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

Background: 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.

Objectives: 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.

Study Design: 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.

Main Limitations: 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.

Conclusion: 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, <1.8 or >2.2), additional cytogenetic investigation is recommended with the aim of detecting the mosaicism.