Comparative genomic mapping of the bovine Fragile Histidine Triad (FHIT) tumour suppressor gene: characterization of a 2 Mb BAC contig covering the locus, complete annotation of the gene, analysis of cDNA and of physiological expression profiles

BACKGROUND: The Fragile Histidine Triad gene (FHIT) is an oncosuppressor implicated in many human cancers, including vesical tumors. FHIT is frequently hit by deletions caused by fragility at FRA3B, the most active of human common fragile sites, where FHIT lays. Vesical tumors affect also cattle, including animals grazing in the wild on bracken fern; compounds released by the fern are known to induce chromosome fragility and may trigger cancer with the interplay of latent Papilloma virus. RESULTS: The bovine FHIT was characterized by assembling a contig of 78 BACs. Sequence tags were designed on human exons and introns and used directly to select bovine BACs, or compared with sequence data in the bovine genome database or in the trace archive of the bovine genome sequencing project, and adapted before use. FHIT is split in ten exons like in man, with exons 5 to 9 coding for a 149 amino acids protein. VISTA global alignments between bovine genomic contigs retrieved from the bovine genome database and the human FHIT region were performed. Conservation was extremely high over a 2 Mb region spanning the whole FHIT locus, including the size of introns. Thus, the bovine FHIT covers about 1.6 Mb compared to 1.5 Mb in man. Expression was analyzed by RT-PCR and Northern blot, and was found to be ubiquitous. Four cDNA isoforms were isolated and sequenced, that originate from an alternative usage of three variants of exon 4, revealing a size very close to the major human FHIT cDNAs. CONCLUSION: A comparative genomic approach allowed to assemble a contig of 78 BACs and to completely annotate a 1.6 Mb region spanning the bovine FHIT gene. The findings confirmed the very high level of conservation between human and bovine genomes and the importance of comparative mapping to speed the annotation process of the recently sequenced bovine genome. The detailed knowledge of the genomic FHIT region will allow to study the role of FHIT in bovine cancerogenesis, especially of vesical papillomavirus-associated cancers of the urinary bladder, and will be the basis to define the molecular structure of the bovine homologue of FRA3B, the major common fragile site of the human genome

Construction of a large collection of small genome variations in French dairy and beef breeds using whole-genome sequences

Background: In recent years, several bovine genome sequencing projects were carried out with the aim of developing genomic tools to improve dairy and beef production efficiency and sustainability.[br/] Results: In this study, we describe the first French cattle genome variation dataset obtained by sequencing 274 whole genomes representing several major dairy and beef breeds. This dataset contains over 28 million single nucleotide polymorphisms (SNPs) and small insertions and deletions. Comparisons between sequencing results and SNP array genotypes revealed a very high genotype concordance rate, which indicates the good quality of our data.[br/] Conclusions: To our knowledge, this is the first large-scale catalog of small genomic variations in French dairy and beef cattle. This resource will contribute to the study of gene functions and population structure and also help to improve traits through genotype-guided selection

C-Nap1 mutation affects centriole cohesion and is associated with a Seckel-like syndrome in cattle

Caprine-like Generalized Hypoplasia Syndrome (SHGC) is an autosomal-recessive disorder in Montbéliarde cattle. Affected animals present a wide range of clinical features that include the following: delayed development with low birth weight, hind limb muscular hypoplasia, caprine-like thin head and partial coat depigmentation. Here we show that SHGC is caused by a truncating mutation in the CEP250 gene that encodes the centrosomal protein C-Nap1. This mutation results in centrosome splitting, which neither affects centriole ultrastructure and duplication in dividing cells nor centriole function in cilium assembly and mitotic spindle organization. Loss of C-Nap1-mediated centriole cohesion leads to an altered cell migration phenotype. This discovery extends the range of loci that constitute the spectrum of autosomal primary recessive microcephaly (MCPH) and Seckel-like syndromes

A second generation radiation hybrid map to aid the assembly of the bovine genome sequence

BACKGROUND: Several approaches can be used to determine the order of loci on chromosomes and hence develop maps of the genome. However, all mapping approaches are prone to errors either arising from technical deficiencies or lack of statistical support to distinguish between alternative orders of loci. The accuracy of the genome maps could be improved, in principle, if information from different sources was combined to produce integrated maps. The publicly available bovine genomic sequence assembly with 6× coverage (Btau_2.0) is based on whole genome shotgun sequence data and limited mapping data however, it is recognised that this assembly is a draft that contains errors. Correcting the sequence assembly requires extensive additional mapping information to improve the reliability of the ordering of sequence scaffolds on chromosomes. The radiation hybrid (RH) map described here has been contributed to the international sequencing project to aid this process. RESULTS: An RH map for the 30 bovine chromosomes is presented. The map was built using the Roslin 3000-rad RH panel (BovGen RH map) and contains 3966 markers including 2473 new loci in addition to 262 amplified fragment-length polymorphisms (AFLP) and 1231 markers previously published with the first generation RH map. Sequences of the mapped loci were aligned with published bovine genome maps to identify inconsistencies. In addition to differences in the order of loci, several cases were observed where the chromosomal assignment of loci differed between maps. All the chromosome maps were aligned with the current 6× bovine assembly (Btau_2.0) and 2898 loci were unambiguously located in the bovine sequence. The order of loci on the RH map for BTA 5, 7, 16, 22, 25 and 29 differed substantially from the assembled bovine sequence. From the 2898 loci unambiguously identified in the bovine sequence assembly, 131 mapped to different chromosomes in the BovGen RH map. CONCLUSION: Alignment of the BovGen RH map with other published RH and genetic maps showed higher consistency in marker order and chromosome assignment than with the current 6× sequence assembly. This suggests that the bovine sequence assembly could be significantly improved by incorporating additional independent mapping information

Du phénotype à la mutation causale : le cas des anomalies récessives bovines

Cet article présente la méthodologie utilisée pour identifier la mutation responsable d’une anomalie génétique à partir de cas d’animaux affectés. Dans un premier temps, une collection de cas aussi homogènes que possible est constituée, de la même race et avec les mêmes signes cliniques, complétée par une population témoin apparentée mais non atteinte. Une analyse de pedigree est possible pour rechercher l’ancêtre commun qui a pu transmettre l’anomalie à chacun des cas. Le génotypage par puce permet de mettre en évidence très rapidement une petite région du génome homozygote et identique à tous les marqueurs qui contient la mutation recherchée. La mutation est ensuite identifiée par séquençage du génome de quelques cas, filtrage des variants observés sur la base d’une part, de leur présence chez d’autres animaux, et d’autre part, de leur annotation fonctionnelle. Une validation statistique est ensuite pratiquée par génotypage à grande échelle, pour vérifier l’association totale entre génotype et phénotype. Enfin, la causalité de la mutation est étudiée par analyse fonctionnelle, incluant l’analyse des ARN et des protéines, l’imagerie cellulaire, voire la création de modèles transgéniques.This article presents the methodology used to identify the mutation responsible for a genetic defect from the observation of cases. In the first step, a set of homogeneous cases are collected, from the same breed and with the same clinical signs. This collection is completed by a related but unaffected control population. A pedigree analysis is possible to point towards a common ancestor who may have transmitted the defect to all cases. A genotyping step using a SNP chip is used to display a small genomic region homozygous and identical at all markers and including the mutation. The mutation is then identified by genome sequencing of a few cases followed by the filtering of the variants against a large sequence database of unaffected animals. The best candidate variants are retained on the basis of their functional annotation. The mutation is then statistically confirmed on the basis of large scale genotyping, to verify the complete association between the mutation and the phenotype. Finally, the causality of the mutation is proven by functional analysis, including RNA and protein analysis, cellular imaging, and even through transgenic models carrying the mutation

L’Observatoire National des Anomalies Bovines, son action et ses résultats pour une aide efficace à la gestion des anomalies génétiques

Le facteur limitant de l’identification des anomalies génétiques dans les populations animales d’élevage est la phase de détection des émergences et de caractérisation clinique. En effet, ces évènements sont rares et mal répertoriés dans les bases nationales d’information génétique. Après différentes expériences passées montrant que des anomalies n’avaient pas été détectées, un Observatoire National des Anomalies Bovines a été mis en place en 2002, rassemblant l’INRA, les acteurs de la sélection animale et les acteurs du monde vétérinaire. L’objectif de cet observatoire est avant tout de collecter les déclarations de cas sur une base normalisée, ainsi que les échantillons biologiques associés, mais aussi d’assurer une veille et une information scientifique et une coordination des actions (www.onab.fr). Les déclarations sont loin d’être exhaustives avec quelques centaines de dossiers par an. Mais par la bonne couverture géographique, raciale et partenariale de l’ONAB, elles sont sans doute suffisantes pour détecter la plupart des nouvelles émergences. Cet article présente un bilan des activités, en mettant l’accent sur les différentes anomalies rencontrées ainsi que les succès en termes d’identification des mutations causales correspondantes, ouvrant la voie à leur gestion en sélection.The limiting factor of genetic defect identification in farm animal populations is the outbreak detection and clinical characterization. Indeed these events are rare and poorly recorded in animal breeding databases. After several experiences in the past showing that outbreaks can be missed, a National Observatory for Bovine Abnormalities was implemented in 2002, jointly by INRA, the main actors in animal breeding, and veterinarian organizations. The goal of this observatory is primarily to collect cases in a central database as well as their corresponding epidemiologic context and biological samples. Another goal is to ensure a scientific watch, to provide information, and to coordinate actions between partners (www.onab.fr). Records are by far not exhaustive with several hundred files per year but are probably sufficient, through a good geographical and breed coverage, to detect most new outbreaks. This article presents an overview of ONAB’s activities, focusing on different defects encountered as well as on the identification of the corresponding causative mutations, paving the way for their management in selection

Ruminants genome no longer contains Whey Acidic Protein gene but only a pseudogene

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Co-invalidation of Prnp and Sprn in FVB/N mice affects reproductive performances and highlight complex biological relationship between PrP and Shadoo

International audienceShadoo and PrP belongs to the same protein family, whose biological function remains poorly understood. Previous experiments reported potential functional redundancies or antagonisms between these two proteins, depending on the tissue analysed. While knockdown experiments suggested the requirement of Shadoo in the absence of PrP during early mouse embryogenesis, knockout ones, on the contrary, highlighted little impact, if any, of the double-knockout of these two loci. In the present study, we reinvestigated the phenotype associated with the concomitant knockout of these two genes using newly produced FVB/N Sprn knockout mice. In this genetic background, the combined two genes & rsquo; knockout induces intra-uterine growth retardations, likely resulting from placental failures highlighted by transcriptomic analyses that revealed potential redundant or antagonist roles of these two proteins in different developmental-related pathways. It also induced an increased perinatal-lethality and ascertained the role of these two loci in the lactation process

Construction of a large collection of small genome variations in French dairy and beef breeds using whole-genome sequences

International audienceAbstractBackgroundIn recent years, several bovine genome sequencing projects were carried out with the aim of developing genomic tools to improve dairy and beef production efficiency and sustainability.ResultsIn this study, we describe the first French cattle genome variation dataset obtained by sequencing 274 whole genomes representing several major dairy and beef breeds. This dataset contains over 28 million single nucleotide polymorphisms (SNPs) and small insertions and deletions. Comparisons between sequencing results and SNP array genotypes revealed a very high genotype concordance rate, which indicates the good quality of our data.ConclusionsTo our knowledge, this is the first large-scale catalog of small genomic variations in French dairy and beef cattle. This resource will contribute to the study of gene functions and population structure and also help to improve traits through genotype-guided selection

CEP250 is Required for Maintaining Centrosome Cohesion in the Germline and Fertility in Male Mice

International audienceMale gametogenesis involves both mitotic divisions to amplify germ cell progenitors that gradually differentiate and meiotic divisions. Centrosomal regulation is essential for both types of divisions, with centrioles remaining tightly paired during the interphase. Here, we generated and characterized the phenotype of mutant mice devoid of Cep250/C-Nap1 , a gene encoding for a docking protein for fibers linking centrioles, and characterized their phenotype. The Cep250 -/- mice presented with no major defects, apart from male infertility due to a reduction in the spermatogonial pool and the meiotic blockade. Spermatogonial stem cells expressing Zbtb16 were not affected, whereas the differentiating spermatogonia were vastly lost. These cells displayed abnormal γ H2AX-staining, accompanied by an increase in the apoptotic rate. The few germ cells that survived at this stage, entered the meiotic prophase I and were arrested at a pachytene-like stage, likely due to synapsis defects and the unrepaired DNA double-strand breaks. In these cells, centrosomes split up precociously, with γ -tubulin foci being separated whereas these were closely associated in wild-type cells. Interestingly, this lack of cohesion was also observed in wild-type female meiocytes, likely explaining the normal fertility of Cep250 -/- female mice. Taken together, this study proposes a specific requirement of centrosome cohesion in the male germline, with a crucial role of CEP250 in both differentiating spermatogonia and meiotic spermatocytes