WO2013160474A2 - miRNAs EXPRESSION IN HEMATOLOGICAL DISEASES - Google Patents

Description

miRNAs EXPRESSION IN HEMATOLOGICAL DISEASES

FIELD OF THE INVENTION The invention relates to the field of molecular biology. Certain aspects of the invention include the application of microRNA (miRNA) molecules in diagnostics and/or prognostics of hematological diseases, preferably in myelodysplastic syndromes (MDS).

BACKGROUND OF THE INVENTION

The myelodysplastic syndromes (MDSs) are a heterogeneous group of disorders characterized by impaired peripheral blood (PB) cell production (cytopenias) and most commonly a hypercellular, dysplastic-appearing bone marrow (BM) (Steensma and Bennett, 2006). According to the Myelodysplastic Syndromes Foundation, approximately 10,000 to 15,000 patients are newly diagnosed with MDS annually in the United States (The Myelodysplastic Syndromes Foundation. Available at URL: http://www.mds-foundation.org. Accessed July 15, 2006). MDS occur most frequently in older people (median age at diagnosis is 65 years). The French- American-British (FAB) group proposed a classification system based on morphologic features of dysplasia, the presence of ringed sideroblasts, and the number of blasts and monocytes (Bennett et al., 1982). Their classification consists of 5 subcategories: refractory anemia (RA), refractory anemia with ringed sideroblasts (RARS), refractory anemia with excess blasts (RAEB), refractory anemia with excess blasts in transformation (RAEB-T) and chronic myelomonocytic leukemia (CMML). In recent years, the FAB system has been succeeded by the WHO (World Health Organization) classification, to achieve progressively greater specifhy regarding cytogenetics, MDS cell morphology, the risk of death, or the risk of the evolution of acute leukemia (Vardiman et al., 2009). The World Health Organization, comprises eight subtypes based on biological, genetic and morphological features. These include refractory anemia (RA), RA with ringed sideroblasts, RA with multilineage dysplasia (RCMD), RA with multilineage dysplasia and ringed sideroblasts (RCMD-RS), RA with excess blasts- 1, RA with excess blasts-2, MDS unclassified and MDS associated with isolated deletion chromosome 5q (del(5q)) (also known as 5q- syndrome). The International Prognostic Scoring System (IPSS), which was proposed originally in 1997, is being used in parallel with increasing frequency because of its focus on risk-adapted therapeutic strategy based on karyotype, percentage of BM blasts, cytopenias, life expectancy and the risk of progression to acute myeloid leukemia (AML) (Greenberg et al., 1997). The basis for the selective outgrowth of MDS clones and the decrease in the normal hematopoietic stem/progenitor cells patients over time are unknown, probably due to imperfect interactions between hematopoietic progenitors and marrow stromal cells (Cazzola and Malcovati, 2005). In recent years, a new class of small non-coding RNA molecules, designated as miRNAs (Bartel, 2004), have been identified as a novel mechanism in gene regulation, including modulation of hematopoiesis and cell differentiation in mammals (Chen et al., 2004). miRNAs are single stranded RNAs (usually 19-25 nt) processed from long hairpin-shaped transcripts precursors (primary miRNAs and pre-miRNAs) and evolutionarily conserved, which are transcribed from the miRNA gene. Generally, miRNAs are known to regulate gene expression at the post-transcriptional level by complementariety-mediated binding to target mRNAs resulting in the repression of translation (Doench and Sharp, 2004) or in the cleavage of the target transcript (Mansfield et al., 2004). Deregulation of miRNAs expression has been shown in cancer, where they are thought to act as oncogenes (oncomirs) or as tumor suppressors (Esquela-Kerscher and Slack, 2006). Although aberrant expression of miRNAs can induce tumourigenesis, miRNAs are also involved in the tumour progression and in the metastatic process (Nicoloso et al., 2009). A proportion of human miRNA genes is reported to be located in regions involved in cancer (Calin et al., 2004) and several examples of an association between disrupted expression of specific miRNAs and cancer have been shown in a variety of tissues (Calin et al., 2002; Michael et al., 2003; Metzler et al., 2004; Takamizawa et al., 2004). Importantly, miRNA expression profiles are now recognized to be more accurate and informative than the mRNA expression profiles, allowing the classification of poorly differentiated tumors that were not discriminated by mRNA profiles (Lu et al., 2005). The importance of miRNAs in MDS has recently been emphasized by several authors: Hussein et al (Hussein et al., 2010a) carried out the first systematic analysis on the miRNA expression profile in BM cells of different MDS entities with distinct karyotype aberrations. In MDS with associated del(5q), a specific pattern of non-chromosome 5 miRNAs was expressed which was not found in -7 and +8. In another study, three miRNA genes map within the CDR (commonly deleted region) of the 5q- syndrome were studied in BM cells (Boultwood et al., 2007). No significant difference was observed between patients with the 5q- syndrome and healthy controls or patients with RA and a normal karyotype, although the expression levels of miR-145 were slightly increased in the 5q- syndrome patients, probably reflecting increased expression from the retained allele. However, Starczynowski et al (Starczynowski et al., 2010) showed that deletion of chromosome 5q correlates with loss of miR-145 and miR-146 that are abundant in hematopoietic stem/progenitor cells. In the same sense, another study performed in BM CD34+ cells of 5q- syndrome have shown a down- expression of miR-378 mapped within the commonly deleted region (CDR) at 5q32 (Dostalova Merkerova et al 2011).

In another study, 25 hematopoiesis-related miRNAs in the BM and PB of patients with MDS were studied (Pons et al., 2009). Twelve miRNAs were over-expressed in BM, six of which were also over-expressed in PB. Also they identified two miRNAs (miR-181a and miR-222) with progressively higher expression from controls to early-stage MDS to advanced MDS to post-MDS AML. Other miRNAs have been related to MDS: For example, miR-337-5p, which might take part in leukemic transformation (Hussein et al., 2009), the myeloid cell differentiation is arrested by miR-125b-l in MDS and AML with the t(2;l I)(p21 ;q23) translocation (Bousquet et al., 2008) and there is a significant inverse correlation of miR- 150/MYB (myeloblastosis virus oncogene, a transcription factor) and miR-222/p27 (tumor suppressor) in MDS (Hussein et al., 2010b).

In spite of considerable research into the importance of miRNAs in MDS, studies of miRNA expression profiling in MDS are still lacking. Moreover, it has not yet been found a standardized in vitro method for diagnosis/prognosis of MDS patients, based on the analysis of the miRNA expression profiling in samples obtained from MDS patients, preferably, samples of blood and more preferably samples of peripheral blood. In addition, there is need for prognostic tests that can identify the stage or progression of MDS patients and that can be used to develop an adequate therapeutic response in different patients. DESCRIPTION OF THE INVENTION

Brief description of the invention

The present invention, overcomes these problems in the art by identifying, in a sample obtained from a subject, preferably a sample of blood or plasma and more preferably a sample of peripheral blood or plasma, a miRNAs fingerprint involved in the diagnosis, prognosis and development of MDS. Moreover, the invention also discloses a fingerprint of miRNAs that allows the identification of the stage or progression disease and it might be used to develop an adequate therapeutic response in different patients.

The miRNAs disclosed in the present invention, which are involved in the diagnosis/prognosis of MDS, are differentially expressed in subjects with MDS in comparison to healthy controls.

Names of mature miRNAs and their sequences related to the present invention are provided in Table 1. The methods, uses and kits should not be limited to miRNAs identified in the application, as they are provided as examples, not necessarily as limitations of the invention.

Thus, in a first aspect the present invention refers to a miRNA selected from the group comprising a sequence selected from SEQ ID Nos: 1-20 (Table 1) or variants, precursors, orthologues or combinations thereof for use in the diagnosis/prognosis of MDS.

Table 1. miRNAs involved in the diagnosis/prognosis of MDS. miRNAs Accesion number Sequence hsa-miR-26a-5p MIMAT0000082 SEQ ID NO:l hsa-miR-451 a MIMAT0001631 SEQ ID NO:2 hsa-miR-99b-5p MIMAT0000689 SEQ ID NO:3 hsa-miR-24-3p MIMAT0000080 SEQ ID NO:4 hsa-miR-625-3p MIMAT0004808 SEQ ID NO:5 hsa-miR-19b-3p MIMAT0000074 SEQ ID NO:6 hsa-let-7e-5p MIMAT0000066 SEQ ID NO:7 hsa-miR-140-3p MIMAT0004597 SEQ ID NO: 8 hsa-miR-361-3p MIMAT0004682 SEQ ID NO:9 hsa-miR-378a-3p MIMAT0000732 SEQ ID NO: 10 hsa-miR-15b-5p MIMAT0000417 SEQ ID NO: 11 hsa-miR-16-5p MIMAT0000069 SEQ ID NO:12 hsa-miR-942 MIMAT0004985 SEQ ID NO: 13 hsa-miR-378a-5p MIMAT0000731 SEQ ID NO:14 hsa-miR-625-5p MIMAT0003294 SEQ ID NO: 15 hsa-miR-25-3p MIMAT0000081 SEQ ID NO: 16 miRNAs Accesion number Sequence

hsa-miR-30b-5p MIMAT0000420 SEQ ID NO: 17 hsa-let-7a-5p MIMAT0000062 SEQ ID NO: 18 hsa-miR-15a-5p MIMAT0000068 SEQ ID NO: 19 hsa-miR-17-5p MIMAT0000070 SEQ ID NO:20

The expression levels of miRNAs disclosed herein have been analyzed by Real Time PCR in a sample obtained from a subject. The sample can be selected from: saliva, blood, plasma, serum, bone marrow, or urine, more preferably, samples of saliva, blood, plasma, serum or bone marrow, and still preferably, samples of peripheral blood or plama. The ability to identify miRNAs that are differentially expressed, over-expressed or under-expressed, in pathological samples compared to control samples can provide high-resolution and high- sensitivity datasets, which may be used in diagnostics, therapeutics, drug development, pharmacogenetics, biosensor development, and other related areas. An expression profile generated by the methods disclosed herein may be a "fingerprint" or "signature" (terms used interchangeably throughout the present invention) of the state of the sample with respect to a number of miRNAs. While two states may have any particular miRNA similarly expressed, the evaluation of a number of miRNAs simultaneously allows the generation of a gene expression profile that is characteristic of the state of the sample. That is, normal sample obtained by healthy controls may be distinguished from samples obtained from MDS patients. By comparing expression profiles of samples in different disease states, information regarding which miRNAs are associated in each of these states of disease development may be obtained. Then, diagnosis may be performed or confirmed to determine whether a sample has the expression profile of normal or disease tissue. This may provide for molecular diagnosis of related conditions.

Several authors have described aberrant expression of miRNAs in MDS patients using micro- array-based platforms (Table 2 and 3). In a preferred embodiment of the invention, the miRNAs disclosed in Table 2 and 3, alone or combinations thereof are used together with the miRNAs disclosed in the present invention for the diagnosis/prognosis of MDS. Table 2. Overexpressed miRNAs in MDS known in the art. miRNAs Sequence Reference

Hsa-miR-34a-5p SEQ ID NO:21 Votavova H et al. 2010

Hsa-miR-9-5p SEQ ID NO:22 Hussein K. et al. 2010a

Hsa-miR-214-3p SEQ ID NO:23 Hussein K. et al. 2010a

Hsa-miR- 181 a-5p SEQ ID NO:24 Sokol L. et al. 2011 ; Pons A. et al. 2009

Hsa-miR-181b-5p SEQ ID NO:25 Sokol L. et al. 2011 ; Pons A. et al. 2009

Hsa-miR-181c-5p SEQ ID NO:26 Sokol L. et al. 2011 ; Pons A. et al. 2009

Hsa-miR-181d SEQ ID NO:27 Sokol L. et al. 2011 ; Pons A. et al. 2009

Hsa-miR-29c-3p SEQ ID NO:28 Votavova H. et al. 2010

Hsa-miR- 133b SEQ ID NO:29 Hussein K. et al. 2010a

Hsa-miR-206 SEQ ID NO:30 Hussein K. et al. 2010a, Sokol L. et al. 2011 ;

Hsa-miR- 148a-3p SEQ ID NO:31 Hussein K. et al. 2010a; Votavova H. et al. 2010

Hsa-miR-335-5p SEQ ID NO:32 Votavova H. et al. 2010

Hsa-miR-29a-3p SEQ ID NO:33 Hussein K. et al. 2010a

Hsa-miR-486-5p SEQ ID NO:34 Votavova H. et al. 2010

Hsa-miR-320a SEQ ID NO:35 Sokol L. et al. 2011

Hsa-miR-548d-3p SEQ ID NO:36 Hussein K. et al. 2010a

Hsa-miR- 151 a-3p SEQ ID NO:37 Votavova H. et al. 2010

Hsa-miR- 199b-5p SEQ ID NO:38 Votavova H. et al. 2010

Hsa-miR- 126-5p SEQ ID NO:39 Dostalova M.M. et al. 2011

Hsa-miR- 130a-3p SEQ ID NO:40 Sokol L. et al. 2011, Dostalova M.M. et al. 2011

Hsa-miR- 100-5p SEQ ID NO:41 Sokol L. et al. 2011

Hussein K. et al. 2010a, Sokol L. et al. 2011 ;

Hsa-miR- 125b-5p SEQ ID NO:42

Dostalova M.M. et al. 2011

Hsa-miR- 15a-5p SEQ ID NO:43 Pons A. et al. 2009

Hsa-miR-342-5p SEQ ID NO:44 Hussein K et al. 2010a, Erdogan B. et al. 2011

Hsa-miR-299-3p SEQ ID NO:45 Dostalova M.M. et al. 2011

Hsa-miR-299-5p SEQ ID NO:46 Dostalova M.M. et al. 2011

Hsa-miR-329 SEQ ID NO:47 Dostalova M.M. et al. 2011

Hsa-miR-370 SEQ ID NO:48 Dostalova M.M. et al. 2011

Hsa-miR-376b SEQ ID NO:49 Sokol L. et al. 2011

Hsa-miR-409-3p SEQ ID NO:50 Dostalova M.M. et al. 2011 miRNAs Sequence Reference

Hsa-miR-431-5p SEQ ID NO:51 Dostalova M.M. et al. 2011

Hsa-miR-432-5p SEQ ID NO:52 Dostalova M.M. et al. 2011

Hsa-miR-494 SEQ ID NO:53 Dostalova M.M. et al. 2011

Hsa-miR-654-5p SEQ ID NO:54 Dostalova M.M. et al. 2011

Hsa-miR-665 SEQ ID NO:55 Dostalova M.M. et al. 2011

Hsa-miR-497-5p SEQ ID NO:56 Hussein K. et al. 2010a

Cluster hsa-miR- 17-92 Pons A. et al. 2009

Hsa-miR- 196a-5p SEQ ID NO:57 Sokol L. et al. 2011

Sokol L. et al. 2011, Dostalova M.M. et al. 2011;

Hsa-miR- 10a-5p SEQ ID NO:58

Votavova H. et al. 2010; Hussein K. et al. 2011

Hsa-miR- 1 SEQ ID NO:59 Hussein K. et al. 2010a

Hsa-miR- 133a SEQ ID NO:60 Hussein K. et al. 2010a

Hsa-miR- 199a-5p SEQ ID NO:61 Hussein K. et al. 2010a, Votavova H. et al. 2010

Hussein K. et al. 2010a, Dostalova M.M. et al.

Hsa-miR- 125a-5p SEQ ID NO:62

2011

Sokol L. et al. 2011, Erdogan B. et al. 2011;

Hsa-miR- 150-5p SEQ ID NO:63

Hussein K. et al. 2010b

Hsa-miR- 155-5p SEQ ID NO:64 Sokol L. et al. 2011 ; Pons A. et al. 2009

Hsa-miR-221-3p SEQ ID NO:65 Sokol L. et al. 2011

Hsa-miR-222-3p SEQ ID NO:66 Sokol L. et al. 2011, Pons A. et al. 2009

Hsa-miR-452-5p SEQ ID NO:67 Hussein K. et al. 2010a

Hsa-miR-323a-3p SEQ ID NO:68 Dostalova M.M. et al. 2011

Hsa-miR- 17-5p SEQ ID NO:69 Pons A. et al. 2009

Hsa-miR- 10b-5p SEQ ID NO:70 Pons A. et al. 2009

Hsa-miR- 17-3p SEQ ID N0:71 Pons A. et al. 2009

Hsa-miR- 18a-5p SEQ ID NO:72 Pons A. et al. 2009

Hsa-miR-21-5p SEQ ID NO:73 Pons A. et al. 2009

Hsa-miR- 142-3p SEQ ID NO:74 Pons A. et al. 2009

Hsa-miR-210 SEQ ID NO:75 Lee DW. et al. 2012

Hsa-miR- 127 SEQ ID NO:76 Dostalova M.M. et al. 2011

Cluster hsa-miR- 14q32 Dostalova M.M. et al. 2011 The term "miRNA cluster" refers to a set of miRNAs which are encoded in polycistronic transcripts. The miRNAs hsa-let-7e-5p (SEQ ID No. 7) and has-miR-99b-5p (SEQ ID No: 3) are included in the same cluster (www.mirbase.org). Table 3. Downregulated miRNAs in MDS known in the art. miRNAs Sequence Reference

Hsa-miR-197-3p SEQ ID NO:77 Sokol L. et al. 2011

Hsa-miR-128 SEQ ID NO:78 Dostalova M.M. et al. 2011

Hsa-miR-1284 SEQ ID NO:79 Dostalova M.M. et al. 2011

Hsa-miR-95 SEQ ID NO:80 Votavova H. et al. 2010

Hsa-miR-1305 SEQ ID NO:81 Dostalova M.M. et al. 2011

Hsa-miR-583 SEQ ID NO: 82 Dostalova M.M. et al. 2011

Hsa-miR-143-3p SEQ ID NO:83 Hussein K. et al. 2010a

Hussein K. et al. 2010a, Starczynowski

Hsa-miR-145-5p SEQ ID NO: 84

D.T. et al. 2010

Sokol L. et al. 2011, Votavova H. et al.

Hsa-miR- 146a-5p SEQ ID NO:85

2010, Starczynowski D.T. et al. 2010

Hsa-miR-93-5p SEQ ID NO:86 Hussein K. et al. 2010a

Hsa-miR- 182-5p SEQ ID NO:87 Hussein K et al. 2010a

Hsa-miR- 124-3p SEQ ID NO:88 Sokol L. et al. 2011

Hsa-miR-875-5p SEQ ID NO:89 Sokol L. et al. 2011

Hsa-miR-30d-5p SEQ ID NO:90 Hussein K. et al. 2010a

Hsa-miR-661 SEQ ID NO:91 Hussein K. et al. 2010a

Hsa-let-7a-5p SEQ ID NO:92 Sokol L. et al. 2011, Zuo Z. et al. 2011

Hsa-miR-326 SEQ ID NO:93 Sokol L. et al. 2011

Hsa-miR-940 SEQ ID NO:94 Dostalova M.M. et al. 2011

Hsa-miR-423-5p SEQ ID NO:95 Dostalova M.M. et al. 2011

Hsa-miR- 196a-3p SEQ ID NO:96 Dostalova M.M. et al. 2011

Hsa-miR-520c-3p SEQ ID NO:97 Votavova H. et al. 2010

Hsa-miR-525-5p SEQ ID NO:98 Dostalova M.M. et al. 2011

Hsa-miR-507 SEQ ID NO:99 Dostalova M.M. et al. 2011

Hsa-miR- 143 -5p SEQ ID NO: 100 Dostalova M.M. et al. 2011

Hsa-miR-632 SEQ ID NO:101 Erdogan B. et al. 2011 miRNAs Sequence Reference

Hsa-miR-636 SEQ ID NO: 102 Erdogan B. et al. 2011

Hsa-miR-337-5p SEQ ID NO: 103 Hussein K. et al. 2009

Hsa-miR-206 SEQ ID NO:30 Hussein K. et al. 2010a, Sokol L. et al.

2011 ;

Hsa-miR-335-5p SEQ ID NO:32 Votavova H. et al. 2010

Sokol L. et al. 2011, Dostalova M.M. et al.

Hsa-miR-10a-5p SEQ ID NO:58 2011 ; Votavova H. et al. 2010; Hussein K.

et al. 2011

Hsa-miR-150-5p SEQ ID NO:63 Sokol L. et al. 2011, Erdogan B. et al.

2011 ; Hussein K. et al. 2010b

Hsa-miR-221-3p SEQ ID NO:65 Hussein K. et al. 2010b

In a second aspect, the invention refers to the use of at least one of the miRNA or their nucleotide sequences disclosed in the present invention or variants, precursors, orthologues or combinations thereof for the manufacture of a kit for the diagnosis/prognosis of MDS.

In a third aspect, the invention refers to a kit which comprises at least one probe that hybridizes with at least one of the miRNA or with their nucleotide sequence, disclosed in the present invention or variants, precursors, orthologues or combinations thereof.

In another aspect, the present invention refers to the use of the kit disclosed herein for the in vitro diagnosis/prognosis of MDS.

In still another aspect, the invention refers to an in vitro method of diagnosis/prognosis of MDS which comprises determining in a sample from a subject, the expression level of at least one of the miRNAs disclosed herein or their nucleotide sequences, or variants, precursors, orthologues or combinations thereof and the comparison of said expression level with respect to the expression values obtained from healthy controls. The miRNAs expression levels are analyzed in a sample of peripheral blood of subjects and the miRNAs expression levels of MDS patients are differentially expressed over the miRNAs expression levels of healthy controls.

The present invention also relates to an in vitro method of determining the expression level of a disease-associated miRNA, particularly MDS, comprising contacting a sample with a probe or kit of the invention and measuring the amount of hybridization. The expression level of a disease-associated miRNA is information in a number of ways. For example, a differential expression of a disease-associated miRNA compared to a control may be used as a diagnostic that a patient suffers from the disease. Expression levels of a disease-associated miRNA may also be used to monitor the treatment and disease state of a patient. Furthermore, expression levels of the disease-associated miRNA may allow the screening of drug candidates for altering a particular expression profile or suppressing an expression profile associated with the disease.

The present invention also relates to a method of diagnosis comprising detecting a differential expression level of a disease-associated miRNA in a sample. The sample may be derived from a patient. Diagnosis of a disease state in a patient allows prognosis and selection of therapeutic strategy.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. However, for ease of reference, some of these terms will now be defined.

The term "sample" refers to a sample of a body fluid, to a sample of isolated cells or to a sample from a tissue or an organ. Samples of body fluids can be obtained by well-known techniques and include, preferably, samples of saliva, blood, plasma, serum, bone marrow, or urine, more preferably, samples of peripheral blood or plasma.

The term "subject" as used herein refers to an animal, preferably a mammal, most preferably a human being, including both young and old human beings of both sexes which may suffer from or are predisposed to a pathology. The subject according to this aspect of the present invention may suffer from a pathology associated with abnormal expression of miRNAs, preferably MDS. The terms "subject" and "patient" could be used interchangeably throughout the present invention.

The term "comprise" or "comprising", all along present patent description, means the presence of the stated features, integers, steps, or components, specially, miRNAs or complementary DNA sequences derived from the transcript of the corresponding miRNAs, as referred to in the claims, but that it does not preclude the presence or addition of one or more other features, integers, steps, components or groups thereof. The term "comprising" is intended to include embodiments encompassed by the terms "consisting essentially of and "consisting of. Similarly, the term "consisting essentially of is intended to include embodiments encompassed by the term "consisting of. The term "differential expression or differentially expressed" may mean qualitative or quantitative differences in the genes, proteins, peptides or polypeptides, specially, miRNAs, expression patterns within and among cells and/or tissue. Thus, a differentially expressed gene, protein, peptides or polypeptides can qualitatively have its expression altered, including an activation-inactivation, or an under-expression-over-expression in, e.g., normal versus diseased subjects. Genes, proteins, peptides or polypeptides, specially, miRNAs, may be turned on or turned off in a particular state, relative to another state thus permitting comparison of two or more states. A qualitatively regulated gene or protein or peptide or polypeptide, specially, miRNA, will exhibit an expression pattern within a state or cell type which may be detectable by standard techniques. Alternatively, the difference in expression may be quantitative, e.g., in that expression is modulated, either up-regulated, resulting in an increased amount of transcript (over-expression), or down-regulated, resulting in a decreased amount of transcript (under-expression). The term "transcripts" as used throughout the present invention refers to a sequence of RNA produced by transcription from a DNA template. In this sense the expression "complementary DNA derived from a transcript" refers to DNA synthesized from a mature RNA template. In another sense, the expression "complementary RNA" (cRNA) refers to a synthetic RNA produced by transcription from a specific DNA single stranded template.

The term "variant" as used throughout the specification is to be understood to mean an nucleotide sequence of a miRNA or amino acid sequence of a protein or polypeptide, preferably, a miRNA that is altered by one or more nucleotides or amino acids, respectively. The variant may have "conservative" changes, wherein a substituted nucleotide or amino acid has similar structural or chemical properties to the replaced nucleotide or amino acid. A variant may also have "non-conservative" changes or a deletion and/or insertion of one or more nucleotides or amino acids. The term also includes within its scope any insertions/deletions of nucleotides or amino acids for a particular miRNA or protein or polypeptide. A "functional variant" will be understood to mean a variant that retains the functional capacity of a reference nucleotide sequence or a protein or polypeptide, specifically a miRNA.

The term "complement" or "complementary" as used herein may mean that each strand of double-stranded nucleic acids such as, DNA and RNA, is complementary to the other in that the base pairs between them are non-covalently connected via two or three hydrogen bonds. For DNA, adenine (A) bases complement thymine (T) bases and vice versa; guanine (G) bases complement cytosine (C) bases and vice versa. With RNA, it is the same except that adenine (A) bases complement uracil (U) bases instead of thymine (T) bases. Since there is only one complementary base for each of the bases found in DNA and in RNA, one can reconstruct a complementary strand for any single strand.

The term "orthologue" as used throughout the specification is to be understood as homologous gene or miRNAs sequences found in different species. The term "pri-miRNA" refers to a reverse-complement DNA sequence that is transcribed into a single-stranded RNA molecule. The miRNA sequence and its reverse-complement base pair to form a double stranded RNA hairpinloop; this pairing form a primary miRNA structure referred to as pri-miRNA. The term "pre-miRNA" refers to the precursors to miRNA generated from pri-miRNA. The sequence of the pre-miRNA may comprise a miRNA and/or a miRNA*. The pre-miRNA may also comprise a miRNA or miRNA* and complements or variants thereof. The sequence of pre-miRNA may comprise from 45-90, more preferably 60- 80 or still more preferably 60-70 nucleotides. The sequence of the pre-miRNA may comprise any of the sequences included at the miRBase registry (http://microrna.sanger.ac.uk/sequences/), or variants thereof. The term "miRNA" refers to a sequence of a miRNA, miRNA* or a variants thereof. miRNA and miRNA* are derived from the same precursor and miRNA* is the minor product of miRNA biogenesis. miRNA* sequences may be found in libraries of cloned miRNAs but typically at lower frequency than the miRNAs. The miRNA sequence may comprise from 13-33, more preferably 18-24 or still more preferably 21-23 nucleotides. The sequence of the miRNA may comprise the sequence of a miRNA referred to in Table 1, or variants thereof, or any sequence of miRNAs included at the miRBase registry (http://microrna.sanger.ac.uk/sequences/). The sequences of the miRNAs comprise the sequences listed in Table 1, along with, further, any other sequences of miRNAs known in the prior art and which are used for the diagnosis/prognosis of MDS (see Tables 2 and 3) as the have a differential expression in MDS patients compared to control subjects.

An expression profile generated by the methods disclosed in the present invention may be a "signature/fingerprint" of the state of the sample with respect to a number of miRNAs. While two states may have any particular miRNA similarly expressed, the evaluation of a number of miRNAs simultaneously allows the generation of a gene expression profile that is characteristic of the state of the cell or patient in general. That is, normal tissue or sample may be distinguished from diseased tissue or sample. By comparing expression profiles of tissue or sample in known different disease states, information regarding which miRNAs are associated in each of these states may be obtained. Then, diagnosis may be performed or confirmed to determine whether a tissue sample has the expression profile of normal or disease tissue. This may provide for molecular diagnosis of related conditions. When comparing the fingerprints between an individual and a standard, the skilled artisan can make a diagnosis, a prognosis, or a prediction based on the findings. It is further understood that the genes which indicate the diagnosis may differ from those which indicate the prognosis, and molecular profiling of the condition of the cells may lead to distinctions between responsive or refractory conditions or may be predictive of outcomes. In this sense, the term prognosis describes the likely outcome of a disease.

The present invention also relates to kits comprising probes and/or primers that hybridize with at least one miRNA, or variants, precursors, orthologues or combinations thereof disclosed in the present invention together with any or all of the following: assay reagents, buffers and sterile saline or another pharmaceutically acceptable emulsion and suspension base. In addition, the kits may include instructional materials containing directions (e.g., protocols) for the practice of the methods of this invention.

The present invention also relates to a method of identifying miRNAs that are associated with a disease or a pathological condition, preferably MDS, comprising contacting a sample taken from a subject with a probe or kit of the invention and detecting the amount of hybridization. PCR may be used to amplify nucleic acids in the sample, which may provide higher sensitivity.

The present invention also provides orthologues of the above human miRNAs, which may be identified by any method known in the art. A database of miRNAs is found at the miRBase registry (http ://microrna. Sanger, ac.uk/sequences/).

Description of the Figures.

Figure 1 : Analysis of miRNAs expression (median of LOGIO(RQ)) in plasma-MDS patients versus plasma-healthy controls. Figure 2. Analysis of miRNAs expression (median of LOGIO(RQ)) in low, intermediate and high risk plasma-MDS patients versus plasma-healthy controls.

Detailed description of the invention

Present invention relates to at least one of the miRNA selected from those comprising a sequence selected from SEQ ID Nos: 1-11, 13-20, or variants, or precursors, or orthologues, or combinations thereof, for use in the diagnosis and/or prognosis of MDS. In a particular embodiment of the invention, the miRNAs are selected from those comprising a sequence selected from SEQ ID Nos: 1, 5, 6, 8, 9, 13, 15, 17 or variants, or precursors, or orthologues, or combinations, thereof.

In another particular embodiment of the invention, the miRNAs are selected from those comprising a sequence selected from SEQ ID Nos: 2, 3, 4, 7, 10, 11, 14, 16, 18, 19, 20 or variants, or precursors, or orthologues, or combinations, thereof.

In another particular embodiment of the invention, the miRNAs are selected from those comprising a sequence selected from SEQ ID Nos: 2, 3, 4, 7, 10, 14, or variants, or precursors, or orthologues, or combinations, thereof.

In another particular embodiment of the invention, the miRNAs disclosed in the present invention, alone or in combination, may be combined with the miRNAs disclosed in Tables 2 and/or 3 or variants, or precursors, or orthologues, or combinations, thereof, for use in the diagnosis and/or prognosis of MDS.

In another particular embodiment of the invention, the miRNAs are selected from those consisting in a sequence selected from SEQ ID Nos: 1-11, 13-20, or variants, or precursors, or orthologues, or combinations, thereof.

In another particular embodiment of the invention, the miRNAs are selected from those consisting in a sequence selected from SEQ ID Nos: 1, 5, 6, 8, 9, 13, 15, 17 or variants, or precursors, or orthologues, or combinations, thereof. In another particular embodiment of the invention, the miRNAs are selected from those consisting in a sequence selected from SEQ ID Nos: 2, 3, 4, 7, 10, 11, 14, 16, 18, 19, 20 or variants, or precursors, or orthologues, or combinations, thereof.

In another particular embodiment of the invention, the miRNAs are selected from those consisting in a sequence selected from SEQ ID Nos: 2, 3, 4, 7, 10, 14, or variants, or precursors, or orthologues, or combinations, thereof.

Present invention also relates a combination of miRNAs sequences consisting in SEQ ID Nos: 1-11, 13-20, or variants, or precursors, or orthologues, or combinations thereof, for use in the diagnosis and/ or prognosis of MD S .

In a particular embodiment of the invention, the combination of miRNAs sequences consisting in SEQ ID Nos: 1, 5, 6, 8, 9, 13, 15, 17 or variants, or precursors, or orthologues, or combinations thereof.

In another particular embodiment of the invention, the combination of miRNAs sequences consisting in SEQ ID Nos: 2, 3, 4, 7, 10, 11, 14, 16, 18, 19, 20 or variants, or precursors, or orthologues, or combinations thereof. In another particular embodiment of the invention, the combination of miRNAs sequences consisting in SEQ ID Nos: 2, 3, 4, 7, 10, 14 or variants, or precursors, or orthologues, or combinations thereof.

In another particular embodiment of the invention, the combination of miRNAs sequences disclosed above could be further combined with at least one of miRNAs selected from those comprising a sequence selected from SEQ ID Nos: 21-103

Diagnosis of a disease state in a patient allows for prognosis and selection of therapeutic strategy. In this sense, differences in miRNA expression levels in low, intermediate and high risk MDS patients shows that four of the miRNAs analyses in the present invention were significantly associated with low, intermediate and high risk groups (p<0.01) (Figure 2). Therefore, another object of the present invention refers to miRNA selected from those comprising a sequence selected from SEQ ID Nos: 3, 7, 8, or 10, or variants, or precursors, or orthologues, or combinations, thereof for use in the prognosis of MDS. In another particular embodiment of the invention the miRNA is SEQ ID No. 3, which was significantly associated (p<0.05) with progression to AML.

Another object of the present invention refers to a combination of miRNA sequences consisting in SEQ ID Nos: 3, 7, 8, or 10, or variants, or precursors, or orthologues, or combinations, thereof, for use in the prognosis of MDS. In another particular embodiment of the invention the miRNA is SEQ ID No. 3, which was significantly associated (p<0.05) with progression to AML. Present invention also relates to the use of at least one of miRNA selected from those comprising a sequence selected from SEQ ID Nos: 1-11, 13-20, or variants, or precursors, or orthologues, or combinations, thereof for the manufacture of a kit for the diagnosis and/or prognosis of MDS. In a particular embodiment of the invention said use for manufacturing a kit for the diagnosis and/or prognosis of MDS is characterized by miRNAs are selected from the group comprising a sequence selected from SEQ ID Nos: 1, 5, 6, 8, 9, 13, 15, 17, or variants, or precursors, or orthologues, or combinations, thereof. In a particular embodiment of the invention said use for manufacturing a kit for the diagnosis and/or prognosis of MDS is characterized by miRNAs are selected from the group comprising a sequence selected from SEQ ID Nos: 2, 3, 4, 7, 10, 11,14, 16, 17, 18, 19, 20, or variants, or precursors, or orthologues, or combinations, thereof. In a particular embodiment of the invention said use for manufacturing a kit for the diagnosis and/or prognosis of MDS is characterized by miRNAs are selected from the group comprising a sequence selected from SEQ ID Nos: 2, 3, 4, 7, 10, 14, or variants, or precursors, or orthologues, or combinations, thereof. In a particular embodiment of the invention said use for manufacturing a kit for the diagnosis and/or prognosis of MDS is characterized in that at least one miRNAs selected from SEQ ID Nos: 1-15 or variants, or precursors, or orthologues, or combinations, thereof is further combined with those comprising a sequence selected from SEQ ID Nos: 21-103 (Tables 2 and 3) or variants, or precursors, or orthologues, or combinations, thereof. In another particular embodiment of the invention said use for manufacturing a kit for the diagnosis and/or prognosis of MDS is characterized by miRNAs are selected from the group consisting in a sequence selected from SEQ ID Nos: 1-11, 13-20, or variants, or precursors, or orthologues, or combinations, thereof.

In another particular embodiment of the invention said use for manufacturing a kit for the diagnosis and/or prognosis of MDS is characterized by miRNAs are selected from the group consisting in a sequence selected from SEQ ID Nos: 1, 5, 6, 8, 9, 13, 15, 17, or variants, or precursors, or orthologues, or combinations, thereof.

In another particular embodiment of the invention said use for manufacturing a kit for the diagnosis and/or prognosis of MDS is characterized by miRNAs are selected from the group consisting in a sequence selected from SEQ ID Nos: 2, 3, 4, 7, 10, 11, 14, 16, 17, 18, 19, 20, or variants, or precursors, or orthologues, or combinations, thereof.

In another particular embodiment of the invention said use for manufacturing a kit for the diagnosis and/or prognosis of MDS is characterized by miRNAs are selected from the group consisting in a sequence selected from SEQ ID Nos: 2, 3, 4, 7, 10, 14, or variants, or precursors, or orthologues, or combinations, thereof.

Another object of the present invention refers to the use of a combination of miRNAs sequences consisting in SEQ ID Nos: 1-11, 13-20, or variants, or precursors, or orthologues, or combinations thereof, for the manufacture of a kit for the diagnosis and/or prognosis of MDS.

In a particular embodiment of the invention said use of a combination of miRNAs sequences further comprises the combination with those miRNAs comprising a sequence selected from SEQ ID Nos: 21-103 or variants, or precursors, or orthologues, or combinations, thereof. In a particular embodiment of the invention said use of a combination of miRNAs sequences is characterized by the combination consisting in SEQ ID Nos: 1, 5, 6, 8, 9, 13, 15, 17 or variants, or precursors, or orthologues, or combinations thereof.

In a particular embodiment of the invention said use of a combination of miRNAs sequences is characterized by the combination consisting in SEQ ID Nos: 2, 3, 4, 7, 10, 11 , 14, 16, 17, 18, 19, 20, or variants, or precursors, or orthologues, or combinations thereof.

In a particular embodiment of the invention said use of a combination of miRNAs sequences is characterized by the combination consisting in SEQ ID Nos: 2, 3, 4, 7, 10, 14 or variants, or precursors, or orthologues, or combinations thereof.

Another object of the present invention refers to the use of at least one miRNA selected from those comprising a sequence selected from SEQ ID Nos: 3, 7, 8, or 10, or variants, or precursors, or orthologues, or combinations, thereof for the manufacture of a kit for the prognosis of MDS. In another particular embodiment of the invention the miRNA is SEQ ID No. 3, which was significantly associated (p<0.05) with progression to AML.

Another object of the present invention refers to the use of a combination of miRNAs sequences consisting in SEQ ID Nos: 3, 7, 8, or 10, or variants, or precursors, or orthologues, or combinations, thereof, for the manufacture of a kit for the prognosis of MDS.

Present invention also relates a kit comprising at least one probe that hybridizes with at least one miRNAs selected from those comprising a sequence selected from SEQ ID Nos: 1-11, 13- 20, or variants, or precursors, or orthologues, or combinations, thereof.

In a preferred embodiment of the invention, the kit disclosed herein is characterized in that comprises at least one probe that hybridizes with at least one of miRNAs selected from those comprising a sequence selected from SEQ ID Nos: 1, 5, 6, 8, 9, 13, 15, 17, or variants, or precursors, or orthologues, or combinations, thereof.

In another preferred embodiment of the invention, the kit disclosed herein is characterized in that comprises at least one probe that hybridizes with at least one miRNA selected from those comprising a sequence selected from SEQ ID Nos: 2, 3, 4, 7, 10, 11, 14, 16, 17, 18, 19, 20, or variants, or precursors, or orthologues, or combinations, thereof.

In another preferred embodiment of the invention, the kit disclosed herein is characterized in that comprises at least one probe that hybridizes with at least one miRNA selected from those comprising a sequence selected from SEQ ID Nos: 2, 3, 4, 7, 10, 14, or variants, or precursors, or orthologues, or combinations, thereof. In another particular embodiment of the invention, the kit disclosed herein is characterized in that further could be comprises at least one probe that hybridizes with at least one miRNA selected from those disclosed in Tables 2 and/or 3. In another preferred embodiment of the invention, the kit disclosed herein is characterized in that comprises at least one probe that hybridizes with at least one miRNA selected from those consisting in a sequence selected from SEQ ID Nos: 1-11, 13-20, or variants, or precursors, or orthologues, or combinations thereof. In a preferred embodiment of the invention, the kit disclosed herein is characterized in that comprises at least one probe that hybridizes with at least one of miRNAs selected from those consisting in a sequence selected from SEQ ID Nos: 1, 5, 6, 8, 9, 13, 15, 17, or variants, or precursors, or orthologues, or combinations, thereof. In another preferred embodiment of the invention, the kit disclosed herein is characterized in that comprises at least one probe that hybridizes with at least one miRNA selected from those consisting in a sequence selected from SEQ ID Nos: 2, 3, 4, 7, 10, 11, 14, 16, 17, 18, 19, 20, or variants, or precursors, or orthologues, or combinations, thereof. In another preferred embodiment of the invention, the kit disclosed herein is characterized in that comprises at least one probe that hybridizes with at least one miRNA selected from those consisting in a sequence selected from SEQ ID Nos: 2, 3, 4, 7, 10, 14, or variants, or precursors, or orthologues, or combinations, thereof. In another preferred embodiment of the invention, the kit disclosed herein is characterized in that the probe comprises a sequence that specifically hybridizes with at least one complementary DNA derived from the transcript of the corresponding miRNA selected from those comprising in a sequence selected from SEQ ID Nos: 1-103, or variants, or precursors, or orthologues, or combinations, thereof.

In another preferred embodiment of the invention, the kit disclosed herein is characterized in that the probe comprises a sequence that specifically hybridizes with at least one complementary DNA derived from the transcript of the corresponding miRNA selected from those consisting in a sequence selected from SEQ ID Nos: 21-103, or variants, or precursors, or orthologues, or combinations, thereof. In another preferred embodiment of the invention, the kit disclosed herein is characterized in that the probe comprises a sequence that specifically hybridizes with at least one complementary RNA derived from the transcript of a specific DNA derived from the transcript of the corresponding miRNA selected from those comprising a sequence selected from SEQ ID No: 1-103, or variants, or precursors, or orthologues, or combinations, thereof.

In another preferred embodiment of the invention, the kit disclosed herein is characterized in that the probe comprises a sequence that specifically hybridizes with at least one complementary RNA derived from the transcript of a specific DNA derived from the transcript of the corresponding miRNA selected from those consisting in a sequence selected from SEQ ID No: 21-103, or variants, or precursors, or orthologues, or combinations, thereof.

Present invention also disclosed a kit comprising a combination of probes that hybridizes with a combination of miRNAs sequences consisting in SEQ ID Nos: 1-11, 13-20, or variants, or precursors, or orthologues, or combinations thereof.

In a preferred embodiment the kit further comprises at least one probe that hybridizes with at least one miRNA selected from those comprising a sequence selected from SEQ ID Nos: 21- 103 or variants, or precursors, or orthologues, or combinations, thereof.

In another preferred embodiment the kit comprises a combination of probes that hybridizes with a combination of miRNAs sequences consisting in SEQ ID Nos: 1, 5, 6, 8, 9, 13, 15, 17 or variants, or precursors, or orthologues, or combinations, thereof.

In another preferred embodiment the kit comprises a combination of probes that hybridizes with a combination of miRNAs sequences consisting in SEQ ID Nos: 2, 3, 4, 7, 10, 11, 14, 16, 17, 18, 19, 20, or variants, or precursors, or orthologues, or combinations, thereof.

In another preferred embodiment the kit comprises a combination of probes that hybridizes with a combination of miRNAs sequences consisting in SEQ ID Nos: 2, 3, 4, 7, 10, 14, or variants, or precursors, or orthologues, or combinations, thereof.

Present invention also disclosed a kit comprising at least one probe that hybridizes with at least one miRNA selected from those comprising a sequence selected from SEQ ID Nos: 3, 7, 8 or 10, or variants, or precursors, or orthologues, or combinations thereof. In a preferred embodiment the kit further comprises at least one probe that hybridizes with at least one miRNA selected from those comprising a sequence selected from SEQ ID Nos:21- 103 or variants, or precursors, or orthologues, or combinations thereof. Present invention also disclosed a kit comprising a combination of probes that hybridizes with a combination of miRNAs sequences consisting in SEQ ID Nos: 3, 7, 8 or 10, or variants, or precursors, or orthologues, or combinations thereof

In a preferred embodiment the kit further comprises at least one probe that hybridizes with at least one miRNA selected from those comprising a sequence selected from SEQ ID Nos:21- 103 or variants, or precursors, or orthologues, or combinations, thereof.

Present invention also disclosed the use of the kit disclosed herein for the in vitro diagnosis and/or prognosis of MDS.

Present invention also disclosed an in vitro method of diagnosis and/or prognosis of MDS which comprises determining in a sample from a subject, the expression level of at least one miRNA selected from those comprising a sequence selected from SEQ ID Nos: 1-11, 13-20, or variants, or precursors, or orthologues, or combinations, thereof and the comparison of said expression level with respect to the expression values obtained from healthy controls.

In a preferred embodiment of the invention, the in vitro method further comprises the determination of the expression level of at least one miRNAs selected from those disclosed in Table 2 and/or 3, or variants, or precursors, or orthologues, or combinations, thereof.

In a preferred embodiment of the invention, the in vitro method comprises the determination of the expression level of a group of miRNAs selected from those comprising a sequence selected from SEQ ID Nos: 1, 5, 6, 8, 9, 13, 15, 17, or variants, or precursors, or orthologues, or combinations, thereof.

In another preferred embodiment of the invention, the in vitro method comprises the determination of the expression level of a group of miRNAs selected from those comprising a sequence selected from SEQ ID Nos: 2, 3, 4, 7, 10, 11, 14, 16, 17, 18, 19, 20, or variants, or precursors, or orthologues, or combinations, thereof. In another preferred embodiment of the invention, the in vitro method comprises the determination of the expression level of a group of miRNAs selected from those comprising a sequence selected from SEQ ID Nos: 2, 3, 4, 7, 10, 14, or variants, or precursors, or orthologues, or combinations, thereof.

In another preferred embodiment of the invention, the in vitro method comprises the determination of the expression level of a group of miRNAs consisting in a sequence selected from SEQ ID Nos: 1-11, 13-20, or variants, or precursors, or orthologues, or combinations, thereof.

In another preferred embodiment of the invention, the in vitro method comprises the determination of the expression level of a group of miRNAs consisting in a sequence selected from SEQ ID Nos: 1, 5, 6, 8, 9, 13, 15, 17, or variants, or precursors, or orthologues, or combinations, thereof.

In another preferred embodiment of the invention, the in vitro method comprises the determination of the expression level of a group of miRNAs consisting in a sequence selected from SEQ ID Nos: 2, 3, 4, 7, 10, 11, 14, 16, 17, 18, 19, 20, or variants, or precursors, or orthologues, or combinations, thereof.

In another preferred embodiment of the invention, the in vitro method comprises the determination of the expression level of a group of miRNAs consisting in a sequence selected from SEQ ID Nos: 2, 3, 4, 7, 10, 14, or variants, or precursors, or orthologues, or combinations, thereof.

In another preferred embodiment of the invention, the in vitro method disclosed herein is characterized in that the sample of the subject is selected from: saliva, blood, plasma, serum, bone marrow, or urine, more preferably, samples of saliva, plasma and blood and still preferably, samples of peripheral blood and plasma.

Other object of the present invention refers to an in vitro method of diagnosis and/or prognosis of MDS which comprises determining in a sample from a subject, the expression level of a combination of miRNA sequences consisting in SEQ ID Nos: 1-11, 13-20, or variants, or precursors, or orthologues, or combinations thereof and the comparison of said expression level with respect to the expression values obtained from healthy controls. In a particular embodiment, the in vitro method further comprises the determination of the expression level of at least one miRNAs selected from those comprising a sequence selected from SEQ ID Nos: 21-103 or variants, or precursors, or orthologues, or combinations thereof. In another particular embodiment, the in vitro method comprises the determination of the expression level of the combination of miRNAs sequences consisting in SEQ ID Nos: 1, 5, 6, 8, 9, 13, 15, 17, or variants, or precursors, or orthologues, or combinations thereof.

In another particular embodiment, the in vitro method comprises the determination of the expression level of the combination of miRNAs sequences consisting in SEQ ID Nos: 2, 3, 4, 7, 10, 11, 14, 16, 17, 18, 19, 20, or variants, or precursors, or orthologues, or combinations thereof.

In another particular embodiment, the in vitro method comprises the determination of the expression level of the combination of miRNAs sequences consisting in SEQ ID Nos: 2, 3, 4, 7, 10, 14, or variants, or precursors, or orthologues, or combinations thereof.

The in vitro method according to the present invention is characterized by the miRNAs of subjects with MDS are differentially expressed with respect to the corresponding miRNA expression levels of healthy controls. In a particular embodiment, the miRNAs sequences SEQ ID No. 1, 3-7, 9-11, 13-18 and 20 are over-expressed with respect to the corresponding miRNA expression levels of healthy controls and the miRNAs sequence SEQ ID No. 2 is down-expressed with respect to the corresponding miRNA expression levels of healthy controls.

In another preferred embodiment of the invention, the in vitro method it is characterized in that the expression levels are obtained by Real Time-PCR. The miRNA may be further analyzed by using microarrays, locked nucleic acid in situ hybridization, fluorescence in situ hybridization, and other RNA processing methods known in the art. In addition to, or in place of, the RNA processing methods, the RNA may be converted to complementary DNA by subjecting the mature miRNA to reverse transcription PCR. The RNA may also be converted to cDNA by any methods known in the art. Non-limiting examples are readily available. The expression profile may be standardized by background subtraction and normalization using a set of housekeeping genes before comparing the expression profile of the at least one miRNA to those obtained from control subjects. Once the RNA or complementary DNA has been processed, the RNA or DNA is analysed to determine the status of the patient. The method may also be used to determine whether a patient is at risk to develop MDS.

Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. It will be appreciated by those of skill in the art that, in light of the present disclosure, numerous modifications and changes can be made in the particular embodiments exemplified without departing from the intended scope of the invention. The disclosures of all publications cited above are expressly incorporated herein by reference, each in its entirety, to the same extent as if each were incorporated by reference individually.

The following examples illustrate the invention and should not be considered in a limiting sense, but rather in an illustrative sense of the invention.

EXAMPLE 1. Identification of a miRNA profile/fingerprint in peripheral blood from MDS patients.

Patient samples and controls

For the study, 40 samples of peripheral blood obtained from MDS patients and 40 age- matched controls were collected in PAXgene tubes (PreAnalytiX, Qiagen/BD Company, Switzerland). All procedures were approved by the Ethics Committee of Aragon (CEICA) and were conducted in accordance with the Helsinki declaration of 1975, as revised in 1983. All patients were diagnosed at Miguel Servet University Hospital of Zaragoza (Spain). All patients gave their informed consent. Clinical characteristics of patients included in the present study are disclosed in Table 4. The control group included 40 healthy controls individuals. Table 4. Clinical characteristics of 40 patients with MDS included in the present study.

Patient Age (years) Sex WHO classification Cytogenetics

1 62 M RA Normal karyotype

2 78 M RA Normal karyotype

3 48 M RA Normal karyotype

4 85 M RA Normal karyotype

F : female; M: male; RA: Refractory Anemia; RARS: Refractory Anemia with ringed sideroblasts; RCMD: Refractory Cytopenia with multilineage dysplasia; RAEB: Refractory Anemia with excess blast; AML: Acute Myeloid Leukemia; CMML: Chronic myelomonocytic leukemia. RNA extraction, reverse transcription and quantitative real-time PCR assay

Total RNA was extracted from cryopreserved samples using PAXgene Blood miRNA kit (PreAnalytiX, Qiagen/BD Company, Switzerland) according to manufacturer's instructions. Samples were reverse transcribed using the Taqman®MicroRNA Reverse Transcription Kit (P/N 4366596, Applied Biosystems, USA) and mature miRNAs were quantified by real-time PCR using the Taqman®MicroRNA Assays (Applied Biosystems, USA) according to the manufacture's instructions (Chen et al., 2005). First, a screening to identify a miRNA signature/fingerprint associated with diagnosis/prognosis in MDS was determined by quantitative real-time PCR using Megaplex™ Primers Human Pool AV2.1 and B (P/N 4399966 and 4399968 respectively; Applied Biosystems, USA). The kit contained assays for 754 miRNAs of the 1000 currently listed in the Sanger miRBase database (Griffiths-Jones et al., 2006). PCR reactions were performed using an ABI 7900 Fast (Sequence Detection System software version 2.4) (Applied Biosystems, USA).

Normalization was performed with hsa-miR-30b-5p (SEQ ID NO: 17) and hsa-miR-25-3p (SEQ ID NO: 16). The data presented correspond to the Ct value by the 2^~AACt method (Livak and Schmittgen, 2001) (AACt = (Ct of microRNA of interest - Ct of RNU24)_patient - (Ct of microRNA of interest - Ct of RNU24)_caiib_rator- Data analysis was performed at Genomics Unit in Scientific Park from Madrid using Integromics 'RealTime Statminer® (Goni R, 2009).

In this sense, it has been identified 47 candidate MDS-associated miRNAs by first profiling the expression of 754 known miRNAs in PB of patients compared to age-matched controls by using Taqman®MicroRNA qRT-PCR (Applied Biosystems, USA). Afterwards, it has been analyzed PB samples from controls and patients with MDS for the levels of these miRNAs by TaqMan qRT-PCR. For this study, Taqman® Custom Plating (Applied Biosystems, USA) in 96 well-plates was used. PCR reactions were performed using an ABI 7900 HT (Sequence Detection System software version 2.2.2, Applied Biosystems, USA). The mean Ct value of each duplicate was used for analysis by the 2^~AACt method disclosed above. The results obtained from this first study showed the differential expression of 15 miRNAs in PB-MDS patients compared with PB-healthy controls (Table 5).

Table 5. miRNAs differentially expressed in samples of peripheral blood obtained from MDS patients versus healthy control.

In the present example it has been identified a miRNA signature/fingerprint characteristic of MDS composed of 15 differentially expressed miRNAs. Therefore, these miRNAs disclosed in the present invention are biological markers for the diagnosis/prognosis of MDS. Additionally, a differential expression of a disease-associated miRNA, specifically MDS, compared to a control may be used as a diagnostic that a patient suffers from the disease. Expression levels of a disease-associated miRNA may also be used to monitor the treatment and disease state of a patient. Diagnosis of a disease state in a patient allows for prognosis and selection of therapeutic strategy. Moreover, in the present invention, the sequences of miRNAs known in the prior art (Tables 2 and 3) can be combined together with the miRNAs of the invention, for a greater reliability in the diagnosis and prognosis of MDS. Example 2. Validation and analysis of the miRNA expression profile/fingerprint in plasma from patients diagnosed with MDS.

To demonstrate that the identified miRNA signature/fingerprint characteristic of 40 MDS 5 patients analyzed in Example 1 could be extrapolated to a wide MDS population, a total of 242 patients diagnosed with MDS were analyzed. In this sense, 192 plasma samples were derived from the project INBIOMED HEMA-001/2006 and 50 corresponding to patients diagnosed in the Haematology department of Miguel Servet University Hospital (2008-2011). Samples were deposited in the Aragon Biobank. Recruited patients were informed of their 0 legal and ethical rights through Informed Consent (IC). Clinical characteristics of patients included in the present study are disclosed in Table 6. The control group included 40 healthy individuals.

Table 6. Clinical characteristics of 242 patients with MDS included in the present study. 5

Sample Age Gender FAB WHO-2008 Cytogenetics Transformation Risk

(Years) classification classification (IPSS-R) to AML

CLI-001 75 1 3 6 1 0 3

CLI-002 78 2 2 3 1 0 2

CLI-003 87 1 2 3 1 0 2

CLI-004 79 1 3 6 3 1 3

CLI-005 78 1 3 5 2 1 3

CLI-006 76 2 1 3 2 0 2

CLI-007 73 2 3 5 2 1 3

CLI-008 71 1 2 3 2 0 2

CLI-009 76 2 3 5 ¹ 0 2

CLI-010 68 2 3 5 ¹ 0 2

CLI-011 81 1 1 3 ¹ 0 2

CLI-012 84 2 2 2 ¹ 0 1

CLI-014 83 1 2 3 ¹ 0 2

CLI-016 80 2 3 5 ¹ 1 3

CLI-017 75 2 1 1 ¹ 0 1

CLI-018 36 1 3 5 ¹ 0 2

CLI-019 81 1 1 3 ¹ 0 2

CLI-020 82 1 1 1 ¹ 0 1 Sample Age Gender FAB WHO-2008 Cytogenetics Transformation Risk

(Years) classification classification (IPSS-R) to AML

CLI-021 66 1 1 3 ¹ 0 2

CLI-022 76 2 2 2 ¹ 0 1

HUS-002 65 2 3 5 ¹ 1 3

HUS-003 66 ¹ 3 5 ¹ 1 3

HUS-005 79 ^{1 1} 3 ¹ 1 3

HUS-006 76 ^{1 1} 3 0 2

HUS-008 82 ^{1 1} 3 ¹ 0 2

HUS-009 68 ^{1 1} 3 ¹ 0 2

HUS-010 72 ¹ 3 ¹ 0 2

HUS-011 84 ^{1 1} 3 ¹ 1 3

HUS-012 70 ^{1 1} 1 1 0 2

HUS-015 60 ^{1 1} 3 0 2

HUS-016 77 ^{1 1} 3 ¹ 0 2

HUS-017 77 ¹ 10 0 3

HUS-020 81 6 ¹ 1 3

LEO-001 78 ^{1 1} 3 ¹ 0 2

LEO-003 63 ¹ 10 ¹ 1 3

LEO-004 76 ¹ 3 ¹ 0 2

LEO-005 82 ^{1 1} 3 ¹ 0 2

LEO-006 74 ^{1 1} 3 0 2

LEO-009 80 3 ¹ 0 2

LEO-010 72 ^{1 1} 3 ¹ 1 3

LEO-011 79 ^{1 1} 3 ¹ 0 2

LEO-012 81 ¹ 5 0 2

LEO-013 71 ^{1 1} 3 ¹ 1 3

LEO-014 52 ^{1 1} 3 ¹ 0 2

LEO-015 80 ^{1 1} 10 ¹ 0 1

LEO-016 84 ¹ 3 ¹ 0 2

LEO-017 70 ^{1 1} 1 ¹ 1 3

LEO-019 86 ^{1 1} 3 ¹ 0 2

LEO-021 68 ¹ 3 5 2 0 2

LEO-022 80 2 1 3 2 0 2 Sample Age Gender FAB WHO-2008 Cytogenetics Transformation Risk

(Years) classification classification (IPSS-R) to AML

LEO-026 83 2 2 2 ¹ 0 1

LEO-027 70 1 ¹ 3 ¹ 0 2

LEO-028 82 1 ¹ 3 ¹ 0 2

LEO-030 64 2 ¹ 3 ¹ 0 2

LEO-031 81 1 ¹ 3 0 2

LEO-034 80 1 ¹ 3 ¹ 0 2

LEO-039 79 2 ¹ 3 ¹ 0 2

LEO-040 81 2 2 3 ¹ 0 2

LEO-041 76 1 2 2 ¹ 0 1

LEO-042 82 2 2 3 ¹ 0 2

LEO-043 86 2 1 3 ¹ 0 2

LEO-044 73 1 2 3 ¹ 0 2

LEO-046 88 1 1 1 1 ¹ 0 1

LEO-047 86 1 1 1 ¹ 0 1

LEO-048 79 2 1 3 ¹ 0 2

LEO-050 84 1 3 5 ¹ 0 2

LEO-051 72 2 3 5 ¹ 1 3

LEO-052 51 2 3 6 0 3

LEO-053 76 1 2 3 ¹ 0 2

LEO-054 82 1 2 3 ¹ 0 2

LEO-056 57 1 2 10 ¹ 1 3

LEO-058 76 1 1 3 ¹ 0 2

LEO-059 81 2 1 10 ¹ 0 1

LEO-060 54 1 2 3 ¹ 0 2

LEO-061 86 2 1 10 ¹ 0 1

LEO-062 77 2 3 5 ¹ 0 2

LEO-064 76 1 2 3 ¹ 0 2

LEO-065 72 2 2 3 ¹ 0 2

LEO-070 75 1 1 3 0 2

LEO-071 64 1 1 3 ¹ 0 2

LEO-072 63 2 1 3 ¹ 0 2

LEO-073 85 1 1 3 ¹ 0 2 Sample Age Gender FAB WHO-2008 Cytogenetics Transformation Risk (Years) classification classification (IPSS-R) to AML

MAR- 81 2 1 10 1 0 1 001

MAR- 73 1 1 3 1 0 2 002

MAR- 81 1 1 1 1 1 3 003

MAR- 81 2 1 1 1 1 0 1 004

MAR- 78 1 2 3 1 0 2 005

MAR- 75 2 3 5 1 1 3 006

MAR- 78 1 3 6 1 0 3 007

MAR- 91 1 3 6 1 1 3 008

MAR- 64 2 1 1 1 0 1 009

MAR- 74 1 2 3 1 1 3 013

MAR- 73 1 3 6 1 0 3 014

MAR- 81 1 3 6 4 0 3 015

MAR- 80 1 1 3 1 0 2 016

MAR- 82 1 1 1 1 0 0 1 017

MAR- 61 2 2 3 1 0 2 018

MAR- 81 2 3 6 1 1 3 019

MAR- 68 1 1 3 2 0 2 021

MAR- 79 2 1 1 1 0 1 022 Sample Age Gender FAB WHO-2008 Cytogenetics Transformation Risk (Years) classification classification (IPSS-R) to AML

MAR- 70 1 3 5 0 2

¹

023

MAR- 70 1 1 3 0 2

¹

024

MAR- 59 2 2 2 0 1

¹

025

MAR- 81 2 2 3 0 2

¹

027

MAR- 79 1 1 3 0 2 030

MAR- 82 1 1 3 0 2

¹

032

MAR- 75 2 1 3 0 2

¹

034

MAR- 73 1 3 5 0 2

¹

035

MAR- 82 2 1 10 0 1

¹

037

MAR- 82 1 1 3 0 2

¹

038

MAR- 89 2 1 10 0 1

¹

039

MAR- 82 1 1 3 0 2

¹

041

MAR- 81 1 1 1 0 1

¹

043

MAR- 80 1 3 6 1 3

¹

044

MAR- 85 1 2 3 0 2

¹

045

MAR- 78 1 1 3 0 2

¹

047

MAR- 75 2 2 2 0 1

¹

048

MAR- 82 2 1 3 2 0 2 049 Sample Age Gender FAB WHO-2008 Cytogenetics Transformation Risk (Years) classification classification (IPSS-R) to AML

MAR- 72 1 1 3 3 0 3 050

MAR- 82 1 3 5 2 0 2 051

MAR- 77 1 2 2 1 0 1 052

MAR- 69 1 3 5 1 0 2 053

MUR- 80 1 1 12 3 0 3 002

MUR- 61 1 2 10 1 0 1 005

MUR- 65 1 4 6 3 1 3 006

PAZ-007 75 2 3 5 2 1 3

PAZ-010 68 1 3 5 4 0 3

PAZ-012 85 1 1 3 1 0 2

PAZ-013 50 1 3 5 3 1 3

PAZ-016 64 2 3 5 1 0 2

PAZ-018 80 1 1 3 2 0 2

PAZ-019 71 1 2 3 1 0 2

PAZ-020 63 1 1 1 1 0 1

PAZ-021 86 2 2 3 1 0 2

PAZ-022 69 2 1 3 1 1 3

PAZ-024 79 2 2 3 1 0 2

PAZ-026 78 1 1 3 1 0 2

PAZ-027 79 2 1 1 1 1 0 1

PAZ-028 63 1 3 6 1 0 3

PAZ-029 85 2 1 3 2 0 2

PAZ-030 63 2 2 2 1 0 1

PAZ-032 42 1 3 6 2 1 3

PAZ-033 73 2 1 1 1 0 1

PAZ-035 83 1 1 10 1 0 1

PAZ-038 72 2 1 3 1 0 2 Sample Age Gender FAB WHO-2008 Cytogenetics Transformation Risk (Years) classification classification (IPSS-R) to AML

PAZ-039 82 ¹ 1 3 1 0 2

PAZ-040 80 ¹ 1 1 1 0 1

PAZ-042 77 ¹ 1 3 1 0 2

PAZ-043 85 ¹ 4 6 3 1 3

PAZ-044 39 ¹ 3 5 3 0 3

PAZ-045 73 ¹ 1 3 0 0 2

PAZ-046 81 2 2 1 0 1

PAZ-047 89 ¹ 2 2 1 0 1

PAZ-048 74 ¹ 1 3 2 0 2

PAZ-049 80 ¹ 1 3 1 0 2

PAZ-050 67 ¹ 1 3 1 0 2

PAZ-052 80 1 3 1 0 2

PAZ-053 55 ¹ 4 6 1 0 3

PAZ-055 61 ¹ 1 3 1 1 3

PAZ-057 79 2 3 6 2 1 3

TRIAS- 74 2 1 3 1 1 3 001

TRIAS- 67 2 4 6 3 0 3 002

TRIAS- 31 2 4 6 2 0 3 003

TRIAS- 62 1 1 3 0 1 3 004

TRIAS- 75 2 3 6 2 1 3 005

TRIAS- 77 2 2 3 1 0 2 007

TRIAS- 52 1 1 3 1 0 2 008

VAL-002 78 2 3 5 1 0 2

VAL-003 46 2 3 6 2 0 3

VAL-004 82 1 1 3 0 0 2

VAL-005 61 1 2 3 2 1 3

VAL-006 71 1 1 3 1 0 2 Sample Age Gender FAB WHO-2008 Cytogenetics Transformation Risk

(Years) classification classification (IPSS-R) to AML

VAL-007 84 ¹ 2 2 0 0 1

VAL-008 75 ¹ 3 5 3 1 3

VAL-009 50 ¹ 3 5 4 0 3

VAL-010 79 3 6 3 1 3

VAL-011 76 ¹ 2 3 2 0 2

VAL-012 88 3 5 2 1 3

VAL-013 77 ¹ 2 3 2 0 2

VAL-014 78 ¹ 2 2 1 0 1

VAL-015 61 1 10 0 0 1

VAL-016 76 ¹ 1 3 4 1 3

VAL-017 86 ¹ 1 3 1 0 2

VAL-018 53 ¹ 3 5 4 0 3

VAL-019 73 4 6 4 1 3

VAL-020 85 ¹ 1 3 4 0 3

VAL-021 76 ¹ 3 5 1 1 3

VAL-022 72 ¹ 1 3 1 0 2

VAL-024 58 ¹ 2 3 2 0 2

VAL-025 87 ¹ 3 6 3 1 3

VAL-026 63 2 1 3 4 1 3

VAL-027 80 2 1 10 1 0 1

NH009 61 2 4 6 3 1 3

NH020 65 2 1 3 1 0 2

NH034 81 2 1 3 1 0 2

NH042 85 1 1 1 1 0 1

NH049 76 1 4 6 1 1 3

NH054 40 2 4 6 N/A 1 3

NH094 58 2 1 1 N/A 0 1

NH109 74 2 1 1 N/A 0 1

NH204 62 1 1 1 1 0 1

NH206 19 2 1 1 3 0 3

NH207 47 2 1 3 2 0 2

NH208 82 2 3 6 1 0 3 Sample Age Gender FAB WHO-2008 Cytogenetics Transformation Risk

(Years) classification classification (IPSS-R) to AML

NH209 79 1 4 5 4 1 3

NH210 52 1 4 6 1 1 3

NH211 69 2 3 5 N/A 0 2

NH212 64 1 3 6 1 0 3

NH213 64 2 3 6 1 0 3

NH214 47 2 1 10 1 0 1

NH215 86 1 1 1 N/A 0 1

NH217 77 1 3 5 2 0 2

NH218 33 1 1 1 1 0 1

NH221 73 1 5 13 1 0 2

NH228 78 1 1 3 0 0 2

NH253 73 2 3 6 1 0 3

NH269 78 1 1 1 1 0 1

NH280 48 1 1 1 1 0 1

NH291 64 2 4 6 1 1 3

NH294 66 1 5 13 1 0 2

NH301 67 1 4 6 1 1 3

NH302 77 1 1 3 1 0 2

NH306 85 2 2 3 1 0 2

NH308 80 2 2 3 1 0 2

NH311 84 1 1 12 2 0 2

NH313 78 1 1 3 1 0 2

NH322 66 1 3 5 4 0 3

NH331 79 2 5 13 N/A 0 2

NH337 78 2 1 10 1 0 1

NH341 69 1 1 3 1 0 2

NH345 53 2 2 2 1 0 1

NH348 46 2 1 3 1 0 2

NH353 44 2 4 6 2 1 3

NH359 66 2 3 5 1 0 2

NH381 82 1 5 13 N/A 0 2

NH397 76 1 1 3 1 0 2 Sample Age Gender FAB WHO-2008 Cytogenetics Transformation Risk

(Years) classification classification (IPSS-R) to AML

NH399 71 1 1 3 1 0 2

NH422 69 1 4 6 1 1 3

NH424 77 1 5 13 1 0 2

NH434 72 1 3 6 3 0 3

NH435 65 2 2 2 1 0 1

NH439 75 1 1 3 1 0 2

Gender: 1 - Male; 2- Female. FAB classification: 1 - RA; 2- RARS; 3- RAEB; 4- RAEB-t; 5- CMML. WHO 2008 Classification: 1 - RCUD; 2- RARS; 3- RCMD; 5- RAEB-I; 6- RAEB-II; 10- 5q- syndrome; 1 1 - MDS unclassified; 12- Hypoplastic MDS; 13- CMML. Cytogenetics (IPSS-R): 0- Very good; 1 - Good; 2- Intermediate; 3- Poor; 4- Very poor; N/A (non- available). Transformation to AML: 0- No; 1 -Yes. Risk: 1 - Low; 2- Intermediate; 3- High.

RNA extraction, reverse transcription and quantitative real-time PCR assay

Plasma samples were collected in EDTA tubes (BD Vacutainer®). Total RNA was extracted from cryopreserved plasma samples using miRCURY™ RNA Isolation kit- Biofluids (Exiqon) according to manufacturer^'s instructions. Samples were reverse transcribed and 0 quantified by real-time PCR using miRCURY LNA™ Universal RT microRNA PCR (Exiqon).

PCR reactions were performed using an ABi 7900HT (Sequence Detection System software version 2.4) (Applied Biosystems, USA). Normalization was performed with has-miR-16 (SEQ ID No. 12) because it had the lowest expression variability in the miRNA expression 5 patient dataset. The data presented correspond to the Ct value by the 2^"AACt method as named Relative Quantity (RQ) (Livak and Schmittgen, 2001) (AACt = (Ct of microRNA of interest - Ct of hsa-miR-16)_patient - (Ct of microRNA of interest - Ct of hsa-miR-16)_caiib_rator ·

Data analysis was performed at Genomics Unit in Scientific Park from Madrid using Integromics ^'RealTime Statminer® v.4.5.0.7 (Goni R, 2009) and statistical analysis was 0 performed at Statistical Methods Department from University of Zaragoza using SPSS 19.0.

The data obtained by the present example show that seventeen miRNAs were differentially expressed between plasma-MDS samples and plasma-healthy controls (Figure 1). Sixteen miRNAs were over-expressed and only one was under-expressed (Figure 1). As indicated above, the expression levels of a disease-associated miRNA may also be used to monitor the treatment and disease state of a patient. Diagnosis of a disease state in a patient allows for prognosis and selection of therapeutic strategy. In this sense, differences in miRNA levels in low, intermediate and high risk MDS patients were analysed. A non-parametric study (Kruskal-Wallis) was used. The data obtained show that four of these miRNAs were significantly associated with low, intermediate and high risk groups (p<0.01) (Figure 2).

Although hsa-miR-140-3p (SEQ ID No. 8) was not deregulated when we compared the data obtained in plasma-MDS samples and plasma-healthy controls (Figure 1), this miRNA could be used to further stratify patients in each risk group into MDS. These findings might serve as a basis for a discriminant model to be used in the classification of patients based on the miRNAs and additional covariates (sex, age...).

Moreover, it is important to note that hsa-let-7e-5p (SEQ ID No. 7) and hsa-miR-99b-5p (SEQ ID No. 3) miRNAs are located in chromosome 19 and are included in the same cluster

(www.mirbase.org).

In addition, the present invention analysed the progression to AML of patients with MDS by a non-parametric study (Mann- Whitney U). Only has-miR-99b-5p (SEQ ID No. 3) was significantly associated (p<0.05) with progression to AML.

Therefore, these extracellular miRNAs disclosed in the present invention are biological markers for the diagnosis/prognosis of MDS and progression to AML. Additionally, a differential expression of a disease-associated miRNA, specifically MDS, compared to a control may be used as a diagnostic that a patient suffers from the disease. Expression levels of a disease-associated miRNA may also be used to monitor the treatment and disease state of a patient. Diagnosis of a disease state in a patient allows for prognosis and selection of therapeutic strategy. Moreover, in the present invention, the sequences of miRNAs known in the prior art (Tables 2 and 3) can be combined together with the miRNAs of the invention, for a greater reliability in the diagnosis and prognosis of MDS. Bibliography

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Claims

C L A I M S miRNA selected from those comprising a sequence selected from SEQ ID Nos: 1-11 , 13- 20, or variants, or precursors, or orthologues, or combinations thereof, for use in the diagnosis and/or prognosis ofMDS. miRNA according to claim 1 selected from those comprising a sequence selected from SEQ ID Nos: 1, 5, 6, 8, 9, 13, 15, 17, or variants, or precursors, or orthologues, or combinations thereof. miRNA according to claim 1 selected from those comprising a sequence selected from SEQ ID Nos: 2, 3, 4, 7, 10, 11, 14, 16, 17, 18, 19, 20, or variants, or precursors, or orthologues or combinations, thereof. miRNA according to claim 1 selected from those comprising a sequence selected from SEQ ID Nos: 2, 3, 4, 7, 10,14 or variants, or precursors, or orthologues, or combinations thereof. miRNA according to any of claims 1 -4 characterized in that could be further combined with at least one of miRNAs selected from those comprising a sequence selected from SEQ ID Nos: 21-103 or variants, or precursors, or orthologues, or combinations thereof.

Combination of miRNAs sequences consisting in SEQ ID Nos: 1-11, 13-20, or variants, or precursors, or orthologues, or combinations thereof, for use in the diagnosis and/or prognosis of MDS.

Combination of miRNAs sequences according to claim 6 consisting in SEQ ID Nos: 1, 5, 6, 8, 9, 13, 15, 17, or variants, or precursors, or orthologues, or combinations thereof.

Combination of miRNAs sequences according to claim 6 consisting in SEQ ID Nos: 2, 3, 4, 7, 10, 11, 14, 16, 17, 18, 19, 20, or variants, or precursors, or orthologues, or combinations thereof.

9. Combination of miRNAs sequences according to claim 6 consisting in SEQ ID Nos: 2, 3, 4, 7, 10, 14 or variants, or precursors, or orthologues, or combinations thereof.

10. Combination of miRNAs sequences according to any of claims 6-9 characterized in that could be further combined with at least one of miRNAs selected from those comprising a sequence selected from SEQ ID Nos: 21-103 or variants, or precursors, or orthologues, or combinations thereof.

11. miRNA selected from those comprising a sequence selected from SEQ ID Nos: 3, 7, 8, or 10, or variants, or precursors, or orthologues, or combinations, thereof for use in the prognosis of MDS.

12. miRNA according to claim 11 wherein the sequence is SEQ ID NO: 3, or variants, precursors, or orthologues, or combinations thereof.

13. Combination of miRNAs sequences consisting in SEQ ID Nos: 3, 7, 8, or 10, or variants, or precursors, or orthologues, or combinations, thereof, for use in the prognosis of MDS.

14. Use of at least one miRNA selected from those comprising a sequence selected from SEQ ID Nos: 1-11, 13-20, or variants, or precursors, or orthologues, or combinations, thereof for the manufacture of a kit for the diagnosis and/or prognosis of MDS. 15. Use according to claim 14 wherein at least one miRNAs selected from SEQ ID Nos: 1-11, 13-20 or variants, or precursors, or orthologues, or combinations, thereof is further combined with those comprising a sequence selected from SEQ ID Nos: 21-103 or variants, or precursors, or orthologues, or combinations, thereof. 16. Use according to any of claims 14 or 15 where at least one miRNA is selected from those comprising a sequence selected from SEQ ID Nos: 1, 5, 6, 8, 9, 13, 15, 17, or variants, or precursors, or orthologues, or combinations, thereof.

17. Use according to any of claims 14 or 15where at least one miRNA is selected from those comprising a sequence selected from SEQ ID Nos: 2, 3, 4, 7, 10, 11, 14, 16, 17, 18, 19, 20, or variants, or precursors, or orthologues, or combinations, thereof.

18. Use according to any of claims 14 or 15where at least one miRNA is selected from those comprising a sequence selected from SEQ ID Nos: 2, 3, 4, 7, 10, 14, or variants, or precursors, or orthologues, or combinations, thereof.

19. Use of a combination of miRNAs sequences consisting in SEQ ID Nos: 1-11, 13-20, or variants, or precursors, or orthologues, or combinations thereof, for the manufacture of a kit for the diagnosis and/or prognosis of MDS. 20. Use according to claim 19 wherein the combination of miRNAs sequences consisting in SEQ ID Nos: 1-11, 13-20, or variants, or precursors, or orthologues, or combinations thereof are further combined with those comprising a sequence selected from SEQ ID Nos: 21-103 or variants, or precursors, or orthologues, or combinations, thereof.

21. Use according to any of claims 19 or 20 where the combination consisting in SEQ ID Nos: 1, 5, 6, 8, 9, 13, 15, 17 or variants, or precursors, or orthologues, or combinations thereof.

22. Use according to any of claims 19 or 20 where the combination consisting in SEQ ID Nos: 2, 3, 4, 7, 10, 11, 14, 16, 17, 18, 19, 20 or variants, or precursors, or orthologues, or combinations thereof.

23. Use according to any of claims 19 or 20 where the combination consisting in SEQ ID Nos: 2, 3, 4, 7, 10, 14 or variants, or precursors, or orthologues, or combinations thereof.

24. Use of at least one miRNA selected from those comprising a sequence selected from SEQ ID Nos: 3, 7, 8, or 10, or variants, or precursors, or orthologues, or combinations, thereof for the manufacture of a kit for the prognosis of MDS.

25. Use of a combination of miRNAs sequences consisting in SEQ ID Nos: 3, 7, 8, or 10, or variants, or precursors, or orthologues, or combinations, thereof, for the manufacture of a kit for the prognosis of MDS.

26. A kit comprising at least one probe that hybridizes with at least one miRNA selected from those comprising a sequence selected from SEQ ID No: 1-11, 13-20, or variants, or precursors, or orthologues, or combinations thereof.

27. Kit according to claim 26 which further comprises at least one probe that hybridizes with at least one miRNA selected from those comprising a sequence selected from SEQ ID Nos: 21-103 or variants, or precursors, or orthologues, or combinations thereof.

28. Kit according to any of claims 26 or 27 which comprises at least one probe that hybridizes with at least one miRNA selected from those comprising a sequence selected from SEQ ID Nos: 1, 5, 6, 8, 9, 13, 15, 17, or variants, or precursors, or orthologues, or combinations thereof.

29. 29 Kit according to any of claims 26 or 27 which comprises at least one probe that hybridizes with at least one miRNA selected from those comprising a sequence selected from SEQ ID Nos: 2, 3, 4, 7, 10, 11, 14, 16, 17, 18, 19, 20, or variants, or precursors, or orthologues, or combinations thereof.

30. Kit according to any of claims 26 or 27 which comprises at least one probe that hybridizes with at least one miRNA selected from those comprising a sequence selected from SEQ ID Nos: 2, 3, 4, 7, 10, 14, or variants, or precursors, or orthologues, or combinations, thereof.

31. Kit, according to anyone of claims 26-30 wherein the probe comprises a sequence that specifically hybridizes with at least one complementary DNA derived from the transcript of the corresponding miRNA selected from those comprising a sequence selected from SEQ ID No: 1-103 or variants, or precursors, or orthologues, or combinations, thereof.

32. Kit, according to anyone of claims 26-30, wherein the probe comprises a sequence that specifically hybridizes with at least one complementary RNA derived from the transcript of a specific DNA derived from the transcript of the corresponding miRNA selected from those comprising a sequence selected from SEQ ID No: 1-103, or variants, or precursors, or orthologues, or combinations, thereof.

33. A kit comprising a combination of probes that hybridizes with a combination of miRNAs sequences consisting in SEQ ID Nos: 1-11, 13-20, or variants, or precursors, or orthologues, or combinations thereof

34. Kit according to claim 33 which further comprises at least one probe that hybridizes with at least one miRNA selected from those comprising a sequence selected from SEQ ID Nos: 21-103 or variants, or precursors, or orthologues, or combinations, thereof.

35. Kit according to any of claims 33 or 34 which comprises a combination of probes that hybridizes with a combination of miRNAs sequences consisting in SEQ ID Nos: 1, 5, 6, 8, 9, 13, 15, 17, or variants, or precursors, or orthologues, or combinations, thereof.

36. Kit according to any of claims 33 or 34 which comprises a combination of probes that hybridizes with a combination of miRNAs sequences consisting in SEQ ID Nos: 2, 3, 4,

7, 10, 11, 14, 16, 17, 18, 19, 20, or variants, or precursors, or orthologues, or combinations, thereof.

37. Kit according to any of claims 33 or 34 which comprises a combination of probes that hybridizes with a combination of miRNAs sequences consisting in SEQ ID Nos: 2, 3, 4, 7, 10, 14, or variants, or precursors, or orthologues, or combinations, thereof.

38. Kit, according to anyone of claims 33-37 wherein the combination of probes consisting in a sequence that specifically hybridizes with at least one complementary DNA derived from the transcript of the corresponding combination of miRNAs sequences consisting in

SEQ ID No: 1-103 or variants, or precursors, or orthologues, or combinations thereof.

39. Kit, according to anyone of claims 33-38, wherein the combination of probes comprises a sequence that specifically hybridizes with at least one complementary RNA derived from the transcript of a specific DNA derived from the transcript of the corresponding combination of miRNA sequence consisting in SEQ ID No: 1-103, or variants, or precursors, or orthologues, or combinations thereof.

40. A kit comprising at least one probe that hybridizes with at least one miRNA selected from those comprising a sequence selected from SEQ ID Nos: 3, 7, 8 or 10, or variants, or precursors, or orthologues, or combinations thereof.

41. Kit according to claim 40 which further comprises at least one probe that hybridizes with at least one miRNA selected from those comprising a sequence selected from SEQ ID Nos: 21-103 or variants, or precursors, or orthologues, or combinations thereof.

42. A kit comprising a combination of probes that hybridizes with a combination of miRNAs sequences consisting in SEQ ID Nos: 3, 7, 8 or 10, or variants, or precursors, or orthologues, or combinations thereof Kit according to claim 42 which further comprises at least one probe that hybridizes with at least one miRNA selected from those comprising a sequence selected from SEQ ID Nos: 21-103 or variants, or precursors, or orthologues, or combinations, thereof. 44. Use of the kit according to anyone of claims 26-43 for the in vitro diagnosis and/or prognosis of MDS.

45. An in vitro method of diagnosis and/or prognosis of MDS which comprises determining in a sample from a subject, the expression level of at least one miRNA selected from those comprising a sequence selected from SEQ ID Nos: 1-11, 13-20, or variants, or precursors, or orthologues, or combinations thereof and the comparison of said expression level with respect to the expression values obtained from healthy controls.

46. An in vitro method according to claim 45 which further comprises the determination of the expression level of at least one miRNAs selected from those comprising a sequence selected from SEQ ID Nos: 21-103 or variants, or precursors, or orthologues, or combinations thereof.

An in vitro method according to any of claims 45-46 which comprises the determination of the expression level of the group of miRNAs selected from those comprising a sequence selected from SEQ ID Nos: 1, 5, 6, 8, 9, 13, 15, 17, or variants, or precursors, or orthologues, or combinations thereof.

An in vitro method according to any of claims 45-46 which comprises the determination of the expression level of the group of miRNAs selected from those comprising a sequence selected from SEQ ID Nos: 2, 3, 4, 7, 10, 11, 14, 16, 17, 18, 19, 20, or variants, or precursors, or orthologues, or combinations thereof.

49. An in vitro method according to any of claims 45-46 which comprises the determination of the expression level of the group of miRNAs selected from those comprising a sequence selected from SEQ ID Nos: 2, 3, 4, 7, 10, 14, or variants, or precursors, or orthologues, or combinations, thereof.

50. An in vitro method of diagnosis and/or prognosis of MDS which comprises determining in a sample from a subject, the expression level of a combination of miRNA sequences consisting in SEQ ID Nos: 1 -11, 13-20, or variants, or precursors, or orthologues, or combinations thereof and the comparison of said expression level with respect to the expression values obtained from healthy controls. 51. An in vitro method according to claim 50 which further comprises the determination of the expression level of at least one miRNAs selected from those comprising a sequence selected from SEQ ID Nos: 21-103 or variants, or precursors, or orthologues, or combinations thereof. 52. An in vitro method according to any of claims 50-51 which comprises the determination of the expression level of the combination of miRNAs sequences consisting in SEQ ID Nos: 1, 5, 6, 8, 9, 13, 15, 17, or variants, or precursors, or orthologues, or combinations thereof. 53. An in vitro method according to any of claims 50-51 which comprises the determination of the expression level of the combination of miRNAs sequences consisting in SEQ ID Nos: 2, 3, 4, 7, 10, 11, 14, 16, 17, 18, 19, 20, or variants, or precursors, or orthologues, or combinations thereof. 54. An in vitro method according to any of claims 50-51 which comprises the determination of the expression level of the combination of miRNAs sequences consisting in SEQ ID Nos: 2, 3, 4, 7, 10, 14, or variants, or precursors, or orthologues, or combinations thereof.

55. An in vitro method according to any of claims 45-54 wherein the sample of the subject is selected from: saliva, blood, plasma, serum, bone marrow, or urine.

56. An in vitro method according to claim 55 wherein the sample is selected from peripheral blood and/or plasma. 57. An in vitro method according to any of claims 45-56 wherein the expression levels are obtained by Real Time-PCR.

58. An in vitro method according to any of claims 45-57 wherein the miRNAs of subjects with MDS are differentially expressed with respect to the corresponding miRNA expression levels of healthy controls. An in vitro method according to claim 58 wherein the miRNAs sequences SEQ ID No. 1, 3-7, 9-11 and 13-18 and 20 are over-expressed with respect to the corresponding miRNA expression levels of healthy controls. 60. An in vitro method according to claim 58 wherein the miRNAs sequence SEQ ID No. 2 is under-expressed with respect to the corresponding miRNA expression levels of healthy controls.