Short abstract
Objective
The aim of this study was to determine whether a novel polymorphism (Tru9I) in the low penetrance vitamin D receptor (VDR) gene is associated with risk of premenopausal breast cancer (BC).
Methods
This case-control study included 228 patients with BC and 503 healthy women living in Pakistan who were analyzed for the VDR Tru9I (rs757343) single nucleotide polymorphism. BC cases were histopathologically confirmed, and all healthy controls were age-matched with patients (age range, 20–45 years). DNA was extracted, and the polymerase chain reaction and restriction fragment length polymorphism assays were performed.
Results
The VDR Tru9I polymorphism was not significantly associated with premenopausal BC. However, the risk of BC was associated with the ‘uu’ genotype (odds ratio [OR], 1.141; 95% confidence interval [95% CI], 0.206–6.317). Further, mutant Tru9I was significantly associated with Grade IV carcinoma (OR, 5.36; 95% CI, 1.181–24.338).
Conclusion
The VDR Tru9I ‘uu’ genotype may increase the risk of premenopausal BC.
Keywords: Vitamin D receptor, Tru9I, rs757343, premenopausal, breast cancer, Pakistan
Introduction
Breast cancer (BC) is the most prevalent cancer and a major cause of death among women worldwide. In Pakistan, the incidence of BC is approximately 34.6% in women.1 Among risk factors, lifestyle, alcoholism, high birth weight, and abdominal adiposity intensify the risk of BC.2–4 Diet may modify the incidence of BC,5 although many dietary components such as vitamin D are categorized as “Limited or no conclusion.”6 Vitamin D plays a major role in mineral homeostasis;6 however, evidence indicates that its deficiency exacerbates the risk of BC.7,8 Moreover, higher vitamin D levels are weakly associated with the low BC risk”.9,10
Vitamin D derived from diet and sun exposure of the skin undergoes activation within the liver through 25 hydroxylation, and in the kidney through 1-α hydroxylation, to form 1,25(OH)2D3 (active vitamin D). The classical role of vitamin D is to regulate calcium and phosphate metabolism to maintain bone mineral density (BMD). However, there are wide ranges of nonclassical functions of vitamin D within the body. Vitamin D is activated by forming a complex with the vitamin D receptor (VDR) that is involved in the regulation of many target genes that contribute to cell differentiation, cell growth, programmed cell death, angiogenesis, inflammation, and immune responses.11,12 Vitamin D, in concert with growth factors, arrests the cell cycle by preventing entry into S phase.13 Vitamin D induces apoptosis through down-regulation of the antiapoptotic protein B-cell lymphoma 2 (BCL2),13 inhibits the expression of P-cadherin14 and increases the expression E-cadherin,15 which may contribute to the anti-invasive or antimetastatic effects of vitamin D. Further, vitamin D induces the expression of the tumor suppressor BRCA1, because the binding sites for the vitamin D/VDR/retinoid X receptor heterodimer complex are present within the BRCA1 promoter region.16 Although VDR is present in many body tissues, including normal and cancerous breast tissues, its expression is low in mammary cancers cells,17 which may be caused by variations in VDR.
There are many polymorphic regions in VDR, among which Fok1, Bsm1, Apa1, and Taq1 are the most extensively studied in American (Hispanic white, nonhispanic white, and Caucasian) and Asians (Iranian and Egyptian) populations.19–24 Attention focuses on the role of single nucleotide polymorphisms (SNPs) in VDR that are associated with the development of a wide range of pathological conditions, including type 2 diabetes mellitus, prostate cancer, and BC18–20. However, limited studies are available on the Pakistani population.25 Studies of Jordanian populations reveal a statistically significant difference between VDR Taq1 variants and 25(OH)D among women with BC.21 Bsm1 and Cdx2 polymorphisms are significantly associated with women with BC living in Southeastern Iran.20 The Fok1 SNP in BC plays a protective role, whereas the Bsm1 SNP is not associated with BC among women living in the Iranian city of Urmia.22 Among Egyptian women, the Bsm1 “B” allele and “Bb” variants of VDR may represent a risk factor for susceptibility to the development of BC.23 In contrast, a meta-analysis shows no association of VDR SNPs (Fok1, Bsm1, Taq1, and Apa1) with risk of BC in a general population or in a Caucasian population.24 Among Pakistanis who are BRCA1/2 non-carriers, the Bsm1 “b” allele is associated with an increased risk of BC”.25 Although the associations of VDR Fok1 and Bsm1 SNPs with the risk of BC have been investigated, no study, to our knowledge, investigated the relationship between the VDR Tru9I polymorphism and the premenopausal risk of BC. Therefore, the present study was designed detect an association between the novel low penetrance VDR Tru9I SNP and BC among premenopausal women of Pakistan.
Materials and methods
Ethics statement
The study was approved by the Bioethics Committee, Board of Advance Studies & Research, University of Karachi (ethics approval number [10(27) 28032012] and conducted in accordance with the Helsinki Declaration. Each subject provided written informed consent before providing a blood sample.
Study design and study population
The study included 228 patients with BC and 503 control subjects who were premenopausal ethnic Pakistanis aged 20–45 years. The sample size was calculated using OpenEpi (http://www.openepi.com/Menu/OE_Menu.htm) with 80% statistical power and 95% confidence intervals (95% CIs). Patients with BC, who were randomly selected from two tertiary hospitals in Sindh, Pakistan from 2012 to 2015, were histopathologically diagnosed. The medical information (types, grades, estrogen receptor, progesterone receptor, human epidermal growth factor receptor 2, and TNM classification) of patients with BC was obtained from histopathological reports and hospital records. All healthy volunteers were age-matched (1:2) to premenopausal healthy subjects acquired from the general population. Whole blood (3 mL) was collected from each subject. We used a self-structured questionnaire to acquire along with information about personal demographics, detailed history, and BC-related risk factors.
DNA isolation
DNA was extracted using a DNA isolation kit (Gene JET Genomic DNA Purification Kit, Thermo Fisher Scientific Baltics UAB, and Vilnius, Lithuania) following the manufacturer’s protocols. Samples were stored at –86°C.
Primer selection and genotyping
VDR Tru9I (rs757343) genotyping was performed using polymerase chain reaction-restriction fragment length polymorphism analysis. The forward primer F: 5’-GCAGGGTACAAAACTTTGGAG-3' and the reverse primer R: 5’-CCTCATCACCGACATCATGTC-3' were used26 to amplify the isolated DNA. Amplification was performed using a thermal cycler (Veriti; Applied Biosystems, Foster City, CA, USA) in a final reaction volume (50 µL) containing 25 µL of 2X GoTaq Green Master Mix (Promega, Madison, WI, USA), 4 µL of 12 pmol of both primers, 5 µL of DNA, and 12 µL of nuclease-free water. Polymerase chain reaction analysis was performed as follows: 94°C, initial denaturation (5 minutes); 40 cycles of denaturation at 94°C (30 s); annealing at 59°C (30 s), and extension at 72°C (30 s). The final extension was performed at 72°C for 7 minutes. The 177-bp amplicons were digested using 5 units of Mse1 (37°C for 1 hour). Amplicons and restriction fragment length polymorphism fragments, which were separated using 2.5% agarose gel electrophoresis, were detected using ethidium bromide and visualized using a ChemiDoc-It2 imaging system with VisionWorks LS Analysis Software (version 7.1) (UVP, Cambridge, UK). The results were coded as U-u, where the Upper case letter is denoted as the absence and lowercase letter is designated as the presence of the restriction site, respectively. The call and concordance rates of the VDR genotypes were 98% and 100%, respectively. The accuracy of the genotypes was determined by subjecting samples ( 15 cases and 15 controls) to nucleotide sequence analysis.
Data analysis
Statistical analysis was performed using SPSS software version 22 (IBM Corporation, Armonk, NY, USA) with significance indicated by P < 0.05. The Shapiro–Wilk and Kolmogorov–Smirnov tests were used to determine whether the variables were normally distributed. The goodness-of-fit (χ2) test was used to test whether the controls exhibited Hardy–Weinberg equilibrium to determine deviation of their genotype distribution. Comparisons between groups of VDR Tru9I genotypic and allelic distributions were calculated using Person’s χ2 test. Odds ratios (ORs) along with 95% CIs were determined using binary and multinomial logistic regression analysis of the Tru9I SNP to determine the risk of BC. The significance of ORs was determined using Wald’s statistics and defined as P < 0.05.
Results
Characteristics of patients with BC
Patients’ characteristics are presented in Table 1, which shows that the parameters were not normally distributed among patients according to the Shapiro–Wilk and Kolmogorov–Smirnov tests (P < 0.01).
Table 1.
Characteristics of patients with BC (N = 228).
| Characteristics | Patients |
|---|---|
| n (%) | |
| Affected area | |
| Right* | 123 (53.9) |
| Left* | 99 (43.2) |
| Both* | 6 (2.6) |
| Types of BC | |
| Invasive ductal carcinoma (IDC)* | 197 (93.4) |
| Invasive lobular carcinoma (ILC)* | 13 (5.7) |
| Ductal carcinoma in situ (DCIS)* | 6 (2.6) |
| Others* | 12 (5.2) |
| Grades of BC | |
| I* | 6 (2.6) |
| II* | 164 (71.9) |
| III* | 45 (19.7) |
| IV* | 7 (3) |
| Both II &III* | 2 (1.3) |
| None* | 4 (1.7) |
| Immunohistochemical classification | |
| Estrogen receptor status | |
| Positive (0 to +9)* | 126 (55.3) |
| Negative (−1 to −2)* | 34 (14.9) |
| Unknown | 68 (29.8) |
| Progesterone receptor status | |
| Positive (0 to +8)* | 114 (5) |
| Negative (−1 to −2)* | 38 (16.6) |
| Unknown | 76 (33.33) |
| Human epidermal growth factor receptor 2 status | |
| Positive (0 to +3)* | 98 (42.9) |
| Negative (−1 to −2)* | 20 (8.7) |
| Unknown | 110 (48.2) |
| TNM classification | |
| (For invasive cases only) | |
| Size of primary tumor (T stage) | |
| Tx* | 2 (0.8) |
| Tis* | 4 (1.7) |
| T1* | 0 (0) |
| T2* | 35 (15.3) |
| T3* | 31 (13.5) |
| T4* | 26 (11.4) |
| No involvement* | 199 (56.5) |
| Metastasis to regional lymph node | |
| (N stage) | |
| Nx* | 6 (2.6) |
| N0* | 23 (10) |
| N1* | 36 (15.7) |
| N2* | 19 (8.3) |
| N3* | 15 (6.5) |
| No involvement* | 129 (56.5) |
| Distant metastasis (M stage) | |
| Mx* | 39 (17.1) |
| M0* | 48 (21.0) |
| M1* | 12 (5.2) |
| Unknown | 129 (56.5) |
BC, breast cancer. *Statistically significant difference (P < 0.05, Shapiro–Wilk or Kolmogorov–Smirnov tests).
Association of the VDR Tru9I SNP with BC
The genotype distributions of Tru9I among patients (228/228) and healthy controls (497/503) are presented in Table 2. The distribution of Tru9I in the control population did not deviate (χ2 = 0.416, P > 0.05) from the Hardy–Weinberg equilibrium. There was no significant difference (χ2 = 0.238) between the VDR Tru9I genotype among patients and controls. However, an 11.2% increase in OR (1.112; 95% CI, 0.202–6.125) was observed among ‘uu’ carriers compared with ‘UU and Uu’ carriers. After adjusting for age, the VDR Tru9I ‘uu’ genotype exhibited a 14.1% increase in the OR (1.141; 95% CI, 0.206–6.317) (Table 2).
Table 2.
VDR Tru9I genotype frequencies and associations with the risk of premenopausal BC.
| Genotypes | Alternate designation | BC cases n (%) |
Controls n (%) |
Χ2 | P value | UOR (95% CI) | AOR (95% CI) |
|---|---|---|---|---|---|---|---|
| GG | UU | 179 (78.5) | 398 (80) | 0.238 | 0.888 | Ref | Ref |
| GA | Uu | 47 (20.6) | 95 (19.1) | 1.1 (0.744–1.627) | 1.085 (0.731–1.611) | ||
| AA | uu | 2 (0.88) | 4 (0.8) | 1.112 (0.202–6.125) | 1.141 (0.206–6.317) | ||
| Total | 228 | 497 | |||||
| GG | UU | 179 (78.5) | 398 (80) | Ref | Ref | ||
| GA+AA | Uu+uu | 49 (21.4) | 99 (19.6) | 1.101 (0.749–1.617) | 1.087 (0.737–1.604) | ||
| Total | 228 | 497 | |||||
| G allele | U allele | 405 (88.8) | 891 (89.6) | 0.223 | 0.637 | Ref | Ref |
| A allele | u allele | 51 (11.1) | 103 (10.3) | 1.089 (0.763–1.554) | 1.086 (0.76–1.55) | ||
| Total | 456 | 994 |
DNA was amplified from 228/228 patients’ samples and 497/503 control subjects’ samples. BC, breast cancer; UOR, unadjusted odds ratio; AOR, odds ratio adjusted for age and time of blood collection, CI, confidence interval.
Association of the VDR Tru9I SNP with specific pathological characteristics of patients with BC
We next investigated the association of the VDR Tru9I SNP with BC risk through immunohistochemical subtyping of BC tissues and TNM classification (Table 3). A direct association with risk of BC was found for at least one ‘u’ allele, which was more evident and significant for G-IV stage carcinoma (OR, 5.36; 95% CI, 1.181–24.338).
Table 3.
Associations of the VDR Tru9I single nucleotide polymorphism and risk of premenopausal BC according to the immunohistochemical and pathological characteristics of patients with BC.
|
Tru9I genotype |
||||
|---|---|---|---|---|
| UU (GG) |
Uu+uu (GA+AA) |
|||
| BC characteristics | Cases/ Controls |
OR (95% CI) |
Cases/ Controls |
OR (95% CI) |
| Types of BC | ||||
| IDC | 152/398 | Ref | 45/99 | 1.19 (0.799–1.774) |
| ILC | 13/398 | Ref | 0/99 | 1.67 × 10−9
(1.67 × 10−9–1.67 × 10−9) |
| DCIS | 5/398 | Ref | 1/99 | 0.804 (0.93–6.96) |
| Other | 9/398 | Ref | 3/99 | 1.34 (0.356–5.042) |
| Grades of BC | ||||
| I | 5/398 | Ref | 1/99 | 0.804 (0.093–6.96) |
| II | 131/398 | Ref | 33/99 | 1.02 (0.66–1.578) |
| III | 35/398 | Ref | 10/99 | 1.149 (0.55–2.399) |
| IV | 3/398 | Ref | 4/99 | 5.36 (1.181–24.338)* |
| II & III | 2/398 | Ref | 0/99 | 12 × 10−9
(12 × 10−9–12 × 10−9) |
| Unknown | 3/398 | Ref | 1/99 | 1.34 (0.138–13.02) |
| Immunohistochemical classification | ||||
| Estrogen receptor status | ||||
| Positive (0 to +9) | 103/398 | Ref | 23/99 | 0.898 (0.543–1.484) |
| Negative (−1 to −2) | 25/398 | Ref | 9/99 | 1.447 (0.655–3.199) |
| None | 51/398 | Ref | 17/99 | 1.34 (0.742–2.421) |
| Progesterone receptor status | ||||
| Positive (0 to +8) | 92/398 | Ref | 22/99 | 0.961 (0.575–1.608) |
| Positive (−1 to −2) | 30/398 | Ref | 8/99 | 1.072 (0.477–2.411) |
| None | 57/398 | Ref | 19/99 | 1.34 (0.762–2.355) |
| Human epidermal growth factor receptor 2 status |
||||
| Positive (0 to +3) | 78/398 | Ref | 20/99 | 1.031 (0.602–1.766) |
| Negative (−1 to −2) | 18/398 | Ref | 2/99 | 0.447 (0.102–1.957) |
| None | 83/398 | Ref | 27/99 | 1.308 (0.804–2.128) |
| TNM classification (For invasive cases) | ||||
| Size of primary tumor (T stage) | ||||
| Tx | 1/398 | Ref | 1/99 | 4.681 (0.277–79.215) |
| Tis | 3/398 | Ref | 1/99 | 1.281 (0.13–12.598) |
| T1 | 0/398 | Ref | 0/99 | NC |
| T2 | 27/398 | Ref | 9/99 | 1.412 (0.639–3.121) |
| T3 | 25/398 | Ref | 6/99 | 0.896 (0.354–2.267) |
| T4 | 23/398 | Ref | 3/99 | 0.521 (0.152–1.783) |
| No involvement | 100/398 | Ref | 29/99 | 1.135 (0.706–1.823) |
| Metastasize to regional lymph node (N stage) |
||||
| Nx | 4/398 | Ref | 2/99 | 1.821 (0.325–10.218) |
| N0 | 20/398 | Ref | 3/99 | 0.593 (0.172–2.048) |
| N1 | 25/398 | Ref | 11/99 | 1.796 (0.847–3.806) |
| N2 | 16/398 | Ref | 3/99 | 0.737 (0.209–2.598) |
| N3 | 14/398 | Ref | 1/99 | 0.297 (0.038–2.304) |
| No involvement | 100/398 | Ref | 29/99 | 1.134 (0.706–1.822) |
| Distant metastasis (M stage) | ||||
| Mx | 29/398 | 10/99 | 1.386 (0.654–2.94) | |
| M0 | 39/398 | Ref | 9/99 | 0.928 (0.435–1.979) |
| M1 | 11/398 | Ref | 1/99 | 0.365 (0.047–2.864) |
| No involvement | 100/398 | Ref | 29/99 | 1.166 (0.73–1.862) |
BC, breast cancer; CI, confidence interval; OR, odds ratio. VDR Tru9I “Uu” and “uu” genotypes were combined, because there were very few subjects with “uu.” ORs were calculated using multinomial logistic regression analysis adjusted for age and time of blood collection. *P < 0.05.
Relationship of the VDR Tru9I SNP and risk factors associated with BC
The possible relationship of the Tru9I SNP and other contributing factors to the risk of BC are presented in Table 4. Those with the mutant (Uu+uu) genotype along with Sindhi and Baluchi ethnicity and marital age greater than 20 years were at significantly increased risk of developing BC; however, there were no significant associations with other contributing factors and BC.
Table 4.
Combined associations of the VDR Tru9I single nucleotide polymorphism and factors contributing to the risk of premenopausal BC.
| BC risk factors | Combined |
UU |
Uu+uu |
|||
|---|---|---|---|---|---|---|
| Cases/ Controls |
OR (95% CI) | Cases/ Controls |
OR (95% CI) | Cases/ Controls |
OR (95% CI) | |
| Age (years)# | ||||||
| 20–26 (Younger) | 20/58 | Ref | 16/51 | Ref | 4/5 | Ref |
| 27–33 | 34/78 | 1.264 (0.661–2.418) | 23/53 | 1.383 (0.667–2.914) | 11/23 | 0.598 (0.134–2.675) |
| 34–40 | 110/225 | 1.418 (0.812–2.475) | 83/179 | 1.478 (0.796–2.745) | 27/44 | 0.767 (0.189–3.109) |
| 41–45 (Older) | 64/142 | 1.307 (0.726–2.352) | 57/115 | 1.58 (0.829–3.012) | 7/27 | 0.324 (0.068–1.535) |
| Total | 228/503 | 179/398 | 49/99 | |||
| Ethnicity/Race | ||||||
| Urdu-Speaking | 94/252 | Ref | 78/199 | Ref | 16/51 | Ref |
| Sindhi | 41/12 | 9.41 (4.719–18.767)* | 29/9 | 8.456 (3.809–18.772)* | 12/3 | 14.934 (3.573–62.418)* |
| Panjabi | 20/104 | 0.522 (0.305–0.891)* | 16/86 | 0.493 (0.272–0.897)* | 4/17 | 0.675 (0.193–2.335) |
| Pukhtoon | 18/47 | 0.996 (0.549–1.806) | 15/39 | 0.946 (0.492–1.821) | 3/8 | 0.97 (0.22–4.283) |
| Baluchi | 19/4 | 12.926 (4.265–39.171)* | 12/3. | 10.875 (2.963–39.908)* | 7/1 | 19.323 (2.15–173.641)* |
| Others | 36/84 | 1.17 (0.738–1.854) | 29/62 | 1.188 (0.709–1.992) | 7/19 | 1.179 (0.408–3.41) |
| Total | 228/503 | 179/398 | 49/99 | |||
| Marital status | ||||||
| Yes | 208/363 | Ref | 162/286 | Ref | 45/75 | Ref |
| No | 20/140 | 0.139 (0.071–0.273)* | 16/112 | 0.15 (0.071–0.318)* | 4/24. | 0.111 (0.024–0.524)* |
| Total | 228/503 | 179/398 | 49/99 | |||
| Marital age (years) | ||||||
| <20 | 105/244 | Ref | 84/186 | Ref | 21/56 | Ref |
| 20 to 30 | 95/117 | 3.343 (2.157–5.18)* | 71/98 | 2.48 (1.525–4.033)* | 24/19 | 10.645 (3.473–32.623)* |
| >30 | 8/2. | 20.996 (4.15–106.236)* | 8/2. | 15.914 (3.138–80.698)* | 0/0 | NC |
| Total | 208/363 | 163/286 | 45/75 | |||
| Parity | ||||||
| Parous | 191/342 | Ref | 149/267 | Ref | 40/71 | Ref |
| Nulliparous | 37/161 | 0.347 (0.216–0.557)* | 30/131 | 0.366 (0.216–0.619)* | 9/28 | 0.395 (0.145–1.071) |
| Total | 228/503 | 179/398 | 45/99 | |||
| Age at first child birth (years) | ||||||
| Below 20 | 49/63 | Ref | 41/55 | Ref | 8/8 | Ref |
| 20 to 24 | 81/118 | 0.528 (0.33–0.845)* | 64/87 | 0.515 (0.302–0.879)* | 17/30 | 0.582 (0.208–1.627) |
| 25 to 29 | 39/115 | 0.621 (0.328–1.179) | 30/88 | 0.458 (0.215–0.977)* | 9/26 | 1.533 (0.41–5.731) |
| >29 | 17/43 | 1.218 (0.753–1.969) | 11/36 | 1.086 (0.639–1.846) | 6/7 | 2.006 (0.604–6.661) |
| Total | 186/339 | 146/266 | 40/71 | |||
| History of breast feeding | ||||||
| Yes | 186/339 | Ref | 146/266 | Ref | 40/71 | Ref |
| No | 22/24 | 1.674 (0.901–3.107) | 17/20 | 1.54 (0.773–3.068) | 5/4 | 2.269 (0.528–9.748) |
| Total | 208/363 | 163/286 | 45/75 | |||
| Age at menarche (years) | ||||||
| 12 to 14 | 186/423 | Ref | 148/334 | Ref | 38/83 | Ref |
| >14 | 28/45 | 0.913 (0.479–1.74) | 13/29 | 0.995 (0.5–1.978) | 1/6 | 0.306 (0.035–2.662) |
| <12 | 14/35 | 1.389 (0.838–2.303) | 18/35 | 1.111 (0.606–2.035) | 10/10 | 2.567 (0.946–6.966) |
| Total | 228/503 | 179/398 | 49/99 | |||
| Use of hormone replacement therapy | ||||||
| No | 209/481 | Ref | 168/382 | Ref | 41/93 | Ref |
| Yes | 19/22 | 1.954 (1.034–3.691)* | 11/16. | 1.547 (0.702–3.412) | 8/6 | 3.114 (0.99–9.798) |
| Total | 228/503 | 179/398 | 49/99 | |||
| Use of oral contraceptive | ||||||
| No | 197/454 | Ref | 155/361 | Ref | 42/87 | Ref |
| Yes | 31/49 | 1.473 (0.91–2.383) | 24/37 | 1.521 (0.878–2.635) | 7/12 | 1.17 (0.423–3.234) |
| Total | 228/503 | 179/398 | 49/99 | |||
| Waist to hip ratio | ||||||
| Good (<0.8) | 27/92 | Ref | 20/71 | Ref | 7/19 | Ref |
| Average (0.8–0.85) | 15/173 | 0.296 (0.15–0.585)* | 12/134 | 0.317 (0.146–0.685)* | 3/37 | 0.252 (0.057–1.12) |
| High (>0.85) | 186/238 | 2.687 (1.672–4.317)* | 147/193 | 2.727 (1.58–4.707)* | 39/43 | 2.568 (0.949–6.944) |
| Total | 228/503 | 179/398 | 49/99 | |||
| BMI (kg/m2) | ||||||
| Normal/healthy weight (18.6–23) |
56/157 | Ref | 43/127 | Ref | 13/29 | Ref |
| Underweight (>18.5) | 22/64 | 0.966 (0.544–1.717) | 19/48 | 1.196 (0.633–2.261) | 3/14 | 0.383 (0.09–1.637) |
| Overweight (23.1–27) | 79/158 | 1.381 (0.909–2.097) | 63/132 | 1.374 (0.858–2.2) | 16/23 | 1.689 (0.654–4.364) |
| Obese (>27) | 71/124 | 1.589 (1.028–2.456) | 54/91 | 1.708 (1.036–2.815)* | 17/33 | 1.183 (0.474–2.953) |
| Total | 228/503 | 179/398 | 49/99 | |||
| Family history of BC | ||||||
| No | 191/455 | 149/361 | Ref | 42/88 | Ref | |
| Yes | 37/48 | 1.83 (1.153–2.906)* | 30/37 | 1.947 (1.156–3.28)* | 7//11 | 1.348 (0.473–3.841) |
| Total | 228/503 | 179/398 | 49/99 | |||
VDR Tru9I “Uu” and “uu” genotypes were combined because there were very few subjects in the “uu” category. DNA was amplified from 228/228 patients’ samples and 497/503 control subjects’ samples. ORs were adjusted for age and time of blood collection. *P <0.05.
#Crude/unadjusted ORs for age were calculated. BC, breast cancer; BMI, body mass index; NC, not calculated, OR, odds ratio.
Nine models of contributing factors were prepared in which the Tru9I genotype was adjusted by sequentially adding individual contributing factors (Appendix 1). This analysis shows that the Tru9I ‘uu’ genotype in model 8 significantly increased the risk of BC (OR, 1.288; 95% CI, 0.16–10.398).
Discussion
Among Asian countries, Pakistan has the highest incidence of BC,27 which is a heterogeneous group of diseases associated with various risk factors. The most common risk factor is exposure to endogenous hormones such as estradiol.28 Approximately 15% of BC is caused by genetic changes,29 including those associated with high penetrance genes such as BRCA 1/2,30 and low-to-moderate penetrance genes such as checkpoint kinase-2 (CHEK2) and VDR.20,31 VDR contributes to the prevention of BC, and the vitamin D/VDR complex induces apoptosis, reduces angiogenesis, proliferation, and invasion and increases cell differentiation.32
Evidence indicates that the VDR mediates most of the activities of vitamin D. Analyses of common VDR SNPs (Fok1, Bsm1, Apa1, Taq1, and Poly-A) show associations between VDR SNPs and BC, although the results are inconsistent among different types of exposure.20,22,33,34 The Fok1 ‘ff’ genotype may increase the risk of BC.35,36 However, evidence suggests that Fok1 significantly correlates with human epidermal growth factor receptor 2 status20 and is significantly associated with estrogen receptor status of patients with BC.37 One study shows that the Bsm1 ‘b’ allele is associated with risk of BC;20 although another study reports the opposite results.22 To our knowledge, the present study is the first to determine the association of the low penetrance VDR Tru9I polymorphism with premenopausal BC. The VDR Tru9I is caused by an A to G substitution that was first identified in 2000.38 VDR Tru9I resides in intron 8, +443-bp from the end of exon 8, and may be involved in the regulation of gene expression through its affect on enhancer affinity and target area.39
The present study shows that the Tru9I mutant ‘uu’ genotype was associated with an increased risk of developing premenopausal BC. There are few association studies of Tru9I in other diseases such as coronary heart disease (CHD), colorectal adenoma, and prostate cancer.26,39,40 However, Tru9I is not associated with CHD39 and prostate cancer40 among the Chinese Han population, whereas Tru9I lowers the risk of colorectal carcinoma among women living in North Carolina.26 We show further that mutant Tru9I may be associated with the development of Grade IV carcinoma, whereas another study shows the opposite relationship with colorectal adenoma, which is more pronounced for multiple, larger, and sessile adenomas, and perhaps with dysplasia.26
This study has certain limitations. First, the sample size was relatively small. Large-scale studies are therefore required to support our conclusions. Second, we did not study environmental factors including diet and lifestyle, which may modify the association between the VDR Tru9I polymorphism and BC. Finally, other VDR SNPs and serum vitamin D levels were not studied. However, further research is required to confirm our findings and explore the possible mechanisms of BC underlying the association of VDR Tru9I with oncogenesis to facilitate the development of more effective treatment.
We conclude that the VDR Tru9I ‘uu’ genotype may predict a high risk for premenopausal BC.
Declaration of conflicting interest
The authors declare that there is no conflict of interest.
Funding
This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.
References
- 1.Shaukat U Ismail M andMehmood N.. Epidemiology, major risk factors and genetic predisposition for breast cancer in the Pakistani population. Asian Pac J Cancer Prev 2013; 14: 5625–5629. [DOI] [PubMed] [Google Scholar]
- 2.Ferlay J, Soerjomataram I, Dikshit R, et al. Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012. Int J Cancer 2015; 136: E359–E386. DOI: 10.1002/ijc.29210. [DOI] [PubMed] [Google Scholar]
- 3.Hirko KA, Chen WY, Willett WC, et al. Alcohol consumption and risk of breast cancer by molecular subtype: Prospective analysis of the nurses' health study after 26 years of follow-up. Int J Cancer 2016; 138: 1094–1101. DOI: 10.1002/ijc.29861. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Nagrani R, Mhatre S, Rajaraman P, et al. Central obesity increases risk of breast cancer irrespective of menopausal and hormonal receptor status in women of South Asian Ethnicity. Eur J Cancer 2016; 66: 153–161. DOI: http://dx.doi.org/10.1016/j.ejca.2016.07.022. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Bassett JK, Hodge AM, English DR, et al. Plasma phospholipids fatty acids, dietary fatty acids, and breast cancer risk. Cancer Causes Control 2016; 27: 759–773. DOI: 10.1007/s10552–016-0753–2. [DOI] [PubMed] [Google Scholar]
- 6.Eisman JA andBouillon R.. Vitamin D: direct effects of vitamin D metabolites on bone: lessons from genetically modified mice. BoneKEy Rep 2014; 3: 499. DOI: 10.1038/bonekey.2013.233. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Bauer SR, Hankinson SE, Bertone-Johnson ER, et al. Plasma vitamin D levels, menopause, and risk of breast cancer: dose-response meta-analysis of prospective studies. Medicine (Baltimore) 2013; 92: 123–131. DOI: 10.1097/MD.0b013e3182943bc2. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Williams JD, Aggarwal A, Swami S, et al. Tumor autonomous effects of Vitamin D deficiency promote breast cancer metastasis. Endocrinology 2016; 157: 1341–1347. DOI: 10.1210/en.2015–2036. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Kim Y andJe Y.. Vitamin D intake, blood 25(OH)D levels, and breast cancer risk or mortality: a meta-analysis. Br J Cancer 2014; 110: 2772–2784. DOI: 10.1038/bjc.2014.175. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Kuhn T, Kaaks R, Becker S, et al. Plasma 25-hydroxyvitamin D and the risk of breast cancer in the European prospective investigation into cancer and nutrition: a nested case-control study. Int J Cancer 2013; 133: 1689–1700. DOI: 10.1002/ijc.28172. [DOI] [PubMed] [Google Scholar]
- 11.Lin R andWhite JH.. The pleiotropic actions of vitamin D. BioEssays 2004; 26: 21–28. DOI: 10.1002/bies.10368. [DOI] [PubMed] [Google Scholar]
- 12.Rai V, Abdo J, Agrawal S, et al. Vitamin D Receptor Polymorphism and Cancer: An Update. Anticancer Research 2017; 37:3991–4003. DOI: 10.21873/anticanres.11784. Available at: https://www.ncbi.nlm.nih.gov/pubmed/28739681 [DOI] [PubMed] [Google Scholar]
- 13.Deeb KK Trump DL andJohnson CS.. Vitamin D signalling pathways in cancer: potential for anticancer therapeutics. Nat Rev Cancer 2007; 7: 684–700. DOI: 10.1038/nrc2196. [DOI] [PubMed] [Google Scholar]
- 14.Pendas-Franco N, Gonzalez-Sancho JM, Suarez Y, et al. Vitamin D regulates the phenotype of human breast cancer cells. Differentiation 2007; 75: 193–207. DOI: 10.1111/j.1432-0436.2006.00131.x. [DOI] [PubMed] [Google Scholar]
- 15.Lopes N, Carvalho J, Duraes C, et al. 1Alpha,25-dihydroxyvitamin D3 induces de novo E-cadherin expression in triple-negative breast cancer cells by CDH1-promoter demethylation. Anticancer Res 2012; 32: 249–257. [PubMed] [Google Scholar]
- 16.Campbell MJ, Gombart AF, Kwok SH, et al. The anti-proliferative effects of 1alpha,25(OH)2D3 on breast and prostate cancer cells are associated with induction of BRCA1 gene expression. Oncogene 2000; 19: 5091–5097. DOI: 10.1038/sj.onc.1203888. [DOI] [PubMed] [Google Scholar]
- 17.Lopes N, Sousa B, Martins D, et al. Alterations in Vitamin D signalling and metabolic pathways in breast cancer progression: a study of VDR, CYP27B1 and CYP24A1 expression in benign and malignant breast lesions. BMC Cancer 2010; 10: 483. DOI: 10.1186/1471–2407-10–483. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Bertoccini L, Sentinelli F, Leonetti F, et al. The vitamin D receptor functional variant rs2228570 (C>T) does not associate with type 2 diabetes mellitus. Endocr Res 2017; 42: 1–5. DOI: 10.1080/07435800.2017.1305965. [DOI] [PubMed] [Google Scholar]
- 19.Torkko KC, van Bokhoven A, Mai P, et al. VDR and SRD5A2 polymorphisms combine to increase risk for prostate cancer in both non-Hispanic White and Hispanic White men. Clin Cancer Res 2008; 14: 3223–3229. DOI: 10.1158/1078–0432.ccr-07–4894. [DOI] [PubMed] [Google Scholar]
- 20.Hashemi SM, Arbabi N, Hashemi M, et al. Association between VDR Gene Polymorphisms (rs 1544410, rs 7975232, rs 2228570, rs 731236 and rs 11568820) and Susceptibility to Breast Cancer in a Sample of Southeastern Iranian Population. Int J Cancer Manag 2017; 10: e8807. [Google Scholar]
- 21.Atoum MF andAl-Khatib YM.. Association between Serum 25-hydroxy Vitamin D Concentration and TaqI Vitamin D Receptor Gene Polymorphism among Jordanian females with breast cancer. Chin Med J (Engl) 2017; 130: 1074–1078. DOI: 10.4103/0366–6999.204933. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Talaneh S, Ghorbani A, Oghabi Bakhshaiesh T, et al. FokI and BsmI Polymorphisms of the VDR Gene and Breast Cancer Risk. MCI 2017; 1: 21–25. [Google Scholar]
- 23.Elzehery RR, Baiomy AA, Hegazy MAF, et al. Vitamin D status, receptor gene BsmI (A/G) polymorphism and breast cancer in a group of Egyptian females. Egyptian Journal of Medical Human Genetics 2017; 18: 269–273. DOI: https://doi.org/10.1016/j.ejmhg.2016.11.003. [Google Scholar]
- 24.Lu D Jing L andZhang S.. Vitamin D Receptor Polymorphism and Breast Cancer Risk: A Meta-Analysis. Medicine 2016; 95: e3535. DOI: 10.1097/md.0000000000003535. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.Rashid MU, Muzaffar M, Khan FA, et al. Association between the BsmI Polymorphism in the Vitamin D Receptor Gene and Breast Cancer Risk: Results from a Pakistani Case-Control Study. PloS One 2015; 10: e0141562. DOI: 10.1371/journal.pone.0141562. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26.Gong YL, Xie DW, Deng ZL, et al. Vitamin D receptor gene Tru9I polymorphism and risk for incidental sporadic colorectal adenomas. World J Gastroenterol 2005; 11: 4794–4799. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Sarwar MR andSaqib A.. Cancer prevalence, incidence and mortality rates in Pakistan in 2012. Cogent Medicine 2017; 4: 1288773. DOI: 10.1080/2331205X.2017.1288773. [Google Scholar]
- 28.Wu H, Chen Y, Liang J, et al. Hypomethylation-linked activation of PAX2 mediates tamoxifen-stimulated endometrial carcinogenesis. Nature 2005; 438: 981–987. DOI: doi:10.1038/nature04225 [DOI] [PubMed] [Google Scholar]
- 29.Saslow D, Boetes C, Burke W, et al. American Cancer Society guidelines for breast screening with MRI as an adjunct to mammography. CA Cancer J Clin 2007; 57: 75–89. [DOI] [PubMed] [Google Scholar]
- 30.Peterlongo P, Chang-Claude J, Moysich KB, et al. Candidate genetic modifiers for breast and ovarian cancer risk in BRCA1 and BRCA2 mutation carriers. Cancer Epidemiol Biomarkers Prev 2015; 24: 308–316. DOI: 10.1158/1055–9965.epi-14–0532. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 31.Desrichard A, Bidet Y, Uhrhammer N, et al. CHEK2 contribution to hereditary breast cancer in non-BRCA families. Breast Cancer Res 2011; 13: R119. DOI: 10.1186/bcr3062. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 32.Krishnan AV andFeldman D.. Mechanisms of the anti-cancer and anti-inflammatory actions of vitamin D. Annu Rev Pharmacol Toxicol 2011; 51: 311–336. DOI: 10.1146/annurev-pharmtox-010510–100611. [DOI] [PubMed] [Google Scholar]
- 33.Guo B, Jiang X, Hu X, et al. Association between vitamin D receptor gene polymorphisms and breast cancer in a Chinese population. Int J Clin Exp Med 2015; 8: 8020–8024. [PMC free article] [PubMed] [Google Scholar]
- 34.Reimers LL, Crew KD, Bradshaw PT, et al. Vitamin D-related gene polymorphisms, plasma 25-hydroxyvitamin D, and breast cancer risk. Cancer Causes Control 2015; 26: 187–203. DOI: 10.1007/s10552–014-0497–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 35.Engel LS, Orlow I, Sima CS, et al. Vitamin D receptor gene haplotypes and polymorphisms and risk of breast cancer: a nested case-control study. Cancer Epidemiol Biomarkers Prev 2012; 21: 1856–1867. DOI: 10.1158/1055–9965.epi-12–0551. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 36.Mishra DK, Wu Y, Sarkissyan M, et al. Vitamin D receptor gene polymorphisms and prognosis of breast cancer among African-American and Hispanic women. PloS One 2013; 8: e57967. DOI: 10.1371/journal.pone.0057967. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 37.Nemenqani DM, Karam RA, Amer MG, et al. Vitamin D receptor gene polymorphisms and steroid receptor status among Saudi women with breast cancer. Gene 2015; 558: 215–219. DOI: 10.1016/j.gene.2014.12.065. [DOI] [PubMed] [Google Scholar]
- 38.Ye WZ Reis AF andVelho G.. Identification of a novel Tru9 I polymorphism in the human vitamin D receptor gene. J Hum Genet 2000; 45: 56–57. DOI: 10.1007/s100380050011. [DOI] [PubMed] [Google Scholar]
- 39.He L andWang M.. Association of vitamin d receptor-a gene polymorphisms with coronary heart disease in Han Chinese. Int J Clin Exp Med 2015; 8: 6224–6229. [PMC free article] [PubMed] [Google Scholar]
- 40.Bai Y, Yu Y, Yu B, et al. Association of vitamin D receptor polymorphisms with the risk of prostate cancer in the Han population of Southern China. BMC Med Gen 2009; 10: 125–125. DOI: 10.1186/1471–2350-10–125. [DOI] [PMC free article] [PubMed] [Google Scholar]