A homozygous FANCM mutation underlies a familial case of non-syndromic primary ovarian insufficiency

Abstract

Primary Ovarian Insufficiency (POI) affects ~1% of women under forty. Exome sequencing of two Finnish sisters with non-syndromic POI revealed a homozygous mutation in FANCM, leading to a truncated protein (p.Gln1701*). FANCM is a DNA-damage response gene whose heterozygous mutations predispose to breast cancer. Compared to the mother's cells, the patients' lymphocytes displayed higher levels of basal and mitomycin C (MMC)-induced chromosomal abnormalities. Their lymphoblasts were hypersensitive to MMC and MMC-induced monoubiquitination of FANCD2 was impaired. Genetic complementation of patient's cells with wild-type FANCM improved their resistance to MMC re-establishing FANCD2 monoubiquitination. FANCM was more strongly expressed in human fetal germ cells than in somatic cells. FANCM protein was preferentially expressed along the chromosomes in pachytene cells, which undergo meiotic recombination. This mutation may provoke meiotic defects leading to a depleted follicular stock, as in Fancm^-/- mice. Our findings document the first Mendelian phenotype due to a biallelic FANCM mutation.

Keywords: FANCM; Mitomycin C; exome sequencing; human; human biology; medicine; mutation; primary ovarian insufficiency; replication inhibitor.

Figure 1.. Molecular analysis of the POI family.

(A) Pedigree of the Finnish family with two sisters affected with POI. (B) Exome data visualization in IGV (Integrative Genomics Viewer) shows a high coverage of the variant position and a large number of reads in the three individuals. (C) Sanger sequencing confirmed the presence of the variant at a homozygous state in both affected sisters and at a heterozygous state in both parents and their brother. (D) Structure of the FANCM gene and protein, and position of the causal variant. (E) Western blots of FANCM in the POI family. HEK293 cell transfected with a FANCM-specific siRNA were used to validate the specificity of the anti-FANCM antibody. To transiently deplete FANCM, HEK293 cells were transfected with 20 nmol/L of small interfering RNA (siRNA) targeting FANCM, 5'-GGC-UAC-GUC-CAG-GAG-CGC-3' with the CaCL₂ method. Panel present the result of a representative experiment on at least three independent analysis.

Figure 2.. FANCM expression in human fetal ovaries.

(A) Relative FANCM mRNA abundance was measured by RT-qPCR in human fetal ovaries from 5 to 32 weeks post-fertilization (wpf). (B) Germ cells (D2-40+) and somatic cells (D2-40-) were sorted from three ovaries ranging from 8 to 12 wpf and FANCM expression was measured. ACTB was used to normalize FANCM expression in all samples. Dots represent different ovaries and the mean is indicated by the line. (C) Immunohistochemistry of FANCM in human fetal and adult ovaries. Fetal ovaries at 8 and 22 wpf and adult ovaries were studied. FANCM positive cells appear in yellow/brown color (monoclonal FANCM CV5.1 antibody, Novus Biologicals, Abingdon, UK). Ovarian sections were counterstained with hematoxylin (blue staining). Oo, oogonia; Pa, oocyte at the pachytene stage of meiosis I, D, oocyte at the diplotene stage of meiosis I; Pr, oocyte in primordial follicle. (D) Co-staining in 22 wpf ovaries, for FANCM (purple) and DDX4 (brown) confirmed the germ cell identity of FANCM-positive cells (left). Successive staining for FANCM and SYCP3 in the same section (panels a and b). Negative control performed with non-immune mouse IgG (right). Scale bar: 10 μm.

Figure 3.. Chromosomal breakage and hypersensitivity to Mitomycin C associated to the FANCM 5101C > T mutation.

(A) Spontaneous and mitomycin C-induced chromosome breakage in primary lymphocytes from the mother and the two POI sisters. Data presented in table are the result of a single experiment. A minimum of 50 metaphases were scored for each sample. (B and B') Examples of untreated and MMC-treated metaphases from patient-2. Arrows indicate breaks and chromosome rearrangements, that is radial figures. (C) Western blot showing FANCD2 expression and monoubiquitination in the same lymphocytes than in A and B. Immunoblot analysis were performed using mouse monoclonal anti-FANCM antibody (CV5.1), mouse monoclonal anti-FANCD2, (Santa-Cruz Biotechnology, Dallas, Texas, USA), rabbit anti-FANCA (Abcam, Cambridge, UK), mouse anti-vinculin (Abcam). Panel present the result of a representative experiment on at least three independent analysis. (D) MMC-induced growth inhibition in lymphoblasts from POI patient-2 and her mother compared to the response of cells from a FANCA (HSC-72), a FANCC (HSC-536), a healthy donor (HSC-93, WT) as well as to the patient cells transduced with a WT FANCM-cDNA. The points on the lines represent the means of 3 to 7 independent experiments ± S.D. *p<0,05, **p<0.01, Unpaired Student's T test. (E) The recovery of the MMC-induced FANCD2-monoubiquitination following FANCM expression in cells from patient 2 is shown. (1) and (2) indicate two independent experiments. Cells were treated with MMC (500 ng/ml) and proteins extracted 24 hr later.

Figure 4.. The FANCM 5101C>T mutation leads to altered FANCD2 monoubiquitination in response to DNA damage but not to replication inhibition.

Western blot showing FANCD2 monoubiquitination, CHK1 and H2AX phosphorylation in response to MMC (A), or HU or APH (B) in cells from patients 2 and her mother. FANCA (HSC-72) and FANCD2 (GM16756) lymphoblasts were used for comparison. Each panel presents the result of a representative experiment on at least three independent analysis. Proteins were extracted 24 hr after exposure to genotoxins.