Functional polymorphisms in the P2X7 receptor gene are associated with osteoporosis.

UNLABELLED: The P2X(7) receptor is an ATP-gated cation channel. We investigated the effect of both loss-of-function and gain-of-function polymorphisms in the P2X(7) receptor gene on BMD and risk of vertebral fractures and found that five polymorphisms and haplotypes containing three of these polymorphisms were associated with BMD and fracture risk. INTRODUCTION: The P2X(7) receptor is an ATP-gated cation channel. P2X(7) receptor knockout mice have reduced total bone mineral content, and because several functional polymorphisms have been identified in the human P2X(7) receptor gene, we wanted to investigate the effect of these polymorphisms on BMD and risk of vertebral fractures in a case-control study including 798 individuals. METHODS: Genotyping was carried out using TaqMan assays. BMD was measured using dual energy X-ray absorptiometry, and vertebral fractures were assessed by lateral spinal X-rays. RESULTS: The rare allele of a splice site polymorphism, 151 + 1: G-T, was associated with increased fracture risk and reduced BMD in women. Two other loss-of-function polymorphisms, Glu496Ala and Gly150Arg, were also associated with BMD. The Glu496Ala variant allele was associated with decreased lumbar spine BMD in women and decreased total hip BMD in men. The 150Arg allele was associated with decreased total hip BMD in women and men combined. The minor allele of the gain-of-function polymorphism, Ala348Thr, was associated with reduced fracture risk and increased BMD at all sites in men. The Gln460Arg variant allele, which has been associated with increased receptor function in monocytes, was associated with increased total hip BMD in women. With the exception of His155Tyr for which we found conflicting results in men and women, our results are consistent with the phenotype of the knockout mouse. Analysis of a haplotype containing Ala348Thr, Gln460Arg, and Glu496Ala showed that the effects of the haplotypes on BMD and fracture were driven by Ala348Thr in men and by Gln460Arg and Glu496Ala in women. CONCLUSION: In conclusion, we found that functional polymorphisms in the P2X(7) receptor gene and haplotypes containing three of these polymorphisms are associated with osteoporosis.

The expression and regulation of bone-acting cytokines in human peripheral adipose tissue in organ culture.

The humoral cross-talk between bone and fat is an area of increasing interest. We investigated the expression and regulation of the bone-acting cytokines; bone morphogenetic protein 2 (BMP2), connective tissue growth factor (CTGF), osteoprotegerin (OPG), and transforming growth factor beta (TGFB1). Subcutaneous adipose tissue was aspirated from lean, healthy women. Tissue samples were incubated with interleukin 1-ß (IL1-ß), tumor necrosis factor-α (TNF-α), cortisol, troglitazone, IL1-ß + troglitazone, or vehicle. Gene expression in the adipose tissue was analyzed using qPCR and protein levels in the incubation media were analyzed using ELISA. OPG expression and secretion was diminished by 40.8% and 43.1% respectively, by cortisol, and OPG expression was diminished by 67.5% by troglitazone (p<0.05). The proinflammatory cytokines IL1-ß and TNF-α significantly increased the expression of CTGF (p<0.05) by 65.1% and 101.3%, respectively, and the expression and secretion of OGP by 62.3-165.8% (p<0.05). This interleukin 1-ß mediated increase in CTGF- and OPG expression and secretion was ameliorated by troglitazone. Troglitazone and related drugs are known to have adverse effects on bone. We suggest that this could be mediated via altered cytokine production in adipose tissue. Moreover, obese individuals have a low-grade inflammation in their adipose tissue and have higher bone mineral density than lean individuals. We suggest that this inflammation may increase the expression and secretion of OPG and CTGF and thereby increase BMD. In conclusion, bone acting cytokines are produced in the adipose tissue and may affect bone through endocrine mechanisms.

Polymorphisms of the peroxisome proliferator-activated receptor γ (PPARγ) gene are associated with osteoporosis.

UNLABELLED: Stimulation of PPARγ turns mesenchymal stem cells into adipocytes instead of osteoblasts. We investigated the effect of polymorphisms in the PPARγ gene on BMD and fracture risk in two Danish cohorts and found opposing effects of certain SNPs and haplotypes in the two cohorts probably owing to environmental factors. INTRODUCTION: Stimulation of PPARγ causes development of mesenchymal stem cells to adipocytes instead of osteoblasts leading to decreased osteoblast number and BMD. The aim of this study was to examine the effect of PPARG polymorphisms on BMD and fracture risk in two Danish cohorts: AROS, a case-control population comprising 809 individuals and DOPS, a population comprising 1,716 perimenopausal women allocated to hormone therapy or not at baseline and followed for 10 years. On the basis of linkage disequilibrium between SNPs throughout the gene and previous studies we chose 10 polymorphisms for investigation. METHODS: In AROS, individuals heterozygous for the polymorphisms rs12497191, rs4135263, and rs1151999 had an increased risk of vertebral fractures (OR = 1.48-1.76, p = 0.005-0.04) compared with individuals homozygous for the common allele. In DOPS, individuals heterozygous for rs1151999 had an increased BMD at the hip sites (p ≤ 0.02). An interaction between rs1151999 and diet was found on BMD in both cohorts. RESULTS: For the polymorphism rs1152003 there was an interaction with body weight on BMD at all sites in both cohorts (p ≤ 0.07). Stratified analyses revealed that in the high weight group in AROS individuals homozygous for the variant allele had a decreased BMD (p ≤ 0.02), whereas the same pattern was found in the low weight group in DOPS (p ≤ 0.03). A number of haplotype associations were found as well, the direction of which was opposite in the two cohorts. CONCLUSION: Our study suggests an association SNPs in PPARG and haplotypes thereof and BMD and fracture risk. The effect however appears to be modifiable by environmental factors.

Polymorphisms in the ALOX12 gene and osteoporosis.

UNLABELLED: ALOX12 produces ligands for PPARγ thereby turning mesenchymal stem cells into adipocytes instead of osteoblasts. We investigated the effect of polymorphisms in the ALOX12 gene on BMD and fracture risk in two Danish cohorts and found four polymorphisms and a haplotype thereof to be associated with BMD and fracture risk. INTRODUCTION: Stimulation of the PPARγ with ligands produced by the ALOX enzymes drives mesenchymal stem cells in an adipocyte direction at the expense of osteoblasts leading to decreased osteoblast number and BMD. Previously, polymorphisms in the ALOX12 gene have been associated with osteoporosis. METHODS: We examined the effect of ALOX12 polymorphisms on BMD and the risk of fractures in two Danish cohorts: AROS, a case-control population comprising 809 individuals and DOPS, a population comprising 1,716 perimenopausal women allocated to hormone therapy or not at baseline and followed for up to 10 years. On the basis of linkage disequilibrium (LD) between SNPs throughout the gene and previous genetic association studies we chose ten polymorphisms for investigation. Genotyping was carried out using the Sequenom MassARRAY genotyping system and TaqMan assays. RESULTS: In AROS, individuals heterozygous for the polymorphisms rs3840880, rs9897850, rs2292350 and rs1126667 had a 3.0-4.7% decreased lumbar spine BMD (p = 0.02-0.06) and an increased risk of vertebral fractures (p < 0.05) compared with individuals homozygous for either allele. In DOPS, none of the individual SNPs were associated with BMD or incident fractures. In both cohorts, the above-mentioned SNPs comprised an LD-block (pairwise D´ = 1.0, r (2) = 0.45-0.97). A haplotype comprising all the common alleles (frequency 9%) was associated with decreased bone loss at the hip (p < 0.05) and decreased incidence of osteoporotic fractures (p < 0.05) in DOPS and increased femoral neck BMD in AROS (p < 0.05). CONCLUSION: Our study suggests that genetic variants in ALOX12 may influence BMD and fracture risk.

Genotypes and haplotypes of the estrogen receptor genes, but not the retinoblastoma-interacting zinc finger protein 1 gene, are associated with osteoporosis.

Osteoporosis is a common age-related disease with a strong genetic influence. Polymorphisms of ESR1 have consistently been shown to be associated with bone mineral density (BMD) and fracture; however, in regulating bone metabolism, ESR1 interacts with both ESR2 and RIZ1. We therefore examined the effects of polymorphisms in the ESR1, ESR2, and RIZ1 genes and their haplotypes on vertebral fractures and BMD in a case-control study comprising 462 osteoporotic patients and 336 controls. In ESR1, we found the variant C allele of the XbaI polymorphism to be associated with decreased risk of vertebral fractures in women (P < 0.01), whereas in men, the T allele seemed protective (P = 0.05). The variant G allele of the PvuII polymorphism decreased the risk of vertebral fractures independently of lumbar spine BMD in women (P = 0.04) but had no effect in men. Haplotype X-P-H (XbaI:C, PvuII:G, and a high number of TA repeats) was associated with decreased risk of vertebral fractures in women (P = 0.04) but not men. In ESR2, the G allele of the AluI polymorphism was associated with increased fracture risk (P = 0.04), and the haplotype that comprises rs1256031:T and AluI:A increased lumbar spine BMD by 0.04 +/- 0.02 g/cm(2) (P < 0.05) and decreased the risk of vertebral fractures (P = 0.04). There was no effect of the RIZ1 polymorphism on BMD or fracture risk and no evidence of interaction between the polymorphisms and haplotypes thereof. We confirm that genetic variants in ESR1 and ESR2, but not RIZ1, are important in osteoporosis. We found no evidence of interaction between polymorphisms, but we found that the effects of genetic variants in ESR1 might be sex dependent.

Haplotypes of promoter and intron 1 polymorphisms in the COLIA1 gene are associated with increased risk of osteoporosis.

Osteoporosis is a common age-related disease with a strong genetic influence. COLIA1 is one of the most extensively studied candidate genes and has consistently been associated with BMD and fracture. We examined the effects of the polymorphisms -1997G>T, -1663indelT, and +1245G>T and their haplotypes on vertebral fractures and bone mineral density (BMD) in a case-control study comprising 462 osteoporotic patients and 336 controls. The -1663indelT polymorphism was associated with a decreased lumbar spine (ls) BMD, 0.75 +/- 0.14 g/cm(2), in individuals with the del/del genotype versus 0.83 +/- 0.18 and 0.85 +/- 0.18 g/cm(2) in individuals with the ins/del and ins/ins genotypes, respectively (p = 0.02). The T-allele of the +1245G>T polymorphism, which was in strong linkage disequilibrium (LD) with -1663indelT, was also associated with a decreased lsBMD (p = 0.02). -1997G>T was not significantly associated with lsBMD. The three most common haplotypes accounted for 98.5% of the alleles. Individuals with one or two copies of haplotype 1 (-1997G/-1663ins/+1245G) had a significantly higher lsBMD, 0.84 +/- 0.18 and 0.85 +/- 0.15 g/cm(2), respectively, versus 0.78 +/- 0.15 g/cm(2) in noncarriers (p = 0.01). Individuals with two copies of haplotype 2 (-1997G/-1663del/+1245T) had a significantly lower lsBMD, 0.76 +/- 0.14 g/cm(2), versus 0.85 +/- 0.18 and 0.82 +/- 0.18 g/cm(2), respectively, in individuals with zero or one copy (p = 0.03). The odds ratio for vertebral fracture in individuals carrying the variant T-allele of the -1997G>T polymorphism was 1.49 (CI, 1.03-2.16; p = 0.03). Logistic regression revealed that this effect was partly independent of BMD. In conclusion, the -1663del and +1245T alleles influence BMD negatively, whereas the -1997T-allele has a minor effect on BMD but increases the risk of vertebral fractures. These findings are in agreement with functional studies showing that these polymorphisms influence gene expression.

The effect of interleukin-1alpha polymorphisms on bone mineral density and the risk of vertebral fractures.

Interleukin-1alpha (IL-1alpha) stimulates bone resorption via osteoclasts. Mononuclear cells from patients with osteoporosis show increased IL-1alpha production, and IL-1alpha mRNA is more often detected in bone biopsies from osteoporotic compared to normal postmenopausal women. Polymorphisms have been identified in the IL-1alpha gene; however, none of these has been examined for an effect on bone phenotypes in Caucasians. We investigated if the polymorphisms in the IL-1alpha gene affect the risk of osteoporotic fractures, bone mineral density (BMD), and bone turnover in 462 osteoporotic patients and 336 normal controls. Based on previous studies of polymorphisms in the gene and data from the International Hap-Map Project, four polymorphisms needed examination in order to investigate the effect of known polymorphisms in the IL-1alpha gene. We examined C(-1202)-T(rs1800794), C(-889)-T(rs1800587), T(155 + 209)-C(rs2071373), C(155 + 320)-T(rs2856838), and G(398)-T(rs 17561) by Taqman and restriction fragment-length polymorphism assays. BMD was examined by dual-energy X-ray absorptiometry. Bone turnover was evaluated by serum osteocalcin, serum carboxy-terminal propeptide of human type I procollagen, serum bone-specific alkaline phosphatase, serum carboxy-terminal telopeptide of type I collagen, and urinary hydroxyproline/creatinine. Genotype distributions were in Hardy-Weinberg equilibrium. All polymorphisms were in strong linkage disequilibrium. The C allele of the C(155 + 320)-T polymorphism tended to be more common among patients with vertebral fractures (P = 0.06) and patients with BMD T score <-2.5 (P = 0.05). Furthermore, haplotype 1 was associated with reduced risk of having BMD T score <-2.5 (P = 0.02). None of the other polymorphisms or haplotypes was associated with fracture risk or BMD T score <-2.5. BMD and bone turnover were not associated with any of the genetic variants. In conclusion, all the polymorphisms within the IL-1alpha gene are in strong linkage disequilibrium and not convincingly associated with fracture risk, BMD, or bone turnover.

A CAG repeat polymorphism in the androgen receptor gene is associated with reduced bone mass and increased risk of osteoporotic fractures.

Osteoporosis is a common disease with a strong genetic component. Hypogonadism results in low bone mass and it increases significantly the risk of osteoporosis in both sexes. The estrogen and androgen receptor genes are therefore strong candidates for mediating the genetic influence on bone mass and risk of osteoporosis. A CAG repeat in the first exon of the androgen receptor (AR) is associated with reduced transcriptional activity of the AR. We therefore examined whether this CAG repeat polymorphism is associated with changes in bone mass and risk of osteoporotic fractures in 284 osteoporotic patients with vertebral fractures and 327 normal individuals. The number of CAG repeats varied between 13 and 30 in men and between 7 and 34 in women. The short and long alleles comprised 19.2 +/- 2.5 and 19.0 +/- 2.3 repeats (ns) and 22.7 +/- 2.4 and 21.9 +/- 2.4 (P < 0.01) in women with vertebral fractures and normal women, respectively. This difference was also reflected in the average number of CAG repeats: 21.0 +/- 2.0 in osteoporotic women vs. 20.5 +/- 2.0 in normal women (P < 0.05). 54.8% of women with osteoporotic fractures vs. 45.9% of normal women had average number of CAG repeats of 21 and more (chi2 = 3.11, P = 0.08). Logistic regression analyses revealed that both the average number of CAG repeats and the length of the long allele were significant predictors of osteoporotic fractures in women (P < 0.05 and P < 0.01, respectively). Men with vertebral fractures had 20.0 +/- 2.8 CAG repeats compared with 20.7 +/- 2.5 CAG repeats in normal men (ns). Linear regression analysis revealed that the length of the long allele was negatively correlated with BMD of the lumbar spine (P < 0.05) and femoral neck (P < 0.05) in women. In men, linear regression analyses demonstrated that BMD of the lumbar spine (P < 0.05), femoral neck (P < 0.05) and total hip (P < 0.05) was positively correlated with length of the CAG repeat polymorphism. In conclusion, we have demonstrated that the CAG repeat polymorphism in the first exon of the AR gene is associated with reduced bone mass and increased risk of osteoporotic fractures in women.

A TA repeat polymorphism in the estrogen receptor gene is associated with osteoporotic fractures but polymorphisms in the first exon and intron are not.

Estrogen and the estrogen receptor (ER) play a central role in bone metabolism as illustrated by the loss of bone mass after menopause and the osteopenia in individuals with defect aromatase or ER. We therefore wanted to investigate the effect of polymorphisms in the ER-alpha gene on bone mass, bone turnover, and the prevalence of osteoporotic fractures in a study of 160 women and 30 men with vertebral fractures and 124 women and 64 men who are normal. Three previously described polymorphisms, G261-C in exon 1 and T-C and A-G in intron 1, in the ER gene were determined by restriction fragment length polymorphism (RFLP) using BstUI, Pvu II, and Xba I after polymerase chain reaction (PCR). A TA repeat polymorphism in the promoter region was examined by PCR and electrophoresis. The distribution of BstUI, Pvu II, and Xba I RFLPs was similar in the osteoporotic patients and the normal controls. No significant differences could be shown in bone mass or bone turnover between the genotypes. The mean number of TA repeats was lower in patients with osteoporotic fractures, 17.3+/-2.8 versus 18.6+/-2.8 in the normal controls (p < 0.01). This also was reflected in a significantly increased odds ratio of osteoporotic fractures in individuals with 11-18 repeats of 2.64 (95% CIs, 1.61-4.34). Furthermore, bone mineral density (BMD) of the lumbar spine was lower in individuals with low mean number of repeats than in individuals with high mean number of repeats (0.790+/-0.184 g/cm2 vs. 0.843+/-0.191 g/cm2; p < 0.05). This difference also was found in BMD of the total hip. Using multiple linear regression, mean number of TA repeats was a predictor of lumbar spine BMD (p < 0.05) and a BMD-independent predictor of fractures (p < 0.05). Mean number of TA repeats was not associated with levels of biochemical markers of bone turnover. All four polymorphisms were in linkage disequilibrium. A TA repeat polymorphism in the ER gene is associated with increased risk of osteoporotic fractures and a modest reduction in bone mass. Polymorphisms in the first exon and first intron of the ER gene are not associated with osteoporotic fractures, bone mass, or bone turnover.

Osteoporotic fractures are associated with an 86-base pair repeat polymorphism in the interleukin-1--receptor antagonist gene but not with polymorphisms in the interleukin-1beta gene.

Interleukin-1beta (IL-1beta) is a potent stimulator of bone resorption, and has been implicated in the pathogenesis of high bone turnover and osteoporosis. IL-1 receptor antagonist (IL-1ra) is a competitive inhibitor of IL-1beta effects and the biological effects of IL-1beta are therefore proportional to the ratio IL-1beta/IL-1ra. The coding regions of IL-1beta were examined for sequence variations by SSCP and sequencing after polymerase chain reaction (PCR) of genomic DNA. Three previously described polymorphisms (C(-511)-T, G(3877)-A and C(3954)-T) in the IL-1beta gene were determined by restriction fragment length polymorphism (RFLP) using Ava I, Aci I, and Taq I after PCR. The 86-base pair repeat polymorphism in IL-1ra was examined by PCR and electrophoresis and the T11100-C polymorphism in the IL-1ra gene was examined by RFLP using MspA1I after PCR. All polymorphisms were related to bone mass, biochemical markers of bone turnover, and presence of fracture in a study including 389 osteoporotic patients with vertebral fractures and normal controls. Two normal women were heterozygous for a shift from cytosine to thymine (C3263-T) in exon 4 of the IL-1beta gene. This substitution did not affect the amino acid sequence. We did not find other sequence variations in the IL-1beta gene apart from the already known polymorphisms. The distribution of C(-511)-T, G(3877)-A, and C(3954)-T genotypes was similar in the osteoporotic and the normal controls. No significant differences could be shown in bone mass or bone turnover. In the IL-1ra gene almost complete linkage was confirmed between the already known polymorphisms: G(1731)-A, G(1821)-A, A(1868)-G, G(1887)-C, T(8006)-C, C(8061)-T, 86 base pair variable number tandem repeat (VNTR), A(9589)-T, and a new polymorphism: T(1934)-C. The A1A1/A3 genotypes of the IL-1ra VNTR polymorphism were significantly more frequent in osteoporotic patients (56.2%) compared with age-matched normal controls (43.3%) (chi2 = 4.09; p = 0.043). The relative risk of osteoporotic fractures was increased to 1.68 (95% CI, 1.01-2.77) in individuals with A1A1/A3 genotypes. Bone mineral density (BMD) of the lumbar spine was reduced in individuals with A1A1/A3 genotypes (p = 0.014, analysis of variance [ANOVA]). The difference in bone mass between A1A1/A3 and A2A1/A2 tended to increase with increasing age. T1100-C genotypes were distributed similarly in osteoporotic patients and normal controls and the polymorphism was without effect on bone mass and biochemical markers of bone turnover. In conclusion, an 86-base pair repeat polymorphism in the IL-lra gene is associated with increased risk of osteoporotic fractures. Other polymorphisms in the IL-1ra and the IL-1beta genes are not associated with osteoporotic fractures or alterations in bone mass or bone turnover.