Alagille Syndrome

Alagille Syndrome Pathogenesis and Clinical Management Binita M. Kamath Kathleen M. Loomes  Editors

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Alagille Syndrome

Binita M. Kamath  ·  Kathleen M. Loomes Editors

Alagille Syndrome Pathogenesis and Clinical Management

Editors Binita M. Kamath, MBBChir MRCP MTR Division of Gastroenterology, Hepatology and Nutrition The Hospital for Sick Children Toronto, ON Canada

Kathleen M. Loomes, MD Division of Gastroenterology, Hepatology and Nutrition Children’s Hospital of Philadelphia Philadelphia, PA USA

ISBN 978-3-319-94570-5    ISBN 978-3-319-94571-2 (eBook) https://doi.org/10.1007/978-3-319-94571-2 Library of Congress Control Number: 2018953718 © Springer International Publishing AG, part of Springer Nature 2018 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, express or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Switzerland AG The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland

I dedicate this book to my parents for their unfailing support and encouragement and to Maya and Anika who keep me going with their love and laughter. Binita M. Kamath I dedicate this book to the memory of my mother Joan, for her constant love and support. Kathleen M. Loomes

Preface

Alagille syndrome (ALGS) is a fascinating disorder that challenges us to comprehend multi-organ disease, molecular genetics, and developmental biology. Our understanding of this disorder has blossomed since its first description almost 50 years ago. We now appreciate that there are two disease-causing genes associated with ALGS and the phenotypic spectrum encompasses at least eight organ systems. The advances in elucidating the genetics of this condition have actually improved our understanding of intrahepatic bile duct development. This explosion of knowledge is encapsulated in the following book. We have the privilege of having the amalgamated knowledge from the experts and leaders in ALGS. Each author is a respected and recognized authority in his or her field that has been especially handpicked for the topics presented. Currently, there is no textbook entirely devoted to the pathophysiology and management of ALGS.  This textbook is designed to provide a comprehensive and current overview of the important issues specific to the field of ALGS. Care of these patients and their multisystem disease can be quite complex, and materials have been collected from the most current, evidence-based resources, providing an overview of all aspects of ALGS, from the developmental and genetic perspectives, to liver transplantation, to the most innovative, molecular advances that will launch our management of this complex disease forward in the near future. Gaps in knowledge about ALGS challenge us for the present and future. Specific understanding about how JAG1/NOTCH2 mutations lead to bile duct paucity, why some patients have resolution or stabilization of their cholestasis, the identification of genetic modifiers, and the lack of specific targeted therapies, to name a few, remains unknown. Finally, issues surrounding nomenclature also persist. Daniel Alagille described a condition characterized by hepatic involvement with multisystem disease – it may not be appropriate to describe an individual with no overt liver involvement as having ALGS, and we are lacking more detailed molecular-based terminologies that better reflect disease states.

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On behalf of all the authors and ourselves, we sincerely hope this text will serve as a valuable and useful guide of better understanding and management strategies for those who are interested in providing the very best approaches to the care of our patients with ALGS. Toronto, ON, Canada Philadelphia, PA, USA

Binita M. Kamath Kathleen M. Loomes

Acknowledgements

We acknowledge the tremendous perseverance of individuals with Alagille syndrome and their families, who inspire us daily. Toronto, ON, Canada Philadelphia, PA, USA

Binita M. Kamath Kathleen M. Loomes

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Contents

1 Alagille Syndrome: Overview and Introduction ���������������������������������������� 1 David A. Piccoli 2 Bile Duct Development and the Notch Signaling Pathway���������������������� 11 Stacey S. Huppert and Kathleen M. Campbell 3 Genetics of Alagille Syndrome�������������������������������������������������������������������� 33 Melissa A. Gilbert and Nancy B. Spinner 4 Liver Disease in Alagille Syndrome������������������������������������������������������������ 49 Alyssa Kriegermeier, Andrew Wehrman, Binita M. Kamath, and Kathleen M. Loomes 5 Transplant Considerations in Alagille Syndrome ������������������������������������ 67 Evelyn Hsu and Elizabeth Rand 6 Cardiac, Aortic, and Pulmonary Vascular Involvement in Alagille Syndrome������������������������������������������������������������������������������������ 77 Justin T. Tretter and Doff B. McElhinney 7 Vascular Manifestations in Alagille Syndrome ���������������������������������������� 91 Shannon M. Vandriel, Rebecca N. Ichord, and Binita M. Kamath 8 The Renal Sequelae of Alagille Syndrome as a Product of Altered Notch Signaling During Kidney Development���������������������� 103 René Romero 9 Skeletal Involvement in Alagille Syndrome �������������������������������������������� 121 Yadav Wagley, Troy Mitchell, Jason Ashley, Kathleen M. Loomes, and Kurt Hankenson 10 Immune Dysregulation in Alagille Syndrome: A Feature of the Evolving Phenotype������������������������������������������������������ 137 Alastair Baker

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11 Health-Related Quality of Life and Neurocognition in Alagille Syndrome���������������������������������������������������������������������������������� 159 Saeed Mohammad and Estella M. Alonso 12 Future Therapeutic Approaches for Alagille Syndrome������������������������ 167 Emma R. Andersson Index�������������������������������������������������������������������������������������������������������������������� 195

Contributors

Estella  M.  Alonso  Department of Pediatrics, Northwestern University, Feinberg School of Medicine and The Ann & Robert H. Lurie Children’s Hospital of Chicago, Chicago, IL, USA Emma R. Andersson  Karolinska Institutet, Solna, Sweden Jason  Ashley  Department of Biology, Eastern Washington University, Cheney, WA, USA Alastair Baker  King’s College Hospital, London, UK Kathleen M. Campbell  Division of Gastroenterology Hepatology and Nutrition, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH, USA Melissa  A.  Gilbert  Department of Pathology, The Children’s Hospital of Philadelphia and the Perelman School of Medicine at The University of Pennsylvania, Philadelphia, PA, USA Kurt  Hankenson  Department of Orthopaedic Surgery, University of Michigan Medical School, Ann Arbor, MI, USA Evelyn  Hsu  Department of Pediatrics, University of Washington School of Medicine, Seattle Children’s Hospital, Seattle, WA, USA Stacey  S.  Huppert  Division of Gastroenterology Hepatology and Nutrition, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA Rebecca N. Ichord  Division of Neurology, The Children’s Hospital of Philadelphia, Philadelphia, PA, USA Binita M. Kamath  Division of Gastroenterology, Hepatology and Nutrition, The Hospital for Sick Children, Toronto, ON, Canada Department of Pediatrics, University of Toronto, Toronto, ON, Canada xiii

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Alyssa  Kriegermeier  Department of Pediatrics, Division of Gastroenterology, Hepatology and Nutrition, The Children’s Hospital of Philadelphia, Philadelphia, PA, USA Kathleen  M.  Loomes  Department of Pediatrics, Division of Gastroenterology, Hepatology and Nutrition, The Children’s Hospital of Philadelphia, Philadelphia, PA, USA Department of Pediatrics, Children’s Hospital of Philadelphia and Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA Doff  B.  McElhinney  Departments of Cardiothoracic Surgery and Pediatrics (Cardiology), Stanford University Medical School, Lucile Packard Children’s Hospital, Betty Irene Moore Children’s Heart Center, Clinical and Translational Research Program, Palo Alto, CA, USA Troy  Mitchell  Department of Orthopaedic Surgery, University of Michigan Medical School, Ann Arbor, MI, USA Saeed  Mohammad  Ann & Robert H.  Lurie Children’s Hospital of Chicago, Chicago, IL, USA David  A.  Piccoli  Division of Gastroenterology, Hepatology and Nutrition, University of Pennsylvania Perelman School of Medicine, The Children’s Hospital of Philadelphia, Philadelphia, PA, USA Elizabeth Rand  Department of Pediatrics, The University of Pennsylvania School of Medicine, Philadelphia, PA, USA René Romero  Department of Pediatrics, Division of Pediatric Gastroenterology, Hepatology, and Nutrition, Emory University School of Medicine, Children’s Healthcare of Atlanta-Egleston, Atlanta, GA, USA Nancy  B.  Spinner  Department of Pathology, The Children’s Hospital of Philadelphia and the Perelman School of Medicine at The University of Pennsylvania, Philadelphia, PA, USA Justin  T.  Tretter  The Heart Institute, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA Shannon  M.  Vandriel  Division of Gastroenterology, Hepatology and Nutrition, The Hospital for Sick Children, Toronto, ON, Canada Yadav  Wagley  Department of Orthopaedic Surgery, University of Michigan Medical School, Ann Arbor, MI, USA Andrew  Wehrman  Department of Pediatrics, Division of Gastroenterology, Hepatology and Nutrition, The Children’s Hospital of Philadelphia, Philadelphia, PA, USA

Chapter 1

Alagille Syndrome: Overview and Introduction David A. Piccoli

Alagille syndrome (ALGS) is an autosomal dominant multisystem disorder that is due to mutations in the Notch signaling pathway. It is characterized by a diverse group of manifestations that can affect the liver, heart, skeleton, eyes, face, vasculature, and kidneys. While the causes and the phenotypes of Alagille syndrome are now well characterized, the progression over the last 50 years of the understanding of ALGS clinical features parallels the advances in pediatric hepatology. Although it is now clear that many individuals with a mutation in Notch signaling can have minimal or no clinical hepatic disease, the liver is the most commonly and most severely affected organ in ALGS.  In 1969, French hepatologist Daniel Alagille reported that a number of patients with paucity of the interlobular bile ducts also have a constellation of findings that include severe cholestasis, cardiovascular disease (a murmur), a characteristic facies, posterior embryotoxon, butterfly vertebrae, and a number of other features. Alagille was able to distinguish this syndromic disease from a large and diverse group of rare disorders that either caused or were associated with paucity of the bile ducts. Alagille first recognized the strong genetic inheritance of syndromic bile duct paucity and postulated dominant inheritance. The relationship of liver disease to congenital cardiovascular disease was also noted in 1973 by Watson and Miller, who coined the term arteriohepatic dysplasia. Alagille was responsible for the defining diagnostic criteria, which required bile duct paucity in association with at least three of the five major clinical features. The role of the biopsy was paramount, and a normal number of ducts were felt to exclude the diagnosis. At that time the histopathology of the neonatal and infant liver was felt to generally fall into four categories: bile duct proliferation, neonatal giant cell D. A. Piccoli Division of Gastroenterology, Hepatology and Nutrition, University of Pennsylvania Perelman School of Medicine, The Children’s Hospital of Philadelphia, Philadelphia, PA, USA e-mail: [email protected] © Springer International Publishing AG, part of Springer Nature 2018 B. M. Kamath, K. M. Loomes (eds.), Alagille Syndrome, https://doi.org/10.1007/978-3-319-94571-2_1

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hepatitis, bile duct paucity, and features of storage disease. The diagnosis of paucity depended on the number of portal tracts evaluated, and most early studies relied on hepatic wedge biopsy. The diagnosis of paucity was felt to be reliably inferred only when ten or more portal tracts were available for examination. It was also recognized, however, that the normal number of bile ducts per portal tract varies with gestational and postnatal age in normal infants, and importantly the number of bile ducts per portal tract typically decreases over time in infants with Alagille syndrome. As needle biopsy became more feasible, wedge biopsy fell into disfavor, and thus pediatric pathologists were required to estimate paucity on a smaller number of portal tracts. Nevertheless, demonstration of paucity for decades remained a critical part of the ALGS diagnostic criteria. Alagille defined the cardinal features of the syndrome, and these features were required to support the accurate diagnosis of syndromic paucity. The frequency of these manifestations varies significantly from study to study, but each feature was felt to be quite common in ALGS. The requirement for at least three clinical features for diagnosis led to a profoundly biased clinical phenotype, such that early studies recorded (by definition) frequent clinical manifestations for all patients with the syndrome. This led to a period of significant underdiagnosis of ALGS, due to exclusion of patients either without paucity or with insufficient number of syndromic features. There was significant clinical confusion around the diagnosis of a patient with paucity and cholestasis but otherwise an inadequate number of cardinal clinical manifestations. Likewise the patient with numerous features in the absence of paucity or liver disease might be excluded. Following the identification of Jagged1 as the gene causing Alagille syndrome, it has been clearly shown that patients with limited clinical manifestations can carry the mutation. They can pass on severe Alagille syndrome to their progeny, and some individuals with Jagged1 mutations have no evident clinical liver disease or bile duct paucity. Early studies reflect the lack of a cohesive name for the syndrome, including (1) syndromic bile duct paucity, (2) paucity of the interlobular bile ducts, (3) arteriohepatic dysplasia, (4) intrahepatic biliary atresia, (5) Watson-Alagille or Alagille-­ Watson syndrome, and ultimately (6) the Alagille syndrome, as a tribute to the profound, seminal observations of Daniel Alagille. Likewise, the initials for the syndrome have migrated (from PILBD, AHD, AS, and AGS) to ALGS. The term Alagille syndrome also serves to redirect the emphasis from hepatic or cardiac disease to a broader systemic disorder. Numerous case reports greatly expanded the diversity of ALGS manifestations. The features of ALGS are well detailed in subsequent chapters in this book. The clinical abnormalities are extraordinarily diverse but can be stratified into abnormalities that occur in the prenatal period, abnormalities that progress over time, abnormalities that are consequences of the structural disease, and problems that are a consequence of malabsorption and nutritional compromise (Table 1.1). Another classification scheme divides features into those that are direct consequences of JAG1 expression versus those that are consequences of malabsorption, vitamin deficiency, malnutrition, cholestasis, and pruritus. These latter features are more amenable to current medical therapy (Table 1.2).

1  Alagille Syndrome: Overview and Introduction Table 1.1  Patterns of abnormalities in ALGS

Table 1.2  Advances in the therapy for ALGS

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1. Early and late embryologic abnormalities 2. Progressive postnatal developmental abnormalities 3. Consequences of cholestasis 4. Consequences of malabsorption 5. Consequences of undernutrition 6. Consequences of chronic illness

Heart  1. Cardiac surgery  2. Pulmonary vascular therapy: dilatation, stents, cutting balloons, surgery Liver  1. Choleretic therapy  2. Antipruritic therapy  3. Fat-soluble vitamin preparations  4. Partial external biliary diversion, internal ileal exclusion  5. Liver transplantation and outcomes  6. Natural history  7. Malignancy, risk, and detection Kidney  1. Renal tubular acidosis  2. Angioplasty for renal artery stenosis  3. Antihypertensive therapy Neurovascular disease  1. Noninvasive detection  2. Surgical therapy for moyamoya, congenital anomalies  3. Interventional radiology, embolization  4. Injury prevention Genetics  1. Identification of Notch signaling in human disease    (a) Jagged 1    (b) Notch 2    (c) Other modifier genes  1. Improved diagnosis  2. Expansion of the Jagged1 (ALGS) phenotype  3. Identification of mutation carriers “at risk”  4. Precision genetic counseling  5. Preimplantation genetic diagnosis  6. Future possibilities for genetic therapy Growth  1. Historical ALGS growth curve  2. Supplemental nutrition components – macro- and micronutrient  3. Role of ALGS confounding features: acidosis, hypothyroidism  4. Gastrostomy supplementation

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Diagnosis of Alagille Syndrome and Jagged1 Mutation Status Neonatal liver diseases are caused by a bewildering array of etiologies, including infectious, toxic, immunologic, metabolic, genetic, maternal, and idiopathic causes. The clinician classifies these diseases according to various patterns and phenotypes. Neonatal liver disease may be dramatically fulminant or lifelong asymptomatic. A common pattern is neonatal cholestasis without significant synthetic compromise. Cholestasis is defined as a lack of bile flow, and while commonly accompanied by jaundice in infants, these disorders can be quite distinct. Conjugated or unconjugated hyperbilirubinemia can occur in the absence of compromised bile flow, particularly in the neonatal period. Included within the cholestatic histopathology pattern are disorders of bile duct paucity, bile duct proliferation, bile duct obstruction, and disorders with no significant or distinct abnormal histopathology. The clinician may also be guided by clinical patterns of disease, such as hepatic disease associated with (1) CNS disease or microcephaly, (2) heart disease or heterotaxy, (3) structural renal disease, renal tubular acidosis or renal Fanconi, (4) splenomegaly, or (5) other systemic features. Many neonatal liver diseases are characterized by a molecular defect that is present only in the hepatocyte or occasionally associated with other cell types (cholangiocyte, renal tubular cell, etc.). Notch signaling is involved in the embryologic development of a majority of organs in humans. The activity of Jagged1 expression and its consequences appear to be highly variable in their effects, depending on a number of as yet uncharacterized molecular events. Alagille and others recognized early on the highly variable expression of affected family members, without any clear pattern of manifestations. Over time, the diagnostic criteria for ALGS were modified such that family members of a documented proband were considered to be “affected” if they had a lesser number of features or even an absence of hepatic disease. The timely and accurate diagnosis of neonatal cholestasis is required for a number of disorders that have specific or life-altering therapy. While hepatic ALGS does not yet have a singular early therapy, it is commonly confused with biliary atresia, for which timely diagnosis and therapy are essential. In infants with cholestasis, a diagnostic sequence that includes physical examination, biochemistry, ultrasound, and nuclear scintiscan would lead to hepatic biopsy to differentiate these relatively common disorders. While ALGS is characterized by bile duct paucity and biliary atresia by bile duct proliferation, the histologic features (particularly in a biopsy performed early in life) can be non-discriminating. In those situations, the clinician faces the option of either performing repeat biopsy in a few weeks or advancing to an operative cholangiogram. A delay can have adverse consequences for biliary atresia outcome. However, the results from operative cholangiograms have also led to misdiagnosis, as the extreme hypoplasia and paucity of the intrahepatic bile ducts can give a picture misdiagnosed as intrahepatic biliary atresia, due to a lack of apparent dye in the liver but with normal egress into the duodenum. The evidence for concurrent overlap of intrahepatic biliary atresia and Alagille syndrome is controversial, but if it does occur, it is extraordinarily rare. Much more common is the

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misdiagnosis of Alagille syndrome as biliary atresia, leading to a Kasai hepatoportoenterostomy. Alagille syndrome patients receiving a Kasai may do less well than other ALGS patients, but it is unclear whether this is due to the consequences of the operation itself or to the severity of the disease, which leads to the misdiagnosis. Regardless, a Kasai procedure is unlikely to improve flow for these patients and should be avoided if possible. Alagille syndrome is also diagnosed in a number of other clinical scenarios. ALGS may present in a proband with severe fetal or newborn cardiac disease. Although diagnosis may be delayed in an infant with a suggestive cardiac lesion, cardiologists are increasingly aware that mutations in Jagged1 are an important cause of autosomal dominant inherited congenital heart disease. Tetralogy of Fallot with or without pulmonary atresia can be a particularly challenging lesion. The congenital heart disease of Alagille syndrome has been seen in isolation, in combination with other features, and in family pedigrees without apparent hepatic disease. Other patterns of cardiovascular disease, including familial pulmonic stenosis, have been seen in the absence of hepatic disease. The importance of Notch signaling in fetal development is also underscored by the recognition that defects in Notch1 and occasionally Notch2 result in dominantly inherited congenital heart disease. Alagille syndrome is occasionally diagnosed later in infancy or in childhood, with abnormalities ranging from elevated aminotransferases to splenomegaly to poor growth. Prior to gene discovery, these abnormalities might not have not been sufficient for diagnosis, even in a proband’s sibling or parent. It has been recognized in many families that characteristic childhood facies or distinct but characteristic adult facies are commonly present and correctly predict mutation status. In the office or at a clinical symposium, a completely asymptomatic parent can be inferred to have a gene abnormality based on facial characteristics alone. In a family with two (nonidentical twin) affected children, it can be assumed that one parent is a mutation carrier, even in the absence of any symptoms. The availability of timely and inexpensive JAGGED1/NOTCH2 gene testing has greatly expanded the clinical phenotype and has provided a basis for reliable genetic counseling. This is particularly true for families with apparently unaffected parents. Gene identification has allowed precise calculation of the frequency of de novo mutations in the child and also specific information for an individual family. Studies of mutation status in all family members have led to a dramatically increased estimated incidence of JAGGED1 mutation in the general population, by including apparently unaffected individuals, but this same inclusion has led to a decreased predicted risk of producing a severely affected child which, while still substantial, should also include the possibility of a child with minimal features. Nearly half of JAGGED1 carriers have a level of disease that would not, in isolation, lead to a diagnosis of Alagille Syndrome. The identification of these “mutation carriers” has however led to some controversy about whether they do indeed have “Alagille syndrome” given that the definition of the syndrome is based on clinical manifestations rather genetic identity. Regardless, these unaffected or minimally affected individuals have the risk of producing progeny with extensive life-threatening ALGS and

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therefore should be considered as falling within the “syndrome spectrum.” Whether these apparently unaffected individuals are at risk for other life-threatening complications, such as vascular disease, is not yet understood.

Discovery of JAGGED1 Mutations as the Cause of ALGS: Implications for ALGS Patients The molecular basis of hepatic disease occurs via the full spectrum of inheritance patterns: (1) autosomal recessive (commonly), (2) X-linked, (3) mitochondrial, and very rarely (4) contiguous gene deletions/insertions or (5) autosomal dominant. Daniel Alagille predicted dominant inheritance for syndromic paucity. The first clues to the causative gene were provided by a number of reported ALGS cases with a cytogenetically visible multigene deletion of chromosome 20. Clinically, ALGS has many features that are similar to the 22q (contiguous gene) deletion syndrome, such as cardiac disease, butterfly vertebrae, posterior embryotoxon, and characteristic facial characteristics, although the syndromes have clearly unique features as well. The region theoretically containing the causative abnormality was progressively reduced, and it seemed more likely that a single gene mutation might cause the diverse manifestations of ALGS. In 1997, Li et al. and Oda et al. reported the discovery that mutations in Jagged1 cause ALGS. This marked a turning point for diagnosis and for understanding the mechanisms of pathogenesis of ALGS.  The sequence and the ramifications of the gene discovery and the role of Notch signaling are fully detailed in the chapter on the molecular basis of ALGS.  An overview would be incomplete without summarizing the remarkable advances made possible by the gene identification. Notch signaling was discovered in Drosophila nearly a century ago and subsequently has been found in essentially all species. The reliance of humans on Notch signaling seems to be more profound than in other species. Despite the redundancy of ligands and receptors, mutations in Notch pathway genes cause a diverse group of (mostly) multisystem human diseases, none more profound than Jagged1/ALGS, and thus far at least, none more common than ALGS. Over the past two decades, there have been remarkable advances in genetic techniques and diagnostics, and these have contributed substantially to the understanding of ALGS and its manifestations. Unlike most other hepatic diseases, ALGS is dominantly inherited, and patients carry one copy of a normal gene (which may be important for future gene therapy). The number of unique mutations in JAGGED1 is quite large and includes terminating mutations, missense mutations, and occasional large deletions across all exons. Unlike cystic fibrosis, alpha1-antitrypsin, or hereditary hemochromatosis, there is no overwhelmingly common mutation (or characteristic biochemical assay), so early genetic diagnosis was cumbersome, slow, rarely available, and not covered by insurance. Currently, mutation analysis is easily available, cost-effective, timely, and covered and now may precede or preclude hepatic biopsy. Mutations are now detected in about 95% of ALGS patients, and about 1% have a mutation in Notch2. When a mutation is identified, other family members can be

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efficiently screened. The analysis of genotype-phenotype analyses is extensively covered in the genetics chapter, but clear patterns do not appear to occur commonly. However, genetic counseling is now significantly more informed. For parents of a child with a de novo mutation, subsequent risk is extremely low, and for apparently unaffected parents who do carry a mutation, the increased risk can be identified and discussed. For family counseling, an informed discussion of egg or sperm donation, prenatal diagnosis, or preimplantation genetic diagnosis can be arranged according to the family’s beliefs and intentions. The discussion of Notch signaling is extensively covered in subsequent chapters, but it is clear that its role in ALGS and its window on human embryogenesis are only a part of its profound implications for human disease and therapy. There have been significant advances in the diagnostic precision of ALGS and in the understanding of the clinical spectrum of patients who carry a mutation in Jagged1. There have also been significant improvements in effective vitamin therapy and nutritional regimens, although some of these medications are unfortunately extraordinarily expensive and not covered by insurance in the USA. Nevertheless, the consequences of severe vitamin deficiency are at least theoretically preventable. There has been less success with the medical therapy for choleresis and for pruritus therapy. Many of the available therapies for intense itching have been available for decades, and none are uniformly successful. Newer therapies with apical bile salt transporter inhibitors and nuclear receptors hold promise for the future. Some patients respond dramatically to partial external biliary diversion as do some with internal ileal exclusion, but many others have only modest improvements. There are newer large studies of the natural history of ALGS, and over time liver transplantation has become more available and more successful, even in patients with pre-­existing cardiac or renal disease. With an evolving understanding of neurocognitive outcomes and an increased appreciation for the profound impairments of quality of life due to pruritus, hepatic transplantation offers a pathway for potential dramatic improvement. There are, however, a wide variety of important, unmet needs for ALGS patients (Table 1.3). In many areas, there is limited access to a pediatric gastroenterologist or hepatologist, some of whom have little experience caring for patients with this rare disease. Even less common are the pediatric specialists in cardiology, nephrology, orthopedics, ophthalmology, nutrition, and others who have significant clinical experience with ALGS patients. Transition to adult medical providers poses an even larger problem. There are no guidelines for care and monitoring of adults, despite the fact that more and more patients survive into adulthood. Many adult patients are cared for by their transplant hepatologists, but few of them offer a holistic approach to the multisystem problems of ALGS. The long-term consequences of pulmonary vascular obstruction, right heart strain, elevated cholesterol, bone disease, kidney disease, and the increased risk of hepatic malignancy are essentially unknown. The natural history of the large cohort of hepatic ALGS patients followed in the Childhood Liver Disease Research Network has greatly increased the understanding of the early progression of disease, but there is limited capacity to follow these patients well into adulthood. It is not surprising that gene discovery has not led to gene therapy for ALGS. Many of the features of the disease are developmental defects that cannot be reversed, such

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Table 1.3  Unmet needs for the ALGS patient population Diagnosis Hepatic therapy Cardiac therapy Vascular therapy Pruritus therapy Transition to adulthood Natural history pediatric Care plan adulthood Family planning Maternal care proband Gene therapy proband Quality of life Availability of pediatric MD expertise Availability of adult medicine expertise Neurocognitive outcomes Prevention in proband

Met +++

Partially met

Unmet

+++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++

as severe intracardiac disease. Bile ducts, however, with their associated portal tracts undergo a developmental maturation sequence that extends at least through the first few years of life. The window for altering this sequence opens at the time of diagnosis and extends through a few years of dramatic hepatocellular and duct growth and maturation. There may be therapeutic opportunities for modulating this development. Although not well studied, it appears unlikely that choleretic therapy induces bile duct development. However, patients do have one normal copy of Jagged1, while the other is commonly a terminating mutation. Haploinsufficiency appears to be the mechanism for Notch signaling abnormalities in humans. It has been suggested that some ALGS patients have signaling capacity partway between half and full ligand expression and that some of these patients may have less dramatic hepatic manifestations. This and other information have led to the hypothesis that increasing Jagged1 expression in the neonatal and infant stages may improve the development of interlobular bile ducts and thereby ameliorate the profound cholestasis or its consequences. There are a number of theoretical possibilities for stimulating increased expression, but the effects and the consequences of this approach are as yet unknown. The development of a mouse model that phenocopies human disease may help to provide valuable information and an experimental platform for discovery.

Family Considerations and Support The Alagille Syndrome Alliance (ALGSA) was founded in 1993 and since that time has evolved into a large and successful foundation dedicated to patient and family support, networking, education, and research. Starting in 1999, the Alagille

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Syndrome Alliance has sponsored international family symposia in various regions throughout the USA, giving families an opportunity to meet together and attend educational and support programs. For many families, these symposia provide a first introduction to others affected by ALGS. The ALGSA has also been active in research participation, including the NIH-sponsored Childhood Liver Research and Education Network (ChiLDREN).

Summary The manifestations of Alagille syndrome include the most complex and most interesting of all pediatric liver diseases. The hepatic disease of ALGS ranges from inconsequential to failure, and the manifestations in many other organs also vary from trivial to catastrophic. The dominant mode of inheritance and the common occurrence of de novo mutations have led to fascinating genetic discoveries, and the presence of a normal gene in all affected individuals may lead to novel genetic therapies based on regulation of gene expression. The ALGS is the most extensively studied of any single gene defect that is involved in human embryogenesis. Notch signaling is an important mechanisms for cell to cell communication, having developed early on in evolution, and it is highly conserved in all species studied. While much has been learned about the Alagille syndrome over the 50 years since its first description, there are many current clinical challenges and opportunities for the development of novel and effective therapies. In the subsequent chapters of this book, the extensive compilation of the knowledge about Alagille syndrome and Notch signaling will serve as a framework for investigation and discovery into the mechanisms and therapies of this extremely important and fascinating disease.

Suggested Reading 1. Alagille D, Odievre M, Gautier M, Dommergues JP. Hepatic ductular hypoplasia associated with characteristic facies, vertebral malformations, retarded physical, mental and sexual development, and cardiac murmur. J Pediatr. 1975;86(1):63–71. 2. Li L, Krantz ID, Deng Y, et al. Alagille syndrome is caused by mutations in human Jagged1, which encodes a ligand for Notch1. Nat Genet. 1997;16(3):243–51. 3. Oda T, Elkahloun AG, Pike BL, Okajima K, Krantz ID, Genin A, Piccoli DA, Meltzer PS, Spinner NB, Collins FS, Chandrasekharappa SC. Mutations in the human Jagged1 gene are responsible for Alagille syndrome. Nat Genet. 1997;16(3):235–42. PMID: 9207787.

Chapter 2

Bile Duct Development and the Notch Signaling Pathway Stacey S. Huppert and Kathleen M. Campbell

At its most basic, the biliary system is a branching network of tubes that routes bile and its components from the liver to the gallbladder and, subsequently, releases this fluid into the small bowel. But to think of the biliary system as a mere drainage, structure belies the true importance and elegance of the structure. The biliary system is a complex configuration which parallels and is intimately related to the portal venous and hepatic arterial supply, as represented by the classic portal triad (Fig. 2.1a). This tubular network is lined by a heterogeneous population of metabolically active epithelial cells, cholangiocytes, which vary in size, gene expression, and proliferative capacity based on location within the biliary system. These specialized cells establish an apicobasal epithelial polarity that forms the connected intrahepatic bile duct (IHBD) system. The smallest components of the biliary system are the narrow bile canalicular channels formed by the apical surfaces of adjacent hepatocytes (Fig.  2.1a). Hepatocytes secrete bile from their apical surface into the canaliculi, which form a connected network extending throughout the parenchyma [1, 2]. Bile flows from the canaliculi to the canals of Hering, conduits lined partly by hepatocytes and partly by cholangiocytes. The canals of Hering are eventually connected to ductules, the smallest ramification of the IHBD system (Fig. 2.1b) [3]. The IHBD system relies on its intricate three-dimensional structure to access all of the hepatocytes and effectively clear bile out of the liver (Fig. 2.1c). IHBD architectural formation is a highly complex and regulated process that occurs in a coordinated fashion along the portal vein network to configure a connected and highly branched network that drains the hepatic parenchyma. S. S. Huppert (*) · K. M. Campbell Division of Gastroenterology Hepatology and Nutrition, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH, USA e-mail: [email protected]; [email protected] © Springer International Publishing AG, part of Springer Nature 2018 B. M. Kamath, K. M. Loomes (eds.), Alagille Syndrome, https://doi.org/10.1007/978-3-319-94571-2_2

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a

S. S. Huppert and K. M. Campbell

b

c

Fig. 2.1  Overall IHBD architecture. (a) BSEP localization highlighting the bile canalicular channels in mouse liver. (b) schematic of canalicular channel formed by apical surfaces of adjacent hepatocytes (blue), communicating with IHBD network composed of cholangiocytes (green). (c) schematic of IHBD hierarchical network structure

Although cholangiocytes comprise only 3–5% of the total cell mass of the liver, they play critical roles in liver health and disease. Not only do they modify biliary flow, composition, and volume in response to a variety of hormonal and other stimuli; cholangiocytes interact with other resident and nonresident cells of the biliary system via the release of inflammatory and fibrotic cytokines and participate in tissue homeostasis by modulating biliary apoptosis, senescence, and proliferation. Perturbations in these critical functions can lead to cholestasis, fibrosis, ductopenia, and/or malignancy [4]. The biliary system receives its arterial supply from the hepatic artery, which forms a delicate peribiliary capillary plexus at the level of the smallest intrahepatic bile ducts, and drains directly into adjacent portal vein branches [5]. In addition to the cholangiocytes and vascular endothelial cells, the IHBD and periportal region contains lymphatic tissue, stromal tissue, and peribiliary glands, which proposed an important reservoir for biliary stem cells in the extrahepatic and large intrahepatic branches of the IHBD system [6].

Normal Intrahepatic Bile Duct Development Most mammalian organs with a branched multicellular epithelial network (such as neural tube, lung, kidney, pancreas, and salivary gland) form tubes by processes that involve wrapping of an epithelial sheet, budding/branching via subdivision or proliferation from an existing cellular compartment, or cavitation of a cylindrical cluster of cells [7]. In contrast to these organs, the liver forms the branched IHBD network through a multistep process that begins with specification and ends with morphogenesis of the specified cholangiocytes into a tube [8, 9]. This alternative process of tubulogenesis allows the liver to continually generate a connected biliary system coordinated with an enlarging parenchymal mass (“the liver” per se) and potentially enables the unique ability of the liver to regenerate upon alleviation of injury [10]. A stark contrast is observed in organs where the process of tubulogenesis is used to form tubes with terminally differentiated cells (alveoli, glomeruli, and beta cells), in which there is no potential for regeneration.

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Cholangiocyte Specification Extensive analysis of human fetal and neonatal liver samples along with examination of genetic mouse models exhibiting abnormal IHBD formation has provided critical insights into the process of IHBD development [11–15]. The cells that contribute to the IHBD system are a subpopulation of liver progenitors (i.e., hepatoblasts) located in close proximity to portal veins. Hepatoblasts are bipotential cells with the capacity to differentiate into hepatocytes and/or cholangiocytes. Around embryonic day 11–14 in mice, and 7–10 weeks of gestation in humans, a subpopulation of hepatoblasts forms a band of potential cholangiocytes, termed the ductal plate, which encircles the portal veins [13–16] (Fig. 2.2a). This first step in IHBD formation is cholangiocyte specification, in which hepatoblasts enter into the cholangiocyte transcriptional program, characterized by expression of specific cytokeratins (e.g., CK19) and the transcription factor Sry-related HMG box 9 (SOX9). Hepatoblasts entering into the cholangiocyte program not only induce cholangiocyte-specific genes but also repress hepatocyte genes and undergo growth arrest [17].

Cholangiocyte Morphogenesis The specified cholangiocytes then undergo morphogenesis, during which the cells become polarized and generate a lumen adjacent to the portal vein. Remodeling of ductal plates into IHBDs begins with the oldest ductal plates surrounding the larger portal veins near the liver hilum, and is thought to move outward, toward the periphery of the liver, following the branches of the portal venous system. Therefore, all steps of biliary morphogenesis progress in a hilum-to-periphery direction, allowing several stages of IHBD formation to be analyzed within a single liver during hepatogenesis [14, 16]. Cholangiocyte morphogenesis begins around embryonic day 15–17 in mice and gestational week 11–15 in humans [13–16]. This step delineates

a

b

c

d

Cholangiocyte – Sox9+ Hepatocyte – Hnf4+ Hepatoblasts Portal myofibroblasts Endothelium

Fig. 2.2  Two-dimensional schematic of temporal cholangiocyte specification and morphogenesis process

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the structure surrounding a forming lumen as either a primitive ductal structure (PDS) or a mature duct. Transient asymmetrical PDS are observed in mice and humans [12]. The PDS is composed of two distinct cell types as distinguished by the presence or absence of marker expression (SOX9, hepatocyte nuclear factor 4 [HNF4], and transforming growth factor receptor type 2 [TGFBR2]) compared to a mature duct. The PDS is asymmetrical; cells on the portal vein side of the lumen express the marker SOX9, compared to cells on the parenchymal side that express HNF4 and TGFBR2 (Fig. 2.2b). A mature duct is symmetrical, composed of cells expressing SOX9 [12, 16] (Fig.  2.2c). Additionally, the portal vein side of PDS displays higher levels of E-cadherin expression and is in contact with laminin. Upon symmetrical mature duct formation, the lumen displays equal levels of E-cadherin expression and is surrounded by extracellular matrix and mesenchyme (Fig. 2.2d) [16]. Detailed analysis using immunostaining suggests that there is a radial progression of differentiation – mature cells on the portal vein side of the lumen promote differentiation of the neighboring less mature cells on the parenchymal side of the lumen [16]. However, it remains unclear if cells from the ductal plate move and contribute to the less mature cells of the forming lumen or if additional hepatoblasts are recruited to contribute to lumen formation. Given the expression of HNF4 in the less mature cells on the parenchymal side of the PDS, the more plausible explanation is that hepatoblasts are recruited to contribute to the forming lumen (Fig. 2.2b, arrows). Nevertheless, sandwich cultures of hepatoblasts suggest that monolayers of progenitors can fold up to form tubular structures in a wrapping process [18]. To map cholangiocyte specification and morphogenesis at a high resolution in three dimensions, serial sections of mouse liver samples from mid-gestation to adult age were immunostained with CK19, and digital three-dimensional reconstruction of images was performed. Using quantitative morphometric analyses, length, number of branch points, and distance from the portal vein were analyzed. Information garnered from the high-resolution three-dimensional maps suggests that CK19positive cholangiocytes are specified in clusters, rather than in a cell layer as suggested by two-dimensional analyses [19]. As IHBD development proceeds, the clusters of CK19-positive cholangiocytes are further increased and form dense networks encircling the portal vein (Fig. 2.3a) [19]. Thus, the ductal plate structure, previously assumed to be a cell layer of specified cholangiocytes surrounding the portal vein, may instead be a three-dimensional dense network of cholangiocytes. Finally, the clusters of CK19-positive cells surrounding the portal vein decrease with age as specific segments of the network are selected to become larger diameter CK19-positive tubes which remain parallel to the portal vein, but at an increasing distance from the portal vein as development progresses (Fig. 2.3c) [19].

Refinement of Intrahepatic Bile Duct Architecture The CK19-positive cellular clusters of the ductal plate that remain unincorporated into an IHBD either undergo apoptosis [20] or turn off expression of some of the cholangiocyte markers and contribute to periportal hepatocytes or the canal of

2  Bile Duct Development and the Notch Signaling Pathway

a

b

15

c Distal peripheral

Bile production Proximal hilar E17

E18

Adult

Cholangiocyte – Sox9+ Bile canaliculi Portal myofibroblasts

Fig. 2.3  Three-dimensional model of IHBD formation

Hering (Fig. 2.2d, SOX9-positive hepatocytes) [17]. Lineage tracing SOX9-positive cells in mouse strongly suggests that the unincorporated cholangiocytes convert into hepatocytes without apoptosis or proliferation [17]. If the unincorporated ductal plate cells do not receive or are deaf to the proper signals, they may contribute to ductal plate malformations [11, 12]. Thus, a high level of coordination is required for regulation of sequential tubulogenesis and regression of the ductal plates along portal veins within the three-dimensional structure of the liver such that the entire IHBD system connects seamlessly to the extrahepatic ductal system. This indicates that careful orchestration of signals between epithelial and mesenchymal cells is required to guide IHBD formation [8, 21]. Another model that provides a global view of IHBD formation without the caveats of three-dimensional reconstruction (such as best guesses for alignment and gaps in images, as well as variable antibody sensitivity in detection of cholangiocytes) involves a retrograde injection of carbon ink into the luminal space from the common bile duct. Ink fills the continuous luminal space of IHBDs but does not leak into the portal vein or the hepatic parenchyma [22]. This procedure was performed at time points between mid-gestation and 1 week after birth and was followed by clearing the liver tissue to expose the ink-filled IHBD structure at different developmental time points [22, 23]. Immunostaining was used in combination with the ink injections to detect the correlation between cholangiocyte specification and communicating luminal IHBD structures. The main findings are consistent with three-dimensional reconstruction models; however, a greater appreciation for the process of cholangiocyte morphogenesis and IHBD formation is revealed. Visualizing the communicating IHBD system as it forms provides a new level of comprehension that cholangiocyte specification and morphogenesis proceed in a hilar to peripheral direction [23]. The homogenous communicating network of luminal structures encircling the portal vein begins to be rearranged into a hierarchical network between embryonic day 17 and embryonic day 18 in mice (Fig. 2.3b). Interestingly, in mice, this timing correlates with lengthening of the bile canalicular network, synthesis of direct bilirubin, and potential influx of bile into the intestine [23]. Using the MRP2 inhibitor

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benzbromarone to block bile canaliculi formation and thereby decrease the flow of bile between mouse embryonic day 16 and embryonic day 18, the structural rearrangement of IHBDs was inhibited [23, 24]. In other words, the homogenous communicating network was not rearranged into a hierarchical network as observed in controls. These results indicate that the influx of bile drives the structural transition of IHBDs from the homogenous tubular network into the mature hierarchical network. In this model, any communicating luminal duct that is part of the homogeneous network and connected to a bile canaliculus has the potential to receive more bile secreted from hepatocytes compared to the other communicating lumen, and thereby its luminal space may enlarge. Even a week after birth, a homogenous network surrounding the portal vein is still visible in the liver periphery of mice [23]. The incomplete IHBD architecture at birth is also true in humans where the IHBD system is still forming during the first years of life [14]. Therefore, the process by which the IHBD system forms allows for progressive assembly of a communicating IHBD architecture coincident with the enlargement of the liver during normal growth in childhood. The final IHBD hierarchical architecture consists of large ducts running along large portal veins and small channels forming a mesh-like network around portal venules (Fig. 2.3c). This is truly a beautiful structure that is formed by a very complicated dance between cells that need to know their place in three-dimensional space to form connections in order to perform the function of draining bile out of the liver. Therefore, the model for IHBD formation can be summarized as follows: (1) cholangiocyte specification initially occurs as clusters of cells surrounding the portal vein, (2) cholangiocyte morphogenesis forms a homogeneous communicating luminal network of small ducts, and (3) coincident with hepatocyte bile production and secretion, the homogeneous luminal IHBD network is rearranged into a hierarchical IHBD system.

The Notch Pathway The Notch pathway is an evolutionary conserved intercellular signaling pathway required for lineage commitment, cell specification, and maintenance of progenitor cells during embryogenesis, adult cell population renewal, and response to injury [25]. Stem/progenitor cell populations in diverse tissues such as the neural, pancreas, intestine, blood, skin, hair, and skeletal muscle require Notch. The mechanism of Notch signal transduction is unique in that the canonical pathway transmits a signal directly to the nucleus via the receptor (Fig.  2.4) [26]. The mammalian Notch genes (N1–4) encode single-pass type I transmembrane receptors that transduce extracellular signals from their canonical ligands Delta-like (DLL1, 3, and 4) and Jagged (Jag1 and 2) present on the cell surface of neighboring cells. Ligand binding triggers activation of the Notch receptor through a series of proteolytic events, culminating in a gamma-secretase, presenilin-dependent, proteolytic release of the Notch intracellular domain (NICD) from the membrane. NICD translocates

2  Bile Duct Development and the Notch Signaling Pathway

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NICD + RBP-JK + co-activators

Signal sending cell

Pro-cholangiocyte Transcriptional program

JAG1 16 EGF-like repeats PDZL CRD

MNNL/C2 DSL

NRR

TAD

RAM ANK

Notch2 36 EGF-like repeats

S1

S3/S4

S2

PEST

NICD

Signal receiving cell

Fig. 2.4  Canonical Notch signaling pathway. The majority of Notch receptors are presented on the cell surface as a heterodimer held together by non-covalent interactions of the HD (heterodimerization domain) after proteolytic cleavage by furin convertase at S1 (site 1). The HD and three cysteine-rich LNRs (Lin12/Notch repeats) comprise the NRR (negative regulatory region) that is critical in preventing receptor activation in the absence of ligand. Ligand binding to the Notch receptor exposes S2 within the HD and allows cleavage by a metalloprotease of the ADAM (a disintegrin and metalloprotease) family. The truncated Notch receptor then becomes instantly susceptible to subsequent intramembranous cleavages by the gamma-secretase complex at S3/S4, thereby releasing the Notch intracellular domain (NICD). NICD has multiple conserved domains: a RAM (RBP-JK-associated molecule) domain, an unstructured linker containing one NLS (nuclear localizing sequence), seven highly conserved ANK (ankyrin) repeats, two NLS and lessconserved variable TAD (trans-activation domain), and a conserved PEST (proline/glutamic acid/ serine/threonine-rich) motif containing degrons that regulate the stability of NICD.  The Jag1 ligand and Notch receptor interaction interface (red-shaded domains) consists of two engagement sites. The first contains the classic ligand DSL (Delta-Serrate-Lag-2) domain and the MNNL (module at the N-terminus of Notch ligands), which is a C2 domain often involved in binding phospholipids [104], and the Notch EGF-like repeats 11 and 12. The second consists of the Jag1 EGF-like repeats 1–3 and the Notch EGF-like repeats 8–10 [105]. The Jag1 ligand also contains a CRD (cysteine-rich domain) with sequence similarity to von Willebrand factor C, distinguishing Jagged/Serrate from the DELTA family of Notch ligands. In addition, Jag1 contains an intracellular PDZ domain which is associated with protein interactions at the adherens junction. A more in-depth description of the Notch pathway and associated protein domains has been reviewed by Kovall et al. [106]

to the nucleus where it associates with RBP-JK/CBF1/Su(H)/Lag1 (hereafter referred to as RBP-JK) to activate transcription of downstream targets. Only a limited number of target genes have been identified, the most familiar belonging to the HES and HEY gene family of basic helix-loop-helix transcription factors [27]. Therefore, the majority of lineage decisions regulated by the Notch pathway are based solely on the context of the unique molecular signature of each cell at the time the Notch pathway is activated. Given the ubiquity of the Notch signaling pathway, it is no surprise that defects involving either core components of the pathway or signaling targets have been associated with a variety of human diseases. Most of these disorders involve axial

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skeletal defects, vascular or cardiovascular abnormalities, or neurologic degeneration [28, 29]. Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is caused by mutations in the Notch3 gene, which encodes the Notch3 receptor expressed in vascular smooth muscle cells [30]. Mutations in the Notch1 gene leading to unregulated activation of the Notch1 receptor in T-cells are involved in more than 60% of human T-cell acute lymphoblastic leukemia [31], and several genes in the Notch pathway have been associated with spondylocostal dysostosis and spondylothoracic dysostosis [32].

 he Requirement of Notch during Intrahepatic Bile Duct T Development The strongest evidence of Notch pathway involvement in liver development is the finding of mutations in Jagged1 (Jag1), the gene encoding a key Notch receptor ligand, in more than 94% of patients with a well-defined clinical diagnosis of Alagille syndrome (ALGS) [33]. Mutations in the Notch2 receptor have been identified in 40–50 >50–60 Total

No of patients 13 10 17 11 13 11 9 2 86

Otitis media 6 8 12 9 5 4 3 0 47

URTI 4 6 5 1 1 0 0 0 17

LRTI 5 8 6 7 3 0 1 1 31

Gastroenteritis 0 2 1 0 0 0 0 0 3

No of patients presenting with Other FUO infections infection 1 0 0 0 0 0 0 0 0 1 0 0 0 1 0 0 1 2 49 (56.97%)

Age range = 0.5–57 years. Median = 19 years

Evidence for Immune Dysregulation in ALGS There will inevitably be difficulty in providing evidence of syndromic consequences of immune dysfunction given that ALGS itself is rare, the manifestations of the condition are variable and complex and sometimes severe, the immune consequences described are protean, overlapping with events seen among normal children, and only a minority, though perhaps a sizable one, are affected with immune features. Our own experience of our ALGS population, similar to that described by Quiros-Teijera et al. [14], recorded an increase in various inflammatory conditions, apparently infection based or triggered. A summary of our patients at King’s College appears in Table 10.1. Infections were noted at all ages but appeared to involve particularly a subgroup of ALGS patients who suffered recurrent and/or persistent infections [15]. A denominator for comparison of these patients to determine the prevalence and clinical significance is difficult. Infections are common in the general population, especially among children, while patients with ALGS have reasons such as their malnutrition and liver disease that could explain more frequent or worse infections. As yet we have been unable to study our ALGS cohort to look for associations between immune dysfunction in vitro and clinical events. In the context of causation of inflammatory bowel disease in the genetic-environmental spectrum, Kellermayer has considered ALGS as a monogenic condition where nongenetic factors being environmentally labile in contrast to the genetic predisposition will be an important modulator of IBD.  Genetically less clearly defined diseases such as celiac disease have a greater environmental etiological contribution [47]. In fact, 4 of our 192 ALGS patients have IBD including a pair of siblings who developed the condition soon after liver transplantation. There is no literature on ALGS among IBD patients or vice versa. Mannion et al. described a young African child with ALGS and granulomatous disease compatible with aggressive sarcoid or Blau syndrome [48]. The child proved

10  Immune Dysregulation in Alagille Syndrome: A Feature of the Evolving Phenotype

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to have refractory vasculitis requiring treatment with steroids, to which the condition rapidly became resistant, and then mycophenolate mofetil and infliximab. They note two other comparable ALGS cases: a young girl with Takayasu arteritis and a patient with a cystic mandibular mass, which on biopsy revealed multiple central giant cell granulomata that were nonmalignant [49, 50]. They reflect that “…overexpression of Jagged1 on APC promotes Treg and TH-2 CD4 T cell development and intact Notch signaling is required for appropriate populations of TCRγδ T cells and TCRαβ T cells, it follows that Jagged1/Notch interactions are critical to immune homeostasis,” and they predict an association with granulomatous conditions. As mentioned in the text, one of our four studied children has developed tracheopathia osteoplastica, a chronic inflammatory and occasionally granulomatous condition of the upper airway (see beneath). The mechanism of cholestatic pruritus remains unclear. There is a moderate but indirect association with elevation of total serum bile acid levels. An inflammatory non-histamine pathway involving autotaxin and lysophosphatidic acid has been proposed as the local neurological trigger of the sensation of itch [51]. Secondary skin damage from scratching and introduction of antigens across the skin barrier set up inflammation with possible histamine-induced itch. Histamine is not a primary cause of cholestatic itch [52]. The pruritus of ALGS tends to be severe, of early onset and disproportionate to other markers of cholestasis with exacerbations associated with intercurrent infections and after surgery. It remains to be demonstrated if TH2-based pro-inflammatory immune responses in the skin may worsen or prolong itch in some ALGS patients in particular. We studied four patients from our ALGS cohort that at King’s College Hospital have diagnostic mutations in JAG1, one being in the recognized CD46-binding domain. All had experienced previous persistent, severe, and recurrent infections, including various respiratory infections, gastrointestinal symptoms, and prolonged fever of unknown origin, features characteristic of but at the severe end of the spectrum of the immune-related features of the syndrome according to our data (Table 10.2) [53]. The patients were well at the time of the studies. They had mutations in either exon 3, 4, 18, or 19 of the JAG1 gene being AP3, 4, 2, and 1, respectively (Fig. 10.3a). Although key lymphocyte populations were normal, we showed a series of consistent abnormalities [53] (Figs.  10.3b and 10.4). There was, surprisingly, increased Jagged1 expression on resting T cells. CD4+ T cells from patients with ALGS and Jagged1 mutations were unable to mount appropriate Th1 responses in  vitro and in vivo. Further, the inability of T cells from ALGS patients to produce normal amounts of IFN-γ extended into a failure to then switch into an IL-10-secreting regulatory phenotype. This effect has also been observed in CD46-deficient patients. Importantly, T cells from all four patients presented with normal cell division and normal or even exaggerated Th2 responses, excluding a general defect in cell cycle progression. Also, similar to T cells isolated from CD46-deficient patients, T cells from ALGS patients had deficient regulation of CD127 and/or CD132 by T cells with high resting levels and supranormal cell surface expression on stimulation. CD127 and CD132 together form the IL-7 receptor required for homeostatic expansion of Th1 and Th17 populations.

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A. Baker

Table 10.2  Features of four patients with ALGS with immune dysfunction and two controls Gender & age (years) Ethnicity Mutation Recurrent otitis media

Recurrent chest infections Recurrent gastrointestinal symptoms Recurrent PUO Allergies/ atopy Other

HD1

HD2

AP1

AP2

AP3

AP4

Male 8 Caucasian – –

Male 10 Caucasian – –

Female 2 Caucasian Exon 18 +

Male 4 Caucasian Exon 3 +

Female 11 Caucasian Exon 4 +

–

–

Female 6 Caucasian Exon 19 + Severe when 4–6 year hospitalized +

+ URTI (tonsillitis)

+ 6-Byr hospitalised

+ URTI (tonsillitis)

–

–

–

–

+ + Gastroenteritis Hospitalized 1year

–

–

–

–

–

–

–

–

+ hospitalised –

–

–

–

+ Tooth abscess

Food allergies & Eczeme –

–

–

AP1 became extremely unwell after MMR + UTI. Recurrent gastrointestinal symptoms include diarrhea, vomiting, abdominal pain/tenderness, blood in stools, and +/− fever AP2 has since developed tracheopathia osteoplastica, a segmental degenerative disorder of the tracheobronchial tree characterized by multiple, variably sized submucosal cartilaginous and osseous nodules that cause upper respiratory tract narrowing. It may be associated with chronic inflammation AP3 – Evidence of food intolerance but negative pin-prick tests

CD132 also is required for responsiveness to the IL-2 cytokine family of molecules, (IL-2, IL-4, IL-7, IL-9, IL-15, IL-21). Patients’ AP1 and AP2 produced no IFN-γ (and did not switch to IL-10 production) upon either CD3, CD3 + CD28 (not shown), or CD3 + CD46 activation, while AP3 and AP4 presented with a marked decrease in TH1 induction (≤50%). T cells from these last two patients also gave a noticeably increased TH2 response that would be predicted to lead to prolonged immune activation. In further experiments T cells from our ALGS patients failed to engraft and to induce an in vivo Th1 response into immune-deficient mice in a graft versus host disease model, implying a possible defect of the homing capacity of T cells. We noted overexpression of Jagged1 protein by our four immune-deficient ALGS patients. When organisms heterozygous for a loss-of-function allele maintain the phenotypic characteristic, it is generally attributed to the metabolic theory of dominance that the phenotypic consequence of heterozygous loss-of-function alleles are masked by the activity of one wild-type allele due to redundancy of cellular physiology [54]. Exceptions to this rule are recognized where deletion of a single gene copy leads to an abnormal phenotype as is the case for ALGS, considered either codominance or partial penetrance and explained in that they result in transcription defects in downstream products [55].

10  Immune Dysregulation in Alagille Syndrome: A Feature of the Evolving Phenotype

a

SP

EGF-like repeats

DSL domain

?

protein

1

exons 1 2 3

4

AP3

5

2

6

3

4

5

6

7

8

9 10

7 11

12

8

9 10

13 14

CR TM domain domain

145 PDZ domain

11 12 13 14 15 16

15

AP4

19 16 17 18 20 21 22 23 24 25 AP2

26

AP1

b

IL-10 (ng/ml)

15 10 5 nd

nd

nd

nd

nd

nd

6 4 2

120

4

100 IL-4 (ng/ml)

5

3 2 1

nd

nd

nd

nd

nd

nd

nd

80 60 40 20

nd

–

nd

nd

NA α – CD3 α – CD3 – CD46 NA α – CD3 α – CD3 + CD46 NA α – CD3 α – CD3 + CD46 NA α – CD3 α – CD3 + CD46 NA α – CD3 α – CD3 + CD46 NA α – CD3 α – CD3 + CD46

0

nd

0

HD1

HD2

AP1

AP2

AP3

AP4

0 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0

nd

NA α – CD3 α – CD3/CD46 NA α – CD3 α – CD3/CD46 NA α – CD3 α – CD3/CD46 NA α – CD3 α – CD3/CD46 NA α – CD3 α – CD3/CD46 NA α – CD3 α – CD3/CD46

0

TNF (ng/ml)

8

IL-5 (ng/ml)

INF-g (ng/ml)

20

HD1

HD2

AP1

AP2

AP3

AP4

Fig. 10.3 (a) The domains in the Alagille syndrome gene where abnormalities were found for each of our four patients. (b) T cells from Alagille syndrome patients with defective in vitro TH1 induction. Data shown are the mean value of each condition performed in duplicate of patients in comparison with the two age-matched normal children, HD1 and HD2

Haploinsufficient genes can be highly expressed. In haploinsufficiency studies in yeasts, for example, the protein levels of haploinsufficient genes are higher than those of essential genes, nonessential genes conferring slow growth as homozygotes, and the entire genome (Fig. 10.5) [56]. A similar trend is observed for mRNA abundance where a fivefold higher mean mRNA expression level is observed for haploinsufficient genes compared to the genome as a whole (t-test, p = 1e–127).

146

A. Baker Jagged1

NA

100 80 α-CD3/ 60 CD46 40 20 0 0 10

HD1 (MF1 11) HD2 (MF1 10) AP1 (MF1 21) AP2 (MF1 44) AP3 (MF1 15) AP4 (MF1 15) Isot. (MF1 1-2)

101

CD127

NA

100 80 α-CD3/ 60 CD46 40 20 0 100

Isot (MFI 4-6)

HD1 (MFI 47) HD2 (MFI 35) AP1 (MFI 212) AP2 (MFI 67) AP3 (MFI 45) AP4 (MFI 872)

Isot (MFI 5-7)

101

Isot. (MFI 5-7)

102 103 104 Jagged1

HD1 (MFI 191) HD2 (MFI 182) AP1 (MFI 203) AP2 (MFI 255) AP3 (MFI 129) AP4 (MFI 513)

102 103 104 CD127

CD46

Notch1

HD1 (MFI 3) HD2 (MFI 4) AP1 (MFI 10) AP2 (MFI 14) AP3 (MF1 9) AP4 (MFI 15) Isot. (MFI 1-2)

HD1 (MFI 8) HD2 (MFI 84) AP1 (MFI 58) AP2 (MFI 72) AP3 (MFI 46) AP4 (MFI 41) Isot. (MFI 4-6)

Isot. (MFI 1-2)

HD1 (MFI 211) HD2 (MFI 209) AP1 (MFI 189) AP2 (MFI 289) AP3 (MFI 215) AP4 (MFI 231)

Isot. (MFI 1-2)

Notch1

HD1 (MFI 92) HD2 (MFI 93) AP1 (MFI 69) AP2 (MFI 89) AP3 (MFI 79) AP4 (MFI 99)

HD1 (MFI 6) HD2 (MFI 5) AP1 (MFI 8) AP2 (MFI 34) AP3 (MFI 68) AP4 (MFI 9)

CD46

CD132 Isot (MFI 4-6)

HD1 (MFI 13) HD2 (MFI 18) AP1 (MFI 92) AP2 (MFI 46) AP3 (MFI 33) AP4 (MFI 49, 158)

HD1 (MFI 77) HD2 (MFI 71) AP1 (MFI 75,503) AP2 (MFI 83) AP3 (MFI 68, 1055) AP4 (MFI 168) Isot (MFI 5-7)

CD132

Fig. 10.4  Alagille syndrome patients present with deregulated expression of Jagged1, CD132, and CD127. T cells from all four ALGS patients had deregulated expression of Jagged1, CD127, and/ or CD132 by T cells with high resting levels and supranormal cell surface expression on CD3/ CD46 co-stimulation and normal Notch1 and CD46 expression. T cells from AP2 and AP3 were unable to downregulate CD46 upon CD3 + CD46 activation efficiently with deviations comparable to those observed in the CD46-deficient patients in the regulation of CD127 and CD132 by T cells from the ALGS patients

An explanatory theory for the effect on the abnormal phenotype proposes that deviations from the normal stoichiometry of the components of a protein complex cause the phenotype of haploinsufficiency. This “balance hypothesis” [57] predicts that the haploinsufficient phenotype will be the same as the overexpression phenotype, as both scenarios should result in an imbalance of subunits of a protein complex with similar functional consequences (Fig. 10.5). An opposing theory states that haploinsufficiency is due to reduced levels of protein produced in the ­heterozygous state implying that overexpression of haploinsufficient genes would not be deleterious and should result in the wild-type phenotype. Our findings are compatible with the former hypothesis in ALGS.

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15 10 5

Log2 (protein abundance)

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HI

Genome

Essentials

Slow Hom

Fig. 10.5  Mean protein abundance of different gene classes defined by fitness in YPD: haploinsufficient (HI), whole genome (genome), essential genes (essentials), and slow-growth homozygous genes (slow hom)

Therefore, in summary, all four patients had a similar defect in TH1 induction. Two patients had concurrent TH2 overactivation/induction. The features are compatible with immune dysregulation in favor of a defective pro-inflammatory state, in turn suggesting a mechanism for the previously unexplained recurrent pyrexias or infections in some ALGS patients. They might also be expected to predispose to symptoms of asthma, eczema, food allergies, and airway atopy with otitis media, which are Th2-driven [58, 59].

ALGS and the Immune System Previously, no specific defects of immune function had been shown to be associated with the persistent infective features recognized in some cases of ALGS or in ALGS patients generally. Our description of immune responses that are abnormal and in keeping with what is known about the role of Jagged1-Notch signaling in the immune system is also compatible with the role of CD46  in this pathway [53]. Moreover, defective CD46/Notch system crosstalk delivers a mechanistic explanation for the infection-associated features beginning to be recorded in ALGS subpopulations including the phenomenon proposed by Mannion et  al. at the more severe end of the spectrum. The prevalence and natural history of these abnormalities in the ALGS population, as well as the optimal treatments, for example, intravenous immunoglobulins, immunosuppression, or indeed anti-C5 biologics, on ALGS patients including during vaccination schedules and transplantation immunosuppression need to be explored [48].

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No description of clinical immunological problems specific to ALGS after transplant has been published, and ALGS transplant outcomes after 30 days are comparable to the pediatric benchmark, biliary atresia, but there is no series closely examining posttransplant complications in ALGS [60]. Infections are frequent for all patients after transplant, being treated diligently and coinciding with immunosuppression. ALGS is an infrequent transplant indication, and not all patients may suffer from immune dysregulation, which in any case may be diverse in its manifestations. Increase in infection rate might easily be overlooked unless it leads to increased posttransplant mortality, and low-level immunosuppression as after transplantation may ameliorate inappropriate or excessive inflammation. Apart from the understanding of immune function in ALGS, the description of ALGS as a model of pro-inflammatory immune dysregulation yields opportunities to explore cell signaling in what is emerging as a key component of phasic immune homeostasis. We have noted other patients similar to the four studied with prolonged fever of unknown origin (FUO), while in our cohort of over 190 patients with ALGS, specific inquiry reveals prevalent dermatitis previously attributed to scratching their itch, food intolerances, and evidence that although persisting in some, the severity of the immune-activated phenotype exhibited by our study patients tends to improve with age. Bee-sting antigen immune tolerance is an example of memory-dependent tolerance where repeated antigen exposure leads to persistence of the IFN-γ to IL-10 switch [61]. ALGS patients could exhibit impaired tolerance of exogenous antigens including at their interfaces with the environment that could improve with maturity or exposure. Changes with age in ALGS immune cell populations and cytokine profiles and responses may elucidate the milieu associated with remission of other uncontrolled inflammatory pathologies and offer key targets for immunotherapy. The observation of apparent improvement in these symptoms in some adds to the ongoing controversy as to whether ALGS patients improve as they mature physically. Conversely, immune memory might be expected to be affected adversely in ALGS.  IL-7 formed from CD127 and CD132 is a key growth factor for T cell homeostasis and T cell memory induction. Our data suggest that the effector and central memory cell pools in our patients with ALGS are likely to be functionally abnormal and should be further assessed. It will be intriguing to discover how ALGS patients respond to IL-7 and to assess if their T helper and T regulatory “half-life” is altered. Jagged-Notch interactions are also involved in the life cycle of other immune-competent cells, such as antigen presenting dendritic cells as well as B cells. As CD46 expression is ubiquitous, a functional link between the Notch and complement system on these latter cells is likely and should be addressed in future studies. Finally, of course, ALGS patients have one defective Jagged1 ligand with evidence of haplotype insufficiency. We may therefore be able to study the effects of JAG1 polymorphisms on immune function through their effects on ALGS patients identified to have a single immunologically functioning JAG1 gene. In summary, our discovery of a novel mechanism underlying defective T cell immunity in ALGS delivers a platform to extend our understanding of coordination of the phases of immune responses mediated through the Jagged1-Notch signaling system.

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Clinical Significance The implications of immune dysregulation for the management of the ALGS population remain unclear. The majority of patients do not exhibit infections so far noticeably different to normal children or children with other chronic liver diseases. The outcome for liver transplantation in terms of survival has not yet been demonstrably impacted. This suggests that the consequences of immune dysregulation are not great in the total ALGS population compared with the symptoms associated with liver, cardiac, and other system disease and the anticipated complications of liver transplantation. Nevertheless there are a cohort of perhaps as many as one third of ALGS patients who exhibit pro-inflammatory or persistent inflammatory features following infections or apparently similar immune exacerbations. The symptomatic consequences may be significant, and the immune features may impact treatment of other system disease, such that recognition of immune dysregulation might lead to change in treatment in individual patients. By far the commonest infections seen in our cohort were otitis media and lower and upper respiratory infections, common in the pediatric population generally but tending to be severe and prolonged [15]. We recognize the consequences for hearing and speech development and the impact on education. Currently our treatment approach remains conservative. However, there may be a case for immune modulatory treatment for those who exhibit significant immune dysregulation such as unexplained FUO lasting for weeks. As mentioned above, pruritus is particularly severe and problematic to treat in ALGS and may be complicated by secondary eczema from scratching. Conventional treatment is only partially successful [62] so that persistent itch is the major symptom reported by the majority of families adversely impacting quality of life for the whole family. Such severe symptoms can justify potent local or systemic immunomodulation, using steroids, immunoglobulins, or cytapheresis, as illustrated by the use of the invasive molecular adsorbent recirculating system (MARS) dialysis temporarily in small numbers of children with some limited benefit [63]. Nephropathy is seen in about 40% of ALGS patients (19–74%) [64, 65]. In a recent series, only four patients (10% of those with nephropathy) had renal biopsies which did not show immunologically driven pathology. Otherwise renal histopathology evidence for ALGS is sparse. Immunofluorescence of one of four cases revealed extensive accumulation of IgG and IgM [66]. Thus, while there is no major body of evidence in favor of complement-mediated nephropathy in ALGS, such as is available is inconclusive. Over 50% of our own series of patients who suffered recurrent infections had recurrent otitis media. The mechanism is unclear, but apart from the immune dysfunction seen among our four patients studied, there may be mucosal defects or local anatomical anomalies/dysmorphisms that compromise secretions and their drainage. The otorhinolaryngeal complications of ALGS remain a major opportunity to improve quality of life for patients.

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Referral to Immunology Services Current practice does not require referral of patients with ALGS for immunological assessment because of recognized aspects of the condition. In general terms threshold for referral to an immunologist is met by features in Table 10.3. These features apply equally to patients with ALGS, but in some instances, a judgment of the contribution of the underlying syndrome versus possible immune dysfunction is required. Indications for suspecting high risk for presence of immune deficiency are shown in Table 10.4. Indications for referral for immune deficiency specifically are taken from the Jeffrey Modell Foundation courtesy of the PiA. Table 10.3  Indications for referral of patients with ALGS to immunology services 1. Immunodeficiency [ICD-10 code: D84.8, D84.9]  Patients with a history of recurrent and/or unusual infections of the respiratory tract, skin, blood, CNS, or internal organs  Patients with two or more months on antibiotic with little or no effect or need for IV antibiotics to clear infections  Patients with a history of antibody, T cell, phagocytic or complement deficiency  Patients or family with a history of immune deficiency 2. Allergic rhinoconjunctivitis [ICD-10 code: J30.1, J30.81, J30.2, J30.89]  Patients with persistent rhinoconjunctivitis unresponsive to medications after 1 month (e.g., nasal steroids) and simple environmental measures (e.g., allergy-proof encasements, etc.)  Children with AR should be referred to allergist since new evidence suggests allergen immunotherapy has potential preventative role in progression of allergic disease and control of asthma as well as dermatitis symptoms  Patients with comorbidity such as bronchial asthma and recurrent sinusitis. 3. Sinusitis and respiratory infections [ICD-10 code: J32.9]  Patients with nasal polyps  Patients with chronic or recurrent infectious sinusitis equal or greater than four times a year  Patients with symptoms suspicious of chronic sinusitis longer than 3 months  Patients with evidence of fungal sinusitis 4. Food allergy [ICD-10 code: T78.1XXA]  Persons who have experienced allergic symptoms (anaphylaxis, urticaria, angioedema, itch, wheezing, gastrointestinal responses) in association with food exposure  Patients with a diagnosed food allergy for ongoing guidance  Infants with recalcitrant gastroesophageal reflux, dysphagia, or known eosinophilic inflammation of the gut  Infants with gastrointestinal symptoms including vomiting, diarrhea (particularly with blood), poor growth, and/or malabsorption. These latter features would be considered disproportionate to the severity of other aspects of the ALGS in the patient These criteria are based on the AAAAI guidelines modified for the features of ALGS – https:// www.aaaai.org/practice-resources/consultation-and-referral-guidelines

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Table 10.4  Indications for referral for immune deficiency   1. Eight or more ear infections in 1 year   2. Two or more serious sinus infections in 1 year   3. Two or more pneumonias in 1 year   4. More than one deep skin or organ abscess   5. Two or more deep-seated infections such as meningitis, osteomyelitis, cellulitis, or septicemia   6. Surgical intervention for chronic infection such as lobectomy for bronchiectasis, tonsillectomy, adenoidectomy, recurrent incision of cutaneous abscesses or boils, recurrent insertion of grommets   7. Two or more months on antibiotics without effect   8. Persistent oral or mucosal candidiasis after 1 year of age   9. Failure to thrive associated with infections 10. A family history of immune deficiency

 reatment of Immune Dysregulation with Immune T Modulation and Immunosuppression A universal solution for therapeutic management of disease secondary to complement dysfunction is unlikely. However, the complement system has intervention points at molecules that detect pathogen and tissue damage epitopes as well as proteins such as activation cascade proteases, opsonins, and anaphylatoxins. This means that with understanding of disease processes, a more specific therapeutic effect may be achieved with less risk of over-immunosuppression so preventing lifethreatening infections by ensuring optimal functioning of the host immune system. To date, inflammatory conditions with immune dysregulation have been treated with corticosteroids either orally or intravenously followed by steroid-sparing agents such as azathioprine or methotrexate. Immunophyllines such as cyclosporine and tacrolimus or a are also employed. The first complement-directed drugs are now clinically available, in the form of purified or recombinant C1 inhibitor concentrates and the anti-C5 antibody eculizumab (Alexion), which binds to C5 and prevents its activation by C5 convertase and, consequently, the generation of C5a and the membrane attack complex (MAC). Recent consensus guidelines support that despite its cost it has become the treatment of choice for patients of all ages with atypical HUS [67]. It is being evaluated in clinical trials including for age-related macular degeneration and organ transplantation. Many other compounds are in development including C1, C3, and C5 inhibitors, C3 convertase and C5a receptor, properdin, and MBL-associated serine protease inhibitors variously for the management of hereditary angioedema, sepsis/inflammation, vasculitis, asthma, atypical HUS, dense deposit disease, ischemia/reperfusion injury, transplantation, etc. [68]. The nature of the optimal treatment for patients with ALGS and infections is currently unknown, neither in terms of selection of patients and timing nor treatment

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modality. The sibling patients with CD46 deficiency and poorly defined common variable immune deficiency and atypical hemolytic uremic syndrome [44] responded to IV immunoglobulin.

 reparation for OLT and Modification of OLT P Immunosuppression A condition resulting in immune dysregulation should lead to special consideration when planning immunosuppression for organ transplantation. Recurrent and persistent infections can delay liver transplant assessment particularly when general anesthesia is required for cardiac catheterizations, which are part of the assessment protocol and particularly during winter. Recognition of what amounts to an exceptional frequency or severity of such infections is more evident with the benefit of hindsight. Nevertheless we ensure such children are fully immunized and take a more aggressive approach to active treatment of intercurrent infections than for other listed children. In our own institution all ALGS patients receive a tailored immunosuppression using early introduction of mycophenolate mofetil and reduced tacrolimus levels from 3 months or earlier because of risk of renal dysfunction particularly renal tubular acidosis exacerbated by tacrolimus [69]. Further consideration may need to be given to immune function including risk of prolonged infections or excessive immune responses particularly among patients with a similar history.

Future Research Opportunities The evidence for immune dysregulation in ALGS is both intriguing and incomplete and much remains to be done. The spectrum of the profile of immune dysregulation in ALGS including various possible phenotypes in the patient population would be valuable starting information, with insight into the natural history with more robust data on the population prevalence and prospective frequency of infections, inflammatory conditions, and autoimmunity in ALGS. These findings need to be correlated with immunological responses both in the quiescent and active phases in anticipation of an association between stimulated immune profile and symptoms and their severity and duration. Genetic and gene modifier associations with immune dysregulation in JAG1, Notch receptor, and CD46 gene polymorphisms combined with immune markers might be expected to predict patients at risk. The development of a biomarker of immune dysregulation in ALGS ideally applicable from birth would allow appropriate and timely therapeutic interventions and permit immunological components of this multisystemic syndrome to be treated in their own right.

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More immediately there are requirements for the development of protocols for clinical management of inflammatory responses in the small number of non-transplanted and transplanted ALGS patients. Consistent definitions of immune dysregulation and common prospective cohort treatment of symptoms can show the value or otherwise in focusing on immune dysregulation as a phenomenon affecting a proportion of ALGS patients. Finally, treatments for complement-mediated inflammatory immune dysregulation are becoming available. Starting with rare but highrisk cases such as the one described by Mannion et al., it would be appropriate to treat such ALGS patients in systematic international trials.

Conclusions ALGS has been recognized sequentially as a pathological phenomenon of the cardiac, hepatobiliary, skeletal, opthalmological, renal, endocrine, and vascular systems of genetic origin and highly variable phenotype, as described above. More recently, insights into the Jagged-Notch system have shown their role in control of adult functions such as tissue repair and healing and responses in innate immunity and immune cell development. A key point of the immune system where the complement system meets the T cell lineage combines Jagged1 with Notch2 and CD46, with evidence coming in part from ALGS syndrome. Crosstalk between T cells and the complement system alternative pathway controls the progress and completion of inflammatory immune responses and may contribute to immune memory as cell fate in terms of tolerance. Despite this relationship, abnormh8alities of mature immune response or regulation had not been recognized as part of the ALGS spectrum. It is now proposed that the syndromic description of ALGS should include the possibility of immune dysregulation whose prevalence, clinical significance, and treatment require extensive further study.

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45. Mohlin FC, Mercier E, Fremeaux-Bacchi V, Liszewski MK, Atkinson JP, Gris JC, et  al. Analysis of genes coding for CD46, CD55, and C4b-binding protein in patients with idiopathic, recurrent, spontaneous pregnancy loss. Eur J Immunol. 2013;43(6):1617–29. 46. Bora NS, Matta B, Lyzogubov VV, Bora PS.  Relationship between the complement system, risk factors and prediction models in age-related macular degeneration. Mol Immunol. 2015;63(2):176–83. 47. Kellermayer R. Epigenetics and the developmental origins of inflammatory bowel diseases. Can J Gastroenterol. 2012;26(12):909–15. 48. Mannion M, Zolak MK, Beukelman DR, T. and Cron RQ. Sarcoidosis in a young child with Alagille syndrome: a case report. Pediatr Rheumatol. 2012;10:32. 49. Kavukçu S, Demir K, Soylu A, Anal O, Saatçi O, Göktay Y. A case of Takayasu disease with findings of incomplete Alagille syndrome. Rheumatol Int. 2005;25:555–7. 50. Emerick KM, Rand EB, Goldmuntz E, Krantz ID, Spinner NB, Piccoli DA.  Features of Alagille syndrome in 92 patients: frequency and relation to prognosis. Hepatology. 1999;29: 822–9. 51. Oude Elferink RP, Kremer AE, Martens JJ, Beuers UH. The molecular mechanism of cholestatic pruritus. Dig Dis. 2011;29(1):66–71. 52. Rishe E, Azarm A, Bergasa NV. Itch in primary biliary cirrhosis: a patients’ perspective. Acta Derm Venereol. 2008;88(1):34–7. 53. Le Friec G, Sheppard D, Whiteman P, Karsten CM, Shamoun SA, Laing A, Bugeon L, Dallman MJ, Melchionna T, Chillakuri C, Smith RA, Drouet C, Couzi L, Fremeaux-Bacchi V, Köhl J, Waddington SN, McDonnell JM, Baker AJ, Handford PA, Lea SM, Kemper C. The CD46Jagged1 interaction is critical for human T(H)1 immunity. Nat Immunol. 2012;13(12):1213–21. 54. Kacser H, Burns JA. The molecular basis of dominance. Genetics. 1981;97:639–66. 55. Seidman JG, Seidman C.  Transcription factor haploinsufficiency: when half a loaf is not enough. J Clin Invest. 2002;109:451–5. 56. Jaramillo DF, Proctor M, Kumm J, Hillenmeyer ME, Davis RW, Nislow C, Giaever G. Mechanisms of Haploinsufficiency Revealed by Genome-Wide Profiling in Yeast. Genetics. 2005;169(4):1915–25. 57. Papp B, Pal C, Hurst LD.  Dosage sensitivity and the evolution of gene families in yeast. Nature. 2003;424:194–7. 58. Robinson DS. The role of the T cell in asthma. J Allergy Clin Immunol. 2010;126(6):1081–91. 59. Kim JS, Sampson HA. Food allergy: a glimpse into the inner workings of gut immunology. Curr Opin Gastroenterol. 2012;28(2):99–103. 60. Kamath BM, Yin W, Miller H, Anand R, Rand EB, Alonso E, Bucuvalas J, Studies of Pediatric Liver Transplantation. Outcomes of liver transplantation for patients with Alagille syndrome: the studies of pediatric liver transplantation experience. Liver Transpl. 2012;18(8):940–8. https://doi.org/10.1002/lt.23437. 61. Meiler F, Zumkehr J, Klunker S, Ruckert B, Akdis CA, Akdis M. In vivo switch to IL-10 secreting T regulatory cells in high dose allergen exposure. J Exp Med. 2008;205(12):2887–98. 62. Kronsten V, Fitzpatrick E, Baker A. Management of cholestatic pruritus in paediatric patients with Alagille syndrome: the King’s College Hospital experience. J Pediatr Gastroenterol Nutr. 2013;57(2):149–54. 63. Schaefer B, Schaefer F, Wittmer D, Engelmann G, Wenning D, Schmitt CP.  Molecular Adsorbent Recirculating System dialysis in children with cholestatic pruritus. Pediatr Nephrol. 2012;27(5):829–34. 64. Kamath BM, Podkameni G, Hutchinson AL, Leonard LD, Gerfen J, Krantz ID, Piccoli DA, Spinner NB, Loomes KM, Meyers K. Renal anomalies in Alagille syndrome: a disease-defining feature. Am J Med Genet A. 2012;0(1):85–9. 65. Kamath BM, Spinner NB, Rosenblum ND. Renal involvement and the role of Notch signalling in Alagille syndrome. Nat Rev Nephrol. 2013;9(7):409–18. 66. Russo PA, Ellis D, Hashida Y. Renal histopathology in Alagille's syndrome. Pediatr Pathol. 1987;7(5–6):557–68.

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Chapter 11

Health-Related Quality of Life and Neurocognition in Alagille Syndrome Saeed Mohammad and Estella M. Alonso

Introduction A growing number of studies have focused on the health related quality of life (HRQOL) and neurocognitive development of children with cholestatic liver diseases such as biliary atresia (BA) [1, 2] and children who have received liver transplantation (LT). [3–6] These studies have demonstrated that children with chronic liver disease as well as liver transplant recipients have lower HRQOL and cognitive outcomes than healthy children. [7, 8] Studying patients with a specific chronic liver disease is difficult due to the small number of patients and their heterogeneous presentations. Alagille syndrome (ALGS) is a disease with an expansive clinical variability caused by variable penetrance, with some patients being severely affected, while others may be unaware of their diagnosis. Not everyone with ALGS has significant liver disease; in a selected group of patients and their affected relatives, the frequency was 61% [9]. There are likely patients with undiagnosed ALGS whose HRQOL and neurocognition may be impaired by their disease. The many manifestations of ALGS which make it difficult to study also increase the importance of measuring HRQOL and neurocognitive outcomes. The cholestasis-related pruritus of ALGS is severe, often resulting in cutaneous mutilation and disrupted sleep and school activities affecting both neurocognition and HRQOL.  Disfiguring xanthomas, linear growth retardation, and poor weight gain are also common in addition to jaundice. Significant cardiac involvement may cause hypoxia, which limits physical and social activities. Repeated surgical S. Mohammad (*) Ann & Robert H. Lurie Children’s Hospital of Chicago, Chicago, IL, USA e-mail: [email protected] E. M. Alonso Department of Pediatrics, Northwestern University, Feinberg School of Medicine and The Ann & Robert H. Lurie Children’s Hospital of Chicago, Chicago, IL, USA © Springer International Publishing AG, part of Springer Nature 2018 B. M. Kamath, K. M. Loomes (eds.), Alagille Syndrome, https://doi.org/10.1007/978-3-319-94571-2_11

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i­nterventions and hospitalizations also negatively affect neurocognitive outcomes and HRQOL.  These symptoms may exponentially worsen as the multisystemic effects of end-stage liver disease wear on.

Health-Related Quality of Life (HRQOL) HRQOL is a subjective patient (or proxy)-reported perception of the functional outcomes and impact of a chronic health condition. This multidimensional construct encompasses physical, cognitive, and psychosocial functioning. HRQOL studies in children with liver disease have usually included patients with multiple diagnoses, with few studies reporting on specific patient groups. A variety of health measures have been used, with the PedsQL™ scales being the most common, allowing the assessment of HRQOL from 1 month to 25 years. There have been two large studies of patient-reported outcomes in pediatric ALGS patients in the last 10 years comparing them to children with other chronic diseases as well as a normal population. Elisofon et  al. studied 71 ALGS patients aged 5–18  years with the Child Health Questionnaire-Parent Form 50 (CHQ-PF50) and compared them to normal children and those other chronic disease states [10]. The Child Health Questionnaire-Parent Form 50 (CHQ-PF50) is validated to measure physical and psychosocial functioning of children aged 5–18  years. The authors also included subjective questions regarding pruritus and sleep and collected data on whether the patient had a mental health diagnosis such as attention deficit hyperactivity disorder (ADHD), obsessivecompulsive disorder, or depression. Children with ALGS had a lower HRQOL in comparison with a normal pediatric population across all subscales of the CHQ except family cohesion. In comparison with children with ADHD, children with ALGS had lower physical function scores on the bodily pain and general health subscales but had higher psychosocial function scores. When compared to children with arthritis, ALGS patients had similar physical function and lower emotional/ behavioral, mental health, and self-esteem subscale scores. This suggests they may have a greater psychosocial burden related to their specific disease. ALGS patients also had lower physical HRQOL when compared to patients transplanted for all types of liver disease. Kamath et  al. have reported similar findings in the largest cohort of Alagille patients studied to date using the PedsQL™ generic core scale. The PedsQL™ is a validated, 23-item modular instrument designed to measure HRQOL in children and adolescents [11]. Ninety eight children were assessed, 70 of whom completed their own HRQOL scores and were compared to healthy children, children with alpha-1 antitrypsin deficiency (A1ATD) and other cholestatic liver diseases. ALGS patients had lower PedsQL™ 4.0 scores by self- and parent report vs. matched healthy controls and A1ATD patients [11]. The greatest decrement was seen in the physical domain scores. When compared to only those with A1ATD, children with ALGS reported worse HRQOL than their A1ATD counterparts, with the largest differences again seen in the physical domain (Fig. 11.1). Parents, however, reported

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Fig. 11.1  Comparison of self-reported PedsQL generic scale scores between ALGS, A1ATD, and normal controls. (Adapted from Kamath et al. [11])

significantly lower scores with higher effect sizes in patients with ALGS across all PedsQL™ 4.0 subscales. Compared to children with other cholestatic liver diseases, HRQOL for subjects with ALGS was modestly impaired by child self-report. The ALGS PedsQL™ 4.0 parent proxy-report scores were lower in almost all domains with small to medium effect sizes. This discrepancy may be due to the multisystemic nature of ALGS resulting in limited physical functioning and decreased social engagement due to bullying or teasing from other children whose effects may weigh more heavily on parents rather than the affected children. In both these reports, patients with ALGS had worse HRQOL scores when compared with other chronic disease groups. They were most similar to patients who had a cholestatic liver disease which suggests the degree of liver function impairment or elevated bilirubin level contributes to changes in HRQOL. It is important to note that the two studies differed in their patient recruitment. Elisofon et al. contacted subjects enrolled in a patient registry provided by the Alagille Syndrome Alliance (https://www.alagille.org/), while the subjects from Kamath et  al. were enrolled through a prospective NIH study and likely represented those with more severe liver disease presenting to a hepatology clinic. Pruritus is one of the most common but difficult to treat symptoms of ALGS. It has been described in 59–98% of all patients and may range from active scratching to destruction of the skin, bleeding, or scarring. Ursodeoxycholic acid is the most commonly prescribed therapy; other options include rifampicin, cholestyramine, and naltrexone. Patients are often on multiple therapies simultaneously, with minimal relief [12]. The association between pruritus and HRQOL is mixed. Two large studies of Alagille patients did not find significant associations between HRQOL and itch despite parents reporting that itch was a major concern. This may be due to the difficulty of quantifying itch as many scales rely on physical signs of itch such as abrasions or mutilation [10, 11].

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Sleep problems affecting the psychosocial and physical domains on the CHQ-PF50 were reported in 30% of patients, much higher than the expected 2% of the normal population. Fifteen percent stated that sleep problems affected their everyday life with over two thirds reporting itching as the primary factor disturbing sleep. Additional data and measurement modalities are needed to study itching and sleep health simultaneously in these patients to provide physicians and caregivers’ insights that will lead to more effective treatment approaches. The presence of significant cardiac disease exemplified by the need for cardiac catheterization or the presence of a cardiac defect other than peripheral pulmonic stenosis was significantly associated with lower HRQOL scores as measured by CHQ and PedsQL™ 4.0 [10, 11]. This has also been previously well described in children with isolated congenital heart disease [13]. The presence of cardiac defects was found to be a predictor for lower parental but not for self HRQOL, possibly due to the additional hospital and physician encounters, operative procedures, or the perception of heart disease as being a more serious concern by the parents. Physical signs of disease have mixed effects on HRQOL.  A greater degree of cholestasis was significantly associated with negative HRQOL scores. This may be either as a physical stigma or as a marker for worsening liver disease [11]. Short stature, a hallmark of ALGS, is associated with lower HRQOL in other chronic disease groups [14, 15]. In multivariate analysis, the only factor in ALGS patients associated with HRQOL was weight z-score. Weight and height z-scores were positively associated with self-reported HRQOL summary scores, but another study reported no significant associations between HRQOL scores and poor growth [10, 11]. Xanthomas affecting physical appearance may cause distress to children though these have not reported to be associated with impaired HRQOL in ALGS.  Bone fractures, which may occur in 28–39% of patients, were also not reported to affect activities of daily life [10, 16]. Children who limit physical and social activities due to medical, family, or social reasons may suffer reduced peer interactions that are a usual part of childhood development which may adversely affect social HRQOL. ALGS patients are at particularly high risk due to their concomitant cardiac disease, xanthomas, and facial dysmorphism. These factors may not have been detected as being determinants of HRQOL in ALGS in the available limited literature due to the use of generic QOL assessment tools and the tremendous variability in the clinical features of patients being assessed.

Posttransplant Outcomes It has been well established that HRQOL in pediatric LT recipients is lower by patient and parent report compared to matched healthy controls in all areas but especially in school functioning (p