Identification and biochemical characterization of a novel mutation in DDX11 causing Warsaw breakage syndrome - PubMed (original) (raw)

Case Reports

doi: 10.1002/humu.22226. Epub 2012 Oct 17.

Sanjay Kumar Bharti, Joshua A Sommers, Tony Yammine, Eliane Chouery, Lysanne Patry, Guy A Rouleau, Mark E Samuels, Fadi F Hamdan, Jacques L Michaud, Robert M Brosh Jr, André Mégarbane, Zoha Kibar

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Case Reports

Identification and biochemical characterization of a novel mutation in DDX11 causing Warsaw breakage syndrome

José-Mario Capo-Chichi et al. Hum Mutat. 2013 Jan.

Abstract

Mutations in the gene encoding the iron-sulfur-containing DNA helicase DDX11 (ChlR1) were recently identified as a cause of a new recessive cohesinopathy, Warsaw breakage syndrome (WABS), in a single patient with severe microcephaly, pre- and postnatal growth retardation, and abnormal skin pigmentation. Here, using homozygosity mapping in a Lebanese consanguineous family followed by exome sequencing, we identified a novel homozygous mutation (c.788G>A [p.R263Q]) in DDX11 in three affected siblings with severe intellectual disability and many of the congenital abnormalities reported in the WABS original case. Cultured lymphocytes from the patients showed increased mitomycin C-induced chromosomal breakage, as found in WABS. Biochemical studies of purified recombinant DDX11 indicated that the p.R263Q mutation impaired DDX11 helicase activity by perturbing its DNA binding and DNA-dependent ATP hydrolysis. Our findings thus confirm the involvement of DDX11 in WABS, describe its phenotypical spectrum, and provide novel insight into the structural requirement for DDX11 activity.

© 2012 Wiley Periodicals, Inc.

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Conflict of interest statement

Disclosure Statement: The authors confirm that they have no conflicts of interest.

Figures

Figure 1

Figure 1

A pathogenic mutation identified in DDX11 in a family affected with WABS. A, B: Pedigree of the consanguineous Lebanese family studied here and their facial features. The proband is indicated by an arrow. Patient V-2 was deceased hence certain analyses could not be extended to biological sampling of this child. C: Schematic representation of DDX11 showing the helicase motifs (I–VI) as well as the Fe–S motif of DDX11. p.R263Q identified herein as well as the two previously published DDX11 mutations (c.2271+2T>C and p.K897del) are shown. D: Integrated Genomics Viewer (IGV) tracks of exome sequencing reads showing the presence of the homozygous DDX11 c.788G>A (p.R263Q) mutation in patient V-1. E: Sanger sequencing validation of c.788G>A (p.R263Q) DDX11 mutation. Representative chromatograms of the parents (both heterozygous for c.788G>A) and the three probands (all homozygous for c.788G>A) are shown. Nucleotide numbering reflects cDNA numbering with +1 corresponding to the A of the ATG translation initiation codon. F: Amino acid alignment of the beginning of Fe–S domain region of DDX11 in different species as well as the corresponding regions in the human-related DNA helicases: XPD (ERCC2), FANCJ, and RTEL1. R263 in human DDX11 is well conserved (in bold). R112 residue in human XPD whose mutation (c.335G>A, p.R112H) causes trichothiodystrophy is italicized and underlined. G: Mitomycin C (MMC)-induced chromosomal breakage. Representative metaphase from blood cultures treated with MMC showed increased chromosomal defects (arrows), including breakage, radial formations, centromeric heterochromatin repulsion (“railroads”) from patient V-1 (i), in contrast to the metaphases prepared from a similarly treated blood cultures prepared from a healthy control individual (ii). Table showing the representation of cells with chromosomal (chr) breaks in presence or absence of MMC. (NT, not tested.) A total of 200 cells (metaphases) were screened for each individual (iii).

Figure 2

Figure 2

Purification and biochemical analyses of recombinant DDX11-WT and DDX11-R263Q for helicase activity and DNA binding. A: The purity of DDX11-WT and DDX11-R263Q was evaluated by Coomassie stained SDS PAGE (predicted MW approximately 110 kD). (M, molecular weight protein standards.) B,C: Helicase activity of DDX11-WT(B) and DDX11-R263Q (C) on forked duplex DNA substrate. Helicase reactions (20 _μ_l) were performed by incubating the indicated DDX11 protein with 0.5 nM forked duplex DNA substrate at 37°C for 15 min under standard helicase assay conditions as described in the methods section in the Supporting Information. Products were resolved on native 10% polyacrylamide gels and representative phosphorimages are shown. Triangle denotes heat-denatured DNA substrate control, and asterisk denotes 5′-32P end label. D: Quantitative analysis of data from helicase activity of DDX11-R263Q and DDX11-WT on forked duplex DNA substrate is shown. Data represent the mean of at least three independent experiments with standard deviations (SD) indicated by error bars. E, F: DNA binding by DDX11-WT and DDX11-R263Q as detected by gel mobility shift assays. The indicated concentrations of DDX11-WT (E) and DDX11-R263Q (F) were incubated with 0.5 nM forked duplex DNA substrate on ice for 30 min under standard gel shift assay conditions as described in the Supporting Information. The DNA-protein complexes were resolved on native 5% polyacrylamide gels. Representative phosphorimages for DNA-binding assays are shown. Asterisk denotes 5′-32P end label.

References

    1. Botta E, Nardo T, Lehmann AR, Egly JM, Pedrini AM, Stefanini M. Reduced level of the repair/transcription factor TFIIH in trichothiodystrophy. Hum Mol Genet. 2002;11:2919–2928. - PubMed
    1. Daoud H, Suhail H, Szuto A, Camu W, Salachas F, Meininger V, Bouchard JP, Dupre N, Dion PA, Rouleau GA. UBQLN2 mutations are rare in French and French–Canadian amyotrophic lateral sclerosis. Neurobiol Aging. 2012;33:2230e1–2230e5. - PubMed
    1. Ding H, Schertzer M, Wu X, Gertsenstein M, Selig S, Kammori M, Pourvali R, Poon S, Vulto I, Chavez E, et al. Regulation of murine telomere length by Rtel: an essential gene encoding a helicase-like protein. Cell. 2004;117:873–886. - PubMed
    1. Dubaele S, Proietti De Santis L, Bienstock RJ, Keriel A, Stefanini M, Van Houten B, Egly JM. Basal transcription defect discriminates between xeroderma pigmentosum and trichothiodystrophy in XPD patients. Mol Cell. 2003;11:1635–1646. - PubMed
    1. Hirota Y, Lahti JM. Characterization of the enzymatic activity of hChlR1, a novel human DNA helicase. Nucleic Acids Res. 2000;28:917–924. - PMC - PubMed

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