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

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. 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. Hereditary 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. 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.1Wolkoff A.W. Goresky C.A. Sellin J. et al.Role of ligandin in transfer of bilirubin from plasma into liver.Am J Physiol. 1979; 236: E638-E648PubMed Google Scholar 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.2Hagenbuch B. Gui C. Xenobiotic transporters of the human organic anion transporting polypeptides (OATP) family.Xenobiotica. 2008; 38: 778-801Crossref PubMed Scopus (226) Google Scholar The human SLCO superfamily comprises 11 members grouped into 6 families encoded by SLCO genes. Bilirubin is a substrate for OATP1B1 (Online Mendelian Inheritance in Man [OMIM]*604843) and OATP1B3 (OMIM*605495).2Hagenbuch B. Gui C. Xenobiotic transporters of the human organic anion transporting polypeptides (OATP) family.Xenobiotica. 2008; 38: 778-801Crossref PubMed Scopus (226) Google Scholar Human OATP1B1 and OATP1B3 can transport conjugated and, possibly, unconjugated bilirubin in vitro.3Cui Y. Konig J. Leier I. et al.Hepatic uptake of bilirubin and its conjugates by the human organic anion transporter SLC21A6.J Biol Chem. 2001; 276: 9626-9630Crossref PubMed Scopus (351) Google Scholar, 4Briz O. Serrano M.A. MacIas R.I. et al.Role of organic anion-transporting polypeptides, OATP-A, OATP-C and OATP-8, in the human placenta-maternal liver tandem excretory pathway for foetal bilirubin.Biochem J. 2003; 371: 897-905Crossref PubMed Scopus (111) Google Scholar 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.5Sanna S. Busonero F. Maschio A. et al.Common variants in the SLCO1B3 locus are associated with bilirubin levels and unconjugated hyperbilirubinemia.Hum Mol Genet. 2009; 18: 2711-2718Crossref PubMed Scopus (87) Google Scholar, 6Zhang W. He Y.J. Gan Z. et al.OATP1B1 polymorphism is a major determinant of serum bilirubin level but not associated with rifampicin-mediated bilirubin elevation.Clin Exp Pharmacol Physiol. 2007; 34: 1240-1244Crossref PubMed Scopus (49) Google Scholar 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.7van de Steeg E. Wagenaar E. van der Kruijssen C.M. et al.Organic anion transporting polypeptide 1a/1b-knockout mice provide insights into hepatic handling of bilirubin, bile acids, and drugs.J Clin Invest. 2010; 120: 2942-2952Crossref PubMed Scopus (84) Google Scholar Serum bilirubin in these mice is mostly conjugated, probably (see the following text) because of defective reuptake of bilirubin glucuronide.8van de Steeg E. Stranecky V. Hartmannova H. et al.Complete OATP1B1 and OATP1B3 deficiency causes human Rotor syndrome by interrupting conjugated bilirubin reuptake into the liver.J Clin Invest. 2012; 122: 519-528Crossref PubMed Scopus (102) Google Scholar However, serum levels of unconjugated bilirubin are 2-fold higher in Oatp1a/1b knockout mice than in their wild-type counterparts.7van de Steeg E. Wagenaar E. van der Kruijssen C.M. et al.Organic anion transporting polypeptide 1a/1b-knockout mice provide insights into hepatic handling of bilirubin, bile acids, and drugs.J Clin Invest. 2010; 120: 2942-2952Crossref PubMed Scopus (84) Google Scholar 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 μmol/L, respectively.3Cui Y. Konig J. Leier I. et al.Hepatic uptake of bilirubin and its conjugates by the human organic anion transporter SLC21A6.J Biol Chem. 2001; 276: 9626-9630Crossref PubMed Scopus (351) Google Scholar However, this observation could not be reproduced with unconjugated bilirubin in HeLa or HEK293 cells transfected with OATP1B1.9Wang P. Kim R.B. Chowdhury J.R. et al.The human organic anion transport protein SLC21A6 is not sufficient for bilirubin transport.J Biol Chem. 2003; 278: 20695-20699Crossref PubMed Scopus (62) Google Scholar Further studies are thus needed to clarify the respective roles of passive diffusion and carrier-mediated transport in unconjugated bilirubin uptake by hepatocytes. 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.10Owens I.S. Basu N.K. Banerjee R. UDP-glucuronosyltransferases: gene structures of UGT1 and UGT2 families.Methods Enzymol. 2005; 400: 1-22Crossref PubMed Scopus (47) Google Scholar, 11Rowland A. Miners J.O. Mackenzie P.I. The UDP-glucuronosyltransferases: their role in drug metabolism and detoxification.Int J Biochem Cell Biol. 2013; 45: 1121-1132Crossref PubMed Scopus (123) Google Scholar, 12Wolkoff A.L. Berk P.D. Bilirubin metabolism and jaundice.in: Schiff E. Maddrey W.C. Sorrell M.E. Diseases of the liver. 11th ed. Wiley-Blackwell, New York, NY2012: 120-151Google Scholar 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.11Rowland A. Miners J.O. Mackenzie P.I. The UDP-glucuronosyltransferases: their role in drug metabolism and detoxification.Int J Biochem Cell Biol. 2013; 45: 1121-1132Crossref PubMed Scopus (123) Google Scholar 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.11Rowland A. Miners J.O. Mackenzie P.I. The UDP-glucuronosyltransferases: their role in drug metabolism and detoxification.Int J Biochem Cell Biol. 2013; 45: 1121-1132Crossref PubMed Scopus (123) Google Scholar Glucuronic acid is derived from uridine diphospho-glucuronic acid, which itself is derived 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.Figure 2Schematic view of bilirubin transport by the hepatocyte. (Inset) Localization of UGT1A1 in the reticulum endoplasmic membrane and transport of uridine diphospho-glucuronic acid (UDPGA), bilirubin, and bilirubin glucuronide across the endoplasmic reticulum membrane. UCB, unconjugated bilirubin; Alb, albumin; BG, bilirubin glucuronide.View Large Image Figure ViewerDownload Hi-res image Download (PPT) 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.13Jemnitz K. Heredi-Szabo K. Janossy J. et al.ABCC2/Abcc2: a multispecific transporter with dominant excretory functions.Drug Metab Rev. 2010; 42: 402-436Crossref PubMed Scopus (42) Google Scholar, 14Keppler D. Multidrug resistance proteins (MRPs, ABCCs): importance for pathophysiology and drug therapy.Handb Exp Pharmacol. 2011; : 299-323Crossref PubMed Scopus (124) Google Scholar 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.13Jemnitz K. Heredi-Szabo K. Janossy J. et al.ABCC2/Abcc2: a multispecific transporter with dominant excretory functions.Drug Metab Rev. 2010; 42: 402-436Crossref PubMed Scopus (42) Google Scholar, 14Keppler D. Multidrug resistance proteins (MRPs, ABCCs): importance for pathophysiology and drug therapy.Handb Exp Pharmacol. 2011; : 299-323Crossref PubMed Scopus (124) Google Scholar Other transporters, particularly Abcg2,15Vlaming M.L. Pala Z. van Esch A. et al.Functionally overlapping roles of Abcg2 (Bcrp1) and Abcc2 (Mrp2) in the elimination of methotrexate and its main toxic metabolite 7-hydroxymethotrexate in vivo.Clin Cancer Res. 2009; 15: 3084-3093Crossref PubMed Scopus (57) Google Scholar 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, Abcc3.8van de Steeg E. Stranecky V. Hartmannova H. et al.Complete OATP1B1 and OATP1B3 deficiency causes human Rotor syndrome by interrupting conjugated bilirubin reuptake into the liver.J Clin Invest. 2012; 122: 519-528Crossref PubMed Scopus (102) Google Scholar From there, it can be taken up again by hepatocytes via Oatp1b1/3.8van de Steeg E. Stranecky V. Hartmannova H. et al.Complete OATP1B1 and OATP1B3 deficiency causes human Rotor syndrome by interrupting conjugated bilirubin reuptake into the liver.J Clin Invest. 2012; 122: 519-528Crossref PubMed Scopus (102) Google Scholar 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).8van de Steeg E. Stranecky V. Hartmannova H. et al.Complete OATP1B1 and OATP1B3 deficiency causes human Rotor syndrome by interrupting conjugated bilirubin reuptake into the liver.J Clin Invest. 2012; 122: 519-528Crossref PubMed Scopus (102) Google Scholar A schematic representation of bilirubin transport and conjugation by hepatocytes is provided in Figure 2. 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 milk jaundice; or (4) defective bilirubin canalicular secretion, such as in Dubin–Johnson syndrome (DJS). 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. Rare cases of unconjugated hyperbilirubinemia have been reported in which plasma clearance of cholephilic dyes such as BSP and ICG was markedly impaired,16Martin J.F. Vierling J.M. Wolkoff A.W. et al.Abnormal hepatic transport of indocyanine green in Gilbert's syndrome.Gastroenterology. 1976; 70: 385-391Abstract Full Text PDF PubMed Google Scholar, 17Metreau J.M. Dhumeaux D. Gisselbrecht C. et al.Constitutional unconjugated hyperbilirubinaemia.Lancet. 1977; 1: 1319Abstract PubMed Google Scholar, 18Gentile S. Persico M. Tiribelli C. Abnormal hepatic uptake of low doses of sulfobromophthalein in Gilbert's syndrome: the role of reduced affinity of the plasma membrane carrier of organic anions.Hepatology. 1990; 12: 213-217Crossref PubMed Scopus (12) Google Scholar 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).17Metreau J.M. Dhumeaux D. Gisselbrecht C. et al.Constitutional unconjugated hyperbilirubinaemia.Lancet. 1977; 1: 1319Abstract PubMed Google Scholar Genetic and molecular analyses suggest that such cases of unconjugated hyperbilirubinemia could be linked to polymorphisms in SLCO1B1 and SCLO1B3.5Sanna S. Busonero F. Maschio A. et al.Common variants in the SLCO1B3 locus are associated with bilirubin levels and unconjugated hyperbilirubinemia.Hum Mol Genet. 2009; 18: 2711-2718Crossref PubMed Scopus (87) Google Scholar, 6Zhang W. He Y.J. Gan Z. et al.OATP1B1 polymorphism is a major determinant of serum bilirubin level but not associated with rifampicin-mediated bilirubin elevation.Clin Exp Pharmacol Physiol. 2007; 34: 1240-1244Crossref PubMed Scopus (49) Google Scholar, 8van de Steeg E. Stranecky V. Hartmannova H. et al.Complete OATP1B1 and OATP1B3 deficiency causes human Rotor syndrome by interrupting conjugated bilirubin reuptake into the liver.J Clin Invest. 2012; 122: 519-528Crossref PubMed Scopus (102) Google Scholar, 19Watchko J.F. Genetics and pediatric unconjugated hyperbilirubinemia.J Pediatr. 2013; 162: 1092-1094Abstract Full Text Full Text PDF PubMed Google Scholar However, given the modest role of OATPs in bilirubin uptake,8van de Steeg E. Stranecky V. Hartmannova H. et al.Complete OATP1B1 and OATP1B3 deficiency causes human Rotor syndrome by interrupting conjugated bilirubin reuptake into the liver.J Clin Invest. 2012; 122: 519-528Crossref PubMed Scopus (102) Google Scholar 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 glucose-6-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.20Skierka J.M. Kotzer K.E. Lagerstedt S.A. et al.UGT1A1 genetic analysis as a diagnostic aid for individuals with unconjugated hyperbilirubinemia.J Pediatr. 2013; 162 (1152 e1–2): 1146-1152Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar The disorder first described by Rotor et al in 1948 is a rare and benign disease (OMIM*237450) characterized by low-grade (40–100 μmol/L), chronic or fluctuating, predominantly conjugated hyperbilirubinemia.21Rotor A. Manahan L. Florentin A. Familial non-hemolytic jaundice with direct Van den Bergh reaction.Acta Med Phil. 1948; 5: 37-49Google Scholar It is a familial disorder with autosomal recessive transmission.22Wolkoff A.W. Wolpert E. Pascasio F.N. et al.Rotor's syndrome. A distinct inheritable pathophysiologic entity.Am J Med. 1976; 60: 173-179Abstract Full Text PDF PubMed Google Scholar 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,22Wolkoff A.W. Wolpert E. Pascasio F.N. et al.Rotor's syndrome. A distinct inheritable pathophysiologic entity.Am J Med. 1976; 60: 173-179Abstract Full Text PDF PubMed Google Scholar 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, contrary to DJS.23Berthelot P. Dhumeaux D. New insights into the classification and mechanisms of hereditary, chronic, non-haemolytic hyperbilirubinaemias.Gut. 1978; 19: 474-480Crossref PubMed Google Scholar 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.24Dhumeaux D. Berthelot P. Chronic hyperbilirubinemia associated with hepatic uptake and storage impairment. A new syndrome resembling that of the mutant Southdown sheep.Gastroenterology. 1975; 69: 988-993PubMed Google Scholar The hepatic relative storage capacity of BSP and dibromosulphthalein was markedly impaired, whereas the biliary transport maximum was only slightly affected.24Dhumeaux D. Berthelot P. Chronic hyperbilirubinemia associated with hepatic uptake and storage impairment. A new syndrome resembling that of the mutant Southdown sheep.Gastroenterology. 1975; 69: 988-993PubMed Google Scholar 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.25Delage Y.S.T. Hadchouel P. Touboul J.P. et al.Rotor's syndrome: evidence for an impairment of hepatic uptake and storage of cholephilic organic anions (abstr).Digestion. 1977; 15: 228-229Google Scholar, 26Namihisa T. Nambu M. Kobayashi N. et al.The constitutional indocyanine green excretory defect—report of four cases.Gastroenterol Jpn. 1975; 10: 70-76PubMed Google Scholar, 27Wolpert E. Pascasio F.M. Wolkoff A.W. et al.Abnormal sulfobromophthalein metabolism in Rotor's syndrome and obligate heterozygotes.N Engl J Med. 1977; 296: 1099-1101Crossref PubMed Google Scholar From that time onward, hepatic uptake and storage disease and RS were considered to be the same entity.23Berthelot P. Dhumeaux D. New insights into the classification and mechanisms of hereditary, chronic, non-haemolytic hyperbilirubinaemias.Gut. 1978; 19: 474-480Crossref PubMed Google Scholar 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.28Hrebicek M. Jirasek T. Hartmannova H. et al.Rotor-type hyperbilirubinaemia has no defect in the canalicular bilirubin export pump.Liver Int. 2007; 27: 485-491Crossref PubMed Scopus (20) Google Scholar However, despite these advances, it remained unclear why serum bilirubin is predominantly conjugated in patients with RS. The recent work of van de Steeg et al probably solves this mystery.8van de Steeg E. Stranecky V. Hartmannova H. et al.Complete OATP1B1 and OATP1B3 deficiency causes human Rotor syndrome by interrupting conjugated bilirubin reuptake into the liver.J Clin Invest. 2012; 122: 519-528Crossref PubMed Scopus (102) Google Scholar 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.7van de Steeg E. Wagenaar E. van der Kruijssen C.M. et al.Organic anion transporting polypeptide 1a/1b-knockout mice provide insights into hepatic handling of bilirubin, bile acids, and drugs.J Clin Invest. 2010; 120: 2942-2952Crossref PubMed Scopus (84) Google Scholar 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, 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 transporters5Sanna S. Busonero F. Maschio A. et al.Common variants in the SLCO1B3 locus are associated with bilirubin levels and unconjugated hyperbilirubinemia.Hum Mol Genet. 2009; 18: 2711-2718Crossref PubMed Scopus (87) Google Scholar), as well as their increased urinary coproporphyrin excretion, in line with the interaction of several porphyrins with OATP1B1.29Campbell S.D. Lau W.F. Xu J.J. Interaction of porphyrins with human organic anion transporting polypeptide 1B1.Chem Biol Interact. 2009; 182: 45-51Crossref PubMed Scopus (7) Google Scholar 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.8van de Steeg E. Stranecky V. Hartmannova H. et al.Complete OATP1B1 and OATP1B3 deficiency causes human Rotor syndrome by interrupting conjugated bilirubin reuptake into the liver.J Clin Invest. 2012; 122: 519-528Crossref PubMed Scopus (102) Google Scholar 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.30Konig J. Seithel A. Gradhand U. et al.Pharmacogenomics of human OATP transporters.Naunyn Schmiedebergs Arch Pharmacol. 2006; 372: 432-443Crossref PubMed Scopus (231) Google Scholar, 31Morimoto K. Oishi T. Ueda S. et al.A novel variant allele of OATP-C (SLCO1B1) found in a Japanese patient with pravastatin-induced myopathy.Drug Metab Pharmacokinet. 2004; 19: 453-455Crossref PubMed Google Scholar, 32Tirona R.G. Kim R.B. Pharmacogenomics of organic anion-transporting polypeptides (OATP).Adv Drug Deliv Rev. 2002; 54: 1343-1352Crossref PubMed Scopus (80) Google Scholar, 33Trevino L.R. Shimasaki N. Yang W. et al.Germline genetic variation in an organic anion transporter polypeptide associated with methotrexate pharmacokinetics and clinical effects.J Clin Oncol. 2009; 27: 5972-5978Crossref PubMed Scopus (147) Google Scholar 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,7van de Steeg E. Wagenaar E. van der Kruijssen C.M. et al.Organic anion transporting polypeptide 1a/1b-knockout mice provide insights into hepatic handling of bilirubin, bile acids, and drugs.J Clin Invest. 2010; 120: 2942-2952Crossref PubMed Scopus (84) Google Scholar and this might be an additional cause of drug-induced conjugated hyperbilirubinemias. The hepatic transport abnormalities seen in RS resemble those of mutant Sout