SOST Genes Research Articles

Radiotherapy-induced bone deterioration is exacerbated in diabetic rats treated with streptozotocin.

Following radiotherapy, patients have decreased bone mass and increased risk of fragility fractures. Diabetes mellitus (DM) is also reported to have detrimental effects on bone architecture and quality. However, no clinical or experimental study has systematically characterized the bone phenotype of the diabetic patients following radiotherapy. After one month of streptozotocin injection, three-month-old male rats were subjected to focal radiotherapy (8 Gy, twice, at days 1 and 3), and then bone mass, microarchitecture, and turnover as well as bone cell activities were evaluated at 2 months post-irradiation. Micro-computed tomography results demonstrated that DM rats exhibited greater deterioration in trabecular bone mass and microarchitecture following irradiation compared with the damage to bone structure induced by DM or radiotherapy. The serum biochemical, bone histomorphometric, and gene expression assays revealed that DM combined with radiotherapy showed lower bone formation rate, osteoblast number on bone surface, and expression of osteoblast-related markers (ALP, Runx2, Osx, and Col-1) compared with DM or irradiation alone. DM plus irradiation also caused higher bone resorption rate, osteoclast number on bone surface, and expression of osteoclast-specific markers (TRAP, cathepsin K, and calcitonin receptor) than DM or irradiation treatment alone. Moreover, lower osteocyte survival and higher expression of Sost and DKK1 genes (two negative modulators of Wnt signaling) were observed in rats with combined DM and radiotherapy. Together, these findings revealed a higher deterioration of the diabetic skeleton following radiotherapy, and emphasized the clinical importance of health maintenance.

Polymorphisms of FDPS, LRP5, SOST and VKORC1 genes and their relation with osteoporosis in postmenopausal Romanian women.

This study aimed to assess the relationship between bone mineral density and genotypes of four polymorphisms in previously detected osteoporosis-candidate genes (FDPS rs2297480, LRP5 rs3736228, SOST rs1234612, VKORC1 rs9934438) in postmenopausal Romanian women with primary osteoporosis. An analytical, prospective, transversal, observational, case-control study on 364 postmenopausal Romanian women was carried out between June 2016 and August 2017 in Cluj Napoca, Romania. Clinical data and blood samples were collected from all study participants. Four polymorphisms were genotyped using TaqMan SNP Genotyping assays, run on a QuantStudio 3 real-time PCR machine. Women with TT genotype in FDPS rs2297480 had significantly lower bone mineral density values in the lumbar spine and total hip, and the presence of the T allele was significantly associated with the osteoporosis. Women carrying the CC genotype in LRP5 rs3736228 tend to have lower bone mineral density values in the femoral neck and total hip. No significant association was found for the genotypes of SOST rs1234612 or VKORC1 rs9934438. Our study showed a strong association between bone mineral density and polymorphisms in the FDPS gene, and a borderline association with LRP5 and SOST polymorphisms in postmenopausal Romanian women with osteoporosis. No association was found for VKORC1.

Gene–gene interaction and association of Wnt/Β-catenin signalling pathway gene polymorphisms with ankylosing spondylitis susceptibility in the Chinese Han population

Objectives: To explore the genetic interaction between Wnt/β-catenin signalling pathway genes and ankylosing spondylitis (AS) in the Chinese population.Methods: Six single-nucleotide polymorphisms (SNPs) in DKK1, LRP5, LRP6, and SOST genes were genotyped in 673 AS patients and 687 healthy controls by using SNPs can Technic. Single marker genetic association analysis was performed. Haplotypes were constructed after linkage disequilibrium analysis; additive, multiplicative, and higher-order interactions were analysed.Results: The DKK1 gene rs1569198 polymorphism was significantly associated with AS susceptibility in females (χ2 = 4.55, p = .03), but the association disappeared after Bonferroni correction. Moreover, a haplotype (T-G) in the DKK1 gene showed a protective role in AS susceptibility in females (p = .04). Significant additive interactions were observed between DKK1: rs1896368 and LRP5: rs3736228, relative excess risk due to interaction (RERI) = 0.40, 95% CI = 0.08 – 0.71; attributable proportion due to interaction (AP) = 51%, 95% CI = 0.07 – 0.94, DKK1: rs1569198 and LRP5: rs3736228 (RERI = 0.49, 95% CI = 0.12 – 0.86; AP = 49%, 95% CI = 0.17 – 0.82), LRP5: rs3736228 and SOST: rs4792909 (RERI = 0.33, 95% CI = 0.002 – 0.65; AP = 41%, 95% CI = 0.01 – 0.81) in the dominant model.Conclusions: Our research implies a potential gene–gene interaction, thus revealing the importance of the Wnt/β-catenin signalling pathway for understanding the genetic architecture of AS.

Bone metabolism genes variation and response to bisphosphonate treatment in women with postmenopausal osteoporosis.

IntroductionLong-term treatment is used in patients with osteoporosis, and bisphosphonates (BPs) are the most commonly prescribed medications. However, in some patients this therapy is not effective, cause different side effects and complications. Unfortunately, at least one year is needed to identify and confirm an ineffectiveness of BPs therapy on bone mineral density (BMD). Among other factors, a response to BPs therapy may also be explained by genetic factors. The aim of this study was to analyze the influence of SOST, PTH, FGF2, FDPS, GGPS1, and LRP5 gene variants on the response to treatment with aminobisphosphonates.Materials and methodsWomen with postmenopausal osteoporosis were included to this study if they used aminobisphosphonates for at least 12 months. Exclusion criteria were: persistence on BPs therapy less than 80%, bone metabolic diseases, diseases deemed to affect bone metabolism, malignant tumours, using of any medications influencing BMD. The study protocol was approved by the local ethics committee. The BMD at the lumbar spine and femoral neck were measured using dual x-ray absorptiometry (GE Lunar) before and at least 12 months after treatment with BPs. According to BMD change, patients were divided in two groups–responders and non-responders to BPs terapy. Polymorphic variants in SOST, PTH, FGF2, FDPS, GGPS1, and LRP5 genes were determined using PCR analysis with TaqMan probes (Thermo Scientific).ResultsIn total, 201 women with BPs therapy were included in the study. No statistically significant differences were observed in age, age at menopause, weight, height, BMI and baseline BMD levels between responders (122 subjects) and non-responders (79 subjects).As single markers, the SOST rs1234612 T/T (OR = 2.3; P = 0.02), PTH rs7125774 T/T (OR = 2.8, P = 0.0009), FDPS rs2297480 G/G (OR = 29.3, P = 2.2×10−7), and GGPS1 rs10925503 C/C+C/T (OR = 2.9; P = 0.003) gene variants were over-represented in non-responders group. No significant association between FGF2 rs6854081 and LRP5 rs3736228 gene variants and response to BPs treatment was observed. The carriers of T-T-G-C allelic combination (constructed from rs1234612, rs7125774, rs2297480, and rs10925503) were predisposed to negative response to BPs treatment (OR = 4.9, 95% CI 1.7–14.6, P = 0.005). The C-C-T-C combination was significantly over-represented in responders (OR = 0.1, 95% CI 0.1–0.5, P = 0.006).ConclusionsOur findings highlight the importance of identified single gene variants and their allelic combinations for pharmacogenetics of BPs therapy of osteoporosis. Complex screening of these genetic markers could be used as a new strategy for personalized antiresorptive therapy.

Nanoparticles to Knockdown Osteoporosis-Related Gene and Promote Osteogenic Marker Expression for Osteoporosis Treatment.

Osteoporosis is the most common disease involving bone degeneration. Current clinical treatments are not able to offer a satisfying curative effect, so the development of effective treatments is desired. Gene silencing through siRNA delivery has gained great attention as a potential treatment in bone diseases. SOST gene inhibits the Wnt signaling pathway reducing osteoblast differentiation. Consequently, silencing SOST genes with a specific siRNA could be a potential option to treat osteoporosis. Generally, siRNAs have a very short half-life and poor transfection capacity, so an effective carrier is needed. In particular, mesoporous silica nanoparticles (MSNs) have attracted great attention for intracellular delivery of nucleic acids. We took advantage of their high loading capacity to further load the pores with osteostatin, an osteogenic peptide. In this study, we developed a system based on MSNs coated with poly(ethylenimine), which can effectively deliver SOST siRNA and osteostatin inside cells, with the consequent augmentation of osteogenic markers with a synergistic effect. This established the potential utility of MSNs to co-deliver both biomolecules to promote bone formation, this being a potential alternative to treat osteoporosis.

Differential expression of periostin, sclerostin, receptor activator of nuclear factor-κB, and receptor activator of nuclear factor-κB ligand genes in severe chronic periodontitis.

The aim of the present study was to determine and compare the expression profile of periostin (POSTN), sclerostin (SOST), receptor activator nuclear factor-κB (RANK), and RANK ligand (RANKL) genes in gingival tissue samples collected from healthy gingiva (control) and severe chronic periodontitis sites. Fifty systemically-healthy individuals was enrolled in the present case-control study. Gingival tissue samples were obtained from healthy gingiva (N=25) and sites with severe chronic periodontitis (N=25). Total RNA was isolated from all the tissues. cDNA conversion was then performed using a reverse transcription polymerase chain reaction (PCR) program. Real-time PCR and SYBR green method were used to determine the expression levels of SOST, POSTN, RANK, and RANKL genes. An elevated expression (3.5-4-fold) of SOST, RANK, and RANKL genes, with a concomitant reduced expression of the POSTN gene, was identified in severe chronic periodontitis. The intergroup difference between the mean delta cyclic threshold values showed statistical significance at P<.001. The expression profile of SOST, RANK, RANKL, and POSTN genes observed in gingival tissue samples from sites with severe chronic periodontitis and healthy gingiva suggests that the differential level of the gene expression could serve as an indicator of periodontitis progression/severity.

Interaction between LRP5 and periostin gene polymorphisms on serum periostin levels and cortical bone microstructure

The purpose of this study is to investigate the interaction between periostin gene polymorphisms (SNPs) and other genes potentially responsible for modulating serum periostin levels and bone microstructure in a cohort of postmenopausal women. In 648 postmenopausal women from the Geneva Retirees Cohort, we analyzed 6 periostin SNPs and another 149 SNPs in 14 genes, namely BMP2, CTNNB1, ESR1, ESR2, LRP5, LRP6, PTH, SPTBN1, SOST, TGFb1, TNFRSF11A, TNFSF11, TNFRSF11B and WNT16. Volumetric BMD and bone microstructure were measured by high-resolution peripheral quantitative computed tomography at the distal radius and tibia. Serum periostin levels were associated with radial cortical porosity, including after adjustment for age, BMI, and years since menopause (p=0.036). Sixteen SNPs in the ESR1, LRP5, TNFRSF11A, SOST, SPTBN1, TNFRSF11B and TNFSF11 genes were associated with serum periostin levels (p range 0.03-0.001) whereas 26 SNPs in 9 genes were associated with cortical porosity at the radius and/or at the tibia. WNT 16 was the gene with the highest number of SNPs associated with both trabecular and cortical microstructure. The periostin SNP rs9547970 was also associated with cortical porosity (p=0.04). In particular, SNPs in LRP5, ESR1 and near the TNFRSF11A gene were associated with both cortical porosity and serum periostin levels. Eventually, we identified an interaction between LRP5 SNP rs648438 and periostin SNP rs9547970 on serum periostin levels (interaction p=0.01) and on radial cortical porosity (interaction p=0.005). These results suggest that periostin expression is genetically modulated, particularly by polymorphisms in the Wnt pathway, and is thereby implicated in the genetic variation of bone microstructure.

Ex vivo construction of human primary 3D–networked osteocytes

A human bone tissue model was developed by constructing ex vivo the 3D network of osteocytes via the biomimetic assembly of primary human osteoblastic cells with 20–25μm microbeads and subsequent microfluidic perfusion culture. The biomimetic assembly: (1) enabled 3D–constructed cells to form cellular network via processes with an average cell-to-cell distance of 20–25μm, and (2) inhibited cell proliferation within the interstitial confine between the microbeads while the confined cells produced extracellular matrix (ECM) to form a mechanically integrated structure. The mature osteocytic expressions of SOST and FGF23 genes became significantly higher, especially for SOST by 250 folds during 3D culture. The results validate that the bone tissue model: (1) consists of 3D cellular network of primary human osteocytes, (2) mitigates the osteoblastic differentiation and proliferation of primary osteoblast-like cells encountered in 2D culture, and (3) therefore reproduces ex vivo the phenotype of human 3D–networked osteocytes. The 3D tissue construction approach is expected to provide a clinically relevant and high-throughput means for evaluating drugs and treatments that target bone diseases with in vitro convenience.

Estrogen Modulates Bone Morphogenetic Protein-Induced Sclerostin Expression Through the Wnt Signaling Pathway.

Clinical data show that estrogen levels are inversely associated with the production of sclerostin, a Wnt antagonist that recently attracted great attention over the use of its antibody in the anabolic treatment of osteoporotic conditions. However, the molecular link between sclerostin expression and estrogen signaling is not yet known. We investigated the mechanisms by which estrogen modulates sclerostin (SOST) gene expression at the cellular level in human osteoblast cells in association with bone morphogenetic protein (BMP)2 signaling given that BMP2 is a potential inducer of SOST in human mesenchymal stromal cells (hMSCs). 17β-Estradiol (E2) alone had no effect on SOST expression, which was significantly induced by treatment with BMP2 in hMSCs and osteoblasts derived from the mandibles of female donors. However, E2 suppressed the induction of SOST and other BMP2 target genes by BMP2 in hMSCs. E2 signaling was independent of the Smad pathway, which plays a critical role in SOST induction mediated by BMP2. Instead, E2 increased the transcriptional expression of β-catenin and levels of its activated form. Silencing of the gene encoding estrogen receptor (ER)α decreased E2 activity in β-catenin activation and the suppression of SOST induction by BMP2, but had no influence on BMP2-mediated SOST induction in the same conditions. Similar results were obtained after treatment with ERα antagonist as a Wnt inhibitor. In human osteoblasts, the effect of E2 on SOST expression was either suppressive or absent, depending on the cell donor. Interestingly, the SOST expression pattern after treatment with BMP2 or BMP2/E2 in human osteoblasts showing a pattern of E2 suppression on SOST induction by BMP2 correlated with the ratio of receptor activator of nuclear factor kappa-B ligand (RANKL) to osteoprotegerin (OPG) expression. These results demonstrate that estrogen signaling in osteoblasts negatively regulates SOST expression in an indirect manner through interaction with BMP2 signaling and that this regulation involves the Wnt/ERα and β-catenin pathways. This study highlights several interactions between estrogen and BMP cascades in osteoblasts that may provide a basis for therapeutic intervention for the modification of bone mass density.

Analysis of association of MEF2C, SOST and JAG1 genes with bone mineral density in Mexican-Mestizo postmenopausal women.

BackgroundOsteoporosis, a disease characterized by low bone mineral density (BMD), is an important health problem in Mexico. BMD is a highly heritable trait, with heritability estimates of 50-85%. Several candidate genes have been evaluated to identify those involved in BMD variation and the etiology of osteoporosis. This study investigated the possible association of single-nucleotide polymorphisms (SNPs) in the MEF2C, SOST and JAG1 genes with bone mineral density (BMD) variation in postmenopausal Mexican-Mestizo women.MethodsFour hundred unrelated postmenopausal women were included in the study. Risk factors were recorded and BMD was measured in total hip, femoral neck and lumbar spine using dual-energy X-ray absorptiometry. In an initial stage, a total of twenty-five SNPs within or near SOST gene and seven SNPs in the JAG1 gene were genotyped using a GoldenGate assay. In a second stage, three MEF2C gene SNPs were also genotyped and SOST and JAG1 gene variants were validated. Real time PCR and TaqMan probes were used for genotyping.ResultsLinear regression analyses adjusted by age, body mass index and ancestry estimates, showed that five SNPs in the SOST gene were significantly associated with BMD in total hip and femoral neck but not lumbar spine. The lowest p value was 0.0012, well below the multiple–test significance threshold (p = 0.009), with mean effect size of -0.027 SD per risk allele. We did not find significant associations between BMD and MEF2C/JAG1 gene variants [rs1366594 “A” allele: β = 0.001 (95% CI -0.016; 0.017), P = 0.938; rs2273061 “G” allele: β = 0.007 (95% CI -0.007; 0.023), p = 0.409].ConclusionsSOST polymorphisms may contribute to total hip and femoral neck BMD variation in Mexican postmenopausal women. Together, these and prior findings suggest that this gene may contribute to BMD variation across populations of diverse ancestry.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2474-15-400) contains supplementary material, which is available to authorized users.

Expression of genes related to energy metabolism (osteocalcin, FOXO1, insulin receptor, and SOST) in bone cells of Goto-Kakizaki rats and response to bariatric surgery

ObjectivesThe aim of this experimental study was to evaluate the expression of 4 genes (osteocalcin, sclerostin (SOST), insulin receptor, and transcription factor forkhead box protein (FOXO1) in the bone cells of Goto-Kakizaki (GK) rats and the expression of these genes in response to bariatric surgery. MethodsWe designed an experimental study with 3 arms (Wistar rats, nonoperated GK rats, and GK rats with gastrojejunal bypass). Gene expression (osteocalcin, insulin receptor, FOXO1, SOST) was measured at baseline and after surgery. Plasma levels of glucose, insulin, homeostasis model assessment (HOMA), glucagon-like peptide 1 (GLP-1), and the production of insulin were measured at baseline and at 60 minutes by pancreatic islets. ResultsGK rats had decreased levels of expression of osteocalcin (50.86±19.21 versus 16.78±22.11, P = .031), insulin receptor (1.45±.44 versus .54±.35, P = .020), and SOST (.92±.05 versus .43±.47, P = .048) compared with Wistar rats. Gene expression in GK rats, operated and nonoperated, was similar. In nonoperated GK rats, there was a negative correlation between the SOST gene and plasma insulin (r:−.786, P = .021) and the production of insulin by pancreatic islets at 60 minutes (r:−0.857, P = .014). GLP-1 increased after surgery. ConclusionDiabetic GK rats presented a reduced expression of the osteocalcin, insulin receptor, and SOST genes. There was an inverse relationship between SOST and plasma and local insulin. We found no changes in the expression of bone genes that regulate energy metabolism after gastrojejunal bypass.

Genetic Association Study of Dickkopf-1 and Sclerostin Genes with Paget Disease of Bone

Increased expression of DKK1 gene was reported in pagetic osteoblasts and stromal cells, and increased serum levels of DKK1 and SOST proteins were reported in patients with Paget disease of bone (PDB). This study aimed at identifying rare genetic variants of the DKK1 and SOST genes and at testing for genetic association with PDB in the French-Canadian population. Exons, promoters, and exon-intron junctions of these genes were sequenced in patients with PDB and healthy controls. An association study of Tag SNPs of both genes was also performed in 239 pagetic patients and 297 healthy individuals. Three rare variants were identified in this study, all located in the DKK1 gene: one variant in the second exon leading to alteration in a highly conserved amino acid (p.R120L), one in the 5'-untranslated region (-50 C/A), and one in a splice site of intron 1 (IVS1 184 T/C), although none of these rare variants were associated with PDB. A genetic association of a Tag SNP of the DKK1 gene was found: the G allele of rs1569198 was significantly decreased in patients in comparison to controls (42 vs. 49 %, uncorrected P = 0.03, OR = 0.77, 95 % CI 0.61-0.98). In conclusion, this study identified three rare genetic variants in DKK1 in the French-Canadian population. In addition, a weak genetic association of a common variant of DKK1, rs1569198, which is located on a predicted new acceptor site for splicing of this gene, was observed in PDB, whereas no rare variant or genetic association was found in the SOST gene.

Novel SOST gene mutation in a sclerosteosis patient and her parents

IntroductionSclerosteosis (OMIM 269500) is a rare autosomal recessive condition characterized by increased bone density associated with syndactyly. It is linked to a genetic defect in the SOST gene coding for sclerostin. So far, six different loss-of-function mutations in SOST have been reported in patients with sclerosteosis. Our objective was to sequence and identify mutation in the SOST and LRP5 genes which are known to be causal for craniotubular hyperostosis in a patient from India. Patient and methodsA 22year old woman presented with typical features of sclerosteosis in form of progressive visual and hearing loss, syndactyly and radiographs revealing increased density of bone. Genomic sequencing of the SOST gene as well as exons 2, 3 and 4 of the LRP5 gene was performed. ResultsWe identified a novel homozygous mutation in the SOST gene, characterized as one nucleotide insertion resulting in a frame shift mutation and loss of functional sclerostin. Her parents were also found to have a similar but heterozygous mutation in the SOST gene. ConclusionA novel frame shift mutation in the SOST gene causing loss of functional sclerostin was identified in a patient with sclerosteosis and her parents.

The virulence gene and clinical phenotypes of osteopetrosis in the Chinese population: six novel mutations of the CLCN7 gene in twelve osteopetrosis families

Osteopetrosis is a heritable bone disorder resulting from a deficiency of or a functional defect in osteoclasts. We aimed to characterize the molecular defects and clinical manifestations in Chinese patients with osteopetrosis by studying 12 unrelated osteopetrosis families. The entire coding region and adjacent splice sites of the CLCN7, TCIRG1, LRP5 and SOST genes were amplified and directly sequenced. X-rays of hip and lumbar spine, bone mineral density and bone turnover markers were examined simultaneously. Family history and fracture history were collected using a questionnaire. Among 12 unrelated families, 10 families were diagnosed with autosomal dominant osteopetrosis type II (ADOII) with 10 probands and 3 affected subjects. Two individuals in the other two families were diagnosed with uncategorized osteopetrosis because no mutations were detected in any of the four studied genes. Eight mutations, including two reported mutations (R767W and E798FS) and six novel mutations (E313K, A316G, R743W, G741R, W127G and S290F), were detected in the CLCN7 gene from 12 living ADOII patients. Among them, R767W and R743W mutations were two common mutations that were each found in 20% of 10 ADOII probands. In CLCN7-related ADOII patients, long bone fractures and elevated serum CK level were two major clinical phenotypes, especially in patients younger than 18 years. Further functional studies of the above eight mutations in the CLCN7 gene are needed in the future.

Relationship of sclerostin and secreted frizzled protein polymorphisms with bone mineral density

Secreted frizzled-related protein and sclerostin, encoded by FRZB and SOST genes, respectively, are extracellular Wnt inhibitors that tend to decrease bone formation. The purpose of this study was to explore the association of the sets of polymorphisms capturing common variations of these genes with bone mineral density (BMD). Twelve polymorphic loci of the FRZB gene and 7 of the SOST gene were genotyped in postmenopausal women from two Spanish regions (Cantabria, n = 1043, and Valencia, n = 342). The polymorphisms included tagging single nucleotide polymorphisms and single nucleotide polymorphisms with possible functional consequences assessed in silico. The rs4666865 polymorphism of the FRZB gene was associated with spine BMD in the Cantabria cohort in the single-locus (P = 0.008) and the haplotypic analysis. However, the results were not replicated in the Valencia cohort. Several polymorphisms at the 5' region of the SOST gene and, particularly, rs851056 were associated with BMD in women from both cohorts (P = 0.002 in Cantabria and P = 0.005 in Valencia). When the results of both cohorts were combined, the mean (SD) BMD difference across rs851056 genotypes was 47 (0.31) mg/cm(2) (P < 0.001). No differences in FRZB and SOST expression in femoral trabecular bone tissue were detected across genotypes. Polymorphisms in the 5' region of SOST gene are associated with BMD in postmenopausal women. They may contribute to explain, in part, the hereditary influence on bone mass.

Identification of the first deletion in the LRP5 gene in a patient with Autosomal Dominant Osteopetrosis type I

In the last decade, the low-density lipoprotein receptor-related protein 5 (LRP5) gene, coding for a coreceptor in the canonical Wnt signalling pathway, has been shown to play an important role in regulating bone mass and to be involved in the pathogenesis of several bone disorders. Here we describe a patient who presented with a clinical picture of Autosomal Dominant Osteopetrosis type I (ADO I), in whom we could identify the first deletion in the LRP5 gene causing increased bone mass. This mutation caused the in-frame deletion of two amino acids in the fourth blade of the first propeller of the protein, namely the highly conserved glycine at position 171 and the following glutamate residue. In vitro studies suggested that the pathogenic effect of this novel mutation could be due to a decreased inhibition of Wnt signalling by the antagonistic proteins sclerostin and Dickkopf-1, encoded respectively by the SOST and DKK1 genes, in the presence of mutated LRP5. Our results highlight an increasing molecular heterogeneity in LRP5-related bone diseases.

Analysis of Association of LRP5, LRP6, SOST, DKK1, and CTNNB1 Genes with Bone Mineral Density in a Slovenian Population

The Wnt pathway has a bifunctional role in bone mass regulation, influencing osteoblasts and osteoclasts. The Wnt pathway genes are therefore candidate genes for susceptibility to osteoporosis. In our study, we focused on the effects of polymorphisms in selected Wnt pathway genes: low-density lipoprotein receptor-related proteins 5 and 6 (LRP5 and LRP6), Dickkopf1 (DKK1), sclerostin (SOST), and beta-catenin (CTNNB1). We genotyped 652 subjects for the following polymorphisms: A1330V in LRP5; I1062V in LRP6; E232K in DKK1; D32Y, G34V, and N287S in CTNNB1; and -1397_-1396insGGA in SOST. Bone mineral density (BMD) was also measured. The allele frequencies were as follows: for A1330V C:T = 87%:13%, for I1062V C:T = 20%:80%, and for -1397_-1396insGGA-:GGA = 64%:36%. The studied nucleotide changes in the DKK1 and CTNNB1 genes were shown not to be polymorphic. In a Slovenian population, no association was shown between lumbar spine and femoral neck BMD in A1330V (P = 0.151 and 0.243) and in I1062V (P = 0.209 and 0.405). We observed a difference between SOST genotypes, corresponding to an allele dose effect, which was borderline significant for lumbar spine and femoral neck BMD (P = 0.047 and 0.085); but this did not survive the adjustment for multiple testing. These results indicate that a larger sample size would be necessary to detect these subtle effects. Our results suggest that A1330V in LRP5, I1062V in LRP6, and -1397_-1396insGGA in SOST are not associated with BMD in the Slovenian population.

Wnt pathway genes in osteoporosis and osteoarthritis: differential expression and genetic association study

In comparison with hip fractures, increased expression of genes in the Wnt pathway and increased Wnt activity were found in bone samples and osteoblast cultures from patients with osteoarthritis, suggesting the involvement of this pathway in subchondral bone changes. No consistent differences were found in the genetic association study. This study aims to explore the allelic variations and expression of Wnt pathway genes in patients with osteoporosis and osteoarthritis. The expression of 86 genes was studied in bone samples and osteoblast primary cultures from patients with hip fractures and hip or knee osteoarthritis. The Wnt-related activity was assessed by measuring AXIN2 and in transfection experiments. Fifty-five SNPs of the LRP5, LRP6, FRZB, and SOST genes were analyzed in 1,128 patients. Several genes were differentially expressed in bone tissue, with the lowest values usually found in hip fracture and the highest in knee osteoarthritis. Overall, seven genes were consistently upregulated both in tissue samples and in cell cultures from patients with knee osteoarthritis (BCL9, FZD5, DVL2, EP300, FRZB, LRP5, and TCF7L1). The increased expression of AXIN2 and experiments of transient transfection of osteoblasts with the TOP-Flash construct confirmed the activation of Wnt signaling. Three SNPs of the LRP5 gene and one in the LRP6 gene showed marginally significant differences in allelic frequencies across the patient groups, but they did not resist multiple-test adjustment. Genes in the Wnt pathway are upregulated in the osteoarthritic bone, suggesting their involvement not only in cartilage distortion but also in subchondral bone changes.

Genetic analysis and effect of triiodothyronine and prednisone trial on bone turnover in a patient with craniotubular hyperostosis

Craniotubular hyperostosis are a group of high bone mass disorders related to mutations in the LRP5 and SOST genes, although other causative genes remain to be identified. Little is known about the bone turnover and the response to T3 or glucocorticoids in these patients. We describe a patient with craniotubular hyperostosis, including mutation analyses of the LRP5, SOST, DKK1 and KRM1 genes. We also studied bone turnover and bone mineral density (BMD), before and after a trial with T3 (75 µg/d for 28 weeks) and T3 and prednisone (T3 100 µg/d for 2 weeks, followed by 10 weeks on prednisone 10 mg/d, and a final 2 weeks period off of medicactions, completing 3 cycles in 42 weeks. Mutation analysis of the complete coding region and flanking highly conserved sequences of SOST, evaluation of the presence of the 52-kb deletion associated with Van Buchem disease in Dutch patients and mutation analysis of exons 2–4 of LRP5, and the coding regions of DKK1 and KRM1 did not reveal any disease-causing mutations. A baseline 5 to 7 fold increase in osteocalcin and in deoxypiridinoline was detected. After 4 weeks on 75 µg/d of T3, osteocalcin decreased 36%, but at week 28, it returned to basal. Deoxypiridinoline did not change. After the first cycle on T3 and prednisone, osteocalcin decreased 72%, and at the end of the third cycle it remained 44% below basal value. Deoxypiridinoline was stable and high during the three cycles; no changes in BMD were observed. As we failed to identify any disease-causing mutations in our patient with craniotubular hyperostosis, we suggest that another gene must be involved in the pathogenesis of his condition. This study provides additional data about the high bone turnover described in craniotubular hyperostosis, and also suggests an abnormal response to T3 excess in this condition.