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Case Reports
. 2010 Nov 16;5(11):e13659.
doi: 10.1371/journal.pone.0013659.

The effect of gentamicin-induced readthrough on a novel premature termination codon of CD18 leukocyte adhesion deficiency patients

Amos J Simon  1 , Atar Lev, Baruch Wolach, Ronit Gavrieli, Ninette Amariglio, Ester Rosenthal, Ephraim Gazit, Eran Eyal, Gideon Rechavi, Raz Somech
Affiliations
Case Reports

The effect of gentamicin-induced readthrough on a novel premature termination codon of CD18 leukocyte adhesion deficiency patients

Amos J Simon et al. PLoS One. .

Abstract

Background: Leukocyte adhesion deficiency 1 (LAD1) is an inherited disorder of neutrophil function. Nonsense mutations in the affected CD18 (ITB2) gene have rarely been described. In other genes containing such mutations, treatments with aminoglycoside types of antibiotics (e.g., gentamicin) were reported to partially correct the premature protein termination, by induction of readthrough mechanism.

Methodology/principal findings: Genetic analysis was performed on 2 LAD1 patients. Expression, functional and immunofluorescence assays of CD18 in the patients were used to determine the in-vivo and in-vitro effects of gentamicin-induced readthrough. A theoretical modeling of the corrected CD18 protein was developed to predict the protein function.

Results: We found a novel premature termination codon, C562T (R188X), in exon 6 of the CD18 gene that caused a severe LAD1 phenotype in two unrelated Palestinian children. In-vivo studies on these patients' cells after gentamicin treatment showed abnormal adhesion and chemotactic functions, while in-vitro studies showed mislocalization of the corrected protein to the cytoplasm and not to the cell surface. A theoretical modeling of the corrected CD18 protein suggested that the replacement of the wild type arginine by gentamicin induced tryptophan at the position of the nonsense mutation, although enabled the expression of the entire CD18 protein, this was not sufficient to stabilize the CD18/11 heterodimer at the cell surface.

Conclusion: A novel nonsense mutation in the CD18 gene causing a complete absence of CD18 protein and severe LAD1 clinical phenotype is reported. Both in vivo and in vitro treatments with gentamicin resulted in the expression of a corrected full-length dysfunctional or mislocalized CD18 protein. However, while the use of gentamicin increased the expression of CD18, it did not improve leukocyte adhesion and chemotaxis. Moreover, the integrity of the CD18/CD11 complex at the cell surface was impaired, due to abnormal CD18 protein and possibly lack of CD11a expression.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. R188X nonsense mutation in ITGβ2/CD18.
a. DNA sequences of the mutation region in exon 6 of CD18 gene in the two patients, their parents, one sister of patient #2 and one brother of patient #2. b. cDNA sequences of the mutation region in CD18 transcripts in the two patients c. Schematic structure of ITGβ2/CD18 protein, its domains and the position of the premature stop mutation. PSI indicates the plexin-semaphorin-integrin domain; HD, hybrid domain; EGFD, EGF-like domain; βTD, β-tail domain; PM, plasma membrane.
Figure 2
Figure 2. CD18 and CD11b cell surface expression upon gentamicin treatment in vivo.
Whole blood samples from patient #1 (a), patient #2 (b) and healthy control (c) were incubated with anti-CD18 or anti-CD11b antibodies (Coulter Diagnostics). Lymphocytes (Lymph.) and granulocytes (Granul.) were gated and the expression of CD18 or CD11b on their cell surface was measured using flow cytometry (Epics V; Coulter Electronics, Hialeah, FL or Becton Dickinson CANTO II, BD Biosciences, NJ, USA, Diva software). Unstained cells obtained from patient #1 (a) were used as the control to set the M1 threshold. The percent of cells expressing the relevant cell surface marker in each case is indicated.
Figure 3
Figure 3. In vitro induction of CD18 protein expression by gentamicin treatment.
The two patients' Epstein-Barr virus-transformed cells were treated with increasing concentrations of gentamicin for 3 days. Proteins were extracted from whole cell lysates for western blot analysis (upper panels) using anti-CD18 antibodies (abcam) and anti-tubulin antibody (Sigma) for equal loading control. Intensities of the CD18 bands were calculated using Image EZ-Quant software package (EZ-Quante LTD., Israel), normalized to the untreated samples (lower panels).
Figure 4
Figure 4. Immuno-localization of CD18 upon treatment with gentamicin.
The two patients' Epstein-Barr virus-transformed cells were treated with gentamicin (1000 µg/ml) for 5 days. Cells were cytospun, fixed and immunostained with mouse monoclonal anti integrin β2 (CD18) antibody (Santa Cruz Biotechnology Inc., USA). Slides were mounted using immunofluorescence and DAPI for nuclear staining and analyzed by optic grid fluorescence microscopy (Olympus). a. 1000-fold magnification of healthy control's Epstein-Barr virus-transformed lymphocytes b. 600-fold magnification of gentamicin untreated (upper panel) and treated (lower panel) lymphoblastoids derived from patients 1 and 2. c. 1000-fold magnification of gentamicin untreated (left panel) and treated (right panel) lymphoblastoids derived from patient 1. N indicates nucleus; PM, plasma membrane; C, cytoplasm.
Figure 5
Figure 5. Theoretical model of human CD18.
A. A 3-D model of CD18 (blue) was built by homology modeling using ITGβ3 (CD61) structure as a template (red, PDB code 3ije). B. Superposition of the model with αvβ3 (CD51/CD61) complex (PDB code 1l5g). The β-I domains (CD18 in the model [blue]), CD61 in the template [red]) are given in cartoon representation with the arrow pointing to the position of the R188 residue, mutated in the LAD1 patients. The ITGαv subunit (CD51) is displayed in a surface representation.
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