Tumor Biol. DOI 10.1007/s13277-014-1621-x
RESEARCH ARTICLE
Transforming growth factor beta1 (TGFβ1) polymorphisms and breast cancer risk Davar Amani & Ahad Khalilnezhad & Abbas Ghaderi & Norrio Niikawa & Ko-ichiro Yoshiura
Received: 1 October 2013 / Accepted: 3 January 2014 # International Society of Oncology and BioMarkers (ISOBM) 2014
Abstract Transforming growth factor β1 (TGFβ1) is suggested to be involved in the pathogenesis of and in complications with breast cancer (BC). Polymorphisms in TGFβ1 gene (TGFβ1) have been suggested by many investigators to have a role in susceptibility to BC; however, many discordant data have been reported. Considering the role of ethnic variations, we performed an association study between TGFβ1 polymorphisms and BC among Iranian women. We sequenced DNA samples of 110 BC and 110 normal control women for the exons and their adjacent intronic regions of TGFβ1 using PCR. The allele, genotype, and haplotype frequencies were calculated using PowerMarker V3.25 and R 3.0.2 softwares. Ten single nucleotide polymorphisms (SNPs) were detected. Statistical analysis on the frequency of seven most frequent SNPs, including the three coding SNPs (cSNPs) revealed no significant difference between BC and control women. Moreover, among 11 constructed haplotypes, “GTGCCGC” was significantly different between two study groups. In conclusion, we found no association between the studied SNPs of TGFβ1 and BC among Iranian women, but a possible association between “GTGCCGC” haplotype and BC was seen. D. Amani (*) : A. Khalilnezhad Department of Immunology, Medical School, Shahid Beheshti University of Medical Sciences, Tehran 1985717443, Iran e-mail: [email protected] A. Ghaderi Department of Immunology, Medical School, Shiraz University of Medical Sciences, Shiraz, Iran A. Ghaderi Shiraz Institute of Cancer Research, Medical School, Shiraz University of Medical Sciences, Shiraz, Iran N. Niikawa : K.0.05). Neither AA-homozygosity at the −14G→A polymorphic site nor deletion-homozygosity at the IVS4 + 131-133delC deletion site was found among BC and control women. Comparison of haplotype frequencies between BC and control women
[15, 16, 34, 40–43]. The potential involvement of TGFβ1 in BC pathogenesis was also suggested by many genetic studies on its polymorphisms; however, the reports are controversial and inconclusive. In the present study, we sequenced DNA samples from BC and control Iranian women for the detection of possible polymorphisms of TGFβ1, and found ten SNPs, two (IVS5 + 18G→C and IVS6 + 910G→A) of which were novel, that we had previously reported (Table 2) [18]. In the present study, statistical analysis on the frequency of the seven most frequent SNPs, including −14G→A, 29T→ C, 74G→C, 788C→T, IVS4 + 131-133delC, IVS5 + 18G→ C, and IVS5 + 51C→T revealed no significant difference between BC and control Iranian women (Table 3). Although our findings are in agreement with some other similar studies, the role of these polymorphisms in BC seems to be more complicated, as have been indicated by various investigations among different populations [24–27]. −14G→A SNP
At the seven polymorphic sites among BC and control women, eight and ten effective haplotypes were constructed, respectively, that two of them, including “GTGCCGC” and “GCGCCGC,” were the most frequent haplotypes in both groups (Table 4). Frequency distribution of the haplotype “GTGCCGC” was significantly different between BC and control women (P0.05) (Table 4).
The −14G→A SNP (rs9282871) is located upstream of exon 1 of TGFβ1 and may play a role in the regulation of transcription and splicing events. As far as our knowledge supports, there is no study carried out on the role of this SNP in BC. Previously, we found no evidence on the role of this SNP in women with RSA [18]. In the current investigation, we also failed to show any association between −14G→A polymorphism and BC. 29T→C cSNP
Discussion Several studies have shown that there might be association between polymorphisms in TGFβ1, TGFβ2, and TGFβ signaling pathways genes and susceptibility to many diseases Fig. 1 Schematic illustration of positions of detected SNP region of TGFβ1 in studied Iranian subpopulation by automated sequencing. Exa Exon
The 29T→C cSNP, which leads to the substitution of Leu to Pro at amino acid position 10 of the signal peptide and thereby can potentially affect the secretion and the amount of circulating TGFβ1, and may have potential influences on overall
Tumor Biol. Table 2 Detected SNPs in screening of exons and adjacent intronic regions of TGFβ1 in studied Iranian subpopulation by automated sequencing
Table 3 Distribution of detected TGFB1 coding region SNP genotypes and alleles in breast cancer (BC) patients and control groupsa by automated sequencing
SNPs
NCBI SNP database
Position
TGFβl genotype and alleles
−14G→A
rs 9282871
Upstream 0.99 (G)
0.01 (A)
29 T→C (Leu 10 Pro) 74G→C (Arg 25 Pro) IVS1 + 4210G→T IVS4 + 131-133delC 788C→T (Thr 263 Ile) IVS5 + 18G→C IVS5 + 51C→T IVS5 + 9582C→T IVS6 + 910G→A
rs 1982073 rs 1800471 rs 11466324 rs 8179 182 rs 1800474 Nc rs 11466 334 rs 8179 1821 N
Exon 1 Exon 1 Intron 1 Intron 4 Exon 5 Intron 5 Intron 5 Intron 5 Intron 6
0.41a (C) 0.08 (C) 0.01 (T) 0.05 (*b) 0.04 (T) 0.01 (C) 0.01 (T) 0.14 (T) 0.01 (A)
Major Minor allele allele frequency frequency
0.59 (T) 0.92 (G) 0.99 (G) 0.95 (C) 0.96 (C) 0.99 (G) 0.99 (C) 0.86 (C) 0.99 (G)
a
Four out of ten polymorphisms had minor allele frequency >5 % (indicated by underline)
b
*deletion
c
N The two novel SNPs are novel and not submitted to NCBI SNP database yet
TGFβ signaling in many diseases, is mostly studied and the most prevalent polymorphism of TGFβ1, according to many in vitro and in vivo studies [16, 17, 40, 44–46]. Prior studies have shown increases, decreases, and or no changes in BC risk associated with the 29T→C cSNP [11, 12, 22–33, 35, 36]. For instances, Yokota et al. showed that C allele of 29T→C are associated with increased TGFβ1 serum levels [41]. Dunning et al. indicated that this allele is associated with increased rates of TGFβ1 secretion and with increased incidence of invasive BC [11]. In line with these studies, Shu et al. have reported that among genetic polymorphisms in the TGFβ1, 29T→C and −509C→T SNPs, but not 74G→C cSNP, may be implicated in BC [27]. Recently, the evidence from 10,392 cases and 11,697 controls strongly suggested an active and low penetrance role for 29T→C cSNP in BC susceptibility in Caucasian [33]. In the opposite side of the abovementioned studies, Ziv et al. found that the women with T/T genotype at the 29T→C cSNPs are in higher risk of BC as compared to the C/C genotype at the same cSNP [23]. On the other hand, the Breast Cancer Association Consortium pooled data suggested a very moderate per-allele increase in BC risk associated with 29T→C polymorphism [31], as well did another pooled analysis of case–control studies [36]. In order to provide an explanation for these controversial findings, several studies have evaluated 29T→ C cSNP association by cancer stages and have proposed a dual and stage-dependant role for this polymorphism in BC invasiveness [12, 35]. For example, Shin et al. [12] and Mu et al. [35] have reported that the association of TGFβ1 29T→C cSNP with disease progression is under control of disease
BC patients (n=110)b
Genotype −14G→A GG 100 (100) GA 0(0) AA 0 (0) Allele G 1.000 A 0.000 Genotype 29 T→C (Leu 10 Pro) TT 45 (45) TC 28 (28) CC 27 (27) Allele T 0.590 C 0.410 Genotype 74G→C (Arg 25 Pro) GG 92 (92) GC 8 (8) CC 0 (0) Allele G 0.960 C 0.040 Genotype IVS4 + 131-133delC CC 97 (96.04) C*d 4 (3.96) ** 0 (0) Allele C 0.980 * 0.020 Genotype 788C→T (Thr 263 Ile) CC 94 (93.07) CT 7 (6.93) TT 0 (0) Allele C 0.965 T 0.035 Genotype IVS5 + 18G→C GG 100 (99.01) GC 1 (0.99) CC 0 (0) Allele G 0.995 C 0.005 Genotype IVS5 + 51C→T CC 99 (98.02) CT 2 (1.98) TT 0 (0) Allele C 0.990
Controls (n=110)c
P value
103 (99.03) 1 (0.97) 0 (0)
P=0.32
0.995 0.005 33 (31.73) 41 (39.42) 30 (28.85)
P=0.21
0.514 0.486 91 (87.5) 13 (12.5) 0 (0)
P=0.38
0.937 0.063 103 (93.64) 7 (6.36) 0 (0)
P=0.55
0.968 0.032 98 (89.91) 11 (10.09) 0 (0)
P=0.49
0.950 0.050 108 (99.08) 1 (0.92) 0 (0)
P=0.98
0.995 0.005 108 (99.08) 1 (0.92) 0 (0) 0.995
P=0.54
Tumor Biol. Table 3 (continued) TGFβl genotype and alleles T a
BC patients (n=110)b
Controls (n=110)c
0.010
0.005
P value
Value are shown in absolute numbers (percentage)
b
In some cases, the analyzed BC patients were less than 110 because of technical problems c
In some, the control samples were less than 110 because of technical problems
d
These observations are in agreement with those of Feigelson et al. [47] who did not found any evidence supporting the TGFβ1 29T→C polymorphism role in postmenopausal BC, neither alone nor in combination with TGF-βR1 gene polymorphism. In contrast, Joshi et al. have found that the higher frequency of C allele of TGFβ1 29T→C polymorphism and lower frequency of 6A allele of TGFβR1 6A/9A polymorphism may together contribute to a lower risk of BC in western Indian women, representing complicated genetic determinants for susceptibility to BC [50].
* Deletion
74G→C and 788C→T cSNPs stage. In other words, T/T genotype confers higher and lower risk of disease relapse in early and late stages of BC, respectively [12, 35]. As we also did in present study, many investigations failed to find any role for 29T→C cSNP in susceptibility to BC. For example, Le Marchand et al. in a multiethnic cohort study, Krippl et al., Jin et al., and Feigelson et al. reported no association between 29T→C polymorphism in the TGFβ1 and BC risk [24, 25, 30, 47]. More recently, Barnett et al. have reported no association between this cSNP and late radiotherapy toxicity to the breast [48]. Presumably, the population differences and ethnicityrelated variations, hormonal state, combination of other factor, or unknown polymorphisms in other genes would explain the heterogeneity between the data [34, 49]. For instances, Lee et al. have reported that polymorphisms of TGFβ1 in parallel with those of TNFβ gene may affect susceptibility of Korean women to BC [26]. Another study revealed that the CC genotype confers low BC risk for premenopausal, but not for postmenopausal, Japanese women [32]. Moreover, it was proposed that a combination of genetic assessment of TGFβ signaling pathway variants might help predict BC risk [29].
Although the 29T→C SNP is the most studied polymorphism of TGFβ1, some studies have focused on other polymorphisms of this gene, and on their relation with BC risk and other diseases. 74G→C cSNP, which changes the 25th codon from Arg to Pro, and 788C→T cSNP, which changes the 263th codon from Thr to Ile, are other detected polymorphisms in TGFβ1 affecting the sequence, and probably the function, of TGFβ1 peptide [13, 15]. The possible and potential involvement of 74G→C and 788C→T cSNPs in BC pathogenesis remains inconclusive because the association studies supporting their hypothetical role are not sufficient. Awad et al. reported that the C allele of the 74G→C polymorphism may be associated with lower TGFβ1 production, whereas the GG genotype (encoding Arg) may in contrast produce high level of this cytokine [13]. Another investigation revealed that the 74G→C and 788C→T, not the 29T→C, polymorphisms in TGFβ1 might play an important role in occurrence of clefts of the lip, alveolus, and palate [15]. In addition, according to Vuong et al., these cSNPs are associated with susceptibility to IgA nephropathy [40]. Moreover,
Table 4 Comparison of haplotype frequency distributions between BC patients and controls No.
1 2 3 4 5 6 7 8 9 10 11 Total a
Haplotype
Frequency
−14G→A
29 T→C
74G→C
IVS4 + 131-133delC
788C→T
IVS5 + 18G→C
IVS5 + 51C→T
BC
Control
G G G G G G G G G G A
T C C C T T C T T C C
G G C G G G G G C C G
C C C C *a C C C C C C
C C C T C T C C C C C
G G G G G G G C G C G
C C C C C C T C C C C
0.55 0.34 0.04 0.02 0.02 0.01 0.01 0.01 0.00 0.00 0.00 1.00
0.45 0.39 0.04 0.02 0.03 0.03 0.01 0.00 0.01 0.01 0.01 1.00
* Deletion
P value
0.036 0.276 1.00 1.00 0.502 0.134 1.00 0.137 0.137 0.137 0.137
Tumor Biol.
a large-scale analysis indicated weak associations between 788C→T and risk of incident vertebral fractures of osteoporosis [51]. In contrast to 29T→C cSNP, Shu et al. found no DNA sequence variation at codon 25 of the TGFβ1 and no association between this cSNP and BC survival [27]. A case– control study that evaluated the possible association of TGFβ1 polymorphisms with BC as well as TGFβRI and TGFβRII genes reported that neither 29T → C nor 74G → C and 788C→T cSNPs were likely to be associated with BC [24]. This study is in accordance with our findings in the present study. Intronic SNPs Although only few studies have been performed to investigate the role of the intronic polymorphisms of TGFβ1 in diseases, some of these SNPs are also thought to be involved in the implications of various diseases [18, 52–54]. Previously, we found no association between any of these SNPs and RSA [18]; however, there might be an association between IVS5 + 9582C→T SNP and a decrease of hip bone mineral density (BMD), according to Keen et al. [52]. In contrast, Langdahl et al. suggested that TT genotype at this position is associated with increased BMD [53]. Moreover, a positive association between IVS4 + 131-133delC and BMD was seen among Italian women [54], which is later reported as 131insC by Weinshenker et al. [55]. To date, there has been no association study between IVS1 + 4210G→T, IVS4 + 131-133delC, IVS5 + 18G→C, IVS5 + 51C→T, IVS5 + 9582C→T, and BC, at which we aimed in the present study for the first time and could not find any evidence supporting their role in BC, though. Haplotype analysis Our haplotype analysis revealed several haplotypes. Two of these haplotypes including “GTGCCGC” and “GCGCCGC” were more frequent than the others. Interestingly, we found that the frequency distribution of “GTGCCGC” was significantly different between BC and control women, and therefore, there might be an association between this haplotype and BC. In addition, frequency distribution of the haplotype “GCGCCGC” differed among control and BC women; however, frequency distributions of this haplotype and other observed haplotypes were not significantly different between two study groups. To our knowledge, the haplotype “GTGCCGC” and its frequency have not been reported among other populations, and we were the first to study this haplotype and to report its association with BC. However, this needs to be approved by further investigations. Also, as far as we know, there was no previous study on association of “GCGCCGC” and other observed haplotypes with BC, and although in our study the
differences in distribution of them among control and BC were insignificant, investigation of their distribution by large population-based studies may suggest more precise and helpful information in this respect. In conclusion, according to the results of the present study among Iranian women with BC, the SNPs located in the exons and adjacent intronic regions of the TGFβ1 did not appear to have a role in implications of this disease. However, there are several discordant reports that have proposed a functional contribution of these polymorphisms. Importantly, we also found the haplotype “GTGCCGC” might be associated with BC, although this is the first report and subsequent studies are to be performed. Finally, in order to clarify the role of TGFβ1 SNPs and pertinent haplotypes, especially “GTGCCGC” in BC, they must be taken into account together with other factors and other gene polymorphisms in future studies among various large populations and with different stages of BC. Acknowledgments This study was financially supported by Shiraz University of Medical Sciences and in a part by Shiraz Institute for Cancer Research. The sequencing of TGFβ1 gene was done in the Department of Human Genetics, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan. Conflicts of interest The authors declare that there is no conflict of interest.
References 1. Porter PL. Global trends in breast cancer incidence and mortality. Salud Publica Mex. 2009;51:s141–6. 2. Ikushima H, Miyazono K. TGFbeta signaling: a complex web in cancer progression. Nat Rev Cancer. 2010;10:415–24. 3. Marcoe JP, Lim JR, Schaubert KL, Fodil-Cornu N, Matka M, McCubbrey AL, et al. TGF-beta is responsible for NK cell immaturity during ontogeny and increased susceptibility to infection during mouse infancy. Nat Immunol. 2012 4. Ling E, Robinson DS. Transforming growth factor-beta1: its antiinflammatory and pro-fibrotic effects. Clin Exp Allergy. 2002;32: 175–8. 5. van Meeteren LA, ten Dijke P. Regulation of endothelial cell plasticity by TGF-beta. Cell Tissue Res. 2012;347:177–86. 6. Ikushima H, Miyazono K. Biology of transforming growth factorbeta signaling. Curr Pharm Biotechnol. 2011;12:2099–107. 7. Fujii D, Brissenden JE, Derynck R, Francke U. Transforming growth factor beta gene maps to human chromosome 19 long arm and to mouse chromosome 7. Somat Cell Mol Genet. 1986;12:281–8. 8. Wakefield LM, Piek E, Bottinger EP. TGF-beta signaling in mammary gland development and tumorigenesis. J Mammary Gland Biol Neoplasia. 2001;6:67–82. 9. Babyshkina N, Malinovskaya E, Stakheyeva M, Volkomorov V, Slonimskaya E, Maximov V, et al. Association of functional −509C>T polymorphism in the TGF-beta1 gene with infiltrating ductal breast carcinoma risk in a Russian Western Siberian population. Cancer Epidemiol. 2011;35:560–3. 10. Woo SU, Park KH, Woo OH, Yang DS, Kim AR, Lee ES, et al. Association of a TGF-beta1 gene −509 C/T polymorphism with
Tumor Biol. breast cancer risk: a meta-analysis. Breast Cancer Res Treat. 2010;124:481–5. 11. Dunning AM, Ellis PD, McBride S, Kirschenlohr HL, Healey CS, Kemp PR, et al. A transforming growth factorbeta1 signal peptide variant increases secretion in vitro and is associated with increased incidence of invasive breast cancer. Cancer Res. 2003;63:2610–5. 12. Shin A, Shu XO, Cai Q, Gao YT, Zheng W. Genetic polymorphisms of the transforming growth factor-beta1 gene and breast cancer risk: a possible dual role at different cancer stages. Cancer Epidemiol Biomarkers Prev. 2005;14:1567–70. 13. Awad MR, El-Gamel A, Hasleton P, Turner DM, Sinnott PJ, Hutchinson IV. Genotypic variation in the transforming growth factor-beta1 gene: association with transforming growth factorbeta1 production, fibrotic lung disease, and graft fibrosis after lung transplantation. Transplantation. 1998;66:1014–20. 14. Berndt SI, Huang WY, Chatterjee N, Yeager M, Welch R, Chanock SJ, et al. Transforming growth factor beta 1 (TGFB1) gene polymorphisms and risk of advanced colorectal adenoma. Carcinogenesis. 2007;28:1965–70. 15. Stoll C, Mengsteab S, Stoll D, Riediger D, Gressner AM, Weiskirchen R. Analysis of polymorphic TGFB1 codons 10, 25, and 263 in a German patient group with non-syndromic cleft lip, alveolus, and palate compared with healthy adults. BMC Med Genet. 2004;5:15. 16. Wei YS, Xu QQ, Wang CF, Pan Y, Liang F, Long XK. Genetic variation in transforming growth factor-beta1 gene associated with increased risk of esophageal squamous cell carcinoma. Tissue Antigens. 2007;70:464–9. 17. Zhang P, Di JZ, Zhu ZZ, Wu HM, Wang Y, Zhu G, et al. Association of transforming growth factor-beta 1 polymorphisms with genetic susceptibility to TNM stage I or II gastric cancer. Jpn J Clin Oncol. 2008;38:861–6. 18. Amani D, Dehaghani AS, Zolghadri J, Ravangard F, Niikawa N, Yoshiura K, et al. Lack of association between the TGF-beta1 gene polymorphisms and recurrent spontaneous abortion. J Reprod Immunol. 2005;68:91–103. 19. Amani D, Zolghadri J, Dehaghani AS, Pezeshki AM, Ghaderi A. The promoter region (−800, −509) polymorphisms of transforming growth factor-beta1 (TGF-beta1) gene and recurrent spontaneous abortion. J Reprod Immunol. 2004;62:159–66. 20. Derynck R, Rhee L, Chen EY, Van Tilburg A. Intron-exon structure of the human transforming growth factor-beta precursor gene. Nucleic Acids Res. 1987;15:3188–9. 21. Feizollahzadeh S, Taheripanah R, Khani M, Farokhi B, Amani D. Promoter region polymorphisms in the transforming growth factor beta-1 (TGFbeta1) gene and serum TGFbeta1 concentration in preeclamptic and control Iranian women. J Reprod Immunol. 2012;94: 216–21. 22. Skerrett DL, Moore EM, Bernstein DS, Vahdat L. Cytokine genotype polymorphisms in breast carcinoma: associations of TGF-beta1 with relapse. Cancer Investig. 2005;23:208–14. 23. Ziv E, Cauley J, Morin PA, Saiz R, Browner WS. Association between the T29→C polymorphism in the transforming growth factor beta1 gene and breast cancer among elderly white women: The Study of Osteoporotic Fractures. JAMA. 2001;285:2859– 63. 24. Jin Q, Hemminki K, Grzybowska E, Klaes R, Soderberg M, Zientek H, et al. Polymorphisms and haplotype structures in genes for transforming growth factor beta1 and its receptors in familial and unselected breast cancers. Int J Cancer. 2004;112:94–9. 25. Le Marchand L, Haiman CA, van den Berg D, Wilkens LR, Kolonel LN, Henderson BE. T29C polymorphism in the transforming growth factor beta1 gene and postmenopausal breast cancer risk: the Multiethnic Cohort Study. Cancer Epidemiol Biomarkers Prev. 2004;13:412–5.
26. Lee KM, Park SK, Hamajima N, Tajima K, Yoo KY, Shin A, et al. Genetic polymorphisms of TGF-beta1 & TNF-beta and breast cancer risk. Breast Cancer Res Treat. 2005;90:149–55. 27. Shu XO, Gao YT, Cai Q, Pierce L, Cai H, Ruan ZX, et al. Genetic polymorphisms in the TGF-beta 1 gene and breast cancer survival: a report from the Shanghai Breast Cancer Study. Cancer Res. 2004;64: 836–9. 28. Cox DG, Penney K, Guo Q, Hankinson SE, Hunter DJ. TGFB1 and TGFBR1 polymorphisms and breast cancer risk in the Nurses’ Health Study. BMC Cancer. 2007;7:175. 29. Kaklamani VG, Baddi L, Liu J, Rosman D, Phukan S, Bradley C, et al. Combined genetic assessment of transforming growth factorbeta signaling pathway variants may predict breast cancer risk. Cancer Res. 2005;65:3454–61. 30. Krippl P, Langsenlehner U, Renner W, Yazdani-Biuki B, Wolf G, Wascher TC, et al. The L10P polymorphism of the transforming growth factor-beta 1 gene is not associated with breast cancer risk. Cancer Lett. 2003;201:181–4. 31. Cox A, Dunning AM, Garcia-Closas M, Balasubramanian S, Reed MW, Pooley KA, et al. A common coding variant in CASP8 is associated with breast cancer risk. Nat Genet. 2007;39:352–8. 32. Hishida A, Iwata H, Hamajima N, Matsuo K, Mizutani M, Iwase T, et al. Transforming growth factor B1 T29C polymorphism and breast cancer risk in Japanese women. Breast Cancer. 2003;10:63–9. 33. Ma X, Chen C, Xiong H, Li Y. Transforming growth factorbeta1 L10P variant plays an active role on the breast cancer susceptibility in Caucasian: evidence from 10,392 cases and 11,697 controls. Breast Cancer Res Treat. 2010;124:453–7. 34. Scollen S, Luccarini C, Baynes C, Driver K, Humphreys MK, Garcia-Closas M, et al. TGF-beta signaling pathway and breast cancer susceptibility. Cancer Epidemiol Biomarkers Prev. 2011;20: 1112–9. 35. Mu L, Katsaros D, Lu L, Preti M, Durando A, Arisio R, et al. TGFbeta1 genotype and phenotype in breast cancer and their associations with IGFs and patient survival. Br J Cancer. 2008;99:1357–63. 36. Breast Cancer Association C. Commonly studied single-nucleotide polymorphisms and breast cancer: results from the Breast Cancer Association Consortium. J Natl Cancer Inst. 2006;98:1382–96. 37. Amani D, Farjadian S, Ghaderi A. The frequency of transforming growth factor-beta1 gene polymorphisms in a normal southern Iranian population. Int J Immunogenet. 2008;35:145–51. 38. Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. 1988;16:1215. 39. Liu K, Muse SV. PowerMarker: an integrated analysis environment for genetic marker analysis. Bioinformatics. 2005;21:2128–9. 40. Vuong MT, Lundberg S, Gunnarsson I, Wramner L, Seddighzadeh M, Hahn-Zoric M, et al. Genetic variation in the transforming growth factor-beta1 gene is associated with susceptibility to IgA nephropathy. Nephrol Dial Transplant. 2009;24:3061–7. 41. Yokota M, Ichihara S, Lin TL, Nakashima N, Yamada Y. Association of a T29→C polymorphism of the transforming growth factor-beta1 gene with genetic susceptibility to myocardial infarction in Japanese. Circulation. 2000;101:2783–7. 42. Beisner J, Buck MB, Fritz P, Dippon J, Schwab M, Brauch H, et al. A novel functional polymorphism in the transforming growth factorbeta2 gene promoter and tumor progression in breast cancer. Cancer Res. 2006;66:7554–61. 43. Ma X, Beeghly-Fadiel A, Lu W, Shi J, Xiang YB, Cai Q, et al. Pathway analyses identify TGFBR2 as potential breast cancer susceptibility gene: results from a consortium study among Asians. Cancer Epidemiol Biomarkers Prev. 2012;21:1176–84. 44. Yamada Y, Miyauchi A, Goto J, Takagi Y, Okuizumi H, Kanematsu M, et al. Association of a polymorphism of the transforming growth factor-beta1 gene with genetic susceptibility to osteoporosis in postmenopausal Japanese women. J Bone Miner Res. 1998;13:1569–76.
Tumor Biol. 45. Pelletier R, Pravica V, Perrey C, Xia D, Ferguson RM, Hutchinson I, et al. Evidence for a genetic predisposition towards acute rejection after kidney and simultaneous kidney-pancreas transplantation. Transplantation. 2000;70:674–80. 46. Gewaltig J, Mangasser-Stephan K, Gartung C, Biesterfeld S, Gressner AM. Association of polymorphisms of the transforming growth factor-beta1 gene with the rate of progression of HCVinduced liver fibrosis. Clin Chim Acta. 2002;316:83–94. 47. Feigelson HS, Patel AV, Diver WR, Stevens VL, Thun MJ, Calle EE. Transforming growth factor beta receptor type I and transforming growth factor beta1 polymorphisms are not associated with postmenopausal breast cancer. Cancer Epidemiol Biomarkers Prev. 2006;15: 1236–7. 48. Barnett GC, Coles CE, Burnet NG, Pharoah PD, Wilkinson J, West CM, et al. No association between SNPs regulating TGF-beta1 secretion and late radiotherapy toxicity to the breast: results from the RAPPER study. Radiother Oncol. 2010;97:9–14. 49. Band AM, Laiho M. Crosstalk of TGF-beta and estrogen receptor signaling in breast cancer. J Mammary Gland Biol Neoplasia. 2011;16:109–15. 50. Joshi NN, Kale MD, Hake SS, Kannan S. Transforming growth factor beta signaling pathway associated gene polymorphisms may
51.
52.
53.
54.
55.
explain lower breast cancer risk in western Indian women. PLoS One. 2011;6:e21866. Langdahl BL, Uitterlinden AG, Ralston SH, Trikalinos TA, Balcells S, Brandi ML, et al. Large-scale analysis of association between polymorphisms in the transforming growth factor beta 1 gene (TGFB1) and osteoporosis: the GENOMOS study. Bone. 2008;42:969–81. Keen RW, Snieder H, Molloy H, Daniels J, Chiano M, Gibson F, et al. Evidence of association and linkage disequilibrium between a novel polymorphism in the transforming growth factor beta 1 gene and hip bone mineral density: a study of female twins. Rheumatology (Oxford). 2001;40:48–54. Langdahl BL, Carstens M, Stenkjaer L, Eriksen EF. Polymorphisms in the transforming growth factor beta 1 gene and osteoporosis. Bone. 2003;32:297–310. Bertoldo F, D’Agruma L, Furlan F, Colapietro F, Lorenzi MT, Maiorano N, et al. Transforming growth factor-beta1 gene polymorphism, bone turnover, and bone mass in Italian postmenopausal women. J Bone Miner Res. 2000;15:634–9. Weinshenker BG, Hebrink D, Kantarci OH, Schaefer-Klein J, Atkinson E, Schaid D, et al. Genetic variation in the transforming growth factor beta1 gene in multiple sclerosis. J Neuroimmunol. 2001;120:138–45.
RESEARCH ARTICLE
Transforming growth factor beta1 (TGFβ1) polymorphisms and breast cancer risk Davar Amani & Ahad Khalilnezhad & Abbas Ghaderi & Norrio Niikawa & Ko-ichiro Yoshiura
Received: 1 October 2013 / Accepted: 3 January 2014 # International Society of Oncology and BioMarkers (ISOBM) 2014
Abstract Transforming growth factor β1 (TGFβ1) is suggested to be involved in the pathogenesis of and in complications with breast cancer (BC). Polymorphisms in TGFβ1 gene (TGFβ1) have been suggested by many investigators to have a role in susceptibility to BC; however, many discordant data have been reported. Considering the role of ethnic variations, we performed an association study between TGFβ1 polymorphisms and BC among Iranian women. We sequenced DNA samples of 110 BC and 110 normal control women for the exons and their adjacent intronic regions of TGFβ1 using PCR. The allele, genotype, and haplotype frequencies were calculated using PowerMarker V3.25 and R 3.0.2 softwares. Ten single nucleotide polymorphisms (SNPs) were detected. Statistical analysis on the frequency of seven most frequent SNPs, including the three coding SNPs (cSNPs) revealed no significant difference between BC and control women. Moreover, among 11 constructed haplotypes, “GTGCCGC” was significantly different between two study groups. In conclusion, we found no association between the studied SNPs of TGFβ1 and BC among Iranian women, but a possible association between “GTGCCGC” haplotype and BC was seen. D. Amani (*) : A. Khalilnezhad Department of Immunology, Medical School, Shahid Beheshti University of Medical Sciences, Tehran 1985717443, Iran e-mail: [email protected] A. Ghaderi Department of Immunology, Medical School, Shiraz University of Medical Sciences, Shiraz, Iran A. Ghaderi Shiraz Institute of Cancer Research, Medical School, Shiraz University of Medical Sciences, Shiraz, Iran N. Niikawa : K.0.05). Neither AA-homozygosity at the −14G→A polymorphic site nor deletion-homozygosity at the IVS4 + 131-133delC deletion site was found among BC and control women. Comparison of haplotype frequencies between BC and control women
[15, 16, 34, 40–43]. The potential involvement of TGFβ1 in BC pathogenesis was also suggested by many genetic studies on its polymorphisms; however, the reports are controversial and inconclusive. In the present study, we sequenced DNA samples from BC and control Iranian women for the detection of possible polymorphisms of TGFβ1, and found ten SNPs, two (IVS5 + 18G→C and IVS6 + 910G→A) of which were novel, that we had previously reported (Table 2) [18]. In the present study, statistical analysis on the frequency of the seven most frequent SNPs, including −14G→A, 29T→ C, 74G→C, 788C→T, IVS4 + 131-133delC, IVS5 + 18G→ C, and IVS5 + 51C→T revealed no significant difference between BC and control Iranian women (Table 3). Although our findings are in agreement with some other similar studies, the role of these polymorphisms in BC seems to be more complicated, as have been indicated by various investigations among different populations [24–27]. −14G→A SNP
At the seven polymorphic sites among BC and control women, eight and ten effective haplotypes were constructed, respectively, that two of them, including “GTGCCGC” and “GCGCCGC,” were the most frequent haplotypes in both groups (Table 4). Frequency distribution of the haplotype “GTGCCGC” was significantly different between BC and control women (P0.05) (Table 4).
The −14G→A SNP (rs9282871) is located upstream of exon 1 of TGFβ1 and may play a role in the regulation of transcription and splicing events. As far as our knowledge supports, there is no study carried out on the role of this SNP in BC. Previously, we found no evidence on the role of this SNP in women with RSA [18]. In the current investigation, we also failed to show any association between −14G→A polymorphism and BC. 29T→C cSNP
Discussion Several studies have shown that there might be association between polymorphisms in TGFβ1, TGFβ2, and TGFβ signaling pathways genes and susceptibility to many diseases Fig. 1 Schematic illustration of positions of detected SNP region of TGFβ1 in studied Iranian subpopulation by automated sequencing. Exa Exon
The 29T→C cSNP, which leads to the substitution of Leu to Pro at amino acid position 10 of the signal peptide and thereby can potentially affect the secretion and the amount of circulating TGFβ1, and may have potential influences on overall
Tumor Biol. Table 2 Detected SNPs in screening of exons and adjacent intronic regions of TGFβ1 in studied Iranian subpopulation by automated sequencing
Table 3 Distribution of detected TGFB1 coding region SNP genotypes and alleles in breast cancer (BC) patients and control groupsa by automated sequencing
SNPs
NCBI SNP database
Position
TGFβl genotype and alleles
−14G→A
rs 9282871
Upstream 0.99 (G)
0.01 (A)
29 T→C (Leu 10 Pro) 74G→C (Arg 25 Pro) IVS1 + 4210G→T IVS4 + 131-133delC 788C→T (Thr 263 Ile) IVS5 + 18G→C IVS5 + 51C→T IVS5 + 9582C→T IVS6 + 910G→A
rs 1982073 rs 1800471 rs 11466324 rs 8179 182 rs 1800474 Nc rs 11466 334 rs 8179 1821 N
Exon 1 Exon 1 Intron 1 Intron 4 Exon 5 Intron 5 Intron 5 Intron 5 Intron 6
0.41a (C) 0.08 (C) 0.01 (T) 0.05 (*b) 0.04 (T) 0.01 (C) 0.01 (T) 0.14 (T) 0.01 (A)
Major Minor allele allele frequency frequency
0.59 (T) 0.92 (G) 0.99 (G) 0.95 (C) 0.96 (C) 0.99 (G) 0.99 (C) 0.86 (C) 0.99 (G)
a
Four out of ten polymorphisms had minor allele frequency >5 % (indicated by underline)
b
*deletion
c
N The two novel SNPs are novel and not submitted to NCBI SNP database yet
TGFβ signaling in many diseases, is mostly studied and the most prevalent polymorphism of TGFβ1, according to many in vitro and in vivo studies [16, 17, 40, 44–46]. Prior studies have shown increases, decreases, and or no changes in BC risk associated with the 29T→C cSNP [11, 12, 22–33, 35, 36]. For instances, Yokota et al. showed that C allele of 29T→C are associated with increased TGFβ1 serum levels [41]. Dunning et al. indicated that this allele is associated with increased rates of TGFβ1 secretion and with increased incidence of invasive BC [11]. In line with these studies, Shu et al. have reported that among genetic polymorphisms in the TGFβ1, 29T→C and −509C→T SNPs, but not 74G→C cSNP, may be implicated in BC [27]. Recently, the evidence from 10,392 cases and 11,697 controls strongly suggested an active and low penetrance role for 29T→C cSNP in BC susceptibility in Caucasian [33]. In the opposite side of the abovementioned studies, Ziv et al. found that the women with T/T genotype at the 29T→C cSNPs are in higher risk of BC as compared to the C/C genotype at the same cSNP [23]. On the other hand, the Breast Cancer Association Consortium pooled data suggested a very moderate per-allele increase in BC risk associated with 29T→C polymorphism [31], as well did another pooled analysis of case–control studies [36]. In order to provide an explanation for these controversial findings, several studies have evaluated 29T→ C cSNP association by cancer stages and have proposed a dual and stage-dependant role for this polymorphism in BC invasiveness [12, 35]. For example, Shin et al. [12] and Mu et al. [35] have reported that the association of TGFβ1 29T→C cSNP with disease progression is under control of disease
BC patients (n=110)b
Genotype −14G→A GG 100 (100) GA 0(0) AA 0 (0) Allele G 1.000 A 0.000 Genotype 29 T→C (Leu 10 Pro) TT 45 (45) TC 28 (28) CC 27 (27) Allele T 0.590 C 0.410 Genotype 74G→C (Arg 25 Pro) GG 92 (92) GC 8 (8) CC 0 (0) Allele G 0.960 C 0.040 Genotype IVS4 + 131-133delC CC 97 (96.04) C*d 4 (3.96) ** 0 (0) Allele C 0.980 * 0.020 Genotype 788C→T (Thr 263 Ile) CC 94 (93.07) CT 7 (6.93) TT 0 (0) Allele C 0.965 T 0.035 Genotype IVS5 + 18G→C GG 100 (99.01) GC 1 (0.99) CC 0 (0) Allele G 0.995 C 0.005 Genotype IVS5 + 51C→T CC 99 (98.02) CT 2 (1.98) TT 0 (0) Allele C 0.990
Controls (n=110)c
P value
103 (99.03) 1 (0.97) 0 (0)
P=0.32
0.995 0.005 33 (31.73) 41 (39.42) 30 (28.85)
P=0.21
0.514 0.486 91 (87.5) 13 (12.5) 0 (0)
P=0.38
0.937 0.063 103 (93.64) 7 (6.36) 0 (0)
P=0.55
0.968 0.032 98 (89.91) 11 (10.09) 0 (0)
P=0.49
0.950 0.050 108 (99.08) 1 (0.92) 0 (0)
P=0.98
0.995 0.005 108 (99.08) 1 (0.92) 0 (0) 0.995
P=0.54
Tumor Biol. Table 3 (continued) TGFβl genotype and alleles T a
BC patients (n=110)b
Controls (n=110)c
0.010
0.005
P value
Value are shown in absolute numbers (percentage)
b
In some cases, the analyzed BC patients were less than 110 because of technical problems c
In some, the control samples were less than 110 because of technical problems
d
These observations are in agreement with those of Feigelson et al. [47] who did not found any evidence supporting the TGFβ1 29T→C polymorphism role in postmenopausal BC, neither alone nor in combination with TGF-βR1 gene polymorphism. In contrast, Joshi et al. have found that the higher frequency of C allele of TGFβ1 29T→C polymorphism and lower frequency of 6A allele of TGFβR1 6A/9A polymorphism may together contribute to a lower risk of BC in western Indian women, representing complicated genetic determinants for susceptibility to BC [50].
* Deletion
74G→C and 788C→T cSNPs stage. In other words, T/T genotype confers higher and lower risk of disease relapse in early and late stages of BC, respectively [12, 35]. As we also did in present study, many investigations failed to find any role for 29T→C cSNP in susceptibility to BC. For example, Le Marchand et al. in a multiethnic cohort study, Krippl et al., Jin et al., and Feigelson et al. reported no association between 29T→C polymorphism in the TGFβ1 and BC risk [24, 25, 30, 47]. More recently, Barnett et al. have reported no association between this cSNP and late radiotherapy toxicity to the breast [48]. Presumably, the population differences and ethnicityrelated variations, hormonal state, combination of other factor, or unknown polymorphisms in other genes would explain the heterogeneity between the data [34, 49]. For instances, Lee et al. have reported that polymorphisms of TGFβ1 in parallel with those of TNFβ gene may affect susceptibility of Korean women to BC [26]. Another study revealed that the CC genotype confers low BC risk for premenopausal, but not for postmenopausal, Japanese women [32]. Moreover, it was proposed that a combination of genetic assessment of TGFβ signaling pathway variants might help predict BC risk [29].
Although the 29T→C SNP is the most studied polymorphism of TGFβ1, some studies have focused on other polymorphisms of this gene, and on their relation with BC risk and other diseases. 74G→C cSNP, which changes the 25th codon from Arg to Pro, and 788C→T cSNP, which changes the 263th codon from Thr to Ile, are other detected polymorphisms in TGFβ1 affecting the sequence, and probably the function, of TGFβ1 peptide [13, 15]. The possible and potential involvement of 74G→C and 788C→T cSNPs in BC pathogenesis remains inconclusive because the association studies supporting their hypothetical role are not sufficient. Awad et al. reported that the C allele of the 74G→C polymorphism may be associated with lower TGFβ1 production, whereas the GG genotype (encoding Arg) may in contrast produce high level of this cytokine [13]. Another investigation revealed that the 74G→C and 788C→T, not the 29T→C, polymorphisms in TGFβ1 might play an important role in occurrence of clefts of the lip, alveolus, and palate [15]. In addition, according to Vuong et al., these cSNPs are associated with susceptibility to IgA nephropathy [40]. Moreover,
Table 4 Comparison of haplotype frequency distributions between BC patients and controls No.
1 2 3 4 5 6 7 8 9 10 11 Total a
Haplotype
Frequency
−14G→A
29 T→C
74G→C
IVS4 + 131-133delC
788C→T
IVS5 + 18G→C
IVS5 + 51C→T
BC
Control
G G G G G G G G G G A
T C C C T T C T T C C
G G C G G G G G C C G
C C C C *a C C C C C C
C C C T C T C C C C C
G G G G G G G C G C G
C C C C C C T C C C C
0.55 0.34 0.04 0.02 0.02 0.01 0.01 0.01 0.00 0.00 0.00 1.00
0.45 0.39 0.04 0.02 0.03 0.03 0.01 0.00 0.01 0.01 0.01 1.00
* Deletion
P value
0.036 0.276 1.00 1.00 0.502 0.134 1.00 0.137 0.137 0.137 0.137
Tumor Biol.
a large-scale analysis indicated weak associations between 788C→T and risk of incident vertebral fractures of osteoporosis [51]. In contrast to 29T→C cSNP, Shu et al. found no DNA sequence variation at codon 25 of the TGFβ1 and no association between this cSNP and BC survival [27]. A case– control study that evaluated the possible association of TGFβ1 polymorphisms with BC as well as TGFβRI and TGFβRII genes reported that neither 29T → C nor 74G → C and 788C→T cSNPs were likely to be associated with BC [24]. This study is in accordance with our findings in the present study. Intronic SNPs Although only few studies have been performed to investigate the role of the intronic polymorphisms of TGFβ1 in diseases, some of these SNPs are also thought to be involved in the implications of various diseases [18, 52–54]. Previously, we found no association between any of these SNPs and RSA [18]; however, there might be an association between IVS5 + 9582C→T SNP and a decrease of hip bone mineral density (BMD), according to Keen et al. [52]. In contrast, Langdahl et al. suggested that TT genotype at this position is associated with increased BMD [53]. Moreover, a positive association between IVS4 + 131-133delC and BMD was seen among Italian women [54], which is later reported as 131insC by Weinshenker et al. [55]. To date, there has been no association study between IVS1 + 4210G→T, IVS4 + 131-133delC, IVS5 + 18G→C, IVS5 + 51C→T, IVS5 + 9582C→T, and BC, at which we aimed in the present study for the first time and could not find any evidence supporting their role in BC, though. Haplotype analysis Our haplotype analysis revealed several haplotypes. Two of these haplotypes including “GTGCCGC” and “GCGCCGC” were more frequent than the others. Interestingly, we found that the frequency distribution of “GTGCCGC” was significantly different between BC and control women, and therefore, there might be an association between this haplotype and BC. In addition, frequency distribution of the haplotype “GCGCCGC” differed among control and BC women; however, frequency distributions of this haplotype and other observed haplotypes were not significantly different between two study groups. To our knowledge, the haplotype “GTGCCGC” and its frequency have not been reported among other populations, and we were the first to study this haplotype and to report its association with BC. However, this needs to be approved by further investigations. Also, as far as we know, there was no previous study on association of “GCGCCGC” and other observed haplotypes with BC, and although in our study the
differences in distribution of them among control and BC were insignificant, investigation of their distribution by large population-based studies may suggest more precise and helpful information in this respect. In conclusion, according to the results of the present study among Iranian women with BC, the SNPs located in the exons and adjacent intronic regions of the TGFβ1 did not appear to have a role in implications of this disease. However, there are several discordant reports that have proposed a functional contribution of these polymorphisms. Importantly, we also found the haplotype “GTGCCGC” might be associated with BC, although this is the first report and subsequent studies are to be performed. Finally, in order to clarify the role of TGFβ1 SNPs and pertinent haplotypes, especially “GTGCCGC” in BC, they must be taken into account together with other factors and other gene polymorphisms in future studies among various large populations and with different stages of BC. Acknowledgments This study was financially supported by Shiraz University of Medical Sciences and in a part by Shiraz Institute for Cancer Research. The sequencing of TGFβ1 gene was done in the Department of Human Genetics, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan. Conflicts of interest The authors declare that there is no conflict of interest.
References 1. Porter PL. Global trends in breast cancer incidence and mortality. Salud Publica Mex. 2009;51:s141–6. 2. Ikushima H, Miyazono K. TGFbeta signaling: a complex web in cancer progression. Nat Rev Cancer. 2010;10:415–24. 3. Marcoe JP, Lim JR, Schaubert KL, Fodil-Cornu N, Matka M, McCubbrey AL, et al. TGF-beta is responsible for NK cell immaturity during ontogeny and increased susceptibility to infection during mouse infancy. Nat Immunol. 2012 4. Ling E, Robinson DS. Transforming growth factor-beta1: its antiinflammatory and pro-fibrotic effects. Clin Exp Allergy. 2002;32: 175–8. 5. van Meeteren LA, ten Dijke P. Regulation of endothelial cell plasticity by TGF-beta. Cell Tissue Res. 2012;347:177–86. 6. Ikushima H, Miyazono K. Biology of transforming growth factorbeta signaling. Curr Pharm Biotechnol. 2011;12:2099–107. 7. Fujii D, Brissenden JE, Derynck R, Francke U. Transforming growth factor beta gene maps to human chromosome 19 long arm and to mouse chromosome 7. Somat Cell Mol Genet. 1986;12:281–8. 8. Wakefield LM, Piek E, Bottinger EP. TGF-beta signaling in mammary gland development and tumorigenesis. J Mammary Gland Biol Neoplasia. 2001;6:67–82. 9. Babyshkina N, Malinovskaya E, Stakheyeva M, Volkomorov V, Slonimskaya E, Maximov V, et al. Association of functional −509C>T polymorphism in the TGF-beta1 gene with infiltrating ductal breast carcinoma risk in a Russian Western Siberian population. Cancer Epidemiol. 2011;35:560–3. 10. Woo SU, Park KH, Woo OH, Yang DS, Kim AR, Lee ES, et al. Association of a TGF-beta1 gene −509 C/T polymorphism with
Tumor Biol. breast cancer risk: a meta-analysis. Breast Cancer Res Treat. 2010;124:481–5. 11. Dunning AM, Ellis PD, McBride S, Kirschenlohr HL, Healey CS, Kemp PR, et al. A transforming growth factorbeta1 signal peptide variant increases secretion in vitro and is associated with increased incidence of invasive breast cancer. Cancer Res. 2003;63:2610–5. 12. Shin A, Shu XO, Cai Q, Gao YT, Zheng W. Genetic polymorphisms of the transforming growth factor-beta1 gene and breast cancer risk: a possible dual role at different cancer stages. Cancer Epidemiol Biomarkers Prev. 2005;14:1567–70. 13. Awad MR, El-Gamel A, Hasleton P, Turner DM, Sinnott PJ, Hutchinson IV. Genotypic variation in the transforming growth factor-beta1 gene: association with transforming growth factorbeta1 production, fibrotic lung disease, and graft fibrosis after lung transplantation. Transplantation. 1998;66:1014–20. 14. Berndt SI, Huang WY, Chatterjee N, Yeager M, Welch R, Chanock SJ, et al. Transforming growth factor beta 1 (TGFB1) gene polymorphisms and risk of advanced colorectal adenoma. Carcinogenesis. 2007;28:1965–70. 15. Stoll C, Mengsteab S, Stoll D, Riediger D, Gressner AM, Weiskirchen R. Analysis of polymorphic TGFB1 codons 10, 25, and 263 in a German patient group with non-syndromic cleft lip, alveolus, and palate compared with healthy adults. BMC Med Genet. 2004;5:15. 16. Wei YS, Xu QQ, Wang CF, Pan Y, Liang F, Long XK. Genetic variation in transforming growth factor-beta1 gene associated with increased risk of esophageal squamous cell carcinoma. Tissue Antigens. 2007;70:464–9. 17. Zhang P, Di JZ, Zhu ZZ, Wu HM, Wang Y, Zhu G, et al. Association of transforming growth factor-beta 1 polymorphisms with genetic susceptibility to TNM stage I or II gastric cancer. Jpn J Clin Oncol. 2008;38:861–6. 18. Amani D, Dehaghani AS, Zolghadri J, Ravangard F, Niikawa N, Yoshiura K, et al. Lack of association between the TGF-beta1 gene polymorphisms and recurrent spontaneous abortion. J Reprod Immunol. 2005;68:91–103. 19. Amani D, Zolghadri J, Dehaghani AS, Pezeshki AM, Ghaderi A. The promoter region (−800, −509) polymorphisms of transforming growth factor-beta1 (TGF-beta1) gene and recurrent spontaneous abortion. J Reprod Immunol. 2004;62:159–66. 20. Derynck R, Rhee L, Chen EY, Van Tilburg A. Intron-exon structure of the human transforming growth factor-beta precursor gene. Nucleic Acids Res. 1987;15:3188–9. 21. Feizollahzadeh S, Taheripanah R, Khani M, Farokhi B, Amani D. Promoter region polymorphisms in the transforming growth factor beta-1 (TGFbeta1) gene and serum TGFbeta1 concentration in preeclamptic and control Iranian women. J Reprod Immunol. 2012;94: 216–21. 22. Skerrett DL, Moore EM, Bernstein DS, Vahdat L. Cytokine genotype polymorphisms in breast carcinoma: associations of TGF-beta1 with relapse. Cancer Investig. 2005;23:208–14. 23. Ziv E, Cauley J, Morin PA, Saiz R, Browner WS. Association between the T29→C polymorphism in the transforming growth factor beta1 gene and breast cancer among elderly white women: The Study of Osteoporotic Fractures. JAMA. 2001;285:2859– 63. 24. Jin Q, Hemminki K, Grzybowska E, Klaes R, Soderberg M, Zientek H, et al. Polymorphisms and haplotype structures in genes for transforming growth factor beta1 and its receptors in familial and unselected breast cancers. Int J Cancer. 2004;112:94–9. 25. Le Marchand L, Haiman CA, van den Berg D, Wilkens LR, Kolonel LN, Henderson BE. T29C polymorphism in the transforming growth factor beta1 gene and postmenopausal breast cancer risk: the Multiethnic Cohort Study. Cancer Epidemiol Biomarkers Prev. 2004;13:412–5.
26. Lee KM, Park SK, Hamajima N, Tajima K, Yoo KY, Shin A, et al. Genetic polymorphisms of TGF-beta1 & TNF-beta and breast cancer risk. Breast Cancer Res Treat. 2005;90:149–55. 27. Shu XO, Gao YT, Cai Q, Pierce L, Cai H, Ruan ZX, et al. Genetic polymorphisms in the TGF-beta 1 gene and breast cancer survival: a report from the Shanghai Breast Cancer Study. Cancer Res. 2004;64: 836–9. 28. Cox DG, Penney K, Guo Q, Hankinson SE, Hunter DJ. TGFB1 and TGFBR1 polymorphisms and breast cancer risk in the Nurses’ Health Study. BMC Cancer. 2007;7:175. 29. Kaklamani VG, Baddi L, Liu J, Rosman D, Phukan S, Bradley C, et al. Combined genetic assessment of transforming growth factorbeta signaling pathway variants may predict breast cancer risk. Cancer Res. 2005;65:3454–61. 30. Krippl P, Langsenlehner U, Renner W, Yazdani-Biuki B, Wolf G, Wascher TC, et al. The L10P polymorphism of the transforming growth factor-beta 1 gene is not associated with breast cancer risk. Cancer Lett. 2003;201:181–4. 31. Cox A, Dunning AM, Garcia-Closas M, Balasubramanian S, Reed MW, Pooley KA, et al. A common coding variant in CASP8 is associated with breast cancer risk. Nat Genet. 2007;39:352–8. 32. Hishida A, Iwata H, Hamajima N, Matsuo K, Mizutani M, Iwase T, et al. Transforming growth factor B1 T29C polymorphism and breast cancer risk in Japanese women. Breast Cancer. 2003;10:63–9. 33. Ma X, Chen C, Xiong H, Li Y. Transforming growth factorbeta1 L10P variant plays an active role on the breast cancer susceptibility in Caucasian: evidence from 10,392 cases and 11,697 controls. Breast Cancer Res Treat. 2010;124:453–7. 34. Scollen S, Luccarini C, Baynes C, Driver K, Humphreys MK, Garcia-Closas M, et al. TGF-beta signaling pathway and breast cancer susceptibility. Cancer Epidemiol Biomarkers Prev. 2011;20: 1112–9. 35. Mu L, Katsaros D, Lu L, Preti M, Durando A, Arisio R, et al. TGFbeta1 genotype and phenotype in breast cancer and their associations with IGFs and patient survival. Br J Cancer. 2008;99:1357–63. 36. Breast Cancer Association C. Commonly studied single-nucleotide polymorphisms and breast cancer: results from the Breast Cancer Association Consortium. J Natl Cancer Inst. 2006;98:1382–96. 37. Amani D, Farjadian S, Ghaderi A. The frequency of transforming growth factor-beta1 gene polymorphisms in a normal southern Iranian population. Int J Immunogenet. 2008;35:145–51. 38. Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. 1988;16:1215. 39. Liu K, Muse SV. PowerMarker: an integrated analysis environment for genetic marker analysis. Bioinformatics. 2005;21:2128–9. 40. Vuong MT, Lundberg S, Gunnarsson I, Wramner L, Seddighzadeh M, Hahn-Zoric M, et al. Genetic variation in the transforming growth factor-beta1 gene is associated with susceptibility to IgA nephropathy. Nephrol Dial Transplant. 2009;24:3061–7. 41. Yokota M, Ichihara S, Lin TL, Nakashima N, Yamada Y. Association of a T29→C polymorphism of the transforming growth factor-beta1 gene with genetic susceptibility to myocardial infarction in Japanese. Circulation. 2000;101:2783–7. 42. Beisner J, Buck MB, Fritz P, Dippon J, Schwab M, Brauch H, et al. A novel functional polymorphism in the transforming growth factorbeta2 gene promoter and tumor progression in breast cancer. Cancer Res. 2006;66:7554–61. 43. Ma X, Beeghly-Fadiel A, Lu W, Shi J, Xiang YB, Cai Q, et al. Pathway analyses identify TGFBR2 as potential breast cancer susceptibility gene: results from a consortium study among Asians. Cancer Epidemiol Biomarkers Prev. 2012;21:1176–84. 44. Yamada Y, Miyauchi A, Goto J, Takagi Y, Okuizumi H, Kanematsu M, et al. Association of a polymorphism of the transforming growth factor-beta1 gene with genetic susceptibility to osteoporosis in postmenopausal Japanese women. J Bone Miner Res. 1998;13:1569–76.
Tumor Biol. 45. Pelletier R, Pravica V, Perrey C, Xia D, Ferguson RM, Hutchinson I, et al. Evidence for a genetic predisposition towards acute rejection after kidney and simultaneous kidney-pancreas transplantation. Transplantation. 2000;70:674–80. 46. Gewaltig J, Mangasser-Stephan K, Gartung C, Biesterfeld S, Gressner AM. Association of polymorphisms of the transforming growth factor-beta1 gene with the rate of progression of HCVinduced liver fibrosis. Clin Chim Acta. 2002;316:83–94. 47. Feigelson HS, Patel AV, Diver WR, Stevens VL, Thun MJ, Calle EE. Transforming growth factor beta receptor type I and transforming growth factor beta1 polymorphisms are not associated with postmenopausal breast cancer. Cancer Epidemiol Biomarkers Prev. 2006;15: 1236–7. 48. Barnett GC, Coles CE, Burnet NG, Pharoah PD, Wilkinson J, West CM, et al. No association between SNPs regulating TGF-beta1 secretion and late radiotherapy toxicity to the breast: results from the RAPPER study. Radiother Oncol. 2010;97:9–14. 49. Band AM, Laiho M. Crosstalk of TGF-beta and estrogen receptor signaling in breast cancer. J Mammary Gland Biol Neoplasia. 2011;16:109–15. 50. Joshi NN, Kale MD, Hake SS, Kannan S. Transforming growth factor beta signaling pathway associated gene polymorphisms may
51.
52.
53.
54.
55.
explain lower breast cancer risk in western Indian women. PLoS One. 2011;6:e21866. Langdahl BL, Uitterlinden AG, Ralston SH, Trikalinos TA, Balcells S, Brandi ML, et al. Large-scale analysis of association between polymorphisms in the transforming growth factor beta 1 gene (TGFB1) and osteoporosis: the GENOMOS study. Bone. 2008;42:969–81. Keen RW, Snieder H, Molloy H, Daniels J, Chiano M, Gibson F, et al. Evidence of association and linkage disequilibrium between a novel polymorphism in the transforming growth factor beta 1 gene and hip bone mineral density: a study of female twins. Rheumatology (Oxford). 2001;40:48–54. Langdahl BL, Carstens M, Stenkjaer L, Eriksen EF. Polymorphisms in the transforming growth factor beta 1 gene and osteoporosis. Bone. 2003;32:297–310. Bertoldo F, D’Agruma L, Furlan F, Colapietro F, Lorenzi MT, Maiorano N, et al. Transforming growth factor-beta1 gene polymorphism, bone turnover, and bone mass in Italian postmenopausal women. J Bone Miner Res. 2000;15:634–9. Weinshenker BG, Hebrink D, Kantarci OH, Schaefer-Klein J, Atkinson E, Schaid D, et al. Genetic variation in the transforming growth factor beta1 gene in multiple sclerosis. J Neuroimmunol. 2001;120:138–45.
