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The potential usefulness of computerized chromosome analysis in human genetics
Cumpn~.Biol. .\l&.
I’erg:lmon Press 1972. Vol. 2, pp. 99-106.
Printed in Circa1 Britain.
The Potential Usefulness of Computerized Chromosome Analysis in Human Genetics* LILLIAN
Y. F. HSU and KURT
HIRSCHHORN
Division of Medical Genetics, Department of Pediatrics, Mount Sinai School of Medicine, New York, New York 10029, U.S.A. (Received 30 October 1970) Abstract-The potential usefulness of computerized chromosome analysis in human genetics is mainly in studies aimed at population cytogenetics, epidemiology and preventive medicine. Three major studies are emphasized, i.e. (1) measurement studies, (2) incidence studies of chromosomal aberrations which include studies of (a) numerical and structural aberrations and (b) chromosome mosaicism, and (3) studies of chromosome damage by various environmental agents.
INTRODUCTION THE
DEMONSTRATION of the normal human chromosome number and morphology by and LEVAN in 1956,(l) not only opened a new era but also a new field of human genetics, i.e. cytogenetics. The rapid progress in this field during the past decade has further shown its importance in medicine. Identification of chromosomal abnormalities in individuals with certain congenital abnormalities has become critical in diagnosis, prognosis, and genetic counselling. The development of the technique of prenatal chromosome diagnosis by culturing the fetal cells from the amniotic fluid has made it possible to prevent the birth of a child with abnormal chromosomes by therapeutic abortion. However, due to the conventional time-consuming procedure in karyotype analysis and the limited manpower, most of the cytogenetic studies have been restricted to phenotypically abnormal individuals. The knowledge of human population cytogenetics is scanty, although it is being explored at a slow pace. It is the purpose of this paper to bring forth the potential usefulness of computerized chromosome analysis in human genetics.
TJIO
I.
MEASUREMENT
STUDIES
The first proposed system of nomenclature of human mitotic chromosomes was put forward by the Denver Conference in 1960.(*) It was generally agreed that the autosomes should be serially numbered l-22 in descending order of length and be classified into seven groups according to the different location of the centromere. The sex chromosomes are referred to as X and Y. The basic measurements including the relative length of each chromosome, arm ratio and the centromere index were also reported by several investigators and summarized in the Denver conference. *Supported by U.S. Public Health Service Grants (HD-02552) and (HD-00210). Kurt Hirschhorn career scientist of the Health Research Council of the City of New York (I-513). 99
is a
100
LILLIAN Y. F. Hsu and KURTHIRSCHHORN
The second conference on normal human karyotype was held in London in 1963 mainly to assess the validity of the Denver classification of human chromosomes. Minor changes were made in the classification of several individual chromosomes.(2) Emphasis was made on the recognition ofsecondaryconstrictions ofcertain specific chromosomes and differential DNA replication patterns in identification of individual chromosomes. However, not all chromosomes can be identified even by these methods. Conventional karyotypic analysis is usually made by the visual pattern-recognition faculties of the cytogeneticist. Measurement, although remaining desirable, has been rarely carried out because it is tedious and time consuming. With computerized chromosome analysis, each metaphase will not only be counted accurately but each chromosome measured as well. Furthermore, a great number of normal cells can be analysed and the data of the measurements can be used for standardization of the human chromosome complement. Cells with minor chromosomal abnormalities will be also detected. Recently, ARRIGHI and Hsut3) described a differential chromosome staining technique in identification of human chromosomes. With this technique, the stain is differentially bound to the repetitive (“satellite”) DNA of the chromosome which is generally associated with constitutive heterochromatin. Promising results have been obtained by T. C. Hsu’s group and our laboratory in identification of several chromosomes in the C group, D group, E group, and G group. Since the computer techniques can discriminate staining density, it would be possible to utilize this technique to recognize the differential staining characteristics for more accurate karyotype analysis.
11. INCIDENCE
STUDIES
1. Incidence of numerical and structural chromosome abnormalities It has been realized that studies of a large, unbiased population is necessary to determine the significance of chromosome abnormalities in medicine for both epidemiological studies and preventive medicine. The latter is now possible by prenatal chromosome analysis of the cells cultured from the amniotic fluid and therapeutic abortion of the fetus with abnormal chromosomal constitution. Since 1969, chromosome surveys of selected live newborns have been reported by COURT BROWNand S~%ITH,(*) and RATCLIFFEet al.,(j) in sizable samples of male live newborns; WALZERet CA.,(~)in 2400 normal newborns. Up to present, there have been only two reports of chromosome survey in unbiased and unselected samples of newborn infants, i.e. studies of 2159 consecutive newborns by SERGOVICH et al.,(‘) and studies of 4500 consecutive newborns by LUBSand RUDDLE.@)In both studies, the frequency of infants with major abnormalities of their chromosomes was found to be about 0.5 per cent and most of these infants would not have been detected if the chromosome surveys were not undertaken. 21.8 per cent of the infants with the chromosome abnormalities were carriers of reciprocal balanced translocations and were phenotypically normal; more than half of the remaining infants with chromosomal abnormalities were found to have sex chromosome aberrations which also presented with no clinical difficulties in the neonatal period. A search for XYY males in all these newborn studies revealed approximately 1 in 562 male consecutive live newborns. (RATCLIFFEet al.15) found 5 in 3496; SERGOVICHet a1.t’) found 4 in 1066; LUBSand RIJDDLE@) found 3 in 2184.) However, in the study reported by WALZERet el.@) in 2400 normal (but not consecutive) newborns, no case of XYY was found.
The potential usefulness of computerized
chromosome
analysis in human genetics
101
It is clear that more extensive studies of unbiased, unselected newborn infants should be carried out on a large scale so as to determine the true incidence of every different kind of chromosomal abnormality. Only with computerized chromosome programs, will this be possible. Tn addition to the newborn study, if a concomitant survey is undertaken in a unselected sample of adolescents in the same area, it would be possible to compare the incidence of chromosome abnormalities in these two different age groups. Although we know that autosomal trisomies mostly die before reaching adolescence, we do not know the difference of the incidence of the other kinds of chromosomal abnormalities in these two age groups. With computerized chromosome analysis, chromosome.surveys can also be carried out extensively in selected samples such as mental institutions, prisoners, juvenile delinquents, etc., each with a matched age and sex control, since the incidence of sex chromosome abnormality, especially XYY, in these specific populations is needed to determine their significance. The detection of structural chromosomal abnormalities in the reported chromosome surveys, so far, has been done mostly on a few metaphase cells analysed without measurements. Therefore, only the obvious gross chromosomal structural abnormalities would be detected. Although SERGOVKHet a(.(‘) found one case of partial deletion of a B group chromosome and WALZER et ~1.c~)detected 3 instances of pericentric inversion in their series. According to WARBURTONet o!.,(~) minute deletions of a chromosome can be detected by careful measurements. They were able to identify 5 cases of cri du chat whose chromosomes would have appeared normal without measurements. The accurate measurements by computer of each chromosome will definitely detect more cases with minute structural abnormalities. We all know that numerous infants with multiple congenital abnormalities were found to have grossly normal chromosomes by conventional karyotyping methods. With the computer measuring each chromosome of every metaphase examined of these individuals, it would be possible to detect a minute chromosomal abnormality which would have been otherwise overlooked. Detection of structural aberrations will be especiaIly useful in studies of couples with subfertility and multiple spontaneous abortions. Since it has been suggested that subfertility, multiple spontaneous abortions, and stillbirths may result from a balanced structural chromosomal abnormality including translocation and inversion (structural heterozygote). In fact, several such cases have been reported.(1°-12) Studies of unselected series of spontaneous abortions have been carried out by several different investigators.(13-“) Chromosomal abnormalities were found in approximately 20 per cent (range 10-60 per cent) of first trimester spontaneous abortions. The abnormal chromosome constitutions consisted of triploidy in approximately l/3 ; trisomies in l/3 ; 45, X constitution in l/3. Cytogenetic studies on therapeutic abortions showed abnormal chromosomes in only 2.8 per cent of the abortuses.( 17) In the studies of triploidy in abortuses the collected data for early aborted fetuses are: 17 XXX; 30 XXY; and 4 XYY. The frequency of XXXXXY is almost 1:2. This will favor the possibility that triploidy in man is most likely caused by diandry rather than digyny. Polyspermy usually results from overripeness of eggs due to delayed fertilization. Therefore, further study in abortus materials, including hydatidiform mole, will yield better understanding of the effect of overripeness or aging of the eggs and chromosomal abnormalities. Chromosome studies in abortuses are also specially important in determination of the
102
LILLIANY. F. Hsu and KURT HIRSCHHORN
possible effect of oral contraceptives or intrauterine devices, drugs, viruses, and ionizing radiation. With the computerized chromosome program, chromosome studies can be done in both unselected large samples of abortuses and specially selected abortuses with a matched control. It will be therefore possible to see the true incidence of chromosome aberration in spontaneous and/or therapeutic abortions and to determine the effects of the above-mentioned possible factors. 2. Incidence of chromosomai mosaicism Since chromosomal mosaicism means presence of two or more cell lines of different chromosomal constitutions in one individual, the mosaicism will not be detected in such individuals if only a few cultured metaphase cells are examined and analysed. In most of the newborn chromosome surveys the basic number of metaphases counted and analysed per individual was 2 cells( 4- 6,8)except in one study with 5-10 cells.(‘) In a total number of 14269 newborns studied by these investigators, 75 infants were found to have chromosomal aberration and only one of them was found to have mosaicism (45, D-, E-, mar+/46, E-, mar+).t4) Cases of mosaicism can be easily missed if only 2 cells are counted. In our laboratory, we routinely count 20 cells in each case, but when a cell with different chromosomal constitution is found in the 20 cells, further examination of 30 more cells is then required to assess the validity of the possible existence of the second cell line. In 840 patients on whom we have performed chromosome studies, 192 cases were found to have abnormal chromosomal constitutions and 32 of 192 cases (i.e. 16.7 per cent) were mosaic. The true incidence of chromosome mosaicism is not known and such studies have not been carried out due to the obvious lack of manpower. With the computer chromosome analyses, it will be possible to count 50 or 100 cells in each individual studied. If this is carried out in unselected, consecutive live newborns, the true incidence of mosaicism in newborns will be determined. Similar studies in different age groups will be useful to find out the difference in incidence of chromosomal mosaicism in the different age groups and to determine its significance. It has been shown in cases of chromosomal mosaicism, that one cell line in vitro may have a selected advantage over the other with disappearance of the mosaicism as time lapses.(ls) It is also known that increased numbers of 45, X cells has been found in aging females.(20~a1) Therefore, studies of different age groups for chromosome mosaicism are warranted. Studies of chromosomal mosaicism will also be extremely important in selected populations, such as families with multiple affected members of Down’s syndrome or parents of multiple children with Down’s syndrome. Most of familial Down’s syndrome is associated with trisomy 21; 20-25 per cent were found to have translocations. Although almost all the studied parents of familial trisomy 21 were found to have normal chromosomes in leukocyte cultures, nevertheless, there have been seven known instances of trisomy 21 Down’s syndrome associated with maternal trisomy 21 mosaicism,(2’) and one report of mosaic trisomy 21 in both a father and his daughter.ta3) We have studied 33 couples of young parents (age less than 30) of children with Down’s syndrome and found 4 fathers carrying a minor cell line with the extra No. 21 chromosome material. In 24 couples with children with 47, 21+, three fathers were found to have 46/47, 2l+mosaicism. In one of these 3 families, there were two children born-with trisomy 21 Down’s syndrome in 3 years. In 9 couples of the infants with translocation Down’s syndrome, one father of a child with G/G translocation trisomy 21 was mosaic for 46146, G-, t(Gq, Gq)+. Therefore, it appears that parental mosaicism can be an important cause for producing children with
The potential usefulness of computerized chromosome analysis in human genetics
103
Down’s syndrome. It would be desirable to study all parents of Down’s syndrome regardless of the parental age so as to determine the real incidence of mosaicism in the parents. Familial mosaicism with multiple members having different or similar chromosomal mosaicism has been reported in several instances. We reported one family with 4 sibs having chromosomal mosaicism of 3 different types(*j) (2 with 45, X/46, XY/47, XYY; 1 with 46, XY/47, XYY; 1 with 46/47, B+/47, Gf). In this family the parents were first cousins and therefore the familial mosaicism is likely due to a genetically determined factor. Families with multiple individuals with congenital abnormalities, if studied extensively by the computer program, will possibly yield more significant results. III.
STUDIES
OF CHROMOSOMES ENVIRONMENTAL
DAMAGE AGENTS
BY VARIOUS
It is now established that when a human being is given a large enough dose of ionizing radiation, an increased frequency of chromosomal aberrations will be detectable in the peripheral lymphocyte culture immediately after the exposure.(2s) The chromosome aberrations consist of two different types, i.e. unstable aberrations including acentric fragments, ring chromosomes and dicentric chromosomes; and stable aberrations including reciprocal translocation, inversion and partial deletion. The cells with unstable chromosomal aberrations quickly decrease and finally disappear by 3-5 years, but the stable aberrations will usually persist for over 20 years. It has been speculated that it is the chromosomally abnormal ceils which predispose to the susceptibility for neoplastic transformation, since there is a significantly increased incidence of leukemias developed from the individuals receiving large doses of irradiation. In fact, TODARO et u~.(~Q was able to demonstrate that cells from individuals with abnormal chromosomal constitutions, such as trisomy 21, or individuals with abnormal chromosomal breakage and rearrangement, such as Fanconi’s anemia, have a greater potential for neoplastic transformation by SV 40, an oncogenic virus. In addition to ionizing radiation, the other two major classes of exogenous agents capable of producing chromosomal damage are chemicals and viruses. The chemicals include antibiotics such as streptonigrin, actinomycin, mitomycin, daunomycin, alkylating agents such as myleran, cytoxan, nitrogen mustard; and others in a heterogenous group.(27v28) These chemicals can be generally subdivided into those affecting DNA and RNA synthesis, or DNA precursors and related substances. Some hallucinogenic drugs such as lysergic acid diethylamide (LSD), have been found to break human chromosomes both in vitro and in vivo by several different investigators,(2g,30) however, negative results were obtained by the others.(31*32) Recently, the artificial sweeteners, sodium and calcium cyclamate were found to be capable of inducing chromosome breaks in vitro in human cells,(33) although the effect in viwois not yet known. The viruses which have been found to induce chromosome damage are: oncogenic viruses such as SV 40,(34) and Rous-sarcoma virus,(s5) and other common viruses such as measles,@*) varicella,@‘) mumps,(37) rubella,(S8) herpes simplex type 1 and 2,(39) etc. A marker chromosome with an isochromatid gap close to the telomere of the long arm of a C group chromosome, most likely No. 10, has been identified in Burkitt’s lymphoma and infectious mononucleosis.(40*41) Since there is a close relationship between chromosome damage (i.e. breakage and rearrangement), carcinogenesis and teratogenesis, well designed in vitro and in vivo studies with matched controls are extremely important to determine the effect of induced chromosome aberrations by various environmental agents. However, up to now, the true incidence
104
LILLIANY. F. Hsu and KURT HIRSCHHORN
of chromosomal breakage and rearrangement in either the general population or in different age groups is not known. With automation of chromosome analysis, not only such control studies, but also extensive studies for detection of chromosome damage by various environmental agents will be possible. CONCLUDING
REMARKS
The rapid progress in cytogenetics during the last decade has provided a great deal of knowledge of the relationships between chromosomal aberrations and congenital abnormalities, abnormal sexual differentiations, fetal wastage, infertility, mutagenesis and carcinogenesis. However, with the vast expansion in cytogenetic research, the conventional timeconsuming karyotypic analysis has become a hampering factor. The development of automated programs for chromosome analysis has brought us a promising future. With the computers, various prospective studies will then be possible. An attempt was made in this paper to define the potential usefulness of computerized chromosome analysis in human genetics. One must realize that, as time lapses, one will find many other studies which can be aided by the computerized chromosome program. Three major studies, i.e. measurement studies, studies of incidence of chromosome aberration, and studies of chromosome damage by environmental agents, have been emphasized. Large scale prospective studies in unselected newborns and fetuses will provide information for the epidemiology of chromosome aberrations. In the incidence studies, the necessity for the study of the incidence of chromosome mosaicism appears to be the most immediate one, since no such information has been available. Studies of chromosome damage by various environmental agents will help to clarify some of the epidemiological uncertainties presently apparent and it thus would be most helpful for preventive medicine. It is thus hoped that the automation of chromosome analysis will be used as a master key to open many unexplored as well as many poorly explored areas in human genetics. SUMMARY The rapid progress and expansion in cytogenetics during the past decade has demanded a rapid method for karyotype analysis. The development of computerized chromosome analysis will certainly make many unexplored areas in human genetics approachable. Three major studies are emphasized in this paper, i.e. (1) measurement studies, (2) incidence studies of chromosomal aberrations which include studies of (a) numerical and structural aberrations, and (b) chromosome mosaicism, and (3) studies of chromosome damage by various environmental agents. With computerized chromosome analysis, the data on chromosome measurement can be used for standardization of the human chromosome complement. Studies of large scale unselected newborns and fetuses will determine the true incidence of every different type of chromosomal abnormality and chromosome mosaicism. Studies in selected samples will then determine their significance in comparison to the general population. Studies of chromosome damage by various environmental agents will help to clarify many uncertainties in epidemiology and make preventive medicine possible. REFERENCES 1. J. H. TJIO and A. LEVAN, The chromosome number of man, Heriditus 42, 1 (1956). 2. Chicago Conference: Standardization in human cytogenetics, Birth Defects: Original Article Series, Vol. II, No. 2, Dec. (1966).
The potential usefulness of computerized
chromosome
analysis in human genetics
105
3. F. E. ARRIGHI and T. C. Hsu, Localization of heterochromatin in human chromosomes, CI/ogelrafics 10, 81 (1971). 4. W. M. COURTBROWN and P. G. SMITH,Human population cytogenetics, Br. Med. Ball. 25, 74 (196Y). 5. S. G. RATCLIFFE,A. L. STEWART,M. M. MELVILLE,P. A. JACOBSand A. J. KEAY, Chromosome studies on 3500 newborn male infants, Lancer I, 121 (1970). 6. S. WALZER. G. BREAU and P. S. GERALD, A chromosome survey of 2400 normal newborn inbnts, J. Ped, 74, 438 (1969). 7. F. SERGOVICH,G. H. VALENTINE,A. T. L. CHEN, R. A. H. KII*;CHand M. S. &OUT, Chromosome aberrations in 2159 consecutive newborn babies. New E~tal. J. Med. 280, 851 (1969).
8. H. A. LUBS and F. H. RU~DLE, Chromosomal hbnormafities in the human population: Estimation of rates based on New Haven newborn study, Science 169,495 (1970). 9. D. WARBURTON,D. A. MILLER, 0. J. MILLER, P. W. ALLDERDICEand A. DECAPOA, Detection of minute deletions in human karyotypcs, Cylogenetics 8, 97 (1969). IO. T. KADOTANI, K. OHAMA, T. SOFUNI and H. B. HAMILTON,Aberrant karyotypes and spontaneous abortions in a Japanese family, Nar~re 225, 735 (1970). . I I. T. KADOTANI,K. OHAMA, T. NAIAYAMAand A. TABUCHI, Chromosome studies in primary sterility. Am. J. Obsr. Gynec. 106, 489 (1970). I?. L. Y. F. Hsu, M. BARCINSKI, L. R. SHAPIRO,E. VALDERAMA, M. GERTNERand K. HIRSCHHOR~.Parental chromosomal aberrations associated with multiple abortions and an abnormal infant, Obsr. G.wwc. 36,
723 (1970). 13. A. E. SZULMAN,Chromosomal aberrations in spontaneous human abortions, N<,w EngI. J. .\I~~cl.272, 81 I (1965). 14. D. J. CARR, Chromosome anomalies as a cause of spotaneous abortion, Am. J. Obsr. G.wrrc. 97, 283 (1967). 15. J. G. BOUT and A. Bou& Les aberrations chromosomiques dans les avortements spontanes humains. C.r. Acad. Sci., Paris 263D, 2054 (1966). 16. M. SMITH,J. MACNA~and M. A. FERCUSON-SMITH,Cell culture technics from cytogenetic investigation of human abortus material: Analysis of 45 cases and report of 3 specimens with gross chromosomal aberrations, Obst. Gynec. 33, 313 (1969). 17. K. MIKAMO, Anatomic and chromosomal anomalies in spozaneous abortion, .-l/rt. J. Ohs!. Guwc. 106, 243 (1970). 18. A. M. SCHINDLERand K. MIKAMO,Triploidy in man, Cytogcnrrics 9, 1 I6 (1970). 19. R. L. NEU. G. J. BARGWANand L. I. GARDNER.DisaDoearance of a 47. X.X. C+ leukocvte cell hne in an infant who had previously exhibited 46, Xx)47, X.2, C+ mosaici&, Pcharrics 43. 654 (1969). 20. P. A. JACOBS,W. M. COURT BROWN and R. DOLL, Distribution of human chromosome counts in relation to age, Nafrrrc 191, 1178 (1961). 21. J. L. HAMERTON,A. I. TAYLOR,R. ANGELLand V. M. MCGUIRE, Chromosome investigations of a small isolated human population: chromosome abnormalities and distribution of chromosome count5 according to age and sex among the population of Tristan Da Cumha, Natrrre 206, 1231 (1965). 22. D. AARSKOG,Down’s syndrome transmitted through maternal mosaicism, Acra Pazdiaf Stand. 58, 609 (1969). 23. F. A. WALKERand R. ISING,Mosaic Down’s syndrome
in a father and daughter, Lnrlcrr I, 374 (1969). 24. 1. Y. F. Hsu, K. HIRSCHHORN,A. GOLDSTEINand M. A. BARCINSKI,Familial chromosomal mosnicisni. genetic aspects, Ann. Hlcm. Genet., Load. 33, 343 (1970). 25. K. E. BucKToN, P. A. JACOBS,W. M. COURTBROWNand R. DOLL, A study of the chromosome damage persisting after X-ray therapy for ankylosing spondylitis, Lancet II, 676 (1962). 26. G. J. TODARO,H. GREEN and M. R. SWIFT, Susceptibility of human diploid fibroblast strains to transformation by SV 40 virus, Science 153, 1252 (1966). 27. K. HIRSCHHORFI and M. M. COHEN.Drue-induced chromosomal aberrations. .&,I. N. Y. dcati. Sci. 151. 977 (1968). 28. J. WHANG-PENG,B. G. LEVENTHAL,J. W. ADAM~~Nand S. PERRY,The effect of Daunomycin on human cells in viva and in vifro, Cancer 23, 113 (1969). 29. M. M. COHEN,K. HIRSCHHORNand W. A. FROSCH,111viva and br vi/r0 chromosomal damage induced by LSD-25, New Engl. J. Med. 277, 1043 (1967). 30. J. EGOZCUE, S. IRWIN and C. A. MARUFFO, Chromosomal damage in LSD users. J..4..V.A. 204, v
214 (1968). 31. R. G. SMARTand K. BATEMAN,The chromosomal and teratogenic effects of lysergic acid diethylamide: A review of the current literature, Can. Med. Assoc. J. 99, 805 (1968).
32. J. H. TJIO, W. N. PAHNKEand A. A. KLRLAND, LSD and chromosomes. J.A.M.A. 210, 849 (1969). 33. D. STONE,E. LAMSON,Y. S. CHANG and K. W. PICKERING,Cytogenetic cells in vitro, Science 164, 568 (1969).
A controlled
experiment,
effects of Cyclamates on human
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34. S. R. WOLMAN,K. HIR~CHHORNand G. J. TODARO, Early chromosomal changes in SV 40-infected human fibroblast cultures, Cyrogenerics 3, 45 (1964). 35. W. W. NICHOU, Relationships of viruses, chromosomes and carcinogenesis, Herediras 50, 53 (1963). 36. W. W. NICHOU, A. LEVAN, P. AULA and E. NORRBY,Chromosome damage associated with measles virus in vitro, Heriditus 54, 101 (1965). 37. P. AULA, Virus-associated chromosome breakage: cytogenetic study of chicken pox, measles, and mumps patients and of cell cultures injected with measles virus, Ann. Acad. SC. Fenniae (Series A IV. Biologica) 89, 1 (1965). 38. J. NUSBACHER,K. HIRSCHHORNand L. Z. COOPER,Chromosomai abnormalities in congenital rubella, New Engl. J. Med. 276, 1409 (1967). 39. F. J. O’NEILLand C. P. MILFS, Chromosome changes induced by Herpex Simplex type 1 and 2, in human cells, Narlrre 223, 851 (1969). 40. W. HENLE, V. DIEHL, G. KOHN, H. ZUR HAUSEXand G. HENLE, Herpes-type virus and chromosome marker in normal leukocytes after growth with irradiated Burkitt cells, Science 157, 1064 (1967). 41. G. KOHN, V. DIEHL, W. J. MELLMAN,W. HENLEand G. HENLE,C-group chromosome marker in iongterm leukocytes cultures, J. Nat. Can. Ins~. 41, 795 (1968).
ADDENDUM Since we submitted this manuscript, there have been several differential staining techniques for identification of individual chromosomes reported. CASPERSSON et a/.(j”) demonstrated 24 fluorescence patterns of the human metaphase chromosomes. HECHT et a/.cJ3) has reviewed the banding patterns produced by the fluorescent staining technique, the other Giemsa denaturation techniques and reverse Giemsa method. A more simple method using trypsin digestion to produce chromosome bandings was reported by SEABRIGHT. A combination of all these newer techniques and the computerized chromosome analysis will be most helpful in cytogenetic diagnosis. ADDITIONAL
REFERENCES
42. T. CASPERSSON, G. LOMAKKAand L. ZECH,The 24 fluorescence patterns of the human metaphase chromosomes-distinguishing characters and variability, Heredirus 67, 89 (1971). 43. F. HECHT,H. E. WYANDTand R. W. ERBE,Revolutionary cytogenetics, New Eng. J. Med. 285,1482 (1971). 44. M. SEABRIGF~~, A rapid banding technique for human chromosomes, Lancer II, 971 (1971).
I’erg:lmon Press 1972. Vol. 2, pp. 99-106.
Printed in Circa1 Britain.
The Potential Usefulness of Computerized Chromosome Analysis in Human Genetics* LILLIAN
Y. F. HSU and KURT
HIRSCHHORN
Division of Medical Genetics, Department of Pediatrics, Mount Sinai School of Medicine, New York, New York 10029, U.S.A. (Received 30 October 1970) Abstract-The potential usefulness of computerized chromosome analysis in human genetics is mainly in studies aimed at population cytogenetics, epidemiology and preventive medicine. Three major studies are emphasized, i.e. (1) measurement studies, (2) incidence studies of chromosomal aberrations which include studies of (a) numerical and structural aberrations and (b) chromosome mosaicism, and (3) studies of chromosome damage by various environmental agents.
INTRODUCTION THE
DEMONSTRATION of the normal human chromosome number and morphology by and LEVAN in 1956,(l) not only opened a new era but also a new field of human genetics, i.e. cytogenetics. The rapid progress in this field during the past decade has further shown its importance in medicine. Identification of chromosomal abnormalities in individuals with certain congenital abnormalities has become critical in diagnosis, prognosis, and genetic counselling. The development of the technique of prenatal chromosome diagnosis by culturing the fetal cells from the amniotic fluid has made it possible to prevent the birth of a child with abnormal chromosomes by therapeutic abortion. However, due to the conventional time-consuming procedure in karyotype analysis and the limited manpower, most of the cytogenetic studies have been restricted to phenotypically abnormal individuals. The knowledge of human population cytogenetics is scanty, although it is being explored at a slow pace. It is the purpose of this paper to bring forth the potential usefulness of computerized chromosome analysis in human genetics.
TJIO
I.
MEASUREMENT
STUDIES
The first proposed system of nomenclature of human mitotic chromosomes was put forward by the Denver Conference in 1960.(*) It was generally agreed that the autosomes should be serially numbered l-22 in descending order of length and be classified into seven groups according to the different location of the centromere. The sex chromosomes are referred to as X and Y. The basic measurements including the relative length of each chromosome, arm ratio and the centromere index were also reported by several investigators and summarized in the Denver conference. *Supported by U.S. Public Health Service Grants (HD-02552) and (HD-00210). Kurt Hirschhorn career scientist of the Health Research Council of the City of New York (I-513). 99
is a
100
LILLIAN Y. F. Hsu and KURTHIRSCHHORN
The second conference on normal human karyotype was held in London in 1963 mainly to assess the validity of the Denver classification of human chromosomes. Minor changes were made in the classification of several individual chromosomes.(2) Emphasis was made on the recognition ofsecondaryconstrictions ofcertain specific chromosomes and differential DNA replication patterns in identification of individual chromosomes. However, not all chromosomes can be identified even by these methods. Conventional karyotypic analysis is usually made by the visual pattern-recognition faculties of the cytogeneticist. Measurement, although remaining desirable, has been rarely carried out because it is tedious and time consuming. With computerized chromosome analysis, each metaphase will not only be counted accurately but each chromosome measured as well. Furthermore, a great number of normal cells can be analysed and the data of the measurements can be used for standardization of the human chromosome complement. Cells with minor chromosomal abnormalities will be also detected. Recently, ARRIGHI and Hsut3) described a differential chromosome staining technique in identification of human chromosomes. With this technique, the stain is differentially bound to the repetitive (“satellite”) DNA of the chromosome which is generally associated with constitutive heterochromatin. Promising results have been obtained by T. C. Hsu’s group and our laboratory in identification of several chromosomes in the C group, D group, E group, and G group. Since the computer techniques can discriminate staining density, it would be possible to utilize this technique to recognize the differential staining characteristics for more accurate karyotype analysis.
11. INCIDENCE
STUDIES
1. Incidence of numerical and structural chromosome abnormalities It has been realized that studies of a large, unbiased population is necessary to determine the significance of chromosome abnormalities in medicine for both epidemiological studies and preventive medicine. The latter is now possible by prenatal chromosome analysis of the cells cultured from the amniotic fluid and therapeutic abortion of the fetus with abnormal chromosomal constitution. Since 1969, chromosome surveys of selected live newborns have been reported by COURT BROWNand S~%ITH,(*) and RATCLIFFEet al.,(j) in sizable samples of male live newborns; WALZERet CA.,(~)in 2400 normal newborns. Up to present, there have been only two reports of chromosome survey in unbiased and unselected samples of newborn infants, i.e. studies of 2159 consecutive newborns by SERGOVICH et al.,(‘) and studies of 4500 consecutive newborns by LUBSand RUDDLE.@)In both studies, the frequency of infants with major abnormalities of their chromosomes was found to be about 0.5 per cent and most of these infants would not have been detected if the chromosome surveys were not undertaken. 21.8 per cent of the infants with the chromosome abnormalities were carriers of reciprocal balanced translocations and were phenotypically normal; more than half of the remaining infants with chromosomal abnormalities were found to have sex chromosome aberrations which also presented with no clinical difficulties in the neonatal period. A search for XYY males in all these newborn studies revealed approximately 1 in 562 male consecutive live newborns. (RATCLIFFEet al.15) found 5 in 3496; SERGOVICHet a1.t’) found 4 in 1066; LUBSand RIJDDLE@) found 3 in 2184.) However, in the study reported by WALZERet el.@) in 2400 normal (but not consecutive) newborns, no case of XYY was found.
The potential usefulness of computerized
chromosome
analysis in human genetics
101
It is clear that more extensive studies of unbiased, unselected newborn infants should be carried out on a large scale so as to determine the true incidence of every different kind of chromosomal abnormality. Only with computerized chromosome programs, will this be possible. Tn addition to the newborn study, if a concomitant survey is undertaken in a unselected sample of adolescents in the same area, it would be possible to compare the incidence of chromosome abnormalities in these two different age groups. Although we know that autosomal trisomies mostly die before reaching adolescence, we do not know the difference of the incidence of the other kinds of chromosomal abnormalities in these two age groups. With computerized chromosome analysis, chromosome.surveys can also be carried out extensively in selected samples such as mental institutions, prisoners, juvenile delinquents, etc., each with a matched age and sex control, since the incidence of sex chromosome abnormality, especially XYY, in these specific populations is needed to determine their significance. The detection of structural chromosomal abnormalities in the reported chromosome surveys, so far, has been done mostly on a few metaphase cells analysed without measurements. Therefore, only the obvious gross chromosomal structural abnormalities would be detected. Although SERGOVKHet a(.(‘) found one case of partial deletion of a B group chromosome and WALZER et ~1.c~)detected 3 instances of pericentric inversion in their series. According to WARBURTONet o!.,(~) minute deletions of a chromosome can be detected by careful measurements. They were able to identify 5 cases of cri du chat whose chromosomes would have appeared normal without measurements. The accurate measurements by computer of each chromosome will definitely detect more cases with minute structural abnormalities. We all know that numerous infants with multiple congenital abnormalities were found to have grossly normal chromosomes by conventional karyotyping methods. With the computer measuring each chromosome of every metaphase examined of these individuals, it would be possible to detect a minute chromosomal abnormality which would have been otherwise overlooked. Detection of structural aberrations will be especiaIly useful in studies of couples with subfertility and multiple spontaneous abortions. Since it has been suggested that subfertility, multiple spontaneous abortions, and stillbirths may result from a balanced structural chromosomal abnormality including translocation and inversion (structural heterozygote). In fact, several such cases have been reported.(1°-12) Studies of unselected series of spontaneous abortions have been carried out by several different investigators.(13-“) Chromosomal abnormalities were found in approximately 20 per cent (range 10-60 per cent) of first trimester spontaneous abortions. The abnormal chromosome constitutions consisted of triploidy in approximately l/3 ; trisomies in l/3 ; 45, X constitution in l/3. Cytogenetic studies on therapeutic abortions showed abnormal chromosomes in only 2.8 per cent of the abortuses.( 17) In the studies of triploidy in abortuses the collected data for early aborted fetuses are: 17 XXX; 30 XXY; and 4 XYY. The frequency of XXXXXY is almost 1:2. This will favor the possibility that triploidy in man is most likely caused by diandry rather than digyny. Polyspermy usually results from overripeness of eggs due to delayed fertilization. Therefore, further study in abortus materials, including hydatidiform mole, will yield better understanding of the effect of overripeness or aging of the eggs and chromosomal abnormalities. Chromosome studies in abortuses are also specially important in determination of the
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possible effect of oral contraceptives or intrauterine devices, drugs, viruses, and ionizing radiation. With the computerized chromosome program, chromosome studies can be done in both unselected large samples of abortuses and specially selected abortuses with a matched control. It will be therefore possible to see the true incidence of chromosome aberration in spontaneous and/or therapeutic abortions and to determine the effects of the above-mentioned possible factors. 2. Incidence of chromosomai mosaicism Since chromosomal mosaicism means presence of two or more cell lines of different chromosomal constitutions in one individual, the mosaicism will not be detected in such individuals if only a few cultured metaphase cells are examined and analysed. In most of the newborn chromosome surveys the basic number of metaphases counted and analysed per individual was 2 cells( 4- 6,8)except in one study with 5-10 cells.(‘) In a total number of 14269 newborns studied by these investigators, 75 infants were found to have chromosomal aberration and only one of them was found to have mosaicism (45, D-, E-, mar+/46, E-, mar+).t4) Cases of mosaicism can be easily missed if only 2 cells are counted. In our laboratory, we routinely count 20 cells in each case, but when a cell with different chromosomal constitution is found in the 20 cells, further examination of 30 more cells is then required to assess the validity of the possible existence of the second cell line. In 840 patients on whom we have performed chromosome studies, 192 cases were found to have abnormal chromosomal constitutions and 32 of 192 cases (i.e. 16.7 per cent) were mosaic. The true incidence of chromosome mosaicism is not known and such studies have not been carried out due to the obvious lack of manpower. With the computer chromosome analyses, it will be possible to count 50 or 100 cells in each individual studied. If this is carried out in unselected, consecutive live newborns, the true incidence of mosaicism in newborns will be determined. Similar studies in different age groups will be useful to find out the difference in incidence of chromosomal mosaicism in the different age groups and to determine its significance. It has been shown in cases of chromosomal mosaicism, that one cell line in vitro may have a selected advantage over the other with disappearance of the mosaicism as time lapses.(ls) It is also known that increased numbers of 45, X cells has been found in aging females.(20~a1) Therefore, studies of different age groups for chromosome mosaicism are warranted. Studies of chromosomal mosaicism will also be extremely important in selected populations, such as families with multiple affected members of Down’s syndrome or parents of multiple children with Down’s syndrome. Most of familial Down’s syndrome is associated with trisomy 21; 20-25 per cent were found to have translocations. Although almost all the studied parents of familial trisomy 21 were found to have normal chromosomes in leukocyte cultures, nevertheless, there have been seven known instances of trisomy 21 Down’s syndrome associated with maternal trisomy 21 mosaicism,(2’) and one report of mosaic trisomy 21 in both a father and his daughter.ta3) We have studied 33 couples of young parents (age less than 30) of children with Down’s syndrome and found 4 fathers carrying a minor cell line with the extra No. 21 chromosome material. In 24 couples with children with 47, 21+, three fathers were found to have 46/47, 2l+mosaicism. In one of these 3 families, there were two children born-with trisomy 21 Down’s syndrome in 3 years. In 9 couples of the infants with translocation Down’s syndrome, one father of a child with G/G translocation trisomy 21 was mosaic for 46146, G-, t(Gq, Gq)+. Therefore, it appears that parental mosaicism can be an important cause for producing children with
The potential usefulness of computerized chromosome analysis in human genetics
103
Down’s syndrome. It would be desirable to study all parents of Down’s syndrome regardless of the parental age so as to determine the real incidence of mosaicism in the parents. Familial mosaicism with multiple members having different or similar chromosomal mosaicism has been reported in several instances. We reported one family with 4 sibs having chromosomal mosaicism of 3 different types(*j) (2 with 45, X/46, XY/47, XYY; 1 with 46, XY/47, XYY; 1 with 46/47, B+/47, Gf). In this family the parents were first cousins and therefore the familial mosaicism is likely due to a genetically determined factor. Families with multiple individuals with congenital abnormalities, if studied extensively by the computer program, will possibly yield more significant results. III.
STUDIES
OF CHROMOSOMES ENVIRONMENTAL
DAMAGE AGENTS
BY VARIOUS
It is now established that when a human being is given a large enough dose of ionizing radiation, an increased frequency of chromosomal aberrations will be detectable in the peripheral lymphocyte culture immediately after the exposure.(2s) The chromosome aberrations consist of two different types, i.e. unstable aberrations including acentric fragments, ring chromosomes and dicentric chromosomes; and stable aberrations including reciprocal translocation, inversion and partial deletion. The cells with unstable chromosomal aberrations quickly decrease and finally disappear by 3-5 years, but the stable aberrations will usually persist for over 20 years. It has been speculated that it is the chromosomally abnormal ceils which predispose to the susceptibility for neoplastic transformation, since there is a significantly increased incidence of leukemias developed from the individuals receiving large doses of irradiation. In fact, TODARO et u~.(~Q was able to demonstrate that cells from individuals with abnormal chromosomal constitutions, such as trisomy 21, or individuals with abnormal chromosomal breakage and rearrangement, such as Fanconi’s anemia, have a greater potential for neoplastic transformation by SV 40, an oncogenic virus. In addition to ionizing radiation, the other two major classes of exogenous agents capable of producing chromosomal damage are chemicals and viruses. The chemicals include antibiotics such as streptonigrin, actinomycin, mitomycin, daunomycin, alkylating agents such as myleran, cytoxan, nitrogen mustard; and others in a heterogenous group.(27v28) These chemicals can be generally subdivided into those affecting DNA and RNA synthesis, or DNA precursors and related substances. Some hallucinogenic drugs such as lysergic acid diethylamide (LSD), have been found to break human chromosomes both in vitro and in vivo by several different investigators,(2g,30) however, negative results were obtained by the others.(31*32) Recently, the artificial sweeteners, sodium and calcium cyclamate were found to be capable of inducing chromosome breaks in vitro in human cells,(33) although the effect in viwois not yet known. The viruses which have been found to induce chromosome damage are: oncogenic viruses such as SV 40,(34) and Rous-sarcoma virus,(s5) and other common viruses such as measles,@*) varicella,@‘) mumps,(37) rubella,(S8) herpes simplex type 1 and 2,(39) etc. A marker chromosome with an isochromatid gap close to the telomere of the long arm of a C group chromosome, most likely No. 10, has been identified in Burkitt’s lymphoma and infectious mononucleosis.(40*41) Since there is a close relationship between chromosome damage (i.e. breakage and rearrangement), carcinogenesis and teratogenesis, well designed in vitro and in vivo studies with matched controls are extremely important to determine the effect of induced chromosome aberrations by various environmental agents. However, up to now, the true incidence
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of chromosomal breakage and rearrangement in either the general population or in different age groups is not known. With automation of chromosome analysis, not only such control studies, but also extensive studies for detection of chromosome damage by various environmental agents will be possible. CONCLUDING
REMARKS
The rapid progress in cytogenetics during the last decade has provided a great deal of knowledge of the relationships between chromosomal aberrations and congenital abnormalities, abnormal sexual differentiations, fetal wastage, infertility, mutagenesis and carcinogenesis. However, with the vast expansion in cytogenetic research, the conventional timeconsuming karyotypic analysis has become a hampering factor. The development of automated programs for chromosome analysis has brought us a promising future. With the computers, various prospective studies will then be possible. An attempt was made in this paper to define the potential usefulness of computerized chromosome analysis in human genetics. One must realize that, as time lapses, one will find many other studies which can be aided by the computerized chromosome program. Three major studies, i.e. measurement studies, studies of incidence of chromosome aberration, and studies of chromosome damage by environmental agents, have been emphasized. Large scale prospective studies in unselected newborns and fetuses will provide information for the epidemiology of chromosome aberrations. In the incidence studies, the necessity for the study of the incidence of chromosome mosaicism appears to be the most immediate one, since no such information has been available. Studies of chromosome damage by various environmental agents will help to clarify some of the epidemiological uncertainties presently apparent and it thus would be most helpful for preventive medicine. It is thus hoped that the automation of chromosome analysis will be used as a master key to open many unexplored as well as many poorly explored areas in human genetics. SUMMARY The rapid progress and expansion in cytogenetics during the past decade has demanded a rapid method for karyotype analysis. The development of computerized chromosome analysis will certainly make many unexplored areas in human genetics approachable. Three major studies are emphasized in this paper, i.e. (1) measurement studies, (2) incidence studies of chromosomal aberrations which include studies of (a) numerical and structural aberrations, and (b) chromosome mosaicism, and (3) studies of chromosome damage by various environmental agents. With computerized chromosome analysis, the data on chromosome measurement can be used for standardization of the human chromosome complement. Studies of large scale unselected newborns and fetuses will determine the true incidence of every different type of chromosomal abnormality and chromosome mosaicism. Studies in selected samples will then determine their significance in comparison to the general population. Studies of chromosome damage by various environmental agents will help to clarify many uncertainties in epidemiology and make preventive medicine possible. REFERENCES 1. J. H. TJIO and A. LEVAN, The chromosome number of man, Heriditus 42, 1 (1956). 2. Chicago Conference: Standardization in human cytogenetics, Birth Defects: Original Article Series, Vol. II, No. 2, Dec. (1966).
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chromosome
analysis in human genetics
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3. F. E. ARRIGHI and T. C. Hsu, Localization of heterochromatin in human chromosomes, CI/ogelrafics 10, 81 (1971). 4. W. M. COURTBROWN and P. G. SMITH,Human population cytogenetics, Br. Med. Ball. 25, 74 (196Y). 5. S. G. RATCLIFFE,A. L. STEWART,M. M. MELVILLE,P. A. JACOBSand A. J. KEAY, Chromosome studies on 3500 newborn male infants, Lancer I, 121 (1970). 6. S. WALZER. G. BREAU and P. S. GERALD, A chromosome survey of 2400 normal newborn inbnts, J. Ped, 74, 438 (1969). 7. F. SERGOVICH,G. H. VALENTINE,A. T. L. CHEN, R. A. H. KII*;CHand M. S. &OUT, Chromosome aberrations in 2159 consecutive newborn babies. New E~tal. J. Med. 280, 851 (1969).
8. H. A. LUBS and F. H. RU~DLE, Chromosomal hbnormafities in the human population: Estimation of rates based on New Haven newborn study, Science 169,495 (1970). 9. D. WARBURTON,D. A. MILLER, 0. J. MILLER, P. W. ALLDERDICEand A. DECAPOA, Detection of minute deletions in human karyotypcs, Cylogenetics 8, 97 (1969). IO. T. KADOTANI, K. OHAMA, T. SOFUNI and H. B. HAMILTON,Aberrant karyotypes and spontaneous abortions in a Japanese family, Nar~re 225, 735 (1970). . I I. T. KADOTANI,K. OHAMA, T. NAIAYAMAand A. TABUCHI, Chromosome studies in primary sterility. Am. J. Obsr. Gynec. 106, 489 (1970). I?. L. Y. F. Hsu, M. BARCINSKI, L. R. SHAPIRO,E. VALDERAMA, M. GERTNERand K. HIRSCHHOR~.Parental chromosomal aberrations associated with multiple abortions and an abnormal infant, Obsr. G.wwc. 36,
723 (1970). 13. A. E. SZULMAN,Chromosomal aberrations in spontaneous human abortions, N<,w EngI. J. .\I~~cl.272, 81 I (1965). 14. D. J. CARR, Chromosome anomalies as a cause of spotaneous abortion, Am. J. Obsr. G.wrrc. 97, 283 (1967). 15. J. G. BOUT and A. Bou& Les aberrations chromosomiques dans les avortements spontanes humains. C.r. Acad. Sci., Paris 263D, 2054 (1966). 16. M. SMITH,J. MACNA~and M. A. FERCUSON-SMITH,Cell culture technics from cytogenetic investigation of human abortus material: Analysis of 45 cases and report of 3 specimens with gross chromosomal aberrations, Obst. Gynec. 33, 313 (1969). 17. K. MIKAMO, Anatomic and chromosomal anomalies in spozaneous abortion, .-l/rt. J. Ohs!. Guwc. 106, 243 (1970). 18. A. M. SCHINDLERand K. MIKAMO,Triploidy in man, Cytogcnrrics 9, 1 I6 (1970). 19. R. L. NEU. G. J. BARGWANand L. I. GARDNER.DisaDoearance of a 47. X.X. C+ leukocvte cell hne in an infant who had previously exhibited 46, Xx)47, X.2, C+ mosaici&, Pcharrics 43. 654 (1969). 20. P. A. JACOBS,W. M. COURT BROWN and R. DOLL, Distribution of human chromosome counts in relation to age, Nafrrrc 191, 1178 (1961). 21. J. L. HAMERTON,A. I. TAYLOR,R. ANGELLand V. M. MCGUIRE, Chromosome investigations of a small isolated human population: chromosome abnormalities and distribution of chromosome count5 according to age and sex among the population of Tristan Da Cumha, Natrrre 206, 1231 (1965). 22. D. AARSKOG,Down’s syndrome transmitted through maternal mosaicism, Acra Pazdiaf Stand. 58, 609 (1969). 23. F. A. WALKERand R. ISING,Mosaic Down’s syndrome
in a father and daughter, Lnrlcrr I, 374 (1969). 24. 1. Y. F. Hsu, K. HIRSCHHORN,A. GOLDSTEINand M. A. BARCINSKI,Familial chromosomal mosnicisni. genetic aspects, Ann. Hlcm. Genet., Load. 33, 343 (1970). 25. K. E. BucKToN, P. A. JACOBS,W. M. COURTBROWNand R. DOLL, A study of the chromosome damage persisting after X-ray therapy for ankylosing spondylitis, Lancet II, 676 (1962). 26. G. J. TODARO,H. GREEN and M. R. SWIFT, Susceptibility of human diploid fibroblast strains to transformation by SV 40 virus, Science 153, 1252 (1966). 27. K. HIRSCHHORFI and M. M. COHEN.Drue-induced chromosomal aberrations. .&,I. N. Y. dcati. Sci. 151. 977 (1968). 28. J. WHANG-PENG,B. G. LEVENTHAL,J. W. ADAM~~Nand S. PERRY,The effect of Daunomycin on human cells in viva and in vifro, Cancer 23, 113 (1969). 29. M. M. COHEN,K. HIRSCHHORNand W. A. FROSCH,111viva and br vi/r0 chromosomal damage induced by LSD-25, New Engl. J. Med. 277, 1043 (1967). 30. J. EGOZCUE, S. IRWIN and C. A. MARUFFO, Chromosomal damage in LSD users. J..4..V.A. 204, v
214 (1968). 31. R. G. SMARTand K. BATEMAN,The chromosomal and teratogenic effects of lysergic acid diethylamide: A review of the current literature, Can. Med. Assoc. J. 99, 805 (1968).
32. J. H. TJIO, W. N. PAHNKEand A. A. KLRLAND, LSD and chromosomes. J.A.M.A. 210, 849 (1969). 33. D. STONE,E. LAMSON,Y. S. CHANG and K. W. PICKERING,Cytogenetic cells in vitro, Science 164, 568 (1969).
A controlled
experiment,
effects of Cyclamates on human
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34. S. R. WOLMAN,K. HIR~CHHORNand G. J. TODARO, Early chromosomal changes in SV 40-infected human fibroblast cultures, Cyrogenerics 3, 45 (1964). 35. W. W. NICHOU, Relationships of viruses, chromosomes and carcinogenesis, Herediras 50, 53 (1963). 36. W. W. NICHOU, A. LEVAN, P. AULA and E. NORRBY,Chromosome damage associated with measles virus in vitro, Heriditus 54, 101 (1965). 37. P. AULA, Virus-associated chromosome breakage: cytogenetic study of chicken pox, measles, and mumps patients and of cell cultures injected with measles virus, Ann. Acad. SC. Fenniae (Series A IV. Biologica) 89, 1 (1965). 38. J. NUSBACHER,K. HIRSCHHORNand L. Z. COOPER,Chromosomai abnormalities in congenital rubella, New Engl. J. Med. 276, 1409 (1967). 39. F. J. O’NEILLand C. P. MILFS, Chromosome changes induced by Herpex Simplex type 1 and 2, in human cells, Narlrre 223, 851 (1969). 40. W. HENLE, V. DIEHL, G. KOHN, H. ZUR HAUSEXand G. HENLE, Herpes-type virus and chromosome marker in normal leukocytes after growth with irradiated Burkitt cells, Science 157, 1064 (1967). 41. G. KOHN, V. DIEHL, W. J. MELLMAN,W. HENLEand G. HENLE,C-group chromosome marker in iongterm leukocytes cultures, J. Nat. Can. Ins~. 41, 795 (1968).
ADDENDUM Since we submitted this manuscript, there have been several differential staining techniques for identification of individual chromosomes reported. CASPERSSON et a/.(j”) demonstrated 24 fluorescence patterns of the human metaphase chromosomes. HECHT et a/.cJ3) has reviewed the banding patterns produced by the fluorescent staining technique, the other Giemsa denaturation techniques and reverse Giemsa method. A more simple method using trypsin digestion to produce chromosome bandings was reported by SEABRIGHT. A combination of all these newer techniques and the computerized chromosome analysis will be most helpful in cytogenetic diagnosis. ADDITIONAL
REFERENCES
42. T. CASPERSSON, G. LOMAKKAand L. ZECH,The 24 fluorescence patterns of the human metaphase chromosomes-distinguishing characters and variability, Heredirus 67, 89 (1971). 43. F. HECHT,H. E. WYANDTand R. W. ERBE,Revolutionary cytogenetics, New Eng. J. Med. 285,1482 (1971). 44. M. SEABRIGF~~, A rapid banding technique for human chromosomes, Lancer II, 971 (1971).