The role of PRKG2 in Angus dwarfism

Dwarfism is an undesirable phenotype in beef cattle that has reduced profitability for both commercial and pure bred producers since the 1950.s. In 2002, a flare up of dwarfism on several ranches across the US led to new genomic research designed to find the genetic mutation. Linkage-mapping of Bos taurus autosome 6 (BTA6) indicated a LOD score of 7.34 between microsatellite markers AFR227 and BM4311. To expedite the fine-mapping process, the BLAST Extension and Alignment Program BEAP was developed to assist with sequence assembly of the Angus dwarfism locus prior to completion of the bovine genome project. Four positional candidate genes, bone morphogenetic protein (BMP) 2 kinase, bone morphogenetic protein (BMP) 3, fibroblast growth factor 5, and cGMP-dependant, type II, protein kinase (PRKG2 ), were re-sequenced using BEAP output as template. Within PRKG2, a C/T nonsense mutation was discovered (R678Z) that truncates PRKG2 by 85 amino acids (AA), including 25 AA of the kinase domain. A mouse knockout model and naturally occurring PRKG2 mutants demonstrate that lack of PRKG2 function causes dwarfism. Recent work in the rat indicates that PRKG2 signals SOX9 to regulate the transition from proliferative to hypertrophic growth in the growth plate. Gene expression data in the rat demonstrated that PRKG2 mutants lose the ability to regulate SOX9 mediated collagen (Col) 2 and Col10 transcription. To determine if the Angus R678Z PRKG2 mutation altered collagen expression, wild-type and R678Z bovine vectors were assayed in a cell culture transfection experiment. Real-time PCR results confirmed that COL2 was inappropriately over-expressed in cells transfected with the R678Z mutation as compared to cells transfected with wild-type PRKG2 (p\u3c0.01). Over-expression of COL2 in dwarf cattle was confirmed by real-time PCR of dwarf and normal cattle growth plate tissue samples (p\u3c0.0001). Expression of COL10 was found to be upregulated in dwarf animals as well (p=0.05). These experiments indicate that the R678Z mutation is a functional mutation, resulting in a loss of PRKG2 regulation of COL2 and COL10 mRNA expression. To determine if the R678Z marker was an accurate predictor of Angus dwarfism, we conducted a breeding experiment. Six calves were produced by embryo transfer and their phenotype was predicted with 100% accuracy prior to expression of the phenotype. Four animals were predicted to be dwarves, homozygous for the R678Z PRKG2 marker, and two were predicted to be normal, heterozygous for the R678Z PRKG2 marker. Differences in skeletal growth were characterized by measurements of gross differences in height, long bone and vertebral column length. Animals homozygous for the R678Z PRKG2 marker had shorter metatarsals (reduced 4.5cm; p\u3c0.01), and fused ulna/radius (reduced 7.5cm; p\u3c0.01) bones. No significant differences were observed between these animals for head length, calvarium (skull) width, or bone circumference (p\u3e0.25). Stature and body length were also compared between animals homozygous for the R678Z PRKG2 marker and the R678Z PRKG2 heterozygotes at approximately 210 days of age. Animals homozygous for the R678Z PRKG2 marker were 15.8cm shorter (p\u3c0.0001) and had spinal lengths reduced by 20.7 cm (p\u3c0.0001) (data not shown). Transcriptional profiling of the null PRKG2 dwarf versus wild-type animals was performed using a 70 mer cDNA microarray to assess global changes in gene expression. Both COL2 and COL10 were found to be differentially expressed in the same direction and similar magnitude as observed by real-time PCR (p\u3c0.05). In addition, non-sense mediated decay of dwarf PRKG2 and up regulation of Wnt5a, and collagen9, markers of proliferation, was observed (p\u3c0.05). Multiple genes were found to be differentially expressed in the MAPK and Wnt5a signaling cascades. In addition, many Golgi transport and neuronally expressed genes were observed as differentially expressed. These genes provide interesting candidates for understanding the intermediate processes in the PRKG2 intracellular signaling pathway that regulate proliferation and developmental timing of the growth plate. Implications of these discoveries will allow Angus breeders to eliminate dwarfism and present a model to study PRKG2 function in the growth plate

BEAP: The BLAST Extension and Alignment Program- a tool for contig construction and analysis of preliminary genome sequence

<p>Abstract</p> <p>Background</p> <p>Fine-mapping projects require a high density of SNP markers and positional candidate gene sequences. In species with incomplete genomic sequence, the DNA sequences needed to generate markers for fine-mapping within a linkage analysis confidence interval may be available but may not have been assembled. To manually piece these sequences together is laborious and costly. Moreover, annotation and assembly of short, incomplete DNA sequences is time consuming and not always straightforward.</p> <p>Findings</p> <p>We have created a tool called BEAP that combines BLAST and CAP3 to retrieve sequences and construct contigs for localized genomic regions in species with unfinished sequence drafts. The rational is that a completed genome can be used as a template to query target genomic sequence for closing the gaps or extending contig sequence length in species whose genome is incomplete on the basis that good homology exists. Each user must define what template sequence is appropriate based on comparative mapping data such as radiation hybrid (RH) maps or other evidence linking the gene sequence of the template species to the target species.</p> <p>Conclusion</p> <p>The BEAP software creates contigs suitable for discovery of orthologous genes for positional cloning. The resulting sequence alignments can be viewed graphically with a Java graphical user interface (GUI), allowing users to evaluate contig sequence quality and predict SNPs. We demonstrate the successful use of BEAP to generate genomic template sequence for positional cloning of the Angus dwarfism mutation. The software is available for free online for use on UNIX systems at <url>http://www.animalgenome.org/bioinfo/tools/beap/</url>.</p

Transcriptional Profiling of PRKG2-Null Growth Plate Identifies Putative Down-Stream Targets of PRKG2

Kinase activity of cGMP-dependent, type II, protein kinase (PRKG2) is required for the proliferative to hypertrophic transition of growth plate chondrocytes during endochondral ossification. Loss of PRKG2 function in rodent and bovine models results in dwarfism. The objective of this study was to identify pathways regulated or impacted by PRKG2 loss of function that may be responsible for disproportionate dwarfism at the molecular level. Microarray technology was used to compare growth plate cartilage gene expression in dwarf versus unaffected Angus cattle to identify putative downstream targets of PRGK2. Pathway enrichment of 1284 transcripts (nominal p \u3c 0.05) was used to identify candidate pathways consistent with the molecular phenotype of disproportionate dwarfism. Analysis with the DAVID pathway suite identified differentially expressed genes that clustered in the MHC, cytochrome B, WNT, and Muc1 pathways. A second analysis with pathway studio software identified differentially expressed genes in a host of pathways (e.g. CREB1, P21, CTNNB1, EGFR, EP300, JUN, P53, RHOA, and SRC). As a proof of concept, we validated the differential expression of five genes regulated by P53, including CEBPA, BRCA1, BUB1, CD58, and VDR by real-time PCR (p \u3c 0.05). Known and novel targets of PRKG2 were identified as enriched pathways in this study. This study indicates that loss of PRKG2 function results in differential expression of P53 regulated genes as well as additional pathways consistent with increased proliferation and apoptosis in the growth plate due to achondroplastic dwarfism

Prenatal and Postnatal Dietary n-3 Fatty Acid Supplementation Alters Buffy Coat DNA Methylation Profile in Pigs

The objective of this study was to determine the effect of maternal long chain n-3 fatty acid supplementation on the DNA methylation profile of offspring buffy coats. We demonstrated several methylated regions across the genome were influenced by maternal n-3 dietary treatment. Therefore, feeding n-3 fatty acids to sows during gestation and lactation may result in a DNA methylation imprint on offspring buffy coats that persists beyond the nursery phase and may alter the phenotype of the growing pig

SNP Discovery and Genomic Architecture of Highly Inbred Leghorn and Fayoumi Chicken Breeds Using Whole Genome Resequencing

Advances in the use of next generation sequencing (NGS) and ability to pool individuals into groups that represent distinct livestock populations has made it possible to examine trait differences between breeds of chicken. The breeds examined are very divergent when compared on their history of laying ability and immune response. The long-term objective is to understand the genetic differences between the Leghorn and Fayoumi breeds for use in developing more productive and disease resistant chickens. Statistical testing of the sequence of the two breeds along with Gene set enrichment analysis (GSEA) to make connections between the genetic variation seen in the NGS data and the breed specific traits of egg laying and heightened immune response can be used to characterize these two breeds. Genetic terms having the highest level of differentiation between the lines appear to group into metabolic processes, with terms over-enriched for immune system process, sexual reproduction, and growth for variants examined between lines. Terms for functions within the Fayoumi and Leghorn populations aligned to immune function and reproductive function, respectively. These results are consistent with known breed phenotypes and provide a means to focus on specific DNA variations and the birds’ genetic diversity that are potentially of more commercial importance

High-performance epistasis detection in quantitative trait GWAS

epiSNP is a program for identifying pairwise single nucleotide polymorphism (SNP) interactions (epistasis) in quantitative-trait genome-wide association studies (GWAS). A parallel MPI version (EPISNPmpi) was created in 2008 to address this computationally expensive analysis on large data sets with many quantitative traits and SNP markers. However, the falling cost of genotyping has led to an explosion of large-scale GWAS data sets that challenge EPISNPmpi’s ability to compute results in a reasonable amount of time. Therefore, we optimized epiSNP for modern multi-core and highly parallel many-core processors to efficiently handle these large data sets. This paper describes the serial optimizations, dynamic load balancing using MPI-3 RMA operations, and shared-memory parallelization with OpenMP to further enhance load balancing and allow execution on the Intel Xeon Phi coprocessor (MIC). For a large GWAS data set, our optimizations provided a 38.43× speedup over EPISNPmpi on 126 nodes using 2 MICs on TACC’s Stampede Supercomputer. We also describe a Coarray Fortran (CAF) version that demonstrates the suitability of PGAS languages for problems with this computational pattern. We show that the Coarray version performs competitively with the MPI version on the NERSC Edison Cray XC30 supercomputer. Finally, the performance benefits of hyper-threading for this application on Edison (average 1.35× speedup) are demonstrated

Deriving Gene Networks from SNP Associated with Triacylglycerol and Phospholipid Fatty Acid Fractions from Ribeyes of Angus Cattle

The fatty acid profile of beef is a complex trait that can benefit from gene-interaction network analysis to understand relationships among loci that contribute to phenotypic variation. Phenotypic measures of fatty acid profile from triacylglycerol and phospholipid fractions of longissimus muscle, pedigree information, and Illumina 54 k bovine SNP genotypes were utilized to derive an annotated gene network associated with fatty acid composition in 1,833 Angus beef cattle. The Bayes-B statistical model was utilized to perform a genome wide association study to estimate associations between 54 k SNP genotypes and 39 individual fatty acid phenotypes within each fraction. Posterior means of the effects were estimated for each of the 54 k SNP and for the collective effects of all the SNP in every 1-Mb genomic window in terms of the proportion of genetic variance explained by the window. Windows that explained the largest proportions of genetic variance for individual lipids were found in the triacylglycerol fraction. There was almost no overlap in the genomic regions explaining variance between the triacylglycerol and phospholipid fractions. Partial correlations were used to identify correlated regions of the genome for the set of largest 1 Mb windows that explained up to 35% genetic variation in either fatty acid fraction. SNP were allocated to windows based on the bovine UMD3.1 assembly. Gene network clusters were generated utilizing a partial correlation and information theory algorithm. Results were used in conjunction with network scoring and visualization software to analyze correlated SNP across 39 fatty acid phenotypes to identify SNP of significance. Significant pathways implicated in fatty acid metabolism through GO term enrichment analysis included homeostasis of number of cells, homeostatic process, coenzyme/cofactor activity, and immunoglobulin. These results suggest different metabolic pathways regulate the development of different types of lipids found in bovine muscle tissues. Network analysis using partial correlations and annotation of significant SNPs can yield information about the genetic architecture of complex traits

Fatty Acid SNP Interaction Analysis in Angus Sired Beef Cattle

The triacylglyceride (TAG) fatty acid content in meat from Angus-sired cattle was analyzed for non-additive genetic effects. A total of 11,482 significant DNA marker interactions (false discovery rate [FDR] \u3c 0.05) were detected across thirty-seven different TAG fatty acids. Interactions were not evenly distributed amongst all fatty acids analyzed, and types of interactions (additive-by-additive, additive-by-dominance, and dominance-by-dominance) varied within each individual fatty acid. These results indicate that it may be possible to account for additional genetic variance amongst TAG fatty acids over and above individual markers

Characterizing Haplotype Diversity in Ten US Beef Cattle Breeds

The distributions of haplotype diversity across the whole genome among 10 US beef cattle breeds were constructed. In most chromosomes for all the breeds, consistent low haplotype diversity were observed in some specific regions, 55% of which was found to match the positions of reported gene duplications. Further work is required to determine whether the low haplotype diversity is real, or a result of problems in sequencing which have limited our ability to identify informative markers in those regions. Haplotype diversity will be the subject of ongoing work to identify haplotypes that are under-represented as homozygotes, to fine-map regions with major gene effects, and to fit haplotype rather than SNP models for genomic prediction

Development of New Measurements and Tools to Mitigate Fescue Toxicosis in Beef Cattle

The objective of this study was to identify new ways to determine the severity of fescue toxicosis and identify genetic differences in fescue impacted traits as a basis in understanding how cattle could be selected for tolerance to fescue toxicosis. We identified across breed and within breed differences in heat stress related traits and growth rate in pregnant cows exposed to toxic fescue. In addition, new biomarkers were identified to differentiate susceptible and tolerant cattle in the form of specific fecal and vaginal microbes. Finally, we identified differentially expressed (DE) genes in high versus low tolerant cattle on toxic fescue. These findings may allow more accurate diagnosis of fescue toxicosis and provide a glimpse into the genes and microorganisms that may impact tolerance or susceptibility to toxic fescue