A single quantitative trait locus on mouse chromosome 11 modifies in vitro hepatocyte susceptibility to TGF-β and in vivo fibrogenesis following CCl4 injections

Background:

Hepatocyte damage triggers the release of cytokines and chemokines, thus initiating wound healing and immune cell recruitment. TGF-β is the central pro-fibrogenic cytokine, and exerts autocrine effects on neighbouring hepatocytes as well as attracting and activating myofibroblasts and macrophages. We speculated that genetic factors causing differenzial sensitivity of hepatocytes to TGF-β signalling may predispose to fibrosis in various aetiologies.

Hypothesis:

Genetic variants modulating the hepatocellular response to TGF-β induced damage in vitro might underly differenzial susceptibility to fibrogenesis in response to chronic hepatocellular damage.

Methods:

We subjected cultured primary hepatocytes from two different inbred laboratory mouse strains, C57BL/6J (B6) and DBA/2J (D2) and 21 BXD lines, recombinant inbred F2 offspring generated from these strains, to TGF-β stimulation (5ng/ml) for 48 hours. Total hepatocellular damage was assayed by measuring the increase of lactate dehydrogenase (LDH) in the tissue culture supernatant relative to untreated cells.

For comparison, fibrogenesis in vivo was quantified by F-score in 30 BXD lines following 12 CCl4 injections over 6 weeks. Total hepatocellular damage in vitro and fibrosis score in vivo were mapped to the mouse genome by quantitative trait linkage analysis using >14.000 SNP markers.

Expression data of 30.000 transcripts in 30 BXD lines following liver damage by CCl4 or ethanol, respectively, were used as traits for quantitative trait locus mapping.

Results:

QTL analysis identified a locus on mouse chromosome 11 spanning a region of 6 megabases that is associated with both phenotypes. Expression quantitative trait locus analysis identified four genomic variants within the region that were associated with gene expression in response to liver damage by CCl4 or ethanol.

Non-conservative exonic variants near expression-associated loci delineate two genes as potential causes for both pro-fibrogenic phenotypes.

Conclusions:

Phenotype QTL mapping of hepatocellular damage in vitro and fibrogenesis in vivo have delineated a common region.

Expression QTL mapping has specified four genomic loci, two of which harbour creedal susceptibility variants as potential drivers of fibrogenesis.

We are presently analysing one of the candidate genes in a knockout mouse model and the second in a cohort of patients with liver fibrosis.