Wilson Disease: Novel Diagnostic and Therapeutic Approaches

Abstract

The Wilson disease (WD) research field is rapidly evolving, and new diagnostic and therapeutical approaches are expected to be change-gamers in the disease for the incoming years, after decades of slow changing options. Non–ceruloplasmin-bound copper assays for circulating bioavailable copper are being tested for use in monitoring therapy and may also help in the diagnosis of new cases of WD. Other diagnostic advances include the use of quantitative detection of ATP7B peptides in dried blood spots, a method that is being tested for use in the newborn screening for WD, and the use of metallothionein immunostaining of liver biopsy specimens to differentiate WD from other liver diseases. Ongoing and future trials of gene therapy and use of methanobactin are expected to restore biliary copper excretion from the liver, thus making a cure for WD a plausible therapeutic objective. With the aim of helping updating physicians, this review summarizes the novel methods for WD diagnosis and future therapies. Advancing understanding of the scientific advances that can be applied to WD will be critical for ensuring that our patients will receive the best current and future care.

Keywords

Wilson disease - research - diagnosis - treatment - copper

Wilson disease (WD) is a rare autosomal recessive disorder of copper metabolism caused by mutations in the ATP7B gene. This gene encodes an important transmembrane copper-dependent ATPase known as the ATP7B protein which is mainly expressed in hepatocytes. In the liver, ATP7B is required for two main critical pathways in copper metabolism: (1) copper incorporation into ceruloplasmin (Cp) and (2) biliary excretion of the excess of copper by the hepatocytes. Whenever there is loss or reduced function of ATP7B in hepatocytes, copper will progressively and pathologically accumulate, primarily in the liver, but also in other tissues, particularly in the central nervous system and the cornea. Because of this broad systemic involvement, the large number of potential pathologic ATP7B mutations, differences in dietary copper intake, and other host factors that govern response to injury, the clinical phenotype is widely variable.[1] The most common age of clinical presentation occurs in children, adolescents, or young adults. WD-associated liver disease may vary from asymptomatic abnormalities in liver tests to acute liver failure (ALF) with poor transplant-free survival and to advanced liver disease with cirrhosis without or with portal hypertension.[2] Neurologic involvement is more frequent in adults and clinically mimics movement disorders. Psychiatric involvement may happen at all stages of the disease, though more commonly in adults, and may lead to a delay in diagnosis when patients lack overt hepatic or neurologic disease.[3] Many other clinical presentations occur with less frequency, and the diagnosis of WD is often very challenging in clinical practice. Clinical suspicion and a proper interpretation of the available diagnostic tools, with prompt patient referral to expert centers, remain critical for preventing patients from unfortunate late diagnosis or disease progression, which are sometimes associated with irreversible changes and severe disability. In the last few years, the development of new strategies for the diagnosis of WD has resulted from advances in research and would potentially simplify our diagnostic approach in the future. With these advances, we hope for earlier disease detection when the clinical manifestations of WD are less severe and potentially reversible or preventable altogether. This review will summarize most of the novel strategies directed at improving the early and accurate diagnosis of WD.

Therapeutic strategies for WD have remained essentially unchanged for several decades, relying on two groups of drugs with differing mechanisms of action[2]: zinc salts (reducing gastrointestinal dietary copper absorption) or chelation therapy (either penicillamine or trientine, which promotes high urinary copper excretion [UCE]). Neither of these strategies entirely restore physiologic hepatic copper metabolism and intrahepatic copper overload is known to persist over time regardless of therapy, leaving transplantation as the only current option for curative treatment in WD. Recently, two new therapeutic agents have reached preclinical or clinical testing. Both treatment options converge on the common mechanism of promoting physiologic copper excretion into bile. Whether removal of this excess copper in the tissue could be considered a real cure of the disease or not remains to be determined. Their potential for successful treatment of WD is likely to change the therapeutic paradigm for WD in the years to come. These novel approaches to the treatment of WD will also be discussed in the following review.

Novel Diagnostic Approaches for WD

Classic Approach—Overview

The imperative for developing and adopting new approaches to improve our diagnosis of WD is the desire to detect this disease at the earliest phase of the disease, preferably before serious symptoms or signs of disease are detectable. Achieving this goal would reduce the burden of liver, neurologic, or psychiatric disease that may result from delayed identification and treatment of WD. The following overview discusses the limitations of the current methods for diagnosing WD and discusses alternatives for the diagnosis of this disorder that are likely to impact our real-life clinical practice. However, it is fair to say that following the initial clinical suspicion of WD, early referral of complex patients to experienced centers with multidisciplinary teams and the proactive and universal screening of a patient's relatives whenever a case is diagnosed remain critical regardless of the diagnostic tools to be used.

The Leipzig score was developed by an expert panel of physicians and scientists back in 2001.[4] This scoring system was designed to help clinicians establish a diagnosis of WD using available clinical, biochemical, and, for the first time, molecular testing. The use of a matrix of clinical and biochemical parameters was needed since no single test on its own (save the genetic testing) was sufficiently sensitive or specific for WD diagnosis. The Leipzig score provides a graduated score for seven items that have been classically used to define WD patients: low Cp serum levels, high 24-hour UCE, elevated hepatic copper content, the presence of Kayser-Fleischer corneal rings, the detection of pathogenic ATP7B mutations, the presence of hemolytic anemia, or the presence of abnormal findings on brain magnetic resonance imaging typical for WD ([Table 1]). When the Leipzig score is equal to or higher than 4 points, a diagnosis of WD is established. Since its introduction, the Leipzig score has been broadly used in clinical practice[5] [6] and represents a good, objective, and standardized approach for physicians worldwide. However, clinical suspicion remains a mandatory requirement to initiate an evaluation and utilize the Leipzig score. This constitutes a limitation itself as WD is a rare disease with a wide spectrum of phenotypic presentations. Additionally, there may be a lack of clarity in the scoring of some elements and even issues of scarce resources, thus limiting the performance of other items in the scoring system (e.g., testing for ATP7B mutations and copper quantitation from biopsy specimens) in less advantaged areas of the world.

Table 1 Current Leipzig score for Wilson disease diagnosis[4] and potential novel proposals for diagnosis

Evaluation	Comments	Score −1 point	Score 0 points	Score 1 point	Score 2 points	Score 4 points
I. Copper-associated measurements
Serum ceruloplasmin (mg/dL)	Based on nephelometric assay, may change if enzymatic assays are used	NA	Normal levels (guidance: > 20 mg/dL)	Intermediate levels (guidance: 10–20 mg/dL)	Low levels (guidance: < 10 mg/dL)	NA
24-hour urinary copper excretion (UCE) (μg)	Refers to spontaneous UCE (no ongoing acute hepatitis)	NA	Normal (guidance: < 40 μg)	Indeterminate 1–2 × ULN (guidance: ≈ 40–80 μg)	Elevated > 2 ULN (guidance: > 80 μg)	NA
24-hour UCE after DPA challenge (1,000 mg)	If normal UCE but high suspicion; validated in children	NA	NA	NA	Elevated > 5 ULN—frequently higher (guidance > 200 μg)	NA
Hepatic copper content (μg/g dry hepatic tissue)[a]	Minimum requirements in tissue samples must be accomplished	Normal (guidance <50 μg/g)	NA	Up to > 5 ULN (guidance 75–250 μg/g)	> 5 ULN (guidance > 250 μg/g)	NA
Hepatic IHC staining (rhodamine)	In case copper quantification not available	NA	Negative staining	Positive staining	NA	NA
Serum copper (µg/dL)	Usually low in WD; represents the ceruloplasmin-bound copper. Not part of the current Leipzig score
NCC (µg/dL) or NCC ratios (%) [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22])	Usually high in WD; different methodologies have been proposed (see [Table 2]), represents the non–ceruloplasmin-bound copper fraction. Not part of the current Leipzig score
Metallothionein (MT) staining in tissue [29] [30]	Positive in most of the naive WD patients. Might be iatrogenically induced in zinc-treated patients. Not part of the current Leipzig score
ATP7B peptides [23]	Reduced in most WD patients. Proposed as a screening method for newborns and measured in dried blood spots. Not part of the current Leipzig score
64 Copper PET-CT ratio [33] [36] [37]	Usually reduced in WD. High costs and high demanding isotope certifications. Not part of the current Leipzig score
II. Clinical signs and symptoms
Hemolytic anemia	Coombs negative, usually in acute presentations only	NA	Absence	Presence	NA	NA
Kayser-Fleischer rings	Corneal evaluation with slit lamp examination, frequent among neuropsycho presentations	NA	Absence	NA	Presence	NA
Compatible neuropsychological symptoms	Or compatible MRI changes if neurological evaluation is not clear	NA	No symptoms (normal MRI)	NA	Presence of either typical neuropsychological or MRI changes	NA
III. Mutational analysis
ATP7B gene	Complete ATP7B sequencing for naive patients, guided by proband's mutations in relatives. More than 900 pathogenic/likely pathogenic mutations described –expertise needed	NA	No pathogenic mutations detected	One pathogenic mutation detected in one chromosome (carrier)	NA	Two pathogenic mutations detected (compound heterozygous) or same mutation in both chromosomes (homozygous)
Interpretation of the Leipzig score system (minimum −1 to maximum 14) Not available items score 0 points
• 0–1 points: Wilson disease can be excluded  • 2–3 points: Requires additional evaluations  • ≥ 4 points: Wilson disease can be established

Abbreviations: DPA, D-penicillamine; IHC, immunohistochemical staining; MRI: magnetic resonance imaging; NA, not applicable; NCC, non–ceruloplasmin-bound copper; UCE, urinary copper excretion; ULN, upper limit of normal.

Notes: The current included Leipzig items are marked in bold letters.

^a Liver biopsy is not mandatory for WD diagnosis and histology is unspecific.

Thus, the successful early identification of patients with WD remains challenging and the time to diagnosis of this disorder is frequently delayed. Fortunately, research on novel diagnostic tools has been crucial to help advance the field in the last few years. Different new tools have been developed so far and could be proposed to be included as part of a new diagnostic system still to be defined ([Table 1]).

Novel Diagnostic Strategies

New Methods for Direct Nonceruloplasmin Copper Measurement

It has been extensively described how the measurement of total serum copper may not reflect the state of copper metabolism among patients with WD. However, this biochemical characteristic might not be so evident for nonexperienced physicians. Total serum copper includes both a Cp-bound copper fraction (which typically represents about 90% of copper in the circulation of healthy individuals) and the remaining fraction of non–ceruloplasmin-bound copper (NCC). In clinical practice, total serum copper eventually measures the Cp-bound copper component. The NCC fraction, on the other hand, comprises copper which is circulating free or loosely bound to other circulating proteins and peptides (mainly to albumin via a histidine moiety near the N-terminus of the protein), and it is this NCC pool of copper that is bioavailable or exchangeable under physiologic conditions which is expanded in untreated patients with WD.

Novel assays have been developed in the last several years with the common objective of detecting and quantifying NCC for its use in the diagnosis and/or monitoring of WD treatment. The success and limitations of these novel assays are discussed next and summarized in [Table 2].

Table 2 NCC assays: benefits and pitfalls

NCC assays	Methodology	Evaluated population	Main results	Limitations
Exchangeable copper (CuEX) Reported in μg/dL and mmol/L[7] [8] [9] [10] [11] [12] [13] [14] [15]	• EDTA chelation and ultrafiltering serum throughout a 30-KDa cut-off membrane. It is assumed that all NCC will be filtered but Cp-bound copper will not trespass the membrane. • NCC result quantified by ICP-MS. • Relative exchangeable copper (REC) is expressed as a ratio (CuEX/total copper, %)	• 109 adult and pediatric WD patients. • 132 carriers (one mutation only) • 78 healthy individuals. • 152 non-Wilsonian patients with liver diseases (103 adult and 49 pediatric)	• REC values >18% have shown to accurately identify naive WD patients. • WD-treated patients may have lower REC values.  • CuEX might be helpful for monitoring WD patients	• CuEX normal ranges established for healthy individuals only (4.1–7.1 µg/dL), may not be applicable for WD patients. • CuEX target for monitoring still to be defined.  • Results may be over- or underestimating the NCC real values as methodology is not precise (assumptions are considered)
Labile-bounded copper (LBC) Reported in ng/mL[19]	• EDTA chelation and ultrafiltering serum throughout two consecutive cut-off membranes (100 and 30 KDa, respectively).  • NCC result quantified by ICP-MS. • LBC fraction expressed as a ratio (LBC/total copper, %)	• 214 healthy individuals	• Sex-specific reference intervals for LBC were established: female (13–105 ng/mL), male (12–107 ng/mL) • Sex-specific reference intervals for LBC fraction were established: female (1–8.1%), male (1.2–10.5%)	• No WD patients evaluated so far
Acurate NCC (ANCC) Reported in μg/L and μmol/L[16] [20] [21]	• Precise measurement of Cp's copper content by SAX-ICP-MS (internal standards used) • ANCC was calculated by subtraction of the Cp-bound copper from the total serum copper (CuANCC = Cutotal − CuCp). • Relative ANCC (RelANCC) expressed as a ratio (ANCC/total copper, %)	• 31 non-WD individuals • 71 WD-treated patients • 1 naive WD patient • 23 WD-ALF • 49 non-WD ALF	• RelANCC was higher among WD patients compared to non-WD individuals. • RelANCC was higher in naive vs. treated WD patients. • ANCC was higher among WD-ALF compared to other mixed ALF causes. • A score including ANCC and ALT-improved accuracy for WD-ALF diagnosis	• Internal standards of normality for protein peaks were established. • Limited WD cohort, especially for naive cases. • More complex and expensive technology for implementation in clinical practice
Speciation NCC (Sp-NCC) Reported in μg/L[17] [18] [22]	• Separation of the main copper species by using standardized protein peaks and HPLC-ICP-MS. • Sp-NCC was the total fraction of copper not bound to Cp	• 53 stable WD-treated patients (Chelate study)	• Sp-NCC was shown to be stable for monitoring. • Mean changes of Sp-NCC were nonsignificantly different between DPA-treated and TETA4-treated patients	• Reference standards for NCC were set.  • Limited number of WD patients included. • No naive WD evaluated

Abbreviations: ALF, acute liver failure; ALT, alanine aminotransferase; ANCC, accurate NCC; CuEX, exchangeable copper; DPA, D-penicillamine; HPLC, anion-exchange high-performance liquid chromatography; ICP-MS, inductively coupled plasma mass spectrometry; LBC, labile bound copper; NCC, non–ceruloplasmin-bound copper; REC, ratio of exchangeable copper; SAX-ICP-MS, strong anion exchange chromatography coupled to triple quadrupole inductively plasma mass spectrometry; Sp, speciation; TETA4, trientine tetrahydrochloride.

Exchangeable Copper

The concept of exchangeable copper (CuEX) was introduced in 2009 by El Balkhi and collaborators.[7] The proposed assay was based on ultrafiltering plasma through a 30-kDa cut-off membrane in the presence of the chelator ethylenediamine tetraacetic acid (EDTA) under conditions where copper should remain bound to Cp (which would not pass through the membrane due to its higher molecular weight [MW]) but where it would be released from other proteins and peptides (such as albumin).[7] [8] [9]

CuEX was shown to be very high among naive (nontreated) WD patients, especially those with extrahepatic presentations[10] but slightly lower among WD-treated patients, suggesting its potential use as a monitoring tool.[11] This method has been broadly utilized in France where it was originally developed and is also in use in other European countries.[12] Its value for monitoring treated WD patients is still a matter of discussion and research, as the target levels of CuEX to be reached among patients remain to be defined.[11] [13] [14] The authors also described that the ratio of CuEX to total copper (or percent relatively exchangeable copper [REC]) identified naive WD patients and accurately separated them from carriers or healthy individuals,[8] especially when REC was higher than 18%.[8] [11] [15] Recent updates on REC suggest the best cut-off to be over 15% (Dujardins et al, unpublished data, WD Aarhus Meeting 2024).

The methodology of determining CuEX has received some criticism as well. Solovyev and collaborators[16] showed that the ultrafiltration method with EDTA could be in fact overestimating the real NCC, as some of the copper bound to Cp was being leached from Cp and measured as part of the NCC pool instead. In addition, the CuEX assay[7] showed to lack repeatability in their hands. Another group[17] also suggested that CuEX method was not accurate enough and showed that CuEX measurement could be underestimating the real NCC fraction, as some of this labile Cu pool could be retained in the filter as part of a high-MW Cu-EDTA–protein complex. The consequence of these studies[16] [17] led to FDA regulators rejecting the use of CuEX as an accurate primary NCC measurement and endpoint in clinical trials[18] and supported the need for the development of new assays instead.

Labile Bound Copper

A variation from the CuEX assay was also proposed by Bitzer et al in 2023.[19] This methodology aimed to obtain the fraction of labile bound copper (LBC) to total copper (LBC/total copper) by means of a dual-filtration–based method coupled to inductively coupled plasma mass spectrometry (ICP-MS) used for copper determination. LBC is isolated from total copper through a series of centrifugation steps using two different MW cut-off filters (100 and 30 KDa, respectively) and an EDTA chelation process. These membranes retain high MW copper-binding proteins such as Cp, while EDTA would remove copper from the remaining proteins (mainly albumin) and peptides and appear in the filtrate. The second filter retains these low-MW products and isolates the total LBC fraction in serum, which is then quantified via ICP-MS.

By measuring both total copper (ng/mL) and LBC (ng/mL) in serum in a large cohort of 214 healthy individuals, the authors were able to calculate the normal reference ranges (as the 2.5th to 97.5th percentiles) for the proposed LBC fraction (%). Interestingly, the reference range was shown to be significantly lower for women (1.0–8.1%) compared to men (1.2–10.5%), although this was not associated with differences in age. Once these sex-specific LBC fraction reference intervals are set, they could constitute the baseline metric for use in the evaluation of copper-related abnormalities. This methodology has not been evaluated in WD patients as yet.

Accurate NCC

This method was based on the use of strong anion exchange chromatography (SAX) to separate serum proteins, and coupling this to triple quadrupole inductively plasma mass spectrometry (ICP-MS-MS) for measurement of copper.[16] Accurate measurement of copper and protein was accomplished using internal standards that took advantage of naturally occurring isotopic variants of copper and sulfur to help standardize recovery. This new approach (SAX-ICP-MS-MS) described in 2019 overcame the limitations of previous methods by directly measuring the amount of Cp (avoiding imprecise measurements with enzymatic assays) and its copper content and does permit further study of protein–copper distribution under different conditions. Thus, ANCC could be directly calculated as the subtraction of Cp-bound copper from the total serum copper (CuANCC = Cu_total − Cu_Cp).

An additional parameter, the relative ANCC (RelANCC), has also been recently published, representing the percentage of the total copper which is present as ANCC (RelANCC = ANCC / Cu_total × 100%).[20] This RelANCC would be equivalent to the REC previously mentioned.[7] [8] [15] In this recent work, ANCC and RelANCC were calculated from a cohort of 71 WD-treated patients (accounting for a total of 126 samples of serums), 1 naive WD patient, and 31 non-Wilsonian individuals. ANCC was significantly lower in treated WD patients compared to the naive WD patients and the non-Wilsonian individuals (p < 0.05). Conversely, RelANCC was significantly higher in the naive WD patient (37.07%), or in the treated WD patients (median value: 30.64%) when compared to the non-WD group (median: 9.66%, p < 0.05). Interestingly, up to 74% of the treated WD patients had RelANCC values greater than the maximum for the non-WD patient group, potentially suggesting that in the remainder, there was potential noncompliance to medication, treatment dosages were inadequate to control the copper excess, or treatment was only very recently added.

This ANCC assay was also reported to be useful in the setting of ALF.[21] Median ANCC at presentation was significantly higher in WD-ALF patients (n = 23, ANCC = 1,601 μg/L) compared to other patients with varied causes of ALF (n = 49, ANCC = 157 μg/L). ANCC showed a very high accuracy (AUROC = 0.94) in differentiating WD acute presentations from other causes of ALF when a cut-off of >484 μg/L (sensitivity of 73.9%, specificity of 100%) was utilized. This accuracy was improved to AUC 0.98 by combining ANCC and ALT into one score (sensitivity of 85% and specificity of 100%) and using a cut-off of 1.92. These results, presented as an abstract previously (Sandahl et al, AASLD, The Liver Meeting),[21] are expected to be published soon.

Speciation-Based NCC

A similar approach to ANCC for NCC measurement came from the speciation strategy[17] developed as a fit-per-purpose strategy during the phase III Chelate study.[18] This clinical trial (NCT03539952) aimed at evaluating the efficacy and safety of trientine tetrahydrochloride (TETA4) as a noninferior alternative to penicillamine (DPA) for the maintenance treatment of 53 adult patients with stable WD. The primary outcome of efficacy was initially based on the CuEX-NCC method. However, the U.S. Food and Drug Administration (FDA) raised concerns about its reliability and therefore a new speciation method was developed by the sponsor (Orphalan).[17] Technically, it is based on the separation of the main copper (Cu) species in human serum (basically Cu-albumin and Cu-Cp) by anion-exchange high-performance liquid chromatography coupled to inductively coupled plasma mass spectrometry (HPLC-ICP-MS) for total Cu determination and using a relative peak area quantification strategy afterward. Standards for each protein were utilized (as opposed to using internal standards as performed in the ANCC method described earlier) and laboratory validation of the method was performed. Essentially, although the CuEX measurements were provided to the clinicians for decision guidance, Sp-NCC was the used method for all the efficacy assessments during follow-up. Importantly, the mean difference in Sp-NCC evaluated by the speciation assay between patients treated with TETA4 or DPA was nonsignificant, and therefore the primary aim of the study was met.

In a recent post hoc analysis performed on baseline samples of the 53 WD patients included in the trial,[18] the Sp-NCC and CuEX results were compared.[22] Although a positive correlation between both measurements (Sp-NCC and CuEX) was observed (r ² = 0.66), there was a significant deviation of CuEX related to the Sp-NCC: basically, CuEX was higher than Sp-NCC when values were below 60 μg/L, but CuEX was lower when above this cut-off. Similar to CuEX, the ranges for NCC in the speciation assays remain to be defined.

Measurement of ATP7B Peptides

With the aim of developing an assay to be used for neonatal screening for WD, another diagnostic proposal was developed.[23] Peptide measurements from digested protein could be performed by using immunoaffinity enrichment coupled to selected reaction monitoring (immune-SRM) mass spectrometry. This technology uses antipeptide antibodies to concentrate and help quantify extremely low-concentration peptide targets, as is the case for circulating ATP7B in WD. ATP7B present in the circulation is thought to be produced by peripheral blood cells and is considered a surrogate of the ATP7B protein in the liver. Importantly, liver impairment should not affect the results of this assay which utilizes dried-blood spots (DBS) for specimen collection. The use of DBS also allows for easy sample preparation and storage.

Results were obtained after the measurement of ATP7B peptides in specimens from 206 WD patients, 48 carriers, and 150 healthy individuals. The signature peptide concentration was obtained from a control individual, and the cut-offs were set accordingly with the −2.5 standard deviation (SD) of the mean values. Those peptides recovered with monoclonal antibodies with the best performance (ATP7B 1056 and 887) were selected. Most of the WD patients (90.2%) in the study had levels of both peptides that were either undetectable or significantly low in value. In up to 92.1% of patients, at least one of the peptides had detectable levels below the cut-off. This proportion increased to 94% among those patients with available and confirmed pathogenic WD genotypes. Conversely, up to 17 WD individuals (7.8%) were classified as false negative, with 13 being patients in whom genotype had been previously associated with a decreased activity but not reduced concentration of ATP7B.

Altogether, the quantitative assessment of ATP7B in dried-blood spots was proposed by the authors to represent a good alternative or complementary methodology for use in patients in whom there was clinical uncertainty as to the diagnosis of WD. There is an ongoing project evaluating the use of this assay in newborn screening for WD in a pilot study in Washington in the United States (Sihoun et al, unpublished data, WD Aarhus Meeting 2024). These results are expected to be published soon.

Metallothionein Staining in Liver Tissue

Obtaining a sample of liver tissue by liver biopsy, obtained by either the percutaneous or transjugular route or by surgical biopsy, has long been used for aiding the diagnosis of WD. The quantification of the hepatic copper content in dry liver tissue constitutes one of the current items of the Leipzig score,[4] which has been validated over the years.[24] [25] However, liver biopsy is not universally possible, and even when it is, an adequate specimen must be obtained for accurately determining the tissue copper content.

Several histochemical methods were developed to reveal copper overload, as an adjunct to copper quantification. Staining with orcein, rhodamine, or rubeanic acid may reveal copper protein deposition in the liver. However, due to the typically patchy copper distribution from differences in tissue content that may arise from hepatic regeneration in response to injury, and the absence or minimal content of copper within severely fibrotic livers, a negative result on these staining methods cannot exclude a diagnosis of WD.[26] Timm's silver stain,[27] showing deposits of copper protein (mainly metallothionein due to its large sulfur content) seen as black granules in the cytoplasm of hepatocytes, was an alternative staining that was abandoned many years ago, as it involved preparation with sulfur gas and was unpleasant for pathologists. However, it might be considered the precursor of a currently proposed strategy for hepatic copper evaluation based on immunohistologic staining of metallothionein.

Metallothionein (MT) is a relatively small (10-KDa) intracellular molecule capable of binding metals (such as copper) due to its high sulfur content. MT is highly induced by high concentrations of many metals.[28] Recently, Rowan et al[29] identified the type-1 MT by mass spectrometry proteomics as the best potential protein target for staining WD in liver samples. In this study, MT immunohistochemistry (IHC) was performed on 223 liver samples from liver disease patients of different etiologies, including 20 genetically confirmed patients with WD. All cases of WD showed diffuse (>50%), moderate to strong cytoplasmic MT immunoreactivity within hepatocytes versus no observed diffuse cytoplasmic presence among samples from the different control groups. Moreover, by quantifying the degree of staining by means of a MT IHC H-score revealed that a threshold of 165 had 100% sensitivity and 100% specificity for the diagnosis of WD. This was validated by Wiethoff and collaborators[30] in another recent paper, including 69 WD liver samples and 160 control liver specimens. Again, MT positivity (defined as at least moderate staining in >50% of the hepatocytes) was seen in 81.2% of the WD samples (56/69) versus only 3.1% of other diagnosis (5/160, all cases among patients with chronic cholestatic diseases) (p < 0.01). The sensitivity, specificity, and accuracy for diagnosis of WD in this work were 81.3, 96.9, and 92.4%, respectively, and this accuracy increased to 94.9% if only naive WD patients were selected.

Both groups suggested the introduction of MT staining as a novel diagnostic approach to exclude WD whenever a liver biopsy was performed in the clinical setting ([Table 1]). However, special consideration for the interpretation of the results of MT staining should be given whenever a patient is exposed to other inductive metals, such as occurs in zinc-treated individuals,[31] as MT might be induced iatrogenically.

⁶⁴Cu PET-CT Scan

The incorporation of 64-radiolabeled copper (⁶⁴Cu) into Cp is not entirely a novel tool for the diagnosis of WD.[32] In fact, this technology was used in the past in some centers at the time in which genetic studies were either not developed enough or were not available. Basically, the index of radioactivity in blood was calculated over time after the administration of the isotope. This blood radioactivity ratio was characteristically low among WD patients compared to unaffected individuals in part because copper was defectively incorporated into Cp in WD, and in addition the liver had a high content of copper. This copper-enriched hepatic parenchyma effectively diluted any newly added copper; so, it remained in the liver and was not available for export, in particular into the bile (the main defect in WD). The radiocopper incorporation test is rarely used now as a diagnostic approach to WD, partially because of the availability of other better diagnostic tests and because of the costs and demanding certifications of isotope laboratories.

However, this theoretical scenario constituted an excellent opportunity to be visualized by radioisotopic detection of the real physiopathology of the disease: the mutated ATP7B protein, unable to excrete the excess copper into the bile, and copper accumulating (and damaging) the liver tissue. Sandahl et al[33] were able to elegantly show this process by using intravenous ⁶⁴Cu in a cohort of nine WD patients, five carriers, and eight healthy individuals, by performing positron emission tomography (PET) at different time points (1.5, 6, and 20 hours) after the radiocopper administration. By visual examination of PET images, the severely impaired hepatobiliary excretion of copper was shown in WD patients, together with the relative retention of radioactivity in the liver that persisted over time. Altogether, ⁶⁴Cu dynamics and distribution could discriminate WD patients from heterozygote and healthy subjects, therefore constituting a novel diagnostic (although complex) approach with interesting opportunities that can be exploited for drug development (to be continued in the next section).

Genetics—Novel Technologies and Clinical Scenarios

Genetic analysis has evolved significantly in the last decades. Testing for ATP7B variants has become part of the standard diagnostic approach for the disease.[1] [2] [4] [5] [6] It is particularly useful in family screening and in challenging cases where clinical and biochemical data may not yield a definitive diagnosis ([Table 1]). As more than 900 pathogenic or likely-pathogenic mutations have been identified in WD, interpretation of ATP7B mutation analysis in WD is challenging and requires expertise. The methods utilized include analysis of the 21 coding exons and intronic flanking sequences, in which exons with recurrent variants would be prioritized depending on the mutation frequency in the local population.[34]

In the years to come, an increasing number of purely genetic WD cases are expected to be diagnosed. This might occur as part of the exome-based next-generation sequencing strategies developed in regular clinical settings (e.g., in the advent of an unknown disorder) or from parents' mutational analysis on fertility treatments and periconceptional embryo's studies. These new circumstances will lead to new challenging clinical scenarios where we must decide whether anti-copper treatment is required or not, or even when/how/for how long to treat these genetic cases. We could also have to face and deal with some potential bioethical considerations. The absence of a clear genotype–phenotype correlation in WD and the absence of a single still-to-define biomarker to ensure copper monitoring within time will also play a critical role for accurate medical advice. These open questions will need to be considered and common strategies for clinical and genetic counselling will need to be established. In particular, the decision as to whether all patients diagnosed in this manner will need to undergo either liver biopsy for characterization of the phenotype (histology, copper quantitation, MT1 immunostaining) or testing, such as PET-CT using radiocopper, to define the phenotype further before committing to life-time medical therapy.

Novel Therapeutic Approaches for Treating WD

Once a patient is diagnosed with WD, there are two main types of medical therapies that can be offered depending on the clinical presentation and availability of drug on site: chelators (named D-penicillamine, DPA, or trientine, TRI) versus nonchelators (zinc salts). Whatever the chosen therapy, medication must be used every day, preferably administered when the patient is fasting, and its use is lifelong. According to current guidelines,[1] [5] first-line therapy should be DPA or TRI for patients presenting with any degree of organ involvement (either mild or severe hepatic injury at presentation and/or if there is a neuropsychiatric phenotype), whereas patients diagnosed on a screening family approach or with pure asymptomatic presentations could be considered for zinc therapies.[1] [5] Maintenance therapy with zinc salts after the initial treatment can also be considered for use to reducing the potential long-term adverse events of chelators.[6] This sequential therapeutic approach is based on the drugs' known mechanism of action: zinc reduces copper gastrointestinal absorption, whereas chelators actively promote chelation of copper within the bloodstream and increase UCE. These drugs have been used for decades for the treatment of patients with WD with a common therapeutical objective: improve clinical symptoms and blood tests, or at least avoid clinical progression of the disease.[35] However, these drugs could have significant drawbacks (lifelong use, posology or high price) and may exert significant short- and long-term safety concerns that require consideration.[2]

The aim of any therapeutic intervention is to achieve a cure for a certain disease. For obvious reasons, “cure” might be conceptually different in the context of genetic disorders. In the case of WD, classical drugs which restored health and prevented disease progression had no or very little effect on previous tissue copper deposition. Treatment aims were based on reducing or stopping copper toxicity, but not correcting the underlying pathophysiology of WD. New therapeutic strategies might change this approach, with new treatment objectives expected to follow. Whether these therapies will require restoration of normal levels of tissue copper to be considered effective is still to be determined.

What is clear is that the development of new drugs with improved safety profiles and new mechanisms of action (MoA) remains a priority for current WD research. These novel treatment strategies are summarized herein, and drugs' MoA can be graphically seen in [Fig. 1] and summarized in [Table 3].

Table 3 Drugs in the pipeline for WD

Drug	Phase of development/NCT	Mechanism of action	Comments
Trientine tetrahydrochloride (TETA4)	Approved by FDA/EMA. NCT03539952 (Phase III): completed	Multiple: decrease intestinal copper absorption and promote urinary copper excretion	Sponsor: Orphalan First trial utilizing NCC-Sp as primary endpoint, showed noninferiority of TETA4 to DPA in stable treated WD. Trial NCT05783687 (Phase IV, noninterventional real-world evidence study to describe NCC-Sp in relation to standard-of-care copper measurements) is ongoing
Methanobactin	Preclinical	Promote biliary copper excretion by the liver	Sponsor: Arbormed Clinical trials in humans are expected to begin in 2025
Genetic therapy (associated adenovirus AAV infusion)	Phase I–III NCT04884815 (active) NCT04537377 (trial suspended in 10/2024)	Promote biliary excretion in the liver by restoring ATP7B protein production in hepatocytes of WD patients	Sponsor: Ultragenyx (NCT04884815) and Vivet Therapeutics (NCT04537377). Results on safety and efficacy of Ultragenyx trial are expected soon
Bis-choline tetrathiomolybdate (TTM)	NCT04422431 (Phase II): completed NCT03403205 (Phase III): completed Extension program suspended in 04/2023	Multiple: Reduce copper absorption in the intestine and copper sequestration in blood by formation of an inert circulating tripartite complex (copper–albumin–TTM)	Sponsor: Alexion Pharmaceutics The drug development program was stopped by the sponsor in 2023 due to the inability to show biliary excretion as a primary mechanism of action or lowering of intrahepatic copper in treated patients

Abbreviations: AAV: associated adenovirus; TETA4, trientine tetrahydrochloride; TTM, tetrathiomolybdate.

Fig. 1 Copper metabolism in WD and how current and future drugs interfere with it. Note that trientine and TTM have multiple mechanisms of action. Cu, copper; Cp, ceruloplasmin; WD, Wilson disease; TTM, tetrathiomolybdate.

New Data on Current Therapies

A better understanding of the mechanism of action of the current drugs is important for aiding the best utilization of these medications. This knowledge will help us use these treatments most effectively and safely. The use of ⁶⁴Cu PET-CT scan has recently been able to directly visualize how all the current drugs work[36] [37] and how they might influence copper metabolic pathways.

Despite being equally effective for copper control,[18] [35] TRI has been shown to have a reduced cupriuretic effect compared to DPA, frequently requiring higher doses to achieve similar UCE levels. This was postulated to be related with a secondary effect of TRI reducing copper absorption from the gut,[38] although the magnitude of this effect had not been defined previously. The use of zinc salts as therapy for WD is based on its ability to induce the metallothionine peptide in intestinal cells where copper is absorbed. The zinc-induced MT binds the copper taken up from the diet, retaining it within the enterocytes which are shed into the stool over time, thus indirectly reducing copper absorption from the diet.[39] However, different salt combinations for zinc are available[40] and different clinical responses have been described since its use,[41] therefore suggesting variability between patients in the efficacy of the zinc in treating WD.

The effect on copper metabolism of two different zinc preparations given as two different oral regimens were evaluated in a cohort of 37 healthy individuals by Munk and collaborators.[36] The authors measured the change in hepatic ⁶⁴Cu radioactivity after 4 weeks of therapy with four randomized regimens (either zinc acetate or gluconate, three times a day or once daily). This work nicely demonstrated that zinc salts significantly reduced copper absorption from the intestines by around 50% of baseline values. It also reported that zinc acetate and gluconate were noninferior compounds, and that the standard-of-care dose of three times daily was better in terms of reducing copper absorption when compared to a once-daily dose. In addition, in a significant proportion of individuals (14%), zinc had no significant effect on copper absorption, thus suggesting there might be a group of pure nonresponders to this therapy in the real-world setting as well. This is in line with the original data reported by Brewer on 60 WD patients in whom a radiocopper-absorption study was performed under different zinc formulations.[42] This work also reported nonresponse to zinc therapy in approximately 10% of individuals, an important point and a reminder that all patients must be evaluated for treatment efficacy regardless of choice of therapy.

Regarding the chelators' MoA, Kirk et al[37] compared the effects of DPA and TRI on copper metabolism among a cohort of 16 healthy individuals. Participants had a PET-CT scan performed at baseline and 15 hours after receiving a first oral dose of ⁶⁴Cu. They were next randomized to receive either a treatment with TRI (trientine tetrahydrochloride) or DPA for 7 days, followed by a repeat PET-CT after a new oral ⁶⁴Cu dose. In this study, treatment with TRI but not with DPA reduced hepatic and venous ⁶⁴Cu radioactivity by ≈50%, strongly suggesting that TRI reduced intestinal uptake of ⁶⁴Cu, as previously postulated in the chelate study,[18] after the clinical observation of significantly reduced UCE levels on TETA4 therapy while NCC remained stable over time. Moreover, renal ⁶⁴Cu activity was significantly lower in participants on TRI compared to DPA, as was expected if copper absorption was reduced. These results demonstrate the reduced cupriuretic effect of TRI compared to DPA, and why some adjustments in the urinary copper targets for monitoring TRI therapy were required in the latest guidelines.[1]

By these recent efforts, the MoA of the currently approved drugs are now clearer, and should aid physicians in better understanding how to use and interpret testing results for patients on these therapies.

Novel Therapies

Trientine Tetrahydrochloride

In the last several years, a new TRI-based compound (tetrahydrochloride trientine [TETA4]) was developed with improved pharmacokinetic and pharmacodynamic profiles. This latest formulation of TETA4 was developed with the purpose of providing room-temperature stability in a tablet form, overcoming limitations of the classical dihydrochloride trientine, which had been discovered in the late 1960s.[43] TETA4 showed to be more rapidly absorbed than TRI, providing greater systemic exposure of the active compound, when evaluated in a phase I single-dose study including 24 healthy individuals ([Table 3]).[44]

More recently, TETA4 was also shown to be noninferior to DPA as an efficacious maintenance treatment for WD patients in the first phase III randomized clinical trial comparing both chelators (NCT03539952, the Chelate trial).[18] The study included 53 clinically stable WD patients treated with long-term stable doses of DPA, where half of them were randomly assigned to receive TETA4 on a mg-to-mg basis, while the others remained on DPA. The primary endpoint of the study was maintaining stable NCC levels over a 24-week follow-up time (plus a 24-week extension period), measured with the speciation method (NCC-Sp), as mentioned earlier.[17] Patients receiving TETA4 had no significant changes in NCC measurements during follow-up, there were no safety concerns for the new drug, clinical stability in patients was maintained, and UCE remained on the defined target range, although significantly reduced under TETA4, consistent with its multiple mechanism of action.[37]

Trientine is known to have a global better safety profile compared with DPA.[2] [5] [35] [45] The use of the new TETA4 formulation has been recently approved by the FDA and EMA for patients with WD. Interestingly, FDA currently allows TETA4 to be used among DPA-tolerant patients in line with the results from the Chelate study,[18] whereas in Europe, TETA4 can be prescribed if patients do not tolerate or have formal contraindications to a first-line treatment with DPA. Real-life experiences on the use of TETA4 have been shown to be safe and efficacious.[46]

Some years ago, a once-daily dosage of trientine was shown to improve compliance in a small prospective study performed among eight WD patients on maintenance therapy.[47] These patients remained clinically stable after dosage modification, while the number of missed doses was reduced during the study period. With the same objective, a recent phase I trial evaluating pharmacokinetics, safety, and tolerability of a once single dose TETA4 formulation has been performed in 26 healthy individuals, and results are expected to be released soon (NCT06128954). If positive results are obtained, the rationale for a new multicentric phase III study for WD patients treated with a once-daily formulation of TETA4 will be set. This could be an excellent strategy to improve compliance and minimize multiple-dose requirements in a disease requiring life-long medical therapy.

Methanobactin

In 2011, methanobactin (MB) was described for the first time as a group of molecules of bacterial origin with impressive copper chelating properties, and preclinical data support its use as a future potential therapeutical opportunity for WD.[48]

These small peptides produced by the methanotrophic bacterium Methylosinus trichosporium OB3b are essential for bacterial copper uptake and protection against copper toxicity.[49] Summer et al[48] successfully used this MB in the Long–Evans Cinnamon (LEC) rat. This rat is an excellent animal model for WD, as it shows impaired copper excretion, hypoceruloplasminemia, and hepatic copper toxicosis.[50] Intraperitoneal treatment with MB was given in two dosing schedules when the copper storage was expected to be at a maximum and the animals already presented with liver damage (ALT elevations). MB was shown to significantly reduce liver copper levels in the animals by chelation of copper from hepatic MT, and interestingly, the metal was shown to be excreted in bile as MB–copper complexes. This was afterward visualized using ⁶⁴Cu PET-CT technology.[51] By means of the ⁶⁴Cu radioactivity measurement, the authors were able to show how MB (now ARBM101) depleted WD hepatic copper from the rats at a dose-dependent manner. Most importantly, this depletion occurred via fecal (thus biliary) excretion, therefore reversing the effects of the original dysfunction of the ATP7B protein by restoring the normal biliary pathway as a route for additional copper elimination. Hepatic copper levels decreased in MB-treated animals to physiological levels within a few days of treatment, but accumulated again with time, leading to recurrent liver injury. A repeat treatment was again successful in restoring liver tests to normal. Thus, the intermittent administration of therapy was shown to be safe and efficacious in these animals and opens the door to a future of new paradigm in WD therapy where individual treatment and “personalized doses” may arise.

ARBM101 is now part of the pipeline of a pharmaceutical company (Arbormed, https://www.arbormed.com/en/). Its clinical development program for humans is expected to be launched soon.

Gene Therapy

WD, as a monogenic-affecting disease, is an a priori excellent candidate for gene therapy. Gene therapy aims to correct the original defect in native cells by transfecting a corrected transgene capable of integrating and produce a corrected functional protein.

The early proof-of-principle for gene therapy in WD came from hepatocyte transplantation assays using Long-Evans Cinnamon (LEC) rat model of WD,[52] showing that a partial restoration of cells could be enough for achieving significant changes in copper metabolism. Indeed, when WD animals were transplanted with normal hepatocytes from healthy rats at early stages of the disease, liver repopulation was shown to occur in most of the animals (in 70% there was detection of ATP7B mRNA in hepatic tissue). In these successfully transplanted animals, copper homeostasis could be restored, liver histology was improved, and liver disease was partially prevented when compared to untreated LEC rats. Some years later, the use of first-generation viral vectors transfecting the ATP7B gene into WD rodent models showed short and transient correction of Cu excretion and adequate incorporation of copper into Cp.[53] In 2016, Murillo et al[54] were able to provide a long-term restitution of copper metabolism in WD mice and rats by using adeno-associated serotype 8 vector (AAV8), a known non-replicative but hepatotropic virus containing DNA encoding the human ATP7B gene. More importantly, as the gene size was too large and compromised the vector's packaging and delivery capacity, the authors generated a new ATP7B construct by deleting four out of the six metal-binding domains (the final construct called “ATP7B minigene”).[55] This new engineered version of the gene gave rise to a miniATP7B protein, which was completely functional and corrective of the original copper defect in the animal models. Moreover, this shorter gene was more easily packaged and transported by the AAV8 vectors, and the mini-ATP7B protein could be produced at high titers in transfected cells. WD rodents were shown to be safely treated, and copper metabolism restored long-term, regardless of their sex or stage of disease. Again, PET evaluation allowed investigators to directly visualize how ⁶⁴Cu biodistribution and excretion occurred in the animals.[56] Copper radioactivity was measured at different time points after therapy, showing a significant reduction of ⁶⁴Cu hepatic accumulation, a restoration of the biliary excretion by means of its high detection in feces, and the correction of blood physiological pharmacokinetics. All these preclinical data confirmed the potential for efficacy of gene therapy and supported the initiation of phase I trials in patients with WD.

At this moment, two pharmaceutical companies have ongoing clinical trials with AAV-ATP7B gene therapy for WD patients. Vivet Therapeutics (https://www.vivet-therapeutics.com) uses the original AAV8-ATP7B serotype (formal drug: VTX-801) and has presented interim safety results from the first two patients included in their trial (NCT04537377, Gateway).[57] Unfortunately, the program has been prematurely discontinued due to insufficient therapeutic effects observed in the recruited patients treated with the initial and second dose escalation. Ultragenyx (https://www.ultragenyx.com/) started a clinical trial for gene therapy for WD about the same time as the above study (NCT04884815, Cyprus2 + ). They utilized their version of the ATP7B minigene packaged in AAV-9 and are actively enrolling patients into their dose escalation study. Currently, we await release and publication of their trial results.

Bis-choline Tetrathiomolybdate

This drug appeared some years ago as a promising therapy for WD.[58] The bis-choline tetrathiomolybdate was a stable oral copper–protein-binding molecule derived from the ammonium tetrathiomolybdate (TTM) formulation, which had been used as a rescue therapy for very severe neurological presentations of WD.[59] [60] This oral bis-choline drug was given as a once-daily dose and had a favorable profile when evaluated in phase II and III multicentric international studies including WD patients.[58] [61] It was shown to lower bioavailable copper levels over time, improving disability and neurological status, while maintaining stable liver function. Its favorable posology was a potential additional benefit for patients. In previous animal models with WD, TTM had been shown to promote copper mobilization from the liver cells, by increasing its excretion through the bile.[62] Nevertheless, this same mechanism of action was not seen in humans. Sixteen healthy individuals and 4 WD patients were recruited for the performance of an absorption and excretion study, by using oral and intravenous TTM, respectively, and being scanned with the PET-CT for ⁶⁴Cu radioactivity afterward.[63] This study failed to show any increase of biliary excretion of ⁶⁴Cu in WD patients, therefore excluding the first mechanistic hypothesis supporting its development. However, it was positively shown that oral TTM inhibited most of the intestinal copper intake and retained ⁶⁴Cu at the blood, therefore limiting the exposure of organs to copper toxicity. Unfortunately, the development of the drug was stopped by the sponsor (Alexion, https://alexion.com/) in April 2023. Results of the phase II and III trials should be available very soon. Despite the halting of the trial, there remains some interest in the development of TTM as a clinical therapy for WD.

Summary and Conclusions

New diagnostic approaches for WD, led by NCC quantification, may soon be implemented in clinical practice and could change the diagnostic approach for this disorder. More accurate diagnostic approaches would ensure earlier diagnosis, therefore limiting the hepatic and extrahepatic burden of the disease and allowing early access to therapy. Additional genetic WD diagnosis is expected to occur more frequently at preclinical stages, as we expand the use of next-generation sequencing and gene periconceptional studies. These new genetic WD cases will lead us to new clinical scenarios in terms of surveillance and counselling, further stimulating clinical research to identify accurate biomarkers for WD diagnosis and monitoring. Moreover, new therapeutic strategies that restore some aspects of the underlying defect in biliary copper excretion may overcome the limitations of current drugs and help aid future therapy to achieve a cure. In the meanwhile, better tools for disease monitoring and a better knowledge of the mechanism of action of the currently available drugs will help clinicians to individualize and optimize treatment for their patients.

Conflict of Interest

None declared.

Acknowledgments

We would like to thank the Wilson Disease Association for their ongoing support of research and patient care, and NIH grant R01 GR124795 to M.L.S. for the support of research on Wilson disease. Z.M. would like to thank Hospital Clínic Barcelona for her sabbatical support, AEEH (Asociación Española para el Estudio del Hígado) for the Joan Rodès 2024 grant and CIBERehd.

Disclosures

• Z.M.: Speaker fees from Gilead and Orphalan; consultancy fees from Alexion, DeepGenomics, and Orphalan; grants from Gilead.

• M.L.S.: Grants from Vivet Therapeutics, Orphalan, Alexion, Wilson Disease Association, National Institute of Health; consultant to DepYmed, Arbomed, Orphalan.

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Publication History

Accepted Manuscript online:
04 November 2024

Article published online:
26 November 2024