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INTRODUÇÃO: A doença de Wilson é um distúrbio autossômico recessivo, decorrente de mutações no gene ATP7B, resultando em acúmulo tóxico de cobre no organismo. Devido a sua raridade, séries com grande casuística e longo seguimento são escassas na literatura. Nesse estudo relatamos a experiência do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (HC-FMUSP) com os pacientes diagnosticados com doença de Wilson entre o período de 1946 a 2010. MÉTODOS: Realizamos análise retrospectiva de 262 casos, descrevendo as formas de apresentação clínica, os achados dos exames diagnósticos, os padrões de resposta terapêutica e a evolução clínica da doença. RESULTADOS: A idade média do início dos sintomas foi 17,4 anos (7-49 anos). Os pacientes foram acompanhados em média 9,6 anos (0-45 anos). As apresentações clínicas mais frequentes foram hepática (36,3%), neurológica (34,7%), assintomática (16,8%), neuro-psiquiátrica (8,3%) e hematológica (1,9%). Outras formas menos comuns foram renal, neuro-hepática, e osteoarticular. Disartria e manifestações clínicas de cirrose hepática descompensada foram as principais características neurológicas e hepáticas, respectivamente. Os parâmetros diagnósticos observados foram anéis de Kayser-Fleisher 78,3%, reduzidos níveis séricos de ceruloplasmina 98,3%, níveis elevados de cuprúria basal de 24 horas 73,0%, teste da D-penicilamina positivo em 54,0% e comprometimento nos dois alelos do gene ATP7B 84,4%. O exame de ressonância magnética encefálica mostrou alterações nos núcleos da base em 77,7% dos examinados. D-penicilamina foi prescrita inicialmente em 93,6% dos 245 casos tratados, e 53% relataram efeitos adversos. Houve necessidade de substituição em 50 indivíduos. Outras medicações utilizadas foram os sais de zinco e trientina. Não evidenciamos diferença significativa entre os resultados terapêuticos dessas três drogas (p=0,2). Os casos de má aderência à terapia evoluiram com pior desfecho quando comparados aos usuários regulares (p <0,0001). Nove pacientes realizaram transplante hepático. Durante o seguimento 82 casos faleceram. As principais causas dos óbitos foram descompensação hepática 41,5% (hemorragia digestiva, peritonite bacteriana espontânea, encefalopatia) e pneumonia 20,7%. Três pacientes cometeram suicídio. CONCLUSÕES: Não existe um exame padrão ouro ou achado patognomônico da doença. Todo paciente jovem com manifestações hepáticas, neurológicas, neuropsiquiátricas, hematológicas, renais ou osteoarticulares de causa indefinida deverá ser investigado para doença de Wilson. Além da farmacoterapia específica é necessária a avaliação psiquiátrica para detectar precocemente sintomas depressivos. / INTRODUCTION: Wilson\'s disease is an autosomal recessive disorder, caused by mutations in the gene ATP7B, leading to toxic copper accumulation in the body. Because it is a rare disease, large series with long-term follow-up are limited in literature. We reported the experience of Hospital das Clínicas of Sao Paulo University School of Medicine (HC-FMUSP) with Wilson\'s disease patients diagnosed between 1946 and 2010. METHODS: A retrospective analysis of 262 cases was performed describing the clinical presentation, the results of diagnostic tests, the patterns of treatment response and outcome. RESULTS: The mean age at the onset of symptoms was 17.4 years (7- 49 years). Patients were followed for a mean of 9.6 years (0-45 years). The most frequent clinical presentations were hepatic (36.3%), neurological (34.7%), asymptomatic (16.8%), neuro-psychiatric (8.3%) and hematologic (1.9%). Other less common forms were renal, neurological-hepatic, and musculoskeletal. Dysarthria and clinical manifestations of decompensated liver cirrhosis were the main neurological and liver features, respectively. The diagnostic parameters observed in this cohort of patients were Kayser-Fleischer rings in 78.3%, low serum ceruloplasmin in 98.3%, high 24-h urinary excretion of copper in 73.0%, positive challenge test with d-pencillamine in 54.0% and detection of two mutations of ATP7B gene in 84.4%. The magnetic resonance of brain showed abnormalities in the basal ganglia in 77.7% of those examined. D-penicillamine was prescribed in 93.6% of 245 cases as the first drug, and 53% reported adverse effects. It was need to replace it in 50 individuals. Other drugs used were salts of zinc and trientine. There was no significant difference between the therapeutic results of these three drugs (p = 0.2). The cases with poor adherence to therapy evolved with a worse outcome when compared to regular users (p <0.0001). Nine patients underwent liver transplantation. During the follow-up 82 patients died. The main causes of death were hepatic decompensation 41.5% (variceal hemorrhage, spontaneous bacterial peritonitis, encephalopathy) and pneumonia 20.7%. Three patients committed suicide. CONCLUSIONS: There is no gold standard or pathognomonic test for diagnosing this disease. Any young patient with hepatic, neurological, neuropsychiatric, hematologic, renal, or osteoarticular manifestations of unknown cause should be investigated for Wilson\'s disease. In addition to the classical pharmacotherapy, specific psychiatric evaluation is necessary to detect early symptoms of depression.

INTRODUÇÃO: A doença de Wilson é um distúrbio autossômico recessivo, decorrente de mutações no gene ATP7B, resultando em acúmulo tóxico de cobre no organismo. Devido a sua raridade, séries com grande casuística e longo seguimento são escassas na literatura. Nesse estudo relatamos a experiência do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (HC-FMUSP) com os pacientes diagnosticados com doença de Wilson entre o período de 1946 a 2010. MÉTODOS: Realizamos análise retrospectiva de 262 casos, descrevendo as formas de apresentação clínica, os achados dos exames diagnósticos, os padrões de resposta terapêutica e a evolução clínica da doença. RESULTADOS: A idade média do início dos sintomas foi 17,4 anos (7-49 anos). Os pacientes foram acompanhados em média 9,6 anos (0-45 anos). As apresentações clínicas mais frequentes foram hepática (36,3%), neurológica (34,7%), assintomática (16,8%), neuro-psiquiátrica (8,3%) e hematológica (1,9%). Outras formas menos comuns foram renal, neuro-hepática, e osteoarticular. Disartria e manifestações clínicas de cirrose hepática descompensada foram as principais características neurológicas e hepáticas, respectivamente. Os parâmetros diagnósticos observados foram anéis de Kayser-Fleisher 78,3%, reduzidos níveis séricos de ceruloplasmina 98,3%, níveis elevados de cuprúria basal de 24 horas 73,0%, teste da D-penicilamina positivo em 54,0% e comprometimento nos dois alelos do gene ATP7B 84,4%. O exame de ressonância magnética encefálica mostrou alterações nos núcleos da base em 77,7% dos examinados. D-penicilamina foi prescrita inicialmente em 93,6% dos 245 casos tratados, e 53% relataram efeitos adversos. Houve necessidade de substituição em 50 indivíduos. Outras medicações utilizadas foram os sais de zinco e trientina. Não evidenciamos diferença significativa entre os resultados terapêuticos dessas três drogas (p=0,2). Os casos de má aderência à terapia evoluiram com pior desfecho quando comparados aos usuários regulares (p <0,0001). Nove pacientes realizaram transplante hepático. Durante o seguimento 82 casos faleceram. As principais causas dos óbitos foram descompensação hepática 41,5% (hemorragia digestiva, peritonite bacteriana espontânea, encefalopatia) e pneumonia 20,7%. Três pacientes cometeram suicídio. CONCLUSÕES: Não existe um exame padrão ouro ou achado patognomônico da doença. Todo paciente jovem com manifestações hepáticas, neurológicas, neuropsiquiátricas, hematológicas, renais ou osteoarticulares de causa indefinida deverá ser investigado para doença de Wilson. Além da farmacoterapia específica é necessária a avaliação psiquiátrica para detectar precocemente sintomas depressivos. / INTRODUCTION: Wilson\'s disease is an autosomal recessive disorder, caused by mutations in the gene ATP7B, leading to toxic copper accumulation in the body. Because it is a rare disease, large series with long-term follow-up are limited in literature. We reported the experience of Hospital das Clínicas of Sao Paulo University School of Medicine (HC-FMUSP) with Wilson\'s disease patients diagnosed between 1946 and 2010. METHODS: A retrospective analysis of 262 cases was performed describing the clinical presentation, the results of diagnostic tests, the patterns of treatment response and outcome. RESULTS: The mean age at the onset of symptoms was 17.4 years (7- 49 years). Patients were followed for a mean of 9.6 years (0-45 years). The most frequent clinical presentations were hepatic (36.3%), neurological (34.7%), asymptomatic (16.8%), neuro-psychiatric (8.3%) and hematologic (1.9%). Other less common forms were renal, neurological-hepatic, and musculoskeletal. Dysarthria and clinical manifestations of decompensated liver cirrhosis were the main neurological and liver features, respectively. The diagnostic parameters observed in this cohort of patients were Kayser-Fleischer rings in 78.3%, low serum ceruloplasmin in 98.3%, high 24-h urinary excretion of copper in 73.0%, positive challenge test with d-pencillamine in 54.0% and detection of two mutations of ATP7B gene in 84.4%. The magnetic resonance of brain showed abnormalities in the basal ganglia in 77.7% of those examined. D-penicillamine was prescribed in 93.6% of 245 cases as the first drug, and 53% reported adverse effects. It was need to replace it in 50 individuals. Other drugs used were salts of zinc and trientine. There was no significant difference between the therapeutic results of these three drugs (p = 0.2). The cases with poor adherence to therapy evolved with a worse outcome when compared to regular users (p <0.0001). Nine patients underwent liver transplantation. During the follow-up 82 patients died. The main causes of death were hepatic decompensation 41.5% (variceal hemorrhage, spontaneous bacterial peritonitis, encephalopathy) and pneumonia 20.7%. Three patients committed suicide. CONCLUSIONS: There is no gold standard or pathognomonic test for diagnosing this disease. Any young patient with hepatic, neurological, neuropsychiatric, hematologic, renal, or osteoarticular manifestations of unknown cause should be investigated for Wilson\'s disease. In addition to the classical pharmacotherapy, specific psychiatric evaluation is necessary to detect early symptoms of depression.

La maladie de Wilson est une atteinte génétique rare associée à des mutations du gène codant pour l’ATP7B, protéine de transport et d’excrétion du cuivre dans l’organisme, entrainant une accumulation toxique de cuivre dans l’organisme au niveau du foie et du cerveau. La difficulté d’établir une corrélation génotype-phénotype et la grande hétérogénéité du tableau clinique conduisent à des difficultés de prise en charge, notamment concernant le diagnostic et le suivi biologique et thérapeutique des patients. Dans cette étude, nous avons orienté nos recherches vers la découverte et l’évaluation de candidats biomarqueurs de diagnostics et de pronostics précoce de l’évolution vers des formes neurologiques de la maladie. L’accessibilité au modèle préclinique murin Atp7b-/- nous a permis d’engager une étude préclinique permettant dans un premier temps d’optimiser la partie expérimentale pour l’identification la plus fiable de nouveaux candidats biomarqueurs plasmatiques. Cette étude a permis l’identification d’un panel de 7 candidats biomarqueurs. Ces résultats nous ont permis de susciter l’intérêt des équipes médicales du Centre National de Référence Wilson (CNR) à Lyon et à Paris et d’engager une étroite collaboration pour débuter l’étude clinique. L’obtention d’une première cohorte d’échantillons plasmatiques provenant de patients atteints de la maladie de Wilson a permis d’évaluer la valeur translationnelle et clinique des candidats biomarqueurs identifiés et de débuter l’étude clinique d’exploration du protéome plasmatique de patients atteints de la maladie de Wilson. En outre, la compréhension des mécanismes moléculaires associés au développement de la physiopathologie hépatique a été étudiée et a permis de mettre en évidence de nouvelles cibles pour, à terme, améliorer la prise en charge clinique des patients atteints de la maladie de Wilson. / Wilson’s disease is a rare genetic disorder triggered by mutations in the ATP7B gene, which encodes a transport protein involved in copper transport and excretion, triggering toxic copper overloads in the liver and the brain. The lack of genotype-phenotype correlation and phenotype variability lead to clinical care difficulties, especially for the diagnosis and biological follow up of patients. In this study, we initiated the discovery and evaluation of biomarker candidates for the diagnosis of Wilson’s disease and for early prognosis towards neurological manifestation. With the availability of the Atp7b-/- mice model, we engaged a preclinical study leading to the qualification of a panel of 7 biomarker candidates. These results allowed us to raise the interest of the National Reference Center for Wilson’s disease (CNR) medical teams in Lyon and Paris and to engage a close collaboration to initiate clinical study. Using a first plasma cohort from Wilson’s disease patients, we assessed the translational and clinical value of the 7 biomarker candidates and engage discovery study on patients’ plasma samples. Furthermore, we also studied the molecular mechanisms involved in liver pathophysiology using the Atp7b-/- mice model using discovery proteomics. These investigations led to the identification of a new potential therapeutic target.

The role of metal accumulation in the pathophysiology of neurodegenerative diseases has been a hot topic in recent years due to the possibility of its treatment by chelating agents. Although the mechanisms of neurodegeneration are well known, the role of metal accumulation is still unclear. The main limitation are unsatisfactory methods for in vivo metal imaging; the most widely used technique is magnetic resonance imaging (MRI). Our aim was to assess the possibility of using transcranial sonography (TCS) in differential diagnosis of neurodegenerative diseases and to further explore the underlying factors of echogenicity. In the first study, using TCS fusion with MRI, we focused on location verification of the commonly assessed structures (substantia nigra and nucleus lentiformis) and exclusion of possible focal structural changes affecting the echogenicity in WD and PD patients. Moreover, obtained MRI were used for semi-quantitative comparison with TCS images. Although TCS has been confirmed to be highly beneficial in differential diagnosis of Wilson's disease and it should be recommended as a screening method for extrapyramidal patients with atypical course of the disease, the direct relationship between TCS and metal deposits could not be proven. The obtained results from the ultrasound fusion...

Hintergrund: Der Morbus Wilson ist eine seltene, angeborene Störung des Kupferstoffwechsels, bei welcher es zu Akkumulationen von Kupfer und infolgedessen zu Schäden in verschiedenen Organen des menschlichen Körpers kommt. Die Therapie besteht vor allem darin, den Kupferspiegel medikamentös zu senken. In einzelnen Fällen wurde der Kupferspiegel während der Therapie so weit gesenkt, dass bei den Patient*innen neurologische Symptome auftraten (sogenannte Kupfermangel-Myeloneuropathien). Kupfer ist ein essenzieller Kofaktor mehrerer Enzyme im menschlichen Körper, so auch der Cytochrom-c-Oxidase, welche einen wichtigen Bestandteil der mitochondrialen Atmungskette und damit der zellulären Energiegewinnung darstellt. Die Bestimmung ihrer Aktivität ist bisher für verschiedene Zellen und Gewebe etabliert worden, ein standardisierter Assay für die Bestimmung in Thrombozyten existiert jedoch nicht. Fragestellung: Für die optimale Bestimmung der Cytochrom-c-Oxidase-Aktivität in Thrombozyten sollen bereits existierende Methoden angepasst werden. Ziel dieser Arbeit ist es, die Aktivität der Cytochrom-c-Oxidase bei Morbus Wilson-Patient*innen unter kupfersenkender Therapie zu untersuchen und auf einen Zusammenhang zum Serum-Kupferspiegel zu prüfen. Die Frage, ob eine zu starke Kupfersenkung durch die Therapie des Morbus Wilson zu einer verringerten Cytochrom-c-Oxidase-Aktivität führt und ob diese Myeloneuropathien hervorruft, soll somit beantwortet werden. Material und Methodik: Es wurden 36 Morbus Wilson-Patient*innen unter kupfersenkender Therapie und 20 gesunde Kontrollproband*innen untersucht. Es erfolgte eine Blutabnahme für die Gewinnung der Thrombozyten sowie für die Bestimmung des Serum-Kupferspiegels. Die Bestimmung der Aktivität der Cytochrom-c-Oxidase erfolgte spektralphotometrisch in Thrombozyten. Des Weiteren wurde die Aktivität des Komplex-II der Atmungskette bestimmt, da dieser nicht kupferabhängig ist und seine Aktivität daher bei Kupfermangel nicht eingeschränkt sein sollte. Zusätzlich ermöglichte die Berechnung des Quotienten der Cytochrom-c-Oxidase-Aktivität und der Komplex-II-Aktivität die Erfassung sehr geringer Aktivitätseinschränkungen der Cytochrom-c-Oxidase. Im Rahmen dieser Dissertation wurde die spektralphotometrische Messung dieser beiden Enzymaktivitäten in Thrombozyten entwickelt und optimiert. Zur Justierung der Enzymaktivitäten bei unbekannter Mitochondrienmenge diente die Aktivität der ausschließlich in Mitochondrien vorkommenden Citratsynthase. Die so bestimmten Enzymaktivitäten wurden mittels SPSS zwischen Wilson-Patient*innen und Kontrollproband*innen verglichen und untereinander sowie mit dem Serum-Kupferspiegel auf Zusammenhänge untersucht. Ferner wurden die Morbus Wilson-Patient*innen klinisch auf Anzeichen für Myeloneuropathien untersucht, um die Untersuchungsergebnisse anschließend auf einen Zusammenhang zu der Cytochrom-c-Oxidase-Aktivität zu prüfen. Ergebnisse: Der auf den Untersuchungen von Kirby et al. beruhende Assay für die spektralphotometrische Bestimmung der Cytochrom-c-Oxidase-Aktivität in isolierten Mitochondrien konnte durch die Zugabe von 0,3 mM Dodecylmaltosid für die Messung in Thrombozyten erfolgreich optimiert werden. Ebenso wurde der Assay für die Komplex-II-Aktivität durch die Zugabe von 1 mg/ml BSA für die Bestimmung in Thrombozyten erweitert (Kirby et al., 2007). Die Aktivität der Cytochrom-c-Oxidase der Wilson-Patient*innen war signifikant niedriger als die der Kontrollgruppe, während die Kontrollgruppe eine signifikant höhere Komplex-II-Aktivität aufwies. Der Quotient von Cytochrom-c-Oxidase-Aktivität und Komplex-II-Aktivität war in der Patient*innengruppe folglich ebenfalls signifikant erniedrigt. In der Analyse aller untersuchten Proben zeigte sich ein signifikanter Zusammenhang zwischen Serum-Kupferspiegel und Cytochrom-c-Oxidase-Aktivität, welcher in der Betrachtung der Subgruppen (Wilson Patient*innen und Kontrollproband*innen) jedoch nicht nachgewiesen werden konnte. Keiner der untersuchten Patient*innen wies klinische Anzeichen für Myeloneuropathien auf. Schlussfolgerung: Der optimierte Assay der Cytochrom-c-Oxidase-Aktivität und Komplex-II-Aktivität in Thrombozyten erlaubt die zuverlässige Bestimmung der Atmungskettenaktivität in einem einfach zugänglichen Gewebe und ist damit für vielfältige Fragestellungen einsetzbar, wenn Einflüsse medizinischer Maßnahmen auf die mitochondriale Funktion untersucht werden sollen. Mit 36 Morbus Wilson-Patient*innen umfasst diese Arbeit eine der bisher größten untersuchten Patient*innengruppen dieses seltenen Krankheitsbildes. Der erniedrigte Quotient der Cytochrom-c-Oxidase-Aktivität und Komplex-II-Aktivität ist als Bestätigung einer Cytochrom-c-Oxidase-Einschränkung bei Morbus Wilson-Patient*innen unter kupfersenkender Therapie zu werten. Die Korrelation zwischen dem Serum-Kupferspiegel und der Cytochrom-c-Oxidase-Aktivität sowie die Aktivitätsreduktion der Cytochrom-c-Oxidase in der Patient*innengruppe ist eine wichtige Erkenntnis für die zukünftige Überwachung und gegebenenfalls Anpassung der Therapie von Morbus Wilson. Der Zusammenhang zwischen der Cytochrom-c-Oxidase-Aktivität und Myeloneuropathien sollte an Patient*innen mit Myeloneuropathien weiter untersucht werden. Es wurde jedoch gezeigt, dass Kupfermangel und niedrige Cytochrom-c-Oxidase-Aktivitäten nicht unbedingt mit Myeloneuropathien einhergehen. Therapie-induzierte Kupfermangel-Myeloneuropathien gilt es weiterhin zu vermeiden. / Background: Wilson’s disease is a rare, congenital disorder of copper metabolism, which leads to accumulations of copper and consequent damage in various organs of the human body. The therapy consists mainly in lowering the copper level by medication. In individual cases, the copper level was lowered during the therapy to such an extent that the patients developed neurological symptoms (so-called copper deficiency myeloneuropathies). Copper is an essential cofactor of several enzymes in the human body, including cytochrome c oxidase, which is an important component of the mitochondrial respiratory chain and thus of cellular energy production. The determination of its activity has been established so far for various cells and tissues, but a standardized assay for its determination in platelets does not exist. Purpose: For the optimal determination of cytochrome c oxidase activity in platelets, existing methods will be adapted. The aim of this work is to investigate the activity of cytochrome c oxidase in Wilson’s disease patients under copper-lowering therapy and to test for a correlation to serum copper levels. The question of whether excessive copper lowering by Wilson’s disease therapy leads to reduced cytochrome c oxidase activity and whether this possibly causes myeloneuropathies will thus be answered. Material and Methods: 36 Wilson’s disease patients under copper-lowering therapy and 20 healthy control subjects were studied. Blood was drawn for platelet collection and determination of serum copper levels. The activity of cytochrome c oxidase was determined spectrophotometrically in platelets. Furthermore, the activity of complex II of the respiratory chain was determined, since this is not copper-dependent and its activity should therefore not be limited in copper deficiency. In addition, calculation of the quotient of cytochrome c oxidase activity and complex II activity allowed detection of very low activity limitations of cytochrome c oxidase. In this dissertation, the spectrophotometric measurement of these two enzyme activities in platelets was developed and optimized. The activity of citrate synthase, which occurs exclusively in mitochondria, was used to adjust the enzyme activities when amount of mitochondria was unknown. The enzyme activities determined in this way were compared between Wilson’s disease patients and control subjects using SPSS and examined for correlations with each other and with serum copper levels. Furthermore, the Wilson’s disease patients were clinically examined for signs of myeloneuropathies, in order to subsequently examine the examination results for a correlation to the cytochrome c oxidase activity. Results: The assay for spectrophotometric determination of cytochrome c oxidase activity in isolated mitochondria, based on studies of Kirby et al, was successfully optimized for measurement in platelets by the addition of 0.3 mM dodecylmaltoside. Similarly, the assay for complex II activity was enhanced by the addition of 1 mg/ml BSA for determination in platelets (Kirby et al., 2007). The activity of cytochrome c oxidase of Wilson patients was significantly lower than that of the control group, with the control group had a significantly higher complex II activity. Consequently, the quotient of cytochrome c oxidase activity and complex II activity was also significantly lower in the Wilson patient group. A significant correlation between serum copper level and cytochrome c oxidase activity was found in the analysis of all samples examined, which, however, could not be proven in the examination of the subgroups (Wilson patients and control subjects). None of the patients examined showed clinical signs of myeloneuropathies. Conclusion: The optimized assay of cytochrome c oxidase activity and complex II activity in platelets allows reliable determination of respiratory chain activity in an easily accessible tissue and is thus applicable to a variety of questions when influences of medical interventions on mitochondrial function are to be investigated. With 36 Wilson's disease patients, this work includes one of the largest groups of patients of this rare disease studied so far. The decreased quotient of cytochrome c oxidase activity and complex II activity is a confirmation of cytochrome c oxidase impairment in Wilson’s disease patients on copper-lowering therapy. The correlation between serum copper level and cytochrome c oxidase activity as well as the reduction of cytochrome c oxidase activity in the patient group is an important finding for future monitoring and, if necessary, adjustment of Wilson’s disease therapy. The relationship between cytochrome c oxidase activity and myeloneuropathies should be further investigated in patients with myeloneuropathies. However, it has been shown that copper deficiency and low cytochrome c oxidase activities are not necessarily associated with myeloneuropathies. Therapy-induced copper deficiency myeloneuropathies should continue to be avoided.