Am. J. Hum. Genet. 46:35S-357, 1990
Enhanced G2 Chromatid Radiosensitivity in Dyskeratosis Congenita Fibroblasts David M. DeBauche, G. Shashidhar Pai, and Wayne S. Stanley Departments of Pathology and Laboratory Medicine, and Pediatrics, Medical University of South Carolina, Charleston
Summary Dyskeratosis congenita (DC) is an inherited disorder characterized by reticular pigmentation of the skin, dystrophic nails, mucosal leukoplakia, and a predisposition to cancer in early adult life. In the majority of cases, DC is an X-linked recessive trait. However, one or more autosomal form(s) of DC may exist. Although excessive spontaneous chromatid breakage has been reported in DC, it is not a consistent cytological marker for this disorder. We examined the frequency and specificity of X-irradiation-induced G2 chromatid breakage in fibroblasts from three unrelated DC patients (two males and one female). Metaphase cells from DC patients had significantly more chromatid breaks (16-18-fold and 17-26-fold at 50 and 100 rad X-irradiation, respectively) and chromatid gaps (10-12-fold and 6-7-fold at 50 and 100 rad, respectively) than those from two different controls. Analysis of banded chromosomes revealed a nonrandom distribution of chromatid aberrations in DC but not in controls, a distribution corresponding to some of the known breakpoints for cancer-specific rearrangements, constitutive fragile sites, and/or loci for cellular proto-oncogenes. The significance of this finding for cancer predisposition in DC patients is uncertain, but the increased susceptibility of X-irradiation-induced chromatid breakage may serve as a cellular marker of diagnostic value.
Introduction
Dyskeratosis congenita (DC [or Zinsser-Cole-Engman syndrome]) is a rare, heritable, multisystem disorder. Since its initial description by Zinsser (1910), more than 100 cases have been reported. Sirinavin and Trowbridge (1975) have reviewed both the clinical and genetic aspects of this disease. An X-linked recessive mode of inheritance has been shown in a majority of the cases (Sirinavin and Trowbridge 1975). Recently, linkage in one large family by using X chromosome-specific RFLP markers has assigned the gene for DC to chromosome band Xq28 (Connor et al. 1986). However, several families with apparent autosomal forms of DC have been reported, suggesting etiologic heterogeneity in this disorder (Scoggins et al. 1971; Fudenberg et al. 1979; Received March 21, 1989; final revision received October 9, 1989. Address for correspondence and reprints: Wayne S. Stanley, Ph.D., Department of Pathology and Laboratory Medicine, Medical University of South Carolina, 171 Ashley Avenue, Charleston, SC 29425. © 1990 by The American Society of Human Genetics. All rights reserved. 0002-9297/90/4602-0015$02.00
350
Gasparini et al. 1985; Menon et al. 1986; Juneja et al. 1987; Pai et al. 1989). The characteristic clinical triad observed in DC consists of nail dystrophy, cutaneous and mucosal pigmentary changes, and chronic, progressive bone marrow failure. Although subtle dermatologic manifestations have been observed at birth, they usually develop after infancy but prior to puberty (Sirinavin and Trowbridge 1975). An increased frequency of infection and a predisposition to malignancy are the most serious and often fatal complications of this disease (Sirinavin and Trowbridge 1975; Womer et al. 1983). Controversy exists concerning the cytogenetic findings in DC. Several reports have suggested increased chromosome breakage in DC lymphocytes and/or fibroblasts (Milgrom et al. 1964; Addison and Rice 1965; Bryan and Nixon 1965; Ortega et al. 1972; Aguilar et al. 1974; Morrison 1974; Gutman et al. 1978; Schneider et al. 1988). In addition, an increased level of sister-chromatid exchange (SCE), both spontaneous (Burgdorf et al. 1977) and induced (Carter et al. 1979), has been reported. However, other investigators have
Radiosensitivity in Dyskeratosis Congenita
demonstrated normal levels of breakage and SCE in studies of their patients (Kano and Fujiwara 1982; Womer et al. 1983; Juneja et al. 1987). These studies indicate, then, that chromosome breakage is not a consistent finding in DC. This might be explained if different complementation groups for DC exist. Recently, enhanced G2 chromatid radiosensitivity has been observed in fibroblasts from individuals genetically predisposed to cancer (Parshad et al. 1983, 1985b, 1985c). Because DC is also characterized by a predisposition to cancer, we investigated the sensitivity that fibroblasts derived from two DC males and one DC female had to X-irradiation, in an effort to determine whether G2 chromatid radiosensitivity was a consistent feature of this disorder. Material and Methods Cell Lines
Cell line GM1774, established from a known X-linked recessive DC male, was obtained from the NIGMS Human Genetic Mutant Cell Repository (Camden, NJ). A second cell line (RB-1), derived from a male patient, was established by one of us (W.S.S.) at Henry Ford Hospital (Detroit). The third cell line, SB-1, was derived in this laboratory from a 7-year-old black female currently being followed by us. This patient represents a sporadic case of DC and has been clinically reported by Pai et al. (1989). RR-1, a newly established cell line from a DC male, was used in the analysis of the progression of G2 cells into metaphase after X-irradiation. Two normal skin fibroblast lines were used in the present study. Cell line UM107 was obtained from Dr. Ernest H. Y. Chu (University of Michigan Medical School) and served as an age- and sex-matched control for DC line GM1774. A second fibroblast cell line, C1, was established in this laboratory and served as a sex-matched control for SB-1. Both normal fibroblast lines were derived from individuals free of known genetic defects and without evidence of neoplastic disease. All of the cell lines used in the present study had a normal karyotype. X-lrradiation and Breakage Analysis All cells were grown in 25-cm2 culture flasks (Corning) by using Dulbecco's modified Eagle's medium supplemented with 15% FCS (Gibco). Each culture was passaged 24 h prior to exposure to insure that a sufficient number of cells in the G2 phase of the cell cycle would be present at the time of irradiation. Two replicates for
351
each cell line were irradiated with 0, 50, or 100 rad by using a General Electric 250-kV X-ray machine fitted with a 15-cm x 15-cm compression cone and a Thorium III filter. Immediately following exposure, the culture medium was replaced with fresh medium and cells were incubated for 1.5 h at 37°C according to a method described by Parshad et al. (1983). Colcemid (0.1 jg/ml) was added during the final 45 min of the incubation period. Harvesting the cultures within 1.5 h after X-irradiation ensured that all analyzed metaphase cells were in G2 phase at the time of exposure (Parshad et al. 1983). Cultures were harvested, and slides were prepared and stained with Wright's stain by using conventional cytogenetic techniques. Breakage analysis data were averaged from at least three independent experiments for each cell line by scoring a minimum of 100 metaphase cells for each exposure. A chromosome break was scored when discontinuity was observed at the same site in both chromatids of a single chromosome. A chromatid break was scored when discontinuity of a single chromatid was found in which there was clear misalignment of one of the chromatids and/or a nonstaining region greater than the width of the chromatid. A chromatid gap was scored when a nonstaining region less than or equal to the chromatid width was observed with minimal misalignment of the chromatid. The distribution of chromatid aberrations was determined from banded preparations. Approximately 50% of all analyzed metaphase cells in the present study had a G-band pattern, produced by staining with Wright's stain (Sanchez et al. 1973), of sufficient quality to allow identification of the site of aberration. The aberration frequency at each of the involved chromosomal locations was determined by dividing the total number of aberrations at a specific site by the total number of aberrations (gaps and breaks) at all chromosomal sites from every analyzed metaphase in a single cell line. An average of 150 metaphase cells from each cell line exposed to 50-rad X-irradiation were used in this analysis. The influence of X-irradiation on the progression of G2 cells into metaphase was examined in the following manner: Cells from both DC (SB1 and RR-1) and control (C-1) cell lines were cultured in chambered slides (Lab-Tek, Miles Scientific) and exposed to 100-rad X-irradiation. Slides were harvested, in situ, at specific time intervals and stained with Giemsa. The number of postirradiation mitotic cells was compared with the number from their nonirradiated control at each time interval. Each determination was based on three independent experiments.
352
DeBauche
Statistical Treatment In the statistical analysis of the data, the mean number of gaps or breaks was determined for each level of exposure from three experiments scoring a minimum of 100 metaphase cells. Comparison of the data within each cell line from experiment to experiment was performed using the t-test (Snedecor and Cochran 1980). The t-test was also used to compare the data from one
et
al.
50-rad X-irradiation were increased an average of 11.4-fold above those in normal fibroblast controls. This observed increase ranged from a low of 10.7-fold (RB1) to a high of 12.1-fold (SB-1). After 100-rad X-irradiation, DC fibroblast lines had a cumulative mean increase in chromatid gaps that was 6.9-fold higher than the increase in control values. Similarly, the cumulative mean increase in chromatid breaks observed in DC lines was 17.2-fold (50 rad) and 21.4-fold (100 rad) higher than the increase in control fibroblasts. The increased chromatid breakage ranged from 16.1-fold (RB-1) to 18.7-fold (SB-i) at 50 rad and from 17.8-fold (GM1774) to 26.9-fold (SB-1) at 100 rad. The incidence of chromatid damage at 0-, 50-, and 100-rad X-irradiation for the two normal human fibroblast cell lines as well as for the three independent DC lines were similar with respect to each group (table 1). SB-1 demonstrated a slight increase in chromatid breaks at 50 and 100 rad when compared with results from GM1774 and RB-1. Although the maximum observed increase in gaps of 1.1-fold (P < .01) at 50 rad and the maximum increase in breaks of 1.45-fold (P < .01) at 100 rad were statistically significant, the biological importance of this difference is unknown. The percentage of cells that had at least one chromosome or chromatid aberration was also calculated for each cell line (table 2). As expected from the data presented in table 1, the percentage of cells with gaps or breaks from each line with 0-rad exposure was very low, with an average of 5.05% and 8.3% for normal and DC fibroblasts, respectively. In every DC line, 100% of the analyzed cells had at least one chromatid gap to
cell line with those from another. The cumulative mean number of gaps and breaks was determined for both normal and DC fibroblasts by averaging the means within each group. All P values reported are two-sided. Results Spontaneous and X-lrradiation-induced Chromatid Damage
Spontaneous chromatid breakage frequencies were obtained at 0-rad X-irradiation (table 1). The DC lines and the normal human fibroblast lines were homogeneous with respect to spontaneous chromatid gaps (P > .2). The incidence of spontaneous chromatid breaks was somewhat higher in the DC fibroblast lines as a group compared with normal fibroblasts (0.041/cell vs 0.01/cell, respectively). Although the P value was slightly less than .05, this increase was judged to be functionally insignificant because the mean chromatid break frequencies in UM107 and GM1774 were identical. The incidence of X-ray-induced G2 chromatid gaps and breaks (table 1) was significantly increased in all DC fibroblast lines compared with normal controls (P < .001). Chromatid gaps in DC fibroblast lines exposed Table I X-Irradiation-induced G2 Phase Chromatid Damage MEAN ± SEM GAPS/CELL
CELL LINE
0 rad
Normal fibroblasts: Ci ............... .020 ± .019 UmlO7 . 080 ± .0 Mean ± SEM .... .050 ± .029 DC fibroblasts: GM1774 ........... . 140 ± .020 RB-1 ...............055 ± .025 .082 ± .028 SB-1 ............... Mean + SEM .... .092 ± .024 P valueb ........ >20 ............
MEAN ± SEM Breaks/CELL
50 rad
100 rad
0 rad
50 rad
100 rad
.442 ± .089 0.440 ± .039 0.441 ± .0
1.386 + .113 1.360 ± .199 1.373 ± .012
.0 ± .0 .020 ± .019 .010 ± .009
.154 ± .021 .120 ± .039 .137 ± .016
.198 ± .072 .260 ± .019 .229 ± .030
4.967 ± .121 4.725 ± .225 5.350 ± .501 5.014 ± .181
Enhanced G2 Chromatid Radiosensitivity in Dyskeratosis Congenita Fibroblasts David M. DeBauche, G. Shashidhar Pai, and Wayne S. Stanley Departments of Pathology and Laboratory Medicine, and Pediatrics, Medical University of South Carolina, Charleston
Summary Dyskeratosis congenita (DC) is an inherited disorder characterized by reticular pigmentation of the skin, dystrophic nails, mucosal leukoplakia, and a predisposition to cancer in early adult life. In the majority of cases, DC is an X-linked recessive trait. However, one or more autosomal form(s) of DC may exist. Although excessive spontaneous chromatid breakage has been reported in DC, it is not a consistent cytological marker for this disorder. We examined the frequency and specificity of X-irradiation-induced G2 chromatid breakage in fibroblasts from three unrelated DC patients (two males and one female). Metaphase cells from DC patients had significantly more chromatid breaks (16-18-fold and 17-26-fold at 50 and 100 rad X-irradiation, respectively) and chromatid gaps (10-12-fold and 6-7-fold at 50 and 100 rad, respectively) than those from two different controls. Analysis of banded chromosomes revealed a nonrandom distribution of chromatid aberrations in DC but not in controls, a distribution corresponding to some of the known breakpoints for cancer-specific rearrangements, constitutive fragile sites, and/or loci for cellular proto-oncogenes. The significance of this finding for cancer predisposition in DC patients is uncertain, but the increased susceptibility of X-irradiation-induced chromatid breakage may serve as a cellular marker of diagnostic value.
Introduction
Dyskeratosis congenita (DC [or Zinsser-Cole-Engman syndrome]) is a rare, heritable, multisystem disorder. Since its initial description by Zinsser (1910), more than 100 cases have been reported. Sirinavin and Trowbridge (1975) have reviewed both the clinical and genetic aspects of this disease. An X-linked recessive mode of inheritance has been shown in a majority of the cases (Sirinavin and Trowbridge 1975). Recently, linkage in one large family by using X chromosome-specific RFLP markers has assigned the gene for DC to chromosome band Xq28 (Connor et al. 1986). However, several families with apparent autosomal forms of DC have been reported, suggesting etiologic heterogeneity in this disorder (Scoggins et al. 1971; Fudenberg et al. 1979; Received March 21, 1989; final revision received October 9, 1989. Address for correspondence and reprints: Wayne S. Stanley, Ph.D., Department of Pathology and Laboratory Medicine, Medical University of South Carolina, 171 Ashley Avenue, Charleston, SC 29425. © 1990 by The American Society of Human Genetics. All rights reserved. 0002-9297/90/4602-0015$02.00
350
Gasparini et al. 1985; Menon et al. 1986; Juneja et al. 1987; Pai et al. 1989). The characteristic clinical triad observed in DC consists of nail dystrophy, cutaneous and mucosal pigmentary changes, and chronic, progressive bone marrow failure. Although subtle dermatologic manifestations have been observed at birth, they usually develop after infancy but prior to puberty (Sirinavin and Trowbridge 1975). An increased frequency of infection and a predisposition to malignancy are the most serious and often fatal complications of this disease (Sirinavin and Trowbridge 1975; Womer et al. 1983). Controversy exists concerning the cytogenetic findings in DC. Several reports have suggested increased chromosome breakage in DC lymphocytes and/or fibroblasts (Milgrom et al. 1964; Addison and Rice 1965; Bryan and Nixon 1965; Ortega et al. 1972; Aguilar et al. 1974; Morrison 1974; Gutman et al. 1978; Schneider et al. 1988). In addition, an increased level of sister-chromatid exchange (SCE), both spontaneous (Burgdorf et al. 1977) and induced (Carter et al. 1979), has been reported. However, other investigators have
Radiosensitivity in Dyskeratosis Congenita
demonstrated normal levels of breakage and SCE in studies of their patients (Kano and Fujiwara 1982; Womer et al. 1983; Juneja et al. 1987). These studies indicate, then, that chromosome breakage is not a consistent finding in DC. This might be explained if different complementation groups for DC exist. Recently, enhanced G2 chromatid radiosensitivity has been observed in fibroblasts from individuals genetically predisposed to cancer (Parshad et al. 1983, 1985b, 1985c). Because DC is also characterized by a predisposition to cancer, we investigated the sensitivity that fibroblasts derived from two DC males and one DC female had to X-irradiation, in an effort to determine whether G2 chromatid radiosensitivity was a consistent feature of this disorder. Material and Methods Cell Lines
Cell line GM1774, established from a known X-linked recessive DC male, was obtained from the NIGMS Human Genetic Mutant Cell Repository (Camden, NJ). A second cell line (RB-1), derived from a male patient, was established by one of us (W.S.S.) at Henry Ford Hospital (Detroit). The third cell line, SB-1, was derived in this laboratory from a 7-year-old black female currently being followed by us. This patient represents a sporadic case of DC and has been clinically reported by Pai et al. (1989). RR-1, a newly established cell line from a DC male, was used in the analysis of the progression of G2 cells into metaphase after X-irradiation. Two normal skin fibroblast lines were used in the present study. Cell line UM107 was obtained from Dr. Ernest H. Y. Chu (University of Michigan Medical School) and served as an age- and sex-matched control for DC line GM1774. A second fibroblast cell line, C1, was established in this laboratory and served as a sex-matched control for SB-1. Both normal fibroblast lines were derived from individuals free of known genetic defects and without evidence of neoplastic disease. All of the cell lines used in the present study had a normal karyotype. X-lrradiation and Breakage Analysis All cells were grown in 25-cm2 culture flasks (Corning) by using Dulbecco's modified Eagle's medium supplemented with 15% FCS (Gibco). Each culture was passaged 24 h prior to exposure to insure that a sufficient number of cells in the G2 phase of the cell cycle would be present at the time of irradiation. Two replicates for
351
each cell line were irradiated with 0, 50, or 100 rad by using a General Electric 250-kV X-ray machine fitted with a 15-cm x 15-cm compression cone and a Thorium III filter. Immediately following exposure, the culture medium was replaced with fresh medium and cells were incubated for 1.5 h at 37°C according to a method described by Parshad et al. (1983). Colcemid (0.1 jg/ml) was added during the final 45 min of the incubation period. Harvesting the cultures within 1.5 h after X-irradiation ensured that all analyzed metaphase cells were in G2 phase at the time of exposure (Parshad et al. 1983). Cultures were harvested, and slides were prepared and stained with Wright's stain by using conventional cytogenetic techniques. Breakage analysis data were averaged from at least three independent experiments for each cell line by scoring a minimum of 100 metaphase cells for each exposure. A chromosome break was scored when discontinuity was observed at the same site in both chromatids of a single chromosome. A chromatid break was scored when discontinuity of a single chromatid was found in which there was clear misalignment of one of the chromatids and/or a nonstaining region greater than the width of the chromatid. A chromatid gap was scored when a nonstaining region less than or equal to the chromatid width was observed with minimal misalignment of the chromatid. The distribution of chromatid aberrations was determined from banded preparations. Approximately 50% of all analyzed metaphase cells in the present study had a G-band pattern, produced by staining with Wright's stain (Sanchez et al. 1973), of sufficient quality to allow identification of the site of aberration. The aberration frequency at each of the involved chromosomal locations was determined by dividing the total number of aberrations at a specific site by the total number of aberrations (gaps and breaks) at all chromosomal sites from every analyzed metaphase in a single cell line. An average of 150 metaphase cells from each cell line exposed to 50-rad X-irradiation were used in this analysis. The influence of X-irradiation on the progression of G2 cells into metaphase was examined in the following manner: Cells from both DC (SB1 and RR-1) and control (C-1) cell lines were cultured in chambered slides (Lab-Tek, Miles Scientific) and exposed to 100-rad X-irradiation. Slides were harvested, in situ, at specific time intervals and stained with Giemsa. The number of postirradiation mitotic cells was compared with the number from their nonirradiated control at each time interval. Each determination was based on three independent experiments.
352
DeBauche
Statistical Treatment In the statistical analysis of the data, the mean number of gaps or breaks was determined for each level of exposure from three experiments scoring a minimum of 100 metaphase cells. Comparison of the data within each cell line from experiment to experiment was performed using the t-test (Snedecor and Cochran 1980). The t-test was also used to compare the data from one
et
al.
50-rad X-irradiation were increased an average of 11.4-fold above those in normal fibroblast controls. This observed increase ranged from a low of 10.7-fold (RB1) to a high of 12.1-fold (SB-1). After 100-rad X-irradiation, DC fibroblast lines had a cumulative mean increase in chromatid gaps that was 6.9-fold higher than the increase in control values. Similarly, the cumulative mean increase in chromatid breaks observed in DC lines was 17.2-fold (50 rad) and 21.4-fold (100 rad) higher than the increase in control fibroblasts. The increased chromatid breakage ranged from 16.1-fold (RB-1) to 18.7-fold (SB-i) at 50 rad and from 17.8-fold (GM1774) to 26.9-fold (SB-1) at 100 rad. The incidence of chromatid damage at 0-, 50-, and 100-rad X-irradiation for the two normal human fibroblast cell lines as well as for the three independent DC lines were similar with respect to each group (table 1). SB-1 demonstrated a slight increase in chromatid breaks at 50 and 100 rad when compared with results from GM1774 and RB-1. Although the maximum observed increase in gaps of 1.1-fold (P < .01) at 50 rad and the maximum increase in breaks of 1.45-fold (P < .01) at 100 rad were statistically significant, the biological importance of this difference is unknown. The percentage of cells that had at least one chromosome or chromatid aberration was also calculated for each cell line (table 2). As expected from the data presented in table 1, the percentage of cells with gaps or breaks from each line with 0-rad exposure was very low, with an average of 5.05% and 8.3% for normal and DC fibroblasts, respectively. In every DC line, 100% of the analyzed cells had at least one chromatid gap to
cell line with those from another. The cumulative mean number of gaps and breaks was determined for both normal and DC fibroblasts by averaging the means within each group. All P values reported are two-sided. Results Spontaneous and X-lrradiation-induced Chromatid Damage
Spontaneous chromatid breakage frequencies were obtained at 0-rad X-irradiation (table 1). The DC lines and the normal human fibroblast lines were homogeneous with respect to spontaneous chromatid gaps (P > .2). The incidence of spontaneous chromatid breaks was somewhat higher in the DC fibroblast lines as a group compared with normal fibroblasts (0.041/cell vs 0.01/cell, respectively). Although the P value was slightly less than .05, this increase was judged to be functionally insignificant because the mean chromatid break frequencies in UM107 and GM1774 were identical. The incidence of X-ray-induced G2 chromatid gaps and breaks (table 1) was significantly increased in all DC fibroblast lines compared with normal controls (P < .001). Chromatid gaps in DC fibroblast lines exposed Table I X-Irradiation-induced G2 Phase Chromatid Damage MEAN ± SEM GAPS/CELL
CELL LINE
0 rad
Normal fibroblasts: Ci ............... .020 ± .019 UmlO7 . 080 ± .0 Mean ± SEM .... .050 ± .029 DC fibroblasts: GM1774 ........... . 140 ± .020 RB-1 ...............055 ± .025 .082 ± .028 SB-1 ............... Mean + SEM .... .092 ± .024 P valueb ........ >20 ............
MEAN ± SEM Breaks/CELL
50 rad
100 rad
0 rad
50 rad
100 rad
.442 ± .089 0.440 ± .039 0.441 ± .0
1.386 + .113 1.360 ± .199 1.373 ± .012
.0 ± .0 .020 ± .019 .010 ± .009
.154 ± .021 .120 ± .039 .137 ± .016
.198 ± .072 .260 ± .019 .229 ± .030
4.967 ± .121 4.725 ± .225 5.350 ± .501 5.014 ± .181
