The results from our high-resolution genome-wide scans for structural variants provide evidence that rare CNVs contribute to breast cancer susceptibility. When compared to controls, the studied breast cancer cases showed a slight but consistent increase in the frequency of rare CNVs. The difference was not as profound as seen in psychiatric disorder studies where the observed changes, typically involving large genomic regions and numerous genes, can have very severe effects on patients' phenotype and many of which are de novo
. However, in our study the biological networks affected by the disrupted genes differed between breast cancer cases and controls, supporting their role in cancer predisposition.
The genes disrupted in familial cases showed a significant overrepresentation in functions involving the maintenance of genomic integrity. This included DSB repair, which is consistent with the prevailing paradigm that defects in this pathway contribute to breast cancer predisposition 
. The three DSB repair genes, BLM
, disrupted in the case group all represent attractive breast cancer susceptibility genes. Moreover, IPA analysis demonstrated that the genes disrupted by rare CNVs in the studied breast cancer cases formed a network centered on TP53
and β-estradiol, a notion confirmed in two independent cohorts. Both networks are coherent and biologically meaningful, and their identification through the used genome-wide approach provides strong evidence for a role in breast cancer predisposition.
network genes encode proteins functioning in pathways with potential role in malignancy prevention, including DNA damage response and apoptosis 
, but also RNA interference 
. They all represent attractive susceptibility genes, which could harbor also other cancer predisposing mutations; thus being excellent candidates for re-sequencing studies. Of the disrupted TP53
network genes DAB2IP
were particularly interesting. DAB2IP
is a member of the Ras GTPase-activating gene family and has been reported to act as a tumor suppressor. Inactivation of DAB2IP by promoter methylation occurs in several malignancies, including prostate and breast cancer 
, and it has been shown to modulate epithelial-to-mesenchymal transition and prostate-cancer metastasis 
is an apoptosis related gene, which encodes a member of a highly conserved caspase protease family, caspase 3. Caspases are key intermediaries of the apoptotic process, failure of which can lead to cancer 
. Various molecular epidemiological studies have suggested that SNPs in caspases may contribute to cancer risk, and a common coding variant in caspase 8 has been associated with breast cancer susceptibility 
. Curiously, apoptosis is also one of the numerous genomic integrity maintenance functions of BRCA1. Caspase 3 has been reported to mediate the cleavage of BRCA1 during UV-induced apoptosis, and the cleaved C-terminal fragment triggers the apoptotic response through activation of BRCA1 downstream effectors 
. The rare CNVs disrupting the DAB2IP
genes were both predicted to result in null alleles ().
For estrogen, there are multiple lines of evidence for its profound role in breast cancer development, and disruptions in estrogen signaling and metabolism have long been considered to affect breast cancer risk. The estrogen network was largely explained by the genes under β-estradiol regulation, but two of the disrupted genes, ESR2
, had a more straightforward role in estrogen signalling. ESR2
encodes the estrogen receptor β, which is one of the main mediators of estrogen actions within the cell 
. It binds estrogens with a similar affinity as estrogen receptor α, and activates expression of estrogen response element containing genes 
has previously been suggested to harbor common breast cancer predisposing variants 
, and ESR2 variation has been suggested to influence the development of breast cancer also by in vitro
. In contrast, striatin acts as molecular scaffold in non-genomic estrogen-mediated signaling 
. It physically interacts with calmodulin 1 
and estrogen receptor α, and also forms a complex with protein phosphatase 2A, which also regulates the function of estrogen receptor α 
. The identification of a recurrent deletion allele in CYP2C19
, encoding an enzyme involved in estrogen metabolism 
and with an increased frequency in familial cases (Table S2
), further emphasizes the role of estrogen in breast cancer predisposition. One CYP2C19
*17, defining an ultra-rapid metabolizer phenotype, has previously been associated with a decreased risk for breast cancer. This suggests that increased catabolism of estrogens by CYP2C19 may lead to decreased estrogen levels and therefore reduced breast cancer risk 
. Correspondingly, decreased activity of CYP2C19 through haploinsufficiency might potentially increase the risk of breast cancer. Curiously, based on their function both ESR2
have long been considered strong candidate genes for breast cancer susceptibility. However, no structural variants have previously been reported in either of them, and it is possible that CNVs might represent a new class of cancer predisposing variation in both genes. Functionally relevant structural variants might be present also in other CYP
genes that locate in gene clusters, like CYP2C19
. The clustering of similar genes increases the potential for unequal crossing-over between sister chromatids and thus for creation of CNV alleles.
The genes disrupted in both studied breast cancer cohorts were also significantly overrepresented among genes connected to diabetes mellitus. This unexpected result likely represents shared risk factors predisposing to both breast cancer and diabetes. Indeed, these two diseases have already been reported to share several non-genetic risk factors, including obesity and a sedentary lifestyle. The hormonal factors altered in diabetes include several hormonal systems that may also affect the development of breast cancer, including insulin, insulin-like growth factors, and other growth factors as well as estrogen 
. Our results support estrogen being the key link in the association between diabetes and breast cancer, as over one third of the diabetes associated genes in the two studied breast cancer cohorts were part of the β-estradiol network.
In conclusion, rare CNVs should be recognized as an alternative source of genetic variation influencing breast cancer risk. This notion is further supported by a recent study which also provided evidence for rare CNVs' contribution to familial and early-onset breast cancer 
. The results from the current network analysis with two independent breast cancer cohorts provide strong evidence for the role of estrogen mediated signaling in breast cancer predisposition and reinforce the concept of TP53
centered tumor suppression in the prevention of malignancy. The variety of disrupted genes belonging to these networks underscores that diverse mechanisms are likely to be relevant to breast cancer pathogenesis.