Association of the VDR translation start site polymorphism and fracture risk in older women.

UNLABELLED: We evaluated the association between the VDR translation start site polymorphism and osteoporotic phenotypes among 6698 older white women. Women with the C/C genotype had lower wrist BMD and an increased risk of wrist and all non-spine/low-trauma fractures. The high frequency of this variant confers a population attributable risk that is similar to several established risk factors for fracture. INTRODUCTION: The vitamin D receptor (VDR) is a nuclear receptor that regulates bone formation, bone resorption, and calcium homeostasis. A common C to T polymorphism in exon 2 of the VDR gene introduces a new translation start site and a protein that differs in length by three amino acids (T = 427aa, C = 424aa; rs10735810). MATERIALS AND METHODS: We conducted genetic association analyses of this polymorphism, BMD, and fracture outcomes in a prospective cohort of 6698 white American women >or=65 years of age. Incident fractures were confirmed by physician adjudication of radiology reports. There were 2532 incident nontraumatic/nonvertebral fractures during 13.6 yr of follow-up including 509 wrist and 703 hip fractures. RESULTS: Women with the C/C genotype had somewhat lower distal radius BMD compared with those with the T/T genotype (CC=0.358 g/cm(2), CT=0.361 g/cm(2), TT=0.369 g/cm(2), p=0.003). The C/C genotype was also associated with increased risk of non-spine, low traumatic fractures (HR: 1.18; 95% CI: 1.04, 1.33) and wrist fractures (HR: 1.33; 95% CI: 1.01, 1.75) compared with the T/T genotype in age-adjusted models. Further adjustments for distal radius BMD only slightly attenuated these associations. The VDR polymorphism was not associated with hip fracture. The population attributable risk (PAR) of the C/C genotype for incident fractures was 6.1%. The PAR for established risk factors for fracture were: low femoral neck BMD (PAR=16.3%), maternal history of fracture (PAR=5.1%), low body weight (PAR=5.3%), corticosteroid use (PAR=1.3%), and smoking (PAR=1.6%). Similar PAR results were observed for wrist fractures. CONCLUSIONS: The common and potentially functional VDR translation start site polymorphism confers a modestly increased relative risk of fracture among older white women. However, the high frequency of this variant confers a population attributable risk that is similar to or greater than several established risk factors for fracture.

Nuclear receptor coactivator-3 alleles are associated with serum bioavailable testosterone, insulin-like growth factor-1, and vertebral bone mass in men.

CONTEXT: Nuclear receptor coactivator-3 (NCOA3) is a member of the steroid receptor coactivator family that interacts with nuclear hormone receptors to enhance their transcriptional activation function and may play a role in somatic growth. OBJECTIVE: The aim of this study was to examine the relationships between the CAG/CAA (glutamine) length variation at the NCOA3 locus, sex steroid hormone, and IGF-I levels and bone mineral density (BMD) in a cohort of older Caucasian men. DESIGN AND METHODS: We analyzed the association between potentially functional alleles at this locus, serum sex steroid hormone, and IGF-I levels and lumbar spine and proximal femur BMD (Hologic QDR) in 263 community-dwelling Caucasian men (age 66 +/- 7 yr, mean +/- sd; range 51-84 yr). Men were genotyped for a CAG/CAA repeat polymorphism in NCOA3, which encodes a polyglutamine tract of variable length in the C-terminal transcriptional activation domain of the protein. RESULTS: We found a significant monotonic decrease in lumbar spine, but not hip, BMD with increasing copies of the most common allele (29 repeats, 53%). For example, men with the 29/29 genotype had 6% or nearly 0.5 sd lower spine BMD than men without this genotype, and NCOA3 genotype explained 3.2% of the phenotypic variation in this trait. Serum levels of bioavailable testosterone and IGF-I paralleled genotype-related differences in lumbar spine BMD. CONCLUSION: Allelic variation at the NCOA3 locus may contribute to the genetic control of androgenic hormone and IGF levels and vertebral bone mass among older men.

Association of endothelial nitric oxide synthase genotypes with bone mineral density, bone loss, hip structure, and risk of fracture in older women: the SOF study.

Nitric oxide (NO) is an important bone-signaling molecule. We examined the associations between the Glu298Asp polymorphism of NOS3, indices of bone strength, and the incidence of fracture among 6691 women aged 65 years and older enrolled in the Study of Osteoporotic Fractures. Calcaneal BMD was measured at an initial exam and after an average of 5.9 years of follow-up. Hip BMD was measured at an initial exam and after 3.7 years of follow-up. Baseline spine BMD and hip structural parameters were measured. Incident hip fractures were confirmed by review of radiographic reports; follow-up was greater than 98% complete. Incident vertebral fractures were defined by morphometry using lateral spine radiography at baseline and an average of 3.7 years later. The frequencies of the NOS3 Glu298Asp genotypes were Glu/Glu=46.2%, Glu/Asp=42.7%, and Asp/Asp=11.1%. There were no significant associations between NOS3 genotypes and initial calcaneal BMD, hip BMD, or rate of change in hip or calcaneal BMD. None of the hip structural parameters differed substantially by genotype. NOS3 genotype was not significantly associated with either incident or prevalent radiographic vertebral fractures. Women with the heterozygous Glu/Asp genotype had a borderline statistically significantly lower rate of hip fracture than either the Glu/Glu genotype (HR=0.87, 95% CI: 0.74, 1.01) or the Asp/Asp genotype (HR=0.78, 95% CI: 0.62, 0.98). In conclusion, the Glu298Asp polymorphism does not contribute substantially or consistently to indices of bone strength in this sample of older white women, although our findings suggest allelic variation at the NOS3 locus maybe associated with hip fracture risk. Confirmation of these findings is needed in other populations and with additional markers within and flanking the NOS3 gene region.

The C161-->T polymorphism in peroxisome proliferator-activated receptor gamma, but not P12A, is associated with insulin resistance in Hispanic and non-Hispanic white women: evidence for another functional variant in peroxisome proliferator-activated receptor gamma.

The P12A variant in the peroxisome proliferator-activated receptor gamma (PPARgamma) gene has been intensely studied for association with obesity-related or type-2 diabetes-related traits; however, the results have been somewhat inconsistent in different populations. We genotyped a large cohort of Hispanic and non-Hispanic white individuals from the San Luis Valley Diabetes Study for P12A and another common variant, C161-->T, in the PPARgamma gene to determine if these sites were associated with fasting glucose, insulin, free fatty acid levels, insulin sensitivity, or body fat. There were no statistically significant frequency differences at these two sites between Hispanic and non-Hispanic individuals. No significant association with the metabolic phenotypes was observed for either of the polymorphisms in men; however, in women, significant associations were shown between the C161-->T variant and fasting insulin (P=.008) and the homeostasis model assessment of insulin resistance (HOMA IR; P=.007). After adjusting for age, smoking, fat mass, and skin reflectance, linear regression showed that C161-->T explained 1.5% of the variation in both fasting insulin (P=.031) and HOMA IR (P=.028) whereas P12A contributed only 0.04% (fasting insulin, P=.268) and 0.02% (HOMA IR, P=.418) to the total trait variation. In the San Luis Valley Diabetes Study female patients, C161-->T appears to be a better predictor of fasting insulin levels and insulin resistance than P12A although the effect of this variant is small. These results support the hypothesis that C161-->T is in linkage disequilibrium with unidentified functional variation in PPARgamma or in a linked gene. This could explain some of the inconsistencies in the P12A association studies as the allele frequency and level of linkage disequilibrium of another functional polymorphism in the region could vary in different populations.

Association of the G-174C variant in the interleukin-6 promoter region with bone loss and fracture risk in older women.

UNLABELLED: We analyzed the association between the IL-6 G-174C polymorphism and osteoporosis phenotypes in 3376 older women. Women with the C/C genotype had a significantly slower rate of decline in hip BMD and a 33% lower risk of wrist fracture than women with the G/G genotype. Variation at the IL-6 locus may contribute to the genetic susceptibility to bone fragility. INTRODUCTION: Interleukin 6 (IL-6) promotes osteoclast formation and bone resorption. The C allele of the G-174C polymorphism in the IL-6 promoter region has been related to lower gene transcription and plasma IL-6 levels. MATERIALS AND METHODS: In this study, we evaluated the relationship between the IL-6 G-174C polymorphism and BMD, the rate of decline in BMD, and the risk of fracture in 3376 women 65 years of age and older participating in the Study of Osteoporotic Fractures. BMD was measured at the distal and proximal radius using single photon absorptiometry and at the hip using DXA. Hip BMD was measured again an average of 3.5 years later. Incident fractures over an average of 10.8 years of follow-up were confirmed by physician adjudication of radiology reports. RESULTS: Distal and proximal radius BMD was lowest among women with the G/G genotype, intermediate in the heterozygotes, and highest in women with the C/C genotype (p = 0.016 and p = 0.049, respectively), although the differences between the genotypes were small. While there were no differences by genotype with initial hip BMD, women with the C/C genotype experienced a slower rate of decline in total hip and femoral neck BMD compared with women with the G/G genotype (p = 0.004 and p = 0.029, respectively). Women with the C/C genotype also had 33% lower risk of wrist fracture compared with women with the G/G genotype, independent of age, body mass index, estrogen use, and study center (RR, 0.67; 95% CI, 0.45, 1.00; p = 0.048), whereas heterozygous women had a more intermediate risk (RR, 0.85; 95% CI, 0.65, 1.12; p = 0.256). No association was found between genotype and risk of hip or all non-spine fractures. CONCLUSIONS: These results suggest that the IL-6 G-174C promoter polymorphism may be a genetic marker for bone loss and wrist fracture among older women.

Genetic variation in uncoupling protein 3 is associated with dietary intake and body composition in females.

The uncoupling proteins (UCPs) are a family of mitochondrial transport proteins that promote proton leakage across the inner mitochondrial membrane, uncoupling oxidative phosphorylation from adenosine triphosphate (ATP) production and releasing energy as heat. Variation in these genes may disrupt biochemical pathways influencing thermogenesis, energy metabolism, and fuel substrate partitioning and oxidation, which may in turn predispose to obesity. We genotyped polymorphisms in UCP2 and UCP3 in a sample of nondiabetic participants (n = 722) of the San Luis Valley Diabetes Study (SLVDS) and found female-specific associations between UCP3 polymorphisms and measures of dietary intake and body composition. The UCP3-5 variant was statistically significantly associated with total caloric intake (P =.012), fat intake (P =.011), fat mass (P =.004), and lean mass (P =.013), with the C allele corresponding to higher dietary intake and lower fat mass and lean mass. The UCP3p-55 and the UCP3-3 polymorphisms, which were in high linkage disequilibrium (D' = 0.9776), showed similar patterns of association with total caloric intake (P =.031 and P =.042, respectively) and lean mass (P =.035 and P =.059, respectively), with the rare alleles corresponding to higher total intake and lean mass. No statistically significant associations were detected between the outcome variables and polymorphisms in UCP2. Two-way analysis of covariance (ANCOVA), used to evaluate the multi-locus effects and interactions between UCP3-5 and UCP3p-55, showed association with the main effect terms, but no evidence for statistically significant interaction between UCP3-5 and UCP3p-55 in regard to dietary intake. The UCP3-5 polymorphism was the only statistically significant genetic predictor of fat mass. The lean mass model showed no statistically significant association with either UCP3 variant. These results support a role for UCP3 in fuel substrate management and energy metabolism, which may influence body weight regulation.

Genetic variation in fatty acid-binding protein-4 and peroxisome proliferator-activated receptor gamma interactively influence insulin sensitivity and body composition in males.

Obesity and type 2 diabetes are closely related, multifactorial metabolic conditions characterized by alterations in energy metabolism and glucose homeostasis, respectively. Peroxisome proliferator-activated receptor gamma (PPARgamma) is a ligand-dependent transcription factor that regulates genes involved in lipid and glucose homeostasis, including the adipocyte-specific fatty acid-binding protein (FABP4). In turn, FABP4 binds fatty acids and transports them to the nucleus where the FABP4/fatty acid complex activates PPARgamma in a positive feedback loop. In this study, we tested the hypothesis that the polymorphisms, FABP4-376 and PPARgamma Pro12Ala, interactively influence insulin sensitivity and body composition in nondiabetic, Hispanic and non-Hispanic white males (n = 314) participating in the San Luis Valley Diabetes Study (SLVDS). Although the individual sites were not statistically significantly associated with any of the outcomes, we found statistically significant interaction terms in 2-way analysis of covariance (ANCOVA) models for homeostasis model assessment of insulin resistance (HOMA-IR) (P =.014) and lean mass (P =.019). While the PPARgamma Pro12Ala site was the only statistically significant predictor of fat mass in the 2-way model (P =.012), the FABP4 and PPARgamma main effect terms individually became stronger when considered in one model compared with the analysis of each polymorphism separately. These findings provide evidence that FABP4 and PPARgamma work together to influence a biologic pathway affecting insulin sensitivity and body composition, illustrating the importance of investigating the joint effect of genes in determining susceptibility for complex disease.

Candidate gene analysis of femoral neck trabecular and cortical volumetric bone mineral density in older men.

In contrast to conventional dual-energy X-ray absorptiometry, quantitative computed tomography separately measures trabecular and cortical volumetric bone mineral density (vBMD). Little is known about the genetic variants associated with trabecular and cortical vBMD in humans, although both may be important for determining bone strength and osteoporotic risk. In the current analysis, we tested the hypothesis that there are genetic variants associated with trabecular and cortical vBMD at the femoral neck by genotyping 4608 tagging and potentially functional single-nucleotide polymorphisms (SNPs) in 383 bone metabolism candidate genes in 822 Caucasian men aged 65 years or older from the Osteoporotic Fractures in Men Study (MrOS). Promising SNP associations then were tested for replication in an additional 1155 men from the same study. We identified SNPs in five genes (IFNAR2, NFATC1, SMAD1, HOXA, and KLF10) that were robustly associated with cortical vBMD and SNPs in nine genes (APC, ATF2, BMP3, BMP7, FGF18, FLT1, TGFB3, THRB, and RUNX1) that were robustly associated with trabecular vBMD. There was no overlap between genes associated with cortical vBMD and trabecular vBMD. These findings identify novel genetic variants for cortical and trabecular vBMD and raise the possibility that some genetic loci may be unique for each bone compartment.

Association analysis of WNT10B with bone mass and structure among individuals of African ancestry.

Wnts comprise a family of secreted growth factors that regulate the development and maintenance of many organs. Recently, Wnt10b was shown to stimulate osteoblastogenesis and bone formation in mice. To evaluate further the role of Wnt10b in bone health in humans, we performed bidirectional sequencing of approximately 8 kb of the WNT10B gene region in 192 individuals (96 African, 96 white) to identify single nucleotide polymorphisms (SNPs). We identified 19 SNPs with minor allele frequency (MAF) > or =0.01. Ten of these SNPs were not present in the NCBI dbSNP database (build 127), whereas 10 of the 20 SNPs (50%) reported in dbSNP were not verified. We initially genotyped seven tagging SNPs that captured common (MAF > or = 0.05) variation in the region with r (2) > 0.80 and a potentially functional SNP in exon 5 in 1035 Afro-Caribbean men > or =40 yr of age. Association analysis showed three SNPs in a 3' region of linkage disequilibrium that were associated with DXA measures of hip BMD. Associations between two of these three SNPs (rs1051886, rs3741627) with hip BMD were replicated in an additional 980 Afro-Caribbean men (p < 0.05), in the combined sample of 2015 men (p < or = 0.006), and in 416 individuals > or =18 yr of age (mean, 44 yr) belonging to eight extended, multigenerational Afro-Caribbean families with mean family size >50 (3535 relative pairs; p < 0.05). Further analysis showed that rs1051886 and rs3741627 were associated with cortical cross-sectional area, periosteal circumference, and BMC in the radius, such that individuals with the minor alleles had lower biomechanical indices of long-bone bending strength. This analysis implicates the WNT10B locus as a genetic element in the regulation of bone mass and structural geometry.

Functional characterization of genetic variation in the Frizzled 1 (FZD1) promoter and association with bone phenotypes: more to the LRP5 story?

WNT signaling is an important determinant of bone formation. The WNT co-receptor, Frizzled homolog 1 (FZD1), initiates WNT signal transduction. To study the influence of FZD1 genetic variation on measures of bone health, we first sequenced a 6.8-kb region surrounding FZD1 in 48 samples of African ancestry. We genotyped all common polymorphisms and performed association analysis with bone phenotypes in a larger sample. Only 3 of 35 SNPs identified were present in >or=5% of the sample and assayed further in 1084 men of African ancestry. Two of these SNPs were in the FZD1 promoter (rs2232157, rs2232158) and were associated with femoral neck areal BMD (p = 0.041 and 0.009, respectively). The minor alleles of these two SNPs were also associated with larger bone size at the radius (p < 0.05 for both), and rs2232158 was associated with greater strength-strain index, an indicator of bone's ability to withstand torsion. Functional experiments were completed to assess the influence of the rs2232158 promoter polymorphism on transcriptional regulation of FZD1. The minor C allele in rs2232158 creates a binding site for the transcription factor Egr1, has higher Egr1 binding affinity, and has greater FZD1 promoter activity in MG63 and SaOS-2 cells, providing a plausible molecular mechanism for the population associations. This study indicates that a cis-regulatory polymorphism in the FZD1 promoter region may have a functional role in determining bone structural geometry.

Identification and association analysis of single nucleotide polymorphisms in the human noggin (NOG) gene and osteoporosis phenotypes.

Noggin, an extracellular bone morphogenic protein (BMP) antagonist, blocks BMP signaling and decreases osteoblastogenesis. The purpose of this study was to identify novel sequence variations in the human noggin gene and to perform association analyses of these variations with phenotypes related to osteoporosis. Novel single nucleotide polymorphisms (SNPs) were identified by resequencing 7 kb of the noggin gene region in 24 randomly selected Afro-Caribbean men without regard to their bone mineral density (BMD) level. We identified 22 SNPs in the 7 kb noggin gene region, only 2 of which were previously described in dbSNP (build 126). There were also 11 unvalidated SNPs from dbSNP that could not be verified in our sequence analysis. Ten of the 22 identified SNPs showed a minor allele frequency greater than 0.05. Seven of these common SNPs were genotyped in 2060 Afro-Caribbean men age 40 and older. None of the 7 SNPs were associated with BMD at the proximal femur or lumbar spine. Our analysis suggests that a common variation in the noggin gene is unlikely to have a major impact on BMD among older men of African ancestry.

High-density association study of 383 candidate genes for volumetric BMD at the femoral neck and lumbar spine among older men.

Genetics is a well-established but poorly understood determinant of BMD. Whereas some genetic variants may influence BMD throughout the body, others may be skeletal site specific. We initially screened for associations between 4608 tagging and potentially functional single nucleotide polymorphisms (SNPs) in 383 candidate genes and femoral neck and lumbar spine volumetric BMD (vBMD) measured from QCT scans among 862 community-dwelling white men >or=65 yr of age in the Osteoporotic Fractures in Men Study (MrOS). The most promising SNP associations (p < 0.01) were validated by genotyping an additional 1156 white men from MrOS. This analysis identified 8 SNPs in 6 genes (APC, DMP1, FGFR2, FLT1, HOXA, and PTN) that were associated with femoral neck vBMD and 13 SNPs in 7 genes (APC, BMPR1B, FOXC2, HOXA, IGFBP2, NFATC1, and SOST) that were associated with lumbar spine vBMD in both genotyping samples (p < 0.05). Although most associations were specific to one skeletal site, SNPs in the APC and HOXA gene regions were associated with both femoral neck and lumbar spine BMD. This analysis identifies several novel and robust genetic associations for volumetric BMD, and these findings in combination with other data suggest the presence of genetic loci for volumetric BMD that are at least to some extent skeletal-site specific.

Peroxisome proliferator-activated receptor-gamma polymorphism, body mass and prostate cancer risk: evidence for gene-environment interaction.

BACKGROUND: Peroxisome proliferator-activated receptor (PPAR)-gamma has been implicated in prostate cancer. In the present case-control study, we tested if a common Pro12Ala polymorphism in PPAR-gamma is associated with the risk of prostate cancer. METHODS: Ninety-one adult Caucasians with prostate cancer were recruited between 1994 and 1998. Blood samples were also obtained from 237 community-based Caucasian men without prostate cancer. RESULTS: Twenty-six percent of cases and 19% of controls carried at least one Ala allele (p = 0.16). There was a significant interaction between the PPAR-gamma polymorphism and body mass index (BMI) in age-adjusted analyses (p < 0.05). Among the subgroup of men with BMI above 27.2 kg/m2 (median in controls), carriers of the Ala allele had over 2-fold greater risk of prostate cancer compared to those with the Pro12Pro genotype (odds ratio, OR: 2.77; 95% confidence interval, CI: 1.25-6.16). No association was observed between the PPAR-gamma genotype and prostate cancer among men with BMI below the median (OR: 0.68; 95% CI: 0.23-1.97). CONCLUSIONS: Our results suggest a novel gene-environment interaction between the PPAR-gamma Pro12Ala polymorphism and body mass in prostate cancer. Further research, particularly prospective studies, is needed to confirm these findings and to clarify the underlying mechanisms involved.

Genetic epidemiology of osteoporosis: past, present, and future.

Research during the past several decades has unequivocally established a role of heredity in the etiology of osteoporosis. Major efforts are currently underway to identify the genes and allelic variants that confer genetic susceptibility to this common and disabling condition. Genome-wide linkage mapping in families, candidate gene association studies in unrelated individuals, and quantitative trait locus mapping in animal models are the primary strategies being used to search for the genetic contributors to osteoporosis. Genome-wide mapping efforts have identified the low-density lipoprotein receptor-related protein 5, bone morphogenetic protein 2, and 15-lipoxygenase as potential susceptibility genes for osteoporosis in the past few years, providing a rich new base for understanding bone biology. Candidate gene association analyses have also provided evidence for a modest role of allelic variants in several additional genes including collagen type Ialpha1, vitamin D receptor, and estrogen receptor-alpha. With the development of a high-density genome-wide polymorphism and haplotype map and continued improvements in high-throughput and cost-effective genotyping technologies, many more genetic contributors to osteoporosis will probably be identified in the near future. The results of this research should facilitate the development of new methods for diagnosing, preventing, and treating the growing clinical and public health problem of osteoporosis.

Variation in the FABP2 promoter alters transcriptional activity and is associated with body composition and plasma lipid levels.

The fatty acid-binding proteins (FABPs) are cytoplasmic proteins involved in intracellular fatty acid transport and metabolism. FABP2, the intestinal-type FABP, is expressed exclusively in enterocytes in the small intestine. In previous studies of an Ala54Thr substitution in FABP2, the Thr-allele showed association with increased lipid oxidation, elevated plasma lipids, and impaired insulin sensitivity. We screened roughly 1 kb 5' of the FABP2 initiation codon and identified three insertion/deletion polymorphisms and four single nucleotide polymorphisms (SNPs). Three of the SNPs were in complete linkage disequilibrium with the three insertion/deletion polymorphisms, defining exactly two haplotypes (FABP2p-ID). We tested the hypothesis that this variation alters gene expression by transfecting Caco-2 cells with pGL3-Basic constructs containing opposite FABP2p-ID haplotypes. Luciferase assays showed a statistically significant two-fold increase in gene expression of the pGL3-insertion construct over the pGL3-deletion construct (P<0.001; n=5). We also tested for association between three FABP2 variants and measurements of body composition, plasma lipids, and insulin sensitivity in non-diabetic control subjects from the San Luis Valley Diabetes Study (n=714). The only informative variant, FABP2p-ID, was statistically significantly associated with body mass index (P=0.042) and marginally associated with fat mass (P=0.084), cholesterol (P=0.066), and HOMA IR (a derived measure of insulin resistance; P=0.062) in the entire cohort. Similar associations were seen only in non-Hispanics when the analysis was stratified by ethnicity. Within the non-Hispanic subgroup, the effects of FABP2p-ID on plasma lipids were sex-specific. These results suggest that genetic variation in the 5' region of FABP2 affects transcriptional activity, presumably leading to alterations in body composition and lipid processing.