| Genetic variation is an important determinant in phenotypic diversity, including disease and disease susceptibility. While single nucleotide polymorphisms are now well recognized as genetic determinants for such variation, the effects of structural variation in genomes has been poorly investigated. Furthermore, the mechanisms by which genetic variation affects phenotypes remain largely unexplored. While many traits are currently linked to a wide variety of genomic regions in Whole Genome Association studies, causal relationships and molecular pathways underlying complex traits and diseases are extremely difficult to deduce from these studies, partially due to the genetic heterogeneity of the human population as well as the experimental inaccessibility of the human system. Here, we propose to employ a genetic rat model system to study the effects of genetic variation (SNPs, CNVs, structural variation) on phenotypic diversity. The rat recombinant inbred (RI) panel HXB/BXH consists of 29 inbred lines (over 80 backcrosses) derived from an outcross of the BN-Lx (Brown Norway and SHR (Spontaneous Hypertensive Rat) inbred lines. As a result, this panel reflects a renewable small outbred population with defined genetic background. The genetic make-up of each line has been determined by high-resolution genotyping of all lines. The SHR line is currently being resequenced using next-generation sequencing (NGS) technology. We propose to resequence the BN-Lx strain (using ABI SOLiD technology) as well to obtain a complete insight in the genetic variation between these strains and thus within the RI panel, at the nucleotide level. As the sequencing of simple short-read libraries with NGS technology does not allow for the detection and discovery of structural variants (Copy Number Variation, translocations, inversions, complex events), we will extend our current efforts in this area (arrayCGH, optical mapping) with massively paired-end mapping using next generation sequencing technology. Ultimately, these efforts should result in a complete inventory of genomic variation between the RI founder strains, which is unprecedented in resolution and size range (from single nucleotides to genomic rearrangements in the megabase range). For decades, the HXB/BXH rat RI panel has been widely studied and large amounts of phenotypic as well as molecular data are publicly available. While genome-wide expression data for 7 different tissues (kidney, spleen, brain, heart, skeletal muscle, adrenal gland, fat pads) from all 29 lines is already available, we propose to extend this resource with small RNA discovery and expression profiling, as well as total RNA sequencing to discover alternative transcript use and noncoding RNA expression differences. Small RNA as well as total RNA (mRNA and noncoding RNA, excluding ribosomal and tRNA) will be cloned from the aforementioned 7 tissues from the RI founder strains and sequenced using next-generation sequencing. The expression of selected miRNA and mRNA genes (quantitatively or qualitatively) will be followed up in all 29 RI strains. Finally, we will use the existing and newly generated genetic and genomic resources to build a transcriptional regulatory network to allow for the modelling of the direct and indirect effects of genomic variation on gene expression levels. The unique characteristics of an RI panel provide a powerful tool for the robust discovery of trans-eQTLs, i.e. gene expression differences caused by genetic variation elsewhere in the genome which are indicative for presence of regulatory elements (transcription factors, regulatory noncoding (small) RNAs). The model that is expected to result from the proposed work would allow for the hypotheses on the molecular mechanism underlying such trans-eQTLs and provide input for experimental validation approaches. As part of this project, selected pathways will be followed up and validated experimentally (e.g. by using other inbred or congenic/consomic rat strains, as well as by targeted in vitro cell line systems in which gene dosage can be manipulated). The proposed work is expected to result in an increased understanding of tissue-specific miRNA and transcription factor-mediated regulation of genome-wide mRNA expression as well as the effect of genomic variation on it. Eventually, this should provide novel insights in the molecular mechanisms underlying disease and disease susceptibility. |
