Cracking the genetic code for benign recurrent and progressive familial intrahepatic cholestasis

Copyright C European Association for the Study of the Liver 1998

Journal of Hepatology 1998; 29: 317–320 Printed in Denmark ¡ All rights reserved Munksgaard ¡ Copenhagen

Journal of Hepatology ISSN 0168-8278

Editorial

Cracking the genetic code for benign recurrent and progressive familial intrahepatic cholestasis Ronald P. J. Oude Elferink1 and Gerard P. van Berge Henegouwen2 Departments of Hepato-Gastroenterology, 1Academic Medical Centre, Amsterdam, and 2University Hospital Utrecht, The Netherlands

   possible to localise genetic disease loci by screening for linkage disequilibrium between the disease phenotype and marker alleles. The basic principle of the method of microsatellite haplotype analysis is that within a closed community all patients with the same genetic defect will have a common region around the mutated gene that they inherited from a common ancestor, the founder. Using a large set of microsatellite markers spread over the entire genome, one can search for this region. If blood samples are available from a few patients and their parents, or, if unavailable, their siblings, it is possible on the basis of the shared haplotype markers to map the disease locus to a relatively small chromosomal region. The method has proven to be powerful and will become a standard tool in human genetics. With the completion of the Human Genome project in sight, it will become progressively easier to identify the genes responsible for many inherited diseases. Recently, this method was successfully applied to two inherited liver diseases: Progressive Familial Intrahepatic Cholestasis type 1 (PFIC-1) and Benign Recurrent Intrahepatic Cholestasis (BRIC). In four patients with BRIC originating from a small village in The Netherlands, a shared region on chromosome 18q21–22 was discovered and implicated as the BRIC locus (1,2). Using the same approach, it was found that the locus for PFIC/Byler’s disease in two distantly related Amish patients resides in the same chromosomal region (3). In the most recent publication from these collaborating groups (Bull et al. (4)), the localisation of the candidate gene was more refined through examination of haplotype sharing in an expanded set of patients with each disorder. Subsequently, a microdeletion in one PFIC-1 patient helped finally to clone the

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Correspondence: Gerard P. van Berge Henegouwen, Department of Gastroenterology, University Hospital Utrecht, P.O. Box 85500, 3508 GA Utrecht, The Netherlands. Tel: 31-30-2507004. Fax: 31-30-2507371. e-mail: G.P.vanBergehenegouwen&digd.azu.nl

responsible gene, which was named FIC-1. Within this gene the authors identified five mutations in PFIC-1 patients and two in BRIC patients. This definitely proved that not only is the locus for these two diseases identical, but that both diseases are caused by mutations in the same gene. In normal subjects the expression of FIC-1 was found to be highest in the pancreas and small intestine, with lower expression in a number of other tissues, including the liver. In this editorial, we would like to draw attention to these findings and try to explain the pathophysiological relevance, as well as to link these findings to phenotypic expression of the BRIC/PFIC syndromes. BRIC is characterised by recurrent episodes of intrahepatic cholestasis, lasting weeks to months. It may start in early childhood or in adulthood. Usually, the cholestatic attacks resolve spontaneously, leaving no detectable liver damage. In contrast, PFIC-1 is manifested by progressive cholestasis starting in early childhood and leading to end-stage liver disease, usually before adulthood. PFIC-1 is so termed because a second PFIC (number 2) locus has been mapped to chromosome 2q24. PFIC-2 is most probably caused by a mutation in the sister of P-glycoprotein (sPgp) gene ((5) and personal communication R.J. Thompson, AASLD Chicago, November 1997), which has recently been characterised as a canalicular bile salt transporter (6). Both PFIC-1 and PFIC-2 are manifested by severe intrahepatic cholestasis but normal serum g-GT activity. In addition, a third PFIC-like syndrome, PFIC-3, is characterised by a similar progressive cholestasis but with high serum g-GT activity and is caused by mutations in the MDR3 gene on chromosome 7q21, leading to absent biliary phospholipid secretion (7). While BRIC and PFIC-1 vary greatly in severity, they may start with similar clinical and biochemical features. In their article, Bull et al. hypothesise that PFIC-1 mutations severely or completely compromise FIC-1 function, while BRIC mutations lead only to partial inactivation of FIC-1, which might explain the different expression of the two diseases.

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The identified FIC-1 gene turns out to be a member of the family of P-type ATPases (8). This family of membrane proteins is divided into several subfamilies: the P1 ATPases mediate transport of (heavy) metal ions and also include the Cu-transporting ATPases involved in Wilson’s and Menke’s disease. The P2 ATPases are transporters for various cations such as Ca2π, Kπ and Naπ, and include, for example, the gastric proton pump (Hπ,Kπ-ATPase). The FIC-1 gene, however, shows highest homology with yet another subfamily of P-ATPases. This subfamily was recognised with the isolation of the gene for the bovine aminophospholipid flippase (9). This protein is responsible for the inward translocation of the aminophospholipids, phosphatidylserine (PS) and phosphatidylethanolamine (PE), thereby generating lipid asymmetry with PE and PS in the inner leaflet of the plasma membrane and leaving phosphatidylcholine (PC) and sphingomyelin (SM) in the outer leaflet. This subfamily also includes the drs2 gene from yeast. In a drs2 yeast null mutant, it was demonstrated that this gene too encodes for a protein that mediates inward phosphatidylserine translocation (9).

What is the Function of the FIC-1 Gene Product? Although the function of the identified FIC-1 gene is still unknown, it is clear that mutations in this gene lead to severe impairment of biliary bile salt secretion. In PFIC-1 patients, as well as in BRIC patients during a cholestatic episode, the serum bile salt concentration is high, while the biliary bile salt concentration is low (10–12). This is, however, also the case in PFIC-2, where the sister of P-glycoprotein is defective. Thus, any explanation of the functional defect in PFIC-1 patients should take into consideration that sPgp has been characterised as a canalicular bile salt transporter, and mutations in sPgp in PFIC-2 patients also lead to a near complete loss of canalicular bile salt secretion. It is therefore possible that FIC-1 is important for bile salt secretion in an indirect mechanism. An obvious first possibility is that FIC-1 has the same function as its homologue and that it is an (inward) aminophospholipid flippase. It should be noted that a high aminophospholipid flippase activity has been reported for the hepatoma cell line HepG2 (13). If FIC-1 is present in the canalicular membrane, it could act in conjunction with MDR3 P-glycoprotein, which catalyses the outward translocation of phosphatidylcholine (14). The function of FIC-1 could be to counteract the MDR3-induced phospholipid imbalance by flipping PS and/or PE to the inner membrane. These combined flippase functions would increase the asymmetry of the

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canalicular membrane. In this way more PC is available for canalicular secretion. It does not explain, however, why the absence of FIC-1 leads to an impairment of bile salt secretion, unless one assumes that the sister of Pgp can only function properly in a highly asymmetric membrane. The latter is not very likely to be the case, as Gerloff et al. (6) expressed sPgp in baculovirusinfected Sf9 cells and were able to measure bile salt transport in membrane vesicles from these cells. The lipids in these vesicles are probably not very asymmetrically localised due to scrambling (loss of lipid asymmetry) during isolation and storage of the membranes. FIC-1 could also be a canalicular flippase for sphingolipids or for cholesterol. In that case it should be an outward flippase, but again there is no obvious reason why the absence of this function impairs bile salt secretion. Alternatively, as a putative aminophospholipid flippase, FIC-1 might be of importance for binding auxiliary proteins to the inner leaflet of the canalicular membrane or for fusion of exocytotic vesicles with the canalicular membrane. This is a reasonable possibility, since vesicle fusion requires binding of annexin and Ca2π to anionic phospholipids (PS). It has been suggested that inward PS flipping is important for this process (15). This is in fact the reason why ‘‘conventional’’ aminophospholipid activity was identified not only in (erythrocyte) plasma membranes, but also in chromaffin granules and synaptic vesicles. In the absence of FIC-1 function, fusion of vesicles with the canalicular membrane could be impaired. This might lead to decreased insertion of canalicular proteins, and the most important of these, with regard to bile formation, is the bile salt transporter. Cholestasis may then be caused by insufficient insertion of the newly synthesised and recycled sister of P-glycoprotein. Clearly, this defect should affect more functions than the bile salt transporter alone. Apical exocytotic function is of general importance to many epithelia and this could explain why the protein is highly expressed in the pancreas and small intestine. Loss of FIC-1 function should, however, also affect these tissues. Diarrhoea and fat malabsorption are usually observed in patients with PFIC, which suggests impaired absorptive functions of the intestine. It has also been suggested that bile salt absorption in the intestine is impaired in patients with BRIC because a highly contracted bile salt pool has been reported in these patients (16). Ileal absorption of bile salts is thought to be mediated by the sodium-dependent transporter iBAT, which has recently also been cloned (17) but it is not known whether other proteins are also involved. There seems to be no overt malfunction of the pancreas in patients with PFIC, but two out of ten BRIC patients

Genetic code for PFIC-I and BRIC

on the Far Oer islands seem to have suffered from spontanous pancreatitis (R. Houwen, pers. commun.). A second possibility is that FIC-1 is not an aminophospholipid flippase. Because the homology of the gene with the bovine aminophospholipid flippase is not extremely high (53% similarity), this is a possibility that should be seriously considered. The gene does have the consensus sequences of a P-type ATPase; it might therefore be an ion transporter. The high rate of anionic bile salt secretion into bile is associated with considerable flux of negative charges across the canalicular membrane. It is generally accepted that counterions (mainly Naπ) permeate through the tight junctions (18). The flux of bile salts could be too high to be compensated by passive permeation of cations. If FIC-1 plays a role in this process, it would need to be a cation extrusion pump or an anion import pump, and it should be tightly regulated by bile salt transport. This hypothesis could possibly explain the anecdotal data on deviating sweat tests and the observed diarrhoea in FIC-1-deficient patients. One problem with this hypothesis is, however, that it does not explain the (near) complete absence of bile salt transport in FIC1-deficient patients. It would be more likely that these patients have problems only with high fluxes of bile salts. A third possibility is that FIC-1 is an (outward) bile salt pump. FIC-1 could have affinity for hydrophobic bile salts such as (cheno)deoxycholate, lithocholate and/or their sulphate- and glucuronide-conjugates. These secondary bile salts are produced from primary bile salts (cholate and chenodeoxycholate) by bacteria in the gut. Normally, only minor amounts of lithocholate are present in plasma. It is known that hydrophobic bile salts easily enter phospholipid bilayers and it may therefore be that they need to be transported by a flippase-like transporter. The bile salt transporting capacity of sPgp has so far only been reported in preliminary form for the hydrophilic bile salt taurocholate. It could be demonstrated that sPgp is able to transport taurocholate in an ATP-dependent manner with a Km of 4.3 mM. The affinity of sPgp for hydrophobic bile salts such as deoxycholate and lithocholate compared to its affinity for taurocholate is as yet unknown. This hypothesis would clearly require that sPgp has a relatively low affinity for these bile salts. In the absence of FIC-1, hydrophobic bile salts would accumulate in plasma, and it is known that these bile salts can induce cholestasis (19). In this hypothesis the cholestasis in FIC-1 deficiency would be a secondary inhibition of sPgp by hydrophobic bile salts. In line with this hypothesis, it has been observed that bile from FIC-1-deficient patients contains very low amounts of bile salts,

with a disproportionately low content of chenodeoxycholate compared to cholate (11,12). In this context, it is also interesting to note that chronic bile diversion has been successfully applied to relieve cholestasis in PFIC patients (20). The high expression of FIC-1 in the gut might serve as a direct defense against the hydrophobic bile salts produced by bacteria. The observed contraction of the bile salt pool in BRIC patients and the high expression of FIC-1 in the pancreas of normal subjects are not explained by this hypothesis. It is clear that none of the proposed functions of FIC-1 easily explains the phenotypic symptoms of BRIC and PFIC-1 patients. Clearly, further research is needed to elucidate the function of this protein. Transfection of the cDNA in relevant cell lines will open up the possibility of testing for the various proposed transport functions. Cultured cells may, on the other hand, be too simple to explain the phenotypic characteristics of the patients. It is therefore of utmost importance to produce transgenic animals in which the various inherited defects are mimicked. Knockout mice for the Fic-1 gene will most probably serve as a model for PFIC-1 patients. With these knockouts, new transgenic mice can be produced which express FIC-1 harbouring typical BRIC mutations, and these animals will serve as a model for BRIC patients. It will be particularly interesting to find the agents or conditions that cause cholestatic attacks in these patients. These studies are likely to lead to new therapeutic approaches for BRIC patients.

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