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Mutations in SPRTN cause early onset hepatocellular carcinoma, genomic instability and progeroid features


Age-related degenerative and malignant diseases represent major challenges for health care systems. Elucidation of the molecular mechanisms underlying carcinogenesis and age-associated pathologies is thus of growing biomedical relevance. We identified biallelic germline mutations in SPRTN (also called C1orf124 or DVC1)1,2,3,4,5,6,7 in three patients from two unrelated families. All three patients are affected by a new segmental progeroid syndrome characterized by genomic instability and susceptibility toward early onset hepatocellular carcinoma. SPRTN was recently proposed to have a function in translesional DNA synthesis and the prevention of mutagenesis1,2,3,4,5,6,7. Our in vivo and in vitro characterization of identified mutations has uncovered an essential role for SPRTN in the prevention of DNA replication stress during general DNA replication and in replication-related G2/M-checkpoint regulation. In addition to demonstrating the pathogenicity of identified SPRTN mutations, our findings provide a molecular explanation of how SPRTN dysfunction causes accelerated aging and susceptibility toward carcinoma.

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Figure 1: Identification of causative SPRTN mutations.
Figure 2: Severe DNA damage in hepatocellular carcinoma biopsies and focal nuclear accumulation of SPRTN.
Figure 3: Genomic instability and cell proliferation defects.
Figure 4: DNA replication stress and leakage of the G2/M cell cycle checkpoint as the origin of genomic instability.
Figure 5: Characterization of patients' mutations in DNA replication and G2/M-checkpoint regulation.

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We are thankful to the family members for participation, G. Gillies for assistance with patient samples, J. Schäfer for zebrafish care and Z. Garajova for technical assistance. We thank A. Ray Chaudhuri for initial help with the DNA fiber assay. We thank F. Böhm, Y. Böge and A. Weber from the University of Zurich and L. Campo and K. Myers from the University of Oxford for providing healthy and HCC human liver biopsies and performing histological and immunohistochemical staining. The zebrafish γ-H2AX antibody was a kind gift of J. Amatruda (University of Texas Southwestern). This work was supported by grants from Deutsche Forschungsgemeinschaft, the Cluster of Excellence 'Macromolecular Complexes' of Goethe University Frankfurt (EXC115), the Landes-Offensive zur Entwicklung Wissenschaftlich-ökonomischer Exzellenz program Ubiquitin Networks of the State of Hesse, Germany and the European Research Council under the European Union's Seventh Framework Programme (FP7/2007-2013)/European Research Council grant agreement number 250241-LineUb to I.D., the European Commission (Marie Curie Reintegration Grant 268333 to M.P.), the Deutsche Stiftung für Herzforschung (M.P.), the Medical Research Council (MC_PC_12001/1) and the Swiss National Science Foundation (31003A_141197) to K.R., grants from the US National Institutes of Health (NIH) National Cancer Institute (R24CA78088 and R24AG042328) to G.M.M., the NIH National Institute on Aging (R21AG033313) to J. Oshima, the Ellison Medical Foundation to J. Oshima, the German Research Foundation (DFG) in the framework of the Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases to C.K., an EMBO long-term fellowship to J.L.-M., a grant from the Croatian Ministry of Science, Education and Sport (216-0000000-3348) and a City of Split grant to J.T. and I.D. K.R.S. is supported by a PhD scholarship funded by the Pratt Foundation. M.B. is supported by an Australian Research Council Future Fellowship (FT100100764). P.J.L. is supported by a National Health and Medical Research Council (NHMRC) Career Development Fellowship (APP1032364). This work was made possible through Victorian State Government Operational Infrastructure Support and the Australian Government NHMRC Independent Research Institutes Infrastructure Support Scheme.

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Authors and Affiliations



D.L., B.V., S.H., P.J.L., I.M.-T., J.L.-M., M.P., J.C.H.S., K.R.S., J. Oehler, K.P., A.N., F.N., R.J.L., M.B.D., G.B., S.v.A., J.H., M.D., R. Fertig, M.D.B., K.H., H.T., J.A., G.N., P.N. and M.B. performed the experiments and did data analysis. E.C., R. Freire, J. Oshima, G.M.M. and C.M.A. contributed materials and reagents used in the study. D.L., K.R. and C.K. wrote the manuscript. J.T., D.J.A., I.D., K.R. and C.K. led and coordinated the entire project.

Corresponding authors

Correspondence to Janos Terzic, David J Amor, Ivan Dikic, Kristijan Ramadan or Christian Kubisch.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Parametric genome-wide linkage analysis.

(a) LOD scores obtained from parametric linkage analysis of family A. Linkage was calculated using 20,044 selected SNP markers from the Affymetrix SNP Array 6.0. LOD scores calculated with ALLEGRO are given along the y-axis relative to genomic position in cM (centiMorgan) on the x-axis. Chromosomes are concatenated from p-ter to q-ter from left to right. Red arrow indicates position of SPRTN. (b) LOD scores obtained from parametric linkage analysis of Family B under a recessive genetic model. LOD scores are on the y-axis and cumulative genomic position in cM (centiMorgan) on x-axis. The vertical lines depict the boundaries of each chromosome. The maximum theoretical autosomal LOD score of 0.85 is achieved at 29 sites, while the maximum chromosome X LOD score of 0.30 is achieved at four regions. The red arrow indicates the location of SPRTN.

Supplementary Figure 2 RT-PCR analysis and Sanger sequencing.

(a) RT-PCR study of SPRTN. M indicates molecular marker. Reaction A, amplifying isoform 1, with primers in exon 2 and 5, revealed a full length allele of 1205 bp (blue arrow) in both a healthy individual (CTRL) and B-II:1. A very weak band (red arrow) corresponding to skipping of exon 4 (1205 bp-268 bp=937 bp) is observed in B-II:1. Reaction B, amplifying isoform 2, with primers in exon 2 and intron 4 revealed a band at 640 bp (pink arrow). (b) Sequence trace of the B-II:1. Reaction A amplified only the allele with the missense p.Tyr117Cys variant. Reaction B preferentially amplified the allele without the missense variant. (c) Sequencing of the reaction B in B-II:1 with reverse primer revealed the c.717_718+2delAGGT mutation, confirming that reaction B amplified only the allele including the 4 bp deletion. Therefore the consequence of this deletion is mostly intron 4 inclusion causing p.Lys239LysfsX7. (d) Sequence chromatograms of all three identified mutations, after PCR amplification of genomic DNA. The amino acid translation is shown in the three-letter code above the chromatograms.

Supplementary Figure 3 Representative images of peripheral blood metaphase spreads of B-II:4.

(a) G-banded analysis of lymphocyte cells showed random, spontaneous breaks and unbalanced rearrangements, including deletions, acentric fragments and marker chromosomes in 13 of 30 cells analyzed. The remaining cells showed an apparently normal male karyotype. A representative abnormal karyotype shows 46,XY,del(5)(?q31.3). (b) Examination of solid stained metaphase chromosomes showed an abnormal level of spontaneous chromosome breakage (arrows). There were 39 breaks in 45 analyzed cells, while the control sample showed 3 breaks in 45 cells. A similar level of spontaneous breakage was observed in the other proband (B-II:1). G-banded analysis of lymphocyte cells showed random, spontaneous breaks and rearrangements, including translocations and deletions, in 14 of 25 cells analyzed (data not shown).

Supplementary Figure 4 Representative images of fibroblast metaphase spreads of B-II:1.

G-banded analysis of two subsequent B-II:1 fibroblast passages, 15 and 16, showed random, spontaneous breaks, balanced and unbalanced rearrangements, including deletions, acentric fragments and marker chromosomes in 9 of 30 cells analyzed from passage 15, and in 9 of 42 cells analyzed from passage 16. The remaining cells showed an apparently normal male karyotype. Depicted are two cytogenetically defined clones in both subsequent passages, 46,XY,del(9)(?p21) and 45,XY,-21,del(6)(?q13), the latter was identified in three metaphases of passage 16. Examination of solid stained metaphase chromosomes also showed an abnormal level of spontaneous chromosome breakage. There were 43 breaks in 47 analysed cells, while the control sample showed 1 break in 30 cells (data not shown).

Supplementary Figure 5 Depletion of SPRTN by siRNA results in chromosomal instability.

(a) Representative images of metaphase spreads of siRNA transfected HEK293T cells. Black arrows indicate chromatid breaks. Red arrows indicate open multiradial figures each representing several chromatid breaks. (b) Mean number of aberrations per cell observed in 100 metaphases of HEK293T cells transfected with siRNA alone or additionally treated either with 40 ng/ml of MMC or 200 ng/ml of 4-NQO. Bar graphs summarize three independent experiments. p-values are given above the graphs. (c) Depletion of SPRTN was confirmed by western blot. Tubulin was used as a loading control.

Supplementary Figure 6 SPRTN depletion suppresses cell proliferation.

(a) 50’000 of U2OS cells were seeded at day 0 and normalized to 1 (y-axis). Every 24 hours cells (x-axis) were washed, trypsinized, resuspended in 1 mL medium and counted. The graph represents the mean +/- standard deviation of 3 independent experiments. Each experiment was performed in triplicate. (b) Efficacy of SPRTN depletion.

Supplementary Figure 7 SPRTN has an evolutionary conserved role in the DNA damage response.

(a) Morpholino specificity and efficacy demonstrated by knockdown of GFP reporter expression. Co-injection of the translation blocking antisense MO targeted against the 5′-UTR inhibited all GFP fluorescence, while coinjection of a 5 bases mismatch control MO did not repress fluorescence. (b) Lateral view of the head region of 27 hpf zebrafish embryos stained with zebrafish anti γ-H2AX (FITC). Nuclei were counterstained with DAPI (blue). Knockdown of sprtn with 1.5 ng ATG MO significantly increases the staining for γ-H2AX, indicating increased DDR. (c) Quantification of γ-H2AX foci after sprtn downregulation in zebrafish. Bar graphs summarize three independent experiments. Statistical significance was determined using Fisher's exact test, p-values are given above the graphs. NI = non injection.

Supplementary Figure 8 Mutations in SPRTN cause elevated number of DNA double-strand breaks (DSBs) in S phase.

(a) Primary skin fibroblasts stained with the S-phase cell cycle marker (Cyclin A) and antibodies against 53BP1 (DNA double strand breaks marker). (b) The number of 53BP1 foci per cell in cyclin A positive cells was quantified in three independent experiments with greater than 100 cells scored per condition per experiment.

Supplementary Figure 9 Overexpression of WT SPRTN rescues DNA replication and proliferation phenotypes in patient LCLs and fibroblasts.

(a) The overexpression of EGFP-SPRTN-wt in stable transfected LCLs was confirmed by fluorescence microscopy (upper panel) and flow cytometry (lower panel). Note that only 20-24% of stable-transfected LCLs express EGFP-SPRTN. (b) Flag-SPRTN-wt or Flag-empty vector transiently transfected B-II:1 skin fibroblasts were seeded and growth curve was measured as indicated in Supplementary Fig. 6; three independent experiments, error bars; s.e.m. (c) Flag-SPRTN-wt expression in primary skin fibroblasts was confirmed by immunofluorescence analysis.

Supplementary Figure 10 Depletion of SPRTN causes DNA replication stress.

a) U2OS cells were treated with two independent siRNAs against SPRTN (siRNA-SPRTN#1 or siRNA-SPRTN#2) or control siRNA (siCTRL) and DNA replication forks were analyzed. The quantification from three independent experiments using the same methodology as in Figure 4 Panels a-d. (b) Representative cells with huge increase in the number of 53BP1 (markers of DSBs) foci in S-phase (EdU-positive; cells were grown in the presence of EdU for 30 min before fixation) of SPRTN-depleted U2OS cells. % of cells with depicted phenotypes when more than 100 cells were scored per condition in three independent experiments. (c) Efficacy of SPRTN depletion.

Supplementary Figure 11 Depletion of DNA polymerase η is unable to rescue the DNA replication defect in patient LCLs or SPRTN-depleted U2OS cells.

Analysis of DNA replication fork progression in patient cells or SPRTN-depleted U2OS cells +/- co-depletion of DNA polymerase η by DNA fiber assay as described earlier in the text. (a) DNA fiber analysis of patient LCLs in the presence of APH. (b) Efficacy of siRNA depletion as indicated. (c) DNA fiber analysis with (right panel) or without APH (left) in siRNA-depleted U2OS cells. Two independent experiments with 100 fibers per condition per experiment. (d) The efficacy of depletion.

Supplementary Figure 12 Cell cycle profile of unchallenged or CPT-challenged patients’ LCL cells.

(a) Graphic representation of the cell cycle distribution in control and patient LCLs. G1, S, G2 and mitotic (M) cells were determinated by flow cytometry based on the triple staining with propidium iodide (PI: DNA content), EdU (S-phase) and phosph-Ser10 histone H3 (marker for mitotic cells).(b) Representative histograms for DNA content (PI). The y-axis represents cell counts and the x-axis represents DNA area. The first peak indicates cells in G1 and the second peak indicates cells in G2-M. Cells between two indicated peaks represent S-phase. (c) Representative dotplot distribution of EdU (left graph) or phospho-Ser10 histone H3 (right graph) positive cells (y axis) versus DNA content (x axis). (d) Graphic representation of the cell cycle distribution following induction of replication-related DSB (1µM CPT treatment for 1 hour and recovery for 16 hours). (e) Representative dotplot distribution of mitotic cells detected by p-H3(S10) and depicted in pink rectangles (% of M cells).

Supplementary Figure 13 G2/M checkpoint leakage following replication-related DSB but not ionizing radiation (IR)-induced DSB of patient LCLs.

(a) Cell cycle profile of patient cells before DNA damage or after UV-irradiation (10 J/m2) or IR (6 Gy) treatment. The cells were allowed to recover for 16 hours in the presence of nocodazole and analysed by PI profile. (b) Representative dotplot distribution of phospho-Serine-10 histone H3 positive cells. (c) Due to proliferation defects observed in patient LCL’s, G2-M checkpoint efficiency was expressed as the mitotic ratio between the number of mitotic cells after and before DNA damage. Note that G2/M-checkpoint is not properly functional in UV-treated patient LCLs (about 3-fold less mitotic cells after UV) when compared to control cells (7-fold less mitotic cells after UV). In contrary to UV, IR treatment (b and c) is equally efficient in both patient and control LCLs (about 12 fold).

Supplementary Figure 14 Patient cells are hypersensitive to replication-related DNA damaging agents but not to IR.

Cell viability of control and patient LCL’s after treatment with different DNA damaging agents: (a) Camptothecin (CPT), (b) Mitomycin C (MMC), (c) UV-radiation, and (d) ionizing radiation (IR), using a commercial MTT assay. The cells were treated for 48 hours with the indicated genotoxic agents and allowed to recover for 48 hours. (a-d) Three independent experiments, and each experiment was performed in triplicate. n.s. = not significant, unpaired t-test.

Supplementary Figure 15 Depletion of SPRTN causes G2/M-checkpoint leakage after UV in U2OS cells.

(a) Cell cycle profile analysed by DNA content (DAPI) of siRNA-depleted cells without or with UV-irradiation in the presence of nocodazole (NOC). (b) Western blot analysis of siRNA-depletion efficacy. (c) A representative dotplot distribution of mitotic cells detected by p-H3(S10) and depicted in red rectangles (% of M cells). In control cells (siCTRL) there is approximately 8-fold less mitotic cells after UV comparing to unchallenged conditions (efficient G2/M-arrest), whereas in SPRTN-depleted cells there is only about 3-fold (SPRTN# 2) or 5-fold less (SPRTN#1). (d) To better visualize the G2/M-defect after UV-damage in DVC1-depleted cells mitotic cells were calculated and presented as a mitotic ratio after and before UV-treatment. Depletion of Chk1 was used as a positive control for G2/M leakage.

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Lessel, D., Vaz, B., Halder, S. et al. Mutations in SPRTN cause early onset hepatocellular carcinoma, genomic instability and progeroid features. Nat Genet 46, 1239–1244 (2014).

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