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. Author manuscript; available in PMC: 2019 Mar 13.
Published in final edited form as: N Engl J Med. 2018 Sep 13;379(11):1042–1049. doi: 10.1056/NEJMoa1800024

Essential Role of BRCA2 in Ovarian Development and Function

Ariella Weinberg-Shukron 1,#, Mariana Rachmiel 1,#, Paul Renbaum 1, Suleyman Gulsuner 1, Tom Walsh 1, Orit Lobel 1, Amatzia Dreifuss 1, Avital Ben-Moshe 1, Sharon Zeligson 1, Reeval Segel 1, Tikva Shore 1, Rachel Kalifa 1, Michal Goldberg 1, Mary-Claire King 1, Offer Gerlitz 1,#, Ephrat Levy-Lahad 1,#, David Zangen 1,#
PMCID: PMC6230262  NIHMSID: NIHMS1507279  PMID: 30207912

SUMMARY

The causes of ovarian dysgenesis remain incompletely understood. Two sisters with XX ovarian dysgenesis carried compound heterozygous truncating mutations in the BRCA2 gene that led to reduced BRCA2 protein levels and an impaired response to DNA damage, which resulted in chromosomal breakage and the failure of RAD51 to be recruited to double-stranded DNA breaks. The sisters also had microcephaly, and one sister was in long-term remission from leukemia, which had been diagnosed when she was 5 years old. Drosophila mutants that were null for an orthologue of BRCA2 were sterile, and gonadal dysgenesis was present in both sexes. These results revealed a new role for BRCA2 and highlight the importance to ovarian development of genes that are critical for recombination during meiosis. (Funded by the Israel Science Foundation and others.)

GENES THAT ARE CRITICAL FOR OVARIAN DEVELOPMENT INCLUDE WNT4, RSPO1, and FOXL2, which encode the proteins involved in early development, and LHR and FSHR, which encode the proteins involved in hormonal signaling.1 Recently, several additional genes (HFM1, MCM8, MCM9, SYCE1, STAG3, and SPIDRI) have been identified.2,3 However, many genes critical for ovarian development and maintenance remain unknown.4

CASE REPORTS

Two sisters, 20 and 15 years of age, from a nonconsanguineous family of Ethiopian ancestry presented with short stature, primary amenorrhea, and absence of spontaneous pubertal development (Fig. 1A). Both sisters had a normal female karyo-type (46,XX) and hypergonadotrophic hypogonadism (luteinizing hormone levels of >25 IU per liter and follicle-stimulating hormone levels of >85 IU per liter) and had no detectable uterus or ovaries on initial imaging studies — findings that are consistent with complete XX ovarian dysgenesis. Thyroid, adrenal, and ovarian antibodies and serum antimüllerian hormone were undetectable. Cortisol levels and findings from adrenal imaging were normal. After 18 months of receiving oral estradiol, in doses that were increased gradually from 0.25 mg daily to 2.00 mg daily, followed by ongoing treatment with a combined estrogen–progesterone oral contraceptive, both sisters had grown to mean familial height (158 cm and 160 cm) and had secondary sexual characteristics, adult-sized uteri (as confirmed by pelvic ultrasonography), and regular menstrual periods. Physical examination revealed microcephaly (head circumference, <1st percentile for age and sex) and a few small café au lait spots in both sisters; the sisters had normal intelligence (both sisters had above-average performance in high school, and the older sister had entered a university). The medical history of the proband (the older sister) revealed long-term remission (>14 years) of acute myelocytic leukemia, which had been diagnosed when she was 5 years of age and had been managed successfully with a regimen of all-trans-retinoic acid, idarubicin, and cytarabine — agents that generally do not cause subsequent ovarian failure. An older brother of the sisters had died from acute promyelocytic leukemia at 13 years of age. Three other siblings (two female and one male) were healthy and had normal puberty, including the initiation of menstrual cycles in the two female siblings and the development of secondary sexual characteristics in all three siblings. When the two affected sisters were first seen, both parents were healthy, and there was no family history of breast or ovarian cancer.

Figure 1. Gene Discovery and Characterization.

Figure 1.

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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 karyo-type (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 pro-band (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).

METHODS

HUMAN GENOMICS AND FUNCTIONAL STUDIES

This study was approved by the institutional review boards at Shaare Zedek Medical Center, Assaf Harofeh Medical Center, the University of Washington, and the Israel National Review Committee for Genetics Studies. All family members provided written informed consent and were evaluated by means of whole-exome sequencing, as described previously.5 Functional studies were performed in both affected sisters, in relatives, and in unrelated controls. Chromosomal breakage was measured in lymphocytes exposed to mitomycin C,2 and formation of γ-H2AX and RAD51 foci was measured after fibroblasts were exposed to neocarzinostatin, as described previously.2,6 BRCA2 transcript expression and protein levels of BRCA2 and RAD51 were evaluated in lympho-blasts.

DROSOPHILA STUDIES

A drosophila mutant strain that was null for Dmbrca2 (the fly gene orthologous to BRCA27) was used to evaluate gonadal morphologic features. Crosses of this null strain with a stock that was homozygous for wild-type Dmbrca2 (yw) were used to evaluate egg and progeny production.

STATISTICAL ANALYSIS

Statistical analyses were performed with the use of two-tailed, unpaired Student’s t-tests. A Holm–Bonferroni correction was used for multiple inferences.

RESULTS

GENE DISCOVERY AND FUNCTIONAL CHARACTERIZATION OF THE MUTATIONS

In the analysis of the exome sequences of the affected sisters, their unaffected siblings, and their parents, we searched for genes that harbored damaging variants that cosegregated with the phenotype of the affected sisters in recessive, de novo, and mosaic models. Only one gene, with two variants, fulfilled these criteria (Table S1 in the Supplementary Appendix, available with the full text of this article at NEJM.org): BRCA2 c.7579delG_p.V2527X (GenBank accession number, NM_000059) at chromosome 13 (position 32,930,708; aligned to hg19 human genome reference sequence, ClinVar database accession identifier: SCV000803632) and BRCA2 c.9693delA_p. S3231fs16* at chromosome 13 (position 32,972,343; aligned to hg19 human genome reference sequence, ClinVar database accession identifier: SCV000803633). Both affected sisters were compound heterozygous, and all unaffected relatives were either singly heterozygous or homozygous for the reference sequence (Fig. 1A and 1B, and Fig. S1A and S1B in the Supplementary Appendix). We were unable to find either variant listed in any public database. When the mother (48 years of age) was identified as a carrier of BRCA2 p.V2527X, she was offered breast cancer and ovarian cancer surveillance, and ovarian cancer stage III was detected by ultrasonography.

Biallelic loss-of-function mutations in BRCA2 cause Fanconi’s anemia type D1, which is characterized by an increased number of chromosomal breaks after cells are exposed to DNA-damaging agents.8 We observed many chromosomal breaks in the lymphocytes from the two affected sisters after exposure to mitomycin C; the number of breaks observed in the peripheral lymphocytes from the proband was 50 times as great as the number observed in the control lymphocytes (those obtained from unaffected relatives or from an unrelated person) (Fig. 1C and 1D, and Table S3 in the Supplementary Appendix).

Cellular levels of BRCA2 messenger RNA and BRCA2 protein were significantly lower in the affected sisters than in their unaffected relatives and unrelated controls (Fig. 2, and Fig. S2 in the Supplementary Appendix). The levels of BRCA2 transcript expression in the affected sisters were lower than those in two unrelated, unaffected normal persons by a factor of 3.5 (P = 2.29×10−6). The levels of BRCA2 transcript expression in the heterozygous relatives were intermediate but were also significantly lower than those in two unrelated, unaffected controls (P≤9.6×10−5). Similarly, cellular levels of BRCA2 protein were lower in the affected sisters than in an unrelated, unaffected control by a factor of 7.0 (P = 6.7×10−12). Cellular levels of BRCA2 protein in heterozygous relatives were intermediate but were also significantly lower than the level in an unrelated, unaffected control (P<0.002).

Figure 2. Characterization of the Effect of BRCA2 Mutations in the Cells from the Enrolled Persons.

Figure 2.

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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.

A critical role of BRCA2 in homologous recombination repair of DNA damage is to recruit RAD51 to double-stranded DNA breaks.9,10 We assessed the formation of RAD51 foci at sites of neocarzinostatin-induced DNA damage in cells with compound heterozygous, heterozygous, and nonmutant genotypes. We observed fewer RAD51-positive foci at the sites of DNA damage (indicated by γ-H2AX positivity) in the fibroblasts from one of the affected sisters (family member III-2) than in the fibroblasts from an unrelated person and from the mother (Fig. 2C and 2D and Fig. S3A in the Supplementary Appendix). This difference in the formation of RAD51 foci was not due to differences in the cellular levels of RAD51 protein, which did not differ significantly between the affected sisters and their unaffected relatives or an unrelated person (Fig. S3B through S3D in the Supplementary Appendix).

DROSOPHILA MELANOGASTER MODEL

DNA repair pathways have been well conserved during evolution and are remarkably similar between mammals and drosophila.11 Similar to its mammalian counterpart, the protein encoded by Dmbrca2 (the drosophila orthologue of BRCA2) interacts with RAD51 (spindle A) and functions in meiotic and mitotic repair of double-stranded breaks in DNA through homologous recombination.7,12 Dmbrca2-null drosophila are thus an appropriate model for the BRCA2 loss-of-function mutations in our patients. Dmbrca2−/− flies (also referred to as Dmbrca2-null flies) have been reported to be viable, and female Dmbrca2−/− flies to be sterile.7 To examine sterility in these flies in more detail, we crossed female and male Dmbrca2−/− flies with wild-type controls (Fig. 3A). Egg production by the Dmbrca2−/− female flies that were crossed with wild-type control male flies was less than 5% of egg production by wild-type female controls (P = 0.001). We also observed that the few eggs laid by these mutant female flies had abnormal morphologic features, including eggshell transparency, round or flattened shape, and fused or no dorsal appendages, as described previously.7 In crosses of Dmbrca2−/− male flies with wild-type control female flies, egg production was not significantly altered, and the morphologic features of the eggs were normal. However, virtually no progeny survived from either cross of Dmbrca2−/− flies with wild-type flies, which indicates complete sterility in both female and male Dmbrca2-null flies. In contrast, we observed that the intercrossing of male and female Dmbrca2+/− heterozygotes had no effect on the numbers of eggs or progeny (Fig. S4 in the Supplementary Appendix).

Figure 3. Drosophila Model of the BRCA2-Null Genotype.

Figure 3.

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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.

We observed a spectrum of phenotypes in the ovaries and testes of Dmbrca2-null flies (Fig. 3B, and Fig. S4 in the Supplementary Appendix). The morphologic findings in the ovaries of the Dmbrca2-null female flies ranged from complete ovarian dysgenesis (69% of ovaries) to underdeveloped ovaries that had fewer ovarioles, with small, disordered, and misshapen egg chambers, and few mature eggs (4% of ovaries). Immunostaining revealed extensive disintegration of egg chambers starting at stage 7, including destruction of cell envelopes, DNA condensation, and extensive apoptosis in Dmbrca2-null female flies, whereas the ovaries and ovarioles of Dmbrca2+/− and wild-type flies were normal (Fig. 3C). The morphologic findings in the testes of the Dmbrca2-null male flies showed that nearly all testes (92%) were moderately to severely small and underdeveloped and lacking spermatogenesis stages of elongation, individualization, and coiling (Fig. 3B, and Fig. S4 in the Supplementary Appendix). Immunostaining revealed aberrant DNA location, lack of actin structures, and abnormal expression of cleaved caspase-3 in Dmbrca2-null male flies, whereas Dmbrca2+/− and wild-type flies had normal testes with normal apoptosis in differentiating spermatids (Fig. 3C).

DISCUSSION

We report that loss of BRCA2 function in the two sisters caused XX ovarian dysgenesis, which manifested as the absence of spontaneous pubertal development and as primary amenorrhea. The loss of BRCA2 function led to reduced recruitment of RAD51 to double-stranded breaks in DNA. Recruitment of RAD51 to double-stranded breaks in DNA is necessary both for the repair of breaks that occur during recombination in meiosis and for the repair of DNA in mitotic cells.13,14

In principle, an alternative explanation for ovarian dysgenesis in the two affected sisters could be that the loss of DNA repair capacity caused by their BRCA2 genotype led to the occur-rence of specific somatic mutations affecting the ovaries. However, because ovarian dysgenesis occurred in both sisters, such hypothetical somatic mutations would have to have occurred independently in the two ovarian lineages. We could not test for somatic mutations in the ovaries by genotyping DNA from ovarian tissue in the sisters, because no ovarian tissue was detectable. The hypothesis that loss-of-function variants in BRCA2 directly cause ovarian dysgenesis is the most parsimonious explanation for our results. The phenotype of Dmbrca2-null drosophila, in which both female and male flies have severe gonadal dys-genesis and produce no progeny, supports the notion that ovarian dysgenesis is the direct consequence of loss of function of BRCA2. We also note that in zebrafish, brca2-null mutants have small gonads and develop only as sterile adult male zebrafish; female zebrafish undergo sex reversal as a result of oocyte death during sex determination.15

Complete loss of function of BRCA2 causes early embryonic lethality in humans and in mice. Biallelic damaging mutations in BRCA2 that are not both fully null are compatible with life but result in Fanconi’s anemia D1,16,17 which is characterized by chromosomal instability, as reflected by extreme hypersensitivity to cross-linking agents.14 Most patients with Fanconi’s anemia have developmental abnormalities including microcephaly, short stature, skeletal malformations, abnormal skin pigmentation, early bone marrow failure, and acute myelocytic leukemia and are at higher risk for some solid tumors.17 However, the patients in our study presented primarily with isolated XX ovarian dysgenesis. Only with detailed physical examinations did we observe microcephaly and a few café au lait spots. The sister who had received a diagnosis of leukemia in childhood had a good response to therapy, and the leukemia has been in remission for nearly 15 years. Her older brother, who was probably also compound heterozygous for the two BRCA2 loss-of-function alleles, was healthy until he presented with promyelocytic leukemia at 13 years of age. In contrast, in all previously reported cases of Fanconi’s anemia D1, cancer developed in the patients in the first decade of life, and death occurred before puberty. The relatively mild pheno-type in our patients most likely reflects that one of the alleles is predicted to result in a mutant protein that is only 171 residues (5%) shorter than the full-length BRCA2 protein. The cells from the two affected sisters were not completely depleted of BRCA2 protein but expressed 14% of the level of BRCA2 protein observed in an unrelated control.

Given the critical role of meiosis in germ-cell survival, endocrine and fertility problems often occur in persons with defects in DNA repair. Among the patients with Fanconi’s anemia who survive to puberty, genital malformations and hypoplastic gonads are common in male patients,18 and premature ovarian failure is common in female patients19,20 as they are in female mouse models.21 Although pregnancy is occasionally possible in women with Fanconi’s anemia, it is often associated with complications.22 Male patients are rarely fertile.18 Descriptions of fertility problems in patients with Fanconi’s anemia D1 (who carry pathogenic variants in both copies of BRCA2) are lacking, because patients described in previous reports did not survive past childhood.23–27

Our results indicate that ovarian development depends on the normal repair of double-stranded DNA breaks that occur at recombination during meiosis. These results highlight an emerging concept of a critical role for DNA repair genes (e.g., MCM-8,2 MCM-9, and, as we report here, BRCA2) in ovarian development and function. This concept has implications for the clinical evaluation of gonadal dysgenesis. In particular, as-says for chromosomal breaks are a useful screening tool in the evaluation of patients who present with ovarian dysgenesis, since they may indicate in some patients that defects in DNA repair are the underlying genetic basis for gonadal dysgenesis and that these patients have a predisposition for cancer that warrants surveillance.28,29

Supplementary Material

Supplement1

Acknowledgments

Supported by a grant (1788/15) from the Legacy Heritage Biomedical Program of the Israel Science Foundation (to Drs. Gerlitz and Zangen), by grants from the Breast Cancer Research Foundation (to Drs. King and Levy-Lahad), by a grant (R35CA197458) from the National Institutes of Health (to Dr. King), by a grant (1353/12) from the Israel Science Foundation (to Dr. Goldberg), and by a donation from the Hassenfeld family to Shaare Zedek Medical Center.

Disclosure forms provided by the authors are available with the full text of this article at NEJM.org.

We thank the family for participating in the project, Tehila Klopstock and Tzvia Rosen for technical assistance, Dr. Mark Baker and Alissa Magwood for advice in performing Western blots of the BRCA2 protein, Drs. Trudi Schupbach and Uri Abdu for providing the drosophila strains and reagents, and Dr. Amnon Lahad for assistance with the statistical analysis.

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Supplementary Materials

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