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n engl j med 361;14 nejm.org october 1, 2009 1359

brief report

Recurrence of Bile Salt Export Pump

Deficiency after Liver TransplantationPaloma Jara, M.D., Loreto Hierro, M.D., Pilar Martnez-Fernndez, Ph.D.,Rita Alvarez-Doforno, Ph.D., Francisca Ynez, B.S., Mara C. Diaz, M.D.,

Carmen Camarena, M.D., Angela De la Vega, M.D., Esteban Frauca, M.D.,Gema Muoz-Bartolo, M.D., Manuel Lpez-Santamara, M.D.,

Javier Larrauri, M.D., and Luis Alvarez, Ph.D.

From the Pediatric Liver Service (P.J.,L.H., M.C.D., C.C., A.D.V., E.F., G.M.-B.),Research Unit (P.M.-F., L.A.), Service of

Immunology (R.A.-D., F.Y.), Departmentof Pathology (J.L.), and Department ofPediatric Surgery (M.L.-S.), La Paz Uni-versity Hospital, Madrid. Address reprintrequests to Dr. Alvarez at the ResearchUnit, La Paz University Hospital-FIBHULP,Paseo de La Castellana 261, 28046Madrid, Spain, or at [email protected].

Drs. Jara, Hierro, and Martnez-Fernndezcontributed equally to this article.

N Engl J Med 2009;361:1359-67.Copyright 2009 Massachusetts Medical Society.

SUMMARY

Severe bile salt export pump (BSEP) deficiency is a hereditary cholestatic condition

that starts in infancy and leads to end-stage liver disease. Three children who un-

derwent orthotopic liver transplantation for severe BSEP deficiency had post-trans-plantation episodes of cholestatic dysfunction that mimicked the original disease.

Remission of all episodes was achieved by intensifying the immunosuppressive regi-

men. The phenotypic recurrence of the disease correlated with the presence of cir-

culating high-titer antibodies against BSEP that inhibit transport by BSEP in vitro.

When administered to rats, these antibodies targeted the bile canaliculi and im-

paired bile acid secretion.

C

holestatic disorders are amongthe most severe liver diseases

in infancy and childhood.1 For some patients, orthotopic liver transplanta-

tion is the only effective therapy, resulting in favorable outcomes and no recur-

rence of the original disease.2,3 Severe BSEP deficiency, also referred to as progres-

sive familial intrahepatic cholestasis type 2, is one such disorder. It is caused by

recessive mutations inABCB11, the gene encoding BSEP.4,5 BSEP is expressed at the

canalicular membrane of the hepatocytes and transports bile acids into the canali-

cular space, using ATP as an energy source.6 Hepatocyte canaliculi in most patients

carryingABCB11 mutations express little or no detectable BSEP.5

Children with severe BSEP deficiency typically have jaundice and pruritus within

the first few months of life. Early-onset cholestasis progresses to hepatic fibrosis,

cirrhosis, and end-stage liver disease.7 Affected children are also at increased risk

for liver cancer.5,8 Biochemical and histopathological features of this disorder in-

clude elevated serum concentrations of bile acids, intrahepatic cholestasis, and often,

giant-cell transformation. Serum values of -glutamyltransferase (GGT) activity arenormal, despite the degree of conjugated hyperbilirubinemia.9-11 Liver disease

caused by severe BSEP deficiency is usually resistant to medical treatment; therefore,

for most patients, transplantation becomes necessary.7,11

We report on a rare phenotypic recurrence of BSEP deficiency that takes place after

liver replacement. The recurrence correlates with the presence in serum of block-

ing antibodies against BSEP.

The New England Journal of Medicine

Downloaded from nejm.org on December 17, 2011. For personal use only. No other uses without permission.

Copyright 2009 Massachusetts Medical Society. All rights reserved.

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Methods

We obtained approval for the study from a local

ethics committee. Written informed consent was

provided by the patients or, if the patients were

under 18 years of age, their parents.

Sequence Analysis of ABCB11

We isolated genomic DNA from peripheral-blood

leukocytes, using the PureGene DNA Isolation kit

(Gentra Systems), and amplified all 28 exons of

ABCB11 and flanking intronexon boundaries by

performing polymerase-chain-reaction assays.

(Primer sequences and assay conditions are avail-

able on request.) We sequenced both strands us-

ing the 1.1 Big Dye Terminator RRMix (Applied

Biosystems).

Immunohistochemical Analysis

Immunohistochemical studies were carried out on

paraff in-embedded explanted liver samples and

needle-biopsy specimens obtained during post-

transplantation cholestatic episodes. Detection of

BSEP and multidrug resistanceassociated pro-

tein 2 (MRP2) was performed by means of the

antigen-retrieval method, as previously described,12

with a mouse monoclonal anti-MRP2 antibody

(clone M2III-6, Chemicon International) and a goat

polyclonal anti-BSEP antibody (Santa Cruz Bio-technology) as primary antibodies.

Indirect Immunofluorescence Analysis

Serum samples from the patients were screened at

dilutions ranging from 1:40 to 1:5120, with the use

of commercially prepared sections from rat liver,

kidney, and stomach (Euroimmun, Lbeck). Bound

antibodies were detected with the use of fluorescein

isothiocyanate (FITC)conjugated rabbit antihuman

IgA, IgG, and IgM antibodies (DakoCytomation).

Table 1. Characteristics of the Three Patients with Recurrent Severe Bile Salt Export Pump Deficiency after LiverTransplantation.

Characteristic Patient 1 Patient 2 Patient 3

Sex M F F

Year of birth 1982 1991 1995

Age at transplantation (yr) 5.2 3.7 2.2

ABCB11 mutation

Nucleotide mutation Homozygote, 907AG Compound heterozygote,1741CT and IVS12+1GT

Homozygote, IVS17+1TA

Predicted amino acidmutation

R303G L581F and splice-site disruption Splice-site disruption

Figure 1 (facing page). Characteristic Findings of Episod-

ic Cholestasis after Liver Transplantation for SevereBile Salt Export Pump (BSEP) Deficiency in Patient 2.

Panel A shows immunohistochemical detection of BSEPand multidrug resistanceassociated protein 2 (MRP2)

in a liver specimen from a child without cholestasis (con-trol) and the explanted liver from Patient 2. Paraffin-

embedded sections were stained with primary antibod-ies at a 1:20 dilution. Sections were counterstained with

hematoxylin. BSEP staining was also absent in Patients 1

and 3. Panel B shows the results of liver-function testingduring the course of the f irst post-transplantation chole-

static episode in Patient 2. Serum aspartate aminotrans-ferase (AST), alanine aminotransferase (ALT), bilirubin,

and -glutamyl transpeptidase (GGT) levels were moni-tored from the time of onset of the cholestatic attack

(day 0) to the time of recovery. GGT activity remainedrelatively constant despite the long-lasting period of jaun-

dice. Panel C shows morphologic characteristics of the

liver-biopsy specimen obtained from Patient 2 at 26 daysafter the onset of the first post-transplantation cholestat-ic attack. Multinucleated giant hepatocytes (arrows) are

visible (hematoxylin and eosin). The inset shows a portal

tract in the same specimen, with a moderately denselymphocytic infiltrate but no evidence of damage to the

bile duct or portal vessels. The left-hand image in PanelD shows the pattern of immunofluorescence staining of

rat-liver sections incubated with serum (dilution, 1:640)obtained at the onset of the third episode of cholestatic

dysfunction in Patient 2. No reaction was found in sec-tions of rat kidney or stomach. Positive bile-canalicular

staining was also detected with the use of serum sam-ples from Patients 1 and 3. The right-hand image in Pan-

el D shows the result of indirect immunofluorescenceanalysis of serum from a control (Patient 4) obtainedduring the second episode of acute cellular rejection (see

the Supplementary Appendix for details). Panel E showsserum antibody titers during the fifth cholestatic attack

in Patient 2. The intensity of the immunosuppressive reg-imen was increased on day 0. Resolution of pruritus was

achieved on day 15. On complete recovery of normal liverfunction, the anticanalicular antibody titer was 6.25% of

that at the peak of the cholestatic attack. (Titers are indi-cated within the plot.) For Panels B and E, to convert val-

ues for bilirubin to micromoles per liter, multiply by 17.1.




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Brief Report

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Western-Blot Analysis

Membrane vesicles (containing a total of 30 g of

protein) from Sf9 cells (derived from Spodoptera

frugiperda) expressing either human BSEP (Sigma-

Aldrich) or human MRP1 (BD Biosciences) were

fractionated by means of 10% sodium dodecyl sul-

fatepolyacrylamide-gel electrophoresis. The gels

were transferred onto polyvinylidene dif luoride

membranes and cut into strips, which were probed

with serum samples from patients (at a 1:100 di-

lution) or with the goat antibody against human

BSEP (at a 1:200 dilution). Immune complexes weredetected by means of horseradish peroxidase

conjugated goat antihuman immunoglobulins

(1:16,000 dilution, Nordic Immunological Labora-

tories) or rabbit antigoat immunoglobulins (1:6000

dilution, DakoCytomation).

Evaluation of Functional Transport

Uptake of3H-labeled taurocholate (American Ra-

diolabeled Chemicals) in membrane vesicles from

Sf9 cells expressing human BSEP was measured

B

D

C

A

1000

AST,

ALT,andGGT(U/liter)

Bilirubin(mg/dl)

800

900

700

600

400

300

100

500

200

0

10

8

9

7

6

4

3

1

5

2

00 30 60 90 120 150 180 210 240

AST

ALT

Total bilirubin

Direct bilirubin

GGT

DayssinceOnsetofAttack

Control Patient2

Staining for MRP2 Staining for MRP2Staining for BSEP

IndirectImmunofluorescenceAnalysis

Patient 2 Control

Staining for BSEP

E

650

AST,

ALT,andGGT(U/liter)

TotalBilirubin(mg/dl)

550

600

500

450

350300

10050

150

400

250

200

0

6

4

3

1

5

2

00 20 40 60 80 100 120

AST

ALT

Total bilirubin

Anticanalicularantibody (titer)

GGT

DayssinceOnsetofAttack

1:5120

1:1280

1:640

1:320




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Table2.Clinical,Biochemical,a

ndHistologicFeaturesandOutcomeofEp

isodesofRecurrentCholestaticGraftDysfu

nctionintheThreePatients.*

Characteristic

Patient1

Patient2

Patient3

Ep

isode1

Episode1

Episode2

Episode3

Episo

de4

Episode5

Episode1

Episode2

Timeaftertransplanta-

tion(yr)

12.0

3.5

5.2

8.1

12.1

13.0

2.1

4.0

Precipitatingfactors

Unknow

n

EBVinfection

Unknown

Withdrawalofcor-

ticosteroids

Unknown

Unknown

Lowcyclosporine

(troughlevel,

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