Interval history. It used to be easy (? Easier) 3/12/2012. The best D--- Teaching Case in my Specialty (Henry Appelman Theme) F.Q.

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1 F.Q. 21 yo F (2011) The best D--- Teaching Case in my Specialty (Henry Appelman Theme) Or An Elderly Pathologist Tries to Assimilate Molecular Genetics into his Practice 1998 Age 7 abnormal LFT, high cholesterol cholecystectomy for stones ascites KF rings; liver biopsy 2003 biopsy 860 ug/g copper Interval history Chelation Rx (penicillamine, trientene) 2005 AST 85, ALT 80, AlkP 774, Bu 0.4, Bc 0; Pre-Alb 10.3 Serum bile acids x2 = 50; 149 (nl 4-16) A1AT M1,M3 lipids normal AFP normal normal Ceruloplasmin; low serum Cu; elevated 24 hr urine copper; Mutation analysis (Mayo) NEGATIVE for WD It used to be easy (? Easier) 19 y.o college student presented with GI symptoms (nausea, vomiting, abdominal distress) for 3-4 days; became jaundiced In hospital had striking serum transaminase elevations, hypoglycemia, coagulopathy despite all supportive care available at that time, became combative, stuporous, with elevated serum ammonia, and died in 4 d 1

2 The parents granted permission for an autopsy limited to the liver, with observation Bellevue Hospital After the Autopsy Review of patient s history with family revealed no GI, liver or neuropsychiatric symptoms But, examination of his school notebooks revealed changes in his handwriting attributable to early toxicity to basal ganglia And 24 hour urine sample from his 10 year old sister contained excess Copper Clinical Presentations Age dependent: younger patients manifest liver dysfunction, some as early as 2 years May be subtle, with signs of chronic disease, cirrhosis, gallstone disease, or resemble acute hepatitis; Up to 25% may have fulminant hepatic failure, hemolysis, even in adults (69% FHF are female) Pathology may resemble Auto-immune hepatitis 3 children had anti SM, 1 anti LKM (E Roberts) CNS signs reflect basal ganglia damage (ataxia, Parkinson slike), headache, seizures, psychiatric disturbances, dysautonomia may present in the 70s Renal Fanconi syndrome, nephrolithiasis 2

3 Alkaline Phos is exceptionally low in FHF of WD versus other causes (A Dhawan) X 3 children (ages 7,7, 14) from Italy with transaminase elevation and WD mutations; NO Cu tests positive; Rhodanine negative in Bx with microsteatosis, fibrosis (J Pediatr 2006) Transplant 4 weeks after onset of symptoms 15 y female Acute onset diarrhea 3 d later -TBil 10, Bc 4.3 (history of Doxycycline for acne 6 mo ago) Bc 34; Bu 7.6,,...Bc maximum 65 4 wk after onset; ALT 36; Alk Phos 42 Ceruloplasmin 4.6 (>18); Urine copper 250 (<8) Coagulopathic requiring plasma exchange Liver copper 1954 ug/ gm WD Mutation (Emory) c delcccaact, exon 2 - frameshift c.3955c>t(p.r1319x) premature translation stop Back to our Patient Age biopsy Copper 191 ug / gm (Nl 15-55) 3

4 Interval history 2008 lichen amyloidosis Feb 2009 AST 185, ALT 115, GGT 583, Alb 2.0, Bu 0.5, Bc 0.1, Nl PT, PTT, NH3 69 (<26); Cholesterol 207, Trigly nl Feb PFIC 3: novel nonsense and missense mutations of ABCB4 (MDR 3) exon 21 c.2563 C>T (p.q855x); exon 12 c.1283 T>C (p.v428a) Functions of MDR3 MDR = Multidrug resistance protein ABCB4 - ATP binding casette transporter (p glycoprotein family) coded on 7q21.1 Apical plasma membrane and Golgi Floppase - moves Phospholipids [phosphatidylcholine] from inner to outer plasma membrane and then into bile for solubilization of cholesterol via micelles Groen A, et al. Gastroenterol 2011; Aug 4. Interval history Apr sudden onset Nephrotic syndrome; MPGN type 1 responsive to Prednisone Feb 2010 lipase 383 (<110), nl amylase Nov 2010 secondary amenorrhea low estradiol, elevated testosterone; US: small ovaries, uterus; MRI nl pituitary, basal ganglia 2009 May 2011 AFP 22.8 (<10) Sept 15, 2011: AST 129, ALT 87, Alk P 249, GGT 391, Bu 0.2, Bc 0.5, Alb 2.0, PT, PTT nl 4

5 2 yr old` ABCB4 mutations Homozygous novel c.3170t>c (p.l1057p) Age explant Alexis Biochemicals MAb (P3II-26) --- amino acids Copper age 8 6 year old Wilson s 6 yr old boy jaundice AST 192 u/l, ALT 139 u/l, GGT 608 u/l Albumin 4.5 gm/dl, Conjugated bilirubin 0.6 mg/dl. Ceruloplasmin normal at 44.6 mg/dl Liver copper 863 ug/g, 24 hr urinary copper (66 mg/day) ABCB4 c.490t>g(p.w164g); c G>C 5

6 1. c,2563 C>T (p.q855x) Right in the middle of transmembrane domain 2. c.1283t>c (p.v428a) 7 AA into the 1 st ATPase domain 3. c.490t>g(p.w164g) Very N-terminal 4. c G>C aa1027 shortly before the 2nd ATPase domain Flens MJ et al. Cancer Res 1994; 54: 4557 Monoclonal Antibody to MDR3 P-gp fusion gene Glutathione S-transferase and coding sequence for aa Antibody: MDR3 P-glycoprotein fragment (aa ) denoted by red rectangle Scheffer GL et al. Cancer Res 2000; 60: 5269 Predictions WARNING: ALWAYS READ THE FINE PRINT 1. EXON 21, c,2563 C>T (p.q855x) Right in the middle of transmembrane domain This should produce a truncated protein without the last transmembrane domains and without the 2 nd ATPase domain; as a result, unlikely to be functional. However, because the antibody recognition site is N-terminal to the mutation, the antibody may still detect it. 2. EXON 12 c.1283t>c (p.v428a) 7 AA into the 1 st ATPase domain N-terminal to the antibody binding site, and because it is a substitution, protein should be made and possibly detected by antibody; however, the first ATPase domain may not work efficiently so the pump would be affected 3. c.490t>g(p.w164g) Very N-terminal Very N-terminal, but it is a substitution, so it should not produce a truncated protein. As a result, the antibody may be able to recognize it. 4. c G>C aa1027 Close to 2 nd ATPase domain This is very C-terminal, so would expect protein to be made and recognized by antibody; however, without the second ATPase domain, should not function properly Conclusions: The antibody we use could recognize proteins produced by all of these mutations. It is unclear whether the mutant proteins would be correctly localize in the canalicular membrane or whether or not the mutations affect the signal Fluorescent DNA sequence analysis was used to test for mutations in the 21 exons of the ATP7B gene. A mutation was not detected. Although unlikely, the result does not rule out the diagnosis of Wilson disease. We predict that some individuals...may have mutations that are not identified by the methods described above... Mayo Medical Laboratories The c.2563c>t (p.q855x) is predicted to result in a premature stop codon, thus it is categorized as a deleterious mutation (ACMG recommendations for standards for interpretation of sequence variation, Genetics in Medicine (4).394). Valine at position 428 of ABCB4 protein is evolutionarily conserved from C elegans to human. Although not validated for clinical uses, computerbased algorithms, SIFT and PolyPhen, predict p.v428a to be deleterious. Medical Genetics Laboratory Baylor College of Medicine The many manifestations of ABCB4 mutation Transient neonatal cholestasis Progressive familial intrahepatic cholestasis, type 3 ( elevated GGT ) Low phospholipid cholelithiasis syndrome Intrahepatic cholestasis of pregnancy (promoter region splicing mutations) Drug-induced liver toxicity Biliary fibrosis, cirrhosis in adults Davit-Sproul a, et al. Semin Liver Dis 2010; 30: 134. Wendum D, Barbu V, Rosmorduc O, Arrivé L, Fléjou JF, Poupon R. Aspects of liver pathology in adult patients with MDR3/ABCB4 gene mutations. Virchows Arch 2012 Feb 14. [Epub ahead of print] Predominant histological features: ductular reaction with no or mild fibrosis without cholangitis. Liver lesions previously unreported: biliary dysplasia, cholangiocarcinoma, small duct sclerosing cholangitis. Lipid crystals in bile ducts are suggestive. MDR3 immunostaining on formalin-fixed paraffinembedded sections does not seem to be sensitive for the diagnosis of heterozygous mutations. 6

7 Copper retention in cholestasis and hepatotoxicity Treatment for PSC (Nov 2011) Excess copper is thought to damage mitochondria via free radicals, lipid peroxidation. Medications that may be used include Cholestyramine; Ursodeoxycholic acid (ursodiol); Fat-soluble vitamins (D, E, A, K); Antibiotics for infections in the bile ducts Medications that quiet the immune system (prednisone, azathioprine, cyclosporine, methotrexate) Lessons? Final Lesson Many Mutations with variable manifestations 1. Excess Copper =/= Wilson s 2. Chronic cholestasis =/= Jaundice (GGT elevated, Bc normal) 3. ABCB4 - MDR3 defect has many guises 3. Immunohistochem =/= Mutations 4. serum AFP = pre- or actual Neoplasia in chronic cholestasis 5. Defining Ductular hepatocyte / Stem cell 6. Role for Copper chelation in all chronic cholestatic diseases Wilson s Age dependent: younger patients manifest liver dysfunction, some as early as 2 years May be subtle, with signs of chronic disease, cirrhosis, gallstone disease, or resemble acute hepatitis CNS signs reflect basal ganglia damage (ataxia, Parkinson s-like), headache, seizures, psychiatric disturbances, dysautonomia may present in the 70s PFIC3 Transient neonatal cholestasis Progressive familial intrahepatic cholestasis, type 3 ( elevated GGT ) Low phospholipid cholelithiasis syndrome Intrahepatic cholestasis of pregnancy (promoter region splicing mutations) Drug-induced liver toxicity Biliary fibrosis, cirrhosis in adults 7