I
33
Volume 69 January 1976
Section of Comparative Medicine President R J W Rees FRcPath
Meeting 16 April 1975
Genetics and Disease Ms Jacqueline A Robinson (MRC Clinical and Population Cytogenetics Unit, Western General Hospital, Edinburgh, EH4 2XU)
Recent Advances in Cytogenetics and their Relevance to Medicine From the time the first modern studies of the human karyotype were published in 1958 (Tijo & Puck 1958, Ford et al. 1958) until the new banding techniques were developed in 1970, there were only four of the 23 pairs of human chromosomes that could be positively identified. Conventional staining techniques using orcein or Giemsa stains gave no sort of longitudinal differentiation along the chromosomes, which were thus classified by overall size and relative lengths of the two arms (Fig 1). On this basis, the human chromosomes were divided into seven groups of chromosomes of similar morphology, with pairs 1, 2, 3, 16 and sometimes the Y chromosome being individually recognized. Any alteration in the chromosomes such as a deletion, or a translocation (in which a part of one chromosome is exchanged for a part of another) could only be recognized if the altered chromosomes were unambiguously changed to look quite different from any normal chromosome. Then, in 1969 and 1970 several papers were published (Zech 1969, Pearson et al. 1970, George 1970) showing that, on staining with a quinacrine dye, and viewing under blue light through a fluorescence microscope, the Y chromosome could be unequivocally identified by the brilliant fluorescence at the distal ends of its long arms. So intense was this fluorescence that it could also be seen in undividing cells (such as buccal mucosa or sperm) (Fig 2) as a bright spot or Y body (Sumner, Robinson & Evans 1971, Robinson 1971). It was realized that not only did this fluorescence technique show the Y chromosome but it produced transverse banding patterns of bright and dull fluorescence
on all chromosomes (Caspersson et al. 1971, O'Riordan et al. 1971); as these patterns are unique for each chromosome pair it is now possible to identify positively all the chromosomes in the human karyotype (Fig 3) and, further, the technique demonstrates that seven pairs of chromosomes have polymorphic regions, areas that can vary in size and fluorescence intensity
:. ... $.J-x.;4;0: 4.
# ,ff~ilila ...~
~ ~~~~~~~~~~~~~~~~~~~~~~.
A_:*A
.. .......
*.
Fig I Karyotype ofnormal male, 46, XY, stained with orcein
34
Proc. roy. Soc. Med. Volume 69 January 1976
2
between members of a pair, and between individuals, but which are constant within any one individual (Fig 6). A number of other banding techniques were shortly published. The ASG technique (Sumner, Evans & Buckland 1971) (Fig 4) developed in our laboratory shows essentially the same bands as does the fluorescence technique, but it gives permanent preparations which can be viewed through an ordinary microscope, thus overcoming the two main difficulties of the fluorescence technique. The R-banding technique (Dutrillaux & Lejeune 1971) produces, as it were, the mirror image of ASG banding with dark bands where ASG gives light ones, and so is of particular use in showing the ends of chromosomes that are lightly stained by the ASG technique. The other technique in routine use is the C-band technique (Arrighi & Hsu 1971) which stains blocks of heterochromatin around the centromere of each chromosome and demonstrates that those blocks on chromosomes number 1, 9 and 16 are polymorphic for size (Fig 5). These techniques have two uses in medicine; an immediate use in clinical cytogenetics, and a prospective use in medical research.
Clinical Cytogenetics Fig 3 Karyotype ofnormal male, 46, XY,fluorescence The significance of these new banding techniques technique can be summarized by saying that they 'bring a new order of precision to human cytogenetics' -altered; with this in mind we re-examined, using (Buckton 1973). We can now accurately identify the banding techniques, those individuals found virtually all chromosome abnormalities - and it by conventional analysis in our laboratory to should be remembered that 1/150 newborns has have a chromosomal abnormality; and a number such an abnormality. We can, for instance, of misdiagnoses have been discovered. For instance, one case had been thought to be a differentiate the 4p- syndrome from the 5p- and can link the phenotypic abnormalities to the loss deletion of a D group chromosome; although a of a particular part of a particular chromosome. translocation had been suspected since the With conventional staining techniques one could individual was phenotypically normal, no other only recognize translocations as such if both altered chromosome could be found. When involved chromosomes were unambiguously reanalysed with banding techniques, it could be seen (Fig 4) that there was indeed a piece missing from one D group chromosome - the 14- but there was also an extra piece on the ends of the long arms of chromosome 1. While it makes little difference to an individual that his chromosome abnormality can now be accurately described, it is of extreme importance, should a pregnancy ensue, that the fetal chromosomes can be accurately identified. In this case, the 14q- chromosome can be identified by orcein staining but the lq + cannot, so a fetus with the lq+ chromosome and a normal number 14 (trisomic for part of the long aims of number 14) would have an apparently nbrmal karyotype; but the use of a banding technique would allow a correct prediction of the child's normality. Lindsten (Lindsten et al. 1973) investigated a family with two mentally-retarded Fig 2 X- and Y-bearing spermatozoa, the latter with sons both of whom had an abnormally long a Ybody
3
Fig 4 Karyotype offemale 46, XX, t (1:14) ASG technique
G-group chromosome, but their phenotypicallynormal father also had the same chromosome, and no other abnormal chromosome could be detected. Fluorescence studies, however, revealed that a small piece of the distal end of chromosome 1 was missing - so small that on this the longest chromosome its loss made no observable difference, but when translocated on to a very small chromosome - in fact the 22- it made an appreciable difference to its size. When the mother became pregnant again, antenatal diagnosis revealed that the fetus had the balanced translocation, like the father, and a healthy child was later born. One type of translocation in which definite identification of the involved chromosomes is important is the Robertsonian translocation, which is found in 1/1000 individuals. These are translocations involving chromosomes which have the centromere very near the chromosome end so that the short arms are very short. The translocation involves the loss of the short arms of two such chromosomes followed by a joining of the centromeres to produce one chromosome made (up of the long arms of the two original chromosomes. The loss of the short arm material seems relatively unimportant; the majority of
Section ofComparative Medicine
35
cases have no phenotypic abnormalities but there is the risk of producing gametes with the unbalanced translocation which can lead to recurrent abortion or the birth of abnormal children. But the risks of such events depend on the chromosomes involved and so it is important to identify the chromosomes before counselling is given. A 13/14 translocation could theoretically lead to a child with trisomy 13 or trisomy 14; but the latter is not found in live born babies and the risk of a trisomy 13 child appears to be only about 1 in 160. But if the translocation is between two number 13 chromosomes (superficially identical by conventional staining), then any offspring will have either trisomy 13 or monosomy 13 since the parent with the translocation can only pass on either both 13s or no 13 to the gamete. Monosomy 13 is incompatible with fetal development and children with trisomy 13 are either aborted, stillborn or die shortly after birth. Similarly a 14/21 translocation carrier has a definite risk of having a child with Down's syndrome (trisomy 21); the risk is 1 in 10 for a female carrier and 1 in 50 for a male. But for the 15/22 translocation, while there is a risk of recurrent abortion, there is apparently a negligible risk of an abnormal liveborn child. For a female with a 21/22 translocation the risk of having a trisomy 21 child would seem to be about 1 in 16, but the available data are too few to estimate the risk for a male carrier. A 21/21 translocation carrier can, however, have no normal children; all conceptions will result in just abortion or birth of a mongol child. Furbetta et al. (1973) have published the case of a woman who had twelve pregnancies - 4 spontaneous abortions and 8 mongol children - before her chromosomes were examined and she was found to be a 21/21 translocation carrier.
Fig S Polymorphisms of C-bands, chromosomes 1, 9, 16
36 Proc. roy. Soc. Med. Volume 69 January1976
4
There is another use of the fluorescence technique in antenatal diagnosis. If the karyotype of the amniotic fluid cells is female then it is necessary to exclude the possibility that they are maternal and not fetal cells; and it is important to check that no error in identification has been made. We analyse the fluorescence polymorphisms of both the mother's chromosomes and those obtained from the amniotic fluid cells to check that the polymorphisms are not identical and that they are compatible with a mother-child relationship.
were found. It may be possible eventually to distinguish karyologically different subgroups of tumours; for instance, subcutaneous sarcomas in rats which are induced by different oncogenic agents cannot be distinguished histopathologically, but they can be distinguished by their karyotypes. Rowley (1973) has proposed a similar model for the karyological changes found in the haematological disorders in man: that perhaps one etiologic agent is involved when the karyotype shows an additional number 8 chromosome, while another agent is associated with a different karyotype, even though both etiologic agents are capable of producing the same spectrum of clinical diseases. This may well not be the whole answer, but certainly the accurate identification of the karyological changes is a prerequisite for an understanding of their significance.
Malignant Disease Leukemia and myeloproliferative disorders: Acquired chromosome abnormalities are frequently found in such diseases, but whether they are the cause or effect of the disorder is unknown. The best-known example is the Philadelphia chromosome found in the proliferating marrow cells of patients with chronic myeloid leukaemia (CML). The. presence of the Ph' chromosome is to a large extent diagnostic and those cases without it have a less good prognosis. The Ph' chromosome has been known for many years and was thought to be a simple deletion of a G-group chromosome, but we now know that it is in fact a translocation of part of a 22, usually on to a number 9 chromosome (Rowley 1973). On entering blast crisis, CML patients may acquire an isochromosome 17 and in general as the disease progresses other varied karyological changes occur. Many patients with myelopr6liferative diseases show karyotypic abnormalities involving C group chromosomes in their marrow cells. A number of these abnormalities have now been identified and they have been found to show a considerably less random pattern than had been thought. Out of 50 patients with various haematological disorders and C group abnormalities, 7 had lost a number 7 chromosome, 28 had gained a number 8 and 6 had gained a number 9 chromosome (Rowley 1975). As yet, the significance of these and other more individual changes is a matter of conjecture, but now that the changes can be accurately classified, we hope that they may identify subgroups within the disorders, indicate the progression of the disease and suggest suitable therapy.
Solid tumours: Solid tumours also show chromosome changes, with chromosome evolution during tumour development being correlated with significant functional properties of the tumour. Granberg (1971), working on cervical tumours, showed that the normal diploid karyotype disappeared with the development of invasiveness. In 32 cases of carcinoma in situ there were 64 normal cells out of 91; in 5 cases of microinvasion, only 1 out of 20 cells was normal, and in 10 invasive carcinomas no normal cells
Gene Mapping This is the process of discovering the positions of the genes on the chromosomes. At first sight this may seem somewhat academic but there are good reasons for mapping the human genome. First, a knowledge of the location of genes is necessary for understanding gene interaction, especially during development, and for investigating the control mechanisms which turn genes on and off - and this information is potentially very important in medicine. Then, mapping can reveal genetic heterogeneity within syndromes ofphenotypic similarity, such as elliptocytosis; one form of the disease is caused by a gene which, on mapping studies, is seen to be close to the RhesPs gene, but another form maps quite separately from Rhesus (Morton 1956). Once this had been discovered it could be seen that the symptoms of the two forms, although similar, were in some respects distinct. When genetic heterogeneity has been detected, there can be greater accuracy of description of the syndromes, with a more reliable prognosis and, one hopes, more effective treatment. Another advantage of detailed knowledge of the human genome lies in genetic counselling, especially in those diseases that cannot be diagnosed in utero, or which are late in onset. It may be possible to tell indirectly if a fetus or child carries a particular deleterious gene by examining for the presence of genes close to that in question. Obviously this is not nearly so good as testing directly for the presence of the particular gene, but there are, and probably always will be, cases where this is not possible. So far this type of indirect diagnosis has not been successfully carried out, but attempts have been made to discover whether individuals are carrying the dystrophia myotonica gene by examining their secretor status (Schrott et al. 1973).
5
Section of Comparative Medicine
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Family studies: For many years gene mapping has involved family studies, which in general give information as to which genes are linked - that is, close together on the same chromosome so that they pass together from parent to child. Only now are we beginning to identify which chromosomes these genes are on; previously, the only chromosomes which could be followed through families together with the genes were the rare variant or abnormal chromosomes. But now with the fluorescence and C-band techniques we have 10 pairs of chromosomes which show polymorphisms and the extremes of these can easily be followed through a family (see Fig 6), together with all the easily tested gene markers such as blood group antigens and any disease genes, e.g. thalassmmia. Essentially one looks for some sort of pattern in the family: for instance, all the family members of blood group A might have the chromosome number 13 with intensely bright satellites and also have thalassemia. This is of course an oversimplification, but it gives the general idea. Obviously one needs to collect a great deal of data frQm many families, but already some results are beginning to emerge. Our own work indicates that the gene for esterase D may be on chromosome 13 and that for adenyl kinase on chromosome 15but these are by no means definite assignments.
Somatic cell genetics: This other technique of mapping involves cell culture, and is based upon the discovery that cells from either the same or different species can be made to fuse together so that both sets of chromosomes are within a single cell. If the original cells are from different species then the chromosomes from one of them are selectively lost during the growth of the culture. Conveniently, in man/mouse and man/Chinese hamster hybrids it is the human chromosomes which are lost. One can continually test these cultures for the presence or absence of various human enzymes and chromosomes. If an enzyme and a chromosome are always lost together or retained together then presumptively the gene for that enzyme is located on that chromosome. In this way we can gradually discover which genes are on which chromosomes; at the moment, between the two techniques, about 40 genes have been assigned to their autosomes and 60 to the X chromosome. It is also possible by the use of translocations, either present in the original cell line or spontaneously occurring during culture, to say whereabouts on a chromosome a particular gene is located since translocations split chromosomes into two pieces which are then lost or retained independently of each other. Finally, the use of the fluorescence technique has demonstrated how in researrch one may discover something quite different from what was expected. As explained before, by the fluorescence
Fig 6 Inheritance offluorescence polymorphisms
technique the Y chromosome can be seen as a bright Y body even in sperm, so that X- and Ybearing sperm can be identified reliably. It would be of great agricultural importance, and useful in medicine, if the X- and Y-bearing sperm could be separated and offspring of the required sex be produced by artificial insemination. The fluorescence technique offers a quick and reliable method of assessing the efficiency of possible methods of separating sperm. Many techniques have been tried but so far none has been found to be successful, but a technique claiming to have achieved separation using passage through bovine serum albumin has been reported by Ericsson (Ericsson et al. 1973). We repeated his experiments and, although we failed to confirm his separation results, in our final isolate the sperm was of exceptionally high motility and morphologically very homogeneous compared with the
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original ejaculate. We think that an extension of this technique may be of value in the treatment of some types of male subfertility. The problem of separating X- and Y-bearing sperm has apparently not yet been solved but doubtless, when it is, the method will utilize the fluorescence technique.
normal breeding procedures, to improve profitability of our cattle industry. The amylase I locus in cattle is a possible example of such a genetic marker. It has been established that there is an apparent excess of heterozygotes at this locus in populations -of dairy cows, whereas no excess exists in dairy calves, or beef animals. The excess within a herd is related to the mean herd yield, and recent findings indicate some association between the yield of an individual, and its amylase genotype. However, the excess appears to be established in heifer populations which have not yet been milked. If farmers are selecting heterozygote animals as herd replacements, serum amylase could be, to some extent, a predictor of the economic worth of a dairy calf.
38
REFERENCES Arrighi F E & Hsu T C (1971) Cytogenetics 10, 81 Buckton K E (1973) In: Nobel Symposia: Medicine and Natural Sciences, Chromosome Identification. Ed. T Caspersson & L Zech. Nobel Foundation, Stockholm; p 196 Caspersson T, Lomakka G & Zech L (1971) Hereditas 67,89 Dutrillaux B & Lejeune J (1971) Comptes Rendus de l'Academie des Sciences 272, 2638 Eriesson R J, Langerin C N & Nishino M (1973) Nature (London) 246, 421 Ford C E, Jacobs P A & Lajtha L G (1958) Nature (London) 181, 1565 Furbetta M, Faloroni A, Antiguain D & Cao A (1973) Journal of Medical Genetics 10, 371 George K P (1970) Nature (London) 226, 80 Granberg I (1971) Hereditas 68, 165 Lindsten J, Holmberg M, Hulton M, Jonasson J, Licznerski G & Therkelsen A J (1973) In: Nobel Symposia: Medicine and Natural Sciences, Chromosome Identification. Ed. T Caspersson & L Zech. Nobel Foundation, Stockholm; p 230 Morton NE (1956) American Journal of Human Genetics 8, 80 O'Riordan M L, Robinson J A, Bucktod K E & Evans H J (1971) Nature (London) 230, 167 Pearson P L, Bobrow M & Vosa C G (1970) Nature (London) 226, 78 Robinson J A (1971) Annals of Human Genetics (London) 35, 61
Rowley J (1973) Nature (London) 243, 290 (1975) Proceedings of the National Academy of Sciences ofthe USA, 72, 152 Schrott H G, Karp L & Omenn G S (1973) Clinical Genetics 4, 38 Sumner A T, Evans H J & Buckland R (1971) Nature New Biology 232, 31 Sumner A T, Robinson J A & Evans H J (1971) Nature New Biology 229, 231 Tijo J M & Puck T T (1958) Proceedings of the National Academy of Sciences of the USA 44, 1229 Zech L (1969) Experimental Cell Research 58, 463
Dr J S Bradley (ARC Animal Breeding Research Organization, West Mains Road, Edinburgh, EH9 3JQ)
Genetic Aspects of Disease in Cattle [Abstract] Disease has a major effect on the longevity and profitability of our dairy cattle. There is evidence which indicates that resistance to many disease conditions is, in part, genetically controlled. The widespread use of artificial insemination in cattle breeding had made the long-term genetic improvement of health in cattle stock a possibility. The question remains, however, whether such breeding programmes are economically viable, since they must compete with the genetic improvement of production. Specific genes have been found in experimental and domestic animals, and in man, which directly affect viability, and resistance to disease. It may be possible to utilize such genes, as an adjunct to
REFERENCE Spooner R L, Bradley J S & Young G B (1975) Veterinary Record 97, 125-130
Dr C 0 Carter (MRC Clinical Genetics Unit, Institute of Child Health, 30 Guilford Street, London WCJ)
Genetics of Common Congenital Malformations in Man
Malformations Determined by Single Genes A great variety of monogenically determined single malformations or syndromes of multiple malformations occur in man, but none of these is common in the sense of having a birth frequency of 1 in 1000 total births or more. This is to be expected, since if the malformation is at all severe, then the birth frequency will, as the result of the balance between fresh mutation and selection, be at a level equivalent to some small multiple of the mutation rate. Gene mutations mostly occur at a frequency of 1 in 5000 or less. Monogenic inheritance is readily recognized by the simple mendelian family patterns it gives dominant, recessive or X-linked. Chromosome Anomalies It has recently been discovered that chromosomal anomalies are relatively frequent at conception. Some 400% of first trimester spontaneous miscarriages are associated with chromosomal anomalies, and-if one accepts that some 15 % of known conceptions miscarry, then about 6 %, or 1 in 16, of zygotes have a major chromosomal anomaly causing miscarriage. The three most common chromosomal anomalies found in spontaneous miscarriages are triploidy (69 chromosomes instead of 46), the 45 XO genotype, which is characteristic of Turner's syndrome, and
33
Volume 69 January 1976
Section of Comparative Medicine President R J W Rees FRcPath
Meeting 16 April 1975
Genetics and Disease Ms Jacqueline A Robinson (MRC Clinical and Population Cytogenetics Unit, Western General Hospital, Edinburgh, EH4 2XU)
Recent Advances in Cytogenetics and their Relevance to Medicine From the time the first modern studies of the human karyotype were published in 1958 (Tijo & Puck 1958, Ford et al. 1958) until the new banding techniques were developed in 1970, there were only four of the 23 pairs of human chromosomes that could be positively identified. Conventional staining techniques using orcein or Giemsa stains gave no sort of longitudinal differentiation along the chromosomes, which were thus classified by overall size and relative lengths of the two arms (Fig 1). On this basis, the human chromosomes were divided into seven groups of chromosomes of similar morphology, with pairs 1, 2, 3, 16 and sometimes the Y chromosome being individually recognized. Any alteration in the chromosomes such as a deletion, or a translocation (in which a part of one chromosome is exchanged for a part of another) could only be recognized if the altered chromosomes were unambiguously changed to look quite different from any normal chromosome. Then, in 1969 and 1970 several papers were published (Zech 1969, Pearson et al. 1970, George 1970) showing that, on staining with a quinacrine dye, and viewing under blue light through a fluorescence microscope, the Y chromosome could be unequivocally identified by the brilliant fluorescence at the distal ends of its long arms. So intense was this fluorescence that it could also be seen in undividing cells (such as buccal mucosa or sperm) (Fig 2) as a bright spot or Y body (Sumner, Robinson & Evans 1971, Robinson 1971). It was realized that not only did this fluorescence technique show the Y chromosome but it produced transverse banding patterns of bright and dull fluorescence
on all chromosomes (Caspersson et al. 1971, O'Riordan et al. 1971); as these patterns are unique for each chromosome pair it is now possible to identify positively all the chromosomes in the human karyotype (Fig 3) and, further, the technique demonstrates that seven pairs of chromosomes have polymorphic regions, areas that can vary in size and fluorescence intensity
:. ... $.J-x.;4;0: 4.
# ,ff~ilila ...~
~ ~~~~~~~~~~~~~~~~~~~~~~.
A_:*A
.. .......
*.
Fig I Karyotype ofnormal male, 46, XY, stained with orcein
34
Proc. roy. Soc. Med. Volume 69 January 1976
2
between members of a pair, and between individuals, but which are constant within any one individual (Fig 6). A number of other banding techniques were shortly published. The ASG technique (Sumner, Evans & Buckland 1971) (Fig 4) developed in our laboratory shows essentially the same bands as does the fluorescence technique, but it gives permanent preparations which can be viewed through an ordinary microscope, thus overcoming the two main difficulties of the fluorescence technique. The R-banding technique (Dutrillaux & Lejeune 1971) produces, as it were, the mirror image of ASG banding with dark bands where ASG gives light ones, and so is of particular use in showing the ends of chromosomes that are lightly stained by the ASG technique. The other technique in routine use is the C-band technique (Arrighi & Hsu 1971) which stains blocks of heterochromatin around the centromere of each chromosome and demonstrates that those blocks on chromosomes number 1, 9 and 16 are polymorphic for size (Fig 5). These techniques have two uses in medicine; an immediate use in clinical cytogenetics, and a prospective use in medical research.
Clinical Cytogenetics Fig 3 Karyotype ofnormal male, 46, XY,fluorescence The significance of these new banding techniques technique can be summarized by saying that they 'bring a new order of precision to human cytogenetics' -altered; with this in mind we re-examined, using (Buckton 1973). We can now accurately identify the banding techniques, those individuals found virtually all chromosome abnormalities - and it by conventional analysis in our laboratory to should be remembered that 1/150 newborns has have a chromosomal abnormality; and a number such an abnormality. We can, for instance, of misdiagnoses have been discovered. For instance, one case had been thought to be a differentiate the 4p- syndrome from the 5p- and can link the phenotypic abnormalities to the loss deletion of a D group chromosome; although a of a particular part of a particular chromosome. translocation had been suspected since the With conventional staining techniques one could individual was phenotypically normal, no other only recognize translocations as such if both altered chromosome could be found. When involved chromosomes were unambiguously reanalysed with banding techniques, it could be seen (Fig 4) that there was indeed a piece missing from one D group chromosome - the 14- but there was also an extra piece on the ends of the long arms of chromosome 1. While it makes little difference to an individual that his chromosome abnormality can now be accurately described, it is of extreme importance, should a pregnancy ensue, that the fetal chromosomes can be accurately identified. In this case, the 14q- chromosome can be identified by orcein staining but the lq + cannot, so a fetus with the lq+ chromosome and a normal number 14 (trisomic for part of the long aims of number 14) would have an apparently nbrmal karyotype; but the use of a banding technique would allow a correct prediction of the child's normality. Lindsten (Lindsten et al. 1973) investigated a family with two mentally-retarded Fig 2 X- and Y-bearing spermatozoa, the latter with sons both of whom had an abnormally long a Ybody
3
Fig 4 Karyotype offemale 46, XX, t (1:14) ASG technique
G-group chromosome, but their phenotypicallynormal father also had the same chromosome, and no other abnormal chromosome could be detected. Fluorescence studies, however, revealed that a small piece of the distal end of chromosome 1 was missing - so small that on this the longest chromosome its loss made no observable difference, but when translocated on to a very small chromosome - in fact the 22- it made an appreciable difference to its size. When the mother became pregnant again, antenatal diagnosis revealed that the fetus had the balanced translocation, like the father, and a healthy child was later born. One type of translocation in which definite identification of the involved chromosomes is important is the Robertsonian translocation, which is found in 1/1000 individuals. These are translocations involving chromosomes which have the centromere very near the chromosome end so that the short arms are very short. The translocation involves the loss of the short arms of two such chromosomes followed by a joining of the centromeres to produce one chromosome made (up of the long arms of the two original chromosomes. The loss of the short arm material seems relatively unimportant; the majority of
Section ofComparative Medicine
35
cases have no phenotypic abnormalities but there is the risk of producing gametes with the unbalanced translocation which can lead to recurrent abortion or the birth of abnormal children. But the risks of such events depend on the chromosomes involved and so it is important to identify the chromosomes before counselling is given. A 13/14 translocation could theoretically lead to a child with trisomy 13 or trisomy 14; but the latter is not found in live born babies and the risk of a trisomy 13 child appears to be only about 1 in 160. But if the translocation is between two number 13 chromosomes (superficially identical by conventional staining), then any offspring will have either trisomy 13 or monosomy 13 since the parent with the translocation can only pass on either both 13s or no 13 to the gamete. Monosomy 13 is incompatible with fetal development and children with trisomy 13 are either aborted, stillborn or die shortly after birth. Similarly a 14/21 translocation carrier has a definite risk of having a child with Down's syndrome (trisomy 21); the risk is 1 in 10 for a female carrier and 1 in 50 for a male. But for the 15/22 translocation, while there is a risk of recurrent abortion, there is apparently a negligible risk of an abnormal liveborn child. For a female with a 21/22 translocation the risk of having a trisomy 21 child would seem to be about 1 in 16, but the available data are too few to estimate the risk for a male carrier. A 21/21 translocation carrier can, however, have no normal children; all conceptions will result in just abortion or birth of a mongol child. Furbetta et al. (1973) have published the case of a woman who had twelve pregnancies - 4 spontaneous abortions and 8 mongol children - before her chromosomes were examined and she was found to be a 21/21 translocation carrier.
Fig S Polymorphisms of C-bands, chromosomes 1, 9, 16
36 Proc. roy. Soc. Med. Volume 69 January1976
4
There is another use of the fluorescence technique in antenatal diagnosis. If the karyotype of the amniotic fluid cells is female then it is necessary to exclude the possibility that they are maternal and not fetal cells; and it is important to check that no error in identification has been made. We analyse the fluorescence polymorphisms of both the mother's chromosomes and those obtained from the amniotic fluid cells to check that the polymorphisms are not identical and that they are compatible with a mother-child relationship.
were found. It may be possible eventually to distinguish karyologically different subgroups of tumours; for instance, subcutaneous sarcomas in rats which are induced by different oncogenic agents cannot be distinguished histopathologically, but they can be distinguished by their karyotypes. Rowley (1973) has proposed a similar model for the karyological changes found in the haematological disorders in man: that perhaps one etiologic agent is involved when the karyotype shows an additional number 8 chromosome, while another agent is associated with a different karyotype, even though both etiologic agents are capable of producing the same spectrum of clinical diseases. This may well not be the whole answer, but certainly the accurate identification of the karyological changes is a prerequisite for an understanding of their significance.
Malignant Disease Leukemia and myeloproliferative disorders: Acquired chromosome abnormalities are frequently found in such diseases, but whether they are the cause or effect of the disorder is unknown. The best-known example is the Philadelphia chromosome found in the proliferating marrow cells of patients with chronic myeloid leukaemia (CML). The. presence of the Ph' chromosome is to a large extent diagnostic and those cases without it have a less good prognosis. The Ph' chromosome has been known for many years and was thought to be a simple deletion of a G-group chromosome, but we now know that it is in fact a translocation of part of a 22, usually on to a number 9 chromosome (Rowley 1973). On entering blast crisis, CML patients may acquire an isochromosome 17 and in general as the disease progresses other varied karyological changes occur. Many patients with myelopr6liferative diseases show karyotypic abnormalities involving C group chromosomes in their marrow cells. A number of these abnormalities have now been identified and they have been found to show a considerably less random pattern than had been thought. Out of 50 patients with various haematological disorders and C group abnormalities, 7 had lost a number 7 chromosome, 28 had gained a number 8 and 6 had gained a number 9 chromosome (Rowley 1975). As yet, the significance of these and other more individual changes is a matter of conjecture, but now that the changes can be accurately classified, we hope that they may identify subgroups within the disorders, indicate the progression of the disease and suggest suitable therapy.
Solid tumours: Solid tumours also show chromosome changes, with chromosome evolution during tumour development being correlated with significant functional properties of the tumour. Granberg (1971), working on cervical tumours, showed that the normal diploid karyotype disappeared with the development of invasiveness. In 32 cases of carcinoma in situ there were 64 normal cells out of 91; in 5 cases of microinvasion, only 1 out of 20 cells was normal, and in 10 invasive carcinomas no normal cells
Gene Mapping This is the process of discovering the positions of the genes on the chromosomes. At first sight this may seem somewhat academic but there are good reasons for mapping the human genome. First, a knowledge of the location of genes is necessary for understanding gene interaction, especially during development, and for investigating the control mechanisms which turn genes on and off - and this information is potentially very important in medicine. Then, mapping can reveal genetic heterogeneity within syndromes ofphenotypic similarity, such as elliptocytosis; one form of the disease is caused by a gene which, on mapping studies, is seen to be close to the RhesPs gene, but another form maps quite separately from Rhesus (Morton 1956). Once this had been discovered it could be seen that the symptoms of the two forms, although similar, were in some respects distinct. When genetic heterogeneity has been detected, there can be greater accuracy of description of the syndromes, with a more reliable prognosis and, one hopes, more effective treatment. Another advantage of detailed knowledge of the human genome lies in genetic counselling, especially in those diseases that cannot be diagnosed in utero, or which are late in onset. It may be possible to tell indirectly if a fetus or child carries a particular deleterious gene by examining for the presence of genes close to that in question. Obviously this is not nearly so good as testing directly for the presence of the particular gene, but there are, and probably always will be, cases where this is not possible. So far this type of indirect diagnosis has not been successfully carried out, but attempts have been made to discover whether individuals are carrying the dystrophia myotonica gene by examining their secretor status (Schrott et al. 1973).
5
Section of Comparative Medicine
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Family studies: For many years gene mapping has involved family studies, which in general give information as to which genes are linked - that is, close together on the same chromosome so that they pass together from parent to child. Only now are we beginning to identify which chromosomes these genes are on; previously, the only chromosomes which could be followed through families together with the genes were the rare variant or abnormal chromosomes. But now with the fluorescence and C-band techniques we have 10 pairs of chromosomes which show polymorphisms and the extremes of these can easily be followed through a family (see Fig 6), together with all the easily tested gene markers such as blood group antigens and any disease genes, e.g. thalassmmia. Essentially one looks for some sort of pattern in the family: for instance, all the family members of blood group A might have the chromosome number 13 with intensely bright satellites and also have thalassemia. This is of course an oversimplification, but it gives the general idea. Obviously one needs to collect a great deal of data frQm many families, but already some results are beginning to emerge. Our own work indicates that the gene for esterase D may be on chromosome 13 and that for adenyl kinase on chromosome 15but these are by no means definite assignments.
Somatic cell genetics: This other technique of mapping involves cell culture, and is based upon the discovery that cells from either the same or different species can be made to fuse together so that both sets of chromosomes are within a single cell. If the original cells are from different species then the chromosomes from one of them are selectively lost during the growth of the culture. Conveniently, in man/mouse and man/Chinese hamster hybrids it is the human chromosomes which are lost. One can continually test these cultures for the presence or absence of various human enzymes and chromosomes. If an enzyme and a chromosome are always lost together or retained together then presumptively the gene for that enzyme is located on that chromosome. In this way we can gradually discover which genes are on which chromosomes; at the moment, between the two techniques, about 40 genes have been assigned to their autosomes and 60 to the X chromosome. It is also possible by the use of translocations, either present in the original cell line or spontaneously occurring during culture, to say whereabouts on a chromosome a particular gene is located since translocations split chromosomes into two pieces which are then lost or retained independently of each other. Finally, the use of the fluorescence technique has demonstrated how in researrch one may discover something quite different from what was expected. As explained before, by the fluorescence
Fig 6 Inheritance offluorescence polymorphisms
technique the Y chromosome can be seen as a bright Y body even in sperm, so that X- and Ybearing sperm can be identified reliably. It would be of great agricultural importance, and useful in medicine, if the X- and Y-bearing sperm could be separated and offspring of the required sex be produced by artificial insemination. The fluorescence technique offers a quick and reliable method of assessing the efficiency of possible methods of separating sperm. Many techniques have been tried but so far none has been found to be successful, but a technique claiming to have achieved separation using passage through bovine serum albumin has been reported by Ericsson (Ericsson et al. 1973). We repeated his experiments and, although we failed to confirm his separation results, in our final isolate the sperm was of exceptionally high motility and morphologically very homogeneous compared with the
Proc. roy. Soc. Med. Volume 69 January 1976
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original ejaculate. We think that an extension of this technique may be of value in the treatment of some types of male subfertility. The problem of separating X- and Y-bearing sperm has apparently not yet been solved but doubtless, when it is, the method will utilize the fluorescence technique.
normal breeding procedures, to improve profitability of our cattle industry. The amylase I locus in cattle is a possible example of such a genetic marker. It has been established that there is an apparent excess of heterozygotes at this locus in populations -of dairy cows, whereas no excess exists in dairy calves, or beef animals. The excess within a herd is related to the mean herd yield, and recent findings indicate some association between the yield of an individual, and its amylase genotype. However, the excess appears to be established in heifer populations which have not yet been milked. If farmers are selecting heterozygote animals as herd replacements, serum amylase could be, to some extent, a predictor of the economic worth of a dairy calf.
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REFERENCES Arrighi F E & Hsu T C (1971) Cytogenetics 10, 81 Buckton K E (1973) In: Nobel Symposia: Medicine and Natural Sciences, Chromosome Identification. Ed. T Caspersson & L Zech. Nobel Foundation, Stockholm; p 196 Caspersson T, Lomakka G & Zech L (1971) Hereditas 67,89 Dutrillaux B & Lejeune J (1971) Comptes Rendus de l'Academie des Sciences 272, 2638 Eriesson R J, Langerin C N & Nishino M (1973) Nature (London) 246, 421 Ford C E, Jacobs P A & Lajtha L G (1958) Nature (London) 181, 1565 Furbetta M, Faloroni A, Antiguain D & Cao A (1973) Journal of Medical Genetics 10, 371 George K P (1970) Nature (London) 226, 80 Granberg I (1971) Hereditas 68, 165 Lindsten J, Holmberg M, Hulton M, Jonasson J, Licznerski G & Therkelsen A J (1973) In: Nobel Symposia: Medicine and Natural Sciences, Chromosome Identification. Ed. T Caspersson & L Zech. Nobel Foundation, Stockholm; p 230 Morton NE (1956) American Journal of Human Genetics 8, 80 O'Riordan M L, Robinson J A, Bucktod K E & Evans H J (1971) Nature (London) 230, 167 Pearson P L, Bobrow M & Vosa C G (1970) Nature (London) 226, 78 Robinson J A (1971) Annals of Human Genetics (London) 35, 61
Rowley J (1973) Nature (London) 243, 290 (1975) Proceedings of the National Academy of Sciences ofthe USA, 72, 152 Schrott H G, Karp L & Omenn G S (1973) Clinical Genetics 4, 38 Sumner A T, Evans H J & Buckland R (1971) Nature New Biology 232, 31 Sumner A T, Robinson J A & Evans H J (1971) Nature New Biology 229, 231 Tijo J M & Puck T T (1958) Proceedings of the National Academy of Sciences of the USA 44, 1229 Zech L (1969) Experimental Cell Research 58, 463
Dr J S Bradley (ARC Animal Breeding Research Organization, West Mains Road, Edinburgh, EH9 3JQ)
Genetic Aspects of Disease in Cattle [Abstract] Disease has a major effect on the longevity and profitability of our dairy cattle. There is evidence which indicates that resistance to many disease conditions is, in part, genetically controlled. The widespread use of artificial insemination in cattle breeding had made the long-term genetic improvement of health in cattle stock a possibility. The question remains, however, whether such breeding programmes are economically viable, since they must compete with the genetic improvement of production. Specific genes have been found in experimental and domestic animals, and in man, which directly affect viability, and resistance to disease. It may be possible to utilize such genes, as an adjunct to
REFERENCE Spooner R L, Bradley J S & Young G B (1975) Veterinary Record 97, 125-130
Dr C 0 Carter (MRC Clinical Genetics Unit, Institute of Child Health, 30 Guilford Street, London WCJ)
Genetics of Common Congenital Malformations in Man
Malformations Determined by Single Genes A great variety of monogenically determined single malformations or syndromes of multiple malformations occur in man, but none of these is common in the sense of having a birth frequency of 1 in 1000 total births or more. This is to be expected, since if the malformation is at all severe, then the birth frequency will, as the result of the balance between fresh mutation and selection, be at a level equivalent to some small multiple of the mutation rate. Gene mutations mostly occur at a frequency of 1 in 5000 or less. Monogenic inheritance is readily recognized by the simple mendelian family patterns it gives dominant, recessive or X-linked. Chromosome Anomalies It has recently been discovered that chromosomal anomalies are relatively frequent at conception. Some 400% of first trimester spontaneous miscarriages are associated with chromosomal anomalies, and-if one accepts that some 15 % of known conceptions miscarry, then about 6 %, or 1 in 16, of zygotes have a major chromosomal anomaly causing miscarriage. The three most common chromosomal anomalies found in spontaneous miscarriages are triploidy (69 chromosomes instead of 46), the 45 XO genotype, which is characteristic of Turner's syndrome, and
