Identification and functional study of cattle deleterious mutations – BOVANO

The project includes five workpackages (WP) plus one for coordination. Sequence and genotyping data mining to detect a priori deleterious variants are the goal of WP1. Variants with strong annotations and genomic regions showing some deficits in homozygotes are targeted. This WP represents the major source of candidate variants to be studied in the other WP. In WP2, well clinically characterized syndromes provided by ONAB are studied in details, up to the identification of the putative causal mutations, taking advantage of WP1 results. WP3 deals with some mutations responsible for embryonic losses, as initially evidenced by homozygote deficit and fertility data. In vivo and in vitro embryos are produced from mating at risk between carrier parents to define the altered development stage and study impacted molecular mechanisms. WP4 focusses on very strong candidates with no obsered impact on gestation. Homozygous calves are identified and then further clinically characterized in details to catch putative defects, with specific but also possibly common clinical signs. Finally, WP5 performs deep functional analyses of gene functions and molecular physiopathology on some confirmed mutations with a number of tools, including mice transgenesis.

Approximately 35 genetic abnormalities have been identified and characterized and all three approaches have been shown to be productive. Progressive ataxia and junctional epidermolysis bullosa in Charolais, SHGC and CHARGE syndromes in Montbéliarde, «turning« calf syndrome in Rouge des Prés, glass eyes or Truncus Arteriosis in Holstein, axonopathy in Blonde, epilepsy in Parthenaise, are examples of the Bottom-Up approach. Some anomalies have been experimentally validated in cellular or mouse models. The homozygous deficiency approach is based on a periodic reanalysis of the database. Seven mutations have been identified in six breeds. Mutations generally concern genes essential to life (cell division, synthesis of nucleic bases, etc.). Functional validation, which is relatively complex, consists of monitoring genotyped embryos from carrier parents. Such a study was carried out for the MH1 and HABH1 locuses. For the approach based on the selection of harmful variants on the sequences, a major upstream work was carried out to develop computer pipelines for the detection and annotation of variants. We detected all variants inducing loss of function or severe deleterious effects and nearly 1000 such variants were added to the genotyping chip, as well as 1000 structural variants. Among the results, we should note the identification of the RP1 gene responsible for retinal degeneration, which is very present in the Normandy breed; EDAR responsible for the «hairless and toothless« syndrome in the Charolais breed, MTCP, a mutation in the Montbéliarde breed leading to calf mortality; CAD and CENPU, two genes responsible for embryonic mortality.

* Clinical observation of progeny born alive from matings at risk between carriers of variants assumed to be strongly deleterious
* Clinical and genetic characterization of the emergences detected by ONAB
* Detailed characterization of several embryonic lethal variants.
* Refined analysis of several defects through transgenic mice models and cell culture
* Scientific communication and dissemination to the industry

Michot P et al. 2015. Whole-genome sequencing identifies a homozygous deletion encompassing exons 17 to 22 of the Integrin Beta 4 Gene in a Charolais calf with Junctional Epidermolysis Bullosa. Genet Sel Evol 47, 37
Floriot S et al. 2015. C-Nap1 mutation affects centriole cohesion and is associated with a Seckel-like syndrome in cattle. Nat Comm 6, 6894
Michot P et al. 2016. A reverse genetic approach identifies an ancient frameshift mutation in RP1 causing recessive progressive retinal degeneration in European cattle breeds. Genet Sel Evol 48, 56
Duchesne A et al. 2017. Bovine and murine models highlight novel roles for SLC25A46 in mitochondrial dynamics and metabolism, with implications for human and animal health. Plos Genet 13, e1006597
Michot P et al. 2017. A missense mutation in PFAS is likely causal for embryonic lethality associated with the MH1 haplotype in Montbeliarde dairy cattle. J Dairy Sci 100, 8176–8187
Bourneuf E et al. 2017. Rapid Discovery of De Novo Deleterious Mutations in Cattle Using Genome Sequence Data: Enhancing the Value of Farm Animals as Model Species. Sci Rep 7: 11466
Fritz S. et al. 2018. An initiator codon mutation in SDE2 causes recessive embryonic lethality in Holstein cattle. J Dairy Sci 101, 6220-6231
Duchesne A et al. 2018. Progressive ataxia of Charolais cattle highlights a role of KIF1C in sustainable myelination. Plos Genet 14: e1007550
Uddin MM et al. 2019. A missense mutation (p.Tyr452Cys) on CAD gene compromises reproductive success in dairy cattle. J Dairy Sci 102:6340–6356
Hozé C et al. 2019. A splice site mutation in CENPU is associated with recessive embryonic lethality in Holstein cattle. J Dairy Sci, accepté le 2/9/19
Escouflaire C et al. A de novo 3.8-Mb inversion affecting the EDA and XIST genes causes generalized Hypohidrotic Ectodermal Dysplasia in a heterozygous female calf. BMC Genom 20, 715
Book: INRA Prod Anim 29-5, 2016. 6 chapters
Secret know-how 16022SF, 2 genotyping licences with the EuroGenomics chip

Bovano aims at identifying mutations responsible for genetic abnormalities in French bovine breeds and, for some of them, to carry out a functional validation. Cattle breeds are a model of choice to identify deleterious mutations and study the corresponding physiopathological mechanisms because of their limited genetic effective size (around 100), the high-quality phenotypic information over a very large number of animals, and major genomic resources available. Because emergent anomalies due to recessive mutations are detected very late, one of the innovations of this project is to identify them as early as possible using a large panel of bull genomic sequences. This project also takes advantage of three on-going initiatives: (a) ONAB (Observatoire National des Anomalies Bovines) is a national observatory of genetic defects, associating research institutions, veterinarian organization and stakeholders for early emergence detection; (b) a huge database of pangenomic genotypings generated for genomic selection purpose, with hundred of thousand genotyped animals ; and (c) a large whole-genome sequence database built up from several research projects carried out by the partners and through international collaboration in the framework of the “1000 bull genome” project. This multidisciplinary proposal gathers three academic partners with complementary skills and know-how in bovine genetics and genomics, reproduction and development, bioinformatics and genome annotation, NGS sequencing platform, as well as a professional organization federating breeding and AI cooperatives, which is directly concerned by genetic defects and is experienced in genetics, genomics and reproductive biotechnologies.. The project includes five workpackages: (1) in silico identification of candidate deleterious mutations from whole genome sequence bioinformatical analysis, and their statistical validation using a large population of bulls and heifers routinely genotyped with a custom chip including these mutations; research of candidate recessive mutations responsible for embryonic mortality, based on fertility data and detection of a deficit in homozygotes in our large genomic selection population; (2) mapping and identification through genotyping and sequencing of genetic defects observed in bovine commercial populations and detected by ONAB; (3) validation and characterization of embryonic lethal mutations detected in (1) by an accurate monitoring of embryos produced from matings at risk; (4) characterization of the phenotypes associated with non-lethal deleterious mutations detected in (1), through the monitoring of homozygous mutated young animals born from matings at risk; (5) detailed functional analysis of some mutations with deep investigations including transcriptomics on bovine tissues and transgenesis in mice. Both academic and applied results are expected. Indeed, the project will improve the bovine genome annotations through newly developed annotation tools and will bring new insight into the role of several genes with unknown function as well as novel knowledge in terms of early embryonic development. For stakeholders, the project will provide an enlarged list of genetic defects segregating in bovine breeds, including a number of them detected before any emergence, and molecular genotyping tools to manage or eradicate these defects.