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Postgrad Med J: first published as 10.1136/pgmj.40.462.193 on 1 April 1964. Downloaded from POSTGRAD. MED. J. (i 964), 40, 193

ELEMENTS OF ABNORMALITIES N. ISLAM, B.Sc., M.B.B.S. Department of Pathology, Dudley Road Hospital, Birmingham

OVER the past 6o years the science of cell INTERPHASE PROPHASE METAPHASE has made profound contributions to an understanding of the hereditary process. It was only seven years ago, however, that geneticists were able to establish the number of n fl in the human cell. These are the structures in the cell nucleus that encode the plan. To look through the microscope at the chromo- somes of man is to see the very stuff that human life is made of. The study of chromosomes can INTERPHASE be said to have been started by Walter S. Sutton TELOPHASE at Columbia University in 1903 and subsequent investigations by Thomas Hunt Morgan and A. H. Sturtevant showed that the units of heredity, ANAPHASE the ' factor' of Gregor Mendel, must be arranged by copyright. in linear order on the chromosomes. These findings stimulated study of the KeN> 0 chromosomes in various animals and plants. It was soon discovered that whereas the total number of chromosomes varies in different organisms, from two in some of worm CENTRIOLES to 300 in certain protozoa, the number in the cell is constant in any species. It was a relatively simple matter to count http://pmj.bmj.com/ the chromosomes of FIG. I.-Diagrammatic representation of in a organisms, notably the fruitfly, in which they hypothetical cell with four chromosomes. are large and few. Where they are small and numerous, however, the task was burdensome and different results were reported by different so that each daughter cell has the same number of investigators. For example, J. H. Tjio and chromosomes as the mother cell (Fig. i). On Albert Levan (1956) of the Institute of Genetics the other hand the germ cell divides in a two-step at Lund found that in the human cell the number process called , the chromosomes being on September 26, 2021 by guest. Protected of chromosomes was 46 and not 48 as had been duplicated only once before the first division. previously believed. In the second division the two sets of chromosomes pull apart or segregate to produce or ova Chromosome Structure (gametes) with only one set of chromosomes To understand the chromosome aberrations ('haploid '), in other words half the number of which occur in different conditions, we must chromosomes of the precursor cells (Fig. 2). have a basic knowledge of their normal structure The subsequent union of the gametes produces a and behaviour. Each cell, including the germ fertilized egg () with the full (' diploid') cells that give rise to and ova, bears two number of chromosomes. Each chromosome sets of chromosomes. The 'homologous' chromo- is a rod-shaped structure, split longitudinally somes of each set (with the exception of the into two which were destined to chromosomes which determine sex) are sirmiilar in become the daughter chromosomes of the two appearance and carry affecting the same new cells. The two chromatids are held together trait: one set is contributed by each parent. at a constricted region, the , which The 'somatic' or body cells divide by mitosis does not take up any stain and looks like a vacuole. Postgrad Med J: first published as 10.1136/pgmj.40.462.193 on 1 April 1964. Downloaded from I94 POSTGRADUATE MEDICAL JOURNAL April I964

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IN FIG. 3.-Photomicrograph of blood culture after treat- 69 ment with colchicine. the total female diploid length (sum of the absolute length and the length of the one ) arnd the arm ratio (ratio of the long arm to the length of the short arm) (Denver Nomerclature). t909 Techniques FIG. 2.-Diagrammatic representation of meiosis of Chromosomes assume their most visible form germ cell giving rise to 'haploid' in a hypothetical at the metaphase of mitosis, when they are ready

cell with four chromosomes. to separate. Colchicine has the unique propertyby copyright. of stopping mitosis at this stage and has been utilized in the study of chromosomes. The position of the centromere is constant for There are three chief sources of mitosis which each chromosome and according to its site is have been utilized so far: termed median, submedian or subterminal. Thus (i) Classical tissue culture methods, aiming it is an important identifying feature. at the production of a single layer of Human chromosomes can be arranged in cells growing rapidly on a flat glass order of decreasing size, which includes 22 pairs surface. This gives good results, but of autosomal homologous pairs (i to 22) and a is time-consuming and needs skill andhttp://pmj.bmj.com/ pair of sex chromosomes of equal length in the labour. female but of unequal length in the male (XX and (2) Marrow cell culture in tissue culture media. XY-Fig. 5). The longest chromosome is about These two methods are still of great value 7-8 microns in length and the shortest about but successive samples cannot be obtained without 1.5 microns. For identification they have been considerable discomfort to the patient, and in classified into groups according to their absolute long-term cultures it is sometimes difficult to lengths, their ' relative lengths ' in relation to in avoid alteration the chromosome complement. on September 26, 2021 by guest. Protected (3) Blood is the most convenient, tissue sample. Denver Nomenclature There is ordinarily no division in usual tissue culture media, but by adding Size and Centromere Idiogram No. in Dip- phytoagglutinin extract from French Group Position No. loid Cell. kidney beans, culture can be obtained (A) Large, median or satisfactorily. The red cells are preci- submedian -..I 3 6 pitated from a blood sample with the (B) Large, submedian .. 4- 6 6 extract added and the white cells in- (C) Medium, submedian 7-12 & X I 3 (male) or 14 (female) cubated in the remaining plasma with (D) Medium I-.... I3 5 6 added synthetic tissue culture medium. (E) Small i...... 6-i8 6 An unexplained burst of mitosis occurs (F) Smallest, median .. I9-20 4 (G) Small, subterminal 21 & 22 & Y 5 (male) or about the third day. This method not 4 (female) only avoids unnecessary skin biopsy or Total sternal puncture, but gives better results _____46 and is almost universally used at present. Postgrad Med J: first published as 10.1136/pgmj.40.462.193 on 1 April 1964. Downloaded from April [964 ISLAM: Elements of Chromosome Abnormalities I95

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S:. ff,S FIG. 4.-Photomicrograph of blood culture (white cell) 7 8 9 if 1 2 after treatment with colchicine and hypnotic saline. In the latter stages of all methods colchicine is added to stop mitosis at metaphase, when the chromosomes are ready for separation by 1i, I the mitotic apparatus and have assumed their most visible form. It also breaks up the spindle and allows the chromosomes to separate, instead 1 14 16 1717 of lying bunched together in the familiar star- shaped mass. This spreading process is helped by copyright. by making the cells swell (just before fixation) with hypotonic solutions, and by flattening them 00n1ii !i out on the slide either by judicious squashing or by air-drying. The cells are stained and then 1'9 20 2122 examined under the oil immersion lens (Fig. 3-4). FIG. 5.--Diagram showing absolute and relative lengths They may be analysed from a photographic of chromosomes with primarv and secondarv print at a suitable enlargement (x 3,000-4,000) constrictions (one from each pair has been shown). or from a camera lucida drawing. In order to exclude errors due to artefacts arising in these the largest of the smallest group of chromosomes http://pmj.bmj.com/ preparations, a number of mitotic cells (usually with subterminal . Unfortunately up to ioo) are analysed. The chromosomes the X chromosome is not so easily identified. are first arranged in order of decreasing size and It is generally the seventh largest chromosome, classified into groups according to the Denver but may prove to be difficult to tell apart from classification. By this method accurate identifica- the sixth and seventh pair of . In such tion of at least six pairs of autosomes (i, 2, 9, i6, a situation we rely very heavily on the nuclear sex

17 and i8) the and sometimes an diagnosis. on September 26, 2021 by guest. Protected additional four autosomal pairs (I9, 20, 2I and 22) can reasonably be made. For more specific Nuclear sex detection identification of the remaining pairs one must The sex chromatin body can be identified look for secondary constrictions and ' satellite' in 30-60% of cells in females. Vaginal smears formations. (Fig. 5) stained with aniline dyes or differential stains With the present degree of precision one can give the best results, though buccal smears, be sure of reliable identification of I4 of the 23 sections of skin and white blood cells can also be pairs at the most. A large proportion of the used. human chromosome abnormalities involve the There is now considerable evidence to support sex chromosomes, which means that it is most the view that the sex chromatin is formed from important to be able to identify the X and Y at least part of one of the two X chromosomes chromosomes accurately in somatic cells. The in the female cell. It is not present in cells which Y chromosomes present no difficulty, although carry only one X chromosome, as in XY (normal there is considerable variation in size in normal male) and XO (Turner's syndrome). In the individuals; they are readily distinguished as Triple X syndrome (XXX) two sex chromatin Postgrad Med J: first published as 10.1136/pgmj.40.462.193 on 1 April 1964. Downloaded from I96 POSTRGADUATE MEDICAL JOURNAI, April I964

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FIG. 6.-Diagrammatic representation of non-dis- junction by means of which a cell can come to have the wrong number of chromosomes (shown in the case of one homologous pair of chromosomes). xx3 XX o ZYOCZYOTE bodies are present. The number of sex chromatin FIG. 7.-Non-dis.iunction in meiosis. bodies increases with the number of X chromo- somes. These observations relate the sex chromatin The mechanism by which an individual cell directly to the constitution might come to have an extra chromosome or lose and thus make the nuclear sex diagnosis an one of its normal number is known as non- exceedingly important aid to accurate chromosome disjunction (Fig. 6). In these rare conditions analysis. two X chromosomes of a female germ cell failsby copyright. to ' disjoin ' in the first stage of meiosis. One of CHROMOSOME ABNORMALITIES the ova produced by the next division accordingly The chromosome abnormalities which can be receives both X chromosomes and the other recognised by present microscopic methods are no X chromosomes. The same may happen fairly gross. The number is smaller still when in the case of male germ cells and auto- we exclude lesions which are incompatible with somes. The most commonly affected survival. iS 2I. The ultimate cause of non-disjunction Simple variation in count is, of course, the remains to be established, although over- most easily identified anomaly. We can usually accumulation of some metabolic products inhttp://pmj.bmj.com/ recognise the particular chromosome which is the ovum of the older woman or the activation in excess or is missing. The number of such of a ' latent ' virus have been blamed. anomalies is finite in each species. A structural deformity of a chromosome can be identified Clinical manifestations only if it is relatively massive. We are able to i. Sex Chromosomes. (Fig. 7) distinguish loss of half a micron from the smallest (a) XXX syndrome ( X; superfemale). chromosomes (say 22) because this is nearly This occurs in one in iooo live female births on September 26, 2021 by guest. Protected half its long arm, but a much larger loss (say and in less than I% institutionalized mental from i) may pass unnoticed. cases. They are often fertile, producing normal offspring and are difficult to diagnose clinically. Aberration in number Vaginal and buccal mucosa show duplicated sex Loss or gain of the sex chromosomes seems chromatin bodies. Cases with four X chromosomes to be especially well tolerated. Sex chromosome occur, but much more rarely. The abnormality (a single X, XO in Turner's syndrome) is greater than with simple XXX. Buccal smear is the only form of monosomy yet recognised, shows the sex-chromatin body for each extra X and nearly all the cases with more than one extra chromosome-the total being one less than the chromosome have involved the sex chromosomes. number of X-chromosomes in each case. Loss, for instance, of one whole chromosome (b) XXY syndrome (Trisomy XY; Klinefelter's (monosomy) is always fatal in cases of autosomes. syndrome). This occurs once in about every Gain of an extra chromosome is damaging, but 400 live male births and is characterized in adults several varieties at least are compatible with by tallness, enlargement of the breasts, atrophy life and one even with reproduction. of the testicles and virtual non-production of Postgrad Med J: first published as 10.1136/pgmj.40.462.193 on 1 April 1964. Downloaded from April I964 ISLAM: Elements of Chromosome Abnormalities 197 sperms. It may be associated with mental defect (b) Trisomy of Group E. The next most and constitutes about I% of institutionalized frequent and best established entity is due to an male mental cases. Such patients are sterile, extra chromosome in the i6-i8 group, which is and testicular biopsy shows tubular atrophy probably chromosome i8. The syndrome includes and Leydig cell hyperplasia. The sex chromatin mental defect, low-set malformed ears, micro- body is present in buccal mucosa. Mosaicism gnathia, flexion deformities of the fingers and may occur (see below). congenital heart disease (V.S.D.). The syndrome (c) XO syndrome (Monosomy X; Turner's can be diagnosed clinically by experienced syndrome). These patients are female in clinicians. appearance, although they lack many secondary (c) Trisomy ofGroup D. An extra chromosome sexual characteristics. They may also display in the I3-15 group has been found on several congenital defects, such as unusually short occasions. The syndrome includes eye defects stature, a ' webbed' neck, malformation of the ( or colobomata), hare-lip, cleft aorta, deafness and mental deficiency. This is palate, polydactyly and congenital heart disease. the only monosomy yet recognised. Clinical Trisomy of Grousp F (19-20) and of 22 have diagnosis is usually easy, at least after puberty. been reported, but none have been conclusively Sex chromatin body is absent in buccal mucosa, differentiated as regards their individual clinical but mosaics with intermediate patterns may status. Autosomal trisomy, however, may be occur. entirely asymptomatic. Chromosomal defects (d) A final possibility would be fertilization involving large autosomes (Group A-B) are of an ovum with no X chromosome by a extremely rare. Y-bearing sperm. This would produce a YO zygote, but such a chromosome complex is Alterations in structure apparently incompatible with life-at least no The number of aberrations in the structure of such individuals have been recognised. chromosomes is infinite. The two principle types are deletions and translocations. In deletions 2. Autosomes: part of a chromosome breaks off and is lost. by copyright. (a) Trisomy 2I (Mongolism; Down's syndrome). Such abnormalities, particularly if they involve They occur once in 6oo live births and are sizeable amounts of the genetic material, could congenital mental defectives. The cause is now be expected to be lethal in many cases, and demonstrated to be due to an extra chromosome- large deletions are in fact not commonly found. either 21 or 22, though conventionally taken to In translocation, breakage is followed by be 2I-the total chromosome number being 47. relocation of the fragments. A reciprocal Statistically the risk of bearing a mongoloid translocation involves breaks in two non- child rises steeply with the age of the mother. homologous chromosomes and thereafter a mutual A very few mongols have the normal number of exchange of the fragments. Depending on how http://pmj.bmj.com/ 46 chromosomes and sometimes 45 chromosomes, the chromosomes behave during meiosis, some but these are the ' translocation ' cases, in which gametes will be found with a normal, although the bulk of the extra iS still displaced, content, i.e. a balanced transloca- present but attached to another chromosome tion. But translocation may also give rise to (see below). The important practical application gametes that have deficiencies of certain genes in relation to Down's syndrome is in predicting and duplications of others. Some offspring of the chances of the parents having a second gametes that carry translocations may, therefore, on September 26, 2021 by guest. Protected similarly affected child. It has recently been be normal and some may not. The germ cells shown that this risk is higher than in the general of an apparently normal person who bears a population, and this is particularly the case with balanced translocation may undergo meiosis in young mothers, for whom the general risk is low. such a way that occasional members of the Parents with detectable chromosomal abnorma- family acquire an extra 2I and so appear as lities also have an increased risk of having an mongols, thus giving rise to the most important affected child. The most common such abnor- variety (even if rare) of familial mongolism. mality appears to be the possession by the mother of a translocated chromosome comprising the Clinical manifestation major part of IS and 21. Women with such an (i) The first recognizable translocation was abnormality have a risk as high as i in 3 of having found in I959 in a mentally deficient boy with a child with the syndrome at any individual associated abnormalities of the spine. The cells pregnancy. Men with this translocation appear had only 45 chromosomes including one that rarely to have affected children, though Down's was longer than its homologue. It was suggested syndrome may appear in their daughters' children. that part of the missing chromosome had been Postgrad Med J: first published as 10.1136/pgmj.40.462.193 on 1 April 1964. Downloaded from I98 POSTGRADUATE MEDICAL JOURNAL AAril I964 relocated, thus explaining the extra length of can go awry at any time and result the abnormal chromosome, the remaining frag- in cells with the wrong complement of chromo- ment being eliminated. somes. Presumably these cells are usually at a (2) Recent reports that some patients suffering disadvantage and are eliminated. Nevertheless, from Down's syndrome have a normal number there may be a small number of aberrant cells of 46 and others 45 chromosomes may be explained in every normal individual. by translocation. In these cases the extra material, usually present as an extra ' free' chromosome Comments (trisomy of chromosome 2I) is translocated to The identification of constitutional defects another chromosome or a chromosome has been with chromosomal abnormalities is perhaps of deleted. The disease would seem, therefore, more direct interest to medicine than to genetics. to be related not so much to the number of Many fundamental genetic studies of the cells chromosomes in the cells as to the extra chromo- of other organisms with fewer and larger chromo- somal material. somes will continue to serve investigators better (3) Patients with chrdnic myelogenous than human cells, but the questions relating leukaimia have what appears to be a unique specifically to man can only be settled by the chromosomal aberration: of a small study of his cells. part of one of the smallest chromosomes tenta- The present methods of studying chromosomes tively identified as 21, the same as that associated are crude. We do not yet know the structure with Down's syndrome (Philadelphia chromo- of chromosomes and there is need for higher some). The association of abnormal white cell magnification in microscopic studies. There is formation with chromosome 21 is strengthened no known way of producing meiosis artificially by the fact that acute leukamia is many times which might help with pairing-off problems. more common in patients with Down's syndrome Ovarian meiosis is hardly ever accessible in man than in the general population. Malignant and, though testicular biopsy is a simple enough cells frequently have abnormal chromosomes procedure, a large proportion of the most interest-by copyright. of various types. So far the abnormalities in ing examples have disorders of cancer cells have given little help in diagnosis which make it valueless. Even given an active except in chronic myeloid leukxmia. In this testicular biopsy, the technical problems of case the presence of 'ph-chromosome' is an obtaining a preparation comparable to modern almost constant feature in cultures of leucocytes mitotic culture have not yet been overcome. from the peripheral blood and marrow. So this chromosome anomaly of the myeloid series Present techniques are unable to demonstrate is a useful guide for and the course of the genetic lesions of traditional Mendel-inherited diagnosis disorders like hiemophilia or phenylketonuria. treatment in this condition. http://pmj.bmj.com/ These probably depend on a disorder of the desoxyribonucleic acid chain, perhaps the mis- Mosaicism placement of a single base in the chain. Auto- The term mosaicism, in cytogenic usage, radiography of human cells after they have been describes a condition in which a substantial supplied with radioactive nucleic acids shows minority of cells differ from the majority in their that different segments of the chromosomes chromosomal content. In man the condition replicate at different times. By studying the was first described in Klinefelter's syndrome pattern and significance of the sequence of re- on September 26, 2021 by guest. Protected and it has been reported in several other condi- plication we may one day be able to explain the tions involving the sex chromosomes, and also alteration in the DNA chain which directs the in mongolism. It was anticipated that her- nature and activity of cells. Long-term tissue- maphrodites might turn out to be 'mosaics', cultures of cells, in which the chromosome some of whose cells are XX and some XY. In complement has been altered, could be studied nearly all instances, however, examination of for the biochemical consequence of such changes. the cells of hermaphrodites has disclosed 46 In this way it might be possible to determine chromosomes and the XX constitution character- which chromosome directs the synthesis of istic of a normal female. which enzyme and so obtain a clue to the main It is none-the-less clear that human mosaics defects in inherited diseases. do occur; they are the products of non-disjunc- tion, not in meiosis of parent germ cells but in I am grateful to Dr. A. Paton, Consultant Physician, mitosis of the somatic cells at some in Dudley Road Hospital, for his guidance, encouragement, point and valuable suggestions in preparing this paper. embryonic development soon after fertilization. I would also like to give thanks to Mrs. M. K. Mason Mosaicism is after all a statistical concept, since for her secretarial assistance. Postgrad Med J: first published as 10.1136/pgmj.40.462.193 on 1 April 1964. Downloaded from April I964 ISLAM: Elements of Chromosome Abnormalities FURTHER READING ATKINS, L. and ROSENTHAL, M. K. (I96I): Multiple Congenital Abnormalities Associated with Chromosomal Trisomy, New Engl. 3r. Med., 265, 3 I4. BAIKIE, A. G., BUCKTON, K. E., COURT BROWN, W. M. and HARDEN, D. G. (I96I): Two Cases of L!uk2emia and a Case of Sex Chromosome Abnormality in the Sibling, Lancet, ii, IOO3. BEARON, A. G. and GERMAN, III, J. L. (I96I): Chromosomes and Disease, Scientific Amer., Nov., 66. C.ARR, D. H. (I963): Chromosomal Abnormalities and Their Relation to Disease, Canad. med. Ass. X., 88, 456. EGGEN, R. R. (I963): Review of Recent Advances in a New Field of Clinical Pathology, Amer. Y. clin. Path., 39, 3. FERGUSON-SMITH, M. A. (I962): Chromosome Abnormalities, Proc. roy. Soc. Med., 55, 471. GUARD, H. R. (1959): A New Technique for Differential Staining of the Sex Chromatin and the Determination of its Incidence in Exfoliated Vaginal Epithelial Cells, Amer. Y. clin. Path., 32, I45. HERNIDON, C. N. Basic Contributions to Medicine by Research in Genetics, 3'. Amer. med. Ass., 177, 695. LENNOX, B. (I96I): Chromosomes for Beginners, Lancet, i, 1046. SPRIGGS, A. I., BODDINGTON, M. M. and CLARK>, C. M. Chromosomes of Human Cancer Cells, Brit. med. Y., ii, 1431 by copyright. http://pmj.bmj.com/ on September 26, 2021 by guest. Protected