A method for accurately determining age from nuclear DNA by specifying the N6-methyladenine level in a specific genomic region

Abstract

The subject of the present invention is a molecular biological method for accurately determining the relative (normalized against an internal control) N6-methyladenine (6mA) levels at selected specific genomic sites within a tissue sample containing multiple individual cell genomes, and projecting this level onto a previously determined reference "relative 6mA level - age" curve. The relative 6mA level of the subject being examined assigns the person's (biological) age on the curve. The reference curve was previously established by measuring the 6mA levels of a large number (over 1000) of healthy people of known age. In the reference curve, the relative 6mA level is correlated with age, and the higher the 6mA level of the genomic site being examined, the higher the age of the subject being examined. The accuracy of the measurement depends on the accuracy of the method for determining the 6mA level. The present invention is based on the recent biological discovery that the 6mA level in specific genomic regions is correlated (proportional) with age. Thus, the epigenetic process of N6-adenine methylation occurs continuously in these specific genomic regions throughout adulthood, thereby functioning as a reliable sign of the aging rate and biological age. Such specific nuclear regions contain active (mobile) transposable elements, also called mobile genetic elements or "jumping genes". For example, in the human genome, the 6mA level of the LINEI sequence functions as a notable marker for age determination. This method can be applied to the following main areas: i) age determination in forensic procedures, ii) prediction of life expectancy (how long an individual can live), and iii) identification of the initial stages of neurodegenerative processes.

Description

Technical Field

[0001] The subject of the present invention is to accurately determine the relative (normalized against an internal control) N6-methyladenine (6mA) levels at selected specific genomic sites within a tissue sample containing multiple individual cell genomes, and project this level onto a previously determined reference "relative 6mA level - age" curve. The 6mA level of an unidentified subject is revealed, and the person's (biological) age is assigned on the curve. The reference curve was previously established by measuring the 6mA levels of a large number (over 1000) of healthy people of known age. In the reference curve, the relative 6mA level correlates with age, and the higher the 6mA level at the genomic site being examined, the higher the age of the subject. The accuracy of the measurement depends on the accuracy of the method for determining the 6mA level. To date, there is only one method capable of accurately determining the relative 6mA level at a selected genomic site (patent application titled "PCR-based accurate determination method" in file numbers P2100409 and W2200015). In this method, individual genomes isolated from a tissue sample of the subject are enzymatically digested with a 6mA-dependent restriction endonuclease, the resulting genomic fragments are ligated to linker DNA (deoxyribonucleic acid) fragments, and finally, sequence-specific PCR (polymerase chain reaction)-based DNA amplification of the digested (target) site is performed using a forward primer specific for both the downstream part of the linker DNA fragment and the target genomic site adjacent to the linker. Ligation of the linker enables direct amplification of the selected methylated (digested) adenine nucleobase (target site). The amount of the PCR product is proportional to the relative 6mA level determined at a specific genomic location.

[0002] Specifically, this innovation is a molecular biology technique that accurately determines the relative 6mA level at specific genomic sites within a tissue sample and projects this level onto a previously established reference "relative 6mA level - age" curve to accurately determine the age of the individual being tested. This technique is also suitable for predicting the life expectancy (how long an individual will live) of individuals whose age is known. The present invention is based on the recent biological discovery that the 6mA level in specific genomic regions is correlated (directly proportional) with the age of the individual being tested. Thus, the epigenetic process of N6-adenine methylation occurs continuously in these specific genomic regions throughout adulthood and thereby functions as a reliable sign of the aging rate and biological age. Such specific nuclear regions contain active (mobile) transposable elements, also known as mobile genetic elements or "jumping genes". For example, in the human genome, the 6mA level of the LINEI (Ll) sequence functions as a notable marker for age determination. This method can be applied to the following major fields: 1. Age determination in forensic procedures (Since the biological traces of an unknown offender, i.e., the remaining tissue sample, can accurately determine the age, the number of suspects can be significantly narrowed down, leading to the success of the investigation process). 2. Life expectancy prediction (The life expectancy of individuals whose age is known can be predicted, and it can be found that there are significant changes in lifestyle and administrative issues (e.g., property) in light of this data). 3. Identification of the early stages of neurodegenerative processes (The rate of the aging process is different between normal individuals and patients suffering from neurodegenerative pathologies. Therefore, if the age estimated from the determined 6mA level is significantly different from the actual age of an otherwise seemingly normal individual, this may indicate the early stage of the neurodegenerative process before the onset of cognitive decline, and this early detection can facilitate the selection of effective treatment methods).

Background Art

[0003] Genetics of the Aging Process Aging refers to the gradual decline of an organism's fitness and physiological functions over time, which is mainly caused by the gradual accumulation of cellular damage, including misfolded, oxidized, and aggregated, i.e., non-functional proteins, throughout life (Kirkwood, 2008). Such components interfere with cellular processes, cause aging (decline in cellular function), and ultimately result in the loss of affected cells. Massive cell loss can lead to tissue / organ dysfunction and may manifest as the onset of degenerative pathologies associated with aging, such as cancer, various neurodegenerative diseases, diabetes, tissue atrophy and fibrosis, and immunodeficiency. Such (fatal) pathologies can ultimately cause the death of the organism. Despite being medically, socially, and economically important, the mechanisms (major genetic basis) of the aging process, i.e., the factors that generate cellular damage, remain largely unexplained (Kenyon, 2010).

[0004] The rate of cellular aging is influenced by several environmental factors such as temperature, oxygen levels in the atmosphere, nutrient availability, and numerous endogenous regulatory pathways and proteins (Kenyon, 2010). Such longevity pathways include, for example, insulin / IGFl (insulin-like growth factor) signaling (Kenyon et al., 1993), the TOR (target of rapamycin) kinase-mediated molecular cascade (Vellai et al., 2003), and the mitochondrial respiratory system (Dillin et al., 2002). Among the known regulatory proteins that determine lifespan, the p53 tumor suppressor and the FoxA transcription factor are worth mentioning (Kenyon, 2010). However, these regulatory factors only affect the rate of the aging process and do not function as its fundamental basis. Mutations or pharmacological inactivation of such proteins / pathways can extend lifespan, but the affected organisms still age and ultimately die. Autophagy (cellular self-eating), the main self-degradation process in eukaryotic cells, plays a central role in aging control; molecular damage that causes aging can be mainly degraded (removed) by the autophagy process (Vellai, 2009). In this way, cell components can be rejuvenated and the homeostasis (stable function) of the cell can be maintained.

[0005] Genomic instability has emerged as a cell property that is characteristic of essentially all aging cells (López-Otin et al., 2013). Recent findings indicate that the main cause of genomic instability is the activity of transposable elements (TEs), which constitute a major part of the eukaryotic genome (Sturm et al., 2015; 2017; Gorbunova et al., 2021). Most TEs (also called mobile genetic elements or "jumping genes") can move from one part of the genome to another and cause insertion mutations at new locations. If the acceptor genomic site contains a functional (coding or regulatory) DNA sequence, the insertion can result in a functional outcome (mutant phenotype). Therefore, TEs are considered powerful biological mutagens. Their importance is also brought about by the fact that a large part of the eukaryotic genome is composed of repetitive sequences such as TEs. For example, more than half of the human genome is constructed from TE-related sequences (Nurk et al., 2022). Indeed, pharmacological inhibition of mobile TE sequences has recently been shown to extend the lifespan of Drosophila (Wood et al., 2016) and delay the aging of mammalian cells (De Cecco et al., 2019).

[0006] However, it is still not understood why TEs become gradually mobilized during adulthood. Since DNA 5-cytosine methylation occurs mainly in TE-like sequences and contributes to their inactivation (Yoder et al., 1997), and DNA N6-adenine methylation plays a major role in TE mobilization (Sturm et al., unpublished results), epigenetic control may be the solution. Generally, it is noteworthy that the 5-methylcytosine (5mC) epigenetic mark suppresses gene expression, while the N6-methyladenine (6mA) epigenetic mark promotes gene expression. The presence of 6mA has only recently been discovered in the genomes of animals and mammals (Greer et al., 2015; Zhang et al., 2015; Wu et al., 2016), but according to some new studies, it is thought to be the result of methodological artifacts in these genetic systems (Schiffers et al., 2017; OBrown et al., 2019; Douvlataniotis et al., 2020).

[0007] Techniques for demonstrating DNA methylation (epigenetic mark) The DNA methylation process is catalyzed by specific DNA methyltransferase enzymes, and the demethylation of methylated adenine and cytosine nucleobases is carried out by specific DNA demethylase enzymes. Therefore, DNA methylation is a dynamic process, and the 6mA and 5mC contents across the genome vary depending on the actual age of the organism and various environmental and physiological factors.

[0008] To prove the presence of methylated DNA nucleobases (6mA and 5mC epigenetic marks), several techniques have been developed. These techniques include single molecule real-time sequencing (SMRT-seq), bisulfite sequencing (the genome is treated with sodium bisulfite and cytosine nucleobases are converted to uracil nucleobases, but 5mC is not altered by this compound), 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 costly (requiring expensive equipment and advanced expertise), time-consuming, and difficult to obtain (e.g., SMRT-seq is provided by only a few companies worldwide), so their application to diagnosis has not yet been established. However, the biggest drawback is that artifacts are frequently generated (OBrown et al. 2019: Schiffers et al., 2017: Douvlataniotis et al., 2020). This is mainly due to the fact that the epigenetic marks of 6mA and 5mC are widely present in the genomes of bacteria that often infect eukaryotic tissues and in the eukaryotic RNA contained in cell samples. Also, artifacts may be generated due to insufficient specificity of antibodies against 6mA and 5mC or due to technical limitations of SMRT-seq. Furthermore, the above techniques cannot determine the amount (relative level) of methylated nucleobases at specific genomic sites within tissue samples (genomic DNA is isolated from a large number of cells) and can only provide a "yes" or "no" answer regarding the methylation status of the nucleobases examined.

[0009] Alternatively, attempts have been made to determine 5mC levels using 5mC-dependent / sensitive restriction enzymes followed by PCR-based amplification of target sites (Luo et al., 2016, Yao et al., 2017). However, even these methods cannot accurately determine the methylation levels of specific DNA sites within tissue samples. These DNA methylation analysis techniques are still rarely used in the fields of medicine and forensic science. Recently, a new method has provided a solution, in which genomic DNA isolated from tissue samples is digested with 6mA- or 5mC-specific restriction endonucleases, the resulting DNA fragments are ligated to linker DNA fragments, and finally, representative sites are amplified by a PCR reaction mediated by a forward primer specific for both the linker sequence (10 - 15 nucleotides) and the adjacent genomic sequence (10 - 15 nucleotides). The application of linker DNA fragments enables the direct amplification of digested (methylated) DNA sequences. This is important because the methylation rate of specific nucleobases is a very rare event; only a very small number of genomes within a tissue sample are methylated at a specific genomic position at a specific time. Using this method, the amount of PCR product reliably reflects the relative 6mA or 5mC level of the selected genomic site (Patent Application: "PCR-based method for accurate determination of...", File Numbers: P2100409 and W2200015, and the inventors have granted us permission to review the application content under the condition of strictly maintaining IP rights (NDA)). Among the individual genomic sites that make up the tissue sample being examined, the more genomes that are methylated at the selected genomic site, the greater the amount of PCR product generated.

[0010] Epigenetic clock for age determination In recent years, Dr. Steve Horvath (University of California, Los Angeles, USA) developed an algorithm that can determine the biological age of a test tissue by measuring the 5mC content of specific genomic regions. This is the Horvath clock (Horvath, 2013). The 5mC content is mainly determined by bisulfite sequencing, which is a rather costly and time-consuming procedure. However, the biggest drawback of this method is its inaccuracy. In the case of human samples, this method can determine the biological age with an error of 7 to 10 years. Due to this limitation, this method is still not applied in medicine and forensic genetics. Some companies (such as Chronomics, Altos Labs, Zymo Research, Elysium Health es Ra Pharmaceuticals Ltds, etc.) are using this method to determine an individual's age in the diagnostic industry, but they only provide useful results for lay procurement agents. The cost and time required for such measurements are approximately 500 euros per sample and one month. Furthermore, it has been found that the 5mC level increases at specific genomic positions throughout life, but decreases at other genomic positions, and the 5-cytosine methylation process is greatly affected by environmental and physiological factors. Finally, there are organisms (e.g., the nematode (Caenorhabditis elegans) and the fruit fly (Drosophila melanogaster)) that essentially lack the 5-cytosine methylation phenomenon. Considering these facts, it can be said that determining the 5mC level in specific genomic regions is not sufficient to accurately determine an individual's age.

Summary of the Invention

[0011] Problems Solved by the Innovation In Caenorhabditis elegans and humans, which are convenient genetic models for studying the regulation and mechanisms of the aging process, it has been shown that the relative 6mA level in activated TE sequences (Figure 1) gradually increases during aging (Figures 2 and 3). Therefore, this epigenetic mark functions as a signature of the biological age of an organism. It is thought that due to the methylation of N6 adenine, TE sequences that were originally inactive in the genome (juvenile stage) gradually become mobile during the adult lifespan. In the C. elegans genome, the TCI, Tc3, and Tc14 sequences were tested. These are the most active TESs in this organism and are present in relatively high copy numbers in its genome (for example, TCI has 31 copies in the haploid genome). Furthermore, a PCR-based approach enables the simultaneous analysis of the methylation status of specific adenine nucleobases in each copy of the TE family. For each TE family investigated, it was found that the relative 6mA level gradually increases with age throughout the adult lifespan (Figure 2). Therefore, this innovation is based on our new biological discovery that as an organism ages, the 6mA level in activated TE sequences Gradually increases. Therefore, the relative 6mA level in activated TE sequences serves as a reliable marker for determining the (biological) age from tissue samples. To the best of our knowledge, such data have not been published in the literature.

[0012] In addition, changes in 6mA levels with aging in the LINEI (Ll) sequences found in the human genome were also examined (Ll is one of the most active TE families in humans). This was carried out using primer pairs specific to the Ll sequence (amplification of Ll-specific genomic fragments was achieved by PCR reaction). In good agreement with the results obtained from C. elegans, the relative (normalized to an internal control) 6mA levels gradually increased at the Ll locus with aging (Figure 3). Therefore, this age-related DNA methylation process is thought to be evolutionarily conserved. Therefore, the method presented in the current patent application is suitable for accurately determining the (biological) age of various organisms from nematodes to humans (Drosophila and dogs have also been tested).

[0013] Using a large number of tissue (genomic DNA) samples from humans of known age, a "relative 6mA level - age" curve can be created. This curve shows a direct correlation between the relative 6mA level in a specific genomic region and the age of the subject being tested (Figure 4). This is called the reference "relative 6mA level - age" curve. When the relative 6mA level in a specific genomic region (e.g., the human L1 sequence) is determined from a tissue sample of an individual of unknown identity (age), this level (the value on the Y-axis) indicates a point on the reference curve. Next, a normal line from the intersection point to the X-axis (age) is extrapolated to assign the age of the subject being tested (Figure 4). This method is artifact-free (sequence-specific) and uses only enzyme digestion, ligation, and PCR-based DNA amplification processes, so it is relatively fast and cost-effective and can be widely used as a diagnostic tool.

[0014] This method can be applied to the following major diagnostic fields: 1. Forensic genetics. In the field of criminal behavior, biological traces of the perpetrator (evidence, such as hair or blood droplets) are often collected. In such cases, genomic DNA is isolated from the remaining traces, and the perpetrator's DNA profile is determined. The profile is often based on individual DNA polymorphisms (e.g., the number of repeats of short DNA repeats called microsatellites). Next, the results (profile) are compared with a criminal DNA database containing known perpetrator DNA profiles, and if there is a match, the identity of the perpetrator can be recognized. However, in most cases, the DNA database does not contain a profile that matches the actually identified DNA polymorphism (i.e., the perpetrator's profile has not yet been introduced into the DNA database). In such cases, by specifying the relative 6mA level of a specific genomic locus from the biological trace and comparing that value with a reference "relative 6mA level - age" curve, the age of the perpetrator can be accurately specified (e.g., 56 ± 2 years - i.e., an error limit of 2 years) (Figure 4). Considering the specified age, the number of suspects can be significantly narrowed down, greatly facilitating the success of the investigation process.

[0015] 2. Prediction of the lifespan of an individual of known age. Even within the same species, lifespan varies from individual to individual. For example, if three 50-year-old humans are randomly selected, the first person lives for 56 years, the second person lives for 66 years, and the third person dies at the age of 84. That is, even people of the same age can have significantly different lifespans. Using the above method, the predicted lifespan (the lifespan of that person) of an individual of known age can be predicted. If the relative 6mA level of a specific genomic site being tested is significantly higher than the level predicted from the reference curve for a specific age, that value predicts a shorter lifespan than expected. Therefore, the higher the relative 6mA level at a specific age, the shorter the predicted lifespan of the individual. It is important to note that this is a "mere" prediction. This is because, for example, due to a fatal accident, that person may die much earlier than predicted from the 6mA level. A person who receives an unfavorable prediction (short predicted lifespan) in the test can significantly change their lifestyle (e.g., start physical activity, avoid stress, have better nutrition) to reverse the rate of cell aging and extend their lifespan. Additionally, an unfavorable predicted lifespan can also be a trigger to start preparing a will.

[0016] 3. Early detection of neurodegenerative processes. If the relative 6mA level identified in a person of known age is significantly different from the level predicted from the reference curve at a given age, that value may indicate the initial stage of the neurodegenerative process (before the onset of cognitive decline) (Figure 5). This is because the rate of the aging process in people suffering from neurodegenerative diseases (such as Alzheimer's disease, Parkinson's disease, Huntington's disease, or ALS) is different (usually faster) from that in people of the same age who do not have the disease, and this difference can be accurately identified by the method introduced in this patent application (the higher the relative 6mA level at a given age, the faster the rate of the aging process). Therefore, the relative 6mA level in mobile TE arrays can be used as an early marker for various neurodegenerative pathologies. This is an important discovery. Because when a neurodegenerative disease appears (manifesting as obvious cognitive impairment), it has been found that the main parts of the brain have already been affected by neuron death, so the applied pharmacological treatments are mostly ineffective (dead neurons cannot be regenerated). An early (well before the onset of cognitive decline) diagnosis can provide a solution, but an appropriate marker for this purpose has not yet been generated. If the measured relative 6mA level indicates involvement in an otherwise apparently normal (healthy) individual, it is recommended that the patient undergo further PET (positron emission tomography) or NMR (nuclear magnetic resonance) examinations to recognize the presence of the degenerative process and immediately initiate drug therapy.

Brief Description of the Drawings

[0017]

Figure 1

[0018]

Figure 2

[0019]

Figure 3

[0020]

Figure 4

[0021]

Figure 5

[0022] Detailed description of the innovation To determine age from DNA, the following steps need to be carried out: i) Determine the relative 6mA level at specific genomic sites within a tissue sample of an unknown individual using a PCR-based approach (semi-quantitative PCR or real-time quantitative PCR); ii) Assign a value on the reference “relative 6mA level - age” curve from the determined 6mA level; iii) Extrapolate this value (intersection point) to the X-axis (age) to assign the age of the individual. To carry out this series of experiments, the following steps are to be executed: i) Isolation of genomic DNA from the tissue, ii) Digestion of the genomic DNA with a 6mA-specific restriction endonuclease (e.g., DpnI which cuts DNA at the -GATC- sequence only when A is methylated: -GAMeTC-), iii) Ligation of linker DNA fragments to the digested genomic DNA fragments, iv) PCR amplification of the target (digested) site using a forward primer specific to both the linker and the adjacent genomic sequence simultaneously, v) Quantification of the PCR products, vi) Comparison of the amount of the product with that of an internal control (normalization - relative 6mA level). In this case, the PCR products accurately reflect the relative 6mA level of the selected genomic sites. This method is described in a recent patent application titled “PCR-based accurate determination method” with file numbers P2100409 and W2200015. The owner (inventor) of this application has given our company permission to review the file (protocol) on the condition that all aspects of the IP rights are maintained.

[0023] Using this technique, we previously determined the relative 6mA levels at the Ll locus (specific adenine nucleobase) in hundreds of individuals of known age (identity). From the data obtained, a reference "relative 6mA level - age" curve was created. The people tested were healthy and of various ages. Next, the relative 6mA levels identified in individuals of unknown identity were projected onto the reference curve, and the age on the X-axis was assigned to the intersection point (Figure 4). Based on our measurements, the deviation of the data was within 2 - 3 years of the error range (±2 - 3 years). If the relative 6mA level of the human L1 locus is determined in an individual with a known identity (age) and the value obtained is significantly different from the value indicated by the regression curve at a specific age, that difference may reflect the presence of the initial stage of a neurodegenerative process (Figure 5). In this case, the affected individual is recommended to undergo an imaging examination such as PET or NMR.

Example

[0024] Method (the basis of which is described in a patent application entitled "PCR-based accurate determination method". File numbers: P2100409 and W2200015. The inventors have given us permission to view the file (protocol) on the condition that we retain all aspects of intellectual property rights - confidentiality agreement): Isolation of genomic DNA Isolation from blood samples was performed using the Thermo Scientific GeneJET Genomic DNA Purification Kit (♯K0721).

[0025] Digestion of genomic DNA with DpnI restriction endonuclease Add the following components to an Eppendorf tube: 1 μg of Dpnl enzyme (10 U / μl, ThermoFisher Scientific, ERI 701) 4 μl of Tango buffer (10x) 15 μl of H20 20 μl of genomic DNA solution (minimum concentration: 10 ng / μl) (Final volume: 40 μl / sample) → Incubate at 37 °C for 20 minutes Inactivation of DpnI: Incubate the sample at 80 °C for 20 minutes.

[0026] Ligation of linker DNA and digested genomic DNA fragments Add the following components to the inactivation mixture (40 μl): 4 μl of T4 ligase (5 U / μl, ThermoFisher Scientific, EL001 l) 5 μl of ATP (2 mM) (ThetnoFisher Scientific, 100 mM, R0441) 5 μl of genomic linker (100x) 4 μl of Tango buffer 22 μl of H20 Final volume: 80 μl Incubate the sample at 40 °C overnight, Inactivation of ligase: Incubate the sample at 800 °C for 20 minutes DNA sequence of linker: 5'-TAG ATC TGA CCT AAC GGT AAG AGA GTT TCA TAA TAt ttt ttt ttt ttT AT-TAT GAA ACT CTC TTA CCG TTA GGT CAG ATC TA-3'

[0027] PCR reaction 6mA sample (Dpnl) - Forward (left) primer: 5'-ATG AAA CTC TCT TAC CGT TAG GTC AGA TCT Atc aac-3' and Reverse (right) primer: 5'-tga acg ttg gcc tgc ctt gc-3' Reaction mixture: 10 μl of ABI master mix (2x - Catalog number: K0171) 3.5 μl of digested / ligated DNA solution (template) 3 μl of primer mix (5 - 5 μM) 3.5 μl of H20 Final volume: 20 μl / sample

[0028] PCR conditions (for DpnI digested samples): 1. Initial denaturation: 95°C, 30 seconds 2. Denaturation: 95°C, 10 seconds 3. Annealing and extension: 71°C, 30 seconds 4. Repeat steps 2 and 3 forty times 5. Store samples at 4°C

[0029] Control (Pvull digested) samples - Forward (left) primer: 5'-tga atg aaa tga agc gag aag gga agt tta gag-3' and Reverse (right) primer 5'- tga acg ttg gcc tgc ctt gc-3' Reaction mixture: 10 μl of ABI master mix (2x) 1 μl of DNA solution (template) 3 μl of primer mix (5 - 5 μM) 6 μl of H20 Final volume: 20 μl

[0030] PCR conditions (for Pvull digested samples): 1. Initial denaturation: 95°C, 60 seconds 2. Denaturation: 95°C, 10 seconds 3. Annealing and extension: :60°C, 45 seconds 4. Repeat steps 2 and 3 fifty-five times 5. Store samples at 4°C

[0031] Gel documentation Analyze DNA samples (PCR products) on a 1% agarose gel at 80 mV and photograph with a Kodak camera.

[0032] Novelty of the Invention 1. Recognition that the relative 6mA level in active TE sequences (e.g., L1 of the human genome) gradually increases with aging. Based on this discovery, the age can be accurately determined from nuclear DNA isolated from an individual of unknown identity. 2. Recognition that this process is evolutionarily conserved (in C. elegans, the TCI sequence, the most active TE family in this organism, is gradually methylated at N6-adenine with aging). 3. Recognition that the relative 6mA level identified in active TE sequences predicts the life expectancy of an individual of known age. 4. Recognition that the relative 6mA level identified in active TE sequences may indicate the presence of a neurodegenerative process.

[0033] References Dahl C, Guldberg P. DNA methylation analysis techniques. Biogerontology 4, 233 - 250 (2003). PMID: 14501188 De Cecco M, Ito T, Petrashen AP, Elias AE, Skvir NJ, Criscione SW, Caligiana A, Brocculi G, Adney EM, Boeke JD, Le O, Beausejour C, Ambati J, Ambati K, Simon M, Seluanov A, Gorbunova V, Slagboom PE, Helfand SL, Neretti N, Sedivy JM. Ll drives IFN in senescent cells and promotes age - associated inflammation. Nature 2019 Feb; 566(7742): 73 - 78. PMID: 30728521 Dillin A, Hsu AL, Arantes-Oliveira N, Lehrer-Graiwer J, Hsin H, Fraser AG, Kamath RS, Ahringer J, Kenyon C. Rates of behavior and aging specified by mitochondrial function during development. Science. 2002 Dec 20; 298(5602): 2398-2401. PMID: 12471266 Douvlataniotis K, Bensberg M, Lentini A, Gylemo B, Nestor CE. No evidence for DNA N 6methyladenine in mammals. Sci Adv. 2020 Mar 1 8; 6(12): eaay3335. PMID: 32206710 Flusberg BA, Webste DR, Lee JH, Travers KJ, Olivares EC, Clark TA, Koriach J, Turner SW. Direct detection of DNA methylation during single-molecule, real-time sequencing. Nat Methods 7, 461-465 (2010). PMID: 20453866 Gorbunova V, Seluanov A, Mita P, McKerrow W, Fenyo D, Boeke JD, Linker SB, Gage FH, Kreiling JA, Petrashen AP, Woodham TA, Taylor JR, Helfand SL, Sedivy JM. The role of retrotransposable elements in ageing and age-associated diseases. Nature. 2021 Aug; 596(7870): 43-53. PMID: 34349292 Greer EL, Blanco MA, Gu L, Sendinc E, Liu J, Aristizabal-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 Horvath S. DNA methylation age of human tissues and cell types. Genome Biol. 2013; 1 4(10): RI 15. PMID: 24138928 JPEG2025520358000001.jpg10157Kenyon C, Chang J, Gensch E, Rudner A, Tabtiang R. A C. elegans mutant that lives twice as long as wild type. Nature. 1993 Dec 2; 366(6454): 461-464. PMID: 8247153 Kirkwood TB. A systematic look at an old problem. Nature. 2008 Feb 7; 45 1 (7179): 644-647. PMID: 18256658 L6pez-Otin C, Blasco MA, Partridge L, Serrano M, Kroemer G. The hallmarks of aging. Cell. 2013 Jun 6; 153(6): 1194-1217. PMID: 23746838 Luo G-z, wang F, Weng X, Chen K, Hao Z, Yu M, Deng X, Liu J, He C. Characterization of eukaryotic DNA N(6)-methyladenine by a highly sensitive restriction enzyme-assisted sequencing. Nat. Commun. 7, 11301 (2016). PMID: 27079427 Nurk S, Koren S, Rhie A, Rautiainen M, Bzikadze AV, Mikheenko A, Vollger MR, Altemose N, Uralsky L, Gershman A, Aganezov Hoyt SJ, Diekhans M, Logsdon GA, Alonge M, Antonarakis SE, Borchers M, Bouffard GG, Brooks S Y, Caldas GV, Chen NC, Cheng H, Chin CS, Chow W, de Lima LG, Dishuck pc, Durbin R, Dvorkina T, Fiddes IT, Formenti G, Fulton RS, Fungtammasan A, Garrison E, Grady PGS, Graves-Lindsay TA, Hall 1M,Hansen NF, Hartley GA, Haukness M, Howe K, Hunkapiller MW.Jain C, Jain M, Jarvis ED, Kerpedjiev P, Kirsche M, Kolmogorov M, Koriach J, Kremitzki M, Li H, Maduro VV, Marschall T, McCartney AM, McDaniel J, Miller DE, Mullikin JC, Myers EW, Olson ND, Paten B, Peluso P, Pevzner PA, Porubsky D, Potapova T, Rogaev El, Rosenfeld JA, Salzberg SL, Schneider VA, Sedlazeck FJ, Shafm K, Shew CJ, Shumate A, Sims Y, Smit AFA, Soto DC, Sovie I, Storer JM, Streets A, Sullivan BIA, ThibaudNissen F, Torrance J, Wagner J, Walenz BP, Wenger A, Wood JMD, Xiao C, Yan SM, Young AC, Zarate S, surti U, McCoy RC, Dennis MY, Alexandrov IA, Gerton JL, O'Neill RJ, Timp W, zook JM, Schatz MC, Eichler EE, Miga KH, Phillippy AM. The complete sequence of a human genome. Science. 2022 Apr; 376(6588): 44-53. PMID: 35357919. O'Brown ZK, Boulias K, wang J, wang sy, O'Brown NM, Hao Z, Shibuya H, Fady P-E, Shi Y, He C, Megason SG, Liu T, Greer EL. Sources of artifact in measurements of 6mA and 4mC abundance in eukaryotic genomic DNA* BMC Genomics. 2019 Jun 3; 20(1): 445. PMID: 31159718 Rocha MS, Castro R, Rivera 1, Kok R, Smulders YM, Jakobs C, de Almeida IT, Biom HJ. Global DNA methylation: comparison of enzymatic- and non-enzymatic-based methods. Clin. Chem. Lab. Med. 48, 1793-1798 (2010). PMID: 20979561 Schiffers S, Ebert C, Rahimoff R, Kosmatchev O, Steinbacher J, Bohne A-V, Spada F, Michalakis S, Nickelsen J, Muller M, Carell T. Quantitative LC-MS Provides No Evidence for m 6 dA or m 4 dC in the Genome of Mouse Embryonic Stem Cells and Tissues. Angew Chem Int EdEng1. 2017 sep 4; 56(37): 11268-1 1271. PMID: 28371147 Sturm A, Ivics Z, Vellai T. The mechanism of ageing: primary role of transposable elements in genome disintegration. Cell Mol Life Sci. 2015 May; 72(1 0): 1839-1847. PMID: 25837999 Sturm A, Perczel A, Ivics Z, Vellai T. The Piwi-piRNA pathway: road to immortality. Aging cell. 2017 Oct; 16(5): 906-911. PMID: 28653810 Vellai T. Autophagy genes and ageing. Cell Death Differ. 2009 Jan; 16(1): 94-102. PMID: 19079287 Vellai T, Takacs-Vellai K, Zhang Y, Kovacs AL, Orosz L, Muller F. Genetics: influence of TOR kinase on lifespan in C. elegans. Nature. 2003 Dec I l ; 426(6967): 620. PMID: 14668850 Wood JG, Jones BC, Jiang N, Chang C, Hosier S, Wickremesinghe P, Garcia M, Hartnett DA, Burhenn L, Neretti N, Helfand SL. Chromatin-modifying genetic interventions suppress age-associated transposable element activation and extend lifespan in Drosophila. Proc. Natl. Acad. sci. USA. 2016 Oct 4; 1 13(40): 11277-1 1282. PMID: 27621458 Wu TP, Wang T, Seetin MG, Lai Y, Zhu S, Lin K, Liu Y, Byrum SD, Mackintosh SG, Zhong M, Tackett A, Wang G, Hon LS, Fang G, Swenberg JA, Xiao AZ. DNA methylation on N(6)-adenine in mammalian embryonic stem cells. Nature. 2016 Apr 21; 532(7599): 329-333. PMID: 27027282 Yoder JA, Walsh CP, Bestor TH. Cytosine methylation and the ecology of intragenomic parasites. Trends Genet. 1997 Aug; 13(8): 335-340. PMID: 9260521 Zhang G, Huang H, Liu D, Cheng Y, Liu X, Zhang W, Yin R, Zhang D, Zhang P, Liu J, Li C, Liu B, Luo Y, Zhu Y, Zhang N, He S, He C, Wang H, Chen D. N6-methyladenine DNA modification in Drosophila. cell 161, 893-906 (2015). PMID: 25936838 Yao B, Cheng Y, Wang Z, Li Y, Chen L, Huang L, Zhang W, Chen D, Wu H, Tang B, Jin P. DNA N6-methyladenine is dynamically regulated in the mouse brain following environmental stress. Nat. Commun. 8, 1122 (2017). PMID: 29066820

Claims

**Claim 1** A method / procedure for accurately determining the age from human nuclear DNA by identifying the relative N6-methyladenine level in the sequence of the active transfer factor family. **Claim 2** The method / procedure according to claim 1, wherein the organism to be examined is any eukaryotic species. **Claim 3** The method / procedure according to claim 1 or 2, wherein the measurement of the N6-methyladenine level is achieved by an appropriate molecular technique. **Claim 4** The method / procedure according to any one of claims 1 to 3, wherein the target sequence is a repetitive sequence such as a transposable element, and further a nuclear DNA sequence. **Claim 5** The method / procedure according to any one of claims 1 to 4, wherein the type of tissue for separating genomic DNA for analysis is arbitrary. **Claim 6** The method / procedure according to any one of claims 1 to 5, wherein the determination of the reference "relative N6-methyladenine level - age" curve is achieved in any population of organisms. **Claim 7** The method / procedure according to any one of claims 1 to 6, wherein the purpose of the analysis is to identify the age of an unknown person. **Claim 8** The method / procedure according to any one of claims 1 to 7, wherein the purpose of the analysis is to predict the life expectancy of an individual whose identity (age) is known. **Claim 9** The method / procedure according to any one of claims 1 to 8, wherein the purpose of the analysis is to identify the initial stage of the neurodegenerative process (before the signs of cognitive impairment appear).

Images