ObjectivePLOD1-related kyphoscoliotic Ehlers-Danlos syndrome (kEDS) is a rare autosomal recessive connective tissue disorder characterized by generalized joint laxity, severe congenital hypotonia, progressive kyphoscoliosis, hyperextensible and easily bruised skin, ocular abnormalities, and significant vascular complications.MethodsWe report on nine patients from seven families, eight of them carrying the common homozygous duplication of exons 10-16 in PLOD1. Longitudinal clinical assessments included muscle ultrasound (n=6) and vascular ultrasound (n=4). Genetic diagnostics varied, with most patients ultimately undergoing trio exome or genome sequencing. Urine pyridinoline analysis was performed in seven of nine patients. A literature review and age-stratified recalculation of vascular incidence, including our cohort, were conducted.ResultsDiagnosis was challenging in five families, as the exon 10-16 duplication often escaped detection due to its high allele frequency. Seven patients were initially diagnosed with congenital myopathy. Muscle ultrasound revealed abnormalities in five of six sonographically examined cases. Severe vascular events included neonatal intracranial hemorrhage, fatal aortic aneurysm rupture at the age of 13 years, multiple aneurysms/dissections (ages 14-19 years), and mesenteric dissection at the age of 10 years. Four younger patients (aged 3-9 years have had no vascular complications to date. Urine pyridinoline analysis was abnormal in all tested cases.DiscussionPLOD1-related kEDS often presents with a phenotype of congenital myopathy, complicating genetic diagnosis and potentially leading to underdiagnosis - especially in cases where the common PLOD1 duplication may be missed by strict frequency filters in exome or genome sequencing data. Literature and our data indicate a vascular event incidence from childhood age of ∼25%.

During early osteogenesis, osteoblasts deposit collagen and subsequently mineralize the extracellular matrix (ECM). Disruption of this process underlies skeletal disorders such as osteogenesis imperfecta (OI). Advancing the mechanistic understanding of OI requires in vitro models that enable analysis of cell-secreted collagen and its mineralization. However, commonly used collagen hydrogels obscure newly deposited ECM, limiting direct assessment of nascent collagen deposition. We present an in vitro model using patient-derived human osteoblasts (hOBs) embedded in synthetic microporous poly(ethylene glycol) (PEG) hydrogels to recapitulate matrix defects in OI. Cell-secreted collagen serves as a de novo organic template for biomineralization through the polymer-induced liquid precursor (PILP) process, in which poly(aspartic acid) stabilizes amorphous calcium phosphate precursors, thereby mimicking the function of non-collagenous proteins in vivo. Acellular experiments indicate that PILP promotes intrafibrillar mineralization of collagen hydrogels, whereas PEG hydrogels exhibit negligible mineral deposition, enabling background-free analysis of cell-secreted ECM. Both healthy and OI hOBs embedded in PEG hydrogels maintained high viability (>90%) and secreted type I collagen. After 14 days of culture, confocal and second harmonic generation microscopy revealed disease-specific matrix alterations, including an increased α1(I):α2(I) chain ratio and impaired collagen fibril organization in OI cultures. Following 3 days of mineralization, both healthy and OI cultures exhibited increased mineral deposition, with significantly greater mineralization in OI cultures, consistent with the hypermineralization matrix characteristic of OI bone. Together, these findings establish a human-relevant in vitro model for mechanistic studies of collagen mineralization using patient-derived cells, providing a complementary alternative to animal models for investigating OI.

Osteogenesis imperfecta (OI), characterised by low bone mass and bone fragility, is a heritable disorder with a heterogeneous genetic cause. Type I collagen is the most predominant type of collagen in the bone. Hence, the vast majority of patients with OI carry genetic variants in the genes that encode for type I collagen. However, a fraction of patients have defects in genes that either participate in collagen synthesis and maturation, in osteoblast maturation and functions including bone mineralisation, or with yet fully-understood mechanisms. An intriguing example is MBTPS2, a gene in which missense variants cause two non-overlapping clinical spectrums - either OI or a dermatological spectrum condition (IFAP/KFSD). Our work in the past decade aimed at molecular profiling of MBTPS2-OI using patient-derived fibroblasts. Here, we expand on this by generating induced pluripotent stem cells (iPSCs) from patient-derived fibroblasts and subsequently differentiated step-wise through the sclerotome and into osteoblasts. We also developed a Fiji-based image analysis pipeline to examine extracellular collagen misfolding and fibril organization in 2-dimensional (2D) fibroblast cultures in vitro, which will complement existing methods to qualitatively assess collagen. Together, qualitative assessment of type I collagen and iPSC-based in vitro bone modelling revealed differences in pathomechanisms underlying MBTPS2-OI and classical COL1-OI This showcases diverging pathologies underlying different genetic forms of OI and highlights the need for better molecular characterization of each genetic form to optimise approaches for patient management and treatment.

Osteogenesis imperfecta (OI) is a genetically heterogeneous connective tissue disorder marked by bone fragility and deformities. This study aimed to define the clinical and molecular characteristics of 21 OI patients from 15 unrelated Egyptian families. Most probands were analyzed by exome sequencing. In three consanguineous cases, variants were identified through SNP array-based homozygosity mapping followed by direct sequencing of a candidate gene. Genotype-phenotype correlations were additionally explored. Parental consanguinity was documented in 66.7% (10/15) of the total cohort and in 100% (8/8) of the families with autosomal recessive OI. Pathogenic or likely pathogenic variants were identified in 14 families, five of which were novel. A variant of uncertain significance was identified in the remaining family. COL1A1 and COL1A2 (n = 7) were the most commonly mutated genes, followed by CRTAP (n = 4), while variants in P3H1, WNT1, CREB3L1, and SEC24D were each identified in a single patient. The present study highlights the molecular heterogeneity of OI. In total, 15 distinct variants in seven OI-related genes were identified. We also report a particularly high number of OI lethal forms affecting 10 patients out of 21. The study adds further evidence for the utility of ES in the genetic diagnosis of OI, which facilitates counseling and personalized care.

Patient-derived organotypic bone models offer insights into the pathomechanisms of genetic skeletal disorders and can act as platforms to study the osteoanabolic effect of drug therapies toward precision medicine. Here, we present a mechanically stimulated 3D bioprinted organotypic bone model developed via a two-step 3D bioprinting and cell seeding process of autologous cells from clinical bone samples, replicating both osteoblastic and osteocyte-like phenotypes. Osteoblasts were isolated from patient bone, encapsulated within a bioink for extrusion 3D bioprinting, and subjected to mechanical loading for 4 weeks until mineralized. These mineralized cell-laden constructs were top-seeded with fresh osteoblasts and cultured for an additional 4 weeks. Using osteogenesis imperfecta (OI) patient-derived osteoblasts and osteoblasts from a metabolically healthy patient undergoing femur osteotomy (FO), we investigated the effects of Dickkopf-1 antibody (DKK1Ab), a Wnt pathway modulator, on gene expression, collagen dynamics, mineralization, and mechanical properties. Time-lapsed micro-computed tomography revealed hypermineralization, increased fragility and structural deformities in OI constructs, while transcriptomics, collagen quantification, and immunohistochemistry demonstrated the model’s ability to capture differential patient-specific responses to treatment. This model offers a powerful platform for predicting disease progression and treatment outcomes in skeletal disorders, with the potential to transform orthopaedic care from reactive fracture management to proactive, personalized intervention strategies. ### Competing Interest Statement The authors have declared no competing interest.

ABSTRACTPatient registries play a crucial role in advancing our understanding of rare diseases, enabling the collection of comprehensive clinical and molecular data that inform diagnosis, treatment, and management strategies and advance our understanding of rare diseases. We showcase the first Swiss registry of 796 patients with suspected or confirmed connective tissue disorders (CTD) who were referred to our center over a period of 26 years between 1995 and 2022. The registry contains information on the natural history, anthropometrics, biochemical, histological, and genetic analyses. 61.3% of patients were referred by other hospitals or genetic specialists, with the primary reasons for referral being suspicion of Ehlers–Danlos syndrome (EDS) (53.6%) and osteogenesis imperfecta (OI) (28.1%). Molecular confirmation of these diagnoses was obtained in 60 cases of EDS and 98 cases of OI through genetic testing. In‐depth analyses of 173 OI patients revealed that the majority of OI cases were caused by mutations in COL1A1 or COL1A2. Rarer variants were identified in genes involved in collagen synthesis and bone regulation. Genotype–phenotype correlations were observed in a small subset of patients, with a high prevalence of glycine substitutions in COL1A1 and COL1A2 variants associated with severe phenotypes. This registry offers insights into the molecular underpinnings of EDS and OI and underscores the importance of genetic testing for accurate diagnosis and management.

We have identified 252 inherited disorders of the extracellular matrix (ECM) caused by 154 different gene defects and have proposed a classification system in 8 categories based on their mode of action: 1. Disorders of ECM glycoproteins, 2. Disorders of ECM proteoglycans, 3. Disorders of proteins in TGF-beta signaling pathway, 4. Disorders of fibrillar collagens, 5. Disorders of fibrillar collagen processing and maturation, 6. Disorders of non-fibrillar collagens, 7. Other disorders of connective tissue with bone fragility and 8. Other disorders of connective tissue. Additionally, using information from IEMbase, we have described the clinical involvement of 18 organs and systems, as well as essential laboratory investigations for each type of ECM disorder. Skeletal, ocular, neurological and dysmorphic manifestations were the most prevalent, occurring in 18 %, 12 %, 10 %, and 10 % of ECM disorders, respectively. This was followed by cardiovascular, dermatological, ear-related, muscular, digestive, endocrine, and hematological symptoms (3-7 %). Among the skeletal symptoms, those affecting joints, spine, upper limbs, lower limbs and mineralization were the most common with rates of 25.8 %, 18.0 %, 14.3 %, 14.1 % and 11.5 %, respectively. 27.4 % of the disorders display a single phenotype, with skeletal issues being the most common at 17.8 % and ocular abnormalities 12.2 %. Conversely, 72.6 % of disorders have multiple phenotypes, with LTBP4-related Cutis laxa (10 phenotypes) and SMAD4- related Myhre Syndrome (gain of function) at the end of the spectrum with up to 11 phenotypes. The information provided in this study, including our proposed dyadic classification system for ECM disorders, may be useful for healthcare providers caring for individuals with conditions associated with ECM problems.

Bone-forming therapies often fail in genetic skeletal disorders, highlighting critical gaps in mechanistic understanding and therapy evaluation. We developed 3D bioprinted organotypic bone models using primary cells from a patient with FKBP10-related osteogenesis imperfecta (OI) and from metabolically healthy controls obtained via femoral osteotomy (FO). Cyclic me-chanical loading and reseeding generated patient-derived bone-like tissue for structural, molecu-lar, and transcriptomic characterization of the engineered donor-specific tissue material. Dick-kopf-1 antibody (DKK1Ab) was then administered as a therapeutic perturbation. OI constructs showed a bidirectional interferon-stimulated gene (ISG) signature and hypermineralization with structural fragility, hallmark features of OI. Unlike FO constructs, DKK1Ab administration in OI resulted in a limited transcriptional response marked by ISG downregulation and increased MKI67 expression. Therapeutic perturbation with DKK1Ab increased early procollagen I se-cretion, and was associated with lower fracture scores, although the within-OI difference was not statistically significant. This proof-of-concept demonstrates multimodal donor-specific char-acterization of engineered patient-derived bone models following DKK1Ab perturbation.

Protein biogenesis within the endoplasmic reticulum (ER) is crucial for organismal function. Errors during protein folding necessitate the removal of faulty products. ER-associated protein degradation and ER-phagy target misfolded proteins for proteasomal and lysosomal degradation. The mechanisms initiating ER-phagy in response to ER proteostasis defects are not well understood. By studying mouse primary cells and patient samples as a model of ER storage disorders (ERSDs), we show that accumulation of faulty products within the ER triggers a response involving SESTRIN2, a nutrient sensor controlling mTORC1 signaling. SESTRIN2 induction by XBP1 inhibits mTORC1's phosphorylation of TFEB/TFE3, allowing these transcription factors to enter the nucleus and upregulate the ER-phagy receptor FAM134B along with lysosomal genes. This response promotes ER-phagy of misfolded proteins via FAM134B-Calnexin complex. Pharmacological induction of FAM134B improves clearance of misfolded proteins in ERSDs. Our study identifies the interplay between nutrient signaling and ER quality control, suggesting therapeutic strategies for ERSDs.

Osteogenesis imperfecta (OI) is a heterogeneous group of rare genetic diseases characterized by increased bone fragility and deformities. The pathomechanisms of OI are poorly understood, hindering the development of disease-specific therapy. Addressing the limited understanding of OI and the lack of targeted treatments remains a challenge, given its varied symptoms and large clinical spectrum. Animal models have greatly advanced the understanding of the disease; however, the heterogeneity and subtype-specific symptoms are difficult to translate to humans. In vitro models offer a promising tool for translational medicine, as they have the potential to yield patient-specific insights in a controlled environment using patient derived-cells. We used mechanically loaded 3D-bioprinted patient-specific organotypic bone models and time-lapsed micro-computed tomography to demonstrate dysregulation of mineralization in FKBP10 -related OI compared to healthy controls. In contrast to healthy controls, tissue mineral density and stiffness were decoupled, such that hypermineralization observed in OI samples did not lead to increased stiffness. Additionally, we were able to replicate experimental stiffness using sample specific micro-finite element analysis. This allowed us to show mineral formation in regions of high local strain, suggesting mechanoregulation in FKBP10 -related OI organotypic bone models is comparable to healthy controls. Regional analysis of mineralization showed increased heterogeneous mineralization, microarchitectural inhomogeneities and scaffold microporosity of OI samples compared to healthy controls. Our results suggest that the observed dysregulation of mineralization is the main driver for the altered mineral-mechanics properties observed in FKBP10 -related organotypic bone models.One Sentence Summary Organotypic bone models demonstrate dysregulated mineralization in osteogenesis imperfecta samples compared to healthy controls.### Competing Interest StatementThe authors have declared no competing interest.

Human organotypic bone models are an emerging technology that replicate bone physiology and mechanobiology for comprehensive in vitro experimentation over prolonged periods of time. Recently, we introduced a mineralized bone model based on 3D bioprinted cell-laden alginate-gelatin-graphene oxide hydrogels cultured under dynamic loading using commercially available human mesenchymal stem cells. In the present study, we created cell-laden scaffolds from primary human osteoblasts isolated from surgical waste material and investigated the effects of a previously reported optimal cell printing density (5 × 10 6 cells/mL bioink) vs. a higher physiological cell density (10 × 10 6 cells/mL bioink). We studied mineral formation, scaffold stiffness, and cell morphology over a 10-week period to determine culture conditions for primary human bone cells in this microenvironment. For analysis, the human bone-derived cell-laden scaffolds underwent multiscale assessment at specific timepoints. High cell viability was observed in both groups after bioprinting (>90%) and after 2 weeks of daily mechanical loading (>85%). Bioprinting at a higher cell density resulted in faster mineral formation rates, higher mineral densities and remarkably a 10-fold increase in stiffness compared to a modest 2-fold increase in the lower printing density group. In addition, physiological cell bioprinting densities positively impacted cell spreading and formation of dendritic interconnections. We conclude that our methodology of processing patient-specific human bone cells, subsequent biofabrication and dynamic culturing reliably affords mineralized cell-laden scaffolds. In the future, in vitro systems based on patient-derived cells could be applied to study the individual phenotype of bone disorders such as osteogenesis imperfecta and aid clinical decision making.

Human bone organoids are an emerging technology that replicate bone physiology and mechanobiology for comprehensive in vitro experimentation over prolonged periods of time. Recently, we introduced a mineralized bone organoid based on 3D bioprinted cell-laden alginate-gelatin-graphene oxide hydrogels using commercially available human mesenchymal stem cells. In the present study, we isolated primary human osteoblastic bone cells to create human bone organoids based on our biofabrication technology and studied their mechanics and mineralization. First, we established a workflow from bone sample collection during orthopedic surgeries, osteoblast isolation, expansion, and extrusion 3D bioprinting of lattice structures, to subsequent dynamic culturing of constructs in a purpose-built compression bioreactor. Then, we investigated the effects of a previously reported optimal cell printing density (5x106 cells/ml bioink) vs. a higher density (10x106 cells/ml bioink) that matched the physiological density of the human donor. We studied mineral formation, organoid stiffness, and cell morphology over a 10-week period to determine optimal culture conditions for human bone cells in this microenvironment. For analysis, the organoids underwent multiscale assessment at specific timepoints. High cell viability was observed in both groups after bioprinting (>90%) and after 2 weeks of daily mechanical loading (>85%). In addition, bioprinting at higher cell density resulted in a significant increase of organoid stiffness and mineral density, and positively impacted cell spreading and formation of dendritic interconnections. We conclude that our methodology of processing patient-specific human bone cells, subsequent biofabrication and dynamic culturing reliably affords mineralized bone organoids that can be applied in the emerging field of personalized medicine to study a wide variety of bone disorders and could replace animal models in pharmaceutical research.

(i) PCR-sequencing: Chromatograms were generated by PCR-sequencing of a region within exon 4 of MBTPS2 using DNA extracted from a healthy control and the proband's fibroblasts (ii) Gene expression was quantified by qRT-PCR using RNA extracted from fibroblasts. Transcript levels of each gene of interest was calculated using the 2^-deltaCt method with normalization to the average Ct values of endogenous control genes GAPDH, IPO8 and TBP. (iii) Cellular fatty acid content was quantified by GC-MS/MS. Each table represents one technical replicate. Absolute values of each fatty acid are listed in the tables; relative ratios of various fatty acids are calculated at the bottom of each table. (iv) Immunocytochemistry images of ECM proteins (COL1 = collagen type I; COL4 = collagen type IV; COL5 = collagen type V; a2b1 = integrin a2b1) and binding of collagen-hybridising peptide (R-CHP).

Osteogenesis imperfecta (OI) is a heritable and chronically debilitating skeletal dysplasia. Patients with OI typically present with reduced bone mass, tendency for recurrent fractures, short stature and bowing deformities of the long bones. Mutations causative of OI have been identified in over 20 genes involved in collagen folding, posttranslational modification and processing, and in bone mineralization and osteoblast development. In 2016, we described the first X-linked recessive form of OI caused by MBTPS2 missense variants in patients with moderate to severe phenotypes. MBTPS2 encodes site-2 protease, a Golgi transmembrane protein that activates membrane-tethered transcription factors. These transcription factors regulate genes involved in lipid metabolism, bone and cartilage development, and ER stress response. The interpretation of genetic variants in MBTPS2 is complicated by the gene's pleiotropic properties; MBTPS2 variants can also cause the dermatological conditions Ichthyosis Follicularis, Atrichia and Photophobia (IFAP), Keratosis Follicularis Spinulosa Decalvans (KFSD) and Olmsted syndrome (OS) without skeletal abnormalities typical of OI. Using control and patient-derived fibroblasts, we previously identified gene expression signatures that distinguish MBTPS2-OI from MBTPS2-IFAP/KFSD and observed stronger suppression of genes involved in fatty acid metabolism in MBTPS2-OI than in MBTPS2-IFAP/KFSD; this was coupled with alterations in the relative abundance of fatty acids in MBTPS2-OI. Furthermore, we observed a reduction in collagen deposition in the extracellular matrix by MBTPS2-OI fibroblasts. Here, we extrapolate our observations in the molecular signature unique to MBTPS2-OI to infer the pathogenicity of a novel MBTPS2 c.516A>C (p.Glu172Asp) variant of unknown significance in a male proband. The pregnancy was terminated at gestational week 21 after ultrasound scans showed bowing of femurs and tibiae and shortening of long bones particularly of the lower extremity; these were further confirmed by autopsy. By performing transcriptional analyses, gas chromatography-tandem mass spectrometry-based quantification of fatty acids and immunocytochemistry on fibroblasts derived from the umbilical cord of the proband, we observed perturbations in fatty acid metabolism and collagen production similar to what we previously described in MBTPS2-OI. These findings support pathogenicity of the MBTPS2 variant p.Glu172Asp as OI-causative and highlights the value of extrapolating molecular signatures identified in multiomics studies to characterize novel genetic variants.

Purpose: This study aimed to describe a multisystemic disorder featuring cardiovascular, facial, musculoskeletal, and cutaneous anomalies caused by heterozygous loss-of-function variants in TAB2. Methods: Affected individuals were analyzed by next-generation technologies and genomic array. The presumed loss-of-function effect of identified variants was assessed by luciferase assay in cells transiently expressing TAB2 deleterious alleles. In available patients' fibroblasts, variant pathogenicity was further explored by immunoblot and osteoblast differentiation assays. The transcriptomic profile of fibroblasts was investigated by RNA sequencing. Results: A total of 11 individuals from 8 families were heterozygotes for a novel TAB2 variant. In total, 7 variants were predicted to be null alleles and 1 was a missense change. An additional subject was heterozygous for a 52 kb microdeletion involving TAB2 exons 1 to 3. Luciferase assay indicated a decreased transcriptional activation mediated by NF-kappa B signaling for all point variants. Immunoblot analysis showed a reduction of TAK1 phosphorylation while osteoblast differentiation was impaired. Transcriptomic analysis identified deregulation of multiple pleiotropic pathways, such as TGF beta-, Ras-MAPK-, and Wnt-signaling networks. Conclusion: Our data defined a novel disorder associated with loss-of-function or, more rarely, hypomorphic alleles in a restricted linker region of TAB2. The pleiotropic manifestations in this disorder partly recapitulate the 6q25.1 (TAB2) microdeletion syndrome and deserve the definition of cardio-facial-cutaneous-articular syndrome. (C) 2021 American College of Medical Genetics and Genomics. Published by Elsevier Inc. All rights reserved.