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Cholestatic Liver Disease: Working Group Report of the First

World Congress of Pediatric Gastroenterology, Hepatology,

and Nutrition

*Frederick J. Suchy, Martin Burdelski, Balvir S. Tomar, *Ronald J. Sokol

From the *North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition; the European Society for

Paediatric Gastroenterology, Hepatology, and Nutrition; and the Asian Pan-Pacific Society of Pediatric Gastroenterology

and Nutrition.

Some of the diseases manifesting as cholestasis duringearly life represent true birth defects, such as cysts of thebiliary tree and congenital hepatic fibrosis. In othercases, immaturity of hepatic structure and function in-fluences how the liver reacts to injury from viruses and

drugs. Injury to the liver during critical periods of de-velopment may adversely affect its growth and capacityto perform vital functions, such as processing of nutri-ents, provision of energy, and excretion of wastesthrough biliary secretion (110).

Owing to an immaturity of hepatic function and theinitial presentation of inborn errors of metabolism andcongenital anomalies, there are more disorders manifest-ing as cholestasis in the neonate than at any other time oflife. The full spectrum of these disorders is beyond thescope of this report. We have focused on those disordersthat are most common or have the potential to yieldinformation of general importance to understanding liverdisease during early life.

SECTION 1: LIVER DEVELOPMENT

I. SUMMARY OF THE PROBLEM

Knowledge of the biochemical and molecular eventsthat affect liver development is critical to an understand-ing of how developmental defects of the liver and bileducts are likely to occur and why the infant is unusuallyprone to develop cholestasis during bacterial infection,intravenous feeding, and with malfunction of other or-gans such as the heart. Recent studies indicate that livercells and cholangiocytes may be derived from a commonprecursor or stem cell. Specific genes are expressed at

precise developmental stages and under the influence ofthe local environment, leading to a final differentiatedcell. The mechanisms underlying the activation or sup-pression of specific genes during this process have onlybeen partially defined. The differential gene expressionin these two types of epithelial cells must be better de-fined to understand how the liver and biliary tree developand how certain functions, particularly related to bile

formation, are restricted either to hepatocytes or cholan-giocytes.

Many disorders unique to childhood result in progres-sive injury and destruction of the intrahepatic and extra-hepatic bile ducts. Paucity of the intrahepatic bile ducts

and biliary atresia are several examples. The process ofthe morphogenesis of the intrahepatic and extrahepaticbiliary tree must be better defined to understand howthese disorders affect the liver of the infant and child. Itis known that the intrahepatic bile ducts are derived froma primitive streak of endodermal cells destined to be-come the liver. During embryonic development, theseprimordial cells express genes that are later found exclu-sively in the liver (hepatocytes) or bile duct cells. Thefactors that induce these hepatoblasts to develop intohepatocytes or cholangiocytes have not been completelydefined, but this knowledge is essential to the under-standing of pediatric disorders such as bile duct paucity,some cases of biliary atresia, and cystic diseases of the

biliary tree. The development of the extrahepatic bileducts and gall bladder is even more poorly understood.There is a stage in early development of the human em-bryo when the primitive gall bladder and common bileducts consist of a solid cord of cells without an openchannel. The process that controls remodeling of thesestructures to form open channels has not been defined. Itis likely that some forms of biliary atresia, particularlythose forms occurring with birth defects of the intestineand other organs, could be related to this developmentalprocess. Basic research must focus on the developmentalbiology of these structures.

The normal infant undergoes a period of physiologiccholestasis that is related to immature pathways for the

formation of bile. Owing to this immaturity of liver func-tion, the infant is more susceptible to developing chole-static liver disease during infection or with the adminis-tration of drugs or parenteral nutrition. Considerableprogress has been made in understanding the transportmechanisms that contribute to bile formation at the levelof the liver cell and cholangiocyte. However, much re-mains to be discovered as to how the specific transport-

Journal of Pediatric Gastroenterology and Nutrition35:S89S97 August 2002 Lippincott Williams & Wilkins, Inc., Philadelphia

S89 DOI: 10.1097/01.MPG.0000026996.95204.FD


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ers develop and are altered by disease in the infant. Thediscovery of several inherited defects in canalicularmembrane transporters has served to stress their potentialimportance to bile formation. Acquired dysfunction ofthese transporters may occur in other liver diseases, lead-ing to significant morbidity and even mortality. Howthese inherited defects progress to liver injury is onlypartially understood. Understanding these mechanismswill potentially provide therapeutic interventions to re-duce injury caused by cholestasis.

The intrahepatic and extrahepatic bile ducts are fre-quently involved in many inherited and acquired liverdiseases of childhood. There has been recent progress inour ability to study the functional properties of cholan-giocytes in the adult liver in isolated cell preparations,cultured cell lines, and isolated bile duct units. However,there is little understanding of how these cells functionduring development and contribute to the process of bileformation. We know little about the genes that are ex-pressed in cholangiocytes of the developing versus theadult liver.

Overall, this is an era of great opportunity in beingable to identify and define the molecular and biochemi-cal pathogenesis of many liver disorders affecting infantsand children. Prevention and treatment strategies tar-geted to this age-group are likely to be especially cost-effective. Basic insight into the development of liverstructure and function will be essential to our effort totreat many of the hepatic disorders affecting infants andchildren. A chronic liver disease prevented or success-fully cured in a child will also restore a healthy, produc-tive citizen to the workforce for many decades.

II. MAJOR ISSUES IN NEED OF

INVESTIGATION OR IMPLEMENTATION

What are the array of genes that are activated orsuppressed during development leading to the fullydifferentiated hepatocyte and cholangiocyte? Thefully differentiated hepatocyte and cholangiocyte arederived from a common precursor stem cell. An arrayof genes, many of which are transcription factors, isactivated or suppressed during development leading tothe mature cell. The process is poorly understood andrequires intensive study to define how the process oc-curs during normal development.

What are the molecular mechanisms underlying

the structural development of the liver and biliarytract and what is their relationship to birth defects,such as some forms of biliary atresia and fibrocys-tic diseases of the liver? The liver in the embryo isderived from a primitive streak of endodermal cells inthe foregut that form hepatocytes and bile ducts. Themolecular mechanisms underlying the formation of theliver are only partially understood. In particular, thefactors leading to the formation and remodeling of the

so-called ductal plate to produce the intrahepatic bili-ary tree should be defined, including genes that deter-mine cell fate, proliferation, and programmed celldeath. We now know that the gene responsible forautosomal dominant polycystic kidney disease (poly-cystin) is expressed in liver and likely contributes tothe cystic abnormalities of the biliary tree. The role ofthis gene product in the morphogenesis of bile ductshas not been defined. It is also unknown how the genedefective in Alagille syndrome, the JAGGED 1 gene,leads to loss of interlobular bile ducts and small size ofthe extrahepatic bile ducts.

How do the liver transport mechanisms that con-tribute to bile formation develop and how aretransporters for organic and inorganic solutes af-fected by cholestasis? Cholestasis occurs more fre-quently and earlier in the course of liver disease in theinfant than at any other time of life. It has recentlybeen established that inherited defects in several aden-osine triphosphate (ATP)-dependent transporters lo-calized to bile canalicular membrane, including trans-porters for bile acids and phospholipids, lead to pro-gressive cholestatic liver disease. Little is known abouthow these transport mechanisms are regulated and de-velop in the fetus and neonate. Moreover, how thesetransporters function in acquired cholestasis due tobile duct obstruction and intrahepatic disease has notbeen well defined.

Do the functional properties of cholangiocytes andtheir contribution to bile formation differ duringearly life? The bile ducts are a frequent site of injuryin pediatric liver disease. However, there is a lack ofknowledge about the functional properties of bile ductcells or cholangiocytes during development. Recent

studies have shown the ability to isolate and culturecholangiocytes and small segments of the biliary tree(isolated bile duct units.) However, such research hasnot been done in developing animals. Determination ofthe genes that are specifically expressed in developingversus mature cholangiocytes will be essential to un-derstand cholangiocyte biology.

III. PROPOSED PLAN TO ACHIEVE GOALS

Define the array of genes that are activated or sup-pressed during development leading to the fully dif-ferentiated hepatocyte and cholangiocyte. There is a

major gap in our knowledge of the factors regulatingthe formation the liver and intrahepatic bile ducts. Thefactors leading to the formation and remodeling of theso-called ductal plate to produce the intrahepatic bili-ary tree should be defined, including genes that deter-mine cell fate, proliferation, and programmed celldeath. Future research should focus on defining factorscontrolling the differentiation of hepatoblasts intocholangiocytes and hepatocytes, the signals that in-

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duce proliferation to form the bilayered ductal plateand remodeling to form the ductal lumen. The role ofthe embryonic microenvironment in hepatogenesismust be better understood. Further study of the com-plex hierarchical control of liver development at thetranscriptional level is another priority.

Understand the molecular mechanisms underlyingthe structural development of the liver and biliarytract and their relationship to birth defects such assome forms of biliary atresia and fibrocystic dis-eases of the liver. It is now know that the gene re-sponsible for autosomal dominant polycystic kidneydisease (polycystin) is expressed in liver and likelycontributes to the cystic abnormalities of the biliarytree. The role of this gene product in the morphogen-esis of bile ducts has not been defined. It is also un-known how the gene defective in Alagille syndrome,the JAG1 gene, leads to loss of interlobular bile ductsand small size of the extrahepatic bile ducts. It ishighly expressed is the developing biliary tree but its

role in hepatic development has not been determined.Whether genes involved in situs determination, such asthe ivn gene and which are associated with biliary tractanomalies in knock-out animals, are involved in somecases of biliary atresia must be defined.

Determine the normal development of liver trans-port mechanisms that contribute to bile formationand how transporters for organic and inorganicsolutes are affected by cholestasis. Research isneeded to define how the function of hepatic trans-porters contributing to bile formation is altered in ac-quired cholestasis due to bile duct obstruction and in-trahepatic disease. It is feasible to study the propertiesof these transport systems in animal models as well as

in the human. Overexpression of transport proteins intransgenic animals and targeted deletion studies willhelp to define the importance of each system to theprocess of bile formation and will significantly con-tribute to our understanding of the pathophysiology ofcholestasis.

Define the functional properties of cholangiocytesand their contribution to bile formation duringearly life. It is critically important to determine thefunctional properties of cholangiocytes from develop-ing liver, including the localization and expression oftransporters and whether, like the adult biliary system,there is functional heterogeneity between large and

small cholangiocytes. It is also unknown how thesecells respond to hormonal agonists such as secretin.Determination of the genes that are specifically ex-pressed in developing vs. mature cholangiocytes willbe essential to understand cholangiocytes biologythrough the use of DNA microarray chip technology.Strategies to target genes specifically to the biliary treewill be important in developing gene replacement forbiliary disorders such as cystic fibrosis.

SPECIFIC DISORDERS

Biliary Atresia


Biliary atresia is a disorder characterized by a fibro-sclerosing obliteration of the extrahepatic bile ducts thatuniquely presents in the first months of life. The condi-tion occurs in approximately 1 in 8,000 to 1 in 15,000live births, and accounts for approximately one third ofall cases of cholestasis in the young infant. Biliary atresiais the most frequent cause of chronic end-stage liverdisease in children and contributes over half of all pedi-atric patients for liver transplantation. Two forms of thisdisease have been recognized. In an embryonic type,which occurs in about 10% to 15% of cases, cholestasisis present at birth, often in association with other extra-hepatic anomalies including polysplenia, portal veinanomalies, malrotation, abdominal situs inversus, andcongenital heart disease. In this form, the common bileduct is often absent on surgical exploration. In the ap-proximately 80% to 90% of infants with the perinataltype biliary atresia, jaundice and acholic stools developwithin the first weeks of life. Other anomalies occur lessfrequently. In this subtype, complete obstruction to bileflow develops as a result of an idiopathic progressivesclerosing fibro-obliteration of the extrahepatic bile duct.

The initial treatment of biliary atresia is surgical, in-volving resection of the obliterated extrahepatic bile ductand the creation of a hepatoportoenterostomy. This op-eration or Kasai procedure should be performed before 2months of age to successfully reestablish bile flow. Inspite of timely diagnosis and surgery, most cases (70% to80%) will eventually progress to develop end-stage bil-iary cirrhosis and require liver transplantation. Delayeddisease recognition and referral still remains a majorproblem in the successful management. The annual costfor this disease has been estimated to be $65 million inthe United States.

The cause and pathogenesis of either the embryonic orperinatal type of biliary atresia are not known. Genetic,infectious and host immune factors are putative etio-pathogenic mechanisms of disease. There is also an im-mediate need to institute more formalized educationalprograms in the medical and lay communities to allowfor earlier recognition of this condition. Research has

been hampered by the lack of suitable animal models.Furthermore, the paucity of cases at any one center haslimited both the availability of sufficient human tissuesamples to study pathogenic mechanisms, and the devel-opment of amply sized clinical trials to test novel treat-ment strategies. Research efforts should be directed to-ward defining the disease cause and pathogenesis, devel-oping methods of early detection, and for creating moreeffective therapeutic interventions.

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What is the contribution of genetic factors to thepathogenesis of biliary atresia? Although there areseveral reports of families in which male and femalesiblings have the disease, HLA-identical twins discor-dant for the condition have been described. Generally,biliary atresia is not considered to be an inherited dis-ease. However, with the less common embryonic typeof biliary atresia, in which other congenital malforma-tions occur, genetic mutations resulting in defectivemorphogenesis may be important to the disease patho-genesis. For example, it is unknown whether there is ahuman correlate of the ivn mouse, a transgenic animalin which a recessive deletion of the inversin gene re-sults in situs inversus and jaundice due to lack of con-tinuity between the extrahepatic biliary tree and thesmall intestine. It is likely that the two forms have adifferent genetic predisposition and etiopathogenesis.

Does an infectious agent cause biliary atresia? Thefibrosclerosing process present in most cases of biliaryatresia has long suggested the possibility that an in-fectious agent with a trophism for the biliary tract maybe causal. There have been a number of contradictorystudies focusing on a possible role for Reovirus type 3in biliary atresia. The most recent detected reovirustype 3 RNA in cases of choledochal cysts as well inbiliary aresia. There has also been evidence for andagainst the possibility that strains of Rotavirus may beinvolved. Sporadic cases of CMV or EBV infection inbiliary atresia have also been reported. No specificvirus has been definitively identified as the etiologiccause for either subtype of biliary atresia.

What is the contribution of host immune factors?Host immune factors likely have a role in the patho-genesis of biliary atresia. With the perinatal type bil-iary atresia, a significant increase in the HLA B12antigen has been noted, suggesting a role for host im-munogenic factors in the disease progression. Aberrantexpression of the MHC class I molecules, ICAM-1,but not MHC class II determinants has been found inintrahepatic bile ductular epithelial cells on analysis ofliver biopsy tissue samples from patients with biliaryatresia. An increased surface expression of HLA DRantigen in addition to ICAM-1 on intrahepatic bileductular epithelial cells has been found in liver biopsysamples from patients with biliary atresia. It remains

uncertain whether these changes are importance in me-diating damage to the biliary tract or reflect a second-ary effect of tissue injury.

What are the remaining diagnostic challenges? Be-cause the physical and biochemical findings may besubtle or ambiguous in cholestatic infants, more sen-sitive, specific, and less invasive methods for earlydiagnosis of biliary atresia are needed. Such testswould help to predict which patients may not benefit

from an initial hepatoportoenterostomy. The most re-liable test for the diagnosis of biliary atresia, asidefrom exploratory laparotomy, is a percutaneous liverbiopsy. A specimen containing five to seven portaltracts is over 95% sensitive in indicating large bileduct obstruction. Although endoscopic retrogradecholangiopancreatography (ERCP) may be of diagnos-tic value in selected cases, the procedure is new to thisage-group, invasive, technically challenging in younginfants, and not readily available. MR cholangiogra-phy shows promise as an approach to image the biliarytract of the neonate. There are no serologic tests orimaging studies that are diagnostic for the condition.The economic and medical benefits of screening allnewborn infants for elevated serum direct bilirubin orbile acids remain uncertain.

Can the results of the portoenterostomy operationbe improved? The Kasai procedure remains the cor-nerstone of therapy, but better indices to predict out-come are needed. Correlation of biochemical and his-tologic features may identify patients unlikely tobeneft from portoenterostomy. For example, the pres-ence of syncytial giant cells, lobular inflammation, fo-cal necrosis, bridging necrosis, and cholangitis hasbeen associated with failure of the portoenterostomy,whereas bile in zone 1 has been associated with clini-cal success. The care of the patient postoperativelypresents many challenges. Repeated episodes of bac-terial cholangitis after portoenterostomy surgery areassociated with progressive fibrosis of the intrahepaticbiliary tree and a worse prognosis. It is not resolvedwhether anti-inflammatory drugs, prophylactic antibi-otics, or choleretics given individually or in combina-tion affect the outcome. Other novel treatment strate-

gies coupled with the Kasai operation need to beevaluated. These would include an evaluation of theefficacy of antivirals, anticytokines, antifibrogenesisagents, and cytoprotective agents for intrahepatic bileducts.


The specific research areas and goals are:

Identify the genetic factors involved in the patho-genesis of biliary atresia: To identify and define thegenetic factors that may be responsible for diseasepathogenesis of either the embryonic or perinatal type

biliary atresia. A search for mutations of human ho-mologues of murine genes determining the laterality inembryonic biliary atresia needs to be performed.

Determine if viral or other infectious agents causebiliary atresia: Although a specific viral infection hasnot yet been identified as the cause of biliary atresia,continued research in this area is required. Use of ani-mal models to delineate the mechanisms of virallyinduced bile duct injury is necessary. Careful collec-

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tion of sera and tissues from multiple centers in astandardized fashion for viral studies is essential.Thorough epidemiologic and virologic evaluation oftemporal or geographic clustering of cases should behigh priority. Animal models of virally induced biliaryatresia, including the rotavirus A and reovirus 3 mod-

els, need further study to help characterize diseasepathogenesis. Define the role of host immune factors in biliary

atresia: The possibility that immunologic processesleading to the fibrosclerosing bile duct injury in biliaryatresia requires further study. Possible maternal im-mune factors need to be explored. The role of theimmune system in the mediation of the progressivefibrosclerosing lesion of the extrahepatic bile duct inbiliary atresia should be defined. Studies identifyingthe phenotype of the extrahepatic bile duct cellularinfiltrate and cytokines and the determinants of im-mune recognition of bile duct epithelial cells should bea priority. A genetic predilection in MHC classes I, II,

or III needs to be sought. Parallel studies should beconducted in vivo by using existing animal models ofeither virally or immunologically induced bile ductinjury. In vitro studies using human or animal bile ductepithelia in cell culture should be employed to specifi-cally characterize mechanisms of epithelial cellimmunocyte interaction. The cellular and molecularevents that mediate bile duct fibrosis during infancyneed to be better elucidated. How hepatic stellate cellsare activated to produce collagen in the developingliver and the role of the cholangiocyte in triggeringthis fibrotic process require investigation.

Develop better diagnostic and prognostic diseaseindicators: Clinical data should be collected in stan-

dardized fashion from multiple centers. To this end, itis critical to establish a central registry for patientswith biliary atresia to collect epidemiologic data andgenerate outcome statistics. Multicenter clinical thera-peutic trials and repositories for human sera and tis-sues from affected patients should be a part of thiseffort. There is a need to develop better diagnosticmarkers for the disease to identify patients early ininfancy who are most likely to benefit from the por-toenterostomy. More precise predictors of outcome forthe various treatment options are required. A costbenefit analysis of universal postnatal screening of se-rum direct bilirubin or bile acid levels should be un-

dertaken to examine its effect on outcome. Improve the results of the portoenterostomy opera-tion and treatment of postoperative complications:Novel treatment strategies to reduce destruction of in-trahepatic bile ducts, ongoing intrahepatic injury, andprogressive fibrosis need to be developed to improvethe outcome after portoenterostomy. These therapiesshould reduce, delay, or prevent ongoing intrahepaticbile duct destruction and fibrosis. The role of multiple

factors alone or in combination including toxic bileacids, oxidant stress, ATP depletion, endotoxins andbacterial cell wall antigens, activation or recruitmentof inflammatory mediators, activation of degradativehydrolases, and mitochondrial damage needs to be ex-amined. Because ascending bacterial cholangitis is amajor cause of morbidity in this patients, role of bileacids and bacterial cell wall products, and their inter-actions with hepatocytes, Kupffer cells, and stellatecells, in the mediation of the progressive liver diseasemust be assessed. Any intervention in infants withbiliary atresia will require a multicenter effort to re-cruit adequate numbers of patients.

Intrahepatic Disorders


Intrahepatic cholestasis includes a heterogenous sub-set of cholestatic diseases that, with or without bile ductalterations (paucity), represent many specific syndromeswith differing pathogenesis, causes, and outcome. Thereis also a high degree of variability in both their presen-tation and prognosis. Progressive, familial forms such asBylers disease are often fatal. In contrast, in patientswith Alagille syndrome or syndromic paucity of inter-lobular bile ducts, the prognosis is much more favorable.Owing in part to a poor understanding of the underlyingpathophysiology, the current nomenclature system forthe various forms of intrahepatic cholestasis is imperfect.

Alagille Syndrome (Arteriohepatic Dysplasiaor Syndromic Paucity of the Intrahepatic BileDucts) Alagille sydrome (AGS) is characterized by cho-lestasis, a decreased number of interlobular ducts, andvarious congenital malformations (e.g., dysmorphic fa-cies, peripheral pulmonic stenosis, vertebral arch defects,renal disease, posterior embryotoxon). Serum levels ofalkaline phosphatase, -GTP, and bile acids are high,indicative of a defective biliary excretion. The mecha-nism leading to progressive loss of interlobular bile ductsand cholestasis is not known. Therapy directed againstthe complications of severe choestasispruritus, cutane-ous xanthoma, and growth failureis often ineffective.Liver transplantation may be required for these compli-cations as well as for progressive liver disease.

AGS is inherited in autosomal dominant fashion and isone of the more common genetic causes of cholestasis in

infancy, with an estimated frequency of 1:70,000 livebirths. The site of the gene responsible for AGS was firstsuggested by the identification of visible gene deletionson the long arm of chromosome 20. Subsequently, in1997 two groups identified mutations in the gene encod-ing Jagged1, one of the ligands for Notch signaling path-way. Well over 200 different mutations have now beendetected in these patients. Approximately 56% to 70% ofmutations are sporadic. Haploinsufficiency, a decrease in




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the amount of the normal protein, appears to be themechanism causing Alagille syndrome. AlthoughJagged1 is expressed in many tissues affected in Alagillesyndrome including the developing and adult biliary tree,it is unknown how mutations in Jagged1 lead the bileduct paucity and liver disease. No phenotypic differenceshave been identified based on type or location of muta-tion. Because there is extreme variability of AGS phe-notype within families, other genetic or environmentalfactors likely contribute to the clinical manifestations ofthe disease.

Nonsyndromic Paucity of Interlobular Bile Ducts Pau-city of interlobular bile ducts can also occur without theclassic features of the Alagille syndrome. Liver biopsyshows an absence or reduction of interlobular bile ducts.The neonate is either more susceptible to damage andloss of bile ducts because of the biology of these struc-tures during early life or are more likely to be affectedwith infections with a tropism for the biliary tree. Bileduct loss can occur in a diverse array of disorders such ascytomegalovirus infection and 1-antitypsin deficiency.The prognosis in nonsyndromic paucity is thought to beworse than in Alagille syndrome. The mechanisms un-derlying bile duct injury and loss have not been welldefined.

Progressive Familial Intrahepatic Cholestasis(PFIC) PFIC represent a group of diseases involvingmembrane transport proteins thought to be involved inbile formation. Onset is typically in the first 6 months oflife with cholestasis, hepatomegaly, severe pruritus,growth failure, and fat-soluble vitamin deficiency. Pro-gressive familial intrahepatic cholestasis type 1 (PFIC1)or Bylers disease is characterized by chronic, unremit-ting cholestasis that develops early in life. Jaundice, se-vere pruritus, and growth failure are typical features in asetting of a low serum -glutamyl transpeptidase andcholesterol levels. The disorder is inherited as an auto-somal recessive trait. The gene for the disorder, FIC1,mapped to the same locus as benign recurrent intrahe-patic cholestasis (chromosome 18q21-q22). FIC1 hasbeen cloned and encodes for a P-type ATPase that mayfunction as an ATP-dependent aminophospholipid flip-pase. FIC1 is highly expressed in intestine and cholan-giocytes and at a lower level in hepatocytes. The functionof FIC1 has not been defined or how defects in the pro-tein lead to progressive liver disease. Progression to cir-rhosis and liver failure usually occurs by 3 to 4 years ofage. Partial external biliary diversion has been used suc-

cessfully to treat intractable pruritus in these patients; insome cases the progressive cholestasis also improves af-ter the procedure. It is unknown why biliary diversion isbeneficial in this disorder. Orthotopic liver transplanta-tion is required in patients with decompensated cirrhosis.Diarrhea often continues after transplantation.

A second locus for PFIC was mapped to chromosome2q24. Patients with this variant (PFIC 2) present withsevere cholestasis in the neonatal period with a normal

serum -glutamyl transpeptidase concentration. Liverhistology initially shows giant cell hepatitis; there israpid progression to cirrhosis. Mutations in the ATP-dependent canalicular bile salt excretory pump (BSEP)have been found in these patients, consistent with thephenotype of decreased canalicular excretion of bile ac-ids described in this form of PFIC.

Another subtype of progressive familial intrahepaticcholestasis (PFIC 3) has been identified in which patientshave high serum -glutamyltranspeptidase levels. Thedisorder shares histologic, biochemical, and genetic fea-tures with mice in which the mdr2 gene has been inac-tivated (mdr2 / mice). Mdr2 and the human homologMDR3 encode a phosphatidylcholine flippase located onthe bile canalicular membrane, which mediates biliaryphospolipid excretion. Patients with PFIC3 have lowconcentrations of phospholipids in bile and develop se-vere liver disease characterized by inflammation of por-tal tracts, bile ductular proliferation, and fibrosis. In theabsence of biliary phospolipids, the biliary tree is subjectto progressive injury from hydrophobic bile salts. Theabsence of immunohistochemical canalicular staining forMDR3 (human mdr2 homolog) has been found in livertissue from affected patients. Several mutations in theMDR3 gene have been demonstrated on analysis of ge-nomic DNA.



What are the mechanisms underlying bile ductpaucity and why is the infant more susceptible tobile duct injury? The mechansims causing bile ductinjury and progressive loss leading to paucity must bebetter understood. Infectious, immunologic, and toxicfactors are likely to be operative. The segmental de-structive changes or progressive decrease in the num-ber of bile ducts on serial sectioning of biopsy speci-mens from the early features of bile duct inflammationto the later observation of paucity, suggests immuno-logic injury to existing ductssimilar to other syn-dromes of disappearing intrahepatic bile ducts in theposttransplantation settingrather than from a failureof ducts to develop. It must be defined whether ductalinjury occurs via necrosis of cholangiocytes or throughprogrammed cell death (apoptosis). The factors regu-lating these processes should be defined.

How do Jag1 mutations lead to the characteristicgroup of anomalies and bile duct paucity? The iden-tification of mutations in Jag1 gene as the cause ofmost cases of Alagille syndrome was one of the mostimportant advances in the decade of the 1990s. How-ever, in spite of its potential intriguing role in tissuegrowth and differentiation, it is not known how muta-tions in this gene lead to the characteristic array ofbirth defects and propensity for progressive loss of

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interlobular bile ducts. There are also approximately30% of Alagille patients who do not harbor a mutationin Jag1. Whether defects in other components of theNotch signaling pathway could be responsible forthese cases remains unknown. Insight into the patho-physiology of Alagille syndrome could be advanced if

a knockout animal model of the disorder could bedeveloped. How do the defective genes in various forms of pro-

gressive familial intrahepatic cholestasis (PFIC)cause liver disease, and do alterations in these pro-teins contribute to acquired cholestatic liver dis-ease? Progressive familial intrahepatic cholestasis(PFIC) occurs in at least three forms caused by muta-tions in ATP-dependent transport proteins. The patho-physiology of PFIC 2 appears to be straightforwardbecause defects in the canalicular membrane BSEPlead to a failure of bile salt-dependent bile secretionand retention of bile salts and other potentially noxiouscompounds within hepatocytes. In contrast, the func-

tion of the abnormal gene in Byler disease, so-calledFIC1, has not been defined. Its localization within theliver has not even been definitively resolved. Expres-sion is highest in the intestine. FIC1 is also present incholangiocytes, albeit at levels less than the gut, butinitially was not detected in hepatocytes. Preliminarystudies now suggest some expression in hepatocytes.How mutations in FIC1 lead to progressive cholestasisis unknown. In the case of PFIC3 the absence of acanalicular membrane flippase leads to failure of bil-iary phospholipid secretion. Damage to the canalicularmembrane and to bile duct epithelia is thought to occurthrough the detergent effect of hydrophobic bile salts.Because inherited defects in these disorders lead to

cholestasis, it will be important to determine how thesetransporters are altered during acquired cholestasis asoccurs after bile duct obstruction or exposure to endo-toxin.

Can management strategies be developed for com-plications of cholestasis (e.g., pruritus, growth fail-ure, osteoporosis, and hypercholesterolemia) in pa-tients with Alagille syndrome and PFIC to improvequality of life and possibly improve survival with-out liver transplantation? Intrahepatic cholestaticdisorders are associated with malabsorption of knownfat-soluble nutrients, contributing to poor growth andspecific micronutrient deficiencies. Secondary accu-

mulations of copper and manganese have also beenassociated with possible increased hepatic injury andlesions in the basal ganglia. Pruritus is a major factorin these disorders and appears related to central opioidpathways. Although partial biliary diversion is of ben-efit, other novel strategies to reduce pruritus (e.g., opi-oid antagonists) need to be developed. Metabolic bonedisease and osteoporosis may be severe despite ad-equate vitamin D and calcium intake, and are poorly

understood. The consequences of hypercholesterol-emia of Alagille syndrome have not been well defined;autopsy studies suggest increased arterial wall choles-terol plaques; however, atherosclerotic vascular symp-toms have not been reported. Finally, improved cyto-protective and choleretic agents, to reduce the effectsof toxic, hydrophobic bile acids, will potentially im-prove hepatocyte function and bile flow, and ulti-mately retard the progression to cirrhosis and liverfailure. Ultimately, gene replacement or modulatorytherapy could correct the underlying molecular andbiochemical defects that underlie these disorders. Thiswould require targeting of vectors to the hepatocyte orcholangioctye.


The specific research areas and goals are:

Understand the pathogenesis of bile duct paucity inboth syndromic and nonsyndromic forms. Paucitysyndromes may have a direct developmental basis insome cases, representing congenital absence or failureto form or may ensue through progressive injury (im-mune, viral, ischemic,or toxic) and disappearance. Un-derstanding cholangiocyte-specific gene expression inthe adult and developing animal as well under patho-logic conditions leading to paucity will be critical tounderstanding the biology and pathobiology of the bil-iary tract. Subtraction hybridization methods as well asnewer microarray chip technology will be particularlyinformative in this effort. It is also uncertain whetherbile ducts disappear via cell necrosis or apoptosis. Abetter understanding of the mechanisms of cholangio-cyte cell death could result in strategies for therapy.Immune, toxic, and infectious factors should be con-sidered as possible causal agents. Careful study of bil-iary epithelial cell physiology during developmentshould be a priority. The role of growth factors, cyto-kines, and other effectors of the immune system inmaintaining ductal integrity and in mediating cell in-jury should be defined. Our understanding of thesedisorders would be greatly enhanced with develop-ment of isolated cholangiocyte models and relevantanimal models.

Undertand how Jag1 mutations lead to the charac-teristic group of anomalies and bile duct paucity inAlagille sydrome. Alagille syndrome exhibits a

highly variable and complex phenotype despite its au-tosomal dominant inheritance. Many sibs and parentsof probands are often found to be minimally affected,with only one or two abnormalities. The frequency ofnew mutations appears to be high (15% to 50%). Closeto 200 different mutations have been found so far,making routine genetic analysis a challenge. Approxi-mately one third of patients do not have a mutation inJag1, indicating that genetic abnormalities, possibly




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involving other components of the Notch signalingpathway, may cause some cases of Alagille syndrome.Further characterization of the responsible gene(s) andmolecular testing must be carried out to better under-stand the pathophysiology and to precisely diagnoseAlagille syndrome. These studies are essential in

avoiding unnecessary surgery in the cholestatic infant,in properly initiating therapy, and providing accurategenetic counseling.

Members of the Notch gene family that encode evo-lutionarily conserved transmembrane receptors are in-volved in cell fate specification during embryonic de-velopment. The Notch locus encodes a receptor medi-ating cellcell interactions. Future studies must focuson the Notch signaling pathway, which appears to de-fine a fundamental mechanism controlling cell fate inearly development, perhaps controlling the ability ofnonterminally differentiated cells to respond todifferentiation/proliferation signals. Although it islogical to envisage how the broad spectrum of organ

involvement in Alagille syndrome could be caused byJAG1 mutations, the molecular mechanisms underly-ing the pathogenesis of the disorder remain undefined.Research should also focus on how this genetic abnor-mality leads to a paucity of interlobular bile ductsthrough a process involving progressive injury andloss of ducts. An animal model of the Alagille syn-drome made by targeted disruption of Jag1 gene wouldbe of considerable value in such an effort. It shouldalso be feasible to selectively turn off expression ofJag1 in the liver at various times postnatally throughuse of the Cre/Lox technology to assess effects on thebiliary tree.

Define how the genes involved in various forms of

progressive familial intrahepatic cholestasis (PFIC)cause liver disease and are altered in acquired cho-lestatic liver disease. The genes for three forms ofprogressive familial intrahepatic cholestasis have beencloned, but the pathophysiology underlying these dis-orders is not well understood. PFIC1 is caused bymutations in a P-type ATPase now called FIC1. Thefunction of the polypeptide encoded by this gene isunknown, but proteins of the same family function asaminophospholipid flippases in transporting amino-phospholipids from the outer to the inner leaflet ofplasma membranes. In this capacity, these transportersserve to maintain the structure of plasma membranes

and thereby contribute to their role as a semipermeablebarrier. FIC1 is highly expressed in the intestine andpancreas and to a lesser extent in cholangiocytes. It hasbeen controversial as to whether the gene is expressedin hepatocytes but recent preliminary studies suggestthe possibility that the transporter may be present onthe canalicular membrane. How defects in this trans-porter lead to progressive cholestatic liver disease re-mains unknown. It is also of interest that the same

gene is mutated in benign, recurrent, intrahepatic cho-lestasis. Much additional work must be done to deter-mine how specific mutations in one case lead to pro-gressive liver disease and in the other are associatedwith a relatively benign disorder. Research will berequired to precisely define the cell types and mem-

brane domains on which FIC1 is expressed. It is alsoessential that the precise function of this protein bedetermined to understand how defects in the proteinlead to cholestasis. PFIC1 is a disorder in which liverdisease may be cured by liver transplantation, but pa-tients may continue to have difficulties with diarrheaand malabsorption after transplantation. The expres-sion of the abnormal gene product in intestine andpancreas may explain the continued symptomatologyin these patients. Creation of transgenic mice in whichFIC1 is over expressed or disrupted by gene targetingwould also be useful in understanding the role of thistransport protein in health and human disease.

The mechanisms responsible for liver disease in

PFIC2 appear to be more straightforward. Mutations inthe gene encoding the canalicular bile salt excretorypump (BSEP) have been found. This protein is a mem-ber of the ATP-binding cassette (ABC) family oftransporters. It appears to be the predominant trans-porter responsible for concentrative excretion of bilesalts into the canalicular lumen. Patients with muta-tions in BSEP have markedly impaired secretions ofbile salts into bile and a failure of bile saltdependentbile secretion. Additional studies on BSEP are re-quired to determine its mechanism of regulation dur-ing normal development and during disease. It wouldalso be of value to study the phenotype and biliaryphysiology of animals in which BSEP has been dis-

rupted by gene targeting.PFIC3 is caused by mutations in the MDR3 gene,

which is responsible for phospholipid secretion intobile. This member of the ABC family of ATP-dependent transporters mediates transfer of phospho-lipids from the inner to the outer leaflet of the cana-licular plasma membrane, where, under the detergentaction of bile salts, phospholipids are incorporated intomixed micelles. Knockout mice lacking MDR2, thehomologue of human MDR3, are also unable to se-crete phospholipids into bile and develop a progressiveliver disease, which is characterized by bile ductularproliferation and progressive biliary cirrhosis. Further

studies of these valuable models as well as the humandisease will be required to develop effective treatmentstrategies.

Efforts should be directed to develop methods toestablish a precise genetic diagnosis for each one ofthese disorders. In each case, different therapies, suchas biliary diversion or replacement with the hydro-philic bile salt ursodeoxycholic acid, may be benefi-cial. It will also be of importance to precisely define

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the specific type of PFIC so as to better predict prob-lems that may continue after liver transplantation.These disorders will eventually be amenable to treat-ment with gene therapy.

It is clear that other types of progressive familialintrahepatic cholestasis exist beyond the forms so fardescribed. Whether disorders such as that occurring inGreenland Eskimos or in hereditary cholestasis withlymphedema involve any of the genes so far describedfor PFIC1, 2, or 3 is unknown. It will be of greatimportance to understand the genetic basis of thesedisorders, which should contribute significantly to ourunderstanding of these diseases as well as normalhepatobiliary physiology.

Develop treatment strategies for complications ofcholestasis. Studies need to be performed to define thecellular and biochemical cause of growth failure inchildren with cholestasis and improved nutritionaltherapies developed to counteract malabsorption, es-sential fatty acid deficiency, and fat-soluble vitamindeficiencies. The mechanisms for pruritus of cholesta-sis need to be better defined and more effective treat-ment strategies developed. Use of central opioid re-ceptor antagonists and partial biliary disease need tobe carefully studied in this regard. The cause of themetabolic bone disease of cholestasis that may lead torecurrent fractures needs to be characterized. Thisshould lead to more effective preventive therapies and

a better understanding of bone metabolism during liverdisease. Areas such as magnesium status, effect ofcirculating cytokines on bone growth, and hormonalstatus should be explored.

Acknowledgment: The input of Tomoo Fujisawa to thisreport is gratefully acknowledged.

References

1. Zaret K. Early liver differentiation: genetic potentiation and mul-tilevel growth control. Curr Opin Genet Dev 1998;5:52631.

2. Bezerra JA.: Liver development: a paradigm for hepatobiliary dis-ease in later life. Semin Liver Dis 1998;18(3):20316.

3. Jacquemin E, Hadchouel M. Genetic basis of progressive familialintrahepatic cholestasis. J Hepatol 1999;31(2):37781.

4. Knisely AS. Progressive familial intrahepatic cholestasis: a per-sonal perspective. Pediatr Dev Pathol 2000;3(2):11325.

5. Birnbaum A, Suchy FJ. The intrahepatic cholangiopathies. SeminLiver Dis 1998;18(3):2639.

6. Arrese M, Ananthananarayanan M, Suchy FJ. Hepatobiliary trans-port: molecular mechanisms of development and cholestasis. Pe-diatr Res 1998;44(2):1417.

7. Bates MD, Bucuvalas JC, Alonso MH, et al. Biliary atresia: patho-genesis and treatment. Semin Liver Dis 1998;18(3):28193.

8. Middlesworth W, Altman RP: Biliary atresia. Curr Opin Pediatr1997;(3):2659.

9. Howard ER, Davenport M. The treatment of biliary atresia inEurope 1969-1995. Tohoku J Exp Med 1997;181(1):7583.

10. Sokol RJ. Fat-soluble vitamins and their importance in patientswith cholestatic liver diseases. Gastroenterol Clin North Am1994;(4):673-705.



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