WO2011144791A1

WO2011144791A1 - Method for the prognosis and diagnosis of aortic stenosis, comprising alpha-1-antichymotrypsin as a marker

Info

Publication number: WO2011144791A1
Application number: PCT/ES2011/070365
Authority: WO
Inventors: Tatiana MARTÍN-ROJAS; Félix GIL-DONES; Verónica MORAL DARDÉ; Luís RODRÍGUEZ PADIAL; Fernando De La Cuesta Marina; Gloria Alvarez-Llamas; Fernando VIVANCO MARTÍNEZ; Mª Eugenia GONZÁLEZ BARDERAS
Original assignee: Fundación Hospital Nacional De Parapléjicos Para La Investigación Y La Integración (Fuhnpaiin)
Priority date: 2010-05-20
Filing date: 2011-05-20
Publication date: 2011-11-24
Also published as: ES2370671B1; ES2370671A1

Abstract

The invention relates to a method for obtaining data that can be used to determine the risk of developing aortic stenosis (AS), the presence of AS and/or the prognosis of the development of an AS, by means of the detection and/or quantification of protein α-1-antichymotrypsin. The invention also relates to an AS diagnosis and prognosis kit.

Description

METHOD OF PROGNOSIS AND DIAGNOSIS OF AORTIC STENOSIS THAT INCLUDES ALPHA-1-ANTICHEMYOTHYRPSIN AS A MARKER

The present invention relates to a method of obtaining useful data for the determination of the risk of suffering aortic stenosis (AD), the presence of AD and / or the prognosis of the development of an AD, by detecting and / or quantifying the α-1-antichymotrypsin protein. Likewise, the present invention relates to a diagnosis and prognosis kit for AD.

STATE OF THE PREVIOUS TECHNIQUE

AD is a disease of high prevalence in developed countries and is responsible for the largest number of aortic valve replacements (Passik et al. Mayo Clin. Proc. 1987; 62: 1 19-23). In the United States alone, more than 70,000 aortic valve replacements are performed annually. 2-3% of adults over 65 suffer from calcified AD, and about 25% of these people suffer from aortic sclerosis (thickening of the aortic valve without obstructing the outflow of the left ventricle) (Otto and O'Brien. Heart 2001; 85: 601-602). Therefore, a large increase in AD is expected, due to the progressive aging of the population that lead to improvements in health.

For years, degenerative AD has been considered an inevitable consequence of aging and was thought of as a passive process secondary to calcium deposition in the aortic valve. At present, although its etiology is not known with certainty, several authors have indicated that it shares the same risk factors as atherosclerosis, since AD is more frequent in patients with hypercholesterolemia, arterial hypertension, smoking and diabetes (Nalini et al Circulation 2003; 107: 2181-84).

In addition, histological similarities have recently been found between the stenotic aortic valve and the atheroma plate, which has led to the hypothesis that degenerative AD is an inflammatory process similar to atherosclerotic. In different studies, it has been shown that the aortic valve develops atherosclerosis with subsequent calcium deposition and expression of specific markers of bone matrix, such as osteopondine (Stewart et al. J Am Coll Cardiol. 1997; 29: 630-4, Emile et al. Arterioscler Thromb Vasc Biol. 1997; 17: 547-52, Kevin et al. Circulation. 1995; 92: 2163-68, Srivatsa et al. J Clin Invest. 1997; 99: 996-1009, Feldman et al. Cathet Cardiovasc Diagn. 1993; 29: 1-7, Fernández- González et al. Tex Heart Inst J. 1997; 24: 232). Similarly, it has been documented that endothelial dysfunction, with accumulation of lipids and proteins (apolipoprotein (apo) B, apo (a) and apoE) (O'Brien et al. 1996; 16: 523-32, Mohler et al. Circulation, 2001; 103: 1522-8) plays an important role in the onset of AD, as oxidative stress with modification of nitric oxide enzymes also plays an important role (Arumugam et al. J Cardiovasc Surg. 1995; 10: 610-1, Rajamannan et al. J Am Coll Cardiol. 2003; 41: 842). However, some data suggest that its pathogenic mechanism is not the same as that of atherosclerosis; Thus, in adults with severe AD, only 50% have significant coronary artery disease. In turn, most patients with coronary artery disease do not have AD (Otto and O'Brien. Heart 2001; 85: 601-602). In addition, some studies aimed at assessing the effect of statins on degenerative AD have described a decrease in coronary disease without change in the progression of degenerative AD (Rossebo et al. N Engl J Med. 2008. 25; 359: 1343- 56, Cowell et al. N Engl J Med. 2005. 9; 352: 2389-97, Chan et al. Circulation 2010; 121: 306-14).

Importantly, although advances have been made in the knowledge of the mechanisms involved in this process, no marker is currently used in the diagnosis / prognosis of AD. Α-1-anti-chymotrypsin is an alpha globulin glycoprotein that is a member of the serpine family. The α-1-antichymotrypsin inhibits some proteases such as cathepsin and is protective of the damage caused by proteolytic enzymes. In recent years it has been shown the participation of this protein in Alzheimer's disease and its deficiency, with liver disease.

The early identification of patients at risk of developing an aortic valve stenosis is of great importance as it would reduce the mortality and morbidity associated with this disease. The existence in the hospital of specific tests for the study of risk of suffering from AD, could be of great help for the prevention and possible prophylaxis of said disease.

DESCRIPTION OF THE INVENTION

The present invention provides a method of obtaining useful data for the determination of aortic stenosis (AD) or of the risk of suffering from AD or of the evolution of an AD quickly, easily and efficiently. Also, the present The invention relates to the use of a kit to determine the risk of suffering from AD or to determine the presence of an AD or to predict the progression of an AD, in an isolated biological sample.

The present invention therefore provides a response to the need for biomarkers that allow diagnosis and prognosis of AD.

The present invention solves the technical problem of finding a good biomarker for AD, if possible that it can be monitored in plasma or blood serum, that is, it does not imply obtaining a more invasive biological sample.

In the present invention, a surprising effect is noteworthy: a-1 - antichymotrypsin has high levels in the stenotic aortic valve tissue, in its secrecy as well as in the blood plasma of patients with AD, in relation to the levels of individuals control.

Thus, a first aspect of the invention relates to a method of obtaining useful data (hereinafter method of the invention) for the determination of AD or the risk of suffering from AD or the evolution of an AD comprising:

a) obtain an isolated biological sample from a subject, and

b) detect and / or quantify the expression product of a nucleotide sequence of an α-1-antichymotrypsin gene encoding an amino acid sequence that has at least 50% identity with

SEQ ID NO: 1, or a fragment of said expression product, in the sample obtained in step (a).

The term "aortic stenosis (AS)" as used herein, refers to aortic valve stenosis, which is a valvulopathy (valvular heart disease) characterized by abnormal narrowing of the aortic valve orifice of the heart . This reduction of the valvular orifice can be congenital or acquired.

The term "determination of AD", as used in the present description, refers to, but is not limited to, the finding of the presence of AD in a subject.

The term "risk of suffering from AD", as used in the present description, refers to, but is not limited to, the predisposition of a subject to suffer or Develop an EA. Therefore, the method refers to obtaining useful data to determine if a subject is developing or can develop an EA.

The term "evolution of AD," as used herein, refers to, but is not limited to, the progression of AD or the likelihood of having heart failure or death from heart failure, as well as the prediction of the response to a certain treatment of AD.

The term "% identity" as used in the present description refers to the% identity between two amino acid sequences. The% identity is the result of counting the number of positions along the alignment of two sequences where all amino acids in the same position are identical.

The expression product or fragment thereof, detected and / or quantified in step (b) may encode for an amino acid sequence at least 55, 60, 65, 70, 75, 80, 85, 90 or 95% identical to SEQ ID NO: 1.

The term "expression product", as used in the description, refers to the product resulting from the transcription (RNA) or translation (protein) of a gene or a nucleotide sequence of DNA, or any resulting form of the processing of the product resulting from the transcription or expression of a gene or a nucleotide sequence of DNA.

The term "α-1-antichymotrypsin gene" refers to the DNA sequence encoding the α-1-antichymotrypsin protein, a variant of the α-1-antichymotrypsin protein or a fragment thereof, as long as said variant or said fragment is functionally equivalent.

The amino acid sequence SEQ ID NO: 1 is the amino acid sequence of the Homo sapiens (human) α-1-antichymotrypsin protein with accession number NP_001076.2 from the NCBI database.

In a preferred embodiment, the expression product or fragment thereof, detected and / or quantified in step (b) encodes an amino acid sequence having at least 70% identity with SEQ ID NO: 1. In a more preferred embodiment, said expression product encodes for the amino acid sequence SEQ ID NO: 1 or for a fragment thereof. In a preferred embodiment, the method of the invention further comprises comparing the data obtained in step (b) with standard values to find a significant deviation.

In a more preferred embodiment, the method of the invention further comprises attributing the significant deviation to the presence of AD or the risk of suffering from AD in the subject of (a).

Preferably, the biological sample from step (a) is a biological fluid. More preferably, the biological fluid is blood or serum or blood plasma.

Preferably, the biological sample from step (a) comprises cells. Preferably, it comprises a fabric.

The biological sample from step (a) is an isolated sample of an organism such as the human or animal body and may come from a physiological fluid and / or any cell or tissue of an organism.

Preferably, the biological sample isolated in step (a) of the method of the invention comprises cells. More preferably, these cells are aortic valve. The biological sample may be a tissue, for example, but not limited to, a biopsy or a fine needle aspirate. Even more preferably, the biological sample isolated in step (a) may be an aortic valve tissue.

In a preferred embodiment, the biological sample isolated in step (a) of the method of the invention is a biological fluid. The biological fluid may include excreted or secreted fluids from the body, as well as fluids that are not normally. Biological fluid may include, but is not limited to, amniotic fluid surrounding the fetus, aqueous humor, interstitial fluid, lymph, breast milk, mucus (including nasal drainage and phlegm), saliva, sebum (skin fat), serum , sweat, tears, urine, pericardial fluid, blood and blood plasma. In a more preferred embodiment, the biological fluid is blood, blood plasma or blood serum.

The biological sample isolated in step (a) of the method of the invention can be, for example, but not limited to, fresh, frozen, fixed or embedded in paraffin.

The term "subject", as used in the description, refers to animals, preferably mammals, and more preferably, humans. The expression "detect and / or quantify the expression product" in the sample obtained, as used in the description of the method of the invention, refers to the detection of the presence and / or the measurement of the quantity or the concentration, preferably semi-quantitatively or quantitatively. The measure can be carried out directly or indirectly. Direct measurement refers to the measure of the quantity or concentration of the expression product of the α-1 -antiquimotripsin gene based on a signal that is obtained directly from said expression product and that is directly correlated with the number of product molecules of gene expression, present in the sample. Said signal can be obtained, for example, by measuring an intensity value of a chemical or physical property of the α-1-anti-chymotrypsin gene expression product. The indirect measure includes the measure obtained from a secondary component (for example, a component other than expression products) or a biological measurement system (for example the measurement of cellular responses, ligands, "tags" or enzymatic reaction products) .

The term "compare," as used in the description of the first method of the invention, refers to, but is not limited to, the comparison of the amount of expression product of the α-1-antichymotrypsin gene of the biological sample. to be analyzed, also called a biological problem sample, with an amount of expression product of the α-1-antichymotrypsin gene of a desirable reference sample, described elsewhere in the present description. The reference sample can be analyzed, for example, simultaneously or consecutively together with the problem biological sample. The comparison of step (c) of the method of the invention can be performed manually or assisted by a computer.

The term "standard values", as used in the description of the method of the invention, refers to the absolute or relative amount of expression product of the α-1-antichymotrypsin gene that allows discriminating the presence of AD. Suitable standard values can be determined by the method of the present invention from a reference sample that can be analyzed, for example, simultaneously or consecutively, together with the problem biological sample. More preferably, the standard values may be derived from the normal distribution limits of a physiological amount found in a population of control subjects. Said physiological amount can be determined by several well-known techniques, such as, for example, the calculation of the average amount of expression product of the α-1-antichymotrypsin gene in a population of control subjects determined by the method of the present invention.

The significant deviation can be established by one skilled in the art by using different statistical tools, for example, but not limited, by determining confidence intervals, determining the p-value, Student's test or Fisher's discriminant functions. Preferably, the confidence intervals are at least 90%, at least 95%, at least 97%, at least 98% or at least 99%. Preferably, the value of p is less than 0.1, 0.05, 0.01, 0.005 or 0.0001. Preferably, the present invention allows the disease to be correctly detected in at least 60%, in at least 70%, in at least 80%, or in at least 90% of the subjects of a certain group or population analyzed.

In accordance with the present invention, the detection of the amount of expression product of the α-1-antichymotrypsin gene can be carried out by any method known in the state of the art.

In a preferred embodiment of the first method of the invention, the detection of the gene expression product is performed by analyzing the level of messenger RNA (mRNA) derived from its transcription, where the mRNA level analysis can be performed, for example, but not limited, by amplification by polymerase chain reaction (PCR), back transcription in combination with polymerase chain reaction (RT-PCR), back transcription in combination with ligase chain reaction (RT-LCR) , or any other method of nucleic acid amplification; DNA microarrays made with oligonucleotides deposited by any mechanism; DNA microarrays made with oligonucleotides synthesized in situ by photolithography or by any other mechanism; in situ hybridization using specific probes labeled with any method of marking; by electrophoresis gels; by membrane transfer and hybridization with a specific probe; by nuclear magnetic resonance or any other imaging technique using paramagnetic nanoparticles or any other type of detectable nanoparticles functionalized with antibodies or by any other means.

In a preferred embodiment, the expression product of the nucleotide sequence of the α-1-antichymotrypsin gene or the fragment of said expression product, which is detected and / or quantified in step (b) of the method of the invention, is a amino acid sequence having at least 50% identity with SEQ ID NO: 1, or a fragment thereof. More preferably, it is an amino acid sequence that has at least 70% identity with SEQ ID NO: 1, or a fragment thereof. Even more preferably, it is SEQ ID NO: 1, or a fragment thereof.

Preferably, the detection and / or quantification of step (b) of the method of the invention is performed by electrophoresis, immunoassay, chromatography, microarray technology and / or mass spectrometry.

The detection and / or quantification of step (b) of the method of the invention can be carried out by any combination of any of the mentioned techniques. Detection can be performed by evaluating the presence or absence of the expression product or fragment thereof.

Electrophoresis is an analytical separation technique based on the movement or migration of macromolecules dissolved in a medium (the electrophoresis buffer), through a matrix or a solid support, as a result of the action of an electric field. The behavior of the molecule depends on its electrophoretic mobility, which in turn depends on the charge, size and shape of the molecule. There are numerous variants of this technique depending on the equipment used to carry out the separation. The electrophoresis can be chosen from the list comprising: capillary electrophoresis, paper electrophoresis, agarose gel electrophoresis, polyacrylamide gel electrophoresis, isoelectric or electro-focused focusing or two-dimensional electrophoresis.

An immunoassay is a biochemical assay that measures the concentration of a substance in a biological sample using a reaction of an antibody or antibodies against its antigen. The assay is based on the specific binding of an antibody with its antigen. The detection and / or quantification of the antibody and / or the antigen can be carried out with various methods. One of the most common is the antigen or antibody mapping. This tide may consist, although without be limited, in an enzyme, a radioisotope (radioimmunoassay), a magnetic marker (magnetic immunoassay) or a fluorophore. In addition there are other techniques such as agglutination, nephelometry, turbidimetry or immunoblot. Heterogeneous immunoassays can be competitive or non-competitive. In a competitive immunoassay the response is inversely proportional to the antigen concentration of the sample. In a non-competitive assay, also called a sandwich assay, the results are directly proportional to the antigen concentration. An immunoassay technique that can be used in the present invention is the enzyme-linked immunoadsorbent assay or ELISA ("Enzyme-Linked ImmunoSorbent Assay").

In a preferred embodiment, the immunoassay is an immunoblot. To carry out the immunoblot technique, also called Western blot or membrane immunodetection, a protein extract is obtained from an isolated biological sample of a subject and the proteins are separated by electrophoresis in a support medium capable of retaining them. Once the proteins are separated, they are transferred to a different support or membrane where they can be detected through the use of specific antibodies that recognize the α-1-antichymotrypsin protein, its variants or fragments thereof. This membrane is hybridized with a first specific antibody (or primary antibody) that recognizes the α-1-anti-chymotrypsin protein, its variant or a fragment thereof. The membrane is then hybridized with a second antibody (or secondary antibody) capable of specifically recognizing the primary antibody and which is conjugated or bound with a marker compound. In an alternative embodiment, it is the primary antibody that recognizes the α-1-antichymotrypsin protein, its variant or a fragment thereof, that is conjugated or bound to a marker compound, and the use of a secondary antibody is not necessary. Different formats, supports and techniques that can be used for the realization of this preferred aspect of the method of the invention are known in the state of the art.

In another preferred embodiment, the immunoassay is an immunohistochemistry.

(IHQ). Immunohistochemical techniques allow the identification, on tissue or cytological samples of characteristic antigenic determinants. Immunohistochemical analysis is performed on tissue cuts, either frozen or included in paraffin, from an isolated biological sample of a subject. These sections hybridize with a specific antibody or primary antibody that recognizes the α-1-antichymotrypsin protein, its variants or fragments thereof. The sections are then hybridized with a secondary antibody capable of specifically recognizing the primary antibody and which is conjugated or bound with a marker compound. In an alternative embodiment, it is the primary antibody that recognizes the α-1-antichymotrypsin protein, its variant or a fragment thereof, that is conjugated or bound to a marker compound, and the use of a secondary antibody is not necessary.

By means of chromatographic techniques the molecules can be separated according to their load, their size or their molecular weight, their polarity or their redox potential, among others. The chromatographic technique can be, but not limited to, liquid chromatography (partition, absorption, exclusion, ion exchange), gas chromatography or supercritical fluid chromatography.

The microarray technology of the present invention is based, for example, but not limited to the fixation on a solid support of a molecule that recognizes the expression product of the present invention. The antibody microarray is one of the most common protein microarrays. In this case the antibodies are placed in small points, fixed on the solid support (called a protein chip) and used to capture molecules and thus detect the proteins in a biological sample, such as a cell lysate, a conditioned medium, serum or urine

The term "solid support" as used herein refers to a wide variety of materials, for example, but not limited to, ion exchange resin or adsorption, glass, plastic, latex, nylon, gel, esters or Cellulose, paramagnetic spheres or a combination of some of them.

More preferably, the detection and / or quantification of step (b) of the method of the invention is performed by liquid chromatography coupled to tandem mass spectrometer (LC-MS / MS).

Another aspect of the invention relates to the use of the expression product of the α-1-anti-chymotrypsin gene or a fragment thereof, as a marker: a) for the determination of the risk of suffering from AD,

b) to determine the presence of an EA, or

c) to predict the progression of an EA.

The expression product may encode for an amino acid sequence at least 55, 60, 65, 70, 75, 80, 85, 90 or 95% identical to SEQ ID NO: 1 or a fragment thereof.

Another aspect of the invention relates to the use of an amino acid sequence having at least 50% identity with SEQ ID NO: 1, or a fragment thereof, as a marker:

a) for the determination of the risk of suffering from AD,

b) to determine the presence of an EA, or

c) to predict the progression of an EA.

The amino acid sequence is at least 55, 60, 65, 70, 75, 80, 85, 90 or 95% identical to SEQ ID NO: 1 or a fragment thereof.

In a preferred embodiment, the amino acid sequence has at least one

70% identity with SEQ ID NO: 1 or a fragment thereof. In a more preferred embodiment, the amino acid sequence is SEQ ID NO: 1 or a fragment of SEQ ID NO: 1.

Another aspect of the invention relates to the use of a kit comprising primers and / or probes that hybridize with the sequence of an α-1-antichymotrypsin gene encoding an amino acid sequence having at least 50% or at least 70 % identity with SEQ ID NO: 1, or coding for SEQ ID NO: 1, to determine the risk of suffering from AD or to determine the presence of an EA or to predict the progression of an EA, in an isolated biological sample.

The term "primers" refers to oligonucleotides with identical or complementary reverse nucleotide sequences that allow amplification of some fragment of the α-1-antichymotrypsin gene by, for example, polymerase chain reaction (PCR). The term "primer" refers to sequences of between 15-30 nucleotides that hybridize with one of the template nucleic acid chains and allow amplification of a DNA sequence by a PCR reaction. The term "probe" refers to an identical or complementary reverse RNA or DNA sequence to the a-1 gene sequence - anti-chymotrypsin, capable of hybridizing with said sequence and which may or may not be labeled with any marker compound known in the state of the art,.

Another aspect of the invention relates to the use of a kit comprising antibodies capable of binding to an amino acid sequence that has at least 50% or at least 70% identity with SEQ ID NO: 1, or that codes for SEQ ID NO: 1, or a fragment thereof, to determine the risk of AD or to determine the presence of an EA or to predict the progression of an EA, in an isolated biological sample.

The term "antibody", as used herein, refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, that is, molecules that contain a binding site and binding of antigen that specifically binds (immunoreacts ) with a protein. There are five major isotypes or classes of immunoglobulins: immunoglobulin M (IgM), immunoglobulin D (IgD), immunoglobulin G (IgG), immunoglobulin A (IgA) and immunoglobulin E (IgE).

Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following examples and drawings are provided by way of illustration, and are not intended to be limiting of the present invention.

DESCRIPTION OF THE FIGURES

Figure 1. A) Shows a representative image of a two-dimensional gel of aortic valve tissue made using 2D-DIGE and subsequently stained with silver. Surrounded by a circle, the protein spot is shown whose expression varies when comparing patients with AD with healthy individuals. B) It shows a 3-dimensional scheme of the variation in the expression of the protein highlighted in (A) between individuals with AD and control individuals.

Figure 2. A) Shows an immunohistochemistry of healthy and stenotic aortic valves against a-1 -antiquimotripsin. 6 μηι cryosections of valve tissue with EA and healthy valve tissue were used. The results show increased expression of α-1-antichymotrypsin in valve sections with AD when compared to healthy valves. As a negative control, an immunohistochemistry was performed without incubation with the antibody against a-1-anti-chymotrypsin. B) Shows the number of positive cells for α-1 anti-chymotrypsin per μηπ ² in control aortic valve tissue versus stenotic aortic valve tissue: the number of cells expressing a-1 -antichymotrypsin is significantly higher in valve tissue stenotic aortic.

Figure 3. It shows immunoblots where a greater expression of α-1-antichymotrypsin is confirmed in samples from patients with AD compared to control patients. A) Two-dimensional electrophoresis of aortic valve tissue samples from patients with AD and from control individuals. In the anti-a-1-antichymotrypsin screening, an over-expressed protein spot of 70 kDa was detected in patients with AD versus healthy individuals. Graphical representation of the amount of protein in the control sample (C) and in the sample of patients with AD (AD) in arbitrary units. B) Immunoblot against a-1 - anti-chymotrypsin in 4 samples of control secrets (C1-C4) and 4 secrets of patients with AD (P1-P4). Graphical representation of the amount of protein in the control samples (C) and in those of patients with AD (AD) in arbitrary units and "p-value" of the Student's t-test. C) Immunoblot against a-1 - anti-chymotrypsin in 4 samples of control plasma (C1-C4) and 4 plasma of patients with AD (P1-P4). Graphical representation of the amount of protein in the control samples (C) and in those of patients with AD (AD) in arbitrary units and "p-value" of the Student's t-test.

Figure 4. A) Shows the MRM chromatograms where the most characteristic transitions are observed for each of the two proteotypic peptides selected from the α-1-antichymotrypsin. B) Shows the graphical representation of the mean values and standard deviation for each transition corresponding to three independent injections of each sample in the 4000-Qtrap (Applied Biosystems) mass spectrometer. C). It shows the graphical representation of the quantification results by MRM for each of the monitored transitions. "P" are patients and "C" controls.

EXAMPLES The following examples, which illustrate but do not limit the present invention, describe tests performed by the inventors that demonstrate the specificity and effectiveness of α-1-antichymotrypsin as a biomarker of AD.

EXAMPLE 01: Detection and quantification of α-1-antichymotrypsin in samples of patients with AD and control individuals

Selection of subjects for the study

The stenotic aortic valves (n = 20) were obtained from patients of both sexes (55% men, 45% women) with a mean age of 74 ± 3.9 years who underwent valve replacement with degenerative aortic stenosis (AD). All patients (100%) had hypertension, 50% had hyperlipemia and 60% had diabetes. The surgery was performed according to the usual clinical practice guidelines. Coronary artery disease was present in 60% of the subjects included in the study. Those patients with aortic insufficiency, mitral valve disease or any suspicion of rheumatic disease were not included in the study (Tablal).

Table 1: Clinical characteristics of the individuals in the control group.

The aortic valves of control subjects were obtained at autopsies of individuals killed by non-cardiovascular diseases, without coronary disease or diabetes mellitus and whose aortic valves had a macroscopically normal appearance (n = 20). (Table 2). Table 2. Clinical characteristics of individuals with aortic stenosis.

The study was carried out according to the recommendations of the Declaration of Helsinki and was approved by the Ethical Committee of the Virgen de la Salud Hospital (Toledo, Spain). Informed consent was obtained from all subjects included in the study.

Preparation of tissue samples for proteomic analysis

The valves with EA were processed within a maximum period of 2 hours since the surgery was performed, maintaining the tissue at 4 ⁰ C in RPMI medium. They were washed 3 times with phosphate buffer to reduce blood contaminants. The control valves were processed in a maximum time of 8 hours from the death of the individual. To perform protein extraction, a valve fragment was frozen in liquid nitrogen and homogenized with mortar. Between 0.1-0.3 g of the powder obtained was resuspended in 400μΙ of protein extraction buffer (10 mM Tris pH 7.5, 500mm NaCI, 0.1% Triton X-100, 1% β-mercaptoethanol, 1 mM PMSF). The homogenate was centrifuged at 21,000 g for 15 minutes at 4 ⁰ C. The supernatant (E1), with most of the soluble proteins, was collected and stored at -20 ⁰ C. The pellet was solubilized in lysis buffer (7 M Urea, 2M Tiourea, 4% CHAPS) and centrifuged again at 21,000 g. This second supernatant (E2) was enriched in hydrophobic proteins, mainly membrane proteins. Cell debris and lipids were eliminated. Both protein extracts E1 and E2 were analyzed together for the analysis of differential two-dimensional electrophoresis (2D-DIGE). The protein concentration was determined by the Bradford-Lowry method.

Two-dimensional electrophoresis (2-DE) Protein extracts were resuspended in rehydration buffer (7M urea, 2M thiourea, 4% CHAPS, 1% TBP, 2% ampholytes) and applied to IPG strips of pH 4-7. The isoelectroenfoque (IEF) was developed horizontally and at a constant temperature of 20 ° C. Once the IEF was finished, the strips were balanced to facilitate the passage of the proteins to the second dimension. Protein separation was performed according to the molecular weight in 12% SDS-PAGE gels, using Protean II system (Bio-Rad) at 25mA / gel at 4 ⁰ C. Next, the gels were stained with silver. This protocol is compatible with mass spectrometry. Finally, the gels were scanned with the GS-800 Calibrated Densitometer (Bio-Rad).

Differential two-dimensional electrophoresis (DIGE)

Sample mapping

The samples were labeled according to the instructions of the commercial house (GE Healthcare, Piscataway, NJ), 50 μg of protein extract of stenotic aortic valves and 50 μg of protein extract of aortic control valves were marked with 400 pmol (1 μΙ) of fluorochrome corresponding, Cy3 or Cy5, depending on the experimental design. In addition, an internal control with all the samples of the experiment was prepared by placing in a single tube 50 μg of each of the samples and marking the mixture with Cy2. The samples were incubated on ice for 30 minutes in the dark to allow the tidal reaction. This reaction was stopped by adding 1 μΙ of 10 mM lysine for every 50 μg of sample and incubating again on ice for 10 minutes and in the dark.

Two-dimensional electrophoresis

Once the samples were labeled, the stenotic valve sample, the control valve sample and the pool of all the samples object of the experiment were mixed (150 μg of total protein), according to experimental design, diluted in rehydration buffer (30 mM TRIS, 7m urea, 2M thiourea, CHAPS 4%) and applied to the 24 cm IPG strips pH 4-7. The first dimension was made in the IPGphor III System (GE Helathcare) following the following IEF program: 500 V for 30 minutes, 1000 V for 1 h, 2000V in 1 h in linear gradient, 5000V in 2 h in linear gradient, 8000V in 1 h in linear gradient, and from 8000V to 88000v / h. After the first dimension, the strips were equilibrated with equilibration buffer (TriCI pH 8.8 1, 5 M, 6M urea, 87% glycerol, 2% SDS). Subsequently, the proteins were separated on 12% acrylamide / bisacrylamide 12% SDS-PAGE gels.

Capture and image analysis

The gels in SDS-PAGE were scanned using Typhoon 9400 (Typhoon 9400 Variable Mode Imager, GE Healthcare) applying the specific excitation and emission wavelengths of each fluorochrome, filters and photomultiplier sensitivity (PTM) values for each fluorochrome, Cy3, Cy5 and Cy2 (PTM values; 480 V, 490 V, 500 V, respectively) (Figure 1A).

The relative protein quantification in the samples of stenotic and healthy valves was carried out through the DeCyder v6.5 program (Figure 1 B) and with the multivariate statistical module EDA (from English, "Extended data analysis") using the DIA module (from English, "Differential in-gel analysis"), which detects and quantifies the protein spots of the three images corresponding to each gel. The algorithm used by this module for the detection of stains is based on the co-detection of the three fluorescent signals (2 samples and 1 internal control) and makes it possible to differentiate the true protein spots from the gel artifacts. On the other hand, the quantification is based on the calculation of the Cy3 / Cy2 and Cy5 / Cy2 ratios, which allows for a standardized intensity value for each spot.

Once this phase was completed, the data generated by DIA were imported into the BVA module ("Biological! Variation Analysis"), to match the spots detected between the images of the different gels in the study to obtain statistical data on Stain expression levels in different study groups. It can be concluded that there is variation when protein expression levels are altered more than 1.5 times and said variation is considered statistically significant when it is within the 95% confidence interval (p value <0.05) determined by the test. t-Student

Finally, a multivariate analysis through Principal Component Analysis (PCA) was performed using the algorithm included in the EDA module of the DeCyder v 6.5 program, based on the protein spots that are paired in all gels. The hierarchical analysis was performed using the Pearson coefficient, based on the protein spots found in 90% of the study gels. The gels were stained with silver solution (GE-Healthcare) after being scanned.

Protein identification by MALDI TOF / TOF

Differentially expressed protein spots were trimmed from the gels manually, automatically digested using the "Ettan Digester" (GE Healthcare) and identified. The gel fragments were reduced using 10 mM dithiothreitol (DTT) in 50 mM ammonium bicarbonate (99% purity) and alkylated with 55 mM iodoacetamide in 50 mM ammonium bicarbonate. The gel fragments were then washed with 50 mM ammonium bicarbonate, 50% methanol (for HPLC) and acetonitrile (for HPLC) after which they were dried in "speedvac" (Thermo Fisher). Was added modified trypsin pig at a final concentration of 20 ng / μΙ in ammonium bicarbonate 20 mM to gel fragments were digested overnight at 37 ⁰ C. Finally, peptide extraction was performed in 60% acetonitrile in water and 0.1% formic acid (99.5% purity).

Once the digestion was finished, an aliquot of each reaction was mixed with a matrix aliquot of a-cyano-4-hydroxycinnamic acid in 50% acetonitrile and 0.1% trifluoroacetic acid. The mixture was deposited in a thin layer on a plate of the spectrometer 384 Opti-TOF 123x81 mm MALDI (Applied Biosystems) and dried at room temperature. MALDI-MS / MS data was obtained in automatic mode using a 4800 Plus MALDI-TOF / TOF Analyzer (Applied Biosystems) (MALDI) mass spectrometer from the English "matrix assisted laser desortion ionization"; TOF flight ").

The spectra were acquired in ion-positive mode with an Nd: YAG laser of 355 nm wavelength, at a frequency of 200 Hz. From 100-2000 individual spectra were obtained. For the analysis of ion fragments in tandem flight time mode (TOF / TOF), the precursors were accelerated to 8 kV and selected at the ion gateway. The collision-generated fragments of the precursors with air (CID) in the collision-induced dissociation "were also accelerated to 15kV at source 2 and their masses were analyzed after passing through the ion reflector. Automatic mass data analysis was performed using the "4000 Series Explorer" version 3.5.3 software (Applied Biosystems). The internal calibration of the mass spectra of the MALDI-TOF was performed using 2 ions of the trypsin autolysis (m / z = 842,510 and m / z = 221 1, 105, respectively). For the calibrations of MALDI-MS / MS, fragment ion spectra obtained from the "Glub-fibrinopeptide" (4800MALDI / TOF-TOF, Applied Biosystem) were used. The MALDI-MS and MS / MS data were combined using the "GPS Explorer" version 3.6 program, which allows non-redundant searches in the Swissprot 56.2 protein database using the Mascot version 2.2 (Matrix science) program, with a mass tolerance of 50 ppm and allowing the loss of a cutting place. MALDI-MS / MS spectra and searches were manually reviewed in detail using this program.

EXAMPLE 2: Detection and quantification of α-1-antichymotrypsin by immunoblot

Antibodies

The antibody against α-1-antichymotrypsin with reference ab9374 of Abcam was used to perform immunohistochemistry (Fig. 2) and immunoblots (Fig. 3). Preparation of secretoma samples for proteomic study

For the study of the secretoma, a valve leaflet with EA and a valve valve leaflet were dissected and cultured separately in RPMI-free amino acid medium and supplemented with labeled arginine and lysine, at 37 ⁰ C.

Culture medium samples were collected at 24, 48, 72, 96 and 120 hours to find the maximum protein secretion time. This time turned out to be 96 hours.

Preparation of plasma samples for proteomic analysis

Approximately 28 ml of blood was taken from each patient or control individual in sterile tubes with EDTA. The blood was centrifuged at 750 g for 10 minutes at room temperature to obtain the plasma, which was divided into aliquots and stored at - 80 ⁰ C.

Immunodetection

One-dimensional electrophoresis of proteins obtained from samples of stenotic and control aortic valves (Figure 3A) and samples of the secrets of said valves (Figure 3B) and blood plasma samples from patients with AD and controls (Figure 3C), It was performed on 12% SDS-PAGE gels using the Bio-Rad Miniprotean II electrophoresis system, applying a Initial voltage of 80 V for 5-10 minutes, followed by a constant voltage of 25 mA for 1 hour. After SDS-PAGE the proteins were transferred to nitrocellulose membranes (Bio-Rad) by semi-dry transfer for 40 minutes at 20 V, using the following transfer buffer: 25 mM Tris, 151, 8 mM Glycine and 20% methanol. (v / v). The proteins were then stained by dipping the nitrocellulose membranes in a solution of 0.2% Ponceau Red in 30% trichloroacetic acid (30%) and 30% (w / v) sulfosalicylic acid for 1 minute, after which membranes were washed with distilled water to remove excess dye. A digitized image of each membrane was taken with the GS-800 calibrated densitometer. The proteins were blocked using a phosphate buffer with 0.1% Tween20 (v / v) and with 7.5% skimmed milk powder (w / v) for 1 hour under stirring at room temperature. The membranes were washed 3 times for 10 minutes in a phosphate buffer with 0.1% Tween20 (v / v). Next, the membranes were incubated for 1 hour at room temperature with the specific antibody against α-1-antichymotrypsin, diluted 1: 1000 in the wash buffer with 5% (w / v) skimmed milk powder. After performing another 3 10-minute washes in the wash buffer, the membranes were incubated for 1 hour at room temperature with the corresponding secondary antibody (anti-rabbit antibody developed in goat and bound to peroxidase), diluted 1: 5,000 in buffer Wash with 5% (w / v) skimmed milk powder. Finally, after another 3 washes of 10 minutes, the membranes were revealed using the chemiluminescent substrate ECL (GE Healthcare). The intensities of the bands were quantified by performing a densitometry of each of them and subtracting the background noise using the Quantity One 4.6.5 program. The results obtained, measured in intensity * mm ² , were analyzed with the GraphPad Prism 4.0 statistical analysis software, using a non-parametric T test to determine significant differences with p <0.05 values.

EXAMPLE 3: Immunohistochemistry against α-1 -antichymotrypsin in healthy and stenotic aortic valve tissue

Immunohistochemistry

Tissue sections, both of stenotic aortic valves and control aortic valves, of 6 μηι thickness fixed in formalin were used, previously descaled in Shandon-TBD1 (Thermo Scientific), embedded in OCT and frozen in liquid nitrogen. Sections were blocked in phosphate buffer with Tween 20 (0.1%) with 1% BSA for 1 hour. Subsequently, the tissue sections were incubated with the primary antibody 1 hour at room temperature. Finally, the sections were incubated with the secondary antibody IgG-HRP-conjugate. The development was performed by adding the 3-3 diaminobenzidine chromogen. The cuts were stained with hematoxylin and dehydrated in a gradient of alcohols (70%, 95%, absolute alcohol). Finally, the cuts were washed in xylol and mounted with DPX mounting medium, to seal the glass sheets with the fabric. As a negative control, the entire immunohistochemical procedure was performed in the absence of the primary antibody (Figure 2).

EXAMPLE 4: Detection and quantification of α-1-antichymotrypsin in plasma samples of aortic valves of patients with AD and of MRM control individuals ("Multiple Reaction Monitorinq")

Proteins plasma samples were reduced and alkylated with 100 mM DTT in 50 mM ammonium bicarbonate for 30 minutes at 37 ⁰ C, followed by iodoacetamide in 550 mM ammonium bicarbonate 50 mM at room temperature in the dark for 20 minutes. After that time, 60% 50 mM ammonium bicarbonate and 15% acetonitrile were added to the samples and then a modified porcine trypsin solution (1 g / l) with a ratio of ^ g trypsin / 50μg protein was added. Digestion was incubated at 37 ⁰ C for at least 6 hours (usually overnight). The reaction was stopped by adding 2% formic acid and the samples were cleaned using centrifuge micro columns (spin-columns) with C18 resin (Pep-Clean, Pierce). The triptych digested was vacuum dried in a "speed-vac" centrifuge and resuspended in a 2% acetonitrile solution with 2% formic acid.

The LC-MS / MS system consists of a nano-LC TEMPO (Applied Biosystems) system combined with a nano-autosampler. Three injections of each digested (2 μg in each injection) were performed using mobile phase A (2% acetonitrile, 0.1% formic acid) with a flow of 10 μΙ / minute for 5 minutes. The peptides were thus loaded into a C18 pre-column to pre-concentrate and desalt the samples. Then the peptides were separated by Reverse phase nanochromatography on a C18 capillary column, using mobile phase A (2% acetonitrile, 0.1% formic acid) and mobile phase B (98% acetonitrile, 0.1% formic acid), with a flow of 300 nl / minute, applying the following gradient: 2- 15% B in the first 2 minutes, increasing to 50% B during the following 38 minutes, 50-90% B in 2 minutes, maintaining 95% B for 3 minutes, returning in 1 minute to initial conditions (2% B) and keeping them for 14 more minutes. The LC-MS / MS analysis was performed on a hybrid triple quadrupole / linear ion trap AB / MDS Sciex 4000 Q TRAP with NanoSprayl l (Applied Biosystems) source. Both this system and the TEMPO nano-LC system are controlled by Analyst software v.1 .4.2. The acquisition was carried out in positive mode, with an ionization voltage of 2800 V, with a "curtain gas" of 10, a "nebulizer gas" of 20 and an "interface temperature" of 150 ⁰ C. Nitrogen was used both in the case of collision gas as in that of "curtain gas". MRM transitions were optimized for maximum transmission and sensitivity. The parameters were optimized for each of the transitions and are summarized in Table 3. A total of 3 MRM transitions (diagnostic fragments) per peptide were monitored for each individual sample, and with three replicates per sample (Table 4, Figure 4A) . Table 3 Proteotypic peptides and transitions analyzed by MRM.

Due to the complexity of the sample and to maximize the specificity, a "unit" resolution was used in the first and third quadrules (Q1 and Q3). A "dwell time" of 50 ms was used. During this time, the quadrules monitor each of the transitions, cyclically. The abundances were obtained based on the areas of the peaks Crómate-graphics (Figure 4B), which were calculated as integral under the curve defined by each peak using the IntelliQuant algorithm, included in the Analyst 1 .4.2 software. These areas were imported into the SPSS program, where the corresponding statistical comparisons were made (Figure 4C, Table 4).

Table 4. Results of the quantification using MRM and statistical analysis of the data obtained for each of the monitored transitions.

Mann-Whitney U Patient Controls

Dev. Dev.

Average Average Value p

Standard Standard

AACT_1_T1 22938.09 33345.90 418327.08 693093.38 0.064957265

AACT_1_T2 77287.36 1 17790.78 1395650.78 2296282.07 0.04988345

AACT_1_T3 42469.24 64666.08 792452.81 1302423.44 0.064957265

AACT_2_T1 32992.15 28726.46 77826.88 32231, 67 0.01041 181

AACT_2_T2 42504.96 41674.52 97227.83 40853.41 0.01041 181

AACT_2_T3 31 132.20 25199.84 75803.50 28997.80 0.006993007

Claims

Method of obtaining useful data for the determination of aortic stenosis (AD) or of the risk of suffering from AD or of the evolution of an AD comprising:

a) obtain an isolated biological sample from a subject, and

b) detecting and / or quantifying the expression product of a nucleotide sequence of an α-1-antichymotrypsin gene encoding an amino acid sequence having at least 50% identity with SEQ ID NO: 1, or a fragment thereof expression product, in the sample obtained in step (a).

Method according to claim 1, wherein the expression product or a fragment thereof, detected and / or quantified in step (b) encodes an amino acid sequence having at least 70% identity with SEQ ID NO: 1.

Method according to any of claims 1 or 2, wherein the expression product or a fragment thereof, detected and / or quantified in step (b) encodes the amino acid sequence SEQ ID NO: 1.

Method according to any of claims 1-3 which further comprises comparing the data obtained in (b) with standard values to find a significant deviation.

Method according to claim 4 which further comprises attributing the significant deviation to the presence of EA or the risk of suffering from AD in the subject of (a).

Method according to claims 1-5, wherein the biological sample from step (a) is a biological fluid.

7. Method according to claims 1-6, wherein the biological fluid is blood or serum or blood plasma.

8. Method according to claims 1-6, wherein the biological sample from step (a) comprises cells.

9. Method according to claims 1-6, wherein the biological sample from step (a) comprises a tissue. 10. A method according to any of claims 1-9, wherein the expression product, or a fragment thereof, of the nucleotide sequence of the α-1-antichymotrypsin gene is an amino acid sequence having at least 50% or at least one 70% identity with SEQ ID NO: 1, or SEQ ID NO: 1. 11. Method according to any one of claims 1-9, wherein the expression product is detected by PCR.

12. Method according to claim 10, wherein the amino acid sequence is detected by electrophoresis, immunoassay, chromatography, microarray technology and / or mass spectrometry.

13. Method according to claim 12 wherein the amino acid sequence is detected by liquid chromatography coupled to tandem mass spectrometry.

14. Use of the expression product of the α-1-anti-chymotrypsin gene or a fragment thereof, as a marker:

a) for the determination of the risk of suffering from AD,

b) to determine the presence of an EA, or

c) to predict the progression of an EA.

15. Use of an amino acid sequence that has at least 50% identity with SEQ ID NO: 1, or a fragment thereof, as a marker:

a) for the determination of the risk of suffering from AD,

b) to determine the presence of an EA, or

c) to predict the progression of an EA.

16. Use according to claim 15, wherein the amino acid sequence has at least 70% identity with SEQ ID NO: 1, or a fragment thereof.

17. Use according to any of claims 15 or 16, wherein the amino acid sequence is SEQ ID NO: 1, or a fragment thereof.

18. Use of a kit comprising primers and / or probes that hybridize with the sequence of an α-1-antichymotrypsin gene encoding an amino acid sequence having at least 50% or at least 70% identity with SEQ ID NO: 1, or coding for SEQ ID NO: 1, to determine the risk of having EA or to determine the presence of an EA or to predict the progression of an EA, in an isolated biological sample.

19. Use of a kit comprising antibodies capable of binding to an amino acid sequence that has at least 50% or at least 70% identity with SEQ ID NO: 1, or that codes for SEQ ID NO: 1, or a fragment thereof, to determine the risk of suffering from AD or to determine the presence of an AD or to predict the progression of an AD, in an isolated biological sample.