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Functioning of BRCA2

Functioning of BRCA2



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I know that BRCA2 interacts with RAD51 to repair DNA damage.

But how exactly does it function ? What are the other proteins that interact with it ?


I'm just skimming through it now, but this 2011 review in Nature Reviews Cancer looks like it has everything you could possibly want to know about the BRCA1/BRCA2 pathway. If you don't have access to the journal, the first page of this Google search should have a PDF link to Researchgate down near the bottom, or you can try this direct link.

I apologize for the "link-only" answer, but there is a ton of info in the paper, and it would be rather difficult to summarize it in just a few paragraphs. This is a rather broad question, so perhaps you could read through the review and references, and if you still have specific questions about the pathways or interactions you can ask another question.


Here is an image of BRCA2 and RAD51 oriented.( I produced this using PyMOL!)

The chocolate coloured part is some BRCA repeats. You can see how BRCA repeats and RAD51 are oriented. Here, the association is shown only for one of the 7 subunits of RAD51

The exact mechanism in brief : .

  1. BRCA2 binds to RAD51 subunits within the ring via BRC repeat mimicry of the RAD51 polymerization motif.

2.BRC repeats disassembles the ring.

3.The RAD51:BRCA2 comple is recruited to a DNA double strand break.

4.BRCA2 helps displace a protective protein, Replication protein A- RPA, and binds the primary ssDNA substrate by its B folds.

5.It loads RAD51 onto DNA. The handoff reaction might be facilitated by attraction of DNA by the positively charged BRC repeat helical arches.The BRCA2 helix-turn-helix domain(red) at the end of the tower may bind dsDNA in cis at the ssDNA/dsDNA intra-DNA junction (3) or in trans to the dsDNA that later serves as the homologous DNA template (6) and the positively charged arch may also help to attract the dsDNA template.

Source : Yang etal. 2002 , Shin et al 2003

Thanks to Nicholas Provert for teaching me basics of PyMOL on Coursera.


The BRCA1 and BRCA2 Genes

The genes most commonly affected in hereditary breast and ovarian cancer are the breast cancer 1 (BRCA1) and breast cancer 2 (BRCA2) genes. About 3% of breast cancers (about 7,500 women per year) and 10% of ovarian cancers (about 2,000 women per year) result from inherited mutations in the BRCA1 and BRCA2 genes.

Normally, the BRCA1 and BRCA2 genes protect you from getting certain cancers. But some mutations in the BRCA1 and BRCA2 genes prevent them from working properly, so that if you inherit one of these mutations, you are more likely to get breast, ovarian, and other cancers. However, not everyone who inherits a BRCA1 or BRCA2 mutation will get breast or ovarian cancer.

Everyone has two copies of the BRCA1 and BRCA2 genes, one copy inherited from their mother and one from their father. Even if a person inherits a BRCA1 or BRCA2 mutation from one parent, they still have the normal copy of the BRCA1 or BRCA2 gene from the other parent. Cancer occurs when a second mutation happens that affects the normal copy of the gene, so that the person no longer has a BRCA1 or BRCA2 gene that works properly. Unlike the inherited BRCA1 or BRCA2 mutation, the second mutation would not be present throughout the person&rsquos body, but would only be present in the cancer tissue.

Breast and ovarian cancer can also be caused by inherited mutations in genes other than BRCA1 and BRCA2. This means that in some families with a history of breast and ovarian cancer, family members will not have mutations in BRCA1 or BRCA2, but can have mutations in one of these other genes. These mutations might be identified through genetic testing using multigene panels, which look for mutations in several different genes at the same time.

You and your family members are more likely to have a BRCA1 or BRCA2 mutation if your family has a strong history of breast or ovarian cancer. Family members who inherit BRCA1 and BRCA2 mutations usually share the same mutation. If one of your family members has a known BRCA1 or BRCA2 mutation, other family members who get genetic testing should be checked for that mutation.

If you are concerned that you could have a BRCA1, BRCA2, or other mutation related to breast and ovarian cancer, the first step is to collect your family health history of breast and ovarian cancer and share this information with your doctor.


Contents

Although the structures of the BRCA1 and BRCA2 genes are very different, at least some functions are interrelated. The proteins made by both genes are essential for repairing damaged DNA (see Figure of recombinational repair steps). BRCA2 binds the single strand DNA and directly interacts with the recombinase RAD51 to stimulate [27] and maintain [28] strand invasion, a vital step of homologous recombination. The localization of RAD51 to the DNA double-strand break requires the formation of the BRCA1-PALB2-BRCA2 complex. PALB2 (Partner and localizer of BRCA2) [29] can function synergistically with a BRCA2 chimera (termed piccolo, or piBRCA2) to further promote strand invasion. [30] These breaks can be caused by natural and medical radiation or other environmental exposures, but also occur when chromosomes exchange genetic material during a special type of cell division that creates sperm and eggs (meiosis). Double strand breaks are also generated during repair of DNA cross links. By repairing DNA, these proteins play a role in maintaining the stability of the human genome and prevent dangerous gene rearrangements that can lead to hematologic and other cancers.

BRCA2 has been shown to possess a crucial role in protection from the MRE11-dependent nucleolytic degradation of the reversed forks that are forming during DNA replication fork stalling (caused by obstacles such as mutations, intercalating agents etc.). [31]

Like BRCA1, BRCA2 probably regulates the activity of other genes and plays a critical role in embryo development.

Certain variations of the BRCA2 gene increase risks for breast cancer as part of a hereditary breast–ovarian cancer syndrome. Researchers have identified hundreds of mutations in the BRCA2 gene, many of which cause an increased risk of cancer. BRCA2 mutations are usually insertions or deletions of a small number of DNA base pairs in the gene. As a result of these mutations, the protein product of the BRCA2 gene is abnormal, and does not function properly. Researchers believe that the defective BRCA2 protein is unable to fix DNA damage that occurs throughout the genome. As a result, there is an increase in mutations due to error-prone translesion synthesis past un-repaired DNA damage, and some of these mutations can cause cells to divide in an uncontrolled way and form a tumor.

People who have two mutated copies of the BRCA2 gene have one type of Fanconi anemia. This condition is caused by extremely reduced levels of the BRCA2 protein in cells, which allows the accumulation of damaged DNA. Patients with Fanconi anemia are prone to several types of leukemia (a type of blood cell cancer) solid tumors, particularly of the head, neck, skin, and reproductive organs and bone marrow suppression (reduced blood cell production that leads to anemia). Women having inherited a defective BRCA1 or BRCA2 gene have risks for breast and ovarian cancer that are so high and seem so selective that many mutation carriers choose to have prophylactic surgery. There has been much conjecture to explain such apparently striking tissue specificity. Major determinants of where BRCA1- and BRCA2-associated hereditary cancers occur are related to tissue specificity of the cancer pathogen, the agent that causes chronic inflammation, or the carcinogen. The target tissue may have receptors for the pathogen, become selectively exposed to carcinogens and an infectious process. An innate genomic deficit impairs normal responses and exacerbates the susceptibility to disease in organ targets. This theory also fits data for several tumor suppressors beyond BRCA1 or BRCA2. A major advantage of this model is that it suggests there are some options in addition to prophylactic surgery. [32]

In addition to breast cancer in men and women, mutations in BRCA2 also lead to an increased risk of ovarian, Fallopian tube, prostate and pancreatic cancer. In some studies, mutations in the central part of the gene have been associated with a higher risk of ovarian cancer and a lower risk of prostate cancer than mutations in other parts of the gene. Several other types of cancer have also been seen in certain families with BRCA2 mutations.

In general, strongly inherited gene mutations (including mutations in BRCA2) account for only 5-10% of breast cancer cases the specific risk of getting breast or other cancer for anyone carrying a BRCA2 mutation depends on many factors. [33]

The gene was first cloned by scientists at Myriad Genetics, Endo Recherche, Inc., HSC Research & Development Limited Partnership, and the University of Pennsylvania. [36]

Methods to diagnose the likelihood of a patient with mutations in BRCA1 and BRCA2 getting cancer were covered by patents owned or controlled by Myriad Genetics. [37] [38] Myriad's business model of exclusively offering the diagnostic test led from Myriad's beginnings as a startup in 1994 to its being a publicly traded company with 1200 employees and about $500M in annual revenue in 2012 [39] it also led to controversy over high test prices and the unavailability of second opinions from other diagnostic labs, which in turn led to the landmark Association for Molecular Pathology v. Myriad Genetics lawsuit. [40]


BRCA1 in Complex With BARD1 Is an E3 Ubiquitin Ligase Critical for Genome Integrity

Breast cancer susceptibility gene 1 (BRCA1) is a tumor suppressor gene, germline mutations of which are linked to familial breast and ovarian cancers (Hall et al., 1990 Futreal et al., 1994 Godwin et al., 1994 Miki et al., 1994). More than two decades of research has implicated BRCA1 function in multiple cellular pathways, including transcriptional regulation, DNA damage signaling, cell cycle checkpoints, centrosome regulation and in the repair of DNA DSBs through HR (Moynahan et al., 1999 Xu et al., 1999 Deng, 2002, 2006 Yarden et al., 2002 Caestecker and Van de Walle, 2013 Hill et al., 2014 Hatchi et al., 2015). Of critical importance, its role in promoting HR is directly linked to maintenance of genome integrity (Roy et al., 2011 Prakash et al., 2015).

In humans, the 1,863 amino acid BRCA1 protein has an N-terminal RING ( R eally I nteresting N ew G ene) domain that coordinates two zinc cations in a cross-braced arrangement, a largely unstructured central region encoded by exon11, followed by a coiled coil domain and two C-terminal BRCT repeats ( Figure 1 ). RING domains create a platform for binding to E2 ubiquitin conjugating enzymes and facilitate the transfer of ubiquitin from the E2 to substrates, thereby specifying E3 ubiquitin ligase activity (Deshaies and Joazeiro, 2009). The BRCT repeats are phosphopeptide interaction modules for binding to phosphorylated proteins (Manke et al., 2003 Rodriguez et al., 2003 Yu et al., 2003). BRCA1 forms a heterodimer with its obligate binding partner BARD1 ( B RCA1- A ssociated R ING D omain protein 1) through their N-terminal regions and the heterodimer exhibits efficient ubiquitin transfer activity (Wu et al., 1996 Meza et al., 1999 Brzovic et al., 2001 Hashizume et al., 2001 Baer and Ludwig, 2002). The BARD1 protein is 777 amino acids in length and similar to BRCA1, contains a RING domain at its N-terminus and two BRCT repeats at its C-terminus ( Figure 1 ). In addition, four ankyrin repeats involved in chromatin recognition of newly replicated sister chromatids are present in the middle of the protein (Fox III, Le Trong et al., 2008 Nakamura et al., 2019). Most studies indicate that BARD1 is indispensable for BRCA1 function and depletion of BARD1 leads to highly similar phenotypes as observed for BRCA1 mutants. Mutations in BARD1 have been identified in patients with breast, ovarian and other cancer types, although at a lower frequency than BRCA1 mutations (Thai et al., 1998 Ghimenti et al., 2002). Further, as with BRCA1, loss of BARD1 results in embryonic lethality in mice as well as defects in HR leading to chromosomal instability (McCarthy et al., 2003).

Domain structure of BRCA1 and BARD1 proteins. Human BRCA1 contains an N-terminal RING domain, an unstructured central region encoded by the large exon11 followed by a coiled coil (CC) domain and two BRCA1 C-terminal (BRCT) repeats. Both human and mouse express an alternatively spliced variant BRCA1𹐑 that contains the N-terminal RING domain and C-terminal BRCT repeats but lacks the unstructured central region (Thakur et al., 1997 Huber et al., 2001). This truncated protein is expressed in the hypomorphic Brca1 Δ1111 mouse. C. elegans BRC-1 is structurally similar to the BRCA1𹐑 splicing variant with the presence of an N-terminal RING domain and two BRCT repeats at its C terminus. A. thaliana encodes a similarly structured BRCA1 ortholog that has a N-terminal RING and two C-terminal BRCT repeats. Human BARD1 and C. elegans BRD-1 are similar in size and domain structure, containing an N-terminal RING domain, ankyrin repeats in the middle and two C-terminal BRCT repeats. A. thaliana BARD-1 has a similar domain structure but appears to lack ankyrin repeats, which were not predicted by sequence alignment. BRCA1 interacts with BARD1 through their RING domains to form a heterodimer with E3 ubiquitin ligase activity.

The mechanisms by which BRCA1-BARD1 promotes HR during DSB repair involve multiple steps. First, BRCA1 promotes DNA end resection by antagonizing 53BP1, a DNA damage response protein that promotes error-prone non-homologous end joining (NHEJ) (Bunting et al., 2010 Daley and Sung, 2014). Two, BRCA1 regulates the MRE11-RAD50-NBS1-CtIP complex essential for DNA end processing (Cruz-Garcia et al., 2014 Aparicio et al., 2016). There is also evidence that BRCA1 removes a chromatin barrier for DNA resection through ubiquitylation of histone H2A (Densham et al., 2016). In addition to promoting resection, BRCA1-BARD1 binds to DNA and interacts with RAD51 directly, enhancing RAD51 recombinase activity by promoting homologous strand invasion and synaptic complex formation (Zhao et al., 2017). However, whether BRCA1 functions by similar mechanisms to promote HR during meiosis for the repair of SPO11-induced DSBs has remained elusive.


Functional Interaction of Monoubiquitinated FANCD2 and BRCA2/FANCD1 in Chromatin

FIG. 1 . Colocalization, cofractionation, and coimmunoprecipitation of monoubiquitinated FANCD2 and BRCA2 in chromatin. (A) Colocalization of FANCD2 and BRCA2 in DNA damage-inducible foci. HeLa cells were either untreated, exposed to IR (2 or 15 Gy), or treated with MMC (80 ng/ml), as indicated. HeLa cells were double stained with polyclonal anti-FANCD2 (E35) (red) and monoclonal anti-BRCA2 (Ab-1) (green) antibodies after the indicated time of treatment. Magnification, ×630. (B) Protocol utilized for nuclear fractionation of cells. Cytoplasm and nucleoplasm were extracted by permeabilization with detergent, the resulting nuclei were DNase I digested, and chromatin was extracted with ammonium sulfate (NH2SO4). (C) U2OS cells, either untreated or exposed to IR (15 Gy) or MMC (170 ng/ml), were fractionated 15 h after the initiation of DNA damage. Supernatants (S) and pellets (P) were subjected to Western blot analysis with the indicated antibodies: E35 for FANCD2 and Ab-1 for BRCA2. The presence of chromatin in the S4 fraction was confirmed by blotting with anti-histone H4 antibody. (D) S2 (soluble nuclear proteins) and S4 (chromatin) fractions of U2OS cells were subjected to immunoprecipitation with mouse monoclonal anti-BRCA2 antibody (Ab-1) or a control mouse antibody (mIgG) and then immunoblotted with either polyclonal anti-BRCA2 (Ab-2) or anti-FANCD2 (E35) antibodies. Heavy chain IgG was used as a loading control. (E) HeLa cells were transfected with a cDNA encoding HA-ubiquitin, as indicated. After transfection, cells were treated with the indicated dose of IR or MMC. S2 (soluble nuclear proteins) and S4 (chromatin) fractions of HeLa cells were immunoprecipitated (IP) with a polyclonal antibody (E35) to FANCD2, as indicated. Immune complexes were run on SDS-PAGE and immunoblotted with anti-FANCD2 (FI-17) or anti-HA (HA.11) monoclonal antibodies. WCE, whole-cell extract. FIG. 2 . Monoubiquitinated FANCD2 promotes IR-induced assembly of BRCA2 foci. (A) Whole-cell extracts of PD20 (FA-D2) fibroblasts stably expressing empty vector (PD20F + Vec) and PD20 fibroblasts corrected with FANCD2 (PD20F + FANCD2) were subjected to immunoprecipitation with mouse monoclonal anti-BRCA2 (Ab-1) or a control mouse antibody (mIgG) and then immunoblotted with either anti-FANCD2 (E35) or anti-BRCA2 (Ab-2) antibodies. Heavy chain IgG was used as a loading control. (B) Formation of subnuclear FANCD2 and BRCA2 foci in response to IR treatment. PD20 (FA-D2) fibroblasts, stably expressing empty vector alone (upper panel) or full-length FANCD2 cDNA (lower panel), were either untreated or treated with IR (15 Gy) and fixed 4 h later. Cells were double stained with polyclonal anti-FANCD2 (E35) (red) and monoclonal anti-BRCA2 (Ab-1) (green) antibodies and analyzed by immunofluorescence microscopy. Magnification, ×630. (C) Quantification of BRCA2 foci. PD20 (FA-D2) fibroblasts stably expressing empty vector alone (PD20F + Vec), FANCD2 (PD20F + FANCD2), or FANCD2-K561R mutant (PD20F + K561R), GM6914 (FA-A) fibroblasts and corrected GM6914 fibroblasts stably expressing FANCA (GM6914 + FANCA), EUFA130 (FA-E) lymphoblasts and corrected EUFA130 lymphoblasts (EUFA130 + HA-FANCE), and HeLa cells were either untreated or treated with IR (15 Gy) and fixed 4 h later. Cells with more than four distinct foci were counted as positive. A total of 200 cells/sample were analyzed. The values shown are the mean ± standard deviation from three separate experiments. FIG. 3 . FANCD2 and BRCA2 foci fail to associate in FA-D1 (BRCA2 −/− ) cells. (A) Schematic diagram of human BRCA2 protein, indicating mutations in EUFA 423 (FA-D1) cells. The positions of epitopes for the anti-BRCA2 antibodies Ab-1 and Ab-2 are shown. (B) EUFA423 (FA-D1) fibroblasts and corrected cells stably transfected with human chromosome 13 (EUFA423 + BRCA2) were exposed to IR, either at different doses (left) or for different periods of time (right). Western blotting was performed with anti-BRCA2 (Ab-1 or Ab-2) or anti-FANCD2 (E35) antibodies. (C) Immunofluorescent localization of BRCA2 and FANCD2 following treatment with IR (15 Gy) was examined in EUFA423 (FA-D1) fibroblasts, EUFA423 fibroblasts transiently transfected with pcDNA3 HA-BRCA2 (EUFA423 + HA-BRCA2), and EUFA 423 fibroblasts stably transfected with human chromosome 13 (EUFA423 + BRCA2). Cells were double stained with the indicated antibodies and analyzed by immunofluorescence microscopy. Magnification, ×630. (D and E) Quantification of the percentage of cells with FANCD2 foci (D) and the percentage of cells with colocalization of FANCD2 and BRCA2 foci (E) in HeLa cells, EUFA423 fibroblasts, EUFA423 fibroblasts transiently expressing HA-BRCA2 (EUFA423 + HA-BRCA2), and EUFA423 fibroblasts stably expressing human chromosome 13 (EUFA423 + BRCA2). Cells were either untreated or treated with IR (15 Gy) and fixed 4 h later for immunofluorescence microscopy. Cells with more than four distinct foci were counted as positive. Two hundred and 100 cells/sample were analyzed in panels D and E, respectively. The values shown are the mean ± standard deviation from three separate experiments. (F) S2 (soluble nuclear proteins) and S4 (chromatin) fractions of irradiated EUFA423 cells and EUFA423 cells stably expressing human chromosome 13 (EUFA423 + BRCA2) were subjected to immunoprecipitation with rabbit polyclonal anti-FANCD2 antibody (E35) or a control nonimmunized rabbit serum (Pre-imm) and then immunoblotted with either monoclonal anti-BRCA2 (Ab-1) or anti-FANCD2 (FI-17) antibodies. Heavy chain IgG was used as a loading control. WCE, whole-cell extract. FIG. 4 . FA-D1 and FA-D2 cells are defective in the assembly of IR-inducible RAD51 foci. (A) Quantification of RAD51 foci in EUFA423 (FA-D1) fibroblasts, EUFA423 fibroblasts stably transfected with human chromosome 13 (EUFA423 + BRCA2), and HeLa cells. Cells were either untreated or treated with IR (2 or 15 Gy) and fixed 15 h later. Cells were stained with monoclonal anti-RAD51 antibody and analyzed by immunofluorescence microscopy. Cells with more than four distinct foci were counted as positive. Two hundred cells/sample were analyzed. The values shown are the mean ± standard deviation from three separate experiments. (B) Quantification of RAD51 foci in PD20 (FA-D2) fibroblasts stably expressing empty vector alone (PD20F + Vec), full-length FANCD2 cDNA (PD20F + FANCD2), or the FANCD2 K561R mutant (PD20F + K561R), and HeLa cells. Cells were either untreated or treated with IR (2 or 15 Gy) and fixed 8 h later. Immunofluorescence microscopy and counts were performed as described above for panel A. FIG. 5 . Monoubiquitination of FANCD2 promotes IR-inducible accumulation of BRCA2 in chromatin. (A) S2 (soluble nuclear proteins) and S4 (chromatin) fractions were prepared from U2OS and FA cells, which were either untreated or treated with IR (15 Gy, fractionated after 15 h). Fractions were subjected to Western blot analysis with anti-FANCD2 (E35) or anti-BRCA2 (Ab-1) antibodies. The extraction of chromatin in the S4 fraction was confirmed by blotting with anti-histone H4 antibody, as indicated. The FA cells are represented by GM6914 (FA-A) fibroblasts, EUFA423 (FA-D1) fibroblasts, and HSC230 (FA-B) lymphoblasts. (B) S2 (soluble nuclear proteins) and S4 (chromatin) fractions were prepared from PD20 (FA-D2) fibroblasts stably expressing empty vector alone (PD20F + HA-PMMP), HA-FANCD2 (PD20F + HA-FANCD2), or FANCD2-HA-K561R mutant (PD20F + HA-K561R), which were either untreated or treated with IR (15 Gy, fractionated after 6 h). Fractions were subjected to Western blot analysis with anti-FANCD2 (E35) or anti-BRCA2 (Ab-1) antibodies. The extraction of chromatin in the S4 fraction was confirmed by blotting with anti-histone H4 antibody. FIG. 6 . ATM is required for IR-activated phosphorylation of BRCA2 in chromatin. (A) BRCA2 immunoprecipitated (Ab-1) from chromatin fractions was treated either with or without λ-phosphatase and phosphatase inhibitors, as indicated. Chromatin fractions were derived either from untreated U2OS cells or 15 h following IR treatment (15 Gy). The mobility shift of BRCA2 is shown in lanes 4 and 6. WCE, whole-cell extract. (B) BRCA2 derived from the chromatin fraction of corrected AT (AT + ATM) fibroblasts at time points after treatment with IR (15 Gy) undergoes a mobility shift, while BRCA2 from the chromatin fraction of AT (ATM −/− ) fibroblasts does not. Chromatin fractions were immunoblotted with either anti-BRCA2 (Ab-1), anti-ATM, or anti-FANCD2 (E35) antibodies. (C) EUFA423 (FA-D1) and PD20 (FA-D2) cells are defective in an IR-inducible S-phase checkpoint. RDS was assessed 30 min after delivery of IR to the indicated cell lines. FIG. 7 . Interaction of FANCE and BRCA2. (A) Whole-cell extracts (WCE) from HeLa cells, either untreated or exposed to IR (15 Gy, harvested 15 h later) or MMC (40 ng/ml, harvested 24 h later), were subjected to immunoprecipitation with mouse monoclonal anti-BRCA2 antibody (Ab-1) or a control mouse antibody (mIgG). Heavy chain IgG was used as a loading control. (B) Whole-cell extracts from unirradiated or irradiated EUFA130 (FA-E), and EUFA130 cells stably expressing HA-FANCE (EUFA130 + HA-FANCE), were subjected to immunoprecipitation with polyclonal anti-BRCA2 antibody (H-300) or a control rabbit antibody (rIgG) and then immunoblotted with anti-BRCA2 (Ab-1) or anti-HA antibodies. Heavy chain IgG was used as a loading control. (C) EUFA130 (FA-E) lymphoblasts and EUFA130 cells stably expressing HA-FANCE (EUFA130 + HA-FANCE) were either untreated or exposed to IR at different doses, as indicated, and harvested after 4 h. Western blotting was performed with anti-BRCA2 (Ab-1), anti-FANCD2 (E35), or anti-HA antibodies. (D) Reciprocal coimmunoprecipitation of FANCD2 and BRCA2 with other antibodies. The indicated fractions (S2 and S4), prepared from irradiated U2OS cells, were immunoprecipitated with antibody to FANCD2 (E35), FANCE, or a control nonimmunized rabbit serum (Pre-imm), and the immune complexes were immunoblotted with anti-BRCA2 (Ab-1) or anti-FANCD2 (FI-17) antibodies. Heavy chain IgG was used as a loading control. (E) Schematic model of the ATM-BRCA2-FANCD2-mediated DNA damage response. IR activates ATM, resulting in the phosphorylation of BRCA2, FANCD2, and several other protein substrates. Activated BRCA2 is then recruited to chromatin by FANCD2, which is activated by monoubiquitination. The interaction of BRCA2 and FANCD2 requires both the C terminus of BRCA2 and FANCD2 monoubiquitination. Following its loading onto chromatin, BRCA2 then functions downstream of monubiquitinated FANCD2.

BRCA Gene Mutations: Cancer Risk and Genetic Testing

BRCA1 (BReast CAncer gene 1) and BRCA2 (BReast CAncer gene 2) are genes that produce proteins that help repair damaged DNA. Everyone has two copies of each of these genes—one copy inherited from each parent. BRCA1 and BRCA2 are sometimes called tumor suppressor genes because when they have certain changes, called harmful (or pathogenic) variants (or mutations), cancer can develop.

People who inherit harmful variants in one of these genes have increased risks of several cancers—most notably breast and ovarian cancer, but also several additional types of cancer. People who have inherited a harmful variant in BRCA1 and BRCA2 also tend to develop cancer at younger ages than people who do not have such a variant.

A harmful variant in BRCA1 or BRCA2 can be inherited from either parent. Each child of a parent who carries any mutation in one of these genes has a 50% chance (or 1 in 2 chance) of inheriting the mutation. Inherited mutations—also called germline mutations or variants—are present from birth in all cells in the body.

Even if someone has inherited a harmful variant in BRCA1 or BRCA2 from one parent, they would have inherited a normal copy of that gene from the other parent (that’s because in most cases, embryos with a harmful variant from each parent cannot develop). But the normal copy can be lost or change in some cells in the body during that person’s lifetime. Such a change is called a somatic alteration. Cells that don’t have any functioning BRCA1 or BRCA2 proteins can grow out of control and become cancer.

How much does an inherited harmful variant in BRCA1 or BRCA2 increase a woman’s risk of breast and ovarian cancer?

A woman’s lifetime risk of developing breast and/or ovarian cancer is markedly increased if she inherits a harmful variant in BRCA1 or BRCA2, but the degree of increase varies depending on the mutation.

Breast cancer: About 13% of women in the general population will develop breast cancer sometime during their lives (1). By contrast, 55% – 72% of women who inherit a harmful BRCA1 variant and 45% – 69% of women who inherit a harmful BRCA2 variant will develop breast cancer by 70–80 years of age (2–4). The risk for any one woman depends on a number of factors, some of which have not been fully characterized.

Like women with breast cancer in general, those with harmful BRCA1 or BRCA2 variants also have an increased risk of developing cancer in the opposite (contralateral) breast in the years following a breast cancer diagnosis (2). The risk of contralateral breast cancer increases with the time since a first breast cancer, reaching 20%–30% at 10 years of follow-up and 40%–50% at 20 years, depending on the gene involved.

Ovarian cancer: About 1.2% of women in the general population will develop ovarian cancer sometime during their lives (1). By contrast, 39%–44% of women who inherit a harmful BRCA1 variant and 11%–17% of women who inherit a harmful BRCA2 variant will develop ovarian cancer by 70–80 years of age (2–4).

What other cancers are linked to harmful variants in BRCA1 and BRCA2?

Harmful variants in BRCA1 and BRCA2 increase the risk of several additional cancers. In women, these include fallopian tube cancer (5, 6) and primary peritoneal cancer (7), both of which start in the same cells as the most common type of ovarian cancer. Men with BRCA2 variants, and to a lesser extent BRCA1 variants, are also at increased risk of breast cancer (8) and prostate cancer (9–11). Both men and women with harmful BRCA1 or BRCA2 variants are at increased risk of pancreatic cancer, although the risk increase is low (12–14).

In addition, certain variants in BRCA1 and BRCA2 can cause subtypes of Fanconi anemia, a rare syndrome that is associated with childhood solid tumors and development of acute myeloid leukemia (15–17). The mutations that cause these Fanconi anemia subtypes have a milder effect on protein function than the mutations that cause breast and ovarian cancer. Children who inherit one of these variants from each parent will develop Fanconi anemia.

Are harmful variants in BRCA1 and BRCA2 more common in certain racial/ethnic populations than others?

Yes. The likelihood of carrying an inherited mutation in BRCA1 or BRCA2 (the prevalence) varies across specific population groups. While the prevalence in the general population is about 0.2%–0.3% (or about 1 in 400), about 2.0% of people of Ashkenazi Jewish descent carry a harmful variant in one of these two genes and the variants are usually one of three specific variants, called founder mutations. Other populations, such as Norwegian, Dutch, and Icelandic peoples, also have founder mutations (18).

Different racial/ethnic and geographic populations also tend to carry different variants in these genes. For instance, African Americans have BRCA1 variants that are not seen in other racial/ethnic groups in the United States (19–21). Most people of Ashkenazi Jewish descent in the United States who carry a BRCA variant have one of three specific variants (two in BRCA1 and one in BRCA2). In the Icelandic population, a different variant in BRCA1 is common among those who inherit a mutation in BRCA1.

Who should consider genetic counseling and testing for BRCA1 and BRCA2 variants?

Anyone who is concerned about the possibility that they may have a harmful variant in the BRCA1 or BRCA2 gene should discuss their concerns with their health care provider or a genetic counselor.

Tests are available to see if someone has inherited a harmful variant in BRCA1 and BRCA2. However, testing is not currently recommended for the general public. Instead, expert groups recommend that testing be focused on those who have a higher likelihood of carrying a harmful BRCA1 or BRCA2 variant, such as those who have a family history of certain cancers. Testing can be appropriate for both people without cancer as well as people who have been diagnosed with cancer. If someone knows they have a mutation in one of these genes, they can take steps to reduce their risk or detect cancer early. And if they have cancer, the information about their mutation may be important for selecting treatment.

Before testing is done, a person will usually have a risk assessment, in which they meet with a genetic counselor or other health care provider to review factors such as which of their relatives had cancer, what cancers they had, and at what ages they were diagnosed. If this assessment suggests that someone has an increased risk of carrying a harmful BRCA1 or BRCA2 gene variant, their genetic counselor can discuss the benefits and harms of testing with them and order the appropriate genetic test, if the individual decides to have genetic testing (22).

Some people may choose to have genetic testing via direct-to-consumer (DTC) testing. Genetic counseling is recommended for those people as well to help them understand the test results and to make sure the most appropriate test was done. People should be aware that DTC tests may not be comprehensive, in that some tests do not test for all of the harmful mutations in the two genes. So receiving a negative result with a DTC test may not mean that they don’t have a harmful variant in BRCA1 or BRCA2.

The United States Preventive Services Task Force recommends risk assessment for women who have a personal or family history of breast, ovarian, fallopian tube, or peritoneal cancer or whose ancestry is associated with having harmful BRCA1 and BRCA2 variants, as well as follow-up genetic counseling as appropriate.

The National Comprehensive Cancer Network (NCCN) has criteria for genetic testing of BRCA1 and BRCA2 as well as for several other genes (including CDH1, PALB2, PTEN, and TP53) that are associated with increased risk of breast and/or ovarian cancer (23). NCCN recommends risk assessment for people who have a blood relative with a known or likely harmful variant in any of these genes who have certain personal and/or family histories of cancer (cancer diagnosed at a younger age, certain types of cancer, people with two or more cancer diagnoses, or families with multiple cases of cancer) or who have certain inherited cancer predisposition disorders, such as Cowden syndrome, Peutz-Jeghers syndrome, Li-Fraumeni syndrome, or Fanconi anemia.

The American Society of Clinical Oncology recommends that all women diagnosed with epithelial ovarian cancer be offered genetic testing for inherited variants in BRCA1, BRCA2, and other ovarian cancer susceptibility genes, regardless of the clinical features of their disease or their family history (24).

Professional societies do not recommend that children under age 18 undergo genetic testing for BRCA1 and BRCA2 variants. This is because there are no risk-reduction strategies that are specifically meant for children, and children are very unlikely to develop a cancer related to an inherited BRCA variant.

Testing for inherited BRCA1 and BRCA2 variants may be done using a blood sample or a saliva sample. That is because blood cells and cells that are present in saliva, like every cell in the body, contain the BRCA1 and BRCA2 genes. Sometimes people with cancer find out that they have a BRCA1 or BRCA2 mutation when their tumor is tested to see if they are a candidate for treatment with a particular targeted therapy. Because harmful BRCA variants reported in the tumor may be of somatic or germline origin, someone with such a variant in their tumor should consider having a germline genetic (blood) test to determine if the variant was inherited.

When a family history suggests the possibility that someone without cancer may have inherited a harmful variant in BRCA1 or BRCA2, it is best for a family member who has already been diagnosed with cancer to be tested, if such a person is alive and willing to get tested. If such testing reveals a known harmful variant, then testing the individual for that variant will provide a clear indication of whether they also carry it. If all family members with cancer are deceased or are unwilling or unable to have genetic testing, testing family members who have not been diagnosed with cancer may still be of value and provide good information.

Does health insurance cover the cost of genetic testing for BRCA1 and BRCA2 variants?

People considering BRCA1 and BRCA2 variant testing may want to confirm their insurance coverage for genetic counseling and testing. Genetic counselors can often help answer questions about insurance coverage for genetic testing.

Some genetic testing companies may offer testing for inherited BRCA1 and BRCA2 variants at no charge to patients who lack insurance and meet specific financial and medical criteria.

What do BRCA1 and BRCA2 genetic test results mean?

BRCA1 and BRCA2 mutation testing can give several possible results: a positive result, a negative result, or a variant of uncertain significance (VUS) result.

Positive result. A positive test result indicates that a person has inherited a known harmful variant in BRCA1 or BRCA2 (these are typically called “pathogenic” or “likely pathogenic” variants on laboratory test reports) and has an increased risk of developing certain cancers. However, a positive test result cannot tell whether or when the tested individual will develop cancer. Some people who inherit a harmful BRCA1 or BRCA2 variant never develop cancer.

A positive test result may also have important implications for family members, including future generations.

  • Both men and women who inherit a harmful BRCA1 or BRCA2 variant, whether or not they develop cancer themselves, may pass the variant to their children. Each child has a 50% chance of inheriting a parent’s variant.
  • All blood relatives of a person who has inherited a harmful BRCA1 or BRCA2 variant are at some increased risk of having the variant themselves. For example, each of that person’s full siblings has a 50% chance of having inherited the variant as well.
  • Very rarely, an individual may test positive for a harmful variant not inherited from either parent. This is called a de novo (or “new”) variant. Such a variant is one that arose in a germ cell (sperm or egg) of one of the parents and is present in all the cells of the person who grew from that cell. The children of someone with a de novo variant (but not his or her siblings) are at risk of inheriting the variant.

Negative result. A negative test result can have several meanings, depending on the personal and family medical history of the person who is tested and whether or not a harmful mutation has already been identified in the family. If a close blood relative of the tested person is known to carry a harmful BRCA1 or BRCA2 variant, a negative test result is clear: it means the tested person did not inherit the harmful variant that is present in the family and cannot pass it to their children. A person with such a test result, called a true negative, has a risk of cancer that is similar to that of someone in the general population. However, there are other factors besides genetic factors that may increase the risk of cancer, such as radiation exposures at an early age, and those factors should be considered in assessing their risk of cancer.

If the tested person has no personal history of cancer and their family isn’t known to carry a harmful variant, then in this case, a negative test result is considered to be “uninformative.” There are several possible reasons why someone could have an uninformative negative test result:

  • Without testing family members who have had cancer, it is uncertain whether the negative test means that the person did not inherit a BRCA1 or BRCA2 mutation that is present in the family or whether the family history might be due to a mutation in another gene that was not tested or to other, nongenetic risk factors.
  • The individual may have a harmful variant that is not detectable by current testing technologies.
  • Rarely, there could be an error in the testing, either because inappropriate tests were recommended or ordered, genetic variants were interpreted incorrectly, or the wrong results were relayed to patients (25).

Variant of Uncertain Significance (VUS) result. Sometimes, a genetic test finds a change in BRCA1 or BRCA2 that has not been previously associated with cancer and is uncommon in the general population. This type of test result is called “a variant of uncertain significance,” or VUS, because it isn’t known whether this specific genetic change is harmful.

As more research is conducted and more people are tested for BRCA1 and BRCA2 variants, scientists will learn more about uncertain changes and cancer risk. Clinicians and scientists are actively working to share information on these mutations so that they can be reclassified as either clearly harmful or clearly not harmful (26, 27).

Genetic counseling can help a person understand what a VUS in BRCA1 or BRCA2 may mean in terms of their cancer risk. Until the interpretation of the variant is clarified, management of risk should be based on family history and other risk factors. However, it is important that a person who has a VUS test result regularly obtains updated information from the testing provider in case that VUS is reclassified as a harmful or likely harmful variant. Testing providers have different policies about notifying a tested person of a change in the interpretation of a VUS test result. Some will contact the tested person directly, whereas others place the responsibility on the tested person to check back in on a regular basis to learn of updates to the interpretation of their VUS test result.

How can a person who has inherited a harmful BRCA1 or BRCA2 gene variant reduce their risk of cancer?

Several options are available for reducing cancer risk in individuals who have inherited a harmful BRCA1 or BRCA2 variant. These include enhanced screening, risk-reducing surgery (sometimes referred to as prophylactic surgery), and chemoprevention.

Enhanced screening. Some women who test positive for harmful BRCA1 and BRCA2 variants may choose to start breast cancer screening at younger ages, have more frequent screening than is recommended for women with an average risk of breast cancer, or have screening with magnetic resonance imaging (MRI) in addition to mammography.

No effective ovarian cancer screening methods are known. Some groups recommend transvaginal ultrasound, blood tests for the CA-125 antigen (which can be present at higher-than-normal levels in women with ovarian cancer), and clinical examinations for ovarian cancer screening in women with harmful BRCA1 or BRCA2 variants. However, none of these methods appear to detect ovarian tumors at an early enough stage to improve long-term survival (28).

The benefits of screening men who carry harmful variants in BRCA1 or BRCA2 for breast and other cancers are not known. Some expert groups recommend that such men undergo regular annual clinical breast exams starting at age 35 (23). The National Comprehensive Cancer Network (NCCN) guidelines recommend that men with harmful germline variants in BRCA1 or BRCA2 consider having a discussion with their doctor about prostate-specific antigen (PSA) testing for prostate cancer screening starting at age 40 (29).

Some experts recommend the use of ultrasound or MRI/magnetic retrograde cholangiopancreatography to screen for pancreatic cancer in people who are known to carry a harmful BRCA1 or BRCA2 variant and who have a close blood relative with pancreatic cancer (30). However, it is not yet clear whether pancreatic cancer screening and early pancreatic cancer detection reduces the overall risk of dying from a pancreatic cancer.

All of these screening approaches have potential harms as well as possible benefits. For example, MRI is more likely than mammography to result in false-positive findings. And there is some concern that women who have a harmful BRCA variant might be particularly sensitive to the DNA-damaging effects of tests that involve radiation (such as mammography) because they already have a defect in DNA repair (31).

Risk-reducing surgery. Risk-reducing, or prophylactic, surgery involves removing as much of the "at-risk" tissue as possible. Women may choose to have both breasts removed (bilateral risk-reducing mastectomy) to reduce their risk of breast cancer. Surgery to remove a woman's ovaries and fallopian tubes (bilateral risk-reducing salpingo-oophorectomy) can help reduce her risk of ovarian cancer. (Ovarian cancers often originate in the fallopian tubes, so it is essential that they be removed along with the ovaries.) Removing the ovaries may also reduce the risk of breast cancer in premenopausal women by eliminating a source of hormones that can fuel the growth of some types of breast cancer.

These surgeries are irreversible, and each has potential complications or harms. These include bleeding or infection, anxiety and concerns about body image (bilateral risk-reducing mastectomy), and early menopause in premenopausal women (bilateral risk-reducing salpingo-oophorectomy).

Risk-reducing surgery does not guarantee that cancer will not develop because not all at-risk tissue can be removed by these procedures. That is why these surgical procedures are described as “risk-reducing” rather than “preventive.” Some women have developed breast cancer, ovarian cancer, or primary peritoneal carcinomatosis (a type of cancer similar to ovarian cancer) even after risk-reducing surgery. Nevertheless, these surgical procedures greatly reduce risk. For example, in several studies women who underwent bilateral salpingo-oophorectomy had a nearly 80% reduction in risk of dying from ovarian cancer, a 56% reduction in risk of dying from breast cancer (32), and a 77% reduction in risk of dying from any cause during the studies’ follow-up periods (33).

The reduction in breast and ovarian cancer risk from removal of the ovaries and fallopian tubes appears to be similar for carriers of BRCA1 and BRCA2 variants (33).

Chemoprevention. Chemoprevention is the use of medicines to reduce the risk of cancer. Two chemopreventive drugs (tamoxifen [Nolvadex] and raloxifene [Evista]) have been approved by the Food and Drug Administration (FDA) to reduce the risk of breast cancer in women at increased risk, but the role of these drugs in women with harmful BRCA1 or BRCA2 variants is not yet clear. Data from three studies suggest that tamoxifen may be able to help lower the risk of breast cancer in women who carry harmful variants in BRCA2 (34) and of cancer in the opposite breast among BRCA1 and BRCA2 variant carriers previously diagnosed with breast cancer (35, 36). Studies have not examined the effectiveness of raloxifene in BRCA1 and BRCA2 variant carriers specifically.

However, these medications may be an option for women who choose not to, or who cannot, undergo surgery. The potential harms of these drugs include menopausal symptoms, blood clots, stroke, increased risk of endometrial cancer (tamoxifen), and allergic reactions (raloxifene).

Both women in the general population, as well as those with harmful BRCA1 or BRCA2 variants, who have ever used oral contraceptives (birth control pills) have about a 50% lower risk of ovarian cancer than women who have never used oral contraceptives (37). Potential harms of oral contraceptives include increased risk of breast cancer, increased risk that a human papillomavirus (HPV) infection will become cervical cancer, and possible cardiovascular effects among older reproductive-age women.

What are the benefits of genetic testing for BRCA1 and BRCA2 variants?

There can be benefits to genetic testing, regardless of whether a person receives a positive or a negative result.

The potential benefits of a true negative result include a sense of relief regarding the future risk of cancer, learning that one's children are not at risk of inheriting the family's cancer susceptibility, and the possibility that special check-ups, tests, or risk-reducing surgeries may not be needed.

A positive test result may allow people to make informed decisions about their future health care, including taking steps to reduce their cancer risk.

What are the possible harms of genetic testing for BRCA1 and BRCA2 variants?

The direct medical harms of genetic testing are minimal, but knowledge of test results, whether positive or negative, may have harmful effects on a person’s emotions, social relationships, finances, and medical choices.

Dealing with uncertainty of an uninformative negative or a VUS test result is another potential harm. For this reason, it is important to have genetic counseling before undergoing genetic testing.

Results of genetic tests are normally included in a person’s medical records, particularly if a doctor or other health care provider has ordered the test or has been consulted about the test results. Therefore, people considering genetic testing must understand that their results may become known to other people or organizations that have legitimate, legal access to their medical records, such as their insurance company or employer, if their employer provides the patient’s health insurance as a benefit.

What are the treatment implications of having a harmful BRCA1 or BRCA2 variant for patients who have already developed cancer?

Because the BRCA1 and BRCA2 genes are involved in DNA repair, tumors with alterations in either gene are particularly sensitive to anticancer agents that act by damaging DNA, such as cisplatin (38).

A class of drugs called PARP inhibitors, which block the repair of DNA damage, have been found to arrest the growth of cancer cells that have harmful BRCA1 or BRCA2 variants. Four PARP inhibitors—olaparib [Lynparza], rucaparib [Rubraca], niraparib [Zejula], and talazoparib [Talzenna]—are approved by the FDA to treat certain cancers bearing harmful variants in BRCA1 or BRCA2. (In some cases, these are used whether or not a BRCA1 or BRCA2 mutation is present.)

Breast cancers with harmful BRCA1 variants are more likely to be "triple-negative cancers" (that is, the breast cancer cells do not have estrogen receptors, progesterone receptors, or large amounts of HER2/neu protein) than sporadic breast cancers or breast cancers with harmful BRCA2 variants. Triple-negative cancers are harder to treat and have a poorer prognosis than other types of breast cancers.

If someone has tumor genetic testing that reveals the presence of a harmful BRCA1 or BRCA2 variant in the tumor, they should consider having a germline genetic (blood) test to determine if the variant was inherited. Knowing if the variant was inherited is important for that individual to understand their risks to potentially develop other cancers in the future. It can also determine if other family members may be at risk of inheriting the harmful variant.

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Memorial Sloan-Kettering scientists uncover function of BRCA2 protein

BRCA2 was already known to be a tumor suppressor -- a protective protein that prevents the development of cancer -- but exactly how the protein does its job was not understood. Now MSKCC scientists have been able to map out the structure of the protein, showing that it interacts directly with DNA and helps to repair genetic damage. An inability to correct genetic damage leads to unstable chromosomes and often to cancer.

"If BRCA2 is altered or missing, it leads to a dangerous accumulation of genetic errors," said the study's senior author Nikola P. Pavletich, PhD, head of MSKCC's Laboratory of Structural Biology of Oncogenes and Tumor Suppressors and an investigator in the Howard Hughes Medical Institute. "By studying the normal function of BRCA2, we can understand how changes in the protein contribute to the development of cancer."

BRCA2 is an unusually large molecule, which has made it difficult for researchers to study. But Dr. Pavletich's team, including first author Haijuan Yang, found a way around that problem and was able to crystallize the protein. Those crystals were then bombarded with high-energy X-rays, a process called X-ray crystallography, and the diffraction patterns created by the X-rays were used to calculate the three-dimensional picture of the protein. This picture revealed that BRCA2 is similar in structure to other proteins known to bind DNA. The researchers then took the work a step further, showing that BRCA2 does indeed bind to DNA in special regions that are commonly found around broken DNA strands.

Researchers showed that BRCA2 participates in the repair of "double-strand" breaks: These breaks are a particularly lethal type of damage because if both strands of the DNA double helix break at the same time, cells can permanently lose genetic information. The structure revealed that BRCA2 binds the broken strands and enables the recovery of lost information via a process called homologous recombination -- in which the missing DNA is copied from another part of the cell.

"We are now a step closer to understanding this particular type of inherited breast and ovarian cancers," Dr. Pavletich said.

Mutations in the BRCA2 gene have been implicated in hereditary breast and ovarian cancers since 1995. The other gene commonly linked to hereditary breast and ovarian cancers is called BRCA1.

Researchers from the University of Texas Health Science Center in San Antonio also contributed to the study, which was funded by the National Institutes of Health, the Howard Hughes Medical Institute, the Dewitt Wallace Foundation, the Samuel and May Rudin Foundation, and the Arthur and Rochelle Belfer Foundation.

Memorial Sloan-Kettering Cancer Center is the world's oldest and largest institution devoted to prevention, patient care, research, and education in cancer. Our scientists and clinicians generate innovative approaches to better understand, diagnose, and treat cancer. Our specialists are leaders in biomedical research and in translating the latest research to advance the standard of cancer care worldwide.

Disclaimer: AAAS and EurekAlert! are not responsible for the accuracy of news releases posted to EurekAlert! by contributing institutions or for the use of any information through the EurekAlert system.


How Do BRCA Mutations Cause Cancer?

Genes are the body’s sets of genetic instructions. Mutations within BRCA genes cause them to not work as well as they should. “When BRCA genes are mutated or altered, cells are unable to repair themselves, causing an increased risk to develop specific types of cancer,” says Clayback.

Mutations in the two kinds of BRCA genes will affect patients’ risk differently.

BRCA1 mutations are associated with an increased risk for:

  • Breast cancer, including an aggressive form called Triple Negative Breast Cancer
  • Ovarian cancer
  • Pancreatic cancer
  • Prostate cancer

BRCA2 mutations are associated with an increased risk for:

  • Breast cancer
  • Ovarian cancer
  • Melanoma
  • Pancreatic cancer
  • Prostate cancer

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Citing Articles (38)

Panel A shows the pedigree of the family the arrow indicates the proband. Circles indicate female family members, and squares male family members the number 3 inside the square and circle indicates the numbers of brothers and sisters, respectively. The current ages are shown below the circles or squares. The slash denotes a deceased family member. Asterisks indicate the family members who were enrolled in the study, from whom the samples were obtained. Solid circles indicate the two sisters who had a normal female karyotype (46,XX), ovarian dysgenesis, microcephaly (head circumferences of 48.7 cm in family member III-2 when she was 19 years of age and 47.5 cm in family member III-4 when she was 14 years of age), and café au lait spots. The older sister (III-2) also had received a diagnosis of leukemia at 5 years of age. The half-solid square indicates the brother who died from acute promyelocytic leukemia at 13 years of age. The gray circle indicates fetal death. V1 denotes the BRCA2 c.7579delG variant, V2 the BRCA2 c.9693delA variant, and N normal. Panel B is a depiction of the BRCA2 protein. The RAD51-binding domain includes eight repeated motifs called BRC repeats (blue). The DNA-binding domain contains a helical domain (H, green), three oligonucleotide binding folds (OB, purple), and a tower domain (T, orange). The nuclear localization signal (NLS) is near the C-terminal of the protein (red). The red arrow indicates the position of V1 and the blue arrow the position of V2 on the BRCA2 protein. BRCA2 p.V2527X is predicted to lack most of the DNA-binding domain and the NLS. BRCA2 p.S3231fs16*, with a predicted truncation of 171 residues, retains these domains. Panel C shows chromosomal breakage in the peripheral lymphocytes obtained from the proband (III-2 [V1/V2]), the mother (II-1 [V1/N]), and an unrelated control (N/N). Representative chromosomal breaks (Br) are marked by red arrows. Triradial (Tra), quadriradial (Qra), and complex rearrangements (cRa) are marked by dashed red arrows. Panel D shows the effect of increasing exposure to mitomycin C on chromosomal breaks in the cells of the two affected sisters (III-2 and III-4), the mother (II-1), a healthy sister (III-3), and an unrelated control (N/N). Chromosomes were first exposed to mitomycin C at increasing concentrations of 0 nM, 150 nM, and 300 nM according to a standard protocol. Because of the large number of chromosomal breaks observed at the 150 nM and 300 nM concentrations, the chromosomes were also exposed to mitomycin C at concentrations of 50 nM and 100 nM. Asterisks indicate more than 100 breaks per cell. The P values in the bottom row are comparisons between the affected sisters and V1/N persons (the mother and a healthy sister), and the P values in the top row are comparisons between the affected sisters and a control (Table S3 in the Supplementary Appendix).

Panel A shows the results of the quantitative real-time reverse-transcriptase–polymerase-chain-reaction (RT-PCR) assays of BRCA2 transcripts. The bars represent the mean levels of BRCA2 RNA expression (shown as the percentage of wild-type) for each genotype from six RT-PCR assays performed in each person T bars indicate the standard deviations. The results show that BRCA2 expression was significantly lower in the cells of the affected sisters (III-2 and III-4 [V1/V2]) than in those of their unaffected relatives (II-1 and III-3 [V1/N] and II-2 and III-5 [V2/N]) and of unrelated controls (N/N). NS denotes not significant. Panel B shows the quantification of Western blots of BRCA2 protein, with the use of the ImageJ image processing program from the National Institutes of Health representative images are shown in Fig. S2 in the Supplementary Appendix. Lysates were obtained from lymphoblasts of all the enrolled family members and unrelated controls. The bars represent the mean levels of BRCA2 protein expression (shown as the percentage of wild-type) from six Western blot analyses performed for each person T bars indicate the standard deviations. The results show that the levels of BRCA2 protein expression differed significantly among the compound heterozygotes (V1/V2), the heterozygotes (V1/N and V2/N), and the unrelated controls (N/N). The levels of BRCA2 protein expression in the samples from the compound heterozygotes were lower — by a factor of more than seven — than those in the samples from the controls who had no BRCA2 mutation. Panel C shows the effect of DNA damage induction by exposure to neocarzinostatin. Fibroblasts from an unrelated control (i though iv), from the mother (v through viii), and from the proband (ix through xii) were stained by immunofluorescence. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (blue [i, v, and ix]). γ-H2AX was detected with anti-phospho-Histone H2A.X (JBW301, Millipore) (green [ii, vi, and x]). RAD51 was detected with anti-RAD51 (N1C2, GeneTex) (red [iii, vii, and xi]). Merged staining of DAPI, γ-H2AX, and RAD51 is shown in iv, viii, and xii. Cells were visualized with the use of an Olympus fluorescent microscope with a ×60 objective lens. Panel D shows quantification of RAD51 foci formation after neocarzinostatin exposure (shown in Panel C). Bars represent the proportion of RAD51-positive γ-H2AX foci in mutant and nonmutant cells. T bars indicate the standard deviations. Foci were counted by a person who was unaware of the cellular genotype. The number of RAD51-positive foci was lower in the compound heterozygote cells (V1/V2) than in the cells from a carrier (V1/N) and in normal cells (N/N) by a factor of at least six.

Panel A shows that homozygous deletion of the drosophila orthologue of BRCA2 leads to sterility in female and male flies. Female and male Dmbrca2 −/− flies were crossed with wild-type controls to evaluate egg and progeny production. The results are presented as the mean daily number of eggs and progeny produced by each of the crosses indicated below the x axis (as averaged across three to five replicates per cross) T bars indicate the standard deviations. Eggs and progeny were counted daily for 3 days. The mean daily number of eggs laid by Dmbrca2-null female flies (−/− female) crossed with wild-type control male flies (yw male) was lower than the mean daily number of eggs laid by wild-type female flies (yw female) crossed with wild-type male flies (yw male) by a factor of 19.5 (mean daily number, 22 vs. 428, P=0.001). In comparison with the mean daily number of eggs hatched when wild-type female flies were crossed with wild-type male flies (388 of 428 eggs hatched [91%]), only 1 of a total of 214 eggs hatched among the Dmbrca2-null female flies that were crossed with wild-type male flies and 1 of a total of 2116 eggs hatched among the Dmbrca2-null male flies that were crossed with wild-type female flies. Panel B shows the percentages of abnormal ovary and testes phenotypes in Dmbrca2-null flies. Among 62 female flies tested, most ovarioles (69%) were severely dysgenic, 27% were moderately abnormal, and 4% were mildly abnormal. Among 59 male flies tested, nearly all testes (92%) were severely or moderately abnormal 8% were mildly abnormal. Panel C shows the morphologic features and immunostaining of the drosophila ovaries and testes. The panels on the left for the wild-type control flies show normal, healthy morphologic features of both the ovaries and testes, as compared with the panels on the left for the Dmbrca2-null flies that show shrunken, underdeveloped ovaries and testes. The panels on the right for both the wild-type control flies and the Dmbrca2-null flies show the results of immunostaining. Nuclear DNA is green, actin is blue, and cleaved caspase-3 (indicating apoptosis) is red. Dmbrca2 +/− (Fig. S4 in the Supplementary Appendix) and wild-type control flies had normal ovarioles, with normal nuclear DNA and actin and no staining of cleaved caspase-3. Dmbrca2-null flies had ovarioles with disintegrated egg chambers, as indicated by nuclear DNA condensation, disappearance of actin structures, and marked staining of cleaved caspase-3. Dmbrca2 +/− (Fig. S4 in the Supplementary Appendix) and wild-type control flies had normal testes, with normal appearance of cleaved caspase-3 in differentiating spermatids. Dmbrca2-null flies had abnormal testes without differentiation of spermatids and with abnormal appearance of cleaved caspase-3.


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