This Clinical Practice Guideline (CPG) has been developed to assist physicians and other healthcare providers in the diagnosis and management of patients with Wilson’s disease. The goal is to describe a number of generally accepted approaches for diagnosis, prevention, and treatment of Wilson’s disease. Recommendations are based on a systematic literature review in the Medline (PubMed version), Embase (Dialog version), and the Cochrane Library databases using entries from 1966 to 2011. The Grades of Recommendation, Assessment, Development, and Evaluation (GRADE) system used in other EASL CPGs was used and set
against the somewhat different grading system used in theAASLD guidelines (Table 1A and B). Unfortunately, there is not a single randomized controlled trial conducted in Wilson’s dis-
ease which has an optimal design. Thus, it is impossible to assign a high or even a moderate quality of evidence to any of the questions dealt with in these guidelines. The evaluation is mostly based on large case series which have been reported within the last decades.
 2011 European Association for the Study of the Liver. Published
by Elsevier B.V. All rights reserved.
Normal dietary consumption and absorption of copper exceed the metabolic need, and homeostasis of this element is maintained exclusively by the biliary excretion of copper. Wilson’s disease is an inherited disorder in which defective biliary excretion of copper leads to its accumulation, particularly in liver and brain [1,2]. Wilson’s disease is due to mutations of the ATP7B gene on chromosome 13 [3,4], which encodes a copper-transporting P-type ATPase (ATP7B) residing in the trans-Golgi network of hepatocytes. ATP7B is responsible for transporting copper from intracellular chaperone proteins into the secretory pathway, both
for excretion into bile and for incorporation into apo-ceruloplasmin for the synthesis of functional ceruloplasmin [3,4]. The development of Wilson’s disease is due to the accumulation of copper in affected tissues. Clinical presentation can vary widely, but the key features of Wilson’s disease are liver disease and cirrhosis, neuropsychiatric disturbances, Kayser–Fleischer rings in Desçemet’s membrane of the cornea, and acute episodes of hemolysis often in association with acute liver failure. Wilson’s disease is not just a disease of
children and young adults, but may present at any age . Wilson’s disease is a genetic disorder that is found worldwide. Wilson’s disease is recognized to be more common than previously thought, with a gene frequency of 1 in 90–150 and an incidence (based on adults presenting with neurologic symptoms ) that may be as high as 1 in 30,000 . More than 500 distinct mutations have been described in the Wilson gene, from which 380 have a conﬁrmed role in the pathogenesis of the disease .
The most common presentations are with liver disease or neuropsychiatric disturbances. Asymptomatic patients are most often detected by family screening.
Age at onset of symptoms
Wilson’s disease may present symptomatically at any age, although the majority presents between ages 5 and 35. The youngest patient reported with cirrhosis due to Wilson’s disease was 3-years-old . About 3% of patients present beyond the fourth decade, either with hepatic or neurologic disease . The oldest patients diagnosed were in their eighth decade [10,11].
The clinical hallmark of Wilson’s disease is the Kayser–Fleischer ring, which is present in 95% of patients with neurologic symptoms and somewhat over half of those without neurologic
symptoms [12,13]. In children presenting with liver disease, Kayser–Fleischer rings are usually absent . Kayser–Fleischer rings are caused by deposition of copper in Desçemet’s mem-
brane of the cornea. A slit-lamp examination by an experienced observer is required to identify Kayser–Fleischer rings. They are not entirely speciﬁc for Wilson’s disease, since they may be found in patients with chronic cholestatic diseases including children with neonatal cholestasis. Other ophthalmologic changes are rare and include sunﬂower cataracts, which are caused by deposits of copper in the center of the lens. They can also be found by slit lamp examination . Neurologic signs are variable, most often tremor, ataxia, and dystonia. Signs of liver disease are nonspeciﬁc, but any liver disease of unknown origin should be considered as Wilson’s disease until proved otherwise. Diagnostic vigilance is important because Kayser Fleischer rings may be absent in up to 50% of patients with Wilson’s disease affecting the liver .
Any type of liver disease may be encountered in patients with Wilson’s disease. Clinically evident liver disease may precede neurologic manifestations by as much as 10 years and most patients with neurologic symptoms have some degree of liver disease at presentation. Presenting symptoms of liver disease can be highly variable, ranging from asymptomatic, with only
biochemical abnormalities, to overt cirrhosis with all its complications. Wilson’s disease may also present as acute hepatic failure sometimes associated with Coombs-negative hemolytic anemia
and acute renal failure. Patients diagnosed with Wilson’s disease who have a history of jaundice may have previously experienced an episode of hemolysis. Clinical symptoms are summarized in
Acute liver failure due to Wilson’s disease (former: ‘‘fulminant Wilson’s disease’’)
Wilson’s disease enters into the differential diagnosis of any young patient presenting with acute hepatitis. Its clinical presentation may be indistinguishable from that of acute viral hepatitis,
with jaundice and abdominal discomfort. In some patients symptoms resolve spontaneously, but once the diagnosis is made, lifelong treatment is necessary. On the other hand, rapid deterioration can occur with acute liver failure.
Wilson’s disease accounts for 6–12% of all patients with acute liver failure who are referred for emergency transplantation [16,17]. Although cirrhosis is already present in most cases, the
clinical presentation is acute and progresses rapidly to hepatic and renal failure and, when untreated, carries an almost 95% mortality. Acute liver failure due to Wilson’s disease occurs pre-
dominantly in young females (female:male ratio 4:1) . an acute presentation with rapid deterioration may also occur in patients who were previously treated but stopped their medica-
tions . Suspicion for acute Wilson’s disease should be particularly high in patients with deep jaundice, low haemoglobin, low cholinesterase , only mildly increased transaminases, and low
Chronic hepatitis and cirrhosis
Many patients present with signs of chronic liver disease and evidence of cirrhosis, either compensated or decompensated. Patients may present with isolated splenomegaly due to clinically inapparent cirrhosis with portal hypertension. The presentation may be indistinguishable from other forms of chronic active hepatitis, with symptoms including jaundice, malaise, and vague abdominal complaints.
Coombs-negative haemolytic anemia may be the only initial symptom of Wilson’s disease. However, marked hemolysis is commonly associated with severe liver disease. Decay of liver
cells may result in the release of large amounts of stored copper, which further aggravates hemolysis. In one series, hemolysis was a presenting feature in 25 out of 220 cases (12%); in these
patients hemolysis occurred either as a single acute episode or recurrently or was low-grade and chronic . In a series of 283 Japanese patients with Wilson’s disease, only three presented
with acute hemolysis alone . One quarter of the patients presenting with jaundice also had hemolysis. Acute liver disease and hemolysis as a presenting symptom can occur during delivery,
mimicking HELLP syndrome . Low-grade hemolysis may be associated with Wilson’s disease even when liver disease is not clinically evident. Some patients presenting with neurologic
symptoms report that they have experienced transient episodes of jaundice previously, probably due to hemolysis . On the other hand, rapid deterioration can occur with acute liver failure.
Wilson’s disease can manifest with an impressive spectrum of neurological, behavioral or psychiatric disorders, which may be its ﬁrst clinical manifestation, appearing simultaneously with
hepatic signs, or some years later.
Neurological presentation can be extremely subtle, and inter-mitted for many years, but may also develop very rapidly, leading within a few months to complete disability. The neurological
abnormalities can be classiﬁed as: (1) Akinetic-rigid syndrome similar to Parkinson’s disease; (2) Pseudosclerosis dominated by tremor; (3) Ataxia; and (4) Dystonic syndrome. In many cases, neu-rological signs are very difﬁcult to classify as patients can have more than one abnormality, each with different levels of severity.
The characteristic tremor is a coarse, irregular proximal trem-ulousness with a ‘‘wing beating’’ appearance. Dystonia can be focal, segmental or very severe, involving all parts of the body,
leading to severe contractures. Very common motor impairments involve the cranial region, and manifest clinically as dysarthria (can be cerebellar or extrapyramidal leading to aphonia), drooling
or oropharyngeal dystonia. Facial grimacing, open jaw, running saliva, and lip retraction are characteristic manifestations. Speech changes and drooling are often early neurologic symptoms. A tre-mor-rigidity syndrome (‘‘juvenile Parkinsonism’’) should raise suspicion of Wilson’s disease [22–24]. Because of an increasing difﬁculty in controlling movement or progressive dystonia, patients become bedridden and unable to care for themselves. Ultimately, the patient becomes severely dis-abled, usually alert, but unable to talk. In patients presenting with advanced liver disease, neurologic symptoms can be mis-taken for signs of hepatic encephalopathy.
Behavioral and psychiatric symptoms are common and some of them may precede neurologic or hepatic signs and symptoms. About one-third of patients initially present with psychiatric abnormalities. In children with Wilson’s disease, declining school performance, personality changes, impulsiveness, labile mood, sexual exhibitionism, and inappropriate behavior are observed [24,25]. The initial symptoms are frequently misdiagnosed as behavioral problems associated with puberty. In older persons, psychotic features resembling paranoia, schizophrenia or depres-sion can be observed but behavioral changes are also common.
Severe cognitive deterioration is observed in patients with advanced neurological disease, but in general, cognitive function is not markedly impaired . A delay in diagnosing Wilson’s disease in patients with neuro-psychiatric presentations is frequent and was in one case as long as 12 years . Patients presenting with neuropsychiatric symp-toms may have concurrent symptomatic liver disease, but in most patients liver disease can only be detected by laboratory evaluation, imaging studies of the liver or by liver histology. About half of the patients have advanced ﬁbrosis or frank cirrho-sis. On the other hand, signs of liver disease may be even com-pletely absent at biopsy .
Other clinical manifestations
Less common presentations include gigantism, lunulae, renal abnormalities including aminoaciduria and nephrolithiasis, hypercalciuria and nephrocalcinosis [29,30], cardiomyopathy
, myopathy , chondrocalcinosis and osteoarthritis , hypoparathyroidism , pancreatitis , infertility or repeated miscarriages [36,37]
Untreated Wilson’s disease is universally fatal, with most patients dying from liver disease and a minority from complications of progressive neurologic disease. With chelation treatment and
liver transplantation, prolonged survival has become the norm [27,38,39], although mortality has not been assessed prospec-tively. In general, prognosis for survival depends on the severity
of liver and neurological disease and compliance with drug treat-ment. Liver function becomes normal over 1–2 years of treatment in most patients with no or compensated cirrhosis at presenta-tion, and then remains stable without progressive liver disease with adherence to treatment. At the other end of the spectrum, medical therapy is rarely effective in patients presenting with acute liver failure due to Wilson’s disease, mainly due to the time
required to remove toxic copper from the organism. A prognostic index has been developed , and later modiﬁed by Dhawan et al. . A score greater than 11 is always fatal without liver
transplantation (Table 3). Patients presenting with neurologic symptoms fare better with respect to life expectancy, especially if liver disease is limited. However, neurologic symptoms appear
to be only partly reversible with treatment and may even worsen following initiation of treatment. In patients undergoing orthotopic liver transplantation, sur-vival may be slightly reduced early on, but appears normal (for transplant population) thereafter .
Acute hepatitis with Wilson’s disease presents similarly to any other acute cases of hepatitis. Similarly, Wilson’s disease should enter into the diagnosis of all patients with chronic hepatitis and cirrhosis, as routine histologic changes are nonspeciﬁc. Wilson’s disease should be considered when acute hepatitis is
accompanied by rapid onset of jaundice and hemolytic anemia. During adolescence, Wilson’s disease presenting with neurologic symptoms may be misdiagnosed as a behavioural problem because initial symptoms may be subtle. More advanced move-ment disorders in a young person should provoke consideration of Wilson’s disease, but the diagnosis may be overlooked where the presentation suggests a primarily psychological or psychiatric disorder.
Typically, the combination of Kayser–Fleischer rings and a low serum ceruloplasmin (<0.1 g/L) level is sufﬁcient to establish a diagnosis. When Kayser–Fleischer rings are not present (as is
common in the hepatic manifestation of Wilson’s disease), ceru-loplasmin levels are not always reliable because they may be low for reasons other than Wilson’s disease (e.g. autoimmune hepati-tis, severe hepatic insufﬁciency in advanced liver disease, celiac disease, familial aceruloplasminemia)  or in heterozygous carriers of ATP7B mutations who do not show copper overload disease. On the other hand, inﬂammation in the liver or else-where may cause the ceruloplasmin concentration to rise to nor-mal levels, reﬂecting its identity as an acute phase protein. This is also true for treatment with estrogens. Thus, for many patients, a
combination of tests reﬂecting disturbed copper metabolism may be needed. Not a single test is per se speciﬁc and, thus, a range of tests has to be applied (Table 4). A diagnostic score based on all available tests was proposed by the Working Party at the 8th International Meeting on Wilson’s disease, Leipzig 2001  (Table 5). The Wilson’s disease scoring system provides a good
diagnostic accuracy . The diagnostic algorithm based on this score is shown in Fig. 1
Ceruloplasmin is the major carrier of copper in the blood. It contains six copper atoms per molecule (holoceruloplasmin) but may be present just as the protein without the copper (apoceruloplasmin). Ceruloplasmin is an acute phase reactant possessing a ferroxidase activity . Levels of serum ceruloplasmin may be measured enzymatically by its copper-dependent
oxidase activity towards speciﬁc substrates, or by antibody dependent assays such as radioimmunoassay, radial immunodiffusion, or nephelometry. Immunologic assays may overestimate ceruloplasmin concentrations since they do not discriminate between apoceruloplasmin and holoceruloplasmin. The normal concentration of ceruloplasmin measured by the enzymatic assay varies among laboratories (with a lower limit between 0.15 and
0.2 g/L). In Wilson’s disease, it is usually lower than 0.1 g/L. Serum ceruloplasmin concentrations are elevated by acute inﬂammation, in states associated with hyperestrogenemia such as pregnancy and estrogen supplementation. Serum ceruloplas min is typically decreased in patients with neurologic Wilson’s disease, but may be in the low normal range in about half of patients with active Wilson’s liver disease. On the other hand, serum ceruloplasmin may be low in other conditions with marked renal or enteric protein loss, malabsorption syndromes or with severe end-stage liver disease of any etiology. Approxi-mately 20% of heterozygotes have decreased levels of serum ceruloplasmin [1,47]. Patients with aceruloplasminemia lack the protein entirely due to mutations in the ceruloplasmin gene on chromosome 3. These patients may exhibit hemosiderosis but do not have copper accumulation . Thus, serum cerulo-plasmin alone is not sufﬁcient to diagnose or to exclude Wilson’s disease. A prospective study on serum ceruloplasmin, as a screen-ing test for Wilson’s disease in patients referred with liver dis-ease, showed that subnormal ceruloplasmin had a positive predictive value of only 6%. In children with Wilson’s disease, 15–36% had ceruloplasmin in the normal range [14,49]. In one
series, 12 out of 55 Wilson’s disease patients had normal cerulo-plasmin and no Kayser–Fleischer rings . The predictive value of ceruloplasmin for diagnosis of Wilson’s disease in acute liver
failure is poor . In one recently published study, measure-ment of serum ceruloplasmin oxidase activity was superior to immunologic assays for diagnosing Wilson’s disease, but these assays are generally not available in routine labs .Serum copper Although a disease of copper overload, the total serum copper (which includes copper incorporated in ceruloplasmin) in Wilson’s disease is usually decreased in proportion to the decreased ceruloplasmin in the circulation. In patients with severe liver injury, serum copper may be within the normal range, independent of whether serum ceruloplasmin levels are elevated or low. In the setting of acute liver failure due to Wilson’s disease, levels of serum copper may even be markedly elevated due to the sudden release of the metal from liver tissue stores. Normal or elevated serum copper levels, in the face of decreased levels of ceruloplasmin, indicate an increase in the concentration of copper which is not bound to ceruloplasmin in the blood (non-ceruloplasmin-bound copper). Non-ceruloplasmin-bound copper (or ‘‘free copper’’) can be calculated by subtracting ceruloplasmin-bound copper (3.15 ceruloplasmin in mg/L equals the amount of ceruloplasmin-bound copper in lg/ L) from the total serum copper concentration (in lg/L; serum copper in lmol/L 63.5 equals serum copper in lg/L) . The serum non-ceruloplasmin-bound copper concentration has been proposed as a diagnostic test for Wilson’s disease . In most untreated patients, it is elevated above 200 lg/L. The serum non-ceruloplasmin copper concentration may be elevated in acute liver failure of any etiology, in chronic cholestasis ,
and in cases of copper intoxication. The major problem with non-ceruloplasmin-bound copper as a diagnostic test for Wilson’s disease is that it is dependent on the adequacy of the methods for
measuring both serum copper and ceruloplasmin. It is of more value in monitoring pharmacotherapy than in the diagnosis of Wilson’s disease.
Urinary copper excretion
The amount of copper excreted in the urine in a 24-hour period may be helpful for diagnosing Wilson’s disease and for monitor-ing treatment. In untreated patients, the 24-hour urinary excre-tion of copper reﬂects the amount of non-ceruloplasmin-bound copper in the circulation. The exact urine volume and the total creatinine excretion per 24 h are important for accurate determi-nation of urinary copper excretion. In case of renal failure, the test is not applicable. In untreated symptomatic patients, ‘‘base-line’’ copper excretion greater than 1.6 lmol/24 h (100 lg/24 h) is taken as diagnostic of Wilson’s disease . However, basal 24-hour urinary copper excretion may be less than 1.6 lmol/ 24 h at presentation in 16–23% of patients, especially in children and asymptomatic siblings [12,14,55]. Since urinary copper excretion is negligible in healthy individuals , a urinary cop-per excretion above 0.64 lmol/24 h can be suggestive of Wilson’s disease in asymptomatic children. The problems of measuring 24-hour copper excretion include incomplete urine collection, and, on the other hand, copper contamination of the collection device (this being less problematic with the advent of disposable containers). Interpreting 24-hour urinary copper excretion can be difﬁcult due to the overlap with ﬁndings in other types of liver disease (e.g. autoimmune hepatitis, chronic active liver disease or cholestasis and in particular during acute hepatic failure of any origin). Heterozygotes may also have higher copper excretion than controls, rarely exceeding the normal range levels .
Urinary copper excretion with D-penicillamine administration was thought to be a useful diagnostic test. This test has only been standardized in a pediatric population in which 500 mg of D-pen-icillamine was administered orally at the beginning and again 12 h later during the 24-hour urine collection, irrespective of body weight . Compared with a spectrum of other liver dis-eases, including autoimmune hepatitis, primary sclerosing cho-langitis, and acute liver failure, a clear differentiation was found when more than 25 lmol/24 h was excreted. A reassessment of
this test in paediatric patients reconﬁrmed the value in the diag-nosis of Wilson’s disease with active liver disease, but was unre-liable to exclude the diagnosis in asymptomatic siblings . In
comparison to children with other liver diseases, the D-penicilla-mine test had only a sensitivity of 12.5%. However, data by Dha-wan et al . and by Nicastro et al . now suggest that using a lower
threshold for urinary copper excretion (without D-penicillamine stimulation) of only 0.64 lmol/24 h increases sensitivity of the test and eliminates the need for the stimulation testing with
D-penicillamine [41,45]. The penicillamine challenge test has been used in adults, but many of the reported results of this test utilized different dosages and timing for administration of the D-penicillamine [12,53,56]. Thus, this test is not recommended for diagnosis of Wilson’s dis-ease in adults.
Hepatic parenchymal copper concentration
Hepatic copper accumulation is the hallmark of Wilson’s disease. However, speciﬁc stains like rhodamine or orcein reveal focal cop-per stores in less than 10% of patients because they detect only lysosomal copper depositions. Thus, hepatic copper overload can-not be excluded by histochemical evaluation of a liver biopsy alone. Therefore, the measurement of hepatic parenchymal copper concentration is the method of choice for the diagnosis of Wilson’s
disease. Biopsies for quantitative copper determination should be placed dry in a copper-free container. Shipment for quantitative copper determination does not require special precautions like freezing. In general, the accuracy of measurement is improved with adequate specimen size: at least 1 cm of biopsy core length should be submitted for analysis . Parafﬁn-embedded speci-mens may also be analyzed for copper content, but may be less reli-able if the specimen is small. Hepatic copper content >4 lmol/g dry weight is considered as the best biochemical evidence for Wilson’s disease. Lowering the threshold from 4 lmol/g dry weight to
1.2 lmol/g dry weight improved sensitivity from 83.3% to 96.5%, while speciﬁcity remained acceptable (95.4% vs. 98.6%) . The major problem with hepatic parenchymal copper concentration is the inhomogeneous distribution of copper within the liver in later stages of Wilson’s disease. Thus, the concentration can be underestimated due to sampling error. In about 18% of adult patients, hepatic copper concentrations are only between 0.8 and 4 lmol/g dry weight with a few even in the normal range . In a pediatric study, sampling error was sufﬁciently common to ren-der this test unreliable in patients with cirrhosis . On the other
hand, in long-standing cholestatic disorders, hepatic copper con-tent may also be increased. Markedly elevated levels of hepatic copper may also be found in idiopathic copper toxicosis syndromes such as Indian childhood cirrhosis .
For diagnostic purposes, a liver biopsy is only required if the clin-ical signs and noninvasive tests do not allow a ﬁnal diagnosis or if there is suspicion of other or additional liver pathologies .
The earliest histologic abnormalities in the liver include mild steatosis (both microvesicular and macrovesicular), glycogenated nuclei in hepatocytes, and focal hepatocellular necrosis [62,63].
Frequently, these changes are misdiagnosed as nonalcoholic fatty liver disease (NAFLD) or nonalcoholic steatohepatitis (NASH). The liver biopsy may show classic histologic features of autoimmune hepatitis (the so-called ‘‘chronic active hepatitis’’ picture). With progressive parenchymal damage, ﬁbrosis and subsequently cirrhosis develop. About half of the patients have cirrhosis at the time of diagnosis . There are a few older patients with Wilson’s disease who do not have cirrhosis or even signs of liver disease [5,12]. In the setting of acute liver failure due to Wilson’s disease, there is a marked hepatocellular degeneration and parenchymal collapse, typically on the background of cirrhosis. Apoptosis of hepatocytes is a prominent feature during the acute injury .
Detection of copper in hepatocytes by routine histochemical evaluation is highly variable. Especially in early stages of the dis-ease, copper is mainly present in the cytoplasm bound to metal-lothionein and is not histochemically detectable . The amount of copper varies from nodule to nodule in the cirrhotic liver and may vary from cell to cell in pre-cirrhotic stages. The
absence of histochemically identiﬁable copper does not exclude Wilson’s disease. Lysosomal copper complexes can be stained by various methods, including the rhodanine or orcein stain.
Ultrastructural analysis of liver specimens at the time steato-sis is present reveals speciﬁc mitochondrial abnormalities . Typical ﬁndings include variability in size and shape, increased
density of the matrix material, and numerous inclusions includ-ing lipid and ﬁne granular material that may be copper. The most striking alteration is increased intracristal space with dilatation
of the tips of the cristae, creating a cystic appearance . In the absence of cholestasis, these changes are considered to be essentially pathognomonic of Wilson’s disease. At later stages of the disease, dense deposits within lysosomes are present. Ultrastructural analysis may be a useful adjunct for diagnosis.
Neurologic ﬁndings and radiologic imaging of the brain
Neurologic evaluation should be performed also on patients with presymptomatic and hepatic Wilson’s disease. Consultation with a neurologist should be sought for evaluation of patients with evident neurologic symptoms before treatment or soon after treatment is initiated.
Neurologic disease may manifest as motor abnormalities with Parkinsonian characteristics of dystonia, hypertonia and rigidity, choreic or pseudosclerotic, with tremors and dysarthria. Due to the great variability of neurological signs, differences in their severity and concomitant presence of different signs in one patient, clinical description is very difﬁcult. There is not yet a commonly accepted scale which describes neurological signs and their severity. One recent proposal is the Uniﬁed Wilson’s disease Rating Scale (UWDRS) [67,68].
Magnetic resonance imaging (MRI) or computerized tomogra-phy of the brain may detect structural abnormalities in the basal ganglia . The most frequent ﬁndings are an increased density on computerized tomography or hyperintensity on T2 MRI in the region of the basal ganglia. MRI may be more sensitive in detecting these lesions. Abnormal ﬁndings are not limited to this region, and other abnormalities have been described. A characteristic ﬁnding in Wilson’s disease is the ‘‘face of the giant panda’’ sign [70,71], but is found only in a minority of patients. Besides this sign, hyperin-tensities in tectal-plate and central pons (CPM-like), and simulta-neous involvement of basal ganglia, thalamus, and brainstem are virtually pathognomonic of Wilson’s disease . Signiﬁcant abnormalities on brain imaging may even be present in some indi-viduals prior to the onset of symptoms .
Other neuroimaging techniques as magnetic resonance spec-troscopy  and single-photon emission computed tomography (SPECT) might be useful in detecting early brain damage in Wilson’s disease, not only in the perspective of assessing and treating motor impairment but also in better evaluating the less investigated disorders in the cognitive domain . Transcranial brain parenchyma sonography (TCS) detects lenticular nucleus hyperechogenicity even when in MRI no abnormalities are observed , but it must be conﬁrmed in further studies .
Auditory-evoked brainstem potentials are helpful to docu-ment the degree of functional impairment and the improvement by treatment [76,77].
Direct molecular-genetic diagnosis is difﬁcult because of more than 500 possible mutations; except for a few more frequent mutations, each of which is rare . Furthermore, most patients
are compound heterozygotes (i.e. carry two different mutations). Comprehensive molecular-genetic screening takes several months, which makes this an impractical method. Nevertheless,it is reasonable to perform molecular analysis of the ATP7B gene in any patient who has a provisional diagnosis of Wilson’s dis-ease, both for conﬁrmation purposes and to facilitate the subse-quent screening of family members. By contrast, allele-speciﬁc probes allow direct identiﬁcation of a mutation and this can be rapid and clinically very helpful. How-ever, this can only be accomplished if a mutation occurs with a rea-sonable frequency in the population (e.g. H1069Q in Central Europe , –441/–427 del. in Sardinia [80,81], R778L in the Far East [82–84]). In those cases, identiﬁcation of a mutation can sup-port the diagnosis, while identiﬁcation of two mutations will con-ﬁrm the diagnosis. With the advancement of DNA-based diagnostics, such as the development of a single chip that is able to identify the most common mutations, these recommendationsmay change.
Acute liver failure due to Wilson’s disease
The most challenging aspect is the diagnosis of acute liver failure due to Wilson’s disease, since mortality without emergency liver transplantation is very high. Readily available laboratory tests,
including alkaline phosphatase (AP), bilirubin, and serum amino-transferases, provide the most rapid and accurate method for diagnosis of acute liver failure due to Wilson’s disease . Com-bination of an AP elevation/total bilirubin elevation ratio <4 and an AST:ALT ratio >2.2 yielded a diagnostic sensitivity and speci-ﬁcity of 100% . However, these ﬁndings were challenged by other authors. Therefore, these parameters should be considered in case acute Wilson’s disease is suspected, but should be used in combination with other signs and symptoms suggesting Wilson’s disease. The combination of clinical symptoms and the conven-tional Wilson’s disease diagnostic parameters (ceruloplasmin, serum or urinary copper) are less sensitive and speciﬁc but important for the diagnosis . The diagnosis has to be ascer-tained by liver biopsy if possible or at least after transplantation (hepatic copper content, mutation analysis) to enable screening of asymptomatic siblings.
It is essential to screen the family of patients presenting with Wil-son’s disease because the chance of a sibling being a homozygote and therefore developing clinical disease – is 25%. Amongst off spring, the chance is 0.5%. Although this risk is low, analysis of the ATP7B gene for mutations in the children of an index patient is justiﬁed given the potential devastating course of Wilson’s dis-ease. There is difﬁculty in diagnosing heterozygote carriers with certainty, but siblings of an index case with a documented muta-tion can be screened by mutational analysis. If the mutation(s) of the index case are not detected, pedigree analysis using haplotypes based on polymorphisms surrounding the Wilson’s disease gene is available. This analysis requires the
identiﬁcation of an index patient with the unquestionable diag-nosis of Wilson’s disease within the family. DNA is required from both parents. Then the haplotype, based on the pattern of dinu-cleotide and trinucleotide repeats around ATP7B, is determined in the index patient and his/her family. The inheritance of the ‘‘disease-associated’’ haplotypes allows determining whether
they are unaffected, heterozygous, or indeed patients . Genetic testing is the only reliable method to separate heterozy-gote from homozygote siblings.
A number of drugs are available for the treatment of Wilson’s dis-ease, including D-penicillamine, trientine, zinc, tetrathiomolyb-date, and dimercaprol. Once the diagnosis has been made, treatment needs to be life-long. There is a lack of high-quality evidence to estimate the relative treatment effects of the avail-able drugs in Wilson’s disease. Therefore, multicentre prospective randomized controlled comparative trials are necessary .
The major effect of D-penicillamine in Wilson’s disease is to promote the urinary excretion of copper. D-penicillamine may also act by inducing metallothionein . The maintenance dose is usually 750–1500 mg/day administered in two or three divided doses. Dosing in children is 20 mg/kg/day rounded off to the nearest 250 mg and given in two or three divided doses.
D-Penicillamine is best administered 1 h prior to meals, because food inhibits its absorption. Since D-penicillamine tends to interfere with pyridoxine action, supplemental pyridoxine should be provided (25–50 mg/day). D-penicillamine interferes with collagen cross-linking  and has some immunosuppres-sant actions [90,91]. Adequacy of treatment can be monitored by measuring 24-hour urinary copper excretion while on treatment. This is highest immediately after starting treatment and may exceed 16 lmol (1000 lg) per 24 h at that time. For long-term treatment, the most important sign of efﬁcacy is a maintained clinical and labo-ratory improvement. Serum ceruloplasmin may decrease after initiation of treatment. Urinary copper excretion should run in the vicinity of 3–8 lmol per 24 h on treatment. To document therapeutic efﬁciency, urinary copper excretion after 2 days of D-penicillamine cessation should be 61.6 lmol/24 h. In addition, the estimate of non-ceruloplasmin bound copper shows normal-ization of non-ceruloplasmin bound copper concentration with effective treatment . Values of urine copper excretion >1.6 lmol/24 h after two days of D-penicillamine cessation may indicate non-adherence to therapy (in those patients non-cerulo-plasmin-bound copper is elevated >15 lg/L).
D-penicillamine is rapidly absorbed from the gastrointestinal tract with a double-peaked curve for intestinal absorption [93,94]. If D-penicillamine is taken with a meal, its absorption is decreased overall by about 50%. Once absorbed, 80% of D-penicillamine circulates bound to plasma proteins. Greater than 80% of D-penicillamine excretion is via the kidneys. The excretion half-life of D-penicillamine is on the order of 1.7–7 h, but there is considerable inter-individual variation.Numerous studies attest to the effectiveness of D-penicilla-mine as treatment for Wilson’s disease [95–97]. In patients with symptomatic liver disease, recovery of synthetic liver function and improvement in clinical signs occur typically during the ﬁrst 2–6 months of treatment, but further recovery can occur during the ﬁrst year of treatment. Failure to comply with therapy leads to signiﬁcant progression of liver disease and liver failure within 1–12 months following discontinuation of treatment. In patients with neurologic Wilson’s disease, improvement of symptoms is slower and may be observed even after three years . Worsening of neurologic symptoms has been reported in 10–50% of patients treated with D-penicillamine during the initial phase of treatment. In a recent series, neurologic worsening occurred on all three treatments used for Wilson’s disease (D-penicillamine, trientine, zinc), but mainly with D-penicillamine, where 13.8% were adversely affected . Tolerability of D-penicillamine may be enhanced by starting with incremental doses, 125–250 mg/day increased by 250 mg increments every 4–7 days to a maximum of 1000–1500 mg/day in 2–4 divided dos-ages. Administration of doses 1500 mg per day or higher at once may lead to rapid and often irreversible neurological deterioration. Rapid re-administration of the treatment in patients who stopped it for longer time may also evoke irreversible neurological signs. D-penicillamine is associated with numerous side effects. Severe side effects requiring the drug to be discontinued occur in approximately 30% of patients [95,98]. Early sensitivity reac-tions marked by fever and cutaneous eruptions, lymphadenopa-thy, neutropenia or thrombocytopenia, and proteinuria may occur during the ﬁrst 1–3 weeks. Signiﬁcant bone marrow toxicity includes severe thrombocy-topenia or total aplasia. In these conditions, D-penicillamine should be discontinued immediately. Late reactions include nephrotoxicity, usually heralded by proteinuria or the appear-ance of other cellular elements in the urine, for which discontin-uation of D-penicillamine should be immediate. Other late reactions include a lupus-like syndrome marked by hematuria, proteinuria, and positive antinuclear antibody, and with higher dosages of D-penicillamine no longer typically used for treating Wilson’s disease, Goodpasture syndrome. Dermatological toxici-ties reported include progeric changes in the skin and elastosis perforans serpingosa , and pemphigous or pemphigoid lesions, lichen planus, and aphthous stomatitis. Very late side effects are rare and include nephrotoxicity, myasthenia gravis , polymyositis, loss of taste, immunoglobulin A depression,and serous retinitis. Hepatic siderosis has been reported in trea-ted patients with reduced levels of serum ceruloplasmin and non-ceruloplasmin bound copper . Overtreatment with pen-icillamine may lead to a reversible sideroblastic anemia and hemosiderosis.
Trientine (triethylene tetramine dihydrochloride or 2,2,2-tetra-mine) was introduced in 1969 as an alternative to D-penicilla-mine. Trientine is a chelator with a polyamine-like structure
chemically distinct from D-penicillamine. It lacks sulfhydryl groups and copper is chelated by forming a stable complex with the four constituent nitrogens in a planar ring. Like D-penicilla-mine, trientine promotes urinary copper excretion. Few data exist about the pharmacokinetics of trientine. It is poorly absorbed from the gastrointestinal tract, and what is absorbed is metabolized and inactivated . About 1% of the administered trientine and about 8% of the biotransformed trientine metabolite, acetyltrien, ultimately appear in the urine.
The amounts of urinary copper, zinc and iron increase in par-allel with the amount of trientine excreted in the urine . The potency of trientine as copper chelator in compari-son to D-penicillamine is controversial [95,104]. Trientine and D-penicillamine may mobilize different pools of body copper . Typical dosages of trientine are 900–2700 mg/day in two or three divided doses, with 900–1500 mg/day used for mainte-nance therapy. In children, the weight-based dose is not estab-lished, but the dose generally used is 20 mg/kg/day rounded off to the nearest 250 mg, given in two or three divided doses. Trien-tine should be administered 1 h before or 3 h after meals. Taking it closer to meals is acceptable if this ensures compliance. Trien-tine tablets may not be stable for prolonged periods at high ambi-ent temperature, which is a problem for patients travelling to warm climates.
Trientine is an effective treatment for Wilson’s disease [106,107]. Trientine, while being developed for use in patients who are intolerant of penicillamine, has also been shown to be
an effective initial therapy, even with patients with decompen-sated liver disease at the outset [108,109]. In general, adverse effects due to D-penicillamine resolve when it is substituted for
trientine and do not recur during prolonged treatment with trientine.
Neurological worsening after beginning of treatment with tri-entine has been reported but appears less common than with D-penicillamine. Trientine also chelates iron, and co-dministration of trientine and iron should be avoided because the complex with iron is toxic. A reversible sideroblastic anemia may be a consequence of overtreatment and resultant copper deﬁciency.
Lupus-like reactions have also been reported in some Wilson’s disease patients treated with trientine. However, these patients were almost all uniformly treated previously with D-penicilla-mine, so the true frequency of this reaction when trientine is used de novo is unknown.
Adequacy of treatment is monitored by measuring 24-hour urinary copper excretion (after 2 days of cessation of therapy) and by measuring non-ceruloplasmin bound copper.
Ammonium tetrathiomolybdate (TM) is a very strong decoppering agent. TM complexes with copper; in the intestinal tract it prevents absorption, and in the circulation renders copper unavailable for cellular uptake . TM can directly and reversibly down-regu-late copper delivery to secreted metalloenzymes . At low doses, TM removes copper from metallothionein, but at higher doses it forms an insoluble copper complex, which is deposited
in the liver . TM remains an experimental therapy, and it is not commercially available. As yet, clinical experience with this drug is limited. The control of free copper was prospectively stud-ied as initial anti-copper treatment in neurologically presenting Wilson’s disease patients . Patients were treated for 8 weeks with TM, and thereafter with zinc. In an open-label trial, TM
showed very strong control of free copper levels. In a double-blind trial, TM signiﬁcantly better controlled free copper levels than tri-entine. On trientine, ﬁve patients worsened neurologically and this was associated with signiﬁcant spikes in serum free copper levels. Other data also indicate its utility because it may less likely cause neurological deterioration [114,115]. Potential adverse effects include bone marrow depression , hepatotoxicity , and overly aggressive copper removal, which causes neurological dys-function. TM also has anti-angiogenic effects due to its extensive decoppering effect .
Zinc was ﬁrst used to treat Wilson’s disease by Schouwink in Holland in the early 1960s . Its mechanism of action is dif-ferent from that of penicillamine and trientine: zinc interferes with the uptake of copper from the gastrointestinal tract. Zinc induces enterocyte metallothionein, a cysteine-rich protein that is an endogenous chelator of metals. Metallothionein has greater
afﬁnity for copper than for zinc and, thus, preferentially binds copper present in the enterocyte and inhibits its entry into the portal circulation. Once bound, the copper is not absorbed but
is lost into the fecal contents as enterocytes are shed by normal turnover . Because copper also enters the gastrointestinal tract from saliva and gastric secretions, zinc treatment can
generate a negative balance for copper and thereby remove stored copper [121,122]. Zinc may also act by inducing levels of hepatocellular metallothionein [123,124], thus binding excess
of toxic copper to prevent hepatocellular injury.
Different zinc salts (sulphate, acetate, gluconate) are used. The recommended dose is 150 mg elemental zinc/day (for children <50 kg in body weight 75 mg) administered in three divided
doses, 30 min before meals. Whether a combination therapy with chelators has advantages is not yet known. However, to avoid the neutralization of zinc efﬁciency by chelators, different times of
dosing have to be considered. The compliance with the three times per day dosage may be problematic. The zinc salt used does not make a difference with respect to efﬁcacy but may affect tol-erability. Taking the zinc medication with food interferes with its absorption . Adequacy of treatment with zinc is judged by clinical and biochemical improvement and by measuring 24-hour urinary excretion of copper, which should be less than 1.6 lmol per 24 h on stable treatment. Additionally, non-ceruloplasmin-bound copper should drop with effective treatment. Urinary excretion of zinc may be measured from time to time to check compliance.
Zinc has few side effects. Gastric irritation is a common prob-lem and may be dependent on the salt employed. Zinc may have immunosuppressant effects and reduce leukocyte chemotaxis.
Elevations in serum lipase and/or amylase may occur, without clinical or radiologic evidence of pancreatitis. Neurological dete-rioration is uncommon with zinc [96,126,127]. Whether high-dose zinc is safe for patients with impaired renal function is not yet established.
Most data on zinc come from uncontrolled studies of dosages ranging from 75 to 250 mg per day [87,128]. Zinc is probably less effective than chelating agents in the treatment of established Wilson’s disease, although data are limited and uncontrolled . Although zinc is currently reserved for maintenance treat-ment, it has also been used as ﬁrst-line therapy, most commonly
for asymptomatic or presymptomatic patients. It appears to be equally effective as D-penicillamine but better tolerated .
Reports of large studies in adults with Wilson’s disease indicate good efﬁcacy . While zinc monotherapy appears to be effec-tive and safe in neurologic Wilson’s disease and in asymptomatic siblings, great caution is needed in patients with hepatic Wilson’s disease. Hepatic deterioration has been occasionally reported when zinc was commenced and was fatal in one case . Thus, exclusive monotherapy with zinc in symptomatic Wilson’s liver disease is controversial. In the Netherlands, 17 symptomatic patients with Wilson’s disease were treated with zinc only with a median follow-up of 14 years . The outcome of exclusive zinc therapy was generally good in cases of neurologic disease.
A less satisfactory outcome in hepatic disease may relate to less efﬁcient de-coppering. Two patients with hepatic Wilson’s dis-ease progressed to a decompensated state and two patients with neurologic Wilson’s disease developed symptomatic liver disease.
Long-term outcomes of different treatments in 288 German and Austrian Wilson’s disease patients indicated that, in the majority of patients, treatment with chelating agents or zinc salts was effective. However, there was an advantage for chelating agents to prevent hepatic deterioration . In contrast, in a Polish cohort of 164 patients there were no differences in survival of patients who started therapy with zinc sulfate or D-penicillamine . Current guidelines recommend that all symptomatic patients with Wilson disease should receive a chelating agent (penicillamine or trientine) [130,131]. Zinc may have a role as a ﬁrst line therapy in neurological patients.
Antioxidants, mainly vitamin E, may have a role as adjunctive treatment [132,133]. Serum and hepatic vitamin E levels have been found to be low in Wilson’s disease [134–136]. Symptom-atic improvement when vitamin E was added to the treatment regimen has been occasionally reported but no rigorous studies have been conducted. One study suggests no correlation of anti-oxidant deﬁciency with clinical symptoms .
Animal data suggest a role for amitriptyline in impending liver failure due to Wilson’s disease, as it reduces the copper-induced apoptosis of liver cells, and thereby increases survival of ATP7B-deﬁcient rats . However, no human data are available yet.
In vitro, treatment with pharmacological chaperones 4-phenylbutyrate and curcumin, partially restored protein expres-sion of most ATP7B mutants and might enable novel treatment strategies in Wilson’s disease, by directly enhancing the protein expression of mutant ATP7B with residual copper export activity Furthermore, curcumin is an ideal antioxidant and na effective scavenger of reactive oxygen species  and can act as a copper-chelating agent . Clinical data in patients with Wilson’s disease are not yet available.
Transplant ation is frequently nec ess ar y for patients presenting wi th acute liver failure orde compensated ci rrhosis due to Wilson ’s disease [ 14 1] . Becausethebioc hemic al defect resides mainly in the liver, orthot opi c liver transplant ation ( OL T) corrects the underlying problem.
Successful treatment means that women with Wilson’s disease can become pregnant [148,149]. Counseling should indicate that the likelihood of ﬁnding a homozygote amongst children is 0.5%; haplotype analysis of the partner is justiﬁed. The patient’s copper status should be optimized prior to pregnancy. Although there is some concern over the teratogenicity of D-penicillamine, the risks of withdrawing treatment outweigh those of continuing it. A compilation of published case series on 161 pregnancies in 83 women with Wilson’s disease (one of them after successful in vitro fertilization) treated with D-penicillamine during pregnancy showed 122 births with 119 normal newborns . A high abortion rate was only observed in a study from India .
Fonte: Journal of Hepatology 2012 vol. 56 j 671–685