Complete OATP1B1 and OATP1B3 deficiency causes human Rotor syndrome by interrupting conjugated bilirubin reuptake into the liver

Bilirubin, a breakdown product of heme, is normally glucuronidated and excreted by the liver into bile. Failure of this system can lead to a buildup of conjugated bilirubin in the blood, resulting in jaundice. The mechanistic basis of bilirubin excretion and hyperbilirubinemia syndromes is largely understood, but that of Rotor syndrome, an autosomal recessive disorder characterized by conjugated hyperbilirubinemia, coproporphyrinuria, and near-absent hepatic uptake of anionic diagnostics, has remained enigmatic. Here, we analyzed 8 Rotor-syndrome families and found that Rotor syndrome was linked to mutations predicted to cause complete and simultaneous deficiencies of the organic anion transporting polypeptides OATP1B1 and OATP1B3. These important detoxification-limiting proteins mediate uptake and clearance of countless drugs and drug conjugates across the sinusoidal hepatocyte membrane. OATP1B1 polymorphisms have previously been linked to drug hypersensitivities. Using mice deficient in Oatp1a/1b and in the multispecific sinusoidal export pump Abcc3, we found that Abcc3 secretes bilirubin conjugates into the blood, while Oatp1a/1b transporters mediate their hepatic reuptake. Transgenic expression of human OATP1B1 or OATP1B3 restored the function of this detoxification-enhancing liver-blood shuttle in Oatp1a/1b-deficient mice. Within liver lobules, this shuttle may allow flexible transfer of bilirubin conjugates (and probably also drug conjugates) formed in upstream hepatocytes to downstream hepatocytes, thereby preventing local saturation of further detoxification processes and hepatocyte toxic injury. Thus, disruption of hepatic reuptake of bilirubin glucuronide due to coexisting OATP1B1 and OATP1B3 deficiencies explains Rotor-type hyperbilirubinemia. Moreover, OATP1B1 and OATP1B3 null mutations may confer substantial drug toxicity risks.

Rotor syndrome (RS; OMIM %237450) is a rare, benign hereditary conjugated hyperbilirubinemia, also featuring coproporphyrinuria and strongly reduced liver uptake of many diagnostic compounds, including cholescintigraphic tracers (1–6). RS is an autosomal recessive disorder that clinically resembles another conjugated hyperbilirubinemia, the Dubin-Johnson syndrome (DJS; OMIM #237500) (7, 8). In both RS and DJS, mild jaundice begins shortly after birth or in childhood. There are no signs of hemolysis, and routine hematologic and clinical-biochemistry test results are normal, aside from the primarily conjugated hyperbilirubinemia. RS is, however, distinguishable from DJS by several criteria (1, 2, 9, 10): (a) it lacks the hepatocyte pigment deposits typical of DJS; (b) in RS, but not DJS, there is delayed plasma clearance of unconjugated bromsulphthalein (BSP), an anionic diagnostic dye, and no conjugated BSP appears in plasma (4); (c) the liver in RS is scarcely visualized on ^99mTc-N[2,6-dimethylphenyl-carbamoylmethyl] iminodiacetic acid (^99mTc-HIDA) cholescintigraphy, with slow liver uptake, persistent visualization of the cardiac blood pool, and prominent kidney excretion (5); and (d) total urinary excretion of coproporphyrins is greatly increased in RS, with coproporphyrin I being the predominant isomer (11).

DJS is caused by mutations affecting ABCC2/MRP2, a canalicular bilirubin glucuronide and xenobiotic export pump, thus disrupting bilirubin glucuronide excretion into bile (7, 8). Excretion of bilirubin glucuronides is then redirected into plasma by the action of ABCC3/MRP3, a homolog of ABCC2 that is present in the sinusoidal membrane and is upregulated in DJS (12, 13). The molecular mechanism of DJS is in line with the generally accepted paradigm of normal hepatic bilirubin excretion, according to which a unidirectional elimination pathway is postulated: first, uptake of unconjugated bilirubin (UCB) from blood into hepatocytes; subsequent glucuronidation; and finally, secretion of bilirubin glucuronide into bile via ABCC2. Individuals with RS, however, lack ABCC2 mutations (14), and the mechanistic basis of RS is unknown.

Organic anion transporting polypeptides (OATPs, genes: SLCOs) contain 12 plasma membrane–spanning domains and mediate sodium-independent cellular uptake of highly diverse compounds, including bilirubin glucuronide, bile acids, steroid and thyroid hormones, and numerous drugs, toxins, and their conjugates (15, 16). Human OATP1B1 and OATP1B3 localize to the sinusoidal membrane of hepatocytes and mediate the liver uptake of, among other compounds, many drugs (15–19). Various SNPs in SLCO1B1 cause reduced transport activity and altered plasma and tissue levels of statins, methotrexate, and irinotecan in patients, potentially resulting in life-threatening toxicities (20–24).

In a Slco1a/1b^–/– mouse model recently generated by our group, the importance of Oatp1a/1b proteins in hepatic uptake and clearance of drugs was confirmed, but the mice also displayed marked conjugated hyperbilirubinemia (25). We therefore hypothesized that sinusoidal Oatps in the normal, healthy mouse liver function in tandem with the sinusoidal efflux transporter Abcc3 to mediate substantial hepatic secretion and reuptake of bilirubin glucuronides and other conjugated compounds (25).

Here we describe how a combination of functional studies in mice to address this hypothesis and independent genetic studies in humans has resulted in elucidation of the genetic and mechanistic basis of Rotor syndrome.

To test our hypothesis regarding the involvement of Abcc3 in the sinusoidal cycling of bilirubin glucuronides, and to assess a possible interplay with Abcc2, we generated Slco1a/1b^–/–Abcc3^–/– (Slco1a/1b;Abcc3^–/–), Slco1a/1b^–/–Abcc2^–/– (Slco1a/1b;Abcc2^–/–), and Slco1a/1b^–/–Abcc2^–/–Abcc3^–/– (Slco1a/1b;Abcc2;Abcc3^–/–) mice by crossbreeding of existing strains. All strains were fertile, with normal life spans and body weights. As previously found for Abcc2^–/– and Abcc2^–/–Abcc3^–/– mice (26, 27), liver weights of Slco1a/1b;Abcc2^–/– and Slco1a/1b;Abcc2;Abcc3^–/– mice were significantly increased (~30% and ~50%, respectively) compared with wild-type mice (data not shown). Quantitative RT-PCR analysis of functionally relevant uptake and efflux transporters in liver, kidney, and intestine of the single and combination knockout strains revealed only some modest expression changes (Supplemental Table 1 and Supplemental Results; supplemental material available online with this article; doi: 10.1172/JCI59526DS1). Hepatic UDP-glucuronosyltransferase 1a1 (Ugt1a1) expression was not significantly altered in any of the strains.

Importantly, the markedly increased plasma bilirubin monoglucuronide (BMG) and bilirubin diglucuronide (BDG) levels observed in Slco1a/1b^–/– mice were substantially reduced in Slco1a/1b;Abcc3^–/– mice, demonstrating that Abcc3 is necessary for most of this increase (Figure 1, A and B). Plasma BMG levels in Slco1a/1b;Abcc2^–/– mice, even further increased owing to strongly reduced biliary BMG excretion (Figure 2, A and B), were similarly decreased in Slco1a/1b;Abcc2;Abcc3^–/– mice (Figure 1, A and B). Thus, Abcc3 secretes bilirubin glucuronides back into blood, and Oatp1a/1b proteins mediate their efficient hepatic reuptake, thereby together establishing a sinusoidal liver-blood shuttling loop. The incomplete reversion of plasma bilirubin glucuronide levels in the Oatp1a/1b/Abcc3-deficient strains (Figure 1, A and B) suggests that additional sinusoidal exporter(s), e.g., Abcc4 (28), can partly take over the sinusoidal bilirubin glucuronide extrusion function of Abcc3.

Figure 1

Increased plasma bilirubin glucuronide in Slco1a/1b^–/– mice is in part dependent on Abcc3. (A) BMG, (B) BDG, and (C) UCB levels in plasma of male wild-type, Abcc3^–/–, Abcc2^–/–, Abcc2^–/–Abcc3^–/–, Slco1a/1b^–/–, Slco1a/1b;Abcc3^–/–, Slco1a/1b;Abcc2^–/–, and Slco1a/1b;Abcc2;Abcc3^–/– mice (n = 4–7). +Oatp1a/1b denotes strains possessing Oatp1a/1b proteins, and –Oatp1a/1b denotes strains lacking Oatp1a/1b proteins. Data are mean ± SD. *P < 0.05, ***P < 0.001 compared with wild-type mice. Bracketed comparisons: ^†P < 0.05, ^††P < 0.01, ^†††P < 0.001. ND, not detectable; detection limit was 0.1 μM.

Figure 2

In the presence of Oatp1a/1b, but not in its absence, Abcc3 enhances biliary excretion of bilirubin glucuronides. (A) BMG, (B) BDG, and (C) UCB output in bile of male wild-type, Abcc3^–/–, Abcc2^–/–, Abcc2^–/–Abcc3^–/–, Slco1a/1b^–/–, Slco1a/1b;Abcc3^–/–, Slco1a/1b;Abcc2^–/–, and Slco1a/1b;Abcc2;Abcc3^–/– mice. Bile collected during the first 15 minutes after gall bladder cannulation was analyzed. +Oatp1a/1b denotes strains possessing Oatp1a/1b proteins, and –Oatp1a/1b denotes strains lacking Oatp1a/1b proteins. Data are shown as mean ± SD (n = 4–7). **P < 0.01, ***P < 0.001 compared with wild-type mice. Bracketed comparisons: ^†P < 0.05, ^††P < 0.01.

The biliary output of bilirubin glucuronides in the single and combination knockout mice showed that, as long as Oatp1a/1b was functional, Abcc3 improved the efficiency of biliary bilirubin glucuronide excretion, even though it transports its substrates initially from liver to blood, not bile (Figure 2, A and B, strains +Oatp1a/1b). This suggests that, within liver lobules, the bilirubin glucuronide extruded by Abcc3 in upstream hepatocytes is efficiently taken up in downstream hepatocytes via Oatp1a/1b and then excreted into bile. The resulting relief of possible saturation of (or competition for) biliary excretion in the upstream hepatocytes may explain why the overall biliary excretion is enhanced by this transfer to downstream hepatocytes. However, when Oatp1a/1b was absent, Abcc3 instead decreased biliary bilirubin glucuronide excretion (Figure 2, strains –Oatp1a/1b) and redirected excretion toward urine via the increased plasma bilirubin glucuronide levels (Supplemental Figure 1). Obviously, in the absence of Oatp1a/1b-mediated hepatic reuptake, Abcc3 activity can only decrease hepatocyte levels of bilirubin glucuronide in upstream and downstream hepatocytes alike, and will therefore reduce overall biliary excretion. Thus, both components of the Abcc3 and Oatp1a/1b shuttling loop are necessary to improve hepatobiliary excretion efficiency.

Human hepatocytes express only two OATP1A/1B proteins at the sinusoidal membrane, OATP1B1 and OATP1B3 (15). To test whether these could mediate the identified Oatp1a/1b functions, and in a liver-specific manner, we generated Slco1a/1b^–/– mice with liver-specific expression of either human OATP1B1 or OATP1B3. Liver-specific expression was obtained using an apoE promoter (29). These strains were viable and fertile, and displayed normal life spans and body weights. Liver levels of transgenic OATP1B1 and OATP1B3 proteins were similar to those seen in pooled human liver samples (data not shown). Both of the transgenic rescue strains displayed a virtually complete reversal of the increases in plasma and urine levels of BMG and BDG seen in Slco1a/1b^–/– mice (Figure 3, A and B, and Supplemental Figure 2). This indicates that both human OATP1B1 and OATP1B3 effectively reabsorb bilirubin glucuronides from plasma into the liver, in line with their demonstrated in vitro role in bilirubin glucuronide uptake (30). The modest (~1.8-fold) increase in plasma UCB in Slco1a/1b^–/– mice was also reduced in the rescue strains (Figure 3C), suggesting an ancillary role of these proteins in hepatic UCB uptake.

Figure 3

Increased plasma bilirubin glucuronide in Slco1a/1b^–/– mice is reversed by human OATP1B1 and OATP1B3. (A) BMG, (B) BDG, and (C) UCB levels in plasma of male wild-type and Slco1a/1b^–/– mice, and of the derived OATP1B1- and OATP1B3-transgenic strains (Slco1a/1b^–/–;1B1^tg and Slco1a/1b^–/–1B3^tg, respectively) (n = 5–8). Data are mean ± SD. **P < 0.01, ***P < 0.001 compared with wild-type mice. Bracketed comparisons: ^†††P < 0.001. Detection limit was 0.1 μM.

These findings collectively raised the question as to whether humans with a severe deficiency in OATP1B1 and OATP1B3, possibly leading to a conjugated hyperbilirubinemia, might exist. A literature search suggested RS as a candidate inborn metabolic disorder. A search for RS subjects by part of the present group led to collaboration with another team already working on mapping of the RS gene(s).

In an unbiased approach, scanning the whole genome, we mapped the genomic candidate intervals for RS in 11 RS index subjects from 8 different families, 4 Central European (CE1–CE4), 3 Saudi-Arabian (A1–A3), and 1 Filipino (P1) (Figure 4A and Supplemental Table 2). Homozygosity mapping identified a single genomic region on chromosome 12 for which 8 tested index subjects and no healthy siblings or parents were homozygous (Figure 4B), suggesting inheritance of both alleles from a common ancestor. Three distinct homozygous haplotypes (R1–R3) segregated with RS: R1 in families CE1, CE2, and CE4; R2 in families CE3, A1, A2, and A3; and R3 in family P1 (Figure 4B; for genotyping details, see Methods). Intersection of these haplotypes defined a candidate genomic region spanning the SLCO1C1, SLCO1B3, SLCO1B1, SLCO1A2, and IAPP genes (Figure 4B). A parallel genome-wide copy number analysis detected a homozygous deletion within the SLCO1B3 gene in the R1 haplotype and a homozygous approximately 405-kb deletion encompassing SLCO1B3 and SLCO1B1 and the LST-3TM12 pseudogene in the R2 haplotype (Figure 4B and Supplemental Figure 3).

Figure 4

RS families display deficiencies in SLCO1B1 and SLCO1B3. (A) Pedigrees of the investigated families. Black symbols denote RS index subjects. Parents in family CE4 had a documented common ancestor. Families CE1–CE3 (only single individuals analyzed) are not shown. (B) Homozygosity regions in 8 RS index subjects and overview of detected mutations and polymorphisms. The genome map shows number and location of overlapping homozygosity regions in RS index subjects, gene content of the top candidate region on chromosome 12, and the genotypes forming all 3 identified RS haplotypes. Mutations crucial for RS are shown in red. chr, chromosome. (C) Sequences and electropherograms of the R1 and R2 deletion breakpoints. (D) Pathogenic point mutations in R1 and R3 haplotypes. Electropherograms indicate the c.1738C→T (p.R580X) mutation in SLCO1B1 in probands CE1, CE2, and CE4 II.1 and the c.757C→T (p.R253X) and c.1747+1G→A mutations in SLCO1B1 and SLCO1B3, respectively, in family P1.

Sequence analysis revealed predictably pathogenic mutations affecting both SLCO1B3 and SLCO1B1 in each of the haplotypes (Figure 4, B–D, Table 1, Supplemental Figure 3, and Supplemental Table 3). In the R1 haplotype, a 7.2-kb deletion removes exon 12 of SLCO1B3, encoding amino acids 500–560 of OATP1B3 (702 aa long) and introduces a frameshift and premature stop codon, thus removing the C-terminal 3 transmembrane domains. Furthermore, a nonsense mutation in exon 13, c.1738C→T, introduces a premature stop codon (p.R580X) in R1-linked OATP1B1 (691 aa long), removing the C-terminal one-and-a-half transmembrane domains. The 405-kb R2 deletion encompasses exons 3–15 of SLCO1B3 (sparing only a small N-terminal region) and the whole of SLCO1B1, but not SLCO1A2. The R3 haplotype harbors a splice donor site mutation, c.1747+1G→A, in intron 13 of SLCO1B3. If SLCO1B3 is still yielding functional mRNA, this would truncate OATP1B3 after amino acid 582, deleting the C-terminal one-and-a-half transmembrane domains. A nonsense mutation, c.757C→T, in exon 8 of R3-linked SLCO1B1 introduces a premature stop (p.R253X), truncating OATP1B1 before the C-terminal 7 transmembrane domains. All of these mutations would severely disrupt or annihilate proper protein expression and function. Moreover, they all showed consistent autosomal recessive segregation with the RS phenotype in the investigated families (Table 1). No SLCO1A2 sequence variation was found in probands representing the 3 haplotypes, rendering involvement of OATP1A2 in RS unlikely. The severity of the identified mutations affecting SLCO1B3 and SLCO1B1 and their strict cosegregation with the RS phenotype indicate that RS is caused by co-inherited complete functional deficiencies in both OATP1B3 and OATP1B1.

Table 1

Mutations in SLCO1B genes detected in RS subjects and their family members

The severity of the mutations was independently supported by immunohistochemical studies of the sparse RS liver biopsy material available. Given their sparseness, immunostaining of these liver biopsies was performed using one antibody recognizing the N terminus of both OATP1B1 and OATP1B3 (31). This revealed absence of detectable staining in probands representing each haplotype (Figure 5). In controls, basolateral membranes of centrilobular hepatocytes stained crisply, as previously reported (31). Thus, the SLCO1B1 and SLCO1B3 mutations in each haplotype result in absence of a detectable signal for OATP1B protein in the liver.

Figure 5

Liver expression of OATP1B proteins in RS subjects and control. With an anti-OATP1B1/3 antibody, basolateral membrane immunostaining of hepatocytes in centrilobular areas was intense in control. Asterisks indicate central veins, arrowheads bile canaliculi, and crosses sinusoids. OATP1B proteins were not detectable in RS subjects CE1 (haplotype R1), CE3 (haplotype R2), and P1 II.1 (haplotype R3). Scale bars: 25 μm (original magnification of CE1 and CE3, ×400; original magnification of P1 II.1 and control, ×200); inset: 5 μm (original magnification, ×1,000).

In family A2, a heterozygous splice donor site mutation, c.481+1G→T, in intron 5 of SLCO1B1 would result in dysfunctional RNA or protein. Its co-occurrence with the 405-kb R2 deletion in two asymptomatic family members (Table 1) indicates that a single functional SLCO1B3 allele can prevent RS.

A search for copy number variations (CNVs) in existing databases and CNV genotyping of more than 2,300 individuals from various populations (see Supplemental Results) revealed additional heterozygous small and large deletions predicted to disrupt SLCO1B1 or SLCO1B3 function, including several approximately 400-kb deletions similar or identical to the R2 haplotype–linked deletion. One individual without jaundice, heterozygous for the R1 haplotype–associated c.1738C→T (p.R580X) mutation in SLCO1B1, was also homozygous for the R1 haplotype–associated deletion in SLCO1B3. Thus, a single functional SLCO1B1 allele can also prevent RS. Combined with the findings in family A2 described above, this demonstrates that only a complete deficiency of both alleles of SLCO1B1 and SLCO1B3 will result in RS.

We demonstrate here that RS is an obligate two-gene disorder, caused by a complete deficiency of the major hepatic drug uptake transporters OATP1B1 and OATP1B3. We further identified individuals with a complete deficiency of either OATP1B1 or OATP1B3, which was not recognizable by obvious jaundice. In spite of the documented important functions of especially OATP1B1 in drug detoxification, apparently such deficiencies are compatible with relatively normal life.

Using Oatp1a/1b-knockout mice, which can, retrospectively, be considered to be a partial model for RS, we showed that Abcc3 is an important factor for the RS-like conjugated hyperbilirubinemia. Our data imply that in the normal human liver ABCC3, OATP1B1, and OATP1B3 may form a liver-blood shuttling loop for bilirubin glucuronide, similar to that driven by Oatp1a/1b and Abcc3 in the mouse (Figure 6). A substantial fraction of bilirubin conjugated in hepatocytes is secreted back into the blood by ABCC3 and subsequently reabsorbed in downstream hepatocytes by OATP1B1 and OATP1B3. In RS this reuptake is hampered, causing increased plasma bilirubin glucuronide levels and jaundice. The flexible “hepatocyte hopping” afforded by this loop facilitates efficient detoxification, presumably by circumventing saturation of further detoxification processes in upstream hepatocytes, including excretion into bile. Indeed, we could show that, counterintuitively, but in accordance with the hepatocyte hopping model, loss of Abcc3 in mice resulted in decreased biliary excretion of bilirubin glucuronide, as long as Oatp1a/1b was present (Figure 2). This process likely also enhances hepatic detoxification of numerous drugs and drug conjugates (e.g., glucuronide, sulfate, and glutathione conjugates) transported by OATP1B1/3 and ABCC3. Moreover, this principle may also apply to other saturable hepatocyte detoxifying processes, such as phase I and phase II metabolism, as long as the substrate compounds involved are transported by ABCC3 and OATP1B proteins. Additional sinusoidal efflux and uptake transporters (e.g., ABCC4, OATP2B1, NTCP) will further widen the scope of compounds affected by this hepatocyte hopping process. Results obtained with the Slco1a/1b;Abcc3-knockout mice indeed show that in addition to Abcc3 there must be other sinusoidal efflux processes for bilirubin glucuronides. Preventing accumulation of drug glucuronides may be particularly important, since protein adduction by acyl-glucuronides is a well-established cause of drug (hepato)toxicity (32).

Figure 6

Hepatocyte hopping distributes the biliary excretion load of bilirubin glucuronides across the liver lobule. (A) Schematic of liver lobule. Hepatocytes are organized around portal tracts, with branches of the portal vein (PV), hepatic artery (HA), and bile ducts (BD). The PV and HA deliver nutrient- and oxygen-rich blood, respectively, which flows through the sinusoids toward the central vein (CV). Basolateral (sinusoidal) membranes of hepatocytes are flushed with perisinusoidal plasma. Bile flows in the opposite direction toward bile ducts through canaliculi lined by canalicular membranes of hepatocytes. (B) Hepatocyte hopping cycle. UCB enters the hepatocytes via passive diffusion and/or transporters, which may include OATP1B1 and/or OATP1B3 in non-Rotor subjects. Conjugation with glucuronic acid by UGT1A1 to bilirubin glucuronides (BG) takes place in endoplasmic reticulum. BG is secreted into bile mainly by ABCC2. ABCG2 also can contribute to this process. Even under physiological conditions, a substantial fraction of the intracellular BG is rerouted by ABCC3 to the blood, from which it can be taken up by downstream hepatocytes via OATP1B1/3 transporters. This flexible off-loading of BG to downstream hepatocytes prevents saturation of biliary excretion capacity in upstream hepatocytes. Relative type sizes of UCB and BG represent local concentrations. Schematic modified, with permission, from ref. 54.

One should exercise caution when extrapolating mouse data to humans, and the individual Oatp1a/1b proteins are not straightforward orthologs of human OATP1B1 and OATP1B3. However, there is a strong analogy between the bilirubin phenotypes of Oatp1a/1b-knockout mice and human Rotor subjects. Moreover, the hepatic transgenic expression of human OATP1B1 or OATP1B3 resulted in virtually complete rescue of the Oatp1a/1b-knockout phenotype for bilirubin handling (Figure 3). This strongly supports that the principles governing bilirubin handling by Oatp1a/1b in mouse liver also apply to OATP1B1 and OATP1B3 in human liver.

Analogous to the mouse data for Oatp1a/1b (25), the extensive glucuronidation of bilirubin in Rotor subjects suggests that OATP1B1 and/or OATP1B3 are not strictly essential for uptake of UCB into the liver. Passive transmembrane diffusion is one likely candidate to take over this process, in hepatocytes and probably many other cell types as well (e.g., ref. 33), but we do not exclude that additional uptake transporters (perhaps OATP2B1) can also contribute to UCB uptake. However, OATP1B1 and/or OATP1B3 probably do contribute to hepatic UCB uptake, since in RS subjects a significant increase in plasma UCB is usually observed and reduced clearance of UCB has been reported (34, 35). Moreover, polymorphisms in SLCO1B1 and SLCO1B3 have been associated with increased serum UCB levels (36, 37). There was also a significant, nearly 2-fold increase in plasma UCB in the Slco1a/1b^–/– mice, and this was partially reversed by both human OATP1B1 and OATP1B3 expression (Figure 3C).

It should be noted that UGT1A1-mediated glucuronidation may also occur in extrahepatic tissues, for instance, colon (38), and we cannot exclude that some of the bilirubin glucuronide observed in RS plasma has resulted from such extrahepatic glucuronidation, possibly enhanced by the increased plasma UCB levels. It seems unlikely, however, that all bilirubin glucuronide in RS subjects would derive from extrahepatic glucuronidation. This would require a complete block of hepatic UCB uptake (due to the OATP1B1 and OATP1B3 deficiency), but at the same time require efficient uptake of UCB into UGT1A1-containing extrahepatic cells (e.g., colonocytes) that do not normally express OATP1B1 and OATP1B3, and certainly not in Rotor subjects. If UCB transmembrane diffusion can do this efficiently, it is hard to see why this would not mediate substantial uptake into the liver as well. Only if hepatic diffusion uptake is negligible (which seems physically unlikely) and an unknown efficient UCB uptake system would function in colonocytes (and not in liver), could one envisage such a situation. On balance, this seems rather implausible.

Elucidation of OATP1B1 and OATP1B3 deficiency as the cause of RS can also readily explain the other diagnostic traits of the disorder. Absence of OATP1B1/3-mediated liver uptake would cause the decreased plasma clearance of anionic diagnostic dyes such as indocyanine green and BSP, an excellent substrate of OATP1B1 and OATP1B3 (15), and the greatly reduced or delayed visualization of the liver by anionic cholescintigraphic radiotracers such as ^99mTc-HIDA and ^99mTc-mebrofenin (5, 6). ^99mTc-mebrofenin, for instance, is efficiently transported by both OATP1B1 and OATP1B3 (39).

The markedly increased urinary excretion of coproporphyrins, and the increased preponderance of isomer I over III in urine of RS subjects, could be simply explained by reduced (re)uptake of these compounds into the liver, partly shifting the excretion route from hepatobiliary/fecal to urinary, especially for isomer I. Coproporphyrin I and III thus most likely are transported substrates of OATP1B1 and OATP1B3. Indeed, interaction of several porphyrins with OATP1B1 has recently been demonstrated (40).

Phenotypic abnormalities in RS subjects are surprisingly moderate. Perhaps OATP1B1 and OATP1B3 functions are partly taken over by other sinusoidal uptake transporters, such as OATP2B1. Nevertheless, since even reduced-activity OATP1B1 polymorphisms can result in life-threatening drug toxicities (20–24, 41, 42), such risks are likely increased substantially in RS subjects. Their evident jaundice, however, may have been a warning sign for physicians to prescribe drugs with caution.

The obligatory deficiency in two different, medium-sized genes explains the rarity of RS, with a roughly estimated frequency of about 1 in 10⁶, although it might be several-fold lower or higher in different populations. Complete deficiency of either OATP1B1 or OATP1B3 alone will occur much more frequently but will not cause jaundice. For instance, the p.R580X mutation in OATP1B1 occurred at an allele frequency of 0.008 (3 of 354) in a Japanese population (43), suggesting that about 1 in 14,000 individuals in this population would be homozygous for this full-deficiency mutant. Such individuals might demonstrate idiosyncratic hypersensitivity to OATP1B1 substrate drugs, including statins or irinotecan. Similarly, in the present study we identified a non-jaundiced individual homozygously deficient for SLCO1B3 in our CNV screening of approximately 2,300 individuals, in line with a non-negligible incidence of fully OATP1B3-deficient individuals.

Some drugs, such as high-dose cyclosporine A, can transiently increase plasma levels of conjugated bilirubin without evoking other markers for liver damage (44, 45). Until now, such increases were thought to be primarily mediated by inhibition of ABCC2 as the main biliary excretion factor for bilirubin glucuronide. However, given the insights from the present study, direct inhibition of OATP1B1 and/or OATP1B3 by the applied drug may be an additional or even the main cause of such drug-induced conjugated hyperbilirubinemias. This might for instance apply to cyclosporine A, rifampin, rifamycin SV, or other drugs that are established inhibitors of OATP1B proteins (23). Moreover, heterozygous carriers of the various full-deficiency mutations in OATP1B1/3 might be more susceptible to such inhibitory effects. This also applies to drug-drug interactions mediated through OATP1B1/3 inhibition.

The molecular mechanism we identified in RS may also underlie a similar disorder called hepatic uptake and storage syndrome, or conjugated hyperbilirubinemia type III (OMIM %237550) (46). This hypothesis can now be tested by mutational analysis of OATP1B1 and OATP1B3 in the only reported family to date. Furthermore, a mutant strain of Southdown sheep has also been described as displaying a similar hepatic uptake and storage syndrome (46), and it would not surprise us if these animals would likewise have a deficiency of one or more hepatic sinusoidal OATPs. The observation that mutant Southdown sheep, like the Slco1a/1b^–/– mice (25), also display strongly reduced clearance of (unconjugated) cholic acid, but not of (conjugated) taurocholic acid (47), further supports this idea.

Collectively, our findings explain the genetic and molecular basis of RS. The demonstration of an Abcc3-, OATP1B1-, and OATP1B3-driven detoxification-enhancing liver-blood shuttling loop in mice and, by implication, most likely also in humans challenges the view of one-way excretion from blood through liver to bile of bilirubin and drugs detoxified by conjugation. Furthermore, the identified full-deficiency alleles of SLCO1B1 and SLCO1B3 may contribute to various “idiosyncratic” drug hypersensitivities.

View Supplemental data

The human study was supported in part by the Ministry of Education of the Czech Republic (projects MSM0021620806 and 1M6837805002) and by the Institute for Clinical and Experimental Medicine (MZO 00023001). The mouse work was supported in part by grant S2918 from GlaxoSmithKline and grant NKI 2007-3764 from the Dutch Cancer Society. The authors thank L. Budiaová and M. Boučková for technical assistance and L. Vítek, M. Mikulecký, J. Horák, and A. Šuláková for referring patients CE1–CE4.

Address correspondence to: Alfred H. Schinkel, Division of Molecular Biology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands. Phone: 31.20.5122046; Fax: 31.20.6961383; E-mail: a.schinkel@nki.nl. Or to: Milan Jirsa, Department of Experimental Medicine, Institute for Clinical and Experimental Medicine, Vídenská 1958/9, 140 00 Prague 4 — Krč, Czech Republic. Phone: 420.261362773; Fax: 420.241721666; E-mail: miji@ikem.cz.

Conflict of interest: The research group of Alfred H. Schinkel receives revenues from commercial distribution of some of the mouse strains used in this study.

Reference information: J Clin Invest. 2012;122(2):519–528. doi:10.1172/JCI59526.

1. Chowdhury JR, Chowdhury NR, Jansen PLM. Bilirubin metabolism and its disorders. In: Boyer TD, Wright TL, Manns MP, Zakim D, eds. Zakim and Boyer’s Hepatology. A Textbook of Liver Diseases . Vol. 2. Philadelphia, Pennsylvania, USA: Saunders Elsevier; 2006:1449–1474.
2. Chowdhury JR, Wolkoff AW, Chowdhury NR, Arias IM. Hereditary jaundice and disorders of bilirubin metabolism. In: Scriver CR, Beaudet AL, Sly WS, Valle D, eds. The Metabolic and Molecular Bases of Inherited Disease . Vol. 2. New York, New York, USA: McGraw Hill; 2001:3063–3101.
3. Rotor AB, Manahan L, Florentin A. Familial nonhemolytic jaundice with direct van den Bergh reaction. Acta Med Phil. 1948;5:37–49.
4. Wolpert E, Pascasio FM, Wolkoff AW, Arias IM. Abnormal sulfobromophthalein metabolism in Rotor‘s syndrome and obligate heterozygotes. N Engl J Med. 1977;296(19):1099–1101.
   View this article via: PubMed CrossRef Google Scholar
5. Bar-Meir S, Baron J, Seligson U, Gottesfeld F, Levy R, Gilat T. 99mTc-HIDA cholescintigraphy in Dubin-Johnson and Rotor syndromes. Radiology. 1982;142(3):743–746.
   View this article via: PubMed Google Scholar
6. LeBouthillier G, Morais J, Picard M, Picard D, Chartrand R, Pommier G. Scintigraphic aspect of Rotor‘s disease with Technetium-99m-mebrofenin. J Nucl Med. 1992;33(8):1550–1551.
   View this article via: PubMed Google Scholar
7. Kartenbeck J, Leuschner U, Mayer R, Keppler D. Absence of the canalicular isoform of the MRP gene-encoded conjugate export pump from the hepatocytes in Dubin-Johnson syndrome. Hepatology. 1996;23(5):1061–1066.
   View this article via: PubMed Google Scholar
8. Paulusma CC, et al. A mutation in the human canalicular multispecific organic anion transporter gene causes the Dubin-Johnson syndrome. Hepatology. 1997;25(6):1539–1542.
   View this article via: PubMed CrossRef Google Scholar
9. Nowicki MJ, Poley JR. The hereditary hyperbilirubinaemias. Baillieres Clin Gastroenterol. 1998;12(2):355–367.
   View this article via: PubMed CrossRef Google Scholar
10. Strassburg CP. Hyperbilirubinemia syndromes (Gilbert-Meulengracht, Crigler-Najjar, Dubin-Johnson, and Rotor syndrome). Best Pract Res Clin Gastroenterol. 2010;24(5):555–571.
    View this article via: PubMed CrossRef Google Scholar
11. Wolkoff AW, Wolpert E, Pascasio FN, Arias IM. Rotor’s syndrome. A distinct inheritable pathophysiologic entity. Am J Med. 1976;60(2):173–179.
    View this article via: PubMed CrossRef Google Scholar
12. König J, Rost D, Cui Y, Keppler D. Characterization of the human multidrug resistance protein isoform MRP3 localized to the basolateral hepatocyte membrane. Hepatology. 1999;29(4):1156–1163.
    View this article via: PubMed CrossRef Google Scholar
13. Lee YM, et al. Identification and functional characterization of the natural variant MRP3-Arg1297His of human multidrug resistance protein 3 (MRP3/ABCC3). Pharmacogenetics. 2004;14(4):213–223.
    View this article via: PubMed CrossRef Google Scholar
14. Hrebicek M, et al. Rotor-type hyperbilirubinaemia has no defect in the canalicular bilirubin export pump. Liver Int. 2007;27(4):485–491.
    View this article via: PubMed CrossRef Google Scholar
15. Hagenbuch B, Gui C. Xenobiotic transporters of the human organic anion transporting polypeptide (OATP) family. Xenobiotica. 2008;38(7–8):778–801.
    View this article via: PubMed CrossRef Google Scholar
16. Hagenbuch B, Meier PJ. Organic anion transporting polypeptides of the OATP/ SLC21 family: phylogenetic classification as OATP/ SLCO superfamily, new nomenclature and molecular/functional properties. Pflügers Arch. 2004;447(5):653–665.
    View this article via: PubMed Google Scholar
17. König J, Cui Y, Nies AT, Keppler D. A novel human organic anion transporting polypeptide localized to the basolateral hepatocyte membrane. Am J Physiol Gastrointest Liver Physiol. 2000;278(1):G156–G164.
    View this article via: PubMed Google Scholar
18. König J, Cui Y, Nies AT, Keppler D. Localization and genomic organization of a new hepatocellular organic anion transporting polypeptide. J Biol Chem. 2000;275(30):23161–23168.
    View this article via: PubMed CrossRef Google Scholar
19. Abe T, et al. LST-2, a human liver-specific organic anion transporter, determines methotrexate sensitivity in gastrointestinal cancers. Gastroenterology. 2001;120(7):1689–1699.
    View this article via: PubMed CrossRef Google Scholar
20. Takane H, et al. Life-threatening toxicities in a patient with UGT1A1*6/*28 and SLCO1B1*15/*15 genotypes after irinotecan-based chemotherapy. Cancer Chemother Pharmacol. 2009;63(6):1165–1169.
    View this article via: PubMed CrossRef Google Scholar
21. Link E, et al. SLCO1B1 variants and statin-induced myopathy — a genomewide study. N Engl J Med. 2008;359(8):789–799.
    View this article via: PubMed CrossRef Google Scholar
22. Treviño LR, et al. Germline genetic variation in an organic anion transporter polypeptide associated with methotrexate pharmacokinetics and clinical effects. J Clin Oncol. 2009;27(35):5972–5978.
    View this article via: PubMed CrossRef Google Scholar
23. Kalliokoski A, Niemi M. Impact of OATP transporters on pharmacokinetics. Br J Pharmacol. 2009;158(3):693–705.
    View this article via: PubMed CrossRef Google Scholar
24. König J, Seithel A, Gradhand U, Fromm MF. Pharmacogenomics of human OATP transporters. Naunyn Schmiedebergs Arch Pharmacol. 2006;372(6):432–443.
    View this article via: PubMed CrossRef Google Scholar
25. Van de Steeg E, 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(8):2942–2952.
    View this article via: JCI PubMed CrossRef Google Scholar
26. Vlaming ML, et al. Carcinogen and anticancer drug transport by Mrp2 in vivo: studies using Mrp2 (Abcc2) knockout mice. J Pharmacol Exp Ther. 2006;318(1):319–327.
    View this article via: PubMed CrossRef Google Scholar
27. Vlaming ML, et al. Impact of Abcc2 (Mrp2) and Abcc3 (Mrp3) on the in vivo elimination of methotrexate and its main toxic metabolite 7-hydroxymethotrexate. Clin Cancer Res. 2008;14(24):8152–8160.
    View this article via: PubMed CrossRef Google Scholar
28. Rius M, Nies AT, Hummel-Eisenbeiss J, Jedlitschky G, Keppler D. Cotransport of reduced glutathione with bile salts by MRP4 (ABCC4) localized to the basolateral hepatocyte membrane. Hepatology. 2003;38(2):374–384.
    View this article via: PubMed Google Scholar
29. Van de Steeg E, et al. Methotrexate pharmacokinetics in transgenic mice with liver-specific expression of human OATP1B1 (SLCO1B1). Drug Metab Dispos. 2009;37(1):1–5.
    View this article via: PubMed CrossRef Google Scholar
30. Cui Y, König J, Leier I, Buchholz U, Keppler D. Hepatic uptake of bilirubin and its conjugates by the human organic anion transporter SLC21A6. J Biol Chem. 2001;276(13):9626–9630.
    View this article via: PubMed CrossRef Google Scholar
31. Cui Y, et al. Detection of the human organic anion transporters SLC21A6 (OATP2) and SLC21A8 (OATP8) in liver and hepatocellular carcinoma. Lab Invest. 2003;83(4):527–538.
    View this article via: PubMed Google Scholar
32. Zhou S, Chan E, Duan W, Huang M, Chen YZ. Drug bioactivation, covalent binding to target proteins and toxicity relevance. Drug Metab Rev. 2005;37(1):41–213.
    View this article via: PubMed Google Scholar
33. Zucker SD, Goessling W, Hoppin AG. Unconjugated bilirubin exhibits spontaneous diffusion through model lipid bilayers and native hepatocyte membranes. J Biol Chem. 1999;274(16):10852–10862.
    View this article via: PubMed CrossRef Google Scholar
34. Kawasaki H, Kimura N, Irisa T, Hirayama C. Dye clearance studies in Rotor‘s syndrome. Am J Gastroenterol. 1979;71(4):380–388.
    View this article via: PubMed Google Scholar
35. Fedeli G, et al. Impaired clearance of cholephilic anions in Rotor syndrome. Z Gastroenterol. 1983;21(5):228–233.
    View this article via: PubMed Google Scholar
36. Zhang W, 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(12):1240–1244.
    View this article via: PubMed CrossRef Google Scholar
37. Sanna S, et al. Common variants in the SLCO1B3 locus are associated with bilirubin levels and unconjugated hyperbilirubinemia. Hum Mol Genet. 2009;18(14):2711–2718.
    View this article via: PubMed CrossRef Google Scholar
38. Strassburg CP, Manns MP, Tukey RH. Expression of the UDP-glucuronosyltransferase 1A locus in human colon. Identification and characterization of the novel extrahepatic UGT1A8. J Biol Chem. 1998;273(15):8719–8726.
    View this article via: PubMed CrossRef Google Scholar
39. Ghibellini G, Leslie EM, Pollack GM, Brouwer KL. Use of tc-99m mebrofenin as a clinical probe to assess altered hatobiliary transport: integration of in vitro, pharmacokinetic modeling, and simulation studies. Pharm Res. 2008;25(8):1851–1860.
    View this article via: PubMed CrossRef Google Scholar
40. Campbell SD, Lau WF, Xu JJ. Interaction of porphyrins with human organic anion transporting polypeptide 1B1. Chem Biol Interact. 2009;182(1):45–51.
    View this article via: PubMed CrossRef Google Scholar
41. Morimoto K, Oishi T, Ueda S, Ueda M, Hosokawa M, Chiba K. A novel variant allele of OATP-C (SLCO1B1) found in a Japanese patient with pravastatin-induced myopathy. Drug Metab Pharmacokinet. 2004;19(6):453–455.
    View this article via: PubMed CrossRef Google Scholar
42. Tirona RG, Kim RB. Pharmacogenomics of organic anion-transporting polypeptides (OATP). Adv Drug Deliv Rev. 2002;54(10):1343–1352.
    View this article via: PubMed CrossRef Google Scholar
43. Kim SR, et al. Genetic variations and frequencies of major haplotypes in SLCO1B1 encoding the transporter OATP1B1 in Japanese subjects: SLCO1B1*17 is more prevalent than *15. Drug Metab Pharmacokinet. 2007;22(6):456–461.
    View this article via: PubMed CrossRef Google Scholar
44. Yahanda AM, et al. Phase I trial of etoposide with cyclosporine as a modulator of multidrug resistance. J Clin Oncol. 1992;10(10):1624–1634.
    View this article via: PubMed Google Scholar
45. List AF, et al. Phase I/II trial of cyclosporine as a chemotherapy-resistance modifier in acute leukemia. J Clin Oncol. 1993;11(9):1652–1660.
    View this article via: PubMed Google Scholar
46. Dhumeaux D, Berthelot P. Chronic hyperbilirubinemia associated with hepatic uptake and storage impairment. A new syndrome resembling that of mutant southdown sheep. Gastroenterology. 1975;69(4):988–993.
    View this article via: PubMed Google Scholar
47. Engelking LR, Gronwall R. Bile acid clearance in sheep with hereditary hyperbilirubinemia. Am J Vet Res. 1979;40(9):1277–1280.
    View this article via: PubMed Google Scholar
48. Zelcer N, et al. Mice lacking Mrp3 (Abcc3) have normal bile salt transport, but altered hepatic transport of endogenous glucuronides. J Hepatol. 2006;44(4):768–775.
    View this article via: PubMed CrossRef Google Scholar
49. Van Waterschoot RA, et al. Midazolam metabolism in cytochrome P450 3A knockout mice can be attributed to up-regulated CYP2C enzymes. Mol Pharmacol. 2008;73(3):1029–1036.
    View this article via: PubMed CrossRef Google Scholar
50. Van Herwaarden AE, et al. The breast cancer resistance protein (Bcrp1/Abcg2) restricts exposure to the dietary carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine. Cancer Res. 2003;63(19):6447–6452.
    View this article via: PubMed Google Scholar
51. Spivak W, Carey MC. Reverse-phase h.p.l.c. separation, quantification and preparation of bilirubin and its conjugates from native bile. Quantitative analysis of the intact tetrapyrroles based on h.p.l.c. of their ethyl anthranilate azo derivatives. Biochem J. 1985;225(3):787–805.
    View this article via: PubMed Google Scholar
52. Abecasis GR, Cherny SS, Cookson WO, Cardon LR. Merlin — rapid analysis of dense genetic maps using sparse gene flow trees. Nat Genet. 2002;30(1):97–101.
    View this article via: PubMed CrossRef Google Scholar
53. Thiele H, Nurnberg P. HaploPainter: a tool for drawing pedigrees with complex haplotypes. Bioinformatics. 2005;21(8):1730–1732.
    View this article via: PubMed CrossRef Google Scholar
54. Van de Steeg E, Iusuf D, Schinkel AH. Physiological and pharmacological functions of OATP1A/1B transporters: insights from knockout and transgenic mice. In: Van de Steeg E. Physiological and pharmacological functions of OATP1A/1B transporters. PhD thesis, University of Utrecht. Enschede, the Netherlands: Gildeprint drukkerijen; 2010:9–37 .