A homozygous FANCM frameshift pathogenic variant causes male infertility

Purpose Fanconi anemia (FA) genes play important roles in spermatogenesis. In mice, disruption of Fancm impairs male fertility and testicular integrity, but whether FANCM pathogenic variants (PV) similarly affect fertility in men is unknown. Here we characterize a Pakistani family having three infertile brothers, two manifesting oligoasthenospermia and one exhibiting azoospermia, born to first-cousin parents. A homozygous PV in FANCM (c.1946_1958del, p.P648Lfs*16) was found cosegregating with male infertility. Our objective is to validate that FANCM p.P648Lfs*16 is the PV causing infertility in this family. Methods Exome and Sanger sequencing were used for PV screening. DNA interstrand crosslink (ICL) sensitivity was assessed in lymphocytes from patients. A mouse model carrying a PV nearly equivalent to that in the patients (FancmΔC/ΔC) was generated, followed by functional analysis in spermatogenesis. Results The loss-of-function FANCM PV increased ICL sensitivity in lymphocytes of patients and FancmΔC/ΔC spermatogonia. Adult FancmΔC/ΔC mice showed spermatogenic failure, with germ cell loss in 50.2% of testicular tubules and round-spermatid maturation arrest in 43.5% of tubules. In addition, neither bone marrow failure nor cancer/tumor was detected in all the patients or adult FancmΔC/ΔC mice. Conclusion These findings revealed male infertility to be a novel phenotype of human patients with a biallelic FANCM PV.

Whole exome sequencing of gDNA from the patient (IV:1) and his father (III:             Supplementary Table S6.

Primer information for constructing FANCM_CDS-EGFP and FANCM-MUT_CDS-EGFP vectors
Transfections were performed using Lipofectamine ® 3000 Reagent (Thermo Fisher Scientific) according to the manufacturer's instructions, 12 hours after plating of cells.

Immunostaining and Western blot of cultured cells
Cells were grown on coverslips and treated with MMC (50 ng/ml for 12 hrs for γH2AX detection and 200 ng/ml for 24 hrs for FANCD2 detection). Immunostaining with the antibodies described in Supplementary Table S3 was performed as we previously described. 3 Antibodies used in this study are listed in. For Western blot, HEK293T cells were treated with MMC as described above and cells were then lyzed with SDS lysis buffer and boiled for 10 min. The proteins were separated on a 10% SDS polyacrylamide gel by electrophoresis for Western blotting as we described. 3 Antibodies used are listed in Supplementary Table S3.

Chromosomal breakage assay in HEK293T cells
For MMC-induced chromosomal breakages analysis, HEK293T cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin (Gibco), and treated with MMC (20 ng/ml) for 24 hr. Metaphase spreads were prepared using standard cytogenetic techniques described previously. 4 Chromosomal breaks were quantified by scoring at least 35 metaphases per culture.

Generation of Fancm ΔC/ΔC mice
Fancm ΔC/ΔC mice with a truncated FANCM protein analogous to that in our patients were generated by one-cell embryo injection using CRISPR/Cas9 genome editing tools. 1

Sperm analysis
One cauda epididymis was removed from each 10-week-old mouse, incised with scissors, and incubated in 1 ml buffer containing 75 mM NaCl, 24 mM EDTA and 0.4% bovine serum albumin (Sigma) at 37°C for 1 hr to allow sperm release. Sperm were then counted with a hemocytometer. Sperm motility was analyzed as previously described. 10 Briefly, extracted sperm were centrifuged and incubated in HTF medium (Millipore) supplemented with 10% FBS (Hyclone) at 37°C for 5 min. Sperm samples were then diluted and analyzed using Hamilton Thorne's Ceros II system for sperm motility.

Hematoxylin & Eosin (H&E) staining of sperm
Sperm were extracted from epididymis and concentrated in FBS.
Sperm smears were made onto microscope slides, air-dried, fixed in 4% paraformaldehyde in PBS, and exposed to 1% Triton X-100, followed by staining with hematoxylin and eosin. Images were captured using a microscope (Nikon Eclipse 80i) equipped with a digital camera (Nikon DS-Ri1). The numbers of sperm with abnormalities in head, neck, tail, head & neck, or head & tail were quantified by examining at least 250 sperm per mouse.

Chromosome breakage assay in spermatogonial metaphase cells
10-week-old male mice were intraperitoneally injected with MMC at 0.1, 1.0 or 2.5 mg/kg. Twenty-four hours later, they were intraperitoneally injected with 4 mg/kg of colchicine and sacrificed 5 hr later. 11 Then, the spermatogonial chromosome preparations were prepared as previously described. 12 Briefly, the testes were removed, peeled of the testicular capsule. Cell suspensions were prepared from isolated seminiferous tubule fragments in 2.2% (w/v) trisodium citrate dihydrate (isotonic solution) and centrifuged for 10 min at 800 rpm, followed by treatment with 1% (w/v) trisodium citrate dihydrate (hypotonic solution) for 12 min at 37°C and fixation in Carnoy's solution (75% methanol, 25% acetic acid) at 4°C. After three washes in fixative, chromosome preparations were made by dropping the cell suspension onto cold slides. Slides were dried and stained with Giemsa. Chromosomal breaks in spermatogonial metaphases (cells with 40 pairs of chromosomes) were quantified by scoring at least 31 cells for each group.

Routine blood test in mice
Peripheral bloods were sampled from lateral tail veins of 10-week-old mice and collected in EDTA-2K anticoagulant tubes (Anhui Heer). Bloods were analyzed using XT-1800i Automated Hematology Analyzer (Sysmex, Japan).
Parameters that were collected are hemoglobin, red blood cell count (RBC), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), white blood cell count (WBC) (and percentages of neutrophils, lymphocytes, eosinophils and monocytes) and platelet count. At least four mice were examined for each group.