METHODS FOR PREDICTING AGE AND IDENTIFYING AGENTS THAT INDUCE OR INHIBIT PREMATURE AGING

• US 20150259742A1
• Filed: 11/12/2013
• Published: 09/17/2015
• Est. Priority Date: 11/09/2012
• Status: Active Grant

• Current Assignee: Sage Bionetworks, Regents of the University of California (University of California)
• Original Assignee: Sage Bionetworks, Regents of the University of California (University of California)
• Inventors: Ideker, Trey, Friend, Stephen H., Zhang, Kang, Hannum, Gregory, Guinney, Justin
• Granted Patent: US 10,087,486 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/6881 for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2600/118 Prognosis of disease development
• CPC Class Codes: C12Q 2600/136 Screening for pharmacological compounds
• CPC Class Codes: C12Q 2600/154 Methylation markers
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays
• CPC Class Codes: G01N 2500/00 Screening for compounds of potential therapeutic value
• CPC Class Codes: G01N 33/5308 for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
• CPC Class Codes: G01N 33/57484 involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites

Abstract

The invention provides for methods for predicting age of a subject based on the epigenome of the subject.

Claims

1. A method for predicting age of a subject based on the epigenome of the subject comprising:

(a) obtaining a biological sample of the subject;

(b) determining the methylation status of a set of age-associated epigenetic marker(s) in the epigenome of the subject selected from FIG. 9, Tables S3, S4 and/or S5; and

(c) comparing the methylation status of a set of age-associated epigenetic marker(s) of the subject with the methylation status of the same markers from an age correlated reference population so as to obtain a value or a range of values for the predicted age of the subject,

thereby predicting the age of a subject based on the epigenome of the subject.

2. The method of claim 1, wherein in (b), determining the methylation status comprises the following steps:

(a) isolating genomic DNA or nuclear DNA from the sample;

(b) reacting the isolated genomic DNA or nuclear DNA with one or more probes which differentially reacts with unmodified cytosine so that the cytosine is converted to uracil;

(c) analyzing the methylation status at the C position of a CpG dinucleotide in the isolated genomic DNA or nuclear DNA of the sample by detecting the presence of a cytosine or uracil, the presence of cytosine or uracil indicating presence of a 5-methylcytosine or unmodified cytosine, respectively, in the original CpG dinucleotide; and

(d) determining proportion of 5-methylcytosine or unmodified cytosine initially present at each age-associated epigenetic marker from (c);

or alternatively, determining the ratio of 5-methyl-cytosine to unmodified cytosine or the ratio of unmodified cytosine to 5-methyl-cytosine initially present at each age-associated epigenetic marker from (c).

5. The method of claim 1, wherein in (b), the methylation status is determined from five or more age-associated epigenetic marker(s) in the epigenome of the subject from FIG. 9 or Table S3;

ten or more age-associated epigenetic marker(s) in the epigenome of the subject from FIG. 9 or Table S3;

fifteen or more age-associated epigenetic marker(s) in the epigenome of the subject from FIG. 9 or Table S3;

twenty or more age-associated epigenetic marker(s) in the epigenome of the subject from FIG. 9 or Table S3;

twenty-five or more age-associated epigenetic marker(s) in the epigenome of the subject from FIG. 9 or Table S3;

thirty or more age-associated epigenetic marker(s) in the epigenome of the subject from FIG. 9 or Table S3;

thirty-five or more age-associated epigenetic marker(s) in the epigenome of the subject from FIG. 9 or Table S3;

forty or more age-associated epigenetic marker(s) in the epigenome of the subject from FIG. 9 or Table S3;

forty-five or more age-associated epigenetic marker(s) in the epigenome of the subject from FIG. 9 or Table S3;

fifty or more age-associated epigenetic marker(s) in the epigenome of the subject from FIG. 9 or Table S3;

fifty-five or more age-associated epigenetic marker(s) in the epigenome of the subject from FIG. 9 or Table S3;

sixty or more age-associated epigenetic marker(s) in the epigenome of the subject from FIG. 9 or Table S3;

sixty-five or more age-associated epigenetic marker(s) in the epigenome of the subject from FIG. 9 or Table S3;

or seventy or more age-associated epigenetic marker(s) in the epigenome of the subject from FIG. 9 or Table S3.

6. The method of claim 1, wherein the age of a subject is a chronological age or a biological age.

7. The method of claim 1, wherein the epigenome is deoxyribonucleic acid (DNA) in which the DNA is subjected to epigenetic modification.

8. The method of claim 7, wherein epigenetic modification is the methylation of CpG residues.

10. The method of claim 1, wherein the biological sample is blood, lymphocyte, monocyte, neutrophil, basophil, eosinophil, myeloid lineage cell, lymphoid lineage cell, bone marrow, saliva, buccal swab, nasal swab, urine, fecal material, hair, breast tissue, ovarian tissue, uterine tissue, cervical tissue, prostate tissue, testicular tissue, brain tissue, neuronal cell, astrocyte, liver tissue, kidney, thyroid tissue, stomach tissue, intestine tissue, pancreatic tissue, vascular tissue, skin, lung tissue, bone tissue, cartilage, ligament, tendon, fat cells, muscle cells, neurons, astrocytes, cultured cells with different passage number, cancer/tumor cells, cancer/tumor tissue, normal cells, normal tissue, any tissue(s) or cell(s) with a nucleus containing genetic material, or genetic material in the form of DNA.

13. The method of claim 1, wherein the set of age-associated epigenetic marker(s) comprises individual CpG residues subject to age-dependent methylation at C-5 position of cytosine in the genome of a subject.

14. The method of claim 13, wherein the set of markers comprise about 70 distinct CpG residue-containing age-associated epigenetic markers.

15. The method of claim 13, wherein the set of markers comprises any one or more of markers as shown in FIG. 9, Table S3, Table S4 or Table S5.

16. The method of claim 15, wherein the set of markers comprises five or more age-associated epigenetic marker(s) as shown in FIG. 9 or Table S3;

ten or more age-associated epigenetic marker(s) as shown in FIG. 9 or Table S3;

fifteen or more age-associated epigenetic marker(s) as shown in FIG. 9 or Table S3;

twenty or more age-associated epigenetic marker(s) as shown in FIG. 9 or Table S3;

twenty-five or more age-associated epigenetic marker(s) as shown in FIG. 9 or Table S3;

thirty or more age-associated epigenetic marker(s) as shown in FIG. 9 or Table S3;

thirty-five or more age-associated epigenetic marker(s) as shown in FIG. 9 or Table S3;

forty or more age-associated epigenetic marker(s) as shown in FIG. 9 or Table S3;

forty-five or more age-associated epigenetic marker(s) as shown in FIG. 9 or Table S3;

fifty or more age-associated epigenetic marker(s) as shown in FIG. 9 or Table S3;

fifty-five or more age-associated epigenetic marker(s) as shown in FIG. 9 or Table S3;

sixty or more age-associated epigenetic marker(s) as shown in FIG. 9 or Table S3;

sixty-five or more age-associated epigenetic marker(s) as shown in FIG. 9 or Table S3;

or seventy or more age-associated epigenetic marker(s) as shown in FIG. 9 or Table S3.

19. The method of claim 13, wherein the set of markers having individual CpG residues subject to methylation of C-5 position of cytosine in the genome of a subject comprises any one or more of methylation marker PENK of Table S3, FLH2 of Table S3, cg05652533 of Table S4, cg27367526 of Table S4, cg18404041 of Table S4, cg23606718 of FIG. 9, Tables S3 and S5, cg16867657 of FIG. 9, Tables S3 and S5, cg04474832 on chromosome 3 at position 52008487, cg05442902 on chromosome 22 at position 21369010, cg06493994 on chromosome 6 at position 25652602, cg09809672 on chromosome 1 at position 236557682, cg19722847 on chromosome 12 at position 30849114, cg22736354 on chromosome 6 at position 18122719, cg05652533 of Table S4, cg27367526 of Table S4, cg18404041 of Table S4, cg23606718 on chromosome 2 at position 131513927, and/or cg16867657 of chromosome 6 at position 11044877.

21. The method of claim 13, wherein the set of markers having individual CpG residues subject to methylation at C-5 position of cytosine in the genome of a subject comprises PENK of Table S3 and FLH2 of Table S3.

23. A method for diagnosing the presence of tumor in a subject comprising:

(a) obtaining biological sample suspected to contain tumor and a second biological sample of the same type but known not to contain tumor;

(b) predicting age of the each biological sample by the method of claim 1;

(c) comparing the ages predicted for the two samples, such that a biological sample with tumor will have an older predicted age than biological sample without tumor;

thereby diagnosing the presence of tumor in a subject.

25. The method of claim 23, wherein the set of age-associated epigenetic marker(s) in the epigenome of the subject comprises any of methylation marker PENK of Table S3, FLH2 of Table S3, cg23606718 of FIG. 9, Tables S3 and S5 and/or cg16867657 of FIG. 9, Tables S3 and S5.

35. The method of claim 2, wherein the ratio of unmodified cytosine to 5-methylcytosine initially present at each age-associated epigenetic marker is the ratio of thymidine to cytosine at pyrimidine position of the CpG dinucleotide after exposure to one or more probe and following analysis of products of nucleic acid amplification.

43. The method of claim 1, wherein in (b), determining the methylation status comprises the following steps:

(a) isolating genomic DNA or nuclear DNA from the sample;

(b) probing the isolated genomic DNA or nuclear DNA with one or more probes which differentially reacts with unmodified and 5-methyl-modified cytosine;

(c) amplifying the DNA of step (b);

(d) digesting the amplified DNA of step (c) with one or more restriction enzyme that recognizes a restriction enzyme site that contains a CpG dinucleotide but fails to digest the restriction enzyme site mutated to TpG dinucleotide from a CpG dinucleotide;

(e) determining the proportion of 5-methylcytosine or unmodified cytosine initially present at each age-associated epigenetic marker based on the fraction or percentage of restriction enzyme sites sensitive or resistant to digestion in step (d), respectively;

or,

alternatively, determining the ratio of 5-methylcytosine to unmodified cytosine initially present at each age-associated epigenetic marker based on the ratio of number or concentration of sensitive restriction enzyme sites to number or concentration of resistant restriction enzyme sites in step (d);

or

alternatively, determining the ratio of unmodified cytosine to 5-methylcytosine initially present at each age-associated epigenetic marker based on the ratio of number or concentration of resistant restriction enzyme sites to number or concentration of sensitive restriction enzyme sites to digestion in step (d).

44. The method of claim 1, wherein in (b), determining the methylation status of the set of age-associated epigenetic marker(s) in the epigenome of the subject selected from FIG. 9, Tables S3, S4, and/or S5 comprises the following steps:

(a) isolating genomic DNA or nuclear DNA;

(b) fragmenting the genomic DNA or nuclear DNA;

(c) exposing the fragmented DNA of step (b) to a 5-methylcytosine-binding protein;

(d) separating 5-methylcytosine-binding protein-bound DNA fragments from 5-methylcytosine-binding protein-free DNA fragments of step (c);

(e) determining for each age-associated epigenetic marker, the proportion of 5-methyl-cytosine-containing DNA fragments or unmodified cytosine-containing DNA fragments by determining the fraction or percent of 5-methylcytosine-binding protein bound or free DNA fragments, respectively, for each age-associated epigenetic marker;

or

alternatively, determining for each age-associated epigenetic marker, the ratio of 5-methylcytosine-containing DNA fragments to unmodified cytosine-containing DNA fragments by determining the ratio of number or concentration of 5-methylcytosine-binding protein-bound DNA fragments to the number or concentration of 5-methylcytosine-binding protein-free DNA fragments;

or

alternatively, determining for each age-associated epigenetic marker, the ratio of unmodified cytosine-containing DNA fragments to 5-methylcytosine-containing DNA fragments by determining the ratio of number or concentration of 5-methylcytosine-binding protein-free DNA fragments to the number or concentration of 5-methylcytosine-binding protein-bound DNA fragments for each age-associated epigenetic marker,

thereby determining the methylation status.

51. A method for screening whether an agent of interest can retard or accelerate aging process comprising:

(a) obtaining a biological sample from a living organism, and optionally, culturing cells, tissue, or organ derived from a living organism;

(b) predicting age or AMAR of the organism, following the method of claim 1 or 46, using organism appropriate age-associated epigenetic marker(s) such that the age-associated epigenetic marker(s) provided in claim 1 for a human subject may need to be substituted with age-associated epigenetic marker(s) for the organism being examined;

(c) exposing the living organism or cultured living cells, tissue, or organ from the living organism of step (a) to an agent of interest in a single dose, multiple doses, or continuous doses;

(d) obtaining a biological sample from the living organism or the cultured living cells, tissue, or organ of step (c);

(e) predicting age or AMAR of the organism from the biological sample of step (d), following the method of claim 1 or 47, using organism appropriate age-associated epigenetic marker(s) such that the age-associated epigenetic marker(s) provided in claim 1 for a human subject may need to be substituted with age-associated epigenetic marker(s) for the organism being examined;

(f) performing the same steps (a)-(e) on another individual from the same organism or a duplicate cultured living cells, tissue, or organ from the same individual or organism but not treated with any agent of interest or treated with a placebo at step (c); and

g) comparing, for biological sample of the same predicted age or AMAR in step (b), the predicted age or AMAR from step (e) with the predicted age or AMAR from step (f), such that a lower value or range of values for step (e) indicates that an agent of interest can retard an aging process whereas a higher value or range of values indicates an agent of interest can accelerate aging process,

thereby screening whether an agent of interest can retard or accelerate aging process.

38. The method of claim 3, wherein analyzing the methylation status in step (c) comprises DNA amplification and analysis of age-associated epigenetic marker (a) for specific DNA end(s) or fragment(s) due to cleavage by the restriction enzyme(s) and (b) for intact restriction enzyme cleavage site associated at a particular age-associated epigenetic marker.