Jagged1 (JAG1) mutations in patients with tetralogy of Fallot or pulmonic stenosis

Abstract

Mutations in the Notch pathway ligand Jagged1 (JAG1) cause Alagille syndrome (AGS), as well as cardiac defects in seemingly nonsyndromic individuals. To estimate the frequency of JAG1 mutations in cases with right-sided cardiac defects not otherwise diagnosed with AGS, we screened 94 cases with tetralogy of Fallot (TOF) and 50 with pulmonic stenosis/peripheral pulmonary stenosis (PS/PPS) or pulmonary valve atresia with intact ventricular septum (PA) for mutations. Sequence changes were identified in three TOF and three PS/PPS/PA patients, that were not present in 100 controls. We identified one frameshift and two missense mutations in the TOF cases, and one frameshift and two missense mutations in cases with PS/PPS/PA. The four missense mutations were assayed for their effect on protein localization, posttranslational modification, and ability to activate Notch signaling. The missense mutants displayed heterogeneous behavior in these assays, some with complete haploinsufficiency, suggesting that there are additional modifiers leading to organ specific features. We identified functionally significant mutations in 2% (2/94) of TOF patients and 4% (2/50) of PS/PPS/PA patients. Patients with right-sided cardiac defects should be carefully screened for features of AGS or a family history of cardiac defects that might suggest the presence of a JAG1 mutation.

Figure 1

A: EndoH sensitivity of JAG1 missense mutations. The post-translational modification of the mutant JAG1 proteins was analyzed via treatment with the glycosidase EndoH and subsequent visualization by western blot with a JAG1 antibody (H114, Santa Cruz). Controls include wildtype JAG1 (not sensitive to EndoH), p.L37S (sensitive), and p.G274D (partially sensitive). p.C234Y and p.P810L appear to be EndoH-sensitive, whereas p.R937Q is insensitive, and p.C664S is partially sensitive. Arrows indicate the untreated or EndoH insensitive JAG1 proteins (top arrow) and the EndoH sensitive proteins (bottom arrows). B: Trypsin sensitivity as a measure of cell surface expression. Cells expressing variant proteins were treated with trypsin for 10 minutes, and JAG1 was visualized by western blot. Proteins present on the cell surface are degraded, with disappearance of the full length product and appearance of lower bands (wildtype, p.R37Q), whereas p.L37S, p.C234Y and p.P810L are insensitive. p.G274D and p.C664S are partially sensitive, consistent with two species of protein. Arrows indicate the full length, undigested JAG1 protein (top arrow) as well as the lower molecular weight degradation products (bottom 3 arrows).

Figure 2

Sub-cellular localization of JAG1 wild-type and missense mutants in NIH3T3 cells by immunofluorescence. Wild-type JAG1 and p.R937Q show localization to the cell surface, while p.C234Y and p.P810L are localized to the perinuclear region. p.C664S shows both intense perinuclear staining as well as cell surface staining, corresponding to the two species of protein observed following EndoH treatment.

Figure 3

Notch signaling analysis. We used a firefly luciferase reporter construct with 4 Notch sensitive CBF binding sites upstream of the luciferase gene. RLU readings were normalized to an internal Renilla luciferase control. p.L37S and p.G274D were included as null and leaky controls, respectively. p.C664S and p.R937Q were able to activate Notch signaling to wild type levels. p.C234Y and p.P810L were unable to activate Notch signaling. Values reported are fold-change over the vector alone (pBabe) readings. Error bars represent standard error for technical triplicates.

Figure 4

A: The locations of the five sequence variants indentified in the JAG1 protein are shown, as is the domain structure of JAG1. Domains are labeled as such; SP, Signal Peptide; NT, N-Terminal Region; DSL, Delta-Serrate-Lag2 conserved region; EGF, Epidermal Growth Factor-like Repeat; CR, Cysteine Rich region; TM, Transmembrane region. B: We demonstrate the conservation of the 5 altered residues of JAG1 investigated in this study amongst a variety of species. In Drosophila, we compared human JAG1 to both the Serrate and Delta ligands.