Methods for characterizing organ damage

Abstract

The present invention relates to a kit and an in vitro method for detecting and characterizing damage to a specific organ or tissue based on the analysis of the presence of at least one specific methylated self-lying DNA in a biological sample. More specifically, the present invention relates to a kit and a method for detecting and characterizing damage to an organ selected from the brain, lung, and kidney, and / or tissue present in this organ. Another object of the present invention is a method for the in vitro diagnosis of a disease or condition involving the dissolution of an organ or tissue.

Description

[Technical Field]

[0001] The present invention provides a kit and an in vitro method for detecting and characterizing degradation of specific organs or tissues based on the analysis of the presence of at least one specific methylated self-leading DNA in a biological sample. More specifically, the present invention provides a kit and a method for detecting and characterizing 1) brain damage during an episode of stroke, 2) lung damage during an episode of acute respiratory distress syndrome, and 3) kidney damage during an episode of acute kidney injury and / or kidney transplant rejection and / or dysfunction. Another object of the present invention is a method for the in vitro diagnosis of diseases or conditions involving organ or tissue dissolution.

[0002] The present invention is thus in the field of molecular biology applied to medical diagnostics. [Background technology]

[0003] During the treatment of patients with acute disease, medical imaging analyses such as CT scans, X-rays, or MRIs are performed to establish the overall level of the patient's suffering and the lesion's damage. However, this type of examination cannot distinguish between inflammatory disease and organic lysis itself. However, the prognosis and therapeutic interventions made by medical staff vary greatly depending on the nature of the organ or tissue involved. Furthermore, in acute cases, treatment decisions must be made within a very short time frame.

[0004] Several acute and relatively widespread diseases share common characteristics, including the dissolution of large-scale tissues.

[0005] This is particularly the case in stroke, where brain tissue is damaged; acute respiratory distress syndrome, where lung tissue is damaged; acute kidney injury (AKI), where kidney tissue is damaged, of the native kidney, and of the transplanted kidney, where transplant rejection or kidney transplant failure occurs.

[0006] Furthermore, in the case of kidney transplants, it is important to monitor the possible occurrence of graft rejection / dysfunction. Currently, postoperative follow-up of transplant patients involves monitoring that is both painful and burdensome for the patient.

[0007] Due to the common occurrence of all these diseases in the population, there is a need for tools that allow reliable, rapid, simple, and inexpensive detection of potential tissue damage in the setting of these various acute diseases.

[0008] Circulating self-lying DNA, also known as "free DNA," is commonly known as a tumor marker, but is also known as a marker of inflammation and tissue stress. Immune cells contribute largely to the total amount of circulating DNA.

[0009] It has been shown that circulating DNA could potentially be used as a potential biomarker for autoimmune diseases such as the emphysematous form of systemic lupus or rheumatoid arthritis (Duvvuri et al., "Cell-free DNA as a biomarker in autoimmune rheumatic diseases", Front. Immunol. 10, 2019).

[0010] The use of characterization of the methylation profile of circulating DNA to identify the tissue origin of said circulating DNA in healthy subjects 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).

[0011] An increased proportion of circulating DNA derived from hepatocytes has been described in patients with inflammatory liver disease, with these immune cells primarily contributing to the total amount of circulating DNA (Liu et al. ("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).

[0012] International Patent Publication No. 2021 / 216985, "Methods for detecting tissue damage, graft versus host disease and infections using cell-free DNA profiling," discloses the detection of tissue damage by establishing circulating DNA profiles in the context of allogeneic hematopoietic cell transplantation.

[0013] Chinese Patent Publication No. 113999901, "Myocardial specific methylation marker", describes the identification and use of specific cardiac methylated cell DNA markers from plasma samples for the diagnosis of acute myocardial infarction.

[0014] International Patent Publication No. "DUAL-PROBE DIGITAL DROPLET PCR STRATEGY FOR SPECIFIC DETECTION OF TISSUE-SPECIFIC CIRCULATING DNA MOLECULES" describes a droplet PCR method for analyzing the methylation status of methylation sites in double-stranded DNA molecules containing at least two single-stranded methylation sites from a sample of a biological fluid such as plasma or urine.

[0015] International Patent Publication No. 2019012543, "DNA TARGETS AS TISSUE-SPECIFIC METHYLATION MARKERS," describes a general method for determining the state of tissue damage during pathological processes from the study of methylation sites of circulating DNA molecules from samples of biological fluids such as plasma or urine.

[0016] Disclosure of the Invention The present inventors have developed a kit and method for the specific and targeted detection of circulating methylated self-lying DNA present in an individual's biological sample and specific to the brain, lung, or kidney. The method of the present invention makes it possible to specifically detect the presence of massive lysis of one of these organs or the specific tissues that compose it, at the level of the vascular or epithelial fraction. Furthermore, this process makes it possible to distinguish organ lysis from immune-derived inflammatory phenomena. The distinction between epithelial and vascular damage and the identification of organic lysis as opposed to purely immune-derived inflammatory phenomena are important for patient diagnosis and cannot be distinguished by medical imaging.

[0017] For two of these organs, namely lung and brain, the inventors have indeed selected at least one region from various differentially hyper- or hypomethylated regions of circulating DNA that allows the development of a sensitive and specific test for damage in said organs.

[0018] For one of these organs, the kidney, we further selected at least three fractions that were differentially methylated in the kidney epithelium, kidney endothelium, and whole kidney from the various fractions of circulating DNA that were specifically hypermethylated or hypomethylated. These regions can be designated "whole kidney," "renal vascular fraction," and "renal epithelial fraction," respectively, and may enable the development of a sensitive and specific test for damage in the organ.

[0019] Therefore, the present inventors designed selective amplification primer pairs, amplification conditions, and probes that specifically detect the amplification products generated using the primer pairs for each of these organs.

[0020] The present inventors have also identified a genetic region of interest containing the amplified nucleotide sequence surrounding each of the nucleotide sequences selectively amplified by the above primer pairs. The genetic region is larger than the region of the marker. The present invention describes the above "genetic region of interest."

[0021] For each of the markers, detection kits and methods are thus provided.

[0022] Another object of the present invention is a kit and a method for detecting at least one specific marker of organ lysis. A further object of the present invention is a kit and a method for simultaneously detecting at least two markers for specifically detecting organ lysis, thereby making it possible to further improve the specificity and sensitivity of the test. The test according to the present invention also has strong diagnostic capabilities.

[0023] The object of the present invention is therefore a kit and a method for detecting lysis of organs selected from the brain, lungs, and kidneys from a biological sample of a patient. The kit and the method for detecting brain lysis can be used in particular for the in vitro diagnosis of stroke. The kit and the method are based on the detection of at least one marker selected from "APC2" and "NBR1". The "NBR1" marker is also called "KRT33B".

[0024] Another object of the present invention is a kit and a method for detecting lung lysis, which can be used in particular for the in vitro diagnosis of acute respiratory distress syndrome or possible lung transplant rejection. The kit and the method are based on the detection of the "SYNE1" marker, which is also called "LINC00336".

[0025] Another object of the present invention is a kit and a method for detecting nephrolysis, which can be used for the in vitro diagnosis of, in particular, acute kidney injury and, in the case of transplant patients, transplant rejection, based on the detection of at least one marker selected from PAX2, PDE4D, CTDP1, ALDH1A1, ARID3A, GATA2, LOC124903692, NRN1, SEPT5-GP1BB, TNS2-AS1 RHBDF2, LINC01599, FLJ12825, ACSL5.

[0026] In certain embodiments, another object of the present invention is to provide a method for producing a pharmaceutical composition comprising at least: Selected from one "whole kidney" marker: CTDP1 one "vascular" marker selected from: ALDH1A1, ARID3A, GATA2, LOC124903692, NRN1, SEPT5-GP1BB, TNS2-AS1 RHBDF2 and LINC01599, and One "epithelial" marker: selected from FLJ12825, ACSL5, PAX2 and PDE4D The present invention provides a kit and method for detecting kidney lysis based on the multiplex detection combination of:

[0027] Increasing the number of markers simultaneously detected by multiplex analysis will ensure a good clinical outcome of the test in terms of diagnosis, screening, optimization of therapy, and patient follow-up.

[0028] Solid biopsy or renal aspiration is the standard method for diagnosing transplant rejection. It allows for the examination of specific anatomical structures of the graft in the renal cortex, located at the periphery of the kidney. Depending on the Banff score, graft lesions can be classified as "vascular" or "epithelial" types. Examination of these lesions in combination with findings of immune infiltration allows for the classification of kidney transplant rejection as follows: "no rejection," "T-cell-mediated rejection (TCMR)," "antibody-mediated rejection (ABMR)," and "mixed rejection, which combines both types of rejection." Depending on the type of rejection, treatment options vary.

[0029] Diagnostic tests for transplant rejection are commercially available, such as Allosure (CareDX) and Prospera™ (Natera). New methodologies for detecting transplant rejection have only demonstrated promise for predicting kidney graft rejection or dysfunction, but are unable to characterize and diagnose the latter, and thus kidney puncture is still performed.

[0030] The kit and method according to the invention thus have the great advantage of providing a minimally invasive test, since it is performed from biological samples that are easily collected, reliable, rapid and inexpensive. Due to its minimally invasive nature, the method according to the invention can be easily repeated, if necessary, at various stages of the disease, which makes it possible to reliably monitor the progression of the disease and ensure medical treatment adapted to each of these stages.

[0031] The kits and methods of the present invention pose a significant challenge to specialists and anatomical pathologists, as the type of organ lesion, particularly the type of renal graft lesion, determines the ultimate diagnosis and therapeutic treatment of the patient.

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

[0033] Another object of the present invention is the use of methylated self-lying DNA as a marker of brain, kidney, or lung damage, or as a marker of brain, kidney, or lung tissue damage.

[0034] Another object of the present invention are primers and probes that can be used in the kits and methods according to the invention. In particular, the object of the present invention is a sense primer 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 having at least 80% identity to said sequences; an antisense primer comprising or consisting of a nucleotide sequence selected 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 thereto; and A probe, optionally labeled, comprising or consisting of a nucleotide sequence selected 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 thereto. is.

[0035] Finally, an object of the present invention is an in vitro method for detecting a disease or condition selected from stroke, acute respiratory distress syndrome, renal injury, and transplant rejection, in particular kidney or lung transplant rejection.

[0036] Detailed Description of the Invention In a first aspect, the object of the present invention is a kit for detecting at least one methylated self-lying DNA target nucleotide sequence in a biological sample, said nucleotide sequence being specific to an organ selected from the brain, lung and kidney, or to a tissue present in the brain, lung or kidney, said kit comprising at least one primer pair consisting of a sense primer and an antisense primer, each primer comprising or consisting of a nucleotide sequence capable of hybridizing to a nucleotide sequence of said self-lying DNA in order to selectively amplify a specific methylated self-lying DNA sequence of said organ or tissue.

[0037] In one embodiment of this first aspect, the object of the present invention is a kit for detecting at least one methylated self-lying DNA target nucleotide sequence in a biological sample, said kit comprising at least: - one primer pair consisting of a sense primer and an antisense primer, each primer comprising or consisting of a nucleotide sequence capable of hybridizing with at least one nucleotide sequence of the self-linked DNA in order to selectively amplify specific methylated self-linked DNA sequences of the organ or tissue; and - one probe comprising or consisting of a nucleotide sequence capable of hybridizing with a target nucleotide sequence of the amplified DNA; Includes.

[0038] "Biological sample" means an element from the human or animal body, such as, inter alia, blood, urine, saliva, plasma, or organ fragments.

[0039] The probes, so-called "detection probes", are preferably coupled to markers whose detection is well known to those skilled in the art. When more than one pair of probes is used (multiplex test), the combination of markers is selected to distinguish between the various amplified nucleotide sequences.

[0040] In a first particular aspect, the object of the present invention is a kit for detecting a methylated self-lying DNA target nucleotide sequence in a biological sample, said nucleotide sequence being specific for a cell type of an organ selected from the brain, lung and kidney, said kit comprising: i) for detecting specific self-lying DNA target sequences of cell types present in the brain, at least one sense primer comprising the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence having at least 80% identity to SEQ ID NO: 1, and one antisense primer comprising the nucleotide sequence of SEQ ID NO: 2 or a nucleotide sequence having at least 80% identity to SEQ ID NO: 2, and / or one sense primer comprising the nucleotide sequence of SEQ ID NO: 4 or a nucleotide sequence having at least 80% identity with SEQ ID NO: 4, and one antisense primer comprising the nucleotide sequence of SEQ ID NO: 5 or a nucleotide sequence having at least 80% identity with SEQ ID NO: 5; ii) at least one method for detecting specific self-referencing DNA target sequences of cell types present in the lung; one sense primer comprising the nucleotide sequence of SEQ ID NO: 7 or a nucleotide sequence having at least 80% identity to SEQ ID NO: 7, and one antisense primer comprising the nucleotide sequence of SEQ ID NO: 8 or a nucleotide sequence having at least 80% identity to SEQ ID NO: 8; and iii) at least one method for detecting specific self-repeated DNA target sequences of cell types present in the kidney, one sense primer comprising the sequence SEQ ID NO: 10 or a sequence having at least 80% identity with SEQ ID NO: 10, and one antisense primer comprising the sequence SEQ ID NO: 11 or a sequence having at least 80% identity with SEQ ID NO: 11, and / or one sense primer comprising the sequence SEQ ID NO: 13 or a sequence having at least 80% identity with SEQ ID NO: 13, and one antisense primer comprising the sequence SEQ ID NO: 14 or a sequence having at least 80% identity with SEQ ID NO: 14, and / or one sense primer comprising the sequence SEQ ID NO: 19 or a sequence having at least 80% identity with SEQ ID NO: 19 and one antisense primer comprising the sequence SEQ ID NO: 20 or a sequence having at least 80% identity with SEQ ID NO: 20, and / or one sense primer comprising the sequence SEQ ID NO: 22 or a sequence having at least 80% identity with SEQ ID NO: 22 and one antisense primer comprising the sequence SEQ ID NO: 23 or a sequence having at least 80% identity with SEQ ID NO: 23, and / or one sense primer comprising the sequence SEQ ID NO: 25 or a sequence having at least 80% identity with SEQ ID NO: 25 and one antisense primer comprising the sequence SEQ ID NO: 26 or a sequence having at least 80% identity with SEQ ID NO: 26, and / or one sense primer comprising the sequence SEQ ID NO: 28 or a sequence having at least 80% identity with SEQ ID NO: 28 and one antisense primer comprising the sequence SEQ ID NO: 29 or a sequence having at least 80% identity with SEQ ID NO: 29, and / or one sense primer comprising the sequence SEQ ID NO: 31 or a sequence having at least 80% identity with SEQ ID NO: 31 and one antisense primer comprising the sequence SEQ ID NO: 32 or a sequence having at least 80% identity with SEQ ID NO: 32, and / or one sense primer comprising the sequence SEQ ID NO: 34 or a sequence having at least 80% identity with SEQ ID NO: 34 and one antisense primer comprising the sequence SEQ ID NO: 35 or a sequence having at least 80% identity with SEQ ID NO: 35, and / or one sense primer comprising the sequence SEQ ID NO: 37 or a sequence having at least 80% identity with SEQ ID NO: 37 and one antisense primer comprising the sequence SEQ ID NO: 38 or a sequence having at least 80% identity with SEQ ID NO: 38, and / or one sense primer comprising the sequence SEQ ID NO: 40 or a sequence having at least 80% identity with SEQ ID NO: 40 and one antisense primer comprising the sequence SEQ ID NO: 41 or a sequence having at least 80% identity with SEQ ID NO: 41, and / or one sense primer comprising the sequence SEQ ID NO: 43 or a sequence having at least 80% identity with SEQ ID NO: 43 and one antisense primer comprising the sequence SEQ ID NO: 44 or a sequence having at least 80% identity with SEQ ID NO: 44, and / or one sense primer comprising the sequence SEQ ID NO: 46 or a sequence having at least 80% identity with SEQ ID NO: 46 and one antisense primer comprising the sequence SEQ ID NO: 47 or a sequence having at least 80% identity with SEQ ID NO: 47, and / or one sense primer comprising the sequence SEQ ID NO: 49 or a sequence having at least 80% identity with SEQ ID NO: 49 and one antisense primer comprising the sequence SEQ ID NO: 50 or a sequence having at least 80% identity with SEQ ID NO: 50, and / or one sense primer comprising the sequence of SEQ ID NO: 52 or a sequence having at least 80% identity with SEQ ID NO: 52, and one antisense primer comprising the sequence of SEQ ID NO: 53 or a sequence having at least 80% identity with SEQ ID NO: 53; Includes. In more particular embodiments, the kits of the present invention comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 primer pairs, each consisting of at least a sense primer of the present invention and an antisense primer of the present invention.

[0041] In a more particular embodiment, the kit according to the invention comprises a "sense" primer 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 to one of the 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, SEQ ID NO:52 above.

[0042] In another more particular embodiment, the kit according to the invention comprises an "antisense" primer comprising or consisting of a nucleotide sequence selected 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 to one of the 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.

[0043] In another more particular embodiment, the kit according to the invention comprises a probe comprising or consisting of a nucleotide sequence selected 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 to one of said nucleotide sequences selected 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.

[0044] "At least 80% identity" means that the sequences have at least 80% identity after optimal global alignment, i.e., by global alignment between the two sequences that gives the highest percentage of identity between them. Optimal global alignment of two sequences can be performed, in particular, by the Needleman-Wunsch algorithm, which is 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). The nucleotide sequences of the present invention comprise or consist of a nucleotide sequence having at least 80%, preferably at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a reference nucleotide sequence after optimal global alignment. Preferably, the nucleotide sequences of the present invention comprise or consist of 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 to a reference nucleotide sequence after optimal global alignment.

[0045] In this embodiment, the nucleotide sequence of the present invention comprising or consisting of a sequence having at least 80% identity to a reference nucleotide sequence is functional, i.e. it is capable of hybridizing on a target nucleotide sequence with sufficient stability to allow amplification or detection of said target nucleotide sequence.

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

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

[0048] In another more particular aspect, the object of the present invention is a kit for detecting a methylated self-lying DNA target nucleotide sequence in a biological sample, said nucleotide sequence being specific for a cell type present in the brain, said kit comprising at least: one sense primer comprising or consisting of the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence having at least 80% identity to SEQ ID NO: 1, and one antisense primer comprising or consisting of the nucleotide sequence of SEQ ID NO: 2 or a nucleotide sequence having at least 80% identity to SEQ ID NO: 2, and / or one sense primer comprising or consisting of the nucleotide sequence of SEQ ID NO: 4 or a nucleotide sequence having at least 80% identity to SEQ ID NO: 4, and one antisense primer comprising or consisting of the nucleotide sequence of SEQ ID NO: 5 or a nucleotide sequence having at least 80% identity to SEQ ID NO: 5; Includes.

[0049] In another more particular aspect, the object of the present invention is a kit for detecting a methylated self-lying DNA target nucleotide sequence in a biological sample, said nucleotide sequence being specific for a cell type present in the lung, said kit comprising at least: It comprises at least one sense primer comprising or consisting of the nucleotide sequence of SEQ ID NO:7 or a nucleotide sequence having at least 80% identity to SEQ ID NO:7, and at least one antisense primer comprising or consisting of the nucleotide sequence of SEQ ID NO:8 or a nucleotide sequence having at least 80% identity to SEQ ID NO:8.

[0050] The nucleotide sequences of the specific primers for the cell types present in the kidney are designated as shown in Table 3 below: Table 3 [Table 3]

[0051] The nucleotide sequences of probes specific for cell types present in the kidney are designated as shown in Table 4 below: Table 4 [Table 4]

[0052] Tissues present in the kidney include: renal epithelial tissue, and renal vascular tissue.

[0053] The specific markers for monitoring kidney damage are characterized as shown in Table 5 below: Table 5 [Table 5]

[0054] In another more particular aspect, the object of the present invention is a kit comprising the reagents (primers and probe) necessary for the amplification of at least one PCR amplicon of less than 85 bp (preferably less than 70 bp) in a genomic region consisting of the nucleotide sequence of 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 the genomic region.

[0055] Thus, the markers used in the kits and methods of the present invention may be classified as follows: "Vascular" markers: selected from the markers ALDH1A1, ARID3A, GATA2, LOC124903692, NRN1, SEPT5-GP1BB, TNS2-AS1 RHBDF2, and LINC01599; "Epithelial" markers: selected from the markers FLJ12825, ACSL5, PAX2, and PDE4D; "Kidney total type" marker: Selected from CTDP1.

[0056] In another particular aspect, the object of the present invention is a kit for detecting a methylated self-lying DNA target nucleotide sequence in a biological sample, said nucleotide sequence being specific for a cell type present in the kidney, said kit comprising at least: one sense primer comprising or consisting of a nucleotide sequence capable of detecting the "kidney whole type" marker, and one antisense primer comprising or consisting of a nucleotide sequence capable of detecting the "kidney whole type" marker, and / or one sense primer comprising or consisting of a nucleotide sequence capable of detecting a "vascular" marker, and one antisense primer comprising or consisting of a nucleotide sequence capable of detecting a "vascular" marker, and / or One sense primer containing or consisting of a nucleotide sequence that may detect an "epithelial" marker, and one antisense primer containing or consisting of a nucleotide sequence that may detect an "epithelial" marker. Includes.

[0057] Preferably, in this more particular aspect, the object of the present invention is a kit for detecting a methylated self-lying DNA target nucleotide sequence in a biological sample, said nucleotide sequence being specific for a cell type present in the kidney, said kit comprising at least: one sense primer comprising or consisting of a nucleotide sequence capable of detecting the "kidney whole type" marker, which is CTDP1, and one antisense primer comprising or consisting of a nucleotide sequence capable of detecting the "kidney whole type" marker; One sense primer comprising or consisting of a nucleotide sequence capable of detecting a "vascular-type" marker selected from GATA2, TNS2-AS1, and one antisense primer comprising or consisting of a nucleotide sequence capable of detecting a "vascular-type" marker; One sense primer containing or consisting of a nucleotide sequence capable of detecting an "epithelial-type" marker selected from PAX2, ACSL5, or PDE4D, and one antisense primer containing or consisting of a nucleotide sequence capable of detecting an "epithelial-type" marker. Includes.

[0058] The markers used in the kits and methods of the present invention will now be described in more detail. The names of the markers correspond to the names of the genes in which they are located.

[0059] The genetic region "PDE4D" of the present invention refers to a sequence encoding a protein of the phosphodiesterase family. The DNA sequence is located at positions 58,969,038-60,522,128 on chromosome 5, more specifically at locus 5q11.2-q12.1. It contains 42 exons. The DNA sequence is referred to as NC_000005.10. It is transcribed to the sequence NM_006203.5. The inventors have identified a DNA region contained at positions 59,039,300-59,039,420 on chromosome 5 (SEQ ID NO: 55) as being specifically hypermethylated in the epithelial fraction. The inventors propose the use of this gene in a non-invasive, sensitive, and reliable method for diagnosing or identifying kidney transplant rejection, particularly in kidney transplant patients, or in a kit useful for such purpose.

[0060] The genetic region "PAX2" of the present invention represents the coding sequence for a protein of the box-paired family 2. The DNA sequence is located at positions 100,735,396-100,829,944 on chromosome 10, more specifically at locus 10q24.31. It contains 14 exons. The DNA sequence is referred to as NC_000010.11. It is transcribed to the sequence NM_000278.5. The inventors have identified a DNA region contained at positions 100,828,800-100,829,020 on chromosome 10 (SEQ ID NO: 56) as being specifically hypermethylated in the epithelial fraction. In particular, the inventors propose the use of 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 purpose.

[0061] The genetic region "FLJ12825" of the present invention represents a complete non-protein-coding genomic molecule. It allows transcription of a non-coding long RNA and is located at the uncharacterized locus LOC440101 of the FLJ12825 gene. The DNA sequence is located at positions 54,058,254-54,122,234 on chromosome 12, more specifically at the intronic locus 12q13.13. The DNA sequence is referred to as NC_000012.12. It is transcribed into one RNA variant designated NR_026655.1. The inventors have identified a DNA region contained in chromosome 12 at positions 54,106,357-54,106,526 (SEQ ID NO: 57) that is specifically hypermethylated in renal epithelial fractions. In particular, the inventors 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 purpose.

[0062] The genetic region "ALDH1A1" of the present invention represents the complete genomic molecule encoding the aldehyde dehydrogenase family type 1 protein (member A1). Its DNA sequence is located at positions 72,900,671-72,953,063 on chromosome 9, more specifically at locus 9q21.13. It contains 13 exons. The DNA sequence is referred to as NC_005224.3. It is transcribed into one RNA variant designated NM_005224.3. The inventors have identified a DNA region contained in chromosome 9, positions 72,952,933-72,953,059 (SEQ ID NO: 58), as being specifically hypermethylated in the renal vascular fraction. The inventors 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 purpose.

[0063] The genetic region "ARID3A" of the present invention represents the complete genomic molecule encoding the ARID (AT-rich interacting domain) DNA-binding protein family. The DNA sequence is located at positions 925,732-975,939 on chromosome 19, more specifically at locus 19p13.3. It contains 12 exons. The DNA sequence is referred to as NC_000019.10. It is transcribed into one RNA variant designated NM_000689.5. The inventors have identified a DNA region contained in chromosome 19 at positions 940,667-941,160 (SEQ ID NO: 59) that is specifically hypermethylated in the renal vascular fraction. The inventors therefore propose the use of this gene in a non-invasive, sensitive, and reliable method for diagnosing or identifying kidney transplant rejection, particularly in kidney transplant patients, or in a kit useful for such purpose.

[0064] The genetic region "ACSL5" of the present invention represents the coding sequence for a protein of the ligase family. The DNA sequence is located at positions 112,374,116-112,428,376 on chromosome 10, more specifically at locus 10q25.2. It contains 23 exons. The DNA sequence is referred to as NC_000010.11. It is transcribed to the sequence NM_016234.4. The inventors have identified a DNA region contained in chromosome 10 at positions 12,376,275-112,376,398 (SEQ ID NO: 59) that is specifically hypermethylated in the epithelial fraction. The inventors therefore propose the use of this gene in a non-invasive, sensitive, and reliable method for diagnosing or identifying kidney transplant rejection, particularly in kidney transplant patients, or in a kit useful for such purpose.

[0065] The genetic region "CTDP1" of the present invention refers to an uncharacterized intron sequence located between the CTDP1 gene and the KCNG2 gene. The DNA sequence is located on chromosome 18 between the location of the CTDP1 gene (chr18:79,756,625-79,787,722) and the location of the KCNG2 gene (chr18:79,797,938-79,900,100). It contains 0 exons. The DNA sequence is not referenced in NCBI. No transcripts have been identified. The present inventors have identified the DNA region 79,791,700-79,791,820 (SEQ ID NO: 61) contained in chromosome 18 as being specifically hypermethylated in the total kidney fraction, and therefore propose to use this gene in a non-invasive, sensitive, and reliable method for diagnosing or identifying kidney transplant rejection, particularly in kidney transplant patients, or in a kit useful for such purpose.

[0066] The genetic region "GATA2" according to the present invention represents a sequence encoding a protein of the zinc finger transcription factor family. The DNA sequence is located at positions 128,479,422-128,493,201 on chromosome 3, more specifically at locus 3q21. It contains eight exons. The DNA sequence is referred to as NC_000003.12. It is transcribed to the sequence NM_032638.5. The inventors have identified a DNA region contained in chromosome 3 at positions 128,491,794-128,492,245 (SEQ ID NO: 62) that is specifically hypermethylated in the vascular fraction. The inventors therefore propose the use of this gene in a non-invasive, sensitive, and reliable method for diagnosing or identifying kidney transplant rejection, particularly in kidney transplant patients, or in a kit useful for such purpose.

[0067] The genetic region "LOC124903692" of the present invention represents a non-coding sequence that has not yet been characterized for protein. The DNA sequence is located at positions 54,936,014-54,938,671 on chromosome 16, more specifically at locus 16q12.2. It contains three exons. The DNA sequence is referred to as NC_000016.10. It is transcribed to the sequence XR_007065074.1. The inventors have identified a DNA region contained in chromosome 16 at positions 54,937,300-54,937,500 (SEQ ID NO: 63) that is specifically hypermethylated in the vascular fraction. The inventors therefore propose the use of this gene in a non-invasive, sensitive, and reliable method for diagnosing or identifying kidney transplant rejection, particularly in kidney transplant patients, or in a kit useful for such purpose.

[0068] The genetic region "NRN1" of the present invention represents the coding sequence for a protein of the neuritin family. The DNA sequence is located at positions 5,997,999-6,007,518 on chromosome 6, more specifically at locus 6p25.1. It contains seven exons. The DNA sequence is referred to as NC_000006.12. It is transcribed to the sequence NM_016588.3. The inventors have identified a DNA region contained in chromosome 6 at positions 5,998,924-5,999,075 (SEQ ID NO: 64) that is specifically hypermethylated in the vascular fraction. The inventors therefore propose the use of this gene in a non-invasive, sensitive, and reliable method for diagnosing or identifying kidney transplant rejection, particularly in kidney transplant patients, or in a kit useful for such purpose.

[0069] The genetic region "SEPT5-GP1BB" of the present invention represents a non-coding sequence for a protein. The DNA sequence is located at positions 19,717,220-19,724,774 on chromosome 22, more specifically at locus 22q11.21. It contains 12 exons. The DNA sequence is referred to as NC_000022.11. It is transcribed to the sequence NR_037611.1. The inventors have identified a DNA region contained in chromosome 22 at positions 19,724,235-19,724,340 (SEQ ID NO: 65) that is specifically hypermethylated in the vascular fraction. The inventors therefore propose the use of this gene in a non-invasive, sensitive, and reliable method for diagnosing or identifying kidney transplant rejection, particularly in kidney transplant patients, or in a kit useful for such purpose.

[0070] The genetic region "TNS2-AS1" of the present invention represents a non-coding sequence for a protein. The DNA sequence is located at positions 53,043,189-53,054,438 on chromosome 12, more specifically at locus 12q13.13. It contains four exons. The DNA sequence is referred to as NC_000012.12. It is transcribed to the sequence NR_033854.1. The inventors have identified a DNA region contained in chromosome 12 at positions 53,054,207-53,054,329 (SEQ ID NO: 66) that is specifically hypermethylated in the vascular fraction. The inventors therefore propose the use of this gene in a non-invasive, sensitive, and reliable method for diagnosing or identifying kidney transplant rejection, particularly in kidney transplant patients, or in a kit useful for such purpose.

[0071] The genetic region "RHBDF2" of the present invention represents the coding sequence of a protein that activates the activity of a protein transporter. The DNA sequence is located at positions 76,470,893-76,501,427 on chromosome 17, more specifically at locus 17q25.1. It contains 21 exons. The DNA sequence is referred to as NC_000017.11. It is transcribed to the sequence NM_024599.5. The inventors have identified a DNA region contained in chromosome 17 at positions 76,500,822-76,500,937 (SEQ ID NO: 67) that is specifically hypermethylated in the vascular fraction. The inventors therefore propose the use of this gene in a non-invasive, sensitive, and reliable method for diagnosing or identifying kidney transplant rejection, particularly in kidney transplant patients, or in a kit useful for such purpose.

[0072] The genetic region "LINC01599" of the present invention represents a sequence that allows transcription of a non-coding intergenic RNA related to a protein. The DNA sequence is located at positions 50,007,313-50,105,043 on chromosome 14, more specifically at locus 14q21.3. It contains nine exons. The DNA sequence is referred to as NC_000014.9. It is transcribed to the sequence NR_131171.1. The inventors have identified a DNA region contained in chromosome 14 at positions 50,056,666-50,056,758 (SEQ ID NO: 68) that is specifically hypomethylated in the vascular fraction. The inventors therefore propose the use of this gene in a non-invasive, sensitive, and reliable method for diagnosing or identifying kidney transplant rejection, particularly in kidney transplant patients, or in a kit useful for such purpose.

[0073] The genetic region "APC2" of the present invention represents the complete genomic molecule encoding a protein of the APC regulator family of the Wnt pathway. Its DNA sequence is located at positions 1,446,230-1,473,244 on chromosome 19, more specifically at locus 19p13.3. It contains 17 exons. The DNA sequence is referred to as NC_000019.10, which is transcribed to the sequence NM_005883.3. The inventors have identified a DNA region contained in chromosome 19 at positions 1,467,853-1,468,054 (SEQ ID NO: 69) that is specifically hypermethylated in neurons. The inventors therefore propose the use of this gene in a non-invasive, sensitive, and reliable method for diagnosing stroke, in particular, or in a kit useful for such purpose. The genetic region "NBR1" of the present invention represents a complete genomic molecule encoding a specific protein of the autophagic receptor family. Its DNA sequence is located at positions 43,170,409-43,211,900 on chromosome 17, more specifically at locus 17q21.31. It contains 23 exons. The DNA sequence is referred to as NC_000017.11. It is transcribed to the sequence NM_005899.5. The inventors have identified the DNA region contained in chromosome 17, 43,211,655-43,211,856 (SEQ ID NO: 70), as being specifically hypermethylated in neurons. The inventors therefore propose the use of this gene in a non-invasive, sensitive, and reliable method for diagnosing stroke, in particular, or in a kit useful for such purpose.

[0074] The genetic region "SYNE1" of the present invention represents a complete genomic molecule encoding a nuclear envelope protein containing spectrin repeats. Its DNA sequence is located at positions 152,121,687-152,637,362 on chromosome 6, more specifically at locus 6p25.2. It contains 153 exons. The DNA sequence is referred to as NC_000006.12. It is transcribed to the sequence NM_033071.5. The inventors have identified a DNA region contained in chromosome 6 at positions 152,302,152-152,302,353 (SEQ ID NO: 71) that is specifically hypermethylated in pneumocytes / alveoli. The inventors therefore propose the use of this gene in a non-invasive, sensitive, and reliable method for diagnosing or identifying ARDS, in particular, or in a kit useful for such purpose.

[0075] The kit of the present invention optionally includes one or more primer pairs consisting of a sense primer and an antisense primer intended to detect the presence of a control element in a biological sample. The control element can be any appropriate element selected by those skilled in the art. The control element can be, in particular, albumin. More specifically, the kit of the present invention can include one sense primer comprising or consisting of the nucleotide sequence of SEQ ID NO: 16 or a nucleotide sequence having at least 80% identity to SEQ ID NO: 16, and one antisense primer comprising or consisting of the nucleotide sequence of SEQ ID NO: 17 or a nucleotide sequence having at least 80% identity to SEQ ID NO: 17.

[0076] In a second aspect, the object of the present invention is a method for identifying specific self-lying DNA target sequences of a cell type present in a first organ selected from the lung, the brain and the kidney, said method comprising: a) determining the location and degree of methylation of CpG islands of specific sequences of said cell types of said organ in at least one genomic DNA methylation database; b) identifying differentially hypermethylated CpG islands in the genomic DNA of said cell types of said healthy organ; c) comparing 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 said first cell type; d) after the comparison performed in step c), selecting only hypermethylated CpG islands in the genomic DNA of said cell type of said first organ; e) comparing the degree of methylation of the hypermethylated CpG islands of said cell types of said first organ with the degree of methylation of the same CpG islands in genomic DNA of other organs of said first organ; f) comparing 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 genomic DNA of healthy leukocytes; g) comparing 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 self-lying DNA of all biological matrices combined from healthy subjects; h) selecting only those CpG islands that are hypermethylated in the genomic DNA of said cell type of said first organ after the comparisons made in steps e, f and g); i) identifying one or more targeted hypermethylated sequences of the specific self-lying DNA of the cell type of the first organ from the comparison performed in step h); Includes.

[0077] Another object of the present invention are methylated self-lying DNA target nucleotide sequences identified by the method according to the invention for their use in detecting lysis of an organ selected from the brain, lung, and kidney from a biological sample of a patient.

[0078] In a third aspect, the present invention provides a method for detecting a methylated self-lying DNA target nucleotide sequence in a biological sample, said nucleotide sequence being specific for an organ selected from the lung, the brain, and the kidney, or for tissue present in the brain, the lung, or the kidney, said method comprising: a) combining, under conditions suitable for nucleic acid amplification, self-linked DNA previously extracted from the biological sample and a primer pair consisting of a sense primer and an antisense primer, each of the primers comprising or consisting of a nucleotide sequence capable of hybridizing to a nucleotide sequence of a target sequence of the self-linked DNA; b) amplifying the target sequence; c) detecting the presence of the sequence amplified during step b). Includes.

[0079] Self-DNA is extracted using commercially available kits that are well known to those skilled in the art and require the presence of at least 0.15 ng / mL of target DNA in the urine or plasma sample.

[0080] The amplification reaction is carried out by any technique or protocol familiar to those skilled in the art. Among the non-limiting examples of these techniques, the following may be mentioned: PCR amplification, digital PCR amplification (dPCR), next-generation sequencing.

[0081] More specifically, the object of the present invention is a method for detecting methylated self-lying DNA target nucleotide sequences in a biological sample, said nucleotide sequences being specific for cell types present in the brain.

[0082] Even more specifically, the object of the present invention is a method for detecting a methylated self-lying DNA target nucleotide sequence in a biological sample, said nucleotide sequence being specific for a cell type present in the brain, said method comprising at least the following steps: a) combining, under conditions suitable for amplifying nucleic acids, self-linked DNA previously extracted from the biological sample and a primer pair consisting of one sense primer comprising or consisting of the nucleotide sequence of 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 the nucleotide sequence of 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 the nucleotide sequence of 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 the nucleotide sequence of 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 to the nucleotide sequence of a target sequence of the self-linked DNA; b) amplifying the sequence of said target reaction; c) detecting 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, depending on the primer pair used. Includes.

[0083] Even more specifically, the object of the present invention is a method for detecting a methylated self-lying DNA target nucleotide sequence in a biological sample, said nucleotide sequence being specific for a cell type present in the lung, said method comprising at least the following steps: and self-released DNA previously extracted from the biological sample under conditions suitable for nucleic acid amplification. a sense primer comprising or consisting of the nucleotide sequence of 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 the nucleotide sequence of SEQ ID NO: 8 (or a nucleotide sequence having at least 80% identity with SEQ ID NO: 8); combining a primer pair consisting of each of the primers comprises or consists of a nucleotide sequence capable of hybridizing with a nucleotide sequence of a target sequence of the self-linked DNA sequence; b) amplifying the target sequence; c) detecting the presence of the sequence amplified during step b) by means of a probe comprising or consisting of SEQ ID NO: 9 Includes.

[0084] Even more specifically, the object of the present invention is a method for detecting a methylated self-lying DNA target nucleotide sequence in a biological sample, said nucleotide sequence being specific for a cell type present in the kidney, said method comprising at least the following steps: a) under conditions suitable for nucleic acid amplification, with self-released DNA previously extracted from said biological sample; a sense primer comprising or consisting of the nucleotide sequence of SEQ ID NO: 10 (or a nucleotide sequence having at least 80% identity to SEQ ID NO: 10) and an antisense primer comprising or consisting of the nucleotide sequence of SEQ ID NO: 11 (or a nucleotide sequence having at least 80% identity to SEQ ID NO: 11); or a sense primer comprising or consisting of the nucleotide sequence of SEQ ID NO: 13 (or a nucleotide sequence having at least 80% identity with SEQ ID NO: 13) and an antisense primer comprising or consisting of the nucleotide sequence of SEQ ID NO: 14 (or a nucleotide sequence having at least 80% identity with SEQ ID NO: 14); wherein each of said primers is capable of hybridizing to a nucleotide sequence of a target sequence of self-ligated DNA; b) amplifying the target sequence; c) detecting 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 pair of primers used. Includes.

[0085] The described diagnostic test is part of an analytical process consisting of two preliminary steps: extraction of circulating DNA and epigenetic conversion (by bisulfite and enzymatic reactions). The diagnostic test is compatible with different circulating DNA extraction kits, such as the QIAamp Circulating Nucleic Acid Kit (50) Cat. No. / ID: 55114 (Qiagen); EZ1&2 ccfDNA Kit (48) Cat. No. / ID: 954854 (Qiagen); MagMAX™ cell-free DNA Isolation Kit (Reference: A29319, Thermofisher); and Automated High-Throughput Extraction of Cell-Free DNA from Plasma, Serum, Urine, and CSF (Reference: A6030, Promega). Once extracted, the self-reconstituted DNA is analyzed by Qubit 4.0™ Fluorometry (Invitrogen™ db HS Qubit™ DNA Assay Kits (Reference: Q32851)) and then subjected to bisulfite conversion using the commercially available Zymo Research EZ DNA Methylation-Lightning Kit (Reference D5031) or an enzymatic equivalent (NEBNext® Enzymatic Methyl-seq Conversion Module (Reference E7125S / E7125L)). These samples are then analyzed by droplet digital PCR (System Naica (STILLA Technologies)) (also compatible with commercially available equivalents such as the QIAcuity Digital PCR System - QIAGEN and the QX ONE Droplet Digital PCR (ddPCR) System - Bio-Rad).

[0086] The described diagnostic test is compatible with all commercially available digital PCR platforms (microdroplets and / or microcompartments). In particular, the inventors have found commercially available equivalents such as the QIAcuity Digital PCR System - QIAGEN and the QX ONE Droplet Digital PCR (ddPCR) System - Bio-Rad.

[0087] We determine typical metrological parameters, especially the repeatability, reproducibility, and sensitivity of the test. At least one marker is required to quantify renal damage. Detection using two markers enhances the biological data.

[0088] Preferably, to quantify renal damage, it is preferable to detect at least one "renal whole-type" marker, one "vascular" marker, and one "epithelial" marker quantified in multiple tests. The greater the number of markers in each of these categories, the higher the clinical performance of the test. Detection of multiple markers per category from "renal whole-type," "vascular," and "epithelial" markers strengthens the biological data.

[0089] In another aspect, another object of the present invention is the use of a kit or a method according to the invention for the specific detection of brain damage and / or for the in vitro diagnosis and / or prognosis and / or monitoring of the progression of stroke and / or for the optimization of the treatment of patients suffering from stroke.

[0090] In another aspect, another object of the present invention is the use of a kit or a method according to the invention for the specific detection of lung damage and / or for the in vitro diagnosis and / or prognosis and / or monitoring of the progression of acute respiratory distress syndrome and / or for the optimization of the treatment of patients suffering from acute respiratory distress syndrome.

[0091] In another aspect, another object of the present invention also relates to the use of a kit or a method according to the present invention for specifically detecting kidney damage and / or for the in vitro diagnosis and / or prognosis and / or screening and / or monitoring the progression and / or episodes of kidney transplant rejection / failure and / or acute kidney injury and / or for the optimization of the treatment of patients suffering from kidney transplant rejection / failure and / or acute kidney injury.

[0092] In another aspect, another object of the present invention is to provide a method for producing a pharmaceutical composition comprising: a sense primer 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 having at least 80% identity to the above sequences; an antisense primer comprising a nucleotide sequence selected 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 thereto; and A probe comprising a nucleotide sequence selected 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 to the above sequences. is a nucleotide sequence selected from

[0093] In another aspect, the object of the present invention is to provide a method for the preparation of a pharmaceutical composition comprising: extracting methylated self-lying DNA from a biological sample of the individual; converting the methylated self-lying DNA (bisulfite and / or enzymatic conversion); combining the DNA with an appropriate primer pair under conditions that allow hybridization; amplifying said sequence; detecting said amplified sequences with an appropriate detection probe. The present invention is directed to diagnosing or monitoring damage to organs selected from the group consisting of the brain, lungs, and kidneys, including the liver, kidneys, and the brain.

[0094] Other advantages and features will become apparent upon review of the detailed description of non-limiting embodiments and the accompanying drawings. [Brief explanation of the drawings]

[0095] [Figure 1] FIG. 1 shows the percentage of neurons labeled using the anti-NeuN marker (left), the APC2 marker (center), and the NBR1 marker (right). [Figure 2] FIG. 2 shows normalized data of mean values ​​of NBR1 and APC2 markers on a logarithmic scale in cases of TAI (left) and stroke (right). [Figure 3] Figure 3 shows the amount of renal markers (ng / mL) in plasma for PDE4D (white) and PAX2 (black) markers in patients at high risk of transplant rejection (left histogram) or low risk of transplant rejection (right histogram). [Figure 4] Figure 4 shows the percentage of circulating DNA of renal origin out of the total circulating DNA in plasma for patients at high risk of transplant rejection (left histogram) or low risk of transplant rejection (right histogram) for the PDE4D (white) and PAX2 (black) markers. [Figure 5]Figure 5 shows the amount of urinary renal markers in ng / mL in patients with (left histogram) or without (right histogram) transplant rejection for the PDE4D (white) and PAX2 (black) markers. [Figure 6] FIG. 6 shows the correlation between histological labeling of TTF-1 (x-axis) and the use of the SYNE1 marker according to the present invention (y-axis). [Figure 7] Figure 7 shows the differentiation between subgroups of patients with moderate ARDS (left), severe ARDS (center), or critical ARDS (right) based on the results obtained with the SYNE marker (vertical axis). [Figure 8A] and [Figure 8B] Figures 8A and 8B show, respectively, (A) detection of markers SEPT5 / GATA2, PDE4D, CTDP1, PAX2, and TNS2-AS1 / ACSL5 in 100% methylated DNA; (B) detection of markers SEPT5 / GATA2, PDE4D, CTDP1, PAX2, and TNS2-AS1 / ACSL5 in unmethylated DNA, i.e., the negative control, demonstrating no detection in unmethylated DNA. [Figure 9] Figure 9 shows the relative quantification of the markers SEPT5, GATA2, TNS2-AS1, PDE4D, ACSL5, PAX2, and CTDP1 normalized by the albumin universal marker (an internal standard corresponding to the total genome count present in the sample) in genomic DNA (gDNA) samples extracted from healthy tissues: kidney, liver, leukocytes, plasma circulating DNA, and urinary circulating DNA. [Figure 10] FIG. 10 shows the correlation between normalized relative quantification (with albumin internal marker) of PAX2 and ACSL markers in gDNA from n=22 kidneys (R2=0.5). [Figure 11] FIG. 11 shows the correlation between normalized relative quantification (with albumin internal marker) of PAX2 and ACSL markers in urinary cfDNA from n=15 (R2=0.8087). [Figure 12A] and [Figure 12B] Figures 12A and 12B show a comparison of the two quantitative methods for the renal epithelial fraction in n=22 healthy kidney samples, respectively. CD10 is a histological marker of renal epithelium. PAX2 and ACSL5 are molecular markers that specifically target renal epithelium. [Figure 13] Figure 13 shows the significant difference in quantification of urinary CTDP1 marker between patients with transplant rejection and patients without kidney transplant rejection (left histogram), as well as a logistic regression model pooling quantitative data of biomarkers CTDP1, PAX2, PDE4D, ACSL5, and SEPT5 to predict the presence or absence of transplant rejection (right graph). [Figure 14] Figure 14 shows significant differences in the quantification of CTDP1 marker in plasma between patients with kidney transplant rejection (with tissue lesions on the graft) and patients without kidney transplant rejection (without tissue lesions on the graft) (left histogram), as well as a logistic regression model pooling quantitative data of biomarkers CTDP1, GATA2, TNS2-AS1, PAX2, and PDE4D to predict the presence or absence of transplant rejection (right graph). [Figure 15] Figure 15 shows significant differences in the quantification of the GATA2 biomarker in plasma between patients with and without vascular graft lesions (left graph), as well as a logistic regression model pooling quantitative data from the biomarkers CTDP1, GATA2, and TNS2-AS1 to predict the presence or absence of vascular lesions on the graft (right graph). DETAILED DESCRIPTION OF THE INVENTION

[0096] Of course, it should be understood that the embodiments described herein below are in no way limiting. Variants of the present invention that include only a selection of the characteristics described herein below, separated from other described characteristics, are conceivable, especially if this selection of characteristics is sufficient to provide the present invention with a technical advantage over the prior art or to distinguish the present invention. The present invention will be better understood by reading the following examples, which are provided to illustrate the present invention and not to limit its scope.

[0097] Example Example 1: Biomarker Identification CpG islands present in the promoters of specific genes are found to have different methylation levels depending on the tissue type. Using the methylation databases TCGA (The Cancer Genome Atlas) and DeepBlue (Epigenomic Data Server - DeepBlue), Gene Expression Omnibus (ID: GSE186458), the location and degree of methylation of numerous CpG islands have been enumerated for many different tissue types, including pulmonary (lung) cell subtypes, neuronal (brain), and kidney (renal) cells (Moss et al. Nat Commun. Dec. 2018;9(1):5068; Liu X et al. Clin Epigenetics. Dec. 2019;11(1):93; Silva TC et al. Bioinformatics. 1 June 2019;35(11):1974-7; Zhou W et al. Nucleic Acids Res. 24 Oct 2016;gkw967; Albrecht F et al. Nucleic Acids Res. 8 July 2016; 44(W1):W581-6). Through a bioinformatics process, the inventors identified various hypermethylated positions in healthy brain, lung, and kidney tissues. This identification involves five steps and is described below in the example of detecting lung damage.

[0098] For the detection of lung damage, we selected positions that were unmethylated in DNA from leukocytes, PBMCs, and immune cells based on the analysis of data from whole blood.

[0099] We then detected hypermethylated and / or hypomethylated CpG islands in healthy tissues in a database established from healthy lung tissues, which we divided into a discovery dataset ("training") and a validation dataset ("testing").

[0100] Differentially methylated CpG islands in lung tissue were then identified compared to CpG islands in healthy tissues of other organs (kidney, brain, muscle, stomach, liver, heart, intestine). The most differentially methylated positions were selected, and thus 432 positions were retained.

[0101] From these 432 positions, refinement was performed by manual selection. Positions were retained according to their average methylation in pulmonary and non-pulmonary tissues. Four candidate positions were selected.

[0102] The performance of these four locations to distinguish between pulmonary and non-pulmonary tissues was analyzed using a penalized logistic regression model with the LASSO method. The AUROC test of the obtained statistics allows us to conclude that multiple CpG islands allow for the identification of pulmonary tissues with a strong predictive performance.

[0103] Table 6 below summarizes the markers selected for each target organ. [Table 6]

[0104] Various amplicons were generated to distinguish each identified position. The technical characteristics of the primers and probes are listed in Table 4 below. The hybridization temperatures of the probes were selected at 40-54°C for the probes and 52-60°C for the primers, with a hybridization temperature difference of 5-10°C between the primers and the probes.

[0105] Table 7 [Table 7]

[0106] A multiplex assay was developed for each organ, and an internal standard for the amount of total circulating DNA was included in the assay, which detects the amount of DNA encoding albumin.

[0107] Example 2: Identification of biomarkers of neuronal damage in the treatment of acute stroke Validation of a biomarker according to the present invention comprises the following steps: Molecular analysis of DNA conversion in droplet digital PCR assays - Validating digital PCR assays on 100% methylated synthetic DNA and commercially available unmethylated genomic DNA Determine the metrological parameters Negative control demonstrating the specificity of the test A positive control for genomic DNA samples derived from sections of healthy tissue, and Comparison with reference histological signs.

[0108] Molecular analysis of DNA conversion in droplet digital PCR testing is common to various biomarkers. Self-contained DNA is first extracted using a commercially available extraction process specifically for self-contained DNA using the QIAamp Circulating Nucleic Acid Kit (Reference Cat. No. / ID: 55114, Qiagen). Self-contained DNA is assayed using Qubit 4.0™ Fluorimetry (Invitrogen™ Kit db HS Qubit™ DNA Assay Kits (Reference: Q32851)) and then subjected to bisulfite conversion using the Zymo Research EZ DNA Methylation-Lightning Kit (Reference D5031) or a commercially available equivalent. Samples are then analyzed by droplet digital PCR (System Naica (STILLA Technologies)).

[0109] The design of the primers and probes required for the PCR reaction on the APC2 and NBR1 targets is mentioned in Table 7. Probes of the Taqman-Mgb type are used (References: PB-MGBEF-006, PB-MGBEC-006, and PB-MGBEH-006, Eurogentec). The PCR amplification reaction is carried out in a manner that includes three main steps, presented below, and which will be referred to as "PCR amplification according to the invention" in the remainder of the text of this application.

[0110] The preparation of specific primer-probe cocktails is presented in Table 8 below. [Table 8]

[0111] The preparation of the PCR reaction mixture is presented in Table 9 below. [Table 9]

[0112] The PCR reactions are presented in Table 10. Table 10 [Table 10]

[0113] The plate is read according to the following acquisition settings: FAM channel (blue) at 120ms - HEX / VIC channel (green) at 180ms - Cy5 channel (red) at 38ms for reading on a STILLA Technologies Naica system.

[0114] Validation of the digital PCR test: The digital PCR test was then validated on 100% methylated synthetic DNA and commercially available unmethylated genomic DNA. Three synthetic DNA samples (100% methylated, Universal Methylated DNA Standard, Zymo Research Reference: D5011) and three commercially available genomic DNA samples (unmethylated, Promega G1521) were analyzed as follows: Step 1: bisulfite conversion using the Zymo Research EZ DNA Methylation-Lightning Kit (Reference D5031), followed by Step 2: analysis of NBR1 and APC2 neurogenic markers using the Naica System digital PCR.

[0115] The results for the 100% methylated DNA control show that epigenetic markers targeting the NBR1 and APC2 genes can be successfully detected and quantified by the test of the present invention. The results for the unmethylated DNA control show that the expanded NBR1 and APC2 markers can be successfully detected when, and only when, the target regions are hypermethylated. No markers are detected in the unmethylated DNA control.

[0116] The specificity of the test was verified by controls in the presence of DNA from peripheral blood mononuclear cells (PBMC) of healthy subjects.

[0117] Ten genomic DNA samples were extracted from leukocyte pellets of healthy subjects using the Qiagen QIAamp DNA Blood Mini Kit (50) Cat. No. / ID: 51104. The samples were then subjected to a bisulfite conversion step using the Zymo Research EZ DNA Methylation-Lightning Kit (Reference D5031) and then subjected to analysis of the NBR1 and APC2 neural markers by Naica System digital PCR according to the present invention as described.

[0118] Negative test results for the tested epigenetic markers indicate that this test is specific because it does not have biological noise in genomic DNA from leukocytes. "Biological noise" refers to background noise from epigenetic markers for samples considered negative or unmethylated. In other words, detection of NBR1 and APC2 neural markers results in negligible signals for samples considered non-negative or unmethylated.

[0119] The specificity of this test was also verified in the presence of plasma DNA from healthy subjects. Ten plasma DNA samples were extracted from 1 mL of plasma from healthy subjects using the Qiagen QIAamp Circulating Nucleic Acid Kit (50) Cat. No. / ID: 55114. The samples were then subjected to a bisulfite conversion step using the Zymo Research EZ DNA Methylation-Lightning Kit (Reference D5031) and then subjected to analysis of the NBR1 and APC2 neural markers of the present invention by Naica System digital PCR, as described herein. The negative test results for the tested epigenetic markers indicate that the latter do not exhibit any biological noise in self-reliant / plasma DNA derived from plasma from healthy subjects.

[0120] Finally, the specificity of this test was determined in the presence of genomic DNA from other tissues. N = 27 healthy tissue samples were collected (transfer of biological samples MTA number CT 69HCL22_0234 from the Department of Anatomic Pathology at the Hospices Civiles de Lyon Sud et Est). For each sample, three 8-μm-thick tissue sections prepared in paraffin were explanted. Genomic DNA from brain-derived tissue sections was extracted using the Qiagen QIAamp DNA FFPE Advanced Kit (50) (Cat. No. / ID: 56604). The DNA samples were then subjected to a bisulfite conversion step using the Zymo Research EZ DNA Methylation-Lightning Kit (Reference D5031) and subsequently subjected to analysis of the NBR1 and APC2 neural markers described in the present invention by Naica System digital PCR. These results demonstrate very low biological noise in genomic DNA from tissues other than brain-derived tissue. Considering the two biomarkers APC2 and NBR1, the biological noise is less than 2%. Studies of the epigenetic markers tested indicate that they have negligible or no biological noise in genomic DNA from various tissue sections (except brain).

[0121] Positive controls are performed with genomic DNA samples derived from tissue sections of healthy brain tissue by comparison with anti-NeuN histological staining.

[0122] Twelve healthy brain tissue samples were collected (transfer of biological samples MTA no. VAL 2022 / 2022-234 / 01 from the Anatomic Pathology Department of the hopital Lariboisière du Pr Homa Adle Biassette): 1 sample = white slides labeled with anti-neuN antibody (neuronal antibody, Ref ABN91, Aldricht-Merck sigma) and 3 8 μm thick tissue sections prepared in paraffin.

[0123] Neurons labeled with anti-NeuN antibodies were counted using HALO® image analysis precision software. Genomic DNA from brain tissue sections was extracted using the Qiagen QIAamp DNA FFPE Advanced Kit (50) (Cat. No. / ID: 56604). DNA samples were then subjected to a bisulfite conversion step using the Zymo Research EZ DNA Methylation-Lightning Kit (Reference D5031) and subsequently subjected to analysis of the NBR1 and APC2 neuronal markers by Naica System digital PCR as described herein.

[0124] The APC2 and NBR1 neuronal markers are identified in 12 of the 12 samples tested. Identical ratios of neurons in healthy brain tissue sections are observed between the anti-NeuN histological marker and the APC2 and NBR1 epigenetic markers. A correlation is observed between the APC2 / NBR1 epigenetic marker and the anti-NeuN histological marker (Pearson's coefficient R is 0.44 and 0.46 for the APC2 and NBR1 epigenetic markers compared to the anti-NeuN marker, respectively). These results are shown in Table 11 below and in Figure 1. [Table 11]

[0125] Correlation data between quantitative assays of brain markers (neurons) and anti-NeuN histological labeling (neuronal cell markers) showed a correlation between molecular and histological labeling (Pearson coefficient R of 0.46 and 0.44 for NBR1 and APC2, respectively), supporting the neuronal specificity (brain cells) of the test.

[0126] In clinical applications, the test according to the present invention allows stratification of stroke patients according to their severity index. The results show that the quantitative mean values ​​of APC2 and NBR1 markers are higher in patients with acute ischemic stroke (AIS) compared to patients with transient ischemic stroke (TIA) (mean values ​​of 11.32 copies / mL of plasma for TIA vs. 75 copies / mL of plasma for AIA, respectively). The results are shown in Figure 2.

[0127] Example 3: Validation of the first group of biomarkers for kidney damage in the treatment of kidney transplant patients Validation of PDE4D and PAX2 biomarkers involves molecular analysis of DNA conversion to droplet digital PCR tests, and the design of Taqman-mgb primers and probes (Reference: PB-MGBEF-006, PB-MGBEC-006, and PB-MGBEH-006, Eurogentec) is identical to that described in Example 2 (Reference: Common parts of analytical methods). Implementation of PCR amplification reactions involves the preparation of specific primer-probe cocktails, which are presented in Table 12 below. [Table 12]

[0128] The preparation of the PCR reaction mixture is presented in Table 13 below. [Table 13]

[0129] PCR reactions are performed as shown in Table 10 (Example 2). Plates are read as shown in Example 2.

[0130] Next, we validated the digital PCR test with 100% methylated synthetic DNA and commercially available unmethylated genomic DNA. Three synthetic DNA samples (100% methylated, Universal Methylated DNA Standard, Zymo Research Reference: D5011) and three commercially available genomic DNA samples (unmethylated, Promega G1521) were analyzed as follows: Step 1: bisulfite conversion using the Zymo Research EZ DNA Methylation-Lightning Kit (Reference D5031), followed by Step 2: Naica System digital PCR analysis of PDE4D and Pax2 kidney markers. The results for the synthetic DNA were: For the 1 / 100% methylated DNA control: Epigenetic markers targeting the PDE4D and Pax2 genes were successfully detected and quantified by our test. 2 / Regarding the unmethylated DNA control: The deployed PDE4D and Pax2 markers are well detectable if and only if the target region is hypermethylated. In the unmethylated DNA control, the markers are not detectable.

[0131] These results indicate that the PDE4D and PAX2 targets are not detected in commercially available unmethylated DNA.

[0132] We determined typical metrological parameters, especially the reproducibility and sensitivity of this test. The test is reproducible with coefficients of variation of 4.5% and 0.6% for the Pax2 and PDE4D markers, respectively. The test is sensitive down to 0.01% and allows quantification down to limits of 0.15 ng and 0.3 ng of DNA for the PDE4D and Pax2 markers, respectively.

[0133] The specificity of this test was verified in the presence of DNA from PBMCs (white blood cells) of healthy subjects: the marker is not detected during analysis in the DNA of PBMCs.

[0134] Ten genomic DNA samples were extracted from leukocyte pellets of healthy subjects using the Qiagen QIAamp DNA Blood Mini Kit (50) Cat. No. / ID: 51104. The samples were then subjected to bisulfite conversion using the Zymo Research EZ DNA Methylation-Lightning Kit (Reference D5031) and then subjected to analysis of the PDE4D and Pax2 kidney markers of the present invention by Naica System digital PCR as described. The markers are not detectable (= no biological background noise) when analyzing genomic DNA from leukocyte pellets of healthy subjects.

[0135] The specificity of this test was also verified in the presence of plasma DNA from healthy subjects. Ten 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. / ID: 55114. The samples were then subjected to a bisulfite conversion step using the Zymo Research EZ DNA Methylation-Lightning Kit (Reference D5031) and then subjected to analysis of the PDE4D and Pax2 kidney markers described in the present invention by Naica System digital PCR. The markers were not detected during analysis of plasma DNA (cellular DNA) from healthy subjects.

[0136] Finally, the specificity of this test was determined in the presence of genomic DNA from other tissues. N = 27 healthy tissue samples were collected (transfer of biological samples MTA number CT 69HCL22_0234 from the Department of Anatomic Pathology of the Hospices Civiles de Lyon Sud et Est and of the Hospital Lariboisiere). For each sample, three 8 μm-thick tissue sections prepared in paraffin were explanted. Genomic DNA from the tissue sections was extracted using the Qiagen QIAamp DNA FFPE Advanced Kit (50) (Cat. No. / ID: 56604). These samples were then subjected to bisulfite conversion using the Zymo Research EZ DNA Methylation-Lightning Kit (Reference D5031) and subsequently subjected to analysis of the PDE4D and Pax2 kidney markers by Naica System digital PCR according to the present invention. The negative test results for PDE4D and Pax2 epigenetic markers indicate that they were not detected in genomic DNA from tissue sections other than kidney, confirming the absence of biological noise.

[0137] A positive control was performed using genomic DNA samples derived from tissue sections of healthy kidney tissue. Twelve samples of healthy kidney tissue were collected from the Department of Anatomic Pathology at the Hospices Civils de Lyon Sud et Est. Genomic DNA from the tissue sections was extracted using the Qiagen QIAamp DNA FFPE Advanced Kit (50) (Cat. No. / ID: 56604). The samples were then subjected to a bisulfite conversion step using the Zymo Research EZ DNA Methylation-Lightning Kit (Reference D5031), followed by analysis of the PDE4D and Pax2 kidney markers by Naica System digital PCR according to the present invention.

[0138] PDE4D and Pax2 kidney markers are identified in 12 of 12 samples tested. Thus, this study supports the kidney specificity of the described PDE4D and Pax2 digital PCR test.

[0139] The amount of circulating DNA of renal origin is higher in the plasma of patients at high risk of transplant rejection compared with the plasma of patients at low risk of transplant rejection during the first few days after kidney transplantation (Figure 3).

[0140] The fraction of circulating DNA of renal origin is higher in the plasma of patients at high risk of transplant rejection compared with the plasma of patients at low risk of transplant rejection during the first few days after kidney transplantation (Figure 4).

[0141] Quantitative assay of urinary PD4ED and Pax2 markers by the described test is higher in patients with biopsy-confirmed transplant rejection than in patients without transplant rejection. This assay allows stratification of patients with biopsy-confirmed transplant rejection from those without transplant rejection (Figure 5).

[0142] Example 4: Validation of SYNE1 biomarkers for lung damage in the treatment of acute respiratory failure In the method of molecular analysis of DNA conversion into droplet digital PCR test, the design of Taqman-mgb primers and probes (Reference: PB-MGBEF-006, PB-MGBEC-006, and PB-MGBEH-006, Eurogentec) is identical to that described in Example 2. The PCR amplification reaction is carried out as follows: The preparation of specific primer-probe cocktails is presented in Table 14 below. [Table 14]

[0143] The preparation of the PCR reaction mixture is presented in Table 15 below. [Table 15]

[0144] PCR reactions are performed as shown in Table 10 (Example 2). Plates are read as shown in Example 2.

[0145] Next, the digital PCR test was validated with 100% methylated synthetic DNA and commercially available unmethylated genomic DNA. Three synthetic DNA samples (100% methylated, Universal Methylated DNA Standard, Zymo Research Reference: D5011) and three commercially available genomic DNA samples (unmethylated, Promega G1521) were analyzed as follows: Step 1: bisulfite conversion using the Zymo Research EZ DNA Methylation-Lightning Kit (Reference D5031), followed by Step 2: Naica System digital PCR analysis of the SYNE1 pulmonary marker.

[0146] The results for the 100% methylated DNA control demonstrate that epigenetic markers targeting the SYNE1 gene can be fully detected and quantified by the test of the present invention implementing this marker. The results for the unmethylated DNA control demonstrate that the deployed SYNE1 marker is well detectable when and only when the target region is hypermethylated. There is no detection of the marker in the unmethylated DNA control.

[0147] The specificity of this test was verified in the presence of genomic DNA derived from PBMCs (white blood cells) of healthy subjects. Ten genomic DNA samples were extracted from the white blood cell pellets of healthy subjects using the Qiagen QIAamp DNA Blood Mini Kit (50) Cat. No. / ID: 51104. The samples were then subjected to a bisulfite conversion step using the Zymo Research EZ DNA Methylation-Lightning Kit (Reference D5031) and then subjected to analysis of the SYNE1 pulmonary marker of the present invention by Naica System digital PCR, as described herein. The marker was not detected during analysis of genomic DNA derived from PBMCs.

[0148] The specificity of this test was also verified in the presence of plasma DNA from healthy subjects. Ten plasma samples were extracted from 1.5 mL of healthy subject plasma using the Qiagen QIAamp Circulating Nucleic Acid Kit (50) Cat. No. / ID: 55104. The samples were then subjected to a bisulfite conversion step using the Zymo Research EZ DNA Methylation-Lightning Kit (Reference D5031) and then subjected to analysis of the SYNE1 pulmonary marker of the present invention described by the Naica System digital PCR. The marker was not detected during analysis with genomic DNA from healthy subjects.

[0149] Finally, the specificity of the test was determined in the presence of genomic DNA from other tissues. N = 27 healthy tissue samples were collected (transfer of biological samples MTA number CT 69HCL22_0234 from the Department of Anatomic Pathology of the Hospices Civiles de Lyon Sud et Est and of the Hospital Lariboisiere). For each sample, three 8 μm-thick tissue sections prepared in paraffin were explanted. Genomic DNA from brain-derived tissue sections was extracted using the Qiagen QIAamp DNA FFPE Advanced Kit (50) (Cat. No. / ID: 56604). The samples were then subjected to a bisulfite conversion step using the Zymo Research EZ DNA Methylation-Lightning Kit (Reference D5031) and subsequently subjected to analysis of the SYNE1 pulmonary marker of the present invention by Naica System digital PCR, as described herein. The negative test result for the SYNE1 epigenetic marker indicates that it was not detected in genomic DNA from tissue sections other than lung, confirming the absence of biological noise.

[0150] Positive control: A positive control was performed using genomic DNA samples derived from tissue sections of healthy lung tissue by comparison with histological labeling. Eighteen healthy lung tissue samples (one white slide and three 8-μm-thick sections prepared in paraffin) were collected from the Department of Anatomic Pathology at the Hospices Civils de Lyon Sud et Est. For each sample, labeling with anti-TTF1 antibody was performed on white slides (antibody TTF1, Reference: DB089-0.5 Rabbit Monoclonal Anti-TTF-1 Clone (G21-G), DB Biotech), and the number of TTF1-labeled cells (=pulmonary lung cells) was counted using "HALO® image analysis" software. Samples were labeled with antibody TTF1. Simultaneously, genomic DNA from 8-μm-thick tissue sections prepared in paraffin was prepared using the Qiagen QIAamp DNA FFPE Advanced Kit (50) (Cat. No. / ID: 56604). The samples were then subjected to a bisulfite conversion step with the Zymo Research EZ DNA Methylation-Lightning Kit (Reference D5031) and then subjected to analysis by Naica System digital PCR of the SYNE pulmonary marker according to the invention as described.

[0151] These results demonstrate that hypermethylated targets of SYNE1 are detected and quantified exclusively in genomic DNA from healthy lung sections, confirming lung specificity. The correlation between histological labeling of TTF1 (= pneumocytes) and the described lung assay was 0.6 (R-Pearson coefficient), demonstrating the lung and pneumocyte specificity of the analysis (Figure 6).

[0152] In clinical applications, the test according to the present invention allows stratification of patients with acute respiratory distress syndrome (ARDS) according to the severity index, designated as low, severe, or critical (Berlin classification). These results indicate that hypermethylated targets of SYNE1 can distinguish between subgroups of patients with moderate, severe, and critical ARDS. A significant difference was observed between these three groups (p-value = 0.0073, Jonckheere-Terpstra test) (Figure 7).

[0153] Furthermore, the results of the study according to the present invention make it possible to monitor the pulmonary status of ARDS patients by correlation indices between the quantitative results of hypermethylated targets of SYNE1 and the ventilation markers O2 max / FiO2 / PAO2 (Pearson correlation coefficients of 0.44, 0.29, and 0.29, respectively).

[0154] Example 5: Validation of a second group of biomarkers for kidney damage in the treatment of kidney transplant patients Two diagnostic tests targeting kidney transplant rejection and types of kidney lesions (total, vascular, and epithelial) were developed by selecting from the markers identified by the present inventors.

[0155] In prototype 1, the markers SEPT5 (glomerular endothelial marker), PDE4D / PAX2 / ACSL5 (tubular epithelial marker), and CTDP1 (pan-kidney marker) are tested in urine samples.

[0156] In prototype 2, the markers GATA2 (renal endothelial marker); TNS2-AS1 (renal capillary endothelial marker) / PDE4D / PAX2 (tubular epithelial marker); CTDP1 (total kidney marker) will be tested in plasma samples.

[0157] For the two prototypes, the "albumin" marker is used as a universal marker to quantify all genomes present in the sample. Biological samples were obtained through the Nephrology Department of Necker and its hospital partners at Hôpital Tenon-Pitie-Salpetriere.

[0158] The concentrations of the different relevant biomarkers in the multiplex (here in a PCR mix) constituting a dedicated kit for monitoring the state of kidney tissue damage in the context of kidney transplantation, at the level of the whole organ, its epithelial fraction and its vascular fraction, are used according to the concentrations provided in Table 16 below. [Table 16]

[0159] After validating these two prototypes at a quantitative level (precision, repeatability, reproducibility), the two prototypes were tested with various so-called "positive" biological controls (genomic DNA from kidney) and so-called "negative" controls (genomic DNA from liver, leukocytes, and plasma and urinary circulating DNA from healthy subjects) (Figure 9). Liver and leukocyte genomic DNA were the controls evaluated in the design of this study and methodology because they contribute to urinary and plasma circulating DNA in healthy subjects.

[0160] These analyses allowed us to confirm that our biomarkers were not detected or were detected with very low noise in negative control biological samples: N=20 genomic DNA samples extracted from cell pellets of healthy subjects, N=30 plasma DNA samples from healthy subjects, N=15 urinary DNA samples from healthy subjects, N=5 genomic DNA samples extracted from tissue sections (5 liver gDNA).

[0161] These gDNAs: urinary cfDNA and plasma cfDNA5, are mentioned as potential low-noise contributions to circulating DNA in healthy subjects.

[0162] result The above-mentioned markers are detected in 100% of the genomic DNA extracted from kidney tissue sections (N=22).

[0163] The markers proposed in this in vitro diagnostic test prototype allow the detection of kidney DNA with very low biological noise, especially in genomic DNA of liver and leukocytes (the major contributors to circulating DNA) (Figure 8). In plasma circulating DNA of healthy subjects, there is very low biological background noise (slightly higher for the CATA2 endothelial marker, consistent with the literature). In urinary circulating DNA of healthy subjects, pan-kidney and renal epithelial markers are found, in accordance with the literature and presumably corresponding to the renal fraction physiologically present in urine.

[0164] The CTDP1 "whole kidney" marker allows for quantification of the endothelial and epithelial fractions of the kidney. It is found in higher abundance in kidney genomic DNA compared to markers that target only the epithelial and endothelial fractions. Similarly, a marker that targets the endothelial fraction of the kidney (GATA2) is found in higher abundance than the TNS2-AS1 and SEPT5 markers (restricted to glomeruli and tubules, respectively), which target the smaller vascular fractions.

[0165] Example 6: Verification of the specificity of renal epithelial markers: Comparison of CD10, molecular markers of renal epithelium (PAX2 / ACSL5), and histological markers We have shown that PAX2 and ACSL5 markers specifically target kidney epithelium. Therefore, these markers were tested to quantify kidney epithelium. In total kidney gDNA (n=22) and in urine plasma circulating DNA, these markers were significantly higher in R, as shown in Figures 10 and 11, respectively. 2 = 0.5 and R 2 A good correlation of =0.8 was observed.

[0166] A publication by Erger et al. showed that a variable fraction of 20-50% of the total urinary circulating DNA in healthy subjects is of renal origin (Erger et al. Genome Medicine, (2020), doi.org / 10.1186 / s13073-020-00750-5) by proposing a bioinformatic deconvolution model (cfNOMe) of urinary circulating DNA by NGS sequencing. This fraction is confirmed by the results of the present invention, suggesting a good specificity of the markers in terms of detecting circulating DNA of renal origin. Furthermore, urinary circulating DNA of renal origin is rather of epithelial origin (detection of vascular marker signals, e.g., GATA2, TNS2-AS1, and SEPT-5, as well as absence of detection of markers PAX2, ACSL5, and PDE4D (for n=15 urinary circulating DNA samples extracted from healthy subjects, R 2 Correlation (PAX2 / ACSL5) = 0.80).

[0167] These results indicate that the PAX2 and ACSL5 markers specifically target renal epithelium. To strengthen the results, a comparison was made between the molecular data of the PAX2 and ACSL5 markers and histological data (labeling of renal epithelial cells on kidney white slides with anti-CD10 antibodies). Genomic DNA from 22 healthy kidneys, for which PAX2 and ACSL5 were quantified, was subjected to immunohistochemical labeling of renal epithelial cells with anti-CD10 antibodies on paired white slides. CD10 is a surface marker that specifically targets epithelial cells within kidney sections. The histological and molecular data indicate that the proportion of gDNA paired with epithelial cells and kidney on these slides varied from 11 to 40%, and there was no significant difference between the molecular and histological markers targeting renal epithelium (Figures 12A and 12B).

[0168] Example 7: Analysis of urine samples from kidney transplant patients To validate the clinical relevance of the first prototype, a clinical collaboration in partnership with the Nephrology / Transplantation Department of the Hopital Necker and Tenon-Pitie Salpetriere enabled the analysis of N=50 urine samples from kidney transplant patients with and without graft rejection, supported by anatomopathological analysis of solid biopsies, and N=55 plasma samples from kidney transplant patients with biopsies with and without cellular lesions, supported by anatomopathological analysis. The aim of these biomarkers is to specifically detect the cellular origin of kidney lysis and therefore the type of rejection according to the Bannff classification of kidney transplant rejection.

[0169] Fifty biological samples (2 ml urine samples) from kidney transplant patients with or without kidney transplant rejection (solid biopsy-confirmed rejection) were analyzed. Of these patients, 20 had T cell-mediated rejection (CTMR) and 30 had no transplant rejection.

[0170] Two milliliters of urine was collected on the day of kidney biopsy. Circulating DNA was extracted and analyzed simultaneously for a set of biomarkers (SEPT-5, ACSL5, PAX2, PDE4D, CTDP1) by multiplex digital PCR (6 colors, one color for each marker and an albumin internal control).

[0171] A significant increase in biomarker levels was observed in urine from patients with T-cell rejection (Wilcoxon test, p-value <0.001). The regression model applied to the biomarkers showed good sensitivity and specificity of the diagnostic test of 0.83 and 0.85, respectively, with an AUC score of 0.88.

[0172] Figure 13 shows significant differences in quantification of urinary CTDP1 biomarker between patients with and without kidney transplant rejection (left graph). Logistic regression model pooling quantitative data from biomarkers CTDP1, PAX2, PDE4D, ACSL5, and SEPT5 to predict the presence or absence of transplant rejection (right graph).

[0173] Example 8: Analysis of plasma samples from kidney transplant patients Fifty-five biological samples from kidney transplant patients with or without kidney transplant rejection (solid biopsy-confirmed rejection in biopsies with glomerular (g), tubular (t), vascular (v), and capillary (ptc) type graft lesions) were analyzed.

[0174] 0.6–1.5 mL of plasma was collected on the day of kidney biopsy. Circulating DNA was extracted and analyzed simultaneously for a set of biomarkers (GATA2, TNS2-AS1, PAX2, PDE4D, and CTDP1) by multiplex digital PCR (6 colors, one color for each marker plus an albumin internal control).

[0175] A significant increase in the amount of biomarkers was also observed in the plasma of transplant patients with kidney graft failure. The regression model applied to the biomarkers showed good sensitivity and specificity of the diagnostic test of 0.96 and 0.75, respectively, with an AUC score of 0.82. These results emphasize the robustness of this test in analyzing transplant rejection in plasma samples. A significant increase in the amount of vascular biomarkers (specifically, GATA2 and TNS2-AS1 markers) was also observed, as expected, in the plasma of patients with vascular lesions of the kidney graft (Figure 14).

[0176] A regression model applied to the "whole kidney" and "vascular" biomarkers (specifically the markers CTDP1, GATA2, and TNS2-AS1) had an AUC of 0.878, indicating good sensitivity and specificity of the diagnostic test (Figure 15).

[0177] In summary, these results are the technical reliability of the developed test in detecting the biomarkers of interest in the urine and plasma of patients; the sensitivity and technical specificity of the developed test; Clinical interest of the developed test in detecting kidney transplant rejection; Clinical interest of the developed test in characterizing epithelial and / or vascular lesions in renal allografts Shows.

Claims

1. 1. A kit for detecting a methylated self-lying DNA target nucleotide sequence in a biological sample, wherein the nucleotide sequence is specific to a cell type of an organ selected from the group consisting of brain, lung, and kidney, the kit comprising: i) for detecting specific self-reliant DNA target sequences of cell types present in the brain, at least one sense primer comprising the nucleotide sequence of SEQ ID NO:1 or a nucleotide sequence having at least 80% identity to SEQ ID NO:1, and one antisense primer comprising the nucleotide sequence of SEQ ID NO:2 or a nucleotide sequence having at least 80% identity to SEQ ID NO:2, and / or one sense primer comprising the nucleotide sequence of SEQ ID NO:4 or a nucleotide sequence having at least 80% identity with SEQ ID NO:4, and one antisense primer comprising the nucleotide sequence of SEQ ID NO:5 or a nucleotide sequence having at least 80% identity with SEQ ID NO:5; ii) at least one method for detecting specific self-repeated DNA target sequences of cell types present in the lung; one sense primer comprising the nucleotide sequence of SEQ ID NO:7 or a nucleotide sequence having at least 80% identity to SEQ ID NO:7, and one antisense primer comprising the nucleotide sequence of SEQ ID NO:8 or a nucleotide sequence having at least 80% identity to SEQ ID NO:8; and iii) at least one method for detecting specific self-repeated DNA target sequences of cell types present in the kidney, one sense primer comprising the sequence SEQ ID NO: 10 or a sequence having at least 80% identity with SEQ ID NO: 10, and one antisense primer comprising the sequence SEQ ID NO: 11 or a sequence having at least 80% identity with SEQ ID NO: 11, and / or one sense primer comprising the sequence SEQ ID NO: 13 or a sequence having at least 80% identity with SEQ ID NO: 13, and one antisense primer comprising the sequence SEQ ID NO: 14 or a sequence having at least 80% identity with SEQ ID NO: 14, and / or one sense primer comprising the sequence SEQ ID NO: 19 or a sequence having at least 80% identity with SEQ ID NO: 19, and one antisense primer comprising the sequence SEQ ID NO: 20 or a sequence having at least 80% identity with SEQ ID NO: 20, and / or one sense primer comprising the sequence SEQ ID NO: 22 or a sequence having at least 80% identity with SEQ ID NO: 22, and one antisense primer comprising the sequence SEQ ID NO: 23 or a sequence having at least 80% identity with SEQ ID NO: 23, and / or one sense primer comprising the sequence SEQ ID NO:25 or a sequence having at least 80% identity with SEQ ID NO:25, and one antisense primer comprising the sequence SEQ ID NO:26 or a sequence having at least 80% identity with SEQ ID NO:26, and / or one sense primer comprising the sequence SEQ ID NO:28 or a sequence having at least 80% identity with SEQ ID NO:28, and one antisense primer comprising the sequence SEQ ID NO:29 or a sequence having at least 80% identity with SEQ ID NO:29, and / or one sense primer comprising the sequence SEQ ID NO: 31 or a sequence having at least 80% identity with SEQ ID NO: 31, and one antisense primer comprising the sequence SEQ ID NO: 32 or a sequence having at least 80% identity with SEQ ID NO: 32, and / or one sense primer comprising the sequence SEQ ID NO: 34 or a sequence having at least 80% identity with SEQ ID NO: 34, and one antisense primer comprising the sequence SEQ ID NO: 35 or a sequence having at least 80% identity with SEQ ID NO: 35, and / or one sense primer comprising the sequence SEQ ID NO: 37 or a sequence having at least 80% identity with SEQ ID NO: 37, and one antisense primer comprising the sequence SEQ ID NO: 38 or a sequence having at least 80% identity with SEQ ID NO: 38, and / or one sense primer comprising the sequence SEQ ID NO: 40 or a sequence having at least 80% identity with SEQ ID NO: 40, and one antisense primer comprising the sequence SEQ ID NO: 41 or a sequence having at least 80% identity with SEQ ID NO: 41, and / or one sense primer comprising the sequence SEQ ID NO: 43 or a sequence having at least 80% identity with SEQ ID NO: 43, and one antisense primer comprising the sequence SEQ ID NO: 44 or a sequence having at least 80% identity with SEQ ID NO: 44, and / or one sense primer comprising the sequence SEQ ID NO: 46 or a sequence having at least 80% identity with SEQ ID NO: 46, and one antisense primer comprising the sequence SEQ ID NO: 47 or a sequence having at least 80% identity with SEQ ID NO: 47, and / or one sense primer comprising the sequence SEQ ID NO: 49 or a sequence having at least 80% identity with SEQ ID NO: 49, and one antisense primer comprising the sequence SEQ ID NO: 50 or a sequence having at least 80% identity with SEQ ID NO: 50, and / or one sense primer comprising the sequence of SEQ ID NO:52 or a sequence having at least 80% identity with SEQ ID NO:52, and one antisense primer comprising the sequence of SEQ ID NO:53 or a sequence having at least 80% identity with SEQ ID NO:53; Includes a kit.

2. i) 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 for detecting a specific self-lying DNA target sequence of a cell type present in the brain; ii) 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 for detecting a specific self-reliant DNA target sequence of a cell type present in the lung; and iii) 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 to 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 for detecting a specific self-lying DNA target sequence of a cell type present in the kidney; 10. The kit of claim 1, further comprising a probe comprising:

3. 3. A kit according to claim 1 or 2 for detecting in a biological sample a self-repeated DNA target sequence specific for a cell type present in the brain.

4. 3. A kit according to claim 1 or 2 for detecting in a biological sample a self-repeated DNA target sequence specific for a cell type present in the lung.

5. 3. The kit according to claim 1 or 2, for detecting in a biological sample a self-repeated DNA target sequence specific for a cell type present in the kidney.

6. 1. A method for identifying specific self-repeated DNA target sequences of a cell type present in a first organ selected from the group consisting of lung, brain, and kidney, comprising at least: a) determining the location and degree of methylation of CpG islands of specific sequences of said cell type of said organ in at least one genomic DNA methylation database; b) identifying differentially hypermethylated CpG islands in the genomic DNA of said cell types of said healthy organ; c) comparing the degree of methylation of the hypermethylated CpG islands in said cell type of said first organ with the degree of methylation of the same CpG islands in a second cell type different from said first cell type; d) selecting only hypermethylated CpG islands in the genomic DNA of said cell type of said first organ after the comparison performed in step c); e) comparing the degree of methylation of the hypermethylated CpG islands of said cell types of said first organ with the degree of methylation of the same CpG islands in genomic DNA of other organs of said first organ; f) comparing the degree of methylation of hypermethylated CpG islands in said cell type of said first organ with the degree of methylation of the same CpG islands in genomic DNA of healthy leukocytes; g) comparing 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 self-lying DNA of all biological matrices combined from healthy subjects; h) selecting only those CpG islands that are hypermethylated in the genomic DNA of said cell type of said first organ after the comparison performed in steps e, f, and g); i) identifying one or more targeted hypermethylated sequences of the specific self-lying DNA of the cell type of said first organ from the comparison performed in step h); A method comprising:

7. 1. A method for detecting a target sequence of self-lying DNA in a biological sample, wherein the sequence is specific to a cell type present in an organ selected from: lung, brain, and kidney, and comprises at least the following: a) combining, under conditions suitable for nucleic acid amplification, self-ligated DNA previously extracted from a biological sample and a pair of primers consisting of a sense primer and an antisense primer, each of said primers comprising: a sense primer and an antisense primer that may hybridize to a nucleotide sequence selected 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) amplifying the target sequence; c) detecting the presence of the sequence amplified during step b). A method comprising:

8. The sequence is specific for a cell type present in an organ selected from the group consisting of lung, brain, and kidney, and includes at least the following: a) combining, under conditions suitable for amplifying said nucleic acid, self-ligated DNA previously extracted from a biological sample and a pair of primers consisting of a sense primer and an antisense primer, each of said primers comprising: i) for detecting specific target sequences of cell types present in the brain, at least: one sense primer comprising the sequence SEQ ID NO: 1 or a sequence having at least 80% identity with SEQ ID NO: 1, and one antisense primer comprising the sequence SEQ ID NO: 2 or a sequence having at least 80% identity with SEQ ID NO: 2, and / or a sense primer comprising the sequence of SEQ ID NO: 4 or a sequence having at least 80% identity with SEQ ID NO: 4, and one antisense primer comprising the sequence of SEQ ID NO: 5 or a sequence having at least 80% identity with SEQ ID NO: 5; ii) at least one antibody for detecting specific target sequences of cell types present in the lung; one sense primer comprising the sequence of SEQ ID NO: 7 or a sequence having at least 80% identity with SEQ ID NO: 7, and one antisense primer comprising the sequence of SEQ ID NO: 8 or a sequence having at least 80% identity with SEQ ID NO: 8; and iii) at least one antibody for detecting specific target sequences of cell types present in the kidney; one sense primer comprising the sequence SEQ ID NO: 10 or a sequence having at least 80% identity with SEQ ID NO: 10, and one antisense primer comprising the sequence SEQ ID NO: 11 or a sequence having at least 80% identity with SEQ ID NO: 11, and / or one sense primer comprising the sequence SEQ ID NO: 13 or a sequence having at least 80% identity with SEQ ID NO: 13, and one antisense primer comprising the sequence SEQ ID NO: 14 or a sequence having at least 80% identity with SEQ ID NO: 14, and / or one sense primer comprising the sequence SEQ ID NO: 19 or a sequence having at least 80% identity with SEQ ID NO: 19, and one antisense primer comprising the sequence SEQ ID NO: 20 or a sequence having at least 80% identity with SEQ ID NO: 20, and / or one sense primer comprising the sequence SEQ ID NO: 22 or a sequence having at least 80% identity with SEQ ID NO: 22, and one antisense primer comprising the sequence SEQ ID NO: 23 or a sequence having at least 80% identity with SEQ ID NO: 23, and / or one sense primer comprising the sequence SEQ ID NO:25 or a sequence having at least 80% identity with SEQ ID NO:25, and one antisense primer comprising the sequence SEQ ID NO:26 or a sequence having at least 80% identity with SEQ ID NO:26, and / or one sense primer comprising the sequence SEQ ID NO:28 or a sequence having at least 80% identity with SEQ ID NO:28, and one antisense primer comprising the sequence SEQ ID NO:29 or a sequence having at least 80% identity with SEQ ID NO:29, and / or one sense primer comprising the sequence SEQ ID NO: 31 or a sequence having at least 80% identity with SEQ ID NO: 31, and one antisense primer comprising the sequence SEQ ID NO: 32 or a sequence having at least 80% identity with SEQ ID NO: 32, and / or one sense primer comprising the sequence SEQ ID NO: 34 or a sequence having at least 80% identity with SEQ ID NO: 34, and one antisense primer comprising the sequence SEQ ID NO: 35 or a sequence having at least 80% identity with SEQ ID NO: 35, and / or one sense primer comprising the sequence SEQ ID NO: 37 or a sequence having at least 80% identity with SEQ ID NO: 37, and one antisense primer comprising the sequence SEQ ID NO: 38 or a sequence having at least 80% identity with SEQ ID NO: 38, and / or one sense primer comprising the sequence SEQ ID NO: 40 or a sequence having at least 80% identity with SEQ ID NO: 40, and one antisense primer comprising the sequence SEQ ID NO: 41 or a sequence having at least 80% identity with SEQ ID NO: 41, and / or one sense primer comprising the sequence SEQ ID NO: 43 or a sequence having at least 80% identity with SEQ ID NO: 43, and one antisense primer comprising the sequence SEQ ID NO: 44 or a sequence having at least 80% identity with SEQ ID NO: 44, and / or one sense primer comprising the sequence SEQ ID NO: 46 or a sequence having at least 80% identity with SEQ ID NO: 46, and one antisense primer comprising the sequence SEQ ID NO: 47 or a sequence having at least 80% identity with SEQ ID NO: 47, and / or one sense primer comprising the sequence SEQ ID NO: 49 or a sequence having at least 80% identity with SEQ ID NO: 49, and one antisense primer comprising the sequence SEQ ID NO: 50 or a sequence having at least 80% identity with SEQ ID NO: 50, and / or one sense primer comprising the sequence of SEQ ID NO:52 or a sequence having at least 80% identity with SEQ ID NO:52, and one antisense primer comprising the sequence of SEQ ID NO:53 or a sequence having at least 80% identity with SEQ ID NO:53; and b) amplifying the target sequence; c) detecting the presence of the sequence amplified during step b). The method of claim 7, comprising:

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

10. 8. The method of claim 7 for detecting in a biological sample a self-repeated DNA target sequence specific for a cell type present in the lung.

11. 8. The method of claim 7 for detecting in a biological sample a self-repeated DNA target sequence specific for a cell type present in the kidney.

12. 12. A method according to any one of claims 1 to 11 for detecting damaged kidney cell types, wherein said cell types are selected from epithelial cells and vascular cells.

13. 13. The method of claim 11 or 12 for detecting kidney transplant rejection in a kidney transplant patient.

14. 13. A method according to claim 11 or 12 for the in vitro diagnosis of the type of rejection lesion, wherein the rejection is epithelial or vascular depending on the histological analysis of a solid biopsy of the kidney transplant.

15. Use of a kit according to any one of claims 1 to 3 or a method according to claim 9 for specifically detecting brain damage and / or for diagnosing or monitoring the progression of stroke in vitro.

16. 11. Use of the kit of claim 1, 2 or 4 or the method of claim 10 for specifically detecting lung damage and / or for diagnosing or monitoring the progression of respiratory distress syndrome in vitro.

17. Use of a kit according to claim 1, 2 or 5 or a method according to claim 7, 8 or 11 for specifically detecting kidney damage and / or for diagnosing or monitoring the progression of kidney damage or transplant rejection in vitro.

18. a sense primer comprising 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 and SEQ ID NO:52, or a nucleotide sequence having at least 80% identity to said sequence; an antisense primer comprising a nucleotide sequence selected 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 thereto; and A probe comprising a nucleotide sequence selected 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 to said sequence. A nucleotide sequence selected from the group consisting of: for use in a kit according to any one of claims 1 to 5, for use in a method according to any one of claims 7 to 11, or for use according to any one of claims 12 to 14.

Images