Methods of using canine dna methylation profiles

Abstract

The present invention provides a method for determining the risk of death and / or the probability of a healthy lifespan of a dog; the method comprising a) providing a DNA methylation map from a sample obtained from the dog; and b) using the DNA methylation map to determine the risk of death and / or the probability of a healthy lifespan of the dog; wherein the DNA methylation map contains at least one methylation site as listed in Table 1.

Description

Cross-references to related applications

[0001] This application claims the benefit and priority of U.S. Provisional Application Serial No. 63 / 604,324, filed November 30, 2023, the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0002] This invention relates to a method for determining the health status of dogs using DNA methylation mapping. In particular, this invention relates to a method for selecting lifestyle programs, dietary programs, or therapeutic interventions for dogs based on the health status determined by DNA methylation mapping, or for determining the effectiveness of such lifestyle programs, dietary programs, or therapeutic interventions. Background Technology

[0003] The ability to determine information about a dog's health is what is expected in informing a dog about its overall health and well-being.

[0004] It is well known that chronological age is a primary indicator of overall health status, with increasing chronological age associated with declining health. However, depending on genetics, nutrition, and lifestyle, an individual may age at a rate that is slower or faster than their chronological age. Therefore, chronological age may not always reflect an individual's rate of aging or risk of declining health. On the other hand, an individual's biological age (based on, for example, clinical biochemistry and cell biology measures) may differ from that of other individuals of the same chronological age. Methods used to determine biological age may help identify individuals at risk of age-related conditions earlier than expected based on their chronological age (see, for example, WO2019 / 046725).

[0005] Epigenetic clocks used to predict chronological age and infer health status as an indicator of biological age are described in WO2022 / 272120. These epigenetic clocks are primarily based on chronological age as a training parameter.

[0006] However, there is a need for other methods to determine the biological age of dogs and to use measurements of biological age to improve canine health outcomes. Summary of the Invention

[0007] This invention relates to a method for quantifying the health status of dogs based on DNA methylation mapping. The method is capable of determining the dog's risk of death and / or probability of healthy lifespan by assessing the DNA methylation map from the dog.

[0008] Existing methods for assessing canine health status determine biological age based on the correlation between DNA methylation and chronological age (see, for example, WO2022 / 272120). Calculating an animal's biological age may involve determining a DNA methylation profile compared to a predicted DNA methylation profile for a given chronological age. Therefore, this approach is based on using chronological age as the primary indicator of overall health.

[0009] In contrast, this invention considers the direct predictive value of DNA methylation profiling for mortality risk and / or healthy lifespan probability. For example, a given DNA methylation biomarker may not be directly related to chronological age, but may indicate a specific pathological condition, thereby indicating the probability of increased mortality risk and / or reduced healthy lifespan. Therefore, this method can be described as identifying the mortality risk and / or healthy lifespan probability in dogs. Thus, the DNA methylation biomarkers and DNA methylation profiling of this invention are not necessarily related to chronological age, but are related to the difference between the dog's phenotypic age and chronological age.

[0010] In a first aspect, the present invention provides a method for determining the risk of death of a dog; the method comprising: a) providing a DNA methylation map from a sample obtained from the dog; and b) using the DNA methylation map to determine the risk of death of the dog; wherein the DNA methylation map contains at least one methylation site as listed in Table 1.

[0011] Advantageously, the method of the present invention can be performed using commercially available DNA methylation arrays (e.g., those from Illumina).

[0012] Determining mortality risk can refer to determining the likelihood that a dog will live a longer or shorter lifespan compared to, for example, dogs of the same age, sex, and breed. Therefore, this method can determine the probability of a dog's lifespan, health span, and / or longevity compared to, for example, dogs of the same age, sex, and breed. Furthermore, methods for improving a dog's mortality risk and / or healthy lifespan probability can improve the dog's likely lifespan, health span, and / or longevity.

[0013] As used in this article, "lifespan" can refer to the length of time a subject has lived (e.g., several years). "Health span" can refer to the length of time a subject has lived without disease (e.g., several years). "Longevity" can refer to the length of time a subject has lived beyond their expected lifespan (e.g., several years).

[0014] Suitablely, the risk of death can be equated to the probability of a dog's healthy lifespan; wherein a decrease in the risk of death is equivalent to an increase in the probability of a dog's longer healthy lifespan, or an increase in the risk of death is equivalent to a decrease in the probability of a dog's longer healthy lifespan. The risk of death can be expressed as the difference between a dog's determined age (i.e., biological age) and its chronological age. For example, a difference between biological ages determined by this method relative to an increase in chronological age may indicate an increased risk of death for the dog. A difference between biological ages determined by this method relative to a decrease in chronological age may indicate a decreased risk of death for the dog. Suitablely, the risk of death and / or the probability of a healthy lifespan can be described as the dog's biological age. Suitablely, the risk of death and / or the probability of a healthy lifespan can be described as the dog's epigenetic age. Suitablely, the biological clock of the present invention may be referred to as an epigenetic clock.

[0015] Appropriately, determining that a dog's biological age is greater than its actual age indicates a higher risk of death. Appropriately, determining that a dog's biological age is less than its actual age indicates a reduced risk of death. Appropriately, determining that a dog's biological age is greater than its actual age indicates a reduced probability of a longer healthy lifespan. Appropriately, determining that a dog's biological age is less than its actual age indicates an increased probability of a longer healthy lifespan.

[0016] Appropriately, this method can be used to determine the biological age of a dog based on its risk of death and / or probability of healthy lifespan.

[0017] Therefore, in another aspect, the present invention provides a method for determining the biological age of a dog; the method comprising: a) providing a DNA methylation profile from a sample obtained from the dog; and b) using the DNA methylation profile to determine the biological age of the dog, wherein the DNA methylation profile is associated with the dog’s risk of death and / or probability of healthy lifespan, and wherein the DNA methylation profile contains at least one methylation site as listed in Table 1.

[0018] In all of these methods, determining or improving a dog's risk of death and / or probability of healthy lifespan is also applicable to determining or improving a dog's biological age; wherein a DNA methylation profile associated with a dog's risk of death and / or probability of healthy lifespan is used to determine the dog's biological age, and wherein the DNA methylation profile contains at least one methylation site as listed in Table 1.

[0019] In another aspect, the present invention provides a method for selecting a lifestyle program, dietary program, or therapeutic intervention for a dog, the method comprising: a) providing a DNA methylation profile from a sample obtained from the dog; b) using the DNA methylation profile to determine the dog's risk of death and / or probability of healthy lifespan, wherein the DNA methylation profile contains at least one methylation site as listed in Table 1; and c) selecting an appropriate lifestyle program, dietary program, or therapeutic intervention for the dog based on the risk of death determined in step b).

[0020] As used in this article, “choosing a suitable lifestyle program, dietary program or therapeutic intervention for a dog” can also encompass “recommending a lifestyle program, dietary program or therapeutic intervention for a dog” or “providing a recommended lifestyle program, dietary program or therapeutic intervention for a dog”.

[0021] In another aspect, the present invention provides a method for determining the efficacy of a lifestyle program, dietary program, or therapeutic intervention in improving the mortality risk and / or healthy lifespan probability of a dog, the method comprising: a) applying the lifestyle program, dietary program, or therapeutic intervention to the dog, wherein the lifestyle program, dietary program, or therapeutic intervention has been selected according to the foregoing aspects of the present invention; b) after applying the lifestyle program, dietary program, or therapeutic intervention to the dog for a period of time; determining the dog's mortality risk and / or healthy lifespan probability using a DNA methylation profile from a sample obtained from the dog, wherein the DNA methylation profile contains at least one methylation site as listed in Table 1; c) determining whether there is a change in the dog's mortality risk and / or healthy lifespan probability after following the lifestyle program, dietary program, or therapeutic intervention for a period of time.

[0022] In another aspect, the present invention provides a method for determining the efficacy of a lifestyle program, dietary program, or therapeutic intervention in improving the mortality risk and / or probability of healthy lifespan in dogs, the method comprising: a) determining the mortality risk of a dog using a DNA methylation profile from a sample obtained from the dog, wherein the DNA methylation profile contains at least one methylation site as listed in Table 1; b) applying a lifestyle program, dietary program, or therapeutic intervention selected based on the mortality risk determined in step a) to the dog; c) after applying the lifestyle program, dietary program, or therapeutic intervention to the dog for a period of time; determining the mortality risk of the dog using a DNA methylation profile from a sample obtained from the dog, wherein the DNA methylation profile contains at least one methylation site as listed in Table 1; d) determining whether there is a change in the mortality risk of the dog between steps a) and c).

[0023] In another aspect, the present invention provides a method for determining the efficacy of a lifestyle program, dietary program, or therapeutic intervention in improving the mortality risk and / or probability of healthy lifespan in a dog, the method comprising: a) determining the mortality risk of the dog using a DNA methylation profile from a sample obtained from the dog, wherein the DNA methylation profile contains at least one methylation site as listed in Table 1; b) applying a lifestyle program, dietary program, or therapeutic intervention selected based on the mortality risk determined in step a) to the dog; c) after applying the lifestyle program, dietary program, or therapeutic intervention to the dog for a period of time; determining the mortality risk of the dog using a DNA methylation profile from a sample obtained from the dog; d) determining whether the mortality risk of the dog changes between steps a) and c).

[0024] In another aspect, the present invention provides a method for determining the efficacy of a lifestyle program, dietary program, or therapeutic intervention in improving the probability of mortality risk and / or healthy life in a dog, the method comprising: a) determining the mortality risk of the dog using a DNA methylation profile from a sample obtained from the dog; b) applying a lifestyle program, dietary program, or therapeutic intervention selected based on the mortality risk determined in step a) to the dog; c) after applying the lifestyle program, dietary program, or therapeutic intervention to the dog for a period of time; determining the mortality risk of the dog using a DNA methylation profile from a sample obtained from the dog, wherein the DNA methylation profile contains at least one methylation site as listed in Table 1; d) determining whether there is a change in the mortality risk of the dog between steps a) and c).

[0025] Suitablely, improving a dog's risk of death and / or probability of healthy lifespan can refer to a reduction in the difference between the dog's biological age and chronological age, wherein the dog's biological age is greater than its chronological age. Alternatively, improving a dog's risk of death and / or probability of healthy lifespan can refer to maintaining or further increasing the difference between the dog's biological age and chronological age, wherein the dog's biological age is less than its chronological age. Alternatively, worsening a dog's risk of death and / or probability of healthy lifespan can refer to an increase in the difference between the dog's biological age and chronological age, wherein the dog's biological age is greater than its chronological age. Worsening a dog's risk of death and / or probability of healthy lifespan can also refer to a reduction in the difference between the dog's biological age and chronological age, wherein the dog's biological age is less than its chronological age.

[0026] Appropriately, an improvement in a dog's risk of death and / or probability of healthy lifespan can refer to a reduction in the rate of change between a dog's biological age and its chronological age, where the dog's biological age is greater than its chronological age. For example, for every 1-year increase in chronological age, a dog's biological age may have increased by 1.5 years. Following lifestyle and dietary interventions, a reduction in the rate of change results in a subsequent increase of 1.25 years in the biological age of a dog for every 1-year increase in chronological age, which could provide an improvement in the dog's risk of death and / or probability of healthy lifespan.

[0027] Improving mortality risk and / or the probability of healthy lifespan can also refer to maintaining or increasing the rate of change between a dog's biological age and its chronological age, where the dog's biological age is less than its chronological age. For example, for every 1-year increase in chronological age, the dog's biological age may have increased by less than 1 year (e.g., 0.9 years). The rate of change can alter after lifestyle, dietary, or therapeutic interventions, such that for every 1-year increase in chronological age, the dog's biological age subsequently increases by, for example, 0.8 years or less, which can provide an improvement in the dog's biological age.

[0028] This method for determining the efficacy of lifestyle programs, dietary programs, or therapeutic interventions in improving a dog's risk of death and / or probability of healthy life expectancy advantageously allows for continuous monitoring of the effectiveness of lifestyle programs, dietary programs, or therapeutic interventions in improving or maintaining a dog's health. The use of this method advantageously allows for the identification of particularly effective lifestyle programs, dietary programs, or therapeutic interventions. In contrast, if a lifestyle program, dietary program, or therapeutic intervention is determined to be ineffective based on the dog's risk of death and / or probability of healthy life expectancy, an alternative lifestyle program, dietary program, or therapeutic intervention can be implemented.

[0029] Therefore, this method enables the selection of appropriate lifestyle programs, dietary programs, or therapeutic interventions for dogs based on their mortality risk and / or healthy lifespan probability, as determined by DNA methylation profiling. For example, a highly digestible and high-quality protein diet is typically recommended based on the dog's chronological age. For instance, it might be recommended to switch the dog to a senior dog diet around 7 or 8 years of age. However, in the context of this invention, determining an increased mortality risk and / or decreased healthy lifespan probability (i.e., increased biological age) compared to the dog's chronological age allows for the determination of switching the dog to a senior dog diet at an earlier age. In contrast, dogs with a decreased mortality risk and / or increased healthy lifespan probability (i.e., decreased biological age) compared to their chronological age may be able to continue on an adult diet for a longer period.

[0030] Suitablely, this approach may include selecting and / or applying lifestyle programs, dietary programs, or therapeutic interventions to dogs after determining that they have an increased risk of death and / or a reduced probability of healthy lifespan compared to their actual age.

[0031] In another aspect, the present invention provides a method for preventing or reducing the risk of canine disease development; the method comprising:

[0032] a) Determine the mortality risk and / or healthy lifespan probability of dogs using DNA methylation profiles from samples obtained from dogs, wherein the DNA methylation profile contains at least one methylation site as listed in Table 1, and wherein the mortality risk and / or healthy lifespan probability determined for the dogs is associated with an increased likelihood of developing disease; and

[0033] b) Select a lifestyle program, dietary program or therapeutic intervention for the dog based on the mortality risk and / or healthy life probability determined in step a);

[0034] The lifestyle program, dietary program, or therapeutic intervention described herein prevents or reduces the risk of the dog developing the disease.

[0035] Appropriately, the disease is an age-related disease. For example, age-related diseases include osteoarthritis, dementia, cognitive impairment, prediabetes, diabetes, cancer, heart disease, obesity, gastrointestinal disorders, incontinence, kidney disease, sarcopenia, vision loss, hearing loss, osteoporosis, cataracts, cerebrovascular disease, and / or liver disease.

[0036] This method may also optionally include applying a lifestyle program, dietary program, or therapeutic intervention to the dog. Suitablely, the lifestyle program may be a dietary intervention or a therapeutic approach.

[0037] In another aspect, the present invention provides a method for selecting dogs suitable for receiving an anti-aging lifestyle program, dietary program, or therapeutic intervention; the method comprising: a) determining the dog's risk of death and / or probability of healthy lifespan using a DNA methylation profile from a sample obtained from the dog, wherein the DNA methylation profile contains at least one methylation site as listed in Table 1; and b) selecting the dog as suitable for receiving an anti-aging lifestyle program, dietary program, or therapeutic intervention if the dog has an increased risk of death and / or a decreased probability of healthy lifespan compared to its age.

[0038] Appropriately, while anti-aging lifestyle programs, dietary programs, or therapeutic interventions may be effective for dogs based on chronological age, they may be particularly effective when applied to dogs with an increased risk of death and / or a reduced probability of healthy lifespan compared to their chronological age. Therefore, this approach advantageously allows for the selection of dogs for whom an anti-aging lifestyle program, dietary program, or therapeutic intervention has an increased likelihood of response or an improved amount of response.

[0039] Lifestyle programs, dietary programs, or therapeutic interventions can be selected based on the determination that the dog has an increased risk of death and / or a reduced probability of healthy lifespan compared to its actual age (i.e., an increased biological age).

[0040] Lifestyle programs, dietary programs, or therapeutic interventions can be dietary interventions. Dietary interventions can include calorie-restricted diets, diets for older dogs, or low-protein diets.

[0041] DNA methylation profiles may be associated with increased biological age in the following areas: (i) tissues; (ii) organs; or (iii) physiological systems such as the immune system, gastrointestinal system, urinary system, muscular system, cardiovascular system, and / or nervous system.

[0042] The present invention also provides dietary interventions for reducing the risk of death in dogs and / or increasing the probability of a healthy lifespan in dogs, wherein the dietary interventions are applied to dogs, and wherein the risk of death and / or the probability of a healthy lifespan are determined by the method of the present invention.

[0043] The present invention further relates to the use of dietary interventions in reducing the risk of death and / or increasing the probability of a healthy lifespan in dogs, wherein the dietary interventions are applied to the dogs, and wherein the risk of death and / or the probability of a healthy lifespan are determined by the method of the present invention.

[0044] In another aspect, the present invention provides a computer-readable medium comprising instructions that, when executed, cause one or more processors to perform the methods of the present invention.

[0045] In another aspect, the present invention provides a computer system for determining the risk of death of a dog; the computer system is programmed to use the dog's DNA methylation profile to determine the dog's risk of death.

[0046] In another aspect, the present invention provides a computer system for selecting a suitable lifestyle program, dietary program, or therapeutic intervention for a dog, the computer system being programmed to perform one or more of the following steps: a) determining the dog's risk of death using a DNA methylation profile from the dog, wherein the DNA methylation profile contains at least one methylation site as listed in Table 1; and b) selecting a suitable lifestyle program, dietary program, or therapeutic intervention for the dog based on the risk of death determined in step a).

[0047] In another aspect, the present invention provides a computer system for determining the efficacy of a lifestyle program, dietary program, or therapeutic intervention in improving the mortality risk of dogs, the computer system being programmed to perform one or more of the following steps: a) determining the mortality risk of dogs using DNA methylation profiles from samples obtained from dogs before and after the lifestyle program, dietary program, or therapeutic intervention, wherein the DNA methylation profiles contain at least one methylation site as listed in Table 1; and b) determining whether there is a change in the mortality risk of dogs between samples obtained before and after the application of the lifestyle program, dietary program, or therapeutic intervention.

[0048] In another aspect, the present invention provides a computer system for determining the likelihood that a dog will benefit from an anti-aging lifestyle program, dietary program, or therapeutic intervention; the computer system is programmed to perform one or more of the following steps: a) determining the dog's risk of death using a DNA methylation profile from a sample obtained from the dog, wherein the DNA methylation profile contains at least one methylation site as listed in Table 1; b) if the dog has an increased risk of death compared to its age, identifying the dog as potentially responsive to an anti-aging lifestyle program, dietary program, or therapeutic intervention.

[0049] In another aspect, the present invention provides a computer program product comprising computer-implementable instructions for causing a programmable computer to use a DNA methylation profile of a dog to determine the dog’s risk of death; wherein the DNA methylation profile contains at least one methylation site as listed in Table 1.

[0050] In another aspect, the present invention provides a computer program product comprising computer-implementable instructions for causing a programmable computer to determine the mortality risk of a dog using a DNA methylation profile from a dog, wherein the DNA methylation profile contains at least one methylation site as listed in Table 1; and for selecting appropriate lifestyle programs, dietary programs or therapeutic interventions for the dog based on the mortality risk determined using the DNA methylation profile.

[0051] In another aspect, the present invention provides a computer program product comprising computer-implementable instructions for causing a programmable computer to: a) determine the mortality risk of a dog using DNA methylation profiles from samples obtained from the dog before and after a lifestyle, dietary, or therapeutic intervention, wherein the DNA methylation profiles contain at least one methylation site as listed in Table 1; and b) determine whether there is a change in the mortality risk of the dog between samples obtained before and after the application of the lifestyle, dietary, or therapeutic intervention.

[0052] In another aspect, the present invention provides a computer program product comprising computer-implementable instructions for causing a programmable computer to: a) determine the dog’s risk of death using a DNA methylation profile from a sample obtained from the dog, wherein the DNA methylation profile contains at least one methylation site as listed in Table 1; and b) identify the dog as potentially responsive to anti-aging lifestyle programs, dietary programs, or therapeutic interventions if the dog has an increased risk of death compared to its age.

[0053] Advantageously, the present invention allows for the determination of mortality risk and / or healthy lifespan probability based on biomarkers of multiple organ systems and functions. Therefore, this method can advantageously cover a range of potential organ dysfunctions.

[0054] Evaluating a dog's risk of death and / or probability of healthy life allows one to test several aspects of animal well-being. First, it can predict whether the animal is more likely to require dietary or supplement-based interventions. It can also be used to test the efficacy of dietary or supplement-based interventions on aging. Attached Figure Description

[0055] Figure 1 The correlation between biological age and actual age determined by the epigenetic clock of the present invention.

[0056] Figure 2 The hazard ratios of the Cox model, which explains survival by sex and delta and is stratified by breed category, are shown. Delta_res is obtained as the residuals of a linear model between DNAmAgeCoxRegression and actual age.

[0057] Figure 3 The epigenetic clock of the present invention is demonstrated using lifetime calorie restriction studies.

[0058] Figure 4 Illustrative epigenetic clocks are shown, containing A) the first 2, B) the first 5, C) the first 10, and D) the first 20 methylation sites from a complete epigenetic clock, which are associated with actual age. Detailed Implementation

[0059] Preferred features and embodiments of the invention will now be described by way of non-limiting examples. Those skilled in the art will understand that they can combine all the features of the invention disclosed herein without departing from the scope of the invention as disclosed.

[0060] It must be noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural references, unless the context clearly specifies otherwise.

[0061] As used herein, the terms “comprising” and “consisting of” are synonymous with “including” or “containing”, and are inclusive or open-ended, and do not exclude additional unlisted members, elements or method steps. The terms “comprising” and “consisting of” also include the term “composed of”.

[0062] The range of numbers includes the numbers that define that range.

[0063] The publications discussed herein are provided only for their disclosure prior to the filing date of this patent application. Nothing herein should be construed as an admission that such publications constitute prior art to the claims appended herein.

[0064] The methods and systems disclosed in this article can be used by veterinarians, healthcare professionals, laboratory technicians, pet care providers, and others.

[0065] Subjects

[0066] This method is designed for canine subjects. Therefore, the subjects of this invention are dogs.

[0067] In an alternative aspect, the subject may be a feline subject. Therefore, in an alternative aspect of the invention, the subject is a cat. Unless otherwise stated, all disclosure herein applies equally to cats.

[0068] variety

[0069] This method can utilize information about dog breeds. For example, dogs can be classified as toy, small, medium, large, or giant breeds. Suitablely, dog breeds can be classified based on weight. Suitablely, dog breeds can be classified based on the average weight of dogs of a given breed.

[0070] Dogs may be classified as small or medium-sized breeds. Classification is determined by the average weight of adult dogs of that breed. Breeds with an average weight of less than 10 kg are classified as small breeds and / or breeds with an average weight of more than 10 kg are classified as medium breeds.

[0071] In the alternative of the subject being a cat, the cat may be a domestic cat. Suitablely, the cat may be a domestic shorthaired cat.

[0072] gender

[0073] Appropriately, dogs can be classified as male or female.

[0074] Actual age

[0075] Chronological age can be defined as the amount of time elapsed from a subject's birth to a given date. Chronological age can be expressed in years, months, days, etc.

[0076] Suitablely, this method can be applied to dogs of any actual age. In some implementations, the dog may be at least about 2 years old. Suitablely, the dog may be at least about 2 years old, at least about 3 years old, at least about 4 years old, at least about 5 years old, at least about 6 years old, at least about 7 years old, at least about 8 years old, at least about 9 years old, or at least about 10 years old.

[0077] Appropriately, the dog can be at least about 7 years old.

[0078] sample

[0079] The present invention includes the step of providing or determining a DNA methylation profile from one or more samples obtained from a subject.

[0080] Appropriately, the sample may be blood, hair follicle, oral swab, saliva, or tissue sample.

[0081] Suitable samples are hair follicle, oral swab, or saliva samples. Such sample types are particularly suitable if, for example, the sample is provided outside a veterinary setting—for example, using a kit according to the invention.

[0082] Suitablely, the sample is derived from blood. The sample may contain blood components or may be whole blood. The sample preferably includes whole blood. The sample may include peripheral blood mononuclear cell (PBMC) or lymphocyte samples. Techniques for collecting samples from subjects and extracting DNA (e.g., genomic DNA) from the samples are well known in the art.

[0083] This method can be performed on one or more samples obtained from the subject. For example, the method can be performed using a first sample obtained at a given time point and a second sample obtained after a time interval following the acquisition of the first sample. The method can be performed more than once on samples obtained from the same dog over a period of time. For example, samples can be obtained repeatedly monthly, annually, or every two years. Suitablely, samples can be obtained approximately once a year (e.g., during an annual veterinary health check). This may help determine the effects of specific treatments or lifestyle changes, such as dietary interventions or changes in exercise programs.

[0084] In one implementation, the method can be applied to samples obtained from subjects prior to lifestyle changes (e.g., dietary product interventions or exercise program changes). In another implementation, the method can be applied to samples obtained from subjects both before and after, for example, dietary product interventions or exercise program changes. The method can also be applied to samples obtained at predetermined times throughout, for example, the entire dietary product intervention or exercise program change process. These predetermined times throughout, for example, the entire dietary product intervention or exercise program change process can be periodic, such as daily or every three days, or may depend on the subjects being tested.

[0085] DNA methylation

[0086] DNA methylation is the process of covalently adding a methyl group (CH3) to a cytosine base that is part of a DNA molecule. In vivo, this process is catalyzed by the DNA methyltransferase (Dnmt) family, which produces modified cytosine by transferring a methyl group from S-adenosylmethionine (SAM). The cytosine is modified at the 5th carbon atom, and the modified residue is called 5-methylcytosine (5mC). DNA methylation may also include 5-hydroxymethylcytosine (5hmc).

[0087] DNA methylation is an example of an epigenetic mechanism that can modify gene expression without altering the underlying DNA sequence. DNA methylation can suppress gene expression, for example, by acting as a recruitment signal for repressors or by directly blocking the recruitment of transcription factors. DNA methylation primarily occurs in the genome of mammalian somatic cells at sites where dinucleotides (CpGs) are adjacent to cytosine and guanine. While non-CpG methylation is observed in embryonic development, these modifications are significantly reduced in most cell types in adults. CpG islands are DNA segments with high CpG density but are typically unmethylated. These regions are associated with promoter regions, particularly those of housekeeping genes, and are thought to be kept in a permissive state that allows gene expression.

[0088] DNA methylation has been found to change with age in humans and other animals. Aging mammalian tissues show overall DNA hypomethylation, thought to be due to the gradual loss or mistargeting of DMNT1 methyltransferase activity, but with localized hypermethylation of CpG islands. Localized hypermethylation can lead to the repression of certain genes, and this can contribute to age-related diseases. The link between epigenetic changes in DNA methylation and age allows the use of “DNA methylation clocks” to estimate “biological age.” Typically, these clocks have been trained against chronological age using supervised machine learning methods, and the deviation of the “clock age” from an individual’s actual chronological age is considered an indicator of “biological” age. This is related to an individual’s chronological age, but deviations from this correlation can indicate a potential risk of age-related diseases or disorders in the individual.

[0089] Detection of specifically methylated DNA can be performed using a variety of methods (see, for example, Zuo et al., 2009; Epigenomics. 1(2): 331-345) and Rauluseviciute et al.; Clinical Epigenetics; 2019; 11(193)). Many methods can be used to detect differentially methylated DNA at specific loci in samples such as blood, urine, feces, or saliva. These methods are able to distinguish 5-methylcytosine or methylated DNA from unmethylated DNA and subsequently quantify the ratio of methylated to unmethylated DNA at specific genomic sites.

[0090] This method may include using any suitable method to determine the DNA methylation profile of a dog. Suitable methods include, but are not limited to, those described below.

[0091] Enzyme-mediated methylation sequencing (EM-seq)

[0092] Suitable for this purpose, enzymatic methods are used to detect 5mC and 5hmC. For example, enzymatic methylation sequencing (EM-seq) can be used.

[0093] In EM-seq, typically in the first enzymatic step, 5mC is oxidized to 5hmC, then to 5fC, and finally to 5caC by the activity of Tet methylcytosine dioxygenase 2 (TET2). Additionally, the use of T4-BGT enzyme glycosylates both the pre-existing 5hmC and the 5hmC generated by TET2 activity. In the second enzymatic step, after double-stranded DNA denaturation, the enzyme apolipoprotein B mRNA editing enzyme catalyzes peptide-like 3A (APOBEC3A) to deaminate cytosine, but not to the oxidized or glycosylated forms of 5mC and 5hmC. Only unmethylated cytosine is deaminated to form uracil bases. Prior to the first enzymatic step, DNA fragments can be generated by mechanical shearing and end repair, A-tailing, and ligation into sequencing adaptors, which can be achieved using, for example, NEBNext. ® DNA Ultra II reagent (NEB) is used. After the second enzymatic step, PCR can be performed using a polymerase that amplifies a template containing uracil (such as NEBNext). ® Q5U ™ EM-seq amplifies deamination-bound single-stranded DNA and can sequence or analyze the resulting library in the same manner as DNA samples produced by bisulfite sequencing. EM-seq output is typically the same as whole-genome bisulfite sequencing, but uses fewer DNA-damaging agents, thus reducing sample loss and offering superior coverage, sensitivity, and accuracy in cytosine methylation recall compared to bisulfite-converted samples. An illustrative EM-seq method is described by Vaisvila et al. (Genome Research; 2021; 31:1-10).

[0094] Methods based on bisulfite conversion

[0095] When treated with sodium bisulfite, bisulfite conversion utilizes the selective conversion of unmethylated cytosine to uracil. Denatured DNA is treated with sodium bisulfite, which converts all unmodified cytosine to uracil, and subsequent PCR amplification converts these residues to thymine. Analysis of the resulting DNA sequences can be performed using a variety of methods, examples of which include, but are not limited to: denaturing gel electrophoresis, single-strand conformation polymorphism, melting curves, real-time fluorescence PCR (MethyLight), MALDI mass spectrometry, array hybridization, and sequencing (e.g., whole-genome bisulfite sequencing, WGBS). Recently developed techniques (such as SeqCap Epi) enrich the sequences of interest before sequencing, enabling deeper coverage of more concentrated regions. Comparing the sequence abundance in bisulfite-converted samples with that in untreated controls allows analysis of methylation at target sites, where the proportion of converted sequences indicates the level of methylation at the target site.

[0096] Other variations of the bisulfite conversion method are available that can distinguish 5mC from its oxidized form, 5-hydroxymethylcytosine (5hmC), which behaves identically to 5mC under standard bisulfite conversion, and can detect further modifications such as 5-formylcytosine (5fC). These methods, such as oxBS-Seq and redBS-Seq, utilize the oxidation and reduction of these markers to alter the sensitivity of each substance to bisulfite conversion and quantify the amount of each modification at the target locus through comparative analysis.

[0097] Selective restriction endonuclease digestion method

[0098] Existing methods for analyzing DNA methylation patterns may involve the use of restriction enzymes. These methods include, for example, Restriction Marker Genome Scan (RLGS) (Costello et al., 2000; Nat Genet.; 24(2):132-8), methylation sensitivity representative differential analysis (MS-RDA) (Ushijima et al., Proc Natl Acad Sci US A. 18 March 1997; 94(6):2284-9), and differential methylation hybridization (DMH) (Huang et al., Cancer Res. 15 March 1997; 57(6):1030-4). The digestive activity of restriction endonucleases can be methylation-dependent. This specificity can be used to distinguish between methylated and unmethylated sequences. Some restriction enzymes (e.g., BstUI, HpaII, and NotI) are sensitive to methylated recognition sequences. Other restriction enzymes (such as McrBC) are specific to methylated sequences.

[0099] For example, differential methylation hybridization (DMH) (Huang et al., as described above) requires initial fragmentation of the genome using a large number of genomic restriction enzymes (such as MseI) to fragment the genome into lengths less than 200 bp. Following this step, the genomic fragments are digested using methylation-sensitive restriction endonucleases (MREs) or, in some versions of the technique, a mixture of MREs to improve coverage. Depending on the specificity of one or more enzymes used, methylated or unmethylated sequences will be degraded. The digested sequences are not amplified in subsequent PCR steps. The resulting PCR products are suitable for further processing and analysis by sequencing or a combination of microarray hybridization and fluorescent dyes.

[0100] Suitablely, this method utilizes DNA methylation maps generated by methods including the use of one or more MREs.

[0101] Suitable comparators can be used to study methylation status between conditions. DNA from healthy subjects can be compared with that from older or diseased subjects to detect changes in methylation status (Huang et al., Hum Mol Genet. 1999 Mar; 8(3):459-70). Alternatively, methylation-insensitive forms of secondary digestive enzymes (such as HpaII isoschizase MspI) can be used to generate control samples, enabling intragenomic or intergenomic comparisons of DNA methylation (Khulan et al., Genome Res. 2006 Aug; 16(8):1046-55).

[0102] In some embodiments, methods for detecting methylation include randomly shearing or fragmenting genomic DNA, cutting the DNA with a methylation-dependent or methylation-sensitive restriction enzyme, and subsequently selectively identifying and / or analyzing the cut or uncut DNA. Selective identification may include, for example, separating the cut and uncut DNA (e.g., by size) and quantifying the cut or alternatively uncut sequences of interest. Alternatively, the method may encompass amplifying intact DNA after restriction enzyme digestion, thereby amplifying only the DNA that has not been cut by the restriction enzyme in the amplified region. In some embodiments, gene-specific primers may be used for amplification. Alternatively, an adaptor may be added to the end of the randomly fragmented DNA, the DNA may be digested with a methylation-dependent or methylation-sensitive restriction enzyme, and the intact DNA may be amplified using primers that hybridize to the adaptor sequence. In this case, a second step may be performed to determine the presence, absence, or amount of a specific gene in the DNA amplification pool. In some embodiments, real-time quantitative PCR is used to amplify the DNA.

[0103] Suitable methods for detecting nucleic acid digestion include selective hybridization of a probe or primer with undigested nucleic acids. Alternatively, the probe selectively hybridizes with both digested and undigested nucleic acids, but the distinction between the two forms is facilitated, for example, by electrophoresis. Suitable detection methods for achieving selective hybridization with the hybridization probe include, for example, Southern blotting or other nucleic acid hybridization.

[0104] Suitable hybridization conditions can be determined based on the melting temperature (Tm) of the nucleic acid duplex containing the probe. Those skilled in the art will recognize that optimal hybridization reaction conditions for each probe should be determined empirically, but some general principles can be applied. Preferably, hybridization with short oligonucleotide probes is performed with low to moderate stringency. High stringency hybridization and / or washing is preferred in the case of GC-rich probes or primers, or longer probes or primers. High stringency is defined herein as hybridization and / or washing performed in about 0.1×SSC buffer and / or about 0.1% (w / v) SDS or lower salt concentration and / or at a temperature of at least 65°C or equivalent conditions. Specific stringency levels mentioned herein cover equivalent conditions using wash / hybridization solutions other than SSC known to those skilled in the art.

[0105] Simplified representation of bisulfite sequencing (RRBS)

[0106] Simplified representation of bisulfite sequencing (RRBS) uses the MspI restriction enzyme to enrich CpG-rich genomic regions—which cuts DNA at all CpG sites regardless of their DNA methylation status at CG sites—and is able to measure 5% to 10% of DNA methylation levels at all CpG sites in the mammalian genome.

[0107] Therefore, this method involves digesting DNA with a methylation-insensitive MspI prior to bisulfite conversion and sequencing. Digesting genomic DNA with MspI produces fragments that always begin with C (if cytosine is methylated) or T (if cytosine is not methylated and is converted to uracil during the bisulfite conversion reaction). This results in a non-random base pair composition. Furthermore, the basic composition is skewed due to the skewed frequencies of C and T within the sample. Various software programs are available for alignment and analysis, such as Maq, BS Seeker, Bismark, or BSMAP. Alignment with a reference genome allows the procedure to identify methylated base pairs within the genome.

[0108] Affinity-based enrichment methods

[0109] Methylated DNA can be distinguished from unmethylated DNA by using antibodies containing a methyl-CpG-binding domain (MBD), such as anti-5mC and / or methylated CpG-binding proteins. Antibodies with MBD-domain proteins can specifically separate methylated DNA relative to unmethylated DNA. The antibody-based method is commonly referred to as MeDIP, while the method using methylated CpG-binding proteins is commonly referred to as the MBD or MIRA method.

[0110] These methods require initial fragmentation of the genome, which can be achieved through mass genome digestion with frequently cleaving enzymes such as MseI, followed by affinity purification of the methylated fragments. The input DNA can be compared with the purified methylated DNA via microarray hybridization or sequencing to obtain comparative analysis of methylation levels at specific sites.

[0111] Other variations of affinity-based enrichment methods are available, such as MethylCap-Seq or MBD-Seq. These methods reduce sample complexity by using a salt gradient to elute methylated DNA fragments in a methyl-CpG abundance-dependent manner, separating CpG islands and other highly methylated loci from loci with lower CpG density. These fragments can then be sequenced separately, thereby improving sequence coverage.

[0112] Based on single-molecule sequencing and de novo methylation sequencing methods

[0113] Modern sequencing methods can sequence individual molecules directly. Single-molecule real-time (SMRT) DNA sequencing is available, such as the Sequel system from Pacific Biosciences, and has been shown to identify modified bases (such as methylated cytosine) based on polymerase kinetics. Nanopore sequencing devices capable of sequencing long DNA chains individually (such as the MinION nanopore sequencer from Oxford Nanopore Technologies) can also detect de novo base modifications, including methylation.

[0114] DNA methylation sites

[0115] Appropriately, DNA methylation sites may refer to the presence or absence of 5mC at a single cytosine, or appropriately a single CpG dinucleotide.

[0116] Suitablely, a DNA methylation site can refer to the presence or absence of methylation at multiple CpG sites across a DNA region (i.e., the number or percentage of 5mC). Suitablely, a DNA methylation site can refer to the methylation level at multiple CpG sites across a DNA region (i.e., the number or percentage of 5mC). A “DNA region” can refer to a specific portion of genomic DNA. These DNA regions can be designated by reference gene names or a set of chromosomal coordinates. Both gene names and chromosomal coordinates are well known and understood by those skilled in the art.

[0117] Where appropriate, gene names and / or coordinates may be based on the “CanFam3.1” canine reference genome (https: / / www.ncbi.nlm.nih.gov / datasets / genome / GCF_000002285.3 / , Lindblad-Toh et al.; Nature 438, 803–819 (2005)).

[0118] For example, a DNA region can define the portion of DNA near the promoter of a gene. Promoter regions are known to be CpG-rich. For instance, a DNA region could refer to approximately 3 kb upstream and 3 kb downstream of the promoter; approximately 2 kb upstream and 2 kb downstream; approximately 2 kb upstream and 1 kb downstream; approximately 2 kb upstream and 0.5 kb downstream; approximately 1 kb upstream and 0.5 kb downstream; or approximately 0.5 kb upstream and 0.5 kb downstream. Suitablely, a DNA region could refer to approximately 1 kb upstream and 0.5 kb downstream of the promoter.

[0119] DNA regions can define other DNA segments that may be located therein – including but not limited to CpG islands, enhancers, open chromatin, transcription factor binding sites, and miRNA promoter regions.

[0120] Suitable, the DNA region may contain or consist of CpG sites spaced less than about 5,000, 4,000, 3,000, 2,000, 1,000, 500, or 200 bases apart.

[0121] Suitable, the DNA region may contain or consist of CpG sites spaced about 200 to about 5,000, about 200 to about 4,000, about 200 to about 3,000, about 200 to about 2,000, or about 200 to about 1,000 bases apart.

[0122] Suitablely, a DNA region may contain one or more CpG islands. Suitablely, a DNA region may consist of CpG islands.

[0123] “CpG islands” can refer to DNA regions containing at least 200 bp, a GC percentage greater than 50%, and an observed CpG ratio greater than 60% of the expected CpG.

[0124] Suitable, the DNA methylation site does not contain CpGs that are known to contain SNPs at the CpG site.

[0125] References to each gene / DNA region detailed above should be understood as references to all forms of these molecules and their fragments or variants. As will be understood by those skilled in the art, some genes are known to exhibit allelic variations or single nucleotide polymorphisms among individuals. Variants include nucleic acid sequences from the same region sharing at least 90%, 95%, 98%, or 99% sequence identity, i.e., having one or more deletions, additions, substitutions, reverse sequences, etc., relative to the DNA region described herein. Therefore, the invention should be understood to extend to such variants, which, for the purposes of this application, can achieve the same results despite minor genetic variations in the actual nucleic acid sequences between individuals. Thus, the invention should be understood to extend to all forms of DNA resulting from any other mutation, polymorphism, or allelic variation.

[0126] Regarding the screening of methylation in these gene regions, it should be understood that the assay can be designed to screen specific DNA. Selecting which strand to analyze and targeting based on chromosome coordinates is entirely within the skill of those skilled in the art. In some cases, assays can be developed to screen two strands.

[0127] "Methylation state" can be understood as the presence, absence, and / or amount of methylation at one or more specific nucleotides within a DNA region. The methylation state of a particular DNA sequence (e.g., a DNA region as described herein) may indicate the methylation state of each base in the sequence, or the methylation state of a subset of base pairs within the sequence (e.g., the methylation state of cytosine or the methylation state of one or more specific restriction enzyme recognition sequences), or it may indicate information about the regional methylation density within the sequence without providing precise information about where methylation occurs within the sequence. Methylation state may optionally be represented or indicated by a "methylation value."

[0128] Suitablely, an EM-Seq strategy can be used to determine DNA methylation. In this method, the methylation level can be determined as the fraction of "C" bases in the total "C"+"U" bases at the target CpG site "i" after enzyme and APOBEC3A transformation treatment. In other embodiments, the methylation level can be determined as the fraction of "C" bases in the total "C"+"T" bases at site "i" after enzyme and APOBEC3A transformation treatment and subsequent nucleotide amplification. The average methylation level at each site can then be evaluated to determine whether one or more thresholds are met.

[0129] In some implementations, particularly when using bisulfite conversion and sequencing methods, the methylation level can be determined as the fraction of "C" bases in the total "C"+"U" bases at the target CpG site "i" after bisulfite treatment. In other implementations, the methylation level can be determined as the fraction of "C" bases in the total "C"+"T" bases at site "i" after bisulfite treatment and subsequent nucleotide amplification. The average methylation level at each site can then be evaluated to determine whether one or more thresholds are met.

[0130] Alternatively, methylation values ​​can be generated, for example, by quantifying the amount of intact DNA present after restriction digestion with a methylation-dependent restriction enzyme. In this example, if a specific sequence in the DNA is quantified using quantitative PCR, an amount of template DNA approximately equal to that of the simulated treatment control indicates that the sequence is not highly methylated, while a amount substantially less than that of the template present in the simulated treatment sample indicates the presence of methylated DNA at that sequence. Therefore, values ​​from the example above (i.e., methylation values) represent the methylation status and can thus be used as a quantitative indicator of methylation status. This is particularly useful when it is necessary to compare the methylation status of a sequence in a sample with a threshold.

[0131] This invention is not limited to the exact number of methylated residues considered to indicate biological age, as some variation will occur between samples. This invention is also not necessarily limited to the location of methylated residues (e.g., specific methylation sites).

[0132] In one implementation, a screening method may be employed that is specifically designed to assess the methylation status of one or more specific cytosine residues or the corresponding cytosine at position n+1 on the DNA strand.

[0133] Enrichment and Detection Methods

[0134] Determining a DNA methylation profile may include the step of enriching selected DNA regions in a DNA sample. For example, the method may include the step of enriching DNA regions in a DNA sample that contain DNA methylation sites, which constitute a DNA methylation profile.

[0135] Suitable enrichment methods are known in the art and include, for example, amplification-based or hybridization-based methods. Amplification enrichment typically refers to PCR-based enrichment, for example, using primers targeted at the DNA region to be enriched. Any suitable form of amplification can be used, such as polymerase chain reaction (PCR), rolling circle amplification (RCA), reverse polymerase chain reaction (iPCR), in situ PCR, strand displacement amplification, or cycling probe techniques.

[0136] Hybridization enrichment, or capture-based enrichment, typically refers to the use of hybridization probes (or capture probes) that hybridize with the DNA region to be enriched.

[0137] Hybridization probes can be directly attached to a solid vector, or they can contain a portion, such as biotin, to allow binding to a solid vector (e.g., beads coated with streptavidin) suitable for capturing the biotin portion. In either case, DNA containing a sequence complementary to the probe can be captured, allowing the separation of DNA containing the region of interest from DNA not containing that region. Therefore, such a capture step allows for the enrichment of the region of interest. For example, the DNA region could be a region adjacent to a gene promoter.

[0138] The arrays used in this article can vary depending on the probe composition and the intended use of the array. For example, the number of nucleic acids (or CpG sites) detected in the array can be at least 10, 100, 1,000, 10,000, 100,000, 1 million, 10 million, 100 million, or more. Alternatively or additionally, the number of nucleic acids (or CpG sites) detected can be selected to be no more than 100 million, 10 million, 1 million, 100,000, 10,000, 1,000, 100, or fewer. Similar ranges can be obtained using nucleic acid sequencing methods, such as those known in the art; for example, next-generation or massively parallel sequencing.

[0139] Appropriately, the enrichment step can be performed before or after the step of separating or differentiating methylated and unmethylated DNA.

[0140] As used herein, the term "enrichment" or "DNA" or "DNA region" refers to the process of increasing the (absolute) amount and / or proportion of DNA containing the desired sequence compared to the amount and / or proportion of DNA containing the desired sequence in the starting material. In this respect, enrichment by amplification increases the amount and proportion of the desired sequence. Enrichment by capture-based enrichment increases the proportion of DNA containing the desired sequence.

[0141] After processing the DNA to distinguish between methylated and unmethylated sites, this method may also include a step of identifying methylated or unmethylated sites (i.e., in the original sample).

[0142] The identification steps may include any suitable method known in the art, such as array detection or sequencing (e.g., next-generation sequencing).

[0143] The sequencing qualification step preferably includes next-generation sequencing (massively parallel or high-throughput sequencing). Next-generation sequencing methods are well known in the art, and in principle, any method can be considered for use in this invention. Next-generation sequencing technology can be performed according to the manufacturer's instructions (e.g., provided by Roche, Illumina, or Applied Biosystems).

[0144] In a preferred embodiment, the sample is processed by using an enzymatic reaction to convert DNA methylation, preparing a whole-genome library, and measuring the methylation profile by sequencing (EM-Seq).

[0145] In one embodiment, the sample is processed by using an enzymatic reaction to convert DNA to methylation, preparing a whole-genome library, hybridizing the converted library with a capture probe (preferably a capture probe capable of capturing DNA regions near gene promoters), and measuring the methylation profile by sequencing (EM-Seq).

[0146] Advantageously, the method of the present invention can be performed using commercially available DNA methylation arrays. In one embodiment, DNA methylation is converted by bisulfite conversion, the converted DNA is optionally amplified, and then labeled (e.g., with a fluorescent dye) and hybridized with a methylation array (e.g., a mammalian methylation array) to process the sample. Suitable methylation arrays are available, for example, from Illumina and are described in WO20150705 and Arneson et al. (Nature Communications; 13(782); 2022).

[0147] DNA methylation map

[0148] A “DNA methylation profile” or “methylation map” can refer to the presence, absence, amount, or level of 5mC at one or more DNA methylation sites. Preferably, a “methylation profile” refers to the presence, absence, amount, or level of 5mC at multiple DNA methylation sites. Therefore, the presence, absence, amount, or level of 5mC at each individual DNA methylation site within multiple sites can be assessed, and this helps determine the risk of death and / or the probability of healthy lifespan in dogs. Therefore, the quality and / or efficacy of this method can be improved by combining values ​​from multiple DNA methylation markers.

[0149] Suitablely, the biological clock of the present invention includes methylation maps from multiple methylation sites.

[0150] Suitablely, the presence or absence of 5mC from at least 3, at least 5, at least 10, at least 20, at least 50, at least 100, at least 200, at least 500, at least 1000, at least 2000, at least 5000, at least 10000, at least 50000, at least 10000, at least 250000, or at least 500000 DNA methylation sites can be used to determine the probability of a dog’s risk of death and / or healthy lifespan (i.e., biological age).

[0151] Suitablely, a methylation profile can refer to the presence or absence of 5mC from at least 100, at least 200, at least 500, at least 1000, or at least 2000 DNA methylation sites.

[0152] Appropriately, a methylation profile can refer to the presence or absence of 5mC from approximately 100, 200, 500, 1000, or 2000 DNA methylation sites.

[0153] To generate a biological clock for determining mortality risk and / or the probability of healthy lifespan, an initial methylation map can be processed or simplified to produce a restricted methylation map, which can then be used to generate the biological clock.

[0154] For example, the initial methylation map can be processed or simplified by, for instance, using DNA regions instead of individual cytosines, selecting a subset of methylation sites associated with specific physiological or biochemical pathways, performing correlation analysis and retaining one or more representative DNA methylation sites for each cluster, or performing differential analysis to pre-select DNA methylation sites or retain DNA methylation sites that vary more between young and older dogs.

[0155] For example, a DNA region can be any DNA region as defined herein.

[0156] Suitablely, a methylation map can point to DNA methylation sites of genes associated with specific physiological or biochemical pathways. Therefore, a methylation map can enable the determination of the biological age of a specific tissue, organ, or physiological system. Determining the biological age of a specific tissue, organ, or physiological system advantageously allows the method to be used in a manner focused on the pathology and disease of that tissue, organ, or physiological system. For example, if a particular breed of dog is known to be associated with muscular or cardiovascular disease, determining the biological age of that physiological system may be advantageous.

[0157] Appropriately, the physiological system can be the inflammatory system, the muscular system, the cardiovascular system, and / or the nervous system.

[0158] The biological age of a specific tissue, organ, or physiological system can be determined using a DNA methylation map, which contains or is composed of methylation sites of genes preferentially or specifically expressed from that tissue, organ, or physiological system. Gene classification by specific tissue, organ, or physiological system is publicly available at, for example, Gene Ontology (http: / / geneontology.org / ), the KEGG pathway database (https: / / www.genome.jp / kegg / ), or MSIgDB (https: / / www.gsea-msigdb.org / gsea / msigdb / index.jsp).

[0159] In some implementations, the threshold is selected for those sites that have the highest average methylation values ​​for epigenetic age predictors. For example, the threshold could be those sites with the highest average methylation levels, which are the top 50%, top 40%, top 30%, top 20%, top 10%, top 5%, top 4%, top 3%, top 2%, or top 1% of the average methylation levels of all sites “i” tested for predictors such as biological clocks.

[0160] Alternatively, the threshold may be those sites where the average methylation level is at or above the percentile of 50, 60, 70, 80, 90, 95, 96, 97, 98, or 99. In other embodiments, the threshold may be based on the absolute value of the average methylation level. For example, the threshold may be those sites where the average methylation level is greater than 99%, greater than 98%, greater than 97%, greater than 96%, greater than 95%, greater than 90%, greater than 80%, greater than 70%, greater than 60%, greater than 50%, greater than 40%, greater than 30%, greater than 20%, greater than 10%, greater than 9%, greater than 8%, greater than 7%, greater than 6%, greater than 5%, greater than 4%, greater than 3%, or greater than 2%. Relative and absolute thresholds may be applied alone or in combination to the average methylation level at each site “i”. As an illustration of the combined threshold application, a subset of sites may be selected from the top 3% of all sites that pass the average methylation level test and also have an absolute average methylation level greater than 6%. The result of this selection process is a DNA methylation profile of a specific hypermethylation site (e.g., a CpG site), which is considered to be the most informative for determining the probability of death risk and / or healthy lifespan.

[0161] Suitablely, the DNA methylation profile used to determine the risk of death and / or the probability of healthy lifespan according to the present invention may include at least one methylation site as listed in Table 1.

[0162] Suitablely, a methylation site can be defined as a methylation marker present in any one or more of SEQ ID NO: 1-49. SEQ ID NO: 1-49 shows sequences near methylation markers in the “CanFam3.1” canine reference genome (https: / / www.ncbi.nlm.nih.gov / datasets / genome / GCF000002285.3 / , Lindblad-Toh et al.; Nature 438, 803–819 (2005)), where the “CG” methylation marker is located at the end of the sequence (at the beginning or end of the sequence, depending on whether the site is on the positive or negative strand in the reference genome). The location of the “CG” methylation marker is provided in Table 1. In addition, the corresponding CGid is provided for each “CG” methylation marker (see Arneson et al.; Nature Communications; 13(783); 2022 and https: / / github.com / shorvath / MammalianMethylationConsortium / tree / v1.0.0).

[0163] Suitablely, methylation sites can be defined using the CG start and CG end columns in Table 1. For example, for DNA methylation site number 1 (SEQ ID NO: 1), the provided sequence is chr10:63518903-63518952, and the methylation marker is chr10:63518903-63518904.

[0164] Suitablely, the DNA methylation map may contain at least 3, at least 5, at least 10, at least 20, at least 30, at least 40, or preferably each of the methylation sites listed in Table 1.

[0165] Appropriately, the DNA methylation map includes at least one methylation site selected from sites numbered 1-40 as listed in Table 1.

[0166] Suitable, the DNA methylation map includes at least 3, at least 5, at least 10, at least 20, at least 30 methylation sites or each methylation site from the methylation sites numbered 1-40 as listed in Table 1.

[0167] Appropriately, DNA methylation maps may include methylation sites as listed in Table 2.

[0168] Suitablely, a DNA methylation map may include methylation sites as listed in Table 3. Suitablely, a DNA methylation map may include methylation sites 1 to 4 as listed in Table 3.

[0169] Suitablely, a DNA methylation map may include methylation sites as listed in Table 4. Suitablely, a DNA methylation map may include methylation sites 1 to 4 and 5 to 8 as listed in Table 4.

[0170] Suitablely, a DNA methylation map may include methylation sites as listed in Table 5. Suitablely, a DNA methylation map may include methylation sites 1 to 4, 5 to 9, and 15 to 17 as listed in Table 5.

[0171] Determination of DNA methylation sites / DNA methylation maps indicating mortality risk and / or probability of healthy lifespan.

[0172] This invention includes using DNA methylation mapping to determine the risk of death and / or the probability of healthy lifespan in dogs, wherein the DNA methylation map contains at least one methylation site as listed in Table 1. Therefore, this invention includes using DNA methylation mapping to generate a biological clock associated with the risk of death and / or the probability of healthy lifespan. The biological clock of this invention may also be referred to as an "epigenetic clock".

[0173] For example, DNA methylation sites or DNA methylation maps indicating mortality risk and / or the probability of healthy lifespan can be provided through training datasets and machine learning methods. Suitablely, the machine learning method can be a supervised machine learning method.

[0174] For example, DNA methylation sites or DNA methylation maps can be trained on a dataset that includes dogs with known mortality outcomes (survival or death) and actual age. Suitablely, DNA methylation sites or DNA methylation maps can be trained on a dataset that includes dogs with known mortality outcomes and combinations of actual age and known breed and / or sex.

[0175] For example, a model of DNA methylation sites or DNA methylation maps that indicates mortality risk and / or the probability of healthy lifespan can be provided by using a machine learning framework to train a dataset of methylation states at multiple DNA methylation sites on a training dataset of dogs with known mortality outcomes (survival or death) and full age, and testing the model against a retained cohort to validate the model's accuracy.

[0176] Machine learning frameworks can include fitting a penalty model to a training dataset of dogs with known mortality outcomes (survival or death) and actual age (and optional breed and / or sex); for example, using the glmnet R package.

[0177] Machine learning frameworks can include fitting a penalized model to a training dataset of dogs with known mortality outcomes (survival or death, age at death) and actual age (and optional breed and / or sex); for example, using the glmnet R package.

[0178] Appropriately, the penalty model can be, for example, a penalty Cox regression, a minimum angle regression path (LARS) Cox regression, or a penalty survival model.

[0179] Machine learning frameworks may include fitting penalized Cox regression to a training dataset of dogs with known mortality outcomes (survival or death) and actual age (and optional breed and / or sex); for example, using the glmnet R package.

[0180] Suitable, the machine learning framework may include a penalized model, preferably penalized Cox regression, that fits known mortality outcomes (survival or death) / survival rates as interpreted by DNA methylation profiles and full age (and optionally breed and / or sex).

[0181] Suitable, the machine learning framework may include a penalized model, preferably penalized Cox regression, that fits known mortality outcomes (survival or death) / survival rates as interpreted by DNA methylation profiles, full age, breed, and sex.

[0182] As used in this article, “known mortality outcome (survival or death)” or “known mortality outcome (survival or death) and age at death” can also be referred to as “survival rate”.

[0183] Appropriately, machine learning frameworks can be used to determine models that include a set of DNA methylation sites or DNA methylation maps that indicate the risk of death and / or the probability of healthy lifespan.

[0184] Appropriately, machine learning frameworks can generate predicted risks (e.g., predicted risk ratios); for example, through penalized Cox regression. This can be converted to biological / epigenetic age using methods known in the art; for example, by fitting a linear model to interpret actual age using predicted risks.

[0185] The model may include the methylation state at multiple DNA methylation sites; where the methylation state at each site is considered by multiplying by a coefficient value in the model.

[0186] Appropriately, sex can be encoded as a numerical value, where 0 represents female and 1 represents male.

[0187] Appropriately, breed can be encoded as a numerical value, where 0 represents a small breed and 1 represents a medium breed.

[0188] A dog's biological age can be expressed in years, months, days, etc.

[0189] The coefficient values ​​for each parameter typically depend on the units of measurement of all variables in the model. As those skilled in the art will understand, the value of each coefficient will therefore depend on, for example, the number and nature of the different parameters used in the model and the nature of the training data provided. Therefore, conventional statistical methods can be applied to the training dataset to obtain the coefficient values. This approach involves, for example, computing two gompertz or weibull functions on the training set (e.g., given the dog's state (survival or death)), one function modeling survival as a function of methylation profile, actual age, breed type (small or medium-sized dog), and sex (Model 1), while the second function considers only actual age, breed type, and sex (Model 2). These models can be fitted in the R software environment using the flexsurv package (v 2.1).

[0190] Biological age can be defined as a time variable (“actual age”), under which the survival probability of an animal given by Model 2 is equal to the survival probability given by Model 1 at its actual age.

[0191] A dog's biological age can be expressed in years, months, days, etc.

[0192] Preferably, the risk of death and / or the probability of healthy lifespan are expressed as the difference between the dog's biological age and its actual age.

[0193] Compare with reference or control

[0194] This method may also include the step of comparing differences in DNA methylation at one or more sites in the test sample with one or more references or controls. The presence or absence of DNA methylation at one or more sites in the reference or control may be associated with a predefined probability of mortality risk and / or healthy lifespan (i.e., biological age). In some embodiments, the reference value is a value previously obtained for subjects or subject groups with a known probability of mortality risk and / or healthy lifespan (i.e., biological age). The reference value may be based on the known DNA methylation status at one or more sites from subject groups with a known mortality status (survival or death), full-time age, breed, and / or sex, such as mean or median levels.

[0195] Combining DNA methylation mapping with other measurements and / or features

[0196] Suitablely, this method also includes combining the DNA methylation profile with one or more of the dog's actual age, breed, and / or sex. By combining this information, the biological age associated with mortality risk and / or probability of healthy lifespan can be determined.

[0197] Subject classification

[0198] The biological age determined by the method of this invention can also be compared to one or more predetermined thresholds (i.e., the difference from chronological age). Using such thresholds, subjects can be stratified into categories indicating a defined risk (e.g., low, medium, or high defined risk). The degree of divergence from the thresholds can be used to determine which individuals will benefit most from certain interventions. In this way, dietary interventions and lifestyle changes can be optimized.

[0199] Methods for selecting / monitoring lifestyle programs, dietary plans, or therapeutic interventions for subjects.

[0200] In another aspect, the present invention provides methods for selecting lifestyle programs, dietary programs, or therapeutic interventions for subjects. Lifestyle changes can be any changes described herein, such as changes in dietary interventions and / or exercise programs. Lifestyle changes can also include the application of therapeutic methods.

[0201] Lifestyle programs, dietary programs, or therapeutic interventions can be applied to dogs at any suitable time period. After the stated time period, this method can be used to reassess the dog's risk of death and / or probability of healthy life expectancy in order to determine the efficacy of the lifestyle program, dietary program, or therapeutic intervention in reducing the dog's risk of death and / or increasing the dog's probability of healthy life expectancy. For example, lifestyle programs, dietary programs, or therapeutic interventions can be applied for at least 2 weeks, at least 4 weeks, at least 8 weeks, at least 16 weeks, at least 32 weeks, or at least 64 weeks. Lifestyle programs, dietary programs, or therapeutic interventions can be applied for at least 3 months, at least 6 months, at least 12 months, at least 24 months, at least 36 months, at least 48 months, or at least 60 months.

[0202] Lifestyle programs, dietary programs, or therapeutic interventions can be referred to as anti-aging lifestyle programs, dietary programs, or therapeutic interventions.

[0203] Preferably, the change is a dietary intervention as described herein. The term "dietary intervention" refers to an external factor applied to a subject and causing a change in the subject's diet. More preferably, the dietary intervention includes the administration of at least one dietary product, dietary protocol, or nutritional supplement.

[0204] Dietary interventions can be diets, dietary plans, supplements, or supplement plans, or a combination of diets and supplements, or a combination of diets and multiple supplements.

[0205] The dietary interventions or dietary products described herein can be any suitable dietary program, such as calorie-restricted diets, senior dog diets, low-protein diets, phosphorus diets, low-protein diets, potassium-supplemented diets, polyunsaturated fatty acid (PUFA) supplemented diets, antioxidant supplemented diets, vitamin B supplemented diets, liquid diets, selenium-supplemented diets, omega-3-6 ratio diets, or diets supplemented with carnitine, branched-chain amino acids or derivatives, nucleotides, nicotinamide precursors (such as nicotinamide mononucleotide (MNM) or nicotinamide nucleoside (NR)), or any combination thereof.

[0206] Appropriately, the dietary intervention or dietary product may be a calorie-restricted diet, a senior dog diet, or a low-protein diet. Appropriately, the dietary intervention or dietary product may be a calorie-restricted diet. Appropriately, the dietary intervention or dietary product may be a low-protein diet.

[0207] Dietary interventions can be determined based on the dog's baseline maintenance energy requirement (MER). Appropriately, MER can be the amount of food required to maintain the dog's stable weight (a change of less than 5% over three weeks).

[0208] For example, young, growing dogs are generally considered to benefit from a high-energy / high-protein diet; however, older dogs may have lower energy requirements, and therefore their diets can be adjusted accordingly. In particular, many manufacturers produce "senior" dog foods that are lower in calories and higher in fiber, but with protein and fat levels suitable for older dogs.

[0209] Appropriately, a calorie-restricted diet may comprise approximately 50%, 55%, 60%, 65%, 75%, 80%, 85%, or 90% of a dog's MER. Appropriately, a calorie-restricted diet may comprise approximately 60% or 75% of a dog's MER.

[0210] Appropriately, a low-protein diet may contain less than 20% protein (dry matter %). For example, a low-protein diet may contain less than 19% protein (dry matter %).

[0211] These diets are typically recommended based on the dog's actual age. For example, it might be recommended to switch the dog to a senior dog diet around 7 or 8 years of age. However, in the context of this invention, an increased risk of death for the dog, as anticipated at a given actual age, may allow for the decision to switch the dog to a senior dog diet at an earlier age. In contrast, dogs with a lower risk of death compared to their actual age may be able to continue on an adult diet for a longer period.

[0212] Dietary interventions may include foods, supplements, and / or beverages that contain nutrients and / or bioactive agents that mimic the benefits of calorie restriction (CR) without restricting daily calorie intake. For example, foods, supplements, and / or beverages may contain functional ingredients with similar CR benefits. Suitablely, foods, supplements, and / or beverages may contain autophagy inducers. Suitablely, foods, supplements, and / or beverages may contain fruits and / or nuts (or extracts thereof). Suitable examples include, but are not limited to, pomegranate, strawberry, blackberry, camu camu, walnut, chestnut, pistachio, and pecan. Suitablely, foods, supplements, and / or beverages may contain probiotics, with or without fruit or nut extracts.

[0213] Modifying a subject's lifestyle also includes instructing them to make lifestyle changes, such as prescribing more exercise. Similar to dietary interventions, determining an increased risk of death in dogs compared to what is expected at a given apex age may allow for the identification of an appropriate exercise program for the dog.

[0214] Modifying a subject's lifestyle also includes selecting or recommending treatment methods or protocols. These treatment methods or protocols can be used to treat and / or prevent conditions such as arthritis, dental diseases, endocrine disorders, heart disease, diabetes, liver disease, kidney disease, prostate disorders, cancer, and behavioral or cognitive impairments. Suitablely, preventative treatment may be administered to dogs identified as being at risk for such conditions due to an increased risk of death and / or based on specific biomarkers known to be associated with disease-related pathways. In other implementations, dogs identified as being at risk for certain conditions (due to an increased risk of death and / or based on specific biomarkers known to be associated with disease-related pathways) may be monitored more regularly so that diagnosis and treatment can be initiated as early as possible.

[0215] This invention also relates to monitoring and / or determining the efficacy of anti-aging therapies or developing anti-aging therapies. Anti-aging therapies may include, for example, "rejuvenation" interventions. Rejuvenation interventions are designed to induce a reduction in the epigenetic or biological age of a subject. Suitably, a rejuvenation intervention may reprogram the epigenetic age to that of a very young dog. Examples of such rejuvenation interventions include, but are not limited to, gene therapies that suitably reprogram the epigenetic age to that of a very young dog. This method is particularly suitable for monitoring and / or determining the efficacy of lifestyle programs, dietary programs, or therapeutic interventions or developing lifestyle programs, dietary programs, or therapeutic interventions to reduce biological age.

[0216] Therefore, the present invention can advantageously identify dogs that are expected to respond particularly well to a given intervention (e.g., a lifestyle program, dietary program, or therapeutic intervention). Thus, the intervention can be applied in a more targeted manner to dogs that are expected to respond.

[0217] In one aspect, the present invention provides a method for determining the efficacy of a lifestyle program, dietary program, or therapeutic intervention in reducing the risk of death and / or increasing the probability of a healthy lifespan in a dog, the method comprising: a) applying the lifestyle program, dietary program, or therapeutic intervention to the dog, wherein the lifestyle program, dietary program, or therapeutic intervention has been selected according to the method of the present invention; b) after applying the lifestyle program, dietary program, or therapeutic intervention to the dog for a period of time; determining the dog's risk of death and / or probability of healthy lifespan using a DNA methylation profile from a sample obtained from the dog, wherein the DNA methylation profile contains at least one methylation site as listed in Table 1; c) determining whether there is a change in the dog's risk of death after following the lifestyle program, dietary program, or therapeutic intervention for a period of time.

[0218] In another aspect, the present invention provides a method for determining the efficacy of a lifestyle program, dietary program, or therapeutic intervention for reducing the risk of death and / or increasing the probability of a healthy lifespan in a dog, the method comprising: a) determining the dog's risk of death and / or probability of healthy lifespan using a DNA methylation profile from a sample obtained from the dog, wherein the DNA methylation profile contains at least one methylation site as listed in Table 1; b) applying a lifestyle program, dietary program, or therapeutic intervention selected based on the risk of death and / or probability of healthy lifespan determined in step a) to the dog; c) after a period of time following the application of the lifestyle program, dietary program, or therapeutic intervention to the dog; determining the dog's risk of death and / or probability of healthy lifespan using a DNA methylation profile from a sample obtained from the dog, wherein the DNA methylation profile contains at least one methylation site as listed in Table 1; d) determining whether there is a change in the dog's risk of death and / or probability of healthy lifespan between steps a) and c).

[0219] Appropriately, a lifestyle program, dietary program, or therapeutic intervention may have been applied to a dog for a period of time before the first mortality risk and / or healthy life probability is determined; however, the effectiveness of the lifestyle program, dietary program, or therapeutic intervention in improving the mortality risk and / or healthy life probability (i.e., reducing mortality risk and / or increasing healthy life probability) can still be monitored by determining the mortality risk and / or healthy life probability at two or more times during the application of the lifestyle program, dietary program, or therapeutic intervention.

[0220] Suitablely, this method may include an “ecosystem”; particularly a digital ecosystem. Suitablely, this method may include providing a sample obtained from a dog, optionally using a kit according to the invention; and (b) providing a sample for subsequent DNA extraction (e.g., by mail) for measuring DNA methylation in DNA extracted from the sample, thereby obtaining a DNA methylation profile.

[0221] The DNA methylation map can then be used according to any of the methods described herein; preferably, a computer system or computer program product according to the invention is used.

[0222] Then, the computer system or computer program can prepare and share reports detailing the results of the analysis / method or any other results of the method, for example, in the form of selecting or recommending suitable lifestyle programs, dietary programs or therapeutic interventions for dogs.

[0223] Suitable samples can be those that can be obtained by the dog owner at home (e.g., without the need for a veterinarian or healthcare professional). Suitable samples can be hair follicles, oral swabs, or saliva samples.

[0224] Uses of dietary intervention

[0225] In one aspect, the present invention provides a dietary intervention for reducing the risk of death in dogs and / or increasing the probability of a healthy lifespan in dogs, wherein the dietary intervention is applied to the dogs, and wherein the risk of death and / or the probability of a healthy lifespan are determined by the method herein.

[0226] In another aspect, the present invention provides the use of dietary intervention in reducing the risk of death and / or increasing the probability of a healthy lifespan in dogs, wherein the dietary intervention is applied to the dogs, and wherein the risk of death and / or the probability of a healthy lifespan are determined by the method herein.

[0227] As described in this article, dietary interventions can be dietary products, dietary plans, or nutritional supplements.

[0228] Computer program products

[0229] This method can be executed using a computer. Therefore, this method can be executed on a computer.

[0230] Where appropriate, the computer can prepare and share a report detailing the results of this method.

[0231] The methods described herein can be implemented as a computer program running on general-purpose hardware such as one or more computer processors. In some embodiments, the functionality described herein can be implemented via a device such as a smartphone, tablet terminal, or personal computer.

[0232] In one aspect, the present invention provides a computer program product comprising computer-implementable instructions for causing a programmable computer to determine, as described herein, the mortality risk and / or healthy lifespan probability of a dog.

[0233] In one implementation, the user inputs the level of one or more DNA methylation markers as defined herein into the device, optionally along with the dog's age, breed, and sex. The device then processes this information and provides a determination of the dog's biological age. Alternatively, the device then processes this information and determines an appropriate lifestyle program, dietary program, or therapeutic intervention for the dog based on its biological age.

[0234] The device can typically be a server on a network. However, any device can be used as long as it can process biomarker data and / or additional parameters or characteristics using a processor, central processing unit (CPU), etc. For example, the device can be a smartphone, tablet terminal, or personal computer, and output information indicating the determined biological age of the dog or, based on the biological age of the dog, appropriate lifestyle programs, dietary programs, or therapeutic interventions.

[0235] Those skilled in the art will understand that they are free to combine all the features of the invention described herein without departing from the scope of the invention disclosed herein.

[0236] Example

[0237] The present invention will now be further described by way of examples, which are intended to help those skilled in the art to implement the invention, without limiting the scope of the invention in any way.

[0238] Example 1 - An exemplary method for generating epigenetic biological clocks

[0239] Whole blood samples from a canine cohort, including data from blood and oral swab samples, were analyzed as follows: DNA extraction, DNA methylation conversion using bisulfite conversion, and amplification of the converted DNA. The DNA was then hybridized to a mammalian methylation array (Illumina) and labeled with a fluorescent dye. After hybridization, the array was washed and scanned using the iScan microarray scanner. The raw data were read and normalized using the sesame R package (Zhou W, Triche TJ, Laird PW, Shen H (2018). “SeSAMe: ​​reducing artifactual detection of DNA methylation by Infinium BeadChips in genomic deletions.” Nucleic Acids Research, gky691.doi:10.1093 / nar / gky691.)

[0240] Several steps were taken to process the array data:

[0241] • Removed outliers from inter-array correlations

[0242] • Samples with incorrectly predicted species were excluded from the dataset.

[0243] • Misclassified samples and technically duplicated samples are also removed to maintain data accuracy.

[0244] In survival analysis, the time variable represents the duration between the sample collection date and the occurrence of the event. In this case, the event could be death (N = 359) or adoption / transfer / activation (N = 412).

[0245] Beta value preparation:

[0246] To reduce the dimensionality of the Beta value matrix, a filtering method was applied based on the reliability of the probes in technically replicated samples. This involved training 13 pairs of technically replicated samples and performing regression analysis using the model beta ~ ReplicateID. This process removed probes that exhibited greater variation in methylation levels among biologically replicated samples than among technically replicated samples. This analysis was performed using the limma package.

[0247] Probes with a detection p-value greater than 0.05 in 10% of the samples were also removed. This filtering process aims to eliminate probes with lower reliability.

[0248] Finally, all CpG IDs that did not match the canine genome were removed.

[0249] A total of 12,263 probes were selected through this process.

[0250] To obtain a reliable estimate of the predictor accuracy, a rigorous approach was employed involving 10 distinct data partitions, each containing unique training and testing datasets (90% of the samples in each partition were used for training the model, and 10% for testing). The test subset was constructed in such a way that each sample appeared in only one validation dataset.

[0251] Optimal parameters were identified using a regularized Cox regression model with 10-fold cross-validation, stratified by variety category. Partial likelihood bias was obtained by evaluating the bias based on the partial likelihood function. This quantifies the difference between observed survival time and survival time predicted by the Cox regression model. A lower partial likelihood bias value indicates a better fit of the model to the data.

[0252] Then, the final predictor is trained using all the samples.

[0253] The model selected 49 methylation sites for the biological clock.

[0254] The Cox model is penalized to predict hazard ratios, which are then converted to biological age by linear regression fitting of the hazard ratios to chronological age. For the Cox model, calibration is performed to convert hazard to age. The biological age is calculated as follows:

[0255] coxAge = lmIntercept+ lmcoeff*(+coeff*meth_value)

[0256] Figure 1 The correlation between biological age and actual age determined by the epigenetic clock of the present invention is shown.

[0257] Figure 2 The hazard ratios of the Cox model, which explains survival by sex and delta and is stratified by breed, are shown. Delta_res is obtained as the residuals of a linear model between DNAmAgeCoxRegression and chronological age. A positive value of delta indicates that the subject is biologically older than their chronological age. Figure 2 In this study, the hazard ratio was significantly greater than 1, indicating that biologically older subjects would have a higher risk of death.

[0258] Figure 3 The epigenetic clock of the present invention is demonstrated using lifetime calorie restriction studies. Figure 3 The results showed that the biological age of the calorie restriction group (R) was consistently lower than that of the control group (C).

[0259] Additional biological clocks were generated using only the first 2, 5, 10, and 20 sites from the complete site list shown in Table 1; and each showed a correlation with biological age (see Table 1). Figure 4 These clocks were generated by selecting the first n sites based on the absolute values ​​of the coefficients of the full clock (in descending order, with the largest coefficients taken first). The first n sites were used as predictors to fit linear models that account for full-age, respectively. Details of the first 3, 5, 10, and 20 clocks are shown in Tables 2 through 5.

[0260] All publications mentioned in the foregoing description are incorporated herein by reference. Various modifications and variations of the methods, compositions, and uses disclosed herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. While the invention has been disclosed in conjunction with specific preferred embodiments, it should be understood that the invention protected by the claims should not be unduly limited to such specific embodiments. In fact, various modifications to the modes disclosed for practicing the invention that are apparent to those skilled in the art are intended to fall within the scope of the following claims.

[0261] Table 1

[0262]

[0263]

[0264]

[0265]

[0266]

[0267]

[0268] Table 2 – Top 2 Clocks

[0269]

[0270] Table 3 – Top 5 Clocks

[0271]

[0272]

[0273] Table 4 – Top 10 Clocks

[0274]

[0275]

[0276]

[0277] Table 5 – Top 20 Clocks

[0278]

[0279]

[0280]

[0281]

[0282]

[0283]

Claims

1. A method for determining the risk of death and / or the probability of a healthy lifespan in a dog; said method comprising: a) Provide a DNA methylation profile from a sample obtained from the dog; as well as b) Use the DNA methylation profile to determine the dog's risk of death and / or probability of healthy lifespan; The DNA methylation map described therein contains at least one methylation site as listed in Table 1.

2. A method for determining the biological age of a dog; said method comprising: a) Provide a DNA methylation profile from a sample obtained from the dog; as well as b) Use the DNA methylation profile to determine the biological age of the dog, wherein the DNA methylation profile is associated with the dog’s risk of death and / or probability of healthy lifespan, and wherein the DNA methylation profile contains at least one methylation site as listed in Table 1.

3. A method for selecting a lifestyle program, dietary program, or therapeutic intervention for a dog, said method comprising: a) Provide a DNA methylation profile from a sample obtained from the dog; b) Use the DNA methylation profile to determine the dog’s risk of death and / or probability of healthy lifespan, wherein the DNA methylation profile contains at least one methylation site as listed in Table 1; as well as c) Select appropriate lifestyle programs, dietary programs or therapeutic interventions for the dog based on the mortality risk and / or healthy lifespan probability determined in step b).

4. A method for determining the efficacy of a lifestyle program, dietary program, or therapeutic intervention in improving the risk of death in dogs, said method comprising: a) Applying a lifestyle program, dietary program, or therapeutic intervention to the dog, wherein the lifestyle program, dietary program, or therapeutic intervention has been selected according to the method of claim 3; b) After the lifestyle program, dietary program or therapeutic intervention has been applied to the dog for a period of time; use DNA methylation profiles from samples obtained from the dog to determine the dog’s risk of death and / or probability of healthy lifespan, wherein the DNA methylation profiles contain at least one methylation site as listed in Table 1; c) After the period of time in which the lifestyle program, dietary program, or treatment intervention was followed, determine whether there was any change in the dog's risk of death and / or probability of healthy lifespan.

5. A method for determining the efficacy of a lifestyle program, dietary program, or therapeutic intervention in improving the risk of death and / or the probability of healthy life expectancy in dogs, said method comprising: a) Use DNA methylation profiles from samples obtained from the dogs to determine the dogs’ risk of death and / or probability of healthy lifespan, wherein the DNA methylation profiles contain at least one methylation site as listed in Table 1. b) Apply the lifestyle program, dietary program or therapeutic intervention selected based on the mortality risk and / or healthy lifespan probability determined in step a) to the dog; c) After a period of time, a lifestyle program, dietary program or therapeutic intervention has been applied to the dog; the dog’s risk of death and / or probability of healthy lifespan is determined using DNA methylation profiles from samples obtained from the dog, wherein the DNA methylation profiles contain at least one methylation site as listed in Table 1. d) Determine whether there is a change in the dog’s risk of death and / or probability of healthy life between steps a) and c).

6. A method for developing anti-aging lifestyle programs, dietary programs, or therapeutic interventions; said method includes; a) Use DNA methylation profiles from samples obtained from dogs to determine the dog’s first risk of death and / or probability of healthy lifespan, wherein the DNA methylation profile contains at least one methylation site as listed in Table 1. b) Apply lifestyle programs, dietary programs, or therapeutic interventions to the dogs; c) After the lifestyle program, dietary program or therapeutic intervention has been applied to the dog for a period of time; determine the dog’s second mortality risk and / or healthy life probability using a DNA methylation profile from a second sample obtained from the dog, wherein the DNA methylation profile contains at least one methylation site as listed in Table 1; d) After the period of time in which the lifestyle program, dietary program or treatment intervention is followed, determine whether there is a change in the dog’s first risk of death and / or probability of healthy life and the second risk of death and / or probability of healthy life; Where the lifestyle program, dietary program, or treatment intervention reduces the risk of death and / or increases the probability of healthy lifespan; If the dog is determined to be anti-aging if it reduces the rate of increase in the dog's risk of death and / or increases the rate of decrease in the dog's probability of healthy lifespan, then it is considered to be anti-aging.

7. A method for preventing or reducing the risk of disease in dogs; said method comprising: a) Use DNA methylation profiles from samples obtained from the dog to determine the dog’s risk of death and / or probability of healthy lifespan, wherein the DNA methylation profile contains at least one methylation site as listed in Table 1, and wherein the risk of death and / or probability of healthy lifespan determined for the dog is associated with an increased likelihood of developing the disease. as well as b) Select a lifestyle program, dietary program or therapeutic intervention for the dog based on the mortality risk and / or healthy life probability determined in step a); The lifestyle program, dietary program, or therapeutic intervention described herein prevents or reduces the risk of the dog developing the disease; preferably, the disease described herein is an age-related disease.

8. A method for selecting dogs suitable for receiving anti-aging lifestyle programs, dietary programs, or therapeutic interventions; said method comprising: a) Use DNA methylation profiles from samples obtained from the dogs to determine the dogs’ risk of death and / or probability of healthy lifespan, wherein the DNA methylation profiles contain at least one methylation site as listed in Table 1. b) If the dog has an increased risk of death and / or a reduced probability of healthy lifespan compared to its actual age, the dog will be selected as suitable for receiving an anti-aging lifestyle program, dietary program or therapeutic intervention.

9. The method according to any preceding claim, wherein the method for determining the mortality risk and / or healthy life probability of the dog further comprises combining the DNA methylation profile with one or more of the dog's actual age, breed, and / or sex.

10. The method according to any one of claims 3 to 9, wherein a lifestyle program, dietary program, or therapeutic intervention is selected based on determining that the dog has an increased risk of death and / or a reduced probability of healthy lifespan compared to its actual age.

11. The method according to any one of claims 3 to 10, wherein the lifestyle program, dietary program, or therapeutic intervention is a dietary intervention.

12. The method of claim 11, wherein the dietary intervention is a calorie-restricted diet, an old dog diet, or a low-protein diet.

13. The method according to any of the preceding claims, wherein the sample is a blood sample.

14. The method according to any one of claims 1 to 3 or 5 to 8, wherein step a) further comprises determining a DNA methylation profile from a sample obtained from the dog.

15. The method according to any of the preceding claims, wherein the DNA methylation map comprises at least one methylation site selected from the sites numbered 1-40 as listed in Table 1.

16. The method according to any one of claims 1 to 14, wherein the DNA methylation map comprises at least 3, at least 5, at least 10, at least 20, at least 30, at least 40 methylation sites or each methylation site as listed in Table 1.

17. The method of claim 15, wherein the DNA methylation map comprises at least 3, at least 5, at least 10, at least 20, at least 30 methylation sites or each methylation site from the sites numbered 1-40 as listed in Table 1.

18. The method of claim 16, wherein the DNA methylation map comprises methylation sites as listed in Table 2.

19. The method of claim 16, wherein the DNA methylation map comprises methylation sites as listed in Table 3.

20. The method of claim 16, wherein the DNA methylation map comprises methylation sites as listed in Table 4.

21. The method of claim 16, wherein the DNA methylation map comprises methylation sites as listed in Table 5.

22. The method according to any one of claims 14 to 21, wherein a method comprising one or more of the following steps is used to determine DNA methylation: (i)(a) treating sample DNA with APOBEC or bisulfite to deamination of cytosine; (b) enrichment based on capture; and / or (c) high-throughput sequencing; (ii) (a) treating the sample DNA with bisulfite conversion to deaminate cytosine; and (b) microarray hybridization detection; or (iii) De novo methylation sequencing.

23. The method according to any of the preceding claims, wherein the DNA methylation profile is associated with the incidence or predicted incidence of: (i) tissues; (ii) organs; or (iii) physiological systems, such as the immune system, gastrointestinal system, urinary system, muscular system, cardiovascular system, and / or nervous system.

24. The method of claim 23, further comprising applying to the dog a lifestyle program, dietary program, or therapeutic intervention suitable for improving the morbidity or predicted morbidity of the tissue, organ, or physiological system identified in claim 23.

25. A dietary intervention or treatment for reducing the risk of death in dogs and / or increasing the probability of a healthy lifespan in dogs, wherein the dietary intervention is applied to the dogs, and wherein the risk of death is determined by the method according to any one of claims 1, 7 to 10 or 13 to 24.

26. Use of a dietary intervention in reducing the risk of death in dogs and / or increasing the probability of a healthy lifespan in dogs, wherein the dietary intervention is applied to the dogs, and the risk of death is determined by the method according to any one of claims 1, 7 to 10 or 13 to 24.

27. A computer-readable medium comprising instructions that, when executed, cause one or more processors to perform the method of any one of claims 1, 2, 3, 7 to 13 or 15 to 24.

28. A computer system for determining the risk of death and / or the probability of a healthy lifespan of a dog; said computer system being programmed to determine the risk of death of the dog using a DNA methylation profile of said dog, said DNA methylation profile containing at least one methylation site as listed in Table 1.

29. A computer system for selecting a suitable lifestyle program, dietary program, or therapeutic intervention for a dog, said computer system being programmed to perform one or more of the following steps: a) Use a DNA methylation profile from the dog to determine the dog's risk of death and / or probability of healthy lifespan, wherein the DNA methylation profile contains at least one methylation site as listed in Table 1; and b) Select appropriate lifestyle programs, dietary programs or therapeutic interventions for the dog based on the mortality risk and / or healthy life probability determined in step a).

30. A computer system for determining the efficacy of a lifestyle program, dietary program, or therapeutic intervention in improving the risk of death and / or the probability of healthy life expectancy in dogs, said computer system being programmed to perform one or more of the following steps: a) Determine the dog's risk of death and / or probability of healthy life using DNA methylation profiles from samples obtained from the dog prior to the lifestyle program, dietary program, or treatment intervention, and from samples obtained from the dog after the lifestyle program, dietary program, or treatment intervention, wherein the DNA methylation profiles contain at least one methylation site as listed in Table 1; and b) Determine whether there is a change in the mortality risk and / or healthy lifespan probability of dogs between samples obtained before and after the application of the lifestyle program, dietary program, or treatment intervention.

31. A computer system for determining the likelihood that a dog will benefit from an anti-aging lifestyle program, dietary program, or therapeutic intervention; said computer system is programmed to perform one or more of the following steps: a) Use DNA methylation profiles from samples obtained from the dogs to determine the dogs’ risk of death and / or probability of healthy lifespan, wherein the DNA methylation profiles contain at least one methylation site as listed in Table 1. b) If the dog has an increased risk of death and / or a reduced probability of healthy lifespan compared to its actual age, the dog is identified as potentially responsive to anti-aging lifestyle programs, dietary programs, or therapeutic interventions.

32. A computer program product comprising computer-implementable instructions for causing a programmable computer to use a DNA methylation profile of a dog to determine the dog’s risk of death and / or probability of healthy lifespan, wherein the DNA methylation profile contains at least one methylation site as listed in Table 1.

33. A computer program product comprising computer-implementable instructions for causing a programmable computer to use a DNA methylation profile from a dog to determine the dog's risk of death and / or probability of healthy lifespan; and to select an appropriate lifestyle program, dietary program, or therapeutic intervention for the dog based on the risk of death and / or probability of healthy lifespan determined using the DNA methylation profile, wherein the DNA methylation profile contains at least one methylation site as listed in Table 1.

34. A computer program product comprising computer-implementable instructions for causing a programmable computer to: a) determine the dog's risk of death and / or probability of healthy life using DNA methylation profiles from samples obtained from the dog prior to and after the lifestyle, dietary, or therapeutic intervention, wherein the DNA methylation profiles contain at least one methylation site as listed in Table 1; and b) determine whether there is a change in the dog's risk of death and / or probability of healthy life between samples obtained from the dog before and after the application of the lifestyle, dietary, or therapeutic intervention.

35. A computer program product comprising computer-implementable instructions for causing a programmable computer to: a) determine the dog's risk of death and / or probability of healthy lifespan using a DNA methylation profile from a sample obtained from the dog, wherein the DNA methylation profile contains at least one methylation site as listed in Table 1; and b) identify the dog as potentially responsive to anti-aging lifestyle programs, dietary programs, or therapeutic interventions if the dog has an increased risk of death and / or a decreased probability of healthy lifespan compared to its age.

36. A computer system or computer program product according to any one of claims 27 to 35; wherein the computer system or computer program prepares and shares a report detailing the results of step (b).

37. A method for determining the mortality risk and / or healthy lifespan probability of a dog using a DNA methylation profile; the method comprising (a) providing a sample from the dog; (b) providing the sample for subsequent DNA extraction to measure DNA methylation in the extracted DNA from the sample, thereby obtaining a DNA methylation profile; (c) determining the mortality risk and / or healthy lifespan probability of the dog using a computer system according to claim 28 or a computer program product according to claim 32, wherein the DNA methylation profile contains at least one methylation site as listed in Table 1; wherein the computer system prepares and shares a report detailing the results of step (c).

38. A method for determining the mortality risk and / or healthy lifespan probability of a dog using a DNA methylation profile; and for selecting a suitable lifestyle program, dietary program, or therapeutic intervention for the dog based on the mortality risk and / or healthy lifespan probability determined using the DNA methylation profile; the method comprising (a) providing a sample from the dog; (b) providing the sample for subsequent DNA extraction to measure DNA methylation in the extracted DNA from the sample, thereby obtaining a DNA methylation profile; (c) determining the mortality risk and / or healthy lifespan probability of the dog and selecting a suitable lifestyle program, dietary program, or therapeutic intervention for the dog using a computer system according to claim 29 or a computer program product according to claim 33, wherein the DNA methylation profile contains at least one methylation site as listed in Table 1; wherein the computer system prepares and shares a report detailing the results of step (c).

39. A method for determining the efficacy of lifestyle programs, dietary programs, or therapeutic interventions in improving the risk of death and / or the probability of healthy lifespan in dogs using DNA methylation profiling, said method comprising: (a) Providing samples obtained from the dog prior to the lifestyle program, dietary program, or treatment intervention, and samples obtained from the dog after the lifestyle program, dietary program, or treatment intervention; (b) Providing the samples for subsequent DNA extraction to measure DNA methylation in the extracted DNA from the samples, thereby obtaining a DNA methylation profile; (c) Using the computer system of claim 30 or the computer program product of claim 34, determining whether there is a change in the dog's mortality risk and / or healthy lifespan probability between samples obtained before and after the application of the lifestyle program, dietary program, or treatment intervention, wherein the DNA methylation profile contains at least one methylation site as listed in Table 1; wherein the computer system prepares and shares a report detailing the results of step (c).

40. A method for identifying dogs that may respond to anti-aging lifestyle programs, dietary programs, or therapeutic interventions using DNA methylation mapping; said method comprising: (a) Provide a sample obtained from the dog; (b) Providing the sample for subsequent DNA extraction to measure DNA methylation in the extracted DNA from the sample, thereby obtaining a DNA methylation profile; (c) Using the computer system of claim 31 or the computer program product of claim 35, identifying the dog as potentially responsive to an anti-aging lifestyle or diet if the dog has an increased risk of death and / or a reduced probability of healthy lifespan compared to its age, wherein the DNA methylation profile contains at least one methylation site as listed in Table 1; wherein the computer system prepares and shares a report detailing the results of step (c).

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