GB2519732A - Composition for canine animals - Google Patents

Abstract

The invention relates to compositions for prevention or treatment of canine hip dysplasia. The use of human muscular dystrophy prophylactics or treatments in canine hip dysplasia, as well as other compositions comprising HDAC or myostatin inhibitors, muscle growth enhancers, anti-inflammatories, antioxidants, cross linking targets, alpha 7 integrin upregulators, steroids or antibiotics, and/or oxygenation proinflammatories is discussed. Also discussed are the potential gene mutations responsible for the symptoms of canine hip dysplasia and the targeting of the gene products as a preventative measure.

Description

Intellectual Property Office Application No. GB1310552.3 RTN4 Date:25 February 2015 The following terms are registered trade marks and should be read as such wherever they occur in this document: M atlab (registered) Intellectual Property Office is an operating name of the Patent Office www.gov.uk/ipo Compositions for Canine Animals The present invention relates to addressing canine hip dysplasia.

Canine hip dysplasia (CHD) is a polygenic disease of abnormal hip joint formation. It is common in many dog breeds, particularly the larger breeds such as the Labrador, Newfoundland, German Shepherd, Golden Retriever, Rottweiler and Mastiff and also some smaller breeds, such as Spaniels and Pugs. Hip dysplasia is one of the most studied veterinary conditions in dogs and is the most common single cause of arthritis of the hip in canine animals.

Canine hip dysplasia can cause mild to extreme pain as well as mobility problems. Dogs exhibit signs of stiffness or soreness after rising from rest, reluctance to exercise, bunny hopping or other abnormal gait, tenderness, pain, reluctance to stand on rear legs, jump up or climb stairs, subluxation or dislocation of the hip joint or wasting away of the muscle mass in the hip area. Radiographs (X-rays) often confirm the presence of hip dysplasia, but radiographic features may not be present until two years of age in some dogs.

Diagnosis is with x-rays or hip score tests. If done at too young an age, the signs may not be revealed.

There is no easy preventioniprophylactic and there is no cure for canine hip dysplasia.

Surgery may be used to reshape the joint to reduce pain or help movement. Hip replacement surgery is expensive and requires significant recovery periods. Often, such extreme measures are not available to canine owners.

The causes of hip dysplasia are considered heritable, although environment may also pay a role. Previously, some genetic variants have been associated with the disease but no specific causative mutations have been identified.

The present invention provides means to prevent development of canine hip dysplasia and to ameliorate the symptoms of canine hip dysplasia. As used herein, prcvcntion includes slowing the development or progression of the dysplasia and!or reducing hip dysplasia.

Accordingly, the present invention provides, according to a first aspect, a human muscular dystrophy prophylactic or treatment for use in the prevention of canine hip dysplasia. Such prophylactic or treatments include compositions which include one or more of sulphoraphane, Creatine, branched-chain amino acids, green tea, CoQIO, giutamine, argine, utrophin, curcumin or selenium.

In particular, the canine is a breed represented by Labrador Retriever, German Shepherd, Rottweilcr, Golden Retriever, Chow Chow, Boxer, Retriever, Pit Bull, Australian Shepherd, Border Coffie, English Bulldo& Beagle, Husky, Pug, Akita, Shih Tzu, Mastiff, Saint Bernard, American Bulldog, Collie, Great Dane, Dalmatian, Basset Hound, Doberman Pinscher, American Cocker Spaniel, Terrier, Siberian Husky or is related to or bred from such a breed.

According to a second aspect, the present invention provides a composition fbr use in the prevention of canine hip dysplasia, wherein the composition ameliorates the symptoms caused by a mutation in any one or more of the following genes: * laminin alpha 2 subunit fragment * collagen, type VI, alpha 3 * histone deacetylase 4 * leucine rich repeat protein 1 * LSM4 homolog, U6 small nuclear RNA associated * CDP-diacylgiycerol synthase (phosphatidate cytidylyltransferase)1 * inositol polyphosphate-5-phosphatase * SH3-domain binding protein 4 * v-ski sarcoma viral oncogene homolog * prostaglandin D2 receptor (PTGDR) Suitable compositions include!br example, laminin-ifi injections to ameliorate the symptoms caused by one or more mutations in the lanñnin alpha 2 submit fragment.

A third aspect of the invention provides composition comprising one or more of the following ingredients, for use in the prevention of canine hip dysplasia: * An HDAC inhibitor or myostatin inhibitor, for example sulforaphanc, curcumin, vaiproic acid, butyrate, biotin, lipoic acid, garlic, Vitamin E or L-argine butyrate.

* A muscle growth enhancer, for example high protein, folic acid (vitamin B9), Calcium, vitamin D, casein, whey protein, branched chain amino acids, essential amino acids (e.g.: glutamine, tyrosine, Arginine, pycnogenol, simple sugars, creatine, creatine phosphate, creatine ethyl ester, creatine monohydrate.

* An anti-inflammatory, for example green lipped muscle, omega 3, curcamin nitric oxide, ehondroitin sulphate in combination with glucatonin-HCL, or dietary nitrates.

* An anti-oxidant, for example green tea extract, glutaminc, flavourals, pcnicillaminc (Vitamin E combination), tairine, chloride, methionine betaine or sulphur amino acids.

* A cross-linking target I such as Vitamin E or branched chain amino acids.

* An alpha 7 intcgrin up-regulator, * A steroid or anabolic * An oxygenation proinflammatory.

A fourth aspect of the invention relates to a method of preventing canine hip dysplasia in a canine (said canine preferably identified as having a risk of developing hip dysplasia). The canine is preferably in need of such prophylaxis. The prevention of canine hip dysplasia according to the present invention is as discussed in the description and the claims. The prevention includes the step of administering a composition of the invention wherein canine hip dysplasia is reduced, the progression or development slowed or in comparison to a canine at the same or similar risk (same or similar level of identification) not receiving the composition. Unexpectedly, it has been determined that the therapeutic use of the compositions described herein are effective in preventing canine hip dysplasia (as defined herein). In particular, it has been determined that the therapeutic usc of the composition decreases the risk of the animal (preferably identified as having a risk of developing canine hip dysplasia and therefore in need of preventative measures) developing canine hip dysplasia. Moreover, the therapeutic use of the composition of the invention as described herein and what comprises the appropriate ingredient/s reduces the cause/development of canine hip dysplasia, thereby enabling the usc of the compositions to prevent canine hip dysplasia. All features of the first aspect described above also relate to the fourth aspect comprising administering to said animal a muscular dystrophy prophylactic or trcatmcnt.

The canine can be identified as having a risk of developing hip dysplasia by being a breed of caninc such as Labrador Rctricvcr, Gcrman Shcphcrd, Rottweiler, Golden Retriever, Chow Chow, Boxer, Retriever, Pit Bull, Australian Shepherd, Border Collie, English Bulldog, Bcaglc, Husky, Pug, Akita, Shih Tzu, Mastiff, Saint Bcrnard, Amcrican Bulldog, Collic, Great Dane, Dalmatian, Basset Hound, Doberman Pinscher, American Cocker Spaniel, Terrier, Sibcrian Husky or is rclatcd to or brcd from such a brccd or by scrccning by the hip-scores or related dogs, (cg. parcnts, cousins, grandparcnts).

A fifth aspect of the invention relates to a method of preventing hip dysplasia in a canine (said caninc identificd as having a risk of developing hip dysplasia, as above), thc mcthod comprising administcring a composition for usc in thc prcvcntion of caninc hip dysplasia, wherein the composition ameliorates the symptoms caused by a mutation in any one or more of the following genes: * laminin alpha 2 subunit fragment; * collagen, type VI, alpha 3; * histone deacetylase 4; and * leucine rich repeat protein I * LSM4 homolog, U6 small nuclear RNA associated * CDP-diacylglyccrol synthase (phosphatidatc cytidylyltransfcrase) 1 * inositol polyphosphate-5-phosphatasc * SH3-domain binding protein 4 * v-ski sarcoma viral oneogene homolog * prostaglandin D2 receptor All aspects of the second aspect also refer to the fifth aspect A sixth aspect of the invention relates to a method of preventing hip dysplasia in a canine (said canine identified as having a risk of developing hip dysplasia, the method comprises administering a composition comprising one or more of the following ingredients, for use in the prevention of canine hip dysplasia: * An HDAC inhibitor * A muscle growth enhancer * An anti-inflammatory * An anti-oxidant * A cross-linking target * An alpha 7 integrin up-regulator * A steroid or anabatic I 0 * An oxygenation proinflammatory All aspects of the third aspect of the invention also relate to the sixth aspect.

The canine may have been identified as having a risk of developing hip dysplasia. Such identification includes X-ray identification according to common general knowledge, a hip score test or canine breed, family history, sex of breed. A definition of CUD can be by the hip joint laxity measured as Norberg Angle or distraction index and hip scores according to the Fédération Cynologique International (FCI) for CHD grading. The risk may be determined by an animal professional, such as a veterinarian or a veterinarian nurse.

Any composition of the invention may be a pet food product in particular a dog food. The pet food product may be a dry food, a wet food, a semi-moist food or a drink, the food may be a snack or treat.

The dog food is preferably packaged. In this way, the consumer is able to identify, from the packaging, the ingredients in the food and is able to confirm that the food is a dog food. The packaging may be metal (usually in the form ofa tin or flexifoil), plastic, paper or card. The dog food contains up to 90% moisture. Wet dog food includes dog food which is usually sold in tins or packages and has a moisture content of from 70 to 90%. Semi-moist dog food contains from over 15 and below 70% moisture. Dry food contains up to and including 15% moisture and is often sold in the form of individual bite-sized pieces, commonly known as kibbles. The amount of moisture in any product may influence the type of packaging which can be used or is required.

The food of this invention encompasses any food which a dog consumes in its diet. Thus, the invention covers standard dry, semi-moist and wet food, as well as dog food snacks and/or treats. Preferably, the food/drink contains the ingredients according to all aspects of the invention for every administration to the dog (that is, every food/drink contains one or more ingrcdicnts which address the problem of eaninc hip dysplasia). Other options include food/drink which is fed daily, weekly, monthly or at any regular or irregular interval.

The food is preferably a cooked product. It may contain gclatiniscd starch. It may be in the form of chunks in gravy, jelly, loaf or water. It may incorporate meat or animal derived material (such as beef, chicken, turkey, lamb, port, fish, blood plasma, marrow bone etc or one or more thereof). The food may include a meat substitute, such as soya, maize g'uten, or a soya product or another non-meat protein source, such as soya protein concentrate, milk proteins, gluten etc. The food may also contain a starch source, such as one or more grains (e.g. wheat, con, rice, oats, barley etc) or may be starch free. The food may contain one or more prebiotics and/or one or more probiotic micro-organisms. The food may contain one or more of colourants, minerals, vitamins, antioxidants and aroma modifiers.

The food may include one or more fibre sources, including digestible, indigestible, soluble, insoluble, fermentable or non-fermentable. Fibre sources include one or more of beet pulp (e.g. sugar or beet pulp), gum arabic, chicory, chicory extract, pysillium, gum talha, rice bran, carob bean gum, citrus pulp, fruetooligosaccharides, insulin, short chain oligofructose, pectin, soy fiber, etc. The food of the invention maybe a complete and nutritionally balanced food. Such a food can be defined as a diet which alone is capable of maintaining life without the need for additional substances, except water.

A supplement includes any form of the invention as defined which is provided to the dog in addition to its usual food. Such a supplement may be added to or mixed into the dog's food or drink or provided separately. In particular, a supplement may be in the form of a powder.

Such a powder may be sprinkled into or mixed with a food or may be in a capsule for administration to the dog. Any dog snack can be used as part of any aspect of the invention.

Any snack, such as "choccy drops", bite-size pieces or e.g. Dentastix® are included. A snack will be one of a dry food, wet food or semi-moist food as defined above.

The ingredient in the composition will vary. Any one or more can be included. The range depends on the dog breed, size, age, energy need, preferred diet, owner requirements etc. Typically, the food/drink will include a range of the ingredient of from 0.01 to 5mg/kg food/drink or an "as is" (by weight) basis. The range maybe from 0.01 to 5mg/kg food, from 0.1 to 1mg/kg food on the same basis.

The present invention also relates to a method of making the food products according to the present invention comprising admixing all the ingredients of the foodstuff/drink and forming the foodstuff/drink. The food/drink may comprise a base wet or dry diet ingredient mix with additional ingredients added. The ingredients may be added together or in any order. The foodstuff'drink may be produced by any method known in the art, including as described in Small Animal Clinical Nutrition, 4th Edition, Hand Thatcher Ressilland Roudebush, 2000, and The Technology of Extrusion Cooking, Blactic Academic & Professional, 1994, Chapman and Hall.

In addition to the aspects of the invention described above, exercise can be implemented alongside the composition for use and the methods of treatment.

A regime of exercise alongside the composition for use and methods of treatment may be of benefit.

The exercise may include one or more of: * walking, for example once or twice a day (between 5 minutes and one hour) * running/jogging, for example once a week or once a day (between 5 minutes and one hour).

Ideally, such exercise is carried out between 0-2 years of age of the dog. Low impact exercise is preferred.

Additional muscle exercises include stretches, use of a vibration exercise machine, electrical muscle stimulation and other non-active muscle stimulation.

Each aspect of the invention can be in combination with any one or more of the muscle exercises described abovc. Thc muscle cxcrcisc can bc prior to, during or after the usc of thc compositions for all aspects of the invention. Preferably, the muscle exercise is for at least 1, 2,3,4,5,6,7,8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20,21,22,23 or24 months of the first 2 ycars of life.

The invention will now be described with reference to the following example:

Example

1.1 Purpose of Example This cxamplc lays out thc results from thc sequcncing projcct and thc subscqucnt analysis.

Following from this it describes the etiology of canine hip dysplasia (CHD) via a combination of minor defects in muscle and variability in growth rates.

By targeting thesc muscle dcfects, a higher muscle growth ratc and a lower bone growth rate the disease may be reduced in severity or even eliminated. This document lays out the current support for this theory, alternative hypotheses and modes of intervention.

2. The Sequencing Project 2.1 Sample collection and plienotyping Blood samples from a population of unrelated Dutch Labradors were collected. The FCI hip scoring system was used to phenotype the dogs. Dogs of score A' were designated as controls and C D or E as cases. Once phylogenetic outliers were removed, 79 phenotyped samplcs (49 CHD cascs and 30 controls) rcmaincd.

In some eases DNA levels were too low for sequencing. A whole genome amplification approach was taken, using three parallel reactions and a pooling of the products. Multiple poois were used to reduce the selection for specific areas of the genome. This produced enough DNA for array-capture.

2.2 Illumina 22k chip genotyping The Illumina Infinium chip was uscd to genotype the samples on approximately 22,000 SNPs.

(Illumina, 2007). Regions were identified by association analysis using the PLINK software.

All coding and regulatory elements within the associated regions could have an effect on CHD development. Therefore all genes within the associated regions are considered candidate genes. 1-lowever, based on the known functions of these genes, a priority set was selected which consisted of genes associated with cartilage of bone development, synthesis or maintenance. This priority set was sequenced in all the samples from the association studies, while the other (non-priority) genes were sequenced in 15 cases and 15 controls. Nine of the 79 samples were of insufficient quality for sequencing, and were replaced by samples positive for CHD from a sample database.

To be able to incrcase the number of genes for sequencing, an exome sequencing approach was adopted. In this case we chose to sequence only exons, intronicxon boundaries (30 bp) and the intergenie regions flanking both sides of each gene (2 x 500 bp). To facilitate haplotype analysis, when the gene was smaller than 3 kb, the 5' (upstream) flanking region was increased until this criterion was met.

To minimize the chance of missing unannotated genes or exons not yet in the current dog genome build, the syntenic regions in the human genome were compared and human eDNA sequences from genes in these regions were BLASTed against the dog genome. When the orientation of the human alignment was correspond to the homologous gene in the dog and located within 100 kb of the gene, this alignment was considered a possible unannotated exon.

Canine expressed sequence tags (EST5) were also BLASTed against the dog genome and alignments were added to the non-priority gene set.

2.3 Sequencing Analysis 2.3.1 SNP mining and genotype assignment Sequencing data was delivered in pileup format. A custom was then used to identify SNPs in the data and extract genotype calls for them.

The custom program performed the following steps: 1. Remove untargetted regions.

Due to repetitive sequence and innefficiency in the capture methodoloy, untargetted DNA is sequenced. All sequence more than 200bp from a targetted base was removed from the data set. Sequence with less than lOOx total coverage over all samples was also removed.

2. For each location in the genome: a. The genotype for each sample is identified, ifan allele is present in more than 10% of the sequences and at least 3 sequences it is counted for that sample b. If at least one sample has a different genotype to the others the location is considered to be a SNP and the genotypes outputted for that location 2.3.2 Significance analysis SNPs were assessed for significance using a chi squared test with two degrees of freedom, testing for independence of phenotype and genotype. The experimental power of non-priority regions is reduced; for this reason SNPs were also considered interesting if they had large odds ratios (>6).

Significant (p-value <0.01) SNPs were checked for significance in the Ensembl Variant Effect Predictor (Ensembl). All other identified SNPs were also processed using this tool and SNPs with interesting predicted effects were noted such as splice site mutations, premature or removed stop codons, non-synonymous coding mutations and mutations in the 3-prime UTR.

Tables IA and lB shows the genes these SNPs are associated with.

Table lÀ: Genes with identified significant mutations lame, aEp1 nthu Eejt& ch eltothe)k is Thm let] £5 C eji«=3t Lfl>Ut 1a4L -1bCôt mHe19 It TiM hr, IogT6 mllnu na R3A Os mars e20 -0cc: gm Ton ----c te 47j714,464-47,721,t]O5 a Ce-4- 5s,561te5 ci let] y lyc rc th Se Os mosrs e 32 cue syncH us cdi Se L,Q E-03 o Ii d t ytTdyM r nsfer 3 1 331, 6 -.s 375,761 C-s hCepbC1e Os msCe3lne 25' te«=3 05 altt tied fC 4 a main binding am -Es urns, c'me 25 -cii -Hears, mutatE ---741 03 9 423 9,155,33 ltsto eeeylth 4 ---sM MUt1 tTll ---42 S: a scalEs peat roles I chum cme& e Ira, IdlyT UTO a2E-oa 9,23 40929,24,995 * Emsart ma es a aaaaJiss a Fag thremesnnse & --one an tanym -t6ss a lairs -- -4 a-q 213 $ a Table I B: Part 2 of Genes with identified significant mutations.

Ensembi iDs: ______________ ____________________________________

Ensernbi. Gene ID Associated Description

_________________________ Gene Name ____________________________________ ENSO.AFGO 0000001 106 LAMA2 laminin, alpha 2 [Souree:HGNC ______________________ ____________ Symbol;Acc:6482] EN SGlEG0 0000-0::.: 9106 CDS 1 CDP-diaeylglyeerol synthase (phosphatidate cyti dyl yliransferase) I ______________________ ____________ [Source:HGNC Symbol;Acc: 1800] ENS CAFGO-0 0 0 0-0 1 1 650: INPPSD inositol polyphosphate-5-phospl-iatase, I 45kDa [Source:HGNC ______________________ ____________ Symbol;Acc:6079] ENSCAFGO 0000012014 S1-13BP4 SH3-domain binding protein 4 ______________________ ____________ [Souree:HGNC Syrnbol;Aec: 10826] ENS CAFGO 0 0 00 0 1222 6 Q9GLN3_CA collagen alpha-3(VI) chain precursor NFA [Souree:RefSeq ______________________ ____________ peptide;Acc:NP 001096685] ENHSCAFG0 0000 0i.25'l 6 AC4 histone deacetylase 4 [Source:HGNC ______________________ ____________ Symbol;Acc: 14063] FNSrFG:) C 0 0 C 0 1 C 7 7 LRR I leucine rich repeat protein I ______________________ ____________ [Source:IIGNC Symbol;Acc: 19742] ET:TscAFG:D. C C ID (C C 14 90 0 LSM4 LSM4 liornolog, U6 small nuclear RNA associated [Source:Refseq ______________________ ____________ peptide;Acc:NP 001104275] F° CAF(; 1) 3 3 ( 3 C: 0 1 9 3 S 7 SM v-ski sarcoma viral oncogene hornolog (avian) [Source:HGNC ______________________ ____________ Symbol;Acc: 10896] ENSCFG.O C Cl 0 0 0 1 1! 692 PTGDR prostaglandin D2 receptor 2.4 Modelling receptor chromosome 8: 31,485,692 -31,494,066 Significant SNPs were taken forward to a Stepwise modelling in MATLAB. A p-value cut-off of 0.00 1 was used for addingircmoving factors in thc process. Thc modc! ling proccss produced a 4 SNP model.

Table 2: Stepwise Linear modelling results _2924 7021 -0 21 Leuc.me uch iepeat protein I $ 2551006290 038 collsgen typeVi alpha3 The odds ratio achieved from the model is not quantifiable because there is 100% NI'V at the bcst cut-off location. Wc can say thc valuc cxcccds 200 though. Whcn a singlc obscrvation of each binary test statistic (true positive, false positive, true negative, false negative) is added, the estimation of odds ratio is 208 with a 95% confidence interval of 23.75 to 1821.97. The accuracy of the model is 94% (95% C.I of 88%-99%), positive predictive value of 90%, negative predictive value of 100%, sensitivity of 100% and specificity of 83%.

3. Gene discussion The following sections discuss the biological relevance and implications of the genes with identified mutations. Results were shared with an experienced veterinarian and an expert in in vitro models of cartilage disease.

3.1.1 Laminin alpha 2 subunit Fragment Mutations in Laminin could therefore be responsible for the non-ideal growth of the joint.

Mutations in the laminin gene have also been shown to cause congenital merosin-deficient muscular dystrophy'. The problem occurs because of a defect in a gene coding a protein that binds muscle fibres together.

The most significant mutation discovered in the gene was a synonymous coding mutation.

Synonymous coding mutations arc mutations in the sequence effecting a protein but not directly changing the protein sequence. However the mutation could still effect protein function as mutations that do not directly change the protein can still have a significant effect.

Up-regulating u7 integrin in a transgenic mouse model of the disease was able to correct the phenotype. This is thought to work by generating more of another protein complex that binds muscles together, "Enhunced exnression of the 7 ntegri regtoreu sirco!emma! iocu lizution at the 7 1 integrin to laminin 2-deficient myofibers, chanced the composition of the muscle extraceUuar matrix, reduced muscle oathelogy, maintained muscle strength and iuncton and imoroved the life expectancy of dy(W/ mice". This suggests that if cC integrin can be stimulated through nutrition, a potential solution might be reached for this ctiological process.

It is also possible that as Laminin is involved in calcium metabolism, the etiology may be partially corrected through nutritionally targeting calcium handling.

The table below shows the mutations detected with p-values and potential for consequence.

The most significant finding was that the synonymous coding mutation does not change amino acid sequence but does sit near a splice site. It is potentially possible that this mutation impacts splice site choice. The principal alternative splice site is 4 bases downstream and would cause a frame-shift being deleterious to protein function. This could be followed up with RNA sequencing. The non-synonymous mutation is in exon 16 changing a serinc at codon 716 to and arginine.

l_70997779 1 70997779 AT,JO116 SYNONYMOUS_CODING I 1_70938018 1 70938018 TA INTRONIC A 1_70976949 1 70976949 AT 0.004028234 INTRONIC A 1_70793904 1 70793904 AG 0.005463992 INTRONIC A 1 70931272 1 70931272 CA Oi721S43t 1_71093063 1 71093063 CT FM0879i52d. INTRONIC 171058059 1 71058059 CT < I426 INTRONIC I

__________________________________ ____________ _____________________________ __________________________________________________________________________ _______ _________________

3.1.2 LSM4 homolog, U6 small nuclear RNA associated In yeast and C.elegans, LSM4 has been associated with growth rate (OMIM, OMIM entry: LSM4 PROTEIN; LSM4, 2003). In meta-studics genes associated with phenotypes such as growth-rate often show orthologous actions in other species. Controlling wowth rate is a known method for delaying the development of canine hip dysplasia.

The most significant of the SNPs (20_477 14388) is less than 100 bases from the gene and could modulate gene expression. The mutation is at a site that is predicted by the TFSEARCH tool to be required for the binding of the FISF and GATA-1 transcription factors.

247714388 20 47714388 GA 0.00021811 UPSTREAM C 20_48071192 20 48071192 GA 0.0DS14i4 DOWNSTREAM G 20_47714336 20 47714336 61 0.0016715 UPSTREAM G 20_47714335 20 47714335 GI 0.003063477 UPSTREAM G 47716669 20 47716669 GA.Q.QO9S98.Q INIRONIC G _____________ _____ __________ _______ \.i*. .VTh _________________________ ______ 3.1.3 Collagen, type VI, alpha 3 Type 6 collagen is located at the epiphyseal owth plate.

Collagen 6A3 (along with Laminin) has also been linked with congenital merosin-defieient muscular dystrophy'.

25_51040259 25 51040259 AG 0.00094Q44 SYNONYMOUS_CODING G 25_51046607 25 51046607 AG OOOW4a7S SYNONYMOUS_CODING A 25_51031100 25 51031100 AC, a00134/90Z SYNONYMOUS_CODING 6 25_51006290 25 51006290 AG 0.001512579 INTRONIC G 25_51028472 25 51028472 CC a00153794& INTRONIC G 25_51030165 25 51030165 AG 0.002222301 INTRONIC G 7_c1o6s4c7 ?S c10(4? A( 0.007408(R1 INTRONIC 25_51034440 25 51034440 CT 0.004776734 INTRONIC C 25_51023068 25 51023068 CT 0.004095797 SYNONYMOUS_CODING T 25_51038074 25 51038074 CT 0.005289604 SYNONYMOUS_CODING C 25_51029326 25 51029326 AG 0.006052002 SPUcF_SLTF.INTRONIIC A 25_51042357 25 51042357 CT 0.006096905 INTRONIC I 51040505 25 51040505 AG 11.02 SYNONYMOUS CODING A 3.1.4 CDP-diacylglycerol synthase (phosphatidate cytidylyltransferase) 1 (CDS1) CDSI is associated with phospholipid processes (specifically phosphatidic acid).

Phospholipids are known to be present in the epiphysis during bone development and important for the process of ossification.

32_11331413 32 11331413 AC 0.001075223 UPSTREAM A 32_11360412 32 11360412 TG 1 0.001075223: INTRONIC I 32_11362156 32 11362156 AG 1 0.001075223 INTRONIC G 32_11366128 32 11366128 CT 1 aoo10m223: SYNONYMOUS_CODING C 32_1137646& 32 11376468 TG 1 0.0010752231 DOWNSTREAM I 32_11376837 32 11376837 TG 1 0,001075223: DOWNSTREAM G 32_12479099 32 12479099 AG 0.00310853 INTRONIC A 32_11365955 32 11365955 AG 0.003405422 INTRONIC G 32_11366052 32 11366052 CT 0.003405422 INTRONIC T 32_11371139 32 11371139 16 0.00357688 INTRONIC G 32_11365924 32 11365924 CT 0.004943424 INTRONIC T 32_11360206 32 11360206 TG 0.006457899 INTRONIC T 3)11.3/gui / { 1 H/80i / AG DOWNSI HFAM A 3.1.5 inositol polyphosphate-5-phosphatase (INPP5D) INIPP5D has been associated with longer lived osteoclasts and altered bone makeup.

47684060 25 47684060 TC a000sf32515 NTRONIC C 25_47664131 25 47664131 CT 0.001731421 SYNONYMOUS_CODING C 25_47684091 25 47684091 TC 0.007982372 INTRONIC C 25_47685188 25 47685188 CT 0.003885197 INTRONIC T - 25_47666842 25 47666842 AG 006492169 SYNONYMOUS_CODING G - 3.1.6 SH3-domain binding protein 4 Unlike many of the other identified mutations in genes, SH3BP4 does not have a direct connection to bone growth. SH3BP4 is however "involved in cargo-specific control of elathrin-mediated endocytosis, specifically controlling the internalization of a specific protein receptor" (NCBT, 2011).

naaaaaaa 25_49142073 25 49142073 CT 0.001744908 UPSTREAM T 3.1.7 histone deacetylase 4 In mouse models, EIDAC4 impacts ehondrocyte hypertrophyby inhibiting the activity of Runx2. HDAC4-null mice displayed premature ossification of developing bones due to eetopie and early chondroeyte hypertrophy.

If the HDAC4 mutation is functional nose morphology changes may occur, as mutations in Runx2 have been associated with nose bridge length across mammalian species.

____ __

Mflflpjj.jkfljj\$j S%1 [zts»=4020z7 25 52402027 CI j 0003226052 INTRONIC iT 3.1.8 Leucine rich repeat protein 1 LRR 1, also known as PPI L5 is known to regulate the 4-1 BB-mediated signalling cascade that results in activation of NFKB and INK 1.

194fl'I 5 294701 IC ft00452 flOW.clRLAM 3.1.9 v-ski sarcoma viral oncogene homolog Mutations in this gene have been associated with many bone morphology and growth speed phenotypes.

5_60210935 5 60210935 AC 0005210815 NON5YNONVMQUSCCDING C 3.1.10 PTGDR Risk allele SNP chr bc bases Consequence _________ 831494277 8 31494277 CT DOWNSTREAM C 8 31494760 8 31494760 CA fl DOWNSTREAM C 4. A Potential Etiology 4.1 CHD, a disease of mild congenital muscle pat Iwlogy? The mutations discovered in the sequencing project are mainly within genes linked to growth rate and a form of muscular dystrophy, Congenital Merosin Deficient Muscular Dystrophy' or CuD (see Table 3).

Table 3: Summary of the genes found to have functional mutations.

tamr -aLpha2subu regiplo chworecome, oneyrTonyrT3o o I 20 au.-tou pl Forfl,ro by fl y U,b to g horn,IJ rnhnq]a Chrornesome2 n C pnroutaipn3 2 S-cowhrte RNAo 0it 7,7,$-7,7 1 90 134e type-VI atpha3 Chrorno once-5 kxSy nyn000 c I gir tatloFt o 940 Mu ular stio hy pJhesito to os CDI> diecylgly ml y ti> Se C rome once one yn y oLson gmu ho 3 Crow h te ho hai1 tecytiyP Ir, er e) 2.332.6-I 75,76 Itla wi-I 9alt41Osph»=t.s-Chrollia 0wi 5 tWO nOhytnOLts 0 log L59wi10514 73E. 3 C 4iwth rate jto photos 7 29,437- 3 Omain binding 911s Clirotil ann s h ups ass a ion i t. nkh Vs 9, 42 306-tkSto doetyl p4 Chrotitamo5 jntthtahs 323-U Molatytrt5h 52 34 0t3-/Gmwth rate-52 43,632 Leucine rich repeat pmtein 1 Chromo orne 8. one down tream potentially in UTR 4.828-03 DQluscular dy trophy 29236 409-v-s -3arccrna ¶0 aIel]coge e Clviorno otne5. one non svnonymouscod pe mutation 5.2 -03 Crowth rate h rooF 60,249 103-213,250 also PTGDR as described above.

Claims (10)

1. Claims 1. A human muscular dystrophy prophylactic or treatment for use in the prevention of canine hip dysplasia.
2. 2. A composition for use in the prevention of canine hip dysplasia, wherein the composition ameliorates the symptoms caused by a mutation in any one or more of the following genes: * laminin alpha 2 subunit fragment * collagen, type VI, alpha 3 * histone deacetylase 4 * eucine rich repeat protein I * LSM4 homolog, U6 small nuclear RNA associated * CDP-diacylglycerol syuthase (phosphatidate cytidylyltransferase) 1 * inositol polyphosphate-5-phosphatase * SH3-domain binding protein 4 * v-ski sarcoma viral oncogene homolog * prostaglandin D2 receptor.
3. 3. A composition comprising one or more of the following ingredients, for use in the prevention of canine hip dysplasia: * An HDAC inhibitor or niyostatin inhibitor * A muscle growth enhancer * An anti-inflammatory * An anti-oxidant * A cross-linking agent * An alpha 7 Tntegrin upregulator * A steroid or antibiotic An oxygenation proinflammatory.
4. 4. A composition, as claimed in claim 2 or claim 3, which is a pet food product.
5. 5. A composition as claimed in claim 4 wherein the pet food is a dry food, wet food, a semi-moist food, or a supplement, or a drink.
6. 6. A method of preventing canine hip dysplasia in a canine (said canine identified as having a risk of developing hip dysplasia) the method comprising administering to said animal a muscular dystrophy prophylactic or treatment.
7. 7. A method of preventing hip dysplasia in a canine (said canine identified as having a risk of developing hip dyspasia), the method comprising administering a composition for use in the prevention of canine hip dysplasia, wherein the composition ameliorates the symptoms caused by a mutation in any one or more of the following genes: * laminin alpha 2 subunit fragment * coflagen, type VI, ifipha 3 * histone deacetylase 4 * eucirie rich repeat protein I * LSM4 honiolog, 1J6 small nuclear RNA associated * CDP-diacylglycerol synthase (phosphatidate cytidylyltransferase) 1 * inositol polyphosphate-5-phosp]iatase * SH3-domain binding protein 4 * v-ski sarcoma viral oncogene honiolog * prostaglandin D2 receptor.
8. 8. A method of preventing hip dysphasia in a canine (said canine identified as having a risk of developing hip dyspiasia), tile method comprises administering a composition comprising one or more of the following ingredients, for use in the prevention of canine hip dysplasia: * An HDAC inhibitor or myostatin inhibitor * A muscle growth enhancer * An anti-inflammatory * An anti-oxidant * Cross-linking agent 1 0 * An alpha 7 integrm upregulator * A steroid or antibiotic * An oxygenation proinfiammatory.
9. 9. A composition as claimed in claim 7 or claim 8, which is a pet food product.
10. 10. A composition as daimed in claim 9, which the pet food in a dry food, a wet food, a semi-moist food, a supplement or drink.