Large-scale detection and characterization of interchromosomal rearrangements in normozoospermic bulls using massive genotype and phenotype data sets.

In this paper, we developed a highly sensitive approach to detect interchromosomal rearrangements in cattle by searching for abnormal linkage disequilibrium patterns between markers located on different chromosomes in large paternal half-sib families genotyped as part of routine genomic evaluations. We screened 5571 families of artificial insemination sires from 15 breeds and revealed 13 putative interchromosomal rearrangements, 12 of which were validated by cytogenetic analysis and long-read sequencing. These consisted of one Robertsonian fusion, 10 reciprocal translocations, and the first case of insertional translocation reported in cattle. Taking advantage of the wealth of data available in cattle, we performed a series of complementary analyses to define the exact nature of these rearrangements, investigate their origins, and search for factors that may have favored their occurrence. We also evaluated the risks to the livestock industry and showed significant negative effects on several traits in the sires and in their balanced or aneuploid progeny compared with wild-type controls. Thus, we present the most comprehensive and thorough screen for interchromosomal rearrangements compatible with normal spermatogenesis in livestock species. This approach is readily applicable to any population that benefits from large genotype data sets, and will have direct applications in animal breeding. Finally, it also offers interesting prospects for basic research by allowing the detection of smaller and rarer types of chromosomal rearrangements than GTG banding, which are interesting models for studying gene regulation and the organization of genome structure.

A recurrent de novo missense mutation in COL1A1 causes osteogenesis imperfecta type II and preterm delivery in Normande cattle.

BACKGROUND: Nine male and eight female calves born to a Normande artificial insemination bull named "Ly" were referred to the French National Observatory of Bovine Abnormalities for multiple fractures, shortened gestation, and stillbirth or perinatal mortality. RESULTS: Using Illumina BovineSNP50 array genotypes from affected calves and 84 half-sib controls, the associated locus was mapped to a 6.5-Mb interval on chromosome 19, assuming autosomal inheritance with germline mosaicism. Subsequent comparison of the whole-genome sequences of one case and 5116 control genomes, followed by genotyping in the affected pedigree, identified a de novo missense substitution within the NC1 domain of the COL1A1 gene (Chr19 g.36,473,965G > A; p.D1412N) as unique candidate variant. Interestingly, the affected residue was completely conserved among 243 vertebrate orthologs, and the same substitution in humans has been reported to cause type II osteogenesis imperfecta (OI), a connective tissue disorder that is characterized primarily by bone deformity and fragility. Moreover, three COL1A1 mutations have been described to cause the same syndrome in cattle. Necropsy, computed tomography, radiology, and histology confirmed the diagnosis of type II OI, further supporting the causality of this variant. In addition, a detailed analysis of gestation length and perinatal mortality in 1387 offspring of Ly and more than 160,000 progeny of 63 control bulls allowed us to statistically confirm in a large pedigree the association between type II OI and preterm delivery, which is probably due to premature rupture of fetal membranes and has been reported in several isolated cases of type II OI in humans and cattle. Finally, analysis of perinatal mortality rates and segregation distortion supported a low level of germ cell mosaicism in Ly, with an estimate of 4.5% to 7.7% of mutant sperm and thus 63 to 107 affected calves born. These numbers contrast with the 17 cases reported and raise concerns about the underreporting of congenital defects to heredo-surveillance platforms, even for textbook genetic syndromes. CONCLUSIONS: In conclusion, we describe a large animal model for a recurrent substitution in COL1A1 that is responsible for type II OI in humans. More generally, this study highlights the utility of such datasets and large half-sib families available in livestock species to characterize sporadic genetic defects.

Integrin alpha 6 homozygous splice-site mutation causes a new form of junctional epidermolysis bullosa in Charolais cattle.

BACKGROUND: Inherited epidermolysis bullosa (EB) is a group of painful and life-threatening genetic disorders that are characterized by mechanically induced blistering of the skin and mucous membranes. Congenital skin fragility resembling EB was recently reported in three Charolais calves born in two distinct herds from unaffected parents. Phenotypic and genetic analyses were carried out to describe this condition and its molecular etiology. RESULTS: Genealogical, pathological and histological investigations confirmed the diagnosis of recessive EB. However, the affected calves showed milder clinical signs compared to another form of EB, which was previously reported in the same breed and is caused by a homozygous deletion of the ITGB4 gene. Homozygosity mapping followed by analysis of the whole-genome sequences of two cases and 5031 control individuals enabled us to prioritize a splice donor site of ITGA6 (c.2160 + 1G > T; Chr2 g.24112740C > A) as the most compelling candidate variant. This substitution showed a perfect genotype-phenotype correlation in the two affected pedigrees and was found to segregate only in Charolais, and at a very low frequency (f = 1.6 × 10-4) after genotyping 186,154 animals from 15 breeds. Finally, RT-PCR analyses revealed increased retention of introns 14 and 15 of the ITGA6 gene in a heterozygous mutant cow compared with a matched control. The mutant mRNA is predicted to cause a frameshift (ITGA6 p.I657Mfs1) that affects the assembly of the integrin α6ß4 dimer and its correct anchoring to the cell membrane. This dimer is a key component of the hemidesmosome anchoring complex, which ensures the attachment of basal epithelial cells to the basal membrane. Based on these elements, we arrived at a diagnosis of junctional EB. CONCLUSIONS: We report a rare example of partial phenocopies observed in the same breed and due to mutations that affect two members of the same protein dimer, and provide the first evidence of an ITGA6 mutation that causes EB in livestock species.

Analysis of Polycerate Mutants Reveals the Evolutionary Co-option of HOXD1 for Horn Patterning in Bovidae.

In the course of evolution, pecorans (i.e., higher ruminants) developed a remarkable diversity of osseous cranial appendages, collectively referred to as "headgear," which likely share the same origin and genetic basis. However, the nature and function of the genetic determinants underlying their number and position remain elusive. Jacob and other rare populations of sheep and goats are characterized by polyceraty, the presence of more than two horns. Here, we characterize distinct POLYCERATE alleles in each species, both associated with defective HOXD1 function. We show that haploinsufficiency at this locus results in the splitting of horn bud primordia, likely following the abnormal extension of an initial morphogenetic field. These results highlight the key role played by this gene in headgear patterning and illustrate the evolutionary co-option of a gene involved in the early development of bilateria to properly fix the position and number of these distinctive organs of Bovidae.

Mutation of the MYH3 gene causes recessive cleft palate in Limousine cattle.

BACKGROUND: The palate is a structure separating the oral and nasal cavities and its integrity is essential for feeding and breathing. The total or partial opening of the palate is called a cleft palate and is a common malformation in mammals with environmental or hereditary aetiologies. Generally, it compromises life expectancy in the absence of surgical repair. A new form of non-syndromic cleft palate arose recently in Limousine cattle, with animals referred to the French National Observatory of Bovine Abnormalities since 2012. Since the number of affected animals has increased steadily, this study was undertaken to identify the cause of this disease. RESULTS: Based on pedigree analysis, occurrence of cleft palate in Limousine cattle was concordant with an autosomal recessive mode of inheritance. Genotyping of 16 affected animals and homozygosity mapping led to the identification of a single disease-associated haplotype on Bos taurus chromosome (BTA)19. The genome of two affected animals was sequenced, and their sequences were compared to the ARS-UCD1.2 reference genome to identify variants. The likely causal variants were compared to the variant database of the 1000 bull genome project and two fully linked mutations in exon 24 of the MYH3 (myosin heavy chain) gene were detected: a 1-bp non-synonymous substitution (BTA19:g.29609623A>G) and a 11-bp frameshift deletion (BTA19:g.29609605-29609615del). These two mutations were specific to the Limousine breed, with an estimated allele frequency of 2.4% and are predicted to be deleterious. The frameshift leads to a premature termination codon. Accordingly, mRNA and protein analyses in muscles from wild-type and affected animals revealed a decrease in MYH3 expression in affected animals, probably due to mRNA decay, as well as an absence of the MYH3 protein in these animals. MYH3 is mostly expressed in muscles, including craniofacial muscles, during embryogenesis, and its absence may impair palate formation. CONCLUSIONS: We describe a new form of hereditary cleft palate in Limousine cattle. We identified two fully linked and deleterious mutations, ultimately leading to the loss-of-function of the MYH3 protein. The mutations were included on the Illumina EuroG10k v8 and EuroGMD v1 SNP chips and are used to set up a reliable eradication strategy in the French Limousine breed.

Identification of the ABCC4, IER3, and CBFA2T2 candidate genes for resistance to paratuberculosis from sequence-based GWAS in Holstein and Normande dairy cattle.

BACKGROUND: Bovine paratuberculosis is a contagious disease, caused by Mycobacterium avium subsp. paratuberculosis (MAP), with adverse effects on animal welfare and serious economic consequences. Published results on host genetic resistance to MAP are inconsistent, mainly because of difficulties in characterizing the infection status of cows. The objectives of this study were to identify quantitative trait loci (QTL) for resistance to MAP in Holstein and Normande cows with an accurately defined status for MAP. RESULTS: From MAP-infected herds, cows without clinical signs of disease were subjected to at least four repeated serum ELISA and fecal PCR tests over time to determine both infected and non-infected statuses. Clinical cases were confirmed using PCR. Only cows that had concordant results for all tests were included in further analyses. Positive and control cows were matched within herd according to their birth date to ensure a same level of exposure to MAP. Cows with accurate phenotypes, i.e. unaffected (control) or affected (clinical or non-clinical cases), were genotyped with the Illumina BovineSNP50 BeadChip. Genotypes were imputed to whole-genome sequences using the 1000 Bull Genomes reference population (run6). A genome-wide association study (GWAS) of MAP status of 1644 Holstein and 649 Normande cows, using either two (controls versus cases) or three classes of phenotype (controls, non-clinical and clinical cases), revealed three regions, on Bos taurus (BTA) chromosomes 12, 13, and 23, presenting significant effects in Holstein cows, while only one of those was identified in Normande cows (BTA23). The most significant effect was found on BTA13, in a short 8.5-kb region. Conditional analyses revealed that only one causal variant may be responsible for the effects observed on each chromosome with the ABCC4 (BTA12), CBFA2T2 (BTA13), and IER3 (BTA23) genes as good functional candidates. CONCLUSIONS: A sequence-based GWAS on cows for which resistance to MAP was accurately defined, was able to identify candidate variants located in genes that were functionally related to resistance to MAP; these explained up to 28% of the genetic variance of the trait. These results are very encouraging for efforts towards implementation of a breeding strategy aimed at improving resistance to paratuberculosis in Holstein cows.

Α de novo 3.8-Mb inversion affecting the EDA and XIST genes in a heterozygous female calf with generalized hypohidrotic ectodermal dysplasia.

BACKGROUND: In mammals, hypohidrotic ectodermal dysplasia (HED) is a genetic disorder that is characterized by sparse hair, tooth abnormalities, and defects in cutaneous glands. Only four genes, EDA, EDAR, EDARADD and WNT10A account for more than 90% of HED cases, and EDA, on chromosome X, is involved in 50% of the cases. In this study, we explored an isolated case of a female Holstein calf with symptoms similar to HED. RESULTS: Clinical examination confirmed the diagnosis. The affected female showed homogeneous hypotrichosis and oligodontia as previously observed in bovine EDAR homozygous and EDA hemizygous mutants. Under light microscopy, the hair follicles were thinner and located higher in the dermis of the frontal skin in the affected animal than in the control. Moreover, the affected animal showed a five-fold increase in the number of hair follicles and a four-fold decrease in the diameter of the pilary canals. Pedigree analysis revealed that the coefficient of inbreeding of the affected calf (4.58%) was not higher than the average population inbreeding coefficient (4.59%). This animal had ten ancestors in its paternal and maternal lineages. By estimating the number of affected cases that would be expected if any of these common ancestors carried a recessive mutation, we concluded that, if they existed, other cases of HED should have been reported in France, which is not the case. Therefore, we assumed that the causal mutation was dominant and de novo. By analyzing whole-genome sequencing data, we identified a large chromosomal inversion with breakpoints located in the first introns of the EDA and XIST genes. Genotyping by PCR-electrophoresis the case and its parents allowed us to demonstrate the de novo origin of this inversion. Finally, using various sources of information we present a body of evidence that supports the hypothesis that this mutation is responsible for a skewed inactivation of X, and that only the normal X can be inactivated. CONCLUSIONS: In this article, we report a unique case of X-linked HED affected Holstein female calf with an assumed full inactivation of the normal X-chromosome, thus leading to a severe phenotype similar to that of hemizygous males.

An initiator codon mutation in SDE2 causes recessive embryonic lethality in Holstein cattle.

Researching depletions in homozygous genotypes for specific haplotypes among the large cohorts of animals genotyped for genomic selection is a very efficient strategy to map recessive lethal mutations. In this study, by analyzing real or imputed Illumina BovineSNP50 (Illumina Inc., San Diego, CA) genotypes from more than 250,000 Holstein animals, we identified a new locus called HH6 showing significant negative effects on conception rate and nonreturn rate at 56 d in at-risk versus control mating. We fine-mapped this locus in a 1.1-Mb interval and analyzed genome sequence data from 12 carrier and 284 noncarrier Holstein bulls. We report the identification of a strong candidate mutation in the gene encoding SDE2 telomere maintenance homolog (SDE2), a protein essential for genomic stability in eukaryotes. This A-to-G transition changes the initiator ATG (methionine) codon to ACG because the gene is transcribed on the reverse strand. Using RNA sequencing and quantitative reverse-transcription PCR, we demonstrated that this mutation does not significantly affect SDE2 splicing and expression level in heterozygous carriers compared with control animals. Initiation of translation at the closest in-frame methionine codon would truncate the SDE2 precursor by 83 amino acids, including the cleavage site necessary for its activation. Finally, no homozygote for the G allele was observed in a large population of nearly 29,000 individuals genotyped for the mutation. The low frequency (1.3%) of the derived allele in the French population and the availability of a diagnostic test on the Illumina EuroG10K SNP chip routinely used for genomic evaluation will enable rapid and efficient selection against this deleterious mutation.

Deciphering the Wisent Demographic and Adaptive Histories from Individual Whole-Genome Sequences.

As the largest European herbivore, the wisent (Bison bonasus) is emblematic of the continent wildlife but has unclear origins. Here, we infer its demographic and adaptive histories from two individual whole-genome sequences via a detailed comparative analysis with bovine genomes. We estimate that the wisent and bovine species diverged from 1.7 × 106 to 850,000 years before present (YBP) through a speciation process involving an extended period of limited gene flow. Our data further support the occurrence of more recent secondary contacts, posterior to the Bos taurus and Bos indicus divergence (∼150,000 YBP), between the wisent and (European) taurine cattle lineages. Although the wisent and bovine population sizes experienced a similar sharp decline since the Last Glacial Maximum, we find that the wisent demography remained more fluctuating during the Pleistocene. This is in agreement with a scenario in which wisents responded to successive glaciations by habitat fragmentation rather than southward and eastward migration as for the bovine ancestors. We finally detect 423 genes under positive selection between the wisent and bovine lineages, which shed a new light on the genome response to different living conditions (temperature, available food resource, and pathogen exposure) and on the key gene functions altered by the domestication process.

Identification of copy number variation in French dairy and beef breeds using next-generation sequencing.

BACKGROUND: Copy number variations (CNV) are known to play a major role in genetic variability and disease pathogenesis in several species including cattle. In this study, we report the identification and characterization of CNV in eight French beef and dairy breeds using whole-genome sequence data from 200 animals. Bioinformatics analyses to search for CNV were carried out using four different but complementary tools and we validated a subset of the CNV by both in silico and experimental approaches. RESULTS: We report the identification and localization of 4178 putative deletion-only, duplication-only and CNV regions, which cover 6% of the bovine autosomal genome; they were validated by two in silico approaches and/or experimentally validated using array-based comparative genomic hybridization and single nucleotide polymorphism genotyping arrays. The size of these variants ranged from 334 bp to 7.7 Mb, with an average size of ~ 54 kb. Of these 4178 variants, 3940 were deletions, 67 were duplications and 171 corresponded to both deletions and duplications, which were defined as potential CNV regions. Gene content analysis revealed that, among these variants, 1100 deletions and duplications encompassed 1803 known genes, which affect a wide spectrum of molecular functions, and 1095 overlapped with known QTL regions. CONCLUSIONS: Our study is a large-scale survey of CNV in eight French dairy and beef breeds. These CNV will be useful to study the link between genetic variability and economically important traits, and to improve our knowledge on the genomic architecture of cattle.