Ceruloplasmin (Cp) is a member of the multi copper oxidase family of enzymes. It is also an acute phase protein. Cp itself is not essential for normal copper metabolism; it is for iron metabolism in which it is an important ferroxi-dase. It seems likely that the reason for the low Cp in WD is not the lack of hepatic expression of apoceruloplasmin (due to a low copper in the secretory pathway) but the change in the proportion of apo- to holoceruloplasmin in the circulation and the lower stability of apoceruloplasmin [7].

Molecular pathogenesis

In WD there are mutations in the ATP7B gene. This gene was identified by three groups in 1993 [8-11].

The ATP7B gene is highly expressed in the liver, kidney and placenta. It encodes a transmembrane ATPase which is a copper-dependent P type ATPase.

Mutations in ATP7B are associated with lack of entry of copper into bile and into the Golgi. This leads to hepatic copper accumulation which, depending on its severity, leads to hepatic and neurological features of WD.

Mutations within the ATP7B gene variously affect the function of the ATP7B protein. The H1069Q mutation (which results in histidine to glutamate substitution at amino acid 1069) is the most common mutation found in patients of European origin. Studies on liver tissue from patients homozygous for this mutation with WD show that the ATP7B resided in the endoplasmic reticulum rather than the trans-Golgi network [12]. This mutation is associ-ated with defective phosphorylation of ATP7B by adenos-ine triphosphate (ATP). This finding parallels the fact that the histidine 1069 is in a conserved motif within the ATP binding domain of ATP7B [13].

Techniques are now available for the analysis of various APT7B mutations and show their effect on copper transport activity [14].

Molecular genetics

Phenotype/genotype relationship

Over 400 disease-related mutations in the ATP7B have been reported in WD (www.wilsondisease.med.ualberta.ca/database.aspdatabase). Most patients are compound heterozygotes. This makes phenotype/genotype correlation problematic.

In some populations, particular mutations predominate [15]. Because of this, screening of specific mutations may be fruitful. Thus, around 50–80% of patients with WD from Central, Eastern and Northern Europe carry at least one allele with the H1069Q mutation [15]. There is a wide range of mutations in the Mediterranean region and their frequency varies between different countries. In some countries, for example Sardinia and Spain, other particular mutations are common.

Studies of genotype/phenotype correlations of WD have been more made difficult by the large number of mutations that exist. Because of the prominence of the H1069Q mutation this has received most attention. Homozygosity for this mutation has been reported to correlate with late onset neurological disease in individual studies and in a meta-analysis [16]. There have been studies of genotype/phenotype associations with mutations in other genes but there is as yet no clinically applicable result [17].

Family screening using genetics

Family screening for WD is covered later in this chapter. Clinical and biochemical tests should be done, but molecular genetics with identification of mutations is an invaluable tool. If both mutations are known in the index patient with WD, testing of indeterminate siblings for the same mutations will define whether or not they are either affected, heterozygote carriers or normal.

Clinical manifestations

The clinical spectrum of manifestations of Wilson’s disease is wide (Table 31.1). Patients can present acutely with liver failure, hemolysis or both. They may also present with chronic liver disease or neuro-psychiatric problems or a combination of the two. Patients who present initially with neurological or psychiatric features tend to be older than those who present with clinical manifestations of liver disease or hepatic decompensation.

Table 31.1 Clinical features of Wilson’s disease.

Classically, WD is recognized as presenting most often in children and young adults, and indeed it had been considered that it did not occur in individuals aged more than 40 years. However, it is now recognized that later presentation does occur, with a recent report of neurological presentation in a group aged 40–52, and hepatic presentation at up to 58 years of age [18]. This emphasizes the need to have WD within the differential diagnosis of patients with unexplained liver disease and particular neurological symptoms presenting anywhere from teenage years to the fifth decade, and beyond.

Most patients with central nervous system involvement are believed to have liver disease at the time of presentation but they may not be symptomatic from their liver disease. However, hepatic histology is not generally available for these patients because the diagnosis is usually established solely on the basis of the presence of Kayser Fleischer rings with a decreased ceruloplasmin concentration.

It is generally the case that those presenting with acute liver disease, even in the younger age group, already have cirrhosis. Deterioration at this stage appears to be based upon the toxic effect of free copper causing decompensation of liver disease [19], or hemolysis [20].

Hepatic disease

Pathology

In the early stages of the disease, diffuse cytoplasmic copper accumulation can only be seen with special immu-nological stains detecting copper. These are not routinely available. The early accumulation of cytoplasmic copper is associated with macrosteatosis, microsteatosis and glyco-genated nuclei.

Ultra structural abnormalities (on electron microscopy) include enlargement and separation of the mitochondrial inner and outer membranes, with widening of the inter-crystal spaces, to increases in the density of granularity of the matrix, or presence of larger vacuoles. In the absence of cholestasis, these changes are regarded as pathogno-monic of Wilson’s disease.

These initial stages of WD progress to an intermediate stage which is characterized by periportal inflammation, mononuclear cellular infiltrates, erosion of the limiting plate, lobular necrosis and bridging fibrosis. These features may be indistinguishable from those of an autoimmune hepatitis [21, 22]. Mallory bodies are seen in up to 50% of liver biopsy specimens [23]. Cirrhosis almost invariably follows with either a micro-nodular or mixed macro-micro nodular pattern.

In patients with fulminant hepatic failure, parenchymal apoptosis, necrosis and collapse may be prominent, often, as already mentioned, on a background of cirrhosis [24]. One series of results of liver biopsy in WD showed cirrhosis in 54% of 83 patients with a hepatic presentation, and 41% in 34 with a neurological presentation [25].

Confirmation of excess copper histologically (rhodanine, rubeanic acid) may be helpful in diagnosis but if absent does not exclude Wilson’s disease. The lack of immuno-reactivity to copper binding protein (orcein stain) may occur because of the diffuse presence of copper in the cytoplasm and because of the stain’s low sensitivity.

Clinical features

Wilson’s disease can present as fulminant hepatic failure. There is worsening coagulopathy and encephalopathy. There may be an associated Coombs’ negative hemolytic anemia, when there is a substantial increase in serum total and free copper as well as a high urinary copper concentra-tion. This complication is recognized as potentially leading to renal failure. Around 5% of patients present in this manner [26], with most patients being in the second decade of life, when KF rings may not yet be formed. Almost all such patients already have cirrhosis, although some might show evidence of massive necrosis with only bridging fibro-sis, which clearly would progress to cirrhosis with time. Concentration of serum alkaline phosphatase is frequently low and this feature has led to the suggestion that a ratio of alkaline phosphatase concentration (in IU/L) to serum bilirubin concentration (mg/dl) is diagnostically useful. A ratio of less than 2 [27,28] or 4 [29], may be diagnostic or very suggestive of Wilsonian fulminant hepatic disease.

In those with a more chronic hepatic presentation, the clinical picture can be similar to other forms of chronic hepatitis, which, as already noted, emphasizes the need for WD to be in the differential diagnosis of such patients.

Some patients present with an asymptomatic cirrhosis. These would have the usual clinical features, including spider nevi, splenomegaly, portal hypertension and ascites. Cirrhosis may be well compensated.

Hepatocellular carcinoma is very rarely associated with Wilson’s disease, but as in other causes of cirrhosis it may occur [30, 31]. In a series of 11 patients reported (from a total cohort of 363) there were cholangiocarcinomas (three cases) and malignancies of uncertain origin (three cases) as well as three hepatocellular carcinomas [30].

If neurological features occur in patients with liver disease, they usually do so 2–5 years later [32].

Eye changes

The classic finding in patients with WD is the Kayser Fleischer (KF) ring. KF rings are caused by the granular deposition of copper on the inner surface of the cornea in Descemet’s membrane. They are most apparent at the periphery of the cornea. The upper pole is affected first. Although sometimes visible to the naked eye as a golden-brown ring on the periphery of the cornea, slit lamp examination by an experienced ophthalmologist is necessary to confirm the presence or absence of KF rings. Appearances indistinguishable from KF rings have also been seen in other forms of chronic liver disease, in particular chronic cholestasis, and cryptogenic cirrhosis [33].

Sunflower cataracts, also only visible by slit lamp examination, are brilliantly multi-coloured [34]. Interestingly, they do not impair vision.

Both of these findings disappear with successful medical therapy or after liver transplantation. The re-appearance in “the medically treated” patient suggests non-compliance with therapy [35].

Other less common ophthalmological findings include night blindness, exotropic strabismus, optic neuritis and optic disc pallor.

Neurological and neuro-psychiatric disease

Pathology

Histologically, there is an increase in astrocytes in the grey matter, associated with swollen glia, liquefaction, and appearances of spongiform degeneration. Neuronal loss is often accompanied by gliosis and active glial fibrillary protein. The characteristic astrocytes are Alzheimer type I and type II cells. Opalski cells are distinctive for WD [36]. These cells are thought to be derived from from degenerating astrocytes [37].

Clinical features

Neuropsychiatric signs are present in 40–50% of patients with WD [26].

The neurological changes have been classified as: (1) an akinetic-rigid syndrome similar to Parkinson’s disease; (2) pseudosclerosis dominated by a tremor; (3) ataxia; and (4) a dystonic syndrome [38].

Subtle changes can appear before the characteristic neurological features. These include changes in behavior, deterioration of schoolwork or an inability to carry out activities that require good hand-eye coordination. There may be deterioration of hand writing and consequent micrographia – as in Parkinson’s disease. Other neurological features include tremor, plasticity, lack of motor coordination, dysarthria, drooling and dystonia. Seizures, migraine, headaches and insomnia have also been reported. As well as behavioral changes already described, depression, anxiety and frank psychosis are seen [39].

Cognitive dysfunction in patients with WD may be present with neurological changes, often in the absence of detectable cortical changes or hepatic encephalopathy. This lends support to pathological changes of basal ganglia as being the primary cause in WD of cognitive deficit [40].

All patients with WD should have a neurological examination. Patients with obvious symptoms or signs need to be seen by a neurologist, preferably with a special interest in movement disorders, before treatment. A specific assessment tool with neurological features (based on that for Huntingdon’s disease) has been used in clinical trials to assess patients [41]. However, a new Unified Wilson’s Disease Rating Scale (UWDRS) has value in assessing disease severity [42].

Imaging

Structural brain MRI for WD has shown widespread lesions in the putamen, globus pallidus, caudate, thalamus, mid brain, pons and cerebellum as well as cortical atrophy and white matter changes. Broadly, these lesions show high signal intensity on T2 weighted images and low intensity on T1 scanning [43]. These changes tend to be more severe and widespread in patients with neurological WD [44], although MRI changes may be present in those without neurological symptoms or signs.

Other clinical changes

There may be bone and peri-articular abnormalities, including osteomalacia, osteoporosis, spontaneous fractures, adult rickets, osteoarthritis, osteochondritis dissecans, chondrocalcinosis, subchondrial cyst formation and azure lunulae of the fingernails. The most common sites for skeletal and articular abnormalities are the knee joints and spine [45]. Myocardial copper accumulation can cause cardiomyopathy and arrhythmias although these are clinically rare [46].

Other rare extra hepatic /neural manifestations include hypoparathyroidism, infertility, repeated miscarriages [47] and renal abnormalities [48], including renal tubular acido-sis with aminoaciduria and nephrocalcinosis.

Diagnostic approach

Certain tests are essential when the possibility of WD has been raised. No single one of these on its own has 100% specificity or sensitivity for WD. A combination of features is necessary for a definitive diagnosis to be made [49].

The important observations or biochemical measurements used in the work-up of patients with suspected WD are: serum ceruloplasmin, serum total and free copper, ophthalmological examination (for KF rings); 24-hour urine excretion of copper, and measurement of liver copper content. One might add to this the presence of two mutations found on molecular analysis of the ATP7B gene, but since some of these may be polymorphisms caution is needed in making a diagnosis based on molecular analysis alone.

Other useful measures include: (1) urinary excretion of copper after penicillamine challenge (this test has been described and validated in the pediatric age group) [50]; (2) the presence of rhodamine staining on liver biopsy histology, if hepatic copper measurement has not been done.

There are potential problems with all the observations and measurements described above. Serum ceruloplasmin concentrations are well recognized to be normal in a proportion of patients presenting particularly with hepatic WD [51]. Urinary copper excretion can be increased in some other types of liver disease. The same is true for liver copper concentration, particularly in cholestatic disease.

However, if an individual with neurological symptoms consistent with Wilson’s disease has Kayser Fleischer rings and a low serum ceruloplasmin concentration, the diagnosis is made. Kayser Fleischer rings are usually present in patients presenting with neurological Wilson’s disease – 18 of 20 patients in one report [51].

In patients presenting with liver disease, diagnosis can be more difficult [51]. Roberts and Schilsky [49] have provided several valuable algorithms for the diagnostic approach for patients with different initial features suggesting WD. They provide specific recommendations on the basis of previous published work and the authors’ experience in caring for pediatric and adult patients with WD.

To provide further help with the diagnostic route, Ferenci et al. [52] have proposed a scoring system (Table 31.2). Within this, clinical, biochemical, histological and molecular genetic data are allocated a score. The cumulative total gives an indication of the possibility of a particular individual having WD. This scoring system may be helpful if diagnosis of the disease is being considered [53]. The recent report of a group of pediatric patients as part of the analysis of the Euro Wilson’s Group of patients gives some information on this scoring system [54].

Liver function tests

Serum aminotransferase activity is in general abnormal in WD except at a very early stage. The rise in aminotransferase activity is often mild and does not necessarily reflect the severity of liver disease.

The ratio of alkaline phosphatase to serum bilirubin in mg/dl has already been discussed. This may be valuable in raising the possibility of WD in patients with fulminant hepatic failure but again is not 100% specific and may only be valuable in particular patient groups [55].

Ceruloplasmin

A low serum ceruloplasmin concentration (normal range 0.2–0.5g/l) should raise the question as to whether WD is a possible diagnosis. However, it should be emphasized that many patients presenting to a hepatology clinic can have a low ceruloplasmin, but in only a small fraction is WD diagnosed [56].

On the other hand, a low ceruloplasmin may be found in hypoproteinemic states due, for example, to the nephrotic syndrome, in copper deficient states (due to mal-absorption) and in inherited diseases such as aceruloplas-minemia [57]. In the latter two situations, the serum ceruloplasmin may be so low as to be undetectable by laboratory methods.

A normal serum ceruloplasmin in WD patients presenting with active liver disease is interpreted as due to an acute phase reaction. The oral contraceptive may also falsely raise the ceruloplasmin.

Table 31.2 Proposed scoring system for diagnosis of Wilson’s disease (from Ferenci et al, 2003; ref 52).

A further confounding factor in the measurement of serum ceruloplasmin is the technique used. Most laboratories use an immunological reaction which measures both apo as well as holo ceruloplasmin. In WD it is the level in particular of holo ceruloplasmin which is low. This would be measured more accurately by a biological method (oxidase activity), but this method appears only to be used in a small number of laboratories. Thus, the technology used for measuring ceruloplasmin may be responsible for the finding of normal levels in some patients with Wilson’s disease, and also the variability between laboratories [58].

Steindl et al. found in their group of hepatic patients, that serum ceruloplasmin (measured immunologically) was low in only 65% of patients, but in patients with neurological or neuropsychiatric presentation, low ceruloplasmin was found in 85% [51].

These findings indicate that if the serum ceruloplasmin is low then other investigations are necessary in order to define whether this is related to WD or not. Conversely, in patients in whom WD has been suspected but who have a normal ceruloplasmin, other tests are required to investigate this further.

Kayser Fleischer rings

Examination of the eyes using a slit lamp should be done by an experienced ophthalmologist. Steindl et al.

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