Methods and treatments for diabetes in dogs

Abstract

The present disclosure relates to methods for screening dogs for a predisposition to diabetes and methods for determining the likelihood that a dog will produce offspring genetically predisposed to diabetes. The disclosure also relates to methods for selecting a therapy for diabetes in a dog, methods for preventing, delaying, or treating diabetes in a dog using an oral hypoglycemic agent, and compositions comprising an oral hypoglycemic agent for use in methods for preventing, delaying, or treating diabetes in a dog. The disclosure further relates to methods for preventing or delaying diabetes in a dog using an antihyperglycemic diet or by spaying, and antihyperglycemic diets for use in methods for preventing or delaying diabetes in a dog.

Description

[Technical Field]

[0001] Field of Disclosure The present disclosure relates to methods for screening dogs for a predisposition to diabetes and methods for determining the likelihood that a dog will produce offspring genetically predisposed to diabetes. The disclosure also relates to methods for selecting a therapy for diabetes in a dog, methods for preventing, delaying, or treating diabetes in a dog using an oral hypoglycemic agent, and compositions comprising an oral hypoglycemic agent for use in methods for preventing, delaying, or treating diabetes in a dog. The disclosure further relates to methods for preventing or delaying diabetes in a dog using an antihyperglycemic diet or by spaying, and antihyperglycemic diets for use in methods for preventing or delaying diabetes in a dog. [Background technology]

[0002] background Diabetes mellitus is characterized by chronically elevated blood glucose levels as a result of insufficient insulin secretion or insulin sensitivity. Not only is diabetes common in humans, but veterinarians recognize naturally occurring diabetes in dogs, with a prevalence of approximately 1 in 300 dogs. The median age of onset of diabetes in dogs is 7 years, but in some breeds, such as Labrador Retrievers, onset can occur as early as 8 weeks of age. Affected dogs typically rely on twice-daily insulin injections, which is burdensome for owners and uncomfortable for the animals.

[0003] The insulin dependence and pancreatic β-cell loss typically observed in canine diabetes suggest some similarities to human autoimmune type 1 diabetes (T1D) rather than type 2 diabetes (T2D), which is characterized by insulin resistance and β-cell dysfunction. However, unlike human T1D, evidence for an autoimmune etiology in canine diabetes is very limited.

[0004] In humans, rare monogenic forms of diabetes have been identified that present in newborns and early adulthood. Approximately 50% of these mutations affect the ATP-sensitive potassium (K ATPIt is found in genes encoding the pore-forming subunits (KCNJ11, Kir6.2) and regulatory subunits (ABCC8, SUR1) of the KCNJ channel. Common variants in KCNJ11 also predispose to type 2 diabetes in humans. ATP The channel plays an important role in glucose-stimulated insulin secretion from pancreatic β-cells by regulating membrane electrical excitability. Glucose uptake and metabolism by β-cells generates ATP, which is converted into K ATP K closes the channel, causing membrane depolarization, electrical activity, calcium influx, and insulin secretion. ATP Activating channel mutations impair the ability of ATP to close the channel, which inhibits insulin secretion.

[0005] While the genetic and pathophysiological basis of human diabetes has been well characterized, the basis of canine disease remains unclear. A better understanding of canine diabetes may provide improved treatments that are tolerated by owners and animals, and breeding programs that reduce the likelihood of diabetic offspring. Summary of the Invention [Means for solving the problem]

[0006] Summary of the Disclosure The present inventors have identified the first diabetes-associated missense mutation in the canine KCNJ11 gene and further characterized this mutation as a monogenic cause of diabetes in dogs.

[0007] This mutation can be used to screen dogs for predisposition to diabetes. This can advantageously allow for early diagnosis and treatment at the onset of clinical disease. Preventive measures can also be implemented before the onset of clinical disease, for example, in a prediabetic state. For example, measures can be taken to minimize the occurrence of hyperglycemia in dogs. Because hyperglycemia is damaging to insulin-producing pancreatic beta cells, minimizing the occurrence of hyperglycemia can help preserve beta cells and / or their ability to produce insulin.

[0008] Human neonatal diabetes caused by KCNJ11 mutations is treated by administering oral hypoglycemic drugs such as sulfonylureas. Diabetic dogs with the KCNJ11 gene mutations disclosed herein can be similarly treated. Such oral treatment is advantageous compared to traditional twice-daily injectable insulin therapy. For example, oral medications are easier to administer and may be better tolerated by dogs. Furthermore, oral sulfonylurea treatment can reduce blood glucose fluctuations, lower HbA1c, and enhance meal-stimulated insulin secretion (by enabling incretin action). Thus, the present disclosure provides a valuable new treatment regimen for canine diabetes. The present disclosure also provides a method for identifying dogs likely to benefit from this regimen through genetic testing for KCNJ11 gene mutations.

[0009] The KCNJ11 gene mutations disclosed herein can also be used to determine the likelihood that a dog will produce offspring that are genetically predisposed to diabetes, allowing carrier dogs to be identified so that these dogs can be removed from the breeding pool to reduce the incidence of offspring with diabetes.

[0010] Thus, the present disclosure provides a method of screening a dog for a predisposition to diabetes, the method comprising determining whether the genotype of the dog's KCNJ11 gene includes the D274N allele. - a method for determining the likelihood that a dog will produce offspring genetically predisposed to diabetes, the method comprising the step of determining whether the genotype of the dog's KCNJ11 gene comprises the D274N allele; - a method for selecting a therapy for diabetes in a dog, the method comprising determining whether the genotype of the KCNJ11 gene of the dog includes the D274N allele, and selecting the therapy based on the determined genotype; - a method for preventing, delaying or treating diabetes in a dog whose KCNJ11 gene contains one or more D274N alleles, said method comprising administering to said dog an oral hypoglycemic agent; - a composition comprising an oral hypoglycemic agent for use in a method for preventing, delaying or treating diabetes in a dog whose KCNJ11 gene comprises one or more D274N alleles, said method comprising administering to said dog an oral hypoglycemic agent; - a method for preventing or delaying diabetes in a dog whose KCNJ11 gene contains one or more D274N alleles, the method comprising the steps of: (a) providing an antihyperglycemic diet to the dog; and / or (b) neutering the dog, wherein the dog is female and the neutering occurs before the dog's first estrus cycle; - An antihyperglycemic diet for use in a method for preventing or delaying diabetes in a dog whose KCNJ11 gene contains one or more D274N alleles. [Brief explanation of the drawings]

[0011] [Figure 1] Association of KCNJ11 rs851344999 with diabetes in UK Labrador Retrievers. (A) Choropleth map of a sample of diabetic Labrador Retrievers (representative from 33 of the 41 UK regions) submitted to the study by contacting UK veterinary practices (left). Donut plot showing study samples by phenotype and sex; M = male, F = female, NA = not applicable (right). (B) Stacked bar plot showing the association of KCNJ11 rs851344999 genotype with diabetes in Labrador Retrievers; χ2 p = 0.004838. [Figure 2-1] Functional studies of the D274N mutation. (A) Representative recordings of whole-cell currents measured in oocytes expressing wild-type (WT) or homomeric D274N mutant (D274N) KATP channels in control solution, after the addition of 3 mM Na azide, and then after the addition of 0.5 mM tolbutamide (Tolb). [Figure 2-2](B) Current amplitudes of WT and D274N recorded in control solution (Ctrl: ●, ○), 3 mM Na azide (Az: ▲, △), and 3 mM Na azide + 0.5 mM tolbutamide (Tb: filled squares, □). Mean ± SEM and individual data points. (C) Whole-cell currents in control solution minus tolbutamide-blocked currents for WT (●, n = 10) and D274N (○, n = 17) channels. ****P < 0.0001 (Student's t-test). Mean ± SEM and individual data points. (D) Average tolbutamide block of WT (light green bar, n = 10) and D274N (dark green bar, n = 17) KATP channels, calculated as the percent block of azide-induced currents (ns: not significant, Student's t-test). For comparison, the mean tolbutamide block for 19 human PNDM mutations is shown (white bars). The horizontal gray bars indicate the level of tolbutamide block that separates sulfonylurea treatment responders from sulfonylurea treatment non-responders in human patients with activating KCNJ11 mutations. [Figure 3-1] The D274N mutation reduces ATP inhibition of KATP channels. (A) Representative recordings of wild-type (A, WT) KATP currents from inside-out patches excised from transfected HEK cells exposed to different [ATP]s. Holding potential: -60 mV. The dashed line indicates the zero current level. (B) Representative recordings of homomeric D274N mutant (B, D274N) KATP currents from inside-out patches excised from transfected HEK cells exposed to different [ATP]s. Holding potential: -60 mV. The dashed line indicates the zero current level. [Figure 3-2](C) Dose-response relationship for ATP inhibition of WT (filled squares, ●) or D274N (□, ○) KATP channels in the absence of Mg2+. The KATP current (I) in the presence of nucleotide is expressed as a fraction of the KATP current in the absence of nucleotide (IC, control). The line is a best fit of the Hill equation to the average data (C; WT, IC50 = 10.2 μmol / L, h = 1.2, n = 7; D274N, IC50 = 15.4 μmol / L, h = 1.3, n = 5). (D) Dose-response relationship for ATP inhibition of WT (filled squares, ●) or D274N (□, ○) KATP channels in the presence of Mg2+. The KATP current (I) in the presence of nucleotide is expressed as a fraction of the KATP current in the absence of nucleotide (IC, control). The line is a best fit of the Hill equation to the average data (D: WT, IC50 = 18.6 μmol / l, h = 1.1, n = 8; D274N, IC50 = 29.0 μmol / l, h = 1.0, n = 7). [Figure 3-3] (E) IC50 values for current inhibition derived from Hill fits to individual dose-response relationships. Mean ± SEM and individual data points (E, WT, IC50 = 10 ± 1 μmol / L, h = 1.24 ± 0.05, n = 7; D274N, IC50 = 15 ± 1 μmol / L, h = 1.19 ± 0.06, n = 5), **P < 0.01 (Student's t-test). (F) IC50 values for current inhibition derived from Hill fits to individual dose-response relationships. Mean ± SEM and individual data points (F, WT, IC50 = 19 ± 2 μmol / L, h = 1.12 ± 0.04, n = 8; D274N, IC50 = 32 ± 2 μmol / L, h = 0.99 ± 0.03, n = 7), **P < 0.01 (Student's t-test). (G) Percentage of unblocked current at 3 mmol / l MgATP for WT (●) and D274N (○) KATP channels, ***P<0.001 (Student's t-test). [Figure 4-1]Predicted effect of the D274N mutation on the Kir6.2 protein and its interaction with SUR1. (A) Overall three-dimensional structure of the ATP-sensitive potassium (KATP) channel, composed of four pore-forming subunits (Kir6.2 - indigo, green, purple, and yellow) and four regulatory sulfonylurea receptor SUR ATP-binding cassette subunits (SUR1 - gray, teal, red, and pink). The location of D274 is indicated by a black box. (B) Location of the ATP-binding site relative to D274. [Figure 4-2] (C) The Kir6.2-SUR1 interface in wild-type D274 protein and mutant N274 protein. N274 does not affect the hydrogen bonds (black) or cation-Pi interactions (red) between H276 and H278 of Kir6.2 (green) and R1352, S1356, and S1357 of SUR1 (pink). (D) The Kir6.2-SUR1 interface in wild-type D274 protein and mutant N274 protein. N274 does not affect the hydrogen bonds (black) or cation-Pi interactions (red) between H276 and H278 of Kir6.2 (green) and R1352, S1356, and S1357 of SUR1 (pink). [Figure 4-3] (E) Close-up of D274, which forms one hydrogen bond with A271 and two with Q279. (F) Substitution with the mutant N274 residue is predicted to disrupt one of the two polar contacts with Q279 and the polar contact with A271. [Figure 5A] Association of KCNJ11 rs851344999 with diabetes in UK Labrador Retrievers. (A) Choropleth map with corresponding bar graph of Labrador Retrievers diagnosed with diabetes as adults at average age across the UK. [Figure 5B] (B) Correlation matrix (top) of age, weight, insulin dose, fructosamine, and HbA1c in Labrador Retrievers with adult-onset diabetes. Scatterplots (bottom) of statistically significant correlations: weight and insulin dose (R = 0.45, p = 9e-06); fructosamine and HbA1c (R = 0.59, p = 3.7e-10). [Figure 5C] (C) Stacked bar graph showing the significant difference in the proportion of neutered female KCNJ11 rs851344999 heterozygotes (CT) with diabetes compared to neutered male heterozygotes (Fisher's p=0.0293) (top), while the proportion of neutered female Labrador retrievers with diabetes that are homozygous (CC) for the reference allele of KCNJ11 rs851344999 is not significantly different from homozygous neutered males (Fisher's p=0.811) (bottom). [Figure 6] Histogram of percent Labrador (2 copy dogs; total study population). The histogram shows the percent of Labrador Retrievers among dogs identified by the Wisdom Panel as homozygous for rs851344999. [Figure 7] Histogram of Percent Labrador (1 copy dogs; total study population). The histogram shows the percent of Labrador Retrievers among dogs identified by the Wisdom Panel as heterozygous for rs851344999. [Figure 8] Histogram of Percent Labrador (1 copy dogs; subset). The histogram shows the percent of Labrador retrievers in the subset of dogs identified by the Wisdom Panel as heterozygous for rs851344999 and included in the Banfield Optimal Wellness Plan. [Figure 9] Histogram of Percent Labrador (2 Copy Dogs, Subset). The histogram shows the percent of Labrador Retrievers in the subset of dogs identified by the Wisdom Panel as homozygous for rs851344999 and included in the Banfield Optimal Wellness Plan. [Figure 10A]Ancestry information for two-copy dogs. This figure provides complete ancestry information for 10 representative dogs identified by the Wisdom Panel as homozygous for rs851344999 and included in the Banfield Optimal Wellness Plan. A) Plot showing selected populations. [Figure 10B] B) Ancestry table. In the table, (B) indicates both sides of the family tree. DETAILED DESCRIPTION OF THE INVENTION

[0012] Detailed Description It is to be understood that various applications of the disclosed methods and products may be adapted to meet particular needs in the art. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the disclosure only and is not intended to be limiting.

[0013] All publications, patents, and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0014] General definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0015] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to a "cell" includes "cell(s)," reference to an "antisense oligonucleotide" includes two or more such antisense oligonucleotides, and so forth.

[0016] In general, the term "comprising" is intended to mean inclusive, but not limited to. For example, the phrase "a composition comprising an oral hypoglycemic agent" should be interpreted to mean that the composition contains an oral hypoglycemic agent, but that the composition may contain additional nucleic acids.

[0017] In some embodiments of the present disclosure, the term "comprising" is replaced with the phrase "consisting of." The term "consisting of" is intended to be limiting. For example, the phrase "a composition consisting of an oral hypoglycemic agent" should be understood to mean that the composition contains an oral hypoglycemic agent and no additional ingredients.

[0018] Screening for predisposition to diabetes Disclosed herein is a method of screening a dog for a predisposition to diabetes, comprising determining whether the dog's genotype of the KCNJ11 gene comprises the D274N allele.

[0019] As explained above, it can be advantageous to determine whether a dog has a predisposition to diabetes. For example, knowledge that a dog has a predisposition to diabetes can facilitate monitoring for the onset of clinical disease. That is, dog owners and / or veterinarians can be alert to the onset of diabetes. This can allow for early diagnosis and treatment, thereby improving the dog's well-being. Early diagnosis and treatment can also improve clinical outcomes, as persistent hyperglycemia adversely affects beta cell function and / or number. For example, persistent hyperglycemia can impair insulin secretion, reduce insulin gene expression, and ultimately cause beta cell apoptosis. Loss of beta cells, or loss of beta cell function, can make the dog more dependent on insulin therapy and / or lead to more unstable disease.

[0020] Knowledge that a dog has a predisposition to diabetes may also allow preventative measures to be taken before the onset of clinical disease. For example, treatment can be initiated in the prediabetic state to minimize the pathophysiological changes that lead to clinical diabetes. Such therapy may, for example, aim to delay the onset of hyperglycemia, minimize the frequency of hyperglycemic episodes, and / or reduce the severity of hyperglycemia. As noted above, hyperglycemia adversely affects the number and function of beta cells. Such adverse effects may contribute to the onset of clinical diabetes. Therefore, therapy to combat hyperglycemia can prevent or delay the onset of clinical diabetes. Such therapy may include, for example, the administration of oral hypoglycemic agents (such as sulfonylureas) and / or dietary management.

[0021] Dog The method screens dogs for a predisposition to diabetes. The dog can be any individual dog of any breed. In one embodiment, the dog is a Labrador Retriever or has Labrador Retriever ancestry.

[0022] A dog with Labrador Retriever ancestry can be defined as a dog with Labrador Retriever bloodline. A dog with Labrador Retriever ancestry can have one or more alleles inherited from Labrador Retrievers. The one or more alleles can include an allele typically limited to Labrador Retrievers. The one or more alleles can include the D274N allele.

[0023] Dogs of Labrador Retriever ancestry can include other retriever breeds, such as flat-coated retrievers or golden retrievers. Dogs of Labrador Retriever ancestry can be, for example, hybrids of a Labrador Retriever with one or more other dog breeds. Labrador hybrids can include the Labradoodle (Labrador x Poodle), Springador (Labrador x Springer Spaniel), Goldador (Labrador x Golden Retriever), and Borador (Labrador x Border Collie).

[0024] The dog may be of any age. The dog may be, for example, an adult dog. The dog may be, for example, a young dog or a puppy.

[0025] The dog may be of any gender. For example, the dog may be a full (i.e., intact) female. The dog may be a full (i.e., intact) male. The dog may be a spayed female. The dog may be a neutered male.

[0026] Predisposing factors The purpose of this method is to identify a predisposition to diabetes. A predisposition to diabetes can be defined as an increased risk of developing diabetes. A dog with a predisposition to diabetes may have an increased risk of developing diabetes compared to the population average. That is, a dog with a predisposition to diabetes may be more likely to develop diabetes than other dogs. For example, a Labrador Retriever with a predisposition to diabetes may be more likely to develop diabetes than other Labrador Retrievers.

[0027] The predisposition factor that this method seeks to identify is a predisposition factor associated with the D274N mutation in the canine KCNJ11 gene. Therefore, the predisposition factor is a genetic predisposition factor. Therefore, this method screens dogs for a genetic susceptibility to diabetes. Other genetic predisposition factors to diabetes associated with other mutations and / or other genes may also exist.

[0028] diabetes Diabetes is a well-known disease in which blood glucose levels are chronically elevated as a result of insufficient insulin secretion or insufficient insulin sensitivity. Clinical signs of diabetes are associated with hyperglycemia and, in dogs, can typically include increased water drinking (polydipsia), increased urination (polyuria), and / or weight loss. Appetite may also increase or decrease. Canine diabetes may also be associated with the development of cataracts (i.e., clouding of the lens of the eye) and / or recurrent infections, such as urinary tract infections.

[0029] Canine diabetes can be diagnosed using the "ALIVE" criteria. Using the ALIVE criteria, diabetes can be diagnosed in the following patients: 1) Patients with random (fasting or non-fasting) blood glucose levels of 200 mg / dL (11.1 mmol / L) or greater who have classic clinical signs of hyperglycemia (without other plausible causes) or hyperglycemic crisis. In some cases, clinical signs may not have been reported by the owner. If the presence or absence of clinical signs is uncertain, the diagnosis can be confirmed by repeated blood glucose measurements and / or documentation of surrogate glycemic parameters such as increased glycated proteins and / or glycosuria. 2) Some patients with fasting blood glucose levels between 7mmol / L and 11mmol / L, with or without clinical signs of hyperglycemia or hyperglycemic crisis. Diabetes is distinguished from stress hyperglycemia by documented fasting hyperglycemia or elevated glycated proteins that persist for more than 24 hours.

[0030] Canine diabetes is typically type 1-like. That is, canine diabetes tends to be phenotypically similar to human type 1 diabetes (T1D). In humans, T1D is characterized by insulin dependence, pancreatic beta cell dysfunction, and / or pancreatic beta cell loss. Thus, the disclosed methods can screen for a predisposition to diabetes characterized by, for example, (i) insulin dependence, (ii) pancreatic beta cell dysfunction, and / or (iii) pancreatic beta cell loss. The disclosed methods can also screen for a predisposition to diabetes characterized by, for example, (i); (ii); (iii); (i) and (ii); (i) and (iii); (ii) and (iii); or (i), (ii), and (iii).

[0031] Genotyping To screen for a predisposition to diabetes, the method includes determining whether the genotype of the dog's KCNJ11 gene includes the D274N allele.

[0032] The canine KCNJ11 gene is a known gene with NCBI gene ID 485401. The canine KCNJ11 gene encodes the protein Kir6.2. The amino acid sequence of canine Kir6.2 is represented by SEQ ID NO: 1.

[0033] The D274N allele of canine KCNJ11 encodes a mutant Kir6.2 protein in which aspartic acid (D) at position 274 is replaced by asparagine (N). The location of this substitution is indicated by underlining in SEQ ID NO: 1. The D274N allele may result from a single nucleotide polymorphism resulting in a thymine (T) instead of a cytosine (C) found in the canine reference genome Canis Familiaris 3.1. In other words, the D274N allele may result from a C>T polymorphism in the canine KCNJ11 gene. The C>T polymorphism may be a polymorphism known as rs851344999. SEQ ID NO: 2 shows the primary transcript of KCNJ11 (KCNJ11-201) containing rs851344999 (see underlined position 820). The nucleotide "N" at position 820 of SEQ ID NO:2 can be G (the complement of the C nucleotide found at the corresponding position in the canine reference genome Canis Familiaris 3.1) or A (the complement of the T nucleotide found at the corresponding position in the D274N allele of canine KCNJ11). For completeness, SEQ ID NO:2 contains a number of additional "N" nucleotides at positions 357, 486, and 930, which can be either C or T, respectively, although these polymorphisms are not the subject of this disclosure.

[0034] To determine whether the genotype of dog's KCNJ11 gene contains D274N allele, the genotype of KCNJ11 gene can be determined.The method for determining genotype is well known in the art.Can use any known genotyping method to determine whether the genotype of dog's KCNJ11 gene contains D274N allele.

[0035] Typically, the genotype of the KCNJ11 gene is determined by analyzing a sample obtained from the dog. The analysis can be performed in vitro. The sample can be any sample containing genetic material from the dog. The sample can, for example, contain cells from the dog. In one embodiment, the sample is a blood sample. In another embodiment, the sample is a tissue sample. Preferably, the tissue sample is a sample that can be obtained non-invasively. The tissue sample can, for example, contain hair, skin cells, or mucosal cells. For example, the tissue sample can be a buccal swab.

[0036] The genotype of the KCNJ11 gene can be determined, for example, by genome sequencing. In this case, the sequence of all or part of the dog's genome is determined. The determined sequence can then be examined to determine whether the KCNJ11 gene contains the D274N allele. Numerous sequencing methods are known in the art and can be used in the methods of the present disclosure. Sequencing can be, for example, Sanger sequencing. Sequencing can be, for example, next-generation sequencing (NGS; e.g., Illumina). Next-generation sequencing can also be known as high-throughput sequencing. Sequencing can be, for example, long-read sequencing. Examples of long-read sequencing technologies include nanopore sequencing and single-molecule real-time sequencing (SMRT; e.g., PacBio). Sequencing can be, for example, DNA nanoball sequencing (e.g., DNBSEQ).

[0037] The genotype of the KCNJ11 gene can be determined, for example, by single nucleotide polymorphism (SNP) genotyping. In the context of the present disclosure, SNP genotyping refers to a method of distinguishing alleles that differ by a single base substitution without the need to sequence the alleles. SNP genotyping methods are known in the art and include 5'-nuclease allele discrimination assays (e.g., TaqMan assays), restriction fragment length polymorphism analysis, allele-specific PCR (also known as KASP), and SNP arrays (a type of DNA microarray).

[0038] Genotyping of the canine KCNJ11 gene can determine the following: (1) the canine KCNJ11 gene contains two D274N alleles (i.e., the gene / dog is homozygous for the D274N allele); (2) the canine KCNJ11 gene contains one D274N allele and one reference allele (i.e., the gene / dog is heterozygous for the D274N allele); or (3) the canine KCNJ11 gene contains two reference alleles (i.e., the gene / dog is homozygous for the reference allele). The reference allele is a KCNJ11 allele that encodes wild-type canine Kir6.2 that does not contain the D274N mutation. The reference allele can encode canine Kir6.2 of SEQ ID NO: 1. The reference allele does not contain the C>T polymorphism in the canine KCNJ11 gene that encodes the D274N mutation in canine Kir6.2.

[0039] Genotype-predisposition associations The genotype of dog KCNJ11 gene can be used to determine the predisposition to diabetes.As shown in the example, there is a significant association between rs851344999 genotype and canine diabetes.Therefore, the dog whose KCNJ11 gene contains one or more D274N alleles can have a predisposition to diabetes.

[0040] More specifically, the example demonstrates that the D274N mutation of KCNJ11 has a single gene recessive effect on diabetes phenotype.Therefore, the KCNJ11 genotype that is homozygous for the D274N allele can indicate that the dog has a predisposition to diabetes.In other words, the dog whose KCNJ11 contains two D274N alleles may have a predisposition to diabetes.Therefore, the method can include determining that the genotype of the KCNJ11 gene of the dog contains two D274N alleles, and thereby concluding that the dog has a predisposition to diabetes.

[0041] Homozygosity of the D274N allele may be sufficient to confer a predisposition to diabetes. That is, in the absence of other diabetogenic factors, diabetes may occur in dogs homozygous for the D274N allele. Homozygosity of the D274N allele, for example, may cause diabetes. The rationale for the causal relationship is explained in detail in the Examples. Briefly, KCNJ11 encodes the protein Kir6.2. As mentioned above, Kir6.2 encodes the ATP-sensitive potassium (K ATP ) channel pore-forming subunit. In pancreatic β cells, glucose uptake generates ATP, which is then converted into K ATP Close the channel. This K ATP ATP-mediated inhibition of the channel leads to membrane depolarization, electrical activity, calcium influx, and insulin secretion. ATP The channel is less sensitive to ATP-mediated inhibition. Accordingly, insulin secretion is impaired in β-cells from dogs homozygous for the D274N allele of KCNJ11.

[0042] A KCNJ11 genotype that is heterozygous for the D274N allele may indicate that the dog has a predisposition to diabetes. In other words, a dog whose KCNJ11 gene contains one D274N allele and one reference allele may be predisposed to diabetes. Thus, the method may include determining that the genotype of the dog's KCNJ11 gene contains one D274N allele, and thereby concluding that the dog has a predisposition to diabetes. The method may include determining that the genotype of the dog's KCNJ11 gene contains one D274N allele and one reference allele, and thereby concluding that the dog has a predisposition to diabetes. As described above, the reference allele is a KCNJ11 allele that encodes wild-type canine Kir6.2 that does not contain the D274N mutation. The reference allele may encode the canine Kir6.2 of SEQ ID NO: 1. The reference allele does not include the C>T polymorphism of the canine KCNJ11 gene that encodes the D274N mutation in canine Kir6.2.

[0043] Heterozygosity for the D274N allele can only lead to diabetes in the presence of other diabetogenic factors. That is, heterozygosity for the D274N allele alone may not be sufficient to cause diabetes. Rather, additional diabetogenic factors may be required to cause (i.e., lead to the onset of) diabetes in dogs heterozygous for the D274N allele. Thus, a KCNJ11 genotype heterozygous for the D274N allele may indicate that the dog has a predisposition to diabetes when combined with one or more other diabetogenic factors. Thus, the method may include determining that the genotype of the dog's KCNJ11 gene contains a D274N allele, and thereby concluding that the dog has a predisposition to diabetes when exposed to one or more other diabetogenic factors. The method may include determining that the genotype of the dog's KCNJ11 gene comprises one D274N allele and one reference allele, and thereby concluding that the dog has a predisposition to diabetes when exposed to one or more other diabetogenic factors.

[0044] In the context of the present disclosure, a diabetogenic factor is a factor that contributes to the onset of diabetes. Many diabetogenic factors are known in the art. For example, female gender is known to contribute to the onset of diabetes. In particular, the diestrus phase of the canine estrous cycle can result in pancreatic β-cell stress due to the levels of mammary gland-derived growth hormone and luteal progesterone. Therefore, heterozygosity for the D274N allele may predispose female dogs to diabetes. This is demonstrated in the examples showing that a higher proportion of heterozygous females was present in the diabetic group than in the control group.

[0045] Heterozygosity for the D274N allele may also predispose dogs to diabetes in dogs that are exposed to one or more other diabetogenic factors.Other diabetogenic factors may include, for example, obesity.Therefore, heterozygosity for the D274N allele may predispose obese dogs to diabetes.

[0046] Other diabetogenic factors may include, for example, medical conditions. For example, pancreatitis or pancreatic stress may be diabetogenic factors. Therefore, heterozygosity for the D274N allele may predispose dogs with pancreatitis or pancreatic stress or a history of pancreatitis or pancreatic stress to diabetes. Endocrine diseases may also be diabetogenic factors. Therefore, heterozygosity for the D274N allele may predispose dogs with endocrine diseases or a history of endocrine diseases to diabetes. In some cases, the endocrine disease is Cushing's disease. Cushing's disease is a condition associated with high levels of blood cortisol, which can increase blood glucose levels and thus cause diabetes. Viral diseases may be an additional diabetogenic factor. Therefore, heterozygosity for the D274N allele may predispose dogs with viral diseases or a history of viral diseases to diabetes.

[0047] Other diabetogenic factors include, for example, treatment with certain drugs. For example, corticosteroid treatment increases blood glucose levels and can thus induce diabetes (in a manner similar to Cushing's disease). Therefore, heterozygosity for the D274N allele may predispose dogs that have been treated with corticosteroids or that have a history of corticosteroid treatment to diabetes. Progestogens may cause pancreatic beta cell stress (e.g., by impairing insulin release and / or insulin sensitivity), thus inducing diabetes similar to diestrus luteal progesterone. Therefore, heterozygosity for the D274N allele may predispose dogs that have been treated with progestogens or that have a history of progestogen treatment to diabetes.

[0048] Other diabetogenic factors can also include, for example, modifier genetic factors. For example, the presence of a modifier locus can cause diabetes in dogs heterozygous for the D274N allele. In other words, heterozygosity for the D274N allele can confer a predisposition to diabetes in dogs with a particular genetic landscape or combination of traits.

[0049] Thus, a KCNJ11 genotype heterozygous for the D274N allele may indicate that the dog has a predisposition to diabetes if the dog is exposed to one or more other diabetogenic factors, which may be selected from female sex, obesity, pancreatitis, pancreatic stress, progestogen treatment, corticosteroid treatment, endocrine disease (e.g., Cushing's disease), viral disease, and modifying genetic factors, alone or in any combination.

[0050] In one embodiment of the present disclosure, it may be determined that the D274N allele is not present in the genotype of the canine KCNJ11 gene. That is, the method can determine that the genotype of the canine KCNJ11 gene does not contain the D274N allele. In other words, the method can determine that the genotype of the canine KCNJ11 gene contains two reference alleles, that is, the genotype of the canine KCNJ11 gene is homozygous for the reference alleles. As described above, the reference allele is the KCNJ11 allele that encodes wild-type canine Kir6.2 that does not contain the D274N mutation. The reference allele may encode the canine Kir6.2 of SEQ ID NO: 1. The reference allele does not contain the C>T polymorphism of the canine KCNJ11 gene that encodes the D274N mutation in canine Kir6.2.

[0051] A KCNJ11 genotype that is homozygous for the reference allele may indicate that the dog does not have a predisposition to diabetes associated with mutations in the KCNJ11 gene. Thus, the method may include determining that the genotype of the dog's KCNJ11 gene contains two reference alleles, and thereby concluding that the dog does not have a predisposition to KCNJ11-related diabetes. However, a dog whose KCNJ11 genotype is homozygous for the reference allele may subsequently develop diabetes. A dog whose KCNJ11 genotype is homozygous for the reference allele may develop diabetes, for example, as a result of environmental factors. A dog whose KCNJ11 genotype is homozygous for the reference allele may have, for example, one or more predispositions to diabetes that are not associated with KCNJ11. One or more other predispositions may be associated with mutations (e.g., SNPs) in one or more genes other than KCNJ11.

[0052] Determining the likelihood of producing predisposed offspring Disclosed herein is a method for determining the likelihood that a dog will produce offspring with a genetic predisposition to diabetes, the method comprising determining whether the dog's genotype of the KCNJ11 gene contains the D274N allele. Determining the likelihood that a dog will produce offspring with a genetic predisposition to diabetes is advantageous because it allows dogs with such a likelihood to be excluded from breeding programs. In this way, the incidence of offspring with diabetes can be reduced.

[0053] Dog The method screens dogs for the likelihood of producing offspring that are genetically predisposed to diabetes. The dog can be any individual dog of any breed, age, or sex. Any of the exemplary breeds, ages, and sexes listed above in connection with the method of screening dogs for a predisposition to diabetes can also be applied to the method of determining the likelihood that a dog will produce offspring that are genetically predisposed to diabetes.

[0054] Predisposition to diabetes The purpose of the method is to identify the likelihood that a dog will produce offspring that are genetically predisposed to diabetes. Predisposition and diabetes are described above in connection with the method of screening dogs for a predisposition to diabetes. Any of the above characteristics or definitions may also apply to the method of determining the likelihood that a dog will produce offspring that are genetically predisposed to diabetes.

[0055] Genotyping To determine the likelihood that a dog will produce offspring genetically predisposed to diabetes, the method includes determining whether the genotype of the dog's KCNJ11 gene contains the D274N allele. To determine whether the genotype of the dog's KCNJ11 gene contains the D274N allele, the genotype of the KCNJ11 gene can be determined. The D274N allele, the corresponding KCNJ11 reference allele, and means for determining the genotype of the KCNJ11 gene are described above in connection with the method of screening dogs for a predisposition to diabetes. Any of the above characteristics or definitions may also apply to the method of determining the likelihood that a dog will produce offspring genetically predisposed to diabetes.

[0056] Associations between genotype and genetic potential Genotyping of a dog's KCNJ11 gene can determine: (1) that the dog's KCNJ11 gene contains two D274N alleles (i.e., the dog / gene is homozygous for the D274N allele); (2) that the dog's KCNJ11 gene contains one D274N allele and one reference allele (i.e., the dog / gene is heterozygous for the D274N allele); or (3) that the dog's KCNJ11 gene contains two reference alleles (i.e., the dog / gene is homozygous for the reference allele).

[0057] A KCNJ11 genotype that is homozygous or heterozygous for the D274N allele (depending on (1) or (2)) may indicate that the dog has the potential to produce offspring that are genetically predisposed to diabetes. This is because the dog's genome contains one or more D274N allele(s), and therefore at least some of the dog's gametes contain the D274N allele of KCNJ11. Specifically, if a dog has a KCNJ11 genotype that is homozygous for the D274N allele, all of the dog's gametes will contain the D274N allele, and if the dog has a KCNJ11 genotype that is heterozygous for the D274N allele, approximately half of the dog's gametes will contain the D274N allele. Therefore, the offspring of the dog may inherit the D274N allele. As explained above and shown in Examples, dogs whose KCNJ11 gene contains at least one D274N allele may be predisposed to diabetes.Specifically, the D274N mutation of KCNJ11 can be the single gene cause of diabetes in dogs whose KCNJ11 gene contains two D274N alleles (i.e., in dogs that are homozygous for the D274N allele).The D274N mutation of KCNJ11 can be the predisposing factor to diabetes in dogs whose KCNJ11 gene contains one D274N allele and one reference allele (i.e., in dogs that are heterozygous for the D274N allele), which are exposed to other diabetogenic factors.

[0058] A KCNJ11 genotype homozygous for the KCNJ11 reference allele (per (3)) may indicate that the dog does not have the genetic potential to produce offspring with a predisposition to diabetes associated with mutations in the KCNJ11 gene. This is because the dog's genome does not contain the D274N allele, and therefore the D274N allele is not present in the dog's gametes. Therefore, the offspring of the dog cannot inherit the D274N allele or the associated predisposition to diabetes described above and in the Examples. However, the offspring of the KCNJ11 dog may subsequently develop diabetes due to other factors, such as environmental factors and / or mutations (e.g., SNPs) in one or more genes other than KCNJ11.

[0059] Treatment Options for Diabetes Disclosed herein is a method for selecting a therapy for diabetes in a dog, the method comprising determining whether the genotype of the dog's KCNJ11 gene includes the D274N allele, and selecting a therapy based on the determined genotype.

[0060] Traditionally, canine diabetes has been treated with injectable insulin therapy, typically administered twice daily. Administration of insulin injections can be difficult for owners and poorly tolerated by dogs. Therefore, oral treatment for canine diabetes is desirable. Because human neonatal diabetes caused by KCNJ11 mutations is treated with oral hypoglycemic agents, diabetic dogs with the KCNJ11 D274N mutation can be similarly treated. The selection method advantageously determines whether a given diabetic dog has the D274N mutation and, therefore, whether the dog is likely to benefit from oral hypoglycemic drug treatment. By administering oral therapy to dogs likely to benefit from oral hypoglycemic drug treatment, owners avoid the inconvenience of insulin injections and dogs avoid the discomfort of insulin injections. Furthermore, treatment with oral hypoglycemic drugs, such as sulfonylureas, can reduce blood glucose fluctuations, lower HbA1c, and enhance meal-stimulated insulin secretion.

[0061] Dog The method selects a therapy for diabetes in a dog. In other words, the method selects a therapy for a dog with diabetes. The dog is a diabetic dog.

[0062] The dog can be any canine individual of any breed, age, or sex. Any of the exemplary breeds, ages, and sexes described above in connection with the method of screening dogs for a predisposition to diabetes can also apply to the method of selecting a therapy for diabetes in a dog.

[0063] diabetes The purpose of the method is to select a therapy for diabetes in a dog. Diabetes is described above in connection with the method of screening dogs for a predisposition to diabetes. Any of the above characteristics or definitions may also apply to the method of selecting a therapy for diabetes in a dog.

[0064] Genotyping To select a therapy for diabetes in a dog, the method includes determining whether the genotype of the KCNJ11 gene of the dog contains the D274N allele. To determine whether the genotype of the KCNJ11 gene of the dog contains the D274N allele, the genotype of the KCNJ11 gene can be determined. The D274N allele, the corresponding KCNJ11 reference allele, and means for determining the genotype of the KCNJ11 gene are described above in connection with the method for screening dogs for a predisposition to diabetes. Any of the above characteristics or definitions may also apply to the method for selecting a therapy for diabetes in a dog.

[0065] Treatment options A treatment for diabetes is selected based on the determined genotype of the KCNJ11 gene. In particular, a treatment is selected based on whether the genotype of the KCNJ11 gene includes one or more D274N alleles.

[0066] In the example, the D274N mutant K ATPThe channel is wild-type K ATP channel (i.e., K ATP In dogs with a KCNJ11 gene homozygous for the D274N allele, the K ATP Impaired insulin secretion associated with decreased channel sensitivity may lead to the development of diabetes. ATP Impaired insulin secretion associated with reduced channel sensitivity can lead to the development of diabetes even in dogs with a KCNJ11 gene heterozygous for the D274N allele, for example, if the dog is exposed to additional diabetogenic factors. ATP Countering impaired insulin secretion associated with decreased channel sensitivity may be of therapeutic benefit in dogs whose KCNJ11 gene contains one or more D274N alleles (i.e., either homozygous or heterozygous for the D274N allele). Oral hypoglycemic agents, such as K ATP It can be used to counteract insulin secretion disorders associated with decreased channel sensitivity. Therefore, oral hypoglycemic agents can be selected as a treatment when the genotype of the KCNJ11 gene includes one or more D274N alleles (i.e., when the KCNJ11 gene is homozygous or heterozygous for the D274N allele).

[0067] Oral hypoglycemic drugs include, for example, K ATP They can bind to and / or close the channel. Such binding and closure can stimulate insulin secretion by pancreatic beta cells. ATP Oral hypoglycemic drugs capable of binding to and closing the channel are known in the art, and include, for example, sulfonylureas. Examples include sulfonylureas that bind to wild-type K ATP Channel and D274N K ATPIt is confirmed that both channels can be blocked, achieving greater than 95% inhibition (see, e.g., FIG. 2D). Thus, the oral hypoglycemic agent can be a sulfonylurea. The sulfonylurea can be, for example, tolbutamide, glibenclamide, or glipizide.

[0068] The amount of oral hypoglycemic agent administered and frequency of administration will depend on the dog being treated. The precise amount of oral hypoglycemic agent administered and the administration regimen will depend on the judgment of the practitioner and may be particular to each subject.

[0069] When an oral hypoglycemic agent is selected as a treatment, the selected treatment may further include an injectable insulin. In other words, the selected treatment may include a combination of an oral hypoglycemic agent and an injectable insulin. Such a combination therapy can, for example, reduce fluctuations in blood glucose levels. The amount and frequency of administration of the injectable insulin to be administered will depend on the dog being treated. The exact amount and administration regimen of the injectable insulin to be administered can be determined by the judgment of the practitioner and may be specific to each subject.

[0070] If the KCNJ11 gene is homozygous for the reference allele (i.e., does not contain at least one D274N allele), the treatment of choice may include injectable insulin. Administering oral hypoglycemic agents to dogs whose KCNJ11 gene is homozygous for the reference allele may reduce the risk of developing KCNJ11. ATP Therefore, the administration of oral hypoglycemic drugs may not be appropriate because they may not reduce the sensitivity of the channel. ATP It may not have an effect on counteracting channel sensitivity and therefore not be able to stimulate insulin secretion, therefore insulin supplementation may be required.

[0071] Preventing, delaying or treating diabetes Disclosed herein is a method for preventing, delaying, or treating diabetes in a dog whose KCNJ11 gene contains one or more D274N alleles, the method comprising administering to the dog an oral hypoglycemic agent. Also disclosed herein is a composition comprising an oral hypoglycemic agent for use in a method for preventing, delaying, or treating diabetes in a dog whose KCNJ11 gene contains one or more D274N alleles, the method comprising administering to the dog an oral hypoglycemic agent.

[0072] Further disclosed herein is a method for preventing or delaying diabetes in a dog whose KCNJ11 gene contains one or more D274N alleles, the method comprising: (a) providing an antihyperglycemic diet to the dog; or (b) neutering the dog, wherein the dog is female. Also disclosed herein is an antihyperglycemic diet for use in the method for preventing or delaying diabetes in a dog whose KCNJ11 gene contains one or more D274N alleles.

[0073] As described above, human neonatal diabetes caused by KCNJ11 mutations is treated by administering oral hypoglycemic agents such as sulfonylureas. Examples of the present disclosure identify the D274N mutation in the canine KCNJ11 gene as a monogenic cause of canine diabetes. Examples also describe the D274N mutant KCNJ11. ATP The channel is wild-type K ATP channel (i.e., K encoded by the KCNJ11 reference allele) ATP The present study also demonstrates that dogs with KCNJ11 genes containing one or more D274N alleles are less sensitive to ATP-mediated inhibition than dogs with KCNJ11 genes containing one or more D274N alleles. The examples demonstrate that oral hypoglycemic drugs, such as tolbutamide (a sulfonylurea), inhibit the D274N mutant KCNJ11 channels. ATPThe results further demonstrate that oral hypoglycemic agents inhibit the channel by more than 95%. Therefore, oral hypoglycemic agents used to treat human neonatal diabetes, likely caused by KCNJ11 mutations, can also be used to treat diabetes associated with the D274N mutation in the canine KCNJ11 gene. Such oral treatments are advantageous compared to traditional twice-daily injectable insulin therapy because oral medications are easier to administer and may be better tolerated by dogs. Furthermore, oral hypoglycemic agent treatment can reduce blood glucose fluctuations, lower HbA1c, and enhance meal-stimulated insulin secretion. Thus, the present disclosure provides a valuable new treatment regimen for canine diabetes.

[0074] The present disclosure also provides a means for preventing or delaying diabetes in dogs whose KCNJ11 gene contains one or more D274N alleles. As explained above, the presence of one or more D274N alleles can indicate that the dog has a predisposition to diabetes. Therefore, by screening dogs for the presence of one or more D274N alleles, dogs at risk for developing diabetes can be identified. In these dogs, preventative measures can be implemented before the onset of clinical disease (i.e., in a prediabetic state) to delay or prevent the onset of diabetes. For example, measures can be taken to minimize the occurrence of hyperglycemia in dogs. Because hyperglycemia is damaging to insulin-producing pancreatic beta cells, minimizing the occurrence of hyperglycemia can help preserve beta cells and / or their ability to produce insulin. The occurrence of hyperglycemia can be minimized by (1) administering oral hypoglycemic agents, (2) providing an antihyperglycemic diet, and / or (3) spaying female dogs. The occurrence of hyperglycemia can be minimized by (1); (2); (3); (1) and (2); (1) and (3); (2) and (3); or (1), (2) and (3).

[0075] Dog The method includes administering an oral hypoglycemic agent to the dog to prevent, delay, or treat diabetes, or providing an antihyperglycemic diet to the dog to prevent or delay diabetes. The dog is a dog whose KCNJ11 gene contains one or more D274N alleles. The KCNJ11 gene can, for example, contain one D274N allele and one reference allele. In other words, the KCNJ11 gene can be heterozygous for the D274N allele. The KCNJ11 gene can, for example, contain two D274N alleles. In other words, the KCNJ11 gene can be homozygous for the D274N allele.

[0076] As described above, dogs whose KCNJ11 gene contains one or more D274N alleles may be predisposed to diabetes. Therefore, the above-mentioned dogs may be dogs predisposed to diabetes. A dog may be predisposed to diabetes based solely on its KCNJ11 genotype. For example, a KCNJ11 genotype that is homozygous for the D274N allele may be sufficient to confer a predisposition to diabetes. A dog may be predisposed to diabetes when its KCNJ11 genotype is combined with other diabetogenic factors such as those described above. For example, a KCNJ11 genotype that is heterozygous for the D274N allele may result in a predisposition to diabetes when combined with other diabetogenic factors such as female sex, obesity, pancreatitis, pancreatic stress, progestogen treatment, corticosteroid treatment, endocrine disease, viral disease, or modifying genetic factors.

[0077] A dog with a predisposition to diabetes may not have (or may not yet have) diabetes. In this case, the method provides prophylactic treatment for the onset of diabetes. That is, the method prevents or delays the onset of diabetes. A dog with a predisposition to diabetes but not diabetes may be in a prediabetic state. Thus, a dog treated prophylactically may be in a prediabetic state. A dog treated prophylactically may have prediabetes. A dog in a prediabetic state or with prediabetes may have impaired glucose tolerance, which may result in inappropriately high blood glucose levels after a meal or after ingestion or injection of glucose. A fasting blood glucose level of 7 mmol / L to 11 mmol / L, for example, may indicate prediabetes in a dog. Treating a dog in a prediabetic state can halt the progression of the prediabetic state to clinical diabetes or symptomatic diabetes. Treating a dog in a prediabetic state can improve glucose tolerance, for example, so that blood glucose is not inappropriately high after a meal or after ingestion or injection of glucose. Treatment of pre-diabetic dogs can, for example, restore fasting blood glucose to below 7 mmol / l.

[0078] A dog whose KCNJ11 gene contains one or more D274N alleles may be a diabetic dog. In other words, a dog whose KCNJ11 gene contains one or more D274N alleles may have diabetes. That is, the dog may already have clinical disease (rather than simply having a predisposition to diabetes). In this case, the method provides a therapeutic treatment for diabetes. That is, the method treats diabetes. Treating diabetes alleviates the above-mentioned clinical signs of diabetes. Clinical signs usually become apparent only when blood glucose levels exceed the renal threshold for glucose reabsorption (about 12 mmol / L). Therefore, treating diabetes can restore blood glucose levels to 12 mmol / L or less.

[0079] The dog can be any canine individual of any breed, age, or sex. Any of the exemplary breeds, ages, and sexes described above in connection with the method of screening dogs for a predisposition to diabetes can also apply to the method of selecting a therapy for diabetes in a dog.

[0080] diabetes The purpose of the method is to prevent, delay, or treat diabetes in dogs whose KCNJ11 gene contains one or more D274N alleles. Diabetes is described above in connection with the method of screening dogs for a predisposition to diabetes. Any of the above characteristics or definitions may also apply to aspects of the disclosure relating to preventing, delaying, or treating diabetes.

[0081] Oral antihyperglycemic drugs The method may include administering an oral hypoglycemic agent to the dog to prevent, delay, or treat diabetes. The oral hypoglycemic agent may be any drug that can be administered orally to reduce blood glucose levels. Oral hypoglycemic agents include, for example, K ATP They can bind to and / or close the channel. Such binding and closure can stimulate insulin secretion by pancreatic beta cells, thereby lowering blood glucose levels. ATP Oral hypoglycemic agents capable of binding to and closing the channel are known in the art, and include, for example, sulfonylureas. Thus, the oral hypoglycemic agent can be a sulfonylurea. The sulfonylurea can be, for example, tolbutamide, glibenclamide, or glipizide.

[0082] The amount and frequency of administration of oral hypoglycemic agent will depend on the dog being treated. The exact amount of oral hypoglycemic agent administered and the administration regimen will depend on the judgment of the practitioner and may be specific to each subject. Typically, oral hypoglycemic agent is administered once a day or twice a day.

[0083] The administration of oral hypoglycemic drugs can prevent diabetes. In other words, the administration of oral hypoglycemic drugs can prevent the onset of diabetes in dogs. In this way, dogs with a predisposition to diabetes can be permanently protected from clinical disease.

[0084] Administration of oral hypoglycemic agents can delay diabetes. In other words, administration of oral hypoglycemic agents can extend the time until the onset of diabetes. In this way, dogs with a predisposition to diabetes can achieve a longer clinical disease-free period. The onset of diabetes can be delayed, for example, by 1 month or more, e.g., 2 months or more, 3 months or more, 4 months or more, 5 months or more, 6 months or more, 7 months or more, 8 months or more, 9 months or more, 10 months or more, 11 months or more, or 12 months or more. The onset of diabetes can be delayed, for example, by 1 to 12 months, 2 to 11 months, 3 to 10 months, 4 to 9 months, 5 to 8 months, or 6 to 7 months. The onset of diabetes can be delayed, for example, by 1 year or more, e.g., 2 years or more, 3 years or more, 4 years or more, 5 years or more, 6 years or more, 7 years or more, 8 years or more, 9 years or more, 10 years or more, 11 years or more, 12 years or more, 13 years or more, 14 years or more, or 15 years or more. The onset of diabetes can be delayed, for example, by 1 to 15 years, 2 to 14 years, 3 to 13 years, 4 to 12 years, 5 to 11 years, 6 to 10 years, or 7 to 9 years.

[0085] Administration of oral hypoglycemic agents can treat diabetes. In other words, administration of oral hypoglycemic agents can alleviate the signs or symptoms of diabetes in clinically ill dogs. Typically, administration of oral hypoglycemic agents reduces blood glucose levels in dogs.

[0086] The method may further include providing one or more other therapies to the dog. For example, the method may include providing an antihyperglycemic diet to the dog. Antihyperglycemic diets are described in more detail below. The method may further include administering injectable insulin to the dog. The method may further include providing an antihyperglycemic diet to the dog and administering injectable insulin to the dog. Typically, injectable insulin therapy is only used in dogs that already have diabetes (as opposed to dogs predisposed to diabetes), and in such dogs, injectable insulin therapy provides a therapeutic effect. Combination therapy with oral hypoglycemic agents and injectable insulin can, for example, reduce blood glucose fluctuations. The amount and frequency of injectable insulin administered will depend on the dog being treated. The exact amount of injectable insulin administered and the administration regimen can be determined by the practitioner's judgment and may be specific to each subject.

[0087] The method may further include providing one or more other therapies to the dog. For example, the method may include (i) providing an antihyperglycemic diet to the dog, and / or (ii) neutering the dog if the dog is female. Antihyperglycemic diets and neutering are described below.

[0088] Administration of antihyperglycemic diet The method may include providing the dog with an antihyperglycemic diet to prevent or delay diabetes. The nature of KCNJ11-associated diabetes in dogs is such that dietary management alone is unlikely to be successful. However, dietary management of dogs predisposed to KCNJ11-associated diabetes (i.e., dogs whose KCNJ11 gene contains one or more D274N alleles) can reduce progression to clinical disease.

[0089] An antihyperglycemic diet can be defined as a diet that minimizes the rise in blood glucose levels after a meal. In other words, an antihyperglycemic diet can stabilize blood glucose levels or limit spikes and drops in blood glucose levels. Antihyperglycemic diets are known in the art and can be formulated, for example, to have a low glycemic index (GI). An antihyperglycemic diet can be, for example, (a) low in carbohydrates, (b) high in protein, and / or (c) high in soluble fiber. An antihyperglycemic diet can be, for example, (a); (b); (c); (a) and (b); (a) and (c); (b) and (c); or (a), (b) and (c). An antihyperglycemic diet can include complex carbohydrates. An antihyperglycemic diet can be deficient (or substantially deficient) in simple carbohydrates. An antihyperglycemic diet can include more complex carbohydrates than simple carbohydrates.

[0090] The antihyperglycemic diet can be, for example, a complete dog food. A complete dog food is a food that provides all nutrients in the amounts and proportions required by a dog. A dog fed a complete dog food does not need to be fed any other food to meet its nutritional requirements. Therefore, the antihyperglycemic diet can be formulated to be fed to a dog as an exclusive diet. The antihyperglycemic diet can be a dry dog food or a wet dog food.

[0091] Providing an antihyperglycemic diet can prevent diabetes. In other words, providing an antihyperglycemic diet can prevent the onset of diabetes in dogs. In this way, dogs with a predisposition to diabetes can be permanently protected from clinical disease.

[0092] Providing an antihyperglycemic diet can delay diabetes. In other words, providing an antihyperglycemic diet can extend the time until diabetes onset. In this way, dogs with a predisposition to diabetes can achieve a longer clinical disease-free period. The onset of diabetes can be delayed, for example, by 1 month or more, e.g., 2 months or more, 3 months or more, 4 months or more, 5 months or more, 6 months or more, 7 months or more, 8 months or more, 9 months or more, 10 months or more, 11 months or more, or 12 months or more. The onset of diabetes can be delayed, for example, by 1 to 12 months, 2 to 11 months, 3 to 10 months, 4 to 9 months, 5 to 8 months, or 6 to 7 months. The onset of diabetes can be delayed, for example, by 1 year or more, e.g., 2 years or more, 3 years or more, 4 years or more, 5 years or more, 6 years or more, 7 years or more, 8 years or more, 9 years or more, 10 years or more, 11 years or more, 12 years or more, 13 years or more, 14 years or more, or 15 years or more. The onset of diabetes can be delayed, for example, by 1 to 15 years, 2 to 14 years, 3 to 13 years, 4 to 12 years, 5 to 11 years, 6 to 10 years, or 7 to 9 years.

[0093] The method may further include providing one or more other therapies to the dog. For example, the method may include (i) providing the dog with an oral hypoglycemic agent, and / or (ii) if the dog is female, spaying the dog before its first estrous cycle. Suitable oral hypoglycemic agents are described above. The amount and frequency of administration of the oral hypoglycemic agent will depend on the dog being treated. The exact amount and administration regimen of the oral hypoglycemic agent administered will depend on the judgment of the practitioner and may be specific to each subject. The step of spaying before its first estrous cycle is described below.

[0094] Spaying before the first heat cycle If the dog is female, the method may include neutering the dog to prevent or delay diabetes. Estrus can cause hyperglycemia due to the insulin-antagonizing effects of progesterone and growth hormone during the diestrus phase. Neutering minimizes the effects of these hormones, thus preventing hyperglycemia.

[0095] Spaying a female dog may refer to an ovariohysterectomy or ovariectomy. Thus, the method may include performing an ovariohysterectomy or ovariectomy on the dog to prevent or delay diabetes.

[0096] The effect of spaying on minimizing the above-mentioned hormonal influences may be greater the earlier the spaying is performed. Therefore, spaying is preferably performed before the dog's first estrous cycle. Dogs typically have their first estrous cycle at 6 to 12 months of age, and the signs of estrus are well recognized. Therefore, for dogs with a known medical history, determining whether the dog has had its first estrous cycle should be within the routine skill set of a person skilled in the art. If the dog's medical history is unknown (e.g., if the dog is a rescue dog), spaying is preferably performed at the earliest practical opportunity. Spaying can also be performed at the earliest practical opportunity if the first estrous cycle has already passed, for example, if the dog did not show signs until after its first estrous cycle, or if there was a medical reason why spaying before the first estrous cycle was not possible.

[0097] Neutering can prevent diabetes. In other words, spaying can stop the onset of diabetes in dogs. In this way, dogs with a predisposition to diabetes can be permanently protected from clinical disease.

[0098] Spaying can delay diabetes. In other words, spaying can extend the time until diabetes develops. In this way, dogs with a predisposition to diabetes can achieve a longer clinical disease-free period. The onset of diabetes can be delayed, for example, by one month or more, e.g., two months or more, three months or more, four months or more, five months or more, six months or more, seven months or more, eight months or more, nine months or more, ten months or more, eleven months or more, or twelve months or more. The onset of diabetes can be delayed, for example, by one to twelve months, two to eleven months, three to ten months, four to nine months, five to eight months, or six to seven months. The onset of diabetes can be delayed, for example, by one year or more, e.g., two years or more, three years or more, four years or more, five years or more, six years or more, seven years or more, eight years or more, nine years or more, ten years or more, eleven years or more, 12 years or more, 13 years or more, 14 years or more, or 15 years or more. The onset of diabetes can be delayed, for example, by 1 to 15 years, 2 to 14 years, 3 to 13 years, 4 to 12 years, 5 to 11 years, 6 to 10 years, or 7 to 9 years.

[0099] The method may further include providing one or more other therapies to the dog. For example, the method may include providing the dog with (i) an oral hypoglycemic agent and / or (ii) an antihyperglycemic diet. Suitable oral hypoglycemic agents are described above. The amount and frequency of administration of the oral hypoglycemic agent will depend on the dog being treated. The exact amount and administration regimen of the oral hypoglycemic agent administered will depend on the judgment of the practitioner and may be specific to each subject. Antihyperglycemic diets are also described above.

[0100] The following examples illustrate the invention. [Example]

[0101] Example 1 During exome sequencing of four Labrador retrievers that developed diabetes before 6 months of age and one dog that developed diabetes as an adult, a homozygous nonsynonymous variant in KCNJ11, rs851344999, was identified in two of the diabetic individuals. The presence of the alternative T allele was predicted to cause a D274N amino acid change in the canine Kir.2 protein. Subsequent genotyping of an additional 117 diabetic Labrador retrievers and Labrador retriever crossbreds from the UK Canine Diabetes Database and Archive using various techniques identified an additional five diabetic individuals homozygous for the same mutation and 28 heterozygous individuals (Figure 1).

[0102] Genotyping of a total of 200 adult Labrador retrievers with no reported clinical signs of diabetes identified only one homozygous dog and 56 heterozygous dogs for the D274N mutation. The association between the rs851344999 genotype and diabetes in Labrador retrievers was significant and likely due to recessive monogenic inheritance (χ 2 p=0.004838). Clinical records suggested that the homozygous control dog had suffered from pancreatitis in adulthood but not diabetes mellitus before euthanasia at age 10 years, although blood glucose measurements were not recorded at that time. The finding of one control dog homozygous for a diabetes-associated variant with no reported diabetic phenotype at the time of sampling could be the result of very late onset or unrecognized diabetes in this patient.

[0103] Samples from juvenile-onset diabetic dogs in this study (n = 13) were submitted primarily by veterinary practices clustered in Herefordshire, Worcestershire, Warwickshire, and the West Midlands. Where signalment and clinical metadata were available for diabetic dogs, there was no association between the presence or absence of the D274N mutation and age at onset of diabetes. Among diabetic Labrador retrievers, the proportion of spayed females that were homozygous (CC) for the reference allele of KCNJ11 rs851344999 was not significantly different from the proportion of homozygous neutered males. However, there was a significant difference in the proportion of neutered female KCNJ11 rs851344999 heterozygotes (CT) compared to neutered male heterozygotes in the diabetic Labrador retriever population (Fisher's p = 0.0293) ( Figure 5 ).

[0104] Significant association between rs851344999 genotype and diabetes in Labrador Retrievers (χ 2 Considering the significant difference in the D274N mutation (p = 0.004838) and the potential monogenic recessive effects of the variant on diabetic phenotype and β-cell function, we performed a functional study of the D274N mutation. First, we demonstrated that the Kir6.2-D274N mutation inhibits Kβ-cell function by measuring whole-cell currents in Xenopus oocytes. ATP We investigated the effect of the channel on metabolic regulation. In control solution, wild-type (WT) channels reacted with adenosine triphosphate (ATP) i ), but this channel is blocked by the metabolic inhibitor Na azide, which inhibits [ATP]. i The homozygous Kir6.2-D274N / SUR1 current (D274N) was similarly activated. ATPThe channel inhibitor tolbutamide (0.5 mM, also used as an oral sulfonylurea hypoglycemic agent) blocked both the WT and D274N channels by >95% (Figure 2D). WT currents were comparable in control and tolbutamide solutions, indicating that resting [ATP] in oocytes was significantly higher than that in control and tolbutamide solutions. i In contrast, in the presence of tolbutamide, the D274N current was significantly smaller, indicating that the D274N channel was closed at resting [ATP] i This suggests that glucose-stimulated K ATP It could be predicted that this would reduce channel closure and insulin secretion, thereby contributing to diabetes in dogs.

[0105] We investigated the ATP sensitivity of the WT and D274N channels by measuring the Mg 2+ The measurements were performed in both the absence and presence of the former (Mg 2+ The former (in the absence of Mg) isolated ATP binding in Kir6.2, whereas the latter (in the presence of Mg) more closely approximated physiological conditions. The ATP concentration that produced half-maximal current inhibition was slightly, but significantly, higher than that of Mg. 2+ In the absence of (WT, IC 50 =10±1μmol / l, n=7;D274N, IC 50 = 15 ± 1 μmol / l, n = 5; p = 0.0044; Figures 3C and 3E), and Mg 2+ In the presence of (WT, IC 50 =19±2μmol / l, n=8;D274N, IC 50 Both the D274N and D274N channels showed increased current at 3 mmol / L MgATP (=32 ± 2 μmol / L, n = 7; p = 0.0011; Figures 3D and 3F). Furthermore, the percent unblocked current at 3 mmol / L MgATP was significantly increased (Figure 3G). Thus, the D274N channels were slightly less sensitive to ATP inhibition.

[0106] K ATPThe inhibitory ATP-binding site of the channel resides at the interface between two adjacent Kir6.2 subunits and includes a contribution from SUR1. Because D274N resides far from this site (Figure 4), it is unlikely that the D274N mutation directly impairs ATP binding. Nevertheless, because D274N is highly conserved, it may play an important structural and / or functional role, and its mutation may indirectly reduce ATP binding. For example, the D274N mutation could allosterically impair ATP binding, increase the intrinsic open probability of the channel, or impair the mechanism by which ATP binding is converted into closure of the channel gate. Interestingly, D274 is followed by a triple-histidine motif, which has been suggested as a potential polar contact between Kir6.2 and SUR1-NBD2. Therefore, the D274N mutation may affect ATP binding by disrupting the interaction between Kir6.2 and SUR1.

[0107] The change in ATP sensitivity caused by the D274N mutation is relatively small, but this is consistent with the finding that small differences in ATP sensitivity can cause neonatal diabetes in humans. The magnitude of the change caused by the D274N mutation is similar to the difference in ATP sensitivity that causes neonatal diabetes in humans. This is not surprising, as small changes in current amplitude translate into large changes in β-cell membrane potential and electrical activity, thereby altering insulin secretion. The small magnitude of the change may explain why heterozygosity did not appear to be associated with an increased risk of diabetes; carriers develop diabetes only if they have additional genetic burden or lifestyle factors. Notably, there was a higher proportion of heterozygous females in the diabetic group than in the control group, suggesting that heterozygosity may contribute to disease risk in certain circumstances. The unique diabetogenic state of the diestrus phase of the estrous cycle in female dogs, associated with mammary-derived growth hormone and luteal progesterone, may contribute to pancreatic β-cell stress. Because information on the number of estrous cycles each female had experienced before spaying was unavailable, we were unable to further investigate this within our dataset.

[0108] D274N mutation is K ATP The effect on the channel structure was modeled (Fig. 4). ATP The inhibitory ATP-binding site of the channel resides at the interface between two adjacent Kir6.2 subunits, with a contribution from SUR1. Although D274 is highly conserved across species, it is located far from this site, making it unlikely that the D274N mutation directly alters ATP binding. However, the D274N mutation may have indirect effects, allosterically impairing ATP binding, increasing the probability that the channel is open, or impairing the mechanism by which ATP binding translates into channel gating closure. Interestingly, D274 is followed by a triple-histidine motif, which has been suggested as a potential polar contact between Kir6.2 and SUR1-NBD2. Thus, the D274N mutation may affect ATP binding by disrupting the interaction between Kir6.2 and SUR1 (Figure 4).

[0109] Sulfonylureas are ATP It is routinely used to treat human diabetes because it binds to and closes the SUR1 subunit of the channel, thereby stimulating insulin secretion. The sulfonylurea tolbutamide inhibited both WT and D274N channels by >95% (Figure 2D). Human neonatal diabetes patients with Kir6.2 mutations that inhibit this level can be successfully treated with oral sulfonylureas, provided that drug therapy is initiated early after diagnosis. Therefore, oral sulfonylureas may reasonably be an effective treatment for canine diabetes associated with the D274N mutation.

[0110] Twice-daily injectable insulin therapy is the mainstay of treatment for diabetes in dogs. However, oral medications are easier for owners to administer and may be better tolerated by dogs. Furthermore, in humans with KCNJ11 mutations, early treatment with oral sulfonylureas has clear clinical benefits: they reduce blood glucose excursions, lower HbA1c, and enhance meal-stimulated insulin secretion (by enabling incretin action), optionally in combination with insulin. Similar benefits may be expected in dogs.

[0111] Genetic screening of diabetic Labrador retrievers for the KCNJ11 mutation could identify heterozygous and homozygous animals that may be suitable for oral drug therapy as part of a precision medicine approach to canine diabetes management. Genotyping also allows for informed avoidance of outbreeding of carrier Labrador retrievers within the breeding pool, potentially reducing the incidence of diabetes in this breed.

[0112] Materials and Methods Molecular Genetics animal All blood samples used in this study were collected for clinical veterinary purposes, and the remaining samples available for research were surplus to clinical needs. Samples were stored and used with permission from the Royal Veterinary College Clinical Research Ethical Review Board (URN 2017 1685-3) and with the consent of the animal owners.

[0113] All diabetic Labrador retriever and Labrador retriever cross blood samples were obtained from the UK Canine Diabetes Database and Archive, established in 1999 at the Royal Veterinary College. Diabetes was diagnosed based on the presence of appropriate clinical signs of polyuria and polydipsia, as well as the presence of persistent hyperglycemia. When available, data regarding age, breed, sex, neuter status, insulin dose, and weight at the time of sample submission were recorded. Serum fructosamine and HbA1c measurements were also performed on submitted samples for clinical monitoring purposes. Control (nondiabetic) Labrador retriever samples were obtained from the Clinical Investigation Center Research Archive, established at the Royal Veterinary College and containing residual samples from patients treated at the Queen Mother Animal Hospital. An additional cohort of nondiabetic control Labrador retriever DNA was provided by Dr. Catherine Mellersh of the University of Cambridge following the completion of another study at the UK Animal Health Trust.

[0114] The control group was age-restricted to minimize the possibility of the controls developing diabetes later in life: all control Labrador retrievers had reached a minimum age of 7 years without clinical signs of diabetes.

[0115] DNA extraction and purification DNA was extracted from stored residual EDTA blood, clots, or buffy coats using the Qiagen DNEasy Blood and Tissue kit according to the manufacturer's instructions, including an RNAse treatment step. Further purification and concentration were performed using the DNA Clean and Concentrator kit (Zymo Research). The concentration of DNA in 1 μL of eluate was measured using a spectrophotometer (Nanodrop), and DNA integrity was assessed using agarose gel electrophoresis and / or TapeStation (Agilent Technologies, Inc.).

[0116] Mutant discovery For variant discovery, canine exome sequencing was performed on 10 dogs at Otogenetics (Atlanta, USA) using DNA extracted from archived blood samples. Data were analyzed using a custom-built bioinformatics pipeline based on best practices in the Genome Analysis Toolkit (GATK3) on a high-performance computer cluster (BMRC Cluster, Wellcome Centre for Human Genetics, University of Oxford). Variant effect prediction was performed using SNPEff, and a candidate gene approach was used to identify nonsynonymous variants present in diabetic dogs and located in genes known to be associated with monogenic diabetes in humans.

[0117] Genotyping follow-up Genotypes of the KNCJ11 variants were confirmed in additional Labrador retrievers by various techniques: targeted PCR and TaqMan sequencing were performed specifically for this study, but the genotypes of diabetic and control dogs were available in an unpublished high-throughput sequencing dataset generated by the authors for another project.

[0118] Additionally, custom TaqMan genotyping assays were used to genotype individual samples. Illumina Whole Genome Sequencing data was performed at Edinburgh Genomics using a HiSeqX sequencer with 150-bp paired-end reads at 30X coverage. Targeted genotyping data were also available from two different custom high-throughput sequencing-based panels, both of which captured variants of interest. The first panel was TWIST Bioscience Target Enrichment (Twist Bioscience, CA, USA), and the second panel was SeqSNP Targeted Genotyping (LGC Genomics, Teddington, UK).

[0119] External Reference Data Sources Additional external data sources were consulted to determine alternative allele frequencies of the KCNJ11 variants of interest in other populations. These included unpublished data from the "Give a Dog a Genome" project, as well as publicly available data from the NHGR Dog Genome Project (https: / / research.nhgri.nih.gov / dog_genome / ) and the Dog BioMedical Variant Database Consortium.

[0120] Mutagenesis and Expression Human Kir6.2 and SUR1 were subcloned into pcDNA4 / TO or pBF expression vectors. Site-directed mutagenesis was performed using the QuikChange XL system (Stratagene; San Diego, CA) and confirmed by sequencing (DNA Sequencing and Services; Dundee, Scotland). mRNA preparation was performed as described (2). HEK-293T cells were maintained in Dulbecco's modified Eagle's medium (DMEM, Sigma) supplemented with 10% (v / v) fetal bovine serum (Life Technologies Ltd), 100 U / mL penicillin, and 100 μg / mL streptomycin (Thermo Fisher Scientific; Waltham, MA) at 37°C and 5% CO2 / 95% air. When approaching confluence, cells were trypsinized and plated on 25cm plates. 2 The cells were seeded into flasks. Three hours later, the cells were transfected with wild-type (WT) SUR1 and WT or mutant Kir6.2 using TransIT-LT1 (Mirus Bio LLC). 48 hours after transfection, the cells were replated onto poly-L-lysine-treated 35 mm Petri dishes (Corning). Measurements were performed 72 hours after transfection.

[0121] Xenopus oocytes were prepared as previously described (Gribble et al., 1997), injected with 0.8 ng wild-type or mutant Kir6.2 mRNA and approximately 4 ng SUR1 mRNA, and maintained at 18°C in Barth's solution (mM: 88 NaCl, 1 KCl, 1.68 MgSO, 0.41 CaCl, 0.47 Ca(NO), 2.4 NaHCO, 10 HEPES, pH adjusted to 7.4 with NaOH). Currents were recorded 2–4 days after injection.

[0122] electrophysiology Two-electrode voltage-clamp recording Using a two-electrode voltage clamp (GeneClamp 500B amplifier, Molecular Devices), whole-cell WT currents or mutant K currents were measured. ATP Currents were recorded at 22–24°C in response to voltage steps of ±20 mV from a holding potential of −10 mV. They were filtered at 500 Hz and sampled at 4 kHz using a Digidata 1440A acquisition system (Molecular Devices). Oocytes were continuously perfused with control solution (in mM): 90 KCl, 1 MgCl2, 1.8 CaCl2, 5 HEPES (pH 7.4 with KOH), plus 3 mM Na azide or 0.5 mM tolbutamide, as indicated.

[0123] Patch clamp recording WT or mutant K ATP Currents were recorded from large inside-out patches excised from HEK cells expressing the channels at a holding potential of -60 mV. Data were recorded with an Axopatch 200B amplifier (Molecular Devices), filtered at 1 kHz, and digitized at 10 kHz using a Digidata 1322A A / D interface driven by pClamp9 software (Molecular Devices).

[0124] For MgATP dose-response curves, the extracellular (pipette) solution contained (in mM): 140 KCl, 1.2 MgCl, 2.6 CaCl, 10 HEPES (pH 7.4 with KOH). The intracellular solution contained (in mM): 107 KCl, 2 MgCl, 1 CaCl, 10 EGTA, 10 HEPES (pH 7.3 with KOH). For ATP dose-response curves, the extracellular (pipette) solution contained (in mM): 140 KCl, 1 EDTA, 10 HEPES (pH 7.4 with KOH). The intracellular solution contained (in mM): 140 KCl, 1 EDTA, 1 EGTA, 10 HEPES (pH 7.3 with KOH).

[0125] ATP sensitivity was determined by expressing the current in the test solution as a fraction of the mean of the current in the control solution before and after each ATP application (3). ATP dose-response curves were calculated using the following Hill equation: I / I C = 1 / (1+([ATP] / IC 50 ) h (where [ATP] is the ATP concentration; I C and I are the currents in the absence and presence of ATP, respectively; IC 50 is K ATP where h is the Hill coefficient).

[0126] Data were analyzed using ClampFit (pClamp10; Molecular Devices) and Prism9 (GraphPad). Results are reported as mean ± SEM and statistical significance determined using Student's t test.

[0127] 3D modeling A reported human K bound to ATP and ADP in a quatrefoil configuration. ATP Three-dimensional modeling of Kir6.2 was performed using the protein data set (Protein Data Bank accession number 6C3O) (4). ATPBecause no crystal structures were available, we used human SUR1 and Kir6.2, which share 95% and 97% identity at the amino acid level with their canine homologs, respectively. Molecular modeling was performed using the PyMOL Molecular Graphics System (version 2.4.0, Schroedinger, LL Pymol).

[0128] Example 2 The presence of rs851344999 was investigated in dogs outside the UK. The diabetes-associated T allele was absent in 9 of 230 non-diabetic controls.

[0129] The rs851344999 SNP was then included in a proprietary SNP chip ("Wisdom Panel") to establish alternative allele frequencies across various U.S. dog breeds undergoing breed validation. Among 203,229 dogs of various breeds, 267 homozygous and 4,307 heterozygous individuals were identified in a random U.S.-based population, yielding an overall alternative allele frequency of 0.012. While the majority of dogs carrying the alternative allele were of purebred Labrador Retriever ancestry, mixed-breed dogs with Labrador Retriever ancestry also carried the allele. The allele frequency for dogs classified as Labrador Retrievers was 0.238.

[0130] Of the 267 rs851344999 homozygotes, not a single dog had detectable Labrador Retriever ancestry. The majority of dogs homozygous for the SNP had a majority of their genome consistent with Labrador ancestry (Figure 6). As shown in Figure 7, dogs heterozygous for the rs851344999 SNP included not only Labrador Retrievers but also Labrador Retriever crossbreeds (see the median peak in the histogram), with many dogs showing a smaller proportion of Labrador Retriever ancestry (toward the left of the histogram). Community science data for 434 homozygous and heterozygous dogs indicate that the median age at sampling was 2.8 years (range, 0.2-16.5 years).

[0131] A subset of dogs analyzed using the Wisdom Panel are included in the Banfield Optimal Wellness Plan for puppies. Data from this subset may be more representative of the overall U.S. dog population. Ancestry histograms are shown in Figure 8 (for heterozygous dogs) and Figure 9 (for homozygous dogs). Community science data for 434 homozygous and heterozygous dogs indicate that the median age at sampling was 0.46 years (range, 0.1 to 11.7 years). Complete ancestry information for 10 representative homozygotes is shown in Figure 10.

[0132] No information was available regarding the lifetime diabetic status of these dogs.

[0133] [Sequence table] SEQ ID NO: 1 - Amino acid sequence of canine Kir6.2 TIFF2025526868000002.tif51160 SEQ ID NO: 2 - Primary transcript of KCNJ11 (KCNJ11-201) TIFF2025526868000003.tif147161TIFF2025526868000004.tif2316135N at position 7 can be C or T N at position 486 can be C or T N at position 820 can be G or A N at position 930 can be C or T

Claims

1. 10. A method of screening a dog for a predisposition to diabetes, comprising determining whether the genotype of the dog's KCNJ11 gene comprises the D274N allele.

2. 2. The method of claim 1, wherein a KCNJ11 genotype homozygous for the D274N allele indicates that the dog has a predisposition to diabetes.

3. 3. The method of claim 1 or 2, wherein a KCNJ11 genotype heterozygous for the D274N allele, when combined with one or more other diabetogenic factors, indicates that the dog has a predisposition to diabetes.

4. 4. The method of claim 3, wherein the one or more other diabetogenic factors are selected from female gender, obesity, pancreatitis, pancreatic stress, progestogen treatment, corticosteroid treatment, endocrine disease, viral disease, and modifying genetic factors.

5. The method of any one of claims 1 to 4, wherein a KCNJ11 genotype homozygous for the reference allele indicates that the dog does not have a predisposition to diabetes associated with mutations in the KCNJ11 gene.

6. 10. A method for determining the likelihood that a dog will produce offspring that are genetically predisposed to diabetes, comprising the step of determining whether the dog's genotype of the KCNJ11 gene includes the D274N allele.

7. 7. The method of claim 6, wherein a KCNJ11 genotype that is homozygous or heterozygous for the D274N allele indicates that the dog has the potential to produce offspring that are genetically predisposed to diabetes.

8. 8. The method of claim 6 or 7, wherein a KCNJ11 genotype homozygous for the reference allele indicates that the dog does not have the genetic potential to produce offspring with a predisposition to diabetes associated with mutations in the KCNJ11 gene.

9. A method for selecting a treatment for diabetes in a dog, comprising the steps of determining whether the genotype of the dog's KCNJ11 gene includes the D274N allele, and selecting a treatment based on the determined genotype.

10. 10. The method of claim 9, wherein an oral hypoglycemic agent is selected as a treatment if the KCNJ11 genotype is homozygous or heterozygous for the D274N allele.

11. 11. The method of claim 10, wherein the selected therapy further comprises injectable insulin.

12. 10. The method of claim 9, wherein the selected therapy comprises injectable insulin if the KCNJ11 genotype is homozygous for the reference allele.

13. The method according to any one of claims 1 to 12, wherein the genotype of the KCNJ11 gene is determined by analyzing a sample obtained from the dog.

14. 13. The method of claim 12, wherein the sample is a blood sample or a tissue sample.

15. The method of claim 13 or 14, wherein the genotype of the KCNJ11 gene is determined by genome sequencing, and optionally the sequencing is Sanger sequencing, next-generation sequencing (NGS), long-read sequencing, single molecule real-time sequencing (SMRT), or DNA nanoball sequencing.

16. 15. The method of claim 13 or 14, wherein the genotype of the KCNJ11 gene is determined by single nucleotide polymorphism (SNP) genotyping, optionally wherein the SNP genotyping comprises a 5'-nuclease allele discrimination assay, restriction fragment length polymorphism analysis, allele-specific PCR, or an SNP array.

17. 1. A method for preventing, delaying or treating diabetes in a dog whose KCNJ11 gene contains one or more D274N alleles, the method comprising administering to said dog an oral hypoglycemic agent.

18. A composition comprising an oral hypoglycemic agent for use in a method for preventing, delaying or treating diabetes in a dog whose KCNJ11 gene contains one or more D274N alleles, said method comprising administering an oral hypoglycemic agent to said dog.

19. 19. The method of claim 17, or the composition for use of claim 18, wherein the KCNJ11 gene comprises two D274N alleles of the KCNJ11 gene.

20. 20. A method according to any one of claims 1 to 17 and 19, or a composition for use according to claim 18 or 19, wherein the dog is a Labrador Retriever or has Labrador Retriever ancestry, and optionally the dog is a Flat-Coated Retriever, a Golden Retriever, or a crossbreed comprising a Labrador Retriever.

21. 21. The method according to any one of claims 1 to 17, 19 and 20, or the composition for use according to any one of claims 18 to 20, wherein the diabetes is characterized by insulin dependence, pancreatic beta cell dysfunction and / or pancreatic beta cell loss.

22. The method of any one of claims 10, 11, 13-17, and 19-21, or the composition for use of any one of claims 18-21, wherein the oral hypoglycemic agent is a sulfonylurea.

23. The method according to any one of claims 10, 11, 13-17 and 19-22, or the composition for use according to any one of claims 18-22, wherein the oral hypoglycemic agent is tolbutamide, glibenclamide or glipizide.

24. 1. A method for preventing or delaying diabetes in a dog whose KCNJ11 gene contains one or more D274N alleles, comprising: (a) providing an antihyperglycemic diet to the dog; and / or (b) neutering the dog, wherein the dog is female, and optionally the neutering occurs before the dog's first estrous cycle; The method comprising:

25. 10. An antihyperglycemic diet for use in a method for preventing or delaying diabetes in a dog whose KCNJ11 gene contains one or more D274N alleles.

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