In this study, we have tried to address the molecular mechanisms that underlie the associations that have previously been reported between COL1A1
alleles, BMD, and osteoporotic fracture. A meta-analysis of data from 16 published studies including a total of 4,965 individuals showed a significant association between carriage of the COL1A1
“s” allele and low BMD. This was apparent at both the lumbar spine and femoral neck with evidence of an allele-dose effect. The “s” allele was also associated with reduced BMI, but the association between genotype and BMD was still highly significant in a subgroup of five studies in which BMD data had been adjusted for BMI and other confounding factors. This indicates that the association between COL1A1
alleles and BMD is direct and not simply the result of reduced BMI. Publication bias also needs to be considered as a possible explanation for the findings observed. Although it is difficult to absolutely exclude this possibility, funnel plots of BMD values were symmetrical and, as such, did not suggest the presence of publication bias (20
Osteoporotic fractures were also strongly associated with the COL1A1
“s” allele in the meta-analysis, and the magnitude of risk was too great to be accounted for by the genotype-specific differences in BMD and BMI that we observed. These data support previous work which has shown COL1A1
genotype predicts osteoporotic fractures after correcting for important confounding factors such BMD, BMI, and age (6
) and raises the possibility that COL1A1
alleles act as a marker for reduced bone quality. Other variables such as ethnic background and sex may also influence fracture risk, but neither of these can readily be invoked as an explanation for the findings observed. Ethnic differences were excluded by the fact that all studies included in the meta-analysis were conducted in white Caucasian subjects and sex differences were excluded by the fact that only one study included men and, in this study, the male cases were matched with male controls.
Studies of DNA-binding, collagen gene regulation, and the material properties of bone showed clear evidence of functional differences between COL1A1 Sp1 alleles. Gel shift assays showed that the COL1A1 “s” allele had increased affinity for Sp1 protein binding in vitro when compared with the “S” allele, and this was accompanied by increased abundance of “s”-derived transcripts in unspliced RNA extracted from bone samples in individuals who were heterozygous for the polymorphism. These observations suggest either that allele-specific transcription is increased in relation to the osteoporosis-associated “s” allele or that transcripts from the “s” allele are spliced or processed differently within the nucleus. Further experiments will be required to determine which of these mechanisms is responsible for the findings observed.
Analysis of collagen protein production by metabolic labeling showed an altered production of the collagen α1(I) chain relative to the α2(I) chain in osteoblasts derived from “Ss” heterozygotes. The ratio of collagen α1(I) to α2(I) was 2.3 to 1 in “Ss” heterozygotes compared with the expected value of 2 to 1 that was observed in “SS” homozygotes. These differences in ratio of α1(I) to α2(I) protein chains were accompanied by relative overexpression of the COL1A1 mRNA relative to the COL1A2 mRNA in osteoblasts cultured in vitro derived from a subgroup of individuals in which samples of both RNA and protein were available for analysis.
Since only intact trimeric collagen molecules are analyzed by the PAGE technique used in this study, the above observation is consistent with the hypothesis that some of the collagen produced by “Ss” heterozygous individuals is composed of [α1(I)3
], although we were unable to formally demonstrate the presence of [α1(I)3
], owing to the limited amounts of patient material available (31
). Increased ratios of α1(I) relative to α2(I) collagen consistent with [α1(I)3
] production have previously been found by other workers in fetal tissues and material derived from tumors and chronic fibrotic conditions (32
). The abnormal ratio of collagen α1(I) relative to α2(I) reported here is potentially relevant for the pathogenesis of osteoporotic fractures in the light of previous studies which have shown [α1(I)3
] is associated with impaired mechanical strength of bone. The mechanism by which [α1(I)3
] impairs bone strength is unclear, but cases of severe osteogenesis imperfecta have been reported in association with inactivating mutations in the COL1A2
gene, which results in production of type I collagen that comprises solely [α1(I)3
). A similar phenotype has been observed in the oim/oim
mouse, which has a null mutation of the COL1A2
gene and type I collagen composed entirely of [α1(I)3
). Mice that are heterozygous for the oim
mutation have a milder increase in bone fragility, however (33
); and here, the bone collagen comprises a mixture of [α1(I)3
] and normal collagen α1(I)2
α2(I). Although we have been unable to demonstrate the presence of [α1(I)3
] in bone derived from patients who carry the “s” allele, the biomechanical data support the hypothesis that individuals who carry the “s” allele have reduced bone strength independent of differences in BMD. Analysis of bone cores from four separate sites in the femoral head of patients with the “Ss” genotype showed evidence of a modest reduction in yield strength when compared with similar cores derived from “SS” heterozygotes, after correcting for differences in the density of the cores and site specific differences in bone strength. Although we cannot absolutely exclude the possibility that these differences were due to differences in bone trabecular structure, this seems unlikely given the consistency of the observations over four separate sites. We also observed slight differences between genotypes in composition of bone such that “Ss”-derived cores had a reduced inorganic content (mainly reflecting mineral content) and an increased organic content when compared with “SS”-derived cores. Although this may reflect subtle abnormalities of mineralization in the “Ss” genotype, further work with more sensitive techniques such as backscatter electron imaging (35
) will be required to confirm this observation.
In summary, the evidence presented here indicates that the COL1A1 Sp1 binding site polymorphism has functional effects on collagen gene regulation that leads to abnormal production of the α1(I) collagen chain relative to α2(I) and reduced bone strength by mechanisms that are partly independent of bone mass. Our studies therefore provide evidence to suggest that the Sp1 polymorphism is a functional genetic variant that predisposes to osteoporotic fractures by mechanisms that involve a reduction in bone quality and quantity.