The HXB/BXH family of recombinant inbred rat strains is a unique genetic resource that has been extensively phenotyped over 25 years, resulting in a vast dataset of quantitative molecular and physiological phenotypes. We built a pangenome graph from 10x Genomics Linked-Read data for 31 recombinant inbred rats to study genetic variation and association mapping. The pangenome includes 0.2Gb of sequence that is not present the reference mRatBN7.2, confirming the capture of substantial additional variation. We validated variants in challenging regions, including complex structural variants resolving into multiple haplotypes. Phenome-wide association analysis of validated SNPs uncovered variants associated with glucose/insulin levels and hippocampal gene expression. We propose an interaction between Pirl1l1, chromogranin expression, TNF-α levels, and insulin regulation. This study demonstrates the utility of linked-read pangenomes for comprehensive variant detection and mapping phenotypic diversity in a widely used rat genetic reference panel.

• Publikační typ
• časopisecké články MeSH

Post transcriptional modifications of RNA are powerful mechanisms by which eukaryotes expand their genetic diversity. For instance, researchers estimate that most transcripts in humans undergo alternative splicing and alternative polyadenylation. These splicing events produce distinct RNA molecules, which in turn yield distinct protein isoforms and/or influence RNA stability, translation, nuclear export, and RNA/protein cellular localization. Due to their pervasiveness and impact, we hypothesized that alternative splicing and alternative polyadenylation in brain can contribute to a predisposition for voluntary alcohol consumption. Using the HXB/BXH recombinant inbred rat panel (a subset of the Hybrid Rat Diversity Panel), we generated over one terabyte of brain RNA sequencing data (total RNA) and identified novel splice variants (via StringTie) and alternative polyadenylation sites (via aptardi) to determine the transcriptional landscape in the brains of these animals. After establishing an analysis pipeline to ascertain high quality transcripts, we quantitated transcripts and integrated genotype data to identify candidate transcript coexpression networks and individual candidate transcripts associated with predisposition to voluntary alcohol consumption in the two-bottle choice paradigm. For genes that were previously associated with this trait (e.g., Lrap, Ift81, and P2rx4) (Saba et al., Febs. J., 282, 3556-3578, Saba et al., Genes. Brain. Behav., 20, e12698), we were able to distinguish between transcript variants to provide further information about the specific isoforms related to the trait. We also identified additional candidate transcripts associated with the trait of voluntary alcohol consumption (i.e., isoforms of Mapkapk5, Aldh1a7, and Map3k7). Consistent with our previous work, our results indicate that transcripts and networks related to inflammation and the immune system in brain can be linked to voluntary alcohol consumption. Overall, we have established a pipeline for including the quantitation of alternative splicing and alternative polyadenylation variants in the transcriptome in the analysis of the relationship between the transcriptome and complex traits.

• Publikační typ
• časopisecké články MeSH

The role of alternative promoter usage in tissue-specific gene expression has been well established; however, its role in complex diseases is poorly understood. We performed cap analysis of gene expression (CAGE) sequencing from the left ventricle of a rat model of hypertension, the spontaneously hypertensive rat (SHR), and a normotensive strain, Brown Norway to understand the role of alternative promoter usage in complex disease. We identified 26,560 CAGE-defined transcription start sites in the rat left ventricle, including 1,970 novel cardiac transcription start sites. We identified 28 genes with alternative promoter usage between SHR and Brown Norway, which could lead to protein isoforms differing at the amino terminus between two strains and 475 promoter switching events altering the length of the 5' UTR. We found that the shift in Insr promoter usage was significantly associated with insulin levels and blood pressure within a panel of HXB/BXH recombinant inbred rat strains, suggesting that hyperinsulinemia due to insulin resistance might lead to hypertension in SHR. Our study provides a preliminary evidence of alternative promoter usage in complex diseases.

• MeSH
• genetická transkripce genetika   MeSH
• hypertenze * genetika   metabolismus   MeSH
• krysa rodu rattus MeSH
• potkani inbrední SHR MeSH
• promotorové oblasti (genetika) genetika   MeSH
• sekvenční analýza RNA metody   MeSH
• stanovení celkové genové exprese metody   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

BACKGROUND: A statistical pipeline was developed and used for determining candidate genes and candidate gene coexpression networks involved in 2 alcohol (i.e., ethanol [EtOH]) metabolism phenotypes, namely alcohol clearance and acetate area under the curve in a recombinant inbred (RI) (HXB/BXH) rat panel. The approach was also used to provide an indication of how EtOH metabolism can impact the normal function of the identified networks. METHODS: RNA was extracted from alcohol-naïve liver tissue of 30 strains of HXB/BXH RI rats. The reconstructed transcripts were quantitated, and data were used to construct gene coexpression modules and networks. A separate group of rats, comprising the same 30 strains, were injected with EtOH (2 g/kg) for measurement of blood EtOH and acetate levels. These data were used for quantitative trait loci (QTL) analysis of the rate of EtOH disappearance and circulating acetate levels. The analysis pipeline required calculation of the module eigengene values, the correction of these values with EtOH metabolism rates and acetate levels across the rat strains, and the determination of the eigengene QTLs. For a module to be considered a candidate for determining phenotype, the module eigengene values had to have significant correlation with the strain phenotypic values and the module eigengene QTLs had to overlap the phenotypic QTLs. RESULTS: Of the 658 transcript coexpression modules generated from liver RNA sequencing data, a single module satisfied all criteria for being a candidate for determining the alcohol clearance trait. This module contained 2 alcohol dehydrogenase genes, including the gene whose product was previously shown to be responsible for the majority of alcohol elimination in the rat. This module was also the only module identified as a candidate for influencing circulating acetate levels. This module was also linked to the process of generation and utilization of retinoic acid as related to the autonomous immune response. CONCLUSIONS: We propose that our analytical pipeline can successfully identify genetic regions and transcripts which predispose a particular phenotype and our analysis provides functional context for coexpression module components.

• MeSH
• ethanol aplikace a dávkování   metabolismus   MeSH
• játra účinky léků   metabolismus   MeSH
• krysa rodu rattus MeSH
• metabolická clearance účinky léků   fyziologie   MeSH
• multifaktoriální dědičnost účinky léků   fyziologie   MeSH
• pití alkoholu genetika   metabolismus   MeSH
• potkani inbrední BN MeSH
• potkani inbrední SHR MeSH
• potkani transgenní MeSH
• strojové učení bez učitele * MeSH
• systémová biologie metody   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH