GASTROENTEROLOGY 1999;117:1370–1379
LIVER, PANCREAS, AND BILIARY TRACT Hepatocanalicular Bile Salt Export Pump Deficiency in Patients With Progressive Familial Intrahepatic Cholestasis PETER L. M. JANSEN,* SANDRA S. STRAUTNIEKS,‡ EMMANUEL JACQUEMIN,§ MICHELLE HADCHOUEL,§ ETIENNE M. SOKAL,\ GUIDO J. E. J. HOOIVELD,* JOHAN H. KONING,* ALIE DE JAGER–KRIKKEN,* FOLKERT KUIPERS,¶ FRANS STELLAARD,¶ CHARLES M. A. BIJLEVELD,¶ ¨ LLER* ANNETTE GOUW,# HARRY VAN GOOR,# RICHARD J. THOMPSON,‡ and MICHAEL MU Departments of *Gastroenterology, ¶Pediatrics, and #Pathology, University Hospital Groningen, Groningen, The Netherlands; ‡Paediatric Liver Service, Department of Child Health, Guy’s, King’s and St. Thomas’ School of Medicine, London, England; §Department of Pediatrics and INSERM Unite ´ 347, Ho ˆ pital de Bice ˆ tre, Paris, France; and \Department of Pediatrics Universite´ Catholique de Louvain, Cliniques St. Luc, Brussels, Belgium
See editorial on page 1496. Background & Aims: Progressive familial intrahepatic cholestasis (PFIC), an inherited liver disease of childhood, is characterized by cholestasis and either normal or increased serum g-glutamyltransferase activity. Patients with normal g-glutamyltransferase activity have mutations of the FIC1 locus on chromosome 18q21 or mutations of the BSEP gene on chromosome 2q24. Also, patients with bile acid synthesis defects have low g-glutamyltransferase activity. We investigated expression of the bile salt export pump (BSEP) in liver samples from patients with a PFIC phenotype and correlated this with BSEP gene mutations. Methods: BSEP and multidrug resistance protein 2 (MRP2) expressions were studied by immunohistochemistry in liver specimens of 28 patients and BSEP gene mutation analysis in 19 patients. Bile salt kinetics were studied in 1 patient. Results: Sixteen of 28 liver samples showed no canalicular BSEP staining. Staining for MRP2 showed a normal canalicular pattern in all but 1 of these samples. Ten of 19 patients showed BSEP gene mutations; BSEP protein expression was lacking in all 10 patients. No mutations were found in 9 of 19 patients, and in all except 1, BSEP protein expression was normal. Bile salt concentration in bile of BSEP-negative/MRP2-positive PFIC patients was 0.2 6 0.2 mmol/L (n 5 9; F1% of normal) and in BSEP-positive PFIC patients 18.1 6 9.9 mmol/L (n 5 3; 40% of normal). The kinetic study confirmed the dramatic decrease of bile salt secretion in BSEP-negative patients. Conclusions: The findings show a close correlation between BSEP gene mutations and canalicular BSEP expression. Biliary secretion of bile salts is greatly reduced in BSEP-negative patients.
P
rogressive familial intrahepatic cholestasis (PFIC) presents in the first year of life with cholestasis and
jaundice. In patients belonging to the Byler kindred (descendants of the 18th century Amish settler Jacob Byler), the disease is called Byler disease,1 or more recently PFIC type 1. Positional cloning studies revealed that in these patients the defect is linked to a mutation in a gene at chromosome 18q21, FIC1.2 The gene defect in benign recurrent intrahepatic cholestasis has also been mapped to the FIC1 locus.3,4 FIC1 encodes a P-type adenosine triphosphatase.5 How mutations in this protein cause cholestasis is unclear. Byler syndrome has been defined as a disease with clinical manifestations similar to those of Byler disease but occurring in patients not belonging to the Byler family.6–10 The FIC1 gene is implicated in some patients with the Byler syndrome.6 In other Byler syndrome patients, the defect does not map to chromosome 18q21.6,7,10 A locus on chromosome 2q24 has been identified in some of these families.11,12 Despite the cholestasis in both subgroups of patients, g-glutamyltransferase (GGT) concentrations in serum are disproportionally low. Histologically, both PFIC subtypes are characterized by cholestasis and only slight bile duct proliferation. This contrasts with a PFIC subtype with elevated serum GGT activity, wherein marked bile duct proliferation dominates the histology. This PFIC subtype is caused by mutations in the PGY3 gene, encoding the canalicular phosphatidylcholine-translocating protein MDR3.
Abbreviations used in this paper: bsep and BSEP, bile salt export pump; FTR, fractional turnover rate; GAPDH, glyceraldehyde-3phosphate dehydrogenase; GGT, g-glutamyltransferase; MRP2, multidrug resistance protein 2; PFIC, progressive familial intrahepatic cholestasis; SSCP, single-strand conformation polymorphism. r 1999 by the American Gastroenterological Association 0016-5085/99/$10.00
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In vitro studies have shown that canalicular bile salt transport is mediated by an adenosine triphosphate– dependent transporter of the P-glycoprotein family, initially called ‘‘sister of P-glycoprotein’’ but renamed ‘‘bile salt export pump, BSEP.’’13–15 We have generated polyclonal antibodies against a synthetic peptide from the carboxy terminus of pig bsep (sequence data from Childs et al.14). This bsep C-terminus is highly conserved across species but diverges from other members of the Pglycoprotein family. With this antibody, and with monoclonal antibodies against MRP2 (multidrug resistance protein 2, the canalicular multispecific organic anion transporter) and C219 (an antibody against a common epitope in the P-glycoproteins MDR1, MDR3, and BSEP), we have studied the expression of these proteins in frozen sections of livers from patients with PFIC. Mutations were identified by sequence analysis of genomic DNA or complementary DNA (cDNA) polymerase chain reaction (PCR) products. In 1 patient, kinetic studies were performed using the stable isotopes [2,2,4,42H]cholate and [2,2,4,4-2H]chenodeoxycholate.
Materials and Methods Patients Patients from the Groningen center. Patient A is a female born to nonconsanguineous parents who presented with neonatal cholestasis. In the first year of life, the serum alkaline phosphatase (ALP) activity was 393 U/L (normal [N] , 220 U/L), GGT 27 U/L (N , 45 U/L), total bilirubin 83 µmol/L (N , 17 µmol/L), conjugated bilirubin 54 µmol/L (N , 5 µmol/L), and serum bile salts 530 µmol/L (N , 9 µmol/L). The patient has 1 affected and 1 nonaffected sibling. At the age of 5 years, her serum bilirubin value was 48 µmol/L, conjugated bilirubin 38 µmol/L, and serum bile salts 263 µmol/L; ALP activity was 329 U/L and GGT 14 U/L. At the age of 10 she was treated for a short period with ursodeoxycholate (1500 µmol/day). At this age a bile salt kinetic study was performed after informed consent was obtained. The patient suddenly died at the age of 14 years. Autopsy was not performed. Patient B is a male born to nonconsanguineous parents. He presented with neonatal cholestasis. A 2 years older sister with a PFIC-like disease underwent transplantation at the age of 4 years. The patient’s clinical course was characterized by persistent neonatal cholestasis. In the first year, serum ALP level was 623 U/L, GGT 7 U/L, total bilirubin 27 µmol/L, conjugated bilirubin 19 µmol/L, and bile salts 224 µmol/L. Stools contained only trace amounts of bile salts. Before transplantation, at the age of 3 years, the patient’s serum ALP activity was 473 U/L, GGT 10 U/L, total bilirubin 429 µmol/L, and conjugated bilirubin 327 µmol/L. Patient C is a male born to nonconsanguineous parents who presented with neonatal hepatitis. A 5 years younger sister had a similar type of cholestasis. Both received a liver transplant at the age of 4 years. Before transplantation, the patient’s serum
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ALP activity was 1025 U/L, GGT 24 U/L, total bilirubin 168 µmol/L, and conjugated bilirubin 117 µmol/L. Serum bile salts varied from 60 to 300 µmol/L. Patient D is a female born to nonconsanguineous parents who initially was thought to have benign recurrent intrahepatic cholestasis. During cholestatic periods, serum ALP levels increased to 477–994 U/L; total serum bilirubin levels varied from slightly elevated (between the cholestatic periods) to 590 µmol/L, conjugated bilirubin levels increased to 403 µmol/L, and serum bile salt levels to 228–418 µmol/L. Serum GGT activity was maximal at 19 U/L. At the age of 10 years she developed persistent cholestasis. The patient received a liver transplant at the age of 17 years. Patient E is a female with nonconsanguineous parents. She had pruritus without jaundice since the age of 9 months. At the age of 3 years, the laboratory values were as follows: ALP, 328 U/L; GGT, 170 U/L; total bilirubin, 22 µmol/L; and serum bile salts, 50 µmol/L. The cholestasis was slowly progressive with, at the age of 5 years, a serum bilirubin of 285 µmol/L, conjugated bilirubin of 228 µmol/L, and bile acids of 370 µmol/L. The patient received a liver transplant at the age of 5 years. A specimen of normal liver came from a reduced liver graft. This served as a control for immunostaining with BSEP, MRP2, and C219 antibodies. Patients from the Paris and Brussels centers. Fourteen patients from the Paris center and 9 patients from the Brussels center with a PFIC phenotype were studied. Liver samples from these 23 patients were scored blindly for BSEP expression in Groningen. BSEP gene mutations were assessed blindly in 19 patients in London. BSEP expression and BSEP gene mutations were correlated after both analyses had been completed. From 4 patients, no samples were available for mutation analysis.
Antibodies A polyclonal antibody against bsep (k12) was raised by immunizing rabbits with a peptide coupled to keyhole limpet hemocyanin. The sequence of the peptide was KGAYYKLVTTGAPIS, corresponding to the carboxy terminal of the pig bsep sequence. The antibody was used as crude antiserum and has
Figure 1. Western blot analysis of BSEP in canalicular subfractions from humans, mice, and rats using rabbit immunoglobulin G anti-BSEP antibodies. Samples from liver subfractions enriched in canalicular membranes were subjected to Western blot analysis.
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Figure 2. Localization of BSEP and MRP2 in normal liver. Staining with BSEP antibody (Control-1); staining with MRP2 antibody (Control-2).
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b Figure 3. Localization of BSEP and MRP2 in liver sections of PFIC patients from the Groningen center. Staining for BSEP is shown in panels A-1 to E-1, staining for MRP2 in panels A-2 to E-2. The letters denote the various patients (Table 1).
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been described previously.16 A mouse monoclonal antibody C219 (Signet Laboratories Inc., Dedham, MA) was used to detect all P-glycoproteins. Monoclonal antibodies against MRP2 (M2III-5) were obtained from Dr. R. Scheper (Department of Pathology, Free University, Amsterdam, The Netherlands).
Immunohistochemistry The expressions of BSEP, MRP2, and P-glycoproteins were studied in cryostat sections using the antibodies described above. The sections were air-dried, fixed in acetone for 10 minutes at room temperature, and incubated with the primary antibody (1:50 in phosphate-buffered saline [PBS] containing 1% bovine serum albumin) for 1 hour at room temperature. Endogenous peroxidase activity was blocked by incubation with 0.075% H2O2 in PBS for 30 minutes. Secondary and tertiary antibodies were horseradish peroxidase–conjugated goat anti-rabbit (1:100) and rabbit anti-goat (1:100) for antibody k12, horseradish peroxidase–conjugated rabbit antimouse (1:50) and goat anti-rabbit (1:50) for antibody C219, and horseradish peroxidase–conjugated rabbit anti-mouse (1: 50), goat anti-rabbit (1:50), and rabbit anti-goat (1:50) for antibody against MRP2 (M2III-5). All secondary and tertiary antibodies were diluted in PBS containing 1% bovine serum albumin and 1% normal human serum. After each incubation, the sections were rinsed with PBS. The sections were finally incubated with filtered 3-amino-9-ethylcarbazole (10 mg/2.5 mL dimethylformamide in 50 mL of 0.1 mol/L acetate buffer, pH 5.0) containing 0.03% H2O2 for 10 minutes at room temperature. Counterstaining was performed with hematoxylin, and the slides were covered with Kaiser’s glycerin-gelatin.
Reverse-Transcription PCR Total RNA was isolated from frozen human liver sections. Single-stranded cDNA was synthesized from 2.5 µg messenger RNA (mRNA) using 0.5 nmol random primers (Pharmacia, Uppsala, Sweden) and 50 U AMV ReverseTranscriptase (Promega, Madison, WI). With the cDNA obtained, a PCR reaction was performed using sense and antisense primers. The number of cycles were 24 for BSEP and 22 for MRP2 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). For every PCR reaction, GAPDH was used as internal control. Primer sequences are available from the authors (M.M.).
BSEP Gene Mutation Detection BSEP gene mutation analysis was performed for the patients from Paris and Brussels centers. If fresh frozen liver was available, reverse-transcription PCR was performed; otherwise, PCR products from genomic template were used. The patients’ PCR products were compared with controls using single-strand conformation polymorphism (SSCP) analysis (Genephor, Pharmacia). PCR products showing band shifts on SSCP were sequenced using an ABI 373A sequencer (Applied Biosystems, Foster City, CA). The primer sequences used are available from the authors (R.J.T. and S.S.S.). A total of 100
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control chromosomes were screened for missense mutations by restriction enzyme analysis or by sequencing.
Bile Salt Analysis Total bile salt concentrations in serum, urine, and bile were measured as described previously.17,18 Individual bile salt species were quantified after enzymic deconjugation by capillary gas chromatography on a Hewlett Packard HP8559 gas chromatograph equipped with a CP Sil 19 CB column (Chrompack, Middelburg, The Netherlands) as methyl ester trimethylsilyl ether derivatives. Biliary phospholipid and cholesterol levels were measured after lipid extraction.
Bile Salt Kinetics Fractional turnover rates (FTRs) and pool sizes of primary bile salts were determined after simultaneous oral administration of 10 mg each of [2,2,4,4-2H]cholate and [2,2,4,4-2H]chenodeoxycholate (Merck Sharpe and Dohme, Montreal, Canada; .98 atom% 2H) in 0.25% sodium bicarbonate solution as described by Stellaard et al.19,20 Six blood samples were collected during a 3-day period after administration of the 2H-labeled bile salts. Natural abundances were measured in blood samples obtained before administration of the labels. Serum samples were prepared as described previously.21 After separation of the trimethyl silyl ether derivatives by gas-liquid chromatography, the samples were introduced into the ion source of a Finnigan Mass Spectrometer (Finnigan MAT, Bremen, Germany) in the selected ion monitoring mode. The 2H/1H ratio was calculated and converted into atom percent excess values. Pool sizes and FTRs were calculated from the ln atom % excess vs. time curves. The apparent synthesis rate (in µmol · kg21 · day21) was calculated by multiplying pool size (µmol/kg) and FTR (1/day).
Results Characterization of BSEP Antibodies The rabbit polyclonal BSEP antibody detects a 170-kilodalton protein band in Western blots from canalicular enriched subfractions prepared from human, mouse, and rat liver (Figure 1). The molecular mass of BSEP has been reported to be 170 kilodaltons.14,15 In control liver, this antibody recognizes a canalicular protein (Figure 2). BSEP Expression by Immunohistochemistry The expression of BSEP and MRP2 in liver sections from patients A–E is shown in Figure 3. Using BSEP antibodies, no protein was detected in liver sections from patients A–C (A-1, B-1, C-1), whereas sections from patients D and E show a normal canalicular staining pattern. Staining for MRP2 and with C219 antibodies (data not shown) revealed a normal canalicular pattern in all samples. Table 1 shows the biochemical, histochemical, and clinical features of patients A–E, all from the Groningen center.
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Table 1. Patients With PFIC Phenotype From Groningen Serum chemistry Immunohistochemistry GGT (U/L)
ALP (U/L)
Bilirubin (mmol/L)
Genotype
Phenotype a
1
27
393
83
ND
7
623
27
ND
1
24
1025
117
ND
1
1
19
994
590
ND
1
1
321
288
27
ND
Congenital cholestasis, short stature; died suddenly at age 14 yr; affected sister; Cons2 Congenital cholestasis; Tx at 3 yr; affected sister; Cons2 Initial diagnosis neonatal hepatitis; later persistent cholestasis; Tx at 4 yr; affected sister; Cons2 Recurrent intrahepatic cholestasis at age 1 yr; later persistent jaundice; Tx at 17 yr; no family history; Cons2 Congenital cholestasis, jaundice, pruritus; Tx at 5 yr; no family history; Cons2
1
1
2
1
F
1
F
1
Patient
Sex
BSEP
MRP2
C219
A
F
2
1
B
M
2
C
M
D
E
NOTE. Normal values: GGT, ,45 U/L; ALP, ,120 U/L; and bilirubin, ,17 µmol/L. ND, not done. aOnset, signs, transplantation (Tx), consanguinity (Cons).
Bile Salt Kinetics in Patient A Duodenal bile from patient A was obtained before and during administration of ursodeoxycholic acid (UDCA, 1500 µmol/day). Table 2 shows that the bile salt concentration in bile is very low. Cholate is the predominant species, and secondary bile salts could not be detected. Phospholipid and cholesterol concentrations in bile were severely reduced. During UDCA treatment, UDCA appeared in bile in very low concentrations. Serum bile salt concentrations were elevated and ranged from 300 to 500 µmol/L (N , 50 µmol/L), with ,60% as cholate and ,40% as chenodeoxycholate. During UDCA treatment, serum bile salt concentrations increased to levels approaching 800 µmol/L with UDCA as the dominant species (,60%) (Table 3). Urinary bile salt output, already clearly elevated under control conditions, dramatically increased during UDCA administration. This increase was almost entirely caused by increased excretion of UDCA. In fact, the daily urinary excretion rate of UDCA (1167 6 68 µmol/day) almost equaled the oral dose (1500 µmol/day). Table 2. Composition of Duodenal Bile From Patient A
Total bile salts (mmol/L ) Cholate (% ) Chenodeoxycholate (% ) Deoxycholate (% ) Lithocholate (% ) UCDA (% ) Phospholipids (mmol/L) Cholesterol (mmol/L) ND, not detected.
No treatment
UDCA therapy
Reference values
0.36 85.3 12.9 ND 0.2 1.6 ,0.2 0.3
0.12 35.4 15.6 ND 0.2 48.9 0.04 1.46
44 6 19 44 6 12 29 6 6 25 6 11 161 Trace 8.2 6 3.9 4.4 6 2.2
Cholate and chenodeoxycholate pool sizes determined in patient A were of the same order of magnitude as pool sizes in healthy adults, but in patient A the entire bile salt pool was in the ‘‘central’’ compartment, consisting of blood, liver, and extravascular space (Table 3). The FTR for cholate was reduced, whereas the FTR for chenodeoxycholate was in the low range of normal. Likewise, synthesis rates for cholate and chenodeoxycholate were reduced and low normal, respectively. UDCA administration did not affect these parameters. Phenotype-Genotype Correlations The correlation between histochemistry and clinical manifestations was studied in detail in specimens from 23 patients from the Paris and Brussels centers. In 19 patients, BSEP gene mutations were assessed. The data are shown in Table 4. The data in Tables 1 and 4 show that 16 of 28 patients are BSEP negative and 12 are BSEP positive. In all but 1 patient (no. 1, Table 4), staining for MRP2 and with C219 antibodies showed a normal canalicular pattern. In Table 3. Bile Salt Metabolism in Patient A No treatment
UDCA therapy
Reference values Cholate
Pool size FTR Synthesis rate
27.1 0.07 1.9
10.3 0.13 1.5
11–52 mol/kg 0.17–0.82 L/day 6.8–22.0 mol · kg21 · day21
Chenodeoxycholate Pool size FTR Synthesis rate
30.3 0.18 5.5
53.6 0.22 11.8
16–40 mol/kg 0.10–0.45 L/day 3.6–13.0 mol · kg21 · day21
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Table 4. Genotype-Phenotype Analysis in Patients With PFIC From the Paris and Brussels Centers Serum chemistry
Bile
Immunohistochemistry Sex
BSEP
MRP2
C219
GGT (U/L)
ALP (U/L)
Bilirubin (mmol/L)
Bile salts (mmol/L)
1
F
2
6
6
54
403
260
3.6
No; BSEP mRNA6
2
M
2
1
1
22
342
59
0.02
Homozygous nonsense mutation R1090X
3
F
2
1
1
17
234
36
0.1
Heterozygous missense mutation in transmembrane domain G982R
4
F
2
1
1
41
338
100
0.06
Homozygous missense mutation in first nucleotide-binding fold G238V
5
M
1
1
1
47
496
285
ND
No
6
M
2
1
1
20
261
207
ND
ND
7
F
1
1
1
45
501
55
ND
No; BSEP mRNA1
8
M
1
1
1
34
558
490
ND
ND
9
F
2
1
1
N
271
139
ND
Heterozygous for a 1-bp deletion at position 695 in cDNA
10
M
1
1
1
26
678
112
ND
No; BSEP mRNA1
11
M
1
1
1
35
469
195
ND
No; BSEP mRNA1
12
M
2
1
1
22
498
334
ND
ND
13
F
2
1
1
24
523
147
0.5
14
M
1
1
1
20
796
70
ND
Patient no.
Genotype a
Heterozygous missense mutation S114R
No
Phenotype b Persistent jaundice since age 1 mo; hepatomegaly, spherocytosis, pruritus; Tx at 7 mo; Cons? Persistent jaundice since age 1–2 mo; hepatomegaly, pruritus; Tx at 4.4 yr; Cons1 Recurrent then persistent jaundice since age 1 mo; hepatomegaly, pruritus; Tx at 9.5 yr; Cons2 Persistent jaundice since age 1 mo; hepatomegaly, pruritus; Tx at 2.8 yr; Cons1 Persistent jaundice, hepatomegaly, diarrhea since age 5 mo; pruritus; Tx at 5.6 yr; Cons1 Persistent jaundice since age 1 mo; hepatomegaly, pruritus; Tx at 2.8 yr; Cons1 Persistent jaundice, hepatomegaly since age 5 mo; pruritus, deafness; Tx at 4.5 yr; Cons1 Persistent jaundice, hepatomegaly since age 2 mo; deafness, diarrhea, pruritus; Tx at 4 yr; Cons2 Recurrent then persistent jaundice, pruritus, hepatomegaly since age 5 yr; Tx at 13.5 yr; Cons2 Persistent jaundice, hepatomegaly since age 1 mo; pruritus, myelofibrosis; Tx at 2.6 yr; Cons2 Persistent jaundice since age 1 mo; hepatomegaly, pruritus; Tx at 7.6 yr; Cons2 Persistent jaundice since age 1 mo; hepatomegaly, pruritus; Tx at 1 yr; Cons2 Recurrent then persistent jaundice and pruritus since age 4 mo; hepatomegaly; Tx at 8.6 yr; Cons? Persistent jaundice, hepatomegaly since age 2 mo; pruritis; Tx at 3.2 yr; Cons2
(Continued on following page)
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Table 4 (Cont’d). Genotype-Phenotype Analysis in Patients With PFIC From the Paris and Brussels Centers Serum chemistry
Bile
Immunohistochemistry GGT (U/L)
ALP (U/L)
1
26
319
29
1
1
12
314
73
0.06
Heterozygous missense mutation in transmembrane domain C336S
2
1
1
17
770
32
ND
F
1
1
1
11
1314
146
31.3
Heterozygous missense mutation in transmembrane domain C336S ND
19
F
1
1
1
132
2390
108
7.4
No
20
M
2
1
1
14
363
92
0.6
Heterozygous nonsense mutation R575X
21
F
2
1
1
21
354
65
0.08
Compound heterozygote for R1057X and E297G
22
F
2
1
1
18
198
148
0.05
Compound heterozygote for S593R and a 1-bp deletion at position 3213.
23
M
1
1
1
85
370
85
ND
No
Patient no.
Sex
BSEP
MRP2
C219
15
M
1
6
16
F
2
17
M
18
Bilirubin (mmol/L)
Bile salts (mmol/L) 15.6
Genotype a No
Phenotype b Cholestasis since birth; jaundice disappeared during UDCA treatment Affected sibling; cholestasis since birth; cirrhosis, intractable pruritus; biliary BS (during UDCA therapy): cholate 0.02, chenodeoxycholate 0, UDCA 0.04 mmol/L; Tx at 8.6 yr Brother of TA; pruritus since 6 mo; no Tx yet (8 yr) Persistent cholestasis since birth; pruritus; no Tx yet One affected sibling; progressive cholestasis since birth; pruritus; BS in duodenal bile (during UDCA acid therapy): CA 0.2, CDCA 3.4, UDCA 3.8 mmol/L; Tx at 3.6 yr Jaundice since birth; biliary derivation at 3 yr; UDCA therapy resulted in a serum concentration of 14,000 mol/L UDCA and in bile: CA 0.2, CDCA 0.44, UDCA 0.025 mmol/L; no Tx yet Persistent jaundice and pruritus since birth; failure to thrive; BS in bile during UDCA therapy: CA 0, CDCA 0, UDCA 0.08 mmol/L; Tx waiting list Jaundice and pruritus at 1 yr; BS in bile during UDCA therapy: CA 0.02, CDCA 0, UDCA 0.03 mmol/L; no Tx yet Neonatal cholestasis, pruritus, normal cholangiogram ductular proliferation on biopsy; Tx at 18 mo
NOTE. Normal values: GGT, ,45 U/L; ALP, ,360 U/L; bilirubin, ,17 µmol/L; and bile salt concentration in bile (BS), .20 mmol/L. BSEP mRNA and MRP2 mRNA was tested using reverse-transcriptase polymerase chain reaction. Data are from samples with a normal GAPDH signal. Patients 1–14 are from the center in Paris, 15–23 from the Brussels center. ND, not done; Tx, liver transplantation; No, no mutation found; 1, normal; 6, weak or dubious signal; CA, cholic acid; CDCA, chenodeoxycholic acid; bp, base pair. aBSEP gene mutations and BSEP mRNA. bOnset, clinical signs, transplantation (Tx), consanguinity (Cons) (1, positive; 2, negative).
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patient 1, BSEP staining was negative and staining for MRP2 and with C219 was also very weak, indicating loss of canaliculi. This precludes the reliable assessment of BSEP expression. Biliary bile salt concentrations were measured in 13 patients and were 0.2 6 0.2 mmol/L (n 5 9; ,1% of normal) in bile of BSEP-negative/MRP2-positive patients and 18.1 6 9.9 mmol/L (n 5 3; 40% of normal) in BSEP-positive patients. In patient 1, biliary bile salt concentration was 3.6 mmol/L. For all patients this is below normal (normal controls, 44 6 19 mmol/L), but for the BSEP-negative/MRP2-positive patients the values are one order of magnitude lower than the biliary bile salt concentrations of the BSEP-positive patients. BSEP gene mutations were studied in 19 patients. The absence of BSEP staining correlates with BSEP gene mutations in all but 1 (patient 1) of 11 tested BSEPnegative patients. When we only consider the 10 BSEPnegative/MRP2-positive patients, the correlation between BSEP gene mutations and the absence of canalicular BSEP expression is 100%. Also, no BSEP gene mutations were found in any of the 8 patients with positive canalicular BSEP staining. In 4 patients, liver RNA samples were available for BSEP and MRP2 mRNA analysis. Three BSEP-positive patients showed a normal BSEP mRNA signal on reverse-transcription PCR. In patient 1, BSEP mRNA was weak (compared with the housekeeping GAPDH signal) but of normal length.
Discussion PFIC is a heterogeneous group of diseases with various clinical manifestations and causes, including defects of hepatocanalicular transport of bile salts or phospholipids and defects of bile acid synthesis. Bile acid synthesis defects seem to be rare. The frequency of transport defects is not known, but this study shows that the combined cohorts of 3 liver transplantation centers yielded 28 patients with a PFIC phenotype with predominantly low serum GGT activity. Liver samples from 16 of 28 patients showed no BSEP staining, whereas staining for MRP2 and with C219 showed a normal characteristic canalicular pattern with the exception of 1 patient. Twelve patients had a normal canalicular BSEP staining pattern. Therefore, in 15 of 16 patients, negative BSEP staining is not caused by the absence of canaliculi but by the absence of BSEP in the canaliculi. In liver samples in which canaliculi are severely damaged or have disappeared, BSEP cannot reliably be assessed. Therefore, control staining with canalicular marker proteins other than BSEP is mandatory. The clinical implications of having no canalicular BSEP expression are clear: when BSEP is absent, there can
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be no normal bile salt secretion. Indeed, the bile salt concentration in bile of BSEP-negative/MRP2-positive patients was less than 1% of normal. Seven of these patients were treated with UDCA. Bile analyzed during treatment showed a very low UDCA concentration. This suggests that, in addition to the primary bile salts, cholic acid and chenodeoxycholic acid, secretion of UDCA also seems to be mediated by BSEP. This is particularly evident from the bile salt kinetic study in patient A. In this patient, UDCA could quantitatively be recovered from the urine and only traces of the administered dose appeared in bile. Bile salt kinetics in this patient showed that the FTRs and calculated synthesis rates of cholate and chenodeoxycholate were reduced and normal, respectively. This indicates that cholate synthesis is depressed but the synthesis of chenodeoxycholate is less affected. This inhibition of cholate synthesis must be caused by the hepatic accumulation of bile salts, whereas chenodeoxycholate synthesis apparently escapes this inhibition to a certain degree. This is further evidence for an earlier finding that, under cholestatic conditions, the classical or acidic pathway of bile salt synthesis is suppressed but the alternative or neutral pathway, leading to chenodeoxycholate, is less affected (Koopen et al., unpublished results). The increased proportion of chenodeoxycholate in the serum of patients with PFIC probably results from a strongly depressed synthesis of cholate and a less affected chenodeoxycholate synthesis. This is not restricted to PFIC but may occur in any cholestatic condition. Both cholesterol and phospholipid secretion were severely reduced in patient A. Phospholipid secretion is mediated by MDR3, a phospholipid translocator in canalicular membranes.22 This transporter acts as a flippase, moving phospholipids from the cytosolic surface of the canalicular membrane to the surface facing the bile canalicular lumen. Bile salts are required to extract phosphatidylcholine from the membrane and to dissolve it in bile. In the absence of bile salt secretion, as is the case in the BSEP-negative patients, phospholipids cannot be secreted. Cholesterol secretion also depends on bile salt secretion. The salient finding of the current study is the close correlation between the absence of canalicular BSEP protein and mutations in the BSEP gene. No mutation was found in BSEP-positive patients, whereas BSEPnegative/MRP2-positive patients all have a BSEP gene mutation. Importantly, patients with missense mutations on 1 or 2 alleles do not show any canalicular BSEP expression. The lack of canalicular BSEP staining in these patients may be caused by either a transcriptional/ translational defect or by abnormal protein conformation, with consequent inability of BSEP to reach the plasma membrane.
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In several BSEP-negative patients, a mutation has only been found on 1 allele. However, screening was performed by SSCP and the entire coding region has not been sequenced. In all cases, ‘‘carrier’’ parents are phenotypically normal. It is therefore assumed that the second disease-causing mutation remains to be defined. In conclusion, this study shows that in a subgroup of patients with PFIC and low serum GGT activity, the BSEP protein in the canalicular domain of hepatocytes is lacking. This transporter deficiency strongly correlates with mutations in the BSEP gene. BSEP gene mutations have been described in a similar subgroup of PFIC patients.11,12 However, BSEP expression was not studied in these patients. The disease characterized by BSEP gene mutations and lack of canalicular BSEP expression is best named ‘‘PFIC with BSEP deficiency.’’ An alternative name is PFIC type 2, but this might be confusing because this name has been used before for patients with PGY3 gene mutations. Immunohistochemical staining for BSEP is a simple test to diagnose PFIC with BSEP deficiency.
BSEP EXPRESSION IN PFIC PATIENTS
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Received August 18, 1998. Accepted August 17, 1999. Address requests for reprints to: Peter L. M. Jansen, M.D., Division of Hepatology and Gastroenterology, University Hospital Groningen, Hanzeplein 1, 9713 GZ Groningen, The Netherlands. e-mail:
[email protected]; fax: (31) 50-3619306. Supported by the Netherlands Organization for Scientific Research, NWO Program 902-23-191. E.J. and M.H. were supported by the Association Franc ¸aise contre les Myopathies, and the Assistance Publique-Ho ˆpitaux de Paris (CRC no. 97001), Paris, France. R.J.T. is a Wellcome Advanced Fellow; S.S.S. is supported by grants from the Medical Research Council and the Children’s Liver Disease Foundation. The authors thank Micheline Dumont (INSERM, Ho ˆpital Beaujon, Paris, France) for the bile salt analysis of the patients at Ho ˆpital de Bice ˆtre, Paris, France; Gerard Dijkstra for his help with the photography; and Renze Boverhof (University Hospital Groningen) for the bile salt analysis of the patients in Groningen.
