Identification of Transcripts with Shared Roles in the Pathogenesis of Postmenopausal Osteoporosis and Cardiovascular Disease

Epidemiological evidence suggests existing comorbidity between postmenopausal osteoporosis (OP) and cardiovascular disease (CVD), but identification of possible shared genes is lacking. The skeletal global transcriptomes were analyzed in trans-iliac bone biopsies (n = 84) from clinically well-characterized postmenopausal women (50 to 86 years) without clinical CVD using microchips and RNA sequencing. One thousand transcripts highly correlated with areal bone mineral density (aBMD) were further analyzed using bioinformatics, and common genes overlapping with CVD and associated biological mechanisms, pathways and functions were identified. Fifty genes (45 mRNAs, 5 miRNAs) were discovered with established roles in oxidative stress, inflammatory response, endothelial function, fibrosis, dyslipidemia and osteoblastogenesis/calcification. These pleiotropic genes with possible CVD comorbidity functions were also present in transcriptomes of microvascular endothelial cells and cardiomyocytes and were differentially expressed between healthy and osteoporotic women with fragility fractures. The results were supported by a genetic pleiotropy-informed conditional False Discovery Rate approach identifying any overlap in single nucleotide polymorphisms (SNPs) within several genes encoding aBMD- and CVD-associated transcripts. The study provides transcriptional and genomic evidence for genes of importance for both BMD regulation and CVD risk in a large collection of postmenopausal bone biopsies. Most of the transcripts identified in the CVD risk categories have no previously recognized roles in OP pathogenesis and provide novel avenues for exploring the mechanistic basis for the biological association between CVD and OP.</p

In silico discovery of blood cell macromolecular associations

Background Physical molecular interactions are the basis of intracellular signalling and gene regulatory networks, and comprehensive, accessible databases are needed for their discovery. Highly correlated transcripts may reflect important functional associations, but identification of such associations from primary data are cumbersome. We have constructed and adapted a user-friendly web application to discover and identify putative macromolecular associations in human peripheral blood based on significant correlations at the transcriptional level. Methods The blood transcriptome was characterized by quantification of 17,328 RNA species, including 341 mature microRNAs in 105 clinically well-characterized postmenopausal women. Intercorrelation of detected transcripts signal levels generated a matrix with > 150 million correlations recognizing the human blood RNA interactome. The correlations with calculated adjusted p-values were made easily accessible by a novel web application. Results We found that significant transcript correlations within the giant matrix reflect experimentally documented interactions involving select ubiquitous blood relevant transcription factors (CREB1, GATA1, and the glucocorticoid receptor (GR, NR3C1)). Their responsive genes recapitulated up to 91% of these as significant correlations, and were replicated in an independent cohort of 1204 individual blood samples from the Framingham Heart Study. Furthermore, experimentally documented mRNAs/miRNA associations were also reproduced in the matrix, and their predicted functional co-expression described. The blood transcript web application is available at http://app.uio.no/med/klinmed/correlation-browser/blood/index.php and works on all commonly used internet browsers. Conclusions Using in silico analyses and a novel web application, we found that correlated blood transcripts across 105 postmenopausal women reflected experimentally proven molecular associations. Furthermore, the associations were reproduced in a much larger and more heterogeneous cohort and should therefore be generally representative. The web application lends itself to be a useful hypothesis generating tool for identification of regulatory mechanisms in complex biological data sets.publishedVersio

Identification of Transcripts with Shared Roles in the Pathogenesis of Postmenopausal Osteoporosis and Cardiovascular Disease

Epidemiological evidence suggests existing comorbidity between postmenopausal osteoporosis (OP) and cardiovascular disease (CVD), but identification of possible shared genes is lacking. The skeletal global transcriptomes were analyzed in trans-iliac bone biopsies (n = 84) from clinically well-characterized postmenopausal women (50 to 86 years) without clinical CVD using microchips and RNA sequencing. One thousand transcripts highly correlated with areal bone mineral density (aBMD) were further analyzed using bioinformatics, and common genes overlapping with CVD and associated biological mechanisms, pathways and functions were identified. Fifty genes (45 mRNAs, 5 miRNAs) were discovered with established roles in oxidative stress, inflammatory response, endothelial function, fibrosis, dyslipidemia and osteoblastogenesis/calcification. These pleiotropic genes with possible CVD comorbidity functions were also present in transcriptomes of microvascular endothelial cells and cardiomyocytes and were differentially expressed between healthy and osteoporotic women with fragility fractures. The results were supported by a genetic pleiotropy-informed conditional False Discovery Rate approach identifying any overlap in single nucleotide polymorphisms (SNPs) within several genes encoding aBMD- and CVD-associated transcripts. The study provides transcriptional and genomic evidence for genes of importance for both BMD regulation and CVD risk in a large collection of postmenopausal bone biopsies. Most of the transcripts identified in the CVD risk categories have no previously recognized roles in OP pathogenesis and provide novel avenues for exploring the mechanistic basis for the biological association between CVD and OP.</p

Genetic polymorphism of miR-196a-2 is associated with bone mineral density (BMD)

MicroRNAs (miRNAs) are small non-coding RNA molecules that post-transcriptionally regulate the translation of messenger RNAs. Given the crucial role of miRNAs in gene expression, genetic variants within miRNA-related sequences may affect miRNA function and contribute to disease risk. Osteoporosis is characterized by reduced bone mass, and bone mineral density (BMD) is a major diagnostic proxy to assess osteoporosis risk. Here, we aimed to identify miRNAs that are involved in BMD using data from recent genome-wide association studies (GWAS) on femoral neck, lumbar spine and forearm BMD. Of 242 miRNA-variants available in the GWAS data, we found rs11614913:C > T in the precursor miR-196a-2 to be significantly associated with femoral neck-BMD (p-value = 9.9 × 10-7, β = −0.038) and lumbar spine-BMD (p-value = 3.2 × 10-11, β = −0.061). Furthermore, our sensitivity analyses using the Rotterdam study data showed a sex-specific association of rs11614913 with BMD only in women. Subsequently, we highlighted a number of miR-196a-2 target genes, expressed in bone and associated with BMD, that may mediate the miRNA function in BMD. Collectively, our results suggest that miR-196a-2 may contribute to variations in BMD level. Further biological investigations will give more insights into the mechanisms by which miR-196a-2 control expression of BMD-related genes

POLD2 and KSP37 (FGFBP2) Correlate Strongly with Histology, Stage and Outcome in Ovarian Carcinomas

BACKGROUND:Epithelial ovarian cancer (EOC) constitutes more than 90% of ovarian cancers and is associated with high mortality. EOC comprises a heterogeneous group of tumours, and the causes and molecular pathology are essentially unknown. Improved insight into the molecular characteristics of the different subgroups of EOC is urgently needed, and should eventually lead to earlier diagnosis as well as more individualized and effective treatments. Previously, we reported a limited number of mRNAs strongly upregulated in human osteosarcomas and other malignancies, and six were selected to be tested for a possible association with three subgroups of ovarian carcinomas and clinical parameters. METHODOLOGY/PRINCIPAL FINDINGS:The six selected mRNAs were quantified by RT-qPCR in biopsies from eleven poorly differentiated serous carcinomas (PDSC, stage III-IV), twelve moderately differentiated serous carcinomas (MDSC, stage III-IV) and eight clear cell carcinomas (CCC, stage I-IV) of the ovary. Superficial scrapings from six normal ovaries (SNO), as well as biopsies from three normal ovaries (BNO) and three benign ovarian cysts (BBOC) were analyzed for comparison. The gene expression level was related to the histological and clinical parameters of human ovarian carcinoma samples. One of the mRNAs, DNA polymerase delta 2 small subunit (POLD2), was increased in average 2.5- to almost 20-fold in MDSC and PDSC, respectively, paralleling the degree of dedifferentiation and concordant with a poor prognosis. Except for POLD2, the serous carcinomas showed a similar transcription profile, being clearly different from CCC. Another mRNA, Killer-specific secretory protein of 37 kDa (KSP37) showed six- to eight-fold higher levels in CCC stage I compared with the more advanced staged carcinomas, and correlated positively with an improved clinical outcome. CONCLUSIONS/SIGNIFICANCE:We have identified two biomarkers which are markedly upregulated in two subgroups of ovarian carcinomas and are also associated with stage and outcome. The results suggest that POLD2 and KSP37 might be potential prognostic biomarkers

Distinct Subsets of Noncoding RNAs Are Strongly Associated With BMD and Fracture, Studied in Weight-Bearing and Non–Weight-Bearing Human Bone

We investigated mechanisms resulting in low bone mineral density (BMD) and susceptibility to fracture by comparing noncoding RNAs (ncRNAs) in biopsies of non–weight-bearing (NWB) iliac (n = 84) and weight bearing (WB) femoral (n = 18) postmenopausal bone across BMDs varying from normal (T-score > −1.0) to osteoporotic (T-score ≤ −2.5). Global bone ncRNA concentrations were determined by PCR and microchip analyses. Association with BMD or fracture, adjusted by age and body mass index, were calculated using linear and logistic regression and least absolute shrinkage and selection operator (Lasso) analysis. At 10% false discovery rate (FDR), 75 iliac bone ncRNAs and 94 femoral bone ncRNAs were associated with total hip BMD. Eight of the ncRNAs were common for the two sites, but five of them (miR-484, miR-328-3p, miR-27a-5p, miR-28-3p, and miR-409-3p) correlated positively to BMD in femoral bone, but negatively in iliac bone. Of predicted pathways recognized in bone metabolism, ECM-receptor interaction and prote

Identification of a novel locus on chromosome 2q13, which predisposes to clinical vertebral fractures independently of bone density

OBJECTIVES: To identify genetic determinants of susceptibility to clinical vertebral fractures, which is an important complication of osteoporosis. METHODS: Here we conduct a genome-wide association study in 1553 postmenopausal women with clinical vertebral fractures and 4340 controls, with a two-stage replication involving 1028 cases and 3762 controls. Potentially causal variants were identified using expression quantitative trait loci (eQTL) data from transiliac bone biopsies and bioinformatic studies. RESULTS: A locus tagged by rs10190845 was identified on chromosome 2q13, which was significantly associated with clinical vertebral fracture (P=1.04×10-9) with a large effect size (OR 1.74, 95% CI 1.06 to 2.6). Bioinformatic analysis of this locus identified several potentially functional SNPs that are associated with expression of the positional candidate genes TTL (tubulin tyrosine ligase) and SLC20A1 (solute carrier family 20 member 1). Three other suggestive loci were identified on chromosomes 1p31, 11q12 and 15q11. All these loci were novel and had not previously been associated with bone mineral density or clinical fractures. CONCLUSION: We have identified a novel genetic variant that is associated with clinical vertebral fractures by mechanisms that are independent of BMD. Further studies are now in progress to validate this association and evaluate the underlying mechanism.Funding: ORCADES was supported by the Chief Scientist Office of the Scottish Government (CZB/4/276, CZB/4/710), the Royal Society, the MRC Human Genetics Unit, Arthritis Research UK and the European Union framework programme 6 EUROSPAN project (contract no. LSHG-CT-2006-018947). DNA extractions were performed at the Wellcome Trust Clinical Research Facility in Edinburgh. We would like to acknowledge the invaluable contributions of Lorraine Anderson and the research nurses in Orkney, the administrative team in Edinburgh and the people of Orkney. CABRIO was supported by the Instituto de Salud Carlos III and Fondos FEDER from the EU (PI 11/1092 and PI12/615). The AOGC study was funded by the Australian National Health and Medical Research Council (Project grant 511132). Lothian Birth Cohort 1921 phenotype collection was supported by the UK’s Biotechnology and Biological Sciences Research Council (BBSRC), The Royal Society and The Chief Scientist Office of the Scottish Government. Phenotype collection in the Lothian Birth Cohort 1936 was supported by Age UK (The Disconnected Mind project). Genotyping of the cohorts was funded by the BBSRC. The work was undertaken by the University of Edinburgh Centre for Cognitive Ageing and Cognitive Epidemiology, part of the cross council Lifelong Health and Wellbeing Initiative (MR/K026992/1). Funding from the BBSRC and Medical Research Council (MRC) is gratefully acknowledged. Research work on Slovenian case and control samples was funded by Slovenian Research Agency (project no. P3-0298 and J3-2330). The Danish National Birth Cohort (DNBC) is a result of major grants from the Danish National Research Foundation, the Danish Pharmacists’Fund, the Egmont Foundation, the March of Dimes Birth Defects Foundation, the Augustinus Foundation and the Health Fund of the Danish Health Insurance Societies. The DNBC biobank is a part of the Danish National Biobank resource, which is supported by the Novo Nordisk Foundation. Dr Bjarke Feenstra is supported by an Oak Foundation Fellowship. The Framingham Study was funded by grants from the US National Institute for Arthritis, Musculoskeletal and Skin Diseases and National Institute on Aging (R01 AR 41398 and R01 AR061162; DPK and R01 AR 050066; DK). The Framingham Heart Study of the National Heart, Lung, and Blood Institute of the National Institutes of Health and Boston University School of Medicine were supported by the National Heart, Lung, and Blood Institute’s Framingham Heart Study (N01-HC-25195) and its contract with Affymetrix, Inc. for genotyping services (N02-HL-6-4278). Analyses reflect intellectual input and resource development from the Framingham Heart Study investigators participating in the SNP Health Association Resource (SHARe) project. A portion of this research was conducted using the Linux Cluster for Genetic Analysis (LinGA-II) funded by the Robert Dawson Evans Endowment of the Department of Medicine at Boston University School of Medicine and Boston Medical Center. This research was performed within the Genetic Factors for Osteoporosis (GEFOS) consortium, funded by the European Commission (HEALTH-F2-2008-201865-GEFOS).Acknowledgments: The authors are grateful to the patients and controls from the different centres who agreed to participate in this study. We would like to thank Ms Dilruba Kabir at the Rheumatology and Bone Disease Unit, CGEM-IGMM, Edinburgh, UK; Mr Matt Sims at the MRC Epidemiology Unit, University of Cambridge, UK; Ms Mila Jhamai and Ms Sarah Higgins at the Genetics Laboratory of Erasmus MC, Rotterdam, The Netherlands; Ms Johanna Hadler, Ms Kathryn A Addison and Ms Karena Pryce of the University of Queensland Centre for Clinical Genomics, Brisbane, Australia, for technical support on the genotyping stage; and Mr Marijn Verkerk and Dr Anis Abuseiris at the Genetics Laboratory of Erasmus MC, Rotterdam, for assistance on the data analysis. We would like to acknowledge the invaluable contributions of Lorraine Anderson and the research nurses in Orkney, the administrative team in Edinburgh and the people of Orkney. We would also like to thank Professor Nick Gilbert and Dr Giovanny Rodriguez-Blanco for their comments and advice on the manuscript preparation. This study makes use of data generated by the Wellcome Trust Case Control Consortium. A full list of the investigators who contributed to the generation of the data is available at www.wtccc.org.uk

Skeletal Site-Related Variation in Human Trabecular Bone Transcriptome and Signaling

BACKGROUND: The skeletal site-specific influence of multiple genes on bone morphology is recognised, but the question as to how these influences may be exerted at the molecular and cellular level has not been explored. METHODOLOGY: To address this question, we have compared global gene expression profiles of human trabecular bone from two different skeletal sites that experience vastly different degrees of mechanical loading, namely biopsies from iliac crest and lumbar spinal lamina. PRINCIPAL FINDINGS: In the lumbar spine, compared to the iliac crest, the majority of the differentially expressed genes showed significantly increased levels of expression; 3406 transcripts were up- whilst 838 were down-regulated. Interestingly, all gene transcripts that have been recently demonstrated to be markers of osteocyte, as well as osteoblast and osteoclast-related genes, were markedly up-regulated in the spine. The transcriptome data is consistent with osteocyte numbers being almost identical at the two anatomical sites, but suggesting a relatively low osteocyte functional activity in the iliac crest. Similarly, osteoblast and osteoclast expression data suggested similar numbers of the cells, but presented with higher activity in the spine than iliac crest. This analysis has also led to the identification of expression of a number of transcripts, previously known and novel, which to our knowledge have never earlier been associated with bone growth and remodelling. CONCLUSIONS AND SIGNIFICANCE: This study provides molecular evidence explaining anatomical and micro-architectural site-related changes in bone cell function, which is predominantly attributable to alteration in cell transcriptional activity. A number of novel signaling molecules in critical pathways, which have been hitherto not known to be expressed in bone cells of mature vertebrates, were identified

Genetic sharing with cardiovascular disease risk factors and diabetes reveals novel bone mineral density loci

Bone Mineral Density (BMD) is a highly heritable trait, but genome-wide association studies have identified few genetic risk factors. Epidemiological studies suggest associations between BMD and several traits and diseases, but the nature of the suggestive comorbidity is still unknown. We used a novel genetic pleiotropy-informed conditional False Discovery Rate (FDR) method to identify single nucleotide polymorphisms (SNPs) associated with BMD by leveraging cardiovascular disease (CVD) associated disorders and metabolic traits. By conditioning on SNPs associated with the CVD-related phenotypes, type 1 diabetes, type 2 diabetes, systolic blood pressure, diastolic blood pressure, high density lipoprotein, low density lipoprotein, triglycerides and waist hip ratio, we identified 65 novel independent BMD loci (26 with femoral neck BMD and 47 with lumbar spine BMD) at conditional FDR &lt; 0.01. Many of the loci were confirmed in genetic expression studies. Genes validated at the mRNA levels were characteristic for the osteoblast/osteocyte lineage, Wnt signaling pathway and bone metabolism. The results provide new insight into genetic mechanisms of variability in BMD, and a better understanding of the genetic underpinnings of clinical comorbidity.</p

Lowering of Circulating Sclerostin May Increase Risk of Atherosclerosis and Its Risk Factors: Evidence From a Genome-Wide Association Meta-Analysis Followed by Mendelian Randomization

OBJECTIVE: In this study, we aimed to establish the causal effects of lowering sclerostin, target of the antiosteoporosis drug romosozumab, on atherosclerosis and its risk factors. METHODS: A genome-wide association study meta-analysis was performed of circulating sclerostin levels in 33,961 European individuals. Mendelian randomization (MR) was used to predict the causal effects of sclerostin lowering on 15 atherosclerosis-related diseases and risk factors. RESULTS: We found that 18 conditionally independent variants were associated with circulating sclerostin. Of these, 1 cis signal in SOST and 3 trans signals in B4GALNT3, RIN3, and SERPINA1 regions showed directionally opposite signals for sclerostin levels and estimated bone mineral density. Variants with these 4 regions were selected as genetic instruments. MR using 5 correlated cis-SNPs suggested that lower sclerostin increased the risk of type 2 diabetes mellitus (DM) (odds ratio [OR] 1.32 [95% confidence interval (95% CI) 1.03-1.69]) and myocardial infarction (MI) (OR 1.35 [95% CI 1.01-1.79]); sclerostin lowering was also suggested to increase the extent of coronary artery calcification (CAC) (β = 0.24 [95% CI 0.02-0.45]). MR using both cis and trans instruments suggested that lower sclerostin increased hypertension risk (OR 1.09 [95% CI 1.04-1.15]), but otherwise had attenuated effects. CONCLUSION: This study provides genetic evidence to suggest that lower levels of sclerostin may increase the risk of hypertension, type 2 DM, MI, and the extent of CAC. Taken together, these findings underscore the requirement for strategies to mitigate potential adverse effects of romosozumab treatment on atherosclerosis and its related risk factors