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1.
Am J Hum Genet ; 107(5): 989-999, 2020 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-33053334

RESUMO

Osteogenesis imperfecta (OI) is characterized primarily by susceptibility to fractures with or without bone deformation. OI is genetically heterogeneous: over 20 genetic causes are recognized. We identified bi-allelic pathogenic KDELR2 variants as a cause of OI in four families. KDELR2 encodes KDEL endoplasmic reticulum protein retention receptor 2, which recycles ER-resident proteins with a KDEL-like peptide from the cis-Golgi to the ER through COPI retrograde transport. Analysis of patient primary fibroblasts showed intracellular decrease of HSP47 and FKBP65 along with reduced procollagen type I in culture media. Electron microscopy identified an abnormal quality of secreted collagen fibrils with increased amount of HSP47 bound to monomeric and multimeric collagen molecules. Mapping the identified KDELR2 variants onto the crystal structure of G. gallus KDELR2 indicated that these lead to an inactive receptor resulting in impaired KDELR2-mediated Golgi-ER transport. Therefore, in KDELR2-deficient individuals, OI most likely occurs because of the inability of HSP47 to bind KDELR2 and dissociate from collagen type I. Instead, HSP47 remains bound to collagen molecules extracellularly, disrupting fiber formation. This highlights the importance of intracellular recycling of ER-resident molecular chaperones for collagen type I and bone metabolism and a crucial role of HSP47 in the KDELR2-associated pathogenic mechanism leading to OI.


Assuntos
Osso e Ossos/metabolismo , Colágeno Tipo I/metabolismo , Proteínas de Choque Térmico HSP47/metabolismo , Osteogênese Imperfeita/genética , Proteínas de Transporte Vesicular/metabolismo , Adulto , Alelos , Sequência de Aminoácidos , Animais , Sítios de Ligação , Osso e Ossos/patologia , Galinhas , Pré-Escolar , Colágeno Tipo I/química , Colágeno Tipo I/genética , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/patologia , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Expressão Gênica , Complexo de Golgi/metabolismo , Complexo de Golgi/patologia , Proteínas de Choque Térmico HSP47/química , Proteínas de Choque Térmico HSP47/genética , Humanos , Lactente , Masculino , Osteogênese Imperfeita/diagnóstico , Osteogênese Imperfeita/metabolismo , Osteogênese Imperfeita/patologia , Linhagem , Cultura Primária de Células , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Transporte Proteico , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/genética
2.
J Endovasc Ther ; 28(4): 604-613, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33902345

RESUMO

INTRODUCTION: Abdominal aortic aneurysms (AAAs) are associated with overall high mortality in case of rupture. Since the pathophysiology is unclear, no adequate pharmacological therapy exists. Smooth muscle cells (SMCs) dysfunction and extracellular matrix (ECM) degradation have been proposed as underlying causes. We investigated SMC spatial organization and SMC-ECM interactions in our novel 3-dimensional (3D) vascular model. We validated our model for future use by comparing it to existing 2-dimensional (2D) cell culture. Our model can be used for translational studies of SMC and their role in AAA pathophysiology. MATERIALS AND METHODS: SMC isolated from the medial layer of were the aortic wall of controls and AAA patients seeded on electrospun poly-lactide-co-glycolide scaffolds and cultured for 5 weeks, after which endothelial cells (EC) are added. Cell morphology, orientation, mechanical properties and ECM production were quantified for validation and comparison between controls and patients. RESULTS: We show that cultured SMC proliferate into multiple layers after 5 weeks in culture and produce ECM proteins, mimicking their behavior in the medial aortic layer. EC attach to multilayered SMC, mimicking layer interactions. The novel SMC model exhibits viscoelastic properties comparable to biological vessels; cytoskeletal organization increases during the 5 weeks in culture; increased cytoskeletal alignment and decreased ECM production indicate different organization of AAA patients' cells compared with control. CONCLUSION: We present a valuable preclinical model of AAA constructed with patient specific cells with applications in both translational research and therapeutic developments. We observed SMC spatial reorganization in a time course of 5 weeks in our robust, patient-specific model of SMC-EC organization and ECM production.


Assuntos
Aneurisma da Aorta Abdominal , Células Endoteliais , Matriz Extracelular , Humanos , Miócitos de Músculo Liso , Resultado do Tratamento
3.
Hum Mutat ; 39(5): 653-665, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29363216

RESUMO

We aimed to determine the diagnostic yield of a targeted-exome panel in a cohort of 74 Dutch primary ciliary dyskinesia (PCD) patients. The panel consisted of 26 PCD-related and 284 candidate genes. To prioritize PCD candidate genes, we investigated the transcriptome of human airway cells of 12 healthy volunteers during in vitro ciliogenesis and hypothesized that PCD-related genes show significant upregulation. We compared gene expression in epithelial precursor cells grown as collagen monolayer and ciliated cells grown in suspension by RNA sequencing. All genes reported as PCD causative, except NME8, showed significant upregulation during in vitro ciliogenesis. We observed 67.6% diagnostic yield when testing the targeted-exome panel in our cohort. There was relatively high percentage of DNAI and HYDIN mutations compared to other countries. The latter may be due to our solution for the problem of the confounding HYDIN2 pseudogene. Candidate genes included two recently published PCD-related genes DNAJB13 and PIH1D3; identification of the latter was a direct result of this study. In conclusion, we demonstrate 67.6% diagnostic yield by targeted exome sequencing in a Dutch PCD population and present a highly sensitive and moderately specific approach for identification of PCD-related genes, based on significant upregulation during in vitro ciliogenesis.


Assuntos
Síndrome de Kartagener/diagnóstico , Síndrome de Kartagener/genética , Adulto , Alelos , Exoma/genética , Regulação da Expressão Gênica , Humanos , Mutação/genética , Análise de Sequência de RNA
4.
Sci Rep ; 12(1): 14686, 2022 08 29.
Artigo em Inglês | MEDLINE | ID: mdl-36038602

RESUMO

Inherited bone disorders account for about 10% of documented Mendelian disorders and are associated with high financial burden. Their study requires osteoblasts which play a critical role in regulating the development and maintenance of bone tissue. However, bone tissue is not always available from patients. We developed a highly efficient platelet lysate-based approach to directly transdifferentiate skin-derived human fibroblasts to osteoblast-like cells. We extensively characterized our in vitro model by examining the expression of osteoblast-specific markers during the transdifferentiation process both at the mRNA and protein level. The transdifferentiated osteoblast-like cells showed significantly increased expression of a panel of osteogenic markers. Mineral deposition and ALP activity were also shown, confirming their osteogenic properties. RNA-seq analysis allowed the global study of changes in the transcriptome of the transdifferentiated cells. The transdifferentiated cells clustered separately from the primary fibroblasts with regard to the significantly upregulated genes indicating a distinct transcriptome profile; transdifferentiated osteoblasts also showed significant enrichment in gene expression related to skeletal development and bone mineralization. Our presented in vitro model may potentially contribute to the prospect of studying osteoblast-dependent disorders in patient-derived cells.


Assuntos
Transdiferenciação Celular , Osteoblastos , Calcificação Fisiológica/genética , Diferenciação Celular/genética , Transdiferenciação Celular/genética , Fibroblastos , Humanos , Osteoblastos/metabolismo , Osteogênese/genética
5.
Biomedicines ; 9(2)2021 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-33572801

RESUMO

The immunophilin FKBP12 is a known inhibitor of type I BMP and TGF-ß receptors that competes for binding with their substrate SMADs. FKBP12 and the close paralog FKBP12.6 additionally assemble with ryanodine receptors to control Ca2+ release. Binding of FKBP12.6 to BMP/TGF-ß receptors has yet to be investigated, but appears plausible given its high sequence similarity to FKBP12. Here, we found that FKBP12.6 can assemble with BMP and TGF-ß-family type I receptors, but not with type II receptors. Cellular immunoprecipitation confirmed similar binding of FKBP12 and FKBP12.6 to the BMP receptor ALK2 (ACVR1), a known target of mutations in the congenital syndrome fibrodysplasia ossificans progressiva (FOP), as well as the pediatric brain tumor diffuse intrinsic pontine glioma (DIPG). SEC-MALS analyses using purified proteins indicated a direct 1:1 interaction between FKBP12.6 and the receptor's cytoplasmic domains. The 2.17 Å structure of this ALK2-FKBP12.6 complex bound to the inhibitor dorsomorphin showed FKBP12.6 binding to the GS domain of ALK2 in a manner equivalent to the FKBP12 complex, with ALK2 residues Phe198 and Leu199 extending into the FK506-binding pocket of FKBP12.6. These findings suggest a level of redundancy in FKBP-family regulation of BMP and TGF-ß signaling.

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