Biallelic Germline BRCA1 Frameshift Mutations Associated with Isolated Diminished Ovarian Reserve

Abstract

The use of next-generation sequencing (NGS) has recently enabled the discovery of genetic causes of primary ovarian insufficiency (POI) with high genetic heterogeneity. In contrast, the causes of diminished ovarian reserve (DOR) remain poorly understood. Here, we identified by NGS and whole exome sequencing (WES) the cause of isolated DOR in a 14-year-old patient. Two frameshift mutations in BRCA1 (NM_007294.4) were found: in exon 8 (c.470_471del; p.Ser157Ter) and in exon 11 (c.791_794del, p.Ser264MetfsTer33). Unexpectedly, the patient presented no signs of Fanconi anemia (FA), i.e., no developmental abnormalities or indications of bone marrow failure. However, high chromosomal fragility was found in the patient's cells, consistent with an FA diagnosis. RT-PCR and Western-blot analysis support the fact that the c. 791_794del BRCA1 allele is transcribed and translated into a shorter protein (del11q), while no expression of the full-length BRCA1 protein was found. DNA damage response (DDR) studies after genotoxic agents demonstrate normal activation of the early stages of the DDR and FANC/BRCA pathway. This is consistent with the maintenance of residual repair activity for the del11q BRCA1 isoform. Our observation is the first implication of bi-allelic BRCA1 mutations in isolated ovarian dysfunction or infertility in humans, without clinical signs of FA, and highlights the importance of BRCA1 in ovarian development and function.

Keywords: BRCA1 mutation; DNA repair; Fanconi anemia; diminished ovarian reserve; genetic counseling; meiosis; primary ovarian insufficiency.

Figure 1

Clinical feature of the family of the proband. Cancer family history including age at the first diagnostic is reported. St. = stomach cancer, Ov. = ovarian cancer, Blad. = bladder cancer, Col. = colorectal cancer, TBT. = temporal benign tumor, Adreno. = adrenocortical adenoma.

Figure 2

Confirmation of the two BRCA1 variants identified in the patient by Sanger Sequencing. The two variants in the patient were confirmed by Sanger sequencing as shown by the two electropherograms centered around the first variant (c.470_471del; p.Ser157Ter) located in exon 8 (left panel) and the second variant (c.791_794del, p.Ser264MetfsTer33) located in exon 11 (right panel). The deleted nucleotides (two and four nucleotides respectively) are highlighted by a red rectangle.

Figure 3

Chromosomal fragility in response to mitomycin C on metaphase chromosomes. Chromosomal breakages and radial figures are shown, respectively, by red and green arrows.

Figure 4

Survival curve after genotoxic treatment. (a) Survival curve after mitomycin C (MMC) and (b) Olaparib treatment during the 3 days. (n = 3 for MMC tests; mean ± SEM; GraphPad unpaired t test *** p < 0.001 and n = 1 for Olaparib test).

Figure 5

BRCA1 transcripts analysis. (a) Partial genomic organization of BRCA1 with both mutations identified in the patient’s DNA. The dashed line at BRCA1 c.787 indicate an alternative splice site that yields an in-frame truncated transcript, BRCA1 del11q [35]. (b) Localization of the primers and size of the amplicons generated to distinguish the full-length (FL) and the del11q BRCA1 transcripts. (c) BRCA1 transcripts: RT-PCR from lymphoblasts cells of a control (WT) and the patient using three pairs of primers amplifying the reference transcript (FL) or the del11q isoform. (d) Relative quantity of BRCA1 transcript isoforms from three replicate experiments with two different amplicons for the full-length and del11q transcripts. (mean ± SEM).

Figure 6

BRCA1 protein analysis. (a) Total protein extract from immortalized lymphoblasts from a control (WT) or the patient were loaded on 3–8% Tris-acetate SDS-PAGE gel and revealed with an anti-BRCA1 antibody recognizing the C terminal extremity. MCM7 is an internal loading control. (b) Relative amounts of BRCA1 protein isoforms from replicate experiments. (n = 7; mean ± SEM).

Figure 7

DDR and FANC/BRCA pathway activation after genotoxic agents’ overnight treatment. (a) Detection of DSB signaling by γ-H2AX, DNA resection revealed by p-RPA32 expression (p-RPA/RPA total) in patient and control cells after overnight treatment. (b) FANC/BRCA pathway activation revealed by FANCD2 monoubiquitination. (Aph: Aphidicolin at 0.6 µM; HU: Hydroxyurea at 5 mM or MMC: mitomycin C at 200 ng/mL).

Figure 8

BRCA1 recruitment on chromatin. Soluble (S1) and chromatin fractions (P2) were analyzed by Western-blot (6% and 15% Tris-Glycine SDS-PAGE), after two culture conditions (treated with HU at 5 mM overnight versus untreated). An enrichment of histone H4 in chromatin extracts confirmed the fractionation. Gray bands mask a sample that cannot be publicly presented (A,B) in different acquisition times).

Figure 9

Cell cycle: (a) cell cycle determined by flux cytometry for each individual by EdU and propidium iodide (PI) staining. (b) Cell distribution by cycle phases (n = 2; mean).