ABCB11 Gene Mutations in Children with Progressive Familial Intrahepatic Cholestasis (PFIC) and Low GGT

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1 Med. J. Cairo Univ., Vol. 84, No. 1, March: , ABCB11 Gene Mutations in Children with Progressive Familial Intrahepatic Cholestasis (PFIC) and Low GGT MARIANNE S. MAKBOOL, M.D.*; HANAA M. EL-KARAKSY, M.D.**; AMAAL A. ABD EL-AAL, M.D.* and NORA M. SELIM, M.D.* The Departments of Clinical & Chemical Pathology* and Pediatrics**, Kasr Al-Ainy School of Medicine, Cairo University, Egypt Abstract Background: Progressive Familial Intrahepatic Cholestasis (PFIC) refers to a heterogeneous group of autosomal recessive disorders of childhood that disrupt bile formation and present with cholestasis of hepatocellular origin. The ABCB 11 gene encodes the ATP-dependent canalicular Bile Salt Export Pump (BSEP) of human liver which is the major exporter of bile acids against extreme concentration gradient. Mutations in this protein leads to decreased bile flow (PFIC2) with accumulation of bile salts inside hepatocytes with ongoing severe hepatocellular damage. Aim of Work: To detect mutations in exon 9 of ABCB 11 gene in patients with suspected PFIC2 among studied Egyptian population in order to confirm diagnosis of PFIC2 and to detect mutations that are the cause of marked B SEP deficiency. Patients and Methods: This study was conducted on 33 subjects including 11 suspected PFIC2 patients and 22 healthy control subjects. ABCB 11 genotyping was performed by DNA extraction followed by PCR amplification, purification then sequencing of exon 9 of the gene. Results: No mutations or variations in sequence results involving Exon 9 of the ABCB 11 gene of studied Egyptian PFIC2 patients and phenotypically healthy subjects. Conclusion: In spite of considering exon 9 mutations of ABCB 11 gene are common worldwide, these mutations are not that common among Egyptian ethnic population, however larger sample size is needed. Key Words: Progressive Familial Intrahepatic Cholestasis (PFIC2) Bile Salt Export Pump (BSEP) ABCB11 gene Exon 9. Introduction PROGRESSIVE Familial Intrahepatic Cholestasis (PFIC) is a group of inherited liver disorders of childhood in which cholestasis of hepatocellular origin mostly presents in the neonatal period and Correspondence to: Dr. Marianne S. Makbool, The Department of Clinical & Chemical Pathology, Kasr Al-Ainy School of Medicine, Cairo University, Egypt leads to death from liver failure at ages usually ranging from infancy to adolescence [1]. Molecular and genetic studies provided the identification of genes responsible for three types of PFIC and exhibited that they were related to mutations in hepatocellular transport system genes involved in bile formation [2]. Progressive familial intrahepatic cholestasis represents 10% to 15% of causes of cholestasis in children and 10% to 15% of indications of liver transplantation in children. PFIC 1 and PFIC2 represent 2/3 of PFIC cases, and PFIC3 represents 1/3 of cases [3]. The exact prevalence of PFIC is not accurately known, but PFIC is considered a rare disease with an incidence of 1/5 0,000 to 1 / 100,000 births. All types of PFIC exist worldwide [4]. Secretion of bile acids is the main driving force for bile flow in humans. The described Adenosine Triphosphate (ATP)-dependent bile acid transporter, Bile Salt Export Pump (BSEP), is responsible for transport of bile acids actively through the hepatocellular canalicular membrane into bile. It was recognized that mutations in the gene that encode this protein (ABCB 11) are responsible for a subgroup of infants and children with Progressive Familial Intrahepatic Cholestasis-2 (PFIC-2) [5]. PFIC2 patients present with severe jaundice, hepatomegaly, failure to thrive, and pruritis. Laboratory investigations show direct hyperbilirubinemia, high level of serum aminotransferases, and unexpectedly normal serum gamma glutamyl trans-peptidase. There is rapidly progressive course to liver cirrhosis, failure and hepatocellular carcinoma, which can only be treated by liver transplantation [6]. 249

2 250 ABCB11 Gene Mutations in Children with PFIC & Low GGT Severe phenotypes are usually associated with mutations leading to premature protein truncation or failure of protein production. Insertion, deletion, missense, non sense and splice site mutations result in destructive effects and patients showed little or no detectable BSEP at the hepatocyte canaliculus [7]. Genotyping of ABCB 1 1 gene mutations is the only way to confirm diagnosis of PFIC2 in suspected patients and close follow-up of these patients that usually progress to liver cirrhosis and hepatocellular carcinoma is mandatory [8]. Patients and Methods The study was conducted on 1 1 children previously diagnosed as suspected PFIC2 and 22 non diseased subjects (control group). They were selected from the Pediatric Hepatology Unit at Cairo University Children's Hospital, after approval by the Ethical Committee of Faculty of Medicine, Cairo University. The study was done over the years 2011 to A written informed consent was taken from parents of pediatric patients undergoing the study. PFIC2 was suspected in children with a clinical history of cholestasis of unknown origin after exclusion of the other main causes of cholestasis (e.g. biliary atresia, Alagille syndrome, (x -1antitrypsine deficiency, cystic fibrosis, sclerosing cholangitis, viral or autoimmune hepatitis, and extra hepatic bile duct obstruction). Diagnosis of PFIC2 was made in patients with chronic cholestasis in the form of recurrent episodes of jaundice from first months of life that become permanent later in the course of the disease. Severe pruritis is usually observed. Clinical manifestations together with normal (GGT) activity, very high serum bile acid concentration, radiological and histological approaches helped the diagnosis. Data collected from the patients' files included: Demographic data: Current age, sex, age of presentation, consanguinity and family history. Clinical features: Presence of jaundice, pruritus, bleeding tendency, hepatomegaly or spleenomegaly. Results of GGT, AST, ALT, bilirubin, serum bile acids, alpha feto protein. Results of abdominal U/S and liver biopsy. All subjects were subjected to: DNA sequencing for exon 9 of ABCB 1 1 gene for the total number of 33 samples. Specimen collection: Three ml of venous blood were collected from each subject in a sterile EDTA vaccutainer for the genotyping technique. DNA was extracted from fresh samples to be used for performing the PCR for ABCB 1 1 gene study. Sequencing of exon 9 of ABCB 1 1 gene required the following steps: DNA extraction, amplification using the Polymerase Chain Reaction, detection of PCR amplification products, DNA purification, cycle sequencing, long read capillary electrophoresis and finally analysis of data. Amplification of exon 9 of ABCB11 gene by Polymerase Chain Reaction (PCR): Genomic DNA was extracted from peripheral blood EDTA samples by QIAamp DNA extraction kit (QIAGEN GmbH. Germany) according to manufacturer's instructions. Amplification of the extracted DNA was done according to the protocol proposed by Thompson et al., [9]. Followed by detection of PCR amplification products using 1.5% agarose gel electrophoresis containing ethidium bromide and ultraviolet light transillumination. Using HOT FIREPol (10X) Master Mix (Solis BioDyne, Tartu.Estonia) which contain: Ready to use HotStart Taq DNA polymerase (recombinant) mixture, optimized HotStart PCR buffer, MgCl2 and dntps. Forward and reverse primers (supplied by Bioscience GmbH. Jena, Germany) and annealing temperatures for exon 9 of ABCB 1 1 gene are described in (Table 1). Table (1): Primers sequence and the protocol of amplification. Exon Exon 9 Forward primer Reversed primer Amplification protocol Number of nucleutides 5-TGAGAATCTAATATTGTATTA AACCCATGCC -3` 5`-CAAGGTGGGTCTGCCGCTT-3` - Initial activation at 95ºC for 15 minute - 34 cycles of: 94ºC for 45 seconds. 62ºC for 45 seconds. 72ºC for 1 minute. - Final elongation at 72ºC for 1 minute 340 base Reactions were performed in a total volume of 25gL containing: 2gl PCR HotStart Master Mix (10X), 1 g l forward primer (100pmol/g l), 1gl reverse primer, 14gl nuclease-free water and 7 g l purified DNA solution. Gradient thermal cycler (Professional Thermocycler, Biometra; Applied Biosystems, Foster City, CA, USA) was programmed according to the following conditions: Initial activation at 95ºC for 15 minutes, then 34 cycles of denaturation at 94ºC for 45 seconds,

3 Marianne S. Makbool, et al. 251 annealing at 62ºC for exon 9 for 45 seconds, extension at 72ºC for 1 minute. Final elongation was done at 72ºC for 1 minute. The DNA quality was examined using 1.5% agarose gel electrophoresis, the amplified DNA ran as a single bright band on an agarose gel which indicated successful amplification. Cycle sequencing and capillary electrophoresis (on the ABI 3500 genetic analyzer): The amplified DNA was purified using QIAquick PCR purification kit supplied by Qiagen, Germany. Then Frederick Sanger's enzymatic dideoxy DNA sequencing technique was applied which was based on the chain-terminating dideoxynucleotide analogues [10] using ABI PRISM Big Dye Terminator cycle Sequencing Ready Reaction Kit v (contain dideoxynucleotides) and BigDye 5X dilution buffer supplied by Applied biosystem (Life Technologies Corporation, California, USA). For each reaction the following reagents were added to a separate tube: 2.0gl of BigDye Terminator, 1.0g l of 5X Dilution buffer, 2.0 g L of the forward Primer used for PCR reactions, 2.0 g L of template DNA, and 3.0 gl Nuclease free water to complete the total volume to 1 0 gl. The thermal cycler was adjusted to the following conditions: Initial denaturation at 96ºC for 1 minute followed by 25 cycles of: Denaturation at 95ºC for 10 seconds, annealing temperature 62ºC for exon 9 for 5 seconds, and extension at 72ºC for 4 minutes. Then purification step was performed using BigDye X terminator purification kit supplied by Applied Biosystem to sequester unincorporated dye terminators and dntps to prevent their co-injection with dye-labeled extension products. The cleaned up sequencing reactions were resuspended in 15 gl of Hi Di formamide for denaturing DNA before injection, then it was injected in Applied Biosystems 3500 Genetic Analyzers Fig. (1). Fig. (1): The sequence results of PCR product of Exon 9 of suspected PFIC2 patient number (10). Analysis of data: Sequences were compared to the published sequence (NM ). According to NM exon 9 is from 910 to 1034 nucleotide base. According to Homo sapiens ATP-binding cassette, sub-family B (MDR/TAP), member 1 1 (ABCB 11), mrna by NCBI (National Centre of Biotechnology Information) [11]. Reference Sequence: NM which is 4775 bp mrna and its translated protein (bile salt export pump) NP which is 1321 amino acid analysis was done by BLAST [Basic Local Alignment Search Tool] [11] ( and the CLC-BIO sequence viewer 6 program ( com). Statistical methods: Data obtained from the study was coded and entered using the software SPSS (Statistical package for social science) version 17. (SPSS, Inc, Chicago, IL, USA) Parametric data were summarized using mean and standard deviation, while non-parametric data were summarized as median and percentiles for quantitative variables. Frequency

4 252 ABCB11 Gene Mutations in Children with PFIC & Low GGT and percentages were used for qualitative variables. Comparison between groups was done using Chi square and Fischer exact test for qualitative variable, t-test and non-parametric Mann Whitney test were used to compare two groups. p-value was considered significant if <0.05. Results Clinical data: This cross-sectional study was performed in the molecular biology unit in chemical pathology department. Eleven suspected PFIC2 patients (their current age 1-6 years), and twenty two control subjects were enrolled in this study. They were suspected to be diagnosed as PFIC2 before the age of 3 years; (9/11) at less than 6 month, 1 /1 1 at the age of 1 year, and 1/ 1 1 at the age of 3 years. Ten cases (90.9%) were males and 1/11 (9.1%) was a female. Eight of cases were positively consanguinous (72.7%), one of these families there was one sibling died at 6 month and he was jaundiced. The (control group) consisted of 22 subjects, 1 1 males (50%) and 11 (50%) females. Demographic data of subjects are presented in (Table 2). All suspected PFIC2 patients presented by jaundice, Seven of them (63%) presented by recurrent episodes of jaundice, while four cases (37%) developed continous jaundice from the start. Eight of cases (72%) presented by pruritis, mostly severe. Five patients had associated bleeding (45%) and three of them (27.3%) had dysmorphic features. Nine patients had hepatomegally (81.8%) and two of them had hepatosplenomegally (18.18%). Biochemically, all PFIC2 patients 1 1/ 1 1 (100%) had normal GGT activity, elevated transaminases (AST, ALT), bilirubin (total & direct) and very high serum bile acid concentration. Radiologically, U/S wise two patients (18.2%) had average size liver and spleen, seven of them (63.6%) had hepatomegally, and two had hepatospleenomegally (18.2%). Pathology wise, six of cases 6/11 (54.54%) had blurred lobular architecture, intracellular and canalicular cholestasis together with diffuse hydropic and ballooning degeneration of hepatocytes with association of multinucleated giant cell forms (neonatal giant cell hepatitis), one of the patients (number 10 among subjects), his current age 6 years, showed developed chronic hepatitis with impending fibrosis. Two of patients 2/11 (18.2%) showed Paucity of Intralobular Bile Ducts (PIBD). Clinical data are presented in (Table 3). DNA sequence analysis of exon 9 of the ABCB 1 1 gene revealed no mutations or variations in sequence results involving phenotypically normal control group and suspected PFIC2 patients as shown in (Table 4). Table (2): Demographic data of subjects. Data Frequency Percent Groups: Patients 11/ Control 22/ Sex (among cases): Males 10/ Females 1/ Familial illness 1/ Familial pedigree: +Ve consanguinity 8/ No consanguinity 3/ Table (3): Clinical data of patients. Data Frequency Percent Hepatomegally 9/ Spleenomegally 2/ Joundice: Intermittent 7/ Continous 4/ Pruritis 8/ Vitamin deficiency (A,D,E,K) manifestation: Bleeding 5/ Rickets or dysmorphic features 3/ Normal (GGT) level 11/ U/S Findings: Hepatomegally 7/ Hepatosplenomegally 2/ Average size liver & spleen 2/ Liver biopsy result: Neonatal hepatitis 6/ PIBD 2/ Not available 3/ Table (4): Variation distribution among subjects involving Exon 9 of ABCB 11 gene. Data Frequency Percent Groups: Patient 11/ Control 22/ Nucleotide/amino acid changes: Exon 9 0/ Discussion Bile salt export pump deficiency is caused by mutations in ABCB 11. The severity of BSEP deficiency varies from progressive early-onset to remitting and late-onset phenotypes. Severe BSEP deficiency falls into the descriptive category of progressive familial intrahepatic cholestasis type 2 [12,13].

5 Marianne S. Makbool, et al. 253 Bile salt export pump is a member of the adenosine triphosphate-binding cassette (ABC) super family and P-glycoprotein/multidrug resistance (MDR/ABCB) subfamily of transporters. It is expressed at the hepatocyte canalicular membrane and it is the major exporter of primary bile acids. It actively transports conjugated bile salts into biliary canaliculi against extreme concentration gradients. Liver disease in BSEP deficiency (PFIC 2) is ascribed to failed secretion and intrahepatocytic accumulation of toxic bile salts. Patients with PFIC2 present as infants with high serum bile salt levels, pruritis, malabsorption, failure to thrive, jaundice, and cholestasis. They develop fibrosis and end-stage liver disease before adulthood. Partial external biliary diversion and ileal exclusion can relieve pruritis and, in some cases, slow disease progression. However, most patients ultimately need liver transplantation [6,14]. It was detected that in HCC patients the decrease in BSEP expression is associated with an alteration in FXR isoform expression induced by inflammation in HCC [15]. The 1321-amino acid B SEP protein is encoded by ABCB 1 1 on chromosome 2q ABCB 1 1 spans a 108 kilobase genomic region and is composed of 27 coding exons and one 5 ' untranslated exon (designated 1-28) [8]. Eighty-six polymorphisms in ABCB 1 1 have been described in a population of Caucasians, Koreans, and African Americans and showed substantial ethnic differences with respect to allele number, frequency of common and populationspecific sites. These polymorphisms are located in exons and introns, as well as in 5 -flanking regions, but no effect on the mrna or protein has been determined. However, population genetic analysis suggested some selective pressure against changes in the protein, supporting the important endogenous role of these transporters [16]. More than 100 different ABCB 1 1 disease causing variants have been identified worldwide and the more frequent mutations are grouped as missense, nonsense, deletions and insertions, and splice-site mutations. A common result of these various gene mutations is the reduction or total loss of expression of the BSEP protein on the canalicular membrane [17]. Thirty tree Egyptian subjects were enrolled in our study; twenty two control subject (22/33), eleven subjects (50%) were males and 11/22 (50%) were females, and eleven suspected PFIC2 patients (11/33), their age ranged from 1 to 6 years. All patients were subjected to full history taking including: Age at presentation, familial pedigree, familial illness, current age, clinical features in- cluding: Continuous jaundice or intermittent, pruritis and its severity, hepatomegally, spleenomegally, diahrrea, hearing lose, manifestation of vitamins (A, D, E, K) deficiency, results of GGT, AST, ALT, Bilirubin, serum bile acids, radiological, histological examination and laboratory investigation included DNA sequencing of ABCB 1 1 gene (Exon 9) for patients and controls. The study revealed no mutations or variations in sequence results involving Exon 9 of the ABCB 1 1 gene of studied Egyptian PFIC2 patients and phenotypically healthy subjects. The choice of studying exon 9 mutations was based on that these mutations are causes of severe BSEP protein deficiency and are considered sites for common mutations worldwide. Missense substitutions are the commonest mutations that either affect protein processing and trafficking or disrupt functional domains and protein structure, the most frequent, are p.e297g (located in intracellular span between TMD4 and TMD5 and encoded by Exon 9) and p.d482g (located in NBD 1 and encoded by Exon 14) specially among European PFIC2 patients [8]. Our results were similar to the results of Lang et al., [16] who studied genetic variability and haplotype structures of ABCB 1 1 in 292 healthy populations of different ethnic backgrounds in which they detected 28 genetic variants in 27 coding exons and 1 noncoding exon; among these were 10 missense mutations, 17 silent mutations and 1 mutation in the un translated exon, with the two Cocasian- specific variants in two exons not including Exon 9. The two Cocasian-specific variants were in Exon 13 (c.1331t > C; 59.4%) and exon 17 (c.2029a > G; 4.2%) coded for amino acid substitutions p.v444a and p.m677v respectively. All amino acid polymorphisms of ABCB 1 1 were predicted to be located in the extracellular region. On the contrary, many collaborative studies have identified mutations in Exon 9 of ABCB 1 1 producing marked BSEP protein deficiency [18-20]. Strautnieks et al., [18] were the first identified in affected members of 7 families segregating PFIC2 homozygosity for an 890A-G transition in the ABCB 1 1, resulting in a glu297-to-gly (p. E297G) substitution in the intracellular loop between transmembrane spans 4 and 5. Affected members of 6 other families had a heterozygous p.e297g mutation, but a second ABCB 1 1 mutation on the other allele was not identified. Also in affected members of a polish family segregating PFIC2 a heterozygous 1 -bp deletion was identified

6 254 ABCB11 Gene Mutations in Children with PFIC & Low GGT (908delG) in the ABCB 11, resulting in a change of amino acid 303 and the introduction of 17 novel amino acids followed by termination of the protein. One year later, Jansen et al., [19] using immunohisto-chemistry, found absence of the BSEP protein in 16 of 28 liver biopsy samples from patients with PFIC. All 10 of the B SEP-negative patients tested were found to have mutations in the ABCB 1 1 gene. Six patients had heterozygous mutations, among them identified compound heterozygosity in a patient with PFIC2 in the ABCB 1 1 gene: ARG to-ter (R1057X) and p.e297g. Later, Knisely et al., [20] studied 10 patients with PFIC2 in whom hepatocellular carcinoma was diagnosed between the ages of 13 and 52 months. Severe BSEP deficiency was demonstrated by immunohistochemical analysis, and 13 different mutations in the ABCB 1 1 gene were identified. These mutations were confirmed in the parents. Among these mutations 3 patients had the common p.e297g (2 homozygous E297G/E297G and one patient with Splice site disruption/e297g). A large scale study of 109 families in patients having PFIC2, Strautnieks et al., [8] identified Eighty-two different mutations (52 novel) (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 p.e297g and/or p.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 p.e297g and p.d482g, with some BSEP detected in 45% of patients (19/42) with these mutations. In addition, Soroka and Boyer, [13] examined differences in protein maturation, plasma membrane localization and transport activity in mutants of BSEP representing two PFIC2 ( p.d482g and p.e297g), and it was found that all but the PFIC2 mutation, p.e297g, retained the ability to transport taurocholate. The reduction in plasma membrane expression correlated equally with reduction in the total amount of BSEP protein, suggesting a defect in protein stability rather than in trafficking. Conclusion: In conclusion, this work detected no mutations or variations involving Exon 9 of ABCB 1 1 gene in studied Egyptian patients with PFIC2 as a cause of severe BSEP deficiency, so further investigation of other exons of the gene are necessary in order to confirm diagnosis of PFIC2 and to prove that inspite of considering Exon 9 mutations are common worldwide, but these mutations are not that common among Egyptian ethnic population, however larger sample size is needed. 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