Determining the status of an individual in respect of alzheimer's disease

Abstract

The present invention relates to the field of diagnostics of neurodegenerative diseases. The present invention more particularly relates to a diagnostic method for evaluating the status ("predisposition before the subject develops the disease", "evaluation of the disease", "predicted age of the onset of signs of the disease") of a subject in respect of Alzheimer's disease, based on the analysis of the level of expression of qualified biomarkers.

Description

Determining an individual's status with regard to Alzheimer's disease

[0001] The present invention relates to the field of characterizing the predisposition and risk of developing neurodegenerative diseases. It relates more particularly to a method for qualifying the status based on indicators relating to the predisposition before the subject develops the pathology, to the early qualification of the pathology, or to a probabilistic indicator of the predicted age of occurrence of pathological signs or of the age at which the patient will present a risk of developing the pathology greater than a threshold value, and more generally to a probabilistic indicator of a subject with respect to Alzheimer's disease, based on the analysis of the level of expression of qualified biomarkers. Field of invention

[0002] The present invention relates to the characterization of a subject's predisposition to a neurodegenerative disease, and in particular Alzheimer's disease. Degenerative diseases are diseases that develop gradually and that cause physical, physiological or mental deficiencies in the affected individuals.

[0003] These progressive and serious diseases include a variety of conditions such as dementia (including Alzheimer's disease), Parkinson's disease, multiple sclerosis, paraplegia, myopathy or myasthenia gravis, epilepsy, stroke, and other neurological conditions. The best known degenerative diseases, and certainly the most widespread, are neurodegenerative diseases, which cause degeneration of the nervous system.

[0004] These diseases result in a progressive loss of cognitive function and dementia affecting one in eight people before the age of 65. These diseases can strike people at an earlier age of 40 to 50 years.

[0005] Degenerative diseases are generally genetic in origin, meaning they are hereditary and passed down from one generation to the next. But these pathologies can also be caused by significant and prolonged exposure to biological and toxic substances throughout life, or by other disorders such as severe sleep apnea.

[0006] Early diagnosis of these diseases is a major challenge in anticipating the onset of clinical signs and the appearance of a serious physical and mental disability, in order to help the subject organize a safe living environment adapted to the probable loss of autonomy, to practice physical and intellectual exercises likely to delay the loss of autonomy, and to slow the progression of the disease by organizing actions that can relieve or reduce some of the symptoms. Early diagnosis makes it possible to encourage the subject to best stimulate cognitive and memorization faculties to anticipate, and to make arrangements for the future while he still has all his faculties of discernment and decision-making.

[0007] Yet, currently, approximately 1 in 2 patients are unaware that they have the disease and will only learn about it at an advanced stage. As a result, nearly half a million people in France do not receive appropriate care. This has dramatic consequences for patients and their families.

[0008] This diagnosis was long practiced empirically, for example by the general practitioner alerted by the subject's relatives about apparent disorders, such as anosognosia, which is characterized by forgetfulness: the subject does not realize that he is in a difficult situation and his entourage encourages him to consult a doctor, a neurologist or a geriatrician in order to characterize the origin of these disorders.

[0009] The usual diagnosis of a possible or probable degenerative disease is essentially based on clinical criteria and neuropsychological tests which aim first to establish whether an individual presents a dementia syndrome, generally according to the criteria of the DSM IV (Diagnostic and Statistical Manual for Mental Disorders, published by the "American Psychiatric Association"), then to determine the etiology of dementia. The criteria of dementia essentially include the association of a memory disorder and other cognitive functions impacting on socio-professional activities and leading to a decline compared to previous functioning. The diagnosis is reinforced by monitoring the patient's progress, which makes it possible to specify the causes of dementia, in particular by ruling out other causes of cognitive decline.

[0010] Effective but highly invasive diagnostic methods have also been developed based on the examination of brain tissue, which are primarily suitable for post-mortem assessment. Slices of brain tissue from Alzheimer's disease victims show the presence of amyloid in the form of extracellular protein nuclei of the neuritic plaques characteristic of Alzheimer's disease.

[0011] Research efforts have also focused on the development of non-invasive in vivo methods using imaging techniques or biochemical or genomic methods for the detection of biomarkers.

[0012] Imaging techniques have been available for many years. Brain imaging, for example, can assess hippocampal regional atrophy, which can also be present in other diseases affecting the elderly, and exclude other causes of cognitive impairment, such as the after-effects of strokes.

[0013] For imaging brain diseases such as amyloid fibril or plaque-forming neurological diseases, a series of uncharged thioflavin T derivatives have been developed as amyloid imaging agents and radiotracers that exhibit high affinity for amyloid deposits and high permeability across the blood-brain barrier. Extensive in vitro and in vivo studies of these amyloid imaging agents represented by thioflavin BTA-1 suggest that they bind specifically to amyloid deposits at concentrations typical of those detectable in positron emission tomography studies. In the complex milieu of the human brain, nonspecific binding of amyloid imaging compounds is low, even in control brains lacking amyloid deposits.

[0014] The most anticipated avenues involve a test that can be performed on a biological fluid taken from the subject, for example a simple blood test.

[0015] A number of recent studies (O'Bryant, SE, Xiao, G., Barber, R., Reisch, J., Hall, J., Cullum, CM, Doody, R., Fairchild, T., Adams, P., Wilhelmsen, K., Diaz-Arrastia, R., & Texas Alzheimer's Research and Care Consortium (2011). A blood-based algorithm for the detection of Alzheimer's disease. Dementia and geriatric cognitive disorders, 32(1), 55–62 https: / doi.org / 10.1159 / 000330750) proposed panels of biomarkers.

[0016] This test is performed on fasting blood samples collected in serum separator tubes, then allowed to clot at room temperature for 30 minutes, centrifuged, aliquoted, and stored at -80°C in plastic vials. Pooled samples from baseline or one-year follow-up examinations were thawed for analysis without additional freeze-thaw cycles using their human Multi-Analyte Profile (humanMAP) multiplexed immunoassay. Individual proteins were quantified using stained microsphere immunoassays. Information on least detectable dose, run-to-run coefficient of variation, dynamic range, overall recovery of spiked standard, and cross-reactivity with other analytes was obtained from other centers. However, these recent tests have been found to be non-reproducible.

[0017] Some commercial kits with specific biomarker panels are now available, the selection of which is based on studies outside King's College London (Proteomics Sciences, Thambisetty et al, PLoS ONE, 6, 2001). However, these kits use expensive instruments such as mass spectrometry and, in some cases, require the use of CSF.

[0018] However, it is generally considered that only about 85% of cases of probable Alzheimer's disease are confirmed postmortem. The 15% of false positives generally result from other neurodegenerative diseases, such as frontotemporal dementias, including dementia with Lewy bodies. Current methodologies for diagnosing neurodegenerative diseases therefore lack specificity. State of the art

[0019] More recently, certain biological markers have been discovered that appear to be specific to Alzheimer's disease.

[0020] Patent application US2012283114 proposes novel biomarker sets for the diagnosis of Alzheimer's disease (AD) and related disorders including early diagnosis of MCI and early prodrome of AD, identification of disease subtypes, prediction of their response to disease management procedures, drugs and their combinations and monitoring of response to these treatments, including validation of biomarkers for clinical trials.This method is intended to detect the presence or risk of Alzheimer's disease in a mammal, or to facilitate the diagnosis and subclassification of Alzheimer's disease, the method comprising determining the (relative) amount or presence, absence or alteration of a set of target biomarkers in a sample of biological fluid from said mammal and comparing the measured level of the biomarker(s) to a reference value, wherein a deviation from said reference value is indicative of the presence, risk, progression or severity of AD, and wherein said set of biomarkers comprises 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, ROB01, SEMA3E, TGFB1, THBS1, VEGFR1 and WWOX.

[0021] Patent application EP2924124 relates to the use of mitochondrial DNA as a quantitative biomarker of neurodegenerative diseases, preferably Alzheimer's disease, as well as a method and kit for diagnosing and / or prognosing these diseases using this biomarker.

[0022] Patent application US2021 / 311076 relates to methods, compositions and uses for counteracting the effects of aberrant lymphatic drainage of the meninges and / or for modulating the lymphatic vessels of the central nervous system. In certain embodiments, the invention relates to methods, compositions and uses for treating, preventing or ameliorating symptoms of a neurodegenerative disease associated with aberrant lymphatic drainage of the meninges. Modulating lymphatic vessels, or counteracting the effects of aberrant meningeal lymphatic drainage, according to certain embodiments, is used to diagnose, treat, prevent or ameliorate symptoms of neurodegenerative diseases such as Alzheimer's disease (AD) and dementia.

[0023] The article by Huabin Zhao et al: “Identification of ferroptosis-related hub genes and diagnostic model in Alzheimer's disease”, Front. Mol. Neuroscie 16:1280639 (2023), presents the identification of genes of the ferroptosis process involved in Alzheimer's disease from an in silico analysis of data available on the GEO database (Barette et al, 2013). The article describes the use of mathematical models for the evaluation of these biomarkers.

[0024] We also know the article by the authors MCDERMOTT JASON E ET AL: "Challenges in biomarker discovery: combining expert insights with statistical analysis of complex amis data",

[0025] EXPERT OPINION ON MEDICAL DIAGNOSTICS, vol. 7, no. 1, January 18, 2013 (2013-01-18), pages 37-51, XP093095314 presenting different statistical approaches to assembling biomarker panels focused on the use of sophisticated statistical models on large quantitative data sets. This article describes possible approaches to this problem, including measures for biomarker assessment. Solution provided by the invention

[0026] In order to overcome these drawbacks, the present invention relates, in its most general sense, to a method for automatically classifying the status of a subject with regard to Alzheimer's disease, the technical specificities of which are set out in claim 1.

[0027] This classification is reflected, for example, by belonging to a category representative of the predisposition before the subject develops the pathology, of the qualification of the pathology, of the expected age of occurrence of pathological signs, of the age from which the probability of appearance of pathological signs exceeds a threshold value.

[0028] The process involves the following steps:

[0029] A. an initial step consisting of collecting reference data including:

[0030] a) a base of VT control values x reference for healthy subjects pour chacun des gènes ATM, APOE, GSNOR, PARKIN2, STOX2, d’ADN mitochondrial

[0031] b) classes of known statuses, determined from CR normalized n-tuples xobtained from biological samples on homogeneous cohorts with respect to one or more classification criteria of patients presenting a common reference R status to determine an R subspace corresponding to a given status

[0032] B. processing steps, for a new subject, consisting of:

[0033] c. carry out on genomic material of said new subject a quantification (by a known technique, for example PCR or western Blot) comprising at least part of the following markers: of the ATM gene and its expression to establish a first value V ATM of the APOE gene and its expression to establish a second V value APOE of the GSNOR gene and its expression to establish a third V value GSNOR of the PARKIN2 gene and its expression to establish a fourth V value PARKIN2 of the STOX2 gene and its expression to establish a fifth V value STOX2of the amount of mitochondrial DNA to establish a sixth value V ADNM

[0034] d. carry out a standardization of the said V values x to determine six standardized coefficients C x = IV x - VT x I / VT x

[0035] e. determine the closest subspace (in the sense of Euclidean distance) of said tuple C x to determine the status of the said subject.

[0036] The result obtained is the automatic obtaining of the status automatic classification of the status representative of the status of a subject in relation to the predisposition of future damage by Alzheimer's disease. The status is characterized by the subject's belonging to a class, determined according to a classification model of the different predisposition situations established by a community of users of the invention. These classes

[0037] ("predisposition before the subject develops the pathology" "qualification of the pathology" "expected age of occurrence of pathological signs") of a subject with regard to Alzheimer's disease

[0038] A class corresponds to a subset of subjects presenting a similar state of predisposition to Alzheimer's disease, the entire population being segmented into a number of classes decided by the community of users of the invention. This segmentation can be determined: according to the probability of occurrence of Alzheimer's disease in any temporality, with classes ranging from "zero probability" to "certainty" according to the temporal duration from which the probability of occurrence of Alzheimer's disease will exceed a threshold or any other mode of segmentation.

[0039] “VT control values” are understood to mean x » a measurable indicator, in particular: the level of expression of the gene of interest (typically zero, low, medium, high) the level of expression of the protein of interest the methylation profile of the gene of interest and more generally any indicator known to those skilled in the art.

[0040] Biological samples are blood samples, or skin samples, or brain cell samples, or cells from a cerebrospinal fluid puncture.

[0041] The method according to the invention optionally comprises an additional step consisting of isolating the cells to characterize the morphology of the cell nuclei and the progerin level, to determine the P value of the proportion of cells presenting a deformed nucleus and to determine the aging status according to the class to which this P value belongs to weight by analysis of the morphology of the nuclei (80% of normal nuclei) by immunofluorescence and antibodies and or a fluorescent DNA intercalator, to confirm the probability of AD, even asymptomatic and / or a progerin level higher than normal.

[0042] According to one variant, the method comprises: a step of analyzing the cells to verify the perinuclear presence of the phosphoAPOE protein and / or quantify the phosphoAPOE protein. and / or a step of analysis by a regression method on an N-uplet of values ​​V xcorresponding to the subject to be diagnosed and an N-tuple of control values ​​VT x. and / or a step of assaying a pATM-pAPOE biomarker in a skin sample from the subject.and / or a step of quantifying the percentage of fibroblasts exhibiting spontaneous and / or post-irradiation perinuclear accumulations of pATM and pAPOE on a significant sample of cells.

[0043] The invention also relates to a kit for qualifying the status (“predisposition before the subject develops the pathology” “qualification of the pathology” “predicted age of occurrence of pathological signs”) of a subject with regard to Alzheimer’s disease, in accordance with the method which is the subject of claim 1, characterized in that it comprises at least part of the following markers: of the ATM gene to establish a first value V ATM of the APOE gene to establish a second V value APOE of the GSNOR gene to establish a third V valueGSNOR of the PARKIN2 gene to establish a fourth V value PARKIN2 of the STOX2 gene to establish a fifth V value STOX2 of the amount of mitochondrial DNA to establish a sixth VADNM value

[0044] and a calculator for:

[0045] a. carry out a standardization of the said V values x to determine six standardized coefficients C x = IV x - VT x I / VT x

[0046] b. determine the closest subspace (in the sense of Euclidean distance) to said n-tuple C x to determine the status of the said subject.

[0047] Detailed description of a non-limiting example of embodiment

[0048] The present invention will be better understood on reading the following description, concerning a non-limiting example of embodiment illustrated by the appended drawings where:

[0049] represents the distribution of APOE genotypes of patients according to age.

[0050] represents the ROC curve of the detection of APOE4 genotype patients via ATM quantification by qPCR.

[0051] represents the ROC curve of the detection of patients with APOE4 genotype via cytoplasmic quantification of ATM protein.

[0052] represents the distribution of normalized relative ATM gene expression according to the age of patients as a binary variable (<60 years = 1, >60 years = 0).

[0053] represents the distribution of the quantification of ATM protein foci 24 hours post-irradiation as a function of the age of the patients in the form of a binary variable (<60 years = 1, >60 years = 0).

[0054] represents the % of cells with normal and abnormal shaped nuclei from Alzheimer's patients.

[0055] represents the analysis of the nuclear morphology of cells from Alzheimer's patients.

[0056] represents a western blot of quantification of pATM and Progerin proteins from protein extracts of Alzheimer's patient cells.

[0057] represents the quantification of ATM, GSNOR and mitochondrial DNA in cells from Alzheimer's patients. General principle of the invention

[0058] The aim of the invention is to automatically and early establish the status of a subject in relation to the probability of occurrence of Alzheimer's disease, for example, to automatically determine the existence of a predisposition to Alzheimer's disease, before the subject develops the pathology or the predicted age of occurrence of the pathological signs of a subject with regard to Alzheimer's disease. Selection of biomarker combination

[0059] The first step is to select a set of biomarkers including at least three of the following biomarkers, known as genetic risk factors: ATM (Ataxia telangiectasia mutated), a serine / threonine kinase that belongs to the phosphatidylinositol 3-kinase-related kinase family contributing to the regulation of the DNA damage response. APOE, (apolipoprotein E) GSNOR (S-nitrosoglutathione reductase), or ADH5, an enzyme of the alcohol dehydrogenase (ADH) family PARKIN2, a gene located on human chromosome 6, a ubiquitin ligase-like enzyme. STOX2, a gene from macaca nemestrina Mitochondrial DNA.

[0060] The selected combination includes at least three and preferably at least 4 of the above six biomarkers, and may include additional biomarkers.

[0061] This combination of N bio forms an N-dimensional space which will be used to determine specific subsets characterizing a state of predisposition of a subject to a neurodegenerative disease.

[0062] This step also consists of defining the dosage protocol for each of the N biomarkers selected.

[0063] Preparation of a reference framework.

[0064] The next step consists of testing cohorts of subjects whose predisposition to a neurodegenerative disease is known, by taking biological samples to then apply the dosage protocol for the N selected biomarkers.

[0065] The result of these dosages determines, for each subject, an N-uplet of values, associated with a class of predisposition to a known neurodegenerative disease.

[0066] The set of N-tuples associated with the same predisposition class defines a domain forming a homogeneous subset of the aforementioned N-dimensional space.

[0067] This reference system is not fixed but is gradually enriched with new qualified samples as well as with old samples concerning subjects whose predisposition becomes verifiable over time.

[0068] Preparation of a reference framework

[0069] To determine a subject's predisposition, a biological sample is taken.

[0070] The sample is preferably a blood sample, but can also be a skin sample or a sample of cerebrospinal fluid.

[0071] The N biomarkers mentioned above are measured, and an N-tuple defining a point in the N-dimensional space is determined. The state of predisposition to a neurodegenerative disease in the closest domain is associated with this point.

[0072] Processing a sample

[0073] As a non-limiting example, the biomarker assay is performed from a sample of healthy tissue, preferably lymphoblasts / lymphocytes or fibroblasts. The former are preferably taken from the blood and the latter from the connective tissue or skin of an individual. This sample can be taken by blood sampling or biopsy.

[0074] The dosage of biomarkers can be carried out by a procedure well known to those skilled in the art consisting of carrying out:

[0075] 1- a qPCR analysis of the expression of the different biomarkers on the coding DNA and including:

[0076] a) means for extracting RNA and synthesizing coding DNA from the cell sample, i.e. reagents such as a lysis buffer and an RT-PCR KIT known to those skilled in the art,

[0077] b) means for determining at the level of the DNA coding said cell sample the expression of certain genes in the basal state (in the state in which the DNA is found in the cell, without external modification), by the use of qPCR reagents known to those skilled in the art and a qPCR apparatus.

[0078] The expression information is then analyzed in silico to determine the expressions of the genes tested for each individual.

[0079] 2- An analysis of the nuclear morphology of fibroblasts using specific biomarkers including:Means to visualize DNA (DAPI) and the nuclear lamina (anti-lamin A / C antibodies)Means to confirm the accumulation of biomarkers in the cell (anti-pATM antibodies, anti-progerin antibodies)

[0080] Carrying out a dosage

[0081] A first object of the invention is a method for characterizing the severity of Alzheimer's disease from a sample taken from a patient in which: DNA extraction is carried out from a blood or skin sample and the APOE allele is sequencing in order to know the patient's status

[0082] An RNA extraction is carried out followed by a synthesis of coding DNA called a “genomic sample”. The expression of ATM, APOE, GSNOR, PARKIN2, SOX2 markers and the quantification of mitochondrial DNA are determined on this genomic sample. The severity of the pathology is determined by an association of the expressions of these markers with the APOE genotype which makes it possible to separate “young Alzheimer’s” patients from “old Alzheimer’s” patients.

[0083] A second subject of the invention is a method for characterizing the “Alzheimer’s patient” status from a sample taken from a patient’s skin in which:The cells taken are amplifiedThe morphology of the cell nuclei is determined using markings made with DAPI or with a nuclear lamina marker (anti-lamin A / C antibodies)A specific accumulation of biomarkers in the perinuclear region for the ATM protein (presence of perinuclear accumulation) and inside the nuclear lamina by immunofluorescence is determined.

[0084] Examples: Fibroblasts from 10 patients with the disease were amplified, then the genetic material (DNA and RNA) was extracted and a number of analyses were carried out by sequencing, qPCR and immunofluorescence.

[0085] Figures 1 to 4 illustrate ATM quantification curves according to patient genotypes. ATM quantification allows the detection of patients with an APOE4 genotype.

[0086] Figures 1 and 2 illustrate the detection of more severe APOE4 cases via ATM quantification by qPCR) on cell lines from 10 Alzheimer's patients.

[0087] We first observed that all patients with an APOE4 genotype (results obtained by sequencing) were under 60 years old (). In addition, the quantity of ATM detected by qPCR allows us to identify with a performance of 80% (: ROC curve on the right) patients with an APOE4 genotype and by extension, the youngest patients (therefore having a more severe phenotype).

[0088] Illustrates the detection of more severe APOE4 cases via the quantification of perinuclear ATM accumulation by immunofluorescence on cell lines from 10 Alzheimer's patients. In addition, the quantity of cells with perinuclear pATM accumulations detected by immunofluorescence allows the identification with a performance of 80% ( : ROC curve on the right) of patients with an APOE4 genotype and by extension, younger patients (therefore with a more severe phenotype).

[0089] Figures 5 and 6 illustrate the distribution of patients according to the age of symptom reporting (n=10) via ATM quantification by qPCR or the number of pATM foci at 24h quantified by immunofluorescence. Alzheimer's patients were divided into 2 classes:Patients < 60 years (1 in the figure)Patients >= 60 years (0 in the figure)

[0090] It is observed that patients over 60 years old are those for whom ATM expression is the strongest (). Similarly, the distribution of the number of pATM foci at 24 hours post-irradiation differs according to age (). The youngest patients are those who have the most foci and therefore defective repair.

[0091] Figures 6 to 8 illustrate the graphs of increase in morphological abnormalities observed as a function of the age of the patients.

[0092] Illustrates the counts of the number of cells with nuclear abnormalities detected by DAPI labeling.

[0093] Illustrates nuclear lamina abnormalities by lamin A / CA staining. % of cells with nuclear lamina abnormalities (visualized by immunofluorescence with an anti-lamin A / C antibody). 100 nuclei counted. **P=0.01 B. Nuclear lamina staining in fibroblast nuclei from an Alzheimer's patient. The arrow indicates nuclear abnormalities. C. Distribution of % Nuclear Lamina abnormalities in fibroblasts from Alzheimer's patients according to sex (p=0.0078).

[0094] It was observed that these nuclear lamina abnormalities increased significantly depending on the age of the patients (Wilcoxon test p = 0.0076) and the sex of the patients (Wilcoxon test p = 0.0078).

[0095] Illustrates the quantification of progerin by Western blot in Alzheimer's cells. We were able to observe that all patients with Alzheimer's disease expressed progerin, which is an abnormal version of the protein Lamin A / C contained in the nuclear lamina and which is responsible for the premature aging disease, Hutchinson Gilford Progeria syndrome (Varela et al, Nature Medicine 2008).

[0096] Figure 1 illustrates the expression of ATM and GSNOR genes, and the quantification of mitochondrial DNA in 10 patients with Alzheimer's disease.

[0097] It highlights a significant correlation between patients with an APOE4 genotype and the decrease in the quantity of these 3 biomarkers in patients under 60 years old.

[0098] The combination of all these factors makes it possible to detect, through several biomarkers, patients under 60 years of age, with an APOE4 phenotype and affected by Alzheimer's disease.

Claims

– Method for the automatic classification of the status of a subject with respect to Alzheimer's disease, comprising:A. an initial step consisting of collecting reference data comprising:a base of VT control values x reference for healthy subjects pour chacun des gènes ATM, APOE, GSNOR, PARKIN2, STOX2, d’ADN mitochondrial classes of known statuses, determined from CR normalized n-tuples x obtained from biological samples on homogeneous cohorts with respect to one or more classification criteria of patients presenting a common reference R status to determine a subspace R corresponding to a given statusB. steps, for a new subject, consisting of:a. carrying out, from genomic material extracted from a biological sample taken from said new subject, a quantification of the following markers:of the ATM gene to establish a first value V ATM of the APOE gene to establish a second V value APOEof the GSNOR gene to establish a third V value GSNOR of the PARKIN2 gene to establish a fourth V value PARKIN2 of the STOX2 gene to establish a fifth V value STOX2 of the amount of mitochondrial DNA to establish a sixth value V ADNM b. carry out a standardization of said V values x to determine six standardized coefficients C x = IV x - VT x I / VTxc. déterminer le sous-espace le plus proche au sens de distance euclidienne dudit n-uplet Cxpour déterminer le statut dudit sujet. – Method for the automatic classification of the status of a subject with regard to Alzheimer's disease, according to claim 1 characterized in that said biological samples are blood samples. – Method for the automatic classification of the status of a subject with regard to Alzheimer's disease, according to claim 1 characterized in that said biological samples are skin samples. – Method for the automatic classification of the status of a subject with regard to Alzheimer's disease, according to claim 1 characterized in that said biological samples are samples of brain cells or cells originating from a puncture of cerebrospinal fluid. – Method for the automatic classification of the status of a subject with respect to Alzheimer's disease, according to claim 1 characterized in that it comprises an additional step consisting of isolating the cells to characterize the morphology of the cell nuclei and the progerin level, to determine the P value of the proportion of cells presenting a deformed nucleus and to determine the aging status according to the class to which this P value belongs to weight by analysis of the morphology of the nuclei (80% of normal nuclei) by immunofluorescence and antibodies and or a fluorescent DNA intercalator, to confirm the probability of AD, even asymptomatic and / or a progerin level higher than normal. – Method for the automatic classification of the status of a subject with regard to Alzheimer's disease, according to claim 1, characterized in that it comprises a step of analyzing the cells to verify the perinuclear presence of the phosphoAPOE protein and / or quantify the phosphoAPOE protein. – Method for the automatic classification of the status of a subject with regard to Alzheimer's disease, according to claim 1 characterized in that it comprises a step of analysis by a regression method on an N-tuple of values V x corresponding to the subject to be diagnosed and an N-uplet of control values VT x . – Method for the automatic classification of the status of a subject with regard to Alzheimer's disease, according to claim 1, characterized in that it comprises a step of dosing a pATM-pAPOE biomarker in a skin sample from the subject. – Method for the automatic classification of the status of a subject with regard to Alzheimer's disease, according to the preceding claim, characterized in that said assay comprises the quantification of the percentage of fibroblasts presenting spontaneous and / or post-irradiation perinuclear accumulations of pATM and pAPOE on a significant sample of cells. - Kit for qualifying the status of a subject with respect to Alzheimer's disease, in accordance with the method which is the subject of claim 1, characterized in that it comprises at least part of the following markers: of the ATM gene and its expression to establish a first value V ATM of the APOE gene and its expression to establish a second V value APOE of the GSNOR gene and its expression to establish a third V value GSNOR of the PARKIN2 gene and its expression to establish a fourth V value PARKIN2 of the STOX2 gene and its expression to establish a fifth V valueSTOX2 of the amount of mitochondrial DNA to establish a sixth value V ADNM and a calculator for:a. carrying out a normalization of said values V x to determine six standardized coefficients C x = IV x - VT x I / VT x Ib. determine the closest subspace (in the sense of Euclidean distance) to said n-tuple C x to determine the status of the said subject.

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