Molecular Mechanisms of Cholestasis
Date Issued
2004
Date
2004
Author(s)
DOI
zh-TW
Abstract
Introduction and Background
Cholestasis, or impaired bile flow, is one of the most common and devastating manifestations of hereditary and acquired liver diseases. Cholestasis of infancy, manifesting as conjugated hyperbilirubinemia, accounts 50% of the work load in tertiary referral centers for pediatric hepatology and is a common disorder for children in Taiwan. Chinese infants have a higher incidence of biliary atresia than Japanese, Philippino, and white infants. Intrahepatic cholestasis is even more common than biliary atresia in Chinese infants. Patients usually manifest by conjugated hyperbilirubinemia within the first 2 months of age. Clinical manifestations include jaundice, light-yellowish or clay-color stool, small for age, poor feeding, and hepatosplenomegaly.
Infantile cholestasis consisted of extrahepatic cholestasis and intrahepatic cholesatsis. Extrahepatic cholestasis includes biliary atresia, choledochal cyst, and inspissated bile syndrome. Intrahepatic cholestasis consists of a diversity of etiologies, termed as “neonatal hepatitis syndrome”. The etiologies includes congenital infections (TORCHES), chromosomal anomalies (Trisomy 18, 21, etc), endocrine disorders (hypopituitarism, hypothyroidism), structural anomalies (neonatal sclerosing cholangigis, Caroli disease, ductal paucity), genetic diseases (Alagille syndrome, progressive familial intrahepatic cholestasis), and metabolic diseases (tyrosinemia, glycogen storage disease, Niemann-Pick disease, lysosomal disorders, inborn errors of bile acid synthesis).
Evaluation of the cholestatic infants remains a difficult task, owing to the diversity of cholestatic syndromes and to the obscure pathogenesis of many of these disorders. Prompt identification and diagnostic assessment of the infant with cholestasis is imperative to recognize disorders amenable either to institute specific medical therapy or effective nutritional and medical support to allow optimal growth and development.
PFIC is a group of inherited, autosomal recessive disorders with infantile onset cholestasis. Patients progress to liver cirrhosis and hepatic failure in the early childhood. Three genes causing PFIC have been cloned. Defects in FIC1 (formal genetic name ATP8B1) that encodes a P-type ATPase, and BSEP (formal genetic name ABCB11) that encodes an ABC transporter, are found in patients with low serum GGT levels. Mutations in class III-multidrug-resistance P-glycoprotein (its formal genetic name ABCB4) are responsible for a distinct type of PFIC characterized by high serum GGT levels. Patients with phenotype of PFIC have been described in Japan but no reports about Chinese patients.
There has been no report about children of PFIC in Taiwan. About half of our cases with neonatal hepatitis were attributed to CMV infection and the other half were idiopathic. Following the clinical course of patients with neonatal hepatitis revealed that most cases recovered within 6 months. However, some patients progressed to liver cirrhosis and died in the early childhood. Only a few of them had a family history of infantile cholestatic jaundice. It is not known whether hereditary cholestatic syndrome with the above mentioned genetic abnormalities existed in our patients. Comprehensive studies are impeded by the laborious genetic analysis, which is available in only a few research centers in the world.
Our study started with identifying the patients with genetic disorders in Taiwan. These patients were previously classified as idiopathic. The diagnosis would directly improve the treatment and management to patients. Secondly, we determined the expression patterns of human fetal and postnatal stage, to correlate with physiological cholestasis in premature and normal infants. Thirdly we apply the primary hepatocyte culture systems to identify the effect of bile acids on the expression of canalicular transporters. The system may be applied to all forms of cholestatic diseases causing by insults such as sepsis and parenteral nutrition. Because current treatment of genetic cholestatic diseases has been unsatisfactory, most patients eventually would need liver transplantation. We have tried cell therapy in a mouse model of PFIC type 2, the sPgp knockout mice, for possible new therapeutic approaches to cholesatic liver diseases.
Genetic Studies in patients with PFIC
PFIC
From 1980 to 1998, a total of 295 patients had been admitted to the Department of Pediatrics in National Taiwan University Hospital for infantile-onset intrahepatic cholestasis. Among them, 46 (15.6%) developed to chronic cholestasis. Among the 46 patients, Alagille syndrome was diagnosed in 18, inborn errors of bile acid synthesis in four, neonatal Dubin-Johnson disease in two, and tyrosinemia in one patient. No identifiable etiology was found in the remaining 21 patients, including 6 patients with high serum GGT levels (above 94U/L according to the normal range of infant in our hospital) and 15 with normal serum GGT levels.
High GGT patients
To investigate the etiology of the 6 patients with high GGT levels, either endoscopic retrograde cholangiography, magnetic retrograde cholangiography, or operative cholangiography was performed and revealed no structural anomalies of biliary trees. Needle biopsy of the liver was performed in all patients under informed consent from the parents. These patients were followed for 17 months to 16 years. The study has been approved by the institutional review board.
Neonatal onset of cholestasis was found in five patients except case 5 who presented pruritus at one year of age. The GGT levels ranged from 1.2 x to 9.8 x of normal value. Liver histology showed significant widening and fibrosis of the portal areas, with progression to liver cirrhosis. In the remaining one patients, liver histology showed frequent spotty necrosis and occasional giant cell transformation. There were no significant changes in portal areas and bile ducts in this patient.
Analyses for MDR3 gene. MDR3 gene was analyzed in all the 6 patients. Only one (case 5) had MDR3 mutation. A homozygous 719-bp deletion (nucleotide 287 to 1005 of coding sequence) of liver cDNA was found which encompassed exon 5 to 9 and led to a frameshift. We found that MDR3 mutations accounted for about 2% (1/47) of patients with infantile-onset chronic intrahepatic cholestasis in Taiwan. The only case with novel mutation in MDR3 manifested pruritus at 1 year instead of neonatal jaundice. This implies that MDR3 defect is not a common cause of high-GGT intrahepatic cholestasis in infancy, especially in neonatal cholestasis. Those patients presenting high GGT-PFIC with early onset cholestasis but without MDR3 mutation probably had inheritable disorders remaining to be clarified.
Low GGT patients
Thirteen patients whose serum GGT levels were normal or low (below 94 U/L according to the normal range of infant in our hospital were included.
Phenotype characterization
Two distinct histopathological phenotypes were identified. Group 1 included five patients (cases 1-5) and had liver histology characterized by bland cholestasis with minimal lobular disarray. Portal fibrosis was minimal to mild. Bile ducts were normal or slightly reduced in number. Group 2 included eight patients (case 6-13) and showed evident lobular disarray, marked giant cell transformation, or confluent cell necrosis accompanied by pericellular fibrosis. The grouping results from the two pathologists were identical. Group 1 patients had chronic or intermittent cholestasis and pruritus, with mild elevation of transaminase and bilirubin levels. The associated extrahepatic manifestations included diarrhea, rickets, and failure to thrive.
Group 2 patients had marked elevation of transaminase and bilirubin levels. Peak serum bilirubin, aspartate aminotransferase, and alanine aminotransferase levels were higher than those in group 1 patients, p=0.02, p=0.001, and p=0.001, respectively. All the 8 patients in group 2 had high AFP levels (range, 2.4 to 16.2 SD score). Whereas all the 5 patients in group 1 had normal AFP levels as compared with age-specific normal range. (p<0.01). Pruritus and failure to thrive were frequent.
Genotype characterization
Analyses for FIC1 gene. Seven patients had frozen liver tissues available for analyses of FIC1 gene alterations. These included 4 patients in group 1 (bland cholestasis) and 3 patients in group 2 (giant cell transformation). All the 4 group 1 patients had mutations in FIC1 gene.
Analyses for BSEP gene. cDNA sequencing was performed in 3 group 2 patients who had no mutations in FIC1 gene. A homozygous 850G→C(V284L), and a heterozygous 1-bp deletion at position 1145 was found in patient 7. The V284L mutation occurs in the intracellular loop between transmembrane spans 4 and 5. The 1145 deletion resulted in frameshift after codon 382 followed by 15 novel amino acids and protein truncation. A homozygous missense mutation 3137 G→A (G1004D) was found in case 8, which located in extracellular loop between transmembrane spans 11 and 12.
In this study, genetic cholestasis disease was found in 86% (6/7) of the patients with infantile onset chronic intrahepatic cholestasis and low serum GGT levels. All the mutations were different from that reported from the Amish, European, or Arabic descents. It is a significant finding since the frequency of genetic diseases can vary greatly in people of different ethnic background. For example, alpha-1-antitrypsin deficiency, tyrosinemia, and cystic fibrosis were rarely seen in Asian children whereas Alagille syndrome is frequent in our children. 19 This finding prompt a specific direction of clinical awareness and research effort toward Asian patients of infantile cholestasis in the future.
We found that the phenotypic diagnosis, based on clinical, serologic, and histologic features, correlated very well with genetic changes in chronic cholestatic patients with serum low-GGT levels. Two groups of patients were identified. Group 1 was characterized by normal AFP level and bland cholestasis in histology, and high frequency of FIC1 mutation (4 of 4 patients examined). In contrast, group 2 was characterized by high AFP level, giant cell transformation with lobular disarray, and high frequency of BSEP mutations (2 of 3 patients examined). We suggest that phenotypic characterization is a simple method to identify non-familial patients suspected to have FIC1 and BSEP mutations. Subsequent cDNA sequencing is useful to confirm the genetic diagnosis, and serve as a basis for genetic counseling or future gene therapy. We also have found that AFP may serve as an additional laboratory marker to differentiate patient with BSEP mutations from FIC1 mutation patients.
Dubin-Johnson Syndrome (DJS)
Four patients were included according to the clinical characteristics and hepatic pathology compatible with the diagnosis of Dubin-Johnson syndrome in the National Taiwan University Hospital. Two of the patients had an onset of cholestasis since the neonatal period, while the other two had a history of neonatal hyperbilirubinemia, which was documented since early adolescence.
Mutational analysis (DJS)
Genomic DNA analysis
Biphasic pattern of jaundice attack was observed in one patient followed for 20 years. Mutations of ABCC2 gene, including 3 compound heterozygous, and 1 heterozygous mutations were found, including deletions (2748del136, 3615del229, and Del3399-3400) and missense mutations (L441M, E1352Q, and R393W). We found that all patients diagnosed as DJS before 10 years old have mutations involve one of the two ATP-binding cassettes (ABCs) of the MRP2 (multidrug-resistance protein 2) protein.
To our knowledge, this is the first article reporting genetic analysis and long-term follow-up of neonatal DJS. DJS is rarely diagnosed in the neonatal period and only a few cases have been reported. The clinical characteristics and diagnostic investigations are similar with adolescent- or adult-onset DJS, except that more severe cholestasis and hepatomegaly may be found. All of the mutations found in our patients are novel. This is the first report of MRP2 mutations in patients of Chinese descent. The mutations are different from those reported from Japanese patients. Most reported mutations of the ABCC2 gene in DJS involve one or both ABCs. These ABCs are important for ATP binding and are highly conserved.
Together with genotype-phenotype correlations in reported childhood cases of DJS, we suggested that disruptions of functionally important ABCs domains in the MRP2/ABCC2 protein maybe related to the earlier onset of the disease. Long-term follow-up of a neonatal-onset case is mandated because DJS may have a biphasic pattern of jaundice attack, with a second attack occurring after adolescence. Careful evaluation of all cases of neonatal jaundice to rule out DJS is suggested, especially in patients with conjugated hyperbilirubinemia with mild or minimally elevated transaminase levels.
Developmental Expression of Canalicular transporters
BSEP, MRP2, and MDR3 are major hepatic canalicular transporters mediating bile salts, conjugated bilirubin, and phospholipid secretion. Before birth, BSEP and MRP2 expressions have been reported to be either undetectable or at a much lower level than the adults in rats. This study aimed to understand the expression of canalicular transporters during human development,
We investigated the expression of genes participating in bile formation and regulation. Human liver samples from fetus at gestational age 14 to 20 weeks, adult livers and liver samples of infants with biliary atresia patients were tested for mRNA expression of BSEP, MDR3, MRP2, NTCP, FIC1, and FXR genes by using real-time RT-PCR. Immunohistochemical staining of BSEP, MDR3, and MRP2 were performed on fetal and adult livers.
Fetal expression levels of all the genes tested were lower than adult levels. The mean expression levels of BSEP, MRP2 and FIC1 were 30 to 50% of adult levels. The mean expression levels of FXR were 75% of adult levels, and the mean expression levels of MDR3 and NTCP were only 6.4% and 1.8 % of adult levels, respectively. The immunohistochemical staining of the fetal liver had distinct patterns from the adult liver. In the adult liver the characteristic canalicular staining of BSEP, MRP2, and MDR3 could be seen. In the fetal liver, different staining patterns in the three proteins were noted. MRP2 showed mainly canalicular pattern, slightly fuzzier than adult staining; BSEP showed partially intracellular and partially canalicular pattern; and MDR3 showed faint intracellular pattern, only occasionally canalicular stain could be seen.
Our study presents that canalicular transporters are expressed early in the second trimester in human, which is different from the studies in rats showing that BSEP was not expressed until after birth. Many of the bile physiology studies have been performed in rodents. However, physiological differences between human and rodents should be considered. Our results showed that the time of BSEP expression correlates with the time when bile starts to flow to the intestine before 14 weeks of gestation. Although BSEP, MRP2, as well as MDR3 genes were readily expressed before birth, the localization pattern was yet distinct from mature hepatocytes. The reasons may be immature polarization of heaptocytes during development and inadequate targeting of specific proteins to the canalicular membrane.
Regulation of canalicular transporters by bile acids in primary hepatocyte cultures
In primary hepatocyte culture systems, we found that expression of canalicular transporters gradually decreased with time in primary hepatocyte cultures after 7~14 days. After adding bile satls into culture medium, expression of BSEP (bile salt export), FIC1, and NTCP was induced, implicating that these canalicular transporters may be regulated by intrahepatic bile acid levels.
Cell therapy of sPgp knockout mice, a model of chronic cholestasis and progressive familial intrahepatic cholestasis type II
Current therapy for PFIC includes palliative therapy: medical therapy (Urso), surgical therapy (partial biliary diversion); and curative therapy: liver transplantation. Major problems of liver transplantation are lack of donors and surgical risks. Cell therapy using hepatocytes or bone marrow cells in treating liver diseases had been successful in a few animal models including metabolic liver disease and partial hepatectomy model. There has been no research on cell therapy on chronic cholestasis animal model. We used green fluorescent protein (GFP) transgenic mice as donors for cell therapy. Isolated hepatocytes were infused into the spleen of sPgp knockout mice; in another study , and bone marrow cells were infused into tail vein in sPgp knockout mice with lethal dose irradiation (700~900 rad). We found that 4 to 8 weeks after hepatocyte transplantation, scattered GFP-positive cells were visible in the liver, especially peri-portal areas. sPgp immunofluorescent staining revealed no positive-stained cells. In mice with bone marrow transplantation, donor cells comprosed about 10 % of recipient liver DNA. Most cells were spindle shaped and were Kupffer cells. Some scattered hepatocytes could be identified. In sPgp knockout mice approximately 1 in 3520 hepatocytes were donor origin, but only 1 in 13863 hepatocytes were donor origin in wild type mice, indicating a higher rate of donor cell incorporation or cell fusion in sPgp knockout mice.
The efficiency of bone marrow cells and hepatocytes repopulated in sPgp knockout mice liver was low, probably due to high endogenous regeneration activity. Further efforts in increasing donor cell repopulation and investigate the function restored by transplanted cell is mandatory.
Subjects
膽汁滯留
肝臟
發育
細胞治療
基因
ABC傳送蛋白
Cholestasis
liver
development
cell therapy
gene
ABC transporter
SDGs
Type
text
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