Clinical Case Conference

HPI
17 year old female presents with a 6 day history of increasing fatigue and diffuse abdominal pain
Dark colored urine, icteric sclera and recent onsent of pruritis
Denies fever, emesis, diarrhea and chills.

PMHx
Epilepsy (GTC) since 8 years old
Medications: Zonisamide, Trileptal
Allergies: Tegretol (rash)
Family Hx
Non-contributory
Social Hx
Increasingly poor school performance
Denies sexual activity, alcohol and drugs

Laboratory
Total bilirubin 25.2, conjugated 13
Total protein 5.1, albumin 2.8
Amylase 53, Lipase 45
Ammonia 68
AST 86, ALT 42, ALP 82
Hemoglobin 11.7
PT 24.1, PTT 43, INR 2.26
Abdominal Ultrasound
Gallbladder wall thickening
Normal CBD
Unremarkable liver and pancreas

Physical Exam
General: tired, easily aroused
HEENT: PERRLA, NCAT, icteric sclera
CV: I/VI SEM at LSB, no radiation
Resp: CTA bilaterally
Abdomen: soft, hepatomegaly 4 cm below rcm, no palpable spleen, NABS, no mass
Derm: jaundice
Neuro: answering questions appropriately

Viral, bacterial and stool cultures sent
Started Vitamin K and Ursodiol
Repeat abdominal ultrasound
Hepatosplenomegaly
Normal doppler
Became more lethargic, developed peripheral edema, tachycardia, tachypnea and hypotension
Hospital Course

DIFFERENTIAL DIAGNOSIS

Acute Liver Failure: Infant
Infections Drug/Toxin Cardiovascular Metabolic
Herpes simplex
Echovirus
Adenovirus
EBV
Hepatitis B
Parvovirus
CMV
ECMO
Hypo. left heart
Shock
Asphyxia
Myocarditis
Galactosemia
Tyrosinemia
Iron storage
Mitochondrial
HFI
Fatty Acid Ox.
Niemann-Pick
No clear etiology in at least 63% of cases
Tylenol

Acute Liver Failure: Child
Infections Drug/Toxin Cardiovascular Metabolic
Hep A,B,C,D
EBV
CMV
Herpes
Leptospirosis
Valproic acid
Isoniazid
Halothane
Tylenol
Mushroom
Phosphorus
ASA
Myocarditis
Heart surgery
Cardiomyopathy
Budd-Chiari
FA oxidation
Reye Syndrome
Leukemia
Autoimmune
α1-Antitrypsin

Acute Liver Failure: The Adult
Infections Drug/Toxin Cardiovascular Metabolic
Hep A,B,C,D,E
Yellow fever
Dengue Fever
Lassa Fever
Tylenol
Tetracycline
Halothane
Valproic Acid
MAO Inhibitors
Isoniazid
Bacillus cereus
Budd-Chiari
Acute failure
Heat stroke
Shock
Fatty Liver
Wilson’s
Autoimmune

Hospital Course
Hemoglobin steadily falling 11.7 to 8.8
Reticulocyte count 6.6%
Increasing total bilirubin, peak 43.7
GGT peaked at 325

What would you do?

CMV, EBV negative
Hepatitis A,B,C negative
Autoimmune markers negative
Copper 120 (nl)
Ceruloplasmin 18 (low)
Low factor levels
Further Analysis

Pathology – H&E
Peri-portal fibrosis
Peri-portal glycogenated hepatic nuclei
Kupffer cell hyperplasia
Hepatocyte enlargement

Pathology – H&E
Hepatic steatosis
Microvesicular then macrovesicular
Cholestasis
False positive
alcoholic steatohepatitis

Copper Stain
False positive
Cholestatic liver disorders
Indian Childhood Cirrhosis
False negative
Copper not present in hepatocyte
Released secondary to cell injury
Cytosolic copper more difficult to appreciate than granular lysosomal copper
Copper = 1153 μg/g dry weight

WILSON’S DISEASE

Copper Physiology
Most abundant in unprocessed wheat, dried beans, peas, shellfish, chocolate, liver, kidney
Impair copper absorption
Zinc, cadmium, ascorbic acid
Vegetarian diet
Aid copper absorption
Gastrointestinal secretions
Absorbed copper is bound to the protein metallothionein or complexed to amino acids and transported into portal system

Copper Metabolism

Copper Metabolism
Copper is transported into hepatocytes by the human copper transporter (hCTR)
In hepatocyte, copper interacts with the ligands metallothionein, glutathione and HAH1
Bind and detoxify excess copper
Transfer or store copper
Provide copper to chaperones
Chaperones incorporate copper into essential proteins or assist in copper excretion into bile
CCS
COX17
ATOX/HAH1

Copper Metabolism
ATP7B sorts copper and incorporates into secretory vesicles and ceruloplasmin

ATP7B
Copper transporting P-type ATPase
13q14-13q21
21 exons
6 cysteine-rich copper binding sites
8 transmembrane domains
CPC (cysteine-proline-cysteine) common to other metal transporters
Copper transporting P-type ATPase

ATP7B
>200 mutations identified
Most small deletions or missense mutations
Missense: neurologic and later presentation
Deletions: hepatic and earlier presentation
Highly expressed in liver and kidney

ATP7B
Makes copper available for ceruloplasmin synthesis and transport of copper into vesicles
Long-Evans Cinnamon (LEC) rat
ATP7B defects involve transport of copper into the vesicular pathway from Golgi apparatus to the canaliculus

Clinical Presentation
Combined date from Walshe and Scheinberg & Sternleib

Hepatic
Acute hepatitis
25%
Fulminant hepatic failure
Liver transplant
May also present after discontinuing copper chelation
Chronic active hepatitis
10-30%
Absence of other symptoms in Wilson’s patients should prompt biochemical screening in those <40 years
Cirrhosis
Absence of other symptoms in Wilson’s patients should prompt evaluation >4 years

Laboratory
Mildly elevated serum aminotransferase levels
Low alkaline phosphatase
Serum alkaline phosphatase to total bilirubin ratio ratio < 2

Neuropsychiatric
40-45% as presentation
Most common 2nd to 3rd decades of life
Extrapyramidal and cerebrellar dysfunction
Migraine headaches
Seizures
Denning et al found 13 of 200 patients with Wilson’s disease had seizures
Prevalence rate 6.2%, exceeding epilepsy frequency by tenfold
Gait disturbances secondary to tremor and dystonia

13 year old boy presents to a neurologist for evaluation of a
deterioration in his handwriting.
Grade 5
Grade 6

Imaging
CT abnormalities
73% ventricular dilation
63% cortical atrophy
55% brainstem atrophy
45% hypodensity in basal ganglia
10% posterior fossa atrophy
Williams and Walshe

Kayser-Fleischer Ring
Superior poles of cornea, then inferior involvement
Copper chelators result in resolution over 3-5 years
Occurs after hepatic saturation of copper
Virtually always present when neurological or psychiatric symptoms develop
Frequently absent in children without neurolgic involvement but with hepatic symptoms
False positive: hepatitis, cholestasis, primary biliary cirrhosis, TPN

Sunflower Cataract
Copper deposition in anterior and posterior lens capsule
False positive with foreign body lodged intraocularly (chalcosis)

Other
Renal
Proximal renal tubular dysfunction (Fanconi’s syndrome)
Renal insufficiency
Nephrocalcinosis
Hematologic
Coombs negative hemolytic anemia
Cardiac
Autonomic dysfunction
Cardiomyopathy
Skeletal
Bone demineralization
Dermatologic
Acanthosis nigrans

Diagnostic Studies

Genetic Analysis
Genetic studies are becoming more available but limited
Kumar et al characterized ATP7B mutations by restriction fragment length polymorphism (RFLP)
3 mutations, Q1256R, A1003T and I1102T were characterized associated with restriction sites for AccII, Bsh1236I and EcoRI
Mutation analysis in combination with RFLP is useful for positive diagnosis of asymptomatic Wilson’s disease and elucidation of carrier status

Treatment Goals
Reduce copper accumulation by
Enhancing urinary excretion
Decreasing intestinal absorption

Therapy

Liver Transplant
Life-saving
Acute fulminant hepatic failure
Decompensated cirrhosis with progressive end stage liver disease

Long-Evans Cinnamon Rats
A deletion of 900 base pairs at the 3’ end of ATP7B gene, eliminating normal gene product
Previous adenoviral gene transfer resulted in successful but transient gene expression
In vivo administration of HIV-1-derived lentiviral vectors (LV) expressing the ATP7B gene
Transplantation of lentivirally modified autologous hepatocytes

Results

Conclusions
Liver copper levels were lowered in all treatment groups compared to untreated LEC rats
Histological analysis showed only fibrous septa after LV-ATP7B treatment but still with round-cell infiltration
Lentiviral ATP7B gene transfer is feasible in Wilson’s disease

Wilson’s Disease
Wilson disease is a disorder of copper transport resulting in copper deposition in multiple organs
Clinical manifestations may be severe, but the disease is treatable if diagnosed early

Bibliography
Kumar et al. Familial gene analysis for Wilson disease from north-west Indian patients. Annals of Human Biology, March-April 2006; 33(2): 177-186.
Goyal and Tripathi. Sunflower Cataract in Wilson’s disease. J Neurol Neurosurg Psychiatry, July 2000; 69:133.
Merle et al. Lentiviral gene transfer ameliorates disease progression in Long-Evans cinnamon rats: An animal model for Wilson disease. Scandinavian Journal of Gastroenterology, 2006; 41: 974-982.
Suchy et al. Liver Disease in Children, 2nd Ed. Copper and Iron Storage Disorders. Jan 2001; 595-691.
Walker et al. Pediatric Gastrointestinal Disease, 4th Ed. Acute Liver Failure. 2006. 1491-1507.
http://www.ohsu.edu/biochem/lutsenko/chaperones.cfm