Wilson’s disease in children: Recent update on pathophysiology and management

Abstract

Wilson’s disease (WD) is an autosomal recessive disease caused by mutations in the ATP7B gene, which plays a fundamental role in copper metabolism, leading to the accumulation of copper in the liver and other vital organs. Major functions of ATP7B are the incorporation of copper into apoceruloplasmin and the excretion of copper into bile. From simple asymptomatic elevation of liver enzymes to acute liver failure, as well as chronic hepatitis, portal hypertension may be the initial presentation of this disease. Approximately 800 different mutations of the ATP7B gene have been recognized. WD has wide phenotypic disease expressions. Genotypic expression does not always correlate with phenotypic disease expression or disease severity. Environmental and epigenetic factors may play a role in disease expression. No single test is diagnostic for WD. Initial testing includes ocular slit-lamp examination, 24-hour urinary copper excretion, and serum ceruloplasmin. The Leipzig scoring system for diagnosis is widely used. Relative exchangeable copper is the new noninvasive biomarker for WD. It gives a quick result before awaiting genetic testing. Timely diagnosis of WD is important, as it can halt the clinical progression of the liver and neurological disease. D-penicillamine and trientine remain the main chelators for WD treatment. Patients need lifelong chelation therapy until liver transplantation. Research on new treatment modalities, such as methanobactin, is ongoing for further human use. This review discusses the genetic aspects of WD, copper metabolism, a new diagnostic method, and a research molecule for future management of WD.

Key Words: Wilson’s disease; Pathophysiology; ATP7B; Copper; Penicillamine

Core Tip: Wilson’s disease (WD) is an autosomal recessive disease caused by mutations in ATP7B gene which has a fundamental role in copper metabolism, leading to accumulation of copper in the liver and other vital organs. Major functions of ATP7B are incorporation of copper into apoceruloplasmin and the excretion of copper into bile. From simple asymptomatic elevation of liver enzyme to acute liver failure, chronic hepatitis, portal hypertension may be the initial hepatic presentation of this disease. No single test is diagnostic for WD. Initial testing includes ocular slit-lamp examination, 24-hour urinary copper excretion and serum ceruloplasmin. Leipzig scoring system for diagnosis is used widely. Relative exchangeable copper is the new non-invasive biomarker for WD diagnosis with the highest sensitivity and specificity. Patients need lifelong chelation therapy until liver transplantation.

INTRODUCTION

Wilson’s disease (WD) is a rare genetic disease of defective Cu metabolism. This disease is caused by mutations in the Cu (I)-transporting ATPase beta polypeptide (ATP7B), leading to Cu accumulation in the liver and other vital organs[1]. Although the main defect is in the ATP7B gene, there is a presence of other modifying factors for the expression of the disease. A systematic literature search on WD using MEDLINE/PubMed, Scopus, and other published databases from 1990 to 2026 is performed, focusing on retrospective, prospective studies, meta-analyses, and clinical trials. Recent and previous guidelines are studied, including the position paper (2018) by European Society for Pediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN), European Association for the Study of the Liver (EASL)-ERN Clinical Practice Guidelines on Wilson’s Disease (2025), Practice Guidance on Wilson’s Disease (2022) from American Association for the Study of Liver Diseases, and others. Recent research on the pathophysiology of WD, the scope of newer tests in diagnostic algorithms, and new therapeutic interventions are described in this narrative review, providing an overall understanding of the pathophysiology and management of WD.

Epidemiology

The prevalence of WD is approximately 1 in 30000 worldwide. About 1 in 90 people is a heterozygote carrier[1]. The incidence is higher in Asian countries where consanguinity is more common. Although there are similarities in disease onset and clinical findings, there may be significant differences in disease severity and organ involvement in affected siblings. This finding suggests that epigenetic and environmental factors play an important role in the expression of the disease.

Genetics

Common mutations in the ATP7B gene are missense and nonsense mutations. Mutations can occur in almost all 21 exons. Compound heterozygous mutations are the most common form. There are about 700 mutations related to WD. The H1069Q missense mutation in exon 14 is the most common in Western populations, while the R778 L mutation in exon 8 is more common (14%-49%) in the Far East and South Asian countries[2]. Positive genotype/phenotype correlation is lacking in WD, indicating that other genetic, epigenetic, and environmental factors play a role in WD phenotypes. Severity of the disease, age of presentation, and primary organ involvement do not correlate with types of genetic mutation. Studies suggest that the H1069Q homozygous mutation causes adult-onset and more frequent neurological presentation than the H1069Q compound heterozygous mutation[3].

Other genes

Patatin-like phospholipase domain-containing protein 3, which is commonly involved in non-alcoholic fatty liver disease (NAFLD), has also been associated with WD. A PNPLA3 mutation has been associated with steatosis in WD. The copper chaperone ATOX1 interacts with ATP7B. ATOX1 has been studied to detect any linkage.

Epigenetic factors

The enzyme S-adenosylhomocysteine (SAH) hydrolase plays a role in the pathogenesis of the disease. SAH activity is decreased in hepatic copper buildup, which causes changes in methionine metabolism[4]. Notably, mouse models of WD showed dysregulation of methionine metabolism and global DNA hypomethylation in hepatocytes with subsequent gene dysregulation involving liver damage. During embryonic development in a mouse model of WD, major changes in gene transcript levels related to cell cycle and replication were observed compared to control animals. The mouse fetal liver is a site of major methionine metabolism. The delivery of additional methyl-donor choline improved gene expression to levels comparable to the control group, indicating that fetal livers are susceptible to nutritional factors with possible consequences for disease phenotype, severity, and progression.

Cu metabolism in humans

Cu is an essential micronutrient for human life. Cu functions as a key cofactor for enzymes in essential biological processes, including mitochondrial respiration, antioxidant defense, compound protein synthesis, tyrosine, and neurotransmitter metabolism. The liver is the major organ for copper metabolism. Excess copper is harmful as it can generate free radicals and increase oxidative stress. The concentration of copper ions within the human body must be maintained at a crucial physiological level. Above and below this homeostatic level, there are two main genetic disorders (Menke’s disease and WD)[5]. Therefore, copper ion absorption from a proper dietary source, distribution in plasma and other organs, storage in the liver, and biliary excretion are strictly controlled.

Cuproptosis is a recently identified form of programmed cell death triggered by excess copper accumulation in the cell[6]. The typical morphological features of cuproptosis are mitochondrial contraction, plasma membrane disruption, damage of the endoplasmic reticulum, and chromatin fragmentation[7]. Cuproptosis plays a pathophysiological role in various diseases, such as WD, and may have therapeutic potential in malignancy. The concept of cuproptosis is further studied to develop novel therapeutic interventions for WD.

The absorption of dietary Cu occurs mostly in the proximal small intestine. Cu is transported into enterocytes through Cu transport protein 1 (CTR1), positioned on the apical side of the enterocytes. This transportation is helped by the activity of the metalloreductases six-transmembrane epithelial antigen of the prostate and duodenal cytochrome b, which reduce Cu²⁺ to Cu⁺; the only ionic state of copper absorption. Within the enterocyte, copper is stored in intestinal metallothionein (MT) and utilized for normal physiological functions. Afterward, most of the copper is transported into portal circulation via the ATP7A transporter protein located in the basolateral membrane of the enterocyte. In portal circulation, copper is loosely bound to albumin, less commonly to transcuprein and histidine, and is delivered to various organs. After reaching the liver sinusoid, Cu is taken up via CTR1 at the hepatocyte’s sinusoidal side. Then, within the cytoplasm of a hepatocyte, Cu is either carried by Cu chaperones to specific proteins or stored in hepatic MT for further utilization. Important Cu chaperones comprise COX17 (Cu chaperones for cytochrome c oxygenase, which delivers Cu to cytochrome c oxygenase), CCS (Cu chaperone for superoxide dismutase, which delivers Cu to superoxide dismutase 1), and ATOX1 (which delivers Cu to ATP7B[8]. In case of WD, ATOX1 is an important chaperone. Upon reaching its target tissues, Cu exerts various physiological effects to maintain homeostasis. ATP7B is located in the hepatocyte trans-Golgi network (TGN). ATP7B has two functions within hepatocytes. One function is the transportation of sequestered excess copper into vesicles bound for lysosomes, followed by the excretion of copper into the bile canaliculus. This represents the main excretion pathway for copper metabolism. Cu conjugated to glutathione is a less important pathway of Cu excretion. The other function of ATP7B is to activate ceruloplasmin. Activation of ceruloplasmin involves the packaging of six copper molecules into apo-ceruloplasmin, which is then called holo-ceruloplasmin. This holo-ceruloplasmin is then secreted into plasma. When the ATP7B gene is mutated in WD, both copper secretion into bile and copper incorporation into ceruloplasmin are impaired, resulting in copper overload within the cell. Organ damage is characterized by DNA damage, lipid peroxidation, and mitochondrial respiratory dysfunction. Mitochondrial changes are widening of the interstitial space, separation, and enlargement of the inner and outer membranes[8].

The liver is the primary storage site for copper in the body. The primary route of Cu elimination is via the feces, either via biliary excretion or as unabsorbed copper ions. Cu elimination through urine plays a little role in Cu loss for normal children. To maintain Cu homeostasis at the cellular level, intracellular Cu content is controlled by Cu chaperones and membrane transporters. These proteins work in a coordinated manner to regulate the intake, transfer, and intracellular utilization of Cu, thereby maintaining homeostatic Cu levels within the cell and preventing the pathological consequences of Cu overload and Cu deficiency.

Cu-ATPases ATP7B protein

The Cu-ATPases ATP7B act as the major transporter for exporting cellular Cu into the bloodstream. ATP7A/7B localization and function are regulated tightly[8]. Under physiological conditions, these transporters are found to be located in the TGN, where they transport Cu from the cytoplasm into the lumen of the TGN. When intracellular Cu levels rise, these transporters relocate from the TGN to vesicular compartments and fuse with the plasma membrane to excrete Cu via the biliary channel; when Cu levels return to normal, they are recycled back to the TGN for further use. ATP7A is present in most tissues, except for the liver, where ATP7B is found to be predominantly expressed. ATP7A is expressed at the basolateral membrane of enterocytes, facilitating the export of Cu into the portal circulation for delivery to the liver. In the placenta and the blood-brain barrier, ATP7A mediates Cu transport for fetal development and brain function. Mutations in ATP7A and ATP7B cause diseases such as Menkes disease and WD, respectively.

Clinical manifestations of WD

WD causes a wide range of clinical manifestations in both children and adults[9]. Clinical features are shown in Table 1[9-12]. Hepatic manifestations range from mild to severe. It may be manifested as incidental findings of abnormal liver tests or features of chronic liver disease or fulminant acute liver failure. When the disease progresses, patients may present with decompensated chronic liver disease and its complications, like ascites and variceal bleeding.

Table 1 Clinical features of Wilson’s disease.

Hepatic	Neurologic	Psychiatric	Eye	Hematologic	Renal	Skin	Osteoarticular	Endocrine	Reproductive system
Asymptomatic increased serum transaminases. Acute hepatitis. Hepatomegaly. Fatty liver. Acute liver failure with hemolysis. Portal hypertension. Decompensated cirrhosis with ascites	Deterioration of handwriting school performance. Dysarthria. Drooling, hypersalivation. Tremor. Dystonia. Coordination defect. Choreoathetosis. Ataxic gait. Fixed grin seizure	Organic dementia. Depression, anxiety. Psychosis. Behavioral changes. Emotional lability. Schizophrenia. Bipolar disorder. Obsessive- compulsive disorder. Antisocial behavior	Kayser-Fleischer ring. Sunflower cataract	Acute hemolytic anemia	Tubular dysfunction. Nephrolithiasis. Nephrocalcinosis. Hypercalciuria. Hyperphosphaturia	Hyperpigmentation. Azure lunulae of nails. Acanthosis nigricans	Osteoporosis. Rickets. Osteomalacia. Pathological fracture	Glucose intolerance. Hypoparathyroidism. Panhypopituitarism	Menstrual irregularity. Delayed puberty. Infertility. Miscarriage

Before the age of 10, 83% of children presented with hepatic symptoms, and 17% presented with neuropsychiatric symptoms. Between 10 and 18 years of age, the percentages of hepatic and neuropsychiatric manifestations are similar, and after 18 years of age, 74% of patients presented with neuropsychiatric manifestations and 24% with hepatic symptoms[10]. Hepatic presentations are acute hepatitis, asymptomatic elevation of serum aminotransferase, acute liver failure, chronic hepatitis, steatohepatitis, portal hypertension, decompensated cirrhosis, cholelithiasis, hepatocellular carcinoma. WD may present at any age between 3 and 74 years, but before age 5, it is rarely diagnosed. A pediatric series of 100 patients from Bangladesh showed that increased serum transaminases was the most common presenting feature of hepatic manifestations[11]. Antinuclear antibodies may be found in patients with WD, and cases of WD with concomitant autoimmune hepatitis (AIH) have been reported[12]. Kayser-Fleischer (KF) rings, caused by copper deposition on Descemet’s membrane, are usually absent on slit-lamp examination in children with asymptomatic or mild liver disease, but are almost always present in children with neurological involvement. Acute hemolysis can be the primary presentation of WD and is prominent in Wilsonian liver failure. The prevalence of acute hemolysis was 6.9% in a retrospective study of 321 patients with WD, with an average age at onset of 12.6 years[12]. A case report of an earlier onset of WD at 3 years of age has been described in the literature[13,14].

Ocular involvement

The KF ring was first discovered in 1902 and 1903, respectively, by two German ophthalmologists, Benhard Kayser and Bruno Fleischer. The ring is caused by copper deposition in the internal corneal layer of the Descemet’s membrane. The sulfur-copper complexes, which form the visible copper deposits, are found only in Descemet’s membrane. On slit lamp examination, the KF ring seems like a golden-brown, green-yellow, golden-yellow coloring ring in the limbal area of the cornea. It first matures in the superior part of the cornea (at the 12 o’clock position), then inferiorly, and eventually in the horizontal position. Thus, it takes time to mature. The KF ring is less commonly found in younger children. The KF ring indicates excess free copper in the blood but is not pathognomonic for WD, as it may occur in any disease with reduced biliary copper excretion. The presence of a KF ring can be identified by a skilled examiner or, rarely, by the naked eye. It usually fades progressively with effective treatment of WD, disappearing initially from the lateral and medial aspects of the cornea, then finally from the superior part, in the opposite direction of its appearance. Follow-up is necessary annually, as its recurrence indicates noncompliance with treatment. Sunflower cataract is another ocular sign of WD, with its frequency reported widely in the literature (ranging from 2% to 20%). Langwińska-Wośko et al[15] studied 81 WD patients and reported that sunflower cataract was observed in 1.2% of patients, suggesting that it is a rare ocular sign of WD. It was first described by Siemerling and Oloff[16] in 1922. After a year of treatment, the cataract can disappear with chelation therapy. Sunflower cataract is caused by copper deposition in the third posterior of the lens’ anterior capsule, sparing the lens cortex or nucleus itself. It has the characteristic of a central disk with radiating petal-like spokes that look like sunflower petals. Energy-dispersive X-ray spectroscopy revealed copper and sulfur in all of these electron-dense granules. Sulfur is present in ocular MT in the lens. Sunflower cataract is caused by the accumulation of heterogeneous compounds, including copper, sulfur, and/or binding-copper proteins. Vision is not disturbed by sunflower cataract. It requires slit-lamp examination for detection.

Neurological manifestations

Neuropsychiatric features are more common in older children and adolescents[17,18].

Dysarthria: Dysarthria is a motor speech disorder manifested by inaccurate, slow, and uncoordinated speech movements. It is one of the earliest neurological manifestations of WD in children. It can result from any condition involving lower motor neuron lesions of cranial nerves IX, X, and XII, or from cerebellar or basal ganglia disorders. Speech components may be affected due to a defect in articulation, phonation, respiration, and resonance.

Tremor: Tremor is an involuntary, rhythmic, oscillatory movement of any part of the body. Tremors are postural, rubral (wing-beating), and resting tremors. It usually starts in one limb, then spreads to the whole body. Wing-beating tremor is a characteristic neurological feature of WD. Postural tremor occurs when a specific posture is voluntarily maintained by the patient. The action tremor in WD occurs due to copper deposition principally in the cerebellum. The tremor starts unilaterally in the distal upper extremities. As the disease progresses, the head, both limbs, and the whole body are involved. Another important tremor seen in WD is essential tremor, which results from neurodegeneration in the cerebellar dentate nucleus.

Dystonia: Dystonia is characterized by involuntary, sustained, or intermittent muscle contractions that cause abnormal postures. It starts with mild symptoms, then gradually becomes a severe disease if left untreated. It could be evident as focal, segmental, multifocal, or generalized symptoms. Focal manifestations include torticollis, blepharospasm, and risus sardonicus (factitious smile). Risus sardonicus is a sustained spasm of the facial muscles that produces fixed grinning. Focal dystonia of the vocal cords and muscles of articulation produces dysphonia, dysarthria, and dysphagia. The putamen is the main structure involved in the dystonia. Damage to the basal ganglia-thalamocortical motor circuits disrupts normal physiological mechanisms and fails to produce an inhibitory signal in cortical neurons, leading to excessive motor output. Dystonia is mainly a disease of the basal ganglia, but it has cerebellar and cortical components.

Choreoathetosis: Chorea is characterized by rapid, unpredictable, dance-like movements, usually involving the distal limbs. Athetosis refers to slow writhing movements involving the distal limbs and other body parts. Choreoathetosis refers to the coexistence of chorea and athetosis. The basal ganglia are the main structures involved in producing choreoathetosis. Excess copper within the cell initiates oxidative damage from free radicals and lipid peroxidation, leading to neuronal damage. Magnetic resonance imaging (MRI) is a diagnostic and prognostic tool during chelation therapy that detects structural changes, particularly in the basal ganglia, thalami, and brainstem. Mild T2 hyperintensities refer to initial changes that may be reversible with chelation treatment. However, in case of more severe disease, T1 hypointensities, atrophy, and T2 hypointensities (iron deposits) occur[19].

Cognitive impairment: Cognitive changes have been reported in WD resulting from frontal lobe degeneration, indicating deficits in executive function and behavioral changes. There may be personality changes, mood disturbances, and slowness of mentation. Copper deposition causes hypertrophy of the astrocytes, cerebral edema, cystic changes, and demyelination of nerves. The findings are usually both symmetrical and bilateral, where both white and gray matter are affected.

Parkinsonism: Parkinsonism is a clinical condition that is a combination of bradykinesia, postural instability, resting tremor, and cogwheel rigidity. In WD, it is symmetrical. Nigrostriatal dopaminergic deficits are the cause of Parkinsonism.

Ataxia: Ataxia is characterized by abnormalities of posture and gait, dysdiadochokinesia, dysmetria, hypotonia, and oculomotor abnormalities. It results from cerebellar dysfunction. In WD, profound demyelination occurs due to copper toxicity. Cerebellar dentate nucleus T2 hypointensity on MRI is a distinguishing finding. Focal thalamic lesions may be associated with this finding.

Dysphagia: One of the most important symptoms found in WD patients is dysphagia. Dysphagia is caused by impairment of muscle tone, uncoordinated movements, or hypotonia. Drooling is also associated with this condition.

Other presentations of WD

Renal manifestations: Renal disease is rare as the first clinical presentation. Proximal renal tubular dysfunction and decreased glomerular filtration rate are the main features. Proximal renal tubular dysfunction is characterized by proteinuria, glucosuria, phosphaturia, uricosuria, aminoaciduria, and hematuria. Distal renal tubular acidosis can rarely occur, leading to nephrocalcinosis. With chelation therapy, renal abnormalities are improved. However, there are other forms of renal involvement. Drug-induced (Penicillamine) nephrotic syndrome may occur. Acute kidney injury can occur in Wilsonian acute liver failure or in decompensated liver disease as a part of hepatorenal syndrome.

Hematological manifestations: Copper-induced intravascular hemolysis is a rare primary presentation (15%) without hepatic or neurological manifestations. However, it may manifest in the later course of the disease. WD may be the cause of Coombs-negative hemolytic anemia in adolescents. Hemolysis is considered the sudden release of copper from the liver, which initiates oxidative stress and lipid peroxidation in red blood cells. Hemolysis is a poor prognostic factor when presented with acute liver failure.

Cardiac involvements: WD causes arrhythmia and, cardiomyopathy. ECG findings may show abnormalities like left ventricular hypertrophy, T inversion, and ST depression. Skeletal manifestations may occur due to hypercalciuria and hypophosphaturia resulting from renal tubular dysfunction. Radiological changes of rickets, osteomalacia, osteoporosis, and pathological fractures may be present. Skin pigmentation may be increased, resulting from melanin deposition, and acanthosis nigricans may be present. There are infrequent endocrine complications like diabetes mellitus, hypoparathyroidism, and exocrine pancreatic insufficiency.

Diagnosis of WD

The diagnosis of WD is usually not straightforward in children due to the wide range of disease presentations. In case of more advanced liver disease, diagnosis can be made more accurately in a short time. But in the early stages of disease, diagnosis is challenging, as the usual tests may be equivocal.

Diagnostic approach to WD should be stepwise according to position paper (2018) by the hepatology committee of the ESPGHAN and EASL-ERN Clinical Practice Guideline (2025), The first step comprises evaluation of clinical features, liver function tests (alanine aminotransferase/alanine aminotransferase, serum bilirubin, prothrombin time, serum albumin), if hepatic features are initial manifestations and measurement of serum ceruloplasmin, urinary copper, and slit-lamp examination (KF ring)[12,20]. The second step includes mutational analysis (common mutations, whole exome sequencing). The third step comprises liver copper estimation when the diagnosis is inconclusive. The Leipzig score should be calculated during the performance of different tests. If the score is 4 or higher, a diagnosis is possible without confirmatory tests (such as mutational analysis or liver copper estimation). In a resource-limited setting, not all tests can be performed. So, whenever scoring is adequate for WD, treatment should be started without delay[12]. The diagnostic approach is illustrated in Figure 1.

Figure 1 Diagnostic algorithm for Wilson’s disease. WD: Wilson’s disease; REC: Relative exchangeable copper; KF: Kayser-Fleischer.

Leipzig diagnostic criteria: The Leipzig scoring system makes the diagnosis of WD simple. Nagral et al[21] produced a rationalized, modified version of the Leipzig scoring system in 2019. Scores of 4 or higher in Leipzig confirm the diagnosis of WD in children. Modified Leipzig score is attached in Table 2[21].

Table 2 The modified Leipzig scoring system.

Scoring items	Score
KF rings
Present	2
Absent	0
Serum ceruloplasmin
Normal > 20 (mg/dL)	0
0 to 5 (mg/dL)	3
6 to 11 (mg/dL)	2
11 to 20 (mg/dL)	1
24 hours urinary copper
> 100 (μg)	2
40-100 (μg)	1
< 40 (μg)	0
Coomb’s-negative hemolytic anemia with liver disease
Present	1
Absent	0
Mutational analysis
On both chromosomes	4
On one chromosome detected	1
No mutation detected/test not done	0
Liver biopsy for histology suggestive of WD
Orcein or rhodamine negative granules	0
Orcein or rhodamine positive granules	1
Neurobehavioral symptom
Present	2
Absent	0
Typical features on MRI brain
Present	1
Absent	0
History of WD in a family member/sibling death from liver disease/neurological disease
Suggestive of WD	1
Absent	0

Interpretation of the score: Score 4 or more: Confirmed Wilson disease, score 3: Diagnosis possible, score 2 or less: Unlikely (the score is taken from Wilson’s disease: Clinical Practice Guidelines of the Indian National Association for Study of the Liver, the Indian Society of Pediatric Gastroenterology, Hepatology and Nutrition, and the Movement Disorders Society of India). WD: Wilson’s disease; MRI: Magnetic resonance imaging; KF: Kayser-Fleischer.

Serum ceruloplasmin

Normal serum ceruloplasmin levels range from 20 mg/dL to 40 mg/dL. Low serum ceruloplasmin levels are typically found in patients with WD[22]. Notably, serum ceruloplasmin levels may rise to normal values during acute phases of concurrent infection. Low ceruloplasmin levels may be present when protein deficiencies occur due to protein-losing enteropathy, malabsorption/malnutrition, and nephrotic syndrome, so they are not an absolute screening tool for WD. Different thresholds of serum ceruloplasmin level (below 20 mg/dL, 14 mg/dL, and 10 mg/dL) exhibited progressively increasing positive predictive values of 48.3%, 100%, and 100%, respectively, and negative predictive values of 98.7%, 97.1%, and 91.9%, respectively. Serum ceruloplasmin measurement (enzymatic assay) demonstrated that the threshold of 14 mg/dL achieved 100% sensitivity and specificity in the validation cohort. Some heterozygote carriers had low serum ceruloplasmin levels; however, in this study, all simple heterozygotes had values > 14 mg/dL[23]. In the Leipzig score, the cutoffs of 10 mg/dL and 5 mg/dL show a higher score.

24-hour urine copper excretion test

The 24-hour urine copper excretion test quantifies the total amount of copper excreted in urine over 24 hours. The urinary copper diagnostic cutoff value is 40 μg/24 hours, with a sensitivity of 78.9% and a specificity of 87.9%[24]. The penicillamine challenge test is unreliable to rule out the diagnosis of WD to avoid contamination with copper, as it increases copper content in urine. Urine should be collected in plastic or glass containers to avoid contamination with copper, as this can increase copper content in urine.

Newer test

Total serum copper (TSC) denotes the amount of copper in the blood that is bound in ceruloplasmin, as well as free copper that is not incorporated in ceruloplasmin. TSC levels cannot measure tissue copper, so they do not reflect the actual amount of body copper, making them unreliable for diagnosing WB. Relative exchangeable copper (REC; exchangeable copper-to- total copper ratio: REC = CuEXC/total % of copper) is used as a newer test. Exchangeable copper resembles the free copper fraction in the serum bound to albumin and amino acids. The percentage of exchangeable to TSC (TSC means copper bound to ceruloplasmin, not bound to ceruloplasmin) is called the REC, which indicates the toxic blood copper fraction. El Balkhi et al[25] investigated the REC and compared its diagnostic efficacy with that of classical WD tests in three populations (WD patients, heterozygous carriers, and normal individuals). The REC test with a 18.5% cutoff value was shown to be more sensitive and specific than other classical tests[24,25]. REC level is not influenced by disease severity, disease stage, or other associated conditions.

The REC determination is presently not available worldwide. In recent studies, REC > 18.5% revealed sensitivity of 79%-100% and specificity of 100% in differentiating patients with WD from healthy controls and from those with other liver diseases and was unaffected by low ceruloplasmin levels detected in patients with advanced cirrhosis[26]. Ultrafiltration coupled with atomic absorption spectrometry is a direct method for measuring plasma-exchangeable copper or free copper[27]. Then, REC is calculated. REC was first measured in healthy individuals to establish a reference value. REC is a useful tool for discriminating between heterozygous ATP7B carriers and individuals without ATP7B mutations and between patients with WD and those without WD, at a cutoff of 15%. Thus, it has a significant role in family screening of WD[27]. REC assay is routinely used in France, Spain, and Denmark. The results of the studies from France and Spain demonstrated that REC values were below 15% in all controls and above 14% in all patients with WD. REC is not yet included in the Leipzig scoring system as it is not widely available.

Mutational analysis

The identification of a mutation of one allele of the ATP7B gene seems acceptable to confirm the diagnosis of WD only in the presence of suspected clinical features and biochemical signs of impaired copper metabolism. In case of asymptomatic children, identification of homozygous mutations is needed to confirm the diagnosis of WD. The mutational analysis helps identify affected siblings of probands with homozygous ATP7B mutations. It is effective in family screening[12].

Histopathology of WD

Liver biopsy is performed to determine the grade of hepatic inflammation and to estimate hepatic copper levels. The main histopathological features are microvesicular and macrovesicular steatosis, glycogenated hepatocyte nuclei, and inflammation. There is a wide variety in liver histology depending on the clinical stage of the disease. In the earliest stage, hepatocyte injury may be evident as simple steatosis, which may mimic NAFLD. In case of Wilsonian liver failure, there may be massive hepatic necrosis. There are other distinct findings in WD, such as glycogenated nuclei and Mallory-Denk bodies (MDBs). Features of metabolic dysfunction-associated steatotic liver disease (MASLD) include steatosis, glycogenated nuclei, and MDBs, which are more prominent in children than in adults. In adults, features of cirrhosis are more prominent in MASLD[28].

The main difference between fatty liver disease and WD is the demonstration of copper accumulation in hepatocytes by histochemical stains. The more advanced stage of the disease displays histological findings similar to those of chronic hepatitis of viral or AIH. The features are periportal inflammation composed of plasma cells and lymphocytes, resulting in destruction of the limiting plate (interface hepatitis), and, sometimes, parenchymal inflammation with bridging fibrosis. Thus, histological features of AIH may mimic with WD. However, the combination of steatosis, glycogenated nuclei, and MDBs help distinguish WD from other causes of chronic hepatitis. In the later stage, macronodular cirrhosis is most common, but it can be mixed or even micronodular. The distribution of copper is patchy in both lobes of the liver, so some areas contain more copper, whereas others contain less. Defining widespread copper deposits by histochemistry can help with the diagnosis. For histochemical staining, two liver specimens are needed to prevent a false-negative result. Rhodamine staining has a trivial role for diagnosing WD due to low sensitivity (11%-56%), depending on disease stage, and very low sensitivity in early stages[29].

Estimation of hepatic copper content is an important diagnostic tool for WD[23]. Due to a troublesome technique, copper estimation is excluded from the modified Leipzig score. Special histochemical stains for copper include rhodanine, rubeanic acid, Timm’s silver stains, and orcein. Orcein reveals dark-brown granules, indicating the accumulation of sequestered copper bound to MT. With rhodanine stain, copper accumulation appears as small red granules. Copper content > 250 μg/g dry weight (normal value < 50 μg/g dry weight) is recognized as diagnostic for WD[19]. Due to patchy copper distribution in the liver, lower values may be found and we cannot solely depend on hepatic copper content. The biopsy sample should be > 1 cm long and placed on a small piece of paper for drying, then placed in a dry plastic, copper-free holder for analysis. Comparison of different diagnostic tests is presented in Table 3[12].

Table 3 Usefulness and drawbacks of different diagnostic tests performed in Wilson’s disease.

Investigations	Normal value	Diagnostic value	Usefulness	Drawbacks
Serum ceruloplasmin	20 mg/dL to 40 mg/dL	Less than 20 mg/dL	Traditionally, it is first line investigation for WD (Recommended in previous and current guidelines and position papers)	It cannot be recommended as a diagnostic tool for children aged less than one year as serum ceruloplasmin level is low in early infancy. The serum level of ceruloplasmin can be affected by other liver diseases, malnutrition, and acute inflammatory state. Ceruloplasmin is acute-phase protein. It may rise in acute inflammatory condition or decrease in any condition of protein losing enteropathy. So, it may give false positive or false negative result. Value of serum ceruloplasmin may differ by different methods like enzymatic assays and immunologic assays. Enzymatic assays are the preferred, rarely available method as it detects only holo-ceruloplasmin while immunologic assays measure both apoceruloplasmin and holo-ceruloplasmin, which could overestimate serum ceruloplasmin
Urinary copper test	< 40 μg/24 hour	> 100 μg/24 hour-high suspicion. 40-100 μg/24 hour-need for further evaluation	Traditionally, it is first line investigation for WD. (Recommended in previous and current guidelines and position papers)	Correct measurement needs exact collection timing and copper free container. It may increase in cholestatic liver disease
Relative exchangeable copper	3.4%-8%	> 15% (high suspicion)	It is recommended as highly useful test. This test does not depend on serum ceruloplasmin level, as ceruloplasmin may vary in different conditions	It is not widely available. It is costly. Toxic free copper fraction can be identified
Genetic test/mutational analysis	No mutation in ATP7B gene. Healthy heterozygote carrier of ATP7B gene	Mutation detected in both allele of ATP7B gene-confirmatory. Mutation detected in one allele of ATP7B gene-need further evaluation	The test is confirmatory. It can differentiate healthy heterozygote carriers from presymptomatic WD children. It is helpful for family screening	Rarely, children with WD may not have any identifiable mutation. Different diagnostic scores are established for minimizing the situation. WD cannot be excluded, if no mutation is found in one or both alleles
Liver copper estimation	Normal value < 50 μg/g dry weight	250 μg/g dry weight liver is recognized as a cut-off value for diagnosis of WD. The value between 50-250 μg/g dry weight liver is not specific for WD	It is done when Leipzig score is ≤ 3 or diagnosis is not certain	It is invasive procedure. Adequate size of liver specimen (> 1 cm) is needed. Inhomogeneous distribution of copper in liver makes the diagnosis less reliable
KF ring	Absent in WD	Present in WD	KF ring on slit lamp examination should always be checked	KF ring may be absent in younger children as it requires time to mature. KF ring is not pathognomonic for WD, as it is found in other cholestatic liver disease

WD: Wilson’s disease; KF: Kayser-Fleischer.

Treatment of WD

The goal of treatment is to eliminate copper by chelating agents such as D-penicillamine or trientine, or by blocking intestinal copper absorption with zinc salts. Copper-rich food (shellfish, nuts, chocolate, mushrooms, and organ meats) is advised to be restricted lifelong. Treatment should be initiated as soon as possible, just after diagnosis, through family screening, even when there are no symptoms. Presymptomatic children can be identified through family screening as early as 2 to 3 years of age. The treatment should be started promptly in symptomatic children to prevent progression of hepatic and/or neurological disease. Treatment should be lifelong without interrupting chelation therapy. Follow-up is needed to monitor drug compliance and early detection complications. Prognosis is excellent as long as the diagnosis is made early and therapy is adequately adhered to. The first-line treatment is the copper chelator D-penicillamine. It is initiated with close follow-up of adverse effects. Zinc acetate is given orally as adjunct therapy along with D-Penicillamine. Then, treatment is maintained with D-penicillamine at the same or reduced dose as given initially, depending on biochemical evidence of copper chelation (serum copper, urinary copper), and is continued lifelong. If any neurological deterioration or other adverse effect occurs, trienine should be used as a second line treatment. Liver transplantation (LT) is reserved for end-stage liver disease and acute liver failure on discontinuation of the copper chelator[12,20,30]. Medications used in WD are illustrated in Table 4[12,20].

Table 4 Medications used in Wilson’s disease.

Medication	Mechanism of action	Dose	Side effects
D-penicillamine	Cu chelator	Initial: 20 mg/kg/day, 3 times a day (one hour before and two hours after meal). Maintenance: 10-20 mg/kg/day. Oral pyridoxine: 25-50 mg/day. This drug is used as initial and maintenance	Early: Hypersensitivity reaction manifested as fever, rash, lymphadenopathy, pancytopenia. Late: Proteinuria, nephrotic syndrome, drug associated systemic lupus erythematosus, agranulocytosis, thrombocytopenia, Dermatopathy (cutis laxa)
Trientine	Cu chelator	Same as D-penicillamine	Less toxic than D-penicillamine. Toxicity includes sideroblastic anemia, nephrotoxicity, skin and mucosal lesion
Zinc	Inhibit Cu absorption. Stimulate hepatic metallothionein synthesis	< 50 kg-75 mg/day three times daily. > 50 kg-150 mg/day three times daily. It is used as maintenance and adjunctive therapy	Headache, gastrointestinal upset, iron deficiency
Ammonium tetrathiomolybdate	Inhibit Cu absorption. Cu chelator	100-200 mg/day. It is not yet Food and Drug Administration approved	Elevation of aminotransferase

D-penicillamine: D-penicillamine was first introduced in 1956 and remains the standard treatment for WD to this day. It chelates copper, and the compound is excreted through urine, which is a physiologically minor route of copper excretion. D-penicillamine induces hepatic endogenous MT, which sequesters copper and thereby reduces oxidative liver damage. D-penicillamine can efficiently prevent the progression of disease in asymptomatic children and produce symptomatic improvement in more than 80% of children with advanced disease within a mean time of 16 months[31]. D-penicillamine has major drawbacks, like worsening of neurologic symptoms. Early adverse effects include hypersensitivity reactions such as fever, rash, arthralgia, and lymphadenopathy. In the later course of disease, serious adverse effects may develop, such as hematuria, proteinuria, bone marrow depression with severe thrombocytopenia or aplasia. Significant adverse effects result in drug withdrawal in up to 30% of cases in children or adults[32]. In children, the starting dose of D-penicillamine should be low, then gradually increased to 20 mg/kg/day given in two or three divided doses, with close monitoring for serious adverse events such as hypersensitivity and proteinuria, as well as hematologic toxicity that requires immediate discontinuation and switching to trientine. D-penicillamine should be administered 1 hour before or 2 hours after meals, as its absorption is hampered by food.

Trientine: Trientine hydrochloride is also a chelating agent that is used to chelate copper and excrete it through urine. It is used by those who are intolerant to penicillamine[32]. It is taken orally on an empty stomach, at least 1 hour before or 2 hours after meals. Before starting Trientine, Penicillamine should be stopped as both are chelating agents. Trientine has fewer side effects than penicillamine. The side effects of trientine are fever, rash, abdominal pain, and bone marrow suppression.

Zinc salt therapy: Zinc can act as an intracellular inducer of the MT enzyme in enterocytes. As MT bind copper more readily than zinc, this action can prevent copper absorption from the intestine. Moreover, zinc may help reduce oxidative liver injury that can occur due to excess copper deposition[12,20,32].

Ammonium tetrathiomolybdate: It binds dietary copper to prevent intestinal absorption and also complexes with copper and proteins in the blood. It acts as a copper chelator. Recently, another compound, bis-choline tetrathiomolybdate, has gained attention. Currently, the phase 3 trial is being conducted in 100 patients receiving this molecule. It effectively reduces non-ceruloplasmin-bound copper and improves neurological features in a phase 2 open-label trial. It can be administered orally and as a once-daily monotherapy[33,34].

Methanobactin: Methanobactin is a copper-binding chromophoric peptide initially identified in bacteria Methylococcus capsulatus and Methylosinus trichosporium OB3b. It is a type of methane-oxidizing bacterium that can convert meAthane into methanol with the assistance of methane monooxygenase (MMO) enzymes. Copper is needed for the proper functioning of MMO enzymes. After extensive research, a peptide is found that can chelate copper in bacterium’s environment[35]. This copper chelating agent is called methanobactin. Methanobactin can reduce toxic Cu (II) to the less toxic Cu (I). Since 2011, research on methanobactin in mouse models of WD has continued. Excess copper is reduced in a mouse model, and its elimination via feces via biliary excretion is detected. However, there are some difficulties for human trials. There is no suitable technique for commercial production. Methanobactin cannot be administered orally, as peptide bonds will be broken in the GI tract. In the animal model, the agent is administered intraperitoneally. In the absence of copper, it can chelate other important metals, such as iron and zinc.

Indications for LT in children

LT for WD in children is rare (1%), including patients with ALF or those with end-stage liver disease despite adequate drug therapy. The United Network for Organ Sharing database of 170 children with WD who underwent LT between 1987 and 2008 was analyzed in a study, which revealed 1- and 5-year survival rates of 90.1% and 89%, respectively[12,36]. The outcome of LT was better in patients transplanted for end-stage chronic liver disease than in those transplanted for ALF. Neurological features may improve after LT. A previous study showed that neurological involvement may demonstrate little improvement with transplantation, although LT cannot be a suitable and worthy therapy for patients with severe neuropsychiatric involvement[37,38].

Follow-up

Follow-up is needed to monitor efficacy, safety, and compliance with treatment. Physical examination and laboratory investigations (liver function tests, complete blood cell count, urine specific gravity/osmotic pressure measurement) should be performed at regular intervals, especially during initiation of therapy. At first, follow-up should be done once weekly while increasing the penicillamine dosage. Afterward, monthly monitoring should be performed for 1 to 3 months, until near-normal laboratory findings are achieved. Further follow-up plan should be directed depending upon the emergence of new symptoms and adherence to drugs. Usually, a 3-6 months monitoring interval is adequate when disease progression is unremarkable. Counseling about drug adherence is necessary as nonadherence to therapy can lead to life-threatening deterioration. Liver function tests usually tend to be normal or near normal within 3-12 months. The value of 24-hour urinary copper excretion should increase after starting therapy with D-penicillamine as a marker of good chelation effect, followed by a decrease in excretion once symptomatic and laboratory improvement indicates a reduction in total body copper. Urinary copper excretion should be between 200 and 500 mg/24 hours during maintenance therapy with D-penicillamine or trientine according to all recent guidelines[12,20,30]. Brief withdrawal of therapy, with close monitoring, is necessary, followed by reintroduction of the drug at a decreased dose. An annual slit-lamp examination should be performed to assess the disappearance of the KF ring, indicating proper chelation therapy. Non-disappearance of KF rings along with continuing abnormalities of liver function tests on chelation therapy indicates noncompliance.

Current research gaps and future directions

Recent literature showed significant differences in disease presentation despite the same genotype of two siblings indicating a substantial effect on phenotype by epigenetic/environmental factors. Even monozygotic twins showed different disease presentations at the same time of their life[39]. One of the twins may be presented as a case of acute liver failure and another twin may only be presented as asymptomatic liver disease. Malnutrition and dietary habit are the important factors for different disease presentations. Another important aspect of our narrative review is the future diagnostic tool, REC. It requires sophisticated equipment. So, most parts of the world still lack this test. It cannot be integrated in diagnostic algorithm in low resource countries. Methanobactin is a promising chelating agent but it is still in preclinical trials. Tetrathiomolybdate is used in small clinical trials in Europe. A new drug, bis-choline tetrathiomolybdate is being used on a developmental trial for neurological WD, however, it is still not Food and Drug Administration approved.

CONCLUSION

WD has a variable clinical presentation. Clinicians should be cautious about varied presentations. A proper understanding of the pathophysiology of WD is needed to develop newer molecules. An easy, cost-effective diagnostic tool is necessary to enable early diagnosis and halt the progression of the disease. A newer copper chelator is under clinical trial. Appropriate steps for diagnostic workup, management, and follow-up of children with WD are essential for better outcomes.