Polymorphisms in Genes Involved in Osteoblast Differentiation and Function Are Associated with Anthropometric Phenotypes in Spanish Women.

Much of the genetic variance associated with osteoporosis is still unknown. Bone mineral density (BMD) is the main predictor of osteoporosis risk, although other anthropometric phenotypes have recently gained importance. The aim of this study was to analyze the association of SNPs in genes involved in osteoblast differentiation and function with BMD, body mass index (BMI), and waist (WC) and hip (HC) circumferences. Four genes that affect osteoblast differentiation and/or function were selected from among the differentially expressed genes in fragility hip fracture (FOXC1, CTNNB1, MEF2C, and EBF2), and an association study of four single-nucleotide polymorphisms (SNPs) was conducted in a cohort of 1001 women. Possible allelic imbalance was also studied for SNP rs87939 of the CTNNB1 gene. We found significant associations of SNP rs87939 of the CTNNB1 gene with LS-sBMD, and of SNP rs1366594 of the MEF2C gene with BMI, after adjustment for confounding variables. The SNP of the MEF2C gene also showed a significant trend to association with FN-sBMD (p = 0.009). A possible allelic imbalance was ruled out as no differences for each allele were detected in CTNNB1 expression in primary osteoblasts obtained from homozygous women. In conclusion, we demonstrated that two SNPs in the MEF2C and CTNNB1 genes, both implicated in osteoblast differentiation and/or function, are associated with BMI and LS-sBMD, respectively.

Comparative transcriptome analysis identifies CARM1 and DNMT3A as genes associated with osteoporosis.

To identify new candidate genes in osteoporosis, mainly involved in epigenetic mechanisms, we compared whole gene-expression in osteoblasts (OBs) obtained from women undergoing hip replacement surgery due to fragility fracture and severe osteoarthritis. Then, we analyzed the association of several SNPs with BMD in 1028 women. Microarray analysis yielded 2542 differentially expressed transcripts belonging to 1798 annotated genes, of which 45.6% (819) were overexpressed, and 54.4% (979) underexpressed (fold-change between - 7.45 and 4.0). Among the most represented pathways indicated by transcriptome analysis were chondrocyte development, positive regulation of bone mineralization, BMP signaling pathway, skeletal system development and Wnt signaling pathway. In the translational stage we genotyped 4 SNPs in DOT1L, HEY2, CARM1 and DNMT3A genes. Raw data analyzed against inheritance patterns showed a statistically significant association between a SNP of DNMT3A and femoral neck-(FN) sBMD and primarily a SNP of CARM1 was correlated with both FN and lumbar spine-(LS) sBMD. Most of these associations remained statistically significant after adjusting for confounders. In analysis with anthropometric and clinical variables, the SNP of CARM1 unexpectedly revealed a close association with BMI (p = 0.000082), insulin (p = 0.000085), and HOMA-IR (p = 0.000078). In conclusion, SNPs of the DNMT3A and CARM1 genes are associated with BMD, in the latter case probably owing to a strong correlation with obesity and fasting insulin levels.

Polymorphisms in genes involved in T-cell co-stimulation are associated with blood pressure in women.

In recent years, conclusive data have emerged on a relationship between immune system, especially the T-cell, and blood pressure (BP). The objective of the present study was to determine the association between BP and four polymorphisms in CD80, CD86, CD28 and CTLA4 genes that code for key proteins in the T-cell co-stimulation process, in a female cohort. To that end, an association study in a cohort of 934 women over 40 years old from two hospitals was done. Raw data showed a significant association between the SNP rs1129055 of CD86 gene and BP. Analyzing this association against inheritance patterns, higher SBP (p < 0.000) and DBP (p = 0.005) values were observed in AA than in GG/GA genotype subjects in the largest sample cohort (Hospital 1). In multivariate linear regression studies, with adjustment for presumed independent predictors of BP, the SNP of the CD86 gene remained a predictor of SBP (p = 0.001) and DBP (p = 0.006), as did the SNP rs867234 of the CD80 gene for DBP (p < 0.000), both resisting the Bonferroni correction for multiple comparisons. As conclusion, we report a robust association between the SNP rs1129055 of CD86 gene and BP. The SNP rs867234 of CD80 gene was also shown to be a strong predictor of DBP.

The role of CD40 and CD40L in bone mineral density and in osteoporosis risk: A genetic and functional study.

Compelling data are revealing that the CD40/CD40L system is involved in bone metabolism. Furthermore, we have previously demonstrated that polymorphisms in both genes are associated with bone phenotypes. The aim of this study is to further characterize this association and to identify the causal functional mechanism. We conducted an association study of BMD with 15 SNPs in CD40/CD40L genes in a population of 779 women. In addition, we assessed the functionality of this association through the study of the allele-dependent expression of CD40 and CD40L in peripheral blood leukocytes (PBLs) and in human osteoblasts (OBs) obtained from bone explants by qPCR and by sequencing. When an allelic imbalance (AI) was detected, studies on allele-dependent in vitro transcription rate and on CpG methylation in the gene promoter were also performed. Our results confirm the genetic association between SNP rs116535 (T>C) of CD40L gene with LS-BMD. Regarding CD40 gene, two SNPs showed nominal P-values<0.05 for FN- and LS-BMD (Z-scores), although the association was not significant after correcting for multiple testing. Homozygous TT women for SNP rs1883832 (C>T) of CD40 gene showed a trend to have lower levels of OPG (Q-value=0.059), especially when women of BMD-quartile ends were selected (P<0.05). Regarding functionality, we detected an AI for rs1883832 with the C allele the most expressed in OBs and in PBLs. Since the rs116535 of CD40L gene did not show AI, it was not further analyzed. Finally, we described a differential methylation of CpGs in the CD40 promoter among women of high in comparison to low BMD. Our results suggest that the CD40/CD40L system plays a role in regulating BMD. Effectively, our data suggest that a decreased production of OPG could be the cause of the lower BMD observed in TT women for rs1883832 of the CD40 gene and that the degree of methylation of CpGs in the CD40 promoter could contribute to the acquisition of BMD. One possibility that deserves further study is whether the degree of methylation of the CD40 gene affects the level of CD40 expression and, consequently, the level of OPG.

Serum Circulating MicroRNAs as Biomarkers of Osteoporotic Fracture.

Osteoporosis is a common skeletal disorder characterized by increased risk of bone fracture (BF) due to fragility. BFs, particularly hip fracture, are a major concern in health care because of the associated morbidity and mortality, mainly in the elderly. Lately the involvement of epigenetic mechanisms in the pathophysiology of many diseases has been recognized. In this context, the identification of microRNAs (miRNAs) specific to BF should represent a substantial step forward in diagnostics and therapeutics. The present study aimed to identify specific miRNAs in osteoporotic BF patients compared to those in osteoarthritic controls. In the profiling stage, total RNA was extracted from serum, two pools were prepared, and then retro-transcribed in triplicate. Levels of 179 serum miRNAs were analyzed by real-time PCR, and 42 of them showed significance (P < 0.05), and 12 passed the false discovery rate test for multiple comparisons. Six miRNAs were selected for the replication stage and individually analyzed in sera from 15 BF patients and 12 controls. Results showed that 3 miRNAs (miR-122-5p, miR-125b-5p, and miR-21-5p) were valuable upregulated biomarkers in BF with respect to controls and, significantly, their levels were not affected by hemolysis. For miR-21-5p, the difference detected between groups was independent of age (P = 0.005) and its levels correlated to those of CTx (r = 0.76; P < 0.00001), a marker of bone resorption. In conclusion, several miRNAs may be biomarkers of BF, particularly miR-21-5p. Further studies are needed in order to better characterize the levels of these miRNAs in other bone diseases and to elucidate the mechanism involved in the association of these three miRNAs with osteoporotic BF.

Gene expression profile induced by ovariectomy in bone marrow of mice: a functional approach to identify new candidate genes associated to osteoporosis risk in women.

Osteoporosis is a multifactorial skeletal pathology with a main genetic component. To date, however, the majority of genes associated with this pathology remain unknown since genes cataloged to date only explain a part of the heritability of bone phenotypes. In the present study, we have used a genome-wide gene expression approach by means of microarrays to identify new candidate genes involved in the physiopathology of osteoporosis, using as a model the ovariectomized (OVX) mice by comparing global bone marrow gene expression of the OVX mice with those of SHAM operated mice. One hundred and eighty transcripts were found to be differentially expressed between groups. The analysis showed 23 significant regulatory networks, of which the top five canonical pathways included B-cell development, primary immunodeficiency signaling, PI3K signaling in B-cells, phospholipase C signaling, and FcgRIIB signaling in B-cells. Twelve differentially expressed genes were validated by MALDI-TOF mass spectrometry with good reproducibility. Finally, the association to bone phenotypes of SNPs in genes whose expression was increased (IL7R and CD79A) or decreased (GPX3 and IRAK3) by OVX in mice was analyzed in a cohort of 706 postmenopausal women. We detected an association of a SNP in a gene involved in the detoxification of free radicals like glutathione peroxidase 3 (GPX3) with femoral neck BMD (rs8177447, P=0.043) and two SNPs in the Ig-alpha protein of the B-cell antigen component gene (CD79A) with lumbar spine BMD (rs3810153 and rs1428922, P=0.016 and P=0.001, respectively). These results reinforce the role of antioxidant pathways and of B-cells in bone metabolism. Furthermore, it shows that a genome-wide gene expression approach in animal models is a useful method for detecting genes associated to BMD and osteoporosis risk in humans.

Replication study of three functional polymorphisms associated with bone mineral density in a cohort of Spanish women.

Gene candidate and genome-wide association studies have revealed tens of loci of susceptibility for osteoporosis. Some limitations such as sample size, use of confounding variables, and control for multiple testing and for population stratification, however, represent common problems in these studies that make replication in independent cohorts desirable and even necessary. The main objective of the present study is to replicate previous data on three functional polymorphisms in a cohort of Spanish women. To that end, we performed an association study of three functional polymorphisms previously associated with bone phenotypes in the LRP5, TNFRSF11B, and FGFBP1 genes with low bone mineral density (BMD) in a cohort of 721 Spanish women, most of them postmenopausal. We detected a strong significant association, even when correcting for multiple comparisons, for polymorphism rs312009 in the LRP5 gene with low BMD at the lumbar-spine site. These were women with the CC genotype, which showed the worst bone parameters. Moreover, these women had a higher risk of osteoporosis (adjusted odds ratio 2.82, P = 0.001) than women with the TT/TC genotype. This association seems to be caused because the rs312009 single nucleotide polymorphism (SNP) is located at a binding site for the transcription factor RUNX2 at the 5' region of the LRP5 gene, and the T allele seems to be a better transcriber than the C allele. Regarding the other two SNPs, only the rs4876869 SNP in the TNFRSF11B gene showed a suggestive trend for both skeletal sites. These results underscore the significance of the LRP5 gene in bone metabolism and emphasize the significance of the replication of previous results in independent cohorts.