A common variant in fibroblast growth factor binding protein 1 (FGFBP1) is associated with bone mineral density and influences gene expression in vitro

Abstract

We previously detected strong evidence for linkage of forearm bone mineral density (BMD) to chromosome 4p (lod=4.3) in a set of 29 large Mexican American families. Fibroblast growth factor binding protein 1 (FGFBP1) is a strong candidate gene for bone homeostasis in this region. We sequenced the coding region of FGFBP1 in a subset of our Mexican American study population and performed association studies with BMD on SNPs genotyped in the entire cohort. We then attempted to replicate these findings in an independent study cohort and performed in vitro functional studies on replicated, potentially functional polymorphisms using a luciferase reporter construct to evaluate influence on gene expression. Several SNPs spanning the gene, all in one large block of linkage disequilibrium, were significantly associated with BMD at various skeletal sites (n=872, p=0.001-0.04). The associations were then replicated in an independent population of European ancestry (n=972; p=0.02-0.04). Sex-stratified association analyses in both study populations suggest this association is much stronger in men. Subsequent luciferase reporter gene assays revealed marked differences in FGFBP1 expression among the three common haplotypes. Further experiments revealed that a promoter polymorphism, rs12503796, results in decreased expression of FGFBP1 and inhibits upregulation of the gene by testosterone in vitro. Collectively, these findings suggest that sequence variation in FGFBP1 may contribute to variation in BMD, possibly influencing osteoporosis risk.

Fig.1

Polymorphisms identified in Mexican Americans and the Amish. Exons are numbered below the gene schematic; black regions correspond to UTR and white regions correspond to coding sequence. Direction of transcription is indicated by arrow. Abbreviations: SNP, single nucleotide polymorphism; N/A, not genotyped due to multiplex restrictions; UTR, untranslated region *Positions given are cDNA coordinates.

Fig. 2

Linkage disequilibrium (D' top, r² bottom) pattern for all SNPs genotyped in Mexican Americans (a) and the Amish (b). For both D' and r², values range from 0 (no LD) to 100 (complete LD). Schematic of the FGFBP1 gene is shown above to indicate approximate locations of each SNP. Arrow indicates direction of transcription; white box indicates coding region. Value in each box indicates D' or r² value between 2 SNPs (intersection). For D' figures, no value indicates complete LD (100). Rs2531174 and rs2072313 were nonpolymorphic in the Amish and are therefore not shown in 2b

Fig. 3

P-values reflecting association of FGFBP1 SNPs with BMD in Mexican Americans and Amish. P-values indicated correspond to a recessive genetic model. Covariates include age, age squared, sex, BMI (and in Mexican Americans diabetes status and physical activity levels. P-values not shown are not significant (p > 0.05)

Fig. 4

Mean BMD (± standard error) at the trochanter according to rs16892645 genotype in Mexican Americans and the Amish. BMD is adjusted for age, sex, BMI, diabetes status, and metabolic equivalents in Mexican Americans, and for age, sex, and BMI in the Amish. P-values shown are for recessive genetic mode. N=572 (GG), 245 (GA), and 32 (AA) for Mexican Americans and N=848 (GG), 110 (GA), and 7 (AA)

Fig. 5

A. Diagram of native haplotype expression vector constructs. Differences from HapA are shown in red. B. Luciferase activity values (adjusted for Renilla luciferase activity and normalized to HapA) for native haplotypes (n = 11 wells). Bars represent standard deviation. All data is normalized to Renilla luciferase activity and adjusted relative to HapA expression (1). *p = 6.2 × 10⁻⁶ for HapB vs HapA, **p = 0.017 for HapC vs HapA. C. A polymorphism in the promoter of FGFBP1 inhibits up-regulation of luciferase reporter gene expression (adjusted for Renilla luciferase activity) by testosterone. Bars represent standard error (n = 6 wells). All data is normalized to Renilla luciferase activity and adjusted relative to HapA expression without testosterone (1). *p = 0.00087; with versus without testosterone, **p = 0.0098; with versus without testosterone.