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J Clin Invest. 2012 February 1; 122(2): 519–528.
Published online 2012 January 9. doi:  10.1172/JCI59526
PMCID: PMC3266790

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 (16). 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 (1519). 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 (2024).

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 (Figure1,1, A and B). Plasma BMG levels in Slco1a/1b;Abcc2–/– mice, even further increased owing to strongly reduced biliary BMG excretion (Figure (Figure2,2, A and B), were similarly decreased in Slco1a/1b;Abcc2;Abcc3–/– mice (Figure (Figure1,1, 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 (Figure1,1, 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.
Figure 2
In the presence of Oatp1a/1b, but not in its absence, Abcc3 enhances biliary excretion of bilirubin glucuronides.

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 (Figure2,2, 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 (Figure2,2, 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 (Figure3,3, 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 (Figure3C),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.

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 (Figure4A4A 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 (Figure4B),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 (Figure4B;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 (Figure4B).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 (Figure4B4B and Supplemental Figure 3).

Figure 4
RS families display deficiencies in SLCO1B1 and SLCO1B3.

Sequence analysis revealed predictably pathogenic mutations affecting both SLCO1B3 and SLCO1B1 in each of the haplotypes (Figure (Figure4,4, B–D, Table Table1,1, 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 (Table1).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 (Figure5).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.

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 (Table1)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 (Figure6).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 (Figure2).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.

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 (Figure3).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 (Figure3C). 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 (2024, 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.


Mouse strains and conditions.

Mice were housed and handled according to institutional guidelines complying with Dutch legislation. Slco1a/1b–/–, Abcc2–/–, Abcc3–/–, and Abcc2–/–Abcc3–/– mice have been described (2527, 48). Human OATP1B1 transgenic mice have been described (29), and human OATP1B3 transgenic mice were generated in an analogous manner, using an apoE promoter to obtain liver-specific expression of the transgene. Each transgene was crossed back into an Slco1a/1b–/– background to obtain the corresponding humanized rescue strains. Routine mouse conditions and analyses of mouse samples are described in Supplemental Methods.

Western blot analysis.

Isolation of crude membrane fractions from mouse liver, kidney, and small intestine and Western blotting were as described previously (29). For detection of Abcc2 and Abcc3 primary antibodies, M2III-5 (dilution 1:1,000) and M3-18 (dilution 1:25) were used, respectively. For detection of transgenic OATP1B1 and OATP1B3 in mouse liver, the rabbit polyclonal antibodies ESL and SKT, provided by D. Keppler (Deutsches Krebsforschungszentrum, Heidelberg, Germany) were used (17, 18).

RNA isolation, cDNA synthesis, and RT-PCR.

RNA isolation from mouse liver, kidney, and small intestine and subsequent cDNA synthesis and RT-PCR were as described previously (49). Specific primers (QIAGEN) were used to detect expression levels of Slco1a1, Slco1a4, Slco1a6, Slco1b2, Slco2b1, Slc10a1, Slc10a2, Abcc2–4, Abcb1a, Abcb1b, Abcb11, Abcg2, Osta, Ostb, and Ugt1a1.

Analysis of bilirubin in mouse plasma, bile, and urine.

Gallbladder cannulations and collection of bile and urine in male mice of the various strains (n = 4–7) as well as bilirubin detection were as described (25, 50, 51). For details, see Supplemental Methods.

RS families.

We examined 11 RS index subjects (8 probands, 3 siblings of probands) of 8 families and 21 clinically healthy members of 5 of these 8 families. Family members of 3 probands (CE1–CE3) were not available. Families CE1–CE4 are of mixed Central European descent by family report. Three families (A1–A3) are Saudi Arabs, and one family (P1) is from the Philippines. Central European families were ascertained at the Institute for Clinical and Experimental Medicine, Prague, and Saudi Arab and Filipino families at the Saudi Aramco Dhahran Health Center. Medical histories were obtained by referring consultants. Subjects CE1 and CE2 were reported as case 1 and case 2, respectively (14).

ABCC2 mutation screening.

ABCC2 mutation screening was performed in 8 probands representing all studied families as described previously (14).


Genotyping was performed using Affymetrix GeneChip Mapping 6.0 Arrays (Affymetrix) according to the manufacturer’s protocol. Raw feature intensities were extracted from Affymetrix GeneChip Scanner 3000 7G images using GeneChip Control Console Software 2.01. Individual SNP calls were generated using Affymetrix Genotyping Console Software 3.02. Details of the experiment and individual genotyping data are available at the GEO repository ( under accession number GSE33733.

Multipoint nonparametric and parametric linkage analysis.

Multipoint nonparametric and parametric linkage analysis along with determination of the most likely haplotypes was performed with version 1.1.2 of Merlin software (52). Parametric linkage was carried out assuming an autosomal recessive mode of inheritance with a 1.00 constant, age-independent penetrance, 0.00 phenocopy rate, and 0.0001 frequency of disease allele. Results were visualized in version 1.032 of HaploPainter software (53) and in version 2.9.2 of R-project statistical software (

Homozygosity mapping.

Extended homozygosity regions were identified in Affymetrix Genotyping Console Software version 3.02 using the algorithm comparing values from the user’s sample set and SNP-specific distributions derived from a reference set of 200 ethnically diverse individuals. Distribution of extended homozygosity regions in affected and healthy individuals was analyzed and visualized using custom R-script.

Copy number changes.

Copy number changes were identified in Affymetrix Genotyping Console Software version 3.02. Data from both SNP and copy number probes were used to identify copy number aberrations compared with built-in reference. Only regions larger than 10 kb containing at least 5 probes were reported.

Quantitative PCR.

Quantitative PCR was carried out in duplicate on a LightCycler 480 System (Roche Applied Science). Data were analyzed by LightCycler 480 Software, release 1.5.0. Absolute quantification was used to determine copy number status of a given fragment in analyzed samples. Genomic positions of the analyzed fragments and control genes, corresponding primer sequences, and Universal ProbeLibrary probes used for amplification and quantitation are provided in Supplemental Table 4.

Mutation analysis.

Long-range PCR products encompassing the genomic regions of deletion breakpoint boundaries were gel-purified and sequenced using a primer walking approach. DNA sequencing of PCR products and genomic fragments covering 1 kb of the promoter regions and all of the exons, with their corresponding exon-intron boundaries, of SLCO1B1, SLCO1B3, and SLCO1A2 was performed. For details, see Supplemental Methods. Confirmation and segregation of both identified copy number changes and missense mutations in the families, as well as frequency of the mutations in a control population of mixed European descent, were assessed by PCR, PCR-RFLP, and direct sequencing of corresponding genomic DNA fragments. For primer sequences, see Supplemental Table 4.

Histology and immunohistochemistry.

Archival liver biopsy specimens were available from 5 unrelated RS index subjects (probands, families CE1, CE2, CE3, and P1; brother [A3 II.9] of proband from family A3). Sections of paraffin-embedded material (formalin or Carnoy solution fixative; 4–6 μm thick) were routinely stained with hematoxylin and eosin and periodic acid–Schiff techniques. For OATP1B1, OATP1B3, and ABCC2 immunostaining, routine techniques were applied (see Supplemental Methods). OATP1B1 and OATP1B3 detection was performed with a primary mouse anti-OATP1B antibody (clone mMDQ, GeneTex; recognizing the N terminus of both OATP1B1 and OATP1B3), 1:100 dilution, overnight at 4°C (31).


One-way ANOVA followed by Tukey’s multiple comparison test was used to assess statistical significance of differences between data sets. Results are presented as mean ± SD. Differences were considered statistically significant when P was less than 0.05.

Study approval.

All mouse studies were ethically reviewed and carried out in accordance with European directive 86/609/EEC and Dutch legislation and the GlaxoSmithKline policy on the Care, Welfare and Treatment of Laboratory Animals. Experiments were approved by the Animal Experimentation Committee (DEC) of the Netherlands Cancer Institute. Investigations involving humans were approved by the Institutional Review Boards of the Institute for Clinical and Experimental Medicine, Prague, Czech Republic, and the Saudi Aramco Dhahran Health Centre, with written informed consent received from participants or their guardians, and conducted according to Declaration of Helsinki principles.

Supplementary Material

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.


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.

Citation for this article: J Clin Invest. 2012;122(2):519–528. doi:10.1172/JCI59526.


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