Dhanpat Jain Yale University School of Medicine, New Haven, CT

Dhanpat Jain Yale University School of Medicine, New Haven, CT

Case history 15 years old female presented with fatigue. Found to have features suggestive of cirrhosis with esophageal varices, splenomegaly and mild thrombocytopenia Labs: Liver transaminases and alkaline phosphatase were mildly elevated AST 141 u/l, ALT 114 u/l, alkaline phosphatase 596 u/l, GGT 734 u/l, albumin 4.2 gm/dl and conjugated bilirubin 1.67 mg/dl Viral (Hepatitis A, B and C) were negative and autoantibody screen was also negative. Serum immunoglobulin levels were normal Increased 24h urine copper [> 100 mcg] Ceruloplasmin level was normal [38 mg/dl (ref 18 46)}, normal serum free copper [0.6 μg/dl (ref 0.0 10.0)] Liver biopsy

Case history Liver biopsy: Chronic hepatitis with moderate inflammatory activity, bridging fibrosis, moderate bile duct proliferation Rare glycogenated nuclei Rare Mallory Denk bodies Markedly elevated hepatic copper content (1471 μg/g).? Diagnosis

Wilson Disease (Leipzig criteria, Liv International 2003:23 139 142) Parameter Score Points K F rings Present Absent 2 0 Neurophychiatric symptoms (or typical MRI changes) Present Absent 2 0 Coombs + hemolytic anemia Present Absent 1 0 Urinary copper (in the absence of acute hepatitis Normal 1 2X ULN >2X ULN N, 5X ULN after challenge with 2X0.5 D penicillamine 0 1 2 2 2 Liver copper quantitative Normal Up to 5X ULN >5X ULN 1 1 2 2 Rhodanine + hepatocytes (if quantitative Cu not available Present Absent 1 0 Serum ceruloplasmin Mutation analysis Normal 10 20 <10 Dis causing mutation on both chromosome Dis causing mutation on one chromosome No mutation detected Total 4 0 1 2 4 1 0 > 4 = Wilson disease; 2-3 probable Wilson diseae; 0-1 Wilson disease unlikley

Case history Diagnosis: Wilson disease with cirrhosis Treatment : Started on zinc therapy, but was switched to Trientine due to GI side effects. After >12 months of treatment her transaminase levels did not improve and urine copper remained elevated. Mutations for Wilson disease (ATP7B)were negative Wilson disease unlikely Another liver biopsy was performed at this time, which is being presented

? Diagnosis Cryptogenic cirrhosis

Cryptogenic cirrhosis Incidence varies between 5 30% Constitutes about 5 10% of liver transplants Etiology of cryptogenic cirrhosis: NASH Occult AIH Occult viral hepatitis: C, B, G and E, Hepatitis X Celiac disease Hepatic vascular disorders Occult biliary disorders Occult alcohol/tash

Cryptogenic cirrhosis Metabolic/ inherited disorders Bile salt transporter defects Bile salt synthetic defects Mitochondrial disorders Short telomere syndrome Keratin 8 and 18 mutations Glutathione S transferase mutations A1At disease Wilson disease Iron overload disorders (HFE or non HFE)

Cryptogenic cirrhosis Molecular diagnostics

ABCB4 mutations (MDR3) Nucleotide change Amino acid change Location Zygosity Reference/Co mments c984t>g py328 Exon 9 heterozygous Nonsense mutation c3218g>a pc1073y Exon 25 heterozygous Unclassified novel variant

Tranuner M, et at. Semin Liv Dis 2007:27:77-98

?? Diagnosis Progressive familial intrahepatic cholestasis (PFIC 3)

PFIC3: MDR3 deficiency/defect Mutations in ABCB4 gene leading to MDR3 protein Highly variable disease presentation 1. Neonatal cholestasis (PFIC3) 2. Intra hepatic cholestasis of pregnancy 3. Drug induced liver disease 4. Low phospholipids associated cholelithiasis (LPAC) 5. Adult biliary cirrhosis 6. TPN associated cholestasis

MDR3 deficiency (PFIC3) Histology: (Highly variable depending on the phenotype) Preserved lobular architecture or minimal disarray Giant cell transformation and ballooning are absent or infrequent Diffuse hepatocellular and occasional canalicular cholestasis Ductular proliferation and portal fibrosis usually with progression of disease EM: Cholesterol crystal may be seen bile IHC: Usually absent or MDR3 staining

BSEP MDR3

PFIC3: MDR3 deficiency/defect Progression is variable some develop cirrhosis as early as 5 th month of life while some in adulthood Infants and young children: Hepatomegaly and acholic stools during 1 st month of life, or cholestasis or growth failure Older children and adults: Slower progression to cirrhosis without overt clinical cholestasis (cryptogenic cirrhosis)

Aspects of liver pathology in adult patients with MDR3/ABCB4 gene mutations Wendum D, et al. Virchows Arch 2012: 460:291 298 Patient group Age range M/F Liv pathology Cirrhosis MDR3 IHC LPAC (n=4) 26 31 2/2 Mild ductular reaction, periductal edema, intraductal cholestrol crystals 0 N Recurrent cholangitis( n=4) Hepatolithi asis with cholangitis (n=2) 24 57 2/2 Mild ductular reaction,sclerosing cholangitis, intraductal cholestrol crystals 54, 63 0/2 Mild ductular reaction, intraductal cholesterol crystals, cirrhosis 0 1 weak staining 1 N Chronic cholestasis (n=3) 34 55 1/2 Mild ductular reaction, cirrhosis 1 N (All patients were heterozygous for MDR3 mutations)

Diagnosis of PFIC 3 High serum bile salts and high GGT Histology variable Chronic non specific cholestasis Biliary cirrhosis IHC for MDR 3 Useful screening tool Preserved staining doesn t exclude the diagnosis ABCB4 sequencing

Treatment and prognosis Ursodeoxycholic acid (URSO) Cirrhosis: Liver transplantation

Key points Need to carefully evaluate cases of Cryptogenic cirrhosis carefully for subtle histologic clues Correlation with other lab data Molecular diagnostics Second chance: Disease recurrence in the allograft

Thank You

Contribution of NGS to the genotyping of patients with hereditary cholestasis and cholelithiasis: Barbu VD, et al. Hepatology, Oct 2014: 289A. 57 Patients with suspected hereditary intrahepatic cholestasis or cholelithiasis M:F= 1:2 37 Patients had variants in ABCB4 (14) ABCB11 (9) ABCC2 (14) ATP8B1 (5)

Case history 15 years old female presented with fatigue Found to have features suggestive of cirrhosis with esophageal varices, splenomegaly and mild thrombocytopenia Labs: Liver transaminases and alkaline phosphatase were mildly elevated AST 141 u/l, ALT 114 u/l, alkaline phosphatase 596 u/l, GGT 734 u/l, albumin 4.2 gm/dl and conjugated bilirubin 1.67 mg/dl Viral (Hepatitis A, B and C) were negative and autoantibody screen was also negative. Serum immunoglobulin levels were normal Increased 24h urine copper [> 100 mcg] Ceruloplasmin level was normal [38 mg/dl (ref 18 46)}, normal serum free copper [0.6 μg/dl (ref 0.0 10.0)] Liver biopsy