Method for improving the healthy life span of dogs

Abstract

The present invention provides a method for evaluating the risk of death and / or the probability of healthy life span 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 evaluate the risk of death and / or the probability of healthy life span of the dog.

Description

Technical Field

[0001] The present invention relates to a method for evaluating the health status of dogs using DNA methylation profiles. In particular, the present invention relates to a method for selecting a lifestyle plan, diet plan, or therapeutic intervention for a dog based on the health status evaluated from the DNA methylation profile, or a method for evaluating the effectiveness of a lifestyle plan, diet plan, or therapeutic intervention.

Background Art

[0002] The ability to evaluate information regarding the health of dogs is desirable for obtaining information about the health status and well-being of dogs.

[0003] Chronological age is known to be a major indicator of health status, and an increase in chronological age is associated with a decline in health. However, due to genetics, nutrition, and lifestyle, an individual may age more slowly or more quickly than their chronological age. Therefore, chronological age may not necessarily reflect the aging rate or the risk of health decline of an individual. On the other hand, the biological age of an individual (e.g., based on clinical biochemistry and cell biology metrics) can vary compared to other individuals of the same chronological age. Methods for evaluating biological age can help identify individuals who are at risk of age-related disorders earlier than predicted based on their chronological age (see, for example, WO 2019 / 046725).

[0004] However, there is a need for further methods for evaluating the biological age of dogs and using biological age metrics to improve the health outcomes of dogs.

Summary of the Invention

[0005] The present invention relates to a method for quantifying the health status of dogs based on DNA methylation profiles. By evaluating the DNA methylation profile of a dog, the risk of death and / or the probability of a healthy lifespan of the dog can be evaluated.

[0006] In existing methods for evaluating the health status of dogs, biological age is evaluated based on the correlation between DNA methylation and chronological age. Calculating the biological age of an animal can involve assessing the DNA methylation profile by comparing it to the DNA methylation profile predicted for a given chronological age. Thus, such methods are based on using chronological age as a major indicator of overall health.

[0007] In contrast, the present invention takes into account the direct predictive value of the DNA methylation profile in terms of the risk of death and / or the probability of a healthy lifespan. As an example, a particular DNA methylation marker may not directly correlate with chronological age but may indicate a particular pathological condition and thus an increased risk of death and / or a probability of a reduced healthy lifespan. Thus, the method can be described as identifying the risk of death and / or the probability of a healthy lifespan in dogs. Accordingly, 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 the dog.

[0008] In a first aspect, the present invention provides a method for evaluating the risk of death 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 evaluate the risk of death in the dog.

[0009] Evaluating the risk of death can refer to assessing the likelihood that a dog will survive for a longer or shorter period compared to, for example, equivalent dogs of the same chronological age, sex, and breed. Thus, the method of the present invention can evaluate the lifespan, healthy lifespan, and / or probability of longevity of a dog compared to, for example, equivalent dogs in terms of the same age, sex, and breed. Further, methods for improving the risk of death and / or the probability of a healthy lifespan in a dog can improve the estimated lifespan, healthy lifespan, and / or longevity of the dog.

[0010] As used herein, "lifespan" can refer to the length of the period (e.g., number of years) during which a subject survives. "Health span" can refer to the length of the period (e.g., number of years) of a life free of disease. "Longevity" can refer to the length of the time (e.g., number of years) that a subject survives beyond its predicted lifespan.

[0011] Preferably, the risk of death can be equivalent to the probability of a dog's health span, a decrease in the risk of death can be equivalent to an increase in the probability of a dog's long-term health span, or an increase in the risk of death can be equivalent to a decrease in the probability of a dog's long-term health span. The risk of death can be represented as the difference between the dog's evaluated age (i.e., biological age) and its chronological age. For example, an increase in the difference between the biological age and the chronological age evaluated by this method can indicate an increase in the risk of death of the dog. A decrease in the difference between the biological age and the chronological age evaluated by this method can indicate a decrease in the risk of death of the dog. Appropriately, the risk of death and / or the probability of health span can be described as the biological age of the dog. Appropriately, the risk of death and / or the probability of health span can be described as the epigenetic age of the dog. Preferably, this biological clock can also be referred to as an epigenetic clock.

[0012] Preferably, an evaluation that a dog's biological age exceeds its chronological age indicates a high risk of death. Preferably, an evaluation that a dog's biological age is less than its chronological age indicates a low risk of death. Preferably, an evaluation that a dog's biological age exceeds its chronological age indicates a low probability of long-term health span. Preferably, an evaluation that a dog's biological age is less than its chronological age indicates a high probability of long-term health span.

[0013] Appropriately, this method can be used to evaluate a dog's biological age based on the risk of death and / or the probability of health span.

[0014] Accordingly, in a further aspect, the present invention provides a method for assessing 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 assess the biological age of the dog, wherein the DNA methylation profile is associated with the dog's risk of death and / or probability of healthy lifespan.

[0015] In all aspects of the method, assessing or improving the dog's risk of death and / or probability of healthy lifespan is applicable to assessing or improving the dog's biological age, and the dog's biological age is assessed using a DNA methylation profile that is related to the dog's risk of death and / or probability of healthy lifespan.

[0016] In a further aspect, the present invention provides a method for selecting a lifestyle plan, diet plan, 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 assess the dog's risk of death and / or probability of healthy lifespan; and c) selecting a lifestyle plan, diet plan, or therapeutic intervention suitable for the dog based on the risk of death assessed in step b).

[0017] As used herein, the step of "selecting a lifestyle plan, diet plan, or therapeutic intervention suitable for the dog" may also include the step of "recommending a lifestyle plan, diet plan, or therapeutic intervention for the dog" or the step of "providing a lifestyle plan, diet plan, or therapeutic intervention recommended for the dog".

[0018] In another aspect, the present invention provides a method for evaluating the effectiveness of a lifestyle plan, diet plan, or therapeutic intervention for improving the risk of death and / or the probability of healthy lifespan in dogs, the method comprising: a) applying a lifestyle plan, diet plan, or therapeutic intervention to a dog, wherein the lifestyle plan, diet plan, or therapeutic intervention is selected according to the previous aspects of the present invention; b) evaluating the risk of death and / or the probability of healthy lifespan in the dog using a DNA methylation profile from a sample obtained from the dog after a period of applying the lifestyle plan, diet plan, or therapeutic intervention; and c) evaluating whether there has been a change in the risk of death and / or the probability of healthy lifespan in the dog after a period according to the lifestyle plan, diet plan, or therapeutic intervention.

[0019] In a further aspect, the present invention provides a method for evaluating the effectiveness of a lifestyle plan, diet plan, or therapeutic intervention for improving the risk of death and / or the probability of healthy lifespan in dogs, the method comprising: a) evaluating the risk of death in a dog using a DNA methylation profile from a sample obtained from the dog; b) applying to the dog a lifestyle plan, diet plan, or therapeutic intervention selected based on the risk of death evaluated in step a); c) evaluating the risk of death in the dog using a DNA methylation profile from a sample obtained from the dog after a period of applying the lifestyle plan, diet plan, or therapeutic intervention; and d) evaluating whether there has been a change in the risk of death in the dog between step a) and step c).

[0020] Appropriately, improving the risk of death and / or the probability of healthy lifespan in a dog may refer to a decrease in the difference between the biological age and the calendar age of the dog, in which case the biological age of the dog is greater than its calendar age. Further, improving the risk of death and / or the probability of healthy lifespan in a dog may refer to maintaining or even increasing the difference between the biological age and the calendar age of the dog, in which case the biological age of the dog is less than its calendar age. Alternatively, worsening the risk of death and / or the probability of healthy lifespan in a dog may refer to an increase in the difference between the biological age and the calendar age of the dog, in which case the biological age of the dog is greater than its calendar age. Worsening the risk of death and / or the probability of healthy lifespan in a dog may also refer to a decrease in the difference between the biological age and the calendar age of the dog, in which case the biological age of the dog is less than its calendar age.

[0021] Appropriately, improving the risk of death and / or the probability of healthy lifespan in a dog may refer to a decrease in the rate of change between the biological age and the calendar age of the dog, in which case the biological age of the dog is greater than its calendar age. For example, the biological age of a dog may increase by 1.5 years for every 1-year increase in calendar age. A decrease in the rate of change after an intervention in lifestyle and diet plan, for example, a decrease in the rate of change such that the biological age of the dog after the intervention increases by 1.25 years for every 1-year increase in calendar age, may provide an improvement in the risk of death and / or the probability of healthy lifespan in the dog.

[0022] When the biological age of a dog is less than its calendar age, improving the risk of death and / or the probability of healthy lifespan may also refer to maintaining or increasing the rate of change between the biological age and the calendar age of the dog. For example, the biological age of a dog may increase by less than 1 year (e.g., 0.9 year) for every 1-year increase in calendar age. The rate of change after an intervention in lifestyle, diet plan, or treatment may change, for example, such that the biological age of the dog after the intervention increases by 0.8 year or less for every 1-year increase in calendar age, and this change may provide an improvement in the biological age of the dog.

[0023] The present method for evaluating the effectiveness of a lifestyle plan, diet plan, or therapeutic intervention for improving the risk of death and / or probability of healthy life span in dogs can advantageously enable continuous monitoring of the effectiveness of a lifestyle plan, diet plan, or therapeutic intervention for improving or maintaining the health of dogs. The use of such a method can advantageously enable identification of particularly effective lifestyle plans, diet plans, or therapeutic interventions. In contrast, if a lifestyle plan, diet plan, or therapeutic intervention is evaluated as ineffective based on the risk of death and / or probability of healthy life span in dogs, then an alternative lifestyle plan, diet plan, or therapeutic intervention can be implemented.

[0024] Accordingly, the present method enables selection of a lifestyle plan, diet plan, or therapeutic intervention suitable for a dog based on the risk of death and / or probability of healthy life span evaluated from the DNA methylation profile. For example, a highly digestible and high-quality protein diet is generally recommended based on the chronological age of the dog. For example, a dog may be recommended to switch to a senior diet around 7 or 8 years of age. However, in the context of the present invention, when it is evaluated that the risk of death is increasing and / or the probability of healthy life span is decreasing (i.e., the biological age is increasing) compared to the chronological age of the dog, it becomes possible to decide to switch the dog to a senior diet at an earlier age. In contrast, a dog with a low risk of death and / or a high probability of healthy life span (i.e., a decreasing biological age) compared to its chronological age may be able to continue an adult diet for a longer period.

[0025] Suitably, the method may include selecting and / or applying a lifestyle plan, diet plan, or therapeutic intervention to a dog after an evaluation that the dog has a high risk of death and / or a low probability of healthy life span compared to its chronological age.

[0026] In another aspect, the present invention provides a method for preventing or reducing the risk of disease onset in dogs, the method comprising a) Using a DNA methylation profile from a sample obtained from a dog to evaluate the dog's risk of death and / or probability of healthy lifespan, wherein the risk of death and / or probability of healthy lifespan evaluated for the dog is associated with an increased likelihood of developing a disease, the step of evaluating; b) Based on the risk of death and / or probability of healthy lifespan evaluated in step a), selecting a lifestyle plan, diet plan, or therapeutic intervention for the dog, wherein the lifestyle plan, diet plan, or therapeutic intervention prevents or reduces the dog's risk of developing a disease, the step of selecting, comprising.

[0027] Suitably, the disease is an age-related disease. For example, age-related diseases are osteoarthritis, dementia, cognitive impairment, prediabetes, diabetes, cancer, heart disease, obesity, gastrointestinal disorders, incontinence, kidney disease, sarcopenia, vision loss, hearing loss, osteoporosis, cataracts, cerebrovascular disease, and / or liver disease.

[0028] The method may optionally further comprise performing a lifestyle plan, diet plan, or therapeutic intervention on the dog. Suitably, the lifestyle plan may be a dietary intervention or a therapeutic modality.

[0029] In another aspect, the present invention provides a method of selecting a dog suitable for receiving an anti-aging lifestyle plan, diet plan, or therapeutic intervention, the method comprising: a) using a DNA methylation profile from a sample obtained from the dog to evaluate the dog's risk of death and / or probability of healthy lifespan; and b) when the risk of death is increased and / or the probability of healthy lifespan is decreased compared to chronological age, selecting the dog as being suitable for receiving an anti-aging lifestyle plan, diet plan, or therapeutic intervention.

[0030] Suitably, a lifestyle plan, diet plan, or therapeutic intervention to prevent aging may be effective for dogs based on chronological age, but may be particularly effective when applied to dogs with an increased risk of death and / or a decreased probability of healthy lifespan compared to that chronological age. Thus, the method can advantageously also enable selection of dogs that are likely to respond, or have an improved degree of response, to a lifestyle plan, diet plan, or therapeutic intervention to prevent aging.

[0031] A lifestyle plan, diet plan, or therapeutic intervention may be selected based on an assessment that the dog has a high risk of death and / or a low probability of healthy lifespan (i.e., an increase in biological age) compared to its chronological age.

[0032] The lifestyle plan, diet plan, or therapeutic intervention may be a dietary intervention. Examples of dietary interventions can include calorie-restricted diets, geriatric diets, low-protein diets, and the like.

[0033] DNA methylation profiles can be associated with an increase in biological age in (i) tissues, (ii) organs, or (iii) biological systems such as, for example, the immune system, gastrointestinal system, urinary system, muscular system, cardiovascular system, and / or nervous system.

[0034] The present invention further provides a dietary intervention for use in reducing the risk of death and / or increasing the probability of healthy lifespan in dogs, wherein the dietary intervention is performed on dogs whose risk of death and / or probability of healthy lifespan has been evaluated by the method of the present invention.

[0035] The present invention further provides the use of a dietary intervention for reducing the risk of death in dogs and / or increasing the probability of healthy lifespan in dogs, the dietary intervention being performed on dogs whose risk of death and / or probability of healthy lifespan has been evaluated by the method of the present invention.

[0036] In another aspect, the present invention provides a computer-readable medium comprising instructions that, when executed, cause one or more processors to execute the method of the present invention.

[0037] In another aspect, the present invention provides a computer system for assessing the risk of death of a dog, and such computer system is programmed to assess the risk of death of a dog using the DNA methylation profile of the dog.

[0038] In another aspect, the present invention is a computer system for selecting a lifestyle plan, diet plan, or therapeutic intervention suitable for a dog, and is programmed to perform one or more of the following steps: a) assessing the risk of death of a dog using the DNA methylation profile obtained from the dog; and b) selecting a lifestyle plan, diet plan, or therapeutic intervention suitable for the dog based on the risk of death assessed in step a).

[0039] In another aspect, the present invention is a computer system for evaluating the effectiveness of a lifestyle plan, diet plan, or therapeutic intervention for improving the risk of death of a dog, and is programmed to perform one or more of the following steps: a) assessing the risk of death of a dog using the DNA methylation profiles from samples obtained from the dog before the lifestyle plan, diet plan, or therapeutic intervention and samples obtained from the dog after the lifestyle plan, diet plan, or therapeutic intervention; and b) evaluating whether there has been a change in the risk of death of the dog between the samples obtained from the dog before and after the application of the lifestyle plan, diet plan, or therapeutic intervention.

[0040] In another aspect, the present invention provides a computer system for assessing the likelihood that a dog can benefit from a lifestyle plan, diet plan, or therapeutic intervention to prevent aging, the computer system comprising: a) using a DNA methylation profile from a sample obtained from the dog to assess the dog's risk of death; and b) if the dog has a high risk of death compared to its chronological age, identifying the dog as likely to respond to a lifestyle plan, diet plan, or therapeutic intervention to prevent aging. The computer system is programmed to perform one or more of the steps described above.

[0041] In another aspect, the present invention provides a computer program product comprising computer-executable instructions for causing a programmable computer to assess a dog's risk of death using the dog's DNA methylation profile.

[0042] In another aspect, the present invention provides a computer program product comprising computer-executable instructions for causing a programmable computer to assess a dog's risk of death using the dog's DNA methylation profile and to select a lifestyle plan, diet plan, or therapeutic intervention suitable for the dog based on the risk of death assessed using the DNA methylation profile.

[0043] In another aspect, the present invention provides a computer program product comprising computer-executable instructions for causing a programmable computer to: a) assess a dog's risk of death using DNA methylation profiles from samples obtained from the dog before and after a lifestyle plan, diet plan, or therapeutic intervention; and b) assess whether there has been a change in the dog's risk of death between the samples obtained from the dog before and after the lifestyle plan, diet plan, or therapeutic intervention has been applied.

[0044] In another aspect, the present invention provides a computer program product comprising computer-executable instructions for causing a programmable computer to a) evaluate the risk of death of a dog using a DNA methylation profile from a sample obtained from the dog, and b) identify the dog as likely to respond to a lifestyle program, diet plan, or therapeutic intervention to prevent aging if the dog has a high risk of death compared to its chronological age.

[0045] Advantageously, the present invention may enable the assessment of the risk of death and / or the probability of healthspan based on markers of multiple organ systems and functions. Thus, the methods of the present invention may advantageously encompass various possible organ dysfunctions.

[0046] Evaluating the risk of death and / or the probability of healthspan of a dog enables the examination of several aspects of the animal's well-being. First, the measurement / evaluation can predict whether this animal is more likely to require an intervention based on diet or nutritional supplements. The measurement / evaluation can also be used to test the effectiveness of an intervention based on diet or nutritional supplements against aging. BRIEF DESCRIPTION OF THE DRAWINGS

[0047]

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[0048] Next, various preferred features and embodiments of the present invention will be described by way of non-limiting examples. It should be understood that those skilled in the art can combine all the features of the present invention disclosed herein without departing from the scope of the present invention disclosed.

[0049] It should be noted that, as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0050] As used herein, the terms "comprising", "comprises" and "comprised of" are synonymous with "including", "includes", or synonymous with "containing", "contains", and can include others or are open-ended and do not exclude additional, unrecited members, elements or method steps. The terms "comprising", "comprises", and "comprised of" also include the term "consisting of".

[0051] A numerical range includes the numbers defining that range.

[0052] The publications discussed herein are provided solely because they were publicly available prior to the filing date of the present application. Any document cited herein should not be construed as an admission that such publications constitute prior art to the claims appended hereto.

[0053] The methods and systems disclosed herein can be used by veterinarians, medical professionals, laboratory technicians, pet care providers, and the like.

[0054] Subject This method is directed to canine subjects. Thus, the subject of the present invention is a dog.

[0055] In another aspect, the subject may be a feline subject. Thus, in another aspect of the present invention, the subject is a cat. All disclosures herein are equally applicable to cats unless otherwise stated.

[0056] Breed The methods of the present invention can also utilize information regarding the breed of a dog. A dog may be classified, for example, as a toy breed, small breed, medium breed, large breed, or giant breed. Preferably, the breed of a dog may be classified based on the weight of the dog. Preferably, the breed of a dog may be classified based on the average weight of a dog for a given breed.

[0057] Preferably, a dog may be classified as a small breed or a medium breed. Preferably, the classification is evaluated by the average weight of an adult dog of this breed. Preferably, a breed with an average weight of less than 10 kg is classified as a small breed and / or a breed with an average weight exceeding 10 kg is classified as a medium breed.

[0058] In another aspect where the subject is a cat, the cat may be a domestic cat. Preferably, the cat may be a domestic short-haired cat.

[0059] Gender Preferably, the gender of a dog may be classified as male or female.

[0060] Calendar age Calendar age may be defined as the amount of time elapsed from the birth of the subject to a given date. Calendar age may be expressed in years, months, days, etc.

[0061] Preferably, the method of the present invention can be applied to dogs of any chronological age. In certain embodiments, the dog may be at least about 2 years old. Preferably, the dog may be at least about 2 years old, at least about 3 years old, at least about 4 years old, at least about 5 years old, at least about 6 years old, at least about 7 years old, at least about 8 years old, at least about 9 years old, or at least about 10 years old.

[0062] Preferably, the dog may be at least about 7 years old.

[0063] Sample The present invention includes providing or assessing a DNA methylation profile from one or more samples obtained from a subject.

[0064] Preferably, the sample is blood, a hair follicle, a buccal swab, saliva, or a tissue sample.

[0065] Preferably, the sample is a hair follicle, a buccal swab, or a saliva sample. Such sample types are particularly applicable when the sample is provided outside a veterinary setting, for example, using a kit according to the present invention.

[0066] Preferably, the sample is blood-derived. The sample may contain a blood fraction or may be whole blood. The sample preferably comprises whole blood. The sample may include peripheral blood mononuclear cells (PBMCs) or a lymphocyte sample. Techniques for collecting a sample from a subject and extracting DNA (e.g., genomic DNA) from the sample are well known in the art.

[0067] The method of the present invention can be carried out on one or more samples obtained from a subject. For example, the method of the present invention may be carried out using a first sample obtained at a given time point and a second sample obtained after a time interval following the obtaining of the first sample. The method of the present invention can be carried out two or more times on samples obtained from the same dog over a period of time. For example, the samples may be obtained repeatedly once a month, once a year, or once every two years. Preferably, the samples may be obtained approximately once a year (e.g., during an annual veterinary health check). This can be useful, for example, in evaluating the effect of a specific treatment, such as a dietary intervention or a change in exercise therapy, or the effect of a change in lifestyle habits.

[0068] In one embodiment, the method of the present invention can be applied to a sample obtained from a subject prior to a change in lifestyle habits (e.g., an intervention with a dietary product or a change in exercise therapy). In another embodiment, the method of the present invention can be applied to samples obtained from a subject, for example, before and after an intervention with a dietary product or a change in exercise therapy. The method of the present invention can also be applied to samples taken at a plurality of predetermined time points, for example, during an intervention period with a dietary product or during a period of change in exercise therapy. These plurality of predetermined time points may be, for example, periodic during an intervention period with a dietary product or during a period of change in exercise therapy, for example, daily or every three days, or may vary depending on the subject being tested.

[0069] DNA methylation DNA methylation is a process in which a methyl group (CH 3 ) is added by a covalent bond to a cytosine base, which is part of a DNA molecule. In vivo, this process is catalyzed by a family of DNA methyltransferases (Dnmt) that generate modified cytosine by the transfer of a methyl group from S-adenyl methionine (SAM). Cytosine is modified on the fifth carbon atom, and the modified residue is known as 5-methylcytosine (5mC). DNA methylation can also include 5-hydroxymethylcytosine (5hmc).

[0070] DNA methylation is an example of an epigenetic mechanism, i.e., it can alter gene expression without modifying the underlying DNA sequence. DNA methylation can inhibit gene expression, for example, by acting as a recruitment signal for repressors or by directly blocking the recruitment of transcription factors. In the mammalian somatic genome, DNA methylation mainly occurs at sites (CpG) where cytosine and guanine form adjacent dinucleotides. While non-CpG methylation is observed during embryogenesis, in adults, these modifications are significantly reduced in most cell types. CpG islands are DNA regions with high CpG density that are generally not methylated. These regions are associated with promoter regions, particularly those of housekeeping genes, and are thought to be maintained in a permissive state to allow gene expression.

[0071] DNA methylation has been found to change with age in humans and other animals. Aging mammalian tissues generally show global DNA hypomethylation, which is thought to be due to a progressive decrease or mistargeting of DNMT1 methyltransferase activity, while local hypermethylation is shown in CpG islands. Local hypermethylation can lead to the repression of specific genes, and this repression may contribute to age-related diseases. The relationship between epigenetic changes in DNA methylation and age enables the estimation of "biological age" using a "DNA methylation clock". Generally, these clocks are trained against chronological age using supervised machine learning techniques, and the deviation of the "clock age" from an individual's actual chronological age is regarded as an indicator of "biological" age. Biological age correlates with an individual's chronological age, but the deviation from the correlation can indicate the potential risk of age-related diseases or illnesses in an individual.

[0072] Detection of specific methylated DNA can be achieved by multiple methods (see, e.g., Zuo et al., 2009; Epigenomics. 1(2):331-345 and Rauluseviciute et al.; Clinical Epigenetics; 2019; 11(193)). Many methods are available for the detection of differentially methylated DNA at specific loci in samples such as blood, urine, feces, or saliva. These methods can distinguish between unmethylated DNA and 5-methylcytosine or methylated DNA and then quantify the proportions of methylated and unmethylated DNA in a specific genomic region.

[0073] The method may include evaluating the DNA methylation profile of a dog using any suitable method. Suitable methods include, but are not limited to, those described below.

[0074] EM-seq (Enzymatic Methyl-seq) Preferably, an enzymatic approach is used to detect 5mC and 5hMC. As an example, EM-seq (Enzymatic Methyl-seq) can be used.

[0075] Typically, in EM-seq, in the first enzymatic step, the activity of TET2 (Tet methylcytosine dioxygenase 2) oxidizes 5mC to 5hMC, then to 5fC, and finally to 5caC. Further, the T4-BGT enzyme is used to glucosylate both the originally present 5hmC and the 5hmC produced by TET2 activity. In the second enzymatic step following denaturation of double-stranded DNA, APOBEC3A (apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3A) is used to deaminate cytosine, but cannot deaminate oxidized or glucosylated 5mC and 5hMC. Only non-methylated cytosine is deaminated to form uracil bases. Prior to the first enzymatic step, DNA fragments can be generated by mechanical shearing, end-repaired, A-tailed, and ligated to sequencing adapters, which can be performed, for example, using the NEBNext® DNA Ultra II reagent (NEB). After the second enzymatic step, the deaminated single-stranded DNA can be amplified by a PCR reaction using a polymerase such as NEBNext® Q5U™ that can amplify uracil-containing templates, and the resulting library can be sequenced or analyzed in the same manner as a DNA sample prepared by bisulfite sequencing. The output of EM-seq is generally the same as whole-genome bisulfite sequencing, but uses fewer reagents that damage DNA, resulting in reduced sample loss and better performance than bisulfite-converted prepared samples in terms of cytosine methylation call coverage, sensitivity, and accuracy. An exemplary EM-seq method is described in Vaisvila et al. (Genome Research; 2021; 31:1-10).

[0076] Methods based on bisulfite conversion Conversion by bisulfite utilizes the selective conversion of non-methylated cytosine to uracil when treated with sodium bisulfite. When denatured DNA is treated with sodium bisulfite, all unmodified cytosines are converted to uracil, and these residues are then converted to thymine by subsequent PCR amplification. Analysis of the resulting DNA sequences can be performed via various methods, examples of which include denaturing gel electrophoresis, single-stranded conformational polymorphism, melting curve, fluorescence real-time PCR (MethyLight), MALDI mass spectrometry, array hybridization, and sequencing (e.g., whole-genome bisulfite sequencing WGBS), but are not limited thereto. Recently developed techniques (e.g., SeqCap Epi) enable deeper coverage over a more restricted region by enriching the target sequence prior to sequencing. By comparing the abundance of sequences in the bisulfite-converted sample with that of the untreated control, analysis of methylation at the target site becomes possible, and the proportion of the converted sequences indicates the methylation level of the target site.

[0077] Further modifications of the bisulfite conversion method are available that can distinguish oxidized 5-hydroxymethylcytosine (5hMC), which behaves similarly to 5mC under standard bisulfite conversion, from 5mC, and can detect additional modified 5-formylcytosine (5fC). These methods, such as oxBS-Seq and redBS-Seq, utilize the oxidation and reduction of these markers to modify the sensitivity of each species to bisulfite conversion and quantify the amount of each modification at the target locus by comparative analysis.

[0078] Digestion method using a selective restriction endonuclease Methods for analyzing existing DNA methylation patterns may include the use of restriction enzymes. These include, for example, the RLGS method (restriction landmark genomic scanning) (Costello et al., 2000; Nat Genet.; 24(2):132-8), the MS-RDA method (methylation-sensitive representational difference analysis) (Ushijima et al., Proc Natl Acad Sci U S A. 1997 Mar 18;94(6):2284-9), and the DMH method (differential methylation hybridization) (Huang et al. 1997 Mar 15;57(6):1030-4). The digestion activity of restriction endonucleases can be methylation-dependent. Utilizing this specificity, methylated and unmethylated sequences can be distinguished. Certain restriction enzymes, such as BstUI, HpaII, and NotI, are sensitive to methylated recognition sequences. Others, such as McrBC, are specific for methylated sequences.

[0079] As an example, in the DMH method (differential methylation hybridisation) (Huang et al., supra), it is necessary to first fragment the genome with a bulk genome restriction enzyme such as MseI that fragments the genome into lengths less than 200 bp. After the fragmentation step, the genomic fragments are digested using a methylation-sensitive restriction endonuclease (MRE), or in some versions of this technique, a cocktail of MREs is used for digestion to improve coverage. Depending on the specificity of the one or more enzymes used, either methylated or unmethylated sequences are degraded. The digested sequences are not amplified in the subsequent PCR step. The resulting PCR products are suitable for further processing and analysis by sequencing or by microarray hybridization combined with fluorescent dyes.

[0080] Suitably, the method utilizes a DNA methylation profile generated by a method that includes the use of one or more MREs.

[0081] An appropriate comparator can be used to investigate the methylation status between conditions. To detect changes in methylation status, DNA from healthy subjects can be compared to DNA from aged or diseased subjects (Huang et al., Hum Mol Genet. 1999 Mar;8(3):459-70). Alternatively, control samples can be generated using a secondary digestion enzyme such as the methylation-insensitive HpaII isoschizomer MspI, allowing for comparison of methylation within or between genomes (Khulan et al., Genome Res. 2006 Aug;16(8):1046-55).

[0082] In some embodiments, a method for detecting methylation includes randomly shearing or fragmenting genomic DNA, cutting the DNA with a methylation-dependent or methylation-sensitive restriction enzyme, and then selectively identifying and / or analyzing the cut or uncut DNA. Selective identification can include, for example, separating cut DNA from uncut DNA (e.g., by size) and quantifying the target sequences that were cut or not cut. Alternatively, the method can include amplifying the undigested DNA after restriction enzyme digestion, thereby amplifying only the DNA in the amplified regions that was not cut by the restriction enzyme. In some embodiments, the amplification can be performed using primers that are gene-specific. Alternatively, adapters can be added to the ends of randomly fragmented DNA, the DNA can be digested with a methylation-dependent or methylation-sensitive restriction enzyme, and the undigested DNA can be amplified using primers that hybridize to the adapter sequences. In this case, the second step can be performed to evaluate the presence, absence, or amount of a specific gene in the amplified DNA pool. In some embodiments, the DNA is amplified using real-time quantitative PCR.

[0083] Suitably, the presence or absence of nucleic acid digestion is detected by selective hybridization of a probe or primer to undigested nucleic acid. Alternatively, the probe selectively hybridizes to both digested and undigested nucleic acid, for example, facilitating discrimination between the two forms by electrophoresis. Suitable detection methods to achieve selective hybridization to a hybridization probe include, for example, Southern hybridization or other nucleic acid hybridizations.

[0084] Appropriate hybridization conditions can be determined based on the melting temperature (Tm) of the nucleic acid duplex containing the probe. Those skilled in the art are aware that the optimal hybridization reaction conditions should be determined empirically for each probe, but some general rules can apply. Preferably, hybridization using short oligonucleotide probes is performed at low to moderate stringency. For 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 approximately 0.1× SSC buffer and / or approximately 0.1% (w / v) SDS, or lower salt concentration, and / or at a temperature of at least 65° C., or equivalent conditions. References to specific levels of stringency herein include equivalent conditions using wash / hybridization solutions other than SSC known to those skilled in the art.

[0085] Methods based on affinity enrichment The discrimination between methylated DNA and unmethylated DNA can be achieved by the use of antibodies such as anti-5mC, and / or methylated CpG-binding proteins containing a methylated CpG-binding domain (MBD). Antibodies to MBD domain proteins can specifically isolate methylated DNA over unmethylated DNA. The method of using antibodies is generally called MeDIP, and the method of using methylated CpG-binding proteins is often known as the MBD or MIRA approach.

[0086] In these methods, it is necessary to first fragment the genome, which can be done using bulk genome digestion with an enzyme that cuts frequently, such as MseI, followed by affinity purification of the methylated fragments. By comparing the input DNA with the purified methylated DNA by microarray hybridization or sequencing, a comparative analysis of the methylation levels at specific sites can be obtained.

[0087] Further variations of affinity enrichment-based methods such as MethylCap-Seq or MBD-Seq are available. In these methods, a salt gradient is used to elute methylated DNA fragments in a manner that depends on the abundance of methylated CpGs, reducing the complexity of the sample by separating CpG islands and other highly methylated loci from loci with lower CpG density. The fractions can then be sequenced separately to improve sequence coverage.

[0088] Approaches based on single molecule sequencing With current sequencing methods, it is possible to directly sequence a single molecule. Single molecule real-time (SMRT) DNA sequencing, for example, the Sequel system from Pacific Biosciences, is available 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 manufactured by Oxford Nanopore Technologies, which can sequence individual long strands of DNA, can also detect base modifications including methylation.

[0089] DNA methylation site Preferably, a DNA methylation site can refer to the presence or absence of 5mC in a single cytosine, preferably a single CpG dinucleotide.

[0090] Preferably, a DNA site methylation site can refer to the presence or absence of methylation (i.e., the number or proportion of 5mC) across multiple CpG sites within a DNA region. Preferably, a DNA methylation site can refer to the methylation level (i.e., the number or proportion of 5mC) across multiple CpG sites within a DNA region. A "DNA region" can refer to a specific portion of genomic DNA. These DNA regions can be identified by reference to either a gene name or a set of chromosomal coordinates. Both gene names and chromosomal coordinates will be well known and understood by those skilled in the art.

[0091] Suitably, the gene name and / or coordinates can 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).

[0092] A DNA region can define, for example, a portion of DNA proximate to a gene promoter. The promoter region is known to be rich in CpG. By way of example, the DNA region can refer to about 3 kb upstream to about 3 kb downstream of the promoter, 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, the DNA region can refer to about 1 kb upstream to about 0.5 kb downstream of the promoter.

[0093] Suitably, the DNA region may contain or consist of CpG sites that are less than about 5000 bases, less than about 4000 bases, less than about 3000 bases, less than about 2000 bases, less than about 1000 bases, less than about 500 bases, or less than about 200 bases apart.

[0094] Suitably, the DNA region may contain or consist of CpG sites that are about 200 to about 5000 bases, about 200 to about 4000 bases, about 200 to about 3000 bases, about 200 to about 2000 bases, or about 200 to about 1000 bases apart.

[0095] Suitably, the DNA region may contain one or more CpG islands. Suitably, the DNA region may consist of CpG islands.

[0096] “CpG island” can refer to a DNA region that contains at least 200 bp, contains a GC percentage of more than 50%, and has an observed to predicted CpG ratio of more than 60% for CpG.

[0097] Preferably, the DNA methylation site does not contain CpG on the X chromosome and / or Y chromosome.

[0098] Preferably, the DNA methylation site does not contain CpG that is known to contain an SNP in the CpG.

[0099] References to each of the gene / DNA regions detailed above are to be understood as references to all forms of these molecules and their fragments or variants. As will be understood by those skilled in the art, some genes are known to exhibit allelic variation or single nucleotide polymorphisms between individuals. Variants include nucleic acid sequences from the same region that share at least 90%, 95%, 98%, 99% sequence identity, i.e., such regions have one or more deletions, additions, substitutions, inverted sequences, etc. compared to the DNA regions described herein. Thus, although there is a fact that small genetic variations may exist between individuals in the actual nucleic acid sequences, it should be understood that for the purposes of this application, the invention extends to variants that achieve the same result. Thus, it should be understood that the invention extends to all forms of DNA resulting from any other mutations, polymorphisms or allelic variations.

[0100] Regarding screening for methylation of these gene regions, it should be understood that an assay can be designed to screen for a specific DNA. Selection of the strand to be analyzed and targeting based on the chromosomal coordinates of such strand are well within the skill of those in the art. In some situations, an assay can be established to screen both strands.

[0101] “Methylation state” can be understood as a reference to the presence, absence and / or amount of methylation at one or more specific nucleotides within a DNA region. The methylation state of a particular DNA sequence (e.g., a DNA region described herein) can indicate the methylation state of all bases in the sequence, or the methylation state of a subset of base pairs within the sequence (e.g., the methylation state of cytosine or one or more specific restriction enzyme recognition sequences), or can indicate information regarding the methylation density of a region within the sequence without providing exact information as to where methylation occurs within the sequence. The methylation state can optionally be represented or indicated by a “methylation value”.

[0102] Suitably, DNA methylation can be evaluated using an EM-Seq strategy. In such a method, the methylation level can be evaluated as the proportion of "C" bases out of the total of "C" bases + "U" bases at a target CpG site "i" after enzymatic treatment and after conversion treatment with APOBEC3A. In other embodiments, the methylation level can be evaluated as the proportion of "C" bases out of the total of "C" bases + "T" bases at site "i" after enzymatic treatment, conversion treatment with APOBEC3A, and subsequent amplification by diffusion. The average methylation level at each site can then be evaluated to assess whether one or more thresholds are met.

[0103] In some embodiments, particularly when bisulfite conversion and sequencing methods are used, the methylation level can be evaluated as the proportion of "C" bases out of the total of "C" bases + "U" bases at a target CpG site "i" after bisulfite treatment. In other embodiments, the methylation level can be evaluated as the proportion of "C" bases out of the total of "C" bases + "T" bases at site "i" after bisulfite treatment and subsequent amplification. The average methylation level at each site can then be evaluated to assess whether one or more thresholds are met.

[0104] Alternatively, the methylation value can be generated, for example, by quantifying the amount of undigested DNA present after restriction digestion with a methylation-dependent restriction enzyme. In this example, when a specific sequence in the DNA is quantified using quantitative PCR, if the amount of template DNA is approximately equal to that of a mock treated control (i.e., untreated with the restriction enzyme), it indicates that the sequence is not highly methylated. In contrast, if the amount of template DNA is substantially less than that present in the sample untreated with the restriction enzyme, it indicates the presence of methylated DNA in that sequence. Thus, 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. The methylation value is particularly useful when it is desirable to compare the methylation state of a sequence in a sample to a threshold value.

[0105] Because some variation occurs between samples, the present invention is not limited by the exact number of methylated residues that are thought to indicate biological age. The present invention is also not necessarily limited by the arrangement of methylated residues (e.g., specific methylation sites).

[0106] In one embodiment, a screening method can be used that specifically targets the evaluation of the methylation state of one or more specific cytosine residues or the corresponding cytosine at the n+1 position on the opposite DNA strand.

[0107] Enrichment and Detection Methods Evaluating a DNA methylation profile may include enriching a DNA sample for a selected DNA region. For example, the method may include enriching a DNA sample for a DNA region that includes DNA methylation sites that make up the DNA methylation profile.

[0108] Suitable enrichment methods are known in the art and include, for example, methods based on amplification or hybridization. Amplification enrichment typically refers to PCR-based enrichment using, for example, 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 cycling probe method.

[0109] Enrichment based on hybridization or capture by hybridization typically refers to the use of a hybridization probe (or capture probe) that hybridizes to the DNA region to be enriched.

[0110] The hybridization probe may be directly bound to a solid support or may contain a moiety (e.g., biotin) that allows binding to a solid support suitable for capturing the biotin moiety (e.g., beads coated with streptavidin). In either case, DNA containing a sequence complementary to the probe can be captured, thus enabling separation of DNA containing the target DNA region from DNA not containing the target DNA region. Thus, such a capture step enables enrichment for the target DNA region. For example, the DNA region can be a DNA region proximal to a gene promoter.

[0111] The arrays used in this specification can vary depending on the probe composition and the desired use of the array. For example, the nucleic acids (or CpG sites) detected in the array can be at least 10, 100, 1,000, 10,000, 100,000, 1,000,000, 10,000,000, 100,000,000, or more. Alternatively, or additionally, the nucleic acids (or CpG sites) detected can be selected to be 100,000,000, 10,000,000, 1,000,000, 100,000, 10,000, 1,000, 100 or less. Similar ranges can be achieved using nucleic acid sequencing approaches known in the art, such as next-generation or massively parallel sequencing.

[0112] Suitably, the enrichment step may be carried out before or after the step of separating or differentially processing methylated DNA and unmethylated DNA.

[0113] As used herein, the term "enrich" or "enrichment" with respect to "DNA" or "DNA region" means a process in which the (absolute) amount and / or proportion of DNA containing the desired sequence is increased compared to the amount and / or proportion of DNA containing the desired sequence in the starting material. In this regard, enrichment by amplification increases the amount and proportion of the desired sequence. Enrichment by capture-based enrichment increases the proportion of DNA containing the desired sequence.

[0114] Following the processing of the DNA to distinguish methylated sites from unmethylated sites, the method of the invention may further comprise the step of identifying methylated sites or unmethylated sites (i.e., in the original sample).

[0115] The identifying step may include any suitable method known in the art, such as array detection or sequencing (e.g., next-generation sequencing).

[0116] The specific step of array determination preferably includes next-generation sequencing (massively parallel or high-throughput sequencing). Next-generation sequencing methods are well known in the art, and in principle, any method can be contemplated for use in the present invention. The next-generation sequencing technique can be carried out according to the manufacturer's instructions (e.g., provided by Roche, Illumina or Applied Biosystems).

[0117] In a preferred embodiment, the sample is processed by using an enzymatic reaction to convert DNA methylation, performing whole-genome library preparation, and measuring the methylation profile by sequencing (EM-Seq).

[0118] In a particularly preferred embodiment, the sample is processed by using an enzymatic reaction to convert DNA methylation, performing whole-genome library preparation, hybridizing the whole-genome converted library preparation to a capture probe (preferably a capture probe capable of capturing a DNA region proximal to a gene promoter), and measuring the methylation profile by sequencing (EM-Seq).

[0119] DNA methylation profile The "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, the "methylation profile" refers to the presence, absence, amount or level of 5mC at a plurality of DNA methylation sites. Thus, the presence, absence, amount or level of 5mC at each individual DNA methylation site within a plurality of sites may be evaluated and may contribute to the assessment of the risk of death and / or the probability of healthspan in dogs. Thus, the quality and / or ability of the method can be improved by combining values from a plurality of DNA methylation markers.

[0120] Preferably, the biological clock comprises a methylation profile from a plurality of methylation sites.

[0121] Preferably, 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 100000, at least 250000, or at least 500000 DNA methylation sites can be used to evaluate the risk of death and / or the probability of healthy lifespan (i.e., biological age) in dogs.

[0122] Preferably, 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.

[0123] 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.

[0124] To create a biological clock for evaluating the risk of death and / or the probability of healthy lifespan, the initial methylation profile may be processed or rationalized to generate a restricted methylation profile, which is then used to generate a biological clock.

[0125] As an example, the initial methylation profile may be processed or rationalized by, for example, selecting a subset of methylation sites related to a specific physiological or biochemical pathway by using DNA regions instead of individual cytosines, performing a correlation analysis, retaining one or more representative DNA methylation sites per cluster, or performing a differential analysis to select or retain DNA methylation sites that vary more between young and old dogs.

[0126] For example, one or more DNA regions can be any one or more DNA regions as defined herein.

[0127] Suitably, the methylation profile can refer to the DNA methylation sites of genes associated with a particular physiological or biochemical pathway. Thus, the methylation profile can enable the assessment of the biological age of a particular tissue, organ, or biological system. Assessing the biological age of a particular tissue, organ or biological system can advantageously enable the method to be utilized in a manner that focuses on the pathology and disease of that tissue, organ, or biological system. For example, if a particular breed of dog is known to be associated with muscle or cardiovascular disease, it may be advantageous to assess the biological age of that biological system.

[0128] Preferably, the biological system can be the inflammatory system, the muscular system, the cardiovascular system, and / or the nervous system.

[0129] The biological age of a particular tissue, organ or biological system may be evaluated using a DNA methylation profile that includes or consists of methylation sites from genes preferentially or specifically expressed by that tissue, organ or biological system. Classification of genes by a particular tissue, organ, or biological system is publicly available, for example, from Gene Ontology (http: / / geneontology.org / ), KEGG pathway database (https: / / www.genome.jp / kegg / ), or MSIgDB (https: / / www.gsea-msigdb.org / gsea / msigdb / index.jsp).

[0130] In some embodiments, the threshold selects sites having the highest ranked average methylation values for epigenetic age predictors. For example, the threshold can be the top 50%, top 40%, top 30%, top 20%, top 10%, top 5%, top 4%, top 3%, top 2%, or top 1% of the average methylation levels across all sites “i” tested for a predictor, such as a biological clock. The threshold can alternatively be a site having an average methylation level at the 50, 60, 70, 80, 90, 95, 96, 97, 98, or 99th percentile rank or higher. In other embodiments, the threshold can be based on the absolute value of the average methylation level. For example, the threshold can be a site having an average methylation level 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%. The relative and absolute thresholds can be applied individually or in combination to the average methylation level at each site “i”. As an example of the application of combined thresholds, a subset of sites that are in the top 3% of all sites assayed by average methylation level and also have an absolute average methylation level greater than 6% can be selected. The result of this selection process is the DNA methylation profile of certain hypermethylated sites (e.g., CpG sites) that are thought to be most beneficial for the assessment of the risk of death and / or the probability of healthspan.

[0131] Alternatively, the threshold can be a site having an average methylation level at the 50, 60, 70, 80, 90, 95, 96, 97, 98, or 99th percentile rank or higher. In other embodiments, the threshold can be based on the absolute value of the average methylation level. For example, the threshold can be a site having an average methylation level 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%. The relative and absolute thresholds can be applied individually or in combination to the average methylation level at each site “i”. As an example of the application of combined thresholds, a subset of sites that are in the top 3% of all sites assayed by average methylation level and also have an absolute average methylation level greater than 6% can be selected. The result of this selection process is the DNA methylation profile of certain hypermethylated sites (e.g., CpG sites) that are thought to be most beneficial for the assessment of the risk of death and / or the probability of healthspan.

[0132] Preferably, the DNA methylation profile can include at least one methylated site listed in Table 3.

[0133] Suitably, one or more methylation sites may be defined as methylation markers present in any one or more of SEQ ID NOs: 1 to 261. SEQ ID NOs: 1 to 261 show the sequences on both sides of the methylation markers 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 preceding the methylation marker and 25 nucleotides following the methylation marker).

[0134] Suitably, the methylation site may be defined as an intervening position in the column labeled "Site" in Table 3. For example, for the site chr1:3844418-3844420, the methylation marker is chr1:3844419. Suitably, the DNA methylation profile may include 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 each of the methylation sites listed in Table 3.

[0135] Suitably, the DNA methylation profile may include the methylation sites chr2.32494387.32494389, chr22.46374563.46374565, and chr6.45773846.45773848. These sites are shown in Table 4.

[0136] Suitably, the DNA methylation profile may include the 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.

[0137] Suitably, the DNA methylation profile may include 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.

[0138] Suitably, the DNA methylation profile may include 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; chr20.57347921.57347923; chr24.21051024.21051026; chr33.26512711.26512713; chr23.37987526.37987528; chr5.32347978.32347980; chr6.60675998.60676000; chr19.44679975.44679977; chr10.7411293.7411295; chr33.26512692.26512694; chr1.121182146.121182148, and chr17.62156690.62156692. These sites are shown in Table 7.

[0139] Preferably, the DNA methylation profile is at 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; chr20.57347921.57347923; chr24.21051024.21051026; chr33.26512711.26512713; chr23.37987526.37987528; chr5.32347978.32347980; chr6.60675998.60676000; chr19.44679975.44679977; chr10.7411293.7411295; chr33.26512692.26512694; chr1.121182146.121182148; chr17.62156690.62156692; chr35.23853863.23853865; chr17.49222883.49222885; chr14.5949013.5949015; chr5.67427228.67427230; chr20.43366718.43366720; chr24.12518910.12518912; chr29.19479611.19479613; chr4.36180013.36180015; chr35.23852932.23852934; chr1.58324050.58324052; chr9.12522577.12522579; chr5.32711703.32711705; chr33.23473772.23473774; chr2.51831832.51831834; chr20.46098568.46098570; chr11.24476652.24476654; chr1.100289525.100289527; chr17.33851462.33851464; chr6.12827818.12827820; chr20.54301453.54301455; chr1.19541665.19541667; chr5.It may include 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.

[0140] Evaluation of DNA methylation sites / DNA methylation profiles indicating the risk of death and / or the probability of healthy life span The present invention includes evaluating the risk of death and / or the probability of healthy life span of dogs by using a DNA methylation profile. Accordingly, the present invention includes using a DNA methylation profile to create a biological clock related to the risk of death and / or the probability of healthy life span. This biological clock may also be referred to as an "epigenetic clock".

[0141] The provision of DNA methylation sites or DNA methylation profiles indicating the risk of death and / or the probability of healthy life span can be achieved, for example, through a training data set and a machine learning approach. Preferably, the machine learning approach may be a supervised machine learning approach.

[0142] As an example, the DNA methylation sites or DNA methylation profiles may be trained on a data set including dogs with known death outcomes (survived or died) and chronological age. Appropriately, the DNA methylation sites or DNA methylation profiles can be trained on a data set including dogs with known death outcomes and chronological age in combination with known breeds and / or genders.

[0143] For example, a model of DNA methylation sites or DNA methylation profiles indicative of a risk of death and / or a healthy life expectancy probability may be provided by using a machine learning framework to train a dataset of methylation states at a plurality of DNA methylation sites against a training dataset of dogs for which the death outcome (survive or die) and chronological age are known, and testing against a treatment-withheld cohort to verify the accuracy of the model.

[0144] The machine learning framework may include fitting a penalized model against a training dataset of dogs for which the death outcome (survive or die) and chronological age (and optionally breed and / or gender) are known, for example using the glmnet R package.

[0145] Preferably, the penalized model can be, for example, a penalized Cox regression, a LARS (Least Angle Regression path of solution) Cox regression, or a penalized survival model.

[0146] The machine learning framework may include fitting a penalized Cox regression against a training dataset of dogs for which the death outcome (survive or die) and chronological age (and optionally breed and / or gender) are known, for example using the glmnet R package.

[0147] Suitably, the machine learning framework may include fitting a known penalized model of death outcome (survive or die) / survival explained by the DNA methylation profile and chronological age (and optionally breed and / or gender), preferably a penalized Cox regression.

[0148] Suitably, the machine learning framework may include fitting a known penalized model of death outcome (survive or die) / survival explained by the DNA methylation profile, chronological age, breed, and gender, preferably a penalized Cox regression.

[0149] As used herein, "known death outcome (survival or death)" may also be referred to as "survival".

[0150] Suitably, a machine learning framework can be used to evaluate a model that includes a set of DNA methylation sites or a DNA methylation profile indicative of the risk of death and / or the probability of healthspan.

[0151] The model may include methylation states at multiple DNA methylation sites, where the methylation state at each site is considered in the model by multiplying by a coefficient value.

[0152] Suitably, gender may be encoded as a numerical value with females as 0 and males as 1.

[0153] Suitably, breed may be encoded as a numerical value with small breeds as 0 and medium breeds as 1.

[0154] The biological age of a dog may be expressed in years, months, days, etc.

[0155] The coefficient value of each parameter typically depends on the measurement units of all variables in the model. Thus, as will be understood by those skilled in the art, the value of each coefficient value will vary depending on, for example, the number and nature of the different parameters used in the model, and the nature of the training data provided. Thus, conventional statistical techniques may be applied to the training data set to arrive at the coefficient values. Such methods may include, for example, the calculation of two Gompertz functions or Weibull functions for a training set (e.g., when the status (survival or death) of a dog is known), one of which models survival as a function of the methylation profile, chronological age, breed class (small or medium dog), and gender (Model 1), and the other of which considers only chronological age, breed class, and gender (Model 2). These models can be fitted using the flexsurv package (v 2.1) in the R software environment.

[0156] Biological age can be defined as the time variable (the "chronological age") at which the survival probability of an animal given by Model 2 is equal to the survival probability at those chronological ages given by Model 1.

[0157] A model of DNA methylation sites or methylation profiles indicative of mortality risk and / or healthspan probability may be provided by training a dataset of methylation states at multiple DNA methylation sites against PhenoAge predicted at the age at DNA sample collection and assaying against a treatment-naive cohort to verify the accuracy of the model.

[0158] Methods for assessing PhenoAge in dogs or cats are described in PCT / EP2023 / 061058 and PCT / EP2023 / 061059, respectively. The calculation of PhenoAge takes into account direct predictors of blood biomarkers in mortality risk and / or healthspan probability. As an example, a given biomarker may not be directly correlated with chronological age but may indicate a particular pathological condition and thus an increased mortality risk and / or an increased probability of reduced healthy lifespan.

[0159] The assessment of a dog's PhenoAge may include measuring the value 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.

[0160] Preferably, the PhenoAge of a dog is a. Measuring the values 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 dogs; b. Evaluating the PhenoAge of dogs using formula (1), which may be provided by:

Number

Number

[0161] The coefficient values of each parameter typically depend on the measurement units of all variables in the model. Therefore, as understood by those skilled in the art, the value of each coefficient value may vary depending on, for example, the number and nature of different parameters used in the model, and the nature of the training data provided. Therefore, conventional statistical methods may be applied to the training data set in order to arrive at the coefficient values for use in the above equations. Such methods may include, for example, the calculation of two Gompertz functions or Weibull functions for a training set (e.g., when the status (alive or dead) of a dog is known), where the first function models survival as a function of a selected biomarker, chronological age, breed class (small dog or medium dog), and gender (Model 1), and the second function considers only chronological age, breed class, and gender (Model 2). These models can be fitted using the flexsurv package (v2.1) in the R software environment.

[0162] Preferably, a negative coefficient for a given biomarker means that the higher the value of the biomarker, the more positive the effect on the reduction of the risk of death, and / or the lower the value of the biomarker, the more negative the effect on the reduction of the risk of death. Preferably, a positive coefficient for a given biomarker means that the higher the value of the biomarker, the more negative the effect on the reduction of the risk of death, and / or the lower the value of the biomarker, the more positive the effect on the reduction of the risk of death.

[0163] Exemplary coefficients and γ and γ breed values are provided in the following table.

Table 1

[0164] The phenotypic age can be defined as the time variable ("chronological age") at which the survival probability of an animal given by Model 2 is equal to the survival probability at their chronological age given by Model 1.

[0165] The phenotypic age of a dog (i.e., PhenoAge) may be expressed in years, months, days, etc.

[0166] The biomarkers used for the assessment of PhenoAge can be measured using standard methods in the art and are typically measured as part of a standard blood test for evaluating the disease state of an animal. For example, biomarkers are generally measured as part of a standard complete blood count (cbc) and a standard clinical blood chemistry analysis.

[0167] Preferably, a model of DNA methylation sites or a DNA methylation profile indicating a risk of death and / or a probability of healthspan trained on PhenoAge can be provided in a two-step process.

[0168] In a first step, the machine learning framework may include fitting a penalized model of phenotypic age (PhenoAge) and chronological age (and optionally sex and / or breed) explained by one or more of the blood biomarkers described herein, for example using the glmnet R package. Preferably, the machine learning framework may include fitting a penalized model of phenotypic age (PhenoAge) explained by one or more of the blood biomarkers, chronological age, sex, and breed described herein.

[0169] Preferably, the penalized model can be, for example, a penalized Cox regression, LARS (Least Angle Regression path of solution) Cox regression, or a penalized survival model.

[0170] The machine learning framework may include fitting a penalized Cox regression of phenotypic age (PhenoAge) explained by one or more of the blood biomarkers, chronological age, sex, and breed described herein.

[0171] In the second step, the machine learning framework may include applying a penalized regression of PhenoAge explained by DNA methylation. Preferably, the machine learning framework may include applying a penalized regression of phenoage explained by a DNA methylation profile.

[0172] The penalized regression may be elastic net regression.

[0173] As used herein, the term "one or more biomarkers" may include at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, or at least 13 biomarkers.

[0174] As used herein, the term "one or more biomarkers" may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 biomarkers.

[0175] Suitably, a DNA methylation site or DNA methylation profile may be combined with the values of one or more of the blood biomarkers described herein to generate a model indicative of the risk of death and / or the probability of healthspan. For example, a model comprising a combination of a DNA methylation profile described herein and the values of one or more blood biomarkers may be provided by training a dataset of methylation states at multiple DNA methylation sites and the values of one or more blood biomarkers against a training dataset of dogs with known death outcome (survive or die) and chronological age, and testing against a treatment-naive cohort to verify the accuracy of the model.

[0176] The machine learning framework may include applying penalized regression to a training dataset of dogs where the death outcome (survive or die) and chronological age (and optionally breed and / or gender) are known, for example, applying it using the glmnet R package.

[0177] The machine learning framework may include applying penalized Cox regression to a training dataset of dogs where the death outcome (survive or die) and chronological age (and optionally breed and / or gender) are known, for example, applying it using the glmnet R package.

[0178] Preferably, the machine learning platform may comprise one or more deep neural networks. A neural network is a collection of neurons (also called units) connected in an acyclic graph. A neural network model is often composed of distinct layers of neurons. The most common type of layer for most neural networks is the fully connected layer, where neurons between two adjacent layers are fully pairwise connected, 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 non-linear activation functions. Combining non-linearly with a deep architecture enables more complex combinations of the input features, ultimately leading to a broader understanding of the relationships between them and, as a result, a more reliable final output. Deep neural networks have been applied to many types of data ranging from structured data to chemical descriptors or transcriptomics data.

[0179] Preferably, the machine learning platform includes one or more adversarial networks. Appropriately, the machine learning platform includes an adversarial autoencoder architecture. Preferably, the machine learning platform includes a feature importance analysis for ranking DNA methylation sites by their importance in the assessment of biological age.

[0180] The biological age of a dog may be expressed in years, months, days, etc.

[0181] Preferably, the risk of death and / or the probability of healthy life are expressed as the difference between the biological age and the chronological age of the dog.

[0182] Comparison with a reference or control The method of the present invention may further include comparing the difference in DNA methylation at one or more sites in a 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 predetermined risk of death and / or probability of healthy life (i.e., biological age). In some embodiments, the reference value is a value previously obtained for a subject or group of subjects for which the risk of death and / or probability of healthy life (i.e., biological age) is known. The reference value may be based on the known DNA methylation status, e.g., the average or median level, at one or more sites from a group of subjects for which the mortality status (alive or dead), chronological age, breed, and / or sex are known.

[0183] Combination of additional measured values and / or features with biomarker values Preferably, the method of the present invention further includes combining the DNA methylation profile with one or more of the chronological age, breed, and / or sex of the dog. By combining this information, the biological age related to the risk of death and / or probability of healthy life may be evaluated.

[0184] Stratification of subjects The biological age evaluated by the method of the present invention may also be compared with one or more predetermined thresholds (i.e., differences relative to chronological age). Such thresholds may be used to classify subjects into categories indicating specific risks, e.g., low, medium, high, etc. The degree of deviation from the threshold is useful for identifying which subjects will most benefit from a particular intervention. In this way, dietary interventions and lifestyle modifications can be optimized.

[0185] A method for selecting / monitoring a lifestyle plan, diet plan, or therapeutic intervention for a subject In a further aspect, the present invention provides a method for selecting a lifestyle plan, diet plan, or therapeutic intervention for a subject. Modification of the lifestyle may be any of the changes described herein, such as, for example, a dietary intervention and / or a change in exercise therapy. Modification of the lifestyle may also be the administration of a therapeutic modality.

[0186] A lifestyle plan, diet plan, or therapeutic intervention may be applied to a dog over any suitable period. After such a period, the risk of death and / or the probability of healthy lifespan of the dog may be re-evaluated using the method of the present invention to assess the effectiveness of the lifestyle plan, diet plan, or therapeutic intervention for reducing the risk of death of the dog and / or increasing the probability of healthy lifespan. By way of example, a lifestyle plan, diet plan, or therapeutic intervention may 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. A lifestyle plan, diet plan, or therapeutic intervention may 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.

[0187] A lifestyle plan, diet plan, or therapeutic intervention may be referred to as a lifestyle plan, diet plan, or therapeutic intervention for preventing aging.

[0188] The modification is preferably a dietary intervention as described herein. The term "dietary intervention" means an external factor applied to a subject to change the subject's diet. More preferably, the dietary intervention includes at least providing a food product or diet plan or dietary supplement.

[0189] The dietary intervention may be a diet, diet plan, dietary supplement or dietary supplement plan, or a combination of a diet and a dietary supplement, or a combination of a diet and a plurality of dietary supplements.

[0190] The dietary intervention or dietary product described in this specification may be any suitable diet plan, such as a calorie-restricted diet, a geriatric 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 omega 3-6 ratio diet, or a diet supplemented with a nicotinamide precursor such as carnitine, branched-chain amino acids or derivatives, nucleotides, nicotinamide mononucleotide (MNM) or nicotinamide riboside (NR), or any combination of the above.

[0191] Preferably, the dietary intervention or dietary product may be a calorie-restricted diet, a geriatric diet, or a low-protein diet. Preferably, the dietary intervention or dietary product may be a calorie-restricted diet. Preferably, the dietary intervention product or dietary product may be a low-protein diet.

[0192] The dietary intervention can be determined based on the dog's baseline maintenance energy requirement (MER). Preferably, the MER may be the amount of food that stabilizes the dog's body weight (change less than 5% over 3 weeks).

[0193] As an example, it is generally understood that young growing dogs benefit from a high-energy / high-protein diet. However, since older dogs may have lower energy requirements, the diet can be appropriately modified. In particular, many manufacturers produce "senior" ranges of dog food that are low in calories, high in fiber, but have appropriate amounts of protein and fat for older dogs.

[0194] Preferably, the calorie-restricted diet may include about 50%, about 55%, about 60%, about 65%, about 75%, about 80%, about 85%, or about 90% of the dog's MER. Preferably, the calorie-restricted diet may include about 60% or about 75% of the dog's MER.

[0195] Preferably, the low-protein diet may contain less than 20% protein (% dry matter). For example, the low-protein diet may contain less than 19% protein (% dry matter).

[0196] These diets are generally recommended based on the dog's chronological age. For example, a dog may be recommended to switch to a senior diet around 7 or 8 years of age. However, in the context of the present invention, an assessment of the increased risk of death compared to what is predicted for a given chronological age of a dog may enable the decision to switch the dog's diet to a senior diet at an earlier age. In contrast, dogs with a reduced risk of death compared to their chronological age may be able to continue an adult diet for a longer period.

[0197] Dietary interventions may include foods, nutraceuticals, and / or beverages that contain nutrients and / or bioactive substances that mimic the benefits of calorie restriction (CR) without restricting the daily calorie intake. For example, the foods, nutraceuticals, and / or beverages may contain functional raw materials that have benefits such as CR. Preferably, the foods, nutraceuticals, and / or beverages may contain autophagy-inducing factors. Preferably, the foods, nutraceuticals, and / or beverages may contain fruits and / or nuts (or extracts thereof). Suitable examples include, but are not limited to, pomegranate, strawberry, blackberry, camu camu, walnut, chestnut, pistachio, pecan. Preferably, the foods, nutraceuticals, and / or beverages may contain probiotics with or without fruit extracts or nut extracts.

[0198] Also, modifying the subject's lifestyle habits includes showing the subject the need to change their lifestyle habits, for example, instructing the subject to exercise more. Similar to dietary interventions, an assessment of the increased risk of death in dogs compared to what is predicted for a given chronological age may enable the decision to switch the dog to a more appropriate exercise program.

[0199] Modifying the target lifestyle also includes selecting or recommending a treatment modality or treatment plan. The treatment modality or treatment plan may be, for example, a modality useful for treating and / or preventing arthritis, dental diseases, endocrine disorders, heart diseases, diabetes, liver diseases, kidney diseases, prostate disorders, cancer, and behavioral or cognitive disorders. Preferably, prophylactic treatment can also be performed on dogs identified as being at risk of such disorders, based on an increased risk of death and / or on specific biomarkers known to be associated with disease-related pathways. In other embodiments, dogs evaluated as being at risk of a particular condition (based on an increased risk of death and / or on specific biomarkers known to be associated with disease-related pathways) may be monitored more regularly so that diagnosis and treatment can be initiated as early as possible.

[0200] The present invention also relates to monitoring and / or evaluating the effectiveness of a therapy for preventing aging, or to developing a therapy for preventing aging. A therapy for preventing aging may include, for example, a "rejuvenation" intervention. A rejuvenation intervention is aimed at causing a decrease in the epigenetic age or biological age of the subject. Suitably, a rejuvenation intervention may reprogram the epigenetic age to that of a very young dog. Examples of such rejuvenation interventions include, but are not limited to, gene therapy that reprograms the epigenetic age, preferably to that of a very young dog. The method for monitoring and / or evaluating the effectiveness of a lifestyle plan, diet plan, or therapeutic intervention, or for developing a lifestyle plan, diet plan, or therapeutic intervention for decreasing biological age, is particularly applicable in this aspect.

[0201] Thus, the present invention can advantageously enable the identification of dogs that are predicted to respond particularly well to a given intervention (e.g., a lifestyle plan, diet plan, or therapeutic intervention). Thus, the intervention can be applied in a more targeted manner to dogs predicted to respond.

[0202] In one aspect, the present invention provides a method for evaluating the effectiveness of a lifestyle plan, diet plan, or therapeutic intervention for reducing the risk of death and / or increasing the probability of a healthy lifespan in dogs, the method comprising: a) applying a lifestyle plan, diet plan, or therapeutic intervention selected according to the method of the present invention to a dog; b) after a period of applying the lifestyle plan, diet plan, or therapeutic intervention to the dog, evaluating the risk of death and / or the probability of a healthy lifespan of the dog using a DNA methylation profile from a sample obtained from the dog; and c) after a period according to the lifestyle plan, diet plan, or therapeutic intervention, evaluating whether there has been a change in the risk of death of the dog.

[0203] In a further aspect, the present invention provides a method for evaluating the effectiveness of a lifestyle plan, diet plan, or therapeutic intervention for reducing the risk of death and / or increasing the probability of a healthy lifespan in dogs, the method comprising: a) evaluating the risk of death and / or the probability of a healthy lifespan of the dog using a DNA methylation profile from a sample obtained from the dog; b) applying to the dog a lifestyle plan, diet plan, or therapeutic intervention selected based on the risk of death and / or the probability of a healthy lifespan evaluated in step a); c) after a period of applying the lifestyle plan, diet plan, or therapeutic intervention to the dog, evaluating the risk of death and / or the probability of a healthy lifespan of the dog using a DNA methylation profile from a sample obtained from the dog; and d) evaluating whether there has been a change in the risk of death and / or the probability of a healthy lifespan of the dog between step a) and step c).

[0204] Preferably, the lifestyle plan, diet plan, or therapeutic intervention may be applied to the dog during a period prior to the evaluation of the first risk of death and / or probability of a healthy lifespan. However, the effectiveness of the lifestyle plan, diet plan, or therapeutic intervention for improving (i.e., reducing the risk of death and / or increasing the probability of a healthy lifespan) the risk of death and / or the probability of a healthy lifespan of the dog may be monitored by evaluating the risk of death and / or the probability of a healthy lifespan two or more times during the application of the lifestyle plan, diet plan, or therapeutic intervention.

[0205] Suitably, the method may include an "ecosystem", particularly a digital ecosystem. Appropriately, the method includes the steps of: (a) optionally using a kit according to the invention to provide a sample obtained from a dog; and (b) providing the sample (e.g., by mail) for subsequent DNA extraction and measuring DNA methylation in the DNA extracted from the sample to obtain a DNA methylation profile.

[0206] The DNA methylation profile may then be used according to any of the methods of the invention, preferably using a computer system or computer program product according to the invention.

[0207] The computer system or computer program may then create and share a report detailing the results of the analysis / method, for example, in the form of selecting or recommending a lifestyle plan, diet plan, or therapeutic intervention suitable for the dog, or any other result of the method of the invention.

[0208] Suitably, the sample can be a sample that can be obtained by the dog owner at home (without the need for a veterinarian or medical professional). Suitably, the sample can be a hair follicle, oral swab, or saliva sample.

[0209] Use of dietary intervention In one aspect, the invention provides a dietary intervention for use in reducing the risk of death and / or increasing the probability of a healthy lifespan in dogs, the dietary intervention being performed on dogs whose risk of death and / or probability of a healthy lifespan has been evaluated by the method of the invention.

[0210] In another aspect, the invention provides the use of a dietary intervention for reducing the risk of death in dogs and / or increasing the probability of a healthy lifespan, the dietary intervention being performed on dogs whose risk of death and / or probability of a healthy lifespan has been evaluated by the method of the invention.

[0211] As described herein, the dietary intervention may be a dietary product or a dietary plan or a dietary supplement.

[0212] Computer program product The method of the present invention may be performed using a computer. Thus, the method may be performed in silico.

[0213] Suitably, the computer may create and share a report detailing the results of the method.

[0214] The method described herein may be implemented as a computer program operating on general-purpose hardware such as one or more computer processors. In some embodiments, the functions described herein may be performed by a device such as a smartphone, a tablet terminal, or a personal computer.

[0215] In one aspect, the present invention provides a computer program product comprising computer-executable instructions for causing a programmable computer to evaluate the risk of death and / or the probability of healthspan of a dog, as described herein.

[0216] In one embodiment, the user optionally inputs into the device one or more values of the DNA methylation markers defined herein, along with the chronological age, breed, and gender. The device then processes this information and provides an assessment of the biological age of the dog. Alternatively, the device then processes this information and provides an assessment of a lifestyle plan, a dietary plan, or a therapeutic intervention suitable for the dog based on the biological age.

[0217] The device may generally be a server on a network. However, any device may be used as long as it can process biomarker data and / or additional parameter or feature data using a processor, a central processing unit (CPU), or the like. The device may be, for example, a smartphone, a tablet terminal, or a personal computer, and may output information indicating the biological age evaluated for a dog, or information indicating an evaluation of a lifestyle plan, a diet plan, or a therapeutic intervention suitable for a dog based on the biological age.

[0218] Those skilled in the art will understand that all features of the present invention described herein can be freely combined without departing from the scope of the disclosed present invention.

Example

[0219] Next, the present invention will be further described by way of examples, which are meant to assist those skilled in the art in practicing the present invention and are not intended to limit the scope of the present invention in any way.

[0220] Example 1 An exemplary method for creating an epigenetic biological clock Identification of DNA methylation sites Whole blood samples from a dog cohort were analyzed by performing DNA extraction, converting DNA methylation using an enzymatic reaction, preparing a whole genome library, hybridizing the whole genome conversion library preparation to a capture probe for a gene promoter, and determining the methylation profile by sequencing (EM-Seq).

[0221] The capture probe targeted approximately 40,000 targets (promoter regions - approximately 1 kb upstream and 0.5 kb downstream of the promoter). These target regions contain potential methylation sites (individual cytosine residues that can be methylated) of interest.

[0222] Before array determination and before further analysis, perform the following bioinformatics steps: Use fastQC to perform quality checks on fastq - https: / / www.bioinformatics.babraham.ac.uk / projects / fastqc / Use trimGalore to trim adapters - https: / / www.bioinformatics.babraham.ac.uk / projects / trim_galore / Align against the dog genome using bwa-meth (https: / / github.com / brentp / bwa-meth) or Bismark - https: / / www.bioinformatics.babraham.ac.uk / projects / bismark / Use Picard to mark duplicates - https: / / gatk.broadinstitute.org / hc / en-us / articles / 360037052812-MarkDuplicates-Picard- Call methylation using Methyldackel - https: / / github.com / dpryan79 / MethylDackel An approach for filtering sites to create a restricted DNA methylation profile for training the circadian clock Following the assessment of the methylation status of methylation sites in each sample, the initial methylation profile may be filtered / processed to generate a restricted methylation profile containing fewer distinct methylation sites. This filtering aims to provide a restricted methylation profile containing, for example, 50,000 to 500,000 methylation sites that can be used to train the circadian clock.

[0223] The method for filtering the initial methylation profile includes the following steps: 1) The step of removing sites that are methylated (not methylated) in all samples 2) Removing sites that do not have five or more counts in at least 90% of the samples 3) Removing X chromosome sites and / or Y chromosome sites

[0224] Further promising filtering steps for reducing the number of distinct methylation sites include the following steps: 1) Defining methylation sites as DNA regions (CpG islands, for example, sites that are less than 1000 bp apart are considered the same) 2) Restricting the target to, for example, inflammatory sites, such as genes related to inflammation by GO / KEGG pathways, and selecting sites on these genes 3) Correlation analysis and retention of one or more representative values for each cluster (for example, weighted gene co-expression network analysis (WGCNA) (Langfelder & Horvath; BMC Bioinformatics; 9(559); 2008 or EBModules (Zollinger et al.; Biostatistics, 19(2), 153 - 168; (2018)) 4) Performing differential analysis for methylation to pre-select DNA methylation sites (for example, using logistic regression) or to identify sites that vary more between young and old dogs (for example, using analysis of variance). When differential analysis for methylation is performed (using the function preprocessQuantile in R), normalization (quantile) is performed before the filtering step.

[0225] Construction of the biological clock A dataset containing information on the death status (survived or died), chronological age, breed, and sex of a cohort of dogs is divided into a training set and a test set (for example, ensuring a good split for metadata, 2 / 3 of the data for training and 1 / 3 of the data for testing; for example, similar proportions of each breed / each sex in the training set and the test set).

[0226] Use methylation sites, breed / breed class, gender, and chronological age as predictors to fit a penalized Cox model to survival. The model parameters as well as the penalty parameters are estimated using the glmnet package within R.

[0227] Execute the calculation of two Gompertz functions or Weibull functions. The first function is a function that models survival as a function of methylation profile, chronological age, breed class (small or medium dogs), and gender (Model 1), and the second function is a function that considers only chronological age, breed class, and gender (Model 2). Such models are fitted using the flexsurv package (v2.1) in the R software environment.

[0228] The mortality risk and / or healthy lifespan probability can be defined as the time variable ("chronological age") at which the survival probability of the animals given by Model 2 is equal to their survival probability at that chronological age given by Model 1.

[0229] The penalty parameter is selected to provide a reasonable number of DNA methylation sites (e.g., up to 1000) that have a good model fitting the mortality risk and / or healthy lifespan probability.

[0230] Next, evaluate the model of biological age based on DNA methylation sites in the test set.

[0231] In this example, the following steps were performed: 1) The step of setting all methylation sites with coverage less than 15 counts as "missing" 2) The step of performing imputation using the "Boostme" algorithm (Zou et al.; BMC Genomics 19, 390 (2018)) and setting the sizes of the training, test, and validation sets to 200,000 randomly selected sites 3) The step of removing ChrX sites 4) Step of performing site filtering using the EB module (Zollinger et al.; Zollinger et al.; Biostatistics, 19(2), 153 - 168; (2018)); The dataset was separated into blocks of approximately 5000 sites by grouping target - specific sites (1500 bp around the TSS) and chromosome - specific sites. Next, a correlation matrix to which the EB module was applied was calculated as described by Zollinger et al. Next, each module was represented by a medoid site. Some sites did not belong to any module and were called scattered sites (definition as described in Zollinger et al.). 5) Step of removing sites present on mammalian CpG arrays (Arneson et al.; Nature Communications; 13; 783; 2022); 6) Step of adjusting Elastic net regression at 500K sites using phenoAge_pred (predicted value of PhenoAge at the age of DNA collection) as the response variable; 261 sites were selected as those forming the biological clock (see Table 3).

[0232] The clock was verified using the evaluation range. Figure 3 shows the verification of the clock under calorie restriction, and dogs under calorie restriction have a lower biological age (lower delta) compared to dogs on the control diet. The delta in Figure 3 corresponds to the residual of the regression model comparing chronological age and predicted phenoDNAmAge.

[0233] Further verification is shown in Figure 4, where there is a difference in delta (phenoDNAmAge) between the two diets, and dogs fed a restricted diet are biologically 6 years younger.

[0234] Figure 5 shows that there is a significant difference between the two diets when adjusting the linear mixed - effects model (DogID as a random effect).

[0235] Figure 6 shows the significant differences in survival between biologically young dogs and biologically old dogs.

[0236] Figure 7 shows that when using gender and ΔphenoDNAmAge (the residual obtained by comparing chronologicalAge with the predicted phenoDNAmAge) to fit a Cox proportional hazards model and stratifying by breed class (small or medium) on the training set, an increase in ΔphenoDNAmAge is significantly associated with an increase in the risk of death.

[0237] Figure 8 shows that the biological clock is highly reproducible between technical replicates.

[0238] Univariate analysis was performed to evaluate whether each of the 261 sites was statistically significant with respect to the relationship with biological age (see Table 3).

[0239] From the complete list of sites shown in Table 3, additional biological clocks were also created using only the top 3, top 5, top 10, top 20, and top 50 sites. Each was shown to be correlated with biological age (see Figures 9 - 13). These biological clocks were created by selecting the top n sites based on the absolute value of the coefficients of the complete biological clock (in descending order, taking the larger coefficients first). A linear model was fitted to explain chronological age using the top n sites as predictors. The details of the top 3, top 5, top 10, and top 20 clocks are shown in Tables 4 - 7.

[0240] Measurement of blood biomarkers related to the risk of death in Example 2 dogs Predictive blood biomarkers were determined from a biomarker panel consisting of a standard clinical complete blood count (cbc) and a standard clinical blood chemistry analysis. Serum samples were collected after an overnight fast and measured by standard veterinary clinical practice.

Table 2

[0241] The inventors conducted a longitudinal study of dogs and repeatedly measured these parameters, as well as information on the dog's status (alive or dead), sex, and breed. First, the inventors classified the breeds as small or medium (less than 10 kg or more than 10 kg, respectively) based on the average weight of adult dogs of this breed. Next, the data were organized using the R programming language. For each dog, the inventors recorded the biomarker as a time-dependent covariate using a time interval where the left side was open and the right side was closed (i.e., (tstart, tstop]). The biomarker information corresponded to the start of this time interval, and the event (alive or dead) was recorded as the last tstop value. For this purpose, the inventors used the tmerge function of the survival package in R (v. 3.2 - 13). Next, the cox proportional hazards model was applied to the data of each of the 28 biomarkers, including sex and breed class (small or medium). Then, considering multiple comparisons (by false discovery rate (fdr)), the p-values of each parameter were corrected, and the features with a corrected fdr less than 0.05 were selected (Figure 1).

[0242] By this method, the inventors identified 13 biomarkers for individually predicting the survival probability in dogs.

[0243] White blood cell count (10^3 / μL) Serum albumin (g / dL) Serum alkaline phosphatase (U / L, natural logarithm transformation) Serum creatine kinase (IU / L, natural logarithm transformation) Hemoglobin (g / dL) Hematocrit (%) Mean corpuscular hemoglobin (pg) Serum sodium (mmol / L) Mean corpuscular volume (fL) Serum globulin (g / dL) Serum calcium (mg / dL) Serum platelet count (10^3 / μL) Red blood cell count (10^3 / μL)

[0244] Example 3 Multi-parameter model for predicting the risk of death Next, the inventors constructed the best model that simultaneously considers multiple parameters as being more likely to cover the various organ dysfunctions that occur with age. However, selecting some features that can be correlated with each other is subject to bias. To avoid this problem, a penalized regression method using the glmnet package (v4.1-3) was used. The data was fitted to the LASSO of the penalized cox proportional hazard model, and 20-fold cross-validation was used to compare different values of the penalty parameter λ. This approach results in the selection of the top 10 most predictive blood biomarkers for survival in the order of importance shown below.

[0245] · White blood cell count (10^3 / μL) · Serum albumin (g / dL) · Serum alkaline phosphatase (U / L, ln-transformed) · Serum creatine kinase (IU / L, ln-transformed) · Hemoglobin (g / dL) · Hematocrit (%) · Mean corpuscular hemoglobin (pg) · Serum glucose (mg / dL) · Mean corpuscular volume (fL) · Serum globulin (g / dL)

[0246] The inventors also found that the first three biomarkers in this list are the most predictive, and that the performance can be improved by incorporating each of the next seven biomarkers.

[0247] To extract the phenotypic age of animals, the inventors calculated two different Gompertz functions for their training set, namely, a first function (Model 1) that models survival as a function of the selected biomarkers, age, breed class (small or medium dogs), and gender, and a second function (Model 2) that considers only age, breed class, and gender. These models were fitted using the flexsurv package (v2.1). The phenotypic age was defined as the time variable ("age") at which the survival probability of the animals given by Model 2 is equal to the survival probability at their chronological age given by Model 1. This yielded a mathematical function that relates the blood biomarkers to phenoage and is given by the following equation.

Equation

[0248] As an example, the coefficients, as well as the γ and γ breed values were measured from the inventors' training set for the complete list of biomarkers and are shown in Table 2.

Table 3

Table 3

[0249] Furthermore, starting from the top of the list and systematically removing one biomarker at a time to reduce the set of ten biomarkers, the inventors observed a decrease in the strength of the survival prediction (p-value). The decrease was most prominent with the first parameter, confirming that it had the largest contribution, but the inventors also observed the change in the quality of prediction by reducing each of the sets, indicating that each parameter contributed to the overall prediction (Figure 2).

[0250] All publications mentioned in the above specification are hereby incorporated by reference into this specification. Various modifications and variations of the disclosed methods, compositions, and uses of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been disclosed in connection with specific preferred embodiments, it should be understood that the claimed invention should not be unduly limited to such specific embodiments. Indeed, various modifications in the manner disclosed for practicing the present invention will be apparent to those skilled in the art and are intended to be within the scope of the following claims.

Table 4

Table 5

Table 6

Table 7

Table 8

Claims

1. A method for evaluating the mortality risk and / or healthy life expectancy probability of dogs, wherein the method is a) A step of providing a DNA methylation profile from a sample obtained from the dog, b) A method comprising the step of evaluating the mortality risk and / or probability of healthy life of the dog using the DNA methylation profile.

2. A method for determining the biological age of a dog, wherein the method is a) A step of providing a DNA methylation profile from a sample obtained from the dog, b) A method comprising the step of evaluating the biological age of the dog using the DNA methylation profile, wherein the DNA methylation profile is associated with the risk of death and / or the probability of healthy lifespan of the dog.

3. A method for selecting a lifestyle plan, dietary plan, or therapeutic intervention for dogs, wherein the method is a) A step of providing a DNA methylation profile from a sample obtained from the dog, b) A step of evaluating the mortality risk and / or healthy life expectancy probability of the dog using the DNA methylation profile, c) A method comprising the step of selecting a lifestyle, dietary plan, or therapeutic intervention appropriate for the dog based on the mortality risk and / or healthy life expectancy probability assessed in step b).

4. A method for evaluating the effectiveness of a lifestyle plan, dietary plan, or therapeutic intervention to improve the risk of death in dogs, wherein the method is a) the step of applying a lifestyle plan, dietary plan, or therapeutic intervention selected according to the method of claim 3 to the dog, b) After a period of applying the lifestyle plan, dietary plan, or therapeutic intervention to the dog, the step of evaluating the dog's mortality risk and / or healthy life expectancy probability using a DNA methylation profile from a sample obtained from the dog, c) A method comprising the step of evaluating whether there has been any change in the dog's mortality risk and / or healthy life expectancy probability after a period of following the lifestyle plan, dietary plan, or therapeutic intervention.

5. A method for evaluating the effectiveness of lifestyle plans, dietary plans, or therapeutic interventions to improve the mortality risk and / or the probability of healthy lifespan in dogs, wherein the method is a) A step of evaluating the mortality risk and / or healthy life expectancy probability of the dog using the DNA methylation profile from a sample obtained from the dog, b) Applying a lifestyle plan, dietary plan, or therapeutic intervention selected based on the mortality risk and / or healthy life expectancy probability assessed in step a) to the dog, c) After a period of applying the lifestyle plan, dietary plan, or therapeutic intervention to the dog, the step of evaluating the dog's mortality risk and / or healthy life expectancy probability using a DNA methylation profile obtained from a sample of the dog, d) A method comprising the step of evaluating whether there has been a change in the dog's mortality risk and / or probability of healthy lifespan between step a and step c.

6. A method for developing a lifestyle plan, dietary plan, or therapeutic intervention to prevent aging, wherein the method is a) A step of evaluating the first mortality risk and / or healthy life expectancy probability of the dog using the DNA methylation profile from a sample obtained from the dog, b) The step of applying a lifestyle plan, dietary plan, or therapeutic intervention to the dog, c) After a period of applying the lifestyle plan, dietary plan, or therapeutic intervention to the dog, the step of evaluating the second mortality risk and / or healthy life expectancy probability of the dog using a DNA methylation profile from a second sample obtained from the dog, d) The step of evaluating whether there has been any change in the first and second mortality risk and / or healthy life expectancy probability of the dog after the period of following the lifestyle plan, dietary plan or therapeutic intervention, A method by which a lifestyle plan, dietary plan, or therapeutic intervention is evaluated as preventing aging if the lifestyle plan, dietary plan, or therapeutic intervention reduces the risk of death of the dog and / or increases the probability of healthy life expectancy and / or reduces the rate of increase in the risk of death and / or increases the rate of decrease in the probability of healthy life expectancy.

7. A method for preventing or reducing the risk of disease development in dogs, wherein the method is a) A step of evaluating the mortality risk and / or healthy life expectancy probability of a dog using a DNA methylation profile obtained from a sample from the dog, wherein the mortality risk and / or healthy life expectancy probability evaluated for the dog is associated with an increased likelihood of developing the disease; b) A step of selecting a lifestyle plan, dietary plan, or therapeutic intervention for the dog based on the mortality risk and / or healthy life expectancy probability assessed in step a), A method comprising the step of selecting a lifestyle plan, dietary plan, or therapeutic intervention that prevents or reduces the risk of the dog developing the disease, wherein the disease is an age-related disease.

8. A method for selecting a dog as suitable for receiving a lifestyle plan, dietary plan, or therapeutic intervention to prevent aging, wherein the method is a) A step of evaluating the mortality risk and / or healthy life expectancy probability of the dog using the DNA methylation profile from a sample obtained from the dog, b) A method comprising the step of selecting a dog that is suitable to receive a lifestyle plan, dietary plan, or therapeutic intervention to prevent aging if the risk of death is increased and / or the probability of healthy life expectancy is decreased compared to its chronological age.

9. The method according to any one of claims 1 to 8, wherein the method for evaluating the mortality risk and / or healthy life expectancy probability of the dog further comprises combining the DNA methylation profile with one or more of the dog's chronological age, breed, and / or sex.

10. The method according to any one of claims 3 to 8, wherein a lifestyle plan, dietary plan, or therapeutic intervention is selected based on an assessment that the dog has a high mortality risk and / or a low probability of healthy life expectancy compared to its chronological age.

11. The method according to any one of claims 3 to 8, wherein the lifestyle plan, dietary plan, or therapeutic intervention is a dietary intervention.

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

13. The method according to any one of claims 1 to 8, wherein the sample is a blood sample.

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

15. The method according to any one of claims 1 to 8, wherein the DNA methylation profile includes at least one methylation site listed in Table 3. Table 1

16. The method according to claim 15, wherein the DNA methylation profile includes 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 each of the methylation sites listed in Table 3.

17. The method according to claim 15, wherein the DNA methylation profile includes the methylation sites listed in Table 4. Table 2

18. The method according to claim 15, wherein the DNA methylation profile includes the methylation sites listed in Table 5. Table 3

19. The method according to claim 15, wherein the DNA methylation profile includes the methylation sites listed in Table 6. Table 4

20. The method according to claim 15, wherein the DNA methylation profile includes the methylation sites listed in Table 7. Table 5

21. The method according to claim 14, wherein DNA methylation is evaluated using a method comprising one or more of the following steps: (a) treating sample DNA with APOBEC to deaminate cytosine, (b) enriching based on capture, and / or (c) performing high-throughput sequencing.

22. The method according to any one of claims 1 to 8, wherein the DNA methylation profile is associated with disease or predicted disease in (i) a tissue, (ii) an organ, or (iii) a biological system, such as the immune system, gastrointestinal system, urinary system, muscular system, cardiovascular system, and / or nervous system.

23. The method according to claim 22, further comprising applying to the dog a lifestyle plan, dietary plan, or therapeutic intervention suitable for improving the disease or anticipated disease in the tissue, organ, or biological system described in claim 22.

24. A dietary intervention or therapeutic modality for use in reducing the risk of death and / or increasing the probability of healthy lifespan in dogs, wherein the dietary intervention is performed on dogs whose risk of death has been assessed by the method of claim 1 or any one of claims 7 to 8, or is a therapeutic modality administered to dogs.

25. Use of a therapeutic modality by dietary intervention to reduce the risk of death in dogs and / or increase the probability of healthy lifespan in dogs, wherein the dietary intervention is performed on dogs whose risk of death and / or probability of healthy lifespan has been assessed by the method of claim 1 or any one of claims 7 to 8.

26. A computer-readable medium comprising instructions that, when executed, cause one or more processors to perform the method according to any one of claims 1, 2, 3, 7, or 8.

27. A computer system for evaluating the mortality risk and / or healthy life expectancy probability of a dog, wherein the computer system is programmed to evaluate the mortality risk of the dog using the dog's DNA methylation profile.

28. A computer system for selecting a lifestyle plan, dietary plan, or therapeutic intervention suitable for a dog, wherein the computer system a) A step of evaluating the mortality risk and / or healthy life expectancy probability of the dog using the DNA methylation profile from the dog, and b) A computer system programmed to perform one or more steps of the following: selecting a lifestyle plan, dietary plan, or therapeutic intervention appropriate for the dog based on the mortality risk and / or healthy life expectancy probability assessed in step a).

29. A computer system for evaluating the effectiveness of lifestyle plans, dietary plans, or therapeutic interventions in order to improve the mortality risk and / or healthy life expectancy probability of dogs, wherein the computer system a) A step of evaluating the mortality risk and / or healthy life expectancy probability of the dog using DNA methylation profiles from samples obtained from the dog before the lifestyle plan, diet plan, or therapeutic intervention, and from samples obtained from the dog after the lifestyle plan, diet plan, or therapeutic intervention, and b) A computer system programmed to perform one or more steps of the following: evaluating whether there has been a change in the mortality risk and / or healthy life expectancy probability of the dogs between the samples obtained from the dogs before and after the lifestyle plan, dietary plan, or therapeutic intervention has been applied.

30. A computer system for evaluating the likelihood that a dog may benefit from a lifestyle plan, dietary plan, or therapeutic intervention to prevent aging, wherein the computer system a) A step of evaluating the mortality risk and / or healthy life expectancy probability of the dog using the DNA methylation profile from a sample obtained from the dog, and b) A computer system programmed to perform one or more of the following steps: identifying the dog as likely to respond to an aging prevention lifestyle plan, dietary plan, or therapeutic intervention if the risk of death is increased and / or the probability of healthy life expectancy is decreased compared to its chronological age.

31. A computer program product comprising a computer-executable instruction for causing a programmable computer to evaluate the mortality risk and / or probability of healthy lifespan of a dog using the dog's DNA methylation profile.

32. A computer program product comprising computer-executable instructions for causing a programmable computer to evaluate the mortality risk and / or healthy life expectancy probability of a dog using a DNA methylation profile from the dog, and to select a lifestyle plan, dietary plan, or therapeutic intervention appropriate for the dog based on the mortality risk and / or healthy life expectancy probability evaluated using the DNA methylation profile.

33. A computer program product comprising computer-executable instructions for causing a programmable computer to: a) evaluate the mortality risk and / or healthy life expectancy probability of a dog using DNA methylation profiles from samples obtained from the dog before a lifestyle plan, dietary plan, or therapeutic intervention and from samples obtained from the dog after the lifestyle plan, dietary plan, or therapeutic intervention; and b) evaluate whether there has been a change in the mortality risk and / or healthy life expectancy probability of the dog between samples obtained from the dog before and after the lifestyle plan, dietary plan, or therapeutic intervention is applied.

34. A computer program product comprising computer-executable instructions for a programmable computer to a) assess the mortality risk and / or healthy life expectancy of a dog using a DNA methylation profile obtained from a sample of the dog, and b) identify dogs that are more likely to respond to lifestyle, dietary, or therapeutic interventions to prevent aging if they have an increased mortality risk and / or a decreased healthy life expectancy compared to their chronological age.

35. A method for constructing an epigenetic clock for evaluating the mortality risk and / or healthy lifespan probability of a dog, the method comprising: a) providing a DNA methylation profile from a sample obtained from the dog; and b) calculating two Gompertz functions or Weibull functions: (i) a first Gompertz function or Weibull function that models survival as a function of the methylation profile, chronological age, and optionally breed class and / or sex; and (ii) a second Gompertz function or Weibull function that models survival as a function of chronological age, and optionally breed class and / or sex only, wherein the mortality risk and / or healthy lifespan probability is evaluated based on a time variable in which the survival probability of the dog given by the second Gompertz function or Weibull function is equal to the survival probability of the dog at chronological age given by the first Gompertz function 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 evaluating the mortality risk and / or healthy lifespan probability of dogs, comprising means and reagents for collecting and optionally stabilizing a sample from a subject, and instructions for collecting and optionally stabilizing the sample, wherein the sample is mailed for subsequent DNA extraction, and DNA methylation in the DNA extracted from the sample is measured to obtain a DNA methylation profile.

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

39. A method for evaluating the mortality risk and / or healthy life expectancy probability of a dog using a DNA methylation profile from the dog, and for selecting a lifestyle plan, diet plan, or therapeutic intervention appropriate for the dog based on the mortality risk and / or healthy life expectancy probability evaluated using the DNA methylation profile, the method comprising: (a) optionally providing a sample from the dog using the kit described in claim 37; (b) providing the sample for subsequent DNA extraction and measuring DNA methylation in the DNA extracted from the sample to obtain a DNA methylation profile; and (c) evaluating the mortality risk and / or healthy life expectancy probability and selecting a lifestyle plan, diet plan, or therapeutic intervention appropriate for the dog using the computer system described in claim 28 or the computer program product described in claim 32, wherein the computer system prepares and shares a report detailing the results of step (c).

40. A method for evaluating the effectiveness of a lifestyle, dietary, or therapeutic intervention to improve the mortality risk and / or healthy life expectancy probability of a dog using a DNA methylation profile, comprising: (a) optionally providing a sample obtained from the dog before the lifestyle, dietary, or therapeutic intervention and a sample obtained from the dog after the lifestyle, dietary, or therapeutic intervention using the kit described in claim 37; (b) providing the sample for subsequent DNA extraction and measuring DNA methylation in the DNA extracted from the sample to provide a DNA methylation profile; and (c) using the computer system described in claim 29 or the computer program product described in claim 33 to evaluate whether there has been a change in the mortality risk and / or healthy life expectancy probability of the dog between the sample obtained from the dog before and after the lifestyle, dietary, or therapeutic intervention is applied, wherein the computer system prepares and shares a report detailing the results of step (c).

41. A method for identifying dogs that are likely to respond to an age-preventing lifestyle, dietary, or therapeutic intervention using a DNA methylation profile, the method comprising: (a) providing a sample obtained from the dog, optionally using the kit described in claim 37; (b) providing the sample for subsequent DNA extraction and measuring DNA methylation in the DNA extracted from the sample to obtain a DNA methylation profile; and (c) using the computer system described in claim 30 or the computer program product described in claim 34 to identify the dog as likely to respond to an age-preventing lifestyle or diet if the dog has a higher mortality risk and / or reduced probability of healthy life compared to its chronological age, wherein the computer system prepares and shares a report detailing the results of step (c).

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