阅读说明：本技术 人类个体的年龄确定 (Age determination of human individuals ) 是由 马克·温尼菲尔德 伊丽莎白·伍尔泽 鲍里斯·克里斯托夫 拉尔斯·卡德雷利 于 2018-06-12 设计创作，主要内容包括：本发明涉及一种用于确定人类皮肤的生物学年龄的方法,所述方法包括：提供人类皮肤细胞,确定所述皮肤细胞的至少一条染色体的特定区域的至少两个CpG-二核苷酸的甲基化水平,以及通过将所述确定的甲基化水平与经验确定的数据进行比较来确定所述皮肤细胞的生物学年龄,所述经验确定的数据表示至少一个人类个体的CpG-核苷酸的甲基化水平和实足年龄之间的相关性。(The present invention relates to a method for determining the biological age of human skin, said method comprising: providing human skin cells, determining the methylation level of at least two CpG-dinucleotides of a specific region of at least one chromosome of said skin cells, and determining the biological age of said skin cells by comparing said determined methylation level with empirically determined data representing a correlation between the methylation level and chronological age of at least one human individual.)

1. A method for determining the biological age of human skin, the method comprising:

a) determining the methylation level of at least two CpG-dinucleotides of human skin cells; and

b) determining the biological age of said skin cells by comparing said determined methylation level to empirically determined data representing a correlation between the methylation level of said CpG-nucleotide and chronological age of at least one human individual.

2. The method according to claim 1, wherein each CpG-dinucleotide for determining the methylation level is part of a nucleotide sequence selected from the group consisting of SEQ ID NO 1 to SEQ ID NO 205.

3. The method according to any of the preceding claims, wherein the methylation level of two CpG-dinucleotides of human skin cells is determined.

4. The method according to any of the preceding claims, wherein at least one of the CpG-dinucleotides that determine the methylation level is part of a nucleotide sequence selected from the group consisting of SEQ ID NO 51, SEQ ID NO 91, SEQ ID NO 110, SEQ ID NO 179, SEQ ID NO 202, SEQ ID NO 203, SEQ ID NO 204 and SEQ ID NO 205.

5. The method according to any of the preceding claims, wherein each CpG-dinucleotide determining the methylation level is part of a nucleotide sequence selected from the group consisting of SEQ ID NO 51, SEQ ID NO 91, SEQ ID NO 110, SEQ ID NO 179, SEQ ID NO 202, SEQ ID NO 203, SEQ ID NO 204 and SEQ ID NO 205.

6. The method according to any of the preceding claims, wherein the data comprises at least one linear regression equation, preferably one equation comprising at least two linear regressions.

7. The method of any one of the preceding claims, further comprising the step of estimating the chronological age of the human subject.

8. The method of claim 7, wherein the estimated chronological age is within 5.25 years of biological age.

9. The method of claims 1 to 8, further comprising the step of calculating a value h indicative of the health of a human individual, wherein h is calculated based on a biological age and a chronological age.

10. The method of claim 9, wherein h is calculated based on the difference between the biological age and the chronological age.

11. The method of any one of the preceding claims, further comprising the step of contacting the skin cells with an active agent.

12. The method of claim 11, wherein the biological age is determined before and after contacting the skin cells with an active agent.

13. A method of testing an active agent, the method comprising the method of any one of claims 1 to 10, further comprising the steps of:

c) contacting skin cells with an active agent;

d) determining the biological age of the skin cells of step c) according to the method of any one of claims 1 to 10; and

e) comparing the biological ages determined in steps a) and b) with the biological age determined in step d).

14. The method of claim 13, wherein step c) is performed in vivo or in vitro.

15. The method of claim 13 or 14, wherein the active agent is a cosmetic and/or therapeutic agent.

16. The method according to any one of claims 13 to 15, for identifying an agent for preventing and/or treating the signs of phenotypic aging of human skin.

17. Use of a nucleic acid molecule for determining the biological age of human skin according to the method of any one of claims 1 to 10, wherein the nucleic acid molecule comprises at least one nucleotide sequence selected from the group consisting of:

a) a nucleotide sequence comprising at least one sequence selected from SEQ ID NO 1 to SEQ ID NO 205;

b) a nucleotide sequence which differs from the nucleotide sequence of a) by replacing at most 10% of the nucleotides, preferably at most 5% of the nucleotides, of the nucleotide sequence of a) other than the CpG-dinucleotide;

c) a nucleotide sequence corresponding to the complementary strand of said nucleotide sequence a) and/or b).

18. The use according to claim 17, wherein two nucleotide sequences are selected.

19. A computer-readable medium having stored computer-executable instructions for causing a computer to perform a method of determining a biological age of human skin, the method comprising:

a) inputting at least two values of the determined methylation levels of at least two CpG-dinucleotides of human skin cells;

b) comparing said value of said determined methylation level with stored data representing a correlation between methylation level of said CpG-dinucleotide and chronological age of at least one human individual; and

c) showing the biological age.

20. The computer readable medium of claim 19, wherein the stored data comprises at least one linear regression equation, preferably one equation comprising at least two linear regressions.

21. A kit for determining the biological age of human skin according to the method of any one of claims 1 to 10, comprising at least one oligonucleotide primer for amplifying and/or sequencing at least two CpG nucleotides in at least one nucleotide sequence selected from the group consisting of SEQ ID No.1 to SEQ ID No. 205.

Technical Field

The present invention relates to determining the biological age of human skin by analyzing epigenetic patterns at specific sites of DNA. The level of methylation at selected CpG sites of a skin sample is evaluated to accurately determine the biological age of human skin. The measured biological age was then correlated with chronological age with a mean absolute error of less than 5.05 years.

Background

Epigenetic changes in DNA are known to occur over time and have been recognized as an indicator of the extent of the aging process in human individuals. In particular, the degree of methylation of CpG dinucleotides has been identified as being age-related.

Methods for determining the biological age by analyzing various CpG-dinucleotides are known. For example, in Weidner et al, Genome Biology, 201415R 24, 102 age-related CpG sites in blood have been identified as being suitable for assessing the biological age of an individual. The described method uses data from 3 CpG sites to achieve a mean absolute deviation from chronological age of less than 5 years. EP 2711431 also analyzed blood cells of the subject and predicted chronological age based on 6 of the 102 identified age-related CpG sites. The prediction of biological age based on the determination of 6 CpG sites was found to differ from chronological age by about 4.53 years up to 10.30 years. In another study, Horvath, Genome Biology, 2013, 14: R115, by characterizing 353 CpG sites from blood cells, found a difference between chronological and biological ages of ± 3.6 years.

Because the various cell types found in blood have different DNA methylation patterns, the use of blood samples in analyzing epigenetic changes is not optimal. In addition, bacterial and viral infections can significantly alter cellular composition in the blood, which can alter methylation patterns.

Human skin has also been used as a model to analyze age-related epigenetic changes and to correlate with chronological age of an individual. In Bormann et al, Aging Cell, 2016, 15, 563 to 571, a protein according to Horvath, genobiology 2013, 14: known predictors of R115 were used on the human epidermis and the mean absolute prediction error was found to be 14.5 years. This indicates that the published predictors underestimate the chronological age of the epidermal samples. In Bormann et al, Aging Cell, 2016, 15, 563 to 571, another predictor was developed that utilizes methylation of 450,000 CpG dinucleotides that were simultaneously analyzed by an array-based approach. The error in biological age determined using the method therein is within 5.25 years of less than chronological age. However, this method has the disadvantage of requiring analysis of a large number of sites, which is both time consuming and cost consuming.

Accordingly, it would be desirable to provide improved and/or alternative methods for determining the biological age of an individual. Preferably, it would be desirable to provide a method using a reduced number of CpG sites.

Disclosure of Invention

The invention is defined in the appended claims.

According to a first aspect, there is provided a method for determining the biological age of human skin, the method comprising:

a) determining the methylation level of at least two CpG-dinucleotides of human skin cells; and

b) determining the biological age of said skin cells by comparing said determined methylation level to empirically determined data representing a correlation between the methylation level of said CpG-nucleotide and chronological age of at least one human individual.

According to a second aspect, there is provided a method of testing an active agent, the method comprising a method according to the first aspect, further comprising the steps of:

c) contacting skin cells with an active agent;

d) determining the biological age of the skin cells of step c) according to the method of the first aspect; and

e) comparing the biological ages determined in steps a) and b) with the biological age determined in step d).

According to a third aspect, there is provided the use of a nucleic acid molecule for determining the biological age of human skin according to the method of the first aspect, wherein the nucleic acid molecule comprises at least one nucleotide sequence selected from the group consisting of:

a) a nucleotide sequence comprising at least one sequence selected from SEQ ID NO 1 to SEQ ID NO 205;

b) a nucleotide sequence which differs from the nucleotides of a) by replacing at most 10% of the nucleotides of a) other than said CpG-dinucleotide, preferably at most 5% of the nucleotides;

c) a nucleotide sequence corresponding to the complementary strand of said nucleotide sequence a) and/or b).

According to a fourth aspect, there is provided a computer-readable medium having stored computer-executable instructions for causing a computer to perform a method of determining a biological age of human skin, the method comprising:

a) inputting at least two values of the determined methylation levels of at least two CpG-dinucleotides of human skin cells;

b) comparing said determined methylation level value to stored data representing a correlation between methylation level of said CpG-dinucleotide and chronological age of at least one human individual; and

c) showing the biological age.

According to a fifth aspect, a kit for determining the biological age of human skin according to the method of the first aspect is provided, comprising at least one oligonucleotide primer for amplifying and/or sequencing at least two CpG nucleotides selected from the nucleotide sequences of SEQ ID No.1 to SEQ ID No. 205.

Certain embodiments of the invention may provide one or more of the following advantages:

the desired ease of collecting a sample from an individual;

desired speed of obtaining the biological information data;

desired ease of obtaining biological information data;

desired cost-effective data collection;

expected age prediction accuracy.

The details, examples and preferences provided in connection with any particular one or more of the stated aspects of the invention apply equally to all aspects of the invention. Any combination of the embodiments, examples, and preferences described herein in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Drawings

The invention will be further explained with reference to the following figures:

FIG. 1 is a table of CpG sites and nucleotide sequences of SEQ ID Nos 1 to 6;

FIG. 2 is a table of CpG sites and nucleotide sequences of SEQ ID Nos. 7 to 12;

FIG. 3 is a table of CpG sites and nucleotide sequences of SEQ ID Nos. 13 to 18;

FIG. 4 is a table of CpG sites and nucleotide sequences of SEQ ID Nos. 19 to 24;

FIG. 5 is a table of CpG sites and nucleotide sequences of SEQ ID Nos. 25 to 30;

FIG. 6 is a table of CpG sites and nucleotide sequences of SEQ ID Nos. 31 to 36;

FIG. 7 is a table of CpG sites and nucleotide sequences of SEQ ID Nos 37 to 42;

FIG. 8 is a table of CpG sites and nucleotide sequences of SEQ ID Nos 43 to 48;

FIG. 9 is a table of CpG sites and nucleotide sequences of SEQ ID Nos. 49 to 54;

FIG. 10 is a table of CpG sites and nucleotide sequences of SEQ ID Nos. 55 to 60;

FIG. 11 is a table of CpG sites and nucleotide sequences of SEQ ID Nos 61 to 66;

FIG. 12 is a table of CpG sites and nucleotide sequences of SEQ ID Nos 67 to 72;

FIG. 13 is a table of CpG sites and nucleotide sequences of SEQ ID Nos. 73 to 78;

FIG. 14 is a table of CpG sites and nucleotide sequences of SEQ ID Nos. 79 to 84;

FIG. 15 is a table of CpG sites and nucleotide sequences of SEQ ID Nos. 85 to 90;

FIG. 16 is a table of CpG sites and nucleotide sequences of SEQ ID Nos 91 to 96;

FIG. 17 is a table of CpG sites and nucleotide sequences of SEQ ID Nos 97 to 102;

FIG. 18 is a table of CpG sites and nucleotide sequences of SEQ ID Nos 103 to 108;

FIG. 19 is a table of CpG sites and nucleotide sequences of SEQ ID Nos 109 to 114;

FIG. 20 is a table of CpG sites and nucleotide sequences of SEQ ID Nos. 115 to 120;

FIG. 21 is a table of CpG sites and nucleotide sequences of SEQ ID Nos. 121 to 126;

FIG. 22 is a table of CpG sites and nucleotide sequences of SEQ ID Nos 127 to 132;

FIG. 23 is a table of CpG sites and nucleotide sequences of SEQ ID Nos. 133 to 138;

FIG. 24 is a table of CpG sites and nucleotide sequences of SEQ ID Nos 139 to 144;

FIG. 25 is a table of CpG sites and nucleotide sequences of SEQ ID Nos. 145 to 150;

FIG. 26 is a table of CpG sites and nucleotide sequences of SEQ ID Nos 151 to 156;

FIG. 27 is a table of CpG sites and nucleotide sequences of SEQ ID Nos 157 to 162;

FIG. 28 is a table of CpG sites and nucleotide sequences of SEQ ID Nos. 163 to 168;

FIG. 29 is a table of CpG sites and nucleotide sequences of SEQ ID Nos 169 to 174;

FIG. 30 is a table of CpG sites and nucleotide sequences of SEQ ID Nos 175 to 180;

FIG. 31 is a table of CpG sites and nucleotide sequences of SEQ ID Nos. 181 to 186;

FIG. 32 is a table of CpG sites and nucleotide sequences of SEQ ID Nos. 187 to 192;

FIG. 33 is a table of CpG sites and nucleotide sequences of SEQ ID Nos. 193 to 198;

FIG. 34 is a table of CpG sites and nucleotide sequences of SEQ ID Nos. 199 to 204;

FIG. 35 is a table of CpG sites and nucleotide sequences of SEQ ID No. 205;

FIG. 36 represents two exemplary CpG labels: cg06335867 and cg13848598, age-dependent methylation changes;

FIG. 37 shows the correlation of chronological age with biological age using markers cg06335867 and cg13848598 using the following formula: 70.002+8.992 cg06335867+12.998 cg 13848598.

It should be understood that the following description and references to the accompanying drawings relate to exemplary embodiments of the present invention and should not limit the scope of the claims.

Detailed Description

The present invention is based on the discovery that the biological age of human skin can be determined by the methylation level of selected CpG nucleotides.

Aging (aging) is a process of aging. Age can be seen in a number of ways, such as chronological age and biological age.

As used herein, the term "chronological age" refers to the amount of time that has elapsed since birth of an individual. The term "subject" as used herein refers to a human subject.

As used herein, "biological age" refers to the extent of physical changes in an individual, such as epigenetic changes in DNA. The extent of epigenetic changes in DNA is determined in skin samples. The biological age is determined from the skin sample and correlated with the biological age of the individual. The biological age disclosed herein is determined using the method of the first aspect of the invention. In particular, the methylation level of specific CpG-dinucleotides is assessed as part of the present invention. The methylation level can be determined, for example, by: methylation specific PCR, sequence analysis of bisulfite treated DNA, CHIP sequencing (IlluminationBeadChip technology), molecular inversion Probe assay, methyl-CAP sequencing, Next Generation sequencing, COBRA assay, methylation specific restriction map, or MassARRAY assay.

In certain embodiments, the biological age of human skin is equal to the biological age of the individual.

Biological age can be affected by many parameters such as genetic background, disease, and lifestyle.

The biological age may be different from the chronological age. Biological age may provide an indication of an individual's health when compared to chronological age. If, for example, the biological age is below the chronological age, it can be concluded that, at the biological level, the signs of aging are not as significant as expected. This may be indicative of the individual's physical health. Alternatively, if the biological age is higher than the chronological age, it may be an indication that the individual is in poor health.

In certain embodiments, the human skin cells used in accordance with the methods of the present invention are obtained by collecting a desired whole skin sample from an individual. In certain embodiments, the human skin cells used according to the methods of the invention are obtained by culturing the skin cells using an in vitro method.

The collection of a sample from an individual may be performed using suction foaming, coring biopsy, scraping biopsy, or during any surgical procedure, such as plastic surgery, skin pulling, transplantation, and the like.

The skin sample may be taken from the epidermis or dermis.

Human skin cells can be cultured from a small sample of skin cells taken from an individual. The harvested human skin cells can be grown in vitro in a container such as a petri dish in a medium or substrate supplied with essential nutrients.

The human skin cells used may be a mixture of harvested cells and cultured cells.

In certain embodiments, a particular region of a chromosome that comprises a CpG-dinucleotide of the invention may be a coding region or a non-coding region. In certain embodiments, the CpG dinucleotide may be present in a coding region and/or a non-coding region. The CpG dinucleotides may be found in a single specific region or in different specific regions.

In certain embodiments, the specific region of the chromosome comprises at least one nucleotide sequence from SEQ ID No.1 to SEQ ID No.205 as shown in fig. 1 to 35. The correlation of each SEQ ID with the CpG dinucleotides of the invention is shown in FIGS. 1 to 35.

In certain embodiments, the method of the first aspect may comprise determining the methylation level of two CpG dinucleotides. The two CpG dinucleotides that determine the methylation level can be on the same nucleotide sequence, for example on SEQ ID NO.9, SEQ ID NO.13, SEQ ID NO.16, SEQ ID NO.19, SEQ ID NO.21, SEQ ID NO.25, SEQ ID NO.35, SEQ ID NO.36, SEQ ID NO.43, SEQ ID NO.45, SEQ ID NO.65, SEQ ID NO.71, SEQ ID NO.77, SEQ ID NO. 9497, SEQ ID NO.102, SEQ ID NO.111, SEQ ID NO.129, SEQ ID NO.151 and SEQ ID NO. 168. The two CpG dinucleotides may be in different dinucleotide sequences, each sequence being independently from SEQ ID No.1 to SEQ ID No. 205.

In certain embodiments, the method of the first aspect may comprise determining the methylation level of three CpG dinucleotides. In certain embodiments, the method of the first aspect may comprise determining the methylation level of four CpG dinucleotides. In certain embodiments, the method of the first aspect may comprise determining the methylation level of five CpG dinucleotides. In certain embodiments, the method of the first aspect may comprise determining the methylation levels of six CpG dinucleotides. In certain embodiments, the method of the first aspect may comprise determining the methylation level of seven CpG dinucleotides. In certain embodiments, the method of the first aspect may comprise determining the methylation level of eight CpG dinucleotides. In certain embodiments, the method of the first aspect may comprise determining the methylation level of nine CpG dinucleotides. In certain embodiments, the method of the first aspect may comprise determining the methylation level of ten CpG dinucleotides. In certain embodiments, the method of the first aspect may comprise determining the methylation level of at least two and no more than three CpG dinucleotides. In certain embodiments, the method of the first aspect may comprise determining the methylation level of at least two and no more than four CpG dinucleotides. In certain embodiments, the method of the first aspect may comprise determining the methylation level of at least two and no more than five CpG dinucleotides. In certain embodiments, the method of the first aspect may comprise determining the methylation level of at least two and no more than six CpG dinucleotides. In certain embodiments, the method of the first aspect may comprise determining the methylation level of at least two and no more than seven CpG dinucleotides. In certain embodiments, the method of the first aspect may comprise determining the methylation level of at least two and no more than eight CpG dinucleotides. In certain embodiments, the method of the first aspect may comprise determining the methylation level of at least two and no more than nine CpG dinucleotides. In certain embodiments, the method of the first aspect may comprise determining the methylation level of at least two and no more than ten CpG dinucleotides.

Certain embodiments relate to a method of estimating chronological age of a human individual, the method comprising the method of the first aspect. For example, a method of estimating chronological age of a human individual comprises: 1) determining the methylation level of at least two CpG-dinucleotides of human skin cells; 2) determining a biological age of said skin cells by comparing said determined methylation level to empirically determined data representing a correlation between methylation level and chronological age of said CpG-nucleotide in at least one human individual; and 3) estimating chronological age.

In certain embodiments, the method of the first aspect further comprises the step of estimating the chronological age of the human individual. Using the methods of the invention, the correlation between biological age and chronological age is high, wherein the difference between biological age and chronological age, i.e., the Mean Absolute Error (MAE) determined by the methods described herein, is no more than about 7 years old, no more than about 6 years old, no more than about 5.5 years old, no more than about 5 years old, no more than about 4.5 years old, no more than about 4 years old, no more than about 3.5 years old, no more than about 3 years old, no more than about 2 years old, no more than about 1.5 years old, no more than about 1 year old, no more than about 0.5 years old, no more than 0 years old.

The difference between the biological age and chronological age of the individual can be determined. The deviation of a single data point from the best fit line superimposed on all persons' data points may vary from about age 0 to about age 15. For example, the deviation of a single data point from the best fit line would be about 0 years old, about 1 year old, about 2 years old, about 3 years old, about 4 years old about 5 years old, about 6 years old, about 7 years old, about 8 years old, about 9 years old, about 10 years old, about 11 years old, about 12 years old, about 13 years old, about 14 years old, about 15 years old. This is an indication of the health of the human individual and the human skin.

In certain embodiments, the biological age of the unknown individual is determined as described above. From this biological age, the chronological age of the individual was estimated by applying the predicted MAE to the CpG data points used.

In certain embodiments, the correlation between biological age and chronological age is better than expected when using a method for determining the methylation level of two CpG dinucleotides. The correlation between biological age and chronological age can be further improved by additionally determining the methylation level in more CpG dinucleotides. Such as a total of three CpG dinucleotides, or a total of four CpG dinucleotides, or a total of five CpG dinucleotides, or a total of six CpG dinucleotides, or a total of seven CpG dinucleotides, or a total of eight CpG dinucleotides, or a total of nine CpG dinucleotides, or a total of ten CpG dinucleotides. It is noted, however, that the results of the method using the methylation levels of two CpG nucleotides are widely accepted in practice, whereas considering that the methylation level of additional CpG dinucleotides may lead to an improvement of the correlation, wherein, however, this improvement decreases with each additional considered CpG dinucleotide.

In certain embodiments, the biological age and chronological age are used to calculate a value h that is indicative of the health of the human subject and the human skin. In certain embodiments, the value h is calculated according to the following formula (I)

h biological age-chronological age (I)

Certain embodiments relate to a method of assessing the health of an individual, the method comprising the method of the first aspect and determining the value h. For example, a method of assessing the health of an individual comprises: 1) determining the methylation level of at least two CpG-dinucleotides of human skin cells; 2) determining a biological age of said skin cells by comparing said determined methylation level to empirically determined data representing a correlation between methylation level and chronological age of said CpG-nucleotide in at least one human individual; 3) estimating the age of the foot; and 4) subtracting the chronological age from the biological age to determine the value h.

Without wishing to be bound by theory, it is believed that if the value h obtained from formula (I) is positive, the human subject is less healthy. In this case, a larger h value indicates that the health of the human individual is worse than a smaller h value. If the value of h obtained from formula (I) is negative, then the individual may be assumed to be healthy.

If the value of h is positive, it may be desirable to reduce the value, thereby improving the health of the individual. The h-value can be reduced by various means, such as exercise, a healthy diet, a proper amount of sleep, drinking enough water, and/or avoiding stress.

The h value can also be reduced by applying the active agent to the skin cells in the form of a pharmaceutical and/or cosmetic agent. The active agent may contact the skin cells in vitro or in vivo. Using an in vitro method, the active agent is added to the culture medium of skin cells. Using in vivo methods, skin cells of a human subject may be contacted with an active agent using topical, subcutaneous, or intradermal administration.

As used herein, an "active agent" is any agent that has a therapeutic and/or cosmetic effect on an individual. The therapeutic effect is the treatment and/or prevention of a disease. The cosmetic effect is an improvement in appearance, for example, the treatment and/or prevention of signs of molecular aging. Signs of molecular aging are, for example, stem cell depletion, altered cell-cell communication, genomic instability, telomere abrasion, epigenetic changes, loss of protein homeostasis, dysregulation of nutrient sensing, mitochondrial dysfunction, cellular senescence and altered proliferative capacity. In certain embodiments, the active agent is a cosmetic agent. When applied to an individual, cosmetic agents may promote appeal, change appearance, beautify, and/or clean. The cosmetic agent can also prevent and/or treat signs of human skin phenotypical aging, such as wrinkle formation, pale complexion, reduced wound healing capacity, loss of elasticity and firmness.

In certain embodiments, the biological age is determined before and after contacting the skin cells with the active agent. For example, the biological age is determined, skin cells are contacted with an active agent, and then the biological age is determined. The biological age of the skin cells before and after contact with the active agent is compared. A decrease in biological age after treatment indicates a therapeutic and/or cosmetic effect of the active agent. The time between contacting the skin cells with the active agent and determining the biological age can vary. The steps of contacting skin cells with an active agent and determining the biological age can be repeated to provide information about the effect of the active agent on the skin cells over time. An advantage of this method may be a fast and cost-effective way of identifying active cosmetic and/or pharmaceutical compounds and/or extracts.

In certain embodiments, the methods of the invention may have one or more of the following effects:

an increase in accuracy in estimating chronological age from biological age;

increased reliability of the correlation between chronological age and biological age;

determining cost reduction of biological age;

reduction in the time required to determine the biological age;

an effective method of determining the effect of a pharmaceutical or cosmetic agent on biological age.

It should be noted that the present invention may include any combination of features and/or limitations mentioned herein, unless such combinations of features are mutually exclusive. The foregoing description is directed to particular embodiments of this invention for the purpose of illustrating the invention. However, it will be apparent to those skilled in the art that many modifications and variations to the embodiments described herein are possible. All such modifications and variations are intended to be within the scope of the present invention as defined in the appended claims.

For the avoidance of doubt, the present application relates to subject matter described in the following numbered paragraphs.

1. A method for determining the biological age of human skin, the method comprising:

a) providing human skin cells;

b) determining the methylation level of at least two CpG-dinucleotides of a specific region of at least one chromosome of said skin cell; and

c) determining the biological age of said skin cells by comparing said determined methylation level to empirically determined data representing a correlation between the methylation level of said CpG-nucleotide and chronological age of at least one human individual.

2. The method of paragraph 1, wherein the skin cells are collected from a human subject.

3. The method of paragraph 1, wherein the skin cells are cultured in vitro.

4. The method of any preceding numbered paragraph, wherein methylation of two CpG-dinucleotides of a particular region of one or both chromosomes of the human skin cell is determined.

5. The method of any one of the preceding paragraphs, wherein each specific region of the chromosome comprises at least one nucleotide sequence selected from SEQ ID No.1 to SEQ ID No. 205.

6. The method of any preceding paragraph, wherein one or both of the nucleotide sequences is selected from SEQ ID NO 51 and SEQ ID NO 110.

7. The method of any preceding paragraph, wherein at least one of the nucleotide sequences is selected from the group consisting of SEQ ID NO 91, SEQ ID NO 179, SEQ ID NO 202, SEQ ID NO 203; 204 in SEQ ID NO; and SEQ ID NO 205.

8. The method of any one of the preceding paragraphs, wherein the data comprises at least one linear regression equation, preferably one equation comprising at least two linear regressions.

9. The method of any preceding paragraph, further comprising the step of estimating the chronological age of the human subject.

10. The method of paragraph 9, wherein the estimated chronological age is within 5.25 years of biological age.

11. The method of any preceding paragraph, further comprising the step of calculating a value h indicative of the health of the human individual, wherein h is calculated based on the biological age and the chronological age.

12. The method of paragraph 11, wherein h is calculated based on the difference between the biological age and the chronological age.

13. The method of any one of the preceding claims, further comprising the step of contacting the skin cells with an active agent.

14. The method of paragraph 13, wherein the biological age is determined before and after contacting the skin cells with the active agent.

15. A method of testing for an active agent, the method comprising the method of any one of paragraphs 1 to 10, further comprising the steps of:

d) contacting the skin cells of step a) with an active agent;

e) determining the biological age of the skin cells of step d) according to the method of any one of paragraphs 1 to 10; and

f) comparing the biological ages determined in steps a) and c) with the biological age determined in step e).

16. The method of paragraph 15, wherein step d) is performed in vivo or in vitro.

17. The method of paragraphs 15 or 16 wherein the active agent is a cosmetic and/or therapeutic agent.

18. The method of any of paragraphs 15 to 17, for identifying an agent that prevents and/or treats signs of phenotypic aging of human skin.

19. Use of a nucleic acid molecule for determining the biological age of human skin according to the method of any of paragraphs 1 to 18, wherein the nucleic acid molecule comprises at least one nucleotide sequence selected from the group consisting of:

d) a nucleotide sequence comprising at least one sequence selected from SEQ ID NO 1 to SEQ ID NO 205;

e) a nucleotide sequence which differs from the nucleotide sequence of a) by replacing at most 10% of the nucleotides, preferably at most 5% of the nucleotides, other than the CpG-dinucleotide, in the nucleotide sequence of a);

f) a nucleotide sequence corresponding to the complementary strand of said nucleotide sequence a) or b).

20. The use of paragraph 19, wherein two nucleotide sequences are selected.

21. A computer-readable medium having stored computer-executable instructions for causing a computer to perform a method of determining a biological age of human skin, the method comprising:

d) inputting at least two values and no more than 10 values of the determined methylation levels of at least two and no more than 10 CpG-dinucleotides for a specific region of at least one chromosome of said skin cell;

e) comparing said determined methylation level value to stored data representing a correlation between methylation level of said CpG-dinucleotide and chronological age of at least one human individual; and

f) showing the biological age.

22. A computer readable medium according to paragraph 21, wherein the stored data comprises at least one linear regression equation, preferably one equation comprising at least two linear regressions.

23. A kit for determining the biological age of human skin according to the method of any of paragraphs 1 to 10, comprising at least one oligonucleotide primer for amplifying and/or sequencing at least two CpG nucleotides in at least one nucleotide sequence selected from the group consisting of SEQ ID NO:1 to SEQ ID NO: 205.