Improved functional JAG1 and NOTCH2 variant testing in patients with clinical or suspected Alagille syndrome using new Low-Notch Activity cells

The autosomal dominant multisystemic Alagille Syndrome (ALGS) is an important cause of pediatric cholestasis. ALGS is associated with pathogenic variants in JAG1 (encoded by JAG1) or NOTCH2 (encoded by NOTCH2), ligand and receptor components of the Notch-signaling pathway, respectively. The detected missense variants are most commonly classified as variants of uncertain significance (VUS), hindering ALGS diagnosis. To overcome this issue, we have developed a set of in vitro assays allowing for selective testing of the JAG1-NOTCH2 activity. We tested this approach on 9 pediatric patients with hepatopathy with phenotypes ranging from the full clinical ALGS spectrum to isolated neonatal cholestasis and atypical ALGS abnormalities carrying 5 JAG1 and 3 NOTCH2 missense variants of interest. Western blot analyses showed an effect on protein expression for two JAG1 missense variants, one with altered glycosylation, which may indicate pathogenic effects. For this JAG1 and one NOTCH2 de novo missense variant, luciferase activity was significantly reduced in the luciferase-based Notch reporter assay employing newly developed Low-Notch activity cells, suggesting an effect on Notch activity. All detected results allow a classification of these two variants as likely pathogenic. Collectively, we provide evidence that selective testing of JAG1-NOTCH2 interaction, combined with glycosylation assay, allows for a robust classification of ALGS - associated JAG1 and NOTCH2 variants.