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Does aneuploidy per se cause developmental abnormalities?
Mutation Research 396 Ž1997. 195–203
Does aneuploidy per se cause developmental abnormalities? Judith Helen Ford
)
Genetic Consulting and Testing (G.C.A.T.) Pty Ltd., P.O. Box 210, Port Adelaide, South Australia 5011, Australia Received 19 December 1996; revised 22 June 1997; accepted 5 July 1997
Abstract The following questions are addressed: ŽA. What are aneuploidogens and how do they act? ŽB. Is there any evidence that aneuploidy per se causes malformations? ŽC. What examples are there of abnormalities, apparently attributable to aneuploidogens acting as teratogens? ŽD. Do abnormalities of cell division cause both teratogenesis and aneuploidy? Considerable research has addressed question ŽA., but there is little which addresses the other three questions. The question of whether aneuploidy per se causes malformations remains open. Some suggestions for further research are made. q 1997 Elsevier Science B.V.
1. Malformation and aneuploidy Syndromes of malformation in animals and humans are often associated with chromosome specific aneuploidy. In humans, three trisomies, namely trisomy 13, trisomy 18 and trisomy 21 frequently survive to full gestation. Trisomy 13 and 18, however, are both associated with early infant death. Trisomies in mouse for chromosomal regions syntenic to the human chromosomal regions show some similar patterns of malformation. Thus there is significant evidence that many of the phenotypic effects of aneuploidy are chromosome-specific and are due to the imbalance of certain critical genes. The question of whether aneuploidy per se induces malformation has rarely been addressed. The evidence that is available is presented in Section 3. Data which demonstrates that aneuploid cells are more error-prone than normal cells indicate that aneuploidy per se has significance. ) Corresponding author. Tel.: q61 8 82443042; Fax: q61 8 82443407; E-mail: [email protected]
Further importance of knowing whether aneuploidy per se causes malformation may emerge if certain types of genetic therapy are considered in the future. One possible example would be: in early foetal therapy it may be considered that the introduction of an extra genetically engineered micro-chromosome could be the preferred method of introducing a new gene construct. This might be considered to prevent the effects of some serious metabolic disorder. The introduction of the extra centromere might disturb the cell sufficiently to cause malformation. A second example would be: early gene therapy might be considered to correct the genetic imbalance in an aneuploid individual, especially in Žsay. trisomy 21. If the syndromic malformation is at least partially caused by aneuploidy per se, then this type of therapy would be at best only partly effective.
2. What are aneuploidogens and how do they act? An aneuploidogen is any factor which can induce aneuploidy. The list is large and includes not only
0027-5107r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 2 7 - 5 1 0 7 Ž 9 7 . 0 0 1 8 4 - X
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Table 1 Aneuploidogens which tested positive in at least two testing systems and their probable sites of action Žafter w5x. Site of action
Effect on cell division
Affect microtubular polymerization
‘‘c-mitosis’’™metaphase arrest
Crystallize tubulin Enhances tubulin polymerization Blocks pole-to-pole spindle elongation Inhibits DNA synthesis
‘‘c-mitosis’’™metaphase arrest Inhibits mitosis Inhibits anaphase B and thus induces polyploidy Disturbs synchronicity ™ multipolar spindles andr or chromosome lagging. Inhibits cell division Causes multipolar spindles andror chromosome lagging
Colchicine, cytosine, arabinoside
Disrupts polymerized microtubules Damages centriolesrcentrosomes Damage kinetochore Binds to calmodulin Damage cell membranes Affect chromosome pairing and crossing-over
Alters rate of separation of sister chromatids Žmitosis. Metaphase arrest Disturb distribution of chromosomes Žspatial orientation., leading to misdivision Meiotic error
J.H. Ford r Mutation Research 396 (1997) 195–203
AneuploidogenŽs. Colchicine, colcemid, podophyllotoxin, benzimidazole derivatives Žbenomyl, MBC, nocodazole. diethylstilbestrol, griseofulvin, sulfhydryl reagents Žmercury compounds, arsenic compounds, diamide., para-fluorophenylalanine Vinca alkaloids Žvinblastine, vincristine. Taxol Chloral hydrate Imbalances in the nucleotide pool or nucleotide analogues, e.g. BUdr, FUdr. Hypothermia or hyperthermia Diazepam, diethylstilbestrol, ethidium bromide, actinomycin D Colchicine, mitomycin C Chlorpromazine Amphotericin B, fenarimol, miconazole
J.H. Ford r Mutation Research 396 (1997) 195–203
chemicals and X-radiation, but other factors such as pH which are capable of disturbing the cellular milieu w1x. In other words, any factor which can disturb the dynamic process of cell division or its spatial orientation, has the potential to be an aneuploidogen. It has been convincingly demonstrated that there are many chemicals whose action is primarily aneuploidogenic Žsee Table 1.. There are, however, those whose primary action is usually regarded as a clastogenic, but which nevertheless also have significant aneuploidogenic action w2,3x. Each part of the cell division machinery is vulnerable to disturbance. In gross terms, the following elements make up the machinery w4x: Ø The kinetochore region of the chromosomes Ø The centrioles and peri-centriolar particles Ø The spindle fibres composed of microtubules and MAPS Žmicrotubular associated proteins. Ø The cytoplasm and its content of microtubular protein Ø The chromosomal telomeres and the nuclear membrane Ø Cytoplasmic microfilaments The process of mitotic cell division requires not only that each component be intact and functional but that all the different processes work together in synchrony. Agents which adversely affect the timing of one or more of the critical processes can induce aneuploidy just as surely as an agent which binds to one of the components. Table 1 shows some examples of aneuploidogens and their probable sites of action w5x. The aneuploidogenic properties of most of these chemicals has been further confirmed by the human lymphocyte micronucleus test ŽMN., in combination with fluorescent in situ hybridisation ŽFISH. of a centromeric DNA probe Že.g. w3,6x.. Just as critical as the elements of the spindle and the chromosomes is the cellular milieu. The maintenance of cellular pH, salt concentration, electro-magnetic forces, GTP and ATP levels, pressure and temperature are critical to the maintenance of proper cell division. These aspects of cell division were often studied in the early years of cell biology, 1930 to 1950, but now rarely receive much attention. Drugs andror physiological conditions which disturb
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any of these parameters can disturb cell division and cause aneuploidy w7x. Meiosis I, in addition to all the factors above, involves the complex process of chromosome pairing and crossing over. It has been established that some chromosomes involved in maternal nondisjunction involve reduced or altered recombination w8–11x. Most discussions of the significance of this look to mechanisms which might generate these ‘altered gametes’ in older women. An alternative hypothesis, which in the view of this author is more likely, is the following. Altered gametes, those with altered or lowered rates of recombination, occur at equal frequencies at all ages but are more prone to error in the ageing oocyte. The distribution of chiasmata alters the three-dimensional structure of a bivalent, and certain structures take more time to align on the spindle. ‘Marker bivalents’ are less stable on the meiotic spindle and are more prone to error w12x. This effect of bivalent instability is enhanced in ageing oocytes w13x. Studies of ageing mouse oocytes have shown that intrinsic differences in the cell cycle occur with age. There is an earlier onset of chromosome separation in oocytes with a premature anaphase transition w14x. It is argued that these altered physiological conditions are the major cause for the increased incidence of nondisjunction of marker bivalents in ageing mice w13x. Altered bivalents do not lead to aneuploidy in younger women because of the greater proficiency of the meiotic 1 division process. It is possible that environmental factors also affect meiotic recombination w15x. However, in humans this would be restricted to effects in the male, or in the female during intra-uterine life. There is little evidence at this stage to support this contention. Aneuploidy frequencies show little variation between human males w16,17x and no appropriate studies have been undertaken of exposures, other than diethylstilbestrol, in human foetal life. Both males and females exposed to diethylstilbestrol ŽDES. in utero have decreased fertility w18x but no study of aneuploidy in their gametes has been reported. Aneuploidogens can thus be extrinsic or intrinsic factors. The list of chemicals which can induce aneuploidy is large. However, the available data in
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J.H. Ford r Mutation Research 396 (1997) 195–203
humans suggest that the effect of ageing far outweighs that of external factors. More research is required to define the precise effects of ageing and to discover whether any of the declining cellular functions can be preserved or stabilised.
3. Is there any evidence that aneuploidy per se causes malformations? 3.1. Possible role of aneuploidy in common malformations In humans, aneuploidy plays a significant role in early embryonic lethality w19x and is the basis of several well-defined congenital abnormalities. Since each type of aneuploid e.g. monosomy X, trisomy 21, trisomy 18, has its own phenotype, the syndrome of abnormalities is interpreted to be the result of each of the specific chromosome imbalances. In order to determine whether aneuploidy per se causes developmental abnormalities, it is necessary to look beyond the effects of the specific chromosomal imbalances. Are there abnormalities common to the aneuploid state, including sex chromosome aneuploidy? Data which contributes to the answer to this question come from chromosome analyses in children with multiple malformations and mental retardation of unknown origin. Several studies were summarised w20x and all gave similar results. The study summarised in w21x excluded cases where a syndrome such as Down’s syndrome was suspected before the analysis. This exclusion was used to minimise the bias of known chromosome-specific effects.
7.6% Ž54r710. infants with abnormalities had chromosome abnormalities compared with 0.7% chromosome abnormalities in controls. The results of the study are summarised in Table 2. Two frequent findings in this group are congenital heart defects and mental retardation. Of the former, 13% have a chromosomal abnormality and up to 48% of the mentally retarded have chromosomal abnormalities. The allocation of a causative role to aneuploidy in the etiology of these malformations is still difficult because of the large impact of chromosomal defects which do not involve changes in chromosome number. However, the inclusion of sex chromosome aneuploidy suggests that there could be an influence of aneuploidy per se. 3.2. Effect of chromosomal anomalies on cell multiplication Cell lines initiated from embryos with aneuploid chromosomal abnormalities exhibited significantly slower generation times and decreased lifespans w22x. These changes seemed to be common to the aneuploid state rather than chromosome specific and were true of monosomic X cells as well as trisomic cells. When cell lines transform, these relationships between chromosome number and cell cycle time are often lost. It is important to note that these studies were undertaken on primary embryonic fibroblast cultures. Further evidence of reduced cell multiplication in human aneuploidy comes from the study of dermatoglyphics. A typical example of the many studies in this field confirms that the total ridge count in palms and soles of children with sex chromosomal abnormalities is decreased with increasing number of sex chromosomes w23x.
Table 2 Results of chromosomal analysis of 710 individuals without suspected syndrome but with mental retardation and three independent malformations w21x Type of abnormality Aneuploid Ž n s 7. Aneuploid and structural Ž n s 8. Structural Ž n s 20. Structural Ž n s 19.
Sub-classification
Karyotype
Apparently balanced Unbalanced
47,q 18; 47,XXX Ž n s 2.; 47,XXY; 46r47,q Er92,XXYY; 47,q D; 47,q G 47,q der Ž n s 2.; 46,XXr47,q r Ž n s 4.; 47,q mar Ž n s 2. Inversions Ž n s 11.; Robertsonian Ž n s 2.; other Ž n s 7. Deletions Ž n s 7.; rings Ž n s 1.; other Ž n s 11.
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Table 3 Chromosome segregation at late anaphase in cells with 2 and 3 X chromosomes Ratio of X centromere-specific silver grains
No. of cells
‘Pre-micronuclei’ — Autosome
‘Pre-micronuclei’ — X chromosome
2:2 3:1 1:1 Total
1359 15 2 1376
19 1 0 20
0 0 2 2
3:3 3:2 4:2 Total
7 1 9 17
2 0 0 2
0 1 0 1
3.3. Effect of aneuploidy and triploidy on embryonic deÕelopment In a quantitative analysis of the number of nuclei in embryonic cells, it was found w24x that both normal and abnormal specimens which had normal chromosomes had a similar distribution of placental cells. However embryonic specimens with trisomy 16, monosomy X or triploidy, all of which were developmentally abnormal, revealed analogous paucity of the number of cytotrophoblastic nuclei. It was concluded that the paucity of nuclei was a direct effect of the aneuploid state. Recent studies on the placentas of pregnancies with intra-uterine growth retardation has led to the recognition that some placentas are mosaic for diploid and trisomic cells. The term ‘‘confined placental mosaicism’’ has been coined for this situation in which there can be a trisomic foetus supported by a diploid placenta or a diploid foetus supported by a trisomic foetus. In the former, a trisomic foetus can survive a pregnancy. In the latter, a diploid foetus might not survive. Whilst these findings fit the general concept expressed above w24x, there is one major difference. The outcome of pregnancies with a trisomic placenta and diploid foetus largely depends on first, the specific chromosome involved and second, the presence or absence of uniparental disomy in the foetus w25x. Both these conditions argue for chromosome specific effects rather than aneuploidy per se. 3.4. A human syndrome of mosaic aneuploidy? A recent paper w26x reports an abnormality syndrome which is associated with a variegated mo-
saicism of normal cells with three cells lines carrying different numbers, one, two or three, of an unidentified marker chromosome. The syndrome has optic atrophy, mental retardation, microcephaly and short stature. This paper argues that that aneuploidy per se accounts for these abnormality rather than the specific genetic imbalance. Our own laboratory had an interesting case which contributes to this discussion Žunpublished data.. A man was referred for extremely short stature; as a mature adult he was 4 foot, 6 inches tall but was not a dwarf. He had normal intelligence and normal secondary sexual characteristics. Chromosome analysis of his lymphocytes showed that 50% of his cells were 47,XY,q 21 and the other 50% were 45,X. Presumably he had enough cells which were XY to account for the normal male development and presumably the 45,X cells inhibited the development of the stigmata of Down syndrome. Both syndromes are associated with short stature. Is this a result of the aneuploidy?
3.5. Are aneuploid cells are more prone to mitotic and meiotic error? Two types of studies show that it is very likely that aneuploid cells are unstable. In both cases the aneuploidy involves the X chromosome but the instability shows interchromosomal effects. Mitotic instability. Previous results from my laboratory were obtained in experiments of mitotic anaphase w27x. The methodology is explained in detail in the manuscript but these data were not in-
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cluded. The subject on whom the analysis was performed was an apparently normal female in whom 2.9% of her lymphocytes were 47,XXX. Table 3 shows the segregations at anaphase which were observed in cells which commenced division with either 2 or 3 X chromosomes. Chromosomes which were completely separate from the anaphase rings were defined as ‘pre-micronuclei’. In cells commencing division with 2 X chromosomes, the rate of X error was 1.5% and the rate of autosomal error was 1.2%. In cells commencing division with 3 X chromosomes, the rate of X error was 58.8% and the rate of autosomal error was 11.8%. These results were scored from the same slides so no experimental bias could have occurred. Meiotic instability. These data which were published in 1981 w28x indicate that comparable instability occurs at meiosis. There could possibly be an hormonal explanation for these data, and further investigation should be undertaken. The data are shown in Table 4. This group of 30 women who had 61 pregnancies bore 17 abnormal babies. Six of the babies in the stillbornrabnormal column had severe developmental abnormalities. Four of the babies had trisomy 21. The rate of X chromosome abnormality was 6.6% and the rate of chromosome 21 abnormality was also 6.6%. These two studies show that the secondary effects of aneuploidy on cell division are profound.
4. What examples are there of abnormalities, apparently attributable to aneuploidogens acting as teratogens? 4.1. Confirmed human teratogens [29,30] Two aneuploidogens fit this category, namely al-
cohol and diethylstilbestrol. The most common major defects for these two agents are: Alcohol Žfoetal alcohol syndrome.
Diethylstilbestrol
Growth retardation Reduced palpebral fissures Microcephaly, mental retardation Vaginal adenosis Cervical erosion and ridges Adenocarcinoma of the vagina Žrare. Abnormalities of male genitalia
Since the effects of DES are very localised and DES is an oestrogen analogue, it is possible that the teratogenic effects result from direct hormonal disturbances rather than through aneuploidy. If aneuploidy is involved then the gametes of affected males and females should have a high rate of aneuploidy. An analysis of the incidence of aneuploidy in the sperm of DES-affected males could demonstrate such an increased rate of aneuploidy. Since numerous studies have demonstrated that there is little between male variation in the rate of aneuploidy, a difference in the rate of aneuploidy in DES males should be easily demonstrated.
4.2. Confirmed animal and suspected human teratogen [29,31,32] The following four aneuploidogens are confirmed teratogens in animals. There is some evidence to suggest that all four are also human teratogens.
Table 4 pregnancy outcome in fertile women with 45,X or 45,X mosaic karyotype Karyotype
no.
no.p
i.a.
s.a.
s.b. or abnormal
Normal
Chromosome abnormality
45,X 45,Xr46,XX 45,Xr47,XXX 45,Xr46,XXr47,XXX
9 8 5 8
12 20 6 23
0 2 0 0
0 8 0 9
2 3 0 3
9 5 6 7
1: 47,q 21 1: 47,q 21; 45,Xr46,XY 0 2: 47,q 21; 1: 45,X; 3: X mosaics
no.p, number of pregnancies; i.a., induced abortions; s.a., spontaneous abortions; s.b., stillborn.
J.H. Ford r Mutation Research 396 (1997) 195–203
Benomyl
Maternal hypoxia Diabetic ketoacidosis Hyperthermia
Microphthalmia or anopthalmia and cleft liprpalate Cleft lip and various malformations Cleft lip and various malformations Microphthalmia, microencephaly and skeletal defects
Although all four teratogens are known aneuploidogens, there is no data to show that the teratogenic effects have arisen from aneuploidy. In the case of hyperthermia, the induction of abnormality is very rapid and probably results from cell death. The minimum studies needed to show that the teratogenic effect was related to aneuploidy would be to demonstrate that the affected progeny had an increased rate of cellular aneuploidy in more than one tissue.
5. Evaluation of teratogenic effects With the exception of DES, the teratogens have overlapping, though not consistent effects. Restricted growth, especially of the brain, is a feature and micropthalmia Žsmall eyes. and cleft lip and palate are recurrent features. These suggest that the substances may be acting in a similar manner although it could be that development canalisation makes these processes sensitive to any disturbance. These features are also those reported in the syndrome of variegated mosaic aneuploidy w26x. Since common features of aneuploid cells are a reduced rate of cell division and reduced numbers of cell nuclei, these teratogenic features of restricted growth are consistent with the action of aneuploidy. However Microphthalmia is not found in all human aneuploid conditions so it is likely that this is caused by some other disturbance in organogenesis. DES has a very specific effect on genital growth w32x. This suggests that this substance acts only on those cells which have oestrogen receptors. The finding of adenocarcinoma as a teratogenic effect provides a strong argument for the involvement of aneuploidy in the initiation of this cancer in DES-exposed individuals.
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6. Do abnormalities of cell division cause both teratogenesis and aneuploidy? Without a study of the chromosome complements of the tissues involved in the abnormalities induced by the aneuploidogens, there is no answer to this question. A study of the affected tissues would indeed provide the answer to the question as to whether aneuploidy has a significant role in producing the teratogenic effect. Given that the primary action of the aneuploidogens is to disturb some aspect of cell division and that arrest is a more common outcome than aneuploidy w4x, it is possible that the primary teratogenic effect of the aneuploidogenic substances is through arrest of cell division. However, it is also very likely that some aneuploidy is induced in the exposed tissues and it is possible that some of the abnormalities occur as a direct result of the induced mosaic aneuploidy. The available data suggest that much of the effect of aneuploidy is chromosome-specific and is due to genetic imbalances. The most compelling role for aneuploidy per se is an effect on the rate of cell multiplication and consequent cell number. Secondary divisional error is also likely to be very important and could lead to considerable cell death. These aspects of aneuploidy need to be thoroughly investigated in different test systems in non-transformed cells and in the progeny of individuals Žanimal or human. who have been exposed to aneuploidogens. The data from pregnancies in 45,X and 45,X mosaic human females suggests that aneuploidy per se may cause significant second generation developmental abnormalities and interchromosomal effects. These need to be examined in controlled two-generation experiments in mice. Adenocarcinoma is only an occasional outcome of diethylstilbestrol induced teratogenesis. Since cancers are typically induced by a mutational event, it is most likely that at least this outcome is a result of DES-induced aneuploidy rather than arrested cell division w33x. The long time lag in the development of the tumour makes this conclusion even more compelling. Without further study of the affected tissues in the abnormalities induced by aneuploidogens, it is not possible to distinguish whether the effects are due directly to arrest of cell division or through disturbed
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cell division leading to aneuploidy. Since both would lead to reduced cell numbers and the observed retardation of organ size, either could produce the majority of the observed effects. There is insufficient evidence yet to conclude whether aneuploidy per se has a teratogenic effect. Specific experiments need to be undertaken to address the questions raised here before the answer will be revealed. References w1x J.H. Ford, A.T. Correll, Factors affecting the attachment of chromosomes to spindles and the implications for errors of division, in: M. de Brabander, J. De Mey ŽEds.., Microtubules and Microtubule Inhibitors, Elsevier, Amsterdam, 1985, pp. 335–344. w2x A. Elhajouji, P. Van Hummelen, M. Kirsch-Volders, Indications for a threshold of chemically-induced aneuploidy in vitro in human lymphocytes, Environ. Mol. Mutagen. 26 Ž1995. 292–304. w3x F. Darroudi, C.M. Meijers, V. Hadjidekova, A.T. Natarajan, Detection of aneugenic and clastogenic potential of X-Rays, directly and indirectly acting chemicals in human hepatoma ŽHep G2. and peripheral blood lymphocytes, using the micronucleus assay and fluorescent in situ hybridization with a DNA centromeric probe, Mutagenesis 11 Ž1996. 425–433. w4x T.C. Hsu, K.L. Satya-Prakash, Aneuploidy induction by mitotic arrestants in animal systems: possible mechanisms, in: V.L. Dellarco, P.E. Voytek, A. Hollaender ŽEds.., Aneuploidy Etiology and Mechanisms. Basic Life Sciences Vol. 36, Plenum, New York, 1985, pp. 279–289. w5x J.C. Liang, B.R. Brinkley, Chemical probes and possible targets for the induction of aneuploidy, in: V.L. Dellarco, P.E. Voytek, A. Hollaender ŽEds.., Aneuploidy Etiology and Mechanisms. Basic Life Sciences Vol. 36, Plenum, New York, 1985, pp. 491–505. w6x L. Migliore, L. Cocchi, R. Scarpato, Detection of the centromere in micronuclei by fluorescent in situ hybridization: its application to the human lymphocyte micronucleus assay after treatment with four suspected aneugens, Mutagenesis 11 Ž1996. 285–290. w7x T. Shimada, G. Watanabe, T.H. Ingalls, Trisomies and triploidies in hamster embryos: induction by low-pressure hypoxia and pH imbalances, Arch. Environ. Health 35 Ž1980. 101–105. w8x I. Lorda-Sanchez, F. Binkert, M. Maechler, W.P. Robinson, A.A. Schinzel, Reduced recombination and paternal age effect in Klinefelter syndrome, Hum. Genet. 89 Ž1992. 524– 530. w9x W.P. Robinson, F. Bernasconi, A. Mutirangura, D.H. Ledbetter, S. Malcolm, M.A. Morris, A.A. Schinzel, Nondisjunction of chromosome 15: origin and recombination, Am. J. Hum. Genet. 53 Ž1993. 740–751.
w10x S.L. Sherman, M.B. Petersen, S.B. Freeman, J. Hersy, D. Pettay, L. Taft, M. Frantzen, M. Mikelsen, T.J. Hassold, Non-disjunction of chromosome 21 in maternal meiosis 1: evidence for a maternal age-dependent mechanism involving reduced recombination, Hum. Mol. Genet. 3 Ž1994. 1529– 1535. w11x T. Hassold, M. Merrill, K. Adkins, S. Freeman, S. Sherman, Recombination and maternal age-dependent nondisjunction: molecular studies of trisomy 16, Am J. Hum. Genet. 57 Ž1995. 867–874. w12x P.J. Wauben-Penris, F.A. van der Hoeven, P. de Boer, Chiasma frequency and nondisjunction in heteromorphic bivalents: meiotic behaviour in TŽ1;13.70HrTŽ1;13.1Wa mice as compared to TŽ1;13.Ž70HrTŽ1;13.70H mice, Cytogenet. Cell Genet. 36 Ž1983. 547–553. w13x P. de Boer, F.A. van der Hoeven, The use of translocationderived ‘marker-bivalents’ for studying the origin of meiotic instability in female mice, Cytogenet. Cell Genet. 26 Ž1980. 49–58. w14x U. Eichenlaub-Ritter, A.C. Chandley, R.G. Gosden, The CBA mouse as a model for age-related aneuploidy in man: studies of oocyte maturation, spindle formation and chromosome alignment during meiosis, Chromosoma 96 Ž1988. 220–226. w15x L.R. Ferguson, J.W. Allen, J.M. Mason, Meiotic recombination and germ cell aneuploidy, Environ. Mol. Mutagen. 28 Ž1996. 192–210. w16x B. Brandiff, L. Gordon, L. Ashworth, G. Watchmaker, D. Moore, A.J. Wyobek, A.V. Carrano, Chromosomes of human sperm: Variability among normal individuals, Hum. Genet. 70 Ž1985. 18–24. w17x R.H. Martin, A. Rademaker, Reliability of aneuploidy estimates in human sperm: results of fluorescence in situ hybridization studies using two different scoring criteria, Mol. Reprod. Dev. 42 Ž1995. 89–93. w18x M. Bibbo, W.B. Gill, F. Azizi, R. Blough, V.S. Fang, R.L. Rosenfield, G.F. Schumacher, K. Sleeper, M.G. Sonek, G.L. Wied, Follow-up study of male and female offspring of DES-exposed mothers, Obstet. Gynecol. 49 Ž1977. 1–8. w19x J.H. Ford, H.Z. Wilkin, P. Thomas, C. McCarthy, A 13 year cytogenetic study of spontaneous abortion: Clinical applications of testing, Aust. N.Z. J. Obstet. Gynaecol. 36 Ž1996. 314–318. w20x E.B. Hook, The impact of aneuploidy upon public health: Mortality and morbidity associated with human chromosome abnormalities, in: V.L. Dellarco, P.E. Voytek, A. Hollaender ŽEds.., Aneuploidy Etiology and Mechanisms. Basic Life Sciences Vol. 36, Plenum, New York, 1985, pp. 7–33. w21x A.T. Therapel, R.L. Summitt, A cytogenetic survey of 200 unclassifiable mentally retarded children with congenital anomalies and 200 normal controls, Hum. Genet. 37 Ž1977. 329–338. w22x S. Cure, A. Boue, J. Boue, Consequence of chromosomal anomalies on cell multiplication, in: A. Boue, C. Thibault ŽEds.., Les Accidents Chromosomiques de la Reproduction, INSERM, 1973, pp. 95–109. w23x T.A. Hreczko, B.A. Sigmon, The dermatoglyphics of a
J.H. Ford r Mutation Research 396 (1997) 195–203
w24x
w25x
w26x
w27x w28x w29x
Toronto sample of children with XXY, XXYY and XXX aneuploidies, Am. J. Phys. Anthropol. 52 Ž1980. 33–41. E. Philippe, Consequences des anomalies chromosomiques sur le developpement, in: A. Boue, C. Thibault ŽEds.., Les Accidents Chromosomiques de la Reproduction, INSERM, 1973, pp. 119–126. D.K. Kalousek, The effect of confined placental mosaicism on development of the human aneuploid conceptus, Birth Defects 29 Ž1993. 39–51. P. Bitoun, B. Martin-Pont, E. Tamboise, J. Gaudelus, Optic atrophy, microcephaly, mental retardation and mosaic variegated aneuploidy: A human mitotic mutation, Ann. Genet. 37 Ž1994. 75–77. J.H. Ford, A.T. Correll, Chromosome errors at mitotic anaphase, Genome 35 Ž1991. 702–705. J.L. Simpson, Antenatal diagnosis of cytogenetic abnormalities, Clin. Obstet. Gynecol. 24 Ž1981. 1023–1039. J.L. Sever, R.L. Brent, Summary table of human teratogens,
w30x
w31x
w32x
w33x
203
in: J.L. Sever, R.L. Brent ŽEds.., Teratogen Update Environmentally Induced Birth Defect Risks, Alan R. Liss, New York, 1986, pp. xiv–xv. M.H. Kaufman, The chromosome complement of single-pronuclear haploid mouse embryos following activation by ethanol treatment, J. Embryol. Exp. Morphol. 71 Ž1982. 139–154. J. Warkany, Hyperthermia, in: J.L. Sever, R.L. Brent ŽEds.., Teratogen Update Environmentally Induced Birth Defect Risks, Alan R. Liss, New York, 1986, pp. 181–187. W.G. Ellis, J.L. Semple, E.R. Hoogenboom, R.J. Kavlock, F.J. Zeman, Benomyl-induced craniocerebral anomalies in fetuses of adequately nourished and protein-deprived rats, Teratogen. Carcinogen. Mutagen. 7 Ž1987. 357–375. T. Tsutsui, M. Heiji, J.A. McLachlan, J.C. Barrett, Aneuploidy induction and cell transformation by diethylstilbestrol: A possible chromosomal mechanism in carcinogenesis, Cancer Res. 43 Ž1983. 3814–3821.
Does aneuploidy per se cause developmental abnormalities? Judith Helen Ford
)
Genetic Consulting and Testing (G.C.A.T.) Pty Ltd., P.O. Box 210, Port Adelaide, South Australia 5011, Australia Received 19 December 1996; revised 22 June 1997; accepted 5 July 1997
Abstract The following questions are addressed: ŽA. What are aneuploidogens and how do they act? ŽB. Is there any evidence that aneuploidy per se causes malformations? ŽC. What examples are there of abnormalities, apparently attributable to aneuploidogens acting as teratogens? ŽD. Do abnormalities of cell division cause both teratogenesis and aneuploidy? Considerable research has addressed question ŽA., but there is little which addresses the other three questions. The question of whether aneuploidy per se causes malformations remains open. Some suggestions for further research are made. q 1997 Elsevier Science B.V.
1. Malformation and aneuploidy Syndromes of malformation in animals and humans are often associated with chromosome specific aneuploidy. In humans, three trisomies, namely trisomy 13, trisomy 18 and trisomy 21 frequently survive to full gestation. Trisomy 13 and 18, however, are both associated with early infant death. Trisomies in mouse for chromosomal regions syntenic to the human chromosomal regions show some similar patterns of malformation. Thus there is significant evidence that many of the phenotypic effects of aneuploidy are chromosome-specific and are due to the imbalance of certain critical genes. The question of whether aneuploidy per se induces malformation has rarely been addressed. The evidence that is available is presented in Section 3. Data which demonstrates that aneuploid cells are more error-prone than normal cells indicate that aneuploidy per se has significance. ) Corresponding author. Tel.: q61 8 82443042; Fax: q61 8 82443407; E-mail: [email protected]
Further importance of knowing whether aneuploidy per se causes malformation may emerge if certain types of genetic therapy are considered in the future. One possible example would be: in early foetal therapy it may be considered that the introduction of an extra genetically engineered micro-chromosome could be the preferred method of introducing a new gene construct. This might be considered to prevent the effects of some serious metabolic disorder. The introduction of the extra centromere might disturb the cell sufficiently to cause malformation. A second example would be: early gene therapy might be considered to correct the genetic imbalance in an aneuploid individual, especially in Žsay. trisomy 21. If the syndromic malformation is at least partially caused by aneuploidy per se, then this type of therapy would be at best only partly effective.
2. What are aneuploidogens and how do they act? An aneuploidogen is any factor which can induce aneuploidy. The list is large and includes not only
0027-5107r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 2 7 - 5 1 0 7 Ž 9 7 . 0 0 1 8 4 - X
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Table 1 Aneuploidogens which tested positive in at least two testing systems and their probable sites of action Žafter w5x. Site of action
Effect on cell division
Affect microtubular polymerization
‘‘c-mitosis’’™metaphase arrest
Crystallize tubulin Enhances tubulin polymerization Blocks pole-to-pole spindle elongation Inhibits DNA synthesis
‘‘c-mitosis’’™metaphase arrest Inhibits mitosis Inhibits anaphase B and thus induces polyploidy Disturbs synchronicity ™ multipolar spindles andr or chromosome lagging. Inhibits cell division Causes multipolar spindles andror chromosome lagging
Colchicine, cytosine, arabinoside
Disrupts polymerized microtubules Damages centriolesrcentrosomes Damage kinetochore Binds to calmodulin Damage cell membranes Affect chromosome pairing and crossing-over
Alters rate of separation of sister chromatids Žmitosis. Metaphase arrest Disturb distribution of chromosomes Žspatial orientation., leading to misdivision Meiotic error
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AneuploidogenŽs. Colchicine, colcemid, podophyllotoxin, benzimidazole derivatives Žbenomyl, MBC, nocodazole. diethylstilbestrol, griseofulvin, sulfhydryl reagents Žmercury compounds, arsenic compounds, diamide., para-fluorophenylalanine Vinca alkaloids Žvinblastine, vincristine. Taxol Chloral hydrate Imbalances in the nucleotide pool or nucleotide analogues, e.g. BUdr, FUdr. Hypothermia or hyperthermia Diazepam, diethylstilbestrol, ethidium bromide, actinomycin D Colchicine, mitomycin C Chlorpromazine Amphotericin B, fenarimol, miconazole
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chemicals and X-radiation, but other factors such as pH which are capable of disturbing the cellular milieu w1x. In other words, any factor which can disturb the dynamic process of cell division or its spatial orientation, has the potential to be an aneuploidogen. It has been convincingly demonstrated that there are many chemicals whose action is primarily aneuploidogenic Žsee Table 1.. There are, however, those whose primary action is usually regarded as a clastogenic, but which nevertheless also have significant aneuploidogenic action w2,3x. Each part of the cell division machinery is vulnerable to disturbance. In gross terms, the following elements make up the machinery w4x: Ø The kinetochore region of the chromosomes Ø The centrioles and peri-centriolar particles Ø The spindle fibres composed of microtubules and MAPS Žmicrotubular associated proteins. Ø The cytoplasm and its content of microtubular protein Ø The chromosomal telomeres and the nuclear membrane Ø Cytoplasmic microfilaments The process of mitotic cell division requires not only that each component be intact and functional but that all the different processes work together in synchrony. Agents which adversely affect the timing of one or more of the critical processes can induce aneuploidy just as surely as an agent which binds to one of the components. Table 1 shows some examples of aneuploidogens and their probable sites of action w5x. The aneuploidogenic properties of most of these chemicals has been further confirmed by the human lymphocyte micronucleus test ŽMN., in combination with fluorescent in situ hybridisation ŽFISH. of a centromeric DNA probe Že.g. w3,6x.. Just as critical as the elements of the spindle and the chromosomes is the cellular milieu. The maintenance of cellular pH, salt concentration, electro-magnetic forces, GTP and ATP levels, pressure and temperature are critical to the maintenance of proper cell division. These aspects of cell division were often studied in the early years of cell biology, 1930 to 1950, but now rarely receive much attention. Drugs andror physiological conditions which disturb
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any of these parameters can disturb cell division and cause aneuploidy w7x. Meiosis I, in addition to all the factors above, involves the complex process of chromosome pairing and crossing over. It has been established that some chromosomes involved in maternal nondisjunction involve reduced or altered recombination w8–11x. Most discussions of the significance of this look to mechanisms which might generate these ‘altered gametes’ in older women. An alternative hypothesis, which in the view of this author is more likely, is the following. Altered gametes, those with altered or lowered rates of recombination, occur at equal frequencies at all ages but are more prone to error in the ageing oocyte. The distribution of chiasmata alters the three-dimensional structure of a bivalent, and certain structures take more time to align on the spindle. ‘Marker bivalents’ are less stable on the meiotic spindle and are more prone to error w12x. This effect of bivalent instability is enhanced in ageing oocytes w13x. Studies of ageing mouse oocytes have shown that intrinsic differences in the cell cycle occur with age. There is an earlier onset of chromosome separation in oocytes with a premature anaphase transition w14x. It is argued that these altered physiological conditions are the major cause for the increased incidence of nondisjunction of marker bivalents in ageing mice w13x. Altered bivalents do not lead to aneuploidy in younger women because of the greater proficiency of the meiotic 1 division process. It is possible that environmental factors also affect meiotic recombination w15x. However, in humans this would be restricted to effects in the male, or in the female during intra-uterine life. There is little evidence at this stage to support this contention. Aneuploidy frequencies show little variation between human males w16,17x and no appropriate studies have been undertaken of exposures, other than diethylstilbestrol, in human foetal life. Both males and females exposed to diethylstilbestrol ŽDES. in utero have decreased fertility w18x but no study of aneuploidy in their gametes has been reported. Aneuploidogens can thus be extrinsic or intrinsic factors. The list of chemicals which can induce aneuploidy is large. However, the available data in
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J.H. Ford r Mutation Research 396 (1997) 195–203
humans suggest that the effect of ageing far outweighs that of external factors. More research is required to define the precise effects of ageing and to discover whether any of the declining cellular functions can be preserved or stabilised.
3. Is there any evidence that aneuploidy per se causes malformations? 3.1. Possible role of aneuploidy in common malformations In humans, aneuploidy plays a significant role in early embryonic lethality w19x and is the basis of several well-defined congenital abnormalities. Since each type of aneuploid e.g. monosomy X, trisomy 21, trisomy 18, has its own phenotype, the syndrome of abnormalities is interpreted to be the result of each of the specific chromosome imbalances. In order to determine whether aneuploidy per se causes developmental abnormalities, it is necessary to look beyond the effects of the specific chromosomal imbalances. Are there abnormalities common to the aneuploid state, including sex chromosome aneuploidy? Data which contributes to the answer to this question come from chromosome analyses in children with multiple malformations and mental retardation of unknown origin. Several studies were summarised w20x and all gave similar results. The study summarised in w21x excluded cases where a syndrome such as Down’s syndrome was suspected before the analysis. This exclusion was used to minimise the bias of known chromosome-specific effects.
7.6% Ž54r710. infants with abnormalities had chromosome abnormalities compared with 0.7% chromosome abnormalities in controls. The results of the study are summarised in Table 2. Two frequent findings in this group are congenital heart defects and mental retardation. Of the former, 13% have a chromosomal abnormality and up to 48% of the mentally retarded have chromosomal abnormalities. The allocation of a causative role to aneuploidy in the etiology of these malformations is still difficult because of the large impact of chromosomal defects which do not involve changes in chromosome number. However, the inclusion of sex chromosome aneuploidy suggests that there could be an influence of aneuploidy per se. 3.2. Effect of chromosomal anomalies on cell multiplication Cell lines initiated from embryos with aneuploid chromosomal abnormalities exhibited significantly slower generation times and decreased lifespans w22x. These changes seemed to be common to the aneuploid state rather than chromosome specific and were true of monosomic X cells as well as trisomic cells. When cell lines transform, these relationships between chromosome number and cell cycle time are often lost. It is important to note that these studies were undertaken on primary embryonic fibroblast cultures. Further evidence of reduced cell multiplication in human aneuploidy comes from the study of dermatoglyphics. A typical example of the many studies in this field confirms that the total ridge count in palms and soles of children with sex chromosomal abnormalities is decreased with increasing number of sex chromosomes w23x.
Table 2 Results of chromosomal analysis of 710 individuals without suspected syndrome but with mental retardation and three independent malformations w21x Type of abnormality Aneuploid Ž n s 7. Aneuploid and structural Ž n s 8. Structural Ž n s 20. Structural Ž n s 19.
Sub-classification
Karyotype
Apparently balanced Unbalanced
47,q 18; 47,XXX Ž n s 2.; 47,XXY; 46r47,q Er92,XXYY; 47,q D; 47,q G 47,q der Ž n s 2.; 46,XXr47,q r Ž n s 4.; 47,q mar Ž n s 2. Inversions Ž n s 11.; Robertsonian Ž n s 2.; other Ž n s 7. Deletions Ž n s 7.; rings Ž n s 1.; other Ž n s 11.
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Table 3 Chromosome segregation at late anaphase in cells with 2 and 3 X chromosomes Ratio of X centromere-specific silver grains
No. of cells
‘Pre-micronuclei’ — Autosome
‘Pre-micronuclei’ — X chromosome
2:2 3:1 1:1 Total
1359 15 2 1376
19 1 0 20
0 0 2 2
3:3 3:2 4:2 Total
7 1 9 17
2 0 0 2
0 1 0 1
3.3. Effect of aneuploidy and triploidy on embryonic deÕelopment In a quantitative analysis of the number of nuclei in embryonic cells, it was found w24x that both normal and abnormal specimens which had normal chromosomes had a similar distribution of placental cells. However embryonic specimens with trisomy 16, monosomy X or triploidy, all of which were developmentally abnormal, revealed analogous paucity of the number of cytotrophoblastic nuclei. It was concluded that the paucity of nuclei was a direct effect of the aneuploid state. Recent studies on the placentas of pregnancies with intra-uterine growth retardation has led to the recognition that some placentas are mosaic for diploid and trisomic cells. The term ‘‘confined placental mosaicism’’ has been coined for this situation in which there can be a trisomic foetus supported by a diploid placenta or a diploid foetus supported by a trisomic foetus. In the former, a trisomic foetus can survive a pregnancy. In the latter, a diploid foetus might not survive. Whilst these findings fit the general concept expressed above w24x, there is one major difference. The outcome of pregnancies with a trisomic placenta and diploid foetus largely depends on first, the specific chromosome involved and second, the presence or absence of uniparental disomy in the foetus w25x. Both these conditions argue for chromosome specific effects rather than aneuploidy per se. 3.4. A human syndrome of mosaic aneuploidy? A recent paper w26x reports an abnormality syndrome which is associated with a variegated mo-
saicism of normal cells with three cells lines carrying different numbers, one, two or three, of an unidentified marker chromosome. The syndrome has optic atrophy, mental retardation, microcephaly and short stature. This paper argues that that aneuploidy per se accounts for these abnormality rather than the specific genetic imbalance. Our own laboratory had an interesting case which contributes to this discussion Žunpublished data.. A man was referred for extremely short stature; as a mature adult he was 4 foot, 6 inches tall but was not a dwarf. He had normal intelligence and normal secondary sexual characteristics. Chromosome analysis of his lymphocytes showed that 50% of his cells were 47,XY,q 21 and the other 50% were 45,X. Presumably he had enough cells which were XY to account for the normal male development and presumably the 45,X cells inhibited the development of the stigmata of Down syndrome. Both syndromes are associated with short stature. Is this a result of the aneuploidy?
3.5. Are aneuploid cells are more prone to mitotic and meiotic error? Two types of studies show that it is very likely that aneuploid cells are unstable. In both cases the aneuploidy involves the X chromosome but the instability shows interchromosomal effects. Mitotic instability. Previous results from my laboratory were obtained in experiments of mitotic anaphase w27x. The methodology is explained in detail in the manuscript but these data were not in-
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cluded. The subject on whom the analysis was performed was an apparently normal female in whom 2.9% of her lymphocytes were 47,XXX. Table 3 shows the segregations at anaphase which were observed in cells which commenced division with either 2 or 3 X chromosomes. Chromosomes which were completely separate from the anaphase rings were defined as ‘pre-micronuclei’. In cells commencing division with 2 X chromosomes, the rate of X error was 1.5% and the rate of autosomal error was 1.2%. In cells commencing division with 3 X chromosomes, the rate of X error was 58.8% and the rate of autosomal error was 11.8%. These results were scored from the same slides so no experimental bias could have occurred. Meiotic instability. These data which were published in 1981 w28x indicate that comparable instability occurs at meiosis. There could possibly be an hormonal explanation for these data, and further investigation should be undertaken. The data are shown in Table 4. This group of 30 women who had 61 pregnancies bore 17 abnormal babies. Six of the babies in the stillbornrabnormal column had severe developmental abnormalities. Four of the babies had trisomy 21. The rate of X chromosome abnormality was 6.6% and the rate of chromosome 21 abnormality was also 6.6%. These two studies show that the secondary effects of aneuploidy on cell division are profound.
4. What examples are there of abnormalities, apparently attributable to aneuploidogens acting as teratogens? 4.1. Confirmed human teratogens [29,30] Two aneuploidogens fit this category, namely al-
cohol and diethylstilbestrol. The most common major defects for these two agents are: Alcohol Žfoetal alcohol syndrome.
Diethylstilbestrol
Growth retardation Reduced palpebral fissures Microcephaly, mental retardation Vaginal adenosis Cervical erosion and ridges Adenocarcinoma of the vagina Žrare. Abnormalities of male genitalia
Since the effects of DES are very localised and DES is an oestrogen analogue, it is possible that the teratogenic effects result from direct hormonal disturbances rather than through aneuploidy. If aneuploidy is involved then the gametes of affected males and females should have a high rate of aneuploidy. An analysis of the incidence of aneuploidy in the sperm of DES-affected males could demonstrate such an increased rate of aneuploidy. Since numerous studies have demonstrated that there is little between male variation in the rate of aneuploidy, a difference in the rate of aneuploidy in DES males should be easily demonstrated.
4.2. Confirmed animal and suspected human teratogen [29,31,32] The following four aneuploidogens are confirmed teratogens in animals. There is some evidence to suggest that all four are also human teratogens.
Table 4 pregnancy outcome in fertile women with 45,X or 45,X mosaic karyotype Karyotype
no.
no.p
i.a.
s.a.
s.b. or abnormal
Normal
Chromosome abnormality
45,X 45,Xr46,XX 45,Xr47,XXX 45,Xr46,XXr47,XXX
9 8 5 8
12 20 6 23
0 2 0 0
0 8 0 9
2 3 0 3
9 5 6 7
1: 47,q 21 1: 47,q 21; 45,Xr46,XY 0 2: 47,q 21; 1: 45,X; 3: X mosaics
no.p, number of pregnancies; i.a., induced abortions; s.a., spontaneous abortions; s.b., stillborn.
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Benomyl
Maternal hypoxia Diabetic ketoacidosis Hyperthermia
Microphthalmia or anopthalmia and cleft liprpalate Cleft lip and various malformations Cleft lip and various malformations Microphthalmia, microencephaly and skeletal defects
Although all four teratogens are known aneuploidogens, there is no data to show that the teratogenic effects have arisen from aneuploidy. In the case of hyperthermia, the induction of abnormality is very rapid and probably results from cell death. The minimum studies needed to show that the teratogenic effect was related to aneuploidy would be to demonstrate that the affected progeny had an increased rate of cellular aneuploidy in more than one tissue.
5. Evaluation of teratogenic effects With the exception of DES, the teratogens have overlapping, though not consistent effects. Restricted growth, especially of the brain, is a feature and micropthalmia Žsmall eyes. and cleft lip and palate are recurrent features. These suggest that the substances may be acting in a similar manner although it could be that development canalisation makes these processes sensitive to any disturbance. These features are also those reported in the syndrome of variegated mosaic aneuploidy w26x. Since common features of aneuploid cells are a reduced rate of cell division and reduced numbers of cell nuclei, these teratogenic features of restricted growth are consistent with the action of aneuploidy. However Microphthalmia is not found in all human aneuploid conditions so it is likely that this is caused by some other disturbance in organogenesis. DES has a very specific effect on genital growth w32x. This suggests that this substance acts only on those cells which have oestrogen receptors. The finding of adenocarcinoma as a teratogenic effect provides a strong argument for the involvement of aneuploidy in the initiation of this cancer in DES-exposed individuals.
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6. Do abnormalities of cell division cause both teratogenesis and aneuploidy? Without a study of the chromosome complements of the tissues involved in the abnormalities induced by the aneuploidogens, there is no answer to this question. A study of the affected tissues would indeed provide the answer to the question as to whether aneuploidy has a significant role in producing the teratogenic effect. Given that the primary action of the aneuploidogens is to disturb some aspect of cell division and that arrest is a more common outcome than aneuploidy w4x, it is possible that the primary teratogenic effect of the aneuploidogenic substances is through arrest of cell division. However, it is also very likely that some aneuploidy is induced in the exposed tissues and it is possible that some of the abnormalities occur as a direct result of the induced mosaic aneuploidy. The available data suggest that much of the effect of aneuploidy is chromosome-specific and is due to genetic imbalances. The most compelling role for aneuploidy per se is an effect on the rate of cell multiplication and consequent cell number. Secondary divisional error is also likely to be very important and could lead to considerable cell death. These aspects of aneuploidy need to be thoroughly investigated in different test systems in non-transformed cells and in the progeny of individuals Žanimal or human. who have been exposed to aneuploidogens. The data from pregnancies in 45,X and 45,X mosaic human females suggests that aneuploidy per se may cause significant second generation developmental abnormalities and interchromosomal effects. These need to be examined in controlled two-generation experiments in mice. Adenocarcinoma is only an occasional outcome of diethylstilbestrol induced teratogenesis. Since cancers are typically induced by a mutational event, it is most likely that at least this outcome is a result of DES-induced aneuploidy rather than arrested cell division w33x. The long time lag in the development of the tumour makes this conclusion even more compelling. Without further study of the affected tissues in the abnormalities induced by aneuploidogens, it is not possible to distinguish whether the effects are due directly to arrest of cell division or through disturbed
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cell division leading to aneuploidy. Since both would lead to reduced cell numbers and the observed retardation of organ size, either could produce the majority of the observed effects. There is insufficient evidence yet to conclude whether aneuploidy per se has a teratogenic effect. Specific experiments need to be undertaken to address the questions raised here before the answer will be revealed. References w1x J.H. Ford, A.T. Correll, Factors affecting the attachment of chromosomes to spindles and the implications for errors of division, in: M. de Brabander, J. De Mey ŽEds.., Microtubules and Microtubule Inhibitors, Elsevier, Amsterdam, 1985, pp. 335–344. w2x A. Elhajouji, P. Van Hummelen, M. Kirsch-Volders, Indications for a threshold of chemically-induced aneuploidy in vitro in human lymphocytes, Environ. Mol. Mutagen. 26 Ž1995. 292–304. w3x F. Darroudi, C.M. Meijers, V. Hadjidekova, A.T. Natarajan, Detection of aneugenic and clastogenic potential of X-Rays, directly and indirectly acting chemicals in human hepatoma ŽHep G2. and peripheral blood lymphocytes, using the micronucleus assay and fluorescent in situ hybridization with a DNA centromeric probe, Mutagenesis 11 Ž1996. 425–433. w4x T.C. Hsu, K.L. Satya-Prakash, Aneuploidy induction by mitotic arrestants in animal systems: possible mechanisms, in: V.L. Dellarco, P.E. Voytek, A. Hollaender ŽEds.., Aneuploidy Etiology and Mechanisms. Basic Life Sciences Vol. 36, Plenum, New York, 1985, pp. 279–289. w5x J.C. Liang, B.R. Brinkley, Chemical probes and possible targets for the induction of aneuploidy, in: V.L. Dellarco, P.E. Voytek, A. Hollaender ŽEds.., Aneuploidy Etiology and Mechanisms. Basic Life Sciences Vol. 36, Plenum, New York, 1985, pp. 491–505. w6x L. Migliore, L. Cocchi, R. Scarpato, Detection of the centromere in micronuclei by fluorescent in situ hybridization: its application to the human lymphocyte micronucleus assay after treatment with four suspected aneugens, Mutagenesis 11 Ž1996. 285–290. w7x T. Shimada, G. Watanabe, T.H. Ingalls, Trisomies and triploidies in hamster embryos: induction by low-pressure hypoxia and pH imbalances, Arch. Environ. Health 35 Ž1980. 101–105. w8x I. Lorda-Sanchez, F. Binkert, M. Maechler, W.P. Robinson, A.A. Schinzel, Reduced recombination and paternal age effect in Klinefelter syndrome, Hum. Genet. 89 Ž1992. 524– 530. w9x W.P. Robinson, F. Bernasconi, A. Mutirangura, D.H. Ledbetter, S. Malcolm, M.A. Morris, A.A. Schinzel, Nondisjunction of chromosome 15: origin and recombination, Am. J. Hum. Genet. 53 Ž1993. 740–751.
w10x S.L. Sherman, M.B. Petersen, S.B. Freeman, J. Hersy, D. Pettay, L. Taft, M. Frantzen, M. Mikelsen, T.J. Hassold, Non-disjunction of chromosome 21 in maternal meiosis 1: evidence for a maternal age-dependent mechanism involving reduced recombination, Hum. Mol. Genet. 3 Ž1994. 1529– 1535. w11x T. Hassold, M. Merrill, K. Adkins, S. Freeman, S. Sherman, Recombination and maternal age-dependent nondisjunction: molecular studies of trisomy 16, Am J. Hum. Genet. 57 Ž1995. 867–874. w12x P.J. Wauben-Penris, F.A. van der Hoeven, P. de Boer, Chiasma frequency and nondisjunction in heteromorphic bivalents: meiotic behaviour in TŽ1;13.70HrTŽ1;13.1Wa mice as compared to TŽ1;13.Ž70HrTŽ1;13.70H mice, Cytogenet. Cell Genet. 36 Ž1983. 547–553. w13x P. de Boer, F.A. van der Hoeven, The use of translocationderived ‘marker-bivalents’ for studying the origin of meiotic instability in female mice, Cytogenet. Cell Genet. 26 Ž1980. 49–58. w14x U. Eichenlaub-Ritter, A.C. Chandley, R.G. Gosden, The CBA mouse as a model for age-related aneuploidy in man: studies of oocyte maturation, spindle formation and chromosome alignment during meiosis, Chromosoma 96 Ž1988. 220–226. w15x L.R. Ferguson, J.W. Allen, J.M. Mason, Meiotic recombination and germ cell aneuploidy, Environ. Mol. Mutagen. 28 Ž1996. 192–210. w16x B. Brandiff, L. Gordon, L. Ashworth, G. Watchmaker, D. Moore, A.J. Wyobek, A.V. Carrano, Chromosomes of human sperm: Variability among normal individuals, Hum. Genet. 70 Ž1985. 18–24. w17x R.H. Martin, A. Rademaker, Reliability of aneuploidy estimates in human sperm: results of fluorescence in situ hybridization studies using two different scoring criteria, Mol. Reprod. Dev. 42 Ž1995. 89–93. w18x M. Bibbo, W.B. Gill, F. Azizi, R. Blough, V.S. Fang, R.L. Rosenfield, G.F. Schumacher, K. Sleeper, M.G. Sonek, G.L. Wied, Follow-up study of male and female offspring of DES-exposed mothers, Obstet. Gynecol. 49 Ž1977. 1–8. w19x J.H. Ford, H.Z. Wilkin, P. Thomas, C. McCarthy, A 13 year cytogenetic study of spontaneous abortion: Clinical applications of testing, Aust. N.Z. J. Obstet. Gynaecol. 36 Ž1996. 314–318. w20x E.B. Hook, The impact of aneuploidy upon public health: Mortality and morbidity associated with human chromosome abnormalities, in: V.L. Dellarco, P.E. Voytek, A. Hollaender ŽEds.., Aneuploidy Etiology and Mechanisms. Basic Life Sciences Vol. 36, Plenum, New York, 1985, pp. 7–33. w21x A.T. Therapel, R.L. Summitt, A cytogenetic survey of 200 unclassifiable mentally retarded children with congenital anomalies and 200 normal controls, Hum. Genet. 37 Ž1977. 329–338. w22x S. Cure, A. Boue, J. Boue, Consequence of chromosomal anomalies on cell multiplication, in: A. Boue, C. Thibault ŽEds.., Les Accidents Chromosomiques de la Reproduction, INSERM, 1973, pp. 95–109. w23x T.A. Hreczko, B.A. Sigmon, The dermatoglyphics of a
J.H. Ford r Mutation Research 396 (1997) 195–203
w24x
w25x
w26x
w27x w28x w29x
Toronto sample of children with XXY, XXYY and XXX aneuploidies, Am. J. Phys. Anthropol. 52 Ž1980. 33–41. E. Philippe, Consequences des anomalies chromosomiques sur le developpement, in: A. Boue, C. Thibault ŽEds.., Les Accidents Chromosomiques de la Reproduction, INSERM, 1973, pp. 119–126. D.K. Kalousek, The effect of confined placental mosaicism on development of the human aneuploid conceptus, Birth Defects 29 Ž1993. 39–51. P. Bitoun, B. Martin-Pont, E. Tamboise, J. Gaudelus, Optic atrophy, microcephaly, mental retardation and mosaic variegated aneuploidy: A human mitotic mutation, Ann. Genet. 37 Ž1994. 75–77. J.H. Ford, A.T. Correll, Chromosome errors at mitotic anaphase, Genome 35 Ž1991. 702–705. J.L. Simpson, Antenatal diagnosis of cytogenetic abnormalities, Clin. Obstet. Gynecol. 24 Ž1981. 1023–1039. J.L. Sever, R.L. Brent, Summary table of human teratogens,
w30x
w31x
w32x
w33x
203
in: J.L. Sever, R.L. Brent ŽEds.., Teratogen Update Environmentally Induced Birth Defect Risks, Alan R. Liss, New York, 1986, pp. xiv–xv. M.H. Kaufman, The chromosome complement of single-pronuclear haploid mouse embryos following activation by ethanol treatment, J. Embryol. Exp. Morphol. 71 Ž1982. 139–154. J. Warkany, Hyperthermia, in: J.L. Sever, R.L. Brent ŽEds.., Teratogen Update Environmentally Induced Birth Defect Risks, Alan R. Liss, New York, 1986, pp. 181–187. W.G. Ellis, J.L. Semple, E.R. Hoogenboom, R.J. Kavlock, F.J. Zeman, Benomyl-induced craniocerebral anomalies in fetuses of adequately nourished and protein-deprived rats, Teratogen. Carcinogen. Mutagen. 7 Ž1987. 357–375. T. Tsutsui, M. Heiji, J.A. McLachlan, J.C. Barrett, Aneuploidy induction and cell transformation by diethylstilbestrol: A possible chromosomal mechanism in carcinogenesis, Cancer Res. 43 Ž1983. 3814–3821.