Cancer Susceptibility Genes BRCA1 Research Articles

Identification of a Danish breast/ovarian cancer family double heterozygote for BRCA1 and BRCA2 mutations

Mutations in the two breast cancer susceptibility genes BRCA1 and BRCA2 are associated with increased risk of breast and ovarian cancer. Patients with mutations in both genes are rarely reported and often involve Ashkenazi founder mutations. Here we report the first identification of a Danish breast and ovarian cancer family heterozygote for mutations in the BRCA1 and BRCA2 genes. The BRCA1 nucleotide 5215G > A/c.5096G > A mutation results in the missense mutation Arg1699Gln, while the BRCA2 nucleotide 859 + 4A > G/c.631 + 4A > G is novel. Exon trapping experiments and reverse transcriptase (RT)–PCR analysis revealed that the BRCA2 mutation results in skipping of exon 7, thereby introducing a frameshift and a premature stop codon. We therefore classify the mutation as disease causing. Since the BRCA1 Arg1699Gln mutation is also suggested to be disease-causing, we consider this family double heterozygote for BRCA1 and BRCA2 mutations.

Abstract LB-102: The p21Cip1/Waf1 cyclin-dependent kinase inhibitor is required for the activation of the FA-BRCA pathway

Abstract Fanconi anemia (FA) is a rare recessive disorder characterized in part by pronounced cancer susceptibility. The FA proteins, and the protein products of the major breast cancer susceptibility genes BRCA1 and BRCA2, function cooperatively in the FA-BRCA pathway, to repair damaged DNA and to prevent cellular transformation. A major step in the activation of the FA-BRCA pathway is the mono-ubiquitination of the FANCD2 and FANCI proteins, targeting these proteins to discrete chromatin-associated nuclear foci. Importantly, the regulation of FANCD2 (and FANCI) mono-ubiquitination remains poorly understood. To gain further insight into this important post-translational modification step, here we have examined the roles of p53 tumor suppressor as well its downstream target p21Cip1/Waf1 in the regulation of the mono-ubiquitination of FANCD2. In this study we have used the isogenic HCT116 p53+/+, p53−/−, p21+/+, and p21−/− cell lines, to determine that p21, and not p53, plays a key role in the regulation of the mono-ubiquitination of FANCD2 following exposure to DNA damaging agents. For example, robust FANCD2 mono-ubiquitination is observed in HCT116 p21+/+ cells 2 h following exposure to 20 J/m2 UV irradiation, while this effect is markedly attenuated in p21−/− cells. Using immunofluorescence microscopy we also demonstrate that p21 is necessary for the efficient recruitment of FANCD2 to nuclear foci following exposure to UV irradiation. Furthermore, preliminary protein-protein interaction analyses in COS-7 cells demonstrate that FLAG-p21 and 6xHis/V5-FANCD2 physically interact. Interestingly, despite a failure to efficiently activate the mono-ubiquitination of FANCD2 following exposure to DNA damaging agents, p21−/− cells are not hypersensitive to the cytotoxic effects of the DNA crosslinking agent mitomycin C (MMC). However, elevated levels of distinct chromosome aberrations, including telomere associations and dicentric chromosomes, are observed in p21−/− cells compared with p21+/+ cells following exposure to MMC. Taken together, our results demonstrate an important upstream role for the p21 cyclin-dependent-kinase inhibitor in the activation of the FA-BRCA pathway. A greater understanding of the regulation of this important tumor suppressor pathway will lead to improved diagnostic and therapeutic approaches to FA and enhance our understanding of the elevated cancer susceptibility of FA patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-102.

Abstract 1858: BRCA1 and BRCA2 sequence variants in Algerian breast/ovarian cancer families

Abstract Abstract: Background: Breast cancer is currently the leading cause of cancer morbidity and mortality among Algerian women. To date, very few reports have been published about the spectrum of BRCA1 and BRCA2 mutations in the Algerian population. Here, we describe analysis of BRCA1 and BRCA2 genes in 57 individuals from 52 families from an Algerian cohort with a personal and family history suggestive of genetic predisposition to breast/ovarian cancer. Methods: The screening for variants in the breast and ovarian cancer susceptibility genes BRCA1 and BRCA2 in Algerian patients was performed by High-Resolution Melting (HRM) curve analysis followed by direct sequencing. All samples for which no pathogenic mutation was found were analyzed by MLPA (Multiplex Ligation-dependent Probe Amplification) for large deletions or duplications. To identify no synonymous amino acid changes likely to disrupt BRCA1 and BRCA2 genes function, we used a comparative evolutionary bioinformatic program, Polymorphism Phenotyping (Polyphen program). We used GeneSplicer program to identify the splice site alterations of new unclassified variants occurring in intron-exon boundaries of BRCA1 and BRCA2. Results: Three distinct pathogenic mutations, c.83_84delTG/p.Leu28ArgfsX12, c.181T>G/p.Cys61Gly, c.798_799delTT/p.Ser267LysfsX19 and two large rearrangements involving exon 2 (c.-19-? 80+?del/p.?) and exon 8 (c.442-? _547+?del/p.?) respectively, were detected in BRCA1 gene. Interestingly, the c.798_799delTT/p.Ser267LysfsX19 BRCA1 pathogenic mutation has been detected in two unrelated families. The BRCA1 exon 2 deletion reported here in this study seems has been never described before. Seventeen unclassified variants and polymorphisms were detected in BRCA1 gene (6 described for the first time). The new unclassified variant c.302-3C>T/p.? located near the acceptor splice site of BRCA1 intron 6 shows no splice alteration site. Two pathogenic mutations, c.1310_1313delAAGA/p.Lys437IlefsX22 and c.5722_5723delCT/p.Leu1908ArgfsX2 and 40 unclassified variants and polymorphisms (15 never described before) were identified in BRCA2 gene. Moreover, five new unclassified variants identified in the present study: one BRCA1 (c.4066C>A/p.Gln1356Lys) located in exon 11 and four BRCA2 (c.3868T>A/p.Cys1290Ser, c.4423A>G/p.Met1475Val, c.5472T>A/p.Asn1824Lys and c.5985C>A/ p.Asn1995Lys) located in exon 11, show a damaging PSIC score yielded by PolyPhen program and could be pathogenic. Conclusions: In this study, we attempted to delineate genetic component of hereditary breast/ovarian cancer among the Algerian population. This is the first screening of BRCA1 and BRCA2 mutations in Algerian breast/ovarian cancer families by using HRM and MLPA. These new findings will contribute to the assessment of the necessity of the preventive program for mutation carriers as part of the national public health policy in Algeria Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1858.

Identification of Filamin A as a BRCA1-interacting protein required for efficient DNA repair

The product of the breast and ovarian cancer susceptibility gene BRCA1 has been implicated in several aspects of the DNA damage response but its biochemical function in these processes has remained elusive. In order to probe BRCA1 function we conducted a yeast two-hybrid screening to identify interacting partners to a conserved motif (Motif 6) in the central region of BRCA1. Here we report the identification of the actin-binding protein Filamin A (FLNA) as BRCA1 partner and demonstrate that FLNA is required for efficient regulation of early stages of DNA repair processes. Cells lacking FLNA display a diminished BRCA1 IR-induced focus formation and a delayed kinetics of Rad51 focus formation. In addition, our data also demonstrate that FLNA is required to stabilize the interaction between components of the DNA-PK holoenzyme, DNA-PKcs and Ku86 in a BRCA1-independent fashion. Our data is consistent with a model in which absence of FLNA compromises homologous recombination and non-homologous end joining. Our findings have implications for the response to irradiation induced DNA damage.

BRCA1 Protein and Nucleolin Colocalize in Breast Carcinoma Tissue and Cancer Cell Lines

The breast and ovarian cancer susceptibility gene BRCA1 encodes a tumor suppressor. BRCA1 protein, which is involved in DNA damage response, has been thought to be found primarily in cell nuclei. In the present investigation, immunohistological studies of BRCA1 protein in frozen breast cancer tissue and MCF7 and HeLa cell lines revealed BRCA1 expression in both nucleoli and nucleoplasmic foci. Immunoelectron microscopic studies of estrogen-stimulated MCF7 cells demonstrated BRCA1 protein localization in the granular components of the nucleolus. Moreover, immunofluorescence of BRCA1 and nucleolin double-labeling showed colocalization in both nucleoli and nucleoplasmic foci in breast tumor cells and asynchronously growing MCF7 and HeLa cells. Multiparameter analysis of BRCA1 and nucleolin in relation to cell cycle position (DNA content) showed expression during G1-S and persistence of BRCA1 during G2/M. After gamma-irradiation of MCF7 cells, BRCA1 protein dispersed from nucleoli and nucleoplasmic foci to other nucleoplasmic sites, which did not colocalize with nucleolin. Small interfering RNA-mediated knockdown of BRCA1 protein resulted in decreased immunofluorescence staining, which was confirmed by Western blotting. The observed colocalization of BRCA1 and nucleolin raises new possibilities for the nucleoplasm-nucleolus pathways of these proteins and their functional significance.

ATM Deficiency Sensitizes Mantle Cell Lymphoma Cells to Poly(ADP-Ribose) Polymerase-1 Inhibitors

Poly(ADP-ribose) polymerase-1 (PARP-1) inhibition is toxic to cells with mutations in the breast and ovarian cancer susceptibility genes BRCA1 or BRCA2, a concept termed synthetic lethality. However, whether this approach is applicable to other human cancers with defects in other DNA repair genes has yet to be determined. The ataxia telangiectasia mutated (ATM) gene is altered in several human cancers including mantle cell lymphoma (MCL). Here, we characterize a panel of MCL cell lines for ATM status and function and investigate the potential for synthetic lethality in MCL in the presence of small-molecule inhibitors of PARP-1. We show that Granta-519 and UPN2 cells have low levels of ATM protein, are defective in DNA damage-induced ATM-dependent signaling, are radiation sensitive, and have cell cycle checkpoint defects: all characteristics of defective ATM function. Significantly, Granta-519 and UPN2 cells were more sensitive to PARP-1 inhibition than were the ATM-proficient MCL cell lines examined. Furthermore, the PARP-1 inhibitor olaparib (known previously as AZD2281/KU-0059436) significantly decreased tumor growth and increased overall survival in mice bearing s.c. xenografts of ATM-deficient Granta-519 cells while producing only a modest effect on overall survival of mice bearing xenografts of the ATM-proficient cell line, Z138. Thus, PARP inhibitors have therapeutic potential in the treatment of MCL, and the concept of synthetic lethality extends to human cancers with ATM alterations.

BRCA1 regulates acetylation and ubiquitination of estrogen receptor-alpha.

Inherited mutations of the breast cancer susceptibility gene BRCA1 confer a high risk for breast cancer development. The (300)RXKK and (266)KXK motifs have been identified previously as sites for acetylation of the estrogen receptor-alpha (ER-alpha), and (302)K was also found to be a site for BRCA1-mediated mono-ubiquitination of ER-alpha in vitro. Here we show that ER-alpha proteins with single or double lysine mutations of these motifs (including K303R, a cancer-associated mutant) are resistant to inhibition by BRCA1, even though the mutant ER-alpha proteins retain the ability to bind to BRCA1. We also found that BRCA1 overexpression reduced and knockdown increased the level of acetylated wild-type ER-alpha, without changing the total ER-alpha protein level. Increased acetylation of ER-alpha due to BRCA1 small interfering RNA was dependent upon phosphatidylinositol 3-kinase/Akt signaling and on up-regulation of the coactivator p300. In addition, using an in vitro acetylation assay, we found that in vitro-translated wild-type BRCA1 but not a cancer-associated point mutant (C61G) inhibited p300-mediated acetylation of ER-alpha. Furthermore, BRCA1 overexpression increased the levels of mono-ubiquitinated ER-alpha protein, and a BRCA1 mutant that is defective for ubiquitin ligase activity but retains other BRCA1 functions (I26A) did not ubiquitinate ER-alpha or repress its activity in vivo. Finally, ER-alpha proteins with mutations of the (300)RXKK or (266)KXK motifs showed modest or no BRCA1-induced ubiquitination. We propose a model in which BRCA1 represses ER-alpha activity, in part, by regulating the relative degree of acetylation vs. ubiquitination of ER-alpha.

Synthetic lethal targeting of PTEN mutant cells with PARP inhibitors.

The tumour suppressor gene, phosphatase and tensin homolog (PTEN), is one of the most commonly mutated genes in human cancers. Recent evidence suggests that PTEN is important for the maintenance of genome stability. Here, we show that PTEN deficiency causes a homologous recombination (HR) defect in human tumour cells. The HR deficiency caused by PTEN deficiency, sensitizes tumour cells to potent inhibitors of the DNA repair enzyme poly(ADP-ribose) polymerase (PARP), both in vitro and in vivo. PARP inhibitors are now showing considerable promise in the clinic, specifically in patients with mutations in either of the breast cancer susceptibility genes BRCA1 or BRCA2. The data we present here now suggests that the clinical assessment of PARP inhibitors should be extended beyond those with BRCA mutations to a larger group of patients with PTEN mutant tumours.

337 Targeting DNA-repair deficiency in mouse models for BRCA- associated breast cancer

The identification of the breast cancer susceptibility genes BRCA1 and BRCA2 a few years ago has been greeted with great excitement and has raised hopes that they might illuminate the common mechanisms of this disease. Today we have to recognize that these expectations remain unfulfilled. Mutations in BRCA1 and BRCA2 account only for a relatively small proportion of breast cancers, even within the group of familiar clusters, they seem to be virtually non-existing in sporadic breast cancers. A substantial proportion of familiar breast cancer clusters has failed to provide evidence for an association with mutations in either BRCA1 or BRCA2, thus we have to look forward to the identification of additional breast cancer susceptibility genes. What has been most disappointing is that the mutation status of BRCA1/2 can provide only limited information for cancer risk. Initial assessments had indicated a risk of close to 90% for mutation carriers to develop breast cancer until age 75 – a value that turned out to be restricted to high-risk families in which the BRCA1 and BRCA2 genes had been genomically mapped. In unselected clusters the risk appears much lower, some estimates suggest less than 40%. Both BRCA1 and BRCA2 large encode proteins that appear to have a plethora of functions, with a conspicuous association to DNA repair and DNA recombination, and probably transcription activation. Defects in DNA repair can result in cancer predisposition syndromes and are recognized as being instrumental in cancer progression. Central questions have remained unanswered: What is the function of damaged BRCA1 and BRCA2 genes in breast cancer risk? What is the basis of large variations of risk conferred to the patients by identical mutations? How can the predictive value of mutation surveys be increased?

Controversial BRCA1 allelotypes in commonly used breast cancer cell lines

To the editor: The breast and ovarian cancer susceptibility gene BRCA1 has been extensively investigated for its biological function and potential for targeted therapy. Cell lines derived from human tissues have been employed as research reagents in such studies, and one of those most commonly used is the breast cancer cell line MCF-7. Recently, the notion that MCF-7 carries a hemizygous wild-type allele of BRCA1 has been referred to in a growing number of research articles [1–9], with some studies fundamentally based upon this concept. This idea seems to originate from an article published in 1996 by Holt et al [10]. In the article, Holt et al. noted that MCF-7 has only one allele at each of seven polymorphic DNA markers encompassing the BRCA1 gene locus, indicating genomic loss of one copy of the 2 Mb region containing BRCA1. This description implies that MCF-7 carries only one copy of the BRCA1 gene and seemingly has been interpreted as such in subsequent publications. In agreement with Holt et al., recent data from Genome-Wide Human Single Nucleotide Polymorphism (SNP) Array 6.0 analysis at the Wellcome Trust Sanger Institute (http://www.sanger.ac.uk/genetics/CGP/) revealed loss of heterozygosity (LOH) at the BRCA1 gene locus at chromosome 17q21.31 in MCF-7. However, in contrast to the above-mentioned interpretation, these SNP array data suggest the presence of two copies of the BRCA1 gene in MCF-7, with a normalized absolute copy number score 1.93 ± 0.412 (mean ± S.D.) at the markers within the BRCA1 gene locus as calculated by Sanger Institute’s PICNIC algorithm. In addition, we conducted a fluorescence in situ hybridization (FISH) analysis of the BRCA1 gene locus using RP11-242D8, a BAC probe overlapping with the BRCA1 5′ region. This assay was performed with two strains of MCF-7 cell line independently grown at distinct institutions (termed MCF-7-J maintained at Johns Hopkins University, and MCF-7-Aat Aichi Cancer Center Research Institute), and demonstrated that the modes of the BRCA1 gene copy number in two strains were four and three, respectively (Fig. 1; b and c and Table 1). The same FISH result was obtained using another BAC probe, RP11-948G15, which was assigned at a close proximity to the 3′ end of BRCA1 (data not shown). The clonal variation between separate cultures of MCF-7 observed in our study is consistent with previous studies by others conducting spectral karyotype (SKY) analysis with MCF-7 [11–13]. Overall, MCF-7 cells were revealed to have multiple copies of BRCA1 despite of the apparent LOH of the corresponding genomic region. This allelotype is generally considered to occur when one of the two alleles is initially deleted and the remaining allele is subsequently duplicated. Holt et al. additionally stated that another breast cancer cell line MDA-MB-157 was also subjected to genomic loss of one copy of the 2 Mb region containing BRCA1 [10]. Similar to MCF-7, however, SNP array 6.0 data at the Sanger Institute indicates a copy number score 1.93 ± 0.412 (mean ± S.D.) at the BRCA1 locus, suggesting that MDA-MB-157 has two copies of the BRCA1 gene, although it also suggests LOH of the entire chromosome 17 in this cell line. Our FISH analysis suggested the presence of two major cell populations within our MDA-MB-157 culture, which carry two and four copies of BRCA1, respectively (Fig. 1d and Table 1). These results are again consistent with previous reports describing the karyotypic heterogeneity of MDA-MB-157 cell line [11, 12]. Figure 1 Representative FISH photographs showing BRCA1 gene copy numbers in two different cultures of MCF-7 (b, c) and MDA-MB-157 (d). Red signals generated by a BAC probe RP11-242D8 represent BRCA1 gene loci, while green signals by RP11-443G13 represent Chromosome ... Table 1 FISH analysis of the BRCA1 locus in MCF-7 and MDA-MB-157 cell lines. Collectively, these findings indicate that MCF-7 and MDA-MB-157 carry multiple copies of the BRCA1 gene. Since the majority of cancer cells exhibit a chromosomal instability phenotype in which chromosomes are actively gained and lost, it should be noted that LOH does not necessarily mean the reduction of copy number at the correspond ing genomic locus, particularly in cancer cells.

The Effect of BRCA Gene Testing on Family Relationships: A Thematic Analysis of Qualitative Interviews

Discovery of mutations in the breast and ovarian cancer susceptibility genes BRCA1 and BRCA2 can have emotional consequences for both the tested individual and his or her relatives. This secondary analysis study investigated how BRCA testing impacts family dynamics and relationships. For the original study, a grounded theory inquiry, participants were recruited from a hereditary breast/ovarian cancer syndrome support website and open-ended interviews were performed asking about individual and family experiences after BRCA testing. All 12 participants whose interviews were included in the secondary analysis had a BRCA mutation. For the secondary analysis, thematic analysis was conducted and revealed three main themes characterizing the effect of BRCA testing on family relationships: 1. That the first in the family to have testing or seek genetic counseling takes on a special family role that can be difficult for them; 2. That discussions in the family often change; and 3. That individuals may feel more or less connected to certain family members. These changes seemed to relate to family cancer history, relationships, coping strategies, communication patterns, and mutation status. Genetic counselors might find it useful to explore these issues in order to prepare clients before BRCA testing and to support them through shifts in family dynamics after disclosure of results.

Brca1 is required for embryonic development of the mouse cerebral cortex to normal size by preventing apoptosis of early neural progenitors

The extent of apoptosis of neural progenitors is known to influence the size of the cerebral cortex. Mouse embryos lacking Brca1, the ortholog of the human breast cancer susceptibility gene BRCA1, show apoptosis in the neural tube, but the consequences of this for brain development have not been studied. Here we investigated the role of Brca1 during mouse embryonic cortical development by deleting floxed Brca1 using Emx1-Cre, which leads to conditional gene ablation specifically in the dorsal telencephalon after embryonic day (E) 9.5. The postnatal Brca1-ablated cerebral cortex was substantially reduced in size with regard to both cortical thickness and surface area. Remarkably, although the thickness of the cortical layers (except for the upper-most layer) was decreased, cortical layering as such was essentially unperturbed. High levels of apoptosis were found at E11.5 and E13.5, but dropped to near-control levels by E16.5. The apoptosis at the early stage of neurogenesis occurred in both BrdU pulse-labeled neural progenitors and the neurons derived therefrom. No changes were observed in the mitotic index of apical (neuroepithelial, radial glial) progenitors and basal (intermediate) progenitors, indicating that Brca1 ablation did not affect cell cycle progression. Brca1 ablation did, however, result in the nuclear translocation of p53 in neural progenitors, suggesting that their apoptosis involved activation of the p53 pathway. Our results show that Brca1 is required for the cerebral cortex to develop to normal size by preventing the apoptosis of early cortical progenitors and their immediate progeny.

Absence of the BRCA1 del (exons 9–12) mutation in breast/ovarian cancer families outside of Mexican Hispanics

The frequency and spectrum of germ line mutations in the high-penetrance breast cancer susceptibility genes BRCA1 and BRCA2 shows considerable variation by ethnic group. Most genetic epidemiological studies of the BRCA genes have been performed among Caucasian populations [1], with the exception of a few studies involving other ethnic groups, such as Hispanics [2–4], Asians [5, 6], and African Americans [7–9]. In most of these studies only the frequencies of sequence detectable BRCA mutations were reported and large genomic rearrangements including deletions and insertions of one or more exons, which account for 6–15% of all deleterious mutations in these genes have infrequently been considered [10–12]. Most BRCA mutations are unique; however, few recurrent mutations with founder effects have been found in European, Hispanic, American and Asian populations [13]. In a recent study on Hispanic high-risk breast/ovarian cancer families of mainly Mexican descent from South California, Weitzel and colleagues reported the identification of a novel deletion of BRCA1 exons 9 through 12 using multiplex quantitative differential polymerase chain reaction (PCR) analysis. The deletion was detected in 3.8% of families negative for sequence detectable BRCA mutations [14]. The large genomic rearrangement mutation is considered deleterious as it results in loss of critical functional domains as well as premature truncation of the BRCA1 protein. Given the relatively high prevalence of the deletion in this cohort, the authors suggest to include the screening for this mutation in the genetic testing strategy in Hispanic women without sequence detectable BRCA mutations. The spectra of recurrent sequence detectable BRCA mutations, including some with founder effects in a cohort of Hispanic breast/ovarian cancer families of predominantly Mexican descent from the United States and in a cohort from Colombia have previously been shown to differ completely, with different common mutations being identified in the two populations [2, 4]. In order to investigate, whether the novel BRCA1 founder deletion is also specific for Hispanic families of Mexican descent or whether it also occurs in other Hispanic cohorts, we screened 229 cancer Diana Torres and Muhammad U. Rashid contributed equally to this work.

A novel deletion of BRCA1 gene that eliminates the ATG initiation codon without affecting the promoter region

Background Point mutations in the highly penetrant cancer susceptibility gene BRCA1 are responsible for the majority of hereditary breast and/or ovarian cancer. We describe a novel large rearrangement of the BRCA1 gene identified in an Italian woman affected by an early onset bilateral breast cancer and a family history of hereditary breast cancer. The proband and her parents were negative for the presence of point mutations in BRCA1 and BRCA2 genes. Methods Multiplex ligation-dependent probe amplification (MLPA) was used to detect rearrangements in the BRCA1 gene. The breakpoint of the rearrangement identified in the proband was defined by restriction mapping and PCR amplification. BRCA1 mRNA encoded by the mutant allele was isolated from peripheral blood. Results The proband was heterozygous for a 9.1 kb deletion spanning from intron 1 to intron 3 (g.1238_10350del) that eliminates exons 2 and 3 in the mature mRNA. In mutant mRNA exon 1a joins directly to exon 5 with no disruption of the reading frame. Conclusions This deletion that eliminates the ATG initiation site in exon 2 and the sequence located in exons 2 and 3 encoding part of the RING finger domain of BRCA1 protein, is expected to abolish the function of this protein.

Gold Nanoparticle-Based Label-Free Detection of BRCA1 Mutations Utilizing DNA Ligation on DNA Microarray

Here we describe a label-free detection strategy for point mutation in breast cancer susceptibility gene BRCA1 utilizing ligation chain reaction (LCR) and zip-code array. We amplified the genomic regions containing mutation sites by polymerase chain reaction (PCR) to prepare the template for LCR. Then we ligated a primary probe extended by zip-code complementary at the 5' end with a 3'-thiol modified secondary probe. The resulting ligated product was labeled with gold nanoparticles and allowed to hybridize with corresponding zip-code sequences on the microarray. Finally, we applied silver enhancement to amplify the signals. Using this strategy, we successfully genotyped BRCA1 mutation, which was detected by naked eye.

Linking the Cellular Functions ofBRCAGenes to Cancer Pathogenesis and Treatment

Single-gene disorders that predispose to cancer afford a unique window into the mechanisms of carcinogenesis. I argue that the instability in chromosome structure and number provoked by inactivation of the breast cancer-susceptibility genes BRCA1 and BRCA2 arises from the distinct functions served by their products in DNA repair or mitosis, explains many features of cancer pathogenesis in this setting, and has important implications for treatment. The chromosomal instability model proposed here suggests a conceptual framework for the connections between chromosomal aberrations and cancer.

Understanding the Genetics of Breast Cancer: A Clinical Overview

Breast cancer is the second leading cause of death among women in the United States, exceeded only by lung cancer.1 An estimated 5 to 10% of all breast cancers are inherited, and the breast cancer susceptibility genes BRCA1 and BRCA2 have been identified as being responsible for 21 to 40% of these cases. Furthermore, women who carry a germline mutation in BRCA1 have a cumulative lifetime risk for developing breast cancer of 50 to 85%.2 Comprehensive genetic counseling that includes risk assessment, genetic testing, surveillance, and preventative strategies are essential for women who have been identified as being at high risk for inherited breast cancer. Accordingly, this paper will review the genetic component of breast cancer and the recommendations for screening and clinical management in this population of patients.

Identification and functional analysis of novel BRCA1 transcripts, including mouse Brca1-Iris and human pseudo-BRCA1

Recent characterization of the mammalian transcriptome has confirmed its predicted complexity, with many loci encoding multiple splice variants and pseudogenes. The breast cancer susceptibility gene BRCA1 is a tumour suppressor gene that produces multiple functional transcripts. For example, BRCA1-IRIS is a splice variant of BRCA1, which encodes a protein that is functionally distinct from BRCA1. Here we describe the identification of ten novel Brca1 splice variants including Brca1-Iris, the mouse orthologue of human BRCA1-IRIS. We show that Brca1-Iris is differentially expressed during mammary epithelial differentiation and regulates survival of mammary epithelial cells. Another transcript, Brca1-Delta22, expressed in both mouse and human cells, was found to be defective in transcriptional activation capacity. Finally, we show that the human BRCA1 pseudogene produces a spliced pseudoBRCA1 transcript. The identification of these transcripts has implications for the understanding of the role of BRCA1 in biology and disease and for the interpretation of mouse knockout models.

Direct and nondestructive verification of PNA immobilization using click chemistry

The fluorogenic 1,3-Huisgen dipolar cycloaddition reaction was used as part of a novel immobilization strategy of PNA capture probes on a microarray. By using this click chemistry, azidocoumarin-anchored PNA probes were immobilized on phenyl acetylene-modified glass slides with the simultaneous generation of the fluorescent triazolylcoumarin moiety. Since the emitting moieties are generated in the immobilization reaction itself, fluorescent signals can be used to directly monitor the integrity of immobilization in a nondestructive manner. By using this strategy, PNA microarrays were prepared and successfully employed to perform microarray-based diagnosis of selected mutations in the breast cancer susceptibility gene BRCA1.

CHEK2 1100delC and male breast cancer in the Netherlands

Mutations in the breast cancer susceptibility genes BRCA1, BRCA2, and CHEK2 are known risk factors for female breast cancer. Mutations in BRCA1 and BRCA2 also are associated with male breast cancer (MBC). Similarly, it had been suggested in the original CHEK2 identification report that the CHEK2 1100delC mutation confers an increased risk for MBC. Here, we have evaluated the risk of CHEK2 1100delC for MBC by genotyping CHEK2 1100delC in 23 familial and 71 unselected Dutch MBC cases. None of the 23 familial MBC cases carried the CHEK2 1100delC mutation. In contrast, CHEK2 1100delC was present in 3 of the 71 (4.2%) unselected MBC cases, which was significantly more prevalent than the 1.1% Dutch population frequency assessed in 1,692 individuals (P = 0.05, OR = 4.1, 95% CI 1.2-14.3). Our data suggest that, in the Netherlands, CHEK2 1100delC is associated with an increased risk for MBC.