WO2023281167A1 - Procédés de prédiction de performances athlétiques chez des animaux de course - Google Patents

Procédés de prédiction de performances athlétiques chez des animaux de course Download PDF

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Publication number
WO2023281167A1
WO2023281167A1 PCT/FI2022/050491 FI2022050491W WO2023281167A1 WO 2023281167 A1 WO2023281167 A1 WO 2023281167A1 FI 2022050491 W FI2022050491 W FI 2022050491W WO 2023281167 A1 WO2023281167 A1 WO 2023281167A1
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Prior art keywords
racing
sample
athletic performance
expression level
gene
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PCT/FI2022/050491
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English (en)
Inventor
Nathalie CONTE GARCON
Manu TAMMINEN
Emine LUNDSTEN
Shane HOGLE
Maria JENSEN
Jaakko HOLLMÉN
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Aavagen Oy
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Priority to EP22743848.8A priority Critical patent/EP4367265A1/fr
Publication of WO2023281167A1 publication Critical patent/WO2023281167A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/124—Animal traits, i.e. production traits, including athletic performance or the like
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the invention relates to the field of estimating athletic performance of racing animals, in particular racing horses, for example for the purposes of select ing animals for breeding, racing or training. More specifically, the invention relates to a method of predicting or monitoring athletic performance in racing animals, in particular racing horses, and a method for breeding racing animals, in particular racing horses, and selecting racing animals, in particular racing horses, for training or racing.
  • An object of the present invention is to provide improved methods and means for predicting or monitoring athletic performance in a racing animal.
  • the present invention thus provides a method of predicting or monitor ing athletic performance in a racing animal, wherein the method comprises provid ing a sample from said racing animal, determining in the sample expression level of at least one gene selected from Family with sequence similarity 107 member B (FAM107B), C-type lectin domain family 4 member E (CLEC4E) and Transmem brane protein 39a (TMEM39A), comparing the determined expression level to a respective control level, and predicting or monitoring athletic performance on the basis of said comparison.
  • FAM107B Family with sequence similarity 107 member B
  • CLEC4E C-type lectin domain family 4 member E
  • TMEM39A Transmem brane protein 39a
  • the present invention provides a method of selecting a racing animal for training, wherein the method comprises providing a sample from said racing animal, determining in the sample expression level of at least one gene selected from FAM107B, CLEC4E and TMEM39A, comparing the determined expression level to a control level, predicting athletic performance on the basis of said comparison, and selecting the racing animal for training based on the predic tion.
  • the present invention provides a method of se lecting a racing animal for racing, wherein the method comprises providing a sam ple from said racing animal, determining in the sample expression level of at least one gene selected from FAM107B, CLEC4E and TMEM39A, comparing the deter mined expression level to a control level, predicting athletic performance on the basis of said comparison, and selecting the racing animal for racing based on the prediction.
  • the present invention provides a method of selecting a racing animal for racing, said method comprising providing a sample from said racing animal, determining in the sample expression level of at least one gene se- lected from FAM107B, CLEC4E and TMEM39A, comparing the determined expres sion level to a control level, predicting athletic performance on the basis of said comparison, and selecting the racing animal for racing based on the prediction.
  • the invention provides use of FAM107B, CLEC4E or TMEM39A as a biomarker for predicting or monitoring athletic performance in a racing animal, selecting a racing animal for training or racing, or breeding racing animals.
  • the present invention provides a kit for use in the present method, wherein the kit comprises one or more testing agents capable of specifically detecting the expression level of at least one of FAM107B, CLEC4E or TMEM39A in a biological sample obtained from a racing animal.
  • Figure 1 illustrates the correlation between the expression levels of CLEC4E and FAM107B.
  • the symbol corresponds with gender (triangle - stallion, circle - mare) and the size of the symbol is proportional to the amount of money won by the horse;
  • Figure 2 illustrates the correlation between the expression levels of CLEC4E and TMEM39A.
  • the symbol corresponds with gender (triangle - stallion, circle - mare) and the size of the symbol is proportional to the amount of money won by the horse;
  • Figure 3 illustrates the correlation between the expression levels of FAM107B and TMEM39A.
  • the symbol corresponds with gender (triangle - stallion, circle - mare) and the size of the symbol is proportional to the amount of money won by the horse;
  • Figure 4 illustrates the linear response between the expression level of
  • FAM107B and the percentage of races won by the horse (p ⁇ 0.05).
  • the symbol corresponds with gender (triangle - stallion, circle - mare) and the size of the sym bol is proportional to the percentage of races won by the horse;
  • Figure 5 illustrates the linear response between the expression level of FAM107B and the amount of money won by the horse (p ⁇ 0.1).
  • the symbol corre sponds with gender (triangle - stallion, circle - mare) and the size of the symbol is proportional to the amount of money won by the horse.
  • upkeep and training may cost up to 10,000 euros/month per individual. If the horse is never able to race or breed successfully, it may incur a significant financial loss during its lifetime. It is therefore desirable to be able to assess and predict performance of a horse or other racing animal that may be expected to have high performance capabilities.
  • Genetic tools may be utilized as one part of breeders’ toolkits in their search for high performing animals such as elite horses having good athletic per formance.
  • a good athletic performance is above average athletic performance, and may manifest as for example the amount of prize money gathered by a racing ani mal and/or percentage of races won by the racing animal (percentage of races won out of total number of races) and/or placement percentage.
  • a placement percent- age describes the relation between all the races the horse has started in and the amount of races in which the horse has placed first, second or third.
  • a MSTN gene may be linked to muscle strength and best racing distance in the thorough bred horse, as well as a mutation in a DMRT3 gene, which may affect locomotion in horses and have a favourable effect on harness racing performance.
  • Non-genetic monitoring tools may also be utilized.
  • one or more sensors may be used to gather information regarding training of a racing an imal such as a horse or a dog.
  • one or more blood tests may be utilized to monitor health and performance potential of the animal. Such blood tests may be used to investigate for example lactate metabolism and inflam- mation. Heart monitoring data and indicators regarding the current performance potential of a racing animal may also be utilized.
  • Some genetic tests may be utilized for athletic performance prediction. For example, such genetic tests may detect variation on the DNA level by looking at certain genes and presence of mutations in these genes. Yet, they may not provide indications regarding gene activity. While genetic mutations and overall genotype are important factors underlying the characteristics of an individual, it may be that the genotype alone underperforms in predicting complex phenotypes such as ath letic performance. Certain genes and genetic variants have previously been linked to good or improved athletic performance, but presence or absence of a single gene or mutant gene may have limited potential in predicting this trait.
  • Such method may provide a comprehensive analysis and/or prediction of athletic performance potential by gene activity assays deter mining expression level of one or more genes. In this way it may be possible to determine athletic performance potential, monitor the performance potential over time and/or predict future performance potential.
  • the present invention is based, at least in part, on determining gene ex pression profiles of horses. Genes may be regulated both spatially and temporally by a process called gene expression, via which information contained in genes is translated into a functional gene product, such as a protein. Gene expression is a level on which the genotype gives rise to a phenotype, i.e. an observable trait.
  • the invention relates to different aspects of the FAM107B, CLEC4E and TMEM39A genes as predictive markers of athletic performance in racing animals. Accordingly, in some aspects, the invention relates to different uses of said markers and to different in vitro methods of predicting athletic performance in racing ani mals. These genes can be used to create a gene expression test to identify racing animals that have a better athletic performance than others. The methods and as says described herein are performed ex vivo and can be considered to be ex vivo or in vitro methods and assays.
  • the present invention is, at least partly, based on a surprising finding that changes in expression levels of FAM107B, CLEC4E and TMEM39A genes in a sample obtained from a racing animal indicates athletic performance of said ani mal. That is, gene expression levels of FAM107B, CLEC4E and TMEM39A correlate with athletic performance.
  • gene expression profiling data of horses was combined with an objective performance metric constructed to objectively compare the performance of indi vidual horses.
  • an objective performance metric constructed to objectively compare the performance of indi vidual horses.
  • FAM107B is Family with sequence similarity 107 member B gene. Re cently, FAM107B has been identified to influence heat stress response in dairy cat tle. In humans, FAM107B plays a role in genetic interactions for the causes of car diovascular diseases.
  • CLEC4E is C-type lectin domain family 4 member E gene. In general, animal lectins act as recognition molecules within the immune system, their functions involving defence against pathogens, cell trafficking, immune regulation and the prevention of autoimmunity. In humans, CLEC4E is a calcium-dependent lectin that recognizes damage-associated molecular patterns (DAMPs) and interacts with signaling adapter Fc receptor gamma chain/FCERIG to activate an immune response.
  • DAMPs damage-associated molecular patterns
  • TMEM39A is Transmembrane protein 39a. In humans, TMEM39A is known to be involved in inflammation, dysregulated type 1 interferon responses, and other immune processes. In C. elegans, the TMEM39A ortholog TMEM-39 has recently been discovered to regulate stress response in the endoplasmic reticulum, collagen secretion as well as lysosome distribution and accumulation.
  • the present invention relates to a method for pre dicting athletic performance in racing animals on the basis of expression level of one or more of FAM107B, CLEC4E and TMEM39A genes.
  • the method comprises determining in a sample obtained from said racing animal the level of expression of one or more of FAM107B, CLEC4E and TMEM39A, comparing the determined expression level to a control level, and predicting athletic performance on the basis of said comparison.
  • decreased expres- sion of one or more of FAM107B, CLEC4E and TMEM39A indicates good athletic performance
  • increased or non-decreased expression of one or more of FAM107B, CLEC4E and TMEM39A indicates poor or average athletic performance.
  • the present invention relates to a method for predicting athletic performance in racing animals on the basis of expression level of the FMA107B gene and/or CLEC4E gene.
  • the method comprises determining in a sample obtained from said racing animal the level of expression of FAM107B and/or CLEC4E, comparing the determined expression level to a control level, and predicting athletic performance on the basis of said comparison.
  • the method for predicting athletic per formance in racing animals comprising determining expression level of FAM107B and/or CLEC4E in a sample may further comprise determining in said sample also expression level of TMEM39A, comparing the determined expression level to a con trol level, and predicting athletic performance on the basis of said comparison.
  • co-expression of CLEC4E and/or TMEM39A with FAM107B indicate(s) athletic performance in that decreased expression of FAM107B, and/or decreased expression of CLEC4E, and/or decreased expression of TMEM39A indicate(s) good athletic performance, whereas increased or non-de- creased expression of FAM107B, and/or increased or non-decreased expression of CLEC4E, and/or increased or non-decreased expression TMEM39A indicate(s) poor or average athletic performance.
  • the present invention allows for predicting and monitoring athletic performance in racing animals in a minimally invasive fashion (i.e. from a sample of e.g. blood, tissue or a bodily fluid).
  • the basis of prediction decision-making in the method of the invention may be in the form of a level of expression of one or more of the genes FAM107B, CLEC4E and TMEM39A indicative of athletic performance.
  • the method may further be relevant for predicting athletic performance based on inherited traits related to athletic performance or charac teristics affecting athletic performance that are completely or in part due to exter nal and environmental factors.
  • "a" or "an” may mean one or more.
  • the term “comprising” means “including principally, but not necessarily solely”. Variations of the word “comprising”, such as “comprise” and “comprises”, have correspondingly similar meanings.
  • the term "gene expression profiling” or “transcriptional profiling” may refer to analysis of the transcriptome of the organism.
  • the transcrip- tome is the set of all RNA molecules in one cell or a population of cells. It may be used to refer to all RNAs, or just mRNA.
  • the transcriptome in this exemplary em bodiment, includes only those RNA molecules that are found in a specified cell pop ulation, and may include the amount or concentration of each RNA molecule in ad dition to the molecular identities.
  • Gene expression profiling may thus indicate which genes are actively transcribed, what their level of expression is, and at what times they are activated or silenced.
  • gene expression profiling as used herein may refer to determining of expression level(s) of one or more target genes instead of the entire transcriptome.
  • DNA microarrays and quantitative PCR alone or in combination may be used for gene expression profiling.
  • next generation sequencing (NGS) methods maybe used for perform ing gene expression profiling.
  • Gene expression profiling may also be performed via determining levels of one or more target gene products such as proteins.
  • any means known to a skilled person may be used included ing mass spectrometry and antibody-based methods.
  • sample may be considered as a biological 5 sample, typically a clinical sample collected or obtained from a racing animal.
  • the sample may be for example a tissue sample, a biopsy, a cell sample, a skin sample, a blood sample such as serum, plasma or peripheral blood, a semen sample, a saliva sample, a bodily fluid sample, a stool sample, a swab, a hair cell sample and/or a urine sample.
  • the sample is a blood sample. Gener ic) ally, obtaining the sample to be analyzed from a racing animal is not part of the present method.
  • sample also includes samples that have been manipulated or treated in any appropriate way after their procurement, including but not lim ited to centrifugation, filtration, precipitation, dialysis, chromatography, treatment 15 with reagents, washing, or enriching for a certain component of the sample such as a cell population.
  • the sample may be collected or obtained from any racing animal includ ing, without limitation, ungulates, carnivores and birds.
  • Exemplary racing animals include horses, camels, mules, donkeys, elephants, Homo sapiens, hares, kangaroos, 20 ostriches, racing pigeons, hawks, falcons, owls and dogs such as greyhounds.
  • the racing animal is a horse.
  • Horses include thoroughbred and non-throughbred horses, Arab endurance horses, polo ponies, quarter horses, trotting horses and standardbreds.
  • biomarker and “marker” are interchange- 25 able, and refer to a molecule that is differentially present or expressed in a sample taken from a racing animal individual having or predicted to have good athletic performance, as compared to a comparable sample taken from control individual, such as a racing animal individual having or predicted to have poor or average ath letic performance.
  • present biomarkers such as FAM107B, CLEC4E and 30 TMEM39A provide information regarding athletic performance and associate with good athletic performance.
  • present biomarker refers to any individual biomarker set forth above, preferably FAM107B or CLEC4E, or to any biomarker combination thereof.
  • the term encompasses not only FAM107B and/or CLEC4E but also any combinations of FAM107B and/or CLEC4E and one or more 35 biomarkers set forth herein that are co-expressed with FAM107B and/or CLEC4E.
  • level or “expression level”, when applied to a biomarker, may be used interchangeably with the terms “amount” and “concentra tion”, and can refer to an absolute or relative quantity of the biomarker.
  • the term "athletic performance” refers to qualities of racing animals that may manifest as for example earnings i.e. the amount of prize money gathered by a racing animal and/or percentage of races won by the racing animal (percentage of races won out of total number of races) and/or placement percentage.
  • racing animals with "good athletic performance” may win an above average amount of prize money as compared to other racing animals of the same group, wherein the same group may refer to e.g. racing animals com- peting in the same racing category or racing animals of the same bloodline or of the same owner or trainer or breeder.
  • racing animals with “average athletic performance” or “poor athletic performance” may win an average or below average amount of prize money as compared to other racing an imals of the same group, wherein the same group may refer to e.g. racing animals competing in the same racing category or racing animals of the same bloodline or of the same owner or trainer or breeder.
  • racing animals with good athletic perfor mance may have an above average percentage of races won out of all races the an imal competed in, or above average placement percentage.
  • the per- centage of races won may be for example at least 30%, preferably at least 40%, more preferably at least 50% even more preferably at least 60%.
  • the placement percentage may be at least 30%, preferably at least 40%, more preferably at least 50% even more preferably at least 60%.
  • rac ing animals with average or poor athletic performance may have an average or be- low average percentage of races won out of all races the animal competed in, or below average placement percentage.
  • the percentage of races won may be for example less than 30%, preferably 25% or less, more preferably 20% or less, even more preferably 15% or less.
  • the placement per centage may be less than 30%, preferably 25% or less, more preferably 20% or less, even more preferably 15% or less.
  • a racing ani mal with good athletic performance is able to perform at a high level in terms of endurance, speed and/or strength (e.g. such that the racing animal is capable of competing at national and/or international level(s) in the racing sport).
  • “Racing sport” and the like as used herein includes racing such as com- petitive racing and equestrian sports such as racing, showjumping, eventing, dres sage, endurance events, riding, hunting, polo and the like.
  • the equestrian sports may be competitive sports.
  • the term “elite” refers to racing animals having or pre dicted to have good athletic performance. Additionally or alternatively, as used herein, the term “non-elite” refers to racing animals having or predicted to have average or poor racing performance. In a non-limiting example, an "elite racing an imal” or variants thereof, refers to a racing animal that performs at the very highest levels in terms of endurance, speed and/or strength (e.g. such that the racing ani mal is capable of competing at national and/or international level(s) in the racing sport). In an embodiment, samples may be collected from various categories, such as elite and non-elite animals, to facilitate comparisons between for example two or more types of animals.
  • elite racing animals may further have a good athletic performance in terms of one or more features including: racing performance, which may be understood as for example track record including, but not limited to, features such as amount of earnings, win per centage, placement percentage, record time, top speed, average speed, age, and/or number of competitions, characteristics such as age, will to win, capability to tolerate pain (e.g. lactic acid build-up), mental soundness (equable temper, stress tolerance), good appetite (easy to make adjustments to diet, efficient liquid uptake), confor mation, and/or pedigree, or lack of undesirable characteristics such as a high inbreeding coefficient, challenging temper, diseases, hereditary diseases and/or other physical de fects.
  • racing performance which may be understood as for example track record including, but not limited to, features such as amount of earnings, win per centage, placement percentage, record time, top speed, average speed, age, and/or number of competitions, characteristics such as age, will to win, capability to tolerate pain (e.g. lactic acid build-
  • expression level of one or more of FAM107B, CLEC4E and TMEM39A genes and optionally other physiological and/or genetic parameters may be measured in racing animals such as horses in order to more accurately access the athletic performance of a racing animal at an early age. It is contemplated that racing animals may be pre-screened before using them for breeding purposes to identify a more satisfactory genetic match. In addition, it is possible that an offspring in utero may be screened to assess the athletic perfor mance potential of the offspring before it is born. The information generated from such screenings would save the breeders and investors of racing animals a tremen dous amount of time and money as well as identify the potential ability of an animal at an early stage of development.
  • control may refer to a comparable sample obtained from a control individual or a pool of control individuals with a known athletic performance.
  • individual refers to a racing animal.
  • appropriate control individuals include racing animals or pools of racing animals having good athletic performance.
  • preferred control individuals are racing animals or pools of racing animals having an average or poor athletic performance.
  • control may also refer to a predetermined threshold or con trol value, originating from a single control individual or a pool of control individ uals set forth above, which value is indicative of athletic performance.
  • Statistical methods for determining appropriate threshold or control values will be readily apparent to those of ordinary skill in the art, and the statistically validated thresh- old or control values can take a variety of forms.
  • a statistically vali dated threshold can be a single cut-off value, such as a median or mean.
  • a statistically validated threshold can be divided equally (or unequally) into groups, such as good, average and poor athletic performance groups, optionally the good athletic performance group being individuals having and/or predicted to have good athletic performance and the poor athletic performance group being in dividuals having and/or predicted to have poor athletic performance.
  • the threshold may be an absolute value or a relative value. However, if an absolute value is used for the level of the assayed biomarker, then the threshold value is also based upon an absolute value. The same applies to relative values, which must be comparable.
  • the biomarker levels are nor malized using standard methods prior to being compared with a relevant control.
  • the threshold or control value may be determined or set for example by a breeder or owner or trainer of racing animals or it may be obtained based on computer aided analysis, for example.
  • racing animal individuals of the same or similar amount of earnings, win percentage, placement percentage, pedigree or bloodline, age and/or disease status, etc. may be employed as appropriate control individuals for obtaining comparable control samples or determining a statistically validated threshold value.
  • the levels of the assayed biomarkers in the sample may be compared with one or more single control values or with one or more ranges of control values, regardless of whether the control value is a predetermined value or a value ob tained from a control sample upon practicing the prediction method.
  • the signifi cance of the difference of biomarker levels in the sample and the control can be assessed using standard statistical methods.
  • the term "positive control” refers to a control from an individual or pool of individuals having good athletic performance.
  • the term “negative control” refers to a control from an individual or pool of individuals having average or poor athletic performance.
  • a statistically significant decrease or non-increase between the assayed biomarker level and a positive con trol level indicates that the individual is even more likely to have good athletic per formance than an individual with biomarker levels comparable to or above the sta tistically validated positive control value. In such cases, decreased or non-in- creased biomarker levels are indicative of good athletic performance.
  • a statistically significant increase between the assayed biomarker level and a positive control level indicates that the racing animal individual is not likely to have a good athletic performance or indicates that the individual has average or poor athletic performance.
  • a statistically significant decrease be tween the assayed biomarker level and a negative control level indicates that the individual is likely to have a good athletic performance.
  • a statis tically significant increase or non-decrease between the assayed biomarker level and a negative control level indicates that the racing animal individual is not likely to have a good athletic performance or indicates that the individual has average or poor athletic performance.
  • a control may be from a racing animal or a pool of racing animals that has undergone one or more changes in environmental fac tors that may affect phenotype to monitor effect of these change (s) on athletic per formance potential.
  • the control may be from the same one or more racing animal or pool of racing animals before undergoing the change (s) in environmental factors and/or from one or more racing animal or pool of racing animals that have not un dergone the change(s) in environmental factors.
  • the environmental factors may include one or more factors including health, disease, medication, nutrition, train ing, ageing, racing, breeding, diet, and hoof care.
  • expressions like "indicative of good athletic perfor mance" and the like refer, at least in some embodiments, to a biomarker which, using routine statistical methods and optionally setting confidence levels at a min imum of 95%, is predictive of good athletic performance such that the biomarker is found significantly more seldom, or in lower levels, in individuals with good ath letic performance than in individuals with average or poor athletic performance.
  • a predictive biomarker which is indicative of a good athletic perfor- mance is found at an expression level ratio which is 0.95 or less or 0.9 or less, or 0.8 or less, or 0.7 or less, or 0.6 or less, or 0.5 or less, or 0.4 or less, or 0.3 or less, or 0.2 or less.
  • the expression level ratio is calculated as expression level in sample divided by control, wherein the control level may be a negative or a positive con trol.
  • expressions like "indicative of poor athletic performance” and the like refer, at least in some embodiments, to a biomarker which, using rou tine statistical methods and optionally setting confidence levels at a minimum of 95%, is predictive of poor athletic performance such that the biomarker is found significantly more often, or in lower levels, in individuals with poor or average ath letic performance than in individuals with good athletic performance.
  • a predictive biomarker which is indicative of a poor or average athletic performance is found at an expression level ratio which is at least 1.05, or at least 1.1, or at least 1.2, or at least 1.3, or at least 1.4, or at least 1.5, or at least 2.0, or at least 3.0, or at least 4.0, or at least 5.0 or more.
  • the expression level ratio is calculated as expres sion level in sample divided by control, wherein the control level may be a negative or a positive control.
  • the term “decreased level” refers to a decrease in the amount of a biomarker in a sample as compared with a relevant control.
  • the de crease may be a decrease in expression level. Said decrease can be determined qualitatively and/or quantitatively according to standard methods known in the art.
  • the term “decreased” encompasses a decrease at any level but refers more spe cifically to a decrease between about 10% and about 250% as compared with a relevant control.
  • the biomarker is decreased by at least 10%, by at least 15%, by at least 20%, by at least 25%, by at least 30%, by at least 35%, by at least 40%, by at least 45%, by at least 50%, by at least 55%, by at least 60%, by at least 65%, by at least 70%, by at least 75%, by at least 80%, by at least
  • the term "decreased level” refers to a statistically sig nificant decrease in the level or amount of the biomarker as compared with that of a relevant control.
  • the term “increased level” refers to an increase in the amount of a biomarker in a sample as compared with a relevant control.
  • the in crease may be an increase in expression level. Said increase can be determined qualitatively and/or quantitatively according to standard methods known in the art.
  • the term “increased” encompasses an increase at any level but refers more specifically to an increase between about 10% and about 250% as compared with a relevant control.
  • the biomarker is increased by at least 10%, by at least 15%, by at least 20%, by at least 25%, by at least 30%, by at least 35%, by at least 40%, by at least 45%, by at least 50%, by at least 55%, by at least 60%, by at least 65%, by at least 70%, by at least 75%, by at least 80%, by at least
  • the term "increased level” refers to a statistically sig- nificant increase in the level or amount of the biomarker as compared with that of a relevant control.
  • non-increased or “non-decreased” or “nor mal” refers to a detected or assayed biomarker level that is essentially the same or essentially non-altered as compared with that of a relevant control sample or a pre- determined threshold value.
  • the invention is useful to various aspects of the sport of racing, for ex ample horse racing. These aspects include gambling, training, breeding, veterinary or nutritional fields.
  • the invention facilitates analysis of genetic expression profiles so as to identify positive outcomes resulting in or contributing to elite athletic per- formance and optionally other characteristics which affect athletic performance or ability.
  • FAM107B, CLEC4E and TMEM39A genes it is possible to rank indi viduals in relation to athletic performance.
  • the pre sent invention permits a classification of the population of racing animals also for breeding, training and racing suitability. For example, for particular races, contest ants can be ranked based on analysis of expression level of one or more of FAM107B, CLEC4E and TMEM39A genes.
  • the applications of the present invention would be e.g. in planned breeding and the selection of mates for breeding stock, the identification of superior animals prior to the start of their racing careers (either in selection of yearlings to buy or as selection of which colts to leave as entires and which to geld), and in the creation of specific, individually designed training pro grams that are tailored to genetic potential of individual horses.
  • the present invention relates to a method of breeding a racing animal on the basis of expression level of one or more of FAM107B, CLEC4E and TMEM39A genes.
  • the method comprises providing a sample from one or more racing animal(s), determining in the sample(s) expression level of at least one gene selected from FAM107B, CLEC4E and TMEM39A, comparing the determined ex- pression level to a control level, predicting athletic performance on the basis of said comparison, selecting a first breeding racing animal from the one or more rac ing animal(s) based on the prediction, optionally selecting a second breeding rac ing animal from the one or more racing animal(s) based on the prediction, breeding the first breeding racing animal, wherein optionally the first breeding racing ani- mal is bred with the second breeding racing animal.
  • a racing animal predicted to have good athletic performance based on decreased expression level of the at least one gene is se lected as the first breeding racing animal. In a further embodiment, a racing animal predicted to have good athletic performance based on decreased expression level of the at least one gene is selected as the second breeding racing animal.
  • the present invention is also useful for trainers of racing animals be cause it will allow them to target the training regimes of racing animals to their genetic potential. Similarly, it may also aid trainers involved in the purchase of rac ing animals for clients in their decision-making processes.
  • FAM107B, CLEC4E and TMEM39A genes may be linked with particular physiological condi tions or processes, which may need particular handling with regard to training ap proaches.
  • laboratory based biochemical or biological tests such as measurement of heart rates or V02 Max, etc., employing mechanical apparatus (e.g. treadmills etc.), or white blood cell counts to estimate 'immunolog- ical' health for racing may be correlated with the expression level of one or more of FAM107B, CLEC4E and TMEM39A genes for the assessment of physical and rac ing/breeding capabilities.
  • database products detailing the expected racing performance based on FAM107B, CLEC4E and/or TMEM39A expression level types and biochemical and/or biological laboratory tests at any one time would be valuable.
  • a very important use will be where racing animals varying in their expression levels can be shown to respond differently to certain drugs, hor mones or manual treatment. Identification of these predispositions through a test or a database will enhance the effects of these processes or treatments. Planning of reproductive or rehabilitation processes following injury or trauma may also be aided by pro-gramme targeting through expression level definition.
  • racing animals of particular FAM107B, CLEC4E and/or TMEM39A expression level may respond differently to specific feeding or nutri tional regimes. It is likely that they will vary with regard to such features as live weight gain or increases in muscle mass.
  • Other uses include use studying the inter- action of diet, such as responses to the use of probiotics, nutraceuticals and other compounds.
  • labora tory based tests such as measurement of heart rates or V02 Max, white blood cells etc., employing mechanical apparatus (e.g. treadmills etc.)
  • FAM107B, CLEC4E and/or TMEM39A expression level for the assessment of veter inary treatments, processes or use of pharmaceuticals.
  • the invention is also particularly useful because it improves the welfare of racing animals in training. For example, by identifying types which are suscepti ble to breakdown or other failures in training also reduces the risk of an animal from being trained in areas for which is it unsuitable.
  • the present invention may even help to select racing animals which are more physically capable of surviving specific training regimes.
  • the present invention relates to a method of selecting a racing animal for training on the basis of expression level of one or more of FAM107B, CLEC4E and TMEM39A genes.
  • the method comprises providing a sam- pie from a racing animal, determining in the sample expression level of at least one gene selected from FAM107B, CLEC4E and TMEM39A, comparing the determined expression level to a control level, predicting athletic performance on the basis of said comparison, and selecting the horse for training based on the prediction.
  • the gene the expression level of which is determined in the method of selecting a racing animal for training is FAM107B and/or CLEC4E.
  • a racing animal predicted to have good ath letic performance based on decreased expression level FAM107B, CLEC4E and/or TMEM39A is selected for training.
  • the present invention relates to a method of select ing a racing animal for racing on the basis of expression level of one or more of FAM107B, CLEC4E and TMEM39A genes. The method comprises providing a sam ple from a racing animal, determining in the sample expression level of at least one gene selected from FAM107B, CLEC4E and TMEM39A, comparing the determined expression level to a control level, predicting athletic performance on the basis of said comparison, and selecting the horse for racing based on the prediction.
  • the gene the expression level of which is determined in the method of selecting a racing animal for racing is FAM107B and/or CLEC4E.
  • a racing animal predicted to have good ath letic performance based on decreased expression level FAM107B, CLEC4E and/or TMEM39A is selected for racing,
  • the present invention provides ranking the genetic profile of an individual racing animal or a group of racing animals, or predicting the relative outcome of racing involving several indi viduals, on the basis of expression level of one or more of FAM107B, CLEC4E and TMEM39A genes. Further, the resulting analysis may be applied so as to rank the individuals according to preferred racing category or distance.
  • the present inven tion further provides determining suitable odds for gambling, optionally taking into account information on other race specific factors.
  • the information provided by the method of invention can be used to prepare a database, literature or a test system. Such databases are a yet further aspect of the invention and their use in consultancy or advice businesses.
  • the invention has application to all types of national and international rac ing systems and amateur as well as professional/top class racing.
  • the present invention is useful in the betting and gambling industry.
  • the information provided can supplement traditional information (going, recent form etc.) in assisting settling of odds for any particular race.
  • Fur thermore more accurate information can be assembled on racing animals appear ing during any one season.
  • the present invention has applications to both the lay- ing and placing of bets and offers useful tools for the bookmaking organisation in providing useful information when deciding odds.
  • the invention enables the devel opment of databases that can provide pre-race genetic data which can be used as supplementary information for the compiling of odds and laying of bets.
  • Horses-in-training operating costs can be reduced and racing strat- egy can be fine-tuned by identifying the most precocious two-year olds having best athletic performance, and horses can be trained and raced for optimal racing dis tance
  • Broodmares breeding outcomes can be optimised by focusing on op timal breeding mares and selecting compatible stallions, both with good athletic performance.
  • Stallions a stallion’s potential can be promoted by predicting good athletic performance of young stallions to attract compatible mares to enhance stallion profile.
  • the predictive tests described herein may be applied to select individ- uals with genetic potential for good athletic performance. These tests can be per formed on an individual at any stage in the life cycle e.g. before birth in utero, Day 1 (birth), prior to sales (i.e. yearlings, 2 year olds etc), during racing career (i.e. from 2 years old), during breeding (i.e. up to approximately 25 years).
  • the expression level of any one of the present biomarkers may be de termined by a variety of techniques.
  • the expression at the nucleic acid level may be determined by measuring the quantity of RNA, preferably mRNA or any other RNA species representing the biomarker in question, using standard methods well known in the art.
  • suitable methods include digital PCR and real-time (RT) quantitative or semi-quantitative PCR. Primers suit able for these methods may be easily designed by a skilled person.
  • Further suitable techniques for determining the expression level of any one of the present biomarkers at nucleic acid level include, but are not limited to, fluorescence-activated cell sorting (FACS) and in situ hybridization.
  • FACS fluorescence-activated cell sorting
  • RNA preferably mRNA or any other RNA species representing the biomarker in question
  • transcriptome approaches in particular, DNA microarrays.
  • test and control mRNA samples are re verse transcribed to cDNA which is labeled e.g. with a fluorescent tag to generate labeled targets.
  • the labeled targets are then hybridized to an array of complemen tary nucleic acid probes immobilized on a solid support.
  • the array is configured such that the sequence and position of each unique probe is known. Hybridization of a unique probe with a labeled target indicates that the sample from which the probe was derived expresses that gene.
  • Non-limiting examples of commercially available microarray systems include Affymetrix GeneChipTM and lllumina Bead- Chip. Furthermore, bulk RNA sequencing, single-cell RNA sequencing or cDNA sequencing, e.g. by Next Generation Sequencing (NGS) methods, may also be used for determining the expression level of any one of the present biomarkers.
  • NGS Next Generation Sequencing
  • the quantity of RNA may also be determined or measured by conventional hybridization-based assays such as Northern blot analysis, as well as by mass cytometry using e.g. Metal In Situ Hybridization (M1SH).
  • M1SH Metal In situ Hybridization
  • Changes in the regulation of activity of a gene encoding the biomarker in question can be determined through epigenetic analysis, such as histone modi fication analysis, for example by chromatin immunoprecipitation (ChlP) followed by sequencing or quantitative PCR, or quantitation of DNA methylation levels, for example by bisulfite sequencing or capture based methods, at the intergenic regulatory sites or gene region of the biomarker in question.
  • epigenetic analysis such as histone modi fication analysis, for example by chromatin immunoprecipitation (ChlP) followed by sequencing or quantitative PCR, or quantitation of DNA methylation levels, for example by bisulfite sequencing or capture based methods, at the intergenic regulatory sites or gene region of the biomarker in question.
  • a variety of techniques may be employed for determining the expression level of any one of the present bi omarkers at the protein level.
  • suitable methods include mass spectrometry-based quantitative proteomics techniques, such as isobaric Tags for Relative and Absolute Quantification reagents (iTRAQ) and label-free anal ysis, as well as selected reaction monitoring (SRM) mass spectrometry and any other techniques of targeted proteomics.
  • iTRAQ isobaric Tags for Relative and Absolute Quantification reagents
  • SRM selected reaction monitoring
  • the level or amount of a protein marker may be determined by e.g.
  • an immunoassay such as ELISA or LUMINEX®
  • Western blotting spectrophotometry, an enzymatic assay, an ultraviolet assay, a kinetic assay, an electro-chemical assay, a colorimetric assay, a turbidimetric assay, an atomic absorption assay, flow cytometry, mass cytometry, or any combination thereof.
  • suitable analytical techniques include, but are not limited to, liquid chromatography such as high performance/pressure liquid chromatography (HPLC), gas chromatography, nuclear magnetic resonance spectrometry, related techniques and combinations and hybrids thereof, for example, a tandem liquid chromatography-mass spectrometry (LC-MS).
  • the present disclosure also relates to an in vitro kit for predicting ath letic performance of racing animals.
  • the kit may be used in any implementation of the present method or its embodiments.
  • the kit comprises one or more testing agents or reagents that are capable of detecting one or more of the present biomarkers, preferably at least FAM107B, or determining its expression level.
  • the kit may comprise a pair of primers and/or a probe specific to FAM107B and/or CLEC4E.
  • a skilled person can easily design suit able primers and/or probes taking into account specific requirements of a tech nique to be applied.
  • the kit may further comprise means for detecting the hybrid ization of the probes with nucleotide molecules, such as mRNA or cDNA, represent ing FAM107B or CLEC4E in a test sample and/or means for amplifying and/or de- tecting the nucleotide molecules representing FAM107B or CLEC4E in the test sam ple by using the pairs of primers.
  • the kit may also comprise one or more testing agents or reagents for detecting one or more genes co-regulated with FAM107B and/or CLEC4E or interaction partners of FAM107B and/or CLEC4E in accordance with the disclosure above.
  • kits include a compartmentalized carrier means, one or more buffers (e.g. block buffer, wash buffer, substrate buffer, etc.), other reagents, positive or negative control samples, etc.
  • buffers e.g. block buffer, wash buffer, substrate buffer, etc.
  • other reagents e.g. positive or negative control samples, etc.
  • the kit may also comprise a computer readable medium comprising computer-executable instructions for performing any method of the present dis- closure.
  • a method of predicting or monitoring athletic performance in a racing animal comprising providing a sample from said racing animal, determining in the sample expression level of at least one gene selected from Family with sequence similarity 107 member B (FAM107B), C-type lectin do main family 4 member E (CLEC4E) and Transmembrane protein 39a (TMEM39A), comparing the determined expression level to a respective control level, and predicting or monitoring athletic performance on the basis of said com parison.
  • FAM107B Family with sequence similarity 107 member B
  • CLEC4E C-type lectin do main family 4 member E
  • TMEM39A Transmembrane protein 39a
  • control level is determined in a control sample or the control level is a predetermined threshold value, optionally wherein the control sample is from a control individual or a pool of control individuals with poor or average athletic performance.
  • a method of selecting a racing animal for training said method com prising providing a sample from said racing animal, determining in the sample expression level of at least one gene selected from FAM107B, CLEC4E and TMEM39A, comparing the determined expression level to a control level, predicting athletic performance on the basis of said comparison, and selecting the racing animal for training based on the prediction.
  • a method of selecting a racing animal for racing said method com- prising providing a sample from said racing animal, determining in the sample expression level of at least one gene selected from FAM107B, CLEC4E and TMEM39A, comparing the determined expression level to a control level, predicting athletic performance on the basis of said comparison, and selecting the racing animal for racing based on the prediction.
  • a method of breeding racing animals comprising providing a sample from one or more racing animal(s), determining in the sample(s) expression level of at least one gene se lected from FAM107B, CLEC4E and TMEM39A, comparing the determined expression level to a control level, predicting athletic performance on the basis of said comparison, selecting a first breeding racing animal from the one or more racing an imals) based on the prediction, optionally selecting a second breeding racing animal from the one or more racing animal(s) based on the prediction, breeding the first breeding racing animal, wherein optionally the first breeding racing animal is bred with the second breeding racing animal.
  • control level is determined in a control sample or the control level is a predeter mined threshold value, optionally wherein the control sample is from a control in dividual or a pool of control individuals with poor or average athletic performance.
  • a racing animal predicted to have good athletic performance based on decreased expression level of the at least one gene is selected as the first breeding racing animal, optionally wherein a racing animal predicted to have good athletic performance based on de creased expression level of the at least one gene is selected as the second breeding racing animal.
  • the sample is a tissue sample, a biopsy, a cell sample, a skin sample, a blood sample such as serum, plasma or peripheral blood, a semen sample, a saliva sam ple, a bodily fluid sample, a stool sample, a swab, a hair cell sample and/or a urine sample.
  • FAM107B, CLEC4E or TMEM39A as a biomarker for predict- ing or monitoring athletic performance in a racing animal, selecting a racing animal for training or racing, or breeding racing animals.
  • kit in the method according to any of the embodiments 1 to 12, wherein the kit comprises one or more testing agents capable of specifically detecting the expression level of at least one of FAM107B, CLEC4E or TMEM39A in a biological sample obtained from a racing animal.
  • cDNA sequencing library To produce a gene expression profile, we prepared a complementary DNA (cDNA) sequencing library by converting the population of single-stranded RNA into cDNA using a reverse transcriptase enzyme. To facilitate cDNA sequenc- ing, adapters including barcodes were added to each end of the cDNA fragments. The cDNA library was then analysed by short read sequencing. The reads i.e. re solved cDNA fragment sequences were demultiplexed i.e. barcodes were used to reassemble the separate samples, and the sequences were trimmed to remove adapter sequences. The reads were further subjected to quality control including filtering out of reads having anomalous GC content, low average quality and low total read counts.
  • the quality-controlled reads were then mapped i.e. aligned to a genome of reference to produce a gene expression profile. Mapped reads were sub jected to further quality control to remove anomalous mapping distribution.
  • Ob jective performance metric was constructed to objectively compare the perfor- mance of individual horses. This metric was constructed by taking the amount of money won by a horse as the first variable and the percentage of races won by the horse as the second variable, and after scaling and normalization, performing a principal component analysis to reduce their variation into a single dimension.
  • DESeq2 DESeq2 package (implemented in Bioconductor; https://bi- oconductor.org) which estimates variance-mean dependence in count data and tests for differential expression based on a model using the negative binomial dis tribution.
  • DESeq2 identifies the sub-set of genes, which, based on their transcriptional activity, exhibit a significant response to certain variable, in this case the performance metric. Because DESeq2 requires the performance metric to be divided into discrete categories, we prepared five alternative binary groupings of the performance metric and used these for DESeq2. These analyses identified the three genes FAM107B, CLEC4E and TMEM39A with a significant (p ⁇ 0.001), bimodal response to the performance categories (Table 1).
  • Figures 1 to 3 where figure 1 illus- trates the correlation between the expression levels of CLEC4E and FAM107B.
  • the symbol corresponds with gender (triangle - stallion, circle - mare) and the size of the symbol is proportional to the amount of money won by the horse.
  • Figure 2 il lustrates the correlation between the expression levels of CLEC4E and TMEM39A.
  • the symbol corresponds with gender (triangle - stallion, circle - mare) and the size of the symbol is proportional to the amount of money won by the horse.
  • Figure 3 illustrates the correlation between the expression levels of FAM107B and TMEM39A.
  • the symbol corresponds with gender (triangle - stallion, circle - mare) and the size of the symbol is proportional to the amount of money won by the horse.
  • the symbol corresponds with gender (triangle - stal lion, circle - mare) and the size of the symbol is proportional to the percentage of races won by the horse.
  • Figure 5 illustrates the linear response between the ex pression level of FAM107B and the amount of money won by the horse (p ⁇ 0.1).
  • the symbol corresponds with gender (triangle - stallion, circle - mare) and the size of the symbol is proportional to the amount of money won by the horse.

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Abstract

La présente invention concerne des biomarqueurs utiles comme marqueurs prédictifs pour des performances athlétiques chez des chevaux de course. L'invention concerne également un procédé de prédiction de performances athlétiques chez des chevaux de course et l'utilisation d'un kit dans ledit procédé.
PCT/FI2022/050491 2021-07-06 2022-07-05 Procédés de prédiction de performances athlétiques chez des animaux de course WO2023281167A1 (fr)

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Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BEATRICE A MCGIVNEY ET AL: "MSTN genotypes in Thoroughbred horses influence skeletal muscle gene expression and racetrack performance", ANIMAL GENETICS, BLACKWELL SCIENTIFIC PUBLICATIONS, LONDON, GB, vol. 43, no. 6, 27 February 2012 (2012-02-27), pages 810 - 812, XP071536040, ISSN: 0268-9146, DOI: 10.1111/J.1365-2052.2012.02329.X *
GHOSH MRINMOY ET AL: "Comparative Transcriptomic Analyses by RNA-seq to Elucidate Differentially Expressed Genes in the Muscle of Korean Thoroughbred Horses", APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY, HUMANA PRESS INC, NEW YORK, vol. 180, no. 3, 28 June 2016 (2016-06-28), pages 588 - 608, XP036069515, ISSN: 0273-2289, [retrieved on 20160628], DOI: 10.1007/S12010-016-2118-4 *
KYUNG-DO PARK ET AL: "Whole transcriptome analyses of six thoroughbred horses before and after exercise using RNA-Seq", BMC GENOMICS, BIOMED CENTRAL LTD, LONDON, UK, vol. 13, no. 1, 12 September 2012 (2012-09-12), pages 473, XP021106739, ISSN: 1471-2164, DOI: 10.1186/1471-2164-13-473 *
STEFANIUK MONIKA ET AL: "RNA sequencing as a powerful tool in searching for genes influencing health and performance traits of horses", JOURNAL OF APPLIED GENETICS: AN INTERNATIONAL JOURNAL OF GENETICS AND BREEDING, SPRINGER, GERMANY, vol. 57, no. 2, 7 October 2015 (2015-10-07), pages 199 - 206, XP035968929, ISSN: 1234-1983, [retrieved on 20151007], DOI: 10.1007/S13353-015-0320-7 *

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