Healthy age

Abstract

The invention relates to the creation of a platform developed for use in the fields of medicine, health, biotechnology, public health, where individuals can determine their DNA methylation patterns and single nucleotide polymorphisms associated with metabolism, share the relevant epigenetic and genetic information with professionals (experts such as doctors, dietitians, psychologists) who monitor the health of individuals, and receive individual sports and dietary recommendations for their metabolism in line with their epigenomic and genomic characteristics.

Description

[0001] Healthy Age

[0002] Field of the Invention

[0003] The invention relates to the creation of a platform developed for use in the fields of medicine, health, biotechnology, public health, where the DNA methylation patterns of individuals can determined and relevant epigenetic information can be shared with professionals (experts such as doctors, dietitians, psychologists) who monitor the health of individuals, and receive individual sports and dietary recommendations in line with their epigenomic characteristics and genetic differences acquired afterwards. The invention which is named HealthyAge by the inventors includes two specific genetic panels where both epigenetic and genetic changes are analyzed and the healthy aging support platform formed after these two panels being evaluated together.

[0004] State of the Art

[0005] During the aging process, the methylation status of the cells changes. Some areas are more methylated, while others are demethylated. DNA methylation in the promoter of a gene often leads to the silencing of that gene. While methylation of DNA is mediated by DNMTs, the methyl group on DNA is removed by TET enzymes. In mammalian cells, TET enzymes oxidize 5-mC to form 5-mC derivatives, including 5- hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-fC), and 5-carboxylcytosine (5- caC). Age prediction models such as Horvath's clock, Hannum's clock, PhenoAge, GrimAge, and single cell age clock (scAge) are based on DNA methylation changes in the genome.

[0006] Over the course of life, the methylation status of the cells change, with some regions becoming more methylated and some demethylated. During embryogenesis, most of the genome is methylated, but the CpG islands are not. Two important changes occur in the aging process. Firstly, regions associated with the nuclear lamina undergo demethylation, while secondly, selected CpG islands bound by polycomb undergo de- novo methylation. This process takes place in all cells of the body, but at a different rate for each tissue. By using an algorithm that samples methylation change as an indicator, an age index can be generated for any DNA sample from a given tissue. DNAm age is the epigenetic biomarker of biological aging, reflecting age related cumulative changes in DNA methylation affected by both environmental and genetic risk factors. Research shows that the aging process is driven by epigenetic factors in many respects, and DNAm age is influenced by both genetic and environmental factors over time on cellular functions.

[0007] Epigenetic mechanisms play an important role in regulating the reflection of the genotype on the phenotype and modulating the aging process of environmental factors. DNA methylation is the most fundamental and most studied type of chemical modification among epigenetic mechanisms. The close relationship between the change of DNA methylation patterns and aging can be demonstrated by extensive research done with new generation sequencing technologies. In this context, it is now known that the DNA methylation process, which is one of the epigenetic change mechanisms in line with scientific research and current literature information, is one of the fundamental mechanisms involved in human aging.

[0008] Age is one of the most important risk factors for many diseases such as cardiovascular, metabolic and neurological diseases and cancer [1], However, individuals do not age to the same extent and at the same rate. There are significant differences between people in the rate of aging and individual differences in their susceptibility to disease and death. As individuals age, it is well known that there are a number of molecular changes that occur in cells and tissues. Changes in DNA methylation patterns have been shown to occur in this process [2], Epigenetic biomarkers of aging, also known as epigenetic clocks, have been developed using DNA methylation measurements and are specifically defined as 'DNA methylation age'. This situation provided an accurate age estimation in individuals of different age groups and enabled the identification of the susceptibility to diseases of individuals who showed significant deviations from their chronological ages [3]. In a study conducted in 2017, it was observed that there were remarkable correlations between epigenetic age and some lifestyle factors such as physical activities and healthy eating [4], In the study, it was stated that these observational results provide the basis for further research on lifestyle interventions that reverse biological age. Early studies have identified age related methylated CpG positions in genes related to chronic diseases and aging. Most of these studies presented limited data due to the low coverage of CpG regions across the genome. Today, with the developing new generation sequencing technologies, genome wide methylation analyzes can be performed more easily and a more comprehensive data can be obtained.

[0009] The use of whole genome bisulfite sequencing (WGBS) enables genome wide identification and quantification of DNA methylation patterns at single-based resolution. This reaction is the gold standard for the DNA methylation analysis [5]. The whole genome bisulfite sequencing technique can do precise cytosine-methylation detection under certain sequences throughout the whole genome. Thus, it creates the potential to determine specific DNA methylation regions and their relationship to specific gene expressions.

[0010] With current studies, the critical role of epigenetic changes in aging is understood better every day. Today, the most up to date tool used for the determination of biological age is DNA methylation-based epigenetic clock models. Horvath's clock, Hannum's clock, DNA PhenoAge, and DNA GrimAge are some of the most well-known of these epigenetic clock models.

[0011] When the current patents on biological age determination are examined, it is seen that they are limited to methods based on DNA methylation.

[0012] For example, the patent application titled CN111763742A "Methylation marker, method for determining age of individual and application" aims to determine biological age by analyzing the methylation status of certain regions in DNA.

[0013] Patent application CA3140124A1 titled ‘EPIAGING: NOVEL ECOSYSTEM FOR MANAGING HEALTHY AGING' aims to determine biological age by analyzing the methylation of specific regions in genomic DNA obtained from individuals, in conjunction with analyzing their personal data.

[0014] The patent application WO2018139826A1 titled “AGE PREDICTING METHOD USING DNA METHYLATION” aims to create an epigenetic age estimation model by evaluating the methylation status in certain DNA regions.

[0015] The patent application WO2023175019A1 titled “METHOD DETERMINING THE DIFFERENCE BETWEEN THE BIOLOGICAL AGE AND THE CHRONOLOGICAL AGE OF A SUBJECT” aims to create a model in which the lifestyle characteristics of individuals are also evaluated by comparing the methylation levels in CpG regions to determine the difference between the biological age and the chronological age of the individual.

[0016] However, when the relevant patents are examined, it is seen that there is no study that presents the whole genome methylation analysis, as well as information such as physical features, lifestyle, clinical parameters of individuals and genetic variations affecting sports and dietary metabolisms as a whole.

[0017] When the existing patents for nutrigenomic analyzes are examined, it is seen that they are limited to epigenetic analyzes only.

[0018] For example, the patent application W02020210487A1 titled "Systems and methods for nutrigenomics and nutrigenetic analysis" aims at systems and methods to facilitate nutrigenomics and nutrigenetic analysis.

[0019] The patent KR102268059B1 titled "Composition, kit for predicting weight control according to exercise, and method using the same" aims at a composition and kit containing a polynucleotide that can detect a single nucleotide polymorphism marker, for predicting or diagnosing motion sensitivity.

[0020] However, upon examining the relevant patents, it is observed that they consist only of nutrigenomic analyzes and do not include population specific rs. For this reason, there is a need for a holistic approach in which both genetic and epigenetic analyzes are presented together. However, the genetic panel should include rs that allow for the evaluation of not only nutrigenomic analyzes but also sports performance. It is necessary that all diet and sports performance evaluating rs included in the genetics panel are selected specific to the relevant population and that they can serve as a whole as a nutrition and sports genetic panel.

[0021] As a result due to the said disadvantages and the insufficiency of the current solutions regarding the subject matter, a development is required to be made in the relevant technical field.

[0022] The Purpose of the Invention

[0023] The object of the invention is to solve said disadvantages by being inspired from the current conditions. The main purpose of the invention is to provide individuals with personalized information and recommendations in the healthy aging process with both the epigenetic panel and the nutrition and sports genetic panel.

[0024] Another purpose of the invention is to provide the analysis of more comprehensive data by whole genome methylation sequencing and the creation of a platform that offers diet and sports recommendations specific to individuals with genetic analyzes made after biological age determination by evaluating individual characteristics.

[0025] A purpose of the invention is to provide a holistic healthy aging recommendation platform that includes methylation analyzes with specific genes determined within the scope of the invention and analyzes of specific rs unique to the population of Turkey regarding diet and sports genetics, unlike SNP analyzes including epigenetic age analyzes, nutrigenetics and sports genetics existing in the current art. The invention differs from the epigenetic analyzes in the present art in that it includes the methylation analysis of the specific gene regions determined by the inventors in the epigenetic panel part. However, the invention differs from existing applications involving SNP analysis within the scope of nutrigenomics and physical activity analysis by enabling the simultaneous analysis of both nutrition related and sports related rs and being designed specifically for the Turkish population in the genetic panel part. In addition, the invention gains authenticity by providing a support platform for a healthy aging process that offers personalized recommendations with the combined use of two different panels that are unique both epigenetically and genetically.

[0026] A purpose of the invention is to provide two different panels in which both epigenetic and genetic changes are examined and a healthy aging support platform is created by evaluating these two panels together. These two panels in turn form a workflow that enables evaluation. Methylation analyzes of specific selected genes with the epigenetic panel include an analysis process in which individuals' habits such as age, gender, physical characteristics, clinical parameters, daily activity, diet and alcohol and smoking are evaluated together. As a result of this analysis, the biological age determination of the individuals is done, and the rs in which nutrition and sports performance are evaluated are analyzed in order to create nutrition and physical activity programs specific to individuals with the genetic panel in line with the biological age information obtained. In order to fulfill said purposes, the invention is a method of creating a HealthyAge platform and comprises the following method steps: i. Determination of the parameters of the physical and clinical characteristics of the individuals which will participate in the evaluation during the analysis stage and the obtaining of biological material from the individuals; ii. DNA isolation; iii. Methyl-MaxiSeq Bisulfite transformation, PCR analyzes, Library preparation; iv. Sequence alignment and methylation analysis; v. Determination of biological age by bioinformatic analysis of data and evaluation of demographic characteristics, development of the epigenomic platform; vi. SNP (Single nucleotide polymorphisms) analysis associated with metabolism; vii. Statistical analyzes and interpretation of results.

[0027] In a preferred embodiment of the invention; the genetic panel comprising the following rs is used relating to the metabolism in the SNP analysis at process step (vi): rs2071045 (LEP); rs1121980(FTO); rs22296(MC4R); rs7799039 (LEP); rs1421085(FTO); rs3751812 (FTO); rs14383 (GDF5); rs1778213(MC4R); rs1522813 (IRS1 ); rs7903146 (TCF7L2); rs964184 (APOA5); rs1815739(ACTN3); rs4880 (SOD2); rs1800012 (COL1A1); rs2228145 (IL6R); rs1141718 (SOD2); rs4295 (ACE).

[0028] In a preferred embodiment of the invention; the epigenetic panel comprising the following genes is used in the methylation analysis at process step (iv): In a preferred embodiment of the invention; in the personal information form in process step (i) the following information is taken into consideration Age; Gender; Height; Weight; Body mass index; Diet (Mediterranean diet, ketogenic diet) / daily calorie intake / specific dietary preference; Cigarette consumption (Less / more than 10 per day); Alcohol consumption (Less / more than two glasses at once per week); Stress (Loss of a family member, loss of employment, migration etc.); Traumatic events in the Childhood / Adolescence (e.g. exposure to poverty in childhood / childhood socioeconomic status / exposure to victimization during childhood); Viral infections (2 or less per year, more than 2 per year); Insomnia (Insomnia is present / not present); Physical Activity (3000 or less steps on average per day / 3000-7000 steps / more than 7000)

[0029] In a preferred embodiment of the invention; in process step (i) Peripheric blood material preferably in a volume of 2ml is taken as biological material.

[0030] In a preferred embodiment of the invention; in process step (ii)QIAamp DNA Mini kit (cat no: 51304, 50 preps isolation Kit) protocol is followed.

[0031] In a preferred embodiment of the invention; in process step (iii) Zymo-Seq WGBS Library Kit and Zymo Research dsDNA ShearaseTM Plus Kit is used for library preparation and sequencing.

[0032] In a preferred embodiment of the invention; in process step (iv) the library is sequenced using Illumina Novaseq 6000 platform.

[0033] In a preferred embodiment of the invention; in process step (v) t test (for those that comprise 2 groups) or ANOVA (for those that comprise >2 groups) is used for the comparison of data.

[0034] In a preferred embodiment of the invention; in process step (vi) SNP analyzes are performed with optimized Taqman probes in RT-PCR device, Taqman Universal master mix and specific SNP primers specific to the Turkish population. In a preferred embodiment of the invention; in process step (vii) GraphPad Prism 6.0 and SPSS (20.0) programs are used for statistical analyzes.

[0035] The structural and characteristic features and all the advantages of the invention will be understood more clearly thanks to the detailed description given below, and therefore the evaluation should be made by taking this detailed description into consideration.

[0036] Detailed Description of the Invention

[0037] In this detailed description, the HealthyAge platform and the preferred embodiments of the invention are described only for a better understanding of the subject.

[0038] The invention is based on whole genome bisulfite sequencing and subsequent analysis of genetic variations (single nucleotide changes). The selected SNP rs include changes that may make a difference in the sports and diets of individuals seen in the Turkish population. Epigenetic analyzes are performed using new generation sequencing technologies and SNP analyzes are performed on RT-PCR device with optimized Taqman probes. The personal and clinical information parameters of the individuals to be used in the analyzes are the parameters that are determined to be particularly important. Analyzes are evaluated considering these parameters. A platform is created where the data obtained from the analyzes can be shared with experts in the field with the individual's knowledge and consent, while ensuring the protection of the individual's personal data.

[0039] In the invention, by whole genome bisulfite sequencing and genome wide methylation patterns are analyzed by doing DNA isolation from peripheric blood samples taken from individuals and methylation pattern differences between individuals are compared. Bioinformatic and biostatistical analyzes can be used to do biological age determination of individuals. Since knowing the epigenomic information and the determined biological age of the individual will not create an original output alone, an epigenomic identity can be revealed within the request and approval of the individual (with the knowledge and permission that he / she has the right to protect his / her personal information) that can be shared with experts in the field of data that can be used in order to increase the quality of life, to be useful to the individual in different areas related to his / her health and to direct his / her health in the future of life. The invention provides a follow-up analysis of the change in the methylation patterns of individuals as a result of the comparative analyzes of the subgroups divided according to the lifestyle characteristics of individuals and provides important information in order to increase the quality of life of individuals and to contribute to healthy aging processes. The innovative approach of the invention is to provide more comprehensive information than the biological age determination kits available on the market in this sense and then transforming this into a platform that aims to follow the changing methylation patterns in parallel with the differentiating habits of individuals. In addition, with the genetic analyzes (gene panel that can analyze the change of 17 regions of 12 different genes) performed after DNA methylation based biological age analysis, unlike biological age determination kits, the following can be formed:

[0040] Information on susceptibility to muscle injuries, susceptibility to muscle damage

[0041] Recommendations for the elimination of oxidative stress and oxidative stress

[0042] Exercise-induced muscle breakdown rate

[0043] Energy metabolism of the individual

[0044] Optimal Fat, carbohydrate and protein intake for genetic structure

[0045] Insulin resistance

[0046] Dietary recommendations suitable for the genetic characteristics of the individual

[0047] After the epigenomic identities of the individuals are established, the SNR (single nucleotide polymorphisms) genetic variation characteristics determined specific to the Turkish population are analyzed and the scope to include the specified diet and sports recommendations reveals the authenticity of the invention.

[0048] The invention is a HealthyAge platform; specific method steps are described below:

[0049] (i) Determination of the parameters of the physical and clinical characteristics of the individuals which will participate in the evaluation during the analysis stage and the obtaining of biological material from the individuals:

[0050] The personal information form is important for determining the parameters of the physical characteristics and clinical characteristics of the individuals who will participate in the evaluation during the analysis stage. Individuals' characteristics such as age, gender, height, weight, body mass index, diet, cigarette consumption, alcohol consumption, stress, viral infections, insomnia, and physical activity should be included in the personal information form.

[0051] In the preferred embodiment of the invention; the evaluation criteria included in the personal information form created by obtaining consent from the individuals include the following Age; Gender; Height; Weight; Body mass index; Diet (Mediterranean diet, ketogenic diet) / daily calorie intake / specific dietary preference; Cigarette consumption (Less / more than 10 per day); Alcohol consumption (Less / more than two glasses at once per week); Stress (Loss of a family member, loss of employment, migration etc.); Traumatic events in the Childhood / Adolescence (e.g. exposure to poverty in childhood I childhood socioeconomic status / exposure to victimization during childhood); Viral infections (2 or less per year, more than 2 per year); Insomnia (Insomnia is present / not present); Physical Activity (3000 or less steps on average per day / 3000-7000 steps / more than 7000).

[0052] In the preferred embodiment of the invention; Peripheric blood material taken in a volume of 2ml is the most important material to determine the genomic DNA of the individuals.

[0053] In addition to the obtaining of peripheric blood material of individuals, the supply of any other biological material from which the genomic DNA of individuals can be obtained is also considered within the scope of the invention.

[0054] (ii) DNA isolation:

[0055] In the preferred embodiment of the invention, isolations are performed by following the QIAamp DNA Mini Kit (cat no: 51304, 50 preps isolation Kit) protocol for DNA isolation from peripheric blood obtained from individuals.

[0056] The procedure performed in accordance with the kit protocol is described below:

[0057] Before starting the isolation, the heater block is set to 560C and preheated. 20 pl Proteinase K is placed in a 1 .5ml eppendorf tube. 200 pl of peripheric blood sample is added on it. 200 pl of Buffer AL (lysis buffer solution) is added and vortexed for 1 minute. At this stage, it is important that the mixture is thoroughly homogeneous. It is incubated at 560C for 10 minutes in a pre-set heater block. After the incubation, 200 pl (96%EtOH) Ethanol is added. It is vortexed for 1 minute. The mixture is transferred to the Mini spin column. It is centrifuged at 6000g for 1 minute. The spin column is switched to a new collection tube. 500 pl AW1 buffer solution is added to the spin column. It is centrifuged at 6000g for 1 minute. The lower collection tube is discarded and a new collection tube is used. 500 pl AW2 buffer solution is added to the spin column. It is centrifuged at 6000g for 1 minute. The spin column is switched to a new collection tube. Once it is centrifuged at the highest speed for 1 minute, the spin column membrane is made to dry. The spin column is taken to 1 .5 ml eppendorf and a maximum of 200 pl of sterile distilled water is added. It is incubated for 5 minutes and centrifuged at 6000 g for 1 minute at the highest speed. Spectrophotometric A260 / A280 ratio is used in the quality determination of the DNA to be obtained in a maximum elution volume of 200 pl, and A260 value is used to determine their concentrations in pg / ml. DNA with a concentration of 50nq / pl-150nq / pl with a ratio of 260 / 280 and an OD value of 1 .8 are included in the study.

[0058] Table 1. DNA isolation:

[0059] The DNA isolation kit is necessary to obtain genomic DNA from the biological material of individuals, and it is important to have an optimized kit in which sufficient quantity and quality of DNA can be obtained for further analyzes.

[0060] The use of different commercial kits for DNA isolation is also considered within the scope of the invention.

[0061] (Hi) Methyl-MaxiSeq Bisulfite transformation, PCR analyzes, Library preparation:

[0062] In the preferred embodiment of the invention, library preparation is made for the samples converted to bisulfite using the Zymo-Seq WGBS Library Kit. Within the scope of the kit protocol, (1) second strand synthesis, (2) labeling and (3) library amplification and indexing processes are performed respectively. 200 ng of the starting genomic DNA is fragmented using Zymo Research dsDNA ShearaseTM Plus (Cat#: E2018-50). The ends of the produced fragments are cut to be blunt ends and extended with 3'-terminalA. The DNA is then purified using Clean & Concentrator TM-5 (Cat#: D4003). The A-spliced fragments bind to adapters containing 5'-methyl-cytosine instead of cytosine according to Illumina's stated guidelines. The >50 bp sized adapter linked fragments are recovered using DNA Clean & ConcentratorTM-5 (Cat#: D4003). The fragments are then processed with bisulfite using the EZ DNA Methylation-LightningTM Kit (Cat#: D5030). PCR is performed with Illumina indices and the resulting products are purified with DNA Clean & ConcentratorTM-5 (Cat#: D4003). The size and concentration of the fragments are verified with Agilent 2200 TapeStation. The prepared library is sequenced using the Illumina Novaseq 6000 platform.

[0063] Zymo-Seq WGBS Library Kit and Zymo Research dsDNA ShearaseTM Plus kit are optimized kits for library preparation and sequencing in methylation analyzes. In case of analysis on different platforms, replacing the existing kits with different optimized kits is also considered within the scope of the invention.

[0064] The use of different new generation sequencing platforms optimized outside of Illumina for whole genome methylation analyzes is also considered within the scope of the invention.

[0065] (iv) Sequence Alignment and Methylation Analysis:

[0066] In the preferred embodiment of the invention; Raw FASTQ files are trimmed using TrimGalore 0.5.0. FastQC 0.11.8 is used to evaluate the impact of data trimming and overall quality distributions. Alignment to the human reference genome is performed using Bismark 0.19.0. The total counts of methylated and unmethylated reads for each CpG region are evaluated using MethylDackel 0.3.0. Differences in methylation patterns are analyzed with web-based programs. (v) Determination of biological age by bioinformatic analysis of data and evaluation of demographic characteristics, development of the epigenomic platform:

[0067] In the preferred embodiment of the invention; the comparison of data is carried out using t test (for those that comprise 2 groups) or ANOVA (for those that comprise >2 groups). The threshold value for the results is accepted as 10% and there is a p value of <0.05 with a minimum methylation difference between the groups. For ANOVA results, it is required that there is at least one pairwise methylation difference of >10% between the included groups.

[0068] Pathway analyzes of different known and / or newly determined methylation data to be obtained in profiling analysis are performed. The determined methylation pattern differences are evaluated statistically by comparing the disease status and personal and clinical parameters of individuals. By creating a special epigenomic identity for each analyzed individual, it is envisaged to form the basis for the development of a platform where individuals can follow their biological age processes changing depending on DNA methylation pattern changes if they have this analysis done at different times, and with the knowledge and permission that they have the right to protect their personal information, they can open this information to experts who provide professional support they receive in health, and make suggestions that contribute to the healthy aging process in line with their epigenomic characteristics.

[0069] (vi) SNP (Single nucleotide polymorphisms) analysis:

[0070] Using isolated DNA for the methylation analysis;

[0071] The regions determined specifically for the Turkish population numbered rs1815739, rs4880, rs1141718, rs2228145, rs1800012, rs143383, rs4295, rs17782313, rs2229616, rs1421085, rs3751812, rs1121980, rs7799039, rs2071045, rs1522813, rs7903146, rs964184 are analyzed to determine the single nucleotide changes in the ACTN3, SOD2, IL6R, COL1A1 , GDF5, ACE, MC4R, FTO, LEP, IRS1 , TCF7L2, APOA5 genes.

[0072] In the preferred embodiment of the invention; SNP analyzes are performed on RT-PCR device with optimized Taqman probes. Taqman Universal master mix is required for the creation of the appropriate reaction mix of Taqman snp primers for SNP analyzes. Table2. Reaction mix content for SNR (Single nucleotide polymorphisms) analysis

[0073] Table 3: PCR conditions for SNR Genotyping

[0074] The SNR primers (rs1815739, rs4880, rs1141718, rs2228145, rs1800012, rs143383, rs4295, rs17782313, rs2229616, rs1421085, rs3751812, rs1121980, rs7799039, rs2071045, rs1522813, rs7903146, rs964184) were chosen to analyze the changes seen in the Turkish population, which constitutes the authenticity of the invention.

[0075] (vii) Statistical Analyzes and Interpretation of Results.

[0076] In the preferred embodiment of the invention; GraphPad Prism 6.0 and SPSS (20.0) programs are used for all statistical analyzes. Descriptive statistics of the data are given as number, percentage, mean and standard deviation, and the differences between the groups are investigated by one way variance analysis (One Way ANOVA).

[0077] Posthoc Tukey test is used to determine which group or groups the difference originates from. p<0.05 is considered statistically significant.

[0078] The invention has two stages, the first stage which is epigenetic analysis that involves determining the methylation status of the identified regions using the bisulfite sequencing method and establishing the biological age of individuals based on the evaluation criteria included in their personal information form. Table 4 provided below shows the The epigenetic panel content comprises the relevant CpG islets of the Genes, which were found to be important in biological age determination as a result of the studies carried out within the scope of the invention with the Whole Genome Bisulfite Sequencing analysis.

[0079] The second step is to analyze the genes related to the determined metabolism of individuals whose biological age is older than their chronological age and the changes in the rs determined for these genes (Table 5) and to make recommendations to individuals in accordance with their genetic structure about getting a healthy age. After the completion of all these processes, it includes creating a platform where individuals can share this information with professionals (such as personal dietitian, sports coach, family physician, etc.) within their own wishes and consent.

[0080]

[0081] The rs in the Genetic panel shown in Table 5 above were included in the panel by conducting allele frequencies and literature research in the studies conducted within the scope of the invention. Below are the tables of allele frequencies seen in the populations of the relevant rs.

[0082] 5 rs2228145 QL.6R) The biological ages of the individuals were determined by methylation analyzes performed during the development of the invention. During the development of the epigenetic panel, methylation analyzes were performed for 336 CpG regions of individuals, and as a result of statistical analyzes, the epigenetic panel within the scope of the invention was developed. In the genetic analyzes, which is the other part of the invention, the single nucleotide polymorphisms in 17 gene regions were analyzed for these individuals. As a result of the analysis, the polymorphisms of each individual were evaluated separately. In line with epigenetic and genetic analyzes, this information is arranged in a format that individuals can share with professionals who monitor their health (experts such as doctors, dietitians, psychologists).

[0083] REFERENCES

[0084] [1] Seale, K., Horvath, S., Teschendorff, A., Eynon, N., & Voisin, S. (2022). Making sense of the ageing methylome. Nature reviews. Genetics, 23(10), 585-605. https: / / doi.Org / 10.1038 / S41576-022-00477-6

[0085] [2] Zhang, W., Qu, J., Liu, G. H., & Belmonte, J. C. I. (2020). The ageing epigenome and its rejuvenation. Nature reviews. Molecular cell biology, 21 (3), 137-150. https: / / doi.Org / 10.1038 / S41580-019-0204-5

[0086] [3] Fransquet, P. D., Wrigglesworth, J., Woods, R. L., Ernst, M. E., & Ryan, J. (2019). The epigenetic clock as a predictor of disease and mortality risk: a systematic review and meta-analysis. Clinical epigenetics, 11 (1 ), 62. https: / / doi.org / 10.1186 / s13148-019- 0656-7

[0087] [4] Quach, A., Levine, M. E., Tanaka, T., Lu, A. T., Chen, B. H., Ferrucci, L., Ritz, B.,

[0088] Bandinelli, S., Neuhouser, M. L., Beasley, J. M., Snetselaar, L., Wallace, R. B., Tsao, P. S., Absher, D., Assimes, T. L., Stewart, J. D., Li, Y., Hou, L., Baccarelli, A. A., Whitsei, E. A., ... Horvath, S. (2017). Epigenetic clock analysis of diet, exercise, education, and lifestyle factors. Aging, 9(2), 419-446. https: / / doi.org / 10.18632 / aging.101168

[0089] [5] Shareef, S.J., Bevill, S.M., Raman, A.T. et al. Extended-representation bisulfite sequencing of gene regulatory elements in multiplexed samples and single cells. Nat Biotechnol 39, 1086-1094 (2021). https: / / doi.org / 10.1038 / s41587-021 -00910-x

Claims

CLAIMS1. HealthyAge platform creating method, characterized by comprising; the following method steps: i. Determination of the parameters of the physical and clinical characteristics of the individuals which will participate in the evaluation during the analysis stage and the obtaining of biological material from the individuals; ii. DNA isolation; iii. Methyl-MaxiSeq Bisulfite transformation, PCR analyzes, Library preparation; iv. Sequence alignment and methylation analysis; v. Determination of biological age by bioinformatic analysis of data and evaluation of demographic characteristics, development of the epigenomic platform; vi. SNP (Single nucleotide polymorphisms) analysis associated with metabolism; vii. Statistical analyzes and interpretation of results.

2. The method according to claim 1 , characterized in that; in process step (vi) a genetic panel comprising the rs below relating to the metabolism in SNP analysis are used: rs2071045 (LEP); rs1121980(FTO); rs22296(MC4R); rs7799039 (LEP); rs1421085(FTO); rs3751812 (FTO); rs14383 (GDF5); rs1778213(MC4R); rs1522813 (IRS1 ); rs7903146 (TCF7L2); rs964184 (APOA5); rs1815739(ACTN3); rs4880 (SOD2); rs1800012 (COL1A1); rs2228145 (IL6R); rs1141718 (SOD2); rs4295 (ACE).

3. The method according to claim 1 , characterized in that; in process step (iv) the epigenetic panel comprising the following genes is used in the methylation analysis:

4. The method according to claim 1 , characterized in that; in the personal information form in process step (i) the following information is taken into consideration Age; Gender; Height; Weight; Body mass index; Diet (Mediterranean diet, ketogenic diet) / daily calorie intake / specific dietary preference; Cigarette consumption (Less / more than 10 per day); Alcohol consumption (Less / more than two glasses at once per week); Stress (Loss of a family member, loss of employment, migration etc.); Traumatic events in the Childhood / Adolescence (e.g. exposure to poverty in childhood / childhood socioeconomic status / exposure to victimization during childhood); Viral infections (2 or less per year, more than 2 per year); Insomnia (Insomnia is present / not present); Physical Activity (3000 or less steps on average per day / 3000-7000 steps / more than 70000).

5. The method according to claim 1 , characterized in that; in process step (i) preferably Peripheric blood material preferably in a volume of 2ml is taken as biological material.

6. The method according to claim 1 , characterized in that; in process step (ii) QIAamp DNA Mini kit (cat no: 51304, 50 preps isolation Kit) protocol is followed.

7. The method according to claim 1 , characterized in that; in process step (iii) Zymo-Seq WGBS Library Kit and Zymo Research dsDNA ShearaseTM Plus Kit is used for library preparation and sequencing.

8. The method according to claim 1 , characterized in that; in process step (iv) the library is sequenced using Illumina Novaseq 6000 platform.

9. The method according to claim 1 , characterized in that; in process step (v) t test (for those that comprise 2 groups) or ANOVA (for those that comprise >2 groups) is used for the comparison of data.

10. The method according to claim 1 , characterized in that; in process step (vi)SNP analyzes are performed with optimized Taqman probes in RT-PCR device, Taqman Universal master mix and specific SNP primers unique to the Turkish population.

11. The method according to claim 1 , characterized in that; in process step (vii)GraphPad Prism 6.0 and SPSS (20.0) programs are used for statistical analyzes.

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