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. 2015 Nov 14;16:932.
doi: 10.1186/s12864-015-2119-7.

Functional Analysis and Transcriptional Output of the Göttingen Minipig Genome

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Free PMC article

Functional Analysis and Transcriptional Output of the Göttingen Minipig Genome

Tobias Heckel et al. BMC Genomics. .
Free PMC article

Abstract

Background: In the past decade the Göttingen minipig has gained increasing recognition as animal model in pharmaceutical and safety research because it recapitulates many aspects of human physiology and metabolism. Genome-based comparison of drug targets together with quantitative tissue expression analysis allows rational prediction of pharmacology and cross-reactivity of human drugs in animal models thereby improving drug attrition which is an important challenge in the process of drug development.

Results: Here we present a new chromosome level based version of the Göttingen minipig genome together with a comparative transcriptional analysis of tissues with pharmaceutical relevance as basis for translational research. We relied on mapping and assembly of WGS (whole-genome-shotgun sequencing) derived reads to the reference genome of the Duroc pig and predict 19,228 human orthologous protein-coding genes. Genome-based prediction of the sequence of human drug targets enables the prediction of drug cross-reactivity based on conservation of binding sites. We further support the finding that the genome of Sus scrofa contains about ten-times less pseudogenized genes compared to other vertebrates. Among the functional human orthologs of these minipig pseudogenes we found HEPN1, a putative tumor suppressor gene. The genomes of Sus scrofa, the Tibetan boar, the African Bushpig, and the Warthog show sequence conservation of all inactivating HEPN1 mutations suggesting disruption before the evolutionary split of these pig species. We identify 133 Sus scrofa specific, conserved long non-coding RNAs (lncRNAs) in the minipig genome and show that these transcripts are highly conserved in the African pigs and the Tibetan boar suggesting functional significance. Using a new minipig specific microarray we show high conservation of gene expression signatures in 13 tissues with biomedical relevance between humans and adult minipigs. We underline this relationship for minipig and human liver where we could demonstrate similar expression levels for most phase I drug-metabolizing enzymes. Higher expression levels and metabolic activities were found for FMO1, AKR/CRs and for phase II drug metabolizing enzymes in minipig as compared to human. The variability of gene expression in equivalent human and minipig tissues is considerably higher in minipig organs, which is important for study design in case a human target belongs to this variable category in the minipig. The first analysis of gene expression in multiple tissues during development from young to adult shows that the majority of transcriptional programs are concluded four weeks after birth. This finding is in line with the advanced state of human postnatal organ development at comparative age categories and further supports the minipig as model for pediatric drug safety studies.

Conclusions: Genome based assessment of sequence conservation combined with gene expression data in several tissues improves the translational value of the minipig for human drug development. The genome and gene expression data presented here are important resources for researchers using the minipig as model for biomedical research or commercial breeding. Potential impact of our data for comparative genomics, translational research, and experimental medicine are discussed.

Figures

Fig. 1
Fig. 1
Multi-species sequence comparisons and assessment on drug binding. a Sequence identities between 1:1 orthologous transcripts and proteins of human, Rhesus macaque, Cynomolgus macaque, minipig, rat, and mouse. The 5’ UTR, CDS, and 3’ UTR of ~35,700 orthologous mRNAs (including splice variants) and of ~28,400 orthologous proteins were considered separately for the calculation of pairwise sequence identities in comparison to human. The relative number of 1:1 orthologous sequences was plotted against the sequence identities. Note that the peak sequence identities for the UTRs are significantly lower between humans and non-primates than for the coding regions. b Peroxisome proliferator-activated receptor alpha (PPARα) small molecule binding pocket analysis across multiple species. X-ray crystal structure of the ligand binding domain of human PPARα (magenta) with the dual PPARα/γ agonist aleglitazar (cyan) and with a 13-residue fragment of the SRC1 receptor co-activator motif 3 (green). Sequence alignments of PPARα orthologs from multiple species indicate that the contact residues (*) are fully conserved between human, macaques, and pigs while mouse and rat have sequence differences at three positions (P272, M279, I332) in comparison to the other species (I272, T279, V332). The inset shows the binding cavity in more detail and the non-conserved amino acids highlighted in a stick representation. PDB code: 3G8I. c Vascular endothelial growth factor (VEGF) epitope analysis for four different antibodies across multiple species. Depicted is the surface of human VEGF homodimer (light grey/dark grey) with residues relevant for antibody binding colored in red. Sequence alignments of VEGF orthologs from multiple species indicate for each antibody good conservation of contact residues (*) in human, macaques, and pigs, but not in rodents. Therefore cross-species reactivity is poor for mAb 4.6.1, the parent antibody of Avastin, and Y0317 (Lucentis) which are a product of immune response against hVEGF in mouse, and for the single-chain variable fragment (scFv) L3H6, targeting a different less conserved epitope. G6-Fab, derived from a synthetic antibody phage library, however shows good cross-reactivity due to full conservation of the functional epitope. PDB code: 1FLT
Fig. 2
Fig. 2
Global Comparison of human and minipig tissue expression profiles. a Principal component analysis of whole transcriptome expression profiles of human (n = 3 - 9) and minipig tissues (n = 9 - 12) based on 16’032 orthologous genes. A common set of 13 tissue types is represented for each species. Tissues types were colored according to organ systems. Principal component 1 accounts for 29 %, principal component 2 for 11 %, and principal component 3 for 6 % of the variation of the data set. Related tissues cluster together between species. b Comparison of minipig whole transcriptome expression profiles to human tissue signatures. Enrichment scores are indicated by a relative color scale (by row); with red representing high similarity and white no similarity of minipig gene expression profiles to human tissue specific gene expression signatures
Fig. 3
Fig. 3
Correlation of transcript abundance and metabolic activity for major drug metabolizing enzymes in human and minipig liver. Liver mRNA abundance of all major phase I and II drug metabolizing enzymes was determined with minipig and human microarray data (left panel). Metabolite formation rates (pmol/min/mg protein) were determined for phase I and II enzyme substrates for human and minipig primary hepatocyte suspension cultures (right panel). Transcript abundance and metabolic activities of human and minipig major cytochrome p450 enzymes (CYPs), aldoketoreductases (AKR/CR), aldehyde oxidase (AOX), N-acetyltransferases (NATs), and sulfotransferases (SULT) are shown as black dots. UDP-glucuronosyltransferase (UGT) family members are shown as red dots, UGT1A1 as separate blue dots, and flavin-monooxygenase 1 (FMO1) as green dots. Liver mRNA expression levels show higher expression for FMO1, UGTs, SULT1C4, and SULT1E1 in minipig as compared to human. The UGT family expression data is represented by four subtypes detected by isoform specific probes. All major phase I CYPs show very similar expression levels in both species. Higher UGT, AKR, and FMO1 enzyme activities compare well with higher liver gene expression levels in minipig. Bars present average transcript abundance and metabolic activity
Fig. 4
Fig. 4
Gene expression developmentsin minipig organs relevant for juvenile toxicity from young to adult. a Principal component analysis of whole transcriptome expression profiles of minipig tissues based on 17,254 genes. Principal component 1 accounts for 21.8 %, principal component 2 for 12.4 %, and principal component 3 for 11.3 % of total variation of the data set. b The heat map shows enrichment scores for human tissue expression signatures (red = high; white = low) in minipig tissues from young to adult. With exception of reproductive organs pairwise male and female data points for 1 week, 4 weeks, 2 months, 4 months and adult are shown
Fig. 5
Fig. 5
Global variability of tissue gene expression profiles in human and minipig. The left bar chart displays the average log2 expression levels, the middle bar chart the corresponding coefficient of variation (CV) profiles, and the right bar chart high variance genes per tissue and species. High variance genes were identified from transcriptome-wide expression signals using a 10 % CV cutoff. The number of high variance genes per tissue and species indicates that cerebellum is the least variable tissue in expression in both species and stomach is the most variable. Notably, gene expression in minipigs appears in general more variable per tissue than in humans

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