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Non-random chromosome loss in PHA-stimulated lymphocytes from normal individuals
Mutation Research, 122 (1983) 403-406
403
Elsevier MRLett 0484
Non-random chromosome loss in PHA-stimulated lymphocytes from normal individuals T. B r o w n , D . P . F o x , F . W . R o b e r t s o n a n d I. B u l l o c k Department of Genetics, University of Aberdeen (Great Britain)
(Accepted 25 August 1983)
Summary 31 773 lymphocyte metaphase cells from 280 karyotypically normal men aged 18-46 were examined for chromosome gain or loss. C h r o m o s o m e loss was much more c o m m o n than c h r o m o s o m e gain. Frequency of c h r o m o s o m e loss did not conform to a binomial distribution. There is a striking non-linear, inverse relationship between likelihood of loss and chromosome length. C h r o m o s o m e gain shows a near binomial distribution between cells and no clear relationship to chromosome length. These facts indicate that the hypodiploid ceils mostly arose as technical artefacts during slide preparation but that hyperdiploid cells were mainly due to nondisjunctional gain.
Lymphocytes in C-metaphase often exhibit c h r o m o s o m e numbers which differ from the normal diploid number, even in karyotypically-normal individuals. These ceils may arise by non-disjunction or as technical artefacts during fixation and slide preparation. Several reports in the literature show an increase in the frequency of aneuploid lymphocytes with age, especially hypodiploid cells, especially in individuals over the age of 60 years and especially in women (Galloway and Buckton, 1978). However, there is little evidence for such a relationship between aneupioidy and age in young men (Sandberg et al., 1967; Bloom et al., 1967). Perhaps because of the observed correlation between age and frequency of hypodiploid cells it has been implicitly assumed that a significant proportion of the hypodiploid cells arise by non-disjunction, though it remains a possibility that technical artefact is the main source o f such cells. Address for correspondence: Dr. D.P. Fox, Department of Genetics. University of Aberdeen, 2 Tillydrone Avenue, Aberdeen, AB9 2TN (Scotland). 0165-7992/83/$ 03.00 © 1983 Elsevier Science Publishers B.V.
4O4
In hypodiploid cells it is difficult to distinguish between non-disjunction and technical artefact as the cause whereas extra chromosomes in hyperdiploid cells can usually be recognised as belonging to the cell or as 'strays' from another cell by their state of contraction. Hypotonic treatment (which may burst the cell membrane), brief fixation (which leaves the cytoplasm fragile) and air-drying (which subjects the rapidly-drying cells to considerable forces) are all factors which contribute to chromosome loss from metaphase cells. Observed hypodiploidy thus reflects the combined frequencies of technical artefact and non-disjunction. Hyperdipioidy, on the other hand, may be almost entirely due to non-disjunction if recognisable 'strays' are excluded. In an extensive study of 280 normal male subjects aged 18-46 years, data were collected on chromosome gain and loss in 31 773 lymphocyte metaphases sampled at 48 h after stimulation. Analysis of these data largely supports the contention that chromosome loss was mostly due to technical factors while chromosome gain was mostly due to non-disjunction. The basic data are contained in Table 1. TABLE 1 Frequencies of cells with chromosome numbers between 40 and 50. Cells with < 4 6 chromosomes were not selected but cells with > 4 6 chromosomes were rejected if any chromosome was significantly overor under-contracted compared with the other. Chromosome number
40
41
42
43
44
45
46
47
Total cells
129
232
346
535
965
1544
27982 31
48
49
50
7
2
0
Chromosomes lost in 45 chromosome cells were classified into Denver groups, including sub-division of G-group into (21 + 22) and Y sub-sets. There is a striking relationship between frequency of chromosome loss from each Denver group and mean chromosome length in that group (Fig. 1). Chromosomes are certainly not lost at random (X2 t6) = 599.68, P<0.0001). Distribution of chromosome loss and gain between cells would be expected to follow a binomial distribution if independent non-disjunction of individual chromosomes were responsible. On the other hand, bursting of the cell membrane during hypotonic treatment or rupture of the fixed cytoplasm during slide preparation would often lead to the simultaneous loss o f several chromosomes. Reference to Table 1 shows that loss is not binomial. The assumed value of 0.00295 for the probability of loss of a single chromosome is derived from the frequency of cells which have suffered no loss. However, this value seriously underestimates the frequency of chromosome loss in all other categories. It is more difficult to test chromosome gain for its fit to a binomial distribution because of the low frequency
405 30
o
00
t -t l 2 ~ 6 O - I ~ LENGTH pm
;
Fig. 1. The relationship between metaphase chromosome length and the frequency of loss of chromosomes in each Denver group for cells with 45 chromosomes. The equation for the curve fitted is y = 70.73 x - L508. 'Frequency' is frequency of loss x 10 - 4 for each Denver group corrected for number of chromosomes in that group.
of cells which have gained chromosomes. However, a similar calculation suggests a much closer fit to a binomial distribution in this case. Table l also shows that c h r o m o s o m e loss is much more frequent than c h r o m o s o m e gain (frequency of cell, with 45 chromosomes is about 50 × greater than cells with 47 chromosomes). Although non-disjunction is expected to lead to c h r o m o s o m e loss in one daughter cell which is not always balanced by chromosome gain in the other daughter cell, this partial non-disjunction is unlikely to be 50 × as frequent as complete non-disjunction. Fig. 1 shows that chromosomes in different Denver groups have quite different probabilities o f being lost and that the probability of loss is an inverse function of c h r o m o s o m e length. There seems to be no a priori reason why a short chromosome should suffer non-disjunction more frequently than a long chromosome but it is likely that a short c h r o m o s o m e will become detached more readily by technical effects since it is anchored to a smaller volume of cytoplasm. A clear complication arises in the case of non-disjunctional loss in that cell selection may intervene to modify the original pattern of non-disjunction and this would be expected to favour the preferential survival of cells which have lost a short c h r o m o s o m e over those which have lost a long c h r o m o s o m e since there is a smaller gene loss in the former case. However, if such selection were operating, the loss of the Y chromosome should be most easily tolerated, especially compared with chromosomes 21 + 22. This is not the case, the latter being lost more frequently. There is a weak correlation between frequency of loss of one c h r o m o s o m e and subject age (r = 0.154, P -0.05-0.01) which suggests that some c h r o m o s o m e loss is due to non-disjunction. All the other evidence points strongly to technical artefact being the main cause of the observed loss. The approach employed in this paper suggests a series of tests for distinguishing between non-disjunction and technical artefact as causes of c h r o m o s o m e loss.
406
Acknowledgements This w o r k was s u p p o r t e d by c o n t r a c t N o . O T / A / 3 3 8
f r o m the D e p a r t m e n t o f
Ene r g y .
References Bloom, A.D., P.G. Arthur and A.A. Awa (1967) Variation in the human chromosome number, Nature (London), 216, 487--489. Galloway, S.M., and K.E. Buckton (1978) Aneuploidy and ageing: chromosome studies on a random sample of the population using G-banding, Cytogenet. Cell Genet., 20, 78-95. Sandberg, A.A., M.M. Cohen, A.A. Rimm and M.L. Levin (1967) Aneuploidy and age in a population survey, Am. J. Hum. Genet., 19, 633-643.
403
Elsevier MRLett 0484
Non-random chromosome loss in PHA-stimulated lymphocytes from normal individuals T. B r o w n , D . P . F o x , F . W . R o b e r t s o n a n d I. B u l l o c k Department of Genetics, University of Aberdeen (Great Britain)
(Accepted 25 August 1983)
Summary 31 773 lymphocyte metaphase cells from 280 karyotypically normal men aged 18-46 were examined for chromosome gain or loss. C h r o m o s o m e loss was much more c o m m o n than c h r o m o s o m e gain. Frequency of c h r o m o s o m e loss did not conform to a binomial distribution. There is a striking non-linear, inverse relationship between likelihood of loss and chromosome length. C h r o m o s o m e gain shows a near binomial distribution between cells and no clear relationship to chromosome length. These facts indicate that the hypodiploid ceils mostly arose as technical artefacts during slide preparation but that hyperdiploid cells were mainly due to nondisjunctional gain.
Lymphocytes in C-metaphase often exhibit c h r o m o s o m e numbers which differ from the normal diploid number, even in karyotypically-normal individuals. These ceils may arise by non-disjunction or as technical artefacts during fixation and slide preparation. Several reports in the literature show an increase in the frequency of aneuploid lymphocytes with age, especially hypodiploid cells, especially in individuals over the age of 60 years and especially in women (Galloway and Buckton, 1978). However, there is little evidence for such a relationship between aneupioidy and age in young men (Sandberg et al., 1967; Bloom et al., 1967). Perhaps because of the observed correlation between age and frequency of hypodiploid cells it has been implicitly assumed that a significant proportion of the hypodiploid cells arise by non-disjunction, though it remains a possibility that technical artefact is the main source o f such cells. Address for correspondence: Dr. D.P. Fox, Department of Genetics. University of Aberdeen, 2 Tillydrone Avenue, Aberdeen, AB9 2TN (Scotland). 0165-7992/83/$ 03.00 © 1983 Elsevier Science Publishers B.V.
4O4
In hypodiploid cells it is difficult to distinguish between non-disjunction and technical artefact as the cause whereas extra chromosomes in hyperdiploid cells can usually be recognised as belonging to the cell or as 'strays' from another cell by their state of contraction. Hypotonic treatment (which may burst the cell membrane), brief fixation (which leaves the cytoplasm fragile) and air-drying (which subjects the rapidly-drying cells to considerable forces) are all factors which contribute to chromosome loss from metaphase cells. Observed hypodiploidy thus reflects the combined frequencies of technical artefact and non-disjunction. Hyperdipioidy, on the other hand, may be almost entirely due to non-disjunction if recognisable 'strays' are excluded. In an extensive study of 280 normal male subjects aged 18-46 years, data were collected on chromosome gain and loss in 31 773 lymphocyte metaphases sampled at 48 h after stimulation. Analysis of these data largely supports the contention that chromosome loss was mostly due to technical factors while chromosome gain was mostly due to non-disjunction. The basic data are contained in Table 1. TABLE 1 Frequencies of cells with chromosome numbers between 40 and 50. Cells with < 4 6 chromosomes were not selected but cells with > 4 6 chromosomes were rejected if any chromosome was significantly overor under-contracted compared with the other. Chromosome number
40
41
42
43
44
45
46
47
Total cells
129
232
346
535
965
1544
27982 31
48
49
50
7
2
0
Chromosomes lost in 45 chromosome cells were classified into Denver groups, including sub-division of G-group into (21 + 22) and Y sub-sets. There is a striking relationship between frequency of chromosome loss from each Denver group and mean chromosome length in that group (Fig. 1). Chromosomes are certainly not lost at random (X2 t6) = 599.68, P<0.0001). Distribution of chromosome loss and gain between cells would be expected to follow a binomial distribution if independent non-disjunction of individual chromosomes were responsible. On the other hand, bursting of the cell membrane during hypotonic treatment or rupture of the fixed cytoplasm during slide preparation would often lead to the simultaneous loss o f several chromosomes. Reference to Table 1 shows that loss is not binomial. The assumed value of 0.00295 for the probability of loss of a single chromosome is derived from the frequency of cells which have suffered no loss. However, this value seriously underestimates the frequency of chromosome loss in all other categories. It is more difficult to test chromosome gain for its fit to a binomial distribution because of the low frequency
405 30
o
00
t -t l 2 ~ 6 O - I ~ LENGTH pm
;
Fig. 1. The relationship between metaphase chromosome length and the frequency of loss of chromosomes in each Denver group for cells with 45 chromosomes. The equation for the curve fitted is y = 70.73 x - L508. 'Frequency' is frequency of loss x 10 - 4 for each Denver group corrected for number of chromosomes in that group.
of cells which have gained chromosomes. However, a similar calculation suggests a much closer fit to a binomial distribution in this case. Table l also shows that c h r o m o s o m e loss is much more frequent than c h r o m o s o m e gain (frequency of cell, with 45 chromosomes is about 50 × greater than cells with 47 chromosomes). Although non-disjunction is expected to lead to c h r o m o s o m e loss in one daughter cell which is not always balanced by chromosome gain in the other daughter cell, this partial non-disjunction is unlikely to be 50 × as frequent as complete non-disjunction. Fig. 1 shows that chromosomes in different Denver groups have quite different probabilities o f being lost and that the probability of loss is an inverse function of c h r o m o s o m e length. There seems to be no a priori reason why a short chromosome should suffer non-disjunction more frequently than a long chromosome but it is likely that a short c h r o m o s o m e will become detached more readily by technical effects since it is anchored to a smaller volume of cytoplasm. A clear complication arises in the case of non-disjunctional loss in that cell selection may intervene to modify the original pattern of non-disjunction and this would be expected to favour the preferential survival of cells which have lost a short c h r o m o s o m e over those which have lost a long c h r o m o s o m e since there is a smaller gene loss in the former case. However, if such selection were operating, the loss of the Y chromosome should be most easily tolerated, especially compared with chromosomes 21 + 22. This is not the case, the latter being lost more frequently. There is a weak correlation between frequency of loss of one c h r o m o s o m e and subject age (r = 0.154, P -0.05-0.01) which suggests that some c h r o m o s o m e loss is due to non-disjunction. All the other evidence points strongly to technical artefact being the main cause of the observed loss. The approach employed in this paper suggests a series of tests for distinguishing between non-disjunction and technical artefact as causes of c h r o m o s o m e loss.
406
Acknowledgements This w o r k was s u p p o r t e d by c o n t r a c t N o . O T / A / 3 3 8
f r o m the D e p a r t m e n t o f
Ene r g y .
References Bloom, A.D., P.G. Arthur and A.A. Awa (1967) Variation in the human chromosome number, Nature (London), 216, 487--489. Galloway, S.M., and K.E. Buckton (1978) Aneuploidy and ageing: chromosome studies on a random sample of the population using G-banding, Cytogenet. Cell Genet., 20, 78-95. Sandberg, A.A., M.M. Cohen, A.A. Rimm and M.L. Levin (1967) Aneuploidy and age in a population survey, Am. J. Hum. Genet., 19, 633-643.