Fanconi anemia with sun-sensitivity caused by a Xeroderma pigmentosum-associated missense mutation in XPF
Fanconi anemia (FA) is an inherited genomic instability disorder with congenital and developmental abnormalities, bone marrow failure and predisposition to cancer early in life, and cellular sensitivity to DNA interstrand crosslinks.
A fifty-one-year old female patient, initially diagnosed with FA in childhood on the basis of classic features and increased chromosomal breakage, and remarkable sun-sensitivity is described. She only ever had mild haematological abnormalities and no history of malignancy. To identify and characterise the genetic defect in this lady, who is one of the oldest reported FA patients, we used whole-exome sequencing for identification of causative mutations, and functionally characterized the cellular phenotype. Detection of the novel splice site mutation c.793-2A > G and the previously described missense mutation c.1765C > T (p.Arg589Trp) in XPF/ERCC4/FANCQ assign her as the third individual of complementation group FA-Q. Ectopic expression of wildtype, but not mutant, XPF/ERCC4/FANCQ, in patient-derived fibroblasts rescued cellular resistance to DNA interstrand-crosslinking agents. Patient derived FA-Q cells showed impaired nuclear excision repair capacity. However, mutated XPF/ERCC4/FANCQ protein in our patient’s cells, as in the two other patients with FA-Q, was detectable on chromatin, in contrast to XP-F cells, where missense-mutant protein failed to properly translocate to the nucleus.
Patients with FA characteristics and UV sensitivity should be tested for mutations in XPF/ERCC4/FANCQ. The missense mutation p.Arg589Trp was previously detected in patients diagnosed with Xeroderma pigmentosum or Cockayne syndrome. Hence, phenotypic manifestations associated with this XPF/ERCC4/ FANCQ mutation are highly variable.
KeywordsFanconi anemia UV sensitivity XPF ERCC4 FANCQ DNA repair
Fanconi anemia (FA) is a rare inherited genomic instability disorder with remarkable clinical and genetic heterogeneity. Whilst variable, typical features include developmental anomalies and malformations, most commonly growth retardation, cutaneous pigment displacement and radial ray defects. FA features also comprise early-onset bone marrow failure and cancer predisposition, specifically for acute myelogenous leukemia and head and neck squamous cell carcinoma [1, 2]. Causative mutations in any one of 22 FA genes (FANCA, -B, -C, -D1, -D2, -E, -F, -G, -I, -J, -L, -M, -N, -O, -P, -Q, -R, -S, -T, -U -V-and W) have been reported, whose corresponding proteins function together in a replication-dependent DNA interstrand crosslink (ICL) repair pathway [3, 4].
XPF, alias ERCC4 or FANCQ is the catalytic subunit of a heterodimer with ERCC1 that forms a structure-specific DNA repair endonuclease on the SLX4/FANCP scaffold. In the FA/BRCA pathway its activity is responsible for the nucleolytic strand incision 5′ to ICLs, which is a prerequisite for unhooking of a crosslink . XPF/ERCC4/FANCQ also acts in the nucleotide excision repair (NER) pathway, independently of SLX4 , to remove intrastrand helix-distorting lesions from DNA, such as UV light-induced photoadducts. In fact, XPF/ERCC4/FANCQ mutations have mainly been identified in patients with Xeroderma pigmentosum (XP) of complementation group F (XP-F) [5, 7], and later in an individual with the XFE progeroid syndrome . Recently, in two individuals (FA104 and 1333) mutations in XPF/ERCC4/FANCQ have been found to cause classic FA . Clinical features of XP and Cockayne syndrome (CS) were described in FA due to XPF/ERCC4/FANCQ mutations in another patient (XPCS1CD), and reconciled clinically impaired ICL repair with deficient NER .
Here we present one of the oldest reported individuals to be affected by FA, whom we identified as the third FA-Q patient due to bi-allelic XPF/ERCC4/FANCQ mutations. As the missense mutation p.Arg589Trp here implicates FA, opposed to XP or CS in other cases, we investigate and discuss biological and clinical implications.
Clinical manifestation of affected systems and clinical management in the FA-Q patient
Low birthweight 2010 g; Short stature; Microcephaly
Right thumb absent
Left thumb hypoplastic
Pollicizations at age 3 and 4
Internal rotation osteotomy age 25
Fatty liver with persistently
elevated liver enzymes
Crossed kidney fusion ectopia, both kidneys on the right
Surveillance of kidney function only
Thrombocytopenia with macrocytosis
Mild bone marrow hypoplasia without evidence of clonal chromosomal aberrations
Endocrine and reproductive health
Diabetes Type 1, age 42
Menopause age 29
Low dose insulin (~0.2 U/kg/ day)
Hormone replacement therapy since age 32
Lifelong sun sensitivity with severe sunburn and blistering after minimal sun exposure
Meticulous sun avoidance and skin protection
Sensorineural hearing loss increasingly relevant age 45
Cerebral and cerebellar atrophy
Small pituitary gland Microangiopathic white matter signal changes
Cyclosporine A was used to establish Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCL) . Peripheral blood lymphoblasts were grown in RPMI 1640 medium supplemented with GlutaMAX (Gibco) and 15% fetal bovine serum (FBS). Primary fibroblasts of the patient concerned were cultured in Amniopan medium (PAN Biotech), while SV40 large T antigen-immortalized fibroblasts were maintained in MEM with GlutaMAX and 10% FBS. For retrovirally transduced fibroblasts 10% Tet System Approved FBS (Clontech Laboratories) was used. All cultures were maintained in incubators with 5% CO2. Cells were exposed to 40 ng/μl mitomycin C (MMC) for 16 h prior to immunofluorescence analysis of RAD51 foci, immunoblotting or cell fractionation studies. Cell lines from disease and normal controls were maintained as previously reported .
Cytogenetic assays were performed on whole blood cultures to assess spontaneous and MMC-induced breakage rates. MMC at indicated final concentrations was added to blood samples as a G0 pulse before culture initiation. After 1 h the cells were washed and transferred to fresh complete RPMI 1640 medium. Lymphocytes were stimulated with phytohemagglutinin (PHA). The cultures were incubated at 37 °C and harvested after 72 h. Chromosome preparations were made by the air drying method . Solid (Giemsa)-stained metaphases were examined for chromatid and chromosome type damage, and the results were compared to age and sex-matched normal controls.
Cell cycle analysis
PHA-stimulated lymphocytes or primary fibroblasts were cultured untreated for 72 h or continuously exposed to MMC. Mono- or bivariate (BrdU-Hoechst 33,258/Ethidium bromide) cell cycle analysis was performed on a triple-laser-equipped flow cytometer (LSRII, BD Biosciences). Data were analyzed using the MPLUS AV software package (Phoenix Flow Systems) .
Nucleic acid isolation and cDNA synthesis
A modified salting-out technique  or the GeneJET Genomic DNA Purification Kit (Thermo Fisher) were used for isolation of genomic DNA (gDNA). For total RNA isolation we employed the High Pure RNA Isolation Kit (Roche) while transcription into cDNA was performed by SuperScript Reverse Transcriptase (Invitrogen).
For relative quantification of mRNA expression levels, allele-specific primers were designed (WT for: 5’-GACGCAGAGCTAACCTTTGTTC-3′; c.1765C > T MUT for: 5′- GACGCAGAGCTAACCTTTGTTT-3′; WT/MUT rev: 5′- GTTCCTCAGTTGAACCTCCGTA-3′). For the detection of exon 5 skipping due to c.793-2A > G primer sequences were as follows: WT for: 5′- TCTGGAATCTCTGAGAGCAACG-3′, WT rev: 5′- AACATCGAGGTGCTGGAGTC-3′, MUT for: 5′- ATAACCCATCGCTTGAAGTGGA-3′, MUT rev: 5′- CAAGAA\ACAGCCAACCTTGTCA-3′. The PCR reaction was performed with HOT FIREPol® EvaGreen® qPCR Mix Plus (Solis BioDyne) on an ABI ViiaTM 7 System (Applied BiosystemsTM). Evaluation of melting curves and amplification plots, and relative quantification (RQ) was done with the QuantStudioTM Real-Time PCR Software v1.2.4 (Applied BiosystemsTM) using the ΔΔCt method. Each sample was analyzed in technical triplets.
Transduction with different retroviral vectors containing XPF/ERCC4/FANCQ cDNA constructs or mock was performed according to standard protocols [15, 16]. Transduced immortalized fibroblasts were analyzed for their sensitivity. Aliquots of 20,000 cells per well were seeded in 6-well culture plates and grown at the indicated concentrations of MMC. After eight days cell viability was determined by image cytometry using VitaBright-48 and propidium iodide staining on a NucleoCounter NC-250 instrument (ChemoMetec A/S).
LCLs were grown in T25 cell culture flasks at concentrations of 0–1000 nM MMC for eight days. Cell viability was analyzed by propidium iodide exclusion and assayed by flow cytometry.
Nuclear RAD51 focus formation was analyzed in fibroblasts grown on glass chamber slides (Sarstedt). Cells were washed with PBS and subsequently fixed in 4% (vol/vol) paraformaldehyde in PBS for 15 min at room temperature. Thereafter they were exposed to ice-cold 100% methanol and kept on ice for 30 min. Blocking of non-specific antibody binding sites was accomplished with 20% (vol/vol) FBS in PBS for 30 min at room temperature. Rabbit anti-RAD51 (1:800; ab63801, Abcam) served as primary antibody, to be detected by Alexa 594-conjugated anti-rabbit secondary antibody (1:2000; A11037, Molecular Probes/Life Technologies). ProLong Gold Antifade Mountant with DAPI (Thermo Fisher) was used for counter-staining and as mounting medium. Foci-positive cells (>5 foci/nucleus) were scored by fluorescence microscopy (Axiovert 40C, Zeiss).
Aliquots of 40 μg whole protein extracts from cultured cells were loaded on NuPAGE Novex 7% Tris-Acetate protein gels (Invitrogen). Electrophoresis was carried out over night at 70 V with constant cooling. Proteins were transferred using a dry blotting system (iBlot 2, Life Technologies). FANCD2 isoform immunodetection was carried out using primary anti-FANCD2 antibody (1:500; sc-20,022, Santa Cruz) and secondary anti-mouse IgG-HRP (sc-2005, Santa Cruz) diluted 1:3000. FANCJ (1:1000; NB100–416, Novus) or vinculin (1:10,000; ab129002, Abcam) served as loading controls. For visualization of ERCC4/XPF/FANCQ whole protein extracts were separated on NuPAGE 4–12% Bis-Tris gels (Invitrogen). Anti-XPF (1:200; ab17798, Abcam) was used for immunodetection while Anti-Tubulin (1:1000; ab44928, Abcam) was employed as loading control. Luminescence was generated by chemical reaction with a standard ECL reagent (Millipore Corporation).
The NE-PER Nuclear and Cytoplasmic Extraction Kit (Thermo Fisher) was used for extraction of cytoplasmic proteins while nuclear and chromatin-associated proteins were isolated by the Subcellular Protein Fractionation Kit (Thermo Fisher). SDS-PAGE was run on NuPAGE 4–12% Bis-Tris gels (Invitrogen). For XPF, YY1, tubulin and histone H3 immunodetection we employed Anti-XPF (1:200; ab17798, Abcam), Anti-Tubulin (1:1000; ab44928, Abcam), Anti-YY1 (1:5000; ab199998, Abcam) and Anti-Histone H3 (1:800; ab1791, Abcam) antibodies. Secondary antibodies were as above.
Cell pellets were resuspended in 200 μl lysis buffer (20 mM Tris-HCl pH 7.4, 10 mM KCl, 140 mM NaCl, 1 mM EDTA, 0.2 mM EGTA, 0,5% Triton X-100, 5% Glycerol, 0.1 mM PMSF, 0.5 mM DTT, Halt Protease/Phosphatase Inhibitor Cocktail 1:50) and lysed by sonication. After centrifugation (14,000×g for 10 min at 4 °C) and lysate pre-clearing, 500 μl cell lysate with 1 mg/ml cell total protein was incubated with the primary antibody (ERCC1: ab2356, Abcam, 1:150; XPF: ab76948, Abcam, 4 μg/mg total protein in the lysate) at 4 °C overnight. 50 μl of pre-washed Protein G Dynabeads (Thermo Scientific) was added. After incubation for 2 h at 4 °C, the beads were washed five times using 500 μl washing buffer (20 mM Tris-HCl pH 7.4, 10 mM KCl, 140 mM NaCl, 1 mM EDTA, 0.2 mM EGTA, 0.1% Triton X-100, 5% Glycerol, 0.1 mM PMSF, 0.5 mM DTT, Halt Protease/Phosphatase Inhibitor Cocktail 1:100). Beads were taken up in 20 μl elution buffer, 4× LDS sample buffer and 10× reducing agent. Following incubation for 10 min at 70 °C beads were collected and the supernatant was loaded on a 4–12% Bis-Tris gel. Immunodetection was carried out using anti-XPF (1:200; ab17798, Abcam) or anti-ERCC1 (1:100; sc-17,809, Santa Cruz) primary antibodies. YY1 (1:5000; ab199998, Abcam) served as loading control.
Aliquots of 5000 fibroblasts per well were seeded in 6-well plates. After two days cells were washed with PBS and irradiated at the indicated doses in quadruplicate (0 J/m2) or triplicate (others) using an UV-C germicidal lamp (254 nm; Philips). An UVX Digital Radiometer (Serial No. E27846, UVP) was used to quantify the exact UV dose. Control cells were UV irradiated simultaneously to serve as an internal control. After another five days, before the non-irradiated cultures reached confluency, cells were pulse-labeled with [methyl-3H]-thymidine (40–60 Ci/mmol; 5 μCi/ml; Amersham Biosciences)-containing medium for 3 h, washed with PBS and chased for 15 min in medium without 3H–thymidine. Finally cells were lysed in 0.25 M NaOH. Each lysate was counted with 7.5 ml Hionic Fluor scintillation fluid in a liquid scintillation counter (Packard) for 10 min. The results obtained from irradiated plates were expressed as percentages of non-irradiated plates (set 100%) and plotted .
UV-C-induced UDS and RRS
Unscheduled DNA synthesis (UDS) and recovery of RNA synthesis (RRS) experiments after UV-C irradiation were done with mixed cell populations . C5RO wildtype (WT) fibroblasts were incubated and preloaded with polystyrene beads of 2 μm diameter for three days, then mixed with the patient (3104 or 1333) fibroblasts and seeded on coverslips. Two days later adherent cells were washed with PBS and UV-C irradiated at 16 J/m2. Thereafter they were incubated for 3 h in medium containing 0.1 μM 5-ethynyl-2′-deoxyuridine (EdU; Invitrogen). Afterwards they were washed with PBS, incubated with medium without EdU for 15 min, washed with PBS, fixed with 3.7% formaldehyde in PBS containing 0.5% Triton for 15 min and washed two times with PBS again. The cells were incubated for 30 min with fluorescent dye-coupling buffer containing 10 mM CuSO4 and Alexa Fluor 594 azide (Qlick-iTTM; Invitrogen). After washing with PBS, cells were mounted in Vectashield Antifade Mounting Medium with DAPI (Vector Laboratories). For RRS studies cells were treated identically as for UDS experiments, but 16 h after irradiation incubated with culture medium containing 0.1 μM 5-ethynyl-uridine (EU) for 2 h.
Visual light distinguished WT cells that contained beads in their cytoplasm from patient cells that did not. On micrographs DAPI is the blue signal that stains nuclei. Red is the UDS (or RRS) signal; it is much brighter in WT than in patient cells. UDS and RRS levels are expressed as the average fluorescence intensity in the nucleus of patient vs. WT cells (set at 100%).
An unpaired two-tailed Student’s t test was used to compare the results of qPCR analyses. A p value less than 0.05 was considered significant; *p < 0.05, **p < 0.01, ***p < 0.001.
Chromosome breakage studies from peripheral lymphocyte culture of the patient confirmed FA at ages 7, 33 and 49, and demonstrated increased levels of spontaneous damage, and in response to MMC (Additional file 1: Table S1, results of the analysis carried out age 7 are no longer available). In primary fibroblast cultures a spontaneous and MMC-induced G2-phase arrest was observed (Additional file 1: Figure S1A). Also peripheral blood lymphocyte cultures showed G2-phase accumulation within the FA-range, excluding mosaicism in the hematopoietic system (Additional file 1: Figure S1B). Functional analysis of FANCD2 ubiquitination in 3104 fibroblasts demonstrated both FANCD2 isoforms (Additional file 1: Figure S1C), indicating a mutation in an FA gene downstream of those encoding the FA core- and FANCD2/FANCI-complex. RAD51 foci developed normally after exposure to MMC in both 3104 fibroblasts and lymphoblasts (Additional file 1: Figure S1D), excluding the post-FANCD2 groups FA-D1, -N and -O, −R, –S, and -U. WES identified two mutations in the XPF/ERCC4/FANCQ gene, which were confirmed by Sanger sequencing. A single nucleotide substitution located two base pair upstream of exon 5 (c.793-2A > G) was detected on the maternal allele, affecting a canonical splice acceptor (Fig. 1d). cDNA sequencing verified aberrant splicing. A major splice product revealed exon 5 skipping (Fig. 1e). This event is predicted to result in premature termination of translation (p.Thr265Valfs*13). The second change in the XPF/ERCC4/FANCQ sequence is the missense mutation c.1765C > T in exon 8 (Fig. 1f) on the paternal allele, which is listed on the ExAC-Browser with a minor allele frequency of 0.0066%. It substitutes a highly conserved amino acid residue (p.Arg589Trp) in the SF2 helicase-like domain. This mutation has previously been reported in two XP-F patients (XP24BR and XP32BR) , one patient with XP with neurodegeneration (AS871) , and an individual showing an intermediate XP/CS phenotype and features of FA (XPCS1CD) . Expression of both mutations in mRNA was re-confirmed by qPCR (Additional file 1: Figures S1E and F). Complementation of 3104 fibroblasts by wildtype, but not Arg589Trp-mutant XPF/ERCC4/FANCQ rescued MMC resistance, providing evidence that the mutations of XPF/ERCC4/FANCQ cause the cellular FA phenotype (Fig. 1g). Premature termination of translation due to exon 5 skipping would result in a truncated protein of 31.4 kDa. However, on XPF/ERCC4/FANCQ immunoblot analysis of 3104 fibroblasts only one low signal intensity band of approximately 100 kDa was detected (Fig. 1h), suggesting the presence of residual mutant XPF/ERCC4/FANCQ protein of normal size but reduced abundance. Dilution studies demonstrated that XPF/ERCC4/FANCQ residual protein with the Arg589Trp mutation is present at approximately a 1:15th the level of wildtype XPF/ERCC4/FANCQ protein (Additional file 1: Figure S1G). However, it is difficult to tell the individual contributions of mutant transcript and protein instability to this reduction. Fractionation studies of 3104 fibroblasts showed that residual full-length XPF/ERCC4/FANCQ protein was detectable in the nucleus (Fig. 1i), while in cells from XP-F or XFE patients the mutant XPF/ERCC4/FANCQ appears to be mislocated and not to be part of the XPF-ERCC1 complex, presumably as a consequence of XPF protein misfolding .
Discussion and conclusion
Long-term observations and genotype/phenotype correlations in rare variants of DNA-repair disorders unlock a potential for understanding gene function and specific effects in the DNA-damage response network. Here we describe one of the oldest FA patients reported, and the first case who gave a strong history of UV-light sensitivity, which was confirmed on the cellular level. Hence, UV sensitivity can be a feature of FA-Q, and the patient’s perception might be the clinical diagnostic clue for complementation group assignment and mutation detection in unclassified FA patients. From a hematological perspective she has done remarkably well, particularly as she has radial ray malformations which are often associated with early hematological problems .
The Arg589Trp mutation observed in individual 3104 appears to be associated with mislocalization of XPF/FANCQ/ERCC4 protein in XP-F cells, where missense-mutant protein failed to properly translocate to the nucleus . Therefore, the DNA repair defect in XP with this mutation is at least in part likely due to mislocalized protein that still interacts with ERCC1, irrespective of its subcellular localization . We performed co-immunoprecipitation experiments using anti-ERCC1 or anti-XPF antibodies. Both ways, we were able to confirm the interaction in 3104 fibroblasts. In contrast to the literature , residual mutant XPF/FANCQ/ERCC4 of patient 3104 reported here was detectable with chromatin, as previously described for both other reported FA-Q-associated mutations Leu230Pro and Arg689Ser , where an interaction of missense-mutant XPF/FANCQ/ERCC4 proteins with the scaffolding protein SLX4/FANCP and its dimeric partner ERCC1 was identified. Hence, the loss of mutant protein interactions that is critical for the FA-phenotype in FA-Q patients seems unlikely, as Arg589Trp should have the same effect also in XP-F patients. Additional factors modulating the clinical phenotype, which are relevant for FA-Q patients could include a mechanism allowing a proportion of mutant protein to escape misfolding. It will be important to determine if natural chaperones play a role in rescuing XPF/FANCQ/ERCC4 conformational defects, as in other protein-misfolding diseases, to pave the way clinically from XP or CS to FA . The concept of manipulating XP, CS, or XFE therapeutically by targeting nuclear re-localization of mutant XPF-ERCC1 as previously suggested , e.g. by pharmaceutical chaperones, is questioned by our study, as the problem with ICL repair may remain. The reverse, however, targeting interactions or functions of XPF/FANCQ/ERCC4, a key protein at the intersection of DNA repair pathways, could disable NER and ICL repair functions at the same time and might represent a promising approach to overcome chemo-resistance in malignant disease .
In the two other reported FA-Q patients FA104 and 1333 both second XPF/FANCQ/ERCC4 mutations are null alleles . In XPCS1CD the missense mutation p.Cys236Arg confers the XPCS phenotype by conveying insufficient NER activity, but fails to provide endonuclease activity for ICL removal, being a functional null allele in this respect . In the FA patient presented here, the second mutation affects a canonical splice acceptor site (c.793-2A > G) resulting in exon 5 skipping, frameshift and predicted protein truncation. We show that the mutant transcript is relatively unstable and can be stabilized with cycloheximide (Additional file 1: Figure S1F). Moreover, we demonstrate that residual abridged XPF/FANCQ/ERCC4 protein is absent (Fig. 1h) such that this splice site mutation can be regarded as a null allele. In addition there is no cryptic splice acceptor nearby that could restore the reading frame. Pathogenicity of the splice mutation is underscored by the fact that there is neither a listed transcript isoform lacking exon 5 sequence nor a reported alternative ERCC4/XPF/FANCQ splicing product that involves skipping of exon 5. Our findings imply that the common basis of all FA-Q cases to date is compound heterozygosity for one null and one hypomorphic missense mutation in XPF/FANCQ/ERCC4, which facilitates the retention of residual mutant protein capable of chromatin relocation and the provision of NER activity , possibly providing the genetic basis for long-term survival of certain FA-Q patients.
We are extraordinarily grateful to the patient for the provision of detailed information on her life and for agreeing to publication. We express sincere appreciation to Richard Friedl, Christin Berger and Sabine Knaup, Wurzburg, for expert technical support. We thank Dr. Nicolaas G. J. Jaspers, Rotterdam, for initiation of the UV sensitivity studies and Dr. Reinhard Kalb, Wurzburg, for critical reading of the manuscript.
Schroeder-Kurth Fund at the University of Wurzburg (DS); Bloodwise (grant 15038), Kay Kendal Leukaemia Fund, Fanconi Hope, UK (SM).
Availability of data and materials
The material (cell lines, primer sequences) and data generated are available from the corresponding authors on reasonable request.
Ensembl Genome Browser: http://www.ensembl.org/
Exome Aggregation Consortium browser: http://exac.broadinstitute.org/
IP designed, performed and analyzed experiments, prepared figures and wrote the draft. MP, NT, SS, DGE, KC, MM, AC, CH and LH performed clinical investigations. AR and AFT analyzed UDS, RRS and UV sensitivity. SM provided clinical data, and with DS. developed the experimental plan, analyzed data, supervised the study and finalized the manuscript. All authors contributed to final manuscript preparation. All authors read and approved the final manuscript.
Ethics approval and consent to participate
The study was approved by the Institutional Review Board of the Faculty of Medicine at the University of Wurzburg. The patient provided written informed consent for genetic analysis.
Consent for publication
The patient gave written informed consent to studies and publication of clinical and personal data and photographs without eye bars. A copy of the consent form is available to the editor.
The authors declare that they have no competing interests.
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