Fig 1.
Exome sequencing identifies GLI2 as candidate disease gene.
(A) Pedigree of the reported family. Main known phenotypes are mapped to individuals. Samples marked by a red star were available for Exome Sequencing. Proband is indicated with an arrow. (B) Bar plot representing prioritization scores obtained with Phenolyzer for genes identified from Exome Sequencing. GLI2 can be effectively associated with the clinical phenotype.
Fig 2.
GLI2 mutant p.P1167LfsX52 lacks transcriptional activity and inhibits Hedgehog-GLI signaling.
(A) Schematic of GLI2 wild-type and p.P1167LfsX52 mutant lacking the C-terminal region of the activation domain. Other functional motifs are intact, including the N-terminal repressor sequence, the zinc finger DNA-binding domain and nuclear localization signal. (B) Western blotting of total protein lysates of HEK293 cells transfected with plasmids expressing GFP-tagged wild-type human GLI2 (GLI2WT), p.P1167LfsX52 (GLI2MUT) or GFP (mock control) revealed with anti-GFP antibody. GADPH is a loading control. (C-F’) GFP-tagged GLI2WT or GLI2MUT visualized in transfected NIH-3T3 cells before or after stimulation with SHH (16nM) for 5 hrs. The GFP signal extracted from the corresponding merged images is show in C’-F’. Both proteins are found in the cytoplasm (arrowhead) and nucleus (arrow) in untreated cells and become primarily localized to the nucleus after stimulation. Scale bar, 10μm. (G, H) Levels of GLI1 (G) and PTCH1 (H) transcripts detected by quantitative-PCR in NIH-3T3 cells expressing GFP-tagged GLI2WT, GLI2MUT or GFP control, before and after treatment with SHH (16nM) for 40 hrs. All conditions are normalized to untreated control cells (mean ± SEM, n = 2). Unpaired t-test, (**) p<0.01 GLI2MUT vs. GLI2WT either untreated or SHH-treated matching conditions. (I) Luciferase-based reporter assay with GLI-responsive construct 8x-Gli-BS-Luc in NIH-3T3 cells transfected with GFP-tagged GLI2WT, GLI2MUT or GFP control, before and after treatment with SHH (16nM) for 30 hrs. The expression levels of the reporter gene are measured by luciferase activity. All conditions are normalized to untreated control (mean ± SEM, n = 2). Unpaired t-test (***), p<0.001 GLI2MUT vs. control either untreated or SHH-treated matching conditions. (NS, non-significant) p = 0.1264 GLI2MUT untreated vs. treated. (J) Luciferase-based assay with 8x-Gli-BS-Luc reporter in chick spinal cord progenitor cells expressing GFP-tagged GLI2WT, GLI2MUT or GFP control, treated with increasing doses of SHH for 24 hrs. All conditions are normalized to untreated control (mean ± SEM, n = 2–4). Unpaired t-test, (*) p = 0.0137 GLI2MUT vs. control, untreated; (***) p<0.001 GLI2MUT vs. control at corresponding SHH concentrations.