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Middle East Journal of Medical Genetics:
doi: 10.1097/01.MXE.0000414809.51187.d6
Original articles

Cytokinesis-blocked micronucleus assay in a group of Egyptian patients with Fanconi anemia

Eid, Maha M.a; Temtamy, Samia A.b; El-Kamah, Ghadab; Eid, Ola M.a

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Author Information

Departments of aHuman Cytogenetics

bClinical Genetics, National Research Center, Cairo, Egypt

Correspondence to Maha M. Eid, National Research Centre, El-Bohouth street, 12311 Dokki, Cairo, Egypt Tel: +20 105 003 576; e-mail: mahaeid67@gmail.com

Received October 18, 2011

Accepted November 21, 2011

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Abstract

Introduction: Fanconi anemia (FA) is one of the chromosomal instability syndromes characterized by a DNA repair defect. Cells derived from patients are hypersensitive to DNA cross-linking agents. In addition to the chromosomal breakages as a manifestation of the genomic instability, a few studies have detected elevated levels of micronuclei (MN) in patients with FA. The cytokinesis-blocked micronucleus (CBMN) assay was originally developed as a comprehensive test for measuring MN; however, it can also be used to measure nucleoplasmic bridges and nuclear buds.

Aim: The aim of this work is to determine the value of CBMN as a biomarker of genomic instability in patients with FA and its relation to their hematological findings.

Patients and methods: CBMN assay was performed to analyze leukocytes from 12 patients with FA and eight normal individuals as controls to determine the sensitivity of this technique for the evaluation of genomic instability in patients with FA. The CBMN assay was performed for cultures with and without diepoxybutane. The patients with FA showed significantly high frequencies of MN, nuclear buds, and dicentric bridges.

Conclusion: From the results of this study, we can conclude that the CBMN assay in fact represents a comprehensive method to evaluate the genomic instability of patients with FA. It can also be used as an indicator for the hematological condition of the patients.

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Introduction

Fanconi anemia (FA) is a well-recognized autosomal recessive disorder (OMIM: 227650). However, in a small minority of patients, the disorder is X-linked [FANC-B subgroup (OMIM: 300514)]. There is considerable genetic heterogeneity in FA, with 13 subtypes/complementation groups. FA patients are characterized by progressive bone marrow failure and an increased predisposition to malignancy (Alter, 2003; Levitus et al., 2004).

Affected individuals may also have one or more congenital abnormalities, such as microcephaly, short stature, café au lait spots, hypoplastic thumbs and radius, and horseshoe kidneys. FA cells characteristically show an abnormally high frequency of spontaneous chromosomal breakages and hypersensitivity to DNA cross-linking agents such as diepoxybutane (DEB) and mitomycin-C (MMC) (Tischkowitz and Dokal, 2004). Other features of the FA cell phenotype include hypersensitivity to oxygen, abnormal cell cycle kinetics (prolonged G2 phase), increased apoptosis, genomic instability, and accelerated telomere shortening (Dokal and Vulliamy, 2008).

A diagnostic test, based on the increased chromosomal breakage seen in FA cells compared with normal controls after exposure to DEB or MMC (DEB/MMC stress test), has been available for over 20 years (Auerbach, 1993). In addition to this test, some studies have found an increased level of micronuclei (MN) in patients with FA (Maluf and Erdtmann, 2001; Callén et al., 2002).

MN and other nuclear anomalies such as nucleoplasmic bridges (NPBs) and nuclear buds (NBUDs) are biomarkers of genotoxic events and chromosomal instability. These genome damage events can be determined simultaneously in the CBMN cytome assay. MN can originate during anaphase from lagging acentric chromosomes or chromatid fragments caused by misrepaired or unrepaired DNA breaks. Malsegregation of whole chromosomes at anaphase may also lead to the formation of MN. NPB originate from dicentric chromosomes, which may result from misrepair of DNA breaks and telomere end fusions. NBUD represent the process of the elimination of amplified DNA, DNA repair complexes, and possibly excess chromosomes from aneuploid cells (Fenech, 2007; Fenech et al., 2011).

We aimed to evaluate the MN as a marker for instability and the tendency to develop malignancies in patients with FA. Although with cytogenetic studies, there is no correlation between sensitivity to DEB and hematological findings, we aimed to determine the usefulness of the MN test as an additional marker for predicting the hematological status of patients with FA and their response to treatment.

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Patients and methods

Patients

Peripheral blood samples were collected from 12 patients with FA diagnosed clinically and cytogenetically (using DEB for induction of chromosomal breakage) and eight healthy individuals as a control group. Informed consents were obtained from the parents of children included in the study according to the guidelines of the ethical committees of the National Research Centre, Egypt. Complete blood count (CBC) was performed for each patient, including hemoglobin level (Hb), red blood cells (RBCs), white blood cells, and platelets.

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Methods

The DEB was added 24 h after culture initiation to all samples at a concentration of 0.1 μg/ml. Unbanded chromosomes were stained with Giemsa and 50 metaphases were screened for chromosome breakages. Breakages per cell were calculated for each case (Auerbach et al., 1989).

For the CBMN test (Fenech, 2007), two sets of culture were used for all samples: one with and one without DEB. Cytochalasin B (Acros, Thermo Fisher Scientific, New Jersey, USA) was added to both culture sets 44 h after culture initiation at a concentration of 4 μg/ml to obtain binucleated cells. Cell harvesting was carried out using a mild hypotonic (1 : 1v/v culture medium : distilled water) for 5 min at room temperature, followed by fixation in 3 : 1 methanol : acetic acid. The cell suspension was dropped onto clean slides and stained with Giemsa. The slides were examined using a light microscope at a magnification of 10×100. One thousand cells per individual were scored for the presence of MN, NBUDs, and dicentric bridges (NPBs between daughter nuclei).

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Statistics

Student’s t-test was used to compare between NB patients and the control group with respect to chromosome breaks and MN assay data. The relationship between chromosome breaks and the MN frequencies was analyzed using the Pearson correlation (two-tailed test).

The frequencies of the MN, NPB, and NB occurring spontaneously or induced by DEB were compared statistically with the CBC profile of the patients using the Pearson correlation (two-tailed test).

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Results

The study included 12 patients clinically and cytogenetically diagnosed with FA, and eight normal individuals as a control group. The mean and the range values of the CBC are shown in Table 1.

Table 1
Table 1
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The detailed cytogenetics results of the patients and the control groups are shown in Tables 2–4. The FA group showed higher levels of the four cytogenetics outcomes evaluated: the difference in DEB yield breakage (Fig. 1), frequency of MN (Fig. 2a and b), NBUDs (Fig. 2c), and NPBs (Fig. 2d), in both spontaneously and induced cultures, and data showed statistically significant differences when compared with the controls. There was also a direct correlation between the level of DEB breakage and the number of MN, NPB, and NB. The development of MN, whether spontaneous or induced, exceeded the level of NB, which itself exceeded the levels of NPB.

Table 2
Table 2
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Table 3
Table 3
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Table 4
Table 4
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Figure 1
Figure 1
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Figure 2
Figure 2
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An inverse correlation was found between the age of the patients and the hematological parameters, the Hb, RBC, and platelets (r=−0.04661, −0.23931, and −0.51594, respectively). However, there was a direct correlation between the patient’s age and the number of MN (r=0.02716). The statistical analysis also showed an inverse correlation between Hb levels and RBC counts and MN, whether spontaneous or induced.

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Discussion

FA is characterized by DNA repair defects. Cells derived from FA patients are hypersensitive to DNA cross-linking agents. Although the primary defects in FA are not known, biochemical evidence supports either a direct defect in the removal of DNA cross-links or a defect in the ability of cells to respond to oxidative stress, resulting from the interaction with cross-linking agents (Donahue and Campbell, 2002).

In our study, FA patients showed significant differences when compared with the controls in terms of MN, NBUD, and dicentric bridge frequency, a finding in agreement with previously reported data (Maluf and Erdtmann, 2001).

The high incidence of the MN in FA patients could be attributed to the increased chromosomal breakage and/or whole chromosome loss. However, the development of the NBUDs could have been the result of a DNA repair defect (Fenech, 2007; Fenech et al., 2011). Also, the increased frequencies of the MN and NBUD in FA could be attributed to unrepaired chromosome breaks, DNA misrepair, and telomeric breakages, reported previously in FA (Callén et al., 2002; Donahue and Campbell, 2002). The development of the NPB could be attributed to the telomeric end fusion that results from telomeric shortening or deletion in FA (Callén et al., 2002; Fenech et al., 2011).

Interestingly, a statistically significant correlation between the severity of the hematological condition and the frequency of MN was detected despite the relatively low number of patients included in this study. Also, there was an inverse correlation between the age of the patients and the severity of the hematological condition (CBC profile). To summarize these findings, it seems that, with advancing age, the CBC profile decreases and the MN frequencies increase. This could be attributed to a cumulative effect of the oxidative stress, inducing DNA damage, which further leads to apoptosis resulting in aplastic anemia.

FA is a chromosomal instability disorder and it is known that genetic susceptibility to develop cancer is related to genomic instability. Thus, the presence of such elevated levels of MN could be an additional marker for predisposition to cancer. This finding has been supported by a previous report using the CBMN assay as an easy and fast test that can be used in a number of patients, especially those at risk of developing cancer (El-Zein et al., 2011).

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Conclusion

Our results show that the increased severity of the anemia is associated with an increased frequency of the MN level. The micronucleus assay could thus be used as a biomarker for assessment of the progress of the disease. Also, a comprehensive evaluation of the micronucleus assay in patients with FA could provide an insight into the pathobiology of the molecular defect in FA.

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Acknowledgements
Conflicts of interest

There are no conflicts of interest.

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References

Alter BPNathan DG, Orkin SH, Look AT, Ginsburg D. Inherited bone marrow failure syndromes. Nathan and Oski’s hematology of infancy and childhood. 20036th ed Philadelphia, USA Saunders:280–365

Auerbach AD. Fanconi anemia diagnosis and the diepoxybutane (DEB) test. Exp Hematol. 1993;21:731–733

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Donahue SL, Campbell C. A DNA double strand break repair defect in Fanconi anemia fibroblasts. J Biol Chem. 2002;277:46243–46247

El-Zein R, Vral A, Etzel CJ. Cytokinesis-blocked micronucleus assay and cancer risk assessment. Mutagenesis. 2011;26:101–106

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Fenech M, Kirsch-Volders M, Natarajan AT, Surralles J, Crott JW, Parry J, et al. Molecular mechanisms of micronucleus, nucleoplasmic bridge and nuclear bud formation in mammalian and human cells. Mutagenesis. 2011;26:125–132

Levitus M, Rooimans MA, Steltenpool J, Cool NFC, Oostra AB, Mathew CG, et al. Heterogeneity in Fanconi anemia: evidence for 2 new genetic subtypes. Blood. 2004;103:2498–2503

Maluf SW, Erdtmann B. Genomic instability in Down syndrome and Fanconi anemia assessed by micronucleus analysis and single-cell gel electrophoresis. Cancer Genet Cytogenet. 2001;124:71–75

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Keywords:

biomarker; cytokinesis-blocked micronucleus assay; Fanconi anemia; genetic instability; malignancy

© 2012 Middle East Journal of Medical Genetics

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