A case of dyskeratosis congenita associated with impaired DNA repair as shown by the comet assay

ARTICLE

Dyskeratosis congenita (DC) is a rare genodermatosis that is usually inherited in a X-linked recessive mode. Skin manifestations are the following : abnormal skin pigmentation, nail dystrophy and leucoplakia of mucous membranes. Other clinical manifestations include: lacrimal duct stenosis, oesophageal diverticulum, hyperhidrosis of the palms and soles, palmoplantar hyperkeratosis, thin hair, loss of teeth, hypogenitalism, short stature and intellectual impairment [1]. Patients can develop bone marrow failure, and are predisposed to malignancy.

In xeroderma pigmentosum, skin tumor promotion is associated with a defect in the most important DNA repair system: the nucleotide excision-repair system. The photoproducts induced by ultraviolet (UV) B radiations on cellular DNA (pyrimidine dimers) are recognized and incised. The lesions are excised, the gap is filled by DNA polymerase, and finally DNA ligase terminates the repair process.

DNA repair of normal, or xeroderma pigmentosum cells to UV radiations, were studied using alkaline single cell gel electrophoresis assay (or comet assay) [2]. In this technique [3], interrupted DNA strands of agarose-embedded cells may, after lysis and electrophoresis, migrate out of the nucleus toward the anode. Each damaged cell had the appearance of a comet, with a brightly fluorescent hail, and a tail whose length and fluorescence intensity were related to the number of DNA strand breaks induced by UV irradiation. Post-exposure decrease of migration indicates that such DNA lesions are repaired. Undamaged cells appear as intact nuclei without tail.

Little is known about the DNA repair mechanism in DC. Herein, we applied the comet assay to examine DNA repair capacity in a case of DC.

Clinical case

An 18-year old Caucasian boy consulted us in May 1997 with a 7-year history of pigmentary skin changes. Physical examination revealed brownish reticulated pigmentation, confined to the chest, neck, shoulders, and arms (Fig. 1). All nails showed atrophy, onycholysis, and pterygium formation was noted (Fig. 2). The tongue was dystrophic with patches of leucokeratosis (Fig. 3). He was otherwise asymptomatic, and physical and mental development was normal. His parents are not consanguineous. There is no family history of skin or hematological diseases. He does not present sun sensitivity. The laboratory investigations excluded any hematological abnormality.

Histological study was performed on a biopsy specimen from a pigmented area on the upper part of the arms. It showed hyperkeratosis, hyperpigmentation of the basal layer, and some melanophages in the superficial dermis (Fig. 4).

Given the clinical features the patient has been diagnosed as a DC.

Material and method

The alkaline single-cell gel electrophoresis (comet assay)

The assay is performed according to Singh et al. [4]. All stages are conducted in the dark. The lymphocytes are isolated from heparinized blood, using the method of gradient centrifugation. Lymphocytes are suspended in phosphate buffered saline (PBS) and mixed with 0.5% low melting point agarose (75 µl). This cell suspension is applied on a first coverslip of 85 µl of 0.8% normal melting agarose. Finally, cells are covered by 75 µl of 0.8% agarose. The slides are exposed, except for the controls, to UVB radiation (312 nm, 0.05 J/cm²). Then, they are immersed in freshly prepared lysing solution (2.5 mmol/l NaCl, 100 mmol/l Na₂ EDTA, 10 mmol/l tris, 1% N-Lauroyl sarcosine, pH 10, with 1% Triton X-100 and 10% DMSO added just before use) for 1 hr at 4° C. The slides are then placed in an electrophoresis box containing alkaline buffer (1 mmol/l Na₂ EDTA, 300 mmol/l NaOH). After 20 min, electrophoresis is conducted for 24 min at room temperature (25 V, 300 mA). The slides are neutralized three times for 5 min with 0.4 mmol/l Tris, pH 7.5, and stained with 50 µl of ethidium bromide (2 µg/ml). Observation is made at 543 nm, using a confocal laser microscope (equiped with an image analysis system).

This assay was performed on our patient, and 2 healthy subjects of the same age and sex, and compared with non irradiated control slides.

Results

As shown in Figure 5A, untreated control cells from healthy or the DC patient appeared as intact nuclei, indicating that the procedure did not induce DNA damage.

In Figure 5B, the observation is made 2 hrs after irradiation. In both healthy and DC patients, a comet appeared, which demonstrates a normal incision capacity.

In Figure 5C, the observation is made 6 hrs after irradiation. In the case of lymphocytes from the healthy patient, the comet tail had decreased, indicating that the DNA resynthesis had begun. For the DC patient, the comet tail remained unchanged. This last result clearly indicates that DNA repair is impaired.

Comment

In our patient, DC has been diagnosed according to the three classic features such as abnormal skin pigmentation, nail dystrophy, and leucoplakia of mucous membranes. Other clinical features and hematological abnormalities are absent.

X-linked recessive inheritence is seen in the majority of families, however both autosomal recessive and dominant transmission has been suggested in others. In several families with an X-linked recessive trait, the gene for DC has been mapped to Xq28, but this gene and the protein products are not yet characterized [5]. The patient we present has no familial history and his little sister is healthy.

The pathogenetic mechanisms involved in DC are not known. Some authors found abnormal "stem cell" function and normal stroma function in long-term bone marrow culture system. They speculated some similarities with the W/W^v mouse, characterized by mutations of the c-kit proto-oncogene [6].

DC is known to be associated with a higher rate of malignancy, presumably mediated by abnormal DNA metabolism. Some studies reported spontaneous chromosome instability, or excessive sister chromatid exchange [7].

There is as yet no proof of DNA repair deficiency in DC. Alkaline comet assay is performed to investigate DNA metabolism of DC cells after UVB exposure.

UVC radiations are more commonly used for the comet assay. Because the major DNA-damaging and probably carcinogenic component is UVB, and because continuing depletion of stratospheric ozone tends to increase UVB intensity on the earth environment, we chose to use UVB wavelengths. It is important to note that the response to UVB is close to that of UVC [8].

We used lymphocytes cells, a convenient and readily available source of human material. A number of authors found that unstimulated peripheral blood lymphocytes can repair DNA damage caused by UV light, even if they are extremely sensitive to alkaling agents, carcinogens and ultraviolet light [9].

The DNA repair can be investigated using the comet assay, or more commonly using the classic method of unscheduled DNA synthesis. Both are sensitive methods, but the comet assay is more simple and rapid and needs few individual cells, relatively inexpensive equipment, and no radioactivity [3].

The comet assay is particularly useful to differentiate the two principal steps of DNA repair: the incision-excision, and the DNA resynthesis. On the contrary, unscheduled DNA synthesis does not distinguish between these two steps.

Comet assay is used by many authors: in 1996, Alapetite et al. [8] studied cell repair capacities in xeroderma pigmentosum or trichothiodystrophy, and found that the incision step is absent or reduced. In 1997 [10], the same authors compared comet assay to unscheduled DNA synthesis and validated comet assay in prenatal diagnosis of xeroderma pigmentosum or trichothiodystrophy.

In our DC patient, lymphocyte responses two hours after irradiation correspond to the response of normal lymphocytes. These data indicate that in DC, the incision repair step is not altered. Normally, the DNA strand breaks are transient and the repair synthesis and ligation occur rapidly. However, in the case presented, the DNA strand breaks persist.

In the literature, we do not find other pathologies where such an abnormality is described using comet assay.

To explain such DNA repair abnormality, the first hypothesis is a prolonged kinetic of repair. It would be interesting to study prolonged kinetic using unscheduled DNA synthesis, because with the comet assay, cells cannot be maintained in agarose gel more than 6 hrs.

The second hypothesis is an extremely small DNA precursor pool. Yew et al. [9] demonstrated that repair capacity is closely related to the size of the DNA precursor pool, and that this effect can be reversed by supplementing with desoxyribonucleosides.

Finally, we can suspect a defect localized in the following steps: DNA synthesis and ligation. Mutations in DNA polymerase alpha that confer UV sensitivity in vitro are known, but have not yet been found among UV-sensitive patients [11]. In the same way, DNA polymerase delta is necessary for DNA repair synthesis [12]. DNA ligase is required to complete any DNA excision repair process. Cells of Bloom's syndrome exhibit several characteristics indicative of a deficiency in DNA ligation. Partially purified DNA ligase I fractions from Bloom's syndrome cells exhibit altered biochemical properties [13]. Moreover, one patient with sensitivity to a number of DNA-damaging agents, associated with severe immunodeficiency involving IgA and IgG, has been found to have mutations in DNA ligase I [14].

In our case, it would be of interest to achieve an in vitro biochemical complementation assay using extracts from human xeroderma pigmentosum or rodent ERCC (excision repair cross-complementing) mutants to regenerate reaction mixtures with normal repair activity [15].

The relationship between the molecular defect in DNA repair and cancer proneness is not clear.

Among the genodermatoses in which abnormal DNA repair is demonstrated, a risk of cancer is present in xeroderma pigmentosum, Rothmund-Thomson syndrome [16], and DC. This risk is absent for Cockayne syndrome and trichothiodystrophy.

So, it seems that abnormal DNA repair is not sufficient to result in neoplasia.

Multiple genes can be responsive for these different diseases with impaired DNA repair.

The cloning of the gene(s) and analysis of the mutation(s) resulting in those diseases, should help us to recognize the underlying defect, and to understand the pathogenesis of this disease.

Long-term follow-up of our DC patient is necessary to detect malignant changes, and to establish a relationship between DNA repair and cancer proneness.

CONCLUSION

In a case of DC, we showed impaired DNA repair capacity, which seems to be different from xeroderma pigmentosum and trichothiodystrophy.

It may be reasonable to assume that DNA repair deficiency, in at least some of the patients with DC, could be related to cancer proneness. So, it seems of interest to study DNA repair in those patients and to correlate it to clinical presentation and presence of malignancy in long term follow-up. The comet assay may have potential as a simple procedure to investigate DC, or other genodermatoses.

Acknowledgments

The authors thank Dr. Rigaud and Dr. Thibaut for their cooperation on this report.

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