DOI 10.1515/dmdi-2013-0066 Drug Metab Drug Interact 2014; 29(2): 111–114
Simona Mencej-Bedrač*, Janja Zupan, Simona Jurković Mlakar, Andrej Zavratnik, Janez Preželj and Janja Marc
Raloxifene pharmacodynamics is influenced by genetic variants in the RANKL/RANK/OPG system and in the Wnt signaling pathway Abstract Background: Raloxifene is a selective estrogen receptor (ER) modulator (SERM) used for the treatment of osteoporosis. However, its efficacy and also its safety vary greatly among treated patients, and it might be influenced by the individuals’ genetic background. As the receptor activator of the nuclear factor κB (RANK) ligand (RANKL)/RANK/ osteoprotegerin (OPG) system is essential for osteoclastogensis and Wnt signaling pathway for osteoblastogenesis, we decided to evaluate the raloxifene treatment in regard to selected polymorphisms in key genes of these two main bone regulatory pathways. Methods: Fifty-six osteoporotic postmenopausal women treated with raloxifene were genotyped for 11 polymorphisms located in six genes: -290C > T, -643C > T, and -693G > C in tumor necrosis factor receptor superfamily member 11 (TNFSF11), +34694C > T, +34901G > A, and +35966insdelC in tumor necrosis factor receptor superfamily member 11A (TNFRSF11A), K3N and 245T > G in tumor necrosis factor receptor superfamily member 11B (TNFRSF11B), A1330V in LRP5, I1062V in LRP6, and -1397_-1396insGGA in SOST. For evaluation of treatment efficacy, bone mineral density (BMD) and biochemical markers of bone turnover were measured. Results: One-year change in total hip BMD was associated with +34901G > A in TNFRSF11A (p = 0.040), whereas, for lumbar spine BMD, the association was shown for -1397_-1396insGGA in SOST (p = 0.015). C-terminal crosslinking telopeptides of type I collagen (CTX) concentrations showed significant association with -643C > T single nucleotide polymorphism (SNP) in TNFSF11 (p = 0.049) and +34694C > T in TNFRSF11A (p = 0.022). No other association was found between 1-year change in BMDs or biochemical markers and the studied SNPs. Conclusions: We have shown that, in postmenopausal osteoporotic women treated with raloxifene, the efficacy of raloxifene treatment might be influenced by +34901G > A in TNFRSF11A gene and -1397_-1396insGGA in the SOST gene as well as -643C > T in TNFSF11 gene and +34694C > T
in TNFRSF11A gene. However, these findings need additional functional and clinical confirmation for potential pharmacogenetic use in the future. Keywords: osteoporosis; polymorphism; raloxifene; SNP. *Corresponding author: Simona Mencej-Bedrač, Faculty of Pharmacy, Department of Clinical Biochemistry, University of Ljubljana, Askerčeva cesta 7, 1000 Ljubljana, Slovenia, E-mail: [email protected] Janja Zupan, Simona Jurković Mlakar and Janja Marc: Faculty of Pharmacy, Department of Clinical Biochemistry, University of Ljubljana, Ljubljana, Slovenia Andrej Zavratnik: Department of Endocrinology and Diabetes, University Medical Centre Maribor, Maribor, Slovenia Janez Preželj: Department of Endocrinology, Diabetes and Metabolic Diseases, University Medical Centre Ljubljana, Ljubljana, Slovenia
Introduction Osteoporosis, a skeletal disease with a strong genetic background, is characterized by low bone mineral density (BMD) and microarchitectural changes, both leading to an increased risk of osteoporotic fractures. A large majority of available drugs for osteoporosis therapy, such as bisphosphonates, denosumab, and selective estrogen receptor (ER) modulators (SERMs), are antiresorptives acting on osteoclasts and decreasing bone loss and fracture risk [1]. Raloxifene, a widely used SERM for the treatment of osteoporosis, reduces vertebral fracture risk, increases lumbar spine and femoral neck BMDs, and decreases serum concentrations of bone turnover markers [2]. The efficacy and safety of raloxifene vary greatly among treated patients, and it may be influenced by the individuals’ genetic backgrounds or environmental influences. So far, the genetic basis of the differences in the efficacy of raloxifene therapy has only been evaluated for some common osteoporosisrelated genes and in genes coding for proteins involved in raloxifene pharmacokinetics. The association between
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112 Mencej-Bedrač et al.: Influence of genetic variants on the efficacy of raloxifene the efficacy of raloxifene and vitamin D receptor (VDR) polymorphism BsmI has been identified in 75 Italian osteoporotic women showing significantly greater improvement of lumbar spine BMD in BB genotype [3]. A similar conclusion was drawn also from a 1-year multicentric, randomized, controlled trial in 1100 osteoporotic women [4]. Bone remodeling is largely regulated by two key pathways of bone biology: receptor activator of the nuclear factor κB (RANK) ligand (RANKL)/RANK/osteoprotegerin (OPG) system being a key regulator of osteoclastogenesis and the Wnt signaling pathway being a key pathway of osteoblastogenesis. As the association of genetic polymorphisms in the genes, involved in the RANKL/RANK/ OPG system or Wnt signaling pathway, with the efficacy of raloxifene has not been studied yet, the aim of our study was to investigate the potential association of selected polymorphisms in tumor necrosis factor receptor superfamily member 11 (TNFSF11), tumor necrosis factor receptor superfamily member 11A (TNFRSF11A), and tumor necrosis factor receptor superfamily member 11B (TNFRSF11B), and genes for sclerostin (SOST) and low-density lipoprotein receptor-related proteins 5 and 6 (LRP5 and LRP6), with the change of BMDs and serum concentrations of bone turnover markers in Slovenian postmenopausal osteoporotic women after 1 year of treatment with raloxifene.
60 mg, cholecalciferol 400 IU, and calcium carbonate 1000 mg daily. At baseline and after 1 year of treatment, BMDs were measured at the lumbar (L1–L4) spine (BMD-ls), total hip (BMD-tot), and femoral neck (BMD-fn) by dual-energy X-ray absorptiometry (QDR-2000+; Hologic, Inc., Waltham, MA, USA). Blood samples for bone turnover marker measurement were collected at baseline and after 6 and 12 months, each time between 08:00 and 10:00 a.m. after an overnight fast. Serum bone alkaline phosphatase (BALP) was assessed by IRMA (TandemW-R OstaseW; Beckman Coulter, Pasadena, CA, USA). Plasma osteocalcin (OC) was measured using Elecsys N-MID Osteocalcin assay (Roche Diagnostics, Mannheim, Germany). Serum C-terminal crosslinking telopeptides of type I collagen (CTX) was measured using Elecsys Beta CrossLaps assay (Roche Diagnostics). The protocol was approved by the National Medical Ethics Committee of the Republic of Slovenia and informed consent was obtained from all patients. Genomic DNA was isolated from peripheral blood leukocytes using Flexigene DNA kit (Qiagen, Hilden, Germany). The data on analyzed single nucleotide polymorphisms (SNPs) and genotyping methods used are presented in Table 1. The differences in BMD and serum markers of bone turnover between the genotype subgroups were tested using Kruskal-Wallis test. Statistical analyses were carried out using IBM SPSS Statistics version 21.0 (IBM SPSS Statistics, Chicago, IL, USA) and p A in TNFRSF11A and in -1397_-1396insGGA in SOST (Figure 1). For SNP +34901G > A in TNFRSF11A, BMD-tot increased significantly in those carrying GA+AA genotypes compared with those carrying the GG genotype
Materials and methods Fifty-six postmenopausal women with primary osteoporosis, at least 5 years after menopause and not older than 70 years, were included in the study. After the first visit, the participants started with raloxifene
Table 1 Analyzed SNPs in the selected genes of the RANKL/RANK/OPG system and Wnt signaling pathway and genotyping methods used. RANKL/RANK/OPG system Wnt signaling pathway
Gene symbol
Protein symbol
Studied SNP (rs number)
TNFSF11
TNFRSF11A TNFRSF11B SOST
RANKL
SOST
LRP5 LRP6
LRP5 LRP6
-290C > T (rs9525641) -643C > T (rs9533156) -693G > C (rs9533155) +34694C > T (rs1805034) +34901G > A (rs12458117) +35966insC (rs75404003) K3N (rs2073618) 245T > G (rs3134069) -1397_-1396insGGA (rs10534024) A1330V (rs3736228) I1062A (rs2302685)
RANK
OPG
Method
Reference
AD PCR/RFLPb PCR/RFLPb PCR/RFLPb PCR/RFLPb dHPLCc ADa PCR/RFLPb dHPLCc
[5]
ADa ADa
[8] [8]
a
[6]
[5] [7] [8]
a Allelic discrimination (AD) method, using the TaqMan SNP genotyping assays (Applied Biosystems, Foster City, CA, USA) and ABI Prism 7000 sequence detection system (Applied Biosystems), or LightCycler 480 (Roche Diagnostics), was performed. bPolymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analyses with appropriate restriction enzymes were applied. cNondenaturing high-performance liquid chromatography (HPLC) analysis was performed using a Transgenomic WAVE 3500 DNA Fragment Analysis System with a DNASep column (Transgenomic, Omaha, NE, USA).
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Mencej-Bedrač et al.: Influence of genetic variants on the efficacy of raloxifene 113
(p = 0.040). The increase in BMD-ls was also observed, although the difference was not statistically significant (p = 0.073). In SOST promoter insertion of GGA nucleotides, the absence of the insertion was associated with significantly lower gain of BMD-ls (p = 0.015). In the other studied SNPs, no significant association with the 1-year change in BMDs was identified. The association of CTX concentrations with -643C > T SNP in TNFSF11 was shown. Women with TT genotype had significantly higher reduction of CTX concentration after 1 year of raloxifene treatment than women with CC or CT genotype (p = 0.049). Additionally, women with the CC genotype in SNP +34694C > T of TNFRSF11A had a markedly higher 1-year reduction of CTX concentration than women with CT or TT genotype (p = 0.022). No other association was found between biochemical markers and studied SNPs.
Discussion In 56 osteoporotic postmenopausal women treated with raloxifene for 1 year, the association between the change in BMD and bone turnover markers and genetic influence of the RANKL/RANK/OPG system and the Wnt signaling pathway has been evaluated. The RANKL/RANK/OPG system is essential for osteoclast formation, activity, and survival in normal and
3
-/-
-/GGA GGA/GGA
2.5
dBMD (%)
2 1.5
GG
GA+AA
1 0.5 0 -0.5 BMD-tot +34901G>A (TNFRSF11A) p = 0.040
BDM-Is +1397_-1396insGGA (SOST) p = 0.015
Figure 1 Significant changes in BMDs in osteoporotic postmenopausal women after 1 year of treatment with raloxifene in the genotype subgroups of TNFRSF11A and SOST genes. dBMD, percentage of change in BMD after 1 year of treatment with raloxifene. Values are medians for percentage of change in BMDs after 1 year of raloxifene therapy.
pathological states of bone remodeling. Although the majority of the available osteoporosis pharmacological therapies, including raloxifene, primarily decrease bone resorption, no pharmacogenetic studies, investigating the influence of the RANKL/RANK/OPG system genetic variances on the efficacy and safety of drugs for the osteoporosis therapy, are available. We have shown that SNP +34901G > A in TNFRSF11A significantly increases BMD-tot in women carrying GA+AA compared with those carrying GG. On the contrary, GG is associated with higher BMD-ls, although the difference was not statistically significant (p = 0.073). These results suggest that women with GA+AA respond better to the raloxifene therapy in the BMD-tot but not at the BMD-ls. It seems that the genetic influence of SNP +34901G > A on the change in BMD in a year of raloxifene therapy differs among measured skeletal sites. Testing biochemical markers of bone turnover, the change in the CTX concentration was significantly associated with -643C > T SNP in TNFSF11, indicating the influence of the promoter region SNP -643C > T in TNFSF11 on its expression. It has been shown previously that raloxifene influences TNFSF11 and TNFRSF11B expression in peripheral blood mononuclear cells [9] possibly through estrogen, ERα, and estrogen response element-binding proteins [10]. Additionally, regarding the +34694C > T SNP of TNFRSF11A, the CTX concentration decreased to a greater extent in women carrying CC genotype, indicating their better response to raloxifene therapy. The effect is probably not the consequence of modified expression, as no effect of raloxifene on RANK expression has been observed previously [9]. Another crucial signaling pathway in bone biology is the Wnt/β-catenin canonical pathway, which controls osteoblast proliferation, differentiation and function, and bone formation. In women carrying one or two copies of the inserted GGA nucleotides in the SOST promoter, the increase in BMD-ls was higher than in those not carrying the GGA insertion. The promoter insertion in SOST influences the efficacy of raloxifene therapy, although the mechanism is not clear. It has been shown that estrogen levels negatively correlate with serum SOST levels, and higher serum SOST levels were observed in postmenopausal women. It seems that estrogen inhibits the production of SOST, its release from the bone tissue, or clearance of SOST from the circulation [11]. As raloxifene also binds to ERs, it might also be able to modify the expression of SOST and the production of SOST protein.
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114 Mencej-Bedrač et al.: Influence of genetic variants on the efficacy of raloxifene Acknowledgments: This work was supported by grants P3-0298 and J3-2330 provided by the Slovenian Research Agency. Conflict of interest statement Authors’ conflict of interest disclosure: The authors stated that there are no conflicts of interest regarding the publication of this article. Research support played no
role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication. Research funding: None declared. Employment or leadership: None declared. Honorarium: None declared. Received August 22, 2013; accepted January 27, 2014; previously published online March 10, 2014
References 1. Delmas PD. Treatment of postmenopausal osteoporosis. Lancet 2002;359:2018–26. 2. Recker RR, Mitlak BH, Ni X, Krege JH. Long-term raloxifene for postmenopausal osteoporosis. Curr Med Res Opin 2011;27: 1755–61. 3. Palomba S, Orio Jr F, Russo T, Falbo A, Tolino A, Manguso F, et al. BsmI vitamin D receptor genotypes influence the efficacy of antiresorptive treatments in postmenopausal osteoporotic women. A 1-year multicenter, randomized and controlled trial. Osteoporos Int 2005;16:943–52. 4. Palombo S, Numis FG, Mossetti G, Rendina D, Vuotto P, Russo T, et al. Raloxifene administration in post-menopausal women with osteoporosis: effect of different BsmI vitamin D receptor genotypes. Hum Reprod 2003;18:192–8. 5. Mencej-Bedrač S, Preželj J, Kocjan T, Teskač K, Ostanek B, Šmelcer M, et al. The combinations of polymorphisms in vitamin D receptor, osteoprotegerin and tumour necrosis factor superfamily member 11 genes are associated with bone mineral density. J Mol Endocrinol 2009;42:239–47. 6. Zupan J, Mencej-Bedrač S, Jurković-Mlakar S, Preželj J, Marc J. Gene-gene interactions in RANK/RANKL/OPG system influence
bone mineral density in postmenopausal women. J Steroid Biochem Mol Biol 2010;118:102–6. 7. Arko B, Preželj J, Kocijančič A, Komel R, Marc J. Association of the osteoprotegerin gene polymorphisms with bone mineral density in postmenopausal women. Maturitas 2005;51:270–9. 8. Mencej-Bedrač S, Preželj J, Kocjan T, Komadina R, Marc J. Analysis of association of LRP5, LRP6, SOST, DKK1, and CTNNB1 genes with bone mineral density in a Slovenian population. Calcif Tissue Int 2009;85:501–6. 9. Bashir A, Mak YT, Sankaralingam S, Cheung J, Mc Gowan NW, Grigoriadis AE, et al. Changes in RANKL/OPG/RANK gene expression in peripheral mononuclear cells following treatment with estrogen or raloxifene. Steroids 2005;70:847–55. 10. Chen H, Gilbert LC, Lu X, Liu Z, You S, Weitzmann MN, et al. A new regulator of osteoclastogenesis: estrogen response element-binding protein in bone. J Bone Miner Res 2011;26:2537–47. 11. Mirza FS, Desmond Padhi I, Raisz LG, Lorenzo JA. Serum sclerostin levels negatively correlate with parathyroid hormone levels and free estrogen index in postmenopausal women. J Clin Endocrinol Metab 2010;95:1991–7.
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Simona Mencej-Bedrač*, Janja Zupan, Simona Jurković Mlakar, Andrej Zavratnik, Janez Preželj and Janja Marc
Raloxifene pharmacodynamics is influenced by genetic variants in the RANKL/RANK/OPG system and in the Wnt signaling pathway Abstract Background: Raloxifene is a selective estrogen receptor (ER) modulator (SERM) used for the treatment of osteoporosis. However, its efficacy and also its safety vary greatly among treated patients, and it might be influenced by the individuals’ genetic background. As the receptor activator of the nuclear factor κB (RANK) ligand (RANKL)/RANK/ osteoprotegerin (OPG) system is essential for osteoclastogensis and Wnt signaling pathway for osteoblastogenesis, we decided to evaluate the raloxifene treatment in regard to selected polymorphisms in key genes of these two main bone regulatory pathways. Methods: Fifty-six osteoporotic postmenopausal women treated with raloxifene were genotyped for 11 polymorphisms located in six genes: -290C > T, -643C > T, and -693G > C in tumor necrosis factor receptor superfamily member 11 (TNFSF11), +34694C > T, +34901G > A, and +35966insdelC in tumor necrosis factor receptor superfamily member 11A (TNFRSF11A), K3N and 245T > G in tumor necrosis factor receptor superfamily member 11B (TNFRSF11B), A1330V in LRP5, I1062V in LRP6, and -1397_-1396insGGA in SOST. For evaluation of treatment efficacy, bone mineral density (BMD) and biochemical markers of bone turnover were measured. Results: One-year change in total hip BMD was associated with +34901G > A in TNFRSF11A (p = 0.040), whereas, for lumbar spine BMD, the association was shown for -1397_-1396insGGA in SOST (p = 0.015). C-terminal crosslinking telopeptides of type I collagen (CTX) concentrations showed significant association with -643C > T single nucleotide polymorphism (SNP) in TNFSF11 (p = 0.049) and +34694C > T in TNFRSF11A (p = 0.022). No other association was found between 1-year change in BMDs or biochemical markers and the studied SNPs. Conclusions: We have shown that, in postmenopausal osteoporotic women treated with raloxifene, the efficacy of raloxifene treatment might be influenced by +34901G > A in TNFRSF11A gene and -1397_-1396insGGA in the SOST gene as well as -643C > T in TNFSF11 gene and +34694C > T
in TNFRSF11A gene. However, these findings need additional functional and clinical confirmation for potential pharmacogenetic use in the future. Keywords: osteoporosis; polymorphism; raloxifene; SNP. *Corresponding author: Simona Mencej-Bedrač, Faculty of Pharmacy, Department of Clinical Biochemistry, University of Ljubljana, Askerčeva cesta 7, 1000 Ljubljana, Slovenia, E-mail: [email protected] Janja Zupan, Simona Jurković Mlakar and Janja Marc: Faculty of Pharmacy, Department of Clinical Biochemistry, University of Ljubljana, Ljubljana, Slovenia Andrej Zavratnik: Department of Endocrinology and Diabetes, University Medical Centre Maribor, Maribor, Slovenia Janez Preželj: Department of Endocrinology, Diabetes and Metabolic Diseases, University Medical Centre Ljubljana, Ljubljana, Slovenia
Introduction Osteoporosis, a skeletal disease with a strong genetic background, is characterized by low bone mineral density (BMD) and microarchitectural changes, both leading to an increased risk of osteoporotic fractures. A large majority of available drugs for osteoporosis therapy, such as bisphosphonates, denosumab, and selective estrogen receptor (ER) modulators (SERMs), are antiresorptives acting on osteoclasts and decreasing bone loss and fracture risk [1]. Raloxifene, a widely used SERM for the treatment of osteoporosis, reduces vertebral fracture risk, increases lumbar spine and femoral neck BMDs, and decreases serum concentrations of bone turnover markers [2]. The efficacy and safety of raloxifene vary greatly among treated patients, and it may be influenced by the individuals’ genetic backgrounds or environmental influences. So far, the genetic basis of the differences in the efficacy of raloxifene therapy has only been evaluated for some common osteoporosisrelated genes and in genes coding for proteins involved in raloxifene pharmacokinetics. The association between
Brought to you by | Western University Authenticated Download Date | 6/6/15 9:22 PM
112 Mencej-Bedrač et al.: Influence of genetic variants on the efficacy of raloxifene the efficacy of raloxifene and vitamin D receptor (VDR) polymorphism BsmI has been identified in 75 Italian osteoporotic women showing significantly greater improvement of lumbar spine BMD in BB genotype [3]. A similar conclusion was drawn also from a 1-year multicentric, randomized, controlled trial in 1100 osteoporotic women [4]. Bone remodeling is largely regulated by two key pathways of bone biology: receptor activator of the nuclear factor κB (RANK) ligand (RANKL)/RANK/osteoprotegerin (OPG) system being a key regulator of osteoclastogenesis and the Wnt signaling pathway being a key pathway of osteoblastogenesis. As the association of genetic polymorphisms in the genes, involved in the RANKL/RANK/ OPG system or Wnt signaling pathway, with the efficacy of raloxifene has not been studied yet, the aim of our study was to investigate the potential association of selected polymorphisms in tumor necrosis factor receptor superfamily member 11 (TNFSF11), tumor necrosis factor receptor superfamily member 11A (TNFRSF11A), and tumor necrosis factor receptor superfamily member 11B (TNFRSF11B), and genes for sclerostin (SOST) and low-density lipoprotein receptor-related proteins 5 and 6 (LRP5 and LRP6), with the change of BMDs and serum concentrations of bone turnover markers in Slovenian postmenopausal osteoporotic women after 1 year of treatment with raloxifene.
60 mg, cholecalciferol 400 IU, and calcium carbonate 1000 mg daily. At baseline and after 1 year of treatment, BMDs were measured at the lumbar (L1–L4) spine (BMD-ls), total hip (BMD-tot), and femoral neck (BMD-fn) by dual-energy X-ray absorptiometry (QDR-2000+; Hologic, Inc., Waltham, MA, USA). Blood samples for bone turnover marker measurement were collected at baseline and after 6 and 12 months, each time between 08:00 and 10:00 a.m. after an overnight fast. Serum bone alkaline phosphatase (BALP) was assessed by IRMA (TandemW-R OstaseW; Beckman Coulter, Pasadena, CA, USA). Plasma osteocalcin (OC) was measured using Elecsys N-MID Osteocalcin assay (Roche Diagnostics, Mannheim, Germany). Serum C-terminal crosslinking telopeptides of type I collagen (CTX) was measured using Elecsys Beta CrossLaps assay (Roche Diagnostics). The protocol was approved by the National Medical Ethics Committee of the Republic of Slovenia and informed consent was obtained from all patients. Genomic DNA was isolated from peripheral blood leukocytes using Flexigene DNA kit (Qiagen, Hilden, Germany). The data on analyzed single nucleotide polymorphisms (SNPs) and genotyping methods used are presented in Table 1. The differences in BMD and serum markers of bone turnover between the genotype subgroups were tested using Kruskal-Wallis test. Statistical analyses were carried out using IBM SPSS Statistics version 21.0 (IBM SPSS Statistics, Chicago, IL, USA) and p A in TNFRSF11A and in -1397_-1396insGGA in SOST (Figure 1). For SNP +34901G > A in TNFRSF11A, BMD-tot increased significantly in those carrying GA+AA genotypes compared with those carrying the GG genotype
Materials and methods Fifty-six postmenopausal women with primary osteoporosis, at least 5 years after menopause and not older than 70 years, were included in the study. After the first visit, the participants started with raloxifene
Table 1 Analyzed SNPs in the selected genes of the RANKL/RANK/OPG system and Wnt signaling pathway and genotyping methods used. RANKL/RANK/OPG system Wnt signaling pathway
Gene symbol
Protein symbol
Studied SNP (rs number)
TNFSF11
TNFRSF11A TNFRSF11B SOST
RANKL
SOST
LRP5 LRP6
LRP5 LRP6
-290C > T (rs9525641) -643C > T (rs9533156) -693G > C (rs9533155) +34694C > T (rs1805034) +34901G > A (rs12458117) +35966insC (rs75404003) K3N (rs2073618) 245T > G (rs3134069) -1397_-1396insGGA (rs10534024) A1330V (rs3736228) I1062A (rs2302685)
RANK
OPG
Method
Reference
AD PCR/RFLPb PCR/RFLPb PCR/RFLPb PCR/RFLPb dHPLCc ADa PCR/RFLPb dHPLCc
[5]
ADa ADa
[8] [8]
a
[6]
[5] [7] [8]
a Allelic discrimination (AD) method, using the TaqMan SNP genotyping assays (Applied Biosystems, Foster City, CA, USA) and ABI Prism 7000 sequence detection system (Applied Biosystems), or LightCycler 480 (Roche Diagnostics), was performed. bPolymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analyses with appropriate restriction enzymes were applied. cNondenaturing high-performance liquid chromatography (HPLC) analysis was performed using a Transgenomic WAVE 3500 DNA Fragment Analysis System with a DNASep column (Transgenomic, Omaha, NE, USA).
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Mencej-Bedrač et al.: Influence of genetic variants on the efficacy of raloxifene 113
(p = 0.040). The increase in BMD-ls was also observed, although the difference was not statistically significant (p = 0.073). In SOST promoter insertion of GGA nucleotides, the absence of the insertion was associated with significantly lower gain of BMD-ls (p = 0.015). In the other studied SNPs, no significant association with the 1-year change in BMDs was identified. The association of CTX concentrations with -643C > T SNP in TNFSF11 was shown. Women with TT genotype had significantly higher reduction of CTX concentration after 1 year of raloxifene treatment than women with CC or CT genotype (p = 0.049). Additionally, women with the CC genotype in SNP +34694C > T of TNFRSF11A had a markedly higher 1-year reduction of CTX concentration than women with CT or TT genotype (p = 0.022). No other association was found between biochemical markers and studied SNPs.
Discussion In 56 osteoporotic postmenopausal women treated with raloxifene for 1 year, the association between the change in BMD and bone turnover markers and genetic influence of the RANKL/RANK/OPG system and the Wnt signaling pathway has been evaluated. The RANKL/RANK/OPG system is essential for osteoclast formation, activity, and survival in normal and
3
-/-
-/GGA GGA/GGA
2.5
dBMD (%)
2 1.5
GG
GA+AA
1 0.5 0 -0.5 BMD-tot +34901G>A (TNFRSF11A) p = 0.040
BDM-Is +1397_-1396insGGA (SOST) p = 0.015
Figure 1 Significant changes in BMDs in osteoporotic postmenopausal women after 1 year of treatment with raloxifene in the genotype subgroups of TNFRSF11A and SOST genes. dBMD, percentage of change in BMD after 1 year of treatment with raloxifene. Values are medians for percentage of change in BMDs after 1 year of raloxifene therapy.
pathological states of bone remodeling. Although the majority of the available osteoporosis pharmacological therapies, including raloxifene, primarily decrease bone resorption, no pharmacogenetic studies, investigating the influence of the RANKL/RANK/OPG system genetic variances on the efficacy and safety of drugs for the osteoporosis therapy, are available. We have shown that SNP +34901G > A in TNFRSF11A significantly increases BMD-tot in women carrying GA+AA compared with those carrying GG. On the contrary, GG is associated with higher BMD-ls, although the difference was not statistically significant (p = 0.073). These results suggest that women with GA+AA respond better to the raloxifene therapy in the BMD-tot but not at the BMD-ls. It seems that the genetic influence of SNP +34901G > A on the change in BMD in a year of raloxifene therapy differs among measured skeletal sites. Testing biochemical markers of bone turnover, the change in the CTX concentration was significantly associated with -643C > T SNP in TNFSF11, indicating the influence of the promoter region SNP -643C > T in TNFSF11 on its expression. It has been shown previously that raloxifene influences TNFSF11 and TNFRSF11B expression in peripheral blood mononuclear cells [9] possibly through estrogen, ERα, and estrogen response element-binding proteins [10]. Additionally, regarding the +34694C > T SNP of TNFRSF11A, the CTX concentration decreased to a greater extent in women carrying CC genotype, indicating their better response to raloxifene therapy. The effect is probably not the consequence of modified expression, as no effect of raloxifene on RANK expression has been observed previously [9]. Another crucial signaling pathway in bone biology is the Wnt/β-catenin canonical pathway, which controls osteoblast proliferation, differentiation and function, and bone formation. In women carrying one or two copies of the inserted GGA nucleotides in the SOST promoter, the increase in BMD-ls was higher than in those not carrying the GGA insertion. The promoter insertion in SOST influences the efficacy of raloxifene therapy, although the mechanism is not clear. It has been shown that estrogen levels negatively correlate with serum SOST levels, and higher serum SOST levels were observed in postmenopausal women. It seems that estrogen inhibits the production of SOST, its release from the bone tissue, or clearance of SOST from the circulation [11]. As raloxifene also binds to ERs, it might also be able to modify the expression of SOST and the production of SOST protein.
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114 Mencej-Bedrač et al.: Influence of genetic variants on the efficacy of raloxifene Acknowledgments: This work was supported by grants P3-0298 and J3-2330 provided by the Slovenian Research Agency. Conflict of interest statement Authors’ conflict of interest disclosure: The authors stated that there are no conflicts of interest regarding the publication of this article. Research support played no
role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication. Research funding: None declared. Employment or leadership: None declared. Honorarium: None declared. Received August 22, 2013; accepted January 27, 2014; previously published online March 10, 2014
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