Contribution of a Common Variant in the Promoter of the 1-α-Hydroxylase Gene (CYP27B1) to Fracture Risk in the Elderly
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- Clifton-Bligh, R.J., Nguyen, T.V., Au, A. et al. Calcif Tissue Int (2011) 88: 109. doi:10.1007/s00223-010-9434-4
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CYP27B1 encodes mitochondrial 1α-hydroxylase, which converts 25-hydroxyvitamin D to its active 1,25-dihydroxylated metabolite. We tested the hypothesis that common variants in the CYP27B1 promoter are associated with fracture risk. The study was designed as a population-based genetic association study, which involved 153 men and 596 women aged 65–101 years, who had been followed for 2.2 years (range 0.1–5.5) between 1999 and 2006. During the follow-up period, the incidence of fragility fractures was ascertained. Bone ultrasound attenuation (BUA) was measured in all individuals, as were serum 25-hydroxyvitamin D and PTH concentrations; 86% subjects had vitamin D insufficiency. Genotypes were determined for the –1260C>A (rs10877012) and +2838T>C (rs4646536) CYP27B1 polymorphisms. A reporter gene assay was used to assess functional expression of the –1260C>A CYP27B1 variants. The association between genotypes and fracture risk was analyzed by Cox’s proportional hazards model. We found that genotypic distribution of CYP27B1 –1260 and CYP27B1 +2838 polymorphisms was consistent with the Hardy-Weinberg equilibrium law. The two polymorphisms were in high linkage disequilibrium, with D′ = 0.96 and r2 = 0.94. Each C allele of the CYP27B1 –1260 polymorphism was associated with increased risk of fracture (hazard ratio = 1.34, 95% CI 1.03–1.73), after adjustment for age, sex, number of falls, and BUA. In transient transfection studies, a reporter gene downstream of the –1260(A)-containing promoter was more highly expressed than that containing the C allele. These data suggest that a common but functional variation within the CYP27B1 promoter gene is associated with fracture risk in the elderly.
Several epidemiologic studies have consistently demonstrated that fracture tends to cluster within families but the segregation does not follow a Mendelian pattern . Women whose mother had a hip fracture are at increased risk of hip fracture compared to those whose mothers did not sustain a fracture . Approximately 25–35% of the variance of fracture liability is attributable to genetic factors [3, 4]. Genetic factors also account for a large proportion of variance in risk factors of fracture such as bone mineral density (BMD) , bone loss , quantitative ultrasound measurement , and bone turnover markers .
Specific genes involved in the regulation of bone mass and fracture susceptibility have been identified on the basis of candidate gene-association studies, such as collagen 1α1  and lipoprotein receptor–related protein 5 (LRP5) . More recently, with the advance of genomewide studies, a number of single-nucleotide polymorphisms (SNPs) have been associated with fracture and bone density [11–14]. Such studies have shown an association between osteoporosis and SNPs within genes encoding osteoprotegerin, LRP5, estrogen receptor 1, and receptor activator of nuclear factor-κB ligand [11–14]. One genomewide study provided some evidence for an association between vitamin D receptor (VDR) variants and BMD , which was first identified by Morrison and colleagues [15, 16] but has been controversial since .
One limitation, however, of current genomewide studies has been incomplete coverage of genetic variation, such that the replicated SNP associations explain only a small proportion of the variance in BMD and fracture . This suggests the existence of other sequence variants that are associated with osteoporosis, and there is still a role therefore in studying key candidate genes within physiologic pathways known to influence skeletal health. For instance, vitamin D is undoubtedly critical for optimal skeletal mineralization , and its active product is generated by 1α-hydroxylation of 25-hydroxyvitamin D . Coding region mutations in the CYP27B1 gene encoding 1α-hydroxylase cause type I vitamin D–dependent rickets [21, 22]. Moreover in two large, separate studies a SNP in the CYP27B1 promoter (rs10877012) was recently associated with multiple sclerosis  and type 1 diabetes ; the same allele was responsible for the disease association in each case and was also associated with other autoimmune disorders in smaller studies [25–29]. Since vitamin D deficiency is increasingly common in the elderly and a risk factor for fracture in postmenopausal women , we hypothesized that CYP27B1 is associated with fracture risk in the elderly. The present study was therefore designed to test the hypothesis by examining the association between CYP27B1 gene polymorphisms and fracture risk in a cohort of frail elderly subjects, a majority of whom were vitamin D–insufficient.
Study Design and Methods
The Fracture Risk Epidemiology in the Frail Elderly (FREE) Study is an ongoing prospective study of predictors of falls and fractures in a cohort of men and women living in residential care facilities in Sydney . Ethical approval was given by the local institutional Human Research Ethics Committee. Not all of the 2,005 participants in the FREE study were able to give consent for venesection, but all participants who had blood collected at the baseline are reported here. Of these, 760 of 915 subjects had DNA available for genotyping.
Fracture was ascertained through chart review during regular visits to institutions (i.e. once every 6–12 weeks) for 2 years from baseline and validated by review of a radiology report. The diagnosis of clinical vertebral fractures required subjects to have radiologic evidence of vertebral fracture in association with acute back pain at a corresponding level, but a previous normal X-ray at that vertebral level was not required. For the purpose of these analyses, the following fractures were included: hip, clinical vertebral, pelvis, wrist, humeral, rib, and femoral shaft. Fractures of the skull, ankle, finger, and hand were excluded from the analysis. Falls were ascertained from incident reports and medical records (examined every 6–12 weeks).
Clinical biochemistry and calcaneal ultrasound measurements have been previously described . Briefly, morning blood samples were collected at baseline assessment. Serum 25-hydroxyvitamin D was measured by RIA (DiaSorin, Stillwater, MN). Serum PTH was measured by a two-site chemiluminescent ELISA on an Immulite 1000 analyzer (Diagnostic Products, Los Angeles, CA). Bone ultrasound attenuation (BUA) was measured at the calcaneus in all participants using a MCue CUBA Mark II ultrasound device .
We studied the SNP rs10877012, which lies 1.2 kb upstream of the transcription start site of CYP27B1 (–1260C>A) and was strongly associated with either type 1 diabetes or multiple sclerosis in two large genotyping studies [23, 24]. This SNP is predicted to cause a functional change using the bioinformatics tool F-SNP, which makes predictions of functionality of variants located within putative transcription factor binding sites (TFBSs) . Moreover, other SNPs within or up to 10 kb upstream of the CYP27B1 gene locus (HapMap) are either in near-complete linkage disequilibrium with rs10877012 (D′ 1.0 and r2 > 0.98), unlikely to be functional by F-SNP (intronic rs4646537, MAF 0.034; synonymous exon rs8176345, MAF 0.049), or rare (MAF < 0.01). We had also previously genotyped this cohort for another intragenic CYP27B1 SNP rs4646536 (+2838T>C, in intron 6), which some , but not all [25, 27, 28], studies had suggested was in near-perfect linkage disequilibrium with rs10877012.
Genomic DNA was extracted from peripheral blood leucocytes using standard procedures. Genomic DNA was amplified by PCR (with an annealing temperature of 60°C in all cases), and genotyping was conducted by restriction fragment length polymorphism. The CYP27B1 –1260 SNP was amplified using primers forward 5′-GGC ACA GAG CCT GAT AGA GTT G-3′ and reverse 5′-GGC AAC ATA GTC GAA CTG TCT C-3′ and detected with the HinfI enzyme. The CYP27B1 +2838 SNP was amplified using reverse primer 5′-TAG GTT GCA AAG CAC AAA ATG GAG TCA-3′ and a mismatched forward primer 5′-GGA AGC AGG GAG ATA GCA GAG -3′ that introduces a HincII restriction site in the presence of the T allele. PCR products were digested for at least 2 hours at 37°C and electrophoresed on agarose gels. All genotypes were scored by two researchers independently (R. J. C.-B., A. A.). Repeat genotyping was conducted in 158 samples (21% of cohort) with 100% concordance as a quality control.
Reporter Gene Studies
A reporter construct containing the proximal 1.3 kb of the human CYP27B1 promoter upstream of the luciferase gene was a kind gift from Prof. Howard Morris (Hanson Institute, Adelaide, Australia) . Sequencing of this construct revealed the C allele at –1260. Standard site-directed mutagenesis was performed to create another reporter containing the –1260ACYP27B1 allele (Quik-ChangeTM; Stratagene, La Jolla, CA), and mutagenesis was confirmed by direct sequencing. Human embryonic kidney HEK293 cells in 24-well plates were transiently transfected using DMRIE-C (Invitrogen Life Technologies, Carlsbad, CA) with 10, 50, or 100 ng of either CYP27B1 reporter gene (–1260A or –1260C) or empty reporter vector (pGL3-basic, 100 ng). A renilla luciferase reporter (Promega, Madison, WI), 10 ng/well, was used to control for transfection efficiency. Cells were lysed after 48-hour incubation and then assayed for firefly and renilla luciferase activities (Promega). The transcriptional response was normalized for renilla activity, then expressed as a fold change relative to data from cells transfected with the empty pGL3-basic reporter. Data represent mean ± SD of three experiments, each performed in triplicate wells.
The association between the polymorphisms and fracture was assessed by the Cox proportional hazards model , in which the time to fracture was considered outcome, whereas age, weight, BUA, and biochemical markers were covariates. The metric of association was expressed in terms of hazard ratio (HR) per allele for each polymorphism. The hormonal and mineral parameters considered in the model were serum 25-hydroxyvitamin D, PTH, and calcium. Because the distribution of these parameters was skewed, a logarithmic transformation was applied to “normalize” the data and stabilize their variance prior to the modeling process. In order to estimate the proportion of fracture liability that may be hypothetically attributable to the polymorphism, the population-attributable risk fraction (PARF) was calculated according to the “sequential attributable fraction” . Analyses were conducted with the R statistical language (www.r-project.org).
Baseline characteristics of participants stratified by gender and fracture status
31.0 (21, 44.3)
26.0 (17, 42.3)
49.6 (33, 72.5)
42.0 (29, 67.1)
Corrected Ca (mmol/l)
Fall rate (falls/year)*
0.68 (0, 2.65)
2.52 (1, 6.21)
24.0 (17, 36)
27.5 (20, 39.3)
58.0 (39, 90.6)
63.7 (41.2, 95)
Corrected Ca (mmol/l)
Fall rate (falls/year)*
0.59 (0, 2.49)
1.53 (0.84, 3.72)
Baseline characteristics of participants stratified by CYP27B1 –1260C>A genotype
26 (17, 37)
27 (18, 41)
25 (17, 35)
56.6 (34, 98.7)
55.0 (38, 81.3)
60.0 (39, 90.9)
Corrected Ca (mmol/l)
Fall rate (falls/year)*
0.73 (0, 2.50)
0.82 (0, 2.96)
0.95 (0, 2.82)
Frequency distribution of genotypes in fracture and nonfracture groups
Proportion of fracture (%)
Rate of fracture per 100 person-years
Independent predictors of fracture risk
CYP27B1 –1260 allelea
Age (per 5 years)
CYP27B1 +2838 allelea
Age (per 5 years)
Vitamin D is important for skeletal health across all ages. During infancy and childhood, vitamin D deficiency leads to rickets and osteomalacia. In adults, and particularly in the elderly, vitamin D deficiency is a key risk factor for osteoporotic fractures, and the mechanisms are complex and may not be solely translated via the known skeletal effects of vitamin D . The present study represents the first demonstration of an association between SNPs in the CYP27B1 gene and fracture risk in frail elderly subjects. The strongest association was with a SNP in the CYP27B1 promoter (–1260C>A); association with a second, intronic SNP (+2838T>C) could be explained by tight linkage disequilibrium between the two SNPs, consistent with the study by Bailey et al. . Our functional data that the –1260C allele leads to lower gene expression is consistent with another recent study that found lower CYP27B1 mRNA expression in peripheral blood mononuclear cells from type 1 diabetic patients homozygous for the –1260C allele . Moreover, the –1260C allele changes a consensus CDX2 transcription factor binding site within the CYP27B1 promoter , and a CDX2-binding site polymorphism within the VDR promoter [37, 38] has recently been significantly associated with vertebral fracture in a large multicenter study . The functional consequence of the CYP27B1 –1260C allele (Fig. 2) is similar in magnitude to that reported for the VDR CDX2 polymorphism in vitro . Taken together, we propose that the CYP27B1 –1260C SNP is associated with skeletal fragility via reduced expression of 1α-hydroxylase, in particular since the majority of our subjects were vitamin D–insufficient.
Further arguments in favor of a functional effect of this SNP include reports that the same CYP27B1 –1260C allele is associated with type 1 diabetes [24–26] and other endocrine autoimmune disorders [27–29]. In the largest study, CYP27B1 –1260C was associated with an increased risk of type 1 diabetes in 7,854 cases compared with 8,758 controls (OR = 1.07), and in 2,774 affected families (RR = 1.11), with the combined association at P = 3.8 × 10−6 . There is strong evidence that vitamin D deficiency is associated with type 1 diabetes . The association of the CYP27B1 –1260C variant with diabetes has been proposed to be due to lower systemic availability of calcitriol or alternatively to reduced CYP27B1 expression in immune regulatory cells .
The association between the CYP27B1 –1260C allele and fracture in our study might also therefore be mediated via reduced CYP27B1 expression within the kidney leading to reduced circulating calcitriol concentrations. Arguing against this possibility, however, is that serum PTH concentrations were not significantly higher in subjects with the –1260C SNP as might have been expected by virtue of the strong negative feedback relationship between calcitriol and PTH. Other factors, such as age and renal impairment, may have obscured this association in our cohort, but multivariable regression models did not support this conclusion (data not shown).
Three alternate hypotheses might explain the association between CYP27B1 polymorphisms and fracture. Firstly, CYP27B1 is expressed in osteoblasts and has been proposed to mediate osteoblast differentiation . Alteration of this osteoblast CYP27B1 expression might therefore affect skeletal integrity without measurable alteration in circulating calcitriol levels. However, we did not find an association between the –1260 SNP and BUA. Secondly, there is evidence that vitamin D deficiency is associated with the risk of falling . However, we did not find an association between the –1260C CYP27B1 allele and fall risk in our cohort. A third possibility is suggested by the fact that CYP27B1 is expressed in the intestine , where CDX2 is primarily expressed and known to regulate VDR expression . Since the –1260C allele changes a CDX2 binding site within the CYP27B1 promoter, it is conceivable that altered intestinal CYP27B1 expression might affect intestinal calcium absorption, possibly in combination with the VDR promoter CDX2 polymorphism. These hypotheses require further study.
The present findings should be interpreted within the context of strengths and potential weaknesses. This cohort represents an ideal group for the study of genetic associations with fracture since the incidence of fractures in our sample is high, making it possible to identify associations with a smaller number of participants. We note that our study adds to the few genetic markers associated with fracture including the COLIA1 Sp1 polymorphism  and LRP5 polymorphisms . Nevertheless, this is a relatively small sample size, and we would certainly argue that our findings must be replicated in other, larger cohorts. Moreover, the CYP27B1 variant did not affect BUA; therefore, this gene may only be important for fractures in the vitamin D–insufficient frail elderly, and further studies are required to determine its importance in osteoporosis in general.
In conclusion, these data suggest that the 1α-hydroxylase gene CYP27B1 is an important novel candidate contributor to osteoporosis. Given the facts that this enzyme is a critical regulator of vitamin D biosynthesis and that mutations within the coding region of CYP27B1 cause vitamin D–dependent rickets type I [21, 22], the present findings have a strong biologic basis. Since the 1α-hydroxylase gene also represents a therapeutic target, the finding reinforces the concept that this is a candidate for further study, in particular to establish whether the polymorphism might predict clinical response to vitamin D therapy [36, 46].
We are grateful to Prof. Howard Morris (Hanson Institute, Adelaide, Australia) for his generous gift of the CYP27B1 promoter-luciferase construct, and to Assoc. Prof. Emma Duncan (Diamantina Institute, University of Queensland, Woolloongaba, Australia) for her helpful review of the manuscript.
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