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 JAGGED1 (JAG1) or NOTCH2, ligand and receptor components of the Notch signaling pathway, respectively. The detected missense variants are commonly classified as variants of uncertain signifi cance, hindering ALGS diagnosis. To overcome this issue, we have developed a CRISPR/Cas9-engineered Low-Notch activity (LNA) cells allowing for selective testing of JAG1-NOTCH2 signaling activity. We tested this approach on 9 patients with pediatric hepatopathies with phenotypes ranging from the full clinical ALGS spectrum to isolated neonatal cholestasis and atypical ALGS abnormalities who carried 5 JAG1 and 3 NOTCH2 missense variants of interest. Addition ally, western blot analyses revealed an effect on protein expression for two JAG1 missense variants, one of which had altered glycosylation, potentially indicating pathogenic effects. For this JAG1 and one NOTCH2 de novo missense vari ant, luciferase-based Notch reporter activity was significantly reduced in LNA cells, while no change was observed in commonly used HEK293T, Huh7, or Hep2G cells. These findings allow independent confirmation of recently classified c.53T>G p.(Leu18Arg) in JAG1, and a re-classification of c.1235G>T p.(Cys412Phe) in NOTCH2 as likely pathogenic based on ACMG criteria. Collectively, we provide evidence that selective testing of JAG1-NOTCH2 interaction in the newly developed CRISPR-engineered cells, combined with a glycosylation assay, enables robust functional evaluation of ALGS-associated JAG1 and NOTCH2 variants