University of Zurich
Zurich Open Repository and Archive
Severe bile salt export pump deficiency: 82 different ABCB11
mutations in 109 families
Strautnieks, S S; Byrne, J A; Pawlikowska, L; Cebecauerova, D; Rayner, A; Dutton, L;
Meier, Y; Antoniou, A; Stieger, B; Arnell, H; Ozcay, F; Al-Hussaini, H F; Bassas, A
F; Verkade, H J; Fischler, B; Nemeth, A; Kotalova, R; Shneider, B L;
Cielecka-Kuszyk, J; McClean, P; Whitington, P F; Sokal, E; Jirsa, M; Wali, S H;
Jankowska, I; Pawlowska, J; Mieli-Vergani, G; Knisely, A S; Buli, L N; Thompson, R
Strautnieks, S S; Byrne, J A; Pawlikowska, L; Cebecauerova, D; Rayner, A; Dutton, L; Meier, Y; Antoniou, A;
Stieger, B; Arnell, H; Ozcay, F; Al-Hussaini, H F; Bassas, A F; Verkade, H J; Fischler, B; Nemeth, A; Kotalova, R;
Shneider, B L; Cielecka-Kuszyk, J; McClean, P; Whitington, P F; Sokal, E; Jirsa, M; Wali, S H; Jankowska, I;
Pawlowska, J; Mieli-Vergani, G; Knisely, A S; Buli, L N; Thompson, R J (2008). Severe bile salt export pump
deficiency: 82 different ABCB11 mutations in 109 families. Gastroenterology, 134(4):1203-1214.
Postprint available at:
Posted at the Zurich Open Repository and Archive, University of Zurich.
Originally published at:
Gastroenterology 2008, 134(4):1203-1214.
Severe bile salt export pump deficiency: 82 different ABCB11
mutations in 109 families
BACKGROUND & AIMS: Patients with severe bile salt export pump (BSEP) deficiency present as
infants with progressive cholestatic liver disease. We characterized mutations of ABCB11 (encoding
BSEP) in such patients and correlated genotypes with residual protein detection and risk of malignancy.
METHODS: Patients with intrahepatic cholestasis suggestive of BSEP deficiency were investigated by
single-strand conformation polymorphism analysis and sequencing of ABCB11. Genotypes sorted by
likely phenotypic severity were correlated with data on BSEP immunohistochemistry and clinical
outcome. RESULTS: Eighty-two different mutations (52 novel) were identified in 109 families (9
nonsense mutations, 10 small insertions and deletions, 15 splice-site changes, 3 whole-gene deletions,
45 missense changes). In 7 families, only a single heterozygous mutation was identified despite
complete sequence analysis. Thirty-two percent of mutations occurred in >1 family, with E297G and/or
D482G present in 58% of European families (52/89). On immunohistochemical analysis (88 patients),
93% had abnormal or absent BSEP staining. Expression varied most for E297G and D482G, with some
BSEP detected in 45% of patients (19/42) with these mutations. Hepatocellular carcinoma or
cholangiocarcinoma developed in 15% of patients (19/128). Two protein-truncating mutations conferred
particular risk; 38% (8/21) of such patients developed malignancy versus 10% (11/107) with potentially
less severe genotypes (relative risk, 3.7 [confidence limits, 1.7-8.1; P = .003]). CONCLUSIONS: With
this study, >100 ABCB11 mutations are now identified. Immunohistochemically detectable BSEP is
typically absent, or much reduced, in severe disease. BSEP deficiency confers risk of hepatobiliary
malignancy. Close surveillance of BSEP-deficient patients retaining their native liver, particularly those
carrying 2 null mutations, is essential.
Editorial Manager(tm) for Gastroenterology
Manuscript Number: GASTRO-D-07-01239R2
Title: Severe bile salt export pump deficiency: 82 different ABCB11 mutations in 109 families
Article Type: Basic - Liver/Pancreas/Biliary
Section/Category: Human Research - Human Material
Corresponding Author: Sandra S Strautnieks, Ph.D
Corresponding Author's Institution: Institute of Liver Studies, King's College London School of Medicine at
King's College Hospital, London, UK
First Author: Sandra S Strautnieks, Ph.D
Order of Authors: Sandra S Strautnieks, Ph.D; Jane A Byrne, Ph.D; Ludmila Pawlikowska, Ph.D; Dita
Cebecauerová, M.D.; Anne Rayner, BSc, FIBMS; Laura Dutton, BSc; Yvonne Meier, Ph.D; Anthony Antoniou,
M.D.; Bruno Stieger, Ph.D; Henrik Arnell, M.D.; Figen Özçay, M.D.; Hussa F Al-Hussaini, M.D.; Atif F Bassas,
M.D.; Henkjan J Verkade, M.D.; Björn Fischler, M.D.; Antal Németh, M.D.; Radana Kotalová, M.D.; Benjamin L
Shneider, M.D.; Joanna Cielecka-Kuszyk, M.D.; Patricia McClean, M.D.; Peter F Whitington, M.D.; Étienne
Sokal, M.D.; Milan Jirsa, Ph.D; Sami Wali, M.D.; Irena Jankowska, M.D.; Joanna Pawłowska, M.D.; Giorgina
Mieli-Vergani, M.D.; A S Knisely, M.D.; Laura N Bull, Ph.D; Richard J Thompson,
* Revised Manuscript (Tracked Changes)
Severe bile salt export pump deficiency: 82 different ABCB11 mutations in 109 families
Short title: ABCB11 mutations in severe BSEP deficiency
Sandra S. Strautnieks
1, Jane A. Byrne
1¤, Ludmila Pawlikowska
2¤, Dita Cebecauerová
1,3, Anne Rayner
4, Yvonne Meier
5, Anthony Antoniou
1, Bruno Stieger
5, Henrik Arnell
6, Figen Özçay
8, Atif F. Bassas
9, Henkjan J. Verkade
10, Björn Fischler
6, Antal Németh
6, Radana Kotalová
Benjamin L. Shneider
12¤, Joanna Cielecka-Kuszyk
13, Patricia McClean
14, Peter F. Whitington
15, Étienne Sokal
3, Sami H. Wali
17, Irena Jankowska
13, Joanna Pawłowska
13, Giorgina Mieli-Vergani
1, A. S. Knisely
Laura N. Bull
2, Richard J. Thompson
No conflicts of interest exist
*To whom correspondence should be directed
Institute of Liver Studies
King’s College Hospital
Denmark Hill London SE5
9RS United Kingdom
+44 (0)20 3299 4625 telephone, +44
(0)20 3299 3760 facsimile
Institute of Liver Studies, King's College London School of Medicine at King’s College Hospital, London,
2University of California, San Francisco, Liver Center Laboratory and Department of Medicine, San
Francisco General Hospital, San Francisco, CA
3Institute for Clinical and Experimental Medicine, Prague, Czech Republic
4Institute of Liver Studies, King's
College Hospital, London, UK
5Division of Clinical Pharmacology and Toxicology, Department of Medicine,
University Hospital, Zürich,
6Department of Pediatrics, Karolinska University Hospital, Huddinge and Solna, Stockholm,
7Department of Pediatric Gastroenterology, Hepatology, and Nutrition, Başkent University
8Department of Pathology, Riyadh Armed Forces Hospital, Riyadh, Saudi Arabia
9Department of Surgery, Riyadh Armed Forces Hospital, Riyadh, Saudi Arabia
Gastroenterology and Pediatrics, University Hospital Groningen, The Netherlands
Pediatrics, Charles University 2nd Faculty of Medicine, Prague, Czech Republic
12Department of Pediatrics
Mount Sinai School of Medicine, New York, NY
13Department of Pediatric Gastroenterology, Hepatology,
and Immunology, The Children's Memorial
Health Institute, Warsaw, Poland
14Children's Liver and Gastrointestinal Unit, St James's University
Hospital, Leeds, UK
15Department of Pediatrics, Northwestern University Feinberg School of Medicine
Memorial Hospital, Chicago, IL
16Université Catholique de Louvain, Unité PEDI, Pediatric hepatology and cell therapy, Brussels,
17Department of Pediatrics, Riyadh Armed Forces Hospital, Riyadh, Saudi Arabia
Jane A. Byrne: Division of Cancer Sciences and Molecular Pathology, School of Medicine, University of
Glasgow, Glasgow, UK
Ludmila Pawlikowska: Department of Anesthesia and Perioperative Care, Center for Cerebrovascular
Research, San Francisco, CA
Benjamin L. Shneider: Department of Gastroenterology, Children's Hospital of Pittsburgh, Pittsburgh, PA
Guy’s and St Thomas’ Charity, London; Children’s Liver Disease Foundation, Birmingham, both UK (JB,
Swiss National Science Foundation; Grant Number: 31-64140.00 (YM, BS)
National Institutes of Health; Grant Number: R01 DK50697 (LP, LNB)
European Association for the Study of the Liver Fellowship (DC, RT)
Swedish Order of Freemasons (HA, BF, AN)
ABC, ATP-binding cassette; AFP, α-fetoprotein; BSEP, bile salt export pump; CpG, cytosine-guanine; CC,
cholangiocarcinoma; FIC1, familial intrahepatic cholestasis 1; γ-GT, γ-glutamyl transferase; HCC,
hepatocellular carcinoma; IC, intracellular loop; MDR1, multidrug resistance protein 1; MDR3, multidrug
resistance protein 3; MRP2, multidrug resistance-associated protein 2; NBF, nucleotide-binding fold; OLT,
orthotopic liver transplantation; PEBD, partial external biliary diversion; PFIC, progressive familial intrahepatic
cholestasis; PCR, polymerase chain reaction; RE, restriction endonuclease; SSCP, single-strand
conformation polymorphism; TM, transmembrane domain; UDCA, ursodeoxycholic acid
Acknowledgements We thank the families and the Children’s Liver Disease Foundation for support and
encouragement, and those who referred families for analysis, including Drs U Baumann, W Berquist, M de
Vree, K Emerick, G Ferry, M Finegold, W Hardikar, S Horslen, R Houwen, R Jaffe, L Klomp, F Lacaille, K
Mann, P McKiernan, H Sharp, R Sokol, E Sturm, L Szönyi, J Taminou, and J Watkins. We also thank Dr R
Garcia-Kennedy for access to materials illustrated by photomicroscopy.
Background and aims Patients with severe bile salt export pump (BSEP) deficiency present as infants with
progressive cholestatic liver disease. We characterised mutations of ABCB11 (encoding BSEP) in such
patients and correlated genotypes with residual protein detection and malignancy risk.
Methods Patients with intrahepatic cholestasis suggestive of BSEP deficiency were investigated by
single-strand conformational polymorphism analysis and sequencing of ABCB11. Genotypes sorted by likely
phenotypic severity were correlated with data on BSEP immunohistochemistry and clinical outcome.
Results Eighty-two different mutations (52 novel) were identified in 109 families (9 nonsense mutations, 10
small insertions and deletions, 15 splice-site changes, 3 whole-gene deletions, 45 missense changes). In 7
families only a single heterozygous mutation was identified despite complete sequence analysis. Thirty-two
percent of mutations occurred in >1 family, with E297G and/or D482G present in 58% (52/89) of European
families. On immunohistochemical analysis (88 patients), 93% had abnormal or absent BSEP marking.
Expression varied most for E297G and D482G, with some BSEP detected in 45% (19/42) of patients with
these mutations. Hepatocellular carcinoma or cholangiocarcinoma developed in 15% (19/128) of patients. Two
protein-truncating mutations conferred particular risk; 38% (8/21) of such patients developed malignancy
versus 10% (11/107) with potentially less severe genotypes (relative risk 3.7 [CL=1.7-8.1, p=0.003]).
Conclusions With this study, >100 ABCB11 mutations are now identified. Immunohistochemically detectable
BSEP is typically absent, or much reduced, in severe disease. BSEP deficiency confers risk of hepatobiliary
malignancy. Close surveillance of BSEP-deficient patients retaining their native liver, particularly those
carrying 2 null mutations, is essential.
Bile salt export pump (BSEP) deficiency is caused by mutations in ABCB11
1, 2. The severity of BSEP deficiency
varies from progressive early-onset
1 to remitting and late-onset phenotypes
3-7. Severe BSEP deficiency falls into
the descriptive category of “progressive familial intrahepatic cholestasis” (PFIC)
8-12, a heterogeneous group of
autosomal recessive conditions that disrupt bile formation. BSEP deficiency is among disorders with low serum
concentrations of γ-glutamyl transferase (γ-GT) activity despite conjugated hyperbilirubinaemia, as is familial
intrahepatic cholestasis 1 (FIC1) deficiency caused by mutations in ATP8B1
13. Both BSEP deficiency and FIC1
deficiency exist worldwide. Their collective estimated Western European incidence is 1/50-70,000 births/year.
BSEP, previously termed “sister of P-glycoprotein”
14, is a member of the ATP-binding cassette (ABC)
superfamily and P-glycoprotein / multidrug resistance (MDR/ABCB) subfamily of transporters
15, 16. BSEP,
expressed at the hepatocyte canalicular membrane, is the major exporter of primary bile acids
19. It actively transports conjugated bile salts into biliary canaliculi against extreme concentration gradients.
Liver disease in BSEP deficiency is ascribed to failed secretion and intrahepatocytic accumulation of toxic
bile salts. Patients with the progressive form present as infants with high serum bile salts, pruritus,
malabsorption, failure to thrive, jaundice and cholestasis. They develop fibrosis and end-stage liver disease
20-22. Partial external biliary diversion (PEBD) and ileal exclusion can relieve pruritus and, in
some cases, slow disease progression
23-27. However, most patients ultimately need orthotopic liver
We here present the mutations of ABCB11 in 109 families with severe BSEP deficiency.
PFIC families were recruited through referral to King’s College London or the University of California, San
Francisco. All procedures were conducted with informed consent as routine diagnostic investigations or under
an institutional-review-board–approved protocol. Referrers supplied clinical data. No patient had elevated
serum concentrations of γ-GT activity. Other causes of neonatal hepatitis were excluded, including primary
disorders of bile acid synthesis in most cases.
Families were defined as affected by “severe” PFIC and included in the study if at least one family member
presented in infancy with a low γ-GT cholestasis that progressed, with clinical and biochemical markers of
cholestasis persistently abnormal (absent surgical intervention). They were excluded if cholestasis ever
Selection for likely BSEP deficiency was based on clinical and histological data (specifically: Liver disease
without the extrahepatic manifestations (pancreatitis, hearing loss, profound diarrhoea) characteristic of
FIC1 deficiency, and exhibiting giant-cell hepatitis rather than bland cholestasis on histological assessment
Where possible, BSEP immunohistochemical analysis (30%)
2, 28, 29 and/or microsatellite-based haplotype
13, 30, 31 were used. The resultant subgroup was analysed for ABCB11 mutations.
One hundred and nine families (data and/or biopsy material available for 132 individuals) met the inclusion
criteria of genetically proven severe BSEP deficiency (Supplementary Tables 1A-1E). Eighty-nine families were
European (European, Australian, North American), 20 Central Asian/Arab, East Asian (Korean, Japanese,
Chinese), South Asian (Indian, Pakistani) or African. BSEP immunohistochemistry was possible for 88 patients;
clinical follow-up for malignancy was available in 128. In 7 families, clinical outcome and/or
immunohistochemical results from a deceased sibling were included without mutational confirmation. In 5
families only parental DNA was analysed. We have previously reported single
mutations in 8 families
1, 2, 32; the second mutant allele is now identified in each. Clinical observations in
21 families have been reported previously
28, 29, 33. These families are included to retain our population’s mutation
Sequence and genomic structure of ABCB11
The 1321 amino-acid BSEP protein is encoded by ABCB11 on chromosome 2q24-31. ABCB11 spans a 108kb
genomic region and is composed of 27 coding exons and one 5’ untranslated exon (designated 1-28).
ABCB11 cDNA sequence (AF091582; AF136523; NM_003742) and genomic structure
1 are available (National
Center for Biotechnology Information;http://www.ncbi.nlm.nih.gov/). Mutation nomenclature
34 follows Human
Genome Variation Society recommendations (http://www.hgvs.org/mutnomen/). Previously published
mutations have been revised accordingly.
Mutation detection strategy
Patients were initially screened by restriction-endonuclease (RE) digestion for recurrent changes at mutation
hotspots and, depending on ethnicity, for population-associated changes. ABCB11 exons subsequently
underwent single-strand conformation polymorphism (SSCP) analysis in 44 patients followed by sequence
analysis of exons with identified mobility changes. Latterly this was replaced by primary sequencing. All exons
were sequenced until clearly damaging, or previously known, mutations were identified on both alleles.
Samples with novel missense changes were sequenced throughout. Mutations were confirmed using freshly
extracted DNA from affected individuals and parental DNA (as available). Missense changes were
distinguished from polymorphisms by several criteria. First was their absence from ethnically matched control
panels of at least 300 alleles (published
35-38, in public databases [http://pharmacogenetics.ucsf.edu/
39], or within
this study). Also considered was conservation across BSEP orthologues and MDR/ABCB homologues. Finally,
predicted functional effects and differences in physical and chemical properties were assessed.
The Statistics Calculator was used (http://www.cebm.utoronto.ca/practise/ca/statscal/). Chi-squared
testing assessed differences between groups. Using the same data and calculator, relative risk and 95%
confidence limits were similarly calculated.
Sections of formalin-fixed, paraffin-embedded liver, when available, were routinely stained and immunostained
for BSEP using a polyclonal antibody raised in rabbit to the carboxy-terminal 21 amino acids of BSEP as
19. As a control for antigen preservation or protein-expression deficiencies not specific to
BSEP, parallel sections were immunostained for a structurally similar canalicular ABC transporter, multidrug
resistance-associated protein 2, using a monoclonal antibody raised in mouse (Signet/Bioquote, York, UK).
Findings were evaluated by light microscopy as described
28, 29. For 6 families immunohistochemical protocols
used were as described
2. BSEP marking was classified as normal, not detected or abnormal, where abnormal
refers to either reduced intensity or focal absence. Immunohistochemical analysis preceded genetic analysis in
30% of cases and followed it in the remainder. Abnormality was judged by two or more investigators, all blinded
to genetic status.
DNA extraction and polymerase chain reaction (PCR) amplification
DNA was extracted from blood and tissue samples using standard protocols. PCR amplification was
conducted using Taq DNA polymerase (New England Biolabs, Ipswich, MA) and Roche Fast Start PCR
systems (Roche Diagnostics, Basel, Switzerland). Primer details provided in Supplementary Table 2.
Microsatellite-marker typing Microsatellite-marker haplotype analysis was conducted across the ABCB11
(2q24) and ATP8B1 (18q21) chromosomal regions in consanguineous families, or those with >2 affected
children, to determine which gene was likely mutated. Marker loci were selected from genetic maps
developed from polymorphic repeats (Human Genome Mapping Project/Celera reference sequences;
http://www.ncbi.nlm.nih.gov/). Primers were designed to allow multiplex analysis based on product size
and fluorescent label (Supplementary Tables 3A/3B). Amplification products were separated on a 3100
Avant Genetic Analyser, data were analysed using GeneMapper software (all Applied Biosystems,
Foster City, CA) and haplotypes were constructed. Families with non-Mendelian segregation of
mutations were investigated for deletions of the ABCB11 chromosomal region using microsatellite
markers spanning 16.2Mb of chromosome 2.
RE digestion was used to identify common or recurring changes rapidly and to screen ethnically matched
control panels for novel changes. Enzymes were selected using NEBcutter V2.0
(http://tools.neb.com/NEBcutter2/index.php; New England Biolabs or Roche Diagnostics). The common
mutations E297G, D482G, R575X, R1153C and R1153H abolish HphI, FokI, FokI, BsrBI and BsrBI sites
respectively, whilst G982R creates an AlwNI site. PCR-amplified exon-digestion products underwent 35%
agarose gel electrophoresis (Supplementary Table 4).
Single-strand conformation polymorphism analysis
SSCP analysis was conducted using 12.5% acrylamide GeneGel Excel nondenaturing gels on a GenePhor
Electrophoresis system (all Amersham Biosciences, Little Chalfont, UK), initially at 5
oC and, if necessary for
enhanced resolution, at 15
oC. SSCP patterns were visualised by DNA silver staining (Amersham Biosciences).
Products larger than 150bp were digested before analysis (as above).
PCR products were purified using the High Pure PCR purification system (Roche Diagnostics) before direct
sequence analysis using the version 3.1 Dye Terminator cycle sequencing kit (Applied Biosystems) and
electrophoresis on a 3100 Avant Genetic Analyzer. Data were analysed using Sequencher (Gene Codes,
Ann Arbor, MI) or SeqScape (Applied Biosystems) software.
Eighty-two different ABCB11 mutations were identified on 208 alleles in 109 families with severe BSEP
deficiency (Tables 1-3; Figure 1; Supplementary Tables 1A-1E). Homozygosity, or compound heterozygosity,
for ABCB11 mutations was completely concordant with disease expression in all families genotyped. Fifty-two
mutations were novel. Eighteen previously reported severe mutations were not
detected1, 2, 5, 44-47
The 82 mutations identified (Tables 1-3, Figure 1) included 9 (11%; 4 novel) nonsense mutations, 10 (12%; 8
novel) small insertions and deletions, and 15 mutations (18%; 6 novel) predicted to disrupt premRNA splicing.
Sixteen (15%), 15 (14%) and 27 (25%) of the 109 families respectively, carried at least one such change.
Whole-gene deletions occurred on a single allele in 3 families. The affected individuals in families 12 and 51
appeared homozygous for a paternal mutation, the child in family 11 for a maternal mutation. Deletions
confirmed and sized by haplotype analysis across the ABCB11 chromosomal region accounted for apparent
homozygosity. In family 12 the flanking markers were D2S156 and D2S326; the deletion included up to
12.5Mb of sequence
29. In family 51 the deletion occurred between markers D2S399 and LRP2 (encoding low
density lipoprotein-related protein 2) and included up to 2.24Mb of sequence. Exact breakpoints were not
determined but both lay outside the coding and promoter regions of ABCB11. Extended haplotype analysis
was not possible in family 11.
The remaining 55% of mutations were missense substitutions (Table 3, Figure 1), which were present on at
least one allele in 86 (79%) families. Forty-five different mutations (32 novel) were identified.
Thirty-two percent (26) of the 82 mutations occurred, or had been reported to occur, in >2 families, with 16%
(13) in >3 families. Most frequent were E297G and D482G, one or both of which were present in 58% (52/89)
of European families, and 15% (3/20) non-European families. E297G was detected in 34
European families (41 alleles) and on one allele in both an African-American and a South Asian family.
D482G occurred in 20 European families (25 alleles) and on one allele in a Central Asian/Arab family.
In all but 10 families mutations were identified on both alleles. Of the remainder, in 2 families (107, 109), only
maternal DNA was available; in one (108) available material was insufficient for complete sequence analysis;
and in 7 (100-106) a second mutation was not detected despite complete sequence analysis.
Of 99 families in which complete sequence analysis was possible and mutations were identified on both alleles
patients were homozygous for a single mutation in 36% (36/99) of cases and compound heterozygotes for 2
different mutations in the remaining 64% (63/99). In 23 families homozygosity was associated with known
consanguinity, whilst in 9 families, 2 copies of either E297G or D482G were found.
To assess effects of specific ABCB11 genotypes on expression of immunohistochemically detectable BSEP
protein, families were grouped according to whether they carried: 2 likely protein-truncating mutations; at least
one missense mutation (E297G or D482G excluded); at least one copy of E297G; at least one copy of D482G;
or only one identified mutation (Supplementary Tables 1A-1E).
Immunohistochemical analysis was possible in 88 patients. All evaluated patients with 2 predicted
protein-truncating mutations lacked demonstrable BSEP (Supplementary Table 1A). Variability in BSEP
expression was greatest when either of the 2 common European mutations, E297G or D482G, was present on
one or both alleles (Supplementary Tables 1C-1E).
Twenty-nine patients with at least one copy of E297G were immunostained: BSEP was not detected in 16
(55%), was deficient in 12 (41%), and normal in one. For 14 patients with at least one copy of D482G, BSEP
was not detected in 8 (57%), was abnormal in 3 (21%) and normal in 3 (21%). In total 45% (19) of 42
immunostained patients with either of these mutations exhibited some BSEP expression. When all genotypes
were considered, BSEP marking was absent in 72% (63/88), abnormal in 22%
(19/88) and normal in 7% (6/88) of patients. Thus in total 93% (82/88) of all immunostained patients had
abnormal or absent BSEP marking. Representative patterns are illustrated in Figures 2A-H.
Outcome data (Supplementary Tables 1A-1E) confirmed that patients with BSEP deficiency are at
considerable risk of hepatobiliary malignancy. Fifteen percent (19/128) of evaluable patients developed
hepatocellular carcinoma or cholangiocarcinoma. Correlation with genotype identified those with 2
protein-truncating mutations as being at particular risk. Thirty-eight percent (8/21) of patients with 2 predicted
protein-truncating mutations developed malignancy versus 10% (11/107) of patients with potentially less
severe defects, giving a relative risk of 3.7 (CL=1.7-8.1 p=0.003)
In keeping with the severe phenotype for which study subjects were selected, at least 45% (37) of the 82
different mutations identified were predicted to result in premature protein truncation or failure of protein
production (Tables 1, 2). Of the 10 deletions and insertions identified, 8 resulted in a frameshift and the
introduction of a premature termination codon, whilst the other 2, in-frame deletions of 4 and 7 amino acid
residues, were likely to lead to protein misfolding and degradation. Deletions were more frequent than
insertions and typically involved the loss of one or 2 nucleotides. They were uniformly distributed throughout
the gene (Figure 1). Most occurred within repeats or strings of nucleotides, suggesting origin by slippage or
misalignment during DNA replication. The exception was exon 11, with 4 different deletions, 3 identified in the
current study and one previously
44. Of these, 3 arose between nucleotides 1100-1146, suggesting a deletion
Of the 15 splice-site changes identified, all but 2 involved the invariant GT or AG dinucleotides respectively at
positions +1 / +2 of the 5’ donor and -2 / -1 of the 3’ acceptor splice sites. The change c.1435-13_1435-8del is
also predicted to be damaging. The remaining change, c.2012-8T>G, disrupted the pyridimine tract of the
already poorly conserved 3’ splice site of intron 16. Analysis of liver cDNA from a patient with this mutation (64)
demonstrated skipping of exon 17 (unpublished data).
The most common defects were missense mutations. Forty-five different substitutions were identified, with a
missense mutation present on at least one allele in 79% (86/109) of families. However, whilst insertions,
deletions, nonsense and splicing mutations are readily envisaged as damaging, the effect of a missense
substitution is more difficult to predict. Changes in amino acid size, charge, polarity and hydrophobicity can all
disrupt functional domains, protein structure, or affect protein processing and trafficking. At the nucleotide
level, changes within the coding region may disrupt sequences that enhance or repress pre-mRNA splicing
Substitutions were considered detrimental based on usual criteria (cf. Methods, mutation-detection strategy).
In all but one case substitutions were not present in control panels. The exception was E297G, present on a
single allele in 1/200 European Caucasian control chromosomes (http://pharmacogenetics.ucsf.edu)
keeping with the high frequency of this allele among European BSEP-deficiency patients.
Degrees of conservation across orthologous and homologous proteins indicate the likely importance of a given
amino acid. All altered residues were conserved or, in 2 cases, replaced only by conservative substitutions, in
BSEP orthologues in the mouse, rat and skate (Table 3). Eighty-seven percent of missense residues were
conserved or substituted only conservatively in homologous MDR/ABCB family members (multidrug resistance
1 [MDR1] and multidrug resistance 3 [MDR3] proteins).
At the biophysical level, 89% of missense mutations were predicted to change at least one aspect among
charge, secondary- or tertiary-structure interactions, and hydrophobicity/polarity of BSEP (Table 3)
percent of mutations significantly changed amino-acid residue charge, 51% size, and 69%
hydrophobicity/polarity. Forty-three percent introduced or removed residues whose interactions typically
determine secondary and tertiary protein structure. Of these, 22% introduced disulfide-bridge - forming
cysteines or alpha-helix - breaking prolines; the remainder introduced or removed the hydroxylated residues
serine or threonine.
Whilst protein-truncating mutations were distributed uniformly throughout the protein (Figure 1), 60% of missense
changes clustered in the 2 highly conserved NBF domains (residues 414-610, 1072-1321
; 38% in NBF1 and
22% in NBF2). Fourteen changes (31%) occurred within, or immediately adjacent to, the Walker motifs. Of the
remainder, 7 (16%) occurred in the transmembrane (TM) domains; 5 of these introduced a charged residue into a
hydrophobic domain and, in 3 cases, simultaneously removed glycines, which stabilise alpha-helical TM spans.
The intracellular loops contained 6 changes (13%), of
which 3 including E297G, occurred in intracellular loop 2. No changes were identified in the extracellular loops
although 2 have been reported
In the 93% (99/106) of families in which complete sequence analysis was possible, 2 mutant ABCB11 alleles
were identified. In the remaining 7 families only a single mutation was identified despite extensive analysis. In 5
cases the single mutations were splice site changes or E297G/D482G. In only 2 families were novel missense
mutations identified (Q466K, N490D). Consistent with pathogenicity, both were at conserved sites in
homologues and were absent from control populations. Q466K was associated with abnormal BSEP marking
(detrimental). N490D was associated with normal marking; however, this mutation lies within NBF1, a region in
which disease-associated mutations co-existed with retained BSEP marking (see below). Whilst we cannot
exclude mutations in genes other than ABCB11 in this patient, analysis of ATP8B1 has identified no defects
Most cystic fibrosis patients in whom sequencing detects only one mutation harbour microdeletions of one or
53. This is likely also true for ABCB11. Such deletions, unless homozygous, will not be detected by
genomic sequencing; exon-dosage analysis such as multiplex ligation-dependent probe amplification must be
Among the 82 different mutations identified in this study, 32% (26) occurred, or had been reported, in multiple
families, with 16% (13) present in >3 families. These likely represent both recurrent and founder mutations. The
most common natural mutation hotspots are cytosine-guanine (CpG) dinucleotides
Thirty-three percent (18) of 54 missense and nonsense mutations occurred at these sites (Tables 1 and 3).
Ten mutations occurred in multiple families: R470Q, R832C
33, R948C, A1110E and R1231Q
48 have now been
reported in 2 families; R1090X
2 in 3 families; G982R
1, 2, R1153C
1, 47and R1153H in 4 families; and R575X in 6
1, 2, 32, 45 .
Six common missense and nonsense changes occurred at non-CpG sites: R520X and A588V
33 in 2
European families and E1302X and I541L
33, 49 in 3 European families each. By far most common, however, were
E297G and D482G, at least one of which was present in 58% (52/89) of European and in 15% (3/20) of
non-European families. The population distribution of E297G, most frequent along the North European
seaboard, suggests origin in Northern Europe and spread southwards through Central and Eastern Europe.
The mutation was also found in one South Asian and one African-American family. In contrast, D482G likely
originated in Central or Eastern Europe, with cases identified in Polish, Austrian, Slovak, Czech, Hungarian and
Russian families. This mutation was also present in a Greek and an Iranian family.
Five common insertions and deletions were identified, with c.379delA in 3 apparently unrelated Arab families
and c.1145-1165del, c.1583_1584delTA, c.1941delA
28 and c.2787_2788insGAGAT
5 in 2 European families
each. Five common splice-site mutations occurred, with c.611+1G>A
29 and c.3213+1delG
2 in respectively 2, 7, 4, 3 and 2 families. Geographical distributions
suggest that most are likely founder mutations.
Mutation clusters were also observed. For NBF domains this likely reflects functional importance. At other sites
both mutations and polymorphisms clustered within the same or adjacent codons, suggesting sequence
instability or mutagen interaction. Mutation clusters include Y472C, Y472X
28 and I541L
33, 49/ I541T. Four different
changes occurred at, or adjacent to, the 5’ splice site of intron 9: R303K, c.908+1delG
1, 33, c.908+1G>T
c.908+1G>A. Three occurred at the 5’ splice site of intron 18: c.2178+1G>A
4, 28, c.2178+1G>T and
BSEP deficiency represents a phenotypic continuum between progressive early-onset
remitting, and late-onset, phenotypes
. Eleven different mutations have been reported in BSEP deficiency
disease clinically assessed as intermediate
or mild in severity
. Three of these, E297G, A570T, and
c.2178+1G>A, have also been found in PFIC. In milder disease, missense mutations
predominate over those that result in protein truncation or a failure of protein production; more mutations occur
in less conserved regions, e.g., intracellular loops, than in the NBFs; and mutated residues are less likely to be
conserved. Compound heterozygosity for both a severe and a mild mutation may result in mild disease. That
homozygosity for E297G is seen in discrepant phenotypes strongly indicates that environment and genetic
background also play roles.
Immunohistochemistry appears valuable in identifying likely BSEP deficiency, as many patients studied
exhibited little or no detectable BSEP at the hepatocyte canaliculus
2, 28, 29
. An anti-C-terminal antibody can be
expected not to mark when protein is absent or when a misfolded or truncated “partial protein” is expressed.
Immunoreactivity may depend on tissue processing, disease stage, therapeutic intervention and disease
mechanism. These factors are to be considered in interpreting immunohistochemical results.
Immunohistochemically detectable BSEP expression does not exclude functional BSEP deficiency.
Twenty-eight percent (25/88) of patients analysed exhibited some degree of BSEP marking; in 6 expression
was considered normal. Residual marking was most striking in patients with E297G or D482G, with some seen
in 45% (19/42) of patients carrying at least one of these alleles. Ten additional missense mutations were
associated with detectable BSEP marking (Supplementary Tables 1B-1E, Figures 2A-2H). Abnormal marking
was seen with L50S, Q466K, N515T, R517H, I541L, and F548Y, and normal with N490D, G562D, R832C, and
A1110E. As most of these were found in combination with E297G or D482G their individual effects could not be
assessed. Nine of the 12 mutations occurred within the highly conserved NBFs (8 within NBF1, 4 within or
adjacent to Walker motifs) suggesting an effect on protein function. Abnormal (typically reduced) marking may
also result from decreased protein production or defective sorting or instability of an otherwise functional
protein. Such patients in particular may be amenable to therapeutic interventions such as PEBD or the use of
pharmacological agents which enhance BSEP cell-surface expression, e.g., 4-phenylbutyrate
The above notwithstanding not to detect BSEP immunohistochemically, or to demonstrate BSEP
deficiency, is highly useful. Deficiency or absence of BSEP expression could be demonstrated in 93%
(82) of the 88 patients in whom immunohistochemical study was possible. As some patients were selected
for mutational analysis in part because they lacked immunohistochemically detectable BSEP, the exact
sensitivity of this technique could not, however, be assessed.
We have previously shown that BSEP deficiency is associated with increased susceptibility to hepatobiliary
. In this study 38% (8/21) of patients with 2 predicted protein-truncating mutations
(Supplementary Table 1A) developed either hepatocellular carcinoma or cholangiocarcinoma, versus 10%
(11/107) of other patients (Supplementary Tables 1B-1E), giving a relative risk of 3.7 (1.78.1, p=0.003). Of the
other 13 patients with 2 protein-truncating mutations, all but 3 have undergone OLT or died of non-malignant
disease. The 3 who retain their native livers (ages 2.4yrs, 7yrs and 16yrs) are under close observation. Without
OLT the incidence of malignancy in patients with 2 protein-truncating ABCB11 mutations is expected to exceed
that observed in this study. BSEP-deficient patients, in particular those carrying 2 predicted null mutations, who
retain their native liver require close surveillance for hepatobiliary malignancy.
Table 1: Nonsense mutations, deletions and insertions
Abbreviations: EU, European Caucasian; CA/AR, Central Asian/Arab; EA, East Asian; AF, African.
Table 2: Splice site mutations
Abbreviations: EU, European Caucasian; CA/AR, Central Asian/Arab. *Denotes studies in which
mutations are associated with mild disease.
Table 3: Missense mutations
Abbreviations: EU, European Caucasian; CA/AR, Central Asian/Arab; EA, East Asian; SA, South Asian; AF,
African; m/r/s bsep, mouse/rat/skate bsep; IC, intracellular loop; Hyd/Pol, hydrophobicity or polarity; NBF,
nucleotide-binding fold; NBF/TM, location between NBF domain and TM region; NH2 Term, amino terminal;
MDR1/3, multidrug resistance proteins 1/3; TM, transmembrane domain. Unless specified all proteins are
human. *Denotes studies in which mutations are associated with mild disease.
Figure 1: Schematic representation of BSEP with location of mutations identified in this study. BSEP is a full
41 composed of 2 homologous halves, arranged in tandem and joined by a linker region. Each
half comprises a hydrophobic membrane domain, containing 6 hydrophobic transmembrane (TM) spans, and
a cytoplasmic nucleotide binding fold (NBF). The NBFs contain the Walker A/B motifs, characteristic of all
nucleotide-binding proteins, and ABC signature, stacking aromatic, D, H and Q, loops which define ABC
42. The NBFs bind and hydrolyse ATP to generate transport-driving energy, whilst the TM domains form
the pore and define substrate specificity. Schematic generated using TOPO2 software
(http://www.sacs.ucsf.edu/TOPO-run/wtopo.pl). Protein topology predicted by comparison with MDR1
spans predicted to fit the accepted topology using TopPred II (http://www.expasy.ch/tools/). Singly mutated
residues represented as coloured
residues; founder/recurrent mutations indicated by arrows. For insertions/deletions the affected residue
is indicated, for splice sites the adjacent amino acid. Key: Blue-missense; Red-stop; Green
deletion/insertion; Orange-splice-site; Black-several different mutations at this site; Purple-Walker A, B and
Figure 2: Expression patterns on immunostaining for canalicular transporters bile salt export pump
(BSEP) and multidrug resistance-associated protein 2 (MRP2). Avidin/biotin technique; all sections
counterstained with haematoxylin.
A. Normal BSEP marking of control liver without cholestasis. Anti-BSEP antibody marks an orderly
canalicular network. Original magnification 200x.
B. Normal MRP2 marking of control liver without cholestasis. Anti-MRP2 antibody marks an orderly
canalicular network. Original magnification 200x.
C. Absent BSEP marking in patient 4, homozygote for ABCB11 mutation yielding Y472X. Hepatectomy for
cirrhosis with hepatocellular carcinoma, age 1 year. No marking for BSEP is seen. Original magnification
D. MRP2 marking in patient 4 (see C, above). Anti-MRP2 antibody highlights canalicular network.
Original magnification 200x.
E. BSEP marking in patient 29, homozygote for ABCB11 mutation yielding R832C. Hepatectomy for
cirrhosis, age 3 years. The canalicular network is delineated well. Original magnification 200x.
F. MRP2 marking in patient 29 (see E, above). Anti-MRP2 antibody highlights canalicular network.
Original magnification 200x.
G. BSEP marking in patient 47b, compound heterozygote for ABCB11 mutations yielding L50S and A1110E.
Hepatectomy for cirrhosis with hepatocellular carcinoma, age 6 years. Canalicular marking for BSEP is
abnormal; it is present only focally (arrows) and is assessed as both faint and patchy. Original magnification
H. MRP2 marking in patient 47b (see G, above). Reaction product (anti-MRP2 antibody) highlights centres
of hepatocellular rosettes as well as canalicular network. Hepatectomy. Original magnification 200x.
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Nucleotide change Exon Predicted effect Mutation type CpG
Total number of
families in which
c.74C>A 2 p.Ser25X Nonsense No 1 18 E
c.379delA 5 p.Thr127HisfsX6 Deletion 3 1-3 C
c.1101_1102delAG 11 p.Val368ArgfsX27 Deletion 1 52 E
c.1139delT 11 p.Leu380TrpfsX18 Deletion 1 13 E
c.1145_1165del 11 p.Ala382_Ala388del Deletion 2 14, 15 E
c.1416T>A 13 p.Tyr472X Nonsense No 1 4 C
c.1558A>T 14 p.Arg520X Nonsense No 2 59, 81 E
c.1583_1584delTA 14 p.Ile528SerfsX21 Deletion 2 53, 54 E
c.1723C>T 15 p.Arg575X Nonsense Yes 6 E
12, 13, 17,
c.1941delA 16 p.Gly648ValfsX6 Deletion 2 16, 55 E
c.2316T>G 19 p.Tyr772X Nonsense No 1 5 E
c.2787_2788insGAGAT 22 p.Lys930GlufsX79 Insertion 2 36 E
c.2906_2917del 23 p.Lys969_Lys972del Deletion 1 56 E
c.3268C>T 25 p.Arg1090X Nonsense Yes 3 17, 60 E
c.3438delA 26 p.Val1147X Deletion 1 57 E
c.3491delT 26 p.Val1164GlyfsX7 Deletion 1 58 E
c.3643C>T 27 p.Gln1215X Nonsense No 1 38 E
c.3703C>T 27 p.Arg1235X Nonsense Yes 1 6 E
Table 1: Nonsense mutations, deletions and insertions in ABCB11
c.3904G>T 28 p.Glu1302X Nonsense No 3 20, 21, 61
Table 2: Splice site mutations in ABCB11
5’ Intron 24 c.3213+1delG
Splice site Nucleotide change Ethnic origin
Total number of
families in which
3’ Intron 5 c.390-1G>A No 1 8 CA / AR
5’ Intron 7 c.611+1G>A No 2 21, 62 EU 28
5’ Intron 9 c.908+1delG No 1 39 EU 1, 33
5’ Intron 9 c.908+1G>T No 1 107 EU 28
5’ Intron 9 c.908+1G>A No 1 82 CA / AR
3’ Intron 13
3’ Intron 16
5’ Intron 18 c.2178+1G>T No 1 19 EU
5’ Intron 18 c.2178+1G>A No 4 EU 4*, 28
5’ Intron 18 c.2178+1G>C No 1 43 EU
3’ Intron 18 c.2179-2A>G Yes 1 11 EU
5’ Intron 19 c.2343+1G>T Yes 3 EU
44, 66, 67 29
5’ Intron 19 c.2343+2T>C Yes 1 9 EU 28
3’ Intron 21 c.2611-2A>T Yes 1 10 CA / AR
No 2 7 EU, CA / AR 2
of families in
B S E P m b s
e p r b s e p s
b s e p
Change in: S
i z e C h a r g
e H y d / P o l
S h a p e
c.149T>C p.Leu50Ser 4 No NH2 Term Y Y L L L L L 1 47
c.470A>G p.Tyr157Cys 6 No TM2 Y Y Y Y Y Y 1 49
c.725C>T p.Thr242Ile 8 No TM4 Y T T T T T T 1 41
c.890A>G p.Glu297Gly 9 No IC2 Y
Y Y E E E E Q
c.908G>A p.Arg303Lys 9 No IC2 R R R R 1 22
1 83 Y Y Y R R R R c.937C>A p.Arg313Ser 10 Yes IC2
Y Y 1 50 c.980G>A p.Gly327Glu 10 No TM5 G G G G
c.1168G>C p.Ala390Pro 11 No TM / NBF Y A A A A 1 68
c.1229G>A p.Gly410Asp 12 No TM / NBF Y Y G G G G 1 84
c.1238T>G p.Leu413Trp 12 No TM / NBF L L L L 1 45
c.1388C>T p.Thr463Ile 13 No Adj Walker A Y Y T T T T T T 1 38
c.1396C>A p.Gln466Lys 13 No Adj Walker A Y E 1 101
c.1409G>A p.Arg470Gln 13 Yes Adj Walker A Y R
2 23, 85
c.1415A>G p.Tyr472Cys 13 No Adj Walker A Y Y Y
Y Y Y 1 46
Y Y c.1442T>A p.Val481Glu 14 No NBF1 V L L L V I 1 36
c.1445A>G p.Asp482Gly 14 No NBF1 Y Y D
c.1460G>C p.Arg487Pro 14 Yes NBF1 Y Y Y 1 24
1 106 c.1468A>G p.Asn490Asp 14 No NBF1 Y
c.1535T>C p.Ile512Thr 14 No NBF1 Y Y I I I I I I 1 46
c.1544A>C p.Asn515Thr 14 No NBF1 Y Y
1 87 c.1550G>A p.Arg517His 14 Yes NBF1 Y Y
c.1621A>C p.Ile541Leu 14 No NBF1 I I I I I I 3 25, 39
c.1622T>C p.Ile541Thr 14 No NBF1 Y Y I I I I I I 1 44
c.1643T>A p.Phe548Tyr 15 No Adj ABC F F F F F F 1 69
c.1685G>A p.Gly562Asp 15 No ABC Y Y 1 88
c.1708G>A p.Ala570Thr 15 Yes ABC / Walker B Y A
A A A 1 26
c.1763C>T p.Ala588Val 15 No Adj Walker B Y A A A A 2 70, 89
Y Y 1 27 c.2272G>C p.Gly758Arg 19 No NBF / TM
c.2296G>A p.Gly766Arg 19 Yes TM7 Y Y 1 28
c.2494C>T p.Arg832Cys 21 Yes IC3 Y
Y Y Y R
2 29, 90
c.2576C>G p.Thr859Arg 21 No IC3 Y Y Y T T T T T T 1 43
c.2842C>T p.Arg948Cys 23 Yes IC4 Y Y Y 2 42, 71
c.2935A>G p.Asn979Asp 23 No TM11 Y 1 30
c.2944G>A p.Gly982Arg 23 Yes TM11 Y
Y Y G
4 31, 37
1 91 Y Y Y T T T T c.3086C>A p.Thr1029Lys 24 No TM12
c.3329C>A p.Ala1110Glu 25 Yes Adj Walker A Y Y A A A A 2 47, 72
1 32 Y Y Y c.3382C>T p.Arg1128Cys 25 Yes Adj Walker A
c.3457C>T p.Arg1153Cys 26 Yes NBF2 Y
Y Y Y R
4 33, 34, c.3458G>A p.Arg1153His 26 Yes NBF2 Y Y
c.3460T>C p.Ser1154Pro 26 No NBF2 Y S
S S A 1 50
Table 3: Missense mutations in ABCB11
c.3628A>C p.Thr1210Pro 27 No Adj ABC Y T T T T T T 1 35
1 92 c.3691C >T p.Arg1231Trp 27 Yes ABC / Walker B Y Y
c.3692G>A p.Arg1231Gln 27 Yes ABC / Walker B Y 2 48
c.3724C>A p.Leu1242Ile 27 No Walker B L L L L L L 1 45
c.3892G>A p.Gly1298Arg 28 No NBF2 Y Y 1 49
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