The development of a bovine interspecies model for the analysis of genomic imprinting in normal and nuclear transfer derived fetuses

The advent of somatic cell nuclear transfer in cattle has provided the opportunity for researchers to generate genetically identical animals as well as animals that possess precise genetic modifications for agriculture and biomedical purposes. However, in spite of the revolutionary impact this technology presents, problems remain which hinder the production of healthy animals on a consistent basis. Research on cloned mice implicates improper reprogramming of epigenetic modifications and genomic imprinting for the low pregnancy rates and high incidence of abnormalities that are manifested in cloned animals; however, a systematic and comprehensive analysis of nuclear reprogramming in cloned cattle remains undone. The purpose of this research is to assess and characterize the patterns of genomic imprinting in normal and nuclear transfer derived bovine fetuses. To facilitate the identification of imprinted genes in the bovine, a Bos gaurus/Bos taurus interspecies model has been incorporated to maximize the genetic heterozygosity that exists between the alleles of putative imprinted genes for allelic discrimination and parental inheritance. The sequence of twenty-six genes, previously reported as imprinted in mice and humans, was analyzed in Bos gaurus (Gaur) and Bos taurus (Angus) cattle for the presence of single nucleotide polymorphisms (SNP). SNPs were detected in the Gene trap locus 2 (GTL2), Insulin like growth factor 2 (IGF2), Wilms tumor 1 (WT1) and the X chromosome inactivation specific transcript (XIST). Allelic expression analysis in interspecies hybrids indicated maternal genomic imprinting at the IGF2 and XIST loci, paternal genomic imprinting at the GTL2 locus and no imprinting at the WT1 locus. Analysis in cloned hybrids indicated fidelity of allelic expression at the IGF2 and GTL2 loci, however disruption of imprinting was observed at the XIST locus in the placenta of clones. These results are the largest identification of imprinted genes in the bovine and the first identification of the disruption of an imprinted gene in an animal derived from somatic cell nuclear transfer

TRPM2 SNP genotype previously associated with susceptibility to Rhodococcus equi pneumonia in Quarter Horse foals displays differential gene expression identified using RNA-Seq

BACKGROUND: Rhodococcus equi (R. equi) is an intracellular bacterium that affects young foals and immuno-compromised individuals causing severe pneumonia. Currently, the genetic mechanisms that confer susceptibility and/or resistance to R. equi are not fully understood. Previously, using a SNP-based genome-wide association study, we identified a region on equine chromosome 26 associated with culture-confirmed clinical pneumonia. To better characterize this region and understand the function of the SNP located within TRPM2 that was associated with R. equi pneumonia, we performed RNA-Seq on 12 horses representing the 3 genotypic forms of this SNP. RESULTS: We identified differentially expressed genes in the innate immune response pathway when comparing homozygous A allele horses with the AB and BB horses. Isoform analyses of the RNA-Seq data predicted the existence of multiple transcripts and provided evidence of differential expression at the TRPM2 locus. This finding is consistent with previously demonstrated work in human cell lines in which isoform-specific expression of TRPM2 was critical for cell viability. CONCLUSIONS: This work demonstrates that SNPs in TRPM2 are associated with differences in gene expression, suggesting that modulation of expression of this innate immune gene contributes to susceptibility to R. equi pneumonia

Whole-Genome sequencing and genetic variant analysis of a quarter Horse mare

BACKGROUND: The catalog of genetic variants in the horse genome originates from a few select animals, the majority originating from the Thoroughbred mare used for the equine genome sequencing project. The purpose of this study was to identify genetic variants, including single nucleotide polymorphisms (SNPs), insertion/deletion polymorphisms (INDELs), and copy number variants (CNVs) in the genome of an individual Quarter Horse mare sequenced by next-generation sequencing. RESULTS: Using massively parallel paired-end sequencing, we generated 59.6 Gb of DNA sequence from a Quarter Horse mare resulting in an average of 24.7X sequence coverage. Reads were mapped to approximately 97% of the reference Thoroughbred genome. Unmapped reads were de novo assembled resulting in 19.1 Mb of new genomic sequence in the horse. Using a stringent filtering method, we identified 3.1 million SNPs, 193 thousand INDELs, and 282 CNVs. Genetic variants were annotated to determine their impact on gene structure and function. Additionally, we genotyped this Quarter Horse for mutations of known diseases and for variants associated with particular traits. Functional clustering analysis of genetic variants revealed that most of the genetic variation in the horse's genome was enriched in sensory perception, signal transduction, and immunity and defense pathways. CONCLUSIONS: This is the first sequencing of a horse genome by next-generation sequencing and the first genomic sequence of an individual Quarter Horse mare. We have increased the catalog of genetic variants for use in equine genomics by the addition of novel SNPs, INDELs, and CNVs. The genetic variants described here will be a useful resource for future studies of genetic variation regulating performance traits and diseases in equids

Identification of Genomic Loci Associated with Rhodococcus equi Susceptibility in Foals

Pneumonia caused by Rhodococcus equi is a common cause of disease and death in foals. Although agent and environmental factors contribute to the incidence of this disease, the genetic factors influencing the clinical outcomes of R. equi pneumonia are ill-defined. Here, we performed independent single nucleotide polymorphism (SNP)- and copy number variant (CNV)-based genome-wide association studies to identify genomic loci associated with R. equi pneumonia in foals. Foals at a large Quarter Horse breeding farm were categorized into 3 groups: 1) foals with R. equi pneumonia (clinical group [N = 43]); 2) foals with ultrasonographic evidence of pulmonary lesions that never developed clinical signs of pneumonia (subclinical group [N = 156]); and, 3) foals without clinical signs or ultrasonographic evidence of pneumonia (unaffected group [N = 49]). From each group, 24 foals were randomly selected and used for independent SNP- and CNV-based genome-wide association studies (GWAS). The SNP-based GWAS identified a region on chromosome 26 that had moderate evidence of association with R. equi pneumonia when comparing clinical and subclinical foals. A joint analysis including all study foals revealed a 3- to 4-fold increase in odds of disease for a homozygous SNP within the associated region when comparing the clinical group with either of the other 2 groups of foals or their combination. The region contains the transient receptor potential cation channel, subfamily M, member 2 (TRPM2) gene, which is involved in neutrophil function. No associations were identified in the CNV-based GWAS. Collectively, these data identify a region on chromosome 26 associated with R. equi pneumonia in foals, providing evidence that genetic factors may indeed contribute to this important disease of foals.The open access fee for this work was funded through the Texas A&M University Open Access to Knowledge (OAK) Fund

Maternal and paternal genomes differentially affect myofibre characteristics and muscle weights of bovine fetuses at midgestation

Postnatal myofibre characteristics and muscle mass are largely determined during fetal development and may be significantly affected by epigenetic parent-of-origin effects. However, data on such effects in prenatal muscle development that could help understand unexplained variation in postnatal muscle traits are lacking. In a bovine model we studied effects of distinct maternal and paternal genomes, fetal sex, and non-genetic maternal effects on fetal myofibre characteristics and muscle mass. Data from 73 fetuses (Day153, 54% term) of four genetic groups with purebred and reciprocal cross Angus and Brahman genetics were analyzed using general linear models. Parental genomes explained the greatest proportion of variation in myofibre size of Musculus semitendinosus (80–96%) and in absolute and relative weights of M. supraspinatus, M. longissimus dorsi, M. quadriceps femoris and M. semimembranosus (82–89% and 56–93%, respectively). Paternal genome in interaction with maternal genome (P<0.05) explained most genetic variation in cross sectional area (CSA) of fast myotubes (68%), while maternal genome alone explained most genetic variation in CSA of fast myofibres (93%, P<0.01). Furthermore, maternal genome independently (M. semimembranosus, 88%, P<0.0001) or in combination (M. supraspinatus, 82%; M. longissimus dorsi, 93%; M. quadriceps femoris, 86%) with nested maternal weight effect (5–6%, P<0.05), was the predominant source of variation for absolute muscle weights. Effects of paternal genome on muscle mass decreased from thoracic to pelvic limb and accounted for all (M. supraspinatus, 97%, P<0.0001) or most (M. longissimus dorsi, 69%, P<0.0001; M. quadriceps femoris, 54%, P<0.001) genetic variation in relative weights. An interaction between maternal and paternal genomes (P<0.01) and effects of maternal weight (P<0.05) on expression of H19, a master regulator of an imprinted gene network, and negative correlations between H19 expression and fetal muscle mass (P<0.001), suggested imprinted genes and miRNA interference as mechanisms for differential effects of maternal and paternal genomes on fetal muscle.Ruidong Xiang, Mani Ghanipoor-Samami, William H. Johns, Tanja Eindorf, David L. Rutley, Zbigniew A. Kruk, Carolyn J. Fitzsimmons, Dana A. Thomsen, Claire T. Roberts, Brian M. Burns, Gail I. Anderson, Paul L. Greenwood, Stefan Hiendlede

Angelman syndrome antisense treatment

Disclosed herein are antisense oligonucleotides that are capable of inducing expression of ubiquitin-protein ligase E3A (UBE3A) from the paternal allele in animal or human neurons. The oligonucleotides target the suppressor of the UBE3A paternal allele by hybridization to SNHG14 long non-coding RNA at the 5'-end of UBE3A-AS, which is downstream of SNORD115-45 snoRNA. Also disclosed are pharmaceutical compositions and methods for treatment of Angelman syndrome.U

Epigenetic profiling at mouse imprinted gene clusters reveals novel epigenetic and genetic features at differentially methylated regions

Genomic imprinting arises from allele-specific epigenetic modifications that are established during gametogenesis and that are maintained throughout somatic development. These parental-specific modifications include DNA methylation and post-translational modifications to histones, which create allele-specific active and repressive domains at imprinted regions. Through the use of a high-density genomic tiling array, we generated DNA and histone methylation profiles at 11 imprinted gene clusters in the mouse from DNA and from chromatin immunoprecipitated from sperm, heart, and cerebellum. Our analysis revealed that despite high levels of differential DNA methylation at non-CpG islands within these regions, imprinting control regions (ICRs) and secondary differentially methylated regions (DMRs) were identified by an overlapping pattern of H3K4 trimethylation (active chromatin) and H3K9 trimethylation (repressive chromatin) modifications in somatic tissue, and a sperm differentially methylated region (sDMR; sperm ≠ somatic tissue). Using these features as a common signature of DMRs, we identified 11 unique regions that mapped to known imprinted genes, to uncharacterized genes, and to intergenic regions flanking known imprinted genes. A common feature among these regions was the presence of a CpG island and an array of tandem repeats. Collectively, this study provides a comprehensive analysis of DNA methylation and histone H3K4me3 and H3K9me3 modifications at imprinted gene clusters, and identifies common epigenetic and genetic features of regions regulating genomic imprinting

Epigenetic and genomic imprinting analysis in nuclear transfer derived Bos gaurus/Bos taurus hybrid fetuses

Somatic cell nuclear transfer (NT) in cattle is an inefficient process, whereby the production of calves is hindered by low pregnancy rates as well as fetal and placental abnormalities. Interspecies models have been previously used to facilitate the identification of single nucleotide polymorphisms (SNPs) within coding regions of genes to discriminate between parental alleles in the offspring. Here we report the use of a bovine interspecies model (Bos gaurus x Bos taurus) for the assessment and characterization of epigenetic modifications and genomic imprinting in Day 40-old female NT-derived fetuses and placenta. Analysis of NT and control pregnancies indicated disruption of genomic imprinting at the X inactivation-specific transcript (XIST) locus in the chorion, but not the fetus of clones, whereas proper allelic expression of the insulin-like growth factor II (IGF2) and gene trap locus 2 (GTL2) loci was maintained in both the fetus and placenta. Analysis of the XIST differentially methylated region (DMR) in clones indicated normal patterns of methylation; however, bisulfite sequencing of the satellite I repeat element and epidermal cytokeratin promoter indicated hypermethylation in the chorion of clones when compared with controls. No differences were detected in methylation levels in the fetus proper. These results indicate that the nuclear transfer process affects gene expression patterns in the trophectoderm- and inner cell mass-derived tissues to different extents