Leonardo Mellibovsky

Publications of Leonardo Mellibovsky

• Functional relevance of the BMD-associated polymorphism rs312009: novel involvement of RUNX2 in LRP5 transcriptional regulation.

  Authors: Lídia Agueda, Rafael Velázquez-Cruz, Roser Urreizti, Guy Yoskovitz, Patricia Sarrión, Susana Jurado, Roberto Güerri, Natàlia Garcia-Giralt, Xavier Nogués, Leonardo Mellibovsky, Adolfo Díez-Pérez, Pierre J Marie, Susana Balcells, Daniel Grinberg

  Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research. 05/2011; 26(5):1133-44.

  LRP5 is an osteoporosis susceptibility gene. Association analyses reveal that individual single-nucleotide polymorphisms (SNPs) determine variation in bone mineral density (BMD) among individuals asLRP5 is an osteoporosis susceptibility gene. Association analyses reveal that individual single-nucleotide polymorphisms (SNPs) determine variation in bone mineral density (BMD) among individuals as well as fracture risk. In a previous study, we identified a lumbar spine BMD-associated SNP, rs312009, located in the LRP5 5' region. A RUNX2 binding site was identified in this region by gel-shift experiments. Here we test the functionality of this SNP and examine whether RUNX2 is indeed a regulator of LRP5 expression. Gene reporter assays were used to test rs312009 functionality. Bioinformatic predictive tools and gel-shift and gene reporter assays were used to identify and characterize additional RUNX2 binding elements in the 3.3-kb region upstream of LRP5. Allelic differences in the transcriptional activity of rs312009 were observed in two osteoblastic cell lines, the T allele being a better transcriber than the C allele. RUNX2 cotransfection in HeLa cells revealed that the LRP5 5' region responded to RUNX2 in a dose-dependent manner and that the previously identified RUNX2 binding site participated in this response. Also, RUNX2 inhibition by RNAi led to nearly 60% reduction of endogenous LRP5 mRNA in U-2 OS cells. Four other RUNX2 binding sites were identified in the 5' region of LRP5. Luciferase experiments revealed the involvement of each of them in the RUNX2 response. The allelic differences observed point to the involvement of rs312009 as a functional SNP in the observed association. To our knowledge, this is the first time that the direct action of RUNX2 on LRP5 has been described. This adds evidence to previously described links between two important bone-regulating systems: the RUNX2 transcription-factor cascade and the Wnt signaling pathway.

• Vitamin D deficiency and bone mineral density in postmenopausal women receiving aromatase inhibitors for early breast cancer.

  Authors: Xavier Nogues, Sonia Servitja, Maria Jesus Peña, Daniel Prieto-Alhambra, Rosa Nadal, Leonardo Mellibovsky, Joan Albanell, Adolfo Diez-Perez, Ignasi Tusquets

  Maturitas. 07/2010; 66(3):291-7.

  Aromatase inhibitors (AI) treatment leads to an increased risk of bone loss and fractures. In a group of women with early breast cancer (EBC) and baseline Vitamin D deficiency (<30 ng/ml) who areAromatase inhibitors (AI) treatment leads to an increased risk of bone loss and fractures. In a group of women with early breast cancer (EBC) and baseline Vitamin D deficiency (<30 ng/ml) who are treated with AI, we aim to describe: serum levels of Vitamin D, bone mineral density (BMD), calcium intake, and the increase of serum 25(OH)D accomplished in 3 months of treatment with Vitamin D supplements. Prospective, non-randomized clinical trial. In 232 consecutively included women with EBC in treatment with AI, we assessed baseline calcium intake, serum levels of 25(OH)D, BMD and, spine X-ray. All received Calcium and Vitamin D supplements, and those with vitamin deficiency received 16,000 IU Vitamin D every 2 weeks. Serum levels of 25(OH)D were newly assessed after treatment. All the baseline evaluation was performed before starting AI treatment. Mean age at baseline (+/-SD) was 63.2+/-8.8 years. In 150 (64.9%) cases, the women had been treated previously with tamoxifen; 101 (43.7%) started exemestane, 119 (51.5%) letrozole, and 11 (4.8%) anastrozole. The AI were initiated within 6 weeks after surgery or after the last cycle of chemotherapy. At baseline, 88.1% had 25(OH)D levels <30 ng/ml, 21.2% had severe deficiency (<10 ng/ml), and 25% of the participants had osteoporosis. Mean daily calcium intake was low (841+/-338). We found a significant association between 25(OH)D levels and BMD at baseline, which remained significant in femoral neck BMD after multivariate adjustment. Plasma 25(OH)D levels improved significantly at 3 months follow-up in those treated with high dose Vitamin D supplements: mean increase 32.55 ng/ml (95%CI 28.06-37.03). Our study suggests a high prevalence of commonly unrecognized Vitamin D deficiency in women with EBC treated with AI, a known osteopenic agent. Our results support the need for a routine assessment of 25(OH)D levels and, when necessary, supplementation in these patients.

• Analysis of three functional polymorphisms in relation to osteoporosis phenotypes: replication in a Spanish cohort.

  Authors: Lídia Agueda, Roser Urreizti, Mariona Bustamante, Susana Jurado, Natàlia Garcia-Giralt, Adolfo Díez-Pérez, Xavier Nogués, Leonardo Mellibovsky, Daniel Grinberg, Susana Balcells

  Calcified tissue international. 04/2010; 87(1):14-24.

  Osteoporosis is a complex disease involving many putative genetic factors. Association analysis of functional SNPs in candidate genes is an important tool for their identification. However, thisOsteoporosis is a complex disease involving many putative genetic factors. Association analysis of functional SNPs in candidate genes is an important tool for their identification. However, this approach is affected by limited power, population stratification, and other drawbacks that lead to discordant results. Replication in independent cohorts is essential. We performed association analyses of three functional polymorphisms previously associated with bone phenotypes--namely, Ala222Val in MTHFR, Ile1062Val in LRP6, and -13910C>T in LCT--in a cohort of 944 postmenopausal Spanish women, all of them with lumbar spine (LS) bone mineral density (BMD) data and most with femoral neck (FN) BMD and fracture data. We found significant differences between genotypes only for the MTHFR polymorphism and vertebral factures, with an OR of 2.27 (95% CI 1.17-4.38) for the TT vs. CC/CT genotypes, P = 0.018. We present genotype and allele frequency data for LCT -13910C>T for a Spanish population, where the T allele (conferring lactase persistence) has a frequency of 38.6%. Genotype frequencies were consistent with observed clines in Europe and with the prevalence of lactase nonpersistence. The LCT -13910C>T polymorphism was significantly associated with height and weight, such that T allele carriers were 0.88 cm taller (95% CI 0.08-1.59 cm, P = 0.032, adjusted by age) than CC individuals and TT homozygotes were 1.91 kg heavier than CC/CT individuals (95% CI 0.11-3.71 kg, P = 0.038, adjusted by age). In conclusion, no significant association was observed between the studied polymorphisms and LS BMD or FN BMD in postmenopausal Spanish women, and only MTHFR Ala222Val was associated with vertebral fractures.

• Effect of IL-1beta, PGE(2), and TGF-beta1 on the expression of OPG and RANKL in normal and osteoporotic primary human osteoblasts.

  Authors: Susana Jurado, Natalia Garcia-Giralt, Adolfo Díez-Pérez, Pedro Esbrit, Guy Yoskovitz, Lídia Agueda, Roser Urreizti, Lluís Pérez-Edo, Guillem Saló, Leonardo Mellibovsky, Susana Balcells, Daniel Grinberg, Xavier Nogués

  Journal of cellular biochemistry. 03/2010; 110(2):304-10.

  The RANKL/RANK/OPG pathway is essential for bone remodeling regulation. Many hormones and cytokines are involved in regulating gene expression in most of the pathway components. Moreover, anyThe RANKL/RANK/OPG pathway is essential for bone remodeling regulation. Many hormones and cytokines are involved in regulating gene expression in most of the pathway components. Moreover, any deregulation of this pathway can alter bone metabolism, resulting in loss or gain of bone mass. Whether osteoblasts from osteoporotic and nonosteoporotic patients respond differently to cytokines is unknown. The aim of this study was to compare the effect of interleukin (IL)-1beta, proftaglandin E(2) (PGE(2)), and transforming growth factor-beta1 (TGF-beta1) treatments on OPG and RANKL gene expression in normal (n = 11) and osteoporotic (n = 8) primary osteoblasts. OPG and RANKL mRNA levels of primary human osteoblastic (hOB) cell cultures were assessed by real-time PCR. In all cultures, OPG mRNA increased significantly in response to IL-1beta treatment and decreased in response to TGF-beta1 whereas PGE(2) treatment had no effect. RANKL mRNA levels were significantly increased by all treatments. Differences in OPG and RANKL responses were observed between osteoporotic and nonosteoporotic hOB: in osteoporotic hOB, the OPG response to IL-1beta treatment was up to three times lower (P = 0.009), whereas that of RANKL response to TGF-beta1 was five times higher (P = 0.002) after 8 h of treatment, as compared with those in nonosteoporotic hOBs. In conclusion, osteoporotic hOB cells showed an anomalous response under cytokine stimulation, consistent with an enhanced osteoclastogenesis resulting in high levels of bone resorption.

• Microindentation for in vivo measurement of bone tissue mechanical properties in humans.

  Authors: Adolfo Diez-Perez, Roberto Güerri, Xavier Nogues, Enric Cáceres, Maria Jesus Peña, Leonardo Mellibovsky, Connor Randall, Daniel Bridges, James C Weaver, Alexander Proctor, Davis Brimer, Kurt J Koester, Robert O Ritchie, Paul K Hansma

  Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research. 02/2010; 25(8):1877-85.

  Bone tissue mechanical properties are deemed a key component of bone strength, but their assessment requires invasive procedures. Here we validate a new instrument, a reference point indentationBone tissue mechanical properties are deemed a key component of bone strength, but their assessment requires invasive procedures. Here we validate a new instrument, a reference point indentation (RPI) instrument, for measuring these tissue properties in vivo. The RPI instrument performs bone microindentation testing (BMT) by inserting a probe assembly through the skin covering the tibia and, after displacing periosteum, applying 20 indentation cycles at 2 Hz each with a maximum force of 11 N. We assessed 27 women with osteoporosis-related fractures and 8 controls of comparable ages. Measured total indentation distance (46.0 +/- 14 versus 31.7 +/- 3.3 microm, p = .008) and indentation distance increase (18.1 +/- 5.6 versus 12.3 +/- 2.9 microm, p = .008) were significantly greater in fracture patients than in controls. Areas under the receiver operating characteristic (ROC) curve for the two measurements were 93.1% (95% confidence interval [CI] 83.1-100) and 90.3% (95% CI 73.2-100), respectively. Interobserver coefficient of variation ranged from 8.7% to 15.5%, and the procedure was well tolerated. In a separate study of cadaveric human bone samples (n = 5), crack growth toughness and indentation distance increase correlated (r = -0.9036, p = .018), and scanning electron microscope images of cracks induced by indentation and by experimental fractures were similar. We conclude that BMT, by inducing microscopic fractures, directly measures bone mechanical properties at the tissue level. The technique is feasible for use in clinics with good reproducibility. It discriminates precisely between patients with and without fragility fracture and may provide clinicians and researchers with a direct in vivo measurement of bone tissue resistance to fracture.

• A Haplotype-Based Analysis of the LRP5 Gene in Relation to Osteoporosis Phenotypes in Spanish Postmenopausal Women.

  Authors: Lídia Agueda, Mariona Bustamante, Susana Jurado, Natalia Garcia-Giralt, Manel Ciria, Guillem Saló, Ramon Carreras, Xavier Nogués, Leonardo Mellibovsky, Adolfo Díez-Pérez, Daniel Grinberg, Susana Balcells

  Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research. 09/2008;

  Abstract LRP5 encodes the low-density lipoprotein receptor-related protein 5, a transmembrane protein involved in Wnt signaling. LRP5 is an important regulator of osteoblast growth andAbstract LRP5 encodes the low-density lipoprotein receptor-related protein 5, a transmembrane protein involved in Wnt signaling. LRP5 is an important regulator of osteoblast growth and differentiation, affecting bone mass in vertebrates. Whether common variations in LRP5 are associated with normal BMD variation or osteoporotic phenotypes is of great relevance. We employed a haplotype-based approach to search for common disease-associated variants in LRP5 in a cohort of 964 Spanish postmenopausal women. Twenty-four SNPs were selected, covering the LRP5 region, including the missense changes p.V667M and p.A1330V. The SNPs were genotyped and evaluated for association with BMD at lumbar spine (LS) or femoral neck (FN) and with osteoporotic fracture, at single-SNP and haplotype levels, by regression methods. Association with LS BMD was found for SNP#1, rs312009, located in the 5'-flanking region (p=0.011, recessive model). SNP#6, rs2508836, in intron 1, was also associated with BMD, both at LS (p=0.025, additive model) and FN (p=0.031, recessive model). Two polymorphisms were associated with fracture: SNP#11, rs729635, in intron 1 and SNP#15, rs643892, in intron 5 (p=0.007 additive model, p=0.019 recessive model, respectively). Haplotype analyses did not provide additional information, except for haplotype "GC" of the block located at the 3'end of the gene. This haplotype spans intron 22 and the 3'UTR region and was associated with FN BMD (p=0.029, one copy of the haplotype vs. none). In silico analyses revealed that SNP#1 (rs312009) lies in a putative RUNX2 binding site. Electro-mobility shift assays confirmed RUNX2 binding to this site.

• Large-scale analysis of association between polymorphisms in the transforming growth factor beta 1 gene (TGFB1) and osteoporosis: the GENOMOS study.

  Authors: Bente L Langdahl, André G Uitterlinden, Stuart H Ralston, Thomas A Trikalinos, Susanne Balcells, Maria Luisa Brandi, Serena Scollen, Paul Lips, Roman Lorenc, Barbara Obermayer-Pietsch [......] Fiona E A McGuigan, Leonardo Mellibovsky, Leif Mosekilde, Xavier Nogués, Huibert A P Pols, Jonathan Reeve, Wilfried Renner, Fernando Rivadeneira, Natasja M van Schoor, John P A Ioannidis

  Bone. 06/2008; 42(5):969-81.

  INTRODUCTION: The TGFB1 gene which encodes transforming growth factor beta 1, is a strong candidate for susceptibility to osteoporosis and several studies have reported associations between boneINTRODUCTION: The TGFB1 gene which encodes transforming growth factor beta 1, is a strong candidate for susceptibility to osteoporosis and several studies have reported associations between bone mineral density (BMD), osteoporotic fractures and polymorphisms of TGFB1, although these studies have yielded conflicting results. METHODS: We investigated associations between TGFB1 polymorphisms and BMD and fracture in the GENOMOS study: a prospective multicenter study involving 10 European research studies including a total of 28,924 participants. Genotyping was conducted for known TGFB1 polymorphisms at the following sites: G-1639-A (G-800-A, rs1800468), C-1348-T (C-509-T, rs1800469), T29-C (Leu10Pro, rs1982073), G74-C (Arg25Pro, rs1800471) and C788-T (Thr263Ile, rs1800472). These polymorphisms were genotyped prospectively and methodology was standardized across research centers. Genotypes and haplotypes were related to BMD at the lumbar sine and femoral neck and fractures. RESULTS: There were no significant differences in either women or men at either skeletal site for any of the examined polymorphisms with the possible exception of a weak association with reduced BMD (-12 mg/cm2) in men with the T-1348 allele (p<0.05). None of the haplotypes was associated with BMD and none of the polymorphisms or haplotypes significantly affected overall risk of fractures, however, the odds ratio for incident vertebral fracture in carriers of the rare T788 allele was 1.64 (95% CI: 1.09-2.64), p<0.05. CONCLUSIONS: This study indicates that polymorphic variation in the TGFB1 gene does not play a major role in regulating BMD or susceptibility to fractures. The weak associations we observed between the C-1348-T and lumbar spine BMD in men and between C788-T and risk of incident vertebral fractures are of interest but could be chance findings and will need replication in future studies.

• Promoter 2 -1025 T/C polymorphism in the RUNX2 gene is associated with femoral neck bmd in Spanish postmenopausal women.

  Authors: Mariona Bustamante, Xavier Nogués, Lídia Agueda, Susana Jurado, Anke Wesselius, Enrique Cáceres, Ramon Carreras, Manel Ciria, Leonardo Mellibovsky, Susana Balcells, Adolfo Díez-Pérez, Daniel Grinberg

  Calcified tissue international. 11/2007; 81(4):327-32.

  Stimulation of bone formation is a key therapeutic target in osteoporosis. Runx2 is a runt domain transcription factor essential to osteoblast differentiation, bone remodeling, and fracture healing.Stimulation of bone formation is a key therapeutic target in osteoporosis. Runx2 is a runt domain transcription factor essential to osteoblast differentiation, bone remodeling, and fracture healing. Runx2 knockout mice exhibit a complete lack of ossification, while overexpression of this gene in transgenic mice results in an osteoporotic phenotype. Thus, RUNX2 is a good candidate for the genetic determination of osteoporosis. In this association study, the effects of the -330 G/T polymorphism in promoter 1 and the -1025 T/C polymorphism (rs7771980) in promoter 2 of RUNX2 were tested in relation to lumbar spine (LS) and femoral neck (FN) bone mineral density (BMD) in a cohort of 821 Spanish postmenopausal women. The minor allele frequencies for the two polymorphisms were 0.15 and 0.07, respectively. The two polymorphisms, located more than 90 kb apart, were not in linkage disequilibrium (D' = 0.27, r (2) = 0.028). In an ANCOVA test adjusting by weight, height, age, and years since menopause, the -330 G/T polymorphism was not associated with any of the phenotypes analyzed, while we found the -1025 T/C polymorphism to be associated with FN BMD (p = 0.001). In particular, individuals carrying the TC genotype had higher mean adjusted FN BMD values than those bearing the TT genotype. Our results highlight the importance of this RUNX2 promoter 2 polymorphism in FN BMD determination.

• Simvastatin and atorvastatin enhance gene expression of collagen type 1 and osteocalcin in primary human osteoblasts and MG-63 cultures.

  Authors: Silvia Ruiz-Gaspa, Xavier Nogues, Anna Enjuanes, Joan C Monllau, Josep Blanch, Ramon Carreras, Leonardo Mellibovsky, Daniel Grinberg, Susana Balcells, Adolfo Díez-Perez, Juan Pedro-Botet

  Journal of cellular biochemistry. 09/2007; 101(6):1430-8.

  To clarify the mechanism of the stimulatory effect of statins on bone formation, we have assessed the effect of simvastatin and atorvastatin on osteoblast activity by analysing cell proliferation, asTo clarify the mechanism of the stimulatory effect of statins on bone formation, we have assessed the effect of simvastatin and atorvastatin on osteoblast activity by analysing cell proliferation, as well as collagen, osteocalcin, and bone morphogenetic protein-2 (BMP2) gene expression in primary human osteoblast (hOB) and MG-63 cell line cultures. Explants of bone from patients without any metabolic disease under orthopedic hip procedures were used to obtain hOB. Cell cultures were established, synchronized, and different concentrations of simvastatin or atorvastatin were added (10(-9) M, 10(-8) M, 10(-7) M, 10(-6) M) during the experiment. Cell proliferation was analyzed after 24 h. Collagen polypeptide alpha1 type 1 (COL1A1) gene expression, osteocalcin, and BMP2 expression levels were quantified by real-time PCR after 24 h incubation with statins. There was a statistically significant decrease in cell proliferation related to simvastatin or atorvastatin addition at all concentrations in primary hOB compared with those not treated. A significant increase in COL1A1, osteocalcin, and BMP2 gene expression was detected when hOB cultures were treated with simvastatin or atorvastatin at different concentrations. Similar but less significant effects were found on MG-63 cells. After statin treatment we observed both an arrest of proliferation in hOB cells and an increase in collagen, osteocalcin, and BMP2 gene expression, consistent with a stimulatory effect towards mature osteoblast differentiation. These findings support the bone-forming effect of statins, probably through the BMP2 pathway.

• Bone mass of a 113-year-old man.

  Authors: Leonardo Mellibovsky, Mariona Bustamante, Pau Lluch, Xavier Nogues, Daniel Grinberg, Susana Balcells, Adolfo Diez-Perez

  The journals of gerontology. Series A, Biological sciences and medical sciences. 08/2007; 62(7):794-5.

  Osteoporosis is a common disease that affects elderly people. Aging induces loss of bone density and quality resulting in a progressive incidence of fragility fractures. In this study, we report theOsteoporosis is a common disease that affects elderly people. Aging induces loss of bone density and quality resulting in a progressive incidence of fragility fractures. In this study, we report the bone density of one of the oldest men in the world and of several of his first-degree relatives, as well as a genetic screen of these cases. No fractures have been suffered by any of them, and their bone mineral density (BMD) values in terms of z score were normal or lightly decreased. Neither mutations at the longevity-related gene KLOTHO nor the Gly171Val mutation of LRP5 associated with high bone mass was detected in the two centenarian stepbrothers.

• [Effect of tobacco smoking on leptin serum levels and its relationship with steroid hormones and bone mineral density]

  Authors: August Supervía, Anna Enjuanes, Joan Vila, Leonardo Mellibovsky, Xavier Nogués, Adolfo Díez-Pérez

  Medicina clínica. 12/2006; 127(17):645-7.

  BACKGROUND AND OBJECTIVE: Leptin is an hormone resulting from the obesity gene. Their actions could be important in the pathogenesis of the osteoporosis. The aim of this study is to analyse theBACKGROUND AND OBJECTIVE: Leptin is an hormone resulting from the obesity gene. Their actions could be important in the pathogenesis of the osteoporosis. The aim of this study is to analyse the influence of tobacco on serum leptin levels, and its relationship with bone mineral density (BMD) and steroid hormones. SUBJECTS AND METHOD: A group of healthy volunteers were recruited and classified as smokers or non-smokers. A subgroup of smokers ceased smoking during one month. Serum leptin and steroids hormones levels were analysed, and a baseline BMD was measured. In the abstinent group the analysis was repeated at the end of the study. RESULTS: Fifty-nine healthy volunteers were included (22 of which were smokers). Fifteen smokers remained abstinent for a month. Both groups were similar except in age, being smokers older. Male smokers had lower lumbar BMD (p = 0.017). After adjusting by age, serum leptin levels were higher in smokers than in non-smokers, with statistical differences in women (p = 0.049). Abstinence increased leptin levels, though not reaching statistical significance. An inverse correlation between leptin levels and androstendione in men (r = -0.622; p = 0.001), and a positive correlation with testosterone in women (r = 0.405; p = 0.019) were found. After adjusting by body mass index, only the correlation of leptin levels with androstendione persisted. CONCLUSIONS: Leptin negatively correlate with sex hormones in young men and is influenced by smoking in young women. Thus, the hormone could be relevant for bone mass regulation in smoker persons.

• A new SNP in a negative regulatory region of the CYP19A1 gene is associated with lumbar spine BMD in postmenopausal women.

  Authors: Anna Enjuanes, Natalia Garcia-Giralt, August Supervía, Xavier Nogués, Silvia Ruiz-Gaspà, Mariona Bustamante, Leonardo Mellibovsky, Daniel Grinberg, Susana Balcells, Adolfo Díez-Pérez

  Bone. 06/2006; 38(5):738-43.

  Osteoporosis is a common disease of bone possessing a strong genetic component. Cytochrome P450 aromatase, which is encoded by the CYP19A1 gene, converts androgens to estradiol. Considerable evidenceOsteoporosis is a common disease of bone possessing a strong genetic component. Cytochrome P450 aromatase, which is encoded by the CYP19A1 gene, converts androgens to estradiol. Considerable evidence suggests that extragonadal estrogens play an important role in determining bone mineral density (BMD) in postmenopausal women, and, among them, those synthesized in bone cells may also be important for the determination of bone phenotype. Therefore, CYP19A1 is an excellent candidate gene for osteoporosis. Since a region upstream of exon I.3, including exon I.6, was identified as containing repressor elements of promoter pII, we conducted a search for SNPs in this region of CYP19A1. Two SNPs [Aro1(rs4775936) and Aro2] located in exon I.6 and promoter I.6, respectively, were identified and their association with BMD analyzed in a cohort of 256 Spanish postmenopausal women. Aro1(rs4775936), but not Aro2, was associated with lumbar spine BMD (P = 0.029). Homozygotes AA (16% of the women) exhibited significantly higher lumbar spine BMD, compared with GG or GA individuals. Therefore, this study describes the Aro1 polymorphism which lies within a regulatory region and which may be a functional polymorphism, partially responsible for the bone phenotype it is associated with.

• Large-scale evidence for the effect of the COLIA1 Sp1 polymorphism on osteoporosis outcomes: the GENOMOS study.

  Authors: Stuart H Ralston, André G Uitterlinden, Maria Luisa Brandi, Susana Balcells, Bente L Langdahl, Paul Lips, Roman Lorenc, Barbara Obermayer-Pietsch, Serena Scollen, Mariona Bustamante [......] Leonardo Mellibovsky, Francesca Del Monte, Huibert A P Pols, Jonathan Reeve, David M Reid, Wilfried Renner, Fernando Rivadeneira, Natasja M van Schoor, Rachael E Sherlock, John P A Ioannidis

  PLoS medicine. 04/2006; 3(4):e90.

  BACKGROUND: Osteoporosis and fracture risk are considered to be under genetic control. Extensive work is being performed to identify the exact genetic variants that determine this risk. Previous workBACKGROUND: Osteoporosis and fracture risk are considered to be under genetic control. Extensive work is being performed to identify the exact genetic variants that determine this risk. Previous work has suggested that a G/T polymorphism affecting an Sp1 binding site in the COLIA1 gene is a genetic marker for low bone mineral density (BMD) and osteoporotic fracture, but there have been no very-large-scale studies of COLIA1 alleles in relation to these phenotypes. METHODS AND FINDINGS: Here we evaluated the role of COLIA1 Sp1 alleles as a predictor of BMD and fracture in a multicenter study involving 20,786 individuals from several European countries. At the femoral neck, the average (95% confidence interval [CI]) BMD values were 25 mg/cm2 (CI, 16 to 34 mg/cm2) lower in TT homozygotes than the other genotype groups (p < 0.001), and a similar difference was observed at the lumbar spine; 21 mg/cm2 (CI, 1 to 42 mg/cm2), (p = 0.039). These associations were unaltered after adjustment for potential confounding factors. There was no association with fracture overall (odds ratio [OR] = 1.01 [CI, 0.95 to 1.08]) in either unadjusted or adjusted analyses, but there was a non-significant trend for association with vertebral fracture and a nominally significant association with incident vertebral fractures in females (OR = 1.33 [CI, 1.00 to 1.77]) that was independent of BMD, and unaltered in adjusted analyses. CONCLUSIONS: Allowing for the inevitable heterogeneity between participating teams, this study-which to our knowledge is the largest ever performed in the field of osteoporosis genetics for a single gene-demonstrates that the COLIA1 Sp1 polymorphism is associated with reduced BMD and could predispose to incident vertebral fractures in women, independent of BMD. The associations we observed were modest however, demonstrating the importance of conducting studies that are adequately powered to detect and quantify the effects of common genetic variants on complex diseases.

• Functional analysis of the I.3, I.6, pII and I.4 promoters of CYP19 (aromatase) gene in human osteoblasts and their role in vitamin D and dexamethasone stimulation.

  Authors: Anna Enjuanes, Natalia Garcia-Giralt, August Supervía, Xavier Nogués, Silvia Ruiz-Gaspà, Mariona Bustamante, Leonardo Mellibovsky, Daniel Grinberg, Susana Balcells, Adolfo Díez-Pérez

  European journal of endocrinology / European Federation of Endocrine Societies. 01/2006; 153(6):981-8.

  OBJECTIVE: Current evidence suggests that extragonadal estrogens play an important role in bone metabolism. Estrogen biosynthesis is catalyzed by P450aromatase, encoded by the CYP19 gene. The aims ofOBJECTIVE: Current evidence suggests that extragonadal estrogens play an important role in bone metabolism. Estrogen biosynthesis is catalyzed by P450aromatase, encoded by the CYP19 gene. The aims of this paper were to study CYP19 gene expression in human osteoblasts under several hormone and cytokine treatments and to define promoter regions involved in this regulation. METHODS: CYP19 transcript levels were measured from primary human osteoblasts and MG-63 cells by real-time PCR in basal conditions, and in response to seven different hormones and cytokines. Four promoters of CYP19 gene were cloned upstream of the luciferase gene and transfected into MG-63 cells. The effect of vitamin D and dexamethasone in these promoter activities was evaluated. RESULTS: Vitamin D and dexamethasone were potent stimulators of CYP19 transcription, while testosterone and 17beta-estradiol stimulated moderately. Promoter pII proved the most potent in driving transient luciferase expression. Promoter I.4 displayed moderate activity, while promoters I.3 and I.6 were weak. A region upstream of exon I.3, including exon I.6, was identified as containing repressor elements of promoter pII. Promoter I.3 activity was modulated by repressors located within exon I.3, while an enhancer of promoter I.4 was detected within exon I.4. In the absence of fetal calf serum, dexamethasone stimulation was observed on promoters I.3 and I.4, while vitamin D stimulation acted only on promoter I.3. CONCLUSIONS: Four regulatory regions of promoters pII, I.3 and I.4 are relevant to CYP19 expression in human osteoblasts. Vitamin D and dexamethasone modulate transcription through these regions.

• In vitro functional assay of alleles and haplotypes of two COL1A1-promoter SNPs.

  Authors: Natàlia García-Giralt, Anna Enjuanes, Mariona Bustamante, Leonardo Mellibovsky, Xavier Nogués, Ramón Carreras, Adolfo Díez-Pérez, Daniel Grinberg, Susana Balcells

  Bone. 06/2005; 36(5):902-8.

  Osteoporosis is a common disease with a strong genetic component. We previously described two polymorphic sites in the COL1A1 gene promoter, -1997 G/T and -1663indelT, which have been associated withOsteoporosis is a common disease with a strong genetic component. We previously described two polymorphic sites in the COL1A1 gene promoter, -1997 G/T and -1663indelT, which have been associated with bone mineral density (BMD), a surrogate trait for osteoporosis. Here, we explore the molecular mechanisms underlying this association by performing transient transfections in MG-63 cells of constructs bearing different COL1A1 promoter regions, containing different alleles or haplotypes of the polymorphic sites. These promoter regions drove the transcription of a luciferase reporter gene. The main differences in transcriptional activity relied on an inhibitory region localized to the -1284 to -254 interval. Regarding the polymorphisms, reproducible differences were observed between the alleles of each of them: the G allele at -1997 showed a higher transcriptional activity than the T allele, as did the 7T allele of -1663 as compared with 8T. Accordingly, the T-8T haplotype was the weakest transcriber. A functional interaction was found between the -1997 and -1663 polymorphisms, in that the difference in transcriptional activity between the 7T and 8T alleles was dependent on the allele at -1997. This different transcriptional activity of the two -1663indelT alleles correlated with different binding capacities of the corresponding oligonucleotides to osteoblast nuclear proteins. Supershift assays allowed us to identify one of these proteins as the architectural transcription factor Nmp4/CIZ, a protein known to be an inhibitor of BMP/Smad signalling.

• Regulation of CYP19 gene expression in primary human osteoblasts: effects of vitamin D and other treatments.

  Authors: Anna Enjuanes, Natalia Garcia-Giralt, August Supervia, Xavier Nogués, Leonardo Mellibovsky, Jordi Carbonell, Daniel Grinberg, Susana Balcells, Adolfo Díez-Pérez

  European journal of endocrinology / European Federation of Endocrine Societies. 06/2003; 148(5):519-26.

  OBJECTIVE: Extragonadal estrogen biosynthesis is relevant for the regulation of bone metabolism. The aims of this paper were: (i) to examine CYP19 (aromatase) gene expression in primary cultures ofOBJECTIVE: Extragonadal estrogen biosynthesis is relevant for the regulation of bone metabolism. The aims of this paper were: (i) to examine CYP19 (aromatase) gene expression in primary cultures of osteoblasts under several hormone and cytokine treatments and (ii) to study the promoter usage of CYP19 in these cells. METHODS: Primary cultures of osteoblasts were obtained from healthy donors. The effects of vitamin D and other factors on CYP19 expression were analysed by semiquantitative RT-PCR. Furthermore, CYP19 alternative promoter usage under the different treatments was characterized by RT-PCR. RESULTS: CYP19 transcripts were detected in cultured human osteoblasts in serum-free conditions. Vitamin D, dexamethasone, 17beta-estradiol and testosterone increased transcript levels of CYP19, whereas interleukin-1beta or tumor necrosis factor alpha decreased them. Aromatase mRNA produced under treatment with vitamin D was transcribed from promoters I.4 and I.3, while stimulation with dexamethasone or dexamethasone plus vitamin D also involved promoter I.2. Testosterone activated promoters I.2 and I.4. CONCLUSIONS: Our results suggested that vitamin D, testosterone, estrogens and glucocorticoids regulate CYP19 gene expression in human primary osteoblasts and the main promoter used appears to be promoter I.4. Promoters pII and I.3 seem to be related to basal transcription and may mediate estrogen stimulation, while promoter I.2 seems to play a role in the effect of glucocorticoids. These findings indicate that vitamin D and several hormones regulate local estrogen synthesis in human osteoblasts mainly through usage of promoters I.4 and I.3.

• Ataxic form of central pontine myelinolysis.

  Authors: Teresa Garzon, Leonardo Mellibovsky, Jaume Roquer, Xavier Perich, Adolfo Diez-Perez

  Lancet neurology. 01/2003; 1(8):517-8.

• Two new single-nucleotide polymorphisms in the COL1A1 upstream regulatory region and their relationship to bone mineral density.

  Authors: Natalia Garcia-Giralt, Xavier Nogués, Anna Enjuanes, Jordi Puig, Leonardo Mellibovsky, Anne Bay-Jensen, Ramon Carreras, Susana Balcells, Adolfo Díez-Pérez, Daniel Grinberg

  Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research. 04/2002; 17(3):384-93.

  Single-nucleotide polymorphisms (SNPs) in regulatory regions of candidate genes may determine variability in bone mineral density (BMD) because they may be responsible for differences in levels of aSingle-nucleotide polymorphisms (SNPs) in regulatory regions of candidate genes may determine variability in bone mineral density (BMD) because they may be responsible for differences in levels of a gene product in response to external signals. Under this hypothesis, we scanned an 800-base pair (bp) region within the COL1A1 promoter, known to harbor cis elements important for in vivo expression, and we found two new polymorphisms: -1663indelT and -1997 G/T. The G to T transversion at -1997 was associated with lumbar spine BMD (p = 0.015) when tested in a cohort of 256 postmenopausal women after adjusting by age, body weight, and years since menopause; a lower degree of association was detected also for femoral neck BMD in a subgroup of 146 women in univariate analysis and after adjusting by age (p = 0.044). The polymorphism -1663indelT, which corresponds to a deletion of a T in a tract of eight T residues (-1670 to -1663), did not show significant association with BMD. Interestingly, -1663indelT is in strong linkage disequilibrium (LD) with the previously described Sp1 polymorphism of intron 1, which in this study did not show association with BMD either. Significant interaction between -1997 G/T and -1663indelT (p = 0.019), and between -1997 G/T and Sp1 (p = 0.045) was observed also. Individuals heterozygous for the three polymorphisms showed the highest mean BMD value. Gel retardation assays showed that oligonucleotides containing either the -1663 or the -1997 polymorphic sites specifically bind primary osteoblast nuclear proteins. We named these binding sites as PCOL1 and PCOL2, respectively. In summary, this study describes two new SNPs in the COL1A1 promoter, which may affect bone mass determination.

• Correction: Large-Scale Evidence for the Effect of the COLIA1 Sp1 Polymorphism on Osteoporosis Outcomes: The GENOMOS Study

  Authors: Stuart H Ralston, André G Uitterlinden, Maria Luisa Brandi, Susana Balcells, Bente L Langdahl, Paul Lips, Roman Lorenc, Barbara Obermayer-Pietsch, Serena Scollen, Mariona Bustamante [......] Leonardo Mellibovsky, Francesca Del Monte, Huibert A P Pols, Jonathan Reeve, David M Reid, Wilfried Renner, Fernando Rivadeneira, Natasja M van Schoor, Rachael E Sherlock, John P A Ioannidis