WO2013098786A1 - Méthode pour le diagnostic in vitro de la maladie de parkinson - Google Patents

Méthode pour le diagnostic in vitro de la maladie de parkinson Download PDF

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WO2013098786A1
WO2013098786A1 PCT/IB2012/057786 IB2012057786W WO2013098786A1 WO 2013098786 A1 WO2013098786 A1 WO 2013098786A1 IB 2012057786 W IB2012057786 W IB 2012057786W WO 2013098786 A1 WO2013098786 A1 WO 2013098786A1
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spot
found
protein
parkinson
differentially expressed
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PCT/IB2012/057786
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Tiziana ALBERIO
Mauro FASANO
Leonardo LOPIANO
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Universita' Degli Studi Dell'insubria
Universita' Degli Studi Di Torino
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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

Definitions

  • the present description relates to a method for the diagnosis in vitro of Parkinson's disease.
  • Parkinson's disease is a chronic, progressive neurodegenerative disease characterized by motor and non-motor symptoms with a multifactorial aetiology.
  • PD is mainly associated with the degeneration of dopaminergic neurons of the Substantia Nigra pars compacta (SNpc) in the midbrain and constitutes the second most common neurodegenerative disorder after Alzheimer's disease.
  • SNpc Substantia Nigra pars compacta
  • the incidence and prevalence of the disease increase with age; the prevalence is 1.2% in people over the age of 50 and increases up to 4-5% in individuals over the age of 85.
  • the clinical diagnosis of PD is based on the presence of various combinations of the cardinal symptoms: resting tremor, bradykinesia, rigidity and postural instability.
  • the clinical picture of PD has, also a wide variety of non-motor symptoms such as cognitive impairment, hallucinations, depression, anxiety, sleep disturbances, hyposmia and autonomic symptoms such as hypotension, constipation, impotence and abnormal sweating. These symptoms are often already present in the earliest stages of the disease and may even precede the onset of motor symptoms by several years.
  • the diagnosis of PD is still an essentially clinical diagnosis and it the diagnostic error rates in experienced centres can be up to 10-20%, if instead general practitioners perform the diagnosis, the error rate can be as high as 50%.
  • the clinical diagnosis is supported by a good, long-lasting response to dopaminergic drugs; in uncertain cases, functional imaging (SPECT, PET) can be used, however they are very expensive and have a not insignificant proportion of false negatives. Diagnostic confirmation requires autopsy (definite diagnosis of PD) .
  • biomarker as "any characteristic that can be objectively measured and evaluated as an indicator of a normal biological process, pathogenic process or pharmacological response to a therapeutic intervention".
  • biomarkers for PD may lead to the identification of individuals at risk before the onset of motor symptoms, allowing recourse to neuroprotective therapies (capable of slowing disease progression) at an early stage, when the neurodegenerative process is still in an early stage. Indeed, motor symptoms appear when degeneration has already affected 60-70% of the dopaminergic neurons in the midbrain SNpc.
  • Another important clinical application of biomarkers regards the differential diagnosis of PD. Diagnostic error is mainly due to the existence of parkinsonian syndromes that may be very similar to PD at onset, but later manifest different clinical features and disease courses (atypical parkinsonism) . Such forms of the disease have a different prognosis compared to PD and require a different therapeutic approach .
  • PD itself is characterised by several clinical phenotypes that differ in terms of course, response to therapy and prognosis. Also in these cases, biomarkers might allow a differential diagnosis with the possibility of "personalising" the treatment of individual patients.
  • biomarkers Another important application of biomarkers concerns the monitoring of disease progression (influenced by several factors, including the clinical phenotype, the genetic contribution to etiopathogenesis, the various therapies used) .
  • Obtaining biomarkers that characterise the different phases of the disease (early, intermediate, advanced) could allow investigation of the impact of various factors on disease progression (with the possibility of modifying them) and make it possible to study the effect of neuroprotective therapies.
  • DA dopamine
  • DAT dopamine transporter
  • CNS central nervous system
  • SNpc substantia nigra pars compacta
  • MRI magnetic resonance imaging
  • biomarkers include clinical biomarkers, imaging biomarkers or biomarkers present in body fluids.
  • imaging biomarkers include imaging biomarkers or biomarkers present in body fluids.
  • Functional imaging techniques employ various radiotracers able to examine the pre-and post-synaptic sides of dopaminergic synapses; functional magnetic resonance imaging methods and transcranial ultrasound are also used.
  • biomarkers present in body fluids have characteristics that make them interesting from a practical point of view; they can be measured with simpler, less invasive and less expensive tests.
  • circulating lymphocytes (PBL Peripheral Blood Lymphocytes) seem to be particularly promising. They are easy to obtain and some functional alterations in these cells have been observed in patients with PD.
  • the functions of certain immune system cells, including T lymphocytes, are regulated not only by cytokines, but also by several neurotransmitters, including dopamine.
  • T lymphocytes can be considered circulating dopaminergic cells, and consequently may show an increased susceptibility to damage from oxidative stress.
  • T cells may also reflect genetic alterations. As stated above, they express several proteins involved in dopamine metabolism, therefore it can be argued that genetic variants responsible for pathological changes in the CNS have peripheral effects on T cells, although through different mechanisms.
  • the present invention relates to methods for the diagnosis of Parkinson's disease. More specifically, the present invention identifies and describes proteins that are differentially expressed in Parkinson's disease with respect to their expression in the normal state.
  • the present description relates to an in vitro method for diagnosing Parkinson's disease in a subject, the method comprising detection of differentially expressed proteins identified with the methods described herein in a peripheral tissue sample from said subject.
  • the present invention also describes methods to i) determine the stage or monitor progression of Parkinson's disease in a subject, ii) perform a differential diagnosis of Parkinson's disease, iii) to verify the pharmacological response of patients with Parkinson's disease, where the methods comprise the detection of differentially expressed proteins identified with the methods described herein in a sample of tissue from said subject.
  • the method comprises: (a) creating at least one panel of proteins differentially expressed in a sample from a subject with Parkinson's disease;
  • this method allows correlation of the Parkinson's disease subtype of a patient with prophylactic or therapeutic treatment, with disease progression and to discriminate between the EOPD and LOPD forms of the disease.
  • the peripheral tissue sample from the patient used in the methods of the invention is a T lymphocyte protein extract.
  • the differentially expressed proteins are the proteins shown in Tables 3, 4, 8 and 10.
  • the method for in vitro diagnosis of Parkinson's disease in a subject comprises detecting differentially expressed proteins in a biological sample from the subject, in which the differentially expressed proteins belong to a first panel of diagnostic proteins, in which said first diagnostic panel comprises vinculin, talin-1, beta- fibrinogen, filamin A, alpha tubulin, gelsolin.
  • the method comprises detecting at least one further differentially expressed protein, wherein the at least one further differentially expressed protein belongs to a second panel of diagnostic proteins, wherein the second diagnostic panel comprises lymphocyte-specific protein 1, vimentin, moesin, trans-aldolase, 14-3-3 protein epsilon .
  • the method comprises detecting at least one further differentially expressed protein, wherein the at least one further differentially expressed protein belongs to a third panel of diagnostic proteins, wherein the third diagnostic panel comprises vimentin, septin-6, trans- aldolase, twinfilin-2, Rho GDP-dissociation inhibitor 2, fragment of beta-actin.
  • the present description relates to a method for the in vitro differential diagnosis of Parkinson's disease in a subject, wherein the differential diagnosis allows to distinguish between LOPD (late onset Parkinson's disease) and EOPD (early onset Parkinson's disease); the method comprises detecting, in a biological sample from the subject, at least the differentially expressed proteins belonging to a differential diagnosis protein panel, in which the differential diagnosis panel comprises beta tubulin, protein disulfide isomerase A3, vimentin, plastin-2, purine nucleoside phosphorylase, glutathione S- transferase Pi, PDCD6-interacting protein.
  • the differential diagnosis panel comprises beta tubulin, protein disulfide isomerase A3, vimentin, plastin-2, purine nucleoside phosphorylase, glutathione S- transferase Pi, PDCD6-interacting protein.
  • the present description relates to a method for in vitro diagnosis of Parkinson's disease progression in a subject, where the method comprises detecting, in a biological sample from the subject, at least the differentially expressed proteins belonging to a progression protein panel, in which the progression panel comprises beta-fibrinogen, vimentin, lymphocyte-specific protein 1, plastin-2, moesin, gelsolin, 14-3-3 protein epsilon.
  • the present description relates to a method for in vitro diagnosis of response to pharmacological treatment of Parkinson's disease in a subject, where the method comprises detecting, in a biological sample from the subject, the differentially expressed proteins belonging to a pharmacological response protein panel, in which the pharmacological response panel comprises prolidase, mitochondrial ATP synthase beta subunit, actin related protein 2, F-actin capping protein subunit beta, tropomyosin alpha-3 chain, proteasome activator complex subunit-1, peroxiredoxin-6, glyceraldehyde-3-phosphate dehydrogenase, proteasome subunit beta type 2.
  • differentially expressed proteins may be increased in patients with Parkinson's disease compared with control subjects.
  • the expression of other differentially expressed proteins can be decreased in subjects with Parkinson's disease compared to control subjects.
  • Tables 4 and 8 indicate whether the expression of the proteins described herein is increased or decreased in Parkinson's disease compared with control subjects. It will be clear from the symbols ( ⁇ and 4 ) in Tables 4 and 8 and by the log2 values in tables 5 and 9 if an increase or a decrease of expression is indicative of the disease state. It will also be clear from trends reported in Tables 3 and 10 if an increase or decrease of expression is indicative respectively of drug response or disease progression.
  • Differentially expressed proteins can be detected using an antibody specific for each protein, for example in an ELISA or by Western blotting.
  • the differentially expressed proteins can be detected by, among other methods, 2D gel electrophoresis or mass spectrometry techniques, including the LS/MS/MS, MALDI-TOF or SELDI-TOF.
  • the sample may be immobilized on a solid support for analysis .
  • the diagnosis may be based on the spot pattern on a 2D gel prepared of a sample from the subject.
  • the pattern of spots obtained from a subject suffering from Parkinson's disease can be directly compared with the pattern obtained with samples from control subjects, without the need to identify individual proteins.
  • the invention is based, in part, on systematic discovery of proteins that change significantly with two-dimensional electrophoresis.
  • T cells are a good source of biomarkers of PD because: 1) Lymphocytes may reflect genetic alterations; 2) lymphocytes are dopaminergic cells as are the neurons affected by the disease; 3) lymphocytes patrol the central nervous system.
  • Panel A Linear correlation between the relative level of protein and daily dose of L-DOPA for spots 441 and 963.
  • Panel B Distribution of the relative volume of spots 400, 608, 774, 779, 839, 893, 921 in patients not taking dopamine agonists (0) and patients taking dopamine agonists (1).
  • the relative volume is the spot volume relative to the sum of the spot volumes common to all gels.
  • the central black line is the median, the box represents the first and third quartiles, the whiskers extend to the limits of the data distribution.
  • the markers for the in vitro diagnosis of Parkinson's disease object of the present description have been identified by two-dimensional gel electrophoresis of protein extracts of T lymphocytes from 32 subjects, a number fully congruous with other biomarkers discovery efforts in the scientific literature.
  • Biomarkers were validated by the leave-one-out method, obtaining an estimate of their sensitivity and specificity.
  • markers object of the invention showed a linear correlation with disease duration, defined as the time elapsed from onset of clinical symptoms. For this reason, said markers can be regarded as of disease progression.
  • the present disclosure concerns a method for the in vitro diagnosis of Parkinson's disease obtained through the determination of differentially expressed proteins in diseased subjects compared to healthy subjects.
  • the method can be implemented with different levels of elaboration, i.e., by increasing the number of differentially expressed proteins determined.
  • differentially expressed proteins belonging to at least a first panel of diagnostic proteins comprising vinculin, talin-1, beta- fibrinogen, filamin A, alpha tubulin, gelsolin, will be determined .
  • At least one further differentially expressed protein belonging to a second panel of diagnostic proteins comprising lymphocyte-specific protein 1, vimentin, moesin, trans-aldolase, 14-3-3 protein epsilon, will be determined.
  • At least one further differentially expressed protein belonging to a third panel of diagnostic proteins comprising vimentin, septin-6, trans-aldolase, twinfilin-2, rho GDP- dissociation inhibitor isoform 2, beta-actin fragment, will be determined.
  • the method for in vitro diagnosis of Parkinson's disease comprises detecting, in a sample of T lymphocyte protein extract, a first diagnostic panel of differentially expressed proteins, where the first diagnostic panel includes vinculin found in spot ID No.: 86, 87 of Table 4, talin-1, beta-fibrinogen found in spot ID NO: 362, 365, 368, 369 of Table 4, filamin A and alpha tubulin found in spot ID No.: 382 of Table 4, and gelsolin.
  • the method for the in vitro diagnosis of Parkinson's disease comprises detecting a second diagnostic panel of differentially expressed proteins, by combining the results obtained from the second panel with those obtained from analysis of the first panel, where the second diagnostic panel comprises lymphocyte-specific protein 1, vimentin found in spot ID No. : 591 of Table 4, moesin, trans-aldolase found in spot ID No.: 676 of Table 4, and 14-3-3 protein epsilon.
  • the process for in vitro diagnosis of Parkinson's disease in a subject comprises detecting a third diagnostic panel of differentially expressed proteins, by combining the results obtained from the third panel with those obtained from analysis of the first and the second panels, where the third diagnostic panel comprises vimentin found in spot ID No.: 329 of Table 4, septin-6, trans-aldolase found in spot ID No.: 679 of Table 4, twinfilin-2, rho GDP-dissociation inhibitor isoform 2, and beta-actin fragment.
  • the biological sample is a sample of protein extract from T lymphocytes, in which the above listed proteins display a change of expression compared to a control subject.
  • Detection of the differentially expressed proteins listed above may be achieved using an antibody specific for each protein, mass spectrometry or two-dimensional gel electrophoresis.
  • the present description relates to a method for the in vitro differential diagnosis of Parkinson's disease in a subject, wherein the differential diagnosis allows to distinguish between a LOPD state and a EOPD state.
  • the method for differential diagnosis comprises detecting, in a biological sample from a subject, at least the differentially expressed proteins of a differential diagnosis panel, in which the differential diagnosis panel includes beta tubulin, protein disulfide isomerase A3, vimentin, plastin-2, purine nucleoside phosphorylase, glutathione S-transferase Pi, PDCD6 interacting protein.
  • the present description concerns a method of in vitro staging of Parkinson's disease in a subject, the method comprising detecting, in a biological sample from the subject, at least the differentially expressed proteins of a progression panel, in which the progression panel comprises beta- fibrinogen, vimentin, lymphocyte-specific protein 1, plastin-2, moesin, gelsolin, 14-3-3 protein epsilon.
  • the present description relates to a method for in vitro diagnosis of response to pharmacological treatment of Parkinson's disease in a subject, where the method comprises detecting, in a biological sample from the subject, at least the differentially expressed proteins belonging to a pharmacological response protein panel, in which the pharmacological response panel comprises prolidase, mitochondrial ATP synthase beta subunit, actin related protein 2, F-actin capping protein subunit beta, tropomyosin alpha-3 chain, proteasome activator complex subunit-1, peroxiredoxin-6, glyceraldehyde-3-phosphate dehydrogenase, proteasome subunit beta type 2.
  • Protein analysis of peripheral lymphocytes by two- dimensional electrophoresis is especially preferred being a powerful investigative tool for assessing biochemical differences associated with MP.
  • this description has allowed the identification of several differentially expressed proteins belonging to various protein panels (three diagnostic panels, including a differential diagnosis panel, a progression panel and a pharmacological response panel), characterized in terms of their identity and their position in two-dimensional gel electrophoresis; based on the expression of these proteins, it is possible to distinguish patients with PD from controls, and patients with EOPD from patients with LOPD, as well as to obtain an indication of progression or response to drug therapy.
  • three diagnostic panels including a differential diagnosis panel, a progression panel and a pharmacological response panel
  • the present inventors have identified three different panels of diagnostic biomarkers.
  • the first panel comprises 9 spots (86, 87, 335, 362, 365, 368, 369, 382, 657) attributable to 6 proteins, which altogether show discriminating capacity, with an area under the ROC curve equal to 0.992.
  • the sensitivity was 100%, i.e., all patients with PD were correctly classified.
  • 4 spots (362, 365, 368, 369) correspond to the same protein, beta-fibrinogen, and have volumes that correlate.
  • the other proteins that constitute the 9- spot model are cytoskeletal proteins. Although these alone are not sufficient to correctly classify subjects, the association to the 4 beta-fibrinogen spots has allowed a correlation between the expression of such proteins and Parkinson's disease to be obtained.
  • Adding an additional 5 spots (405, 591, 598, 676, 1641) substantially improves the performance of the classification model, obtaining the correct classification of all patients with PD and the false classification of a single control.
  • the performance of the model is described by the area under the ROC curve, which is equal to 0.996.
  • the spots discussed so far are a subset of those selected on the basis of the non-parametric Wilcoxon test.
  • a slight improvement of model performance is achieved by including the 6 remaining spots (329, 414, 679, 842, 871, 1639) in the model.
  • spots 362, 365, 368, 369, 392, 405, 505, 591, 598, 657, 1641) using Pearson correlation analysis were selected, thus allowing identification of possible progression markers.
  • spots were identified that correlate with possible confounding factors, such as age and treatment regimen. Only two spots (441 and 963) correlated linearly with the daily dose of L-DOPA, demonstrating that the extracellular action of plasma L-DOPA on peripheral lymphocytes is extremely limited. In contrast, patients treated with non ergot D3-selective dopamine-agonists show significant differences in the levels of 7 lymphocyte proteins (400, 608, 774, 779, 839, 893 and 921) . This result is an interesting demonstration of the possibility to pharmacologically modulate the dopaminergic system in peripheral lymphocytes.
  • dopaminergic stimulation of lymphocytes may be the basis for the observed effects to the immune system in patients with PD treated with dopamine agonists.
  • the variations induced by dopamine agonist therapy at the peripheral level reflect the therapeutic efficacy of the drug at the central level.
  • lymphocyte biochemical markers for the in vitro diagnosis of PD: a panel of biochemical lymphocyte markers for the in vitro differential diagnosis of EOPD and LOPD forms, a panel of biochemical lymphocyte markers of PD progression, and finally a panel of biochemical markers associated with lymphocyte response to pharmacological therapy of PD.
  • Two groups of patients were selected: a first group of 8 subjects with EOPD (onset before 50 years of age), identified anonymously as EOFJTO048, EOM_NO018, EOM_TO049, EOM_TO008, EOM_TO022, EOF_TO050, EOM_NO028 and EOM_NO017, and a group of 7 patients with LOPD (onset after 50 years of age) , identified anonymously as LOM TO040, LOF TO021, LOF_TO016, LOM TO006, LOM_TO064, LOF_NO023 and LOM_TO066.
  • each subject provided informed consent, in accordance with the protocol approved by the ethics committee of the University of Turin .
  • the following data were collected: age, sex, diagnosis, age at disease onset, family history, therapies and any other recent illnesses.
  • each participant was associated with an alphanumeric code and personal data has been entrusted solely to the practitioner .
  • PBMC Peripheral blood mononuclear cells
  • the buffy coat was collected, using a pasteur pipette, and diluted to 15 ml with MACS buffer (2 mM EDTA, sodium azide 0.09% w/v, BSA 0.5% w/v, in PBS) in two separate tubes. After centrifugation at 400g for 15' at RT, the supernatant was aspirated and the two pellets were combined and resuspended with 10 ml of MACS buffer. Cells were counted with a Biirker chamber and centrifuged again at 400g, for 15' at RT to remove residual platelets (in the supernatant) .
  • MACS buffer 2 mM EDTA, sodium azide 0.09% w/v, BSA 0.5% w/v, in PBS
  • T cells were isolated from PBMCs by means of a commercial kit for immunomagnetic separation, the QuadroMACS (Miltenyi Biotec, Bergisch Gladbach, Germany) . Initially cells were incubated with a mixture of primary antibodies against various antigens from blood cells other than T lymphocytes (i.e., excluding the molecular marker CD3) . To the pellet 40 ⁇ of MACS buffer, 10 ⁇ of biotin-conj ugated antibodies were added for each 10 7 PBMCs and this was incubated at 4°C for 10'. These antibodies bind to all cells except those of interest. This avoids possible alteration/activation of the lymphocyte population of interest due to antibody binding to their surface.
  • the cells were incubated with anti-biotin microbeads, i.e., superparamagnetic nanoparticles conjugated with the secondary antibody that binds to biotin. Then 30 ⁇ of MACS buffer and 20 ⁇ of anti- biotin microbeads were added for every 10 7 cells and the whole was incubated at 4°C for 15'. After incubation, excess antibodies and microbeads were removed by resuspending in 10 ml of MACS buffer and centrifuging at 400g for 10' at RT . Then the supernatant was aspirated and the pellet resuspended with 500 ⁇ of MACS buffer.
  • anti-biotin microbeads i.e., superparamagnetic nanoparticles conjugated with the secondary antibody that binds to biotin.
  • 30 ⁇ of MACS buffer and 20 ⁇ of anti- biotin microbeads were added for every 10 7 cells and the whole was incubated at 4°C for
  • the sample was loaded onto a magnetic column and eluted with 3 additions of 3 ml MACS Buffer. Cells bound to the microbeads are retained (non-T fraction) , while T-lymphocytes pass through the column and can be collected.
  • the fraction obtained was centrifuged at 400g for lO'at RT and washed with 5 ml of PBS. Then the sample was centrifuged again at 400g for 10' at RT, the supernatant removed and total proteins extracted from the T-lymphocyte pellet.
  • each pellet was resuspended in 120 ⁇ of a lysis buffer consisting of UTC (7 M urea, 2 M thiourea, 4% CHAPS) and a mixture of protease inhibitors at the concentrations recommended by the manufacturer (Sigma-Aldrich, Steinheim, Germany) .
  • the pellets were then sonicated immersion (three 5 second bursts) and left 30' at RT, to facilitate extraction.
  • the samples were subjected to centrifugation at lOOOOg for 30' at 10°C, to remove any cellular debris, and the protein extract (supernatant) frozen with liquid nitrogen and stored at -80°C.
  • the absorbance of the solution at 595 nm is proportional to the concentration of protein, which can be estimated by means of a spectrophotometer reading at said wavelength.
  • a portion of protein extract (1 ⁇ ) was added to the suitably diluted Bradford Reagent and the absorbance read at a wavelength of 595 nm by using a Cary 50 Bio single beam UV-Vis spectrophotometer (Varian Inc., Palo Alto, USA).
  • the protein concentration was obtained by interpolation of the absorbance values with a calibration curve previously calculated using as the standard bovine serum albumin (Biorad, Hercules, CA, USA) at various known concentrations (between 1 pg/ml and 5 g/ml) .
  • Two-dimensional gel electrophoresis is an electrophoretic technique that allows separation of proteins according to isoelectric point and molecular mass. With this technique it is possible to distinguish hundreds of proteins at once, allowing subsequent quantitative analysis.
  • the separation of proteins by means of two different physical properties along the two orthogonal directions makes it possible to separate complex protein mixtures; this makes investigation of the cellular proteome possible.
  • the proteins are separated according to their isoelectric point using the isoelectric focusing technique (IEF) .
  • the net charge of a protein depends on the pH of the environment in which it is located; the electrophoretic motility of each protein depends on its amino acid composition and becomes zero when the pH is equal to its isoelectric point.
  • Samples for IEF containing 200 ⁇ q of protein extract in a final volume of 340 ⁇ in UTC were prepared by adding dithiothreitol (DTT) 20 mM, 1% IPG buffer and traces of bromophenol blue. This solution was placed in contact with an 18 cm IPG DryStrip non ⁇ linear gradient pH 3-10 (GE Healthcare, Uppsala, Sweden) and allowed to rehydrate overnight at RT. The next day IEF was conducted using a suitable apparatus, the Ettan IPGphor IITM (GE Healthcare, Uppsala, Sweden) .
  • DTT dithiothreitol
  • the voltage was varied according to the following protocol: 100 V for 8 hours; gradient from 100 V to 500 V in 2 hours; gradient from 500 V to 2000 V in 2 hours; 2000 V for 2 hours; gradient from 2000 V to 5000 V in 1 hour; 5000 V for 2 hours; gradient from 5000 V to 8000 V in 2 hours; 8000 V for 3 hours.
  • Each experiment envisioned the simultaneous focusing of a variable number of strips up to a maximum of 12; the current was limited to a maximum of 75 ⁇ per strip and the temperature maintained constant at 18°C. After focusing, the strip was stored at -20°C.
  • the second dimension consists of SDS-PAGE (Sodium Dodecylsulfate-Poly Acrylamide Gel Electrophoresis) through which the proteins are separated according to their molecular mass.
  • SDS-PAGE Sodium Dodecylsulfate-Poly Acrylamide Gel Electrophoresis
  • the strips were thawed and prepared for the second dimension by immersion in an equilibration solution comprising: 50 mM Tris-HCl pH 8.8, 6 M urea, 30% glycerol, 2% sodium dodecyl sulfate (SDS) and traces of bromophenol blue.
  • the gels with dimensions of 250 ⁇ 200 * 1 mm were prepared the day before use starting from a solution of: acrylamide/bis-acrylamide 14%, 0.1% SDS (Serva Electrophoresis, Heidelberg, Germany), Tris-HCl pH 8.8 375 mM, ammonium persulfate 0.1% (APS, Sigma-Aldrich, Steinheim, Germany) and 0.02% tetramethylethylenediamine (TEMED, Sigma-Aldrich, Steinheim, Germany) . To ensure complete polymerization, after pouring the solution between the glass panes, the gels were allowed to cure overnight .
  • the strips were rinsed quickly with deionised water, placed on top of the gel and immobilized in position by pouring a boiling solution of 0.6% IEF agarose (GE Healthcare, Uppsala, Sweden) in running buffer consisting of: 25 mM Tris, 192 mM glycine, 0.1% SDS.
  • the electrophoretic run is conducted in an appropriate chamber (Hoefer SE900; Hoefer, San Francisco, CA, USA) at a constant temperature of 16 °C maintained by means of a suitable cooling apparatus, in order to avoid distortion of the migration fronts.
  • a voltage of 25 V was applied to allow a slow entrance of the proteins into the gel, then the run was conducted overnight at 20 mA/gel.
  • Ru (II) tris (bathophenanthroline sulfonate ) RuBPS
  • RuBPS bathhophenanthroline sulfonate
  • the staining protocol was optimized to obtain the best signal/noise ratio.
  • the gels were immersed in a fixing solution consisting of 30% ethanol and 10% acetic acid in water for 24 hours. Subsequently they were immersed in a solution of ethanol 30%, 10% acetic acid and 1 ⁇ RuBPs where they remained for at least 6 hours (usually overnight) .
  • the binding of the molecule to the protein is non-covalent , occurring preferentially with basic residues.
  • the gels were decolorized with agitation for 2 hours in a solution similar to that of fixing, before being image acquisition .
  • Gel images were acquired with the GelDoc-It® Imaging System (UVP, Upland, CA, USA) , equipped with a 12-bit CCD sensor (Sony 1/1/8 "ICX274AL) , UV transilluminator (254 - 365 nm) and with optical filter at 590 nm.
  • 10 images were acquired in sequence ⁇ frames) with the acquisition parameters (focus, zoom, aperture, exposure time, gain) optimized and fixed, and 10 additional images were acquired using the same parameters with the empty chamber (to detect the electrical noise and correct it later) .
  • the first part of the image analysis was designed to optimize the elaboration of data and was conducted using the ImageJ software (NIH, version 1.44).
  • NASH ImageJ software
  • the mean intensity value was calculated for each pixel from the 10 images acquired by averaging the intensity values for each pixel; from the resulting image, the average of 10 images acquired with the chamber empty is subtracted. This reduces noise due to the production of free electrons in the CCD sensor (a phenomenon proportional to the operating temperature) , increasing the signal/noise ratio 3-fold.
  • the subtraction of the values recorded with empty chamber moreover, allows removal of problematic "hot" pixels due to defects in the manufacture of the sensor.
  • the image obtained was then processed to improve the subsequent detection of spots.
  • Artefacts from RuBPS precipitates were removed using a median filter that replaces the value of each pixel with the median of the values of neighbouring pixels (3 ⁇ 3).
  • a rolling ball algorithm with a radius of 75 pixels was applied.
  • a linear increase in contrast was applied, to utilize the full 16-bit dynamic range; this step does not increase the information given by the image, but favours the detection of spots by the software used in the next phase: ImageMaster 2D Platinum 6.0 (GE Healthcare, Uppsala, Sweden).
  • the resulting images were inverted and subjected to the automatic spot detection algorithm, and then refined manually. After detection, the operator applies landmarks on the image and the matching algorithm associates the corresponding spots on different gels.
  • the result of the analysis consists of a large number of spots that have associated volume values. The volumes are calculated as the sum of the pixel intensities that describe the spot considered.
  • the volumes of the spots that are present in at least 75% of all gels were exported and further analyzes were conducted using R, an open-source development environment specifically designed for statistical analysis (R Development Core Team (2009) . R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. ISBN 3- 900051-07-0; http: //www. r-project . org/) .
  • the volume of each spot was normalized to the sum of the volumes of a set of reference spots in each gel. This reference set comprises spots present in all images. The sum of these common spots was calculated for each patient and, assuming that most proteins do not have large variations among groups, the volume of each spot was expressed as a fraction of this value (thus correcting for possible differences in loading or staining) .
  • Missing values for each spot were replaced using either its mean value within the group or - if the group mean was less than the second percentile of the total distribution of all values - with the minimum value associated with that spot in the group. This double criterion was necessary to treat differently the values missing due to experimental variability and those missing due to reduced protein expression in a particular group (potential biomarkers) .
  • x is the independent variable (L-DOPA dose, age, age at onset or years from onset)
  • y represents the dependent variable (relative spo t volume)
  • x indicates the average value of x
  • y indicates the average value of y
  • xy indicates the product of x to y
  • xy indicates the product of the average values x and y .
  • spots that vary significantly in each comparison those that show significant linear correlation (r * 0 with p ⁇ 0.05) with disease duration were selected as possible markers of disease progression of PD.
  • LDA linear discriminant analysis
  • EOPD and LOPD groups were combined, thus obtaining a single set of coefficients c ⁇ from which a PD probability score (PD Score) is obtained by summing ( ⁇ ) the relative spot volumes Voli multiplied by the corresponding coefficients (Cj) determined through linear regression.
  • PD Score PD probability score
  • the weights (W) of the individual spots in the model were obtained by multiplying each coefficient Ci by the difference in absolute value between the mean volumes of the spots in the two groups (Volco-Vol p ⁇ , where Vol,CO is the average value of the relative volumes spot in the control group (CO) and Vol PD is the average of the relative volumes of the spot in the group of patients (PD) .
  • Preparative maps were made for cutting the spots.
  • the abundant spots were collected from a map obtained by loading 100 ⁇ iq of total protein from T lymphocytes, derived from various subjects used in the study ⁇ pool) .
  • the less abundant spots were collected from a map obtained by loading 500 g of total protein from T lymphocytes, derived from various subjects used in the study (pool) .
  • the preparative maps were stained with Colloidal Coomassie Blue G250 using the following protocol: fixing overnight (50% ethanol, 3% H 3 P0 4 ); three washes of 20 min with water; pre-staining for one hour (10% H3PO4, 10% (NH 4 ) 2 S0 4 , 20% methanol); staining overnight (0.12 % G250, 10% H 3 P0 4 , 10% (NH 4 ) 2 S0 4 , 20% methanol) .
  • the protein spots of interest were excised from the gel by cutting with a scalpel.
  • each spot was immersed in a solution of ethanol 50% for 2 hours; each spot was then immersed in 100 ⁇ of a solution of 200 mM (NH 4 )HC0 3 for 20' and washed in deionized water for 20' . The water was removed, and each spot dehydrated with acetonitrile for 40' (100 ⁇ /spot) . Finally, acetonitrile was eliminated by vacuum centrifugation in a SpeedVac .
  • spots were treated with 10 ⁇ of a solution of 50 mM NH 4 HC0 3 and trypsin 12.5 ng/ ⁇ (Modified Porcine Trypsin, sequencing grade, Promega, Madison, WI) . After 10' 30 ⁇ of 50 mM NH 4 HC03 were added and the reaction allowed to proceed overnight at 37°C.
  • the supernatants containing the hydrophilic peptides were collected; further extraction of more hydrophobic peptides was obtained in an ultrasonic bath with three 10-minute sonications (two times with 100 ⁇ /spot of a 1:1 solution of acetonitrile and 1% formic acid; a third time with 50 ⁇ acetonitrile/spot) .
  • the supernatants collected in the various steps were combined in one tube, dried by vacuum centrifugation and stored at -80°C.
  • the proteins of interest were identified by mass spectrometry.
  • the peptide mixtures were separated using a nanoflow HPLC-chip (Agilent 1200 series) .
  • a 5 ⁇ sample volume was loaded into the pre-column of the chip (Zorbax 300SB-C18, 5 ⁇ ) at a flow rate of 10 ⁇ /min.
  • Sequential elution of the peptides was carried out with a flow rate of 250 nl/min and a linear gradient of a solution A (composed of 2% acetonitrile, 0.1% formic acid) to 50% of a solution B (composed of 98% acetonitrile, 0.1% formic acid) in 40'.
  • the pre- column chip was interfaced with a separation column (Zorbax 300SB-C18 40 nl) .
  • Peptides were eluted directly into a mass spectrometer with a nano-Electrospray Ionization source (nanoESI) and ion trap analyzer (Esquire 6k, Bruker-Daltonics ) .
  • the parameters used for the nanoESI were voltage 1.5 to 2 kV, 10 1/min flow velocity of the drying gas and a temperature of 200°C. Only signals from peptides with a m/ z from 300 to 1800 were observed.
  • Protein identification was obtained by searching the NCBInr database (National Center for Biotechnology Information non-redundant) using the Mascot program (Perkins, DN, Pappin, DJC, Creasy, DM and Cottrell, JS (1999), Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis, 20: 3551-3567, HYPERLINK "http://www.matrixscience.com”).
  • the margin of error in the accuracy of peptide mass was set to ⁇ 0.9 Da for the first mass analysis and ⁇ 0.9 Da for the MS/MS analysis; the value for "missed cleavages" (i.e., the number of sites not digested by trypsin) has been set to 1 and the acetylation of the N-terminus, the partial oxidation of methionines and the carbamidometylation of cysteines have been set as variable modifications. Identifications with significant Mascot score (Fisher's exact test, p ⁇ 0.05) based on the size of the database interrogated were considered positive.
  • 2-DE maps revealed 2 spots related to the daily dose of L-DOPA (441 and 963) and 7 spots related to assumption of dopamine agonists (400, 608, 774, 779, 839, 893 and 921 ).
  • Figure 3 shows the location of these spots, the identification of which is reported in Table 3 (Identification of proteins that correlate with the daily dose of L-DOPA therapy and with the dopamine agonist (DAGO) in patients with PD)
  • Figure 4 shows the correlation of the levels of protein with the daily dose of L-DOPA (panel A) or with the intake of dopamine agonists (panel B) .
  • T lymphocytes which, as described in the introduction, have all the elements of the dopaminergic system and thus are subject to receptor- and transporter-mediated effects. Because drug treatment induces differences in the expression of these proteins, they are not considered peripheral markers of the disease .
  • Figure 6 shows the difference between the means of these spots in the group of PD patients compared to controls, expressed as log2 values, and the p-value of the Wilcoxon test. All spots were analyzed by linear discriminant analysis (LDA) and to each spot a coefficient C was attributed, as reported in Table 5 together with Vol co and Vol PD means, standard deviations
  • Figure 7 shows the classification of subjects based on these 20 spots, corresponding to a sensitivity of 87% and a specificity of 81%, the distribution of scores compared for the two groups (p ⁇ 0.001) and the ROC curve of model, with an area under the curve equal to 0.906.
  • a simplified model was obtained by eliminating the 6 spots with the smallest W value, thereby reducing the set to 14 (i.e., spots 86, 87, 335, 362, 365, 368, 369, 382, 405, 591, 598, 657, 676 and 1641).
  • Figure 8 shows the locations of these spots.
  • the leave-one-out validation of the model, reported in Figure 9, shows a sensitivity of 100% and specificity of 94%, the distribution of scores compared for the two groups with p ⁇ 0.001 and area under the ROC curve equal to 0.996.
  • Table 6 shows classification of the spots that discriminate patients with PD and controls in the 14- spot model. Table 6
  • a further simplification of the model was obtained by reducing the number of spots in the model to 9 spots locatable on the map in Figure 10 (i.e., 86, 87, 335, 362, 365, 368, 369, 382, 657), based on the value of W.
  • the leave-one-out validation of the model, reported in Figure 11, shows a sensitivity of 100% and specificity of 88%, the distribution of scores compared for the two groups with p ⁇ 0.001 and area under the ROC curve equal to 0.992.
  • Table 7 shows the classification of the spots that discriminate patients with PD and controls in the 9-spot model.
  • Figure 13 shows the difference between the means of these spots in the group of patients with LOPD compared to patients with EOPD, expressed as the log2 value, and the p-value of the Wilcoxon test.
  • Figure 14 shows the classification of the subjects obtained based on the 7 spots, corresponding to sensitivity of 71% and specificity of 100%, the distribution of scores compared for the two groups (p ⁇ 0.001) and the ROC curve of the model, with an area under the curve equal to 0.911.
  • Pearson correlation analysis revealed 11 spots among those previously identified (362, 365, 368, 369, 392, 405, 505, 591, 598, 657, 1641) that correlate linearly with years of disease and could therefore represent markers of progression to be followed over time. Table 10 shows the identity of these spots, the Pearson correlation coefficients (r) and the p-value of the correlation.

Abstract

L'invention concerne une méthode pour le diagnostic in vitro de la maladie de Parkinson chez un sujet, comprenant la détection de protéines différentiellement exprimées dans un échantillon biologique provenant dudit sujet, dans laquelle les protéines différentiellement exprimées appartiennent à un premier panel de protéines de diagnostic, dans laquelle ledit premier panel de diagnostic comprend vinculine, taline-1, bêta-fibrinogène, filamine A, alpha tubuline, gelsoline, et dans laquelle ledit échantillon biologique est un extrait protéique de lymphocytes T.
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JP2020504815A (ja) * 2016-12-14 2020-02-13 オーボ アカデミー ユニヴァーシティー 全体的な翻訳の減少に基づくパーキンソン病の診断
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WO2019211343A1 (fr) * 2018-05-01 2019-11-07 University Of Ulster Méthode de diagnostic ou de pronostic de trouble neurologique
CN112740044A (zh) * 2018-05-01 2021-04-30 阿尔斯特大学 神经病症的诊断或预后方法
JP2020064051A (ja) * 2018-08-29 2020-04-23 国立大学法人 岡山大学 神経変性疾患の診断用ペプチドマーカー
JP7457300B2 (ja) 2018-08-29 2024-03-28 国立大学法人 岡山大学 神経変性疾患の診断用ペプチドマーカー

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