Abstract The understanding of where exactly, and how differently, DNA replication starts and ends in the cancer genomes remain limited. Although DNA sequences that underwent somatic alterations across human primary cancers have been comprehensively studied, the entire accurately replicated sequences of the same primary cancer genomes have not been widely explored. Here, we present a novel in silico framework to assess the tumour replication timing (RT) programme directly using 256 primary cancer whole genomes across three tumour types. We introduce a novel bootstrap-based replication fork directionality (RFD) method to model the bi-directional replication, and simultaneously map the replication origin and terminus domains at 1 kb resolution. Unexpectedly, our high-resolution results suggest that the genome-wide distribution of termination zones (TZs) is tightly coordinated with the initiation zones (IZs) in both normal and cancer genomes, which has not been previously reported using in vitro directional sequencing of Okazaki fragments (OK-seq) method. Overall, our data demonstrate that the cell type specificity of the tumour replication timing domains is preserved in closely related normal tissues and in lineage-specific cancer cell lines. Finally, comparing altered replication initiations between normal and cancer genomes, our data identifies delocalisation of replication initiations near highly expressed proliferation-associated PIF1 gene in small cell lung cancer (SCLC) genome, suggesting a potential cancer-specific target.
The extracellular matrix (ECM) provides structural support for tissue architecture and is a major effector of cell behavior during skin repair and inflammation. Macrophages are involved in all stages of skin repair but only limited knowledge exists about macrophage-specific expression and regulation of ECM components. In this study, we used transcriptome profiling and bioinformatic analysis to define the unique expression of ECM-associated genes in cultured macrophages. Characterization of the matrisome revealed that most genes were constitutively expressed and that several genes were uniquely regulated upon interferon gamma (IFNγ) and dexamethasone stimulation. Among those core matrisome and matrisome-associated components transforming growth factor beta (TGFβ)-induced, matrix metalloproteinase 9 (MMP9), elastin microfibril interfacer (EMILIN)-1, netrin-1 and gliomedin were also present within the wound bed at time points that are characterized by profound macrophage infiltration. Hence, macrophages are a source of ECM components in vitro as well as during skin wound healing, and identification of these matrisome components is a first step to understand the role and therapeutic value of ECM components in macrophages and during wound healing.
Zusammenfassung Die Osteogenesis imperfecta (OI) ist eine angeborene Erkrankung des Knochens und Bindegewebes. Sie geht mit einer erhöhten Frakturneigung, Deformierung der Extremität, aber auch mit extraskelettalen Symptomen einher. Nach einer kurzen Darstellung von Klinik, Diagnostik und aktueller Therapie folgt ein umfassender Überblick über die genetischen und pathophysiologischen Grundlagen der Erkrankung und die daraus abgeleiteten zukünftigen therapeutischen Möglichkeiten. Ungefähr 80 % der Patienten haben eine Mutation in den Kollagen-Genen COL1A1 und COL1A2 . Bei diesen Patienten ist für das Kollektiv keine klare Genotyp-Phänotyp-Korrelation beschrieben. Stoppmutationen führen in der Regel zu einem quantitativen Kollagendefekt, wodurch weniger normales Kollagen gebildet wird und ein eher leichter Phänotyp entsteht. Missense-Mutationen führen zu strukturell verändertem Kollagen (qualitativer Defekt) und zu einem schwereren Phänotyp. Trotzdem gibt es Unterschiede und Vorhersagen über den individuellen Verlauf sind nur sehr eingeschränkt möglich. Neben Veränderungen in den Kollagen-Genen gibt es Mutationen, welche die Kollagenmodifikation und die Kollagensekretion betreffen. Eine eigene Gruppe bilden Gene, welche an der Osteoblastendifferenzierung beteiligt sind. Wie auch bei den weiteren, nicht näher zugeordneten Genen sind dies häufig übergeordnete Gene, deren Funktion in der Osteogenese nicht völlig verstanden ist. Abgeleitet aus den pathophysiologischen Grundlagen, können vorhandene Medikamente zukünftig womöglich zielgerichtet eingesetzt werden. So ist der „Receptor-Activator-of-Nuclear-Factor-Kappa B-Ligand“ (RANKL)-Antikörper Denosumab spezifischer als Bisphosphonate und wird schon heute bei OI-Typ VI ( SERPINF1 ) verwendet. Weitere Medikamente wie Anti-Sklerostin oder Stammzelltherapien werden unter Berücksichtigung der Pathophysiologie aktuell entwickelt.
Wound healing is a coordinated process to restore tissue homeostasis and reestablish the protective barrier of the skin. miRNAs may modulate the expression of target genes to contribute to repair processes, but due to the complexity of the tissue it is challenging to quantify gene expression during the distinct phases of wound repair. Here, we aimed to identify a common reference gene to quantify changes in miRNA and mRNA expression during skin wound healing.Quantitative real-time PCR and bioinformatic analysis tools were used to identify suitable reference genes during skin repair and their reliability was tested by studying the expression of mRNAs and miRNAs.Morphological assessment of wounds showed that the injury model recapitulates the distinct phases of skin repair. Non-degraded RNA could be isolated from skin and wounds and used to study the expression of non-coding small nuclear RNAs during wound healing. Among those, RNU6B was most constantly expressed during skin repair. Using this reference gene we could confirm the transient upregulation of IL-1β and PTPRC/CD45 during the early phase as well as the increased expression of collagen type I at later stages of repair and validate the differential expression of miR-204, miR-205, and miR-31 in skin wounds. In contrast to Gapdh the normalization to multiple reference genes gave a similar outcome.RNU6B is an accurate alternative normalizer to quantify mRNA and miRNA expression during the distinct phases of skin wound healing when analysis of multiple reference genes is not feasible.
Cartilage originates from mesenchymal cell condensations that differentiate into chondrocytes of transient growth plate cartilage or permanent cartilage of the articular joint surface and trachea. MicroRNAs fine-tune the activation of entire signaling networks and thereby modulate complex cellular responses, but so far only limited data are available on miRNAs that regulate cartilage development. Here we characterize a miRNA which promotes the biosynthesis of a key component in the RAF/MEK/ERK pathway in cartilage. Specifically, by transcriptome profiling we identified miR-322 to be upregulated during chondrocyte differentiation. Among the various miR-322 target genes in the RAF/MEK/ERK pathway only Mek1 was identified as a regulated target in chondrocytes. Surprisingly, an increased concentration of miR-322 stabilizes Mek1-mRNA to rise protein levels and dampen ERK1/2 phosphorylation, while cartilage-specific inactivation in mice linked the loss of miR-322 to decreased MEK1 levels and increased RAF/MEK/ERK pathway activation. Such mice died perinatally due to tracheal growth restriction and respiratory failure. Hence, a single miRNA can stimulate the production of an inhibitory component of a central signaling pathway to impair cartilage development.
Correspondence9 June 2023Open Access Source DataTransparent process TAPT1—at the crossroads of extracellular matrix and signaling in Osteogenesis imperfecta Julia Etich Corresponding Author Julia Etich [email protected] orcid.org/0000-0003-3238-6692 Dr. Rolf M. Schwiete Research Unit for Osteoarthritis, Department of Orthopedics (Friedrichsheim), University Hospital Frankfurt, Goethe University Frankfurt/Main, Frankfurt, Germany Department of Pediatrics and Adolescent Medicine, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany Contribution: Conceptualization, Formal analysis, Supervision, Validation, Investigation, Visualization, Methodology, Writing - original draft, Writing - review & editing Search for more papers by this author Oliver Semler Oliver Semler orcid.org/0000-0003-0029-7556 Department of Pediatrics and Adolescent Medicine, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany Center for Rare Diseases, University Hospital Cologne, University of Cologne, Cologne, Germany Center for Family Health, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany Contribution: Conceptualization, Supervision, Funding acquisition, Writing - review & editing Search for more papers by this author Nicola L Stevenson Nicola L Stevenson orcid.org/0000-0001-8967-7277 Cell Biology Laboratories, School of Biochemistry, Faculty of Life Sciences, University Walk, University of Bristol, Bristol, UK Contribution: Formal analysis, Investigation, Writing - review & editing Search for more papers by this author Alice Stephan Alice Stephan orcid.org/0000-0001-7863-7982 Dr. Rolf M. Schwiete Research Unit for Osteoarthritis, Department of Orthopedics (Friedrichsheim), University Hospital Frankfurt, Goethe University Frankfurt/Main, Frankfurt, Germany Department of Pediatrics and Adolescent Medicine, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany Contribution: Formal analysis, Investigation, Writing - review & editing Search for more papers by this author Roberta Besio Roberta Besio orcid.org/0000-0003-1430-2934 Biochemistry Unit, Department of Molecular Medicine, University of Pavia, Pavia, Italy Contribution: Formal analysis, Investigation, Writing - review & editing Search for more papers by this author Nadia Garibaldi Nadia Garibaldi orcid.org/0000-0003-0353-537X Biochemistry Unit, Department of Molecular Medicine, University of Pavia, Pavia, Italy Department of Biomedical Engineering, The City College of New York, New York, NY, USA Contribution: Formal analysis, Investigation, Writing - review & editing Search for more papers by this author Nadine Reintjes Nadine Reintjes orcid.org/0000-0003-3504-7098 Institute of Human Genetics, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany Contribution: Formal analysis, Investigation, Writing - review & editing Search for more papers by this author Claudia Dafinger Claudia Dafinger Department of Pediatrics and Adolescent Medicine, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany Department II of Internal Medicine, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany Contribution: Formal analysis, Investigation, Writing - review & editing Search for more papers by this author Max Christoph Liebau Max Christoph Liebau orcid.org/0000-0003-0494-9080 Department of Pediatrics and Adolescent Medicine, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany Center for Rare Diseases, University Hospital Cologne, University of Cologne, Cologne, Germany Center for Family Health, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany Department II of Internal Medicine, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany Center for Molecular Medicine, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany Contribution: Formal analysis, Investigation, Writing - review & editing Search for more papers by this author Ulrich Baumann Ulrich Baumann Institute of Biochemistry, University of Cologne, Cologne, Germany Contribution: Formal analysis, Investigation, Writing - review & editing Search for more papers by this author Matthias Mörgelin Matthias Mörgelin Colzyx AB, Lund, Sweden Contribution: Formal analysis, Investigation, Writing - review & editing Search for more papers by this author Antonella Forlino Antonella Forlino orcid.org/0000-0002-6385-1182 Biochemistry Unit, Department of Molecular Medicine, University of Pavia, Pavia, Italy Contribution: Formal analysis, Supervision, Writing - review & editing Search for more papers by this author David J Stephens David J Stephens orcid.org/0000-0001-5297-3240 Cell Biology Laboratories, School of Biochemistry, Faculty of Life Sciences, University Walk, University of Bristol, Bristol, UK Contribution: Formal analysis, Supervision, Writing - review & editing Search for more papers by this author Christian Netzer Christian Netzer orcid.org/0000-0002-9416-0712 Center for Rare Diseases, University Hospital Cologne, University of Cologne, Cologne, Germany Institute of Human Genetics, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany Contribution: Formal analysis, Supervision, Writing - review & editing Search for more papers by this author Frank Zaucke Frank Zaucke orcid.org/0000-0002-7680-9354 Dr. Rolf M. Schwiete Research Unit for Osteoarthritis, Department of Orthopedics (Friedrichsheim), University Hospital Frankfurt, Goethe University Frankfurt/Main, Frankfurt, Germany Contribution: Conceptualization, Supervision, Funding acquisition, Writing - review & editing Search for more papers by this author Mirko Rehberg Corresponding Author Mirko Rehberg [email protected] orcid.org/0000-0001-9534-4102 Department of Pediatrics and Adolescent Medicine, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany Center for Family Health, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany Contribution: Conceptualization, Investigation, Visualization, Methodology, Writing - original draft, Writing - review & editing Search for more papers by this author Julia Etich Corresponding Author Julia Etich [email protected] orcid.org/0000-0003-3238-6692 Dr. Rolf M. Schwiete Research Unit for Osteoarthritis, Department of Orthopedics (Friedrichsheim), University Hospital Frankfurt, Goethe University Frankfurt/Main, Frankfurt, Germany Department of Pediatrics and Adolescent Medicine, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany Contribution: Conceptualization, Formal analysis, Supervision, Validation, Investigation, Visualization, Methodology, Writing - original draft, Writing - review & editing Search for more papers by this author Oliver Semler Oliver Semler orcid.org/0000-0003-0029-7556 Department of Pediatrics and Adolescent Medicine, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany Center for Rare Diseases, University Hospital Cologne, University of Cologne, Cologne, Germany Center for Family Health, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany Contribution: Conceptualization, Supervision, Funding acquisition, Writing - review & editing Search for more papers by this author Nicola L Stevenson Nicola L Stevenson orcid.org/0000-0001-8967-7277 Cell Biology Laboratories, School of Biochemistry, Faculty of Life Sciences, University Walk, University of Bristol, Bristol, UK Contribution: Formal analysis, Investigation, Writing - review & editing Search for more papers by this author Alice Stephan Alice Stephan orcid.org/0000-0001-7863-7982 Dr. Rolf M. Schwiete Research Unit for Osteoarthritis, Department of Orthopedics (Friedrichsheim), University Hospital Frankfurt, Goethe University Frankfurt/Main, Frankfurt, Germany Department of Pediatrics and Adolescent Medicine, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany Contribution: Formal analysis, Investigation, Writing - review & editing Search for more papers by this author Roberta Besio Roberta Besio orcid.org/0000-0003-1430-2934 Biochemistry Unit, Department of Molecular Medicine, University of Pavia, Pavia, Italy Contribution: Formal analysis, Investigation, Writing - review & editing Search for more papers by this author Nadia Garibaldi Nadia Garibaldi orcid.org/0000-0003-0353-537X Biochemistry Unit, Department of Molecular Medicine, University of Pavia, Pavia, Italy Department of Biomedical Engineering, The City College of New York, New York, NY, USA Contribution: Formal analysis, Investigation, Writing - review & editing Search for more papers by this author Nadine Reintjes Nadine Reintjes orcid.org/0000-0003-3504-7098 Institute of Human Genetics, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany Contribution: Formal analysis, Investigation, Writing - review & editing Search for more papers by this author Claudia Dafinger Claudia Dafinger Department of Pediatrics and Adolescent Medicine, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany Department II of Internal Medicine, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany Contribution: Formal analysis, Investigation, Writing - review & editing Search for more papers by this author Max Christoph Liebau Max Christoph Liebau orcid.org/0000-0003-0494-9080 Department of Pediatrics and Adolescent Medicine, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany Center for Rare Diseases, University Hospital Cologne, University of Cologne, Cologne, Germany Center for Family Health, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany Department II of Internal Medicine, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany Center for Molecular Medicine, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany Contribution: Formal analysis, Investigation, Writing - review & editing Search for more papers by this author Ulrich Baumann Ulrich Baumann Institute of Biochemistry, University of Cologne, Cologne, Germany Contribution: Formal analysis, Investigation, Writing - review & editing Search for more papers by this author Matthias Mörgelin Matthias Mörgelin Colzyx AB, Lund, Sweden Contribution: Formal analysis, Investigation, Writing - review & editing Search for more papers by this author Antonella Forlino Antonella Forlino orcid.org/0000-0002-6385-1182 Biochemistry Unit, Department of Molecular Medicine, University of Pavia, Pavia, Italy Contribution: Formal analysis, Supervision, Writing - review & editing Search for more papers by this author David J Stephens David J Stephens orcid.org/0000-0001-5297-3240 Cell Biology Laboratories, School of Biochemistry, Faculty of Life Sciences, University Walk, University of Bristol, Bristol, UK Contribution: Formal analysis, Supervision, Writing - review & editing Search for more papers by this author Christian Netzer Christian Netzer orcid.org/0000-0002-9416-0712 Center for Rare Diseases, University Hospital Cologne, University of Cologne, Cologne, Germany Institute of Human Genetics, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany Contribution: Formal analysis, Supervision, Writing - review & editing Search for more papers by this author Frank Zaucke Frank Zaucke orcid.org/0000-0002-7680-9354 Dr. Rolf M. Schwiete Research Unit for Osteoarthritis, Department of Orthopedics (Friedrichsheim), University Hospital Frankfurt, Goethe University Frankfurt/Main, Frankfurt, Germany Contribution: Conceptualization, Supervision, Funding acquisition, Writing - review & editing Search for more papers by this author Mirko Rehberg Corresponding Author Mirko Rehberg [email protected] orcid.org/0000-0001-9534-4102 Department of Pediatrics and Adolescent Medicine, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany Center for Family Health, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany Contribution: Conceptualization, Investigation, Visualization, Methodology, Writing - original draft, Writing - review & editing Search for more papers by this author Author Information Julia Etich *,1,2, Oliver Semler2,3,4, Nicola L Stevenson5, Alice Stephan1,2, Roberta Besio6, Nadia Garibaldi6,7, Nadine Reintjes8, Claudia Dafinger2,9, Max Christoph Liebau2,3,4,9,10, Ulrich Baumann11, Matthias Mörgelin12, Antonella Forlino6, David J Stephens5, Christian Netzer3,8, Frank Zaucke1 and Mirko Rehberg *,2,4 1Dr. Rolf M. Schwiete Research Unit for Osteoarthritis, Department of Orthopedics (Friedrichsheim), University Hospital Frankfurt, Goethe University Frankfurt/Main, Frankfurt, Germany 2Department of Pediatrics and Adolescent Medicine, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany 3Center for Rare Diseases, University Hospital Cologne, University of Cologne, Cologne, Germany 4Center for Family Health, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany 5Cell Biology Laboratories, School of Biochemistry, Faculty of Life Sciences, University Walk, University of Bristol, Bristol, UK 6Biochemistry Unit, Department of Molecular Medicine, University of Pavia, Pavia, Italy 7Department of Biomedical Engineering, The City College of New York, New York, NY, USA 8Institute of Human Genetics, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany 9Department II of Internal Medicine, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany 10Center for Molecular Medicine, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany 11Institute of Biochemistry, University of Cologne, Cologne, Germany 12Colzyx AB, Lund, Sweden *Corresponding author. Tel: +49 (0) 22147832743; E-mail: [email protected] *Corresponding author. Tel: +49 (0) 22147884755; E-mail: [email protected] EMBO Mol Med (2023)15:e17528https://doi.org/10.15252/emmm.202317528 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Osteogenesis imperfecta (OI) is a rare, hereditary connective tissue disorder, clinically characterized by bone fragility, bone deformities, and small stature that can be accompanied by variable extraskeletal symptoms. At the molecular level, OI is caused by a reduced quality and/or quantity of bone matrix. So far, 22 OI types are listed in the Online Mendelian Inheritance in Man (OMIM) database, and even though most of the encoded proteins have been associated with collagen type I structure and/or function, the underlying molecular mechanisms are rarely completely understood (OMIM, phenotypic series #166200). In a previous issue of this journal, a deep intronic variant in TAPT1 was reported to create a protein-null allele and segregated with recessive features of OI and neonatal progeria syndrome (Nabavizadeh et al, 2023). Using integrated analysis of RNA- and SI-NET-seq data, the authors revealed that extracellular matrix (ECM) organization and collagen-related pathways were highly dysregulated, supporting a role for TAPT1 in ECM and collagen dynamics. However, a specific function of the TAPT1 protein remains to be identified, and also the link to collagen type I remains elusive. In this correspondence, we provide further evidence based on our clinical, genetic, and cellular data to corroborate the role of TAPT1 as a novel OI-causing gene and highlight a novel disease-relevant link between OI, ECM, and signaling. Using gene panel sequencing, we identified the homozygous mutation (c.323T>G, p.Leu108Trp) in TAPT1 in a consanguine family (Fig 1A, upper panel). In an earlier study, mutations in TAPT1 were reported to result in a complex and early lethal osteochondrodysplasia disrupting ciliogenesis in patient cells and leading to an altered Golgi morphology and delayed collagen secretion (Symoens et al, 2015). Our patient, an 18-year-old woman at the time of manuscript submission, was clinically diagnosed with OI type III prior to the identification of the disease-causing gene. At the age of five (Fig 1A, middle panel), she had all typical features of classical OI, including multiple fractures, short stature (86 cm, −5.26 SDS), long bone deformities, reduced bone mineral density (0.193 g/cm2, −7.0 SDS, TBLH aBMD), progressive popcorn calcifications and progressive, and severe scoliosis (Fig 1A, lower panel). Remarkably, modeling of the identified mutation into the predicted 3D protein structure of TAPT1 indicated that a central network of salt bridges and hydrogen bonds between the amino acids Glu285, Asp306, Asp353, Lys356, His357, and Tyr371 on different surrounding helices is most likely being disrupted by the point mutation (c.323T>G, p.Leu108Trp), and a severe perturbation of the structural integrity is highly expected (Fig 1B upper panel). None of the tryptophan rotamers can be fitted at this position without severe clashes (Fig 1B lower panel). Prediction of the protein stability change upon mutation by multiple Cutoff Scanning Matrix method yielded an estimated loss of 2.1 kcal/mol in energy and the mutation is classified as "highly destabilizing." Figure 1. Consequences of p.Leu108Trp mutation in TAPT1 on disease phenotype and the underlying pathomechanism(A) Pedigree of the family with the affected individual (black symbol) carrying a homozygous TAPT1 mutation. Heterozygous carriers (half-shaded symbols) and consanguineous relationship (double line) are highlighted (upper panel). In the affected individual (II:3) with the clinical phenotype of OI (middle panel), dual-energy X-ray absorptiometry (DEXA) was used to assess areal bone mineral density (aBMD) and radiographs of tibia and hand/wrist show long bones with thin cortices (bone health index 3.04, −5.68 SDS) as well as disorganized calcifications around the growth plate (also called popcorn calcifications, lower panel). (B) Model for human TAPT1 protein and the c.323 T > G, p.Leu108Trp mutation were visualized employing ChimeraX and the AlphaFold 2 model of human TAPT1 (UniProt entry Q6NXT6). Leu108 is located within an N-terminal alpha helix protruding into the membrane bilayer and projects into a cavity surrounded by several transmembrane helices (upper panel). Glu285, Asp306, Asp353, Lys356, His357 and Tyr371 are located on several helices and are engaged in salt bridges or connected by hydrogen bonds. In-silico substitution of Leu108 by a tryptophane reveals severe clashes for all rotamers (lower panel). (C) Immunoblotting of TAPT1 protein in control and patient fibroblasts (upper panel) and quantification of band intensities by ImageJ analysis (lower panel) revealed slightly reduced TAPT1 levels in patient cells. Fold change difference relative to the mean of controls is plotted as individual values from n = 3 independent experiments. Statistical analysis: One way ANOVA; P-value = 0.06; (D) qPCR analysis of relative gene expression of the collagen genes COL1A1 (upper panel) and COL1A2 (lower panel) in control and patient fibroblasts confirmed that expression was not downregulated in patient cells. Gene expression was normalized to GAPDH, calibrated to the mean of controls and fold changes are plotted on a logarithmic scale form at least n = 5 independent experiments. Statistical analysis: One-way ANOVA with post hoc Bonferroni multiple comparison of patient to controls a, b or c; P-value [COL1A1] = 0.004, **c; P-value [COL1A2] = 0.007, **c. (E) Immunofluorescence analysis of collagen type I in control (upper panel) and patient fibroblasts (lower panel) detected reduced collagen network in patient cells. Representative pictures are shown from at least n = 3 independent experiments with three control cell populations each. Scale bar: 500 μm. (F) Negative staining and transmission electron microscopy of cell culture media from control and patient cells visualized impaired collagen fibril formation in patient cells. Higher magnifications of fibers are shown as inserts. Scale bars: 500 nm (overviews), 100 nm (inserts). (G, H) qPCR analysis of relative gene expression of SFRP1 in control and patient fibroblasts without stimulation (-SAG, G) or upon stimulation with 1 μM smoothened agonist (+SAG, H) for 24 h hours uncovered increased SFRP1 expression in patient cells under both conditions. Fold changes plotted on a logarithmic scale from n = 6 independent experiments are shown. Gene expression was normalized to GAPDH and calibrated to the mean of nonstimulated control cells from (G). Statistical analysis: One-way ANOVA with post hoc Bonferroni multiple comparison of patient to controls a, b or c; P-value [-SAG] = 0.0001, ***a, ***b, ***c; P-value [+SAG] < 0.0001, ***a, ***b, ***c. (I) ELISA of SFRP1 protein levels in the serum of eight healthy controls or two independent serum samples of the patient determined increased SFRP1 levels in patient serum. No statistical analysis could be performed on this dataset due to only two available patient values. Source data are available online for this figure. Source Data for Figure 1 [emmm202317528-sup-0002-SDataFig1.zip] Download figure Download PowerPoint To elucidate the underlying molecular pathomechanism, we used patient-derived fibroblasts. Informed consent was obtained from the parents and the underage patient and the experiments conformed to the principles set out in the WMA Declaration of Helsinki and the Department of Health and Human Services Belmont Report. We detected in vitro that the p.Leu108Trp variant of TAPT1 slightly reduced protein (Fig 1C) but not RNA levels (Appendix Fig S1A). Inhibition of the proteasomal degradation machinery with the inhibitor MG132 restored TAPT1 protein levels to the levels of control cells while TAPT1 levels remained reduced in patient cells when lysosomal degradation was inhibited by Bafilomycin A1 (Appendix Fig S1B). These results indicate that mutant TAPT1 protein is mostly degraded by the proteasome pathway and are in agreement with the in silico analysis of mutant protein structure. We tested whether the mutation-driven decrease in TAPT1 protein abundance will induce changes in collagen type I secretion and deposition generally associated with OI. Although we did not detect downregulated gene expression of either collagen genes, COL1A1 and COL1A2 (Fig 1D), we observed reduction in collagen deposition by immunofluorescence staining (Fig 1E). A slight delay of collagen secretion in patient cells was corroborated by pulse-chase secretion kinetics (Appendix Fig S1C) and quantification of secreted or deposited collagen in cell culture medium or cell layer under steady state conditions (Appendix Fig S1D). Importantly, when analyzing the patient's extracellular quality of collagen fibrils by electron microscopy, secreted collagen was not able to assemble into banded fibrils (Fig 1F). This impaired assembly creates most likely an unorganized collagen network, which might impair the spatial interaction and composition of the ECM in affected individuals. Nabavizadeh et al (2023) determined by integrated pathway enrichment analysis combining RNA-seq and SI-NET-seq that collagen- and ECM-related pathways were most significantly dysregulated. We now prove the ECM, and particularly collagen type I assembly, as a main target of TAPT1 mutations. Thus, our experimental data reinforce TAPT1-associated OI as a collagenopathy. We could detect some anomalies in Golgi morphology (Appendix Fig S2) but no defects in ciliogenesis (Appendix Fig S3A–E) that were previously linked to TAPT1 mutations (Symoens et al, 2015). Thus, we performed functional assays and analyzed the responsiveness of the cilia-associated hedgehog (HH) signaling pathway in smoothened agonist (SAG)-stimulated patient fibroblasts by target gene expression. While we could detect slightly increased induction of GLI1 and PTCH1 expression in patient cells (Appendix Fig S3F–G), secreted frizzled-related protein 1 (SFRP1) gene expression was hardly induced upon SAG stimulation. However, it was strongly upregulated in our patient compared with control cells even without stimulation by SAG (Fig 1G and H). Secreted frizzled-related protein 1 is a secreted glycoprotein that can suppress WNT/β-catenin signaling to regulate osteoblast differentiation and function during endochondral bone development (He et al, 2006). With its amino-terminal cysteine-rich domain SFRP1 antagonistically sequesters WNTs interfering with their binding to the Frizzled receptor to prevent β-catenin-mediated gene transcription, inhibit the pathway and reduce bone formation. Deletion of the SFRP1 gene in mice resulted in increased trabecular bone mineral density and upregulated osteoblast proliferation and differentiation by preferentially activating WNT signaling in osteoblasts (Bodine et al, 2004). Recently, as an attempt to use WNT pathway components as potential drug targets for treating bone diseases, it was shown that inhibition of SFRP1 led to increased total bone area in a murine calvarial organ culture assay (Bodine et al, 2009) and to an elevated bone synthesis activity in ovariectomy-induced osteoporotic mice (García-García et al, 2022). Interestingly, osteoblasts were previously also shown to directly inhibit osteoclastogenesis through the expression, release, and binding of SFRP1 to RANKL (Häusler et al, 2004). The membrane-spanning, cilia-associated TAPT1 protein might be involved in the transcriptional regulation of SFRP1 gene expression. It is increasingly evident that a number of cilia-associated proteins have a nuclear presence and potentially nuclear roles (McClure-Begley & Klymkowsky, 2017). For example, dual roles were previously described for myocardin-related transcription factor and serum response factor that act both as transcription factors and as primary cilia constituents impacting ciliary protein–protein interactions (Speight et al, 2021). It is therefore conceivable that intact TAPT1 at the centrosome/ciliary base could interact with transcription-modulating proteins and regulate their nuclear translocation. Thus, we speculate that mutant TAPT1 protein due to reduced protein stability and diminished protein interactions lacks the ability to regulate SFRP1 silencing and consequently SFRP1 gene expression is induced impairing bone remodeling. Bone formation (e.g., alkaline phosphatase) and bone resorption markers (e.g., deoxypyridinoline) were on the lower end within the age-adjusted reference range in our patient (Table 1). Moreover, low bone health index with almost normal bone length in the palm but extremely thin cortical thickness indicated that the appositional bone growth mediated by the concerted interplay of bone-forming osteoblasts and bone-resorbing osteoclasts might indeed be a causative factor in the pathology of our patient. The importance of WNT1 in regulating remodeling-based bone formation, cortical thickness, and appositional bone growth has been demonstrated earlier in OI-related models (Vollersen et al, 2021; Wang et al, 2021). Strikingly, several clinical features in our patient overlap with those of OI type XV caused by mutations in WNT1, such as reduced cortical bone thickness, normal longitudinal bone growth, low bone turnover, progressive popcorn calcifications, and progressive scoliosis (Pyott et al, 2013; Liu et al, 2017; Nampoothiri et al, 2019). It is therefore possible that the clinical bone phenotype in our patient is at least in part caused by deregulated WNT signaling even though some extraskeletal symptoms do not completely overlap. Currently, we do not understand how exactly TAPT1 regulates SFRP1 gene expression but comparison of sera from healthy donors with two independent serum samples of our patient revealed an increase in SFRP1 protein levels (Fig 1I), which underscores the possibility of persistent WNT signaling malfunction in vivo. This newly elucidated pathomechanism paves the way for new therapeutic approaches like the use of osteoanabolic treatments in OI (Rauch, 2017). Moreover, increased serum levels of SFRP1 might represent a potentially new and easily accessible diagnostic marker for TAPT1-related OI. Table 1. Clinical parameters of the patient over a period of 12 years. Age of patient in years 5 8 12 17 Height (cm) 86 91 101 110 Height SDS −5.26 −6.39 −7.08 −8.72 Weight (kg) 9.0 10.3 13.1 18.8 Weight SDS −6.92 −6.39 −7.97 −11.58 BMI (kg/m2) 12.2 12.4 12.7 15.5 BMI SDS −2.69 −2.52 −3.28 −2.75 TBLH-aBMD (g/cm2) – 0.352 0.428 0.610 TBLH-aBMD aSDS (age adjusted) – −4.4 −4.2 −3.9 LS-aBMD (g/cm2) 0.193 Not followed up due to skoliosis LS-aBMD aSDS (age adjusted) −7.000 BAMF (range 1–10) – 4 4 4 GMFM88 (%) – 46.0 49.8 44.5 Spine morphology severity classification (range 1–5) – 5.0 – – Spine morphology severity Score (range 1–138) – 78.0 – – Nephrocalcinosis No No No No Alkaline phosphatase (U/l) 161 (↔) 136 (↔) 136 (↔) 136 (↔) N-terminal procollagen type 1 propeptide (μg/l) 152 (↔) – – – Osteocalcin (ng/ml) – – – 68.2 (↑*) Deoxypyridinolin/creatinine in urine (μg/g) – – – 90 (↔) Cross-linked carboxy-terminal telopeptide of type I collagen (CTX) (ng/ml) – – – 0.713 (↔) Calcium/creatinine in urine (g/g) – – – 0.05 (↔) Parathyroid hormone (ng/l) 11.0 (↓) 37.0 (↔) 35.0 (↔) 35.6 (↔) Calcium in serum (mmol/l) 2.68 (↑) 2.38 (↔) 2.49 (↔) 2.48 (↔) Vitamin D (μg/l) 19.2 (↓) 12.9 (↓) 41.1 (↔) 34.1 (↔) Insulin-like growth factor I (IGF-I) (μg/l) – – – 340 (↔) Reference values according to local laboratory or most suitable ones in literature; age adjusted if available: alkaline phosphatase: 5 years of age: < 269 U/l, others: < 300 U/l; N-terminal procollagen type 1 propeptide: 49.9–1,200 μg/l (age adjusted); osteocalcin (*not age adjusted, premenopausal women >20): 11–43 ng/ml; deoxypyridinolin/creatinine (urine): 65–380 μg/g (adjusted to bone age); cross-linked carboxy-terminal telopeptide of type I collagen (CTX): 0.146–0.818 ng/ml; calcium/creatinine (urine) in g/g: < 0.21 g/g; parathyroid hormone: 15–65 ng/l; calcium (serum): until 2020: 2.20–2.65 mmol/l, from 2021: 2.10–2.55 mmol/l; vitamin D: 30–70 μg/l; Insulin-like growth factor I (IGF-I): 190–429 μg/l. Abbreviations are as follows: aBMD, areal bone mineral density; aSDS, age adjusted SDS; BAMF, brief assessment of motor function score; BMI, body mass index; cm, centimeters; g, gram; GMFM88, gross motor function measure 88; kg, kilogram; LS-aBMD, lumbar spine areal bone mineral density (L2–L4); m2, square meter; SDS, standard deviation score; TBLH, total body less head. The first fetuses harboring TAPT1 mutations were initially diagnosed with lethal features of OI (Symoens et al, 2015). Other patients with a combination of bone fragility and variable additional symptoms have been described since then, expanding the vast phenotypic spectrum of TAPT1 insufficiency (Jarayseh et al, 2023; Nabavizadeh et al, 2023). The impact of the identified mutations ranges from partial to complete loss-of-function most likely causing the phenotypic variability from early lethal to survivable disease manifestation. The reported phenotypes associating with poor mineralization (Symoens et al, 2015), skeletal osteopenia (Jarayseh et al, 2023), and calcification defects (Nabavizadeh et al, 2023) are similar to those seen in our patient. In summary, we provide a first mechanistic indication that SFRP1 gene expression is induced in patient cells harboring the c.323T>G TAPT1 mutation and potentially other mutations affecting TAPT1 protein levels and/or stability to presumably deregulate bone remodeling. Thus, we propose that mutations in TAPT1 can cause OI of variable severity through impaired collagen fibril formation and deregulation of bone turnover. Acknowledgments This work was supported by grants from the German Science Foundation (Deutsche Forschungsgemeinschaft, DFG) to the Research Unit FOR2722 [grant number 407168728 (SE2373/1-1 to OS, ZA561/3-1 to FZ) and 384170921 (ET144/3-2 to JE, SE2373/1-2 to OS, ZA561/3-2 to FZ)] and from UKRI-BBSRC (BB/T001984/1 to DJS). We are grateful to the staff in the BioEM Lab, Biozentrum, University of Basel, and the Core Facility for Integrated Microscopy (CFIM), Panum Institute, University of Copenhagen, for providing highly innovative environments for electron microscopy. We thank Carola Alampi (BioEM lab), Mohamed Chami (BioEM lab) and Klaus Qvortrup (CFIM) for practical help with electron microscopy. Author contributions Julia Etich: Conceptualization; formal analysis; supervision; validation; investigation; visualization; methodology; writing – original draft; writing – review and editing. Jörg Oliver Semler: Conceptualization; supervision; funding acquisition; writing – review and editing. Nicola Stevenson: Formal analysis; investigation; writing – review and editing. Alice Stephan: Formal analysis; investigation; writing – review and editing. Roberta Besio: Formal analysis; investigation; writing – review and editing. Nadia Garibaldi: Formal analysis; investigation; writing – review and editing. Nadine Reintjes: Formal analysis; investigation; writing – review and editing. Claudia Dafinger: Formal analysis; investigation; writing – review and editing. Max Christoph Liebau: Formal analysis; investigation; writing – review and editing. Ulrich Baumann: Formal analysis; investigation; writing – review and editing. Matthias Mörgelin: Formal analysis; investigation; writing – review and editing. Antonella Forlino: Formal analysis; supervision; writing – review and editing. David John Stephens: Formal analysis; supervision; writing – review and editing. Christian Netzer: Formal analysis; supervision; writing – review and editing. Frank Zaucke: Conceptualization; supervision; funding acquisition; writing – review and editing. Mirko Rehberg: Conceptualization; investigation; visualization; methodology; writing – original draft; writing – review and editing. Disclosure and competing interests statement The authors declare that they have no conflict of interest. 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Combining behavioral, computational and a novel high‐content tailored imaging methodology, the authors identified small molecules restoring the physiological and cellular deficits in GAN. This works identifies the first therapeutic candidates to treat this fatale and incurable disease that can be impactful to various diseases of the neuromuscular system. (Scientific image by Léa Lescouzères, University of Lyon 1, France, transformed with AdobePhotoshop) Volume 15Issue 710 July 2023In this issue FiguresReferencesRelatedDetailsLoading ...
Identification and clearance of apoptotic cells prevents the release of harmful cell contents thereby suppressing inflammation and autoimmune reactions. Highly conserved annexins may modulate the phagocytic cell removal by acting as bridging molecules to phosphatidylserine, a characteristic phagocytosis signal of dying cells. In this study five members of the structurally and functionally related annexin family were characterized for their capacity to interact with phosphatidylserine and dying cells. The results showed that AnxA3, AnxA4, AnxA13, and the already described interaction partner AnxA5 can bind to phosphatidylserine and apoptotic cells, whereas AnxA8 lacks this ability. Sequence alignment experiments located the essential amino residues for the recognition of surface exposed phosphatidylserine within the calcium binding motifs common to all annexins. These amino acid residues were missing in the evolutionary young AnxA8 and when they were reintroduced by site directed mutagenesis AnxA8 gains the capability to interact with phosphatidylserine containing liposomes and apoptotic cells. By defining the evolutionary conserved amino acid residues mediating phosphatidylserine binding of annexins we show that the recognition of dying cells represent a common feature of most annexins. Hence, the individual annexin repertoire bound to the cell surface of dying cells may fulfil opsonin-like function in cell death recognition.
