Orthotopic liver transplantation for Wilson's disease: a single-center experience.

BACKGROUND: Wilson's disease is an inherited disorder of copper metabolism characterized by reduced biliary copper excretion, which results in copper accumulation in tissues with liver injury and failure. Orthotopic liver transplantation (OLT) can be lifesaving for patients with Wilson's disease who present with fulminant liver failure and for patients unresponsive to medical therapy. The aim of this study is to review our experience with OLT for patients with Wilson's disease. METHODS: Between 1988 and 2000, 21 OLTs were performed in 17 patients with Wilson's disease. Patient demographics, pre-OLT laboratory data, operative data, and early and late postoperative complications were reviewed retrospectively. One-year patient and graft survival was calculated. RESULTS: Eleven patients had fulminant Wilson's disease; in six patients the presentation was chronic. Mean patient age at presentation was 28 years (range 4-51 years); mean follow-up was 5.27 years (range 0.4-11.4 years). Neurologic features of Wilson's disease were not prominent preoperatively and did not develop post-OLT except in one patient who developed acute neuropsychiatric illness and seizure. Renal failure, present in 45% of patients with fulminant Wilson's disease, resolved post-OLT with supportive care. One-year patient and graft survivals were 87.5% and 62.5%, respectively. Fifteen survivors have remained well with normal liver function and no disease recurrence. CONCLUSION: Liver transplantation for hepatic complications of Wilson's disease cures and corrects the underlying metabolic defect and leads to long-term survival in patients who present with either acute or chronic liver disease. Acute renal failure develops frequently in patients with fulminant Wilsonian hepatitis and typically resolves postoperatively.

Wilson's disease.

During the last 90 years, Wilson's hepatolenticular degeneration has evolved from a disease presenting insurmountable challenges to the clinician's diagnostic acumen to a metabolic disorder which is diagnosable woth certainty, treatable successfully, and even preventable. It may be long before the genetic defect becomes amenable to correction and a cure of the disease becomes possible.

Identification of B10, an alkaline phosphodiesterase of the apical plasma membrane of hepatocytes and biliary cells, in rat serum: increased levels following bile duct ligation and during the development of cholangiocarcinoma.

Alkaline phosphodiesterase (APDE) is associated with the cellular plasma membrane of many organs. Several isoforms are also detected in normal human serum and their respective amounts vary in liver diseases but their significance is unknown. The aims of this study were: 1) to identify a serum form of B10, an APDE exclusively localized at the apical pole of the plasma membrane of rat hepatocytes and biliary cells; 2) to gain insight into its origin; and 3) to investigate its behavior, in two liver diseases in which an abnormal membrane expression of B10 has been reported, namely cholestasis and cholangiocarcinoma. A soluble form of B10 was immunoprecipitated from normal rat serum, which amounted to 13% of total serum APDE activity. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the size of the serum enzyme was 125 kd, which is slightly lower than that found in the plasma membrane (130 kd). In bile, a 120-kd and a 130-kd form was found. A sixfold and fivefold increase of B10 APDE activity was observed in the serum of bile duct-ligated rats and in the Long-Evans Cinnamon (LEC) rats which spontaneously develop cholangiocarcinoma. The molecular size of the form present in serum was unchanged. A threefold increase was also observed in LEC rats which had not yet developed a cholangiocarcinoma. In conclusion, we identified a soluble form of B10 in normal rat serum. The increase in serum B10 in the experimental and pathological conditions investigated does not seem to result from passage of the biliary form to the serum but seems to be caused by increased cleavage of the membrane form. Its rise early during the onset of cholangiocarcinoma suggests that B10 in the serum might be a marker of carcinogenesis and/or be involved in the development of cholangiocarcinoma.

Spontaneous cholangiofibrosis in Long-Evans Cinnamon rats: a rodent model for Wilson's disease.

The Long-Evans Cinnamon (LEC) rat is a rodent model of Wilson's disease characterized by ceruloplasmin deficiency, hepatic copper accumulation, and hepatocellular injury. So far, the LEC rat appears to be the only strain in which cholangiofibrosis develops spontaneously. The aim of the study reported here was to characterize the time course of development and investigate the structural and ultrastructural features of cholangiofibrosis and their possible relationship to hepatic copper and iron content. The livers of 54 rats (22 males), ages 5 to 113 weeks, were examined by light microscopy and graded for statistical analysis, with respect to extent of replacement of liver tissue by cholangiofibrosis. The study was complemented by electron microscopy, and by measurements of copper and iron contents by atomic absorption spectroscopy. Cholangiofibrosis was present in LEC rats by 20 weeks of age. The hyperplastic biliary epithelial cells varied markedly in size and shape, ranging from flat to cuboidal or elongated. Epithelial cells did not exhibit characteristics of intestinal cells. Some basement membranes had splits, duplications, or multiplications. Cytoplasmic organelles within hyperplastic biliary cells appeared unremarkable in contrast to the characteristic mitochondrial abnormalities present in neighboring hepatocytes. There was a positive correlation between histologic grades of cholangiofibrosis and ages of the animals (r = 0.68, P < 0.001), but no significant correlation between histologic grade and hepatic copper or iron content. We conclude that cholangiofibrosis is the predominant pathologic response to chronic liver injury induced by excess copper in LEC rats. The pathogenic role of copper in the development of cholangiofibrosis requires clearer definition.

Identification and analysis of mutations in the Wilson disease gene (ATP7B): population frequencies, genotype-phenotype correlation, and functional analyses.

Wilson disease (WD) is an autosomal recessive disorder characterized by toxic accumulation of copper in the liver and subsequently in the brain and other organs. On the basis of sequence homology to known genes, the WD gene (ATP7B) appears to be a copper-transporting P-type ATPase. A search for ATP7B mutations in WD patients from five population samples, including 109 North American patients, revealed 27 distinct mutations, 18 of which are novel. A composite of published findings shows missense mutations in all exons-except in exons 1-5, which encode the six copper-binding motifs, and in exon 21, which spans the carboxy-terminus and the poly(A) tail. Over one-half of all WD mutations occur only rarely in any population sample. A splice-site mutation in exon 12 accounts for 3% of the WD mutations in our sample and produces an in-frame, 39-bp insertion in mRNA of patients homozygous, but not heterozygous, for the mutation. The most common WD mutation (His1069Glu) was represented in approximately 38% of all the WD chromosomes from the North American, Russian, and Swedish samples. In several population cohorts, this mutation deviated from Hardy-Weinberg equilibrium, with an overrepresentation of homozygotes. We did not find a significant correlation between His1069Glu homozygosity and several clinical indices, including age of onset, clinical manifestation, ceruloplasmin activity, hepatic copper levels, and the presence of Kayser-Fleischer rings. Finally, lymphoblast cell lines from individuals homozygous for His1069Glu and 4 other mutations all demonstrated significantly decreased copper-stimulated ATPase activity.

Wilson disease and idiopathic copper toxicosis.

The pathogenic agent of both Wilson disease (WD) and non-Indian childhood cirrhosis (which we term idiopathic copper toxicosis, or ICT) is copper accumulating to excess in the liver. Inheritance of a pair of alleles of an autosomal recessive gene on chromosome 13 is necessary and sufficient to cause such copper accumulation in WD; reducing the dietary intake of copper cannot prevent the development of WD. In contrast, the lethal accumulations of copper in children with ICT have been attributed primarily to an increased dietary intake of copper. However, 64 124 child-year exposures of children under the age of 6 y to drinking water containing a copper concentration of approximately 125.9 micromol/L (8 mg/L) produced no deaths from any form of liver disease. Moreover, the ICT of seven infants was attributed primarily to drinking water containing < 110.2 micromol Cu/L (7 mg/L) despite evidence of the presence of a genetic defect in three of the patients, one of whom was exclusively breast-fed. These data suggest that ICT cannot be caused solely by increased dietary intake of copper and occurs only in children with an identified genetic defect.

The application of laser microprobe mass analysis to the study of biological material.

Laser microprobe mass analysis (LAMMA) is an investigational method which is a powerful tool for the identification and quantitation of various elements present in small volumes of tissue. LAMMA is highly sensitive and capable of rapidly detecting concentrations of 1-3 p.p.m. of most metallic elements, in precisely localized cellular compartments. In order to further assess its value, cultured skin fibroblasts and biopsy tissues from human subjects and experimental animals were probed by LAMMA, and the results were correlated with ultrastructural findings. Biopsy samples were obtained from patients suffering from Gaucher disease, and from patients and animals with pathologic iron or copper metabolism. No significant abnormalities were detected in the cultured fibroblasts from patients with Gaucher disease, in contrast to the iron content of tissue biopsy Gaucher cells, which was markedly increased, apparently as a consequence of erythrophagocytosis. Particularly intense iron-related peaks were found in liver cytosiderosis due to neonatal or genetic haemochromatosis, thalassaemia major and in animal models of iron overload. An additional finding was the presence of aluminium accumulation in siderosomes of different cells. In liver biopsy samples from human Wilson's disease and from rats with an inherited disorder causing copper toxicosis, copper-containing compounds were identified and localized, and their relative concentration was estimated by LAMMA. The present study showed that LAMMA is a valuable technique for the localization and estimation of relative abundance of trace elements in various tissues containing excessive amounts of metals.

An array of mitochondrial alterations in the hepatocytes of Long-Evans Cinnamon rats.

In an attempt to identify the cellular targets of copper toxicity, we studied the ultrastructure of hepatocytes in the livers of 23 Long-Evans Cinnamon (LEC) rats ranging in age from 10 to 89 weeks. The hepatic copper concentration ranged from 325 to 2,126 (mean, 930) micrograms/g dry weight. Thirteen rats displayed varying degrees of jaundice at the time of killing. Numerous nuclei were indented by cytoplasmic invaginations. Conspicuous abnormalities were displayed by mitochondria. These included marked pleomorphism; increased or diminished matrical density; rearrangement, elongation, dilatation, stacking, or disappearance of cristae; absence of matrical granules; presence of matrical inclusions of flocculent electron-dense deposits; and degenerative changes. The severity of the ultrastructural changes did not correlate with the hepatic copper concentration but did correlate with the degree of the icterus displayed by the rats. Certain mitochondrial changes resembled those encountered in the hepatocytes of patients with Wilson's disease, confirming that these organelles are important targets of copper toxicity.

Is non-Indian childhood cirrhosis caused by excess dietary copper?

Indian childhood cirrhosis is generally believed to be caused by toxic excesses of hepatic copper derived from milk boiled in copper vessels. Sporadic cases of a disorder indistinguishable from Indian childhood cirrhosis have appeared in other countries where the toxic hepatic copper has been thought to be derived from drinking water. In published reports of seven 2-year-old or younger infants with non-Indian childhood cirrhosis (five of whom died), the copper content of their drinking water--which the authors considered the essential, if not the sole, aetiological factor--ranged from 0.05 to 6.8 mg Cu/L. We identified three Massachusetts towns in which between 1969 and 1991 there were 64,124 child-years of exposure of children under the age of 6 years to drinking water that contained between 8.5 and 8.8 mg Cu/L. Data from the Massachusetts Department of Public Health showed that there were 135 deaths among these children, but no deaths from cirrhosis or any form of liver disease. These data, and evidence of a genetic aetiology in three of the seven infants reported previously, suggest that non-Indian childhood cirrhosis is an inherited disorder.

Pleiotropic effect of LEC mutation: a rodent model of Wilson's disease.

Metabolic studies with 67Cu were undertaken to identify the site of the cellular defect in copper metabolism in the Long-Evans Cinnamon (LEC) rat. The apparent rate of copper uptake by LEC primary hepatocytes was increased [maximal velocity (Vmax) = 259 pmol.min-1.mg protein-1] compared with controls (Vmax = 161 pmol.min-1.mg protein-1); however, Michaelis-Menten constant (Km) values were comparable (11.8 and 12.7 microM, LEC and control, respectively). Efflux of copper from LEC and control hepatocytes was similar from 0 to 15 min, but was reduced from 15 to 60 min in the former. Although hepatic copper contents were markedly elevated in LEC rats compared with controls (658 +/- 199 vs. 21.5 +/- 6.6 micrograms/g dry wt), biliary copper concentration was reduced in LEC rats compared with controls (0.187 vs. 1.39 +/- 0.66 microgram/ml). Subcellular fractionation of LEC liver homogenates revealed approximately 75% of copper to be present in cytosol, with gradients of copper concentration from cytosol to either lysosome or microsomal subcellular fractions. LEC rat bile and hepatic microsome and lysosome fractions contained smaller fractions of 67Cu administered intravenously as cupric acetate compared with control rats. However, recovery of 67Cu in bile and in lysosomal subcellular fractions were similar for LEC and controls following administration of 67Cu-labeled asialoceruloplasmin, which is targeted to lysosomes. This discordance suggests a possible defect in the entry of copper into lysosomes but normal delivery of lysosomal copper to bile. Based on these findings, we conclude that the mutation in LEC rats alters copper transport at more than one cellular site.

Liver transplantation for Wilson's disease: indications and outcome.

The objective of this study was to determine the indications for and results of liver transplantation in patients with Wilson's disease on the basis of results of a survey with retrospective review of data obtained on 55 transplants performed at centers in the United States and Europe. The study group comprised 32 females and 23 males, aged 8.5 to 51 yr, with features diagnostic of Wilson's disease. Indication for orthotopic liver transplantation included hepatic insufficiency (n = 32), wilsonian fulminant hepatitis (n = 21), intractable neurological Wilson's disease (n = 1) and gastrointestinal hemorrhage (n = 1). Forty-three patients have survived, at this writing, from 3 mo to 20 yr. Mean and median survival after orthotopic liver transplantation were 2.7 and 2.5 yr, respectively. Survival at 1 yr was 79%. Nonfatal complications occurred in five patients. Of the seven patients given transplants for hepatic insufficiency who manifested neurological and/or psychiatric manifestations at the time of orthotopic liver transplantation, four showed improvement of these symptoms. One patient given a transplant for intractable neurological disease improved but died of a vascular complication. Our data demonstrate that liver transplantation is life-saving but not without risk for patients with wilsonian fulminant hepatitis or chronic severe hepatic insufficiency unresponsive to medical therapy. Furthermore, neurological or psychiatric symptoms due to Wilson's disease may improve after liver transplantation; however, the role of this procedure in the management of patients with neurological Wilson's disease in the absence of hepatic insufficiency is still uncertain.

Fraternal concordance of types of abnormal hepatocellular mitochondria in Wilson's disease.

Three distinct patterns of structural abnormalities of mitochondria, indicated as types I, II and III and associated with steatosis, were identified in the hepatocytes of 40 of 42 asymptomatic and 8 of 22 symptomatic patients with documented Wilson's disease before treatment. No correlation was seen between the type of mitochondrial abnormality and the patient's age, hepatic copper concentration, degree of hepatic steatosis or serum aminotransferase level. However, comparison of the types of abnormal hepatocellular mitochondria displayed by five pairs and one trio of asymptomatic siblings revealed remarkably similar types of abnormalities in each family. The variety of mitochondrial types encountered in different families and the high degree of type identity in sibling relationships indicate that the structural changes are genetically determined.