A multidrug resistance 3 gene mutation causing cholelithiasis, cholestasis of pregnancy, and adulthood biliary cirrhosis

We describe a 47-year-old patient who developed cholelithiasis in adolescence, followed by recurrent intrahepatic cholestasis of pregnancy, and finally biliary cirrhosis in adulthood. In our patient, the consecutive presentation of the 3 mentioned
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  CASE REPORTS A Multidrug Resistance 3 Gene Mutation CausingCholelithiasis, Cholestasis of Pregnancy, and AdulthoodBiliary Cirrhosis JUAN–FELIPE LUCENA,* J. IGNACIO HERRERO,* JORGE QUIROGA,* BRUNO SANGRO,*JESUS GARCIA–FONCILLAS, ‡ NATALIA ZABALEGUI, ‡ JOSU SOLA, § MAITE HERRAIZ,*JUAN F. MEDINA,* and JESUS PRIETO* *Clinica Universitaria and Medical School, University of Navarra, Medicine and Liver Unit, Pamplona, Navarra, Spain;  ‡ Clinica Universitariaand Medical School, University of Navarra, Biotechnology, Pamplona, Navarra, Spain; and  § Clinica Universitaria and Medical School,University of Navarra, Pathology, Pamplona, Navarra, Spain We describe a 47-year-old patient who developed cho-lelithiasis in adolescence, followed by recurrent intrahe-patic cholestasis of pregnancy, and finally biliary cirrho-sis in adulthood. In our patient, the consecutivepresentation of the 3 mentioned disorders raised thesuspicion of a defect of MDR3, the canalicular proteininvolved in the transport of phospatidylcholine to bile.Mutational analysis in our patient showed a heterozy-gous missense mutation of the MDR3 gene that has notbeen described previously, which occurs in exon 14 atcodon 535, and results in the substitution of glycine foraspartic acid. Further analysis of 7 members of thefamily showed the same mutation in her daughter who,on follow-up, developed cholestasis of pregnancy andpersisting high serum levels of   -glutamyl transpepti-dase and alkaline phosphatase after delivery. Althoughbiliary cirrhosis associated with MDR3 deficiency typi-cally appears before the age of 25 years, in our case, therelatively mild MDR3 dysfunction allowed for a slowerprogression of the disease with established, well-ad-vanced cirrhosis in the fifth decade of life. The presentcase, which accumulates the 3 clinical disorders asso-caited with MDR3 deficiency, shows that this conditionshould be suspected not only in children or young peoplewith high  -glutamyl transpeptidase cholestasis but alsoin middle-aged or older patients with chronic idiopathiccholestasis, especially when there is a previous historyof cholestasis of pregnancy or juvenile cholelithiasis. R ecently, several genetic defects in hepatocanaliculartransport have been identified, and a broad spec-trum of cholestatic syndromes can be explained as aresult of impaired bile formation. 1–4 Dysfunction of spe-cific transport systems, including familial intrahepaticcholestasis gene 1 (FIC1), bile salt export pump (BSEP),and multidrug resistance 3 glycoprotein (MDR3) havebeen described in association with different cholestaticliver diseases, such as benign recurrent intrahepatic cho-lestasis, cholesterol gallstones, Byler’s disease, intrahe-patic cholestasis of pregnancy (ICP), and biliary cirrho-sis. 5–11 Some of the affected patients develop chroniccholestasis of hepatocellular srcin in the neonatal periodor early childhood, leading to end-stage liver diseasewithin the first decade of life and eventually in earlyadulthood. 12 The general term applied to these disordersis “progressive familial intrahepatic cholestasis” (PFIC).The first type (PFIC-1) is caused by mutations in theFIC-1 gene, 6 which appears to encode an aminophospho-lipid transporter responsible for maintaining the phos-pholipid environment on the inner leaflet of the canalic-ular membrane. The second type (PFIC-2; previouslycalled Byler syndrome) is the result of BSEP deficiency, 3,7 a canalicular protein responsible for bile acid secretion.Its defect leads to accumulation of bile salts inside thehepatocyte and secondary hepatocellular damage. Al-though in cholestatic disorders the detergent effect of bile acids liberates   -glutamyl transpeptidase (  -GT)from the canalicular membrane, which leaks back intothe circulation, the activity of   -GT in blood is normalin both PFIC-1 and PFIC-2 as a consequence of thealtered biliary secretion of bile acids.MDR3 glycoprotein is a phospholipid translocatorinvolved in biliary phosphatidylcholine (PC) excretion,which is predominantly expressed in the canalicular  Abbreviations used in this paper:  ICP, intrahepatic cholestasis ofpregnancy; MDR3, multidrug resistance 3 glycoprotein; PFIC, progres-sive familial intrahepatic cholestasis. ©  2003 by the American Gastroenterological Association0016-5085/03/$30.00doi:10.1053/gast.2003.50144 GASTROENTEROLOGY 2003;124:1037–1042  membrane of hepatocytes. 13,14 Defects in the MDR3gene determine the production of bile with a low contentof PC, increased lithogenicity, and high detergent power,which causes damage of the luminal membrane of thehepatobiliary system, resulting in clinical cholestasiswith high  -GT activity in serum. 1,4,8,10 – 12 Here, we describe a female with juvenile cholelithiasiswho later developed recurrent ICP and  fi nally adulthoodbiliary cirrhosis. This patient and one affected daughterwere heterozygous for an as yet undescribed MDR3mutation. To our knowledge, this is the  fi rst report of apatient with 3 consecutive diseases associated with aMDR3 mutation that led to end-stage cirrhosis after thefourth decade of life. This case illustrates the wide clin-ical spectrum of the MDR3 de fi ciency syndrome thatcovers cholestatic disorders presenting from the neonatalperiod of life to late adulthood. Case Report A 47-year-old woman with chronic cholestasisand ascites was referred to our institution for study.Seven years before, abnormal liver function tests, withhigh levels of transaminases and anicteric cholestasis,were found in routine analysis. During the last year, shehad complained of mild fatigue and presented 2 episodesof ascites with partial remission after diuretic treatment.The patient had undergone cholecystectomy for symp-tomatic cholesterol cholelithiasis when she was 19 yearsold. She experienced cholestasis and pruritus during thethird trimester of her pregnancies at ages 27 and 31years, with spontaneous resolution after delivery. She hadno history of alcohol consumption, illicit drug abuse,hepatotoxic medications, or drug allergies.The patient had a daughter 21 years of age and a son26 years of age. Her family history was only remarkablefor the presence of asymptomatic hypertransaminasemiain the daughter. The diagnostic evaluation before admis-sion included a liver biopsy revealing chronic intrahe-patic cholestasis and viral markers and autoimmunitytests that were all negative.Physical examination showed distended, nontenderabdomen with ascites and splenomegaly. There were noother abnormal  fi ndings, and the neurologic examinationwas unremarkable.The analysis showed increased alanine aminotrans-aminase (ALT; 54 UI/L, N   22 UI/L) and aspartateaminotransaminase (AST; 60 UI/L, N   21 UI/L),signs of cholestasis with high alkaline phosphatase(975 UI/L, N 73 – 207 UI/L),  -GT (302 UI/L, N 5 – 25UI/L), total bilirubin (2.82 mg/dL, N 0.3 – 1 mg/dL),conjugated bilirubin (1.33 mg/dL, N 0.1 – 0.4 mg/dL),and a high level of    -globulin with normal albumin.Complete blood cell count revealed hypersplenismwith 52,000 platelets/mm 3 and 2900 leukocytes/mm 3 with normal levels of hemoglobin. The hepatitis Bsurface antigen, hepatitis C antibody, antineutrophilcytoplasmic antibodies, antimitochondrial antibodies,and antiendomissium antibodies were negative, andthe serum levels of cholesterol, creatinine, ceruloplas-min,  1-antitrypsin, iron, prothrombin time, and fer-ritin were normal. Antinuclear and antismooth muscleantibodies were positive at titers of 1/240 and 1/80,respectively. A CT scan of the abdomen showed asmall nodular liver, with right lobe atrophy; absenceof gallbladder; and presence of ascites, splenomegaly,and collateral circulation. There was no evidence of biliary tract obstruction, portal thrombosis, or livertumor. Magnetic resonance cholangiopancreatographydid not show any abnormality of the biliary tree. Adiagnostic paracentesis revealed a serum-ascites albu-min gradient   1.1 g/dL and ruled out bacterial peri-tonitis. Upper gastrointestinal endoscopy showedprominent gastroesophageal varices and signs of con-gestive gastropathy. The sequential presentation of juvenile cholelithiasis, recurrent ICP, and,  fi nally, bil-iary cirrhosis raised the suspicion of MDR3 genemutation, and the patient together with her  fi rst andsecond degree family members were subjected to mu-tational analysis. Patients and Methods Mutational Analysis of the MDR3 Gene Peripheral blood lymphomononuclear cells were col-lected from the patient and from 7  fi rst- and second-degreefamily members. DNA was extracted using the MagnaPure LCDNA Isolation Kit I (Roche Pharmaceuticals) according to themanufacturer ’ s instructions. Polymerase chain reaction (PCR)ampli fi cation was performed in different fragments with in-tronic primers from the sequence previously published (genebank accession number M23234). The products were electro-phoresed on 1.5% agarose gel and then puri fi ed with GFXPCR DNA and gel band puri fi cation Kit (Amersham Phar-macia Biotech Inc., Barcelona, Spain).The PCR products were sequenced using the Big Dyeterminator cycle sequencing ready reaction kit (Applied Bio-systems, Madrid, Spain), according to the protocol supplied bythe manufacturer. The sequencing reactions were carried out ina Gene Amp PCR system 2400 (Applied Biosystems). PCRproducts were puri fi ed through Centri Sep spin columns (Ap-plied Biosystems) and,  fi nally, were processed with the ABIPrism 377 DNA sequencer. The software used to analyze theresults included ABI Prism 377XL collection, SequencingAnalysis 3.4.1., and Sequence Navigator 1.0.1. 1038 LUCENA ET AL. GASTROENTEROLOGY Vol. 124, No. 4  Liver Immunohistochemistry P3II26 monoclonal antibody against human MDR3P glycoprotein was used for detection of canalicular phos-pholipid translocator. 8 The antibody is mouse isotypeIgG2b and reacts in Western blotting and immuno fl uores-cence without cross-reactivity against mouse MDR2. Thecanalicular multispeci fi c organic-anion transporter (MRP2)was detected with monoclonal antibody M2III6 and servedas control for canalicular staining (both P3II26 and M2III6antibodies were the kind gift of Dr. Ronald P.J. Oude Figure 1.  ( A ) Electropherogram of the heterozygous mutation (arrow)of the MDR3 gene in exon 14 at codon 535 (G 3  A), resulting in asubstitution of glycine for aspartic acid, found in the patient (IIa) andher daughter (IIIb). ( B  ) Pedigree of the case report.  Squares   representmale family members and  circles   female family members.  Solid sym- bols   indicate affected members with MDR3 mutation.  Slash  over thesymbol denotes death, and  line   under the symbol denotes memberswho were not evaluated.biliary ducts were markedly reduced in number with extensive prolif-eration of bile ductules. ‹ Figure 2.  ( A ) Liver biopsy of the patient with adulthood biliary cirrho-sis and heterozygous MDR3 mutation, showing bands of connectivetissue separating the regenerative nodules with mild chronic inflam-matory infiltrate, intrahepatic cholestasis, and periportal fibrosis. ( B  )Immunostaining of liver biopsy for cytokeratin 19. The segmentary  April 2003 MULTIDRUG RESISTANCE 3 GENE MUTATION 1039  Elferink from the Academic Medical Center, University of Amsterdam, The Netherlands). Standard immunohisto-chemical procedures were performed. Results Sequence analysis of the patient and her familyrevealed in the proband a heterozygous mutation of the MDR3 gene in exon 14 at codon 535, resulting ina substitution of glycine (GGT) for aspartic acid(GAT) (Figure 1  A ). The same mutation was found inthe patient ’ s daughter and was absent in the otherfamily members tested (Figure 1 B ). Interestingly, thedaughter with the G535D mutation developed in thefollow-up intrahepatic cholestasis of pregnancy at theage of 22 years. She also presented persisting elevationof   -GT (260 UI/L), alkaline phosphatase (538 UI/L),ALT (147 UI/L), and AST (53 UI/L) after spontaneouspremature delivery. Histologic examination of theproband liver showed extensive areas of   fi brous tissueseparating regenerative parenchymal nodules withmild, chronic in fl ammatory in fi ltrate and occasionalneutrophils (Figure 2  A ). The segmentary biliary ductswere markedly reduced within the portal triads withperiportal  fi brosis, marked proliferation of bileductules (Figure 2 B ), and important intrahepatic cho-lestasis. There was no evidence of iron deposits, andthe orcein staining was negative.Immunohistochemical analysis of the liver biopsyspecimen showed positive detection but reduced expres-sion of MDR3 protein, which was mainly localized at thecanalicular membrane with apparent spreading of thesignal to the basolateral membrane, suggesting alteredcellular traf  fi cking of the molecule (Figure 3). Othercanalicular transporters, such as MRP2, showed normalexpression and localization (Figure 3). The biochemicaland histologic features were consistent with high  -GTprogressive familial intrahepatic cholestasis (PFIC3) andbiliary cirrhosis.The patient was placed on a salt-restricted diet andadjusted doses of spironolactone and furosemide thatresulted in gradual resolution of ascites. Ursodeoxycholicacid (UDCA) and propranolol were started at doses of 150 mg, 3 times daily and 20 mg, twice daily, respec-tively. Six months later, the patient is stable, with min-imal ascites and moderate reduction of the cholestasisand waiting for liver transplantation. Figure 3.  Immunohistochemical detection of MDR3 ( A  and  B  ) and MRP2 ( C   and  D  ) in the reported case with MDR3 mutation ( A  and  C  ) and incontrol liver ( B   and  D  ). 1040 LUCENA ET AL. GASTROENTEROLOGY Vol. 124, No. 4  Discussion Recent genetic and molecular studies have iden-ti fi ed genes associated with different cholestatic syn-dromes with clinical manifestations ranging from cho-lesterol lithiasis, PFIC, cholestasis of pregnancy, andbiliary cirrhosis. They have in common defects in bileformation and composition. 2,6,7,9,10,11,15,16 The MDR3 gene, also designated ABCB4, is localizedon chromosome 7q21, and its product is a multidrugresistance glycoprotein belonging to the family of ATP-binding cassette transporters. 17  Jacquemin 10 and de Vreeet al. 8 have reported the association of MDR3 de fi ciencyand PFIC3. This syndrome is characterized by high levelsof   -GT, clinical signs of cholestasis in the late infancy orchildhood, and eventually biliary cirrhosis before thethird decade of life. In these patients, the absence of PCin bile results in the production of highly detergent bilewith secondary injury of bile canaliculi and biliary epi-thelium, leading to cholangitis and cholestatic liverdisease, characterized histologically by ductular prolifer-ation, in fl ammatory in fi ltrate, and intrahepatic cholesta-sis. 1,10,12 MDR3 mutations were also found in mildercholestatic conditions such as ICP. 9,18 In these cases, ithas been proposed that female sex hormones could reducethe expression of the normal allele during pregnancy, 18 resulting in clinical cholestasis in the heterozygous sub-ject. In addition to PFIC3 and ICP, MDR3 de fi ciencymay cause cholesterol lithiasis as result of high lithoge-nicity of bile because of a low PC content. In thesepatients, cholesterol stones may appear early in life, bothin gallbladder and in intrahepatic bile ducts. 11,12 Thenormal serum cholesterol levels found in our patient wereconsistent with other PFIC patients reported 12 and op-posed to other forms of chronic cholestasis in which thenormal function of the canalicular secretion mechanismsare responsible for the formation of biliary/lipid vesiclesthat leak back into the plasma, leading to altered cho-lesterol homeostasis, including the presence of an abnor-mal lipoprotein fraction (lipoprotein X) and  fi nally hy-percholesterolemia. 19 The majority of patients with MDR3 de fi ciency de-scribed in the literature develop end-stage liver diseasebefore the third decade of life. Severity of cholestasis andtime of presentation depend on the homozygous/het-erozygous condition and the type of mutation. Non-sensemutations result in complete abolition of protein expres-sion, whereas missense mutations may cause limitedexpression with residual function of the transporter. 1,10,12 Thus, the immunohistochemical detection of MDR3 atthe canalicular membrane does not exclude the existenceof a defective MDR3 activity.To our knowledge, this is the  fi rst reported case of a patient developing consecutively the 3 different clin-ical manifestations associated with MDR3 de fi ciency,namely, juvenile cholesterol cholelithiasis, recurrent ICP,and biliary cirrhosis. Moreover, our patient is peculiar forthe very late presentation of biliary cirrhosis, which wasdiagnosed at the age of 47 years. In fact, the present caseis the oldest in the literature with biliary cirrhosis asso-ciated with MDR3 de fi ciency. The slow evolution of thecholestatic disease in our patient may be explained by herheterozygous condition and by the singularity of themutation in exon 14. Mutations of the MDR3 gene inexon 14 have been previously reported in 4 patients, 12 but all of them developed symptoms within the  fi rst yearof life with early liver transplantation being required in3 patients. The mutation observed in our patient affect-ing codon 535 has not been previously described eitherin the Human Gene Mutation Database or in previousclinical studies. The  fi nding of the same mutation in theaffected daughter with ICP is a strong evidence of theassociation of this mutation with the cholestatic disease.As indicated above, G535D is a missense mutation thatdoes not prevent the canalicular expression of the pro-tein, but it seems to be associated with a moderatereduction in the levels of the transporter. Immunohisto-chemical staining shows the presence of MDR3 at thecanalicular membrane but with an apparent spreading of the signal to basolateral membrane. These observationsare consistent with previous observations, which havedemonstrated abnormal cellular traf  fi cking of heterozy-gous missense mutations at exon 14. 9 In our case, itseems that the functional defect is suf  fi cient to causelithogenicity of bile, cholestasis of pregnancy, and biliarycirrhosis but relatively mild to allow clinical manifesta-tions of cirrhosis to develop late in life.Therapy with UDCA has been shown to be of bene fi tfor patients with MDR3 missense mutations because thiscompound up-regulates the expression of the glycopro-tein, minimizes the hepatotoxicity of endogenous bileacids, and increases the pool of hydrophilic bile acids,thus reducing the toxicity of bile salts. 10,12,20 Accord-ingly, it has been reported that the administration of UDCA could prevent the development of cirrhosis insome patients with PFIC3 12,20 and may be an alternativeto liver transplantation in certain cases. Moreover, itseems to be useful in the treatment of ICP and choles-terol gallstones due to MDR3 de fi ciency. 11,12,21,22 Sub-jects with non-sense mutations, however, will not re-spond to UDCA treatment, 8,12 and liver transplantationis the  fi rst option, although possibly in the future, cel- April 2003 MULTIDRUG RESISTANCE 3 GENE MUTATION 1041
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