• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
  • 引用文献
  • 法律状态
  • 优先权
    • 专利摘要:
    Method for the early diagnosis of equine infertility. The present invention relates to an in vitro method for the diagnosis of equine infertility of genetic origin by detecting the ecax markers: lex026, tky38, tky270, lex003, ucdeq502, and the ecay markers: eca.yh12 and sry. To the use of said ecax and ecay markers for the diagnosis of equine infertility of genetic origin. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2645684A1
    申请号:ES201630755
    申请日:2016-06-06
    公开日:2017-12-07
    发明作者:Sebastian DEMYDA PEYRAS；Miguel MORENO MILLÁN；Gabriel ANAYA CALVO-RUBIO；Antonio MOLINA ALCALÁ；Alberto MEMBRILLO DEL POZO；Mercedes VALERA CÓRDOBA
    申请人:Universidad de Sevilla；Universidad de Cordoba；
    IPC主号:
    专利说明:

    DESCRIPTION
    Method for the early diagnosis of equine infertility.
    Field of the Invention
    The present invention falls within the general field in the area of equine veterinary medicine and in particular, it relates to a method for the early diagnosis of equine infertility.
    State of the art
    The economic impact of the Equestrian Sector in Spain is very important, generating a volume of € 5,303.6 million, which represents 0.51% of the country's GDP. It is represented by 723,496 animals that are distributed in 175,429 farms, which generate more than 61,000 employees. More than 25% of these horses are from Pure Spanish Breed (PRE), being this breed the most important of the entire Iberian Peninsula, and even one of the most important worldwide. The current census of the breed includes 182,509 animals (55,853 breeding mares and 33,391 stallions) distributed in 22,843 livestock in Spain to which we must add 41,025 individuals (13,118 females and 15 8,917 breeding males) found in the 8,054 farms distributed to throughout 60 countries (Gómez, Valera et al. 2009. "Assessment of inbreeding depression for body measurements in Spanish Purebred (Andalusian) horses." Livest Sci 122 (2-3): 149-155). Its relevance far exceeds the productive interest, since these animals are also recognized throughout the world as a symbol of Spanish culture and traditions.
    The management of the genealogical book and the improvement plan of this breed are in charge of the National Association of Horse Breeders of Pure Spanish Breed (ANCCE), both in the Iberian Peninsula and in the rest of the world.
    Within the sector, the purchase and sale of specimens as well as coverings for reproduction and breeding generate a considerable economic volume. Hence the importance of using animals that do not present any type of impediment or reproductive tare, whether innate, genetic, or acquired, derived from medical problems.
    Congenital infertilities are caused by various causes. These types of alterations usually go unnoticed in animals because most of the 30 animals do not develop an appreciable external symptomatology until they reach sexual maturity, at which point they begin to manifest reproductive problems. In
    Males show abnormal behaviors and lack of libido, while in females there are difficulties when it comes to becoming pregnant, even when there is no presence of regular sexual cycles during their reproductive season.
    Among the diseases of genetic origin are chromosomal alterations, characterized by variation in the number or physical structure in any of the 64 5 chromosomes present in the equine species, which represent the highest percentage of casuistry. These types of pathologies are very poorly studied in this species due mainly to the extreme difficulty of its karyotype as a result of the similarity between many of its chromosomes (Bugno, Słota et al. 2009. "Identification of chromosome abnormalities in the horse using a panel of chromosome-10 specific painting probes generated by microdissection. "Acta Vet Hung 57 (3): 369-381). It has been determined that most cases of horses that have chromosomal abnormalities demonstrate an associated infertility, usually due to disorders related to sexual development (DSD). Within chromosomal alterations in horses, Turner syndrome and reverse sex syndrome are the most common pathologies with 15 more than 70% of the casuistry recorded to date (Lear, TL and RB McGee (2012). "Disorders of sexual development in the domestic horse, Equus caballus. "Sexual Development 6 (1-3): 61-71).
    In Turner syndrome, described for the first time in humans in the 50s, the absence of a sex chromosome is observed, with 62 autosomal chromosomes being normal, resulting in odd chromosomal complement (monosomy in the sexual pair; 2n = 63, X0). The animals carrying this pathology have a normal external appearance, a mild gonadal dysgenesis and therefore, a sterility in most cases.
    On the other hand, there are animals that show a phenotype and sexual behavior 25 as opposed to what they should have according to their chromosomal endowment, known as reverse sex syndrome. Initially, this pathology was presented in only two distinct cases: phenotypically male animals with a female chromosomal endowment (2n = 64, XX), or animals whose phenotype coincided with that of a female but had a male chromosomal endowment (2n = 64, XY). However, there is a region within the genome of the upper mammals that encodes a gene related to the onset of fetal sexual differentiation (SRY, sex determining region), highly involved in cases of reverse sex (Sinclair, Berta et al. (1990). "A gene from the human sex-determining region encodes a protein with homology to a conserved DNA-binding motif." Nature 346 (6281): 240-244). From this it was possible to expand the 35
    classification of this type of pathologies detecting four possible presentations: males 64, XX, positive SRY; males 64, XX, SRY negative and mares 64, XY SRY positive or negative. Female 64, XY, negative SRY is the most common case among equines (Lear, TL and RB McGee (2012). "Disorders of sexual development in the domestic horse, Equus caballus." Sexual Development 6 (1-3): 61-71). Animals that have 5 reverse sex usually show a normal external appearance and sometimes a mild gonadal dysgenesis.
    Another pathology derived from chromosomal abnormalities most commonly observed in production animals is cell chimerism. This pathology occurs due to the exchange of blood tissues and hormones through the placental circulation between dicygotic twin brothers during twin gestations (Mishra, Arora et al. 2009. "Studies on effect of Booroola (FecB) genotype on lifetime ewes 'productivity efficiency, litter size and number of weaned lambs in Garole x Malpura sheep. "Anim Reprod Sci 113 (1–4): 293-298). It is a very strange alteration in horses because the twin gestation in horses ends in a very high percentage of abortions. However, in the case that happens, its presentation in horses is usually also asymptomatic until advanced ages (Padula, AM (2005). "The freemartin syndrome: An update." Animal Reproduction Science 87 (1-2): 93- 109.)
    These types of alterations have been historically diagnosed by cytogenetic techniques of karyotyping. They require a culture of blood cells, which stop at the metaphase stage (phase of cell division in which the cell has organized its DNA into chromosomes) and its subsequent treatment with banding techniques to specifically identify each of the chromosomes present or absent (). These karyotyping techniques present in the horse a high difficulty due to the existence of a large number of chromosomes of similar shape and small size that hinder their correct identification and can be easily confused among which is the Y chromosome (ECAY) ( Bowling, Breen et al. 1997. "Report of the Third International Committee for the standardization of the domestic horse Karyotype, Davis, CA, USA, 1996. International system for cytogenetic nomenclature of the domestic horse." Chromosome Research 5 (7): 433-443). 30
    Subsequently, molecular cytogenetic techniques based on fluorescent in situ hybridization (FISH) have been developed. In them, DNA probes are used to specifically determine the presence or absence of a chromosome, or even a part thereof. However, these types of techniques are not commercially developed in horses, and have a number of disadvantages, such as the lack of probes 35
    specific to each of the 33 chromosomes of the species, the high cost of each determination, the high investment of time needed to analyze each sample, etc. Due to all these factors, these techniques are developed in very few laboratories worldwide (Villagómez, Lear et al. 2011. "Equine disorders of sexual development in 17 seas including XX, SRY-negative, XY, SRY-negative and XY, SRY-positive genotypes. "5 Sex Dev 5 (1): 16-25).
    These technical inconveniences, together with the fact that the majority of horses that have chromosomal alterations do not demonstrate phenotypic changes or in their behavior, mean that the number of individuals who remain undiagnosed is probably very high and therefore, the real prevalence of animals carriers of 10 chromosomal aberrations remain largely underestimated (Lear and Bailey 2008. Lear, TL and E. Bailey (2008). "Equine clinical cytogenetics: The past and future." Cytogenet Genome Res 120 (1-2): 42-49 ).
    Our development is based on the use of specific molecular markers of the Microsatellite or STR type ("Short Tandem Repeats") for the determination of the 15 anomalies described above. These markers are widely used in various scientific areas and have demonstrated a high percentage of reliability in forensic, legal and productive applications, being the most used in investigations of genetic and molecular characterization.
    At present, these types of markers are the ones of choice for performing a paternity test or identifying DNA samples. However, routinely used markers (Dimsoski 2003. "Development of a 17-plex microsatellite polymerase chain reaction kit for genotyping horses." Croatian Medical Journal 44 (3): 332-335) do not meet the conditions necessary to determine anomalies chromosomes due to their lack of coverage on ECAX chromosomes (horse X chromosome) and ECAY 25 (horse Y chromosome).
    There is therefore a need to provide a procedure for the detection of reproductive problems in horses, early, fast, reliable and with high sensitivity and specificity.
    Brief Description of the Invention 30
    The present invention solves the problems described above since it provides a molecular diagnostic system that allows the detection of chromosomal alterations related to reproductive problems in horses. The method of the present invention
    It has the advantage of being a fast and reliable method since it has a 100% sensitivity (probability of giving an affected individual as positive) and a 99.75% specificity (probability of giving an unaffected individual as negative) by what every animal with alteration will be given as positive and only 0.25% of healthy animals can be diagnosed as false positives. 5
    Thus, in a first aspect, the present invention relates to an in vitro method for the diagnosis of equine infertility of genetic origin (hereinafter, method of the present invention) comprising the following steps:
    a) detect, in a sample obtained from an animal, the ECAX markers: LEX026, TKY38, TKY270, LEX003, UCDEQ502, and the ECAY markers: Eca.YH12 and SRY, 10
    b) obtain the genotype of said isolated sample based on homozygosis, heterozygosis of the ECAX markers, and the presence or absence of the ECAY markers detected in stage a), where:
    - the detection of amplification of at least one allele in at least one ECAX marker is indicative of the presence of at least one X chromosome and the heterozygosis of at least one ECAX marker is indicative of the presence of at least two X chromosomes,
    and where
    - the presence of amplification in the EcaYH12 marker is indicative of the presence of at least one Y chromosome, and where the presence of amplification in the SRY marker is indicative of the presence of the gene responsible for the start of the male sexual differentiation chain. In a particular embodiment, the isolated sample of the animal includes any type of isolated sample of the animal that contains DNA, preferably the isolated sample includes but is not limited to blood, saliva, or a hair follicle.
    In a particular embodiment of the present invention, the detection of the markers of step a) is performed by amplification. More preferably the amplification is a simultaneous amplification. Preferably the amplification is performed by multiplex PCR.
    Infertility of genetic origin includes but is not limited to infertility caused by Turner syndrome, chimerism and reverse sex syndrome.
    In a second aspect, the present invention relates to the use of the markers LEX026, TKY38, TKY270, LEX003, UCDEQ502, Eca.YH12 and SRY (markers of the present invention) for the diagnosis of equine infertility of genetic origin.
    In a third aspect, the present invention relates to a kit for the diagnosis of equine infertility of genetic origin (hereinafter kit of the present invention) according to the method of the present invention comprising the probes and primers necessary to detect the Markers LEX026, TKY38, TKY270, LEX003, UCDEQ502, Eca.YH12 and SRY. In a particular embodiment, the kit comprises probes and primers 5 identified as SEQ ID NO: 1 to SEQ ID NO: 14.
    In another aspect, the present invention relates to the use of the kit of the present invention for the diagnosis of equine infertility of genetic origin.
    Detailed description of the invention
    The method of the present invention allows the detection of equine infertility of genetic origin by probabilistic analysis of the results obtained from a panel of microsatellites specifically developed, tested and adjusted for use in the PRE horse. It offers a series of combinations based on the presence or absence of amplification in each of the markers as well as the different possible allelic combinations. On the one hand, the microsatellite amplification of the X chromosome indicates the presence of at least one X chromosome while the amplification of some marker of the Y chromosome indicates the presence of a Y chromosome. On the other hand, the presence of at least one marker of the X chromosome in heterozygosis, it indicates that there are at least two different X chromosomes. In the first instance, through the use of microsatellite markers, this method allows identifying, based on the 20 possible combinations and phenotype of the animal between normal males (XY), normal females (XX), Turner syndrome (X0), syndrome of cell chimerism (XX / XY) and reverse sex syndrome (Males, XX and Females XY). In turn, the presence or absence of the SRY fragment allows the classification of animals with reverse sex syndrome into: Males XX, SRY positive; XX males, negative SRY; Female XY, SRY negative; 25 Female XY, SRY positive.
    Table 1 shows the markers used in the reaction, the expected genomic segment size, the primers used, the type of fluorescent label used in each of them and their physical location in the different sex chromosomes of the equine.
    30

     Marker  Size Primers Location on the chromosome
     LEX026  300-314 SEQ ID NO: 1 (labeled with FAM fluorochrome) Xp
     SEQ ID NO: 2
     TKY38  105-131 SEQ ID NO: 3 (labeled with FAM fluorochrome) Xq23
     SEQ ID NO: 4
     TKY270  154-172 SEQ ID NO: 5 (marked with HEX fluorochrome) X
     SEQ ID NO: 6
     LEX003  194-214 SEQ ID NO: 7 (FAM fluorochrome marking) Xq
     SEQ ID NO: 8
     UCDEQ502  164-176 SEQ ID NO: 9 (labeled with FAM fluorochrome) Xp
     SEQ ID NO: 10
     Eca.YH12  98 SEQ ID NO: 11 (labeled with FAM fluorochrome) Y
     SEQ ID NO: 12
     SRY  249 SEQ ID NO: 13 (marked with HEX fluorochrome) Yq13
     SEQ ID NO: 14

    Table 1: markers used in the present invention
    All primer pairs of the different markers were included in a single multiplexed PCR reaction. The reaction was carried out in a total volume of 25 µl of 5 reaction mixture containing 20-60 ng of genomic DNA, 1.5-7.5 pmol of each pair of primers, 0.33 mmol / L of dNTPs, 2.5 mmol / L of MgCl2, reaction buffer at a concentration of 1X, and 1.5 units of Taq Polymerase. A thermal protocol was applied to the reaction tube, which consisted of an initial denaturation step of 95 ° C for 10 minutes, 33 cycles of 94 ° C for 30 seconds, 57 ° C for 1 minute, and 72 ° C 10 for 30 seconds. Finally, an elongation of 72 ° C was performed for 10 minutes. The resolution of the amplified products was performed by sequencing by
    capillary electrophoresis The analysis of the resulting fragments to determine the size of the alleles was performed using specific software using a standard of size LIZ 500 bp.
    Based on the amplification or not of the markers, the presence or absence of ECAX and ECAY was determined. In turn, the amplification of ECAX markers in heterozygosis or 5 homozygosis determined the presence of one or two X chromosomes in the same individual. You can also know the presence or not of the male sexual differentiation SRY gene. Table 2 details all possible combinations and diagnosis according to our test.
     PHENOTYPE  GENOTYPE
     ECAX microsatellites  ECAY DIAGNOSTIC Fragments
     YH12 SRY
     Male  All in homozygosis + + Normal male (XY)
     Female  At least one in heterozygous - - Normal female (XX) *
     Female  All in homozygosis - - Tumer syndrome (X0)
     Male  At least one in heterozygous + + Male Chimerism **
     Female  At least one in heterozygous + + Female Chimerism **
     Male  At least one in heterozygous - + SRY positive male DSD
     Male  At least one in heterozygous - - Male SRY negative DSD
     Female  All in homozygosis + + SRY positive female DSD
     Female  All in homozygosis + - SRY negative female DSD
    Table 2: results obtained in the test performed 10
    * This result has a 99.75% specificity.
    ** If there are differences between the results obtained in blood and hair samples, the chimerism will be hematopoietic, that is, blood only.
    This diagnostic system was validated on samples of Purebred Spanish Horse (PRE) to determine the level of Sensitivity and Specificity. For this the 15 allelic frequencies and the level of heterozygosity of each of the ECAX markers were calculated. This allowed to detect the probability that in a normal female, the results demonstrated the presence of at least one marker of the X chromosome in heterozygosis indicating the presence of two X chromosomes. In this way, the level of Specificity of this technique was determined in a 99.75%, so only 0.25 of healthy females 20 would be diagnosed as false positives. On the other hand, the technique has 100% of
    sensitivity since any alteration related to sexual markers will be determined in all affected individuals.
    Example 1: infertility diagnostic method
    Samples were received from two PRE mares sent by the expert veterinarian due to the lack of reproductive events in more than two seasons. 5
    The first case was a 4-year-old PRE mare with infertility symptoms without apparent cause. The second case was a copy of 3 years who had not shown zeal in his first reproductive campaign.
    On the day of the reception, DNA extraction was carried out from the blood samples of both animals and the PCR reaction, being sent once finished to the sequencing service of the University of Córdoba. The next day the sequenced results were received and their analysis proceeded.
    The results of the markers were as follows:
     PHENOTYPE  GENOTYPE
     X chromosome microsatellites  Chromosome Fragments and DIAGNOSTI-CO
     LEX003  LEX026 TKY270 TKY38 UCDEQ502 YH12 SRY
     Horse  206 314 168 109 176 + - SRS negative SDS female
     Horse  204 298 12 127 166 + - SDS negative SDS female
    According to the analysis of the data and the presence of an ECAX chromosome and an ECAY chromosome and the absence of the SRY gene in each of the animals, 15 were determined to be cases 64, XY reverse sex of negative SRY type with a 100 % certainty. It should be noted that the result was obtained in 24 hours, which is impossible through the use of other diagnostic methodologies.
    权利要求:
    Claims (9)
    [1]
    REINVINDICATIONS
    1. In vitro method for the diagnosis of equine infertility of genetic origin comprising the following stages:
    a) detect, in a sample obtained from an animal, the ECAX markers: LEX026, TKY38, TKY270, LEX003, UCDEQ502, and the ECAY markers: Eca.YH12 and SRY, 5
    b) obtain the genotype of said isolated sample based on homozygosis, heterozygosis of the ECAX markers, and the presence or absence of the ECAY markers detected in stage a), where:
    - the detection of amplification of at least one allele in at least one ECAX marker is indicative of the presence of at least one X chromosome and the heterozygosis of at least one ECAX marker is indicative of the presence of at least two X chromosomes,
    and where
    - the presence of amplification in the EcaYH12 marker is indicative of the presence of at least one Y chromosome, and where the presence of amplification in the SRY marker is indicative of the presence of the gene responsible for the start of the male sexual differentiation chain.
    [2]
    2. Method according to claim 1, wherein the animal's sample is blood, saliva, or a hair follicle.
    [3]
    3. Method according to any of claims 1-2, wherein the detection of the markers of step a) is performed by amplification. twenty
    [4]
    4. Method according to claim 3, wherein the amplification is a simultaneous amplification.
    [5]
    5. Method according to any of the preceding claims, wherein infertility of genetic origin is selected from Turner syndrome, chimerism and reverse sex syndrome.
    [6]
    6. Use of markers LEX026, TKY38, TKY270, LEX003, UCDEQ502, Eca.YH12 and 25 SRY, for the diagnosis of equine infertility of genetic origin.
    [7]
    7. Kit for the diagnosis of equine infertility of genetic origin according to the method of claims 1-5 comprising the probes and primers necessary to detect the markers LEX026, TKY38, TKY270, LEX003, UCDEQ502, Eca.YH12 and SRY.
    [8]
    8. Kit according to claim 7, comprising the probes and primers identified as SEQ ID NO: 1 to SEQ ID NO: 14.
    [9]
    9. Use of the kit according to any of claims 6-7 for the diagnosis of equine infertility of genetic origin.
     5
    类似技术:
    公开号 | 公开日 | 专利标题
    Kadri et al.2014|A 660-Kb deletion with antagonistic effects on fertility and milk production segregates at high frequency in Nordic Red cattle: additional evidence for the common occurrence of balancing selection in livestock
    Irano et al.2016|Genome-wide association study for indicator traits of sexual precocity in Nellore cattle
    Weinberg-Shukron et al.2015|A mutation in the nucleoporin-107 gene causes XX gonadal dysgenesis
    Yatsenko et al.2015|X-linked TEX11 mutations, meiotic arrest, and azoospermia in infertile men
    Davis et al.2015|Mechanisms underlying mammalian hybrid sterility in two feline interspecies models
    de la Fuente et al.2015|Ancient marine hunter‐gatherers from P atagonia and T ierra D el F uego: Diversity and differentiation using uniparentally inherited genetic markers
    Lear et al.2012|Disorders of sexual development in the domestic horse, Equus caballus
    Zhang et al.2020|Asian water buffalo: domestication, history and genetics
    He et al.2019|A chromosome‐level genome of black rockfish, Sebastes schlegelii, provides insights into the evolution of live birth
    ES2445099T3|2014-02-28|A genetic marker test for braquispine and fertility in cattle
    Whitworth et al.2010|Activation method does not alter abnormal placental gene expression and development in cloned pigs
    Shilton et al.2020|Whole genome analysis reveals aneuploidies in early pregnancy loss in the horse
    Berry et al.2017|Characterization of an X-chromosomal non-mosaic monosomy | dairy heifer detected using routinely available single nucleotide polymorphism genotype data
    Demyda-Peyras et al.2013|The use of molecular and cytogenetic methods as a valuable tool in the detection of chromosomal abnormalities in horses: a case of sex chromosome chimerism in a Spanish purebred colt
    Piro et al.2008|Frequency of the severe combined immunodeficiency disease gene among horses in Morocco
    Miller2014|Sperm RNA as a mediator of genomic plasticity
    ES2645684B1|2018-10-09|METHOD FOR THE EARLY DIAGNOSIS OF EQUINE INFERTILITY
    Szczerbal et al.2020|Application of droplet digital PCR in diagnosing of X monosomy in mares
    Lear et al.2014|Repeated early embryonic loss in a thoroughbred mare with a chromosomal translocation [64, XX, t |]
    Lühken2012|Genetic testing for phenotype-causing variants in sheep and goats
    ES2257139B1|2007-07-16|METHOD AND KIT FOR GENOTIPIFICATION OF HLA-DRB BASED ON REAL-TIME PCR.
    Szczerbal et al.2021|Clinical cytogenetics of the dog: a review
    Ji et al.2014|A novel mutation in MID1 in a patient with X-linked Opitz G/BBB syndrome
    US20180282812A1|2018-10-04|Diagnostic test for skeletal atavism in horses
    JP5897704B2|2016-03-30|Detection of Brachyspina mutation
    同族专利:
    公开号 | 公开日
    ES2645684B1|2018-10-09|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    法律状态:
    2018-10-09| FG2A| Definitive protection|Ref document number: 2645684 Country of ref document: ES Kind code of ref document: B1 Effective date: 20181009 |
    优先权:
    申请号 | 申请日 | 专利标题
    ES201630755A|ES2645684B1|2016-06-06|2016-06-06|METHOD FOR THE EARLY DIAGNOSIS OF EQUINE INFERTILITY|ES201630755A| ES2645684B1|2016-06-06|2016-06-06|METHOD FOR THE EARLY DIAGNOSIS OF EQUINE INFERTILITY|
    [返回顶部]