Analysis of differentially expressed microRNAs in MEN1 parathyroid adenomas.

Multiple Endocrine Neoplasia type 1 (MEN1) syndrome is a rare complex tumor-predisposing hereditary disorder, inherited in an autosomal dominant manner (OMIM 131100). MEN1 is characterized by tumors of the parathyroids, the neuroendocrine cells of the gastro-entero-pancreatic tract, and the anterior pituitary. The molecular mechanisms that control parathyroid tumorigenesis are still poorly understood. Here we studied the global microRNAs (miRNAs) expression profile in MEN1 parathyroid adenomas to understand the role of these regulatory factors in MEN1 parathyroid tumorigenesis. miRNA arrays containing 1890 human miRNAs were used to profile seven different MEN1 parathyroid adenomas (four presenting somatic loss of heterozygosity (LOH) at 11q13 and three still retaining one wild type copy of the MEN1 gene). Eight miRNAs in non-LOH MEN1 parathyroid adenomas and two miRNAs in LOH MEN1 parathyroid adenomas resulted to be differentially expressed, with a significant fold change, with respect to the control pool. Six microRNAs also resulted to be differentially expressed between LOH MEN1 parathyroid adenomas and non-LOH MEN1 parathyroid adenomas. Significantly differentially expressed miRNAs were all validated by SYBR green real-time quantitative RT-PCR. Pearson correlation coefficient indicated miR-4258, miR-664 and miR-1301 as the most significant miRNAs. In silico target-prediction and network analysis showed miR-664 and miR-1301 as organized in predicted GRNs with genes interested in parathyroid adenomas and carcinomas. In conclusion, our study identified three new miRNAs involved in the MEN1 parathyroid neoplasia, directly targeting genes associated with the development of different inheritable forms of parathyroid tumors. These identified miRNAs could be revealed as prognostic and diagnostic biomarkers for parathyroid tumors to improve the diagnosis of MEN1 neoplasia and other syndromes.

Establishment of Cancer Stem Cell Cultures from Human Conventional Osteosarcoma.

The current improvements in therapy against osteosarcoma (OS) have prolonged the lives of cancer patients, but the survival rate of five years remains poor when metastasis has occurred. The Cancer Stem Cell (CSC) theory holds that there is a subset of tumor cells within the tumor that have stem-like characteristics, including the capacity to maintain the tumor and to resist multidrug chemotherapy. Therefore, a better understanding of OS biology and pathogenesis is needed in order to advance the development of targeted therapies to eradicate this particular subset and to reduce morbidity and mortality among patients. Isolating CSCs, establishing cell cultures of CSCs, and studying their biology are important steps to improving our understanding of OS biology and pathogenesis. The establishment of human-derived OS-CSCs from biopsies of OS has been made possible using several methods, including the capacity to create 3-dimensional stem cell cultures under nonadherent conditions. Under these conditions, CSCs are able to create spherical floating colonies formed by daughter stem cells; these colonies are termed "cellular spheres". Here, we describe a method to establish CSC cultures from primary cell cultures of conventional OS obtained from OS biopsies. We clearly describe the several passages required to isolate and characterize CSCs.

Establishment and Characterization of a Human Small Cell Osteosarcoma Cancer Stem Cell Line: A New Possible In Vitro Model for Discovering Small Cell Osteosarcoma Biology.

Osteosarcoma (OSA) is the most common primary malignant bone tumor, usually arising in the long bones of children and young adults. There are different subtypes of OSA, among which we find the conventional OS (also called medullary or central osteosarcoma) which has a high grade of malignancy and an incidence of 80%. There are different subtypes of high grade OS like chondroblastic, fibroblastic, osteoblastic, telangiectatic, and the small cell osteosarcoma (SCO). In this study, for the first time, we have isolated, established, and characterized a cell line of cancer stem cells (CSCs) from a human SCO. First of all, we have established a primary finite cell line of SCO, from which we have isolated the CSCs by the sphere formation assay. We have proved their in vitro mesenchymal and embryonic stem phenotype. Additionally, we have showed their neoplastic phenotype, since the original tumor bulk is a high grade osteosarcoma. This research demonstrates the existence of CSCs also in human primary SCO and highlights the establishment of this particular stabilized cancer stem cell line. This will represent a first step into the study of the biology of these cells to discover new molecular targets molecules for new incisive therapeutic strategies against this highly aggressive OSA.

In vitro effects of extracts of extra virgin olive oil on human colon cancer cells.

The Mediterranean diet is associated with a lower incidence of atherosclerosis, cardiovascular diseases, and some types of cancer. Recent interest has been focused on the biological activity of phenolic compounds present in extra virgin olive oils (EVOOs). Both in vivo and in vitro studies have shown that EVOO components have positive effects on metabolic parameters, such as plasma lipoproteins, oxidative damage, inflammatory markers, platelet function, and antimicrobial activity. We have investigated the possible interactions between 2 extracts of extra virgin olive oil and estrogen receptor ß (ERß) in an in vitro model of colon cancer. The qualification and quantification of the components of the 2 samples tested showed that phenolic compounds-hydroxytyrosol, secoiridoids, and lignans-are the major represented compounds. EVOO extracts were tested on a colon cancer cell line engineered to overexpress ERß (HCT8-ß8). By using custom made Oligo microarray, gene expression profiles of colon cancer cells challenged with EVOO-T extracts when compared with those of cells exposed to 17ß-estradiol (17ß-E2). This study demonstrated that the EVOO extracts tested showed an antiproliferative effect on colon cancer cells through the interaction with estrogen-dependent signals involved in tumor cell growth. Specifically, the ability of EVOO extracts to inhibit cell proliferation was superimposable to the activation of the ERß receptor, similar to what was observed after 17ß-E2 challenge.

In vitro effects of polyphenols on colorectal cancer cells.

AIM: To investigate the effects of quercetin and genistein on colon cancer cell proliferation and their estrogen receptor ß (ERß) expression. METHODS: Colon cancer cells were stably transfected with a mammalian expression vector to overexpress ERß (HCT8-ß8-expressing cells) or a control vector (HCT8-pSV2neo-expressing cells). The proliferation of these cells was examined after treatment with quercetin or genistein (5-100 µmol/L), or 10 nmol/L 17ß-estradiol (17ß-E2). Cell viability was examined by acridine orange staining following treatments for 48 or 144 h. Effects of quercetin and genistein on ERß transcriptional transactivation were examined by luciferase activity in HCT8-ß8-expressing cells transiently transfected with a pEREtkLUC reporter vector. In addition, the regulation of ERß transcription by phytoestrogens and 17ß-E2 was examined by quantitative polymerase chain reaction. RESULTS: Proliferation of HCT8-ß8-expressing cells was not reduced low doses (5 µmol/L) of quercetin and genistein, while it was reduced at 25-50 µmol/L with an effect similar to 10 nmol/L 17ß-E2. Treatment with doses of phytoestrogens ≥ 75 µmol/L completely blocked cell growth and reduced overall cell counts, however no effects at any dose were observed in HCT8-pSV2neo-expressing cells. These results were supported by viability staining that revealed acridine orange-stained lysosomes with high doses or extended treatment periods. Genistein and quercetin (50 µmol/L) significantly increased ER-responsive luciferase activity similar to 10 nmol/L 17ß-E2 (P < 0.05). Furthermore, genistein and quercetin (50 µmol/L), as well as 10 nmol/L 17ß-E2 significantly increased ERß mRNA levels in HCT8-ß8-expressing cells (P < 0.05). In addition, treatment of HCT8-pSV2neo-expressing cells with 50 µmol/L quercetin or 10 nmol/L 17ß-E2 significantly increased ERß mRNA levels compared to untreated controls (P < 0.05), though the absolute levels were much lower than in HCT8-ß8-expressing cells. CONCLUSION: The antitumorigenic effects of the phytoestrogenic compounds quercetin and genistein on colon cancers cells occur through ERß activity and expression.

PTH-C1: a rat continuous cell line expressing the parathyroid phenotype.

The lack of a continuous cell line of epithelial parathyroid cells able to produce parathyroid hormone (PTH) has hampered the studies on in vitro evaluation of the mechanisms involved in the control of parathyroid cell function and proliferation. The PT-r cell line was first established from rat parathyroid tissue in 1987, but these cells were known to express the parathyroid hormone-related peptide (Pthrp) gene, but not the Pth gene. In an attempt to subclone the PT-r cell line, a rat parathyroid cell strain was isolated and named PTH-C1. During 3 years, in culture, PTH-C1 cells maintained an epithelioid morphology, displaying a diploid chromosome number, a doubling time around 15 h during the exponential phase of growth, and parathyroid functional features. PTH-C1 cell line produces PTH and expresses the calcium sensing receptor (Casr) gene and other genes known to be involved in parathyroid function. Most importantly, the PTH-C1 cells also exhibit an in vitro secretory response to calcium. Altogether these findings indicate the uniqueness of the PTH-C1 cell line as an in vitro model for cellular and molecular studies on parathyroid physiopathology.

Role of GSH/GSSG redox couple in osteogenic activity and osteoclastogenic markers of human osteoblast-like SaOS-2 cells.

This study comprised a comprehensive analysis of the glutathione (GSH) redox system during osteogenic differentiation in human osteoblast-like SaOS-2 cells. For the first time, a clear relationship between expression of specific factors involved in bone remodeling and the changes in the GSH/oxidized GSH (GSSG) redox couple induced during the early phases of the differentiation and mineralization process is shown. The findings show that the time course of differentiation is characterized by a decrease in the GSH/GSSG ratio, and this behavior is also related to the expression of osteoclastogenic markers. Maintenance of a high GSH/GSSG ratio due to GSH exposure in the early phase of this process increases mRNA levels of osteogenic differentiation markers and mineralization. Conversely, these events are decreased by a low GSH/GSSG ratio in a reversible manner. Redox regulation of runt-related transcription factor-2 (RUNX-2) activation through phosphorylation is shown. An inverse relationship between RUNX-2 activation and extracellular signal-regulated kinases related to GSH redox potential is observed. The GSH/GSSG redox couple also affects osteoclastogenesis, mainly through osteoprotegerin down-regulation with an increase in the ratio of receptor activator of NF-κB ligand to osteoprotegerin and vice versa. No redox regulation of receptor activator of NF-κB ligand expression was found. These results indicate that the GSH/GSSG redox couple may have a pivotal role in bone remodeling and bone redox-dysregulated diseases. They suggest therapeutic use of compounds that are able to modulate not just the GSH level but the intracellular redox system through the GSH/GSSG redox couple.

Polymorphisms of estrogen receptors: risk factors for invasive melanoma - a prospective study.

Cross-sectional studies have reported associations between a number of polymorphisms in the estrogen receptor alpha (ERα) gene and the body mass index, hypertension, coronary flow reserve, coronary atherosclerosis, and osteoporosis. There are currently no data examining the genetic polymorphisms of the ERα and estrogen receptor beta (ERß) genes in melanoma patients. The aims of this study were to investigate the associations of genetic polymorphisms of the ERα and ERß genes with melanoma risk. The study group consisted of consecutive patients who visited the Department of Dermatology of the University of Florence between March 2005 and July 2007 for surgical excision of melanoma. In our study, homozygosity for the wild-type alleles showed different results at the PvuII, XbaI, and AluI restriction sites. Only the AluI site showed a lower proportion of the A allele in the melanoma group compared to the control group; the P and X alleles were lower in the control group than in the melanoma group. The distribution of wild-type alleles is important because these alleles have a protective role in the expression of altered proteins, which involves the ERs in our case. Because of the phenotypic prevalence of the wild-type allele, the heterozygotes did not express the polymorphism. The homozygosity of the polymorphic-type alleles shows that a alleles are more frequent in the case group than in the control group, with proportions of 43.8 and 39.5%, respectively. These results suggest that a polymorphism at the AluIrestriction site correlates with a higher proportion of melanoma. Thus, the polymorphism of ERß could ascribe to a higher susceptibility to melanoma.

Tissue engineered human tracheas for in vivo implantation.

Two years ago we performed the first clinical successful transplantation of a fully tissue engineered trachea. Despite the clinically positive outcome, the graft production took almost 3 months, a not feasible period of time for patients with the need of an urgent transplantation. We have then improved decellularization process and herein, for the first time, we completely describe and characterize the obtainment of human tracheal bioactive supports. Histological and molecular biology analysis demonstrated that all cellular components and nuclear material were removed and quantitative PCR confirmed it. SEM analysis revealed that the decellularized matrices retained the hierarchical structures of native trachea, and biomechanical tests showed that decellularization approach did not led to any influence on tracheal morphological and mechanical properties. Moreover immunohistological staining showed the preservation of angiogenic factors and angiogenic assays demonstrated that acellular human tracheal scaffolds exert an in vitro chemo-active action and induce strong in vivo angiogenic response (CAM analysis). We are now able to obtained, in a short and clinically useful time (approximately 3 weeks), a bioengineered trachea that is structurally and mechanically similar to native trachea, which exert chemotactive and pro-angiogenic properties and which could be successfully used for clinical tissue engineered airway clinical replacements.

Estrogen receptor expression in cutaneous melanoma: a real-time reverse transcriptase-polymerase chain reaction and immunohistochemical study.

OBJECTIVE: To evaluate estrogen receptor (ER) expression in human melanoma tissues and in the adjacent healthy skin with the aim of explaining whether the ERalpha:ERbeta expression ratio has a role in neoplastic progression. DESIGN: Prospective study. SETTING: Department of Dermatology, University of Florence, Florence, Italy. Patients Fourteen patients, 12 with cutaneous melanoma (6 women and 6 men) and 2 with melanocytic nevi (1 woman and 1 man). MAIN OUTCOME MEASURES: Using quantitative reverse transcriptase-polymerase chain reaction and immunohistochemical analysis, we analyzed ERalpha and ERbeta messenger RNA (mRNA) and ERbeta protein expression in cutaneous melanoma and in the healthy skin surrounding the lesions. RESULTS: All melanocytic lesions expressed detectable levels of ERalpha and ERbeta mRNA as well as ERbeta protein. Dividing melanoma cases into 2 groups according to Breslow thickness, we found lower ERalpha and ERbeta mRNA levels and lower ERbeta protein levels in thicker, more invasive tumors. CONCLUSIONS: These observations suggest a role for ERs in the metastatic process of melanoma cells, pointing at the possibility of using ERbeta expression as a prognostic indicator of melanoma. The possibility of distinguishing proliferative melanomas, which are associated with dismal prognosis, from the so-called dormant melanomas opens up novel avenues in tailoring individual treatments, as already happens for other tumors.

In vitro differentiation of human mesenchymal stem cells on Ti6Al4V surfaces.

Long-term stability of arthroplasty prosthesis depends on the integration between the bone tissue and the implanted biomaterials, which requires the contribution of osteoblastic precursors and their continuous differentiation into the osteoblastic phenotype. Classically, these interactions are tested in vitro using mesenchymal stem cells (MSCs) isolated and ex vivo expanded from bone marrow aspirates. Human adipose tissue-derived stromal cells (AMSCs) may be a more convenient source of MSCs, according to their abundance and accessibility, but no data are available on their in vitro interactions with hard biomaterials. The aim of this work is to compare the osteogenic potential of human AMSCs and bone marrow-derived MSCs (BMMSCs) and to evaluate their response to Ti6Al4V alloy in terms of adhesion, proliferation and differentiation features, using the human osteosarcoma cell line SaOS-2 for comparison. The overall results showed that AMSCs have the same ability to produce bone matrix as BMMSCs and that Ti6Al4V surfaces exhibit an osteoinductive action on AMSCs, promoting their differentiation into functional osteoblasts and increasing bone formation. In conclusion, adipose tissue is a promising autologous source of osteoblastic cells with important clinical implications for bone tissue engineering.

Methodological models for in vitro amplification and maintenance of human articular chondrocytes from elderly patients.

Articular cartilage defects, an exceedingly common problem closely correlated with advancing age, is characterized by lack of spontaneous resolution because of the limited regenerative capacity of adult articular chondrocytes. Medical and surgical therapies yield unsatisfactory short-lasting results. Recently, cultured autologous chondrocytes have been proposed as a source to promote repair of deep cartilage defects. Despite encouraging preliminary results, this approach is not yet routinely applicable in clinical practice, but for young patients. One critical points is the isolation and ex vivo expansion of large enough number of differentiated articular chondrocytes. In general, human articular chondrocytes grown in monolayer cultures tend to undergo dedifferentiation. This reversible process produces morphological changes by which cells acquire fibroblast-like features, loosing typical functional characteristics, such as the ability to synthesize type II collagen. The aim of this study was to isolate human articular chondrocytes from elderly patients and to carefully characterize their morphological, proliferative, and differentiative features. Cells were morphologically analyzed by optic and transmission electron microscopy (TEM). Production of periodic acid-schiff (PAS)-positive cellular products and of type II collagen mRNA was monitored at different cellular passages. Typical chondrocytic characteristics were also studied in a suspension culture system with cells encapsulated in alginate-polylysine-alginate (APA) membranes. Results showed that human articular chondrocytes can be expanded in monolayers for several passages, and then microencapsulated, retaining their morphological and functional characteristics. The results obtained could contribute to optimize expansion and redifferentiation sequences for applying cartilage tissue engineering in the elderly patients.

A novel polymorphism at the GNAS1 gene associated with low circulating calcium levels.

The concentration of calcium in the extracellular fluid is crucial for several physiological functions in humans and in normal conditions its circulating levels are maintained between 8.5-10.5 mg/dl. Among the regulators of calcium homeostasis parathyroid hormone (PTH) acts though the G-protein coupled PTH receptor and a hormone-sensitive adenylate cyclase, with Gsα subunit (stimulatory guanine nucleotide-binding protein alpha-subunit) being responsible for the stimulation of the catalytic complex. Mutations of the Gsα encoding gene, GNAS1, are causal for some forms of congenital hypocalcemia. In the present study genetic variability in the GNAS1 gene was analyzed in a group of hypocalcemic patients collected through the Italian Register of Primary Hypoparathyroidism (RIIP). We identified a new intronic variant of the GNAS1 gene, consisting of a T>C polymorphism. This polymorphism was studied in a group of unrelated healthy subjects for a possible association with bone turnover biomarkers and bone mineral density. The T>C polymorphism was found in 18% of the studied populations, with 15% heterozygous TC and 3% homozygous CC (Pearson χ(2)analysis: p=0.04). A significant association with low serum calcium levels was found in healthy subjects carrying the T > C polymorphism (ANCOVA analysis: p=0.04). These results support segregation of a novel GNAS1 gene intronic variant with low calcium levels in primary hypoparathyroidism, pseudo-hypoparathyroidism and in the general population.

The association between common vitamin D receptor gene variations and osteoporosis: a participant-level meta-analysis.

BACKGROUND: Polymorphisms of the vitamin D receptor (VDR) gene have been implicated in the genetic regulation of bone mineral density (BMD). However, the clinical impact of these variants remains unclear. OBJECTIVE: To evaluate the relation between VDR polymorphisms, BMD, and fractures. DESIGN: Prospective multicenter large-scale association study. SETTING: The Genetic Markers for Osteoporosis consortium, involving 9 European research teams. PARTICIPANTS: 26,242 participants (18,405 women). MEASUREMENTS: Cdx2 promoter, FokI, BsmI, ApaI, and TaqI polymorphisms; BMD at the femoral neck and the lumbar spine by dual x-ray absorptiometry; and fractures. RESULTS: Comparisons of BMD at the lumbar spine and femoral neck showed nonsignificant differences less than 0.011 g/cm2 for any genotype with or without adjustments. A total of 6067 participants reported a history of fracture, and 2088 had vertebral fractures. For all VDR alleles, odds ratios for fractures were very close to 1.00 (range, 0.98 to 1.02) and collectively the 95% CIs ranged from 0.94 (lowest) to 1.07 (highest). For vertebral fractures, we observed a 9% (95% CI, 0% to 18%; P = 0.039) risk reduction for the Cdx2 A-allele (13% risk reduction in a dominant model). LIMITATIONS: The authors analyzed only selected VDR polymorphisms. Heterogeneity was detected in some analyses and may reflect some differences in collection of fracture data across cohorts. Not all fractures were related to osteoporosis. CONCLUSIONS: The FokI, BsmI, ApaI, and TaqI VDR polymorphisms are not associated with BMD or with fractures, but the Cdx2 polymorphism may be associated with risk for vertebral fractures.

Association of estrogen receptor-alpha genepolymorphisms with venous thrombosis.

XbaI and PvuII polymorphisms of the estrogen receptor-alpha gene (ERalpha) have been associated with several multifactorial diseases. We studied the distribution of ERalpha polymorphisms in patients with deep vein thrombosis. PvuII PP and XbaI XX genotypes may be associated with an approximately 2-fold increased DVT risk of deep vein thrombosis in men.

ERbeta is a potent inhibitor of cell proliferation in the HCT8 human colon cancer cell line through regulation of cell cycle components.

Several strands of evidence indicate that oestrogens exert a protective role against the development of colon cancer through indirect and direct effects on colonic epithelium. Oestrogen receptor beta (ERbeta), the predominant ER subtype in human colon, is significantly decreased in colonic tumours compared with normal mucosa suggesting a potential role in the regulation of colon tumour growth. To investigate this hypothesis we engineered human colon cancer ERalpha-negative HCT8 cells in order to obtain ERbeta protein over-expression. Stably transfected cells were cloned and ERbeta expression and functionality were monitored by RT-PCR, Western blotting and transactivation in an assay using oestrogen-responsive reporter constructs. Over-expression of ERbeta inhibited cell proliferation and increased cell adhesion in a ligand-independent manner. Its constitutive activation is possibly due to cross-talk with intracellular signalling pathways, as epidermal growth factor and IGF-I were able to induce ERbeta transactivation. A possible mechanism by which ERbeta over-expression inhibits proliferation in HCT8 cells is by modulation of some key regulators of the cell cycle; there is a decrease in cyclin E and an increase in the cdk inhibitor p21CIP1. In fact, flow cytometry analysis provided evidence for blocking of the G1-S phase progression induced by ERbeta over-expression. The magnitude of this effect was affected by the level of ERbeta expression. These results provide the first direct evidence that ERbeta plays an important role in colon cancer as a regulator of cell proliferation through the control of key cell cycle modulators and arrest in G1-S phase transition. These findings are compatible with the hypothesis that the loss of ERbeta expression could be one of the events involved in the development or progression of colon cancer.

Azidothymidine induces apoptosis and inhibits cell growth and telomerase activity of human parathyroid cancer cells in culture.

UNLABELLED: Telomerase activity has been correlated to parathyroid carcinoma. Because its role in acquisition of a malignant phenotype by parathyroid cells is unclear, we treated telomerase-positive cultured human parathyroid cancer cells with the telomerase inhibitor AZT, evaluating cell telomerase activity, cytotoxic effects, growth, and morphological changes. In vitro exposure of these cells to AZT correlated with inhibition of cell proliferation. INTRODUCTION: Parathyroid carcinoma represents an uncommon cause of primary hyperparathyroidism, whose spectrum of clinical presentation, degree of malignancy, and prognosis are difficult to be properly identified. Neck surgery, specifically an en bloc resection of primary tumor, is the only curative treatment. Alternatively, affected patients could undergo repetitive palliative surgical exeresis of metastatic nodules. It has been previously shown that telomerase activity is specifically present in parathyroid carcinoma cells, being absent in hyperplastic and adenomatous tissues. Thus, determination of telomerase activity could represent either a useful diagnostic molecular marker for human parathyroid carcinoma or a potential target for pharmacological intervention in a malignant neoplasia usually resistant to chemo- and radiotherapeutic interventions. MATERIALS AND METHODS: To further investigate the role of telomerase activity in acquisition of a malignant phenotype by parathyroid cells, we treated telomeric repeat amplification protocol-positive cultured human parathyroid cells with the telomerase inhibitor zidovudine, 3'-azido-3'deoxythymidine (AZT), evaluating cell telomerase activity, growth characteristics, potential cytotoxic effects, and morphological changes. RESULTS: Our findings indicate that in vitro exposure of human parathyroid cancer cells to AZT resulted in intracellular accumulation of AZT-monophosphate (AZT-MP) and inhibition of telomerase, which correlate with inhibition of human parathyroid cancer cell proliferation. Moreover, we also found that AZT induced an apoptotic rather than a necrotic type of cellular death. None of these effects were observed in human adenomatous parathyroid cells in culture. CONCLUSIONS: Altogether these results indicate that AZT may be a highly effective agent against cancer parathyroid cells proliferation, which is an extremely important observation for a neoplasia which shows lack of response to classical pharmacological and physical antiblastic treatments.

Differential genetic effects of ESR1 gene polymorphisms on osteoporosis outcomes.

CONTEXT: Both bone mineral density (BMD) and fracture risk have a strong genetic component. Estrogen receptor alpha (ESR1) is a candidate gene for osteoporosis, but previous studies of ESR1 polymorphisms in this field were hampered by small sample size, lack of standardization, and inconclusive results. OBJECTIVE: To generate large-scale evidence on whether 3 common ESR1 polymorphisms (intron 1 polymorphisms XbaI [dbSNP: rs9340799] and PvuII [dbSNP: rs2234693] and promoter TA repeats microsatellite) and haplotypes thereof are associated with BMD and fractures. DESIGN AND SETTING: Meta-analysis of individual-level data involving standardized genotyping of 18 917 individuals in 8 European centers. MAIN OUTCOME MEASURES: BMD of femoral neck and lumbar spine; all fractures and vertebral fractures by genotype. RESULTS: No between-center heterogeneity was observed for any outcome in any genetic contrast. None of the 3 polymorphisms or haplotypes had any statistically significant effect on BMD in adjusted or unadjusted analyses, and estimated differences between genetic contrasts were 0.01 g/cm2 or less. Conversely, we found significant reductions in fracture risk. In women homozygous for the absence of an XbaI recognition site, the adjusted odds of all fractures were reduced by 19% (odds ratio, 0.81 [95% CI, 0.71-0.93]; P = .002) and vertebral fractures by 35% (odds ratio, 0.65 [95% CI, 0.49-0.87]; P = .003). Effects on fractures were independent of BMD and unaltered in adjusted analyses. No significant effects on fracture risk were seen for PvuII and TA repeats. CONCLUSIONS: ESR1 is a susceptibility gene for fractures, and XbaI determines fracture risk by mechanisms independent of BMD. Our study demonstrates the value of adequately powered studies with standardized genotyping and clinical outcomes in defining effects of common genetic variants on complex diseases.