Although the relevance of mutations in several DNA repair genes (BRCA1, BRCA2, TP53, PTEN, PALB2, BRIP1) in breast cancer susceptibility have been well established, the association of variants in other genes, potentially accounting for the remaining familial clustering, is not well defined. For example, heterozygous carriers of deleterious mutations in the NBN gene, in particular the Slavic founder mutation 657del5, has been associated with a 2- to 3-fold increased risk of cancer . However the impact of other NBN variants on cancer risk is unclear. Nonetheless, studies have demonstrated an increased spontaneous chromosomal instability in cells from heterozygous carriers of NBN mutations [7, 16]. In addition, the presence of a specific pattern of gene expression involving pathways of DNA repair and damage bypass, mitotic checkpoint and apoptosis was demonstrated, suggesting that cells from these carriers may display a much less efficient DNA repair system . In this regard, a recent study by Someya et al.  showed a correlation between persistent radiation-induced NBN foci and both chromosomal instability and sporadic breast cancer risk. To address the possible implication of NBN sequence variants on breast cancer risk, we took advantage of our resource of high-risk breast cancer families drawn from the French Canadian population. In order to increase the statistical power of our investigation, one affected individual from each family was selected for analysis , each thoroughly screened for BRCA1 and BRCA2 mutations or large genomic rearrangements in these genes .
The majority of the coding variants identified in our cohort of breast cancer cases are situated in the forkhead-associated (FHA) domain and the two BRCT domains, and these domains have been demonstrated to be essential to NBN binding to the histone γ-H2AX [34, 35]. One of the rare variants identified in this study, p.Ile171Val, was first described in acute lymphoblastic leukemia (ALL) patients . This variant has been previously reported to be over-represented in some cancer cohorts [9, 10, 37, 38], and has also been previously described at the homozygous state in a Japanese NBS patient affected with aplastic anemia and genomic instability . Interestingly, analysis of the patient's and her father's lymphoblastoid cell lines demonstrated a higher frequency of spontaneous chromosomal aberrations compared with healthy controls (6- and 4-fold increase, respectively). These results suggest that the p.Ile171Val variation may be deleterious, also supported by the fact that this variant alters an amino acid which is conserved in species such as the chicken and the fruitfly . However, the impact of the p.Ile171Val substitution remains to be clarified as a large study, including cases from Germany and the Republic of Belarus, did not find any association with breast cancer in those populations .
Another rare variant, p.Arg215Trp, has been considered pathogenic in previous studies based on its highly conserved position and the change introduced. Moreover, this variant has been identified in monozygotic twins compound heterozygotes for 657del5/p.Arg215Trp and affected with a severe form of NBS . NibrinTrp215 appears to affect the correct nibrin function, and cells carrying this variant shows delayed DNA DSB rejoining . Modelisation of the tandem BRCT domains suggests that the Arg215 residue is required for correct orientation of the BRCT domains and recognition of γ-H2AX . In their experiment, nibrinTrp215 seems to partially interfere with nibrinArg215 activity and may act in a co-dominant fashion. It is also interesting to note that although this variant is relatively frequent in several studies, to our knowledge no NBS cases homozygous for this variant have been reported.
Of the other rare variants present in our cohort of breast cancer cases, the p.Pro266Leu variant was observed in one breast cancer case, and the p.Asp95Asn variant was found in one breast cancer case and one control. Although these variants involve conserved residues, their putative effect on protein function remains unclear. In a previous study on non-Hodgkin lymphoma, both variants were detected only in healthy controls . Regarding the p.Asp95Asn variant first identified in an ALL patient , no association was found in a study of larynx cancer  or ALL . This variant was found in one out of 613 and 121 unselected and familial prostate cancer cases, respectively, but not in controls . However, p.Asp95Asn is not predicted to be highly damaging as its expression in NBS cells seems to have a similar activity to the wild type protein . The only other non-synonymous change identified in our cohort is the p.Glu185Gln common variant (MAF >25%), which has been genotyped in several association studies. Although the majority of the studies analyzing this variant found no association with cancer [12, 38, 42–44], some studies reported an association with breast cancer , basal cell carcinoma in men  and a recent meta-analysis of this variant in bladder cancer revealed a significant association after controlling for potential bias . In addition, Musak et al.  reported that this variant may modulate the frequency of chromatid-type aberrations among tire plant workers. However, our data do not allow us to confirm any positive association.
Among the non-coding variants, a possible association of the c.-242-110delAGTA variant with an increased risk of breast cancer could be observed (OR 3.4 95% CI: 1.1–10.5). This association was further confirmed by the estimation of the haplotype diversity in our dataset, which highlighted the presence of the haplotype bearing the c.-242-110delAGTA variant at a higher frequency among the breast cancer case subset. In silico prediction of putative transcription factor binding sites indicated that of the four transcription factor binding sites potentially affected by the deletion, some are predicted to involve proteins not expressed in the breast. MTBF acts in the muscle regulation of the myostatin gene , while CDX2 is mainly expressed in the gut although its ectopic expression was also reported in cases of non-gastrointestinal carcinomas . As for the transcription factor NKX3-1, it is mainly expressed in the prostate although it is also found at low levels in the mammary gland and breast tumors, and has been suggested to act as a haplo-insufficient tumor suppressor in prostate cancer . The fourth transcription factor potentially affected is ATBF1 (also known as ZFHX3). ATBF1 was shown to suppress the expression of the alpha-fetoprotein  and the MYB oncogene . ATBF1 also cooperates with p53 to activate the CDKN1A promoter and trigger cell cycle arrest . ATBF1 is expressed in the mammary gland and breast tumors (NCBI's UNIGENE data), and the expression of ATBF1 mRNA was correlated with a better prognosis in 153 patients with invasive carcinomas of the breast . This might suggest that deregulation of genes controlled through ATBF1 could have an important impact on breast cancer formation and progression.
Luciferase reporter gene assay using the promoter sequence proximal to the ATG codon indicated a similar level of expression of the c.-242-110delAGTA variant allele as compared to the reference sequence construct in the breast cancer cell line MCF-7 and in HeLa cells. However, a slight decrease in luciferase expression was observed in both HEK293 and LNCaP cells, which might indicate that this variant could have an impact on NBN expression in a cell type manner. The analysis of ATBF1 gene expression indicated that this transcription factor is weakly expressed, in particular in the MCF-7 cells. This is comparable to a recent study performed on a panel of 32 breast cancer cell lines, which indicated that in 75% of these cell lines, ATBF1 is expressed at 50% or less relative to the normal breast . This could in turn explain the lack of effect observed for the luciferase assay in the MCF-7 cell line. On the other hand, one could hypothesize that NBN (or transcription factors affecting its expression) may be regulated following irradiation or other genotoxic stress. Additional research will however be needed to address this possibility, as well as binding experiments to confirm ATFB1 binding to this region of the promoter. However, it is also important to keep in mind that the promoter sequence cloned here, although supporting NBN expression, is unlikely to constitute the entire NBN promoter. It is indeed plausible that transcription factors acting on the proximal sequence might be influenced by other transcription factors acting further upstream. As such, despite the lack of variation in expression induction found in MCF-7 by luciferase assay, it is possible that the c.-242-110delAGTA variant could be in LD with another functional variant located elsewhere in the promoter, as a high degree of LD is present across the whole NBN gene. Unfortunately, the promoter variant could not be associated by LD with another variant in the coding region of the gene, which would have allowed to directly measure NBN allelic expression.