Jagged1 Research Articles

Assessment of multi-trait analysis of GWAS (MTAG) for discovery of novel genetic variants and mechanistic insight in common cardiovascular diseases

Abstract Background Variability in cardiovascular conditions is partly explained by common genomic variation identifiable by genome-wide association studies (GWAS). Genetic substrate may overlap in certain entities, this being quantified by genetic correlation. We aimed to leverage multi-trait analysis of GWAS (MTAG), (1) which boosts statistical power by joint analysis of genetically correlated traits, to improve genetic loci discovery in 3 common cardiovascular diseases: atrial fibrillation (AF), coronary artery disease (CAD), and heart failure (HF). Methods and Results Cardiovascular phenotypes with available GWAS summary statistics most correlated with the disease of interest were analysed jointly by MTAG (workflow depicted in Figure 1). These included body mass index and HF for AF (2); HF, LDL-cholesterol, and systolic blood pressure for CAD (3); and AF, CAD, and dilated cardiomyopathy for HF (4). MTAG increased statistical power for all 3 disease GWAS and allowed the identification of 19 new genomic loci for AF, 89 for CAD and 52 for HF (at a GWAS significance level P<5x10-8) (Figure 2). New loci were defined as not previously identified in the single trait original GWAS. Consistently, 19/19, 85/89 and 48/52 of new loci reached nominal significance (P<0.05) in the original studies; and 37%, 44% and 36% of new loci have been associated with AF, CAD and HF in GWAS and phenome wide association studies (PheWAS) in independent cohorts. MTAG summary statistics annotation and functional analysis integrated the distance to the canonical transcriptional start site, chromatin interaction experiments, quantitative molecular phenotype trait experiments and in silico functional prediction through OpenTargets Genetics. MAGMA gene-set functional analysis of the new MTAG results showed 87, 27 and 24 significant associated pathways to AF, CAD and HF respectively. Among the top associations, cardiac depolarization, repolarization, and muscle contraction in AF; macromolecular complex remodeling, triglyceride, chylomicron and LDL metabolism in CAD; and muscle structure, tissue development and RNA transcription initiation in HF. Among novel relevant genes in AF, SRR on chromosome 16 is involved in central nervous system development through regulation of NMDA receptors. This locus has been previously associated with QRS in another GWAS and SRR is related to reduction in heart rate variability and AF enhancement in rats. In CAD, RSPO3 and JAG1 genes, on chromosomes 6 and 20 respectively, have a relevant role in angiogenesis, where RSPO3 is a crucial regulator of coronary artery formation in the developing heart. Conclusions MTAG allows the discovery of new trait-specific loci involved in the pathogenesis of AF, CAD, and HF congruent with the mechanisms of each disease and consistent with independent external cohort PheWAS and GWAS. Thus, MTAG is an interesting tool to unravel relevant mechanisms and potential therapeutic targets in common cardiovascular diseases.

Optimization of a Three-Dimensional Culturing Method for Assessing the Impact of Cisplatin on Notch Signaling in Head and Neck Squamous Cell Carcinoma (HNSCC).

Head and neck squamous cell carcinoma (HNSCC) is a prevalent cancer type, with cisplatin being a primary treatment approach. However, drug resistance and therapy failure pose a significant challenge, affecting nearly 50% of patients over time. This research had two aims: (1) to optimize a 3D cell-culture method for assessing the interplay between tumor cells and cancer-associated fibroblasts (CAFs) in vitro; and (2) to study how cisplatin impacts the Notch pathway, particularly considering the role of CAFs. Using our optimized "3D sheet model" approach, we tested two HNSCC cell lines with different cisplatin sensitivities and moderate, non-mutated NOTCH1 and -3 expressions. Combining cisplatin with a γ-secretase inhibitor (crenigacestat) increased sensitivity and induced cell death in the less sensitive cell line, while cisplatin alone was more effective in the moderately sensitive line and sensitivity decreased with the Notch inhibitor. Cisplatin boosted the expression of core Notch signaling proteins in 3D monocultures of both lines, which was counteracted by crenigacestat. In contrast, the presence of patient-derived CAFs mitigated effects and protected both cell lines from cisplatin toxicity. Elevated NOTCH1 and NOTCH3 protein levels were consistently correlated with reduced cisplatin sensitivity and increased cell survival. Additionally, the Notch ligand JAG2 had additional, protective effects reducing cell death from cisplatin exposure. In summary, we observed an inverse relationship between NOTCH1 and NOTCH3 levels and cisplatin responsiveness, overall protective effects by CAFs, and a potential link between JAG2 expression with tumor cell survival.

High homocysteine level promotes autophagy and apoptosis of mouse hippocampal HT22 cells through the Notch1/Hes1 signaling pathway

To explore the mechanism of neuronal injury caused by hyperhomocysteinemia. Mouse hippocampal HT22 cells were treated with homocysteine (Hcy, 100 μmol/L), Hcy+folic acid+vitamin B12 (100+fv group) or folic acid+vitamin B12 (0+fv group), and the changes in cell autophagy and apoptosis were detected using transmission electron microscope (TEM) and flow cytometry. The expressions of Hes1, Hes5, Notch1, Jagged1, Bcl-2, Bax, P62 and LC3 in the treated cells were detected with Western blotting and real-time PCR. Treatment with Hcy for 48 h significantly increased the number of dead cells in HT22 cell cultures. Flow cytometry showed that the percentage of apoptotic cells was significantly higher in cells treated with Hcy alone than in other treatment groups (P<0.05). TEM revealed obvious mitochondrial swelling and vacuolation and increased autophagy in Hcy-treated cells. Western blotting showed that the Bax/Bcl-2 ratio was significantly higher in Hcy-treated cells than in the blank control cells and cells in 100+fv group (P<0.05). The Hcy-treated cells showed a significantly lower relative expression of P62 than the blank control cells (P<0.05), a higher LC3II/LC3I ratio than the cells in the blank control and 100+fv groups (P<0.05), and lower expressions of HES1, HES5, Notch1 and Jagged1 proteins than the blank control cells (P<0.05). Interference with Hes1 siRNA significantly lowered the expression levels of Hes1 and Jagged1 without obviously affecting Notch1 expression in HT22 cells (P>0.05). High Hcy levels promote autophagy and apoptosis and down-regulate Hes1 and Jagged1 expressions in HT22 cells.

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.

Sulindac sulfide as a non-immune suppressive γ-secretase modulator to target triple-negative breast cancer.

Triple-negative breast cancer (TNBC) comprises a heterogeneous group of clinically aggressive tumors with high risk of recurrence and metastasis. Current pharmacological treatment options remain largely limited to chemotherapy. Despite promising results, the efficacy of immunotherapy and chemo-immunotherapy in TNBC remains limited. There is strong evidence supporting the involvement of Notch signaling in TNBC progression. Expression of Notch1 and its ligand Jagged1 correlate with poor prognosis. Notch inhibitors, including g-secretase inhibitors (GSIs), are quite effective in preclinical models of TNBC. However, the success of GSIs in clinical trials has been limited by their intestinal toxicity and potential for adverse immunological effects, since Notch plays key roles in T-cell activation, including CD8 T-cells in tumors. Our overarching goal is to replace GSIs with agents that lack their systemic toxicity and ideally, do not affect tumor immunity. We identified sulindac sulfide (SS), the active metabolite of FDA-approved NSAID sulindac, as a potential candidate to replace GSIs. We investigated the pharmacological and immunotherapeutic properties of SS in TNBC models in vitro, ex-vivo and in vivo. We confirmed that SS, a known γ-secretase modulator (GSM), inhibits Notch1 cleavage in TNBC cells. SS significantly inhibited mammosphere growth in all human and murine TNBC models tested. In a transplantable mouse TNBC tumor model (C0321), SS had remarkable single-agent anti-tumor activity and eliminated Notch1 protein expression in tumors. Importantly, SS did not inhibit Notch cleavage in T- cells, and the anti-tumor effects of SS were significantly enhanced when combined with a-PD1 immunotherapy in our TNBC organoids and in vivo. Our data support further investigation of SS for the treatment of TNBC, in conjunction with chemo- or -chemo-immunotherapy. Repurposing an FDA-approved, safe agent for the treatment of TNBC may be a cost-effective, rapidly deployable therapeutic option for a patient population in need of more effective therapies.

Myocardial Remodeling in Early Chronic Kidney Disease—Mineral and Bone Disorder Model with Low Bone Turnover

Chronic kidney disease—mineral and bone disorder (CKD-MBD) plays a significant role in causing cardiovascular morbidity and mortality related to CKD. CKD-MBD has been studied during advanced stages when changes in inorganic phosphate (Pi) and its hormonal regulation are obvious. The initial phases of myocardial remodeling (MR) in early CKD-MBD remain poorly understood. We induced mild CKD-MBD in spontaneously hypertensive rats using 3/4 nephrectomy. Animals were fed standard chow, containing 0.6% phosphate. In each animal, we analyzed indices of chronic kidney injury, bone turnover and Pi exchange, and assessed the myocardial histology and gene expression profile. Applied CKD-MBD models corresponded to human CKD S1-2 with low bone turnover and without an increase in systemic Pi-regulating factors (parathyroid hormone and fibroblast growth factor 23). In mild CKD-MBD models, we found MR features characterized by cardiomyocyte hypertrophy, interstitial and perivascular fibrosis, intramyocardial artery media thickening, along with alterations in Ppp3ca, Mapk1, Jag1, Hes1, Ptch1, Numb, Lgr4 and Bmp4 genes. Among other genes, the down-regulation of Jag1 was most tightly associated with either myocardial hypertrophy or fibrosis. Myocardial alterations concurrently occurred with mild CKD-MBD and comprised fibrosis preceding cardiomyocyte hypertrophy. The histological features of MR were associated with myocardial P accumulation in settings of low bone turnover, prior to a response of systemic Pi-regulating factors and with alterations in calcineurin, ERK1/2, Notch, BMP and Hedgehog genes.

Annexin A2 Stabilizes Oncogenic JAG1 Intracellular Domain by Inhibiting Proteasomal Degradation in Glioblastoma Cells.

Glioblastoma (GBM) is the most lethal brain cancer, causing inevitable deaths of patients owing to frequent relapses of cancer stem cells (CSCs). The significance of the NOTCH signaling pathway in CSCs has been well recognized; however, there is no NOTCH-selective treatment applicable to patients with GBM. We recently reported that Jagged1 (JAG1), a NOTCH ligand, drives a NOTCH receptor-independent signaling pathway via JAG1 intracellular domain (JICD1) as a crucial signal that renders CSC properties. Therefore, mechanisms regulating the JICD1 signaling pathway should be elucidated to further develop a selective therapeutic regimen. Here, we identified annexin A2 (ANXA2) as an essential modulator to stabilize intrinsically disordered JICD1. The binding of ANXA2 to JICD1 prevents the proteasomal degradation of JICD1 by heat shock protein-70/90 and carboxy-terminus of Hsc70 interacting protein E3 ligase. Furthermore, JICD1-driven propagation and tumor aggressiveness were inhibited by ANXA2 knockdown. Taken together, our findings show that ANXA2 maintains the function of the NOTCH receptor-independent JICD1 signaling pathway by stabilizing JICD1, and the targeted suppression of JICD1-driven CSC properties can be achieved by blocking its interaction with ANXA2.

Jagged1 Acts as an RBP-J Target and Feedback Suppresses TNF-Mediated Inflammatory Osteoclastogenesis.

TNF plays a crucial role in inflammation and bone resorption in various inflammatory diseases, including rheumatoid arthritis (RA). However, its direct ability to drive macrophages to differentiate into osteoclasts is limited. Although RBP-J is recognized as a key inhibitor of TNF-mediated osteoclastogenesis, the precise mechanisms that restrain TNF-induced differentiation of macrophages into osteoclasts are not fully elucidated. In this study, we identified that the Notch ligand Jagged1 is a previously unrecognized RBP-J target. The expression of Jagged1 is significantly induced by TNF mainly through RBP-J. The TNF-induced Jagged1 in turn functions as a feedback inhibitory regulator of TNF-mediated osteoclastogenesis. This feedback inhibition of osteoclastogenesis by Jagged1 does not exist in RANKL-induced mouse osteoclast differentiation, as RANKL does not induce Jagged1 expression. The Jagged1 level in peripheral blood monocytes/osteoclast precursors is decreased in RA compared with the nonerosive inflammatory disease systemic lupus erythematosus, suggesting a mechanism that contributes to increased osteoclast formation in RA. Moreover, recombinant Jagged1 suppresses human inflammatory osteoclastogenesis. Our findings identify Jagged1 as an RBP-J direct target that links TNF and Notch signaling pathways and restrains TNF-mediated osteoclastogenesis. Given that Jagged1 has no effect on TNF-induced expression of inflammatory genes, its use may present a new complementary therapeutic approach to mitigate inflammatory bone loss with little impact on the immune response in disease conditions.

The Potential of JAG Ligands as Therapeutic Targets and Predictive Biomarkers in Multiple Myeloma.

The NOTCH ligands JAG1 and JAG2 have been correlated in vitro with multiple myeloma (MM) cell proliferation, drug resistance, self-renewal and a pathological crosstalk with the tumor microenvironment resulting in angiogenesis and osteoclastogenesis. These findings suggest that a therapeutic approach targeting JAG ligands might be helpful for the care of MM patients and lead us to explore the role of JAG1 and JAG2 in a MM in vivo model and primary patient samples. JAG1 and JAG2 protein expression represents a common feature in MM cell lines; therefore, we assessed their function through JAG1/2 conditional silencing in a MM xenograft model. We observed that JAG1 and JAG2 showed potential as therapeutic targets in MM, as their silencing resulted in a reduction in the tumor burden. Moreover, JAG1 and JAG2 protein expression in MM patients was positively correlated with the presence of MM cells in patients' bone marrow biopsies. Finally, taking advantage of the Multiple Myeloma Research Foundation (MMRF) CoMMpass global dataset, we showed that JAG2 gene expression level was a predictive biomarker associated with patients' overall survival and progression-free survival, independently from other main molecular or clinical features. Overall, these results strengthened the rationale for the development of a JAG1/2-tailored approach and the use of JAG2 as a predictive biomarker in MM.

Effect of electroacupuncture on proliferation and differentiation of endogenous neural stem cells and Jagged1/Notch1 pathway in hippocampus of APP/PS1 model mice

To investigate the effect of electroacupuncture(EA) stimulation on proliferation and diffe-rentiation of endogenous neural stem cells as well as Jagged1/Notch1 pathway in AD model mice, so as to explore its mechanism underlying amelioration of AD. A total of 40 6-week-old male APP/PS1 transgenic AD mice were randomly divided into EA group (n=20) and AD model group ( n=20), and other 20 normal C57BL/6J mice of the same age were used as the normal control group. The mice in the EA group received EA (10 Hz, 2 mA) at "Baihui"(GV20), "Fengfu"(GV16) and bilateral "Shenshu" (BL23) for 20 min, once daily, 6 days a week for 16 weeks. The mice's learning-memory ability was detected by Morris water maze tests. The Aβ senile plaques in the hippocampal CA1 region were detected by Congo red staining, the immunofluorescence double label of BrdU, neuronal nuclear antigen (NeuN) and astrocyte specific protein GFAP in dentate gyrus of hippocampus was performed for detecting the proliferation and differentiation of the endogenous neural stem cells. The expression levels of Nestin (neuron specific protein) and GFAP were detected by Western blot, and those of Jagged1 and Notch1 mRNAs and proteins in the hippocampus were detected by real-time fluorescence quantifative PCR and Western blot. Compared with the normal control group, the escape latencies at 2nd, 3rd and 4th day, and Aβ senile plaques were significantly increased (P<0.05, P<0.01), whereas the platform crossing times and time spent in the target quadrant, the expression levels of Jagged1 mRNA and Nestin protein were remarkably down-regulated (P<0.05) in the model group. Following EA intervention, the escape latencies at the 3rd and 4th day, Aβ senile plaques, immunofluorescence density of BrdU/GFAP, and GFAP protein expression were pronouncedly decreased (P<0.05, P<0.01), while the platform crossing times, platform quadrant residence time, immunofluorescence density of BrdU/NeuN, expression levels of Jagged1 and Notch1 mRNAs and proteins and Nestin protein evidently increased (P<0.05, P<0.01), suggesting an enhancement of proliferation and diffe-rentiation of endogenous neural stem cells into neurons and a suppression of the proliferation and differentiation towards astrocytes in the hippocampus. EA at GV20, GV16 and BL23 can improve the learning-memory ability, promote the proliferation and differentiation of endogenous neural stem cells towards neurons and inhibit the proliferation and differentiation of endogenous neural stem cells towards astrocytes in the hippocampus, which may be achieved by regulating Jagged1/Notch1 pathway.

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.

The Curious Case of Alagille Syndrome: A Case Report With NANDA-I Classification, NIC, and NOC Linkage to the Patient Care Plan.

Alagille syndrome is a rare and complex pleiotropic multisystem disorder caused by an autosomal dominant genetic mutation of JAG1 (90%) and NOTCH2 (1%-2%) genes located on the short arm of chromosome 20. This case is reported as per the CAse REports (CARE) guidelines (2013). A 14-year-old boy who is a known case of chronic cholestatic liver disease of neonatal onset, was diagnosed with Alagille syndrome as evident from a NOTCH 2 mutation in genetic analysis and paucity of intrahepatic bile ducts on biopsy. He presented with portal hypertension, growth failure, and persistent hyperbilirubinemia. This case highlights the gamut of multisystem dysfunctions faced by this child. He is currently on conservative management and worked up for liver transplantation. The condition is often rare and challenging due to the multisystem pathogenesis. Thus, the nursing care is also multifaceted. This case study identified relevant North American Nursing Diagnosis Association (NANDA) Classification, Nursing Interventions Classification (NIC), and Nursing Outcomes Classification (NOC) concepts to describe care of children with Alagille syndrome based on actual patient data.

Exosomes from hypoxia-conditioned apical papilla stem cells accelerate angiogenesis in vitro through Notch/JAG1/VEGF signaling

Dental pulp angiogenesis is a committed step in pulp regeneration therapy, and exosomes provide a new cell-free choice for tissue regeneration. This study revealed the underlying regulatory mechanism of exosomes from stem cells of the apical papilla (SCAPs) under hypoxic state on angiogenesis of human umbilical vein endothelial cells (HUVECs) in vitro. Exosomes extracted from normoxia or hypoxia-pretreated SCAPs were co-cultured with HUVECs, and hypoxia pretreatment increased the release of exosomes and the internalization of exosomes by HUVECs. Compared to normoxic SCAPs-derived exosomes, exosomes from hypoxic SCAPs were found to promote cell proliferation and migration in HUVECs, as it was respectively determined by Cell Counting Kit-8, RT-qPCR and Transwell assay. Besides, hypoxia-educated SCAPs-exosomes especially enhanced the angiogenesis abilities of HUVECs in vitro, which were confirmed by tube formation assay and RT-qPCR detection of angiogenesis-related molecular markers. Interestingly, we found that the hypoxia inducible factor-1α (HIF-1α)/Notch1 signaling pathway was activated in hypoxic SCAPs, and protein jagged-1 (JAG1) was delivered by hypoxic SCAPs-derived exosomes to increase vascular endothelial growth factor (VEGF) production in HUVECs. Moreover, exogenous interference of JAG1 expression in HUVECs partially neutralized the activities of hypoxic SCAPs-exosomes in promoting cell proliferation, migration and tube formation of HUVECs. In summary, this study elucidates that exosomes from hypoxic SCAPs shows high potential to promote angiogenesis in vitro through the HIF-1α/JAG1/VEGF signaling cascade, which may provide a new perspective for the development of vascular reconstruction measures during dental regeneration engineering.

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.

Tspan5 promotes the EMT process to regulate the syncytialization of trophoblast cells by activating Notch signalling.

Placental trophoblastic cells play important roles in placental development and fetal health. However, the mechanism of trophoblastic cell fusion is still not entirely clear. The level of Tspan5 in the embryo culture medium was detected using enzyme-linked immunosorbent assay (ELISA). Fusion of BeWo cells was observed by immunofluorescence. Cell fusion-related factors and EMT-related factors were identified by qRT-PCR and western blotting. Notch protein repressor DAPT was used to verify the role of Tspan5 in BeWo cells. The expression of Tspan5 was significantly increased in embryo culture medium. The fusion of BeWo cells was observed after treatment with forskolin (FSK). Cell fusion-related factors (i.e. β-hCG and syncytin 1/2) and Tspan5 were significantly increased after FSK treatment. In addition, FSK treatment promoted EMT-related protein expression in BeWo cells. Knockdown of Tspan5 inhibited cell fusion and EMT-related protein levels. Notch-1 and Jagged-1 protein levels were significantly upregulated, and the EMT process was activated by overexpression of Tspan5 in FSK-treated BeWo cells. Interestingly, blocking the Notch pathway by the repressor DAPT had the opposite results. These results indicated that Tspan5 could promote the EMT process by activating the Notch pathway, thereby causing cell fusion. These findings contribute to a better understanding of trophoblast cell syncytialization and embryonic development. Tspan5 may be used as a therapeutic target for normal placental development.

Clinical Characterization of Alagille Syndrome in Patients with Cholestatic Liver Disease.

Alagille syndrome (ALGS) is a multisystem condition characterized by cholestasis and bile duct paucity on liver biopsy and variable involvement of the heart, skeleton, eyes, kidneys, and face and caused by pathogenic variants in the JAG1 or NOTCH2 gene. The variable expressivity of the clinical phenotype and the lack of genotype-phenotype correlations lead to significant diagnostic difficulties. Here we present an analysis of 18 patients with cholestasis who were diagnosed with ALGS. We used an NGS panel targeting coding exons of 52 genes, including the JAG1 and NOTCH2 genes. Sanger sequencing was used to verify the mutation in the affected individuals and family members. The specific facial phenotype was seen in 16/18 (88.9%). Heart defects were seen in 8/18 (44.4%) patients (pulmonary stenosis in 7/8). Butterfly vertebrae were seen in 5/14 (35.7%) patients. Renal involvement was detected in 2/18 (11.1%) cases-one patient had renal cysts, and one had obstructive hydronephrosis. An ophthalmology examination was performed on 12 children, and only one had posterior embryotoxon (8.3%). A percutaneous liver biopsy was performed in nine cases. Bile duct paucity was detected in six/nine cases (66.7%). Two patients required liver transplantation because of cirrhosis. We identified nine novel variants in the JAG1 gene-eight frameshift variants (c.1619_1622dupGCTA (p.Tyr541X), c.1160delG (p.Gly387fs), c.964dupT (p.C322fs), c.120delG (p.L40fs), c.1984dupG (p.Ala662Glyfs), c.3168_3169delAG (p.R1056Sfs*51), c.2688delG (p.896CysfsTer49), c.164dupG (p.Cys55fs)) and one missense variant, c.2806T > G (p.Cys936Gly). None of the patients presented with NOTCH2 variants. In accordance with the classical criteria, only six patients could meet the diagnostic criteria in our cohort without genetic analysis. Genetic testing is important in the diagnosis of ALGS and can help differentiate it from other types of cholestasis.

Two unrelated Alagille syndrome cases of South Indian origin: Showing multi-exonic deletion and a novel mutation in JAG1 gene

Two unrelated Alagille syndrome cases of South Indian origin: Showing multi-exonic deletion and a novel mutation in JAG1 gene

The Notch-mediated circuitry in the evolution and generation of new cell lineages: the tooth model

The Notch pathway is an ancient, evolutionary conserved intercellular signaling mechanism that is involved in cell fate specification and proper embryonic development. The Jagged2 gene, which encodes a ligand for the Notch family of receptors, is expressed from the earliest stages of odontogenesis in epithelial cells that will later generate the enamel-producing ameloblasts. Homozygous Jagged2 mutant mice exhibit abnormal tooth morphology and impaired enamel deposition. Enamel composition and structure in mammals are tightly linked to the enamel organ that represents an evolutionary unit formed by distinct dental epithelial cell types. The physical cooperativity between Notch ligands and receptors suggests that Jagged2 deletion could alter the expression profile of Notch receptors, thus modifying the whole Notch signaling cascade in cells within the enamel organ. Indeed, both Notch1 and Notch2 expression are severely disturbed in the enamel organ of Jagged2 mutant teeth. It appears that the deregulation of the Notch signaling cascade reverts the evolutionary path generating dental structures more reminiscent of the enameloid of fishes rather than of mammalian enamel. Loss of interactions between Notch and Jagged proteins may initiate the suppression of complementary dental epithelial cell fates acquired during evolution. We propose that the increased number of Notch homologues in metazoa enabled incipient sister cell types to form and maintain distinctive cell fates within organs and tissues along evolution.

Two NOTCH1 O-fucose sites have opposing functions in mouse retinal angiogenesis.

Previous in vitro studies demonstrated that Fringe glycosylation of the NOTCH1 extracellular domain at O-fucose residues in Epidermal Growth Factor-like Repeats (EGFs) 6 and 8 is a significant contributor to suppression of NOTCH1 activation by JAG1 or enhancement of NOTCH1 activation by DLL1, respectively. In this study, we sought to evaluate the significance of these glycosylation sites in a mammalian model by generating 2 C57BL/6J mouse lines carrying NOTCH1 point mutations, which eliminate O-fucosylation and Fringe activity at EGFs 6 (T232V) or 8 (T311V). We assessed changes to morphology during retinal angiogenesis, a process in which expression of Notch1, Jag1, Dll4, Lfng, Mfng, and Rfng genes coordinate cell-fate decisions to grow vessel networks. In the EGF6 O-fucose mutant (6f/6f) retinas, we observed reduced vessel density and branching, suggesting that this mutant is a Notch1 hypermorph. This finding agrees with prior cell-based studies showing that the 6f mutation increased JAG1 activation of NOTCH1 during co-expression with inhibitory Fringes. Although we predicted that the EGF8 O-fucose mutant (8f/8f) would not complete embryonic development due to the direct involvement of the O-fucose in engaging ligand, the 8f/8f mice were viable and fertile. In the 8f/8f retina, we measured increased vessel density consistent with established Notch1 hypomorphs. Overall, our data support the importance of NOTCH1 O-fucose residues for pathway function and confirms that single O-glycan sites are rich in signaling instructions for mammalian development.

Insufficient GDF15 expression predisposes women to unexplained recurrent pregnancy loss by impairing extravillous trophoblast invasion.

Insufficient extravillous trophoblast (EVT) invasion during early placentation has been shown to contribute to recurrent pregnancy loss (RPL). However, the regulatory factors involved and their involvement in RPL pathogenesis remain unknown. Here, we found aberrantly decreased growth differentiation factor 15 (GDF15) levels in both first-trimester villous and serum samples of unexplained recurrent pregnancy loss (URPL) patients as compared with normal pregnancies. Moreover, GDF15 knockdown significantly reduced the invasiveness of both HTR-8/SVneo cells and primary human EVT cells and suppressed the Jagged-1 (JAG1)/NOTCH3/HES1 pathway activity, and JAG1 overexpression rescued the invasion phenotype of the GDF15 knockdown cells. Induction of a lipopolysaccharide-induced abortion model in mice resulted in significantly reduced GDF15 level in the placenta and serum, as well as increased rates of embryonic resorption, and these effects were reversed by administration of recombinant GDF15. Our study thus demonstrates that insufficient GDF15 level at the first-trimester maternal-foetal interface contribute to the pathogenesis of URPL by impairing EVT invasion and suppressing JAG1/NOTCH3/HES1 pathway activity, and suggests that supplementation with GDF15 could benefit early pregnancy maintenance and reduce the risk of early pregnancy.