Systems genetics of liver injury in ABCB4 deficiency: identification of genetic modifiers involved in hepatic cholesterol homeostasis

Background:

Mutations in the ABCB4 transporter gene cause progressive cholestatic liver disease. Modifier genes of familial cholestasis have not been identified systematically. In this study we generated congenic Abcb4-/- knockout mice, in which deficiency of the hepatobiliary phosphatidylcholine translocator causes variable cholangiopathy in different inbred mouse strains. We applied systems genetics to investigate the genetic control of liver fibrosis in an experimental cross of congenic Abcb4-/- lines.

Methods:

The Abcb4 knockout was transferred from the fibrosis-resistant FVB-Abcb4-/- strain to the susceptible BALB/cJ background by repeated backcrossing. To identify genetic modifiers of fibrosis susceptibility linked to ABCB4 deficiency, we crossed the two congenic strains to generate an intercross (F2) population. Phenotypic differences among the 217 F2 progeny were mapped to polymorphic regions across the whole genome using quantitative trait locus (QTL) analysis. Single and two-dimensional QTL scans were applied to identify modifiers and gene interactions. For translation, the effect of genetic variation in the orthologous PCSK9 locus was tested in a cohort of 193 Caucasian patients with primary sclerosing cholangitis (PSC).

Results:

Compared to FVB-Abcb4-/- mice, BALB-Abcb4-/- mice progress to higher fibrosis stages. The heterogenic F2 population shows marked phenotypic variation. Whereas single modifiers demonstrate minor effects, gene-gene interaction scans identified a significant interaction of two QTLs on chromosomes 4 and 17. Underlying these loci we identified the genes Lrp8 (LDL receptor related protein 8), Pcsk9 (proprotein convertase subtilin-kexin type 9) and Scp2 (sterol carrier protein 2) on chromosome 4, as well as Abcg5/g8 and the trigenic region Rhoq-Pigf-Cript on chromosome 17. The specific genes are functionally related to hepatobiliary cholesterol homeostasis and represent creedal modifier genes. The analysis of the PSC cohort revealed that carriers of a variant PCSK9 allele had significantly higher serum alkaline phosphatase activities.

Conclusions:

The congenic Abcb4-/- lines allow the systematic genomic exploration of chronic cholestasis in inbred mice. Analyses of the experimental cross of ABCB4-deficient strains with distinct differences in fibrosis susceptibility resulted in the identification of ABCB4-dependent modifiers of cholestatic liver injury. Several candidate genes identified in mice, such as Pcsk9, are involved in the regulation of hepatic cholesterol metabolism and accordingly, the human PCSK9 gene was associated with serum surrogate markers of liver injury in patients with PSC.