Cara S. Nestlerode

Genetic association of five apolipoprotein polymorphisms with serum lipoprotein-lipid levels in African blacks

Genetic studies carried out mainly in European and European‐derived populations have shown that common polymorphisms in genes coding for apolipoproteins are significant determinants of serum lipoprotein‐lipid levels variation. However, except for a few sporadic studies, the distribution of apolipoprotein polymorphisms and their association with serum lipoprotein‐lipid levels have not been evaluated systematically in African or African‐derived populations. In this investigation we have studied five apolipoprotein polymorphisms, including APOA1/MspI –75 bp, APOA1/MspI +83 bp, APOC3/PvuII, APOE, and APOH in 786 Africans (493 men, 293 women) from Nigeria. The sample is comprised of Nigerian civil servants consisting of 462 junior staff (less affluent) and 324 senior staff (more affluent) where staff status is a correlate of their socioeconomic status. We first examined genetic associations in the total sample stratified by gender to determine the role of apolipoprotein polymorphisms in affecting serum lipid profile in the general population, and then by staff status to evaluate possible gene‐environment interactions. In the total sample, the APOC3/PvuII polymorphism showed significant effect on HDL‐cholesterol (P = 0.029) and HDL3‐cholesterol (P = 0.009) in women, and the APOE polymorphism was significantly associated with total cholesterol (P = 0.031) and LDL‐cholesterol (P = 0.0006) in women. Multiple regression analyses showed that the APOC3/PvuII polymorphism accounts for about 2 and 3% of the variation in HDL‐cholesterol and HDL3‐cholesterol, respectively, in women; while the APOE polymorphism accounted for about 5 and 6% of the variation in total‐ and LDL‐cholesterol, respectively, in women. Whereas the association of the APOE polymorphism was independent of the staff status, the significant affect of the APOC3/PvuII polymorphism on HDL‐ and HDL3‐cholesterol was confined to senior staff women where it explained about 7% of their variation. We also observed an interaction between staff and the APOH polymorphism in affecting cholesterol levels. The APOH polymorphism showed significant association with total cholesterol (P = 0.010) and LDL‐cholesterol (P = 0.016) in senior staff women and explained about 7 and 5% of their phenotypic variations, respectively. These data indicate that gene‐environment interaction may play an important role in affecting serum lipid profile in African populations. Genet. Epidemiol. 16:205–222, 1999.

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 ≥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.

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 ∼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) ≥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 ≥ 0.05) variation in the region with r2 > 0.80 and a potentially functional SNP in exon 5 in 1035 Afro‐Caribbean men ≥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 ≤ 0.006), and in 416 individuals ≥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.

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 β2‐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*3W (present only in whites) and APOH*3B (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*3B allele was found to be identical to the chimpanzees wild type APOH. Novel two‐site or three‐site haplotypes, encoded in the third domain of APOH, explained the molecular basis of the APOH*3B, APOH*3W and APOH*4 alleles. Based on the comparison of the human and chimpanzee APOH DNA sequences, we suggest that the APOH*3W and APOH*4 alleles arose on the ancestral APOH*3B 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.

Association analysis of 33 lipoprotein candidate genes in multi-generational families of African ancestry

African ancestry individuals have a more favorable lipoprotein profile than Caucasians, although the mechanisms for these differences remain unclear. We measured fasting serum lipoproteins and genotyped 768 tagging or potentially functional single nucleotide polymorphisms (SNPs) across 33 candidate gene regions in 401 Afro-Caribbeans older than 18 years belonging to 7 multi-generational pedigrees (mean family size 51, range 21–113, 3,426 relative pairs). All lipoproteins were significantly heritable (P < 0.05). Gender-specific analysis showed that heritability for triglycerides was much higher (P < 0.01) in women than in men (women, 0.62 ± 0.18, P < 0.01; men, 0.13 ± 0.17, P > 0.10), but the heritability for LDL cholesterol (LDL-C) was higher (P < 0.05) in men than in women (men, 0.79 ± 0.21, P < 0.01; women, 0.39 ± 0.12, P < 0.01). The top 14 SNPs that passed the false discovery rate threshold in the families were then tested for replication in an independent population-based sample of 1,750 Afro-Caribbean men aged 40+ years. Our results revealed significant associations for three SNPs in two genes (rs5929 and rs6511720 in LDLR and rs7517090 in PCSK9) and LDL-C in both the family study and in the replication study. Our findings suggest that LDLR and PCSK9 variants may contribute to a variation in LDL-C among African ancestry individuals. Future sequencing and functional studies of these loci may advance our understanding of genetic factors contributing to LDL-C in African ancestry populations.

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 β‐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 ≥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.

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.

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.

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.