NOTCH2 mutations in Alagille syndrome

Abstract

Background: Alagille syndrome (ALGS) is a dominant, multisystem disorder caused by mutations in the Jagged1 (JAG1) ligand in 94% of patients, and in the NOTCH2 receptor in <1%. There are only two NOTCH2 families reported to date. This study hypothesised that additional NOTCH2 mutations would be present in patients with clinical features of ALGS without a JAG1 mutation.

Methods: The study screened a cohort of JAG1-negative individuals with clinical features suggestive or diagnostic of ALGS for NOTCH2 mutations.

Results: Eight individuals with novel NOTCH2 mutations (six missense, one splicing, and one non-sense mutation) were identified. Three of these patients met classic criteria for ALGS and five patients only had a subset of features. The mutations were distributed across the extracellular (N=5) and intracellular domains (N=3) of the protein. Functional analysis of four missense, one nonsense, and one splicing mutation demonstrated decreased Notch signalling of these proteins. Subjects with NOTCH2 mutations demonstrated highly variable expressivity of the affected systems, as with JAG1 individuals. Liver involvement was universal in NOTCH2 probands and they had a similar prevalence of ophthalmologic and renal anomalies to JAG1 patients. There was a trend towards less cardiac involvement in the NOTCH2 group (60% vs 100% in JAG1). NOTCH2 (+) probands exhibited a significantly decreased penetrance of vertebral abnormalities (10%) and facial features (20%) when compared to the JAG1 (+) cohort.

Conclusions: This work confirms the importance of NOTCH2 as a second disease gene in ALGS and expands the repertoire of the NOTCH2 related disease phenotype.

Figure 1

NOTCH2 domain structure and Alagille syndrome (ALGS) mutation locations. The protein domain structure of the human NOTCH2 protein is depicted. NOTCH2 is a transmembrane protein with intracellular and extracellular components. The extracellular part of the protein contains 36 epidermal growth factor (EGF) repeats involved in ligand binding. The 11th and 12th of the EGF repeats (depicted in a different colour from the rest) are known to be required for efficient ligand binding. The intracellular portion of the protein is comprised of multiple domains, including seven ankyrin repeats (ANK) which are required for binding to the CSL (for CBF1 (mammalian C promoter-binding factor 1), suppressor of hairless (fly) Lag2 (worm)) transcription factor in the nucleus and activation of downstream transcription. All of the NOTCH2 mutations identified in ALGS patients are listed in the corresponding region of the protein. Mutations previously reported in McDaniell et al are marked with an asterick. The likely polymorphism in patient 7 is marked with a double asterix. ANK, ankyrin repeats; EGF Repeats, epidermal growth factor-like repeats; LNR, Lin/Notch repeats; NLS, nuclear localisation signal; PEST, proline/glutamic acid/serine/threonine rich domain; RAM, RBP-Jκ-associated module; TMD, Transmembrane domain.

Figure 2

Facial features of NOTCH2 (+) probands. Front and side profiles of two available NOTCH2 (+) probands. The typical Alagille syndrome facial features associated with JAG1 mutations (broad forehead, deep-set eyes, pointed chin) are not present in these individuals.

Figure 3

Notch signalling ability of mutant NOTCH2 proteins. The ability of the mutant NOTCH2 proteins to activate Notch signalling in the presence of JAG1 ligand was examined. Cells transfected with wild-type or mutant NOTCH2 or vector alone, along with a CBF-responsive luciferase reporter, were plated in culture wells coated with soluble JAG1 ligand, or soluble TRAIL (TNF related apoptosis inducing ligand) as a ligand specific control. Forty-eight hours post-transfection lysates were collected and luciferase readings taken. Transfection reactions also included a renilla internal control for transfection efficiency. Luciferase readings were normalised to renilla readings, and values are expressed as fold change over vector alone. No mutant proteins were able to activate Notch signalling to levels significantly different from vector alone or TRAIL control wells, as assayed by Student t test.