Method for characterizing the degradation of an organ

Abstract

The present invention relates to a kit and an in vitro method for detecting and characterizing the degradation of a particular organ or tissue, on the basis of the analysis of the presence of at least one specific methylated cell-free DNA in a biological sample. More particularly, the invention relates to a kit and a method for detecting and characterizing the degradation of an organ selected from: the brain, the lung and the kidney, and / or of a tissue present in this organ. The invention also relates to a method for the in vitro diagnosis of a disease or condition involving the lysis of an organ or tissue.

Description

[0001] DESCRIPTION

[0002] Title: METHOD FOR CHARACTERIZING THE DEGRADATION OF AN ORGAN

[0003] Field of invention

[0004] [1] The present invention relates to a kit and an in vitro method for detecting and characterizing the degradation of a particular organ or tissue, based on the analysis of the presence of at least one specific methylated cell-free DNA in a biological sample. More particularly, the invention relates to a kit and a method for detecting and characterizing the degradation of 1) the brain during an episode of stroke, 2) the lung during an episode of acute respiratory distress syndrome and 3) the kidney during an episode of acute renal failure and / or an episode of rejection and / or dysfunction of the renal graft. The invention also relates to a method for in vitro diagnosis of a pathology or condition involving the lysis of an organ or tissue.

[0005] [2] The present invention is therefore in the field of molecular biology applied to medical diagnosis.

[0006] State of the art

[0007] [3] When treating a patient with an acute phase of illness, medical imaging analysis, such as a CT scan, X-ray or MRI, is performed to establish the patient's overall level of suffering and lesion damage. However, this type of examination does not distinguish between organic lysis itself and inflammatory damage. However, the prognosis and therapeutic intervention carried out by medical staff are very different depending on the nature of the damage to the organ or tissue. Furthermore, in the acute phase, management decisions must be made within an extremely short time.

[0008] [4] Certain acute and relatively widespread pathologies have the common characteristic of involving massive tissue lysis.

[0009] [5] This is particularly the case for cerebrovascular accident (CVA), in which brain tissue is degraded, acute respiratory distress syndrome, in which lung tissue is degraded, acute renal failure (ARF) of a native kidney and of a transplanted kidney when graft rejection or renal graft dysfunction occurs, in which renal tissue is degraded. [6] Furthermore, in the case of kidney transplantation, it is essential to monitor the possible occurrence of graft rejection / dysfunction. Currently, post-operative follow-up of transplant patients involves painful and burdensome monitoring for the patient.

[0010] [7] Due to the incidence of all these pathologies among the population, there is a need for tools that can reliably, quickly, simply and inexpensively detect possible tissue in the context of these different acute pathologies.

[0011] [8] Circulating cell-free DNA, also referred to as "free DNA," is generally known as a tumor marker, but also as a marker of inflammation and a marker of tissue stress. Immune cells contribute mainly to the elevation of the total amount of circulating DNA.

[0012] [9] It has been shown that circulating DNA can possibly be used as a potential biomarker for autoimmune diseases such as systemic lupus erythematosus or rheumatoid arthritis (Duvvuri et al., “Cell-free DNA as a biomarker in autoimmune rheumatic diseases,” Front. Immunol. 10, 2019).

[0013]

[0010] The use, in a healthy subject, of the characterization of the methylation profile of circulating DNA to identify the tissue origin of said circulating DNA has been described (Moss et al “Comprehensive human cell-type methylation atlas reveals origins of circulating cell-free DNA in health and disease”, Nat. Commun., 9, 1-12, 2018).

[0014]

[0011] The increase in the proportion of circulating DNA from hepatocytes in patients with inflammatory liver disease has been described, with immune cells contributing predominantly to the total circulating DNA quantity (Liu et al

[0015] (“Comprehensive DNA methylation analysis of tissue of origin of plasma cell-free DNA by methylated CpG tandem amplification and sequencing”, Clin. Epigenetics, 11, 93 2019).

[0016]

[0012] WO 2021 / 216985 “Methods for detecting tissue damage, graft versus host disease and infections using cell-free DNA profiling” discloses the detection of tissue degradation by profiling circulating DNA in the context of allogeneic hematopoietic cell transplantation.

[0017]

[0013] Document CN 113999901 "Myocardial specific methylation marker" describes the identification and use of a cardiac marker specific for methylated cellular DNA for the diagnosis of an acute myocardial infarction, from a plasma sample.

[0014] WO2019012544 (Al) "DUAL-PROBE DIGITAL DROPLET PCR STRATEGY FOR SPECIFIC DETECTION OF TISSUE-SPECIFIC CIRCULATING DNA MOLECULES" describes a droplet digital PCR method for analyzing the methylation status of methylation sites of a double-stranded DNA molecule that comprises at least two methylation sites per single strand of the double-stranded DNA molecule from a sample of a biological fluid such as plasma or urine

[0018]

[0015] WO2019012543 (Al) “DNA TARGETS AS TISSUE-SPECIFIC METHYLATION MARKERS” describes a general method for determining the state of degradation of a tissue during a pathological process from the study of the methylation sites of a DNA molecule circulating from a biological fluid such as plasma or urine.

[0019] Statement of the invention

[0020]

[0016] The inventors have developed a kit and a method for the specific and targeted detection of circulating methylated cell-free DNA present in a biological sample of an individual and specific to the brain, lung or kidney. A method according to the invention makes it possible to specifically detect the existence of massive lysis of one of these organs or of a particular tissue which composes it at the level of its vascular or epithelial fraction. In addition, this method makes it possible to distinguish the lysis of an organ from an inflammatory phenomenon of immune origin. The distinction of an epithelial or vascular attack as well as the identification of an organic lysis rather than an inflammatory phenomenon of purely immune origin is major for the diagnosis of the patient and cannot be discriminated by medical imaging.

[0021]

[0017] For two of these organs, namely the lung and the brain, the inventors have in fact selected, among different specifically hypermethylated or hypomethylated regions of the circulating DNA, at least one region making it possible to develop a sensitive and specific test for the degradation of said organ.

[0022]

[0018] For one of these organs, namely the kidney, the inventors have further selected, from among different specifically hypermethylated or hypomethylated fractions of the circulating DNA, at least three differentially methylated fractions in the renal epithelium, the renal endothelium and the total kidney, these regions can be designated respectively by "total kidney", the "renal vascular fraction" and the "renal epithelial fraction" making it possible to develop a sensitive and specific test for the degradation of said organ.

[0019] The inventors have thus designed, for each of these regions, a pair of selective amplification primers, amplification conditions and a probe specifically detecting the amplification product generated using said pair of primers.

[0023]

[0020] The inventors also identified, around each of the nucleotide sequences selectively amplified by a pair of primers as described above, a genetic region of interest including the amplified nucleotide sequence, said genetic region being larger than that of the marker. The present invention describes said “genetic regions of interest”.

[0024]

[0021] For each of the markers, a detection kit and a detection method are thus provided.

[0025]

[0022] The invention also relates to a kit and a method for detecting at least one specific marker of organ lysis. The invention further relates to a kit and a method for simultaneously detecting at least two specific detection markers of organ lysis, thereby further improving the specificity and sensitivity of the test. A test according to the invention also has high diagnostic performance.

[0026]

[0023] The invention therefore relates to a kit and a method for detecting the lysis of an organ chosen from the brain, the lung and the kidney, from a biological sample of a patient. A kit and a method for detecting cerebral lysis can be used in particular for the in vitro diagnosis of stroke. This kit and this method are based on the detection of at least one marker chosen from "APC2" and "NBR1". ». The marker

[0027] “NBR1” is a marker also designated as “KRT33B”.

[0028]

[0024] The invention also relates to a kit and a method for detecting pulmonary lysis, which can be used in particular for the in vitro diagnosis of acute respiratory distress or possible lung graft rejection. This kit and this method are based on the detection of the marker "SYNE1". The marker "SYNE1" is a marker also designated by "LINC00336".

[0029]

[0025] The invention also relates to a kit and a method for detecting renal lysis, which can be used in particular for the in vitro diagnosis of acute renal failure and, in the case of a transplanted patient, of graft rejection. This kit and this method are based on the detection of at least one marker chosen from: PAX2, PDE4D, CTDP1, ALDH1A1, ARID3A, GATA2, LOC124903692, NRN1, SEPT5-GP1BB, TNS2-AS1 RHBDF2, LINC01599, FLJ12825, ACSL5.

[0026] According to a particular embodiment, the invention also relates to a kit and a method for detecting renal lysis, based on the combination of multiplexed detection of at least:

[0030] - a “total kidney” marker: chosen from CTDP1

[0031] - a “Vascular” type marker: chosen from the markers ALDH1A1, ARID3A, GATA2, LOC124903692, NRN1, SEPT5-GP1BB, TNS2-AS1 RHBDF2 and LINC01599 and

[0032] - an “epithelial” type marker: chosen from the markers FLJ12825, ACSL5, PAX2 and PDE4D.

[0033]

[0027] Increasing the number of markers detected simultaneously by multiplexed analysis ensures better clinical performance of the test in terms of diagnosis, screening, therapeutic optimization and patient monitoring.

[0034]

[0028] Solid biopsy, or renal puncture, is the reference method for diagnosing graft rejection. It allows the study of certain anatomical structures of the graft at the level of the renal cortex, located on the periphery of the kidney. Depending on the Banff score, graft lesions can be segmented by "vascular" typology or

[0035] "epithelial". The study of these lesions combined with the observation of immune infiltrates allows the typological diagnosis of renal transplant rejection to be made as follows: "no rejection", "Lymphocyte-mediated cellular rejection (TCMR)", "Antibody-mediated humoral rejection (ABMR)" and "Mixed rejection combining both types of rejection". Depending on the typology of rejection, the therapeutic options are different.

[0036]

[0029] Diagnostic tests for transplant rejection are commercially available, namely Allosure (CareDX) and ProsperaTM (Natera). New methodologies for detecting transplant rejection focus their proposal solely on predicting rejection or dysfunction of the renal graft, without being able to characterize and diagnose the nature of the latter. Also, the use of renal puncture remains current.

[0037]

[0030] A kit and a method according to the invention therefore have the great advantage of offering a minimally invasive test, since it is carried out using a biological sample that is easily collected, reliable, rapid and inexpensive. Due to its minimally invasive nature, a method according to the invention can easily, if necessary, be repeated at different stages of the pathology, which allows reliable monitoring of the development of said pathology and ensures medical care adapted to each of these stages.

[0038]

[0031] A kit and a method according to the invention present a major challenge for specialists and anatomopathologists, because the typology of organ lesions, in particular the typology of renal graft lesions, conditions the final diagnosis and the therapeutic management of the patient.

[0039]

[0032] The sensitivity and specificity of the test for the presence of each of the markers have been verified. A kit and a method according to the invention also have the advantage of being reproducible, rapid and inexpensive.

[0040]

[0033] The invention also relates to the use of methylated cell-free DNA as a marker for brain, kidney or lung degradation, or for degradation of brain, kidney or lung tissue.

[0041]

[0034] The invention also relates to the primers and probes that can be used in a kit and a method according to the invention. In particular, the invention relates to:

[0042] - a sense primer comprising, or consisting of, a nucleotide sequence chosen from: SEQ ID No. 1, SEQ ID No. 4, SEQ ID No. 7, SEQ ID No. 10, SEQ ID No. 13, SEQ ID No. 19, SEQ ID No. 22, SEQ ID No. 25, SEQ ID No. 28, SEQ ID No. 31, SEQ ID No. 34, SEQ ID No. 37, SEQ ID No. 40, SEQ ID No. 43, SEQ ID No. 46, SEQ ID No. 49, SEQ ID No. 52, or a nucleotide sequence having at least 80% identity with said sequences;

[0043] - an antisense primer comprising, or consisting of, a nucleotide sequence chosen from: SEQ ID No. 2, SEQ ID No. 5, SEQ ID No. 8, SEQ ID No. 11, SEQ ID No. 14, SEQ ID No. 20, SEQ ID No. 23, SEQ ID No. 26, SEQ ID No. 29, SEQ ID No. 32, SEQ ID No. 35, SEQ ID No. 38, SEQ ID No. 41, SEQ ID No. 44, SEQ ID No. 47, SEQ ID No. 50 or SEQ ID No. 53 or a nucleotide sequence having at least 80% identity with said sequences;

[0044] - and a probe, optionally labeled, comprising, or consisting of a nucleotide sequence chosen from: SEQ ID No. 3, SEQ ID No. 6, SEQ ID No. 9, SEQ ID No. 12, SEQ ID No. 15, SEQ ID No. 21, SEQ ID No. 24, SEQ ID No. 27, SEQ ID No. 30, SEQ ID No. 33, SEQ ID No. 36, SEQ ID No. 39, SEQ ID No. 42, SEQ ID No. 45, SEQ ID No. 48, SEQ ID No. 51 and SEQ ID No. 54, or a nucleotide sequence having at least 80% identity with said sequences.

[0045]

[0035] The invention finally relates to an in vitro method for detecting a pathology or condition chosen from: cerebrovascular accident (CVA), acute respiratory distress syndrome, renal failure and graft rejection, in particular rejection of a renal graft or lung graft.

[0046] Detailed description of the invention

[0047]

[0036] According to a first aspect, the invention relates to a kit for the detection in a biological sample of at least one target nucleotide sequence of methylated cell-free DNA, said nucleotide sequence being specific to an organ chosen from: the brain, the lung and the kidney or to a tissue present in the brain, the lung or the kidney, said kit comprising at least one pair of primers consisting of a sense primer and an antisense primer, each of the primers comprising, or consisting of, a nucleotide sequence capable of hybridizing with a nucleotide sequence of said cell-free DNA to selectively amplify a methylated cell-free DNA sequence specific to said organ or tissue.

[0048]

[0037] According to an embodiment of this first aspect, the invention relates to a kit for the detection in a biological sample of at least one target nucleotide sequence of methylated cell-free DNA, said kit comprising at least:

[0049] - a pair of primers consisting of a sense primer and an antisense primer, each of the primers comprising, or consisting of, a nucleotide sequence capable of hybridizing with at least one nucleotide sequence of said cell-free DNA to selectively amplify a methylated cell-free DNA sequence specific to said organ or tissue, and

[0050] - a probe comprising, or consisting of, a nucleotide sequence capable of hybridizing with the amplified target DNA nucleotide sequence.

[0051]

[0038] By “biological sample” we mean an element from the body, human or animal, in particular for example: blood, urine, saliva, plasma or a fragment of organ.

[0052]

[0039] Said probe, called “detection probe” is preferably associated with a marker whose detection is well known to a person skilled in the art. When more than one pair of probes is used (multiplex test), the combination of markers is chosen in order to distinguish the different amplified nucleotide sequences.

[0053]

[0040] According to a first particular aspect, the subject of the present invention is a kit for the detection in a biological sample of a target nucleotide sequence of methylated cell-free DNA, said nucleotide sequence being specific to a cell type of an organ chosen from: the brain, the lung and the kidney, said kit comprising: i) for the detection of a target sequence of cell-free DNA specific to a cell type present in the brain, at least: a sense primer comprising a nucleotide sequence SEQ ID No. 1, or a nucleotide sequence having at least 80% identity with SEQ ID No. 1 and an antisense primer, comprising a nucleotide sequence SEQ ID No. 2, or a nucleotide sequence having at least 80% identity with SEQ ID No. 2, and / or - a sense primer comprising a nucleotide sequence SEQ ID No. 4, or a nucleotide sequence having at least 80% identity with SEQ ID No. 5, and / or - a sense primer comprising a nucleotide sequence SEQ ID No. 6, or a nucleotide sequence having at least 80% identity with SEQ ID No. 7, and / or - a sense primer comprising a nucleotide sequence SEQ ID No. 8, or a nucleotide sequence having at least 80% identity with SEQ ID No. 9, and / or - a sense primer comprising a nucleotide sequence SEQ ID No. 10, or a nucleotide sequence having at least 80% identity with SEQ ID No. 11, and / or - a sense primer comprising a nucleotide sequence SEQ ID No. 11, or a nucleotide sequence having at least 80% identity with SEQ ID No. 12, and / or - a sense primer comprising a nucleotide sequence SEQ ID No. 12, or a nucleotide sequence having at least 80% identity with SEQ ID No. 13, and / or - a sense primer comprising a nucleotide sequence SEQ ID No. 13, or a nucleotide sequence having at least 80% identity with SEQ ID No. 14, and / or - a sense primer comprising a nucleotide sequence SEQ ID No. 14 identity with SEQ ID No. 4 and an antisense primer,comprising a nucleotide sequence SEQ ID No. 5, or a nucleotide sequence having at least 80% identity with SEQ ID No. 5. ii) for the detection of a cell-free DNA target sequence specific to a cell type present in the lung, at least: a sense primer, comprising a nucleotide sequence SEQ ID No. 7, or a nucleotide sequence having at least 80% identity with SEQ ID No. 7 and an antisense primer, comprising a nucleotide sequence SEQ ID No. 8, or a nucleotide sequence having at least 80% identity with SEQ ID No. 8, and iii) for the detection of a cell-free DNA target sequence specific to a cell type present in the kidney, at least:,

[0054] - a sense primer comprising a sequence SEQ ID No. 10, or a sequence having at least 80% identity with SEQ ID No. 10 and an antisense primer, comprising a sequence SEQ ID No. 11, or a sequence having at least 80% identity with SEQ ID No. 11, and / or

[0055] - a sense primer comprising a sequence SEQ ID No. 13, or a sequence having at least 80% identity with SEQ ID No. 13 and an antisense primer, comprising a sequence SEQ ID No. 14, or a sequence having at least 80% identity with SEQ ID No. 14, and / or

[0056] - a sense primer comprising a sequence SEQ ID No. 19, or a sequence having at least 80% identity with SEQ ID No. 19 and an antisense primer, comprising a sequence SEQ ID No. 20, or a sequence having at least 80% identity with SEQ ID No. 20, and / or

[0057] - a sense primer comprising a sequence SEQ ID No. 22, or a sequence having at least 80% identity with SEQ ID No. 22 and an antisense primer, comprising a sequence SEQ ID No. 23, or a sequence having at least 80% identity with SEQ ID No. 23, and / or

[0058] - a sense primer comprising a sequence SEQ ID No. 25, or a sequence having at least 80% identity with SEQ ID No. 25 and an antisense primer, comprising a sequence SEQ ID No. 26, or a sequence having at least 80% identity with SEQ ID No. 26, and / or

[0059] - a sense primer comprising a sequence SEQ ID No. 28, or a sequence having at least 80% identity with SEQ ID No. 28 and an antisense primer, comprising a sequence SEQ ID No. 29, or a sequence having at least 80% identity with SEQ ID No. 29, and / or

[0060] - a sense primer comprising a sequence SEQ ID No. 31, or a sequence having at least 80% identity with SEQ ID No. 31 and an antisense primer, comprising a sequence SEQ ID No. 32, or a sequence having at least 80% identity with SEQ ID No. 32, and / or

[0061] - a sense primer comprising a sequence SEQ ID No. 34, or a sequence having at least 80% identity with SEQ ID No. 34 and an antisense primer, comprising a sequence SEQ ID No. 35, or a sequence having at least 80% identity with SEQ ID No. 35, and / or

[0062] - a sense primer comprising a sequence SEQ ID No. 37, or a sequence having at least 80% identity with SEQ ID No. 37 and an antisense primer, comprising a sequence SEQ ID No. 38, or a sequence having at least 80% identity with SEQ ID No. 38, and / or

[0063] - a sense primer comprising a sequence SEQ ID No. 40, or a sequence having at least 80% identity with SEQ ID No. 40 and an antisense primer, comprising a sequence SEQ ID No. 41, or a sequence having at least 80% identity with SEQ ID No. 41, and / or

[0064] - a sense primer comprising a sequence SEQ ID No. 43, or a sequence having at least 80% identity with SEQ ID No. 43 and an antisense primer, comprising a sequence SEQ ID No. 44, or a sequence having at least 80% identity with SEQ ID No. 44, and / or

[0065] - a sense primer comprising a sequence SEQ ID No. 46, or a sequence having at least 80% identity with SEQ ID No. 46 and an antisense primer, comprising a sequence SEQ ID No. 47, or a sequence having at least 80% identity with SEQ ID No. 47, and / or

[0066] - a sense primer comprising a sequence SEQ ID No. 49, or a sequence having at least 80% identity with SEQ ID No. 49 and an antisense primer, comprising a sequence SEQ ID No. 50, or a sequence having at least 80% identity with SEQ ID No. 50, and / or

[0067] - a sense primer comprising a sequence SEQ ID No. 52, or a sequence having at least 80% identity with SEQ ID No. 52 and an antisense primer, comprising a sequence SEQ ID No. 53, or a sequence having at least 80% identity with SEQ ID No. 53.

[0068] According to a more particular aspect, a kit according to the invention comprises at least:

[0069] - one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen or twenty pairs of primers, each consisting of a sense primer according to the invention and an antisense primer according to the invention.

[0041] According to a more particular aspect, a kit according to the invention comprises a primer

[0070] "sense" comprising or consisting of a nucleotide sequence selected from SEQ ID No. 1, SEQ ID No. 4, SEQ ID No. 7, SEQ ID No. 10, SEQ ID No. 13, SEQ ID No. 19, SEQ ID No. 22, SEQ ID No. 25, SEQ ID No. 28, SEQ ID No. 31, SEQ ID No. 34, SEQ ID No. 37, SEQ ID No. 40, SEQ ID No. 43, SEQ ID No. 46, SEQ ID No. 49, SEQ ID No. 52, or a nucleotide sequence comprising at least 80% identity with one of said sequences SEQ ID No. 1, SEQ ID No. 4, SEQ ID No. 7, SEQ ID No. 10, SEQ ID No. 13, SEQ ID No. 19, SEQ ID No. 22, SEQ ID No. 25, SEQ ID No. 28, SEQ ID No. 31, SEQ ID No. 34, SEQ ID No. 37, SEQ ID No. 40, SEQ ID No. 43, SEQ ID No. 46, SEQ ID No. 49 or SEQ ID No. 52.

[0071]

[0042] According to another more particular aspect, a kit according to the invention comprises an “antisense” primer comprising or consisting of a nucleotide sequence chosen from SEQ ID No. 2, SEQ ID No. 5, SEQ ID No. 8, SEQ ID No. 11, SEQ ID No. 14, SEQ ID No. 20, SEQ ID No. 23, SEQ ID No. 26, SEQ ID No. 29, SEQ ID No. 32, SEQ ID No. 35, SEQ ID No. 38, SEQ ID No. 41, SEQ ID No. 44, SEQ ID No. 47, SEQ ID No. 50 or SEQ ID No. 53, or a nucleotide sequence comprising at least 80% identity with one of said nucleotide sequences SEQ ID No. 2, SEQ ID No. 5, SEQ ID No. 8, SEQ ID No. 11, SEQ ID No. 14, SEQ ID No. 20, SEQ ID No. 23, SEQ ID No. 26, SEQ ID No. 29, SEQ ID No. 32, SEQ ID No. 35, SEQ ID No. 38, SEQ ID No. 41, SEQ ID No. 44, SEQ ID No. 47, SEQ ID No. 50 or SEQ ID No. 53.

[0072]

[0043] According to another more particular aspect, a kit according to the invention comprises a probe comprising or consisting of a nucleotide sequence chosen from SEQ ID No. 3, SEQ ID No. 6, SEQ ID No. 9, SEQ ID No. 12, SEQ ID No. 15, SEQ ID No. 21, SEQ ID No. 24, SEQ ID No. 27, SEQ ID No. 30, SEQ ID No. 33, SEQ ID No. 36, SEQ ID No. 39, SEQ ID No. 42, SEQ ID No. 45, SEQ ID No. 48, SEQ ID No. 51 and SEQ ID No. 54, or a nucleotide sequence comprising at least 80% identity with one of said nucleotide sequences chosen from SEQ ID No. 3, SEQ ID No. 6, SEQ ID No. 9, SEQ ID No. 12, SEQ ID No. 15, SEQ ID No. 21, SEQ ID No. 24, SEQ ID No. 27, SEQ ID No. 30, SEQ ID No. 33, SEQ ID No. 36, SEQ ID No. 39, SEQ ID No. 42, SEQ ID No. 45, SEQ ID No. 48, SEQ ID No. 51 and SEQ ID No. 54.

[0073]

[0044] By "at least 80% identity" is meant that said sequences have at least 80% identity after optimal global alignment, that is to say by global alignment between two sequences giving the highest percentage of identity between them. The optimal global alignment of two sequences can in particular be carried out according to the Needleman-Wunsch algorithm, well known to those skilled in the art (Needleman & Wunsch, "A general method applicable to the search for similarities in the amino acid sequences of two proteins", J. Mol. Biol., 48(3):443-53). A nucleotide sequence according to the invention comprises, or is constituted by, a nucleotide sequence having at least 80%, advantageously at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the reference nucleotide sequence after optimal global alignment.

[0074] Advantageously, a nucleotide sequence according to the invention comprises, or is constituted by, a nucleotide sequence having at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identity with the reference nucleotide sequence after optimal global alignment.

[0075]

[0045] According to this aspect, a nucleotide sequence according to the invention comprising, or consisting of, a sequence having at least 80% identity with the reference nucleotide sequence is functional, that is to say that it is capable of hybridizing to the target nucleotide sequence with sufficient stability to allow the amplification or detection of said target nucleotide sequence.

[0076]

[0046] The nucleotide sequences of a first group of primers are designated as indicated in Table 1 below:

[0077] Table 1

[0078]

[0047] The nucleotide sequences of a first group of probes are designated as indicated in Table 2 below:

[0079] Table 2

[0048] According to another more particular aspect, the subject of the invention is a kit for the detection in a biological sample of a target nucleotide sequence of methylated cell-free DNA, said nucleotide sequence being specific to a type of cells present in the brain, said kit comprising at least:

[0080] - a sense primer comprising, or consisting of, a nucleotide sequence SEQ ID No. 1, or a nucleotide sequence having at least 80% identity with SEQ ID No. 1 and an antisense primer, comprising, or consisting of, a nucleotide sequence SEQ ID No. 2, or a nucleotide sequence having at least 80% identity with SEQ ID No. 2, and / or

[0081] - a sense primer comprising, or consisting of, a nucleotide sequence SEQ ID No. 4, or a nucleotide sequence having at least 80% identity with SEQ ID No. 4 and an antisense primer, comprising, or consisting of, a nucleotide sequence SEQ ID No. 5, or a nucleotide sequence having at least 80% identity with SEQ ID No. 5.

[0082]

[0049] According to another more particular aspect, the subject of the invention is a kit for the detection in a biological sample of a target nucleotide sequence of methylated cell-free DNA, said nucleotide sequence being specific to a type of cells present in the lung, said kit comprising at least one sense primer comprising, or consisting of, a nucleotide sequence SEQ ID No. 7, or a nucleotide sequence having at least 80% identity with SEQ ID No. 7 and an antisense primer, comprising, or consisting of, a nucleotide sequence SEQ ID No. 8, or a nucleotide sequence having at least 80% identity with SEQ ID No. 8.

[0083]

[0050] The nucleotide sequences of the primers specific to a cell type present in the kidney are designated as indicated in Table 3 below:

[0084] Table 3

[0085]

[0086]

[0051] The nucleotide sequences of the probes specific to a cell type present in the kidney are designated as indicated in Table 4 below:

[0087] Table 4

[0088]

[0052] Among the tissues present in the kidney, we can notably cite: renal epithelial tissue and renal vascular tissue.

[0089]

[0053] The markers dedicated to monitoring the state of renal suffering are characterized as indicated in Table 5 below:

[0090] Table 5

[0054] According to another more particular aspect, the subject of the invention is a kit which contains the necessary reagents (primers and probes) for the amplification of at least one PCR amplicon less than < 85 bp (preferably < 70 bp) in a genomic region included among the nucleotide sequences SEQ ID No. 55, SEQ ID No. 56, SEQ ID No. 57, SEQ ID No. 58, SEQ ID No. 59, SEQ ID No. 61, SEQ ID No. 62, SEQ ID No. 63, SEQ ID No. 64, SEQ ID No. 65, SEQ ID No. 66, SEQ ID No. 67, SEQ ID No. 68, SEQ ID No. 69, SEQ ID No. 70 and SEQ ID No. 71 according to the methylation status of said genomic region.

[0091]

[0055] The markers used in a kit and a method according to the invention are therefore likely to be classified as markers:

[0092] - “vascular type”: chosen from the markers ALDH1A1, ARID3A, GATA2, LOC124903692, NRN1, SEPT5-GP1BB, TNS2-AS1 RHBDF2 and LINC01599,

[0093] - “epithelial type”: chosen from the markers FLJ 12825, ACSL5, PAX2 and PDE4D,

[0094] - “total kidney type”: chosen from CTDP1.

[0095]

[0056] According to another more particular aspect, the subject of the invention is a kit for the detection in a biological sample of a target nucleotide sequence of methylated cell-free DNA, said nucleotide sequence being specific to a type of cells present in the kidney, said kit comprising at least:

[0096] -a sense primer comprising, or consisting of, a nucleotide sequence capable of detecting a “total kidney-like” marker and an antisense primer, comprising, or consisting of, a nucleotide sequence capable of detecting a “total kidney-like” marker, and / or

[0097] - a sense primer comprising, or consisting of, a nucleotide sequence capable of detecting a “vascular-type” marker and an antisense primer, comprising, or consisting of, a nucleotide sequence capable of detecting a “vascular-type” marker, and / or

[0098] - a sense primer comprising, or consisting of, a nucleotide sequence capable of detecting an “epithelial-type” marker and an antisense primer, comprising, or consisting of, a nucleotide sequence capable of detecting an “epithelial-type” marker.

[0099]

[0057] Preferably, according to this more particular aspect, the subject of the invention is a kit for the detection in a biological sample of a target nucleotide sequence of methylated cell-free DNA, said nucleotide sequence being specific to a type of cells present in the kidney, said kit comprising at least:

[0100] -a sense primer comprising, or consisting of, a nucleotide sequence capable of detecting a “total kidney-like” marker and an antisense primer, comprising, or consisting of, a nucleotide sequence capable of detecting a “total kidney-like” marker, said marker being CTDP1 and

[0101] - a sense primer comprising, or consisting of, a nucleotide sequence capable of detecting a “vascular-type” marker and an antisense primer, comprising, or consisting of, a nucleotide sequence capable of detecting a “vascular-type” marker, said marker being chosen from GATA2, TNS2-AS1 and

[0102] - a sense primer comprising, or consisting of, a nucleotide sequence capable of detecting an “epithelial-type” marker and an antisense primer, comprising, or consisting of, a nucleotide sequence capable of detecting an “epithelial-type” marker, said marker being chosen from: PAX2, ACSL5 or PDE4D.

[0103]

[0058] The markers used in a kit and a method according to the invention will now be described in more detail. The names of the markers correspond to the name of the gene on which these markers are located.

[0104]

[0059] The genetic region "PDE4D" according to the invention designates a sequence coding for a protein of the phosphodiesterase family. Its DNA sequence is located on chromosome 5 at position 58,969,038-60,522,128, more specifically at the 5q11.2-q12.1 locus. It contains 42 exons. Its DNA sequence is referred to as NC_000005.10. It is transcribed in the sequence NM_006203.5. The inventors have identified the DNA region included in chromosome 5 position 59,039,300-59,039,420 (SEQ ID No. 55), as being specifically hyper-methylated in the epithelial fraction. They propose to use this gene in a non-invasive, sensitive and reliable method, particularly to diagnose or identify kidney transplant rejection in kidney transplant patients, or in a kit useful for such purposes.

[0105]

[0060] The genetic region "PAX2" according to the invention designates a coding sequence for a protein of the box-paired 2 family. Its DNA sequence is located on chromosome 10 at position 100,735,396-100,829,944, more specifically at the locus 10q24.31. It contains 14 exons. Its DNA sequence is referred to as NC_000010.11. It is transcribed in the sequence NM_000278.5. The inventors have identified the DNA region included in chromosome 10 position 100,828,800-100,829,020 (SEQ ID No. 56), as being specifically hyper-methylated in the epithelial fraction. They propose to use this gene in particular in a non-invasive, sensitive and reliable method for diagnosing or identifying kidney transplant rejection in kidney transplant patients, or in a kit useful for such purposes.

[0106]

[0061] The genetic region "FLJ12825" according to the invention designates a complete genomic molecule not coding for proteins. It allows the transcription of long non-coding RNAs and is present on the uncharacterized locus LOC440101 of the FLJ12825 gene. Its DNA sequence is located on chromosome 12 at position 54,058,254-54,122,234, more specifically at the locus 12ql3.13 in an intronic sequence. Its DNA sequence is referred to as NC_000012.12. It is transcribed into 1 RNA variant named NR_026655.1. The present inventors have identified the DNA region included in chromosome 12: Position 54,106,357- 54,106,526 (SEQ ID NO: 57), as being specifically hyper-methylated in the renal epithelial fraction. In particular, they propose to use this gene in a non-invasive, sensitive and reliable method for diagnosing or identifying renal graft rejection in the kidney transplant patient, or in a kit useful for such purposes.

[0107]

[0062] The genetic region "ALDH1A1" according to the invention designates a complete genomic molecule coding for a protein of the aldehyde dehydrogenase type 1 family (member A1). Its DNA sequence is located on chromosome 9 at position 72,900,671-72,953,063, more specifically at the 9q21.13 locus. It contains 13 exons. Its DNA sequence is referred to as NC_005224.3. It is transcribed into 1 RNA variant named NM_005224.3. The present inventors have identified the DNA region included in chromosome 9:

[0108] Position 72,952,933-72,953,059 (SEQ ID No. 58), as being specifically hypermethylated in the renal vascular fraction. They therefore propose to use this gene in a non-invasive, sensitive and reliable method to diagnose or identify kidney transplant rejection in kidney transplant patients, or in a kit useful for such purposes.

[0109]

[0063] The genetic region "ARID3A" according to the invention designates a complete genomic molecule coding for a protein of the ARID (AT-rich interaction domain) DNA binding protein family. Its DNA sequence is located on chromosome 19 at position 925,732-975,939, more specifically at the 19pl3.3 locus. It contains 12 exons. Its DNA sequence is referred to as NC_000019.10. It is transcribed into 1 RNA variant named NM_000689.5. The present inventors have identified the DNA region included in chromosome 19:

[0110] Position 940,667-941,160 (SEQ ID No. 59), as being specifically hyper-methylated in the renal vascular fraction. They therefore propose to use this gene in a non-invasive, sensitive and reliable method to diagnose or identify kidney transplant rejection in kidney transplant patients, or in a kit useful for such purposes.

[0111]

[0064] The genetic region "ACSL5" according to the invention designates a coding sequence for a protein of the ligase family. Its DNA sequence is located on chromosome 10 at position 112,374,116-112,428,376, more specifically at the 10q25.2 locus. It contains 23 exons. Its DNA sequence is referred to as NC_000010.11. It is transcribed in the sequence NM_016234.4. The present inventors have identified the DNA region included in chromosome 10 position 112,376,275-112,376,398 (SEQ ID No. 60), as being specifically hyper-methylated in the epithelial fraction. They therefore propose to use this gene in a non-invasive, sensitive and reliable method to diagnose or identify kidney transplant rejection in kidney transplant patients, or in a kit useful for such purposes.

[0112]

[0065] The genetic region "CTDP1" according to the invention designates an uncharacterized intronic sequence located between the CTDP1 gene and KCNG2. Its DNA sequence is located on chromosome 18 between the positions of the CTDP1 (chrl8: 79,756,625-79,787,722) and KCNG2 (chrl8: 79,797,938-79,900,100) genes. It contains 0 exons. Its DNA sequence is not referenced in the NCBI. There is no transcript identified.

[0113] The present inventors have identified the DNA region included in chromosome 18 position 79,791,700-79,791,820 (SEQ ID NO: 61), as being specifically hypermethylated in the total fraction of the kidney. They therefore propose to use this gene in a non-invasive, sensitive and reliable method for in particular diagnosing or identifying renal graft rejection in the kidney transplant patient, or in a kit useful for such purposes.

[0114]

[0066] The genetic region "GATA2" according to the invention designates a coding sequence for a protein of the zinc finger DNA transcription factor family. Its DNA sequence is located on chromosome 3 at position 128,479,422-128,493,201, more specifically at the locus 3q21.3. It contains 8 exons. Its DNA sequence is referred to as NC_000003.12. It is transcribed in the sequence NM_032638.5. The present inventors have identified the DNA region included in chromosome 3 position 128,491,794-128,492,245 (SEQ ID NO: 62), as being specifically hyper-methylated in the vascular fraction. They therefore propose to use this gene in a non-invasive, sensitive and reliable method to diagnose or identify kidney transplant rejection in kidney transplant patients, or in a kit useful for such purposes.

[0115]

[0067] The genetic region "LOC124903692" according to the invention designates an uncharacterized and non-protein coding sequence. Its DNA sequence is located on chromosome 16 at position 54,936,014-54,938,671, more specifically at the locus 16ql2.2. It contains 3 exons. Its DNA sequence is referred to as NC_000016.10. It is transcribed in the sequence XR_007065074.1. The present inventors have identified the DNA region included in chromosome 16 position 54,937,300-54,937,500 (SEQ ID NO: 63), as being specifically hyper-methylated in the vascular fraction. They therefore propose to use this gene in a non-invasive, sensitive and reliable method to diagnose or identify kidney transplant rejection in kidney transplant patients, or in a kit useful for such purposes.

[0116]

[0068] The genetic region "NRN1" according to the invention designates a coding sequence for a protein of the neuritin family. Its DNA sequence is located on chromosome 6 at position 5,997,999-6,007,518, more specifically at the locus

[0117] 6p25.1. It contains 7 exons. Its DNA sequence is referred to as NC_000006.12. It is transcribed in the sequence NM_016588.3. The present inventors have identified the DNA region included in chromosome 6 position 5,998,924-5,999,075 (SEQ ID NO: 64), as being specifically hyper-methylated in the vascular fraction. They therefore propose to use this gene in a non-invasive, sensitive and reliable method for diagnosing or identifying kidney transplant rejection in kidney transplant patients, or in a kit useful for such purposes.

[0118]

[0069] The genetic region "SEPT5-GP1BB" according to the invention designates a non-protein coding sequence. Its DNA sequence is located on chromosome 22 at position 19,717,220-19,724,774, more specifically at the locus 22qll.21. It contains 12 exons. Its DNA sequence is referred to as NC_000022.11. It is transcribed in the sequence NR_037611.1. The present inventors have identified the DNA region included in chromosome 22 position 19,724,235-19,724,340 (SEQ ID NO: 65), as being specifically hyper-methylated in the vascular fraction. They therefore propose to use this gene in a non-invasive, sensitive and reliable method to diagnose or identify kidney transplant rejection in kidney transplant patients, or in a kit useful for such purposes.

[0119]

[0070] The genetic region "TNS2-AS1" according to the invention designates a non-protein coding sequence. Its DNA sequence is located on chromosome 12 at position 53,043,189-53,054,438, more specifically at the locus 12ql3.13. It contains 4 exons. Its DNA sequence is referred to as NC_000012.12. It is transcribed in the sequence NR_033854.1. The inventors have identified the DNA region included in chromosome 12 position 53,054,207-53,054,329 (SEQ ID No. 66), as being specifically hyper-methylated in the vascular fraction. They therefore propose to use this gene in a non-invasive, sensitive and reliable method to diagnose or identify kidney transplant rejection in kidney transplant patients, or in a kit useful for such purposes.

[0120]

[0071] The genetic region "RHBDF2" according to the invention designates a coding sequence for a protein enabling the activation of protein transporter activity. Its DNA sequence is located on chromosome 17 at position 76,470,893-76,501,427, more specifically at the locus 17q25.1. It contains 21 exons. Its DNA sequence is referred to as NC_000017.11. It is transcribed in the sequence NM_024599.5. The inventors have identified the DNA region included in chromosome 17 position 76,500,822-76,500,937 (SEQ ID No. 67), as being specifically hyper-methylated in the vascular fraction. They therefore propose to use this gene in a non-invasive, sensitive and reliable method to diagnose or identify kidney transplant rejection in kidney transplant patients, or in a kit useful for such purposes.

[0121]

[0072] The genetic region “LINC01599” according to the invention designates a sequence allowing the transcription of intergenic RNAs not coding for proteins. Its DNA sequence is located on chromosome 14 at position 50,007,313-50,105,043, more specifically at the locus 14q21.3. It contains 9 exons. Its DNA sequence is referred to as NC_000014.9. It is transcribed in the sequence NR_131171.1. The inventors have identified the DNA region included in chromosome 14 position 50,056,666-50,056,758 (SEQ ID No. 68), as being specifically hypomethylated in the vascular fraction. They therefore propose to use this gene in a non-invasive, sensitive and reliable method to diagnose or identify kidney transplant rejection in kidney transplant patients, or in a kit useful for such purposes.

[0122]

[0073] The genetic region “APC2” according to the invention designates a complete genomic molecule sequence coding for a protein of the family of APC regulators of the Wnt pathway. Its DNA sequence is located on chromosome 19 at position 1,446,230-1,473,244, more specifically at the locus 19pl3.3. It contains 17 exons. Its DNA sequence is referred to as NC_000019.10. It is transcribed in the sequence NM_005883.3. The inventors have identified the DNA region included in chromosome 19 position 1,467,853-1,468,054 (SEQ ID No. 69), as being specifically hypermethylated in neurons. They therefore propose to use this gene in a non-invasive, sensitive and reliable method, in particular to diagnose a stroke, or in a kit useful for such purposes. The genetic region "NBR1" according to the invention designates a complete genomic molecule coding for a protein of the family of receptors specific for autophagy.Its DNA sequence is located on chromosome 17 at position 43,170,409-43,211,900, more specifically at the 17q21.31 locus. It contains 23 exons. Its DNA sequence is referenced as NC_000017.11. It is transcribed in the sequence NM_005899.5. The inventors have identified the DNA region included in chromosome 17 at position 43,211,655-43,211,856 (SEQ ID No. 70), as being specifically hypermethylated in neurons. They therefore propose to use this gene in a non-invasive, sensitive and reliable method for diagnosing stroke, in particular, or in a kit useful for such purposes.

[0074] The genetic region “SYNE1” according to the invention designates a complete genomic molecule coding for a nuclear envelope protein containing spectrin repeat sequences. Its DNA sequence is located on chromosome 6 at position 152,121,687-152,637,362, more specifically at the 6p25.2 locus. It contains 153 exons.Its DNA sequence is referenced as NC_000006.12. It is transcribed in the sequence NM_033071.5. The inventors have identified the DNA region included in chromosome 6 position 152,302,152-152,302,353 (SEQ ID NO: 71), as being specifically hypermethylated in pulmonary pneumocytes / alveoli. They therefore propose to use this gene in a non-invasive, sensitive and reliable method for diagnosing or identifying ARDS, in particular, or in a kit useful for such purposes.

[0123]

[0075] A kit according to the invention optionally comprises one or more pairs of primers consisting of a sense primer and an antisense primer, intended to detect the presence of a control element in the biological sample. This control element may be any suitable element chosen by a person skilled in the art. Said control element may in particular be albumin. More particularly, a kit according to the invention may comprise a sense primer comprising, or consisting of, a nucleotide sequence SEQ ID No. 16, or a nucleotide sequence having at least 80% identity with SEQ ID No. 16 and an antisense primer, comprising, or consisting of, a nucleotide sequence SEQ ID No. 17, or a nucleotide sequence having at least 80% identity with SEQ ID No. 17.

[0124]

[0076] According to a second aspect, the invention relates to a method for identifying a target sequence of cell-free DNA specific to a cell type present in a first organ chosen from: the lung, the brain and the kidney, the method comprising at least the following steps: a) Determination, in at least one genomic DNA methylation database, of the position and degree of methylation of the CpG islands of sequences specific to said cell type of said organ, b) Identification, in the genomic DNA of said cell type of said healthy organ, of the specifically hypermethylated CpG islands, c) Comparison of the degree of methylation of the hypermethylated CpG islands of said cell type of said first organ with the degree of methylation of the same CpG islands of a second cell type, different from the first cell type,d) Selection of the CpG islands that are only hypermethylated in the genomic DNA of said cell type of said first organ after comparison carried out in step c), e) Comparison of the degree of methylation of the hypermethylated CpG islands of said cell type of said first organ with the degree of methylation of the same CpG islands in the genomic DNA of organs other than said first organ, f) Comparison of the degree of methylation of the hypermethylated CpG islands of said cell type of said first organ with the degree of methylation of the same CpG islands in the genomic DNA of healthy white blood cells, g) Comparison of the degree of methylation of the hypermethylated CpG islands of said cell type of said first organ with the degree of methylation of the same CpG islands in cell-free DNA of all biological matrices combined from healthy subjects, h) Selection of the CpG islands that are only hypermethylated in the genomic DNA of said cell type of said first organ after comparison carried out in step e,f and g) i) Identification of one or more hypermethylated target sequences of cell-free DNA specific to said cell type of the first organ from the comparison carried out during step h).,

[0125]

[0077] The invention also relates to a target nucleotide sequence of methylated cell-free DNA identified by means of a method according to the invention, for its use in the detection of lysis of an organ chosen from the brain, the lung and the kidney, from a biological sample of a patient.

[0126]

[0078] According to a third aspect, the invention relates to a method for detecting, in a biological sample, a target nucleotide sequence of methylated cell-free DNA, said nucleotide sequence being specific to an organ chosen from: the lung, the brain and the kidney, or to a tissue present in the brain, the lung or the kidney, said method comprising at least the following steps: a) Bringing together, under conditions appropriate for amplification of the nucleic acids, the cell-free DNA previously extracted from said biological sample and a pair of primers consisting of a sense primer and an antisense primer, each of the primers comprising, or consisting of, a nucleotide sequence capable of hybridizing with a nucleotide sequence of said target sequence of cell-free DNA, b) Amplification reaction of said target sequence, c) Detection of the presence of the sequence amplified during step b).

[0127]

[0079] Cell-free DNA is extracted using a commercial kit, well known to those skilled in the art and requires the presence of at least 0.15 ng / mL of target DNA in the urine or plasma sample.

[0128]

[0080] The amplification reaction is carried out using any technique or protocol well known to those skilled in the art. Non-limiting examples of these techniques include: PCR amplification, digital PCR (dPCR) amplification, next-generation sequencing.

[0081] More particularly, the subject of the invention is a method for detecting in a biological sample a target nucleotide sequence of methylated cell-free DNA, said nucleotide sequence being specific to a type of cells present in the brain.

[0129]

[0082] Even more particularly, the subject of the invention is a method for the detection in a biological sample of a target nucleotide sequence of methylated cell-free DNA, said nucleotide sequence being specific to a type of cells present in the brain, said method comprising at least the following steps: a) Bringing together, under conditions appropriate for amplification of the nucleic acids, the cell-free DNA previously extracted from said biological sample and a pair of primers consisting of a sense primer comprising or consisting of a nucleotide sequence SEQ ID No. 1 (or a nucleotide sequence having at least 80% identity with SEQ ID No. 1) and an antisense primer SEQ ID No. 2 (or a nucleotide sequence having at least 80% identity with SEQ ID No. 2),or a sense primer comprising or consisting of a nucleotide sequence SEQ ID No. 4 (or a nucleotide sequence having at least 80% identity with SEQ ID No. 4) and an antisense primer SEQ ID No. 5 (or a nucleotide sequence having at least 80% identity with SEQ ID No. 5), each of the primers comprising, or consisting of, a nucleotide sequence capable of hybridizing with a nucleotide sequence of said target sequence of cell-free DNA, or else brought into contact, under conditions appropriate for amplification of the nucleic acids, b) Amplification reaction of said target sequence, c) Detection of the presence of the sequence amplified during step b), by means of a probe comprising, or consisting of SEQ ID No. 3 or SEQ ID No. 6, respectively according to the pairs of primers used.,

[0130]

[0083] Even more particularly, the subject of the invention is a method for the detection in a biological sample of a target nucleotide sequence of methylated cell-free DNA, said nucleotide sequence being specific to a type of cells present in the lung, said method comprising at least the following steps:

[0131] - Bringing together, under conditions appropriate for nucleic acid amplification, cell-free DNA previously extracted from said biological sample and a pair of primers consisting of

[0132] - a sense primer comprising or consisting of a nucleotide sequence SEQ ID No. 7 (or a nucleotide sequence having at least 80% identity with SEQ ID No. 7) and an antisense primer SEQ ID No. 8 (or a nucleotide sequence having at least 80% identity with SEQ ID No. 8), each of the primers comprising, or consisting of, a nucleotide sequence capable of hybridizing with a nucleotide sequence of said target sequence of cell-free DNA, or else brought into contact, under conditions appropriate for amplification of the nucleic acids, b) Amplification reaction of said target sequence, c) Detection of the presence of the sequence amplified during step b), by means of a probe comprising, or consisting of SEQ ID No. 9.

[0133]

[0084] Even more particularly, the subject of the invention is a method for the detection in a biological sample of a target nucleotide sequence of methylated cell-free DNA, said nucleotide sequence being specific to a type of cells present in the kidney, said method comprising at least the following steps:

[0134] - a) Bringing together, under conditions appropriate for nucleic acid amplification, cell-free DNA previously extracted from said biological sample and a pair of primers consisting of

[0135] - a sense primer comprising or consisting of a nucleotide sequence SEQ ID No. 10 (or a nucleotide sequence having at least 80% identity with SEQ ID No. 10) and an antisense primer SEQ ID No. 11 (or a nucleotide sequence having at least 80% identity with SEQ ID No. 11), or

[0136] - a sense primer comprising or consisting of a nucleotide sequence SEQ ID No. 13 (or a nucleotide sequence having at least 80% identity with SEQ ID No. 13) and an antisense primer SEQ ID No. 14 (or a nucleotide sequence having at least 80% identity with SEQ ID No. 14), each of the primers comprising, or consisting of, a nucleotide sequence capable of hybridizing with a nucleotide sequence of said target sequence of cell-free DNA, or else brought together, under conditions appropriate for amplification of the nucleic acids,

[0137] - b) Amplification reaction of said target sequence,

[0138] - c) Detection of the presence of the sequence amplified during step b), by means of a probe comprising, or consisting of SEQ ID No. 12 or SEQ ID No. 15, respectively depending on the pairs of primers used.

[0139]

[0085] The diagnostic tests described are part of an analytical process composed of 2 preliminary steps: extraction of circulating DNA and epigenetic conversion (by bisulfite, enzymatic reaction). The diagnostic tests are compatible with different circulating DNA extraction kits such as the QIAamp Circulating Nucleic Acid Kit (50) Cat. No. I ID: 55114 (Qiagen); EZ1&2 ccfDNA Kit (48)Cat. No. I ID: 954854 (Qiagen); MagMAX™ Cell-Free DNA Isolation Kit (Reference: A29319, Thermofisher); Automated High-Throughput Extraction of Cell-Free DNA from Plasma, Serum, Urine and CSF (Ref: A6030, Promega). The cell-free DNAs once extracted are assayed by Qubit 4 fluorimetry.0™ (Invitrogen™ Qubit™ HS dsDNA Assay Kits (Part Number: Q32851)) and then undergo bisulfite conversion with the Zymo Research EZ DNA Methylation-Lightning Kit (Part Number D5031) or commercial enzymatic equivalent (NEBNext® Enzymatic Methyl-seq Conversion Module (Part Number E7125S / E7125L)). The samples are then analyzed by droplet digital PCR (Naica System (STILLA Technologies)) (also compatible with commercial equivalents such as the QIAcuity Digital PCR System - QIAGEN and QX ONE Droplet Digital PCR (ddPCR) System - Bio-Rad).

[0140]

[0086] The diagnostic tests described are compatible with all digital PCR platforms on the market (microdroplets and / or micro-compartments). Among them, we find commercial equivalents such as the QIAcuity Digital PCR System - QIAGEN and QX ONE Droplet Digital PCR (ddPCR) System - Bio-Rad).

[0141]

[0087] The inventors determine typical metrological parameters, including repeatability, reproducibility, and sensitivity of the test. To quantify renal degradation, at least 1 of the markers is required. Detection with two markers strengthens the biological data.

[0142]

[0088] Preferably, to quantify renal degradation, it is preferable to detect at least one "whole kidney" marker, one "vascular type" marker and one "epithelial type" marker quantified in a multiplexed test. The more the number of markers in each of these categories is increased, the more the clinical performance of the test is increased. The detection of several markers per category among the "whole kidney", "vascular type" and "epithelial type" markers strengthens the biological data.

[0143]

[0089] According to another aspect, the invention also relates to the use of a kit or a method according to the invention, for the specific detection of brain degradation and / or for in vitro diagnosis and / or prognosis, and / or monitoring of the progression of a stroke and / or therapeutic optimization of the patient suffering from a stroke.

[0144]

[0090] According to another aspect, the invention also relates to the use of a kit or a method according to the invention, for the specific detection of lung degradation and / or for in vitro diagnosis and / or prognosis, and / or monitoring the development of an acute respiratory distress syndrome and / or therapeutic optimization of the patient suffering from an acute respiratory distress syndrome.

[0145]

[0091] According to another aspect, the invention also relates to the use of a kit or a method according to the invention, for the specific detection of kidney degradation and / or for in vitro diagnosis and / or prognosis, and / or screening and / or monitoring of the development of a renal transplant rejection / dysfunction and / or an episode of acute renal failure and / or therapeutic optimization of the patient suffering from renal transplant rejection / dysfunction and / or acute renal failure.

[0146]

[0092] According to another aspect, the invention also relates to a nucleotide sequence chosen from:

[0147] - a sense primer comprising, or consisting of, a nucleotide sequence chosen from: SEQ ID No. 1, SEQ ID No. 4, SEQ ID No. 7, SEQ ID No. 10, SEQ ID No. 13, SEQ ID No. 19, SEQ ID No. 22, SEQ ID No. 25, SEQ ID No. 28, SEQ ID No. 31, SEQ ID No. 34, SEQ ID No. 37, SEQ ID No. 40, SEQ ID No. 43, SEQ ID No. 46, SEQ ID No. 49, SEQ ID No. 52, or a nucleotide sequence having at least 80% identity with said sequences;

[0148] - an antisense primer comprising a nucleotide sequence chosen from: SEQ ID No. 2, SEQ ID No. 5, SEQ ID No. 8, SEQ ID No. 11, SEQ ID No. 14, SEQ ID No. 20, SEQ ID No. 23, SEQ ID No. 26, SEQ ID No. 29, SEQ ID No. 32, SEQ ID No. 35, SEQ ID No. 38, SEQ ID No. 41, SEQ ID No. 44, SEQ ID No. 47, SEQ ID No. 50 and SEQ ID No. 53, or a nucleotide sequence having at least 80% identity with said sequences; and

[0149] - a probe comprising a nucleotide sequence chosen from: SEQ ID No. 3, SEQ ID No. 6, SEQ ID No. 9, SEQ ID No. 12, SEQ ID No. 15, SEQ ID No. 21, SEQ ID No. 24, SEQ ID No. 27, SEQ ID No. 30, SEQ ID No. 33, SEQ ID No. 36, SEQ ID No. 39, SEQ ID No. 42, SEQ ID No. 45, SEQ ID No. 48, SEQ ID No. 51 and SEQ ID No. 54, or a nucleotide sequence having at least 80% identity with said sequences.

[0150]

[0093] According to another aspect, the invention relates to diagnosis, or monitoring of the degradation of an organ chosen from the brain, the lung and the kidney, comprising the following steps:

[0151] - Extraction of methylated cell-free DNA from a biological sample of an individual

[0152] - Conversion of methylated cell-free DNA (bisulfite and / or enzymatic conversion)

[0153] - Bringing together DNA and an appropriate pair of primers under conditions allowing hybridization,

[0154] - Amplification of said sequence,

[0155] - Detection of the amplified sequence, using the appropriate detection probe.

[0156] Description of figures and embodiments

[0157]

[0094] Other advantages and characteristics will appear on examining the detailed description of a non-limiting embodiment, and the attached drawings, in which:

[0158]

[0095] Figure 1 represents the proportion of labeled neurons when using the anti-NeuN marker (left), the APC2 marker (center) and the NBR1 marker (right).

[0096] Figure 2 represents the normalized data of the mean of the NBR1 and APC2 markers, on a logarithmic scale in the case of TIA (left), and stroke (right).

[0159]

[0097] Figure 3 represents the amount of renal markers in plasma (in ng / ml) in the case of patients at high risk of graft rejection (left histograms) or at low risk of graft rejection (right histograms), for the markers PDE4D (white) and PAX2 (black).

[0160]

[0098] Figure 4 represents the fraction of circulating DNA of renal origin, out of the total circulating DNA in the plasma (in %) in the case of patients at high risk of graft rejection (left histograms) or at low risk of graft rejection (right histograms), for the markers PDE4D (white) and PAX2 (black).

[0161]

[0099] Figure 5 represents the amount of renal markers in ng / ml of urine in the case of patients with graft rejection (left histograms) or without graft rejection (right histograms), for the markers PDE4D (white) and PAX2 (black).

[0162]

[0100] Figure 6 represents the correlation between the TTF-1 histological marking (x-axis) and the use of the SYNE1 marker (y-axis) according to the invention.

[0163]

[0101] Figure 7 represents the discrimination between subgroups of patients with moderate ARDS (left), severe ARDS (center) or critical ARDS (right) based on the results obtained with the SYNE1 marker (ordinates).

[0164]

[0102] Figures 8A and 8B respectively represent: (A) the detection of the markers SEPT5 / GATA2, PDE4D, CTDP1, PAX2 and TNS2-AS1 / ACSL5 in 100% methylated DNA; (B) the detection of the markers SEPT5 / GATA2, PDE4D, CTDP1, PAX2 and TNS2-AS1 / ACSL5 in unmethylated DNA, i.e. the negative control, the results showing an absence of detection in unmethylated DNA.

[0165]

[0103] Figure 9 represents the relative quantification of the markers SEPT5, GATA2, TNS2-AS1, PDE4D, ACSL5, PAX2 and CTDP1, with normalization by the ubiquitous marker albumin (internal control corresponding to the number of total genomes present in the sample) in samples of genomic DNA (gDNA) extracted from healthy tissues: kidney, liver, white blood cells, plasma circulating DNA and urinary circulating DNA.

[0166]

[0104] Figure 10 represents the correlation (R 2 = 0.5) between the normalized relative quantifications (via an internal albumin marker) of the PAX2 and ACSL5 markers in n = 22 renal gDNAs.

[0105] Figure 11 represents the correlation (R 2 = 0.8087) between the normalized relative quantifications (via an internal Albumin marker) of PAX2 and ACSL5 markers in n = 15 urinary cf-DNA.

[0167]

[0106] Figures 12A and 12B) respectively represent the comparison of 2 quantitative methods of the renal epithelial fraction on n = 22 healthy kidney samples. CD10 is a histological marker of the renal epithelium. PAX2 and ACSL5 are molecular markers specifically targeting the renal epithelium.

[0168]

[0107] Figure 13 represents the significant difference in quantification of the CTDP1 marker in urine between patients with kidney transplant rejection and those without kidney transplant rejection (left histogram) and a logistic regression model compiling the quantitative data of the biomarkers CTDP1, PAX2, PDE4D, ACSL5 and SEPT5 to predict the presence or absence of kidney transplant rejection (right graph)

[0169]

[0108] Figure 14 represents the significant difference in quantification of the CTDP1 biomarker in plasmas between patients with graft rejection (with tissue damage on the graft) and those without renal graft rejection (without tissue damage on the graft) (left histogram) and a logistic regression model compiling the quantitative data of the biomarkers CTDP1, GATA2, TNS2-AS1, PAX2 and PDE4D to predict the presence or absence of graft rejection (right graph).

[0170]

[0109] Figure 15 represents the significant difference in quantification of the GATA2 biomarker in plasmas between patients with graft vascular lesions and those without graft vascular lesions (left graph) and a logistic regression model compiling the quantitative data of the CTDP1, GATA2, and TNS2-ASl biomarkers to predict the presence or absence of vascular lesions on the graft (right graph).

[0171]

[0110] It is understood that the embodiments which will be described subsequently are in no way limiting. In particular, it will be possible to imagine variants of the invention comprising only a selection of characteristics described subsequently isolated from the other characteristics described, if this selection of characteristics is sufficient to confer a technical advantage or to differentiate the invention from the state of the prior art. The present invention will be better understood by reading the following examples, which are given to illustrate the invention and not to limit its scope. EXAMPLES

[0172] EXAMPLE 1: Identification of biomarkers

[0173]

[0111] The CpG islands present at the level of the promoters of certain genes are found at different levels of methylation depending on the nature of the tissue. Based on the methylation databases TCGA (The Cancer Genome Atlas) and DeepBlue (Epigenomic Data Server - DeepBlue), Gene Expression Omnibus (ID: GSE186458), the positions and methylation degrees of a large number of CpG islands are listed for many different tissue types, including subtypes of pulmonary pneumocytes (lung), neurons (brain), and renal cells (kidney) (Moss et al. Nat Commun, Dec 2018;9(l):5068. ; Liu X et al. Clin Epigenetics. Dec 2019;ll(l):93; Silva TC et al. Bioinformatics. 1 Jun 2019;35(ll):1974-7; Zhou W et al. Nucleic Acids Res. 24 Oct 2016;gkw967. ; Albrecht F et al. Nucleic Acids Res. 8 Jul 2016;44(Wl):W581-6).Using bioinformatics, the inventors identified different hypermethylated positions in healthy brain, lung, and kidney tissues. This identification involves 5 steps and is described below in the example of lung degradation detection.

[0174]

[0112] In the case of the detection of pulmonary degradation, the inventors selected non-methylated positions in DNA from white blood cells, PBMCs and immune cells, based on an analysis of data from whole blood.

[0175]

[0113] The inventors then detected the hypermethylated and / or hypomethylated CpG islands in the healthy tissue, in a database established from healthy lung tissue. These data were separated into a discovery data set (“training”) and a validation data set (“test”).

[0176]

[0114] Differentially methylated CpG islands from lung tissue compared to CpG islands from healthy tissue of other organs (kidney, brain, muscle, stomach, liver, heart, intestine) were then identified. The most differentially methylated positions were selected, thus 432 positions were retained.

[0177]

[0115] Among these 432 positions, refinement was carried out by manual selection. Positions were retained based on their average methylation in lung and non-lung tissues. 4 candidate positions were selected.

[0178]

[0116] The performance of these four positions for discriminating lung tissue from non-lung tissue was analyzed using a penalized logistic regression model using the LASSO method. The AUROC statistical test obtained allows us to conclude that several CpG islands allow us to identify lung tissue with high predictive performance.

[0179]

[0117] Table 6 below lists the markers selected for each of the target organs. Table 6

[0180]

[0118] Different amplicons were generated in order to discriminate each of the identified positions. The technical characteristics of the primers and probes are described in Table 4 below. The hybridization temperatures of the probes were chosen between 40° and 54°C for the probes and 52 and 60°C for the primers, the difference in hybridization temperature between primers and probes is 5 to 10°C.

[0181] Table 7

[0182]

[0119] For each organ, a multiplexed test was constructed. An internal control of the total circulating DNA quantity, detecting the quantity of DNA coding for albumin, is implemented in the test.

[0183] EXAMPLE 2: Validation of biomarkers of neuronal degradation in the management of stroke in the acute phase

[0120] The validation of the biomarkers according to the invention comprises the following steps:

[0184] - molecular analysis of DNA conversion to droplet digital PCR tests, - validation of the digital PCR test on 100% methylated synthetic DNA and on commercial unmethylated genomic DNA,

[0185] - the determination of metrological parameters,

[0186] - a negative control demonstrating the specificity of the test,

[0187] - a positive control on genomic DNA samples from healthy tissue sections, and comparison with the reference histological marking

[0188]

[0121] Molecular analysis of DNA conversion to droplet digital PCR assays is common to the different markers. Cell-free DNA is first extracted by a commercial extraction method dedicated to cell-free DNA via QIAamp Circulating Nucleic Acid Kit (Cat. No. I ID: 55114, Qiagen). Cell-free DNA is assayed by Qubit 4.0™ fluorimetry (Invitrogen™ HS Qubit™ dsDNA Assay Kits (Cat. No. Q32851)) and then undergoes bisulfite conversion with the Zymo Research EZ DNA Methylation-Lightning Kit (Cat. No. D5031) or commercial equivalent. Samples are then analyzed by droplet digital PCR (System Naica (STILLA Technologies)).

[0189]

[0122] The design of the primers and probes required for the PCR reaction for the APC2 and NBR1 targets are mentioned in Table 7. Taqman-Mgb type probes are used (Reference: PB-MGBEF-006, PB-MGBEC-006 and PB-MGBEH-006, Eurogentec). The implementation of the PCR amplification reaction is carried out in a process comprising 3 main steps presented below, this process is designated

[0190] “PCR amplification according to the invention” in the rest of the text of this application.

[0191]

[0123] The preparation of the specific primer-probe cocktail is presented in the following Table 8.

[0192] Table 8

[0193]

[0124] The preparation of the PCR reaction mix is ​​presented in the following Table 9.

[0194] Table 9

[0195]

[0125] The PCR reaction is shown in Table 10. Table 10

[0196]

[0126] The plates are read according to the following acquisition setting: 120 ms FAM channel (blue) - 180 ms HEX / VIC channel (green) - 38 ms Cy5 channel (Red) for reading with the Naica system from STILLA Technologies.

[0127] Validation of the digital PCR test: The digital PCR test is then validated on 100% methylated synthetic DNA and on commercial unmethylated genomic DNA. 3 synthetic DNA samples (100% methylated, Universal Methylated DNA Standard, Zymo Research Reference: D5011) and 3 commercial genomic DNA samples (unmethylated, Promega G1521) were analyzed as follows: Step 1: Bisulfite conversion by the Zymo Research EZ DNA Methylation-Lightning Kit (Reference D5031) then Step 2: System Naica digital PCR analysis of the neurological markers NBR1 and APC2.

[0197]

[0128] The results on the 100% methylated DNA controls show that the epigenetic markers targeted on the NBR1 and APC2 genes are well detected and quantifiable by the test according to the invention. The results on the unmethylated DNA controls show that the developed NBR1 and APC2 markers are well detectable if and only if the targeted regions are hypermethylated. There is no detection of the markers in unmethylated DNA controls.

[0198]

[0129] The specificity of the test was verified by a control in the presence of DNA from peripheral blood mononuclear cells, called “PBMC” (Peripheral Blood Monocyte Cells) from healthy subjects.

[0199]

[0130] 10 genomic DNA samples were extracted from white blood cell pellets of healthy subjects using the Qiagen QIAamp DNA Blood Mini Kit (50) Cat. No. / ID: 51104. The samples then underwent a bisulfite conversion step using the Zymo Research EZ DNA Methylation-Lightning Kit (Reference D5031) followed by System Naica digital PCR analysis of the neurological markers NBR1 and APC2 according to the invention described.

[0200]

[0131] The negative test results for the tested epigenetic markers show that the test is specific because it does not present biological noise at the level of genomic DNA from white blood cells. By "biological noise" we mean a background noise of epigenetic markers on samples considered negative or unmethylated. In other words, the detection of the neurological markers NBR1 and APC2 generates a negligible signal on samples considered non-negative or unmethylated.

[0201]

[0132] The specificity of the test was also verified in the presence of plasma DNA from healthy subjects. 10 plasma DNA samples were extracted from 1 mL of plasma from healthy subjects using the Qiagen QIAamp Circulating Nucleic Acid Kit (50) Cat. No. I ID: 55114. The samples then underwent a bisulfite conversion step using the Zymo Research EZ DNA Methylation-Lightning Kit (Reference D5031) followed by a System Naica digital PCR analysis of the neurological markers NBR1 and APC2 according to the invention described. The negative results of the test for the epigenetic markers tested show that it does not present any biological noise at the level of cell-free / plasma DNA from the plasma of healthy subjects.

[0202]

[0133] Finally, the specificity of the test was determined in the presence of genomic DNA from other tissues. N = 27 samples of healthy tissues were collected (MTA Biological Sample Assignment No. CT 69HCL22_0234 from the anatomopathology department of the Hospices Civiles de Lyon Sud et Est). Per sample, 3 chips of 8 μm thickness of tissues prepared in paraffin were assigned. The genomic DNAs of the tissue chips of cerebral origin were extracted via the Qiagen QIAamp DNA FFPE Advanced Kit (50) (Cat. No. / ID: 56604). The DNA samples then underwent a bisulfite conversion step using the Zymo Research EZ DNA Methylation-Lightning Kit (Reference D5031) followed by a System Naica digital PCR analysis of the neurological markers NBR1 and APC2 according to the invention described. The results show very low biological noise in genomic DNA tissues other than tissues of brain origin.If both biomarkers APC2 and NBR1 are taken into account, the biological noise is less than 2%. The test results for the epigenetic markers tested show that this presents negligible or even absent biological noise at the level of the genomic DNA from the different tissue sections (other than the brain).

[0203]

[0134] A positive control is performed on genomic DNA samples from tissue sections of healthy brain tissue, by comparison with the anti-NeuN histological labeling.

[0204]

[0135] 12 samples of healthy brain tissue were collected (MTA biological sample transfer no. VAL 2022 / 2022-234 / 01 from the anatomopathology department of the Lariboisière hospital of Pr Homa Adie Biassette). 1 sample = White slide marked with the anti-neuN antibody (neuronal antibody, Ref ABN91, sigma Aldricht-Merck) and 3 chips of 8 μm thickness of tissue prepared in paraffin.

[0205]

[0136] Neuronal cells labeled with the anti-NeuN antibody were counted by the precision software "HALO® image analysis". Genomic DNA from brain tissue chips was extracted using the "Qiagen QIAamp DNA FFPE Advanced Kit" (50) (Cat. No. / ID: 56604). The DNA samples then underwent a bisulfite conversion step using the Zymo Research EZ DNA Methylation-Lightning Kit (Reference D5031) followed by a System Naica digital PCR analysis of the neurological markers NBR1 and APC2 according to the invention described.

[0137] The neuronal markers APC2 and NBR1 are identified in 12 samples out of 12 samples tested. An identical proportion of neurons in healthy brain tissue sections is observed between the histological marker anti-NeuN and the epigenetic markers APC2 and NBR1.A correlation between the epigenetic markers APC2 / NBR1 and the histological marker anti-NeuN was noted (Pearson's R coefficient = 0.44 and 0.46 respectively for the epigenetic markers APC2 and NBR1 compared to the anti-NeuN marker). The results are shown in Table 11 below and in Figure 1.

[0206] Table 11

[0207]

[0138] The data of the correlation between the quantitative dosage of brain markers (neurons) and the histological labeling Anti-NeuN (neuronal cell marker) show a correlation between the molecular and histological labeling (Pearson coefficient R = 0.46 and 0.44 respectively for NBR1 and APC2) confirming the neuronal specificity (brain cells) of the test.

[0208]

[0139] In a clinical application, a test according to the invention makes it possible to stratify patients with stroke according to its severity index. The results show an elevation in the quantitative average of the markers APC2 and NBR1 in patients with acute ischemic stroke (AIS) compared to patients with transient ischemic stroke (TIA) (respective average of 11.32 copies / ml of plasma for TIA versus 75 copies / ml of plasma for AIA). The results are presented in Figure 2.

[0209] EXAMPLE 3: Validation of a first group of biomarkers of renal degradation in the management of kidney transplant patients

[0140] The validation of the PDE4D and PAX2 biomarkers includes the molecular analysis of the conversion of DNA to digital PCR tests in droplets, the design of the primers and Taqman-mgb probes (Reference: PB-MGBEF-006, PB-MGBEC-006 and PB-MGBEH-006, Eurogentec) are identical to that described in Example 2 (see: Common part analysis methodology). The implementation of the PCR amplification reaction includes the preparation of the specific primer-probe cocktail is presented in the following Table 12.

[0210] Table 12

[0211]

[0141] The preparation of the PCR reaction mix is ​​presented in the following Table 13. Table 13

[0212]

[0142] The PCR reaction is carried out as indicated in Table 10 (Example 2).

[0213] The plates are read as indicated in Example 2.

[0143] The digital PCR test is then validated on 100% methylated synthetic DNA and on commercial unmethylated genomic DNA. 3 synthetic DNA samples (100% methylated, Universal Methylated DNA Standard, Zymo Research Reference: D5011) and 3 commercial genomic DNA samples (unmethylated, Promega G1521) were analyzed as follows: Step 1: Bisulfite conversion by the Zymo Research EZ DNA Methylation-Lightning Kit (Reference D5031) then Step 2: System Naica digital PCR analysis of the renal markers PDE4D and Pax2. The results on synthetic DNA show that: 1 / On 100% methylated DNA controls: the epigenetic markers targeted on the PDE4D and Pax2 genes are well detected and quantifiable by our test. 2 / On unmethylated DNA controls: The developed PDE4D and Pax2 markers are well detectable if and only if the targeted regions are hypermethylated.There is no detection of markers in unmethylated DNA controls.

[0214]

[0144] The results show that PDE4D and PAX2 targets are not detected in commercial unmethylated DNA.

[0215]

[0145] The inventors determine typical metrological parameters, including the repeatability and sensitivity of the test. The test is repeatable with a coefficient of variation of 4.5% and 0.6% respectively for the Pax2 and PDE4D markers. The test is sensitive to 0.01% and allows quantification up to a limit of 0.15 ng and 0.3 ng of DNA for the PDE4D and Pax2 markers respectively.

[0216]

[0146] The specificity of the test was verified in the presence of DNA from PBMC (white blood cells) of healthy subjects. The markers are not detected during the analysis in the DNA of PBMCs.

[0217]

[0147] 10 genomic DNA samples were extracted from white blood cell pellets of healthy subjects using the Qiagen QIAamp DNA Blood Mini Kit (50) Cat. No. / ID: 51104. The samples then underwent a bisulfite conversion step using the Zymo Research EZ DNA Methylation-Lightning Kit (Reference D5031) followed by a System Naica digital PCR analysis of the renal markers PDE4D and Pax2 according to the invention described. The markers are not detected during the analysis of genomic DNAs from white blood cell pellets of healthy subjects (= absence of biological background noise).

[0218]

[0148] The specificity of the test was also verified in the presence of plasma DNA from healthy subjects. 10 plasma DNA samples were extracted from 1.5 mL of plasma from healthy subjects using the Qiagen QIAamp Circulating Nucleic Acid Kit (50) Cat. No. I ID: 55114. The samples then underwent a bisulfite conversion step using the Zymo Research EZ DNA Methylation-Lightning Kit (Reference D5031) followed by a System Naica digital PCR analysis of the renal markers PDE4D and Pax2 according to the invention described. The markers are not detected when analyzed in plasma DNA (cellular DNA) from healthy subjects.

[0219]

[0149] Finally, the specificity of the test was determined in the presence of genomic DNA from other tissues. N = 27 samples of healthy tissues were collected (MTA Biological Sample Assignment No. CT 69HCL22_0234 from the anatomopathology department of the Hospices Civiles de Lyon Sud et Est and the Lariboisière Hospital). Per sample, 3 chips of 8 μm thickness of tissue prepared in paraffin were assigned. The genomic DNA from the tissue chips was extracted via the Qiagen QIAamp DNA FFPE Advanced Kit (50) (Cat. No. / ID: 56604). The samples then underwent a bisulfite conversion step using the Zymo Research EZ DNA Methylation-Lightning Kit (Reference D5031) followed by a System Naica digital PCR analysis of the renal markers PDE4D and Pax2 according to the invention described.The negative test results for the epigenetic markers PDE4D and Pax2 show that these were not detected in genomic DNA from tissue sections other than the kidney, confirming the absence of biological noise.

[0220]

[0150] A positive control is performed on genomic DNA samples from tissue sections of healthy renal tissue. 12 samples of healthy renal tissue were collected from the anatomopathology department of the Hospices Civils de Lyon Sud et Est. The genomic DNA from the tissue chips was extracted using the Qiagen QIAamp DNA FFPE Advanced Kit (50) (Cat. No. / ID: 56604). The samples then underwent a bisulfite conversion step using the Zymo Research EZ DNA Methylation-Lightning Kit (Reference D5031) followed by System Naica digital PCR analysis of the renal markers PDE4D and Pax2 according to the invention described.

[0221]

[0151] The renal markers PDE4D and Pax2 are identified in 12 samples out of 12 tested samples. Thus, this work confirms the renal specificity of the described PDE4D and Pax2 digital PCR test.

[0222]

[0152] The amount of circulating DNA of renal origin is higher in the plasma of patients at high risk of graft rejection versus the plasma of patients at low risk of graft rejection during the first days post-kidney transplant (Figure 3).

[0223]

[0153] The fraction of circulating DNA of renal origin is higher in the plasma of patients with a high risk of graft rejection versus the plasma of patients with a low risk of graft rejection during the first days after kidney transplantation (Figure 4).

[0154] The quantitative dosage of PD4ED and Pax2 markers in urine by the described test is higher in patients with biopsy-confirmed graft rejection than in patients without graft rejection. This dosage allows stratification of patients with biopsy-confirmed graft rejection and patients without graft rejection (Figure 5).

[0224] EXAMPLE 4: Validation of the SYNE1 biomarker of pulmonary deterioration in the management of acute respiratory failure

[0225]

[0155] The molecular analysis methodology of DNA conversion to digital droplet PCR tests, the design of the primers and Taqman-mgb probes (Reference: PB-MGBEF-006, PB-MGBEC-006 and PB-MGBEH-006, Eurogentec) are identical to that described in Example 2. The implementation of the PCR amplification reaction is carried out as follows. The preparation of the specific primer-probe cocktail is presented in the following Table 14.

[0226] Table 14

[0227]

[0156] The preparation of the PCR reaction mix is ​​presented in the following Table 15.

[0228] Table 15

[0229]

[0157] The PCR reaction is carried out as indicated in Table 10 (Example 2). The plates are read as indicated in Example 2.

[0230]

[0158] The digital PCR test is then validated on 100% methylated synthetic DNA and on commercial unmethylated genomic DNA. 3 synthetic DNA samples (100% methylated, Universal Methylated DNA Standard, Zymo Research Reference: D5011) and 3 commercial genomic DNA samples (unmethylated, Promega G1521) were analyzed as follows: Step 1: Bisulfite conversion by the Zymo Research EZ DNA Methylation-Lightning Kit (Reference D5031) then Step 2: System Naica digital PCR analysis of the lung marker SYNE1.

[0231]

[0159] The results on the 100% methylated DNA controls show that the epigenetic marker targeted on the SYNE1 gene is well detected and quantifiable by a test according to the invention using this marker. The results on the unmethylated DNA controls show that the developed SYNE1 marker is well detectable if and only if the targeted regions are hypermethylated. There is no detection of the marker in the unmethylated DNA controls.

[0232]

[0160] The specificity of the test was verified in the presence of genomic DNA from PBMC (white blood cells) of healthy subjects. 10 samples of genomic DNA were extracted from pelleted white blood cells of healthy subjects using the Qiagen QIAamp DNA Blood Mini Kit (50) Cat. No. / ID: 51104. The samples then underwent a bisulfite conversion step using the Zymo Research EZ DNA Methylation-Lightning Kit (Reference D5031) and then a System Naica digital PCR analysis of the lung marker SYNE1 according to the invention described. The marker is not detected when analyzing genomic DNA from PBMCs

[0233]

[0161] The specificity of the test was also verified in the presence of plasma DNA from healthy subjects. 10 plasma DNA samples were extracted from 1.5 mL of plasma from healthy subjects using the Qiagen QIAamp Circulating Nucleic Acid Kit (50) Cat. No. I ID: 55104. The samples then underwent a bisulfite conversion step using the Zymo Research EZ DNA Methylation-Lightning Kit (Reference D5031) followed by a System Naica digital PCR analysis of the lung marker SYNE1 according to the invention described. The markers are not detected during the analysis in the genomic DNA of healthy subjects.

[0234]

[0162] Finally, the specificity of the test was determined in the presence of genomic DNA from other tissues. N = 27 samples of healthy tissues were collected (MTA Biological Sample Assignment No. CT 69HCL22_0234 from the anatomopathology department of the Hospices Civiles de Lyon Sud et Est and the Lariboisière Hospital). Per sample, 3 chips of 8 μm thickness of tissues prepared in paraffin were assigned. The genomic DNAs of the tissue chips of cerebral origin were extracted via the Qiagen QIAamp DNA FFPE Advanced Kit (50) (Cat. No. / ID: 56604). The samples then underwent a bisulfite conversion step using the Zymo Research EZ DNA Methylation-Lightning Kit (Reference D5031) followed by a System Naica digital PCR analysis of the lung marker SYNE1 according to the invention described.The negative results of the test for the epigenetic marker SYNE1 show that it was not detected in genomic DNA from tissue sections other than the lung, confirming the absence of biological noise.

[0235]

[0163] Positive control: A positive control is performed on genomic DNA samples from tissue sections of healthy lung tissue, by comparison with histological labeling. 18 samples of healthy lung tissue (1 blank slide and 3 chips of 8 μm thickness prepared in paraffin) were collected from the anatomopathology department of the Hospices Civils de Lyon Sud et Est. For each sample, labeling with an anti-TTF1 antibody on the blank slides was performed (TTF1 antibody, Reference: DB089-0.5 Rabbit Monoclonal Anti-TTF-1 Clone (G21-G), DB Biotech) and each TTF1-labeled cell (= pulmonary pneumocytes) was counted using the “HALO® image analysis” software. The samples were labeled with the TTF1 antibody. In parallel, genomic DNA from 8 μm thick tissue chips prepared in paraffin were extracted using the Qiagen QIAamp DNA FFPE Advanced Kit (50) (Cat. No. / ID: 56604).The samples then underwent a bisulfite conversion step using the Zymo Research EZ DNA Methylation-Lightning Kit (Reference D5031) followed by a System Naica digital PCR analysis of the lung marker SYNE1 according to the invention described.

[0236]

[0164] The results show that the hypermethylated target SYNE1 is detected and quantified only in genomic DNA from healthy lung sections confirming the pulmonary specificity. The correlation between the histological TTF1 labeling (=pulmonary pneumocytes) and the described pulmonary test is 0.6 (Pearson R-coefficient) demonstrating the pulmonary and pneumocyte specificity of the analysis (Figure 6).

[0165] In a clinical application, a test according to the invention makes it possible to stratify patients suffering from acute respiratory distress syndrome (ARDS) according to a severity index designated as mild, severe or critical (Berlin classification). The results show that the hypermethylated target SYNE1 makes it possible to discriminate each patient subgroup (moderate, severe, critical ARDS). A significant difference was observed between these 3 groups (p-value = 0.0073, Jonckheere-Terpstra test). (Figure 7)

[0237]

[0166] Furthermore, the results of the test according to the invention make it possible to monitor the pulmonary state of a patient suffering from ARDS with correlation indicators between the quantitative result of the hypermethylated target SYNE1 and the ventilatory markers 02 max / FiO2 / PAO2 (respectively Pearson correlation coefficient of 0.44, 0.29 and 0.29).

[0238] EXAMPLE 5: Validation of a second group of biomarkers of renal degradation in the management of kidney transplant patients

[0239]

[0167] Two types of diagnostic tests targeting renal graft rejection and the typology of renal lesions (total, vascular and epithelial) were developed by selecting from the markers identified by the inventors.

[0240]

[0168] According to prototype 1, the markers SEPT5 (glomerular endothelial marker); PDE4D / PAX2 / ACSL5 (tubular epithelial marker); CTDP1 (total kidney marker) are tested on urine samples.

[0241]

[0169] According to prototype 2, the markers GATA2 (renal endothelial marker); TNS2-AS1 (renal capillary endothelial marker) / PDE4D / PAX2 (tubular epithelial marker); CTDP1 (total kidney marker) are tested on plasma samples.

[0242]

[0170] For both prototypes, the “Albumin” marker is used as a ubiquitous marker quantifying all genomes present in the sample. The biological samples were obtained through hospital partners from the Necker nephrology departments and the Tenon-Pitié-Salpêtrière hospital.

[0243]

[0171] The concentrations of the different biomarkers associated in multiplex (here on a PCR mix) making up the kit dedicated to monitoring the state of renal tissue suffering in the context of renal transplantation at the level of the total organ, its epithelial fraction and its vascular fraction are used according to the concentrations given in table 16 below.

[0244] Table 16

[0245]

[0172] After validation of these 2 prototypes at the metrological level (precision, repeatability, reproducibility), the 2 prototypes were tested on different biological controls called "positive" (genomic DNA from kidney) and controls called "negative" (genomic DNA from liver, white blood cells and circulating plasma and urinary DNA from healthy subjects) (Figure 9). Genomic DNA from the liver as well as white blood cells are controls evaluated in the design of this test and this methodology because they are contributors to circulating urinary and plasma DNA in healthy subjects.

[0173] These analyses confirmed the absence of detection or very low-noise detection of our biomarkers in negative control biological samples: N = 20 genomic DNA samples extracted from cell pellets of healthy subjects, N = 30 plasma DNA samples of healthy subjects, N = 15 urinary DNA samples of healthy subjects, N = 5 genomic DNA samples extracted from tissue sections (5 liver gDNAs).

[0174] These gDNAs were mentioned as potential low-noise contributors to circulating DNA in healthy subjects: respectively urinary cf-DNA and plasma cf-DNA 5.

[0246] Results :

[0247]

[0175] The above-mentioned markers are detected in 100% of genomic DNA extracted from renal tissue sections (N = 22).

[0248]

[0176] The markers proposed in this prototype in vitro diagnostic test allow the detection of renal DNA, specifically with very low biological noise in the genomic DNA of the liver and white blood cells (main contributors of circulating DNA) (Figure 8). In the plasma circulating DNA of healthy subjects, there is a very low biological background noise (a little higher for the endothelial marker GATA2 (consistent with the literature)). Concerning the urinary circulating DNA of healthy subjects, renal and pan-kidney epithelial markers are found, probably corresponding to the renal fraction physiologically present in the urine, in agreement with the literature.

[0249]

[0177] The "total kidney" marker CTDP1 allows quantification of the endothelial and epithelial fractions of the kidney. It is found in higher quantities in kidney genomic DNA compared to markers targeting only the epithelial and endothelial fraction. Similarly, markers targeting the endothelial fraction of the kidney (GATA2) are found in greater quantities than the markers TNS2-AS1 and SEPT5, which respectively target smaller vascular fractions (restricted to the glomerulus and tubule respectively).

[0250] Example 6: Validation of the specificity of renal epithelial markers: Comparison of molecular markers (PAX2 / ACSL5) and the histological marker of renal epithelium CD10

[0251]

[0178] The inventors have shown that the markers PAX2 and ACSL5 specifically target the renal epithelium. These markers were therefore tested to quantify the renal epithelium. In total kidney gDNA (n=22) and in urinary plasma circulating DNA, these markers showed a good correlation of respectively R 2 = 0.5 and R 2 = 0.8, as shown in Figure 10 and Figure 11.

[0252]

[0179] The publication by Erger et al. showed that a fraction varying from 20 to 50% of the total urinary circulating DNA in healthy subjects has a renal origin (Erger et al. Genome Medicine, (2020), doi.org / 10.1186 / sl3073-020-00750-5) by proposing a bioinformatics deconvolution model (cfNOMe) of urinary circulating DNA by NGS sequencing. This proportion is confirmed by the present results, suggesting good specificity of the markers in terms of detection of circulating DNA of renal origin. Furthermore, urinary circulating DNA of renal origin is rather of epithelial origin (absence of signal detection of vascular markers e.g.: GATA2, TNS2-AS1 and SEPT-5 and detection of the markers PAX2, ACSL5 and PDE4D (R 2 correlation (PAX2 / ACSL5) = 0.80 for the n = 15 urinary circulating DNA samples extracted from healthy subjects).

[0253]

[0180] The results show that PAX2 & ACSL5 markers specifically target the renal epithelium. In order to reinforce the results obtained, comparisons between the molecular data of PAX2 and ACSL5 markers and histological data (labeling of renal epithelial cells on white kidney slides by the anti-CD10 antibody) were performed. For the n = 22 genomic DNA from healthy kidneys for which PAX2 and ACSL5 were quantified, immunohistochemical labeling of renal epithelial cells via the anti-CD10 antibody on a paired white slide was performed. CD10 is a surface marker specifically targeting epithelial cells within a kidney section.Histological and molecular data show that the proportion of epithelial cells on these slides and paired kidney gDNAs varies between 11 and 40% and that there is no significant difference between molecular markers and histological markers targeting the renal epithelium (Figures 12A and 12B).

[0254] Example 7: Analysis of urine samples from kidney transplant patients

[0255]

[0181] In order to validate the clinical relevance of the first prototype, a clinical collaboration in partnership with Necker and Tenon-Pitié Salpêtrière Hospital - Nephrology / Transplantation Department made it possible to analyze respectively N = 50 urine samples from kidney transplant patients with / without graft rejection, confirmed by anatomopathological analysis of the solid biopsy, and n = 55 plasma samples from kidney transplant patients with biopsies with and without cellular lesions, confirmed by anatomopathological analysis. These biomarkers aim to specifically detect the cellular origin of renal lysis, and thus the type of rejection in accordance with the classification of renal transplant rejection according to the Banff classification.

[0256]

[0182] 50 biological samples (2 ml of urine samples) from kidney transplant patients, with or without renal graft rejection (rejection confirmed by solid biopsy) were analyzed. Among these patients, 20 presented cellular type graft rejection (TCMR) and 30 did not present graft rejection.

[0257]

[0183] 2 mL of urine was collected on the day of renal biopsy. Circulating DNA was extracted and all biomarkers (SEPT-5, ACSL5, PAX2, PDE4D, CTDP1) were analyzed simultaneously by multiplex digital PCR (6 colors, one for each marker and the internal control albumin).

[0258]

[0184] A significant increase in the amount of biomarkers was observed in the urine of patients with cellular rejection (Wilcoxon test, p-value < 0.001). A regression model applied to the biomarkers showed a good sensitivity and specificity of the diagnostic test of 0.83 and 0.85 respectively, with an AUC score of 0.88.

[0259]

[0185] Figure 13 shows the significant difference in CTDP1 biomarker quantification in urine between patients with kidney transplant rejection and those without kidney transplant rejection (left graph). Logistic regression model compiling the quantitative data of CTDP1, PAX2, PDE4D, ACSL5 and SEPT5 biomarkers to predict the presence or absence of kidney transplant rejection (right graph).

[0260] Example 8: Analysis on plasma samples from kidney transplant patients

[0261]

[0186] 55 biological samples from kidney transplant patients, with or without renal graft rejection (rejection confirmed by solid biopsy with biopsies showing glomerular (g), tubular (t), vascular (v) and capillary (ptc) graft lesions) were analyzed.

[0262]

[0187] 0.6 to 1.5 mL of plasma was collected on the day of renal biopsy. Circulating DNA was extracted and all biomarkers (GATA2, TNS2-AS1, PAX2, PDE4D, CTDP1) were analyzed simultaneously by multiplex digital PCR (6 colors, one for each marker + Internal Control Albumin).

[0263]

[0188] A significant increase in the amount of biomarkers was also observed in the plasmas of transplanted patients with renal graft degradation. A regression model applied to the biomarkers showed a good sensitivity and specificity of the diagnostic test of 0.96 and 0.75, respectively, with an AUC score of 0.82. These results highlight the reliability of the test in the analysis of graft rejection in plasma samples. A significant increase in the amount of vascular biomarkers (specifically GATA2 and TNS2-AS1 markers) was also observed in the plasmas of patients with renal graft vascular lesions as expected (Figure 14).

[0264]

[0189] A regression model applied to the “total kidney” and “vascular” biomarkers (specifically the CTDP1, GATA2 and TNS2-ASl markers) showed good sensitivity and specificity of the diagnostic test, with an AUC of 0.878 (Figure 15).

[0190] Together, these results demonstrate:

[0265] -the technical reliability of the test developed in the detection of biomarkers of interest in the urine and plasma of patients,

[0266] - the sensitivity and technical specificity of the developed test, - the clinical interest of the developed test in the detection of kidney transplant rejection,

[0267] - the clinical interest of the test developed in the characterization of epithelial and / or vascular lesions of the renal graft.

Claims

CLAIMS

1. Kit for the detection in a biological sample of a target nucleotide sequence of methylated cell-free DNA, said nucleotide sequence being specific to a cell type of an organ chosen from: the brain, the lung and the kidney, said kit comprising: i) for the detection of a target sequence of cell-free DNA specific to a cell type present in the brain, at least: a sense primer comprising a nucleotide sequence SEQ ID No. 1, or a nucleotide sequence having at least 80% identity with SEQ ID No. 1 and an antisense primer, comprising a nucleotide sequence SEQ ID No. 2, or a nucleotide sequence having at least 80% identity with SEQ ID No. 2, and / or - a sense primer comprising a nucleotide sequence SEQ ID No. 4, or a nucleotide sequence having at least 80% identity with SEQ ID No. 4 and an antisense primer, comprising a nucleotide sequence SEQ ID No. 5, or a nucleotide sequence having at least 80% identity with SEQ ID No.

5. ii) for the detection of a cell-free DNA target sequence specific to a cell type present in the lung, at least: a sense primer, comprising a nucleotide sequence SEQ ID No. 7, or a nucleotide sequence having at least 80% identity with SEQ ID No. 7 and an antisense primer, comprising a nucleotide sequence SEQ ID No. 8, or a nucleotide sequence having at least 80% identity with SEQ ID No. 8, and iii) for the detection of a cell-free DNA target sequence specific to a cell type present in the kidney, at least: - a sense primer comprising a sequence SEQ ID No. 10, or a sequence having at least 80% identity with SEQ ID No. 10 and an antisense primer, comprising a sequence SEQ ID No. 11, or a sequence having at least 80% identity with SEQ ID No. 11, and / or - a sense primer comprising a sequence SEQ ID No. 13, or a sequence having at least 80% identity with SEQ ID No. 13 and an antisense primer, comprising a sequence SEQ ID No. 14, or a sequence having at least 80% identity with SEQ ID No. 14, and / or - a sense primer comprising a sequence SEQ ID No. 19, or a sequence having at least 80% identity with SEQ ID No. 19 and an antisense primer, comprising a sequence SEQ ID No. 20, or a sequence having at least 80% identity with SEQ ID No. 20, and / or - a sense primer comprising a sequence SEQ ID No. 22, or a sequence having at least 80% identity with SEQ ID No. 22 and an antisense primer, comprising a sequence SEQ ID No. 23, or a sequence having at least 80% identity with SEQ ID No. 23, and / or - a sense primer comprising a sequence SEQ ID No. 25, or a sequence having at least 80% identity with SEQ ID No. 25 and an antisense primer, comprising a sequence SEQ ID No. 26, or a sequence having at least 80% identity with SEQ ID No. 26, and / or - a sense primer comprising a sequence SEQ ID No. 28, or a sequence having at least 80% identity with SEQ ID No. 28 and an antisense primer, comprising a sequence SEQ ID No. 29, or a sequence having at least 80% identity with SEQ ID No. 29, and / or - a sense primer comprising a sequence SEQ ID No. 31, or a sequence having at least 80% identity with SEQ ID No. 31 and an antisense primer, comprising a sequence SEQ ID No. 32, or a sequence having at least 80% identity with SEQ ID No. 32, and / or - a sense primer comprising a sequence SEQ ID No. 34, or a sequence having at least 80% identity with SEQ ID No. 34 and an antisense primer, comprising a sequence SEQ ID No. 35, or a sequence having at least 80% identity with SEQ ID No. 35, and / or - a sense primer comprising a sequence SEQ ID No. 37, or a sequence having at least 80% identity with SEQ ID No. 37 and an antisense primer, comprising a sequence SEQ ID No. 38, or a sequence having at least 80% identity with SEQ ID No. 38, and / or - a sense primer comprising a sequence SEQ ID No. 40, or a sequence having at least 80% identity with SEQ ID No. 40 and an antisense primer, comprising a sequence SEQ ID No. 41, or a sequence having at least 80% identity with SEQ ID No. 41, and / or - a sense primer comprising a sequence SEQ ID No. 43, or a sequence having at least 80% identity with SEQ ID No. 43 and an antisense primer, comprising a sequence SEQ ID No. 44, or a sequence having at least 80% identity with SEQ ID No. 44, and / or - a sense primer comprising a sequence SEQ ID No. 46, or a sequence having at least 80% identity with SEQ ID No. 46 and an antisense primer, comprising a sequence SEQ ID No. 47, or a sequence having at least 80% identity with SEQ ID No. 47, and / or - a sense primer comprising a sequence SEQ ID No. 49, or a sequence having at least 80% identity with SEQ ID No. 49 and an antisense primer, comprising a sequence SEQ ID No. 50, or a sequence having at least 80% identity with SEQ ID No. 50, and / or - a sense primer comprising a sequence SEQ ID No. 52, or a sequence having at least 80% identity with SEQ ID No. 52 and an antisense primer, comprising a sequence SEQ ID No. 53, or a sequence having at least 80% identity with SEQ ID No.

53.

2. Kit according to claim 1, further comprising a probe comprising: i) for the detection of a cell-free DNA target sequence specific for a cell type present in the brain, at least one nucleotide sequence selected from: SEQ ID No. 3, SEQ ID No. 6 and a nucleotide sequence having at least 80% identity with SEQ ID No. 3 or SEQ ID No. 6, ii) for the detection of a cell-free DNA target sequence specific for a cell type present in the lung, at least one nucleotide sequence selected from: SEQ ID No. 9 and a nucleotide sequence having at least 80% identity with SEQ ID No. 9, and iii) for the detection of a cell-free DNA target sequence specific for a cell type present in the kidney, at least one nucleotide sequence selected from: SEQ ID No. 12, SEQ ID No. 15, SEQ ID No. 21, SEQ ID No. 24, SEQ ID No. 27, SEQ ID No. 30, SEQ ID No. 33, SEQ ID No. 36, SEQ ID No. 39, SEQ ID No. 42, SEQ ID No. 45, SEQ ID No. 48, SEQ ID No. 51, SEQ ID No. 54,and a nucleotide sequence having at least 80% identity with SEQ ID No. 12, SEQ ID No. 15, SEQ ID No. 21, SEQ ID No. 24, SEQ ID No. 27, SEQ ID No. 30, SEQ ID No. 33, SEQ ID No. 36, SEQ ID No. 39, SEQ ID No. 42, SEQ ID No. 45, SEQ ID No. 48, SEQ ID No. 51 or SEQ ID No. 54.,

3. A kit according to any one of claims 1 or 2 for the detection in a biological sample of a target sequence of cell-free DNA specific to a cell type present in the brain. Claim 4] A kit according to any one of claims 1 or 2 for the detection in a biological sample of a target sequence of cell-free DNA specific to a cell type present in the lung.

5. Kit according to any one of claims 1 or 2 for the detection in a biological sample of a target sequence of cell-free DNA specific to a type of cells present in the kidney.

6. Method for identifying a cell-free DNA target sequence specific to a cell type present in a first organ chosen from: the lung, the brain and the kidney, the method comprising at least the following steps: a) Determination, in at least one genomic DNA methylation database, of the position and degree of methylation of the CpG islands of sequences specific to said cell type of said organ, b) Identification, in the genomic DNA of said cell type of said healthy organ, of specifically hypermethylated CpG islands, c) Comparison of the degree of methylation of the hypermethylated CpG islands of said cell type of said first organ with the degree of methylation of the same CpG islands of a second cell type, different from the first cell type, d) Selection of the CpG islands only hypermethylated in the genomic DNA of said cell type of said first organ after comparison carried out in step c), e) Comparison of the degree of methylation of the hypermethylated CpG islands of said cell type of said first organ with the degree of methylation of the same CpG islands of the genomic DNA of organs other than said first organ, f) Comparison of the degree of methylation of the hypermethylated CpG islands of said cell type of said first organ with the degree of methylation of the same CpG islands of the genomic DNA of white blood cells healthy,g) Comparison of the degree of methylation of the hypermethylated CpG islands of said cell type of said first organ with the degree of methylation of the same CpG islands of the cell-free DNA of all biological matrices combined from healthy subjects, h) Selection of the CpG islands only hypermethylated in the genomic DNA of said cell type of said first organ after comparison carried out in steps e, f and g) i) Identification of one or more hypermethylated target sequences of cell-free DNA specific to said cell type of the first organ from the comparison carried out during step h).,

7. Method for detecting in a biological sample a target sequence of cell-free DNA, said sequence being specific to a type of cells present in an organ chosen from: the lung, the brain and the kidney, said method comprising at least the following steps: a) Bringing together, under conditions appropriate for amplification of the nucleic acids, cell-free DNA previously extracted from a biological sample and a pair of primers consisting of a sense primer and an antisense primer, each of the primers comprising: - a sense primer and an antisense primer capable of hybridizing with a nucleotide sequence chosen from: SEQ ID No. 55, SEQ ID No. 56, SEQ ID No. 57, SEQ ID No. 58, SEQ ID No. 59, SEQ ID No. 60, SEQ ID No. 61, SEQ ID No. 62, SEQ ID No. 63, SEQ ID No. 64, SEQ ID No. 65, SEQ ID No. 66, SEQ ID No. 67, SEQ ID No. 68, SEQ ID No. 69, SEQ ID No. 70 and SEQ ID No. 71, b) Amplification reaction of said target sequence, c) Detection of the presence of the sequence amplified during step b).

8. Method for detecting in a biological sample a target sequence of cell-free DNA according to claim 7, said sequence being specific to a type of cells present in an organ chosen from: the lung, the brain and the kidney, said method comprising at least the following steps: a) Bringing together, under conditions suitable for amplification of the nucleic acids, cell-free DNA previously extracted from a biological sample and a pair of primers consisting of a sense primer and an antisense primer, each of the primers comprising: i) for the detection of a target sequence specific to a type of cells present in the brain, at least: a sense primer comprising a sequence SEQ ID No. 1, or a sequence having at least 80% identity with SEQ ID No. 1 and an antisense primer, comprising a sequence SEQ ID No. 2, or a sequence having at least 80% identity with SEQ ID No. 2, and / or - a sense primer comprising a sequence SEQ ID No. 4, or a sequence having at least 80% identity with SEQ ID No. 4 and an antisense primer comprising a sequence SEQ ID No. 5, or a sequence having at least 80% identity with SEQ ID No. 5., ii) for the detection of a target sequence specific to a cell type present in the lung, at least: at least one sense primer comprising a sequence SEQ ID No. 7, or a sequence having at least 80% identity with SEQ ID No. 7 and an antisense primer comprising a sequence SEQ ID No. 8, or a sequence having at least 80% identity with SEQ ID No. 8, and iii) for the detection of a target sequence specific to a cell type present in the kidney, at least: - a sense primer comprising a sequence SEQ ID No. 10, or a sequence having at least 80% identity with SEQ ID No. 10 and an antisense primer comprising a sequence SEQ ID No. 11, or a sequence having at least 80% identity with SEQ ID No. 11 and an antisense primer comprising a sequence SEQ ID No. 12, or a sequence having at least 80% identity with SEQ ID No. 12 and an antisense primer comprising a sequence SEQ ID No. 13, or a sequence having at least 80% identity with SEQ ID No. 13 and an antisense primer comprising a sequence SEQ ID No. 14, or a sequence having at least 80% identity with SEQ ID No. 14 and an antisense primer comprising a sequence SEQ ID No. 15, or a sequence having at least 80% identity with SEQ ID No. 15 and an antisense primer comprising a sequence SEQ ID No. 16, or a sequence having at least 80% identity with SEQ ID No. 16 and an antisense primer comprising a sequence SEQ ID No. 17, or a sequence having at least 80% identity with SEQ ID No. 17 and an antisense prime antisense, comprising a sequence SEQ ID No. 11, or a sequence having at least 80% identity with SEQ ID No. 11, and / or - a sense primer comprising a sequence SEQ ID No. 13, or a sequence having at least 80% identity with SEQ ID No. 13 and an antisense primer, comprising a sequence SEQ ID No. 14, or a sequence having at least 80% identity with SEQ ID No. 14, and / or - a sense primer comprising a sequence SEQ ID No. 19, or a sequence having at least 80% identity with SEQ ID No. 19 and an antisense primer, comprising a sequence SEQ ID No. 20, or a sequence having at least 80% identity with SEQ ID No. 20, and / or - a sense primer comprising a sequence SEQ ID No. 22, or a sequence having at least 80% identity with SEQ ID No. 22 and an antisense primer, comprising a sequence SEQ ID No. 23, or a sequence having at least 80% identity with SEQ ID No. 23, and / or - a sense primer comprising a sequence SEQ ID No. 25, or a sequence having at least 80% identity with SEQ ID No. 25 and an antisense primer, comprising a sequence SEQ ID No. 26, or a sequence having at least 80% identity with SEQ ID No. 26, and / or - a sense primer comprising a sequence SEQ ID No. 28, or a sequence having at least 80% identity with SEQ ID No. 28 and an antisense primer, comprising a sequence SEQ ID No. 29, or a sequence having at least 80% identity with SEQ ID No. 29, and / or - a sense primer comprising a sequence SEQ ID No. 31, or a sequence having at least 80% identity with SEQ ID No. 31 and an antisense primer, comprising a sequence SEQ ID No. 32, or a sequence having at least 80% identity with SEQ ID No. 32, and / or - a sense primer comprising a sequence SEQ ID No. 34, or a sequence having at least 80% identity with SEQ ID No. 34 and an antisense primer, comprising a sequence SEQ ID No. 35, or a sequence having at least 80% identity with SEQ ID No. 35, and / or - a sense primer comprising a sequence SEQ ID No. 37, or a sequence having at least 80% identity with SEQ ID No. 37 and an antisense primer, comprising a sequence SEQ ID No. 38, or a sequence having at least 80% identity with SEQ ID No. 38, and / or - a sense primer comprising a sequence SEQ ID No. 40, or a sequence having at least 80% identity with SEQ ID No. 40 and an antisense primer, comprising a sequence SEQ ID No. 41, or a sequence having at least 80% identity with SEQ ID No. 41, and / or - a sense primer comprising a sequence SEQ ID No. 43, or a sequence having at least 80% identity with SEQ ID No. 43 and an antisense primer, comprising a sequence SEQ ID No. 44, or a sequence having at least 80% identity with SEQ ID No. 44, and / or - a sense primer comprising a sequence SEQ ID No. 46, or a sequence having at least 80% identity with SEQ ID No. 46 and an antisense primer, comprising a sequence SEQ ID No. 47, or a sequence having at least 80% identity with SEQ ID No. 47, and / or - a sense primer comprising a sequence SEQ ID No. 49, or a sequence having at least 80% identity with SEQ ID No. 49 and an antisense primer, comprising a sequence SEQ ID No. 50, or a sequence having at least 80% identity with SEQ ID No. 50, and / or - a sense primer comprising a sequence SEQ ID No. 52, or a sequence having at least 80% identity with SEQ ID No. 52 and an antisense primer, comprising a sequence SEQ ID No. 53, or a sequence having at least 80% identity with SEQ ID No. 53, 5 b) Amplification reaction of said target sequence, c) Detection of the presence of the amplified sequence during step b).

9. A method according to claim 7 or 8 for detecting in a biological sample a cell-free DNA target sequence specific to a cell type present in the brain.

10. A method according to claim 7 for detecting in a biological sample a cell-free DNA target sequence specific to a cell type present in the lung.

11. A method according to claim 7 or 8 for detecting in a biological sample a target sequence of cell-free DNA specific to a cell type present in the kidney.

12. Method according to the preceding claim for detecting the type of renal cell which is degrading, said cell type being chosen from epithelial cells and vascular cells.

13. A method according to claim 11 or 12 for detecting kidney transplant rejection in a kidney transplant patient.

14. Method according to claim 11 or 12 for the in vitro diagnosis of the lesion typology of rejection, said rejection being of the epithelial or vascular type, depending on the histological analyses of the solid biopsy of the renal graft.

15. Use of a kit according to one of claims 1 to 3, or of a method according to claim 9, for the specific detection of brain degradation and / or for in vitro diagnosis or monitoring of the development of a stroke.

16. Use of a kit according to one of claims 1, 2 or 4, or of a method according to claim 10, for the specific detection of lung degradation and / or for in vitro diagnosis or monitoring of the development of a syndrome of 30 respiratory distress.

17. Use of a kit according to one of claims 1, 2 or 5, or of a method according to claim 7, 8 or 11, for the specific detection of degradation of the kidney and / or for in vitro diagnosis or monitoring of renal failure or graft rejection.

18. Nucleotide sequence for use in a kit according to one of claims 1 to 5, in a method according to one of claims 7 to 11 or for use according to one of claims 12 to 14, chosen from: - a sense primer comprising a nucleotide sequence chosen from: SEQ ID No. 1, SEQ ID No. 4, SEQ ID No. 7, SEQ ID No. 10, SEQ ID No. 13, SEQ ID No. 19, SEQ ID No. 22, SEQ ID No. 25, SEQ ID No. 28, SEQ ID No. 31, SEQ ID No. 34, SEQ ID No. 37, SEQ ID No. 40, SEQ ID No. 43, SEQ ID No. 46, SEQ ID No. 49 and SEQ ID No. 52, or a nucleotide sequence having at least 80% identity with said sequences; - an antisense primer comprising a nucleotide sequence chosen from: SEQ ID No. 2, SEQ ID No. 5, SEQ ID No. 8, SEQ ID No. 11, SEQ ID No. 14, SEQ ID No. 20, SEQ ID No. 23, SEQ ID No. 26, SEQ ID No. 29, SEQ ID No. 32, SEQ ID No. 35, SEQ ID No. 38, SEQ ID No. 41, SEQ ID No. 44, SEQ ID No. 47, SEQ ID No. 50 and SEQ ID No. 53, or a nucleotide sequence having at least 80% identity with said sequences; and - a probe comprising a nucleotide sequence chosen from: SEQ ID No. 3, SEQ ID No. 6, SEQ ID No. 9, SEQ ID No. 12, SEQ ID No. 15, SEQ ID No. 21, SEQ ID No. 24, SEQ ID No. 27, SEQ ID No. 30, SEQ ID No. 33, SEQ ID No. 36, SEQ ID No. 39, SEQ ID No. 42, SEQ ID No. 45, SEQ ID No. 48, SEQ ID No. 51 and SEQ ID No. 54, or a nucleotide sequence having at least 80% identity with said sequences.

Images