SMAD7 and GREM1 are signaling components on the transforming growth factor-β pathway, which regulates normal mammary gland development and has been implicated in breast tumor invasion and metastasis. Three variants within SMAD7 and two variants in CRAC1 (a colorectal cancer–associated region on chromosome 15 in which GREM1 is located) have been associated with colorectal cancer risks [odds ratios (OR), 0.85-1.26; all P < 10−7]. We genotyped these five variants in a series of 1,267 bilateral breast cancer cases and 900 controls to determine whether they are associated with breast as well as colorectal cancer risk. None of these single nucleotide polymorphisms were associated with breast cancer risk in our study and the 95% confidence limits of our data, pooled with data from the Cancer Genetic Markers of Susceptibility study, exclude per allele ORs of <0.94 or >1.08. One or more of these variants may be associated with a very small OR for breast cancer, but our data suggest that the effects of these alleles are cancer site–specific. (Cancer Epidemiol Biomarkers Prev 2009;18(6):1934–6)

A possible explanation for the surprising observation that familial cancer risks are predominantly site-specific (1) is tissue-specific regulation of some of the crucial genes underlying polygenic susceptibility. This is consistent with recent whole genome association studies of common epithelial cancers, which have identified single nucleotide polymorphisms (SNP) within a 0.5 Mb “gene desert” on chromosome 8q24, which was associated with several cancer sites (2-7). One variant (rs6983267) was associated with colorectal, prostate, ovarian, and possibly breast cancer, but others seem to be cancer site–specific (8, 9). The most significant SNPs identified by Broderick et al. (10) in their colorectal cancer genome-wide association study were rs6983267 at 8q24 and three variants (rs4939827, rs12953717, and rs4464148) that map to a 3 kb LD block within intron 3 of SMAD7. In a classic linkage study (11), Jaeger et al. identified a locus on chromosome 15q13.3-q14 (CRAC1) which was associated with colorectal cancer risk; genotyping of 145 SNPs within this region identified two SNPs (rs4779584 and rs10318) that were associated with colorectal cancer risk, one of which (rs10318) lies in the 3′-untranslated region of GREM1. We have genotyped these three variants within SMAD7 and the two variants in CRAC1 in a series of bilateral breast cancer cases and controls to determine whether they are associated with breast as well as colorectal cancer risk.

The British Breast Cancer Study

Cases. Eight hundred and eighty-two cases with two primary breast cancers were ascertained through the English and Scottish cancer registries as described previously (ref. 12; median age at first diagnosis, 48 years; range 25-65 years old). An additional 385 bilateral breast cancer cases were ascertained through the National Cancer Research Network as described previously (ref. 8; median age at first diagnosis, 50 years; range 24-70 years old).

Controls. Nine hundred controls who were friends or non–blood relatives of index cases were recruited through the cases (12). None of the controls had a personal history of malignancy at the time of ascertainment. All cases and controls were Caucasians resident in the United Kingdom. Written informed consent was obtained from all participants and the study was approved by the UK South East MREC.

Genotyping. DNA was extracted from blood samples and quantified using PicoGreen (Invitrogen). Genotyping of samples was done by KBioscience5

using their proprietary in-house competitive allele-specific PCR SNP genotyping system (KASPar).

Statistical Methods. Genotype frequencies in controls were tested for departure from Hardy-Weinberg equilibrium using a χ2 test. Unadjusted odds ratios (OR) with two-sided exact P values were calculated using standard methods. All five of these SNPs were genotyped in stage I of the Cancer Genetic Markers of Susceptibility (CGEMS) breast cancer genome-wide association study (13). Full details of CGEMS data available at the NCI CGEMS web site.6

The OR for a second primary breast cancer is approximately the square of the OR for a first primary (14, 15), so we used a square root transformation of ORs from the British Breast Cancer (BBC) study for pooling with data from the CGEMS study (13). Pooled estimates are inverse variance-weighted averages of the log OR from the transformed BBC data and the CGEMS data. All statistical analyses were carried out using STATA 10 (Stata Corporation).

Call rates were 98%, 99%, 98%, 97%, and 96% for rs4939827, rs12953717, rs4464148, rs4779584, and rs10318, respectively. Genotype frequencies in controls did not deviate from Hardy-Weinberg equilibrium. We found no association between any of these SNPs and breast cancer in our data (Table 1). Meta-analyses of data from the BBC study and CGEMS are shown in Table 2. The 95% confidence limits exclude per allele ORs of <0.94 or >1.08 for these SNPs.

Table 1.

Risk of breast cancer associated with the SMAD7 and CRAC1 SNPs

Locus/SNPGenotypeMAF controlsCases
Controls
OR (95% CI)P trend
n%n%
rs4939827 TT 0.468 345 27.8 248 28.2 Reference  
18q21 TC  628 50.7 440 50.0 1.03 (0.84-1.26)  
 CC  266 21.5 192 21.8 1.00 (0.78-1.28)  
 Allelic OR      1.00 (0.88-1.13) 0.997 
rs12953717 CC 0.445 375 30.0 277 31.2 Reference  
18q21 CT  630 50.4 431 48.6 1.08 (0.89-1.32)  
 TT  244 19.5 179 20.2 1.01 (0.79-1.29)  
 Allelic OR      1.01 (0.89-1.15) 0.856 
rs4464148 TT 0.317 580 48.8 415 46.9 Reference  
18q21 TC  546 44.0 377 42.6 1.04 (0.86-1.24)  
 CC  114 9.2 92 10.4 0.89 (0.66-1.20)  
 Allelic OR      0.98 (0.85-1.12) 0.718 
rs4779584 CC 0.185 825 66.8 575 66.2 Reference  
15q13 TC  364 29.5 267 30.7 0.95 (0.79-1.15)  
 TT  46 3.7 27 3.1 1.19 (0.73-1.93)  
 Allelic OR      1.00 (0.85-1.17) 0.995 
rs10318 CC 0.180 808 66.7 582 67.4 Reference  
15q13 CT  359 29.6 252 29.2 1.03 (0.85-1.24)  
 TT  44 3.6 29 3.4 1.09 (0.68-1.77)  
 Allelic OR      1.03 (0.88-1.22) 0.684 
Locus/SNPGenotypeMAF controlsCases
Controls
OR (95% CI)P trend
n%n%
rs4939827 TT 0.468 345 27.8 248 28.2 Reference  
18q21 TC  628 50.7 440 50.0 1.03 (0.84-1.26)  
 CC  266 21.5 192 21.8 1.00 (0.78-1.28)  
 Allelic OR      1.00 (0.88-1.13) 0.997 
rs12953717 CC 0.445 375 30.0 277 31.2 Reference  
18q21 CT  630 50.4 431 48.6 1.08 (0.89-1.32)  
 TT  244 19.5 179 20.2 1.01 (0.79-1.29)  
 Allelic OR      1.01 (0.89-1.15) 0.856 
rs4464148 TT 0.317 580 48.8 415 46.9 Reference  
18q21 TC  546 44.0 377 42.6 1.04 (0.86-1.24)  
 CC  114 9.2 92 10.4 0.89 (0.66-1.20)  
 Allelic OR      0.98 (0.85-1.12) 0.718 
rs4779584 CC 0.185 825 66.8 575 66.2 Reference  
15q13 TC  364 29.5 267 30.7 0.95 (0.79-1.15)  
 TT  46 3.7 27 3.1 1.19 (0.73-1.93)  
 Allelic OR      1.00 (0.85-1.17) 0.995 
rs10318 CC 0.180 808 66.7 582 67.4 Reference  
15q13 CT  359 29.6 252 29.2 1.03 (0.85-1.24)  
 TT  44 3.6 29 3.4 1.09 (0.68-1.77)  
 Allelic OR      1.03 (0.88-1.22) 0.684 
Table 2.

Summary ORs for SMAD7 and CRAC1 SNPs in breast cancer and colorectal cancer association studies

Locus/SNPStudyGenotypeAllelic OR (95% CI)P trendP heterogeneity
rs4939827 BBC T/C 1.00 (0.94-1.06)   
18q21 CGEMS*  1.01 (0.90-1.14)   
 BBC and CGEMS pooled  1.00 (0.95-1.06) 0.94 0.88 
 Colorectal pooled (10)  0.85 (0.81-0.89) 1 × 10−12  
rs12953717 BBC C/T 1.01 (0.94-1.07)   
18q21 CGEMS*  1.02 (0.91-1.15)   
 BBC and CGEMS pooled  1.01 (0.95-1.07) 0.77 0.83 
 Colorectal pooled (10)  1.17 (1.12-1.22) 9.1 × 10−12  
rs4464148 BBC T/C 0.99 (0.93-1.06)   
18q21 CGEMS*  1.03 (0.90-1.17)   
 BBC and CGEMS pooled  1.00 (0.94-1.06) 0.95 0.60 
 Colorectal pooled (10)  1.15 (1.09-1.21) 6.68 × 10−8  
rs4779584 BBC T/C 1.00 (0.92-1.08)   
15q13 CGEMS*  1.04 (0.89-1.21)   
 BBC and CGEMS pooled  1.01 (0.94-1.08) 0.82 0.66 
 Colorectal pooled (11)  1.26 (1.19-1.34) 4.44 × 10−14  
rs10318 BBC C/T 1.01 (0.94-1.10)   
15q13 CGEMS*  0.99 (0.85-1.15)   
 BBC and CGEMS pooled  1.01 (0.94-1.08) 0.80 0.78 
 Colorectal pooled (11)  1.19 (1.12-1.26) 7.93 × 10−9  
Locus/SNPStudyGenotypeAllelic OR (95% CI)P trendP heterogeneity
rs4939827 BBC T/C 1.00 (0.94-1.06)   
18q21 CGEMS*  1.01 (0.90-1.14)   
 BBC and CGEMS pooled  1.00 (0.95-1.06) 0.94 0.88 
 Colorectal pooled (10)  0.85 (0.81-0.89) 1 × 10−12  
rs12953717 BBC C/T 1.01 (0.94-1.07)   
18q21 CGEMS*  1.02 (0.91-1.15)   
 BBC and CGEMS pooled  1.01 (0.95-1.07) 0.77 0.83 
 Colorectal pooled (10)  1.17 (1.12-1.22) 9.1 × 10−12  
rs4464148 BBC T/C 0.99 (0.93-1.06)   
18q21 CGEMS*  1.03 (0.90-1.17)   
 BBC and CGEMS pooled  1.00 (0.94-1.06) 0.95 0.60 
 Colorectal pooled (10)  1.15 (1.09-1.21) 6.68 × 10−8  
rs4779584 BBC T/C 1.00 (0.92-1.08)   
15q13 CGEMS*  1.04 (0.89-1.21)   
 BBC and CGEMS pooled  1.01 (0.94-1.08) 0.82 0.66 
 Colorectal pooled (11)  1.26 (1.19-1.34) 4.44 × 10−14  
rs10318 BBC C/T 1.01 (0.94-1.10)   
15q13 CGEMS*  0.99 (0.85-1.15)   
 BBC and CGEMS pooled  1.01 (0.94-1.08) 0.80 0.78 
 Colorectal pooled (11)  1.19 (1.12-1.26) 7.93 × 10−9  
*

CGEMS study comprises genotype data on 1,145 breast cancer cases and 1,142 controls (http://cgems.cancer.gov/data; ref. 13).

Both SMAD7 and GREM1 are signaling components on the transforming growth factor-β pathway, which regulates normal mammary gland development (16) and has been implicated in tumor invasion and metastasis (17). A nonsynonymous coding SNP (L10P) in transforming growth factor-β has previously been associated with a modest breast cancer risk (18). These five SNPs are therefore plausible candidates for association with breast as well as colorectal cancer, but our data and the CGEMS results show no associations with breast cancer risk. The confidence limits from the pooled data from this study and the CGEMS stage I study exclude a breast cancer OR of <0.95 for rs4989327 in SMAD7 compared with an OR of 0.85 (95% CI, 0.81-0.89; Table 2) for colorectal cancer (10). The confidence limits for the other SMAD7 and GREM1 SNPs that we tested exclude breast cancer ORs of >1.08; in contrast, the ORs for colorectal cancer for these SNPs are all >1.15, with lower confidence limits of 1.09 or greater (Table 2; refs. 10, 11). One or more of these variants may be associated with a very small OR for breast cancer, but our data suggests that the effects of these alleles are cancer site–specific.

No potential conflicts of interest were disclosed.

Grant support: Cancer Research UK and Breakthrough Breast Cancer. We acknowledge NHS funding to the NIHR Biomedical Research Centre.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1
Goldgar DE, Fields P, Lewis CM, et al. A large kindred with 17q-linked breast and ovarian cancer: genetic, phenotypic, and genealogical analysis.
J Natl Cancer Inst
1994
;
86
:
200
–9.
2
Easton DF, Pooley KA, Dunning AM, et al. Genome-wide association study identifies novel breast cancer susceptibility loci.
Nature
2007
;
447
:
1087
–93.
3
Amundadottir LT, Sulem P, Gudmundsson J, et al. A common variant associated with prostate cancer in European and African populations.
Nat Genet
2006
;
38
:
652
–8.
4
Gudmundsson J, Sulem P, Manolescu A, et al. Genome-wide association study identifies a second prostate cancer susceptibility variant at 8q24.
Nat Genet
2007
;
39
:
631
–7.
5
Haiman CA, Patterson N, Freedman ML, et al. Multiple regions within 8q24 independently affect risk for prostate cancer.
Nat Genet
2007
;
39
:
638
–44.
6
Haiman CA, Le Marchand L, Yamamato J, et al. A common genetic risk factor for colorectal and prostate cancer.
Nat Genet
2007
;
39
:
954
–6.
7
Zanke BW, Greenwood CM, Rangrej J, et al. Genome-wide association scan identifies a colorectal cancer susceptibility locus on chromosome 8q24.
Nat Genet
2007
;
39
:
989
–94.
8
Fletcher O, Johnson N, Gibson L, et al. Association of genetic variants at 8q24 with breast cancer risk.
Cancer Epidemiol Biomarkers Prev
2008
;
17
:
702
–5.
9
Ghoussaini M, Song H, Koessler T, et al. Multiple loci with different cancer specificities within the 8q24 gene desert.
J Natl Cancer Inst
2008
;
100
:
962
–6.
10
Broderick P, Carvajal-Carmona L, Pittman AM, et al. A genome-wide association study shows that common alleles of SMAD7 influence colorectal cancer risk.
Nat Genet
2007
;
39
:
1315
–7.
11
Jaeger E, Webb E, Howarth K, et al. Common genetic variants at the CRAC1 (HMPS) locus on chromosome 15q13.3 influence colorectal cancer risk.
Nat Genet
2008
;
40
:
26
–8.
12
Johnson N, Fletcher O, Naceur-Lombardelli C, dos Santos Silva I, Ashworth A, Peto J. Interaction between CHEK2*1100delC and other low-penetrance breast-cancer susceptibility genes: a familial study.
Lancet
2005
;
366
:
1554
–7.
13
Hunter DJ, Kraft P, Jacobs KB, et al. A genome-wide association study identifies alleles in FGFR2 associated with risk of sporadic postmenopausal breast cancer.
Nat Genet
2007
;
39
:
870
–4.
14
Fletcher O, Johnson N, Palles C, et al. Inconsistent association between the STK15 F31I genetic polymorphism and breast cancer risk.
J Natl Cancer Inst
2006
;
98
:
1014
–8.
15
Antoniou AC, Easton DF. Polygenic inheritance of breast cancer: implications for design of association studies.
Genet Epidemiol
2003
;
25
:
190
–202.
16
Bierie B, Moses HL. Tumour microenvironment: TGFβ: the molecular Jekyll and Hyde of cancer.
Nat Rev Cancer
2006
;
6
:
506
–20.
17
Derynck R, Akhurst RJ, Balmain A. TGF-β signaling in tumor suppression and cancer progression.
Nat Genet
2001
;
29
:
117
–29.
18
Cox A, Dunning AM, Garcia-Closas M, et al. A common coding variant in CASP8 is associated with breast cancer risk.
Nat Genet
2007
;
39
:
352
–8.