Integrated analysis of whole genome and transcriptome sequencing reveals a frameshift mutation associated with recessive embryonic lethality in Holstein cattle

Abstract

Embryo loss is an important factor affecting fertility in dairy production. HH2 was identified as a haplotype on chromosome 1 associated with embryonic lethality in Holstein cattle. In the current study, both short-and long-read WGS was performed on four carriers and four non-carriers of HH2 to screen for variants in concordance with HH2 haplotype status. Sequence variation analysis revealed five putative functional variants of protein-coding genes, including a frameshift mutation (g.107172616delT) in intraflagellar transport protein 80 (IFT80) gene. Transcriptome analysis of whole blood indicated that no gene exhibited significantly differential expression or allele-specific expression between carriers and non-carriers in the candidate region. This evidence points to g.107172616delT as the highest priority causative mutation for HH2. Protein prediction reveals that the frameshift mutation results in a premature stop codon to reduce the peptide chain from 760 to 383 amino acids and greatly alters the structure and function of IFT80 protein. Our results demonstrate that the use of a combination of multiple high-throughput sequencing technologies is an efficient strategy to screen for the candidate causative mutations responsible for Mendelian traits, including genetic disorders.

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

“…They acknowledged that our methodology provides experimental confirmation of the developmental effects of the variant we discovered (Li et al, 2022). Interestingly, they did not observe allele-specific transcriptional bias in the blood of carriers, which supports our observation that nonsense-mediated RNA decay does not act on the mutant allele's transcript (Yang et al, 2022). However, they did not specifically report the absence of adjacent exons in gene transcripts that we report.…”

“…In the preimplantation mammalian embryo, FGF2 signaling is essential for lineage commitment and blastocyst formation (Lanner and Rossant, 2010;Kuijk et al, 2012), in which disruption of FGF2 signaling coincides with the developmental arrest observed in this study. Yang et al (2022) recently published an independent analysis of whole-genome and transcriptome data from 8 Chinese Holsteins (4 carriers and 4 noncarriers) in which they identified 5 putative functional variants of protein-coding genes associated with HH2. They acknowledged that our methodology provides experimental confirmation of the developmental effects of the variant we discovered (Li et al, 2022).…”

Homozygous haplotype deficiency in Manech Tête Rousse dairy sheep revealed a nonsense variant inMMUTgene affecting newborn lamb viability

“…Initially, this method was developed to detect embryonic lethal variant but the generalization of the method can also finemap deleterious variants leading to neonatal or juvenile lethality and morphological disorders. This reverse genetic screen has successfully identified numerous deficient homozygous haplotypes in several species, cattle [12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27], pigs [28,29], chicken [30], turkey [31] and horses [32], and the whole genome sequencing that followed has revealed the associated causative variants hosted by these regions [13,14,18,19,21,[24][25][26][27][33][34][35][36][37][38][39][40][41]. We recently, and for the first time, validated this approach in sheep with the identification of 8 independent deficient homozygous haplotypes in the Lacaune dairy breed [42].…”

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

“…The x-and y-axes show the molecular weight and fluorescence intensity, respectively. (Khan et al, 2021;Yang et al, 2021) and compared with mf-ARMS PCR genotyping results. Conflicting results were validated by Sanger sequencing using sequencing primers (Table S2).…”

“…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).…”