The techniques of human cytogenetics

The techniques of human cytogenetics MALCOLM Glasgow,

A.

FERGUSON-SMITH,

M.B.,

CH.B.

Scotland

T E c H N I c A L advances in the study of human chromosomes have led in recent years to the recognition of an increasingly large number of developmental disorders caused by gross chromosome aberrations. These aberrations are detected in preparations of dividing cells, using standard cytological procedures and the oil immersion lens of an ordinary laboratory microscope. The means of diagnosing these disorders are thus available to all clinical laboratories, and to anyone willing to spend time in gaining the experience necessary to avoid the pitfalls involved in chromosome analysis. The purpose of this article is to describe some of the techniques of human cytogenetics and give an account of the possibilities and limitations in this new branch of medical genetics. As those techniques concerned with cell culture and the preparation of mitotic cells are mastered, relatively simple and quickly greater attention will be given to the more complicated problems of chromosome identification and karyotype analysis.

With the advent of human cell culture, an ideal type of material became available and it was soon found that the cytological techniques gained from many years of experience with plant and animal chromosomescould also be applied to human cells dividing in vitro. One of the principal steps in the preparation of chromosomesfor examination is the use of colchicine to cause metaphase arrest. This drug, when added to a rapidly growing culture, interferes with the formation of the mitotic spindle so that dividing cells are accumulated at metaphase-the most convenient stage of cell division for the examination of somatic chromosomes.If the cells are then exposedto the osmotic effects of a hypotonic solution they become distended and the chromosomes separate. The cells are fixed at this stage and squash or smear preparations are made for microscopic examination. These principles may be applied to any tissue rapidly dividing in vitro. Fibroblast cultures (obtained for exampIe, from skin biopsy specimens),1 short-term cultures of bone marrow,2 and short-term cultures of lymphocytes from peripheral blood3 have all been extensively used for chromosome analysis in man. The peripheral blood culture technique, using phytohemagglutinin to initiate mitosis in lymphocytes, is the simplest and most reliable method for routine chromosomestudiesin patients. It seemsappropriate to give a brief account of this method here. (Most laboratories have modified the minutiae of the technique to suit local conditions; the method that follows is the one used at the Glasgow University Department of Gcnetics)

Cell culture methods for the study of somatic chromosomes

The first essential in the analysis of a patient’s chromosomeconstitution is to have an adequate sample of actively dividing ceils. The cytologists who first attempted to study human chromosomesfound that tumor and testis materials were the only tissueswhich provided sufficient numbers of dividing cells. But both are unsuitable for patient study.

From the Department of Genetics, The University, Glasgow.

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1036

Blood

Ferguson-Smith

culture

methods

Ten milliliters of peripheral blood is drawn into a sterile syringe, transferred to a sterile tube containing 100 I.U. of heparin and mixed thoroughly. The specimen is centrifuged gently for 5 minutes at approximately 300 r.p.m., and the supernatant plasma containing white cells is transferred to a sterile container. A white blood cell count is made from a sample of the plasma, so that the correct amount of Waymouth’s tissue culture medium (or equivalent medium) without serum4 may be added to make a final concentration of 1,000 to 2,000 cells per cubic millimeter of cell culture. Four to 5 ml. of this is transferred to each of several 2 ounce culture flasks, and 0.5 ml. of phytohemagglutinin added to each flask. The cultures are then refrigerated (+4’ C.) overnight before incubation at 37’ C. At 72 hours incubation, 0.25 ml. of a 80 pg per milliliter solution of Colcemide is added to each flask for a further 2% hours incubation. (At this point aseptic techniques are no longer essential.) The cell cultures are transferred to centrifuge tubes and spun at 300 to 400 r.p.m. for 5 minutes. The supernatant is replaced by 10 ml. of a hypotonic 1.12 per cent aqueous solution of sodium citrate at 3J0 C., incubated for 5 minutes, and centrifuged at 300 to 400 r.p.m. for 5 minutes. Ten milliliters of freshly prepared cold 3: 1 ethanol-glacial acetic acid is added to the cell deposit resuspended in a few drops of the supernatant, and the cells are kept in this fixative for 30 minutes at +4-O C. Thereafter, the cells are centrifuged at 600 r.p.m. and resuspended in several drops of fresh fixative (enough to make a milky suspension) . One drop of this suspension is dropped onto a clean wet microscope slide from a height of 6 inches, drained onto filter paper, and air-dried rapidly over a flame. The slide is stained for 1 hour in 2 per cent natural orcein in 65 per cent acetic acid, dehydrated and mounted in the usual manner. Where only a very small sample of blood

Fig. la, Mitotic cell from peripheral 6, Karyotype analysis of a, ?n = 46(XY).

leukocyte

is available (e.g., from newborn irlfiwts : satisfactory results” may be obtained by adding 2 to 3 drops of heparinized whole blood to 5 ml. of tissue culture medillm containing 10 per cent fetal calf serum and 0.25 ml. of phytohemagglutinin. After incubating for 72 hours, the procedure follows that outlined above. Fibroblast, or other solid cell culture material may be prepared by the same technique after treating the active culture with Colcemide (for 4 to 6 hours) usually 2 to 3 days after subculture. In this case the culture is made into a cell suspension by trypsinization. Skin biopsy is a convenient source of material for fibroblast cultures in patients not undergoing surgical procedures, and this may be very simply and painlessly performed without local anesthesia by means of a high speed punch drill. 37 Direct bone marrow6 and lymph node preparations? may be made by incubating the fresh specimens (suitably cut into small 1 to 2 mm. pieces and trypsinized) for 1 to 3 hours in the presence of Colcemide. Chromosome counts. With this type of air-dried preparation, suitably spread cells at metaphase are visible using the 10 times magnification scanning objective of the ordinary light microscope. The chromosome counts are made with a 90 or 100 times magnification oil immersion lens. At this magnification, the 46 chromosomes in a normal cell (Fig. 1) are seen to be rodshaped structures split longitudinally into two chromatids which are destined to become the chromosomes of two daughter cells. The two chromatids of each chromosome lie side-by-side, held together at a constricted region, the centromere (or primary constriction), which is the site of the spindle fiber attachment. The centromere is constant in position for each chromosome and, for descriptive purposes, conveniently divides the chromosome into long and short arms. The total length of the chromosome and the ratio of the lengths of the short arm to the

culture.

(Aceto-orcein;

~3,000

approx.)

Techniques

Fig. 1. For legend

see opposite

page.

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1038

Ferguson-Smith

long arm are important individual characteristics for each chromosome. Not every metaphase cell in these air-dried preparations is technically suitable for analysis, and the accuracy of the method depends upon the use of rigid criteria for selection. Only well-spread intact cells without undue chromosome overlap or other distortion should be accepted for counting. All cells initially accepted for counting under the scanning objective, are counted under the oil immersion lens and recorded; it need hardly be emphasized that a cell should never be excluded from the results after being counted, as this introduces observer bias. Similarly, the criteria for selection of ceIIs for counting should in no way be compromised because a specimen has few mitoses or is otherwise of poor technical quality. The most suitable cells for accurate chromosome identification are those in early metaphase which neither show the undue contraction and separation of chromatids characteristic of prolonged colchicine treatment, nor the marked variation in relative condensation found in prophase chromosomes. Using these criteria in the chromosomal analysis of peripheral blood cultures it is becoming apparent that the frequency of hyperdiploid counts (i.e., a chromosome count higher than 46, but excluding tetraploid cells) in normal persons is much lower than is often stated-and rarely over 2 per cent. Experience shows that in normal controls, hypodiploid counts, which are usually less than 5 per cent of the total counts, can usually be attributed to artifacts or errors in counting. There is evidence of only one factor which might increase the frequency of nonmodal counts in normal persons. This factor is the advancing age of the subject.* Commonly, about 1 to 2 per cent of all mitotic cells found in peripheral blood cultures are tetraploid, but a much higher frequency is usual in almost all other types of cell culture. Many, if not all, of these tetraploid cells arise in culture, as is demonstrated by the not uncommon finding of cells showing endomitotic reduplication (Fig. 2). These are originahy diploid ceils whose chro-

mosomes have divided twice although the cell has only divided once, so that at metaphase the 92 chromosomes are seen to be arranged in 46 pairs. Extreme degrees of polyploidy (e.g., 16n) is a normal finding in direct bone marrow preparations, such cells presumably representing dividing megakaryocytes. In routine chromosome analysis it is usually sufficient to determine the chromosome number in 50 cells, provided the criteria outlined above are consistently employed. Full karyotype analysis should be made in at least five of the modal cells, and in all nonmodal cells to insure that chromosome mosaicism is not missed. In patients whose initial chromosome counts suggest mosaicism for two or more cell lines with different chromosome constitutions, a larger number of counts should be made, preferably from cultures of two or more different tissues.9

Techniques for karyotype chromosome identification.

analysis and

Chromosome counts by themselves merely demonstrate whether or not a person has an abnormal number of chromosomes (aneuploidy). A structural chromosome aberration may exist in association with either a normal or abnormal chromosome count and, thus, will only be detected by a careful examination of the entire chromosome complement (the karyotype) of the cell. With experience the karyotype may be analyzed to a certain extent by direct microscopy, and with the criteria to be described below, eight pairs of autosomes (1, 2, 3, 6, 9, 16, 17, and 18) and the Y chromosome can be identified by this means, the remaining chromsomes being assigned to one of several small groups (Table I). A more complete analysis is possible either by drawing the karyotype with the aid of a camera lucida, or by photomicrography. In most hands the latter method is satisfactory and more reliable. As there are few accounts of photomicrographic techniques for chromosome analysis, a simple system will be described here: The limiting factor in photomicrography is the resolving power of the optical system

Volume Il;umber

Fig.

Techniques

90 7, part 2

2. Endomitotic

reduplication

(see text).

(Aceto-orcein;

and not the degree of resolution of the negative, so that the advantages of modern 35 mm. negative material far outweigh the advantages of plates and sheet film. Several very excellent automatic 35 mm. microscope cameras are available, either built into the microscope or readily attached to it, but when a good research microscope is already avaiiable it is often more convenient and economical to use a 35 mm. single lens reflex camera with a microscope adaptor. With the latter a sensitive light meter is also required, with a sensing device which can either be placed in the eye piece of the microscope or at a fixed point in the camera-microscope adaptor. Unlessthe camera has a focal-plane shutter with slow speedsof !,$2to 2 seconds, a separate shutter device must be inserted in the optical axis, usually between the microscope and camera, or in front of the light source. The correct exposure time is determined by taking a series of photographs at different exposure times with the light meter reading constant. Once the best shutter speed has been chosen all exposures are made at that speed by regulating the intensity of the microscope light so that the light meter gives the standard reading. Thus a11 the negatives will be identically exposed, and the enlarging and printing processesmuch simplified. A suitable negative material is Kodak

~1,300

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approx.)

high contrast copy film which can be developed in Kodak Dektol developer. The negative contrast may be improved by the use of a narrow-band pass interference filter, appropriate to the stain employed, in the light source of the microscope; (560 to 580 millimicrons filter is suitable for aceto-orcein stain). Photomicrographs of mitotic cells require to be enlarged to an original magnification of 3,000 or 4,000 times to be suitable for karyotype analysis. The procedure of analysis consists in cutting out the chromosomes from such a print, and assembling them in order of decreasing size. When this is done in a normal male cell (Fig. IA), it is immediately apparent that most of the chromosomescan be matched in pairs on the basis of their centromere positions (Fig. tB) . There are 22 different autosomes (non sex chromosomes) which occur in pairs of identical length and centromere position, and one pair of sex chromosomes of unequal length, the X and Y sex chromosomes.The X is usually the seventh largest in size and the Y is about the third smallest. In the normal female karyotype (Fig. 3C) there are two X chromosomesand no Y chromosome. Since the X chromosome is larger than the Y the female has in consequence about 3 per cent more genetic material than the male.

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Ferguson-Smith

The system of numbering the chromosomes is that proposed by human chromosome study groups meeting first in Denver in 196110 and later in London in 1963.ll Thus the autosomes are numbered 1 to 22 in order of decreasing size while the sex chromosomes are labelled with the letters X and Y. The greatest difficulty has been to characterize the chromosomes in the 6 to 12 and X group. At the London ConferenceI it was suggested that the four most metacentric pairs of chromosomes in this group should be numbered 6, 7, 8, and 11; the three submetacentric chromosomes would then be numbered 9, 10, and 12. The system described here, which fits best with the results

Fig.

3A. Normal

female

cell in mitosis.

(Aceto-orcein;

of chromosome measurements made in this Department’” (Table I) differs very sJightJ\ in that chromosome 6 is grouped with chromosomes 4 and 5, and the metacentric chromosomes in the X-7-12 group are numbered X, 7, 9, and 11, while the submetacentric chromosomes are 8, 10, and 12. In these preparations, the largest chromosome in the karyotype, chromosome 1, is about 7 to 8 ,U in length and the shortest, chromosome 22, is about 1 to 2 I*, but these measurements vary enormously from cell to cell depending largely on the stage of mitosis at which the cell is fixed. Comparison of chromosome measurements between cells and between individuals thus relies on esti-

~3,000

approx.)

Techniques

mations of chromosome length relative to the total chromosome length in each cell. The usual method is to express the length of each chromosome as a percentage of the total length of the X-containing haploid set; this is the relative length (Table I). A second index, the arm ratio, is calculated by dividing the long arm length by the short arm length. A scale diagram (Fig. 4) of the chromosome complement based on these indices is referred to as an idiogram, and this is a useful aid in karyotype analysis. Although the measurement of relative lengths and arm ratios might be considered the most precise means of characterizing each chromosome, in practice it is of limited

Fig.

3B. Autoradiograph

of Fig.

3A.

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value because chromosomes, like coiled springs, are very variable in their degree of contraction. Thus, it is rare even to find two obviously homologous chromosomes in the same cell with exactly the same measurements. Also, some chromosomes tend to contract relatively more in late metaphase than others with the result that, for exam@, chromosome 3 may be shorter than chromosome 4 in a minority of cells. Despite these reservations, the crude measurements allow classification of the chromosome complement into seven groups (Table I) and reasonably accurate identification of at least six pairs of autosomes (1, 2> 3, 16, 17, and 181, the Y chromosome, and very rarely art addi-

Late-labeling

X chromosome

heavily

silver

qrain

c~~vcred

1042 Ferguson-Smith

tional four autosomal pairs ( 19, 20, 21, and 22). In routine karyotype analysis, this classification may be made sufficiently accurately by eye, so that actual measurements are only of practical importance in the study of structurally abnormal chromosomes.“” (A useful method of chromosome measuremerit” is to project a negative of a photo-

Fig.

3C.

Karyotype

Fig.

30.

Karotype

analysis

analysis

of Fig.

of Fig.

3 A.

3 B.

micrograph of the cell onto drawing paper, so that a magnification of about 15,000 times is obtained. The individual chromosomes may then be centered, traced onto the paper, and measured with the aid of a sensitive map measurer.) As chromosome measurement allows identification of only a minority of chromosome

Techniques of cytogenetics~43

Volume ‘311 Number 7, pal t ?

Table I.

Chromosome

measurements*

in 20 cells from

three individuals

Chromosome No.

Arm ratio

1

Short

arm

length S. D.

---.-

(% total female haploid ( Long

arm

1

length)

S. D.

2 3

1.13 1.59 1.. 1'5

3.97 2.99 3.05

.20 .22 .1'8

A50 4.77 3.51

.31 .17

4

2.45

1.17

.24

4.36

.27

;

2.52 1.61

2.16 1.58

.2Q .09

4.010 3.49

.24 .2'3

X 7 8

1.49 1.88

10

2.018

1.41

.I8 .16

I1 12

1.51 2.12

1.73 1.33

.?O .16

3.08 3.05 3.25 3.09 2.94 2.62 2.83

.?5 .I6

9

2.06 1.95 1.5’2 1.64

-19

1.56 2.1'3

13,

0.52 0.48 0.49

.16

15

5.85 5,.812 5.39

16 17 181

1.51 2.04 2.60

1.24 0.99 0.76

.lO

1.87

.14

.12 .14

2.03

.16

1.97

.12

14

.15

.16

3.07 2.80 2.62

.ll .ll

.14 .14

1.42 1.31

.lO .ll

21

2.82 2.72 2.34

0.50 0.45 0.63

.13

1.41

.I5

.lO’

1.24

.lrZ

.14

1.47

.31

have

been

calculated

before

pairs, more specific identification in routine karyotype analysis depends on the ability to distinguish secondary constrictionsl’ Secondary constrictions are so-named to distinguish them from the primary constriction or centromere. They are constricted regions at specific chromosomal sites and are thought to indicate sites of nucleolus formation.13 Unfortunately they are only visible under certain conditions which are not well understood. When they are visible, they are useful markers for chromosome identification. The most frequent type of secondary constriction in human somatic chromosomes is one which occurs at the end of a chromosome arm and separates a small terminal part of the chromosome arm from the rest of the chromosome. The terminal mass is called the satellite, and the constricted re,qion the satellite stalk. It has been found that the five pairs of acrocentric chromosomes (Nos. 13, 14, 15, 21, and 22) all have

6.13 5.58 5.65

.12 .16

1.16 1.01

deviations

8.47 7.76 6.56

.21

1.2’2

standard

__^_._ Total

.28 .28 .18 .20

1.30

and

--

.18

19

Y

j

.19

20 212 “Ratios

Relative

I .90 -

1.69 2.09

rounding.

the potential to arms.14 Very notably No. 17, ever, secondary to the ends of

show satellites on their short rarely other chromosomes, may show satellites.” Howconstrictions are not limited these chromosomes, and are seen SUffickntly frequently at specific tJ&ntS in other chromosomes to be useful in chromosome identification.‘? Particularly helpful are the short arm constriction in chromosome 6, the paracentric long arm constriction in chromosomes 9 and 11, and the midlong arm constriction in chromosome 1’7: in one study of 610 cells’” these four constrictions are visible in 23 per cent, 38 per cent, 7 per cent, and 23 per cent of the chromosomes, respectively. The sites of these and other constrictions are shown in Fig. +. It should be emphasized that secondary constrict.ions are very variable in their appearance, and sometimes are associated with extreme elongation of the adjacent part of the chromosome arm. If this possibility is

1044

Ferguson-Smith

not borne in mind it may lead to mismatching in the karyotype analysis. The appearance of the secondary constriction may be so bizarre on some occasions, that the chromosomemay be mistakenly considered to be abnormal. Chromosomes 1, 9 and 16 and the Y chromosome are particularly prone to show extreme variation in the expression of their secondary constrictions. To sum up, the procedure of karyotype analysis from photomicrographs consists in

first arranging the cut-out chromosomes in order of decreasing size into seven groups (Table I). The individual chromosomesare then paired off and numbered into presumedhomologous pairs on the basisof their centromere position. At this point, chromosomes1, 2, 3, 16, 17, 18, and the Y chromosomemay be correctly identified and placed in their correct position. If the cell has been fixed in early metaphase, it may also be possible to distinguish by size, chromosomes

Fig. 4. The normal human idiogram, indicating the sites of the twenty most frequently observed secondary constrictions (From Ferguson-Smith et al.: Cytogenetics 1: 325, 1962.)12

Techniques

19 from 20 and 21 from 22. Even in early metaphase it is not possible to be confident in distinguishing the correct homologues in the 13 to 15 group, although in individual cases the size of the short arms and the prominence of satellites may help. There remain chromosomes 4 to 12. Chromosomes 4 and 5 are readily identified but not so easily separated; in exceptional cells they may be distinguished from one another by size, by the position of the long-arm constriction, and by the fact that the centromere is more centrally placed in chromosome 4. Chromosome 6 is much larger than any of the remainder (it is occasionally larger even than chromosome 5) and can usually be identified, particularly when its short arm constriction is visible. Chromosomes 7 to 12 and the X chromosome are matched primarily according to centromere position into four pairs with more medianly placed cen-

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tromeres, and three pairs with more terminally placed centromeres. Once separated into these two subgroups, the individual size differences are more readily appreciated. The three pairs with more terminally placed centromere are, in order of decreasing size, Nos. 8, 10, and 12. Among the four pairs with more medianly placed centromeres, the smallest, No. 11, is the most readily identified. No. 9 can usually be distinguished by its secondary constriction from the X and No. 7, the two chromosome pairs which probably give the most difficulty. The X is the largest in the X-7-12 group and its centromere is more medianly placed than in 7. However, in female cells, the two X’S frequently show marked asymmetry in that one homologue (the inactivated Xj is usually more contracted than the other. The identifying features of individual chrotnosomes art: summarized in Table I and Fi:. 4.

Fig. 5. A, Nucleus from buccal smear in a normal female. Sex chromatin body of normal size. (Cresyl violet; ~2,500 approx.) B, Nucleus from buccal smear in a patient with gonadal agenesis and an X deleted-X sex chromosome constitution. Small sex chromatin body. (Cresyl violet; ~2,500 approx.) C, Nucleus from buccal smear in a patient with gonadal agenesis and an X isochromosome-X sex chromosome constitution. Large sex chromatin body. (Cresyl violet; ~2,500 approx.) D, Nucleus from section of epidermis in a patient with XXXY Klinefelter’s syndrome. Two sex chromatin bodies of normal size. (Hematoxylin and eosin; X2,500 approx.) (Parts A, B, and C from Ferguson-Smith et al. : Cytogenetics, In press, 1964.) se

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Ferguson-Smith

Th te sex chromatin

It has now been very convincingly shown that one of the two X chromosomes in human female somatic cells becomes condensed in il2termitotic nuclei and is visible under

the microscope as the sex chromatin or Ba rr body. This structure, which is about 1 P in diameter and usually lies close to the nL 1cle ar membrane (Fig. 5, A), is not found in rnzLlC somatic cells. The condensation of tlhe X

Fig. 6. Polymorphonuclear leukocytes showing “drumsticks.” A, Small drumstick from patient with an X deleted-X sex chromosome constitution. B, Normal drumstick from normal female. C, Large drumstick from a patient with an X isochromosome-X sex chromosome constitution. (Leishman; ~2,000 approximately)

Fig. 7A. Primary spermatocyte illustrating the pachytene stage of meiosis. The XY sex bivalent is very condensed compared with the autosomal bivalents. One autosomal bivalent (from 13 to 15 group) is shown with a large nucleolus attached to its terminal chromomere.

Techniques

chromosome appears to he associated with genetic inactivation, and this has been shown to affect the two X chromosomes at random, because females heterozygous for Xlinked genes show a mosaic pattern of ac-

1047

tivity with respect to those genes.‘” The Xinactivation principle, so clearly formulated by Lyon,‘5 very satisfactorily explains how male cells compensate for their 3 per c’ent deficiency in chromosomal material. It also

Fig. 7B. Autosomal bivalents of the 13 to 15 group isolated from ing thr nucleolus attached to the terminal chromomere (satellited

Fig. 7C. Autosomal cells, showing the Smith.*s)

of cytogenetics

bivalents of the 21 to 22 group terminal nucleoli. (Aceto-orcein;

isolated ~3,000.

different region).

cells show-

from different from Ferguson-

1048

Ferguson-Smith

accounts for the phenomenon of “dosage compensation,” which refers to the observation that the product of an X-linked gene is quantitatively about the same when it is

*-

present in sinqlc dose irk tile i male as it is when present in doll the homozygous female. In persons with sex chromosol me aberra-

.% .‘

characteristic end-to-end Fig. 8. Primary spermatocytes at first meiotic metaphase. n, The attachment of the X and Y chromosomes in the sex bivalent. b, The sex chromosomes are present as univalents due either to failure of conjugation or to precocious disjunction. (Feulgen; ~3,000.) (Ferguson-Smith, M. A.: In Steinberg, A. G., editor: Progress in Medical Genetics, New York, 1961, Grune & Stratton, Inc., vol. I, pp. 325-326.)3x

Techniques

tions, it has been found that if a cell contains more than two X chromosomes, the additional X chromosomes are also inactivated, so that the number of X chromosomes is always one more than the maximum number of sex chromatin bodies in interphase diploid nuclei (Fig. 5, D) . AS the X chromosome is one of the most difficult chromosomes to identify, the nuclear sex diagnosis is of great importance in the correct interpretation of karyotype analyses, particularly in cases rvhere a sex chromosome aberration is suspected. The size of the sex chromatin body is also important, for in patients with structural aberrations of the X chromosome, the size of the abnormal X corresponds closely with the size of the sex chromatin body (Fig. 5, B and C) .I9 Furthermore, the frequency of cells showing sex chromatin in a given tissue is a useful observation, as a low frequency may indicate X chromosome mosaic&m; an apparent discrepancy between

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different tissues in the same person also indicates X chromosome mosaicism. Nuclear sex may be determined in almost any tissue fixed and stained by routine histological methods. Two widely used methods are the buccal smear method,l” and the polymorphonuclear drumstick method.‘; The former is very simple and is particularly useful for population surveys.‘8 The latter is rather more time-consuming, but has the advantage that more accurate estimations of sex chromatin size are possible.l!’ The buccal smear method. The inside of the patient’s cheek is scraped firmly with the edge of a tongue depressor and the mucoid deposit containing the surface epithelial cells is smeared thickly onto a clean glass slide. The slide is immediatelv immersed in 1:l ether-ethanol and fixed for 2 hours. The smear is then hydrated through 70 per cent and SO per cent alcohol, 10 distilled water and stained in 1 per cent aque-

Fig. 9. Mitotic cell from peripheral leukocyte culture showing arms of satellited chromosomes and the secondary constriction chromosome 9. (Aceto-orcein; ~2,000.)

associations between the short regions of both homo1oe;ue.s of

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Ferguson-Smith

cresyl violet acetate for 30 minutes. Thereafter it is cleared and mounted in the usual way. The smear is scanned under the oil immersion lens for suitably fixed and stained nuclei. In order to obtain consistent sex chromatin counts, only those nuclei which show a finely granular nucleoplasm and well-defined nuclear membrane are counted. Pyknotic nuclei and nuclei which are folded, distorted, or obscured by bacteria and other debris are excluded. Cells in which the cytoplasm has taken up the stain, should be rejected, as this indicates improper fixation and usually drying of the cell prior to fixation. Sex chromatin masses at the nuclear membrane (Fig. 5, A) are seen in about 30 to 50 per cent of suitable nuclei in normal females. Chromocenters indistinguishable from sex chromatin are rarely seen in normal male cells and never in over 1 per cent. The “drumstick” method. The “drumstick” is the sex chromatin body of the polymorphonuclear leukocyte which projects from one of the nuclear lobes by a narrow stalk (Fig. 6, B). It is a mass of dense chromatin, normally about 1.5 p in diameter, which is visible in about 2 per cent of polymorphs in the normal female, but is absent in male polymorphs. Smaller drumstick-like bodies called nuclear tags are present in both sexes and should not be mistaken for sex chromatin. They stain less intensely than the rest of the nucleus and are usually obviously smaller than the true drumstick, In patients with structural X chromosome aberrations, the size of the true drumstick is directly related to the size of the abnormal X chromosome (Fig. 6, A and C) . “Drumstick” counts (i.e., the frequency with which drumsticks are identified in polymorphonuclear leukocytes) are made from ordinary blood smears stained by standard hematological techniques. It is the usual practice to examine 500 polymorphs scanned consecutively under the oil immersion lens, and compare the results with normal male and female controls. Estimations of drumstick size should be made in relation to the size of the nucleus. ous

The time sequence of deoxyribonucleic acid

(DNA)

synthesis

‘I’he autoradiographic technique for studying DNA synthesis is fast becoming important in human cytogenetics. The principle of the method is to label DNA precursors during cell culture with a suitable radioactive isotope and to study the uptake of the labeled precursor in the chromosomesby autoradiography of the cells at metaphase. Tritium (H3), by virtue of its low-energy beta emissionwith a very short path length, produces autoradiographs of high resolution and is the ideal isotope in this work; tritiated thymidine is the most suitable substance for DNA studies. The technique demonstrates that DNA is synthesized in different chromosomesat different times during the synthetic period of the cell cycle. This indicates yet another way in which individual chromosomes may be characterized. Although these studies are at an early stage, they have convincingly shown that one of the two X chromosomesin female somatic cells finishes DNA replication much later than any of the other chromosomes(Fig. 3B). 2ol21 The late-replicating X is believed to be the inactive, sex chromatinforming X. There is also a possibility that chromosome 4 may be distinguished from chromosome 5, and chromosome 21 from 22, by the pattern and timing of DNA synthesis, and that in the 13 to 15 group, there may be a sequence sufficiently distinctive to separate the three pairs.2’ The technique is relatively simple and may be applied to all cell cultures including peripheral blood cultures: In the blood culture method, H”-thymidine is added during the final 4 to 6 hours of culture so that the final concentration of the isotope is about 0.3 ,u.cper milliliter of culture.” The methods of culture and chromosome preparation are otherwise the same as described previously. The aceto-orcein stained preparations (without cover glass) are scanned for well-spread cells at metaphase, and 30 to 50 of theseare selectedand photographed before applying the autoradiographic emulsion. For this, either stripping

Techniques

film or liquid emulsion may be used. Stripping film is supplied mounted on sheets of glass, from which it is cut in the darkroom, floated onto water, and picked up by the microscope slide so that the emulsion side lies in direct contact with the chromosome preparation. The slide is allowed to dry, and is then left in a light-proof box containing desiccant (e.g., CaSO,) for 1 to 2 weeks before being developed in Kodak D 19 developer. The liquid emulsions are applied by the very simple dipping techniquez3; dipped slides are exposed, developed, and fixed the same as slides with stripping film. Following exposure and development, the slides are scanned under the oil immersion lens and the cells previously photographed for karyotype analysis are relocated and examined for radioactivity as shown by the distribution of silver grains in the overlying emulsion (Fig. 3B). The chromosomes of cells which begin their DNA replication after the time that H3-thymidine was added to the culture are seen to be very heavily covered with silver grains. Sometimes, the chromosomes are completely obscured by silver grains, but they can always be identified from the photographs taken before the emulsion was applied (Fig. 3A). Those cells which started the synthetic period before the addition of H3-thymidine show decreasing degrees of radioactivity depending on the amount of synthesis completed before that time. In the cells which were well advanced in DNA synthesis when the isotope was added, silver grains are seen only over those chromosomes which are the last to finish replication. In the female cell these almost invariably include the more contracted of the two X chromosomes (Fig. 30). Chromosome No. 4, one member of the 13 to 15 group. No. 16, No. 18, one member of the 21 to 22 group, and the Y chromosome, characteristically take up the label later in the synthetic period than do the other chromosomes, although earlier than the latelabeling X.?? At present the application of this technique has been mainly in the elucidation of sex chromosome aberrations. It has been

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found that in patients with structural sberrations of the X chromosome, the abnormal X is invariably the late-replicating X.s” In patients with more than two X chromosomes, the number of late replicating X’s is always one less than the total number of X’s.“j In these respects, the autoradiographic results are complementary to the results of sex chromatin studies. The identification of chromosome aberrations Abnormalities of cell division, such as nondisjunction, anaphase lagging, etc., which have occurred during parental gametogenesis or during the early cleavage divisions of the fertilized egg, may lead to the development of an individual with an abnormal number of normal chromosomes. Thus, there may be a missing chromosome, as in XC) Turner’s Syndrome (article by Miller) where the patient is monosomic for the X chromosome, or there may be an extra normal chromosome, as in Mongolism (article by Smith) where the patient is trisomic for a chromosome in the 21 to 22 group. These numerical chromosome abnormalities are immediately detected by the chromosome count, and the specific identification of the missing or extra chromosome is made by routine karyotype analysis. In the case of the X chromosome, the parental source of the missing or extra X may be indicated by the way in which an X-linked trait segregates in the family”“; useful X-linked traits are color blindness, and the Xg blood group. Other forms of numerical chromosomal aberrations include polyploidy where the number of chromosomes in each cell is an exact multiple of the haploid number, and chromosomal mosaicism, where there are two or more lines of cells each with a different chromosomal constitution. Identification of these aberrations presents no special problem except that mosaicism is always difficrrlt to exclude. Structural chromosome aberrations are usually detected during karyotype analysis by finding a chromosome of abnormal size and arm ratio in place of a normal chromosome.

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A chromosome may be abnormally long due to duplication of part of an arm, or due to reciprocal translocation. The latter results from the exchange of chromosomal fragments between nonhomologous chromosome. When both products of the translocation are present there will be no appreciable change in the genetic constitution of the individual, the translocation is said to be balanced, and karyotype analysis should reveal two abnormal chromosomes. In an unbalanced translocation only one abnormal chromosome is usually present, consequently there is a deficiency of part of one chromosome and duplication of part of another. Other types of identifiable chromosomal aberrations include large deletions, large inverand ring chromosions, isochromosomes, somes. Deletions give rise to abnormally small chromosomes. Large inversions, which include the centromere in the inverted segment, give rise to chromosomes of normal length but abnormal arm ratio. The isochromosome is a metacentric chromosome, most examples of which are thought to arise at a previous cell division by misdivision of the centromere, which was broken transversely instead of longitudinally. Unless the misdivision has occurred during first meiosis and after crossing-over, the two arms of an isochromosome are genetically identical. Large ring chromosomes are usually obvious, but very small ring chromosomes are more difficult to recognize. This diagnosis should be suspected if the small abnormal chromosome appears very heteropyknotic, and is very variable in shape (so that no conclusion can be made about the centromere position). The answer to such a situation will be provided by examination of the abnormal chromosome in early prophase. Chromosomal aberrations occasionally arise in culture, and care should be taken to exclude this possibility before assuming that an aberration is present in vivo. If multiple cultures are set up from the original specimen of tissue, this type of mistake is avoided, as chromosome analysis of each culture will determine whether or not the aberration is consistently present.

‘l’he difficult question as to whether ;I chromosome aberration is or is not causally related to a patient’s developmental disorder is frequently encountered in cytogenetics laboratories. This point is often not settled until the parents and both affected and unaffected siblings and other close relatives have chromosome analysis. If possible, similarly affected but unrelated patients should also be studied before concluding that a chromosome aberration is the usual cause of the disorder. Other techniques for characterizing human

chromosomes

Several very promising lines of research are currently being investigated and these should eventually provide information leading to a much better characterization of the human chromosome constitution. Perhaps the most important of these is the study of chromosome morphology during meiosis. This specialized form of cell division occurs during gametogenesis and has the twofold function of reducing the chromosome number in the gametes to the haploid number (23), and of recombining the genetic material by random assortment and crossing over, so that there is infinite variation in the genetic make-up of the resulting gametes. In order to accomplish these functions the homologous chromosomes pair with one another to form bivalents at the prophase of the first meiotic division. At the pachytene stage of meiotic prophase, these bivalents have a characteristic chromosome pattern by which they may be to some extent identified.” At present, probably only the sex chromosomes and the satellited chromosomes (Figs. 7A, 7B, and 7C) of the human mitotic complement have been distinguished at pachytene in male meiosisz8 but it should not be long before some of the other 17 bivalents are identified. A complete pachytene map of the human complement would be invaluable, not only for genetic linkage studies, but as a means of identifying much smaller structural chromosome aberrations than is possible from mitotic cells. A particular advantage would be obtained in the recogni-

Techniques

tion of inversions, which appear in pachytene as loops, of translocations, which may appear a$ quadriradial figures, and of isochromosomes, in which both arms may pair with one another. Studies of later meiotic stages, including the first meiotic metaphase (Fig. 8, A and B), may be expected to give information about chiasma frequencies and also about the segregation of translocated chromosomes. Attempts are also being made to improve the identification of mitotic chromosomes by cytological techniques which increase the frequency of appearance of secondary constrictions. One group of workers find that this may be accomplished by growing the cells in a culture medium deficient in calcium,‘” another group have observed the same results by using the standard methods of culture but employing a different method of fixation.30 If these results can be confirmed, karyotype analysis in the future will be much simplified. However, using standard techniques, it is useful to note that the sites of secondary constrictions may sometimes be indicated by specific types of chromosome association even if the constrictions themselves are not visible.‘” The most ob\-ious types of association which are useful for this purpose are those which involve the short arms of satellited chromosomes and the secondaq constriction regions of other nonsatellited chromosomes (Fig. 9) .

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Quite apart from the association of’ ct~romosomes at their secondary constriction sites, there appears to be some evidence for two other types of nonrandom distribution of chromosomes in metaphase figures. ‘I’hr titst type, the association of homolopus chrornnsomes, is not a very regular phenomenon, I. ” and may reflect somatic pairing.“” The se<:ond type of nonrandom distribution 1s of unknown cause : it appears that certain chromosomes occupy a more peripheral location on the metaphase plate more frequently than would be expected by chance. The most peripheral chromosome is stated to be the inactive X,31 but the Y chromosome and chromosomes lZ3, 21, and 17 to 18 aiso seem to show the same tendency.“l. ” These observations have so far not been app1it.d as an aid to chromosome identification. Several other cytological techniques ;tre at present being investigated with regard to their application to human cytogenetics, and among these electron microscopy and DXA microdensitometry would seem to be among the most promising. At present full application of both these techniques is prevented by the technical problems involved.

REFERENCES

1. Harnden, 31,

D. G.:

Brit. J. Exper.

Path. 41:

‘7. Ford, C. E., Jacobs, P. A., and Lajtha, L. G.: Nature 181: 1565, 1958. 3. Moorehead, P. S., Nowell, P. C., Mellman, W. J., Batipps, D. M., and Hungerford, D. A.: ExDer. Cell Res. 20: 613. 1960. 4. Paul, j:. Cell and tissue cilture, Baltimore, 1961, Williams & Wilkins’ Company. 5. Arakaki, D. T., and Sparkes, R. S.: Cytogenetics 2: 57. 1962. 6. Tjio, J. H., and Whang, J.: Stain Technol. 37:

10.

1960.

17,

1962.

7. Baker, M. C., and Atkin, N. B.: Lancet 1: 1164, 1963. 8. Jacobs, P. A., Court Brown, W. M.. and Doll. R.: Nature 191: 1178. 1961. 9. Co&t Brown, W. M., Jacobs, P. A.. and Doll, R.: Lancet 1: 160, 1960.

Il. 12.

Chromosomes Study Group: Lancet 1: 1063, 1960. London Conference on the Normal Human Karyotype: Cytogenetics 2: 264, 1963. Human

Ferguson-Smith,

E.. Ellis. genetics

M.

A.,

Frrguson-Smith,

P. M., and Dickson, 1: 325,

M:

M.

Cyto-

1962.

13. Ferguson-Smith, M. A., and Handmaker. S. D.: Ann. Hum. Genet. 2’7: 14-3, 1963. 1.4. Ferguson-Smith, M. A., and Handmakrar, S. D.: Lancet 1: 638, 1961. 15. Lyon, M. F.: Nature 190: 372. 1961. 16. Moore. K. L., and Barr, M. D.: L:+nc.et 2: 1955. Davidson, W. M., and Smith. D. K.: Brit. M. J. 2: 6, 1954. Ferguson-Smith. M. A.: .4rta rytol 6: 73, 1962. Maclean, N.: Lancet: 1: 1154. 19h.l. 57, 17. 18. 19.

1054

20. 21.

22. 23. 24.

25.

26.

27, 28. 29.

Ferguson-Smith

German, J. L.: Tr. New York Acad. SC. 24: 395, 1962. (Series 2.) Morishima, A. Grumbach, M. M., and Taylor, J. H.: Proc. Nat. Acad. SC. 48: 756, 1962. German, J. L.: J. Cell Biol. 21: 37, 1964. Joftes, D. C.: J. Nuclear Med. 4: 143, 1963. Muldal, S., Gilbert, C. W., Lajtha, L. G., Lindsten, J., Rowley, J., and Fraccaro, M.: Lancet 1: 861, 1963. Rowley, J., Muldal, S. Gilbert, Cl. W., Lajtha, L. G., Lindsten, J., Fraccaro, M., and Kaijser, K.: Nature 197: 251. Ferguson-Smith, M. A., Mack, W. S., Ellis, P. M., Dickson, M., Sanger, R., and Race, R. R.: Lancet 1: 46, 1964. Yerganian, G.: Am. J. Hum. Genet. 9: 42, 1957. Ferguson-Smith, M. .4.: Cytogenetics, vol. III, 1964. (In press.) Sasaki, M. S., and Makino, S.: Am. J. Human Genet. 15: 24, 1963.

30. 31. 32. 33. 34.

35.

36.

37. 38.

Saksela, E., and Moorehead, P. S.: Cytogenetics 1: 225, 1962. Barton, D. E., and David, F. N.: Am. Hum. Genet. 25: 323, 1962. Schneiderman, L. J., and Smith, C. A. B.: Nature 195: 1229, 1962. German, J. L.: Science 144: 298, 1964. Miller, 0. J., Mukherjee, B. B., Breg, W. R., and Gamble, A. Van N., Cytogenetics 2: 1, 1963. Miller, 0. J., Breg, W. R., Mukherjee, B. B., Gamble, A. Van N., and Christakos, i\. C.: Cytogenetics 2: 152, 1963. Ferguson-Smith, M. A., Alexander, D. S., Bowen, P., Goodman, R. M., Kaufmann, B. N., Jones, H. W., and Heller, R. H.: Cytogenetics, vol. III, 1964. (In press.) Davidson, R. G., Brusilow, S. W., and Nitowsky, H. M.: Nature 199: 296, 1963. Ferguson-Smith, M. A.: In Steinberg, A. G., editor: Progress in Medical Genetics, New York, 1961, Grune & Stratton, Inc., vol. I, pp. 325-326.