The structural basis for the collagen processing by human P3H1/CRTAP/PPIB ternary complex.

Collagen posttranslational processing is crucial for its proper assembly and function. Disruption of collagen processing leads to tissue development and structure disorders like osteogenesis imperfecta (OI). OI-related collagen processing machinery includes prolyl 3-hydroxylase 1 (P3H1), peptidyl-prolyl cis-trans isomerase B (PPIB), and cartilage-associated protein (CRTAP), with their structural organization and mechanism unclear. We determine cryo-EM structures of the P3H1/CRTAP/PPIB complex. The active sites of P3H1 and PPIB form a face-to-face bifunctional reaction center, indicating a coupled modification mechanism. The structure of the P3H1/CRTAP/PPIB/collagen peptide complex reveals multiple binding sites, suggesting a substrate interacting zone. Unexpectedly, a dual-ternary complex is observed, and the balance between ternary and dual-ternary states can be altered by mutations in the P3H1/PPIB active site and the addition of PPIB inhibitors. These findings provide insights into the structural basis of collagen processing by P3H1/CRTAP/PPIB and the molecular pathology of collagen-related disorders.

Loss of the long form of Plod2 phenocopies contractures of Bruck syndrome-osteogenesis imperfecta.

Bruck syndrome is an autosomal recessive form of osteogenesis imperfecta caused by biallelic variants in PLOD2 or FKBP10 and is characterized by joint contractures, bone fragility, short stature, and scoliosis. PLOD2 encodes LH2, which hydroxylates type I collagen telopeptide lysines, a critical step for collagen crosslinking. The Plod2 global knockout mouse model is limited by early embryonic lethality, and thus, the role of PLOD2 in skeletogenesis is not well understood. We generated a novel Plod2 mouse line modeling a variant identified in two unrelated individuals with Bruck syndrome: PLOD2 c.1559dupC, predicting a frameshift and loss of the long isoform LH2b. In the mouse, the duplication led to loss of LH2b mRNA as well as significantly reduced total LH2 protein. This model, Plod2fs/fs, survived up to E18.5 although in non-Mendelian genotype frequencies. The homozygous frameshift model recapitulated the joint contractures seen in Bruck syndrome and had indications of absent type I collagen telopeptide lysine hydroxylation in bone. Genetically labeling tendons with Scleraxis-GFP in Plod2fs/fs mice revealed the loss of extensor tendons in the forelimb by E18.5, and developmental studies showed extensor tendons developed through E14.5 but were absent starting at E16.5. Second harmonic generation showed abnormal tendon type I collagen fiber organization, suggesting structurally abnormal tendons. Characterization of the skeleton by µCT and Raman spectroscopy showed normal bone mineralization levels. This work highlights the importance of properly crosslinked type I collagen in tendon and bone, providing a promising new mouse model to further our understanding of Bruck syndrome.

Bruck syndrome is a rare disease where individuals have brittle bone as well as contracted or stiff joints. Mutations in two genes are associated with Bruck syndrome and, in this work, we focus on PLOD2. Mice without Plod2 die at an early embryonic stage, before they have a chance to fully develop. In this work, we created a mouse with a PLOD2 mutation seen in people with Bruck syndrome. Some of these new Bruck syndrome model mice survived to a later gestational age, but all died at birth. The Bruck syndrome mice were small and had contracted joints. We found that they were missing tendons in their arms and had structurally abnormal tendons in their knees. Bone mineralization was normal, but there were indications that the modifications needed for normal type I collagen structure were absent. Overall, this is an advantageous new mouse model of Bruck syndrome that can be used to study this rare disease and highlights the importance of Plod2 in tendon.

Multi-omics analyses reveal aberrant differentiation trajectory with WNT1 loss-of-function in type XV osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is a group of severe genetic bone disorders characterized by congenital low bone mass, deformity, and frequent fractures. Type XV OI is a moderate to severe form of skeletal dysplasia caused by WNT1 variants. In this cohort study from southern China, we summarized the clinical phenotypes of patients with WNT1 variants and found that the proportion of type XV patients was around 10.3% (25 out of 243) with a diverse spectrum of phenotypes. Functional assays indicated that variants of WNT1 significantly impaired its secretion and effective activity, leading to moderate to severe clinical manifestations, porous bone structure, and enhanced osteoclastic activities. Analysis of proteomic data from human skeleton indicated that the expression of SOST (sclerostin) was dramatically reduced in type XV patients compared to patients with COL1A1 quantitative variants. Single-cell transcriptome data generated from human tibia samples of patients diagnosed with type XV OI and leg-length discrepancy, respectively, revealed aberrant differentiation trajectories of skeletal progenitors and impaired maturation of osteocytes with loss of WNT1, resulting in excessive CXCL12+ progenitors, fewer mature osteocytes, and the existence of abnormal cell populations with adipogenic characteristics. The integration of multi-omics data from human skeleton delineates how WNT1 regulates the differentiation and maturation of skeletal progenitors, which will provide a new direction for the treatment strategy of type XV OI and relative low bone mass diseases such as early onset osteoporosis.

Osteogenesis imperfecta is a rare disease characterized by low bone mass, frequent fractures, and long bone deformity. Type XV osteogenesis imperfect is an autosomal recessive disorder caused by WNT1 variants, while heterozygous variants of WNT1 result in early onset osteoporosis. In this cohort study, we summarized the clinical features of 25 patients diagnosed with type XV osteogenesis imperfect. The WNT1 variants were confirmed by genetic test. Molecular assays were conducted to reveal the impact of variants on WNT1 protein activity and bone structure. We then compared the protein levels in bone tissues isolated from the type XV patients and patients with mild deformity using proteomic method, and found that the expression of SOST, mainly produced by mature osteoblasts and osteocytes, was dramatically reduced in type XV patients. We further compared the global mRNA expression levels in the skeletal cells using single-cell RNA sequencing. Analyses of these data indicated that more immature progenitors were identified and maturation of osteocytes was impaired with WNT1 loss-of-function. Our study helps to understand the underlying pathogenesis of type XV osteogenesis imperfecta.

Schnurri-3 inhibition rescues skeletal fragility and vascular skeletal stem cell niche pathology in the OIM model of osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is a disorder of low bone mass and increased fracture risk due to a range of genetic variants that prominently include mutations in genes encoding type I collagen. While it is well known that OI reflects defects in the activity of bone-forming osteoblasts, it is currently unclear whether OI also reflects defects in the many other cell types comprising bone, including defects in skeletal vascular endothelium or the skeletal stem cell populations that give rise to osteoblasts and whether correcting these broader defects could have therapeutic utility. Here, we find that numbers of skeletal stem cells (SSCs) and skeletal arterial endothelial cells (AECs) are augmented in Col1a2oim/oim mice, a well-studied animal model of moderate to severe OI, suggesting that disruption of a vascular SSC niche is a feature of OI pathogenesis. Moreover, crossing Col1a2oim/oim mice to mice lacking a negative regulator of skeletal angiogenesis and bone formation, Schnurri 3 (SHN3), not only corrected the SSC and AEC phenotypes but moreover robustly corrected the bone mass and spontaneous fracture phenotypes. As this finding suggested a strong therapeutic utility of SHN3 inhibition for the treatment of OI, a bone-targeting AAV was used to mediate Shn3 knockdown, rescuing the Col1a2oim/oim phenotype and providing therapeutic proof-of-concept for targeting SHN3 for the treatment of OI. Overall, this work both provides proof-of-concept for inhibition of the SHN3 pathway and more broadly addressing defects in the stem/osteoprogenitor niche as is a strategy to treat OI.

Genotype and Phenotype Correlation of Patients with Osteogenesis Imperfecta.

Osteogenesis imperfecta (OI) is the most common inherited connective tissue disease of the bone, characterized by recurrent fractures and deformities. In patients displaying the OI phenotype, genotype-phenotype correlation is used to screen multiple genes swiftly, identify new variants, and distinguish between differential diagnoses and mild subtypes. This study evaluated variants identified through next-generation sequencing in 58 patients with clinical characteristics indicative of OI. The cohort included 18 adults, 37 children, and 3 fetuses. Clinical classification revealed 25 patients as OI type I, three patients as OI type II, 18 as OI type III, and 10 as OI type IV. Fifteen variants in COL1A1 were detected in 19 patients, 9 variants in COL1A2 (n = 19), 5 variants in LEPRE1/P3H1 (n = 7), 3 variants in FKBP10 (n = 4), 3 variants in SERPINH1 (n = 2), 1 variant in IFITM5 (n = 1), and 1 variant in PLS3 (n = 1). In total, 37 variants (18 pathogenic, 14 likely pathogenic, and 5 variants of uncertain significance), including 16 novel variants, were identified in 43 (37 probands, 6 family members) of the 58 patients analyzed. This study highlights the efficacy of panel testing in the molecular diagnosis of OI, the significance of the next-generation sequencing technique, and the importance of genotype-phenotype correlation.

Pamidronate Response in a Novel Biallelic CREB3L1 Gene Mutation-Associated Osteogenesis Imperfecta: A Case Report.

CASE: We report a 15-year-old adolescent boy being followed up for 6 years with osteogenesis imperfecta (OI). Genetic testing of this child revealed a novel missense variant c.925C>T p.Arg309Cys in the CREB3L1 gene. Treatment with regular pamidronate therapy showed increased bone mineral density and a reduced fracture rate. His lower limb rush rodding improved his mobility. His withdrawal from bisphosphonate therapy worsened his mobility status but started improving after he restarted treatment, suggesting a response to pamidronate therapy. CONCLUSION: We report a novel biallelic missense variant c.925C>T, p.Arg309Cys, in the CREB3L1 gene causing OI, which responded to bisphosphonate therapy and corrective surgery.

Effectiveness of whole exome sequencing analyses in the molecular diagnosis of osteogenesis imperfecta.

OBJECTIVES: Osteogenesis imperfecta (OI) is a group of phenotypically and genetically heterogeneous connective tissue disorders that share similar skeletal anomalies causing bone fragility and deformation. This study aimed to investigate the molecular genetic etiology and to determine the relationship between genotype and phenotype in OI patients with whole exome sequencing (WES). METHODS: Multiplex-Ligation dependent Probe Amplification (MLPA) analysis of COL1A1 and COL1A2 and WES were performed on cases between the ages of 0 and 18 whose genetic etiology could not be determined before using a targeted next-generation sequencing panel, including 13 genes (COL1A1, COL1A2, IFITM5, SERPINF1, CRTAP, P3H1, PPIB, SERPINH1, FKBP10, SP7, BMP1, MBTPS2, PLOD2) responsible for OI. RESULTS: Twelve patients (female/male: 4/8) from 10 different families were included in the study. In 6 (50 %) families, consanguineous marriage was noted. The clinical typing based on Sillence classification; 3 (25 %) patients were considered to be type I, 7 (58.3 %) type III, and 2 (16.7 %) type IV. Deletion/duplication wasn't detected in the COL1A1 and COL1A2 genes in the MLPA analysis of the patients. Twelve patients were molecularly analyzed by WES, and in 6 (50 %) of them, a disease-causing variant in three different genes (FKBP10, P3H1, and WNT1) was identified. Two (33.3 %) detected variants in all genes have not been previously reported in the literature and were considered deleterious based on prediction tools. In 6 cases, no variants were detected in disease-causing genes. CONCLUSIONS: This study demonstrates rare OI types' clinical and molecular features; genetic etiology was determined in 6 (50 %) 12 patients with the WES analysis. In addition, two variants in OI genes have been identified, contributing to the literature.

Gnathodiaphyseal dysplasia: Diagnostic clues from two fetal cases and literature review.

This article presents two fetal cases of gnathodiaphyseal dysplasia (GDD), a rare autosomal dominant disorder, and reviews the relevant literature. The cases involved two fetuses exhibiting bone bowing, which led to the diagnosis of GDD. Genetic testing revealed two de novo variants of the ANO5 gene, confirming the diagnosis. A literature review was conducted to explore GDD's clinical and paraclinical presentation, diagnosis, and management. GDD is a rare but frequently inherited cause of bone fragility and jaw lesions characterized by a gain-of-function variant within the ANO5 gene. Clinical manifestations range from recurrent dental infections with mild jaw lesions to severe bone fragility with several fractures associated with large jaw lesions requiring disfiguring surgeries. Diagnostic techniques depend on the context and include targeted genetic testing of ANO5, untargeted molecular analysis with whole-exome sequencing, or whole-genome sequencing. This case report highlights the importance of recognizing GDD as a novel cause of bone bowing and fractures during pregnancy. By summarizing the literature, this article contributes to healthcare professionals' knowledge and improves the recognition, diagnosis, and care of patients with GDD.

The IFITM5 mutation in osteogenesis imperfecta type V is associated with an ERK/SOX9-dependent osteoprogenitor differentiation defect.

Osteogenesis imperfecta (OI) type V is the second most common form of OI, distinguished by hyperplastic callus formation and calcification of the interosseous membranes, in addition to the bone fragility. It is caused by a recurrent, dominant pathogenic variant (c.-14C>T) in interferon-induced transmembrane protein 5 (IFITM5). Here, we generated a conditional Rosa26-knockin mouse model to study the mechanistic consequences of the recurrent mutation. Expression of the mutant Ifitm5 in osteo-chondroprogenitor or chondrogenic cells resulted in low bone mass and growth retardation. Mutant limbs showed impaired endochondral ossification, cartilage overgrowth, and abnormal growth plate architecture. The cartilage phenotype correlates with the pathology reported in patients with OI type V. Surprisingly, expression of mutant Ifitm5 in mature osteoblasts caused no obvious skeletal abnormalities. In contrast, earlier expression in osteo-chondroprogenitors was associated with an increase in the skeletal progenitor cell population within the periosteum. Lineage tracing showed that chondrogenic cells expressing the mutant Ifitm5 had decreased differentiation into osteoblastic cells in diaphyseal bone. Moreover, mutant IFITM5 disrupted early skeletal homeostasis in part by activating ERK signaling and downstream SOX9 protein, and inhibition of these pathways partially rescued the phenotype in mutant animals. These data identify the contribution of a signaling defect altering osteo-chondroprogenitor differentiation as a driver in the pathogenesis of OI type V.

Presentation of Rare Phenotypes Associated with the FKBP10 Gene.

Pathogenic variants in the FKBP10 gene lead to a spectrum of rare autosomal recessive phenotypes, including osteogenesis imperfecta (OI) Type XI, Bruck syndrome Type I (BS I), and the congenital arthrogryposis-like phenotype (AG), each with variable clinical manifestations that are crucial for diagnosis. This study analyzed the clinical-genetic characteristics of patients with these conditions, focusing on both known and newly identified FKBP10 variants. We examined data from 15 patients, presenting symptoms of OI and joint contractures. Diagnostic methods included genealogical analysis, clinical assessments, radiography, whole exome sequencing, and direct automated Sanger sequencing. We diagnosed 15 patients with phenotypes due to biallelic FKBP10 variants-4 with OI Type XI, 10 with BS I, and 1 with the AG-like phenotype-demonstrating polymorphism in disease severity. Ten pathogenic FKBP10 variants were identified, including three novel ones, c.1373C>T (p.Pro458Leu), c.21del (p.Pro7fs), and c.831_832insCG (p.Gly278Argfs), and a recurrent variant, c.831dup (p.Gly278Argfs). Variant c.1490G>A (p.Trp497Ter) was found in two unrelated patients, causing OI XI in one and BS I in the other. Additionally, two unrelated patients with BS I and epidermolysis bullosa shared identical homozygous FKBP10 and KRT14 variants. This observation illustrates the diversity of FKBP10-related pathology and the importance of considering the full spectrum of phenotypes in clinical diagnostics.

Clinical utility of comprehensive gene panel testing for common and rare causes of skeletal dysplasia and other skeletal disorders: Results from the largest cohort to date.

Molecular genetics enables more precise diagnoses of skeletal dysplasia and other skeletal disorders (SDs). We investigated the clinical utility of multigene panel testing for 5011 unrelated individuals with SD in the United States (December 2019-April 2022). Median (range) age was 8 (0-90) years, 70.5% had short stature and/or disproportionate growth, 27.4% had a positive molecular diagnosis (MDx), and 30 individuals received two MDx. Genes most commonly contributing to MDx were FGFR3 (16.9%), ALPL (13.0%), and COL1A1 (10.3%). Most of the 112 genes associated with ≥1 MDx were primarily involved in signal transduction (n = 35), metabolism (n = 23), or extracellular matrix organization (n = 17). There were implications associated with specific care/treatment options for 84.4% (1158/1372) of MDx-positive individuals; >50% were linked to conditions with targeted therapy approved or in clinical development, including osteogenesis imperfecta, achondroplasia, hypophosphatasia, and mucopolysaccharidosis. Forty individuals with initially inconclusive results became MDx-positive following family testing. Follow-up mucopolysaccharidosis enzyme activity testing was positive in 14 individuals (10 of these were not MDx-positive). Our findings showed that inclusion of metabolic genes associated with SD increased the clinical utility of a gene panel and confirmed that integrated use of comprehensive gene panel testing with orthogonal testing reduced the burden of inconclusive results.

A recurrent de novo missense mutation in COL1A1 causes osteogenesis imperfecta type II and preterm delivery in Normande cattle.

BACKGROUND: Nine male and eight female calves born to a Normande artificial insemination bull named "Ly" were referred to the French National Observatory of Bovine Abnormalities for multiple fractures, shortened gestation, and stillbirth or perinatal mortality. RESULTS: Using Illumina BovineSNP50 array genotypes from affected calves and 84 half-sib controls, the associated locus was mapped to a 6.5-Mb interval on chromosome 19, assuming autosomal inheritance with germline mosaicism. Subsequent comparison of the whole-genome sequences of one case and 5116 control genomes, followed by genotyping in the affected pedigree, identified a de novo missense substitution within the NC1 domain of the COL1A1 gene (Chr19 g.36,473,965G > A; p.D1412N) as unique candidate variant. Interestingly, the affected residue was completely conserved among 243 vertebrate orthologs, and the same substitution in humans has been reported to cause type II osteogenesis imperfecta (OI), a connective tissue disorder that is characterized primarily by bone deformity and fragility. Moreover, three COL1A1 mutations have been described to cause the same syndrome in cattle. Necropsy, computed tomography, radiology, and histology confirmed the diagnosis of type II OI, further supporting the causality of this variant. In addition, a detailed analysis of gestation length and perinatal mortality in 1387 offspring of Ly and more than 160,000 progeny of 63 control bulls allowed us to statistically confirm in a large pedigree the association between type II OI and preterm delivery, which is probably due to premature rupture of fetal membranes and has been reported in several isolated cases of type II OI in humans and cattle. Finally, analysis of perinatal mortality rates and segregation distortion supported a low level of germ cell mosaicism in Ly, with an estimate of 4.5% to 7.7% of mutant sperm and thus 63 to 107 affected calves born. These numbers contrast with the 17 cases reported and raise concerns about the underreporting of congenital defects to heredo-surveillance platforms, even for textbook genetic syndromes. CONCLUSIONS: In conclusion, we describe a large animal model for a recurrent substitution in COL1A1 that is responsible for type II OI in humans. More generally, this study highlights the utility of such datasets and large half-sib families available in livestock species to characterize sporadic genetic defects.

A Retrospective Study of the Presentation, Diagnosis, Management, and Outcomes of 27 Patients with Osteogenesis Imperfecta at a Single Center in Türkiye.

BACKGROUND This retrospective study aimed to evaluate the presentation, diagnosis, management, and outcomes of 27 patients diagnosed with osteogenesis imperfecta at a single center in Türkiye between January 2011 and January 2020. MATERIAL AND METHODS We analyzed data from the medical records of 27 patients with osteogenesis imperfecta admitted to Çukurova University Faculty of Medicine, Department of Orthopedics and Traumatology, between January 2011 and January 2020. The data included the clinical examination notes of the cases classified according to the Sillence and Shapiro systems, age, sex, parental consanguinity, genetic analysis (DNA isolation) results, the number and localization of past fractures, treatment methods, complications, hypermobility, and ambulation scoring. RESULTS The mean age of the patients (n=13 male, n=14 female) was 10.4±7.4 years, ranging from 3 to 39 years. Almost half (n=15, 55.6%) had consanguineous parents. The patients had 131 fractures during the 9 years between January 2011 and January 2020, with the femur being the most commonly fractured bone; 13 patients (48.15%) received surgical and conservative treatments, while the remaining 14 underwent only conservative treatments. The results revealed a strong association between the number of fractures and the types of genetic mutations (P=0.004). CONCLUSIONS Study findings indicate that the type of genetic mutation was not significantly correlated with the risk of treatment complications in osteogenesis imperfecta cases. Nevertheless, the study reveals a noteworthy association between the type of mutation and the number of surgeries required. Specifically, patients with the COL1A1 mutation needed more surgeries.

Craniofacial dysmorphism of osteogenesis imperfecta mouse and effect of cathepsin K knockout: Preliminary craniometry observations.

OBJECTIVES: In addition to bone fragility, patients with osteogenesis imperfecta (OI) type III have typical craniofacial abnormalities, such as a triangular face and maxillary micrognathism. However, in the osteogenesis imperfecta mouse (oim), a validated model of OI type III, few descriptions exist of craniofacial phenotype. Treatment of OI mostly consists of bisphosphonate administration. Cathepsin K inhibition has been tested as a promising therapeutic approach for osteoporosis and positive results were observed in long bones of cathepsin K knocked out oim (oim/CatK-/-). This craniometry study aimed to highlight the craniofacial characteristics of oim and Cathepsin K KO mouse. MATERIALS AND METHODS: We analyzed the craniofacial skeleton of 51 mice distributed in 4 genotype groups: Wt (control), oim, CatK-/-, oim/CatK-/-. The mice were euthanized at 13 weeks and their heads were analyzed using densitometric (pQCT), X-ray cephalometric, and histomorphometric methods. RESULTS: The craniofacial skeleton of the oim mouse is frailer than the Wt one, with a reduced thickness and mineral density of the cranial vault and mandibular ramus. Different cephalometric data attest a dysmorphism similar to the one observed in humans with OI type III. Those abnormalities were not improved in the oim/CatK-/- group. CONCLUSION: These results suggest that oim mouse could serve as a complete model of the human OI type III, including the craniofacial skeleton. They also suggest that invalidation of cathepsin K has no impact on the craniofacial abnormalities of the oim model.

Osteogenesis imperfecta type 10 and the cellular scaffolds underlying common immunological diseases.

Osteogenesis imperfecta type 10 (OI10) is caused by loss of function codon variants in the gene SERPINH1 that encodes heat shock protein 47 (HSP47), rather than in a gene specifying bone formation. The HSP47 variants disrupt the folding of both collagen and the endonuclease IRE1α (inositol-requiring enzyme 1α) that splices X-Box Binding Protein 1 (XBP1) mRNA. Besides impairing bone development, variants likely affect osteoclast differentiation. Three distinct biochemical scaffold play key roles in the differentiation and regulated cell death of osteoclasts. These scaffolds consist of non-templated protein modifications, ordered lipid arrays, and protein filaments. The scaffold components are specified genetically, but assemble in response to extracellular perturbagens, pathogens, and left-handed Z-RNA helices encoded genomically by flipons. The outcomes depend on interactions between RIPK1, RIPK3, TRIF, and ZBP1 through short interaction motifs called RHIMs. The causal HSP47 nonsynonymous substitutions occur in a novel variant leucine repeat region (vLRR) that are distantly related to RHIMs. Other vLRR protein variants are causal for a variety of different mendelian diseases. The same scaffolds that drive mendelian pathology are associated with many other complex disease outcomes. Their assembly is triggered dynamically by flipons and other context-specific switches rather than by causal, mendelian, codon variants.

Galunisertib downregulates mutant type I collagen expression and promotes MSCs osteogenesis in pediatric osteogenesis imperfecta.

Qualitative alterations in type I collagen due to pathogenic variants in the COL1A1 or COL1A2 genes, result in moderate and severe Osteogenesis Imperfecta (OI), a rare disease characterized by bone fragility. The TGF-ß signaling pathway is overactive in OI patients and certain OI mouse models, and inhibition of TGF-ß through anti-TGF-ß monoclonal antibody therapy in phase I clinical trials in OI adults is rendering encouraging results. However, the impact of TGF-ß inhibition on osteogenic differentiation of mesenchymal stem cells from OI patients (OI-MSCs) is unknown. The following study demonstrates that pediatric skeletal OI-MSCs have imbalanced osteogenesis favoring the osteogenic commitment. Galunisertib, a small molecule inhibitor (SMI) that targets the TGF-ß receptor I (TßRI), favored the final osteogenic maturation of OI-MSCs. Mechanistically, galunisertib downregulated type I collagen expression in OI-MSCs, with greater impact on mutant type I collagen, and concomitantly, modulated the expression of unfolded protein response (UPR) and autophagy markers. In vivo, galunisertib improved trabecular bone parameters only in female oim/oim mice. These results further suggest that type I collagen is a tunable target within the bone ECM that deserves investigation and that the SMI, galunisertib, is a promising new candidate for the anti-TGF-ß targeting for the treatment of OI.

Clinical features, treatment, and follow-up of OPPG and high-bone-mass disorders: LRP5 is a key regulator of bone mass.

Osteoporosis-pseudoglioma syndrome (OPPG) and LRP5 high bone mass (LRP5-HBM) are two rare bone diseases with opposite clinical symptoms caused by loss-of-function and gain-of-function mutations in LRP5. Bisphosphonates are an effective treatment for OPPG patients. LRP5-HBM has a benign course, and age-related bone loss is found in one LRP5-HBM patient. PURPOSE: Low-density lipoprotein receptor-related protein 5 (LRP5) is involved in the canonical Wnt signaling pathway. The gain-of-function mutation leads to high bone mass (LRP5-HBM), while the loss-of-function mutation leads to osteoporosis-pseudoglioma syndrome (OPPG). In this study, the clinical manifestations, disease-causing mutations, treatment, and follow-up were summarized to improve the understanding of these two diseases. METHODS: Two OPPG patients and four LRP5-HBM patients were included in this study. The clinical characteristics, biochemical and radiological examinations, pathogenic mutations, and structural analysis were summarized. Furthermore, several patients were followed up to observe the treatment effect and disease progress. RESULTS: Congenital blindness, persistent bone pain, low bone mineral density (BMD), and multiple brittle fractures were the main clinical manifestations of OPPG. Complex heterozygous mutations were detected in two OPPG patients. The c.1455G > T mutation in exon 7 was first reported. During the follow-up, BMD of two patients was significantly improved after bisphosphonate treatment. On the contrary, typical clinical features of LRP5-HBM included extremely high BMD without fractures, torus palatinus and normal vision. X-ray showed diffuse osteosclerosis. Two heterozygous missense mutations were detected in four patients. In addition, age-related bone loss was found in one LRP5-HBM patient after 12-year of follow-up. CONCLUSION: This study deepened the understanding of the clinical characteristics, treatment, and follow-up of OPPG and LRP5-HBM; expanded the pathogenic gene spectrum of OPPG; and confirmed that bisphosphonates were effective for OPPG. Additionally, it was found that Ala242Thr mutation could not protect LRP5-HBM patients from age-related bone loss. This phenomenon deserves further study.

Unraveling the genetic collagen connection: clinical and therapeutic insights on genetic connective tissue disorders.

Hereditary connective tissue disorders include more than 200 conditions affecting different organs and tissues, compromising the biological role of the extracellular matrix through interference in the synthesis, development, or secretion of collagen and/or its associated proteins. The clinical phenotype includes multiple signs and symptoms, usually nonspecific but of interest to rheumatologists because of musculoskeletal involvement. The patient´s journey to diagnosis is long, and physicians should include these disorders in their differential diagnoses of diseases with systemic involvement. In this review, insights for the diagnosis and treatment of osteogenesis imperfecta, hypermobility spectrum disorder/Ehlers-Danlos syndrome, Marfan, Loeys-Dietz, and Stickler syndromes are presented.

Exome sequencing identified mutations in the WNT1 and COL1A2 genes in osteogenesis imperfecta cases.

BACKGROUND: Osteogenesis imperfecta (OI) is a heritable connective tissue disorder characterized by bone deformities, fractures and reduced bone mass. OI can be inherited as a dominant, recessive, or X-linked disorder. The mutational spectrum has shown that autosomal dominant mutations in the type I collagen-encoding genes are responsible for OI in 85% of the cases. Apart from collagen genes, mutations in more than 20 other genes, such as CRTAP, CREB3L1, MBTPS2, P4HB, SEC24D, SPARC, FKBP10, LEPRE1, PLOD2, PPIB, SERPINF1, SERPINH1, SP7, WNT1, BMP1, TMEM38B, and IFITM5 have been reported in OI. METHODS AND RESULTS: To understand the genetic cause of OI in four cases, we conducted whole exome sequencing, followed by Sanger sequencing. In case #1, we identified a novel c.506delG homozygous mutation in the WNT1 gene, resulting in a frameshift and early truncation of the protein at the 197th amino acid. In cases #2, 3 and 4, we identified a heterozygous c.838G > A mutation in the COL1A2 gene, resulting in a p.Gly280Ser substitution. The clinvar frequency of this mutation is 0.000008 (GnomAD-exomes). This mutation has been identified by other studies as well and appears to be a mutational hot spot. These pathogenic mutations were found to be absent in 96 control samples analyzed for these sites. The presence of these mutations in the cases, their absence in controls, their absence or very low frequency in general population, and their evaluation using various in silico prediction tools suggested their pathogenic nature. CONCLUSIONS: Mutations in the WNT1 and COL1A2 genes explain these cases of osteogenesis imperfecta.

In Vitro Modelling of Osteogenesis Imperfecta with Patient-Derived Induced Mesenchymal Stem Cells.

(1) Mesenchymal stem cells (MSCs) are a valuable cell model to study the bone pathology of Osteogenesis Imperfecta (OI), a rare genetic collagen-related disorder characterized by bone fragility and skeletal dysplasia. We aimed to generate a novel OI induced mesenchymal stem cell (iMSC) model from induced pluripotent stem cells (iPSCs) derived from human dermal fibroblasts. For the first time, OI iMSCs generation was based on an intermediate neural crest cell (iNCC) stage. (2) Skin fibroblasts from healthy individuals and OI patients were reprogrammed into iPSCs and subsequently differentiated into iMSCs via iNCCs. (3) Successful generation of iPSCs from acquired fibroblasts was confirmed with changes in cell morphology, expression of iPSC markers SOX2, NANOG, and OCT4 and three germ-layer tests. Following differentiation into iNCCs, cells presented increased iNCC markers including P75NTR, TFAP2A, and HNK-1 and decreased iPSC markers, shown to reach the iNCC stage. Induction into iMSCs was confirmed by the presence of CD73, CD105, and CD90 markers, low expression of the hematopoietic, and reduced expression of the iNCC markers. iMSCs were trilineage differentiation-competent, confirmed using molecular analyses and staining for cell-type-specific osteoblast, adipocyte, and chondrocyte markers. (4) In the current study, we have developed a multipotent in vitro iMSC model of OI patients and healthy controls able to differentiate into osteoblast-like cells.