WILSON

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WILSONS DISEASE
Ben Winrow
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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 Wilsons is not a common
disorder, but it is the severity of the condition
if left untreated which prompted design of this
SDL
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Learning objectives

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

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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.
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Aetiology

• The cause of Wilsons 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 Wilsons
  disease. The gene is responsible for
  incorporation of copper into caeruloplasmin and
  its excretion into bile.
• In Wilsons 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.

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Epidemiology
The prevalence of Wilsons 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
Northern India
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Genetics
Chromosome 13 carries the gene which causes
Wilsons 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
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Presentation 1
Hepatic presentation
The presentation of hepatic disease due to
Wilsons 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 Wilsons, 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.
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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 Wilsons- 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
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Presentation 3
Neurological presentation
The patient that presents with neurological
pathologies tends to be older (gt35 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 patients 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
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Presentation4
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 Wilsons disease
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Presentation5
An overview of the possible presentations of
Wilsons 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.
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Diagnosis 1
Copper and caeruloplasmin measurements The
normal range for serum copper is
10-22µmol/L Wilsons patients have a
characteristically high level of copper in the
liver (gt250µg/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
Wilsons. These results have to be taken in light
of the clinical picture as copper-levels can be
near normal in many Wilsons 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).          
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Diagnosis 2
Urinary copper 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
gt0.6µmols/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 gt25 µmols/24hrs is
indicative of Wilsons disease.
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Diagnosis 3
Slit lamp examination of the eye 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.
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Advances in genetic diagnosis 1
Over 300 mutations exist on chromosome 13, the
chromosome which carries the ATP7B gene,
responsible for Wilsons 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 its function
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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 Wilsons 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

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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) Have to measure the free serum caeruloplasmin level as measuring urinary copper is misleading
Zinc 200mg tds Gastritis Induction of intestinal metallothionein Measure urinary copper excretion
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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
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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
LFTs once every 2 weeks any toxicity (anaemia,
leukopaenia) is responsive to lowering the dose
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Summary

• Wilsons disease is a rare autosomal recessive
  condition
• Its effects are due to the inability to excrete
  copper in the bile
• Over 300 mutations on the Wilsons 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
  Wilsons disease

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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

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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 Wilsons
• 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

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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 Wilsons
  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 Wilsons
  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

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References

• Bingham M. Ong T-J. Physiologic function of the
  Wilson disease gene product, ATP7B. AM J Clin
  Nutr. 199867982-7
• Brewer G. Askari F. Wilsons disease clinical
  management and therapy. Journal of hepatology.
  20054213-21
• Caprai S. Loudianos G. et al. Direct diagnosis
  of Wilsons disease by molecular genetics. J
  paediar. 2006148138-40
• Hoogenraad T. Paradigm shift in treatment of
  Wilsons disease Zinc therapy now the treatment
  of choice. Brain and development. 200628141-46
• Lin J. J. Lin K-L. Isolated psychological
  presentation without hepatic involvement.
  Paediatr neurol. 200635284-6
• Roberts E. A. Wilsons disease. Medicine.
  2006111-3
• Roberts E. A. Schilsky M. L. A practice
  guideline on Wilsons disease. Hepatology.
  2003371475-92
• Wilson S. A. K. Progressive lenticular
  degeneration A familial nervous disease
  associated with cirrhosis of the liver. Brain.
  191234295-509

• Some useful websites
• www.wilsonsdisease.org/
• www.wilsons-disease.org.uk/

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Autosomal recessive inheritance Inheritance
which is not sex linked i.e. the mutation is
carried on any chromosome other then the sex
chromosomes. The effect is only seen when both
alleles are the same (both mutated) Bac
k to presentation
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Homozygous describing an individual in whom the
member of a pair of genes determining a
particular characteristic are identical Compound
heterozygous describing an individual in whom
the members of a pair of genes determining a
particular characteristic are dissimilar i.e.
there is more then one mutation affecting the
genes but there is still the presence of the
disease (this make mutation analysis very
difficult) Back to presentation
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Missense types of point mutations where a
nucleotide is changed which results in a
different amino acid. This can render the
resulting protein non-functional Nonsense a
point mutation in a sequence of DNA that results
in a premature stop codon, or a nonsense codon in
the transcribed mRNA, and possibly a truncated
and non-functional protein product Frameshift a
genetic mutation that inserts or deletes a number
of nucleotides that is not evenly divisible by
three from a DNA sequence, this disrupts the
reading frame and results in a completely
different translation from the original,
producing a non-functional protein
product Back to presentation
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Dystonia muscle dysfunction characterised by
spasms or abnormal muscle contraction. Often
associated with basal ganglia damage Dysarthria
a speech disorder in which the pronunciation is
unclear although the language content and meaning
are normal Dysphagia a condition in which the
action of swallowing is either difficult to
perform, painful. In Wilsons disease it is
caused by neurological co-ordination
abnormalities Back to presentation
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Caeruloplasmin
A copper containing protein present in blood
plasma and hepatic tissue. In Wilsons disease it
is in an apo-form due to the fact that copper has
not bound to it (copper stimulates its
formation) When copper does bind it forms a
non-toxic complex When unbound, as in Wilsons,
the copper becomes toxic Back to presentation
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WRONG This is X-linked recessive inheritance,
due to the overlap of the X chromosome over the Y
chromosome (in males) the diseases caused by this
form of inheritance usually present in men as
only 1 copy of the faulty gene is needed to show
symptoms of the disease. An example would be
haemophilia. Back to presentation
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Haplotype a complete set of HLA antigens
(inherited from either parent), for the sake of
mutation analysis however they are di/tri single
stranded repeats used to fish for
mutations. Back to presentation
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WRONG This is autosomal dominant inheritance,
where only 1 copy of the mutated gene is needed
to show symptoms as the allele is
dominant Back to presentation
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CORRECT Autosomal recessive inheritance is where
2 mutations of the same gene are needed to show
symptoms, it is not sex-linked and with parents
that both carry the mutation each child has a ¼
chance of getting the disease
Back to presentation
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WRONG This is a nonsense mutation, the premature
stop codon usually results in a non-functional
protein product
Back to presentation
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CORRECT This is a missense mutation and is the
most common mutation seen in Wilsons disease. It
results in a reduced or non-functional protein
product
Back to presentation
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WRONG This is a frameshift mutation. This alters
the reading frame and causes a completely
different protein product after the mutation
Back to presentation
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WRONG This is an apoprotein (apocaeruloplasmin)
and when it binds to copper it becomes
caeruloplasmin and makes copper non-toxic to the
body HOWEVER it is not the product of the ATP7B
gene
Back to presentation
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CORRECT It is a transporter and transports
copper out of the liver. It is the dysfunction of
this protein that causes the build up of copper
and the symptoms of Wilsons
Back to presentation
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WRONG A G-protein linked second messenger
cascade can be associated with production of ATP
and interaction with many different drugs e.g.
canabinoid receptors
Back to presentation
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CORRECT Some papers have shown that neurological
damage can be seen in as many as 50 of patients
15 of which dont ever recover to baseline
neurological activity before the onset of
treatment. This is due to the mobilisation of
copper from its stores in the body and as it can
cross the blood brain barrier, it causes severe
deterioration.
Back to presentation
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WRONG There is no evidence that the doses are
unpalatable. The doses are given in slide 7.
Back to presentation
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WRONG Efficacy measurement are more difficult to
do when using penicillamine but are possible, the
equation is seen below
Non-Cp-Cu (umol/l) total serum Cu (umol/l)
0.047 x serum Cp (mg/l)
Back to presentation
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Steatosis The infiltration of hepatocytes with
fat. This can occur in pregnancy, alcoholism,
obesity, hepatitis C infection, some drugs and
with Wilsons disease.
A liver showing steatosis (black
arrow pointing to fat deposit)
Back to presentation
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Kayser-Fleischer (K-F) rings A brownish-yellow
ring in the outer rim of the cornea of the eye.
It is caused by the deposition of copper granules
in decemets membrane. When well developed they
can be seen unaided but faint K-F rings need
specialist ophthalmological examination
(Slit-lamp examination)
K-F ring
Back to presentation
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Pro-thrombin time (PTT) The time taken for blood
clotting to occur in a sample of blood to which
calcium and thromboplastin have been added. A
prolonged PTT indicates a deficiency of
coagulation factors, which are required to
convert pro-thrombin to thrombin in the final
stages of the coagulation cascade.
Back to presentation
47
Teratogenic The adjective of teratogen, any
substance, agent or process that induces the
formation of developmental abnormalities in a
foetus.
Back to presentation
48
Maintenance therapy Used in patients that are
asymptomatic but have a mutation on the Wilsons
gene, or for patients that have undergone initial
therapy and are maintaining their copper levels.
Zinc 200mg/tds is used.
Back to presentation
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Tri-partite complex A complex that has three
(tri) parts (partite)
Tetrathiomolybdate
Copper
Protein
Back to presentation
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Leukopaenia A reduction in the number of white
blood cells (leucocytes) in the blood
Back to presentation
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WRONG Although this is an important examination
it cannot be done in isolation as at presentation
only 40 of patients will have Kayser-Fleischer
rings, the majority of these will be
neurologically presenting.
Back to presentation
52
WRONG Although this is a very sensitive test,
copper levels may be normal or near normal and
therefore it cannot be done in isolation. The
appropriate levels are given in slide 14.
Back to presentation
53
WRONG This is a very important test but the
accumulation of copper is not always diffuse
through the liver and therefore may be missed.
The appropriate levels of copper (dry weight) are
given in slide 15.
Back to presentation
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CORRECT All of these tests should be carried
out, along with liver function tests. The
diagnosis of Wilsons disease can sometimes be
difficult to come to when presented with
neurological deterioration with no obvious cause
and therefore a high index of suspicion is
needed. These tests, when done in conjunction
with each other should give the diagnosis of
Wilsons disease.
Back to presentation
55
CORRECT Stopping treatment is dangerous to the
mother as this will cause copper to accumulate in
her tissues causing the complications of Wilsons
disease. It is also noted that levels of copper
that are too low are teratogenic to the unborn
child therefore treatment cannot be too vigorous.
Zinc or Trientine is used as maintenance therapy.
Back to presentation
56
WRONG Only penicillamine is known to be
teratogenic and as most patients are diagnosed by
a child-bearing age, they should already be on
maintenance therapy. In addition to this there
has been a shift away from using penicillamine as
treatment due to its neurotoxic effects.
Back to presentation
57
WRONG There is no evidence that this is the
case. Side effects of treatment are given on
slide 18
Back to presentation