Identification of cis-regulated genes in quantitative trait loci driving liver fibrosis

R Hall; R Müllenbach; S Huss; R Alberts; K Schughart; K Hochrath; SN Weber; RW Williams; F Lammert

doi:10.1055/s-0032-1323960

Zeitschrift für Gastroenterologie, Inhaltsverzeichnis

Identification of cis-regulated genes in quantitative trait loci driving liver fibrosis

R Hall ¹, R Müllenbach ¹, S Huss ², R Alberts ³, K Schughart ³, K Hochrath ¹, SN Weber ¹, RW Williams ⁴, F Lammert ¹

¹Saarland University Medical Center, Department of Medicine II, Homburg, Germany
²Bonn University, Department of Pathology, Bonn, Germany
³Helmholtz Centre for Infection Research, Department of Experimental Mouse Genetics, Braunschweig, Germany
⁴University of Tennessee, Department of Anatomy and Neurobiology, Memphis Tennessee, United States

Abstract

Aims: Liver fibrosis is a complex disease mediated by multiple interacting genes and environmental factors. Our aim is to identify genes that drive profibrogenic expression profiles during liver fibrogenesis. We availed of the genetically well characterised reference panel of BXD recombinant inbred lines and linked phenotypic differences to the genetic variation segregating in this genetic reference population.

Methods: Liver fibrosis was induced in 30 BXD lines (392 mice) by CCl₄ (12 injections i.p.; 0.7mg/kg). Fibrosis progression was assessed by histological fibrosis staging and quantification of hepatic collagen. The hepatic expression profiles of each line were generated using Affymetrix Mouse Gene 1.0 ST arrays (>34000 probes). Genome-wide interval-mapping was performed to identify quantitative trait loci influencing fibrotic phenotypes (pQTLs) and regulating gene expression (eQTLs). In silico analyses were performed using the GeneNetwork database, which allows the comparison of gene regulation in different treatment groups (CCl₄, NaCl or EtOH) and tissues (kidney, lung, leukocytes).

Results: The BXD lines show specific differences in susceptibility to CCl₄-induced fibrosis. Interval mapping identified seven significant (P_G<0.05) fibrosis susceptibility loci (pQTL) on chromosomes (Chr) 4, 5, 7, 12 and 17. The dissection of these pQTL regions by eQTL mapping revealed several cis-regulated genes (cisQTG). In silico analyses of the cisQTG identified genes differenzially regulated during fibrogenesis, as compared to their regulation in healthy animals (e.g., Cxcl10 controlled by a marker on Chr5: 98.2 Mb after CCl₄ vs. Chr2: 64.7 Mb in healthy animals. Similar results were obtained for Nuclear receptor1h2 (Lxr), known to be involved in stellate cell regulation.

Conclusions: Genotype-phenotype correlations in the BXD panel allowed us to identify potential regulatory mechanisms during fibrogenesis. The identification of known profibrogenic molecules Cxcl10 and Nr1h2 is proof of concept for our approach to detect fibrotic mediators. The experimental set-up provides an experimental framework for modelling gene networks that drive liver fibrogenesis.