10 Review Article Alagille syndrome and non-syndromic paucity of the intrahepatic bile ducts Melissa A. Gilbert1, Kathleen M. Loomes2,3 1Division of Genomic Diagnostics, Department of Pathology and Laboratory Medicine, 2Division of Gastroenterology, Hepatology and Nutrition, The Children’s Hospital of Philadelphia, Philadelphia, PA, USA; 3Department of Pediatrics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA Contributions: (I) Conception and Design: All authors; (II) Administrative support: None; (III) Provision of study materials or patients: None; (IV) Collection and assembly of data: All authors; (V) Data analysis and interpretation: All authors; (VI) Manuscript writing: All authors; (VII) Final approval of manuscript: All authors. Correspondence to: Kathleen M. Loomes, MD. Division of Gastroenterology, Hepatology and Nutrition, The Children’s Hospital of Philadelphia, 3401 Civic Center Blvd., Philadelphia, PA 19104, USA. Email: [email protected]. Abstract: The observation of bile duct paucity is an important diagnostic finding in children, occurring in roughly 11% of pediatric liver biopsies. Alagille syndrome (ALGS) is a well-defined syndromic form of intrahepatic bile duct paucity that is accompanied by a number of other key features, including cardiac, facial, ocular, and vertebral abnormalities. In the absence of these additional clinical characteristics, intrahepatic bile duct paucity results in a broad differential diagnosis that requires supplementary testing and characterization. Nearly 30 years after ALGS was first described, genetic studies identified a causative gene, JAGGED1, which spearheaded over two decades of research aimed to meticulously delineate the molecular underpinnings of ALGS. These advancements have characterized ALGS as a genetic disease and led to testing strategies that offer the ability to detect a pathogenic genetic variant in almost 97% of individuals with ALGS. Having a molecular understanding of ALGS has allowed for the development of numerous in vitro and in vivo disease models, which have provided hope and promise for the future generation of gene-based and protein-based therapies. Generation of these disease models has offered scientists a mechanism to study the dynamics of bile duct development and regeneration, and in doing so, produced tools that are applicable to the understanding of other congenital and acquired liver diseases. Keywords: Alagille syndrome (ALGS); bile duct paucity; JAG1; NOTCH2; Notch signaling; liver disease; cholestasis Received: 27 February 2020; Accepted: 28 April 2020; Published: 05 April 2021. doi: 10.21037/tgh-2020-03 View this article at: http://dx.doi.org/10.21037/tgh-2020-03 Alagille syndrome (ALGS) Clinical criteria of arteriohepatic dysplasia, or ALGS as it is more commonly known, were later described by Alagille Overview and clinical manifestations et al., and include bile duct paucity (Figure 1), cholestasis, In 1969, Daniel Alagille first described several families cardiac involvement (heart murmur, peripheral pulmonic manifesting cholestatic jaundice, and went on to publish a stenosis, and/or structural heart defects), characteristic larger series characterizing the clinical phenotype of bile facial appearance, eye findings (most commonly posterior duct hypoplasia with cardiac manifestations (1). Around the embryotoxon), and skeletal anomalies (most commonly same time, Watson and Miller observed a syndrome that butterfly vertebrae) (3). Renal and vascular findings are they called arteriohepatic dysplasia, characterized by familial also relatively common and are considered to be primary neonatal liver disease and pulmonary artery stenosis (2). manifestations of the syndrome. ALGS has a strong © Translational Gastroenterology and Hepatology. All rights reserved. Transl Gastroenterol Hepatol 2021;6:22 | http://dx.doi.org/10.21037/tgh-2020-03 Page 2 of 10 Translational Gastroenterology and Hepatology, 2021 A B Figure 1 Liver biopsy from a 5-month old cholestatic infant with ALGS. (A) Hematoxylin and eosin stain of liver biopsy shows a portal tract (arrow) with branches of hepatic artery and portal vein but no bile duct present. (B) Cytokeratin 19 staining highlights minimal ductular reaction around portal tracts (arrow) throughout the biopsy. Scale bar =200 µM. ALGS, Alagille syndrome. genetic component with dominant inheritance and variable by haploinsufficiency of JAG1, and additional support for penetrance, and in 1997 the gene JAGGED1 (JAG1), was this pathophysiologic mechanism includes the finding that identified as the first disease gene (4,5). Genetic testing individuals with whole gene deletions have been shown is now a key component of the diagnostic algorithm for to have phenotypes indistinguishable from those with ALGS. intragenic variants, including missense variants (5,11-13). Many functional studies have been published to investigate the basis of missense variant pathogenicity, and Genetics they have identified cellular trafficking defects, caused by The genetics of ALGS have been very well-defined and errors in post-translational glycosylation of the mature show a clear pattern of autosomal dominant Mendelian JAG1 protein, and an inability of mutant JAG1 proteins inheritance, with pathogenic variants identified in one of to bind to and activate NOTCH2 (6,14-17). However, two genes in close to 97% of cases (6). These two genes, individual analysis of these missense variants has revealed JAG1 and NOTCH2, encode a ligand-receptor pair, differing degrees of impairment in their functional respectively, that participate in the Notch signaling pathway capacity. For instance, the L37S variant has been well- and are specifically critical in the formation of bile ducts, studied and shows both an inability of the mutant JAG1 where JAG1 expression in the portal vein mesenchyme has to interact with NOTCH2 as well as impaired cellular been shown to be necessary in driving NOTCH2-expressing trafficking, while the G274D variant has been shown to hepatoblasts to adopt a cholangiocyte fate (7,8). Failure to produce both an abnormally-glycosylated protein that is not activate this signaling, which could arise from the presence expressed on the cell surface as well as a protein that can of a pathogenic variant in either JAG1 or NOTCH2, drives effectively induce Notch signaling (15,18). Other proposed hepatoblasts toward the hepatocyte lineage resulting in bile pathogenic missense variants have been studied and found duct paucity, one of the classic features of ALGS (7-10). to retain complete wild type function, resulting in their reclassification as benign polymorphisms (15). JAG1 These studies have highlighted the importance of Pathogenic variants are most commonly identified functionally validating each missense variant independently, in JAG1, occurring in 94.3% of individuals with and they have also led to the hypothesis that an alternate ALGS (6). The overwhelming majority of JAG1 pathogenic disease mechanism could exist for a minority of identified variants are protein-truncating or result in whole or JAG1 variants, namely dominant negative pathogenesis. partial gene deletions or complex structural variants. A Studies to consider a dominant negative disease mechanism smaller percentage of individuals with ALGS have been have shown that in some instances the mutant transcripts found to have JAG1 missense variants (13%) (6). This (mainly arising from missense alleles rather than protein- observation has led to the hypothesis that ALGS is caused truncating alleles) are stably-expressed, and that when the © Translational Gastroenterology and Hepatology. All rights reserved. Transl Gastroenterol Hepatol 2021;6:22 | http://dx.doi.org/10.21037/tgh-2020-03 Translational Gastroenterology and Hepatology, 2021 Page 3 of 10 resultant protein products are co-expressed with wild type support any genotype-phenotype correlations, and even the JAG1 in a NOTCH2 luciferase reporter assay, there is a earliest genetic reports have commented on a general lack reduction in fluorescence readout compared to transfection of distinction between individuals who have whole gene of wild type alone, suggesting that the mutant protein may deletions compared to intragenic variants (4,12,31). be able to compete with wild type protein for NOTCH2 This variable expressivity of ALGS has prompted a binding (19,20). However, the physiological relevance of search for genetic factors that are capable of modifying this in vitro model is debatable since it does not take into the effects of pathogenic variants in JAG1 and NOTCH2 account cellular trafficking defects that would prevent to either alleviate or worsen disease features. A number of JAG1 from interacting with NOTCH2. A separate study genetic modifiers have now been proposed, and this field has shown that induced pluripotent stem cells (iPSCs) represents an ongoing and active area of research. Work containing a heterozygous deletion of JAG1 resulted in has shown that genes involved in the post-translational the establishment of liver organoids that appeared normal addition of sugar moieties (glycosylation and fucosylation) whereas iPSCs containing a heterozygous nonsense variant to JAG1 protein can influence JAG1-mediated Notch were impaired in their ability to generate liver organoids, signaling. The first group of genes identified to function in providing evidence that haploinsufficiency in this model this capacity was a family of genes called the Fringe genes, does not alone result in a deficiency