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Hereditary forms of intrahepatic cholestasis Laura N Bull Several genes that are mutated in hereditary forms of intrahepatic cholestasis have been identified or mapped, providing new insights into the process of enterohepatic bile acid circulation in health and disease and new tools with which to study this process. Murine models of several of these disorders have been generated. Unanticipated genetic heterogeneity has been identified. Addresses Liver Center Laboratory and Department of Medicine, San Francisco General Hospital, University of California San Francisco, San Francisco, California 94110, USA; e-mail: [email protected] Current Opinion in Genetics & Development 2002, 12:336–342 0959-437X/02/$ — see front matter © 2002 Elsevier Science Ltd. All rights reserved. Abbreviations ABC ATP-binding cassette AGS Alagille syndrome BRIC benign recurrent intrahepatic cholestasis GFC Greenland familial cholestasis GGT γ-glutamyl transpeptidase ICP intrahepatic cholestasis of pregnancy JAG1 Jagged 1 LCS lymphedema-cholestasis syndrome NAICC North American Indian childhood cirrhosis PFIC progressive familial intrahepatic cholestasis
recovering from damage, and shows striking regenerative abilities; however, these mechanisms can be overwhelmed such that the liver suffers severe injury and ultimately fails. Cholestasis — or impaired bile flow — is one of the most common and devastating manifestations of both inherited and acquired liver disease. Cholestasis can be classified as either intrahepatic or extrahepatic. There are different functional definitions of cholestasis, with varying degrees of breadth in the types of phenotype they include. In this review, I focus on forms of hereditary intrahepatic cholestasis in which evidence of disordered bile acid circulation is found together with histopathological evidence of cholestasis. Individuals with cholestasis manifest jaundice, severe itching, malabsorption of fats and fat-soluble vitamins and, in many cases, progressive liver damage.
Identified cholestasis genes In recent years, application of genetic and candidate gene approaches has led to a marked increase in our understanding of hereditary cholestasis, and consequently in our knowledge about the normal process of bile circulation. Genes that are mutated in disease are being mapped and identified, and animal models of cholestasis are being created. Alagille syndrome and Jagged 1
Introduction The liver is the largest human solid organ, and it performs a range of essential functions, including clearing waste products, protecting the body from toxins, synthesizing plasma proteins and regulating metabolism. Blood flows directly from the intestine to the liver, so the liver is the first organ that nutrients and chemicals encounter after absorption. The liver filters blood, modifying potential toxins and waste products and/or removing them from the circulation [1]. The liver has a crucial role in the synthesis and transport of bile acids (Figure 1a). These acids are essential for normal digestion to occur; in the small intestine, they aid in emulsification and absorption of dietary fats and fat-soluble vitamins. They are synthesized from cholesterol in the hepatocytes of the liver and secreted across the canalicular membrane of hepatocytes into the canaliculus to form a component of bile. From the canaliculus, bile flows through the intrahepatic ducts and then out of the liver into the gallbladder and common bile duct. When needed for digestion, bile is secreted into the intestine. Bile acids are reabsorbed from the intestine with high efficiency, returned through the portal circulation to the liver, and subsequently resecreted. The liver is exposed to many different potentially damaging substances, including high concentrations of bile acids. The liver has evolved mechanisms for protecting itself and
Alagille syndrome (AGS) is an autosomal dominant disorder in which developmental abnormalities occur in many structures, including the liver, heart, face, eye, kidney and skeleton. Affected individuals often manifest cholestasis and a paucity of bile ducts. The gene mutated in this disorder, Jagged 1 (JAG1), encodes a cell-surface protein that is a ligand for Notch receptors — highly conserved receptors involved in developmentally important intercellular signaling that regulates cell fate decisions [2,3]. A summary of published results of JAG1 mutation screening in 233 individuals with AGS has been published recently [4•]. Roughly 70% of these individuals show mutation of one copy of JAG1, and most of these mutations lead to frameshifts and consequently premature termination. Some missense and splice-site mutations, as well as large deletions, have also been reported. Most mutations (60–70%) are de novo. Two missense mutations in JAG1 have been shown to lead to loss of JAG1 function, abnormal glycosylation of JAG1 and its absence from the cell surface, with abnormal accumulation elsewhere [5•]. Generally, there is little consistent evidence of a correlation between genotype — that is, the presence of a specific mutation — and phenotype, and different people carrying an identical mutation can have widely varying manifestations and severity of disease. One notable exception is the presence of a specific missense mutation in JAG1 in
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Figure 1 Enterohepatic bile acid circulation. (a) The enterohepatic circulation of bile, with an enlarged illustration of the canaliculus and adjacent cells. (b) A depiction of hepatocytes and cholangiocytes, indicating where blood and bile flow, and where GGT (γ-GT) protein is expressed. (c) A schematic, again, of the hepatocytes and cholangiocytes, indicating where BSEP, MDR3, and FIC1 are expressed. BA, bile acids; PC, phosphatidylcholine.
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Cholesterol 0.2g/day
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PC MDR3
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members of a large pedigree who have isolated heart disease, as well as characteristic facial features that are distinct from those typically seen in individuals with AGS [6••]. JAG1 is expressed widely during embryogenesis and in adults [2,7,8]. With regard to hepatic expression, JAG1 is
present in the ductal plate — the ductal plate undergoes remodeling to form the portal duct architecture — in human fetuses. (Fetuses from 14–22 weeks gestation were studied.) In postnatal liver, JAG1 is expressed in the biliary epithelium and zone 3 hepatocytes [9]. Another study, which examined human embryos at 32–52 days post-ovulation,
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has shown that JAG1 is expressed by 41 days in the portal vein and hepatic artery, but is not found in the hepatic parenchyma [7]. A third study has shown that JAG1 is expressed in the liver in human embryos at 28 days and 20 weeks, but only in blood vessels and not in hepatocytes or cholangiocytes [8]. It seems likely that JAG1 is important in morphogenesis and maintaining the biliary tree [9]; bile duct paucity may result from ischemia secondary to abnormal development of the liver vasculature, or JAG1 may be expressed in both vascular structures and developing bile ducts, but at different times during development [8]. Mice with a null Jag1 allele have been generated. Mice homozygous for this allele die from hemorrhage early in embryogenesis and have defects in remodeling of the embryonic and yolk sac vasculature. Heterozygotes for the null allele are grossly normal and fertile, but demonstrate an eye dysmorphology [10]. ATP8B1 deficiency
Individuals suffering from benign recurrent intrahepatic cholestasis (BRIC) experience recurrent bouts of intrahepatic cholestasis that resolve spontaneously, leaving no lasting liver damage. A genomic screen has placed a BRIC locus showing autosomal recessive inheritance on chromosome 18q21–22 [11]. A form of progressive familial intrahepatic cholestasis (PFIC) found in the Amish has subsequently been localized to this same region [12]. PFIC is an autosomal recessive disorder that is more severe than BRIC; individuals with PFIC manifest cholestasis in infancy, develop malnutrition and growth retardation, and usually progress to end-stage liver disease before adulthood. Further studies identified ATP8B1 (which was initially called FIC1 for ‘familial intrahepatic cholestasis 1’) as the gene mutated in both of these disorders [13–15]. ATP8B1 encodes a protein belonging to a recently identified subfamily of P-type ATPases, which are ATP-dependent membrane transporters [13,16]. Genetic studies of the ATP8B1 region in people with Greenland familial cholestasis (GFC) indicated that individuals with GFC from East Greenland might carry mutations in ATP8B1, although the results in individuals from West Greenland were less convincing [17]. Subsequently, homozygosity for a mutation in ATP8B1, corresponding to Asp554→Asn, has been found in all individuals with GFC who have been studied from both East and West Greenland [18]. Intrahepatic cholestasis of pregnancy (ICP) has been reported in mothers of PFIC-affected individuals and in relatives of people with BRIC, suggesting that heterozygous carriers of ATP8B1 mutations may be at elevated risk of developing ICP [19,20].
The function of ATP8B1 is not yet certain, although homology and initial functional studies have suggested that it may function as an aminophospholipid flippase, translocating aminophospholipids between membrane leaflets [13,21•,22]. ATP8B1 is expressed in a broad range of tissues, which may account for possible extrahepatic findings associated with ATP8B1 deficiency; interestingly, expression levels seem higher in the small intestine and pancreas than in the liver [13]. Initial immunohistochemical studies indicate that, within the liver, ATP8B1 is present in the canalicular membrane of hepatocytes and in cholangiocytes [21•,23•]. A mouse model of ATP8B1 deficiency has been generated recently; the mice have a milder hepatic phenotype than is seen in people with the same mutation and seem likely to have a defect in regulating intestinal bile salt absorption [24•] (Figure 1c). ABCB11 deficiency
Progressive familial intrahepatic cholestasis can be divided into two classes, based on the serum levels of γ-glutamyl transpeptidase (GGT) present in affected individuals (Figure 1b). The disorder caused by ATP8B1 deficiency is a form of ‘low-GGT PFIC’; it is also referred to as PFIC type 1 (PFIC1), because locus heterogeneity for low-GGT PFIC was demonstrated after it was mapped [25,26]. In 1997, a second locus for low-GGT PFIC was mapped to chromosome 2q24, and this form of PFIC was termed PFIC type 2 (PFIC2) [27]. The gene mutated in PFIC2 is an ATP-binding cassette (ABC) transporter, ABCB11 (aliases include BSEP for ‘bile salt export protein’ and SPGP for ‘sister of P-glycoprotein’) [28]. ABCB11 is an ATP-dependent membrane transporter that is expressed in the canalicular membrane of hepatocytes. It functions in transporting bile acids out of the hepatocyte into the canaliculus [29,30,31•] (Figure 1c). A close correlation between lack of canalicular ABCB11 expression and the presence of ABCB11 mutations has been observed in individuals with PFIC [32]. The amino acid sequence alignment of ABCB11 orthologs from different species has shown that previously reported missense mutations occur in highly conserved residues. Introduction of two of these mutations into skate ABCB11 resulted in defective expression of the mutant protein in cultured cells [33•]. Recently, an individual affected with BRIC has been reported to be a compound heterozygote for two mutations in ABCB11 [34]. A mouse homozygous for a knockout of the murine ABCB11 ortholog shows mild nonprogressive cholestasis and reduced hepatic bile acid output. The mice secrete unexpectedly high amounts of tetrahydroxylated bile acids, which suggests that they may have an alternative mechanism for bile acid export that protects the liver from damage [35••].
Hereditary forms of intrahepatic cholestasis Bull
ABCB4 deficiency
Functional assays and a mouse knockout model have shown that ABCB4 (also known as MDR3 in humans and Mdr2 in mouse) is expressed in the canalicular membrane of hepatocytes and functions in transport of phosphatidylcholine from the inner to the outer leaflet of this membrane [36,37] (Figure 1c). Large quantities of phosphatidylcholine are normally present in bile and probably help to protect the liver from damage caused by bile acids. It thus seems feasible that some hereditary cholestasis may be caused by defects in ABCB4. In an initial study of two people with high-GGT PFIC, one was found to lack expression of ABCB4 mRNA in her liver, and the other was found to have substantially decreased levels of biliary phospholipid [38]. This preliminary study was followed up by mutation analysis of ABCB4 in two more individuals with high-GGT PFIC: one was found to be homozygous for a frameshift-inducing deletion, and the second for a nonsense mutation. In addition, no canalicular staining for ABCB4 was observed in the liver of these people [39]. This study indicated that some cases of high-GGT PFIC are due to mutation in ABCB4, and PFIC caused by ABCB4 deficiency was thus termed PFIC3. More recently, 31 individuals with high-GGT PFIC have been studied. Mutation screening of ABCB4 using singlestranded conformation polymorphism and sequencing was carried out, as was immunohistochemistry of liver using an antibody against ABCB4 and quantification of biliary phospholipid. Mutations in one or both copies of ABCB4 were found in 17 of these people. This study suggests that individuals with missense mutations may have milder disease than those with protein-truncating mutations [40•]. Heterozygosity for ABCB4 mutations has also been found in some women with ICP [39,40•,41,42]. Most recently, ABCB4 mutations have been identified in people with a form of cholesterol gallstone disease [43•]; gallstones have been also reported in some individuals with PFIC3 and the parents of some PFIC3-affected individuals [40•]. Hepatocyte transplantation has been shown to correct liver disease in the mouse model of PFIC3 [44].
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recently to the mapping of a locus for LCS to a 6.6-cM region on human chromosome 15q [48]. The most probable location of the LCS gene has subsequently been narrowed to an interval of several hundred kilobases, and identification and screening of candidate genes is underway (LN Bull, unpublished data). North American Indian childhood cirrhosis
North American Indian childhood cirrhosis (NAICC) occurs in the Ojibway-Cree population and manifests as transient neonatal jaundice with persistent direct hyperbilirubinemia and progression to biliary cirrhosis. Affected individuals have elevated serum GGT and typically eventually require liver transplant. The disorder is considered probably caused by a problem in the bile ducts and may thus represent a form of ‘progressive familial cholangiopathy’ [49]. A genome screen recently localized the NAICC gene to chromosome 16q22 [50]. Intrahepatic cholestasis of pregnancy
Women with ICP (which is also known as obstetric cholestasis) develop itching, elevated serum concentrations of bile acid, and sometimes jaundice and other manifestations of liver disease during pregnancy; the disorder resolves shortly after delivery. Some women have normal serum levels of GGT, whereas others have elevated concentrations of GGT. Although ICP is unpleasant for mothers, the main danger from the disorder is to the fetus, as the risk of fetal death or premature birth is elevated. ICP is a relatively common disorder, occurring with widely varying frequency in different populations; incidences from 0.1% to 10% of pregnancies have been reported in various studies [51,52]. Susceptibility to ICP is probably influenced by several genetic and environmental factors. As discussed above, heterozygous carrier status for mutation in ATP8B1 or ABCB4 has been reported in some women with ICP. In addition, a recent report suggests that in the Finnish population, an ICP susceptibility locus may lie on chromosome 2p13 [53•]. A second study of Finnish women with ICP raises the possibility that a particular allele of angiotensin-converting enzyme is enriched in ICP-affected women [54]; it will be informative to see whether either of these findings can be replicated.
Mapped genes Lymphedema-cholestasis syndrome
Unmapped forms of PFIC and BRIC
Lymphedema-cholestasis syndrome (LCS; also known as Aagenaes syndrome) is an autosomal recessive disorder characterized by severe neonatal cholestasis with elevated serum GGT. Usually, manifestations of liver disease lessen during early childhood and become episodic. Individuals with LCS also suffer chronic severe lymphedema, which may be apparent from birth or may manifest during childhood [45–47].
Recent findings indicate that additional as yet unmapped loci for both low- and high-GGT PFIC probably exist. In a study of 34 families affected with low-GGT PFIC, data from 10 were inconsistent with linkage to either ABCB11 or ATP8B1, which suggests that there is at least one additional locus for this disorder [55•]. Many individuals with high-GGT PFIC that were screened for mutation in ABCB4 showed no evidence of mutation [40•,56•]; because liver ABCB4 cDNA was fully sequenced for many of these people, this finding suggests the existence of at least one additional high-GGT PFIC locus, although other
Originally, LCS was described in a Norwegian kindred, and a genomic screen of members of this kindred led
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explanations for the negative results cannot be ruled out as yet. At least one unmapped BRIC locus exists, as a large pedigree has been described in which segregation of BRIC is consistent with autosomal dominant inheritance and inconsistent with linkage to either ATP8B1 or ABCB11 [57].
Acknowledgements
Conclusions
References and recommended reading
Genetic characterization of forms of hereditary intrahepatic cholestasis has revealed unanticipated genetic heterogeneity and has resulted in the identification of several proteins necessary for normal enterohepatic bile circulation; further such studies will continue to provide new insights. A consistent theme seen in murine models of these disorders is that a milder hepatic phenotype occurs in mouse than in humans. This may be due, in part, to the lower hydrophobicity of the murine bile acid pool, and to alternative pathways for bile acid transport and metabolism that probably exist in the mouse.
Papers of particular interest, published within the annual period of review, have been highlighted as:
Genes mutated in hereditary cholestasis are promising candidate loci for susceptibility to drug-induced cholestasis. Some sequence variations in such genes could affect the function or expression of the resulting proteins in subtle ways; whereas individuals carrying such sequence changes might not be affected with hereditary cholestasis as typically defined, they might bear an increased risk of adverse cholestatic reactions to certain medications. Identification of such susceptibility loci is becoming feasible and could substantially facilitate prevention of this undesired side-effect of some medications.
Update A recent report [59••] describes mice doubly heterozygous for the Jag1 null allele and a Notch2 hypomorphic allele. The phenotype of these mice is more similar to that seen in people with AGS than is the phenotype of the mice with the heterozygous or homozygous null Jag1 allele alone. The interaction of the Jag1 and Notch2 mutations also suggests the hypothesis that some of the phenotypic variability seen in human AGS may be due to variation in the NOTCH2 alleles present in AGS-affected individuals. Mutations in both ATP8B1 and ABCB11 have now been described in different Taiwanese individuals with PFIC [60]. In this study, clinical, biochemical, and histologic data were also examined to identify features that might distinguish ATP8B1 deficiency from ABCB11 deficiency. In a separate study, several individuals are described who initially presented clinically with BRIC, but whose disease later worsened, becoming chronic and progressive, as is seen in PFIC; this finding supports the sensible hypothesis that BRIC and PFIC represent two extremes along a continuum of possible disease severity [61]. Recently, the promoter region of ABCB11 has been characterized, and the farnesoid X receptor and bile salts are shown to be involved in regulation of expression of ABCB11 [62].
I wish to thank Nancy Spinner, Victoria Carlton and Ludmila Pawlikowska for helpful discussions. This work was supported by National Institutes of Health R01 grants DK50697 and DK58214 to LN Bull. I wish to thank N Lomri and B Scharschmidt for the diagram of the enterohepatic circulation used in Figure 1b. I also thank R Thompson for the schematic used in Figure 1a-c.
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31. Noe J, Hagenbuch B, Meier PJ, St-Pierre MV: Characterization of the • mouse bile salt export pump overexpressed in the baculovirus system. Hepatology 2001, 33:1223-1231. This functional study of murine bile salt export pump (BSEP) characterizes the ability of BSEP to transport different bile acids. The authors provide data supporting the ATP dependence of this activity and suggesting that bile acids may regulate BSEP through signaling mediated by protein kinase C. 32. Jansen PLM, Strautnieks SS, Jacquemin E, Hadchouel M, Sokal E, Hooiveld GJ, Koning JH, Jager-Krikken AD, Kuipers F, Stellaard F et al.: Hepatocanalicular bile salt export pump deficiency in patients with progressive familial intrahepatic cholestasis. Gastroenterology 1999, 117:1370-1379. 33. Cai SY, Wang L, Ballatori N, Boyer JL: Bile salt export pump is • highly conserved during vertebrate evolution and its expression is inhibited by PFIC type II mutations. Am J Physiol Gastrointest Liver Physiol 2001, 281:G316-G322. In this study, the ABCB11 ortholog present in the marine skate Raja erinacea is isolated and characterized functionally. When two mutations associated with ABCB11 deficiency are introduced separately into the skate gene, defective expression of the resulting protein is seen. 34. Kullak-Ublick GA, Kerb R, Mullhaupt B, Renner EI, Penger A, Brinkmann U, Stieger B, Meier PJ: A novel R432T mutation in the bile salt export pump gene (BSEP;ABCB11) is associated with recurrent intrahepatic cholestasis in an adolescent patient. Hepatology 2001, 34:216. 35. Wang R, Salem M, Yousefm IM, Tuchweber B, Lam P, Childs SJ, •• Helgason CD, Ackerley C, Phillips MJ, Ling V: Targeted inactivation of sister of P-glycoprotein gene (spgp) in mice results in nonprogressive but persistent intrahepatic cholestasis. Proc Natl Acad Sci USA 2001, 98:2011-2016. In this study, a knockout model of the murine ortholog of ABCB11 is reported and characterized. The homozygous mutant mice demonstrate mild nonprogressive cholestasis and decreased secretion of bile acids from the liver. Secretion of cholic acid is reduced to 6% of the wild-type value, but total bile salt output is 30% of the wild-type value. An unexpectedly large amount of tetrahydroxylated bile acids are secreted by the mutant animals and are not seen in wild type. These findings suggest that mice have a mechanism for hydroxylation and transport of bile acids out of the hepatocyte that partially compensates for the ABCB11 defect. 36. Smit JJ, Schinkel AH, Oude Elferink RP, Groen AK, Wagenaar E, van Deemter L, Mol CA, Ottenhoff R, van der Lugt NM, van Roon MA et al.: Homozygous disruption of the murine mdr2 P-glycoprotein gene leads to a complete absence of phospholipid from bile and to liver disease. Cell 1993, 75:451-462. 37.
Ruetz S, Gros P: Phosphatidylcholine translocase: a physiological role for the mdr2 gene. Cell 1994, 77:1071-1081.
38. Deleuze JF, Jacquemin E, Dubuisson C, Cresteil D, Dumont M, Erlinger S, Bernard O, Hadchouel M: Defect of multidrug-resistance 3 gene expression in a subtype of progressive familial intrahepatic cholestasis. Hepatology 1996, 23:904-908. 39. de Vree JM, Jacquemin E, Sturm E, Cresteil D, Bosma PJ, Aten J, Deleuze JF, Desrochers M, Burdelski M, Bernard O et al.: Mutations in the MDR3 gene cause progressive familial intrahepatic cholestasis. Proc Natl Acad Sci USA 1998, 95:282-287. 40. Jacquemin E, de Vree JM, Cresteil D, Sokal E, Sturm E, Dumont M, • Scheffer GL, Paul M, Burdelski M, Bosma PJ et al.: The wide spectrum of multidrug resistance 3 deficiency: from neonatal cholestasis to cirrhosis of adulthood. Gastroenterology 2001, 120:1448-1458. This study reports the results of ABCB4 mutation analysis, antibody studies of liver tissue and evaluation of biliary phospholipid levels in individuals with high-GGT PFIC. Three mothers heterozygous for mutations in ABCB4 experience ICP, and some individuals heterozygous or homozygous for ABCB4 mutations have biliary lithiasis. 41. Jacquemin E, Cresteil D, Manouvrier S, Boute O, Hadchouel M: Heterozygous non-sense mutation of the MDR3 gene in familial intrahepatic cholestasis of pregnancy. Lancet 1999, 353:210-211. 42. Dixon PH, Weerasekera N, Linton KJ, Donaldson O, Chambers J, Egginton E, Weaver J, Nelson-Piercy C, de Swiet M, Warnes G et al.: Heterozygous MDR3 missense mutation associated with intrahepatic cholestasis of pregnancy: evidence for a defect in protein trafficking. Hum Mol Genet 2000, 9:1209-1217. 43. Rosmorduc O, Hermelin B, Poupon R: MDR3 gene defect in adults • with symptomatic intrahepatic and gallbladder cholesterol cholelithiasis. Gastroenterology 2001, 120:1459-1467. Six people with a specific form of biliary gallstone disease are studied; all are shown to possess mutations in one or both of their copies of ABCB4.
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44. de Vree JML, Ottenhoff R, Bosma PJ, Smith AJ, Aten J, Oude Elferink RPJ: Correction of liver disease by hepatocyte transplantation in a mouse model of progressive familial intrahepatic cholestasis. Gastroenterology 2000, 119:1720-1730. 45. Aagenaes O, van der Hagen CB, Refsum S: Hereditary recurrent intrahepatic cholestasis from birth. Arch Dis Child 1968, 43:646-657. 46. Aagenaes O, Sigstad H, Bjorn-Hansen R: Lymphoedema in hereditary recurrent cholestasis from birth. Arch Dis Child 1970, 45:690-695. 47.
Aagenaes O: Hereditary cholestasis with lymphoedema (Aagenaes syndrome, cholestasis-lymphoedema syndrome). New cases and follow-up from infancy to adult age. Scand J Gastroenterol 1998, 33:335-345.
48. Bull LN, Roche E, Song EJ, Pedersen J, Knisely AS, van Der Hagen CB, Eiklid K, Aagenaes O, Freimer NB: Mapping of the locus for cholestasis-lymphedema syndrome (Aagenaes syndrome) to a 6.6-cM interval on chromosome 15q. Am J Hum Genet 2000, 67:994-999. 49. Drouin E, Russo P, Tuchweber B, Mitchell GA, Rasquin-Weber A: North American Indian cirrhosis in children: a review of 30 cases. J Pediatr Gastroenterol Nutr 2000, 31:395-404. 50. Betard C, Rasquin-Weber A, Brewer C, Drouin E, Clark S, Verner A, Darmond-Zwaig C, Fortin J, Mercier J, Chagnon P et al.: Localization of a recessive gene for North American Indian childhood cirrhosis to chromosome region 16q22 and identification of a shared haplotype. Am J Hum Genet 2000, 67:222-228. 51. Reyes H: Intrahepatic cholestasis: a puzzling disorder of pregnancy. J Gastrointerol Hepatol 1997, 12:211-216. 52. Lammert F, Marschall H-U, Glantz A, Matern S: Intrahepatic cholestasis of pregnancy: molecular pathogenesis, diagnosis and management. J Hepatol 2000, 33:1012-1021. 53. Heinonen ST, Eloranta ML, Heiskanen JTM, Punnonen KRA, • Helisalmi S, Mannermaa AJ, Hiltunen MJ: Maternal susceptibility locus for obstetric cholestasis maps to chromosome region 2p13 in Finnish patients. Scand J Gastroenterol 2001, 36:766-770. In this study, population-based linkage disequilibrium mapping is used to screen chromosome 2 for loci associated with ICP. The study was originally carried out to evaluate the hypothesis that variants in the HADHA gene might be associated with increased risk of ICP, as suggested by a previous report that mothers that are heterozygous for HADHA mutations have an increased risk of ICP [58]. No evidence of an association of ICP with HADHA is found; instead, evidence suggests the existence of an ICP
susceptibility locus elsewhere on chromosome 2. Follow-up studies will be necessary to confirm this association. 54. Heiskanen JTM, Pirskanen MM, Hiltunen MJ, Mannermaa AJ, Punnonen KRA, Heinonen ST: Insertion–deletion polymorphism in the gene for angiotensin-converting enzyme is associated with obstetric cholestasis but not with preeclampsia. Am J Obstet Gynecol 2001, 185:600-603. 55. Strautnieks S, Byrne J, Knisely AS, Bull LN, Sokal E, Lacaille F, • Vergani G, Thompson R: There must be a third locus for low GGT PFIC [Abstract]. Hepatology 2001, 34:240 Substantial evidence that supports the existence of at least one additional locus for low-GGT PFIC is reported. 56. Chen HL, Chang PS, Hsu HC, Lee JH, Ni YH, Hsu HY, Jeng YM, • Chang MH: Progressive familial intrahepatic cholestasis with high γ-glutamyl transpeptidase levels in Taiwanese infants: role of MDR3 gene defect? Pediatr Res 2001, 50:50-55. Data in this paper support the hypothesis that some cases of high-GGT PFIC may be due to mutation in a gene or genes other than ABCB4. 57.
Floreani A, Molaro M, Mottes M, Sangalli A, Baragiotta A, Roda A, Naccarato R, Clementi M: Autosomal dominant benign recurrent intrahepatic cholestasis (BRIC) unlinked to 18q21 and 2q24. Am J Med Genet 2000, 95:450-453.
58. Tyni T, Ekholm E, Pihko H: Pregnancy complications are frequent in long-chain 3-hydroxyacyl-coenzyme A dehydrogenase deficiency. Am J Obstet Gynecol 1998, 178:603-608. 59. McCright B, Lozier J, Gridley T: A mouse model of Alagille •• syndrome: Notch2 as a genetic modifier of Jag1 haploinsufficiency. Development 2002, 129:1075-1082. Mice doubly heterozygous for mutant Jag1 and Notch2 alleles are described: these mice seem to represent a promising animal model for AGS. 60. Chen HL, Chang PS, Hsu HC, Ni YH, Hsu HY, Lee JH, Jeng YM, Shau WY, Chang MH: FIC1 and BSEP defects in Taiwanese patients with chronic intrahepatic cholestasis with low γ-glutamyltranspeptidase levels. J Pediatr 2002, 140:119-124. 61. van Ooteghem NA, Klomp LW, van Berge-Henegouwen GP, Houwen RH: Benign recurrent intrahepatic cholestasis progressing to progressive familial intrahepatic cholestasis: low GGT cholestasis is a clinical continuum. J Hepatol 2002, 36:439-443. 62. Plass JR, Mol O, Heegsma J, Geuken M, Faber KN, Jansen PL, Muller M: Farnesoid X receptor and bile salts are involved in transcriptional regulation of the gene encoding the human bile salt export pump. Hepatology 2002, 35:589-596.
Hereditary forms of intrahepatic cholestasis Laura N Bull Several genes that are mutated in hereditary forms of intrahepatic cholestasis have been identified or mapped, providing new insights into the process of enterohepatic bile acid circulation in health and disease and new tools with which to study this process. Murine models of several of these disorders have been generated. Unanticipated genetic heterogeneity has been identified. Addresses Liver Center Laboratory and Department of Medicine, San Francisco General Hospital, University of California San Francisco, San Francisco, California 94110, USA; e-mail: [email protected] Current Opinion in Genetics & Development 2002, 12:336–342 0959-437X/02/$ — see front matter © 2002 Elsevier Science Ltd. All rights reserved. Abbreviations ABC ATP-binding cassette AGS Alagille syndrome BRIC benign recurrent intrahepatic cholestasis GFC Greenland familial cholestasis GGT γ-glutamyl transpeptidase ICP intrahepatic cholestasis of pregnancy JAG1 Jagged 1 LCS lymphedema-cholestasis syndrome NAICC North American Indian childhood cirrhosis PFIC progressive familial intrahepatic cholestasis
recovering from damage, and shows striking regenerative abilities; however, these mechanisms can be overwhelmed such that the liver suffers severe injury and ultimately fails. Cholestasis — or impaired bile flow — is one of the most common and devastating manifestations of both inherited and acquired liver disease. Cholestasis can be classified as either intrahepatic or extrahepatic. There are different functional definitions of cholestasis, with varying degrees of breadth in the types of phenotype they include. In this review, I focus on forms of hereditary intrahepatic cholestasis in which evidence of disordered bile acid circulation is found together with histopathological evidence of cholestasis. Individuals with cholestasis manifest jaundice, severe itching, malabsorption of fats and fat-soluble vitamins and, in many cases, progressive liver damage.
Identified cholestasis genes In recent years, application of genetic and candidate gene approaches has led to a marked increase in our understanding of hereditary cholestasis, and consequently in our knowledge about the normal process of bile circulation. Genes that are mutated in disease are being mapped and identified, and animal models of cholestasis are being created. Alagille syndrome and Jagged 1
Introduction The liver is the largest human solid organ, and it performs a range of essential functions, including clearing waste products, protecting the body from toxins, synthesizing plasma proteins and regulating metabolism. Blood flows directly from the intestine to the liver, so the liver is the first organ that nutrients and chemicals encounter after absorption. The liver filters blood, modifying potential toxins and waste products and/or removing them from the circulation [1]. The liver has a crucial role in the synthesis and transport of bile acids (Figure 1a). These acids are essential for normal digestion to occur; in the small intestine, they aid in emulsification and absorption of dietary fats and fat-soluble vitamins. They are synthesized from cholesterol in the hepatocytes of the liver and secreted across the canalicular membrane of hepatocytes into the canaliculus to form a component of bile. From the canaliculus, bile flows through the intrahepatic ducts and then out of the liver into the gallbladder and common bile duct. When needed for digestion, bile is secreted into the intestine. Bile acids are reabsorbed from the intestine with high efficiency, returned through the portal circulation to the liver, and subsequently resecreted. The liver is exposed to many different potentially damaging substances, including high concentrations of bile acids. The liver has evolved mechanisms for protecting itself and
Alagille syndrome (AGS) is an autosomal dominant disorder in which developmental abnormalities occur in many structures, including the liver, heart, face, eye, kidney and skeleton. Affected individuals often manifest cholestasis and a paucity of bile ducts. The gene mutated in this disorder, Jagged 1 (JAG1), encodes a cell-surface protein that is a ligand for Notch receptors — highly conserved receptors involved in developmentally important intercellular signaling that regulates cell fate decisions [2,3]. A summary of published results of JAG1 mutation screening in 233 individuals with AGS has been published recently [4•]. Roughly 70% of these individuals show mutation of one copy of JAG1, and most of these mutations lead to frameshifts and consequently premature termination. Some missense and splice-site mutations, as well as large deletions, have also been reported. Most mutations (60–70%) are de novo. Two missense mutations in JAG1 have been shown to lead to loss of JAG1 function, abnormal glycosylation of JAG1 and its absence from the cell surface, with abnormal accumulation elsewhere [5•]. Generally, there is little consistent evidence of a correlation between genotype — that is, the presence of a specific mutation — and phenotype, and different people carrying an identical mutation can have widely varying manifestations and severity of disease. One notable exception is the presence of a specific missense mutation in JAG1 in
Hereditary forms of intrahepatic cholestasis Bull
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Figure 1 Enterohepatic bile acid circulation. (a) The enterohepatic circulation of bile, with an enlarged illustration of the canaliculus and adjacent cells. (b) A depiction of hepatocytes and cholangiocytes, indicating where blood and bile flow, and where GGT (γ-GT) protein is expressed. (c) A schematic, again, of the hepatocytes and cholangiocytes, indicating where BSEP, MDR3, and FIC1 are expressed. BA, bile acids; PC, phosphatidylcholine.
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Normal bile acid metabolism
Cholesterol 0.2g/day
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Canaliculus γ-GT Cholangiocyte Cholangiocyte Hepatocyte Blood flow
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PC MDR3
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Hepatocyte Blood flow Current Opinion in Genetics & Development
members of a large pedigree who have isolated heart disease, as well as characteristic facial features that are distinct from those typically seen in individuals with AGS [6••]. JAG1 is expressed widely during embryogenesis and in adults [2,7,8]. With regard to hepatic expression, JAG1 is
present in the ductal plate — the ductal plate undergoes remodeling to form the portal duct architecture — in human fetuses. (Fetuses from 14–22 weeks gestation were studied.) In postnatal liver, JAG1 is expressed in the biliary epithelium and zone 3 hepatocytes [9]. Another study, which examined human embryos at 32–52 days post-ovulation,
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has shown that JAG1 is expressed by 41 days in the portal vein and hepatic artery, but is not found in the hepatic parenchyma [7]. A third study has shown that JAG1 is expressed in the liver in human embryos at 28 days and 20 weeks, but only in blood vessels and not in hepatocytes or cholangiocytes [8]. It seems likely that JAG1 is important in morphogenesis and maintaining the biliary tree [9]; bile duct paucity may result from ischemia secondary to abnormal development of the liver vasculature, or JAG1 may be expressed in both vascular structures and developing bile ducts, but at different times during development [8]. Mice with a null Jag1 allele have been generated. Mice homozygous for this allele die from hemorrhage early in embryogenesis and have defects in remodeling of the embryonic and yolk sac vasculature. Heterozygotes for the null allele are grossly normal and fertile, but demonstrate an eye dysmorphology [10]. ATP8B1 deficiency
Individuals suffering from benign recurrent intrahepatic cholestasis (BRIC) experience recurrent bouts of intrahepatic cholestasis that resolve spontaneously, leaving no lasting liver damage. A genomic screen has placed a BRIC locus showing autosomal recessive inheritance on chromosome 18q21–22 [11]. A form of progressive familial intrahepatic cholestasis (PFIC) found in the Amish has subsequently been localized to this same region [12]. PFIC is an autosomal recessive disorder that is more severe than BRIC; individuals with PFIC manifest cholestasis in infancy, develop malnutrition and growth retardation, and usually progress to end-stage liver disease before adulthood. Further studies identified ATP8B1 (which was initially called FIC1 for ‘familial intrahepatic cholestasis 1’) as the gene mutated in both of these disorders [13–15]. ATP8B1 encodes a protein belonging to a recently identified subfamily of P-type ATPases, which are ATP-dependent membrane transporters [13,16]. Genetic studies of the ATP8B1 region in people with Greenland familial cholestasis (GFC) indicated that individuals with GFC from East Greenland might carry mutations in ATP8B1, although the results in individuals from West Greenland were less convincing [17]. Subsequently, homozygosity for a mutation in ATP8B1, corresponding to Asp554→Asn, has been found in all individuals with GFC who have been studied from both East and West Greenland [18]. Intrahepatic cholestasis of pregnancy (ICP) has been reported in mothers of PFIC-affected individuals and in relatives of people with BRIC, suggesting that heterozygous carriers of ATP8B1 mutations may be at elevated risk of developing ICP [19,20].
The function of ATP8B1 is not yet certain, although homology and initial functional studies have suggested that it may function as an aminophospholipid flippase, translocating aminophospholipids between membrane leaflets [13,21•,22]. ATP8B1 is expressed in a broad range of tissues, which may account for possible extrahepatic findings associated with ATP8B1 deficiency; interestingly, expression levels seem higher in the small intestine and pancreas than in the liver [13]. Initial immunohistochemical studies indicate that, within the liver, ATP8B1 is present in the canalicular membrane of hepatocytes and in cholangiocytes [21•,23•]. A mouse model of ATP8B1 deficiency has been generated recently; the mice have a milder hepatic phenotype than is seen in people with the same mutation and seem likely to have a defect in regulating intestinal bile salt absorption [24•] (Figure 1c). ABCB11 deficiency
Progressive familial intrahepatic cholestasis can be divided into two classes, based on the serum levels of γ-glutamyl transpeptidase (GGT) present in affected individuals (Figure 1b). The disorder caused by ATP8B1 deficiency is a form of ‘low-GGT PFIC’; it is also referred to as PFIC type 1 (PFIC1), because locus heterogeneity for low-GGT PFIC was demonstrated after it was mapped [25,26]. In 1997, a second locus for low-GGT PFIC was mapped to chromosome 2q24, and this form of PFIC was termed PFIC type 2 (PFIC2) [27]. The gene mutated in PFIC2 is an ATP-binding cassette (ABC) transporter, ABCB11 (aliases include BSEP for ‘bile salt export protein’ and SPGP for ‘sister of P-glycoprotein’) [28]. ABCB11 is an ATP-dependent membrane transporter that is expressed in the canalicular membrane of hepatocytes. It functions in transporting bile acids out of the hepatocyte into the canaliculus [29,30,31•] (Figure 1c). A close correlation between lack of canalicular ABCB11 expression and the presence of ABCB11 mutations has been observed in individuals with PFIC [32]. The amino acid sequence alignment of ABCB11 orthologs from different species has shown that previously reported missense mutations occur in highly conserved residues. Introduction of two of these mutations into skate ABCB11 resulted in defective expression of the mutant protein in cultured cells [33•]. Recently, an individual affected with BRIC has been reported to be a compound heterozygote for two mutations in ABCB11 [34]. A mouse homozygous for a knockout of the murine ABCB11 ortholog shows mild nonprogressive cholestasis and reduced hepatic bile acid output. The mice secrete unexpectedly high amounts of tetrahydroxylated bile acids, which suggests that they may have an alternative mechanism for bile acid export that protects the liver from damage [35••].
Hereditary forms of intrahepatic cholestasis Bull
ABCB4 deficiency
Functional assays and a mouse knockout model have shown that ABCB4 (also known as MDR3 in humans and Mdr2 in mouse) is expressed in the canalicular membrane of hepatocytes and functions in transport of phosphatidylcholine from the inner to the outer leaflet of this membrane [36,37] (Figure 1c). Large quantities of phosphatidylcholine are normally present in bile and probably help to protect the liver from damage caused by bile acids. It thus seems feasible that some hereditary cholestasis may be caused by defects in ABCB4. In an initial study of two people with high-GGT PFIC, one was found to lack expression of ABCB4 mRNA in her liver, and the other was found to have substantially decreased levels of biliary phospholipid [38]. This preliminary study was followed up by mutation analysis of ABCB4 in two more individuals with high-GGT PFIC: one was found to be homozygous for a frameshift-inducing deletion, and the second for a nonsense mutation. In addition, no canalicular staining for ABCB4 was observed in the liver of these people [39]. This study indicated that some cases of high-GGT PFIC are due to mutation in ABCB4, and PFIC caused by ABCB4 deficiency was thus termed PFIC3. More recently, 31 individuals with high-GGT PFIC have been studied. Mutation screening of ABCB4 using singlestranded conformation polymorphism and sequencing was carried out, as was immunohistochemistry of liver using an antibody against ABCB4 and quantification of biliary phospholipid. Mutations in one or both copies of ABCB4 were found in 17 of these people. This study suggests that individuals with missense mutations may have milder disease than those with protein-truncating mutations [40•]. Heterozygosity for ABCB4 mutations has also been found in some women with ICP [39,40•,41,42]. Most recently, ABCB4 mutations have been identified in people with a form of cholesterol gallstone disease [43•]; gallstones have been also reported in some individuals with PFIC3 and the parents of some PFIC3-affected individuals [40•]. Hepatocyte transplantation has been shown to correct liver disease in the mouse model of PFIC3 [44].
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recently to the mapping of a locus for LCS to a 6.6-cM region on human chromosome 15q [48]. The most probable location of the LCS gene has subsequently been narrowed to an interval of several hundred kilobases, and identification and screening of candidate genes is underway (LN Bull, unpublished data). North American Indian childhood cirrhosis
North American Indian childhood cirrhosis (NAICC) occurs in the Ojibway-Cree population and manifests as transient neonatal jaundice with persistent direct hyperbilirubinemia and progression to biliary cirrhosis. Affected individuals have elevated serum GGT and typically eventually require liver transplant. The disorder is considered probably caused by a problem in the bile ducts and may thus represent a form of ‘progressive familial cholangiopathy’ [49]. A genome screen recently localized the NAICC gene to chromosome 16q22 [50]. Intrahepatic cholestasis of pregnancy
Women with ICP (which is also known as obstetric cholestasis) develop itching, elevated serum concentrations of bile acid, and sometimes jaundice and other manifestations of liver disease during pregnancy; the disorder resolves shortly after delivery. Some women have normal serum levels of GGT, whereas others have elevated concentrations of GGT. Although ICP is unpleasant for mothers, the main danger from the disorder is to the fetus, as the risk of fetal death or premature birth is elevated. ICP is a relatively common disorder, occurring with widely varying frequency in different populations; incidences from 0.1% to 10% of pregnancies have been reported in various studies [51,52]. Susceptibility to ICP is probably influenced by several genetic and environmental factors. As discussed above, heterozygous carrier status for mutation in ATP8B1 or ABCB4 has been reported in some women with ICP. In addition, a recent report suggests that in the Finnish population, an ICP susceptibility locus may lie on chromosome 2p13 [53•]. A second study of Finnish women with ICP raises the possibility that a particular allele of angiotensin-converting enzyme is enriched in ICP-affected women [54]; it will be informative to see whether either of these findings can be replicated.
Mapped genes Lymphedema-cholestasis syndrome
Unmapped forms of PFIC and BRIC
Lymphedema-cholestasis syndrome (LCS; also known as Aagenaes syndrome) is an autosomal recessive disorder characterized by severe neonatal cholestasis with elevated serum GGT. Usually, manifestations of liver disease lessen during early childhood and become episodic. Individuals with LCS also suffer chronic severe lymphedema, which may be apparent from birth or may manifest during childhood [45–47].
Recent findings indicate that additional as yet unmapped loci for both low- and high-GGT PFIC probably exist. In a study of 34 families affected with low-GGT PFIC, data from 10 were inconsistent with linkage to either ABCB11 or ATP8B1, which suggests that there is at least one additional locus for this disorder [55•]. Many individuals with high-GGT PFIC that were screened for mutation in ABCB4 showed no evidence of mutation [40•,56•]; because liver ABCB4 cDNA was fully sequenced for many of these people, this finding suggests the existence of at least one additional high-GGT PFIC locus, although other
Originally, LCS was described in a Norwegian kindred, and a genomic screen of members of this kindred led
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explanations for the negative results cannot be ruled out as yet. At least one unmapped BRIC locus exists, as a large pedigree has been described in which segregation of BRIC is consistent with autosomal dominant inheritance and inconsistent with linkage to either ATP8B1 or ABCB11 [57].
Acknowledgements
Conclusions
References and recommended reading
Genetic characterization of forms of hereditary intrahepatic cholestasis has revealed unanticipated genetic heterogeneity and has resulted in the identification of several proteins necessary for normal enterohepatic bile circulation; further such studies will continue to provide new insights. A consistent theme seen in murine models of these disorders is that a milder hepatic phenotype occurs in mouse than in humans. This may be due, in part, to the lower hydrophobicity of the murine bile acid pool, and to alternative pathways for bile acid transport and metabolism that probably exist in the mouse.
Papers of particular interest, published within the annual period of review, have been highlighted as:
Genes mutated in hereditary cholestasis are promising candidate loci for susceptibility to drug-induced cholestasis. Some sequence variations in such genes could affect the function or expression of the resulting proteins in subtle ways; whereas individuals carrying such sequence changes might not be affected with hereditary cholestasis as typically defined, they might bear an increased risk of adverse cholestatic reactions to certain medications. Identification of such susceptibility loci is becoming feasible and could substantially facilitate prevention of this undesired side-effect of some medications.
Update A recent report [59••] describes mice doubly heterozygous for the Jag1 null allele and a Notch2 hypomorphic allele. The phenotype of these mice is more similar to that seen in people with AGS than is the phenotype of the mice with the heterozygous or homozygous null Jag1 allele alone. The interaction of the Jag1 and Notch2 mutations also suggests the hypothesis that some of the phenotypic variability seen in human AGS may be due to variation in the NOTCH2 alleles present in AGS-affected individuals. Mutations in both ATP8B1 and ABCB11 have now been described in different Taiwanese individuals with PFIC [60]. In this study, clinical, biochemical, and histologic data were also examined to identify features that might distinguish ATP8B1 deficiency from ABCB11 deficiency. In a separate study, several individuals are described who initially presented clinically with BRIC, but whose disease later worsened, becoming chronic and progressive, as is seen in PFIC; this finding supports the sensible hypothesis that BRIC and PFIC represent two extremes along a continuum of possible disease severity [61]. Recently, the promoter region of ABCB11 has been characterized, and the farnesoid X receptor and bile salts are shown to be involved in regulation of expression of ABCB11 [62].
I wish to thank Nancy Spinner, Victoria Carlton and Ludmila Pawlikowska for helpful discussions. This work was supported by National Institutes of Health R01 grants DK50697 and DK58214 to LN Bull. I wish to thank N Lomri and B Scharschmidt for the diagram of the enterohepatic circulation used in Figure 1b. I also thank R Thompson for the schematic used in Figure 1a-c.
• of special interest •• of outstanding interest 1.
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Li L, Krantz ID, Deng Y, Genin A, Banta AB, Collins CC, Qi M, Trask BJ, Kuo WL, Cochran J et al.: Alagille syndrome is caused by mutations in human Jagged1, which encodes a ligand for Notch1. Nat Genet 1997, 16:243-251.
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Oda T, Elkahloun AG, Pike BL, Okajima K, Krantz ID, Genin A, Piccoli DA, Meltzer PS, Spinner NB, Collins FS et al.: Mutations in the human Jagged1 gene are responsible for Alagille syndrome. Nat Genet 1997, 16:235-242.
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Spinner NB, Colliton RP, Crosnier C, Krantz ID, Hadchouel M, Meunier-Rotival M: Jagged1 mutations in Alagille syndrome. Hum Mutat 2001, 17:18-33. A comprehensive review of published JAG1 mutations associated with AGS.
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Morrissette JJD, Colliton RP, Spinner NB: Defective intracellular transport and processing of JAG1 missense mutations in Alagille syndrome. Hum Mol Genet 2001, 10:405-413. Functional and localization studies of two JAG1 missense mutations found in individuals with AGS. 6. ••
Eldadah ZA, Hamosh A, Biery NJ, Montgomery RA, Duke M, Elkins R, Dietz HC: Familial tetralogy of fallot caused by mutation in the Jagged1 gene. Hum Mol Genet 2001, 10:163-169. In this study an incompletely penetrant missense mutation in JAG1 is described in a large kindred in which affected individuals manifest cardiac disease and characteristic facial features that are distinct from those seen in typical AGS-affected individuals. 7.
Jones EA, Clement-Jones M, Wilson DI: Jagged1 expression in human embryos: correlation with the Alagille syndrome phenotype. J Med Genet 2000, 37:658-662.
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Crosnier C, Attie-Bitach T, Encha-Razavi F, Audollent S, Soudy F, Hadchouel M, Meunier-Rotival M, Vekemans M: Jagged1 gene expression during human embryogenesis elucidates the wide phenotypic spectrum of Alagille syndrome. Hepatology 2000, 32:574-581.
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Louis AA, Van Eyken P, Haber BA, Hicks C, Weinmaster G, Taub R, Rand EB: Hapatic Jagged1 expression studies. Hepatology 1999, 30:1269-1275.
10. Xue Y, Gao X, Lindsell CE, Norton CR, Chang B, Hicks C, GendronMaguire M, Rand EB, Weinmaster G, Gridley T: Embryonic lethality and vascular defects in mice lacking the Notch ligand Jagged1. Hum Mol Genet 1999, 8:723-730. 11. Houwen RH, Baharloo S, Blankenship K, Raeymaekers P, Juyn J, Sandkuijl LA, Freimer NB: Genome screening by searching for shared segments: mapping a gene for benign recurrent intrahepatic cholestasis. Nat Genet 1994, 8:380-386. 12. Carlton VE, Knisely AS, Freimer NB: Mapping of a locus for progressive familial intrahepatic cholestasis (Byler disease) to 18q21–q22, the benign recurrent intrahepatic cholestasis region. Hum Mol Genet 1995, 4:1049-1053. 13. Bull LN, van Eijk MJ, Pawlikowska L, DeYoung JA, Juijn JA, Liao M, Klomp LW, Lomri N, Berger R, Scharschmidt BF et al.: A gene encoding a P-type ATPase mutated in two forms of hereditary cholestasis. Nat Genet 1998, 18:219-224. 14. Bull LN, Juijn JA, Liao M, van Eijk MJ, Sinke RJ, Stricker NL, DeYoung JA, Carlton VE, Baharloo S, Klomp LW et al.: Fine-resolution mapping by haplotype evaluation: the examples of PFIC1 and BRIC. Hum Genet 1999, 104:241-248.
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15. Sinke RJ, Carlton VE, Juijn JA, Delhaas T, Bull L, van Berge Henegouwen GP, van Hattum J, Keller KM, Sinaasappel M, Bijleveld CM et al.: Benign recurrent intrahepatic cholestasis (BRIC): evidence of genetic heterogeneity and delimitation of the BRIC locus to a 7-cM interval between D18S69 and D18S64. Hum Genet 1997, 100:382-387. 16. Tang X, Halleck MS, Schlegel RA, Williamson P: A subfamily of P-type ATPases with aminophospholipid transporting activity [erratum Science 1996, 274:1597]. Science 1996, 272:1495-1497. 17.
Eiberg H, Nielsen IM: Linkage of cholestasis familiaris groenlandica/Byler-like disease to chromosome 18. Int J Circumpolar Health 2000, 59:57-62.
18. Klomp LW, Bull LN, Knisely AS, van Der Doelen MA, Juijn JA, Berger R, Forget S, Nielsen IM, Eiberg H, Houwen RH: A missense mutation in FIC1 is associated with greenland familial cholestasis. Hepatology 2000, 32:1337-1341. 19. Clayton RJ, Iber FL, Ruebner BH, McKusick VA: Byler disease. Fatal familial intrahepatic cholestasis in an Amish kindred. Am J Dis Child 1969, 117:112-124. 20. de Pagter AGF, van Berge Henegouwen GP, Huinink tB, Brandt KH: Familial benign recurrent intrahepatic cholestasis. Gastroenterology 1976, 71:202-207. 21. Ujhazy P, Ortiz D, Misra S, Li S, Moseley J, Jones H, Arias IM: Familial • intrahepatic cholestasis 1: Studies of localization and function. Hepatology 2001, 34:768-775. Expression of ATP8B1 in rat is shown to localize to the canalicular membrane of hepatocytes and within the apical membrane of small intestinal epithelium, with enrichment in the jejunum and ileum relative to the duodenum. This is also the first published effort to carry out functional studies of the familial intrahepatic cholestasis 1 (FIC1) protein encoded by ATP8B1, and suggests that FIC1 may have aminophospholipid flippase activity. 22. Harris MJ, Ujhazy P, Ortiz D, Arias IM: Functional analysis of the familial intrahepatic cholestasis protein type 1 (FIC1) [Abstract]. Hepatology 2001, 34:240. 23. Eppens EF, van Mil SWC, de Vree JM, Mok KS, Juijn JA, • Oude Elferink RP, Berger R, Houwen RH, Klomp LW: FIC1, the protein affected in two forms of hereditary cholestasis, is localized in the cholangiocyte and the canalicular membrane of the hepatocyte. J Hepatol 2001, 35:436-443. FIC1 is localized to the canalicular membrane of hepatocytes, and is also present in cholangiocytes, predominantly in the apical membrane. 24. Pawlikowska L, Ottenhoff R, Looije N, Eppens EF, Knisely AS, Bull LN, • Killeen NP, Oude Elferink RP, Freimer NB: FIC1 mutant mice have a defect in the regulation of intestinal bile salt absorption [Abstract]. Hepatology 2001, 34:240. Although as yet published only in abstract form, this study is intriguing because it reports the phenotype of Fic1 mutant mice and suggests that these mice have a defect in regulating intestinal uptake of bile acids, rather than in hepatic secretion of bile acids. 25. Strautnieks SS, Kagalwalla AF, Tanner MS, Gardiner RM, Thompson RJ: Locus heterogeneity in progressive familial intrahepatic cholestasis. J Med Genet 1996, 33:833-836. 26. Arnell H, Nemeth A, Anneren G, Dahl N: Progressive familial intrahepatic cholestasis (PFIC): evidence for genetic heterogeneity by exclusion of linkage to chromosome 18q21–q22. Hum Genet 1997, 100:378-381. 27.
Strautnieks SS, Kagalwalla AF, Tanner MS, Knisely AS, Bull L, Freimer N, Kocoshis SA, Gardiner RM, Thompson RJ: Identification of a locus for progressive familial intrahepatic cholestasis PFIC2 on chromosome 2q24. Am J Hum Genet 1997, 61:630-633.
28. Strautnieks SS, Bull LN, Knisely AS, Kocoshis SA, Dahl N, Arnell H, Sokal E, Dahan K, Childs S, Ling V et al.: A gene encoding a liver-specific ABC transporter is mutated in progressive familial intrahepatic cholestasis. Nat Genet 1998, 20:233-238. 29. Gerloff T, Stieger B, Hagenbuch B, Madon J, Landmann L, Roth J, Hofmann AF, Meier PJ: The sister of P-glycoprotein represents the canalicular bile salt export pump of mammalian liver. J Biol Chem 1998, 273:10046-10050. 30. Green RM, Hoda F, Ward KL: Molecular cloning and characterization of the murine bile salt export pump. Gene 2000, 241:117-123.
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31. Noe J, Hagenbuch B, Meier PJ, St-Pierre MV: Characterization of the • mouse bile salt export pump overexpressed in the baculovirus system. Hepatology 2001, 33:1223-1231. This functional study of murine bile salt export pump (BSEP) characterizes the ability of BSEP to transport different bile acids. The authors provide data supporting the ATP dependence of this activity and suggesting that bile acids may regulate BSEP through signaling mediated by protein kinase C. 32. Jansen PLM, Strautnieks SS, Jacquemin E, Hadchouel M, Sokal E, Hooiveld GJ, Koning JH, Jager-Krikken AD, Kuipers F, Stellaard F et al.: Hepatocanalicular bile salt export pump deficiency in patients with progressive familial intrahepatic cholestasis. Gastroenterology 1999, 117:1370-1379. 33. Cai SY, Wang L, Ballatori N, Boyer JL: Bile salt export pump is • highly conserved during vertebrate evolution and its expression is inhibited by PFIC type II mutations. Am J Physiol Gastrointest Liver Physiol 2001, 281:G316-G322. In this study, the ABCB11 ortholog present in the marine skate Raja erinacea is isolated and characterized functionally. When two mutations associated with ABCB11 deficiency are introduced separately into the skate gene, defective expression of the resulting protein is seen. 34. Kullak-Ublick GA, Kerb R, Mullhaupt B, Renner EI, Penger A, Brinkmann U, Stieger B, Meier PJ: A novel R432T mutation in the bile salt export pump gene (BSEP;ABCB11) is associated with recurrent intrahepatic cholestasis in an adolescent patient. Hepatology 2001, 34:216. 35. Wang R, Salem M, Yousefm IM, Tuchweber B, Lam P, Childs SJ, •• Helgason CD, Ackerley C, Phillips MJ, Ling V: Targeted inactivation of sister of P-glycoprotein gene (spgp) in mice results in nonprogressive but persistent intrahepatic cholestasis. Proc Natl Acad Sci USA 2001, 98:2011-2016. In this study, a knockout model of the murine ortholog of ABCB11 is reported and characterized. The homozygous mutant mice demonstrate mild nonprogressive cholestasis and decreased secretion of bile acids from the liver. Secretion of cholic acid is reduced to 6% of the wild-type value, but total bile salt output is 30% of the wild-type value. An unexpectedly large amount of tetrahydroxylated bile acids are secreted by the mutant animals and are not seen in wild type. These findings suggest that mice have a mechanism for hydroxylation and transport of bile acids out of the hepatocyte that partially compensates for the ABCB11 defect. 36. Smit JJ, Schinkel AH, Oude Elferink RP, Groen AK, Wagenaar E, van Deemter L, Mol CA, Ottenhoff R, van der Lugt NM, van Roon MA et al.: Homozygous disruption of the murine mdr2 P-glycoprotein gene leads to a complete absence of phospholipid from bile and to liver disease. Cell 1993, 75:451-462. 37.
Ruetz S, Gros P: Phosphatidylcholine translocase: a physiological role for the mdr2 gene. Cell 1994, 77:1071-1081.
38. Deleuze JF, Jacquemin E, Dubuisson C, Cresteil D, Dumont M, Erlinger S, Bernard O, Hadchouel M: Defect of multidrug-resistance 3 gene expression in a subtype of progressive familial intrahepatic cholestasis. Hepatology 1996, 23:904-908. 39. de Vree JM, Jacquemin E, Sturm E, Cresteil D, Bosma PJ, Aten J, Deleuze JF, Desrochers M, Burdelski M, Bernard O et al.: Mutations in the MDR3 gene cause progressive familial intrahepatic cholestasis. Proc Natl Acad Sci USA 1998, 95:282-287. 40. Jacquemin E, de Vree JM, Cresteil D, Sokal E, Sturm E, Dumont M, • Scheffer GL, Paul M, Burdelski M, Bosma PJ et al.: The wide spectrum of multidrug resistance 3 deficiency: from neonatal cholestasis to cirrhosis of adulthood. Gastroenterology 2001, 120:1448-1458. This study reports the results of ABCB4 mutation analysis, antibody studies of liver tissue and evaluation of biliary phospholipid levels in individuals with high-GGT PFIC. Three mothers heterozygous for mutations in ABCB4 experience ICP, and some individuals heterozygous or homozygous for ABCB4 mutations have biliary lithiasis. 41. Jacquemin E, Cresteil D, Manouvrier S, Boute O, Hadchouel M: Heterozygous non-sense mutation of the MDR3 gene in familial intrahepatic cholestasis of pregnancy. Lancet 1999, 353:210-211. 42. Dixon PH, Weerasekera N, Linton KJ, Donaldson O, Chambers J, Egginton E, Weaver J, Nelson-Piercy C, de Swiet M, Warnes G et al.: Heterozygous MDR3 missense mutation associated with intrahepatic cholestasis of pregnancy: evidence for a defect in protein trafficking. Hum Mol Genet 2000, 9:1209-1217. 43. Rosmorduc O, Hermelin B, Poupon R: MDR3 gene defect in adults • with symptomatic intrahepatic and gallbladder cholesterol cholelithiasis. Gastroenterology 2001, 120:1459-1467. Six people with a specific form of biliary gallstone disease are studied; all are shown to possess mutations in one or both of their copies of ABCB4.
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44. de Vree JML, Ottenhoff R, Bosma PJ, Smith AJ, Aten J, Oude Elferink RPJ: Correction of liver disease by hepatocyte transplantation in a mouse model of progressive familial intrahepatic cholestasis. Gastroenterology 2000, 119:1720-1730. 45. Aagenaes O, van der Hagen CB, Refsum S: Hereditary recurrent intrahepatic cholestasis from birth. Arch Dis Child 1968, 43:646-657. 46. Aagenaes O, Sigstad H, Bjorn-Hansen R: Lymphoedema in hereditary recurrent cholestasis from birth. Arch Dis Child 1970, 45:690-695. 47.
Aagenaes O: Hereditary cholestasis with lymphoedema (Aagenaes syndrome, cholestasis-lymphoedema syndrome). New cases and follow-up from infancy to adult age. Scand J Gastroenterol 1998, 33:335-345.
48. Bull LN, Roche E, Song EJ, Pedersen J, Knisely AS, van Der Hagen CB, Eiklid K, Aagenaes O, Freimer NB: Mapping of the locus for cholestasis-lymphedema syndrome (Aagenaes syndrome) to a 6.6-cM interval on chromosome 15q. Am J Hum Genet 2000, 67:994-999. 49. Drouin E, Russo P, Tuchweber B, Mitchell GA, Rasquin-Weber A: North American Indian cirrhosis in children: a review of 30 cases. J Pediatr Gastroenterol Nutr 2000, 31:395-404. 50. Betard C, Rasquin-Weber A, Brewer C, Drouin E, Clark S, Verner A, Darmond-Zwaig C, Fortin J, Mercier J, Chagnon P et al.: Localization of a recessive gene for North American Indian childhood cirrhosis to chromosome region 16q22 and identification of a shared haplotype. Am J Hum Genet 2000, 67:222-228. 51. Reyes H: Intrahepatic cholestasis: a puzzling disorder of pregnancy. J Gastrointerol Hepatol 1997, 12:211-216. 52. Lammert F, Marschall H-U, Glantz A, Matern S: Intrahepatic cholestasis of pregnancy: molecular pathogenesis, diagnosis and management. J Hepatol 2000, 33:1012-1021. 53. Heinonen ST, Eloranta ML, Heiskanen JTM, Punnonen KRA, • Helisalmi S, Mannermaa AJ, Hiltunen MJ: Maternal susceptibility locus for obstetric cholestasis maps to chromosome region 2p13 in Finnish patients. Scand J Gastroenterol 2001, 36:766-770. In this study, population-based linkage disequilibrium mapping is used to screen chromosome 2 for loci associated with ICP. The study was originally carried out to evaluate the hypothesis that variants in the HADHA gene might be associated with increased risk of ICP, as suggested by a previous report that mothers that are heterozygous for HADHA mutations have an increased risk of ICP [58]. No evidence of an association of ICP with HADHA is found; instead, evidence suggests the existence of an ICP
susceptibility locus elsewhere on chromosome 2. Follow-up studies will be necessary to confirm this association. 54. Heiskanen JTM, Pirskanen MM, Hiltunen MJ, Mannermaa AJ, Punnonen KRA, Heinonen ST: Insertion–deletion polymorphism in the gene for angiotensin-converting enzyme is associated with obstetric cholestasis but not with preeclampsia. Am J Obstet Gynecol 2001, 185:600-603. 55. Strautnieks S, Byrne J, Knisely AS, Bull LN, Sokal E, Lacaille F, • Vergani G, Thompson R: There must be a third locus for low GGT PFIC [Abstract]. Hepatology 2001, 34:240 Substantial evidence that supports the existence of at least one additional locus for low-GGT PFIC is reported. 56. Chen HL, Chang PS, Hsu HC, Lee JH, Ni YH, Hsu HY, Jeng YM, • Chang MH: Progressive familial intrahepatic cholestasis with high γ-glutamyl transpeptidase levels in Taiwanese infants: role of MDR3 gene defect? Pediatr Res 2001, 50:50-55. Data in this paper support the hypothesis that some cases of high-GGT PFIC may be due to mutation in a gene or genes other than ABCB4. 57.
Floreani A, Molaro M, Mottes M, Sangalli A, Baragiotta A, Roda A, Naccarato R, Clementi M: Autosomal dominant benign recurrent intrahepatic cholestasis (BRIC) unlinked to 18q21 and 2q24. Am J Med Genet 2000, 95:450-453.
58. Tyni T, Ekholm E, Pihko H: Pregnancy complications are frequent in long-chain 3-hydroxyacyl-coenzyme A dehydrogenase deficiency. Am J Obstet Gynecol 1998, 178:603-608. 59. McCright B, Lozier J, Gridley T: A mouse model of Alagille •• syndrome: Notch2 as a genetic modifier of Jag1 haploinsufficiency. Development 2002, 129:1075-1082. Mice doubly heterozygous for mutant Jag1 and Notch2 alleles are described: these mice seem to represent a promising animal model for AGS. 60. Chen HL, Chang PS, Hsu HC, Ni YH, Hsu HY, Lee JH, Jeng YM, Shau WY, Chang MH: FIC1 and BSEP defects in Taiwanese patients with chronic intrahepatic cholestasis with low γ-glutamyltranspeptidase levels. J Pediatr 2002, 140:119-124. 61. van Ooteghem NA, Klomp LW, van Berge-Henegouwen GP, Houwen RH: Benign recurrent intrahepatic cholestasis progressing to progressive familial intrahepatic cholestasis: low GGT cholestasis is a clinical continuum. J Hepatol 2002, 36:439-443. 62. Plass JR, Mol O, Heegsma J, Geuken M, Faber KN, Jansen PL, Muller M: Farnesoid X receptor and bile salts are involved in transcriptional regulation of the gene encoding the human bile salt export pump. Hepatology 2002, 35:589-596.