Familial breast cancer

Breast cancer is the most common malignancy in women; it affects about one in eight women. Familial breast cancer typically presents earlier than sporadic breast cancer, and is more often bilateral than in sporadic cases. Ovarian cancer is more common in familial breast cancer. A large number of studies have confirmed an increased breast cancer risk in patients with a significant family history of breast cancer. The breast cancer genotype has an autosomal dominant pattern of transmission. This article considers familial breast cancer and various aspects of breast cancer management in primary care, including the genetics of familial breast cancer, and guidelines on referral to secondary care.


Introduction
Pathogenic mutations in BRCA1, BRCA2, TP53, PTEN, ATM and CHEK2 account for approximately a third of high-risk breast cancer families, suggesting that other breast cancer susceptibility genes exist [1][2][3][4][5]. Given the number of candidate breast cancer susceptibility genes, any approach to their identification needs to be focussed. Genes whose products are known to interact with BRCA1 and/or BRCA2, or are downregulated in breast tumours, are particularly attractive candidates, and can be prioritised for investigation. FANCD2 is one of eight genes known to cause the fatal human autosomal recessive disorder Fanconi anaemia (FA) [6,7]. FA is a heterogenous condition characterised by progressive bone marrow failure, congenital abnormalities, hypersensitivity to DNA damaging agents and, most importantly, an increased risk of developing cancer [8]. There are currently eight cloned FA genes (FANCA, FANCC, FANCD1/ BRCA2, FANCD2, FANCE, FANCF, FANCG and FANCL), all of which interact with each other in a common cellular pathway [6,7]. Five of the FA proteins (FANCA, C, E, F, and G) form a constitutive complex in the nucleus of normal cells [9]. With the help of the recently identified ubiquitin ligase protein PHF9 (or FANCL), this multisubunit nuclear complex mediates the monoubiquitination of the FANCD2 protein at lysine 561 in response to the S-phase of the cell cycle or DNA damage [6]. The activated FANCD2 protein is then translocated to chromatin and DNA-repair foci, where it co-localises with other DNA repair proteins such as BRCA1, BRCA2, ATM, NBS1 and RAD51 [9]. Interestingly, this translocation has been recently identified to be BRCA1 dependent, suggesting that FANCD2 and BRCA1 interact in this process [6]. In response to ionising radiation, FANCD2 is also phosphorylated by ATM on serine 222, which leads to the activation of an S-phase checkpoint of the cell cycle [9]. FANCD2 is located at 3p25. 3 and consists of 44 exons, encoding a protein of 1,451 amino acids. Houghtaling et al. [10] showed that FANCD2 homozygous and heterozygous mice display a high incidence of epithelial tumours, including mammary and ovarian carcinomas. These mice display other features found in BRCA2 mutant mice, including germ-cell defects, small size, and perinatal lethality [11]. FANCD2, like BRCA2, may, therefore, play an important role in the recombination DNA repair pathways [10]. The FA pathway has also been implicated in ovarian cancer, as the FANC-BRCA pathway was shown to be disrupted in a subset of ovarian tumour lines [12]. Furthermore, the 3p25-26 region of the human genome has been shown to have a high incidence of loss of heterozygosity in ovarian tumours [13]. Analysis of the FA genes (FANCA, B, C, D1, D2, E, F, G) in 88 non-BRCA1, non-BRCA2 breast cancer families failed to identify any penetrant mutations, but none of these families were known to share a haplotype around the relevant FANC genes, or to include cases of ovarian cancer [14]. BRIP1/BACH1 was first isolated and identified by using a glutathione S-transferase fusion protein containing the BRCT motifs and the carboxyl terminus of BRCA1. This protein was originally named BACH1 (for BRCA1-associated carboxy-terminal helicase 1), but is also known as BRIP1 (for BRCA1 interacting protein 1) [15]. The BRIP1/BACH1 gene maps to 17q22 and contains 20 exons, encoding a protein of 1,249 amino acids. Amino acid residues 888 to 1,063 of BRIP1/ BACH1 interact with the BRCT domain of BRCA1 during the process of DNA repair [15]. Cantor et al. [15] screened the BRIP1/BACH1 gene for mutations in 21 sporadic breast/ ovarian cancer cell lines, and 65 individuals with early onset breast cancer. Two germline heterozygous missense variants (p. P47A and M299I) were detected in the germlines of two early onset breast cancer patients but no family members were available for segregation analysis. Both variants are within the helicase domain of BACH1 (residues 1 to 888), with P47A located in the highly conserved nucleotide binding box, and M299I situated between two other conserved motifs [15]. Two other studies looking at variants in the BRIP1/BACH1 gene in breast cancer families failed to find any highly penetrant mutations, although these studies were limited in their sample size, and the number of available samples from additional family members, and none of the families were known to share a haplotype around BRIP1/BACH1 [16,17].
LMO4 is a member of the LIM-only (LMO) family of transcription regulators. The four known members of this group (LMO1 to LMO4) are composed of two LIM domains and are thought to function as transcriptional cofactors via protein-protein interactions (reviewed in [18]). LMO1 and LMO2 overexpression is linked to T-cell tumourigenesis and LMO4 has been associated with breast oncogenesis, where overexpression is observed in approximately 50% of breast cancer cell lines and primary breast cancers [19]. Furthermore, overexpression of LMO4 induces mammary hyperplasia in transgenic mice and may be a predictor of poor outcome in breast cancer [20]. The presence of LMO4 in a complex containing the binding partners Ldb1, CtIP and the familial breast cancer tumour suppressor BRCA1 provides further compelling evidence for LMO4 playing a significant role in breast cancer pathogenesis [21], and activating mutations might be predicted to occur in some tumours and even in the germline of some patients. Although no activation mutations have been found, one somatic truncation mutation of LMO4 has been reported in a sporadic breast tumour [22]. This finding, as well as the deregulation of LMO4 expression in breast cancer and the interaction between LMO4 and the tumour suppressor BRCA1, prompted us to screen non-BRCA1/2 familial breast cancer cases for genetic alterations in LMO4 that may contribute to pathogenesis.
Stratifin (SFN; 14-3-3 σ; HME1) was first identified by serial analysis of gene expression (SAGE) analysis as an epithelial specific marker that was expressed at seven-fold lower levels in breast cancer cells compared to normal breast epithelium [23]. Recently, hypermethylation of SFN was detected in more than 90% of invasive breast cancers and was specifically associated with lack of expression [24]. In addition, methylation of this gene was detected in 83% of ductal carcinoma in situ and 38% of atypical hyperplasias but was unmethylated in all hyperplasias without atypia and normal breast epithelium obtained from patients without breast cancer [25]. Of most interest was the fact that SFN hypermethylation was also detected in the histologically normal adjacent breast epithelium in patients with breast cancer, suggesting that methylation of this gene may be an early event in breast cancer development. SFN is a negative regulator of cell cycle progression and is suggested to have an important function in preventing breast tumour cell growth, particularly at the G2 cell cycle checkpoint [26]. BRCA1 is a co-activator of SFN, and the expression of SFN is modulated by the BRCA1 status of the cell and requires intact BRCA1 and p53 to synergistically induce the optimal level of stratifin required for DNA damage response [27]. Interestingly, there is a nine-fold decreased expression of SFN in BRCA1-and BRCA2-related tumours compared to sporadic breast tumours [28]. SFN is located on 1p36.11 and is encoded by a single 747 base pair (bp) exon; 1p36 is a target of loss of heterozygosity in 16% to 37% of sporadic breast tumours [29,30] and in 32% to 35% of familial tumours [31]. To our knowledge there has been no report of mutation analysis of SFN in familial breast cancer.
We sought to carry out mutation analysis of FANCD2, BRIP1/ BACH1, LMO4 and SFN in a large number of non-BRCA1/2 breast cancer families. For the biggest genes, FANCD2 and BRIP1/BACH1, we screened a smaller number of families, but included those in which the affected family members shared a haplotype around the gene of interest. We also screened additional index cases for mutations in the FANCD2 exons that contain the ATM phosphorylation (S222) and the FANCD2 monoubiquitination regions (K561), and the BRIP1/ BACH1 exons that contained the previously reported breast cancer-association variants, p. P47A and p. M299I.

Multiple-case breast cancer families
Multiple-case breast cancer families were ascertained through the Kathleen Cuningham Foundation Consortium for Research into Familial Breast Cancer (kConFab) [32]. The ascertainment criteria for families without mutations in BRCA1 or BRCA2 were four or more cases of breast or ovarian cancer (Criteria 1), or two or more if one has 'high risk' features, such as breast cancer diagnosis at less than 40 years, male breast cancer, bilateral breast cancer, or ovarian and breast cancer in the same woman (Criteria 1B). In both cases, the criteria also require that two or more affected women are alive and that the families have four or more living, female, unaffected first or second degree relatives over the age of 18. The index cases, defined as the youngest available breast cancer case, were tested by diagnostic laboratories for mutations in BRCA1 and BRCA2 by a variety of methods estimated to be 75% sensitive, and a subset were fully sequenced for BRCA1 and BRCA2.
A subset of the index cases screened for mutations were included in a 10 cM genome-wide search for novel breast cancer susceptibility genes in multiple case breast cancer families from which BRCA1 and BRCA2 mutations had been excluded by high-sensitivity methods and in which no haplotype was shared at either locus (data not shown). The index cases qualified for FANCD2 and BRIP/BACH1 mutation analysis if an individual family logarithm of the odds (LOD) score under heterogeneity or a non-parametric LOD score of ≥0.5 had been obtained at any of the markers closest to or flanking the FANCD2 (D3S1304, D3S1263, D3S2338) or BRIP/BACH (D17S944, D17S949, D17S787) genes.
All 44 coding exons of FANCD2 were evaluated in 33 index cases from 30 non-BRCA1/2 multiple case breast cancer families. Three families contained two cases with the same age of onset of breast cancer and so both cases were screened. The families were selected because they contained one or more cases of ovarian cancer (n = 18), or because all of the affected individuals in the family shared a haplotype around the 3p25 region (n = 12). The entire BRIP1/BACH1 coding sequence (19 exons) was evaluated in the index case of 75 breast cancer families in which all the affected individuals shared a haplotype around BRIP1/BACH1 on chromosome 17q (n = 7), or which had undergone complete sequencing of BRCA1 and BRCA2 (n = 68). All three coding exons of LMO4 were screened in the index cases from 247 non-BRCA1/2 breast cancer families, and the single coding exon of SFN was screened in the index cases from 92 non-BRCA1/ 2 breast cancer families. Index cases from an additional 164 families were screened for just 639 bp of the single SFN exon. Eight index cases were fully screened for FANCD2, BRIP1/ BACH1 and LMO4 genes (and six of these for SFN as well), and 227 individuals from 222 families were screened for both LMO4 and SFN.
In addition, 399 index cases, from 356 non-BRCA1/2 breast cancer families (some had more than one index case because multiple women were affected at the same age), were screened for FANCD2 mutations in the ATM phosphorylation (exon 9) and the FANCD2 monoubiquitination (exon 19) regions. Of these additional index cases (from 231 families) that were used for additional FANCD2 screening, 253 were also screened for BRIP1/BACH1 mutations in exons 3 and 7, where the p. P47A and p. M299I breast cancer-associated variants are located.
We used as controls DNA from 93 unrelated, adult, female monozygotic twins (only one from each pair) selected from a sample of 3,348 twin pairs. The twins were almost exclusively of European origin and had been recruited through the Australian Twin Registry. Approvals were obtained from the Human Research Ethics Committees of the Queensland Institute of Medical Research, and for kConFab from the Peter MacCallum Cancer Centre and all other committees to which kCon-Fab reports.

Mutation analysis
Primers were designed using the web-based program Primer3    Samples that produced a heterozygous peak or an aberrant shift in retention time and/or peak shape were confirmed by DHPLC and re-amplified for sequencing. DNA sequencing was performed with both forward and reverse primers using the ABI Prism Big Dye Terminator cycle Sequencing Ready reaction kit (PE Applied Biosystems) and analysed on an ABI 377 sequencer. Coding variants and variants located near the exon/intron boundary, were analysed in silico for amino acid Table 3 In

FANCD2
DHPLC analysis of FANCD2 in the 33 index cases from 30 breast and ovarian cancer families, and of exons 9 and 19 (containing the ATM phosphorylation site and the FANCD2 monoubiquitination site, respectively) in a further 399 non-BRCA1/2 index cases, identified 32 germline sequence alterations, most of which were novel ( Table 2). Analysis of sequencing results identified 25 intronic variants, 6 silent coding variants, and another variant located within the 3' untranslated region (UTR).
The c.633 C>T and c. 2148 C>G variants did not appear by in silico analyses to affect mRNA folding or the concensus splice site sequences, as predicted by the BDGP Splice Site Prediction, SpliceSiteFinder, and mFOLD web-based programs (Table 3). c.2148 C>G was predicted to change the SF2/ASF exon enhancer sites and gain a SRp55 enhancer site. Because this nucleotide is not conserved in the murine Fancd2 gene, however, the functional significance of these changes remains unclear. The c. 3558 C>G (L1186L) variant, located 3 bp 5' of the end of exon 35, was predicted to result in a gain of a SC35 exonic splicing enhancer site, and a loss of a SF2/ASF site, and also subtly changing the predicted mRNA folding. In addition, the BDGP Splicing program predicted that the variant causes a complete loss of the donor site for exon 35 splicing, although this was not predicted by SpliceSiteFinder, consistent with the more sensitive algorithm of the BDGP splicing program [37]. To address this further we performed RT-PCR analysis with lymphoblastoid cell line RNA but found no evidence for altered splicing of this transcript (data not shown). The c. 3558 C>G variant was found in a family with five cases of breast cancer, of whom two also had ovarian cancer. DNA was available from two additional affected relatives of the index case (her daughter and cousin).  (Table 4). We were unable to calculate the exact frequencies of each of the haplotypes because DHPLC did not distinguish the two homozygotes from each other. Sequencing showed that the rare variants, c. 633 C>T, c. 1828+34 C>T, c. 2148 C>G, c. 2021+10 G>T and c. 3558 C>G, were all found on the common haplotype that corresponds to the reference sequence found on the NCBI database [42].

BRIP1/BACH1
A total of 10 nucleotide variants, four of which have not been previously reported, were identified in BRIP1/BACH1 among 75 non-BRCA1/2 index cases ( Table 5). Six of these variants were exonic, of which one was a single base-pair deletion, four resulted in amino acid substitutions and one was silent ( Table  5). Three of the missense variants, c. 430 G>A (p. A144T), c. 584 T>C (p. L195P) and c. 3464 G>A (p. G1155E), and the deletion variant c. 3401delC were absent in 93 controls. The c. 584 T>C (p. L195P) variant has been reported previously in an early onset breast cancer case, but not in controls [17]. In silico analyses of c. 430 G>A (p. A144T), c. 584 T>C (p. L195P) and c. 3464 G>A (p. G1155E) predicted that they

LMO4
Index cases from 247 families were screened by DHPLC across the three coding exons of LMO4. Using the primers designed to amplify exon 3, two intronic variations were observed in two individuals each, c.237-72T>G and c.237-51_237-46delTTCTTT, but no coding variants were identified.  (Table 3).

Discussion
Previous analyses of FANCD2 and BRIP1/BACH1 in non-BRCA1/2 families failed to identify any pathogenic mutations [14,16,17]; however, these studies did not choose the families to be screened on the basis of haplotype sharing, or the occurrence of other cancers (e.g. ovarian cancer in the case of FANCD2) in the family. Furthermore, some of these studies were limited by the fact that DNA from additional family members was not available for genotyping. To our knowledge, LMO4 and SFN have not been previously examined as BRCAx candidate genes. We therefore hypothesized that germline mutations in the BRCA1-interacting genes, FANCD2, BRIP1/BACH1, LMO4 and SFN, may account for RT-PCR analysis of a lymphoblastoid cell line from the carrier failed, however, to identify any aberrant transcripts, suggesting that this variant is unlikely to be pathogenic. The L1186L variant was not identified in any of 93 controls, but it did not segregate with breast cancer in the single family in which it was found. Therefore, this variant was also assumed to be a rare, neutral SNP.
Two common haplotypes of the FANCD2 gene were identified, one of which (haplotype B) was identical to the reference sequence obtained from the NCBI database. All of the rare variants were found to occur on haplotype B. Even though individually these variants were classified as neutral SNPs, an association study designed to test whether the two haplotypes confer different breast cancer risks would be worthwhile.
In the analysis of the BRIP1/BACH1 gene, we did not observe the two previously reported variants, p. P47A and p. M299I, in the 253 non-BRCA1/2 breast cancer cases [15]. However, we did identify three non-conservative missense variants (p. A144T, p. L195P, and p. G1155E) and one novel frameshift mutation (c. 3401delC) in the 75 selected non-BRCA1/ BRCA2 breast cancer index cases. None of these variants were found in 93 controls. Additional genotyping of a total of 68 family members indicated, however, that these variants are not the underlying cause of breast cancer in these families, as none of the other affected relatives carried the variants. Nevertheless, it is possible that these variants are low-risk breast cancer susceptibility alleles, in which case further investigation may be warranted. The 3782 T>C 3' UTR variant of BRIP1/ BACH1 is predicted to alter the folding of the transcript; however, the biological significance and frequency of this change in the normal population has yet to be determined.
Mutation analysis of 82 sporadic tumours previously revealed one somatic frameshift mutation of LMO4 [22]. No activating or inactivating coding or splice site mutations of LMO4 were found by DHPLC analysis of 247 index cases from non-BRCA1/2 families. Two intronic variants were found, each in two index cases. Their recurrent nature in two families and apparent lack of effect on splicing suggests that they are rare SNPs.
SFN is markedly down-regulated in breast cancer tissue compared to normal mammary epithelium but to our knowledge has not been evaluated for germline mutations in familial breast cancer. We screened the majority of the coding region of the gene in 267 index cases and found one silent change, T207T, in 23 index cases. This silent change has been previously reported (rs11542704) [43]. We also found some variants in the 3' UTR, and three different missense changes in one individual (F198L, L218I and Q244K) that were considered unlikely to be pathogenic because of the multiple occurrences in one individual. None of these variants have been previously reported.

Conclusion
Mutation analysis of the BRCA1-interacting genes FANCD2, BRIP1/BACH, LMO4 and SFN in a large number of non-BRCA1/2 breast cancer families did not identify any highly penetrant, pathogenic mutations. Given that DHPLC is a robust and sensitive screening technique, we consider it unlikely that we missed any coding or splice site pathogenic mutations among the index cases analysed. In particular, we analysed each PCR fragment at all the temperatures recommended by the DHPLC melt algorithm and under these conditions DHPLC has been reported to have a sensitivity of 99.4% [40]. It appears unlikely, therefore, that FANCD2, BRIP1/ BACH, LMO4 and SFN account for more than a small proportion of inherited forms of breast cancer. Many novel SNPs were identified in these genes, however, and large association studies of breast cancer cases and controls is warranted to determine whether any of these variants confer small risks of breast cancer. biospecimens, were provided by kConFab. MAB assisted with analysis of mFOLD data, and with preparation of the manuscript.