Compositions and methods associated with canine atopic dermatitis

Abstract

According to an aspect of the present disclosure, an in vitro method of determining a genotype relating to canine atopic dermatitis (CAD) in a canine mammal may be provided. The method may comprise determining a presence or absence of a splice variant in a signaling lymphocytic activation molecule 1 (SLAMF1) gene of the canine mammal. The presence of the splice variant may indicate that the canine mammal is at increased risk of suffering from CAD and of passing the splice variant to its progeny.

Description

Attorney Docket No.: 00307-0154-00304Mars Ref.: MARS / P / 393656 / WO / SEC / 1 COMPOSITIONS AND METHODS ASSOCIATED WITH CANINE ATOPIC DERMATITISCROSS-REFERENCE TO RELATED APPLICATION(S)

[0001] This patent application claims the benefit of priority to U. S. Provisional Application No. 63 / 737,270, filed on December 20, 2024, the entirety of which is incorporated herein by reference.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in .XML format and is hereby incorporated by reference in its entirety. Said. XML copy, created on December 18, 2025, is named Sequence Listing.xml and is 13,662 bytes in size.TECHNICAL FIELD

[0003] The present disclosure relates to the use of a splice donor variant in canine SLAMF1 (signaling lymphocytic activation molecule 1), an immune system associated gene as a biomarker for predicting the onset and diagnosing canine atopic dermatitis (CAD). The disclosure also relates to in vitro methods of diagnosing and / or assessing a risk for developing CAD in a canine mammal by detecting a splice donor variant in the canine SLAMF1 gene and to related primers and diagnostic kits.BACKGROUND

[0004] Canine atopic dermatitis (CAD) is a life-long inflammatory skin condition. The overall prevalence varies depending on the population but is estimated to affect up to 30% of dogs. Despite its prevalence, the pathogenesis of CAD is complex and not fully known. It is understood that a combination of heritable and environmental factors contributes to immune dysregulation with production of immunoglobulin E (IgE), skinAttorney Docket No.: 00307-0154-00304Mars Ref.: MARS / P / 393656 / WO / SEC / 1 barrier defects and alterations to the cutaneous microbiome, thus allowing an allergic phenotype to develop.

[0005] The initial clinical signs include inflammation (erythema) of the skin and pruritus which can be displayed as scratching, rubbing, chewing, licking and head shaking. Over time, this can lead to self-trauma of the skin, alopecia, crusting, hyperpigmentation, thickening of the skin (lichenification) and secondary’ infections with bacteria and yeast. Often the irritation is directed at the paws, pinnae, face, axillae and groin regions. Diagnosis of CAD is challenging, largely due to a lack of diagnostic test. Currently, diagnosis relies on thorough history taking, physical examination findings, and eliminating other potential causes of pruritus such as ectoparasites, skin infections, food and flea allergies. CAD is typically managed through anti-inflammatory medication, diet, and avoidance of allergens, but there is no definitive cure.

[0006] Several genome-wide association studies (GWAS) looking at CAD have been conducted, and single-nucleotide polymorphisms (SNPs) have been identified. That said, many of these studies are limited in their power due to small cohort size and low genotyping density’. An early GWAS in Golden Retrievers using a low density 22k SNP microarray identified two significant intronic SNPs, Prominin 1 and Ras-related protein Rab-3c. Two separate cohorts of atopic Labrador Retrievers and WHWTs showed association with elevated dust-mite specific IgE on chromosomes 5 and 35 respectively. In a Swedish cohort of German Shepherd Dogs, a GWAS in a set of 91 cases and 88 controls, identified a significant association on chromosome 27 in the region encoding Plakophilin 2, an important protein for skin structure. Although this seemed promising, further analysis has shown no difference in expression of this gene between CAD case and control groups. Conflicting observations have also been seen in WHWTs from different geographical areas with an Australian population showing CAD association with a 1.3 Mb area on chromosomeAttorney Docket No.: 00307-0154-00304Mars Ref.: MARS / P / 393656 / WO / SEC / 1 17 compared to a 2.7 Mb region on chromosome 3 in an American population. Finally, a recent GWAS identified a variant in the interleukin 4 receptor which reduces the risk of CAD in miniature Dachshunds, possibly through impairment of the receptor and reducing downstream inflammatory pathways associated with CAD. Previous studies demonstrate the complexity and genetic heterogeneity of CAD both across and within breeds, and the need for large cohorts to sufficiently power studies.SUMMARY

[0007] This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the detailed description. This summary’ is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.

[0008] According to an aspect of the present disclosure, an in vitro method of determining a genotype relating to canine atopic dermatitis (CAD) in a canine mammal may be provided. The method may comprise determining a presence or absence of a splice variant in a signaling lymphocytic activation molecule 1 (SLAMF1) gene of the canine mammal. The presence of the splice variant may indicate that the canine mammal is at increased risk of suffering from CAD and of passing the splice variant to its progeny.

[0009] According to other aspects of the present disclosure, the method may include one or more of the following features. Detecting the presence or absence of the splice variant may comprise microarray genotyping, DNA sequencing, allele discrimination analysis, restriction fragment length analysis, capillary electrophoresis fragment analysis, melt curve analysis, or KASP genotyping. The splice variant in the SLAMF1 gene may be downstream of SLAMF1's exon 4 (c799+2T> C), being the sequence in SEQ ID NO: 3. The splice variant may result in an amino acid sequence including 83 amino acids different from wild type SLAMF1 amino acid sequence before termination according to SEQ ID NO: 4. DeterminingAttorney Docket No.: 00307-0154-00304Mars Ref.: MARS / P / 393656 / WO / SEC / 1 the presence or absence of the splice variant may include analyzing a sample of genomic DNA or RNA obtained from the canine mammal. The method may further comprise determining a presence or absence of one or more genetic markers associated with a disorder other than CAD. The canine mammal may be a French Bulldog, a Boxer, or a Boston Terrier.

[0010] According to another aspect of the present disclosure, a use of a splice variant in a signaling lymphocytic activation molecule 1 (SLAMF1) gene to identify a canine mammal suffering or at increased risk of suffering from canine atopic dermatitis (CAD) may be provided.

[0011] According to other aspects of the present disclosure, the use may include one or more of the following features. The splice variant in the SLAMF1 gene may be downstream of SLAMFl's exon 4 (c799+2T> C), being the sequence in SEQ ID NO: 3. The splice variant may result in an amino acid sequence including 83 amino acids different from wild type SLAMF1 amino acid sequence before termination according to SEQ ID NO: 4.

[0012] According to another aspect of the present disclosure, a method of treating a canine mammal may be provided. The method may comprise treating the canine mammal based on a presence or absence of a splice variant in a signaling lymphocytic activation molecule 1 (SLAMF1) gene in a sample of genomic DNA obtained from the canine mammal. The presence of the splice variant may indicate that the canine mammal is at increased risk of suffering from CAD, and may pass the risk variant to its progeny.

[0013] According to other aspects of the present disclosure, the method may include one or more of the following features. Treating the canine mammal may include administering an anti-inflammatory medication. The canine mammal may be a French Bulldog, a Boxer, or a Boston Terrier. Treating the canine mammal may be further based on a presence of one or more genetic markers associated with a disorder other than CAD. TireAttorney Docket No.: 00307-0154-00304Mars Ref.: MARS / P / 393656 / WO / SEC / 1 splice variant in the SLAMF1 gene may be downstream of SLAMFl's exon 4 (c799+2T> C), being the sequence in SEQ ID NO: 3. The splice variant may result in an aberrant run of 83 amino acids before termination according to SEQ ID NO: 4.

[0014] The foregoing general description of the illustrative embodiments and the following detailed description thereof are merely exemplary aspects of the teachings of this disclosure and are not restrictive.BRIEF DESCRIPTION OF DRAWINGS

[0015] FIG. 1 depicts a combined single breed CAD GWAS, with genome-wide significant signals on canine chromosomes 12 and 38.

[0016] FIGS 2A-2C depict a single breed CAD GWAS with genome-wide significant signals. FIG. 2A depicts Boxer CAD GWAS with a genome-wide significant signal on canine chromosome 38. FIG. 2B depicts French Bulldog CAD GWAS with a genome-wide significant signal on canine chromosome 38. FIG. 2C depicts Labrador Retriever CAD GWAS, with a genome-wide significant signal on canine chromosome 12.

[0017] FIGS. 3A-3B depict CAD GWAS for all canine breeds analyzed.

[0018] FIG. 4 is a visualization in IGV of deep sequencing data from an atopic dermatitis case showing a splice donor variant in exon 4 of canine SLAMF1 (c799+2T> C).

[0019] FIG. 5 depicts results of RT-PCR spanning exons 3,4 and 5 of canine SLAMF1. FIG. 5, top panel A is a gel electrophoresis image showing C / C homozygotes with additional sequence in comparison to T / T homozygotes, FIG. 5, bottom panel B is a sequence analysis of the large and small fragments identifies an aberrant string of 41 bases to the canine SLAMF1 transcript.DETAILED DESCRIPTION

[0020] There are many embodiments described and illustrated herein. The present disclosure is neither limited to any single aspect nor embodiment thereof, nor to anyAttorney Docket No.: 00307-0154-00304Mars Ref.: MARS / P / 393656 / WO / SEC / 1 combinations and / or permutations of such aspects and / or embodiments. Moreover, each aspect of the present disclosure, and / or embodiments thereof, may be employed alone or in combination with one or more other aspects of the present disclosure and / or embodiments thereof. For the sake of brevity, certain permutations and combinations are not discussed and / or illustrated separately herein. Notably, an embodiment or implementation described herein as “exemplary” is not to be construed as preferred or advantageous, for example, over other embodiments or implementations; rather, it is intended to reflect or indicate that the embodiment(s) is / are “example” embodiment(s).

[0021] A deeper understanding of CAD pathogenesis may assist in the development of new diagnostic and management strategies for this chronic condition. A pronounced breed-specific predisposition to CAD among the Golden and Labrador Retriever, Boxer, West Highland White Terrier (WHWT), French Bulldog and German Shepherd Dog suggests genetics plays a role in the disease. CAD heritability has been estimated at 0.47 and 0.31 in Labrador and Golden Retrievers and WHWTs respectively.

[0022] Introduction of genetic testing using a 100k SNP genotyping array into veterinary clinics at the start of life and as part of large scale longitudinal biobanking studies provided a unique opportunity to accumulate large numbers of diagnosed cases for multiple disorders over time. Using a cohort accumulated over a five-year period and derived from a clinico-genetics dataset of over 1.2 million dogs, a GWAS was performed to search for genetic associations in dogs diagnosed with atopic dermatitis.

[0023] SLAMF1 splice donor variant (5Zzl F7:c799-t-2T> C) associated with allergic dermatitis has been identified using a GWAS approach followed by whole genome resequencing, The splice donor site involved is highly conserved across vertebrate species suggesting that disruption of this site is likely to be deleterious. Furthermore, cDNA sequencing has shown that the splice donor variant results in cryptic splicing, putting theAttorney Docket No.: 00307-0154-00304Mars Ref.: MARS / P / 393656 / WO / SEC / 1 transcript out of frame and predicted to result in a run of aberrant amino acids. In some aspects, the splice donor variant results in cryptic splicing, putting the transcript out of frame, and may result in the translation of a non-wild type polypeptide that includes an amino acid sequence different from those in wild type SLAMF1 amino acid sequence. This results in the replacement of the cytoplasmic tail of canine SLAMF 1, which includes functional ITSM motifs which act as binding sites for cell signaling ligands. SEQ ID NO: 1 and SEQ ID NO:2 show the DNA and amino acid sequences for wild type canine SLAMF1 coding sequence and wild type SLAMF1 amino acid sequence, respectively, while SEQ ID NO:3 and SEQ ID NO:4 show the variant canine SLAMF1 DNA and amino acid sequences. SEQ ID NO: 5 shows a nucleic acid sequence that may form the splice donor variant.

[0024] As can be observed from SEQ ID NO:3, the splice donor variant may include additional 41 nucleic acid sequences compared to the wild type SLAMF1 shown in SEQ ID No: 1. These additional sequences may result in cryptic splicing, which may lead to a frameshift downstream of exon 4, which may result in an amino acid sequence that includes 83 amino acids, as can be seen in SEQ ID NO: 4, that are different from wild type SLAMF 1 amino acid sequence shown in SEQ ID NO: 2. This modification of the cytoplasmic tail may prevent SAP bonding and inhibition of the SLAM-SAP-Fyn-SH3 ternary complex.

[0025] SLAMF1 or signaling lymphocytic activation molecule 1, is an immune system regulatory protein, also designated as CD 150, expressed on the cell surface of T, B, NK, and dendritic cells. SLAMF1 is part of a family of SLAM receptors and SLAM-associated proteins (SAP) intracellular adaptors that play an active role in the immune system. Measles virus entry is reported to be facilitated by SLAMF 1 and CD46 as cellular receptors. A potential link between measles and atopic dermatitis has been established showing that human keratinocytes can be infected by the measles virus, modulating the expression of cytokines involved in allergic conditions such as atopic dermatitis.Attorney Docket No.: 00307-0154-00304Mars Ref.: MARS / P / 393656 / WO / SEC / 1 Furthermore, it was demonstrated that vaccination against measles resulted in a protective effect against development of atopic dermatitis. The canine distemper virus is a single stranded RNA virus and part of the same family of viruses as the measles virus. Given the high frequency of the 5Zzl F7:c799-t-2T> C allele, and the evidence of measle infection and vaccination giving a protective effect, a defective SLAMF1 receptor may provide some protection against canine distemper infection, whilst increasing the risk of atopic dermatitis development. SLAMF1 receptors act as self-ligands. Evidence that SLAM / SLAM interactions inhibit CD40-induced production of inflammatory cytokines in monocyte derived dendritic cells, a specialized immune system cell found in tissues including skin, further suggests that deleterious variants in SLAMF1 could reduce moderation of inflammatory responses after a trigger event.

[0026] SLAMF1 has been associated with several disease processes in humans including rheumatoid arthritis, systemic lupus erythematosus and diabetes. These associations further establish SLAMF1 involvement in autoimmune processes. Studies have identified increased odds of comorbidity for rheumatoid arthritis in atopic dermatitis patients and lupus among patients with atopic dermatitis, so although there has not been a previous direct link between SLAMF 1 and atopic dermatitis, there is reasonable evidence that SLAMF1 is involved in connected disease processes. Pathways involving slam family members have already been investigated as potential therapeutic targets. For example, in a clinical pilot study, treatment with alefacept (a CD8-IgG1 fragment crystallizable (Fc) domain fusion protein), reduced skin inflammation in cases of atopic eczema. Alefacept binds CD2, a member of the CD2 / SLAM gene family, with the results suggesting reduced activation of T cells after therapy. It has also been shown that activation of SLAM by an mAb agonist in IL12 cells derived from the skin of AD patients, results in stable populations of IFN-gamma-producing cells, which do not support IgE synthesis, potentially attenuating theAttorney Docket No.: 00307-0154-00304Mars Ref.: MARS / P / 393656 / WO / SEC / 1 allergic process. These results support the SLAM family as potential therapeutic targets of Th2 allergic disease.

[0027] The SLAMF1:c799+2T>C variant, which has an allele frequency of 0.082 in the Dog 10k genome release, is found in dog breeds such as the French Bulldog, Boxer, and Boston Terrier, and increases tire risk of allergic dermatitis by approximately two-fold. Atopic dermatitis is a disease with a complex etiology and the presence of the variant in both case and control populations supports this, with both environmental factors and additional breed-specific genetic risk factors likely to contribute to disease progression. In addition to the SLAMF1 variant identified, significant associations were also established between atopic dermatitis and the dog leukocyte antigen (DLA) region. DLA involvement has been associated with autoimmune disease in the dog and the link between allergic dermatitis and the DLA region is a logical one, and supported by large scale atopic dermatitis GWAS in humans.

[0028] A clinical manifestation of canine atopic dermatitis may be complicated by secondary infections with yeast or bacteria. Failure to resolve a secondary infection may exacerbate clinical signs of dermatitis. Study of a SLAMF1- / - TCR knock-out mouse has shown SLAMF1 is required for resistance to environmental fungal infection. There may be a possibility that the SLAMF1:c799+2T>C has a dual effect of increasing the risk of an initial presentation of atopic dermatitis and the risk of developing secondary yeast infection.

[0029] The methods herein may include establishing whether or not the canine mammal is homozygous for the ancestral wild-type allele (SLAMF1:c799+2 T / T), heterozygous (SLAMFl:c799+2 T / C) or homozygous for the derived allele(SLAMF1:c799+2 C / C) for the splice variant in the SLAMF1 gene. If the canine mammal is heterozygous for the splice variant within the SLAMF1 gene, for example, the canine mammal may be selected as being suitable for breeding wi th a canine mammal of a sameAttorney Docket No.: 00307-0154-00304Mars Ref.: MARS / P / 393656 / WO / SEC / 1 breed which is homozygous for wild-type SLAMF1 gene. Or if the canine mammal is homozygous for wild-type SLAMF1 gene, for example, the canine mammal may be selected as being suitable for breeding with a canine mammal of a same breed which is homozygous or heterozygous for wild-type SLAMF1 gene.

[0030] The methods described herein may include any genotyping technique known in the art. The genotyping techniques may include but not be limited to microarray genotyping, DNA sequencing, allele discrimination analysis (also known as Taqman), restriction fragment length analysis, capillary electrophoresis fragment analysis, melt curve analysis, and KASP genotyping.

[0031] In some aspects, microarray genotyping may comprise hybridizing labeled DNA fragments to an array of oligonucleotide probes, wherein the probes may be designed to detect specific allelic variants, and wherein signal intensities may be measured to determine genotype calls.

[0032] In other aspects, DNA sequencing may comprise amplifying a target region containing the splice variant, sequencing the amplified product using Sanger sequencing or next-generation sequencing platforms, and analyzing the resulting sequence data to identify the presence or a bsence of the variant. The amplification step may involve polymerase chain reaction (PCR) using primers flanking the region of interest, wherein the reaction may be optimized for specificity and yield. The sequencing data may be processed through quality control pipelines, wherein base calling algorithms may assign confidence scores to each nucleotide position. Sequence alignment software may be employed to compare the obtained sequences against reference sequences, wherein variant calling tools may identify single nucleotide polymorphisms and insertions or deletions,

[0033] In some aspects, allele discrimination analysis may comprise using fluorescently labeled probes that differentially bind to wild-type and variant alleles, whereinAttorney Docket No.: 00307-0154-00304Mars Ref.: MARS / P / 393656 / WO / SEC / 1 real-time PCR amplification may be performed and fluorescence signals may be measured to determine the genotype. The probes may be designed with reporter and quencher dyes, wherein the reporter fluorescence may be released upon probe hybridization and subsequent cleavage during amplification. Thermal cycling parameters may be optimized to ensure specific probe binding, wherein annealing temperatures may be adjusted based on probe melting characteristics. The resulting fluorescence data may be analyzed using clustering algorithms, wherein samples may be assigned to genotype groups based on their fluorescence intensity ratios.

[0034] In other aspects, Restriction fragment length analysis may comprise amplifying a region containing the splice variant, digesting the amplified product w ith a restriction enzyme that differentially cleaves wild-type and variant sequences, and separating the resulting fragments by gel electrophoresis to determine genotype based on fragment size patterns. The restriction enzyme may be selected based on recognition site specificity, wherein the variant nucleotide may create or abolish an enzyme recognition sequence. The digestion reaction may be incubated under optimal temperature and buffer conditions, w herein complete digestion may be verified through appropriate controls.Fragment visualization may be achieved through nucleic acid staining, wherein band patterns may be compared against size standards to confirm expected fragment lengths.

[0035] In some aspects, capillary electrophoresis fragment analysis may comprise amplifying a target region using fluorescently labeled primers, separating the amplified fragments by capillary electrophoresis, and detecting fragment sizes to identify size differences associated with the splice variant. The fluorescent labels may be attached to forward or reverse primers, w'herein different fluorophores may be used for multiplexing multiple targets. The electrophoretic separation may occur in polymer-filled capillaries, wherein an electric field may drive fragment migration based on size. Peak detectionAttorney Docket No.: 00307-0154-00304Mars Ref.: MARS / P / 393656 / WO / SEC / 1 software may analyze the electropherogram data, wherein fragment sizes may be calculated by comparison to internal size standards run concurrently with the samples.

[0036] In other aspects, melt curve analysis may comprise amplifying a target region and gradually increasing the temperature while monitoring fluorescence, wherein differences in melting temperature may indicate the presence of the splice variant. The amplification may be performed in the presence of intercalating dyes, wherein the dye fluorescence may decrease as double-stranded DNA denatures during heating. The temperature ramp rate may be controlled precisely, wherein high-resolution melting instruments may detect subtle differences in melting profiles. The resulting melt curves may be analyzed using derivative plots, wherein the melting temperature peaks may be compared to reference samples of known genotypes.

[0037] In some aspects, KASP genotyping may comprise using allele-specific primers with distinct fluorescent labels, wherein competitive allele-specific PCR may be performed and endpoint fluorescence may be measured to determine the genotype at the splice variant position. The allele-specific primers may contain unique tail sequences, wherein universal fluorescent reporter cassettes may bind to these tails during amplification. The PCR reaction may be performed under stringent conditions, where in only perfectly matched primers may efficiently extend. The endpoint fluorescence data may be plotted on a two-dimensional scatter plot, wherein samples may cluster into distinct genotype groups based on their relative fluorescence intensities for each allele.

[0038] In other aspects, the genotyping techniques may be performed using a sample of genomic DNA obtained from the canine mammal. The genomic DNA sample may be obtained from various biological sources including, but not limited to, whole blood, buccal swabs, saliva, hair follicles, skin tissue, or other tissue samples. In some cases, the sample may comprise RNA obtained from the canine mammal, wherein the RNA may be extractedAttorney Docket No.: 00307-0154-00304Mars Ref.: MARS / P / 393656 / WO / SEC / 1 from whole blood or other tissues expressing SLAMF1. The RNA sample may be used for expression analysis, such as RT-PCR, to detect the presence of aberrant transcripts resulting from the splice variant. In some aspects, DNA extraction from whole blood and buccal swabs may be performed using standard laboratory protocols. The extracted nucleic acid may then be subjected to one or more of the genotyping techniques described herein to determine the presence or absence of the splice vanant in the SLAMF1 gene.

[0039] In some aspects, the genotyping techniques may be performed using a sample of genomic DNA obtained from the canine mammal. The genomic DNA sample may be obtained from various biological sources including, but not limited to, whole blood, buccal swabs, saliva, hair follicles, skin tissue, or other tissue samples. In some cases, the sample may comprise RNA obtained from the canine mammal, wherein the RNA may be extracted from whole blood or other tissues expressing SLAMF1. The RNA sample may be used for expression analysis, such as RT-PCR, to detect the presence of aberrant transcripts resulting from the splice variant. In some aspects, DNA extraction from whole blood and buccal swabs may be performed using standard laboratory protocols. The extracted nucleic acid may then be subjected to one or more of the genotyping techniques described herein to determine the presence or absence of the splice variant in the SLAMF1 gene.

[0040] In some aspects, the methods described herein may be performed as part of a genetic testing panel, wherein the SLAMF1 splice variant genotyping may be combined with detection of one or more additional genetic markers, The genetic testing panel may include markers associated with other canine health conditions, breed identification markers, or markers for traits of interest to breeders and owners. In some cases, the panel may include markers for genetic health conditions not related to CAD, wherein such conditions may include cardiac disorders, musculoskeletal conditions, metabolic diseases, ophthalmologic conditions, neurological disorders, or other hereditary health conditions. The panel may alsoAttorney Docket No.: 00307-0154-00304Mars Ref.: MARS / P / 393656 / WO / SEC / 1 include markers associated with physical traits, wherein such traits may include coat color, coat type, body size, ear shape, tail characteristics, or other morphological features of interest to breeders and owners. In other aspects, the panel may include markers associated with behavioral dispositions, wherein such dispositions may include trainability, activity level, sociability, prey drive, or other behavioral characteristics that may be of interest in breeding selection or ow ner education. The panel may also include markers associated w ith the dog leukocyte antigen (DLA) region, wherein DLA variants may provide additional information regarding immune system function and disease susceptibility. In other aspects, the genetic testing panel may be implemented on a microarray platform, wherein multiple genetic markers may be interrogated simultaneously from a single sample.

[0041] In some cases, the combined results from multiple markers on the panel may¬ be used to generate a composite risk assessment, wherein the presence of multiple risk variants may indicate a higher overall likelihood of disease development. In some aspects, the genetic testing panel may be accompanied by a genetic consultation service, wherein at-risk findings may be reviewed and explained by a qualified professional. The genetic consultation may provide guidance regarding the significance of identified risk variants, wherein recommendations for clinical follow-up, preventive care, or breeding decisions may be offered based on the test results.

[0042] Atopic dermatitis is a common and significant welfare issue encountered at the veterinary clinic. Identification of a genetic risk factor may help improve understanding of disease processes and inform more targeted therapeutics. The identification of the SLAMFl:c799+2T> C variant may also present the opportunity for breeders to breed towards dogs with a lower risk of atopic dermatitis. However, the frequency of the disease-associated variant is high and even potentially fixed in some breeds, as observed in Dog 10k data. Maintenance of diversity should be considered as breeders seek to reduce the risk inAttorney Docket No.: 00307-0154-00304Mars Ref.: MARS / P / 393656 / WO / SEC / 1 their breeding lines. SLAMFl:c799+2T> C association with CAD within a particular breed may be confirmed before test results are used for selective breeding purposes, as CAD is a complex disease with modifiers likely altering the SLAMFl:c799+2T> C associated risk in each breed.

[0043] In another aspect, the disclosure may provide a method for treating canine atopic dermatitis. In one embodiment, the method may include administering an antiinflammatory agent to a canine mammal in need thereof, based on the identification of the presence of a splice variant in a signaling lymphocytic activation molecule 1 (SLAMF1) gene in a sample of genomic DNA or mRNA obtained from the canine mammal, wherein the presence of the splice variant indicates that the canine mammal is at increased risk of suffering from CAD. The method may further comprise determining the genotype of the canine mammal prior to administration of the anti-inflammatory agent, wherein the genotype determination may be performed using any of the genotyping techniques described herein. In some aspects, the treatment may be initiated prophylactically in canine mammals identified as being at increased risk based on the presence of the splice variant, wherein early intervention may help manage or delay the onset of clinical signs associated with CAD.

[0044] Suitable anti-inflammatory agents for use in the methods described herein may include, but are not limited to, corticosteroids such as prednisolone, prednisone, methylprednisolone, dexamethasone, hydrocortisone, and triamcinolone. In some aspects, the anti-inflammatory agent may comprise a Janus kinase (JAK) inhibitor such as oclacitinib. In other aspects, the anti-inflammatory agent may comprise a monoclonal antibody targeting interleukin- 31, such as lokivetmab. The anti-inflammatory agent may also include calcineurin inhibitors such as cyclosporine or tacrolimus. In some cases, the antiinflammatory' agent may comprise a phosphodiesterase-4 (PDE4) inhibitor. Non-steroidalAttorney Docket No.: 00307-0154-00304Mars Ref.: MARS / P / 393656 / WO / SEC / 1 anti-inflammatory drugs (NSAIDs) may also be suitable in some aspects. The antiinflammatory agent may further include antihistamines such as diphenhydramine, cetirizine, hydroxyzine, or chlorpheniramine. In other aspects, the anti-inflammatory agent may comprise essential fatty acid supplements, wherein omega-3 and omega-6 fatty acids may help modulate inflammatory' responses. Topical anti -inflammatory' agents such as hydrocortisone aceponate, betamethasone, or mometasone may also be suitable for localized treatment.

[0045] The anti-inflammatory agent may be administered via various routes depending on the specific agent and the clinical presentation of the canine mammal. In some aspects, the anti-inflammatory' agent may be administered orally, wherein oral formulations may include tablets, capsules, chewable treats, or liquid suspensions. In other aspects, the anti-inflammatory agent may be administered parenterally, wherein parenteral administration may include subcutaneous injection, intramuscular injection, or intravenous infusion. Topical administration may be suitable in some cases, wherein topical formulations may include creams, ointments, gels, sprays, shampoos, or lotions applied directly to affected skin areas. In some aspects, the anti-inflammatory' agent may be administered via otic preparations for cases wherein otitis is a clinical manifestation. The dosage and frequency of administration may be determined based on factors including the weight of the canine mammal, the severity of clinical signs, the specific anti-inflammatory'- agent selected, and the response to treatment. In some cases, combination therapy using multiple anti-inflammatory agents may be employed, wherein different agents may target distinct pathways involved in the inflammatory response. The treatment regimen may also include adjunctive therapies such as medicated baths, allergen avoidance strategies, dietary modifications, or immunotherapy, wherein such adjunctive therapies may complement the anti-inflammatory treatment.Attorney Docket No.: 00307-0154-00304Mars Ref.: MARS / P / 393656 / WO / SEC / 1

[0046] The following examples are intended to illustrate the present disclosure without, however, being limiting in nature. It is understood that the present disclosure encompasses additional embodiments consistent with the foregoing description and following examples.EXAMPLES

[0047] Methods

[0048] DNA Sample Collection

[0049] DNA samples were collected via commercial testing of Wisdom Panel ™ Premium, Wisdom Panel™ Essential, Wisdom Panel™ Health and Optimal Selection™ retail products, and genetic testing performed as a part of Optimum Wellness Plans® for puppies, through Banfield Pet Hospital branches (Vancouver, WA, USA) and as part of the MARS PETCARE BIOBANKIM. Samples were either collected through non-invasive cheek swabbing by dog owners or veterinary professionals or through blood sampling by a veterinary professional at a Banfield Pet Hospital in line with regulations governing diagnostic testing. Consent for use of DNA data in research was obtained through the client’s agreement with terms and conditions of DNA testing through Wisdom Panel.Analysis and sequencing of cDNA were performed using samples collected from dogs participating in the Mars Petcare Biobank. All samples originated from the United States or Mexico.

[0050] Genotyping

[0051] DNA extraction from whole blood and buccal swabs was performed at GeneSeek Laboratories (Neogen Corporation, Lincoln, NE, USA). Genotyping was performed on a custom 100k Illumina Infinium XT SNP microarray (Illumina, Inc., San Diego, CA, USA), also at GeneSeek Laboratories. The microarray was designed and validated for use following the same protocol and principles as previously described.Attorney Docket No.: 00307-0154-00304Mars Ref.: MARS / P / 393656 / WO / SEC / 1 Microarray genotyping analyses were carried out following manufacturer-recommended standard protocols for the Illumina XT platform (Illumina, Inc.). Only samples achieving at least 95% genotyping call rate were included in the study.

[0052] Clinical information

[0053] For DNA samples submitted directly for genotyping through Banfield clinics, data from genotyped dogs was directly linked with clinical records stored in the Banfield EMR (electronic medical records). For DNA samples collected and submitted by general retail consumers of Wisdom Panel products, data from genotyped dogs w as linked with the Banfield EMR by anonymised cross matching of pet and owner information, in accordance with personally identifiable information (PII) regulations.

[0054] Inclusion criteria

[0055] CAD cases and controls were categorized based on labelling provided by general veterinary practitioners in the Banfield EMR and through criteria set by a board-certified veterinary' dermatologist. Given the retrospective nature of the data, this is a diagnosis of atopic dermatitis in the broad sense w hich may include dogs with food allergic dermatitis.

[0056] CAD cases w'ere in the age range of 0.3 to 20 years and must have received at least three months of ectoparasite control. CAD cases had also been diagnosed w ith recurrent (more than once) clinical signs of atopic dermatitis including: Dermati tis, Atopic Allergic; Otitis; Pododermatitis; Pruritus; Pyoderma or Malassezia. In addition, CAD cases could also have been prescribed recurrent systemic anti-inflammatories, antihistamines or medicated ear drops.

[0057] Controls were in the age range of 3 to 20 years and were not diagnosed with of any of the following recurrent symptoms: Acne; Alopecia Undetermined; Dermatitis (of any variety), Folliculitis; Lichenification; Malassezia; Otitis (of any variety); Paronychia;Attorney Docket No.: 00307-0154-00304Mars Ref.: MARS / P / 393656 / WO / SEC / 1 Pododermatitis; Pruritus; Pyoderma (of any variety). Controls were not prescribed recurrent systemic or topical anti-inflammatories, antihistamines or medicated ear drops.

[0058] For breed assignment single breed dogs were defined as dogs with greater or equal to 80% single breed ancestry as defined by the Wisdom Panel breed classification algorithm (BCSYS).

[0059] Genotype Analysis

[0060] Genome-wide association study analysis was performed using a linear mixed- model approach performed in the software package GEMMA v0.98.5 including a centered relatedness matrix, PLINK was used to apply the following quality control: Samples with an overall genotyping success rate of lower than 95% across all tested SNPs were also filtered out; then, variants below 1% minor allele frequency or lower than 95% genotyping success rate were filtered out. All reported genome locations are given based on the CanFam3.1 genome build, unless stated otherwise. Mode of inheritance was assessed by fitting a generalized linear model with a logit link function using the R programming language, testing for the association between allele dose and case-control status as follows: For the additive model, dose was coded as 0, 1 or 2, representing the number of copies of the risk allele. For the dominant model, dose was coded as 0 or 1, with zero representing the non-risk homozygous genotype and 1 representing the heterozygous or risk homozygous genotype. For the recessive model, dose was coded as 0 or 1, with zero representing the non-risk homozygous genotype and the heterozygous genotype, and 1 representing the risk homozygous genotype. A likelihood ratio test was used to identify the best model fit.

[0061] Genome Sequencing

[0062] Genome sequencing was performed using buccal swab DNA collected through DNA testing. Whole genome sequencing using standard methodology, to a target of 30x read depth w as performed on Illumina Novoseq at Neogen Inc, Lincoln,Attorney Docket No.: 00307-0154-00304Mars Ref.: MARS / P / 393656 / WO / SEC / 1 Nebraska, US. Data were analysed using an Illumina Dragen pipeline aligned to the CanFam4 genome assembly. Variants were annotated using SNPeff and statistical analysis performed using SNPsift.

[0063] Extended SLAMF1 genotyping

[0064] Extended genotyping for the SLAMF1 candidate variant was performed by LGC Service Lab UK, using KASP genotyping methodology.

[0065] SLAMF1 cDNA analysis and sequencing

[0066] Expression analysis was performed using RNA extracted from whole blood stored samples in RNAProtect (catalogue #76,554; Qiagen, Germantown, MD, USA). RNA was extracted by Qiagen Genomic Services (Frederick, MD, USA), and expression analysis performed by BioVet Inc. (Antech Diagnostics).

[0067] Results

[0068] Combined single breed GWAS

[0069] An initial GWAS was performed using 14,378 single breed cases and 14,633 breed matched controls to tightly control against potential population stratification. Genome¬ wide significant signals were seen on canine chromosome 12 in the DLA region and canine chromosome 38, with top SNPs at chr12: 1,575,916 (p=6.41xl0-ll) and chr38:21,605,689 (p=1.79x10-12) respectively. FIG. 1 shows results for all single breeds, with breed matched cases and controls (14,378 cases, 14,663 controls, λ 0.978). Coordinates refer to the CanFam3.1 genome build.

[0070] Within breed GWAS

[0071] Within breed GWAS was performed where at least 100 cases were available for a given breed. Genome-wide significant results were observed for the Boxer (FIG. 2A, corresponding to 447 cases, 515 controls λ: 0.973), French Bulldog (FIG. 2B), and Labrador Retriever (FIG, 2C) breeds. The top SNPs for French Bulldog and Boxer were atAttorney Docket No.: 00307-0154-00304Mars Ref.: MARS / P / 393656 / WO / SEC / 1 chr38:21,592,552 (p= 2.04 x10-13) and chr38: 21434822 (p= 2.27x10-8) respectively, replicating the loci identified in combined single breed GWAS. The top SNP for the Labrador Retriever GWAS was at chr12:1752027 (p=9.09x10-08) which is located within the DLA region. Suggestive association to the chromosome 38 region was also shown for Yorkshire terriers. GWAS results with QQ plots for all breed analyzed can be found in FIGS. 3A-3B.

[0072] Whole genome sequence analyses

[0073] Including whole genome sequencing performed for other studies. a total of 78 genomes were available for analyses. Cases were defined using a combination of phenotypic and genotypic information. Three cases were purposely included for analysis based on phenotype and homozygosity across the associated region. In addition, due to tlie expected high frequency of the causal variant, an additional seven dogs were defined as haplotypic cases based on the presence of a shared homozygous interval (CanFam3.1:chr38:21300340-21715689). Final analysis was performed with ten cases and 68 controls, extending the disease-associated region empirically around the top SNP to capture additional variants (CanFam4 chr38: 21805771-22919607). Genome sequences were aligned to the CanFam4 genome build, variants called, and effects predicted using SNPeff. Statistical analysis was performed using SNPsift. Of the 13,154 unique variants identified, the top-ranking variant based on both effect and probability value was a splice donor variant downstream of SLAMF1 exon 4 (c799+2T> C) (FIG. 4). MaxtentScan analysis predicted the splice site to be weakened by the variant (score reduced from 9.65 to 1.90) and GenScan analysis predicted that the splice donor variant causes exon skipping. No other deleterious and segregating variants were identified. Tire region was assessed for structural variants using the Dog10k genome set. No provocative structural variants were identified across the associated region.Attorney Docket No.: 00307-0154-00304Mars Ref.: MARS / P / 393656 / WO / SEC / 1

[0074] Gene expression analysis

[0075] SLAMF1 gene expression was accessed using RNA from canine blood as described above. An exon spanning RT-PCR gave a PCR product larger than predicted for C / C homozygotes suggesting that the wild-type splice site had indeed been disrupted and that an alternative cryptic donor splice site downstream was being adopted. In FIG 5, panel (A), lanes 1, 3, and 4 represent the cDNA samples from the Boston Terriers, and lanes 2, 5, 7, 8, 9, and 10 represent the cDNA samples from the French Bulldogs. Lane L signifies a 100 bp ladder, and lane - represents the no-template control. Panel (B) of FIG. 5, illustrates the use of Sanger sequencing to compare the fragments of different length revealing a 41 bp (SEQ ID NO: 3) addition to exon 4 when compared to wild type SLAMF1 gene (SEQ ID NO: 4), resulting in a predicted aberrant run of 83 amino acids before termination (SEQ ID NO: 4).

[0076] Odds ratio, mode of inheritance and Dog 10k data analysis

[0077] Odd ratios were calculated using the GWAS top SNPs and using SLAMF1 c799+2 T>C genotypes for a subset of cases and controls for the French Bulldog and Boxer. The top labrador SNP showed heterozygosity excess, due to an apparent duplication based on genome studio analysis. Therefore, odds ratios were calculated using the top GWAS SNP and third most significant GWAS SNP for Labradors. Best mode of inheritance (MOI) models with odds ratios are shown in Table 1. Data from the 10k project was used to identify other breeds in which the SLAMF1 c799+2T> C was present. The variant was found in a total of 91 breeds and in free-roaming dogs of eight regions.Table 1Breed Variant Chr: Position MOI (best) OR (95% CI)(CF3)Attorney Docket No.: 00307-0154-00304Mars Ref.: MARS / P / 393656 / WO / SEC / 1 FBD BICF2G63068122 38:21592552 Additive 1.66 [1.46- 1.88J FBD SLAMF1 c799 +2 T> C 38:21,671,895 Additive 1.89 [1.00- 3.71] Boxer BICF2P422110 38:21434822 Additive 1.74 [1.44-2.1] Boxer SLAMF1 c799 +2 T> C 38:21,671,895 Additive 2.75 [1.48- 5.43]Lab DQB1 SNP27 12: 2,252,493 Dominant* 1.91 [1.63- 2.24]Lab BICF2P1419525 12:1,752,027 Additive 1.56 [1.4-1.74]

[0078] Those skilled in the art will appreciate that the conception upon which this disclosure is based may readily be used as a basis for designing other methods and systems for carrying out the several purposes of the present disclosure. Accordingly, the claims are not to be considered as limited by the foregoing description.

Claims

Attorney Docket No.: 00307-0154-00304Mars Ref.: MARS / P / 393656 / WO / SEC / 1 CLAIMS1. An in vitro method of determining a genotype relating to canine atopic dermatitis (CAD) in a canine mammal comprising:determining a presence or absence of a splice variant in a signaling lymphocytic activation molecule 1 SLAMF1) gene of the canine mammal, wherein the presence of the splice variant indicates that the canine mammal is at increased risk of suffering from CAD and of passing the splice variant to its progeny.

2. The method of claim 1, wherein detecting the presence or absence of the splice variant comprises microarray genotyping, DNA sequencing, allele discrimination analysis, restriction fragment length analysis, capillary electrophoresis fragment analysis, melt curve analysis, or KASP genotyping3. The method of claim 1 or 2, wherein the splice variant in the SLAMF gene is downstream of SLAMF1's exon 4 (c799+2T> C), being the sequence in SEQ ID NO: 3.

4. The method of any one of the preceding claims, wherein the splice variant results in an amino acid sequence including 83 amino acids different from wild type SLAMF1 amino acid sequence before termination according to SEQ ID NO: 4.

5. Tire method according to any one of the preceding claims, w herein determining the presence of absence of the splice variant includes analyzing a sample of genomic DNA or RNA obtained from the canine mammal.Attorney Docket No.: 00307-0154-00304Mars Ref.: MARS / P / 393656 / WO / SEC / 1 6. The method of any one of the preceding claims, further comprising determining a presence or absence of one or more genetic markers associated with a disorder other than CAD.

7. The method of any one of the preceding claims, wherein the canine mammal is a French Bulldog, a Boxer, a Boston Terrier.

8. Use of a splice variant in a signaling lymphocytic activation molecule 1 (SLAMF1) gene to identify a canine mammal suffering or at increased risk of suffering from canine atopic dermatitis (CAD).

9. The use according to claim 8, wherein the splice variant in the SLAMF1 gene is downstream of SLAMF1 's exon 4 (c799+2T> C), being the sequence in SEQ ID NO: 3.

10. The use according to claim 8 or 9, wherein the splice variant results in an amino acid sequence including 83 amino acids different from wild type SLAMF1 amino acid sequence before termination according to SEQ ID NO: 4.

11. A method of treating a canine mammal, the method comprising:treating the canine mammal based on a presence or absence of a splice variant in a signaling lymphocytic activation molecule 1 (SLAMF1) gene in a sample of genomic DNA obtained from the canine mammal;wherein the presence of the splice variant indicates that the canine mammal is at increased risk of suffering from CAD, and may pass the risk variant to its progeny.Attorney Docket No.: 00307-0154-00304Mars Ref.: MARS / P / 393656 / WO / SEC / 1 12. The method of claim 11, wherein treating the canine mammal includes administering an anti-inflammatory medication.

13. The method of claim 11, wherein the canine mammal is a French Bulldog, a Boxer, or a Boston Terrier.

14. The method of claim 11, wherein treating the canine mammal is further based on a presence of one or more genetic markers associated w ith a disorder other than CAD.

15. The method of claim 11, wherein the splice variant in the SLAMFl gene is downstream of SLAMFl’s exon 4 (c799+2T> C), being the sequence in SEQ ID NO: 3.

16. The method of claim 11, wherein the splice variant results in an aberrant run of 83 amino acids before termination according to SEQ ID NO: 4.

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