Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

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

Abstract

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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

Prices may be subject to local taxes which are calculated during checkout

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.

Similar content being viewed by others

Accession codes

Primary accessions

NCBI Reference Sequence

Swiss-Prot

References

  1. Centore, R.C., Yazinski, S.A., Tse, A. & Zou, L. Spartan/C1orf124, a reader of PCNA ubiquitylation and a regulator of UV-induced DNA damage response. Mol. Cell 46, 625–635 (2012).

    Article  CAS  Google Scholar 

  2. Davis, E.J. et al. DVC1 (C1orf124) recruits the p97 protein segregase to sites of DNA damage. Nat. Struct. Mol. Biol. 19, 1093–1100 (2012).

    Article  CAS  Google Scholar 

  3. Mosbech, A. et al. DVC1 (C1orf124) is a DNA damage–targeting p97 adaptor that promotes ubiquitin-dependent responses to replication blocks. Nat. Struct. Mol. Biol. 19, 1084–1092 (2012).

    Article  CAS  Google Scholar 

  4. Juhasz, S. et al. Characterization of human Spartan/C1orf124, an ubiquitin-PCNA interacting regulator of DNA damage tolerance. Nucleic Acids Res. 40, 10795–10808 (2012).

    Article  CAS  Google Scholar 

  5. Kim, M.S. et al. Regulation of error-prone translesion synthesis by Spartan/C1orf124. Nucleic Acids Res. 41, 1661–1668 (2013).

    Article  CAS  Google Scholar 

  6. Machida, Y., Kim, M.S. & Machida, Y.J. Spartan/C1orf124 is important to prevent UV-induced mutagenesis. Cell Cycle 11, 3395–3402 (2012).

    Article  CAS  Google Scholar 

  7. Ghosal, G., Leung, J.W., Nair, B.C., Fong, K.W. & Chen, J. Proliferating cell nuclear antigen (PCNA)-binding protein C1orf124 is a regulator of translesion synthesis. J. Biol. Chem. 287, 34225–34233 (2012).

    Article  CAS  Google Scholar 

  8. Burtner, C.R. & Kennedy, B.K. Progeria syndromes and ageing: what is the connection? Nat. Rev. Mol. Cell Biol. 11, 567–578 (2010).

    Article  CAS  Google Scholar 

  9. Fletcher, O. & Houlston, R.S. Architecture of inherited susceptibility to common cancer. Nat. Rev. Cancer 10, 353–361 (2010).

    Article  CAS  Google Scholar 

  10. Martin, G.M. Genetic syndromes in man with potential relevance to the pathobiology of aging. Birth Defects Orig. Artic. Ser. 14, 5–39 (1978).

    CAS  PubMed  Google Scholar 

  11. Navarro, C.L., Cau, P. & Levy, N. Molecular bases of progeroid syndromes. Hum. Mol. Genet. 15, R151–R161 (2006).

    Article  CAS  Google Scholar 

  12. Puente, X.S. et al. Exome sequencing and functional analysis identifies BANF1 mutation as the cause of a hereditary progeroid syndrome. Am. J. Hum. Genet. 88, 650–656 (2011).

    Article  CAS  Google Scholar 

  13. Chen, L. et al. LMNA mutations in atypical Werner's syndrome. Lancet 362, 440–445 (2003).

    Article  CAS  Google Scholar 

  14. Oshima, J. & Hisama, F.M. Search and insights into novel genetic alterations leading to classical and atypical Werner syndrome. Gerontology 60, 239–246 (2014).

    Article  CAS  Google Scholar 

  15. Ruijs, M.W. et al. Atypical progeroid syndrome: an unknown helicase gene defect? Am. J. Med. Genet. A. 116A, 295–299 (2003).

    Article  CAS  Google Scholar 

  16. Vaz, B., Halder, S. & Ramadan, K. Role of p97/VCP (Cdc48) in genome stability. Front. Genet. 4, 60 (2013).

    Article  Google Scholar 

  17. King, K.L. et al. Ki-67 expression as a prognostic marker in patients with hepatocellular carcinoma. J. Gastroenterol. Hepatol. 13, 273–279 (1998).

    Article  CAS  Google Scholar 

  18. Nowsheen, S., Aziz, K., Panayiotidis, M.I. & Georgakilas, A.G. Molecular markers for cancer prognosis and treatment: have we struck gold? Cancer Lett. 327, 142–152 (2012).

    Article  CAS  Google Scholar 

  19. Hoehn, H. et al. Variegated translocation mosaicism in human skin fibroblast cultures. Cytogenet. Cell Genet. 15, 282–298 (1975).

    Article  CAS  Google Scholar 

  20. Aanes, H. et al. Zebrafish mRNA sequencing deciphers novelties in transcriptome dynamics during maternal to zygotic transition. Genome Res. 21, 1328–1338 (2011).

    Article  CAS  Google Scholar 

  21. Shen, J.C. & Loeb, L.A. The Werner syndrome gene: the molecular basis of RecQ helicase–deficiency diseases. Trends Genet. 16, 213–220 (2000).

    Article  CAS  Google Scholar 

  22. Venkatesan, R.N. et al. Mutation at the polymerase active site of mouse DNA polymerase δ increases genomic instability and accelerates tumorigenesis. Mol. Cell. Biol. 27, 7669–7682 (2007).

    Article  CAS  Google Scholar 

  23. Branzei, D. & Foiani, M. Maintaining genome stability at the replication fork. Nat. Rev. Mol. Cell Biol. 11, 208–219 (2010).

    Article  CAS  Google Scholar 

  24. Zeman, M.K. & Cimprich, K.A. Causes and consequences of replication stress. Nat. Cell Biol. 16, 2–9 (2014).

    Article  CAS  Google Scholar 

  25. Costantino, L. et al. Break-induced replication repair of damaged forks induces genomic duplications in human cells. Science 343, 88–91 (2014).

    Article  CAS  Google Scholar 

  26. Lukas, C. et al. 53BP1 nuclear bodies form around DNA lesions generated by mitotic transmission of chromosomes under replication stress. Nat. Cell Biol. 13, 243–253 (2011).

    Article  CAS  Google Scholar 

  27. Sidorova, J.M., Li, N., Folch, A. & Monnat, R.J. Jr. The RecQ helicase WRN is required for normal replication fork progression after DNA damage or replication fork arrest. Cell Cycle 7, 796–807 (2008).

    Article  CAS  Google Scholar 

  28. Davies, S.L., North, P.S. & Hickson, I.D. Role for BLM in replication-fork restart and suppression of origin firing after replicative stress. Nat. Struct. Mol. Biol. 14, 677–679 (2007).

    Article  CAS  Google Scholar 

  29. Löbrich, M. & Jeggo, P.A. The impact of a negligent G2/M checkpoint on genomic instability and cancer induction. Nat. Rev. Cancer 7, 861–869 (2007).

    Article  Google Scholar 

  30. El-Serag, H.B. Hepatocellular carcinoma. N. Engl. J. Med. 365, 1118–1127 (2011).

    Article  CAS  Google Scholar 

  31. Caldwell, S. & Park, S.H. The epidemiology of hepatocellular cancer: from the perspectives of public health problem to tumor biology. J. Gastroenterol. 44 (suppl. 19), 96–101 (2009).

    Article  Google Scholar 

  32. Wilsker, D., Petermann, E., Helleday, T. & Bunz, F. Essential function of Chk1 can be uncoupled from DNA damage checkpoint and replication control. Proc. Natl. Acad. Sci. USA 105, 20752–20757 (2008).

    Article  CAS  Google Scholar 

  33. Borck, G. et al. Loss-of-function mutations of ILDR1 cause autosomal-recessive hearing impairment DFNB42. Am. J. Hum. Genet. 88, 127–137 (2011).

    Article  CAS  Google Scholar 

  34. Bahlo, M. & Bromhead, C.J. Generating linkage mapping files from Affymetrix SNP chip data. Bioinformatics 25, 1961–1962 (2009).

    Article  CAS  Google Scholar 

  35. Abecasis, G.R., Cherny, S.S., Cookson, W.O. & Cardon, L.R. Merlin—rapid analysis of dense genetic maps using sparse gene flow trees. Nat. Genet. 30, 97–101 (2002).

    Article  CAS  Google Scholar 

  36. Leutenegger, A.L. et al. Estimation of the inbreeding coefficient through use of genomic data. Am. J. Hum. Genet. 73, 516–523 (2003).

    Article  CAS  Google Scholar 

  37. DePristo, M.A. et al. A framework for variation discovery and genotyping using next-generation DNA sequencing data. Nat. Genet. 43, 491–498 (2011).

    Article  CAS  Google Scholar 

  38. McKenna, A. et al. The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res. 20, 1297–1303 (2010).

    Article  CAS  Google Scholar 

  39. Wang, K., Li, M. & Hakonarson, H. ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data. Nucleic Acids Res. 38, e164 (2010).

    Article  Google Scholar 

  40. Drmanac, R. et al. Human genome sequencing using unchained base reads on self-assembling DNA nanoarrays. Science 327, 78–81 (2010).

    Article  CAS  Google Scholar 

  41. Bansal, V. & Bafna, V. HapCUT: an efficient and accurate algorithm for the haplotype assembly problem. Bioinformatics 24, i153–i159 (2008).

    Article  Google Scholar 

  42. Hisama, F.M. et al. Coronary artery disease in a Werner syndrome–like form of progeria characterized by low levels of progerin, a splice variant of lamin A. Am. J. Med. Genet. A. 155A, 3002–3006 (2011).

    Article  Google Scholar 

  43. Saha, B. et al. Ethnic-specific WRN mutations in South Asian Werner syndrome patients: potential founder effect in patients with Indian or Pakistani ancestry. Mol. Genet. Genomic Med. 1, 7–14 (2013).

    Article  CAS  Google Scholar 

  44. Jackson, D.A. & Pombo, A. Replicon clusters are stable units of chromosome structure: evidence that nuclear organization contributes to the efficient activation and propagation of S phase in human cells. J. Cell Biol. 140, 1285–1295 (1998).

    Article  CAS  Google Scholar 

  45. Cortez, D., Guntuku, S., Qin, J. & Elledge, S.J. ATR and ATRIP: partners in checkpoint signaling. Science 294, 1713–1716 (2001).

    Article  CAS  Google Scholar 

  46. Zhou, W. et al. FAN1 mutations cause karyomegalic interstitial nephritis, linking chronic kidney failure to defective DNA damage repair. Nat. Genet. 44, 910–915 (2012).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

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.

Author information

Authors and Affiliations

Authors

Contributions

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.

Ethics declarations

Competing interests

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.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–15 and Supplementary Tables 1–5 (PDF 7074 kb)

Source data

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

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). https://doi.org/10.1038/ng.3103

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ng.3103

This article is cited by

Search

Quick links

Nature Briefing: Cancer

Sign up for the Nature Briefing: Cancer newsletter — what matters in cancer research, free to your inbox weekly.

Get what matters in cancer research, free to your inbox weekly. Sign up for Nature Briefing: Cancer