John P. Kemp

Utilising graph-based network analysis frameworks, 'Graphia' is a powerful open source visual analytics application developed to aid the interpretation of large and complex datasets. For more details, see article by Freeman et al. (2022) . 'gRaphia' is an extension of the 'Graphia' application within the R environment, providing tools for network analysis and visualisation. 'gRaphiaExtra' provides additional functionality specifically designed for single-cell RNA-sequencing data, enabling users to seamlessly integrate and utilise existing 'Seurat' analysis outputs in 'gRaphia'. The package also provides supplementary functions to support and enhance the 'gRaphia' analysis framework.

Neutrophils are among the first immune cells recruited to fractures, yet their contribution to bone repair remains poorly understood. Anti-inflammatory use has been linked to increased risk of non-union fractures, while prolonged neutrophil activity or failed clearance of neutrophil-derived DNA in the form of neutrophil extracellular traps (NETs), can also impair fracture healing. Using dynamic live imaging and cellular perturbation in adult zebrafish, we show that neutrophils play a key role in shaping the early fracture microenvironment. Neutrophils rapidly accumulated at fractures where they released NETs and exhibited the activation of gene programmes associated with matrix remodelling. Compromising neutrophil recruitment, via ibuprofen treatment or orthogonal genetic and pharmacological perturbations, dramatically increased incidence of fracture non-union and lead to altered callus architecture through delay of osteoblast differentiation and impaired mineralisation. Mechanistically, neutrophils actively interacted with key structural proteins, including laminin, fibronectin, and collagen I, through uptake, recycling and active de novo secretion pathways. Together, our findings reveal that neutrophils establish a provisional emergency fracture matrix that templates subsequent bone repair, providing a mechanistic link between early non-steroidal anti-inflammatory drug (NSAID) exposure and impaired skeletal healing.

Musculoskeletal diseases are a major health burden. Development of bone-active therapies has been hindered by limited understanding of the cells and genes that regulate the skeleton. We exploited the value of cross-species analysis and developed single-cell methodologies in skeletal tissues to define the critical endosteal compartment that regulates bone turnover. Thirty-four distinct cell types were identified, and disease-relevant cells prioritized using enrichment for rare skeletal disorder genes and bone-mineral-density-associated genes in an extended UK Biobank genome-wide association study. Functional validation was undertaken in over 1,000 genetically modified mouse models. Endothelial cells and vascular smooth muscle cells were identified as new skeletal-disease-relevant cells alongside osteoblast, chondrocyte and osteoclast cell lineages. Hundreds of cell-specific genes with unappreciated roles in skeletal pathophysiology were identified. This comprehensive cellular and molecular framework underpins skeletal physiology and disease and will help prioritize new therapeutic targets to accelerate development of therapies to treat musculoskeletal disease.

Clinical cardiovascular disease (CVD) is often present in frail individuals. However, it remains unclear whether subclinical CVD, e.g., abdominal aortic calcification (AAC), is associated with frailty. This study investigated the cross-sectional relationship between AAC scored using a validated machine learning model (ML-AAC24) and physical frailty. 49,081 participants from the UK Biobank Imaging Study without atherosclerotic CVD (ASCVD) diagnosis were included. ML-AAC24 extent was categorised as low, moderate and high, based on established severity categories. Physical frailty was based on a modified Fried’s frailty phenotype comprising weak hand grip strength, slow walking speed, weight loss, exhaustion, and physical inactivity. Individuals with three or more deficits were considered frail, while one or two deficits was considered pre-frail. Multivariable-adjusted multinominal logistic regression models were used to test the associations between ML-AAC24 extent and frailty status. One in five individuals had moderate or high ML-AAC24. Compared to individuals with low ML-AAC24, those with moderate and high ML-AAC24 had greater odds of being pre-frail (ORs 1.06 95%CI 1.00–1.12 moderate; 1.14 95%CI 1.04–1.26 high) or frail (ORs 1.27 95%CI 1.12–1.44 moderate; 1.58 95%CI 1.31–1.91 high), adjusted for multiple covariates. When stratified by sex, similar results for frailty were recorded. In a population, those with moderate and high ML-AAC24 were more likely to present as pre-frail and frail. AAC identified from lateral spine images obtained during routine bone density testing, could serve as a useful marker for the early detection of frailty, highlighting the importance of multimodality care. Graphical Abstract

Hip shape is thought to be an important causal risk factor for hip osteoarthritis and fracture. We aimed to identify genetic determinants of hip shape and use these to assess causal relationships with hip osteoarthritis. Statistical hip shape modelling was used to derive 10 hip shape modes (HSMs) from DXA images in UK Biobank and Shanghai Changfeng cohorts (n_total = 43 485). Genome-wide association study meta-analyses were conducted for each HSM. Two-sample Mendelian randomisation (MR) was used to estimate causal effects between HSM and hip osteoarthritis using hip fracture as a positive control. Analysis of the first 10 HSMs identified 203 independent association signals (P < 5 × 10⁻⁹). Hip shape SNPs were also associated (P < 2.5 × 10⁻⁴) with hip osteoarthritis (n = 26) and hip fracture (n = 4). Fine mapping implicated SMAD3 and PLEC as candidate genes that may be involved in the development of hip shape and hip osteoarthritis. MR analyses suggested there was no causal effect between any HSM and hip osteoarthritis, however there was evidence that HSM2 (more obtuse neck-shaft angle) and HSM4 (wider femoral neck) have a causal effect on hip fracture (OR_IVW method 1.27 [95% CI 1.12–1.44], P = 1.79 × 10⁻⁴ and OR_IVW 0.74 [0.65–0.84], P = 7.60 × 10⁻⁶ respectively). We report the largest hip shape GWAS meta-analysis that identifies hundreds of novel loci, some of which are also associated with hip osteoarthritis and hip fracture. MR analyses suggest hip shape may not cause hip osteoarthritis but is implicated in hip fractures. Consequently, interventions targeting hip shape in older adults to prevent hip osteoarthritis may prove ineffective.

Femoral neck width (FNW) derived from DXA scans may provide a useful adjunct to hip fracture prediction. Therefore, we investigated whether FNW is related to hip fracture risk independently of femoral neck bone mineral density (FN-BMD), using a genetic approach. FNW was derived from points automatically placed on the proximal femur using hip DXA scans from 38 150 individuals (mean age 63.8 yr, 48.0% males) in UK Biobank (UKB). Genome-wide association study (GWAS) identified 71 independent genome-wide significant FNW SNPs, comprising genes involved in cartilage differentiation, hedgehog, skeletal development, in contrast to SNPs identified by FN-BMD GWAS which primarily comprised runx1/Wnt signaling genes (MAGMA gene set analyses). FNW and FN-BMD SNPs were used to generate genetic instruments for multivariable Mendelian randomization. Greater genetically determined FNW increased risk of all hip fractures (odds ratio [OR] 1.53; 95% CI, 1.29–1.82 per SD increase) and femoral neck fractures (OR 1.58;1.30–1.92), but not trochanteric or forearm fractures. In contrast, greater genetically determined FN-BMD decreased fracture risk at all 4 sites. FNW and FN-BMD SNPs were also used to generate genetic risk scores (GRSs), which were examined in relation to incident hip fracture in UKB (excluding the FNW GWAS population; n = 338 742, 3222 cases) using a Cox proportional hazards model. FNW GRS was associated with increased risk of all incident hip fractures (HR 1.08;1.05–1.12) and femoral neck fractures (hazard ratio [HR] 1.10;1.06–1.15), but not trochanteric fractures, whereas FN-BMD GRS was associated with reduced risk of all hip fracture types. We conclude that the underlying biology regulating FNW and FN-BMD differs, and that DXA-derived FNW is causally related to hip fractures independently of FN-BMD, adding information beyond FN-BMD for hip fracture prediction. Hence, FNW derived from DXA analyses or a FNW GRS may contribute clinically useful information beyond FN-BMD for hip fracture prediction. Femoral neck width (FNW) derived from DXA scans may provide useful information about hip fracture prediction, over and above that provided by BMD measurements. Therefore, we investigated whether FNW is related to hip fracture risk independently of BMD, using a genetic approach. FNW was derived from points automatically placed on the hip in DXA scans obtained from 38 150 individuals (mean age 63.8 yr, 48.0% males) in UK Biobank. Seventy-one distinct genetic factors were found to be associated with FNW. Individuals who were predicted by their genes to have greater FNW had a higher risk of hip but not forearm fractures. In contrast, those with greater genetically determined BMD of the femoral neck had a lower risk of both hip and forearm fractures. We conclude that the underlying biology regulating FNW and BMD of the femoral neck differs, and that FNW derived from DXA analyses may contribute clinically useful information beyond BMD for hip fracture prediction.

Skull bone mineral density (SK-BMD) provides a suitable trait for the discovery of key genes in bone biology, particularly to intramembranous ossification, not captured at other skeletal sites. We perform a genome-wide association meta-analysis (n ~ 43,800) of SK-BMD, identifying 59 loci, collectively explaining 12.5% of the trait variance. Association signals cluster within gene-sets involved in skeletal development and osteoporosis. Among the four novel loci (ZIC1,PRKAR1A,AZIN1/ATP6V1C1,GLRX3), there are factors implicated in intramembranous ossification and as we show, inherent to craniosynostosis processes. Functional follow-up in zebrafish confirms the importance ofZIC1on cranial suture patterning. Likewise, we observe abnormal cranial bone initiation that culminates in ectopic sutures and reduced BMD in mosaicatp6v1c1knockouts. Mosaicprkar1aknockouts present asymmetric bone growth and, conversely, elevated BMD. In light of this evidence linking SK-BMD loci to craniofacial abnormalities, our study provides new insight into the pathophysiology, diagnosis and treatment of skeletal diseases.

Observational epidemiological studies have reported that higher maternal blood pressure (BP) during pregnancy is associated with increased future risk of offspring cardiometabolic disease. However, it is unclear whether this association represents a causal relationship through intrauterine mechanisms. We used a Mendelian randomization (MR) framework to examine the relationship between unweighted maternal genetic scores for systolic BP and diastolic BP and a range of cardiometabolic risk factors in the offspring of up to 29 708 genotyped mother-offspring pairs from the UKB study (UK Biobank) and the HUNT study (Trøndelag Health). We conducted similar analyses in up to 21 423 father-offspring pairs from the same cohorts. We confirmed that the BP-associated genetic variants from the general population sample also had similar effects on maternal BP during pregnancy in independent cohorts. We did not detect any association between maternal (or paternal) unweighted genetic scores and cardiometabolic offspring outcomes in the meta-analysis of UKB and HUNT after adjusting for offspring genotypes at the same loci. We find little evidence to support the notion that maternal BP is a major causal risk factor for adverse offspring cardiometabolic outcomes in later life.

Skull bone mineral density (SK-BMD) provides a suitable trait for the discovery of genes important to bone biology in general, and particularly for identifying components unique to intramembranous ossification, which cannot be captured at other skeletal sites. We assessed genetic determinants of SK-BMD in 43,800 individuals, identifying 59 genome-wide significant loci (4 novel), explaining 12.5% of its variance. Pathway and enrichment analyses of the association signals resulted in clustering within gene-sets involved in regulating the development of the skeleton; overexpressed in the musculoskeletal system; and enriched in enhancer and transcribed regions in osteoblasts. From the four novel loci (mapping to ZIC1, PRKAR1A, ATP6V1C1, GLRX3 ), two ( ZIC1 and PRKAR1A ) have previously been related to craniofacial developmental defects. Functional validation of skull development in zebrafish revealed abnormal cranial bone initiation that culminated in ectopic sutures and reduced BMD in mutated zic1 and atp6v1c1 fish and asymmetric bone growth and elevated BMD in mutated prkar1a fish. We confirmed a role of ZIC1 loss-of-function in suture patterning and discovered ATP6V1C1 gene associated with suture development. In light of the evidence presented suggesting that SK-BMD is genetically related to craniofacial abnormalities, our study opens new avenues to the understanding of the pathophysiology of craniofacial defects and towards the effective pharmacological treatment of bone diseases.