A gene encoding a P-type ATPase mutated in two forms of hereditary cholestasis.

Cholestasis, or impaired bile flow, is an important but poorly understood manifestation of liver disease. Two clinically distinct forms of inherited cholestasis, benign recurrent intrahepatic cholestasis (BRIC) and progressive familial intrahepatic cholestasis type 1 (PFIC1), were previously mapped to 18q21. Haplotype analysis narrowed the candidate region for both diseases to the same interval of less than 1 cM, in which we identified a gene mutated in BRIC and PFIC1 patients. This gene (called FIC1) is the first identified human member of a recently described subfamily of P-type ATPases; ATP-dependent aminophospholipid transport is the previously described function of members of this subfamily. FIC1 is expressed in several epithelial tissues and, surprisingly, more strongly in small intestine than in liver. Its protein product is likely to play an essential role in enterohepatic circulation of bile acids; further characterization of FIC1 will facilitate understanding of normal bile formation and cholestasis.

Analysis of expression of cMOAT (MRP2), MRP3, MRP4, and MRP5, homologues of the multidrug resistance-associated protein gene (MRP1), in human cancer cell lines.

By screening databases of human expressed sequence tags, we have identified three new homologues of MRP1, the gene encoding the multidrug resistance-associated protein, and cMOAT (or MRP2), the canalicular multispecific organic anion transporter gene. We call these new genes MRP3, MRP4, and MRP5. MRP3, like cMOAT, is mainly expressed in the liver. MRP4 is expressed only at very low levels in a few tissues, and MRP5, like MRP1, is expressed in almost every tissue tested. To assess a possible role of these new MRP homologues in multidrug or cisplatin resistance, a large set of resistant cell lines was examined for the (over)expression of MRP1, cMOAT, MRP3, MRP4, and MRP5. We find that even in cells selected for a low level of resistance, several MRP-related genes can be up-regulated simultaneously. However, MRP4 is not overexpressed in any of the cell lines we analyzed; MRP3 and MRP5 are only overexpressed in a few cell lines, and the RNA levels do not seem to correlate with resistance to either doxorubicin or cisplatin. cMOAT is substantially overexpressed in several cell lines, and cMOAT RNA levels correlate with cisplatin but not doxorubicin resistance in a subset of resistant cell lines. Our results emphasize the need for gene-specific blocks in gene expression to define which transporter contributes to resistance in each resistant cell line.

[Recurrent familial intrahepatic cholestasis in the Faroe Islands]. / Recidiverende familiaer intrahepatisk kolestase på Faerøerne.

Recurrent familial intrahepatic cholestasis is an autosomal recessive disorder characterized by episodes of severe pruritus and jaundice lasting for weeks to months without extrahepatic bile duct obstruction. Symptom-free intervals may last for months to years, and chronic liver damage does not develop. We recently studied four of the five patients from the Faeroe Islands described by us 30 years ago (one had recently died), and a further five patients who were identified after the initial report. The episodes of cholestasis were more frequent and severe in patients with early onset, but tended to reduce in frequency with age. The youngest patient, aged 25 years, who had had 16 episodes, each lasting about six months, had a liver transplant after which no further episodes were recorded (one year after surgery). Signs of chronic liver disease were absent in all patients. The FIC1 gene was investigated for mutations in the surviving patients. A single mutation (I661T) was found on both chromosomes in all 9 patients, indicating that they are genetically identical for the disease causing defect. Nevertheless, considerable differences between patients were observed clinically.

Recurrent familial intrahepatic cholestasis in the Faeroe Islands. Phenotypic heterogeneity but genetic homogeneity.

Recurrent familial intrahepatic cholestasis is an autosomal recessive disorder characterized by episodes of severe pruritus and jaundice lasting for weeks to months without extrahepatic bile duct obstruction. Symptom-free intervals may last for months to years, and chronic liver damage does not develop. We recently studied four of the five patients from the Faeroe Islands described by us 30 years ago (one had recently died) and an additional five patients that were identified after the initial report. The episodes of cholestasis were more frequent and severe in patients with early onset, but tended to reduce in frequency with age. The youngest patient, aged 25 years, who had had 16 episodes each lasting about 6 months, had a liver transplant after which no further episodes were recorded (1 year after surgery). Signs of chronic liver disease were absent in all patients. The FIC1 gene was investigated for mutations in the surviving patients. A single mutation (I661T) was found on both chromosomes in all nine patients, indicating that they are genetically identical for the disease-causing defect. Nevertheless, considerable differences among patients were observed clinically.

A missense mutation in FIC1 is associated with greenland familial cholestasis.

Greenland familial cholestasis is a severe form of intrahepatic cholestasis described among indigenous Inuit families in Greenland. Patients present with jaundice, pruritus, bleeding episodes, and steatorrhea, and die in childhood due to end-stage liver disease. We investigated the possibility that Greenland familial cholestasis is caused by a mutation in FIC1, the gene defective in patients with progressive familial intrahepatic cholestasis type 1 and many cases of benign recurrent intrahepatic cholestasis. Using single-strand conformation polymorphism analysis and sequencing of the FIC1 exons, a missense mutation, 1660 G-->A (D554N), was detected and was shown to segregate with the disease in Inuit patients from Greenland and Canada. Examination of liver specimens from 3 Inuit patients homozygous for this mutation revealed bland canalicular cholestasis and, on transmission electron microscopy, coarsely granular Byler bile, as previously described in patients with progressive familial intrahepatic cholestasis type 1. These data establish Greenland familial cholestasis as a form of progressive familial intrahepatic cholestasis type 1 and further underscore the importance of unimpeded FIC1 activity for normal bile formation.

FIC1, the protein affected in two forms of hereditary cholestasis, is localized in the cholangiocyte and the canalicular membrane of the hepatocyte.

BACKGROUND/AIMS: FIC1 (familial intrahepatic cholestasis 1) is affected in two clinically distinct forms of hereditary cholestasis, namely progressive familial intrahepatic cholestasis type 1 (PFIC1) and benign recurrent intrahepatic cholestasis. Here we examined the subcellular localization of this protein within the liver. METHODS: Antibodies raised against different epitopes of human FIC1 were used for immunoblot analysis and immunohistochemical detection of FICI. RESULTS: Immunoblot analysis of intestine and liver tissue extracts from human, rat and mouse origin indicated that the antibodies raised against FIC1 specifically detected FIC1 as a 140-kDa protein. In the liver homogenate of a PFIC1 patient, FIC1 could not be detected. Analysis of isolated rat liver membrane vesicles indicated that this protein is predominantly present in the canalicular membrane fraction. Immunohistochemical detection of the protein in liver sections confirmed that FIC1 was present in the canalicular membrane, whereas no staining was observed in the PFIC1 patients liver. Double label immunofluorescence of murine liver revealed that FIC1 colocalized with cytokeratin 7 in cholangiocytes. CONCLUSIONS: The localization of FIC1 in the canalicular membrane and cholangiocytes suggests that it may directly or indirectly play a role in bile formation since mutations in FICI are associated with severe symptoms of cholestasis.

Benign recurrent intrahepatic cholestasis (BRIC): evidence of genetic heterogeneity and delimitation of the BRIC locus to a 7-cM interval between D18S69 and D18S64.

Benign recurrent intrahepatic cholestasis (BRIC) is an autosomal recessive liver disease characterized by multiple episodes of cholestasis without progression to chronic liver disease. The gene was previously assigned to chromosome 18q21, using a shared segment analysis in three families from the Netherlands. In the present study we report the linkage analysis of an expanded sample of 14 BRIC families, using 15 microsatellite markers from the 18q21 region. Obligate recombinants in two families place the gene in a 7-cM interval, between markers D18S69 and D18S64. All intervening markers had significant LOD scores in two-point linkage analysis. Moreover, we identified one family in which the BRIC gene seems to be unlinked to the 18q21 region, or that represents incomplete penetrance of the BRIC genotype.

Fine-resolution mapping by haplotype evaluation: the examples of PFIC1 and BRIC.

Loci for two inherited liver diseases, benign recurrent intrahepatic cholestasis (BRIC) and progressive familial intrahepatic cholestasis type 1 (PFIC1), have previously been mapped to 18q21 by a search for shared haplotypes in patients in two isolated populations. This paper describes the use of further haplotype evaluation with a larger sample of patients for both disorders, drawn from several different populations. Our assessment places both loci in the same interval of less than 1 cM and has led to the discovery of the PFIC1/BRIC gene, FIC1; this discovery permits retrospective examination of the general utility of haplotype evaluation and highlights possible caveats regarding this method of genetic mapping.