MiRNA profiling of whole trabecular bone: identification of osteoporosis-related changes in MiRNAs in human hip bones

Background MicroRNAs (miRNAs) are important regulators of gene expression, with documented roles in bone metabolism and osteoporosis, suggesting potential therapeutic targets. Our aim was to identify miRNAs differentially expressed in fractured vs nonfractured bones. Additionally, we performed a miRNA profiling of primary osteoblasts to assess the origin of these differentially expressed miRNAs. Methods Total RNA was extracted from (a) fresh femoral neck trabecular bone from women undergoing hip replacement due to either osteoporotic fracture (OP group, n = 6) or osteoarthritis in the absence of osteoporosis (Control group, n = 6), matching the two groups by age and body mass index, and (b) primary osteoblasts obtained from knee replacement due to osteoarthritis (n = 4). Samples were hybridized to a microRNA array containing more than 1900 miRNAs. Principal component analysis (PCA) plots and heat map hierarchical clustering were performed. For comparison of expression levels, the threshold was set at log fold change > 1.5 and a p-value < 0.05 (corrected for multiple testing). Results Both PCA and heat map analyses showed that the samples clustered according to the presence or absence of fracture. Overall, 790 and 315 different miRNAs were detected in fresh bone samples and in primary osteoblasts, respectively, 293 of which were common to both groups. A subset of 82 miRNAs was differentially expressed (p < 0.05) between osteoporotic and control osteoarthritic samples. The eight miRNAs with the lowest p-values (and for which a validated miRNA qPCR assay was available) were assayed, and two were confirmed: miR-320a and miR-483-5p. Both were over-expressed in the osteoporotic samples and expressed in primary osteoblasts. miR-320a is known to target CTNNB1 and predicted to regulate RUNX2 and LEPR, while miR-483-5p down-regulates IGF2. We observed a reduction trend for this target gene in the osteoporotic bone. Conclusions We identified two osteoblast miRNAs over-expressed in osteoporotic fractures, which opens novel prospects for research and therapy

Genetic Analysis of High Bone Mass Cases from the BARCOS Cohort of Spanish Postmenopausal Women

The aims of the study were to establish the prevalence of high bone mass (HBM) in a cohort of Spanish postmenopausal women (BARCOS) and to assess the contribution of LRP5 and DKK1 mutations and of common bone mineral density (BMD) variants to a HBM phenotype. Furthermore, we describe the expression of several osteoblast-specific and Wnt-pathway genes in primary osteoblasts from two HBM cases. A 0.6% of individuals (10/1600) displayed Z-scores in the HBM range (sum Z-score >4). While no mutation in the relevant exons of LRP5 was detected, a rare missense change in DKK1 was found (p.Y74F), which cosegregated with the phenotype in a small pedigree. Fifty-five BMD SNPs from Estrada et al. [NatGenet 44:491-501,2012] were genotyped in the HBM cases to obtain risk scores for each individual. In this small group of samples, Z-scores were found inversely related to risk scores, suggestive of a polygenic etiology. There was a single exception, which may be explained by a rare penetrant genetic variant, counterbalancing the additive effect of the risk alleles. The expression analysis in primary osteoblasts from two HBM cases and five controls suggested that IL6R, DLX3, TWIST1 and PPARG are negatively related to Z-score. One HBM case presented with high levels of RUNX2, while the other displayed very low SOX6. In conclusion, we provide evidence of lack of LRP5 mutations and of a putative HBM-causing mutation in DKK1. Additionally, we present SNP genotyping and expression results that suggest additive effects of several genes for HBM

Genetic analysis of high bone mass cases from the BARCOS cohort of spanish postmenopausal women

The aims of the study were to establish the prevalence of high bone mass (HBM) in a cohort of Spanish postmenopausal women (BARCOS) and to assess the contribution of LRP5 and DKK1 mutations and of common bone mineral density (BMD) variants to a HBM phenotype. Furthermore, we describe the expression of several osteoblast-specific and Wnt-pathway genes in primary osteoblasts from two HBM cases. A 0.6% of individuals (10/1600) displayed Z-scores in the HBM range (sum Z-score >4). While no mutation in the relevant exons of LRP5 was detected, a rare missense change in DKK1 was found (p.Y74F), which cosegregated with the phenotype in a small pedigree. Fifty-five BMD SNPs from Estrada et al. [NatGenet 44:491-501,2012] were genotyped in the HBM cases to obtain risk scores for each individual. In this small group of samples, Z-scores were found inversely related to risk scores, suggestive of a polygenic etiology. There was a single exception, which may be explained by a rare penetrant genetic variant, counterbalancing the additive effect of the risk alleles. The expression analysis in primary osteoblasts from two HBM cases and five controls suggested that IL6R, DLX3, TWIST1 and PPARG are negatively related to Z-score. One HBM case presented with high levels of RUNX2, while the other displayed very low SOX6. In conclusion, we provide evidence of lack of LRP5 mutations and of a putative HBM-causing mutation in DKK1. Additionally, we present SNP genotyping and expression results that suggest additive effects of several genes for HBM

Genetic Polymorphisms of RANK, RANKL and their relation to osteoporosis (Polimorfismos genéticos de RANK y RANKL y su relación con la osteoporosis)

[eng] Osteoporosis is a systemic skeletal disorder and the most common metabolic bone disease. It is recognized as one of the most prevalent problems facing postmenopausal women in western society. The World Health Organization definition of osteoporosis uses bone mineral density (BMD) measurements as the gold standard. The genetic complexity of BMD is incompletely defined. Bone turnover, also called bone remodelling, is a lifelong process that refers to the entire cycle of bone resorption and formation, which determines BMD. In general, the cell biology of an adult bone includes 3 cell types, among others, that have opposite functions: osteoblasts produce the extracellular matrix that becomes mineralized; osteoclasts are responsible for the resorptive actions; and osteocytes are involved in the regulation of both resorption and formation (and are even claimed to dominate the process). A complex signal system between these 3 cell types balances their activities to avoid any over-creation or loss of bone tissue. The bone remodelling equilibrium is in part dominated by a set of protein reactions known as the RANK/RANKL/OPG system. The special importance of the Receptor Activator of NF-kappa-B (RANK) and its interaction with its ligand (RANKL) is that the RANK/RANKL complex is one of the main triggers of osteoclast differentiation and survival. Hence, in-depth analysis of variants in these 2 genes may contribute to the understanding of the genetics of BMD. This study had 4 main objectives: (1) association analysis of putative functional SNPs in evolutionary conserved regions of the RANK and RANKL genes with BMD and the occurrence of fractures in our cohort (BARCOS). (2) Replication of previously associated SNPs, in the BARCOS cohort (3) In-silico study followed by in-vitro functional experiments of the BMD-associated SNP(s) in order to reveal its (their) role(s) in the pathological process of osteoporosis and (4) Characterization of the human RANKL promoter and regulatory regions in-silico and in-vitro. We replicated the association of SNP rs9594738, a genetic variant at 184 kb upstream to RANKL gene, with BMD. Statistical analysis for other SNPs in the RANKL gene failed to be associated with osteoporotic phenotypes. The functional experiments’ results demonstrate that this region surrounding rs9594738, between AKAP11 and RANKL, has the capacity to regulate RANKL, with different effects on its expression in the presence or absence of vitamin D. These results suggest that it may play a role in the RANK/RANKL/OPG equilibrium, and might explain the association between the SNPs in this region and BMD. A transcript of minimum 300 bp (with rs9594738 in a central position) has been detected in this region. The existence of this RNA segment suggests its involvement in alternative functions of the region. We also identified 2 SNPs in the RANK 3’UTR (rs78326403 and rs884205) that are associated with low trauma fractures in our cohort. SNP rs78326403 is associated with wrist/forearm fractures, while SNP rs884205 is associated with spine fractures. In addition, we observed a significant interaction between rs78326403 and the RANKL BMD-associated SNP (rs9594738), highlighting the relevance of both microarchitecture and low BMD as genetically determined predictors of fracture risk that should be assessed using multiple techniques. To conclude, our results highlight the importance of the region between AKAP11 and RANKL on RANKL transcription regulation and suggest that it may play an important role in the RANK/RANKL/OPG equilibrium. Furthermore, the site-dependent associations in the RANK gene might be clinically relevant in the future to better profile a more specific approach to the different types of fractures, both to better understand their underlying mechanisms and to search for site-specific therapeutic strategies.[spa] La osteoporosis es la enfermedad metabólica ósea más frecuente. Está definida como un trastorno esquelético sistémico caracterizado por una disminución de la densidad mineral ósea (DMO) y alteraciones en la microarquitectura del tejido óseo, con un consecuente incremento de la fragilidad ósea y del riesgo de fractura. Su complejidad genética permanece incompletamente definida. La DMO viene marcada por un remodelado óseo basado en ciclos de resorción y formación que suceden a lo largo de toda la vida del organismo. Este proceso, está regulado en parte por un conjunto de reacciones proteicas pertenecientes al sistema conocido como RANK/RANKL/OPG. La especial importancia de RANK, así como la interacción de éste con su ligando RANKL, recae en el hecho que, son factores clave tanto en el desencadenamiento de la diferenciación como de la supervivencia osteoclástica. El estudio se centra en el análisis detallado de variantes pertenecientes a ambos genes, seguido del estudio funcional correspondiente. Se ha replicado la asociación del SNP rs9594738 con la DMO, una variante genética localizada a 184 kb 5' del gen RANKL. Los resultados del estudio funcional muestran que la región que engloba dicha variante actúa como un regulador distal de RANKL, ejerciendo efectos en su expresión que varían en presencia ó ausencia de vitamina D. Además, se identificaron dos SNPs (rs78326403 y rs884205) en el 3’UTR de RANK, asociados con fracturas por bajo traumatismo en nuestra cohorte. Por último, una interacción significativa entre rs78326403 y rs9594738 en la determinación del riesgo de fractura, pone de relieve la importancia de la DMO baja y de la microarquitectura como predictores genéticamente determinados del riesgo de fractura que se deben evaluar con el uso de diversas técnicas

Estudio del patrón de expresión de microRNAs en el hueso osteoporótico

Objetivos: Identificar microRNAs (miRNAs) diferencialmente expresados en muestras óseas con fractura osteoporótica respecto a huesos sanos. Material y métodos: Se extrajo RNA total a partir de hueso trabecular fresco del cuello femoral de mujeres sometidas a reemplazo de cadera, ya sea debido a fractura osteoporótica (n=6) o por artrosis en ausencia de osteoporosis (según la DMO) (n=6). Las muestras se hibridaron en un array de miRNAs y se realizaron diagramas de PCA y de mapa de calor. Para la comparación de los niveles de expresión, se fijó como significativo un umbral de cambio de >1,5 veces y un valor p<0,05 en la t de Student (corregido para múltiples pruebas). Resultados: Tanto los análisis de PCA como el mapa de calor mostraron una agrupación de las muestras según si eran de fractura o no. Se detectaron 790 miRNAs en las muestras de hueso, 82 de los cuales estaban alterados en las muestras osteoporóticas. Tras la validación en otro panel de 6 muestras osteoporóticas y 6 no osteoporóticas mediante PCR a tiempo real de los miRNAs más significativos, y para los que existía un ensayo disponible, se confirmaron los miRNAs miR-320a y miR-22-3p. Estos dos miRNAs se detectaron en cultivos de osteoblastos primarios, aunque no mantenían el mismo patrón de expresión que en las muestras de hueso total. Conclusiones: Hemos demostrado que existen diferencias en la expresión de miRNAs en muestras con fractura osteoporótica, lo que abre nuevas perspectivas para la investigación y diseño de nuevas terapias.Objectives: To identify microRNAs (miRNAs) differentially expressed in bone samples with osteoporotic fracture compared with healthy bones. Methods: Total RNA was extracted from fresh trabecular bone of the femoral neck of women undergoing hip replacement surgery, either because to osteoporotic fracture (n=6) or in the absence of osteoarthritis osteoporosis (based on BMD) (n=6). The samples were hybridized on an array of miRNAs and PCA diagrams and heat map were made. To compare expression levels, >1.5 times and a value p<0.05 Student's T test (corrected for multiple testing) was set as a threshold of significant change. Results: Both PCA analysis and the heat map showed a samples grouping whether there was fracture or not. 790 were detected miRNAs in bone samples, 82 of which were altered in the osteoporotic samples. After validation in another panel of 6 samples 6 osteoporotic and non-osteoporotic by PCR real time of the most significant miRNAs, and for which there was a test available, the miRNAs, miR-320a and miR22-3p were confirmed. These two miRNAs were detected in cultures of primary osteoblasts, although they did not maintain the same pattern of expression in total bone samples. Conclusions: We have shown that there are differences in the expression of miRNAs in samples with osteoporotic fracture. This opens prospects for research and design of new therapies.Este trabajo ha sido financiado por la Red Temática de Investigación Cooperativa en Envejecimiento y Fragilidad (RETICEF; RD12/0043/0022), y la ayuda FIS PI13/00116 (Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación) y los fondos FEDE

Estudio del patrón de expresión de microRNAs en el hueso osteoporótico

Objetivos: Identificar microRNAs (miRNAs) diferencialmente expresados en muestras óseas con fractura osteoporótica respecto a huesos sanos. Material y métodos: Se extrajo RNA total a partir de hueso trabecular fresco del cuello femoral de mujeres sometidas a reemplazo de cadera, ya sea debido a fractura osteoporótica (n=6) o por artrosis en ausencia de osteoporosis (según la DMO) (n=6). Las muestras se hibridaron en un array de miRNAs y se realizaron diagramas de PCA y de mapa de calor. Para la comparación de los niveles de expresión, se fijó como significativo un umbral de cambio de >1,5 veces y un valor p<0,05 en la t de Student (corregido para múltiples pruebas). Resultados: Tanto los análisis de PCA como el mapa de calor mostraron una agrupación de las muestras según si eran de fractura o no. Se detectaron 790 miRNAs en las muestras de hueso, 82 de los cuales estaban alterados en las muestras osteoporóticas. Tras la validación en otro panel de 6 muestras osteoporóticas y 6 no osteoporóticas mediante PCR a tiempo real de los miRNAs más significativos, y para los que existía un ensayo disponible, se confirmaron los miRNAs miR-320a y miR-22-3p. Estos dos miRNAs se detectaron en cultivos de osteoblastos primarios, aunque no mantenían el mismo patrón de expresión que en las muestras de hueso total. Conclusiones: Hemos demostrado que existen diferencias en la expresión de miRNAs en muestras con fractura osteoporótica, lo que abre nuevas perspectivas para la investigación y diseño de nuevas terapias.Objectives: To identify microRNAs (miRNAs) differentially expressed in bone samples with osteoporotic fracture compared with healthy bones. Methods: Total RNA was extracted from fresh trabecular bone of the femoral neck of women undergoing hip replacement surgery, either because to osteoporotic fracture (n=6) or in the absence of osteoarthritis osteoporosis (based on BMD) (n=6). The samples were hybridized on an array of miRNAs and PCA diagrams and heat map were made. To compare expression levels, >1.5 times and a value p<0.05 Student's T test (corrected for multiple testing) was set as a threshold of significant change. Results: Both PCA analysis and the heat map showed a samples grouping whether there was fracture or not. 790 were detected miRNAs in bone samples, 82 of which were altered in the osteoporotic samples. After validation in another panel of 6 samples 6 osteoporotic and non-osteoporotic by PCR real time of the most significant miRNAs, and for which there was a test available, the miRNAs, miR-320a and miR22-3p were confirmed. These two miRNAs were detected in cultures of primary osteoblasts, although they did not maintain the same pattern of expression in total bone samples. Conclusions: We have shown that there are differences in the expression of miRNAs in samples with osteoporotic fracture. This opens prospects for research and design of new therapies.Este trabajo ha sido financiado por la Red Temática de Investigación Cooperativa en Envejecimiento y Fragilidad (RETICEF; RD12/0043/0022), y la ayuda FIS PI13/00116 (Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación) y los fondos FEDE

Erratum to: MiRNA profiling of whole trabecular bone: identification of osteoporosis-related changes in MiRNAs in human hip bones

Background: MicroRNAs (miRNAs) are important regulators of gene expression, with documented roles in bone metabolism and osteoporosis, suggesting potential therapeutic targets. Our aim was to identify miRNAs differentially expressed in fractured vs nonfractured bones. Additionally, we performed a miRNA profiling of primary osteoblasts to assess the origin of these differentially expressed miRNA

MiRNA profiling of whole trabecular bone: identification of osteoporosis-related changes in MiRNAs in human hip bones.

BACKGROUND: MicroRNAs (miRNAs) are important regulators of gene expression, with documented roles in bone metabolism and osteoporosis, suggesting potential therapeutic targets. Our aim was to identify miRNAs differentially expressed in fractured vs nonfractured bones. Additionally, we performed a miRNA profiling of primary osteoblasts to assess the origin of these differentially expressed miRNAs. METHODS: Total RNA was extracted from (a) fresh femoral neck trabecular bone from women undergoing hip replacement due to either osteoporotic fracture (OP group, n = 6) or osteoarthritis in the absence of osteoporosis (Control group, n = 6), matching the two groups by age and body mass index, and (b) primary osteoblasts obtained from knee replacement due to osteoarthritis (n = 4). Samples were hybridized to a microRNA array containing more than 1900 miRNAs. Principal component analysis (PCA) plots and heat map hierarchical clustering were performed. For comparison of expression levels, the threshold was set at log fold change > 1.5 and a p-value < 0.05 (corrected for multiple testing). RESULTS: Both PCA and heat map analyses showed that the samples clustered according to the presence or absence of fracture. Overall, 790 and 315 different miRNAs were detected in fresh bone samples and in primary osteoblasts, respectively, 293 of which were common to both groups. A subset of 82 miRNAs was differentially expressed (p < 0.05) between osteoporotic and control osteoarthritic samples. The eight miRNAs with the lowest p-values (and for which a validated miRNA qPCR assay was available) were assayed, and two were confirmed: miR-320a and miR-483-5p. Both were over-expressed in the osteoporotic samples and expressed in primary osteoblasts. miR-320a is known to target CTNNB1 and predicted to regulate RUNX2 and LEPR, while miR-483-5p down-regulates IGF2. We observed a reduction trend for this target gene in the osteoporotic bone. CONCLUSIONS: We identified two osteoblast miRNAs over-expressed in osteoporotic fractures, which opens novel prospects for research and therapy.This work was supported by grant FIS PI10/01537 and the Red Temática de Investigación Cooperativa en Envejecimiento y Fragilidad (RETICEF) (Carlos III Health Institute, Science and Innovation Ministry), and FEDER funds. Grant SAF2011-25431 and PIB2010AR-00473 (Science and Innovation Ministry), and the support from the Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER, an initiative of ISCIII) are also acknowledged. Grants from the Generalitat de Catalunya (DIUE; 2009 SGR 818, 2009 SGR 971) also supported this work

MiRNA profiling of whole trabecular bone: identification of osteoporosis-related changes in MiRNAs in human hip bones

Background MicroRNAs (miRNAs) are important regulators of gene expression, with documented roles in bone metabolism and osteoporosis, suggesting potential therapeutic targets. Our aim was to identify miRNAs differentially expressed in fractured vs nonfractured bones. Additionally, we performed a miRNA profiling of primary osteoblasts to assess the origin of these differentially expressed miRNAs. Methods Total RNA was extracted from (a) fresh femoral neck trabecular bone from women undergoing hip replacement due to either osteoporotic fracture (OP group, n = 6) or osteoarthritis in the absence of osteoporosis (Control group, n = 6), matching the two groups by age and body mass index, and (b) primary osteoblasts obtained from knee replacement due to osteoarthritis (n = 4). Samples were hybridized to a microRNA array containing more than 1900 miRNAs. Principal component analysis (PCA) plots and heat map hierarchical clustering were performed. For comparison of expression levels, the threshold was set at log fold change > 1.5 and a p-value < 0.05 (corrected for multiple testing). Results Both PCA and heat map analyses showed that the samples clustered according to the presence or absence of fracture. Overall, 790 and 315 different miRNAs were detected in fresh bone samples and in primary osteoblasts, respectively, 293 of which were common to both groups. A subset of 82 miRNAs was differentially expressed (p < 0.05) between osteoporotic and control osteoarthritic samples. The eight miRNAs with the lowest p-values (and for which a validated miRNA qPCR assay was available) were assayed, and two were confirmed: miR-320a and miR-483-5p. Both were over-expressed in the osteoporotic samples and expressed in primary osteoblasts. miR-320a is known to target CTNNB1 and predicted to regulate RUNX2 and LEPR, while miR-483-5p down-regulates IGF2. We observed a reduction trend for this target gene in the osteoporotic bone. Conclusions We identified two osteoblast miRNAs over-expressed in osteoporotic fractures, which opens novel prospects for research and therapy