Elsevier

Bone

Volume 32, Issue 3, March 2003, Pages 297-310
Bone

Original article
Polymorphisms in the transforming growth factor beta 1 gene and osteoporosis

https://doi.org/10.1016/S8756-3282(02)00971-7Get rights and content

Abstract

Transforming growth factor (TGF)-β1 is the most abundant growth factor in human bone. It is produced by osteoblasts and inhibits osteoclast proliferation and activity and stimulates proliferation and differentiation of preosteoblasts. Several polymorphisms have been described in the TGF-β1 gene. Previously, we and others have found associations between some of these polymorphisms and bone mass. We therefore wanted to examine if these polymorphisms are also predictors of osteoporotic fractures. The polymorphisms G−1639-A, C−1348-T, C−765insC, T29-C, G74-C, 713-8delC, C788-T, and T816-20-C were examined using RFLP and sequencing in 296 osteoporotic patients with vertebral fractures and 330 normal individuals. Bone mineral density (BMD) was examined at the lumbar spine and at the femoral neck by DXA. Genotype distributions were in H-W equilibrium. Linkage disequilibrium was found between the polymorphisms. The T816-20-C genotypes were distributed differently among osteoporotic patients and normal controls. The TT genotype was less common in individuals with osteoporotic fractures (χ2 = 6.02, P < 0.05). BMD was higher in individuals with the TT-genotype (T816-20-C) at the lumbar spine, 0.960 ± 0.173 g/cm2 compared with individuals with the TC or CC genotypes: 0.849 ± 0.181 g/cm2 and 0.876 ± 0.179 g/cm2, respectively (P < 0.001, ANOVA). Similar differences between genotypes were found at the different hip regions as well as at the total hip. Individuals with the TT-genotype (C−1348-T) had higher bone mass at the femoral neck: 0.743 ± 0.134 g/cm2 compared with 0.703 ± 0.119 g/cm2 in individuals with TC or CC genotypes (P < 0.05). Individuals with the CC-genotype (T29-C) had higher bone mass at the femoral neck, 0.735 ± 0.128 g/cm2 compared with 0.703 ± 0.120 g/cm2 in individuals with TC or TT genotypes (P < 0.05) and at the total hip: 0.852 ± 0.166 g/cm2 vs. 0.818 ± 0.149 g/cm2, respectively (P < 0.05). None of the other polymorphisms were distributed differently in patients and controls and did not affect BMD. In conclusion, The TT genotype of the T816-20-C polymorphism is less common in patients with osteoporotic fractures and is associated with higher bone mass both at the lumbar spine and at the hip. The C−1348-T and T29-C polymorphisms were distributed similarly in osteoporotic patients and normal controls, however, the rare genotypes were associated with higher bone mass at the hip.

Introduction

Osteoporosis is a common disease affecting one in three women and one in 10 men above the age of 50 years. Osteoporosis is characterized by a combination of low bone mass and deteriorated microarchitecture of the bone. Twin and family studies have revealed that genetic factors are responsible for 50%–85% of the interindividual variation in bone mass [1], [2], [3]. However, the effect is most likely a combined effect of several genes, each with a modest effect [4]. The genes involved can be unraveled by two different approaches, linkage studies in families or candidate gene searches in case-control studies. If there are many genes involved in the pathogenesis of osteoporosis, they may be difficult to detect in linkage studies, and the candidate gene approach will be more powerful [5].

Transforming growth factor β1 (TGF-β1) is by far the most abundant of the three isoforms of TGF β, both in serum and in bone. TGF-β1 is produced by osteoblasts as an inactive propeptide and is incorporated into newly formed bone matrix. During bone resorption, the propeptide is activated by the acid milieu in the resorptive zone under the osteoclast. TGF-β1 inhibits further activity of the osteoclasts and stimulates proliferation and differentiation of preosteoblasts. The overall effect on bone remodeling of TGF-β1 is therefore favoring bone formation [6], [7]. Because of this important role in bone turnover, the TGF-β1 gene is a candidate for mediating genetic influence on bone mass and risk of fracture. We previously performed an examination of the coding parts of the TGF-β1 gene (exon 2–7) and found two polymorphisms. Both were rare, but 713-8delC was more common among osteoporotic patients [8]. This association has recently been confirmed in an Italian population [9]. TGF-β1 has also been implicated in the pathogenesis of other diseases with a complex genetic component such as cancer [10], [11], [12], autoimmunine disease [13], and atherosclerosis [14], [15]. Cambien et al. therefore examined the TGF-β1 gene for polymorphisms and mutations in 20 patients with recent myocardial infarction and found six polymorphisms in the promoter and the first exon [16]. Of the polymorphisms located in the promoter of the gene, only G−1638-A is in a known consensus site. A change from G to A in a CREB half-site would be expected to reduce affinity for the CREB family of transcription factors which has been implicated in regulating transcription of other members of the TGF-β gene family [17]. Both G−1638-A and C−1348-T lie several bases away from consensus DR1 or DR5 nuclear hormone receptor binding sites [18] which is of interest because ligands for nuclear hormone receptors have been shown to regulate TGF-β production in vitro and in vivo [19], [20]. The two polymorphisms in the first exon; G29-C (codon 10) and G74-C (codon 25), are located in the signal peptide sequence that is cleaved from the TGF-β1 precursor at the level of codon 29. Signal peptides allow export of newly formed proteins across the endoplasmic reticulum and consist of a positively charged N-terminal region, a central hydrophobic core, and a polar C-terminal region [21]. The 10th amino acid is located in the hydrophobic core. Both leucine and proline are apolar, and the polymorphism would therefore probably not affect the function of the signal peptide. A change from the big polar amino acid arginine to the small apolar proline in position 25, which is in the hydrophophic core, could potentially affect intracellular trafficking or export efficiency of the preproprotein [22], [23].

Grainger et al. have demonstrated in a twin study that genetic factors are responsible for 54% of the variation in circulating levels of TGF-β1 and that the C−1348-T polymorphism was significantly associated with serum levels of TGF-β1 [24]. The CC-genotype of the T29-C polymorphism has been found to be associated with increased bone mass and reduced risk of vertebral fractures [25], [26]. The G74-C polymorphism was demonstrated to be associated with in vitro production of TGF-β1 [27], myocardial infarction, and blood pressure [16]. A T861-20-C polymorphism in the fifth intron, 20 bases upstream of exon 6, has been found to be associated with reduced bone mass at the hip, but not at the lumbar spine [28].

Recently, Janssens et al. have demonstrated that Camurati–Engelmann disease is caused by mutations in the TGF-β1 gene [29]. Camurati–Engelmann disease or progressive diaphyseal dysplasia is a rare, sclerosing bone dysplasia inherited as an autosomal dominant trait. The mutations are three missense mutations: C652-T (Arg218Cys), T673-C (Cys225Arg), and T241-C(Tyr81His) and a triple insertion of three leucines in the signal peptide 35insGCTGCTGCT.

We therefore wanted to extend our previous examination of exon 2 to 7 of the TGF-β1 gene, by examining the promoter and the first exon for polymorphisms, and by investigating the effect of polymorphisms in this part of the gene alone and in combination with polymorphisms in other parts of the gene on prevalence of osteoporotic fractures and bone mass in men and women.

Section snippets

Study subjects

The study was a case control study. The osteoporotic group consisted of 238 women and 58 men with primary spinal osteoporosis defined by the presence of at least one nontraumatic fracture of the spine, referred to the Department of Endocrinology, Aarhus University Hospital. The diagnosis of primary osteoporosis was made after extensive examination for secondary causes. Spinal fracture was defined as a 20% or more reduction of the anterior, central, or posterior height of a vertebra. The normal

Results

We found five polymorphisms in the in the promoter and first exon (Fig. 1). All have previously been reported [16]. Three were located in the promoter, C− 1639-A, C−1348-T, and C−765insC. G29-C and G74-C are located in the first exon and cause changes in amino acid from leucine to proline and from arginine to proline, respectively. These five polymorphisms, as well as 713-8delC, C788-T, and T861-20-C, were all in Hardy-Weinberg equilibrium (χ2 = 0.00–0.32, ns). Linkage disequilibrium was

Discussion

The aim of this study was to identify genetic variants in the TGF-β1 gene and investigate their role in the pathogenesis of osteoporotic fractures. Genetic variants can either be functional themselves or be in linkage disequilibrium with the causal mutation. When combining the results of our previous and present studies, we have examined 50 osteoporotic patients and 50 normal controls, corresponding to 200 alleles, for polymorphisms in the promoter and exons with surrounding introns, giving a

Acknowledgements

The authors thank The Novo Nordisk Fonden, The Institute of Experimental Clinical Research at University of Aarhus, The Fonden til Lægevidenskabens Fremme, and The Danish Centre for Molecular Gerontology for financial support.

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