In this study, we performed both SNP-based and haplotype-based analyses to assess the association of the DBP gene with CSI in a large sample of nuclear families using FBAT software. The rationale for performing this study was based on the known physiological role of DBP in bone biology [39
] and the relatively high heritability of CSI.
To the best of our knowledge, this is the first study to test for associations between DBP
and CSI. The significant associations with CSI were found to be male specific in our sample. The most promising associations between haplotypes and CSI were detected in block 2 in the male subgroup. Importantly, associations between DBP
and FN BMD and FNW and weight were not significant. Consequently, it is evident that important genetic factors influencing osteoporosis may be missed if BMD or FNW or weight is the only phenotypic trait analyzed for the genetic dissection of osteoporosis. In addition, SNP 3 (rs222029
), which produced the most significant association signal in males and is contained in block 2, lies in a transcription factor binding site revealed by a web-based functional analysis and selection tool for SNP (http://fastsnp.ibms.sinica.edu.tw/pages/input_SNPListAnalysis.jsp
). Therefore, the variation of SNP 3 (rs222029
), or functional mutations in strong LD with it, may influence DBP
gene expression by affecting the binding capacity of transcription factors. Further molecular studies are required to validate this hypothesis. Finally, these sex-specific association results were not unexpected given previous studies demonstrating that DBP
polymorphisms affect plasma levels of DBP, bone density, and fracture risk in males [19
] and that FN BMD correlated with serum DBP levels in elderly Caucasian men [42
]. Besides BMD, another major contributory factor to fracture risk is muscle strength [43
]. There is evidence showing that muscle strength significantly correlated with 25 (OH) vitamin D only in males [44
]. Therefore, the importance of DBP as vitamin D transport molecule on fractures might be more obvious in males.
A bone fracture occurs when stress within the bone exceeds its ultimate strength. The stress effect depends on the geometry, mechanical properties, and spatial distribution of the bone, as well as the direction, magnitude, and position of the force applied [45
]. Based on the structural engineering principles [13
], CSI integrates density and geometric information embedded in DXA scans with mechanical loading effects of total body weight on the skeletal system. Previous studies show that FN BMD, FN geometric parameters, and weight are highly correlated with each other [46
]. They may share common genetic factors and molecular pathways contributing to their variation in human populations. Only one phenotype alone may not be able to predict hip fractures well. This is similar to the situation of metabolic syndrome (Mets), which was defined by multiple concurrent diseases including abdominal obesity, insulin resistance, dyslipidemia, and elevated blood pressure [49
]. To investigate the genetic factors that may have a common contribution to a clustering of hip fracture-related phenotypes and maximize the chance to identify hip fracture-related genetic factors, it is helpful to use an alternative measurement that characterizes the common features of these highly correlated phenotypes that are significant to fractures. Thus, this newly developed CSI phenotype combining FN BMD with FNW and weight was used to find the shared genetic information among its correlated components.
The FBAT software used in our association study is an extension of the transmission disequilibrium test and can deal with incomplete nuclear families [37
]. The statistical power of our study was estimated by an online Genetic Power Calculator (http://pngu.mgh.harvard.edu/~purcell/gpc/qtlassoc.htm
) under a relatively conservative significance level of P
=0.001. Assuming that a marker is in strong LD (|D
′| = 0.9) with a functional mutation that accounts for 4% variation in phenotype, our study design can reach >90% power in both females and males, under additive models. The 90% power was estimated under a rather ideal situation. We note that the power under various conditions may be affected by multiple other factors, such as LD between the markers tested and the functional allele and the phenotypic variation the functional allele may account for. When assuming a marker is in a weaker LD (|D
′| = 0.75) with a functional mutation that accounts for 1% variation in phenotype, the power reduces to 43% and 72% in male and female, respectively.
Several other issues need to be addressed here. First, the FNW (the average diameter of the FN) was obtained by dividing the bone area by the width of the region of interest, usually 1.5 cm. A previous study [27
] has assessed the reliability of this approximation of bone geometry, with the conclusion that FN diameter estimated from bone area had a single measured CV of 10.6% compared with 9.6% (n
=30) obtained by direct measurement with the DXA ruler tool. Second, CSI of the FN studied here was derived from DXA measurement, which is not sensitive to the mineralization quality of bones. Although deterioration in the quality of mineralization with age may contribute to an increased susceptibility to fractures in the elderly, age alone explains only a small part of the total variance in material strength of cadaveric cortical femoral bone [50
]. Therefore, the CSI phenotype based on structural measures can provide good assessment of hip fracture risk [13
]. Third, due to the shape of the fan beam X-ray in our QDR 4500 scanner, an inherent magnification of scanned bone area at FN were generated and hence caused the projected area of FN to decrease linearly with distance above the X-ray source. This change was estimated as 1.6% cm−1
by simulating FN as round solid aluminum rod in Cole's study [51
]. However, there is no good method to correct the magnification error for an individual measured by this scanner because the soft tissue thickness that could be used to correct this error is not available [52
]. Thus, hardware and software modifications for the Hologic fan beam DXA machines would be necessary to allow scanning at different table heights to calculate soft tissue thickness [52
] and thus overcome the limitation.
In summary, our findings suggest the importance of DBP gene polymorphisms on the variation in CSI in Caucasians, especially in men. It is necessary to perform further statistical genetic and functional studies to replicate and confirm these results in other populations in order to assess the generality of the findings.