Inherited Disorders of Bilirubin Transport and Conjugation: New Insights Into Molecular Mechanisms and Consequences

REVIEWS IN BASIC AND CLINICAL GASTROENTEROLOGY AND HEPATOLOGY Robert F. Schwabe and John W. Wiley, Section Editors

Inherited Disorders of Bilirubin Transport and Conjugation: New Insights Into Molecular Mechanisms and Consequences Serge Erlinger,1 Irwin M. Arias,2 and Daniel Dhumeaux3 1 University of Paris 7, Paris, France; 2National Institutes of Health, Bethesda, Maryland; and 3Henri Mondor Hospital, Créteil, University of Paris-Est, Créteil, France

Inherited disorders of bilirubin metabolism might reduce bilirubin uptake by hepatocytes, bilirubin conjugation, or secretion of bilirubin into bile. Reductions in uptake could increase levels of unconjugated or conjugated bilirubin (Rotor syndrome). Defects in bilirubin conjugation could increase levels of unconjugated bilirubin; the effects can be benign and frequent (Gilbert syndrome) or rare but severe, increasing the risk of bilirubin encephalopathy (Crigler–Najjar syndrome). Impairment of bilirubin secretion leads to accumulation of conjugated bilirubin (Dubin–Johnson syndrome). We review the genetic causes and pathophysiology of disorders of bilirubin transport and conjugation as well as clinical and therapeutic aspects. We also discuss the possible mechanisms by which hyperbilirubinemia protects against cardiovascular disease and the metabolic syndrome and the effects of specific genetic variants on drug metabolism and cancer development. Keywords: Crigler–Najjar Syndrome; Hepatic Storage Disease; Glucuronosyl Transferase; Bile Secretion; Kernicterus.

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ereditary hyperbilirubinemias range from benign to lethal, and all are caused by defective bilirubin transport or conjugation by the liver. Bilirubin is the end product of heme catabolism. It belongs to the superfamily of tetrapyrrolic compounds, one of the most highly conserved groups of molecules in living organisms. Bilirubin is poorly water soluble. In blood, it circulates bound to serum albumin, presumably to prevent the toxicity of free (unbound) bilirubin. Unbound bilirubin is rapidly and selectively taken up by hepatocytes and then conjugated to glucuronic acid into bilirubin glucuronides by uridine diphosphate (UDP)glucuronosyl transferase before being secreted into bile through the hepatocyte canalicular membrane via an adenosine triphosphate (ATP)-dependent transporter. In clinical practice, hereditary hyperbilirubinemias can be separated into predominantly unconjugated and predominantly conjugated forms. These conditions result from mutations of transporters or enzymes involved in the hepatic bilirubin elimination pathway. The aim of this review is to describe these inherited disorders, with a particular focus on their molecular mechanisms. Studies of bilirubin metabolism have broader implications, showing the beneficial effects of moderate hyperbilirubinemia (due to the antioxidant

properties of bilirubin) and other consequences of mutations on drug metabolism and cancer susceptibility. We will not discuss hereditary hemolytic unconjugated hyperbilirubinemia, which is caused by bilirubin overproduction, or several genetically mediated cholestatic diseases such as progressive familial intrahepatic cholestasis or Alagille syndrome, which can also lead to hyperbilirubinemia.

Bilirubin Transport and Conjugation by the Liver Uptake by Hepatocytes: Passive Diffusion or Active Transport? Unconjugated bilirubin is lipid soluble and should thus readily cross biological membranes. However, passive diffusion alone would not account for the remarkable specificity of hepatic uptake. One possible explanation for this specificity is the presence in hepatocytes of cytoplasmic proteins with a higher affinity than albumin for bilirubin. One such protein was identified and characterized in the late 1960s and early 1970s by Arias et al and was named Y protein or ligandin.1 Kinetic studies suggested that bilirubin binding to this protein was not involved in initial cellular uptake but rather reduced bilirubin efflux from the cytosol back into the space of Disse, thus resulting in intrahepatocytic bilirubin accumulation. More recent studies have attempted to identify bilirubin transport proteins in the hepatocyte basolateral membrane, particularly among the organic anion transport proteins (OATPs). They belong to the OATP superfamily, which is also called the solute carrier organic anion transporter (SLCO) superfamily.2 The human SLCO superfamily comprises 11 members grouped

Abbreviations used in this paper: ATP, adenosine triphosphate; BSP, bromosulphophthalein; CN, Crigler–Najjar; DJS, Dubin–Johnson syndrome; GS, Gilbert syndrome; ICG, indocyanine green; MRP2, multidrug related protein 2; OATP, organic anion transport protein; OMIM, Online Mendelian Inheritance in Man; RS, Rotor syndrome; SLCO, solute carrier organic anion transporter; UDP, uridine diphosphate; UGT1A1, uridine diphosphate glucuronosyl transferase 1A1. © 2014 by the AGA Institute 0016-5085/$36.00 http://dx.doi.org/10.1053/j.gastro.2014.03.047

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into 6 families encoded by SLCO genes. Bilirubin is a substrate for OATP1B1 (Online Mendelian Inheritance in Man [OMIM]*604843) and OATP1B3 (OMIM*605495).2 Human OATP1B1 and OATP1B3 can transport conjugated and, possibly, unconjugated bilirubin in vitro.3,4 Studies in humans, and particularly genome-wide association studies, suggest that polymorphisms that reduce OATP1B1 or OATP1B3 activity are associated with higher serum levels of both conjugated and unconjugated bilirubin.5,6 Oatp1a and Oatp1b knockout mice lacking the Oatp1a and 1b transporters have serum bilirubin levels more than 40 times higher than those of their wild-type counterparts.7 Serum bilirubin in these mice is mostly conjugated, probably (see the following text) because of defective reuptake of bilirubin glucuronide.8 However, serum levels of unconjugated bilirubin are 2-fold higher in Oatp1a/1b knockout mice than in their wild-type counterparts.7 This suggests that Oatp1a/1b proteins may contribute to unconjugated bilirubin uptake by hepatocytes. Human embryonic kidney cells (HEK293) permanently expressing recombinant OATP1B1 (formerly OATP2) showed uptake of [3H]monoglucuronosyl bilirubin, [3H]bisglucuronosyl bilirubin, and [3H]sulfobromophthalein, with Km values of 0.10, 0.28, and 0.14 mmol/L, respectively.3 However, this observation could not be reproduced with unconjugated bilirubin in HeLa or HEK293 cells transfected with OATP1B1.9 Further studies are thus needed to clarify the respective roles of passive diffusion and carriermediated transport in unconjugated bilirubin uptake by hepatocytes.

Conjugation After its uptake and binding to ligandin, bilirubin is transferred to the smooth endoplasmic reticulum, where it is conjugated into bilirubin glucuronides by UDP–glucuronosyl transferase 1A1 (UGT1A1). The process of bilirubin conjugation and the function of UDP–glucuronosyl transferases have been extensively reviewed recently.10–12 UGT1A1 (OMIM*191740) (Figure 1) appears to be the only enzyme that glucuronidates bilirubin. Indeed, mutations that completely suppress UGT1A1 activity result in a total absence of bilirubin glucuronides.11 UGT1A1 is a transmembrane protein located mainly on the smooth endoplasmic reticulum. It has a binding site for bilirubin and another one for glucuronic acid, with both sites located on the luminal face of the endoplasmic reticulum membrane.11 Glucuronic acid is derived from uridine diphospho-glucuronic acid, which itself is derived

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from UDP glucose. The location of the binding sites implies that both bilirubin and glucuronic acid are transported from the cytosol into the lumen of the endoplasmic reticulum. UGT1A1 catalyzes the conversion of bilirubin to bilirubin monoglucuronide and then to bilirubin diglucuronide. Bilirubin glucuronides then move to the cytoplasm, probably through a specific endoplasmic reticulum membrane transporter (Figure 2, inset), where they bind to ligandin, albeit with far lower affinity than unconjugated bilirubin.

Secretion Into Bile and Plasma Once back in the cytosol, bilirubin diglucuronide can diffuse toward either the canalicular pole or the sinusoidal pole of the hepatocyte. At the canalicular pole, it is efficiently secreted into bile, mostly by the ATP-dependent MRP2/ABCC2 transporter.13,14 This protein mediates the canalicular secretion of several organic anions, including bilirubin glucuronides, dyes such as sulfobromophthalein (BSP) and indocyanine green (ICG), divalent bile salts, and reduced glutathione.13,14 Other transporters, particularly Abcg2,15 may be involved in bilirubin secretion across the canalicular membrane. Interestingly, a substantial fraction of bilirubin glucuronide is rerouted to the sinusoidal pole and secreted back into plasma by another transporter, Abcc3.8 From there, it can be taken up again by hepatocytes via Oatp1b1/3.8 It has been proposed that this reuptake process may take place in downstream hepatocytes (hepatocytes located near the central vein) to prevent saturation of the biliary secretory capacity of upstream hepatocytes (hepatocytes located near the portal tracts).8 A schematic representation of bilirubin transport and conjugation by hepatocytes is provided in Figure 2.

Hereditary Hyperbilirubinemias Hereditary hyperbilirubinemias may be caused by increased bilirubin production, mostly as a result of hyperhemolysis, or decreased bilirubin clearance. This review will be limited to conditions associated with decreased bilirubin clearance. Decreased bilirubin clearance may be caused by (1) defective bilirubin uptake by hepatocytes, leading to unconjugated hyperbilirubinemia; (2) defective conjugated bilirubin reuptake, such as in Rotor syndrome (RS) (also known as hepatic uptake and storage disease; see the following text); (3) defective bilirubin conjugation, such as in Gilbert syndrome (GS), Crigler–Najjar (CN) syndrome, neonatal transient familial hyperbilirubinemia, and breast

Figure 1. Schematic representation of the UGT1A1 locus and UGT1A1 protein.

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Figure 2. Schematic view of bilirubin transport by the hepatocyte. (Inset) Localization of UGT1A1 in the reticulum endoplasmic membrane and transport of uridine diphosphoglucuronic acid (UDPGA), bilirubin, and bilirubin glucuronide across the endoplasmic reticulum membrane. UCB, unconjugated bilirubin; Alb, albumin; BG, bilirubin glucuronide.

milk jaundice; or (4) defective bilirubin canalicular secretion, such as in Dubin–Johnson syndrome (DJS).

Defective Uptake One might expect defective bilirubin uptake to result systematically in unconjugated hyperbilirubinemia. Surprisingly, however, only a few cases of unconjugated hyperbilirubinemia have been attributed to defective uptake. Most defects of bilirubin uptake result in predominantly conjugated hyperbilirubinemia (RS). The reason for this was recently identified, as explained in the following text.

Unconjugated hyperbilirubinemia associated with an uptake defect? Rare cases of unconjugated hyperbilirubinemia have been reported in which plasma clearance of cholephilic dyes such as BSP and ICG was markedly impaired,16–18 suggesting a role of impaired hepatic bilirubin uptake. Among the 39 cases of unconjugated hyperbilirubinemia that we investigated, 3 patients had a significant reduction in the BSP plasma disappearance rate. Interestingly, the patients were 3 brothers (suggesting familial transmission), and liver UGT1A1 activity measured in one of these patients was normal, ruling out GS (if defined by defective conjugation).17 Genetic and molecular analyses suggest that such cases of unconjugated hyperbilirubinemia could be linked to polymorphisms in SLCO1B1 and SCLO1B3.5,6,8,19 However, given the modest role of OATPs in bilirubin uptake,8 the role of these polymorphisms remains to be confirmed.

Although this type of unconjugated hyperbilirubinemia is probably rare, its prevalence may nonetheless have been underestimated; in the absence of BSP plasma kinetics and liver UGT1A1 activity measurement, many cases may have been erroneously classified as GS. Indeed, although glucose6-phosphate dehydrogenase deficiency and other causes of unconjugated hyperbilirubinemia could not be ruled out, Skierka et al recently showed that 39% of their patients with unconjugated hyperbilirubinemia did not have identifiable UGT1A1 variants that explained the disorder.20

Rotor Syndrome: Conjugated Hyperbilirubinemia Due to Defective Reuptake? History and presentation. The disorder first described by Rotor et al in 1948 is a rare and benign disease (OMIM*237450) characterized by low-grade (40–100 mmol/L), chronic or fluctuating, predominantly conjugated hyperbilirubinemia.21 It is a familial disorder with autosomal recessive transmission.22 Except for jaundice when apparent, there are no symptoms and clinical findings are normal. In cases with apparent jaundice, RS is usually detected shortly after birth or during childhood. Other cases are discovered fortuitously or after diagnosis of another family member. Apart from the predominantly conjugated hyperbilirubinemia, liver test results are normal. Urinary excretion of coproporphyrins is markedly elevated because of increased urinary excretion of isomer I and, to a lesser extent, isomer III,22 following a shift from the biliary to the urinary route of coproporphirin excretion. Liver biopsy is not required. When performed, it is normal and shows no abnormal pigment deposits,

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contrary to DJS.23 The prognosis is excellent, and no treatment is necessary. Because of the presence of conjugated bilirubin in blood, RS was initially attributed to defective biliary excretion and was considered a variant of DJS. In 1975, one of us reported a case of hereditary, benign, predominantly conjugated hyperbilirubinemia associated with very slow plasma clearance of BSP and other cholephilic dyes such as dibromosulphthalein and ICG.24 The hepatic relative storage capacity of BSP and dibromosulphthalein was markedly impaired, whereas the biliary transport maximum was only slightly affected.24 After this description (hepatic uptake and storage disease; OMIM*237550), several groups, including ours, reexamined patients with RS and found that they all had defective hepatic uptake and storage, mainly characterized by decreased BSP clearance and storage capacity.25–27 From that time onward, hepatic uptake and storage disease and RS were considered to be the same entity.23 Impressive progress has since been made in our understanding of hepatic transporters and the pathophysiology of hereditary hyperbilirubinemias. In the absence of mutations in ABCC2 (the canalicular export pump for bilirubin glucuronide and other organic anions), it has been confirmed that RS is not caused by defective biliary excretion.28 However, despite these advances, it remained unclear why serum bilirubin is predominantly conjugated in patients with RS. Genetic and molecular basis. The recent work of van de Steeg et al probably solves this mystery.8 These investigators generated knockout mice in which the Slco1a/b genes (encoding the sinusoidal transporters Oatp1a/b, functionally close to human OATP1B1/3) were inactivated. They showed that (1) these knockout mice exhibited marked conjugated hyperbilirubinemia and (2) sinusoidal Oatps in the normal mouse function in “tandem” with the sinusoidal efflux transporter Abcc3 to successively mediate hepatic efflux (by Abcc3) and reuptake (by Oatps) of bilirubin glucuronides. They also found that transgenic expression of human OATP1B1 or OATP1B3 restored the liver-blood shuttle in Oatp1a/1b-deficient mice, indicating that, in humans, both OATP1B1 and OATP1B3 effectively reuptake bilirubin glucuronide from plasma to liver, in line with their bilirubin glucuronide uptake capacity in vitro.7 The same investigators scanned the whole genome and mapped candidate gene intervals in 11 patients with RS from 8 different families. Homozygosity mapping identified a single genomic region on chromosome 12 for which 8 of the tested index patients and none of the healthy siblings were homozygous, suggesting inheritance of both mutated alleles from a common ancestor. Sequence analysis revealed predictably pathogenic mutations affecting both SLCO1B3 and SLCO1B1 in each of the tested subjects. Autosomal recessive segregation was found in all the investigated RS families. The severity of the mutations was supported by the near absence of OATP1B immunostaining in liver biopsy specimens. Although these experimental results need to be confirmed, mouse experiments and studies of patients with RS strongly suggest that homozygous mutations of the human OATP genes SLCO1B1 and SLCO1B3 on chromosome 2, inducing complete OATP1B1 and OATP1B3 deficiency,

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disrupt hepatic reuptake of bilirubin conjugates and lead to accumulation of conjugated bilirubin in blood, thus causing RS. OATP1B1/3 deficiency also explains the marked impairment of BSP uptake and storage capacity in patients with RS (BSP is a substrate of these transporters5), as well as their increased urinary coproporphyrin excretion, in line with the interaction of several porphyrins with OATP1B1.29 Finally, the obligatory deficiency in 2 different genes explains the rarity of RS, which has an estimated frequency of about 1 in 106 overall, although it may be several times lower or higher in specific populations.8 Drug interactions. OATP1B1 plays a major role in drug detoxification. Indeed, reduced-activity OATP1B1 polymorphisms have been shown to reduce drug transport and increase plasma and tissue concentrations of drugs such as anticancer agents, methotrexate, and statins, potentially resulting in toxicity.30–33 Even if no drug accumulation or toxicity has so far been reported in patients with RS, it is advisable to take these findings into account, especially in patients with RS who have jaundice. Some drugs, such as high-dose cyclosporin A and antiviral agents such as alisporivir, can increase the plasma concentration of conjugated bilirubin, with no other evidence of liver damage. Until now, this was believed to be mediated primarily by inhibition of the canalicular transporter ABCC2. However, cyclosporin A and other drugs also inhibit OATP1B,7 and this might be an additional cause of drug-induced conjugated hyperbilirubinemias. Animal model. The hepatic transport abnormalities seen in RS resemble those of mutant Southdown sheep.34 Indeed, like patients with RS, these sheep are characterized by chronic hyperbilirubinemia, impaired plasma clearance of BSP and ICG, and a reduction in the BSP hepatic relative storage capacity with a subsequent reduction in transport maximum.34 Serum bilirubin is predominantly unconjugated in this model, originally suggesting defective uptake. The Oatp1b1/3 functional status of these animals has not been tested.

Defective Conjugation Gilbert Syndrome. First described at the turn of the 20th century,35 GS (OMIM*143500) is characterized by mild, predominantly unconjugated hyperbilirubinemia without hyperhemolysis that usually occurs in young adults with otherwise normal liver test results, including normal serum bile acid levels. Patients sometimes describe right upper abdominal discomfort or dyspepsia, but the relationship with hyperbilirubinemia is unclear. Except for jaundice when apparent, clinical findings are normal, as is liver histology when performed. The serum bilirubin level rarely exceeds 70 mmol/L in these patients. Fasting increases serum bilirubin levels, while phenobarbital, a microsomal enzyme inducer, lowers serum bilirubin levels, often to normal values. Treatment with this drug is rarely necessary, however. The prevalence of GS in the general population is approximately 8%. The condition is benign, and the prognosis is excellent. UGT1A1 mutations. A total of 130 UGT1A1 mutations have been identified to date.36,37 The most common molecular defect in GS is the addition of an extra dinucleotide

sequence, TA, to the promoter TATA box of the conjugating enzyme UGT1A1.38 The resulting genotype is designated A(TA)7TAA (instead of the normal A(TA)6TAA) or UGT1A1*28. Much less common A(TA)5TAA or A(TA)8TAA mutations are also found.39 UGT1A1 activity in those homozygous for one of these mutations is approximately 10% to 35% of normal, owing to decreased synthesis of the functionally normal enzyme. Bilirubin in bile is mostly diglucuronidated, but the proportion of monoglucuronide is >20% instead of the normal 7%.12 Homozygosity for this promoter mutation is necessary but not sufficient for hyperbilirubinemia to occur; population studies suggest that only 40% of A(TA)7TAA homozygotes have hyperbilirubinemia.38 Additional variables such as mild hemolysis (reported in up to 50% of patients with GS),40 dyserythropoiesis, additional defects in bilirubin uptake, or perhaps other mutations41 may be necessary for biochemical or clinical expression of the mutation. The existence of an uptake defect in GS (in addition to UGT1A1 deficiency) has been repeatedly suggested, mostly because the clearance of diagnostic dyes such as BSP and ICG is delayed in some patients.16,18 Unconjugated hyperbilirubinemia without overt hemolysis (the clinical definition of GS) could be attributable to 3 mechanisms: (1) decreased UGT1A1 activity with no uptake defect, (2) both decreased UGT1A1 activity and defective uptake, and (3) defective bilirubin uptake with normal UGT1A1 activity17 (see the preceding text). Although the 3 patient categories were probably covered by the original description of the disease,35 we propose that only patients with decreased UGT1A1 activity and the A(TA)7TAA genotype should be diagnosed with GS, whether or not they have an additional uptake defect. Interestingly, a patient with mutations in both the UGT1A1 promoter and ABCC2 (the bilirubin canalicular transporter gene) was found to have a phenotype corresponding to GS/DJS,42 illustrating the genetic complexity of these disorders. Several mutations in the coding regions of the UGT1A1 gene (rather than the promoter region) have been shown to result in proteins with only mildly reduced enzymatic activity. Such missense mutations resulting in a GS phenotype have thus far been reported only in Japan.43 Patients with GS are either homozygotes or compound heterozygotes, and GS is now regarded as an autosomal recessive disorder.44 More than 100 mutations have so far been reported, and their frequencies differ among countries.45 For example, UGT1A1*28 (þ/þ) is found in 12% of Scottish people, 16% of European people, 12% of Indian people, 8% of Egyptian people, and 23% of black people.45 Frequencies of UGT1A1*28 (þ/þ) are much lower in Asia. Some variants are found in geographically distinct populations. Haplotypes including more than one of these genetic variants have also been described; in particular, variants of the promoter TATA box can coexist with coding region mutations.45 This explains in part why GS is observed in only approximately 8% of the white general population despite a 10% to 16% prevalence of UGT1A1*28 homozygosity. GS and CN syndrome were once considered to be distinct genetic and pathophysiological entities, with GS considered

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autosomal dominant and CN syndrome considered autosomal recessive. Both of these entities are now attributed to homozygous mutations of the same enzyme, UGT1A1, but with quantitatively different consequences. Molecular studies have clearly established that a single normal UGT1A1 allele is sufficient to maintain a normal plasma bilirubin concentration and that almost all cases of both GS and CN syndrome are autosomal recessive. The earlier belief that transmission of GS was dominant was probably attributable to the high frequency of the disease and, hence, the common observation of 2 successive affected generations. Diagnosis and management. The diagnosis of GS is usually based on mild prolonged unconjugated hyperbilirubinemia, without overt hemolysis and with otherwise normal liver tests and normal clinical and hepatic ultrasonographic findings. Hyperbilirubinemia is often discovered on a routine blood test performed for another reason and rarely because of clinical jaundice. A definitive diagnosis can be made by determining the A(TA)7TAA genotype, but such a sophisticated method should only be used in clinical practice when the serum bilirubin level is >70 mmol/L or when treatment with irinotecan is planned (see the following text). The patient should be reassured as to the benign course of the condition and informed that the metabolism of some drugs may be affected (see the following text). Phenobarbital (or another microsomal enzyme inducer) should only be prescribed when the bilirubinemia is high enough to cause overt, unsightly jaundice. GS and drug toxicity. Hepatic handling of a variety of drugs metabolized by glucuronidation may be affected in patients with GS, including menthol, estradiol benzoate, ethinyl estradiol, lamotrigine, tolbutamide, rifamycin SV, acetaminophen, nonsteroidal inflammatory drugs, statins and gemfibrozil,45 and human immunodeficiency virus protease inhibitors.45,46 The human immunodeficiency virus protease inhibitors indinavir and atazanavir produce hyperbilirubinemia by inhibiting UGT1A1.47 This hyperbilirubinemia is more pronounced in patients with preexisting GS. Sorafenib, used in patients with various types of cancer and approved for hepatocellular carcinoma, also inhibits UGT1A1, despite being metabolized by UGT1A9.48,49 It increases the serum bilirubin concentration in patients with GS who are homozygous for A(TA)7TAA as well as in A(TA)7TAA heterozygotes.48 Clinically significant toxicity is rarely ascribed to these pharmacokinetic abnormalities. An increased risk of antitubercular drug toxicity has been reported in people with a compound UGT1A1*27 and UGT1A1*28 genotype.50 The main drug-related danger in adults with GS comes from the antitumor agent irinotecan (CPT-11), the active metabolite of which is glucuronidated by UGT1A1. Administration of CPT-11 to patients with GS has resulted in severe toxicity, including intractable diarrhea and myelosuppression with severe neutropenia. European and American patients at risk are mostly UGT1A1*28,51,52 whereas most affected Japanese patients are UGT1A1*6.53 This field is rapidly expanding, and other drug toxicities may be discovered in the future.

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The possibility of genotype-based dosing is being actively explored. GS and atherosclerosis: a genetic advantage? It is becoming clear that unconjugated bilirubin has a protective effect on cardiovascular disease and also perhaps on cancer. Long considered at best a waste product and at worst toxic in certain circumstances, a moderately elevated serum unconjugated bilirubin level is now known to have a number of beneficial effects on oxidative stress-mediated diseases, particularly vascular disease, diabetes, metabolic syndrome, and obesity.54 This appears to be largely due to its antioxidant action (for review, see Rigato et al55) and antiinflammatory properties.56 These epidemiological observations are supported by experimental evidence obtained both in vitro and in vivo. The negative relationship between serum bilirubin levels and coronary artery disease was first reported in 1994.57 Numerous studies have since confirmed the protective effect of moderately increased serum bilirubin concentrations (particularly in patients with GS) on both coronary and peripheral atherosclerotic disease.56,58–62 It appears that each 1-mmol/L increment in serum bilirubin level is associated with a 6.5% decrease in cardiovascular disease.61 Supporting these epidemiological observations, pathological studies have shown an inverse relationship between serum bilirubin concentration and both coronary artery calcification63 and ischemic stroke64 as well as markedly slower progression of carotid intimomedial thickness in patients with GS compared with normobilirubinemic subjects.65 The benefits of moderately increased serum bilirubin concentrations also extend to diabetes and metabolic syndrome.54,66,67 It appears that UGT1A1 variants resulting in hyperbilirubinemia may confer a strong genetic advantage with respect to major causes of death, including cardiovascular disease as well as overweight, obesity, and metabolic syndrome, the prevalence of which is increasing worldwide. Modulation of bilirubin levels may prove to be an attractive intervention for cardiovascular disease and metabolic syndrome. It is conceivable that the high worldwide allelic frequency of homozygous genetic variants of the UGT1A1 gene might be due in part to an evolutionary advantage. GS and cancer: good or bad?. As previously mentioned, a decrease in UGT1A1 activity impairs the glucuronidation not only of bilirubin but also of a variety of other compounds, including estrogen and benzopyrene metabolites.68 It is thus conceivable that reduced glucuronidation of estrogen and mutagens in tissues carrying UGT1A1*28 or other promoter variants could influence the development of hormone-dependent and carcinogen-induced tumors. In a Chinese study and in a European study, UGT1A1*6 and UGT1A1*7 variants were associated with an increased risk of colorectal cancer.69,70 Antioxidant mechanisms might be expected to protect against cancer initiation. Indeed, some studies have shown a decreased risk of colorectal cancer in patients with GS and in patients with moderate hyperbilirubinemia.71,72 Zucker et al found an inverse correlation between the prevalence of colorectal cancer and serum bilirubin concentrations

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among 176,748,462 American subjects, with each 10-mg/L (17-mmol/L) increase in the bilirubin concentration associated with a significant decrease in the prevalence of colorectal cancer.71 Jirásková et al detected a significant association between the risk of colorectal cancer and UGT1A1*28 allele carrier status.72 This issue clearly needs to be revisited. In a case-control study, low UGT1A1 activity due to UGT1A1*28 was associated with a lower risk of endometrial cancer.73 Low UGT1A1 activity may also improve the prognosis of Hodgkin lymphoma.74 Results for breast cancer are also controversial, probably because of geographic variability.45 A Chinese study showed an increase in the risk of breast cancer among women younger than 40 years of age carrying UGT1A1*28, but other variants were not examined.75 This link was not confirmed in a large recent case-control study from the United States.76 A single variable is unlikely to be associated with a clinically significant risk, and the issue clearly requires further haplotype and multigene analyses. This is true, for instance, of the claimed association between UGT1A1 variants and gallstones; an additional risk factor for gallstones, such as cystic fibrosis, spherocytosis, or sickle cell disease, was found in all reported cases.45 Crigler–Najjar Syndrome. CN syndrome is a rare recessive disorder that was first described in 1952.77 It is characterized by major unconjugated hyperbilirubinemia (100–750 mmol/L) due to UGT1A1 mutations. The estimated prevalence is 0.6 per million.78 There are 2 types of CN syndrome; in CN syndrome type I, bilirubin levels are 350 to 750 mmol/L and UGT1A1 mutations result in a complete or near-complete loss of UGT1A1 enzyme activity. In CN syndrome type II, bilirubin levels are 100 to 400 mmol/L and UGT1A1 activity is <10% of normal but not completely abolished. CN syndrome type I. In CN syndrome type I (OMIM*218800), jaundice is apparent from the first days of life. It increases progressively, and the risk of kernicterus (bilirubin encephalopathy) is high. Kernicterus is a disabling neurological condition characterized by extrapyramidal dystonia and/or choreoathetosis, hearing loss due to auditory neuropathy, and oculomotor pareses. Its pathophysiology and treatment have recently been reviewed and are beyond the scope of this report.79 Apart from bilirubin, liver biochemistry in these patients is normal. If the patient survives beyond the neonatal period, jaundice persists and the child remains at substantial risk for late-onset bilirubin encephalopathy, which is sometimes triggered by a mild febrile illness.80 Phenobarbital does not reduce the serum bilirubin concentration.81 There is a complete lack of bilirubin glucuronidation, and there are no detectable bilirubin conjugates in duodenal secretions.82 In CN syndrome type I, mutations are often found in exons 2 to 5.36 All UGT1A isoforms are affected, and glucuronidation of a broad spectrum of substrates, in addition to bilirubin, may therefore be impaired (type Ia). Mutations can also occur in exon 1,37 in which case only UGT1A1 is affected and the loss of glucuronidation capacity is limited

to bilirubin (type Ib). Most patients are homozygotes or compound heterozygotes. The mutations generally consist of premature truncation or critical amino acid substitution, resulting in abolished or considerably reduced enzyme activity.36 Treatment of CN syndrome type I in the neonate consists of exchange transfusions to maintain serum bilirubin levels below the threshold for kernicterus (approximately 150 mmol/L) plus phototherapy for 12 hours a day. Phototherapy transforms bilirubin into colorless water-soluble derivatives and isomers that are secreted into bile without conjugation and are more easily excreted into urine than bilirubin itself. Isolated hepatocyte transplantation has been used experimentally in a few patients but is not sufficient to correct CN syndrome type I.83,84 Liver transplantation remains the best option to prevent brain injury and death. The largest survey of liver transplantation for CN syndrome was performed by van der Veere et al.78 Fifty-seven patients were included, of whom 21 underwent transplantation (37%). The mean age at transplantation was 9.1  6.9 years (range, 1–23 years). Ten patients remained alive, with some degree of mental or physical handicap. Transplantation should be performed as soon as technically feasible to prevent brain damage, especially when phototherapy is ineffective. Brain damage can be reversed by liver transplantation when not too severe. Smaller series of patients have been reported with similar results.85,86 Much of our understanding of the pathophysiology of CN syndrome type I originates from studies on the Gunn rat, a mutant Wistar rat exhibiting chronic nonhemolytic unconjugated hyperbilirubinemia.87 In these animals, jaundice is inherited as an autosomal recessive character. Liver histology in affected rats is normal. Bilirubin glucuronosyl transferase activity is undetectable in the liver of jaundiced rats. Heterozygotes do not develop jaundice. More recently, a mouse model with a disrupted ugt1 in exon 4 reproduced human CN syndrome type I with a lethal phenotype.88 Ugt(/) mice had 40- to 60-fold higher serum bilirubin concentrations than their wild-type counterparts and Ugt(þ/) mice. The condition was lethal within 2 weeks. The mice had no detectable UGT1A-specific RNA, and UGT1A protein was completely absent from liver microsomes. Even more interestingly, Bortolussi et al constructed a mouse model with a premature stop codon in the Ugta1a1 gene, resulting in an inactive enzyme.89 These mice developed severe jaundice soon after birth and died within 11 days with cerebellar lesions. The mice could be completely rescued when injected at birth with a single dose of an adeno-associated viral vector expressing human UGT1A1. This represented the first demonstration that gene therapy has the potential to prevent the lethal effects of neonatal hyperbilirubinemia. CN syndrome type II. In contrast to CN syndrome type I, UGT1A1 activity is maintained in CN syndrome type II (OMIM# 606785), albeit at a minimal level (<10% of normal). Serum bilirubin concentrations are typically between 100 and 400 mmol/L. Bile contains mostly bilirubin monoglucuronides.90 Induction of UGT1A1 by phenobarbital (60–120 mg/day) reduces the bilirubin concentration by >25%.12 In most cases, basal or phenobarbital-induced

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UGT1A1 activity is sufficient to prevent bilirubin encephalopathy. However, neurological sequelae may occur after an intercurrent illness, fasting, or any other event that temporarily increases the serum bilirubin concentration. In most patients with CN syndrome type II who have normal serum albumin levels, prolonged exposure to bilirubin concentrations <250 mmol/L does not result in neurologic injury. Indeed, the majority of patients in the original reports were healthy adults. CN syndrome type II is caused by substitution of single amino acid residues, markedly reducing but not abolishing the catalytic activity of the enzyme.36,43 Compound heterozygotes with a promoter variant (as found in GS) on one allele and a coding region variant on the other allele have been found to have a CN syndrome type II phenotype.91 Some of these patients are at risk for kernicterus.91 Treatment of CN syndrome type II consists of lifelong phenobarbital therapy.

Neonatal Hyperbilirubinemia and Breast Milk Jaundice. Occasionally, severe neonatal unconjugated hyperbilirubinemia occurs without a clear cause. One such condition, transient familial neonatal hyperbilirubinemia (OMIM*237900), occurs in families and has been linked to the presence in serum of steroids that competitively inhibit UGT1A1, particularly pregnane-3a, 20b diol.92 Jaundice may be severe enough to cause kernicterus. Recently, it was shown that variations at nucleotide 211 (211G / A) of the UGT1A1 gene are a risk factor for this condition, in keeping with the familial nature of the disease.93 Similarly, transient nonhemolytic unconjugated hyperbilirubinemia is observed in some breast-fed babies of mothers whose breast milk contains pregnane-3a, 20b diol but not in their bottle-fed counterparts.94 In other cases, jaundice may result from inadequate calorie provision by breast milk and from enhanced intestinal absorption of bilirubin. Kernicterus has not been reported in this setting, probably because severe jaundice does not develop until the 7th to 10th day of life, when the infant’s blood-brain barrier has become relatively impermeable to unconjugated bilirubin. In some cases, this condition is associated with the 211G / A mutation of UGT1A1.93

Defective Canalicular Secretion Dubin—Johnson syndrome. History and clinical features. In 1954, Dubin and Johnson, and Sprinz and Nelson, who were US Army pathologists, described otherwise healthy young soldiers who had chronic low-grade jaundice associated with a black liver, plasma retention of predominantly conjugated bilirubin and organic anionic compounds such as BSP, and nonvisualization of the gallbladder after administration of an organic anionic contrast agent (iodopanoic acid) but no other features of hepatobiliary disease (OMIM*237500).95,96 A curious finding in DJS is that, after intravenous injection of BSP, a normal initial disappearance curve is followed by a “secondary” rise in plasma BSP, suggesting an excretory defect. BSP is conjugated to glutathione, and this “secondary” rise is related to reflux of conjugated BSP from the liver to plasma. It is not observed after injection of dyes such as ICG and dibromosulphthalein that are not

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conjugated in the liver.97 A reduced secretory maximum for BSP was also found.98 Subsequent studies revealed the phenotype to be inherited, distributed worldwide, and benign (essentially aesthetic). The black liver was linked to a melanin-like pigment in hepatocyte lysosomes.99 In mutant rats, pigment accumulation was shown to result from retention of anionic metabolites of tyrosine, phenylalanine and tryptophan, which polymerized to form a similar melanin-like pigment in vivo.100 A similar phenotype was identified in mutant Corriedale sheep,101,102 Eisai hyperbilirubinemic rats,103 TR() rats,104,105 and golden lion tamarins.106 Studies of Corriedale sheep equipped with a bile fistula revealed the existence of 3 major transport systems: one for bile acids, one for non–bile acid organic anions, and one for organic cations. Mutant sheep showed greatly reduced biliary secretion of organic anions, including bilirubin glucuronide, iodopanoic acid, BSP, and phylloerythrin, but normal maximal secretion of bile acids and an organic cation.107 Subsequent studies using rat canalicular membrane vesicles revealed the presence of an ATP-dependent transport system for non–bile acid organic anions, including glutathione and glucuronide conjugates of endogenous and exogenous substrates.104,105,108 Following the observation that the multidrug resistance protein 1 (ABCB1) resides in the canalicular membrane and transports daunomycin and other cationic anticancer drugs via an ATPdependent system,109 the concept that ATP hydrolysis might provide energy for the secretion of other biliary constituents resulted in the discovery of a family of ATP-binding cassette transporters, including ABCC2 (specific for non–bile acid organic anions), ABCB11 (bile acids), ABCB4 (phospholipids), and ABCG5,8 (sterols).110 Molecular genetics. Although family studies indicated that DJS is inheritable, urinary coproporphyrin isomer studies established its inheritance as an autosomal recessive characteristic.111 Subsequent genomic studies revealed that the DJS phenotype results from mutations in the protein encoded by ABCC2 (previously called MRP2 [multidrugrelated protein 2] or MOAT [multispecific organic anion transporter]), which is a member of the large ABC transporter family.112,113 ABCC2 couples with ATP hydrolysis to actively transport a wide variety of endogenous and exogenous organic anions of <1000 daltons into the bile canaliculus.14,105,108 The endogenous ligands include bilirubin glucuronides, glutathione disulfide, and glucuronide or glutathione conjugates of leukotrienes, prostanoids, and several hormones. The exogenous ligands include conjugates of various drugs, chemicals, and metals. In contrast to ABCB11, the bile acid transporter, which is expressed only in the canalicular membrane, ABCC2 is additionally expressed in the apical plasma membrane domain of epithelial cells in the proximal renal tubules, gallbladder, small intestine, bronchi, and placenta and is an efflux pump that eliminates many toxins and carcinogens into bile, urine, and the intestine.14,114 The human ABCC2 gene is located on chromosome 10q24, spans approximately 45 kilobases, and contains 32 exons. Since the discovery in 1997 of the first mutations of

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the ABCC2 gene responsible for DJS, more than 20 different ABCC2 mutations have been described in patients with DJS, including exon skipping, missense, nonsense, and base deletion.112,113 Point mutations result in stop codons and failure to transcribe MRP2 protein. Less frequent are mutations that result in endoplasmic reticulum retention of MRP2, which is blocked in the secretory pathway and does not appear in the canalicular membrane.115 Additional studies in patients with DJS, obligate heterozygotes, and genetically engineered mice should provide valuable information concerning the cell biology and transcriptional regulation of apical ABC transporters and the role of ABCC2 in cholestasis, drug distribution, and toxicity. As with other canalicular ABC transporters, MRP2 traffics from the trans-Golgi network to the apical membrane, from which it cycles to a large intracellular recycling endosome pool.116,117 Bile acids and adenosine 30 ,50 -cyclic monophosphate enhance transfer of ABC transporters from the recycling pool to the apical membrane, thereby enhancing postprandial biliary secretion.116,118 The cycling process is energy dependent and involves AMP-activated protein kinase–mediated microtubular-based trafficking of rab11amyosin Vb endosomes.119 Retrieval from the apical membrane involves an HAX protein-clathrin-actin mechanism.120 Phosphoinositide-3 kinase, mitogen-activated protein kinase, protein kinase kinase 3, and protein kinase C participate in various steps in the overall process.121–124 MRP2 apical membrane targeting also involves the C-terminus PDZ domain that binds to NERF2. This facilitates linkage to actin.125 When radixin, which cross-links actin filaments and integral membrane proteins, was deleted in mice, Mrp2 was absent from the canalicular membrane, resulting in a DJS-like phenotype; subsequently, other canalicular transporters disappeared.126 In radixin small interfering RNA–treated hepatocyte cultures, the canalicular structure was disrupted.127 Although MRP2 is important in the secretion of anionic drugs, metals, and their conjugates, there is little information on its role in drug distribution, secretion, and interaction, and there are no reports of drug toxicity in patients with DJS. In Abcc2(-/-) genetically engineered mice and Mrp2 small interfering RNA–treated rat hepatocytes, enhanced toxicity and/or altered secretion was observed for various drugs, including erythromycin, human immunodeficiency virus protease inhibitors, and others.128,129 Pharmacokinetic studies in DJS will be of interest particularly regarding potential drug toxicity and interaction. Pregnancy and use of oral contraceptives increase hyperbilirubinemia in patients with DJS.130 It is not known whether heterozygosity for MRP2 mutations predisposes to or accentuates any clinical disorder. The functional defect in DJS is restricted to non–bile acid organic anions; bile acids are secreted normally into the bile by ABCB11. Cholestasis is associated with decreased canalicular and increased intracellular MRP2 and other canalicular ABC transporters. Similar events occur in rats and hepatocytes rendered cholestatic by estrogen, monohydroxy bile acids, oxidative stress, lipopolysaccharide, and AMPactivated protein kinase depletion.131,132 Whether and

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Table 1.Main Characteristics of Hereditary Nonhemolytic Hyperbilirubinemia Syndromes

Disease

Type of Degree of hyperbilirubinemia Molecular defect Inheritance hyperbilirubinemia BSP kinetics

Liver histology Prognosis

Treatment

Uptake defect

Mutant Southdown sheep

Unconjugated — bilirubin Conjugated RS (hepatic bilirubin uptake and storage disease) Conjugation GS defect CN syndrome Type I

Type II Secretion defect

Animal model

DJS

Unconjugated Mainly conjugated

OATP1B1/3, chromosome 2

Low to moderate grade Autosomal Low to moderate recessive grade

Unconjugated

UDPG1A1, chromosome 2

Autosomal Low to moderate recessive grade

Normal PDR

Normal

Good

Phenobarbital (occasionally)

Unconjugated

UDPG1A1, chromosome 2

Autosomal Very high grade recessive

Normal PDR

Normal

Severe

Unconjugated

UDPG1A1, Autosomal High grade chromosome 2 recessive ABCC2, Autosomal Low to moderate chromosome 10 recessive grade

Normal PDR

Normal

Generally good Good

Exchange Gunn rat transfusions Phototherapy Liver transplantation Phenobarbital No

Conjugated

?

?

Reduced PDR

Normal

Good

No

Reduced PDR

Normal

Good

No

No

Mutant Corriedale sheep

PDR, plasma disappearance rate.

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Initially normal Pigment PDR deposit “secondary” rise

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Figure 3. Schematic view of the transporters involved in inherited disorders of bilirubin metabolism. UCB, unconjugated bilirubin; Alb, albumin; BG, bilirubin glucuronide.

how this process, which involves Mrp2 and other ABC transporters, specifically contributes to decreased bile secretory function in cholestasis remains to be elucidated. Resolving these issues may provide new therapeutic targets and agents for treating cholestatic diseases. MRP2 is regulated by nuclear pregnane X receptor, farnesoid X-activated receptor, and constitutive androstane receptor.133 In normal human liver, constitutive hepatic messenger RNA levels for constitutive androstane receptor, hepatocyte nuclear factor 1a, and proliferator-activated receptor a messenger RNA exhibit the greatest correlation with MRP2, 3, and 4.134 Further studies of transcriptional regulation of ABCC2 and other ABC transporters in health and liver disease are warranted. The main characteristics of these disorders are indicated in Table 1, and the defective transporters are shown in Figure 3. Inherited disorders of bilirubin metabolism are yet another example of how spectacular advances in molecular genetics have helped us understand both normal physiology and disease mechanisms. Future research may provide tools to cure even the most severe disorders.

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Received November 23, 2013. Accepted March 23, 2014. Reprint requests Address requests for reprints to: Serge Erlinger, MD, 1422 Route des Mauvares, 13840 Rognes, France. e-mail: [email protected]. Conflicts of interest The authors disclose no conflicts.