THE COPPER PATHWAY

Dietary copper is absorbed in the upper intestine and binds loosely to albumin in serum. Albumin and copper-histidine transport copper to a variety of tissues; most copper is transported to the liver. Trace amounts of copper are required for essential enzymes that affect connective tissue and elastin cross-linking (lysyl oxidase), free radical scavenging (superoxide dismutase), electron transfer (cytochrome oxidase), pigment production (tyrosinase), and neurotransmission (dopamine β-monooxygenase). Copper in hepatocytes and in other cells is bound to metallochaperones, low-molecular-weight proteins that each deliver copper to a specific target molecule.

In the liver, copper is incorporated into the protein apoceruloplasmin to produce ceruloplasmin (also called holoceruloplasmin). More than 90% of the copper in plasma is an integral part of ceruloplasmin, an α₂-glycoprotein that contains six molecules of copper and has a molecular weight of 132 kd. The normal serum concentration of ceruloplasmin in adults, as measured by immunochemical or enzymatic techniques, is 200 to 400 mg/L, rising from a very low level at birth to 300 to 500 mg/L in the first years of life. Ceruloplasmin is an acute-phase reactant that is elevated by inflammation (including inflammatory hepatic disease), pregnancy, and the use of exogenous estrogen. The majority of ingested copper is excreted via the bile; a small fraction is excreted in urine. When intestinal or liver cells are overloaded with copper, the metallothioneins, a class of low-molecular-weight cysteine-rich proteins, are induced and sequester copper in a nontoxic form. The normal pathways of copper transport in the body and in the hepatocyte are shown in Figures 75-1 and 75-2.

Figure 75-1. Simplified overview of the pathways for copper ion transport and steps affected in genetic disorders of copper metabolism. MT, metallothioneins.

(Modified from Cox DW. Genes of the copper pathway. Am J Hum Genet 1995; 56:828-34.)

Figure 75-2. Model of a hepatocyte showing major proteins in the copper transport pathway. Low-molecular-weight copper chaperones (ATOX1, COX17, and CCS) deliver copper to specific target proteins (ATP7B, cytochrome oxidase, and superoxide dismutase, respectively). SCO1 transports copper across the mitochondrial membrane. ATP7B (shown as a channel) traffics from the trans-Golgi network (TGN) to cytoplasmic vesicles that deliver copper to the bile canaliculus. COMMD1 may be involved in excretion of copper into bile.

THE BASIC MOLECULAR DEFECT

Our understanding of the basic defect in Wilson disease increased dramatically with the cloning of the genes, first for X-linked Menkes disease and then for Wilson disease. The gene for Menkes disease (ATP7A), which was cloned by using a chromosomal breakpoint in an affected female patient, was found to be related to bacterial copper resistance genes. Cloning of the Wilson disease gene (ATP7B) was accomplished by a combination of linkage analysis, physical mapping of the relevant region of chromosome 13q14, and recognition of its extensive homology with the Menkes disease gene.^2,³ The coding region of the gene is 4.1 kilobases in length, with messenger RNA (mRNA) of about 8 kilobases. The product, ATP7B (or the Wilson ATPase), is a membrane P-type adenosine triphosphatase (ATPase) that consists of 1443 amino acid residues and has a molecular mass of 160 kd. The predicted structure,² as confirmed by structural studies,⁴ has six copper binding domains, a phosphorylation domain, an ATP-binding region, and eight transmembrane domains. (Fig. 75-3). All functionally important regions of the gene are conserved between bacteria and yeast. Mutations in the ATP7B gene result in retention of copper in the liver as well as impaired incorporation of copper into ceruloplasmin. The Long-Evans cinnamon (LEC) rat and the toxic milk (tx) mouse have mutations in their homologous ATP7B genes and are thus suitable models for the study of Wilson disease mechanisms and therapy.^5,⁶

Figure 75-3. A model of the predicted product of the Wilson disease gene, ATP7B. Functional domains conserved in the Menkes disease gene are indicated as blocks. Numerous mutations occur in functionally important regions. Positions of common missense mutations, H1069Q and R778L, are shown. ATP, adenosine triphosphate.

(Model modified from Bull PC, Cox DW. Wilson disease and Menkes disease: New handles on heavy-metal transport. Trends Genet 1994; 10:246-52.)

Although the gene for Menkes disease is expressed in many tissues, including muscle, kidney, heart, and intestine, the gene for Wilson disease is expressed predominantly in the liver and kidney, with minor expression in brain, lungs, and placenta.² Studies in cultured cells show localization of the Wilson ATPase in the trans-Golgi network, with trafficking from the trans-Golgi network to cytoplasmic vesicles in the presence of increased copper.⁷ When intracellular copper concentrations are elevated, the Wilson ATPase is found near the apical (bile canalicular) membrane in hepatocytes, consistent with its proposed function of facilitating excretion of copper via bile.⁸

Additional proteins are involved in the intracellular transport of copper. Molecular copper is not free in the cell but is transported to specific proteins by copper chaperones.⁹ The chaperone ATOX1 transports copper to the Wilson ATPase. Other chaperones deliver copper to superoxide dismutase in the cytoplasm and various copper-containing proteins in mitochondria.

The study of inherited hepatic copper toxicosis in Bedlington terriers has identified a new component of the copper transport system. Affected dogs show clinical variability that ranges from death or hepatic disease at two to three years of age to less severe chronic disease to a high level of hepatic copper. The proposed defective canine gene was identified by positional cloning through the use of markers to identify a region containing the COMMD1 (initially called the MURR1) gene. The gene has a deletion of one exon in some,¹⁰ but not all,¹¹ affected dogs. Although the gene responsible for the excess copper remains to be identified, the disease in Bedlington terriers has highlighted other genes that may be involved in response to excess copper; COMMD1 interacts not only with the Wilson ATPase,¹² but also with the X-linked inhibitor of apoptosis (XIAP), which can also bind copper.¹³ COMMD1 could be involved in response to, and facilitate elimination of, excess copper.

CLINICAL FEATURES

The clinical presentation of Wilson disease is extremely variable. The age at onset of symptoms is much broader than previously thought, ranging from 3 to 55 years. Wilson disease with hepatic involvement has been identified in one- to two-year-old patients and in patients older than age 60.¹⁴ Patients may present with chronic or fulminant liver disease, a progressive neurologic disorder without clinically prominent hepatic dysfunction, isolated acute hemolysis, or psychiatric illness. The clinical variability often makes confirmation of the diagnosis difficult. Further description of the clinical features and treatment has been published.^14,¹⁵

HEPATIC PRESENTATION

The hepatic presentation of Wilson disease is more common in younger patients than in older patients. Wilson disease should be considered as a possible diagnosis in any child, symptomatic or not, with hepatomegaly, persistently elevated serum aminotransferase levels, or evidence of fatty liver. Symptoms may be vague and nonspecific, such as fatigue, anorexia, or abdominal pain. Occasionally patients present with a self-limited clinical illness that resembles acute hepatitis, with malaise, anorexia, nausea, jaundice, elevated serum aminotransferase levels, and abnormal coagulation test results. Some patients have a history of self-limited jaundice, apparently caused by unexplained hemolysis. Patients may present with severe, established chronic liver disease with hepatosplenomegaly, ascites, congestive splenomegaly, a low serum albumin level, and persistently abnormal coagulation test results. Some patients have isolated splenomegaly without hepatomegaly. Many of these findings relate more to portal hypertension as a consequence of Wilson disease than to the metabolic disorder itself.

Wilson disease may present in children and young adults with clinical liver disease indistinguishable from autoimmune hepatitis.¹⁶ As in autoimmune hepatitis, the onset may be acute. Fatigue, malaise, arthropathy, and rashes may occur; laboratory findings include elevated aminotransferase levels, a greatly increased serum immunoglobulin (IgG) concentration, and detectable nonspecific autoantibodies such as antinuclear antibodies and anti-smooth muscle (anti-actin) antibodies. Wilson disease must be specifically ruled out because the treatment of the two diseases is entirely different. With appropriate treatment, the long-term outlook for patients with Wilson disease that manifests as autoimmune hepatitis appears to be favorable, even if cirrhosis is present.

Wilson disease may present as fulminant hepatic failure, with severe coagulopathy and encephalopathy. Acute intravascular hemolysis is usually present, and renal failure may develop. Because the patient typically has not been suspected of having underlying liver disease, fulminant viral hepatitis is usually the working diagnosis. Unlike fulminant viral hepatitis, fulminant Wilson disease is typically characterized by disproportionately low aminotransferase levels (usually much less than 1500 U/L) at the onset of clinically apparent disease. The serum alkaline phosphatase level is in the normal range or even low for the patient’s age, and the bilirubin level is often disproportionately elevated as a result of hemolysis. In adults who present with Wilsonian fulminant liver failure, the combination of a ratio of the alkaline phosphatase level to the total bilirubin level of less than 4 and a ratio of the aspartate aminotransferase (AST) to alanine aminotransferase (ALT) level of greater than 2.2 can be extremely helpful for making the diagnosis, whereas the serum ceruloplasmin level is not (see later).¹⁷ Slit-lamp examination may reveal Kayser-Fleischer rings (see later). Urinary copper excretion is greatly elevated. These patients require urgent liver transplantation because they do not respond well to chelation treatment; albumin dialysis and related techniques may serve as temporary procedures until liver transplantation can be performed (see later).¹⁸ This presentation of Wilson disease is not rare, and affected patients account for a substantial proportion of persons transplanted for fulminant hepatic failure (see Chapters 93 and 95).

Recurrent bouts of hemolysis may predispose to the development of gallstones. Cirrhosis, if present, may be a further predisposing factor. Children with unexplained cholelithiasis, particularly with small bilirubinate stones, should be tested for Wilson disease. Compared with other chronic liver diseases, Wilson disease is rarely complicated by hepatocellular carcinoma; however, patients may have an increased propensity to abdominal malignancies,¹⁹ and hepatocellular carcinoma has been reported in a child with Wilson disease.

In patients who have predominantly hepatic disease, evidence of subtle neurologic involvement can often be found. Mood disturbance (mainly depression, but sometimes impulsive or neurotic behavior), deterioration in school performance or handwriting, and clumsiness may be identified by careful direct questioning. A soft whispery voice (hypophonia) is another early feature of neurologic involvement.

NEUROLOGIC PRESENTATION

The neurologic presentation of Wilson disease tends to occur in the second and third decades or later but has been reported in children as young as 6 to 10 years old. Most patients with a neurologic presentation have hepatic involvement, albeit often asymptomatic. Neurologic involvement follows two main patterns: movement disorder or rigid dystonia. Movement disorders tend to occur earlier and include tremors, poor coordination, and loss of fine motor control. Spastic dystonic disorders generally develop later, with mask-like facies, rigidity, gait disturbance, and pseudobulbar involvement such as dysarthria, drooling, and swallowing difficulty. Rarely, patients present with peripheral neuropathy or dysautonomia. Seizures are uncommon, and intellect is not impaired.

Imaging of the brain is important for assessing neurologic Wilson disease, and results may be abnormal in the absence of overt neurologic symptoms. Magnetic resonance imaging may be the most sensitive modality.

PSYCHIATRIC PRESENTATION

As many as 20% of patients may present with purely psychiatric symptoms. These symptoms are highly variable, although depression is common. Phobias and compulsive behaviors have been reported; aggressive or antisocial behavior may also be seen.

OCULAR SIGNS

The classic Kayser-Fleischer ring is caused by copper deposition in Descemet’s membrane of the cornea. Copper is actually distributed throughout the cornea, but fluid streaming favors accumulation near the limbus, especially at the superior and inferior poles, and eventually circumferentially around the iris. Kayser-Fleischer rings are visible on direct inspection only when iris pigmentation is light and copper deposition is heavy. A careful slit-lamp examination is mandatory. Copper deposition in the lens (sunflower cataract), which does not interfere with vision, may be seen on slit-lamp examination and, like Kayser-Fleischer rings, disappear with chelation therapy. Kayser-Fleischer rings may be absent in 40% to 60% of patients with exclusively hepatic involvement and in presymptomatic patients. Most patients with a neurologic or psychiatric presentation of Wilson disease have Kayser-Fleischer rings; only 5% do not. Kayser-Fleischer rings are not specific for Wilson disease; they may be found occasionally in patients with other types of chronic liver disease, usually with a prominent cholestatic component, such as primary biliary cirrhosis, primary sclerosing cholangitis, or familial cholestatic syndromes, and rarely in patients with nonhepatic diseases.

INVOLVEMENT OF OTHER SYSTEMS

Wilson disease can be accompanied by various extrahepatic disorders apart from neurologic disease. Episodes of hemolytic anemia can result from the sudden release of copper into the blood. Renal disease, mainly Fanconi’s syndrome, may be prominent. Findings include microscopic hematuria, aminoaciduria, phosphaturia, and defective acidification of the urine. Nephrolithiasis has also been reported. Arthritis, affecting mainly the large joints, may occur as a result of synovial copper accumulation. Other musculoskeletal problems include osteoporosis and osteochondritis dissecans. Vitamin D–resistant rickets may develop as a result of the renal damage. Copper deposition in the heart can lead to cardiomyopathy or cardiac arrhythmias. Sudden death in Wilson disease has been attributed to cardiac involvement but is rare. Copper deposition in skeletal muscle can cause rhabdomyolysis. Endocrine disorders can occur. Hypoparathyroidism has been attributed to copper deposition. Amenorrhea and testicular problems appear to result from Wilson disease itself, not from cirrhosis. Infertility or repeated spontaneous abortion may be a sign of Wilson disease. Pancreatitis, possibly resulting from copper deposition in the pancreas, may also occur.

PATHOLOGY

In the earliest stages before cirrhosis develops, histologic findings in the liver include steatosis, focal necrosis, glycogenated nuclei in hepatocytes, and sometimes apoptotic bodies. As parenchymal damage progresses, possibly through repeated episodes of lobular necrosis, periportal fibrosis develops. Cirrhosis is usually macronodular but may be micronodular.

Early in the course of Wilson disease, hepatocellular copper is bound mainly to metallothionein and is distributed diffusely in the cytoplasm of hepatocytes; therefore, histochemical stains for copper are negative. As the disease progresses, the copper content exceeds the storage capacity of metallothionein, and copper is deposited in lysosomes. Lyosomal aggregates of copper can be detected by special staining techniques for copper or copper-binding protein (such as rubeanic acid or orcein, respectively). In the cirrhotic liver, some areas may have no stainable copper at all. If the clinical presentation mimics autoimmune hepatitis, a liver biopsy specimen may reveal classic histologic features such as interface hepatitis. Inflammation may be severe. Mallory (Mallory-Denk) bodies may be found. In patients who present with fulminant hepatic failure, histologic findings confirm preexisting liver disease; cirrhosis may be present; and parenchymal copper is located mainly in Kupffer cells rather than hepatocytes.

Changes in hepatocellular mitochondria, identified with electron microscopy, are an important feature in Wilson disease.²⁰ The mitochondria vary in size, and the numbers of dense bodies in mitochondria may be increased. The most striking change is dilatation of the tips of the mitochondrial cristae as a result of separation of the inner and outer membranes of the cristae, with widening of the intercristal space until the appearance is irregularly cystic. The crista resembles a tennis racquet if only the tip is dilated. This finding, although not entirely specific for Wilson disease, can be helpful diagnostically, even in young and minimally affected patients. Involvement of hepatocytes may be not uniform, and abnormalities may be found in some hepatocytes in some lobules and not in others. The mitochondrial changes are probably a consequence of oxidative damage from excessive copper in hepatocytes.²¹

DIAGNOSIS

The patient with the classic combination of chronic liver disease, tremor or dystonia, and Kayser-Fleischer rings is readily diagnosed on clinical grounds, but such patients are uncommon. A diagnostic scoring system has been proposed ²² but has been evaluated only in children. Suggestive clinical symptoms are often the main prerequisite for diagnosing Wilson disease, and laboratory investigations may provide confirmation. Kayser-Fleischer rings should be sought through a careful slit-lamp examination and repeated if necessary. Lack of Kayser-Fleischer rings does not exclude the diagnosis of Wilson disease. Routine liver biochemical test levels are usually abnormal, with mild-to-moderate elevations of the serum aminotransferase levels. Serum levels of the ALT may be much lower than those of AST, possibly reflecting damage to hepatocellular mitochondria.

Two major disturbances of copper disposition in Wilson disease are defective incorporation of copper into ceruloplasmin and decreased biliary excretion of copper. A summary of biochemical features in Wilson disease in comparison with normal persons is shown in Table 75-1

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