A mutation in the canalicular phospholipid transporter gene, ABCB4, is associated with cholestasis, ductopenia, and cirrhosis in adults

Authors


  • A portion of this work was presented as a poster at the 58th Meeting of the American Association for the Study of Liver Diseases, Boston, MA, November 2–6, 2007.

  • Potential conflict of interest: Nothing to report.

Abstract

Cholestatic liver disease (CLD) is a major cause of progressive liver damage and liver failure. Several forms of biliary cirrhosis are caused by mutations in specific genes. We sought to identify a genetic defect in a family with CLD impossible to assign to a distinct pathogenic entity. Clinical and histopathological characterization of the family members, microarray-based single-nucleotide polymorphism genotyping, and analysis of candidate genes were performed. Among six of 11 siblings severely affected by idiopathic CLD in a family from a population isolate in Transylvania, three died of cirrhosis (aged 5, 7, and 43 years) and three had adult-onset disease with small duct cholangiopathy, including ductopenia. Others were mildly affected and experienced intrahepatic cholestasis of pregnancy, miscarriages, or stillbirth. Pedigree studies revealed distant parental consanguinity. Genome-wide linkage analysis and autozygosity mapping yielded a single maximal lod-score of 3.88 on chromosome 7q21.1-7q22, excluding other genomic loci. Sequencing of ABCB4 at this locus revealed a novel missense mutation c.2362C>T (p.Arg788Trp) which cosegregated with severity of disease. Bile from a mutation homozygote showed a reduced phosphatidylcholine/bile acid ratio, consistent with reduced ABCB4 phosphatidylcholine transport activity. Conclusion: We show that a missense mutation in ABCB4 is a cause for ductopenic CLD in adulthood. Allelic status correlated with severity of liver disease ranging from intrahepatic cholestasis of pregnancy through fibrosis to cirrhosis and death in childhood and adulthood. Mutational analysis of ABCB4 should be generally considered in all patients with cholestatic liver disease of unknown etiology regardless of age and onset of disease. (HEPATOLOGY 2008.)

Chronic cholestatic liver disease (CLD) is a common cause of progressive liver damage, leading to cirrhosis, liver failure, and death. Its etiologies and clinical characteristics are diverse. Two frequent causes of bile duct destruction are primary biliary cirrhosis and primary sclerosing cholangitis. Whereas in primary biliary cirrhosis immunogenic factors have been suggested to cause progressive destruction of bile ducts, the pathogenesis of primary sclerosing cholangitis is less clear.1, 2 In addition, several genetic defects have recently been described as causative for cholestatic liver disease opening new perspectives for targeted therapies.3 Mutations in TJP2 encoding the tight-junction protein TJP2 and in BAAT, encoding bile acid coenzyme A/amino acid N-acyltransferase can underlie familial hypercholanemia,4 and variants in CLDN1 are associated with neonatal ichthyosis-sclerosing cholangitis.5 Lack of bile ducts in portal tracts (ductopenia) is a hallmark of Alagille syndrome, an autosomal-dominant genetic disorder frequently caused by mutations in JAG1.6 Cholestasis is also observed in certain inborn disorders of bile acid metabolism.7 Moreover, in progressive familial intrahepatic cholestasis (PFIC), which is characterized by biliary cirrhosis in childhood, mutations in genes encoding canalicular transporters have been described. PFIC has been classified into three distinct clinical subtypes (PFIC1-3), attributed respectively to mutations in ATP8B1 (encoding adenosine triphosphatase [ATPase], class I, type 8B, member 1/phospholipid-transporting ATPase IC, FIC1), ABCB11 (encoding ATP-binding cassette, subfamily B [MDR/TAP], member 11/bile salt export pump) and ABCB4 (encoding ATP-binding cassette, subfamily B, member 4/multidrug resistance protein, MDR3, mediating efflux of phospholipids, mainly phosphatidylcholine [PC] into the bile). These genes were identified via linkage analysis (ATP8B1 and ABCB11)8, 9 or as an ortholog of a gene mutated in mice with biliary cirrhosis (ABCB4).10 Genetic causes are assumed for other CLDs, among them biliary cirrhosis of unknown cause and idiopathic adulthood ductopenia (IAD).

Here we describe a family from a population isolate in Transylvania in which 6 of 11 siblings were severely affected by familial CLD of unknown cause. As the disease pattern was unusual for known cholestatic diseases, we performed linkage analysis via single nucleotide polymorphism (SNP) genotyping. We demonstrate that a single sequence variant in ABCB4 as causative for chronic CLD and biliary cirrhosis in adulthood.

Abbreviations

AP alkaline phosphatase; CLD cholestatic liver disease; GGT γ-glutamyltransferase; IAD idiopathic adulthood ductopenia; ICP intrahepatic cholestasis of pregnancy; LFT, liver function test; MRP2, multidrug resistance-associated protein 2; MDR3, multidrug resistance protein 3; PC phosphatidylcholine; PFIC progressive familial intrahepatic cholestasis; SNP, single-nucleotide polymorphism; UDCA ursodeoxycholic acid.

Patients and Methods

Clinical Characterization of the Index Patient and the Family.

We aimed at identifying a genetic locus underlying CLD in the 34-year-old female index patient (I.1, Fig. 1), who suffered from idiopathic adulthood biliary cirrhosis. Liver function tests (LFTs; serum concentrations of bilirubin, bile acids, and transaminase, alkaline phosphatase [AP], and γ-glutamyltransferase [GGT] activities), autoantibodies, and markers of hepatotropic-virus infection were assessed in her, in seven of her siblings, and in her parents. Descriptions of disease in deceased siblings came from oral reports and medical records. Detailed clinical histories allowed classification as (1) severely affected, with abnormal LFTs for >3 months or definite histopathological changes; (2) mildly affected, with intrahepatic cholestasis of pregnancy (ICP) or only subtle histopathological changes; or (3) unaffected, without evidence of CLD. The local ethics committee approved the study, and written informed consent was obtained.

Figure 1.

(A) Family of the index patient (I.1). Siblings I.2-I.6 (black) were considered severely affected by CLD. Siblings I.7–I.11 and the parents (II.1 and II.2) were considered unaffected (II.1) or mildly affected by CLD (II.2; I.7–I.11). (B,C) Selected LFTs in (B) the index patient and (C) the severely affected sister I.6. Note the marked reduction of LFTs upon administration of UDCA (arrows) and later signs of progression to liver failure in the index patient.

Histology and Immunohistochemistry.

Liver tissue samples were fixed in 4% formaldehyde, embedded in paraffin, sectioned (4 μm), and stained with hematoxylin-eosin, diastase/periodic acid–Schiff, Perls', and modified Gömöri's silver techniques. Immunostaining for cytokeratin 7 delineated biliary epithelium. Specialty immunostaining (DAKO Chem-Mate, Ely, UK), as described,11, 12 used antibodies against MDR3 (Alexis Biochemicals ALX-801-028, Nottingham, UK) and its homolog, multidrug resistance-associated protein 2 (MRP2; Signet M2III-6, Signet Bioquote, York, UK).

SNP Genotyping.

GeneChip Human Mapping 50K Xba Array and Assay Kit (Affymetrix, Santa Clara, CA) were used to genotype DNA isolated from the blood of nine subjects (II.1, II.2, I.1, I.5, I.6, I.8–I.11) (Fig. 1). This array covers approximately 50,000 SNPs with annotated chromosomal locations (NCBI Build 36.2), distributed on all chromosomes but the Y chromosome, resulting in median intermarker distance of 8.5 kb. Analyses were performed according to the manufacturer's instructions.13

Statistical Analysis.

We performed genome-wide parametric linkage analysis with SNP genotypes using MERLIN software,14 assuming affected family members were homozygous at putative disease locus for a disease allele inherited from a common ancestor. Due to computational restrictions, for statistical analysis the pedigree was made one generation shorter than in reality, resulting in a lower approximation of the logarithm (base 10) of the odds ratio lod-scores. A rare disease mutation frequency (10−4) was assumed for the background population. To avoid multipoint linkage analysis with markers in linkage disequilibrium, which would lead to inflation of false-positive findings,15 30,840 SNPs with an intermarker distance of 15 kb were selected. Data underwent quality control routines, including graphical representation of relationship errors16 and PedCheck.17 All data analysis was preprocessed by ALOHOMORA software.18

Sequence Analysis of ABCB4.

All exons and exon/intron boundaries of ABCB4 were sequenced in I.1 as described.19 For other individuals and controls, only exon 19 was sequenced. ABCB4 (ENSG00000005471) genomic primer sequences are available upon request. To predict mutation consequences for ABCB4 (ENST00000265723) structure and function, SIFT (http://blocks.fhcrc.org/sift/sift.html), SNP3D (http://www.snps3d.org/), and TOPO2 (http://www.sacs.ucsf.edu/TOPO2) algorithms were applied according to guidelines using standard parameters.20, 21

Immunofluorescence and Confocal Laser Scanning Microscopy.

Cryosections of liver samples were air-dried, methanol-fixed and immunofluorescence-labeled as described,19 using anti-MDR3, polyclonal anti-MRP2 (EAG5), monoclonal anti-MRP2 (M2I4, Alexis, Grünberg, Germany) primary antibodies, with fluorescein or cyanine-3–conjugated secondary antibodies (Jackson Immunoresearch Laboratories, West Grove, PA), and analyzed on a Zeiss LSM 510 META confocal microscope (Oberkochen, Germany).

Bile Sampling and Analysis.

Bile duct bile samples were collected by endoscopic retrograde cholangiography from I.6 and controls. Total bile acid concentrations in bile were determined enzymatically with 3a-hydroxysteroid dehydrogenase (Enzabile; Nycomed, Oslo, Norway). Total serum bile-acids and bile cholesterol were determined via laboratory tests (ecoline S+kit, DiaSys Diagnostic Systems, Holzheim, Germany; Nobical Cholesterin/Nobiflow Cholesterin, Hitado Diagnostic Systems, Möhnensee, Germany). Bile PC was measured enzymatically (MTI Diagnostics, Idstein, Germany) after lipid extraction according to Folch.

Results

Clinical Characterization: A Cholestatic Liver Disease of Unclear Etiology.

The index patient (I.1, Fig. 1A) presented with elevated LFTs and pruritus at 26 years of age. Endoscopic retrograde cholangiography and computerized tomography revealed a normal biliary tract without vascular malformation. Autoantibodies and evidence of viral hepatitis A-E were not found; α-1-antitrypsin values and copper and iron metabolism markers were normal. The patient progressed to cirrhosis and underwent liver transplantation at 34 years of age. Cholestasis parameters were elevated initially (Fig. 1B), decreased to near-normal values on beginning ursodeoxycholic acid (UDCA) treatment, and subsequently deteriorated. The course was typical for biliary cirrhosis, with rising transaminase values as disease advanced and a terminal decrease in hepatocellular synthetic capacity (not shown).

Of her 10 siblings (Fig. 1A), the only brother (I.2) and a sister (I.3) died at age 5 years and 7 years, respectively. Both had jaundice, which deepened as ascites developed, consistent with progressive CLD. Another sister (I.4) first presented with cholestasis aged 25 years. She died at age 43 years of biliary cirrhosis. Her AP values were markedly elevated (527-706 IU/L), whereas her GGT values were only slightly elevated (51–94 IU/L). Both her pregnancies ended in first-trimester miscarriage. Based on clinical and histopathological criteria, IAD was initially diagnosed in I.1 and I.4.

Family members presumed healthy were screened for abnormalities in LFTs (Table 1). One sister (I.6, Fig. 1A) showed prominently elevated AP and only slightly elevated GGT and aminotransferase values at 35 years of age. Over 8 years, her LFTs worsened (AP 5 times the upper limit of normal; Fig. 1C), but without decreased liver synthetic activity. Another sister (I.5, Fig. 1A) had slightly elevated AP and GGT values at 37 years of age that increased thereafter. Both sisters reported symptoms of ICP (marked pruritus and jaundice). LFTs in the five remaining siblings (I.7–I.11, Fig. 1A) and the parents (II.1, II.2, Fig. 1A) were normal. Three other sisters reported symptoms of ICP. In I.8 a pregnancy had ended in stillbirth. I.7 reported an early miscarriage. In the only nonparous sister with normal LFTs (I.11), liver biopsy revealed subtle histopathological changes (see below). The mother (II.2) had had cholelithiasis and ICP. The father (II.1) had no evidence of CLD. Total serum bile acid concentrations were elevated only in I.1.

Table 1. Clinical Presentation of the Family
  1. n/a, not applicable; n/d, not determined.

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In summary, of the 11 siblings, two developed cholestatic cirrhosis in early childhood; four showed signs of progressive cholestatic liver failure in adulthood, resulting in cirrhosis in two; and of the remaining five and the mother, all showed signs of milder liver disease, either ICP or subtle histological changes.

Histopathology Reveals Small Duct Cholangiopathy, Including Ductopenia.

At hepatectomy, the liver of I.1 was cirrhotic. Principal interlobular bile ducts were lacking (ductopenia) with neoductular proliferation (Fig. 2A) and mild patchy portal tract inflammation. Swollen periportal hepatocytes suggested cholate stasis, indicating cholestastis (Fig. 2B). Finely flocculent and angulated, even paracrystalline, sludged material lay in septal bile duct lumina (Fig. 2C) with extravasated bile at portal tract margins (Fig. 2D). Periportal and juxtaseptal hepatocytes contained generous quantities of copper-binding protein, further indicating cholestasis (Fig. 2E). Similar findings (ductopenic, biliary cirrhosis; Fig. 2F) were present in a deceased sister (I.4). Liver biopsy in patient I.6 showed bile duct loss in a few portal tracts and mild ductular proliferation (Fig. 2G), as reported for I.1 early in her course (not shown). Subtle ductular proliferation was observed at liver biopsy in individual I.11, though without loss of bile ducts (Fig. 2H).

Figure 2.

Microscopy of liver tissue from (A-E) the index patient I.1, (F) a sister dying in adulthood (I.4), (G) a severely affected sister (I.6), and (H) a mildly affected sister (I.11). (A) Portal tracts revealing ductopenia (asterisk) and ductular proliferation (solid arrowheads). (Anti–cytokeratin 7/hematoxylin stain.) (B) Mild, patchy mixed inflammatory infiltrate in portal tract with spillover into parenchyma. Solid arrows indicate swollen hepatocytes. (Hematoxylin-eosin stain.) (C) Paracrystalline debris inside septal bile duct (solid asterisk). (Hematoxylin-eosin stain.) (D) Extravasated bile (open arrow). (Diastase/periodic acid–Schiff stain.) (E) Copper-binding-protein within hepatocytes (gray arrowheads). (Orcein stain.) (F) Portal tract. Pronounced fibrosis and ductular reaction. (Trichrome stain.) (G,H) Discrete inflammatory infiltrate (open arrowhead) and neoductules (solid arrowheads). (Anti–cytokeratin 7/hematoxylin stain.)

Genotyping Yields a Common Haploblock on Chromosome 7q21-q22.

The marked clustering of affected family members was unlikely to be explained by chance or multifactorial determination. Thus, a strong genetic component was suspected, and monogenetic inheritance was assumed. Because the German ancestors of both parents originated from a small geographic region in Transylvania (Romania), a highly penetrant autosomal-recessive trait was hypothesized. Church records and family information revealed that the parents shared a common ancestor and were fourth-degree cousins once removed (Fig. 3A).

Figure 3.

(A) Extended pedigree displaying parental consanguinity (fourth-degree cousins once removed). (B) The lod-score distribution relative to chromosomal location upon genotyping nine family members for linkage of CLD. (C) Haplotypes of highest lod-score region on chromosome 7 (98.91027613-107.38497337 cM). Note homozygosity for disease-carrying paternal and maternal alleles (green) in severely affected individuals and heterozygosity in all other family members.

To identify an underlying gene defect, genotyping was performed using Affymetrix 50k Gene Chips. DNA samples from seven siblings and both parents were available. Three subjects (I.1, I.5, I.6) were considered severely affected by CLD, and six were considered unaffected (II.1) or only mildly affected (I.8-I.11, II.2). Taking into account relationship information, we analyzed linkage using SNP arrays. Computational analysis of SNP data demonstrated genome-wide significant evidence of linkage of disease to a segment of chromosome 7q21. Specifically, two loci on 7q21.1-7q22 (bp 86,111,166-88,261,357 and bp 98,993,981-106,630,227) showed a maximal multipoint logarithm (base 10) of the odds ratio lod-score for linkage of CLD of 3.01 and 3.88 (corresponding to an odds ratio of ≈7,500:1 in favor of linkage). No other interval yielded a lod-score greater than 1.5 (Fig. 3B).

Severely affected family members (I.1, I.5, I.6) were homozygous across this interval, implying homozygosity for an underlying disease-causing mutation. Conversely, mildly affected (I.8-11, II.2) or unaffected family members (II.1) were heterozygous (Fig. 3C).

Homozygosity for a Missense Mutation in ABCB4 Is Associated with Cholestatic Liver Disease.

The identified haploblocks contained 149 annotated genes (Supplementary Table 1). Among these, ABCB1 and ABCB4 were noteworthy, because both encode canalicular transporters. Whereas ABCB1 has not been described as associated with known disease, mutations in ABCB4 can cause PFIC3, characterized by cholestasis in early childhood and cirrhotic liver failure before adulthood, and cholesterol cholelithiasis.22 Moreover, mutations in ABCB4 are associated with ICP.23, 24

Direct sequencing of ABCB4 in I.1 revealed homozygosity for a single sequence variant, c.2362C>T (p.R788W, Arg788Trp) in exon 19. c.2362C is the first base of a triplet encoding the polar amino acid arginine. The identified missense mutation exchanges arginine for the aromatic amino acid tryptophan. Sequencing of other family members revealed that c.2362C>T (R788W) precisely cosegregated with CLD. As expected, the mutation was found in homozygous state in all severely affected siblings studied and in heterozygous state in all other family members tested, including I.7, who had not been genotyped via SNP arrays. It was not detected in 96 unrelated German control chromosomes (Fig. 4A–C).

Figure 4.

Sequence analysis of exon 19 of ABCB4 shows homozygosity for the variant c.2362C>T in all severely affected individuals (B) and heterozygosity in all other family members (C). The variant was not present in healthy controls (A). (D) Sequence homology of ABCB4 protein among different species and different ABC protein isoforms. Arg788 is shown in red. (E) Topological model of ABCB4 protein shows site of Arg788 (blue) in conserved intracellular loop domain 1.2 of the second subunit (orange).

Biliary Phosphatidylcholine Levels Are Reduced in a Homozygous Individual.

Amino acid exchange owing to nucleotide change at the first base of the codon of p.R788 is not reported in public databases and is predicted to be deleterious by the SIFT and SNP3ds algorithms. To evaluate if the base pair exchange in ABCB4 had consequences for protein expression, biochemical and microscopic analyses were performed. Relative to controls, amounts of ABCB4 messenger RNA and ABCB4 protein in liver from I.1 were unchanged (not shown). Immunohistochemical studies revealed that mutant ABCB4 localizes to the apical membranes of hepatocytes (Fig. 5A), similar to ABCC2 (MRP2), a close homolog of ABCB4, also expressed at the canalicular membrane (Fig. 5B). Immunofluorescence microscopy confirmed that mutant ABCB4 matured properly and localized to hepatocyte canalicular membranes in the patient (Fig. 5C,D). To test for functional impairment of PC transport activity, bile duct bile from severely affected patient I.6 was analyzed for PC, cholesterol, and total bile acid content. Relative to controls, the PC/bile acid ratio was decreased by 40% (Fig. 6). In contrast, the cholesterol/bile acid ratio was unchanged. These results are consistent with functional impairment, but residual activity of ABCB4 PC transport activity in homozygous mutation carriers.

Figure 5.

Immunostaining of (A) ABCB4 and (B) ABCC2 in liver sections of the index patient shows proper localization to the canaliculi. (C,D) Confocal imaging shows localization of ABCB4 (left) and ABCC2 (middle) to canalicular membranes in (D) the index patient and (C) a healthy control.

Figure 6.

Lipid composition of bile duct bile of I.6 and controls. Total bile acids, cholesterol, and PC content were determined enzymatically. The ratio of cholesterol/bile acids in I.6 was normal compared with controls (right), but the PC/bile acid ratio was reduced in I.6, consistent with impaired PC transport by ABCB4 protein.

Discussion

Chronic cholestasis is a major cause of liver disease, cirrhosis, and progressive liver failure, ending in transplantation or death. However, for chronic CLD in a considerable proportion of patients, assignment to a distinct pathogenic entity is still impossible. We describe a family in which initial diagnostic allocation of CLD to a known category was unsuccessful. Of 11 siblings, two died of liver failure in childhood and one in adulthood. Among the survivors, one sister underwent liver transplantation and two had worsening liver damage. The remaining siblings presented subtle signs of CLD. Whereas AP values were markedly elevated, GGT values were normal or only slightly elevated. Microscopy of liver revealed a cholestatic and ductopenic pattern suggesting no specific etiology.

Onset of severe liver disease in four siblings occurred in adulthood with features of IAD, a rare cholestatic condition by definition not attributable to any known etiology. The histopathological hallmark of IAD is cholangiopathy, including lack of bile ducts in more than 50% of portal tracts.25 IAD has mild or aggressive courses, the latter resulting in cirrhosis and liver failure.26, 27 Because monogenic diseases with cholestasis and/or ductopenia as a leading feature typically begin before adulthood, a factor implicated in childhood CLD has been postulated to cause IAD.28 However, no gene has yet been identified.

In certain pediatric cholestatic disorders, genes encoding canalicular transporters are mutated. PFIC1 and PFIC2 show a similar clinical presentation with onset of disease during infancy and normal serum GGT values, whereas high GGT values are considered obligate in PFIC3.29 Moreover, in PFIC3 serum bile acids are usually elevated, and ductular proliferation is present. PFIC1-3 were dismissed as a diagnostic consideration in the family studied, as GGT values (PFIC3), serum bile acids (PFIC3), and histopathological findings (PFIC1 and PFIC2) were unremarkable, and in several siblings disease onset was not before adulthood (PFIC1-3).

Surprisingly, however, a combined strategy of linkage analysis and genomic sequencing identified a homozygous missense mutation in ABCB4 as plausibly disease-causing. Linkage mapping of autosomal-recessive diseases or autozygosity mapping is a powerful strategy to localize candidate regions even in single pedigrees, provided that many affected members are available for genotyping and/or that families studied are consanguine. In this family, distant consanguinity was revealed by genealogic research. For consanguine families, the number of generations to the common ancestor highly influences the likelihood of identifying a homozygous disease-associated region. If the parents in our family had been either unrelated or more closely related, analysis would not have revealed a region of genome-wide significance. For example, hypothesizing the parents to be first-degree cousins would have yielded 13 genomic regions with a maximal lod-score of only 1.7, which is due to the impossibility of distinguishing regions homozygous by descent from regions being randomly homozygous (data not shown). Using correct pedigree information, however, genome-wide SNP genotyping revealed disease linkage to a region on chromosome 7q21 with a maximal lod-score of 3.88, with all other regions excluded. This underlines the importance of accurate family information when attempting disease gene identification in single-pedigree studies.

Of the 149 genes in the haploblock, ABCB1 and ABCB4 are known to be expressed at the bile canaliculus. Because mutations in ABCB4 are associated with CLD,30 we prioritized ABCB4 for initial molecular studies. A contribution of a sequence variant of ABCB1 to the phenotype of these patients appeared even more unlikely due to the evolving results of this study. But it cannot be entirely excluded, because ABCB1 was not sequenced. Sequencing of ABCB4 revealed the missense mutation c.2362C>T (p.R788W) for which siblings suffering from progressive liver damage were homozygous and family members showing only ICP or subtle histopathological changes were heterozygous. R788 is highly conserved among members of the ABC gene family as well as in ABCB4 orthologs (Fig. 4D), except for TAP1, where a glutamine can be found at this position,31 indicating tolerance for this polar amino acid. A glutamine has also been described at position 788 (c.2363G>A) in ABCB4 in up to 16% of a Nigerian population as a heterozygous nonsynonymous SNP (rs45595532). In Caucasians, this SNP has not been reported, consonant with our sequencing data. R788 lies in the intracellular loop1 domain of the second subunit of the ABCB4 protein (Fig. 4E). This region is essential for coupling with the nucleotide binding domains of ABCB4 and therefore for the conformational change resulting from ATP hydrolysis.31 Modification of R788 to an aromatic amino acid residue, as in this family, is predicted to affect protein PC transport activity considerably. Although ABCB4 messenger RNA expression, proteins levels and subcellular localization were unremarkable, this is in line with our findings of reduced biliary PC levels in homozygous individual I.6. Because PC renders cholesterol soluble in bile,32 impairment of PC transport due to this ABCB4 mutation is further supported by the observation of cholesterol-like paracrystalline sludge within bile ducts of I.1. In contrast to PFIC3 children, in whom PC/bile acid ratios are reduced by more than 80%,29 this value was only reduced by 40% in patient I.6. This indicates residual ABCB4 transport activity in this family, which may account for the onset of CLD symptoms not before adulthood in several members.

Our study shows systematically that in addition to causing cirrhosis in childhood or adolescence, ABCB4 mutations impairing PC transport can cause biliary cirrhosis in adults. Two case reports suggest that this may be more frequent than anticipated: Strautnieks et al.33 described two families with compound heterozygosity for ABCB4 variants in patients with adult-onset CLD. Lucena et al.12 report a woman heterozygous for G535D in ABCB4, who had ICP, cholelithiasis and cholestatic cirrhosis and whose daughter had cholestasis.

From a clinical perspective ICP, PFIC3, IAD, and adult-onset biliary cirrhosis are distinct clinical entities. Although no further information is available on the two siblings reported here who died with cirrhosis as children, it is not unlikely that they also were homozygous for the familial ABCB4 mutation and died of PFIC3.

How might homozygosity for the same ABCB4 mutation result in such broad clinical variability? One likely explanation is that additional genetic and/or environmental factors in some family members influenced the course of disease. Although no additional genomic loci were evident in our analysis, studies are clearly indicated to address the possible existence of modifying genes and changes in bile composition leading to more aggressive bile, which correlates with severity of liver disease.34, 35

In the family reported here, all childbearing family members had ICP, and three suffered miscarriages or stillbirth. This emphasizes the importance of closely monitoring LFTs during pregnancy in known carriers of ABCB4 mutations and their family members, with genetic counseling and ABCB4 mutation analysis considered.

Importantly, UDCA improved LFTs in all affected family members in our study. UDCA renders bile composition less aggressive, thereby protecting hepatocytes and the biliary epithelia.36 UDCA also enhances ABCB4 protein levels,37 suggesting a mechanism by which UDCA benefits ABCB4 mutation carriers. ABCB4 expression is regulated via an FXR-responsive element, and it supports ABCG5/ABCG8-mediated secretion of cholesterol. We believe that better understanding of this intricate interplay among transporters, substrates, and regulators may allow the development of targeted therapies that will specifically modify bile composition relative to the need of the individual patient.

In conclusion, we describe a family in which a single missense mutation in ABCB4 is associated with profound clinical variability of CLD. Using genome-wide SNP genotyping and autozygosity mapping, we excluded other genomic loci and demonstrate that impaired ABCB4 was linked to cholestatic and ductopenic liver disease in adulthood; bile analysis supported this interpretation. Our results show that a common underlying genetic defect should be considered in families with cholestatic phenotypes ranging from ICP through ductopenic liver fibrosis, to cirrhosis with death in childhood and adulthood. Based on our findings, mutational analysis of ABCB4 should be generally considered in all patients with cholestatic liver disease of unknown etiology, regardless of age and onset of disease.

Acknowledgements

We are indebted to the family members participating in this study. We further thank Prof. B. Janssen (Heidelberg) for technical support. The protocol for anti-MDR3 staining in formalin-fixed, paraffin-embedded tissues was derived from one developed by Dr. J. Sola, Department of Pathology, University Clinic of Navarre (Spain).

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