Genomic variant diagnosis

Genomic DNA was extracted from peripheral blood via standard protocols. Molecular karyotyping was conducted using OGT Cytosure 180 k, v3 array. The clinical exome analysis was done using the Nimblegen V4 Panel clinical exome, consisting of 6178 genes. We analyzed the two affected siblings (ii-2 and ii-3) and their parents and data were obtained with massive parallel sequencing on Illumina Hiseq2500. Annotation of the variants was done using Genome build GRCh37, RefSeq78 and Cartagenia Bench Lab NGS (version 5.0). We filtered sequence variants in a stepwise manner to exclude synonymous variants, non-exonic SNVs, indels and variants with a minor allele frequency > 1% in gnomAD (version v2.1.1), the 1000 Genomes Project, and internal exome databases. Using the parental data, we looked for “de novo”, X-linked as well as combined heterozygous and homozygous mutations in the affected siblings. Variants were confirmed by Sanger sequencing in the siblings as well as in the parents.

Chromosomal breakage assays using etoposide or diepoxybutane

Epstein Barr Virus-transformed peripheral-blood lymphocytes (EBV-PBLs) of the two affected siblings, the healthy brother (no carrier), a healthy control line, as well as two established Fanconi cell lines (F1 and F2) were used to study chromosomal breakage. Fanconi patient F1 carries a homozygote pathogenic class 5 variant in FANCG (NM_004629.1), namely c.637_643delTACCGCC (p.Tyr213Lysfs*6). Fanconi patient F2 is combined heterozygote for two mutations in FANCC (NM_000136.2) namely c.520C > T (p.Arg174*) and c.455dupA (p.Asn152Lysfs*9), both class 5 mutations. Peripheral blood lymphocytes were cultured according to standard procedures. After 24 h of incubation, 20 nM Etoposide (Sigma E1383) was added to the culture for 27 h. After 24 h, cells were arrested in metaphase through a 3 h treatment with 10 μg/ml KaryoMAX Colcemid solution (Gibco), treated with 0.075 M KCl, fixed in methanol:acetic acid (3:1), spread onto glass slides and air-dried. Slides were stained with bisBenzimide H 33258 (Sigma), exposed to UV light and finally stained with Giemsa. For DEB analysis, the procedure is similar. Instead of etoposide, a 0.004% diepoxybutane solution was added to the cultures after 24 h of incubation. In both cases, a minimum of 25 cells were analyzed and the number of breakages per mitosis was calculated.

Cell culture

Lymphoblastoid cells from unaffected and affected individuals were grown in RPMI 1640 medium (Gibco) at 10% FCS in the presence of penicillin (10U/mL), streptomycin (10 μg/mL) and 2 mM l-glutamine. Primary fibroblasts from affected individual ii-3 were derived from skin biopsy and grown in MEM (Gibco) at 20% FCS in the presence of penicillin (10U/mL), streptomycin (10 μg/mL) and 2 mM l-glutamine. Fibroblasts and lymphoblasts from previously characterized SCAR23 (TDP2-mutated) patients (850BR, IV-9, IV-14 and IV-16) and controls (1BR, IV-2) were described previously described (Gómez-Herreros et al. 2014; Zagnoli-Vieira et al. 2018). RPE-1 hTERT cas9 cells and RPE-1 h-TERT Cas9 TDP2−/− cells were cultured in DMEM:F12 mix (Gibco) at 10% FCS in the presence of penicillin (10U/mL), streptomycin (10 μg/mL) and 2 mM l-Glutamine. All cells were maintained at 37 °C and 5% CO2.

TDP2−/− RPE-1 hTERT Cas9 cells and mCherry-TDP2/mCherry-TDP2E152K expression

RPE-1 hTERT Cas9 cells were a gift from Prof. Steve Jackson, and have been described previously (Balmus et al. 2019). RPE-1 hTERT Cas 9 TDP2−/− were created by transfecting the sgRNA 5′-CCTGTAGAAATATCACATCT-3′ that targets TDP2 exon 4 into RPE-1 hTERT Cas9 cells. Following clonal selection, gene targeting was confirmed by western blotting. For complementation studies, the human TDP2 ORF was cloned into the HpaI/AfeI sites of the Vector Builder mCherry plamid VB200726-1045daz, using Gibson assembly (NEB) with the indicated forward (5′-AGGATGACGATGACAAGAGCATGGAGTTGGGGAGTTGCCT-3′) and reverse primers (5′-TCGAGGTCGACACGCGTGTTCAATATTATATCTAAGTTGCACAGAAGACC-3′), creating mCherry-TDP2. mCherry-TDP2E152K was generated by site-directed mutagenesis (Q5 Site-directed mutagenesis kit; NEB) using mCherry-TDP2 and the forward/reverse primers 5′-CAGATGTGATATTTCTACAGaAAGTTATTCCCCCATATTA-3′ & 5′-TAATATGGGGGAATAACTTtCTGTAGAAATATCACATCTGGGCT-3′. The final constructs were packaged using psPAX2 (Addgene #12260) and pMD2.G (Addgene #12259) and lentiviral particles transduced into cells for 24 h prior to selection with 1 mg/mL Geneticin (Gibco) for 7 days.

Western blotting

Protein samples were prepared in Laemmli sample buffer (2% SDS, 10% glycerol, 60 mM Tris–HCl pH6.8) and heated for 10 min at 95 °C. Sample proteins were quantified using Bicinchoninic acid assay (BCA) reagent (Pierce) according to manufacturer instructions. Prior to loading, samples were supplemented with 100 mM dithiothreitol and 0.005% bromophenol blue. Following SDS-PAGE electrophoresis and western blotting, TDP2 and KU80 were detected using a rabbit anti-TDP2 primary antibody (Thomson et al. 2013), and KU80 was detected as a loading control using an anti-Ku80 rabbit monoclonal primary antibody (Abcam Ab80592). For overexpressed mCherry-tagged TDP2, anti-mCherry antibody (Abcam ab167453) was employed.

Tyrosyl DNA phosphodiesterase (TDP) assays

TDP assays were performed as previously described (Zagnoli-Vieira et al. 2018). In brief, cell extracts were prepared by resuspension of lymphoblastoid cells in lysis buffer (40 mM Tris/HCl pH 7.5, 100 mM NaCl, 0.1% Tween-20, 1 mM DTT, 1 mM PMSF, 1 × EDTA free protease cocktail inhibitor), followed by 30 min incubation on ice and mild sonication using a BioRuptor (Diagenode) for five cycles of 30 s on/off. The cell extract was then clarified by centrifugation for 10 min at 4 °C at 16,000 × g in a microfuge and the protein concentration quantified using the bicinchoninic acid (BCA) assay reagent (ThermoFisher). 3 μg of clarified cell extract was incubated with 40 nM of TDP2 substrate (Cy5-5′Tyrosine-ssDNA19-BHQ) or TDP1 substrate (BHQ-ssDNA13-3′Tyrosine-Cy5) diluted in reaction buffer (50 mM Tris/HCl pH8.0, 10 mM MgCl2, 80 mM KCl, and 1 mM DTT, 0.05% Tween-20, 6 μM unlabeled 19 bp oligo) in a total volume of 6 µl at room temperature for 45 min. Cy5 fluorescence was measured at 650 nm on a BMG PHERAstar plate reader. Benzonase (Sigma) was used as positive control.

Immunoflourescence

Cells were grown in 24-well plates (Greiner SensoPlate) until confluent and then treated for 30 min with 50 µM etoposide or irradiated with γ-irradiation (2 Gy). After treatment, cells were rinsed with PBS and replenished with fresh media or fixed for 10 min in PBS containing 4% paraformaldehyde at the indicated time points. Following fixation, cells were permeabilised (20 min, 0.2% Triton X-100 in PBS), blocked (1 h in PBS-5% BSA), and incubated with anti-γH2AX (Millipore, 05–636, 1:2500) and anti-CENP-F (Abcam, ab5, 1:2500) antibodies for 3 h in PBS containing 5% BSA. Cells were then washed (3 × 5 min in PBS containing 0.1% Tween-20), incubated for 1 h with the corresponding Alexa Fluor conjugated secondary antibody (1:1000, 5% BSA), and washed again as described earlier. Finally, cells were counterstained with DAPI (Sigma, Gillingham, UK) and imaged on Opera Phenix microscope (PerkinElmer) with 40X water immersion objectives. Image analysis and evaluation was done using Harmony High-Content Imaging and analysis software. Cells were gated to the G1 population according to the CENPF signal.

Clonogenic survival assays

Patient-derived fibroblasts were plated onto feeder layers and 3 h later treated with the indicated concentrations of etoposide or ionising radiation prior to incubation for 21 days to allow the formation of macroscopic colonies. For feeder layers, 1BR cells were irradiated (35 Gy) and plated 24 h before use at 5 × 104 cells/10 cm dish. For RPE-1 hTERT Cas9 cells, clonogenics were performed as above but the feeder layer was omitted, and macroscopic colonies were allowed to form for 10 days. Following colony formation, cells were fixed in 100% ethanol and dishes rinsed with PBS prior to staining with 70% ethanol/1% crystal violet. Dishes were allowed to dry, prior to scoring colonies of > 50 cells. The surviving fraction at each dose was calculated by dividing the average number of colonies in treated dishes by the average number in untreated dishes.