1. Pediatric Cholestatic Jaundice: Differential Diagnosis of Treatable Disorders Saul J. Karpen, MD, PhD Professor of Pediatrics Raymond F. Schinazi Distinguished Biomedical Chair Division Chief, Gastroenterology, Hepatology & Nutrition Emory University School of Medicine Atlanta, Georgia

2. A Common Clinical Challenge: Neonatal Jaundice Is it Physiologic or Pathologic?

3. Case: Full-Term Female Infant Weight: 7 lb, 2 oz Apgar score: 9 Jaundice noted by parents while in hospital Elevated total serum bilirubin level at time of discharge on day 2

4. Neonatal Jaundice Yellowing of the skin and sclerae  Occurs in ~60% [a] of full-term and 85% [b] of preterm babies within 1 to 3 days of birth  Not always easy to determine or gauge a. American Academy of Pediatrics. Pediatrics. 2004;114:297-316. b. NHS National Institute for Health and Clinical Excellence website.

5. Neonatal Jaundice (cont) Often due to elevated bilirubin level in the blood when the immature liver is not efficient enough to remove bilirubin from the blood for biotransformation and fecal elimination Diminishes at ~1 to 2 weeks after birth as baby's liver matures and the production of bilirubin begins to decrease

6. Case (cont) Recurrence of Jaundice After Initial Improvement Normal baby Breastfed Returned to pediatrician for 1-week visit –Normal exam –Total bilirubin level remained elevated, but lower than at discharge from hospital Returned to pediatrician for 2-week visit –Total bilirubin level remained elevated

7. Case (cont) Diagnosis: Prolonged Neonatal Jaundice Evaluate for cholestasis ‒Measure total and direct serum bilirubin in any infant noted to be jaundiced after 2 weeks of age  If direct bilirubin level > 1.0 mg/dL or > 17 µmol/L, infant should be referred for evaluation by a pediatric gastroenterologist or hepatologist

8. Prolonged Neonatal Jaundice Breast Milk-Related Jaundice Persists in otherwise healthy, full-term, breast-fed babies (~0.5% to 2.4% of all newborns [a] ) after physiologic jaundice subsides a. Winfield CR, et al. American Academy of Pediatrics. Archives of Disease in Childhood. 1978;53:506-516. Can last for many weeks after birth Rarely serious

9. Prolonged Neonatal Jaundice: Is It Noncholestatic or Cholestatic in Origin? Differential diagnosis is different for noncholestatic and cholestatic neonatal jaundice Cholestatic jaundice –Prevalence: ~1:2500 full-term infants [a] –Wide range of differential diagnoses  Biliary atresia  Genetic disorders –Important to make correct diagnosis because some disorders can progress rapidly, but can be treated Due to reduced bile formation or flow resulting in retention of biliary substances within the liver that are normally excreted into bile and eliminated into the intestinal tract a. Feldman AG, Sokol RJ. Neonatal cholestasis. Neoreviews. 2013;14:e63.

10. Liver Disease in the Newborn Hoerning A, et al. Front Pediatr. 2014;2:65.

11. Differential Diagnosis of Cholestasis in Newborns Think Anatomic Extrahepatic biliary tree: can make bile normally, but it gets stuck on its way out of the liver Intrahepatic biliary tree: can make bile normally, but can't handle bile formed in the intrahepatic tree Hepatocytes: metabolic disorders

12. Differential Diagnosis of Cholestasis in Newborns (cont) Bile duct obstruction, eg – structural, hepatocellular abnormalities Metabolic disorders, eg – storage diseases Endocrine disorders, eg – hypothyroidism Toxic disorders, eg – due to soy lipid infusion Rare immunologic disorders Infections, eg – CMV Rare vascular malformations Miscellaneous, eg – hematologic, oncologic, intestinal disorders

13. Evaluating Prolonged Neonatal Jaundice Visual Assessment is Observer Dependent Difficult to correlate visual recognition of jaundice with serum bilirubin level Even experienced clinicians cannot visually assess the total serum bilirubin with accuracy –The true color of an infant's skin is not necessarily apparent at birth –Newborns often have their eyes closed so scleral icterus can be missed –Level of bilirubin required for jaundice to be visually apparent is unknown, but thought to be ~5 mg/dL –Cannot determine whether jaundice is due to indirect (cause is physiologic) or direct (cause is liver disease) hyperbilirubinemia Kramer LI. Amer J Dis Child. 1969;118:454-458. Moyer VA, et al. Arch Pediatr Adolesc Med. 2000;154:391-394.

14. NASPGHAN/ESPGHAN: Recommendations for Evaluation of the Potential for Cholestatic Liver Diseases Measurements of serum bilirubin should always be fractionated into unconjugated (indirect) or conjugated (direct) hyperbilirubinemia Any formula-fed infant noted to be jaundiced after 2 weeks of age should be evaluated for cholestasis with measurement of total and conjugated (direct) serum bilirubin Depending upon local practice, breast-fed babies that appear otherwise well may be followed clinically until 3 weeks of age, at which time, if they appear icteric, should then undergo serum evaluation of total and conjugated (direct) serum bilirubin Conjugated (direct) hyperbilirubinemia (> 1.0 mg/dL [17umol/L]) is considered pathological and warrants diagnostic evaluation Fawaz R, et al. J Pediatr Gastroenterol Nut. 2016 Jul 16. [Epub ahead of print]

15. Evaluation of the Baby With Persistent Jaundice Neonatal screening tests –Blood tests*  Organic acid disorders  Fatty acid oxidation disorders  Amino acid disorders (eg, tyrosinemia)  Hemoglobinopathy disorders  Other disorders (eg, cystic fibrosis) –Hearing test –Pulse oximetry for critical congenital heart diseases *Blood tests are state specific, these are required in the state of Georgia Georgia Department of Public Health website.

16. Evaluation of the Baby With Persistent Jaundice (cont) History  Medication history in mother and infant: vitamin K supplementation at birth  Normal behaviors: feeding, normal interactions, sleeping  Direct visualization of stool pigment is a key aspect of a complete evaluation of the jaundiced infant [a]  Changes in stool and urine pigmentation: caregivers should make direct observations  Pale or alcholic stools are a hallmark of cholestasis because bilirubin gives stool its color  Dark urine is indicative of cholestasis  Timing of onset of jaundice Other family history  Liver disease in newborns  Unexplained death a. Fawaz R, et al. J Pediatr Gastroenterol Nut. 2016 Jul 16. [Epub ahead of print]

17. Physical Examination of the Baby With Persistent Jaundice: Head to Toe Examine for presence of dysmorphic features: visual clues to chromosomal abnormalities Assess for abnormal behavioral interactivity Auscultate chest for presence of heart murmur Palpate abdomen just above the pelvic brim –Normal finding is a soft liver edge below the right costal margin –Negative findings: spleen, fluid waves, abdominal tenseness, hard masses, cirrhosis (later in life)

18. Physical Examination of the Baby With Persistent Jaundice: Head to Toe (cont) Perform neurologic exam –Presence of poor (floppy) tone –Unable to suck and swallow normally is a clue to mitochondrial disorders Look at skin for signs of infection, eg – rash A thorough physical examination is crucial to the proper evaluation of the jaundiced infant. Attention to hepatomegaly, splenomegaly, and ill appearance warrants special considerations [a] a. Fawaz R, et al. J Pediatr Gastroenterol Nut. 2016 Jul 16. [Epub ahead of print]

19. Evaluation of the Baby With Persistent Jaundice Additional Laboratory Tests Core liver function tests: ALT, AST, AP (less helpful in infancy), total and direct bilirubin GGTP for determining presence of bile duct damage, cholangiopathy PT, INR for determining presence of coagulopathy Albumin Glucose Ammonia

20. Evaluation of the Baby With Persistent Jaundice Additional Laboratory Tests (cont) Serum metabolites for amino acids, lipids, bile acids Urine metabolites to determine presence of inborn errors of metabolism ‒Succinylacetone (elevated with tyrosinemia) ‒Urine organic acids ‒Acylglycines Bacterial and urine cultures to determine presence of sepsis Viral studies: birth-acquired HSV, CMV Genetic testing is evolving: perhaps order individual gene tests if a single diagnosis is likely, but panels or genome-wide studies are currently being developed

21. Differential Diagnosis: Intrahepatic Cholestasis Red Flags That Require Early Intervention Look for –Failure to thrive –Poor feeding –Lethargy –Hepatomegaly –Splenomegaly –Laboratory tests  Hyperbilirubinemia (direct)  Elevated liver function tests  Hypoglycemia, especially with absent urinary ketones  Hyperammonemia

22. Differential Diagnosis of Treatable Disorders: Single-Gene Defects Identification and Early Intervention Can Make a Difference Tyrosinemia Bile acid synthetic disorders –3-beta-hydroxy-delta-5-C27-steroid oxidoreductase deficiency –Alpha-methylacyl-CoA racemase (AMACR) deficiency –Amino acid n-acyltransferase deficiency –Bile acid CoA ligase deficiency –Cholesterol 7-alpha-hydroxylase deficiency –Delta4-3-oxosteroid 5-beta-reductase deficiency –Oxysterol 7-alpha-hydroxylase deficiency –Sterol 27-hydroxylase deficiency (cerebrotendinous xanthomatosis) –Trihydroxycholestanoic acid CoA oxidase deficiency

23. Origins of Neonatal Cholestasis Extrahepatic Bile DuctIntrahepatic Bile DuctHepatocytes Biliary atresia Bile duct hypoplasia Bile duct duplication Agenesis of extrahepatic ducts Choledocholithiasis Inspissated bile syndrome Miscellaneous Panhypopituitarianism Hypothyroidism Neonatal hemochromatosis TPN-associated cholestasis Bile duct paucity Alagile syndrome (JAG1, Notch2) Nonsyndromic Ductal plate malformation Caroli disease ARPKD, ADPKD Von Meyenburg complexes Indeterminant (neonatal hepatitis) Viral infection (HSV, CMV) Bacterial or parasitic infection Transporter (bile acids, phospholipids) gene defects PFIC1 (ATPB1) PFIC2 (ATPB11) PFIC3 (ABCB4) Metabolic/storage diseases Neimann-Pic Tyrosinemia Galactosemia Zellweger's Mitochondrial enzymopathies OTC deficiency  1 -antitrypsin deficiency Drug toxicity

24. Emory Cholestasis 57 Gene Panel * Only 1 Blood Sample Required to Identify Key Genes: Example of a Gene Panel Genes Included on the Neonatal and Adult Cholestasis Panel ABCB11CC2D2AHNF1BNPC2PEX7PKHD1UGT1A1 ABCB4CFTRHSD3B7NPHP1PEX10POLGVPS33b ABCC2CLDN1INVSNPHP3PEX11BSERPINA1 ABCD1CYP27A1JAG1NPHP4PEX12SLC25A13 ABCG5CYP7A1LIPAPEX1PEX13SLC27A5 ABCG8CYPB1MKS1PEX2PEX14SMPD1 AKR1D1DGUOKMPV17PEX3PEX16TJP2 ATP8B1DHCR7NOTCH2PEX5PEX19TMEM216 BAATFAHNCPC1PEX6PEX26TRMU *CLIA-approved laboratory Emory Genetics Laboratory website.

25. NASPGHAN/ESPGHAN Recommendations The role of imaging –The abdominal ultrasound is useful in excluding choledochal cyst or gallstone disease causing extrahepatic bile duct obstruction (eg, cyst, stone, mass)  It may demonstrate an absent or abnormal gallbladder, or other features suggestive, but not diagnostic, of biliary atresia Fawaz R, et al. J Pediatr Gastroenterol Nut. 2016 Jul 16. [Epub ahead of print]

26. NASPGHAN/ESPGHAN Recommendations (cont) The role of imaging –Limited specificity precludes the use of the HBS scan as a stand-alone test in making a definitive diagnosis of biliary atresia –Definitively-demonstrated bile flow (patency) by selective use of HBS may be of value in excluding biliary atresia –Limited specificity of MRCP, ERCP, PTC has a limited role in the general guidance to caregivers toward diagnosing biliary atresia in the present era Fawaz R, et al. J Pediatr Gastroenterol Nut. 2016 Jul 16. [Epub ahead of print]

27. Diagnosis of biliary atresia –Cannot make diagnosis by lab tests, stool color, or physical examination –Evaluation by intraoperative cholangiogram and histological examination of the duct remnant is considered the gold standard for diagnosis of biliary atresia [a] Diagnosis of diseases other than biliary atresia that cause cholestasis –Can be determined via histologic examination of the liver for evidence of: [a]  Biliary tract obstruction  Storage disease  Other specific pathologic features  Rule out other diagnoses a. Fawaz R, et al. J Pediatr Gastroenterol Nut. 2016 Jul 16. [Epub ahead of print] NASPGHAN/ESPGHAN Recommendations (cont)

28. NASPGHAN/ESPGHAN Recommendation Consider Whether a Liver Biopsy Would Be Helpful In the hands of an experienced pediatric pathologist, histopathological findings of bile duct proliferation, bile plugs, and fibrosis in an appropriately timed liver biopsy is the most supportive test in the evaluation of the infant with protracted conjugated hyperbilirubinemia [a] –Precludes the need for specialized genetic or other tests –Consultation with pediatric surgeon for consideration of intraoperative cholangiogram Fawaz R, et al. J Pediatr Gastroenterol Nut. 2016 Jul 16. [Epub ahead of print]

29. Liver Biopsy for Biliary Atresia Does the Patient Need An Intraoperative Cholangiogram? Hallmark features –Bile duct proliferation –Bile duct fibrosis –Bile duct plugs –Inflammation Infants identified and referred in a timely manner will benefit from specific therapy –Kasai procedure (hepatoportoenterostomy): if done within first 60 days of life, bile flow established in ~70% [a] –Avoid liver transplantation Chardot C, et al. J Hepatol. 2013;58:1209-1217.

30. Biliary Atresia Biliary atresia: one or more bile ducts are abnormally narrow, blocked, or absent –The earlier the diagnosis is made and the earlier the surgical intervention is performed, the better the outcome –50% can benefit from Kasai procedure [a] Chardot C, et al. J Hepatol. 2013;58:1209-1217.

31. Therapies Are Available for Some Cholestatic Disorders No effective therapies for cholestasis in general Early diagnosis with urine tests and gene tests/panels is important because there are therapies for some specific gene defects –Cystic fibrosis, depending on the genotype –Tyrosinemia, depending on the genotype –Bile acid synthesis defects

32. Concluding Remarks Take-Home Messages Observer-assessment of jaundice is not a good or reliable marker of serum bilirubin Conjugated hyperbilirubinemia (>1 mg/dL or > 35 µmol/L) is never normal ‒Should prompt a referral to a pediatric gastroenterologist or hepatologist Early detection, diagnosis, and referral!