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The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 12 6173-6178
Copyright © 2004 by The Endocrine Society

A Deletion Polymorphism in the RIZ Gene, a Female Sex Steroid Hormone Receptor Coactivator, Exhibits Decreased Response to Estrogen in Vitro and Associates with Low Bone Mineral Density in Young Swedish Women

E. Grundberg, T. Carling, H. Brändström, S. Huang, E. L. Ribom, Ö. Ljunggren, H. Mallmin and A. Kindmark

Department of Medical Sciences (E.G., H.B., Ö.L., A.K.) and Department of Surgical Sciences, Section of Orthopedics (E.L.R., H.M.), Uppsala University, SE-751 85 Uppsala, Sweden; Department of Surgery, Yale University School of Medicine (T.C.), New Haven, Connecticut 06510; and Program in Oncogenes and Tumor Suppressor Genes, The Burnham Institute (S.H.), La Jolla, California 92037

Address all correspondence and requests for reprints to: Dr. Elin Grundberg, Department of Medical Sciences, Uppsala University Hospital, Entrance 70, Third Floor, Clinical Research Department 2/3, SE-751 85 Uppsala, Sweden. E-mail: elin.grundberg{at}medsci.uu.se.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Low bone mineral density (BMD) is a major risk factor for osteoporotic fracture, and the trait is under genetic control by a large number of genes. It is recognized that estrogen plays an important role in the maintenance of bone mass by binding to estrogen receptor {alpha} (ER{alpha}). RIZ1 has previously been shown to be a specific ER{alpha} coactivator and strongly enhances its function both in vivo and in vitro. We performed in vitro studies comparing the abilities of RIZ1 P704 polymorphic variants (homozygous presence, P704+; absence, P704–; heterozygosity P704+/– of a proline at position 704) to coactivate the ER{alpha} and also examined the polymorphism associated to BMD of 343 Swedish women, aged 20–39 yr. The expression vector containing P704– RIZ1 showed an impaired response in coactivating ER{alpha} in a ligand- and dose-dependent manner compared with P704+ RIZ (P < 0.0001). The genotype frequencies were 19% (P704+), 32% (P704–), and 49% (P704+/–) and were in Hardy-Weinberg equilibrium. BMD at the heel was higher in the P704+ genotype group than in the P704+/– group (P = 0.02), which was evident also after corrections for fat and lean mass (P = 0.03). We conclude that RIZ1 may be a new candidate gene for involvement in the variation seen in BMD.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
OSTEOPOROSIS IS A complex disease, characterized by reduced bone strength and development of fractures, e.g. of spine, hip, and wrist. Twin and family studies have shown a strong genetic component determining bone mineral density (BMD) and bone quality (1, 2, 3, 4, 5) with heritability (H2) values between 0.50 and 0.90 (6, 7, 8). A number of candidate genes have been investigated for association between allelic variations and BMD, but results have varied depending on the genes studied and in study populations around the world.

The genes investigated previously for an association with BMD are those with known effects on bone metabolism, in steroid hormone pathways, and various growth factors and cytokines. Both estrogens and androgens are thought to influence bone turnover during adolescence and in adult life (9) by binding to specific nuclear receptors, the estrogen (ER) and androgen receptors, respectively. The ERs are ligand-dependent transcription factors. Upon binding of hormones, the receptors bind to their cognate DNA response elements on target genes and recruit coactivators and general transcription factors to form an active transcriptional complex, resulting in enhancement of target gene expression (10, 11). Recent studies have revealed a novel class of coactivators that function as histone/protein methyltransferases (HMTs) (12, 13, 14, 15, 16). One of these HMTs is the retinoblastoma-interacting zinc finger protein RIZ1, which has been demonstrated to function as a specific female sex steroid nuclear hormone receptor coactivator and strongly enhances the function of the receptor both in vivo and in vitro (16, 17). However, RIZ1 was initially characterized as a tumor suppressor gene (18, 19). The gene maps to chromosome 1p36, one of the most commonly deleted region in human cancers (18, 20). However, a genome screen in seven large pedigrees suggested that a candidate region conferring susceptibility to low BMD of the femoral neck was located on chromosome 1p36 (21). This was confirmed and extended by fine-mapping the candidate region in an expanded sample of 42 families (22). In studies of a large unselected nonidentical twin pair cohort, Wilson et al. (23), found evidence of linkage of whole body BMD (linkage of difference, Lod score, 2.4) at chromosome 1p36 (17 cM), supporting previous findings of suggestive linkage to BMD in the region (22).

The coding region in the RIZ gene has been screened, showing seven polymorphisms. Four of these are silent nucleotide substitutions that do not affect the amino acid sequence, two are amino acid substitutions, and one is a deletion polymorphism of a proline at codon 704 (P704) in exon 8 in the RIZ gene (24, 25).

In the present study we performed in vitro studies comparing the abilities of RIZ P704 polymorphic variants to coactivate the ER{alpha} to better understand the functional effect of the polymorphism. We also examined the RIZ P704 deletion polymorphism (homozygous presence, P704+; absence, P704–; heterozygosity, P704+/–) in relation to BMD in a cohort of young Swedish women, containing a total of 343 individuals, aged 20–39 yr.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Plasmids and transient transfections

The human RIZ1 expression vector containing the wild-type RIZ1 has been described previously (16). The vector p3RIZRH{Delta}P-3, homozygous for the P740 deletion polymorphism (P704–),was generated by the Quik-Change mutagenesis kit (Stratagene, La Jolla, CA) and was verified by automated DNA sequencing. Expression vectors of RIZ1 proteins (either P704+ or P704–) and an expression vector containing full-length ER{alpha} were cotransfected into CV-1 cells with an estrogen response element-thymidine kinase-chloramphenicol acetyltransferase (CAT) (26) reporter construct containing a synthetic hormone response element linked to the thymidine kinase-CAT reporter.

A calcium phosphate precipitation procedure was used for transient transfections as described previously (16). Briefly, 0.5–1.0 x 105 cells/well were seeded in 24-well plates with charcoal-stripped medium, and 50–200 ng RIZ1 plasmids, 100 ng of the ER{alpha} expression vector, 100 ng reporter plasmids, and 100 ng of a ß-galactosidase expression vector were mixed with carrier DNA to 1 µg total DNA/well. Cells were treated with or without 50 nM estradiol for 24 h, and CAT activity was measured as described previously (26). CAT values were normalized for transfection efficiency by the corresponding ß-galactosidase activity. All transfections were performed at least three times.

Subjects

The study cohort consisted of women randomly selected from the population registry of the county of Uppsala. In all, 685 women were randomly selected and invited by mail to participate in the study. Of the invited women, 342 did not participate and were excluded; of these 22 were pregnant, 35 had moved, 155 did not contact the research center and were not reached despite repeated letters, three were of non-Swedish origin, and 127 were not interested in participating. In total, 343 (50%) of the invited women, aged 20–39 yr, attended the investigation and were included. Of these 343 individuals, 341 were examined by dual energy x-ray absorptiometry (DXA) of the total body, 340 by DXA of the spine (L2–L4), 341 by DXA of the femur (neck and total hip), and 336 by DXA of the heel, yielding a total of 336 women examined by DXA at all sites. The main reasons for women attending not being examined for bone mass were high body weight, instrument failure, and/or computer failure when analyzing the data. Subjects answered a questionnaire regarding medications, menstrual status, oral contraceptive use, and number of children. General exclusion criteria for the association study were non-Swedish origin and drugs and diseases known to affect bone metabolism (27). Thus, we exclude subjects who had received treatment with corticosteroids (n = 2), antiepileptic drugs (n = 3), or levothyroxine (n = 3) and subjects with endometriosis (n = 1), rheumatoid arthritis (n = 2), multiple sclerosis (n = 1), and scoliosis (n = 1). Blood samples for DNA extraction were obtained from 325 individuals, and the samples were stored at –20 C until processing and analysis. A total of 304 individuals were included in the association study of different genotypes and BMD at the total hip and femoral neck, 303 individuals were included in the association study between genotypes and BMD at the lumbar spine (L2–L4), and 298 individuals were included in the association study between genotypes and BMD at the heel. All individuals gave informed consent upon arrival, in accordance with the Helsinki Declaration and the local ethics committee.

DXA measurements

BMD (grams per square centimeter), bone mineral content (grams), total body fat mass (grams), and total body lean mass (grams) were estimated by DXA, using a DPX-IQ scan (Lunar Corp., Madison, WI). BMD was measured at the lumbar spine (vertebrae L2–L4), hip (neck and total), and total body. BMD (grams per square centimeter) measurements of the heel were performed with a LUNAR Pixi scanner (Lunar Corp.). The stability of the DPX-IQ equipment was checked every morning using a phantom. The long-term precision, expressed as the coefficient of variance (percentage), was determined by daily measurements of a spine phantom and was less than 0.5% during the study period.

Genotyping

Genomic DNA was extracted from leukocytes from 3 ml EDTA blood using a Wizard Genomic DNA purification kit (Promega Corp.), typically yielding approximately 50–75 µg genomic DNA/sample.

The P740 deletion polymorphism of RIZ1, localized in exon 8, was amplified by PCR with the fluorescent 6-tetrachlorofluorescein (TET)-labeled forward primer 5'-ACT CCA TGC TGG TGA GTC-3' and the unlabeled reverse 5'-CCC AAG ATA AAC TAA CTC CT-3' primer (Thermo Hybaid, Interactiva Division, Ulm, Germany). The PCRs were run on an ABI-877 Integrated Thermal cycler PCR robot (Applied Biosystems, Foster City, CA) using 0.1 µg genomic DNA, AmpliTaq Gold kits (Applied Biosystems, Foster City, CA), and standard reagents. The amplification procedure for the RIZ1 gene was carried out according to the following protocol: denaturation for 10 min at 95 C, 36 cycles of denaturation for 30 sec at 95 C, a touch-down annealing at 59–57 C for 30 sec, elongation at 72 C for 1 min, and finally an extension step at 72 C for 10 min. The gene fragments were separated by electrophoresis on a 6% polyacrylamide gel using a 96-well ABI 377 automated sequencer (Applied Biosystems). Gel data and allele sizes were analyzed using the software programs GeneScan Analysis 3.1 and Genotyper 2.0 (Applied Biosystems, Warrington, UK). The RIZ1 deletion polymorphism was verified by sequencing.

Statistical analysis

All statistical analyses for the genetic association study were performed using STATISTIKA software (StatSoft, Inc., Tulsa, OK). ANOVA and Fisher’s least significant differences (LSD) post hoc test compared the means of the genotype groups for the differences in the continuous variables. In addition, multiple regression analysis was used to adjust the data for differences in confounding factors and to define the relative contributions of independent variables to the variation of BMD. The genotype information was entered into the model along with fat mass, lean mass, age, and height. The R2 value is the coefficient of determination and explains the model’s relative contribution to the variation of BMD. For categorical variables, such as RIZ1 genotypes, binary dummy variables were constructed for use in the multiple regression analysis. The P704–/– and P704+/– genotype variables were included in the multiple regression model as dummy variables, and the P704+/+ genotype variable was left out as the reference group. Each dummy variable was coded as 1 for observations that fell into that genotype group and as 0 for the two other genotype groups, which means that observations in the reference group (i.e. P704+/+) were coded as 0 for every dummy. The {chi}2 test was used to assess whether discrete variables were independent or associated.

Regarding the transient transfections, an unpaired t test was used for statistical evaluation of means, with P < 0.05 considered significant. The results are expressed as the mean ± SEM.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Abilities of RIZ1 P704 polymorphic variants to coactivate the ER{alpha} in vitro

RIZ1 P704+ has been shown to enhance the activation of ER{alpha} in a ligand- and dose-dependent manner. To analyze any functional differences between the RIZ1 P704+ and RIZ1 P704– variants, an expression vector containing P704– RIZ was created. In a reporter gene system using vectors expressing either P704+ or P704– RIZ, we compared their respective abilities to enhance ER{alpha} coactivation. As shown in Fig. 1Go, using various concentrations of the two RIZ1 protein variants, both were able to enhance the ER{alpha} in a ligand- and dose-dependent manner. However, at all concentrations (50–200 ng), the P704– RIZ variant showed a reduced enhancing effect in ER{alpha} coactivation vs. the P704+ RIZ (P < 0.0001 at all concentrations).



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  		FIG. 1. The indicated amounts of the RIZ1 P704 deletion construct (3RIZRH{Delta}P-3); RIZ1 wt construct (3RIZRH 4.3) and ER{alpha} expression vectors were cotransfected with a reporter construct containing the mammalian ER{alpha} response element into CV-1 cells. Cells were treated with or without 50 nM estradiol (E2) for 24 h before CAT activity measurements. Data are presented as the mean ± SEM of three independent experiments performed in triplicate, and CAT values are normalized for transfection efficiency by the corresponding ß-galactosidase activity. At all concentrations (50–200 ng), the P704– RIZ variant showed a statistically reduced enhancing effect in ER{alpha} coactivation vs. the P704+ RIZ (P < 0.0001 at all concentrations).

 
RIZ1 genotype analysis

The clinical characteristics of the study population are presented in Table 1Go. Individuals homozygous for the P704 deletion polymorphism were denoted P704–, individuals with absence of the deletion on both alleles were denoted P704+, and those heterozygous for the deletion were denoted P704+/–. The genotype frequencies in the study cohort were: P704–, 32%, P704+/–, 49%; and P704+, 19% and were in Hardy-Weinberg equilibrium ({chi}2 = 0.0075). The frequency and features according to RIZ1 genotype are reported in Table 2Go. The genotype groups were similar in terms of age, weight, height, body mass index, fat mass, and lean mass. There was no difference in the distribution of current and past oral contraceptives in the various genotype groups ({chi}2= 3.814; P = 0.15).


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  		TABLE 1. Subjects’ characteristics

 

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  		TABLE 2. Clinical features of the study population in relation to RIZ1 genotype

 
RIZ1 genotype in relation to BMD

The BMD at the heel was higher in women with absence of the P704 deletion on both alleles compared with that in the heterozygous group (P704+, 0.534 ± 0.011 g/cm2; P704+/–, 0.504 ± 0.006 g/cm2; P = 0.02, by ANOVA and Fisher’s LSD; Fig. 2AGo). The same pattern was shown regarding RIZ1 genotype in relation to total body BMD (Fig. 2BGo) and lumbar spine BMD (Fig. 2CGo), although it was not statistically significant [total body: P704+, 1.23 ± 0.008 g/cm2; P704+/–, 1.21 ± 0.006 g/cm2 (P = 0.13); lumbar spine: P704+, 1.25 ± 0.013 g/cm2; P704+/–, 1.23 ± 0.010 g/cm2 (P = 0.28, using ANOVA and Fisher’s LSD). There were no effects of RIZ1 genotype on either BMD at the femoral neck or BMD at total hip.



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  		FIG. 2. A, The RIZ1 genotypes P704–, P704+/–, and P704+ in relation to left heel (LH) BMD (grams per square centimeter). The P704– group consists of 93 (31%) individuals, the P704+/– group consists of 147 (49%) individuals, and the P704+ group consists of 58 (20%) individuals. B, The RIZ1 genotypes P704–, P704+/–, and P704+ in relation to total body (TB) BMD. The P704– group consists of 96 (32%) individuals, the P704+/– group consists of 149 (49%) individuals, and the P704+ group consists of 59 (19%) individuals. C, The RIZ1 genotypes P704–, P704+/–, and P704+ in relation to lumbar spine (LS; L2–L4) BMD. The P704– group consists of 96 (32%) individuals, the P704+/– group consists of 149 (49%) individuals, and the P704+ group consists of 58 (19%) individuals. D, The RIZ1 genotypes P704–, P704+/–, and P704+ in relation to total hip (TH) BMD. The P704– group consists of 96 (32%) individuals, the P704+/– group consists of 149 (49%) individuals, and the P704+ group consists of 59 (19%) individuals. Data are presented as the mean ± SEM. *, P < 0.05.

 
RIZ1 genotype in relation to BMD after adjustments for confounding factors

Table 3Go demonstrates the multiple regression models for heel BMD with RIZ1 genotype, age, height, fat mass, and lean mass as independent variables. The general P value for the multiple regression analysis was P < 0.00001. It was shown that RIZ1 genotype was also associated with heel BMD after adjustments for age, height, fat mass, and lean mass (P = 0.03).


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  		TABLE 3. Multiple regression analysis for heel BMD against age, height, fat mass, lean mass, and RIZ1 genotype

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
To date, a large number of association studies have been performed to identify genetic factors underlying osteoporosis. The vitamin D receptor gene, the collagen type I {alpha}1 gene, and the ER{alpha} gene are among those most intensively studied candidate genes in relation to BMD. Even though a high number of association studies have demonstrated correlations of different genetic variations in candidate genes with BMD, far less is known about how these genetic variations affect the functional mechanism underlying BMD.

In this study we investigated the functional effect of a P704 deletion polymorphism in the RIZ1 gene in vitro. We also studied the relationship between the deletion polymorphism and BMD in young adult Swedish women. RIZ1 is a novel candidate gene to BMD due to its role as a specific coactivator of the ER{alpha}. The physiological concentration of estradiol is specifically able to induce the binding of ER{alpha} to RIZ protein in both target tissue and cultured cells (28). It has also been shown that RIZ1 is required for efficient estrogen action in vivo (17). It is well known that estrogen plays an important role in bone metabolism by interaction with the ER{alpha}, and it is generally agreed that estrogen deficiency is the most important factor in the genesis of postmenopausal bone loss (29). Our results showed that both RIZ1 protein variants, P704+ and P704–, were able to enhance the ER{alpha} in a ligand- and dose-dependent manner in vitro. These findings are in line with several reports showing that RIZ1 has the capacity to enhance ER{alpha} activation of transcription in a ligand-dependent manner (17, 28, 30). However, this is the first study to demonstrate that the RIZ1 P704– polymorphic variant has a statistically reduced enhancing effect in ER{alpha} coactivation vs. the RIZ1 P704+ polymorphic variant (P < 0.0001 at all concentrations). In addition, an effect of the P704– RIZ1 polymorphic variant was shown in our association study of BMD. After adjustments for age, height, fat mass, and lean mass, individuals in the P704– RIZ1 genotype group had significantly lower BMD at the heel compared with those in the P704+ RIZ1 genotype group (P < 0.05). The same pattern was shown for total body and lumbar spine BMD. The difference in heel BMD between the homozygous individuals in the association study was 0.36 SD units or 0.03 g/cm2, indicating a relatively large effect of the RIZ1 gene on heel BMD. However, the power calculations of the study confirmed that our sample was not large enough to detect minor genotypic differences in BMD in the range 0.10–0.25 SD units, which were shown for the RIZ1 polymorphic variants on total body BMD (0.23 SD), total hip BMD (0.11 SD), and lumbar spine BMD (0.17 SD). To have 80% power to detect the genotypic differences in BMD with SD ranges of 0.10–0.25, approximately 1500–3500 subjects should be included and genotyped in the association study.

The RIZ1 P704 deletion polymorphism is one of seven reported polymorphisms in the coding region of the RIZ1 gene; the deletion polymorphism is located at exon 8 and encodes a proline at position 704 (25). Because of the relatively high rate of heterozygosity of the P704 deletion polymorphism (~50%) Fang et al. (25) used the deletion polymorphism to study whether the loss of heterozygosity at the RIZ locus may be common in human cancers. By analyzing the Pro704– deletion polymorphism in relation to hepatocellular carcinoma (HCC), colon cancer, and breast cancer cases, it was shown that the P704+ allele undergoes preferential loss and has a stronger tumor suppressor role than the P704– allele (25), supporting the functional role of the RIZ P704 deletion polymorphism.

However, no previous studies have been performed on RIZ1 polymorphisms in association with bone phenotypes despite the fact that the RIZ gene maps to chromosome 1p36, which has been shown to be a quantitative trait loci (QTL) controlling BMD (23, 31, 32). Devoto et al. (22) confirmed and extended these QTL findings by analyzing nine microsatellite markers spanning a 40-cM interval across the 1p36 candidate region and found a major QTL controlling BMD located on chromosome 1p36.2-p36.3. This region includes the chromosomal region 1p36.21, where the RIZ gene has been mapped (20, 24). The genes encoding methylene tetrahydrofolate reductase (MTHFR), TNF receptor superfamily member 1B (TNFRSF1B), lysyl hydroxylase (PLOD1), and brain natriuretic peptide (BNP) are other candidate genes located on the chromosome region 1p36.2-p36.3 that have been associated with BMD. A variant form of MTHFR was found to be correlated to reduced BMD in postmenopausal women (33, 34) and with a reduced risk of osteoporotic fractures (35). For the TNFRSF1B gene, Spotila et al. (36) initially demonstrated an association of two polymorphic sites within the gene and low BMD in a group of 159 unrelated individuals. These findings are in agreement with the results from a large cohort study of 1240 perimenopausal women from the United Kingdom (37, 38). Also, recent studies have independently reported associations of polymorphic variants in the genes for PLOD1, BNP, and BMD (39, 40). The underlying mechanisms by which the polymorphic variants in the candidate genes affect bone parameters remain to be elucidated.

In the literature there is an inverse relation between breast cancer risk and postmenopausal osteoporosis. High or prolonged estrogen exposure would, according to this theory, preserve bone and promote the development of breast cancer. Is there a possibility that the functional polymorphism in RIZ is involved in this association? From our data this is not evident. In bone tissue, our data argue that the deletion diminishes estrogen activity, leading to bone loss. A similar mechanism in breast tissue would lead to reduced risk for breast cancer. Instead, RIZ is reported to be a tumor suppressor gene, and the deletion is associated with an increase in breast cancer incidence. These data suggest that the tumor suppressor activity of RIZ is not necessarily dependent on estrogen levels. RIZ1 activates ER{alpha} target gene expression by chromatin remodeling and histone methylation, which is characteristic of HMT cofactors (12, 13). Studies have confirmed that it is the HMT function of RIZ1 that underlies the tumor suppression (41), and RIZ1 gene silencing is common in many types of cancers, including those of breast, colon, and liver (42). Loss of the HMT function of RIZ1 also results in partial hormone resistance in normal and malignant mammary tissues (17), which indicates that RIZ1 may play a role in the development of hormone-resistant breast cancer, because this type of cancer commonly shows RIZ1 silencing. Based on this, the previously reported inverse association of breast cancer and osteoporosis (43, 44) may not be applicable to hormone-resistant cancers. It is also important to acknowledge that the role of estrogen as a cause of breast cancer development might be questioned. The reported data from the Womens Health Initiative suggest that it is the combination of gestagen and estrogens that promotes breast cancer, whereas estrogens alone apparently have no effect.

In conclusion, we found that the two RIZ1 P704 polymorphic variants were both able to enhance ER{alpha} in a ligand- and dose-dependent manner, but the P704– RIZ variant showed a statistically reduced enhancing effect in ER{alpha} coactivation vs. P704+ RIZ. We also showed an association of the P704 deletion polymorphism with low BMD in healthy premenopausal women. To our knowledge, this is the first study to demonstrate that the ER{alpha} coactivator RIZ gene is a strong candidate gene for involvement in the variation seen in BMD based on the functional role of the polymorphism shown in vitro and the association with BMD. This indicates that RIZ1 could mediate proliferation and growth induced by estradiol in some target tissues, e.g. bone.


   Acknowledgments
 
We gratefully acknowledge the research nurses, Ann-Charlotte Adolfsson, Marja Gustavsson, Lena Lundberg, and Katarina Nisser, for work with the cohort, and Anna-Lena Johansson for skilful technical assistance.


   Footnotes
 
This work was supported by grants from the Swedish Society for Medicine, the Swedish Council of Sports Medicine (Centrum för Idrottsforskning), and the County Council of Uppsala.

Abbreviations: BMD, Bone mineral density; BNP, brain natriuretic peptide; CAT, chloramphenicol acetyltransferase; DXA, dual energy x-ray absorptiometry; ER, estrogen receptor; HMT, histone/protein methyltransferase; LSD, least significant differences; QTL, quantitative trait loci.

Received March 5, 2004.

Accepted September 8, 2004.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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E. Grundberg, K. Akesson, A. Kindmark, P. Gerdhem, A. Holmberg, D. Mellstrom, O. Ljunggren, E. Orwoll, H. Mallmin, C. Ohlsson, et al.
The Impact of Estradiol on Bone Mineral Density Is Modulated by the Specific Estrogen Receptor-{alpha} Cofactor Retinoblastoma-Interacting Zinc Finger Protein-1 Insertion/Deletion Polymorphism
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