Mutations germinales en Bos taurus

De novo mutation (dnm) in the germline is a fundamental biological process that is the source of all population genetic variation. In this thesis, we have exploited the unique population structure of cattle to select pedigrees of three (parents, proband and >1 grand-offspring) and four (plus grandparents) generations to identify and characterise germline dnm. We have also looked at the impact of recent dnms on the genetic load of the cattle population, using forward and reverse genetics to identify the causative mutations responsible for major defects in cattle populations including embryonic lethality. In the first study, we utilised five pedigrees to identify SNP and small insertion-deletion (INDEL) dnms, assign them to a parent of origin and determine the stage of development at which they occurred. We determined the dnm rate in cattle to be ~1.2x10-8 per base pair per generation, with 2.5 paternal dnms for each maternal dnm. We showed that 30% and 50% of the dnms in sperm and eggs respectively are mosaic in the parental DNA, occurring early in embryonic development. By simulation we show that this is incompatible with a constant mutation rate through gametogenesis and best fits a 20x higher mutation rate for the first four cell divisions of the fertilised egg. This paper is currently in review with a preprint available on BioRxiv (Harland et al. 2017a). In a second study, we looked at the rate of dnm in the wider population and for the presence of inter- individual variation in the rate. We utilised the complete Damona dataset of 131 three generation pedigrees identifying ~7,500 dnms, confirming the previously observed degree of mosaicism and our dnm rate of 1.2x10-8 per bp per generation in a wider sample. We observe several outliers in the population, with 5-17x the average number of dnms occurring during embryo development, along with distinct mutational signatures. For one outlier pedigree, we identified two candidate causative mutations that are in the process of characterising. In addition, we detect a significant environmental effect from the use of reproductive technologies, such as in vitro fertilisation and maturation, on the rate of dnm during early embryo development. (Harland et al. 2017b, in preparation). In the third study, we turned our attention to alternative forms of dnm. Utilising the full dataset, we identified five cases of de novo transposition of an endogenous retrovirus family K (ERVK) element, with three of the five events occurring within the germ-line of a single individual, and two of the three in the same gamete. This indicates that the ERVK family is presently active in the bovine genome with an average de novo transposition rate of ~1 event per 50 gametes, but there is strong evidence for considerable inter-individual variation. We identified ~1,600 polymorphic ERVs resulting from the activity of these elements in the cattle population. One recent de novo transposition of an ERVK element in the APOB gene is responsible for the lethal monogenic Cholesterol Deficiency disorder in cattle. This transposition has reached a minor allele frequency (MAF) of 2.8% in the European population (Harland et al. 2017c, in preperation). In a fourth study, we describe a dominant deleterious missense dnm in the Prolactin gene (PRL) that caused heat stress, abnormal hair growth and a failure to milk in ~2,000 affected offspring of a single bull. This set of phenotypes is opposite to those observed in ‘slick’ cattle, which show increased heat tolerance, short hair and potentially increased milk production. We demonstrate that the slick phenotype in Senepol cattle is due to a dominant frameshift mutation in the Prolactin receptor gene, and is an example of a beneficial variant that has undergone positive selection within a population (Littlejohn et al. 2014). The large number of affected cattle for the PRL mutation demonstrates how a rare dnm can rapidly increase in frequency within a population when it is present in an elite sire used for artificial insemination. For examples of other recent dnms, we turned to the Belgian Blue population and investigated recessive junctional epidermolysis bullosa. This lead to the identification of a breed specific, premature stop-gain mutation in the laminin, alpha 3 gene with a MAF of 1%, thus allowing the development of a direct genetic test for the disorder (Sartelet & Harland et al. 2015). We then used whole genome sequences (WGS) obtained on Illumina HiSeq’s to search for embryonic lethal (EL) mutations segregating in the New Zealand dairy and Belgian Blue beef cattle populations using a reverse genetic approach. We genotyped >40,000 cattle for 296 loss of function (LOF) and 3,483 potentially deleterious missense variants that were breed specific, and identified variants with a significant deficiency in homozygous mutant animals. Nine of these variants were confirmed to be EL by genotyping 200 carrier x carrier trios and demonstrating the absence of homozygous offspring. The MAF of these variants was between 1.2% and 6.6%. We estimate that 15% of the tested LOF and 6% of the missense events are EL reducing the fertility of dairy cattle (Charlier et al. 2016).Les évènements mutationnels de novo au sein de la lignée germinale constituent un processus biologique fondamental, ils sont la source principale de toute nouvelle variation génétique. Au cours de cette thèse, nous avons tiré parti de la structure particulière des populations bovines (larges familles de demi- frères/sœurs) pour identifier puis caractériser les mutations de novo (dnms) dans la lignée germinale. Nous avons également étudié l’impact phénotypique de certaines de ces récentes mutations en utilisant à la fois des approches de type clonage positionnel et de génétique dite réverse pour identifier les mutations causales d’une série de caractères d’intérêt agronomique chez le bovin, allant de la fertilité au type de pelage. Dans une première étude pilote, nous avons exploité les données de séquence ‘génome entier’ (WGS) de cinq pedigrees multi-générationnels pour identifier les dnms (de type SNP ou petites insertions/délétions), leur attribuer une origine parentale et déterminer le stade de développement auquel elles se sont produites. Nous avons ainsi estimé un taux moyen de mutations de novo chez le bovin à ~1.2X10-8, par paire de bases et par génération, avec un biais d’origine en faveur du père de 2,5 fois. Nous avons démontré que respectivement 30% et 50% des dnms présentes dans le sperme ou les oocytes sont mosaïques dans l’ADN parental, elles sont donc apparues lors du développement embryonnaire précoce du parent. Des simulations ont montré qu’un taux de dnms constant au cours du développement ne permettait pas d’expliquer ces observations, et qu’une augmentation du taux de mutations de ~20X lors des quatre premières divisions cellulaires de l’œuf fécondé était requise. Ce manuscrit est en cours de révision et une version initiale est disponible dans BioRxiv (Harland et al., 2017a). La seconde étude s’est attachée à renforcer et valider ces conclusions préliminaires à l’aide d’un data set étendu, ainsi qu’à quantifier d’éventuelles variations interindividuelles du taux mutationnel. Pour ce faire, un large pedigree (Damona) composé de 131 familles a été analysé. Un total de ~7.500 dnms ont été identifiées, caractérisées et classifiées, ce qui a confirmé le degré élevé de mosaïcisme initialement observé, ainsi que le taux moyen de mutations de novo chez le bovin. Une poignée d’individus extrêmes arborant un nombre significativement plus élevé de mutations apparues durant leur développement embryonnaire précoce ont été repérés. Chacun présente une signature mutationnelle unique. Au moins deux gènes et variations candidates sont actuellement suivis pour tenter d’expliquer ces taux mutationnels extrêmes. Enfin, la mise en évidence d’un effet significatif des technologies reproductives utilisées démontre qu’elles influencent le taux mutationnel lors du développement précoce de l’embryon qui y est soumis (Harland et al., 2017b). La troisième étude a visé d’autres catégories d’évènements de novo, à savoir la mobilisation des éléments transposables du génome et, en particulier, les éléments rétroviraux endogènes (ERV). Exploitant le même pedigree, cinq évènements de novo de transposition d’ERV, tous issus de la famille ERVK, ont été identifiés. Remarquablement, trois de ces évènements se sont produits dans la lignée Abstract 6 germinale d’un même taureau et deux été transmis par le même gamète. Cela indique que la famille ERVK est encore active dans le génome bovin et on peut ainsi estimer le taux de transposition de novo à ~1 événement par 50 gamètes, avec toutefois une grande variation interindividuelle. En outre, nous avons établi un catalogue de ~1.600 ERVs polymorphes qui ségrégent dans les populations bovines étudiées. Ils résultent de l’activité récente de ces éléments. L’un de ces événements a touché la partie codante du gène APOB, il est responsable d’une maladie récessive létale, caractérisée pas une déficience en cholestérol sanguin. Cette mutation est présente en race laitière Holstein à une fréquence de 2,8% et est maintenant activement contre-sélectionnée (Harland et al., 2017c). La quatrième étude décrit deux mutations de novo dominantes à effet phénotypique opposé. La première est une mutation non-synonyme dans le gène de la prolactine (PRL). Cette mutation cause une diminution de la résistance à la chaleur, l’apparition d’un pelage hirsute et a un impact négatif sévère sur la production laitière. Les ~2.000 descendants atteints sont issus d’un seul taureau, mosaïque germinale pour la mutation. Cela illustre comment une mutation délétère peut très rapidement émerger lorsqu’elle se produit chez un taureau d’élite largement disséminé grâce à l’insémination artificielle. La seconde mutation introduit un décalage de la phase de lecture dans le gène du récepteur à la prolactine (PRLR), elle est responsable d’une collection de phénotypes opposés aux précédents (pelage ras, résistance à la chaleur,...). C’est ici un exemple de mutation à effet bénéfique qui a été sélectionnée et fixée dans des races bovines tropicales (Littlejohn et al., 2014). D’autres occurrences de mutations de novo délétères ont été décrites en race Blanc-Bleu belge (BBB), la plus récente étant une mutation qui introduit un codon stop prématuré dans le gène de la laminine alpha 3 (LAMA3), elle y est responsable d’une épidermolyse jonctionnelle bulleuse récessive létale. Les porteurs sont maintenant identifiés en routine grâce au test génétique développé (Sartelet, Harland et al., 2015). Enfin, des données de WGS, en populations laitière Néozélandaise et BBB, ont été exploitées dans des études dites réverses (allant du génome et de la mutation au phénotype) pour identifier et valider neuf mutations, létales pour l’embryon homozygote, et ayant des fréquences de 1,2 à 6,6%. Nous avons démontré que, collectivement, ces mutations avaient un impact négatif non-négligeable sur la fertilité de ces races. Cette information est mise à profit dans des programmes de conseil d’accouplements afin d’éviter les croisements à risque (Charlier et al., 2016).Damon

F-actin and integrin like proteins in Phytophthora cinnamomi

Tip growth is the primary form of growth in hyphal organisms and some plant cells. Tip growth in hyphae is highly dependent on F-actin, which acts to regulate and support growth. One of the models suggested for tip growth, the amebae model of tip growth, suggests that F-actin may also be the primary source of protrusive force for tip growth in some conditions, and that proteins with a similar function to animal integrins would be present an involved in tip growth (Heath and Steinberg 1999). In this thesis we examine the role of F-actin in the growth of the oomycete Phytophthora cinnamomi and the effects on growth of the F-actin disrupting compound Latrunculin B. We demonstrate that F-actin plays a critical role in the tip growth of Phytophthora cinnamomi with it's disruption causing rapid cessation in directional growth, followed by significant subapical swelling. Further more we examine Phytophthora cinnamomi for the presence of an B4 integrin like protein that has been previously reported in the oomycete Achlya bisexualis (Chitcholtan & Garrill 2005) and show that the B4 integrin like protein is not present in Phytophthora cinnamomi. These experiments help further our understanding of tip growth in Phytophthora cinnamomi an economically important plant pathogen

An evaluation of inbreeding measures using a whole-genome sequenced cattle pedigree.

peer reviewedThe estimation of the inbreeding coefficient (F) is essential for the study of inbreeding depression (ID) or for the management of populations under conservation. Several methods have been proposed to estimate the realized F using genetic markers, but it remains unclear which one should be used. Here we used whole-genome sequence data for 245 individuals from a Holstein cattle pedigree to empirically evaluate which estimators best capture homozygosity at variants causing ID, such as rare deleterious alleles or loci presenting heterozygote advantage and segregating at intermediate frequency. Estimators relying on the correlation between uniting gametes (F(UNI)) or on the genomic relationships (F(GRM)) presented the highest correlations with these variants. However, homozygosity at rare alleles remained poorly captured. A second group of estimators relying on excess homozygosity (F(HOM)), homozygous-by-descent segments (F(HBD)), runs-of-homozygosity (F(ROH)) or on the known genealogy (F(PED)) was better at capturing whole-genome homozygosity, reflecting the consequences of inbreeding on all variants, and for young alleles with low to moderate frequencies (0.10 < . < 0.25). The results indicate that F(UNI) and F(GRM) might present a stronger association with ID. However, the situation might be different when recessive deleterious alleles reach higher frequencies, such as in populations with a small effective population size. For locus-specific inbreeding measures or at low marker density, the ranking of the methods can also change as F(HBD) makes better use of the information from neighboring markers. Finally, we confirmed that genomic measures are in general superior to pedigree-based estimates. In particular, F(PED) was uncorrelated with locus-specific homozygosity

GWAS reveals determinants of mobilization rate and dynamics of an active endogenous retrovirus of cattle.

peer reviewedFive to ten percent of mammalian genomes is occupied by multiple clades of endogenous retroviruses (ERVs), that may count thousands of members. New ERV clades arise by retroviral infection of the germline followed by expansion by reinfection and/or retrotransposition. ERV mobilization is a source of deleterious variation, driving the emergence of ERV silencing mechanisms, leaving "DNA fossils". Here we show that the ERVK[2-1-LTR] clade is still active in the bovine and a source of disease-causing alleles. We develop a method to measure the rate of ERVK[2-1-LTR] mobilization, finding an average of 1 per ~150 sperm cells, with >10-fold difference between animals. We perform a genome-wide association study and identify eight loci affecting ERVK[2-1-LTR] mobilization. We provide evidence that polymorphic ERVK[2-1-LTR] elements in four of these loci cause the association. We generate a catalogue of full length ERVK[2-1-LTR] elements, and show that it comprises 15% of C-type autonomous elements, and 85% of D-type non-autonomous elements lacking functional genes. We show that >25% of the variance of mobilization rate is determined by the number of C-type elements, yet that de novo insertions are dominated by D-type elements. We propose that D-type elements act as parasite-of-parasite gene drives that may contribute to the observed demise of ERV elements

Immunospecific Responses to Bacterial Elongation Factor Tu during Burkholderia Infection and Immunization

Burkholderia pseudomallei is the etiological agent of melioidosis, a disease endemic in parts of Southeast Asia and Northern Australia. Currently there is no licensed vaccine against infection with this biological threat agent. In this study, we employed an immunoproteomic approach and identified bacterial Elongation factor-Tu (EF-Tu) as a potential vaccine antigen. EF-Tu is membrane-associated, secreted in outer membrane vesicles (OMVs), and immunogenic during Burkholderia infection in the murine model of melioidosis. Active immunization with EF-Tu induced antigen-specific antibody and cell-mediated immune responses in mice. Mucosal immunization with EF-Tu also reduced lung bacterial loads in mice challenged with aerosolized B. thailandensis. Our data support the utility of EF-Tu as a novel vaccine immunogen against bacterial infection

F-actin and integrin like proteins in Phytophthora cinnamomi

Tip growth is the primary form of growth in hyphal organisms and some plant cells. Tip growth in hyphae is highly dependent on F-actin, which acts to regulate and support growth. One of the models suggested for tip growth, the amebae model of tip growth, suggests that F-actin may also be the primary source of protrusive force for tip growth in some conditions, and that proteins with a similar function to animal integrins would be present an involved in tip growth (Heath and Steinberg 1999). In this thesis we examine the role of F-actin in the growth of the oomycete Phytophthora cinnamomi and the effects on growth of the F-actin disrupting compound Latrunculin B. We demonstrate that F-actin plays a critical role in the tip growth of Phytophthora cinnamomi with it's disruption causing rapid cessation in directional growth, followed by significant subapical swelling. Further more we examine Phytophthora cinnamomi for the presence of an B4 integrin like protein that has been previously reported in the oomycete Achlya bisexualis (Chitcholtan & Garrill 2005) and show that the B4 integrin like protein is not present in Phytophthora cinnamomi. These experiments help further our understanding of tip growth in Phytophthora cinnamomi an economically important plant pathogen

The genomes of precision edited cloned calves show no evidence for off-target events or increased de novo mutagenesis

Abstract Background Animal health and welfare are at the forefront of public concern and the agricultural sector is responding by prioritising the selection of welfare-relevant traits in their breeding schemes. In some cases, welfare-enhancing traits such as horn-status (i.e., polled) or diluted coat colour, which could enhance heat tolerance, may not segregate in breeds of primary interest, highlighting gene-editing tools such as the CRISPR-Cas9 technology as an approach to rapidly introduce variation into these populations. A major limitation preventing the acceptance of CRISPR-Cas9 mediated gene-editing, however, is the potential for off-target mutagenesis, which has raised concerns about the safety and ultimate applicability of this technology. Here, we present a clone-based study design that has allowed a detailed investigation of off-target and de novo mutagenesis in a cattle line bearing edits in the PMEL gene for diluted coat-colour. Results No off-target events were detected from high depth whole genome sequencing performed in precursor cell-lines and resultant calves cloned from those edited and non-edited cell lines. Long molecule sequencing at the edited site and plasmid-specific PCRs did not reveal structural variations and/or plasmid integration events in edited samples. Furthermore, an in-depth analysis of de novo mutations across the edited and non-edited cloned calves revealed that the mutation frequency and spectra were unaffected by editing status. Cells in culture, however, appeared to have a distinct mutation signature where de novo mutations were predominantly C > A mutations, and in cloned calves they were predominantly T > G mutations, deviating from the expected excess of C > T mutations. Conclusions We found no detectable CRISPR-Cas9 associated off-target mutations in the gene-edited cells or calves derived from the gene-edited cell line. Comparison of de novo mutation in two gene-edited calves and three non-edited control calves did not reveal a higher mutation load in any one group, gene-edited or control, beyond those anticipated from spontaneous mutagenesis. Cell culture and somatic cell nuclear transfer cloning processes contributed the major source of contrast in mutational profile between samples

BIRTH OF POLYMORPHIC PIRNA CLUSTERS DRIVEN BY TRANSPOSABLE ELEMENT INSERTIONS IN THE MALE CATTLE GERMLINE

We aim at studying the two sides of the same coin: (i) one major line of host defense against the mobilization of transposable elements (TE): the PIWI-interacting RNA (piRNA) pathway and (ii) the activity of TE in the bovine germline. We previously demonstrated that several TE families are still active in present-day cattle breeds. These include endogenous retroviruses (ERV), long and short interspersed repeats (LINE and SINE) and processed pseudogenes. We have collected testes samples from sexually immature (pre-pachytene stage; n=100) and mature (pachytene stage; n=100) animals. From the host defense side, we are sequencing oxidized piRNA libraries, reads are mapped to the ARS-UCD1.2 bovine reference sequence and piRNA clusters are annotated using proTRAC (Rosenkranz & Zischler, 2012). From the TE side, we developed a bioinformatics tool to mine available whole genome sequences (WGS; n>1000) of the corresponding breeds to establish a catalog of polymorphic (unfixed) TE segregating in these breeds. Custom, array-based, genotyping assays, targeting insertion sites, are developed to directly genotype our testes collection. We will integrate piRNA and TE genotyping data to establish the degree of interplay between host defense and young TE to answer the following question: are unfixed piRNA clusters directly driven by recent TE insertions? Preliminary data on pre-pachytene testes with matched WGS, piRNA and mRNA sequences allowed us to indeed identify striking instances of births of new, allele-specific, polymorphic piRNA clusters derived from unfixed (i) ERVK, (ii) LINE1 and (iii) processed pseudogene intronic insertions. Latest results will be presented

Frequency of mosaicism points towards mutation-prone early cleavage cell divisions in cattle.

It has recently become possible to directly estimate the germ-line de novo mutation (dnm) rate by sequencing the whole genome of father-mother-offspring trios, and this has been conducted in human1–5, chimpanzee6, mice7, birds8and fish9. In these studiesdnm’s are typically defined as variants that are heterozygous in the offspring while being absent in both parents. They are assumed to have occurred in the germ-line of one of the parents and to have been transmitted to the offspring via the sperm cell or oocyte. This definition assumes that detectable mosaïcism in the parent in which the mutation occurred is negligible. However, instances of detectable mosaïcism or premeiotic clusters are well documented in humans and other organisms, including ruminants10–12. We herein take advantage of cattle pedigrees to show that as much as ∼30% to ∼50% ofdnm’s present in a gamete may occur during the early cleavage cell divisions in males and females, respectively, resulting in frequent detectable mosaïcism and a high rate of sharing of multiplednm’s between siblings. This should be taken into account to accurately estimate the mutation rate in cattle and other species.</jats:p

A stop-gain in the laminin, alpha 3 gene causes recessive junctional epidermolysis bullosa in Belgian Bleu cattle

RILOUK