Microdeletions on chromosomes 2 and 20 are associated with equine early pregnancy loss

Copy number variants (CNVs) are chromosomal microdeletions and microduplications greater than 1000 bp and can confer disease risk, if they impact gene dosage. Specific pathogenic CNVs have been described as associated with miscarriage and stillbirth in women but are unexplored in equine pregnancy. This study aimed to identify and characterize pathogenic CNVs associated with equine early embryonic loss (EEL) and early fetal losses collectively referred to as early pregnancy loss (EPL). Conceptuses were submitted to the laboratory by the attending veterinarians following clinical confirmation of pregnancy failure between gestation days 15 and 65. DNA was isolated from the allantochorion (ALC) (n=52) and fetus (n=13) of EPL pregnancies. As a control, DNA was isolated from allantochorion and fetuses of manually terminated clinically normal pregnancies (CNP n=20), and peripheral blood mononuclear cells (PBMCs) were isolated from adult horses (n=371). DNA was hybridized to 96 sample Axiom Equine 670K Arrays. DNAcopy was used to identify autosomal CNVs >1000 bp in length after excluding samples with <95% call rate, MAPD <0.35, log2 intensity between -0.2 and +0.2, and a significance of p>0.05. Two microdeletions were found in at least two EPL conceptuses that were absent in all control samples. A review of the literature confirmed both microdeletions were unreported to date (n=3040 individuals). In this study, PCR confirmed the deletions(del2:91608493-91610830 and del20:30652320-30656312). Bioinformatic analysis of del2:91608493-91610830 using ENSEMBL revealed that the 2.3 kb deletion region contained part of exon1 and intron1 of the mastermind like transcriptional coactivator 3 (MAML3) gene, a co-activator of Notch signaling known to play a key role in early development. This deletion in MAML3 was identified in the ALC of two EPLs lost at 42 and 64 days of gestation. Del20:30652320-30656312 was identified in seven EPLs, including both the fetal and ALC genome of two EPLs and only the fetal genome of an additional five EPLs. Bioinformatic analysis revealed the deletion in ECA20 encompassed a 4 kb region that included exon1 and partial intron1 of both transcripts for tubulin beta class 1 (TUBB), which is essential to neurogenesis. Five of the seven EPLs failed at day 35-40, and three of the fetuses had abnormalities of the central nervous system, including one failure of neural tube closure. Whilst causality can't be confirmed from these experiments, the rarity of microdeletions in viable horses, and the biological plausibility of the deleted gene impacting early development is supportive of a role for both microdeletions in lethality of the conceptus in the first two months of pregnancy. Further experiments are required to determine if the deletions are acquired through the germline.