Maria Rodriguez-Sanz

SNPs in bone-related miRNAs are associated with the osteoporotic phenotype

Biogenesis and function of microRNAs can be influenced by genetic variants in the pri-miRNA sequences leading to phenotypic variability. This study aims to identify single nucleotide polymorphisms (SNPs) affecting the expression levels of bone-related mature microRNAs and thus, triggering an osteoporotic phenotype. An association analysis of SNPs located in pri-miRNA sequences with bone mineral density (BMD) was performed in the OSTEOMED2 cohort (n = 2183). Functional studies were performed for assessing the role of BMD-associated miRNAs in bone cells. Two SNPs, rs6430498 in the miR-3679 and rs12512664 in the miR-4274, were significantly associated with femoral neck BMD. Further, we measured these BMD-associated microRNAs in trabecular bone from osteoporotic hip fractures comparing to non-osteoporotic bone by qPCR. Both microRNAs were found overexpressed in fractured bone. Increased matrix mineralization was observed after miR-3679-3p inhibition in human osteoblastic cells. Finally, genotypes of rs6430498 and rs12512664 were correlated with expression levels of miR-3679 and miR-4274, respectively, in osteoblasts. In both cases, the allele that generated higher microRNA expression levels was associated with lower BMD values. In conclusion, two osteoblast-expressed microRNAs, miR-3679 and miR-4274, were associated with BMD; their overexpression could contribute to the osteoporotic phenotype. These findings open new areas for the study of bone disorders.

CYP11A1 expression in bone is associated with aromatase inhibitor-related bone loss

Aromatase inhibitors (AIs) used as adjuvant therapy in postmenopausal women with hormone receptor-positive breast cancer cause diverse musculoskeletal side effects that include bone loss and its associated fracture. About half of the 391 patients treated with AIs in the Barcelona-Aromatase induced bone loss in early breast cancer cohort suffered a significant bone loss at lumbar spine (LS) and/or femoral neck (FN) after 2 years on AI-treatment. In contrast, up to one-third (19.6% LS, 38.6% FN) showed no decline or even increased bone density. The present study aimed to determine the genetic basis for this variability. SNPs in candidate genes involved in vitamin D and estrogen hormone-response pathways (CYP11A1, CYP17A1, HSD3B2, HSD17B3, CYP19A1, CYP2C19, CYP2C9, ESR1, DHCR7, GC, CYP2R1, CYP27B1, VDR and CYP24A1) were genotyped for association analysis with AI-related bone loss (AIBL). After multiple testing correction, 3 tag-SNPs (rs4077581, s11632698 and rs900798) located in the CYP11A1 gene were significantly associated (P<0.005) with FN AIBL at 2 years of treatment. Next, CYP11A1 expression in human fresh bone tissue and primary osteoblasts was demonstrated by RT-PCR. Both common isoforms of human cholesterol side-chain cleavage enzyme (encoded by CYP11A1 gene) were detected in osteoblasts by western blot. In conclusion, the genetic association of CYP11A1 gene with AIBL and its expression in bone tissue reveals a potential local function of this enzyme in bone metabolism regulation, offering a new vision of the steroidogenic ability of this tissue and new understanding of AI-induced bone loss.

Analyses of RANK and RANKL in the post-GWAS context: functional evidence of vitamin D stimulation through a RANKL distal region.

Over the past decade, many genome‐wide association studies (GWAs) and meta‐analyses have identified genes and regions involved in osteoporotic phenotypes. Nevertheless, the large majority of these results were not tested at any functional level. GWA‐associated single‐nucleotide polymorphisms (SNPs) near candidate genes such as RANK and RANKL suggest that these SNPs and/or other variants nearby may be involved in bone phenotype determination. This study focuses on SNPs along these two genes, which encode proteins with a well‐established role in the bone remodeling equilibrium. Thirty‐three SNPs, chosen for their location in evolutionary conserved regions or replicated from previous studies, were genotyped in the BARCOS cohort of 1061 postmenopausal women and tested for association with osteoporotic phenotypes. SNP rs9594738, which lies 184 kb upstream of the RANKL gene, was the only SNP found to be associated with a bone phenotype (dominant model: beta coefficient = –0.034, p = 1.5 × 10−4, for lumbar spine bone mineral density). Functional experiments exploring a distal region (DR) of 831 bp that harbors this SNP in a centered position (nt 470) demonstrated its capacity to inhibit the RANKL promoter in reporter gene assays. Remarkably, this DR inhibition was significantly reduced in the presence of vitamin D. In conclusion, the GWA‐associated SNP rs9594738 lies in a region involved in transcription regulation through which vitamin D could be regulating RANKL expression and bone mineral density.

AI-related BMD variation in actual practice conditions: A prospective cohort study

The aim of the study was to evaluate the progression of bone mineral density (BMD) during 3 years of aromatase inhibitors (AI) therapy in actual practice conditions. This prospective, clinical cohort study of Barcelona-Aromatase induced Bone Loss in Early breast cancer (B-ABLE) assessed BMD changes during 3 years of AI treatment in women with breast cancer. Patients with osteoporosis (T score < -2.5 or T score ≤ -2.0) and a major risk factor and/or prevalent fragility fractures were treated with oral bisphosphonates (BPs). Of 685 women recruited, 179 (26.1%) received BP treatment. By the third year of AI therapy, this group exhibited increased BMD in the lumbar spine (LS; 2.59%) and femoral neck (FN; 2.50%), although the increase was significant only within the first year (LS: 1.99% and FN: 2.04%). Despite BP therapy, however, approximately 15% of these patients lost more than 3% of their baseline bone mass. At 3 years, patients without BP experienced BMD decreases in the LS (-3.10%) and FN (-2.79%). In this group, BMD changes occurred during the first (LS: -1.33% and FN: -1.25%), second (LS: -1.19% and FN: -0.82%), and third (LS: -0.57% and FN: -0.65%) years of AI treatment. Increased BMD (>3%) was observed in just 7.6% and 10.8% of these patients at the LS and FN, respectively. Our data confirm a clinically relevant bone loss associated with AI therapy amongst nonusers of preventative BPs. We further report on the importance of BMD monitoring as well as calcium and 25-hydroxy vitamin D supplementation in these patients.