WILSON’S DISEASE Ben Winrow

How to use this SDL This SDL is designed to be informative and interactive Every time you see words that are underlined you can click on these to get an explanation of what the word means, then click on ‘back to presentation’ to return to the SDL At the end of the SDL there are a set of questions so be sure to read and remember the information that is contained in the following slides Use the content finder on the left to revert to any section of the SDL Wilson’s is not a common disorder, but it is the severity of the condition if left untreated which prompted design of this SDL 17/09/2018

Learning objectives To understand what Wilson’s disease is To be aware of its prevalence To understand the genetic factors leading to Wilson’s disease To understand the molecular processes that lead to the symptoms of Wilson’s disease To have an appreciation of the presentation of Wilson’s in the young patient and in the older patient To have an awareness of the ways in which Wilson’s disease is diagnosed To understand the role of genetic analysis in the diagnosis of first degree relatives of a Wilson’s patient To be aware of current treatment of Wilson’s disease To understand the shift in treatment options To know the recent advances in treatment 17/09/2018

Definition An inborn error of copper metabolism – characterised by an increase in copper accumulation in tissues of the liver (1), brain (2), cornea (3), skin (4), joints (5) and kidney (6), as a result of decreased hepatobiliary excretion of copper. It is also known as lenticular degeneration. 2 3 4 1 5 6 17/09/2018

Aetiology The cause of Wilson’s has only recently been deduced but the exact mechanism is always changing. The ATP7B gene product is a P-type ATPase transporter, which is part of the trans-Golgi network. The ATP7B gene itself is around 80kb long and has 21 introns. The mutated gene leads to a faulty gene product and this causes the pathologies seen in Wilson’s disease. The gene is responsible for incorporation of copper into caeruloplasmin and its excretion into bile. In Wilson’s disease copper is not bound to the caeruloplasmin, causing toxicity. Characteristically, there is also low serum caeruloplasmin due to poor synthesis. It is known that copper stimulates caeruloplasmin production but the exact mechanism of poor synthesis is not fully understood. The lack of transportation of copper into the bile leads to accumulation of copper in the hepatocytes When hepatocyte storage capacity is reached, free copper is slowly released into the blood stream causing an accumulation of copper in extra-hepatic tissue. Rarely, a massive release of copper can lead to haemolysis and fulminant hepatic failure Genotype-Phenotype relationship is still unknown, however mutations that limit the function of the gene product seem to be related to an early onset of symptoms of the disease. 17/09/2018

Epidemiology The prevalence of Wilson’s in Northern European Caucasian population is 1/30,000 Even though this makes the disease quite rare it is speculated that 1/90 people carry a defective ATP7B gene In some areas of the world the prevalence can be as high as 1/5000, and certain mutations in the gene are associated with these pockets of increased prevalence The areas highlighted below have an increased prevalence: Iceland China South Korea Sicily 17/09/2018 Northern India

The site of the mutation determines the severity of the disease Genetics Chromosome 13 carries the gene which causes Wilson’s There are around 300 known mutations the most common being His1069Gln The pattern of inheritance is Autosomal recessive With 2 carrying parents the child has a ¼ chance of having the disease Patients are usually homozygous or compound heterozygous, this can make it difficult to obtain a diagnosis by mutation analysis Mutations are either nonsense, missense or frameshift, the most common mutation found on the gene being missense. The site of the mutation determines the severity of the disease 17/09/2018

Presentation [1] Hepatic presentation The presentation of hepatic disease due to Wilson’s is diverse. Most patients that present hepatically are young. Typically symptoms will become apparent at around 6 years of age. At this age slit lamp examination may not show Kayser-fleischer (K-F) rings One of the first signs that pathology is present is persistent asymptomatic hepatomegaly or the elevation of serum aminotransferases (as seen in the young patient presenting with Wilson’s, sometimes misdiagnosed as hepatitis) Jaundice with no apparent cause is often a presenting complaint Acute hepatitis is another presentation with positive non-specific autoantibodies and raised serum IgG Non-alcoholic fatty liver disease can also be misdiagnosed as steatosis can often occur A majority of patients however present with chronic liver disease having been undiagnosed and therefore have small, scarred livers, ascites and splenomegaly. 17/09/2018

Presentation [2] Hepatic presentation Fulminant hepatic failure is the ultimate progression, giving rise to characteristic features– renal dysfunction (due to haemolysis by the free copper in the blood stream causing renal tubulopathy), low amino transferases and low alkaline phosphatases. Due to this presentation the diagnosis is often wrong (acute hepatitis), which can be life threatening as the only treatment is liver transplantation. Due to the nature of the presentation it is often confused with other, more common disease processes e.g. viral hepatitis A high index of suspicion is needed to diagnose Wilson’s- if left untreated the copper can deposit in extra hepatic tissues and cause irreversible damage to the brain, causing neurological problems; the kidney, causing tubulopathy; and the joints, causing early onset osteoarthritis. Fig. A liver with acute hepatitis 17/09/2018

Presentation [3] Neurological presentation The patient that presents with neurological pathologies tends to be older (>35 years) and symptoms can often mimic other disorders The majority of patients presenting neurologically will have movement disorders (e.g. tremor) Rigid dystonia can also be a presenting feature and this can be confused with a parkinsonian disorder (drooling, slow walking) There is a deficit in the patient’s handwriting (small and illegible) and unusual clumsiness for age can be shown using direct questioning or specific intelligence testing (mainly testing fluid intelligence) Some patients present with the following symptoms: Dystonia Dysarthria Dysphagia 17/09/2018

Presentation[4] Neurological presentation 20% of patients present with a psychiatric disorder, usually in adolescence, and this can be misdiagnosed as many other mood or affective disorders. Severe depression and neurotic behaviour patterns predominate In the adolescent presenting in this way, attention may be limited and a short temper is usually seen. There may also be an unexplained drop in the level of intelligence and deterioration in school work. Due to this children are often diagnosed as having Attention deficit hyperactivity disorder (ADHD) or, in some cases, as being a ‘problem child’ It is important to diagnose these patients quickly as the damage that is caused in the brain is largely irreversible Basal ganglia damage due to Wilson’s disease 17/09/2018

Presentation[5] An overview of the possible presentations of Wilson’s is given below. As can be seen, these are very diverse and it is important to have a high index of suspicion when presented with the symptoms below with no obvious cause. 17/09/2018

Copper and caeruloplasmin measurements Diagnosis [1] Copper and caeruloplasmin measurements The normal range for serum copper is 10-22μmol/L Wilson’s patients have a characteristically high level of copper in the liver (>250µg/g dry weight) found on biopsy Serum caeruloplasmin should be between 200-600mg/L - the combination of caeruloplasmin below this level and K-F rings in the eyes is diagnostic of Wilson’s. These results have to be taken in light of the clinical picture as copper-levels can be near normal in many Wilson’s patients. Liver function tests Depends on type of presentation – if there is an associated haemolytic anaemia then a rise in AST is seen. If the liver is cirrhotic, ALP and bilirubin will be increased but serum albumin will be decreased. If there is progression to liver failure, pro-thrombin time is also increased).           17/09/2018

Diagnosis [2] Urinary copper Challenge with Penicillamine 24-hour excretion of copper via the urine is an indicator of the amount of ‘free’ copper (unbound to caeruloplasmin) in the blood In affected individuals it is always >0.6µmols/24hrs. Challenge with Penicillamine Penicillamine increases the amount of copper excreted via the urine. Another collection of 24hr urine is collected and analysed An increase to >25 µmols/24hrs is indicative of Wilson’s disease. 17/09/2018

Diagnosis [3] Slit lamp examination of the eye Liver biopsy Absence of K-F rings does not exclude disease. The K-F rings are nearly always associated with neurological presentation of the disease but in total are present only 40% of the time Liver biopsy This is a definitive test. A wide section of the liver needs to be taken for biopsy as accumulation of copper is not always diffuse in the liver. Liver parenchymal levels of copper greater than 250µg/g dry weight is diagnostic. 17/09/2018

Advances in genetic diagnosis [1] Over 300 mutations exist on chromosome 13, the chromosome which carries the ATP7B gene, responsible for Wilson’s ATP7B codes for an ATPase transporter protein, which is part of the trans-golgi network (protein shown below) The mutations cause a variably defective protein, hence the range of ages that present with differing symptoms. The most common mutation is His1069Gln, which is shown on the above structure, this mutation seems to affect ATP binding and therefore the protein cannot carry out it’s function 17/09/2018

Advances in genetic diagnosis [2] When presented with cryptogenic liver disease in the child or isolated neurological problems in the adult, mutation analysis of the DNA (specifically chromosome 13 and the ATP7B gene) should be carried out. Mutation analysis involves the following steps; Obtain haplotypes based on polymorphisms surrounding the Wilson’s gene Obtain DNA and run PCR to amplify Test exons known to carry the most mutations, with haplotypes (di/tri single stranded repeats) This highlights where the mutations are on the gene Then test 1st degree relatives DNA for the same mutations Most common type of mutation is a missense mutation 17/09/2018

Treatment [1] Drug Dose Side effects Mechanism of action Measure of efficacy Penicillamine 500mg bd or 250mg qd + 25mg/day pyridoxine 50% chance of acute neural toxicity at commencement of treatment Reductive copper chelator Have to measure the free serum caeruloplasmin level as measuring urinary copper is misleading Trientine 1.2 - 2.4g/day in 2-4 divided doses before food Proteinuria; 20% chance of neural toxicity at commencement of Rx Reductive copper chelator (less potent then penicillamine) Zinc 200mg tds Gastritis Induction of intestinal metallothionein Measure urinary copper excretion 17/09/2018

Treatment [2] Diet: For obvious reasons a low copper diet is needed- no vitamin supplements containing copper should be taken and analysis of the drinking water in the home is needed. The level of copper in the water should not exceed 0.1ppm. Avoid mushrooms, nuts, shell fish and dried fruit especially Pregnancy: Should stay on anti-copper therapy for the protection of the mothers health, however penicillamine is known to be teratogenic, therefore the patient should be put on trientine or zinc. Therapy should be monitored and the lowest effective dose used as copper deficiency is also teratogenic. Prophylactic treatment of the asymptomatic patient: Asymptomatic patients are usually siblings of an affected patient that have been diagnosed using mutation analysis. These patients should be treated as if they are on maintenance therapy, usually with zinc. Fulminant liver failure The only successful treatment when the patient presents with this condition is liver transplant 17/09/2018

Advances in treatment Tetrathiomolybdate – A novel drug currently undergoing phase III trials and not yet commercially available. To be used on the neurologically presenting patient Dose: 20mg tds + 60mg at bedtime (away from food) Acts by forming a tri-partite complex between itself, copper and protein If taken away from food it enters the bloodstream and binds with copper and albumin – rapidly reducing the toxicity of the high levels of copper Take with a chelating agent (e.g. Trientine) to increase the amount of free copper to form a tri-partite complex with If on Tetrathiomolybdate, only 5% of patients experience neurological worsening compared to 20% and 50% of patients on Trientine and Penicillamine, respectively Test efficacy by carrying out neurological tests once a week (as this drug is used for the neurologically presenting patient Test for toxicity – FBC and LFT’s once every 2 weeks – any toxicity (anaemia, leukopaenia) is responsive to lowering the dose 17/09/2018

Summary Wilson’s disease is a rare autosomal recessive condition It’s effects are due to the inability to excrete copper in the bile Over 300 mutations on the Wilson’s gene are known The young patient usually presents hepatically The older patient usually presents neurologically Due to the amount of mutations that diagnosis by mutation analysis is difficult but techniques are improving Treatment is improving with phase III trials continuing If presented with cryptogenic liver disease or unexplained neurological deficit always consider Wilson’s disease 17/09/2018

Questions What is autosomal recessive inheritance? Inheritance which is sex linked and needs 1 copy of the mutated gene to show symptoms Inheritance which is not sex linked and needs 1 copy of the mutated gene to show symptoms Inheritance which is not sex linked and needs 2 copies of the mutated gene to show symptoms 2. What is a missense mutation? A point mutation in a sequence of DNA that results in a premature stop codon types of point mutations where a nucleotide is changed which results in a different amino acid. a genetic mutation that inserts or deletes a number of nucleotides that is not evenly divisible by three from a DNA sequence 17/09/2018

Questions cont. What is the function of the ATP7B gene product? It is caeruloplasmin, a copper containing apoprotein, which binds copper to make it non-toxic It is a transporter protein, part of the trans Golgi network, transporting copper out of the hepatocytes into bile It is a G-protein linked second messenger cascade, causing copper to be taken up by the liver Why is penicillamine no longer the 1st choice of drug in the treatment of Wilson’s It carries a high risk of neurological damage at the onset of treatment The doses needed for it to be effective are unpalatable to the patient To measure efficacy is too difficult 17/09/2018

Questions cont. What tests should be carried out on a 12 year old that has shown deterioration in school work and has raised aminotranferases? Slit lamp examination of the eye 24 hour urine collection Liver biopsy All of the above What are the dangers of therapy for a Wilson’s patient who is pregnant? Copper levels that are too high are dangerous to the mother and levels that are too low are known to be teratogenic The drugs used in the maintenance of a Wilson’s patient are known to be teratogenic Side effects of treatment in the mother include syncope and fitting putting both the mother and the unborn child at risk 17/09/2018

References Some useful websites www.wilsonsdisease.org/ Bingham M. Ong T-J. Physiologic function of the Wilson disease gene product, ATP7B. AM J Clin Nutr. 1998;67:982-7 Brewer G. Askari F. Wilson’s disease: clinical management and therapy. Journal of hepatology. 2005;42:13-21 Caprai S. Loudianos G. et al. Direct diagnosis of Wilson’s disease by molecular genetics. J paediar. 2006;148:138-40 Hoogenraad T. Paradigm shift in treatment of Wilson’s disease: Zinc therapy now the treatment of choice. Brain and development. 2006;28:141-46 Lin J. J. Lin K-L. Isolated psychological presentation without hepatic involvement. Paediatr neurol. 2006;35:284-6 Roberts E. A. Wilson’s disease. Medicine. 2006;11:1-3 Roberts E. A. Schilsky M. L. A practice guideline on Wilson’s disease. Hepatology. 2003;37:1475-92 Wilson S. A. K. Progressive lenticular degeneration: A familial nervous disease associated with cirrhosis of the liver. Brain. 1912;34:295-509 Some useful websites www.wilsonsdisease.org/ www.wilsons-disease.org.uk/ 17/09/2018