Journal: The Journal of Experimental Medicine

Article Title: Somatic SMAD3 -activating mutations cause melorheostosis by up-regulating the TGF-β/SMAD pathway

doi: 10.1084/jem.20191499

Figure Lengend Snippet: Clinical findings in SMAD3 mutation–positive melorheostosis. (A and B) Radiographs of the (A) humerus and (B) forearm of patient Melo-17 with melorheostosis of the right upper extremity. Note increased density and intramedullary sclerosis extending across joints into lunate and capitate bones following an endosteal pattern without dripping candle wax or heterotopic bone. (C) Clinical appearance of patient Melo-17 with melorheostosis. Note that there is no visible thickening of the affected extremity and normal-appearing skin. The affected bone was found to harbor a SMAD3 mutation, not found in paired unaffected bone from the contralateral extremity. (D) Maximum intensity projection 18 F-NaF positron emission tomography image of the skull and torso showing abnormally increased metabolic activity in the right scapula, humeral head, radius, and hand. Red arrows show areas of increased tracer uptake on NaF bone scan.

Article Snippet: CS2-Flag- SMAD3 was a gift from Joan Massagué (Cancer Biology and Genetics Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY; Addgene plasmid #14052; ).

Techniques: Mutagenesis, Positron Emission Tomography, Activity Assay

Journal: The Journal of Experimental Medicine

Article Title: Somatic SMAD3 -activating mutations cause melorheostosis by up-regulating the TGF-β/SMAD pathway

doi: 10.1084/jem.20191499

Figure Lengend Snippet: Radiographs of three patients with SMAD3 mutation–positive melorheostosis demonstrating the endosteal appearance. (A) Left femur of patient Melo-12. (B) First metatarsal, left foot of patient Melo-8. (C) Left tibia of patient Melo-11.

Article Snippet: CS2-Flag- SMAD3 was a gift from Joan Massagué (Cancer Biology and Genetics Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY; Addgene plasmid #14052; ).

Techniques: Mutagenesis

Journal: The Journal of Experimental Medicine

Article Title: Somatic SMAD3 -activating mutations cause melorheostosis by up-regulating the TGF-β/SMAD pathway

doi: 10.1084/jem.20191499

Figure Lengend Snippet: VAFs of SMAD3 mutations

Article Snippet: CS2-Flag- SMAD3 was a gift from Joan Massagué (Cancer Biology and Genetics Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY; Addgene plasmid #14052; ).

Techniques: Mutagenesis, Amplification

Journal: The Journal of Experimental Medicine

Article Title: Somatic SMAD3 -activating mutations cause melorheostosis by up-regulating the TGF-β/SMAD pathway

doi: 10.1084/jem.20191499

Figure Lengend Snippet: Identification of SMAD3 somatic mutations in four individuals with melorheostosis. (A) Flow chart illustrating genetic analysis strategy leading to identification of SMAD3 somatic mutations in the affected bone of melorheostosis patients. (B) Quantification of mutant allele abundance by ddPCR in unaffected and affected bone biopsies (left), as well as in cultured osteoblasts (passage 1) from the respective biopsies (right), from patient Melo-12 identified by WES to have a SMAD3 p.S264Y mutation in affected bone. Each dot in the 2D fluorescence amplitude plots represents a droplet, with blue being mutant positive, green being wild-type positive, orange being positive for both, and black being negative for both. Color-matched numbers correspond to count of droplets per quadrant. The boxed number (VAF) is the calculated fractional abundance of mutant allele in each sample. Magenta lines are thresholds of negative vs. positive populations.

Article Snippet: CS2-Flag- SMAD3 was a gift from Joan Massagué (Cancer Biology and Genetics Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY; Addgene plasmid #14052; ).

Techniques: Mutagenesis, Cell Culture, Fluorescence

Journal: The Journal of Experimental Medicine

Article Title: Somatic SMAD3 -activating mutations cause melorheostosis by up-regulating the TGF-β/SMAD pathway

doi: 10.1084/jem.20191499

Figure Lengend Snippet: Identification of SMAD3 somatic mutations in melorheostosis . (A) Targeted sequencing of Melo-17 ( SMAD3 p.S264F). (B) Quantification of mutant allele abundance by ddPCR. Genomic DNAs isolated from bone biopsies and cultured cells from Melo-11 ( SMAD3 p.S264Y) were subjected to ddPCR, and VAF was determined. Each dot represents a droplet, with blue being mutant positive, green being wild-type positive, orange being positive for both, and black being negative for both. Magenta lines are thresholds of negative vs. positive populations. (C) Schematic representation of human SMAD3 protein structure. Boxes represent MH1 domain at the N-terminus and MH2 domain at the C-terminus, connected by linker region of SMAD3 protein. On each side of the schematic are the previously reported SMAD3 missense mutations in LDS and CRC. Comprehensive lists of the SMAD3 mutations can be found in and . The SMAD3 mutations identified in this melorheostosis study (MELO) are marked in red. Positions of the mutations are not to scale. (D) SMAD3 protein sequence alignment among the indicated species ( http://www.mutationtaster.org ) around the sites of the missense mutations (p.S264Y or p.S264F; highlighted in red).

Article Snippet: CS2-Flag- SMAD3 was a gift from Joan Massagué (Cancer Biology and Genetics Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY; Addgene plasmid #14052; ).

Techniques: Sequencing, Mutagenesis, Isolation, Cell Culture

Journal: The Journal of Experimental Medicine

Article Title: Somatic SMAD3 -activating mutations cause melorheostosis by up-regulating the TGF-β/SMAD pathway

doi: 10.1084/jem.20191499

Figure Lengend Snippet: Endosteal melorheostosis caused by gain-of-function SMAD3 mutations. (A) SMAD3 phosphorylation upon TGF-β stimulation with or without TGFβRI inhibitor (SB431542) pretreatment was assessed by Western blotting (Melo-11, SMAD3 p.S264Y). Total SMAD2/3 and histone H3 were internal controls. After quantification and normalization against histone H3, band intensities of phos-SMAD3 relative to basal control (lane 1) were presented in the table below the images. (B) TGF-β pathway target gene expression examined by real-time qPCR (Melo-17, SMAD3 p.S264F; n = 3; *, P < 0.05; Student's t test). (C) Mutant SMAD3 (p.S264Y and p.S264F) and wild-type SMAD3 (all Flag-tagged) were overexpressed in MC3T3-E1, and SMAD3 phosphorylation was examined by Western blotting. Histone H3 was used as internal control. After quantification and normalization against histone H3, band intensities of phos-SMAD3 and FLAG-SMAD3 relative to basal control (lane 1) were presented in the table below the images. (D) SMAD3 p.S264Y and wild-type SMAD3 were overexpressed in MC3T3-E1, and nuclear localization was detected by immunofluorescence and quantitated. Scale bars = 100 µm ( n = 4; *, P < 0.05; two-way ANOVA). (E) SMAD3 mutants (p.S264Y and p.S264F) and wild-type SMAD3 were overexpressed in MC3T3-E1, and transcriptional response was analyzed by real-time qPCR ( n = 3; *, P < 0.05; Student's t test). Error bars: SD of replicates. (F) Primary cells isolated from affected and unaffected bone of melorheostosis patient Melo-11 ( SMAD3 p.S264Y, VAF 41%) were subjected to live-cell imaging to measure cell proliferation rate ( n = 8; *, P < 0.05; two-way ANOVA). (G) Cell proliferation rate between affected and unaffected cells from a melorheostosis patient with relatively low VAF (Melo-12, SMAD3 p.S264Y, VAF 1.5%) was comparable ( n = 8; n.s., not significant; two-way ANOVA). Presented are representative data from at least three independent experiments. Aff, affected; Exp, expression; Unaff, unaffected.

Article Snippet: CS2-Flag- SMAD3 was a gift from Joan Massagué (Cancer Biology and Genetics Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY; Addgene plasmid #14052; ).

Techniques: Phospho-proteomics, Western Blot, Control, Targeted Gene Expression, Mutagenesis, Immunofluorescence, Isolation, Live Cell Imaging, Expressing

Journal: The Journal of Experimental Medicine

Article Title: Somatic SMAD3 -activating mutations cause melorheostosis by up-regulating the TGF-β/SMAD pathway

doi: 10.1084/jem.20191499

Figure Lengend Snippet: Effects of SMAD3 mutations on TGF-β signaling. (A and B) Effect of the mutant SMAD3 with low VAF on TGF-β signaling activation. SMAD3 phosphorylation in affected cells of melorheostotic bone in response to TGF-β stimulation was comparable to that in unaffected cells. (A) Melo-12 ( SMAD3 p.S264Y, VAF 1.5%). (B) Melo-17 ( SMAD3 p.S264F, VAF 1.2%). Total SMAD2/3 and Histone H3 were probed as internal controls. (C) SMAD3 p.S264Y resistant to TGF-β receptor kinase inhibitor. SMAD3 p.S264Y and wild-type SMAD3 were overexpressed in MC3T3-E1 cells. Cells were stimulated with recombinant TGF-β1 with or without pretreatment of TGF-β I receptor inhibitor, SB431542. Western blotting showed the increased SMAD3 phosphorylation in SMAD3 (p.S264Y)–expressing cells compared with wild-type SMAD3–expressing cells. SB431542 significantly decreased phos-SMAD3 in wild-type SMAD3, but not in SMAD3 p.S264Y. GAPDH was used as internal control. (D) Increased SMAD3 phosphorylation preserved in SMAD3 p.S264A. The SMAD3 mutants (p.S264Y or p.S264A) and wild-type SMAD3 were overexpressed in MC3T3-E1 cells. Cells were stimulated with recombinant TGF-β1 for the indicated time. Western blotting showed increased SMAD3 phosphorylation in wild-type SMAD3 over time in response to TGF-β1 stimulation. However, SMAD3 p.S264Y– and SMAD3 p.S264A–expressing cells displayed constitutive activation of SMAD3, even in the absence of TGF-β1 stimulation. β-actin was used as internal control. Data are representative of at least two independent experiments.

Article Snippet: CS2-Flag- SMAD3 was a gift from Joan Massagué (Cancer Biology and Genetics Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY; Addgene plasmid #14052; ).

Techniques: Mutagenesis, Activation Assay, Phospho-proteomics, Recombinant, Western Blot, Expressing, Control

Journal: The Journal of Experimental Medicine

Article Title: Somatic SMAD3 -activating mutations cause melorheostosis by up-regulating the TGF-β/SMAD pathway

doi: 10.1084/jem.20191499

Figure Lengend Snippet: Osteogenesis stimulation by SMAD3 gain-of-function mutation in affected cells. (A) Alizarin Red staining of ECM mineralization in osteoblasts (Melo-11, SMAD3 p.S264Y). After 6 wk in osteogenic media containing L-ascorbate, β-glycerophosphate, and dexamethasone, significantly increased mineralization was observed in affected compared with unaffected cells. Scale bar = 10 mm. (B) Real-time qPCR of osteogenic marker expression in osteoblasts ( n = 3; *, P < 0.05; Student's t test). Error bars indicate the SD of replicates. (C) MA plot of DEGs from RNA-Seq (Melo-11, SMAD3 p.S264Y). Red dots represent significantly up- or down-regulated DEGs (FDR < 1e-20). (D) GSEA plot for skeletal genes, with black bars indicating genes ranked by log 2 -fold change. (E) Cluster 2 from degPatterns analysis of RNA-Seq data. Genes in cluster 2 showed relatively higher expression in affected cells (red dots) than in unaffected cells (blue dots) throughout the time points (week 0, week 1, and week 2). (F) Functional and pathway enrichment analysis of cluster 2 (585 genes) with Metascape. Enriched pathway terms depicted as an interaction network and listed according to their P values (<0.01). Circle nodes represent enriched functions for an annotated ontology term, and the circle size indicates the number of genes that fall into that term. Presented are representative data from at least two independent experiments. Aff, affected; Exp, expression; Unaff, unaffected.

Article Snippet: CS2-Flag- SMAD3 was a gift from Joan Massagué (Cancer Biology and Genetics Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY; Addgene plasmid #14052; ).

Techniques: Mutagenesis, Staining, Marker, Expressing, RNA Sequencing, Functional Assay

Journal: The Journal of Experimental Medicine

Article Title: Somatic SMAD3 -activating mutations cause melorheostosis by up-regulating the TGF-β/SMAD pathway

doi: 10.1084/jem.20191499

Figure Lengend Snippet: Transcriptome profiling by RNA-Seq. (A–C) BMP2(−) condition. (A) A bar graph shows total number of DEGs with more than twofold changes (FDR < 1e-20) at 0-, 1-, and 2-wk time points in conventional osteogenic media without recombinant BMP2. (B) Clusters from degPatterns analysis of RNA-Seq data. The number of member genes in each cluster is also shown. (C) Cell growth inhibition by SMAD3 gain-of-function mutation. Heatmap of gene sets in eIF2 pathway displays significant down-regulation of ribosomal protein components (RPS [ribosomal protein S] and RPL (ribosomal protein L]) in affected cells. (D and E) BMP2(+) condition. (D) A bar graph shows total number of DEGs with more than twofold changes (FDR < 1e-20) at 0-, 1-, and 2-wk time points in conventional osteogenic media supplemented with recombinant BMP2. (E) 9 clusters from degPatterns analysis of RNA-Seq data. The number of member genes in each cluster is also shown. Aff, affected; Unaff, unaffected.

Article Snippet: CS2-Flag- SMAD3 was a gift from Joan Massagué (Cancer Biology and Genetics Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY; Addgene plasmid #14052; ).

Techniques: RNA Sequencing, Recombinant, Inhibition, Mutagenesis

Journal: The Journal of Experimental Medicine

Article Title: Somatic SMAD3 -activating mutations cause melorheostosis by up-regulating the TGF-β/SMAD pathway

doi: 10.1084/jem.20191499

Figure Lengend Snippet: BMP2-stimulated osteogenesis dampened by SMAD3 -activating mutation. (A) Alizarin Red staining of mineralization in osteoblasts (Melo-11, SMAD3 p.S264Y). After 6 wk in osteogenic media (L-ascorbate, β-glycerophosphate, and dexamethasone) supplemented with BMP2, significantly decreased mineralization was observed in affected compared with unaffected cells. Scale bar = 10 mm. (B) Real-time qPCR of osteogenic marker expression in osteoblasts undergoing differentiation in the presence of BMP2 ( n = 3; *, P < 0.05, Student's t test). Error bars indicate the SD of replicates. (C) MA plot of DEGs from RNA-Seq (Melo-11, SMAD3 p.S264Y). Genes in red dots are significantly up- or down-regulated (FDR < 1e-20). (D) GSEA plot for skeletal genes, with black bars indicating genes ranked by log 2 -fold change. (E) Cluster 1 from degPatterns analysis of RNA-Seq data (affected shown in red dots and unaffected in blue dots; time points: week 0, week 1, and week 2). (F) Functional and pathway enrichment analysis of cluster 1 (404 genes). Enriched pathway terms depicted as an interaction network and listed according to their P values (<0.01). Circle nodes represent enriched functions for an annotated ontology term, and the circle size indicates the number of genes that fall into that term. Presented are representative data from at least two independent experiments. Aff, affected; Exp, expression; Unaff, unaffected.

Article Snippet: CS2-Flag- SMAD3 was a gift from Joan Massagué (Cancer Biology and Genetics Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY; Addgene plasmid #14052; ).

Techniques: Mutagenesis, Staining, Marker, Expressing, RNA Sequencing, Functional Assay

Journal: The Journal of Experimental Medicine

Article Title: Somatic SMAD3 -activating mutations cause melorheostosis by up-regulating the TGF-β/SMAD pathway

doi: 10.1084/jem.20191499

Figure Lengend Snippet: Graphical summaries. (A) Mosaic-activating SMAD3 mutation accelerates osteogenesis in affected cells cultured in conventional osteogenic media without BMP2. (B) Osteogenesis-stimulating effect of BMP2 is dampened by the mosaic-activating SMAD3 mutation in affected cells that were cultured in osteogenic media supplemented with BMP2. Cell growth–associated gene expression is significantly down-regulated by the gain-of-function SMAD3 mutation, regardless of BMP2 presence. P in red circle denotes phosphorylation on TGF-β receptors, BMP2 receptors, and C-terminal SxxS motif of SMAD3 protein. Thickness of arrows indicates levels of gene expression in response to signaling via TGF-β/SMAD and BMP2/SMAD pathways. Thicker arrows indicate higher gene expression.

Article Snippet: CS2-Flag- SMAD3 was a gift from Joan Massagué (Cancer Biology and Genetics Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY; Addgene plasmid #14052; ).

Techniques: Mutagenesis, Cell Culture, Gene Expression, Phospho-proteomics