Quantitative trait analysis in Abcb4 deficient mice identifies Pcsk9 as a potential modifier gene

 

Mutations in the ABCB4 (ATP-binding casette, subfamily B, member 4) gene cause cholestatic liver diseases. Modifier genes have yet to be identified systematically. We performed a quantitative trait locus (QTL) anaylsis in a cross of FVB- and BALB- Abcb4 knockout (KO) strains to elucidate the genetic control of cholestatic liver diseases. In Abcb4 KO mice, a deficiency of the hepatobiliary phosphatidylcholine floppase causes chronic cholestasis and fibrosis. Apart from low phosphatidylcholine levels, the mice also present with low biliary cholesterol levels. Pcsk9 (proprotein convertase subtilin-kexin type 9) responds to cholesterol concentrations and regulates the degradation of the LDL-receptor. For the QTL analysis, genetic maps were generated from 281 SNP markers and fibrosis was quantified by hydroxyproline measurement. Genetic regions linked to fibrosis susceptibility were identified in single and pairwise QTL scans. Loci were screened for potential candidate genes with nsSNPs and an association with liver disease. Hepatic mRNA expression of Pcsk9 and plasma cholesterol levels was determined in wildtype (WT) and KO mice. Genetic variation in PCSK9 was tested in a cohort of 193 patients with primary sclerosing cholangitis (PSC). While single loci exhibited minor effect sizes, a significant interaction of two QTLs on chromosomes 4 and 17 was identified. We identified genes functionally related to hepatobiliary cholesterol homeostasis. Among these, Pcsk9 expression was significantly higher in FVB WT mice compared to fibrotic BALB mice (p = 0.011). Irrespective of the strain WT mice show higher Pcsk9 expression than KO mice (p = 0.014). Plasma cholesterol levels were higher in FVB compared to BALB and in WT compared to KO mice (p < 0.01). In the PSC cohort, carriers of a PCSK9 variant presented with significantly higher serum alkaline phosphatase activities. The experimental cross of Abcb4 KO strains allowed the identification of Abcb4-dependent modifiers of cholestatic liver disease. Candidate genes such as Pcsk9 are involved in the regulation of hepatic cholesterol metabolism. First experiments indicate that low cholesterol levels correspond to low Pcsk9 mRNA levels in fibrotic mice. Variants of PCSK9 could also be associated with liver injury in PSC patients.