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

2. Paediatric cholestasis: historical background

4. Year 2000
Good prognosis
Bad prognosis

5. 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

6. Liver biopsy in biliary atresia
Portal fibrosis Ductular proliferation Bile plugs

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

8. CAUSES OF NEONATAL CHOLESTASIS
Semin Neonatol Apr;7(2):
McKiernan PJ. Neonatal cholestasis.

10. 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

11. The clinical condition matters

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

13. 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

14. A revolution is approaching: next generation sequencing

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

16. 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

17. 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

18. 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

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

20. 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)= base-pairs.

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

22. 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

23. Grazie