A missense mutation (p.Tyr452Cys) in the CAD gene compromises reproductive success in French Normande cattle

We scanned the genome of 77,815 Normande cattle with different Illumina SNP chips (Illumina Inc., San Diego, CA) to map recessive embryonic lethal mutations using homozygous haplotype deficiency. We detected 2 novel haplotypes on chromosomes 11 and 24 but did not confirm 6 previously reported haplotypes. The one on chromosome 11 showed a marked reduction in conception rates and moderate decrease in nonreturn rate in at-risk versus control mating, supporting late embryonic mortality. After fine mapping and analyzing whole-genome sequences, we prioritized a missense mutation in CAD (g.72399397T>C; p.Tyr452Cys)-a gene encoding a protein (carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase) essential for de novo pyrimidine biosynthesis-as a candidate causal variant. This transition mutation replaces a tyrosine residue, which is perfectly conserved among living organisms, with a cysteine residue in the carbamoyl-phosphate synthetase 2 domain of the protein. A single animal was confirmed to be homozygous for the mutation based on Sanger sequencing. However, large-scale genotyping of the candidate variant with the Illumina EuroG10k BeadChip revealed an absence of live homozygotes in a panel of 33,323 Normande animals and an absence of carriers in 348,593 animals from 19 other cattle breeds. These results support recessive embryonic lethality with nearly complete penetrance, as was previously reported in CAD mutants in several eukaryote species. The only homozygous cow had extremely poor udder conformation, suggesting a potential role of CAD in udder development, but no effect was detected when comparing daughter yield deviations of 250 heterozygous bulls with that of 2,912 homozygotes for the ancestral allele. Together, our results showed the importance of large-scale screening for homozygous haplotype deficiency with hundreds of thousands of animals, validating results with an independent data set, and considering unexpected live homozygotes, to avoid both false-positive and false-negative discoveries. These discoveries will be used primarily in mating decisions to avoid at-risk mating. In addition, we recommend including CAD in the breeding objectives of Normande cattle.