Truncation of IFT80 causes early embryonic loss in Holstein cattle associated with Holstein haplotype 2

Abstract

Recessive alleles represent genetic risk in populations that have undergone bottleneck events. We present a comprehensive framework for identification and validation of these genetic defects, including haplotype-based detection, variant selection from sequence data, and validation using knockout embryos. Holstein haplotype 2 (HH2), which causes embryonic death, was used to demonstrate the approach. Holstein haplotype 2 was identified using a deficiency-of-homozygotes approach and confirmed to negatively affect conception rate and stillbirths. Five carriers were present in a group of 183 sequenced Holstein bulls selected to maximize the coverage of unique haplotypes. Three variants concordant with haplotype calls were found in HH2: a high-priority frameshift mutation resulting, and 2 low-priority variants (1 synonymous variant, 1 premature stop codon). The frameshift in intraflagellar 80 (IFT80) was confirmed in a separate group of Holsteins from the 1000 Bull Genomes Project that shared no animals with the discovery set. IFT80-null embryos were generated by truncating the IFT80 transcript at exon 2 or 11 using a CRISPR-Cas9 system. Abattoir-derived oocytes were fertilized in vitro, and zygotes were injected at the one-cell stage either with a guide RNA and CAS9 mRNA complex (n = 100) or Cas9 mRNA (control, n = 100) before return to culture, and replicated 3 times. IFT80 is activated at the 8-cell stage, and IFT80-null embryos arrested at this stage of development, which is consistent with data from mouse hypomorphs and HH2 carrier-to-carrier matings. This frameshift in IFT80 on chromosome 1 at 107,172,615 bp (p.Leu381fs) disrupts WNT and hedgehog signaling, and is responsible for the death of homozygous embryos.

Multiplex fluorescent amplification‐refractory mutation system PCR method for the detection of 10 genetic defects in Holstein cattle and its comparison with the KASP genotyping assay

“…Genetic defects are currently a concern in the dairy industry. The common genetic defects include Holstein haplotype 1 (HH1) (Adams et al., 2016), Holstein haplotype 2 (HH2) (Ortega et al., 2022; Yang et al., 2021), Holstein haplotype (HH3) (Daetwyler et al., 2014; McClure et al., 2014), Holstein haplotype 4 (HH4) (Fritz et al., 2013), Holstein haplotype 5 (HH5) (Schütz et al., 2016), Holstein haplotype 6 (HH6) (Fritz et al., 2018), haplotype for cholesterol deficiency (HCD) (Kipp et al., 2016; Menzi et al., 2016), bovine leukocyte adhesion deficiency (BLAD) (Shuster et al., 1992), complex vertebral malformation (CVM) (Agerholm et al., 2001; Thomsen et al., 2006) and brachyspina syndrome (BS) (Charlier et al., 2012) (Table 1). Recessive inheritance patterns of these 10 genetic defects cause around 25% embryo loss or neonatal death from the mating between carriers (Cole et al., 2016; Segelke et al., 2016).…”

Biomarker-based human and animal sperm phenotyping: the good, the bad and the ugly

“…In livestock, the focus is on reproductive efficiency with better conception rates, potentially with lower sperm doses per AI service (to max out the number of doses per sire/ejaculate). By taking advantage of the increasing number of genome-sequenced bulls, the AI sire model can address important questions about paternal influence in human fertility disorders such as fertilization failure and recurrent pregnancy loss [ 21 , 22 ].…”