Genetic polymorphism of the OPG gene associated with breast cancer

  • Jasmin Teresa Ney1, 6Email author,

    Affiliated with

    • Ingolf Juhasz-Boess1,

      Affiliated with

      • Frank Gruenhage2,

        Affiliated with

        • Stefan Graeber3,

          Affiliated with

          • Rainer Maria Bohle4,

            Affiliated with

            • Michael Pfreundschuh5,

              Affiliated with

              • Erich Franz Solomayer1 and

                Affiliated with

                • Gunter Assmann5

                  Affiliated with

                  BMC Cancer201313:40

                  DOI: 10.1186/1471-2407-13-40

                  Received: 1 November 2012

                  Accepted: 22 January 2013

                  Published: 31 January 2013

                  Abstract

                  Background

                  The receptor activator of NF-κB (RANK), its ligand (RANKL) and osteoprotegerin (OPG) have been reported to play a role in the pathophysiological bone turnover and in the pathogenesis of breast cancer. Based on this we investigated the role of single nucleotide polymorphisms (SNPs) within RANK, RANKL and OPG and their possible association to breast cancer risk.

                  Methods

                  Genomic DNA was obtained from Caucasian participants consisting of 307 female breast cancer patients and 396 gender-matched healthy controls. We studied seven SNPs in the genes of OPG (rs3102735, rs2073618), RANK (rs1805034, rs35211496) and RANKL (rs9533156, rs2277438, rs1054016) using TaqMan genotyping assays. Statistical analyses were performed using the χ2-tests for 2 x 2 and 2 x 3 tables.

                  Results

                  The allelic frequencies (OR: 1.508 CI: 1.127-2.018, p=0.006) and the genotype distribution (p=0.019) of the OPG SNP rs3102735 differed significantly between breast cancer patients and healthy controls. The minor allele C and the corresponding homo- and heterozygous genotypes are more common in breast cancer patients (minor allele C: 18.4% vs. 13.0%; genotype CC: 3.3% vs. 1.3%; genotype CT: 30.3% vs. 23.5%). No significantly changed risk was detected in the other investigated SNPs. Additional analysis showed significant differences when comparing patients with invasive vs. non-invasive tumors (OPG rs2073618) as well as in terms of tumor localization (RANK rs35211496) and body mass index (RANKL rs9533156 and rs1054016).

                  Conclusions

                  This is the first study reporting a significant association of the SNP rs3102735 (OPG) with the susceptibility to develop breast cancer in the Caucasian population.

                  Keywords

                  Breast cancer Case control study OPG Polymorphism RANK RANKL rs3102735

                  Background

                  Breast cancer is one of the most common malignancies in women, leading to distant metastases in patients with advanced disease, particularly in liver, lung and bone. Bone metastases are associated with hypercalcemia, pathologic fracture, spinal cord compression, pain and reduced quality of life [1]. The discovery of receptor activator of NF-κB (RANK), its ligand RANKL and osteoprotegerin (OPG) has contributed significantly to the understanding of the physiological bone turnover. A functional interaction between RANKL, a member of the tumor necrosis factor (TNF) ligand superfamily and RANK, its cognate TNF-receptor is essential for osteoclast differentiation, survival and activation [2].

                  RANKL, a type II homotrimeric transmembrane protein, is expressed by osteoblasts, osteocytes, bone marrow stromal cells, Tcells and various tumor cells, e. g. myeloma and breast cancer [36]. The type-I homotrimeric transmembrane protein RANK is not only expressed by osteoclast, Tcells, dendritic cells, endothelial cells, and mammary glands but also by cancer cells including prostate and breast [711]. RANKL- or RANK-deficient mice develop osteopetrosis resulting from a lack of osteoclasts and absence of bone resorption [12, 13]. OPG is a secreted homodimeric glycoprotein from the TNF receptor family, lacking a transmembrane domain and has homology to the CD40 protein [14]. OPG neutralizes RANKL, which leads to a reduced RANK-RANKL interaction, thus inhibiting osteoclastogenesis [6, 15]. Transgenic mice overexpressing OPG show increased bone mass (osteopetrosis) as a result of reduced osteoclasts [14], whereas OPG-deficient mice are characterized by massive osteoclast activity and osteoporosis [16]. With regard to tumor development, OPG is discussed to be a positive regulator of microvessel formation and to promote neovascularisation [17] and might therefore have an influence on tumor progression. Moreover OPG overexpression by breast cancer cells increased cell proliferation and tumor growth in vivo[18].

                  A disturbed RANKL/OPG ratio was found in a spectrum of skeletal diseases (e. g. rheumatoid arthritis, osteoporosis, bone metastases) characterized by extensive osteoclast activity. Additionally, the RANK/RANKL pathway has intrinsic functionality in mammary epithelium development. Mice that are deficient for RANK or RANKL did not develop lactating mammary gland [8]. Recently, two groups have found that RANKL has not only a fundamental role in the normal physiology of the mammary gland, but may also be crucial for breast cancer development [19, 20]. These data support earlier results, where RANKL was shown to play a role in breast cancer cell migration into bone [21] and underscore the potential use of RANKL inhibition in the prevention of breast cancer development. Based on its pivotal role in the bone remodeling process, RANKL has become a therapeutic target. A monoclonal antibody against RANKL, denosumab, has been approved for the treatment of postmenopausal osteoporosis and bone metastasis in breast cancer [22, 23].

                  In summary, the functional properties of the RANK/RANKL/OPG pathway suggest an important effect of the genes on the pathogenesis of breast cancer. These findings led us to investigate the link between seven single nucleotide polymorphisms (SNPs) in the genes of RANK, RANKL and OPG, all possibly associated with functional alterations, and breast cancer risk.

                  Methods

                  Study populations

                  A total of 703 participants consisting of 307 female breast cancer patients and 396 gender-matched healthy controls were enrolled in this study (Table 1). All patients and controls were of central European Caucasian ethnicity. Breast cancer patients were collected from the Department of Gynecology, Obstetrics and Reproductive Medicine of Saarland University Medical School, Homburg/Saar, Germany. Controls were either recruited from the Departments of Gynecology, Obstetrics and Reproductive Medicine (n=47), Internal Medicine II (n=163) or the Institute for Transfusion Medicine (n=186) of Saarland University Medical School, Homburg/Saar, Germany. The local ethics committee of the Medical Association from the Saarland (reference number: 162/11) approved the study and all individuals in the study gave written informed consent. The study was carried out in compliance with the Helsinki Declaration.
                  Table 1

                  Characteristics of study population

                  Clinical parameters

                  Breast cancer patients (n=307)

                  Healthy controls (n=396)

                  Age (median) in years k

                  56 (22-91)

                  45 (18-88)

                  Menopausal status

                  n=287

                   

                  Premenopausal

                  88 (31%)

                   

                  Postmenopausal

                  179 (62%)

                   

                  Perimenopausal

                  20 (7%)

                   

                  Unknown

                  20

                   

                  Tumor growth

                  n=303

                   

                  Invasive

                  275 (91%)

                   

                  Non-invasive

                  28 (9%)

                   

                  Unknown

                  4

                   

                  Localization

                  n=306

                   

                  Right

                  123 (40%)

                   

                  Left

                  173 (57%)

                   

                  Bilateral

                  10 (3%)

                   

                  Unknown

                  1

                   

                  Type a, b

                  n=255

                   

                  Ductal

                  189 (74%)

                   

                  Lobular

                  34 (13%)

                   

                  Other types

                  32 (13%)

                   

                  Unknown

                  21

                   

                  Tumor size (T) a, b, c

                  n=229

                   

                  T1 (< 2 cm)

                  142 (62%)

                   

                  T2 (>/= 2 cm – 5 cm)

                  76 (33%)

                   

                  T3 (</= 5 cm)

                  6 (3%)

                   

                  T4 (infiltration of the chest

                  5 (2%)

                   

                  wall/skin)

                    

                  Unknown

                  24

                   

                  Nodal status (N) b, c

                  n=250

                   

                  N+

                  75 (30%)

                   

                  N-

                  175 (70%)

                   

                  Unknown

                  36

                   

                  Distant metastases (M)

                  n=292

                   

                  M+

                  16 (5%)

                   

                  osseous

                  10 (3%)

                   

                  M-

                  276 (95%)

                   

                  Unknown

                  15

                   

                  Tumor grading (G)

                  n=245

                   

                  G1

                  16 (6%)

                   

                  G2

                  161 (63%)

                   

                  G3

                  78 (31%)

                   

                  Unknown

                  49

                   

                  Estrogen receptor (ER) d

                  n=275

                   

                  ER+

                  224 (81%)

                   

                  ER-

                  51 (19%)

                   

                  Unknown

                  32

                   

                  Progesterone receptor (PR) b, d

                  n=274

                   

                  PR+

                  193 (70%)

                   

                  PR-

                  81 (30%)

                   

                  Unknown

                  32

                   

                  Her-2 a, b, e

                  n=208

                   

                  Her2+

                  42 (20%)

                   

                  Her2-

                  166 (80%)

                   

                  Unknown

                  67

                   

                  Ki67 a, b, f

                  n=187

                   

                  Ki67+

                  84 (45%)

                   

                  Ki67-

                  103 (55%)

                   

                  Unknown

                  88

                   

                  CEA f

                  n=107

                   

                  CEA+

                  26 (24%)

                   

                  CEA-

                  81 (76%)

                   

                  Unknown

                  200

                   

                  CA15-3 h

                  n=215

                   

                  CA15-3+

                  81 (38%)

                   

                  CA15-3-

                  134 (62%)

                   

                  Unknown

                  92

                   

                  Body mass index (BMI) m

                  n=219

                   

                  BMI < 28

                  150 (68%)

                   

                  BMI >/= 28

                  69 (32%)

                   

                  Unknown

                  88

                   

                  Subgroup a, i

                  n=249

                   

                  Triple negative

                  22 (9%)

                   

                  Non triple negative

                  227 (91%)

                   

                  Unknown

                  30

                   

                  Subgroup a, j

                  n=262

                   

                  Risk group

                  18 (7%)

                   

                  Non risk group

                  244 (93%)

                   

                  Unknown

                  15

                   

                  aOnly invasive tumors are included; bBilateral tumors are only included if both sides had the same result; cExclusion of cases with neoadjuvant chemotherapy; dImmunoreactive score: 0: negative, 1-12: positive; eHer2 = human epidermal growth factor receptor 2; immunoreactive score 0-2 (FISH negative): negative, 2 (FISH positive)-3: positive; fKi67 = marker for proliferation (< 13%: negative, >/= 13%: positive); gCEA = carcinoembryonic antigen (tumor marker, < 3 ng/ml: negative, >/= 3 ng/ml: positive); hCA15-3 = tumor marker (< 21 U/ml: negative, >/= 21 U/ml: positive); iTriple negative = ER, PR and Her2 negative; jRisk group: T >/= 2, G3, ER negative; FISH = fluorescence in situ hybridization; ksignificant difference (p< 0.001), age-adjusted statistical analysis performed; mBMI >/= 28 was defined as overweight in order to age-adjustment [https://​www.​uni-hohenheim.​de/​wwwin140/​info/​interaktives/​bmi.​htm].

                  Case patients were diagnosed as unambiguously having breast cancer through standard clinical and histological findings. Specific cancer characteristics such as histological subtypes, grading, metastasis were not used as a criterion for the inclusion or exclusion of samples.

                  SNP selection

                  The three genes of interest together span more than 120 kb pairs and show only weak to moderate linkage-disequilibrium patterns according to the HapMap data. We have preferentially selected SNPs which might be functionally relevant, either by their location within a potentially regulatory region (3’ untranslated or promoter region, intron-exon boundary) or by altering the amino acid sequence (missense mutation). A total of seven SNPs were analyzed, two within the OPG (rs3102735, rs2073618) and RANK (rs1805034, rs35211496) gene, respectively, and three within the RANKL gene (rs9533156, rs2277438, rs1054016). Table 2 summarizes the chromosomal position and function of the selected SNPs.
                  Table 2

                  Selected SNPs for genotyping

                  Gene

                  SNP number

                  SNP position

                  Allele [major/minor]

                  Function

                  OPG

                  rs3102735

                  chr8: 119965070

                  T/C

                  Transition substitution (5’ near region)

                  OPG

                  rs2073618

                  chr8: 119964052

                  G/C

                  Missense (p.K3N)

                  RANK

                  rs1805034

                  chr18: 60027241

                  T/C

                  Missense (p.V192A)

                  RANK

                  rs35211496

                  chr18: 60021761

                  C/T

                  Missense (p.H141Y)

                  RANKL

                  rs9533156

                  chr13: 43147671

                  T/C

                  Transition substitution (5’ near region)

                  RANKL

                  rs2277438

                  chr13: 43155168

                  A/G

                  Transition substitution (intron1/exon2 boundary)

                  RANKL

                  rs1054016

                  chr13: 43182002

                  G/T

                  Transversion substitution (3’ UTR)

                  RANK = receptor activator of nuclear factor-κB; RANKL = RANK ligand; SNP = single nucleotide polymorphism; OPG = osteoprotegerin.

                  Genomic DNA extraction and Genotyping

                  Genomic DNA was isolated from peripheral blood lymphocytes using QIAamp DNA Blood Mini Kit according to the manufacturer’s protocols (Qiagen, Hilden, Germany). DNA quantity was assessed spectrophotometrically with the Nanodrop ND 1000 (Peqlab, Erlangen, Germany). All SNPs were genotyped using commercial TaqMan assays (assay IDs: rs3102735: C_1971046_10; rs2073618: C_1971047_1; rs1805034: C_8685532_20; rs35211496: C_25473190_10; rs9533156: C_30009803_10; rs2277438: C_25473654_10; rs1054016: C_7444426_10) with TaqMan Genotyping Master Mix on a 7500 real-time PCR cycler (Life Technologies, Darmstadt, Germany) by following the manufacturer’s instructions.

                  Statistical analyses

                  Hardy-Weinberg equilibrium was assessed in each cohort by comparing the observed genotype distribution with the expected one using a χ2-test (Institute of Human Genetic, Munich, Germany: http://​www.​ihg.​gsf.​de/​). The difference in allele and genotype frequencies between patients and healthy controls (as well as between different subgroups) were analyzed using χ2-tests for 2 x 2 and 2 x 3 tables, respectively, with Fisher’s exact test. Differences in allele frequencies were quantified by odds ratios (OR) and 95% confidence intervals (CI). With regard to significantly elder breast cancer patients than healthy controls age-adjusted covariate analysis was performed. All p-values are two-sided and p-values <0.05 were considered as statistically significant. All statistical analyses were performed using the SPSS statistical software. Finally, a power analysis was performed using the G power 3.1.3 software. To the best of our knowledge no adjustment for multiple testing was made because analyses were considered exploratory and needing confirmation by an independent set of data. Previous studies have demonstrated that the analyzed SNPs only show a weak to moderate linkage-disequilibrium patterns according to the HapMap data.

                  Results

                  Subject characteristics

                  The mean age was 56 years (range 22-91) for the breast cancer patients and 45 (range 18-88) for the healthy controls showing significant difference. Clinical data (e. g. menopausal status, body mass index (BMI)) and specific cancer characteristics such as localization, histological subtypes, tumor size, metastasis, grading, proliferation index as well as hormone receptor and Her2 expression are listed in Table 1. The tumor markers carcinoembryonic antigen (CEA) and CA15-3 were measured routinely in the blood of preoperative patients. Invasive ductal carcinomas (74%) with a size smaller 2 cm (T1, 62%) and without metastases (nodal negative: 70%, no distant metastases: 95%) at first diagnosis were most frequently. Additionally, most tumors expressed estrogen (81%) and progesterone receptors (70%), as expected, while Her2 was negative in most cases (80%) (Table 1).

                  Allele and genotype frequencies and risk of breast cancer

                  The genotype distributions for all seven SNPs were in the Hardy-Weinberg equilibrium. Table 3 summarizes the results of all SNP analyses in the genes encoding for OPG (rs3102735, rs2073618), RANK (rs1805034, rs35211496) and RANKL (rs9533156, rs2277438, rs1054016). Allelic and genotype frequencies in breast cancer patients were compared to healthy controls.
                  Table 3

                  Association of allele and genotype frequencies of OPG , RANK and RANKL in patients with breast cancer and healthy controls

                  SNP

                  Alleles / Genotypes

                  Breast cancer

                  Healthy controls

                  OR (95% CI)

                  p-value*

                  OPG rs3102735

                   

                  n=614 (%)

                  n=784 (%)

                    

                  Alleles

                  C

                  113 (18.4%)

                  102 (13.0%)

                  1.508

                  0.006

                   

                  T

                  501 (81.6%)

                  682 (87.0%)

                  (1.127-2.018)

                   
                    

                  n=307 (%)

                  n=392 (%)

                    

                  Genotypes

                  CC

                  10 (3.3%)

                  5 (1.3%)

                   

                  0.019

                   

                  CT

                  93 (30.3%)

                  92 (23.5%)

                    
                   

                  TT

                  204 (66.4%)

                  295 (75.3%)

                    

                  OPG rs2073618

                   

                  n=614 (%)

                  n=786 (%)

                    

                  Alleles

                  C

                  269 (43.8%)

                  357 (45.4%)

                  0.937

                  0.552

                   

                  G

                  345 (56.2%)

                  429 (54.6%)

                  (0.758-1.159)

                   
                    

                  n=307 (%)

                  n=393 (%)

                    

                  Genotypes

                  CC

                  57 (18.6%)

                  77 (19.6%)

                   

                  0.810

                   

                  CG

                  155 (50.5%)

                  203 (51.7%)

                    
                   

                  GG

                  95 (30.9%)

                  113 (29.7%)

                    

                  RANK rs1805034

                   

                  n=614 (%)

                  n=790 (%)

                    

                  Alleles

                  C

                  291 (47.4%)

                  362 (45.8%)

                  1.065

                  0.590

                   

                  T

                  323 (52.6%)

                  428 (54.2%)

                  (0.862-1.316)

                   
                    

                  n=307 (%)

                  n=395 (%)

                    

                  Genotypes

                  CC

                  73 (23.8%)

                  78 (19.7%)

                   

                  0.334

                   

                  CT

                  145 (47.2%)

                  206 (52.2%)

                    
                   

                  TT

                  89 (29.0%)

                  111 (28.1%)

                    

                  RANK rs35211496

                   

                  n=614 (%)

                  n=792 (%)

                    

                  Alleles

                  T

                  122 (19.9%)

                  141 (17.8%)

                  1.145

                  0.335

                   

                  C

                  492 (80.1%)

                  651 (82.2%)

                  (0.875-1.499)

                   
                    

                  n=307 (%)

                  n=396 (%)

                    

                  Genotypes

                  TT

                  12 (3.9%)

                  9 (2.3%)

                   

                  0.423

                   

                  TC

                  98 (31.9%)

                  123 (31.1%)

                    
                   

                  CC

                  197 (64.2%)

                  264 (66.7%)

                    

                  RANKL rs9533156

                   

                  n=614 (%)

                  n=788 (%)

                    

                  Alleles

                  C

                  280 (45.6%)

                  369 (46.8%)

                  0.952

                  0.666

                   

                  T

                  334 (54.4%)

                  419 (53.2%)

                  (0.770-1.176)

                   
                    

                  n=307 (%)

                  n=394 (%)

                    

                  Genotypes

                  CC

                  68 (22.1%)

                  82 (20.8%)

                   

                  0.387

                   

                  CT

                  144 (46.9%)

                  205 (52.0%)

                    
                   

                  TT

                  95 (30.9%)

                  107 (27.2%)

                    

                  RANKL rs2277438

                   

                  n=614 (%)

                  n=788 (%)

                    

                  Alleles

                  G

                  109 (17.8%)

                  132 (16.8%)

                  1.073

                  0.669

                   

                  A

                  505 (82.2%)

                  656 (83.2%)

                  (0.812-1.418)

                   
                    

                  n=307 (%)

                  n=394 (%)

                    

                  Genotypes

                  GG

                  8 (2.6%)

                  9 (2.3%)

                   

                  0.866

                   

                  GA

                  93 (30.3%)

                  114 (28.9%)

                    
                   

                  AA

                  206 (67.1%)

                  271 (68.8%)

                    

                  RANKL rs1054016

                   

                  n=614 (%)

                  n=786 (%)

                    

                  Alleles

                  T

                  258 (42.0%)

                  345 (43.9%)

                  0.927

                  0.514

                   

                  G

                  356 (58.0%)

                  441 (56.1%)

                  (0.749-1.147)

                   
                    

                  n=307 (%)

                  n=393 (%)

                    

                  Genotypes

                  TT

                  57 (18.6%)

                  73 (18.6%)

                   

                  0.543

                   

                  TG

                  144 (46.9%)

                  199 (50.6%)

                    
                   

                  GG

                  106 (34.5%)

                  121 (30.8%)

                    

                  CI = confidence intervals; RANK = receptor activator of nuclear factor-κB; RANKL = RANK ligand; OPG = osteoprotegerin; OR = odds ratio; *χ2-tests for 2x2 tables (alleles) and for 2x3 tables (genotypes), respectively.

                  The allelic frequencies (OR: 1.508 CI: 1.127-2.018, p=0.006) as well as the genotype distribution (p=0.019) of the OPG SNP rs3102735 differed significantly between breast cancer patients and healthy controls. The minor allele C was more frequent in breast cancer patients (18.4%) compared to the control group (13.0%). In addition, the homozygous genotype CC of the minor allele as well as the heterozygous genotype CT were more frequent in the breast cancer group (3.3% and 30.3%) compared to the controls (1.3% and 23.5%) (Table 3). The power analysis revealed a power of 0.79 for the allele frequency and 0.72 for the genotype distribution to detect dependencies (α = 0.05) (Additional file 1: Figure S1). Further statistical analysis revealed that the heterozygous genotype CT as well as the homozygous genotype CC together with the heterozygous genotype CT versus the homozygous genotype TT of the major allele significantly differed between breast cancer patients and controls (CT vs. TT: OR: 1.462, CI 1.042-2.052, p=0.030; [CC + CT] vs. TT: OR: 1.536, CI 1.104-2.135, p=0.011). Due to significantly differences in the median age between controls and breast cancer patients (Table 1) we confirmed these data with a logistic regression using age as a covariate (p=0.005).

                  No significant differences in the allele frequencies and genotype distributions were found, when the breast cancer patients were compared with the controls for the other SNPs analyzed in this study.

                  Association between SNPs within different breast cancer subgroups

                  Next we examined the association between the distribution of genotypes and allelic frequencies of all analyzed SNPs and clinicopathological data including tumor localization, histological subtypes, tumor size, metastasis, grading, proliferation index, hormone receptor expression, Her2 expression, tumor marker level, menopausal status as well as body mass index at the time of diagnosis (Table 1).

                  Regarding the two OPG SNPs the most interesting result was the significant difference in genotype distribution and allelic frequency of OPG rs2073618 between invasive versus non invasive tumors. The homozygous major genotype GG (31.3% vs. 21.4%, p=0.006) as well as the major allele G (57.5% vs. 39.3%, OR 2.088 CI 1.189-3.663, p=0.011) were more frequent in patients with invasive tumors in contrast to non-invasive ones (Table 4).
                  Table 4

                  Association of allele and genotype frequencies within selected breast cancer subgroups

                  SNP

                  Alleles

                  Genotypes

                  OPG rs2073618

                  G

                  C

                  GG

                  CG

                  CC

                   Invasive tumors

                  316 (57.5%)

                  234 (42.5%)

                  86 (31.3%)

                  144 (52.4%)

                  45 (16.4%)

                   Non-invasive tumors

                  22 (39.3%)

                  34 (60.7%)

                  6 (21.4%)

                  10 (35.7%)

                  12 (42.9%)

                   OR (95%CI) p-value*

                  2.088 (1.189-3.663) p=0.011

                  p=0.006

                  RANK rs35211496

                  T

                  C

                  TT

                  TC

                  CC

                   right breasta

                  62 (25.2%)

                  184 (74.8%)

                  9 (7.3%)

                  44 (35.8%)

                  70 (56.9)

                   left breasta

                  53 (15.3%)

                  293 (84.7%)

                  3 (1.7%)

                  47 (27.2%)

                  123 (71.1%)

                   OR (95%CI) p-value*

                  1.863 (1.236-2.808) p=0.003

                  p=0.009

                  RANKL rs9533156

                  C

                  T

                  CC

                  CT

                  TT

                   BMI >/=28

                  70 (50.7%)

                  68 (49.3%)

                  22 (31.9%)

                  26 (37.7%)

                  21 (30.4%)

                   BMI <28

                  120 (40%)

                  180 (60%)

                  24 (16.0%)

                  72 (48.0%)

                  54 (36.0%)

                   OR (95%CI) p-value*

                  1.543 (1.029-2.315) p=0.038

                  p=0.032

                  RANKL rs1054016

                  T

                  G

                  TT

                  TG

                  GG

                   BMI >/=28

                  66 (47.8%)

                  72 (52.2%)

                  20 (29.0%)

                  26 (37.7%)

                  23 (33.3%)

                   BMI <28

                  108 (36.0%)

                  192 (64.0%)

                  19 (12.7%)

                  70 (46.7%)

                  61 (40.7%)

                   OR (95%CI) p-value*

                  1.630 (1.083-2.453) p=0.021

                  p=0.018

                  BMI = body mass index; CI = confidence intervals; RANK = receptor activator of nuclear factor-κB; RANKL = RANK ligand; OPG = osteoprotegerin; OR = odds ratio; *χ2-tests for 2x2 (alleles) and 2x3 (genotypes) tables, respectively; aExclusion of cases with bilateral tumor involvement.

                  Data not shown concerning the remaining SNPs stratified into further subgroups according to Table 1.

                  Another important difference was found with respect to the genotype distribution as well as the allelic frequency comparing the tumor localization (right breast vs. left breast) for the RANK SNP rs35211496. The homozygous minor allele T (25.2% vs. 15.3% OR 1.863 CI 1.236-2.808, p=0.003) and the minor allele genotype TT (7.3% vs. 1.7%, p=0.009) were more frequent in patients with tumor involvement of the right breast in contrast to the left side (Table 4).

                  The allelic frequencies (rs9533156: OR 1.543 CI 1.029-2.315, p=0.038; rs1054016: OR 1.630 CI 1.083-2.453, p=0.021) as well as the genotype distribution (rs9533156: p=0.032; rs1054016: p=0.018) of the RANKL SNPs rs9533156 and rs1054016 differed significantly between patients with a higher BMI (>/= 28) compared to patients with a lower BMI (< 28) at the first diagnosis. The minor allele C for SNP rs9533156 and T concerning the SNP rs1054016 were more frequent in patients with a BMI >/= 28 (rs9533156: 50.7%; rs1054016: 47.8%) compared to patients with a lower BMI (rs9533156: 40%, rs1054016: 36%; Table 4).

                  No significant differences in the allele frequencies and genotype distributions were found in the different subgroup analyses (including distant metastases) for the remaining analyzed SNPs (data not shown).

                  Discussion

                  To the best of our knowledge, this is the first study showing a significant association between the SNP rs3102735 of the OPG gene and the susceptibility of breast cancer in Caucasian populations. For the SNP rs3102735 containing the minor allele C as well as for the homo- and heterozygous genotype with the minor allele C, we observed a 1.5-fold increased risk of breast cancer. All other SNPs were not associated with an increased risk for breast cancer. These results suggest a role for the OPG gene polymorphism in relation to breast cancer development.

                  Previous studies showed that genetic variants in the OPG locus have been associated with differences in bone mineral density (BMD; [2433], osteoporotic fractures [28, 34], bone turnover [31], bisphosphonate-induced osteonecrosis of the jaw [35], calcaneal quantitative ultrasound (velocity of sound) [36], ankylosing spondylitis development [37] and diabetic charcot neuroarthropathy [38].

                  In detail, concerning the rs3102735 SNP the G allele was more common among fracture patients [28, 34] and patients with lower BMD at the distal radius [30]. Furthermore, there is an association within a subgroup of postmenopausal patients carrying the minor allele and a lower calcaneal velocity of sound [36]. In an earlier study the variation (rs3102735) within the OPG gene showed a trend with higher frequency of the minor allele (p=0.076) and responding genotypes (p=0.097) in patients with psoriasis compared to controls without reaching significance [39].

                  Recently, several genome wide association studies or studies of specific candidate SNPs revealed additional loci to be associated with breast cancer including the same chromosomal region 8q24 as for the OPG gene [4049]. The majority of the association on chromosome 8q24 lies at approximately 128 Mb and is related to several tumor entities (prostate [50], colon [51]) in addition to breast cancer. Each locus within the 128 Mb bears epigenetic enhancer elements and forms chromatin loops with the myc proto-oncogene located several hundred kilobases telomeric [52]. A recent meta-analysis revealed an additional locus around 120 Mb on chromosome 8 associated with cancer development [53]. This region is close to the locus of OPG rs3102735 SNP (chromosome 8q24 119.965.070), which is associated with breast cancer in our study.

                  In this context we found a second genetic variation within the rs2073618 SNP of the OPG gene when stratifying our breast cancer patients into the subgroups of invasive or non-invasive tumors. However, the impact of the SNPs rs3102735 (5’ near promoter region) and rs2073618, located in the first exon, which encodes the signal peptide of OPG, are still unclear. Zhao et al. discussed that the change of the third amino acid from lysine (basic amino acid) to asparagine (uncharged polar amino acid) may have an influence of the OPG secretion from the cells. In their study they found that patients carrying the CC genotype had lower serum level of OPG [33]. In another study, a mutation in a basic amino acid (arginin) in the signal peptide of angiotensinogen drastically affected the secretory kinetics [54]. However, the exact mechanism that the SNP rs2073618 possibly affects the secretory characteristics of OPG needs to be elucidated by further functional studies. Genetic variation within the promoter region of OPG (rs3102735) could have an effect on the OPG gene expression and thus an influence on tumor development.

                  Further subgroup analyses according to clinical parameters showed an association with BMI (<28 or >/=28). In general, increased BMI is associated with the risk of some cancers and might differ between sexes and different ethnic populations such as breast cancer [55]. Combined studies revealed that the increase in breast cancer risk with increasing BMI among postmenopausal women is mostly depending on associated increase in bioavailable estradiol [56]. Here we show that the minor allele as well as the genotype of the minor allele of the RANKL SNPs rs9533156 and rs1054016 were strongly associated with a higher BMI (>/= 28) in the breast cancer group. Whether obese patients carrying the minor allele from one of the two RANKL SNPs have an additionally a higher risk of developing breast cancer remains open in this study due to the lack of BMI data from the control group.

                  Moreover, we confirmed an asymmetry of breast carcinoma to the left side (57% vs. 40%, Table 1) in accordance with several other studies, which revealed asymmetries in paired organs including breast [57, 58], the lungs [59], kidney [60] and testes [61]. Especially for the unsymmetric incidence of breast cancer in favour of the left side, several possible explanations are discussed, including the sleeping habit [62], handedness [63], the preference for breast feeding [64] or breast size [63]. We found that a genetic variation within the rs35211496 RANK SNP could have an influence on the tumor localization. Whether this polymorphism has a direct effect on the unsymmetric incidence or indirectly via the breast size can not be answered from this study.

                  The subgroup analyses stratified into metastatic disease at initial diagnosis showed no significant differences in genotype or allelic distribution. Only 10 of 292 patients were primarily diagnosed with bone metastases. Further studies focusing on skeletal metastases with respect to genetic background are required.

                  Other genetic variants at the RANK locus and/or functionally related genes, including RANKL have been associated with differences in bone mineral density [31], rheumatoid arthritis [65, 66], aortic calcification [67], age at menarche [68] or Paget′s disease of bone [69]. There is one recent study which showed a genetic variant near the 5′-end of RANK (rs7226991) associated with a breast cancer risk [70].

                  Conclusion

                  Our case-control study points to an association of the OPG SNP rs3102735 with an increased risk of developing breast cancer. These results could extend the constellation of possible breast cancer risk and might affect early diagnosis.

                  Future studies are needed, including confirmation of our observation in an independent validation set, to determine the relationship between OPG rs3102735 SNP and breast cancer risk in other ethnic groups. Whether this SNP leads to a functional alteration of OPG expression and consequently to an altered RANKL level remains to be shown.

                  Abbreviations

                  BMD: 

                  bone mineral density

                  BMI: 

                  body mass index

                  CEA: 

                  carcinoembryonic antigen

                  CI: 

                  confidence intervals

                  DF: 

                  degree of freedom

                  ER: 

                  estrogen receptor

                  FISH: 

                  fluorescence in situ hybridization

                  G: 

                  tumor grading

                  Her2: 

                  human epidermal growth factor receptor 2

                  M: 

                  distant metastases

                  N: 

                  nodal status

                  OPG: 

                  osteoprotegerin

                  OR: 

                  odds ratio

                  PR: 

                  progesterone receptor

                  RANK: 

                  receptor activator of NF-κB

                  RANKL: 

                  receptor activator of NF-κB ligand

                  SNP: 

                  single nucleotide polymorphism

                  T: 

                  tumor size

                  TNF: 

                  tumor necrosis factor.

                  Declarations

                  Acknowledgments

                  We thank Wilhelmine Daub for her technical assistance and Miriam Langhirt for her expert advice for the implementation of the genotyping assays. We also thank the Center of Pediatrics and Neonatology, University Medical School of Saarland, especially Dominik Monz, PhD, for providing of laboratory equipment and helpful discussions. We thank Sebastian Wieczorek for providing healthy controls.

                  This work was supported in part by research grants from Abbott (Wiesbaden, Germany) and research grants from the Universitiy of Saarland (Saarbruecken, Germany).

                  Authors’ Affiliations

                  (1)
                  Gynecology, Obstetrics and Reproductive Medicine, University Medical School of Saarland
                  (2)
                  Internal Medicine II, University Medical School of Saarland
                  (3)
                  Institute of Medical Biometry, Epidemiology and Medical Informatics, Saarland University
                  (4)
                  General and Surgical Pathology, University Medical School of Saarland
                  (5)
                  Internal Medicine I, José-Carreras-Center for Immuno- and Gene Therapy, University Medical School of Saarland
                  (6)
                  Geburtshilfe und Reproduktionsmedizin, Kirrbergerstr, Universitätsklinikum des Saarlandes, Klinik für Frauenheilkunde

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                  71. Pre-publication history

                    1. The pre-publication history for this paper can be accessed here:http://​www.​biomedcentral.​com/​1471-2407/​13/​40/​prepub

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