10/3/2012. I have no financial relationships with any commercial entity to disclose. Alagille Syndrome: Research Advances Changing Clinical Care

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1 THE HOSPITAL FOR SICK CHILDREN Alagille Syndrome: Research Advances Changing Clinical Care I have no financial relationships with any commercial entity to disclose Binita M. Kamath, MBBChir MRCP MTR Division of Gastroenterology, Hepatology and Nutrition The Hospital for Sick Children University of Toronto State of the Art Research Lecture: October 2012 LEARNING OBJECTIVES 1. Describe how recent studies in Alagille Syndrome (ALGS) are changing the management of affected patients: a. The expansion of disease phenotype b. The identification of a novel disease-causing gene 2. Provide an update on ongoing research endeavors and their potential impact Background OUTLINE Clinical studies that impact ALGS Management Renal involvement Pancreatic insufficiency Predicting liver disease outcomes Liver Transplantation Genetic advances in ALGS Identification of NOTCH2 Current research BPA st. 76% PVS 8% TOF 12% ASD 5% VSD 5% 1

2 Renal involvement in ALGS Retrospective review of 466 JAG1 mutation-positive ALGS individuals Recent Clinical Studies of ALGS 187 with evaluable renal information 39% with renal involvement Renal dysplasia is most common abnormality (defined by increased echogenicity on ultrasound, with or without renal cysts and reduced size) Implications for liver transplant care Kamath et al, AJMG 2011 Pancreatic insufficiency in ALGS Steatorrhea prevalent in ALGS Pancreatic insufficiency (PI) and/or cholestasis? Data supporting PI limited ChiLDREN consortium study: 42 ALGS children underwent fecal human elastase measurement 95% had normal results 5% indeterminate (n=2) PI is not a clinically important problem in ALGS Kamath et al, JPGN 2012 Predicting Hepatic Outcomes in ALGS ALGS: variable phenotype Unpredictable natural history No known genotypic or histologic predictors of liver disease outcome Study design: Retrospective review of serial biochemistry from 0-5 yrs of age in 33 ALGS patients Patients stratified to mild or severe outcome groups based on data from age 10yrs or above Kamath et al, JPGN

3 Ongoing multicentre study 7 centres, n=101 Liver Transplantation in ALGS Univariate analysis confirms same risk factors Mixed model analysis and recursive partitioning identifies new (lower) thresholds TB of 3.8mg/dL between 1-2 years of age is critical threshold Awaiting validation in separate cohort Goal is to develop predictive model/score Mouzaki et al, Abstract AASLD 2011 Arnon et al, Pediatric Transplantation 2010 & Kamath et al, Liver Transplantation 2012 Complications following Liver Transplantation Linear Growth Post-Transplant in ALGS & BA Change in Height z-score Height z-score Kamath et al, Liver Transplantation 2012 Kamath et al, Liver Transplantation 2012 Human Notch Signaling Ligands (Jag1,2 Delta-like 1,3,4) Genetic Advances in ALGS Notch receptors (Notch 1,2,3,4) cytoplasm nucleus NICD bhlh genes (hes, hey) Inhibition of differentiation 3

4 ALGS Genetics First disease gene, JAGGED1, identified in 1997 >400 mutations reported 75% protein-truncating (nonsense, frameshift) 15% missense Remainder are gene deletions No clear genotype-phenotype correlations Mutation/deletion detection in clinically defined patients = 94% J1N2+/- WT Mouse models of ALGS Jag1 heterozygote Mild inner defect Jag1 homozygote Embryonic lethal due to vascular defect Jag1 homozygote Abnormal bile ducts Heart defects Eye anomalies Hypoplastic kidneys Warthen et al, Hum Mut 2006 McCright et al, Development 2002 NOTCH2 Mutation in ALGS Proband NOTCH2 Mutations in ALGS Notch2 Facies Cholestasis Pulmonic stenosis Neonatal renal failure No JAG1 mutation Protein Truncation c g->a 2 families in original report Renal predominant phenotype? 8 additional probands identified recently (international cohort) 3/6 mutations ti inherited, it 1 de novo Mutations included missense, frameshift and splice site mutations Luciferase reporter assay demonstrates mutations are damaging to Notch signaling in vitro McDaniell et al AJHG, 2006 Kamath et al, Hum Mut 2012 Facial Features in ALGS JAG1-related Kamath et al, Hum Mut 2012 NOTCH2-related 4

5 ALGS Time for a Redefinition Genetically heterogeneous disease 2 disease genes Consider ALGS even if features atypical Expansion of phenotype allows for 7 likely defining criteria Is JAG1 related-disease without hepatic involvement ALGS? ALGS is the tip of the iceberg of diseases associated with disruptions of the Notch signaling pathway Current Research in ALGS A study to identify genetic modifiers of liver disease severity Stratification protocol for hepatic phenotype Classification Clinical Features (liver) Objective: To identify genetic modifiers that are associated with severity of liver disease in ALGS Genome-wide Association study (GWAS) All subjects are JAG1 mutation positive individuals Large cohort required (n=700), patients being recruited internationally and via ChiLDREN ALGS patients stratified according to severity of hepatic phenotype, mild vs. severe A B C1 C2 D No known hepatic involvement Biochemical abnormalities = AST/ALT elevated, but normal bilirubin Biochemical cholestasis = elevated conj bilirubin or elevated cholesterol, or bile salts or GGT without overt clinical manifestations Cholestasis with clinical manifestations including severe pruritus, xanthomata, bone fractures Portal hypertension without cholestasis Spinner Induced Pluripotent Stem (ips) Cells ips cells are derived from fibroblasts and genetically reprogrammed to become pluripotent Major breakthrough in patient-oriented research as it allows Development of a patient-specific p in vitro model Can study developmentally expressed genes Hepatocytes have been successfully differentiated from patients with A1ATD, GSD These cells recapitulate features of the disease Defect in A1ATD can be corrected ips cell models of ALGS Objective: To develop patient-specific ALGS cell based models of ALGS Rationale: To understand developmental mechanisms of bile duct paucity Predict hepatic outcomes? Drug screens? Aims: To differentiate cholangiocytes from human ips cells To apply this differentiation protocol derived from ALGS patients Rashid et al, JCI 2010, Yusa & Rashid et al, Nature 2011 Kamath and Ghanekar 5

6 Wild-type ipsc-derived Hepatoblasts under cholangiocyte differentiation conditions: CK-19 positive staining branching structures (red). Not seen when Notch and TGFbeta signaling are Inhibited. ipsc-derived hepatoblasts cultured under cholangiocyte differentiation conditions. Cholangiocyte-like cells demonstrate branching in 3-D culture system. Branching elements stain positively for CK19. SUMMARY: Advances in ALGS Acknowledgements The clinical assessment of ALGS Should include careful evaluation of kidneys Should not include routine evaluation for PI Careful monitoring of biochemistry (TB) in early years may predict hepatic outcomes Liver transplantation is feasible and successful Kidneys are particularly vulnerable Catch-up growth better than BA Atypical or typical ALGS patients may have NOTCH2 mutations Facial and skeletal features may not be as penetrant Investigators Funding Anand Ghanekar Gordon Keller Shin Ogawa Mina Ogawa Nancy Spinner and her laboratory David Piccoli Kathy Loomes Novel genetic and stem cell studies may further explain disease mechanisms and explain disease variability 6