In this case--control study, which drew participants from a national cohort, SNPs in genes in the vitamin D pathway were found to be associated with breast cancer risk. Of six polymorphisms in CYP24A1, two variants found in independent regions of the gene were significantly associated with breast cancer risk. In VDR, rare alleles for Bsm1 were significantly associated with breast cancer risk.
Germline polymorphisms in CYP24A1
have been the subject of only a few recent epidemiologic studies of cancer risk (18
). The variants studied here are found in two regions of the gene (): two near the proximal promoter region which includes two vitamin D response elements (VDRE) (22
), and four near the 3′ end of the gene, where additional VDRE act as downstream enhancers of expression (23
). While most of the selected SNPs are intronic; rs2762934 is found in the 3′ untranslated region of the CYP24A1
Three studies have investigated associations of CYP24A1
polymorphisms with risk of breast cancer (19
). In a nested case--control study of postmenopausal breast cancer, which included measures of only one polymorphism in CYP24A1
, rs2296241, a well-conserved variant in the fourth exon was not associated with breast cancer risk; this SNP was not among those selected for the present analysis. Recently, Anderson et al
. conducted a large breast cancer case-control study in Ontario, Canada; they assessed four SNPs in CYP24A1
and found no statistically significant associations with risk among studied variants (21
); of these polymorphisms none are examined or in linkage disequilibrium with the variants studied here.
In a population-based breast cancer case--control study in Shanghai, Dorjgochoo et al
. measured 59 SNPs in CYP24A1
in addition to 500 variants in other genes in the vitamin D pathway (20
). They found three markers in CYP24A1
with nominal statistical significance (P
<0.05) but these did not survive correction for multiple comparisons, as was the case for all other SNPs under study, suggesting that genetic variation in the vitamin D pathway does not play an important role in breast cancer etiology in this population. Variants studied by Dorjgochoo et al.
do not coincide with those in the present study however the SNP rs6097809, found to be modestly associated with breast cancer risk with a per allele OR of 0.90, (95% CI=0.83-0.98, Ptrend
=0.02) in their study, is in LD with the variant rs1570669 (r2
=0.25), for which we found effects of similar direction and magnitude (OR=0.84 per rare allele, 95% CI=0.71-1.00, Ptrend
Wang et al
. conducted a genome wide association study to find SNPs associated with vitamin D insufficiency among men and women of European descent (6
). They found that SNP rs6013897, in the intergenic region downstream of CYP24A1,
achieved genome-wide statistical significance as a predictor of vitamin D insufficiency in the pooled sample; in HapMap data this SNP is in weak LD with two SNPs we studied here (rs1570669, rs2762934, with r2
=0.09 and 0.12, respectively), found to be associated with significantly reduced, and significantly increased breast cancer risk, respectively. However, it is not clear that these results are comparable, since vitamin D insufficiency for the analysis of Wang et al.
was based on circulating 25-hydroxyvitamin D, while we have suggested that CYP24A1
polymorphisms might impact breast cancer risk through effects on the availability of its downstream metabolite, 1,25 hydroxyvitamin D. Genome-wide association studies of breast cancer have not identified loci in CYP24A1
, although achieving genome-wide significance may exclude some true risk genes and it may be that additional studies conducted to evaluate interaction may be required to find these loci.
We recorded reduced risk of breast cancer in association with the number of rare alleles for rs1570669 in CYP24A1
. This polymorphism is located in a haplotype block identified in our data that includes polymorphisms in the 8th, 9th and 12th introns. Previous research has shown differences among cancer cell lines in enzyme activities of induced and constitutive CYP24A1 resulting from differential splicing of transcripts between exons 9 and 11 (24
). Investigators have speculated that germline polymorphisms in the ninth intron could influence splicing but this has not yet been shown (25
Finally, in exploratory analyses, we assessed potential interactions between the genetic variants and a number of different measures of sunlight exposure, including average annual sunlight in the state of residence at study baseline and at birth, and retrospectively collected data on time spent outdoors during summers in childhood (<13 years) and during the teen years (13-19 years). Several interactions were observed, each indicating that among women carrying rare alleles of genotypes in the VDR and CYP24A1 genes, greater time spent outdoors during summers in the teen years was associated with reduced risk of breast cancer; in contrast, no inverse associations in association with time outdoors were observed in women homozygous for the same genotypes. While a large number of comparisons were made, the clustering of statistically significant interactions suggest that sun exposure and vitamin D may be most important for women who are genetically susceptible and at a time of life when breast tissue is more susceptible to insult.
Polymorphic variation in the CYP24A1
gene has not been extensively studied but may be important as a determinant of vitamin D availability in tissue. In the breast, available 1,25-hydroxyvitamin D is determined not only by the availability of substrate (25-hydroxyvitamin D), but also by the relative expression and activity of the hydroxylases (CYP27B1 and CYP24A1) which activate and degrade vitamin D. Paracrine mechanisms to regulate expression of these enzymes are complex, tissue-specific, and as yet, poorly understood (26
). SNP variants in CYP24A1
could result in differences in gene expression, transcript stability, or enzyme activity, and so could have direct effects on tissue levels of 1,25-hydroxyvitamin D. Variation in the gene could also affect co-regulation and so modulate the ability to maintain homeostatic tissue levels of activated vitamin D when circulating 25-hydroxyvitamin D undergoes seasonal fluctuations (27
A number of polymorphisms in the VDR
have been studied previously in association with breast cancer risk, however results are not consistent. Epidemiological studies of Bsm1
and breast cancer risk have inconsistent findings and both a meta-analysis (9
) and a pooled study of six prospective studies (8
) found no statistically significant association for this polymorphism. The Bsm1
–related variant has not been associated with VDR expression or function but is in LD with a known poly (A) variant in the 3′ untranslated region of the gene which is thought to modulate the stability of RNA transcripts. Limited data has suggested that differences across populations in patterns of LD near this variant may explain the heterogeneity seen in association studies using this genetic marker (28
Variation in the VDR genotypes for Fok1 suggests that the rare allele results in compromised efficiency of signal transduction. Results of a recent pooled analysis of six prospective studies  suggest a modest increase in breast cancer risk associated with the rare allele for the Fok1-related polymorphism (combined OR for two rare alleles =1.16, 95% CI=1.04-1.28 and Ptrend=0.006), however, there was significant heterogeneity across studies (P=0.03). In our study, no significant main effect was seen for this polymorphism and in fact measures of association for this locus tended towards an inverse association.
While several previous studies have looked at interactions of dietary intake of vitamin D and its interactions with polymorphic variants in the VDR
, only one has looked at interactions of genotypes with exposure to sunlight, which has a greater impact on circulating vitamin D than diet. In a study by Anderson et al
), investigators report that they assessed interactions of variables related to sunlight exposure in adulthood, including geographic location, skin tone and sun-protective behaviors, and found no significant interactions with genetic polymorphisms in predicting breast cancer risk. Animal models of breast cancer and some epidemiological data suggest that prenatal, childhood, and adolescent periods are periods of greater susceptibility to carcinogenic insult since breast tissue remains undifferentiated until the first pregnancy (29
). While exploratory, our findings of significant interactions with time spent outdoors in the teen years suggest that vitamin D exposure may be particularly important for later risk of breast cancer.
Our study has several unique features including the availability of genetic material and detailed information about reproductive, demographic and lifestyle factors derived from interviews of all subjects. Study participants came from states across the U.S. A limitation of the present study is retrospective identification of cases, which required survival from the time of breast cancer diagnosis to blood collection; however, analysis of allelic frequencies in cases with varying lengths of time from diagnosis to blood sampling did not suggest that any SNP was a correlate of survival (data not shown). Comparisons of demographic characteristics did not reveal any significant differences between participating cases and those who did not participate (11
In summary, we have identified a number of variants in the gene that codes for the 1,25-dihydroxyvitamin D3 24-hydroxylase enzyme associated with breast cancer risk. These findings need to be confirmed in other studies. Future studies should include more genes in the vitamin D pathway and greater coverage of these genes, particularly the CYP24A1 gene, and measures of sunlight exposure in early life and adulthood to permit an examination of whether genetic variants interact with vitamin D levels to influence cancer risks.