Characterization of mutations in ATP8B1 associated with hereditary cholestasis.

Progressive familial intrahepatic cholestasis (PFIC) and benign recurrent intrahepatic cholestasis (BRIC) are clinically distinct hereditary disorders. PFIC patients suffer from chronic cholestasis and develop liver fibrosis. BRIC patients experience intermittent attacks of cholestasis that resolve spontaneously. Mutations in ATP8B1 (previously FIC1) may result in PFIC or BRIC. We report the genomic organization of ATP8B1 and mutation analyses of 180 families with PFIC or BRIC that identified 54 distinct disease mutations, including 10 mutations predicted to disrupt splicing, 6 nonsense mutations, 11 small insertion or deletion mutations predicted to induce frameshifts, 1 large genomic deletion, 2 small inframe deletions, and 24 missense mutations. Most mutations are rare, occurring in 1-3 families, or are limited to specific populations. Many patients are compound heterozygous for 2 mutations. Mutation type or location correlates overall with clinical severity: missense mutations are more common in BRIC (58% vs. 38% in PFIC), while nonsense, frameshifting, and large deletion mutations are more common in PFIC (41% vs. 16% in BRIC). Some mutations, however, lead to a wide range of phenotypes, from PFIC to BRIC or even no clinical disease. ATP8B1 mutations were detected in 30% and 41%, respectively, of the PFIC and BRIC patients screened.

The N-terminus of the human copper transporter 1 (hCTR1) is localized extracellularly, and interacts with itself.

We have used indirect immunofluorescense studies and glycosylation-site insertion and deletion mapping to characterize the topology of human copper transporter 1 (hCTR1), the putative human high-affinity copper-import protein. Both approaches indicated that hCTR1 contains three transmembrane domains and that the N-terminus of hCTR1, which contains several putative copper-binding sites, is localized extracellularly, whereas the C-terminus is exposed to the cytosol. Based on previous observations that CTR1 proteins form high-molecular-mass complexes, we investigated directly whether CTR1 proteins interact with themselves. Yeast two-hybrid studies showed that interaction of yeast, mouse, rat and human CTR1 occurs at the sites of their N-terminal domains, and is not dependent on the copper concentration in the growth media. Analysis of deletion constructs indicated that multiple regions in the N-terminus are essential for this self-interaction. In contrast, the N-terminal tail of the presumed low-affinity copper transporter, hCTR2, does not interact with itself. Taken together, these results suggest that CTR1 spans the membrane at least six times, permitting formation of a channel, which is consistent with its proposed role as a copper transporter.