Method for improving healthy life of dogs

Abstract

The present invention provides a method for determining the risk of death and / or the probability of healthy lifetime of a dog; the method comprises a) providing a DNA methylation profile from a sample obtained from the dog; and b) using the DNA methylation profile to determine the probability of mortality risk and / or healthy lifespan of the dog.

Description

Technical Field

[0001] The present invention relates to methods for determining the health status of a dog using a DNA methylation profile. In particular, the present invention relates to methods for selecting a lifestyle regimen, a dietary regimen or a therapeutic intervention for a dog based on the health status determined by the DNA methylation profile, or determining the efficacy of a lifestyle regimen, a dietary regimen or a therapeutic intervention. Background Art

[0002] The ability to determine information about a dog's health is desirable to inform the dog's overall health and well-being.

[0003] As is well known, chronological age is a major indicator of overall health status, wherein an increase in chronological age is associated with a decrease in health status. However, depending on genetics, nutrition, and lifestyle, the rate of individual aging may be slower or faster than chronological age. Therefore, chronological age may not always reflect the risk of reduced aging rate or health status of an individual. On the other hand, the biological age of an individual (based on, for example, clinical biochemistry and cell biology measurements) may be different from that of other individuals of the same chronological age. Methods for determining biological age may help to identify individuals at risk of age-related disorders earlier than expected based on the chronological age of the individual (see, for example, WO2019 / 046725).

[0004] However, other methods of determining a dog's biological age and utilizing measures of biological age to improve health outcomes in dogs are needed. Summary of the invention

[0005] The present invention relates to a method for quantifying the health status of a dog based on a DNA methylation profile. The method can determine the mortality risk and / or probability of healthy life span of a dog by evaluating a DNA methylation profile from the dog.

[0006] Existing methods for assessing the health status of dogs determine biological age based on a correlation between DNA methylation and chronological age. Calculating the biological age of an animal may include determining a DNA methylation profile compared to an expected DNA methylation profile for a given chronological age. Thus, this method is based on using chronological age as a primary indicator of overall health.

[0007] In contrast, the present invention contemplates the direct predictive value of a DNA methylation profile for the risk of death and / or the probability of healthy life span. For example, a given DNA methylation marker may not be directly correlated with chronological age, but may indicate a particular pathological condition, thereby indicating the probability of increased risk of death and / or decreased healthy life span. Thus, the present method can be described as identifying the risk of death and / or the probability of healthy life span for a dog. Thus, the DNA methylation markers and DNA methylation profiles of the present invention are not necessarily correlated with chronological age, but are related to the difference between the phenotypic age and the chronological age of a dog.

[0008] In a first aspect, the present invention provides a method for determining the risk of mortality in 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 risk of mortality in the dog.

[0009] Determining the risk of death can refer to determining the likelihood that a dog will live longer or shorter than, for example, a comparable dog of the same chronological age, sex, and breed. Thus, the present method can determine the probability of a dog's life span, health span, and / or longevity compared to, for example, a comparable dog of the same chronological age, sex, and breed. Additionally, methods for improving the probability of a dog's risk of death and / or health span can improve a dog's likely life span, health span, and / or longevity.

[0010] As used herein, "lifespan" may refer to the length of time (e.g., years) that a subject lives. "Health span" may refer to the length of time (e.g., years) that a subject lives without disease. "Longevity" may refer to the length of time (e.g., years) that a subject lives beyond their life expectancy.

[0011] Suitably, the risk of death may be equated to the probability of a dog's healthy lifespan; wherein a decrease in the risk of death is equated to an increased probability of a longer healthy lifespan for the dog, or an increase in the risk of death is equated to a decreased probability of a longer healthy lifespan for the dog. The risk of death may be expressed as the difference between the dog's determined age (i.e., biological age) and its chronological age. For example, an increase in the difference between the biological ages determined by the present method compared to the chronological age may indicate an increased risk of death for the dog. A decrease in the difference between the biological ages determined by the present method compared to the chronological age may indicate a decreased risk of death for the dog. Suitably, the risk of death and / or the probability of a healthy lifespan may be described as the dog's biological age. Suitably, the risk of death and / or the probability of a healthy lifespan may be described as the dog's epigenetic age. Suitably, the biological clock of the present invention may be referred to as an epigenetic clock.

[0012] Suitably, determining that the biological age of a dog is greater than its actual age indicates a higher risk of death. Suitably, determining that the biological age of a dog is less than its actual age indicates a reduced risk of death. Suitably, determining that the biological age of a dog is greater than its actual age indicates a reduced probability of a longer healthy lifespan. Suitably, determining that the biological age of a dog is less than its actual age indicates an increased probability of a longer healthy lifespan.

[0013] Suitably, the method may be used to determine the biological age of a dog based on its risk of mortality and / or probability of healthy life span.

[0014] 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 correlated with the risk of mortality and / or probability of healthy life span of the dog.

[0015] In all of the present methods, determining or improving a dog's risk of death and / or probability of healthy life span 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 life span is used to determine the dog's biological age.

[0016] In another aspect, the present invention provides a method for selecting a lifestyle regimen, dietary regimen 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 risk of mortality and / or probability of healthy life span for the dog; and c) selecting an appropriate lifestyle regimen, dietary regimen or therapeutic intervention for the dog based on the mortality risk determined in step b).

[0017] As used herein, "selecting an appropriate lifestyle regimen, dietary regimen, or therapeutic intervention for a dog" may also encompass "recommending a lifestyle regimen, dietary regimen, or therapeutic intervention for a dog" or "providing a recommended lifestyle regimen, dietary regimen, or therapeutic intervention for a dog."

[0018] In another aspect, the present invention provides a method for determining the efficacy of a lifestyle regimen, dietary regimen or therapeutic intervention in improving a dog's risk of mortality and / or probability of healthy life span, the method comprising: a) applying a lifestyle regimen, dietary regimen or therapeutic intervention to the dog, wherein the lifestyle regimen, dietary regimen or therapeutic intervention has been selected according to the aforementioned aspects of the present invention; b) after applying the lifestyle regimen, dietary regimen or therapeutic intervention to the dog for a period of time; determining the dog's risk of mortality and / or probability of healthy life span using a DNA methylation profile from a sample obtained from the dog; c) determining whether there is a change in the dog's risk of mortality and / or probability of healthy life span after following the lifestyle regimen, dietary regimen or therapeutic intervention for the period of time.

[0019] In another aspect, the present invention provides a method for determining the efficacy of a lifestyle regimen, dietary regimen or therapeutic intervention in improving a dog's risk of mortality and / or probability of healthy life, the method comprising: a) using a DNA methylation profile from a sample obtained from the dog to determine the dog's risk of mortality; b) applying the lifestyle regimen, dietary regimen or therapeutic intervention selected based on the risk of mortality determined in step a) to the dog; c) after applying the lifestyle regimen, dietary regimen or therapeutic intervention to the dog for a period of time; determining the dog's risk of mortality using a DNA methylation profile from a sample obtained from the dog; d) determining whether the dog's risk of mortality changes between step a) and step c).

[0020] Suitably, improving the dog's risk of death and / or the probability of healthy life span may 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. In addition, improving the dog's risk of death and / or the probability of healthy life span may 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, the deterioration of the dog's risk of death and / or the probability of healthy life span may 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. The deterioration of the dog's risk of death and / or the probability of healthy life span may 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.

[0021] Suitably, improving the dog's risk of death and / or probability of healthy life span may refer to a reduction in the rate of change between the dog's biological age and chronological age, where the dog's biological age is greater than its chronological age. For example, for every additional year of chronological age, the dog's biological age may have increased by 1.5 years. After the lifestyle and dietary regimen intervention, the reduction in the rate of change causes the dog's biological age to subsequently increase by 1.25 years for every additional year of chronological age, which may provide an improvement in the dog's risk of death and / or probability of healthy life span.

[0022] Improving the probability of mortality risk and / or healthy life span can also refer to maintaining or increasing the rate of change between the biological age and the chronological age of the dog, where the biological age of the dog is less than its chronological age. For example, for each additional year of chronological age, the biological age of the dog may have increased by less than 1 year (e.g., 0.9 years). After lifestyle, dietary regimen, or therapeutic intervention, the rate of change can be changed so that for each additional year of chronological age, the biological age of the dog subsequently increases by, for example, 0.8 years or less, which can provide an improvement in the biological age of the dog.

[0023] The present method for determining the efficacy of a lifestyle regimen, dietary regimen, or therapeutic intervention for improving a dog's risk of death and / or probability of healthy life span can advantageously allow for continuous monitoring of the effectiveness of a lifestyle regimen, dietary regimen, or therapeutic intervention for improving or maintaining the health of a dog. The use of such a method can advantageously allow identification of a particularly effective lifestyle regimen, dietary regimen, or therapeutic intervention. In contrast, if a lifestyle regimen, dietary regimen, or therapeutic intervention is determined to be ineffective based on a dog's risk of death and / or probability of healthy life span; an alternative lifestyle regimen, dietary regimen, or therapeutic intervention can be implemented.

[0024] Thus, the present method enables selection of an appropriate lifestyle regimen, dietary regimen, or therapeutic intervention for a dog based on the dog's risk of death and / or probability of healthy life span as determined from the DNA methylation profile. For example, a highly digestible and high-quality protein diet is typically recommended based on the dog's chronological age. For example, it may be recommended that a dog be switched to an elderly diet around the age of 7 or 8. However, in the context of the present invention, determining that a dog has an increased risk of death and / or a decreased probability of healthy life span (i.e., an increased biological age) compared to the dog's chronological age may allow determination to switch the dog to an elderly diet at an earlier age. In contrast, a dog with a reduced risk of death and / or an increased probability of healthy life span (i.e., a decreased biological age) compared to the dog's chronological age may be able to continue to enjoy an adult diet for longer.

[0025] Suitably, the method may comprise selecting and / or applying a lifestyle regimen, dietary regimen or therapeutic intervention to the dog upon determining that the dog has an increased risk of mortality and / or a decreased probability of healthy lifespan compared to the dog's chronological age.

[0026] In another aspect, the present invention provides a method for preventing or reducing the risk of a dog developing a disease; the method comprising:

[0027] a) using a DNA methylation profile from a sample obtained from said dog to determine a risk of mortality and / or a probability of healthy lifespan for said dog; wherein said risk of mortality and / or probability of healthy lifespan determined for said dog is associated with an increased likelihood of developing said disease; and

[0028] b) selecting a lifestyle regimen, dietary regimen or therapeutic intervention for said dog based on said risk of mortality and / or probability of healthy life span determined in step a);

[0029] wherein said lifestyle regimen, dietary regimen or therapeutic intervention prevents or reduces said dog's risk of developing said disease.

[0030] Suitably, the disease is an age-related disease. For example, the age-related disease osteoarthritis, dementia, cognitive dysfunction, pre-diabetic conditions, diabetes, cancer, heart disease, obesity, gastrointestinal disorders, incontinence, kidney disease, sarcopenia, vision loss, hearing loss, osteoporosis, cataracts, cerebrovascular disease and / or liver disease.

[0031] The method may also optionally comprise administering to the dog a lifestyle regimen, a dietary regimen or a therapeutic intervention. Suitably, the lifestyle regimen may be a dietary intervention or a therapeutic regimen.

[0032] In another aspect, the present invention provides a method for selecting a dog suitable for an anti-aging lifestyle regimen, dietary regimen, or therapeutic intervention; the method comprising: a) using a DNA methylation profile from a sample obtained from the dog to determine the dog's risk of mortality and / or probability of healthy lifespan; b) selecting the dog as suitable for an anti-aging lifestyle regimen, dietary regimen, or therapeutic intervention if the dog has an increased risk of mortality and / or a decreased probability of healthy lifespan compared to the dog's chronological age.

[0033] Suitably, while an anti-aging lifestyle regimen, dietary regimen or therapeutic intervention may be effective for a dog based on chronological age, it may be particularly effective when applied to a dog that has an increased risk of mortality and / or a decreased probability of healthy lifespan compared to their chronological age. Thus, the present method may advantageously enable the selection of a dog that has an increased likelihood of responding or an improved amount of responding to an anti-aging lifestyle regimen, dietary regimen or therapeutic intervention.

[0034] A lifestyle regimen, dietary regimen, or therapeutic intervention may be selected based on a determination that the dog has an increased risk of mortality and / or a decreased probability of healthy lifespan compared to the dog's chronological age (ie, increased biological age).

[0035] The lifestyle program, dietary program or therapeutic intervention may be a dietary intervention. The dietary intervention may be a calorie-restricted diet, an elderly diet or a low-protein diet.

[0036] The DNA methylation profile may be associated with increasing biological age of: (i) a tissue; (ii) an organ; or (iii) a physiological system, such as the immune system, gastrointestinal system, urinary system, muscular system, cardiovascular system, and / or nervous system.

[0037] The invention also provides a dietary intervention for reducing the risk of mortality and / or increasing the probability of healthy life span in a dog, wherein the dietary intervention is administered to a dog, wherein the risk of mortality and / or the probability of healthy life span is determined by the method of the invention.

[0038] The invention further relates to the use of a dietary intervention for reducing the risk of mortality and / or increasing the probability of a healthy life span in a dog, wherein the dietary intervention is administered to a dog, wherein the risk of mortality and / or the probability of a healthy life span is determined by the method of the invention.

[0039] In another aspect, the present invention provides a computer-readable medium comprising instructions which, when executed, cause one or more processors to perform the method of the present invention.

[0040] In another aspect, the present invention provides a computer system for determining the risk of mortality of a dog; the computer system is programmed to determine the risk of mortality of the dog using the DNA methylation profile of the dog.

[0041] In another aspect, the present invention provides a computer system for selecting an appropriate lifestyle regimen, dietary regimen, or therapeutic intervention for a dog, the computer system being programmed to perform one or more of the following steps: a) determining the risk of mortality for the dog using a DNA methylation profile from the dog; and b) selecting an appropriate lifestyle regimen, dietary regimen, or therapeutic intervention for the dog based on the risk of mortality determined in step a).

[0042] In another aspect, the present invention provides a computer system for determining the efficacy of a lifestyle regimen, dietary regimen, or therapeutic intervention in improving a dog's risk of mortality, the computer system being programmed to perform one or more of the following steps: a) determining the dog's risk of mortality using DNA methylation profiles from samples obtained from the dog before the lifestyle regimen, dietary regimen, or therapeutic intervention and samples obtained from the dog after the lifestyle regimen, dietary regimen, or therapeutic intervention; and b) determining whether the dog's risk of mortality changes between the samples obtained from the dog before and after the lifestyle regimen, dietary regimen, or therapeutic intervention has been applied.

[0043] In another aspect, the invention provides a computer system for determining the likelihood that a dog will benefit from an anti-aging lifestyle regimen, dietary regimen, or therapeutic intervention; the computer system being programmed to perform one or more of the following steps: a) determining the risk of mortality for the dog using a DNA methylation profile from a sample obtained from the dog; b) identifying the dog as likely to respond to an anti-aging lifestyle regimen, dietary regimen, or therapeutic intervention if the dog has an increased risk of mortality compared to the dog's chronological age.

[0044] In another aspect, the present invention provides a computer program product comprising computer-implementable instructions for causing a programmable computer to determine the risk of mortality of a dog using the DNA methylation profile of the dog.

[0045] In another aspect, the present invention provides a computer program product comprising computer-implementable instructions for causing a programmable computer to determine a risk of mortality for a dog using a DNA methylation profile from the dog; and to select an appropriate lifestyle regimen, dietary regimen, or therapeutic intervention for the dog based on the risk of mortality determined using the DNA methylation profile.

[0046] In another aspect, the present invention provides a computer program product comprising computer-implementable instructions for causing a programmable computer to: a) determine a dog's risk of mortality using DNA methylation profiles from samples obtained from the dog before a lifestyle regimen, dietary regimen, or therapeutic intervention and samples obtained from the dog after the lifestyle regimen, dietary regimen, or therapeutic intervention; and b) determine whether the dog's risk of mortality changes between the samples obtained from the dog before and after the lifestyle regimen, dietary regimen, or therapeutic intervention has been applied.

[0047] In another aspect, the present invention provides a computer program product comprising computer-implementable instructions for causing a programmable computer to: a) determine the risk of mortality for a dog using a DNA methylation profile from a sample obtained from the dog; and b) identify the dog as likely to respond to an anti-aging lifestyle regimen, dietary regimen, or therapeutic intervention if the dog has an increased risk of mortality compared to the dog's chronological age.

[0048] Advantageously, the present invention may allow for the determination of mortality risk and / or probability of healthy life span based on markers of multiple organ systems and functions. Thus, the present method may advantageously cover a range of potential organ dysfunctions.

[0049] Assessing a dog's mortality risk and / or probability of healthy life span allows one to test several aspects of the animal's well-being. First, it can predict whether the animal is more likely to require dietary or supplement-based intervention. It can also be used to test the efficacy of dietary or supplement-based interventions on aging. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 - Identification of blood biomarkers that predict mortality risk. Cox proportional hazards models were fit for each of the 28 biomarkers evaluated, including sex and breed category (small or medium). P values ​​for each parameter were adjusted to account for multiple comparisons (by false discovery rate (FDR)). Parameters shown are those with adjusted FDRs below 0.05.

[0051] Figure 2 - Demonstration that biomarkers contribute to the predictive power of a multiparameter model for determining phenotypic age.

[0052] Figure 3 - Validation of the exemplary clock of the invention in a caloric restriction study. The variance corresponds to the residuals from the regression model of chronological age versus predicted phenoDNAmAge. Dogs on caloric restriction had significantly lower biological age (lower variance) compared to dogs on a control diet.

[0053] Figure 4 - Validation of the exemplary clock of the invention in a caloric restriction study at 6 years of age. The variance corresponds to the residuals from the regression model of chronological age versus predicted phenoDNAmAge. Dogs on caloric restriction had lower biological age (lower variance) compared to dogs on a control diet.

[0054] Figure 5 - Adjusted linear mixed-effects model (DogID as random effect). Dogs on calorie restriction had significantly lower biological age compared with dogs on a control diet.

[0055] Figure 6 - Survival rates between biologically young dogs and biologically old dogs as determined by the exemplary clock of the present invention

[0056] Figure 7 - Cox proportional hazards models were fitted using sex and variance phenoDNAmAge (residual from chronological age relative to predicted phenoDNAmAge), stratified by breed category (small or medium) on the training set. Increased variance was associated with a higher risk of death

[0057] Figure 8 - Assessment of reproducibility between technical replicates

[0058] Fig. 9 - An exemplary biological clock containing the first 3 methylation sites from the complete biological clock is associated with chronological age

[0059] Fig.10 - An exemplary biological clock containing the top 5 methylation sites from the complete biological clock is associated with chronological age

[0060] Fig.11 - An illustrative biological clock containing the top 10 methylation sites from the complete biological clock associated with chronological age

[0061] Fig.12 - An illustrative biological clock containing the top 20 methylation sites from the complete biological clock associated with chronological age

[0062] Fig.13 - An illustrative biological clock containing the top 50 methylation sites from the complete biological clock associated with chronological age DETAILED DESCRIPTION

[0063] Various preferred features and embodiments of the present invention will now be described by way of non-limiting examples. The skilled person will appreciate that they can combine all features of the present invention disclosed herein without departing from the scope of the invention disclosed.

[0064] It must be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0065] 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 unrecited members, elements, or method steps. The terms "comprising" and "consisting of" also include the term "composed of.

[0066] Numeric ranges are inclusive of the numbers defining the range.

[0067] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present patent application. Nothing herein should be construed as an admission that such publications constitute prior art to the claims appended hereto.

[0068] The methods and systems disclosed herein can be used by veterinarians, healthcare professionals, laboratory technicians, pet care providers, and the like.

[0069] Subjects

[0070] The present method is directed to canine subjects. Therefore, the subject of the present invention is a dog.

[0071] In an alternative aspect, the subject can be a feline subject. Thus, in an alternative aspect of the present invention, the subject is a cat. Unless otherwise indicated, all disclosures herein are equally applicable to cats.

[0072] variety

[0073] The method may make use of information about the breed of the dog. For example, the dog may be classified as a toy, small, medium, large or giant breed. Suitably, the dog breeds may be classified based on the weight of the dog. Suitably, the dog breeds may be classified based on the average weight of a dog of a given breed.

[0074] Suitably, a dog may be classified as a small or medium breed. Suitably, the classification is determined by the average weight of an adult dog of the breed. Suitably, a breed having an average weight below 10 kg is classified as a small breed and / or a breed having an average weight above 10 kg is classified as a medium breed.

[0075] In alternative aspects where the subject is a cat, the cat can be a domestic cat. Suitably, the cat can be a domestic shorthair cat.

[0076] gender

[0077] Suitably, the gender of a dog may be classified as male or female.

[0078] Chronological age

[0079] Chronological age can be defined as the amount of time that has passed from the subject's birth to a given date. Chronological age can be expressed in years, months, days, etc.

[0080] Suitably, the present methods can be applied to dogs of any chronological age. In certain embodiments, the dog can be at least about 2 years old. Suitably, the dog can 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.

[0081] Suitably, the dog may be at least about 7 years old.

[0082] sample

[0083] The present invention includes the step of providing or determining a DNA methylation profile from one or more samples obtained from a subject.

[0084] Suitably, the sample is a blood, hair follicle, buccal swab, saliva or tissue sample.

[0085] Suitably, the sample is a hair follicle, a buccal swab or a saliva sample. 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.

[0086] Suitably, the sample is derived from blood. The sample may comprise blood components or may be whole blood. The sample preferably comprises whole blood. The sample may comprise a peripheral blood mononuclear cell (PBMC) or lymphocyte sample. Techniques for collecting samples from subjects and extracting DNA (e.g., genomic DNA) from the samples are well known in the art.

[0087] The method can be performed on one or more samples obtained from a 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 after the first sample is obtained. 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 once a month, once a year, or once every two years. Suitably, samples can be obtained approximately once a year (e.g., during an annual veterinary health checkup). This may help determine the effect of a particular treatment or lifestyle change, such as a dietary intervention or a change in an exercise regimen.

[0088] In one embodiment, the method can be applied to samples obtained from a subject before a lifestyle change (e.g., a dietary product intervention or a change in exercise regimen). In another embodiment, the method can be applied to samples obtained from a subject before and after, for example, a dietary product intervention or a change in exercise regimen. The method can also be applied to samples obtained at predetermined times during, for example, the entire dietary product intervention or change in exercise regimen. These predetermined times can be periodic, for example, every day or every three days, or can depend on the subject being tested, for example.

[0089] DNA methylation

[0090] DNA methylation is the process of covalently adding a methyl group (CH3) to the cytosine base that is part of the 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). Cytosine is modified on the 5th carbon atom, and the modified residue is called 5-methylcytosine (5mC). DNA methylation can also include 5-hydroxymethylcytosine (5hmc).

[0091] DNA methylation is an example of epigenetic mechanism, that is, it can modify gene expression without modifying the potential DNA sequence. DNA methylation can, for example, inhibit gene expression by acting as a recruitment signal of an inhibitor or by directly blocking the recruitment of transcription factors. DNA methylation mainly occurs at the site of adjacent cytosine and guanine forming dinucleotides (CpG) in the genome of mammalian somatic cells. Although non-CpG methylation is observed in embryonic development, these modifications are greatly reduced in most cell types in adults. CpG islands are DNA fragments with high CpG density but usually unmethylated. These regions are associated with promoter regions, particularly promoter regions of housekeeping genes, and are considered to remain in a permissive state that allows gene expression.

[0092] DNA methylation has been found to change with age in humans and other animals. Aging mammalian tissues show overall DNA hypomethylation, which is thought to be due to the gradual loss or mistargeting of DMNT1 methyltransferase activity, but local hypermethylation of CpG islands. Local hypermethylation can lead to the suppression of certain genes, and this can contribute to age-related diseases. The connection between epigenetic changes in DNA methylation and age allows the use of a "DNA methylation clock" to estimate "biological age". Typically, supervised machine learning methods have been used to train these clocks for chronological age, and the deviation of the "clock age" from the actual chronological age of the individual is considered to be an indicator of "biological" age. This is related to the chronological age of the individual, but deviations from the correlation can indicate the potential risk of age-related diseases or disorders in the individual.

[0093] Detection of specifically methylated DNA can be accomplished by 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 non-methylated DNA, and then quantify the ratio of methylated and unmethylated DNA at specific genomic sites.

[0094] The method may include determining the DNA methylation profile of the dog using any suitable method. Suitable methods include, but are not limited to, those described below.

[0095] Enzymatic methylation sequencing (EM-seq)

[0096] Suitably, enzymatic methods are used to detect 5mC and 5hmC. For example, enzymatic methylation sequencing (EM-seq) can be used.

[0097] Typically in EM-seq, 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). In addition, the use of the T4-BGT enzyme glycosylates both pre-existing 5hmC and 5hmC generated by TET2 activity. In the second enzymatic step, after denaturation of double-stranded DNA, the enzyme apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3A (APOBEC3A) is used to deaminize cytosine, but not 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 to sequencing adapters, which can be used, for example DNA Ultra II reagent (NEB) is used for this. After the second enzymatic step, the PCR reaction can be performed using a polymerase that can amplify uracil-containing templates (such as Q5U TM ) to amplify deaminated single-stranded DNA, and the resulting library can be sequenced or analyzed in the same manner as DNA samples generated by bisulfite sequencing. The output of EM-seq is generally the same as whole-genome bisulfite sequencing, but uses less DNA damaging reagents, which therefore reduces sample loss and can outperform samples prepared by bisulfite conversion in terms of coverage, sensitivity, and accuracy of cytosine methylation calls. An exemplary EM-seq method is described by Vaisvila et al. (Genome Research; 2021; 31: 1-10).

[0098] Bisulfite conversion based methods

[0099] 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 cytosines into uracil, and subsequent PCR amplification converts these residues into thymine. Analysis of the resulting DNA sequence can be performed via many different methods, examples of which include, but are not limited to, denaturing gel electrophoresis, single-stranded conformational polymorphism, melting curves, fluorescent real-time PCR (MethyLight), MALDI mass spectrometry, array hybridization, and sequencing (e.g., whole genome bisulfite sequencing WGBS). Recently developed technologies (such as SeqCap Epi) enrich the sequence of interest before sequencing, which enables deeper coverage in more concentrated areas). Comparison of sequence abundance in bisulfite-converted samples with sequence abundance in untreated controls allows analysis of methylation at the target site, wherein the ratio of the converted sequence indicates the methylation level at the target site.

[0100] Other variations of the bisulfite conversion method are available that are able to distinguish 5mC from the oxidized form 5-hydroxymethylcytosine (5hmC), which behaves the same as 5mC under standard bisulfite conversion, and are able to detect a further modification, 5-formylcytosine (5fC). These methods, such as oxBS-Seq and redBS-Seq, exploit the oxidation and reduction of these markers to alter the sensitivity of each species to bisulfite conversion and quantify the amount of each modification at the target locus by comparative analysis.

[0101] Selective restriction endonuclease digestion method

[0102] Existing methods for analyzing DNA methylation patterns may involve the use of restriction enzymes. These methods include, for example, restriction landmark genome scanning (RLGS) (Costello et al., 2000; Nat Genet.; 24 (2): 132-8), methylation sensitivity representative difference analysis (MS-RDA) (Ushijima et al., Proc Natl Acad Sci USA. March 18, 1997; 94 (6): 2284-9) and differential methylation hybridization (DMH) (Huang et al., Cancer Res. March 15, 1997; 57 (6): 1030-4). The digestion activity of restriction endonucleases can be methylation-dependent. This specificity can be used to distinguish methylated sequences from unmethylated sequences. Certain restriction enzymes (e.g., BstUI, HpaII, and NotI) are sensitive to methylated recognition sequences. Other restriction enzymes (such as McrBC) are specific for methylated sequences.

[0103] For example, differential methylation hybridization (DMH) (Huang et al., as described above]) needs to carry out initial fragmentation to genome with a large amount of genomic restriction enzymes (such as MseI), and this enzyme is less than 200bp length by genomic fragmentation.After this step, use methylation sensitive restriction endonuclease (MRE) or use the mixture of MRE in some versions of this technology to digest genomic fragments to improve coverage.According to the specificity of one or more enzymes used, methylated sequence or unmethylated sequence will be degraded.The sequence of digestion can not be amplified in subsequent PCR step.Gained PCR product is suitable for further processing and analysis by the combination of order-checking or microarray hybridization and fluorescent dye.

[0104] Suitably, the method utilises a DNA methylation profile generated by a method comprising the use of one or more MREs.

[0105] Suitable comparators can be used to study the methylation status between conditions. DNA from healthy subjects can be compared with elderly or sick subjects to detect changes in methylation status (Huang et al., Hum Mol Genet. 1999 March; 8 (3): 459-70). Alternatively, a methylation-insensitive form of a secondary digestive enzyme (such as HpaII isoschizomer MspI) can be used to generate a control sample so that intra-genomic or inter-genomic DNA methylation comparisons can be performed (Khulan et al., Genome Res. 2006 August; 16 (8): 1046-55).

[0106] In some embodiments, the method for detecting methylation includes random shearing or random fragmentation of genomic DNA, cutting DNA with methylation-dependent or methylation-sensitive restriction enzymes, and then selectively identifying and / or analyzing cut or uncut DNA. Selective identification may include, for example, separating cut and uncut DNA (e.g., by size) and quantifying the cut or alternatively uncut sequence of interest. Alternatively, the method may encompass amplifying complete DNA after restriction enzyme digestion, thereby only amplifying DNA that is not cut by restriction enzymes in the amplified region. In some embodiments, gene-specific primers may be used for amplification. Alternatively, adapters may be added to the ends of randomly fragmented DNA, DNA may be digested with methylation-dependent or methylation-sensitive restriction enzymes, and complete DNA may be amplified using primers hybridized with adapter sequences. In this case, a second step may be performed to determine the presence, absence or amount of a specific gene in a DNA amplification pool. In some embodiments, DNA is amplified using real-time quantitative PCR.

[0107] Suitably, the digestion of nucleic acids is detected by selective hybridization of probes or primers with undigested nucleic acids. Alternatively, the probe selectively hybridizes with both digested and undigested nucleic acids, but facilitates differentiation between the two forms, for example, by electrophoresis. Suitable detection methods for achieving selective hybridization with hybridization probes include, for example, Southern or other nucleic acid hybridizations.

[0108] Suitable hybridization conditions can be determined based on the melting temperature (Tm) of the nucleic acid duplex comprising the probe. Those skilled in the art will appreciate that for each probe, the optimal hybridization reaction conditions should be determined empirically, but some general principles can be applied. Preferably, hybridization using short oligonucleotide probes is performed with low to medium stringency. In the case of GC-rich probes or primers or longer probes or primers, high stringency hybridization and / or washing is preferred. 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 concentrations and / or at least 65° C. or at equivalent conditions. The specific stringency levels mentioned herein encompass equivalent conditions using washing / hybridization solutions other than SSC known to those skilled in the art.

[0109] Affinity-based enrichment methods

[0110] Differentiation of methylated DNA from unmethylated DNA can be achieved by using antibodies containing a methyl-CpG-binding domain (MBD), such as anti-5mC and / or methylated CpG binding proteins. Antibodies to MBD-domain proteins are able to specifically separate methylated DNA relative to unmethylated DNA. Methods utilizing antibodies are generally referred to as MeDIP, while methods utilizing methylated CpG binding proteins are generally referred to as MBD or MIRA methods.

[0111] These methods require initial fragmentation of the genome, which can be performed by extensive genome digestion with a frequently cutting enzyme (such as MseI), followed by affinity purification of the methylated fragments. Input DNA can be compared to purified methylated DNA by microarray hybridization or sequencing to obtain a comparative analysis of methylation levels at specific sites.

[0112] Other variations of affinity-enrichment-based 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 fractions can then be sequenced separately, thereby increasing sequence coverage.

[0113] Single-molecule sequencing-based methods

[0114] Contemporary sequencing methods can sequence single molecules directly. Single molecule real-time (SMRT) DNA sequencing is available, such as the Sequel system from Pacific Biosciences, and has been shown to be able to identify modified bases (such as methylated cytosine) based on polymerase kinetics. Nanopore sequencing devices (such as the MinION nanopore sequencer from Oxford Nanopore Technologies) that can sequence long strands of DNA individually can also detect base modifications, including methylation.

[0115] DNA methylation sites

[0116] Suitably, a DNA methylation site may refer to the presence or absence of 5mC at a single cytosine, suitably a single CpG dinucleotide.

[0117] Suitably, DNA methylation sites can refer to the presence or absence of methylation of multiple CpG sites in a cross DNA region (i.e., the number of 5mC or the percentage of 5mC). Suitably, DNA site methylation sites can refer to the methylation level of multiple CpG sites in a cross DNA region (i.e., the number of 5mC or the percentage of 5mC). "DNA region" can refer to a specific portion of genomic DNA. These DNA regions can be specified by reference to gene names or a set of chromosome coordinates. Both gene names and chromosome coordinates are well known and understood by those skilled in the art.

[0118] Suitably, gene names and / or coordinates may be based on the 'Tasha' dog reference genome (https: / / www.ncbi.nlm.nih.gov / assembly / GCF_000002285.5; Jagannathan et al.; Genes (Bsael); 2021; 12(6); 847).

[0119] For example, a DNA region can define a portion of DNA near the promoter of a gene. Promoter regions are known to be rich in CpG. For example, a DNA region can refer to about 3 kb upstream of the promoter to about 3 kb downstream; about 2 kb upstream to about 2 kb downstream; about 2 kb upstream to about 1 kb downstream; about 2 kb upstream to about 0.5 kb downstream; about 1 kb upstream to about 0.5 kb downstream; about 0.5 kb upstream to about 0.5 kb downstream. Suitably, a DNA region can refer to about 1 kb upstream of the promoter to about 0.5 kb downstream.

[0120] Suitably, the DNA region may comprise or consist of CpG sites that are less than about 5000, less than about 4000, less than about 3000, less than about 2000, less than about 1000, less than about 500, or less than about 200 bases apart.

[0121] Suitably, the DNA region may comprise or consist of CpG sites that are about 200 to about 5000, about 200 to about 4000, about 200 to about 3000, about 200 to about 2000, or about 200 to about 1000 bases apart.

[0122] Suitably, the DNA region may comprise one or more CpG islands. Suitably, the DNA region may consist of CpG islands.

[0123] A "CpG island" may refer to a DNA region comprising at least 200 bp, a GC percentage greater than 50%, and an observed to expected CpG ratio greater than 60%.

[0124] Suitably, the DNA methylation sites do not comprise X and / or Y chromosome CpGs.

[0125] Suitably, the DNA methylation site does not comprise a CpG known to comprise a SNP at the CpG.

[0126] Reference to each gene / DNA region described in detail above should be construed as reference to all forms of these molecules and fragments or variants thereof. As understood by those skilled in the art, some genes are known to show allele variation or single nucleotide polymorphism between individuals. Variant includes the nucleotide sequence from the same region sharing at least 90%, 95%, 98%, 99% sequence identity, i.e., has one or more deletions, additions, substitutions, reverse sequences, etc. relative to DNA region as described herein. Therefore, the present invention should be construed as extending to such variants, and for the purposes of this application, although there may be a slight genetic variation between the actual nucleotide sequence between individuals, these variants can still achieve identical results. Therefore, the present invention should be construed as extending to all forms of DNA produced by any other sudden change, polymorphism or allele variation.

[0127] With respect to screening for methylation in these gene regions, it will be appreciated that the assay can be designed to screen for a specific DNA. It is well within the skill of those skilled in the art to select which strand to analyze and target based on the chromosome coordinates. In some cases, an assay can be established to screen both strands.

[0128] "Methylation state" can be understood to refer to the presence, absence and / or amount of methylation at one or more specific nucleotides in a DNA region. The methylation state of a specific DNA sequence (e.g., a DNA region as described herein) can indicate the methylation state of each base in the sequence or can indicate the methylation state of a subset of base pairs in the sequence (e.g., the methylation state of cytosine or the methylation state of one or more specific restriction enzyme recognition sequences), or can indicate information about the regional methylation density in the sequence without providing the exact information of where methylation occurs in the sequence. Methylation state can optionally be represented or indicated by a "methylation value".

[0129] Suitably, the 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 the enzyme and APOBEC3A conversion treatment. In other embodiments, the methylation level can be determined as the fraction of "C" bases in the total "C" + "T" bases at the site "i" after the enzyme and APOBEC3A conversion 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] In some embodiments, particularly when bisulfite conversion and sequencing methods are used, the methylation level can be determined as the fraction of "C" bases out of the total "C" + "U" bases at the target CpG site "i" after bisulfite treatment. In other embodiments, the methylation level can be determined as the fraction of "C" bases out of the total "C" + "T" bases at the 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.

[0131] Alternatively, a methylation value 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 the mock-treated control indicates that the sequence is not highly methylated, while an amount substantially less than the amount of template present in the mock-treated sample indicates that methylated DNA is present at the sequence. Therefore, for example, the value from the above example (i.e., the methylation value) represents the methylation state and can therefore be used as a quantitative indicator of the methylation state. This is particularly useful when the methylation state of a sequence in a sample needs to be compared to a threshold value.

[0132] The present invention is not limited by the exact number of methylated residues that are considered indicative of biological age, as some variation will occur between samples. The present invention is also not necessarily limited by the location of the methylated residues (eg, specific methylation sites).

[0133] In one embodiment, a screening method may be employed that is specifically directed toward assessing the methylation status of one or more particular cytosine residues or the corresponding cytosine at position n+1 relative to the DNA strand.

[0134] Enrichment and detection methods

[0135] Determining the DNA methylation profile may include the step of enriching the selected DNA region in the DNA sample. For example, the method may include the step of enriching the DNA sample for a DNA region containing a DNA methylation site, the DNA methylation site containing the DNA methylation profile.

[0136] Suitable enrichment methods are known in the art and include, for example, methods based on amplification or hybridization. Amplification and enrichment generally refer to, for example, PCR-based enrichment using primers for the DNA region to be enriched. Any suitable amplification format can be used, such as polymerase chain reaction (PCR), rolling circle amplification (RCA), inverse polymerase chain reaction (iPCR), in situ PCR, strand displacement amplification or circulating probe technology.

[0137] Hybridization enrichment or capture-based enrichment generally refers to the use of hybridization probes (or capture probes) that hybridize to the region of DNA to be enriched.

[0138] The hybridization probe can be directly attached to a solid support, or can include a portion, such as biotin, to allow binding to a solid support suitable for capturing a biotin portion (e.g., beads coated with streptavidin). In any case, DNA comprising a sequence complementary to the probe can be captured, thereby allowing the DNA comprising the DNA region of interest to be separated from the DNA not comprising the DNA region of interest. Therefore, such capture steps allow enrichment of the DNA region of interest. For example, the DNA region can be a DNA region close to a gene promoter.

[0139] Arrays used herein may vary according to the probe composition and the desired use of the array. For example, the nucleic acids (or CpG sites) detected in the array may be at least 10, 100, 1,000, 10,000, 100,000, 1 million, 10 million, 100 million, 100 million or more. Alternatively or additionally, the nucleic acids (or CpG sites) detected may be selected to be no more than 100 million, 10 million, 1 million, 100,000, 1,000, 100 or less. Using nucleic acid sequencing methods, such as those known in the art; For example, next generation or massively parallel sequencing, similar ranges can be obtained.

[0140] Suitably, the enrichment step may be performed before or after the step of separating or differentially treating methylated and unmethylated DNA.

[0141] As used herein, the term "enrichment" or "enrichment" of "DNA" or "DNA region" means a process in which the (absolute) amount and / or ratio of the DNA comprising the desired sequence is increased compared to the amount and / or ratio of the DNA comprising the desired sequence in the starting material. In this regard, enrichment by amplification increases the amount and ratio of the desired sequence. Enrichment by capture-based enrichment increases the ratio of the DNA comprising the desired sequence.

[0142] After treating the DNA to distinguish between methylated and unmethylated sites, the method may further comprise the step of identifying the methylated or unmethylated sites (ie, in the original sample).

[0143] The identification step may comprise any suitable method known in the art, such as array detection or sequencing (eg, next generation sequencing).

[0144] The sequencing identification step preferably includes next generation sequencing (large-scale 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 the present invention. Next generation sequencing technology can be carried out according to the manufacturer's instructions (for example, provided by Roche, Illumina or Applied Biosystems).

[0145] In a preferred embodiment, samples are processed by converting DNA methylation using an enzymatic reaction, performing whole genome library preparation, and measuring methylation profiles by sequencing (EM-Seq).

[0146] In a particularly preferred embodiment, the sample is processed by converting DNA methylation using an enzymatic reaction, performing whole-genome library preparation, hybridizing the whole-genome converted library preparation with capture probes (preferably capture probes capable of capturing DNA regions near gene promoters); and measuring the methylation profile by sequencing (EM-Seq).

[0147] DNA methylation profile

[0148] A "DNA methylation profile" or "methylation profile" may 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. Thus, the presence, absence, amount or level of 5mC at each individual DNA methylation site within multiple sites may be assessed and help determine the risk of mortality and / or probability of healthy life span for a dog. The quality and / or efficacy of the method may therefore be improved by combining values ​​from multiple DNA methylation markers.

[0149] Suitably, the biological clock of the present invention comprises methylation profiles from a plurality of methylation sites.

[0150] Suitably, 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 risk of mortality and / or probability of healthy lifespan (i.e. biological age) in a dog.

[0151] Suitably, the methylation profile may 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] Suitably, the methylation profile may refer to the presence or absence of 5mC from about 100, about 200, about 500, about 1000 or about 2000 DNA methylation sites.

[0153] To generate a biological clock for determining mortality risk and / or probability of healthy life span, an initial methylation profile may be processed or simplified to generate a restricted methylation profile, which is then used to generate a biological clock.

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

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

[0156] Suitably, a methylation profile may refer to DNA methylation sites of genes associated with a particular physiological or biochemical pathway. Thus, a methylation profile may enable the biological age of a particular tissue, organ, or physiological system to be determined. Determining the biological age of a particular tissue, organ, or physiological system may advantageously allow 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 muscle or cardiovascular disease, determining the biological age of that physiological system may be advantageous.

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

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

[0159] In some embodiments, the threshold selects those sites with the highest ranked average methylation values ​​for the epigenetic age predictor. For example, the threshold can be those sites with an average methylation level that is in 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 level of all sites "i" tested for a predictor (e.g., a biological clock).

[0160] Alternatively, the threshold value can be those sites where the average methylation level is greater than or equal to 50, 60, 70, 80, 90, 95, 96, 97, 98 or 99 percentiles. In other embodiments, the threshold value can be based on the absolute value of the average methylation level. For example, the threshold value can 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 thresholds and absolute thresholds can be applied to the average methylation level at each site "i" alone or in combination. As an illustration of the application of a combined threshold, a subset of sites that are in the top 3% of all sites tested by the average methylation level and also have an absolute average methylation level greater than 6% can be selected. The result of this selection process is a DNA methylation profile of specific hypermethylated sites (e.g., CpG sites) that are considered most informative for the probability of mortality risk and / or healthspan determination.

[0161] Suitably, the DNA methylation profile may comprise at least one methylation site as listed in Table 3.

[0162] Suitably, a methylation site may be defined as a methylation marker present in any one or more of SEQ ID NOs: 1-261. SEQ ID NOs: 1-261 show the sequence on either side of the methylation marker in the "Tasha" dog reference genome (https: / / www.ncbi.nlm.nih.gov / assembly / GCF_000002285.5; Jagannathan et al.; Genes (Bsael); 2021; 12(6); 847). The "CG" methylation marker is the 26th and 27th nucleotides in the sequence (i.e., there are 25 nucleotides before the methylation marker and 25 nucleotides after the methylation marker).

[0163] Suitably, the methylation site may be defined as the insertion position in the column labeled "site" in Table 3. For example, for site chr1:3844418-3844420, the methylation marker is chr1:3844419. Suitably, the DNA methylation profile may comprise at least 3, at least 5, at least 10, at least 20, at least 50, at least 100, at least 150, at least 200 or preferably every methylation site in the methylation sites listed in Table 3.

[0164] Suitably, the DNA methylation profile may comprise methylation sites chr2.32494387.32494389; chr22.46374563.46374565; and chr6.45773846.45773848. These sites are shown in Table 4.

[0165] Suitably, the DNA methylation profile may comprise methylation sites chr2.32494387.32494389; chr22.46374563.46374565; chr6.45773846.45773848; chr5.61645225.61645227; and chr3.70831746.70831748. These sites are shown in Table 5.

[0166] Suitably, the DNA methylation profile may comprise methylation sites chr2.32494387.32494389; chr22.46374563.46374565; chr6.45773846.45773848; chr5.61645225.61645227; chr3.70831746.70831748; chr15.10856498.10856500; chr16.8886545.8886547; chr3.46843750.46843752; chr20.44942213.44942215; and chr20.57347921.57347923. These sites are shown in Table 6.

[0167] Suitably, the DNA methylation profile may comprise methylation sites chr2.32494387.32494389; chr22.46374563.46374565; chr6.45773846.45773848; chr5.61645225.61645227; chr3.70831746.70831748; chr15.10856498.10856500; chr16.8886545.8886547; chr3.46843750.46843752; chr20.44942213.44942215; chr20.57347921.5 These sites are shown in Table 7.

[0168] Suitably, the DNA methylation profile may comprise methylation sites chr2.32494387.32494389; chr22.46374563.46374565; chr6.45773846.45773848; chr5.61645225.61645227; chr3.70831746.70831748; chr15.10856498.10856500; chr16.8886545.8886547; chr3.46843750.46843752; chr20.44942213.44942215; chr20.57347921.57347 923; chr24.21051024.21051026; chr33.26512711.26512713; chr23.3798 7526.37987528;chr5.32347978.32347980;chr6.60675998.60676000;chr 19.44679975.44679977; chr10.7411293.7411295; chr33.26512692.2651 2694; chr1.121182146.121182148; chr17.62156690.62156692; chr35.238 53863.23853865;chr17.49222883.49222885;chr14.5949013.5949015;c hr5.67427228.67427230; chr20.43366718.43366720; chr24.12518910.12 518912; chr29.19479611.19479613; chr4.36180013.36180015; chr35.23 852932.23852934;chr1.58324050.58324052;chr9.12522577.12522579;c hr5.32711703.32711705; chr33.23473772.23473774; chr2.51831832.51 831834; chr20.46098568.46098570; chr11.24476652.24476654; chr1.100 289525.100289527; chr17.33851462.33851464; chr6.12827818.1282782 0; chr20.54301453.54301455; chr1.19541665.19541667; chr5.55848493.55848495; chr10.44524681.44524683; chr9.50976682.50976684; chr33.26512695.26512697; chr24.24968751.24968753; chr10.18237423.18237425; chr35.23853763.23853765; chr9.1046331.1046333; and chr20.49940551.49940553. .

[0169] Determination of DNA methylation sites / DNA methylation profiles that indicate mortality risk and / or probability of healthy life span

[0170] The present invention includes using DNA methylation profiles to determine the risk of death and / or the probability of healthy life span of a dog. Therefore, the present invention includes using DNA methylation profiles to generate a biological clock associated with the risk of death and / or the probability of healthy life span. The biological clock of the present invention may also be referred to as an "epigenetic clock".

[0171] For example, the provision of DNA methylation sites or DNA methylation profiles indicating the probability of mortality risk and / or healthy life span can be achieved through a training data set and a machine learning method. Suitably, the machine learning method can be a supervised machine learning method.

[0172] For example, DNA methylation sites or DNA methylation profiles may be trained on a dataset comprising dogs with known death outcomes (alive or dead) and chronological ages. Suitably, DNA methylation sites or DNA methylation profiles may be trained on a dataset comprising dogs with known death outcomes and chronological ages in combination with known breeds and / or sexes.

[0173] For example, a model of DNA methylation sites or DNA methylation profiles that indicate mortality risk and / or probability of healthy life span can be provided by training a dataset of methylation states at multiple DNA methylation sites on a training dataset of dogs with known mortality outcomes (survival or death) and chronological age using a machine learning framework, and testing on a retained cohort to validate the accuracy of the model.

[0174] The machine learning framework may include fitting a penalized model to a training dataset of dogs with known mortality outcome (survival or death) and chronological age (and optionally breed and / or sex) using, for example, the glmnet R package.

[0175] Suitably, the penalized model may be, for example, a penalized Cox regression, a least angle regression path to solution (LARS) Cox regression or a penalized survival model.

[0176] The machine learning framework may include fitting a penalized Cox regression to a training dataset of dogs with known mortality outcome (alive or dead) and chronological age (and optionally breed and / or sex) using, for example, the glmnet R package.

[0177] Suitably, the machine learning framework may comprise fitting a penalized model, preferably a penalized Cox regression, of known mortality outcomes (survival or death) / survival rates explained by the DNA methylation profile and chronological age (and optionally breed and / or sex).

[0178] Suitably, the machine learning framework may comprise fitting a penalized model, preferably a penalized Cox regression, of known mortality outcomes (survival or death) / survival rates explained by the DNA methylation profile, chronological age, breed and sex.

[0179] As used herein, "known mortality outcome (survival or death)" may also be referred to as "survival rate."

[0180] Suitably, a machine learning framework may be used to determine a model comprising a set of DNA methylation sites or a DNA methylation profile that is indicative of mortality risk and / or probability of healthy life span.

[0181] The model may include methylation states at a plurality of DNA methylation sites; wherein the methylation state at each site is considered in the model by multiplying by a coefficient value.

[0182] Suitably, sex may be encoded as a numeric value where 0 represents female and 1 represents male.

[0183] Suitably, breed may be coded as a numerical value where 0 represents a small breed and 1 represents a medium sized breed.

[0184] A dog's biological age can be expressed in years, months, days, etc.

[0185] The coefficient value of each parameter usually depends on the measurement unit of all variables in the model. As the technician will understand, the value of each coefficient value will therefore depend on the quantity and property of the different parameters used in the model and the property of the training data provided. Therefore, conventional statistical methods can be applied to training data sets to obtain coefficient values. This method includes, for example, calculating two gompertz or weibull functions (for example, in the case of the state (survival or death) of a known dog) on ​​a training set, a function models the survival rate as a function of methylation profiles, chronological age, breed category (small or medium-sized dog) and sex (model 1), and a second function only considers chronological age, breed category and sex (model 2). These models can be fitted using flexsurv package (v2.1) in the R software environment.

[0186] Biological age can be defined as the time variable ("chronological age") at which the probability of survival of an animal given by Model 2 is equal to the probability of survival given by Model 1 at their chronological age.

[0187] A model of DNA methylation sites or DNA methylation profiles that indicate mortality risk and / or probability of healthy life span can be provided by training a dataset of methylation states at multiple DNA methylation sites for a phenotypic age predicted at the age of DNA sample collection, and testing against a retained cohort to validate the accuracy of the model.

[0188] Methods for determining the phenotypic age of a dog or cat are described in PCT / EP2023 / 061058 and PCT / EP2023 / 061059, respectively. The calculation of the phenotypic age takes into account the direct predictive value of blood biomarkers for the risk of death and / or the probability of healthy life span. For example, a given biomarker may not be directly correlated with chronological age, but may indicate a specific pathological condition, thereby indicating an increased risk of death and / or a probability of reduced healthy life span.

[0189] Determining the phenotypic age of the dog may include determining the level of one or more biomarkers in one or more samples obtained from the dog, wherein the one or more biomarkers are selected from white blood cell count, serum albumin, serum alkaline phosphatase, serum creatine kinase, hemoglobin, hematocrit, mean corpuscular hemoglobin, serum glucose, mean corpuscular volume, serum globulin, serum calcium, platelet count, and / or red blood cell count.

[0190] Suitably, the phenotypic age of a dog may be provided by:

[0191] a. determining the levels of the following biomarkers: white blood cell count, serum albumin, serum alkaline phosphatase, serum creatine kinase, hemoglobin, hematocrit, mean corpuscular hemoglobin, serum glucose, mean corpuscular volume, and serum globulin in one or more samples obtained from the dog; and

[0192] b. Use formula (1) to determine the dog's phenotypic age (Phenoage):

[0193]

[0194] where xb is the sum of the values ​​of each biomarker, sex and breed multiplied by their corresponding coefficients according to formula (2):

[0195]

[0196] where sex is encoded as a numeric value, with 0 representing females and 1 representing males.

[0197] The breed is coded as a numeric value, where 0 represents a small breed and 1 represents a medium-sized breed.

[0198] and wherein phenotypic age is used to determine a dog's risk of mortality and / or probability of healthy life span.

[0199] The coefficient value of each parameter usually depends on the measurement unit of all variables in the model. As the technician will understand, the value of each coefficient value will therefore depend on the quantity and property of the different parameters used in the model and the property of the training data provided. Therefore, conventional statistical methods can be applied to training data sets to obtain the coefficient value for the above formula. This method includes, for example, calculating two gompertz or weibull functions (for example, in the case of the state (survival or death) of known dogs) on the training set, a function models the survival rate as a function of selected biomarkers, chronological age, breed category (small or medium-sized dogs) and gender (model 1), and the second function only considers chronological age, breed category and gender (model 2). These models can be fitted using flexsurv package (v 2.1) in the R software environment.

[0200] Suitably, a negative coefficient for a given biomarker means that a higher level of the biomarker has a positive effect on reducing the risk of death, and / or a lower level of the biomarker has a negative effect on reducing the risk of death. Suitably, a positive coefficient for a given biomarker means that a higher level of the biomarker has a negative effect on reducing the risk of death, and / or a lower level of the biomarker has a positive effect on reducing the risk of death.

[0201] Exemplary coefficients and γ and γ 品种 The values ​​are provided in the table below.

[0202]

[0203]

[0204] Phenotypic age can be defined as the time variable ("chronological age") at which the probability of survival of an animal given by Model 2 is equal to the probability of survival given by Model 1 at their chronological age.

[0205] The phenotypic age of a dog can be expressed in years, months, days, etc.

[0206] Biomarkers for determining phenotypic age can be determined using standard methods in the art and are typically measured as part of a standard blood test to determine the disease state of an animal. For example, biomarkers are typically determined as part of a standard clinical complete blood count (CBC) and standard clinical blood chemistry analysis.

[0207] Suitably, a model of DNA methylation sites or a DNA methylation profile indicative of the probability of mortality risk and / or healthy life span trained for phenotypic age may be provided in a two-step process.

[0208] In a first step, the machine learning framework may include fitting a penalized model of phenotypic age (PhenoAge) explained by one or more blood biomarkers as described herein and chronological age (and optionally sex and / or breed) using, for example, the glmnet R package. Preferably, the machine learning framework may include fitting a penalized model of phenotypic age (PhenoAge) explained by one or more blood biomarkers as described herein, chronological age, sex, and breed.

[0209] Suitably, the penalized model may be, for example, a penalized Cox regression, a least angle regression path to solution (LARS) Cox regression or a penalized survival model.

[0210] The machine learning framework may include fitting a penalized Cox regression of phenotypic age (PhenoAge) explained by one or more blood biomarkers as described herein, chronological age, sex, and breed.

[0211] In a second step, the machine learning framework may comprise fitting a penalized regression of the phenotypic age explained by the DNA methylation. Suitably, the machine learning framework may comprise fitting a penalized regression of the phenotypic age explained by the DNA methylation profile.

[0212] The penalized regression can be an elastic net regression.

[0213] As used herein, the term "one or more biomarkers" may include at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, or at least thirteen biomarkers.

[0214] As used herein, the term "one or more biomarkers" may include one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or thirteen biomarkers.

[0215] Suitably, DNA methylation sites or DNA methylation profiles may be combined with the levels of one or more blood biomarkers described herein to generate a model indicative of mortality risk and / or probability of healthy life span. For example, a dataset of methylation states at multiple DNA methylation sites and levels of one or more blood biomarkers may be trained for a training dataset of dogs with known mortality outcomes (survival or death) and chronological age, and tested against a retained cohort to verify the accuracy of the model, to provide a model comprising a combination of the DNA methylation profiles described herein and the levels of one or more blood biomarkers.

[0216] The machine learning framework may include fitting a penalized regression to a training dataset of dogs with known mortality outcome (alive or dead) and chronological age (and optionally breed and / or sex) using, for example, the glmnet R package.

[0217] The machine learning framework may include fitting a penalized Cox regression to a training dataset of dogs with known mortality outcome (alive or dead) and chronological age (and optionally breed and / or sex) using, for example, the glmnet R package.

[0218] Suitably, the machine learning platform may include one or more deep neural networks. A neural network is a collection of neurons (also referred to as units) connected in an acyclic graph. Neural network models are typically organized into different layers of neurons. For most neural networks, the most common layer type is a fully connected layer, in which neurons between two adjacent layers are fully connected in pairs, but neurons within a single layer do not share connections. One of the main features of a deep neural network is that neurons are controlled by a nonlinear activation function. This nonlinearity combined with a deep architecture makes it possible to make more complex combinations of input features, ultimately leading to a broader understanding of the relationships between them, and therefore leading to a more reliable final output. Deep neural networks have been applied to many types of data ranging from structural data to chemical descriptors or transcriptomic data.

[0219] Suitably, the machine learning platform comprises one or more generative adversarial networks. Suitably, the machine learning platform comprises an adversarial autoencoder architecture. Suitably, the machine learning platform comprises a feature importance analysis for ranking DNA methylation sites by their importance in determining biological age.

[0220] A dog's biological age can be expressed in years, months, days, etc.

[0221] Preferably, the risk of mortality and / or probability of healthy life span is expressed as the difference between the biological age and the chronological age of the dog.

[0222] Compare with a reference or control

[0223] The method may also include a step of comparing the DNA methylation differences 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 risk of death and / or probability of healthy life span (i.e., biological age). In some embodiments, the reference value is a value previously obtained for a subject or subject group with a known risk of death and / or probability of healthy life span (i.e., biological age). The reference value can be based on a known DNA methylation state at one or more sites from a subject group with a known death status (survival or death), chronological age, variety, and / or gender, such as an average or median level.

[0224] Combining biomarker levels with additional measures and / or characteristics

[0225] Suitably, the method further comprises combining the DNA methylation profile with one or more of the chronological age, breed and / or sex of the dog. By combining this information, a biological age associated with the risk of death and / or the probability of healthy life span can be determined.

[0226] Subject classification

[0227] The biological age determined by the method of the present invention can also be compared to one or more predetermined thresholds (i.e., differences from chronological age). Using such thresholds, subjects can be stratified into categories indicating a determined risk (e.g., low, medium, or high determined 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.

[0228] Methods for selecting / monitoring a subject's lifestyle regimen, dietary regimen, or therapeutic intervention

[0229] In another aspect, the invention provides methods for selecting a lifestyle regimen, a dietary regimen, or a therapeutic intervention for a subject. The change in lifestyle can be any change described herein, such as a change in a dietary intervention and / or an exercise regimen. The change in lifestyle can be the administration of a therapeutic modality.

[0230] Lifestyle regimen, dietary regimen or therapeutic intervention can be applied to dogs in any suitable time period. After the period of time, the method can be used to determine the risk of death and / or the probability of healthy life span of the dog again, so as to determine the efficacy of the lifestyle regimen, dietary regimen or therapeutic intervention to reduce the risk of death of the dog and / or increase the probability of healthy life span of the dog. For example, lifestyle regimen, dietary regimen or therapeutic intervention 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 regimen, dietary regimen or therapeutic intervention 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.

[0231] The lifestyle regimen, dietary regimen, or therapeutic intervention may be referred to as an anti-aging lifestyle regimen, dietary regimen, or therapeutic intervention.

[0232] 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 comprises the administration of at least one dietary product or dietary regimen or nutritional supplement.

[0233] The dietary intervention may be a diet, a dietary regimen, a supplement, or a supplement regimen, or a combination of a diet and a supplement, or a combination of a diet and multiple supplements.

[0234] The dietary intervention or dietary product described herein can be any suitable dietary regimen, such as a calorie-restricted diet, an elderly diet, a low-protein diet, a phosphorus diet, a low-protein diet, a potassium-supplemented diet, a polyunsaturated fatty acid (PUFA)-supplemented diet, an antioxidant-supplemented diet, a vitamin B-supplemented diet, a liquid diet, a selenium-supplemented diet, an ω3-6 ratio diet, or a diet supplemented with carnitine, branched-chain amino acids or derivatives, nucleotides, nicotinamide precursors (such as nicotinamide mononucleotide (MNM) or nicotinamide riboside (NR)), or any combination of the foregoing.

[0235] Suitably, the dietary intervention or dietary product may be a calorie restricted diet, an elderly diet or a low protein diet. Suitably, the dietary intervention or dietary product may be a calorie restricted diet. Suitably, the dietary intervention or dietary product may be a low protein diet.

[0236] The dietary intervention may be determined based on the dog's baseline maintenance energy requirement (MER). Suitably, the MER may be the amount of food that stabilizes the dog's body weight (varies by less than 5% over three weeks).

[0237] For example, it is generally believed that young, growing dogs benefit from a high-energy / high-protein diet; however, older dogs may have lower energy requirements, so the diet may be adjusted appropriately. In particular, many manufacturers produce "senior" lines of dog foods that are lower in calories and higher in fiber, but with protein and fat levels appropriate for older dogs.

[0238] Suitably, the calorie restricted diet may comprise about 50%, about 55%, about 60%, about 65%, about 75%, about 80%, about 85%, or about 90% of the dog's MER. Suitably, the calorie restricted diet may comprise about 60% or about 75% of the dog's MER.

[0239] Suitably, the low protein diet may comprise less than 20% protein (dry matter %).For example, the low protein diet may comprise less than 19% protein (dry matter %).

[0240] These diets are often recommended based on the dog's chronological age. For example, it may be recommended that a dog be switched to a senior diet around 7 or 8 years old. However, in the context of the present invention, determining that a dog has an increased risk of mortality may allow for a determination to switch the dog to a senior diet at an earlier age than would be expected given the dog's chronological age. In contrast, a dog with a reduced risk of mortality may be able to continue on an adult diet longer than the dog's chronological age.

[0241] Dietary interventions may include foods, supplements and / or beverages that contain nutrients and / or bioactive agents that mimic the benefits of caloric restriction (CR) without limiting daily caloric intake. For example, foods, supplements and / or beverages may contain functional ingredients with benefits similar to CR. Suitably, foods, supplements and / or beverages may contain autophagy inducers. Suitably, foods, supplements and / or beverages may contain fruits and / or nuts (or their extracts). Suitable examples include, but are not limited to, pomegranates, strawberries, blackberries, camu camu, walnuts, chestnuts, pistachios, pecans. Suitably, foods, supplements and / or beverages may contain probiotics, with or without fruit extracts or nut extracts.

[0242] Modifying the subject's lifestyle also includes indicating that the subject needs to make a lifestyle change, such as prescribing more exercise.Similar to dietary intervention, determining that a dog has an increased risk of mortality compared to what would be expected given its chronological age can allow for a determination to switch the dog to an appropriate exercise regimen.

[0243] Changing the lifestyle of the experimenter also includes selecting or recommending a treatment mode or scheme. A treatment mode or scheme can be a mode for treating and / or preventing, for example, arthritis, dental disease, endocrine disorders, heart disease, diabetes, liver disease, nephropathy, prostate disorders, cancer, and behavioral or cognitive disorders. Suitably, preventive treatment can be applied to a dog identified as being at risk of such a condition due to an increased risk of death and / or specific biomarkers associated with a known disease-related pathway. In other embodiments, dogs that are determined to be at risk of certain conditions (due to an increased risk of death and / or specific biomarkers associated with a known disease-related pathway) can be monitored more regularly so that diagnosis and treatment can be started as early as possible.

[0244] The present 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 intended to cause a reduction in the epigenetic or biological age of a subject. Suitably, the 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. The present methods of monitoring and / or determining the efficacy of a lifestyle regimen, dietary regimen, or therapeutic intervention or developing a lifestyle regimen, dietary regimen, or therapeutic intervention to reduce biological age are particularly applicable to this aspect.

[0245] Thus, the present invention can advantageously enable identification of dogs that are expected to respond particularly well to a given intervention (e.g., a lifestyle regimen, a dietary regimen, or a therapeutic intervention). Thus, intervention can be applied in a more targeted manner to dogs that are expected to respond.

[0246] In one aspect, the present invention provides a method for determining the efficacy of a lifestyle regimen, dietary regimen, or therapeutic intervention in reducing the risk of mortality and / or increasing the probability of a healthy lifespan in a dog, the method comprising: a) applying a lifestyle regimen, dietary regimen, or therapeutic intervention to the dog, wherein the lifestyle regimen, dietary regimen, or therapeutic intervention has been selected according to the methods of the present invention; b) after applying the lifestyle regimen, dietary regimen, or therapeutic intervention to the dog for a period of time; determining the risk of mortality and / or the probability of a healthy lifespan in the dog using a DNA methylation profile from a sample obtained from the dog; c) determining whether there is a change in the risk of mortality in the dog after following the lifestyle regimen, dietary regimen, or therapeutic intervention for the period of time.

[0247] In another aspect, the present invention provides a method for determining the efficacy of a lifestyle regimen, dietary regimen or therapeutic intervention in reducing the risk of death and / or increasing the probability of healthy life of a dog, the method comprising: a) using a DNA methylation profile from a sample obtained from the dog to determine the risk of death and / or the probability of healthy life of the dog; b) applying a lifestyle regimen, dietary regimen or therapeutic intervention selected based on the risk of death and / or the probability of healthy life of the dog determined in step a); c) after applying the lifestyle regimen, dietary regimen or therapeutic intervention to the dog for a period of time; determining the risk of death and / or the probability of healthy life of the dog using a DNA methylation profile from a sample obtained from the dog; d) determining whether the risk of death and / or the probability of healthy life of the dog changes between step a) and step c).

[0248] Suitably, the lifestyle regimen, dietary regimen or therapeutic intervention may have been applied to the dog for a period of time prior to determining the first risk of mortality and / or probability of healthy lifespan; however, the effectiveness of the lifestyle regimen, dietary regimen or therapeutic intervention in improving the dog's risk of mortality and / or probability of healthy lifespan (i.e., reducing the risk of mortality and / or increasing the probability of healthy lifespan) may still be monitored by determining the risk of mortality and / or probability of healthy lifespan at two or more times during application of the lifestyle regimen, dietary regimen or therapeutic intervention.

[0249] Suitably, the method may comprise an "ecosystem"; in particular a digital ecosystem. Suitably, the method may comprise providing a sample obtained from a dog, optionally using a kit according to the invention; and (b) providing the sample for subsequent DNA extraction (e.g. by post) for measuring DNA methylation in DNA extracted from the sample, thereby obtaining a DNA methylation profile.

[0250] The DNA methylation profile may then be used according to any of the methods; preferably using a computer system or computer program product according to the invention.

[0251] The computer system or computer program may then prepare and share a report detailing the results of the analysis / method, or any other results of the method, for example in the form of selecting or recommending an appropriate lifestyle regimen, dietary regimen, or therapeutic intervention for the dog.

[0252] Suitably, the sample may be a sample that can be obtained by a dog owner at home (e.g. without the need for a veterinarian or healthcare professional). Suitably, the sample may be a hair follicle, oral swab or saliva sample.

[0253] Uses of dietary intervention

[0254] In one aspect, the invention provides a dietary intervention for reducing the risk of mortality and / or increasing the probability of healthy life span in a dog, wherein the dietary intervention is administered to a dog, wherein the risk of mortality and / or the probability of healthy life span is determined by the present method.

[0255] In another aspect, the invention provides for use of a dietary intervention in reducing the risk of mortality and / or increasing the probability of healthy lifespan in a dog, wherein the dietary intervention is administered to a dog, wherein the risk of mortality and / or the probability of healthy lifespan is determined by the present method.

[0256] As described herein, a dietary intervention can be a dietary product or a dietary regimen or a nutritional supplement.

[0257] Computer program product

[0258] The method can be performed using a computer. Therefore, the method can be performed on a computer.

[0259] Suitably, the computer may prepare and share a report detailing the results of the method.

[0260] The methods described herein can be implemented as computer programs running on general-purpose hardware such as one or more computer processors. In some embodiments, the functions described herein can be implemented by devices such as smart phones, tablet terminals or personal computers.

[0261] In one aspect, the present invention provides a computer program product comprising computer-implementable instructions for causing a programmable computer to determine the risk of mortality and / or probability of healthy life span for a dog as described herein.

[0262] In one embodiment, the user inputs the level of one or more DNA methylation markers as defined herein into the device, optionally along with chronological age, breed, and sex. The device then processes this information and provides a determination of the biological age of the dog. Alternatively, the device then processes this information and provides a determination of an appropriate lifestyle regimen, dietary regimen, or therapeutic intervention for the dog based on the biological age.

[0263] The device may typically be a server on a network. However, any device may be used as long as it can process biomarker data and / or additional parameters or characteristics using a processor, a central processing unit (CPU), etc. For example, the device may be a smart phone, a tablet terminal, or a personal computer, and outputs information indicating the determined biological age of the dog or a determination of an appropriate lifestyle regimen, dietary regimen, or therapeutic intervention for the dog based on the biological age.

[0264] Those skilled in the art will understand that they can freely combine all features of the present invention described herein without departing from the scope of the invention disclosed herein.

[0265] Example

[0266] The present invention will now be further described by way of examples, which are intended to help those skilled in the art to practice the present invention but are not intended to limit the scope of the present invention in any way.

[0267] Example 1 - Exemplary Methods for Generating Epigenetic Biological Clocks

[0268] Identification of DNA methylation sites

[0269] Whole blood samples from the canine cohort were analyzed by performing DNA extraction, converting DNA methylation by using an enzymatic reaction, performing whole-genome library preparation, hybridizing the whole-genome converted library preparation with capture probes against gene promoters, and measuring methylation profiles by sequencing (EM-Seq).

[0270] The capture probes are directed against approximately 40,000 targets (promoter regions - approximately 1 kb upstream to 0.5 kb downstream of the promoter). These target regions contain potential methylation sites of interest (single cytosine residues that can be methylated).

[0271] The following bioinformatics steps were performed after sequencing and before further analysis:

[0272] Fastq quality check using fastQC – https: / / www.bioinformatics.babraham.ac.uk / projects / fastqc /

[0273] Adapter trimming using trimGalore – https: / / www.bioinformatics.babraham.ac.uk / projects / trim_galore /

[0274] Align to the dog genome using bwa-meth (https: / / github.com / brentp / bwa-meth) or Bismark - https: / / www.bioinformatics.babraham.ac.uk / projects / bismark /

[0275] Marking Duplicates with Picard – https: / / gatk.broadinstitute.org / hc / en-us / articles / 360037052812-MarkDuplicates-Picard-

[0276] Calling methylation using Methyldackel - https: / / github.com / dpryan79 / MethylDackel

[0277] Method for filtering sites to generate restricted DNA methylation profiles for training biological clocks

[0278] After determining the methylation status of the methylation sites in each sample, the initial methylation profile can be filtered / processed to generate a restricted methylation profile containing fewer discrete methylation sites. The purpose of this filtering is to provide a restricted methylation profile containing, for example, 50,000 to 500,000 methylation sites that can be used to train the biological clock.

[0279] Methods used to filter the initial methylation profiles include:

[0280] 1) Remove (un)methylated sites in all samples

[0281] 2) Remove sites that do not have at least 5 counts in at least 90% of the samples

[0282] 3) Removal of chromosome X and / or Y loci

[0283] Other potential filtering steps to reduce the number of discrete methylation sites include:

[0284] 1) Define methylation sites as DNA regions (CpG islands, sites separated by less than e.g. 1000 bp are considered identical)

[0285] 2) Limit the target to, for example, inflammatory sites, such as genes associated with inflammation from GO / KEGG pathways, and select sites on these genes

[0286] 3) Correlation analysis and retention of representatives of each cluster (e.g., weighted correlation network analysis (WGCNA) (Langfelder & Horvath; BMC Bioinformatics; 9(559); 2008 or EBModules (Zollinger et al.; Biostatistics, 19(2), 153-168; (2018)).

[0287] 4) Differential methylation analysis to pre-select DNA methylation sites (e.g. using logistic regression) or to identify sites that vary more between young and old dogs (e.g. using ANOVA). If differential methylation analysis is performed (using the function preprocessQuantile in R), normalization (quantile) is performed before the filtering step.

[0288] Generates biological clock

[0289] The dataset, which includes information on the mortality status (alive or dead), chronological age, breed and sex of the dog cohort, is split into training and testing sets (e.g., 2 / 3 of the data is used for training and 1 / 3 of the data is used for testing, thus ensuring a good split with respect to metadata; e.g., similar proportions of each breed / each sex in training and test sets).

[0290] A penalized Cox model was fitted to survival using methylation sites, breed / breed category, sex, and chronological age as predictors. The parameters of the model as well as the penalization parameters were estimated using the glmnet package in R.

[0291] Two gompertz or Weibull functions were calculated, one modeling survival as a function of methylation profile, chronological age, breed category (small or medium dog), and sex (model 1), while the second function only considered chronological age, breed category, and sex (model 2). The models were fitted using the flexsurv package (v 2.1) in the R software environment.

[0292] The risk of death and / or the probability of healthy life span can be defined as a time variable ("chronological age") at which the probability of survival of animals given by Model 2 is equal to the probability of survival given by Model 1 at their chronological age.

[0293] The penalty parameter is chosen to provide a reasonable number of DNA methylation sites (eg, up to 1000) with a good model fit for the risk of death and / or probability of healthy life span.

[0294] The biological age model based on DNA methylation sites was then evaluated on the test set.

[0295] In this embodiment, the following steps are performed:

[0296] 1) All methylation sites with less than 15 counts coverage were set as “missing”;

[0297] 2) imputation was performed using the “Boostme” algorithm (Zou et al.; BMC Genomics 19, 390 (2018)) and the training, test, and validation set sizes were set to 200,000 randomly selected sites;

[0298] 3) Removal of ChrX site;

[0299] 4) Site filtering using EBmodule (Zollinger et al.; Biostatistics, 19(2), 153-168; (2018)); The dataset was divided into blocks of approximately 5000 sites by grouping sites per target (1500 bp around TSS) and per chromosome. The correlation matrix for applying the EB module was then calculated - as described by Zollinger et al. Each module was then represented by a medoid site. Some sites did not belong to any module and were called scattered sites (defined as described by Zollinger et al.);

[0300] 5) Removal of sites present on mammalian CpG arrays (Arneson et al.; Nature Communications; 13; 783; 2022);

[0301] 6) An elastic net regression was fitted on the 500K loci using phenoAge_pred (prediction of phenotypic age at age of DNA collection) as the response variable, which selected 261 loci as loci that form the biological clock (see Table 3).

[0302] A series of evaluations are used to verify the clock. Figure 3 Validation of the clock in caloric restriction is shown, where caloric restricted dogs have a lower biological age (lower variance) compared to dogs on a control diet. Figure 3 The changes in correspond to the residuals from the regression model of chronological age relative to the predicted phenoDNAmAge.

[0303] Further verification in Figure 4 , where there was a difference in the amount of change (phenoDNAmAge) between the two diets, the dogs on the restricted diet were biologically younger at 6 years of age.

[0304] Figure 5 It is shown that there are significant differences between the two diets when adjusting for a linear mixed effects model (DogID as a random effect).

[0305] Figure 6 A significant difference in survival rates between biologically younger dogs and biologically older dogs is shown.

[0306] Figure 7 It is shown that when a Cox proportional hazards model was fit using sex and variance phenoDNAmAge (residual from chronological age relative to predicted phenoDNAmAge), stratified by breed category (small or medium) on the training set, increases in variance phenoDNAmAge were significantly associated with increased risk of death.

[0307] Figure 8 It was shown that the biological clock is highly reproducible between technical replicates.

[0308] Univariate analysis was performed to determine whether each of the 261 loci was individually statistically significant in its association with biological age (see Table 3).

[0309] Further biological clocks were generated using only the first 3, 5, 10, 20 and 50 sites from the complete list of sites shown in Table 3, and each site was shown to correlate with biological age (see Figure 9-13 ). These clocks were generated by selecting the first n sites based on the absolute value of the coefficients of the full clock (in descending order, with the largest coefficients first). Linear models explaining chronological age were fit using the first n sites as predictors. Details of the first 3, first 5, first 10, and first 20 clocks are shown in Tables 4-7.

[0310] Example 2 - Determination of blood biomarkers associated with mortality risk in dogs

[0311] Predictive blood biomarkers were determined from a biomarker panel consisting of a standard clinical complete blood count (CBC) and standard clinical blood chemistry analysis. Serum samples were collected after an overnight fast and measured using standard veterinary clinical practice.

[0312] Table 1 - Clinical complete blood count (CBC) and clinical blood chemistry analysis

[0313]

[0314]

[0315] *Values ​​were log-transformed using the natural logarithm.

[0316] We conducted a longitudinal study of dogs with repeated measures of these parameters as well as on the status (alive or dead), sex, and breed of the dogs. We first classified the breeds as small or medium based on the mean weight of adult dogs of the breed (less than 10 kg or more than 10 kg, respectively). We then organized the data using the R programming language. For each dog, we recorded the biomarkers as time-dependent covariates using time intervals open on the left and closed on the right, i.e., (tstart, tstop), where the biomarker information corresponded to the start of the interval and the event (alive or dead) was recorded as the last tstop value. To do this, we used the tmerge function of the survival package in R (v.3.2-13). We then fitted a Cox proportional hazards model to the data for each of the 28 biomarkers, including sex and breed category (small or medium). We then adjusted the p-value for each parameter to account for multiple comparisons (by the false discovery rate (FDR)), and selected features with an adjusted FDR below 0.05 ( Figure 1 ).

[0317] Using this approach, we identified 13 biomarkers that individually predicted a dog's probability of survival:

[0318] White blood cell count (10^3 / ul)

[0319] Serum albumin (g / dL)

[0320] Serum alkaline phosphatase (U / L, ln conversion)

[0321] Serum creatine kinase (IU / L, ln conversion)

[0322] Hemoglobin (g / dL)

[0323] Hematocrit (%)

[0324] Mean corpuscular hemoglobin (pg)

[0325] Serum sodium (mmol / L)

[0326] Mean corpuscular volume (fL)

[0327] Serum globulin (g / dL)

[0328] Serum calcium (mg / dL)

[0329] Serum platelet count (10^3 / uL)

[0330] Red blood cell count (10^3 / uL)

[0331] Example 3 - Multi-parameter model for predicting mortality risk

[0332] Next, we constructed the best model that considered multiple parameters simultaneously, as this is more likely to cover the various organ dysfunctions that occur with aging. However, selecting several features that may be correlated with each other will be biased. To avoid this problem, we used a penalized regression approach using the glmnet package (v4.1-3). We fit a LASSO penalized cox proportional hazards model on the data and used 20-fold cross-validation to compare different values ​​of the penalty parameter λ. This approach selected the 10 most predictive blood biomarkers for survival, ranked in order of importance as follows:

[0333] White blood cell count (10^3 / ul)

[0334] Serum albumin (g / dL)

[0335] Serum alkaline phosphatase (U / L, ln conversion)

[0336] Serum creatine kinase (IU / L, ln conversion)

[0337] Hemoglobin (g / dL)

[0338] Hematocrit (%)

[0339] Mean corpuscular hemoglobin (pg)

[0340] Serum glucose (mg / dL)

[0341] Mean corpuscular volume (fL)

[0342] Serum globulin (g / dL)

[0343] We also found that the first 3 biomarkers in this list were the most predictive and that performance could be improved by combining each of the next 7 biomarkers.

[0344] To extract the phenotypic age of the animals, we calculated two different gompertz functions on our training set, one that modeled survival as a function of the selected biomarkers, age, breed class (small or medium dog) and sex (model 1), while the second function only considered age, breed class and sex (model 2). These models were fitted using the flexsurv package (v 2.1). Phenotypic age was defined as the time variable (“age”) under which the probability of survival of the animals given by model 2 was equal to the probability of survival given by model 1 at their chronological age. This resulted in a mathematical function that relates blood biomarkers to phenotypic age and is given by the following formula:

[0345]

[0346] where xb is the sum of the values ​​for each biomarker, sex, and breed multiplied by their corresponding coefficients. Sex and breed are encoded as numeric values, with 0 for females and 1 for males for sex and 0 for small breeds and 1 for medium breeds for breeds. The coefficients are given by two gompertz functions trained on our training set.

[0347] As an example, the coefficients γ and γ 品种 Values ​​have been measured for the full list of biomarkers from our training set and are given in Table 2 .

[0348]

[0349] Table 2 - Coefficients and γ and γ 品种 The values ​​have been measured from the training set

[0350] coefficient γ 0.491790219 <![CDATA[β0]]> -6.036261473 Beta cell count 0.091862564 Beta hemoglobin -0.009131623 β mean corpuscular volume -0.007486146 Beta hematocrit -0.018418391 β-mean corpuscular hemoglobin -0.128195615 β serum glucose 0.009169677 Beta serum globulin 0.132755858 Serum creatine kinase beta 0.332818902 Beta serum albumin -0.744060565 Serum alkaline phosphatase β 0.262594338 Beta Varieties 1.138018960 Beta Gender 0.151826455 <![CDATA[γ 品种 ]]> 0.5668399 <![CDATA[β 02 ]]> -9.5204440 <![CDATA[β 品种2 ]]> 1.2299804 <![CDATA[β 性别2 ]]> 0.2678798

[0351] Furthermore, we observed a decrease in the strength of survival prediction (p-value) by reducing the set of 10 biomarkers by systematically removing one biomarker from the top of the list. The decrease was most pronounced for the first parameters, confirming their largest contribution, but we observed changes in the quality of predictions each time the set was reduced, indicating that each parameter contributes to the overall prediction ( Figure 2 ).

[0352] All publications mentioned in the above description are incorporated herein by reference. Various modifications and variations of the methods, compositions and uses disclosed in the present invention will be apparent to the skilled person without departing from the scope and spirit of the present invention. Although the present invention has been disclosed in conjunction with specific preferred embodiments, it should be understood that the present 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 present invention that are apparent to the skilled person are intended to fall within the scope of the following claims.

[0353] Table 3

[0354]

[0355]

[0356]

[0357]

[0358]

[0359]

[0360]

[0361]

[0362]

[0363]

Claims

1. A method for determining the risk of mortality and / or the probability of healthy life span in a dog; the method comprising: a) providing a DNA methylation profile from a sample obtained from said dog; as well as b) using the DNA methylation profile to determine the risk of mortality and / or probability of healthy life span of the dog.

2. A method for determining the biological age of a dog; the method comprising: a) providing a DNA methylation profile from a sample obtained from said dog; as well as b) using the DNA methylation profile to determine the biological age of the dog, wherein the DNA methylation profile is correlated with the risk of mortality and / or the probability of healthy life span of the dog.

3. A method for selecting a lifestyle regimen, dietary regimen or therapeutic intervention for a dog, the method comprising: a) providing a DNA methylation profile from a sample obtained from said dog; b) using the DNA methylation profile to determine the risk of mortality and / or probability of healthy life span of the dog; and c) selecting an appropriate lifestyle regimen, dietary regimen or therapeutic intervention for said dog based on said risk of mortality and / or probability of healthy life span determined in step b).

4. A method for determining the efficacy of a lifestyle regimen, dietary regimen or therapeutic intervention in improving the risk of mortality in a dog, the method comprising: a) applying a lifestyle regimen, a dietary regimen or a therapeutic intervention to the dog, wherein the lifestyle regimen, the dietary regimen or the therapeutic intervention has been selected according to the method of claim 3; b) after applying the lifestyle regimen, dietary regimen or therapeutic intervention to the dog for a period of time; using the DNA methylation profile from a sample obtained from the dog to determine the risk of mortality and / or probability of healthy life span of the dog; c) determining whether there is a change in said risk of mortality and / or probability of healthy life span for said dog after following said lifestyle regimen, dietary regimen or therapeutic intervention for said period of time.

5. A method for determining the efficacy of a lifestyle regimen, dietary regimen or therapeutic intervention on improving the risk of mortality and / or probability of healthy life span in a dog, the method comprising: a) using a DNA methylation profile from a sample obtained from the dog to determine the risk of mortality and / or probability of healthy life span of the dog; b) applying to said dog a lifestyle regimen, dietary regimen or therapeutic intervention selected based on said risk of mortality and / or probability of healthy life span determined in step a); c) after applying the lifestyle regimen, dietary regimen or therapeutic intervention to the dog for a period of time; using the DNA methylation profile from a sample obtained from the dog to determine the risk of mortality and / or probability of healthy life span of the dog; d) determining whether the risk of mortality and / or probability of healthy life span of the dog has changed between step a) and step c).

6. A method for developing an anti-aging lifestyle regimen, dietary regimen, or therapeutic intervention; the method comprising: a) using a DNA methylation profile from a sample obtained from a dog to determine a first mortality risk and / or a probability of healthy life span for said dog; b) applying a lifestyle regimen, dietary regimen or therapeutic intervention to said dog; c) after applying the lifestyle regimen, dietary regimen or therapeutic intervention to the dog for a period of time; using the DNA methylation profile from a second sample obtained from the dog to determine a second mortality risk and / or probability of healthy life span for the dog; d) determining whether there is a change in said first risk of mortality and said second risk of mortality and / or probability of healthy life span for said dog after said period of following said lifestyle regimen, dietary regimen or therapeutic intervention; Wherein if the lifestyle program, dietary program or therapeutic intervention reduces the risk of death and / or increases the probability of healthy life span; and / or reduces the rate of increase of the dog's risk of death and / or increases the rate of decrease of the probability of healthy life span of the dog, it is determined to be anti-aging.

7. A method for preventing or reducing the risk of a dog developing a disease; the method comprising: a) using a DNA methylation profile from a sample obtained from the dog to determine the risk of mortality and / or probability of healthy life span of the dog; wherein the risk of mortality and / or probability of healthy life span determined for the dog is associated with an increased likelihood of developing the disease; and b) selecting a lifestyle regimen, dietary regimen or therapeutic intervention for said dog based on said risk of mortality and / or probability of healthy life span determined in step a); wherein said lifestyle regimen, dietary regimen or therapeutic intervention prevents or reduces the risk of said dog developing said disease; preferably wherein said disease is an age-related disease.

8. A method for selecting a dog suitable for receiving an anti-aging lifestyle regimen, dietary regimen, or therapeutic intervention; the method comprising: a) using a DNA methylation profile from a sample obtained from the dog to determine the risk of mortality and / or probability of healthy life span of the dog; b) selecting the dog as suitable for receiving an anti-aging lifestyle regimen, dietary regimen or therapeutic intervention if the dog has an increased risk of mortality and / or a decreased probability of healthy life span compared to the dog's chronological age.

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

10. The method of any one of claims 3 to 9, wherein a lifestyle regimen, dietary regimen or therapeutic intervention is selected based on a determination of the probability that the dog has an increased risk of mortality and / or a decreased healthy lifespan compared to the dog's chronological age.

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

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

13. A method according to any preceding claim, 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 preceding claim, wherein the DNA methylation profile comprises at least one methylation site as listed in Table 3.

16. The method of claim 15, wherein the DNA methylation profile comprises at least 3, at least 5, at least 10, at least 20, at least 50, at least 100, at least 150, at least 200 or every methylation site listed in Table 3.

17. The method of claim 15, wherein the DNA methylation profile comprises methylation sites as listed in Table 4.

18. The method of claim 15, wherein the DNA methylation profile comprises methylation sites as listed in Table 5.

19. The method of claim 15, wherein the DNA methylation profile comprises methylation sites as listed in Table 6.

20. The method of claim 15, wherein the DNA methylation profile comprises methylation sites as listed in Table 7.

21. The method of any one of claims 14 to 20, wherein DNA methylation is determined using a method comprising one or more of the following steps: (a) treating sample DNA with APOBEC to deaminize cytosine; (b) capture-based enrichment; and / or (c) high-throughput sequencing.

22. A method according to any preceding claim, wherein the DNA methylation profile is associated with the incidence or predicted incidence of: (i) a tissue; (ii) an organ; or (iii) a physiological system, such as the immune system, gastrointestinal system, urinary system, muscular system, cardiovascular system and / or nervous system.

23. The method of claim 22, further comprising applying to said dog a lifestyle regimen, dietary regimen, or therapeutic intervention suitable for improving said incidence or predicted incidence of said tissue; organ; or physiological system of claim 22.

24. A dietary intervention or treatment regimen for reducing the risk of mortality and / or increasing the probability of a healthy life span in a dog, wherein the dietary intervention is administered to a dog, wherein the risk of mortality is determined by the method of any one of claims 1, 7 to 10 or 13 to 23.

25. Use of a dietary intervention treatment to reduce the risk of mortality and / or increase the probability of a healthy life span in a dog, wherein the dietary intervention is administered to a dog, wherein the risk of mortality is determined by a method according to any one of claims 1, 7 to 20 or 13 to 23.

26. A computer readable medium comprising instructions which, when executed, cause one or more processors to perform the method of any one of claims 1, 2, 3, 7 to 16, or 23.

27. A computer system for determining the risk of mortality and / or probability of healthy life span of a dog; the computer system being programmed to use the DNA methylation profile of the dog to determine the risk of mortality of the dog.

28. A computer system for selecting an appropriate lifestyle regimen, dietary regimen, or therapeutic intervention for a dog, the computer system being programmed to perform one or more of the following steps: a) using the DNA methylation profile from the dog to determine the risk of mortality and / or probability of healthy life span of the dog; and b) selecting an appropriate lifestyle regimen, dietary regimen or therapeutic intervention for said dog based on said risk of mortality and / or probability of healthy life span determined in step a).

29. A computer system for determining the efficacy of a lifestyle regimen, dietary regimen, or therapeutic intervention on improving the risk of mortality and / or probability of healthy life span in a dog, the computer system being programmed to perform one or more of the following steps: a) determining the risk of mortality and / or probability of healthy life span of the dog using DNA methylation profiles from samples obtained from the dog before the lifestyle regimen, dietary regimen or therapeutic intervention and samples obtained from the dog after the lifestyle regimen, dietary regimen or therapeutic intervention; and b) determining whether said risk of mortality and / or probability of healthy life span of said dog varies between said samples obtained from said dog before and after said lifestyle regimen, dietary regimen or therapeutic intervention has been applied.

30. A computer system for determining the likelihood that a dog will benefit from an anti-aging lifestyle regimen, dietary regimen, or therapeutic intervention; the computer system being programmed to perform one or more of the following steps: a) using a DNA methylation profile from a sample obtained from the dog to determine the risk of mortality and / or probability of healthy life span of the dog; b) Identifying the dog as likely to respond to an anti-aging lifestyle regimen, dietary regimen, or therapeutic intervention if the dog has an increased risk of mortality and / or a decreased probability of healthy life span compared to the dog's chronological age.

31. 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 risk of mortality and / or probability of healthy life span for the dog.

32. A computer program product comprising computer-implementable instructions for causing a programmable computer to determine the risk of mortality and / or the probability of healthy life span for a dog using a DNA methylation profile from the dog; and to select an appropriate lifestyle regimen, dietary regimen, or therapeutic intervention for the dog based on the risk of mortality and / or the probability of healthy life span determined using the DNA methylation profile.

33. A computer program product comprising computer-implementable instructions for causing a programmable computer to: a) determine the risk of mortality and / or probability of healthy lifespan for a dog using DNA methylation profiles from samples obtained from a dog before a lifestyle regimen, dietary regimen, or therapeutic intervention and samples obtained from the dog after the lifestyle regimen, dietary regimen, or therapeutic intervention; and b) determine whether the risk of mortality and / or probability of healthy lifespan for the dog changes between the samples obtained from the dog before and after the lifestyle regimen, dietary regimen, or therapeutic intervention has been applied.

34. A computer program product comprising computer-implementable instructions for causing a programmable computer to: a) determine the risk of mortality and / or probability of healthy lifespan for a dog using a DNA methylation profile from a sample obtained from the dog; and b) identify the dog as likely to respond to an anti-aging lifestyle regimen, dietary regimen, or therapeutic intervention if the dog has an increased risk of mortality and / or decreased probability of healthy lifespan compared to the dog's chronological age.

35. A method for creating an epigenetic clock to determine a dog's mortality risk and / or probability of healthy life span; the method comprising: a) providing a DNA methylation profile from a sample obtained from said dog; b) calculating two gompertz or weibull functions, (i) a first gompertz or weibull function that models survival as a function of the methylation profile, chronological age and optionally breed category and / or sex; and (ii) a second gompertz or weibull function that models survival as a function of chronological age and optionally only breed category and / or sex; wherein the mortality risk and / or probability of healthy life span is determined based on a time variable, at which the probability of survival of the dogs given by the second gompertz or weibull function is equal to the probability of survival at their chronological age given by the first gompertz or weibull function.

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 kit for determining the risk of mortality and / or probability of healthy life span in a dog, the kit comprising a device and reagents for collecting and optionally stabilizing a sample from a subject; and instructions for collecting and optionally stabilizing the sample, and wherein the sample is mailed for subsequent DNA extraction, for measuring DNA methylation in DNA extracted from the sample, thereby obtaining a DNA methylation profile.

38. A method for determining the dog's risk of mortality and / or probability of healthy life span using a dog's DNA methylation profile; the method comprising (a) providing a sample from the dog, optionally using a kit according to claim 37; (b) providing the sample for subsequent DNA extraction for measuring DNA methylation in DNA extracted from the sample, thereby obtaining a DNA methylation profile; (c) determining the dog's risk of mortality and / or probability of healthy life span using a computer system according to claim 27 or a computer program product according to claim 31; wherein the computer system prepares and shares a report detailing the results of step (c).

39. A method for determining the risk of death and / or the probability of healthy life span of the dog using a DNA methylation profile from the dog; and selecting an appropriate lifestyle regimen, dietary regimen or therapeutic intervention for the dog based on the risk of death and / or the probability of healthy life span determined using the DNA methylation profile; the method comprising (a) providing a sample from the dog, optionally using a kit according to claim 37; (b) providing the sample for subsequent DNA extraction for measuring DNA methylation in DNA extracted from the sample, thereby obtaining a DNA methylation profile; (c) using a computer system according to claim 28 or a computer program product according to claim 32 to determine the risk of death and / or the probability of healthy life span of the dog and select an appropriate lifestyle regimen, dietary regimen or therapeutic intervention for the dog; wherein the computer system prepares and shares a report detailing the results of step (c).

40. A method for using DNA methylation profiling to determine the efficacy of a lifestyle regimen, dietary regimen, or therapeutic intervention on improving mortality risk and / or probability of healthy life span in a dog, the method comprising: (a) providing a sample obtained from the dog before the lifestyle regimen, dietary regimen or therapeutic intervention and a sample obtained from the dog after the lifestyle regimen, dietary regimen or therapeutic intervention, optionally using a kit according to claim 37; (b) providing the sample for subsequent DNA extraction for measuring DNA methylation in DNA extracted from the sample, thereby obtaining a DNA methylation profile; (c) determining, using a computer system according to claim 29 or a computer program product according to claim 33, whether the risk of mortality and / or probability of healthy life span of the dog changes between the samples obtained from the dog before and after the lifestyle regimen, dietary regimen or therapeutic intervention has been applied; Wherein the computer system prepares and shares a report detailing the results of step (c).

41. A method of using DNA methylation profiling to identify a dog as likely to respond to an anti-aging lifestyle regimen, dietary regimen, or therapeutic intervention; the method comprising: (a) providing a sample obtained from said dog; optionally using a kit according to claim 37; (b) providing the sample for subsequent DNA extraction for measuring DNA methylation in DNA extracted from the sample to obtain a DNA methylation profile; (c) using a computer system according to claim 30 or a computer program product according to claim 34, identifying the dog as likely to respond to an anti-aging lifestyle or diet if the dog has an increased risk of mortality and / or a decreased probability of healthy lifespan compared to the dog's chronological age; wherein the computer system prepares and shares a report detailing the results of step (c).

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