NOTCH2 VUS Interpretation in Pediatric Cholestasis: A 365-Variant Landscape and Genotype–Phenotype Analysis of 7 Cases

Background/Objectives: Interpretation of NOTCH2 variants of uncertain significance (VUSs) remains challenging in pediatric cholestasis and suspected Alagille syndrome (ALGS) due to incomplete penetrance, phenotypic variability, and extensive genetic variation. Although disease-specific variant classification criteria have identified pathogenic hotspots within epidermal growth factor (EGF)-like repeats 8–12 and ankyrin repeat domains 3–6, characterizing these VUSs requires a robust framework. Methods: We reviewed aggregate deidentified data from 519 multigene cholestasis panel entries and evaluated genotype–phenotype data from a consortium cohort of 7 pediatric patients with heterozygous NOTCH2 VUSs using a clinical–genomic prioritization framework informed by disease-specific ACMG criteria and domain location. Results: Across the 519 entries, 365 unique NOTCH2 VUSs were identified, none of which overlapped with 69 previously reported ALGS-associated variants or localized to established hotspots, with over 60% occurring outside recognized functional domains. Among the 7 consortium cases, one patient carrying a cysteine-altering variant (p.Cys593Arg) in EGF-like domain 15 demonstrated a strong ALGS-compatible phenotype, while alternative diagnoses in other cases included trisomy 21 and congenital hepatic fibrosis with polycystic kidney disease. Conclusions: Among 365 unique NOTCH2 VUSs identified through pediatric cholestasis panel testing, none overlapped with previously reported ALGS-associated variants or localized to established pathogenic hotspots. However, a cysteine-altering variant outside canonical hotspots occurred in a patient with a strong ALGS-compatible phenotype, highlighting the importance of integrating variant characteristics, domain location, detailed phenotyping, and alternative diagnoses when prioritizing NOTCH2 VUSs for further evaluation.