The extracellular proteinase inhibitor gene Expi is a candidate gene for hepatic fibrosis susceptibility
Roman Liebe, Rabea A. Hall, Steven Dooley, Frank Lammert;
Department of Medicine II, Saarland University, Homburg, Germany
Aim: To identify new contributors towards increased susceptility for hepatic fibrosis progression, we mapped quantitative trait loci (QTLs) that determine hepatocellular susceptibility to profibrogenic transforming growth factor (TGF)-β signalling in cultured primary hepatocytes. We availed of cells from the murine reference population BXD, recombinant inbred offspring of the mouse strains C57BL/6J and DBA/2J, which differ in fibrosis susceptibility (Andreuxet al. Cell 2012). A common 6 Mb locus on chromosome 1 1 modulated TGF-β-induced total cell death in vitro and histological fibrosis stage after CCl4 challenge in vivo. Methods: The effects of genetic loci within a 6 Mb phenotypic QTL on hepatic gene expression following short-term (24 hrs) liver damage by ethanol or chronic CCl4 challenge (6 wks) were assessed using expression QTL (eQTL) analysis. A mouse with targeted Expi knockout was provided by the EUCOMM repository. Homozygous disruption of the Expi gene by insertion of a neo cassette was verified by genomic PCR and resulted in no detectable phenotype in untreated mice. Results: eQTL mapping of correlations between SNPs within the QTL and transcript abundance in liver identified a variant at 83.5 Mb that co-segregated significantly with expression variation. Analysis of amino acid exchanges near the major expression-associated SNP indicated candidacy of the extracellular proteinase inhibitor Expi for both traits. Cellular damage assessment of knockout hepatocytes following 48 hrs treatment with TGF-β suggested higher susceptibility in Expi knockout cells. Quantification of hepatic collagen contents following 6 weeks of CCl4 challenge was indicative of higher fibrosis rates in wild-type mice as compared to knockout animals. Conclusions: The combination of QTL mapping in vitro and in vivo with eQTL analysis identifies novel susceptibility genes for fibrogenesis. Knockout of the candidate gene Expi results in differential susceptibility to TGF-β-induced cell death and hepatic collagen contents after CCl4 challenge, consistent with a potential novel role of this extracellular proteinase inhibitor in acute and chronic liver injury.
The following people have nothing to disclose: Roman Liebe, Rabea A. Hall, Steven Dooley, Frank Lammert