Pediatric Reports 2019; volume 11:8206

Novel mutations the newborn period results in accumulation in NOTCH2 in infants of biliary components and leads to neonatal Correspondence: Nadia Ovchinsky, Division of cholestasis (NC).1 Paucity of intrahepatic Pediatric Gastroenterology, Pediatric with neonatal cholestasis bile ducts (PIBD) is one cause of NC and Hepatology, Children’s Hospital at Montefiore, can be part of a an autosomal dominant 3411 Wayne Ave, 7th Floor, Bronx, NY 10467, Eliana Shaul,1 familial cholestatic syndrome called USA. Tel.: +1.718-741-2332 - Fax 718-515-5426. Debora Kogan-Liberman,2 (ALGS)2 or can be non- E-mail: [email protected] Stephanie Schuckalo,3 Dominique Jan,4 syndromic.3 In 94% of patients with ALGS, 5 2 Michelle Ewart, Trang Nguyen, mutations in the gene for the Notch Key words: Paucity of intrahepatic bile ducts; Mercedes Martinez,6 signaling pathway Jagged1 (JAG1) NOTCH2; Neonatal Cholestasis. Nadia Ovchinsky2,3 are found. Furthermore, mutations in NOTCH2, a in the Notch signaling Contributions: ES: study concept and design, 1Department of Pediatrics, Children’s have been found in patients with ALGS.4 drafting of the manuscript, analysis and interpre- Hospital at Montefiore, Bronx, NY; We report 5 patients, including 2 siblings tation of results, literature review. DKL: drafting 2 Division of Pediatric Gastroenterology, (patients 1&2), with NC without any other the work and revising it critically for important Hepatology and Nutrition, Children’s features of ALGS, who were found to have intellectual content. SS: acquisition of data and Hospital at Montefiore, Bronx, NY; novel variants of unknown chart review, technical support. DJ: Acquisition 3Division of Pediatric Gastroenterology, significance (VOUS) in the NOTCH2 gene. of data and surgical findings. ME: Acquisition of Hepatology, and Nutrition, Goryeb data and pathological findings. TN: Acquisition of data and chart review, technical and adminis- Children’s Hospital - Atlantic Health Methods trative support. MM, critical revision of the man- 4 System, Morristown, NJ; Department This is a retrospective case series of 5 uscript for important intellectual content. NO: of Pediatric Surgery, Children’s patients with NC. Data was obtained study concept and design, critical revision of the Hospital at Montefiore, Bronx, NY; through a chart review, including electronic manuscript for important intellectual content, 5Division of Surgical Pathology, medical records, imaging and pathology final approval of the version published, agree- Montefiore Medical Center, Bronx, NY; specimens. Genetic testing for known pedi- ment to be accountable for all aspects of the onlywork in ensuring that questions related to the 6Division of Pediatric Gastroenterology, atric cholestatic disorders was accuracy or integrity of any part of the work are done via Emory Genetics Laboratory (EGL) Hepatology, and Nutrition, Morgan appropriately investigated and resolved. Stanley Children’s Hospital of New Genetic Cholestasis Panel. Direct sequenc- York, NY, USA ing of the amplified captured regionsuse was Conflict of interest the authors declare no poten- performed using next generation short base tial conflict of interest. pair read sequencing (NGS). Exons with inadequate quality or coverage by NGS Funding: none. were assessed with Sanger sequencing. We Abstract utilized sorting intolerant from tolerant Received for publication: 14 June 2019. One cause of neonatal cholestasis (NC) (SIFT) and polymorphism phenotyping Revision received: 22 June 2019. Accepted for publication: 29 July 2019. is paucity of intrahepatic bile ducts which (PolyPhen), which are silico bioinformatic can be associated with Alagille syndrome or tools that predict the likelihood of patho- 5 This work is licensed under a Creative non- syndromic. Alagille syndrome is genicity of missense variants. SIFT pre- Commons Attribution NonCommercial 4.0 caused by autosomal dominant mutations in dicts whether an amino acid substitution is License (CC BY-NC 4.0). the ligand likely to affect function based on Jagged1 in 94% of patients and mutations in and similarity between ©Copyright: the Author(s), 2019 the NOTCH2 receptor in <1% of patients. the alternate amino acids and then predicts Licensee PAGEPress, Italy This is a retrospective case series studying if the amino acid change is either ‘tolerated’ Pediatric Reports 2019; 11:8206 doi:10.4081/pr.2019.8206 infants with neonatal cholestasis found to or ‘deleterious’. PolyPhen-2 predicts the have variants of unknown significance effect of an amino acid substitution on the (VOUS) in NOTCH2. SortingNon-commercial intolerant structure and function of a protein using from tolerant (SIFT) and polymorphism sequence homology, 3D structures where phenotyping (PolyPhen) were utilized to available, and a number of other databases ed with jaundice, acholic stools and without predict a damaging effect. Five infants with and tools and then provides a qualitative growth failure. Patients 1 and 2 were sib- NC without other features of Alagille syn- prediction of ‘probably damaging’, ‘possi- lings with identical mutations. Liver biopsy drome were found to have one copy of a bly damaging’, ‘benign’ or ‘unknown’. This was done in 3 patients demonstrating pauci- VOUS in NOTCH2, predicted to be damag- study was deemed exempt by The Albert ty of bile ducts in 2 patients (Figure 1) and ing by SIFT and PolyPhen. Our cases sup- Einstein College of Medicine Institutional mild ductular proliferation in the third. In Review Board. port the notion that NOTCH2 mutations all patients SIFT predicted the mutations to may result in hypoplastic biliary system. be deleterious and PolyPhen predicted them Further characterization of these variants is to be probably damaging. Further workup important to assist with our clinical for other features of ALGS, including an approach to NC. Case Report echocardiogram, eye exam and X-Ray of Five male infants with NC between the the spine, were all within normal limits. age of 2 and 8 weeks were found to have Additionally, workup for common etiolo- Introduction one copy of VOUS in NOTCH 2 gene gies of NC including infectious, anatomic, (Table 1). None of the infants had known metabolic, and genetic etiologies was unre- Impaired flow and excretion of bile in risk factors for cholestasis. They all present- markable. All patients demonstrated

[Pediatric Reports 2019; 11:8206] [page 53] Case Report

improvement in liver disease over time and remain asymptomatic.

Discussion NC is caused by diminished flow and excretion of bile.1 Symptoms typically include jaundice, dark urine, acholic stools and hepatomegaly. Cholestasis can occur due to infectious, genetic, anatomic and metabolic causes, generally caused by impairment of hepatobiliary transport, inter- mediary metabolism, storage disorders or bile duct dysgenesis.6 PIBD is one cause of NC and is defined by a specimen from a liver biopsy shows a loss of intrahepatic bile ducts in more than 50% of portal tracts in a specimen that contains at least 10 portal tracts.7 PIBD can be part of a genetic syndrome, ALGS or can caused other rare metabolic diseases, infections such as CMV, or can be idiopathic non-syndromic paucity.2,3 A liver biopsy in patients with ALGS typically demonstrates paucity of the only intrahepatic bile ducts, however in new- borns with ALGS, bile duct paucity is not always present and instead ductal prolifera- tion can be found.8 The diagnosis of ALGS use requires the presence of bile duct paucity with three of five major clinical features including liver disease, vertebral abnormal- ities, congenital heart defects, ocular anom- alies and characteristic facial features.9 ALGS is an autosomal dominant inherited disorder with highly variable expressivity, therefore the disease penetration and sever- ity of the affected organs can vary signifi- cantly.10 Notch pathway interactions are critical for determination of cell fates and differen- tiation in early development. The Notch system includes of four transmembrane Notch receptors (, 2, 3, 4) and two types of ligands Jagged (Jag 1, 2) orNon-commercial Delta- like (Dll 1, 3, 4). The Notch pathway is involved in several stages of bile duct mor- phogenesis including in the expression of cholangiocytes-specific markers commit- ting cells to the biliary lineage.11 In 94% with ALGS, mutations in JAG1 are found,4 while mutations in NOTCH2 have been described in in a small number of patients who met diagnostic cri- teria for ALGS without JAG1 mutations.9,12 In one study, Kamath et al describe a cohort of JAG1-negative individuals with clinical features suggestive of ALGS screened for NOTCH2 mutations. Eight patients with a NOTCH2 mutations were identified and only three met classic criteria for ALGS. Of the remaining five individuals, four had two (weeks) symptoms bilirubin, mg/dL transferase, U/L Biopsy Mutation Biopsy mg/dL U/L (weeks) bilirubin, symptoms transferase, excretion excretion liverwith prompt (p.R1567W) prompt liverwith acholic stools, No dilated intrahepatic or radiotracer c.1847G>T radiotracer or intrahepatic dilated No stools, acholic acholic stools, No dilated intrahepatic or c.2102T>A or intrahepatic dilated No stools, acholic into the bowel bowel the into Gallbladder present. excretion into the bowel the into excretion present. Gallbladder present. present. or extrahepatic bile ducts. Patent intrahepatic and proliferation (p.E1772K) and proliferation intrahepatic Patent ducts. bile extrahepatic or system biliary extrahepatic present. Gallbladder feeding difficulty extrahepatic bile ducts. uptake in the liver with prompt (p.C616F) prompt with liver the in uptake ducts. bile extrahepatic difficulty feeding acholic stools Gallbladder was not visualized. radiotracer uptake in the proliferation c.4699C>T proliferation the in uptake radiotracer visualized. not was Gallbladder stools acholic acholic stools No dilated intrahepatic uptake after 24 hours cholangiogram: and mild ductular c.5314G>A ductular c.5314G>A mild and cholangiogram: hours 24 after uptake intrahepatic dilated No stools acholic acholic stools but present gallbladder uptake after 24 hours Patent intrahepatic and c.5314G>A and intrahepatic Patent hours 24 after uptake gallbladder present but stools acholic extrahepatic biliary system (p.E1772K) system biliary extrahepatic hepatomegaly extrahepatic bile ducts. Gallbladder (p.C701E) Gallbladder ducts. bile extrahepatic hepatomegaly 5 2 Jaundice, 5.7/4.8 217 Normal liver in size and echogenicity. Prompt and homogeneous Not done Mild ductular NOTCH2 VOUS VOUS NOTCH2 ductular Mild done Not homogeneous and echogenicity. Prompt and size in liver Normal 217 5.7/4.8 Jaundice, 2 5 3 2 Jaundice, 10/2.5 296 Normal liver size and echotexture. Prompt and homogeneous Not done Not done NOTCH2 VOUS NOTCH2 done Not done Not homogeneous and Prompt echotexture. and size liver Normal 296 10/2.5 Jaundice, 2 3 Patient Age Presenting Total/direct Gamma-glutamyl Abdominal ultrasound HIDA Scan Cholangiogram Liver NOTCH2 NOTCH2 Liver Cholangiogram Scan HIDA ultrasound Abdominal Gamma-glutamyl Age Total/direct Patient Presenting ducts bile of Paucity VOUS NOTCH2 cholangiogram: bowel or bladder Percutaneous gall No contracted with hepatomegaly Mild 638 6.7/4.5 Jaundice, 8 2 VOUS NOTCH2 done Not done Not done Not echotexture. and size liver Normal 224 4.6/0.9 Jaundice, 4 4 1 3 Jaundice, 6.1/3.4 222 Normal liver size and echotexture. No gall bladder or bowel Intra-operative Paucity of bile ducts NOTCH2 VOUS NOTCH2 ducts bile of Paucity bowel or bladder Intra-operative gall No echotexture. and size liver Normal 222 6.1/3.4 Jaundice, 3 1 typical ALGS diagnostic features and one 1. characteristics. Patient Table

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Pediatrics 2013;23:65-70. 3. Kenny AP, Crimmins NA, Mackay DJ, et al. Concurrent course of transient neonatal diabetes with cholestasis and paucity of interlobular bile ducts: A case report. Pediatr Dev Pathol 2009;12:417- 20. 4. McDaniell R, Warthen DM, Sanchez- Lara PA, et al. NOTCH2 mutations AB cause Alagille syndrome, a heteroge- neous disorder of the notch signaling pathway. Am J Hum Figure 1. Histological findings demonstrating PIBD in patient 1. A) Portal area with mid chronic inflammation with visible hepatic artery branch and portal vein branches with- Genet 2006;79:169-73. out any native bile ducts. There are surrounding giant cell transformation of hepatocytes. 5. Flanagan SE, Patch AM, Ellard B) Another portal area showing hepatic artery and portal vein without a native bile duct. S. Using SIFT and PolyPhen to predict loss-of-function and gain-of-function mutations. Genet Test Mol patient had bile duct paucity with no other tions in patients with NC is warranted and Biomarkers 2010;14:533-77. syndromic features.13 This study found that in those with PIBD even if they only fulfil 6. Suchy FJ. Neonatal cholestasis. Pediatr in their patients the variety of clinical fea- partial criteria for ALGS. Validation of Rev 2004;25:388-96. tures associated with NOTCH2 mutations these finding in a larger human cohort and differed from JAG1 mutations, with a lower further characterization of the NOTCH2 7. Suchy FJ. Clinical problems with devel- prevalence of butterfly vertebrae and facial variants in an animal model is especially opmental anomalies of the biliary tract. features. important to understand the clinical applica- Semin Gastrointest Dis 2003;14:156- tion of these results. Further investigation 64. will need to be done in order to determine only8. Saleh M, Kamath BM, Chitayat whether these NOTCH2 variants are truly D. Alagille syndrome: clinical perspec- Conclusions deleterious as the current data presented are tives. Appl Clin Genet 2016;9:75-82. insufficient to determine causality of these 9. Piccoli DA, Spinner NB. Alagille syn- These five patients, along with one variants in neonatal cholestasis. Increasinguse drome and the Jagged1 gene. Semin patient described by Kamath et al., suggest availability of genetic testing and ability to Liver Dis 2001;21:525-34. that NOTCH2 mutations may be related to link the clinical finding with previously 10. Kamath BM, Bason L, Piccoli DA, et isolated NC or PIBD without other features unrecognized mutations provide a platform al. Consequences of JAG1 mutations. J of ALGS. Since the Notch pathway is for more precise and less invasive approach Med Genet 2003;40:891-5. involved in bile duct morphogenesis, these to achieve a diagnosis in this vulnerable 11. Morell CM, Strazzabosco M. Notch sig- cases stipulate that NOTCH2 mutations population. naling and new therapeutic options in may result in hypoplastic biliary system and liver disease. J Hepatol 2014;60:885- intrahepatic bile duct paucity. Furthermore, 90. this series suggests that NOTCH2 muta- 12. Guegan K, Stals K, Day M, et al. JAG1 tions may provide the genetic basic to References mutations are found in approximately explain the clinical finding in infants with one third of patients presenting with isolated neonatal cholestasis without other 1. Feldman AG, Sokol RJ. Neonatal only one or two clinical features of features of ALGS. It is also crucial to con- cholestasis. Neoreviews 2013;14. Alagille syndrome. Clin sider that the highly variable expression and 2. Zahmatkeshan M, Geramizadeh B, Genet 2012;82:33-40. reduced penetrance seen with ALGS could Haghighat M, Enteshari, H. Paucity of 13. Kamath BM, Bauer RC, Loomes KM, be influencing clinical presentation seen in Intrahepatic Bile Ducts in Neonates: the et al. NOTCH2 mutations in Alagille our patients. Screening for NOTCH2Non-commercialmuta- First Case Series from Iran. Iran J syndrome. J Med Genet 2012;49:138-

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