Codon 325 sequence polymorphism of the estrogen receptor α gene and bone mineral density in postmenopausal women
Introduction
Osteoporosis is a multifactorial disease, characterized by low bone mass and increased fracture risk [1]. Various environmental factors such as dietary calcium, physical activity, smoking habits and alcohol intake together with body weight and menopausal status, influence the bone mass [2]. However, most authors agree that here genetic factors are major determinants [3], [4], [5], [6], [7], [8], [9]. A great deal is known about the factors that regulate bone turnover and the proteins that compose bone matrix. This gives us a wide range of potential, candidate genes which may be involved in the development of osteoporosis. Estrogen receptor α (ERα) gene is one of them because estrogens regulate bone turnover and skeletal growth.
ERα is a nuclear regulatory protein that mediates the actions of estrogens in target cells. Binding of an estrogen causes conformational changes of the receptor protein and increases its affinity for DNA resulting in transcription activation or repression of a target gene [10]. However, in spite of the wide variety of estrogen actions in different tissues, relatively few genes are known which are responsive to the ERα.
The gene coding for ERα is located on chromosome 6q25.1. It extends over more than 140 kb and includes eight exons with sizes of 684, 191, 117, 336, 139, 134, 184 and 4537 bp, respectively (6322 bp in total); 232 nucleotides on the 5′-end and 4305 nucleotides on the 3′-end of the mRNA are not translated [11].
Until now three polymorphisms of the ERα gene have been investigated in connection with bone mineral density (BMD) (15,10,8). PvuII (T → C) and XbaI (A → G) polymorphisms have been found in intron 1 while BstUI polymorphism (silent mutation in codon 87: GCG → GCC; Ala → Ala) has been localized in exon 1. The study, where the relationship between PvuII and XbaI restriction fragment length polymorphisms (RFLP) and BMD has been analyzed in 238 Japanese subjects, showed that some genotypes are associated with low BMD [12]. A group of Korean researchers has carried out a similar research, in which also BstUI RFLP was included [13]. In this studies no significant relationship between ERα genotypes and Z-scores of lumbar spine BMD was found. Some of the research groups have performed studies where correlation of both ERα and vitamin D receptor gene polymorphisms with BMD have been studied [14], [15], [16], [17]. Their results have shown correlation of particular genotypes to low BMD. Furthermore, the newest results of Sowers et al. [18] again confirmed the contribution of sex hormone genes to variations of BMD.
According to the contradictory results of these studies we decided to look for the possibility of a new, yet unknown, polymorphism(s) of the ERα gene which could be associated with BMD. The whole coding region of the ERα gene in a group of 53 postmenopausal women was analyzed using PCR, single-stranded conformation analysis (SSCP), and direct sequencing of DNA fragments.
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Patients
Eighty-five consecutive late postmenopausal female patients (in menopause >6 years), aged 55–74 years (mean 65.5 years), who were referred to the Outpatient Department of Endocrinology at the University Clinical Centre in Ljubljana for measurement of bone mineral density, were evaluated. Each patient was examined clinically and routine biochemical tests were performed to exclude systemic and metabolic bone disease. None had previously taken any drug known to influence bone metabolism.
The study
Results
SSCP analysis showed mobility shift of the samples only in exons 1 and 4 (Fig. 1). Direct DNA sequence analysis of those samples revealed two silent mutations, in codon 87 (GCG → GCC; A87A) and 325 (CCC → CCG; P325P), respectively.
In codon 87 only four heterozygous and no homozygous GCC subjects were found.
In codon 325 42 subjects were homozygous for the normal CCC codon (genotype CC), eight subjects were homozygous for the mutated codon CCG̱ (genotype GG) and 35 subjects were heterozygous CCC̱
Discussion
In the whole coding region of the ERα gene two silent mutations in codon 87 and 325, respectively, were found. The silent mutation in codon 85 of exon 1 (GCG → GCC; A87A) has been described previously, as BstUI polymorphism. The silent mutation in codon 325 (CCC → CCG; P325P), located in exon 4, has not been analyzed so far in correlation with BMD. According to the distribution of genotypes CC:CG:GG=49.4:41.2:9.4, we can affirm the existence of genetic polymorphism in codon 325 in our population of
Acknowledgements
This work was supported by grant J1-8762-0381 provided by the Ministry of Science and Technology of Slovenia. The authors are grateful to L. Strmecki, PhD and K. Vouk, MSc for technical assistance.
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