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[31] Inborn Errors of Biliary Canalicular Transport Systems By RALF KUBITZ, VERENA KEITEL , and DIETER Ha¨ussinger Abstract

Cholestatic syndromes are inborn or acquired disorders of bile formation. In recent years, several inherited cholestatic syndromes were characterized at the molecular level: progressive familial intrahepatic cholestasis (PFIC) and benign recurrent intrahepatic cholestasis (BRIC). Both PFIC and BRIC were divided phenotypically in distinct subtypes; however, at the genotype level, these clinical entities overlap. PFIC starts in early childhood and progresses toward liver cirrhosis, which often requires liver transplantation within the first decade of life. The diagnosis of PFIC is usually made on the basis of clinical and laboratory findings but needs to be confirmed by genetic and histological analysis. Only recently was it recognized that BRIC, which was estimated as a milder form of PFIC‐1, may be caused by more than one gene. Disorders of Hepatobiliary Transport: Cholestatic Syndromes

Cholestatic disorders are characterized by deficient bile formation, which resides in either a disturbance of hepatocellular secretion of cholephilic compounds (intrahepatic or hepatocellular cholestasis) or a mechanical obstruction of bile ducts (obstructive cholestasis). Cholestasis most often is acquired during life but some forms of cholestasis are inherited. The identification of hepatobiliary transporter proteins provided the basis for the molecular understanding of inborn hepatocellular cholestatic syndromes (Fig. 1). Progressive Familial Intrahepatic Cholestasis

Clinically, three types of progressive familial intrahepatic cholestasis are distinguished and their underlying molecular defects identified in recent years. In 1969, Clayton and colleagues described a cholestatic syndrome affecting several siblings of an Amish family, which was named Byler’s disease according to the family’s name. This inherited form of cholestasis was later termed progressive familial intrahepatic cholestasis (PFIC). The underlying gene defect of Byler’s disease, or PFIC type 1 METHODS IN ENZYMOLOGY, VOL. 400 Copyright 2005, Elsevier Inc. All rights reserved.

0076-6879/05 $35.00 DOI: 10.1016/S0076-6879(05)00031-5

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FIG. 1. Expression of hepatobiliary transporters in human liver parenchymal cells. Hepatobiliary transporters are expressed differentially at the sinusoidal (basolateral) or canalicular (apical) membrane. Mutations of certain transporters are associated with distinct hereditary diseases (common names beneath transporter abbreviations). NTCP, Naþ‐ taurocholate cotransporting protein; OAT, organic anion transporter; OCT, organic cation transporter; OATP, organic anion transporting polypeptide; BSEP, bile salt export pump; FIC, familial intrahepatic cholestasis; BRIC, benign recurrent intrahepatic cholestasis; PFIC, progressive familial intrahepatic cholestasis; MRP, multidrug resistance associated protein; MDR, multidrug resistance; ICP, intrahepatic cholestasis of pregnancy; LPAC, low phospholipid associated cholelithiasis; ABCG, ABC‐transporter subfamily G.

(PFIC‐1), involves a defect of the familial intrahepatic cholestasis1 gene (FIC1/ATP8B1). FIC1, a P‐type ATPase, is expressed in liver parenchymal cells and more strongly in enterocytes and may play a role in the enterohepatic circulation of bile acids (Bull et al., 1998). It probably functions as an aminophospholipid translocase (Ujhazy et al., 2001). PFIC‐1 is associated with elevated bile salt concentrations in serum, normal ‐GT serum activities, decreased bile acid secretion, pruritus, jaundice, and diarrhea. Impaired bile secretion causes fat malabsorption and a deficiency of lipophilic vitamins. Therapeutic options for affected patients include partial external biliary diversion (Kurbegov et al., 2003), administration of rifampicin (van Ooteghem et al., 2002), which only seems to be effective in some PFIC‐1 patients, and the HMG‐CoA reductase inhibitor simvastatin, which is applied in order to reduce bile acid synthesis from its precursor cholesterol (van Mil et al., 2001). FIC1 mutations with milder functional defects

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are the molecular basis for ‘‘benign recurrent intrahepatic cholestasis’’ (BRIC), which presents with recurrent cholestatic/icteric attacks but without progression toward liver cirrhosis (Bull et al., 1997, 1998). Greenland familial cholestasis (GFC) is also caused by mutations of FIC1, but represents a more severe form of FIC1 disease (Klomp et al., 2000). The reason for elevated bile acids during attacks of BRIC or in PFIC‐1 and GFC is not fully elucidated yet. However, a decrease in expression of the farnesoid‐X‐ receptor (FXR) (Makishima et al., 1999; Parks et al., 1999), the principal regulator of bile acid homeostasis, which counteracts elevated bile acid levels, has been demonstrated in PFIC‐1 patients (Alvarez et al., 2004; Chen et al., 2004). It has been shown that decreased FXR levels lead to increased bile acid reabsorption in the small intestine and reduced bile acid secretion into bile by inverse regulation of the apical sodium‐dependent bile acid transporter (ASBT) in enterocytes and of the bile salt export pump (BSEP) in hepatocytes. An increase in hydrophobic, that is, more toxic bile acid, is associated with hepatocellular injury at least in part due to bile acid‐dependent apoptosis (Higuchi and Gores, 2003). There are at least two further phenotypes of PFIC. The defect in PFIC‐2 was mapped to the gene of the bile salt export pump BSEP (SPGP, ABCB11) (Jansen et al., 1999; Strautnieks et al., 1998; Thompson and Strautnieks, 2001) located to chromosome 2q24-31 (Strautnieks et al., 1997). BSEP is the major canalicular bile salt transporter that transports conjugated bile salts from the hepatocytes into the bile (Gerloff et al., 1998). It has been documented that some patients with recurrent intrahepatic cholestasis of the BRIC phenotype do not have a FIC1 mutation but also carry a BSEP mutation (van Mil et al., 2004), which was consequently named BRIC‐2. Interestingly, almost two‐thirds of the patients investigated in that study suffered from gallstones (van Mil et al., 2004), which is unusual for ‘‘BRIC‐1’’ patients with a FIC1 mutation. Because bile acid excretion is diminished in PFIC‐2 (and PFIC‐1), bile canaliculi and cholangiocytes experience less contact with toxic bile constituents and are therefore less prone to damage, which may explain the normal serum ‐GT activities in both PFIC‐1 and PFIC‐2. Indeed, elevations of serum ‐GT activities are so far considered a hallmark of PFIC‐3, but not of PFIC‐1 and ‐2. PFIC‐3 is due to a defect of the MDR3 gene product (Deleuze et al., 1996; De Vree et al., 1998; Jacquemin, 2001), which normally brings about phospholipid secretion into bile. Phospholipids are required in bile in order to protect the canaliculi and cholangiocytes from the toxic effects of high bile salt concentrations, which are achieved by mixed micelle formation (Oude Elferink et al., 1997). Liver damage starts later in PFIC‐3 patients, but is more pronounced. Due to the damage of cholangiocytes, strong bile duct proliferation can be detected

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histopathologically. Together with jaundice and pruritus, PFIC‐3 patients develop hepatosplenomegaly and portal hypertension. An important therapy for PFIC‐3 is the administration of ursodeoxycholic acid (UDCA), but it has been recognized that only patients with some remaining MDR3 activity benefit from UDCA (Jacquemin et al., 1997; Jansen et al., 1999). The protective effect of UDCA is explained by its inhibitory effect on hepatocyte apoptosis, which is otherwise triggered by an intrahepatocellular accumulation of hydrophobic bile acids (Faubion and Gores, 1999; Graf et al., 2002; Higuchi and Gores, 2003). All forms of PFIC are inherited in an autosomal recessive way. Other Clinical Manifestations of MDR3 Mutations

Mutations of MDR3 not only cause PFIC‐3, but are also found in about half of the cases of intrahepatic cholestasis of pregnancy (ICP) (Jacquemin, 2001; Jacquemin et al., 1999). ICP is a reversible form of cholestasis that develops in late pregnancy, characterized by jaundice, elevated serum bile acids, and pruritus, while other causes of cholestasis are absent (such as obstruction of the biliary tree). Even though ICP resolves after delivery and is not detrimental to the mother, it is associated with an increased incidence of fetal complications (Lammert et al., 2000). Some mutations of MDR3 predispose for a rare condition of gallstone susceptibility called ‘‘low phospholipid associated cholelithiasis’’ (LPAC) syndrome (Rosmorduc et al., 2001). LPAC characteristically includes two or three of the following features: (1) it occurs in patients below an age of 40 years, (2) the patients experience recurrence of gallstone disease after cholecystectomy, and (3) intrahepatic sludge or stones are found on abdominal ultrasonography (Rosmorduc et al., 2003). Furthermore, a positive family history of gallstone disease is frequently observed. Dubin– Johnson Syndrome

The Dubin–Johnson syndrome is an inherited form of conjugated hyperbilirubinemia (Dubin and Johnson, 1954). It is caused by mutations of canalicular multidrug resistance associated protein 2 (MRP2/cMRP/ cMOAT/ABCC2) (Kartenbeck et al., 1996; Keitel et al., 2000, 2002; Shoda et al., 2003; Suzuki and Sugiyama, 2002). Livers of Dubin–Johnson syndrome patients contain a black pigment within the lysosomes. Affected patients present with chronic jaundice, but have a normal life expectancy and the Dubin–Johnson syndrome has a benign clinical course. The minor impairment of overall liver function may be due to the compensatory upregulation of MRP3 (ABCC3) at the lateral membrane of liver cells of

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Dubin–Johnson syndrome patients (Ko¨ nig et al., 1999), which may mediate reflux of conjugated bilirubin (and other MRP2 substrates) into the blood (Oude Elferink and Groen, 2002). Sitosterolemia

Plant sterols are almost completely excluded from the body due to efficient enteric excretion. In the rare inherited disease called sitosterolemia, mutations of either one of the half‐transporters ABCG5 (Lee et al., 2001) or ABCG8 (Berge et al., 2000) lead to accumulation of the plant sterol sitosterol (24‐ethyl‐cholesterol) due to the impaired secretion of sitosterol (and cholesterol) from the enterocytes back into the gut lumen and from the liver into the bile (Graf et al., 2003). Clinically, sitosterolemia is characterized by atherosclerosis at a young age, tendon xanthomas, hemolytic episodes, and arthralgias (Salen et al., 1992). Mutations of ABCG5/ABCG8, which represent the major hepatic transporter for cholesterol secretion, also affect cholesterol secretion: however, an increase of cholesterol is partly antagonized by a reduction of HMG‐Co reductase activity (Salen et al., 1992). Genetic Abnormalities in Sinusoidal Transporter Proteins

So far no distinct inherited diseases are attributed to defects of sinusoidal transport systems in humans. However, some nonsynonymous single nucleotide polymorphisms (SNP), affecting single amino acids in transporter proteins from this membrane, have been identified. For example, Ho and co‐workers (2004) identified five SNPs in the human sodium‐ dependent taurocholate cotransporting polypeptide (NTCP), the major uptake system for bile acids into hepatocytes. One of these SNPs was associated with a defect in maturation and/or targeting of NTCP (Ho et al., 2004). In addition to NTCP, members of the organic anion transporting polypeptide (OATP) superfamily (Hagenbuch and Meier, 2003) mediate bile acid uptake in addition to many other organic anions. For OATP1B1 (OATP‐C/OATP2/LST‐1), SNPs were correlated with alterations in the transport of estrogen derivatives (Tirona et al., 2001). Another SNP of OATP1B1 was identified, which was associated with a defect in maturation and intracellular retention of OATP1B1 (Michalski et al., 2002). Similarly, several nonsynonymous mutations were identified within OATP1B3 (OATP8), resulting in alterations in targeting and/or transport of OATP1B3 (Letschert et al., 2004). For human MRP3, a member of the ABCC subfamily of ABC transporters, a frequently nonsynonymous SNP, was identified close to the second nucleotide‐binding domain, but without

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functional consequences (Lee et al., 2004). Interestingly, another study reported about polymorphisms of the promoter region of human MRP3, which were associated with decreased MRP3 mRNA expression (Lang et al., 2004). To what extent such SNPs predispose to diseases is currently unknown. Diagnostic Procedures for Progressive Familial Intrahepatic Cholestasis

The diagnosis of PFIC can be suspected on the basis of clinical and laboratory findings (see earlier discussion). PFIC‐1 and PFIC‐2 may be clinically discriminated by the manifestation of extrahepatic symptoms, such as diarrhea or pancreatic insufficiency, which occur in PFIC‐1 but not in PFIC‐2. PFIC‐3 is differentiated more easily from PFIC‐1/2 due to high GT activities; however, some patients are borderline in terms of their GT activities, that is, PFIC‐2 patients can exhibit slightly elevated

GT activities and vice versa, whereas some PFIC‐3 patients have normal

GT values (Keitel et al., 2005). Therefore, accurate diagnosis must rely on criteria distinct from the clinical phenotype, as prognosis and treatment differ between PFIC subtypes. One approach to diagnose PFIC subtypes includes identification of mutations at the genome level. Mutations may be detected by cDNA sequencing when (liver) tissue is available for reverse transcription of the mRNA of the respective gene. Normally, a biopsy cylinder of 1 cm length derived from the standard liver puncture procedure yields enough material for cDNA sequencing. If mRNA is not available, mutation analysis can be performed using genomic DNA. The polymerase chain reaction (PCR)‐based technique of single strand conformation polymorphism allows searching for single nucleotide exchanges within short stretches of DNA in the range of 150 to 250 bp. In order to identify new mutations of a gene, multiple PCR fragments of this size are produced from genomic DNA by the use of specific primers covering the entire coding regions and the adjacent exon/intron boundaries. The PCR fragments are separated by agarose gel electrophoresis at different temperatures. Single nucleotide exchanges within the PCR product result in differences of electrophoretic mobility compared to fragments of the wild‐type gene and give rise to different bands in the agarose gels. These fragments can then be analyzed by sequencing. Direct sequencing of the entire gene is more time‐consuming and expensive. When more disease‐causing mutations (DCM) are identified, a more rapid screen for such DCMs in individual patients will be possible using a plate reader or high‐throughput technologies. Techniques that can be

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applied include hybridization‐based methods (such as TaqMan technology or melting curve analysis) or pyro‐sequencing. Depending on the type of polymorphism/mutation detected, the relevance of the polymorphism/mutation for the disease has to be established. While homozygous deletion or frameshift mutations are correlated easily, it is more difficult with nonsense mutations. Here, direct proof of the

FIG. 2. Immunofluorescence of livers from patients with PFIC. Livers from one patient with PFIC‐2 (A, D) and two patients with PFIC‐3 (B and E; C and F) were examined by immunofluorescence and confocal laser‐scanning microscopy. In the upper row, the bile salt export pump BSEP (red), which is mutated in PFIC‐2, was counterstained by the multidrug resistance protein 2 (MRP2, green). In PFIC‐2 patients, no canalicular BSEP staining was detectable. In contrast, some patients have MDR3 mutations (B, E; middle), which display a normal canalicular MDR3‐staining (MDR3 in red; MRP2 in green). Other MDR3 mutations (such as patient of C and F) have absent canalicular MDR3 (right).

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functional consequence, for example, by insertion of the mutation into a cell system, is often necessary. Possible effects of a mutation are decreased mRNA stability due to nonsense‐mediated decay (Thermann et al., 1998) and defects in protein maturation and targeting (Dixon et al., 2000; Keitel et al., 2000, 2002; Plass et al., 2004; Wang et al., 2002). Other mutations may only affect transporter activity (Hashimoto et al., 2002; Keitel et al., 2002; Mor‐Cohen et al., 2001), but some may also be irrelevant. cDNA and gene sequencing studies need to be complemented by either functional assays or immunohistochemistry in order to assess transporter localization and protein expression levels. For example, PFIC‐2 can be diagnosed by the absence of BSEP immunoreactivity from the canaliculi (Fig. 2) when BSEP‐specific antibodies are used. Thus, BSEP staining in livers of PFIC‐2 patients might be a reliable tool for diagnosis (Thompson and Strautnieks, 2001), as shown in a study on five PFIC‐2 patients (Keitel et al., 2005). Liver immunofluorescence studies may especially be important in cases with borderline GT activities in order to differentiate PFIC‐2 from PFIC‐3. While all BSEP mutations identified so far were associated with a clear targeting defect in human liver, several MDR3 mutations were identified that display normal apical protein targeting (see examples in Fig. 2) (Jacquemin, 2001; Jacquemin et al., 2001). So far, these mutations have not been assessed for their functional impact. Perspectives

During the last decades many transporter proteins were cloned and were characterized at the molecular level. With the help of current technologies, many transporter polymorphisms will be discovered— polymorphisms that account for monogenetic diseases such as PFIC, but also polymorphisms that predispose for or protect from the development of illnesses. Because hepatobiliary transporters are major determinants of pharmocokinetics, individual drug tolerance and drug response may find their explanation in transporter SNPs. However, genetic as well as morphological and functional studies will be necessary to answer upcoming questions. References Alvarez, L., Jara, P., Sanchez‐Sabate, E., Hierro, L., Larrauri, J., Diaz, M. C., Camarena, C., De, L. V., Frauca, E., Lopez‐Collazo, E., and Lapunzina, P. (2004). Reduced hepatic expression of Farnesoid X Receptor in hereditary cholestasis associated to mutation in ATP8B1. Hum. Mol. Genet. 13(20), 2451–2460.

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[32] Epoxide Hydrolases: Structure, Function, Mechanism, and Assay By MICHAEL ARAND, ANNETTE CRONIN, MAGDALENA ADAMSKA , and FRANZ OESCH Abstract

Epoxide hydrolases are a class of enzymes important in the detoxification of genotoxic compounds, as well as in the control of physiological signaling molecules. This chapter gives an overview on the function, structure, and enzymatic mechanism of structurally characterized epoxide METHODS IN ENZYMOLOGY, VOL. 400 Copyright 2005, Elsevier Inc. All rights reserved.

0076-6879/05 $35.00 DOI: 10.1016/S0076-6879(05)00032-7