Causal impact of DNA methylation on refracture in elderly individuals with osteoporosis - a prospective cohort study.

BACKGROUND: Osteoporotic vertebral compression fractures (OVCF) in the elderly increase refracture risk post-surgery, leading to higher mortality rates. Genome-wide association studies (GWAS) have identified susceptibility genes for osteoporosis, but the phenotypic variance explained by these genes has been limited, indicating the need to explore additional causal factors. Epigenetic modifications, such as DNA methylation, may influence osteoporosis and refracture risk. However, prospective cohorts for assessing epigenetic alterations in Chinese elderly patients are lacking. Here, we propose to conduct a prospective cohort study to investigate the causal network of DNA polymorphisms, DNA methylation, and environmental factors on the development of osteoporosis and the risk of refracture. METHODS: We will collect vertebral and peripheral blood from 500 elderly OVCF patients undergoing surgery, extract DNA, and generate whole genome genotype data and DNA methylation data. Observation indicators will be collected and combined with one-year follow-up data. A healthy control group will be selected from a natural population cohort. Epigenome-wide association studies (EWAS) of osteoporosis and bone mineral density will be conducted. Differential methylation analysis will compare candidate gene methylation patterns in patients with and without refracture. Multi-omics prediction models using genetic variants and DNA methylation sites will be built to predict OVCF risk. DISCUSSION: This study will be the first large-scale population-based study of osteoporosis and bone mineral density phenotypes based on genome-wide data, multi-time point methylation data, and phenotype data. By analyzing methylation changes related to osteoporosis and bone mineral density in OVCF patients, the study will explore the feasibility of DNA methylation in evaluating postoperative osteoporosis intervention effects. The findings may identify new molecular markers for effective anti-osteoporosis treatment and inform individualized prevention and treatment strategies. TRIAL REGISTRATION: chictr.org.cn ChiCTR2200065316, 02/11/2022.

LncRNA LINC00205 stimulates osteoporosis and contributes to spinal fracture through the regulation of the miR-26b-5p/KMT2C axis.

BACKGROUND: Osteoporosis (OP) is a common bone disease marked by decreased bone strength. Increasing evidence suggests that long non-coding RNA (lncRNAs) play important roles in the occurrence and progression of OP. This study aimed to investigate the role and mechanism of LINC00205 in the osteogenic differentiation of human mesenchymal stem cells (hMSCs) and OP. METHODS: Bone tissue samples were obtained from healthy controls and patients with osteoporosis with a spinal fracture (OP-Frx) or without a spinal fracture (OP-no-Frx). HMSCs were cultured and induced to undergo osteogenic differentiation. The expression of LINC00205, lysine (K)-specific methyltransferase 2C (KMT2C), and miR-26b-5p in bone tissues and cells was evaluated using western blotting and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). The effects of LINC00205, miR-26b-5p, and KMT2C on calcium deposition, alkaline phosphatase (ALP) activity, and mRNA levels of the osteogenic differentiation marker genes [ALP, osteocalcin (OCN), and runt-related transcription factor 2 (RUNX2)] were investigated using alizarin red S staining, an ALP activity assay, and qRT-PCR, respectively. Dual-luciferase reporter assay was performed to ascertain the binding relationship between miR-26b-5p and LINC00205/KMT2C. RESULTS: LINC00205 and KMT2C were upregulated in patients with OP-Frx and OP-no-Frx, whereas miR-26b-5p was downregulated. Furthermore, LINC00205 and KMT2C expression decreased, whereas that of miR-26b-5p increased over time from day 7 to 21 of the osteogenic differentiation of hMSCs. The knockdown of LINC00205 and KMT2C significantly increased ALP activity, calcium deposition, and the expression of RUNX2, ALP, and OCN. In contrast, the inhibition of miR-26b-5p yielded the opposite result. These data suggest that LINC00205 inhibits the osteogenic differentiation of hMSCs by modulating the miR-26b-5p/KMT2C signaling axis. CONCLUSION: LINC00205 promotes OP and is involved in spinal fractures. LINC00205 is also a potential negative regulator of the osteogenic differentiation of hMSCs.

Correlations of IL-6 and TGF-ß Gene Polymorphisms and Expressions With Osteoporotic, Thoracolumbar, Vertebral Compression Fracture.

Context: Associations between genes and diseases manifest as the influence of gene expression on disease development as well as the impact of variations in the disease-related genes themselves. It's important to determine the genetic variations that can lead to compressed fractures of osteoporotic, thoracic lumbar vertebrae to develop personalized clinical methods to prevent or delay the disease's development. Objective: The study intended to explore the correlations between the gene polymorphisms and gene expressions of the interleukin-6 (IL-6) gene and the transforming growth factor-beta (TGF-ß) gene and osteoporotic, thoracolumbar, vertebral compression fracture. Design: The research team performed an observational study using data from medical records. Setting: The study took place at Xuzhou Medical University in Xuzhou, China. Participants: Participants were 200 patients with an osteoporotic, thoracolumbar, vertebral compression fracture who had been admitted to the hospital at the university between 2019 and 2021 prior to the study and 200 healthy people The research team divided the participants into two groups. The patients became participants in the disease group, and the healthy individuals became participants in the control group. Outcome Measures: The research team: (1) collected peripheral blood from the two groups, (2) extracted genomic deoxyribonucleic acids (DNAs) from karyocytes, (3) examined the IL-6 and TGF-ß gene polymorphisms, and (4) analyzed and correlated participants' clinical data with the gene polymorphisms and expressions. The team used a quantitative polymerase chain reaction (qPCR) to examine the expression levels of IL-6 and TGF-ß. Results: Compared to the control group, the disease group: (1) had allele distributions that were significantly different at the rs2069829 locus of the IL-6 gene (P < .001) and at the rs3087453 of the TGF-ß gene (P = .004); (2) had significantly higher frequencies of allele T at the rs2069829 locus of the IL-6 gene and of allele G at the rs3087453 locus of the TGF-ß gene; (3) had genotype distributions that were significantly different at the rs2069829 locus (P < .001) and the rs2069857 locus (P = .048) of the IL-6 gene and at the rs3087453 locus (P < .001) of the TGF-ß gene; (4) had frequencies that were significantly higher of the TT genotype at the rs2069829 locus, the CC genotype at the rs2069857 locus, and the GC genotype at the rs3087453 locus of the IL-6 gene and the TGF-ß gene; (5) had dominant models that were significantly different at the rs2069829 locus of the IL-6 gene (P = .009) and at rs3087453 locus of the TGF-ß gene (P = .026) and had a recessive model that was significantly different at the rs2069857 locus of the IL-6 gene (P = .040); (6) had significantly different haplotypes CC (P < .001) and TC (P < .001) at the rs2069829 locus and the rs2069857 locus of the IL-6 gene and a significantly different haplotype AC (P = .011) at the rs1800469 locus and the rs3087453 locus of the TGF-ß gene; (7) had an IL-6 gene polymorphism at the rs2069857 locus that was related to the expression of the IL-6 gene (P < .05) and an expression of the IL-6 gene for participants with the AA genotype that was significantly lower than for other genotypes; (8) had a TGF-ß gene polymorphism at the rs1800469 locus that was associated with the expression of the TGF-ß gene (P < .05), and an expression for participants with the GG genotype that was significantly higher than for other genotypes; (9) had an IL-6 gene polymorphism at the rs2069857 locus with an overt correlation with the genotype of osteoporotic, thoracolumbar, vertebral compression fracture (P < .001). Also, participants in the disease group with the genotype CC mainly had type 2 and 3 fractures, while those with genotype AA primarily had type 0 and 1 fractures. Conclusions: IL-6 and TGF-ß gene polymorphisms and expressions are significantly related to osteoporotic, thoracolumbar, vertebral compression fracture.

MicroRNA miR-18a-3p promotes osteoporosis and possibly contributes to spinal fracture by inhibiting the glutamate AMPA receptor subunit 1 gene (GRIA1).

The promoting role that miR-18a-3p plays in osteoporosis (OP) has been previously described. However, the detailed mechanisms remain unclear. Bone tissues were collected from healthy patients, OP patients, and patients with osteoporotic spinal fractures. An osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) was constructed to detect the expression of miR-18a-3p and glutamate AMPA receptor subunit 1 (GRIA1). Alkaline phosphatase (ALP) activity and a qRT-PCR analysis were used to detect ALP content, alizarin red S staining was used to detect calcium deposition, and qRT-PCR was used to evaluate runt-related transcription factor 2 (RUNX2), osteocalcin (OCN), and osteopontin (OPN) expression levels. A dual-luciferase reporter and RNA pull-down assay was used to verify the targeted correlation between miR-18a-3p and GRIA1. We observed an increase in miR-18a-3p expression and a decrease in GRIA1 expression in OP and osteoporotic vertebral fracture patients. Upregulation of miR-18a-3p restrained the activity and expression of ALP in hBMSCs, inhibited the expression of RUNX2, OCN, and OPN, and inhibited calcium deposition. Knockdown of miR-18a-3p or upregulation of GRIA1 promoted osteogenic differentiation. Our findings indicate that miR-18a-3p promotes OP progression by regulating GRIA1 expression, suggesting that targeting miR-18a-3p/GRIA1 may be a therapeutic strategy for OP.

By inhibiting ADCY5, miR-18a-3p promotes osteoporosis and possibly contributes to spinal fracture.

To investigate the influence of miR-18a-3p and ADCY5 on OP and osteogenic differentiation of human Mesenchymal stem cell (hBMSCs) and its possible mechanism. Samples were collected from osteoporotic patients with or without vertebral compression fracture, and without OP volunteers. MiR-18a-3p and ADCY5 mRNA expression levels in the tissue samples and hBMSCs during osteogenic differentiation were detected。MiR-18a-3p mimic and OE-ADCY5 were introduced into hBMSCs to research the effects of miR-18a-3p and ADCY5 on osteogenesis differentiation of hBMSCs. Dual luciferase reporter system and RNA pull-down were applied to determine whether ADCY5 was a target gene of miR-18a-3p. Compared with the control group, ADCY5 expression level was down-regulated in patients with OP-no-Frx and OP-Frx, but that of miR-18a-3p was up-regulated. In addition, ADCY5 increased during osteogenesis differentiation of hBMSCs, whereas miR-18a-3p did not. OE-ADCY5 significantly facilitated calcium deposition, ALP activity, osteoblast protein expression (OSX, ALP and EUNX2), miR-18a-3p mimic inhibited osteogenic differentiation, and partially reversed the effect of OE-ADCY5 on osteogenic differentiation. In general, miR-18a-3p targets ADCY5 to promote OP and may be involved in spinal fracturs.

Is there any association between osteoporotic vertebral fracture and vitamin K epoxide reductase complex subunit-1 polymorphism in Turkish society? A pilot study

OBJECTIVE: In this study, the relationship between osteoporotic vertebral fractures and 9041 Guanine/Adenine and 3673 Guanine/Adenine polymorphisms related to the vitamin K epoxide reductase complex subunit-1 (VKORC1) gene in postmenopausal women with osteoporosis was investigated. METHOD: DNA was isolated from blood samples collected from 150 women with postmenopausal osteoporosis. Genotyping of the two polymorphic regions (9041 Guanine/Adenine and 3673 Guanine/Adenine) in VKORC1 was performed using polymerase chain reaction-restriction fragment length polymorphism analysis. The presence of radiographic fractures among the 150 patients was ascertained by using the Genant method. RESULT: At least one fracture was detected in 98 patients, and no fracture was observed in 52 patients on radiological images. We found no association between the 9041 Guanine/Adenine (p=0.283) and 3673 Guanine/Adenine (p=0.232) polymorphisms of the VKORC1 gene and the development of secondary postosteoporotic fractures in our study. CONCLUSION: There was no relationship between osteoporotic vertebral fracture and VKORC1 gene polymorphism in a postmenopausal Turkish population.

Impaired WNT signaling and the spine-Heterozygous WNT1 mutation causes severe age-related spinal pathology.

BACKGROUND: WNT signaling plays a major role in bone and cartilage metabolism. Impaired WNT/ß-catenin signaling leads to early-onset osteoporosis, but specific features in bone and other tissues remain inadequately characterized. We have identified two large Finnish families with early-onset osteoporosis due to a heterozygous WNT1 mutation c.652T>G, p.C218G. This study evaluated the impact of impaired WNT/ß-catenin signaling on spinal structures. METHODS: Altogether 18 WNT1 mutation-positive (age range 11-76years, median 49years) and 14 mutation-negative subjects (10-77years, median 43years) underwent magnetic resonance imaging (MRI) of the spine. The images were reviewed for spinal alignment, vertebral compression fractures, intervertebral disc changes and possible endplate deterioration. The findings were correlated with clinical data. RESULTS: Vertebral compression fractures were present in 78% (7/9) of those aged over 50years but were not seen in younger mutation-positive subjects. All those with fractures had several severely compressed vertebrae. Altogether spinal compression fractures were present in 39% of those with a WNT1 mutation. Only 14% (2/14) mutation-negative subjects had one mild compressed vertebra each. The mutation-positive subjects had a higher mean spinal deformity index (4.0±7.3 vs 0.0±0.4) and more often increased thoracic kyphosis (Z-score>+2.0 in 33% vs 0%). Further, they had more often Schmorl nodes (61% vs 36%), already in adolescence, and their intervertebral discs were enlarged. CONCLUSION: Compromised WNT signaling introduces severe and progressive changes to the spinal structures. Schmorl nodes are prevalent even at an early age and increased thoracic kyphosis and compression fractures become evident after the age of 50years. Therapies targeting the WNT pathway may be an effective way to prevent spinal pathology not only in those harboring a mutation but also in the general population with similar pathology.

Increased osteoclastogenesis in patients with vertebral fractures following discontinuation of denosumab treatment.

OBJECTIVE: To test the hypothesis that rebound of bone remodeling is responsible for clinical vertebral fractures reported in a few patients with osteoporosis after cessation of denosumab treatment. DESIGN: In this case-control study we compared clinical and biochemical characteristics of postmenopausal women with clinical vertebral fractures 8-16 months after the last injection of denosumab (Dmab/Fx+, n = 5) with those of treatment-naïve women with such fractures (Fx+, n = 5). In addition, 5 women who discontinued denosumab treatment but did not sustain vertebral fractures 18-20 months after the last injection were studied (Dmab/Fx-, n = 5). METHODS: We measured serum microRNAs, gene expression of mRNAs of factors regulating formation and activity of osteoclasts and biochemical markers of bone and mineral metabolism. In Dmab/Fx+ and Fx+ women, blood was taken 4-8 weeks after the fracture. RESULTS: Compared to Fx+ women, Dmab/Fx+ women had higher serum P1NP and CTx levels, and significantly lower serum miR-503 and miR-222-2 that downregulate osteoclastogenesis and osteoclast activity, and higher RANK (13-fold) and CTSK (2.6-fold) mRNA. The respective values of Dmab/Fx- women were in the same direction as those of Dmab/Fx+ women but of a lesser magnitude. CONCLUSIONS: Bone fragility in women with clinical vertebral fractures after stopping denosumab therapy is pathophysiologically different from that of treatment-naïve women with osteoporosis and clinical vertebral fractures and it is associated with upregulation of markers of osteoclast formation and activity. The small number of women with this rare event studied is a limitation.

Novel Genetic Variants Associated With Increased Vertebral Volumetric BMD, Reduced Vertebral Fracture Risk, and Increased Expression of SLC1A3 and EPHB2.

Genome-wide association studies (GWASs) have revealed numerous loci for areal bone mineral density (aBMD). We completed the first GWAS meta-analysis (n = 15,275) of lumbar spine volumetric BMD (vBMD) measured by quantitative computed tomography (QCT), allowing for examination of the trabecular bone compartment. SNPs that were significantly associated with vBMD were also examined in two GWAS meta-analyses to determine associations with morphometric vertebral fracture (n = 21,701) and clinical vertebral fracture (n = 5893). Expression quantitative trait locus (eQTL) analyses of iliac crest biopsies were performed in 84 postmenopausal women, and murine osteoblast expression of genes implicated by eQTL or by proximity to vBMD-associated SNPs was examined. We identified significant vBMD associations with five loci, including: 1p36.12, containing WNT4 and ZBTB40; 8q24, containing TNFRSF11B; and 13q14, containing AKAP11 and TNFSF11. Two loci (5p13 and 1p36.12) also contained associations with radiographic and clinical vertebral fracture, respectively. In 5p13, rs2468531 (minor allele frequency [MAF] = 3%) was associated with higher vBMD (ß = 0.22, p = 1.9 × 10-8 ) and decreased risk of radiographic vertebral fracture (odds ratio [OR] = 0.75; false discovery rate [FDR] p = 0.01). In 1p36.12, rs12742784 (MAF = 21%) was associated with higher vBMD (ß = 0.09, p = 1.2 × 10-10 ) and decreased risk of clinical vertebral fracture (OR = 0.82; FDR p = 7.4 × 10-4 ). Both SNPs are noncoding and were associated with increased mRNA expression levels in human bone biopsies: rs2468531 with SLC1A3 (ß = 0.28, FDR p = 0.01, involved in glutamate signaling and osteogenic response to mechanical loading) and rs12742784 with EPHB2 (ß = 0.12, FDR p = 1.7 × 10-3 , functions in bone-related ephrin signaling). Both genes are expressed in murine osteoblasts. This is the first study to link SLC1A3 and EPHB2 to clinically relevant vertebral osteoporosis phenotypes. These results may help elucidate vertebral bone biology and novel approaches to reducing vertebral fracture incidence. © 2016 American Society for Bone and Mineral Research.

Associations between OPG and RANKL polymorphisms, vertebral fractures, and abdominal aortic calcification in community-dwelling older subjects: the Sao Paulo Ageing & Health Study (SPAH).

This is the first study analyzing concomitantly osteoprotegerin (OPG)/receptor activator of nuclear factor kappa B ligand (RANKL) polymorphisms and OPG/RANKL serum levels and their association with bone mineral density (BMD), vertebral fractures, and vascular aortic calcification in a cohort of 800 subjects in community-dwelling older individuals. INTRODUCTION: Osteoprotegerin (OPG) and RANKL play an important role in osteoclast activation and differentiation as well as in vascular calcification. At present, there are no studies of OPG or RANKL gene polymorphisms in Brazilian older populations. The aim of this study was to evaluate OPG/RANKL polymorphism and their association with vertebral fractures (VFs) and aortic calcification. METHODS: Eight hundred subjects (497 women/303 men) were genotyped for the OPG 1181G>C (rs2073618), 163C>T (rs3102735), 245T>G (rs3134069), and 209G>A (rs3134070) and RANKL A>G (rs2277438) single-nucleotide polymorphisms (SNPs). VFs were evaluated by spine radiography (Genant's method). Aortic calcification was quantified using Kauppila's method. RESULTS: The isolated genotype analyses and single-allele frequency data showed association of OPG 163C, 245G, and 209A alleles with presence of VFs (P < 0.05). Multiple logistic regression of subjects with absence of VFs vs. those with VFs (grades II/III) revealed only OPG 209A homozygosity as a risk factor for higher-grade VFs (odds ratio (OR) = 4.17, 95 % CI 1.03-16.93, P = 0.046). Regarding aortic calcification, the isolated genotype analysis frequency data revealed a significant association of OPG 1181G, 163C, 245G, and 209A alleles with absent aortic calcification (P < 0.05). Multiple logistic regression data confirmed that the OPG 209A allele was protective for aortic calcification (OR = 0.63, 95 % CI 0.45-0.88, P = 0.007) and the OPG 1181C allele was a risk factor for aortic calcification (OR = 1.26, 95 % CI 1.00-1.58, P = 0.046). CONCLUSION: This study showed that the OPG 209AA genotype was a risk factor for higher-grade VFs, the OPG 209A allele was protective for aortic calcification, and the OPG 1181C was a risk factor for aortic calcification, supporting the involvement of OPG polymorphisms in the analyzed phenotypes and the concept that the related pathogenesis is multifactorial.

RANKL and OPG gene polymorphisms: associations with vertebral fractures and bone mineral density in premenopausal systemic lupus erythematosus.

UNLABELLED: Premenopausal women with systemic lupus erythematosus (SLE) have a higher prevalence of low bone mineral density and vertebral fractures. Multiple genetic loci for osteoporotic fracture were identified in recent genome-wide association studies. This study provides a novel data demonstrating that receptor activator of NF-κB ligand (RANKL)/osteoprotegerin (OPG) polymorphisms likely plays an important role in the bone remodeling process in SLE premenopausal women. INTRODUCTION: The purpose of this study was to evaluate single-nucleotide polymorphisms (SNPs) of the RANKL, RANK, and OPG genes in premenopausal SLE patients and their association with sRANKL and OPG serum levels, vertebral fractures, and bone mineral density (BMD). METHODS: A total of 211 premenopausal SLE patients (American College of Rheumatology (ACR) criteria) and 154 healthy controls were enrolled. SNPs of RANKL 290A>G (rs2277438), OPG 1181G>C (rs2073618), 245T>G (rs3134069), 163A>G (rs3102735), and RANK A>G (rs3018362) were obtained by real-time PCR. sRANKL/OPG serum levels were determined by ELISA. BMD and vertebral fractures were evaluated by dual-energy X-ray absorptiometry (DXA). RESULTS: SLE patients and controls had similar frequencies of the RANKL 290 G allele (p = 0.94), OPG 1181 C allele (p = 0.85), OPG 245 G allele (p = 0.85), OPG 163 G allele (p = 0.78), and RANK G allele (p = 0.87). Further analysis of the SLE patients revealed that the frequency of the RANKL 290 G allele was lower in patients with fractures than that in patients without fractures (28.1 vs 46.9%, p = 0.01). In addition, the frequency of the OPG 245 G allele was higher in patients with low BMD than that in patients with normal BMD (31.4 vs 18.1%, p = 0.04). No association of OPG 1181 G>C, OPG 163 A>G, and RANK A>G SNPs with BMD/fractures was found. Additionally, no association was observed between RANKL/OPG/RANK SNPs and sRANKL/OPG serum levels. CONCLUSIONS: Our study provides novel data demonstrating that RANKL/OPG genetic variations appear to play a role in bone remodeling, particularly in its major complication, fracture, in premenopausal patients with SLE.

Interleukin-21 Is Associated with the Pathogenesis of Lumbar Disc Herniation.

Inflammation is an important reaction underlying lumbar disc herniation (LDH). Th17 cells play a critical role in immune activation. Interleukin (IL)-21 controls the functional activity of effector T-helper cells and the differentiation of Th17 cells, and promotes B-cell differentiation. It plays important roles in chronic inflammation and autoimmune diseases. However, little is known about relationship between IL-21 and LDH. This study was aimed to determine the association between IL-21 levels and pain scores in LDH patients compared to healthy controls.We enrolled 34 LDH patients and 20 healthy controls in this study. The LDH patients underwent surgery. Pain intensity was recorded using visual analogue scale (VAS) scores preoperatively. Serum IL-21 and IL-17 levels in the peripheral blood were determined using enzyme-linked immunosorbent assay. Disc tissue was examined using western blot and quantitative reverse-transcription polymerase chain reaction to determine IL-21, IL-17, and cyclooxygenase (COX)-2 expression, and using immunohistochemistry to assess IL-21 expression.LDH patients exhibited significantly higher levels of serum IL-21 and IL-17 than healthy controls. Moreover, higher expression of IL-21, IL-17, and COX-2 was found in the protein and mRNA levels in disc tissues from LDH patients than in normal disc tissues. Different parameters like VAS pain scores, IL-17, and COX-2 were positively correlated with the IL-21 levels. Enhanced production of IL-21 in disc tissues of LDH patients was also confirmed using immunohistochemical analyses.We concluded that inflammation was responsible for the pain associated with LDH, and that increased IL-21 expression may be associated with the pathogenesis of LDH.

Variation in the PTH2R gene is associated with age-related degenerative changes in the lumbar spine.

In the elderly, degenerative changes in the lumbar spine are common, contributing to falsely elevated bone mineral density (BMD) values. The parathyroid hormone (PTH) system plays an important role in the regulation of bone turnover and we explore the hypothesis that polymorphisms (SNPs) within genes in this pathway (PTH, PTHLH, PTH1R and PTH2R) contribute to degenerative manifestations of the spine in elderly women. The study included 1,004 Swedish women aged 75 years from the population-based OPRA cohort who attended follow-up at 5 and 10 years. Lumbar spine BMD was assessed by dual energy X-ray absorptiometry (DXA) and each individual vertebra was evaluated visually on the DXA image for apparent degenerative manifestations. Six SNPs in PTH and 3 SNPs each in PTH1R, PTH2R and PTHLH were analysed. Among women with degenerative manifestations at the lumbar spine, there was an over-representation at baseline of those carrying the PTH2R SNP rs897083 A-allele (p = 0.0021; odds ratio 1.5 95 % CI 1.2-2.0) and across the duration of follow-up (p = 0.0008). No association was observed between degenerative manifestations and variation in the other genes. None of the PTH hormone system genes were associated with vertebral fracture. Variation in the PTH2R gene (Chr2q34, rs897083) may contribute to the age-associated degenerative manifestations that develop at the lumbar spine.

The association between genetic variants of RUNX2, ADIPOQ and vertebral fracture in Korean postmenopausal women.

Contrary to the traditional belief that obesity acts as a protective factor for bone, recent epidemiologic studies have shown that body fat might be a risk factor for osteoporosis and bone fracture. Accordingly, we evaluated the association between the phenotypes of osteoporosis or vertebral fracture and variants of obesity-related genes, peroxisome proliferator-activated receptor-gamma (PPARG), runt-related transcription factor 2 (RUNX2), leptin receptor (LEPR), and adiponectin (ADIPOQ). In total, 907 postmenopausal healthy women, aged 60-79 years, were included in this study. BMD and biomarkers of bone health and adiposity were measured. We genotyped for four single nucleotide polymorphisms (SNPs) from four genes (PPARG, RUNX2, LEPR, ADIPOQ). A general linear model for continuous dependent variables and a logistic regression model for categorical dependent variables were used to analyze the statistical differences among genotype groups. Compared with the TT subjects at rs7771980 in RUNX2, C-carrier (TC + CC) subjects had a lower vertebral fracture risk after adjusting for age, smoking, alcohol, total calorie intake, total energy expenditure, total calcium intake, total fat intake, weight, body fat. Odds ratio (OR) and 95% interval (CI) for the vertebral fracture risk was 0.55 (95% CI 0.32-0.94). After adjusting for multiple variables, the prevalence of vertebral fracture was highest in GG subjects at rs1501299 in ADIPOQ (p = 0.0473). A high calcium intake (>1000 mg/day) contributed to a high bone mineral density (BMD) in GT + TT subjects at rs1501299 in ADIPOQ (p for interaction = 0.0295). Even if the mechanisms between obesity-related genes and bone health are not fully established, the results of our study revealed the association of certain SNPs from obesity-related genes with BMD or vertebral fracture risk in postmenopausal Korean women.

Skeletal characteristics associated with homozygous and heterozygous WNT1 mutations.

Recent reports have shown that homozygous or compound heterozygous mutations in WNT1 can give rise to severe bone fragility resembling osteogenesis imperfecta, whereas heterozygous WNT1 mutations have been found in adults with dominant early-onset osteoporosis. Here we assessed the effects of WNT1 mutations in four children with recessive severe bone fragility and in heterozygous family members. In vitro studies using the Topflash luciferase reporter system showed that two WNT1 missense mutations that were observed in these families, p.Cys143Phe and p.Val355Phe, decreased the ability of WNT1 to stimulate WNT signaling by >90%. Analyses of iliac bone samples revealed no major abnormalities in bone mineralization density distribution, an indicator of material bone properties, whereas a shift towards higher bone mineralization density is characteristic of classical osteogenesis imperfecta caused by mutations in COL1A1/COL1A2. Intravenous bisphosphonate treatment of four children with homozygous or compound heterozygous WNT1 mutations was associated with increasing lumbar spine areal bone mineral density z-scores, as measured by dual energy X-ray absorptiometry, but the effect was smaller than what had previously been reported for children with classical osteogenesis imperfecta. Family members with heterozygous WNT1 mutation tended to have low bone mass. Three of these heterozygous individuals had radiographic signs of vertebral fractures. These observations suggest that more effective treatment approaches are needed for children with recessive WNT1-related bone fragility and that a systematic work-up for osteoporosis is warranted for WNT1 mutation carriers in these families.

Bone complications in patients with Cushing's syndrome: looking for clinical, biochemical, and genetic determinants.

SUMMARY: This is the first study examining the impact of both clinical, biochemical, and genetic determinants in the occurrence of bone complications in patients with overt Cushing's syndrome (CS). It demonstrates that the degree and duration of hypercortisolism seem to play a major role in bone loss and fractures development in these patients. INTRODUCTION: Bone loss and fractures are a common complication of CS. We investigate the role of gender, disease etiology, duration, and degree of hypercortisolism as well as the impact of glucocorticoid receptor (GR) polymorphisms on the development of bone complications in CS. METHODS: Fifty-two patients with active CS (38 Cushing's disease and 14 with cortisol-secreting adrenal adenoma) were genotyped for GR polymorphisms (BclI, N363S, ER22/23EK, and A3669G). In all patients, clinical, hormonal, and biochemical markers of bone turnover, densitometric parameters [lumbar spine and left femur bone mineral density (BMD), T-score, Z-score] as well as the prevalence of bone demineralization and both vertebral and peripheral fractures were assessed. RESULTS: No differences were found in bone complications according to gender, disease etiology, and genetic variants distribution. Fractured patients compared to non-fractured ones showed increased levels of urinary free cortisol (UFC) and a more compromised densitometric profile. UFC levels correlated with the occurrence of vertebral fractures (r = 0.43, p = 0.009) while midnight serum cortisol correlated with L1-L4 BMD values (r = -0.35, p = 0.04). Disease duration correlated with the presence of peripheral fractures (r = 0.36, p = 0.04). CONCLUSIONS: While GR gene variants as well as gender and disease etiology seem not to play a role, the degree and duration of hypercortisolism seem to be the major determinants of bone loss and fractures in this group of patients. More investigations are needed to understand the real impact of these determinants on the development of bone complications in patients with hypercortisolism.

Association of GALNT3 gene polymorphisms with bone mineral density in Chinese postmenopausal women: the Peking Vertebral Fracture study.

OBJECTIVE: GALNT3 gene encodes the glycosyltransferase polypeptide N-acetylgalactosaminyltransferase-3 (ppGalNacT3), which initiates the O-glycosylation of fibroblast growth factor 23 (FGF23) that is important in phosphorous regulation. Inactivating mutations of the GALNT3 gene can cause familial tumoral calcinosis. The aim of present study is to investigate the association of GALNT3 polymorphisms with osteoporosis phenotypes in Chinese postmenopausal women. METHODS: A community-based population of 1,353 postmenopausal women was randomly selected in Beijing. Bone mineral densities (BMDs) of the lumbar spine, femoral neck (FN), and total hip (TH) were measured by dual-energy x-ray absorptiometry. Vertebral fracture phenotypes were ascertained by vertebral x-ray reading. Osteoporotic fracture phenotypes were obtained from questionnaires. Single nucleotide polymorphisms of GALNT3 were determined by TaqMan allelic discrimination assay. Differences in BMD, serum phosphorus, or serum calcium across diverse genotypes or haplotypes were analyzed by general linear model analysis of covariance. Linear regression or logistic regression was used for association analyses of different osteoporosis phenotypes, phosphorous, or calcium. Partial correlation was used to investigate the relationship between phosphorus or calcium and BMD. RESULTS: We found that polymorphisms of rs1863196, rs6710518, and rs13429321 were significantly associated with FN BMD (P values of 0.002, 0.003, and 0.002, respectively). Polymorphisms of rs1863196, rs6710518, rs4667492, rs13429321, and rs6721582 were associated with TH BMD (P values of 0.002, 0.004, 0.037, 0.005, and 0.014, respectively). Haplotype-1 additive and dominant models were found to be associated with TH BMD (P values of 0.035 and 0.024, respectively). Haplotype-2 dominant model was found to be associated with FN BMD (P = 0.003) and TH BMD (P = 0.001). CONCLUSIONS: GALNT3 may play a role in genetic susceptibility to osteoporosis among Chinese postmenopausal women. Efforts should be exerted to replicate our findings in other similar and ethnically diverse populations.

A haplotype of MATN3 is associated with vertebral fracture in Chinese postmenopausal women: Peking Vertebral Fracture (PK-VF) study.

The Matrilin3 gene (MATN3) encodes an extracellular matrix protein, which modulates chondrocyte differentiation. The aim of this study was to test for association of MATN3 polymorphisms with bone mineral density (BMD), fracture, vertebral fracture, bone turnover or 25-hydroxyvitamin D [25(OH)D] in postmenopausal women. A community-based population of 1488 postmenopausal women was randomly selected in Beijing. The history of fracture and vertebral fracture was obtained via questionnaire and vertebral X-ray respectively. BMD of lumbar spine (2-4), femoral neck and total hip were measured by dual energy X-ray absorptiometry. Serum N-terminal procollagen of type 1 collagen (P1NP), ß-isomerized type I collagen C-telopeptide breakdown products (ß-CTX) and 25(OH)D were quantified. Binary logistic regression revealed that Haplotype-4 was significantly associated with vertebral fracture risk in both additive model (p=0.023, OR=1.521) and dominant model (p=0.028, OR=1.623). The significance remained after 10,000 permutation tests to correct multiple testing (p=0.042). Re-selected age matched vertebral fracture case-control groups revealed similar associations in additive model (p=0.014, OR=1.927, 95%CI=1.142-3.253) and in dominant model (p=0.011, OR=2.231, 95%CI=1.200-4.148). However, no significant association was found between MATN3 polymorphisms and serum ß-CTX, P1NP, 25(OH)D levels, or BMD. In linear regression, Haplotype-2 approached marginal significance in association with femoral neck BMD T-score (p=0.050), but this would account for only 0.2% of BMD variation in our sample. This study suggests that Haplotype-4 of MATN3 is associated with vertebral fracture risk independent of BMD in Chinese postmenopausal women. Efforts should be made to replicate our finding in other, similar and ethnically diverse, populations.

Type IX collagen neo-deposition in degenerative discs of surgical patients whether genotyped plus or minus for COL9 risk alleles.

STUDY DESIGN: Immunohistochemical analysis of type IX collagen in disc tissue from spinal fusion patients. OBJECTIVE: To determine if collagen IX can be detected in adult disc tissue removed at spinal fusion surgery from patients either with or without degeneration-associated tryptophan single nucleotide polymorphisms (SNPs) and whether the distribution is associated either with severity of degeneration or incidence of a collagen IX SNP genotype. SUMMARY OF BACKGROUND DATA: Genetic factors are strongly associated with risk of development and/or progression of disc degeneration. Two SNPs that introduce tryptophan polymorphisms in COL9A2 and COL9A3 are independently linked to an increased risk of lumbar disc disease. Although tryptophan variants are associated with accelerated degeneration, it is not known if collagen IX can be detected in adult disc tissue. METHODS: We selected age-matched disc samples from five clinical groups: fracture with Trp(-) (six cases), herniation (six cases), degeneration (five cases), spondylolisthesis with Trp(-) (eight cases), and spondylolisthesis/herniation/fracture with Trp(+) (six cases of Trp3 allele and one case of Trp2 allele). Using hematoxylin and eosin staining and immunohistochemical staining (collagens IX and IIA), 78 sections from 32 patients were analyzed. Selected disc tissues were assayed biochemically for collagen IX. RESULTS: Focal deposition of collagen IX was observed in regions of adult human disc tissue from spines showing degenerative changes in patients whether or not they were positive for a tryptophan SNP. However, in nondegenerative control disc tissue from fracture cases, little or no collagen IX was detected. The latter finding was confirmed by direct biochemical analyses for collagen IX in pooled samples of normal adult human annulus fibrosus or nucleus pulposus. CONCLUSION: During growth and maturation of the disc, collagen IX is presumably removed completely during matrix remodeling so that the protein is absent from normal adult annulus and nucleus but can reappear at sites of degeneration presumably as part of a repair response to mechanical injury.