Nuclear biomarkers of alzheimer's disease, uses thereof and associated methods

Abstract

An in vitro procedure for diagnosing or determining the risk of a person developing Alzheimer's disease (AD), said procedure detecting and quantifying the expression products of the LMNA gene (SEQ. ID: No. 3): lamin A protein (SEQ. ID: No. 1) and its precursor prelamin A (SEQ. ID: No. 2), in a sample of peripheral nerve or smooth muscle.

Description

[0001] Nuclear biomarkers of Alzheimer's disease, their uses and methods

[0002] DESCRIPTION

[0003] Field of the invention

[0004] The invention provides for the reliable detection and identification of biomarkers essential for the diagnosis and prognosis of Alzheimer's disease (AD) in tissue samples from the mesenteric peripheral nervous system and in smooth muscle fiber samples from living subjects. The present invention also relates to an in vitro method for the diagnosis and / or prognosis of AD in tissue samples isolated from living subjects, as well as a method for the diagnosis of AD at its various stages of severity and / or at its various stages of progression.

[0005] Background of the invention

[0006] AD is a neurodegenerative disease and the leading cause of dementia worldwide, with aging being the primary risk factor. The number of people living with dementia worldwide is estimated to increase from 55 million in 2019 to 139 million in 2050, according to the QMS. The costs associated with dementia are also expected to double from US$1.3 trillion per year in 2019 to US$2.8 trillion by 2030.

[0007] Although there have been significant advances in the diagnosis and treatment of dementia in recent years, a cure is still far from being found.

[0008] Reducing the risk of dementia is not a binary choice between developing the disease later in life or never developing it. Delaying the onset of dementia or slowing its progression after diagnosis are equally important goals when addressing the burden of the disease at the societal and individual levels, not to mention the incalculable value of living a few more years in good health for both patients themselves and their loved ones.

[0009] Thus, early diagnosis of the disease acquires incalculable value in health and social policies against AD.

[0010] Clinically, AD is characterized by impaired memory and cognitive function. Most patients present with neuropsychiatric symptoms known as "behavioral and psychological symptoms of dementia," such as depression, excessive activity, psychosis, and aggressive behavior. The histological hallmarks of AD are senile plaques, composed of deposits of the beta-amyloid peptide (A|3) and neurofibrillary tangles (NFTs), which are fibrillar deposits of the hyperphosphorylated tau protein (p-tau). Other pathological events that appear to play a key role in the disease include synaptic dysfunction and inflammation or vascular alterations.

[0011] It is known that the first changes in neurons begin years, even decades before the disease manifests and symptoms become evident, usually in stages III-IV of cytoskeletal pathology (phosphotau tangles in the cytoplasm and beta amyloid plaques in the neuropil) according to the Braak & Braak classification. Thus, the topographic method called Braak & Braak (Braak H, Alafuzoff I, Arzberger T, Kretzschmar H, Del Tredici K. Staging of Alzheimer's disease-associated neurofibrillary pathology using paraffin sections and immunocytochemistry. Acta Neuropathol. 2006 Oct;112(4):389-404), is used to determine the stage of severity of the disease.Briefly, in Braak stages II and III, the accumulation of defective tau protein (neurofibrillary deposits) is mild and begins in a specific region of the nervous system; in Braak stages III–IV, the accumulations begin to become more severe and, thanks to the defective protein's ability to travel from one neuron to another and to disrupt healthy tau proteins, spread to other regions; finally, in Braak stages V–VI, tau accumulations extend throughout the entire nervous system.

[0012] Currently, diagnostic criteria for AD are typically based on criteria established by the American Institute on Aging and the Alzheimer's Association, while neuropathological confirmation—microscopic examination of the brain—is not typically performed during life.

[0013] Currently used diagnostic procedures for the disease include blood tests, which assess general health parameters that may affect cognitive function. Plasma biomarkers (p-tau217) are also currently available, which can aid in the diagnosis of Alzheimer's disease in 70–80% of cases. In addition, brain neuroimaging techniques are used. Different types of technology are used to obtain brain images: computed tomography (CT or CAT) and magnetic resonance imaging (MRI), both of which provide a life-size, still image of the brain, allowing for assessment of significant neuronal loss in areas typically affected by AD or other types of lesions. Positron emission tomography (PET) uses substances labeled with a small dose of radioactivity to study neuronal metabolism (glucose PET) or amyloid deposition (amyloid PET).Cerebrospinal fluid analysis is also considered as a diagnostic technique: Cerebrospinal fluid is the fluid that surrounds the brain and spinal cord. Because it is in contact with the brain, it reflects what is happening in the organ. Thus, the proteins that accumulate in the brain in patients are measured: the protein p-amyloid and the proteins tau and phosphorylated tau. In order to analyze cerebrospinal fluid, a sample must first be obtained through a lumbar puncture. Finally, genetic testing is performed in cases where a genetic form of the disease is suspected.

[0014] However, there remains a need to improve diagnostic and prognostic techniques for the disease, but it is also necessary to establish a biological definition of AD based on biomarkers that reflect the underlying neuropathology. In a syndrome as complex as AD, biomarkers are useful for early diagnosis when the disease is not yet symptomatic, staging the disease, establishing a prognosis, developing new treatment strategies, and assessing treatment response.

[0015] The approved traditional cerebrospinal fluid (CSF) and structural and functional neuroimaging biomarkers have limited clinical application. The main limitations of traditional AD biomarkers in cerebrospinal fluid (CSF)—AB, total tau (t-tau) and phosphorylated tau (p-tau), and PET for glucose metabolism and amyloid deposition—are their invasive nature and excessive cost, which hampers their application in clinical practice.

[0016] WO2021 / 202777 discloses an AD diagnostic method that quantifies biomarkers such as beta amyloid or various phosphoTAU in cerebrospinal fluid samples.

[0017] In addition, the state of the art describes other diagnostic methods that are performed on other biological samples of the subject, such as: US20220283184, which discloses a diagnostic method that measures the concentration of beta-synuclein in blood.

[0018] ES2774519 refers to a method for diagnosing AD in a subject, comprising the step of: determining the presence or absence of one or more circular RNAs (circRNAs) in a blood sample from said subject; wherein the presence or absence of said one or more circRNAs is indicative of the disease.

[0019] ES2581178 describes a method for detecting the presence of AD risk through the detection of a set of biomarkers comprising a protein or ribonucleic acid (RNA) encoded by each of the following genes: APBA1 , ATG7, BECN1 , CD44, CDH2, COL18A1 , ERBB4, F3, FLNA, FYN, GRIN2B, IL20, ITPR1 , LRP8, MTOR, NPPC, NRP1 , PDGFC, ROBO1 , SEMA3E, TGFB1 , THBS1 , VEGFR1 and WWOX.

[0020] Thus, existing diagnostic techniques may not be entirely reliable; some entail significant risk in obtaining samples, and the most effective ones are performed postmortem. As a result, there remains a need to provide an in vitro diagnostic and / or prognostic method for the disease, including early diagnosis when the disease is not yet symptomatic, staging the disease, and its degree of severity. This method is effective and reliable, and can be performed on biological samples isolated from living individuals, thereby enabling a reliable and early diagnosis that will lead to improved treatment.

[0021] Summary of the invention

[0022] The present invention is based on previous work carried out by the inventors of the present application, in which it is demonstrated that AD develops from a nuclear pathology that is maintained throughout all stages of the disease, in neurons of the hippocampus and the entorhinal cortex (Gil et al. 2020, 2021, 2022; Rodríguez-Leyva et al. 2020). Phosphorylated nuclear tau and the abnormal expression of lamin A associated with the loss of pyramidal neurons are involved in this nuclear dysfunction (Gil et al. 2020; Rodríguez-Leyva et al. 2020). In this way, it was demonstrated that the onset of the AD phenotype (Braak l-ll), in the pyramidal and granule neurons of the hippocampus and the entorhinal cortex, is manifested with a nuclear pathology (NP) that persists until the late stages (Braak V-VI).The main features already published in relation to this PN are: (i) the exit of nuclear tau (AT100) to the cytoplasm, (ii) epigenetic changes in histones specific to the onset of AD such as the overexpression of H4K20me3 and (iii) the aberrant expression of lamin A in the nuclear lamina (LN) of neurons (Gil et al. 2020, 2021; Rodríguez-Leyva et al. 2020).

[0023] These previous works confirm that in healthy subjects the lamin A protein is barely expressed in human brain neurons (Gil et al. 2020) in accordance with previous studies in mouse brain, rat brain and rat retinal neurons (Takamori et al. 2018; Wakabayashi et al. 2011). Low lamin A expression is regulated in neurons by m¡R-9, a brain-specific microRNA (Jung et al. 2012, 2013; Nissan et al. 2012). Indeed, in the rare premature aging disorder Hutchinson-Gilford progeria syndrome, neurons do not undergo neurodegeneration because they do not express the mutated lamin A precursor (Gonzalo et al. 2017).

[0024] Furthermore, it has been histologically demonstrated that in brains with AD, neurons in the hippocampus and entorhinal cortex abnormally express lamin A from the early stages of the disease (Gil et al. 2020, 2021, 2022; Rodríguez-Leyva et al. 2020). These results have been supported by RT-PCR (Méndez-López et al. 2019) and confirmed in transgenic mouse models of AD (Gil et al. 2020); Alzheimer's disease has been defined as an acquired laminopathy (Frost B 2016; Frost et al. 2016). These results indicate a direct relationship between the abnormal expression of Lamin A in neuronal nuclei and neurodegeneration.

[0025] The inventors of the present invention have now succeeded in identifying the abnormal expression of the following biomarkers: the proteins lamin A and / or prelamin A, as well as the ribonucleic acid (RNA) encoded by the LMNA gene in the nuclei of smooth muscle tissue cells as well as in the nuclei of neurons in the peripheral nervous system in subjects suffering from AD, while in healthy subjects no expression of said proteins is detected in the aforementioned tissues.

[0026] Likewise, it has been possible to identify and quantify in the aforementioned tissues of subjects suffering from AD the overexpression with different intensities of said biomarkers, such that the overexpression in different intensities of the biomarkers correlates with different stages or severity of AD in the subject. Specifically, the different intensities of overexpression of the biomarkers allow differentiation between at least the stages of AD: early AD (according to the Braak classification I-II) and late AD (according to the Braak classification V-VI). The identification of overexpression of the biomarkers as defined in the previous paragraphs allows the design of an in vitro method for the diagnosis of AD and / or prognosis of (determination of the risk of developing) AD in biological samples of smooth muscle as well as in neuronal tissue of the peripheral nervous system using as a biomarker at least the lamin A protein with SEQ.ID. No. 1 and / or prelamin with SEQ.ID. No.2 and / or the ribonucleic acid (RNA) encoded by the LMNA gene with SEQ.ID. No.3.

[0027] An aspect of the present invention therefore provides an in vitro method for diagnosing or determining the risk of developing AD in a subject, comprising a) detecting or measuring the concentration, quantity or activity of at least one biomarker selected from lamin A with SEQ.ID. No. 1 and / or prelamin with SEQ.ID. No. 2 and / or the ribonucleic acid (RNA) encoded by the LMNA gene with SEQ.ID. No. 3 in a biological sample isolated from a living subject; and b) determining or diagnosing the presence or risk of developing AD based on the detection, concentration, quantity or activity of said biomarker, wherein the detection or increase in the concentration, quantity or activity of said biomarker compared to a reference concentration or quantity of said biomarker in a control sample is indicative of the presence or risk of developing AD, where the biological sample is peripheral nervous tissue and / or smooth muscle of a subject.

[0028] The present invention also provides an in vitro method for diagnosing AD in its various degrees of severity, from early AD to late AD, according to the Braak classification, based on a specific intensity of overexpression of at least one of the biomarkers: lamin A with SEQ.ID. No. 1 and / or prelamin with SEQ.ID. No. 2 and / or the ribonucleic acid (RNA) encoded by the LMNA gene with SEQ.ID. No. 3.

[0029] , in smooth muscle as well as in neuronal tissue of the peripheral nervous system.

[0030] Therefore, another aspect of the present invention is an in vitro method for diagnosing and classifying AD in a subject according to different degrees of severity of the disease, comprising a) detecting and measuring the concentration, quantity or activity of at least one biomarker selected from lamin A with SEQ.ID. No. 1 and / or prelamin with SEQ.ID. No. 2 and / or the ribonucleic acid (RNA) encoded by the LMNA gene with SEQ.ID. No. 3. in a biological sample isolated from a living subject; and b) determining or diagnosing the presence of AD in the subject according to different degrees of severity of the disease, wherein the detection or increase in the concentration, quantity or activity of said biomarker compared to a reference concentration or quantity of said biomarker in a control sample is indicative of AD according to different degrees of severity of the disease, where the biological sample is smooth muscle and / or neuronal tissue of the peripheral nervous system.

[0031] Another object of the present invention is a method as defined in any of the preceding paragraphs, where step a) is performed with one or more of the following techniques: RT-PCR, Northern Blot, Western Blot, microarray analysis and / or immunoassay.

[0032] Another object of the present invention is a method as defined in any of the preceding paragraphs, where in step a) the detection of the biomarker is carried out through a visual analysis using an immunohistochemical technique followed by RT-PCR.

[0033] Another object of the present invention is a test kit and its use to carry out step a) of any of the methods defined in previous paragraphs, where the kit comprises: a) means to detect in a biological sample isolated from the subject the expression levels of the biomarkers: lamin A with SEQ.ID. No. 1 and / or prelamin with SEQ.ID. No. 2 and / or the ribonucleic acid (RNA) encoded by the LMNA gene with SEQ.ID. No. 3., and b) means to compare the expression level of the biomarkers determined in (a) with a reference sample.

[0034] It is also an object of the present invention a kit for use in the procedures defined in the preceding paragraphs where the kit comprises means for carrying out one or more of the following techniques: RT-PCR, Northern Blot, Western Blot, microarray analysis and / or immunoassay.

[0035] It is also an object of the present invention a kit for use in the methods defined in the preceding paragraphs where the kit comprises means for performing an immunoassay followed by RT-PCT.

[0036] Brief description of the figures

[0037] Fig. 1. Immunohistochemical analysis of lamin A protein in mesenteric neuron nuclei from the colon, from healthy tissues (a, b), tissues from patients diagnosed postmortem with early AD (Braak l-ll) (c, d) and late AD (Braak v-vi) (e, f). Scale bar in b, d, f: 100 mm; in D, a, b, e: 10 mm.

[0038] Fig. 2. Using ImageJ software to evaluate the intensity level of nuclear lamin A signal in mesenteric neurons of the samples (NIH, Bethesda, MD (http: / / rsb.info.nih.gov / ij / index.html).

[0039] Fig. 3. Immunohistochemical analysis of lamin A protein in mesenteric neurons from tissues diagnosed postmortem with late AD (Braak V-VI) (a, f). Scale bar = 100 mm.

[0040] Fig. 4. Immunohistochemical analysis of lamin A protein in smooth muscle cell nuclei from healthy tissues (a,b), tissues from patients diagnosed postmortem with early AD (Braak l-ll) (c,d) and late AD (Braak V-VI) (e,f). Scale bar in b,d,f: 100 mm; in D, a,b,e: 10 mm.

[0041] Fig. 5. Immunohistochemical analysis of lamin A protein in nuclei of PNS neurons (a,b,c) and nuclei of smooth muscle cells (d,e,f) of prostate, in tissues from diagnosed

[0042] Fig. 6 The one-way ANOVA test determines that the proportion of positives is different among the three samples with a significance level of 0.05. mm.

[0043] Fig. 7 The ANOVA test in both cases has a significance of 0, clearly below 0.05, in both the variance difference test and the mean difference test. Detailed description of the invention

[0044] The term "biomarker", as used in the present description, refers to a substance, the presence of which can be objectively determined and quantified, which is used as an indicator of the presence / absence of the AD disease or of its prognosis. The biomarker of the invention can be detected and / or quantified, that is, only its presence can be detected or changes in its quantity can be detected and / or quantified. The biomarkers of the invention can be detected directly in the form of protein in patient samples or indirectly from the expression product of the genes that encode said proteins. In the present invention, the biomarker(s) are at least one of: lamin A with SEQ.ID. No. 1 and / or prelamin with SEQ.ID. No. 2 and / or the ribonucleic acid (RNA) encoded by the LMNA gene with SEQ.ID. No. 3.

[0045] In the present invention, "diagnosis" is understood as the procedure by which it is determined that a subject suffers or is suffering from a certain disease, nosological entity, syndrome, or any health-disease condition, by analyzing a series of clinical parameters of said disease, and that distinguish it from other diseases with similar clinical pictures. In the present invention, the disease to be identified is AD, and the clinical parameter is the detection and / or quantification of a biomarker in an isolated sample of peripheral nervous tissue or smooth muscle of a subject, where the biomarker is at least one of: lamin A with SEQ.ID. No. 1 and / or prelamin with SEQ.ID. No. 2 and / or the ribonucleic acid (RNA) encoded by the LMNA gene with SEQ.ID. No. 3.

[0046] Both the diagnostic use of the invention and the predictive use of the invention are made on an isolated sample of peripheral nervous tissue or smooth muscle in a subject, where said sample is obtained by methods and procedures well established and known to the person skilled in the art.

[0047] The term “isolated sample” as used herein refers to a portion or small amount of something that is considered representative of the whole and that is taken or separated from it for study, analysis or experimentation. In the present invention, said study, analysis or experimentation refers to the presence or amount of lamin A with SEQ.ID. No. 1 and / or prelamin with SEQ.ID. No. 2 and / or the ribonucleic acid (RNA) encoded by the LMNA gene with SEQ.ID. No. 3., in an isolated sample of peripheral nervous tissue or smooth muscle. The techniques for obtaining biological samples from an individual are widely known in the state of the art, and any of them can be used in the practice of the present invention.

[0048] The term “peripheral nervous tissue” as used herein refers to the part of the nervous system that is outside the central nervous system (CNS), consisting of nerves, nerve ganglia and sensory receptors that are found in various parts of the body.

[0049] The term “smooth muscle” as used in the present description refers to a type of muscle tissue characterized by having elongated and thin muscle cells, without the regular organization of striated fibers, without transverse bands and with the ability to contract involuntarily, usually located in the walls of internal organs such as the stomach, intestines, blood vessels, bronchi, uterus and other organs.

[0050] A "reference sample," as used herein, means a sample isolated from a group of healthy subjects that does not have a particular disease state or phenotype, in this case AD. The "reference amount / concentration / activity" can be determined by measuring the levels of the biomarkers of the invention and the expression products of said gene in a group of healthy subjects, and those reference levels can be tailored to specific populations. For example, a reference level can be age-related, so that comparisons can be made between expression levels in samples from subjects of a certain age and reference levels for a particular disease state, phenotype, or lack thereof in a certain age group. In a preferred embodiment, the reference sample is obtained from multiple subjects.The person skilled in the art will appreciate that the type of reference sample may vary depending on the specific method to be performed.

[0051] By "accuracy" is meant the proportion of correct results of a diagnosis, by "sensitivity" is meant the proportion of all positive diagnoses that are correctly classified as positive, and by "specificity" is meant the proportion of all negative diagnoses that are correctly classified as negative. The inventors of the present invention have succeeded in identifying the abnormal expression of the proteins lamin A (SEQ. ID. No. 1) and / or prelamin A (SEQ. ID. No. 2) in nuclei of smooth muscle cells as well as in nuclei of neurons of the peripheral nervous system in subjects suffering from AD, while in healthy subjects no expression of said proteins is detected in said neuronal cells. Thus, the method of the invention preferentially uses the difference in expression of lamin A (SEQ. ID. No. 1) and / or prelamin A (SEQ. ID. No. 2) in said tissues, as a biomarker of AD.

[0052] The methods described in this application are performed on samples isolated from a subject. In a preferred embodiment of the diagnostic use of the described methods or predictive methods of the invention, the subject is a human being of any sex, age, or race.

[0053] The inventors of the present invention have also succeeded in identifying and quantifying in the mentioned tissues the overexpression of the proteins lamin A with SEQ. ID. No. 1 and / or prelamin with SEQ. ID. No. 2 and / or the ribonucleic acid (RNA) encoded by the LMNA gene with SEQ. ID. No. 3.

[0054] , where said overexpression occurs with different intensities, such that it allows the diagnosis or detection of AD in a living subject, as well as determining the degree / state / affect / phase of severity of AD in the subject. The method described makes it possible to distinguish different stages or severity of AD in the subject. Specifically, the different intensities of overexpression of the biomarkers allow differentiation between at least the stages of AD: early AD (according to the Braak I-II classification) and late AD (according to the Braak V-VI classification).

[0055] The identification of overexpression of the protein: lamia A and / or prelamin A as well as the RNA encoded by the gene, LMNA, as defined in previous paragraphs, allows the design of an in vitro method for the diagnosis and / or prognosis of AD and / or classification of AD in its different degrees of severity, in biological samples of peripheral nervous tissue or smooth muscle using as biomarkers at least the protein lamin A and / or prelamin A or the RNA that encodes for said proteins, the LMNA gene.

[0056] Depending on the level of expression, degree of accumulation, and extravasation into the perinuclear space of these biomarkers, it is also possible to determine the degree of patient involvement. The lamin A protein regulates gene expression as it is directly or indirectly involved in DNA replication, transcription, and repair, as well as in cell cycle control (Shimi et al. 2008). Consequently, the lamin A protein is closely associated with basic and complex biological processes such as the development, differentiation, and aging of cells and organs (van Steensel et al. 2017; Kim et al. 2019).

[0057] Lamin A is barely expressed in human brain neurons (Gil et al. 2020) in agreement with previous studies in mouse brain, rat brain and rat retinal neurons (Takamori et al. 2018; Wakabayashi et al. 201 1).

[0058] However, the authors of the present invention have demonstrated histologically for the first time that in AD brains, hippocampal and entorhinal cortex neurons aberrantly express lamin A from the early stages of the disease (Gil et al. 2020, 2021, 2022; Rodríguez-Leyva et al. 2020). These results have been supported by RT-PCR (Méndez-López et al. 2019) and confirmed in transgenic mouse models of AD (Gil et al. 2020).

[0059] The lamin A protein (SEQ.ID.No. 1) is initially expressed as prelamin A (SEQ.ID.No.2) which after a cleavage process becomes lamin A. The term lamin A refers to the 572 amino acid protein (UniProt number W8QEH3), and the gene that encodes said protein is called: LMNA (ENSEMBLE accession number: ENSG00000160789), (SEQ.ID.No.3).

[0060] Prelamin A (SEQ. ID. NO. 2) is the precursor form of lamin A, (SEQ. ID. No. 1), it contains a CAAX-box motif at the C-terminal end that is subject to farnesylation in the cisterna, the AAX amino acids being eliminated, and subsequently an endoproteolytic cleavage takes place that eliminates the last 18 amino acids to generate the mature lamin A (SEQ. ID. No. 1), capable of integrating into the nuclear envelope. The term prelamin A (SEQ. ID. No. 2) refers to the 664 amino acid protein with 70kD (UniProt number P02545), and the gene that also encodes said protein is the LMNA gene (ENSEMBLE accession number: ENSG00000160789), (SEQ. ID. No. 3).

[0061] The LMNA gene (SEQ.ID.No.3) encodes prelamin A (SEQ:ID.No.2) to lamin A (SEQ.ID.No.1) which, as already indicated, are nuclear proteins. Among its functions, its role in the assembly of the nucleus, maintenance of chromatin stability and the regulation of gene expression is known (Ovsiannikova NL et al. 2021; Forsberg F et al. 2019; Puckelwartz MJ et al. 201 1; Ghosh S et al. 2015; Fernandez A et al. 2022; Wilson KL et al. 2005; Koch AJ et al. 2014).

[0062] The methods proposed in the invention are specifically developed to be performed on solid tissue samples extracted from smooth muscle tissue and / or peripheral nervous system tissue, that is, peripheral neuronal tissue, such as mesenteric neuronal tissue and / or smooth muscle tissue, such as a colon or prostate biopsy.

[0063] The invention also relates to a kit and its use in any of the methods of the invention, comprising means necessary for the detection and / or quantification of at least one of the biomarkers for the diagnosis and / or prognosis of AD in a subject.

[0064] In a preferred embodiment, the kit or device of the invention comprises: a) means for detecting the expression levels of the biomarkers of the invention in a biological sample isolated from the subject, and b) means for comparing the expression level of the determined biomarkers with a reference sample.

[0065] In particular embodiments, the kit is selected from

[0066] (a) a suitable kit for RT-PCR,

[0067] (b) a suitable kit for Northern Blot / Western Blot,

[0068] (c) a kit suitable for microarray analysis and

[0069] (d) an immunoassay.

[0070] Two or more of these embodiments may also be combined, so that the kit may comprise, for example, (a) and (c).

[0071] Thus, in another aspect, the invention relates to the use of the kit of the invention in any of the methods described above, although a kit comprising means for an immunoassay and RT-PCR is preferably contemplated.

[0072] As understood by one of ordinary skill in the art, the kit may comprise any component useful for practicing the present invention, such as primers, probes, transcriptases, antibodies, buffer solutions, delivery vehicles, material supports, positive and / or negative control components, etc. In addition to the aforementioned components, the kits may also include instructions for practicing the subject matter of the invention. These instructions may be present in the aforementioned kits in a variety of forms, one or more of which may be present in the kit. One form in which these instructions may be present is as printed information on a suitable medium or substrate, e.g., a sheet or sheets of paper on which the information is printed, on kit packaging, on a package insert, etc. Another medium would be a computer-readable medium, for example, a CD, a USB, etc., where the kit has been registered, is a website address that can be used via the Internet to access information at a remote site. Any convenient means may be included in the kits.

[0073] The expression “detect or measure the concentration, quantity or activity of biomarker” of step a) of the diagnostic method or method of predicting AD progression refers to any procedure that enables the quantification of the biomarker mentioned in the present invention, that is, of the nuclear proteins lamin A, prelamin A and / or the expression products of their corresponding LMNA gene.

[0074] Thus, protein markers can be detected and quantified with proteomic techniques such as immunoassays (ELISA), CBA (cytometric Bead assay), immunohistochemistry, western blot, Northern blot, microarrays, or mass spectrometry.

[0075] For its part, genetic techniques that allow the identification and quantification of mRNA, mainly qPCR and RTqPCR, are used to detect the expression product of the LMNA gene that encodes lamin A and prelamin A.

[0076] -PCR (Polymerase Chain Reaction): PCR is a widely used molecular technique in molecular biology. It is based on the in vitro replication of specific DNA segments. It involves the denaturation of the target DNA at high temperature, followed by the hybridization of specific primers and the synthesis of new DNA strands by a polymerase enzyme. This process is repeated in cycles, exponentially amplifying the target DNA. PCR is essential for the detection of genetic diseases, providing an accurate and sensitive tool for the selective amplification of genetic material.

[0077] -RT-PCR (Reverse Transcriptase Polymerase Chain Reaction): RT-PCR combines nucleic acid amplification using PCR with the prior conversion of RNA to DNA using reverse transcriptase. This method is crucial for detecting RNA, such as in messenger RNA diagnostics. Reverse transcriptase converts RNA into complementary DNA (cDNA), which then serves as a template for PCR. RT-PCR is fundamental in biomedical research and clinical diagnostics, providing a valuable tool for studying gene expression.

[0078] Immunohistochemistry (IHC) immunoassays are techniques used in biology and medicine to detect and visualize the presence of specific antigens in tissues or cells. These antigens can be proteins, carbohydrates, or other molecular components. The technique, based on the interaction between specific antibodies and the antigens of interest, is widely used in biomedical research and pathology for disease diagnosis, tumor characterization, and investigation of protein expression in specific tissues.

[0079] -Immunofluorescence Immunoassays: Immunofluorescence immunoassays involve the use of antibodies conjugated with fluorochromes to detect the presence of specific antigens. The antibodies selectively bind to the antigens, and the emitted fluorescence allows direct visualization under a fluorescence microscope. Immunofluorescence allows for quantitative and qualitative analysis, making it a valuable technique in biomedical research and clinical diagnosis. In a preferred embodiment of the invention, the antibody is anti-lamin A.

[0080] Western blotting: Western blotting is a molecular biology technique used to detect specific proteins in a sample. It begins with the separation of proteins by size using electrophoresis and then transfers the proteins to a membrane. The membrane is incubated with specific antibodies, which bind to the proteins of interest. Detection is performed using chemical reactions or fluorochromes. Western blotting is crucial for protein expression studies, the identification of post-translational modifications, and disease diagnosis, providing detailed information about the proteins present in a sample.

[0081] -Northern blot: The Northern blot technique is a methodology used in molecular biology to study gene expression and is used to detect RNA. It involves five steps: RNA extraction, agarose gel electrophoresis, membrane transfer, hybridization, and autoradiography or detection.

[0082] -Direct Visualization: Direct visualization involves the direct microscopic observation of a sample. It is a rapid and simple technique that allows for the detection of morphological characteristics, such as the presence of structural changes. Direct visualization is valuable for analyzing histology and pathology for rapid diagnosis. It can be used on clinical samples and biological tissues, facilitating the identification and characterization of elements of interest.

[0083] -Manual Counting: Manual counting is a quantification method in which observers directly count the number of entities of interest in a sample, such as cells or particles. This technique involves viewing the sample through a microscope or other observation tool, where the elements of interest are identified and counted one by one. Although it is a simple and straightforward approach, manual counting can be laborious and is subject to observer variability and subjectivity.

[0084] In Situ Hybridization: In situ hybridization is a molecular technique that uses nucleic acid probes to detect specific sequences in cells or tissues. The probes are complementary RNA or DNA sequences that bind specifically to the target sequence. During the process, probes labeled with fluorescent molecules or enzymes hybridize with the target nucleic acid molecules, revealing the presence and location of the specific sequences under a microscope. This technique not only provides information on the presence of genetic material but also on its spatial distribution in the tissue, allowing for detailed studies of molecular genetics and pathology.

[0085] Microarrays, also known as microarrays, are technological tools used in molecular biology and genomics to study gene expression, identify genetic variants, and analyze the presence or abundance of specific molecules in biological samples. There are two main types of microarrays: deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

[0086] -Chromatography: Chromatography is an analytical technique that separates and detects individual components in a sample based on their chemical properties. There are several types of chromatography, such as liquid chromatography and gas chromatography. In this method, the sample is placed on a stationary phase and moved through a porous medium. The sample components are separated based on their interactions with the stationary and mobile phases, resulting in different retention times. Detection is achieved by analyzing the output of the system. Chromatography is widely used in analytical chemistry, biochemistry, and pharmacology to separate and quantify substances in complex mixtures.

[0087] Spectroscopy: Spectroscopy is a technique that analyzes the interaction between electromagnetic radiation and a sample. In the context of quantification, spectroscopy provides information about the chemical composition and molecular structure of a sample. Different forms of spectroscopy, such as absorption, emission, and nuclear magnetic resonance spectroscopy, are used to study different properties of molecules.

[0088] Preferably, the relative levels or concentrations of biomarkers can be determined by contacting the sample with probes, preferably immobilized on a substrate, specific for each of the protein biomarkers, either Lamin A or prelamin A. The interactions between the biomarker and its respective probe can be monitored and quantified using various methods that are well known in the art. An example of a suitable method is an enzyme-linked immunosorbent assay (ELISA). Performing an ELISA involves at least one antibody with specificity for at least one of the biomarker proteins: Lamin A and Prelamin A.

[0089] A sample containing an unknown amount of biomarker protein is immobilized on a solid support (usually a polystyrene microtiter plate), either non-specifically (by adsorption to the surface) or specifically (by capture by another antibody specific for the same biomarker protein, in a sandwich ELISA). After the biomarker protein has been immobilized, the detection antibody is added, forming a complex with the biomarker protein(s). The detection antibody may be covalently linked to an enzyme, or it may itself be detected by a secondary antibody that is linked to an enzyme by bioconjugation.

[0090] In the present case and for the detection of lamin A and / or prelamin proteins, reagents can be used that comprise: capture antibodies, detection antibodies, secondary antibodies, conjugated enzymes, substrate for the enzyme, blocking solution, washing solution, coating buffer, dilution buffer, and substrate buffer, etc.

[0091] Preferably, genetic techniques that allow the identification and quantification of mRNA, mainly qPCR, RTqPCR, are used to detect the expression product of the LMNA gene encoding lamin A and prelamin A:

[0092] PCR is a widely used molecular technique in molecular biology. It is based on the in vitro replication of specific DNA segments. It involves the denaturation of the target DNA at high temperature, followed by the annealing of specific primers and the synthesis of new DNA strands by a polymerase enzyme. This process is repeated in cycles, exponentially amplifying the target DNA. PCR is essential for the detection of genetic diseases, providing an accurate and sensitive tool for the selective amplification of genetic material.

[0093] In the present case and for the detection of the RNA encoded by the LMNA gene, at least the following can be used: template DNA, forward primers, reverse primers, Taq DNA polymerase, dNTPs (deoxynucleotides), PCR buffer, stabilizing agents, nuclease-free water, positive control, negative control, staining dyes, etc.

[0094] RT-PCR (Reverse Transcriptase Polymerase Chain Reaction):

[0095] RT-PCR combines nucleic acid amplification using PCR with the prior conversion of RNA to DNA using reverse transcriptase. This method is crucial for detecting RNA, such as in messenger RNA diagnostics. Reverse transcriptase converts RNA into complementary DNA (cDNA), which then serves as a template for PCR. RT-PCR is fundamental in biomedical research and clinical diagnostics, providing a valuable tool for studying gene expression.

[0096] In the present case and for the detection of the RNA encoded by the LMNA gene, at least the following can be used: forward primers, reverse primers, reverse transcriptase, Taq DNA polymerase (or thermostable polymerase), dNTPs (deoxynucleotides), reverse transcriptase buffer, PCR buffer, nuclease-free water, RNase inhibitors, staining dyes, positive control, negative control, etc.

[0097] Preferably, the method of the invention has an accuracy of at least 75%, for example an accuracy of 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. Preferably, the method of the invention has a sensitivity of at least 75%, for example a sensitivity of 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0098] Preferably, the method of the invention has a specificity of at least 75%, for example, a specificity of 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0099] Preferably, as shown in the attached example, the method of the invention achieves a specificity and sensitivity of the test close to 100% with a sample size that is statistically sufficient to demonstrate the usefulness of this diagnostic test due to the excellent results obtained, (Fig. 6 and 7).

[0100] Example

[0101] Detection of lamin A overexpression (SEQ.ID.No. 1) in neurons of the peripheral nervous system and smooth muscle

[0102] Materials

[0103] The invention is preferably carried out from an isolated sample of mesenteric neurons and colon smooth muscle from a patient with AD symptoms with the following characteristics:

[0104] -Be between 60 and 90 years old, since only sporadic AD cases are considered,

[0105] -Meet the diagnostic criteria for dementia according to FM3R, and

[0106] -Not presenting tumorous or inflammatory digestive pathology.

[0107] In this case, two cohorts were compared: one of AD patients with an n of 6 individuals previously diagnosed postmortem through histological analysis of brain samples with specific markers (phospho Tau), and another cohort of individuals previously diagnosed as healthy with an n of 6 (Table 1). The cohort diagnosed with AD was subdivided into:

[0108] (1) Early AD, with very few manifestations of cytoplasmic tangles of the protein fofo-tau and with anomalous expression of Lamin A in the nucleus of its brain neurons (Braak l-ll)

[0109] (2) Late AD where there is a massive presence of cytoplasmic tangles and overexpression of nuclear Lamin A in brain neurons (Braak V-VI). The characteristics of the sample are presented descriptively in Table 1 below, indicating that in all samples the abnormal expression of the lamin A protein is measured in the nuclei of neurons of the mesenteric peripheral nervous system and smooth muscle fibers, in addition to the brain status of each patient.

[0110] Table 1

[0111] Methods The technique used for the detection of lamin A (SEQ.ID.No.1) in the sample under study is the immunoassay technique that highlights the accumulation of lamin A protein as a visual marker of neuronal cell nucleus deterioration. The results that identify the presence of lamin A (SEQ.ID.No.1) will be used to determine the diagnosis and risk of early development of AD, since this protein is not expressed in the normal nucleus of healthy neurons.

[0112] Immunohistochemical localization of lamin A (SEQ.ID. No. 1) was performed using the Lamin A antibody (Thermo Fisher Scientific, Rockford, USA) on 4 mm paraffin-embedded sections. The immunohistochemical (IHC) technique used in the selected tissues was processed using tissue sections using the following steps:

[0113] -Stage 1. Deparaffinization using standard protocols.

[0114] -Stage 2. Antigen retrieval by using a low pH 6.0 citrate buffer (Dakocytomation, Glostrup, Denmark) at 96 C for 20 minutes in a Dako PTLink (Dakocytomation, Glostrup, Denmark).

[0115] -Stage 3. Blocking endogenous peroxidases with Dako.

[0116] -Step 4. Preincubation for 1 h at 37 C with a blocking solution containing bovine serum albumin.

[0117] -Step 5. Incubation with monoclonal and / or polyclonal antilamin A primary antibody (Thermo Fisher Scientific, Rockford, USA) diluted 1:100 in Dako Antibody Diluent (Dakocytomation Glostrup, Denmark) for 30 minutes at room temperature.

[0118] -Step 6. Staining was visualized using EnVision FLEX+ Mouse (linker) (Dakocytomation Glostrup, Denmark), and with 303-diaminobenzidine tetrahydrochloride (DAB) and hematoxylin counterstain.

[0119] All steps were performed at room temperature, and between incubations the sections were washed with Dako wash buffer.

[0120] IHC staining includes a positive control for the anti-Lamin A antibody.

[0121] The total and relative number of mesenteric neuronal and smooth muscle cells with nuclei positive for lamin A expression (SEQ. ID. No. 1) was recorded by manual counting in three optical fields. The number of lamina A-positive muscle cells per section was taken into account.

[0122] Results:

[0123] Mesenteric neuron and colonic smooth muscle samples from subjects with AD show overexpression of Lamin A (SEQ ID NO: 1). The expression levels of these proteins in these samples determine the AD risk level or diagnosis (ICD) if accompanied by symptoms necessary to define AD pathology.

[0124] The present invention presents robust data on its efficacy as a diagnostic method. These data are derived from the analysis of postmortem samples obtained by our research team. The results obtained after the IHC analysis in all cases presented in Table 1, where the biomarker lamin A (SEQ:ID.No.1) was not evident in the mesenteric neurons of healthy samples and the overexpression of the biomarker in the intestinal samples of those diagnosed with AD (Fig. 1).

[0125] The available data show that the relative quantification of lamina A staining was normalized with respect to the EA1 group (Fig. 2). The one-way ANOVA test determined that the intensity level is different between the three samples (healthy, early AD (Braak l-ll) and late AD (Braak V-VI)) and showed, with statistical significance of 0.03, the differential results between healthy subjects with respect to early AD and 0 with respect to late AD, thus determining this diagnostic test as an efficient method to differentiate patients with AD.

[0126] The overexpression of lamin A in mesenteric neurons of digestive samples from deceased patients histologically diagnosed with late AD is presented in Fig. 3, which supports the use of the presented methodology as an IN VITRO diagnosis of Alzheimer's for living patients.

[0127] IHC analysis of smooth muscle cells adjacent to mesenteric neurons did not show the biomarker Lamin A (SEQ.ID. NO.1) in the smooth muscle fibers of healthy samples; however, early AD samples (Braak l-ll) showed abnormal expression in some smooth muscle cell nuclei; and late AD samples (Braak V-VI) showed a high number of smooth muscle fibers with abnormal overexpression of the Lamin A marker (Fig. 4). This same analysis was performed in samples of peripheral neurons and smooth muscle in other organs and systems, such as prosthetic tissue (Fig. 5), with similar results.

[0128] The total and relative number of smooth muscle cells with nuclei positive for Lamin A expression (SEQ.ID. NO.1) was obtained in three optical fields. The number of muscle cells positive for Lamin A (SEQ.ID. NO.1) was taken into account for each section (Figs. 6 and 7). A specificity and sensitivity of the test close to 100% was determined, with a sample size that is statistically sufficient to demonstrate the usefulness of this diagnostic test due to the excellent results obtained.

[0129] The overall quantitative results in the pathological analysis of the anomalous expression of Lamin A in mesenteric neurons (neurons) and fibers of the adjacent smooth muscle (smooth muscle), indicate that this biomarker is shown in 25% of the cell nuclei of the field of tissues studied in samples from individuals with early AD involvement (Braak l-ll), progressively increasing this percentage as the disease develops in the intermediate stages (Braak lll-IV). Determining a specificity and sensitivity of the test close to 100% of the cell nuclei of the studied field of said tissues in the advanced stages (Braak V-VI), with a sample size that is statistically significant (p <0.05) to demonstrate the usefulness of this diagnostic test to determine AD and the relationship with the tissue deterioration of the pathology due to the results obtained.

[0130] References

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Claims

CLAIMS 1. An in vitro method for diagnosing or determining a risk of developing AD in a subject, comprising a) detecting or measuring the concentration, quantity or activity of at least one biomarker selected from lamin A with SEQ.ID. No. 1 and / or prelamin with SEQ.ID. No. 2 and / or the ribonucleic acid (RNA) encoded by the LMNA gene with SEQ.ID. No. 3 in a biological sample isolated from a living subject; and b) determining or diagnosing the presence or risk of developing AD based on the detection, concentration, quantity or activity of said biomarker, wherein the detection or increase in the concentration, quantity or activity of said biomarker compared to a reference concentration or quantity of said biomarker in a control sample is indicative of the presence or risk of developing AD, where the biological sample is peripheral nervous tissue and / or smooth muscle of a subject. 2.- An in vitro method for diagnosing and classifying AD in a subject according to different degrees of severity of the disease, comprising a) detecting and measuring the concentration, quantity or activity of at least one biomarker selected from lamin A with SEQ.ID. No. 1 and / or prelamin with SEQ.ID. No. 2 and / or the ribonucleic acid (RNA) encoded by the LMNA gene with SEQ.ID. No. 3, in a biological sample isolated from a living subject; and b) determining or diagnosing the presence of AD in the subject according to different degrees of severity of the disease, wherein the detection or increase in the concentration, quantity or activity of said biomarker compared to a reference concentration or quantity of said biomarker in a control sample is indicative of AD according to different degrees of severity of the disease, where the biological sample is smooth muscle and / or neuronal tissue of the peripheral nervous system. 3.- A method as defined in any of the preceding claims, wherein in step a) the detection of the biomarker is carried out through one or more of the techniques selected from: RT-PCR, Northern Blot, Western Blot, microarray analysis and / or immunoassay. 4.- A method according to claim 1 to 3, wherein in step a) the detection of the biomarker is carried out through a visual analysis using an immunohistochemical technique followed by RT-PCR. 5.- Test kit for carrying out step a) of any of the methods defined in the preceding claims, wherein the kit comprises: a) means for detecting in a biological sample isolated from the subject the expression levels of the biomarkers: lamin A with SEQ.ID. No. 1 and / or prelamin with SEQ.ID. No. 2 and / or the ribonucleic acid (RNA) encoded by the LMNA gene with SEQ.ID. No. 3, and b) means for comparing the expression level of the biomarkers determined in (a) with a reference sample. 6.- Kit according to claim 5 comprising means for carrying out one or more of the following techniques: RT-PCR, Northern Blot, Western Blot, microarray analysis and / or immunoassay. 7.- Kit according to claim 6 comprising means for performing an immunoassay followed by RT-PCT.

Images