[en] 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.

[es] 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.