Vitamin D receptor, oestrogen receptor-{alpha} and calcium-sensing receptor genotypes, bone mineral density and biochemical markers in Paget's disease of bone

J. Donáth, G. Speer1, G. Poór, P. Gergely, Jr, A. Tabák1 and P. Lakatos1

National Institute of Rheumatology and Physiotherapy and 1 Semmelweis University, Faculty of Medicine, 1st Department of Medicine, Budapest, Hungary.

Correspondence to: J. Donáth, National Institute of Rheumatology and Physiotherapy, 1023 Budapest, Frankel Leó u. 38–40, Hungary. E-mail: orfireuma{at}axelero.hu


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives. The significance of genetic polymorphisms in the development of Paget's disease of bone is unclear at present.

Methods. We analysed the BsmI polymorphism of the vitamin D receptor (VDR) gene, the PvuII and XbaI polymorphisms of the oestrogen receptor-{alpha} (ER{alpha}) gene, and the A986S polymorphism of the calcium-sensing receptor (CaSR) gene in 69 pagetic patients and 120 healthy subjects. We also examined the relationship of these polymorphisms with lumbar spine and femoral neck BMD as well as with biochemical parameters (serum alkaline phosphatase, osteocalcin and parathyroid hormone) in Paget's disease.

Results. The XbaI and PvuII genotype distributions of the ER{alpha} gene were significantly different between patients with Paget's disease and control subjects (P<0.001). Also, the CaSR A986S genotype frequency was significantly different between pagetic patients and controls (P<0.01). No significant effect of gene polymorphisms on BMD or biochemical parameters of bone turnover was observed.

Conclusion. Our results suggest that the ER{alpha} PvuII/XbaI and CaSR A986S polymorphisms may contribute to genetic susceptibility to Paget's disease. However, further studies are required to investigate the underlying pathomechanism and to replicate the associations.

KEY WORDS: Paget's disease, Genetic polymorphism, Oestrogen receptor gene, Vitamin D receptor gene, Calcium-sensing receptor gene.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Paget's disease is characterized by focal areas of increased bone turnover at specific sites throughout the skeleton. Although the biochemical and molecular basis of this process is unknown, alterations in certain laboratory parameters, such as levels of osteocalcin (OC), serum alkaline phosphatase (ALP) and serum parathyroid hormone (PTH), can be used as markers of aberrant bone metabolism in this disease. The disease is complex and both its aetiology and pathogenesis remain unclear. However, recent evidence suggests a significant genetic contribution to the abnormal osteogenesis and development of the disease [1]. Paget's disease has a familial tendency, although few genetic associations have been described [2]. The genetic background seems to involve a variety of genes. Little is known about the exact role of genetic factors in Paget's disease.

The vitamin D receptor (VDR) gene has long been thought to play an important role in calcium and bone metabolism, because vitamin D is required for normal bone mineralization, absorption of calcium from the gut, the control of calcium and phosphate homeostasis and the regulation of parathyroid hormone secretion. Furthermore, genetic studies have also described polymorphic regions, such as BsmI polymorphism at the VDR gene, and found associations between osteoporosis and other diseases of bone and cartilage metabolism with certain alleles of the gene [35]. BMD is also, at least in part, influenced by genetic factors which, according to epidemiological data, may contribute as much as 70–80% to the variability of BMD [6].

The oestrogen receptor-{alpha} (ER{alpha}) gene has also been reported to contribute to pathological conditions of calcium and bone metabolism. The XbaI and PvuII restriction enzyme polymorphisms of the ER{alpha} gene are associated with the pathogenesis of different diseases, such as osteoporosis and osteoarthritis [7, 8]. Certain allelic combinations of the ER{alpha} and VDR genes have been shown to influence BMD more markedly than ER{alpha} or VDR alone [9].

The A986S polymorphism of the calcium-sensing receptor (CaSR) gene has been shown to affect serum calcium homeostasis [10]. Data suggest that CaSR also plays a role in the pathogenesis of primary hyperparathyroidism [11]. Furthermore, an association has been reported between this polymorphism and BMD in healthy subjects [12], although, a recent study could not corroborate this finding in Hungarian postmenopausal women [13].

At present, no data are available on the possible roles of these polymorphisms in Paget's disease of bone. In this study, we investigated the potential relationship between BMD, the characteristic laboratory parameters, such as serum OC, PTH and ALP concentrations, and polymorphisms of these candidate genes in Paget's disease of bone.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Subjects
From the Outpatient Clinic of the National Institute of Rheumatology and Physiotherapy, 69 patients with Paget's disease of bone [34 women, 35 men; mean age 70.0 ± 10.2 (S.D.) yr (range 46–90 yr] were examined. The diagnosis of Paget's disease was established in all patients by clinical, biochemical and radiological criteria. There was no difference in smoking habits, calcium intake, alcohol and caffeine consumption and physical activity among subjects within the pagetic groups. For the genetic studies, 120 sex-matched healthy individuals served as controls. All patients and controls were of Caucasian origin. The study was approved by the local ethics committee (TUKEB, Semmelweis University) and all patients gave written informed consent. The clinical features of our patients are shown in Table 1.


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TABLE 1. Clinical features of the pagetic patients

 
Biochemical markers
Serum ALP levels were measured by colorimetry (Hitachi 912; Roche, Basel, Switzerland), OC and PTH levels were assessed by an electrochemiluminescent immunoassay (Elecsys 2010; Roche).

Bone densitometry
BMD of patients and controls was measured by dual-energy X-ray absorptiometry (DEXA) using a Lunar DPX-L (Lunar Corp, Madison, WI, USA) in posterior–anterior position at the lumbar spine and proximal femur. BMD was expressed as areal density in grams per square centimetre.

Genotyping
Genomic DNA was isolated from peripheral blood. DNA was extracted by Wizzard Genomic DNA Separation Kit (Promega, Madison, WI, USA). The BsmI polymorphic site of the VDR gene and the PvuII and XbaI polymorphic regions of the ER{alpha} gene were amplified by the polymerase chain reaction (PCR).

For the VDR gene, the following primers were used: primer A, 5' AAC CAG CGG GAA GAG GTC AAG GG 3' 23-mer; primer B, 5' CAA CCA AGA CTA CAA GTA CCG CGT CAG TGA 3' 30-mer (10 µmol/l final concentration). PCR was carried out in 50 µl of final volume using the following materials: 5 µl 10x (Mg-free) PCR buffer, 1 µl dNTP (10 mmol/l), 5 µl MgCl2 (25 mmol/l), 10 µl of purified DNA, 1-1 µl primers A and B (10 µmol/l), 0.4 µl Taq (Promega) and 26.6 µl 2D PCR water. The following programme was used: 95°C for 3 min, 35x (72°C for 90 s, 95°C for 45 s) and 72°C for 10 min. The amplified PCR product was digested with the BsmI restriction enzyme (Hybaid-AGS, Teddington, UK; 10 U/µl) for 90 min at 65°C. The BsmI restriction site is missing in the B allele and is present in the b allele.

For the ER{alpha} gene, the following primers were used: primer A, 5' CTG CCA CCC TAT CTG TAT CTT TTC CTA TTC TCC 3' 33-mer; primer B, 5' TCT TTC TCT GCC ACC CTG GCG TCG ATT ATC TGA 3' 33-mer (10 µmol/l final concentration). PCR conditions are the same as for VDR. The PCR product was subsequently digested with the XbaI and PvuII restriction enzymes (Promega; 10 U/µl) at 37°C overnight. The lack of XbaI/PvuII restriction sites corresponds to the X/P alleles, and the sites are present in the x/p alleles. Electrophoretic separation was carried out in a 2% agarose gel containing 10 µg/ml ethidium bromide.

The polymorphic region of the CaSR gene was amplified with an allele-specific PCR. The following primers were used: primer M, 5' ACG GTC ACC TTC TCA CTG ACG TTT GAT GAG CCT CAG AAG TAC T 3' 43-mer; primer W, 5' GCT TTG ATG AGC CTC AGA AGA TCG 3' 24-mer; primer R, 5' CTC TTC AGG GTC CTC CAC CTC T 3' 22-mer (10 µM final concentration). PCR was carried out in 20 µl final volume using the following materials: 2 µl 10x Mg-free reaction buffer, 4 µl dNTP (1 mM), 1.2 µl 25 mM MgCl2, 1 µl DNA (25 ng/ml), 3-2-1 µl (primer R, W, M), 0.1 µl (0.5 U/µl) Taq (Promega) and 5.7 µl 2D PCR water. The PCR programme was as follows: 94°C for 12 min, 35x (94°C for 20 s, 55°C 20 s, 72°C 30 s) and 72°C for 5 min. There are two CaSR alleles, A and S, so the genotypes are AA, AS and SS. Electrophoretic separation was carried out in a 7% Spreadex/acrylamide-bis (29:1) gel (Elchrom, Cham, Switzerland). For all of the PCR we used a Hybaid Express thermocycler (Hybaid, Teddington, UK).

Statistical analysis
Pagetic subjects were divided into subgroups according to their VDR, ER{alpha} or CaSR genotypes. The control group was tested for the Hardy–Weinberg equilibrium for all genotypes. The controls were in Hardy–Weinberg equilibrium unless stated otherwise. Genotype frequencies of patients and controls were compared using standard {chi}2 or Fisher's exact test as appropriate. Analysis of variance (ANOVA) and t-tests were carried out to investigate the relationship between values reflecting BMD or biological parameters (ALP, OC, PTH) and certain genotypes in patients with Paget's disease. Differences were considered significant at P<0.05. All statistical analyses were carried out using the SPSS software, version 7.0 (SPSS 9.0.0, Chicago, IL, USA).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
VDR gene BsmI polymorphism
We found no difference in the distribution of the VDR BsmI genotypes between pagetic and control groups (P = 0.492) (Table 2). Furthermore, no relationship was found between VDR BsmI genotypes and the biochemical parameters (ALP, OC and PTH) or BMD in either group.


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TABLE 2. VDR BsmI, ER{alpha} PvuII and XbaI and CaSR A986S genotypes in patients with Paget's disease (n = 69) and control subjects (n = 120)

 
ER gene PvuII and XbaI polymorphisms
Allele frequencies determined by the ER{alpha} gene PvuII and XbaI polymorphisms in patients with Paget's disease were different from those of the control subjects (P<0.001 for both polymorphisms) (Table 2). The frequency of the pp allele (PvuII) was significantly lower and the xx genotype (XbaI) was absent in patients with Paget's disease. Analysis of the genotypic frequencies revealed no significant correlation between biochemical parameters, BMD data and any of the ER{alpha} genotypes in both study groups.

CaSR A986S gene polymorphism
The CaSR A986S genotype distribution was significantly different between the pagetic and control groups (P<0.01), as indicated by the higher frequency of the SS genotype among patients with Paget's disease (Table 2). No significant effect of the CaSR genotype on BMD was observed either in the pagetic patients or in the control subjects. Moreover, we did not find any association between laboratory parameters and CaSR genotypes.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Paget's disease has a familial tendency, suggesting that there is a genetic predisposition. Data from Morales-Piga et al. [14] support the existence of two distinct groups of Paget's disease of bone cases: familial and sporadic. According to their data, an autosomal dominant mode of inheritance is suggested, with 40% of patients with Paget's disease having at least one other first-degree relative affected with the disease in Spain. Siris et al. [15] found a positive family history in 12.3% of pagetic patients as opposed to only 2.1% among control individuals. The geographical distribution of Paget's disease shows considerable differences, exhibiting high incidences in the US, UK and Spain but low incidences in the rest of Europe [16], including Hungary.

In our study we investigated genetic polymorphisms that might have a role in calcium and bone mineral metabolism and might be associated with Paget's disease. VDR BsmI allele frequencies were similar in pagetic patients and healthy controls. This finding indicates that the VDR BsmI polymorphism does not contribute to the development of Paget's disease in Hungarian patients. However, the sample size was small, so the lack of association may reflect lack of power. Several studies about the relationship between VDR polymorphisms and BMD have been published, but the data provide conflicting results [9, 17]. The differences among reports might be explained by the geographical and ethnic differences that are also characteristic for Paget's disease.

The importance of the ER{alpha} gene in skeletal diseases (such as osteoporosis and rheumatoid arthritis) and in the determination of BMD is supported by several observations [79]. The allele frequencies in our study determined by the PvuII and XbaI polymorphisms of the ER{alpha} gene in patients with Paget's disease were significantly different from those of healthy individuals, suggesting that ER{alpha} gene might contribute to the genetic background of Paget's disease. However, the different alleles did not influence bone mass, serum ALP levels or concentrations of serum PTH directly. The exact role of ER{alpha}, if any, in the development of the disease process remains to be elucidated. Nevertheless, oestrogen appears to play a crucial role in immunological events, including its inhibitory effect on IL-6 secretion [18]. IL-6 has also been suggested to participate in the pathogenic process of Paget's disease [19].

CaSR has a crucial role in the regulation of extracellular calcium that might be of importance at the site of pagetic changes in bone. There are only a limited number of studies available regarding the relationship between the polymorphisms of the CaSR gene and bone metabolism [12, 20]. Cole et al. [10] found increased serum calcium levels in subjects with the AA genotype. Nevertheless, data from Takács et al. [13] do not support the idea that the CaSR gene A986S polymorphism affects bone mass. In our study, the CaSR A986S genotypes of pagetic patients were significantly different from those of controls, without having an effect on bone mass or the biological parameters. The SS genotype was over-represented in our patient population, while it is extremely rare in both healthy Hungarian and Canadian subjects [10, 21]. Our finding suggests that the CaSR gene might be involved in the pathogenesis of Paget's disease, although its effect is unclear at present.

Our study is the first to show an association of ER{alpha} and CaSR gene polymorphisms and Paget's disease of bone. The fact that genotypic differences have no relation to biochemical markers or BMD does not contradict the pathogenic role of these polymorphisms, since these alterations could serve as the genetic basis that initiates processes leading to the disease. Although an association found between certain alleles and a phenotype does not prove that the presence of these alleles has functional implications, further studies in an independent sample might be of value to investigate the role of these polymorphisms in geographical areas where the incidence of Paget's disease is more frequent than in Hungary.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Submitted 15 September 2003; revised version accepted 21 January 2004.



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