Regenerative failure of intrahepatic biliary cells in Alagille syndrome rescued by elevated Jagged/Notch/Sox9 signaling.

Despite the robust healing capacity of the liver, regenerative failure underlies numerous hepatic diseases, including the JAG1 haploinsufficient disorder, Alagille syndrome (ALGS). Cholestasis due to intrahepatic duct (IHD) paucity resolves in certain ALGS cases but fails in most with no clear mechanisms or therapeutic interventions. We find that modulating jag1b and jag2b allele dosage is sufficient to stratify these distinct outcomes, which can be either exacerbated or rescued with genetic manipulation of Notch signaling, demonstrating that perturbations of Jag/Notch signaling may be causal for the spectrum of ALGS liver severities. Although regenerating IHD cells proliferate, they remain clustered in mutants that fail to recover due to a blunted elevation of Notch signaling in the distal-most IHD cells. Increased Notch signaling is required for regenerating IHD cells to branch and segregate into the peripheral region of the growing liver, where biliary paucity is commonly observed in ALGS. Mosaic loss- and-gain-of-function analysis reveals Sox9b to be a key Notch transcriptional effector required cell autonomously to regulate these cellular dynamics during IHD regeneration. Treatment with a small-molecule putative Notch agonist stimulates Sox9 expression in ALGS patient fibroblasts and enhances hepatic sox9b expression, rescues IHD paucity and cholestasis, and increases survival in zebrafish mutants, thereby providing a proof-of-concept therapeutic avenue for this disorder.

ASO silencing of a glycosyltransferase, Poglut1 , improves the liver phenotypes in mouse models of Alagille syndrome.

BACKGROUND AND AIMS: Paucity of intrahepatic bile ducts (BDs) is caused by various etiologies and often leads to cholestatic liver disease. For example, in patients with Alagille syndrome (ALGS), which is a genetic disease primarily caused by mutations in jagged 1 ( JAG1) , BD paucity often results in severe cholestasis and liver damage. However, no mechanism-based therapy exists to restore the biliary system in ALGS or other diseases associated with BD paucity. Based on previous genetic observations, we investigated whether postnatal knockdown of the glycosyltransferase gene protein O -glucosyltransferase 1 ( Poglut1) can improve the ALGS liver phenotypes in several mouse models generated by removing one copy of Jag1 in the germline with or without reducing the gene dosage of sex-determining region Y-box 9 in the liver. APPROACH AND RESULTS: Using an ASO established in this study, we show that reducing Poglut1 levels in postnatal livers of ALGS mouse models with moderate to profound biliary abnormalities can significantly improve BD development and biliary tree formation. Importantly, ASO injections prevent liver damage in these models without adverse effects. Furthermore, ASO-mediated Poglut1 knockdown improves biliary tree formation in a different mouse model with no Jag1 mutations. Cell-based signaling assays indicate that reducing POGLUT1 levels or mutating POGLUT1 modification sites on JAG1 increases JAG1 protein level and JAG1-mediated signaling, suggesting a likely mechanism for the observed in vivo rescue. CONCLUSIONS: Our preclinical studies establish ASO-mediated POGLUT1 knockdown as a potential therapeutic strategy for ALGS liver disease and possibly other diseases associated with BD paucity.

Intrahepatic cholangiocyte regeneration from an Fgf-dependent extrahepatic progenitor niche in a zebrafish model of Alagille Syndrome.

BACKGROUND AND AIMS: Alagille Syndrome (ALGS) is a congenital disorder caused by mutations in the Notch ligand gene JAGGED1, leading to neonatal loss of intrahepatic duct (IHD) cells and cholestasis. Cholestasis can resolve in certain patients with ALGS, suggesting regeneration of IHD cells. However, the mechanisms driving IHD cell regeneration following Jagged loss remains unclear. Here, we show that cholestasis due to developmental loss of IHD cells can be consistently phenocopied in zebrafish with compound jagged1b and jagged2b mutations or knockdown. APPROACH AND RESULTS: Leveraging the transience of jagged knockdown in juvenile zebrafish, we find that resumption of Jagged expression leads to robust regeneration of IHD cells through a Notch-dependent mechanism. Combining multiple lineage tracing strategies with whole-liver three-dimensional imaging, we demonstrate that the extrahepatic duct (EHD) is the primary source of multipotent progenitors that contribute to the regeneration, but not to the development, of IHD cells. Hepatocyte-to-IHD cell transdifferentiation is possible but rarely detected. Progenitors in the EHD proliferate and migrate into the liver with Notch signaling loss and differentiate into IHD cells if Notch signaling increases. Tissue-specific mosaic analysis with an inducible dominant-negative Fgf receptor suggests that Fgf signaling from the surrounding mesenchymal cells maintains this extrahepatic niche by directly preventing premature differentiation and allocation of EHD progenitors to the liver. Indeed, transcriptional profiling and functional analysis of adult mouse EHD organoids uncover their distinct differentiation and proliferative potential relative to IHD organoids. CONCLUSIONS: Our data show that IHD cells regenerate upon resumption of Jagged/Notch signaling, from multipotent progenitors originating from an Fgf-dependent extrahepatic stem cell niche. We posit that if Jagged/Notch signaling is augmented, through normal stochastic variation, gene therapy, or a Notch agonist, regeneration of IHD cells in patients with ALGS may be enhanced.

A Case of Dubin-Johnson Syndrome Presenting as Neonatal Cholestasis With Paucity of Interlobular Bile Ducts.

Dubin-Johnson syndrome (DJS) is a rare autosomal recessive disorder that typically manifests in young adulthood as jaundice with conjugated hyperbilirubinemia. We report a case presenting as neonatal cholestasis with the unexpected histologic finding of paucity of interlobular bile ducts, a feature that is not typically seen in DJS. The diagnosis was confirmed by absent canalicular multidrug-resistance-associated protein 2 (MRP2) immunohistochemical staining on liver biopsy tissue and molecular genetic testing that demonstrated heterozygous mutations in the ATP-Binding Cassette Subfamily C Member 2 (ABCC2) gene, including a novel missense mutation. This report describes a case of DJS with atypical clinicopathologic findings and suggests that DJS should be considered in patients with neonatal cholestasis and bile duct paucity.

Alagille syndrome mutation update: Comprehensive overview of JAG1 and NOTCH2 mutation frequencies and insight into missense variant classification.

Alagille syndrome is an autosomal dominant disease with a known molecular etiology of dysfunctional Notch signaling caused primarily by pathogenic variants in JAGGED1 (JAG1), but also by variants in NOTCH2. The majority of JAG1 variants result in loss of function, however disease has also been attributed to lesser understood missense variants. Conversely, the majority of NOTCH2 variants are missense, though fewer of these variants have been described. In addition, there is a small group of patients with a clear clinical phenotype in the absence of a pathogenic variant. Here, we catalog our single-center study, which includes 401 probands and 111 affected family members amassed over a 27-year period, to provide updated mutation frequencies in JAG1 and NOTCH2 as well as functional validation of nine missense variants. Combining our cohort of 86 novel JAG1 and three novel NOTCH2 variants with previously published data (totaling 713 variants), we present the most comprehensive pathogenic variant overview for Alagille syndrome. Using this data set, we developed new guidance to help with the classification of JAG1 missense variants. Finally, we report clinically consistent cases for which a molecular etiology has not been identified and discuss the potential for next generation sequencing methodologies in novel variant discovery.

Mouse Model of Alagille Syndrome and Mechanisms of Jagged1 Missense Mutations.

BACKGROUND & AIMS: Alagille syndrome is a genetic disorder characterized by cholestasis, ocular abnormalities, characteristic facial features, heart defects, and vertebral malformations. Most cases are associated with mutations in JAGGED1 (JAG1), which encodes a Notch ligand, although it is not clear how these contribute to disease development. We aimed to develop a mouse model of Alagille syndrome to elucidate these mechanisms. METHODS: Mice with a missense mutation (H268Q) in Jag1 (Jag1+/Ndr mice) were outbred to a C3H/C57bl6 background to generate a mouse model for Alagille syndrome (Jag1Ndr/Ndr mice). Liver tissues were collected at different timepoints during development, analyzed by histology, and liver organoids were cultured and analyzed. We performed transcriptome analysis of Jag1Ndr/Ndr livers and livers from patients with Alagille syndrome, cross-referenced to the Human Protein Atlas, to identify commonly dysregulated pathways and biliary markers. We used species-specific transcriptome separation and ligand-receptor interaction assays to measure Notch signaling and the ability of JAG1Ndr to bind or activate Notch receptors. We studied signaling of JAG1 and JAG1Ndr via NOTCH 1, NOTCH2, and NOTCH3 and resulting gene expression patterns in parental and NOTCH1-expressing C2C12 cell lines. RESULTS: Jag1Ndr/Ndr mice had many features of Alagille syndrome, including eye, heart, and liver defects. Bile duct differentiation, morphogenesis, and function were dysregulated in newborn Jag1Ndr/Ndr mice, with aberrations in cholangiocyte polarity, but these defects improved in adult mice. Jag1Ndr/Ndr liver organoids collapsed in culture, indicating structural instability. Whole-transcriptome sequence analyses of liver tissues from mice and patients with Alagille syndrome identified dysregulated genes encoding proteins enriched at the apical side of cholangiocytes, including CFTR and SLC5A1, as well as reduced expression of IGF1. Exposure of Notch-expressing cells to JAG1Ndr, compared with JAG1, led to hypomorphic Notch signaling, based on transcriptome analysis. JAG1-expressing cells, but not JAG1Ndr-expressing cells, bound soluble Notch1 extracellular domain, quantified by flow cytometry. However, JAG1 and JAG1Ndr cells each bound NOTCH2, and signaling from NOTCH2 signaling was reduced but not completely inhibited, in response to JAG1Ndr compared with JAG1. CONCLUSIONS: In mice, expression of a missense mutant of Jag1 (Jag1Ndr) disrupts bile duct development and recapitulates Alagille syndrome phenotypes in heart, eye, and craniofacial dysmorphology. JAG1Ndr does not bind NOTCH1, but binds NOTCH2, and elicits hypomorphic signaling. This mouse model can be used to study other features of Alagille syndrome and organ development.

Clinical features, outcomes, and genetic analysis in Korean children with Alagille syndrome.

BACKGROUND: Alagille syndrome (AGS) is a multisystem autosomal dominant disorder that affects the liver, heart, eyes, face, bone, and other organs. AGS is caused by mutations in one of two genes, JAG1 or NOTCH2. We evaluated clinical features, outcomes, and the presence of JAG1 and NOTCH2 mutations in Korean children with AGS. METHODS: Between January 1997 and December 2013, 19 children were diagnosed with AGS at Asan Medical Center, Seoul, Korea. Their clinical features, outcomes, and JAG1 and NOTCH2 mutation status were retrospectively analyzed. RESULTS: The prevalence of clinical features in the 19 patients was as follows: dysmorphic facial features, 100% (n = 19); liver symptoms, 89% (n = 17); cardiac symptoms, 95% (n = 18); ophthalmologic symptoms, 67% (n = 10); skeletal deformities, 47% (n = 9); and renal symptoms, 21% (n = 4). JAG1 mutations were identified in 14 patients. The 13 different JAG1 mutations, seven of which were novel, included four deletions, three insertions, two missense mutations, three nonsense mutations, and one indel mutation. No NOTCH2 mutations were found. Two patients who received liver transplantation due to liver failure were still alive. Two patients died of comorbidities related to AGS: one of cardiac failure and one of hepatic failure. CONCLUSION: This study describes the clinical characteristics of 19 Korean AGS patients with seven novel JAG1 mutations. Neonatal cholestatic jaundice was the most common initial presenting symptom; thus the presence of neonatal cholestasis warrants screening for syndromic features of AGS. Complex heart anomalies and progressive liver dysfunction resulted in significant morbidity and mortality in AGS.

Notch and disease: a growing field.

Signals through the Notch receptors are used throughout development to control cellular fate choices. Our intention here is to provide an overview of the involvement of Notch signaling in human disease, which, keeping pace with the known biology of the pathway, manifests itself in a pleiotropic fashion. A pathway with such broad action in normal development, a profound involvement in the biology of adult stem cells and intricate and complex controls governing its activity, poses numerous challenges. We provide an overview of Notch related pathologies identified thus far and emphasize aspects that have been modeled in experimental systems in order to understand the underlying pathobiology and, hopefully, help the definition of rational therapeutic avenues.

Notch signaling in human development and disease.

Mutations in Notch signaling pathway members cause developmental phenotypes that affect the liver, skeleton, heart, eye, face, kidney, and vasculature. Notch associated disorders include the autosomal dominant, multi-system, Alagille syndrome caused by mutations in both a ligand (Jagged1 (JAG1)) and receptor (NOTCH2) and autosomal recessive spondylocostal dysostosis, caused by mutations in a ligand (Delta-like-3 (DLL3)), as well as several other members of the Notch signaling pathway. Mutations in NOTCH2 have also recently been connected to Hajdu-Cheney syndrome, a dominant disorder causing focal bone destruction, osteoporosis, craniofacial morphology and renal cysts. Mutations in the NOTCH1 receptor are associated with several types of cardiac disease and mutations in NOTCH3 cause the dominant adult onset disorder CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy), a vascular disorder with onset in the 4th or 5th decades. Studies of these human disorders and their inheritance patterns and types of mutations reveal insights into the mechanisms of Notch signaling.

Splenic hamartomas in Alagille syndrome: case report and literature review.

Alagille syndrome is a rare autosomal dominant disorder with characteristic findings of paucity of intrahepatic bile ducts, congenital heart disease, and vertebral, ocular, and renal abnormalities. We present a unique autopsy case of an 18-year-old female with Alagille syndrome and splenic hamartomas. Autopsy findings included growth restriction, Tetralogy of Fallot, paucity of intrahepatic bile ducts, end-stage renal disease with mesangiolipidosis, and splenomegaly with two well-circumscribed, splenic tumors. Histologic findings of the splenic tumors revealed disorganized vascular channels lined by cells without cytologic atypia. Immunohistochemical analysis demonstrated CD8(+)CD31(+) endothelial cells, consistent with splenic hamartomas. In summary, Alagille syndrome is a rare genetic disorder characterized by JAG1 mutations and disrupted Notch signaling. Review of the literature highlights the importance of Notch signaling in vascular development and disorders. However, to our knowledge this is the first description of splenic hamartomas in Alagille syndrome.

Generation of JAG1 gene c.1615C > T heterozygous mutation human embryonic stem cell line (SDQLCHe001-A) using cytosine base editor.

Pathogenic variants in Jagged-1 (JAG1), which encodes the ligand of the Notch receptor, had been demonstrated to cause Alagille syndrome. However, there is no evidence to support any genotype-phenotype correlations. Here, we generated a gene-edited human embryonic stem cell (hESC) line (H9) carrying the c.1615C > T mutation in JAG1 that was identified in a patient with Alagille syndrome (ALGS). This modified cell line was accomplished by using cytosine base editor (CBE), and may serve as a valuable model for JAG1 mutaion related disease, and facilitate to gain more insight into the biological function of JAG1.

Characterization of an induced pluripotent stem cell line NCHi011-A from a 23-year-old female with Alagille Syndrome harboring a heterozygous JAG1 pathogenic variant.

Alagille syndrome (ALGS) is a multisystem disease with high variability in clinical features. ALGS is predominantly caused by pathogenic variants in the Notch ligand JAG1. An iPSC line, NCHi011-A, was generated from a ALGS patient with complex cardiac phenotypes consisting of pulmonic valve and branch pulmonary artery stenosis. NCHi011-A is heterozygous for a single base duplication causing a frameshift in the JAG1 gene. This iPSC line demonstrates normal cellular morphology, expression of pluripotency markers, trilineage differentiation potential, and identity to the source patient. NCHi011-A provides a resource for modeling ALGS and investigating the role of Notch signaling in the disease.

Generation of an Alagille syndrome (ALGS) patient-derived induced pluripotent stem cell line (TRNDi032-A) carrying a heterozygous mutation (p.Cys682Leufs*7) in the JAG1 gene.

Alagille syndrome (ALGS) is an autosomal dominant, multisystemic disorder due to haploinsufficiency in either the JAG1 gene (ALGS type 1) or the NOTCH2 gene (ALGS type 2). The disease has been difficult to diagnose and treat due to its muti-system clinical presentation, variable expressivity, and prenatal onset for some of the features. The generation of this iPSC line (TRNDi032-A) carrying a heterozygous mutation, p.Cys682Leufs*7 (c.2044dup), in the JAG1 gene provides a means of studying the disease and developing novel therapeutics towards patient treatment.

Generation of iPSC line NCHi012-A from a patient with Alagille syndrome and heterozygous pathogenic variant in the JAG1 gene.

Alagille syndrome (ALGS) is an autosomal dominant disease affecting the liver, heart and other organs with high variability. About 95% of ALGS cases are associated with pathogenic variants in JAG1, encoding the Jagged1 ligand that binds to Notch receptors. The iPSC line NCHi012-A was derived from an ALGS patient with cholestatic liver disease and mild pulmonary stenosis, who is heterozygous for a 2 bp deletion in the JAG1 coding sequence. We report here an initial characterization of NCHi012-A to evaluate its morphology, pluripotency, differentiation potential, genotype, karyotype and identity to the source patient.

Pancreatic insufficiency is not a prevalent problem in Alagille syndrome.

Alagille syndrome (ALGS) is an inherited multisystem disorder in which pancreatic insufficiency (PI) has been regarded a minor but important clinical manifestation. As part of a multicenter prospective study, 42 patients with ALGS underwent fecal elastase (FE) measurement to screen for exocrine PI. FE measurements were normal (>200  µg/g) in 40 (95%) and indeterminate (100-200  µg/g) in 2 (5%). As FE is the most reliable screen for PI, these data suggest that PI is not a prevalent problem in ALGS.

Identifying potential regulators of JAGGED1 expression in portal mesenchymal cells.

OBJECTIVE: Portal mesenchymal cells induce the epithelial differentiation of the bile ducts in the developing liver via one of the Delta-Notch signaling components, JAGGED1. Although this differential induction is crucial for normal liver physiology as its genetic disorder (Alagille syndrome) causes jaundice, the molecular mechanism behind JAGGED1 expression remains unknown. Here, we searched for upstream regulatory transcription factors of JAGGED1 using an integrated bioinformatics method. RESULTS: According to the DoRothEA database, which integrates multiple lines of evidence on the relationship between transcription factors and their downstream target genes, three transcription factors were predicted to be upstream of JAGGED1: SLUG, SOX2, and EGR1. Among these, SLUG and EGR1 were enriched in ACTA2-expressing portal mesenchymal cells in two previously reported human fetal liver single-cell RNA-seq datasets. JAGGED1-expressing portal mesenchymal cells tended to express SLUG rather than EGR1, supporting that SLUG induced JAGGED1 expression. Together with the higher confidentiality of SLUG (DoRothEA level A) over EGR1 (DoRothEA level D), we concluded that SLUG was one of the most important candidate transcription factors upstream of JAGGED1. These results add mechanistic insights into the developmental biology of how portal mesenchymal cells support biliary development in the liver.

Directing Cholangiocyte Morphogenesis in Natural Biomaterial Scaffolds.

Patients with Alagille syndrome carry monogenic mutations in the Notch signaling pathway and face complications such as jaundice and cholestasis. Given the presence of intrahepatic ductopenia in these patients, Notch2 receptor signaling is implicated in driving normal biliary development and downstream branching morphogenesis. As a result, in vitro model systems of liver epithelium are needed to further mechanistic insight of biliary tissue assembly. Here, primary human intrahepatic cholangiocytes as a candidate population for such a platform are systematically evaluated, and conditions that direct their branching morphogenesis are described. It is found that extracellular matrix presentation, coupled with mitogen stimulation, promotes biliary branching in a Notch-dependent manner. These results demonstrate the utility of using 3D scaffolds for mechanistic investigation of cholangiocyte branching and provide a gateway to integrate biliary architecture in additional in vitro models of liver tissue.

Diagnosis of Alagille syndrome-25 years of experience at King's College Hospital.

OBJECTIVE: The aim of the study was to study the clinical and histological features of Alagille syndrome (AGS) at presentation comparing the value of the various modalities before the implementation of genetic diagnosis. PATIENTS AND METHODS: We performed a retrospective analysis of the records of 117 children diagnosed as having AGS after referral to King's College Hospital between 1980 and 2005. RESULTS: Cholestasis was seen in 104 of 117 (89%), characteristic facies in 91 of 117 (77%), posterior embryotoxon in 72 of 117 (61%), butterfly vertebrae in 44 of 117 (39%), heart disease (most often peripheral pulmonary stenosis) in 107 of 117 (91%), and renal disease in 27 of 117 (23%). Serum cholesterol levels of >5 mmol/L were seen in 52 of 86 (60.4%). Liver biopsy showed characteristic features of paucity of interlobular bile ducts in 59 of 77 (76.6%) children younger than 16 weeks of age, in 10 of 14 (71.4%) between 16 weeks and 1 year of age, and in 8 of 12 (66.66%) older than 1 year of age. Other biopsy findings were those of nonspecific hepatitis and biliary features. Iminodiacetic acid scans showed no excretion of isotope into the bowel after 24 hours in 21 of 35 (60%), and small/no gallbladder on ultrasound was seen in 29 of 104 (27.8%). Eleven of 117 (9.4%) had a diagnostic laparotomy and operative cholangiography, 2 proceeding to Kasai portoenterostomy before referral to our unit. CONCLUSIONS: Clinical features of AGS are not as consistently informative as suggested in the literature. Hypercholesterolaemia is nonspecific but may be a helpful pointer. Histology is not characteristic in 25%; hepatobiliary iminodiacetic acid scan and ultrasound may suggest a false diagnosis of biliary atresia in 60% and 28%, respectively, supporting the concept that infants with liver disease warrant early referral to a specialist centre. The advent of genetic diagnosis will redefine the syndrome with likely effects on the prognosis of the defined group.

Bile duct proliferation in Jag1/fringe heterozygous mice identifies candidate modifiers of the Alagille syndrome hepatic phenotype.

UNLABELLED: Alagille syndrome (AGS) is a heterogeneous developmental disorder associated with bile duct paucity and various organ anomalies. The syndrome is caused by mutations in JAG1, which encodes a ligand in the Notch signaling pathway, in the majority of cases and mutations in the NOTCH2 receptor gene in less than 1% of patients. Although a wide array of JAG1 mutations have been identified in the AGS population, these mutational variants have not accounted for the wide phenotypic variability observed in patients with this syndrome. The Fringe genes encode glycosyltransferases, which modify Notch and alter ligand-receptor affinity. In this study, we analyzed double heterozygous mouse models to examine the Fringe genes as potential modifiers of the Notch-mediated hepatic phenotype observed in AGS. We generated mice that were haploinsufficient for both Jag1 and one of three paralogous Fringe genes: Lunatic (Lfng), Radical (Rfng), and Manic (Mfng). Adult Jag1(+/-)Lfng(+/-) and Jag1(+/-)Rfng(+/-) mouse livers exhibited widespread bile duct proliferation beginning at 5 weeks of age and persisting up to 1 year. The Jag1(+/-)Mfng(+/-) livers showed a subtle, yet significant increase in bile duct numbers and bile duct to portal tract ratios. These abnormalities were not observed in the newborn period. Despite the portal tract expansion by bile ducts, fibrosis was not increased and epithelial to mesenchymal transition was not shown in the affected portal tracts. CONCLUSION: Mice heterozygous for mutations in Jag1 and the Fringe genes display striking bile duct proliferation, which is not apparent at birth. These findings suggest that the Fringe genes may regulate postnatal bile duct growth and remodeling, and serve as candidate modifiers of the hepatic phenotype in AGS.

Functional role of Notch signaling in the developing and postnatal heart.

In the developing heart, Notch signaling plays an essential role in several key developmental processes, such as epithelial-to-mesenchymal transition and myocyte proliferation and differentiation. The importance of Notch in cardiac development has been demonstrated in knockout mice carrying null mutations in genes encoding components of the Notch pathway. Furthermore, humans with inactivating mutations in the Notch ligand Jagged1 suffer from Alagille syndrome, a condition characterized by several cardiac defects. Notch1 receptor haploinsufficiency has also been involved in aortic valve disease in humans. In addition, accumulating evidence indicates that Notch may also regulate homeostasis in the adult heart. Notch may protect the heart from an excessive and detrimental hypertrophic response and increase cardiomyocyte survival. Emerging evidence also suggests that Notch could be important for cardiac tissue renewal by controlling the maintenance and commitment of a cardiac stem cell compartment.