Susana Balcells

Large-Scale Analysis of Association Between LRP5 and LRP6 Variants and Osteoporosis

CONTEXT Mutations in the low-density lipoprotein receptor-related protein 5 (LRP5) gene cause rare syndromes characterized by altered bone mineral density (BMD). More common LRP5 variants may affect osteoporosis risk in the general population. OBJECTIVE To generate large-scale evidence on whether 2 common variants of LRP5 (Val667Met, Ala1330Val) and 1 variant of LRP6 (Ile1062Val) are associated with BMD and fracture risk. DESIGN AND SETTING Prospective, multicenter, collaborative study of individual-level data on 37,534 individuals from 18 participating teams in Europe and North America. Data were collected between September 2004 and January 2007; analysis of the collected data was performed between February and May 2007. Bone mineral density was assessed by dual-energy x-ray absorptiometry. Fractures were identified via questionnaire, medical records, or radiographic documentation; incident fracture data were available for some cohorts, ascertained via routine surveillance methods, including radiographic examination for vertebral fractures. MAIN OUTCOME MEASURES Bone mineral density of the lumbar spine and femoral neck; prevalence of all fractures and vertebral fractures. RESULTS The Met667 allele of LRP5 was associated with reduced lumbar spine BMD (n = 25,052 [number of participants with available data]; 20-mg/cm2 lower BMD per Met667 allele copy; P = 3.3 x 10(-8)), as was the Val1330 allele (n = 24,812; 14-mg/cm2 lower BMD per Val1330 copy; P = 2.6 x 10(-9)). Similar effects were observed for femoral neck BMD, with a decrease of 11 mg/cm2 (P = 3.8 x 10(-5)) and 8 mg/cm2 (P = 5.0 x 10(-6)) for the Met667 and Val1330 alleles, respectively (n = 25 193). Findings were consistent across studies for both LRP5 alleles. Both alleles were associated with vertebral fractures (odds ratio [OR], 1.26; 95% confidence interval [CI], 1.08-1.47 for Met667 [2001 fractures among 20 488 individuals] and OR, 1.12; 95% CI, 1.01-1.24 for Val1330 [1988 fractures among 20,096 individuals]). Risk of all fractures was also increased with Met667 (OR, 1.14; 95% CI, 1.05-1.24 per allele [7876 fractures among 31,435 individuals)]) and Val1330 (OR, 1.06; 95% CI, 1.01-1.12 per allele [7802 fractures among 31 199 individuals]). Effects were similar when adjustments were made for age, weight, height, menopausal status, and use of hormone therapy. Fracture risks were partly attenuated by adjustment for BMD. Haplotype analysis indicated that Met667 and Val1330 variants both independently affected BMD. The LRP6 Ile1062Val polymorphism was not associated with any osteoporosis phenotype. All aforementioned associations except that between Val1330 and all fractures and vertebral fractures remained significant after multiple-comparison adjustments. CONCLUSIONS Common LRP5 variants are consistently associated with BMD and fracture risk across different white populations. The magnitude of the effect is modest. LRP5 may be the first gene to reach a genome-wide significance level (a conservative level of significance [herein, unadjusted P < 10(-7)] that accounts for the many possible comparisons in the human genome) for a phenotype related to osteoporosis.

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

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 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/cm 2 (CI, 16 to 34 mg/cm 2) lower in TT homozygotes than the other genotype groups ( p < 0.001), and a similar difference was observed at the lumbar spine; 21 mg/cm 2 (CI, 1 to 42 mg/cm 2), ( 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.

ORMDL proteins are a conserved new family of endoplasmic reticulum membrane proteins

BackgroundAnnotations of completely sequenced genomes reveal that nearly half of the genes identified are of unknown function, and that some belong to uncharacterized gene families. To help resolve such issues, information can be obtained from the comparative analysis of homologous genes in model organisms.ResultsWhile characterizing genes from the retinitis pigmentosa locus RP26 at 2q31-q33, we have identified a new gene, ORMDL1, that belongs to a novel gene family comprising three genes in humans (ORMDL1, ORMDL2 and ORMDL3), and homologs in yeast, microsporidia, plants, Drosophila, urochordates and vertebrates. The human genes are expressed ubiquitously in adult and fetal tissues. The Drosophila ORMDL homolog is also expressed throughout embryonic and larval stages, particularly in ectodermally derived tissues. The ORMDL genes encode transmembrane proteins anchored in the endoplasmic reticulum (ER). Double knockout of the two Saccharomyces cerevisiae homologs leads to decreased growth rate and greater sensitivity to tunicamycin and dithiothreitol. Yeast mutants can be rescued by human ORMDL homologs.ConclusionsFrom protein sequence comparisons we have defined a novel gene family, not previously recognized because of the absence of a characterized functional signature. The sequence conservation of this family from yeast to vertebrates, the maintenance of duplicate copies in different lineages, the ubiquitous pattern of expression in human and Drosophila, the partial functional redundancy of the yeast homologs and phenotypic rescue by the human homologs, strongly support functional conservation. Subcellular localization and the response of yeast mutants to specific agents point to the involvement of ORMDL in protein folding in the ER.

Unusual expression of Gaucher's disease: cardiovascular calcifications in three sibs homozygous for the D409H mutation.

Three sisters suffering from an unusual form of Gauchers disease are described. These patients had cardiovascular abnormalities consisting of calcification of the ascending aorta and of the aortic and mitral valves. Neurological findings included ophthalmoplegia and saccadic eye movements in two patients, and tonic-clonic seizures in the third. The three patients died, two of them after having undergone aortic valve replacement. Tissue was obtained from one of the sibs and fibroblast and liver beta-glucocerebrosidase activity was reduced to 4% and 11% of mean normal values. Genotype analysis indicated that the patient was homozygous for the D409H mutation. It is tempting to relate the phenotype of severe cardiac involvement to the D409H/D409H genotype, although further cases will be needed before this association can be confirmed.

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

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 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.

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

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, 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 α1 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. J. Cell. Biochem. 101: 1430–1438, 2007.

Effect of IL-1β, PGE2, and TGF-β1 on the expression of OPG and RANKL in normal and osteoporotic primary human osteoblasts

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, 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)‐1β, proftaglandin E2 (PGE2), and transforming growth factor‐β1 (TGF‐β1) 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‐1β treatment and decreased in response to TGF‐β1 whereas PGE2 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‐1β treatment was up to three times lower (P = 0.009), whereas that of RANKL response to TGF‐β1 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. J. Cell. Biochem. 110: 304–310, 2010.

Promoter 2 -1025 T/C Polymorphism in the RUNX2 Gene Is Associated with Femoral Neck BMD in Spanish Postmenopausal Women

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, r2 = 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.

Assessment of Gene-by-Sex Interaction Effect on Bone Mineral Density

Sexual dimorphism in various bone phenotypes, including bone mineral density (BMD), is widely observed; however, the extent to which genes explain these sex differences is unclear. To identify variants with different effects by sex, we examined gene‐by‐sex autosomal interactions genome‐wide, and performed expression quantitative trait loci (eQTL) analysis and bioinformatics network analysis. We conducted an autosomal genome‐wide meta‐analysis of gene‐by‐sex interaction on lumbar spine (LS) and femoral neck (FN) BMD in 25,353 individuals from 8 cohorts. In a second stage, we followed up the 12 top single‐nucleotide polymorphisms (SNPs; p < 1 × 10−5) in an additional set of 24,763 individuals. Gene‐by‐sex interaction and sex‐specific effects were examined in these 12 SNPs. We detected one novel genome‐wide significant interaction associated with LS‐BMD at the Chr3p26.1‐p25.1 locus, near the GRM7 gene (male effect = 0.02 and p = 3.0 × 10−5; female effect = −0.007 and p = 3.3 × 10−2), and 11 suggestive loci associated with either FN‐ or LS‐BMD in discovery cohorts. However, there was no evidence for genome‐wide significant (p < 5 × 10−8) gene‐by‐sex interaction in the joint analysis of discovery and replication cohorts. Despite the large collaborative effort, no genome‐wide significant evidence for gene‐by‐sex interaction was found to influence BMD variation in this screen of autosomal markers. If they exist, gene‐by‐sex interactions for BMD probably have weak effects, accounting for less than 0.08% of the variation in these traits per implicated SNP.

A novel mutation in exon 17 of the β-subunit of rod phosphodiesterase in two RP sisters of a consanguineous family

We report the molecular analysis of the β subunit of the rod phosphodiesterase (PDEB) gene in a consanguineous autosomal recessive retinitis pigmentosa family that shows homozygosity for polymorphisms in the genomic region comprising this gene, and positive linkage between a PDEB marker and the diesease. The two affected sisters are homozygous for a T to G transversion in codon 699 of the PDEB gene, leading to the substitution of a leucine by an arginine residue. This change, enclosed in the catalytic domain of the PDEB, could result in a modification of the protein structure preventing the physiological hydrolysis of cGMP.