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Equine veterinary journal2020; 52(4); 627-631; doi: 10.1111/evj.13214

Application of droplet digital PCR in diagnosing of X monosomy in mares.

Abstract: X monosomy is the most common disorder of sex development in horses. Although cytogenetic analysis is still the gold standard in the diagnosis of equine X monosomy, novel molecular techniques are being sought to quickly and reliably detect this chromosome abnormality. Objective: The goal of this study was to evaluate the usefulness of a novel variant of the PCR technique-namely, droplet digital PCR (ddPCR)-in the detection of X monosomy in mares. Methods: A proof of concept of the usefulness of ddPCR in diagnosing an abnormal number of X chromosomes in mares. Methods: We examined an infertile mare using cytogenetic (fluorescent in situ hybridisation-FISH) and molecular (droplet digital PCR-ddPCR) techniques. The X chromosome copy number in ddPCR was estimated via detection of the AMELX gene copy number. In addition, 70 mares homozygous for X-linked microsatellite marker (LEX3) were examined by ddPCR. For all mares, a PCR search for the Y-linked SRY gene was also performed. Results: Cytogenetic analysis and ddPCR gave concordant results, indicating pure X monosomy in the studied mare. Of the 70 additional mares examined by ddPCR, a single copy of the X chromosome was found in two cases. All mares were SRY-negative and thus both freemartinism, manifested by leucocyte XX/XY chimerism, and sex reversal syndrome (XX, SRY-positive) could be excluded. Conclusions: The ddPCR approach does not allow for unequivocal identification of mosaicism (63,X/64,XX or 65,XXX/64,XX), but may give an indication that further cytogenetic analysis is necessary. Conclusions: The ddPCR approach appeared to be useful for diagnosing nonmosaic X monosomy in mares. If the number of X chromosome copies in a mare, as determined by ddPCR, differs from two (in our study, 2.2), additional cytogenetic investigation is recommended with the aim of detecting the mosaicism.
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Publication Date: 2020-02-12 PubMed ID: 31793061DOI: 10.1111/evj.13214Google Scholar: Lookup
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Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

The research evaluates the droplet digital PCR (ddPCR) technique’s utility for the detection of X monosomy, a sex developmental disorder in horses. The results suggest that ddPCR is an effective method for diagnosing non-mosaic X monosomy in mares, and when detected chromosome counts differ from the norm, it indicates the need for further cytogenetic analysis.

Objective and Methodology of the Study

  • The aim of the study was to test the usefulness of droplet digital PCR (ddPCR) in diagnosing X monosomy in mares. X monosomy is a common sex development disorder in horses, typically diagnosed by cytogenetic analysis.
  • The researchers utilised two main techniques—a cytogenetic technique called fluorescent in situ hybridisation (FISH), and a molecular approach, ddPCR. The efficiency of ddPCR in this use case was examined via a proof of concept test.
  • The X chromosome copy number in ddPCR was estimated using the AMELX gene’s copy number. Additionally, 70 mares known to be homozygous for X-linked microsatellite marker (LEX3) were studied via ddPCR.
  • The research also used PCR to search for the SRY gene, a Y-linked genetic factor. This was significant in excluding other potential equine disorders, like freemartinism and sex reversal syndrome.

Results of the Study

  • In their initial case study on an infertile mare, the researcher observed that the results of cytogenetic analysis and ddPCR agreed. It indicated unambiguous X monosomy in the tested mare.
  • Out of the additional 70 mares studied via ddPCR, the ddPCR detected a single copy of the X chromosome in two cases.
  • All mares tested were SRY-negative, hence freemartinism and sex reversal syndrome disorders, which are only observed in SRY-positive mares, were ruled out.

Conclusions of the Study

  • The researchers concluded that while ddPCR does not conclusively identify mosaicism (63,X/64,XX or 65,XXX/64,XX), it may signal that additional cytogenetic investigation is required to verify such conditions.
  • The researchers suggested that ddPCR is beneficial in diagnosing nonmosaic X monosomy in mares. They recommend cytogenetic analysis whenever the X chromosome copies’ number detected by ddPCR diverges from the standard two (i.e., less than 1.8 or more than 2.2). The purpose of this further analysis would be to detect potential cases of mosaicism.

Cite This Article

APA
Szczerbal I, Nowacka-Woszuk J, Kopp-Kuhlman C, Mackowski M, Switonski M. (2020). Application of droplet digital PCR in diagnosing of X monosomy in mares. Equine Vet J, 52(4), 627-631. https://doi.org/10.1111/evj.13214

Publication

Equine veterinary journal
ISSN: 2042-3306
NlmUniqueID: 0173320
Country: United States
Language: English
Volume: 52
Issue: 4
Pages: 627-631

Researcher Affiliations

Szczerbal, Izabela
  • Department of Genetics and Animal Breeding, Poznan University of Life Sciences, Poznan, Poland.
Nowacka-Woszuk, Joanna
  • Department of Genetics and Animal Breeding, Poznan University of Life Sciences, Poznan, Poland.
Kopp-Kuhlman, Christine
  • Veterinary Clinic, Degerby, Finland.
Mackowski, Mariusz
  • Department of Horse Breeding, Poznan University of Life Sciences, Poznan, Poland.
Switonski, Marek
  • Department of Genetics and Animal Breeding, Poznan University of Life Sciences, Poznan, Poland.

MeSH Terms

  • Animals
  • Cattle
  • Female
  • Horses
  • Leukocytes
  • Monosomy
  • Mosaicism
  • Polymerase Chain Reaction / veterinary
  • X Chromosome

Grant Funding

  • Department of Genetics and Animal Breeding
  • 506.534.05.00 / Poznan University of Life Sciences, Poland

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Citations

This article has been cited 6 times.
  1. Demyda-Peyrás S, Laseca N, Anaya G, Kij-Mitka B, Molina A, Karlau A, Valera M. Prevalence of Sex-Related Chromosomal Abnormalities in a Large Cohort of Spanish Purebred Horses. Animals (Basel) 2023 Feb 3;13(3).
    doi: 10.3390/ani13030539pubmed: 36766428google scholar: lookup
  2. Arroyo E, Patiño C, Ciccarelli M, Raudsepp T, Conley A, Tibary A. Clinical and Histological Features of Ovarian Hypoplasia/Dysgenesis in Alpacas. Front Vet Sci 2022;9:837684.
    doi: 10.3389/fvets.2022.837684pubmed: 35400100google scholar: lookup
  3. Laseca N, Anaya G, Peña Z, Pirosanto Y, Molina A, Demyda Peyrás S. Impaired Reproductive Function in Equines: From Genetics to Genomics. Animals (Basel) 2021 Feb 3;11(2).
    doi: 10.3390/ani11020393pubmed: 33546520google scholar: lookup
  4. Switonski M, Szczerbal I, Nowacka-Woszuk J. From cytogenetics to cytogenomics: a new era in the diagnosis of chromosomal abnormalities in domestic animals. J Appl Genet 2025 Sep;66(3):661-673.
    doi: 10.1007/s13353-025-00943-xpubmed: 39869248google scholar: lookup
  5. Valera M, Karlau A, Anaya G, Bugno-Poniewierska M, Molina A, Encina A, Azor PJ, Demyda-Peyrás S. The Use of Genomic Screening for the Detection of Chromosomal Abnormalities in the Domestic Horse: Five New Cases of 65,XXY Syndrome in the Pura Raza Español Breed. Animals (Basel) 2024 Sep 3;14(17).
    doi: 10.3390/ani14172560pubmed: 39272345google scholar: lookup
  6. Cieslak J, Mackowski M, Skrzetuska W, Fidos-Tama E, Siwinska N, Szczerbal I. A case of non-mosaic X trisomy (65,XXX) in a Thoroughbred mare confirmed by cytogenetic and molecular analysis. J Appl Genet 2024 May;65(2):395-398.
    doi: 10.1007/s13353-024-00844-5pubmed: 38368284google scholar: lookup
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