Hot Topics in Liver Disease Wilson Disease

a

-1-Antitrypsin Deficiency

Norman L. Sussman, MD Baylor College of Medicine St. Luke’s Center for Liver Disease S

Copper Absorption and Excretion

• • • CMT1: Copper membrane transporter 1 ATOX1 Metallothionein   Copper is a cofactor for a number of enzymes  Ceruloplasmin (CPN), cytochrome c oxidase, dopamine b hydroxylase, superoxide dismutase, tyrosinase ATP7B (P-type ATPase)  Catalyzes self-phosphorylation of aspartate in the Cu 2+ pump   Excretes copper into the canaliculus Attaches copper to ceruloplasmin

Samuel Alexander Kinnier Wilson 1878 – 1937

     Born in Cedarville, NJ, moved to Edinburgh at one year of age after the death of his father Graduated with MB from University of Edinburgh in 1902 Trained in Paris with Pierre Marie and Joseph Babinski Returned to King’s College in London MD in 1912: "Progressive lenticular degeneration” and introduced the word “extrapyramidal”

Question 1 Which of the following is true of Wilson disease (one answer):

A.

B.

C.

D.

Autosomal dominant disease.

Is caused by a mutation in the ceruloplasmin gene.

Is caused by a mutation in the ATP7B gene.

Is always associated with discoloration of the cornea (Kayser-Fleishcher rings).

23% A.

17% B.

47% C.

13% D.

The Genetic Basis of Wilson Disease

Hepatoleticular Degeneration Autosomal Recessive

  Copper storage disease – caused by defective copper export   Accumulates in the liver, brain, cornea Not added to ceruloplasmin  shortened half-life (low serum level)  Not added to AP  low level Mutations in ATP7B - multiple mutations   The gene was identified in 1993 Prevalence ~30/million population

Kayser–Fleischer Rings

50% in patients with liver disease 98% in patients with neurologic disease

Schrag A, Schott JM. N Engl J Med 2012;366:e18.

Question 2 Which of the following is not true (one answer):

A.

B.

C.

D.

Wilson disease may cause acute hepatitis in children.

Wilson disease may cause acute liver failure Adults with Wilson disease may have no definite liver disease Neurological disease is the most common presentation in newborns

9% A.

14% B.

40% 37% C.

D.

Wilson Disease

Clinical Manifestations

  Hepatic, neurologic, psychiatric, hemolysis (ALF) – presentation may vary, even within a family  Liver: 18-84%   Neurologic: 18-73% Psychiatric: 10-100% Usually present at 5-35 years (up to 70s)   Children typically present with liver disease (9-13 years) Older patients usually present with neurologic and/or psychiatric symptoms (15-21 years)

Wilson Disease

Clinical Manfestations

    Mild: abnormal liver tests, hepatomegaly, NAFLD Advanced: cirrhosis (with all complications) Acute liver failure (ALF) Neurologic  Movement disorders (arms, gait, swallowing)  Psychiatric

Sunflower Cataract

Wilson Disease – Diagnosis

   Classic presentation (about 50% of patients)  5-40 years old, cirrhosis, neurological findings, K-F rings, low ceruloplasmin Genetic diagnosis – better for confirmation  Expensive  Not universally available  Sometimes inconclusive The rest – scoring system

Wilson Disease – Diagnosis

   Very low ceruloplasmin (CPN) is strongly predictive Uric acid – usually low, but not validated Non-CPN bound copper is usually >25 m g/dL (250 m g/L) in untreated patients (normal = 15 m g/dL)  Binding is ~3.15 m g of Cu 2+  Free Cu 2+ = serum Cu 2+ per mg CPN ( m g/dL) – 3x CPN (mg/dL)   Google: ceruloplasmin-free copper calculator Caveat: may be elevated in chronic cholestasis, copper intoxication, and ALF of any cause

Wilson Disease – Diagnosis

24-Hour Urine Copper

  Diagnostic at 100 m g/24 hours (1.6 m mol/24 hours) in symptomatic patients   Many labs use 40 m g as the cutoff May be <100 m g at presentation in 16%-23% Penicillamine challenge (500 mg at 0 and 12 Hr)  1,600 m g (25 mol) copper/24 hours  Validated in children, but not adults

Wilson Disease – Diagnosis

Liver Biopsy

     Early: mild steatosis (micro and macrovesicular) May show classic histological features of AIH Cirrhosis – macronodular and micronodular  May be absent, even with neurological disease Copper stain is unreliable (metallothionein-bound)  Absence of stainable copper does not exclude WD ALF – massive necrosis on a background of cirrhosis

Copper Stains

Rhodanine

Electron Microscopy

Wilson disease with steatosis

    Mitochondria vary in size and shape Increased density of the matrix Inclusions including lipid and fine granular material (copper) Pathognomonic increased intracristal space & dilatation of the tips of the cristae Elongated mitochondria and dense deposits. X15,000 Iancu & Manov, Technion, Haifa

Wilson Disease – Diagnosis

Hepatic Parenchymal Copper Concentration

    Copper copper content >250 m g/g dry weight  8% were <250, and all were >95 m g/g*   Heterozygotes rarely exceed 250 mg/g Chronic cholestasis and copper intoxication Heterogeneous distribution – biopsy size Copper stain – unreliable (metallothionein-bound) Radiocopper labeling – rarely performed *Merle U, Schaefer M, Ferenci P, Stremmel W. Gut 2007;56:115-120

MRI Brain – T2-weighted Images

Hyperintense signal in the bilateral thalami and putamen “Face of the giant panda” in the midbrain with high signal in tegmentum and normal red nuclei (arrow) “Face of miniature panda” in pons with hypointensity of central tegmental tracts (arrow) with hyperintensity of aqueductal (4 th ventricle)

Genetic Analysis

    Screening after the proband is diagnosed Over 300 mutations around ATP7B Not all gene changes cause Wilson Disease Single predominant mutation in some populations   Sardinia, Iceland, Korea, Japan, Taiwan, Spain, Brazil and Canary Islands Eastern Europe –H1069Q mutation

Wilson Disease – Therapy

Uniformly Fatal Without Therapy

 British anti-Lewisite (BAL, dimercaprol) – introduced in1951  Developed to treat arsenic-based chemical warfare agent   Still used for metal poisoning (arsenic, antimony, gold, etc.) Free sulfhydryl groups binds copper

Wilson Disease – Therapy

D-penicillamine – John Walsh, 1956 Chelates, induces metalothionein, prevents collagen cross linking, immunosuppressant

   Initial worsening of neurologic symptoms in 10-50% Intolerance – 30% of patients   Early – fever, eruptions, lymphadenopathy, neutropenia, thrombocytopenia Late – lupus-like (ANA+) – renal injury (hematuria, proteinuria), skin lesions Monitor with 24-hr urine and serum free copper  1,000 m g (16 m mol) (early) to 200-500 m g (3-8 m mol)/24h  Non-ceruloplasmin copper  Non-compliance vs inadequate chelation

Wilson Disease – Therapy

Trientine (triethylene tetramine dihydrochloride or 2,2,2-tetramine, trien

     Polyamine-like structure - copper forms a stable complex with 4 nitrogens in a planar ring Neurological worsening less common then with penicillamine

Do not co-administer with iron:

 Chelates iron  toxic complex Check effectiveness as for penicillamine Non-adherence: non-ceruloplasmin bound copper is over 150 m g/L

Wilson Disease – Therapy

Zinc

   Zinc induces enterocyte metallothionein which has a greater affinity for copper than for zinc  binds copper in the enterocyte  feces Used for maintenance or early cases – less toxic and more specific than chelating agents Dosed in mg elemental zinc – 50 mg TID for adults

Wilson Disease – Therapy

Ammonium Tetrathiomolybdate

     Strong de-coppering agent – two mechanisms: Interferes with intestinal uptake if taken with meals Binds copper from plasma if taken between meals TM remains an experimental therapy in the USA Does not cause neurological deterioration

And Now for Something Completely Different

Question 3 Which of the following is true of AAT

A.

B.

C.

D.

The null mutation causes severe liver disease A mutation in one AAT gene may predispose a patient to alcoholic liver disease Liver disease and lung disease are both caused by low circulating levels of the enzyme Patients with the SS phenotype are particularly predisposed to liver disease

16% 23% 26% 35% A.

B.

C.

D.

a

-1 Antitrypsin (AAT) Deficiency

A Tale of Two Diseases

 Common in Caucasians – 1-3% of pts with COPD   ~ 25 million people carry at least 1 deficient gene.

The mutant protein is not secreted    Liver: gets stuck in the hepatocyte  injury Lung: Inadequate protection from leukocyte proteases e.g. trypsin, elastase, proteinase 3 May affect neonates (hepatitis) or adults (CLD, COPD)

a

-1 Antitrypsin

Serpins – enzyme inhibitors

     Acute phase protein 52-kDa protein - 394 amino acids Produced in the liver – secreted into blood Complex folding – globular tertiary structure Neutrophil elastase binding site

a

-1 Antitrypsin (AAT) Deficiency

a

-1 Antitrypsin (AAT) Deficiency

     Pair of genes at the protease inhibitor (Pi) locus. The SERPINA1 (formerly Pi) gene on chr. 14 More than 100 allelic variants – classified based on serum levels of AAT protein. M alleles (Pi MM) are the most common  Normal serum level Most patients with clinical disease are homozygous ZZ or SS or heterozygous MS, MZ, or SZ

a

-1 Antitrypsin (AAT) Deficiency

PAS-positive, diastase-resistant granules

a

-1 Antitrypsin (AAT) Deficiency

      Normal is Pi MM – mutant alleles are S, Z, null Patients with liver disease typically have Pi ZZ Liver disease has no relationship to serum AAT activity May progress to cirrhosis and/or HCC No specific therapy Cirrhosis and HCC are indications for liver transplantation

a

-1 Antitrypsin (AAT) Deficiency

Frequency

Country Sweden Netherlands France Spain Italy Portugal Australia New Zealand Canada Pi ZZ 0.06% 0.03% 0.01% 0.02% 0.02% 0.02% 0.02% 0.02% 0.02% Pi MZ - - 2% - 2.4% 2.5% 2.7% 4.5% 2.7% Pi SZ 0.05% 0.04% 0.15% 0.2% 0.06% 0.3% 0.13% 0.17% 0.11% Year 1976 1980 2003 2003 2003 2003 2003 2003 2003 Kok et al. Netherlands Journal of Med, 2007

a

-1 Antitrypsin (AAT) Deficiency

Frequency

Region

Rochester,MN Minneapolis, MN Tucson, AZ Long Beach, CA Los Angeles, CA Rochester, NY Rochester, NY St. Louis, MO Total

Pop/No. Tested

Healthy donors/212 Blood donors/904 General pop/2,944 7th-graders/1,380 Blood donors/2,010 Consecutive/500 Random pts/930 Blood donors/1,933 10,813

Pi MZ

5 (2%) 25 (2.8%) 88 (3.0%) 34 (2.5%) 59 (2.9%) 18 (3.6%) 21 (2.3%) 42 (2.2%) 292 (2.7%)

Pi MS

7 (3.3%) 37 (4.1%) 208 (7.1%) 110 (8.0%) 95 (4.7%) 30 (6.0%) 60 (6.4%) 130 (6.7%) 670 (6.2%) Graziadei IW et al. Hepatology 1998 (4):1058-63.

a

-1 Antitrypsin (AAT) Deficiency

Mayo: 641 OLT patients (Mar 1985 - Dec 1996)

  Phenotyping in 599 patients listed for liver transplant – examined for the Z allele 51 patients identified, 49 with MZ allele (8.2%)   2x frequency reported in the US population (2-4%) Pi MZ found in 27% with cryptogenic cirrhosis Graziadei IW et al. Hepatology 1998 (4):1058-63.

a

-1 Antitrypsin (AAT) Deficiency

Heterozygotes Liver Disease Swedish Study (1972-1974) – 200,000 Neonates Screened

 120 Pi ZZ (0.06%), 2 Pi Z-, 54 Pi SZ and 1 Pi S  Only 14 Pi ZZ had prolonged jaundice – nine with severe disease  All appeared healthy at six months of age    Infants with a Pi SZ phenotype had no signs of liver disease.

At 16 years, elevated liver enzymes were found in 17% of Pi ZZ adolescents and in 8% of Pi SZ Adults with liver disease in infancy were clinically healthy Kok et al. Netherlands Journal of Med, 2007

a

-1 Antitrypsin (AAT) Deficiency

Heterozygotes Liver Disease Northern Itlay – Phenotyping in umbilical cord blood

  Early childhood:   Pi SZ: 5% had elevated enzymes Pi MZ: 7% had elevated liver enzymes At the age of 5 and 10 years, none had liver disease.

Kok et al. Netherlands Journal of Med, 2007

a

-1 Antitrypsin (AAT) Deficiency

Liver Transplantation – Mayo Clinic 1987 2012: 5,246 patients

    73 patients – 50 with ZZ and 23 with SZ phenotype Mean age 52.8 years, 76% men Pre-OLT AAT levels were lower in ZZ than SZ patients  28.3 vs 58.0 mg/dl, p=0.0001

Coexistent liver disease    8% in ZZ 43% in SZ 90% in MZ Carey et al. Liver Transplantation, 2013 - epub

Summary

  Wilson disease – copper storage  Caused by defect in ATP7B – low CPN  Workup includes CPL, copper levels, biopsy  Treatment with chelating agents and/or zinc AAT deficiency    Pi ZZ may cause liver disease and HCC Pi MZ and Pi SZ may predispose to liver injury No treatment is available for liver disease besides liver transplantation