Patent Description:
DLB is the second most common cause of dementia worldwide after AD and has several common pathological and clinical features with AD and PD. This overlap between these neurodegenerative disorders implies that just <NUM> of <NUM> DLB cases is correctly diagnosed.

Dementia is defined as the progressive cognitive decline of sufficient magnitude to interfere with normal social or occupational functions or with the usual daily activities. DLB belongs, together with PD, to the group of Lewy body disorders. Besides Lewy body pathology, DLB brains often contain concomitant AD pathology with β-amyloid and tau depositions, thus DLB present an important overlap with both, PD and AD. About <NUM>-<NUM>% of PD patients develop dementia after <NUM> years of PD diagnosis, being diagnosed as Parkinson's disease with dementia (PDD). Although many advances in the field have allowed improving their characterization, it is still a challenge to early and specifically diagnose DLB, AD and PD. In particular, still up to <NUM>% of DLB cases are missed or misdiagnosed, usually as AD; and the treatment of DLB patients with AD or PD specific therapies can adversely affect their cognition and disease course. For this reason, the identification of biomarkers and molecular features involved in the pathobiology of these disorders for its differential and specific diagnosis is urgently needed.

Moreover, trying to minimize the risk and discomfort of patients, the study of blood has been recently growing, being a promising source of circulating molecules and cell-based biomarkers. In this cell-based scenario, platelets are released into the circulation from the bone marrow after megakaryocytic differentiation essential in processes like homeostasis. Though platelets are anucleate cells, they contain a rough endoplasmic reticulum, ribosomes, a complete mitochondrial and apoptotic system, and display an enzymatic pathway similar to neurons. Thus, research in neurodegenerative diseases has also been conducted through investigation of biological changes and characteristics in platelets. In fact, platelets have been considered along the years a suitable peripheral tissue to study neurodegenerative diseases. The presence of microRNA (miRNA, miR-) in these anucleate cells, converts them also into a promising non-invasive source of miRNAs as biomarkers for several disorders.

<CIT> discloses the in vitro use of the increased expression level of miR-<NUM>-5p in blood for the diagnosis of synucleinopathies. <NPL> discloses miR-15a-5p in blood and cerebrospinal fluid for the differential diagnosis of patients with Alzheimer disease and dementia with Lewy bodies. miR-<NUM>-5p was also tested, but provided results that were not significant.

Precisely, the present invention is focused on solving the above cited problems by analyzing miRNAs as promising biomarkers for the diagnosis of neurodegenerative diseases and, more specifically, for the specific and/or differential diagnosis of synucleinopathies.

The present invention refers to an in vitro method for the specific diagnosis of synucleinopathies and/or for the differential diagnosis of synucleinopathies versus Alzheimer disease (AD). In a preferred embodiment, the synucleinopathy is DLB or PD.

In order to implement the invention, the miRNA content of platelets from DLB (n=<NUM>) and healthy controls (n=<NUM>) was analyzed using Next-Generation Sequencing (NGS). This analysis resulted in <NUM> differentially expressed miRNAs between both cohorts, which were validated by quantitative polymerase chain reaction (qPCR) on a different cohort of <NUM> DLB and <NUM> control samples. A second validation of <NUM> best differential miRNAs on independent cohorts, including <NUM> additional healthy control samples, <NUM> DLB patients and <NUM> AD patients was performed. The results showed that, in general, DLB patients have less miRNA expression than controls or AD patients. The <NUM> validated miRNAs appeared down-regulated in DLB samples, specifically hsa-miR-<NUM>-5p. Last, a blind validation using PD samples showed similar expression levels for hsa-miR-<NUM>-5p in DLB and PD distinguishing both synucleinopathies from AD and healthy controls. Target gene prediction for hsa-miR-<NUM>-5p revealed MYB, EGR2, MUC4 and NOTCH3 among the highest prediction scored. So, the present invention shows hsa-miR-<NUM>-5p as a biomarker for the specific diagnosis of synucleinopathies (particularly DLB or PD) versus controls, and/or for the differential diagnosis of synucleinopathies (particularly DLB or PD) versus AD, with an area under the curve of <NUM> and <NUM>, respectively (see <FIG> and <FIG>).

Although hsa-miR-<NUM>-5p, as cited above, is cited in the present invention as a preferred candidate, the present invention offers scientific support (see <FIG>, <FIG>, <FIG> and <FIG>), for the use of any of the following miRNAs: miR-7d-5p, miR-<NUM>-3p, miR-26b-5p, miR-<NUM>-5p, miR-146a-5p, miR-<NUM>-3p, miR-<NUM>-3p, miR-<NUM>-5p, miR-<NUM>-3p, hsa-miR-<NUM>-5p or hsa-miR-26a-5p as individual biomarkers for the specific diagnosis of synucleinopathies (particularly DLB or PD) versus controls, and/or for the differential diagnosis of synucleinopathies (particularly DLB or PD) versus AD. Moreover, the present invention also refers to the use of any combination of the above cited <NUM> biomarkers for the specific diagnosis of synucleinopathies (particularly DLB or PD) versus controls, and/or for the differential diagnosis of synucleinopathies (particularly DLB or PD) versus AD. In a preferred embodiment said combination of miRNAs comprises at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM> or at least <NUM> of the following miRNAs: miR-<NUM>-5p, miR-7d-5p, miR-<NUM>-3p, miR-26b-5p, miR-<NUM>-5p, miR-146a-5p, miR-<NUM>-3p, miR-<NUM>-3p, miR-<NUM>-5p, miR-<NUM>-3p, hsa-miR-<NUM>-5p or hsa-miR-26a-5p.

Thus hsa-miR-<NUM>-5p or, alternatively, any of the following miRNAs: miR-7d-5p, miR-<NUM>-3p, miR-26b-5p, miR-<NUM>-5p, miR-146a-5p, miR-<NUM>-3p, miR-<NUM>-3p, miR-<NUM>-5p, miR-<NUM>-3p, hsa-miR-<NUM>-5p and hsa-miR-26a-5p, or any combination thereof, are identified in the present invention as consistently under-expressed in DLB patients compared to both, AD and controls. Additionally, ROC curves reinforced that hsa-miR-<NUM>-5p could be a promising biomarker for DLB rendering <NUM>% specificity and <NUM>% sensibility to differentiate DLB from AD, and around <NUM>% when distinguishing DLB from healthy controls. When samples from PD patients were included, these also showed decreased hsa-miR-<NUM>-5p expression that was overlapping with DLB. Hence, it is an indication that this platelet-miRNA has a potential relation to the development of synucleinopathies including both DLB and PD.

Consequently, the present invention provides evidence for the use of miRNA content, preferably from platelets, as promising biomarker source, and proposes the use of any of the miRNAs: hsa-miR-<NUM>-5p, miR-7d-5p, miR-<NUM>-3p, miR-26b-5p, miR-<NUM>-5p, miR-146a-5p, miR-<NUM>-3p, miR-<NUM>-3p, miR-<NUM>-5p, miR-<NUM>-3p, hsa-miR-<NUM>-5p or hsa-miR-26a-5p, or any combination thereof, as biomarkers showing a high sensitivity and sensibility, for the differentiation between synucleinopathies, including DLB, and AD. The detection of these miRNAs in platelets or whole blood represents a non-invasive, quick and easy procedure for clinical implementation.

Thus, the first embodiment of the present invention refers to an in vitro method (hereinafter the method of the invention) for the specific diagnosis or prognosis of synucleinopathies, which comprises determining the expression level of at least one of the following miRNAs: hsa-miR-<NUM>-5p, miR-7d-5p, miR-<NUM>-3p, miR-26b-5p, miR-<NUM>-5p, miR-146a-5p, miR-<NUM>-3p, miR-<NUM>-3p, miR-<NUM>-5p, miR-<NUM>-3p, hsa-miR-<NUM>-5p or hsa-miR-26a-5p, or any combination thereof, isolated from platelets obtained from the patient, wherein a reduced expression level of any of the above cited miRNAs, as compared with the expression level of any of the above cited miRNAs in healthy control subjects, is an indication that the patient is suffering from a synucleinopathy.

The second embodiment of the present invention refers to an in vitro method (hereinafter the method of the invention) for the differential diagnosis of synucleinopathies versus AD, which comprises determining the expression level of at least one of the following miRNAs: hsa-miR-<NUM>-5p, miR-7d-5p, miR-<NUM>-3p, miR-26b-5p, miR-<NUM>-5p, miR-146a-5p, miR-<NUM>-3p, miR-<NUM>-3p, miR-<NUM>-5p, miR-<NUM>-3p, hsa-miR-<NUM>-5p or hsa-miR-26a-5p, or any combination thereof, isolated from platelets obtained from the patient, wherein a reduced expression level of any of the above cited miRNAs, as compared with the expression level of any of the above cited miRNAs in control patients suffering from AD, is an indication that the subject is suffering from a synucleinopathy and is not suffering from AD.

In a preferred embodiment the present invention refers to an in vitro method for the specific diagnosis or prognosis of DLB and/or PD, which comprises determining the expression level of at least one of the following miRNAs: hsa-miR-<NUM>-5p, miR-7d-5p, miR-<NUM>-3p, miR-26b-5p, miR-<NUM>-5p, miR-146a-5p, miR-<NUM>-3p, miR-<NUM>-3p, miR-<NUM>-5p, miR-<NUM>-3p, hsa-miR-<NUM>-5p or hsa-miR-26a-5p, or any combination thereof, isolated from platelets obtained from the patient, wherein a reduced expression level of any of the above cited miRNAs, as compared with the expression level of any of the above cited miRNAs measured in healthy control subjects, is an indication that the patient is suffering from DLB and/or PD.

In a preferred embodiment the present invention refers to an in vitro method for the differential diagnosis of DLB and/or PD versus AD, which comprises determining the expression level of at least one of the following miRNAs: hsa-miR-<NUM>-5p, miR-7d-5p, miR-<NUM>-3p, miR-26b-5p, miR-<NUM>-5p, miR-146a-5p, miR-<NUM>-3p, miR-<NUM>-3p, miR-<NUM>-5p, miR-<NUM>-3p, hsa-miR-<NUM>-5p or hsa-miR-26a-5p, or any combination thereof, isolated from platelets obtained from the patient, wherein a reduced expression level of any of the above cited miRNAs, as compared with the expression level of any of the above cited miRNAs measured in control patients suffering from AD, is an indication that the subject is suffering from DLB and/or PD and is not suffering from AD.

The third embodiment of the present invention refers to the in vitro use of the expression level of at least one of the following miRNAs: hsa-miR-<NUM>-5p, miR-7d-5p, miR-<NUM>-3p, miR-26b-5p, miR-<NUM>-5p, miR-146a-5p, miR-<NUM>-3p, miR-<NUM>-3p, miR-<NUM>-5p, miR-<NUM>-3p, hsa-miR-<NUM>-5p or hsa-miR-26a-5p, or any combination thereof, isolated from platelets obtained from the patient, for the specific diagnosis or prognosis of synucleinopathies, or the differential diagnosis of synucleinopathies versus Alzheimer disease.

The fourth embodiment of the present invention refers to the in vitro use of a kit comprising reagents for the determination of the expression level of at least one of the following miRNAs: hsa-miR-<NUM>-5p, miR-7d-5p, miR-<NUM>-3p, miR-26b-5p, miR-<NUM>-5p, miR-146a-5p, miR-<NUM>-3p, miR-<NUM>-3p, miR-<NUM>-5p, miR-<NUM>-3p, hsa-miR-<NUM>-5p or hsa-miR-26a-5p, or any combination thereof, for the specific diagnosis or prognosis of synucleinopathies, or the differential diagnosis of synucleinopathies versus Alzheimer disease.

In a preferred embodiment the present invention refers to the in vitro use of the expression level of at least one of the following miRNAs: hsa-miR-<NUM>-5p, miR-7d-5p, miR-<NUM>-3p, miR-26b-5p, miR-<NUM>-5p, miR-146a-5p, miR-<NUM>-3p, miR-<NUM>-3p, miR-<NUM>-5p, miR-<NUM>-3p, hsa-miR-<NUM>-5p or hsa-miR-26a-5p, or any combination thereof, for the specific diagnosis or prognosis of DLB and/or PD, or the differential diagnosis of DLB and/or PD versus AD.

In a preferred embodiment the present invention refers to the in vitro use of a kit comprising reagents for the determination of the expression level of at least one of the following miRNAs: hsa-miR-<NUM>-5p, miR-7d-5p, miR-<NUM>-3p, miR-26b-5p, miR-<NUM>-5p, miR-146a-5p, miR-<NUM>-3p, miR-<NUM>-3p, miR-<NUM>-5p, miR-<NUM>-3p, hsa-miR-<NUM>-5p or hsa-miR-26a-5p, or any combination thereof, for the specific diagnosis or prognosis of DLB and/or PD, or the differential diagnosis of DLB and/or PD versus AD.

In a preferred embodiment the invention refers to the determination of the expression level of at least one of at least hsa-miR-<NUM>-5p, preferably in combination with any of the miRNAs comprised in the group: miR-7d-5p, miR-<NUM>-3p, miR-26b-5p, miR-<NUM>-5p, miR-146a-5p, miR-<NUM>-3p, miR-<NUM>-3p, miR-<NUM>-5p, miR-<NUM>-3p, hsa-miR-<NUM>-5p or hsa-miR-26a-5p, for any of the above cited purposes.

In preferred embodiment, the miRNAs are derived from whole blood or platelets.

In a preferred embodiment, the results obtained with the above cited method of the invention are clinically confirmed by other techniques such as: the determination of phosphorylated tau/amyloid beta <NUM>-<NUM> ratio in cerebrospinal fluid and amyloid PET. On the other hand, once the differential diagnosis is carried out with the method of the invention, the fact that the patient is suffering from DLB or PD is clinically confirmed by using techniques such as DatScan.

According to the fifth embodiment of the present invention, the patients diagnosed with the method of the invention can be treated with any of the treatments currently used in the general practice for treating or to provide relief to those patients suffering from synucleinopathies, preferably DLB and/or PD. For example, levodopa (precursor of dopamine), sometimes combined with a dopa decarboxylase inhibitor and sometimes also with a COMT (enzyme which degrades dopamine) inhibitor can be used for treating synucleinopathies, particularly PD. Other examples of treatment that could be used for treating synucleinopathies are dopamine agonists or MAO-B (an enzyme which breaks down dopamine) inhibitors (safinamide, selegiline and rasagiline). Importantly, the use of erroneous treatments arising from misdiagnoses of synucleinopathies, especially DLB, such as neuroleptics for antipsychotic treatment usually used in AD patients can be avoided by the use of the method of the invention. On the other hand, the present invention can be used to identify individuals (e.g. iRBD patients, patients with hyposmia, LRRK2 mutation carriers) who are at elevated risk for developing a synucleinopathy as candidates to receive neuromodulation therapies. Since the development of disease specific neuromodulation treatments is on-going in numerous laboratories, patients identified as classified to undergo this kind of therapies are directly eligible at the moment these are available. Aggregation of the protein α-synuclein is a key event in the development synucleinopathies, such as DLB and/or PD. So, any compound (including antibodies, vaccines, etc.) which is able to prevent α-synuclein aggregation could be potentially used for treating synucleinopathies, such as DLB and/or PD. Consequently, in preferred embodiment of the present invention, patients suffering from synucleinopathies, preferably DLB and/or PD, who have been diagnosed with any of the methods of the invention, can be treated with alpha-synuclein antiaggregation compounds, for example (non-exhaustive list): BIIB054, NPT200-<NUM>/ UCB0599, PRX002/RO7046015 or NPT088. So, in a preferred embodiment, the present invention refers to a method for treating patients suffering from synucleinopathies, preferably DLB and/or PD, which comprises the administration of therapeutically effective doses of alpha-synuclein antiaggregation compounds, once they have been diagnosed using any of the methods of the invention, preferably a method which comprises determining the expression level of at least miR-<NUM>-5p isolated from platelets obtained from the patient, wherein a reduced expression level of at least the miR-<NUM>-5p, as compared with the expression level of at least the miR-<NUM>-5p measured in healthy control subjects, is an indication that the patient is suffering from a synucleinopathy. Moreover, the present invention refers to alpha-synuclein antiaggregation therapies for use in the treatment of synucleinopathies, preferably DLB and/or PD, once the patient has been diagnosed by using any of the methods of the invention, preferably a method which comprises determining the expression level of at least miR-<NUM>-5p isolated from platelets obtained from the patient, wherein a reduced expression level of at least the miR-<NUM>-5p, as compared with the expression level of at least the miR-<NUM>-5p measured in healthy control subjects, is an indication that the patient is suffering from a synucleinopathy.

Moreover, the present invention refers to a method for evaluating whether the therapy based on alpha-synuclein antiaggregation compounds is effective, or to a method for assessing whether a patient suffering from synucleinopathies, preferably DLB and/or PD, is responding to a treatment with alpha-synuclein antiaggregation compounds which comprises determining an overexpression of the miRNAs assayed in the present inventions, particularly the miR-<NUM>-5p, as compared to the control. In preferred embodiment, the above cited method of the invention can be performed by determining the expression level of at least one of the miRNAs described above, in combination with any of the biomarkers described in the patent application <CIT>.

Specifically, the method of the invention can be performed by determining the expression level of at least one of the miRNAs described above, in combination with the determination of the genotype of the following alterations in butyrylcholinesterase (BChE) gene: the polymorphic site at position <NUM> in NCBI Accession Number NG_009031 and/or the poly-thymine region at positions <NUM> to <NUM> in NCBI Accession Number NG_00903. The method may further comprise the determination of the genotype of the following alterations in BChE gene: the polymorphic site at position <NUM>, the polymorphic site at position <NUM>, and the polymorphic site at position <NUM>, said positions with reference to NCBI Accession Number NG_009031 (i.e. positions <NUM>, <NUM> and <NUM> respectively in SEQ ID NO: <NUM>). Please note that the above cited sequences are disclosed in the patent application <CIT>.

In a preferred embodiment, the above cited method of the invention can be performed by determining the expression level of at least one of the miRNAs described above, in combination with any of the biomarkers described in the patent application <CIT>.

Specifically, the method of the invention can be performed by determining the expression level of at least one of the miRNAs described above, in combination with the detection of at least one variation in SEQ ID NO: <NUM>, or alternatively at least one variation in SEQ ID NO: <NUM>, or alternatively at least one variation in SEQ ID NO: <NUM>, wherein SEQ ID NO: <NUM>, SEQ ID NO: <NUM> and SEQ ID NO: <NUM> are comprised within position <NUM>. <NUM> to position <NUM>. <NUM> in Homo sapiens chromosome <NUM> according to HapMap data release <NUM> (SEQ ID NO: <NUM> ). Please note that the above cited sequences are disclosed in the patent application <CIT>.

Specifically, the method of the invention can be performed by determining the expression level of at least one of the miRNAs described above, in combination with the determination of the amount of transcripts SNCAtv3 (SEQ ID NO: <NUM>) and SNCAtv2 (SEQ ID NO: <NUM>) of the human alpha-synuclein gene (SNCA) isolated from platelets obtained from the patient, wherein when the amount of both transcripts determined for the patient is reduced with respect to a reference value, this is indicative of the presence of DLB in the patient. Please note that the above cited sequences are disclosed in the patent application <CIT>.

On the other hand the present invention also refers to an in vitro method for the diagnosis of AD, which comprises determining the expression level of at least one miRNA included in Table <NUM>, or any combination thereof, isolated from platelets obtained from the patient, wherein an increased expression level of at least one of the miRNAs included in Table <NUM>, as compared with the expression level measured in healthy control subjects, is an indication that the patient is suffering from AD. Thus the present invention also refers to the use of any of the miRNA included in Table <NUM> for the in vitro diagnosis of AD.

Moreover, the present invention also refers to an in vitro method for the differential diagnosis of DLB from PD, which comprises determining the expression level of at least one miRNA included in Table <NUM>, or any combination thereof, isolated from platelets obtained from the patient, wherein a decreased expression level of at least one of the miRNAs included in Table <NUM>, as compared with the expression level measured in PD patients, is an indication that the patient is suffering from DLB and not from PD. Thus the present invention also refers to the use of any of the miRNA included in Table <NUM> for the in vitro differential diagnosis of DLB from PD.

For the purpose of the present invention the following terms are defined:.

Thirty-three DLB patients (age range <NUM>-<NUM> years; mean <NUM> years; male:female ratio <NUM>:<NUM>) from the Universitary Bellvitge Hospital, (L'Hospitalet de Llobregat, Barcelona), and <NUM> age- and gender-matched healthy control individuals (age-range <NUM>-<NUM>; mean <NUM> years; male:female <NUM>:<NUM>) from the same hospital and the University Hospital Germans Trias i Pujol (Badalona, Barcelona), were included in this study. DLB patients were diagnosed according to the <NUM> DLB Consortium criteria [<NPL>] and age at onset was defined as the age when memory loss or parkinsonism was first noticed by relatives. A third cohort of <NUM> AD patients (age range <NUM>-<NUM>; mean <NUM>; male:female ratio <NUM>) with a Global Deterioration Scale of <NUM>±<NUM> degrees, was also recruited. AD diagnosis was assessed in the University Hospital Germans Trias i Pujol (Badalona, Barcelona) following the <NUM>-revised criteria from the National Institute on Aging and the Alzheimer's Association [<NPL>]. Thirteen non-demented PD patients (age range <NUM>-<NUM> years; mean <NUM> years; male:female ratio <NUM>:<NUM>) were also recruited in the same hospital for the final validation assay. PD diagnosis was assessed by the UK PD Society Brain Bank criteria [<NPL>]. The following protocol was approved by the Clinical Research Ethics Committee of our institution and conducted according to the Declaration of Helsinki Principles [<NPL>]. Written informed consent was obtained from each subject.

Peripheral blood was collected following standard procedures to minimize coagulation and platelet activation [<NPL>; <NPL>; György B, Pálóczi K, Kovács A, Barabás E, Bekő G, Várnai K, et al. Improved circulating microparticle analysis in acid-citrate dextrose (ACD) anticoagulant tube, Thromb Res <NUM>; <NUM>:<NUM>-<NUM>]. Briefly, after venous puncture, <NUM>-<NUM> of blood were collected in sodium citrate pre-treated tubes (BD Vacutainer®, New Jersey, USA), and processed within the <NUM> hours following the collection. After centrifugation at <NUM> x g for <NUM> minutes at room temperature to minimize contamination by red blood cells and leukocytes, centrifugation at <NUM>,<NUM> x g for <NUM> minutes at room temperature [<NPL>)] was performed in order to obtain a platelet-enriched pellet. The pellet was res-suspended in <NUM>µL of PBS and characterized by flow cytometry. <NUM>µL of the samples were incubated for <NUM> at room temperature with <NUM>µL of CD61-FITC antibody, as platelets marker, and <NUM>µL CD45-APC antibodies in order to detect possibly leukocyte contamination. The samples were then frozen and kept at -<NUM> until processing and miRNA isolation.

Platelet-enriched pellet obtained post centrifugation at <NUM>,<NUM> x g for <NUM> minutes were thawed slowly on ice previous miRNA isolation. miRNA extraction was performed using mirVana Paris Kit (Invitrogen) at room temperature as described by the manufacturers. Briefly, <NUM>µL of lysis buffer and <NUM>/<NUM> of miRNA Homogenate Additive Mix were added to each pellet and incubated after vortexing for <NUM> minutes on ice. After adding one volume of phenol-chloroform and mixing, centrifugation at <NUM>,<NUM> x g for <NUM> minutes was performed. One third and <NUM>/<NUM> volume of ethanol was added in <NUM> consecutive steps to the miRNA containing aqueous phase, and passed through a filter column. After corresponding washing steps, miRNAs were eluted with <NUM>µL of elution buffer. The extracted material was kept on ice and frozen at -<NUM> until forthcoming analysis.

RNA isolation was carried out after collection of <NUM> of whole blood in PAXgene Blood RNA tubes (PreAnalytiX, Hombrechtikon, Switzerland) by the use of the PAXgene Blood miRNA Kit <NUM>, v2 (PreAnalytiX) following manufacturer's instructions. RNA quantity, purity and integrity were ascertained by the Agilent <NUM> Bioanalyzer (Agilent Technologies, Santa Clara, USA).

The total volume of the obtained miRNAs from <NUM> DLB and <NUM> control samples was precipitated overnight at -<NUM>° C with <NUM>µL of glycogen (<NUM>µg/ µL), <NUM>% <NUM> AcNa (ph <NUM>) and <NUM> volumes of ethanol. miRNAs were resuspended in <NUM>µL RNase free H<NUM>O and heated at <NUM>° C for <NUM>-<NUM>. Quality control and size distribution of the purified small RNA was assessed by Bioanalyzer <NUM> (Agilent Technologies, Santa Clara, USA).

Six µL of each sample (n=<NUM> DLB samples; n=<NUM> control samples) were used for library preparation with NEBNext Multiplex Small RNA Sample Preparation Set for Illumina (New England Biolabs) following manufacturer's instructions. Individual libraries were subjected to the quality analysis using a D1000 ScreenTape (TapeStation, Agilent Technologies), quantified by fluorimetry and pooled. Clustering and 'sequencing were performed on an Illumina Sequencer (MiSeq, Illumina, San Diego, USA) at <NUM> x 50c single read mode and <NUM>,<NUM> reads were obtained for each sample.

FastQ raw data obtained from the Illumina Platform were analyzed as follow. Firstly, adapter sequences from the obtained reads were removed using Trimmomatic [<NPL>] and reads were mapped to miRNA sequences using the Bowtie2 algorithm [<NPL>]. For each sample, the number of reads matched with a particular miRNA sequence was counted and the final count matrix was normalized through the weighted trimmed means of M-values (TMM) [<NPL>]. For possible biomarker selection the following criteria had to be fulfilled: (a) Minimum of <NUM> reads per sample; (b) Present in all patient samples and absent (less than <NUM> reads) in more than the half of the control samples; (c) Present in all control samples and absent in more than the half of the patient samples. In all the cases, and when a miRNAs was present in both cohorts, differential expression analyses were carried through applying the Lilliefors' composite goodness-of-fit test, Jarque-Bera hypothesis test and Shapiro-Wilk test to test if the samples fitted normal distributions; and the Wilconxon-rank sum test (p-value <<NUM>) [<NPL>] was used to determine whether miRNAs were differentially expressed between both cohorts. Validation process of the differences obtained was analyzed by the methodology Leave-One-Out (LOO) cross-validation.

miRNA was reverse-transcribed using MiRCURY LNA™ Universal cDNA synthesis Kit II (Exiqon, Vedbaek, Denmark) according to the manufacturer's protocol. RNA concentration was adjusted to <NUM> ng/µL with nuclease free water and mixed with the reaction buffer and enzyme mix according to the working volume specified in the instruction manual. Retro-transcription reaction took place at <NUM> for <NUM> minutes and enzymatic activity was stopped at <NUM> for <NUM>. cDNA mixture was diluted <NUM>:<NUM> and <NUM>µL were used for the quantitative PCR (qPCR) reactions with ExiLENT SYBR Green Master Mix (Exiqon, Vedbaek, Denmark) following manufacturer's indications on a LightCycler <NUM> (Roche, Basel, Switzerland). Samples were set up in duplicates, miRNA spike-in UniSp6 was used as control for the retrotransctiption. miRNA LNA technology Pick&Mix PCR pre-designed panels (Exiqon) with miRNA UniSp3 as interplate calibrator control were used.

Values for NGS data and reads are given as mean ± SD. Expression levels of the miRNAs selected for qPCR validation were determined using crossing point (Cp) values. Cp values were averaged between duplicates and normalized against UniSp6 spike-in Cp values for platelet derived miRNA and against hsa-miR-<NUM>-5p in the case of whole blood. Relative expression in DLB, AD and PD was estimated respect to healthy controls and represented as fold expression changes as obtained by <NUM>-ΔΔCp. Statistical analyses were performed using GraphPadPrism <NUM> (GraphPad Software, Inc. , La Jolla, CA, USA). Two-tailed unpaired T-test was applied individually for each miRNA analyzed to compare Cp values between control and DLB groups. When comparing more than two groups (DLB, controls, AD and PD), multiple comparisons were performed applying Kruskal-Wallis non-parametric test. In all cases, confidence interval of <NUM>% and a p-value below <NUM> was considered to be significant. To assess the diagnostic potential, the area under the ROC curve (AUC) was calculated for each miRNA using SPSS Statistics <NUM> (IBM, Armonk, NY, USA) and GraphPadPrism <NUM> in order to determine the diagnostic and characterization sensitivity and specificity (<NUM>% C. , AUC > <NUM> was considered as minimum value for a miRNA to be defined as good biomarker).

Biomarker candidates obtained from NGS were sought in several databases and relation with dementia with Lewy bodies and others neurodegenerative disorders (PD, AD, mild cognitive impairment, vascular dementia, multiple sclerosis, amyotrophic lateral sclerosis, Huntington's disease and progressive supranuclear palsy) was assessed through a manual bibliographic search in PubMed, The Nervous System Disease NcRNAome Atlas (NSDNA) [<NPL>], miR2Disease database [<NPL>] and the Human microRNA Disease Database (HMDD) [<NPL>].

Hsa-miR-<NUM>-5p possible targets were predicted using MirWalk <NUM> [<NPL>], miRGate [<NPL>] and miRTarBase [<NPL>] databases. Relation among the confirmed predicted targets was analyzed with String DataBase [<NPL>] and GO consortium online tool [<NPL>; <NPL>] obtaining related biological processes, cellular components and KEEG Pathways. Gene description and main information were screened through Uniprot database [<NPL>]. As well, DAVID database [<NPL>] was used to obtain a clustered network based on involved KEEG pathways and related diseases with a Fisher's exact p-value assigned to each miRNA-process relation (the cut off EASE -Expression Analysis Systematic Explorer- was set as default at <NUM>).

The whole workflow of this study is shown in <FIG>.

Characterization of the platelet-enriched pellet obtained after serial centrifugations for possibly leukocyte contamination did not show almost staining for the leukocyte marker CD45 in our samples. Instead, a high fluorescent signal for the platelet marker CD61 was obtained (<FIG>).

After small RNA extraction, bioanalyzer analysis showed an enriched profile of <NUM>-<NUM> nucleotides molecules characteristic of small RNA and miRNA which were used to construct libraries by NGS. NGS generated a mean total of <NUM>,<NUM>,<NUM> raw reads sized around <NUM>-<NUM> nucleotides (taking into account the ligated adapters). After raw data processing and adapter removal, the mapping by Bowtie algorithm reported an average of <NUM>,<NUM>,<NUM> ± <NUM>,<NUM> reads per sample in the control group, and <NUM>,<NUM>,<NUM> ± <NUM>,<NUM> reads per DLB sample that mapped to already known <NUM>,<NUM> different mature miRNA molecules. From those, <NUM> miRNAs met the previously established criteria for the minimum number of reads. Taking into account the precursor immature forms of our data set (no -5p or -3p forms consideration), <NUM> different precursors-miRNAs was compared with the available literature in platelet-miRNA content. From those, <NUM> had already been described as associated to platelets. Of all mature miRNAs identified, <NUM>% had been already described in the first platelet-miRNA profiling studies [<NPL>]; [<NPL>]. It was also found that the most representative miRNA families described in platelets, such as let-<NUM> family or miR-<NUM>, miR-<NUM> groups [<NPL>], were also the most representative in our analysis.

The normalized counts from NGS data were analyzed using the Wilconxon-rank sum test (threshold p-value set at <NUM>) and a total of <NUM> miRNAs showing good classifier capability and differentially expressed between DLB and healthy control cohorts, were selected for further validation by qPCR (hsa-miR-<NUM>-3p, hsa-miR-<NUM>-3p, hsa-miR-<NUM>-3p, hsa-miR-<NUM>-5p, hsa-miR-<NUM>-3p, hsa-miR-<NUM>-3p, hsa-miR-<NUM>-5p, hsa-miR-<NUM>-5p, hsa-let-7d-5p, hsa-let-7d-3p, hsa-miR-<NUM>-3p, hsa-miR-<NUM>-5p, hsa-miR-<NUM>-5p, hsa-miR-23a-5p, hsa-miR-26b-5p, hsa-miR-<NUM>-3p, hsa-miR-<NUM>-3p, hsa-miR-146a-5p, hsa-miR-<NUM>-3p, hsa-miR-<NUM>-3p, hsa-miR-<NUM>-5p, hsa-miR-<NUM>-5p).

The selected <NUM> differentially expressed miRNAs were validated by qPCR in independent DLB and control cohorts, each constituted of <NUM> individuals. The majority of the validated miRNAs were down-regulated in the DLB group compared to controls as shown in <FIG> for three of the most representative. Among those, the most important decrease was found for hsa-miR-<NUM>-5p in DLB vs controls (<NUM> ± <NUM> vs. <NUM> ± <NUM>; p<<NUM>). Then, the <NUM> miRNAs more differentially expressed between both cohorts (hsa-miR-<NUM>-5p, hsa-miR-7d-5p, hsa-miR-<NUM>-3p, hsa-miR-26b-5p, hsa-miR-<NUM>-5p, hsa-miR-146a-5p, hsa-miR-<NUM>-3p, hsa-miR-<NUM>-3p, hsa-miR-<NUM>-5p, miR-<NUM>-3p, hsa-miR-<NUM>-5p or hsa-miR-26a-5p) underwent an additional qPCR-validation in independent cohorts of <NUM> DLB and <NUM> controls from a different hospital, and where a group of <NUM> AD patients was also included. Expression levels for all miRNAs tested in AD were similar to control samples and greatly differed from DLB. The previous results for hsa-miR-<NUM>-5p expression levels were confirmed as it was repeatedly significantly decreased in DLB compared to controls, but also compared to AD samples (p<<NUM> and p=<NUM>, respectively). The ensemble of both qPCR-analyses is represented in <FIG>. ROC curves taking into account fold changes for each cohort were calculated. A ROC curve with AUC= <NUM> was obtained for the specificity and sensibility distinguishing between controls (n=<NUM>) and DLB samples (n=<NUM>) (<FIG>). When AD (n=<NUM>) and DLB (n=<NUM>) were considered, AUC=<NUM> ROC curve was obtained (<FIG>). No significant sensibility and specificity was reached for the determination of AD compared to healthy controls (<FIG>).

Then, we analyzed the expression of hsa-miR-<NUM>-5p in a blind qPCR where <NUM> controls (<NUM> new, not previously analyzed), <NUM> DLB (<NUM> independent new samples), <NUM> non-demented PD and <NUM> of the AD patients were included. Results were analyzed by two independent researchers who did not know the identity of the samples and were grouped into low- and high- hsa-miR-<NUM>-5p expressing. After sample identification, DLB and PD were localized within the low- hsa-miR-<NUM>-5p expressing region and AD within the high- hsa-miR-<NUM>-5p expressing region. Hsa-miR-<NUM>-5p expression in controls was overlapping with both, AD and DLB/PD (<FIG>). Expression levels differed significantly between DLB and AD (p=<NUM>), and also between PD and AD (p=<NUM>) (<FIG>). When DLB and PD were grouped as synucleinopathies, significant differences were observed compared to the control and AD groups (p=<NUM> and p=<NUM>, respectively) (<FIG>). ROC curves displayed a high sensitivity and specificity for the differentiation between synucleinopathies and controls (AUC=<NUM>) and AD (AUC=<NUM>) (<FIG>).

Based upon these results, hsa-miR-<NUM>-5p expression in platelet-enriched pellets could be considered a plausible biomarker for the differential diagnosis of DLB vs AD with an easy application in clinical settings. In PCR-based analysis, Cp or crossing point, which inversely correlates with gene expression, corresponds to the number of cycles needed for the amplification-associated fluorescence to reach a specific threshold. Hence, considering qPCR-Cp values after reaction in a LightCycler480 (Initial heat activation at <NUM>° C / <NUM>; <NUM> cycles: <NUM> at <NUM>, <NUM> at <NUM>; and melting curve analysis from <NUM>-<NUM>° C), it is possible to assess a Cp-based stratification panel for samples' classification as exposed in Table <NUM>.

MiRGate online software was used for screening of specific predicted targets of hsa-miR-<NUM>-5p. Possible affected pathways were identified using DAVID software. The prediction revealed <NUM>,<NUM> target genes, but only <NUM> of them (MYB, P2RX7, EGR2, MUC4, ZEB1, ATP13A3, EP300, TP53, NOTCH3) were confirmed by independent tools (miRTarbase, Mirecords and OncomiRBD). These <NUM> target genes together with the most computational predicted ones (at least <NUM> different software identified them) were submitted to String online tool (https://string-db. org/) defining a small cluster involving <NUM> of our proteins and related to transcription factor activity and binding (p=<NUM>). Also, <NUM> of these proteins were associated to generation of neurons and regulation of neurogenesis by GO analysis for biological process (p=<NUM> in both cases). Reactome analysis defined these proteins as related to TP53-negative regulation of cell cycle (p=<NUM>·<NUM>-<NUM>), pre-Notch transcription and translation (p=<NUM>·<NUM>-<NUM>) and to factors involved in the development and production of platelets (p=<NUM>·<NUM>-<NUM>) (<FIG>). Disease relation screening recognized an association of these genes to neuropathy (p=<NUM>) with high involvement in protein sumoylation processes and ubiquitin-protein ligase binding (maximum similarity score obtained - <NUM> - by DAVID online tool analysis). Bibliographic search in several data bases also revealed other target genes, including ELK1, SUMO3 and MAPK1, and their association to prion diseases (p=<NUM>), MAPK signalling pathway (p=<NUM>) and Pl3K-Akt signalling pathway (p=<NUM>) in a String-KEGG pathway analysis (<FIG>).

In the validation study, <NUM> DLB, <NUM> AD, <NUM> PD patients and <NUM> control individuals were included in the analysis. The results of this study were analysed altogether, after joint normalization of all samples (total: <NUM>; comprising <NUM> DLB patients, <NUM> PD patients, <NUM> AD patients and <NUM> control individuals).

Such as it can be seen in <FIG>, relevant results were obtained, especially for miR-<NUM>-5p.

<FIG> shows a statistically significant reduced level of miR-<NUM>-5p in platelets isolated from DLB and PD patients (i.e. patients suffering from a synucleinopathy) as compared with healthy control. This means that miR-<NUM>-5p is a strong biomarker for the diagnosis of synucleinopathies.

Moreover, <FIG> shows a statistically significant reduced level of miR-<NUM>-5p in platelets isolated from DLB and PD patients (i.e. patients suffering from a synucleinopathy) as compared with AD patients. This means that miR-<NUM>-5p is a strong biomarker for the differential diagnosis of synucleinopathies versus AD.

These results are confirmed by ROC curves shown in <FIG>.

Additionally, miRNAs shown in Table <NUM> are significantly diminished in DLB in comparison with AD.

The values in the first line of Table <NUM> represent the relative expression change of each miRNA in DLB vs AD obtained by the deltadeltaCt method. Values below <NUM> represent diminished expression in comparison with the other group (in this case AD). The values in the second line represent the deviation rage. p-value represented in the third line was obtained with the Wilcoxon-Mann-Whitney test.

Six miRNAs were significantly increased in AD when compared to healthy controls. This means that any of the miRNAs included in Table <NUM>, or combinations thereof, could be used for the diagnosis of AD. So, the present invention also refers to an in vitro method for the diagnosis of AD, which comprises determining the expression level of at least one miRNA included in Table <NUM>, or combination thereof, isolated from platelets obtained from the patient, wherein an increased expression level of at least one of the miRNAs included in Table <NUM>, as compared with the expression level measured in healthy control subjects, is an indication that the patient is suffering from AD.

The values in the first line of Table <NUM> represent the relative expression change of each miRNA in AD vs heathy controls and were obtained by the deltadeltaCt method. Values higher than <NUM> represent increased expression in comparison with the other group (in this case controls). The values in the second line represent the deviation rage. p-value represented in the third line were obtained with the Wilcoxon-Mann-Whitney test.

On the other hand, two miRNAs shown in Table <NUM> were significantly decreased in DLB when compared to PD patients. These could therefore serve as diagnostic markers to differentiate DLB from PD patients. So, the present invention also refers to an in vitro method for the differential diagnosis of DLB from PD, which comprises determining the expression level of at least one miRNA included in Table <NUM>, or combination thereof, isolated from platelets obtained from the patient, wherein a decreased expression level of at least one of the miRNAs included in Table <NUM>, as compared with the expression level of measured in PD patients, is an indication that the patient is suffering from DLB and not from PD.

Claim 1:
In vitro method for the diagnosis of synucleinopathies, which comprises determining the expression level of at least miR-<NUM>-5p isolated from platelets obtained from the patient, wherein a reduced expression level of at least the miR-<NUM>-5p, as compared with the expression level of at least the miR-<NUM>-5p measured in healthy control subjects, is an indication that the patient is suffering from a synucleinopathy.