Biallelic mutations in PALB2 cause Fanconi anemia subtype FA-N and predispose to childhood cancer

PALB2 was recently identified as a nuclear binding partner of BRCA2. Biallelic BRCA2 mutations cause Fanconi anemia subtype FA-D1 and predispose to childhood malignancies. We identified pathogenic mutations in PALB2 (also known as FANCN) in seven families affected with Fanconi anemia and cancer in early childhood, demonstrating that biallelic PALB2 mutations cause a new subtype of Fanconi anemia, FA-N, and, similar to biallelic BRCA2 mutations, confer a high risk of childhood cancer.     

Truncating mutations in the Fanconi anemia J gene BRIP1 are low-penetrance breast cancer susceptibility alleles

We identified constitutional truncating mutations of the BRCA1-interacting helicase BRIP1 in 9/1,212 individuals with breast cancer from BRCA1/BRCA2 mutation-negative families but in only 2/2,081 controls (P = 0.0030), and we estimate that BRIP1 mutations confer a relative risk of breast cancer of 2.0 (95% confidence interval = 1.2-3.2, P = 0.012). Biallelic BRIP1 mutations were recently shown to cause Fanconi anemia complementation group J. Thus, inactivating truncating mutations of BRIP1, similar to those in BRCA2, cause Fanconi anemia in biallelic carriers and confer susceptibility to breast cancer in monoallelic carriers.     

Fanconi anemia is associated with a defect in the BRCA2 partner PALB2

The Fanconi anemia and BRCA networks are considered interconnected, as BRCA2 gene defects have been discovered in individuals with Fanconi anemia subtype D1. Here we show that a defect in the BRCA2-interacting protein PALB2 is associated with Fanconi anemia in an individual with a new subtype. PALB2-deficient cells showed hypersensitivity to cross-linking agents and lacked chromatin-bound BRCA2; these defects were corrected upon ectopic expression of PALB2 or by spontaneous reversion.     

Low-penetrance susceptibility to breast cancer due to CHEK2(*)1100delC in noncarriers of BRCA1 or BRCA2 mutations

Mutations in BRCA1 and BRCA2 confer a high risk of breast and ovarian cancer, but account for only a small fraction of breast cancer susceptibility. To find additional genes conferring susceptibility to breast cancer, we analyzed CHEK2 (also known as CHK2), which encodes a cell-cycle checkpoint kinase that is implicated in DNA repair processes involving BRCA1 and p53 (refs 3,4,5). We show that CHEK2(*)1100delC, a truncating variant that abrogates the kinase activity, has a frequency of 1.1% in healthy individuals. However, this variant is present in 5.1% of individuals with breast cancer from 718 families that do not carry mutations in BRCA1 or BRCA2 (P = 0.00000003), including 13.5% of individuals from families with male breast cancer (P = 0.00015). We estimate that the CHEK2(*)1100delC variant results in an approximately twofold increase of breast cancer risk in women and a tenfold increase of risk in men. By contrast, the variant confers no increased cancer risk in carriers of BRCA1 or BRCA2 mutations. This suggests that the biological mechanisms underlying the elevated risk of breast cancer in CHEK2 mutation carriers are already subverted in carriers of BRCA1 or BRCA2 mutations, which is consistent with participation of the encoded proteins in the same pathway.     

ATM mutations that cause ataxia-telangiectasia are breast cancer susceptibility alleles

We screened individuals from 443 familial breast cancer pedigrees and 521 controls for ATM sequence variants and identified 12 mutations in affected individuals and two in controls (P = 0.0047). The results demonstrate that ATM mutations that cause ataxia-telangiectasia in biallelic carriers are breast cancer susceptibility alleles in monoallelic carriers, with an estimated relative risk of 2.37 (95% confidence interval (c.i.) = 1.51-3.78, P = 0.0003). There was no evidence that other classes of ATM variant confer a risk of breast cancer.     

A novel ubiquitin ligase is deficient in Fanconi anemia

Fanconi anemia is a recessively inherited disease characterized by congenital defects, bone marrow failure and cancer susceptibility. Cells from individuals with Fanconi anemia are highly sensitive to DNA-crosslinking drugs, such as mitomycin C (MMC). Fanconi anemia proteins function in a DNA damage response pathway involving breast cancer susceptibility gene products, BRCA1 and BRCA2 (refs. 1,2). A key step in this pathway is monoubiquitination of FANCD2, resulting in the redistribution of FANCD2 to nuclear foci containing BRCA1 (ref. 3). The underlying mechanism is unclear because the five Fanconi anemia proteins known to be required for this ubiquitination have no recognizable ubiquitin ligase motifs. Here we report a new component of a Fanconi anemia protein complex, called PHF9, which possesses E3 ubiquitin ligase activity in vitro and is essential for FANCD2 monoubiquitination in vivo. Because PHF9 is defective in a cell line derived from an individual with Fanconi anemia, we conclude that PHF9 (also called FANCL) represents a novel Fanconi anemia complementation group (FA-L). Our data suggest that PHF9 has a crucial role in the Fanconi anemia pathway as the likely catalytic subunit required for monoubiquitination of FANCD2.     

A common variant in BRCA2 is associated with both breast cancer risk and prenatal viability

Inherited mutations in the gene BRCA2 predispose carriers to early onset breast cancer, but such mutations account for fewer than 2% of all cases in East Anglia. It is likely that low penetrance alleles explain the greater part of inherited susceptibility to breast cancer; polymorphic variants in strongly predisposing genes, such as BRCA2, are candidates for this role. BRCA2 is thought to be involved in DNA double strand break-repair. Few mice in which Brca2 is truncated survive to birth; of those that do, most are male, smaller than their normal littermates and have high cancer incidence. Here we show that a common human polymorphism (N372H) in exon 10 of BRCA2 confers an increased risk of breast cancer: the HH homozygotes have a 1.31-fold (95% CI, 1.07-1.61) greater risk than the NN group. Moreover, in normal female controls of all ages there is a significant deficiency of homozygotes compared with that expected from Hardy-Weinberg equilibrium, whereas in males there is an excess of homozygotes: the HH group has an estimated fitness of 0.82 in females and 1.38 in males. Therefore, this variant of BRCA2 appears also to affect fetal survival in a sex-dependent manner.     

The BRCA1-interacting helicase BRIP1 is deficient in Fanconi anemia

Seven Fanconi anemia-associated proteins (FANCA, FANCB, FANCC, FANCE, FANCF, FANCG and FANCL) form a nuclear Fanconi anemia core complex that activates the monoubiquitination of FANCD2, targeting FANCD2 to BRCA1-containing nuclear foci. Cells from individuals with Fanconi anemia of complementation groups D1 and J (FA-D1 and FA-J) have normal FANCD2 ubiquitination. Using genetic mapping, mutation identification and western-blot data, we identify the defective protein in FA-J cells as BRIP1 (also called BACH1), a DNA helicase that is a binding partner of the breast cancer tumor suppressor BRCA1.     

Mutations of the SLX4 gene in Fanconi anemia

Fanconi anemia is a rare recessive disorder characterized by genome instability, congenital malformations, progressive bone marrow failure and predisposition to hematologic malignancies and solid tumors. At the cellular level, hypersensitivity to DNA interstrand crosslinks is the defining feature in Fanconi anemia. Mutations in thirteen distinct Fanconi anemia genes have been shown to interfere with the DNA-replication-dependent repair of lesions involving crosslinked DNA at stalled replication forks. Depletion of SLX4, which interacts with multiple nucleases and has been recently identified as a Holliday junction resolvase, results in increased sensitivity of the cells to DNA crosslinking agents. Here we report the identification of biallelic SLX4 mutations in two individuals with typical clinical features of Fanconi anemia and show that the cellular defects in these individuals' cells are complemented by wildtype SLX4, demonstrating that biallelic mutations in SLX4 (renamed here as FANCP) cause a new subtype of Fanconi anemia, Fanconi anemia-P.     

Synergistic tumor suppressor activity of BRCA2 and p53 in a conditional mouse model for breast cancer.

Inheritance of one defective BRCA2 allele predisposes humans to breast cancer. To establish a mouse model for BRCA2-associated breast cancer, we generated mouse conditional mutants with BRCA2 and/or p53 inactivated in various epithelial tissues, including mammary-gland epithelium. Although no tumors arose in mice carrying conditional Brca2 alleles, mammary and skin tumors developed frequently in females carrying conditional Brca2 and Trp53 alleles. The presence of one wildtype Brca2 allele resulted in a markedly delayed tumor formation; loss of the wildtype Brca2 allele occurred in a subset of these tumors. Our results show that inactivation of BRCA2 and of p53 combine to mediate mammary tumorigenesis, and indicate that disruption of the p53 pathway is pivotal in BRCA2-associated breast cancer.     

Recurrent gross mutations of the PTEN tumor suppressor gene in breast cancers with deficient DSB repair

Basal-like breast cancer (BBC) is a subtype of breast cancer with poor prognosis. Inherited mutations of BRCA1, a cancer susceptibility gene involved in double-strand DNA break (DSB) repair, lead to breast cancers that are nearly always of the BBC subtype; however, the precise molecular lesions and oncogenic consequences of BRCA1 dysfunction are poorly understood. Here we show that heterozygous inactivation of the tumor suppressor gene Pten leads to the formation of basal-like mammary tumors in mice, and that loss of PTEN expression is significantly associated with the BBC subtype in human sporadic and BRCA1-associated hereditary breast cancers. In addition, we identify frequent gross PTEN mutations, involving intragenic chromosome breaks, inversions, deletions and micro copy number aberrations, specifically in BRCA1-deficient tumors. These data provide an example of a specific and recurrent oncogenic consequence of BRCA1-dependent dysfunction in DNA repair and provide insight into the pathogenesis of BBC with therapeutic implications. These findings also argue that obtaining an accurate census of genes mutated in cancer will require a systematic examination for gross gene rearrangements, particularly in tumors with deficient DSB repair.     

SLX4, a coordinator of structure-specific endonucleases, is mutated in a new Fanconi anemia subtype

DNA interstrand crosslink repair requires several classes of proteins, including structure-specific endonucleases and Fanconi anemia proteins. SLX4, which coordinates three separate endonucleases, was recently recognized as an important regulator of DNA repair. Here we report the first human individuals found to have biallelic mutations in SLX4. These individuals, who were previously diagnosed as having Fanconi anemia, add SLX4 as an essential component to the FA-BRCA genome maintenance pathway.     

A human ortholog of archaeal DNA repair protein Hef is defective in Fanconi anemia complementation group M

Fanconi anemia is a genetic disease characterized by genomic instability and cancer predisposition. Nine genes involved in Fanconi anemia have been identified; their products participate in a DNA damage-response network involving BRCA1 and BRCA2 (refs. 2,3). We previously purified a Fanconi anemia core complex containing the FANCL ubiquitin ligase and six other Fanconi anemia-associated proteins. Each protein in this complex is essential for monoubiquitination of FANCD2, a key reaction in the Fanconi anemia DNA damage-response pathway. Here we show that another component of this complex, FAAP250, is mutant in individuals with Fanconi anemia of a new complementation group (FA-M). FAAP250 or FANCM has sequence similarity to known DNA-repair proteins, including archaeal Hef, yeast MPH1 and human ERCC4 or XPF. FANCM can dissociate DNA triplex, possibly owing to its ability to translocate on duplex DNA. FANCM is essential for monoubiquitination of FANCD2 and becomes hyperphosphorylated in response to DNA damage. Our data suggest an evolutionary link between Fanconi anemia-associated proteins and DNA repair; FANCM may act as an engine that translocates the Fanconi anemia core complex along DNA.     

X-linked inheritance of Fanconi anemia complementation group B

Fanconi anemia is an autosomal recessive syndrome characterized by diverse clinical symptoms, hypersensitivity to DNA crosslinking agents, chromosomal instability and susceptibility to cancer. Fanconi anemia has at least 11 complementation groups (A, B, C, D1, D2, E, F, G, I, J, L); the genes mutated in 8 of these have been identified. The gene BRCA2 was suggested to underlie complementation group B, but the evidence is inconclusive. Here we show that the protein defective in individuals with Fanconi anemia belonging to complementation group B is an essential component of the nuclear protein 'core complex' responsible for monoubiquitination of FANCD2, a key event in the DNA-damage response pathway associated with Fanconi anemia and BRCA. Unexpectedly, the gene encoding this protein, FANCB, is localized at Xp22.31 and subject to X-chromosome inactivation. X-linked inheritance has important consequences for genetic counseling of families with Fanconi anemia belonging to complementation group B. Its presence as a single active copy and essentiality for a functional Fanconi anemia-BRCA pathway make FANCB a potentially vulnerable component of the cellular machinery that maintains genomic integrity.     

BRCA1 regulates the G2/M checkpoint by activating Chk1 kinase upon DNA damage

The breast cancer tumor-suppressor gene, BRCA1, encodes a protein with a BRCT domain-a motif that is found in many proteins that are implicated in DNA damage response and in genome stability. Phosphorylation of BRCA1 by the DNA damage-response proteins ATM, ATR and hCds1/Chk2 changes in response to DNA damage and at replication-block checkpoints. Although cells that lack BRCA1 have an abnormal response to DNA damage, the exact role of BRCA1 in this process has remained unclear. Here we show that BRCA1 is essential for activating the Chk1 kinase that regulates DNA damage-induced G2/M arrest. Thus, BRCA1 controls the expression, phosphorylation and cellular localization of Cdc25C and Cdc2/cyclin B kinase-proteins that are crucial for the G2/M transition. We show that BRCA1 regulates the expression of both Wee1 kinase, an inhibitor of Cdc2/cyclin B kinase, and the 14-3-3 family of proteins that sequesters phosphorylated Cdc25C and Cdc2/cyclin B kinase in the cytoplasm. We conclude that BRCA1 regulates key effectors that control the G2/M checkpoint and is therefore involved in regulating the onset of mitosis.     

Germline mutations in RAD51D confer susceptibility to ovarian cancer

Recently, RAD51C mutations were identified in families with breast and ovarian cancer. This observation prompted us to investigate the role of RAD51D in cancer susceptibility. We identified eight inactivating RAD51D mutations in unrelated individuals from 911 breast-ovarian cancer families compared with one inactivating mutation identified in 1,060 controls (P = 0.01). The association found here was principally with ovarian cancer, with three mutations identified in the 59 pedigrees with three or more individuals with ovarian cancer (P = 0.0005). The relative risk of ovarian cancer for RAD51D mutation carriers was estimated to be 6.30 (95% CI 2.86-13.85, P = 4.8 × 10(-6)). By contrast, we estimated the relative risk of breast cancer to be 1.32 (95% CI 0.59-2.96, P = 0.50). These data indicate that RAD51D mutation testing may have clinical utility in individuals with ovarian cancer and their families. Moreover, we show that cells deficient in RAD51D are sensitive to treatment with a PARP inhibitor, suggesting a possible therapeutic approach for cancers arising in RAD51D mutation carriers.     

Germline BAP1 mutations predispose to malignant mesothelioma

Because only a small fraction of asbestos-exposed individuals develop malignant mesothelioma, and because mesothelioma clustering is observed in some families, we searched for genetic predisposing factors. We discovered germline mutations in the gene encoding BRCA1 associated protein-1 (BAP1) in two families with a high incidence of mesothelioma, and we observed somatic alterations affecting BAP1 in familial mesotheliomas, indicating biallelic inactivation. In addition to mesothelioma, some BAP1 mutation carriers developed uveal melanoma. We also found germline BAP1 mutations in 2 of 26 sporadic mesotheliomas; both individuals with mutant BAP1 were previously diagnosed with uveal melanoma. We also observed somatic truncating BAP1 mutations and aberrant BAP1 expression in sporadic mesotheliomas without germline mutations. These results identify a BAP1-related cancer syndrome that is characterized by mesothelioma and uveal melanoma. We hypothesize that other cancers may also be involved and that mesothelioma predominates upon asbestos exposure. These findings will help to identify individuals at high risk of mesothelioma who could be targeted for early intervention.     

Adaptive evolution of the tumour suppressor BRCA1 in humans and chimpanzees. Australian Breast Cancer Family Study

Mutations in BRCA1 (ref. 1) confer an increased risk of female breast cancer. In a genome-wide scan of linkage disequilibrium (LD), a high level of LD was detected among microsatellite markers flanking BRCA1 (ref. 3), raising the prospect that positive natural selection may have acted on this gene. We have used the predictions of evolutionary genetic theory to investigate this further. Using phylogeny-based maximum likelihood analysis of the BRCA1 sequences from primates and other mammals, we found that the ratios of replacement to silent nucleotide substitutions on the human and chimpanzee lineages were not different from one another (P=0.8), were different from those of other primate lineages (P=0.004) and were greater than 1 (P=0.04). This is consistent with the historic occurrence of positive darwinian selection pressure on the BRCA1 protein in the human and chimpanzee lineages. Analysis of genetic variation in a sample of female Australians of Northern European origin showed evidence for Hardy-Weinberg (HW) disequilibrium at polymorphic sites in BRCA1, consistent with the possibility that natural selection is affecting genotype frequencies in modern Europeans. The clustering of between-species variation in the region of the gene encoding the RAD51-interaction domain of BRCA1 suggests the maintenance of genomic integrity as a possible target of selection.     

NAD(P)H:quinone oxidoreductase 1 NQO1*2 genotype (P187S) is a strong prognostic and predictive factor in breast cancer

NQO1 guards against oxidative stress and carcinogenesis and stabilizes p53. We find that a homozygous common missense variant (NQO1(*)2, rs1800566(T), NM_000903.2:c.558C>T) that disables NQO1 strongly predicts poor survival among two independent series of women with breast cancer (P = 0.002, N = 1,005; P = 0.005, N = 1,162), an effect particularly evident after anthracycline-based adjuvant chemotherapy with epirubicin (P = 7.52 x 10(-6)) and in p53-aberrant tumors (P = 6.15 x 10(-5)). Survival after metastasis was reduced among NQO1(*)2 homozygotes, further implicating NQO1 deficiency in cancer progression and treatment resistance. Consistently, response to epirubicin was impaired in NQO1(*)2-homozygous breast carcinoma cells in vitro, reflecting both p53-linked and p53-independent roles of NQO1. We propose a model of defective anthracycline response in NQO1-deficient breast tumors, along with increased genomic instability promoted by elevated reactive oxygen species (ROS), and suggest that the NQO1 genotype is a prognostic and predictive marker for breast cancer.