Two Case Reports of Successful

Treatment of Cholestasis With Steroids in

Patients With PFIC-2
Guido Engelmann, MDa, Daniel Wenning, MDb, Diran Herebian, PhDc, Oliver Sander, MDd, Carola Drögee,
Stefanie Kluge, PhDe, Ralf Kubitz, MDe,f

Mutations in the gene encoding the canalicular bile salt export pump (BSEP) can abstract
result in progressive familial intrahepatic cholestasis type 2 (PFIC-2). Treatment
options are limited, and PFIC-2 often necessitates liver transplantation. We report
on a young woman and a boy who clinically presented with PFIC-2 phenotypes
and dramatically improved with steroid treatment. Gene sequencing of ABCB11
encoding for BSEP revealed 2 relevant mutations in both patients. The young
woman was compound heterozygous for p.T919del and p.R1235X. At the age of 5
a
years, partial biliary diversion was performed and rescued liver function but left b
Department of Pediatrics, Lukashospital, Neuss, Germany;
Department of General Pediatrics, University Hospital
serum bile salt levels elevated. At age 23 she developed systemic lupus Heidelberg, Ruprecht-Karls-University, Heidelberg, Germany;
c
erythematosus. Unexpectedly, steroid therapy normalized serum bile salt levels, Departments of General Pediatrics, Neonatology and
d
Pediatric Cardiology, Rheumatology, and
with a strong correlation with the steroid dose. She is currently in clinical e
Gastroenterology, Hepatology and Infectious Diseases,
remission. The boy was compound heterozygous for the ABCB11 mutations University Hospital Düsseldorf, Heinrich-Heine-University,
f
Düsseldorf, Germany; and Medical Clinic I, Bethanien
c.150+3A.C and p.R832C and presented with intractable pruritus. When he Hospital, Moers, Germany
developed colitis, he was treated with steroids. The pruritus completely
Drs Engelmann and Wenning treated one of the
disappeared and relapsed when steroids were withdrawn. To date, with low-dose
patients, conceptualized and designed the
budesonide, the boy has been symptom-free for .3 years. In conclusion, the study, and drafted the initial manuscript; Dr
clinical courses suggest that patients with BSEP deficiency and residual BSEP Herebian carried out biochemical analysis; Dr
Sander treated one of the patients, coordinated
activity may benefit from steroid-based therapy, which represents a new treatment
clinical data collection, and critically reviewed
option. the manuscript; Ms Dröge and Drs Kluge and
Kubitz carried out the biochemical and genetic
analyses, collected clinical data, and reviewed
and revised the manuscript; and all authors
approved the final manuscript as submitted.

Familial intrahepatic cholestasis cirrhosis, often necessitating liver www.pediatrics.org/cgi/doi/10.1542/peds.2014-2376

4
syndromes comprise a group of liver transplantation in early childhood. In DOI: 10.1542/peds.2014-2376
diseases that are caused by the failure of large studies, PFIC represented Accepted for publication Feb 4, 2015
hepatobiliary transporter proteins. 10% to 15% of causes of childhood
1– Address correspondence to Guido Engelmann,
Certain mutations in the ABCB11 gene cholestasis and is the reason for up to MD, Lukashospital, Department of Pediatrics,
3 15% of liver transplantations in children. Preussenstrasse 84, D-41464 Neuss,
lead to deficient expression of the Treatment focuses on symptom relief and Germany. E-mail: [email protected]
canalicular bile salt export pump normalization of liver function tests. It is
(BSEP), causing hepatocellular bile salt PEDIATRICS (ISSN Numbers: Print, 0031-
based on ursodeoxycholic acid (UDCA), 4005; Online, 1098-4275).
(BS) accumulation and subsequent liver with improvement of symptoms in 75%
cirrhosis. The residual transport activity 5,6 Copyright © 2015 by the American
of cases, rifampicin, and partial biliary Academy of Pediatrics
of BSEP inversely correlates with the
diversion. We here report on 2 patients FINANCIAL DISCLOSURE: The authors have
severity of the patient’s phenotype.
with PFIC-2 phenotypes and sustained indicated they have no financial relationships
Complete absence of BSEP activity is
clinical relief of symptoms and relevant to this article to disclose.
associated with progressive familial
unexpected improvement in FUNDING: No external funding.
intrahepatic cholestasis type 2 (PFIC-2).
hypercholemia in response to oral
Patients with PFIC-2 suffer from POTENTIAL CONFLICT OF INTEREST: The
steroids.
continuously evolving pruritus, wasting authors have indicated they have no
jaundice, and sequelae of liver potential conflicts of interest to disclose.

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PEDIATRICS Volume 135, number 5, May 2015 CASE REPORT
PATIENT PRESENTATION loss of a single threonine at amino acid At the age of 23 years the patient’s
position 919 (p.T919del, inherited by general condition deteriorated, and she
Patient 1 the mother) and the non-sense mutation presented with polyarthritis,
A girl (born 1989) presented with vitamin c.3703C.T/p. R1235X, resulting in a polyserositis, splenomegaly, anemia,
K deficiency (8 weeks of age), pruritus (8 premature stop-codon at amino acid leukocytopenia, and pruritus.
months), followed by jaundice (10 position 1235 (inherited by the father) Antinuclear antibody titer was .1:
months) with a BS concentration of 583 (Fig 1). The initial liver biopsy revealed 5120, and anti-double-strandedDNA
mmol/L (normal: ,8 mmol/L). an apparently normal expression of antibodies were 6738 U/mL (ELISA).
Aminotransferases and gamma-glutamyl BSEP (Fig 1). The anti-BSEP antibody, Systemic lupus erythematosus was
transferase were normal. A liver biopsy which was used for diagnosed.
revealed severe intrahepatic cholestasis immunofluorescence, was directed
13 Treatment was started with
and PFIC-2 was suspected. UDCA against the C-terminal amino acids of prednisolone at a dose of 30 mg/day
treatment was started, and a partial 7
BSEP. BSEP immunoreactivity in the followed by complete symptom relief.
external biliary diversion procedure was
patient’s liver biopsy sample must Serositis recovered, and the arthritis
performed at the age of 5 years, which led
therefore have been due to expression improved thereafter. Steroids were
to good control of pruritus for 15 years.
of the allele carrying the T919del tapered to 7.5 mg/day, with a flare up of
variant, because in the presence of arthritis and increasing anti-double-
Sequencing of ABCB11 (BSEP) p.R1235X (premature stop codon) the strandedDNA antibodies to 4549
revealed compound heterozygosity for 86 C-terminal amino acids are lacking. U/mL. Prednisolone was increased to
the deletion of a coding triplet 15 mg/day, once again resulting
(c.2756_2758delCCA), resulting in the

FIGURE 1
Immunofluorescence of liver tissue and DNA sequencing in patient 1. Liver tissue of patient 1 (taken at the age of 10 months) reveals a
clear canalicular staining pattern for BSEP and MRP2 (scale bar = 10 mm), as visualized by immunofluorescent staining and confocal laser
scanning microscopy. The young woman is compound heterozygous for a triplet deletion in exon 21 c.2756_2758delCCA (p.T919del) and a
non-sense mutation c.3703C.T (p.R1235X) in coding exon 26 of ABCB11. Sequences are shown on the nucleotide and protein level.

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2 ENGELMANN et al
 At the age of 4.5 years pruritus and
jaundice worsened, with a direct
bilirubin of 230 mmol/L. In addition,
diarrhea and painful bowel movements
developed. Endoscopic retrograde
cholangiography revealed no evidence
of primary sclerosing cholangitis but
showed a secretion of pale-appearing
bile. On colonoscopy,
a left-sided colitis with superficial
granulocyte infiltration of the mucosa
in the proximal parts of the colon was
diagnosed.
Genetic analysis revealed mutations in
the ABCB11 gene. In the 20th coding
exon a heterozygotic missense mutation
c.2494C.T (p.R832C, inherited by the
mother) was detected together with the
heterozygotic splice site mutation
c.150+3A.C distally to exon 3
(inherited by the father). No mutations
of the ATP8B1 gene or
of the JAG1 gene (Alagille syndrome)
were detected. Immunofluorescence
revealed normal distribution of BSEP
and of the bilirubin transporter
multidrug resistance-associated protein
2 (MRP 2) used as a canalicular marker
(Fig 3).
FIGURE 2
Serum BS and bilirubin levels in correlation with steroid therapy in patient 1. Upper panel: (1) The course of the disease was
steroid therapy (30 mg/day) led to normalized BS levels and a decline in bilirubin; (2) dose
progressive, with increasing
reduction to 15 mg/day resulted in slightly increased BSs; (3) further reduction to 7.5 mg/day
entailed a distinct increase in BSs; (4) redoubling the dose led again to a decrease in BSs. Lower hepatomegaly and advancing liver
panel: BSs were measured in serum and bile before (2009) and during (2012) steroid therapy. fibrosis (FibroScan [Echosense, Paris,
Although BS concentration in serum decreased by a factor of 4.5, the concentration in bile France] was 10.4 kPa at the age of
(collected from the biliostoma) increased by a factor of 6.2, from 1.5 to 9.6 mmol/L, suggesting a
strong stimulation of bile acid secretion during steroid therapy.
4.75 years and 14.6 kPa at the age of 5
years). The patient was listed for liver
transplantation and a living
in clinical improvement in arthritis despite adequate therapy with related transplantation was
and pruritus. rifampicin, phenobarbital, and considered.
Serum BS levels normalized during the naloxone; interrupted night sleep; and
fatigue. Blood examinations revealed a The administration of prednisolone led
initial higher dose of systemic steroid to relief of the diarrhea and pruritus.
therapy (Fig 2). Interestingly, total BS mild cholestasis with total bilirubin of
BSs and bilirubin normalized
concentration in bile collected from the 23.4 mmol/L and direct bilirubin of 9
completely (Fig 4). Budesonide had the
partial external biliary diversion mmol/L. Serum BS concentration was
same benefit but without side effects.
increased to 9.56 mmol/L (August 148.6 mmol/L, whereas
Liver stiffness improved to almost
2012) compared with 1.53 mmol/L aminotransferases, gamma-glutamyl- 8
transferase, alkaline phosphatase, and normal values (6.3 kPa, age 7 years).
before steroid treatment (May 2009).
cholesterol were within normal ranges. The patient was taken off the transplant
Three years after the introduction of
PFIC-2 was suspected, and a liver list and is currently free of symptoms
steroids (November 2011), she is still (as of 2014).
free of symptoms of cholestasis. biopsy was obtained. Histology
revealed chronic cholestasis,
disorganization of portal structures, and
Patient 2 degenerative bile duct alterations. DISCUSSION
Patient 2 (born 2004) presented at an Treatment with UDCA and naltrexon We here report on complete symptom
age of 2.5 years with severe pruritus improved pruritus. relief and sustained normalization of

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PEDIATRICS Volume 135, number 5, May 2015 3
FIGURE 3
Immunofluorescence of liver tissue and DNA sequencing in patient 2. BSEP and MRP2 show a clear canalicular and a weak basolateral
hepatocyte staining (scale bar = 10 mm) as uncovered by immunofluorescence and confocal laser scanning microscopy. Patient 2 and his
brother (our patient’s younger brother with the same phenotype but much lesser symptoms has not been treated with steroids so far) are
compound heterozygous for the splice site mutation c.150+3A.C in intervening sequence 4 (IVS4) and the missense mutation c.2494C.T
(p.R832C) in ABCB11 coding exon 20. Sequences are shown on the nucleotide and protein level.

liver function tests in 2 unrelated suggests residual BSEP activity, which of prednisolone was associated with
patients with PFIC-2 phenotypes. In may be an important requirement for worsening of cholestasis and intestinal
most PFIC-2 patients, BSEP successful steroid therapy. It is believed disease. Eventually, budesonide at a
expression is absent or severely that the extent of expression inversely minimal effective dosage of 3 mg
9
reduced in liver tissue. However, our correlates with phenotype severity every second day resulted in complete
patients showed clear (reviewed in ref 10), with some clinical and biochemical remission
5 within
immunoreactivity for BSEP in their exceptions.
livers. Detection of significant 6 months for .3 years.
In patient 2, the course of liver and
amounts of BSs (between 1.5 and 9.5 intestinal disease could be positively The mechanism of action of the
mmol/L) in the bile of patient 1 influenced by prednisolone. Tapering steroids in the context of PFIC-2 is

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4 ENGELMANN et al
FIGURE 4
Progression of serum BS concentrations in patient 2. Prednisolone (Pred) administration was started at 2 mg/kg per day. Decreasing dosages of
prednisolone led to an increase in BS concentrations. Repetitive prednisolone treatment improved serum BS concentrations. Treatment with
2
azathioprine (Aza; 2 mg/kg) and methotrexate (MTX; 15 mg/m ) were inefficient. Budesonide (Bud; 6 mg/day) induced a sustained remission.

not yet clear but could possibly involve 357 mL/minute) independent of BS under cholestatic conditions. An
upregulation of BSEP transporter concentration. essential glucocorticoid response
activity, although experimental data are element (GRE) has been identified
The ABCB11 splice site mutation
conflicting. In primary rat hepatocytes, within the NTCP promoter at
c.150+3A.C results in partial exon
BSEP-mRNA as well as Mrp2-mRNA a nucleotide position of 232 to 212
skipping of exon 3 in vivo, and
and protein expression were 23
approximately one-third of mRNA relative to the transcription start site.
upregulated by dexamethasone in a
11–13 transcripts are correctly spliced (C.D., Therefore, glucocorticoids may
concentration-dependent manner. R.K., D.H., unpublished data). improve transepithelial BS transport by
In line with this finding, treatment of Therefore, in patient 2, steroids may upregulation of NTCP, providing an
rats with glucocorticoids including increased gradient for BSEP, thus
upregulate transcription and could
budesonide was associated with the counteracting cholestasis.
potentially improve correct mRNA
upregulation of BSEP, Mrp2, and
14 splicing or recover transport activity of Budesonide is a steroid with rapid
cytochrome P450 oxidase. On the BSEP
R832C
. intestinal reabsorption and a high first-
other hand, Liu 24,25
pass clearance of ∼90%. Special
15
et al showed that dexamethasone Another steroid-dependent mechanism
of regulated BS transport may involve formulations of budesonide, however,
administration to neonatal rats 26
+ may reach the distal colon. In line
in vivo had a stimulating effect on Na -dependent cellular bile acid uptake
with this, colitis in patient 2 remained
Mdr2 (the ortholog to the human via NTCP, which is localized at the
17 in remission under alternating dosages
+ sinusoidal membrane of hepatocytes.
MDR3) and Na -taurocholate of 3 mg budesonide. It is possible that
cotransporting polypeptide (Ntcp) It has been shown that, under cholestasis might be perpetuated due to
gene expression, but decreased the cholestatic conditions such as biliary extrahepatic inflammation as the
18
gene expression of Bsep, Mrp2, and atresia, inflammation-induced primary insult. On the other hand,
Fic1, a P-type ATPase protein. 19,20
cholestasis, primary biliary patient 1 had a slight decrease in BS
However, most differences were not 21 coincident with active systemic lupus
cirrhosis, or progressive familial
significant in that study. The effect of erythematosus even before initiation of
22
steroids on canine bile flow has been intrahepatic cholestasis, NTCP is
steroid therapy. It is well known that
demonstrated by Brems downregulated in human livers. This
lipopolysaccharides induce
16 downregulation may serve to protect 27
et al. Dogs given high dosages of cholestasis and downregulate BSEP
hepatocytes from toxic BS 19
hydrocortisone at 5 mg/kg per hour concentration but may contribute to the in a model of human liver slices.
showed a significant increase in bile retention of BSs in the blood However, primary sclerosing
flow (from 258 ml/minute to

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PEDIATRICS Volume 135, number 5, May 2015 5
cholangitis in the context of ulcerative children with normal gamma-glutamyl 16. Brems JJ, Reese J, Kane R III,
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PEDIATRICS Volume 135, number 5, May 2015 7
 Two Case Reports of Successful Treatment of Cholestasis With Steroids in
Patients With PFIC-2
Guido Engelmann, Daniel Wenning, Diran Herebian, Oliver Sander, Carola Dröge,
Stefanie Kluge and Ralf Kubitz
Pediatrics originally published online April 6, 2015;

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 Two Case Reports of Successful Treatment of Cholestasis With Steroids in
Patients With PFIC-2
Guido Engelmann, Daniel Wenning, Diran Herebian, Oliver Sander, Carola Dröge,
Stefanie Kluge and Ralf Kubitz
Pediatrics originally published online April 6, 2015;

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services, is located on the World Wide Web at:
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