Despite the high heritability of BMD, efforts to identify individual genes that explain a large proportion of the variation in this trait have been largely unsuccessful. Numerous candidate genes have been proposed, some of which have been associated with BMD in multiple populations [27
], and recent genome-wide association studies have identified a small number of additional loci that appear to be robustly associated with BMD [29
]. However, very few association studies have resulted in the identification of functional variants with demonstrable effects on gene expression. We have performed an association study in two very different populations, a genetically heterogenous population with a high degree of admixture and a genetically homogenous population of European ancestry, and followed up with functional studies of associated haplotypes. The fact that the FGFBP1
associations demonstrated in our study were observed across two populations, taken together with our in vitro
studies showing a dramatic effect on gene expression, make this gene a very convincing candidate for bone homeostasis.
The mechanisms for differences in FGFBP1 expression are not clear; however, we provide evidence that the promoter polymorphism (rs12503796) may be involved. Rs12503796 is located in the promoter approximately 280 bases upstream of the transcription start site. It is not located in a known transcription factor binding site; however, it is a predicted binding site for several transcription factors that may be involved in bone metabolism. In this regard we did, in fact, observe enhanced in vitro luciferase expression when transfected cells were co-cultured with testosterone, although the testosterone-enhancing effect was obliterated in constructs having the rs12503796 T allele, suggesting that this allele may destroy a binding site for the androgen receptor. An attractive interpretation of this hypothesis is that loss of a testosterone-enhancing effect on BMD might be expected to have a greater effect on BMD in men than in women, which is also consistent with our observed association results. However, the “T” allele is predicted to bind the androgen receptor, while the “G” allele is not, so if the prediction is correct, other mechanisms to account for the differences in haplotype expression must be in play. For example, there may be other transcription factors not tested, either alone or through interaction with the androgen receptor, that mediate this effect. In fact, the promoter polymorphism is also a predicted binding site for the general transcription factor TFII-I and the rs12503796 polymorphism might alter TFII-I binding, possibly explaining why basal expression levels of the constructs were different in the absence of hormones. Finally, it is also possible that FGFBP1 expression differences may have nothing to do with the rs12503796 promoter polymorphism because FGFBP1 expression was decreased in HapC, despite the presence of the G allele at this promoter site. It is also possible that this effect is not true as this construct did not have significantly different expression from HapA in the presence of testosterone, which is likely more representative of true physiological conditions in which testosterone is always present at some level.
Despite the parallel associations observed in these data of FGFBP1
SNPs with BMD and of FGFBP1
haplotypes with gene expression, there are some limitations and inconsistencies in our study. First, Mexican Americans are an admixed population and our analysis of FGFBP1
SNPS did not account for population substructure. However, replication in a non-admixed population, the Amish, mitigates this limitation. Second, the expression experiments may not have been performed in the most physiologically relevant tissue. FGFBP1
expression in osteoblasts was measured; expression was detected but at insufficient amounts to carry out in vitro studies (data not shown). However, because FGFBP1 is secreted, it is possible that high expression in tissues other than bone could certainly influence bone mineral density. Third, the allele most strongly associated with higher BMD (e.g. rs16892645 A allele) is not by itself associated with gene expression. Fourth, the T allele at the promoter polymorphism (rs12503796) appears to block the increased expression of this gene in the presence of testosterone. How this potential decrease in activity and/or lack of stimulation leads to increased BMD is unclear since FGFBP1 is thought to enhance FGF-2 binding to its receptors, which should lead to osteoblast stimulation. Therefore, one might expect decreased expression or function of FGFBP1 to lead to decreased BMD. It is possible that in bone, FGFBP1 acts to sequester FGF-2 instead of enhancing binding. Indeed, some data suggests that while FGFBP1 stimulates FGF-2 at low concentrations, it can also inhibit FGF-2 at higher concentrations [7
]. Likewise, FGF-2 has also been found to have dual effects on osteoblasts. FGF-2 induces proliferation and differentiation in immature osteoblasts but has also been found to block mineralization and induce apoptosis in differentiated osteoblasts [13
]. FGF-2 is also thought to influence osteoclast maturation and activity [36
]; hence we would expect in this scenario that decreased expression of FGFBP1 would lead to increased BMD, as we observed, via decreased osteoclast activity. These hypotheses require further testing in order to determine the mechanism of this effect. Additionally, the cell type used in our in vitro experiments may not be representative of the pathway in relevant tissues in vitro
The possibility that the effects of FGFBP1 SNPs may manifest differently between men and women is intriguing, but must be considered speculative. While the intronic rs16892645 variant was more strongly associated in Mexican American men than women, the estimated effect size in men was not statistically greater than that observed in women in a formal sex by SNP interaction analysis. Moreover, we had little power to test for sex differences in the association of this SNP with BMD in the Amish because of the low frequency of the rs16892645 variant in this population. Nevertheless, coupled with the observation that expression was significantly enhanced in the presence of testosterone, the possibility of a sex interaction makes an interesting hypothesis. Future studies of FGFBP1 should consider the possibility of differential effects between men and women.
It seems unlikely that variation in FGFBP1 is responsible for our previously observed linkage peak due to the modest, albeit replicated, associations observed with BMD. Although linkage analysis can detect rare genetic effects, our prior linkage was exclusively with forearm BMD, while the current analyses indicate association across multiple BMD sites, particularly at the hip. It is unclear why our previous linkage study detected little evidence for linkage to hip BMD; likewise, despite modest association of rs2245964 and rs16892645 with forearm BMD, a subsequent (not shown) linkage analysis indicated essentially no change in the evidence for linkage to forearm BMD after accounting for the associated SNPs from this study. Other genes in this region must therefore be considered as candidates for their role in driving the forearm BMD linkage result.
In summary, we have identified several polymorphisms in FGFBP1 that are associated with BMD at several sites in both Mexican Americans and the Old Order Amish. The minor alleles of these polymorphisms tend to be associated with higher BMD in both populations and there is a suggestion that effects may be stronger in males than in females. A promoter polymorphism (rs12503796) was shown to influence gene expression in vitro, possibly due to alteration of a transcription factor binding site as demonstrated by abolishment of response to testosterone in vitro. However, while these data provide evidence that FGFBP1 is involved in bone remodeling, the precise mechanism governing the regulation of gene expression is not clear. This mechanism likely involves an interaction between sequence variation and hormonal or other influences on transcription. Further elucidation of the factors regulating expression of this gene and the downstream effects of altered gene expression of skeletal phenotypes seems warranted.