Detection of dialysis-induced brain ischemia

Abstract

The present invention relates to a method for measuring, haemodialysis-associated brain ischemia, haemodialysis-associated brain hypoxia, and / or haemodialysis-associated neural cell death in a human subject based on the quantification of neuronal and / or glial cfDNA methylation profile. The invention further relates to a kit comprising a set of capture probe and its use.

Description

[0001] DETECTION OF DIALYSIS-INDUCED BRAIN ISCHEMIA

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to a method for measuring haemodialysis-associated brain ischemia, haemodialysis-associated brain hypoxia, and / or haemodialysis-associated neural cell death, and to a kit for its implementation.

[0004] BACKGROUND OF THE INVENTION

[0005] For patients with end-stage renal disease, haemodialysis is the most common form of kidney replacement therapy. A particularly harsh and pervasive complication associated with haemodialysis is cognitive impairment. Patients on haemodialysis are at a 44%- increased risk of cognitive decline, and this often progresses to dialysis dementia. While the etiopathogenesis of haemodialysis-associated cognitive decline is often unclear, several factors are known to contribute to its development. A key trigger for neuronal damage is intradialytic haemodynamic instability. The haemodialysis procedure itself reduces the fluid volume in patients, which causes frequent hypotensive episodes and rapid fluid shifts. This leads to cerebral ischemia, hypoxia, and oedema. This was clearly illustrated in a large US cohort, where patients who started haemodialysis had a 4 to 8- fold increased risk for stroke in their first year on dialysis when compared to the year before dialysis. Dialysis initiation thus imposes an increased risk of ischemic cerebrovascular disease. The importance of the dialysis procedure itself in the pathogenesis is further highlighted by the 30% higher risk for cognitive decline in haemodialysis patients versus peritoneal dialysis patients, even when discounting differences in patient characteristics between both modalities. A key difference between both treatments is that haemodialysis entails removal of excess fluid over a short period of time (3-5 hours, 3 times per week), while peritoneal dialysis is a much more gradual procedure. In line with this, studies have reported that up to 20-30% of haemodialysis sessions are affected by intradialytic haemodynamic instability, with some patients experiencing multiple episodes within a single session. Severe intradialytic haemodynamic instability, defined as a decrease in systolic blood pressure by > 20 mmHg or to < 90 mmHg, occurs in approximately 10-20% of haemodialysis treatments. Knowing that the brain receives 15-20% of the cardiac output, it is unsurprising that patients on haemodialysis frequently have severe white matter disease, lacunae and infarcts, and atrophy on brain imaging. This white matter damage also impacts cognition, as more white matter damage in dialysis patients correlates with poorer cognitive test scores. Factors contributing to intradialytic hemodynamic instability in the dialysis procedure include the rapid fluid removal, inadequate volume management, and the high burden of underlying cardiovascular comorbidities. Guiding haemodialysis to its treatment goals while avoiding neuronal ischemia should be the standard in dialysis care. This is however not achievable due to the lack of validated neuronal ischemia markers. This lack of markers precludes a safe, patient-tailored, treatment.

[0006] Several protein biomarkers have been studied for their potential utility in assessing neuronal damage and neurocognitive function in patients undergoing dialysis (Ref. 1-3). Some biomarkers show promise in this context, but none are validated for clinical significance and utility. The interpretation of biomarker findings is moreover confounded by haemodialysis-related variables such as the molecular weight cut-off of the haemodialysis membrane. The latter can induce removal of the protein during dialysis, thus invalidating its potential as a biomarker for intradialytic complications. At a molecular weight of 21kD, SIOOB for example is anticipated to be cleared rather efficiently during dialysis. Identifying sensitive and specific biomarkers for neurological damage and the associated cognitive decline stand to ameliorate management of neurocognitive complications in dialysis patients by guiding the personalized management strategies. The current lack of such biomarkers however renders these dialysis patient management strategies reactive, rather than aiming to prevent neuronal loss.

[0007] Cell-free DNA (cfDNA) is a class of short double- stranded DNA fragments, typically about 150 base pairs long, that can be found in bodily fluids such as blood, urine and saliva. The release of cfDNA from the cells to the extracellular environment occurs in both physiological and pathological circumstances. cfDNA can be released through multiple different processes, including cell death (apoptosis, necrosis) and active release (NETosis). In healthy individuals, cfDNA concentrations are low and half-life is short due to rapid elimination by the liver and spleen, while elevated cfDNA concentrations are observed in pathogenic conditions resulting from excessive cell death and inflammation. Increased cfDNA concentrations have been described in cancer but also in various ischemic conditions, such as following acute myocardial infarction and ischemic stroke (Ref. 4-6). Whether these increases solely reflect the products of myocardial or neuronal cell death, or also the sequelae of inflammatory reactions, remains unclear. Also in haemodialysis, elevated cfDNA concentrations have been detected after a dialysis session, and both post- and pre-dialysis cfDNA levels can predict mortality in dialysis patients. Also here, both inflammatory processes and organ injury due to comorbidities have been proposed as explanations (Ref. 7), but all studies are based on associations, and the precise origin of the increase is yet to be revealed.

[0008] SUMMARY

[0009] Here the inventors have developed a method to detect and quantify haemodialysis- induced brain ischemia based on the detection of neuron- specific and / or glial cell-specific DNA methylation in cfDNA.

[0010] The present invention relates to a method for measuring haemodialysis-associated brain ischemia, haemodialysis-associated brain hypoxia and / or haemodialysis-associated neural cell death, in a human subject comprising the steps of, a. providing a cell-free DNA sample, previously obtained from said human subject during or after haemodialysis; b. determining the DNA methylation profiles of a plurality of genomic regions in the cfDNA sample provided at step a; c. quantifying neuron- specific and / or glial cell-specific DNA methylation profiles in the DNA methylation profiles obtained at step b; and, d. measuring, based on the quantification performed at step c, hemodialysis- associated brain ischemia, haemodialysis-associated brain hypoxia, and / or haemodialysis-associated neural cell death in said human subject, wherein haemodialysis-associated brain ischemia, haemodialysis-associated brain hypoxia, and / or haemodialysis-associated neural cell death, increases with the level of neuron- specific and / or glial cell-specific DNA methylation in said cfDNA sample.

[0011] In one embodiment, step (b) comprises determining, in the sample provided at step a, the DNA methylation profiles of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 or 49 genomic regions and wherein each of said genomic regions is distinct and defined in reference to one of SEQ ID NO: 1 to 1551, as found in the human genome build GRCh38 / hg38.

[0012] In one embodiment, wherein step (b) comprises determining, in the sample provided at step a, the DNA methylation profiles of at least 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123,

[0013] 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141,

[0014] 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159,

[0015] 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177,

[0016] 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195,

[0017] 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213,

[0018] 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231,

[0019] 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249,

[0020] 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267,

[0021] 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285,

[0022] 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298 or 299 genomic regions and wherein each of said genomic regions is distinct and defined in reference to one of SEQ ID NO: 1 to 1551, as found in the human genome build GRCh38 / hg38.

[0023] In one embodiment, step (b) comprises determining, in the sample provided at step a, the DNA methylation profiles of at least 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327,

[0024] 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345,

[0025] 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363,

[0026] 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381,

[0027] 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399,

[0028] 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417,

[0029] 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435,

[0030] 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453,

[0031] 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471,

[0032] 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489,

[0033] 490, 491, 492, 493, 494, 495, 496, 497, 498, 499 or 500 genomic regions and wherein each of said genomic regions is distinct and defined in reference to one of SEQ ID NO: 1 to 1551, as found in the human genome build GRCh38 / hg38.

[0034] In one embodiment, said cell-free DNA sample was obtained at least 10 minutes after the beginning of haemodialysis, preferably at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 minutes after the beginning of haemodialysis, more preferably at least 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118 or 119 minutes after the beginning of haemodialysis, even more preferably at least 120 minutes after the beginning of haemodialysis.

[0035] In one embodiment, said cell-free DNA sample was obtained in a time interval ranging from 10 minutes after the beginning of haemodialysis to 2 days after the end of haemodialysis, preferably to 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14 or 13 hours after the end of haemodialysis, preferably to 12, 11, 10, 9, 8, 7, 6, 5 or 4 hours after the end of haemodialysis, more preferably to 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 20 or 10 minutes after the end of haemodialysis.

[0036] In one embodiment, said cell-free DNA is circulating cell-free DNA.

[0037] In one embodiment, said sample is a blood, plasma or serum sample. In one embodiment, said sample is a plasma sample.

[0038] In one embodiment, said human subject is affected with kidney failure.

[0039] The invention further relates to a kit, comprising capture probes for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,

[0040] 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 genomic regions and wherein each of said genomic regions is distinct and defined in reference to one of SEQ ID NO: 1 to 1551, as found in the human genome build GRCh38 / hg38.

[0041] The invention further relates to the use of the kit of the invention, for measuring haemodialysis-associated brain ischemia, haemodialysis-associated brain hypoxia, and / or haemodialysis-associated neural cell death.

[0042] DEFINITIONS

[0043] In the present invention, the following terms have the following meaning unless the context clearly dictate otherwise.

[0044] “cell-free DNA” or “cfDNA” are used interchangeably herein in reference to DNA fragments, found in the bodily fluids of the subject outside of said subject’s cells. Examples of bodily fluids wherein cfDNA can be found include, without being limited to, blood, plasma, serum, urine and saliva. cfDNA is typically found in the form of short double stranded DNA fragment. The release of cfDNA from the cells to the extracellular environment occurs in both physiological and pathological circumstances. cfDNA can be released through multiple different processes, including, without being limited to, cell death (e.g. apoptosis, necrosis) and active release (e.g. NETosis). The term “circulating cell-free DNA” refers herein to cell-free DNA found in blood, plasma or serum.

[0045] “DNA methylation” refers to covalent attachment of a methyl or hydroxymethyl group on one more nucleotide present in a deoxyribonucleic acid (DNA) molecule. The term includes, without being limited to, methylation of cytosine, the covalent attachment of a methyl or hydroxymethyl group at the position 5 of a cytosine’s pyrimidine ring thereby forming 5-methylcytosine or 5-hydromethylcytosine. Methylation of cytosine is almost exclusively found in the DNA sequence context of a cytosine (C) nucleotide followed by a guanine (G) nucleotide, referred herein as a CpG or a CG dinucleotide. In one embodiment, said DNA methylation is methylation of cytosine, preferably methylation of cytosine in a CpG dinucleotide.

[0046] The term “DNA methylation profile” is used herein in reference to the ensemble of position- specific (or base-specific) DNA methylation status for a given genomic region.

[0047] The term “neuron- specific DNA methylation profiles” is used herein in reference to a DNA methylation profile characteristic, or specific, of neuronal cells. The term “glial cell-specific DNA methylation profiles” is used herein in reference to a DNA methylation profile characteristic, or specific, of glial cells. The skilled artisan is familiar with methods allowing to determine cell- or tissue- specific DNA methylation profile. Such methods include, without being limited to, differential methylation analysis between one- vs-all cell types in which loci are selected both on FDR-adjusted significance and effect sizes in the form of absolute methylation differences.

[0048] DETAILED DESCRIPTION

[0049] The present invention relates to method for measuring, detecting, and / or quantifying, haemodialysis-associated brain ischemia, haemodialysis-associated brain hypoxia, and / or haemodialysis-associated neural cell death, in a human subject. In one embodiment, the method of the invention is for measuring, detecting, and / or quantifying haemodialysis-associated neuronal ischemia, haemodialysis-associated neuronal hypoxia, and / or haemodialysis-associated neural and / or glial cell death, and / or haemodialysis-associated brain damage. In one embodiment, the method of the invention is for measuring, or predicting, the risk of developing haemodialysis-induced cognitive decline.

[0050] In the context of the invention, the subject is a human subject. In one embodiment, the subject is in need of kidney replacement therapy. In one embodiment, the subject is in need of dialysis. In one embodiment, the subject is in need of haemodialysis. In one embodiment, the subject is affected with kidney failure. Examples of kidney failure include, without being limited to, acute kidney injury and chronic kidney disease. In one embodiment, the subject is affected with acute kidney injury or chronic kidney disease. In one embodiment, the chronic kidney disease is advanced chronic kidney disease or end-stage chronic kidney disease.

[0051] In one embodiment, the subject presents or is affected with, one or several comorbidities increasing the risk of developing a neurological disorder. Example of comorbidities increasing the risk of developing a neurological disorder or disease include, without being limited to, cerebrovascular disease, such as stroke and small vessel disease, cardiovascular diseases, metabolic diseases, malnutrition, vitamin deficiencies, accumulation of uremic toxins such as urea, guanidine compounds, and indoxyl sulfate, age superior or equal to 50 years. Example of neurological disorders relevant in the context of the invention include, without being limited to, cognitive decline and dementia.

[0052] In one embodiment, the method of the invention comprises a step of providing a sample, preferably a cell-free DNA sample (i.e. cfDNA sample).

[0053] In one embodiment, the sample was previously obtained, or collected, from the subject. In other words, in this embodiment, the method of the invention does not comprise a step of obtaining (or collecting) the sample from the subject and / or the method of the invention is an in vitro method. In one embodiment, the cfDNA sample was (previously) obtained during or after haemodialysis. In one embodiment, the sample was (previously) obtained at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 minutes after the beginning of haemodialysis, preferably at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 minutes after the beginning of haemodialysis, more preferably at least 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118 or 119 minutes after the beginning of haemodialysis, even more preferably at least 120 minutes after the beginning of haemodialysis.

[0054] In one embodiment, the sample, preferably the cfDNA sample, was (previously) obtained in a time interval ranging from 10 minutes after the beginning of haemodialysis to 2 days after the end of haemodialysis, preferably to 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14 or 13 hours after the end of haemodialysis, preferably to 12, 11, 10, 9, 8, 7, 6, 5 or 4 hours after the end of haemodialysis, more preferably to 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 20 or 10 minutes after the end of haemodialysis.

[0055] In one embodiment, the sample, preferably the cfDNA sample, was (previously) obtained in a time interval ranging from 15 minutes after the beginning of haemodialysis to 2 days after the end of haemodialysis, preferably to 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14 or 13 hours after the end of haemodialysis, preferably to 12, 11, 10, 9, 8, 7, 6, 5 or 4 hours after the end of haemodialysis, more preferably to 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 20 or 10 minutes after the end of haemodialysis.

[0056] In one embodiment, the sample, preferably the cfDNA sample, was (previously) obtained in a time interval ranging from 30 minutes after the beginning of haemodialysis to 2 days after the end of haemodialysis, preferably to 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14 or 13 hours after the end of haemodialysis, preferably to 12, 11, 10, 9, 8, 7, 6, 5 or 4 hours after the end of haemodialysis, more preferably to 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 20 or 10 minutes after the end of haemodialysis.

[0057] In one embodiment, the sample , preferably the cfDNA sample, was (previously) obtained in a time interval ranging from 60 minutes after the beginning of haemodialysis to 2 days after the end of haemodialysis, preferably to 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14 or 13 hours after the end of haemodialysis, preferably to 12, 11, 10, 9, 8, 7, 6, 5 or 4 hours after the end of haemodialysis, more preferably to 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 20 or 10 minutes after the end of haemodialysis.

[0058] In one embodiment, the sample, preferably the cfDNA sample, was (previously) obtained in a time interval ranging from 90 minutes after the beginning of haemodialysis to 2 days after the end of haemodialysis, preferably to 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14 or 13 hours after the end of haemodialysis, preferably to 12, 11, 10, 9, 8, 7, 6, 5 or 4 hours after the end of haemodialysis, more preferably to 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 20 or 10 minutes after the end of haemodialysis.

[0059] In one embodiment, the sample, preferably the cfDNA sample, was (previously) obtained in a time interval ranging from 120 minutes after the beginning of haemodialysis to 2 days after the end of haemodialysis, preferably to 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14 or 13 hours after the end of haemodialysis, preferably to 12, 11, 10, 9, 8, 7, 6, 5 or 4 hours after the end of haemodialysis, more preferably to 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 20 or 10 minutes after the end of haemodialysis.

[0060] In one embodiment, the sample is a bodily fluid. In one embodiment, the sample is a blood sample, a plasma sample or a serum sample. In one embodiment, the sample is a plasma sample.

[0061] In one embodiment, the sample contains, or comprises, cell-free DNA. In other words, the sample is a cell-free DNA sample. In one embodiment, the sample is a bodily fluid and contains cell-free DNA. In one embodiment, the sample is blood, plasma or serum sample and contain circulating cell-free DNA (ccfDNA).

[0062] In one embodiment, the method of the invention comprises a step of determining the DNA methylation profiles of one, or of a plurality of, genomic region in the cfDNA sample of the subject to be tested.

[0063] The skilled artisan is familiar with techniques allowing to determine the methylation profile of a genomic region. Such techniques include, without being limited to, bisulfite sequencing, enzymatic methylome sequencing, TET-assisted pyridine borane sequencing, DNA immunoprecipitation, direct or native 5 -methylcytosine or 5- hydroxymethylcytosine sequencing and the like.

[0064] In one embodiment, the step of determining the DNA methylation profiles of one, or of a plurality of, genomic region in the cfDNA sample of the subject to be tested comprises a step of extracting and / or purifying cfDNA from said cfDNA sample.

[0065] As used herein, the terms “genomic region” or “genomic DNA region” are used interchangeably to refer to a portion or fragment of the genome, in the context of the invention, of the human nuclear genome. Genomic regions are identified or defined by the location in the genome of where they begin and end. The location of the begin and end points of a given genomic region may, for instance and without limitation, be identified or defined by their coordinates given as the combination of the identification of the reference of the genome sequence used, the chromosome number, the nucleotide coordinate on the reference strand (also known as the top strand, the Watson strand or the + strand) and the base coordinate system used for the reference of the genome sequence used. The location of the begin and end points of a given genomic region may also, for instance and without limitation, defined in reference to, the nucleic acid sequence of said genomic region. It is then within the reach of the skilled artisan provided with the nucleic acid sequence of a genomic region to locate its position within a given species’ genome build using sequence comparison tools such as for instance and without limitation, using BLAT (Kent, Genome Research 4: 656-664). For example the genomic region as found in human genome build GRCh38 / hg38 with the coordinates chr2:237,955,956- 237,956,108 in the one-based coordinate system may also be defined in reference to SEQ ID NO: 1 in the human genome build GRCh38 / hg38.

[0066] As used herein, the term human genome build GRCh38 / hg38 is used in reference to the assembly available under the Genbank Assembly Accession number GCA_000001405.15.

[0067] In one embodiment, said genomic region comprises one or more CpG dinucleotide.

[0068] In one embodiment, the method of the invention comprises a step of determining the DNA methylation profiles (in the cfDNA sample of the subject to be tested) of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 or 49 genomic regions, wherein each of said genomic regions is distinct and defined in reference to one of SEQ ID NO: 1 to 1551, as found in the human genome build GRCh38 / hg38, preferably of at least 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131,

[0069] 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149,

[0070] 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167,

[0071] 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185,

[0072] 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221,

[0073] 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239,

[0074] 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257,

[0075] 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275,

[0076] 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293,

[0077] 294, 295, 296, 297, 298 or 299 genomic regions, wherein each of said genomic regions is distinct and defined in reference to one of SEQ ID NO: 1 to 1551, as found in the human genome build GRCh38 / hg38, more preferably of at least 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323,

[0078] 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341,

[0079] 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359,

[0080] 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377,

[0081] 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395,

[0082] 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413,

[0083] 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431,

[0084] 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449,

[0085] 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467,

[0086] 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485,

[0087] 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498 or 499 genomic regions, wherein each of said genomic regions is distinct and defined in reference to one of SEQ ID NO: 1 to 1551, as found in the human genome build GRCh38 / hg38, even more preferably of at least 500 genomic regions, wherein each of said genomic regions is distinct and defined in reference to one of SEQ ID NO: 1 to 1551, as found in the human genome build GRCh38 / hg38.

[0088] In one embodiment, the method of the invention comprises a step of determining the DNA methylation profiles (in the cfDNA sample of the subject to be tested) of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 or 49 genomic regions, wherein each of said genomic regions is selected from the group of genomic regions indicated in table 1, preferably of at least 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123,

[0089] 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141,

[0090] 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159,

[0091] 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177,

[0092] 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195,

[0093] 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213,

[0094] 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231,

[0095] 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249,

[0096] 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267,

[0097] 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285,

[0098] 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298 or 299 genomic regions, wherein each of said genomic regions is selected from the group of genomic regions indicated in table 1, more preferably of at least 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325,

[0099] 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343,

[0100] 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361,

[0101] 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379,

[0102] 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397,

[0103] 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415,

[0104] 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433,

[0105] 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451,

[0106] 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469,

[0107] 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487,

[0108] 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498 or 499 genomic regions, wherein each of said genomic regions is selected from the group of genomic regions indicated in table 1, even more preferably of at least 500 genomic regions, wherein each of said genomic regions is selected from the group of genomic regions indicated in table 1. In one embodiment, the method of the invention comprises a step of determining the DNA methylation profiles (in the cfDNA sample of the subject to be tested) of all genomic regions defined in reference to one of SEQ ID NO: 1 to 1551, as found in the human genome build GRCh38 / hg38. In one embodiment, the method of the invention comprises a step of determining the DNA methylation profiles (in the cfDNA sample of the subject to be tested) of all genomic regions indicated in table 1.

[0109] In one embodiment, the method of the invention comprises a step of quantifying neuronspecific and / or glial cell- specific DNA methylation profiles (in the methylation profiles of the subject to be tested). In embodiments wherein the method of the invention is for measuring, detecting, and / or quantifying haemodialysis-associated neural cell death, the method of the invention comprises a step of quantifying neuron- specific DNA methylation profiles (in the methylation profiles of the subject to be tested). In embodiment wherein the method of the invention is for measuring, detecting, and / or quantifying haemodialysis-associated glial cell death, the method of the invention comprises a step of quantifying glial cell-specific DNA methylation profiles (in the methylation profiles of the subject to be tested). Quantification of neuron-specific and / or glial cell-specific DNA methylation profiles may be used when the method of the invention is for measuring, detecting, and / or quantifying, haemodialysis-associated brain ischemia, haemodialysis-associated brain hypoxia, haemodialysis-associated brain damage, and / or for measuring, or predicting, the risk of developing haemodialysis- induced cognitive decline.

[0110] In one embodiment, the neuron- specific DNA methylation profiles are specific of Central Nervous System (CNS) neuron. In one embodiment, the glial cell-specific DNA methylation profiles are specific of CNS glial cells. In one embodiment, the method of the invention comprises a step of quantifying CNS neuron- specific and / or CNS glial cellspecific DNA methylation profiles (in the methylation profiles of the subject to be tested).

[0111] In one embodiment, the neuron- specific DNA methylation profiles are specific of brain neuron. In one embodiment, the glial cell-specific DNA methylation profiles are specific of brain glial cells. In one embodiment, the method of the invention comprises a step of quantifying brain neuron- specific and / or brain glial cell- specific DNA methylation profiles (in the methylation profiles of the subject to be tested).

[0112] The skilled artisan is familiar with techniques allowing the quantification of tissues- or cell- specific methylation profiles. Such techniques include, without being limited to, use of genomic region- and cell- / tissue- specific deconvolution matrix built after differential methylation analysis between one-vs-all cell types in which loci are selected both on FDR-adjusted significance and effect sizes in the form of absolute methylation differences.

[0113] In one embodiment, the step of quantifying neuron- specific and / or glial cell- specific DNA methylation profiles (in the methylation profiles of the subject to be tested) comprises a step of comparing the level or amount of neuron- specific and / or glial cellspecific DNA methylation profiles (in the methylation profiles of the subject to be tested) with a reference neuron- specific and / or glial cell- specific DNA methylation level.

[0114] As used herein, the term “reference neuron- specific and / or glial cell DNA methylation level” refers to the level of neuron- specific and / or glial cell- specific DNA methylation for a given control subject, or for a population of control subjects, of known status with respect to haemodialysis. The reference neuron- specific and / or glial cell-specific DNA methylation level may, for example and without limitation, be derived from a cfDNA sample taken from the subject to be tested using the method of the invention before the beginning of haemodialysis and / or derived from a population of comparable control subject that have not been subjected to haemodialysis or from a population of comparable control subjects that have been subjected to haemodialysis. It is within the reach of the skilled artisan to select the control subject(s) in order to determine reference neuronspecific and / or glial cell- specific DNA methylation level appropriate for comparison purpose. For instance, and without limitation, the skilled artisan may account for comorbidities influencing neuronal cell death such as neurodegenerative disease, inflammation, age, and the like. It is to be understood in the context of the comparison step of the invention, that the reference neuron- specific and / or glial cell-specific DNA methylation level comprises information on neuron- specific and / or glial cell-specific DNA methylation of the genomic region considered when determining the methylation profile of the subject to be tested. Therefore, embodiment relating to the genomic regions, or plurality thereof, and / or embodiment relating to the neuron and / or glial cell type, considered for the step of determining the methylation profile of the subject to be tested may apply to the determination of the reference DNA methylation profile mutatis mutandis.

[0115] In one embodiment, the reference neuron- specific and / or glial cell-specific DNA methylation level corresponds to a known haemodialysis status.

[0116] In one embodiment, the method of the invention comprises a step of measuring, detecting, and / or quantifying hemodialysis-associated brain ischemia, haemodialysis-associated brain hypoxia, haemodialysis-associated neural and / or glial cell death, and / or haemodialysis-associated brain damage in said human subject. This step is based on the results of the quantification of neuron- specific and / or glial cell-specific DNA methylation profiles (in the methylation profiles of the subject).

[0117] In one embodiment, haemodialysis-associated brain ischemia, haemodialysis-associated brain hypoxia, haemodialysis-associated neural and / or glial cell death, and / or haemodialysis-associated brain damage increases with the level of, or amount of, neuronspecific and / or glial cell-specific DNA methylation in the cfDNA sample.

[0118] In one embodiment, the method of the invention comprises a step of measuring, detecting, and / or quantifying hemodialysis-associated neuronal ischemia and / or haemodialysis- associated neuronal hypoxia and / or haemodialysis-associated neural cell death in said human subject. This step is based on the results of the quantification of neuron- specific DNA methylation profiles (in the methylation profiles of the subject).

[0119] In one embodiment, hemodialysis-associated neuronal ischemia and / or haemodialysis- associated neuronal hypoxia and / or haemodialysis-associated neural cell death increases with the level of, or amount of, neuron- specific DNA methylation in the cfDNA sample. In one embodiment, the method of the invention comprises a step of measuring, or predicting, the risk of developing haemodialysis-induced cognitive decline. This step is based on the results of the quantification of neuron- specific DNA methylation profiles (in the methylation profiles of the subject). In one embodiment, the risk of developing haemodialysis-induced cognitive decline increases with the level of, or amount of, neuron- specific and / or glial cell-specific DNA methylation in the cfDNA sample.

[0120] The present invention also relates to a kit for implementing the method of the invention. The invention hence also relates to the use of a kit in the method of the invention. In particular the invention relates to the use of the kit of the invention for measuring haemodialysis-associated brain ischemia, haemodialysis-associated brain hypoxia, and / or haemodialysis-associated neural cell death preferably using the method of the invention.

[0121] In one embodiment, the kit of the invention comprises a set of capture probe.

[0122] As used herein, the term capture probes correspond to oligonucleotides that hybridize specifically with a genomic region (whether or not it is bisulfite-converted), and that are modified to perform target enrichment on a genomic (cf)DNA sample and that may hence be used in the step of determining the methylation profile in the cfDNA sample from the subject to be tested. Example of modification include, for instance and without limitation, the addition of a binding site on the oligonucleotide allowing the direct or indirect capture on a solid substrate of the oligonucleotide hybridized to a specific genomic DNA region. Such modification may be for instance, and without limitation, be implemented and used as described in the example section. With the knowledge of the DNA sequence of the targeted genomic region, it is within the reach of the skilled artisan to design capture probes.

[0123] In one embodiment, the kit of the invention comprise a set of capture probes, wherein said set of capture probes consist of, or comprises, capture probes specific for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 or 49 genomic regions, wherein each of said genomic regions is distinct and defined in reference to one of SEQ ID NO: 1 to 1551, as found in the human genome build GRCh38 / hg38, preferably of at least 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131,

[0124] 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149,

[0125] 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167,

[0126] 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185,

[0127] 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203,

[0128] 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221,

[0129] 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239,

[0130] 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257,

[0131] 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275,

[0132] 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293,

[0133] 294, 295, 296, 297, 298 or 299 genomic regions, wherein each of said genomic regions is distinct and defined in reference to one of SEQ ID NO: 1 to 1551, as found in the human genome build GRCh38 / hg38, more preferably of at least 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323,

[0134] 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341,

[0135] 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359,

[0136] 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377,

[0137] 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395,

[0138] 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413,

[0139] 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431,

[0140] 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449,

[0141] 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467,

[0142] 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485,

[0143] 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498 or 499 genomic regions, wherein each of said genomic regions is distinct and defined in reference to one of SEQ ID NO: 1 to 1551, as found in the human genome build GRCh38 / hg38, even more preferably of at least 500 genomic regions, wherein each of said genomic regions is distinct and defined in reference to one of SEQ ID NO: 1 to 1551, as found in the human genome build GRCh38 / hg38.

[0144] In one embodiment, the kit of the invention comprise a set of capture probes, wherein said set of capture probes consist of or comprises capture probes specific for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 or 49 genomic regions, wherein each of said genomic regions is selected from the group of genomic regions indicated in table 1, preferably of at least 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123,

[0145] 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141,

[0146] 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159,

[0147] 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177,

[0148] 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195,

[0149] 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213,

[0150] 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231,

[0151] 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249,

[0152] 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267,

[0153] 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285,

[0154] 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298 or 299 genomic regions, wherein each of said genomic regions is selected from the group of genomic regions indicated in table 1, more preferably of at least 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325,

[0155] 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343,

[0156] 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361,

[0157] 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379,

[0158] 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397,

[0159] 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415,

[0160] 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451,

[0161] 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469,

[0162] 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487,

[0163] 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498 or 499 genomic regions, wherein each of said genomic regions is selected from the group of genomic regions indicated in table 1, even more preferably of at least 500 genomic regions, wherein each of said genomic regions is selected from the group of genomic regions indicated in table 1.

[0164] BRIEF DESCRIPTION OF THE DRAWINGS

[0165] Figure 1 is a graph showing brain oxygenation during dialysis in patient with and without neuronal cfDNA increase during haemodialysis.

[0166] EXAMPLES

[0167] The present invention is further illustrated by the following examples.

[0168] Materials and. Methods

[0169] Sample collection and processing

[0170] All peripheral blood samples, before and after haemodialysis, were collected from dialysis patients at Ziekenhuis Oost-Limburg following informed consent. Blood was sampled directly from the vascular access used for dialysis (either the dialysis catheter or the arteriovenous fistula or graft), right before start of dialysis and immediately after. Standard centrifugation methods were used for plasma isolation. cfDNA extraction was performed automatically using the Maxwell HT cfDNA kit (Promega, no. A6030) on the Hamilton Liquid Handler according to the manufacturer’s recommendations.

[0171] Bisulfite conversion and library preparation cfDNA was treated with bisulfite using the EZ DNAme-Lightning Kit (Zymo Research, no. D5031). Library preparation was carried out within 1 h of bisulfite treatment using the ACCEL-NGS 1 S PLUS DNA Library Kit (Westburg, no. SD 10096), which uses an adaptase-induced tailing step of bisulfite-converted DNA before library preparation. Eollowing amplification using KAPA HiLi HotStart Uracil+ ReadyMix (Roche), DNA library concentrations were quantified using Nanodrop and fragment lengths analyzed with Bioanalyzer HS (Agilent, no. 5067-4626). Up to 16 samples, each uniquely indexed, were pooled in an equimolar fashion before target enrichment.

[0172] Marker selection and capture enrichment

[0173] Targets of interest were captured before sequencing. Pirst, regions were selected that are differentially methylated in one cell type vs all other cell types that were included in the reference. A total of 5,038 regions were selected for which 4,989 custom probes were designed by Roche. Target enrichment was performed according to the standard Roche protocol and the resulting libraries were sequenced in house on HiSeq4000, NextSeq2000 or NovaSeq instruments (Illumina) in 150bp paired-end mode, yielding on average 10 million reads per sample. Resequencing of samples was performed when insufficient numbers of reads were sequenced.

[0174] Sequencing data analysis

[0175] Sequencing reads were first processed using Trim Galore! to remove adapter contamination and trim off adaptase-induced addition of random nucleotides (Pelix Krueger, the Babraham Institute). Then, reads were mapped to the human genome build GRCh38 / Hg38 using Bismark (Ref. 8). Finally, reads were deduplicated and DNA methylation data was extracted.

[0176] Reference-based DNA methylation deconvolution

[0177] A reference matrix was built for deconvolution consisting of the 1551 most cell-specific regions (SEQ ID NO. 1 to 1551; table 1) and 16 tissue / cell types (neuron, skeletal muscle, heart, liver, pancreas, intestine, granulocyte, B-cell, CD4+ T-cell, CD8+ T-cell, natural killer cell, monocyte, prostate epithelium, kidney epithelium, vascular endothelium and adipocytes). Table 1: genomic regions with the highest tissue specificity. Start and end coordinates in the one-based coordinate system are indicated in GRCh38 / Hg38.

[0178] Next, cell type fractions were deconvolved from unnormalized data using EMeth-Laplace (EMeth R-package - Ref. 9).

[0179] Results and conclusions

[0180] More neuron-derived cfDNA was detected after haemodialysis in a subset of patients (n=13 out of 35 in total) compared to before haemodialysis. In the remaining 22 patients, no neuron-derived cfDNA was detectable, either before or after dialysis. The neuronal cfDNA concentrations we detected after haemodialysis were estimated at an average of 0.1% while it was on average equal to 0.01% before dialysis. Furthermore, this subset of patients showed a significant drop in cerebral oxygenation during haemodialysis (P < 0.05; 30min - 135min and 210min -240min into dialysis session) (figure 1). These findings indicate that cerebral neuronal hypoxia / ischemia / cell death induced by haemodialysis is associated with an increase in neuronal circulating cfDNA. REFERENCES

[0181] 1- Park et al., “Serum SIOOB represents a biomarker for cognitive impairment in patients with end-stage renal disease”, Clin Neurol Neurosurg. 2020, Aug: 195: 105902. doi: 10.1016 / j.clineuro.2020.105902.

[0182] 2- Hernandez et al., “Blood-brain barrier and gut barrier dysfunction in chronic kidney disease with a focus on circulating biomarkers and tight junction proteins”, Sci Rep. 2022; 12: 4414. doi: 10.1038 / s41598-022-08387-7.

[0183] 3- Zhu at al., “Differential expression of serum biomarkers in hemodialysis patients with mild cognitive decline: A prospective single-center cohort study”, Sci Rep. 2018 Aug 16;8(1): 12250. doi: 10.1038 / s41598-018-29760-5.

[0184] 4- Grosse, Gerrit et al. "Endogenous deoxyribonuclease activity and cell-free deoxyribonucleic acid in acute ischemic stroke: a cohort study." Stroke 53.4 (2022): 1235-1244.

[0185] 5 Cui et al;. “Cell-free circulating DNA: a new biomarker for the acute coronary syndrome”. Cardiology, 124(2), 76-84.

[0186] 6- Xie et al. (2018). “Correlations of circulating cell-free DNA with clinical manifestations in acute myocardial infarction” The American journal of the medical sciences, 356(2), 121-129.

[0187] 7- Coimbra et al. “Cell-free DNA as a marker for the outcome of end-stage renal disease patients on haemodialysis”, Clin Kidney J. 2020 Aug 24; 14(5): 1371-1378. doi: 10.1093 / ckj / sfaal l5.

[0188] 8- Krueger and Andrews. "Bismark: a flexible aligner and methylation caller for Bisulfite- Seq applications." bioinformatics 27.11 (2011): 1571-1572.

[0189] 9- Zhang et al. “EMeth: An EM algorithm for cell type decomposition based on DNA methylation data”, Sci Rep. 2021 Mar 11 ; 11(1):5717.

Claims

CLAIMS1. A method for measuring haemodialysis-associated brain ischemia, haemodialysis- associated brain hypoxia, and / or haemodialysis-associated neural cell death, in a human subject comprising the steps of, a. providing a cell-free DNA sample, previously obtained from said human subject during or after haemodialysis; b. determining the DNA methylation profiles of a plurality of genomic regions in the cfDNA sample provided at step a; c. quantifying neuron- specific and / or glial cell-specific DNA methylation profiles in the DNA methylation profiles obtained at step b; and, d. measuring, based on the quantification performed at step c, hemodialysis- associated brain ischemia, haemodialysis-associated brain hypoxia, and / or haemodialysis-associated neural cell death in said human subject, wherein haemodialysis-associated brain ischemia, haemodialysis-associated brain hypoxia, and / or haemodialysis-associated neural cell death increases with the level of neuron- specific and / or glial cell-specific methylation in said cfDNA sample.

2. The method according to claim 1 wherein step (b) comprises determining, in the sample provided at step a, the DNA methylation profiles of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 or 49 genomic regions and wherein each of said genomic regions is distinct and defined in reference to one of SEQ ID NO: 1 to 1551, as found in the human genome build GRCh38 / hg38.

3. The method according to claim 1 or 2 wherein step (b) comprises determining, in the sample provided at step a, the DNA methylation profiles of at least 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129,130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145,146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161,162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177,178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193,194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209,210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225,226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241,242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257,258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273,274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289,290, 291, 292, 293, 294, 295, 296, 297, 298 or 299 genomic regions and wherein each of said genomic regions is distinct and defined in reference to one of SEQ ID NO: 1 to 1551, as found in the human genome build GRCh38 / hg38.

4. The method according to any one of claims 1 to 3, wherein step (b) comprises determining, in the sample provided at step a, the DNA methylation profiles of at least 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314,315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330,331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346,347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362,363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378,379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394,395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410,411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426,427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442,443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458,459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474,475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499 or 500 genomic regions and wherein each of said genomic regions is distinct and defined in reference to one of SEQ ID NO: 1 to 1551, as found in the human genome build GRCh38 / hg38.

5. The method according to any one of claims 1 to 4, wherein said cell-free DNA sample was obtained at least 10 minutes after the beginning of haemodialysis, preferably at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 minutes after the beginning of haemodialysis, more preferably at least 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114,115, 116, 117, 118 or 119 minutes after the beginning of haemodialysis, even more preferably at least 120 minutes after the beginning of haemodialysis.

6. The method according to any one of claims 1 to 5, wherein said cell-free DNA sample was obtained in a time interval ranging from 10 minutes after the beginning of haemodialysis to 2 days after the end of haemodialysis, preferably to 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14 or 13 hours after the end of haemodialysis, preferably to 12, 11, 10, 9, 8, 7, 6, 5 or 4 hours after the end of haemodialysis, more preferably to 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 20 or 10 minutes after the end of haemodialysis.

7. The method according to any one of claims 1 to 6, wherein said cell-free DNA is circulating cell-free DNA and / or wherein said sample is a blood, plasma or serum sample.

8. The method according to any one of claims 1 to 7, wherein said human subject is affected with kidney failure.

9. Use of a kit, for measuring haemodialysis-associated brain ischemia, haemodialysis-associated brain hypoxia, and / or haemodialysis-associated neural cell death, wherein said kit comprises capture probes for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 genomic regions and wherein each of said genomic regions is distinct and defined in reference to one of SEQ ID NO: 1 to 1551, as found in the human genome build GRCh38 / hg38.

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