Mammalian DNA methylation measurements based on conserved loci

Abstract

To provide methods and materials useful for observing methylation and phenomena associated with methylation (e.g., aging) across a wide variety of mammalian species.SOLUTION: Provided are methods of observing the effects of a test agent on genomic methylation associated epigenetic aging of mammalian cells (e.g. human primary keratinocytes). Typically these methods comprise the steps of: combining the test agent with mammalian cells; observing methylation status of methylation markers in genomic DNA from the mammalian cells using a DNA methylation array of disclosed herein; and then comparing these observations with observations of the methylation status in genomic DNA from control mammalian cells not exposed to the test agent such that effects of the test agent on genomic methylation associated epigenetic aging in the mammalian cells are observed.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. §119(e) to co-pending and commonly owned U.S. Provisional Patent Application No. 62 / 794,364, filed Jan. 18, 2019, and entitled "DNA METHYLATION MEASUREMENT FOR MAMMALS BASED ON CONSERVED LOCI," which is incorporated herein by reference.

[0002] STATEMENT OF GOVERNMENT RIGHTS This invention was made with Government support under Grant No. 1254200 awarded by the National Science Foundation. The Government has certain rights in this invention.

[0003] Technical Field The present invention relates to methods and materials for investigating genomic DNA methylation in mammals. [Background technology]

[0004] 2. Background of the Invention DNA methylation, the addition of methyl groups to cytosines, is one of the most widely studied epigenetic modifications due to its involvement in regulating gene expression across many biological processes (1, 2). In humans, DNA methylation levels can be used to accurately predict an individual's age, as well as age across tissues and cell types (3).

[0005] The two most widely used techniques to obtain DNA methylation levels are bisulfite sequencing and microarray-based methylation chips. Whole-genome bisulfite sequencing is a costly assay, making reduced representation bisulfite sequencing (RRBS) the commonly performed sequencing approach. RRBS effectively queries only a few nucleotides on the genome, but still provides a genome-wide methylation profile. However, the sequencing depth required even for RRBS can still drive up costs. Due to this, for human samples, array chips containing an increasing number of polynucleotide probes remain the most reliable and widely used technique (4-6). The first human methylation chip (ILLUMINA Infinium 27K) was introduced more than a decade ago. However, similar chips have not been presented for other non-human mammalian species, which may reflect the fact that it is not economical to design conventional methylation chips for non-human mammals, and progress has been delayed. For example, the development and use of conventional species-specific methylation chips / arrays may hinder inter-species comparisons due to different measurement platforms. In light of this, conventional species-specific methylation chips may be suboptimal for inter-species comparisons. As a result, there is a need for methods and materials useful for observing methylation and methylation-associated phenomena (e.g., aging) across a wide variety of mammalian species. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Bernstein, BE, Meissner, A. & Lander, ES The Mammalian Epigenome. Cell 128, 669-681 (2007). [Non-Patent Document 2] Smith, ZD & Meissner, A. DNA methylation: roles in mammalian development. Nature Reviews Genetics 14, 204-220 (2013). [Non-Patent Document 3] Horvath, S. DNA methylation age of human tissues and cell types. Genome Biol. 14, R115 (2013). [Non-Patent Document 4] Genome-wide DNA methylation profiling using Infinium(R) assay | Epigenomics. Available at: https: / / www.futuremedicine.com / doi / abs / 10.2217 / epi.09.14. (Accessed: 28th August 2018) [Non-Patent Document 5] Evaluation of the Infinium Methylation 450K technology. - PubMed - NCBI. Available at: https: / / www.ncbi.nlm.nih.gov / pubmed / 22126295. (Accessed: 28th August 2018) [Non-Patent Document 6] Pidsley, R. et al. Critical evaluation of the ILLUMINA MethylationEPIC BeadChip microarray for whole-genome DNA methylation profiling. Genome Biology 17, 208 (2016). Summary of the Invention [Means for solving the problem]

[0007] Summary of the Invention

[0008] Valuable information can be obtained from the study of methylation patterns in mammalian species other than those that are the representative centers of scientific research (e.g., humans and mice). However, a problem in such studies is the fact that it is technically challenging and expensive to develop methods and materials designed to observe methylation profiles in rarely studied species (e.g., naked mole rats and killer whales). In this context, a single measurement platform useful for studying all mammalian species would provide a solution that would make such endeavors much more efficient and cost-effective. The invention disclosed herein provides this platform in the form of methods and materials that can be used to observe methylation and phenomena associated with methylation in a wide variety of mammalian species. As discussed below, one advantageous aspect of the present invention is the identification and utilization of highly conserved segments of DNA containing CpG methylation sites in the human genome, i.e., segments of the human genome that facilitate interspecies comparisons.

[0009] The invention disclosed herein has many embodiments. One embodiment of the invention is an algorithm called "Conserved Methylation Array Probe Selector" (CMAPS). This algorithm is used to identify DNA sequences useful in embodiments of the invention, such as DNA methylation arrays / chips, by reusing traditional degenerate base techniques used to tolerate intra-human variation in a manner that allows polynucleotides to tolerate inter-species variation. In an embodiment of the invention, the CMAPS algorithm performs a comprehensive sequence search to obtain the maximum number of species that can be targeted using a single probe for CpGs in the human genome based on multiple sequence alignment. The CMAPS algorithm then ranks all sequences / probes and selects a final set such that such sequences can be used to query multiple mammalian species at various genomic locations based on external annotation of exons, CpG islands, and hyper- versus hypo-methylated regions.

[0010] For example, the CMAPS algorithm may be used to facilitate the design of embodiments of the invention, including DNA methylation arrays (e.g., arrays of polynucleotides arranged in a matrix such as a bead or chip). One such embodiment of the invention is a DNA methylation array comprising a plurality of polynucleotides linked to a matrix, wherein the plurality of polynucleotides are selected by: (a) performing polynucleotide sequence alignment to compare the human genome with a plurality of non-human mammalian genomes to identify polynucleotide sequences in the human genome that contain CpG methylation sites that are homologous to polynucleotide sequences in the genome of the non-human mammalian species that contain CpG methylation sites; (b) ranking the polynucleotide sequences in the human genome identified in (a), wherein the ranking criteria include sequence homology to polynucleotide sequences in the genome of the non-human mammalian species, and then (c) using the ranking in (b) to select a plurality of polynucleotides in the human genome that cross-hybridize to a plurality of polynucleotide sequences in the genome of the non-human mammalian species. Other illustrative ranking criteria may include, for example, identifying those CpG-containing polynucleotide sequences that function in the greatest number of different mammalian species; and / or identifying those CpG-containing polynucleotide sequences that have been characterized as significant in other epigenetic biomarker studies (e.g., human aging studies).

[0011] Typically, in these embodiments of the invention, the plurality of human genomic polynucleotide sequences are selected to have no more than 3 base pair mismatches with polynucleotide sequences in the genome of the non-human mammalian species. Optionally, ranking the sequence alignments includes comparing the human genomic sequence with genomic sequences of at least 10, 20, 30, 40 or more non-human mammalian species, and / or comparing the human genomic polynucleotide sequence with genomic polynucleotide sequences in evolutionarily distinct species, such as non-placental and placental mammalian species. In certain embodiments of the invention, the DNA methylation chip comprises at least 10,000, 20,000 or 30,000 unique polynucleotides linked to the matrix. Typically, the polypeptides comprise about 60 nucleotides (e.g., 40-80 nucleotides) that are at least about 95% identical to a DNA segment of the non-human mammalian genome that contains a CpG methylation site (e.g., where 57 nucleotides of the 60 nucleotides of the non-human mammalian genome are identical to a 60 nucleotide DNA segment of the human genome). In certain illustrative working embodiments of the invention disclosed herein, at least one polynucleotide in the plurality of polynucleotides is a polynucleotide having a sequence as set forth in Table 3.

[0012] A related embodiment of the invention is a DNA methylation array comprising a plurality of polynucleotide sequences linked to a matrix, wherein the polynucleotides comprise CpG motifs (or their complements) at their termini. These polynucleotides typically comprise a sequence of about 60 nucleotides that exhibits about 95% homology (e.g., 57 of 60 nucleotides) between the human genomic sequence and the genomic sequence of a non-human mammalian species. In certain embodiments of the invention, at least 2,000 polynucleotides within said plurality of polynucleotide sequences are capable of hybridizing to a 60 nucleotide segment in a genomic polynucleotide sequence of a marsupial mammal species (e.g., a wallaby species) with less than three base pair mismatches; and / or at least 2,000 polynucleotides within said plurality of polynucleotide sequences are capable of hybridizing to a 60 nucleotide segment in a genomic polynucleotide sequence of a monotreme mammal species (e.g., a platypus) with less than three base pair mismatches; and / or at least 2,000 polynucleotides within said plurality of polynucleotide sequences are capable of hybridizing to a 60 nucleotide segment in a genomic polynucleotide sequence of a laurasia mammal species (e.g., a bat species) with less than three base pair mismatches. and / or at least 2,000 polynucleotides within said plurality of polynucleotide sequences may hybridize with less than three base pair mismatches to a 60 nucleotide segment in a genomic polynucleotide sequence of a mammalian species of the superorder Eucaryoptera (e.g., a Rodent species); and / or at least 2,000 polynucleotides within said plurality of polynucleotide sequences may hybridize with less than three base pair mismatches to a 60 nucleotide segment in a genomic polynucleotide sequence of a mammalian species of the superorder Xenarthra (e.g., an Armadillo species); and / or at least 2,000 polynucleotides within said plurality of polynucleotide sequences may hybridize with less than three base pair mismatches to a 60 nucleotide segment in a genomic polynucleotide sequence of a mammalian species of the superorder Afrothea (e.g., a Tenrec species).

[0013] Another embodiment of the present invention is a method of making a DNA methylation array, comprising linking a plurality of polynucleotides to a matrix. Typically, in such an embodiment of the present invention, each of the plurality of polynucleotides is a polynucleotide sequence of about 60 nucleotides that contains a CpG motif (or its complement) and shows about 95% homology (e.g., 57 of 60 nucleotides) between the human genome sequence and the non-human mammalian species. In a representative embodiment of the present invention, the DNA methylation array is designed such that the array contains at least 2,000 unique polynucleotide sequences that hybridize with less than 3 base pair mismatches to 60 nucleotide segments in the genome polynucleotide sequences of non-placental and placental mammalian species. Typically, the plurality of polypeptides used to generate a DNA methylation array are selected by: (i) performing a polynucleotide sequence alignment to compare the human genome with the plurality of non-human mammalian genomes to identify polynucleotide sequences in the human genome comprising CpG methylation sites that are homologous to polynucleotide sequences comprising CpG methylation sites in the genome of the non-human mammalian species; (ii) ranking the polynucleotide sequences in the human genome identified in (a), wherein the ranking criteria comprises sequence homology to polynucleotide sequences in the genome of the non-human mammalian species; and then (iii) using the ranking in (b) to select a plurality of polynucleotides having CpG methylation sites that cross-hybridize with no more than 2, 3 or 4 base pair mismatches to the plurality of polynucleotide sequences having CpG methylation sites in the genome of the non-human mammalian species, thereby generating the DNA methylation array.

[0014] Yet another embodiment of the present invention is a method for observing a methylation profile in a non-human mammal, comprising obtaining genomic DNA from the non-human mammal; and then observing the cytosine methylation of a plurality of CG loci in the genomic DNA using the DNA methylation array disclosed herein, thereby observing a methylation profile in the non-human mammal. Optionally, the method comprises comparing the observed CG loci methylation profile with CG loci methylation observed in genomic DNA derived from an individual of a non-human mammal species having a known age; and then correlating the observed CG loci methylation with the known age of the non-human mammal species, thereby obtaining information useful for determining the age of the non-human mammal. In a representative embodiment of the present invention, the DNA methylation array is used to observe the methylation profile in a plurality of non-human mammal species. Importantly, embodiments of the present invention further enable one of skill in the art to assess whether an intervention (e.g., exposure to a test agent) that affects DNA methylation levels in one species (e.g., mouse) also affects the corresponding DNA methylation levels in another species (e.g., human). Furthermore, the conserved sequences further enable one of skill in the art to develop epigenetic age estimators (epigenetic clocks) for different mammalian species based on highly conserved CpGs.

[0015] As discussed below, a working embodiment of the invention disclosed herein is referred to as "HorvathMammalMethylChip40" and is a DNA methylation array arranged on a chip that contains approximately 38k unique human genomic polynucleotides linked to a matrix as probes of complementary sequences. Among them, 36,000 polynucleotide probes interrogate CpG sites in conserved regions of the mammalian genome, making this embodiment of the invention useful in studies across mammalian species. In this embodiment of the invention, the remaining 2,000 probes were selected due to their particular importance in human epigenetic biomarker studies. As shown by the data presented in Table 2 below, the resulting DNA methylation chip is applicable to all mammals, thus lowering the cost per chip through economies of scale. Moreover, this chip embodiment is specifically tailored for interspecies comparisons.

[0016] For example, certain embodiments provide the following: (Item 1) 1. A method for making a DNA methylation array comprising a plurality of polynucleotides linked to a matrix, the plurality of polynucleotides being selected from the group consisting of the following steps: (a) performing a polynucleotide sequence alignment comprising comparing a human genome with a plurality of non-human mammalian genomes to identify polynucleotide sequences in the human genome that contain CpG methylation sites that are homologous to polynucleotide sequences in the genome of a non-human mammalian species that contain CpG methylation sites; (b) ranking the polynucleotide sequences in the human genome identified in (a), wherein the ranking criteria comprises sequence homology to polynucleotide sequences in the genome of a non-human mammalian species; and (c) using the ranking in (b) to select a plurality of polynucleotides in the human genome that cross-hybridize to a plurality of polynucleotide sequences in the genome of the non-human mammalian species; and (d) ligating the selected sequences from step (c) to a matrix to form a DNA methylation array. The method is selected by the method comprising: (Item 2) 2. The method of claim 1, wherein the plurality of human genomic polynucleotide sequences are selected to have no more than three base pair mismatches with a polynucleotide sequence in the genome of a non-human mammalian species. (Item 3) 3. The method of claim 2, wherein the ranking comprises a homology comparison to genomic polynucleotide sequences in non-placental mammal species and placental mammal species in the supergroups of Laurasian, Eucernotrichia, Xenarthria and Afrotheria. (Item 4) 4. The method of claim 3, wherein the sequence alignment compares the human genome sequence with the genome sequences of at least 10 non-human mammalian species. (Item 5) 2. The method of claim 1, wherein the DNA methylation array comprises at least 30,000 unique polynucleotides linked to the matrix. (Item 6) 6. The method of claim 5, wherein the plurality of unique polynucleotides are between 40 and 80 nucleotides in length. (Item 7) 2. The method according to claim 1, wherein the matrix is ​​a bead or a chip. (Item 8) 1. A method for making a DNA methylation array comprising a plurality of polynucleotides linked to a matrix, wherein the plurality of polynucleotides comprises: (a) below; CpG motifs at least 2,000 unique polynucleotide sequences that hybridize with less than three base pair mismatches to a 60 nucleotide segment in a genomic polynucleotide sequence of a marsupial mammal species, a monotreme mammal species, a Laurasian mammal species, a Eucoptera mammal species, a Xenarthra mammal species, and an Afrotherian mammal species; Including; (b) Below: (i) performing a polynucleotide sequence alignment to compare the human genome with a plurality of non-human mammalian genomes to identify polynucleotide sequences in the human genome that contain CpG methylation sites that are homologous to polynucleotide sequences that contain CpG methylation sites in the genome of the non-human mammalian species; (ii) ranking the polynucleotide sequences in the human genome identified in (a), wherein the ranking criteria comprises the degree of sequence homology to polynucleotide sequences in the genome of a non-human mammalian species; and (iii) using the ranking in (ii) to select a plurality of polynucleotides having CpG methylation sites that cross-hybridize with no more than three base pair mismatches to a plurality of polynucleotide sequences having CpG methylation sites in the genome of the non-human mammalian species; Selected by; and (c) ligating the selected sequences from step (b) to a matrix to form a DNA methylation array; As a result, the DNA methylation array is produced. method. (Item 9) A DNA methylation array produced by the method according to any one of items 1 to 8. (Item 10) 1. A DNA methylation array comprising a plurality of polynucleotide sequences linked to a matrix, wherein: said polynucleotides contain at least 40 nucleotides and CpG motifs at their termini; The polynucleotide comprises a polynucleotide sequence present in the human genome; and at least 2,000 polynucleotides within said plurality of polynucleotide sequences are capable of hybridizing with less than three base pair mismatches to a 40 nucleotide segment in a genomic polynucleotide sequence of a marsupial mammal species; at least 2,000 polynucleotides within said plurality of polynucleotide sequences are capable of hybridizing with less than three base pair mismatches to a 40 nucleotide segment in a genomic polynucleotide sequence of a monotreme mammalian species; at least 2,000 polynucleotides within said plurality of polynucleotide sequences are capable of hybridizing with less than three base pair mismatches to a 40 nucleotide segment in a genomic polynucleotide sequence of a mammalian species of the superorder Laurasiana; at least 2,000 polynucleotides within said plurality of polynucleotide sequences are capable of hybridizing with less than three base pair mismatches to a 40 nucleotide segment in a genomic polynucleotide sequence of a mammalian species of the superorder Eucoptera; At least 2,000 polynucleotides in the plurality of polynucleotide sequences are capable of hybridizing to a 40 nucleotide segment in a genomic polynucleotide sequence of a mammalian species of the superorder Xenarthra with less than three base pair mismatches; and At least 2,000 polynucleotides in the plurality of polynucleotide sequences are capable of hybridizing with less than three base pair mismatches to a 40 nucleotide segment in a genomic polynucleotide sequence of a mammalian species of the superorder Afro-Therian. DNA methylation arrays. (Item 11) said marsupial mammal species is a wallaby species; and / or said monotreme mammalian species being a platypus species; and / or said Laurasian mammal species is a bat species; and / or said mammalian species of the superorder Eucteroptera is a rodent species; and / or said Xenarthra mammal species is an Armadillo species; and / or The Afro-Therian mammal species is a Tenrec species. 11. The DNA methylation array according to item 10. (Item 12) 12. The DNA methylation array according to any one of items 9 to 11, wherein at least one polynucleotide in the plurality of polynucleotides is a polynucleotide having a sequence shown in Table 1. (Item 13) 1. A method for observing a methylation profile in a non-human mammal, the method comprising the steps of: (a) obtaining genomic DNA from said non-human mammal; (b) observing cytosine methylation at a plurality of CG loci in the genomic DNA using the DNA methylation array according to any one of items 9 to 12; whereby a methylation profile in said non-human mammal is observed. (Item 14) (c) comparing the CG locus methylation observed in (b) with the CG locus methylation observed in genomic DNA from individuals of the non-human mammal species of known age; and (d) correlating the CG locus methylation observed in (b) with the known age of the non-human mammalian species; Further encompasses; 14. The method of claim 13, wherein information useful for determining the age of the non-human mammalian species is thereby obtained. (Item 15) Methylation is observed by a process comprising treating genomic DNA of a population of cells derived from said mammal with bisulfite to convert unmethylated cytosines of CpG dinucleotides in said genomic DNA to uracil; The DNA methylation array is used to observe methylation profiles in multiple non-human mammalian species; and / or The genomic DNA is amplified by the polymerase chain reaction process. Item 14. The method according to item 13. (Item 16) 1. A method for observing the effect of a test agent on genome methylation-associated epigenetic aging in a mammalian cell, the method comprising: (a) combining the test agent with mammalian cells; (b) observing the methylation state of the methylation marker in the genomic DNA derived from the mammalian cell using the DNA methylation array according to any one of items 9 to 12; (c) comparing the observations from (b) with observations of the methylation status in genomic DNA from control mammalian cells not exposed to the test agent, such that an effect of the test agent on genome methylation associated epigenetic aging in the mammalian cells is observed; The method includes: (Item 17) 17. The method of claim 16, wherein a plurality of test agents are combined with the mammalian cells. (Item 18) 17. The method of claim 16, wherein the cells are human primary keratinocytes. (Item 19) 17. The method of claim 16, wherein the test agent is a compound having a molecular weight of less than 3,000 g / mol, 2,000 g / mol, 1,000 g / mol, or 500 g / mol. (Item 20) Methylation is observed by a process comprising treating genomic DNA from a population of cells derived from said mammal with bisulfite to convert unmethylated cytosines of CpG dinucleotides in said genomic DNA to uracils; and / or The genomic DNA is amplified by the polymerase chain reaction process. Item 17. The method according to item 16. Other objects, features and advantages of the present invention will become apparent to those skilled in the art from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating certain embodiments of the present invention, are given by way of illustration and not limitation. Many changes and modifications within the scope of the present invention may be made without departing from the spirit of the invention, and the invention includes all such modifications. [Brief description of the drawings]

[0017] [Figure 1] Figure 1 provides graphed data showing CpG sites identified via the CMAPS algorithm targeting high-density CpG islands due to inclusion of Infinium I probes. The representation of selected CpGs (blue) resembles the representation of all CpGs in the human genome (red). [Diagram 2] Figure 2 provides graphed data showing the CpG sites identified through the CMAPS algorithm, which targets both hypermethylated and hypomethylated CpG sites. The methylation histogram of selected probes (red) resembles that of all sites in the human genome (blue). [Diagram 3] FIG. 3 provides graphed data showing an epigenetic aging clock based on 404 highly conserved CpGs from human methylation data. Left panel: weighted average of 404 epigenetic clock CpGs versus chronological age in the training dataset. The rate of change of the red curve can be interpreted as the tick rate. Points are colored and displayed by dataset as described in Horvath, S. DNA methylation age of human tissues and cell types. Genome Biol. 14, R115 (2013 ("Horvath 2013")). Right panel: similar results for the test dataset. Only the test data is suitable for independent validation. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] Detailed Description of the Invention In describing the embodiments, reference may be made to the accompanying drawings, which form a part thereof. Here, specific embodiments in which the present invention may be implemented are shown by way of example. It should be understood that other embodiments may be utilized and structural changes may be made without departing from the scope of the present invention. Many of the techniques and procedures described or referred to herein are well understood and commonly used by those skilled in the art. Unless otherwise defined, all technical terms, annotations and other scientific terms or techniques used herein are intended to have the meanings commonly understood by those skilled in the art to which the present invention belongs. In some cases, terms having commonly understood meanings are defined herein for clarity and / or ease of reference, and the inclusion of such definitions herein should not necessarily be interpreted as indicating a substantial difference to what is commonly understood in the art.

[0019] All publications mentioned herein are incorporated by reference herein to disclose and describe the aspects, methods and / or materials in connection with which the publications are cited. For example, U.S. Patent Application Publication No. 20150259742, U.S. Patent Application No. 15 / 025,185 filed by Stefan Horvath, entitled "METHOD TO ESTIMATE THE AGE OF TISSUES AND CELL TYPES BASED ON EPIGENETIC MARKERS"; U.S. Patent Application No. 14 / 119,145 filed by Eric Villain et al., entitled "METHOD TO ESTIMATE AGE OF INDIVIDUAL BASED ON EPIGENETIC MARKERS IN BIOLOGICAL SAMPLE"; and Hannum et al. "Genome-Wide Methylation Profiles Reveal Quantitative Views Of Human Aging Rates." Molecular Cell. 2013; 49(2):359-367 and patent US2015 / 0259742 are incorporated by reference herein in their entireties.

[0020] As noted above, embodiments of the invention disclosed herein include an algorithm for identifying highly conserved methylation probes (CMAPS), which is useful for observing genome methylation patterns across a wide variety of mammalian species. Polynucleotide probe sequence information, including specific nucleotides within each probe sequence, is designed to tolerate specified variations. Polynucleotide probes identified by the CMAPS algorithm allow for the measurement of cytosine methylation levels in short stretches of DNA that are highly conserved across mammals using polynucleotide arrays such as those sold by ILLUMINA. Embodiments of the invention disclosed herein include gene chips that include multiple human genome sequences identified using the CMAPS algorithm.

[0021] As discussed below, an exemplary working embodiment of the invention disclosed herein is a gene chip that includes a set of 35,988 polynucleotide probes that allows for the assessment of cytosine DNA methylation levels in essentially all mammalian species. The CMAPS algorithm is the basis for the design of this custom-made ILLUMINA Infinium chip (HorvathMammalMethylChip40) that includes approximately 38k of these polynucleotide probes. Among them, 36,000 probes interrogate CpG sites in conserved regions of the human genome, making the gene chip applicable in all mammalian species. The remaining 2,000 probes were selected due to their particular importance in human epigenetic biomarker research. This DNA methylation chip is useful for observing methylation profiles in all mammalian species and is therefore specially made for interspecies comparison.

[0022] The embodiments of the present invention include a method for making a DNA methylation array comprising a plurality of polynucleotides linked to a matrix, such as a bead or chip.Typically, in these methods, the plurality of polynucleotides are selected by a method comprising the steps of: performing polynucleotide sequence alignment, comprising comparing a human genome with a plurality of non-human mammalian genomes to identify polynucleotide sequences in the human genome that comprise CpG methylation sites that are homologous to polynucleotide sequences in the genome of a non-human mammalian species that comprise CpG methylation sites; ranking the polynucleotide sequences in the human genome identified in the polynucleotide sequence alignment, wherein the ranking criteria comprises sequence homology to polynucleotide sequences in the genome of a non-human mammalian species; and using this ranking to select a plurality of polynucleotides in the human genome that cross-hybridize with a plurality of polynucleotide sequences in the genome of a non-human mammalian species; and then linking the selected sequences to a matrix to form a DNA methylation array.In a representative embodiment of the present invention, the DNA methylation array comprises at least 30,000 unique polynucleotides linked to the matrix.

[0023] In certain embodiments of the method for generating a DNA methylation array, the plurality of human genomic polynucleotide sequences are selected to have no more than 3 base pair mismatches with polynucleotide sequences in the genome of a non-human mammalian species. Typically, the plurality of polynucleotides are between 40 and 80 nucleotides in length. In some embodiments of the invention, ranking the polynucleotide sequences comprises a step of homology comparison to genomic polynucleotide sequences in non-placental mammalian species and placental mammalian species in the supergroups of Laurasian, Eucerna, Xenarthra, and Afrotherian. Optionally, the sequence alignment compares the human genomic sequence to genomic sequences of at least 10 non-human mammalian species.

[0024] In another illustrative embodiment of a method of making a DNA methylation array comprising a plurality of polynucleotides linked to a matrix, the plurality of polynucleotides comprises at least 2,000 polynucleotide sequences that comprise a CpG motif and hybridize with less than three base pair mismatches to a 60 nucleotide segment in a genomic polynucleotide sequence of a marsupial mammal species, a monotreme mammal species, a Laurasian mammal species, a Eucoptera mammal species, a Xenarthra mammal species, and a Afrotherian mammal species. Typically, the polynucleotide sequences are selected by performing a polynucleotide sequence alignment to compare the human genome with a plurality of non-human mammalian genomes to identify polynucleotide sequences in the human genome comprising CpG methylation sites that are homologous to polynucleotide sequences in the genome of the non-human mammalian species comprising CpG methylation sites; ranking the polynucleotide sequences in the human genome identified in (a), wherein the ranking criteria include the degree of sequence homology to the polynucleotide sequences in the genome of the non-human mammalian species; using the ranking to select a plurality of polynucleotides having CpG methylation sites that cross-hybridize with no more than 3 base pair mismatches to a plurality of polynucleotide sequences having CpG methylation sites in the genome of the non-human mammalian species; and then linking the selected sequences from step (b) to a matrix to form a DNA methylation array, thereby producing the DNA methylation array.

[0025] An embodiment of the present invention includes a DNA methylation array made by the methods disclosed herein. In certain embodiments of the present invention, at least 1, 10, 100 or more polynucleotides in the plurality of polynucleotides are polynucleotides having sequences shown in Table 3. For example, an embodiment of the present invention includes a DNA methylation array comprising a plurality of polynucleotide sequences linked to a matrix, wherein the polynucleotides comprise at least 60 nucleotides and CpG motifs at their ends; the polynucleotides comprise polynucleotide sequences present in the human genome; and at least 2,000 polynucleotides in the plurality of polynucleotide sequences are capable of hybridizing to a 60 nucleotide segment in a genomic polynucleotide sequence of a marsupial mammal species with less than 3 base pair mismatches; at least 2,000 polynucleotides in the plurality of polynucleotide sequences are capable of hybridizing to a 60 nucleotide segment in a genomic polynucleotide sequence of a monotreme mammal species with less than 3 base pair mismatches; at least 2,000 polynucleotides in the plurality of polynucleotide sequences are capable of hybridizing to a 60 nucleotide segment in a genomic polynucleotide sequence of a monotreme mammal species with less than 3 base pair mismatches; at least 2,000 polynucleotides in the plurality of polynucleotide sequences can hybridize to a 60 nucleotide segment in a genomic polynucleotide sequence of a mammalian species of the superorder Laurasian with less than three base pair mismatches; at least 2,000 polynucleotides in the plurality of polynucleotide sequences can hybridize to a 60 nucleotide segment in a genomic polynucleotide sequence of a mammalian species of the superorder Euceratomorpha with less than three base pair mismatches; at least 2,000 polynucleotides in the plurality of polynucleotide sequences can hybridize to a 60 nucleotide segment in a genomic polynucleotide sequence of a mammalian species of the superorder Xenarthra with less than three base pair mismatches; and at least 2,000 polynucleotides in the plurality of polynucleotide sequences can hybridize to a 60 nucleotide segment in a genomic polynucleotide sequence of a mammalian species of the superorder Afrotherian with less than three base pair mismatches.In certain embodiments, the marsupial mammal species is a wallaby species; and / or the monotreme mammal species is a platypus species; and / or the laurasian mammal species is a bat species; and / or the euprimate mammal species is a rodent species; and / or the xenarthra mammal species is an armadillo species; and / or the afrothea mammal species is a tenrec species.

[0026] Another embodiment of the present invention is a method of observing a methylation profile in a non-human mammal, comprising the steps of obtaining genomic DNA from the non-human mammal; observing the cytosine methylation of a plurality of CG loci in the genomic DNA using a DNA methylation array as disclosed herein, thereby observing a methylation profile in the non-human mammal. Optionally, these methods also comprise the steps of comparing the CG loci methylation observed in the method with the CG loci methylation observed in genomic DNA derived from an individual of the non-human mammal species having a known age; and then correlating the CG loci methylation observed in (b) with the known age of the non-human mammal species, thereby obtaining information useful for determining the age of the non-human mammal species. Typically, in these embodiments, methylation is observed by a process comprising treating genomic DNA of a population of cells derived from the mammal with bisulfite to convert unmethylated cytosines of CpG dinucleotides in the genomic DNA to uracil; the DNA methylation array is used to observe the methylation profile in multiple non-human mammalian species; and / or genomic DNA is amplified by a polymerase chain reaction process.

[0027] Yet another embodiment of the present invention is a method for observing the effect of a test agent (a compound having a molecular weight of less than 3,000 g / mol, 2,000 g / mol, 1,000 g / mol, or 500 g / mol, such as rapamycin) on genome methylation-related epigenetic aging of mammalian cells (e.g., human primary keratinocytes).Typically, these methods include combining the test agent with mammalian cells; observing the methylation status of methylation markers in genomic DNA derived from the mammalian cells using the DNA methylation array disclosed herein; and then comparing these observations with those of the methylation status in genomic DNA of control mammalian cells that are not exposed to the test agent, so as to observe the effect of the test agent on genome methylation-related epigenetic aging in the mammalian cells (e.g., whether the test agent reduces or increases the genome methylation pattern associated with epigenetic aging).Optionally, in these methods, multiple test agents are combined with the mammalian cells. In certain embodiments of these methods, the polynucleotide is linked to a matrix and methylation is observed by a process comprising treating genomic DNA of a population of cells derived from said mammal with bisulfite to convert unmethylated cytosines of CpG dinucleotides in said genomic DNA to uracil; and / or the genomic DNA is amplified by a polymerase chain reaction process.

[0028] Further aspects and embodiments of the invention are discussed in the following sections.

[0029] Further illustrative aspects and embodiments of the present invention DNA methylation refers to the chemical modification of DNA molecules. It has been found that technological platforms such as ILLUMINA Infinium microarrays or DNA sequencing-based methods provide very robust and reproducible measurements of DNA methylation levels in humans. There are more than 28 million CpG loci in the human genome. As a result, certain loci are listed in the ILLUMINA CpG loci database and given unique identifiers such as those used in Table 3 (see, for example, Technical Note: Epigenetics, CpG Loci Identification ILLUMINA Inc. 2010). Certain illustrative CG locus designation identifiers and sequences are used herein. Such sequences can be further characterized using one or more of genome databases that are readily available to those skilled in the art, such as the UCSC Genome Browser (an online and downloadable genome browser hosted by the University of California, Santa Cruz (UCSC)).

[0030] The term "epigenetic" as used herein means relating to, being, or involving chemical modification of DNA molecules. Epigenetic factors include the addition or removal of methyl groups, which results in changes in DNA methylation levels.

[0031] The term "polynucleotide" as used herein may include any polymer or oligomer of pyrimidine and purine bases, preferably cytosine, thymine and uracil, and adenine and guanine, respectively. The present invention contemplates any deoxyribonucleotide, ribonucleotide or peptide nucleic acid component, as well as any chemical variants thereof, such as methylated, hydroxymethylated or glycosylated forms of these bases. The polymer or oligomer may be heterogeneous or homogeneous in composition, isolated from naturally occurring sources, or artificially or synthetically produced. Furthermore, the nucleic acid may be DNA or RNA, or a mixture thereof, and may exist permanently or transiently in single-stranded or double-stranded form, including homoduplex, heteroduplex, and hybrid states.

[0032] The term "methylation marker" as used herein refers to a potentially methylated CpG position. Methylation typically occurs in a CpG-containing nucleic acid. The CpG-containing nucleic acid may be, for example, in a CpG island, or a CpG doublet, promoter, intron, or exon of a gene. For example, in the gene region provided herein, the potential methylation site includes the promoter / enhancer region of the indicated gene. Thus, the region may begin upstream of the gene promoter and extend downstream to the transcribed region.

[0033] The phrase "selectively measuring," as used herein, refers to methods in which only a finite number of methylation markers or genes (including methylation markers) are measured, rather than assaying essentially all potential methylation markers (or genes) in the genome. For example, in some aspects, "selectively measuring" methylation markers or genes that contain such markers can refer to measuring 100, 75, 50, 25, 10, or 5 or more (or less) different methylation markers or genes that contain methylation markers.

[0034] DNA methylation of the above methylation markers (or markers close to them) can be measured using a variety of approaches ranging from commercial array platforms (e.g., ILLUMINA's) to individual gene sequencing approaches, including standard laboratory techniques or array platforms. Various methods for detecting methylation status or patterns are described, for example, in U.S. Pat. Nos. 6,214,556, 5,786,146, 6,017,704, 6,265,171, 6,200,756, 6,251,594, 5,912,147, 6,331,393, 6,605,432, and 6,300,071, and U.S. Patent Application Publication Nos. 20030148327, 20030148326, 20030143606, 20030082609, and 20050009059, each of which is incorporated herein by reference. Other array-based methods of methylation analysis are disclosed in US Patent Application Serial No. 11 / 058,566. For a review of some methylation detection methods, see Oakley, EJ, Pharmacology & Therapeutics 84:389-400 (1999). Available methods include, but are not limited to, reversed-phase HPLC, thin-layer chromatography, SssI methyltransferase with incorporation of a labeled methyl group, chloroacetaldehyde reaction, restriction enzymes with different sensitivities, hydrazine or permanganate treatment (m5C is cleaved by permanganate treatment but not by hydrazine treatment), sodium bisulfite, combined bisulphate-restriction analysis, and methylation-sensitive single nucleotide primer extension.The ILLUMINA method uses sequences flanking CpG loci to generate unique CpG locus cluster IDs using a strategy similar to NCBI's refSNP IDs (rs#) in dbSNP (see, e.g., Technical Note: Epigenetics, CpG Loci Identification ILLUMINA Inc. 2010).

[0035] The methylation level of a subset of DNA methylation markers disclosed herein is assayed (e.g., using ILLUMINA DNA methylation arrays or using PCR protocols with relevant primers). To quantify the methylation level, the beta value of methylation can be calculated according to the standard protocol described by ILLUMINA. This is equal to the percentage of methylated cytosines at that position. The present invention can also be applied to any other approach to quantify DNA methylation at positions close to genes as disclosed herein. DNA methylation can be quantified using many currently available assays.

[0036] In certain embodiments of the present invention, genomic DNA is hybridized to a complementary sequence (e.g., a synthetic polynucleotide sequence) that is linked to a matrix (e.g., arranged in a microarray). Optionally, the genomic DNA is converted from its native state through amplification by a polymerase chain reaction process. For example, prior to or in conjunction with hybridization to the array, the sample is amplified by various mechanisms, some of which may use PCR. See, e.g., PCR Technology: Principles and Applications for DNA Amplification (ed. H.A. Erlich, Freeman Press, NY, NY, 1992); PCR Protocols: A Guide to Methods and Applications (eds. Innis et al., Academic Press, San Diego, Calif., 1990); Mattila et al., Nucleic Acids Res. 19, 4967 (1991); Eckert et al., PCR Methods and Applications 1, 17(1991); PCR (eds. McPherson et al., IRL Press, Oxford); and U.S. Pat. Nos. 4,683,202, 4,683,195, 4,800,159, 4,965,188, and 5,333,675. The samples can be amplified on the array. See, e.g., U.S. Pat. No. 6,300,070, which is incorporated herein by reference.

[0037] The embodiments of the present invention may include various art-accepted technical processes. For example, in certain embodiments of the present invention, a bisulfite conversion process is carried out so that cytosine residues in genomic DNA are converted to uracil, while 5-methylcytosine residues in genomic DNA are not converted to uracil. Kits for DNA bisulfite modification are commercially available, for example, MethylEasy™ (Human Genetic Signatures™) and CpGenome™ Modification Kit (Chemicon™). See also WO04096825A1, which describes the bisulfite modification method, and Olek et al. Nuc. Acids Res. 24:5064-6 (1994), which discloses a method of performing bisulfite treatment and subsequent amplification. Bisulfite treatment allows the methylation status of cytosine to be detected by various methods. For example, any method that can be used to detect SNPs can be used. See, e.g., Syvanen, Nature Rev. Gen. 2:930-942 (2001). Methods such as single base extension (SBE) can be used (or sequence-specific probe hybridization similar to allele-specific hybridization methods). In another aspect, Molecular Inversion Probe (MIP) assays can be used.

[0038] There are many techniques to measure DNA methylation levels in a single species. To measure methylation in human DNA, human ILLUMINA Infinium arrays can be used to measure DNA methylation levels in human DNA samples. A recent paper (Needhamsen et al., BMC Bioinformatics, BMC series -2017, 18:486) showed that it is possible to use the EPIC chip for methylation measurement in mice, but only about 19K of the 850K probes on the EPIC chip are useful in mice. Species more distant from humans may have even fewer useful probes on the EPIC chip, pointing out the need for a platform that can be used in non-human mammals.

[0039] An alternative to chips / arrays for measuring DNA methylation is bisulfite sequencing (see, for example, Meissner et al., Nucleic Acids Research, Volume 33, Issue 18, 1 January 2005, Pages 5868-5877). It applies to all mammalian species, but has not been established as quantitatively reliable. Array technology is particularly valuable for the development of advanced and robust epigenetic biomarkers of aging and development. The present invention provides an algorithm for selecting probes and the results of this algorithm for identifying (non-natural) nucleotide sequences that can be used for methylation arrays / chips that apply to all mammals. The inventors have shown that highly conserved sequences are suitable for building advanced and accurate epigenetic aging clocks (see, for example, U.S. Patent Application Serial No. 15 / 025,185, entitled "METHOD TO ESTIMATE THE AGE OF TISSUES AND CELL TYPES BASED ON EPIGENETIC MARKERS").

[0040] The first human methylation chip (ILLUMINA Infinium 27K) was introduced over a decade ago, but similar chips are not available for other species. This delay may reflect the fact that it is not economical to design methylation chips for non-human species. Even if cost were not an obstacle, the development of species-specific arrays may hinder inter-species comparisons. Because the measurement platforms are different. As noted above, to address these challenges, we developed an algorithm, Conserved Methylation Array Probe Selector (CMAPS). This algorithm reuses the degenerate base technology used to tolerate intra-human variation and tolerate inter-species variation. CMAPS performs a greedy search to obtain the maximum number of species that can be targeted using a probe for any CpG in the human genome based on multiple sequence alignment. CMAPS was used to design nearly 36,000 probes that query CpG sites in conserved regions of the human genome. This makes the chip directly applicable to mammalian species, thus facilitating inter-species comparison. To obtain such a large number of probes for multiple species, CMAPS ranks all the probes and selects a final set so that each Infinium array can query multiple mammalian species and various genomic locations based on external annotation of exons, CpG islands, and hyper- versus hypo-methylated regions. To enhance the usefulness of the chip in human studies, we also added approximately 2,000 probes that are of particular importance in human biomarker studies. In the following, we describe the CMAPS algorithm and the characteristics of the resulting chip (HorvathMammalMethylChip40).

[0041] ILLUMINA Infinium Probes Currently, ILLUMINA-generated methylation arrays can contain two types of probes: Infinium 1 and Infinium 2. Infinium 2 is a newer technology that requires only one bead to query a CG, while Infinium 1 requires two beads.

[0042] For the design and development of the MammalMethyl40 chip, we have taken advantage of a set of all human CG sites that can be interrogated using one or both of these probes. There are two variants in each of the two probes, depending on whether the probe is designed for the forward genomic strand versus the reverse genomic strand. The probe allows up to three degenerate bases, which can allow variations in sequence to be interrogated. The number of degenerate bases allowed is a function of the probe's design score calculated by ILLUMINA, and in the case of the Infinium 2 probe, the number of underlying CpGs (Table 1).

[0043] To be able to interrogate a particular CpG site, an oligonucleotide probe must be synthesized on the array that contains 60 base pairs either upstream or downstream of the CpG site. The degenerate base technique allows the CpG site to be interrogated by the probe even if the individual happens to have a variant in the adjacent region that causes a mismatch with the synthesized probe (Method). We have developed the CMAPS algorithm to reuse this technique to design degenerate bases for each human probe so that the probe can tolerate mutations here and also hybridize to DNA from other species. We applied the CMAPS algorithm to 100 alignments of 99 other species to the human genome, which provides the ability to pick up mutations within the rules specified by the underlying array technology (Infinium technology in this particular case). However, the algorithm can take as input any multiple sequence alignment with any reference genome, along with a set of design considerations, and provide conserved probes and degenerate base selection within those rules.

[0044] Determination of the initial set of 60,000 probes For each CG site in the human genome, we selected the Infinium 1 probe set from the selection that covered the most species based on the algorithm described above, and did the same for Infinium 2. We first included all Infinium 2 probes that targeted the mm10 mouse genome, thus ensuring that the chip was useful for one of the most widely used model organisms. We then sorted the CpG sites in descending order of the number of species covered by the Infinium 2 probes, and added all probes that were not yet selected because they targeted mm10, for a total of up to 53,000 probes. We then ranked the probes on the ILLUMINA EPIC array in descending order of the number of species they could target using the degenerate bases picked up by the CMAPS algorithm, and selected an additional 3,000 probes that had not yet been picked up based on the previous criteria. Finally, we sorted the CpG sites in descending order of the number of species they could target and picked the top 4,000 Infinium 1 probes that targeted CpG sites that had not yet been included. The Infinium 1 probes were chosen to allow us to query denser CG regions because the underlying CpG counting of the Infinium 1 probes does not count against the number of allowed SNVs. This gave us a total of 60,000 probes.

[0045] Probe selection based on mappability Since the probes on the array are only 60 base pairs long, they run the risk of mapping to multiple locations in the genome, which results in confusing signals from multiple CpG sites. This problem can be complicated by the fact that each of our probes can have up to 2^(number of degenerate bases) variants. For the 16 quality genomes, we calculated for each probe how many of its variants map uniquely in that genome. We then filtered the probes by requiring that all variants of a probe must map uniquely in at least 80% of the species they are designed to target, or that the probe must target at least 40 species. This reduced the set of working probes to a final set of 35,988 probes.

[0046] Custom Chip Characteristics The outline of the HorvathMammalMethylChip40 is that it has fewer than 40k probes (hence the "40" suffix).

[0047] 2000 of the 38k probes were selected based on their utility for human biomarker research. These CpGs, which were previously implemented in the human ILLUMINA Infinium array (EPIC, 450K, 27K), were selected due to their relevance for predicting age, blood counts, or percentage of neurons in brain tissue.

[0048] The remaining 35,988 probes were selected to assess cytosine DNA methylation levels in a wide variety of evolutionarily distinct mammalian species. To this end, the CMAPS algorithm was used to identify highly conserved CpGs across 50 mammalian species: 33,493 Infinium II probes and 2,496 Infinium I probes. Not all probes on the array are predicted to work for all species, but rather each probe is designed to cover a specific subset of species, so that overall all species have a large number of probes. The specific subset of species for each probe is provided in the chip manifest file. Of the 50 mammalian species observed, 46 of them have more than 10,000 probes on the array, and 36 have more than 20,000 probes (Table 2).

[0049] Chromosomal environment The CpG sites targeted by these probes represent diverse regions of the genome. In humans, 40% of CpG sites are in exonic regions, as predicted by the known strong conserved signals in exons. The selected set of CpG sites targets high-density CpG islands (Figure 1), due to our choice to include Infinium I probes, and can target both hypermethylated and hypomethylated CpG sites (Figure 2).

[0050] A highly conserved CpG-based epigenetic clock in humans Using 404 highly conserved CpGs on the ILLUMINA 27K array, we developed a novel epigenetic clock using the same data previously used to develop a pan-tissue epigenetic clock disclosed in Horvath, S. DNA methylation age of human tissues and cell types, Genome Biol. 14, R115 (2013).

[0051] To ensure unbiased validation in the test data, we used only the training data to define the age predictor. A transformed version of chronological age was regressed onto CpGs using a penalized regression model (elastic net) as detailed in Horvath 2013. The elastic net regression model automatically selected the covariate CpGs. These highly conserved CpGs are referred to as (epigenetic) clock CpGs because their weighted average (formed by the regression coefficients) spans the epigenetic clock. Although the clock was based on only 404 CpGs, the resulting epigenetic age estimator performs surprisingly well across a wide range of tissues and cell types (Figure 3).

[0052] The linear combination (resulting from the regression coefficients) of the 404 highly conserved epigenetic clock CpGs varies widely over the entire life course (from cradle to grave), as can be seen from Figure 3. The red calibration curve reveals a logarithmic dependence until adulthood, which slows down to a linear dependence later in life. The rate of change (of this red curve) can be interpreted as the tick rate of the epigenetic clock. Similar to the original pan-tissue clock of Horvath 2013, we find that organismal growth leads to a high tick rate, which slows down to a constant tick rate (linear dependence) from adulthood onwards.

[0053] Consideration The CMAPS algorithm facilitated the design of novel mammalian methylation arrays that apply to all mammals, specifically tailored for interspecies comparisons across mammals and for the development of biomarkers that apply to multiple species. Our work shows that the relatively small number of highly conserved CpGs (approximately 400) resulting from the CMAPS algorithm are already suitable for constructing highly accurate epigenetic age estimators (conserved epigenetic clocks).

[0054] Overall, we expect that the mammalian chip is particularly well suited for DNA methylation-based biomarker research in mammals. For example, the present invention allows for the assessment of whether a particular intervention (e.g., therapeutic agent and / or regimen) that affects DNA methylation levels in one species (e.g., mouse) also affects the corresponding DNA methylation levels in another species (e.g., human).

[0055] method Conserved Methylation Array Probe Selector (CMAPS) The CMAPS algorithm was applied to the Multiz alignment of 99 vertebrates with the hg19 human genome downloaded from the UCSC Genome Browser (7). For the purposes of this chip, only mammalian species in this alignment were considered. The design scores of each CpG in the human genome and each possible type of probe at each position were provided by ILLUMINA and taken as input by CMAPS. For each CG site in the human genome, we calculated the maximum number of species that could be targeted by each of the four different possible probe designs in humans, considering each possible arrangement of the maximum number of allowed mutations. For each probe option, we tried all possibilities to arrange the maximum number of potential variants, greedily picking the variant that covers the most species at a particular position. More specifically, the algorithm for selecting the number of species covered by the probe is described in the following pseudocode: The function get_max_species performs a greedy selection for the nucleotide of a particular SNV by picking whatever nucleotide is included in the majority of species in the alignment at that position. Function get_max_species(SNV_pos, num_SNV, multiple_sequence_alignment): max_species = [] for X in {A, C, T, G} count_species = number of species with X at SNV_pos in the multiple_sequence_alignment max_species.append(count_species, x)) sort(max_species, descending = True) # Sort by number of species covered in descending order return max_species[:num_SNV][,1] # Return the top num_SNV nucleotides in order of the number of species they target

[0056] In the pseudocode below, SNV_set iterates over all possible positions of an SNV in a particular probe, given the design score and probe type constraints. Cur_max_species = 1 for SNV_set in all positions in probe: alt_nucleotide_list = [] for SNV_pos in SNV_set: alt_nucleotide_list.append(get_max_species(SNV_pos, multiple_sequence_alignment, count(SNV_pos, SNV_set))) num_species = number of species fully matching human given SNV_set and alt_nucleotide_list if num_species > cur_max_species: cur_max_species = num_species final_SNV_set = SNV_set

[0057] Since the get_max_species function performs a greedy selection, this may not be the true maximal subset of species for the probes, but the method is relatively computationally inexpensive and provided satisfactory species coverage for our purposes.

[0058] Supplementary Data: SupplementMammalianChip36Kprobes The following explanation describes the variables. Forward_Sequence: Sequence on the forward strand Genome_Build: Human genome construction Chromosome: Human chromosome with CG site Coordinate: Human genomic coordinate of "C" at CG site (1-based) TB_Strand_OrigP: TOP / BOTTOM chain Top_Sequence: Sequence on the TOP chain Methyl_Probe_Sequence: The methylation probe sequence that is offset by one from the sequence selected for Infinium 2. Allele_Fr_Strand: forward / reverse strand Allele_TB_Strand: TOP / BOT chain Allele_CO_Strand: Conversion / opposite strand Underlying_CpG_Count: The underlying CpG count for each site. UnMethyl_Probe_Sequence: Unmethylated probe sequence that is offset by one from the sequence selected for Infinium 2 Num_Species: The number of mammalian species probes that are predicted to function. species: Comma-separated genome assembly names of the species for which the probe is predicted to function Probe_Start_Coord: Probe start coordinate on the hg19 forward strand, starting from 1 Probe_End_Coord: Probe end coordinate on the hg19 forward strand, starting from 1 Reference_Probe_Sequence: Probe forward strand reference sequence in hg19 starting from 1 SNV_location: Comma-separated coordinates of the base in hg19 where the SNV exists, starting from 1. designed forSNV_original: comma-separated reference nucleotides in hg19 for each SNV; 1-1 correspondence with the order of coordinates in SNV_location SNV_change: comma-separated alternative nucleotides for each SNV; order of coordinates in SNV_location and 1-1 correspondence with reference nucleotides in SNV_original Infinium_Type: Inf1 / Inf2 Infinium probe type Is_EPIC_site: a 0 / 1 binary variable indicating whether the CG site is also queried by a probe on the EPIC Array. Is_EPIC_design: A 0 / 1 binary variable indicating whether the probes querying this site on the EPIC Array are of the same Infinium type (1 / 2) and the same strand (both forward / reverse and reverse); always 0 if Is_EPIC_site is 0 Nvariants: Number of SNV-based probe variations. Effectively 2^(number of SNVs) Used in mappability analysis.

[0059] table [Table 1]

[0060] [Table 2-1] [Table 2-2]

[0061] [Table 3-1] [Table 3-2]

[0062] References Describing Methods and Materials Useful in Aspects of the Invention All publications mentioned herein are incorporated by reference herein to disclose and describe the aspects, methods and / or materials in connection with which the publications are cited. For example, U.S. Patent Publication 20150259742; U.S. Patent Application Serial No. 15 / 025,185, filed by Stefan Horvath, entitled "METHOD TO ESTIMATE THE AGE OF TISSUES AND CELL TYPES BASED ON EPIGENETIC MARKERS"; U.S. Patent Application Serial No. 14 / 119,145, filed by Eric Villain et al., entitled "METHOD TO ESTIMATE AGE OF INDIVIDUAL BASED ON EPIGENETIC MARKERS IN BIOLOGICAL SAMPLE"; and Hannum et al. "Genome-Wide Methylation Profiles Reveal Quantitative Views Of Human Aging Rates." Molecular Cell. 2013; 49(2):359-367 and Patent US2015 / 0259742 are incorporated by reference herein in their entireties.

[0063] Cited references 1. Bernstein, BE, Meissner, A. & Lander, ES The Mammalian Epigenome. Cell 128, 669-681 (2007). 2. Smith, ZD & Meissner, A. DNA methylation: roles in mammalian development. Nature Reviews Genetics 14, 204-220 (2013). 3. Horvath, S. DNA methylation age of human tissues and cell types. Genome Biol. 14, R115 (2013). 4. Genome-wide DNA methylation profiling using Infinium(R) assay | Epigenomics. Available at: https: / / www.futuremedicine.com / doi / abs / 10.2217 / epi.09.14. (Accessed: 28th August 2018) 5. Evaluation of the Infinium Methylation 450K technology. - PubMed - NCBI. Available at: https: / / www.ncbi.nlm.nih.gov / pubmed / 22126295. (Accessed: 28th August 2018) 6. Pidsley, R. et al. Critical evaluation of the ILLUMINA MethylationEPIC BeadChip microarray for whole-genome DNA methylation profiling. Genome Biology 17, 208 (2016). 7. Rosenbloom, KR et al. The UCSC Genome Browser database: 2015 update. Nucleic Acids Res. 43, D670-681 (2015).

[0064] Further publications describing methods and materials useful in aspects of the invention 1. Horvath S: DNA methylation age of human tissues and cell types. Genome Biol 2013, 14:R115. 2. Hannum G, Guinney J, Zhao L, Zhang L, Hughes G, Sadda S, Klotzle B, Bibikova M, Fan JB, Gao Y, et al: Genome-wide methylation profiles reveal quantitative views of human aging rates. Mol Cell 2013, 49:359-367. 3. Bocklandt S, Lin W, Sehl ME, Sanchez FJ, Sinsheimer JS, Horvath S, Vilain E: Epigenetic predictor of age. PLoS One 2011, 6:e14821. 4. Levine ME, Lu AT, Quach A, Chen BH, Assimes TL, Bandinelli S, Hou L, Baccarelli AA, Stewart JD, Li Y, et al: An epigenetic biomarker of aging for lifespan and healthspan. Aging (Albany NY) 2018. 5. Zhang Y, Wilson R, Heiss J, Breitling LP, Saum KU, Schottker B, Holleczek B, Waldenberger M, Peters A, Brenner H: DNA methylation signatures in peripheral blood strongly predict all-cause mortality. Nat Commun 2017, 8:14617. 6. Bocklandt S, Lin W, Sehl ME, Sanchez FJ, Sinsheimer JS, Horvath S, Vilain E: Epigenetic predictor of age. PLoS One 2011, 6. 7. Weidner CI: Aging of blood can be tracked by DNA methylation changes at just three CpG sites. Genome Biol 2014, 15. 8. Hannum G: Genome-wide methylation profiles reveal quantitative views of human aging rates. Mol Cell 2013, 49. 9. Lin Q, Weidner CI, Costa IG, Marioni RE, Ferreira MRP, Deary IJ: DNA methylation levels at individual age-associated CpG sites can be indicative for life expectancy. Aging 2016, 8:394-401. 10. Marioni R, Shah S, McRae A, Chen B, Colicino E, Harris S, Gibson J, Henders A, Redmond P, Cox S, et al: DNA methylation age of blood predicts all-cause mortality in later life. Genome Biol 2015, 16:25. 11. Christiansen L, Lenart A, Tan Q, Vaupel JW, Aviv A, McGue M, Christensen K: DNA methylation age is associated with mortality in a longitudinal Danish twin study. Aging Cell 2015. 12. Perna L, Zhang Y, Mons U, Holleczek B, Saum KU, Brenner H: Epigenetic age acceleration predicts cancer, cardiovascular, and all-cause mortality in a German case cohort. Clinical Epigenetics 2016, 8:1-7. 13. Horvath S, Pirazzini C, Bacalini MG, Gentilini D, Blasio AM, Delledonne M, Mari D, Arosio B, Monti D, Passarino G: Decreased epigenetic age of PBMCs from Italian semi-supercentenarians and their offspring. Aging (Albany NY) 2015, 7.

[0065] conclusion This concludes the description of the preferred embodiments of the present invention. The foregoing description of one or more embodiments of the present invention has been presented for purposes of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise form disclosed. Many modifications and variations are possible in light of the above teachings.

Claims

1. 1. A method for observing the effect of a test agent on genome methylation-associated epigenetic aging in a mammalian cell, the method comprising: (a) combining the test agent with mammalian cells; (b) observing the methylation status of methylation markers in genomic DNA derived from said mammalian cells using a DNA methylation array comprising a plurality of polynucleotide sequences linked in a matrix, wherein: said polynucleotides are at least 60 nucleotides and contain CpG motifs at their termini; The polynucleotide comprises a polynucleotide sequence present in the human genome; and At least 2,000 polynucleotides in said plurality of polynucleotide sequences are capable of hybridizing with less than three base pair mismatches to a 40 nucleotide segment in a genomic polynucleotide sequence of a monotreme mammalian species; at least 2,000 polynucleotides within said plurality of polynucleotide sequences are capable of hybridizing with less than three base pair mismatches to a 40 nucleotide segment in a genomic polynucleotide sequence of a mammalian species of the superorder Laurasiana; at least 2,000 polynucleotides within said plurality of polynucleotide sequences are capable of hybridizing with less than three base pair mismatches to a 40 nucleotide segment in a genomic polynucleotide sequence of a mammalian species of the superorder Eucoptera; At least 2,000 polynucleotides in the plurality of polynucleotide sequences are capable of hybridizing to a 40 nucleotide segment in a genomic polynucleotide sequence of a mammalian species of the superorder Xenarthra with less than three base pair mismatches; and at least 2,000 polynucleotides within said plurality of polynucleotide sequences are capable of hybridizing with less than three base pair mismatches to a 40 nucleotide segment in a genomic polynucleotide sequence of a mammalian species of the superorder Afro-Therian; and (c) comparing said observations from (b) with observations of the methylation status in genomic DNA from control mammalian cells not exposed to said test agent, so as to observe an effect of said test agent on genome methylation associated epigenetic aging in said mammalian cells; The method includes:

2. The method of claim 1 , wherein a plurality of test agents are combined with the mammalian cells.

3. The method of claim 2 , wherein the cells are human primary keratinocytes.

4. The method of claim 1 , wherein the test agent is a compound having a molecular weight of less than 3,000 g / mol.

5. The method of claim 4 , wherein the test agent is a compound having a molecular weight of less than 1,000 g / mol.

6. 2. The method of claim 1, wherein methylation is observed by a process comprising treating genomic DNA from a population of cells derived from said mammal with bisulfite to convert unmethylated cytosines of CpG dinucleotides in said genomic DNA to uracil.

7. The method of claim 1 , wherein the genomic DNA is amplified by a polymerase chain reaction process.

8. 2. The method of claim 1, wherein at least one polynucleotide in the plurality of polynucleotides is a polynucleotide selected from SEQ ID NO:1 to SEQ ID NO:

50.

9. 2. The method of claim 1, wherein at least 10 polynucleotides in the plurality of polynucleotides are selected from SEQ ID NO:1 through SEQ ID NO:

50.

10. A method for producing a DNA methylation array, comprising the steps of: (a) selecting a plurality of polynucleotides by performing a polynucleotide sequence alignment comprising comparing a human genome with a plurality of non-human mammalian genomes to identify polynucleotide sequences in the human genome that contain CpG methylation sites that are homologous to polynucleotide sequences in the genome of a non-human mammalian species that contain CpG methylation sites, wherein the polynucleotide sequence alignment compares a human genome sequence with genome sequences of three or four non-human mammalian species, including at least one non-placental mammalian species, and wherein the polynucleotides comprise at least 60 nucleotides; (b) ranking the polynucleotide sequences in the human genome identified in (a) according to sequence homology to polynucleotide sequences in genomes of non-human mammalian species, wherein the polynucleotide sequences of the plurality of human genomes are selected to have no more than three base pair mismatches with polynucleotide sequences in the genome sequences of the three or four non-human mammalian species; and (c) using the ranking in (b) to select a plurality of polynucleotides in the human genome that cross-hybridize to a plurality of polynucleotide sequences in the genomes of the three or four non-human mammalian species; and (d) ligating the selected sequences from step (c) to a matrix to form a DNA methylation array. The method includes:

11. 11. The method of claim 10, wherein the ranking comprises a homology comparison to genomic polynucleotide sequences in non-placental mammal species and placental mammal species in the supergroups of Laurasian, Eucernotrichtii, Xenarthria and Afrotheria.

12. 11. The method of claim 10, wherein the DNA methylation array comprises at least 30,000 unique polynucleotides linked to the matrix.

13. The method of claim 12, wherein the unique polynucleotides linked to the matrix are each between 60 and 80 nucleotides in length.

14. The method of claim 10 , wherein the matrix is ​​a bead.

15. The method of claim 10 , wherein the matrix is ​​a chip.

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