ES2787725A1 - METHOD OF OBTAINING USEFUL DATA TO DIAGNOSE HEREDITARY HEMORRHAGIC TELANGIECTASIA (Machine-translation by Google Translate, not legally binding) - Google Patents

Abstract

A method of obtaining useful data to diagnose inherited hemorrhagic telangiectasia. The present invention relates to the use of a set of miRNAs transported in plasma exosomes in obtaining useful data to diagnose hereditary hemorrhagic telangiectasia, as well as for its classification into HHT type 1 or HHT type 2. A kit for carry out an in vitro diagnostic method using the data obtained. (Machine-translation by Google Translate, not legally binding)

Description

DESCRIPTION

METHOD OF OBTAINING USEFUL DATA FOR DIAGNOSING

HEREDITARY HEMORRHAGIC TELANGIECTASIA

FIELD OF THE INVENTION

The present invention is within the field of Molecular Biology and Clinical Medicine. Specifically, it relates to an in vitro method of obtaining data useful for predicting or predicting hereditary hemorrhagic telangiectasia.

BACKGROUND OF THE INVENTION

Hereditary hemorrhagic telangiectasia (HHT)

Hereditary hemorrhagic telangiectasia (HHT), or Rendu-Osler-Weber syndrome (ORPHA: 774), is a vascular dysplasia with autosomal dominant transmission with a prevalence of approximately 1/8000, which is why it is considered a rare, minority or infrequent. The vast majority of HHT patients (~ 85%) present heterozygous mutations in either the endoglin gene (ENG) or the ALK1 gene ( ACVRL1), giving rise to the HHT1 (OMIM: # 187300) and HHT2 types (# 600376), respectively. So far, up to 507 different mutations have been described throughout the endoglin gene and up to 571 in the case of the ALK1 gene (http://www.arup.utah.edu/database/HHT/). There is also a small percentage of patients who do not present mutations in any of these genes, although 2 loci have been identified on chromosomes 5 and 7 that give rise to HHT3 (5q31.3-q32; OMIM: # 601101) and HHT4 ( 7p14; OMIM: # 610655), respectively. Finally, recent studies have identified mutations in the BMP9 (GDF2) gene as a fifth type of HHT (HHT5; OMIM: # 615506) [A. Macri, R. Bermudez, Osler-Weber-Rendu Disease (Hereditary Hemorrhagic Telangiectasia, HHT), StatPearls [Internet], StatPearls Publishing, Treasure Island (FL), 2018].

Generally, for the diagnosis of HHT a consensual clinical profile is used that is known as the Cura? Ao criteria [CL Shovlin, AE Guttmacher, E. Buscaini, ME Faughnan, RH Hyland, CJ Westermann, AD Kjeldsen, H. Plauchu, Diagnostic criteria for hereditary hemorrhagic telangiectasia (Rendu-Osler-Weber syndrome), Am J Med Genet 91 (1) (2000) 66-7], and include the following four features: 1) Epistaxis recurring; 2) Telangiectasias in skin and / or mucosa; 3) Arteriovenous malformations in internal organs; and 4) Family history. The diagnosis of HHT is established if three of these criteria are present. If one or two criteria are met, determination of the genetic mutation is recommended. However, the provision of a sequencing-based diagnostic service is controversial due to the prevalence of new mutations in affected individuals and due to difficulties in detecting heterozygous mutations, especially when they are due to a single base change in the sequence of DNA associated with disease.

Use of exosomes as diagnostic markers

Exosomes are microvesicles of <200 nm in diameter present in all biological fluids that transport molecules of diverse nature from the tissue where they are produced to another receptor where said cargo exerts its function. The exosomes circulating in the blood therefore have a high bioavailability and are relatively easy to obtain. In this way, the analysis of its content can establish new biomarkers associated with a certain pathology such as hereditary hemorrhagic telangiectasia (HHT), or Rendu-Osler-Weber syndrome (ORPHA: 774).

At present, there are numerous studies in which the content of these exosomes is used as biomarkers of a certain pathology, as in different types of cancer [F. Lousada-Fernandez, O. Rapado-Gonzalez, JL Lopez-Cedrun, R. Lopez-Lopez, L. Muinelo-Romay, MM Suarez-Cunqueiro, Liquid Biopsy in Oral Cancer, Int J Mol Sci 19 (6) (2018) E1704 .], [H. Huang, X. Zheng, C. Cai, Z. Yao, S. Lu, X. Meng, Y. Miao, Z. He, F. Zou, Exosomes derived from breast cancer lung metastasis subpopulations promote tumor self-seeding, Biochem Biophys Res Commun (2018) S0006-291X (18) 31321-4], [Y. Toiyama, Y. Okugawa, J. Fleshman, C. Richard Boland, A. Goel, MicroRNAs as potential liquid biopsy biomarkers in colorectal Cancer: A systematic review, Biochim Biophys Acta (2018) S0304-419X (18) 30067-2], autoimmune diseases [Y. Ishibe, M. Kusaoi, G. Murayama, T. Nemoto, T. Kon, M. Ogasawara, K. Kempe, K. Yamaji, N. Tamura, Changes in the Expression of Circulating microRNAs in Systemic Lupus Erythematosus Patient Blood Plasma After Passing Through a Plasma Adsorption Membrane, Ther Apher Dial 22 (3) (2018) 278-289], [I. Manna, E. Iaccino, V. Datodón, S. Barone, E. Vecchio, S. Mimmi, E. Filippelli, G. Demonte, S. Polidoro, A. Granata, S. Scannapieco, I. Quinto, P. Valentino, A. Quattrone, Exosomeassociated miRNA profile as a prognostic tool for therapy response monitoring in multiple sclerosis patients, FASEB J (2018) 2018) fj201701533R], neurological [TT

Yang, C.G. Liu, S.C. Gao, Y. Zhang, P.C. Wang, The Serum Exosome Derived MicroRNA- 135a, -193b, and -384 Were Potential Alzheimer's Disease Biomarkers, Biomed Environ Sci 31 (2) (2018) 87-96], [X.Y. Cao, J.M. Lu, Z.Q. Zhao, M.C. Li, T. Lu, X.S. An, L.J. Xue, MicroRNA biomarkers of Parkinson's disease in serum exosome-like microvesicles, Neurosci Lett 644 (2017) 94-99] or cardiovascular [F. Jansen, Q. Li, Exosomes as Diagnostic Biomarkers in Cardiovascular Diseases, Adv Exp Med Biol 998 (2017) 61-70], [M. Lu, S. Yuan, S. Li, L. Li, M. Liu, S. Wan, The Exosome-Derived Biomarker in Atherosclerosis and Its Clinical Application, J Cardiovasc Transl Res (2018)], [T. Wu, Y. Chen, Y. Du, J. Tao, Z. Zhou, Z. Yang, Serum Exosomal MiR-92b-5p as a Potential Biomarker for Acute Heart Failure Caused by Dilated Cardiomyopathy, Cell Physiol Biochem 46 (5) ( 2018) 1939-1950]. In fact, many of these studies have contributed to the registration of patents aimed at the molecular diagnosis of this disease.

Applications of exosomes for diagnosis can also be found in [WO / 2017/181183] for lymphoma, [WO / 2016/148313] for different types of cancer, [WO / 2012/126531] for acute coronary syndrome, or [WO / 2016/08227] for Kawasaki disease, among others.

The characterization of these biomarkers in exosomes falls within what is called liquid biopsy, a term originally coined for its great potential in cancer diagnosis. A liquid biopsy basically consists of extracting a small volume of blood (or any other biological fluid), isolating the exosomes from serum or plasma and analyzing their molecular content, mainly proteins and / or nucleic acids (DNA, mRNA, microRNA)

OBJECT OF THE INVENTION

The authors of the present invention have been able to obtain by the technique of NGS ( Next Generation Sequencing) RNA-seq for the first time the pattern of differential expression of microRNAs (miRNAs) transported in plasma exosomes characteristic of HHT (n = 30) compared to unaffected individuals (n = 10). Furthermore, they have established a group of up to 66 differentially expressed miRNAs that characterize HHT. They have also been able to differentiate between the HHT1 and HHT2 types (Figure 1) based on the global expression profiles of miRNAs, and based on their studies, 8 miRNAs have been selected for having a clear diagnostic value, as shown by the analysis of ROC ( Receiver Operating Characteristic) curves . This group of 8 miRNAs allows a fast and reliable diagnosis of HHT (Figure 3). In this way, this new molecular diagnostic method included in the present invention represents a great advance that would confirm at a molecular level the diagnosis by the clinical criteria of Cura? a or of patients with HHT.

Therefore, a first aspect of this invention refers to the use of a set of miRNAs transported in plasma exosomes in obtaining useful data to diagnose HHT.

A second aspect of the invention refers to a method for obtaining useful data for the diagnosis of HHT, in an individual, which comprises the detection, in a sample of said individual, of the relative expression levels of one of these miRNAs . A third aspect of the invention refers to a kit or device comprising at least one oligonucleotide (s) capable of hybridizing with any one or more of the miRNAs mentioned.

The invention also extends to computer programs adapted so that any processing means can carry out the methods of the invention.

Currently, the confirmation of the diagnosis of HHT by the clinical criteria of Cura? Ao is carried out by sequencing the target genes responsible for HHT, aimed at identifying the causative mutation. The main advantage of the present invention is therefore that the genetic diagnosis is carried out by determining the relative expression of a set of plasma exosomal miRNAs.

The fact that the present invention uses miRNAs (ie, very short and well defined RNAs) as indicative factors comes with the advantage of easy and reliable detection against some known methods, such as the use of messenger RNA (mRNA). The mRNA molecules are typically several hundred or even several thousand bases in size; molecules so large and at the same time delicate, so they are generally prone to degradation, and their detection depends on the choice (often trial and error) of the appropriate probe, which makes such tests, in In principle, less reliable than the methods of the present invention.

In addition, real-time PCR can be used, which is a very popular technique today that has evolved enormously and whose costs have consequently been significantly reduced, especially those of the required instruments.

Optimization of TaqMan® Advanced probes for miRNAs has further increased the detection limit (~ 10 copies / reaction) and eliminated false positives providing a higher specificity with respect to the classical method based on SYBR chemistry. Therefore, the present invention constitutes a much more efficient and effective diagnostic method than the current one.

There are also a number of other advantages. The symptoms of HHT usually appear during adolescence around 12 years of age and it is not until the age of 50 when it presents full penetrance. Due to this gradual appearance of the symptoms, one of the main problems that a patient with HHT faces is that from the time the symptoms appear and they first go to the doctor until a definitive diagnosis is made, they usually elapse on average ~ 7 years, even more than 10 years for 20% of those affected. This implies a hospital pilgrimage on the part of patients who have to go through the consultation of various specialists frequently in different localities and provinces or even autonomous communities, with the consequent dissatisfaction of these patients with the health system and a high expense related to care of the disease that concludes in a perception of discrimination and disability on the part of the patients. Thus, this invention aims to replace the current genetic diagnostic method and thus reduce the time required for the determination of HHT.

DESCRIPTION OF THE FIGURES

Figure 1. A. RNAseq results showing that a set of exosomal miRNAs differentially expressed in HHT allow to discriminate patients from healthy donors and even the type of HHT ( heatmaps). The control groups (n = 8), HHT1 (n = 13) and HHT2 (n = 7), made up of independent donors with no family relationship, were analyzed. Some samples were eliminated from the analysis by criteria of coherence of the detected signal. B. PCA analysis. Each point represents a sample: Control (green), HHT1 (blue) and HHT2 (red). The samples are grouped according to their healthy or pathological origin.

Figure 2. Workflow diagram. 5 ml of peripheral blood is drawn by venipuncture into tubes with sodium citrate as an anticoagulant. Plasma is separated by centrifugation (1500xg, 10 min.). Exosomes are isolated from plasma and hence the total RNA content. The miRNAs are detected by real-time PCR (qPCR) with specific TaqMan® Advanced probes.

Figure 3. Relative expression profile of miRNAs transported in plasma exosomes. The comparison with the control group was made using the analysis of variance test (ANOVA) with the Bonferroni correction (n = 10, * p <0.05; ** p <0.01; *** p <0.005 )

Figure 4.- ROC curves to determine the diagnostic accuracy of the test. Sensitivity, specificity and area under the curve (AUC,%) are plotted. Biomarkers are considered good (90% <75%, yellow) and excellent (> 90%, green) with a statistical significance with p value <0.005.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

Throughout this document the abbreviation “HHT will be used to refer to hereditary hemorrhagic telangiectasia, also called Rendu-Osler-Weber Syndrome or Rendu-Osler-Weber disease.

Two genes are involved in its pathogenesis, which determine two different forms of the same disease "HHT1" and "HHT2". In HHT1, the affected gene ( ENG) is located on chromosome 9 and encodes a transmembrane protein called endoglin. In HHT2, the alteration affects the ACVRL1 gene is located on chromosome 12, which encodes the receptor with kinase activity similar to the type II receptor for activin A (ALK1, activin A receptor type Il-like kinase 1), and which forms part of the TGF-B receptor complex.

A microRNA (abbreviated as "miRNA") is a small non-coding RNA molecule that is found in both plants and animals, and that frequently exerts a function related to the post-transcriptional regulation of gene expression.

MicroRNAs contain approximately 22 nucleotides (approx. 18-25 nucleotides), are single-stranded RNAs with a certain secondary structure, and negatively regulate (inhibit) gene expression through inhibition of mRNA translation or cleavage. Therefore, miRNAs are post-transcriptional regulators that bind to complementary sequences in messenger RNA (mRNA) transcripts, generally resulting in translational repression or target degradation and gene silencing. In a particular aspect of the invention, understanding the molecular details of the action of the miRNAs of the invention is not critical, since the detected levels of the indicative miRNAs allow to carry out the methods of the invention.

Under a standard naming system, names are assigned to experimentally confirmed miRNAs as follows: the prefix "mir" is followed (by a hyphen and) a number, so the latter can indicate the order of the names. Uncapitalized "mir-" refers to the pre-miRNA, while an uppercase "miR-" refers to the mature form. MiRNAs with nearly identical sequences, except for one or two nucleotides, are annotated with an additional lowercase letter, for example miR-99a. Pre-miRNAs that lead to 100% identical mature miRNAs but that are located at different places in the genome are indicated with an additional dash number suffix. The species of origin can be designated with a three letter prefix, for example hsa-miR-223 is a human miRNA (Homo sapiens). Since in the context of this document, all individualized miRNAs are human miRNAs, the "hsa-" prefix is sometimes omitted. When two mature miRNAs originate from opposite arms of the same pre-miRNA, they are denoted by a suffix -3p or -5p, such as miR-142-3p. When relative expression levels are known, an asterisk after the name indicates a miRNA expressed at low levels relative to the miRNA on the opposite arm of a hairpin. Most of the known miRNA genes are in intergenic regions or oriented antisense to neighboring genes and are therefore believed to be transcribed as independent units. Their genes are generally transcribed using RNA polymerase II, and the transcripts are processed, exported from the nucleus, and further processed by specific mechanisms, as is well known in the art (see, for example, He et al., Nat. Rev. Genet. 2004 Jul; 5 (7): 522-31). The miRNA sequences can be accessed at http://www.mirbase.org.

Thus, the term "miRNA of the invention" will be used to refer to any miRNA selected from the group consisting of miR-142, miR-150, miR-486, miR-106b, miR-183, miR-654, miR-9 and miR-29c (Table 1).

Tablal. MiRNA sequences

The group formed by the miR-142, miR-150 and miR-486 miRNAs will be called "HC group " .

The group formed by the miR-106b, miR-183 and miR-654 miRNAs will be called "group H1" .

The group formed by the miR-9 and miR-29c miRNAs will be called "H2 group" .

The term "control miRNA" refers to a plasma exosomal miRNA whose expression level remains unchanged in healthy individuals and individuals suffering from HHT.

In a particular embodiment, the control miRNA will be the miRNA with Seq ID n ° 9, called "hsa-miR-103a-3p":

The term "identity", as used herein, refers to the ratio of identical nucleotides or amino acids between two nucleotide or amino acid sequences being compared. Sequence comparison methods are known in the art, and include, but are not limited to, the GAG program, including GAP (Devereux et al., Nucleic Acids Research 12: 287 (1984) Genetics Computer Group University of Wisconsin , Madison, (Wl), BLAST, BLASTP or BLASTN, and FASTA (Altschul et al., 1999. J. Mol. Biol. 215: 403-410.

An "isolated biological sample" includes, but is not limited to, cells, tissues and / or biological fluids of an organism, obtained by any method known to one of ordinary skill in the art. Preferably, the isolated biological sample is peripheral venous blood.

The term "individual", as used in the description, refers to animals, preferably mammals, and more preferably humans. The terms "individual""humansubject" and "subject" are used interchangeably herein and are synonymous with "Patient", and is not intended to be limiting in any way, and it can be of any age, sex and physical condition.

The terms "one or more " or "at least one" or "several" as used in this document include one and the individualized specification of any number that is more than one, such as two, three, four, five , six, etc. "

The term "comprises" may also be interpreted, in a particular embodiment, as "consists of". The term "comprises" and its variations are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and characteristics of the invention will emerge partly from the description and partly from the practice of the invention.

Invention markers

Therefore, a first aspect of the invention refers to the use of miR-142, miR-150, miR-486, miR-106b, miR-183, miR-654, miR-9, miR-29c, or any of their combinations, in obtaining useful data to diagnose HHT.

In a particular embodiment of this aspect of the invention, at least two HC group markers are used, preferably the three HC group markers, simultaneously.

In an even more preferred embodiment of this aspect of the invention, all the markers of the invention are used simultaneously (miR-142, miR-150, miR-486, miR-106b, miR-183, miR-654, miR -9 and miR-29c).

Methods of the invention

A second aspect of the invention refers to a method for obtaining useful data, hereinafter "first method of the invention", for the diagnosis of HHT, in an individual, comprising detection, in an isolated biological sample of said individual, of the expression levels of one or more miRNAs of the invention, in particular a miRNA that is selected from the list consisting of: SEQ ID NO: 1 (miR-142); SEQ ID NO: 2 (miR-150); SEQ ID NO: 3 (miR-486); SEQ ID NO: 4 (miR-106b); SEQ ID NO: 5 (miR-183); SEQ ID NO: 6 (miR-654); SEQ ID NO: 7 (miR-9) and SEQ ID NO: 8 (miR-29c). The inventors have shown that the levels of one or more of these miRNAs in an individual can be indicative that said individual suffers from HHT.

Preferably, the sample is peripheral venous blood or plasma from a peripheral venous blood sample.

Preferably, the first method of the invention comprises detecting the expression levels of at least 2 markers of the HC group, more preferably of the 3 markers of the HC group.

In a particular embodiment, the first method of the invention includes the individual in the group of individuals with HHT when they present a decrease in the level of expression of miR-142, a decrease in the level of expression of miR-150 or an increase in the level of miR-486 expression, or any combination thereof.

Preferably, the first method of the invention includes the individual in the group of individuals with HHT when they show a decrease in the level of expression of miR-142, a decrease in the level of expression of miR-150 and an increase in the level of expression of miR-486.

The first method of the invention involves the comparison of said miRNA levels with the levels of said miRNAs from a reference sample or with a median value. In the context of the present invention, "reference sample" is understood as the reference sample that is used to determine the variation of the expression levels of the miRNA genes of the present invention. In one embodiment, the reference value is obtained from the signal provided using a sample obtained from an individual who does not have the disease (ie, who does not have HHT).

Preferably, samples (reference) are taken from several individuals who do not have the disease and combined, so that the reference value reflects the average value of said molecules in the population of individuals who do not have HHT. "Reference value" is the expression level of a miRNA of the invention in a reference sample

In a preferred embodiment, the expression levels are normalized with respect to the control miRNA, in order to alleviate the expression variations of non-biological origin.

In a more preferred embodiment, the control miRNA is called "hsa-miR-103a-3p" (SEQ ID No. 9).

In the invention, the method for determining the result, that is, the expression level of the miRNA need not be particularly limited, and can be selected by a gene profiling method, such as a microarray, and / or a method comprising PCR, such as PCR time; and / or Northern Blot. Real-time quantitative PCR (generally abbreviated as RQ-PCR, RT-qPCR, rt-PCR, or qPCR) is a sensitive and reproducible gene expression quantification technique that can be used particularly to profile miRNA expression in cells and tissues. Any method can be used to evaluate RT-PCR results, and the AACt method may be preferred. The AACt method is described in detail by Livak et al. (Methods 2001, 25: 402-408). (Ct = Cycle threshold values). In practicing the present invention, the AACt method described by Livak et al. (Methods 2001, 25: 402-408) will be used preferably. The AACt-method will include a 'control sample' and a 'subject sample'. The 'subject sample' is a sample of the subject to be tested. For each sample, a target gene (here: miRNA of interest) and an endogenous control gene (as described below) are included for PCR amplification from aliquots (typically serial). Typically, several replicates are used for each diluted concentration to derive the amplification efficiency. The efficiency of PCR amplification can be defined as percent amplification (0 to 1). During the qPCR reaction, software typically measures for each sample the cycle number in which the fluorescence (PCR amplification indicator) crosses an arbitrary line, the threshold. This crossover point is the Ct value.

A microarray is an array on a solid substrate (usually a glass sheet or a silicon thin film cell) that analyzes large amounts of biological material, in the present case a large number of different miRNAs or, preferably, their DNA transcripts. inverse, which are detectable by specific probes immobilized on the solid substrate.

A Northern blot involves the use of electrophoresis to separate RNA samples by size and subsequent detection with a hybridization probe complementary to (part of) the target RNA sequence of interest.

The method of the present invention can be applied with samples from individuals of either sex, that is, men or women, and at any age.

In the method of the present invention, the expression of the miRNA can be normalized, preferably relative to the expression of another RNA molecule. There are standardization methods well known in the state of the art.

In another aspect, the invention also provides a method for assigning a human subject to one of the two groups, hereinafter "second method of the invention", in which group 1 comprises subjects identifiable by the method of the invention; and where group 2 represents the remaining subjects.

It is possible to provide personalized therapy to an individual, depending on whether the individual is assigned to group 1 or group 2. Group 1 comprises subjects identifiable by the method of the invention as individuals suffering from HHT; and where group 2 represents the remaining subjects.

In another aspect, the invention also provides a method for obtaining useful data that allows determining if an individual suffers from type 1 HHT, hereinafter "third method of the invention" which comprises carrying out the first method of the invention and which furthermore it comprises detecting an increase in the level of expression of at least one miRNA of group H1, preferably an increase in the level of expression of the three miRNAs of group H1.

In another aspect, the invention also provides a method for obtaining useful data that allows determining whether an individual suffers from type 2 HHT, hereinafter "fourth method of the invention" which comprises carrying out the first method of the invention and which further comprises the detection of a variation in the level of expression of miRNAs of group H2, particularly a decrease in the level of expression of miR-9 or an increase in the level of expression of miR-29c, preferably the detection of both variations.

Kit or device of the invention

The present invention also provides a kit or device, hereinafter "kit of the invention or " device of the invention ", comprising at least one oligonucleotide (s) capable of hybridizing with any one or more, preferably two or more, and most preferably all, the miRNAs set forth as SEQ ID NOs: 1 to 8. It is preferred that said oligonucleotide (s) be capable of doing so under stringent conditions. In a preferred embodiment, one or more of said one or more oligonucleotides (preferably DNA) are further defined by the following TaqMan® sequences

TaqMan Advanced Assay hsa-miR-143-3 477912_mir TaqMan Advanced Assay hsa-miR-9-5p 478214_mir TaqMan Advanced Assay hsa-miR-654-3 479135 mir TaqMan Advanced Assay hsa-miR-142-3 477910_mir TaqMan Advanced Assay hsa- miR-150-5 477918_mir TaqMan Advanced Assay hsa-miR-29c-3 479229 mir TaqMan Advanced Assay hsa-miR-103a- 478253_mir

478293_mirTaqMan Advanced Assay cel-miR39-3p

478293_mir

Table 2: TaqMan® sequences used

Astringency is a term used in hybridization experiments. The stringency reflects the degree of complementarity between the oligonucleotide and the nucleic acid (which in this case is the miRNA to be detected); the higher the stringency, the higher the percentage of homology between the probe and the nucleic acid bound to the filter. It is well known to the person skilled in the art that temperature and salt concentrations have a direct effect on the results obtained. Hybridization results are recognized to be related to the number of degrees below the Tm (melting temperature) of the DNA in which the experiment is performed. Stringent conditions are often defined as a wash with 0.1X SSC (saline-sodium citrate (SSC) buffer at 65 ° C. (SSC is generally provided as a 20X stock solution, consisting of 3M sodium chloride). and 300 mM trisodium citrate (adjusted to pH 7.0 with HCl)).

In particular embodiments, the kit is selected from (a) a suitable kit for PCR, (b) a suitable kit for Northern Blot and (c) a suitable kit for microarray analysis. Two or more of these embodiments can also be combined, so that the kit can comprise, for example, both (a) and (c).

In the case of (a) a kit suitable for PCR, this PCR is typically a real-time quantitative PCR (RQ-PCR), a sensitive and reproducible gene expression quantification technique. In this case, it is desired that the kit further comprise a polyT oligonucleotide primer in addition to the kit oligonucleotide (s). The polyT oligonucleotide primer can be used together with the oligonucleotide (s) of the invention for priming PCR, after polyadenylation of the isolated miRNAs by methods known to those of skill, such as the use of poly- (A) polymerase and ATP. . These reagents can optionally be included in the kit.

A Northern blot involves the use of electrophoresis to separate RNA samples by size and subsequent detection with an oligonucleotide (s) (hybridization probe) complementary to (part of) the target RNA sequence of interest.

It is also possible that the oligonucleotide (s) is immobilized in spots on a surface (preferably solid). In one embodiment thereof, the kit comprises a microarray. An RNA microarray is a matrix on a solid substrate (usually a glass slide or a silicon thin film cell) that analyzes large amounts of different RNAs (in this case miRNA), which can be detected by specific probes immobilized at points of the solid substrate. Each site contains a specific nucleic acid sequence, typically a DNA sequence, known as probes (or reporters). Although the number of spots is not limited, there is a preferred embodiment in which the microarray is adapted to the methods of the invention. In one embodiment, such a custom microarray comprises fifty dots or less, such as thirty dots or less, including twenty dots or less.

The kit or device of the invention can be used and the use is not particularly limited, although the use in the method of the invention is preferred in any of its embodiments.

In another preferred embodiment of this aspect of the invention, the oligonucleotides, primers, probes, or antibodies are modified or labeled, for example, but not limited to, by radioactive or immunological labeling. Thus, preferably, the oligonucleotides present modifications in some of their nucleotides, such as, for example, but not limited to, nucleotides that have some of their atoms with a radioactive isotope, usually 32P or tritium, immunologically labeled nucleotides, such as with a molecule of digoxigenin, and / or immobilized on a membrane. Several possibilities are known in the state of the art.

The kit or device of the invention may comprise controls, program instructions and information necessary to carry out any of the methods of the invention.

Automation of the methods of the invention

The invention also extends to computer programs adapted so that any processing means can carry out the methods of the invention. Such programs may be in the form of source code, object code, an intermediate source of code and object code, for example, as in partially compiled form, or in any other form suitable for use in implementing the processes according to the invention. . Computer programs also cover cloud applications based on this procedure.

In particular, the invention encompasses computer programs arranged on or within a carrier. The carrier can be any entity or device capable of supporting the program. When the program is incorporated into a signal that can be transported directly by a cable or other device or medium, the carrier can be constituted by said cable or other device or medium. As a variant, the carrier could be an integrated circuit in which the program is included, the integrated circuit being adapted to execute, or to be used in the execution of, the corresponding processes.

For example, the programs could be embedded in a storage medium, such as a ROM memory, a CD ROM memory or a semiconductor ROM memory, a USB memory stick, or a magnetic recording medium, for example a floppy disk or a disk. Lasted. Alternatively, the programs could be supported on a transmittable carrier signal. For example, it could be an electrical or optical signal that could be transported via electrical or optical cable, by radio or by any other means.

The invention also extends to computer programs adapted so that any processing means can carry out the methods of the invention. Such programs may be in the form of source code, object code, an intermediate source of code and object code, for example, as in partially compiled form, or in any other form suitable for use in implementing the processes according to the invention. . Computer programs also cover cloud applications based on this procedure.

Therefore, another aspect of the invention relates to a computer-readable storage medium comprising program instructions capable of causing a computer to carry out the steps of any of the methods of the invention.

Another aspect of the invention relates to a transmittable signal comprising program instructions capable of causing a computer to carry out the steps of any of the methods of the invention.

REALIZATION MODES

The following examples and drawings are provided by way of illustration, and are not intended to be limiting of the present invention.

To carry out this new methodology, 5 ml samples of peripheral blood are extracted from the individual to be diagnosed in BD Vacutainer® tubes with 0.129M sodium citrate by venipuncture and the plasma is separated by centrifugation following the standard protocol (1500xg, 10 minutes). . The exosomes are then isolated from a minimum quantity of 500 µl of plasma using the Total Exosome Isolation Kit (Invitrogen; Vilnius, Lithuania) and then the total RNA present in the exosomes is extracted with the Total Exosome RNA kit. & Protein Isolation kit (Invitrogen), always following the manufacturer's instructions. The isolated total RNA is resuspended in 100 µl of nuclease-free water. Finally, the detection of the miRNAs is carried out by means of real-time PCR (qPCR), after the reverse transcription step with the TaqMan® Advanced miRNA cDNA Synthesis Kit (Applied Biosystems, Carlsbad, CA), using the specific commercial assays of each miRNA called TaqMan ® Advanced Assay (Applied Biosystems) with the TaqMan® Fast Advanced Master Mix reagent (Applied Biosystems), always following the manufacturer's instructions (Figure 2).

This system is the most sensitive and specific in its category, outperforming fluorophore-based technology such as SYBR Green [M. Soltany-Rezaee-Rad, Z. Sepehrizadeh, N. Mottaghi-Dastjerdi, M.T. Yazdi, N. Seyatesh, Comparison of SYBR Green and TaqMan real-time PCR methods for quantitative detection of residual CHO host-cell DNA in biopharmaceuticals, Biologicals 43 (2) (2015) 130-5].

The relative expression of these 8 miRNAs is calculated with respect to the expression of miR-103a, a miRNA that, thanks to previous RNA-seq analysis, we observed was present simultaneously in plasma exosomes and whose expression level remains unchanged in all groups. , a condition that the miRNAs used as endogenous must fulfill to carry out normalization.

Therefore, its expression level is used as an internal reference in order to alleviate expression variations of non-biological origin.

Thus, applying the new methodology described in this invention, the diagnosis of HHT would be made by the differential expression of the miRNAs of the HC group.

At the same time, the differential expression of the H1 or H2 group would indicate the type of HHT of the patient (Figures 3 and 4).

Claims (14)

1. - A method for obtaining useful data for the in vitro diagnosis of HHT in an individual, which comprises detecting the expression levels of SEQ ID NO: 1 (miR-142) in a biological sample isolated from said individual. 2. - A method for obtaining useful data for the in vitro diagnosis of HHT in an individual according to the previous claim, which also comprises the detection of the expression levels of SEQ ID NO: 2 (miR-150) 3. - A method for obtaining useful data for the in vitro diagnosis of HHT in an individual according to claims 1 or 2 that also comprises the detection of the expression levels of SEQ ID NO: 3 (miR-486) 3. - A method for obtaining data useful for the in vitro diagnosis of HHT in an individual according to any of the preceding claims that comprises detecting the expression levels of SEQ ID NO: 4 (miR-106b); SEQ ID NO: 5 (miR-183); SEQ ID NO: 6 (miR-654); SEQ ID NO: 7 (miR-9) and SEQ ID NO: 8 (miR-29c) 4. - A method according to any of the preceding claims wherein the isolated biological sample is peripheral venous blood or plasma obtained from peripheral venous blood. 5. - An in vitro diagnostic method of HHT, which comprises obtaining data according to any of the preceding claims and further comprising: a) Compare the levels obtained in the previous step with the reference, standard and / or values obtained from a reference sample. 6. - An in vitro diagnostic method of HHT, according to the previous claim, characterized in that the comparison is made after the normalization of the expression levels of the miRNAs with respect to a control miRNA. 7. - An in vitro diagnostic method of HHT, according to the previous claim, characterized in that the control miRNA is "hsa-miR-103a-3p" (SEQ ID No. 9). 8. - An in vitro diagnostic method of HHT, which comprises the steps of the method according to any of claims 5 to 7 and further comprises: b1) include the individual in the group of individuals with HHT when they present a decrease in miR-142, miR-150 or / and an increase in miR-486, or any of their combinations. 9. - An in vitro diagnostic method of HHT, comprising the steps of the method according to any of claims 5 to 7 and further comprising: b2) include the individual in the group of individuals with type 1 HHT when they present an increase in miR-106b, miR-183 or / and miR-654, or any of their combinations. 10. - An in vitro diagnostic method of HHT, which comprises the steps of the method according to any of claims 5 to 7, and further comprises: b2) include the individual in the group of individuals with type 2 HHT when they present a decrease in miR-9 and / or an increase in miR-29c. 11. A method according to any of claims 1-10 wherein the detection of expression levels is carried out by: (i) a genetic profiling method, such as a microarray, and / or (ii) a method comprising PCR, such as real-time PCR; I (iii) Northern Blot. 12. The method according to the preceding claim, where the detection of the expression levels is carried out by means of RT-PCR and / or quantitative PCR (qPCR). 13.- A kit or device comprising at least one oligonucleotide (s) capable of hybridizing with any miRNA with any of SEQ ID NOs: 1 to 8 under stringent conditions. 14. Use of the kit or device according to the preceding claim in a method according to any one of claims 1-12.