Mitochondrial markers of neurodegenerative diseases

专利摘要:

公开号:ES2744592T9
申请号:ES15770042T
申请日:2015-03-27
公开日:2020-12-15
发明作者:Castillo Marta Barrachina；Abizanda Isidre Ferrer；Lozano Marta Blanch
申请人:Fundacio Inst Dinvestigacio Biomedica De Bellvitge Idibell；Universitat Autonoma de Barcelona UAB；Universitat de Barcelona UB；Fundacio Privada Institut dInvestigacio Biomedica de Bellvitge；
IPC主号:

专利说明:

[0002] Mitochondrial markers of neurodegenerative diseases
[0004] Field of the invention
[0006] The present invention is classified as part of the diagnostic methods of neurological diseases.
[0008] Background of the invention
[0010] There are a considerable number of neurodegenerative diseases that are caused by or associated with alterations in mitochondrial function.
[0012] Alzheimer's disease (AD) and Parkinson's disease (PD) fall within this group. The pathophysiological characteristics of AD and PD are related to deposits of aggregated proteins. Specifically, AD is associated with the formation of intracellular phosphorylated tau aggregates in neurofibrillary tangles and extracellular aggregates of the p-amyloid peptide in senile plaques, and PD is associated with the formation of abnormal α-synuclein aggregates that constitute the main component of the so-called Lewy bodies and Lewy neurites.
[0014] It is accepted that Alzheimer's patients show reduced levels of the ND4 subunit in their brain tissue and that Parkinson's patients show reduced levels of ND6 in the substantia nigra. Furthermore, genetic studies have identified mutations in several COX genes and in the D-loop region, as well as deletions in mtDNA in the brains of subjects with AD and in the substantia nigra of subjects with PD.
[0016] In technology, different methods and strategies have been developed for the diagnosis, prediction of the onset and development of neurodegenerative diseases and, in particular, AD and PD. Thus, diagnostic methods for neurodegenerative diseases based on the identification of mutations in mitochondrial DNA by using the RFLP technique (restriction fragment length polymorphisms) or other related techniques have been described. Document WO98038334 describes an AD diagnostic method based on the identification of mutations in COX genes. A method of diagnosing PD in a subject by identifying single nucleotide polymorphisms in mitochondrial DNA samples from a subject has also been proposed (WO 2000063441). Other prior art documents describe methods for the diagnosis of Alzheimer's or Parkinson's disease based on the identification of polymorphisms in genes that encode nuclear proteins that control the mitochondrial transcription process. Despite the efforts made to date, there is still a need for reliable methods for diagnosing neurodegenerative diseases such as AD and PD, as well as for diagnosing the stage of these diseases and for predicting their evolution.
[0017] Compendium of the invention
[0019] In a first aspect, the invention relates to an in vitro method for diagnosing or determining the risk of developing a neurodegenerative disease selected from Alzheimer's disease and Parkinson's disease in a subject that contains, in a sample of said subject that contains DNA mitochondrial, the methylation pattern in the D-loop region and / or in the ND1 gene, where the methylation pattern is determined at at least one site selected from the group consisting of:
[0021] i. the CpG sites in the D loop region shown in Table 1
[0023] ii. the CpG sites in the ND1 gene shown in Table 2,
[0025] iii. the CHG sites in the D loop region shown in Table 3,
[0027] iv. the CHG sites in the ND1 gene shown in Table 4, and / or
[0029] v. the CHH sites in the D loop region shown in Table 5,
[0031] wherein a hypermethylation in at least one of said CpG sites in the D loop region, a hypermethylation in at least one of said CHG sites in the D loop region, a hypermethylation in at least one of said CHH sites in the D-loop region loop D, a hypomethylation in at least one of said CpG sites in the ND1 gene and / or a hypomethylation in at least one of said CHG sites in the ND1 gene is indicative that the subject suffers from Alzheimer's disease or that the subject have a high risk of developing Alzheimer's disease or
[0033] wherein a hypomethylation in at least one of said CpG sites in the D loop region, a hypomethylation in at least one of said CHG sites in the D loop region and / or a hypomethylation in at least one of said CHH sites in the D-loop region is indicative that the subject suffers from Parkinson's disease or that the subject is at high risk of developing Parkinson's disease.
[0035] In a second aspect, the invention relates to an in vitro method to select a subject for preventive treatment of a neurodegenerative disease selected from Alzheimer's disease and Parkinson's disease in a subject that contains, in a sample from said subject that contains mitochondrial DNA, the methylation pattern in the D-loop region and / or in the ND1 gene, wherein the methylation pattern is determined in at least one site selected from the group consisting of:
[0036] i. the CpG sites in the D loop region shown in Table 1,
[0037] ii. the CpG sites in the ND1 gene shown in Table 2;
[0038] iii. the CHG sites in the D loop region shown in Table 3,
[0039] iv. the CHG sites in the ND1 gene shown in Table 4, and / or
[0040] v. CHH sites in the D loop region shown in Table 5
[0041] wherein a hypermethylation in at least one of said CpG sites in the D loop region, a hypermethylation in at least one of said CHG sites in the D loop region, a hypermethylation in at least one of said CHH sites in the D-loop region loop D, a hypomethylation in at least one of said CpG sites in the ND1 gene and / or a hypomethylation in at least one of said CHG sites in the ND1 gene is indicative that the subject is eligible to receive a treatment aimed at preventing the Alzheimer's disease or
[0042] wherein a hypomethylation in at least one of said CpG sites in the D loop region, a hypomethylation in at least one of said CHG sites in the D loop region and / or a hypomethylation in at least one of said CHH sites in the D-loop region is indicative that the subject is eligible to receive a treatment aimed at preventing Parkinson's disease.
[0043] In a third aspect, the invention relates to an in vitro method for monitoring the progression of a neurodegenerative disease selected from Alzheimer's disease or Parkinson's disease in a subject, comprising:
[0044] a) determining in a sample of said subject that contains mitochondrial DNA the methylation pattern in the D loop region, and / or in the ND1 gene, where the methylation pattern is determined in at least one site selected from the group formed by :
[0045] i. the CpG sites in the D loop region shown in Table 1,
[0046] ii. the CpG sites in the ND1 gene shown in Table 2;
[0047] iii. the CHG sites in the D loop region shown in Table 3,
[0048] iv. the CHG sites in the ND1 gene shown in Table 4, and / or
[0049] v. CHH sites in the D loop region shown in Table 5
[0050] b) comparing the methylation pattern determined in step a) with said methylation pattern obtained in an earlier stage of the disease,
[0051] wherein a hypermethylation at least one of said CpG sites in the D loop region, a hypermethylation at at least one of the said CHG sites in the D loop region, a hypermethylation at at least one of said CHH sites in the D-loop region D loop, a hypomethylation in at least one of said CpG sites in the ND1 gene and / or a hypomethylation in at least one of said CHG sites in the ND1 gene with respect to said methylation pattern determined at an early stage of the disease is indicative of the progression of Alzheimer's disease; and
[0052] wherein a hypomethylation in at least one of said CpG sites in the D loop region, a hypotemylation in at least one of said CHG sites in the D loop region and / or hypomethylation in at least one of said CHH sites in the region D-loop with respect to said methylation pattern determined in an early stage of the disease, is indicative of the progression of Parkinson's disease.
[0053] In a fourth aspect, the invention relates to an in vitro method for diagnosing or determining the risk of developing Alzheimer's disease in a subject that contains, in a sample comprising mitochondrial DNA from said subject, the nucleotide at polymorphic position 16519 according to the sequence defined with the access number NC_012920 in the NCBI database, where the detection of nucleotide C in said polymorphic position or presence of nucleotide C in said polymorphic position in at least 60% of the mitochondrial DNA molecules of the subject is indicative that the subject suffers from said disease or that the subject is at high risk of developing the disease.
[0054] In a fifth aspect, the invention refers to an in vitro method to select a subject to be presented for a preventive treatment of Alzheimer's disease, which involves determining in a sample that contains DNA mitochondrial of said subject, the nucleotide in polymorphic position 16519 according to the sequence defined with accession number NC_012920 in the NCBI database, where the detection of nucleotide C in said polymorphic position or the presence of nucleotide C in said position polymorphic in at least 60% of the mitochondrial DNA molecules of said subject, is indicative that the subject is eligible to receive a treatment aimed at preventing Alzheimer's disease.
[0055] In a sixth aspect, the invention relates to a nucleic acid selected from the group consisting of:
[0056] (i) a nucleic acid comprising at least 9 contiguous nucleotides in a region of mitochondrial DNA where said region comprises at least one methylation site selected from the group consisting of:
[0057] a) the CpG sites in the D loop region shown in Table 1,
[0058] b) the CpG sites in the ND1 gene shown in Table 2;
[0059] c) the CHG sites in the D loop region shown in Table 3,
[0060] d) the CHG sites in the ND1 gene shown in Table 4, and
[0061] e) the CHH sites in the D loop region shown in Table 5,
[0062] (ii) a nucleic acid comprising at least 9 contiguous nucleotides in a region of mitochondrial DNA, wherein said region comprises at least one methylation site selected from the group consisting of:
[0063] a) the CpG sites in the D loop region shown in Table 1,
[0064] b) the CpG sites in the ND1 gene shown in Table 2;
[0065] c) the CHG sites in the D loop region shown in Table 3,
[0066] d) the CHG sites in the ND1 gene shown in Table 4, and
[0067] e) the CHH sites in the D loop region shown in Table 5,
[0068] where the position corresponding to the cytosine in the CpG, CHG or CHH site is uracil; and
[0069] (iii) a polynucleotide that specifically hybridizes to the nucleic acids of (i) or (ii)
[0070] The present description further refers to a kit comprising at least one oligonucleotide capable of hybridizing specifically and in a methylation-dependent manner with a mitochondrial DNA sequence comprising a methylation site selected from the group consisting of:
[0071] i. the CpG sites in the D loop region shown in Table 1,
[0072] ii. the CpG sites in the ND1 gene shown in Table 2;
[0073] iii. the CHG sites in the D loop region shown in Table 3,
[0074] iv. the CHG sites in the ND1 gene shown in Table 4, and / or
[0075] v. CHH sites in the D loop region shown in Table 5
[0076] The present description further refers to a kit comprising at least one oligonucleotide capable of specifically hybridizing at the 5 'position or at the 3' position with respect to a methylation site in mitochondrial DNA selected from the group consisting of:
[0077] (i) the CpG sites in the D loop region shown in Table 1,
[0078] (ii) the CpG sites in the ND1 gene shown in Table 2;
[0079] (iii) the CHG sites in the D loop region shown in Table 3,
[0080] (iv) the CHG sites in the ND1 gene shown in Table 4, and / or
[0081] (v) CHH sites in the D loop region shown in Table 5
[0082] wherein the cytosine methylated at said position has been converted to uracil or another base that is distinguishable from cytosine in its hybridization properties.
[0083] Finally, in a seventh aspect, the invention relates to the use of the kits defined above to determine the methylation pattern of mitochondrial DNA and to determine the diagnosis of a neurodegenerative disease in a subject selected from Alzheimer's disease or Parkinson's disease. .
[0085] Brief description of the drawings
[0087] Figure 1: Log2 (OR) plots for the CpG (A) CHG (B) and CHH (C) sites in the D-loop amplicon in the entorhinal cortex of cases related to EA pathology. The 5 'to 3' methylation sites are represented on the x-axis. The sites marked with a diamond are the differentially methylated sites (FDR <0.05). The points are the OR estimation values, one for each site, and the band is the joining band of all 95% confidence intervals. C: control samples, AD: Alzheimer's disease.
[0089] Figure 2: Log2 (OR) plots for the CpG (A) CHG (B) and CHH (C) sites in the ND1 amplicon in the entorhinal cortex of cases related to EA pathology. The 5 'to 3' methylation sites are represented on the x-axis. The sites marked with a diamond are the differentially methylated binding sites of all 95% confidence intervals. C: control samples, AD: Alzheimer's disease.
[0091] Figure 3: Log2 (OR) plots for CpG and non-CpG (CHG and CHH) sites in the D-loop amplicon in the substantia nigra of PD patients. The 5 'to 3' methylation sites are represented on the x-axis. The sites marked with a diamond are the differentially methylated sites (FDR <0.05). The points are the OR estimation values, one for each site, and the band is the joining band of all 95% confidence intervals. C: control samples, PD: Parkinson's disease.
[0093] Figure 4: Log2 (OR) plots for CpG (A) and CHG (B) sites in the D-loop amplicon in the frontal cortex of three, six and twelve month old APP / PS1 mice and wild type (WT) mice. The methylation sites arranged from 5 'to 3' are represented on the x-axis. The sites marked with a diamond are the differentially methylated sites (FDR <0.05). The points are the OR estimation values, one for each site, and the band is the joining band of all 95% confidence intervals. C: control samples, WT: wild, TG: Transgenic.
[0095] Figure 5: Log2 (OR) plots for CG (A), CHG (B) and CHH (C) sites in the D loop amplicon in the frontal cortex of APP / PS1 mice aged three, six and twelve months. The methylation sites arranged from 5 'to 3' are represented on the x-axis. The sites marked with a diamond are the differentially methylated sites (FDR <0.05). The points are the OR estimation values, one for each site, and the band is the joining band of all 95% confidence intervals. C: control samples, TG: Transgenic.
[0097] Detailed description of the invention
[0099] The present inventors have developed a method for diagnosing neurodegenerative diseases based on the determination of the methylation pattern in a mitochondrial DNA sample from a subject. The inventors have found that, surprisingly, there are variations in the methylation pattern in the D-loop region and in the ND1 gene in subjects suffering from AD or PD when compared to healthy subjects as demonstrated in the examples. Furthermore, the inventors have discovered differential methylation patterns associated with the development of such diseases.
[0101] First method of the invention
[0103] The first feature of the invention refers to an in vitro method for diagnosing or determining the risk of developing a neurodegenerative disease selected from Alzheimer's disease and Parkinson's disease in a subject (hereinafter, first method of the invention) that involves determining in a sample of said subject that comprises mitochondrial DNA, the methylation pattern in the D loop region and / or in the ND1 gene, where the methylation pattern is determined in at least one site selected from the group consisting of:
[0105] (i) the CpG sites in the D loop region shown in Table 1,
[0107] (ii) the CpG sites in the ND1 gene shown in Table 2;
[0109] (iii) the CHG sites in the D loop region shown in Table 3,
[0111] (iv) the CHG sites in the ND1 gene shown in Table 4, and / or
[0113] (v) the CHH sites in the D loop region shown in Table 5;
[0115] wherein a hypermethylation in at least one of said CpG sites in the D loop region, a hypermethylation in at least one of said CHG sites in the D loop region, a hypermethylation in at least one of said CHH sites in the D-loop region loop D, a hypomethylation in at least one of said CpG sites in the ND1 gene and / or a hypomethylation in at least one of said CHG sites in the ND1 gene is indicative that the subject suffers from Alzheimer's disease or that the subject has a high risk of developing Alzheimer's disease or wherein a hypomethylation in at least one of said CpG sites in the D loop region, a hypomethylation in at least one of said CHG sites in the D loop region and / or a hypomethylation at at least one of said CHH sites in the D loop region is indicative that the subject is suffering from Parkinson's disease or that the subject is at high risk of developing Parkinson's disease.
[0117] The term "diagnosis" as used in this document, refers both to the process of trying to determine and / or identify a possible disease in a subject, that is, the diagnostic procedure, and to the opinion reached through this process, it is that is, the diagnostic opinion. As such, it can also be viewed as an attempt to classify an individual's condition into separate and distinct categories that allow medical decisions about treatment and prognosis to be made. As the person skilled in the art will understand, said diagnosis may not be correct for 100% of the subjects to be diagnosed, although it is preferred that it be.
[0119] However, the term requires that a statistically significant part of the subjects can be identified as having a disease, in particular a neurodegenerative disease selected from Alzheimer's disease and Parkinson's disease 'in the context of the invention, or a predisposition to it. The person skilled in the art can determine whether a part is statistically significant using different well-known statistical evaluation tools, for example, determining confidence intervals, determining the value of the Student's t-test, the Mann-Whitney test, etc. (see Dowdy Wearden, 1983). Preferred confidence intervals are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%. P-values are preferably 0.05, 0.025, 0.001 or less.
[0121] The term "risk of developing a neurodegenerative disease" as used herein refers to the predisposition, susceptibility or propensity of a subject to develop a neurodegenerative disease. The risk of developing a neurodegenerative disease generally implies that there is a high risk or a low risk. Similarly, a subject who has a high risk of developing a neurodegenerative disease, particularly Alzheimer's disease or Parkinson's disease has a probability of developing this disease of at least 50%, or at least 60%, or at least 70 %, or at least 80%, or at least 90, or at least 95%, or at least 97%, or at least 98%, or at least 99%, or at least 100%. Similarly, a subject with a low risk of developing a neurodegenerative disease, particularly Alzheimer's disease, or Parkinson's disease, is a subject who has at least a 0% chance of developing the disease, or at least a 1%, or at least 2%, or at least 3%, or at least 5%, or at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 49% .
[0123] In general, the term "predicted risk", "predicted risk", or the like, refers to the risk that a patient has of developing a neurodegenerative disease selected from Alzheimer's disease or Parkinson's disease, either high or low. As one skilled in the art will understand, the prediction (or risk), although preferred, does not need to be correct for 100% of the subjects to be assessed, but it is preferable that it is. The term, however, requires that a statistically significant part of the subjects can be identified with a higher probability of having a particular outcome. The person skilled in the art can easily determine whether a part is statistically significant using various well-known statistical tools for evaluation, for example, determination of confidence intervals, determination of the p-value, cross-validation classification indices, etc. (more details in "Statistics for research" Dowdy and Wearden, John Wiley & Sons, New York, 1983). Preferred confidence intervals are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%. The P-values are preferably 0.1, 0.05, 0.02, 0.01 or less.
[0125] The term "neurodegenerative disease" as used herein includes chronic and progressive processes that are characterized by selective and symmetric losses of neurons in the motor, sensory, and cognitive systems.
[0127] The term "Alzheimer's disease" or "senile dementia" or AD refers to a mental deterioration associated with a specific degenerative brain disease that is characterized by the appearance of senile plaques, neuritic tangles and progressive neuronal loss that manifests clinically in progressive deficiencies memory problems, confusion, behavior problems, inability to care for himself, gradual physical decline, and finally death. In preferred embodiments, Alzheimer's disease is in any stage according to Braak's staging:
[0129] • Stages I-II: the brain area affected by the presence of neurofibrillary tangles corresponds to the transentorhinal region of the brain
[0131] • Stages III-IV: the affected brain area also extends to areas of the limbic region such as the hippocampus
[0132] • Stages V-VI: the affected brain area also involves the neocortical region
[0134] This classification by neuropathological stages is correlated with the clinical evolution of the existing disease and there is a parallel between memory loss with neurofibrillary changes and plaque formation. neuritic diseases in the entorhinal cortex and hippocampus (stages I to IV). Likewise, the isocortical presence of these changes (stages V and VI) is correlated with clinically severe alterations. The transentorhinal state (I-II) corresponds to clinically silent periods of the disease. The limbic state (III-IV) corresponds to a clinically incipient AD. The necortical state corresponds to a fully developed AD.
[0136] The term "Parkinson's disease" or "idiopathic parkinsonism" or "agitant paralysis" or PD as used herein, refers to a chronic and degenerative disease involving problems with movement control, tremor, stiffness, bradykinesia. in all kinds of movements such as walking, sitting, eating, talking, etc., as well as postural instability. Symptoms of the disease are clearly associated with selective degeneration of dopaminergic neurons in the substantia nigra. The dopaminergic deficit induces a consequent loss of striatal neurons causing a variety of cytological changes that include the aggregation of asinuclein in the so-called Lewy bodies. The “substantia nigra” is a nucleus of the basal ganglia located in the upper portions of the midbrain, under the thalamus and takes its color from neuromelanin. In preferred embodiments, PD is in any of the stages according to Braak's staging:
[0138] • Stage I: the affected area is the dorsal motor nucleus and / or the reticular intermediate zone.
[0140] • Stage II: the affected area extends to the locus ceruleus and the nucleus of the raphe
[0142] • Stage III: the affected area extends to the midbrain, particularly to the substantia nigra pars compacta.
[0144] • Stage IV: the affected area extends to the transentorhinal region of the anteromedial temporal mesocortex and the allocorteza.
[0146] • Stage V: the affected area extends to the insular cortex, the cingulate cortex, and the temporal gyrus.
[0148] • Stage VI: the affected area extends to the frontal and parietal area of the cerebral cortex.
[0150] The term "subject" as used herein refers to a person, such as a human, non-human primate (eg, chimpanzees and other apes and monkey species), farm animals such as birds, fish, cattle, sheep, pigs, goats, and horses; pets such as dogs and cats; mammals, laboratory animals including rodents, such as mice, rats, and guinea pigs. The term does not denote a certain age or gender. In a specific embodiment of the invention, the subject is a mammal. In a preferred embodiment of the invention, the subject is a human.
[0152] The term "sample comprising mitochondrial DNA" as used herein refers to any sample obtainable from a subject in which there is genetic material from the mitochondria suitable for detecting the methylation pattern.
[0154] The term "mitochondrial DNA" or "mtDNA" as used herein refers to genetic material located in the mitochondria of living organisms. It is a double-stranded, circular, closed molecule. In humans it consists of 16,569 base pairs, containing a small number of genes, distributed between the H chain and the L chain. Mitochondrial DNA encodes 37 genes: two ribosomal RNAs, 22 transfer RNAs and 13 proteins that participate in the oxidative phosphorylation.
[0156] In a specific embodiment of the invention, the sample comprising mitochondrial DNA is selected from a biopsy of a solid tissue or a biological fluid. Samples can be obtained by conventional methods known to those skilled in the art.
[0158] In an even more specific embodiment, the biological fluid is selected from peripheral blood or cerebrospinal fluid.
[0160] In an even more specific embodiment, said solid tissue is brain tissue.
[0162] In a preferred embodiment of the invention, if it is desired to diagnose a subject or if it is desired to determine the risk of developing PD, said sample is a sample of brain tissue obtained from the substantia nigra.
[0164] If the material where it is desired to determine the methylation pattern according to the present method, that is, mtDNA, is found in a solid tissue or a biological fluid, preferably, a previous extraction of the nucleic acid from the sample is carried out using any appropriate technique to it. In a preferred embodiment of the invention, the DNA fraction suitable for the practice of the invention is total DNA 35. DNA extraction can be carried out using any method known to those skilled in the art (Sambrock et al ., 2001. "Molecular Cloning: A Laboratory Manual", 3rd ed., Cold Spring Harbor Laboratory Press, NY, Vol. 1-3) including without limitation, density gradient centrifugations, two-phase extraction using aqueous phenol or chloroform with ethanol, column chromatography, methods based on the ability of DNA to bind to glass surfaces and / or silicates, such as diatomaceous earth as preparations or glass beds, using commercial kits, for example, the “Q-Biogene kits fast DNA® ”or the“ QIAamp® (R) DNA Blood Mini Kit ”(Qiagen, Hilden, Germany) the“ G-Spin IIp "(Intron Biotechnology, Korea) or the" Fast Prep System Bio 101 "(Qbiogene®, Madrid, Spain) or the methods described in US5,057,426, US4,923,978 and European patent application EP0512767A1.
[0166] If desired, the present method can be carried out on samples where the mitochondrial fraction has previously been isolated and the DNA thereof has subsequently been isolated. Isolation of the mitochondrial fraction can be carried out using any known method of cell fractionation. Said methods include prior cell disruption by techniques that include physical membrane disruption, application of ultrasound, application of pressure, or enzymatic techniques, followed by differential centrifugation by applying density gradients (such as Ficoll or Percoll gradients). Commercial kits, for example, Qproteome "Mitochondrial isolation kit" (Qiagen, Hilden, Germany) or "Mitochondrial isolation kit for culterd cells" (Thermo Scientific; USA) can also be used. These kits are based on the same basic principle, namely cell lysis and differential centrifugation to isolate or enrich the mitochondrial fraction.
[0168] The first method of the invention involves determining the methylation pattern in a sample from a subject comprising mitochondrial DNA. The term "DNA methylation" as used herein refers to a biochemical process involving the addition of a methyl group (-CH ³ ) to the DNA nucleotides cytosine (C) or adenine (A ). DNA methylation at position 5 of cytosine has the specific effect of gene repression and has been found in all vertebrates examined. The term "methylation pattern", as used herein, refers to, but is not limited to, the presence or absence of methylation of one or more nucleotides. In this way, said one or more nucleotides are comprised in a single nucleic acid molecule.
[0170] Said one or more nucleotides have the ability to be methylated or not. The term "methylation state" can also be used when only a single nucleotide is considered. A methylation pattern can be quantified; in the case where more than one nucleic acid molecule is considered.
[0172] The term "D-loop" or "control region", as used herein, refers to a non-coding mtDNA region containing approximately 1,100 base pairs, visible under electron microscopy, that is generated during replication. of the H chain for the synthesis of a short segment of the heavy chain, 7S DNA.
[0173] The term "ND1" or "NADH dehydrogenase 1" or "ND1 mt", as used herein, refers to the gene located in the mitochondrial genome that encodes the protein NADH dehydrogenase 1 or ND1. The sequence of the human ND1 gene is deposited in the GenBank database (version of January 2, 2014) under the accession number NC_012920. SEQ ID NO: 1. The ND1 protein is part of the enzymatic complex called complex I that is active in the mitochondria and is involved in the oxidative phosphorylation process.
[0175] The term "CpG site", as used herein, refers to regions of DNA, particularly regions of mitochondrial DNA, where a cytosine nucleotide is followed by a guanine nucleotide in the linear sequence of bases along the of its length. "CpG" is an abbreviation for "C-phosphate-G", that is, cytosine and guanine separated by only one phosphate; phosphate binds any two nucleosides in DNA together. The term "CpG" is used to distinguish this linear sequence from the CG base pairing of cytosine and guanine. Cytosine in CpG dinucleotides can be methylated to form 5-methylcytosine.
[0177] The term "CHG site", as used herein, refers to regions of DNA, particularly regions of mitochondrial DNA, where a cytosine nucleotide and a guanine nucleotide are separated by a variable nucleotide (H) that it can be adenine, cytosine, or thymine. The cytosine at the CHG site can be methylated to form 5-methylcytosine.
[0179] The term "CHH site", as used herein, refers to regions of DNA, particularly regions of mitochondrial DNA, where a cytosine nucleotide is followed by a first and second variable nucleotide (H) that it can be adenine, cytosine, or thymine. The cytosine at the CHG site can be methylated to form 5-methylcytosine.
[0181] In a specific embodiment, the first method of the invention comprises determining in a sample from a subject comprising mitochondrial DNA, the methylation pattern in at least one site selected from the CpG sites in the D-loop region, selected from the sites shown. in Table 1.
[0182] Table 1: List of CpG positions 16386 and 256 in loop region D.
[0187] The term "determination of the methylation pattern at a CpG site", as used herein, refers to the determination of the methylation status of a particular CpG site. Determination of the methylation pattern of a CpG site can be performed by multiple processes known to those skilled in the art.
[0188] In a specific embodiment, the first method of the invention involves determining the methylation pattern of at least one CpG site in the D-loop region selected from the sites shown in Table 1. In another specific embodiment, the first method of the invention involves determining the methylation pattern in at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12 , at least 13, at least 14, at least 15 or at least 16 CpG sites selected from Table 1.
[0189] In an even more specific and preferred embodiment of the invention, the first method of the invention comprises determining the methylation pattern at all CpG sites in the D-loop region shown in Table 1.
[0190] In another specific embodiment, the first method of the invention comprises determining in a sample from a subject comprising mitochondrial DNA, the methylation pattern in at least one site selected from the CpG sites of the ND1 gene shown in Table 2.
[0191] Table 2: List of CpG sites in the ND1 gene between positions 3313 and 3686.
[0194] In another particular embodiment, the first method of the invention involves determining the methylation pattern in a selected CpG site of the ND1 gene shown in Table 2. In another specific embodiment, the first method of the invention involves determining the methylation pattern in al least 2, at least 3, at least 4, at least 5 or at least 6 CpG sites selected from Table 2.
[0195] In an even more particular and preferred embodiment of the invention, the first method of the invention involves determining the methylation pattern in all the CpG sites of the ND1 gene shown in Table 2.
[0196] In another particular embodiment, the first method of the invention comprises determining in a sample from a subject comprising mitochondrial DNA, the methylation pattern in at least one site selected from the CHG sites in the D loop region, shown in Table 3 .
[0197] Table 3: List of CHG sites between positions 16386 and 256 in the D loop region.
[0202] The term "determination of the methylation pattern at a CHG site", as used herein, refers to the determination of the methylation status of a particular CHG site.
[0203] Determination of the methylation pattern of a CHG site can be performed by multiple processes known to those skilled in the art.
[0204] In a specific embodiment, the first method of the invention involves determining the methylation pattern in at least one CHG site of the D-loop region selected from the sites shown in Table 3. In another specific embodiment, the first method of the invention involves determining the methylation pattern in at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12 , at least 13 or at least 14 or at least 15 CHG sites selected from Table 3.
[0205] In an even more specific and preferred embodiment of the invention, the first method of the invention involves determining the methylation pattern at all CHG sites in the D-loop region shown in Table 3.
[0206] In another specific embodiment, the first method of the invention comprises determining in a sample from a subject comprising mitochondrial DNA, the methylation pattern in at least one site selected from the CHG sites of the ND1 gene shown in Table 4.
[0207] Table 4: List of CHG sites in the ND1 gene between positions 3313 and 3686.
[0212] In another specific embodiment, the first method of the invention involves determining the methylation pattern in at least one CHG site in the ND1 gene selected from the sites shown in Table 4. In another specific embodiment, the first method of the invention comprises determining the methylation pattern in at least 2, at least 3, at least 4, at least 5 or at least 6 CHG sites selected from Table 4.
[0213] In an even more specific and preferred embodiment of the invention, the first method of the invention involves determining the methylation pattern in all the CHG sites of the ND1 gene shown in Table 4.
[0214] In another specific embodiment, the first method of the invention comprises determining in a sample from a subject comprising mitochondrial DNA, the methylation pattern in at least one site selected from the CHH sites of the D-loop region, selected from the CHH sites shown in table 5.
[0215] Table 5: List of CHH sites between positions 16386 and 256 of the D loop region.
[0220] The term "determination of a methylation pattern at a CHH site", as used herein, refers to the determination of the methylation status of a particular CHH site. Determination of the methylation pattern at a CHG site can be performed by multiple processes known to those skilled in the art.
[0222] In another specific embodiment, the first method of the invention comprises determining the methylation pattern in at least one CHH site of the D-loop region selected from those shown in Table 5. In another specific embodiment, the first method of the invention involves determine the methylation pattern in at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least at least 17, at least 18, at least 19, at least 20, at least 21, at least at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, or at least 43 CHH sites selected from Table 5.
[0223] In an even more specific and preferred embodiment of the invention, the first method of the invention involves determining the methylation pattern at all CHH sites in the D-loop region shown in Table 5.
[0225] In preferred methods of implementation, the first method of the invention includes:
[0227] (i) determine the methylation pattern in all the CpG sites of the D loop region shown in Table 1 and in all the CpG sites of the ND1 gene shown in Table 2
[0229] (ii) determine the methylation pattern in all the CpG sites of the D loop region shown in Table 1 and in all the CHG sites of the D loop region shown in Table 3
[0231] (iii) determine the methylation pattern in all CpG sites of the D loop region shown in Table 1 and in all CHG sites of the ND1 gene shown in Table 4
[0233] (iv) determine the methylation pattern at all CpG sites in the D loop region shown in Table 1 and at all CHH sites in the D loop region shown in Table 5
[0235] (v) determine the methylation pattern in all the CpG sites of the ND1 gene shown in Table 2 1 and in all the CHG sites in the D loop region shown in Table 3
[0237] (vi) determine the methylation pattern in all CpG sites in the ND1 gene shown in Table 2 and in all CHG sites in the ND1 gene shown in Table 4,
[0238] (vii) determine the methylation pattern at all CpG sites in the ND1 gene shown in Table 2 and at all CHH sites in the D loop region shown in Table 5
[0240] (viii) determine the methylation pattern in all CHG sites in the D loop region shown in Table 3 and in all CHG sites of the ND1 gene shown in Table 4
[0242] (ix) determine the methylation pattern at all CHG sites in the D-loop region shown in Table 3 and / or
[0243] (x) determine the methylation pattern at all CHG sites in the ND1 gene shown in Table 4 and at all CHH sites in the D loop region shown in Table 5
[0245] In some embodiments, the determination of the methylation pattern in at least one CpG site, at least one CHG site and / or at least one CHH site according to the first method of the invention is carried out on a whole blood sample, in which case the determination can be made directly. In other embodiments, the mitochondrial DNA-containing sample, preferably a total DNA sample, is extracted from cells that are present in a biological fluid (eg, whole blood, cerebrospinal fluid) as an initial stage and in such cases, the total nucleic acid extracted from these samples represents suitable working material for further analysis. Isolation of total DNA or mitochondrial DNA can be carried out by conventional methods known to the person skilled in the art (cited supra). After isolating and amplifying (if necessary) the nucleic acid containing mitochondrial DNA, the methylation pattern of one or more CpG sites, one or more CHG sites, and / or one or more CHH sites is determined. The person skilled in the art will readily recognize that analysis of the methylation pattern present in one or more of the CpG, CHG and / or CHH sites described herein present in the mitochondrial DNA of a subject, can be performed by any method or technique capable of measuring the methylation pattern present at these sites.
[0247] In another specific embodiment, the first method of the invention involves determining the methylation pattern at said CpG, CHG and / or CHH sites by a technique selected from the group consisting of methylation-specific PCR (MSP), an enrichment-based method ( eg MeDIP, MBD-seq and MethylCap), bisulfite sequencing and by a bisulfite-based method (eg RRB, Infinium, GoldenGate, Cobra, MSP, MethyLight) and a restriction digest method (eg. g., MRE-seq, or HELP assay), pyrosequencing, or differential conversion, differential restriction, differential weight of methylated CpG, CHG and / or CHH sites of DNA.
[0249] In a specific and preferred embodiment of the invention, the methylation pattern of one or more CpG, CHG and / or CHH sites in the D loop region and / or one or more CpG and / or CHG sites in the ND1 gene is determined by pyrosequencing. Briefly, this technique is based on the principle of sequencing by synthesis and on the detection of pyrophosphate (PPi) released during DNA synthesis. This technique uses a series of four enzymes to detect nucleic acid sequences during the synthesis process; DNA polymerase, ATP sufurylase, luciferase and apyrase and uses adenosine 5 'phosphosulfate (APS) and luciferin as substrates.
[0251] To determine the methylation pattern in mitochondrial DNA, it is necessary to chemically treat said sample in such a way that all non-methylated cytosine bases are modified to uracil bases, or another base that is different from cytosine in terms of pairing behavior. bases, while the bases of 5-methylcytosine remain unchanged. The term "modify" as used herein means the conversion of an unmethylated cytosine into another nucleotide that will distinguish unmethylated cytosine from methylated cytosine. The conversion of the unmethylated, but not the methylated, cytosine bases in the sample containing mitochondrial DNA is carried out with a conversion agent. The term "conversion agent" or "conversion reagent", as used herein, refers to a reagent capable of converting unmethylated cytosine to uracil or another base that is differentially detectable from cytosine in hybridization property terms. The converting agent is preferably a bisulfate such as bisulfites or hydrogen sulfite. However, other agents that similarly modify unmethylated cytosine, but not methylated cytosine, such as hydrogen sulfite, can also be used in the method of the invention. The reaction is carried out according to standard procedures (Frommer et al., 1992, Proc. Natl. Acad. Sci. USA 89: 1827-1831; Olek, 1996, Nucleic Acids Res. 24: 5064-6; EP 1394172). It is also possible to carry out the conversion enzymatically, e.g. ex. by specific methylation of cytidine deaminases.
[0253] In a preferred embodiment of the first method of the invention, the sample containing mitochondrial DNA has been treated with a reagent capable of converting an unmethylated cytosine to uracil or another base that is detectably different from cytosine in terms of hybridization properties. In a more preferable embodiment, the sample comprising mitochondrial DNA is treated with bisulfite using an appropriate commercial kit for this, for example "EZ Methylation Kit" (Zymo Research, Ecogen; Barcelona, Spain).
[0255] Once the sample containing mitochondrial DNA has been treated with a bisulfite, the D-loop region and / or the ND1 gene containing one or more CpG, CHG and / or CHH sites shown in Tables 1 to 5 can be amplified. using primers that allow to distinguish the unmethylated sequence (in which the cytosine of the CpG site becomes uracil) from the methylated sequence (in which the cytosine of the CpG site remains cytosine). Many amplification methods are based on an enzyme chain reaction such as, for example, a polymerase chain reaction (PCR), ligase chain reaction (LCR), ligase polymerase chain reaction 35, Gap- CSF, reaction in repair chain, 3SR and NASBA. Furthermore, there is strand displacement amplification (SDA), transcription-mediated amplification (TMA), and Qp-amplification, etc., this list being merely illustrative. Nucleic acid amplification methods are described in Sambrook et al., 2001 (cited supra). Other amplification methods include the methylation-specific PCR (MSP) method, described in US 5,786,146 that combines bisulfite treatment and allele-specific PCR (see, eg, US 5,137,806 , US 5,595,890, US 5,639,611). Uracil is recognized as a thymine by Taq polymerase and, therefore, after PCR, the resulting product contains cytosine only at the position where DNA with 5-methylcytosine exists in the starting template.
[0257] In a preferred embodiment of the invention, once the sample comprising mitochondrial DNA, preferably a total DNA sample, has been treated with a bisulfite, the region containing one or more CpG, CHG and / or CHH sites can be amplified using primers that are not specific for the methylated sequence. For example, the preferred sequence of the primers does not correspond to a nucleotide sequence comprising a CpG dinucleotide.
[0259] The products of amplification are detected according to standard procedures in the prior art. The amplified nucleic acid can be determined by methods known to the person skilled in the art and are described e.g. eg, in Sambrook et al., 2001 (cited above). There may also be additional purification steps before the target nucleic acid is detected, for example a precipitation step. Detection methods can include, but are not limited to, the binding or intercalation of specific dyes such as ethidium bromide that intercalates into double-stranded DNA and changes its fluorescence thereafter. Purified nucleic acids can also optionally be separated by electrophoretic methods after restriction digestion and visualized afterwards. There are also probe-based assays that take advantage of oligonucleotide hybridization to specific sequences and subsequent detection of the hybrid. It is also possible to sequence the target nucleic acid after more steps known to the person skilled in the art. Other methods use various nucleic acid sequences with a silicon chip to which specific probes are attached and produce a signal when a complementary sequence is attached.
[0261] In a preferred embodiment of the invention, after amplification of the region of interest where it is desired to determine the methylation pattern (eg, in the D loop region or in the ND1 gene), pyrosequencing is used to determine in said sequence the CpG, CHG and / or CHH sites modified after bisulfite treatment. The cytosine / thymine ratio at each of the sites can be quantitatively determined based on the amount of cytosine and thymine incorporated during the sequence extension step.
[0263] Alternatively, the methylation pattern of at least one CpG, CHG, and / or CHH site in the D loop region or at least one CpG and / or CHG site of the site's ND1 gene can be confirmed by restriction enzyme digestion and Southern blot analysis. Examples of methylation-sensitive restriction endonucleases that can be used include Smal, SacII, EagI, MspI, HpalI, BstilI, and BssHIl, for example.
[0265] The term "hypermethylation", as used herein, refers to an altered methylation pattern where one or more nucleotides, preferably cytosines from the CpG, CHG, and / or CHH sites, are methylated compared to a sample. reference. Said reference sample is preferably a sample containing mitochondrial DNA obtained from a subject that does not suffer from a neurodegenerative disease selected from AD or PD. In particular, the term refers to a greater number of 5-methylcytosines at one or more CpG sites in the D loop region shown in Table 1, at one or more CpG sites in the ND1 gene shown in Table 2, in one or more CHG sites in the D loop region shown in Table 3, at one or more CHG sites in the ND1 gene shown in Table 4 and / or at one or more CHH sites in the D loop region shown in Table 5, on a mitochondrial DNA sequence when compared to the relative amount of 5-methylcytosines present at said one or more sites in a reference sample.
[0267] The term "hypomethylation", as used herein, refers to an altered methylation pattern where one or more nucleotides, preferably cytosines at the CpG, CHG, and / or CHH sites, are unmethylated compared to a reference sample. The term "reference sample" refers to a sample containing mitochondrial DNA obtained from a subject not suffering from a neurodegenerative disease selected from AD or PD. In particular, said term refers to a reduced number of 5-methylcytosines in one or more CpG sites in the D loop region shown in Table 1, in one or more CpG sites of the ND1 gene shown in Table 2, in one or more CHG sites in the D loop region shown in Table 3, at one or more CHG sites in the ND1 gene shown in Table 4 and / or at one or more CHH sites in the D loop region shown in Table 5 in a mitochondrial DNA sequence when compared to the relative amount of 5-methylcytosines present at said one or more CpG sites, one or more CHG sites and / or one or more CHH sites in a reference sample.
[0269] In a preferred embodiment of the invention, said reference sample containing mitochondrial DNA is selected from tissue samples, or biological fluids, preferably blood or cerebrospinal fluid samples from subjects. In a preferred embodiment, said reference sample is total DNA. The methods for obtaining these samples, as well as the methods for isolating total DNA or mitochondrial DNA from a sample have been detailed above. In a still more preferred embodiment, the reference sample is a sample containing mitochondrial DNA from age-matched subjects.
[0270] In this first method, the invention provides some specific CpG, CHG and CHH sites that are related to the diagnosis or risk of developing a neurodegenerative disease selected from Alzheimer's disease and Parkinson's disease. Thus:
[0272] • a hypermethylation in at least one of the CpG sites in the D-loop region shown in Table 1,
[0274] • a hypermethylation in at least one of the CHG sites in the D-loop region shown in Table 3,
[0276] • a hypermethylation in at least one of the CHH sites in the D-loop region shown in Table 5,
[0278] • a hypomethylation in at least one of the CpG sites in the ND1 gene shown in Table 2, and / or
[0280] • a hypomethylation in at least one of the CHG sites in the ND1 gene shown in Table 4,
[0282] it is indicative that the subject is suffering from Alzheimer's or that the subject is at high risk of developing Alzheimer's disease; or
[0284] • a hypomethylation in at least one of the CpG sites in the D-loop region shown in Table 1,
[0286] • a hypomethylation in at least one of the CHG sites in the D-loop region shown in Table 3, and / or
[0288] • a hypomethylation in at least one of the CHH sites in the D-loop region shown in Table 5 is indicative that the subject suffers from Parkinson's or that the subject is at high risk of developing Parkinson's disease.
[0290] In a specific embodiment, the first method of the invention involves determining the methylation pattern of all CpG sites, all CHG sites, and all CHH sites in the D-loop region shown in Tables 1,3 and 5, and the methylation pattern of all CpG sites and all CHG sites of the ND1 gene shown in Tables 2 and 4.
[0292] The authors of the present invention have found that the degree of methylation of the CpG, CHG and CHH sites in the D-loop region is greater in subjects suffering from Alzheimer's in stages I-II than in subjects suffering from said disease in stages III- IV.
[0294] In a specific embodiment, if the methylation pattern is observed relative to the reference pattern in a sample containing mitochondrial DNA from a subject diagnosed with stage I-II Alzheimer's disease, then a hypomethylation in at least one of the sites CpG in the D loop region shown in Table 1 or a hypomethylation at one of said CHG sites in the D loop region shown in Table 3 indicates that the subject is suffering from stage III-IV Alzheimer's disease.
[0296] Second method of the invention
[0298] In a second aspect, the invention relates to an in vitro method for selecting a subject for preventive treatment of a neurodegenerative disease selected from Alzheimer's disease and Parkinson's disease in a subject (hereinafter second method of the invention) which involves determining in a sample from said subject containing mitochondrial DNA the methylation pattern in the D loop region and / or in the ND1 gene, where the methylation pattern is determined in at least one selected site of the group formed by:
[0299] i. the CpG sites in the D loop region shown in Table 1,
[0301] ii. the CpG sites in the ND1 gene shown in Table 2;
[0303] iii. the CHG sites in the D loop region shown in Table 3,
[0305] iv. the CHG sites in the ND1 gene shown in Table 4, and / or
[0307] v. CHH sites in the D loop region shown in Table 5
[0309] wherein a hypermethylation in at least one of said CpG sites in the D loop region, a hypermethylation in at least one of said CHG sites in the D loop region, a hypermethylation in at least one of said CHH sites in the D-loop region loop D, a hypomethylation in at least one of said CpG sites in the ND1 gene and / or a hypomethylation in at least one of said CHG sites in the ND1 gene is indicative that the subject is a candidate to receive a treatment aimed at preventing the Alzheimer's disease or
[0311] wherein a hypomethylation in at least one of said CpG sites in the D loop region, a hypomethylation in at least one of said CHG sites in the D loop region and / or a hypomethylation in at least one of said CHH sites in the D-loop region is indicative that the subject is a candidate for a treatment aimed at preventing Parkinson's disease.
[0312] The term "preventive treatment", as used herein, refers to the prevention or set of prophylactic measures to prevent a disease to prevent or delay the onset of its symptoms, as well as to reduce or alleviate the clinical symptoms of it. In particular, the term refers to prevention or the set of measures to prevent the onset, to delay or to alleviate the clinical symptoms associated with a neurodegenerative disease selected from Alzheimer's disease and Parkinson's disease. Desired clinical outcomes associated with the administration of such treatment to a subject include, but are not limited to, stabilization of the pathological stage of the disease, delay in the progression of the disease or improvement in the physiological state of the subject.
[0314] Appropriate preventive treatments aimed at preventing or delaying the onset of Alzheimer's disease symptoms include, but are not limited to, cholinesterase inhibitors such as donezepil hydrochloride (Arecept), rivastigmine (Exelon), and galantemin (Reminyl) or N-methyl D-aspartate (NMDA) antagonists. Treatments aimed at preventing or delaying the onset of Parkinson's disease symptoms include, but are not limited to, L-dopa, catechol-o-methyl transferase (COMT) inhibitors such as tolcapone (Tasmar) and entacapone (Comtan), monoamine oxidase B (MAOB) such as selegiline (Eldepryl) and rasagaline (Azilect) and dopamine agonists such as pramipexole, rotigotine and ropinirole.
[0316] The term "select", as used herein, refers to the action of selecting a subject for preventive treatment of a neurodegenerative disease selected from Alzheimer's disease and Parkinson's disease.
[0318] The terms "subject", "neurodegenerative disease", "Alzheimer's disease", "Parkinson's disease", "sample", "mitochondrial DNA", "D-loop region", "ND1 gene", "CpG site", " CHG site ", CHH site", "methylation pattern", "hypermethylation" and "hypomethylation" have been described in detail in the context of the first method of the invention and are used with the same meaning in the second method of the invention.
[0320] In a specific embodiment of the second method of the invention, the sample comprising mitochondrial DNA is selected from a biopsy of a solid tissue or a biological fluid. Samples can be obtained by conventional methods known to those skilled in the art.
[0322] In an even more specific embodiment, the biological fluid is selected from peripheral blood or cerebrospinal fluid.
[0323] In an even more specific embodiment, said solid tissue is brain tissue. In a preferred embodiment of the invention, if it is desired to select a subject for a preventive treatment of Parkinson's disease, said brain tissue sample is a sample obtained from the substantia nigra.
[0325] In a specific embodiment, the second method of the invention involves determining in a sample from a subject containing mitochondrial DNA, the methylation pattern in at least one site selected from the CpG sites in the D-loop region, shown in Table 1 .
[0327] In a specific embodiment, the second method of the invention involves determining the methylation pattern in at least one CpG site in the D-loop region selected from the sites shown in Table 1. In another specific embodiment, the first method of the invention involves determining the methylation pattern in at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12 , at least 13, at least 14, at least 15 or at least 16 CpG sites selected from Table 1.
[0329] In an even more specific and preferred embodiment of the invention, the second method of the invention involves determining the methylation pattern at all CpG sites in the D-loop region shown in Table 1.
[0331] In another specific embodiment, the second method of the invention involves determining in a sample from a subject comprising mitochondrial DNA, the methylation pattern in at least one site selected from the CpG sites in the ND1 gene, shown in Table 2.
[0333] In another specific embodiment, the second method of the invention involves determining the methylation pattern at a CpG site in the ND1 gene selected from the sites shown in Table 2. In another specific embodiment, the second method of the invention involves determining the pattern of methylation in at least 2, at least 3, at least 4, at least 5 or at least 6 CpG sites selected from Table 2.
[0335] In an even more specific and preferred embodiment of the invention, the second method of the invention involves determining the methylation pattern at all CpG sites in the ND1 gene shown in Table 2.
[0337] In another particular embodiment, the second method of the invention involves determining in a sample from a subject containing mitochondrial DNA, the methylation pattern in at least one site selected from the CHG sites in the D loop region, shown in Table 3 .
[0339] In a particular embodiment, the second method of the invention involves determining the methylation pattern in at least one CHG site in the D-loop region selected from the sites shown in Table 3. In another specific embodiment, the second method of the invention involves determining the methylation pattern in at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13 or at least 14 or at least 15 selected CHG sites from Table 3.
[0341] In an even more specific and preferred embodiment of the invention, the second method of the invention involves determining the methylation pattern at all CHG sites in the D-loop region shown in Table 3.
[0343] In another specific embodiment, the second method of the invention involves determining in a sample from a subject containing mitochondrial DNA, the methylation pattern in at least one site selected from the CHG sites in the ND1 gene, shown in Table 4.
[0345] In another specific embodiment, the second method of the invention involves determining the methylation pattern in at least one CHG site in the ND1 gene selected from the sites shown in Table 4. In another specific embodiment, the second method of the invention involves determining the methylation pattern in at least 2, at least 3, at least 4, at least 5 or at least 6 CpG sites selected from Table 4.
[0347] In an even more particular and preferred embodiment of the invention, the second method of the invention involves determining the methylation pattern in all the CHG sites of the ND1 gene shown in Table 4.
[0349] In another specific embodiment, the second method of the invention involves determining in a sample from a subject containing mitochondrial DNA, the methylation pattern at at least one site selected from the CHH sites in the D-loop region, shown in Table 5 .
[0351] In another specific embodiment, the second method of the invention involves determining the methylation pattern in at least one CHH site in the D-loop region selected from those shown in Table 5. In another specific embodiment, the second method of the invention involves determine the methylation pattern in at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least at least 17, at least 18, at least 19, at least 20, at least 21, at least at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42 or at least 43 CHH sites selected from Table 5.
[0352] In an even more specific and preferred embodiment of the invention, the second method of the invention involves determining the methylation pattern at all CHH sites in the D-loop region shown in Table 5.
[0354] In preferred methods of implementation, the second method of the invention includes:
[0356] (i) determine the methylation pattern at all CpG sites in the D loop region shown in Table 1 and at all CpG sites in the ND1 gene shown in Table 2
[0358] (ii) determine the methylation pattern at all CpG sites in the D loop region shown in Table 1 and at all CHG sites in the D loop region shown in Table 3
[0360] (iii) determine the methylation pattern at all CpG sites in the D loop region shown in Table 1 and at all CHG sites in the ND1 gene shown in Table 4
[0362] (iv) determine the methylation pattern at all CpG sites in the D loop region shown in Table 1 and at all CHH sites in the D loop region shown in Table 5
[0364] (v) determine the methylation pattern at all CpG sites in the ND1 gene shown in Table 21 and at all CHG sites in the D loop region shown in Table 3
[0366] (vi) determine the methylation pattern in all CpG sites in the ND1 gene shown in Table 2 and in all CHG sites in the ND1 gene shown in Table 4
[0368] (vii) determine the methylation pattern at all CpG sites in the ND1 gene shown in Table 2 and at all CHH sites in the D loop region shown in Table 5
[0370] (viii) determine the methylation pattern at all CHG sites in the D loop region shown in Table 3 and at all CHG sites in the ND1 gene shown in Table 4
[0372] (ix) determine the methylation pattern at all CHG sites in the D-loop region shown in Table 3 and / or
[0373] (x) determine the methylation pattern at all CHG sites in the ND1 gene shown in Table 4 and at all CHH sites in the D loop region shown in Table 5
[0375] Suitable methods for determining the methylation pattern in a sample from a subject containing mitochondrial DNA have been described in detail in the context of the first method of the invention.
[0377] In a specific embodiment, the second method of the invention involves determining the methylation pattern at said CpG, CHG and / or CHH sites by means of a technique selected from the group consisting of the enrichment method based on methylation-specific PCR (eg MeDIP, MBD-seq and MethylCap), bisulfite sequencing and by a bisulfite-based method (eg RRB, Infinium, GoldenGate, Cobra, MSP, MethyLight) and a method by restriction digestion (e.g., MRE-seq, or HELP assay), pyrosequencing, chIP-on-chip assay, or differential conversion, differential restriction, differential weight of methylated CpG, CHG, and / or CHH sites of the DNA.
[0378] In a specific and preferred embodiment of the invention, the methylation pattern of one or more CpG, CHG and / or CHH sites in the D-loop region and / or one or more CpG and / or CHH sites in the ND1 gene, of According to the second method of the invention, it is determined by pyrosequencing.
[0379] According to the second method of the invention:
[0380] • a hypermethylation in at least one of the CpG sites in the D-loop region shown in Table 1,
[0381] • a hypermethylation in at least one of the CHG sites in the D-loop region shown in Table 3,
[0382] • a hypermethylation in at least one of the CHH sites in the D-loop region shown in Table 5
[0383] • a hypomethylation in at least one of the CpG sites in the ND1 gene shown in Table 2
[0384] • and / or a hypomethylation in at least one of the CHG sites in the ND1 gene shown in Table 4, is indicative that the subject is a candidate to receive a treatment aimed at preventing Alzheimer's disease; or
[0385] • a hypomethylation in at least one of the CpG sites in the D-loop region shown in Table 1
[0386] • a hypomethylation in at least one of the CHG sites in the D-loop region shown in Table 3,
[0387] • and / or a hypomethylation in at least one of the CHH sites in the D-loop region shown in Table 5 is indicative that the subject is a candidate for a treatment aimed at preventing Parkinson's disease. In a specific embodiment, the second method of the invention involves determining the methylation pattern of all CpG sites, all CHG sites, and all CHH sites in the D-loop region shown in Tables 1, 3 and 5, and the methylation pattern of all CpG sites and all CHG sites in the ND1 gene shown in Tables 2 and 4.
[0388] Third method of the invention
[0389] In the third feature, the invention relates to an in vitro method for monitoring the progression of a neurodegenerative disease selected from Alzheimer's disease or Parkinson's disease in a subject (hereinafter, third method of the invention) involving :
[0390] (a) determining in a sample from said subject containing mitochondrial DNA the methylation pattern in the D loop region, and / or in the ND1 gene, wherein the methylation pattern is determined in at least one site selected from the group formed by:
[0391] (i) the CpG sites in the D loop region shown in Table 1,
[0392] (ii) the CpG sites in the ND1 gene shown in Table 2;
[0393] (iii) the CHG sites in the D loop region shown in Table 3,
[0394] (iv) the CHG sites in the ND1 gene shown in Table 4, and / or
[0395] (v) the CHH sites in the D loop region shown in Table 5 and
[0396] (b) comparing the methylation pattern determined in step a) with said methylation pattern obtained in an earlier stage of the disease, in the case of hypermethylation in at least one of said CpG sites in the D-loop region, a hypermethylation in at least one of said CHG sites in the D loop region, a hypermethylation in at least one of said CHH sites in the D loop region, a hypomethylation in at least one of said CpG sites in the ND1 gene and / or a hypomethylation in at least one of said CHG sites in the ND1 gene with respect to said methylation pattern determined in an earlier stage of the disease, is indicative of the progression of Alzheimer's disease; and wherein a hypomethylation in at least one of said CpG sites in the D-loop region, a hypomethylation in at least one of said CHG sites in the D-loop region and / or a hypomethylation in at least one of said CHH sites in the D-loop region with respect to said methylation pattern determined in an earlier stage of the disease is indicative of the progression of Parkinson's disease.
[0397] The term "monitoring progression", which is equivalent to "determining prognosis", refers to determining the progression of a disease in a subject diagnosed with said disease. In particular, the term refers to the determination of the progression of a neurodegenerative disease selected from the disease of Alzheimer's and Parkinson's disease in a subject diagnosed with said disease. As the person skilled in the art knows, there are several suitable parameters to determine the evolution of a disease in a subject, for example, the evolution of a neurodegenerative disease selected from AD and PD, can be determined, for example, by determining survival global.
[0399] In a specific embodiment, the subject under study has been diagnosed with stage I-II AD.
[0401] In another specific embodiment, the subject under study has been diagnosed with PD in stages III-V.
[0403] The term "overall survival", as used herein, refers to the percentage of patients who survive from the time of diagnosis or treatment of a neurodegenerative disease selected from Alzheimer's disease and Parkinson's disease, after a period definite time.
[0405] The terms "subject", "neurodegenerative disease", "Alzheimer's disease", "Parkinson's disease", "sample", "mitochondrial DNA", "D-loop region", "ND1 gene", "CpG site", " CHG site "," CHH site "," methylation pattern "," hypermethylation "and" hypomethylation "have been described in detail in the context of the first method of the invention and are used with the same meaning in the third method of the invention .
[0407] According to the third method of the invention, the first step of determining the prognosis of a neurodegenerative disease selected from AD and PD involves determining in a sample containing mitochondrial DNA from a subject diagnosed with said neurodegenerative disease, the methylation pattern in the D loop region and / or the ND1 gene in at least one site selected from the sites shown in Tables 1 to 5.
[0409] In a second stage, the third method of the invention involves comparing the methylation pattern obtained in said first stage with said methylation pattern obtained in an early stage of the disease. Therefore, the third method of the invention involves determining the methylation pattern in a sample containing mitochondrial DNA (first sample) from a subject diagnosed with said neurodegenerative disease, the methylation pattern in the D-loop region and / or in the ND1 gene in at least one site selected from the CpG, CHG and / or CHH sites shown in Tables 1 to 5 and, after a suitable period of time, determine in a sample containing mitochondrial DNA (second sample) of said subject diagnosed with said neurodegenerative disease, the methylation pattern at these sites. Said second sample can be obtained in a period of one month, two months, three months, four months, five months, six months, one year, two years, three years, four years, five years, ten years or more after obtaining the first sample.
[0411] In a particular embodiment, said first sample is obtained from a subject who is not receiving any suitable treatment for said selected neurodegenerative disease of AD and PD and said sample is obtained after a period of time where the treatment of the disease is taking place. In another specific embodiment, said first sample is obtained at the beginning of the appropriate treatment for said neurodegenerative disease and the second sample is obtained at one or more points during the course of treatment.
[0413] According to the third method of the invention:
[0415] • a hypermethylation in at least one of the CpG sites in the D-loop region shown in Table 1,
[0416] • a hypermethylation in at least one of the CHG sites in the D-loop region shown in Table 3,
[0417] • a hypermethylation in at least one of the CHH sites in the D-loop region shown in Table 5,
[0418] • a hypomethylation in at least one of the CpG sites in the ND1 gene shown in Table 2,
[0420] • and / or a hypomethylation in at least one of the CHG sites in the ND1 gene shown in Table 4, with respect to said methylation pattern determined in an earlier stage of the disease is indicative of the progression of Alzheimer's disease; or
[0422] • a hypomethylation in at least one of the CpG sites in the D-loop region shown in Table 1,
[0424] • a hypomethylation in at least one of the CHG sites in the D-loop region shown in Table 3,
[0426] • and / or a hypomethylation in at least one of the CHH sites in the D-loop region shown in Table 5,
[0427] with respect to said methylation pattern determined in an earlier stage of the disease, it is indicative of the progression of Parkinson's disease.
[0429] The term "progression of Alzheimer's disease", as used herein, refers to the subject being in a more advanced stage of the disease than the stage in which the subject was diagnosed. That is, if the subject was classified as stage I-II of AD (according to brain involvement and / or the symptoms or clinical manifestations of the disease present in said subject) the subject is considered to be in a more advanced stage of the disease if the subject is classified as stage III-IV or stage V- VI or if the subject goes from being classified as stage III-IV to being classified as stage V-VI of AD.
[0431] The term "progression of Parkinson's disease," as used herein, refers to the subject being at a more advanced stage of the disease than the stage at which the subject was diagnosed. That is, if the subject was classified as stage I-II of PD (according to the brain involvement and / or the symptoms or clinical manifestations of the disease present in said subject), the subject is considered to be in a stage more advanced disease if the subject is classified as stage III-IV or stage V-VI or if the subject changes from being classified as stage III-IV to being classified as stage V-VI of the EP.
[0433] In a specific embodiment of the third method of the invention, the sample comprising mitochondrial DNA is selected from a biopsy of a solid tissue or biological fluid. Samples can be obtained by conventional methods known to those of skill in the art.
[0435] In an even more specific embodiment, the bliological fluid is selected from peripheral blood or cerebrospinal fluid.
[0437] In an even more specific embodiment, said solid tissue is brain tissue.
[0439] In a preferred embodiment of the invention, if the progression of Parkinson's disease is monitored, said brain tissue sample is obtained from the substantia nigra.
[0441] In a particular embodiment, the third method of the invention involves determining in a sample containing mitochondrial DNA the methylation pattern in at least one site selected from the CpG sites in the D loop region, shown in Table 1.
[0443] In a specific embodiment, the third method of the invention involves determining the methylation pattern of at least one CpG site in the D-loop region selected from the sites shown in Table 1. In another specific embodiment, the first method of the invention involves determine the methylation pattern in at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15 or at least 16 CpG sites selected from Table 1.
[0445] In an even more specific and preferred embodiment of the invention, the third method of the invention comprises determining the methylation pattern of all CpG sites in the D-loop region shown in Table 1.
[0447] In another specific embodiment, the third method of the invention comprises determining in a sample from a subject containing mitochondrial DNA, the methylation pattern in at least one site selected from the CpG sites in the ND1 gene shown in Table 2.
[0449] In another specific embodiment, the third method of the invention involves determining the methylation pattern at a CpG site in the ND1 gene selected from the sites shown in Table 2. In another specific embodiment, the third method of the invention involves determining the pattern of methylation at at least 2, at least 3, at least 4, at least 5 or at least 6 CpG sites selected from Table 2.
[0451] In an even more specific and preferred embodiment of the invention, the third method of the invention involves determining the methylation pattern at all CpG sites in the ND1 gene shown in Table 2.
[0453] In a specific embodiment, the third method of the invention involves determining in a sample from a subject containing mitochondrial DNA, the methylation pattern at at least one site selected from the CHG sites in the D-loop region, shown in Table 3 .
[0455] In a specific embodiment, the third method of the invention involves determining the methylation pattern in at least one CHG site in the D-loop region selected from the sites shown in Table 3. In another specific embodiment, the third method of the invention involves determining the methylation pattern in at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12 , at least 13 or at least 14 or 15 CHG sites selected from Table 3.
[0457] In an even more specific and preferred embodiment of the invention, the third method of the invention involves determining the methylation pattern at all CHG sites in the D-loop region shown in Table 3.
[0458] In another specific embodiment, the third method of the invention involves determining in a sample from a subject containing mitochondrial DNA, the methylation pattern at at least one site selected from the CHG sites in the ND1 gene shown in Table 4.
[0460] In another specific embodiment, the third method of the invention involves determining the methylation pattern in at least one CHG site in the ND1 gene selected from the sites shown in Table 4. In another specific embodiment, the third method of the invention involves determining the methylation pattern in at least 2, at least 3, at least 4, at least 5, or at least 6 CpG sites selected from Table 4.
[0462] In an even more specific and preferred embodiment of the invention, the third method of the invention involves determining the methylation pattern in all the CHG sites of the ND1 gene shown in Table 4.
[0464] In another specific embodiment, the third method of the invention involves determining in a sample from a subject containing mitochondrial DNA, the methylation pattern in at least one site selected from the CHH sites in the D-loop region, shown in Table 5 .
[0466] In another specific embodiment, the third method of the invention involves determining the methylation pattern at at least one CHH site in the D-loop region selected from the sites shown in Table 5. In another specific embodiment, the third method of the invention involves determining the methylation pattern in at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12 , at least 13, at least 14, at least 15, at least 16, at least at least 17, at least 18, at least 19, at least 20, at least 21, at least at least 22, at least 23, at least least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36 , at least 37, at least 38, at least 39, at least 40, at least 41, at least 42 or at least 43 CHH sites selected from Table 5.
[0467] In an even more specific and preferred embodiment of the invention, the third method of the invention involves determining the methylation pattern at all CHH sites in the D-loop region shown in Table 5.
[0469] In preferred methods of implementation, the third method of the invention includes:
[0471] i. determine the methylation pattern at all CpG sites in the D loop region shown in Table 1 and at all CpG sites in the ND1 gene shown in Table 2,
[0473] ii. determine the methylation pattern in all CpG sites in the D loop region shown in Table 1 and in all CHG sites in the D loop region shown in Table 3,
[0475] iii. determine the methylation pattern at all CpG sites in the D loop region shown in Table 1 and at all CHG sites in the ND1 gene shown in Table 4,
[0477] iv. determine the methylation pattern at all CpG sites in the D loop region shown in Table 1 and at all CHH sites in the D loop region shown in Table 5,
[0479] v. determine the methylation pattern in all the CpG sites of the ND1 gene shown in Table 2 1 and in all the CHG sites in the D loop region shown in Table 3,
[0481] saw. determine the methylation pattern at all CpG sites in the ND1 gene shown in Table 2 and at all CHG sites in the ND1 gene shown in Table 4,
[0483] vii. determine the methylation pattern at all CpG sites in the ND1 gene shown in Table 2 and at all CHH sites in the D loop region shown in Table 5.
[0485] viii. determine the methylation pattern at all CHG sites in the D loop region shown in Table 3 and at all CHG sites in the ND1 gene shown in Table 4 and / or
[0487] ix. determine the methylation pattern at all CHG sites in the D loop region shown in Table 3
[0488] x. determine the methylation pattern at all CHG sites in the ND1 gene shown in Table 4 and at all CHH sites in the D loop region shown in Table 5.
[0490] Suitable methods for determining the methylation pattern in a sample from a subject containing mitochondrial DNA have been described in detail in the context of the first method of the invention.
[0492] In another specific embodiment, the third method of the invention involves determining the methylation pattern at said CpG, CHG and / or CHH sites by a technique selected from the group consisting of methylation specific PCR (MSP), an enrichment-based method ( eg MeDIP, MBD-seq and MethylCap), bisulfite sequencing and by a bisulfite-based method (eg RRBS, Infinium, GoldenGate, Cobra, MSP, MethyLight) and a restriction digest method (eg. g., MRE-seq, or HELP assay), pyrosequencing, or differential conversion, differential restriction, differential weight of methylated CpG, CHG and / or CHH sites of DNA.
[0493] In a specific and preferred embodiment of the invention, the methylation pattern of one or more CpG, CHG and / or CHH sites in the D-loop region and / or one or more CpG and / or CHH sites in the ND1 gene, of According to the third method of the invention, it is determined by pyrosequencing.
[0495] In another specific embodiment, the third method of the invention involves determining the methylation pattern of all CpG sites, all CHG sites, and all CHH sites in the D-loop region shown in Tables 1,3 and 5, and the methylation pattern of all CpG sites and all CHG sites in the ND1 gene shown in Tables 2 and 4.
[0497] Fourth method of the invention
[0499] The authors of the present invention have discovered a single nucleotide polymorphism (SNP) in the D-loop region of mitochondrial DNA that is statistically associated with the development of Alzheimer's disease if said SNP is found in at least 60% of patients. mtDNA molecules from a subject.
[0501] Therefore, in the fourth feature, the invention relates to an in vitro method for diagnosing or determining the risk of developing Alzheimer's disease in a subject (hereinafter the fourth method of the invention) that involves determining in a sample containing mitochondrial DNA from said subject, the nucleotide at polymorphic position 16519 according to the sequence defined with accession number NC_012920 (SEQ ID NO: 1) in the NCBI database, where the detection of nucleotide C in said Polymorphic position or the presence of nucleotide C at said polymorphic position in at least 60% of the mitochondrial DNA molecules of said subject is indicative that the subject suffers from said disease or that the subject has a high risk of developing the disease.
[0502] The terms "diagnosis", "determine risk", "sample" and "mitochondrial DNA" have been defined in the context of the first, second and third methods of the invention and are used with the same meaning in the fourth method of the invention. .
[0504] The presence of a specific nucleotide in a polymorphic position can be defined as the percentage of DNA molecules that have said nucleotide in said polymorphic position with respect to the total number of DNA molecules present in the sample. According to the fourth method of the invention, it is considered that the subject suffers from Alzheimer's disease or that the subject has a high risk of developing said disease if the mtDNA of said subject has nucleotide C in polymorphic position 16519 according to the sequence defined with accession number NC_012920 when the percentage of molecules that have said nucleotide in said position is at least 60%, at least 65%, at least 70%, at least 75%, at least 80% , at least 85%, at least 90% or more.
[0506] In a specific embodiment, it is considered that the subject has Alzheimer's disease or that the subject has a high risk of developing the disease if the mtDNA of said subject has nucleotide C at polymorphic position 16519 according to the sequence defined with the number of access NC_012920 in 60% of mtDNA molecules. In a preferred embodiment of the invention, it is considered that the subject has Alzheimer's disease or that the subject has a high risk of developing said disease if the mtDNA of said subject has nucleotide C at polymorphic position 16519 according to the sequence defined with accession number NC_012920 in 71% of mtDNA molecules.
[0508] In another preferred embodiment of the invention, it is considered that the subject has Alzheimer's disease or that the subject has a high risk of developing said disease if the mtDNA of said subject has nucleotide C at polymorphic position 16519 according to the sequence defined with the accession number Nc_012920 in 74% of the mtDNA molecules. In another preferred embodiment of the invention, it is considered that the subject has Alzheimer's disease or that the subject has a high risk of developing said disease if the mtDNA of said subject has nucleotide C at polymorphic position 16519 according to the sequence defined with accession number NC_012920 in 78% of mtDNA molecules.
[0510] As the person skilled in the art knows, a sample containing mitochondrial DNA can be homoplasmic or heteroplasmic. The term "heteroplasmy" or "heteroplasmic mitochondrial DNA", as used herein, refers to the mitochondrial DNA of a subject consisting of a mixture of DNA from at least two different genotypes of mitochondria. The term "homoplasmy" or "homoplasmic mitochondrial DNA" as used herein refers to the mitochondrial DNA of a subject that is made up of a single DNA genotype of a single mitochondrial genotype.
[0512] In a specific embodiment, in the context of the present invention, the mitochondrial DNA sample is homoplasmic, in which case all the mitochondria present in the subject contain identical genetic material so that all the mitochondria of a subject have the nucleotide in their genome C in polymorphic position 16519 according to the sequence defined with accession number NC_012920. In those embodiments in which the mitochondrial DNA sample is homoplasmic, the fourth method of the invention allows to diagnose or determine the risk of developing Alzheimer's disease in a subject by detecting nucleotide C in polymorphic position 16519 according to the sequence defined with accession number NC_012920 in said mitochondrial DNA sample; that is, the detection of nucleotide C at polymorphic position 16519 according to the sequence defined with accession number NC_012920 in a homoplasmic mitochondrial DNA sample from a subject is indicative that the subject suffers from Alzheimer's disease or that the subject has a high risk of developing the disease. For him On the contrary, the detection of nucleotide T in the polymorphic position 16519 according to the sequence defined with the accession number NC_012920 in a homoplasmic mitochondrial DNA sample, indicates that the subject does not suffer from Alzheimer's disease or that the subject has a low risk of developing the illness.
[0514] In another specific and preferred embodiment of the invention, the mitochondrial DNA sample is heteroplasmic, that is, the subject's mtDNA is from two mitochondrial populations whose genetic material is not identical to each other. According to this invention, heteroplasmy refers to the fact that a percentage of said mitochondria of said subject have oligonucleotide C in their genome in polymorphic position 16519 according to the sequence defined with accession number NC_012920 and that the remaining percentage of Mitochondria of said subject present oligonucleotide T in the polymorphic position 16519 in their genome according to the sequence defined with accession number NC_012920.
[0515] The person skilled in the art will understand that the identification of the presence of the polymorphism in at least 60% of the mtDNA molecules is equally useful in order to select a subject to be subjected to a preventive treatment of Alzheimer's disease, in cases wherein the subject's mtDNA is heteroplasmic. As in the cases in which the subject's mtDNA is homoplasmic, all the molecules will present the T nucleotide or the C nucleotide at position 16519, in which case the percentage of mtDNA molecules with the polymorphisms indicates that the patient is a candidate for preventive treatment for Alzheimer's disease is 100% or 0%. Thus, in the case of subjects whose mtDNA is heteroplasmic, there will be a population of mtDNA molecules that will present nucleotide T at position 16519 and a second population of molecules that will present nucleotide C at position 16519. In this case, it is considered that the patient is a candidate to receive a preventive treatment for Alzheimer's disease when the percentage of mt DNA molecules that present nucleotide C at position 16519 is equal to or greater than 60%.
[0517] The determination of homoplasmy and heteroplasmy as well as the percentage of heteroplasmy or the "degree of heteroplasmy" can be determined by any technique known to the person skilled in the art. Illustrative non-limiting examples of techniques that allow determining whether a mitochondrial DNA sample is heteroplasmic include, but are not limited to, southern blotting, or PCR-RFLP or mtDNA sequencing. Briefly, the PCR-RFLP technique is based on the fact that, normally, the presence of a SNP in a sample is associated with the creation or destruction of specific sequences or targets of one or more restriction enzymes. The detection of heteroplasmy using the PCR-RFLP technique is a first stage in the amplification of the region of the genetic material that contains the polymorphism to be detected, by using specific oligonucleotides, followed by a second stage in which the amplified fragments are undergo an enzymatic digestion reaction in the presence of an appropriate restriction enzyme. Since the presence or absence of the polymorphism in the sample is associated with the presence or absence of a target specific restriction pattern, the sizes of the fragments obtained determine whether the sample consists of a single pattern of bands, in which case the sample it is homoplasmic. However, if the analysis determines the presence of two band patterns, corresponding to two different populations of mitochondrial DNA, then the mtDNA sample is heteroplasmic.
[0519] The term "single nucleotide polymorphism" or "single nucleotide polymorphism" or "SNP," as used herein, refers to a variation in the nucleotide sequence of a nucleic acid that occurs in a single nucleotide (A, C, T or G), where each possible sequence is present in a proportion equal to or greater than 1% of the population. These polymorphisms appear when a single nucleotide in the genome is altered (eg, by substitution, addition, or deletion). Each version of the sequence with respect to the polymorphic site is referred to as an allele of the polymorphic site. SNPs tend to be evolutionarily stable from generation to generation and, as such, can be used to study specific genetic abnormalities in a population.
[0521] The polymorphic variant of the invention is position 16519 based on the numbering defined by the number NC_012920 in the NCBI database. The polymorphic variant contains a C at that position.
[0523] The terms "determining the sequence of a SNP" or "detecting a SNP" are used interchangeably herein, and refer to determining a sequence of a particular SNP in the subject under study. The determination of the sequence of the SNPs can be carried out by means of several processes known to the person skilled in the art.
[0525] In some specific embodiments of the invention, the sample comprising mitochondrial DNA is selected from a biopsy of a solid tissue or a biological fluid. Samples can be obtained by conventional methods known to those skilled in the art.
[0527] In an even more specific embodiment, the biological fluid is selected from peripheral blood or cerebrospinal fluid.
[0528] In an even more specific embodiment, said solid tissue is brain tissue.
[0530] If the material in which it is desired to determine said SNP according to the present method is a solid tissue or a biological fluid, preferably a previous extraction of the nucleic acid from the sample is carried out using any appropriate technique for this.
[0531] In a preferred embodiment of the invention, the DNA fraction suitable for the implementation of the invention is total DNA. DNA extraction can be carried out using any method known to the person skilled in the art as detailed for the first method of the invention.
[0533] If desired, the present method can be carried out on samples where the mitochondrial fraction has previously been isolated and the DNA thereof has subsequently been isolated. The isolation of the mitochondrial fraction can be carried out using any known method of cell fractionation and that have been detailed in the first method of the present invention.
[0535] After isolating and amplifying (if necessary) the nucleic acid, the sequence of the SNP of the invention is detected by any method or technique capable of determining nucleotides present in a SNP or polymorphism. For example, a SNP can be detected by performing sequencing, mini-sequencing, hybridization, restriction fragment analysis, oligonucleotide ligation assay, allele-specific PCR, or a combination thereof. As such, systems and methods limited to nucleic acid sequencing, hybridization methods, and array technology (eg, technology available from BioSciences Aclara, Affymetrix, Agilent Technologies, Inc. Illumina, etc); They can also be used in techniques based on displacement of the mobility of amplified nucleic acid fragments, for example, Conformational Single Strand Polymorphism (SSCP), denaturing gradient gel electrophoresis (DGGE), Chemical Mismatch Cleavage (CMC). ), Restriction Fragment Polymorphisms (RFLP), PCR-RFLP, WAVE analysis and the like (Methods Mol. Med. 2004; 108: 173-88). Of course, this list is merely illustrative and in no way limiting. Those skilled in the art can use any appropriate method to achieve such detection.
[0537] In another specific embodiment, the determination of the sequence of said SNP is carried out by PCR-RFLP.
[0538] In a specific embodiment, the fourth method of the invention corresponds to an in vitro method for diagnosing early stages of AD. The term "early stages of Alzheimer's disease", as used herein, refers to Alzheimer's disease in stages I-II according to the Braak scale defined in the context of the first method of the invention.
[0540] Fifth method of the invention
[0542] In a fifth characteristic, the invention corresponds to an in vitro method to select a subject to undergo a preventive treatment of Alzheimer's disease that involves determining in a sample containing mitochondrial DNA from said subject the nucleotide at polymorphic position 16519 according to the sequence defined with the accession number NC_012920 in the NCBI database, where the detection of nucleotide C in said polymorphic position or the presence of nucleotide C in said polymorphic position in at least 60% of the mitochondrial DNA molecules is indicative that the subject is a candidate for a treatment aimed at preventing Alzheimer's disease.
[0544] The terms "Alzheimer's", "treatment", "sample", mitochondrial DNA ", and" polymorphism "as well as the methods for obtaining the sample and detecting a polymorphism have been detailed in the context of the first, second, third and fourth methods of the invention and are used here with the same meaning.
[0546] According to the fifth method of the invention, the subject is considered to be a candidate to undergo a treatment aimed at preventing Alzheimer's disease if the subject's mtDNA has nucleotide C at polymorphic position 16519 according to the sequence defined with the accession number NC_012920 when the percentage of mtDNA molecules that have said nucleotide in said position is at least 60%, at least 65%, at least 70%, at least 75%, at least 80% , at least 85%, at least 90% or more. In a specific embodiment, the subject is considered eligible to receive a preventive treatment for Alzheimer's disease if said subject's mtDNA has nucleotide C at polymorphic position 16519 according to the sequence defined with accession number nC_012920 at 60 % of mtDNA molecules.
[0548] The person skilled in the art will understand that the identification of the presence of the polymorphism in at least 60% of the mtDNA molecules is equally useful in selecting a subject for a preventive treatment of Alzheimer's disease in cases where the MtDNA of the subject presents heteroplasmy as in the cases in the present homoplasmy. Thus, in the case of subjects whose mtDNA is homoplasmic, all of the molecules will present nucleotide T or nucleotide C at position 16519, in which case the percentage of mtDNA molecules with polymorphisms indicative that the patient is a candidate for receiving preventive treatment for Alzheimer's disease is 100% or 0%. Thus, in the case of subjects whose mtDNA is heteroplasmic, there will be a population of mtDNA molecules that will present nucleotide T at position 16519 and a second population of molecules that will present nucleotide C at position 16519. In this case, it is considered that the patient is a candidate to receive a preventive treatment for Alzheimer's disease when the percentage of mt DNA molecules that present nucleotide C at position 16519 is less than or greater than 60%.
[0550] In some specific embodiments of the invention, the sample comprising mitochondrial DNA is selected from a biopsy of a solid tissue or a biological fluid. Samples can be obtained by conventional methods known to those skilled in the art.
[0551] In a specific embodiment, in the context of the present invention, the mitochondrial DNA sample is homoplasmic, in which case all the mitochondria present in the subject contain identical genetic material so that all the mitochondria of a subject have the nucleotide in their genome C in polymorphic position 16519 according to the sequence defined with accession number NC_012920. In those embodiments where the mitochondrial DNA sample is homoplasmic, the fifth method of the invention allows selecting a patient to be subjected to a preventive treatment of Alzheimer's disease by detecting nucleotide C in polymorphic position 16519 according to the defined sequence with the access number NC_012920 in said mitochondrial DNA sample; that is, the detection of nucleotide C in polymorphic position 16519 according to the sequence defined with accession number NC_012920 in a homoplasmic mitochondrial DNA sample from a patient is indicative that the subject is eligible to receive preventive treatment for the disease of Alzhemier. On the contrary, the detection of nucleotide T in the polymorphic position 16519 according to the sequence defined with accession number NC_012920 in a homoplasmic mitochondrial DNA sample from a patient is indicative that said patient is not a candidate to receive preventive treatment for Alzheimer disease.
[0553] In another specific and preferred embodiment of the invention, the mitochondrial DNA sample is heteroplasmic, that is, the subject's mtDNA is from two mitochondrial populations whose genetic material is not identical to each other. According to the present invention, heteroplasmy refers to a percentage of the mitochondria of said subject that present oligonucleotide C in the polymorphic position 16519 in their genome according to the sequence defined with accession number NC_012920 and the remaining percentage of mitochondria from Said subject has oligonucleotide T in its genome at polymorphic position 16519 according to the sequence defined with accession number NC_012920. The methods for determining whether a sample is homoplasmic or heteroplasmic as well as for determining the degree of heteroplasmy of a sample have been detailed in the context of the fourth method of the invention.
[0555] In an even more specific embodiment, the biological fluid is selected from peripheral blood or cerebrospinal fluid.
[0556] In an even more specific embodiment, said solid tissue is brain tissue.
[0558] If the material in which it is desired to determine said SNP according to the present method is a solid tissue or a biological fluid, preferably a previous extraction of the nucleic acid from the sample is carried out using any appropriate technique for this. In a preferred embodiment of the invention, the DNA fraction suitable for the implementation of the invention is total DNA. DNA extraction can be carried out using any appropriate method known to those skilled in the art as detailed for the first method of the invention.
[0559] In another specific embodiment, the determination of the sequence of said SNP is carried out by PCR-RFLP.
[0560] Polynucleotides of the invention
[0562] In another feature, the present invention corresponds to a nucleic acid (hereinafter "first polynucleotide of the invention") comprising at least 9 contiguous polynucleotides in a region of mtDNA wherein said region comprises at least one methylation site selected from the CpG sites in the D loop region shown in Table 1.
[0564] The term "polynucleotide", as used herein, refers to DNA or RNA molecules greater than 13 bases in length. The polynucleotides of the invention are preferably DNA molecules of at least 14, at least 15, at least 16, at least 18, at least 20, at least 25, at least 30, at least 35, at least 40, at least 50 , at least 60, at least 70, at least 80, at least 90, at least 100 or more bases in length.
[0566] In a specific embodiment, the first polynucleotide of the invention comprises at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30 or more nucleotides contiguous to said at least one CpG site selected from Table 1.
[0568] In another feature, the present invention refers to a nucleic acid (hereinafter "second polynucleotide of the invention") comprising at least 9 contiguous polynucleotides in a region of mitochondrial DNA wherein said region contains at least one methylation site selected from the CpG sites in the ND1 gene shown in Table 2.
[0570] In a specific embodiment, the second polynucleotide of the invention comprises at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30 or more nucleotides contiguous to said at least one CpG site selected from Table 2. In another feature, the present invention refers to a nucleic acid (hereinafter "second polynucleotide of the invention") comprising at least 9 polynucleotides contiguous in a region of mitochondrial DNA wherein said region comprises at least one methylation site selected from the CHG sites in the D-loop region shown in Table 3.
[0572] In a specific embodiment, the third polynucleotide of the invention comprises at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30 or more nucleotides contiguous to said at least one CHG site selected from Table 3.
[0573] In another feature, the present invention refers to a nucleic acid (hereinafter "fourth polynucleotide of the invention") comprising at least 9 contiguous polynucleotides in a region of mitochondrial DNA wherein said region contains at least one methylation site selected from the CHG sites of the ND1 gene shown in Table 4.
[0575] In a specific embodiment, the fourth polynucleotide of the invention comprises at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30 or more nucleotides contiguous to said at least one CHG site selected from Table 4.
[0577] In another aspect, the present invention relates to a nucleic acid (hereinafter "fifth polynucleotide of the invention") comprising at least 9 contiguous polynucleotides in a region of mtDNA wherein said region comprises at least a methylation site selected from the CHH sites in the D loop region shown in Table 5.
[0579] In a specific embodiment, the fifth polynucleotide of the invention comprises at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30 or more nucleotides contiguous to said at least one CHH site selected from Table 5.
[0581] In another feature, the present invention corresponds to a nucleic acid (hereinafter "sixth polynucleotide of the invention") comprising at least 9 contiguous polynucleotides in a region of mitochondrial DNA wherein said region comprises at least one selected methylation site of the CpG sites in the D loop region shown in Table 1, wherein the position corresponding to cytosine in said CpG site is uracil.
[0583] In a particular embodiment, the sixth polynucleotide of the invention comprises at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30 or more nucleotides contiguous to said at least one CpG site selected from Table 1 wherein the position corresponding to cytosine in said CpG site is uracil.
[0585] In another aspect, the present invention relates to a nucleic acid (hereinafter "seventh polynucleotide of the invention") comprising at least 9 contiguous polynucleotides in a region of mitochondrial DNA wherein said region comprises at least one methylation site selected from the CpG sites of the ND1 gene shown in Table 2, where the position corresponding to cytosine in said CpG site is uracil.
[0587] In a particular embodiment, the seventh polynucleotide of the invention comprises at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30 or more nucleotides contiguous to said at least one CpG site selected from Table 2, wherein the position corresponding to cytosine in said CpG site is uracil.
[0589] In another feature, the present invention refers to a nucleic acid (hereinafter "eighth polynucleotide of the invention") comprising at least 9 contiguous polynucleotides in a region of mitochondrial DNA wherein said region comprises at least one methylation site selected from the CHG sites in the D loop region shown in Table 3, wherein the position corresponding to cytosine in said CHG site is uracil.
[0591] In a particular embodiment, the eighth polynucleotide of the invention comprises at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30 or more nucleotides contiguous to said at least one CHG site selected from Table 3, wherein the position corresponding to cytosine in said CHG site is uracil.
[0593] In another feature, the present invention refers to a nucleic acid (hereinafter "ninth polynucleotide of the invention") comprising at least 9 contiguous polynucleotides in a region of mitochondrial DNA wherein said region comprises at least one methylation site selected from the CHG sites of the ND1 gene shown in Table 4, where the position corresponding to cytosine in said CHG site is uracil.
[0595] In a specific embodiment, the ninth polynucleotide of the invention comprises at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30 or more nucleotides contiguous to said at least one CHG site selected from Table 4, wherein the position corresponding to cytosine in said CHG site is uracil.
[0597] In another feature, the present invention refers to a nucleic acid (hereinafter "tenth polynucleotide of the invention") comprising at least 9 contiguous polynucleotides in a region of mitochondrial DNA wherein said region comprises at least one methylation site selected from the CHH sites in the D loop region shown in Table 5, wherein the position corresponding to cytosine in said CHH site is uracil.
[0599] In a specific embodiment, the tenth polynucleotide of the invention comprises at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30 or more nucleotides contiguous to said at least one CHH site selected from Table 5, wherein the position corresponding to cytosine in said CHH site is uracil.
[0600] In another feature, the invention relates to a polynucleotide that specifically hybridizes with said first, second, third, fourth, fifth, sixth, seventh, eighth, ninth and tenth polynucleotides of the invention.
[0602] The term "specifically hybridizing" or "capable of specifically hybridizing" as used herein refers to the ability of an oligonucleotide or polynucleotide to specifically recognize a sequence from a CpG site. , CHG or CHH. As used herein, the term "hybridization" is the process of combining two nucleic acid molecules or single-stranded molecules with a high degree of similarity resulting in a single double-stranded molecule through specific pairing between complementary bases. Hybridization normally occurs under very stringent conditions or moderately stringent conditions.
[0603] As is known in the art, "similarity" between two nucleic acid molecules is determined by comparing the nucleotide sequence of one molecule with the nucleotide sequence of a second molecule. Variants according to the present invention include nucleotide sequences that are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% similar or identical to the sequence of said at least a CpG, CGH, and CHH site selected from the sites shown in Tables 1 to 5. The degree of identity between two nucleic acid molecules is determined using computer algorithms and methods that are widely known to those of skill in the art. The identity between two amino acid sequences is preferably determined by the BLASTN algorithm (BLAST Manual, Altschul et al, 1990, NCBI NLM NIH Bethesda, Md. 20894, Altschul, S., et al, J. Mol Biol. 215: 403- 10).
[0604] The "stringency" of hybridization reactions is readily determined by one of ordinary skill in the art, and is generally an empirical calculation dependent on probe length, wash temperature, and salt concentration. In general, longer probes require higher temperatures for proper hybridization, while shorter probes require lower temperatures. Hybridization generally depends on the ability of denatured DNA to rehybridize when complementary strands are present in an environment below their melting temperature. The higher the degree of homology desired between the probe and the hybridizable sequence, the higher the relative temperature that can be used. As a result, it follows that higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperatures not so much. For additional details and an explanation of the stringency of hybridization reactions, see Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995).
[0606] The term "stringent conditions" or "high stringency conditions", as used herein, typically: (1) employ low ionic strength and high temperature for washing, eg, 0.015M sodium chloride / 0.0015M sodium citrate / 0.1% sodium dodecyl sulfate at 50 ° C; (2) employ during hybridization a denaturing agent, such as formamide, for example 50% (v / v) formamide with 0.1% bovine serum albumin / 0.1% Ficoll / 0.1 polyvinylpyrrolidone % / 50 mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42 ° C; or (3) employ 50% formamide, 5 x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, solution of Denhardt 5x, sonicated salmon sperm DNA (50 mg / ml), 0.1% s Ds, and 10% dextran sulfate at 42 ° C, with washes at 42 ° C in 0.2 x SSC (chloride sodium / sodium citrate) and 50% formamide, followed by a high stringency wash consisting of 0.1 x SSC containing EDTA at 55 ° C.
[0608] "Moderately stringent conditions" can be identified as described by Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, and include the use of wash solution and hybridization conditions (eg ., temperature, ionic strength and% SDS) less astringent than previously described. An example of moderately stringent conditions is incubation overnight at 37 ° C in a solution comprising: 20% formamide, 5 x SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7 , 6), 5x Denhardt's solution, 10% dextran sulfate and 20 mg / ml fragmented denatured salmon sperm DNA, followed by washing the filters in 1 x SSC at approximately 3537-50 ° C. The person skilled in the art will know how to adjust temperature, ionic strength, etc. if necessary to accommodate factors such as probe length and the like.
[0610] Kits
[0612] The present description further refers to a kit (hereinafter "first kit of the description"), which comprises at least one oligonucleotide capable of specifically hybridizing in a methylation-dependent manner with a mitochondrial DNA sequence comprising a methylation site selected from the group consisting of:
[0614] (i) the CpG sites in the D loop region shown in Table 1,
[0616] (ii) the CpG sites in the ND1 gene shown in Table 2,
[0618] (iii) the CHG sites in the D loop region shown in Table 3,
[0620] (iv) the CHG sites in the ND1 gene shown in Table 4, and / or
[0622] (v) CHH sites in the D loop region shown in Table 5
[0623] The terms "CpG site", CHG site "," CHH site "," D-loop region "and" ND1 gene "have been described in detail in the context of the first method of the invention and the term" capable of hybridizing in a specific "has been defined in the context of the polynucleotides of the invention. Said terms are used with the same meaning in the context of the kits of the invention.
[0625] In a preferred embodiment, the oligonucleotides are part of the kit that is capable of specifically hybridizing in a methylation-dependent manner with a mitochondrial DNA sequence comprising a methylation site selected from the group consisting of
[0627] (i) the CpG sites in the D loop region shown in Table 1,
[0629] (ii) the CpG sites in the ND1 gene shown in Table 2,
[0631] (iii) the CHG sites in the D loop region shown in Table 3,
[0633] (iv) the CHG sites in the ND1 gene shown in Table 4, and / or
[0635] (v) CHH sites in the D loop region shown in Table 5
[0637] They constitute at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least minus 90% or at least 100% of the total amount of the oligonucleotides that make up the kit. In further embodiments, said oligonucleotides constitute at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of the total amount of oligonucleotides that make up the kit.
[0639] Suitable kits include various reagents for use in accordance with the present invention, in suitable containers and packaging materials, including shrink and blow molded tubes, vials, and containers. In addition, the kits may contain instructions for the simultaneous, sequential, or separate use of the various components found in the kit. Said instructions may be in the form of printed material or in the form of an electronic medium capable of storing instructions so that they can be read by a subject, such as electronic storage media (magnetic discs, tapes and the like), optical media ( CD-ROM, DVD) and the like. Additionally, or alternatively, the media may contain the Internet addresses that provide such instructions. Suitable materials for inclusion in an exemplary kit according to the present disclosure comprise one or more of the following: reagents capable of amplifying a specific sequence of a domain either total DNA or mtDNA without the need to perform PCR; Reagents required to discriminate between the various possible alleles in the sequence domains amplified by PCR or non-PCR amplification (e.g., restriction endonucleases, oligonucleotides that preferentially hybridize methylated or unmethylated CpG, CHG and / or CHH sites, including those modified to contain enzymes or fluorescent chemical groups that amplify the oligonucleotide signal and make the discrimination between methylated or unmethylated CpG, CHG and / or CHH sites more robust); or reagents required to physically separate the various amplified regions produced (eg, agarose or polyacrylamide and a buffer to be used in electrophoresis, HPLC columns, SSCP gels, formamide gels, or a matrix support for MALDI-TOF) .
[0641] The term "oligonucleotide", as used herein, refers to a short DNA or RNA molecule, up to bases in length. The oligonucleotides of the invention are preferably DNA molecules of at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15 , at least 20, at least 30, at least 40, at least 45 or 50 bases in length.
[0643] As used in the kit of the description, at least one oligonucleotide capable of hybridizing with at least one sequence of a CpG site in the D-loop region selected from the CpG sites shown in Table 1 is used, at least one oligonucleotide capable of hybridizing with at least one sequence of a CpG site in the ND1 gene shown in Table 2, at least one oligonucleotide capable of hybridizing with at least one sequence of a CHG site in the D loop region shown in Table 3 , at least one oligonucleotide capable of hybridizing with at least one sequence of a CHG site in the ND1 gene shown in Table 4 and / or at least one oligonucleotide capable of hybridizing with at least one sequence of a CHH site in the loop region D shown in Table 5, specifically for methylation, as a primer to amplify the region containing said CpG, CHG and / or CHH sites. Alternatively, at least one oligonucleotide can also be used as a probe to detect such methylated or unmethylated CpG, CHG or CHH sites.
[0645] In a preferred embodiment of the first kit of the description, it comprises oligonucleotides capable of specifically hybridizing with all the CpG sites in the D loop region shown in Table 1, with all the CpG sites of the ND1 gene shown in Table 2, with all CHG sites in the D loop region shown in Table 3, with all CHG sites in the ND1 gene shown in Table 4 and / or with all CHH sites in the D loop region shown in Table 5.
[0647] If desired, the first kit of the disclosure may comprise a first oligonucleotide capable of specifically hybridizing with a bisulfite-treated oligonucleotide comprising at least one sequence of a CpG site in the D loop region shown in Table 1 when said CpG site is methylated, and at least one oligonucleotide or polynucleotide capable of specifically hybridizing with the same bisulfite-treated oligonucleotide comprising at least one sequence of a CpG site in the region of D loop when said CpG site is not methylated; and / or the kit may comprise a first oligonucleotide capable of specifically hybridizing to a bisulfite-treated oligonucleotide comprising at least one sequence of a CpG site in the ND1 gene shown in Table 2 when said CpG site is methylated, and at least least one oligonucleotide or polynucleotide capable of specifically hybridizing to the same bisulfite-treated oligonucleotide comprising at least one sequence of a CpG site in the D-loop region when said CpG site is unmethylated; and / or the kit may comprise a first oligonucleotide capable of specifically hybridizing with a bisulfite-treated oligonucleotide comprising at least one sequence of a CHG site in the D-loop region shown in Table 3 when said CHG site is methylated, and at least one oligonucleotide or polynucleotide capable of specifically hybridizing with the same bisulfite-treated oligonucleotide comprising at least one sequence of a CHG site in the D-loop region when said CHG site is not methylated; and / or the kit may comprise a first oligonucleotide capable of specifically hybridizing with a bisulfite-treated oligonucleotide comprising at least one sequence of a CHG site in the ND1 gene shown in Table 4 when said CHG site is methylated, and to least one oligonucleotide or polynucleotide capable of specifically hybridizing to the same bisulfite-treated oligonucleotide that comprises at least one sequence of a CHG site in the D-loop region when said CHG site is unmethylated; and / or the kit may comprise a first oligonucleotide capable of specifically hybridizing to a bisulfite-treated oligonucleotide comprising at least one sequence of a CHH site in the D-loop region shown in Table 5 when said CHH site is methylated and at least one oligonucleotide or polynucleotide capable of specifically hybridizing with the same bisulfite-treated oligonucleotide comprising at least one sequence of a CHH site in the D-loop region when said CHH site is not methylated.
[0649] For hybridization to an unmethylated CpG site, specific primers that hybridize to unmethylated DNA preferably have a T in the '' CG pair to distinguish it from the C retained in methylated DNA. It is preferable that the primers contain relatively few C's or G's in the sequence since C's will be absent in the sense primer and G's absent in the antisense primer (cytosine is converted to uracil, which is amplified as thymidine in the amplification product ). Consequently, for hybridization to a methylated CpG site, primers that specifically hybridize to methylated DNA preferably have a C in the 3'CG pair.
[0651] In another characteristic, the invention refers to a kit (hereinafter "second kit of the description"), which comprises at least one oligonucleotide capable of specifically hybridizing with a region in the 5 'to 3' position with respect to to a mitochondrial DNA methylation site selected from the group consisting of:
[0653] i. the CpG sites in the D loop region shown in Table 1,
[0655] ii. the CpG sites in the ND1 gene shown in Table 2,
[0657] iii. the CHG sites in the D loop region shown in Table 3,
[0659] iv. the CHG sites in the ND1 gene shown in Table 4, and / or
[0661] v. CHH sites in the D loop region shown in Table 5
[0663] wherein the cytosine methylated at said position has been converted to uracil or another base that is distinguishable from cytosine in its hybridization properties.
[0665] In a preferred embodiment, the oligonucleotides that are part of the kit of the description and that are capable of specifically hybridizing with a region or position 5 'or position 3' with respect to a methylation site in the mitochondrial DNA selected from the group formed by
[0667] i. the CpG sites in the D loop region shown in Table 1,
[0669] ii. the CpG sites in the ND1 gene shown in Table 2,
[0671] iii. the CHG sites in the D loop region shown in Table 3,
[0673] iv. the CHG sites in the ND1 gene shown in Table 4, and / or
[0675] v. CHH sites in the D loop region shown in Table 5
[0677] They constitute at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least minus 90% or at least 100% of the total amount of the oligonucleotides that make up the kit. In further embodiments, said oligonucleotides constitute at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of the total amount of oligonucleotides that make up the kit.
[0678] In a specific embodiment, the second kit of the disclosure further comprises one or more reagents for converting an unmethylated cytosine to uracil or another base that is differentially detectable from cytosine in terms of hybridization properties.
[0680] In a preferred embodiment, the one or more reagents for converting an unmethylated cytosine to uracil or another base that is differentially detectable from cytosine in terms of hybridization properties is a bisulfite, preferably sodium bisulfite. The reagent capable of converting an unmethylated cytosine to uracil or another base that is differentially detectable from cytosine in terms of hybridization properties is metabisulfite, preferably sodium metabisulfite.
[0682] The term "conversion reagent" and its details have been described in detail in the context of the diagnostic method of the invention and are used with the same meaning in the context of the kit according to the invention.
[0684] In a specific embodiment, the second kit of the description comprises at least one oligonucleotide comprising a sequence selected from the sequences shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and / or SEQ ID NO: 5.
[0686] In another characteristic, the invention relates to the use of the first and / or second kit of the description to determine the pattern of mitochondrial DNA in a subject or to determine the diagnosis of a neurodegenerative disease in a subject selected from Alzheimer's disease and Parkinson's disease.
[0688] The invention is described by means of the following examples which are to be construed as merely illustrative and not limiting the scope of the invention
[0690] Examples
[0692] Materials and methods
[0694] Study design and subjects included
[0696] The study was carried out on 44 samples, including pathology related to Alzheimer's disease (AD), Parkinson's disease (PD) and control cases. The samples were run on a plate divided into two lanes. In lane 1, loop D and ND1 amplicons were analyzed in entorhinal cortex samples from AD-related pathology cases and their corresponding controls (Table 7). In lane 2 the amplicons of loop D were analyzed in EP black substance samples and their corresponding controls (Table 7). Each patient was identified in the primers used with a MID (multiple identifier) (Tables 7 and 8). Methylation at CpG and non-CpG sites (CHG and CHH, where H = A, T, or C) was analyzed using a 454 GS FLX Titanium pyrosequencer from Roche that generated 569,684 sequences in lane 1, whose lengths ranged from 40-1098 base pairs (bp) with a mean length of approximately 417 bp. In lane 2 the number of sequences obtained was 513,579, whose length varied from 40 to 933 bp (mean length 466 bp). The alignments of the sequences obtained for each MID, amplicon and lane against their respective reference sequences were noted and the percentages of identity between these were close to 100%. The mean and median bisulfite conversion rates for each locus and MID were analyzed.
[0698] The number of unmethylated sequences was greater than the number of methylated sequences identified at each site and some methylation sites were not present. Those sequences that after alignment were found to have at least one methylation pattern site missing were removed from the analysis to avoid any bias at the time of quantification. This bypasses the rough analysis of putative mitochondrial pseudogenes whose amplicons exhibited almost 100% identity to mitochondrial DNA when analyzed in NCBI BLAST. Most of the CpG, CHG, and CHH sites analyzed were unmethylated. However, different methylation sites could be identified.
[0700] Table 6: Number of differentially methylated sites. FRD: p-value adjusted with the Benjamini and Hocheberg (1995) method
[0704] Table 7: Summary of the main clinical characteristics and human neuropathological cases analyzed
[0705]
[0706]
[0709] The Braak stages for Alzheimer's disease (AD) indicate the degree of presence of neurons with fibrillar neurodegeneration (Roman numerals) and senile plaques (letters) following the Braak classification. The Braak stages for Parkinson's disease (PD) refer to the degree of presence of the protein asinuclein (Lewy bodies).
[0710] Table 8: MID sequence associated with each case analyzed
[0715] Human brain samples
[0717] Tissue samples were provided by the Neurological Tissue Bank, University of Barcelona - Hospital de Barcelona and the Bank of the Institute of Neuropathology, HUB-ICO-IDIBELL. The donation and collection of the samples was regulated by the ethics committee of both institutions. Half of each brain was kept in 4% formalin buffered solution for morphological and histological study, while the other half was processed in coronal sections to be frozen at -80C to be available for biochemical studies. The neuropathological examination in all control and pathological cases was carried out on thirty standardized sections of the brain, cerebellum and brainstem, which were stained with hematoxylin and eosin, and Klüver Barrera, or processed for immunohistochemistry for fibrillar glial acid protein, microglial markers, beta-amyloid, phosphorylated tau (AT8 antibody), a-synuclein, p-crystallin, ubiquitin, and TDP-43. Cases with pathology related to AD and PD were classified according to current neuropathological criteria (Braak and Braak 1991, 1999; Braak et al, 2003, 2006). Cases with mixed pathology (including vascular lesions) were excluded from this study. The brains used as controls belonged to individuals without neurological manifestations and without lesions in the neurological study. The medical records were re-examined for each case, and the cases with pathology related to AD (stages I-IV) were reassessed through telephone calls or interviews with relatives, asking if they had evidence of any neurological or cognitive impairment. Only the cases that met these criteria were considered in the present work. All the cases analyzed are summarized in Table 7.
[0719] The average post-mortem interval of the entorhinal cortex samples was 4.98 ± 1.57 hours in controls, 7.51 ± 5.13 hours in stages I-II, and 5.70 ± 2.85 hours in stages III-IV; for the black substance samples the intervals were 5.59 ± 2.46 hours and 9.23 in controls ± 7.07 hours in the case of PD.
[0721] Murine brain samples
[0723] APP / PS1 transgenic mice and wild-type strain were obtained from Jackson Laboratory (USA). The transgenic model expresses a murine / human chimeric Pp (Mo / HuAPP695swe: Swedish mutation APP) molecule and the human variant of presenilin 1 (PS1-DE9), both expressions in neurons of the nervous system product. The animals were housed under standard conditions with 12-hour light and dark cycles, with unlimited access to food and water. The establishment was carried out according to the ethical guidelines (European Communities Council Directive 86/609 / EEC) approved by the local ethics committee.
[0725] Total DNA extraction
[0727] Total DNA was isolated from human samples from the entorhinal cortex and substantia nigra (Table 7) using the DNeasy Blood and Tissue kit (Qiagen, Madrid, Spain) following the manufacturer's instructions. Murine total DNA samples were obtained from the frontal cortex using the same procedure.
[0729] Bisulfite treatment
[0731] Three hundred nanograms of DNA were treated with bisulfite using the KIT EZ DNA Methylation (Zymo Research, Ecogen, Barcelona, Spain) following the supplier's instructions. The bisulfite-treated DNA was resuspended at 30 µl to reach a final concentration of 10ng / µl. All samples were treated with bisulfite in parallel, using the same reagent lot to avoid differences in the bisulfite conversion rate between different commercial lots.
[0732] Design of the FLX primers for loop D and ND1 amplicons
[0734] The primers for the FLX experiment were designed following the technical instructions for the Roche FLX sequencer “Amplicon Fusion Primer Design Guidelines for GS FLX Titanium Series Lib-A Chemistry.” The fusion primers for the amplicons contained a GS FLX Titanium directional primer primer A or primer B (including a key sequence (Key) of four bases) in Lot 5 - rpima oligonucleotide, plus a template specific sequence in the first 3 - end. On the other hand, a MID sequence (identifier multiplex) between primer A (or primer B) and the specific sequence for subsequent automated identification of samples with the software after the pooling / multiplexing and sequencing steps. The primers used contained the following components: Forward primer (Primer A -Key-MID-specific sequence template), 5'-CGTATCGCCTCCCTCGCGCCA (SEQ ID NO: 33) - -MID TCAG - 3 'specific template sequence; Invert primer or (Primer B- Key-MID-specific template sequence): 5'-CTATGCGCCTTGCCAGCCCGC (SEQ ID NO: 34) - MID TCAG- specific template sequence-3 '. The specific template sequences for each of the amplicons: D-direct loop, 5'-TAGGGGTTTTTTGATTATTATTTTT-3 '(SEQ ID NO: 2) and D-reverse loop, 5'-ACAAACATT CAATT ATT ATT ATT ATCCT-3' ( SEQ ID NO: 3); ND1-direct, 5'-ATGGTT AATTTTTT ATTTTTT ATT GT ATTT-3 '(SEQ ID NO: 4) and ND1-reverse, 5'-TAATTTAAATTTAATACTCACCCTAATCAA-3' (SEQ ID NO: 5). The primers used in this study were designed to avoid CpG sites. The specific sequence of MIDs for each patient is indicated in Table 8. The amplified regions (loop D: 16386-256; ND1: 3313-3686) are based on the nucleotide position of the human mtDNA map (www.mitomap. org).
[0736] Preparation of the amplicon library
[0738] PCRs for loop D and ND1 amplicons were performed following Roche's Amplicon Library Preparation Method Manual (GS FLX Titanium Series). Twenty nanograms of bisulfite-treated total DNA were used for the PCRs. Amplification of the bisulfite treated DNA was carried out in a reaction volume of 25pl. Each PCR reaction consisted of: 1x FastStart 10x Buffer no. 2, 0.05U / μl FastStart HiFi Polymerase polymerase (Roche), 200 nM of each dNTP, and 200 nM of each specific forward and reverse primer. The primers were synthesized with a quality HPLC purification (Sigma-Aldrich, Madrid, Spain). Amplifications were carried out in an Applied Biosystems Verity ® thermal cycler (Applied Biosystems, Madrid, Spain) using the following conditions: 94 ° C for 3 min and then 36 cycles of 94 ° C for 15s, hybridization temperature (61 ° C ND1, and 62 ° C loop D) for 45s and 72 ° C for 1 min, followed by a final extension step at 72 ° C for 8 min and a final holding temperature at 4 ° C. Two microliters of each PCR product were tested on an agarose gel stained with SYBR ® Safe DNA Gel Stain 1.5% (Invitrogen, Madrid, Spain).
[0740] PCR purification
[0742] The purification of the PCR products was carried out using the Agencourt®AMPure®XP PCR Purification kit (Beckman Coulter, Madrid, Spain) following the Roche Amplicon Library Preparation Method Manual (GS FLX Titanium Series).
[0744] Quantification of amplicon libraries and FLX sequencing
[0746] The quantification and quality controls of the amplicon libraries and the rest of the FLX sequencing protocol were carried out by the team from the Vall d'Hebron Research Institute Genomics Platform (VHIR, Barcelona, Spain).
[0747] Selection of differentially methylated sites
[0749] Alignment and identification of CpG, CHG, and CHH sites and bisulfite conversion rates were performed using BIQ Analyzer HT software (Lutsik et al, 2011). The quality control of the raw data and all the statistical analyzes were performed using the statistical language R and the bioconductor software, http: /// www bioconductor.org).
[0750] The selection of the differentially methylated sites was based on the calculation of Fisher's exact statistical test, considering those differentially methylated sites with an adjusted p-value using the Benjamini and Hochberg (1995) method below 0.05. The b-value represented in the heat map graphs is the ratio of the methylated sequences to the global sum of the methylated and unmethylated sequences per site (Du et al., 2010, BMC Bioinformatics, 30; 11: 587), that is, py = M / (M OR) where M is the number of methylated sequences at site (i) and MID (j), and U is the number of unmethylated sequences at site (i) and MID (j) .
[0752] Example 1: DNA methylation is increased in the D-loop region and reduced in the ND1 gene in cases with early stages of AD-related pathology
[0754] Increased methylation was observed at CpG and non-CpG sites (CHG and CHH) in the D-loop region in cases with Braak stage I / II and III / IV AD-related pathology (Fig. 1).
[0756] The degree of methylation was higher in the cases of pathology related to AD compared to the control samples, and higher in stages I / II compared to stages III / IV, as represented in the log2 (OR) graphs (Fig. 2). However, no differences were found in the methylation of CHH sites between controls and cases with stage III / IV AD-related pathology.
[0758] ND1 analysis revealed the presence of some less methylated CpG and CHG sites in cases with stage I / II and III / IV AD-related pathology compared to control samples (log2 [O]> 0, Fig. 3). No differences were found for the CHH sites.
[0760] Example 2: DNA methylation is reduced in the D-loop region in the substantia nigra of PD cases.
[0761] In contrast to what was observed in the entorhinal cortex in AD, the D-loop region showed a loss of methylation in almost all CpG and non-CpG sites in the substantia nigra of the PD cases compared to the control samples (Fig. 3 ). However, as in AD, the percentage of DNA methylation represents a small part of the total mitochondrial DNA.
[0763] Example 3: DNA methylation is increased in the D-loop region in murine models of AD.
[0765] In this study, APP / PS1 mice (murine model of AD) of six months of age (n = 4) and control mice of the same age (n = 4) were used. As can be seen in Figure 4A, CpG sites showed hypermethylation in samples obtained from mouse model pathology relative to control samples. These results were also seen at CHG sites (see Figure 4B).
[0767] To determine the methylation pattern as the advancement of AD, they used APP / PS1 mice of three, six and twelve months. At three months, APP / APS1 mice have a low degree of neuritic plaque accumulation, which is increased in six-month-old APP / APS1 mice. Six-month-old model EA mice show cognitive and memory failures. Finally, 12-month-old APP / APS1 animals show symptoms similar to those observed in humans in advanced stages of AD.
[0769] Analysis of the degree of methylation in the D-loop region corroborated the pattern observed in samples of human brain tissue. As shown in Figure 5, a tendency to hypomethylation of the CpG CHG and CHH sites was observed in the model mice in advanced stages of AD with respect to said CpG, CHG and CHH sites in the model mice in early stages of AD. EA (Figures 5A, 5B and 5C).
[0771] Example 4: The presence of polymorphic position 16519 in mitochondrial DNA is associated with the development of Alzheimer's disease.
[0773] The results shown in Table 9 show that the presence of the T16519C polymorphism is associated with Alzheimer's disease. These results were obtained by conventional sequencing and using a chi square test.

权利要求:
Claims (15)
[1]
1. In vitro method for diagnosing or determining the risk of developing a neurodegenerative disease selected from Alzheimer's disease and Parkinson's disease in a subject, comprising determining in a sample from said subject comprising mitochondrial DNA, the methylation pattern in the D loop region, and / or in the ND1 gene, where the methylation pattern is determined in at least one site selected from the group consisting of:
(i) the CpG sites in the D loop region shown in Table 1,
(ii) the CpG sites of the ND1 gene shown in Table 2;
(iii) the CHG sites in the D loop region shown in Table 3,
(iv) the CHG sites in the ND1 gene shown in Table 4, and / or
(v) the CHH sites in the D loop region shown in Table 5,
wherein a hypermethylation in at least one of said CpG sites in the D loop region, a hypermethylation in at least one of said CHG sites in the D loop region, a hypermethylation in at least one of said CHH sites in the D-loop region D loop, a hypomethylation in at least one of said CpG sites in the ND1 gene and / or a hypomethylation in at least one of said CHG sites in the ND1 gene is indicative that the subject suffers from Alzheimer's disease or that the subject have a high risk of developing Alzheimer's disease or
wherein a hypomethylation in at least one of said CpG sites in the D loop region, a hypomethylation in at least one of said CHG sites in the D loop region and / or a hypomethylation in at least one of said CHH sites in the D-loop region is indicative that the subject is suffering from Parkinson's disease or that the subject is at high risk of developing Parkinson's disease.
[2]
The method according to claim 1, wherein a hypomethylation at at least one of said CpG sites in the D-loop region or a hypomethylation at at least one of said CHG sites in the D-loop region relative to the methylation pattern in a subject with stage I-II Alzheimer's disease it is indicative that the subject has stage III-IV Alzheimer's disease.
[3]
3. In vitro method of selecting a subject for treatment to prevent progression of a neurodegenerative disease selected from Alzheimer's disease or Parkinson's disease in a subject comprising:
determine in a sample from said subject that contains mitochondrial DNA the methylation pattern in the D loop region and / or in the ND1 gene, where the methylation pattern is determined in at least one site selected from the group consisting of:
i. the CpG sites in the D loop region shown in Table 1,
ii. the CpG sites in the ND1 gene shown in Table 2;
iii. the CHG sites in the D loop region shown in Table 3,
iv. the CHG sites in the ND1 gene shown in Table 4, and / or
v. CHH sites in the D loop region shown in Table 5
wherein a hypermethylation in at least one of said CpG sites in the D loop region, a hypermethylation in at least one of said CHG sites in the D loop region, a hypermethylation in at least one of said CHH sites in the D-loop region loop D, a hypomethylation in at least one of said CpG sites in the ND1 gene and / or a hypomethylation in at least one of said CHG sites in the ND1 gene is indicative that the subject is a candidate to receive a treatment aimed at preventing Alzheimer's disease; or
wherein a hypomethylation in at least one of said CpG sites in the D loop region, a hypomethylation in at least one of said CHG sites in the D loop region and / or a hypomethylation in at least one of said CHH sites in the D-loop region is indicative that the subject is a candidate for receiving a treatment aimed at preventing Parkinson's disease.
[4]
4. A method of monitoring the progression of a selected neurodegenerative disease of Alzheimer's disease or Parkinson's disease in a subject comprising:
a) determining in a sample of said subject that comprises mitochondrial DNA the methylation pattern in the D loop region, and / or in the ND1 gene, where the methylation pattern is determined in at least one site selected from the group formed by :
(i) the CpG sites in the D loop region shown in Table 1,
(ii) the CpG sites in the ND1 gene shown in Table 2;
(iii) the CHG sites in the D loop region shown in Table 3,
(iv) the CHG sites in the ND1 gene shown in Table 4, and / or the CHH sites in the D loop region shown in Table 5,
b) comparing the methylation pattern determined in step a) with said methylation pattern obtained in an earlier stage of the disease,
wherein a hypermethylation in at least one of said CpG sites in the D loop region, a hypermethylation in at least one of said CHG sites in the D loop region, a hypermethylation in at least one of said CHH sites in the D-loop region loop D, a hypomethylation in at least one of said CpG sites of the ND1 gene and / or a hypomethylation in at least one of said CHG sites in the ND1 gene with respect to said methylation pattern determined in an earlier stage of the disease, is indicative of the progression of Alzheimer's disease; and
wherein a hypomethylation in at least one of said CpG sites in the D loop region, a hypomethylation in at least one of said CHG sites in the D loop region, and / or a hypomethylation in at least one of said CHH sites in the D-loop region with respect to said methylation pattern determined in an earlier stage of the disease is indicative of the progression of Parkinson's disease.
[5]
The method according to claim 4, wherein the subject has been diagnosed with stage I-II Alzheimer's disease or wherein the subject has been diagnosed with stage III-V Parkinson's disease.
[6]
The method according to claims 1 to 5, wherein determining the methylation pattern comprises determining the methylation of all CpG sites in the D-loop region shown in Table 1.
[7]
7. The method according to claims 1 to 6, wherein the determination of the methylation pattern comprises the determination of the methylation in all the CpG sites of the ND1 gene shown in Table 2.
[8]
The method according to claims 1 to 7, wherein determining the methylation pattern comprises determining the methylation of all CHG sites in the D-loop region shown in Table 3.
[9]
9. The method of any of claims 1 to 8, wherein determining the methylation pattern comprises determining the methylation pattern of all CHG sites in the ND1 gene shown in Table 4.
[10]
The method according to claims 1 to 9, wherein determining the methylation pattern comprises determining the methylation of all CHH sites in the D-loop region shown in Table 5.
[11]
The method according to any of claims 1 to 10, wherein the methylation pattern is determined by a technique selected from the group consisting of specific methylation PCR, bisulfite sequencing, restriction-digestion-based techniques, pyrosequencing, assay of ChIP-on-chip, differential conversion, differential restriction and differential weight of the methylated site (s).
[12]
12. The method of claim 11, wherein the methylation pattern is determined by pyrosequencing.
[13]
13. Use of a kit to determine the diagnosis of a neurodegenerative disease in a selected subject from Alzheimer's disease and Parkinson's disease, wherein the kit comprises:
- at least one oligonucleotide capable of specifically and methylation-dependent hybridization with a mitochondrial DNA sequence comprising a methylation site selected from the group consisting of:
(i) the CpG sites in the D loop region shown in Table 1,
(ii) the CpG sites in the ND1 gene shown in Table 2;
(iii) the CHG sites in the D loop region shown in Table 3,
(iv) the CHG sites in the ND1 gene shown in Table 4, and / or
(v) the CHH sites in the D loop region shown in Table 5.
[14]
14. Use of a kit to determine the diagnosis of a neurodegenerative disease in a subject selected from Alzheimer's disease and Parkinson's disease, wherein the kit comprises at least one oligonucleotide capable of specifically hybridizing to a position in the 5 'region or 3 'to a methylation site in mitochondrial DNA selected from the group consisting of:
(i) the CpG sites in the D loop region shown in Table 1,
(ii) the CpG sites in the ND1 gene shown in Table 2;
(iii) the CHG sites in the D loop region shown in Table 3,
(iv) the CHG sites in the ND1 gene shown in Table 4, and / or
(v) the CHH sites in the D loop region shown in Table 5.
wherein the cytosine methylated at said position has been converted to uracil or another base that is distinguishable from cytosine in its hybridization properties.
[15]
15. The use of a kit according to claim 14, wherein said at least one oligonucleotide comprises a sequence selected from SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and / or SEQ ID NO: 5.

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法律状态:

优先权:

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申请号 | 申请日 | 专利标题

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ES201430444A|ES2546743B1|2014-03-28|2014-03-28|Mitochondrial markers of neurodegenerative diseases|

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PCT/ES2015/070230|WO2015144964A2|2014-03-28|2015-03-27|Mitochondrial markers of neurodegenerative diseases|

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