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Journal of Pediatric Gastroenterology & Nutrition:
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Cholestatic Liver Disease: Working Group Report of the First World Congress of Pediatric Gastroenterology, Hepatology, and Nutrition

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

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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 during early life represent true birth defects, such as cysts of the biliary tree and congenital hepatic fibrosis. In other cases, immaturity of hepatic structure and function influences how the liver reacts to injury from viruses and drugs. Injury to the liver during critical periods of development may adversely affect its growth and capacity to perform vital functions, such as processing of nutrients, provision of energy, and excretion of wastes through biliary secretion (1–10).

Owing to an immaturity of hepatic function and the initial presentation of inborn errors of metabolism and congenital anomalies, there are more disorders manifesting as cholestasis in the neonate than at any other time of life. The full spectrum of these disorders is beyond the scope of this report. We have focused on those disorders that are most common or have the potential to yield information of general importance to understanding liver disease during early life.

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SECTION 1: LIVER DEVELOPMENT

I. SUMMARY OF THE PROBLEM

Knowledge of the biochemical and molecular events that affect liver development is critical to an understanding of how developmental defects of the liver and bile ducts are likely to occur and why the infant is unusually prone to develop cholestasis during bacterial infection, intravenous feeding, and with malfunction of other organs such as the heart. Recent studies indicate that liver cells and cholangiocytes may be derived from a common precursor or stem cell. Specific genes are expressed at precise developmental stages and under the influence of the local environment, leading to a final differentiated cell. The mechanisms underlying the activation or suppression of specific genes during this process have only been partially defined. The differential gene expression in these two types of epithelial cells must be better defined to understand how the liver and biliary tree develop and how certain functions, particularly related to bile formation, are restricted either to hepatocytes or cholangiocytes.

Many disorders unique to childhood result in progressive injury and destruction of the intrahepatic and extrahepatic bile ducts. Paucity of the intrahepatic bile ducts and biliary atresia are several examples. The process of the morphogenesis of the intrahepatic and extrahepatic biliary tree must be better defined to understand how these disorders affect the liver of the infant and child. It is known that the intrahepatic bile ducts are derived from a primitive streak of endodermal cells destined to become the liver. During embryonic development, these primordial cells express genes that are later found exclusively in the liver (hepatocytes) or bile duct cells. The factors that induce these hepatoblasts to develop into hepatocytes or cholangiocytes have not been completely defined, but this knowledge is essential to the understanding 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 bile ducts and gall bladder is even more poorly understood. There is a stage in early development of the human embryo when the primitive gall bladder and common bile ducts consist of a solid cord of cells without an open channel. The process that controls remodeling of these structures to form open channels has not been defined. It is likely that some forms of biliary atresia, particularly those forms occurring with birth defects of the intestine and other organs, could be related to this developmental process. Basic research must focus on the developmental biology of these structures.

The normal infant undergoes a period of physiologic cholestasis that is related to immature pathways for the formation of bile. Owing to this immaturity of liver function, the infant is more susceptible to developing cholestatic liver disease during infection or with the administration of drugs or parenteral nutrition. Considerable progress has been made in understanding the transport mechanisms that contribute to bile formation at the level of the liver cell and cholangiocyte. However, much remains to be discovered as to how the specific transporters develop and are altered by disease in the infant. The discovery of several inherited defects in canalicular membrane transporters has served to stress their potential importance to bile formation. Acquired dysfunction of these transporters may occur in other liver diseases, leading to significant morbidity and even mortality. How these inherited defects progress to liver injury is only partially understood. Understanding these mechanisms will potentially provide therapeutic interventions to reduce injury caused by cholestasis.

The intrahepatic and extrahepatic bile ducts are frequently involved in many inherited and acquired liver diseases of childhood. There has been recent progress in our ability to study the functional properties of cholangiocytes 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 function during development and contribute to the process of bile formation. We know little about the genes that are expressed in cholangiocytes of the developing versus the adult liver.

Overall, this is an era of great opportunity in being able to identify and define the molecular and biochemical pathogenesis of many liver disorders affecting infants and children. Prevention and treatment strategies targeted to this age-group are likely to be especially cost-effective. Basic insight into the development of liver structure and function will be essential to our effort to treat many of the hepatic disorders affecting infants and children. A chronic liver disease prevented or successfully cured in a child will also restore a healthy, productive citizen to the workforce for many decades.

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II. MAJOR ISSUES IN NEED OF INVESTIGATION OR IMPLEMENTATION

* What are the array of genes that are activated or suppressed during development leading to the fully differentiated hepatocyte and cholangiocyte? The fully differentiated hepatocyte and cholangiocyte are derived from a common precursor stem cell. An array of genes, many of which are transcription factors, is activated or suppressed during development leading to the mature cell. The process is poorly understood and requires intensive study to define how the process occurs during normal development.

* What are the molecular mechanisms underlying the structural development of the liver and biliary tract and what is their relationship to birth defects, such as some forms of biliary atresia and fibrocystic diseases of the liver? The liver in the embryo is derived from a primitive streak of endodermal cells in the foregut that form hepatocytes and bile ducts. The molecular mechanisms underlying the formation of the liver are only partially understood. In particular, the factors leading to the formation and remodeling of the so-called ductal plate to produce the intrahepatic biliary tree should be defined, including genes that determine cell fate, proliferation, and programmed cell death. We now know that the gene responsible for autosomal dominant polycystic kidney disease (polycystin) is expressed in liver and likely contributes to the cystic abnormalities of the biliary tree. The role of this gene product in the morphogenesis of bile ducts has not been defined. It is also unknown how the gene defective in Alagille syndrome, the JAGGED 1 gene, leads to loss of interlobular bile ducts and small size of the extrahepatic bile ducts.

* How do the liver transport mechanisms that contribute to bile formation develop and how are transporters for organic and inorganic solutes affected by cholestasis? Cholestasis occurs more frequently and earlier in the course of liver disease in the infant than at any other time of life. It has recently been established that inherited defects in several adenosine triphosphate (ATP)-dependent transporters localized to bile canalicular membrane, including transporters for bile acids and phospholipids, lead to progressive cholestatic liver disease. Little is known about how these transport mechanisms are regulated and develop in the fetus and neonate. Moreover, how these transporters function in acquired cholestasis due to bile duct obstruction and intrahepatic disease has not been well defined.

* Do the functional properties of cholangiocytes and their contribution to bile formation differ during early life? The bile ducts are a frequent site of injury in pediatric liver disease. However, there is a lack of knowledge about the functional properties of bile duct cells or cholangiocytes during development. Recent studies have shown the ability to isolate and culture cholangiocytes and small segments of the biliary tree (isolated bile duct units.) However, such research has not been done in developing animals. Determination of the genes that are specifically expressed in developing versus mature cholangiocytes will be essential to understand cholangiocyte biology.

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III. PROPOSED PLAN TO ACHIEVE GOALS

* Define the array of genes that are activated or suppressed during development leading to the fully differentiated hepatocyte and cholangiocyte. There is a major gap in our knowledge of the factors regulating the formation the liver and intrahepatic bile ducts. The factors leading to the formation and remodeling of the so-called ductal plate to produce the intrahepatic biliary tree should be defined, including genes that determine cell fate, proliferation, and programmed cell death. Future research should focus on defining factors controlling the differentiation of hepatoblasts into cholangiocytes and hepatocytes, the signals that induce proliferation to form the bilayered ductal plate and remodeling to form the ductal lumen. The role of the embryonic microenvironment in hepatogenesis must be better understood. Further study of the complex hierarchical control of liver development at the transcriptional level is another priority.

* Understand the molecular mechanisms underlying the structural development of the liver and biliary tract and their relationship to birth defects such as some forms of biliary atresia and fibrocystic diseases of the liver. It is now know that the gene responsible for autosomal dominant polycystic kidney disease (polycystin) is expressed in liver and likely contributes to the cystic abnormalities of the biliary tree. The role of this gene product in the morphogenesis of bile ducts has not been defined. It is also unknown how the gene defective in Alagille syndrome, the JAG1 gene, leads to loss of interlobular bile ducts and small size of the extrahepatic bile ducts. It is highly expressed is the developing biliary tree but its role in hepatic development has not been determined. Whether genes involved in situs determination, such as the ivn gene and which are associated with biliary tract anomalies in knock-out animals, are involved in some cases of biliary atresia must be defined.

* Determine the normal development of liver transport mechanisms that contribute to bile formation and how transporters for organic and inorganic solutes are affected by cholestasis. Research is needed to define how the function of hepatic transporters contributing to bile formation is altered in acquired cholestasis due to bile duct obstruction and intrahepatic disease. It is feasible to study the properties of these transport systems in animal models as well as in the human. Overexpression of transport proteins in transgenic animals and targeted deletion studies will help to define the importance of each system to the process of bile formation and will significantly contribute to our understanding of the pathophysiology of cholestasis.

* Define the functional properties of cholangiocytes and their contribution to bile formation during early life. It is critically important to determine the functional properties of cholangiocytes from developing liver, including the localization and expression of transporters and whether, like the adult biliary system, there is functional heterogeneity between large and small cholangiocytes. It is also unknown how these cells respond to hormonal agonists such as secretin. Determination of the genes that are specifically expressed in developing vs. mature cholangiocytes will be essential to understand cholangiocytes biology through the use of DNA microarray chip technology. Strategies to target genes specifically to the biliary tree will be important in developing gene replacement for biliary disorders such as cystic fibrosis.

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SPECIFIC DISORDERS

Biliary Atresia
I. SUMMARY OF THE PROBLEM

Biliary atresia is a disorder characterized by a fibrosclerosing obliteration of the extrahepatic bile ducts that uniquely presents in the first months of life. The condition occurs in approximately 1 in 8,000 to 1 in 15,000 live births, and accounts for approximately one third of all cases of cholestasis in the young infant. Biliary atresia is the most frequent cause of chronic end-stage liver disease in children and contributes over half of all pediatric patients for liver transplantation. Two forms of this disease have been recognized. In an embryonic type, which occurs in about 10% to 15% of cases, cholestasis is present at birth, often in association with other extrahepatic anomalies including polysplenia, portal vein anomalies, malrotation, abdominal situs inversus, and congenital heart disease. In this form, the common bile duct is often absent on surgical exploration. In the approximately 80% to 90% of infants with the perinatal type biliary atresia, jaundice and acholic stools develop within the first weeks of life. Other anomalies occur less frequently. In this subtype, complete obstruction to bile flow develops as a result of an idiopathic progressive sclerosing fibro-obliteration of the extrahepatic bile duct.

The initial treatment of biliary atresia is surgical, involving resection of the obliterated extrahepatic bile duct and the creation of a hepatoportoenterostomy. This operation or Kasai procedure should be performed before 2 months of age to successfully reestablish bile flow. In spite of timely diagnosis and surgery, most cases (70% to 80%) will eventually progress to develop end-stage biliary cirrhosis and require liver transplantation. Delayed disease recognition and referral still remains a major problem in the successful management. The annual cost for this disease has been estimated to be $65 million in the United States.

The cause and pathogenesis of either the embryonic or perinatal type of biliary atresia are not known. Genetic, infectious and host immune factors are putative etiopathogenic mechanisms of disease. There is also an immediate need to institute more formalized educational programs in the medical and lay communities to allow for 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 has limited both the availability of sufficient human tissue samples to study pathogenic mechanisms, and the development of amply sized clinical trials to test novel treatment strategies. Research efforts should be directed toward defining the disease cause and pathogenesis, developing methods of early detection, and for creating more effective therapeutic interventions.

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II. MAJOR ISSUES IN NEED OF INVESTIGATION OR IMPLEMENTATION

* What is the contribution of genetic factors to the pathogenesis of biliary atresia? Although there are several reports of families in which male and female siblings have the disease, HLA-identical twins discordant for the condition have been described. Generally, biliary atresia is not considered to be an inherited disease. However, with the less common embryonic type of biliary atresia, in which other congenital malformations occur, genetic mutations resulting in defective morphogenesis may be important to the disease pathogenesis. For example, it is unknown whether there is a human correlate of the ivn mouse, a transgenic animal in which a recessive deletion of the inversin gene results in situs inversus and jaundice due to lack of continuity between the extrahepatic biliary tree and the small intestine. It is likely that the two forms have a different genetic predisposition and etiopathogenesis.

* Does an infectious agent cause biliary atresia? The fibrosclerosing process present in most cases of biliary atresia has long suggested the possibility that an infectious agent with a trophism for the biliary tract may be causal. There have been a number of contradictory studies focusing on a possible role for Reovirus type 3 in biliary atresia. The most recent detected reovirus type 3 RNA in cases of choledochal cysts as well in biliary aresia. There has also been evidence for and against the possibility that strains of Rotavirus may be involved. Sporadic cases of CMV or EBV infection in biliary atresia have also been reported. No specific virus has been definitively identified as the etiologic cause for either subtype of biliary atresia.

* What is the contribution of host immune factors? Host immune factors likely have a role in the pathogenesis of biliary atresia. With the perinatal type biliary atresia, a significant increase in the HLA B12 antigen has been noted, suggesting a role for host immunogenic factors in the disease progression. Aberrant expression of the MHC class I molecules, ICAM-1, but not MHC class II determinants has been found in intrahepatic bile ductular epithelial cells on analysis of liver biopsy tissue samples from patients with biliary atresia. An increased surface expression of HLA DR antigen in addition to ICAM-1 on intrahepatic bile ductular epithelial cells has been found in liver biopsy samples from patients with biliary atresia. It remains uncertain whether these changes are importance in mediating damage to the biliary tract or reflect a secondary effect of tissue injury.

* What are the remaining diagnostic challenges? Because the physical and biochemical findings may be subtle or ambiguous in cholestatic infants, more sensitive, specific, and less invasive methods for early diagnosis of biliary atresia are needed. Such tests would help to predict which patients may not benefit from an initial hepatoportoenterostomy. The most reliable test for the diagnosis of biliary atresia, aside from exploratory laparotomy, is a percutaneous liver biopsy. A specimen containing five to seven portal tracts is over 95% sensitive in indicating large bile duct obstruction. Although endoscopic retrograde cholangiopancreatography (ERCP) may be of diagnostic value in selected cases, the procedure is new to this age-group, invasive, technically challenging in young infants, and not readily available. MR cholangiography shows promise as an approach to image the biliary tract of the neonate. There are no serologic tests or imaging studies that are diagnostic for the condition. The economic and medical benefits of screening all newborn infants for elevated serum direct bilirubin or bile acids remain uncertain.

* Can the results of the portoenterostomy operation be improved? The Kasai procedure remains the cornerstone of therapy, but better indices to predict outcome are needed. Correlation of biochemical and histologic features may identify patients unlikely to beneft from portoenterostomy. For example, the presence of syncytial giant cells, lobular inflammation, focal necrosis, bridging necrosis, and cholangitis has been associated with failure of the portoenterostomy, whereas bile in zone 1 has been associated with clinical success. The care of the patient postoperatively presents many challenges. Repeated episodes of bacterial cholangitis after portoenterostomy surgery are associated with progressive fibrosis of the intrahepatic biliary tree and a worse prognosis. It is not resolved whether anti-inflammatory drugs, prophylactic antibiotics, or choleretics given individually or in combination affect the outcome. Other novel treatment strategies coupled with the Kasai operation need to be evaluated. These would include an evaluation of the efficacy of antivirals, anticytokines, antifibrogenesis agents, and cytoprotective agents for intrahepatic bile ducts.

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III. PROPOSED PLAN TO ACHIEVE GOALS

The specific research areas and goals are:

* Identify the genetic factors involved in the pathogenesis of biliary atresia: To identify and define the genetic factors that may be responsible for disease pathogenesis of either the embryonic or perinatal type biliary atresia. A search for mutations of human homologues of murine genes determining the laterality in embryonic biliary atresia needs to be performed.

* Determine if viral or other infectious agents cause biliary atresia: Although a specific viral infection has not yet been identified as the cause of biliary atresia, continued research in this area is required. Use of animal models to delineate the mechanisms of virally induced bile duct injury is necessary. Careful collection of sera and tissues from multiple centers in a standardized fashion for viral studies is essential. Thorough epidemiologic and virologic evaluation of temporal or geographic clustering of cases should be high priority. Animal models of virally induced biliary atresia, including the rotavirus A and reovirus 3 models, need further study to help characterize disease pathogenesis.

* Define the role of host immune factors in biliary atresia: The possibility that immunologic processes leading to the fibrosclerosing bile duct injury in biliary atresia requires further study. Possible maternal immune factors need to be explored. The role of the immune system in the mediation of the progressive fibrosclerosing lesion of the extrahepatic bile duct in biliary atresia should be defined. Studies identifying the phenotype of the extrahepatic bile duct cellular infiltrate and cytokines and the determinants of immune recognition of bile duct epithelial cells should be a priority. A genetic predilection in MHC classes I, II, or III needs to be sought. Parallel studies should be conducted in vivo by using existing animal models of either virally or immunologically induced bile duct injury. In vitro studies using human or animal bile duct epithelia in cell culture should be employed to specifically characterize mechanisms of epithelial cell–immunocyte interaction. The cellular and molecular events that mediate bile duct fibrosis during infancy need to be better elucidated. How hepatic stellate cells are activated to produce collagen in the developing liver and the role of the cholangiocyte in triggering this fibrotic process require investigation.

* Develop better diagnostic and prognostic disease indicators: Clinical data should be collected in standardized fashion from multiple centers. To this end, it is critical to establish a central registry for patients with biliary atresia to collect epidemiologic data and generate outcome statistics. Multicenter clinical therapeutic trials and repositories for human sera and tissues from affected patients should be a part of this effort. There is a need to develop better diagnostic markers for the disease to identify patients early in infancy who are most likely to benefit from the portoenterostomy. More precise predictors of outcome for the various treatment options are required. A cost–benefit analysis of universal postnatal screening of serum direct bilirubin or bile acid levels should be undertaken to examine its effect on outcome.

* Improve the results of the portoenterostomy operation and treatment of postoperative complications: Novel treatment strategies to reduce destruction of intrahepatic bile ducts, ongoing intrahepatic injury, and progressive fibrosis need to be developed to improve the outcome after portoenterostomy. These therapies should reduce, delay, or prevent ongoing intrahepatic bile duct destruction and fibrosis. The role of multiple factors alone or in combination including toxic bile acids, oxidant stress, ATP depletion, endotoxins and bacterial cell wall antigens, activation or recruitment of inflammatory mediators, activation of degradative hydrolases, and mitochondrial damage needs to be examined. Because ascending bacterial cholangitis is a major cause of morbidity in this patients, role of bile acids and bacterial cell wall products, and their interactions with hepatocytes, Kupffer cells, and stellate cells, in the mediation of the progressive liver disease must be assessed. Any intervention in infants with biliary atresia will require a multicenter effort to recruit adequate numbers of patients.

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Intrahepatic Disorders
I. SUMMARY OF THE PROBLEM

Intrahepatic cholestasis includes a heterogenous subset of cholestatic diseases that, with or without bile duct alterations (paucity), represent many specific syndromes with differing pathogenesis, causes, and outcome. There is also a high degree of variability in both their presentation and prognosis. Progressive, familial forms such as Byler's disease are often fatal. In contrast, in patients with Alagille syndrome or syndromic paucity of interlobular bile ducts, the prognosis is much more favorable. Owing in part to a poor understanding of the underlying pathophysiology, the current nomenclature system for the various forms of intrahepatic cholestasis is imperfect.

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Alagille Syndrome (Arteriohepatic Dysplasia or Syndromic Paucity of the Intrahepatic Bile Ducts)

Alagille sydrome (AGS) is characterized by cholestasis, a decreased number of interlobular ducts, and various congenital malformations (e.g., dysmorphic facies, peripheral pulmonic stenosis, vertebral arch defects, renal disease, posterior embryotoxon). Serum levels of alkaline phosphatase, γ-GTP, and bile acids are high, indicative of a defective biliary excretion. The mechanism leading to progressive loss of interlobular bile ducts and cholestasis is not known. Therapy directed against the complications of severe choestasis—pruritus, cutaneous xanthoma, and growth failure—is often ineffective. Liver transplantation may be required for these complications as well as for progressive liver disease.

AGS is inherited in autosomal dominant fashion and is one of the more common genetic causes of cholestasis in infancy, with an estimated frequency of 1:70,000 live births. The site of the gene responsible for AGS was first suggested by the identification of visible gene deletions on the long arm of chromosome 20. Subsequently, in 1997 two groups identified mutations in the gene encoding Jagged1, one of the ligands for Notch signaling pathway. Well over 200 different mutations have now been detected in these patients. Approximately 56% to 70% of mutations are sporadic. Haploinsufficiency, a decrease in the amount of the normal protein, appears to be the mechanism causing Alagille syndrome. Although Jagged1 is expressed in many tissues affected in Alagille syndrome including the developing and adult biliary tree, it is unknown how mutations in Jagged1 lead the bile duct paucity and liver disease. No phenotypic differences have been identified based on type or location of mutation. Because there is extreme variability of AGS phenotype within families, other genetic or environmental factors likely contribute to the clinical manifestations of the disease.

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Nonsyndromic Paucity of Interlobular Bile Ducts

Paucity of interlobular bile ducts can also occur without the classic features of the Alagille syndrome. Liver biopsy shows an absence or reduction of interlobular bile ducts. The neonate is either more susceptible to damage and loss of bile ducts because of the biology of these structures during early life or are more likely to be affected with infections with a tropism for the biliary tree. Bile duct loss can occur in a diverse array of disorders such as cytomegalovirus infection and α1-antitypsin deficiency. The prognosis in nonsyndromic paucity is thought to be worse than in Alagille syndrome. The mechanisms underlying bile duct injury and loss have not been well defined.

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Progressive Familial Intrahepatic Cholestasis (PFIC)

PFIC represent a group of diseases involving membrane transport proteins thought to be involved in bile formation. Onset is typically in the first 6 months of life with cholestasis, hepatomegaly, severe pruritus, growth failure, and fat-soluble vitamin deficiency. Progressive familial intrahepatic cholestasis type 1 (PFIC1) or Byler's disease is characterized by chronic, unremitting cholestasis that develops early in life. Jaundice, severe pruritus, and growth failure are typical features in a setting of a low serum γ-glutamyl transpeptidase and cholesterol levels. The disorder is inherited as an autosomal recessive trait. The gene for the disorder, FIC1, mapped to the same locus as benign recurrent intrahepatic cholestasis (chromosome 18q21-q22). FIC1 has been cloned and encodes for a P-type ATPase that may function as an ATP-dependent aminophospholipid flippase. FIC1 is highly expressed in intestine and cholangiocytes and at a lower level in hepatocytes. The function of FIC1 has not been defined or how defects in the protein lead to progressive liver disease. Progression to cirrhosis and liver failure usually occurs by 3 to 4 years of age. Partial external biliary diversion has been used successfully to treat intractable pruritus in these patients; in some cases the progressive cholestasis also improves after the procedure. It is unknown why biliary diversion is beneficial in this disorder. Orthotopic liver transplantation is required in patients with decompensated cirrhosis. Diarrhea often continues after transplantation.

A second locus for PFIC was mapped to chromosome 2q24. Patients with this variant (PFIC 2) present with severe cholestasis in the neonatal period with a normal serum γ-glutamyl transpeptidase concentration. Liver histology initially shows giant cell hepatitis; there is rapid progression to cirrhosis. Mutations in the ATP-dependent canalicular bile salt excretory pump (BSEP) have been found in these patients, consistent with the phenotype of decreased canalicular excretion of bile acids described in this form of PFIC.

Another subtype of progressive familial intrahepatic cholestasis (PFIC 3) has been identified in which patients have high serum γ-glutamyltranspeptidase levels. The disorder shares histologic, biochemical, and genetic features with mice in which the mdr2 gene has been inactivated (mdr2 −/− mice). Mdr2 and the human homolog MDR3 encode a phosphatidylcholine flippase located on the bile canalicular membrane, which mediates biliary phospolipid excretion. Patients with PFIC3 have low concentrations of phospholipids in bile and develop severe liver disease characterized by inflammation of portal tracts, bile ductular proliferation, and fibrosis. In the absence of biliary phospolipids, the biliary tree is subject to progressive injury from hydrophobic bile salts. The absence of immunohistochemical canalicular staining for MDR3 (human mdr2 homolog) has been found in liver tissue from affected patients. Several mutations in the MDR3 gene have been demonstrated on analysis of genomic DNA.

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II. MAJOR ISSUES IN NEED OF INVESTIGATION OR IMPLEMENTATION

* What are the mechanisms underlying bile duct paucity and why is the infant more susceptible to bile duct injury? The mechansims causing bile duct injury and progressive loss leading to paucity must be better understood. Infectious, immunologic, and toxic factors are likely to be operative. The segmental destructive changes or progressive decrease in the number of bile ducts on serial sectioning of biopsy specimens from the early features of bile duct inflammation to the later observation of paucity, suggests immunologic injury to existing ducts—similar to other syndromes of “disappearing” intrahepatic bile ducts in the posttransplantation setting—rather than from a failure of ducts to develop. It must be defined whether ductal injury occurs via necrosis of cholangiocytes or through programmed cell death (apoptosis). The factors regulating these processes should be defined.

* How do Jag1 mutations lead to the characteristic group of anomalies and bile duct paucity? The identification of mutations in Jag1 gene as the cause of most cases of Alagille syndrome was one of the most important advances in the decade of the 1990s. However, in spite of its potential intriguing role in tissue growth and differentiation, it is not known how mutations in this gene lead to the characteristic array of birth defects and propensity for progressive loss of interlobular bile ducts. There are also approximately 30% of Alagille patients who do not harbor a mutation in Jag1. Whether defects in other components of the Notch signaling pathway could be responsible for these cases remains unknown. Insight into the pathophysiology of Alagille syndrome could be advanced if a knockout animal model of the disorder could be developed.

* How do the defective genes in various forms of progressive familial intrahepatic cholestasis (PFIC) cause liver disease, and do alterations in these proteins contribute to acquired cholestatic liver disease? Progressive familial intrahepatic cholestasis (PFIC) occurs in at least three forms caused by mutations in ATP-dependent transport proteins. The pathophysiology of PFIC 2 appears to be straightforward because defects in the canalicular membrane BSEP lead to a failure of bile salt-dependent bile secretion and retention of bile salts and other potentially noxious compounds within hepatocytes. In contrast, the function of the abnormal gene in Byler disease, so-called FIC1, has not been defined. Its localization within the liver has not even been definitively resolved. Expression is highest in the intestine. FIC1 is also present in cholangiocytes, albeit at levels less than the gut, but initially was not detected in hepatocytes. Preliminary studies now suggest some expression in hepatocytes. How mutations in FIC1 lead to progressive cholestasis is unknown. In the case of PFIC3 the absence of a canalicular membrane flippase leads to failure of biliary phospholipid secretion. Damage to the canalicular membrane and to bile duct epithelia is thought to occur through the detergent effect of hydrophobic bile salts. Because inherited defects in these disorders lead to cholestasis, it will be important to determine how these transporters are altered during acquired cholestasis as occurs after bile duct obstruction or exposure to endotoxin.

* Can management strategies be developed for complications of cholestasis (e.g., pruritus, growth failure, osteoporosis, and hypercholesterolemia) in patients with Alagille syndrome and PFIC to improve quality of life and possibly improve survival without liver transplantation? Intrahepatic cholestatic disorders are associated with malabsorption of known fat-soluble nutrients, contributing to poor growth and specific micronutrient deficiencies. Secondary accumulations of copper and manganese have also been associated with possible increased hepatic injury and lesions in the basal ganglia. Pruritus is a major factor in these disorders and appears related to central opioid pathways. Although partial biliary diversion is of benefit, other novel strategies to reduce pruritus (e.g., opioid antagonists) need to be developed. Metabolic bone disease and osteoporosis may be severe despite adequate vitamin D and calcium intake, and are poorly understood. The consequences of hypercholesterolemia of Alagille syndrome have not been well defined; autopsy studies suggest increased arterial wall cholesterol plaques; however, atherosclerotic vascular symptoms have not been reported. Finally, improved cytoprotective and choleretic agents, to reduce the effects of toxic, hydrophobic bile acids, will potentially improve hepatocyte function and bile flow, and ultimately retard the progression to cirrhosis and liver failure. Ultimately, gene replacement or modulatory therapy could correct the underlying molecular and biochemical defects that underlie these disorders. This would require targeting of vectors to the hepatocyte or cholangioctye.

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III. PROPOSED PLAN TO ACHIEVE GOALS

The specific research areas and goals are:

* Understand the pathogenesis of bile duct paucity in both syndromic and nonsyndromic forms. Paucity syndromes may have a direct developmental basis in some cases, representing congenital absence or failure to form or may ensue through progressive injury (immune, viral, ischemic,or toxic) and disappearance. Understanding cholangiocyte-specific gene expression in the adult and developing animal as well under pathologic conditions leading to paucity will be critical to understanding the biology and pathobiology of the biliary tract. Subtraction hybridization methods as well as newer microarray chip technology will be particularly informative in this effort. It is also uncertain whether bile ducts disappear via cell necrosis or apoptosis. A better understanding of the mechanisms of cholangiocyte cell death could result in strategies for therapy. Immune, toxic, and infectious factors should be considered as possible causal agents. Careful study of biliary epithelial cell physiology during development should be a priority. The role of growth factors, cytokines, and other effectors of the immune system in maintaining ductal integrity and in mediating cell injury should be defined. Our understanding of these disorders would be greatly enhanced with development of isolated cholangiocyte models and relevant animal models.

* Undertand how Jag1 mutations lead to the characteristic group of anomalies and bile duct paucity in Alagille sydrome. Alagille syndrome exhibits a highly variable and complex phenotype despite its autosomal dominant inheritance. Many sibs and parents of probands are often found to be minimally affected, with only one or two abnormalities. The frequency of new mutations appears to be high (15% to 50%). Close to 200 different mutations have been found so far, making routine genetic analysis a challenge. Approximately one third of patients do not have a mutation in Jag1, indicating that genetic abnormalities, possibly involving other components of the Notch signaling pathway, may cause some cases of Alagille syndrome. Further characterization of the responsible gene(s) and molecular testing must be carried out to better understand the pathophysiology and to precisely diagnose Alagille syndrome. These studies are essential in avoiding unnecessary surgery in the cholestatic infant, in properly initiating therapy, and providing accurate genetic counseling.Members of the Notch gene family that encode evolutionarily conserved transmembrane receptors are involved in cell fate specification during embryonic development. The Notch locus encodes a receptor mediating cell–cell interactions. Future studies must focus on the Notch signaling pathway, which appears to define a fundamental mechanism controlling cell fate in early development, perhaps controlling the ability of nonterminally differentiated cells to respond to differentiation/proliferation signals. Although it is logical to envisage how the broad spectrum of organ involvement in Alagille syndrome could be caused by JAG1 mutations, the molecular mechanisms underlying the pathogenesis of the disorder remain undefined. Research should also focus on how this genetic abnormality leads to a paucity of interlobular bile ducts through a process involving progressive injury and loss of ducts. An animal model of the Alagille syndrome made by targeted disruption of Jag1 gene would be of considerable value in such an effort. It should also be feasible to selectively turn off expression of Jag1 in the liver at various times postnatally through use of the Cre/Lox technology to assess effects on the biliary tree.

* Define how the genes involved in various forms of progressive familial intrahepatic cholestasis (PFIC) cause liver disease and are altered in acquired cholestatic liver disease. The genes for three forms of progressive familial intrahepatic cholestasis have been cloned, but the pathophysiology underlying these disorders is not well understood. PFIC1 is caused by mutations in a P-type ATPase now called FIC1. The function of the polypeptide encoded by this gene is unknown, but proteins of the same family function as aminophospholipid flippases in transporting aminophospholipids from the outer to the inner leaflet of plasma membranes. In this capacity, these transporters serve to maintain the structure of plasma membranes and thereby contribute to their role as a semipermeable barrier. FIC1 is highly expressed in the intestine and pancreas and to a lesser extent in cholangiocytes. It has been controversial as to whether the gene is expressed in hepatocytes but recent preliminary studies suggest the possibility that the transporter may be present on the canalicular membrane. How defects in this transporter lead to progressive cholestatic liver disease remains unknown. It is also of interest that the same gene is mutated in benign, recurrent, intrahepatic cholestasis. Much additional work must be done to determine how specific mutations in one case lead to progressive liver disease and in the other are associated with a relatively benign disorder. Research will be required to precisely define the cell types and membrane domains on which FIC1 is expressed. It is also essential that the precise function of this protein be determined to understand how defects in the protein lead to cholestasis. PFIC1 is a disorder in which liver disease may be cured by liver transplantation, but patients may continue to have difficulties with diarrhea and malabsorption after transplantation. The expression of the abnormal gene product in intestine and pancreas may explain the continued symptomatology in these patients. Creation of transgenic mice in which FIC1 is over expressed or disrupted by gene targeting would also be useful in understanding the role of this transport protein in health and human disease.The mechanisms responsible for liver disease in PFIC2 appear to be more straightforward. Mutations in the gene encoding the canalicular bile salt excretory pump (BSEP) have been found. This protein is a member of the ATP-binding cassette (ABC) family of transporters. It appears to be the predominant transporter responsible for concentrative excretion of bile salts into the canalicular lumen. Patients with mutations in BSEP have markedly impaired secretions of bile salts into bile and a failure of bile salt–dependent bile secretion. Additional studies on BSEP are required to determine its mechanism of regulation during normal development and during disease. It would also be of value to study the phenotype and biliary physiology of animals in which BSEP has been disrupted by gene targeting.PFIC3 is caused by mutations in the MDR3 gene, which is responsible for phospholipid secretion into bile. This member of the ABC family of ATP-dependent transporters mediates transfer of phospholipids from the inner to the outer leaflet of the canalicular plasma membrane, where, under the detergent action of bile salts, phospholipids are incorporated into mixed micelles. Knockout mice lacking MDR2, the homologue of human MDR3, are also unable to secrete phospholipids into bile and develop a progressive liver disease, which is characterized by bile ductular proliferation and progressive biliary cirrhosis. Further studies of these valuable models as well as the human disease will be required to develop effective treatment strategies.Efforts should be directed to develop methods to establish a precise genetic diagnosis for each one of these disorders. In each case, different therapies, such as biliary diversion or replacement with the hydrophilic bile salt ursodeoxycholic acid, may be beneficial. It will also be of importance to precisely define the specific type of PFIC so as to better predict problems that may continue after liver transplantation. These disorders will eventually be amenable to treatment with gene therapy.It is clear that other types of progressive familial intrahepatic cholestasis exist beyond the forms so far described. Whether disorders such as that occurring in Greenland Eskimos or in hereditary cholestasis with lymphedema involve any of the genes so far described for PFIC1, 2, or 3 is unknown. It will be of great importance to understand the genetic basis of these disorders, which should contribute significantly to our understanding of these diseases as well as normal hepatobiliary physiology.

* Develop treatment strategies for complications of cholestasis. Studies need to be performed to define the cellular and biochemical cause of growth failure in children with cholestasis and improved nutritional therapies developed to counteract malabsorption, essential fatty acid deficiency, and fat-soluble vitamin deficiencies. The mechanisms for pruritus of cholestasis need to be better defined and more effective treatment strategies developed. Use of central opioid receptor antagonists and partial biliary disease need to be carefully studied in this regard. The cause of the metabolic bone disease of cholestasis that may lead to recurrent fractures needs to be characterized. This should lead to more effective preventive therapies and a better understanding of bone metabolism during liver disease. Areas such as magnesium status, effect of circulating cytokines on bone growth, and hormonal status should be explored.

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Acknowledgment:

The input of Tomoo Fujisawa to this report is gratefully acknowledged.

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References

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© 2002 Lippincott Williams & Wilkins, Inc.

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