Biallelic mutations in BRCA1 cause a new Fanconi anemia subtype

Abstract

Deficiency in BRCA-dependent DNA interstrand crosslink (ICL) repair is intimately connected to breast cancer susceptibility and to the rare developmental syndrome Fanconi anemia. Bona fide Fanconi anemia proteins, BRCA2 (FANCD1), PALB2 (FANCN), and BRIP1 (FANCJ), interact with BRCA1 during ICL repair. However, the lack of detailed phenotypic and cellular characterization of a patient with biallelic BRCA1 mutations has precluded assignment of BRCA1 as a definitive Fanconi anemia susceptibility gene. Here, we report the presence of biallelic BRCA1 mutations in a woman with multiple congenital anomalies consistent with a Fanconi anemia-like disorder and breast cancer at age 23. Patient cells exhibited deficiency in BRCA1 and RAD51 localization to DNA-damage sites, combined with radial chromosome formation and hypersensitivity to ICL-inducing agents. Restoration of these functions was achieved by ectopic introduction of a BRCA1 transgene. These observations provide evidence in support of BRCA1 as a new Fanconi anemia gene (FANCS).

Significance: We establish that biallelic BRCA1 mutations cause a distinct FA-S, which has implications for risk counselling in families where both parents harbor BRCA1 mutations. The genetic basis of hereditary cancer susceptibility syndromes provides diagnostic information, insights into treatment strategies, and more accurate recurrence risk counseling to families.

Figure 1

An individual with early onset breast cancer, clinical features of Fanconi anemia, and biallelic BRCA1 mutations. A, The affected individual at 3 and 23 years old showing hypertelorism, epicanthal folds, ptosis, strabismus, blepharophimosis, broad nasal bridge and nasal tip, and proximally inserted thumbs. B, Sequencing of BRCA1 in family members demonstrated that both BRCA1 alleles in the proband were inherited from the heterozygous parents. III: 2, proband; III: 3, brother; II: 3, father; II: 4, mother

Figure 2

Pedigree and LOH analysis of proband breast cancer. A, Pedigree with all known cancer diagnoses in the family. B, LOH analysis by Sanger sequencing of genomic DNA extracted from paraffin-embedded tumor blocks. Breast tumor was derived from the proband (III: 2), and ovarian cancer was derived from the mother (II: 4). Cancer cells and control non-cancer cells were isolated and enriched by laser capture microdissection (LCM).

Figure 3

Characterization of BRCA1 function in the proband derived cells. A, Immunoblot for BRCA1 from fibroblast cell lysates shows reduced BRCA1 expression in the proband compared to the heterozygous sibling. KFB14-1, fibroblast cells derived from the proband III: 2. KFB14-2, fibroblast cells derived from the sibling III: 3. B, RT-PCR followed by cDNA sequencing showed approximately 80% of BRCA1 mRNA in KFB14-1 cells contains the p.Arg1699Trp mutation, while approximately 50% of BRCA1 mRNA in KFB14-2 cells are wild type. C, Examples of metaphase chromosomes from peripheral blood lymphocytes (treated with 0.2 µg/ml diepoxybutane (DEB)) and fibroblast KFB14-1 cells (treated with 10ng/ml MMC or 0.1 µg/ml DEB). Arrows indicate radial chromosomes or chromatid breaks. D, Quantification of abnormal chromosomes in chromosome breakage tests from peripheral blood samples of the proband and sibling.

Figure 4

Defective BRCA1 DNA repair function in proband derived cells. A, Immunofluorescence showing reduced BRCA1 and Rad51 foci formation at IR induced damage sites. Complementation of KFB14-1 cells with BRCA1Δ512-1283 (KFB14-1/BRCA1Δ) restored BRCA1 and Rad51 foci formation. B, Quantification of the percentage of fibroblasts with BRCA1 and γH2AX colocalization and/or percentage of Cyclin A positive cells with Rad51 foci. KFB14-1, KFB14-2 and KFB14-1/BRCA1Δ cells were exposed to 10 (Gy) ionizing radiation or 0.5 µM MMC for 24 hrs. Cells with ≥ 5 foci are scored as positive cells. C,D, Cell survival (MTT) assay showing the survival curve of fibroblasts treated with PARP inhibitor Olaparib (C) or MMC (D). KFB14-1, KFB14-2 and KFB14-1/BRCA1Δ cells were treated with Olaparib or MMC at the indicated concentrations for 72 hrs. BRCA1Δ512-1283 successfully restored resistance to Olaparib or MMC in KFB14-1 cells. Error bars represent standard error of the mean from 3 independent experiments.