Itteri a bilirubina diretta ad insorgenza precoce Lorenzo D'Antiga Child Health Paediatric Hepatology, Gatroenterology and Transplantation Hospital Papa Giovanni XXIII Bergamo 1

Paediatric cholestasis: historical background

Year 2000 Good prognosis Bad prognosis

Approach to neonatal conjugated jaundice Days of life Outpatient Jaundice and dark urine 20 Test bilirubin and refer Inpatient Confirmation of conjugated jaundice 30 Hypocholic or acholic stool? NO YES No bile in the gut. Suspect biliary atresia Passage of bile in the gut. Biliary atresia ruled out Intrahepatic causes Liver biopsy, direct cholangiogram 40

Liver biopsy in biliary atresia Portal fibrosis Ductular proliferation Bile plugs

Liver biopsy in intrahepatic cholestasis Giant cells No fibrosis No ductular proliferation Hepatocellular cholestasis No bile plugs

CAUSES OF NEONATAL CHOLESTASIS Semin Neonatol. 2002 Apr;7(2):153-65. McKiernan PJ. Neonatal cholestasis.

Prevalence has changed Prevalence of cholestasis 1:2500 1970 2004 Whatever happened to neonatal hepatitis? Balistreri WF, Bezerra JA. Clin Liver Dis 10 (2006) 27– 53

The clinical condition matters

Approach to neonatal cholestasis: well-child Davit-Spraul. Orphanet Journal of Rare Diseases 2009, 4:1

Approach to neonatal cholestasis: unwell-child Sign Diagnosis Severely ill Sepsis/UTI, galactosemia, hypocortisolism, HLH Dysmorfism Alagille, chromosomal abn Eye abnormalities Alagille, galactosaemia, TORCH, storage disorders Heart murmur Cardiac malformations, Alagille Situs inversus Biliary atresia with SM Abdominal mass Tumors, choledochal cyst Splenomegaly Lysosomal storage disease Ascites A1ATD, metabolic, spontaneous perforation of bile duct

A revolution is approaching: next generation sequencing

How many genes causing disease in our DNA? We know only a small part of genes causing diseases

Targeted sequencing Sequencing that specifically targets regions of the genome that are of interest to clinicians Targeted sequencing yields much higher coverage of genomic regions of interest Sequencing panels can be developed to target specific genomic regions or disease-causing mutation hotspots => Bergamo NGS liver panel

Bergamo NGS liver panel Wilson disease: ATP7B ARPKD: FCYT/PKHD1 Fructose intolerance ALDOB Galactosemia GALT Niemann-Pick A-B: SMPD1 Niemann-Pick C: NPC1, NCP2 Citrullinemia II, (NICCD): SLC25A13 MCAD deficiency: ACADM Crigler-Najjar syndrome: UGT1A1 Mitochondrial DNA depletion: DGUOK, POLG LAL deficiency: LIPA PFIC 1, BRIC 1, ICP: ATP8B1 PFIC 2, BRIC 2: ABCB11 PFIC 3: ABCB4 Intrahepatic cholestasis: TJP2 Hypercholanemia, familial: EPHX1, BAAT Alagille syndrome: JAG1,NOTCH2 Bile acid synthesis defect: HSD3B7, AKR1D1 CYP7B1, AMACR A1ATD: SERPINA1

Recognition of mutations by NGS Sequenced fragments Reference genome Sequencing/mapping error The raw sequencing data consists of large computer text files (tens to hundreds of gigabytes) containing several million short (∼35–400 bp) nucleotide sequences called reads. These cryptic files need to undergo complex computational processing in order to become meaningful information. To determine the position of the reads in the genome they must be aligned (mapped) to their most probable location on the reference human genome and possible mismatches or gaps must be taken into consideration Homozygous variant Heterozygous variant Schnekenberg RP Arch Dis Child 2014

Bergamo NGS liver panel Patients tested in the last 3 years: 183 Number of patients diagnosed: 67 Detection rate: 36%

Cost per megabase of DNA sequence Sanger NGS Costs per raw megabase of DNA sequence. Note the logarithmic scale of costs and steep drop after introduction of NGS in 2007. 1 megabase (Mb)=1 000 000 base-pairs. http://www.genome.gov/sequencingcosts

Diagnosis of genetic liver disease: timeline Clinical diagnosis Genetics (NGS) Epigenetics Match genotype-phenotype

A novel algorithm for neonatal cholestasis Acholic stool Yes Liver biopsy Fibrosis, ductular proliferation Giant cell hepatitis No GGT level High Low High serum bile acid Normal serum bile acid A novel algorithm for neonatal cholestasis NGS Cholangiography Biliary atresia Choledocal cyst A1ATD Neon sclerosing cholangitis Urine mass spectrometry Bile acid defects Alagille A1ATD, CF PFIC3 NP-C, Gaucher High ggt PFIC1 PFIC2 PFIC4 ARC, TALDO Low ggt

Grazie