Introduction

Wilson’s disease (WD), also known as hepatolenticular degeneration, is an inherited disorder of copper metabolism caused by its impaired excretion into bile, and caused by the defective function of the hepatocyte enzyme ATPase 7B, encoded on chromosome 13 (13q14.3) [1]. This dysfunction leads to the accumulation of copper in the liver and subsequently in other organs such as the brain, kidneys, and cornea, resulting in liver diseases, as well as neurological and/or psychiatric manifestations [2, 3].

The clinical symptoms of WD are highly variable. With increasing copper deposition several different organs are affected leading to symptoms but the phenotype differs between patients with the same genotype, and even within a single family [4, 5]. The hepatic manifestations are nonspecific and mainly manifested as acute and chronic hepatitis, liver cirrhosis, and fulminant hepatitis [6]. Nevertheless, any liver disease of unknown origin should be considered WD until proven otherwise. Making a rapid and early diagnosis of WD is critical for both therapy of the proband and genetic counseling of family members. Early diagnosis is crucial to disease prognosis, since undiagnosed or untreated hepatic copper accumulation leads to fibrosis, cirrhosis, and end-stage liver disease. In this article, we aim to review the clinical features of WD and highlight the challenges in making a reliable diagnosis.

Copper Metabolism and Pathophysiology of WD

Copper is the third most abundant trace element in the body, after iron and zinc, and is an essential trace element and micronutrient for all the living organisms. It plays a crucial role in many enzymatic reactions and in neurotransmitter biosynthesis. Copper is used as a cofactor for enzymes and proteins, including cytochrome C oxidase, lysyl oxidase, dopamine-oxidase, superoxide dismutase, tyrosinase, ascorbic acid oxidase, ferroxidase, and ceruloplasmin, the main extracellular copper transporter. Dietary copper is absorbed by small intestine enterocytes by a human transporter of copper (CTR1) and is then transported to the bloodstream by a membrane-spanning copper transporter, the ATP7A, at the basolateral aspect of duodenal epithelia. Copper reaches the liver via the portal vein bound to circulating albumin, and there stored in hepatocytes (predominantly bound to metallothionenin). Furthermore, small quantities of copper are bound in the blood by transcuprein and other peptides [7]. Excess copper is removed by excretion into the bile [8] (Fig. 1). Copper is assimilated on the basolateral side of hepatocytes via CTR1. In the hepatocyte cytoplasm, copper is bound to small proteins such as metallothionein and glutathione, or copper-specific chaperones, and in this way, the cell is well protected against the toxic effects of copper. Copper chaperone for superoxide dismutase (CCS), antioxidant 1 copper chaperone (ATOX1), and cytochrome C oxidase copper chaperone (COX17) are the three described copper chaperones and bind the copper ion and guide it to different intracellular locations, ensuring efficient delivery of the metal to the enzymes. ATOX1 is a specific copper chaperone antioxidant protein that delivers copper to the WD protein, ATP7B, by copper-dependent protein–protein interactions [9]. In addition, copper transport is regulated by adaptor proteins (APs) involved in the transmembrane protein and clathrin-coated vesicle transport between the Golgi apparatus, endosomes, and the plasma membrane. AP1 is responsible for the normal function of ATP7A, ATP7B, and clathrin. A mutation in the AP1 gene, sigma complex subunit 1A (AP1S1), results in dysfunction of ATP7A and ATP7B despite intact enzymatic activities [7]. Their mislocalization leads to dysfunction of copper metabolism, reduction of copper-dependent enzymes, and lack of copper transport [10].

Fig. 1
figure 1

Copper metabolism and pathophysiology of Wilson’s disease

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ATP7B encodes a metal-transporting ATPase, a multifunctional membrane-spanning protein that resides especially in the trans-Golgi network (TGN) of hepatocytes, monitoring hepatocellular levels of copper. Indeed, ATP7B ensures two main functions within the TGN network and cytoplasmic hepatocyte vesicles [11]. It firstly activates the ceruloplasmin in TNG by incorporating six copper molecules into apoceruloplasmin, that is subsequently secreted into the bloodstream, carrying 90% of the copper present in plasma. Ceruloplasmin acts as a source of copper for other organs, including the brain and kidney. Secondly, the cytoplasmic ATP7B regulates the excretion of Cu by sequestering the excess copper into vesicles and excretes it via exocytosis across the apical canalicular membrane into the bile [12, 13]. A dysfunction of this copper-regulating protein leads to a defect in the regulation of copper homeostasis in the body. WD is caused by a mutation in the ATP7B causing defective biliary excretion of copper leading to its accumulation, especially in the liver and brain. The activation of apoceruloplasmin, a peptide without copper, that has a shorter half-life, into ceruloplasmin [14] is reduced in WD patients, and holo-ceruloplasmin (holo-Cp) serum concentration is usually low. Total serum copper levels typically are reduced in WD patients, reflecting the reduced copper bound to Cp that normally represents 90% of total serum copper [15]. An increase in hepatic copper with associated liver damage is noted and is associated with the release of free copper from the liver. This increase in non-ceruloplasmin-bound copper (free copper) is associated with an increased urinary copper excretion [16].

Clinical Manifestations

WD is a multisystem disorder that affects principally the liver, the brain, and the musculoskeletal system. During early life (≤ 3 years), the patient is presymptomatic, although the accumulation of copper invariably causes subclinical liver disease. The age of 3 years has been considered the optimal point for mass screening for WD [17]. The hepatic symptoms of WD may precede the onset of neuropsychiatric disorders by up to 10 years [18]. At a median age of 17 years, patients present with hepatic, neurologic, and psychiatric manifestations in roughly equal proportions [19].

WD may present with a wide spectrum of liver disease. The variability in the age of onset of WD probably reflects differences in pathogenic variant and penetrance, extragenic factors, and environmental influences, including diet [16]. In addition, this wide variability may be attributable to ascertainment bias based upon the clinical specialty to which the patient was referred. Neurologists, for example, are likely to see patients with neurologic symptoms and report therefore that the majority of patients with WD have neurologic involvement [20, 21].

The symptoms, clinical course, and outcome of WD are also highly variable. The main features—the hepatic and the neurologic forms—can be distinguished, but many patients present with a mixture of both.

Although WD usually presents in childhood into early adulthood, a later age range of onset (after age 35) is recognized, and indeed later-onset cases continue to be reported [22].

Hepatic Manifestations

Liver dysfunction can be extremely variable in its presentation, and recognized presentations include subtle asymptomatic morphological changes, auto-limited hepatitis and autoimmune hepatitis, recurrent jaundice (in the presence of hemolysis), cirrhosis with or without portal hypertension, and even acute liver failure (Fig. 2). The most common age of hepatic manifestation is between 8 and 18 years [23]. WD rarely presents clinically in children less than younger than 3–5 years old, but there are reports of such, a 13-month-old who was evaluated for transaminitis [24], a 3-year-old with cirrhosis [25], and acute liver failure (ALF) in a 5-year-old patient [26]. In asymptomatic WD, patients may have no signs of clinical liver disease but have abnormalities of liver tests alone. Between 18 and 23% of asymptomatic WD patients presented with only mild elevations in hepatic enzymes [27, 28]. However, a few of these patients may have had more advanced liver disease that was unrecognized [29].

Fig. 2
figure 2

Hepatic presentations in patients with Wilson’s disease

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Symptomatic patients present a wide spectrum of liver disease, ranging from moderate liver damage to acute liver failure. The most common hepatic symptoms are jaundice, anorexia, and vomiting (37–44%), followed by ascites/edema (23–26%) and hepatomegaly/splenomegaly (16–29%) [27, 28, 30]. Jaundice is typical of more severe liver involvement but can be due to intravascular hemolysis. Other presenting symptoms of advanced liver disease include fatigue and confusion due to hepatic encephalopathy, and clinical signs of cirrhosis such as spider angiomas, gynecomastia, palmar erythema, easy bruising, easy bleeding (epistaxis, gingival), and muscle wasting.

Hepatic WD should therefore be considered a possible diagnosis in any child or adolescent with hepatomegaly, elevated serum aminotransferases or signs of hepatic steatosis, or in the presence of evidence of portal hypertension, including splenomegaly, ascites, esophageal and gastric varicose veins, with or without hemorrhage, and hepatic encephalopathy.

Obesity and overweight may be confounding factors in the diagnosis of WD, particularly in the presence of hepatic steatosis, leading to a delayed diagnosis of WD. Bracciamà et al. [31], reported two siblings (sister, 24 years old, presenting moderate hepatic steatosis; and brother, 22 years old, presenting liver cirrhosis) with the same genotype but different clinical evolutions. The brother had episodes of hypertransaminasemia at age 4 and the sister at age 6, without further diagnostic investigations. Unfortunately, appropriate diagnostic investigations were not performed at the time, and WD was initially overlooked in these patients due to their hepatic steatosis and obesity, resulting in a delayed diagnosis of 20 years [31]. Accordingly, it is important to perform screening for WD in patients at risk of developing moderate steatotic liver disease to avoid delays in diagnosis.

The symptomatology of hepatic encephalopathy includes altered sleep patterns, tremor and asterixis, and severe altered mentation that can advance to hepatic coma. Patients with some degree of hepatic encephalopathy may also have exacerbation of other underlying neurologic symptoms, making their evaluation difficult [32].

Cirrhosis is uncommonly found in presymptomatic pediatric patients identified by first-degree-relative screening [6], but its development is highly variable in children diagnosed with WD. In a retrospective study of 229 patients, 62% of patients with hepatic manifestations of WD had cirrhosis [6]. By contrast, only 11% of asymptomatic patients with WD had cirrhosis [29].

Acute Wilsonian hepatitis is indistinguishable from other forms of acute (viral or toxic) liver diseases. In WD, acute liver disease copper that is released from necrotic hepatocytes induces a severe hemolytic anemia that complicates the liver disease. Although WD is rare, it accounts for 6–12% of patients with fulminant hepatic failure referred for emergency liver transplantation. Acute liver failure due to WD occurs more frequently in young females, where they often outnumber males 4:1 [33, 34].

Neurological Manifestations

Copper is essential for brain metabolism, serving as a cofactor to superoxide dismutase, dopamine-β-hydroxylase, amyloid precursor protein, ceruloplasmin, and other proteins required for normal brain function. Although copper physiology in the brain is largely unknown, defects in copper homeostasis have marked consequences for brain function [35]. Even though ATP7B is normally expressed in the central nervous system, its malfunction does not produce clinically significant signs. In WD, the nerve tissue damage that leads to neurological and psychiatric symptoms is principally caused by excess copper. The copper content in the brain and cerebrospinal fluid (CSF) of WD patients with neurologic symptoms are elevated 10- to 15-fold above normal [36]. There is a strong correlation between the severity of neuropathological findings and brain copper content [37], but not always between copper concentration and clinical presentation [36, 38]. Although macroscopically, the WD patient’s brain is not usually greatly altered, there is occasional slight atrophy in long-term cases and the ventricles can be enlarged with a flattened convexity of the caudate nucleus head [39]. Imaging studies have shown that the injuries may occur in several brain structures in symptomatic patients, but are most common in the basal ganglia, since this region of the brain has a high metabolic rate, an increased blood supply, and mitochondrial content, which make it more vulnerable and susceptible to toxins and metal accumulation [40, 41]. The most severe abnormalities are found in the middle zone of the putaminal nuclei, which are characteristically shrunken, soft, and yellowish-brown in appearance. A neuropathological study of metal accumulation in a cohort of 12 brains from WD patients demonstrated that the concentration of copper was increased by almost eightfold in the brains of WD patients compared with controls (0.65 vs. 0.08 µmol/g dry tissue), with a uniform accumulation of copper in the structures examined (putamen, pons, dentate nucleus, and frontal cortex). The putamen had the highest copper concentration 0.79 + 0.44 µmol/g (49.7 + 28 µg/g) and the frontal cortex the lowest 0.54 + 0.28 µmol/g (34.3 + 17.5 µg/g) [36].

Excess copper may cause cell injury and inflammation via several molecular processes including mitochondrial toxicity, oxidative stress, cell membrane damage, crosslinking of DNA, and inhibition of enzymes. Multiple cell culture model studies have revealed that astrocytes are key regulators of copper homeostasis, as they efficiently take up, store, and export this metal in the brain. Astrocytes can play this role as they can upregulate the synthesis of metallothionein and glutathione, which are peptides with the ability to detoxify copper [42, 43]. In the brain, besides astrocytes, oligodendrocytes seem to be particularly sensitive to copper toxicity and may be involved in the regulation of copper overload.

The onset of neurological manifestations of WD usually begins in the second or third decade of life, although there is much variability [44, 45]. Late presenting WD, that presenting over 70 years of age, is recognized to present with mainly neurological symptoms. The patient is never “too old” to have WD [46]. The youngest reported WD patient with neurologic symptoms was aged 9 months [36].

Most neurological presentations of WD are movement disorders including tremor, dystonia, parkinsonism, and ataxia, which are frequently associated with dysphagia, dysarthria, and drooling [45, 47] (Fig. 3). Generally these symptoms overlap and they worsen during the progression of the disease. In any individual patient, one can see many complex combinations of neurological symptoms and signs, which makes it difficult to have a typical picture for the neurology of WD.

Fig. 3
figure 3

Main neurological manifestations of WD

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Numerous large case series studies describe that dysarthria is a typical feature of WD, and occurs in 85–97% of patients with neurological WD. Slow movements of the tongue and orofacial dyskinesia, especially the “risus sardonicus” which describes an involuntary grimace with open mouth and contracted upper lip, are often combined with dysarthria. Dystonia is a movement disorder characterized by uncontrollably sustained or intermittent muscle contractions resulting in repetitive movements and abnormal postures, and it is observed in 11–65% of neuro-WD patients in different cohorts. Dystonia can affect different body parts and could manifest as focal (risus sardonicus, oro-facial dystonia, limb dystonia), segmental (trunk dystonia), multifocal, or even generalized symptoms (status dystonicus) [48]. Isolated cervical dystonia is unlikely to be due to WD [49].

Tremor is a neurological symptom defined as an involuntary, rhythmic, and oscillatory movement of a part of the body that is often persistent and visible in hepatolenticular degeneration and other neurological diseases that include postural, rubral (wing-beating), and rest tremors. It occurs in 55% of WD patients as the first neurological symptom and in up to 90% of patients during disease progression [45, 50, 51]. In WD, this tremor starts in the upper distal extremities unilaterally and reaches the head, legs, and the whole body during the development of the disease. It is caused by the excess of copper accumulated mainly in the cerebellum at the vermis and hemispheres, leading to an ataxic gait, dysdiadochokinesia, and impaired fine hand movements [47, 52]. In Parkinson’s disease, parkinsonism is a common syndrome that includes bradykinesia, gear imbalance, and stiffness [50, 51, 53]; copper toxicity leads to ataxia, that is a signs and symptoms resulting from cerebellar dysfunction and involvement of cerebellar pathways. Ataxia involves abnormalities of the posture, gait, dysdiadochokinesia, dysmetria, hypotonia, oculomotor abnormalities, tremor, and speech disturbances [54]. It has been reported in almost 30% of neuro-WD patients, mostly in combination with other neurologic symptoms. High levels of copper in the striatum, globus pallidus, locus coeruleus, substantia nigra, and cerebral cortex are responsible for choreoathetosis which is characterized by dance-like movements with rapid and unpredictable contractions, usually in distal limbs, but also can involve the proximal limbs, face, and trunk. It is found in approximately 6–16% of neurologic patients [55, 56].

Other neurologic features such as dysphagia, defined as difficulty in any phase of swallowing, occur in about 18% of WD patients and in 50% of patients with neurologic symptoms. Drooling is defined as an involuntary flow of saliva from the mouth and affects approximately 70% of neuro-WD patients. Seizures may also be the principal symptom of WD. Although these can occur during any stage of the disease, they are more frequent in patients receiving treatment [57, 58].

Psychiatric symptoms may occur in the majority of patients with WD during the course of the illness, affecting both untreated and treated individuals. The most common psychiatric features include abnormal behavior (usually increased irritability or disinhibition), personality changes, anxiety, and depression [59].

Ophthalmologic Manifestations

During decades, the classical clinical hallmark of WD was the Kayser–Fleischer (KF) ring which alone was sufficient for the diagnosis of WD [60]. Many studies describe that although the KF ring is present in 90 to 99% of cases with neurologic symptoms it is only present in a little more than half of patients with hepatic presentations [61, 62]. KF rings are caused by the deposition of copper in Desçemet’s membrane of the cornea. They are identified by slit lamp examination performed by an experienced ophthalmologist. KF rings are actually not entirely specific for WD, since they may be found in patients with chronic cholestatic diseases, including children with neonatal cholestasis [63] and in many other forms of liver disease including alcoholic liver disease [64], and the KF ring can no longer be considered as pathognomonic of WD.

Laboratory Diagnosis of WD

Biochemical Diagnosis

The lack of sensitive and specific criteria makes rapid diagnosis of WD difficult. This leads to a delay in the management of patients, including liver transplantation and/or the initiation of medical therapy with chelating agents.

Biochemical diagnosis of WD is usually suggested by the association of an increase of serum transaminase concentrations, low serum ceruloplasmin, high urinary copper concentration, and the presence of a DAT-negative hemolytic anemia.

Since genetic diagnosis is expensive, not universally available, and sometimes inconclusive WD is usually diagnosed using a combination of clinical findings and biochemical testing.

Biochemical Liver Tests

Serum aminotransferase activities are generally abnormal in WD except at a very early age. In many individuals, the degree of elevation of aminotransferase activity may be mild and does not reflect the severity of the liver disease. In the following paragraphs, we will detail the specific biochemical parameters and the techniques adopted to retain or exclude WD disease in suspected patients, and we will try to highlight their strengths and limitations.

Serum Ceruloplasmin (Cp) Determination

Ceruloplasmin (Cp) is a glycoprotein synthesized in hepatic microsomes as an inactive, unstable, non-copper bound form, apoCp. It is the main copper-carrying protein in the blood, binding 70–95% of circulating copper [65]. Six copper molecules are incorporated into newly synthesized apoCp by the ATP7B protein and became mature ceruloplasmin (holoceruloplasmin) [66]. A mutation in the ATP7B gene causes rapid degradation of this precursor of ceruloplasmin due to insufficient copper ions, resulting in the reduction of serum ceruloplasmin level, the most useful test in the diagnosis of WD. The physiological levels of Cp are low between birth and 6 months, compared to those of adults. Serum ceruloplasmin level can be measured by radioimmunoassays (antibody-dependent), immunodiffusion, nephelometry (quantitative immunochemical) or by enzymatic methods. However, immunologic assays may overestimate serum ceruloplasmin, as these methods measure simultaneously inactive apo-Cp and active holo-Cp. The only method available to determine the copper-dependent oxidative activity of Cp (active holo-Cp) is the enzymatic determination that is not performed routinely. In addition to these methodological problems, there are physiological variations in Cp levels that can lead to misdiagnosis. Increased Cp levels are caused by estrogen, pregnancy, and the contraceptive pill, and also with inflammation, infections, and rheumatoid arthritis, and in patients with myocardial disease or cancer. Similarly, low concentrations of Cp may occur in patients with fulminant hepatitis or in cases of acaeruloplasminaemia, Menkes disease, nephritic syndrome, copper deficiencies, or severe malabsorption or malnutrition. Moreover, about 10% of heterozygous carriers (ATP7B variants), who will never have clinical problems, also demonstrate low ceruloplasmin values [67]. Therefore, serum Cp interpretation is not easy and insufficient alone in confirming a WD diagnosis.

Serum Copper and Free Copper

In circulation, copper is both bound to proteins (ceruloplasmin or albumin) or is unbound (free). Nearly 70% of bound copper is with Cp, and less than 20% is with albumin. Total serum copper determination measures copper incorporated in Cp and non-Cp bound copper. In WD it is usually decreased due to the absence of holo-Cp that carries most copper atoms (normal level of total serum copper is estimated around 12.7–22. 2 µmol/L). The concentration of copper in blood can be influenced by several clinical conditions such as acute hemolysis or hepatitis that can lead to a significant release of copper from liver tissue stores. Unbound copper is considered the toxic portion in WD, and is calculated from Cp and total serum copper. The copper associated with Cp is approximately 3.15 µg copper/mg Cp. Therefore, for mass units, the free copper would be the difference between the serum copper concentration in µg/dL and three times the serum ceruloplasmin concentration in mg/dL.A formula for calculating the free copper in µmol/L can be used as follows: Free copper = Total copper (µmol/L) – 0,049 × ceruloplasmin (mg/L) [68]

However, determination of free copper is not recommended by the guidelines for diagnosis of WD due to its limitations [44]. In fact, immunological methods that is used to determine the concentration of serum Cp lead to falsely high values, as these tests do not discriminate between holoCp and apoCp [69, 70]. Measurement of the Cp via oxidase activity could be more promising and resolve these difficulties but this enzymatic determination is not performed routinely.

Urine Copper Excretion

Twenty-four-hour urinary copper excretion in untreated patients corresponds to the amount of non-ceruloplasmin-bound copper in serum. This fraction may be helpful for diagnosing WD and for monitoring treatment. In order to have an accurate interpretation of the urinary copper, urine must be collected correctly, including its exact urine volume, the total creatinine, and that contamination has been prevented. This requirement poses a challenge in the diagnosis of WD in pediatric patients. Twenty-four-hour urinary copper ranges are laboratory-dependent but generally are greater than 1.6 µmol/24 h (> 100 µg/24 h) in patients with WD [71]. However, asymptomatic children with WD or heterozygotes may have basal copper excretion below the threshold of 1.6 µmol/24 h [72]. Thus, some studies suggest that even a lower threshold for basal urinary excretion of copper at 0.64 µmol/24 h (40 µg/24 h) should be considered [62, 73]. D-penicillamine stimulation test has been used for the diagnosis of WD in children if urinary copper level is only mildly increased and consists of measuring urinary copper before and after taking D-penicillamine and assesses whether urinary copper levels increase after the administration of this chelating agent. Although this test has been proposed as a diagnosis test, its reference values have only been validated in children with liver symptoms [72], Furthermore, when the lower threshold for urinary copper excretion of 0.64 µmol/24 h is applied then it might not be required. Furthermore, an increased level of urinary copper is observed in different liver diseases and ATP7B heterozygotes resulting in misdiagnosis of WD.

Liver Copper

Direct determination of hepatic copper content is justified when clinical features and these other biochemical markers, including determination of Cp, serum copper, or 24-h urinary copper excretion, do not allow a definite diagnosis of WD to be reached. This is an invasive procedure that is not required for the diagnosis of WD in all cases [74]. However, a hepatic copper content of ≥ 4 µmol/g (> 250 μg/g)dry weight provides the best biochemical evidence for WD, and this threshold value is included in the diagnostic score, whilst values < 0.8 µmol/g (< 50 μg/g) dry weight are considered to exclude a diagnosis of WD [75]. Copper content in the liver is also increased significantly (more than 4 µmol/g dry weight) in established cholestatic disorders and idiopathic copper toxicosis syndromes [76]. The main limitation of quantitative copper determination is sampling error since the distribution of copper in the liver is not homogenous and a single biopsy specimen can result in underestimation of copper content [77]. Furthermore, several studies have shown that this sampling error depends on the stage of liver disease; in early stages, copper is mainly in the cytoplasm bound to metallothionein and therefore hardly detectable. A lower copper concentration was also found in advanced liver disease, such as cirrhosis with nodules and fibrosis [78, 79]. In a pediatric study, sampling error was sufficiently common to render this test unreliable in patients with cirrhosis [80]. To surmount this sampling problem, it is recommended to increase liver biopsy size to at least 1 cm length of a 1.6 mm diameter of biopsy core [22, 80] or > 1-mg liver tissue, and with two biopsies from various areas [81] or sampling from the deeper regions of the liver. However, in a more recent study by Sintusek and Dhawan, there was no correlation between the size and weight of the liver and the copper measurement for WD patients and controls [82].

The detection of copper in hepatocytes by routine histochemical evaluation is highly variable. The histological findings in WD can mimic those of nonalcoholic fatty liver disease (NAFLD), autoimmune hepatitis (AIH), and multidrug resistance protein 3 (MDR3) deficiency as they include mild steatosis, glycogenated nuclei, focal hepatocellular necrosis, and piecemeal necrosis [74]. A specific stain like rhodamine or orcein may be helpful, but in the early stage of WD, copper may be unstained, and this method reveals focal copper stores in less than 10% of patients since it detects only lysosomal copper deposition and is therefore not sensitive for the diagnosis [83].

Exchangeable Copper and Relative Exchangeable Copper

Exchangeable copper (CuEXC) is the direct determination of labile copper (non-Cp-bound copper) and primarily refers to the copper loosely complexed to albumin in the circulation. Heavy extraction procedures have sometimes been used [84, 85]. However, the exchange is easier using chelators, and ethylene diamine tetra acetic acid (EDTA), a chelator of high copper affinity (incubation for 1 h), and is able to mobilize this exchangeable fraction. The main advantage of CuEXC is that it is not dependent on Cp, and it represents an exact estimation of copper overload [86]. Exchangeable fraction is used to determine the “relative exchangeable copper” (REC) which refers to the ratio of CuEXC to total copper reflecting the toxic-free fraction of copper in the circulation [87].

CuEXC is not able to indicate the severity of liver damage. In patients with WD affecting only the liver, the fractions of CuEXC are normal or moderately increased except in case of acute liver failure associated with Coombs negative hemolytic. CuEXC is therefore an interesting experimental biomarker which still has to interpreted with caution, especially in WD patients with only hepatic manifestations. Additionally, the measurement of CuEX has limitations, including its extreme instability in plasma or serum which require immediate transfer for processing within 15 min of blood collection. The analytical methodology required is furthermore not a routine technique and is not available in all laboratories [88].

Genetic Testing

There is significant genetic heterogeneity among Wilson’s disease (WD) patients, with over 600 variants described. Many patients may present with a novel variant of uncertain significance [29], and in some cases, no mutations have been detected in the promoter and exon regions of the ATP7B gene [21]. Many patients are compound heterozygotes (i.e., carry two different variants), comprehensive molecular-genetic screening can therefore take several months, and although this makes it impractical for urgent diagnosis, molecular analysis of the ATP7B gene is mandatory in any patient who has a provisional diagnosis of WD [44].

Mutations can affect almost all 21 exons and are frequently missense and nonsense. The missense mutation H1069Q in exon 14 is very common [89], and about 50–80% of WD patients from Central, Eastern, and Northern Europe carry at least one allele with this variant [90]. But other variants are common in Southern Europe such as the missense mutation M645R in Spain and R778L in exon 8 is found more frequently in South-eastern Asia [90]. Recently, a large study on phenotype-genotype correlation was reported in 227 WD patients, which showed no significant correlation [91]. This is partly due to the poor phenotypic characterization of patients, late diagnosis, and overlapping neurological, psychiatric, and hepatic signs and symptoms of various severities. Next generation sequencing (NGS) has revolutionized the genetic approach for WD and can rapidly sequence the whole ATP7B gene and detect variants, saving both time and cost compared to the traditional Sanger sequencing, and the current challenge is to make NGS more accessible widely around the world [92].

WD Score (Leipzig Score)

As the diagnosis of WD is challenging for clinicians, the empirical diagnostic approaches have included the development of a scoring system, proposed at the 8th international meeting on WD, to help clinicians evaluate patients with Wilson’s disease, by combining clinical, biochemical, and, for the first time, molecular genetic testing for WD [28]. This has come to be known as the Leipzig criteria (Table 1). It has been validated in adult and pediatric populations and incorporated into the European Association for the Study of the Liver guidelines [44].

Table 1 Scoring system developed at the 8th International Meeting on Wilson’s disease Leipzig 2001
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These diagnostic systems are based on serum ceruloplasmin level (normal, < 200 mg/L), presence or absence of Coombs-negative hemolytic anemia, 24-h urinary copper excretion (normal, < 0.8 µmol/day), presence or absence of Kayser–Fleisher ring, neuropsychiatric symptoms suggestive of WD, liver copper content (normal, 0.8 µmol/g (< 50 μg/g)) and variant analysis for ATP7B [93]. The threshold score of 4 provided a good correlation between sensitivity and specificity for the diagnosis of WD (sensitivity of 98% and specificity of 97% [94]. If the cumulative score of this system is equal to or greater than 4, the diagnosis of WD is highly probable. Nevertheless, this scoring system has some limitations—evaluation by summation of scores relies on each parameter being correctly measured and determined. Even with minor evidence-based modifications for the pediatric age range (Table 1), the parameters may still not be useful for very young children since the relevant data may be difficult to collect from them [95]. Those hepatic conditions with a long-standing cholestasis and other diseases of abnormal copper metabolism may also have high hepatic copper levels and therefore result in a higher score [96, 97]. Therefore, other clinical findings should be consistent with the diagnosis of WD, and conditions that may mimic WD should be excluded.

ATP7B Peptides

Recent investigations regarding the diagnosis of WD have revealed that selected ATP7B peptides are suitable for early diagnosis before the onset of the disease. Using a combination of immunocapture with monoclonal antibodies to ATP7B and targeted mass spectroscopy involving multiple reaction monitoring, these selected ATP7B peptides were quantified in proteolysed dried blood spots [98, 99]. ATP7B peptide levels were lower in patients with WD than in controls. However, additional testing of initial diagnostic thresholds has largely succeeded in distinguishing patients from carriers and unaffected controls. This technology is promising for interpreting the functional effect of ATP7B variants of unknown importance, and could also serve as a complementary non-invasive test for the diagnosis of WD. Furthermore, it could considerably promote the use of proteomics as a rapid screening tool, an area with great potential but still largely unexploited [100].

Challenges in the Clinical Diagnosis

The clinical diagnosis of WD is one of the most challenging in clinical medical practice. The classic clinical picture is of a child or adolescent with personality disorder, motor disturbance, and liver manifestations. However, the variability of phenotypes is extreme, especially regarding the early onset of symptoms, delayed symptoms development, and the ages at which the disease is diagnosed.

Walshe and colleagues analyzed symptom presentations in 136 patients with WD and stated “no two patients are ever the same, even in a sibship” [101]. Although hepatic presentation is common in WD these actual clinical manifestations range from asymptomatic liver diseases through fulminant liver failure to cirrhosis. When diagnosing, several atypical hepatic manifestations of WM should be considered. These symptoms can be extremely variable, ranging from asymptomatic hepatomegaly, persistent or intermittent elevation of serum transaminases, jaundice, and hepatic steatosis, to compensated or decompensated cirrhosis [102].

Differential Diagnosis

The difficult diagnosis of WD has been discussed. This difficulty complicates the potential decisions concerning liver transplantation and the initiation of medical therapy with chelating agents for WD.

two groups of diseases can be evoked next in a patient with WD clinical symptoms: diseases suggested in the differential diagnosis of WD, a larger class, and diseases mimicking WD.

Neurodegenerative Diseases Suggested in the Differential Diagnosis of WD

Based on exhibited symptoms, several common neurologic disorders are in the differential diagnosis of WD including essential tremor, young-onset Parkinson disease, generalized dystonia, rare juvenile genetic extrapyramidal disorders, including Huntington disease, the pediatric disorder Huppke–Brendel–Horvath syndrome [103], neuro-degeneration with brain iron accumulation (NBIA) which is a heterogeneous group of disorders with excessive brain iron deposition including aceruloplasminemia [104], and manganese storage disorder [105]. The main symptoms in such diseases are developmental and psychomotor delay, postural instability, dystonia, spasticity, cognitive dysfunction, dysarthria movement disorders, psychiatric symptoms, and parkinsonism, thus referring to WD.

Cerebral magnetic resonance imaging (MRI) is commonly utilized in the assessment of various extrapyramidal disorders and plays a crucial role in the neurological evaluation of WD, being included in the Leipzig score. Several neuroradiological signs have been proposed as potentially pathognomonic for WD, including the “face of the giant panda,” “miniature panda,” “split thalamus,” “bright claustrum,” and “whorl.” However, these signs are most frequently observed in patients with early and severe neurological symptoms. Nonetheless, because these signs can also appear in brain MRI scans of other disorders, they may not be truly pathognomonic for WD [106].

As WD is primarily a liver disorder, hepatologists are often the primary physicians involved in its management. MRI should be conducted in all patients before treatment initiation, as cerebral MRI changes are observed even in hepatic or presymptomatic cases, irrespective of the presence of neurological symptoms [107].

Fortunately, the diagnosis cannot be based solely on the clinical manifestation but also on the biochemical assays which will make it possible to limit the number and/or rule out these pathologies.

Hepatic Diseases Suggested in the Differential Diagnosis of WD

Patients with WD may present with acute hepatitis that is similarly to any other acute cases of hepatitis. Importantly, WD should be included into differential diagnosis in most patients with various presentations of liver disease from increased transaminases and/or hepatic steatosis through liver cirrhosis to liver failure, as routine histologic changes are nonspecific. An onset of jaundice and hemolytic anemia are factors that further supported this hypothesis [108]. There is therefore a long list of liver diseases that must be considered in the differential diagnosis. The subgroups of these diseases that are associated with copper excess are Indian childhood cirrhosis (ICC) [109], endemic Tyrolean infantile cirrhosis (ETIC) [110], and idiopathic copper toxicosis (ICT) [111]. ICC, ETIC, and ICT form a second class of copper-overload disorders that are easily ruled out by their age of onset and their evolutions, and indeed, most patients with ICC, ETIC, and ICT die at an early age due to liver failure as a consequence of decompensated liver cirrhosis [112].

Another subgroup of pediatric liver diseases had to be considered; nonalcoholic fatty liver disease (NAFLD) which is highly common than WD and autoimmune hepatitis which is almost as rare as WD and sclerosing cholangitis which may lead to hepatic copper retention and requires consideration when stainable copper is found on liver biopsy.

The recently recognized manganese storage disease caused by an autosomal recessive mutation in the manganese transporter gene (SLC30A10) has been described according to some authors as the new WD, in effect, in addition to neurological symptoms, the patients present features similar to the hepatolenticular degeneration seen in the natural history of WD, patients with this condition also develop hepatic cirrhosis [105].

Compared with patients who had acute liver failure (ALF) due to other causes rather than WD, patients with WD had a higher urinary copper (≥ 100 μmol/24 h vs 3.5 ± 1.8 μmol/24 h) [30]. According to these findings, it appears that the copper and ceruloplasmin changes seen in these disorders occur to a similar degree as in WD, but the underlying mechanisms remain to be elucidated.

Diseases Mimicking WD

With such diseases, It is difficult to differentiate and opt for a safe and accurate diagnosis, even based on the specific biochemical diagnosis of WD, namely ceruloplasmin, blood copper, and the urinary excretion of copper over 24 h, qualified as the specific biochemical test for WD (Table 2).

Table 2 Laboratory abnormalities in WD and mimicking diseases
Full size table

MEDNIK Syndrome

The name MEDNIK syndrome is an acronym for some of the main clinical features of the disease, including mental retardation, enteropathy (diarrhea), deafness peripheral neuropathy, ichthyosis, and keratoderma [113]. Other features include hepatopathy with elevated transaminases and cholestasis exhibited in 30% of patients. MEDNIK syndrome combines clinical and biochemical signs of both Menkes disease and WD [114]. It shares some of the neurological, cutaneous and skeletal symptoms, and low plasma copper and ceruloplasmin, with Menkes disease; while hepatic copper accumulation along with increased urinary copper excretion, and mild T2 hyperintensity of bilateral caudate and putamen on brain MRI are consistent with WD. MEDNIK is due to defects in the AP1S1 gene, which encodes a protein that regulates the intracellular copper machinery mediated by ATP7A and ATP7B, the defective copper ATPase in Menkes disease and WD, respectively [10].

MDRIII Deficiency

Several liver diseases are known to be associated with multidrug resistance protein 3 (MDR3) deficiency, it is suggested to result in a disbalanced bile which may damage the luminal membrane of cells of the hepatobiliary system [115] in infancy or adolescence, the deficiency may lead to end-stage liver disease. Affected children usually present with jaundice, hepatomegaly, discolored stools, or pruritus. Gastrointestinal bleeding due to portal hypertension and cirrhosis is the presenting symptom in adolescent or young adult patients. Initial serum liver tests (mean) show: elevated alanine aminotransaminase activity (5xN), conjugated bilirubin concentration (2xN), alkaline phosphatase activity (2xN), GGT activity (13xN), total bile acid concentration (25xN), normal cholesterol concentration and prothrombin time [116], significant copper accretion in liver and increased copper secretion in urine [117].

Congenital Disorders of Glycosylation

Congenital disorders of glycosylation (CDG) are a genetically and clinically heterogeneous group of > 200 diseases caused by defects in various steps along glycan modification pathways. The vast majority of these monogenic diseases are autosomal recessive where patients present a great phenotypic diversity ranging from poly- to mono organ/system involvement and from very mild to extremely severe presentation including growth failure, developmental delay, facial dysmorphisms, liver involvement, variable coagulation, and endocrine abnormalities [118, 119].

The CDG giving rise to liver disease appears grouped at the early steps of the glycosylation pathway (MPI-CDG, PMM2-CDG, PGM1-CDG) and in the asparagine-linked protein glycosylation (ALG) phase of the glycan chain production (ALG-1,3,6,8,9-CDG). Among these subgroup’s CDG diseases; TMEM199-CDG, CCDC115-CDG, ATP6AP1-CDG, PMM2-CDG, and COG2-CDG overlap with WD[120]. One of the first studies that raised a possible confusion between WD and CDG reported that mutations in the V-ATPase Assembly Factor VMA21 cause a congenital disorder of glycosylation with autophagic liver disease, their index patient was treated as a Wilsonian patient during 2 years because of low serum ceruloplasmin and copper and elevated urinary copper excretion. The degeneration of his condition prompted a screening for CDG as part of the routine metabolic investigations that was abnormal [121].

In patients presenting with a partial or complete Wilson hepatocerebral syndrome phenocopy (low serum copper and/or ceruloplasmin, copper accumulation on liver biopsy, increased urinary copper excretion), CDG testing (transferrin and apolipoprotein C-III isoelectric focusing) should be performed[120].

Patients with these diseases usually present with cholestasis, liver failure, and significant copper accretion in the liver, impaired copper secretion, and decreased serum ceruloplasmin and copper. Copper accumulation can be seen in all chronic cholestatic and or hepatic disorders; however, in the disorders mimicking WD, the quantity of copper was significant to meet diagnostic criteria for WD and led to altered copper excretion, the undergo mechanism remains to be elucidated.

A misdiagnosis that may arise due to this simultaneous overlap of clinical and biochemical features may alter treatment, as to date there are no known beneficial effects of chelation in MDR3, CDG, or other non-WD or cholestatic conditions, which underlines the importance of metabolic screening as well as molecular genetic tests in the precise diagnosis of mimicking disorders which directly affect the management decisions.

Conclusion

At any age, individuals presenting with a movement disorder should be considered for WD, as the disease is treatable and must be distinguished from other common and rare neurological diseases that mimic WD.

WD fits into a wide range of internal and neurological disease models with jaundice, anemia, and more or less severe liver damage. The evocation of diseases entering into the differential diagnosis and or mimicking WD discussed above are relevant after having excluded an ATP7B classic variant. On the one hand, it is necessary to start treatment for WD early, but on the other hand, only a reliable diagnosis justifies treatment, which involves side effects. Starting treatment at an early stage, avoiding the wrong medications, and evaluating prognosis are also important for differential diagnoses.