Biallelic germline BRCA1 mutations in a patient with early onset breast cancer, mild Fanconi anemia-like phenotype, and no chromosome fragility

Abstract

Background: Biallelic BRCA1 mutations are regarded either embryonically lethal or to cause Fanconi anemia (FA), a genomic instability syndrome characterized by bone marrow failure, developmental abnormalities, and cancer predisposition. We report biallelic BRCA1 mutations c.181T > G (p.Cys61Gly) and c.5096G > A (p.Arg1699Gln) in a woman with breast cancer diagnosed at the age of 30 years. The common European founder mutation p.Cys61Gly confers high cancer risk, whereas the deleterious p.Arg1699Gln is hypomorphic and was suggested to confer intermediate cancer risk.

Methods and results: Aside from significant toxicity from chemotherapy, the patient showed mild FA-like features (e.g., short stature, microcephaly, skin hyperpigmentation). Chromosome fragility, a hallmark of FA patient cells, was not present in patient-derived peripheral blood lymphocytes. We demonstrated that the p.Arg1699Gln mutation impairs DNA double-strand break repair, elevates RAD51 foci levels at baseline, and compromises BRCA1 protein function in protecting from replication stress. Although the p.Arg1699Gln mutation compromises BRCA1 function, the residual activity of the p.Arg1699Gln allele likely prevents from chromosome fragility and a more severe FA phenotype.

Conclusion: Our data expand the clinical spectrum associated with biallelic BRCA1 mutations, ranging from embryonic lethality to a mild FA-like phenotype and no chromosome fragility.

Keywords: Fanconi anemia; biallelic BRCA1; early onset breast cancer; p.Arg1699Gln.

Figure 1

Pedigree and germline mutation status. (a) Cancer family history of the index patient (III‐1, marked with an arrow), including age at first diagnosis. BC = breast cancer; PC = prostate cancer; y = years. The index patient's mother (II‐2) had no personal history of cancer at the age of 60 years. The maternal grandfather of the index patient was diagnosed with prostate cancer at the age of 80 years. The index patient's father (II‐1) had no personal history of cancer at the age of 59 years. The paternal grandmother of the index patient was diagnosed with breast cancer at the age of 42 years and died at the age of 87 years. No other cancer cases were reported in the family. (b) Sanger sequencing electropherograms show BRCA1 genotypes (BRCA1 reference transcript NM_007294.3) of the index patient III‐1 and both parents (II‐1, II‐2). Red arrows mark the presence of a single nucleotide substitution

Figure 2

(a) Analysis of DSB‐induced HR activities in individuals with wild‐type, mono‐ or biallelic mutations in the BRCA1. To measure the repair of DSBs by HR, we used the EGFP‐based reporter substrate HR‐EGFP/5'EGFP consisting of mutated EGFP genes with an I‐SceI endonuclease recognition sequence replacing 4 bp (HR‐EGFP) and a 3' truncation (5'EGFP), respectively. The DSB repair substrate for the determination of HR frequencies is schematically drawn on top (Akyuz et al., 2002). I‐SceI recognition sequence, red triangle; cross, truncating mutation; white bars, mutated EGFP genes; dark green bars, reconstituted EGFP; blue bar, spacer sequence; gray bar with kinked arrow, transcriptional promoter; scissors, I‐SceI endonuclease. HR measurements were performed 24 hr following transfection of PBLs (left diagram) or LCLs (right diagram) from individuals with BRCA1 (p.Arg1699Gln) or BRCA1 (p.Cys61Gly/p.Arg1699Gln) with HR‐EGFP/5´EGFP substrate plus I‐SceI expression plasmid for substrate cleavage. Measurements for PBLs with wild‐type BRCA1 were obtained from one (left diagram) and two (right diagram) independent control individuals. Percentages of EGFP‐positive live cells were normalized to the individually determined transfection efficiencies for HR frequency calculations. Mean values of wild‐type controls were set to 100% (absolute mean frequency: 0.2%). Mean values and standard errors of the mean (SEM) from 3 to 5 measurements are shown. (b) Immunofluorescence microscopic analysis of DNA damage and RAD51 nucleofilament formation. Nuclear foci of the DNA damage marker γH2AX (left diagram) and of RAD51 (right diagram), indicating nucleofilament formation, were analyzed in PBLs from individuals with wild‐type and mutated BRCA1 (p.Arg1699Gln, p.Cys61Gly/p.Arg1699Gln) 1 and 6 hr after treatment with 2 Gy ionizing radiation (IR) and in untreated samples. Immunolabeled foci were scored by automated quantification from 97 to 115 nuclei per sample. Mean foci/cell and SEM are graphically shown on the left side. Statistically significant differences were determined using nonparametric Mann–Whitney test for unpaired samples with the software GraphPad Prism version 7.03. *p < .05; **p < .01; ***p < .001; ****p < .0001. Representative images of green γH2AX and red RAD51 foci in DAPI‐stained nuclei (blue) 6 hr post‐IR are shown on the right side. The size bar indicates 5 µm. (c) Analysis of replication fork protection following stalling. Single DNA fiber analysis was performed as schematically outlined in the sketch on top. Representative fibers ∓ HU are shown for the patient carrying biallelic BRCA1 mutations (p.Cys61Gly/p.Arg1699Gln). The size bar indicates 5 µm. CldU track length distributions of CldU‐ and IdU‐positive DNA fibers from PBLs derived from individuals with wild‐type and mutated BRCA1 (p.Arg1699Gln, p.Cys61Gly/p.Arg1699Gln) were determined in the presence of HU, indicating replication stalling (right) or absence of HU for unperturbed replication. Mean values were calculated from fiber track lengths of 106–172 single fibers in each sample. Statistically significant differences between PBLs from individuals with wild‐type and mutated BRCA1 (p.Cys61Gly/p.Arg1699Gln) were calculated using Dunn's test. ***p < .001; ****p < .0001