Abstract SATB2-Associated Syndrome (SAS) is an autosomal dominant genetic disorder caused by pathogenic variations in the special AT-rich sequence-binding protein 2 (SATB2) gene. In addition to neurodevelopmental and craniofacial defects, over 90% of patients with SAS manifest biochemical and/or radiographic skeletal abnormalities, and around one third of patients report clinical and/or radiographic fractures. SATB2 protein is a potent transcription factor that promotes osteoblast differentiation and maturation; loss-of-function pathogenic variations of the SATB2 gene result in a wide spectrum of skeletal abnormalities ranging from gross skeletal anomalies to abnormal bone turnover markers, low bone mineral density, and recurrent fractures. There is at present no known effective treatment for bone health in patients with SAS. We present an adult patient with SAS who has recurrent fractures despite long-term treatment with antiresorptive agents. We propose an alternative pharmacotherapy approach utilizing a PTH analogue to stimulate osteoblasts, hence addressing the underlying pathophysiology of bone disease in patients with SAS.

Abstract Although most fractures, and about half of hip fractures, occur outdoors among older women, limited research has uncovered neighborhood predictors for fractures among older women. This study assessed the independent associations of neighborhood socioeconomic status, walkability, and green space with incident any and hip fracture among postmenopausal women. The Women’s Health Initiative recruited a national sample of postmenopausal women (50-79 years) across 40 U.S. clinical centers and conducted yearly assessments from 1993 to 2012 (n = 161 808). Women reporting a history of hip fracture or walking limitations were excluded from the analytic sample, yielding a final sample of 157 583 participants. Fracture events were self-reported and adjudicated annually. Walkability was calculated annually using measures of population density, land use mix, and presence/quantity of nearby high-traffic roadways. Neighborhood green space was calculated annually using measures of exposure to trees/vegetation. Neighborhood SES, walkability, and green space were categorized intro tertiles: high, intermediate, and low. The time-varying relationship between neighborhood environmental factors and age at first fracture (any; hip) was examined using extended Cox proportional hazards modeling with adjustment. Neighborhood socioeconomic status (intermediate vs low: hazard ratio = 1.03, 95% Confidence Interval (CI): 1.01-1.05; high vs low, hazard ratio = 1.01, 95% CI: 0.99-1.03) and green space (intermediate vs low, hazard ratio = 1.15, 95% CI: 1.12-1.18; high vs low hazard ratio = 1.18, 95% CI: 1.15-1.21) were associated with increased any incident fractures, while walkability had a mixed association (intermediate vs low hazard ratio = 1.06, 95% CI: 1.04-1.07; high vs low, hazard ratio = 0.97, 95% CI: 0.95-0.98). Neighborhood socioeconomic status, walkability, and green space did not have a relationship with hip fracture after adjustment for important covariates. Results indicate that macroscale neighborhood features did not protect against fractures. Additional research is needed to investigate more granual neighborhood features that might influence injury risk and support physical activity among postmenopausal women.

Abstract Ossification of the spinal ligament (OSL), including ossification of the posterior longitudinal ligament (OPLL) and ossification of the ligamentum flavum (OLF), is a multifactorial disease that includes genetic predisposition. The association between the rate of ossification in the spinal canal and the severity of myelopathy symptoms is well known, but the degree of progression varies widely among patients. Although many candidate genes and biomarkers have been reported, there are no definitive and quantitative conclusions to date, probably because of low reproducibility due to individual differences. In this study, we focused on exosomes secreted by ossified spinal ligament cells. Exosomes are crucial for intercellular communication during development and progression of disease. In a co-culture study of non-OLF cells with OLF cells, there was increased osteogenic differentiation, including Runx2 and Wnt3a expression, with use of exosome-penetrating filters (1.2 μm) compared to exosome-non-penetrating filters (0.03 μm). Dose-dependent increases in alkaline phosphatase activity and mineral deposition were observed in non-OLF cells treated with OLF-derived exosomes. These results support the hypothesis that OLF-derived exosomes are involved in regulation of osteogenic differentiation. In comparative proteomics analysis, 32 factors were increased and 40 were decreased in OLF-derived exosomes compared to non-OLF-derived exosomes. Molecular network analysis of these 72 factors indicated 10 significant pathways, including the MMP signaling, mTOR signaling, Wnt signaling and VDR-associated pathways. Among the upregulated exosomal membrane proteins in OLF samples, COL IV, FMNL3, mTORC2, and PIP4K showed increased expression with greater ossification, suggesting they may serve as biomarkers of disease activity and therapeutic targets. These factors are involved in the PI3K/Akt/mTOR signaling pathway, and particularly mTOR is known to regulate osteogenic and chondrogenic differentiation. In contrast, FABP5, several KRT family proteins, S100A8, SERPINB3, and TGM, were significantly downregulated in OLF-derived exosomes. These findings provide novel insights into the molecular mechanisms underlying OSL pathogenesis.

Abstract X-linked hypophosphatemia (XLH) is a rare inherited disorder characterized by elevated levels of fibroblast growth factor-23 (FGF23), chronic hypophosphatemia, impaired bone mineralization and chronic long-term manifestations. Treatment for XLH has been mainly focused on normalizing its biochemical abnormalities. Despite treatment, patients with XLH often present impaired physical function and decreased quality of life (QoL). We hypothesize that physical functionality and QoL is more strongly associated with chronic pain and decreased muscle mass than persistent biochemical abnormalities or exposure to conventional treatment. We conducted an observational, cross-sectional study with patients with XLH. Clinical records and biochemical parameters were assessed. QoL surveys SF36v.2 and WOMAC were applied. Functional status was measured by a physiatrist and an occupational therapist. Appendicular lean mass (ALM) was measured and compared to age and sex-matched healthy controls. Enthesopathies and osteoarthritis were evaluated. Pain was assessed using the Brief Pain Inventory, the Visual Analog Scale and the Doleur Neuropathique-4 scales. Muscle strength was evaluated by the Quadriceps Muscle isometric Strength (QMS) and physical performance with the 6-minute walk test (6MWT) and the Functional Independence Measure (FIM) scale. A total of 30 patients were included: 21 females; median age of 32 years. All participants had significant functional deficits, chronic pain and reduced QoL. Limitations in daily activities were significantly associated with higher severity of pain, decreased ALM, lower QMS, and less distance in 6MWT (P<.05). Neither FIM scale, phosphate levels, FGF23, nor the lifetime exposure to conventional treatment were associated with these functional variables. In conclusion, impaired physical functionality in patients with XLH was associated with lower muscle mass, lower muscle strength and severe chronic pain. These findings highlight the importance of, in addition to optimizing the biochemical control of the disease, expanding patient care including pain prevention and management as well as comprehensive physical therapy and rehabilitation.

Abstract Osteogenesis imperfecta (OI) is a genetic condition with improperly or inadequately produced Type I collagen. Manifestations include bowing deformities, fractures, hydrocephalus, respiratory insufficiency and feeding difficulty. Moderate or severe OI is often diagnosed prenatally based on ultrasound findings and genetic testing may be labeled as lethal. Here we present 18 infants with moderate to severely presenting OI who received neonatal care at a single center over a 5 year period, 10 of which were delivered at our institution. All 18 infants survived to neonatal discharge, with seven infants requiring respiratory support and nine infants requiring feeding support at discharge. Through Fisher Exact Test, Mann Whitney U Test and backward elimination regression, we do not observe that lethal or possibly lethal diagnoses prenatally were correlated with medically relevant outcomes such as need for respiratory support at discharge or need for feeding support at discharge. 16/18 individuals are alive, with a minority requiring either respiratory or feeding support. With a multidisciplinary team approach to neonatal care, outcomes may be optimized. Infants formerly diagnosed with lethal OI may survive. Given our findings, and lack of correlation of prenatal assessments with survival and other medical outcomes, we recommend all families be given the option to pursue medical interventions.

Activating internal tandem duplications (ITD) in the juxtamembrane domain of receptor tyrosine kinase FLT3 occur frequently in patients with acute myeloid leukemia (AML). Constitutive active FLT3-ITD mutations induce aberrant signaling and promote leukemic cell transformation. Inactivation of the attenuating receptor protein tyrosine phosphatase CD45 (PTPRC) in FLT3-ITD mice resulted in the development of a severe hematopoietic phenotype with characteristics of AML. In addition, abnormal bone structures and ectopic bone formation were observed in these mice, suggesting a previously unknown role of FLT3 to control bone development and remodeling. While Ptprc knockout and Flt3-ITD mutant mice showed a largely normal bone microarchitecture, micro-CT analysis of femurs from Flt3-ITD Ptprc knockout mice revealed trabecularization of the cortical bone. This resulted in increased trabecular bone volume at the metaphysis, while the cortical bone at the diaphysis was thinner and less dense. In the metaphysis, severely reduced osteoclast and osteoblast numbers were observed. Reduced capacity of ex vivo differentiation of CD11b-positive bone marrow stem cells to mature osteoclast was accompanied by their abnormal morphology and reduced size. Transcriptome analysis revealed reduced expression of osteoclastogenic genes. Unexpectedly, cumulative resorption activity of osteoclasts was increased. Size and structure of resorption pits of differentiated osteoclasts remained similar to those observed in osteoclast cultures derived from control animals. Enhanced proliferation of cells in osteoclast cultures derived from FLT3-ITD-expressing mice was mediated by increased expression of STAT5 target genes. Transcriptome analysis of differentiated osteoclasts showed dysregulated signaling pathways influencing their differentiation as well as the coupling of bone resorption and formation. Taken together, inactivation of attenuating CD45 in mice expressing oncogenic FLT3-ITD resulted in marked abnormalities of the osteo-hematopoietic niche, which can be explained by aberrant STAT5 activation.

Abstract Generalized arterial calcification of infancy (GACI) and autosomal recessive hypophosphatemic rickets type 2 (ARHR2) are age-related phenotypes of the rare genetic mineralization disorder, ENPP1 Deficiency, which evolve on a phenotypic continuum. To date, our understanding of the clinical spectrum of ENPP1 Deficiency is based on small studies or case reports, across which there is significant variability in clinical presentation, and limited duration of follow-up. From a previously published large retrospective natural history study, we performed a subgroup analysis to elucidate the most prevalent signs and symptoms of ENPP1 Deficiency diagnosed as GACI or ARHR2 to illustrate the onset and incidence of these complications over the lifetime, and to characterize the associated medical burden of disease. Of the 84 individuals with ENPP1 Deficiency analyzed, 51 had a recorded diagnosis of GACI, 11 were diagnosed with ARHR2, and 22 were diagnosed with both. We confirmed that those diagnosed with GACI presented predominantly with early-onset arterial calcification, respiratory distress, heart failure, and hypertension, necessitating acute inpatient care and leading to high (44%) infant mortality. Notably, we found that the majority (60.3%) of those with a history of GACI had prenatal ultrasound anomalies, including effusions, polyhydramnios, and hydrops fetalis. We estimated that 70% of individuals with ENPP1 Deficiency who survive to age 10 will have developed musculoskeletal complications, primarily rickets and/or osteomalacia. The clinical picture of ARHR2 in this study extended beyond skeletal deformities to include hearing impairment, joint involvement, and ongoing risk of cardiovascular problems. This study sheds light on the signs and symptoms of ENPP1 Deficiency in the real world, with implications for life-long patient monitoring.

Abstract Supine sagittal abdominal diameter (SAD), also known as abdominal height, has been proposed as a simple measure for assessing abdominal adiposity. We aimed to determine whether SAD from dual-energy X-ray absorptiometry (DXA) performed for osteoporosis assessment predicts major adverse cardiovascular events (MACE) using the population-based DXA registry for the Province of Manitoba, Canada. The study population comprised 72,974 individuals aged 40 years and older with baseline DXA assessment between February 1999 and March 2018. Incident MACE (composite of all-cause mortality, acute myocardial infarction [MI], non-hemorrhagic stroke) was ascertained from linked healthcare databases. During mean 8.4 years follow-up (611,862 person-years), 14,457 (18.8%) individuals experienced incident MACE. Risk stratification was greatest with SAD/weight ratio, with area under the curve (AUC) for MACE and its components ranging from 0.582 for acute MI to 0.620 for death (all p<0.001), all significantly better than with body mass index (BMI) (p<0.001). In multivariable-adjusted models, each SD increase in SAD/weight was associated with increased risk for MACE (hazards ratio [HR] 1.20, 95% CI 1.18-1.22), death (HR 1.22, 95% CI 1.20-1.25), acute MI (HR 1.19, 95% CI 1.14- 1.24), and stroke (HR 1.17, 95% CI 1.12-1.22). A linear gradient was seen across SAD/weight quintiles (all p-trend<0.001), with adjusted HR for MACE 1.61 (95% CI 1.50-1.72) for highest versus lowest quintile. Results were similar when further adjusted for BMI, in non-obese and obese individuals (p-interaction for obesity = 0.141), and in both women and men (p-interaction for sex = 0.471). In conclusion, SAD measured opportunistically at the time of DXA testing is predictive of death and major cardiovascular events in individuals undergoing osteoporosis assessment.

Abstract Recent clinical studies have established a strong association between cigarette smoking and degenerative disc disease. Both in vitro and in vivo research indicated that cigarette smoke disrupts cellular homeostasis in the intervertebral disc (IVD), leading to spatiotemporal remodeling of the extracellular matrix, with a notable reduction in solute diffusivity within the cartilage endplate (CEP). As the CEP serves as a critical mechanical barrier and solute diffusion pathway for the IVD, both roles can be compromised by pathological changes in the tissue. This underscores the need for a more comprehensive examination of endplate remodeling during IVD degeneration, particularly in the context of cigarette smoking and cessation. The objective of this study was to perform a quantitative analysis of the structure-material property relationship changes in the endplate at tissue and cellular levels to determine how endplate mineralization progresses during IVD degeneration in the context of cigarette smoke exposure and cessation, using our previously developed Sprague–Dawley rat model. Our results indicates that cigarette smoke exposure induced endplate remodeling is characterized by a higher CEP histological grade, increased aberrant CEP calcification level, and elevated bony endplate surface flatness score, all of which correlated with an accelerated chondrocyte cell life cycle. Smoke cessation alone was insufficient to reverse the mineralization progression in the endplate. Principal component analysis further identified alterations in endplate morphometry at the tissue level and disruptions in the chondrocyte life cycle at cellular level as key markers of degenerative remodeling. These findings establish endplate remodeling as a key indicator of smoke exposure induced IVD degeneration and inform the development of novel therapeutic strategies aimed at preserving or improving disc health.