Wnt Pathway Gene Expression Is Associated With Arterial Stiffness.

Background Animal and in vitro experiments implicate the Wnt pathway in cardiac development, fibrosis, vascular calcification, and atherosclerosis, but research in humans is lacking. We examined peripheral blood Wnt pathway gene expression and arterial stiffness in 369 healthy African ancestry men (mean age, 64 years). Methods and Results Gene expression was assessed using a custom Nanostring nCounter gene expression panel (N=43 genes) and normalized to housekeeping genes and background signal. Arterial stiffness was assessed via brachial-ankle pulse-wave velocity. Fourteen Wnt genes showed detectable expression and were tested individually as predictors of pulse-wave velocity using linear regression, adjusting for age, height, weight, blood pressure, medication use, resting heart rate, current smoking, alcohol intake, and sedentary lifestyle. Adenomatous polyposis coli regulator of Wnt signaling pathway (APC), glycogen synthase kinase 3ß (GSK3B), and transcription factor 4 (TCF4) were significantly associated with arterial stiffness (P<0.05 for all). When entered into a single model, APC and TCF4 expression remained independently associated with arterial stiffness (P=0.04 and 0.003, respectively), and each explained ≈3% of the variance in pulse-wave velocity. Conclusions The current study establishes a novel association between in vivo expression of the Wnt pathway genes, APC and TCF4, with arterial stiffness in African ancestry men, a population at high risk of hypertensive vascular disease.

Association of Lipopolysaccharide-Binding Protein With Aging-Related Adiposity Change and Prediabetes Among African Ancestry Men.

OBJECTIVE: Cross-sectional studies suggest that lipopolysaccharide-binding protein (LBP) may be associated with obesity and metabolic disorders. However, prospective studies examining LBP are lacking. This prospective study investigated the association between LBP and metabolic abnormalities in 580 African ancestry men (mean age, 59.1 ± 10.5 years). RESEARCH DESIGN AND METHODS: We measured fasting serum LBP at baseline. Changes in adiposity and glucose homeostasis as well as case subjects with new type 2 diabetes and impaired fasting glucose (IFG) were assessed at a follow-up visit ~6 years later. Baseline LBP values were tested across quartiles for linear trend with metabolic measures. Multivariable logistic regression was used to determine the odds of new cases of IFG or diabetes per 1-SD greater baseline LBP. RESULTS: LBP was significantly associated with baseline BMI, waist circumference, whole-body and trunk fat, skeletal muscle density, fasting serum insulin, and HOMA-insulin resistance (IR) (all P < 0.01). Greater baseline LBP was significantly associated with longitudinal increases in the percentage of trunk fat (P = 0.025) and HOMA-IR (P = 0.034), but only borderline so with a decrease in skeletal muscle density (P = 0.057). In men with normal glucose, baseline LBP was associated with increased odds of having IFG at follow-up after adjustment for age, baseline trunk fat, and lifestyle factors (odds ratio per 1-SD LBP: 1.51; 95% CI 1.02-2.21). This association was attenuated after additional adjustment for change in trunk fat (P = 0.067). CONCLUSIONS: LBP may be a marker of prediabetes. Some of this association appears to be mediated through increased central and ectopic skeletal muscle adiposity.

Heritability and genetics of serum dickkopf 1 levels in African ancestry families.

The aim of the study was to determine the heritability of serum dickkopf-1 (DKK1) and its association with DKK1 polymorphisms in African ancestry subjects. Serum DKK1 was measured in 422 Afro-Caribbean men and women aged 18+ from 7 large, multi-generational families (mean family size: 60; 3,215 relative pairs). Twenty-four common single nucleotide polymorphisms (SNPs) were genotyped within an 80 kilobase-pair region encompassing the DKK1 gene. Heritability was estimated and SNPs were tested for association with serum DKK1 using variance components analysis. DKK1 mRNA expression was tested in peripheral blood of 16 individuals from each of the rs7069912 genotypes. Mean serum DKK1 was 1724.1 pg/mL and was significantly lower in women than men (P = 0.043). Residual genetic heritability of serum DKK1 was 0.4460 (P < 0.0001). Six SNPs reached nominal significance with DKK1, with rs7069912 being significant after adjustment for multiple comparisons. Two of these six SNPs represented independent association signals (rs7069912 and rs16928725), which accounted for 4.6% of the phenotypic variation in DKK1. Additionally, carriers of the rs7069912 variant had significantly greater DKK1 expression than non-carriers (P = 0.036). Serum DKK1 levels are highly heritable in the African ancestry families. Two SNPs within the DKK1 region accounted for nearly 5% of the variation in serum DKK1.

Association of circulating sclerostin with vascular calcification in Afro-Caribbean men.

OBJECTIVE: Sclerostin, a Wingless (Wnt) pathway antagonist, is an established regulator of bone mineralization in humans but its potential importance in the regulation of vascular calcification is less clear. Therefore, our objective was to assess the relationship of serum sclerostin levels with coronary and aortic artery calcification (CAC and AAC, respectively) in Afro-Caribbean men on the island of Tobago. METHODS: Serum sclerostin levels and computed tomography of CAC and AAC were measured in 191 men (age mean(SD): 62.9(8.0)years) recruited without regard to health status. Multivariable logistic regression models were used to assess the cross-sectional association of sclerostin with prevalent arterial calcification. RESULTS: Mean(SD) sclerostin was 45.2 pmol/L (15.6 pmol/L). After adjusting for risk factors including age, physical and lifestyle characteristics, comorbidities, lipoproteins and kidney function, 1 SD greater sclerostin level was associated with a 1.61-times (95%CI 1.02-2.53) greater odds of having CAC. Sclerostin was not associated with AAC in any model. CONCLUSIONS: This is the first study to show that, among Afro-Caribbean men, greater serum sclerostin concentrations were associated with prevalence and extent of CAC. Further studies are needed to better define the role of the Wnt signaling pathway in arterial calcification in humans.

Functional and association analysis of frizzled 1 (FZD1) promoter haplotypes with femoral neck geometry.

Frizzleds are receptors for Wnt signaling and are involved in skeletal morphogenesis. Little is known about the transcriptional regulation of frizzleds in bone cells. In the current study, we determined if two common and potentially functional genetic variants (rs2232157, rs2232158) in the frizzled-1 (FZD1) promoter region and their haplotypes influence FZD1 promoter activity in human osteoblast-like cells. We also determined if these variants are associated with femoral neck bone mineral density (BMD) and geometry in 1319 African ancestry men aged > or =40 years. Real-time quantitative PCR and western blot analysis demonstrated FZD1 mRNA and protein expression in the human osteoblast-like cell lines, MG63 and SaOS-2. Promoter activity was next assessed by transient expression of haplotype specific FZD1 promoter reporter plasmids in these cells. In comparison to the common GG haplotype, promoter activity was 3-fold higher for the TC haplotype in both cell lines (p<0.05). We previously demonstrated that rs2232158 is associated with differential FZD1 promoter activity and Egr1 binding and thus focused further functional analyses on the rs2232157 G-to-T polymorphism. Electrophoretic mobility shift assay demonstrated that distinct nuclear protein complexes were associated with rs2232157 in an allele specific manner. Bioinformatics analysis predicted that the G to T transversion creates an E2F1 binding site, further supporting the functional significance of rs2232157 in FZD1 promoter regulation. Individual SNPs and haplotypes were not associated with femoral neck BMD. The TC haplotype was associated with larger subperiosteal width and greater CSMI (p<0.05). These results suggest that FZD1 expression is regulated in a haplotype-dependent manner in osteoblasts and that these same haplotypes may be associated with biomechanical indices of bone strength.

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.

Molecular variation in neuropeptide Y and bone mineral density among men of African ancestry.

Neuropeptide Y (NPY) is a physiological candidate gene for the regulation of body weight and has more recently been implicated in regulating bone mass. The current study sought to test if inherited variation in NPY might influence BMD in a population of African-ancestry men who have high bone mineral density (BMD). We genotyped 17 tagging single-nucleotide polymorphisms (SNPs) across the NPY gene region in 1,113 randomly selected men of African ancestry aged >or=40 years and tested for association with anthropometric characteristics and proximal femur BMD. The homozygous rare genotype of four SNPs was associated with a 0.92-1.59% decrease in stature (corrected P < 0.05). No SNP was associated with body mass index or body weight. Two SNPs in a 5-kb linkage disequilibrium block encompassing exons 3 and 4 were associated with proximal femur BMD, adjusted for age, body weight, and height (corrected P < 0.05). These results suggest that genetic variation at the NPY locus may contribute to bone density, independently of body weight.

Association of the CPT1B gene with skeletal muscle fat infiltration in Afro-Caribbean men.

Skeletal muscle fat is greater in African ancestry individuals compared with whites, is associated with diabetes, and is a heritable polygenic trait. However, specific genetic factors contributing to skeletal muscle fat in humans remain to be defined. Muscle carnitine palmitoyltransferase-1B (CPT1B) is a key enzyme in the regulation of skeletal muscle mitochondrial beta-oxidation of long-chain fatty acids, and as such is a reasonable biological candidate gene for skeletal muscle fat accumulation. Therefore, we examined the association of three nonsynonymous coding variants in CPT1B (G531L, I66V, and S427C; a fourth, A320G, could not be genotyped) and quantitative computed tomography measured tibia skeletal muscle composition and BMI among 1,774 Afro-Caribbean men aged > or =40, participants of the population-based Tobago Health Study. For all variants, no significant differences were observed for BMI or total adipose tissue. Among individuals who were homozygous for the minor allele at G531L or I66V, intermuscular adipose tissue (IMAT) was 87% (P = 0.03) and 54% lower (P = 0.03), respectively. In contrast, subcutaneous adipose tissue (SAT) was 11% (P = 0.017) and 7% (P = 0.049) higher, respectively, than among individuals without these genotypes. These associations were independent of age, body size, and muscle area. Finally, no individuals with type 2 diabetes were found among those who were homozygous for the minor allele of either at G531L and I66V whereas 14-18% of men with the major alleles had type 2 diabetes (P = 0.03 and 0.007, respectively). Our results suggest a novel association between common nonsynonymous coding variants in CPT1B and ectopic skeletal muscle fat among middle-aged and older African ancestry men.

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.

Association of a high mobility group gene (HMGA2) variant with bone mineral density.

High mobility group (HMG) proteins regulate chromatin architecture and gene expression. Constitutional rearrangement of an HMG family member, HMGA2, in an 8-year old boy resulted in extreme overgrowth and advanced bone development. Moreover, a recent genome-wide association study documented an association between a variant in the 3' untranslated region of HMGA2 (rs1042725) and height in otherwise healthy individuals. We attempted to extend these findings by testing if this HMGA2 polymorphism is associated with other skeletal measures in two large population cohorts of diverse race/ethnicity. Genotyping was completed in 1680 Afro-Caribbean men aged > or = 40 years and 1548 Caucasian American men aged > or = 69 years. Bone mineral density (BMD) was assessed with peripheral quantitative computed tomography. The minor allele frequency of rs1042725 was 32% among Afro-Caribbeans and 48% among Caucasians (p<0.0001). No association was observed with height in either study cohort. However, presence of the minor allele of this SNP was associated with decreased tibia trabecular volumetric BMD in both populations (p=0.007 Afro-Caribbean; p=0.0007 Caucasian). Real-time quantitative RT-PCR and Western blot analysis demonstrated HMGA2 mRNA and protein expression in the human fetal osteoblast cell line, hFOB. Our analyses suggest a novel association between a common genetic variant in HMGA2 and trabecular BMD in ethnically diverse older men. Additional research is needed to better understand the role of HMGA2 in the regulation of bone metabolism.

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.

Single nucleotide polymorphisms in the coding region of the apolipoprotein H (beta2-glycoprotein I) gene and their correlation with the protein polymorphism, anti-beta2glycoprotein I antibodies and cardiolipin binding: description of novel haplotypes and their evolution.

Apolipoprotein H (APOH), also known as beta2-glycoprotein I, is a major autoantigen for the production of antiphospholipid antibodies (APA) in autoimmune diseases. APA is also recognized by a cryptic epitope generated following the interaction of APOH with anionic phospholipids (PL). The prevalence of APA in the general U.S. white population is about 10%, but it ranges from 30-70% in patients with lupus and antiphospholipid syndrome. Since the structural characterization of APOH from different mammalian species is important to identify the evolutionary conserved regions that may be critical for its function, we have previously determined the chimpanzee APOH gene structure and the prevalence of APA. There are only two amino acid differences between the chimpanzee and human wild type APOH proteins. Chimpanzees have an unusually high prevalence (64%) of APA. There is a common protein polymorphism in the human APOH gene, with the occurrence of four alleles APOH*1, APOH*2, APOH*3 and APOH*4, the latter being present only in blacks. Based on its differential reactivity with an APOH monoclonal antibody, the APOH*3 allele is further divided into APOH*3(W) (present only in whites) and APOH*3(B) (present only in blacks). In this study we have screened a large African population (n = 755) to determine the prevalence of APA and the molecular basis of the protein polymorphism. Almost 50% of the Africans were found to be positive for APA. The APOH*3(B) allele was found to be identical to the chimpanzee's wild type APOH. Novel two-site or three-site haplotypes, encoded in the third domain of APOH, explained the molecular basis of the APOH*3(B), APOH*3(W) and APOH*4 alleles. Based on the comparison of the human and chimpanzee APOH DNA sequences, we suggest that the APOH*3(W) and APOH*4 alleles arose on the ancestral APOH*3(B) haplotype after the split of human races. We also found that these haplotypes are associated with the occurrence of APA. Recombinant APOH haplotypes, expressed in COS-1 cells, showed that these mutations also affect the binding of APOH to anionic PL.