Method for accurately determining age from mitochondrial DNA by identifying relative N6-methyladenine levels at specific sites

Abstract

The subject matter of the present invention is a molecular biological method by which the relative (relative to internal control normalization) N6-methyladenine (6mA) level at a selected site of mitochondrial (mt) DNA (deoxyribonucleic acid) (mtDNA) in a tissue sample is accurately determined and projected onto a previously constructed reference "relative mtDNA 6mA level-age" curve. On a reference curve, the relative mtDNA 6mA level is in negative correlation with age; the higher the relative mtDNA 6mA level is, the younger the analyzed individual is. When a value relative to the mtDNA 6mA level intersects the reference curve, this intersection specifies a corresponding age on the X-axis. The reference curve is previously determined by measuring a large amount (gt; 1000) is established based on the 6mA level at a specific mtDNA site in a healthy individual with known age. The accuracy of the measurement depends on the accuracy of the method for determining the mtDNA 6mA level. The method is also suitable for predicting the life expectancy of an age-known individual (how long the individual will live). The present invention relies on the recent biological discovery of the present inventors that the 6mA levels at specific mtDNA regions are negatively correlated (inversely proportional) to the age of the analysed individual. The method can be applied to the following main fields: i) age determination in a forensic procedure; ii) life expectancy prediction; iii) identifying an early stage of the neurodegenerative process.

Description

[0001] The subject of the present invention is a molecular biology method by which the relative (normalized to an internal control) N6-methyladenine (6mA) level at a selected site of mitochondrial (mt) DNA (deoxyribonucleic acid) (mtDNA) in a tissue sample can be accurately determined and projected onto a previously constructed reference "relative mtDNA 6mA level-age" curve. On the reference curve, the relative mtDNA 6mA level is negatively correlated with age; the higher the relative mtDNA 6mA level, the younger the individual analyzed. When the value of the relative mtDNA 6mA level intersects the reference curve, the intersection specifies the corresponding age on the X-axis. The reference curve was previously established by measuring the 6mA levels at specific mtDNA sites in a large number (>1000) of healthy individuals whose ages are known. The accuracy of the measurement depends on the accuracy of the method used to measure the mtDNA 6mA level. So far, only one method has been used to accurately determine the relative 6mA level at the selected genomic site (a patent application entitled "A PCR-based method for the accurate determination of...", with file numbers P2100409 and W2200015). In this method, the individual genome isolated from the examined tissue sample is digested with a 6mA-dependent restriction endonuclease, and the resulting genomic fragment is then connected to a linker DNA fragment called an adapter, and finally a sequence-specific PCR- (polymerase chain reaction) DNA amplification is achieved by using a forward primer that is specific to both the downstream portion of the linker DNA fragment and the target mitochondrial genomic site adjacent to the linker. The connection of the linker makes it possible to directly amplify the selected methylated (digested) adenine nucleobase. The amount of the PCR product is proportional to the relative (related to another mtDNA site) 6mA level measured at a given mtDNA position.

[0002] Specifically, the innovation of the present invention is a molecular biological method by which the relative 6mA level at a specific mtDNA site in a tissue sample containing a large number of individual genomes can be accurately determined, and then the level is extrapolated to a previously established reference "mtDNA 6mA level-age" curve to accurately determine the age of the individual analyzed. The method is also applicable to predicting the life expectancy of an individual of known age (how long the individual will live). The present invention relies on the inventor's recent biological discovery that the 6mA level at a specific mtDNA region is negatively correlated (inversely proportional) to the age of the individual analyzed. Therefore, during the adult life span, the epigenetic process N6-adenine methylation continues to weaken in these specific mtDNA regions, thereby serving as a reliable marker for the rate of aging and the age of the organism. The method can be applied to the following major areas:

[0003] 1. Age determination in forensic procedures (the age of an unknown criminal can be accurately determined from biological traces (i.e. tissue samples left behind), which can significantly narrow the number of suspects and thus facilitate the success of the investigation process).

[0004] 2. Life expectancy prediction (the life expectancy of an individual whose age is known can be predicted, and based on this data, significant changes can be made in lifestyle and management issues (such as will design)).

[0005] 3. Identify the early stages of the neurodegenerative process (the rate of the aging process differs between normal individuals and patients affected by neurodegenerative pathologies, so when the age estimated by measuring mtDNA 6mA levels is significantly different from the actual age of an otherwise normal individual, this can indicate the early stages of the neurodegenerative process (before cognitive decline is apparent), and such early detection can facilitate the selection of effective therapies). Background Art

[0006] N6-methyladenine (6mA) epigenetic mark

[0007] The genetic material (genome) of living organisms consists of DNA (deoxyribonucleic acid), and in many organisms the genome is divided into functional units called chromosomes (chromosomes exist as complete DNA chains). DNA is composed of nucleotides (building blocks - each consisting of a ribose sugar, a phosphate group and an organic base called a nucleobase), which differ from each other in the nucleobase, which can be adenine, guanine, cytosine or thymine. The genetic information stored in DNA is essentially determined by the order of the nucleotides - this is called the DNA sequence. The human genetic information (haploid genome) consists of 3.1 billion nucleotides, which are organized into 23 pairs of chromosomes: 22 pairs of autosomes and 1 pair of sex chromosomes.

[0008] The functional units of chromosomes are called genes, whose products (proteins and RNA - ribonucleic acid) operate the cell. At the DNA level, the activity of genes can be changed in two main ways. First, at the genetic level - in this case, certain nucleotides are modified (deleted, added or converted into another nucleotide). Such changes involve mutations and genetic polymorphisms. Second, at the epigenetic level - in this case, the nucleotide sequence remains intact, but small chemical changes (addition of methyl groups (-CH3)) affect specific nucleobases: adenine or cytosine. Adenine can be converted mainly into N6-methyladenine (6mA), while cytosine can be converted mainly into 5-methylcytosine (5mC) ( Figure 1 ). Generally, 6mA has a gene activation effect (when the modification occurs in the genome, the affected gene is activated), while 5mC has a gene repression effect (X chromosome inactivation, genomic imprinting and maintaining genome stability by suppressing transposable elements called "jumping genes"). These epigenetic modifications are usually triggered by environmental factors and can be inherited to daughter cells and subsequent generations. The 6mA mark is generated from adenine and the process is catalyzed by DNA methyladenyl transferase ( Figure 1 ). 6mA can be converted back into adenine through the action of 6mA demethylase ( Figure 1 ).

[0009] DNA N6-adenine methylation has been widely observed in plants and bacteria. Just a few years ago, this process was identified in animal and organelle (mitochondrial) genomes (Greer et al., 2015; Zhang et al., 2015; Wu et al., 2016; Hao et al., 2022). However, recent studies have shown that the presence of 6mA in animal and organelle genomes is only the result of methodological artifacts (Schiffers et al., 2017; O'Brown et al., 2019; Douvlataniotis et al., 2020). However, the biological function of 6mA marks remains largely unexplored.

[0010] Techniques to demonstrate DNA methylation (epigenetic marks)

[0011] Several techniques have been developed to demonstrate the presence of methylated DNA nucleobases (6mA and 5mC epigenetic marks). They include single molecule real-time sequencing (SMRT-seq), bisulfite sequencing (the genomic sequence is treated with sodium bisulfite (Na-bisulfite), which converts cytosine nucleobases to uracil nucleobases, but 5mC remains unmodified by this agent), liquid chromatography-tandem mass spectrometry (LC-MS / MS), and labeling (hybridization) with 6mA / 5mC-specific antibodies (Dahl and Guldberg, 2003; Flusberg et al., 2010; Rocha et al, 2010). These methods are quite expensive (because they rely on expensive instruments and a high degree of expertise), time-consuming, and difficult to access (e.g., SMRT-seq is only provided by a few companies worldwide), so their diagnostic applications in medical and forensic genetics are largely unestablished. However, their biggest drawback is that they often produce artifacts (O'Brown et al., 2019; Schiffers et al., 2017; Douvlataniotis et al., 2020). This is mainly due to the fact that 6mA and 5mC epigenetic marks are widely present in the genomes of bacteria that frequently infect eukaryotic tissues and also in eukaryotic RNA. Artifacts can also be produced when the specificity of antibodies against 6mA and 5mC is insufficient, and due to the technical limitations of SMRT-seq. In addition, the techniques listed above cannot identify the amount (relative level) of methylated nucleobases at a given genomic site in the analyzed tissue sample (genomic DNA is isolated from a large number of cells), that is, how many individual genomes in the sample are methylated at a given genomic site, and they can only provide a "yes" or "no" answer to the methylation status of the examined nucleobases.

[0012] Alternatively, 5mC levels can be determined by using 5mC-dependent / sensitive restriction endonucleases and subsequent PCR-based target site amplification (Luo et al., 2016; Yao et al., 2017). However, this method also cannot accurately determine the methylation level at a certain genomic site in a tissue sample. Therefore, these DNA methylation detection technologies listed above are still not widely used in medical and forensic applications. Recently, a new method provides a solution, in which genomic DNA isolated from tissue samples is digested with 6mA- or 5mC-specific restriction endonucleases, and the resulting DNA fragments are then ligated to linker DNA fragments called adapters, and the target site is amplified by PCR using forward primers that are specific for both the linker sequence (10-15 nucleotides) and the adjacent genomic sequence (10-15 nucleotides). The use of linker DNA fragments makes it possible to directly amplify the digested (methylated) DNA sequence. This is crucial because methylation of the adenine nucleobase at the N6 position is a relatively rare event, and at a given time, only a very small number of individual (cell) genomes in a tissue sample are methylated. Using this method, PCR products reliably reflect the relative 6mA or 5mC levels at the selected genomic loci (patent application "A PCR-based method for the accurate determination of ...", file numbers P2100409 and W2200015 - the inventors of which gave the inventors permission to view the contents of the application under the condition of strictly maintaining intellectual property rights - NDA). The more genomes that are methylated at the selected genomic loci (nucleobases) in the individual genome (tissue sample) analyzed, the higher the amount of PCR products generated.

[0013] DNA methylation in age determination

[0014] In the past decade, it has been demonstrated that the epigenetic mark 5mC is formed in an age-dependent manner in the human genome. 5mC accumulates primarily in transposable element (TE) loci during early development and plays an important role in suppressing these mutagenic sequences, thereby maintaining genome stability (Yoderés mtsi., 1997). Dr. Steve Horvath (UCLA, US) has developed an algorithm by which the biological age of human tissue samples can be determined by identifying the 5mC content of certain genomic regions—the "Horvath's clock" (Horvath, 2013). He demonstrated that the 5mC content in these genomic regions decreases with age. The determination of 5mC content in specific genomic regions is essentially performed by bisulfite sequencing, and the results provided are used to determine the biological age, with an error mean of 7-10 years (±7-10 years), which is not accurate enough to apply the technology to medical and forensic genetics, which require higher accuracy. Although the method is used by several companies in the field of DNA diagnosis (such as Chronomics, Altos Labs, Zymo Research, Elysium Health and Ra Pharmaceuticals Ltds.), the results are only of reference value to non-professional purchasers. In addition, it has been shown that during the life span, 5mC levels increase at certain genomic positions, but decrease at other genomic positions, and the 5-cytosine methylation process is highly affected by environmental and physiological factors. Finally, there are organisms that basically lack the 5-cytosine methylation phenomenon, such as the nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster. According to these facts, it can be said that the 5mC level at certain genomic regions is not enough to accurately determine the (biological) age of an individual.

[0015] Problems to be solved by the present invention

[0016] The present inventors have demonstrated from human mitochondrial DNA (mtDNA) samples that relative 6mA levels at specific sites decrease with age during the adult lifespan ( Figure 2 Although the biological significance of this phenomenon is not known to the inventors at present, the 6mA epigenetic mark at specific mtDNA sites is an effective marker of biological age. The present invention relies on the observation that the relative 6mA level at specific sites of the mitochondrial genome decreases with the aging of the organism. graduallyTo the best of the inventors' knowledge, this finding has not been documented in the literature. Using specific primer pairs, the inventors determined the relative 6mA levels at multiple sites in the mitochondrial genome and found a gradual, age-dependent decrease at each site analyzed.

[0017] Subsequently, the inventors measured the relative 6mA levels at specific sites of mtDNA in a large number (hundreds) of healthy individuals with known identities (ages), and constructed a reference "relative mtDNA 6mA level-age" curve, in which the 6mA level showed a negative correlation with age ( Figure 3 ). The lower the measured 6mA level, the older the individual analyzed. This data set is called the reference "relative mtDNA 6mA level-age" curve. If the relative 6mA level at this particular site is measured in the mtDNA of an individual whose identity is unknown, and the obtained value (on the Y axis) is projected onto the reference curve, the intersection point at right angles to the X axis specifies the age of the individual ( Figure 3 The method produces relatively accurate (error mean 2-3 years) and specific (artifact-free) data, is rapid (PCR-based) and cost-effective, so it can be used to establish epigenetic DNA diagnostics, a potentially important branch of the DNA diagnostics industry.

[0018] The method can be applied in the following main areas:

[0019] 1. Forensic genetics. In the field of criminal behavior, biological traces (evidence, such as hair or blood drops) of criminals can often be collected. In this case, genomic DNA is isolated from the traces left behind to determine the DNA profile of the criminal. The profile is usually based on individual DNA polymorphisms (such as the number of repeats of microsatellites called short DNA repeats). The result (profile) is then compared with a criminal DNA database containing DNA profiles of known criminals, and if there is a match, the identity of the criminal can be identified. However, in most cases, the DNA database does not contain a profile that matches the actually identified DNA polymorphism (i.e., the profile of the criminal has not yet been introduced into the DNA database). In this case, the relative 6mA level at a specific mtDNA locus is identified from the biological trace, and this value is compared with the reference "relative mtDNA 6mA level-age" curve, and the age of the criminal can be accurately determined (e.g., 56 ± 2 years old - therefore, the error limit is 2 years old) ( Figure 4 ). Based on the results of the determination, the number of suspects can be significantly narrowed down, which can greatly facilitate the success of the investigation process.

[0020] 2. Lifespan prediction for individuals of known age. Different individuals of a species live for different times. For example, if three 50-year-olds are randomly selected, theoretically the first will live to 56, the second will live to 66, and the third will die at 84. Therefore, the lifespans of individuals of the same age can be significantly different. Using the method shown above, the life expectancy (how long she or he will live) of an individual of known age can be predicted. If the relative 6mA level at a given mtDNA site in the person tested is significantly higher than the level expected by the reference curve at a given age, the lifespan predicted by this value is shorter than the expected lifespan. Therefore, at a given age, the higher the relative mtDNA 6mA level, the shorter the life expectancy of the individual. It is important to note that this is "only" a prediction, because the person may die much earlier than the lifespan expected by her / his 6mA level due to, for example, a fatal accident. Those who get an unfavorable prediction (short life expectancy) from the test can significantly change their lifestyle (such as starting physical activity, avoiding stress and better nutrition) to reverse the rate of cellular aging. In addition, unfavorable expectations can also trigger will preparation.

[0021] 3. Early detection of neurodegenerative processes. If the relative mtDNA 6mA level identified in a person of known age is significantly different from the level expected from a reference curve at a given age, this value may indicate an early stage of a neurodegenerative process (before cognitive decline is apparent) ( Figure 5 ). This is because the speed of the aging process in people affected by neurodegenerative diseases (such as Alzheimer's disease, Parkinson's disease or Huntington's disease or ALS) is different from the speed of the aging process found in unaffected people of the same age (usually faster), and this difference can be accurately measured by the method introduced in this patent application (the higher the relative mtDNA 6mA level at a given age, the higher the speed at which the aging process occurs). Therefore, the relative 6mA level at a specific mtDNA site can be used as an early marker for various neurodegenerative pathologies. This is an important discovery because when neurodegenerative diseases manifest (obvious cognitive problems), a major part of the brain has been affected by neuronal death, so the pharmacological treatment applied has been proven to be largely ineffective (already dead neurons cannot be revived). Early (long before cognitive decline manifests) diagnosis can provide a solution, but suitable markers for early diagnosis have not yet been generated. When relative mtDNA 6mA levels indicate involvement of apparently normal (healthy) individuals, the patient is advised to undergo further PET (positron emission tomography) or NMR (nuclear magnetic resonance) examinations and, by identifying the presence of a degenerative process, receive drug treatment. DETAILED DESCRIPTION

[0022] In order to determine age from mtDNA, the following steps must be performed: i) the relative 6mA level at a given mtDNA site in a tissue sample obtained from an unknown person is identified by a PCR-based method (semi-quantitative PCR or real-time quantitative PCR); ii) the extrapolation of the identified 6mA level (Y-axis) is assigned a value on a reference "relative mtDNA 6mA level-age" curve; iii) the projection of this value (intersection point) on the X-axis specifies the age of the individual. In order to accurately perform this set of experiments, only one method is available so far, the steps of which are as follows: i) genomic DNA is isolated from tissue; ii) the genomic DNA is digested with a 6mA-specific restriction endonuclease (e.g., DpnI, which only methylates at the -GATC- sequence (-GATC) when A is methylated) Me TC-) cutting DNA); iii) connecting the adapter DNA fragment to the digested genomic DNA fragment; iv) PCR amplification of the target (digested) site by using a forward primer that is specific for both the adapter and the adjacent mtDNA sequence; v) quantification of the PCR product; vi) comparing the amount of the product with the amount of the internal control (this normalization gives the relative 6mA level). In this case, the PCR product reliably reflects the relative 6mA level at the selected mtDNA site. The method is described in a recent patent application entitled "A PCR-based method for the accurate determination of...."; file numbers are P2100409 and W2200015. The owner of the application (inventor) allows the inventor to review the file (scheme) under the condition of maintaining various aspects of its intellectual property rights.

[0023] Using this technique, the inventors previously identified relative 6mA levels at specific mtDNA sites (a given adenine nucleobase) in hundreds of individuals whose age (identity) was known. From the data obtained, the inventors established a reference "relative mtDNA 6mA level-age" curve. The people analyzed were healthy and of different ages. The relative mtDNA 6mA levels identified in people whose identity was unknown were then projected onto the reference curve, and the intersection points designated the age of the individual on the X-axis ( Figure 4 ). Based on the inventors' measurements, the data deviation is within an error range of 2-3 years (±2-3 years). If the relative 6mA level at a human mtDNA locus is identified in an individual whose identity (age) is known, and the value obtained is significantly different from the value indicated by the reference curve at a given age, the difference may reflect the presence of an early stage of a neurodegenerative process ( Figure 5 ). In such cases, it is recommended that affected individuals undergo imaging studies such as PET or NMR.

[0024] Build Example

[0025] Method (the technical basis of which is described in a patent application entitled "A PCR-based method for the accurate determination of . . . "; File No.: P2100409 and W2200015, and its inventors allow the inventors to view the file (scheme) under the condition of maintaining every aspect of intellectual property rights (confidentiality agreement)):

[0026] Isolation of genomic DNA from samples

[0027] Isolation from blood samples was performed by using the Thermo Scientific GeneJET Genomic DNA Purification Kit (#K0721).

[0028] Digestion of genomic DNA with DpnI restriction endonuclease

[0029] Add the following components to an Eppendorf tube:

[0030] 1μl DpnI enzyme (10U / μl, ThermoFisher Scientific, ER1701)

[0031] 4 μl Tango buffer (10x)

[0032] 15 μl HO

[0033] 20μl genomic DNA solution (minimum concentration 10ng / μl)

[0034] (Final volume: 40 μl / sample)

[0035] → Incubate at 37°C for 20 minutes

[0036] Inactivation of DpnI: Incubate at 80 °C for 20 min

[0037] Ligation of adapter DNA to digested genomic DNA fragments

[0038] To the inactivated mixture (40 μl) the following components were added:

[0039] 4 μl T4 ligase (5 U / μL, ThermoFisher Scientific, EL0011)

[0040] 5μl ATP (2mM) (ThermoFisher Scientific, 100mM, R0441)

[0041] 5 μl genomic adapter (100x concentration)

[0042] 4 μl Tango buffer

[0043] 22 μl HO

[0044] Final volume of the mixture: 80 μl

[0045] Incubate the samples overnight at 4 °C

[0046] Inactivation of ligase: Incubate the sample at 80°C for 20 minutes. DNA sequence of adapter:

[0047]

[0048] PCR reaction

[0049] 6mA sample (DpnI) - forward (left) primer:

[0050] Reverse (right) primer:

[0051]

[0052] Reaction mixture:

[0053] 10 μl ABI master mix (2x)

[0054] 3,5μl digested / ligated DNA solution (template)

[0055] 3 μl primer mix (5-5 μM)

[0056] 3,5 μl H2O

[0057] Final volume: 20 μl / sample

[0058] PCR conditions (DpnI):

[0059] 1. Initial denaturation: 95°C, 30 seconds

[0060] 2. Denaturation: 95°, 10 seconds

[0061] 3. Annealing and extension: 60°C, 45 seconds

[0062] 4. Repeat steps 2. and 3. 37 times

[0063] 5. Store samples at 4°C

[0064] Control (PvuII) - Forward (left) Primer:

[0065] 5'-acc gcc atc ttc agc aaa c-3' and reverse (right) primer:

[0066]

[0067] Reaction mixture:

[0068] 10 μl ABI master mix (2x)

[0069] 1μl DNA solution (template)

[0070] 3 μl primer mix (5-5 μM)

[0071] 6 μl HO

[0072] Final volume: 20 μl

[0073] PCR conditions (PvuII):

[0074] 1. Initial denaturation: 95°C, 60 seconds

[0075] 2. Denaturation: 95°C, 10 seconds

[0076] 3. Annealing and extension: 60℃, 45msp

[0077] 4. Repeat steps 2 and 3. 27 times

[0078] 5. Store samples at 4°C

[0079] Gel Recording

[0080] The samples were run on a 1% agarose gel (run at 80 mV) and photographed with a Kodak camera. Novelty of the Invention

[0081] 1. Recognize that relative 6mA levels at specific sites in the mitochondrial genome increase proportionally with age. Based on this discovery, age can be accurately determined from mtDNA isolated from individuals whose identities are unknown.

[0082] 2. Recognize that relative 6mA levels at specific mtDNA loci can predict lifespan.

[0083] 3. Recognize that relative 6mA levels at specific mtDNA loci indicate early stages of the neurodegenerative process.

[0084] References

[0085] Dahl C, Guldberg P. DNA methylation analysis techniques. Biogerontology 4, 233-250 (2003). PMID: 14501188

[0086] Doublataniotis K,Bensberg M,Lentini A,Gylemo B,Nestor CE.No evidencefor DNA N 6-methyladenine in mammals.Sci.Adv.2020 Mar 18;6(12):eaay3335.PMID:32206710

[0087] Flusberg BA,Webste DR,Lee JH,Travers KJ,Olivares EC,Clark TA,KorlachJ,Turner SW.Direct detection of DNA methylation during single-molecule,reaI-time sequen cing.Nat.Methods 7,461-465(2010).PMID:20453866

[0088] Greer EL,Blanco MA,Gu L,Sendinc E,Liu J,Aristizábal-Corrales D,Hsu C-H,Aravind L,He C,Shi Y.DNA Methylation on N6-Adenine in C.elegans.Cell 161、868-878(2015).PMID:25936839

[0089] Hao Z,Wu T,Cui X,Zhu P,Tan C,Dou X,Hsu KW,Lin YT,Peng PH,Zhang LS.etal.(2020)N(6)-deoxyadenosine methylation in mammalian mitochondrialDNA.Mol.Cell 78,382-395 e388.PMID:32183942

[0090] Horvath S.DNA methylation age of human tissues and cell types.GenomeBiol,2013;14(10):R115.PMID:24138928

[0091] Luo G-Z,Wang F,Weng X,Chen K,Hao Z,Yu M,Deng X,Liu J,HeC.Characterization of eukaryotic DNA N(6)-methyladenine by a highly sensitiverestriction enzyme-assisted sequencing.Nat.Commun.7,11301(2016).PMID:27079427

[0092] O′Brown ZK,Boulias K,Wang J,Wang SY,O′Brown NM,Hao Z,Shibuya H,FadyP-E,Shi Y,He C,Megason SG,Liu T,Greer EL.Sources of artifact in measurementsof 6mA and 4mC abundance in eukaryotic genomic DNA.BMC Genomics 2019 Jun 3;20(1):445.PMID:31159718

[0093] Rocha MS,Castro R,Rivera I,Kok R,Smulders YM,Jakobs C,de Almeida IT,Blom HJ.Global DNA methylation:comparison of enzymatic-and non-enzymatic-based methods.Clin.Chem.Lab.Med.48,1793-1798(2010).PMID:20979561

[0094] Schiffers S,Ebert C,Rahimoff R,Kosmatchev O,Steinbacher J,Bohne A-V,Spada F,Michalakis S,Nickelsen J,Mǖller M.Carell T.Quantitative LC-MSProvides No Evidence for m 6 dA or m 4 dC in the Genome of Mouse EmbryonicStem Cells and Tissues.Angew Chem Int Ed Engl.2017 Sep 4;56(37):11268-11271.PMID:28371147

[0095] Wu TP,Wang T,Seetin MG,Lai Y,Zhu S,Lin K,Liu Y,Byrum SD,MackintoshSG,Zhong M,Tackett A,Wang G,Hon LS,Fang G,Swenberg JA,Xiao AZ.DNA methylationon N(6)-adenine in mammalian embryonic stem cells.Nature.2016 Apr 21;532(7599):329-33.PMID:27027282

[0096] Zhang G,Huang H,Liu D,Cheng Y,Liu X,Zhang W,Yin R,Zhang D,Zhang P,LiuJ,Li C,Liu B,Luo Y,Zhu Y,Zhang N,He S,He C,Wang H,Chen D.N6-methyladenine DNAmodification in Drosophila.Cell 161,893-906(2015).PMID:25936838

[0097] Yao B, Cheng Y, Wang Z, LY, Chen L, Huang L, Zhang W, Chen D, Wu H, Tang B, JinP. DNA N6-methyladenine is dynamically regulated in the mouse brain following environmental stress. Nat. Commun. 8, 1122 (2017). PMID: 29066820

[0098] Yoder JA, Walsh CP, Bestor TH. Cytosine methylation and the ecology of intragenomic parasites. Trends Genet. 1997 Aug; 13(8): 335-40. PMID: 9260521 BRIEF DESCRIPTION OF THE DRAWINGS

[0099] Figure 1 .N6-methyladenine (6mA) epigenetic mark. Adenine (A) can be converted to N6-methyladenine (6mA) by adding a methyl group (-CH3). The -CH3 group is transferred to the N atom at position 6 of adenine by DNA N6-adenine methyltransferase. N6-methyladenine demethylase can remove the -CH3 group from 6mA, thereby regenerating adenine. Therefore, the methylation and demethylation processes together affect the methylation state of a single adenine at a given genomic site. The -CH3 group is highlighted.

[0100] Figure 2 .Relative N6-methyladenine (6mA) levels at specific mitochondrial DNA (mtDNA) sites gradually decrease with age. a) Gel photograph shows the relative 6mA levels (PCR products) at specific mtDNA sites in patients aged 22, 35, 58 and 68 years. Top: control band, bottom: 6mA levels. The number of control bands in the samples was almost similar (similar amounts of template mtDNA were used for analysis). a') Quantification of the bands is shown in Figure (a). The samples were normalized to the corresponding controls. b) Relative mtDNA 6mA levels in individuals of different ages. 6mA levels decrease proportionally with age. Top: control band, bottom: 6mA levels of the samples.

[0101] Figure 3. Predicting human life span by identifying relative 6mA levels at specific mtDNA sites. A reference "relative 6mA level-age" curve (light blue line) is constructed by measuring the 6mA level at a specific mtDNA site in an individual whose identity (age) is known. When the relative 6mA level at the mtDNA site is determined in a person whose identity is unknown (dashed red line) and the obtained value is projected onto the reference curve, the intersection (projected to the X-axis) specifies the age of the individual (e.g., 80 years old).

[0102] Figure 4 . Predicting life span in individuals of known age by measuring the relative N6-methyladenine (6mA) level at a specific mitochondrial DNA (mtDNA) site. Measuring the relative 6mA level at a specific mtDNA site can be used to predict the life span of the analyzed individual. Two gel images show the 6mA levels of people of different ages. The levels shown by samples aged 60, 62, 64, 65, 66, 68, 77, 77 and 85 correspond to the levels found in the reference curve at the same age. The 6mA levels shown by samples aged 72, 76 and 80 are much higher than the expected levels of the reference curve at the same age (indicated by the yellow "!" mark). It is expected that the life span of these individuals is much longer than the average life span at the same age. The 6mA levels shown by samples aged 74 and 82 are much lower than those levels found on the reference curve at the same age. These samples are indicated with red "!!" marks. It is expected that the life span of these people is much shorter than the corresponding average life span at the same age.

[0103] Figure 5 .The early stages of a neurodegenerative process are predicted by measuring the relative N6-methyladenine (6mA) levels at specific mitochondrial DNA (mtDNA) sites. Patients affected by neurodegenerative diseases age at rates that differ from those found in age-matched healthy controls. If the relative 6mA level at a specific mtDNA site is measured in a person of known age (50 years old) (dashed red line), and the level is significantly different from the level indicated by the reference curve (blue line) of the same age (50 years old), projecting an age of, for example, 70 years old, the 6mA value indicates the presence of a neurodegenerative process.

Claims

1. A method / scheme to accurately determine age from mitochondrial DNA (mtDNA) by identifying the relative N6-methyladenine (6mA) levels at specific sites in mtDNA.

2. The method / protocol according to claim 1, wherein the organism analyzed is any eukaryotic species.

3. The method / protocol according to any one of claims 1 and 2, wherein the determination of the relative 6mA level at a specific mtDNA locus is achieved by any suitable molecular technique.

4. The method / scheme according to any one of claims 1-3, wherein the target site is an optional mtDNA sequence.

5. The method / protocol according to any one of claims 1 to 4, wherein the type of tissue sample detected is optional.

6. The method / scheme according to any one of claims 1-5, wherein the population in which the determination of relative 6mA levels at a specific mtDNA site is achieved is optional.

7. The method / scheme according to any one of claims 1-6, wherein the purpose of the analysis is to determine the age of an individual whose identity (age) is unknown.

8. The method / protocol according to any one of claims 1 to 7, wherein the purpose of the analysis is to predict the lifespan of an individual of known age.

9. The method / protocol according to any one of claims 1 to 8, wherein the purpose of the analysis is to identify early stages of a neurodegenerative process.

Images