Wilson disease is an autosomal-recessive disorder resulting in hepatic cirrhosis and neuronal degeneration. Although sporadic reports of a similar clinical syndrome appeared in the literature as early as 1850, this disease was recognized as a distinct clinical entity in 1912 by Samuel Alexander Kinnear Wilson, who reported several cases of a new familial disorder resulting in progressive degeneration of the lenticular nuclei invariably associated with hepatic cirrhosis at autopsy.91 Wilson disease is observed in all ethnic groups and occurs worldwide, with an estimated frequency of 1 in 30,000 individuals and a carrier rate of 1 in 90.92,93 This frequency is increased in populations from regions of the world where consanguinity was once a more common practice. In 1985, analysis of one such kindred established linkage of the Wilson disease gene to the esterase D locus on chromosome 13.94
In 1993, the Wilson disease gene was cloned and shown to encode a novel member of the family of cation-transporting P-type ATPases.95– 97 Sequence comparison and hydropathy analysis of the derived amino acid sequence of the Wilson protein reveals a polytopic membrane protein predicted to transport copper across the lipid bilayer in an ATP-dependent manner (Fig. 126-4). Homologous copper-transporting P-type ATPases have now been identified in a wide variety of prokaryotic and eukaryotic species.98,99 The Wilson protein contains a number of amino acid motifs characteristic of such ATPases including MXCXXC copper-binding sequences in the N-terminus, a transmembrane CPC thought to be essential for metal transfer across the membrane, an ITGEA phosphatase domain, a conserved DKTGT sequence that is the site of the aspartyl-phosphate intermediate essential for energy transduction, and a GDGVND sequence forming the ATP-binding domain (Fig. 126-4). In addition to these well-defined functional motifs, a highly conserved sequence, SEHPL, is found proximal to the ATP-binding domain. The histidine residue in this sequence, conserved among all known copper-transporting P-type ATPases,100 is the site of the most common disease mutation (H1069Q) in patients with Wilson disease.101 Although polymerase chain amplification studies suggest the possibility of tissue-specific splicing of the Wilson disease gene, the only isoform of the Wilson protein identified thus far is a truncated species of unknown function generated by alternate promoter usage in the rat pineal gland.102, 102a
Structural model of the Wilson ATPase in the trans-Golgi membrane with conserved functional motifs also present in the homologous Menkes ATPase. Asterisk indicates the conserved histidine, which is the site of the most common mutation in Wilson disease. (Modified with permission from Payne and Gitlin. 169)
The Wilson disease gene is encoded by 21 exons spanning more than 60 kb on chromosome 13q14.3. Analysis of patient mutations reveals an enormous molecular heterogeneity consisting of a very small number of frequent mutations that are population specific, as well as a much greater number of rare individual alleles. To date almost 100 different mutations have been characterized in Wilson disease patients from varying ethnic origins.103– 113 Of these, more than half are missense mutations occurring in transmembrane domains or the ATP-binding region. The remainder of the characterized mutations consist of small deletions or insertions (25 percent), splice site abnormalities (10 percent), and nonsense mutations (10 percent). The inability to identify a specific mutation in about 20 percent of examined alleles suggests the presence of mutations in upstream regions controlling transcription of the Wilson disease gene.101 Of the common mutations, H1069Q accounts for about 40 percent of the alleles in populations of Northern European origin.101 Similarly, A778L, a common missense mutation in transmembrane domain 4, has been identified in up to 30 percent of the alleles in Asian populations.110,114
At first pass it is reasonable to assume that the degree of allelic heterogeneity at the Wilson locus might account, at least in part, for the enormous clinical variability observed in patients with Wilson disease. However, as expected from the large number of different mutations, the majority of affected individuals are compound heterozygotes, making it difficult to study any correlation between genotype and clinical phenotype. Nevertheless, such studies have been conducted in individuals homozygous for the H1069Q mutation, and the results revealed no correlation between this mutation and the age of onset, clinical manifestations, biochemical markers of disease, or disease activity in affected patients.111 These findings are consistent with the observation of marked clinical variation among affected sibs, and even identical twins, and supports the notion that additional genetic and environmental factors may significantly influence the outcome of a given disease mutation at the Wilson locus.115 Although it remains possible that specific mutations such as those leading to variations in splicing may result in a less severe clinical phenotype owing to the presence of residual functional protein, no data are currently available on the abundance of the Wilson protein in tissues from such patients.
The Wilson disease gene encodes a 7.5-kb transcript that is abundantly expressed in the human liver.95– 97 Polyclonal antisera generated against the N-terminus of the Wilson ATPase detect a 165-kDa protein that is synthesized in hepatocytes as a single-chain polypeptide localized to the trans-Golgi network22 (Fig. 126-5). Mutations resulting in the absence or dysfunction of the Wilson protein interrupt the transport of copper into the secretory pathway, interfering with holoceruloplasmin synthesis and biliary copper excretion. In support of this model, expression of wild-type but not mutant Wilson proteins in the ccc2Δ strain of S. cerevisiae restores copper incorporation into the multicopper oxidase FET3,116 providing direct evidence of copper transport by the Wilson protein.22
Immunofluorescent colocalization of the Wilson protein (A) and the trans-Golgi 58-kDa marker (B) in the human liver. Arrowheads focus on regions of colocalization within hepatocytes.
Although the yeast system is useful to directly evaluate the effect of specific mutations on the copper transport function of the Wilson protein, observations on the cell biology of this ATPase suggest a greater complexity to the pathogenesis of the disease. In normal individuals, an increase in the intracellular copper concentration of the hepatocytes results in translocation of the Wilson protein to a cytoplasmic vesicular compartment. As copper is transported into this compartment by the Wilson ATPase, cytoplasmic copper content decreases, resulting in the redistribution of the protein to the trans-Golgi network. This process is rapid and independent of new protein synthesis, providing a mechanism for immediate response of the Wilson protein to changes in the steady-state intracellular copper concentration22,28 (Fig. 126-6). Analogous to what has been observed in cystic fibrosis,117 patient mutations might be predicted to affect Wilson protein function not only at the level of transporter activity, but also through alteration of amino acids necessary for proper localization of the transport protein to a specific compartment. In the case of the Wilson protein, mutations also may affect responsiveness to intracellular copper concentrations. In support of this paradigm, expression of the wild-type Wilson protein and the H1069Q mutant in a Menkes copper transporter–deficient mottled fibroblast cell line revealed that the H1069Q mutation causes a temperature-sensitive defect in protein folding, resulting in mislocalization and rapid degradation of the mutant protein in the endoplasmic reticulum.118, 118a Additional genetic and environmental factors affecting one or more of these steps are likely candidates to explain the clinical heterogeneity of Wilson disease. Although heterozygotes do not develop signs and symptoms of Wilson disease, the paradigm presented above also raises the possibility that the presence of certain specific single mutant alleles may increase the susceptibility of the liver to damage in other more common liver disorders such as alcoholic cirrhosis.119
Immunofluorescent detection of the copper-mediated recycling of the Wilson protein in HepG2 cells incubated in calf serum alone (A and D), or in the presence of excess copper (B and E) or the copper chelator bathocupronine disulfonate (C and F). In some experiments (D–F), cells were preincubated with cycloheximide prior to incubation. (Reprinted with permission from Hung et al.22 )
A spontaneously arising mutant strain, the Long Evans Cinnamon (LEC) rat, develops hepatitis in association with elevated hepatic copper content, decreased serum ceruloplasmin concentration, and impaired biliary copper excretion.120,121 LEC rats lack expression of the rat orthologue of the Wilson disease gene due to an extensive intragenic deletion, and these rodents are therefore considered to be a bona fide animal model of Wilson disease.122,123 Most LEC rats present with acute hepatitis accompanied by jaundice, hemolytic anemia, and fulminant liver failure, with survivors progressing to chronic hepatitis and cirrhosis.124 The hepatic copper toxicosis in LEC rats is responsive to penicillamine chelation therapy,125 and holoceruloplasmin biosynthesis can be restored by adenovirally mediated expression of the human Wilson disease gene in vivo,126 making it likely that this rodent model will be useful in further understanding the pathogenesis and treatment of Wilson disease. In contrast to humans, hepatocellular carcinoma is a common event in the LEC rat and appears to be related to a concomitant disturbance in hepatic iron homeostasis.127
The “toxic milk mouse” was originally identified by the observation that newborn mice from affected mothers are copper deficient when suckled on their mother's milk.128 Although neonatal copper deficiency is not observed in the children of women with Wilson disease, adult toxic milk mice do develop progressive liver disease in association with elevated hepatic copper content and decreased serum ceruloplasmin concentration. An M1356V missense mutation in a highly conserved methionine in the eighth transmembrane domain of the murine orthologue of the Wilson disease gene in these mice suggests that this strain is also a model of Wilson disease although no functional data on this mutation is currently available.129 Presumably the unique phenotypic differences in this murine model reflect species-specific environmental and genetic factors that considerably modify disease presentation in comparison with that observed in LEC rats and humans.
Although the signs and symptoms of Wilson disease can be protean, the overwhelming majority of patients present with hepatic or neuropsychiatric disease.92,115 The diverse clinical manifestations of Wilson disease require alertness to the possibility of this diagnosis in any patient with unusual symptoms and tests indicating abnormal hepatic function.93 Liver dysfunction is the most common initial manifestation of Wilson disease in children.130 Patients presenting with hepatic disease usually do so at an average age of 10 to 13 years, which is at least 10 years earlier than those initially presenting with neurologic symptoms. Nevertheless, there is considerable overlap, and individual patients have presented with hepatic disease as late as the sixth decade of life. The manifestations of liver disease range from asymptomatic individuals with mild elevation of serum transaminases to chronic active hepatitis and cirrhosis.131 In some cases the presentation may be fulminant hepatic failure, suggesting that a viral illness or other external factor has triggered the abrupt onset of hepatic degeneration in the copper-loaded liver. Such a fulminant presentation is more common in women and frequently is accompanied by hemolytic anemia due to excess copper abruptly released into the bloodstream.132 Hepatocellular carcinoma is a rare consequence of cirrhosis in Wilson disease. Regardless of the presenting symptoms, most patients will have evidence of cirrhosis on liver biopsy as a result of many years of hepatocyte copper accumulation prior to clinical symptoms. Initially such biopsy samples may reveal micronodular cirrhosis with variable copper deposition throughout the lobules, but as symptoms worsen, the histology progresses to that of chronic hepatitis with nodular regeneration.133 The eventual hepatocyte dysfunction from copper overload results in cell death, with release of copper into the bloodstream.134
Although neuropsychiatric symptoms are also common at presentation, occurring in upward of 60 percent of all patients, such individuals are generally older than those presenting with liver disease and are most often diagnosed in the third or fourth decade of life. Neurologic manifestations include Parkinsonian symptoms with marked diminution in facial expressions and movement, pseudosclerotic patients with tremors resembling those of multiple sclerosis, and dystonic individuals with hypertonicity and choreoathetosis.135 These clinical symptoms reflect the changes in the basal ganglia observed at autopsy, which include cavitary degeneration, gliosis, and neuronal loss. The presence of copper in these brain regions as well as specific structural changes can be detected by neuroimaging techniques, which at times may be useful in diagnosis.136 The mechanisms resulting in the specific involvement of the basal ganglia as well as relative sparing of the motor and sensory cortex in patients with Wilson disease, despite a diffuse increase in copper throughout the central nervous system, are unknown.
Isolated or combined psychiatric illness also may occur in patients with Wilson disease and can include abnormal behavior, personality changes, depression, and marked impairment in cognition with evidence of schizophrenia.137,138 These neuropsychiatric symptoms are also the result of copper accumulation, although little or no information is available as to the mechanisms leading to specific brain dysfunction in such cases. Because patients initially presenting with psychiatric symptoms will eventually progress to the development of neurologic degeneration and hepatic failure, awareness of Wilson disease as a potential cause of such symptoms is essential to early diagnosis and treatment.
As with hepatic and neuropsychiatric manifestations, signs and symptoms in patients with Wilson disease may arise in any organ in which excess copper is deposited.115 Such copper often may be detected in the limbus of the cornea by slit-lamp examination (Kayser-Fleischer rings). This finding, frequently present in patients with neuropsychiatric disease, is not pathognomonic and occurs in individuals with increased serum copper resulting from other disorders such as cholestatic liver disease. Similarly, excess copper may occasionally be detected as azure lunulae in the fingernails. Less common clinical manifestations related to copper toxicosis include Fanconi syndrome with aminoaciduria, nephrolithiasis, cardiac dysrhythmias, arthritis and arthralgias, rhabdomyolysis, hemolytic anemia, hypoparathyroidism, and amenorrhea. These symptoms are reversed by systemic chelation therapy, confirming the etiology as copper accumulation rather than any associated hepatic disease.92,115
The diagnosis of Wilson disease is based on clinical signs and symptoms in correlation with tests that can detect abnormalities of copper metabolism. Careful diagnostic evaluation should be performed in any individual with isolated elevated serum transaminases, chronic active hepatitis of undetermined etiology, Kayser-Fleischer rings, unexplained psychiatric symptoms including sudden behavioral changes in childhood, or basal ganglia abnormalities on neuroimaging. In patients with Wilson disease, an inability to efficiently transfer copper into the secretory pathway of hepatocytes results in serum ceruloplasmin concentrations that are generally decreased well below normal values (20 mg/dl) owing to secretion and rapid degradation of the apoprotein. Because ceruloplasmin is an acute-phase reactant, the synthesis of this protein will increase during infection or inflammation, so the serum ceruloplasmin concentration may be in the normal range in some 5 percent of patients.139 However, in such cases analysis of the ceruloplasmin oxidase activity will reveal that this is circulating apoprotein devoid of copper.140 Urinary copper concentrations may be elevated (often >100 μg Cu/24 h) and are a cost-effective way of screening individuals. In all cases where it is possible, a liver biopsy should be performed for accurate quantitative measurement of hepatic copper concentration. The normal copper concentration in liver is often increased in affected patients (>250 μg/g dry wt) but it should be remembered that hepatic copper concentration also can be elevated in any condition in which biliary copper excretion is impaired. The measurement of hepatic copper concentration generally permits a definitive diagnosis to be made because even presymptomatic individuals will have elevated levels at the time of diagnosis. Occasionally, when the serum ceruloplasmin concentration is in the normal range and liver biopsy is contraindicated, an oral radioactive copper loading test may be helpful to demonstrate the lack of holoceruloplasmin biosynthesis.93,140
Based on physical examination, slit-lamp examination, and laboratory data, the diagnosis of Wilson disease can be made with accuracy in the majority of patients. Nevertheless, the wide range of age of onset and variable symptomatology can make a diagnosis difficult. Although direct molecular diagnosis is problematic due to the degree of molecular heterogeneity at the Wilson locus, analysis for common mutations in patients of specific ethnic origins may be useful.141 Such analysis is complicated by the fact that most patients will be compound heterozygotes carrying two different mutations of the gene. The identification of unusual haplotypes in affected family members with Wilson disease also may lend support to the diagnosis of this disorder in circumstances where family studies can be used.101 In such cases these may be followed up by direct identification of the mutation in affected individuals.
Given the low heterozygote frequency and the lack of a useful biochemical marker in the newborn period, screening for Wilson disease is confined to sibs and first-degree relatives of affected patients.119 Careful history and physical examination along with ophthalmologic studies and measurement of serum ceruloplasmin, liver transaminases, and urinary copper excretion should be performed. Such an evaluation may suggest the diagnosis of Wilson disease in asymptomatic individuals and may support the need for liver biopsy. In individuals heterozygous for Wilson disease, the results of this evaluation will be entirely normal, with the exception of the serum ceruloplasmin concentration, which may be decreased in 20 percent of heterozygotes. In those cases where the proband mutation has been identified, direct molecular screening can offer a rapid approach to diagnosis. Reproductive genetic counseling should be offered to all identified carriers.
Indian childhood cirrhosis is a disorder of progressive liver failure in young children associated with marked hepatic copper overload (>800 μg/g dry wt) and hepatocellular necrosis.142,143 Serum ceruloplasmin concentrations in affected children are always elevated, suggesting impairment in biliary copper excretion at a step beyond the entry of copper into the hepatocyte secretory pathway. This disorder is confined to India and appears to result from increased dietary copper consumption in genetically susceptible individuals; a history of consanguinity and repetitive use of brass and copper cooking vessels is usually present.143 Consistent with this concept, studies of excess dietary copper intake in otherwise normal individuals reveal no evidence of hepatic copper accumulation or liver disease.144 Similar disorders termed idiopathic copper toxicosis, endemic Tyrolean cirrhosis, and non-Indian childhood cirrhosis also have been reported and here also epidemiologic studies suggest a combination of genetic and environmental factors in disease pathogenesis.145,146 Hepatic copper concentrations also may be elevated in cholestatic liver disease, but in these conditions serum ceruloplasmin is elevated, indicating a defect late in the copper excretory pathway.
The goal of treatment in Wilson disease is to restore normal copper homeostasis through systemic chelation therapy directed at removing or detoxifying accumulated copper. D-penicillamine is the treatment of choice for systemic chelation therapy.115 The precise mechanism by which penicillamine results in detoxification and elimination of copper remains unclear. Long-term treatment promotes urinary copper excretion and is effective for prophylaxis in presymptomatic patients. Long-term evaluation with neuroimaging often reveals a decrease in copper-related abnormalities, which correlates with clinical improvement.147,148 Therapy is initiated with a small test dose that if tolerated is then given orally in four divided doses. Penicillamine is taken an hour before eating to prevent chelation with food, and a gradual response in symptoms should occur within the first several months of therapy. If improvement does not ensue, the dose may be increased twofold. Although most patients will become asymptomatic within 4 months after starting penicillamine, occasionally neurologic symptoms may worsen, presumably as a result of increased deposition of mobilized hepatic copper within brain tissue. Once improvement has occurred and decreased total body copper content has been demonstrated, patients should be placed on maintenance therapy at half the initial dose. Assuring compliance with therapy is essential because rapid deterioration can occur following the abrupt discontinuation of penicillamine related to the sudden release of copper from sites where this metal was sequestered in a nontoxic form.93
Urinalysis and complete blood counts should be followed at regular intervals in all patients taking penicillamine because hypersensitivity reactions, including fever, lymphadenopathy, and blood dyscrasias, may occur in up to 20 percent of patients. Although these side effects are dose related and can be controlled with corticosteroids, autoimmune symptoms are an indication to discontinue therapy immediately. In such cases, triethylenetetramine dihydrochloride (trientine) is a reasonable alternative therapy.149,150 Trientine may be taken orally at a dose of 1 to 2 g divided before meals, and although it is a somewhat less effective copper chelating agent, significant improvement has been reported in a number of patients. Neither chelating agent has been reported to have teratogenic effects in humans, and both drugs have been used successfully in pregnant women with Wilson disease.151
The treatment of patients with Wilson disease also should include prevention by dietary restriction of foods rich in copper, such as nuts, liver, chocolate, and shellfish. Absorption of copper also can be limited by the oral administration of zinc salts. The presumed effect of such treatment is the induction of metallothionein within the enterocyte, with subsequent intracellular chelation of copper impairing absorption.131 Ammonium tetrathiomolybdate also may be used as an adjunct in preventing copper toxicity. Thiomolybdate forms complexes with copper in the diet, resulting in nontoxic complexes of copper within the plasma and tissues. However, the toxicity of molybdate, including bone marrow suppression, has thus far limited the usefulness of this approach in most patients.92
Orthotopic liver transplantation is the treatment of choice in individuals with progressive hepatic insufficiency who do not respond to systemic chelation therapy.93 In addition, this approach is the only reasonable option in patients presenting with fulminant hepatitis. In both cases, patients fare well, with 1-year survival rates exceeding 80 percent in most studies. Hepatic transplantation will result in complete normalization of copper homeostasis within 6 months and usually results in sustained improvement in symptoms.152 Although many of the neurologic deficits as well as psychiatric symptoms in patients with Wilson disease may resolve following liver transplantation, refractory neurologic disease alone is not currently considered an appropriate criterion for hepatic transplantation.93