1. By: Xiaomeng Li, Tiffany Lai, and Eisha Vijay
Wilson’s Disease
By: Xiaomeng Li, Tiffany Lai, and Eisha Vijay
Oct. 10, 2017
PHM Fall 2017
Instructor: Dr. Jeffrey Henderson

2. What is Wilson’s disease?
Also known as progressive hepatolenticular degeneration1
Rare genetic disorder that prevents the body from excreting excess copper
Leads to copper accumulation in the liver, brain, and eyes which can cause organ damage over time
Affects about 1 in 30,000 people worldwide
Fatal if left untreated
Wilson’s disease, also known as progressive hepatolenticular degeneration is a rare genetic disorder that prevents the body from excreting excess copper. In a healthy body, the liver filters out excess copper and releases it through urine, but a person with Wilson’s disease, their liver cannot remove the extra copper properly. This leads to copper accumulation primarily in the liver, brain and the eyes.
Wilson’s disease affects about 1 in 30,000 people worldwide and can be fatal if left untreated due to organ damage over time from copper poisoning.

3. What causes Wilson’s disease?
Autosomal recessive disorder1
If both parents carry an abnormal gene, 25% chance that the child had the condition
Gene involved is ATP7B (on chromosome 13)
Encodes for a transmembrane protein ATPase
Functions in the transport of copper
More than 500 ATP7B mutations have been identified
H1069Q mutation common in patients of European origin
Wilson’s disease is an autosomal recessive genetic disorder, which means that two copies of an abnormal gene must be present in order for the disease to develop. The abnormal gene, ATP7B, which is located on chromosome 13 is responsible for Wilson’s disease. ATP7B gene encodes for a transmembrane protein ATPase that helps to transport copper. So, mutations in the ATP7B gene leads to Wilson’s disease.

4. Mechanistic Route of Action
Gut
ATP7A: transports Cu+ out of tissues2,3
Plasma
Ceruloplasmin: copper-binding protein and ferroxidase activity2
hCRT: passive transporter  & reductase: reduces Cu2+ → Cu+
Liver
GSH: metallochaperone2
Atox1: copper metallochaperone
ATP7B: transports Cu+ out of the liver
Excess copper
Copper’s transport throughout the body is extremely regulated, and requires multiple protein chaperones. Copper leaves the gut via the ATP7A  and enters the portal vein.It is immediately bound to ceruloplasmin in the plasma and shuttled to the liver. Before copper can enter the liver via orexin, it will be reduced into cu+ by an unspecific reductase. To keep intracellular free copper concentrations low, GSH automatically binds to cu+ before being transferred onto Atox1, which is a metallochaperone that will direct copper to ATP7B for excretion out of the liver, either to be delivered to other tissues or processed into bile. If ATP7B is damaged or mutated, then copper will not be able to shunted out of the liver, which will accumulate. --
Figure reference: Huster, D. Wilson Disease. Best Pract Res Clin Gastroenterol. 2005, 24,
General reference:
Fatemi, N.; Sarkar, B. Insights into the mechanism of copper transport by the Wilson and Menkes disease copper-transporting ATPases. Inorganica Chimica Acta. 2002, 339,
Huster et al. 2005
GSH: glutathione
Atox1: antioxidant 1 copper chaperone
hCRT: (human) orexin

5. Protein Structure of ATP7B
Metal binding domain4,5: Binds Cu+
Nucleotide-binding domain: binds with ATP
Phosphorylation domain: receives the  γ-phosphate
Actuator domain: dephosphorylation of the acyl-phosphate intermediate
Physiological concentration: Metal binding domain 5 and 6 are bound → secretory pathway through TGN
Excess concentration: Metal binding domains 1 through 4 are bound → biliary copper excretion
h1069Q
As mentioned previously, copper has two destinations once it leaves the liver through ATP7B. The concentration of intracelllular copper dictates whether or not copper will be excreted as bile or shuttled to another tissue. Firstly, the structure of ATP7B must be analyzed. Functionally, ATP7B can be split into 3 main domains: the metal binding domain, the nucleotide binding domain and the phosphorylation domain, and the actuator domain. The metal binding domain acts as the sensor for intracellular copper concentrations. Under normal physiological concentrations, only the fifth and sixth  domain will be bound to copper. In order for copper to be shuttled out, ATP needs to be bound to the nucleotide binding domain. Once copper at ATP are both bound, ATP gets hydrolyzed and the gamma phosphate gets transferred to the phosphorylation domain. This initiates a conformational change and allows copper to leave the liver to be transported to other tissues. Following, the actuator domain removes the leftover ADP from the nucleotide binding site, which frees up this transporter for another round of copper export. Under high concentrations of copper, the the metal binding domain 1 through 6 will be bound. Therefore, resulting in hyperphosphorylation of the phosphorylation domain, which signals for copper excretion out of the body through bile.
h1069Q, a common mutation in ATP7B, affects the nucleotide domain. It ultimately results in the inability to bind ATP, therefore preventing copper transport out of the liver, thus causing its accumulation.
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Figure ref: Fatemi, N.; Sarkar, B. Insights into the mechanism of copper transport by the Wilson and Menkes disease copper-transporting ATPases. Inorganica Chimica Acta. 2002, 339,
General:
Dmitriev O.; Tsivkovskii, R.; Abildgaard, F.; Morgan, C.T.; Markley, J.L.; Lutsenko, S. Solution structure of the N-domain of Wilson disease protein: Distinct nucleotide-binding environment and effects of disease mutations. PNAS. 2005, 103,
Fatemi et al. 2002

6. Excessive Copper Irving-Williams series:6,7 Fenton chemistry
Cr2+ < Mn2+ < Fe2+ <Co2+ <Ni2+ < Cu2+ > Zn2+
Shows increasing stability of protein complexes with divalent metals
Tight regulation of Cu2+ is required to prevent it from displacing other divalent metals
Dependent on atomic size and coordination
Fenton chemistry
Reactive oxygen species (ROS) generated through the oxidation of Cu+ → Cu2+ will accumulate in the liver and cause hepatic damage
So now, let’s look at why copper is bad for the body.
Here, we have the Irving Williams series. This series shows the reactivity and stability of metal protein complexes in the body. As you can see, Cu2+ has the highest reactivity compared to the other metals. Therefore, if given the opportunity, free cu2+ can displace another metal out of its protein complex, which will ultimately affect the protein’s original function. This is why copper is so tightly regulated in the body by metallochaperones such as GSH.
Next, we have the fenton series for copper. Essentially, copper can create free radicals which will harm the body. In wilson’s disease, there is an excess of copper ions in the liver because of a mutation in ATP7B. There are not enough metallochaperones in the liver to restrict copper from reacting with other substances, therefore, there is a high concentration of unbound and extremely reactive copper ions in the liver. The reactive copper ions will create free radicals, thus damaging the liver (which we see in Wilson’s disease), which ultimately allows for the leakage and escape of copper to the rest of the body, which can then harm other tissues via free radicals or displacement of metal-protein complexes.
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Figure ref:
Shan, Z.; Lu, M.; Wang, L.; MacDonald, B.; MacInnis, J.; Mkandawire, M.; Zhang, X.; Oakes, K.D. Chloride accelerated Fenton chemistry for the ultrasensitive and selective colorimetric detection of copper. Chem. Commun , 52,
General:
Kandanapitiye, M.S.; Wang, F.J.; Valley, B.; Gunathilake, C.; Jaroniec, M.; Huang, S.D. Selective Ion Exchange Governed by the Irving−Williams Series in K2Zn3[Fe(CN)6]2 Nanoparticles: Toward a Designer Prodrug for Wilson’s Disease. Ingor. Chem , 54,
Shan et al. 2016

7. Symptoms
First appear in the second or third decades of a person’s life1
Figure: Healthy liver vs liver with cirrhosis
Figure: K-F rings
Figure: Rigid dystonia
Liver
Eyes
Neurological
Elevated serum aminotransferases
Kayser-Fleischer(K-F) rings
Parkinsonism
Chronic hepatitis
Sunflower cataracts
Dysarthria
Cirrhosis
Seizures
Severe liver failure
Rigid dystonia
Insomnia
Choreiform movements
Most symptoms appear in the first appear in the second and third decades of a person’s life. The copper accumulation primarily occurs in the liver, eyes and the brain. In the liver, due to toxic levels of copper in the liver, this can cause liver failure which can cause further complications. As for the eyes, brown discolouration can form around the cornea which is known as Kayser-Fleischer rings. As for the brain, there are many neurological conditions that can occur as listed on the table.

8. Diagnosis Slit lamp eye examination1 Lab tests Genetic testing
Limited eye movement
Rusty or brown rings around iris
Lab tests
Serum ceruloplasmin concentrations ( < 100 mg/L) , urinary copper concentration (>100 μg/ 24 hr)
Genetic testing
Family screening to detect presymptomatic Wilson’s disease
Physical
Loss of coordination or muscle control
Liver or spleen disorder
Typically, the presence of Kayser-Fleischer rings and serum ceruloplasmin levels less than 100 mg/L are sufficient to establish the diagnosis. As mentioned before, ceruloplasmin, is a copper chaperone in the blood. When there is excess copper trapped in the liver, less copper is present in the blood. And due to this, there will be lower concentrations of copper chaperones, ceruloplasmin. Therefore, low levels of serum ceruloplasmin can be used as an indicator of Wilson’s disease.
As for genetic testing, if a family has a history of Wilson’s disease then the doctor can do genetic screening to help detect Wilson’s disease at an early stage.

9. Treatment is lifelong unless liver transplant is performed
Goal: normalize free plasmatic Cu8,9
Chelating Agents
Dietary Changes
Zinc Salts
Restrict Cu-rich foods
Liver
Chocolate
Mushrooms
Soy
Shellfish
Brain
nuts
Reduce intestinal uptake of Cu
Fewer side-effects
First choice with neurological symptoms or pediatric patients
Remove accumulated Cu
D-penicillamine
Trientine
Wilson Disease. Micromedex Solutions. Truven Health Analytics, Inc. Ann Arbor, MI.  Updated online Nov 16, 2016: (accessed Sep 27, 2017)
Patient compliance is essential to success of treatment, Death in 2-3 years after treatment cessation
CPS [Internet]. Ottawa (ON): Canadian Pharmacists Association; c2017 [updated 2012 Feb 28; cited 2017 Sep 27]. Valeant [product monograph]. Available from:  Also available in paper copy from the publisher.
Treatment is lifelong unless liver transplant is performed

10. Treatment- Chelating Agents
D-penicillamine9,10
2:1 drug: Cu complex => urine
L-isomer inhibits pyridoxine (vitB6), co-supplement
May worsen neurological symptoms
Interferes with collagen crosslinking, delay wound healing, stop 2-3 months prior to surgery
Also chelates other metals: Pb, Zn
CPS [Internet]. Ottawa (ON): Canadian Pharmacists Association; [updated 2012 Feb 28; cited 2017 Sep 27]. Valeant [product monograph]. Available from:  Also available in paper copy from the publisher.
Otomo, S.; Sasajima, M.; Ohzeki, M.; Tanaka, I. Effects of D-penicillamine on vitamin B6 and metal ions in rats (author's transl). Nihon yakurigaku zasshi. Japanese Journal of Pharmacology  76(1), 1-13.
D-penillamine structure:
Worsen neurological symptoms due to sudden increase in free Cu, or possibly reduction of GSH
Paper shows complex structure and hypothesizes why penicillamine worsens neurological symptoms:penicillamine oxidation in presence of Cu produces H2O2 which permeates through the membrane, and puts oxidative stress on human glioblastoma cells to induce apoptosis. (
L-isomer is toxic (

11. ZnSO4, ZnCl2, Zinc acetate, Zinc gluconate11
Treatment-Zinc Salts
ZnSO4, ZnCl2, Zinc acetate, Zinc gluconate11
Same efficacy, anions may effect tolerability
Zn induces enterocyte metallothionein (cysteine-rich protein), an endogenous metal chelator, traps Cu in enterocytes => feces
Inhibit lipid peroxidation, increase GSH in hepatocytes
Food interferes with absorption
Zinc Acetate. Micromedex Solutions. Truven Health Analytics, Inc. Ann Arbor, MI.  Updated online Sep 20, 2017: (accessed Sep 27, 2017) .
Anion doesn’t effect efficacy, maybe tolerability (sulphate, acetate, gluconate)
Dosing
Adults: 150mg of elemental Zn 3c/day, 30mins before each meal
Children (<50kg), 75mg
No alterations during pregnancy
Monitoring
24 hour serum and urine copper levels should be stable over time
Monitor transaminase levels, if increase, switch to chelators, why?
Mechanism of action
induces enterocyte metallothionein, an endogenous chelator of metals, thus favoring copper entrapment into enterocytes and its elimination in the feces with the normal shedding of intestinal cells
zinc is the inhibition of lipid peroxidation and the increase of available glutathione within hepatocytes, reducing oxidative damage
Cautions
Don’t simultaneously administer with chelating agent. Studies show increased efficacy but typically damaged liver can’t handle both, alternate timing if you must

12. Summary Slide
Autosomal recessive genetic disorder caused by mutation on ATP7B gene which prevents the excretion of excess copper by ATP7B transporter in the body
Copper accumulation in the liver, eyes and the brain leads to organ damage over time
Copper’s route through the body: [Gut] ATP7A/Cu+ → [Plasma] Ceruloplasmin (Cu+ → Cu2+) → [Liver] reductase (Cu2+ → Cu+) → hCRT/Cu+ → GSH/Cu+ → Atox1/Cu+ → ATP7B/Cu+ → [Plasma] Ceruloplasmin (Cu+ → Cu2+)
Under physiological conditions, Cu+ binds to MBD 5 and 6 for regular transport from the TGN to other tissues. Under high copper conditions, Cu+ binds to MBD 1 through 6 and the hyperphosphorylation causes biliary excretion of copper. -Nucleotide-binding domain binds ATP and the γ-phosphate is transferred to the Phosphorylation domain. Cu+ is shuttled out and the Actuator domain dephosphorylates the Nucleotide-binding domain to restart the cycle.
Cu2+ is low intracellularly due to the Irving-Williams series while unregulated Cu+ can interact with hydrogen peroxide and produce ROS (as seen in Wilson’s Disease)
Diagnosis
Typically, the presence of Kayser-Fleischer rings in the eyes and serum ceruloplasmin concentrations of less than 100 mg/L are sufficient to establish the diagnosis
Treatment (life-long medication and monitoring)
Reduce dietary intake of Cu-rich foods: liver, chocolate, mushrooms
Reduce gut uptake via zinc salt that increase metallothionein
Chelating agents: D-penicillamine-Cu complexes are excreted in the urine

13. References
1. Ala, A.; Walkeer A.; Ashkam K.; Dooley, J.; Schilsky, M. Wilson’s disease. Lancet. 2007, 369, Fatemi, N.; Sarkar, B. Insights into the mechanism of copper transport by the Wilson and Menkes disease copper-transporting ATPases. Inorganica Chimica Acta. 2002, 339, Huster, D. Wilson Disease. Best Pract Res Clin Gastroenterol. 2005, 24, Dmitriev O.; Tsivkovskii, R.; Abildgaard, F.; Morgan, C.T.; Markley, J.L.; Lutsenko, S. Solution structure of the N-domain of Wilson disease protein: Distinct nucleotide-binding environment and effects of disease mutations. PNAS. 2005, 103, Fatemi, N.; Sarkar, B. Insights into the mechanism of copper transport by the Wilson and Menkes disease copper-transporting ATPases. Inorganica Chimica Acta. 2002, 339, Kandanapitiye, M.S.; Wang, F.J.; Valley, B.; Gunathilake, C.; Jaroniec, M.; Huang, S.D. Selective Ion Exchange Governed by the Irving−Williams Series in K2Zn3[Fe(CN)6]2 Nanoparticles: Toward a Designer Prodrug for Wilson’s Disease. Ingor. Chem , 54, Shan, Z.; Lu, M.; Wang, L.; MacDonald, B.; MacInnis, J.; Mkandawire, M.; Zhang, X.; Oakes, K.D. Chloride accelerated Fenton chemistry for the ultrasensitive and selective colorimetric detection of copper. Chem. Commun , 52, Wilson Disease. Micromedex Solutions. Truven Health Analytics, Inc. Ann Arbor, MI. Updated online Nov 16, 2016: (accessed Sep 27, 2017) 9. CPS [Internet]. Ottawa (ON): Canadian Pharmacists Association; c2017 [updated 2012 Feb 28; cited 2017 Sep 27]. Valeant [product monograph]. Available from: Also available in paper copy from the publisher.\ 10. Otomo, S.; Sasajima, M.; Ohzeki, M.; Tanaka, I. Effects of D-penicillamine on vitamin B6 and metal ions in rats (author's transl). Nihon yakurigaku zasshi. Japanese Journal of Pharmacology (1), Zinc Acetate. Micromedex Solutions. Truven Health Analytics, Inc. Ann Arbor, MI. Updated online Sep 20, 2017: (accessed Sep 27, 2017)
Figure reference:
Huster, D. Wilson Disease. Best Pract Res Clin Gastroenterol. 2005, 24,
General reference:
Fatemi, N.; Sarkar, B. Insights into the mechanism of copper transport by the Wilson and Menkes disease copper-transporting ATPases. Inorganica Chimica Acta. 2002, 339,