfide-isomerase A3 ERp57
  • CypA peptidyl-prolyl cis-trans isomerase A
  • biomarkers Any techniques known by the expert in the field may be used to detect and measure biomarkers, preferred ones are immunochemical techniques or mass spectrometric techniques.
  • a subject is an human or a mammalian animal, also to be use for drug tests.
  • PBMCs Peripheral blood mononuclear cells
  • a diagnostic kit to diagnose and/or to make prognostic predictions and/or to monitor the efficacy of a therapy of Amyotrophic lateral sclerosis (ALS) in a subject comprising selective ligands for the biomarkers or allelic variants or functional fragments or post-translationally modified isoforms or gene transcripts thereof, as above disclosed.
  • the selective ligands are immunoglobulins or synthetic or recombinant fragments thereof.
  • ALS Markers can be identified in PBMCs by different methods known in the art.
  • the invention relates also to diagnostic/prognostic tests.
  • ALS Markers were identified in PBMCs by a proteomic approach (2D DIGE and MALDI TOF/TOF mass spectrometry) and validated by immunochemical assay (dot blot analysis on single samples) comparing healthy individuals, sporadic ALS patients (in two levels of ALS severity) and non-ALS neurological patients that may resemble ALS (Multiple Sclerosis, diabetic peripheral neuropathy, demyelinating polyneuropathy, alcoholic polyneuropathy, axonal peripheral neuropathy, Charcot-Marie-Tooth disease, spinocerebellar ataxia, myelopathy localized at C6-C7, transverse myelitis, polymyositis and Lambert-Eaton myasthenic syndrome).
  • Figure 1 Proteome-based strategy to identify specific biomarkers of ALS in PBMC.
  • the ALS network (the panel of proteins and their direct interactors) was then examined by network analysis to identify correlations with ALS pathogenesis (Fig. 5). Large-scale validation was done on 14 proteins by dot blot analysis with single samples from an independent set of sALS patients, healthy and non- ALS neurological disorder controls (Table 2-3, Fig. 2,3). We obtained five protein biomarkers that significantly distinguish ALS patients from healthy and non ALS neurological controls.
  • the 14 PBMC proteins were also measured in G93A SOD1 rats: 5 of them are similarly regulated in the patients and animal model (translational biomarkers).
  • Figure 4 Analysis of protein biomarkers in the G93A SOD1 transgenic rat model: comparison with sALS patients.
  • FIG. 5 Analysis of the PBMC ALS network. Enrichment of the ALS network in GO molecular function (A), GO biological processes (B) and GO cellular component (C) and distribution in the five modules of the ALS network.
  • A GO molecular function
  • B GO biological processes
  • C GO cellular component
  • Figure 7 A: Anti-nitrated actin (actin NT ) antibody preparation and characterization by dot blot assay. Purified human actin was nitrated in vitro and used as antigen in rabbits for raising polyclonal antibodies. The rabbit polyclonal antiserum was tested by dot blot with actin, actin NX , bovine serum albumin (BSA) and BSA NX , prepared by the same
  • Figure 7 A shows that anti-actin does not efficiently recognize another nitrated protein and has more than three times affinity for actin NT of unmodified actin. 3 ⁇ g of actin, actin NX , BSA and BSA NX were loaded in each slot on the nitrocellulose membrane. The membrane was probed overnight with the polyclonal antiserum diluted 1 :7500. Immunoreactivity was normalized to the actual amount of protein loaded on the membrane as detected after Red Ponceau staining. B: Characterization of the anti-
  • actin antibody The anti-actin antibody was further characterized to determine the specificity of the immunoreactivity against PBMC lysates from controls and ALS patients by Western blotting.
  • Figure 7 B shows that the antiserum recognized a band at 42 kDa, which is the expected Mw for actin NT , but also other bands at higher Mw. These are probably polymerized actin forms.
  • These high-Mw species are essentially only detected in the patients and make this antibody especially useful to distinguish ALS patients from controls, also in a dot blot assay, as shown in Figure 3.
  • Equal amounts of PBMC lysates (30 ⁇ g) from healthy controls and ALS patients were analyzed. A representative experiment is shown.
  • the PVDF membrane was probed first with the anti-actin antibody (mouse monoclonal, 1 : 1000 dilution, Chemicon), and the signal was revealed with a goat anti-mouse Qdot® 800-conjugated secondary antibody (Invitrogen, 1 : 1000 dilution) on a laser scanner Molecular Imager FX (Bio-Rad), then with the anti-actinNT antibody, and the signal was revealed with an anti-rabbit peroxidase-conjugated secondary antibody/chemiluminescent HRP Substrate (Millipore) on a ChemiDoc XRS system (Bio- Rad).
  • the anti-actin antibody mouse monoclonal, 1 : 1000 dilution, Chemicon
  • a goat anti-mouse Qdot® 800-conjugated secondary antibody Invitrogen, 1 : 1000 dilution
  • Bio-Rad Molecular Imager FX
  • the anti-actinNT antibody an anti-rabbit peroxid
  • FIG. 8 Analysis of the ALS network.
  • FIG. 1 schematically shows the proteome-based strategy used to identify and validate protein biomarkers of ALS in PBMC.
  • PBMC of healthy controls and sALS patients with two levels of disease severity (Table 4), low, with a functional rating scale (ALSFRS-R) score(18)>24 (ALS>24), and high, with a ALSFRS-R score ⁇ 24 (ALS ⁇ 24), were analyzed by 2D DIGE (Fig. 1A).
  • the analysis done with 11 pooled samples for each group, detected a total of 129 differential protein spots in the three experimental groups (Table 5). From these spots we used MALDI TOF/TOF mass spectrometry to identify 71 corresponding proteins (Fig.
  • Multivariate logistic analysis showed that CLIC1, actin NT and hnRNPA2/Bl were the proteins most associated with ALS in comparison with healthy controls, with 98% discriminatory power (AUC 0.981) (Fig. 2A). Moreover, CypA, TDP-43 and ERp57 levels significantly differed in ALS>24 and ALS ⁇ 24 patients ( Figure 2B and Table 2, indicating that these proteins can discriminate between patients with high and low disease severity). Multivariate logistic analysis showed that ERp57 was the most associated with more severe ALS, with 89% discriminatory power (AUC 0.893) (Fig. 2B). In all 60 sALS patients the levels of the 14 protein biomarkers were independent of sex and age.
  • CypA, GSTOl, FUBP1, CLIC1 and actin NT are translational biomarkers of ALS
  • Translational biomarkers are molecules that can be assessed in both human and animal models and be useful as disease, efficacy and toxicity biomarkers in preclinical and clinical settings accelerating the discovery and development of new treatments.
  • PBMC proteins were translational biomarkers, their level was measured in PBMC of a transgenic S0D1 G93A rat model of ALS, at pre-symptomatic and symptomatic stages of the disease, in comparison with samples from non-transgenic (nonTg) rats by immunoblot.
  • Five protein biomarkers, CypA, GSTOl, FUBP1, CLIC1 and actin NT showed similar changes in PBMC of ALS patients and S0D1 G93A rats (Fig. 4).
  • FUBPl was one of the least specific ALS biomarkers in PBMC of sporadic patients. It is possible that this protein is more associated with SODl mutation- induced alterations and is more informative for the mutant SODl fALS forms.
  • the proteins changed significantly already at a presymptomatic stage, suggesting that they might underline early disease alterations also in patients.
  • ERp57, PRDX2, PA28a showed an altered expression in PBMC of SOD1G93A rats compared to nonTg rats, but differently from PBMC of sALS patients.
  • ERp57 and PA28a were respectively down- and up-regulated at a presymptomatic stage, while PRDX2 was up-regulated only at a symptomatic stage (data not shown).
  • the remaining proteins IRAK4, PDI, HSC70, CALR, TDP-43 and hnRNPA2/Bl did not change their expression with the progression of the disease and if compared with controls.
  • the five translational biomarkers were also investigated in the rat spinal cord at a presymptomatic and symptomatic stages of the disease (Fig. 4). Lumbar spinal cords were sectioned in ventral and dorsal horns to investigate whether proteins alterations were associated or not with motor neurons. In ventral horns, all of them changed similarly to PBMC already at a presymptomatic stage except for CLIC1, up-regulated only at a symptomatic stage. In dorsal horns, the proteins did not change with the progression of the disease and if compared with controls (Fig. 9), suggesting that protein alterations observed in ventral horns may be associated more specifically with motor neurons.
  • CypA, GSTOl, FUBP1, CLIC1 and actin NT are translational biomarkers that might be markers of multi-cellular/multi-systemic alterations underlining pathogenic events both in the human sporadic and animal mutant SOD 1 -linked disease forms.
  • the PBMC ALS network modularity and functional annotations
  • PBMC ALS network was built by retrieving (from protein interaction databases) the known physical interactions among the proteins reported in Table 1 (core proteins) and their direct interactors (first neighbors). Out of the 43 core proteins (protein fragments and post- translationally modified iso forms were excluded from the 71 identified proteins), 41 are reported to interact with other proteins. In total, 499 proteins (41 core proteins and 458 first neighbors) share 1540 interactions within the ALS network.
  • modules 2, 3 and 5 contain proteins that are mostly localized in membranes
  • modules 1 and 4 are more heterogeneous and with interesting features that did not immediately emerge in the initial analysis.
  • module 1 is enriched in proteins that are mainly localized to either the cytosol or the nucleus, and that are involved in the molecular function of nucleotide (R A and DNA) binding.
  • module 4 proteins are almost equally localized to cytosol and nucleus and transcription regulation is one of the most enriched biological processes.
  • the ideal diagnostic marker should detect disease before clinical diagnosis, which is highly challenging for a rare and sporadic disease. ALS patients very often see the specialized neurologist only after months from the first symptoms, when they are unquestionably ill. It is therefore very difficult to test and validate the applicability of biomarkers in preclinical diagnosis.
  • our PBMC protein biomarkers seems to be promising to support prompt clinical diagnosis, since all the 14 validated proteins can distinguish with high significance ALS patients with ALSFRS-R>24 from healthy controls (Table 9).
  • alterations of the level of CypA, GSTOl, FUBP1, CLIC1 and actinNT are detected before disease onset. It is possible that some of these biomarkers may predict the onset of SOD 1 -linked familial ALS.
  • ALS disease progression There are no precise measures of ALS disease progression that allow for short-term monitoring of the disease and assessment of treatment efficacy. In clinical trials survival time is therefore used as the primary measure of outcome. This requires large number of patients followed over a long period of time making ALS clinical trials very expensive. A panel of biomarkers that can reliably assess disease progression would enable a substantial reduction of the costs of the clinical trial and accelerate therapy development in ALS. ERp57, CypA and TDP-43, that were able to discriminate between patients with high and low disease severity, were selected for a pilot longitudinal study and proved to be good candidates for such applications. Large longitudinal studies are now needed to further validate the use of these proteins in clinical practice.
  • PBMC protein biomarkers that significantly distinguish ALS from neurological disorders, such as chaperones (CALR, CypA), proteins involved in redox homeostasis (GSTOl, CLIO) and immune responses (IRAK4).
  • IRAK4 which has no previous association with ALS, has a central function in innate immunity (40). Its up-regulation may be connected with the high cytokine levels observed in sALS patients (34).
  • HnRNPA2/Bl belongs to the family of heterogeneous nuclear ribonucleoproteins that participates to several RNA-related biological processes such as transcription, pre-mRNA processing, mRNA transport to the cytoplasm and translation (53). It is also a binding partner of TDP- 43 and seems to be crucial for at least one of its putative functions (54).
  • the up-regulation of hnRNPA2/B and TDP-43 in PBMC of both ALS>24 and ALS ⁇ 24 patients may underline aberrant R A processing events that are now emerging as central in neurodegeneration in ALS and other neurological disorders (55). All these data endorse the use of PBMC for further in vitro mechanistic studies and reinforce the theory that ALS is a multi-cellular disease. Experimental models for the sporadic form are not available and all mechanistic studies are done with transgenic cells and animals expressing one of the mutant genes linked to familial ALS. Studies with PBMC would have the advantage to consider the influence of the genetic background of the patient.
  • Translational biomarkers that link responses between human and animal model, are of particular interest because their role in the pathogenesis can be investigated in detail in the animal model where they can also offer important preliminary information for clinical trials.
  • CypA, GSTOl, FUBP1, CLIC1 and actin NT are translational biomarkers and, as well as PDI and ERp57, are altered in the spinal cord of SODl G93A rodents before disease onset (20, 22, 25), suggesting a possible involvement in pathways that trigger the disease. Further mechanistic studies in the animal models with these proteins are now warranted.
  • PBMC are a reliable source of biologically relevant biomarkers for ALS
  • the PBMC ALS network is structurally and functionally connected.
  • the probability of finding ALS proteins linked either directly or indirectly (via a common neighbor) was higher in the ALS network than in random networks.
  • the ALS network was significantly enriched in GO annotations that were also enriched in genomic/proteomic data obtained from spinal cord and motor neurons of familial ALS animal models (56-58).
  • GO terms related to DNA/RNA binding, transcription regulation and nucleus were enriched in two highly-connected groups of nodes (modules 1 and 4) within the ALS network.
  • sALS patients and controls Patients with sALS, according to revised El Escorial criteria(62), were examined and blood samples were collected at the IRCCS Fondazione S. Maugeri, in Milano and Pavia, Italy. The patients were then divided into two groups according to the ALSFRS-R score, ALS>24 or ALS ⁇ 24 patients. None of the sALS patients had systemic inflammatory conditions as detected by the erythrocyte sedimentation rate and total blood cell count. Blood samples of non-ALS neurological disorder controls were provided by the NEMO, Niguarda Ca' Granda Hospital, Milano, Italy and the IRCCS Fondazione S. Maugeri, in Milano and Pavia, Italy. None of the patients was receiving drugs that might interfere with total blood cell count.
  • Tables 4 and 7-8 summarize the characteristics of ALS patients and non-ALS neurological disorder controls. Blood samples of healthy donors were provided by the Transfusion Medical Centre at the IRCCS Policlinico S. Matteo, Pavia, Italy. Informed consent was obtained from all subjects, according to the ethics committee guidelines at the different centers.
  • PBMC peripheral venous blood from patients and controls were collected in EDTA pre-coated vials (Vacuette K3E K3EDTA, Greiner bio-one).
  • PBMC peripheral venous blood from patients and controls were collected in EDTA pre-coated vials (Vacuette K3E K3EDTA, Greiner bio-one).
  • PBMC peripheral venous blood from patients and controls were collected in EDTA pre-coated vials (Vacuette K3E K3EDTA, Greiner bio-one).
  • PBMC peripheral venous blood from patients and controls were collected in EDTA pre-coated vials (Vacuette K3E K3EDTA, Greiner bio-one).
  • PBMC peripheral venous blood from patients and controls were collected in EDTA pre-coated vials (Vacuette K3E K3EDTA, Greiner bio-one).
  • PBMC peripheral venous blood from patients and controls were collected in EDTA pre-coated vials (Vacuette
  • PBMC proteins were obtained by cell lysis in 20 mM Tris-HCl pH 7.5, 0.1% NP40 and 0.1% SDS supplemented with Protease Inhibitors (Sigma). Proteins were quantified by the BCA protein assay (Pierce).
  • PBMC proteins were prepared for 2D DIGE analysis. Three pools of 25 ⁇ g from 1 1 healthy controls, 11 ALS>24 and 11 ALS ⁇ 24 patients were methanol-precipitated. Proteins were then dissolved in 30 mM Tris-HCl pH 8.5, 7 M urea, 2 M thiourea, CHAPS 4% (w/v) and Cydye-labeled according to the manufacturer's instructions (GE Healthcare) with minor modifications. Briefly, 25 ⁇ g of each pool was labeled with 200 pmol of Cy3 or Cy5 dye for 30 min in ice in the dark. To exclude preferential labeling of the dyes, each sample was also reverse labeled.
  • SDS-PAGE was done using precast 10% poly aery lamide SDS gel (Invitrogen).
  • Four 2D gels were run with the three experimental groups: healthy controls and ALS patients, ALS>24, and ALS ⁇ 24.
  • Each gel contained two experimental groups, one Cy3-labelled, the other Cy5-labelled plus the Cy2-labelled internal standard.
  • Gel images were captured by the laser scanner Molecular Imager FX (Bio-Rad). Image analysis was done with Progenesis PG240 v2006 software (Nonlinear Dynamics).
  • the normalized volume was standardized against the intra-gel standard, dividing the value for each spot normal volume by the corresponding internal standard spot normal volume within each gel.
  • the values for each spot in each group were expressed as the mean of the Cy3- and Cy5- labelled analyses.
  • the values for ALS patients were reported as fold change: higher (positive) or lower (negative) spot volume of the samples from ALS patients in comparison with healthy controls.
  • Protein spots were located and excised from 2D gels with the EXQuestTM spot cutter (Bio- Rad). Spots were processed and gel-digested with modified trypsin from bovine pancreas (Roche) and identified by mass spectrometry (MS), essentially as previously described(25). Digestion, desalting and concentration with ZipTip® pipette tips with CI 8 resin (Millipore) and MALDI target deposition were carried out on an automated protein digestor DigestPro MS (Intavis AG). Peptide mass fingerprinting and tandem mass spectrometry (MS/MS) were done on a 4800 MALDI TOF/TOF mass spectrometer (Applied Biosystems).
  • the mass spectra were internally calibrated with trypsin autolysis fragments.
  • the combined MS and MS/MS data were submitted by GPS Explorer v.3.6 software (Applied Biosystems) to the MASCOT database search engine (Version 2.1, Matrix Science) and searched with the following parameters: Uniprot Swissprot 57.8 database over all Homo sapiens protein sequences deposited, no fixed modifications, as possible modifications carboamidomethylation of cysteine and oxidation of methionine, 1 missed trypsin cleavage, a mass tolerance of ⁇ 0.1 Da for the peptide masses and ⁇ 0.3 Da for the MS/MS fragment ion masses.
  • a protein was regarded as identified if the MASCOT protein score, based on the combined MS and MS/MS data, was above the 5% significance threshold for the database(63).
  • Some of the proteins were identified by liquid chromatography- (LC)- MS/MS using the microfluid chip-based technology for nanoelectrospray coupled to an ion trap mass spectrometer (Agilent 1200 LC/MSD Trap XCT) as previously described(64). Data were acquired sequentially in MS mode (scan range 200-2000 amu) and in data- dependent mode, automatically recording the MS/MS spectra of the four most abundant ions in every scan cycle.
  • NonTg and Tg rats expressing a high copy number of mutant human SOD1 with Gly-93- Ala substitution were bred and maintained at the Mario Negri Institute for Pharmacological Research, Milan, Italy. Animals were housed at 21 ⁇ 1°C with relative humidity 55 ⁇ 10% and 12 h of light. Food (standard pellets) and water were supplied ad libitum. Transgenic rats were identified with PCR on DNA from tail biopsies. G93A SOD1 rats were killed at presymptomatic (15 weeks of age) and early-symptomatic (20 weeks of age) stages of disease. NonTg rats were used as controls (20 weeks of age).
  • PBMC Plasma PBMC
  • PBMC proteins were obtained by cell lysis in 50 mM Tris-HCl, pH 7.5, 2% (w/v) CHAPS, 37.5 U benzonase (Merck), supplemented with Protease Inhibitors (Sigma). Proteins were quantified by the BCA protein assay (Pierce).
  • Proteins were separated by SDS-PAGE on precast 12% Criterion XT Bis-Tris gels (Bio-Rad) and immunoblotted, as described(43). The blots were probed with primary and secondary antibodies, as described for dot blot assay of the human samples. Blots were also probed with an antibody that recognizes actin (mouse monoclonal, 1 : 1000 dilution, Chemicon) for loading control and developed by Immobilon Western Chemiluminescent HRP Substrate (Millipore) on the ChemiDoc XRS system (Bio-Rad). Densitometry was done with Progenesis PG240 v2006 software (Nonlinear Dynamics).
  • ALS proteins Like the ALS proteins, the random ones were obtained in proteomic studies (Supporting References) but, unlike the ALS proteins, they were selected randomly, without any bias for a particular experimental setting.
  • the Rives and Galitski algorithm was applied to sub-divide the ALS network into modules (69). Briefly, an adjacency matrix of all-pairs shortest path distances was subjected to average linkage hierarchical clustering (70) with uncentered correlation as similarity metric, using Cluster 3.0 (71). The resulting dendrogram, generated using Java Tree View 1.0.13(72), was parsed into modules, by cutting the tree after the 4 th bifurcation.
  • GO analysis generated using Java Tree View 1.0.13(72), was parsed into modules, by cutting the tree after the 4 th bifurcation.
  • BinGO software(73) was used to assess the enrichment of the test set (the ALS network or modules thereof) in GO terms (along each of the three GO branches), compared to a reference set of similar size, randomly chosen from all the human genes annotated along the same GO branch.
  • the statistical significance of the enriched GO annotations was first assessed with the hyper-geometric test, then the -values were corrected with the Benjamini-Hochberg correction of false discovery rate (74). After adjustment for the significant terms ( ⁇ 0.05), terms were selected with an intermediate level of specificity along the GO hierarchy. Fine-grained terms, i.e. those not included in the GO Slim (generic GO A) dataset (75), were first removed from the BinGO output lists.
  • Chloride intracellular channel protein 1 000299 15
  • Mw ca i c and pl calc calculated Mw and pi
  • Mw sanction bs and pl obs observed Mw and pi
  • * protein with lower than expected Mw, possibly a fragment.
  • Proteins were identified based on combined MS and MS/MS on a 4800 MALDI TOF/TOF mass spectrometer (Applied Biosystems) except those indicated by #. These were identified by LC- MS/MS using the microfluid chip-based technology for nanoelectrospray coupled to an ion trap mass spectrometer (Agilent 1200 LC/MSD Trap XCT); MASCOT score, MASCOT protein score derived from the combination of MS and MS/MS data.
  • Results are expressed as odds ratios (OR) and 95% confidence intervals (95 % CI). A 95% CI not including the value of 1 indicates a statistically significant result. All probability values were two-sided and p ⁇ 0.05 was considered statistically significant (values in bold type).
  • Results are expressed as odds ratios (OR) and 95% confidence intervals (95 % CI). A 95% CI not including the value of 1 indicates a statistically significant result. All probability values were two-sided and p ⁇ 0.05 was considered statistically significant (values in bold type).
  • Amyotrophic lateral sclerosis oxidative energy metabolism and calcium homeostasis in peripheral blood lymphocytes. Neurology 47: 1060-1064.
  • ALSFRS-R a revised ALS functional rating scale that incorporates assessments of respiratory function.
  • Amyotrophic lateral sclerosis mimic syndromes a population-based study. Arch Neurol 57: 109-113.
  • Oxidative stress in ALS a mechanism of neurodegeneration and a therapeutic target. Biochim Biophys Acta 1762: 1051-1067.
  • TDP-43 binds heterogeneous nuclear ribonucleoprotein A/B through its C-terminal tail: an important region for the inhibition of cystic fibrosis transmembrane conductance regulator exon 9 splicing. J Biol Chem 280:37572-
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La présente invention concerne un procédé et un kit de diagnostic pour diagnostiquer et/ou pour effectuer des prévisions pronostiques et/ou pour suivre l'efficacité d'une thérapie de la sclérose latérale amyotrophique (SLA) chez un sujet.
PCT/EP2011/061344 2010-07-06 2011-07-05 Marqueurs biologiques multiprotéines de la sclérose latérale amyotrophique dans les cellules mononucléaires du sang périphérique, procédés et kits de diagnostic WO2012004276A2 (fr)

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WO2019036577A1 (fr) * 2017-08-18 2019-02-21 University Of South Carolina Détection d'une modification dérivée d'ester de fumarate dans un échantillon d'essai
WO2019217916A1 (fr) * 2018-05-10 2019-11-14 The Methodist Hospital Procédés de pronostic et de gestion de maladie
US10501513B2 (en) 2012-04-02 2019-12-10 Modernatx, Inc. Modified polynucleotides for the production of oncology-related proteins and peptides
WO2022189463A1 (fr) * 2021-03-08 2022-09-15 Akershus Universitetssykehus Hf Dosage de clairance

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WO2010148323A2 (fr) 2009-06-18 2010-12-23 Whittemore Peterson Institute For Neuro-Immune Disease Diagnostic et traitement de maladies ou troubles associés au virus de la leucémie murine xénotrope
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