Abstract
Excess copper in the liver is toxic in humans and other mammals, and may lead to acute or chronic hepatitis, steatohepatitis, acute liver failure, cirrhosis and death. Of the several human copper storage diseases that have been described, the molecular basis of only Wilson disease is understood with the discovery of the Wilson disease gene (ATP7B) in 1993. The therapeutic success using oral copper chelating agents and zinc therapy make Wilson disease one of the few treatable genetic metabolic liver diseases. In cases with a fulminant presentation or advanced disease at diagnosis, copper chelation is ineffective and liver transplantation may be lifesaving. Indian Childhood Cirrhosis (ICC) has been defined as a copper-storage disorder precipitated by increased copper intake which affects young children primarily of Indian descent, and which progresses to cirrhosis and death before age three to four years without treatment. Children from North America, Asia, Austria, Germany and other countries have been described with a similar condition, which has been termed idiopathic copper toxicosis (ICT). Several newer disorders of hepatic copper metabolism have been recently described.
Introduction
Excess copper in the liver is toxic in humans and other mammals, and may lead to acute or chronic hepatitis, steatohepatitis, acute liver failure, cirrhosis and death. Of the several human copper storage diseases that have been described, the molecular basis of only Wilson disease is understood with the discovery of the Wilson disease gene (ATP7B) in 1993. The therapeutic success using oral copper chelating agents and zinc therapy make Wilson disease one of the few treatable genetic metabolic liver diseases. In cases with a fulminant presentation or advanced disease at diagnosis, copper chelation is ineffective and liver transplantation may be lifesaving. Indian Childhood Cirrhosis (ICC) has been defined as a copper-storage disorder precipitated by increased copper intake which affects young children primarily of Indian descent, and which progresses to cirrhosis and death before age three to four years without treatment. Children from North America, Asia, Austria, Germany and other countries have been described with a similar condition, which has been termed idiopathic copper toxicosis (ICT). Several newer disorders of hepatic copper metabolism have been recently described. In this chapter, copper physiology and mechanisms of copper hepatotoxicity will be reviewed, followed by descriptions of the major copper-storage diseases of childhood.
Copper Absorption and Metabolism
The normal adult Western diet contains 2–5 mg per day of copper. The efficiency of copper absorption in adults ranges from 40% to 60% [1], with higher absorption at lower intakes (Figure 28.1). Foods containing high amounts of copper include unprocessed wheat, dried beans, peas, shellfish (particularly oysters), chocolate, liver and kidney. The estimated daily copper requirement for adults is approximately 0.9–1.7 mg [1]. Dietary and chemical factors may impair copper absorption. For example, excess intake of zinc, cadmium, and ascorbic acid can interfere with copper bioavailability because of the formation of insoluble copper salts at an alkaline pH [2]. A vegetarian diet as well as ingested raw meat have been associated with decreased copper absorption. Gastrointestinal secretions (e.g., saliva, gastric juice, and duodenal secretions) form low molecular weight soluble complexes that aid in the absorption of copper by preventing the precipitation of copper salts [2], and certain digestive products (e.g., L-amino acids) facilitate copper absorption. It is the balance between these exogenous and endogenous factors that regulate the extent of intestinal absorption of copper. Because dietary intake and absorption generally exceed metabolic needs, a large amount of ingested copper is eventually excreted in bile (Figure 28.1), the major regulator of copper homeostasis.
Figure 28.1 Copper balance in man. Relative amounts of copper arising from 5 mg of daily dietary intake in adults.
Within the small intestine epithelial cells, absorbed copper is bound to metallothionein or is complexed to amino acids and transported into the portal venous circulation by the copper transporting enzyme ATP7A. Although enterocyte metallothionein also binds zinc and cadmium, copper is bound most avidly. Because zinc stimulates metallothionein synthesis, it has been shown that increased dietary zinc impairs copper absorption by causing retention of metallothionein-bound copper in the enterocyte, which is then excreted in the feces following desquamation of the enterocyte. This mechanism forms the basis for oral zinc therapy in Wilson disease.
Once absorbed into the portal venous blood, copper is complexed to albumin and amino acids in equilibrium with a very small fraction of free ionic copper. Copper is then transported into hepatocytes by a specific membrane transport system for albumin-bound copper by the high affinity copper uptake protein 1 (CTR1) [1] (Figure 28.2). Among the amino acids present in blood, the binding affinities for copper in decreasing order are histidine, threonine, glutamine, and asparagine. This amino acid-bound copper is most likely the form in which copper is transported to various tissues other than the liver. Within three hours of absorption, 60–90% of copper has been transported to and taken up by hepatocytes where copper initially interacts with low molecular weight ligands, such as cytosolic metallothionein, glutathione, and HAH1 [3]. The function of these proteins is to store copper for subsequent metabolic needs of the hepatocyte, to bind and detoxify excess copper, and to provide copper to chaperones that assist in incorporating it into essential proteins that are secreted (e.g., ceruloplasmin) or assist in copper excretion in bile (ATP7B). Important hepatic copper metalloenzymes include superoxide dismutase (MW of 32,000 Dalton), mitochondrial monoamine oxidase (MW 195,000 Dalton), cytochrome c oxidase (MW 290,000 Dalton) and ceruloplasmin. In other tissues, copper is incorporated into tyrosinase and lysyl oxidase.
The ATP7B gene (80Kb) is located on the short arm of chromosome 13, contains 21 exons and 20 introns, and is highly expressed in liver and kidney with lower levels of expression in lung, placenta, and other tissues. Splicing variants of the gene may be present in brain. The encoded protein, ATP7B, is a P-type cation-transporting ATPase, homologous to the Menkes disease gene product (ATP7A) and the copper transporting ATPase (copA) in copper-resistant enterococcus hirae. The protein structure (Figure 28.3) includes domains for copper binding (six sites), ATP-binding, a phosphorylation domain, a transmembrane cation channel, and a transduction domain. Within one membrane-spanning domain is a sequence of amino acids (cysteine-proline-cysteine) that is characteristic of all metal-transporting ATPases. ATP7B has two functions within the hepatocyte. ATP7B is located in the hepatocyte trans-Golgi network and, after sequestering excess copper in the cytoplasm, transports copper into vesicles bound for lysosomes and eventual excretion into the bile canaliculus (Figure 28.2). This process represents the main homeostatic mechanism for copper metabolism in man. Copper conjugated to glutathione is a minor pathway of copper excretion into bile. In the trans-Golgi network, ATP7B also activates ceruloplasmin by packaging six Cu molecules into apoceruloplasmin, which is then secreted into the plasma. When ATP7B is mutated in Wilson disease, both copper secretion into bile and copper incorporation into ceruloplasmin are impaired, resulting in copper accumulation within the hepatocyte.
Figure 28.3 Structural features of ATP7B, the Wilson disease gene, include six cysteine-rich metal binding regions (open boxes) near the amino terminus in the cytosol of the cell; eight transmembrane domains, one of which includes the cysteine-proline-cysteine (CPC) sequence common to other metal transporters; the DKTG phosphatase and phosphorylation domain; the TGDN ATP-binding domain; and the hinge region (GDGVND).
Metallochaperones play an essential key role in copper homeostasis in mammalian (and yeast) cells. These proteins control delivery of copper to specific intracellular targets, in which copper is incorporated into synthesis of critical enzymes and proteins [4]. Three mammalian copper chaperones (Figure 28.2) have been well characterized including: human CCS (homologous to yeast Lys7p) which delivers copper to copper/zinc superoxide dismutase (SOD1) in cytosol; human COX17 (homologous to yeast Cox17p) which delivers copper to SCO1 and SCO2 for subsequent incorporation into cytochrome c oxidase in mitochondria [25]; and human ATOX1 (homologous to yeast Atx1p) which shuttles copper to copper transporting ATPases, including ATP7A and ATP7B (homologous to yeast Ccc2p) in the trans-Golgi compartment [5]. Atx1p has also been found to function as an antioxidant.
Ceruloplasmin, a blue-colored copper-containing alpha-2 globulin (MW 134,000 Dalton), is synthesized mainly by hepatocytes and secreted into the systemic circulation [6]. Its major role as a ferroxidase is to promote iron mobilization from tissues by oxidizing ferrous iron for transfer into transferrin. Consequently, patients homozygous for mutations in the ceruloplasmin gene (aceruloplasminemia) do not have copper storage but rather develop iron-overload in the liver, pancreas and brain [7]. Ceruloplasmin may also transport copper to other tissues; as an oxidase toward aromatic amines (e.g., epinephrine, 5-hydroxytryptamine, and dopamine), phenols, cystine, ferrous ion and ascorbic acid; or as an antioxidant [8]. The normal plasma concentration of ceruloplasmin in older children and adults, measured by the oxidase enzymatic assay, is between 20 and 45 mg/dL, and of copper is between 70 and 150 µg/dL. In the human newborn, ceruloplasmin levels are 1.8–13.1 mg/dL and copper levels vary between 12 and 26 µg/dL [9], increasing to adult levels by age two years. This is in contrast to the parturient mother whose ceruloplasmin and copper levels are 40–89 mg/dL and 118–302 µg/dL, respectively [9]. Non-ceruloplasmin-bound plasma copper does not differ between newborn and mother and ranges between 5–15 µg/dL. It should be noted that the classic assays for ceruloplasmin were based on its oxidase activity in vitro, and hence, depended on the presence of copper in the molecule [10]. More recent immuno-assays detect apo-ceruloplasmin (which does not contain copper) as well as holo-ceruloplasmin (which contains copper) and may thus give higher serum values of ceruloplasmin in Wilson disease patients than does the oxidase method, which only detects holo-ceruloplasmin [10]. Thus, many Wilson disease patients may now have low normal plasma concentrations of ceruloplasmin measured by the immunoassays. This may affect the predictive value of ceruloplasmin levels in diagnosing Wilson disease.
Copper bound to albumin and amino acids, not including that contained in ceruloplasmin, is called “non-ceruloplasmin-bound copper” or “free copper,” which normally totals about 10% of total plasma copper, with the remainder being accounted for by non-exchangeable copper bound to ceruloplasmin [11]. When copper accumulates in the liver in Wilson disease, or if severe liver injury occurs for other reasons, copper is released into the circulation and increases the fraction of “free copper” relative to the copper bound to ceruloplasmin. This copper becomes available for renal excretion causing the cupriuria that is characteristic of Wilson disease and patients with other liver injuries, but to a lesser extent.
More than 80% of absorbed copper is excreted in bile (Figure 28.1), totaling approximately 1.2–1.7 mg/day [12]. Exocystosis of lysosomal copper across the canalicular membrane is the major source of biliary copper, which is then complexed to large proteins (e.g., metallothionein) that prevent reabsorption by the small intestine. Identification of a new gene that causes copper overload in the Bedlington terrier [13] suggests that the gene product COMMD1, formerly called MURR1, is required for vesicular copper movement and excretion (Figure 28.2). The human COMMD1 has been mapped to chromosome region 2p13-16 [39]. Human COMMD1 has been excluded as the gene causing non-Wilson disease hepatic copper toxicosis syndromes. This protein interacts with the copper binding N-terminus of ATP7B, providing biochemical evidence in support of the proposed role of the COMMD1 gene product in hepatic copper toxicosis [40]. However, several more recent studies showed that COMMD1 is not involved in Wilson disease or other causes of human toxicosis.
Metallothionein-bound copper does not account for all of biliary copper. An additional pathway for copper secretion into bile may involve glutathione-conjugated copper secreted into the canaliculus by MRP2, the multi-organic ion transporter (Figure 28.2). There is minimal enterohepatic circulation of copper as metallothionein-bound copper in the intestinal lumen is not absorbed. Urine, sweat, and menstrual blood are minor pathways for copper excretion. Urine copper (<40–100 µg daily) is neither indicative of dietary intake, nor is it important in copper homeostasis under normal circumstances. Pathologic processes that interfere with biliary excretion of copper, such as intrahepatic and extrahepatic hepatobiliary cholestatic disorders, produce copper retention in the liver, generally in the lysosomal fraction of hepatocytes, and are associated with elevated plasma copper and ceruloplasmin levels. In fact, hepatic concentrations of copper in intrahepatic cholestatic disorders of childhood and primary biliary cirrhosis in adults may equal or exceed levels found in Wilson disease. The excess hepatic copper in cholestasis does not appear to be hepatotoxic.
The organ distribution of the 80–100 mg of copper in an adult includes 15% in the liver, with lesser amounts in brain, heart, and kidneys in decreasing order. Fifty percent of the body’s copper is stored in muscle and bone, however the concentration is low in these tissues. In fetal liver, copper concentration is several-fold higher than in older children and adults, most of which is bound to metallothionein in hepatocyte lysosomes [16]. During fetal life the amount of copper in the liver declines from over 90% of total body copper during early development to approximately 50–60% at birth. After birth, hepatic copper content falls rapidly, reaching adult levels by three months of age. Because of delayed maturation of ceruloplasmin synthesis in the infant’s liver, plasma levels of both ceruloplasmin and copper remain low during the first 6 to 12 months of life. In conditions with increased serum levels of thyroxine, estrogens, and testosterone [16], plasma ceruloplasmin and copper may be elevated, making laboratory values difficult to interpret. In contrast, insufficiency of corticosteroids decreases biliary copper excretion resulting in elevated plasma copper and ceruloplasmin levels.
Mechanisms of Copper Toxicity
High levels of orally or parenterally administered copper accumulate in the liver in most mammalian species. Excess hepatic copper has been shown to cause liver injury in rodents, chickens, ruminants, sheep, Bedlington terriers, and humans [11]. Endogenous copper detoxification mechanisms, such as sequestration with metallothionein, export via copper translocating ATPases, and biliary secretion, have allowed certain animal species (such as the normal dog, Dominican toad, and mute swan) to tolerate higher hepatic copper content. The human newborn infant has the capacity to tolerate 5- to 100-fold the hepatic copper content of normal adults [17]. Hepatic lysosomal sequestration of excess copper is also an effective mechanism to render nontoxic enormous concentrations of intracellular copper. However, in some species, natural accumulation of dietary copper leads to severe liver injury and death, as observed in sheep and the Bedlington terrier [18].
The precise intracellular target for the toxic action of copper is uncertain. Many cytosolic enzymes that contain sulfhydryl groups may be inhibited in vitro by copper [19]. Copper inhibits polymerization of tubulin, the chief protein of microtubules, possibly perturbing intracellular trafficking of proteins and mitotic spindle formation. Copper also functions as a pro-oxidant, catalyzing the transformation of hydrogen peroxide to the hydroxyl free radical, which, in turn, may react with and damage polyunsaturated fatty acid residues of cell membranes, thiol-rich proteins, and nucleic acids [19], and may activate intrinsic apoptotic cell death pathways. These effects may lead to disturbances in plasma membrane function, mitochondrial oxidative phosphorylation, nuclear control of cell processes, protein synthesis by endoplasmic reticulum, and leakage of lysosomal enzymes into the cytosol. In addition, by-products of lipid peroxidation, such as malondialdehyde and 4-hydroxynonenal, have been shown to stimulate collagen gene expression in hepatic stellate cells and promote fibrogenesis [58], as well as stimulate NFκB and cytokine gene expression [59].
There is considerable evidence that oxidative injury is a key factor in copper toxicity. Lipid peroxidation has been documented in both hepatic lysosomes and mitochondria isolated from copper-overloaded rats [21], and hepatic mitochondria from these rats have abnormal respiration and diminished mitochondrial activity of cytochrome c oxidase in conjunction with increased lipid peroxidation [21]. Elevated hepatic mitochondrial copper concentrations in patients with Wilson disease and copper-overloaded Bedlington terriers have also been associated with excessive lipid peroxidation of these organelles [18] and in Wilson disease patients with oxidative modification of mitochondrial DNA [19]. Furthermore, the abnormal ultrastructure of hepatocyte mitochondria in Wilson disease [1] and ICC patients support the hepatic mitochondrion being one of the target organelles in copper toxicity. Metallothionein has been shown to function as an antioxidant, thus, it may not only chelate excess copper, but may play a role in reducing oxidative stress stimulated by copper. Interestingly, high intracellular copper levels lead to a conformational change in the antiapoptotic protein, X-linked inhibitor of apoptosis (XIAP), which increases its degradation thereby decreasing its ability to inhibit caspase-3, leading to a lower apoptotic threshold and cell death [20]. Apoptosis may be induced by cytochrome c released from damaged mitochondria or through the activation of acid sphingomyelinase by copper and the release of ceramide. Other studies are consistent with hepatocyte apoptosis being the primary mode of cell death in copper toxicity [22], possibly explaining the characteristic mild elevation of serum aminotransferases associated with Wilson disease. In addition, oxidative modification of mitochondria DNA may result in impaired oxidative phosphorylation and amplification of oxidative stress.
Wilson Disease (Hepatolenticular Degeneration)
History
In 1912, Kinnear Wilson, an American neurologist, described the degenerative disease of the central nervous system associated with cirrhosis that now bears his name [23]. He proposed the term “progressive lenticular degeneration” for this rare, familial, invariably fatal disease of young people that was characterized by softening of the lenticular nuclei (putamen and globus pallidus) and hepatic cirrhosis. In 1921 Hall further characterized hepatic involvement and introduced the term “hepatolenticular degeneration” [11]. It was in 1948 that Cumings proposed that copper toxicity caused the tissue injury and suggested the novel use of 2,3-dimercaptopropanol (British Anti-Lewisite (BAL)) to increase urinary copper excretion and thus treat the disorder. In 1952, Scheinberg and Gitlin discovered that a low circulating ceruloplasmin level was a practical diagnostic test for the disorder [11]. In 1956, Walshe reported the successful use of the oral copper chelator, D-penicillamine, for treatment of this condition, and in 1968 Sternlieb and Scheinberg showed that D-penicillamine could prevent neurologic and hepatic injury in asymptomatic affected siblings [11]. Walshe reported in 1982 that triethylene tetramine (trientine) was effective with less toxicity than D-penicillamine. The role of liver transplantation as a treatment option under specific circumstances was defined in the 1980s. In the 1990s, the search for the Wilson disease gene localized the gene to within 13q14-q21 on chromosome 13. Using positional cloning techniques in 1993, three groups independently identified the gene responsible for Wilson disease, designated ATP7B [11, 24].
Epidemiology
Wilson disease is ubiquitous with a worldwide prevalence of approximately 1 in 30,000, and the heterozygote carrier state in approximately one in 90 persons [1]. Prevalence is generally higher in China and Asian countries than in Western countries. There are isolated communities in Japan, Sardinia and Israel with a higher frequency due to consanguinity prevalence. A recent molecular study from the UK estimated that the frequency of Wilson disease may be as high as 1 in 7,000 based on the presence of two pathologic gene variants, with the carrier state being as high as 2.5% of the general population. An underestimation of the prevalence of Wilson disease may be explained by the varying clinical presentations of disease leading to underdiagnosis and misdiagnosis, the low sensitivity of certain copper metabolism tests and the unknown age-related clinical penetrance of ATP7B mutations [1]. Patients present with a variety of clinical manifestations, including hepatic presentations which are common in childhood [26] and a later-onset predominately neurologic form in adults [1]. The youngest age with a neurologic presentation was a six-year old and the oldest a 72-year old [1]. Although there are often similarities in the age of onset and clinical findings of Wilson disease in affected siblings, there may be marked differences in organ system involvement and biochemical findings, suggesting that polygenic or environmental factors may play a role in expression of the disease. Wilson disease has been described as late as the eighth decade of life and occasionally before three years of age [25]. Forty to 60% of patients will present with a primary hepatic presentation in the second decade of life [26]. The remainder of patients come to clinical attention during the third and fourth decades with a primarily neurologic (34%) or psychiatric (10%) presentation [26] (Table 28.1). Other presenting features include hematologic and endocrine abnormalities (e.g., amenorrhea) in 12% and renal symptoms in 1% of patients [26]. All patients have liver involvement, although it may be asymptomatic and well-compensated. Although symptoms attributable to Wilson disease may start in childhood, establishing the diagnosis may be delayed for several years or even decades because of nonspecific symptoms and a low index of suspicion by the clinician. Not infrequently this delay results in advanced hepatic or neurologic manifestations at the time of diagnosis that potentially could have been prevented.
Table 28.1 Clinical Presentation in 802 Patients of All Ages with Wilson Disease
| Series | N | Hepatic (%) | Neuropsych (%) | Hematologic (%) | Endocrine (%) | Asymptomatic (%) |
|---|---|---|---|---|---|---|
| Walshe [26] | 217 | 101 (47) | 90 (42) | 28 (13) | ||
| Scheinberg [27] | 151 | 68 (45) | 85 (56) | 19 (13) | 4 (3) | |
| Saito [57] | 140 | 82 (59) | 58 (41) | |||
| Giagheddu [58] | 68 | 30 (40) | 23 (34) | 15 (22) | ||
| Dobyns [59] | 53 | 25 (47) | 28 (53) | |||
| Stremmel [60] | 51 | 34 (67) | 31 (61) | 5 (10) | ||
| Aksoy [61] | 49 | 14 (29) | 31 (63) | 4 (8) | ||
| Oder [62] | 45 | 27 (60) | 12 (27) | 6 (13) | ||
| Park [63] | 28 | 12 (43) | 10 (36) | 6 (21) | ||
| Total | 802 | 393 (49) | 368 (46) | 24 (3) | 4 (<1) | 59 (7) |
Genetics
Wilson disease is an autosomal recessive disease caused by biallelic mutations in ATP7B which encodes for the protein copper-transporting ATPase2. Genetic linkage of Wilson disease to the locus of the red blood cell esterase D gene indicated that the Wilson disease gene was on the long arm of chromosome 13, which was further mapped to a smaller region, 13q14-13q21. The identification of the Menkes disease gene prompted the search for the Wilson disease gene. Menkes disease is a rare autosomal recessive inherited copper deficiency disorder caused by impaired copper absorption at the intestinal level. Because the Menkes disease gene, ATP7A, was a putative cation-transporting P-type ATPase involved in copper transport, the search began for a homologous gene located in the Wilson disease locus of chromosome 13. In 1993, three groups reported isolation and identification of the gene for Wilson disease (82,83,84), designated ATP7B (Figure 28.3), with mutations unique to patients with Wilson disease. There are over 800 distinct mutations identified from patients with Wilson disease. Most of these are small deletions or missense mutations, the latter requiring confirmation that they are not merely polymorphisms. Missense mutations are generally associated with a predominance of neurological symptoms and a later clinical presentation. Deletions and other mutations causing premature stop codons appear to be associated with an earlier clinical presentation predominated by symptoms of liver disease, although not all studies are in agreement. Specific mutations appear to be more common among certain ethnic groups. The most common mutation in descendants from northern Europe, p.His1069Gln (c.3207C>A), may be present in 26–70% of cases, while in Asian populations a p.Arg778Leu (c.2333G>T) mutation occurs in 57% of affected Asians under 18 years of age. However, over half of all mutations occur rarely in any population. This degree of heterogeneity suggests most affected individuals are compound heterozygotes. Because of the wide variety of mutations, complete ATP7B gene sequencing, which is clinically available, is frequently necessary. A battery of mutations common to a given ethnic group can be screened, however, the absence of one of these mutations does not exclude the diagnosis. Several modifier genes have been proposed to explain the phenotypic variation in presentation, including COMMD1, APOE, ATOX1, and MTHFR, and are under study.
Pathogenesis
Mutations in ATP7B cause impaired biliary copper excretion that leads to progressive accumulation of copper in the liver followed by subsequent deposition in other organs, causing the varied clinicopathologic features of Wilson disease. The initial accumulation of copper in the liver begins in the first few years of life and can be substantial. It has been proposed that there is also failure to clear the high hepatic copper burden that is usually well tolerated in the neonate. By the end of the first or into the second decade of life, the hepatic burden of copper is exceeded, causing increasing free copper in the circulation that becomes deposited in other tissues [26]. During this time hepatic copper may actually decrease in concentration, whilst brain, kidney, and ocular copper increase [26].
It is clear that mutations in ATP7B cause both decreased biliary excretion of copper and defective hepatocyte incorporation of copper into ceruloplasmin [1]. Serum ceruloplasmin levels are low in Wilson disease because of decreased synthesis of holoceruloplasmin and rapid clearance of apoceruloplasmin which is still secreted by the liver in Wilson disease. The ceruloplasmin gene on chromosome 3 is normal in patients with Wilson disease. Aceruloplasminemia, a congenital deficiency of ceruloplasmin caused by lack of synthesis of apopoceruloplasmin due to homozygous mutations in its gene, causes iron deposition (not copper) in liver, brain and spleen, retinal degeneration, diabetes and dementia. Heterozygotes for Wilson disease may have low ceruloplasmin levels yet no pathologic accumulation of copper in tissues. Conversely, 5–30% of Wilson disease patients have normal plasma ceruloplasmin levels [1]. The normal ceruloplasmin levels in Wilson disease result from ceruloplasmin being an acute phase reactant that can elevate in the face of acute liver injury, as well as the fact that circulating apoceruloplasmin may be detected by commonly used immunologic assays for plasma ceruloplasmin. Although a very useful biomarker for this disease, the impairment in ceruloplasmin synthesis appears to be a result of, rather than responsible for, the disturbance of copper metabolism in Wilson disease, and does not, in itself, appear related to any of the manifestations of Wilson disease. Thus, plasma ceruloplasmin concentrations are neither specific nor sensitive for the diagnosis of Wilson disease unless very low (<5 mg/dL).
ATP7B is localized in hepatocytes to the trans-Golgi part of the late secretory pathway. With increasing intracellular copper concentrations, the ATPase traffics to a cytoplasmic vesicular compartment that distributes near the canaliculus where it participates in copper excretion into bile. Once the ATP7B sequestration of copper in vesicles reduces the cytoplasmic copper content, ATP7B is recycled back to the trans-Golgi network (Figure 28.2).
The role of ATP7B in the incorporation of copper into ceruloplasmin has been investigated in yeast. The ATP7B orthologue Ccc2 in yeast transports copper to Fet3, which is analogous to apoceruloplasmin in mammalian tissues. In yeast lacking Ccc2, ATP7B replaced this activity while mutant ATP7B did not. Mutations in the metal binding sites closest to the transmembrane domain 1 are more important for this copper transporting activity than sites closer to the N-terminus. Thus, copper incorporation into ceruloplasmin is dependent on the copper transport function of ATP7B. Mutations in ATP7B result in failure to incorporate copper in ceruloplasmin and the secretion of apoceruloplasmin which is rapidly removed from the circulation.
Clinical Features of Wilson Disease
Wilson disease has a multitude of clinical presentations that can present at almost any age. Although the failure to excrete biliary copper is present from birth, clinical manifestations of Wilson disease are rarely apparent prior to age three years [27]. Clinical symptoms typically develop sequentially based on the pathophysiologic disturbance of copper metabolism in Wilson disease. Copper silently accumulates in the liver during childhood. After the liver storage capacity for copper becomes saturated, circulating non-ceruloplasmin-bound “free copper” levels rise and copper is then redistributed systemically, accumulating in the nervous system, cornea, kidneys and other organs and tissues. This change in organ distribution of copper over time is paralleled by the clinical presentations of Wilson disease. Based on the combined large patient series of Walshe [26] and Scheinberg and Sternlieb [27], prior to age ten years, 83% of patients presented with hepatic symptoms and 17% with neuropsychiatric manifestations; between ten and 18 years, 52% presented with hepatic and 48% with neuropsychiatric symptoms; and after age 18 years, 24% presented with hepatic and 74% with neuropsychiatric symptoms (Figure 28.4). Considering patients of all ages using data derived from nine combined series, approximately 49% of patients present with hepatic and 46% with neuropsychiatric symptoms (Table 28.1). The median delay in establishing the diagnosis of Wilson disease in those with neuropsychiatric symptoms was 18 months, which is considerably longer than those with hepatic presentation (median six months) [122]. During the phase of copper redistribution, other organ systems become involved, with renal, endocrine, and hematologic manifestations appearing after age ten years (Figure 28.4). A combination of liver dysfunction and other organ system involvement, at any age, should suggest Wilson disease.
Figure 28.4 Histogram of age distribution of initial mode of clinical presentation in children and adolescents with Wilson disease. Split vertical bars represent combined clinical presentation.
After hepatic saturation, copper accumulating in the ocular cornea may cause the characteristic Kayser-Fleischer (K-F) ring (Figure 28.5), a greenish-brown ring at the periphery of the cornea on its posterior surface in Descemet membrane [27] that is present in 40–50% of patients with hepatic Wilson disease and 20–30% of pre-symptomatic patients [1]. This is best detected by slit-lamp examination by an ophthalmologist, although a prominent K-F ring can be seen easily if the iris is of light pigmentation. The color of the ring is due to scattering and reflection of light by layers of copper granules. The K-F ring initially appears at the superior poles of the cornea, with subsequent involvement of inferior poles followed by circumferential involvement. Treatment with copper chelators results in gradual resolution of K-F rings over three to five years in reverse order of appearance, occasionally leading to complete disappearance. This pattern of copper deposition has been said to be due to a relative stagnation of solvent flow in the superior poles of the cornea, allowing the precipitation of copper to occur. Analysis by X-ray energy spectroscopy showed that the K-F ring consisted of granules that were rich not only in copper, but also in sulfur. This suggested that metallothionein-bound copper in Descemet membrane was essential for the visual appearance characteristic of K-F rings. The K-F ring does not interfere with visual function. The K-F ring is virtually always present at the time when neurologic or psychiatric symptoms develop, although there are rare exceptions (5% of patients with neurologic symptoms) [1]. Importantly, the K-F ring is frequently absent in children without neurologic involvement but who present with hepatic symptoms. The K-F ring is not pathognomonic for Wilson disease, but also has been reported in patients with prolonged cholestatic liver disease, such as chronic hepatitis, chronic intrahepatic and neonatal cholestasis, cryptogenic cirrhosis and primary biliary cirrhosis [1]. This is likely the result of diminished biliary excretion and secondary copper overload that occurs in cholestasis. K-F rings can also be present in other states of high serum copper levels, such as multiple myeloma, and during estrogen intake [1].
Another characteristic, but less common, ophthalmologic feature of Wilson disease is the grayish-brown “sunflower cataract” that may develop because of deposits of copper in the anterior and posterior lens capsule. Visualizing these cataracts requires an ophthalmoscopic examination; they rarely interfere with vision and resolve with therapy. The other circumstance in which these cataracts may develop is from a copper-containing foreign body lodged intraocularly (chalcosis). Because of these characteristic ocular findings all patients in whom Wilson disease is suspect should undergo a thorough ophthalmologic examination.
Hepatic Presentations
The liver is the major site of the biochemical defect in Wilson disease, the initial target of copper toxicity, and the site of initial clinical manifestations in 40–60% of all patients, but a higher percentage in children. Clinical symptoms of liver disease are rare prior to age three years (Figure 28.4), however asymptomatic mild elevation of serum aminotransferase levels has been reported and a rare patient has developed fulminant liver failure before age five years. Thereafter, symptoms of acute or chronic liver disease may interrupt a pre-symptomatic period at any time in the first two decades of life. Hepatic presentations include steatohepatitis, acute hepatitis, acute liver failure, chronic active hepatitis, gallstone disease, cirrhosis and hepatocellular carcinoma (Table 28.2). Elevated aminotransferase levels may be detected in completely asymptomatic patients for whom a chemistry panel is drawn for unrelated reasons, such as seizure medication monitoring or trauma. Exclusion of more common causes of abnormal liver blood tests should lead to consideration of Wilson disease if the tests do not normalize. It is essential that the clinician maintains a high level of suspicion and thoroughly investigates the child with persisting asymptomatic elevation of aminotransferase levels, to facilitate timely diagnosis and initiation of treatment early in the course of Wilson disease.
Hepatic steatosis is the first finding in many children and young adults with Wilson disease, which can be identified by ultrasonography or liver biopsy, and is frequently accompanied by mildly elevated aminotransferases. Physical findings may include mild hepatomegaly, however most patients in this stage are asymptomatic. The high prevalence of non-alcoholic fatty liver disease associated with the current childhood and adult obesity epidemics emphasizes the need to have a high index of suspicion for Wilson disease in an overweight child or adult with fatty liver disease.
Acute hepatitis is the mode of presentation in approximately 25% of patients. Clinical signs of jaundice, anorexia, nausea, malaise, pale stools and dark urine may mimic acute infectious hepatitis. Laboratory investigation reveals a conjugated hyperbilirubinemia associated with elevated aminotransferases but normal serum albumin and prothrombin time/international normalized ratio (INR). Although serologic testing for viral hepatitis types A, B, C, and Epstein–Barr virus is negative, this presentation can be confused with acute viral hepatitis, particularly if the patient makes a complete, although temporary, recovery.
Acute liver failure (previously called fulminant Wilson disease) is the most severe form of Wilson disease and occurs in up to 12% of patients, usually presenting in adolescent females as an acute icteric hepatitis that rapidly evolves over a few days to several weeks [1, 27]. Symptoms progress to fatigue, hepatic insufficiency, extreme jaundice (because of the accompanying hemolysis), severe coagulopathy, ascites, hepatic encephalopathy, renal failure and death if liver transplantation is not performed [1]. This presentation may be similar to that of acute fulminant viral hepatitis or following ingestion of a hepatotoxin. However, non-immune hemolysis and rapid onset of renal failure with low serum alkaline phosphatase in a female adolescent are characteristic of the Wilson disease presentation.
A similar fulminant presentation has been described in patients who had been successfully treated for up to 20 years with copper chelators or zinc therapy, but who discontinued therapy or became noncompliant for as little as eight months’ time [1]. This rapid development of fatal hepatic disease suggests that the therapeutic action of copper chelation may be due to formation of non-toxic copper-chelator or copper-protein complexes (e.g., copper-metallothionein) rather than the generally accepted action of chelators in removing excess copper from the patient. The abrupt discontinuation of penicillamine or triene may expose the liver to a large load of free toxic copper released by dissociation of this complex. Similar presentations have occurred after the discontinuation of zinc therapy. For this reason, Wilson disease patients must be repeatedly and frequently reminded (especially when they feel healthy) that copper chelation or zinc maintenance therapy is lifelong and discontinuation could be fatal.
Chronic hepatitis is the presentation in 10–30% of Wilson disease patients during adolescence or young adulthood. Cirrhosis is frequently present at the time of this diagnosis, particularly in adults [1, 27]. Malaise, anorexia, fatigue, abdominal pain and nausea may precede the onset of jaundice and hepatic dysfunction. Amenorrhea, delayed puberty, polyarthralgias, edema, gynecomastia, ascites, clubbing or spider angiomata, when present, indicate the chronic nature and likelihood of hepatic fibrosis or cirrhosis. Tender hepatomegaly and splenomegaly are typically found on examination. It is important to note that K-F rings may be absent in up to 50% if they do not have associated neurologic or psychiatric findings. The presence of neurologic or psychiatric symptoms, or a family history of Wilson disease, should immediately raise suspicion for Wilson disease in any patient with chronic hepatitis. Laboratory tests show raised serum aminotransferases, low albumin, elevated gamma globulins, and a variably abnormal prothrombin time/INR. Except for lower aminotransferase levels, Wilsonian chronic hepatitis patients appear clinically no different than other patients with autoimmune hepatitis and may even have positive auto-antibodies (ANA, anti-smooth muscle antibody), however, liver biopsy in Wilson disease may reveal steatohepatitis which would be unusual in autoimmune hepatitis (unless the patient is overweight). With the rising frequency of adolescent obesity, one can expect up to 30% of Wilson disease patients to be obese leading to an incorrect diagnosis of non-alcoholic fatty liver disease when steatohepatitis is found on liver biopsy. Unless appropriate diagnostic studies are performed, the patient may well carry a misdiagnosis of autoimmune, viral, alcoholic or non-alcoholic steatohepatitis or idiopathic chronic hepatitis until neurologic symptoms develop or the liver is examined at transplantation or autopsy. Thus, it is imperative that biochemical screening for Wilson disease be undertaken in all patients with chronic hepatitis without a clearly defined diagnosis. The response to copper chelation therapy is generally excellent even if cirrhosis is present. In a series of 20 chronic hepatitis patients, Schilsky et al. [28] described long-term survival in 90% of cirrhotic patients who were compliant with copper chelation therapy. This compares favorably to survival rates of 55–80% in patients with chronic hepatitis and cirrhosis caused by hepatitis B virus, autoimmune hepatitis or other causes.
The cirrhotic presentation of Wilson disease may be insidious with cutaneous signs of chronic liver disease or splenomegaly being the only clues. Alternatively, anorexia, fatigue, abdominal pain, weight loss, jaundice, ascites, gastrointestinal hemorrhage, hypersplenism, coagulopathy, spontaneous bacterial peritonitis, encephalopathy, poor school function or hepatorenal syndrome may signal the onset [27]. Indeed, none of these features is specific to Wilsonian cirrhosis, so, sadly, patients may be misdiagnosed as post-necrotic, steatohepatitis, cryptogenic cirrhosis, or as alcoholic cirrhosis in adults. Although cirrhosis and its complications are relatively common presenting features of Wilson disease in childhood, cirrhosis may be well compensated and may remain silent and asymptomatic into adulthood when patients present with neurologic, psychiatric, endocrine or other symptoms.
Cholelithiasis is relatively common in adolescents with Wilson disease, resulting from ongoing hemolysis in the presence of cirrhosis. Abdominal pain in a patient with Wilson disease should prompt ultrasonographic evaluation for gallstones. Analysis of gallstones showed twice the level of cholesterol as found in gallstones from children with hemolytic disease, but <30% of that measured in typical cholesterol gallstones. Interestingly, the copper content of gallstones removed from Wilson disease patients (5.2–85.4 µg/gm) was significantly lower than that in pigment gallstones found in non-Wilsonian patients (571.7–1,951.8 µg/gm) [26], illustrating the impaired biliary excretion of copper characteristic of ATP7B mutations.
A retrospective evaluation of 363 Wilson disease patients in the UK and Sweden demonstrated intra-abdominal malignancies (primarily hepatoma, cholangiocarcinoma and poorly differentiated adenocarcinoma) in 4.2–5.3% of patients followed for 10 to 29 years, and 15% for those followed for 30–39 years [29]. Hepatocellular carcinoma may develop in adults but is exceedingly rare in children and adolescents with Wilson disease.
Neuropsychiatric Presentation
In 40–45% of Wilson disease patients, neurologic or psychiatric signs are the first indication of illness (Table 28.1). Neurologic onset has been recorded in children as young as six years [28] and in adults as old as 72 years [1], however typically occurs in the third and fourth decade. Neurologic symptomatology is generally limited to motor manifestations of extrapyramidal or cerebellar dysfunction [1, 28]. Psychiatric symptomatology can take many forms (Table 28.3). Neurologic symptoms most commonly present during the second and third decade of life with the insidious appearance of a single symptom, followed by gradual worsening of the symptom with development of other motor abnormalities. Recently, neurologic and psychiatric symptoms have been correlated with magnetic resonance imaging of the brain. There are three typical neurologic presentations of Wilson disease. The first presentation is one of symmetric bradykinesia, cognitive impairment, cog-wheel rigidity, and an organic mood syndrome, termed “pseudoparkinsonian.” This presentation was associated with dilation of the third ventricle on brain MRI scanning. The presentation termed “pseudosclerosis,” is manifested by tremor, ataxia, and reduced functional capacity, and is characterized by focal thalamic lesions. Tremors may be of the resting, intention, or postural forms and can become incapacitating. The third presentation, termed “dyskinesia” includes patients who exhibit dyskinesia, dysarthria, and organic personality syndrome and correlates with focal lesions in the putamen and globus pallidus. Extrapyramidal symptoms include facial grimaces, stereotypic gestures, drooling, a fixed grin (risus sardonicus), dysphagia, and finally contractures of the jaw or extremities. Dysarthria may be the most frequent neurologic manifestation of Wilson disease, reported in up to 97% of those with neurologic disease [1]. Titubation, dysmetria, scanning speech, illegible handwriting, and rarely choreiform and athetoid movements also occur. The nature of these abnormalities may lead to misdiagnoses of multiple sclerosis, Parkinson disease, Friedreich ataxia, and other conditions. Because intelligence is unaffected, the patient generally experiences frustration and secondary depression.
Table 28.3 Neurologic and Psychiatric Symptoms Associated with Wilson Disease
The neurologic basis for these motor abnormalities is involvement of the basal ganglia and cerebellum, which are well visualized on brain MRI, the most important neuroradiologic examination for the diagnosis of Wilson disease [1]. The corticospinal and corticobulbar pathways are also affected to some extent. There appears to be a critical threshold of brain copper deposition, above which time neurologic injury and symptomatology ensue (40 µg/gm wet weight).
Sensory function and intelligence in Wilson disease patients remain normal; however, there may be mild memory impairment. Lower scores reported on various intelligence tests are most likely the result of impaired ability to perform motor tasks. Other symptoms that may develop include migraine headaches; grand mal, focal motor, or partial complex seizures; and various gait disturbances due to both the tremor and dystonia. Seizures are unusual with a prevalence rate of 6.2%. Seizures rarely present initially but may develop within weeks or months of starting copper chelation therapy. It has thus been proposed that sudden mobilization of large quantities of copper by chelating agents may be responsible for these seizures. Neurological presentations of Wilson disease are summarized in Table 28.2.
At some time during the course of their life, patients may suffer from organic dementia, neurotic behavior, bipolar or schizophrenic psychosis. Behavioral and personality disorders, such as aggressive outbursts, deterioration in school performance or handwriting, or a major change in affect or personality, may be the initial symptoms in an adolescent or college student. Therefore, psychiatric evaluation and ongoing psychotherapy in addition to chelation therapy are important elements in the total care of these patients.
In approximately 10–25% of Wilson disease patients, a psychiatric disturbance is the initial clinical presentation, even before the appearance of any movement disorder. If the diagnosis of Wilson disease is not made, the development of a movement disorder during therapy with drugs for the psychiatric disorder may be attributed to a medication side effect rather than to the possibility of undiagnosed Wilson disease [27]. Psychiatrists must, therefore, maintain a high index of suspicion of Wilson disease, and should measure plasma ceruloplasmin and obtain a slit-lamp examination in patients with psychiatric disorders (even if chronic in nature). This is of particular importance if there is a history of liver disease, a family history of a psychiatric disorder or Wilson disease; if the patient is under age 50 years; or if the patient is not responding satisfactorily to conventional psychiatric treatment [27].
Other Presentations
Renal disturbances may occur in patients with Wilson disease [27]; however, renal disease as a presenting symptom is rare. Copper has been shown to accumulate in the kidneys of Wilson disease patients, with up to 100 times the normal concentration being observed. Proximal renal tubular dysfunction, decreased glomerular filtration rate, and decreased renal plasma flow characterize the resulting renal dysfunction. The renal tubular dysfunction is manifested by proteinuria, glucosuria, phosphaturia, uricosuria, generalized aminoaciduria, and microscopic hematuria. Distal renal tubular acidosis contributes to an increased tendency for formation of renal stones. In one series, 16% of 45 patients developed renal stones. Patients with the acute liver failure presentation of Wilson disease and those with end-stage liver disease may develop severe acute kidney injury, requiring temporary renal dialysis. Finally, isosthenuria has been reported. Many of the renal abnormalities improve during copper chelation therapy; however severe proteinuria and the nephrotic syndrome [27] or a Goodpasture-like syndrome may occur as a consequence of D-penicillamine administration rather than because of copper toxicity.
A variety of hematologic manifestations have been reported in Wilson disease. Intravascular hemolysis is frequent and may be the presenting abnormality in approximately 15% of patients, or may be transient and occur when there are no associated neurologic or hepatic clinical manifestations. Therefore, Wilson disease should be considered in children and adolescents with Coombs-negative hemolytic anemia. When associated with the acute liver failure presentation, hemolysis is a poor prognostic factor, likely contributing to the renal failure by excess hemoglobinuria. Hemolysis is considered secondary to sudden release of copper from the liver, initiating an oxidative stress capable of peroxidation of red cell membrane lipids. If hepatic involvement is advanced, circulating hepatic-derived coagulation factor levels may be low, platelets may have impaired function, and portal hypertension may cause splenomegaly and resultant thrombocytopenia and leukopenia.
Cardiac involvement has been recognized in Wilson disease. Electrocardiographic abnormalities were present in 34% of 53 patients with a mean age of 21 years, including left ventricular hypertrophy, ST wave depression, and T-wave inversion [30]. Arrhythmias were present in 13% of patients and 19% had orthostatic hypotension (although asymptomatic) indicating autonomic dysfunction. Histological findings at autopsy have included cardiac hypertrophy, interstitial fibrosis, intramyocardial small vessel sclerosis, and focal inflammation [30]. Factor et al. postulate that sudden death may be caused by arrhythmias in Wilson disease patients.
Skeletal manifestations of Wilson disease are not uncommon and may even be the first clinical symptoms of the disease. Bone demineralization is the most common feature, possibly caused by the hypercalciuria and hyperphosphaturia resulting from renal tubular dysfunction. Other radiologic changes include rickets and osteomalacia, osteoporosis, spontaneous fractures, bone fragmentation near joints, osteochondritis desiccans, chondromalacia patellae, premature osteoarthrosis, and premature degenerative arthritis of the knees and wrists. Stiffness of larger joints is a complaint of many patients.
Skin pigmentation may be increased, particularly on the anterior aspect of the lower legs, due to deposition of melanin, and acanthosis nigricans may be present. Blue lunulae of the fingernails have also been reported. Other associated dermatologic findings may be caused by cirrhosis and portal hypertension. Hormonal imbalance secondary to chronic liver disease has been thought to lead to amenorrhea in women and gynecomastia in boys. However, more recent studies suggest that primary ovarian dysfunction may be present and that increased androgen levels and abnormalities in the hypothalamic-pituitary-testicular axis in males is probably not a result of liver dysfunction. Additional infrequent associations with Wilson disease include diabetes mellitus, exocrine pancreatic insufficiency and hypoparathyroidism.
Laboratory Findings
Patients with Wilson disease may present with almost any combination of abnormalities in liver blood tests, or even no abnormality at all. Serum aminotransferase levels are characteristically only mild-to-moderately elevated with aspartate aminotransferase (AST) more than alanine transaminase (ALT), even in patients with the acute liver failure presentation. Serum alkaline phosphatase is usually in the low range, particularly when acute liver failure is present. In patients with acute liver failure, the combination of an AST:ALT ratio >2.2 and an alkaline phosphatase:total bilirubin ratio <4 has almost a 100% diagnostic accuracy for Wilson disease as the cause [31], and thus provides very useful information in this clinical situation.
Serum copper is usually low in Wilson disease; however, during acute liver failure, serum copper is elevated due to massive copper release from the necrosing liver as with other causes of acute liver failure but to a greater extent. This released copper contributes to the hemolytic anemia, hemoglobinuria and renal failure that are common during this Wilson disease presentation. The non-ceruloplasmin bound copper (serum “free” copper) is generally elevated in untreated patients (>25 µg/dL; normal <15 µg/dL) and can be estimated by the serum copper (µg/dL) minus 3x plasma ceruloplasmin (mg/dL). Serum free copper may also be elevated in other causes of acute liver failure and in chronic cholestasis or during copper ingestion/poisoning. This calculation depends on accurate serum copper and ceruloplasmin measurements, the latter being subject to variation with the newer immunologic assays currently in use. Thus, the non-ceruloplasmin bound copper level should not be used to establish the diagnosis of Wilson disease, but may be useful in monitoring patients during chelation or zinc therapy. Serum phosphate and uric acid may be low because of renal tubular losses. Recent studies suggest that uric acid may also be oxidized as a result of oxidative stress. A complete Fanconi syndrome, including aminoaciduria and glycosuria, may also be present. Changes on radiologic evaluation of the skeleton may include osteoporosis, rickets, osteomalacia, localized demineralization, osteoarthritis, and other lesions. MRI and CT imaging of the brain may detect changes in the basal ganglia, pons or thalamus, including the “giant panda sign.” Copper cannot be detected or quantified by standard imaging techniques.
Diagnosis of Wilson Disease
Establishing the diagnosis of Wilson disease may be challenging because not all lab tests may be abnormal or obtainable in a given patient (Figure 28.10), however, it is absolutely essential in that therapy is most effective early in the course of the disease (Table 28.4). For example, quantifying urine copper excretion may not be possible if the patient is in oliguric renal failure, and plasma ceruloplasmin may be elevated into the low normal range during acute hepatitis or estrogen therapy. Severe coagulopathy may preclude safe percutaneous liver biopsy. Therefore, the specific criteria used to establish the diagnosis of Wilson disease must be tailored to the patient’s clinical presentation. No single laboratory test result can establish this diagnosis without confirmatory clinical and laboratory data, with the possible exception of genetic testing. An international group has developed a scoring system to assist with the diagnosis of Wilson disease [32]. The following sections discuss diagnostic criteria in classical and problematic clinical presentations.
Hepatic Presentations
In a patient with liver dysfunction, the finding of a plasma or serum ceruloplasmin less than 20 mg/dL suggests the diagnosis of Wilson disease [1]; however, confirmatory studies are necessary because a number of disease states can also yield low ceruloplasmin values. For example, low serum ceruloplasmin levels may also occur with massive protein loss, kwashiorkor, severe copper deficiency, severe hepatic insufficiency, hereditary hypoceruloplasminemia or aceruloplasminemia, acute liver failure, the normal neonate, Menkes syndrome and 10% of heterozygotes for Wilson disease (Table 28.4). Thus, the diagnosis of Wilson disease must be confirmed by either an elevated urine 24-hour copper excretion (normally <40 µg/24 hours) above 40–100 µg/24 hours, an elevated urine copper during a penicillamine challenge, the presence of a K-F ring (and the absence of other cholestatic liver disorders), elevated hepatic copper content (>250 μg/gm dry weight) with consistent liver histology, or the finding of two pathologic ATP7B mutations on genetic testing. A plasma ceruloplasmin <5 mg/dL is very suggestive of Wilson disease in the absence of the above confounding conditions. It should also be stressed that a ceruloplasmin concentration between 20 and 35 mg/dL does not conclusively exclude this diagnosis, so if there is reasonable suspicion, further testing should be undertaken as below.
Urine must be collected in copper-free containers to avoid contamination. Additionally, determining if the collection was a full 24-hour collection should be confirmed by measuring total urinary creatinine excretion (normal 10–20 mg/kg/24 hours). False positive results of a 24-hour urine copper excretion (i.e., >100 µg/24 hours) may be seen if the patient is receiving any type of copper chelation therapy, if the collection is contaminated by exogenous copper, or if the patient has chronic hepatitis, cholestatic cirrhosis, or nephrotic syndrome. In children and young adults with autoimmune hepatitis, acute hepatitis, primary sclerosing cholangitis, acute liver failure, and primary biliary cirrhosis, there can be significant overlap in values with Wilson disease. Recent studies indicate that basal 24-hour urinary copper excretion may be even less than 100 µg at presentation in 16–23% of patients [1], leading some to recommend a threshold of 40 µg for diagnosis in children [1]. Using this threshold would increase the number of false positive urine coppers in patients with other liver diseases. Thus, to improve the diagnostic accuracy of urine copper, the King’s College Hospital group [33] have demonstrated good discrimination between Wilson disease and other liver disorders when 24-hour urine copper excretion was measured after a D-penicillamine challenge (500 mg given orally immediately before and repeated 12 hours into the urine collection); values above 25 µmol/24 hours (1,575 µg) indicated Wilson disease. However, the same group has subsequently reported similar post-D-penicillamine challenge copper excretion in three children with acute persistent hepatitis A virus infection, cautioning against the use of this test as sole criteria for diagnosing Wilson disease. Moreover, this test showed poor sensitivity for excluding the diagnosis in asymptomatic siblings.
If the plasma ceruloplasmin concentration is greater than 20 mg/dL but less than 35 mg/dL, the diagnosis of Wilson disease is not totally excluded if clinical circumstances are suggestive, because up to 30–40% of patients will have ceruloplasmin in this normal range. Steindl et al. [34] reported that 40% of 25 patients presenting with liver disease had ceruloplasmin values in the normal range, with several exceeding 30 mg/dL. In this study, ceruloplasmin was measured by radial immunodiffusion, an immunologic technique that recognizes both the oxidase-active holoprotein that contains six copper atoms per molecule and the enzymatically inactive apoceruloplasmin. Thus, immunologic techniques may yield higher values than those obtained by the older “gold standard” oxidase reaction, confusing the diagnosis. Most commercial laboratories have adopted the more convenient immunoassay, making low ceruloplasmin concentration less valuable as a diagnostic pillar for Wilson disease. If ceruloplasmin is normal but there is a high index of suspicion, then a 24-hour urine copper excretion, slit lamp examination, and liver biopsy or genetic testing should be performed to confirm or exclude the diagnosis.
A percutaneous liver biopsy may be helpful in establishing the diagnosis. Light microscopy, electron microscopy and quantitative copper analysis should be performed. Transjugular liver biopsy can be performed if a significant coagulopathy is present. Characteristic histologic findings of Wilson disease (fatty change, periportal glycogenated nuclei) or of other liver disorders (e.g., primary biliary cirrhosis in an adult) aid in the diagnosis. Ultrastructural mitochondrial changes of Wilson disease are also valuable. Measuring quantitative hepatic copper concentration is absolutely essential. Normal hepatic copper content is less than 50 ug/g dry weight of liver. Assuming an adequate sample has been obtained, values are usually above 250 ug/g dry weight in Wilson disease, and may reach 3,000 ug/g tissue. However, some studies have suggested that the 250 ug/g dry weight threshold can miss cases in which liver copper as low as 70 ug/g dry weight has been reported [1]. Thus, if liver copper is between 70–250 ug/g dry weight, further testing is indicated [1]. In affected asymptomatic siblings of Wilson disease patients, liver copper content may be borderline elevated. Although a normal hepatic copper content excludes the diagnosis of Wilson disease, a false-positive result can occur in other diseases. Specific diagnostic studies will be required to exclude other diseases (Table 28.4), such as autoimmune or infectious chronic hepatitis. The presence of autoimmune markers, plasma ceruloplasmin above 30 mg/dL, and 24-hour urine copper excretion (with or without penicillamine challenge) below the threshold will generally exclude Wilson disease.
If the diagnosis remains uncertain despite the testing already described, or if liver biopsy is contraindicated, the rate of incorporation of radio-labeled copper into ceruloplasmin has been used in the past as a diagnostic study [1]. A dose of 2.0 mg of cupric acetate containing 0.3–0.5 mCi 64Cu is administered orally in 100–150 ml of fruit juice following an eight-hour fast. The concentration of 64Cu in serum is determined serially (at +1, 2, 4, 24, and 48 hours) over 48 hours. Normally, the radiocopper rises at one and two hours, falls thereafter with a secondary rise over the ensuing 24- or 48-hours representing incorporation of the radio-labeled copper into newly synthesized ceruloplasmin. In Wilson disease, the secondary rise in serum copper that normally occurs after four hours is absent. The pattern is intermediate in Wilson disease heterozygotes. This test is only offered in very specialized centers.
Molecular genetic testing is now widely available and may yield valuable diagnostic information, particularly if the diagnosis remains in doubt despite the testing already discussed. Newer more rapid DNA sequencing methods allow for timely genetic diagnosis (despite the many hundreds of possible mutations) in most cases for initiation of therapy. Prenatal diagnosis is also possible, although this has limited application since early postnatal diagnosis allows appropriate timing for treatment. For specific ethnic groups who have a single predominant mutation, rapid genotyping may be particularly useful, including Sardinian, Icelandic, Korean, Japanese and Canary Island populations. It is essential that experienced geneticists be available to interpret the results of genotyping, inasmuch as variants of uncertain significance are common and should not be overinterpreted as pathologic. Interpretation is particularly challenging in the patient with suspected Wilson disease who has only one ATP7B pathologic variant, raising the question of an unidentified second mutation in the promoter region or introns.
Tests that appear to be of no value in establishing the diagnosis of Wilson disease include CT scanning and MRI of the liver, since liver copper content cannot currently be quantitated by these modalities. Although advocated by some experts as useful, the non-ceruloplasmin-bound serum copper fraction (“free copper”) may be elevated in a variety of liver diseases as well as Wilson disease, so its value in establishing the diagnosis is questionable. It is of more value in monitoring treatment.
Figure 28.6 illustrates a suggested approach to diagnosing Wilson disease in patients with a hepatic presentation. Plasma ceruloplasmin, a 24-hour urine collection for copper analysis and an ophthalmology examination for a K-F ring should be obtained. A liver biopsy should be obtained if any of these are abnormal, if there is a high suspicion for Wilson disease and the ceruloplasmin is 20–30 μg/dL or if it was measured by an immunologic method and is in the normal range. In addition to routine histology, electron microscopy and quantitative copper analysis should be obtained. If quantitative hepatic copper exceeds 250 μg/gm dry weight and other diagnoses are excluded by histology and other appropriate laboratory tests, then the diagnosis of Wilson disease is established. Some may substitute molecular testing of ATP7B to avoid the liver biopsy. DNA sequencing can be performed for a defined ATP7B mutation, common mutations in the appropriate ethnic population, or full sequencing can be performed; haplotype (microsatellite) marker analysis can be performed if a first degree relative has Wilson disease.
Related posts:
Chapter 5 – Cirrhosis and Chronic Liver Failure in Children
Chapter 13 – Familial Hepatocellular Cholestasis
Chapter 14 – Alagille Syndrome
Chapter 25 – α1-Antitrypsin Deficiency
Chapter 32 – Lysosomal Storage Disorders in Children
Chapter 43 – Liver Transplantation in Children: Indications and Surgical Aspects
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