61
Mutation Research, 248 (1991) 61-72 © 1991 Elsevier Science Publishers B.V. 0027-5107/91/$03.50 ADONIS 002751079100095F MTX 04958
Sister-chromatid exchanges in lymphocytes from infants with Down's syndrome E . K . S h u b b e r , H . A . H a m a m i 1, B . M . A . A l l a k a n d A . H . K h a l e e l Department of Biochemistry, Faculty of Agriculture and Biology and i A fi-Mustansiriya Medical College, Baghdad (Iraq) (Received 13 October 1988) (Revision received 4 September 1990) (Accepted 28 September 1990)
Keywords: Sister-chromatid exchange; Down's syndrome
Summary Sister-chromatid exchange (SCE) frequencies were studied in blood lymphocytes from 12 patients (3 females and 9 males) with Down's syndrome (DS). The mean frequency of SCE per metaphase for the patients (both sexes) was 9.2 + 0.8 which was significantly higher (P < 0.01) than the mean SCE value (5.1 +_0.2) scored for 16 healthy infants (8 females and 8 males). A significant increase in the mean frequency of SCE in 12 parents of infants with DS (8.7 + 0.9 SCE/ceI1) was noticeable when compared with 20 parents of normal infants (6.3 + 0.1 SCE/cell). Increases in cellular division with reduction in their replication were also observed in patients with DS. Treatment with mitomycin C (0.05 /~g/ml), hycanthone (0.1/~g/ml) and y-radiation (0.1 Gy) revealed a significant (P < 0.01) increase in frequencies of SCE in DS lymphocytes and in those of their parents as compared to controls. These data may reveal a familial hypersensitivity reaction to these agents. The results indicate a genomic instability and deranged DNA-repair mechanisms which are accentuated by exposure to mutagenic agents, the underlying causal factor for which might be genetic.
Down's syndrome, the most common autosomal anomaly in man, is currently the focus of an intensive investigation that promises to yield a wealth of information on gene expression and the molecular basis of disease (Patterson, 1987). The disease is the commonest cause of mental retardation with a frequency of 1/700 live births. Its
Correspondence: Dr. E.K. Shubber, Department of Biochemistry, Faculty of Agriculture and Biology, P.O. Box 765, Baghdad (Iraq).
predisposition to the development of leukemia is well known (Heidemann et al., 1985). Sister-chromatid exchanges (SCEs) are known to be a manifestation of damage to the genome (Latt et al., 1981). SCEs are seemingly related to mutagenesis (Carrano et al., 1979), morphological transformation induction in mammalian cells by viruses, physical and chemical carcinogens (Nichols et al., 1978; Popescu and Dipaolo, 1982) and the teratogenicity of many chemical and physical agents (Kram, 1982). In recent years more attention has been devoted to the study of SCE
62 levels in relation to health and disease. SCE frequency was reported to be significantly higher in lymhocytes of patients with Bloom's syndrome (Chaganti et al., 1974), acute lymphoblastic leukemia (Otter et al., 1979), chronic myeloid leukemia (Shirishi and Sandberg, 1980), and in patients with schistosomiasis (Shubber, 1987). Furthermore, previous studies revealing increased SCEs in infants with neural tube defects (Seshadri, 1982) and in parents with unfavorable reproductive health ( H a m a m y et al., 1990) necessitated further investigations correlating SCE frequencies with reproductive health and congenital malformations. Since the SCE frequency in cells of patients with Down's syndrome (DS) is still controversial (Lezana et al., 1977; Knuutila et al., 1979; Major et al., 1985: Heidemann et al., 1985), the aim of the present study was to further evaluate SCE frequencies in cells from DS infants born to mothers less than 37 years of age, as well as to investigate any correlation between SCE frequencies in DS infants and their parents. Furthermore, the cytogenetic response of cells from patients and controls was tested after their in vitro exposure to DNA-damaging agents. Materials and methods
Cytogenetic analysis Blood lymphocytes were cultured in RPMI-1640 medium for replicative indices (RI) under optimum conditions (Crossen, 1982~ Shubber et al., 1987). Two cultures were initiated from the blood sample of each individual. Cultures from controls and test subjects were made simultaneously. Briefly, 0.5 ml of heparinized blood was inocculated in 4.5 ml of the medium containing 10% heat-inactivated fetal calf serum. PHA (Pharmacia) in 2 # g / m l and BrdUrd in 10 /xg/ml. The cultures were kept in the dark at 3 7 ° C for 68 h. Colchicine (Houde. France) was added at a concentration of 0.1 m g / m l 4 h before harvest. The cells were treated with 0.075 M KCI, fixed with methanol:acetic acid (3:1 v / v ) and spread on slides. Air-dried slides were then stained with a 2% Giemsa solution. The mitotic index was counted as a ratio of mitoses to interphase nuclei in at least 750 cells. For each individual, 10 15 good metaphases were examined for chromosome counting, and 4 - 5 for karyotyping. Identical slides were stained in 4,6-diamidino-2-phenyl indol (DAPI) solution (Lin and Alfi, 1976), and the SCE were counted in 50 well-spread second metaphases. The replicative index was analyzed in 100 consecutive metaphase cells as percentage of first, second and third cell divisions in the presence of BrdUrd (Lamberti et al., 1983).
Population studied
Treatment with selective DNA-damaging agents
28 infants from families living under similar socioeconomic conditions were included in this study. Twelve of those infants (3 females and 9 males) at the time of the test had the clinical picture of Down's syndrome with no superimposed chest infection or leukemia. They had been referred to the Genetic Service Unit, Yarmouk Teaching Hospital for chromosome studies. The remaining infants (8 females and 8 males) were treated as controls. Twelve parents of infants with Down's syndrome and 20 parents of control infants were also included in this study. The mothers of both groups were mostly housewives or primary-school teachers, and the fathers had administrative jobs. All parents included in this study were healthy at the time of testing with irrelevant medical history or drug intake; neither the mothers nor the fathers of both groups smoked or drank.
Three agents were chosen because each has a different interaction with cellular DNA. Ionizing radiation causes primarly single- and doublestranded D N A breaks. It is a weak inducer of SCEs in animal and human cells (Shubber, 1981; Shirishi, 1984; AL-Allak, 1986; Shubber and Salih, 1988). Mitomycin C (MMC), a leukomogenic teratogen, acts as a cross-linking and bifunctional alkylating agent and is a powerful inducer of SCEs in maternal and fetal cells (Harris, 1979: Latt, 1981: Kram, 1982). The third agent is hycanthone (Hyc), an intercalating agent with a wide range of mutagenic activity in bacteria and mammalian cells including human cells (Clive, 1974: Shubber, 1981). It is a carcinogenic-teratogenic antischistosomal drug (Tsuda et al., 1979; Sieber et al., 1974). Finally incorporation of BrdUrd into D N A is required for visualization of SCEs (Latt,
63 1974). Much of the variation in baseline SCEs is undoubtedly due to differing amounts of BrdUrd in the culture medium (Crossen, 1982; Shubber et al., 1987). The doses of radiation and the concentrations of the chemicals were selected as the lowest effective doses that double the spontaneous frequencies of SCEs in human cells (Shubber, 1981; AL-Allak, 1986). Blood samples obtained from 6 normal infants, 6 DS infants, and the parents of both groups were exposed to: (1) y-radiation: lithium-heparinized blood culture tubes containing 1 ml of blood from each sample were exposed at room temperature (25°C + 2) to 0.1 Gy 3,-rays (Gamma cell 40; Atomic Energy, Ltd., Canada, operating with Cesium-137 at a dose rate of 1.25 G y / m i n ) was used. Immediately after irradiation, 9 ml of complete medium was added to each tube and incubated for 72 h. Colchicine was present for the last 4 h. SCE, MI and RI were determined as described above. (2) MMC, Hyc and BrdUrd: blood samples were inoculated in medium containing 0.05 /~g/ml MMC or 0.10 / t g / m l of Hyc in the presence of BrdUrd (10 /~g/ml) for 2 h at 37 o C. The treatments were terminated by washing the cultures 3 times with fresh worm medium and recultured in complete medium for 70 h. For BrdUrd, the blood was cultured in medium containing 20/~g/ml of BrdUrd for 72 h. 4 h before fixation, colchicine was added and slides were prepared and examined for determination of SCE, MI and RI.
Incubation time Blood taken from the same individuals in the above experiments was tested. 3 ml of blood was mixed with 27 ml of complete medium, distributed at equal volumes in 6 tubes (5 ml each) and incubated for 48, 72 or 96 h. Colchicine was added for the last 4 h of each period. Slides were then prepared and examined for scoring MI, RI and SCE. Statistical analysis The method of general linear models was followed to analyze the data (Soper et al., 1984). This method provides an analysis of variance with estimation of the percentage of variation in SCE attributable to each of the following independent
variables: trisomy 21, age and sex as associated with SCE level. Data of radiation and DNAdamaging chemicals were analyzed by Student's t test. Results and discussion
Chromosomal studies were performed on lymphocytes of 60 subjects: 12 infants with Down's syndrome (DS infants), 16 normal infants (N controls), 12 parents of DS infants and 20 parents of normal infants. Karyotypic analysis showed that 9 out of 12 patients with the clinical picture of DS had the regular trisomy-21 (47, +21) and 3 patients had mosaic trisomy-21 (46/47, +21) karyotypes. However, their parents showed normal karyotypes.
Sister-chromatid exchanges The incidences of SCE in blood lymphocytes of normal infants are presented in Table 1. The mean frequency of SCEs for the males was 5.2 + 0.3 (range 3.5-5.6 SCE/cell) and for the females 5.1 + 0.3 (range 3.9-6.6 SCE/ceI1). The spontaneous frequencies of SCEs in the lymphocytes of their parents were significantly higher ( P < 0.05) than the SCE frequency of the infants (Table 2). The SCE mean for the mothers was 6.5 + 0.2 SCE/cell and for the fathers 6.2 + 0.2 SCEs/cell. Differences in SCE frequency between infants and adults have also been observed by other investigators (Funes-Cravioto et al., 1977; HusgafvelPursiainen et al., 1988). This might reflect the difference in exposure to DNA-damaging agents, since infants presumably have had little or no contact with those agents (Lundberg and Livingston, 1983). It is also suggested that the susceptibility of human lymphocytes to BrdUrd increases with advancing age (Dutkowski et al., 1985). The results of 12 patients with DS are summarized in Table 1. The mean SCE frequencies for the male patients was 10.1 + 0.8 and for female patients 8.4 + 0.6 with a mean of 9.2 + 0.7 (range 6.9-16.0 SCE/cell). Although the numbers are small, it was noted that DS infants with a mosaic karyotype (46/47, +21) had a significantly lower mean SCE level (8.2 + 0.7) than DS infants with pure trisomy-21 (mean SCE = 9.8 + 0.9) (t = 3.1, P < 0.01). All values of DS patients
64 TABLE 1 C Y T O G E N E T I C ANALYSIS OF LYMPHOCYTES FROM I N F A N T S WITH DOWN'S S Y N D R O M E A N D HEALTHY CONTROLS Normal infants
Infants with Down's syndrome
Female
Male
Both sexes
Female
Male
Number
8
8
16
3
9
Age (months) Range X+SD
1-13 6.7_+3.6
1 -15 9.3_+4.3
8.0 +1.8
2-10 6.0_+4.0
1 12 5.1_+3.4
5.5-+0.6
Mitotic index (MI) Range X-+SE
3.2-8.0 5.2_+0.7
2.7 7.6 4.8-+0.7
5.0 -+0.2
7.4-9.5 8.5-+0.6
3.0-39.8 14.1-+4.6
11.3_+2.8
Replicative index (RI) Range X_+SE
1.71 2.25 1.96_+0.06
1.82-2.23 1.97-+0.04
1.96_+0.01
1.12-1.91 1.44+0.2
Sister-chromatid exchanges (SCE)/cell Range X_+SE
3.9-6.6 5.1_+0.3
3.5 6.3 5.2_+0.3
5.2 +0.2
7.5-9.7 8.4_+0.6
1.05 2.15 1.75_+0.11
8.2-16.0 10.1_+0.8
Both sexes
Significance *
12
P < 0.01
1.59_+0.15
P < 0.01
9.2-+0.8
P < 0.01
* Significance at means of both sexes of the normal and Down's syndrome infants.
were significantly higher ( P < 0.01) than the mean frequencies of the controls (5.2 + 0.2; range 3.56.6 SCE/cell, for both sexes). In 6 families with DS infants, both parents were willing to cooperate further in our research program. The results of their cytogenetic analysis are presented in Table 2. The mean SCE frequencies for mothers, aged 29.6 _+ 3.1 years, and fathers, aged 35.0_+ 3.0 years, were 7.8 + 0.2 (range 6.7-8.7 SCE/cell) and 9.5 _+ 0.5 (range 7.9-11.1 SCE/cell) respectively. Furthermore, the mean frequency of SCEs for both parents was 8.7 _+ 0.4 SCE/cell. These values were significantly higher ( P
Mutation Research, 248 (1991) 61-72 © 1991 Elsevier Science Publishers B.V. 0027-5107/91/$03.50 ADONIS 002751079100095F MTX 04958
Sister-chromatid exchanges in lymphocytes from infants with Down's syndrome E . K . S h u b b e r , H . A . H a m a m i 1, B . M . A . A l l a k a n d A . H . K h a l e e l Department of Biochemistry, Faculty of Agriculture and Biology and i A fi-Mustansiriya Medical College, Baghdad (Iraq) (Received 13 October 1988) (Revision received 4 September 1990) (Accepted 28 September 1990)
Keywords: Sister-chromatid exchange; Down's syndrome
Summary Sister-chromatid exchange (SCE) frequencies were studied in blood lymphocytes from 12 patients (3 females and 9 males) with Down's syndrome (DS). The mean frequency of SCE per metaphase for the patients (both sexes) was 9.2 + 0.8 which was significantly higher (P < 0.01) than the mean SCE value (5.1 +_0.2) scored for 16 healthy infants (8 females and 8 males). A significant increase in the mean frequency of SCE in 12 parents of infants with DS (8.7 + 0.9 SCE/ceI1) was noticeable when compared with 20 parents of normal infants (6.3 + 0.1 SCE/cell). Increases in cellular division with reduction in their replication were also observed in patients with DS. Treatment with mitomycin C (0.05 /~g/ml), hycanthone (0.1/~g/ml) and y-radiation (0.1 Gy) revealed a significant (P < 0.01) increase in frequencies of SCE in DS lymphocytes and in those of their parents as compared to controls. These data may reveal a familial hypersensitivity reaction to these agents. The results indicate a genomic instability and deranged DNA-repair mechanisms which are accentuated by exposure to mutagenic agents, the underlying causal factor for which might be genetic.
Down's syndrome, the most common autosomal anomaly in man, is currently the focus of an intensive investigation that promises to yield a wealth of information on gene expression and the molecular basis of disease (Patterson, 1987). The disease is the commonest cause of mental retardation with a frequency of 1/700 live births. Its
Correspondence: Dr. E.K. Shubber, Department of Biochemistry, Faculty of Agriculture and Biology, P.O. Box 765, Baghdad (Iraq).
predisposition to the development of leukemia is well known (Heidemann et al., 1985). Sister-chromatid exchanges (SCEs) are known to be a manifestation of damage to the genome (Latt et al., 1981). SCEs are seemingly related to mutagenesis (Carrano et al., 1979), morphological transformation induction in mammalian cells by viruses, physical and chemical carcinogens (Nichols et al., 1978; Popescu and Dipaolo, 1982) and the teratogenicity of many chemical and physical agents (Kram, 1982). In recent years more attention has been devoted to the study of SCE
62 levels in relation to health and disease. SCE frequency was reported to be significantly higher in lymhocytes of patients with Bloom's syndrome (Chaganti et al., 1974), acute lymphoblastic leukemia (Otter et al., 1979), chronic myeloid leukemia (Shirishi and Sandberg, 1980), and in patients with schistosomiasis (Shubber, 1987). Furthermore, previous studies revealing increased SCEs in infants with neural tube defects (Seshadri, 1982) and in parents with unfavorable reproductive health ( H a m a m y et al., 1990) necessitated further investigations correlating SCE frequencies with reproductive health and congenital malformations. Since the SCE frequency in cells of patients with Down's syndrome (DS) is still controversial (Lezana et al., 1977; Knuutila et al., 1979; Major et al., 1985: Heidemann et al., 1985), the aim of the present study was to further evaluate SCE frequencies in cells from DS infants born to mothers less than 37 years of age, as well as to investigate any correlation between SCE frequencies in DS infants and their parents. Furthermore, the cytogenetic response of cells from patients and controls was tested after their in vitro exposure to DNA-damaging agents. Materials and methods
Cytogenetic analysis Blood lymphocytes were cultured in RPMI-1640 medium for replicative indices (RI) under optimum conditions (Crossen, 1982~ Shubber et al., 1987). Two cultures were initiated from the blood sample of each individual. Cultures from controls and test subjects were made simultaneously. Briefly, 0.5 ml of heparinized blood was inocculated in 4.5 ml of the medium containing 10% heat-inactivated fetal calf serum. PHA (Pharmacia) in 2 # g / m l and BrdUrd in 10 /xg/ml. The cultures were kept in the dark at 3 7 ° C for 68 h. Colchicine (Houde. France) was added at a concentration of 0.1 m g / m l 4 h before harvest. The cells were treated with 0.075 M KCI, fixed with methanol:acetic acid (3:1 v / v ) and spread on slides. Air-dried slides were then stained with a 2% Giemsa solution. The mitotic index was counted as a ratio of mitoses to interphase nuclei in at least 750 cells. For each individual, 10 15 good metaphases were examined for chromosome counting, and 4 - 5 for karyotyping. Identical slides were stained in 4,6-diamidino-2-phenyl indol (DAPI) solution (Lin and Alfi, 1976), and the SCE were counted in 50 well-spread second metaphases. The replicative index was analyzed in 100 consecutive metaphase cells as percentage of first, second and third cell divisions in the presence of BrdUrd (Lamberti et al., 1983).
Population studied
Treatment with selective DNA-damaging agents
28 infants from families living under similar socioeconomic conditions were included in this study. Twelve of those infants (3 females and 9 males) at the time of the test had the clinical picture of Down's syndrome with no superimposed chest infection or leukemia. They had been referred to the Genetic Service Unit, Yarmouk Teaching Hospital for chromosome studies. The remaining infants (8 females and 8 males) were treated as controls. Twelve parents of infants with Down's syndrome and 20 parents of control infants were also included in this study. The mothers of both groups were mostly housewives or primary-school teachers, and the fathers had administrative jobs. All parents included in this study were healthy at the time of testing with irrelevant medical history or drug intake; neither the mothers nor the fathers of both groups smoked or drank.
Three agents were chosen because each has a different interaction with cellular DNA. Ionizing radiation causes primarly single- and doublestranded D N A breaks. It is a weak inducer of SCEs in animal and human cells (Shubber, 1981; Shirishi, 1984; AL-Allak, 1986; Shubber and Salih, 1988). Mitomycin C (MMC), a leukomogenic teratogen, acts as a cross-linking and bifunctional alkylating agent and is a powerful inducer of SCEs in maternal and fetal cells (Harris, 1979: Latt, 1981: Kram, 1982). The third agent is hycanthone (Hyc), an intercalating agent with a wide range of mutagenic activity in bacteria and mammalian cells including human cells (Clive, 1974: Shubber, 1981). It is a carcinogenic-teratogenic antischistosomal drug (Tsuda et al., 1979; Sieber et al., 1974). Finally incorporation of BrdUrd into D N A is required for visualization of SCEs (Latt,
63 1974). Much of the variation in baseline SCEs is undoubtedly due to differing amounts of BrdUrd in the culture medium (Crossen, 1982; Shubber et al., 1987). The doses of radiation and the concentrations of the chemicals were selected as the lowest effective doses that double the spontaneous frequencies of SCEs in human cells (Shubber, 1981; AL-Allak, 1986). Blood samples obtained from 6 normal infants, 6 DS infants, and the parents of both groups were exposed to: (1) y-radiation: lithium-heparinized blood culture tubes containing 1 ml of blood from each sample were exposed at room temperature (25°C + 2) to 0.1 Gy 3,-rays (Gamma cell 40; Atomic Energy, Ltd., Canada, operating with Cesium-137 at a dose rate of 1.25 G y / m i n ) was used. Immediately after irradiation, 9 ml of complete medium was added to each tube and incubated for 72 h. Colchicine was present for the last 4 h. SCE, MI and RI were determined as described above. (2) MMC, Hyc and BrdUrd: blood samples were inoculated in medium containing 0.05 /~g/ml MMC or 0.10 / t g / m l of Hyc in the presence of BrdUrd (10 /~g/ml) for 2 h at 37 o C. The treatments were terminated by washing the cultures 3 times with fresh worm medium and recultured in complete medium for 70 h. For BrdUrd, the blood was cultured in medium containing 20/~g/ml of BrdUrd for 72 h. 4 h before fixation, colchicine was added and slides were prepared and examined for determination of SCE, MI and RI.
Incubation time Blood taken from the same individuals in the above experiments was tested. 3 ml of blood was mixed with 27 ml of complete medium, distributed at equal volumes in 6 tubes (5 ml each) and incubated for 48, 72 or 96 h. Colchicine was added for the last 4 h of each period. Slides were then prepared and examined for scoring MI, RI and SCE. Statistical analysis The method of general linear models was followed to analyze the data (Soper et al., 1984). This method provides an analysis of variance with estimation of the percentage of variation in SCE attributable to each of the following independent
variables: trisomy 21, age and sex as associated with SCE level. Data of radiation and DNAdamaging chemicals were analyzed by Student's t test. Results and discussion
Chromosomal studies were performed on lymphocytes of 60 subjects: 12 infants with Down's syndrome (DS infants), 16 normal infants (N controls), 12 parents of DS infants and 20 parents of normal infants. Karyotypic analysis showed that 9 out of 12 patients with the clinical picture of DS had the regular trisomy-21 (47, +21) and 3 patients had mosaic trisomy-21 (46/47, +21) karyotypes. However, their parents showed normal karyotypes.
Sister-chromatid exchanges The incidences of SCE in blood lymphocytes of normal infants are presented in Table 1. The mean frequency of SCEs for the males was 5.2 + 0.3 (range 3.5-5.6 SCE/cell) and for the females 5.1 + 0.3 (range 3.9-6.6 SCE/ceI1). The spontaneous frequencies of SCEs in the lymphocytes of their parents were significantly higher ( P < 0.05) than the SCE frequency of the infants (Table 2). The SCE mean for the mothers was 6.5 + 0.2 SCE/cell and for the fathers 6.2 + 0.2 SCEs/cell. Differences in SCE frequency between infants and adults have also been observed by other investigators (Funes-Cravioto et al., 1977; HusgafvelPursiainen et al., 1988). This might reflect the difference in exposure to DNA-damaging agents, since infants presumably have had little or no contact with those agents (Lundberg and Livingston, 1983). It is also suggested that the susceptibility of human lymphocytes to BrdUrd increases with advancing age (Dutkowski et al., 1985). The results of 12 patients with DS are summarized in Table 1. The mean SCE frequencies for the male patients was 10.1 + 0.8 and for female patients 8.4 + 0.6 with a mean of 9.2 + 0.7 (range 6.9-16.0 SCE/cell). Although the numbers are small, it was noted that DS infants with a mosaic karyotype (46/47, +21) had a significantly lower mean SCE level (8.2 + 0.7) than DS infants with pure trisomy-21 (mean SCE = 9.8 + 0.9) (t = 3.1, P < 0.01). All values of DS patients
64 TABLE 1 C Y T O G E N E T I C ANALYSIS OF LYMPHOCYTES FROM I N F A N T S WITH DOWN'S S Y N D R O M E A N D HEALTHY CONTROLS Normal infants
Infants with Down's syndrome
Female
Male
Both sexes
Female
Male
Number
8
8
16
3
9
Age (months) Range X+SD
1-13 6.7_+3.6
1 -15 9.3_+4.3
8.0 +1.8
2-10 6.0_+4.0
1 12 5.1_+3.4
5.5-+0.6
Mitotic index (MI) Range X-+SE
3.2-8.0 5.2_+0.7
2.7 7.6 4.8-+0.7
5.0 -+0.2
7.4-9.5 8.5-+0.6
3.0-39.8 14.1-+4.6
11.3_+2.8
Replicative index (RI) Range X_+SE
1.71 2.25 1.96_+0.06
1.82-2.23 1.97-+0.04
1.96_+0.01
1.12-1.91 1.44+0.2
Sister-chromatid exchanges (SCE)/cell Range X_+SE
3.9-6.6 5.1_+0.3
3.5 6.3 5.2_+0.3
5.2 +0.2
7.5-9.7 8.4_+0.6
1.05 2.15 1.75_+0.11
8.2-16.0 10.1_+0.8
Both sexes
Significance *
12
P < 0.01
1.59_+0.15
P < 0.01
9.2-+0.8
P < 0.01
* Significance at means of both sexes of the normal and Down's syndrome infants.
were significantly higher ( P < 0.01) than the mean frequencies of the controls (5.2 + 0.2; range 3.56.6 SCE/cell, for both sexes). In 6 families with DS infants, both parents were willing to cooperate further in our research program. The results of their cytogenetic analysis are presented in Table 2. The mean SCE frequencies for mothers, aged 29.6 _+ 3.1 years, and fathers, aged 35.0_+ 3.0 years, were 7.8 + 0.2 (range 6.7-8.7 SCE/cell) and 9.5 _+ 0.5 (range 7.9-11.1 SCE/cell) respectively. Furthermore, the mean frequency of SCEs for both parents was 8.7 _+ 0.4 SCE/cell. These values were significantly higher ( P
