Quantitative trait analysis in Abcb4 deficient mice identifies Pcks9 as a potential modifier gene

Background:

Mutations in the ABCB4 (ATP-binding cassette, subfamily B, member 4) gene cause cholestatic liver diseases. Modifier genes have yet to be investigated systematically. Here we performed a quantitative trait locus (QTL) anaylsis in an experimental cross of the congenic FVB- and BALB-Abcb4-/-knockout strainsto elucidate the genetic control of cholestatic liver diseases. In the Abcb4-/- model, a deficiency of the hepatobiliary phosphatidylcholine translocase causes chronic cholestasis, liver injury, and fibrosis. Apart from low phosphatidylcholine levels, the Abcb4-/- mice also present with low biliary and plasma cholesterol concentrations. Pcsk9 (proprotein convertase subtilin-kexin type 9) responds to cholesterol concentrations and regulates the degradation of the LDL-receptor.

Methods:

For the QTL analysis, genetic maps were generated from 281 single nucleotide polymorphisms spanning the whole genome. Fibrosis progression was quantified by photometric measurement of hydroxyproline. Genetic regions linked to fibrosis susceptibility were identified in single and two-dimensional QTL scans. All loci were screened for potential candidate genes, which contain nsSNPs and are functionally associated with liver metabolism and disease. Hepatic mRNA expression of Pcsk9 and plasma cholesterol levels were determined in wild-type and knockout mice. Genetic variation in the orthologous PCSK9 locus was tested in a cohort of 193 patients with primary sclerosing cholangitis (PSC).

Results:

Single modifier loci exhibited minor effect sizes only. However, a significant interaction of two QTLs on chromosomes (chr) 4 and 17 could be identified. Underlying these loci we identified a group of genes functionally related to hepatobiliary cholesterol homeostasis: chr 4) Lrp8, Pcsk9 and Scp2; chr17) Abcg5/g8 and the tri-genic region Rhoq-Pigf-Cript. In the mouse, Pcsk9 expression is significantly (p = 0.011) higher in FVB mice compared to fibrotic BALB-Abcb4-/- mice; irrespective of the strain, wild-type mice display significantly (p = 0.014) higher Pcsk9 mRNA levels than knockouts. Concomitantly, plasma cholesterol levels were significantly (p < 0.01) higher in controls than in Abcb4 deficient mice and highest in FVB mice. The analysis of the PSC cohort showed that carriers of a variant PCSK9 allele presented with significantly (p = 0.017) higher serum alkaline phosphatase activities as surrogate markers of PSC severity.

Conclusions:

The experimental cross of ABCB4-deficient strains with distinct differences in fibrosis susceptibility allowed the identification of ABCB4-dependent modifiers of cholestatic liver disease. The identified candidate genes such as Pcsk9 are involved in the regulation of hepatic cholesterol metabolism. First experiments indicate that increased cholesterol levels correspond to higher Pcsk9 mRNA levels in fibrotic mice. Variants of PCSK9 might be associated with disease severity in PSC patients.