BMC Cancer 2004, 4 http://www.biomedcentral.com/1471-2407/4/9 Table 3: Cumulative breast cancer risk ratios to age 50 and to age 70 by genotype among 2430 first degree female relatives of breast cancer cases (probands), 190 of whom were reported to have breast cancer (Continued) PP PR or RR TGFß1 T263I TT TI or II IGFBP3 -202 A>C AA AC or CC 0.04 0.53*** 48 8 54 3 11 45 1.0 1.0 1.0 2.1 1.0 1.0 --- 0.1–3.1 --- 0.0–4.9 --- 0.3–3.5 0.952 0.492 0.848 126 20 138 9 36 110 1.0 1.2 1.0 1.7 1.0 0.7 --- 0.6–2.1 --- 0.6–3.1 --- 0.4–1.3 0.554 0.312 0.228 * Amino acids and their symbols: R: Arginine, W: Tryptophan, H: Histidine, Q: Glutamine, D: Aspartic Acid, E: Glutamic Acid, N: Asparagine, P: Proline, S: Serine, L: Leucine, I: Isoleucine, T: Threonine. ** Numbers represent those first degree female relatives who had breast cancer up to age 50 or 70 by the genotype of the case (index proband). The numbers are not the probabilistic assignment of the kin-cohort calculation to a specific genotype. *** Represents the frequency of the C allele, although not technically "rare". directly assess the relationship between the IGFBP3- 202A>C variant and circulating IGFBP3 and IGF-1 levels, we did not observe an association between this SNP (as an indicator of IGF-1 levels) and breast cancer risk in our study. Three SNPs in the TGFß1 gene (L10P, P25R, and T263I) have previously been described [37], with the L10P variant showing some association with increased risk of breast cancer [38,39]. Our data do not support an association between these TGFß1 SNPs and breast cancer risk in this cohort. Of all the SNPs evaluated, BRCA2 N372H had shown the most consistent relationship to breast cancer in previous studies: in both a European study [40] and an Australian study [41], HH homozygotes had significantly elevated odds ratios for breast cancer of 1.3 to 1.5 compared to NN homozygotes, and in a study of ovarian cancer from the UK and Australia [42], risk was similarly elevated (OR = 1.4) for HH homozygotes. In all three of these studies, there was no evidence of increased (or decreased) risk among heterozygotes, with ORs of 1.01 to 1.03, compared to NN homozygotes. In our study, HH homozygotes had a slightly increased RR of 1.5 (95% CI, 0.4–4.4) and 1.3 (95% CI, 0.8 – 2.4) over NN homozygotes up to ages 50 and 70, respectively. Unlike the previous studies, hetero- zygotes had a significantly reduced breast cancer risk com- pared to NN homozygotes. The minor allele frequency was somewhat higher in our study (0.32) of cases, versus approximately 0.28 in the UK\Australian study [42]. In the initial study [40], it was noted that the SNP in female controls was not in Hardy-Weinberg equilibrium (HWE), with an excess of heterozygotes and a deficit of both homozygotes. In our case series, the SNP was not in HWE, with 23 fewer heterozygotes than expected and more than 20% over the expected numbers of HH homozygotes under HWE; this is the trend one might have expected if the H allele increases risk. In addition, the 9.1% HH gen- otype frequency in our breast cancer cases was very similar to that observed in the Australian breast cancer study (9.2%) [41]. Whether the breast cancer risk is lower in het- erozygotes, as is clearly evident in the risk curves from our analyses, or whether the inconsistent findings are still due to chance (even after multiple comparisons adjustment) are not known and will require further study in case-con- trol analyses in this cohort or other large epidemiologic studies. Because the striking heterozygous advantage in BRCA2 N372H and APEX D148E were unexpected and difficult to interpret biologically, we performed analyses stratified by type of relative (mother, sister). We discovered that, in general, the kin-cohort model for three genotype catego- ries is not identifiable when restricted to mothers of the index cases because the matrix of Mendelian genotype probabilities for relatives conditional on the genotype and type of relative of the index case is rank deficient. This meant that calculations restricted to mothers could not be performed, but we could determine the relationship between individual SNPs and breast cancer among sisters. Therefore, we relied on the analysis restricted to sisters to corroborate patterns observed for all relatives combined. For sisters only, the analyses revealed the same patterns as shown in Figure 2, which were based on all female rela- tives, except for APEX D148E, where the results for sisters only showed similar risks for homozygous common and heterozygous genotypes and an increased risk for homozygous variant genotypes (data not shown). Due to the biological inconsistency of the results for APEX D148E and because of the differences between analyses based on sisters only and all relatives, we suggest caution in inter- preting this result, despite the statistical significance. For BRCA2 N372H, results for sisters only were very similar to results for all relatives combined, lending credence to our observations, despite the difficult interpretation. There are several study limitations. Fifty-six per cent of the women eligible donated a blood sample before the arbitrary genotyping cut-off date (December 31, 2001). Page 9 of 12 (page number not for citation purposes)