Goubran et al. BMC Medical Genetics(2020) 21:238
https://doi.org/10.1186/s12881-020-01173-0

CASE REPORTOpen Access

Case report: progressive familial
intrahepatic cholestasis type 3 withcompound heterozygous ABCB4 variants
diagnosed 15 years after livertransplantation
Mariam Goubran1, Ayodeji Aderibigbe1, Emmanuel Jacquemin2, Catherine Guettier3, Safwat Girgis4,
Vincent Bain1 and Andrew L. Mason1,5*

Abstract
Background: Progressive familial intrahepatic cholestasis (PFIC) type 3 is an autosomal recessive disorder arisingfrom mutations in the ATP-binding cassette subfamily B member 4 (ABCB4) gene. This gene encodes multidrug
resistance protein-3 (MDR3) that acts as a hepatocanalicular floppase that transports phosphatidylcholine from theinner to the outer canalicular membrane. In the absence of phosphatidylcholine, the detergent activity of bile salts
is amplified and this leads to cholangiopathy, bile duct loss and biliary cirrhosis. Patients usually present in infancy
or childhood and often progress to end-stage liver disease before adulthood.
Case presentation: We report a 32-year-old female who required cadaveric liver transplantation at the age of 17
for cryptogenic cirrhosis. When the patient developed chronic ductopenia in the allograft 15 years later, we
hypothesized that the patient’s original disease was due to a deficiency of a biliary transport protein and theductopenia could be explained by an autoimmune response to neoantigen that was not previously encounteredby the immune system. We therefore performed genetic analyses and immunohistochemistry of the native liver,which led to a diagnosis of PFIC3. However, there was no evidence of humoral immune response to the MDR3 andtherefore, we assumed that the ductopenia observed in the allograft was likely due to chronic rejection rather than
autoimmune disease in the allograft.
Conclusions: Teenage patients referred for liver transplantation with cryptogenic liver disease should undergo workup for PFIC3. An accurate diagnosis of PFIC 3 is key for optimal management, therapeutic intervention, andavoidance of complications before the onset of end-stage liver disease.
Keywords: Progressive familial intrahepatic cholestasis, ABCB4, MDR3, Case report* Correspondence: andrew.mason@ualberta.ca
1
Department of Medicine, University of Alberta Hospital, Edmonton, Canada
5
Division of Gastroenterology, 7-142 KGR, University of Alberta, Edmonton,
Alberta T6G 2E1, Canada
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Goubran et al. BMC Medical Genetics(2020) 21:238

Background
Progressive Familial Intrahepatic Cholestasis (PFIC) syndromes represent a group of genetic disorders characterized by a defect in the secretion of bile components andbile acids [1]. PFIC disorders are numbered 1 through 5
based on the gene involved. They generally present ininfancy or childhood with growth failure, coagulopathies
due to impaired Vitamin K absorption, and progressive
liver disease leading to cirrhosis before adulthood [2].
PFIC type 3 is an autosomal recessive disorder arisingfrom mutations in the ATP-binding cassette subfamily B
member 4 (ABCB4) gene which is located on chromosome
7 [3]. This gene encodes multidrug resistance protein 3(MDR3), a phosphatidylcholine transporter (“floppase”)
belonging to the family of adenosine-triphosphate-binding
cassettes [4]. MDR3 localizes to the canalicular membraneof hepatocytes where it translocates phosphatidylcholine
from the inner to the outer canalicular membrane [5].
Mutations in the ABCB4 gene lead to impaired biliary
phospholipid secretion. As a result, detergent bile salts are
not inactivated by phospholipids. This causes injury to bile
canaliculi and biliary epithelium that ultimately leads tocholestasis, cholangitis, ductopenia and biliary cirrhosis [6].
Variants in the ABCB4 gene manifest with variabledisease penetrance. For example, heterozygotes withfunctional MDR3 activity may simply manifest with increased serum gamma-glutamyl transferase (GGT), or
present with a range of increasingly penetrant disorders,such as intrahepatic cholestasis of pregnancy, mild
chronic cholangiopathy, or low-phospholipid-associatedcholelithiasis [6]. Whereas a complete deficiency of
MDR3 activity is more clearly linked with PFIC3 and decompensated biliary cirrhosis [6]. PFIC3 is the only PFIC
associated with marked elevations in GGT [7]. The disease presents later in childhood or young adulthood anddoes not typically present with the intractable pruritus
characteristic of both PFIC1 and PFIC2 [8].
Clinicians should suspect PFIC in infants and children
presenting with cholestasis of unknown origin after the
exclusion of more common causes such as Alagillesyndrome, biliary atresia, sclerosing cholangitis, cystic fibrosis, and alpha-1 anti-trypsin deficiency [9]. Diagnosis
of PFIC syndromes can be confirmed by genetic testing
for all types and by immunostaining for PFIC 2 and 3.
Ursodeoxycholic acid is the initial therapy of choice forchildren with all types of PFIC and is especially effectivefor PFIC3 patients with less severe disease [10]. However, liver transplantation is often the only alternative
when pharmacological measures fail [11].
Case presentation
Patient presentation and timeline

We describe a 32-year old female with a retrospective
diagnosis of PFIC type 3 that was made fifteen years

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after receiving liver transplantation for end-stage cryptogenic liver disease (see patient timeline).
The patient was first found to have hepato-splenomegalyat 18 months of age. The proband was otherwise healthy
and had no family history of liver disease. No records could
be found of investigations performed at the time. The patient then presented at age 14 with jaundice and fatigue
and was found to have liver cirrhosis of unknown etiology.
An abdominal ultrasound revealed evidence of cirrhosis,
portal hypertension and incidental cholelithiasis. Liverbiopsy showed micronodular cirrhosis with significant bile
stasis in the hepatocytes. There was no significant ironstaining, nor typical alpha-1 antitrypsin inclusions identified, but there was increased copper staining, consistent
with cholestasis without other evidence of Wilson disease.
A viral etiology was also excluded as the patient was confirmed to be immune to hepatitis B virus, and the hepatitis
C serology was negative.
Over the following 3 years, a hepatic diagnosis was notfound and the patient progressed to end-stage liverdisease. The proband suffered from delayed growth andpuberty as well as multiple complications of liver failure.
These included jaundice, edema, ascites, two episodes of
spontaneous bacterial peritonitis, splenomegaly, esophageal varices, coagulopathy, and hepatic encephalopathy.
The patient was referred to the University of Alberta
Liver Transplant program for a workup. At the time, herlaboratory tests revealed an elevated serum AST of 174(normal < 40 U/L), ALT of 64 (normal < 50 U/L), andtotal bilirubin levels of 491 (normal < 20 μmol/L),
whereas serum GGT was not measured prior to transplantation. At age 17, the patient underwent a cadaveric
liver transplantation and splenectomy for end stage liverdisease. The histological evaluation of the native liverdemonstrated biliary cirrhosis with moderate portal inflammation, ductular reaction, minimal loss of small bileducts and chronic cholestasis. There was no biopsy
evidence suggestive of sclerosing cholangitis such as aconcentric “onion like” fibrosis nor fibro-obliterative scarof the bile ducts (Fig. 1a and b).
Post-operative course was complicated by obstructive
jaundice due to a stricture within the common bile duct,
which required stenting. One month after transplant, thepatient experience elevated hepatic biochemistry tests
and the liver biopsy was consistent with a mild acute
cellular rejection, which was successfully treated with asteroid taper. The patient remained on immunosuppressive therapy post-transplant with tacrolimus to achievelevels of 10–12 μg/L (median tacrolimus level, 11.3 μg/L)
as well as low dose mycophenolate. The latter was discontinued at 1 year following transplantation, due to bonemarrow suppression.
Two years after liver transplantation, at age 19, the patient presented with a new thyroid nodule. A fine needle

Goubran et al. BMC Medical Genetics(2020) 21:238

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Fig. 1 a Histology of explanted native liver showing biliary cirrhosis with annular fibrosis delimiting irregular parenchymal nodules (PicroSirius
staining, × 15) and b moderate septal inflammation with some ductular reaction, canalicular bilirubinostasis and cholestasis changes in periseptal
hepatocytes (H&E staining, × 120).c Immunochemistry study of the patient’s explanted native liver showing typical and even canalicular
distribution of BSEP staining; d whereas the pattern of MDR3 canalicular staining is inconsistent and granular cytoplasmic signal observed withina proportion of hepatocytes. Within the control healthy liver, e regular canalicular BSEP and f MDR3 staining were observed

aspiration revealed papillary thyroid carcinoma, which
was successfully treated with thyroidectomy and iodine
ablation. The patient had no further complications from
immunosuppression.
The proband’s clinical status and liver enzymes werestable until eleven years after liver transplant, at age 29,
when routine follow-up labs revealed rising liver
enzymes and bilirubin. The patient was asymptomatic atthat time. Liver biopsy revealed pathological findings
consistent with late cellular ductopenic rejection. Lowdose prednisone (20 mg daily) and azathioprine 100 mg
daily were subsequently added to the tacrolimus (2.5 mg
BID, median tacrolimus level of 9.1 μg/L). The patientcomplained of increasing fatigue and xerostomia. The
liver enzymes continued to rise despite high dose immunosuppressive therapy and then improved following a
trial of ursodeoxycholic acid (500 mg BID).

Fifteen years post-transplant, further biopsies revealed
the patient has ongoing severe ductopenia (80%) with acomponent of plasma cell rich rejection. Serum markers
for autoimmune hepatitis and primary biliary cholangitis
were repeatedly negative.
At that point, the patient’s pretransplant hepatic
diagnosis was still unknown. The initial histology had revealed biliary cirrhosis but no classical features or serum
markers diagnostic of an immune mediated cholangiopathy such as primary sclerosing cholangitis or primarybiliary cholangitis. We therefore hypothesized that thepatient’s original disease was due to a congenital deficiency of a biliary transporter protein. This hypothesis
raised the possibility that the ductopenia observed in the
allograft could be due to an autoimmune response to
the same biliary protein acting as a neoantigen notpreviously encountered by the immune system. This

Goubran et al. BMC Medical Genetics(2020) 21:238

phenomenon has been demonstrated in some patients
undergoing liver transplantation for PFIC, who subsequently develop alloimmune responses to canalicular
proteins [12]. We therefore investigated whether the
proband harbored a genetic defect to account for thebiliary cirrhosis and sought evidence of alloimmune
serum reactivity to the allograft.
Genetic testing and immunohistochemistry

A DNA sample from the patient was sent to Eurofins
Clinical Diagnostics for evaluated for variants within 76
genes within the Genetic Cholestasis panel [13]. The
patient was found to have variants in the ABCB4 geneand both were described as variants of unknown significance (VOUS).
The first was c.3227G > A (p.S1076N) that was previously reported in neonatal cholestasis and intrahepaticcholestasis of pregnancy [14]. The second variant,c.3431 T > C (p.I1144T), has not been described to date.
To analyse the presence and position of these mutations, we employed nested PCR to clone and sequence
product from both alleles. This was performed using
outer primers (Forward: TCTCTCACCTTCATTTCACACC; Reverse: CTGAAGTATGGTGGTTTTGAGC)
and inner primers (Forward: TTCAACTATCCCAC
CCGAGC; Reverse: TGAAAGGATGTATGTTGGCA
GC). We confirmed that these mutations are found on
alternate alleles by PCR amplification of the ABCB4 region encompassing both variants. These data indicatedthat the patient was indeed a compound heterozygote.
We performed immunohistochemistry to determine
the expression of the MDR3 protein and the bile salt export pump (BSEP) in the patient’s explanted native liver
as compared to a healthy liver sample. BSEP is localized
in the hepatic canaliculi and is encoded by the ABCB11
gene, which is mutated in patients with PFIC2 [15]. A
canalicular expression of BSEP was observed in the
explanted patients’ native liver and this was comparable
to the pattern observed in the healthy liver control (Fig. 1
c and e). In contrast, the MDR3 expression in the
explanted native liver differed from the healthy liver
control with a mixed signal of regular and dispersed canalicular pattern associated with a granular signal fromwithin the hepatocytes (Fig. 1 d and f).
Given the mixed targeting of MDR3 to the canalicularmembrane, the patient was probably not immunologically naïve to MDR3 protein. Nevertheless, we performed
further studies using the patient’s serum to assess foralloimmunization. An immunofluorescence study was
performed using the patient’s post liver transplant serum
on normal human liver that revealed no reactivity to
MDR3. While alloimmunization against MDR3 causing
disease recurrence in allografts is theoretically possible,
it has only been reported to occur in patients undergoing

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liver transplantation for PFIC2 associated with development of antibody reactivity to BSEP [12].
Taken together, the clinical presentation, the genetic
testing, and the MDR3 distribution on both the canalicular membrane and within hepatocytes, were consistent
with a diagnosis of PFIC3 disease. However, the patient
has developed progressive ductopenic rejection with liver
failure and is currently re-listed for a second liver transplantation. By clarifying the original hepatic diagnosis,
we can conclude that the ductopenia in the allograft isdue to chronic rejection rather than autoimmune in the
allograft.

Discussion and conclusions
Herein, we describe a patient with PFIC3 who ultimately
required liver transplantation at the age of 17. This diagnosis was confirmed by retrospective analysis of thepatient’s explanted native liver using immunostaining for
MDR3 protein as well as genetic analysis of the ABCB4
gene. It is probable that both mutations identified had aloss of function and contributed to the patient’s disease.
One missense mutant (p.S1076N) likely leads to correcttranslocation of the protein to the canalicular membrane
but with likely a loss of function. Indeed, it has beenshown previously that the p.S1076C mutant identified iscorrectly targeted but has a significant loss of function[16]. The functional activity of the other missense mutant has not been previously described but this variant
likely encodes a loss of function protein. The granular
intracellular immunostaining suggests that the mutant
protein is probably retained within the endoplasmic
reticulum, as previously shown for a proportion of other
MDR3 missense mutants [17].
Both mutations were previously described as variants of
unknown significance which now may be of interest forbiochemical study as targets for future therapies [16–18].
It has been suggested that gene therapy may become apotentially curative approach for affected individuals, as
we gain a better understanding of the genotype-phenotype
correlation in PFIC syndromes. Recently, two teams havesuccessfully demonstrated stable ABCB4 gene expression
and correction of PFIC3 phenotype in a murine modelusing adeno-associated virus serotype 8 (AAV8)-mediatedgene therapy [19, 20]. Other studies have shown thatmolecular chaperones may be of therapeutic value in PFIC
syndromes, including PFIC3 [21–25].
In retrospect, the clinical picture, laboratory results,
and pathology data were al consistent with PFIC disease.
The copper staining identified on the patient’s liverbiopsies 3 years before transplant is a common findingin patients with cholestasis and PFIC 3 as a result of
prolonged cholestasis [26, 27]. The PFIC3 diagnosis was
not made prior to the patient’s presentation with endstage liver failure and as such the patient did not receive

Goubran et al. BMC Medical Genetics(2020) 21:238

ursodeoxycholic acid prior to transplantation which may
have positively influenced the disease course. Ursodeoxycholic acid is the therapy of choice for children with PFIC
and is especially effective for PFIC 3 with less penetrant
disease [6]. Accordingly, this case report highlights thepotential impact of exploring a diagnosis of PFIC 3 inteenagers referred for liver transplantation with cryptogenic cirrhosis with MDR3 immunostaining and ABCB4
genetic analysis. An early diagnosis is critical for optimal
management, therapeutic intervention, and avoidance ofcomplications before the onset of end-stage liver disease.

Supplementary Information
The online version contains supplementary material available at https://doi.
org/10.1186/s12881-020-01173-0.
Additional file 1. Patient timeline
Additional file 2. CARE Checklist of information to include whenwriting a case report
Abbreviations
PFIC: Progressive familial intrahepatic cholestasis; MDR3: Multidrug resistance
protein 3; GGT: Gamma-glutamyl transferase; VOUS: Variant of unknown
significance; BSEP: Bile salt export pump
Acknowledgements
The authors would like to thank the patient and her family.
Authors’ contributions
MG and AM wrote the manuscript, AA performed the genetic cloning andanalysis, EJ and CG performed the immunohistochemistry, SG performed thepathology on the native liver, VB and AM were involved in the care of thepatient. The authors evaluated the relevant data, edited and approved thefinal manuscript.
Funding
We gratefully acknowledge awards from the Canadian Institutes for HealthResearch and the Canadian Liver Foundation, funds were used to complete
the genetic analysis for this study.
Availability of data and materials
The datasets used and/or analysed during the current study are available
from the corresponding author on reasonable request.
Ethics approval and consent to participate
Ethics approval was obtained from the University of Alberta, Human
Research Ethics Board, Pro00005105. Written consent was obtained from thepatient for sample collection and genetic analyses.
Consent for publication
The patient has provided informed written consent for publication.
Competing interests
The authors declare no competing interests relevant to this publication.
Author details
1
Department of Medicine, University of Alberta Hospital, Edmonton, Canada.
2
Paediatric Hepatology & Paediatric Liver Transplant Department, ReferenceCenter for Rare Paediatric Liver Diseases, FILFOIE, ERN RARE LIVER, Assistance
Publique-Hôpitaux de Paris, Faculty of Medicine and University Paris-Saclay,
CHU Bicêtre, Le Kremlin-Bicêtre, France. 3Pathology Department, Assistance
Publique-Hôpitaux de Paris, Faculty of Medicine and University Paris-Saclay,
CHU Bicêtre, Le Kremlin-Bicêtre, France. 4Department of Laboratory Medicine
and Pathology, University of Alberta Hospital, Edmonton, Canada. 5Divisionof Gastroenterology, 7-142 KGR, University of Alberta, Edmonton, Alberta T6G
2E1, Canada.

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Received: 25 July 2020 Accepted: 12 November 2020

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