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. 2016 Sep 1;126(9):3580-4.
doi: 10.1172/JCI88010. Epub 2016 Aug 8.

Biallelic inactivation of REV7 is associated with Fanconi anemia

Dominique Bluteau, Julien Masliah-Planchon, Connor Clairmont, Alix Rousseau, Raphael Ceccaldi, Catherine Dubois d'Enghien, Olivier Bluteau, Wendy Cuccuini, Stéphanie Gachet, Régis Peffault de Latour, Thierry Leblanc, Gérard Socié, André Baruchel, Dominique Stoppa-Lyonnet, Alan D D'Andrea, Jean Soulier

Biallelic inactivation of REV7 is associated with Fanconi anemia

Dominique Bluteau et al. J Clin Invest. .

Erratum in

  • Biallelic inactivation of REV7 is associated with Fanconi anemia.
    Bluteau D, Masliah-Planchon J, Clairmont C, Rousseau A, Ceccaldi R, d'Enghien CD, Bluteau O, Cuccuini W, Gachet S, de Latour RP, Leblanc T, Socié G, Baruchel A, Stoppa-Lyonnet D, D'Andrea AD, Soulier J. Bluteau D, et al. J Clin Invest. 2017 Mar 1;127(3):1117. doi: 10.1172/JCI92946. Epub 2017 Mar 1. J Clin Invest. 2017. PMID: 28248207 Free PMC article. No abstract available.

Abstract

Fanconi anemia (FA) is a recessive genetic disease characterized by congenital abnormalities, chromosome instability, progressive bone marrow failure (BMF), and a strong predisposition to cancer. Twenty FA genes have been identified, and the FANC proteins they encode cooperate in a common pathway that regulates DNA crosslink repair and replication fork stability. We identified a child with severe BMF who harbored biallelic inactivating mutations of the translesion DNA synthesis (TLS) gene REV7 (also known as MAD2L2), which encodes the mutant REV7 protein REV7-V85E. Patient-derived cells demonstrated an extended FA phenotype, which included increased chromosome breaks and G2/M accumulation upon exposure to DNA crosslinking agents, γH2AX and 53BP1 foci accumulation, and enhanced p53/p21 activation relative to cells derived from healthy patients. Expression of WT REV7 restored normal cellular and functional phenotypes in the patient's cells, and CRISPR/Cas9 inactivation of REV7 in a non-FA human cell line produced an FA phenotype. Finally, silencing Rev7 in primary hematopoietic cells impaired progenitor function, suggesting that the DNA repair defect underlies the development of BMF in FA. Taken together, our genetic and functional analyses identified REV7 as a previously undescribed FA gene, which we term FANCV.

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Figures

Figure 1
Figure 1. Genetic and cellular phenotype of the REV7-mutated patient EGF123.
(A) Metaphase EGF123 lymphoid cells upon MMC exposure; arrows show chromosome breaks. Original magnification, ×630. (B) Immunoblot analysis of FANCD2 monoubiquitination of the REV7-mutated (EGF123), FANCA-mutated (EGF192), and healthy fibroblasts. (C) Quantification of the MMC-induced breaks per cell in REV7-mutated (EGF123) and FANCA-mutated (EGF192) EBV-transformed cells and cells of a healthy subject. (D) Cell-cycle analysis after MMC pulse; arrows show the late S/G2 arrest. (E) Proliferation curves after MMC pulse. (F) Protein immunoblot analysis before (–) and 24 hours after (+) MMC pulse; asterisks underline the absence of REV7 in EGF123 protein extracts. (G) Transcript expression levels of a set of DNA damage response genes analyzed by quantitative reverse-transcriptase PCR (qRT-PCR) relative to HPRT (primers are indicated in Supplemental Table 1). Experiments shown in panels E–H were performed using EBV-transformed cells. (H) Partial REV7 exon5 sequence in gDNA from primary fibroblasts.
Figure 2
Figure 2. Correction of the extended phenotype of the EGF123 cells by WT REV7 expression.
(A) Protein immunoblot analysis 24 hours after MMC pulse of EBV-transformed cells from the patient transduced with REV7 or the empty vector. (B) Quantification of MMC-induced breaks per cell. (C) Proliferation curves after MMC pulse. (D) Cell-cycle analysis after MMC pulse; arrows show late S/G2 arrests. (E) Transcript expression levels of a set of DNA damage response genes analyzed by qRT-PCR relative to HPRT. (F) 53BP1 immunofluorescence analysis of EGF123 fibroblasts transduced with REV7 or empty vector, with or without MMC exposure. Scale bars: 10 μm. (G) Percentage of γH2AX-positive cells by flow cytometry analysis with or without MMC exposure. Experiments shown in panels B–F were performed using EBV-transformed cells.
Figure 3
Figure 3. CRISPR/Cas9 knockout of REV7 recapitulates the FA phenotype.
(A) Western blot of whole cell lysates shows no detectable REV7 protein in the REV7–/– line. FANCD2 is still efficiently monoubiquitylated in response to 100 ng/ml MMC. (B) Western blot showing reconstitution of WT REV7 expression in REV7–/– cells. (C–D) Increase in chromosome aberrations (left graph) and radials (right) after 48-hour treatment with 20 ng/ml MMC as compared with cells complemented with WT REV7 cDNA. Original magnification, ×1,000. (E) Increase in G2/M arrest with and without MMC treatment (20 ng/ml). (F) Impaired clonogenic capacity of REV7–/– cells over 10-day treatment with MMC. (G) REV7 is required for normal hematopoiesis; CFU scoring of bone marrow Lin– cells after lentiviral silencing using REV7 or scramble (scr) shRNAs. Fancg-/- cells are used as FA cell control. CFUs were numbered after 7 days on methylcellulose; 2 passages (P1 and P2) were performed. (H) Cell population percentages in CFUs after 7 days of culture.
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References

    1. Shimamura A, Alter BP. Pathophysiology and management of inherited bone marrow failure syndromes. Blood Rev. 2010;24(3):101–122. doi: 10.1016/j.blre.2010.03.002. - DOI - PMC - PubMed
    1. Soulier J. Fanconi anemia. Hematology Am Soc Hematol Educ Program. 2011;2011:492–497. - PubMed
    1. Kottemann MC, Smogorzewska A. Fanconi anaemia and the repair of Watson and Crick DNA crosslinks. Nature. 2013;493(7432):356–363. doi: 10.1038/nature11863. - DOI - PMC - PubMed
    1. Bogliolo M, Surrallés J. Fanconi anemia: a model disease for studies on human genetics and advanced therapeutics. Curr Opin Genet Dev. 2015;33:32–40. doi: 10.1016/j.gde.2015.07.002. - DOI - PubMed
    1. Ceccaldi R, Sarangi P, D’Andrea AD. The Fanconi anaemia pathway: new players and new functions. Nat Rev Mol Cell Biol. 2016;17(6):337–349. - PubMed

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