Wilson's disease in Sardinian population: The experience of a pediatric referral center.

BACKGROUND AND OBJECTIVES: Wilson's disease (WD) in children and adolescents is predominantly asymptomatic or oligo-symptomatic. The symptoms are nonspecific and difficult to distinguish from other hepatic or neuropsychiatric disorders. In this study, we present the experience of a pediatric referral center for WD diagnosis and treatment. PATIENTS AND METHODS: We retrospectively analyzed clinical and laboratory data from 99 patients with WD of Sardinian origin, including physical examination, laboratory biochemical testing, liver biopsy, and genetic analysis. RESULTS: Patients were prevalently oligo-symptomatic or asymptomatic. The median age of diagnosis was 8.78 years. Ceruloplasmin values were lower than normal values in all analyzed patients. Twenty-four-hour urinary copper levels were higher than 40 µg/24-h in 92/96 patients. In all analyzed patients with the exception of one, liver copper was higher than 250 µg/g of dry weight but all had >75 µg/g of dry weight. Statistical analysis showed correlation between the age at diagnosis, serum copper, and 24-h urinary copper. Correlation was also found between serum copper and 24-h urinary copper. Molecular analysis of ATP7B gene allowed complete characterization in all the analyzed patients. CONCLUSION: A high index of clinical suspicion and biochemical tests including liver tests, serum ceruloplasmin, and basal 24-h urinary copper excretion and genotype determination are key to WD diagnosis. The long experience that a referral center for WD possesses is an important factor in making WD diagnosis a more accurate process. Studies in animal models on WD could be used as a guide to further investigate the molecular mechanisms that regulate copper metabolism and influence the natural history of WD.

Profile of plasma microRNAs as a potential biomarker of Wilson's disease.

BACKGROUND: Wilson's disease (WD) is a rare condition resulting from autosomal recessive mutations in ATP7B, a copper transporter, manifesting with hepatic, neurological, and psychiatric symptoms. Timely diagnosis and appropriate treatment yield a positive prognosis, while delayed identification and/or insufficient therapy lead to a poor outcome. Our aim was to establish a prognostic method for WD by characterising biomarkers based on circulating microRNAs. METHODS: We conducted investigations across three cohorts: discovery, validation (comprising unrelated patients), and follow-up (revisiting the discovery cohort 3 years later). All groups were compared to age- and gender-matched controls. Plasma microRNAs were analysed via RNA sequencing in the discovery cohort and subsequently validated using quantitative PCR in all three cohorts. To assess disease progression, we examined the microRNA profile in Atp7b-/- mice, analysing serum samples from 6 to 44 weeks of age and liver samples at three time points: 20, 30, and 40 weeks of age. RESULTS: In patients, elevated levels of the signature microRNAs (miR-122-5p, miR-192-5p, and miR-885-5p) correlated with serum activities of aspartate transaminase, alanine aminotransferase and gamma-glutamyl transferase. In Atp7b-/- mice, levels of miR-122-5p and miR-192-5p (miR-885-5p lacking a murine orthologue) increased from 12 weeks of age in serum, while exhibiting fluctuations in the liver, possibly attributable to hepatocyte regenerative capacity post-injury and the release of hepatic microRNAs into the bloodstream. CONCLUSIONS: The upregulation of the signature miR-122-5p, miR-192-5p, and miR-885-5p in patients and their correlation with liver disease progression in WD mice support their potential as biomarkers of WD.

Clinical characteristics and prognosis of early diagnosed Wilson's disease: A large cohort study.

BACKGROUND AND AIMS: Few studies have focused on the outcomes of Wilson's disease (WD) diagnosed before age of 5 years. This study aimed to summarize the clinical features of early diagnosed WD and analyse treatment outcomes and the risk factors associated with treatment failure. METHODS: A total of 139 children confirmed with WD before 5 years were enrolled in this study. Only patients with follow-up over 1 year were analysed with Kaplan-Meier survival analysis. The composite outcomes included death, progression to liver failure or acute hepatitis, development of renal or neurological symptoms and persistent elevation of alanine aminotransferase (ALT). The treatment failure was defined as occurrence of at least one of above outcomes. RESULTS: Among 139 WD patients at diagnosis, two (1.4%) WD patients presented with symptomatic liver disease, whereas 137 (98.6%) were phenotypically asymptomatic, including 135 with elevated ALT and 2 with normal liver function. Median serum ceruloplasmin (Cp) was 3.1 mg/dL, and urinary copper excretion was 87.4 µg/24-h. There were 71 variants identified in the the copper-transporting ATPase beta gene, and 29 were loss of function (LOF). 51 patients with LOF variant were younger at diagnosis and had lower Cp than 88 patients without LOF. Among 93 patients with over 1 year of follow-up, 19 (20.4%) received zinc monotherapy, and 74 (79.6%) received a zinc/D-penicillamine combination therapy. 14 (15.1%) patients underwent treatment failure, and its occurrence was associated with poor compliance (p < .01). CONCLUSIONS: Cp is a reliable biomarker for early diagnosis, and zinc monotherapy is an effective treatment for WD during early childhood. Good treatment compliance is critical to achieve a favourable outcome.

Analysis of risk factors for fatty liver disease in children with Wilson's disease.

BACKGROUND AND AIMS: Many children with Wilson's disease are complicated with dyslipidemia. The aim of this study was to investigate the risk factors for the development of fatty liver disease (FLD) in children with Wilson's disease. METHODS: We evaluated sex, age, weight, the disease course, treatment course, clinical classification, alanine transaminase (ALT), aspartate transaminase, γ-glutamyl transpeptidase, total biliary acid, triglyceride, total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, homocysteine, uric acid, fibrinogen (FBG), creatinine, procollagen III N-terminal propeptide, laminin, hyaluronic acid, type IV collagen, and performed receiver operating characteristic curve analysis to investigate the forecast value of individual biochemical predictors and combined predictive indicators to evaluate FLD in Wilson's disease. RESULTS: The multivariate logistic regression analysis revealed that ALT [odds ratio (OR), 1.011; 95% confidence interval (CI), 1.004-1.02; P  = 0.006], uric acid (OR, 1.01; 95% CI, 1.002-1.018; P  = 0.017), FBG (OR, 3.668; 95% CI, 1.145-13.71; P  = 0.037), creatinine (OR, 0.872; 95% CI, 0.81-0.925; P  < 0.001), and laminin (OR, 1.01; 95% CI, 1.002-1.018; P  = 0.017) acted as independent risk factors in Wilson's disease complicated with FLD. The receiver operating characteristic curves for combined predictive indicators demonstrated an area under the curve values of 0.872, which was found to be a significant predictors for FLD in Wilson's disease. CONCLUSIONS: We screened out the most important risk factors, namely ALT, uric acid, creatinine, FBG, and laminin for Wilson's disease complicated with FLD. The joint prediction achieved is crucial for identifying children with Wilson's disease complicated with FLD.

Selective and sensitive CQD-based sensing platform for Cu2+ detection in Wilson's disease.

Excessive Cu2+ intake can cause neurological disorders (e.g. Wilson's disease) and adversely affect the gastrointestinal, liver, and kidney organs. The presence of Cu2+ is strongly linked to the emergence and progression of Wilson's disease (WD), and accurately measuring the amount of copper is a crucial step in diagnosing WD at an early stage in a clinical setting. In this work, CQDs were fabricated through a facile technique as a novel fluorescence-based sensing platform for detecting Cu(II) in aqueous solutions, and in the serum samples of healthy and affected individuals by WD. The CQDs interact with Cu(II) ions to produce Turn-on and Turn-off states at nano-molar and micro-molar levels, respectively, with LODs of 0.001 µM and 1 µM. In fact, the Cu2+ ions can act like a bridge between two CQDs by which the charge and electron transfer between the CQDs may increase, possibly can have significant effects on the spectroscopic features of the CQDs. To the best of our knowledge, this is the first reported research that can detect Cu(II) at low levels using two different complexation states, with promising results in testing serum. The potential of the sensor to detect Cu(II) was tested on serum samples from healthy and affected individuals by WD, and compared to results obtained by ICP-OES. Astonishingly, the results showed an excellent correlation between the measured Cu(II) levels using the proposed technique and ICP-OES, indicating the high potential of the fluorimetric CQD-based probe for Cu(II) detection. The accuracy, sensitivity, selectivity, high precision, accuracy, and applicability of the probe toward Cu(II) ions make it a potential diagnostic tool for Wilson's disease in a clinical setting.

ATP7B variant spectrum in a French pediatric Wilson disease cohort.

BACKGROUND/AIM: The spectrum of ATP7B variants varies significantly according to geographic distribution, and there is insufficient data on the variants observed in the French population. METHODS: Clinical data of 113 children included in the French WD national registry were gathered from March 01, 1995 to July 01, 2020. Data included epidemiological, clinical, laboratory, genetics. RESULTS: Diagnosis was made at a mean age of 11.0 ± 4.1 years (range 1-18 years). At diagnosis, 91 patients (79.8 %) had hepatic manifestations, 18 (15.8 %) presented neurological manifestations, and 4 patients (3.5 %) were asymptomatic. Only 29 patients (25 %) were homozygous for a variant. We have found a total of 102 different variants including 14 novel variants. Recurrent variant p.His1069Gln was the most prevalent, n = 31 alleles (14,2%), with only seven homozygous; in contrast 55% of variants are identified in only one family. 45% were truncating variants. In respect of mutated exon, the three most prevalent were exon 14 (16.5%), exon 8 (13.8%), and exon 3 (11.5%). When considering patients with two Nonsense / Frameshift variants as a group and those with two Missense variants, we found significantly lower ceruloplasmin for the former: 2.8 ± 0.7 mg/dl vs 8.4 ± 5mg/dl (p<0.05). CONCLUSION: p.His1069Gln is the most frequent variant (14,2%) and exons 14, 8, and 2 of the ATP7B gene account for 41.7% of total variants. However, there is significant heterogeneity in the French population concerning the other ATP7B variants. Nonsense / Frameshift variants were associated with lower ceruloplasmin levels.

Identification of mutations in the ATP7B gene in 14 Wilson disease children: Case series.

INTRODUCTION: Wilson Disease (WD) is an autosomal recessive inherited metabolic disease caused by mutations in the ATPase copper transporting beta gene (ATP7B). WD can cause fatal neurological and hepatic disorders if not diagnosed and treated. OBJECTIVE: To analyze the disease-causing mutations of 14 Chinese WD children, 11 of whom are diagnosed with hepatic disorders, 2 with neurological degeneration and 1 with both hepatic and neurological disorders. METHODS: All ATP7B coding regions were analyzed by Sanger sequencing. Single nucleotide polymorphisms (SNPs) functional impacts were assessed by combining the results of four bioinformatics tools (Poly-phen-2, SIFT, PANTHER-PSEP and PhD-SNPs) in an index that reflects the combined probability (cPdel) of an amino acid change to be deleterious to the protein function. RESULTS: Two novel variants involved in WD development, c.1448_1455del (p.Arg483SerfsX19) and c.4144G>T (p.Glu1382Stop), and 11 previously reported mutations were detected. Both new variants result in shortened and dysfunctional ATP7B proteins. cPdel score suggests that SNPs may be deleterious to the ATP7B functionality. CONCLUSIONS: This study enriches the library of the ATP7B mutations that lead to WD and can be used as a basis for genetic counseling, for WD prevention and clinical and prenatal diagnosis. Those SNPs that are believed to be harmless to ATP7B protein may be involved in the pathogenesis of WD.

Reproductive function of long-term treated patients with hepatic onset of Wilson's disease: a prospective study.

RESEARCH QUESTION: Wilson's disease (WD) is a disorder of copper metabolism that can cause hormonal alterations. The impact of WD and its therapies on fertility is not well defined. The aim of this study was to evaluate ovarian reserve and sperm parameters in long-term treated WD patients with hepatic onset. DESIGN: WD patients with hepatic onset treated for at least 5 years were compared with healthy controls. Men underwent spermiogram and sperm DNA fragmentation (SDF) analysis. Women were tested for serum FSH, anti-Müllerian hormone (AMH) and sonographic antral follicle count (AFC) in the early follicular phase. Ovulation was monitored with ultrasound and progesterone serum concentrations in the luteal phase. RESULTS: The WD group included 26 patients (12 males), the control group 19 subjects (9 males). All patients apart from four (one male) were responders to WD treatment. Sperm count and morphology were comparable between cases and controls. Sperm motility (total and after 1 h) was significantly lower in cases (44.78 ± 21.65%; 47.85 ± 21.52%) than controls (61.88 ± 11.03; 69.44 ± 11.02%, P = 0.03 and 0.01, respectively). The only non-responder had severe oligo-astheno-teratozoospermia. SDF values were normal in cases and controls. AMH, AFC and FSH did not differ between cases and controls. LH was significantly lower in cases (3.36 ± 1.65 mIU/ml) than controls (6.25 ± 1.03 mIU/ml, P < 0.0001). A non-responder woman who developed neurological signs had a 7-year history of infertility. CONCLUSIONS: WD patients with hepatic onset, diagnosed early and treated, have no impairment in fertility potential even if males show reduced sperm motility and females lower LH values. Only patients with poor disease control have some evidence of impaired fertility.

Total and Exchangeable Copper Assay Using Inductively Coupled Plasma Mass Spectrometry and Establishment of a Pediatric Reference Interval.

CONTEXT.­: Recently, an exchangeable copper (CuEXC) assay has been suggested as a robust and feasible diagnostic tool for Wilson disease (WD). Although WD is a disorder that requires lifelong treatment and monitoring, few data are currently available regarding the status of copper levels in children. OBJECTIVE.­: To evaluate the performance of copper assays and establish a reference interval for total copper and CuEXC in the pediatric population. DESIGN.­: Serum samples from children aged 1-5 (n = 122), 6-12 (n = 125), and 13-18 years (n = 120) were analyzed. Total copper and CuEXC concentrations were directly measured using inductively coupled plasma mass spectrometry, and relative CuEXC levels were calculated. Total copper reference intervals, CuEXC levels, and relative CuEXC levels were determined based on the 2.5th and 97.5th percentiles of the data with 90% confidence intervals. RESULTS.­: There were significant differences in the median concentrations of total copper and relative CuEXC among the age groups. Reference intervals determined for total copper were 82 to 167, 75 to 139, and 64 to 133 µg/dL for children aged 1 to 5, 6 to 12, and 13 to 18 years, respectively. The reference intervals for CuEXC were 4.29 to 9.79, 4.02 to 9.09, and 3.55 to 8.25 µg/dL for children aged 1 to 5, 6 to 12, and 13 to 18 years, respectively. Among 11 patients with suspected WD, relative CuEXC values were elevated in all 3 diagnosed with WD. CONCLUSIONS.­: The pediatric reference intervals derived in this study are expected to be useful for the diagnosis, differential diagnosis, treatment, and monitoring of pediatric patients with WD.

Wilson disease: revision of diagnostic criteria in a clinical series with great genetic homogeneity.

BACKGROUND: Wilson disease is an autosomal recessive disorder of copper metabolism caused by mutations in the ATP7B gene. An early diagnosis is crucial to prevent evolution of the disease, as implantation of early therapeutic measures fully prevents its symptoms. As population genetics data predict a higher than initially expected prevalence, it was important to define the basic diagnostic tools to approach population screening. METHODS: A highly genetically homogeneous cohort of 70 patients, belonging to 50 unrelated families, has been selected as a framework to analyze all their clinical, biochemical and genetic characteristics, to define the disease in our population, with an estimated prevalence of 1 in 12,369, and determine the most useful features that reach diagnostic value. RESULTS: Serum ceruloplasmin below 11.5 mg/dL and cupremia below 60 µg/mL, were the best analytical predictors of the disease in asymptomatic individuals, while cupruria or hepatic copper determination were less powerful. Genetic analysis reached a conclusive diagnosis in all 65 patients available for complete testing. Of them, 48 were carriers of at least one p.Leu708Pro mutant allele, with 24 homozygotes. Nine patients carried a promoter deletion mutation, revealing that extended sequencing beyond the ATP7B gene-coding region is essential. All mutations caused hepatic damage since early ages, increasing its severity as diagnosis was delayed, and neurological symptoms appear. CONCLUSION: Serum ceruloplasmin determination followed by genetic screening would reduce costs and favor the prioritization of non-invasive procedures to reach a definitive diagnosis, even for asymptomatic cases.

The blood copper isotopic composition is a prognostic indicator of the hepatic injury in Wilson disease.

Wilson disease (WD) is an autosomal recessive disorder of copper (Cu) metabolism. The gene responsible for WD, ATP7B, is involved in the cellular transport of Cu, and mutations in the ATP7B gene induce accumulation of Cu in the liver and ultimately in the brain. In a pilot study, the natural variations of copper stable isotope ratios (65Cu/63Cu) in the serum of WD patients have been shown to differ from that of healthy controls. In the present study, we challenged these first results by measuring the 65Cu/63Cu ratios in the blood of treated (n = 25), naïve patients (n = 11) and age matched healthy controls (n = 75). The results show that naïve patients and healthy controls exhibit undistinguishable 65Cu/63Cu ratios, implying that the Cu isotopic ratio cannot serve as a reliable diagnostic biomarker. The type of treatment (d-penicillamine vs. triethylenetetramine) does not affect the 65Cu/63Cu ratios in WD patients, which remain constant regardless of the type and duration of the treatment. In addition, the 65Cu/63Cu ratios do not vary in naïve patients after the onset of the treatment. However, the 65Cu/63Cu ratios decrease with the degree of liver fibrosis and the gradient of the phenotypic presentation, i.e. presymptomatic, hepatic and neurologic. To get insights into the mechanisms at work, we study the effects of the progress of the WD on the organism by measuring the Cu concentrations and the 65Cu/63Cu ratios in the liver, feces and plasma of 12 and 45 week old Atp7b-/- mice. The evolution of the 65Cu/63Cu ratios is marked by a decrease in all tissues. The results show that 63Cu accumulates in the liver preferentially to 65Cu due to the preferential cellular entry of 63Cu and the impairment of the 63Cu exit by ceruloplasmin. The hepatic accumulation of monovalent 63Cu+ is likely to fuel the production of free radicals, which is potentially an explanation of the pathogenicity of WD. Altogether, the results suggest that the blood 65Cu/63Cu ratio recapitulates WD progression and is a potential prognostic biomarker of WD.

Assay in serum of exchangeable copper and total copper using inductively coupled plasma mass spectrometry (ICP-MS): development, optimisation and evaluation of a routine procedure.

The assay in serum of non-caeruloplasmin copper, as exchangeable copper after complexation with EDTA (ExCu) and total copper has been evaluated and compared in patients with varying c-reactive protein(CRP). Measurement of ExCu and total copper, range 0.2-47.2 µmol/L, was developed using ICP-MS. The chelating agents EDTA and TEPA were compared over 0.0-10 g/L after incubation with serum for 60 mins followed by ultrafiltration with Amicon 10 kDa filter. The assay for ExCu was optimised with EDTA 3 g/L (8.1 mmol/L) maintained at pH 7.0-8.0 before ultrafiltration. TEPA was not as selective in chelation of copper. Patients n = 82 were studied in relation to changes in inflammatory marker CRP and a group of patients n = 37 with normal CRP. The ExCu assay gave excellent recoveries (94-102 % but poor recovery for free uncomplexed copper), good repeatability, limit of quantitation 0.19 µmol/l with a provisional reference range 0.48 to 1.63 µmol/L (n = 37 patients). The range for relative exchangeable copper (exchangeable copper divided by total serum copper) was 2.49 to 9.96 %. ExCu was elevated in conditions with increased CRP greater than 100 mg/L suggesting an effect of inflammation on the free copper fraction. A reliable and reproducible assay for ExCu and total copper has been developed. The upregulated inflammatory state increases the ExCu suggesting excess free copper.

LC-ICP-MS method for the determination of "extractable copper" in serum.

Copper is an essential element for biological functions within humans and animals. There are several known diseases associated with Cu deficiency or overload, such as Menkes disease and Wilson disease, respectively. A common clinical method for determining extractable Cu levels in serum, which is thought to be potentially dangerous if in excess, is to subtract the value of tightly incorporated Cu in ceruloplasmin from total serum Cu. In this work, an automated sample preparation and liquid chromatography (LC) system was combined with inductively coupled plasma-mass spectrometry (ICP-MS) to determine bound Cu and extractable Cu in serum. This LC-ICP-MS method took 250 s for sample preparation and analysis, followed by a column recondition/system reset, thus, a 6 minute sample-to-sample time including sample preparation. The method was validated using serum collected from either control (Atp7b+/-) or Wilson disease rats (Atp7b-/-). The extractable Cu was found to be 4.0 ± 2.3 µM Cu in healthy control rats, but 2.1 ± 0.6 µM Cu in healthy Wilson rats, and 27 ± 16 µM Cu in diseased Wilson rats, respectively. In addition, the extractable Cu/bound Cu ratio was found to be 6.4 ± 3.5%, 38 ± 29%, and 34 ± 22%, respectively. These results suggest that the developed method could be of diagnostic value for Wilson disease, and possibly other copper related diseases.

A study of susceptibility-weighted imaging in patients with Wilson disease during the treatment of metal chelator.

OBJECTIVE: A randomized-controlled trial comparing study of the changes in brain sensitive-weighted imaging (SWI) of Wilson disease (WD) patients during the treatment with metal chelator was done. METHODS: 100 untreated WD patients (80 cases of cerebral type, 20 cases of hepatic type, age 20.13 ± 9.12 years old) and 20 normal controls were selected. Neurological symptoms were scored using the modified Young scale. Liver function tests and copper indices were collected. All study objects received SWI test of the brain. The values of corrected phase (CP) were calculated on SWI. Cerebral-type WD patients were treated with D-penicillamine (DPA) (group 1) or Dimercaptopropane Sulfonate (DMPS) + Dimercaptosuccinic Acid (DMSA) (group 2). Hepatic-type WD patients were treated with DPA (group 3). All patients received annual neurological symptom score, liver function, copper indices, and SWI examination. RESULTS: At the first year of treatment, score of the modified Young scale in group 2 was lower than that in group 1 (P = 0.023) and lower than that before treatment (P = 0.040). After 2 years of treatment, the score of the modified Young scale in group 1 was lower than that before treatment (P = 0.012). At the second year after treatment, the urinary copper in group 2 was higher than that in group 1 (P = 0.014). Urinary copper was maintained at 200 µg/day in group 1 and 300 µg/day in group 2 after 3 years of treatment. At the first year of treatment, serum copper in group 1 was lower than that in group 2 (P = 0.032). At the first year of treatment, CP values of the pallidum and substantia nigra in group 2 were higher than those in group 1 (P = 0.026, 0.040). At the second year of treatment, CP value of substantia nigra in group 2 was higher than that in group 1 (P = 0.037). After 3 years of treatment, there was no difference in CP values between WD patients and normal controls. CONCLUSIONS: Therapy with DMPS and DMSA improves neurological symptoms of WD patients more quickly and leads to less aggravation, compared with therapy with DPA. The metal content in the brain of WD patients was at a low level after 3 years of treatment. DMPS and DMSA can remove metal from brain tissue faster than DPA.

The co-occurrence of Wilson disease and X-linked agammaglobulinemia in one family highlights the promising diagnostic potential of proteolytic analysis.

BACKGROUND: We report the first case of a family with co-occurrence of Wilson disease (WD), an autosomal recessive disorder of copper metabolism, and X-linked agammaglobulinemia (XLA), a primary immunodeficiency disorder (PIDD) that features marked reduction in circulating B lymphocytes and serum immunoglobulins. METHODS AND RESULTS: Through utilization of a multiplexed biomarker peptide quantification method known as the immuno-SRM assay, we were able to simultaneously and independently identify which family members are affected with WD and which are affected with XLA using dried blood spots (DBS). CONCLUSION: Being able to delineate multiple diagnoses using proteolytic analysis from a single DBS provides support for implementation of this methodology for clinical diagnostic use as well as large-scale population screening, such as newborn screening (NBS). This could allow for early identification and treatment of affected individuals with WD or XLA, which have been shown to reduce morbidity and decrease mortality in these two populations.

Biomedical copper speciation in relation to Wilson's disease using strong anion exchange chromatography coupled to triple quadrupole inductively coupled plasma mass spectrometry.

Biomedical analytical methods often rely on indirect measurements, such as immunoassays, which can lack effective metrological traceability. In the nephelometric determination of ceruloplasmin (Cp), an important protein whose circulating level is altered in Wilson's disease (WD), the anti-Cp antibody used is not specific for the biologically active holoprotein so the assay can overestimate the concentration of Cp due to the presence of the apoprotein. By providing quantitation using elemental standards, the use of strong anion exchange chromatography (SAX) coupled to triple quadrupole inductively coupled plasma mass spectrometry (ICP-MS-MS) can overcome the drawbacks of methods for the measurement of metalloproteins reliant on immunoassays. In the current study, a SAX-ICP-MS-MS method for Cp quantification was designed and tested in samples of blood serum of WD patients and healthy controls. Using standards based on a copper-EDTA complex for calibration, the method provides relatively simple quantification of Cp with the limit of detection of 0.1 µg L-1 (limit of quantification 0.4 µg L-1). The method was also used to investigate the copper species separated by using a 30 kDa cut-off ultrafiltration device. The so-called "exchangeable" copper fraction is considered as an alternative clinical biomarker of WD. Using the designed speciation approach, it was shown that the ultrafiltration method can overestimate the "exchangeable" copper fraction due to a removal of copper from Cp. This was confirmed by comparing the enzymatic activity of the fractions. Thus, the specificity of the "exchangeable" copper test can be ensured only under strict maintenance of ultrafiltration conditions.

Comprehensive and comparative exploration of the Atp7b-/- mouse plasma proteome.

Wilson's disease (WD), a rare genetic disease caused by mutations in the ATP7B gene, is associated with altered expression and/or function of the copper-transporting ATP7B protein, leading to massive toxic accumulation of copper in the liver and brain. The Atp7b-/- mouse, a genetic and phenotypic model of WD, was developed to provide new insights into the pathogenic mechanisms of WD. Many plasma proteins are secreted by the liver, and impairment of liver function can trigger changes to the plasma proteome. High standard proteomics workflows can identify such changes. Here, we explored the plasma proteome of the Atp7b-/- mouse using a mass spectrometry (MS)-based proteomics workflow combining unbiased discovery analysis followed by targeted quantification. Among the 367 unique plasma proteins identified, 7 proteins were confirmed as differentially abundant between Atp7b-/- mice and wild-type littermates, and were directly linked to WD pathophysiology (regeneration of liver parenchyma, plasma iron depletion, etc.). We then adapted our targeted proteomics assay to quantify human orthologues of these proteins in plasma from copper-chelator-treated WD patients. The plasma proteome changes observed in the Atp7b-/- mouse were not confirmed in these samples, except for alpha-1 antichymotrypsin, levels of which were decreased in WD patients compared to healthy individuals. Plasma ceruloplasmin was investigated in both the Atp7b-/- mouse model and human patients; it was significantly decreased in the human form of WD only. In conclusion, MS-based proteomics is a method of choice to identify proteome changes in murine models of disrupted metal homeostasis, and allows their validation in human cohorts.

Adjunctive Antioxidant Therapy in Neurologic Wilson's Disease Improves the Outcomes.

Oxidative stress has been reported in Wilson's disease with neurological manifestation (WDNM), but there is a paucity of studies on the role of adjunctive antioxidant therapy. This study aims to evaluate the efficacy of adjunctive vitamin C and E treatment in reducing oxidative stress and improving clinical outcomes. Forty-nine patients with WDNM were included and their clinical details were noted. Glutathione (GSH), total antioxidant capacity (TAC), and malondialdehyde (MDA) were measured using spectrophotometer at baseline and follow-up. All patients received zinc with or without chelating therapy, and 32 of them prescribed vitamin C (500 mg/day) and E (400 mg/day). Clinical outcomes at 6, 12, and 24 months were categorized as improved, static, or worsened based on improvement in Burke-Fahn-Marsden (BFM) score (>10%) and/or severity grade (> 1). Baseline parameters were similar between two groups; except BFM score was higher in the antioxidant group. At follow-up, the antioxidant group had higher GSH, TAC, and lower MDA levels compared with baseline. Patients on antioxidant treatment experienced improvement more frequently at 6 (53.1% vs. 29.4%), 12 (62.5% vs. 29.4%), and 24 months (68.8% vs. 35.3%) compared with those without antioxidant treatment. In WDNM, adjunctive vitamin C and E treatment reduce oxidative stress and improve clinical outcome.

Is it possible to diagnose fulminant Wilson's disease with simple laboratory tests?

BACKGROUND: Wilson's disease is a rare cause of acute liver failure and is highly fatal without liver transplantation. Fast and accurate diagnostic methods are needed for fulminant Wilson's disease (FWD). In this study, we aimed to develop an early, simple and accurate diagnostic method to differentiate FWD from nonwilsonian acute liver failure (NWALF) causes using routine biochemical data. METHODS: The medical records of 24 paediatric FWD and 120 paediatric NWALF cases diagnosed at the Department of Pediatric Gastroenterology, Hepatology, and Nutrition between January 2007 and February 2017 were retrospectively reviewed. RESULTS: Using receiver operator characteristics curve (ROC) analysis, we have determined the best cut-off point for laboratory findings in FWD. Patients meeting these cut-off points were assigned one point and others were assigned zero point. We then formed a new variable consisting of the combination of 14 variables and performed a new ROC analysis. We obtained a cut-off point of ≥4.5 for FWD. The diagnostic performance of the score was characterized by a sensitivity of 0.889, a specificity of 0.879 (P < .001). A scoring system based only on aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase, AST/ALT ratio, uric acid and haemoglobin had a best cut-off point of ≥2.5 for FWD, which had a sensitivity of 0.875, a specificity of 0.867 (P < .001). CONCLUSIONS: Our study demonstrated that biochemical markers offer almost as reliable, fast and accurate diagnosis of FWD as offered by ceruloplasmin and 24-hour urinary copper.