Annotation J Med Genet: first published as 10.1136/jmg.13.2.103 on 1 April 1976. Downloaded from

Journal of Medical Genetics (1975). 12, 103-122.

Classification and relationships of induced chromosomal structural changes JOHN R. K. SAVAGE From the M.R.C. Radiobiology Unit, Harwell, Didcot, Oxon'

Summary. A detailed survey is given ofthe types and classification ofprimary structural changes that can be induced in chromosomes and observed at the first metaphase after the initial damage. Comments upon identification and scoring are given for the benefit of new workers. The annotation concludes with a brief dis- cussion ofthe potential relationships between the primary types, and the secondary or derived types encountered in clinical studies.

1. Introduction as this is the only time when the structural changes Structural changes are being increasingly used in can be observed in their entirety (primary aberra- the screening of drug and environmental hazards, tions). Many kinds lead to mechanical separation and this requires an agreed classification of the problems and genetic loss when the cell attempts copyright. aberrations which may be observed. The ten- division and result in the death of one or both dency seen among some workers coming new to daughter cells. Such aberrations are not seen these fields to invent their own classifications and again. Some kinds of structural change, however, criteria, or to lump together different kinds of may not cause problems at division, and may be aberrations under some blanket name like 'breaks' transmitted to further cell generations. These

or 'fragments' should be avoided. persistent (secondary) aberrations are frequently http://jmg.bmj.com/ The underlying construction of the chromosome modified in various ways, and it is seldom possible makes it unlikely that any structural alteration will to infer with any certainty their primary form (see be found that has not already been described and Section 10). It is the secondary types which are en- documented in the vast literature on radiation- countered in clinical work. induced chromosomal aberrations. Since this There have been numerous descriptions of pri- literature (much of which involves plant chromo- mary chromosome-type aberrations induced in human somes) is not readily accessible to new workers, the cells as these are the kind most frequently studied purpose of this annotation is to provide a compre- (UNSCEAR, 1969; Buckton and Evans, 1973). on September 24, 2021 by guest. Protected hensive survey ofthe kinds ofaberrations observable Chromatid-type aberrations in human cells have with visible light microscopy at the first post-treat- hitherto received only scant attention, but the in- ment metaphase, the principles of their classification, creasing use of chemical mutagens and chemothera- and some characteristics which may be of help in peutic agents in experimental work is bound to identification and scoring. Also included are some bring this kind into prominence in the future. brief comments on current controversies with regard Wherever possible, the descriptions and dis- to the formation of aberrations so that those wishing cussion in the following sections refer to human to consult original papers may have a working cells, but it is clear from the large body of data vocabulary with which to start. available that, although the relative frequencies of In all critical qualitative and quantitative work specific aberrations may differ between organisms, with aberrations, it is necessary to confine attention the kinds observed are the same whatever cells are to the first metaphase following aberration induction, used. 103 104 John R. K. Savage J Med Genet: first published as 10.1136/jmg.13.2.103 on 1 April 1976. Downloaded from 2. General principles of classification of sister chromatids at a particular location, as seen The chromosome as seen at metaphase of cell in chromatid-type aberrations, was taken as an division consists of two parallel threads or chroma- indication that the chromosome region involved had tids, those composing one chromosome being termed already split or duplicated at the time of exposure. sister chromatids. In each chromosome, the sister Such types arise from irradiation of cells in very chromatids are held together at a definite point, the late G1 and throughout the DNA synthesis phase, primary constriction or centromere (synonyms: S and the post-synthesis phase, G2- kinetochore, spindle attachment region), which Classification into these two major categories is may be situated anywhere along the thread. The still valid, though the passage of time has raised centromere partitions the thread into two arms some questions as to their precise relationship to the (often referred to as opposite arms with respect to state of chromosome duplication. the centromere). The ratio of the lengths of these Aberrations may be classified according to their two arms (arm ratio), expressed as long arm (q)/ appearance or according to their presumed method short arm (p) or as short arm x 100/total chromo- of formation. The former 'descriptive' kind of some length (the centromeric index) forms an im- terminology is more usual, and the names and portant diagnostic character in identifying indi- symbols used are intended to be without implications vidual chromosomes. as to the precise mode of origin (for example, Sax, At anaphase of cell division, the sister chromatids 1941; Catcheside et al, 1946; Lea, 1946). The latter of each chromosome are freed by longitudinal divi- kind is exemplified by the scheme of Darlington and sion of the centromere, and they migrate to opposite LaCour (1945) in which emphasis is placed upon poles of the cell, where they regroup to form the numbers of breakage and rejoining events re- daughter nuclei. The key factors in this migration quired for the aberrations within a cell, rather than are (1) the presence in the cell of an organized individual aberration kinds. The full form is spindle, and (2) the possession by the chromosome seldom used these days, though a number of the of a functional centromere which is attached to cer- symbols have been carried over into descriptive tain fibres of the spindle which determine its move- systems. ment to the poles. Any chromosomal piece with- The recognition of the highly complex nature of out a centromere fails to move, and is usually left chromosomal organization (particularly at the copyright. out in the cytoplasm where it may form (alone, or molecular level); of the complex biochemical repair with other fragments) a micronucleus in one or other mechanisms present in the cells; of the great ofthe daughter cells. variety of agents which can produce qualitatively In the interval between successive divisions (inter- identical types of structural change; and, not least, phase, or resting phase), the daughter cells grow, the fact that the limits of optical resolution are such and each chromatid duplicates (or 'splits'), so that it that only a small proportion of structural changes is once again a double structure when the cell re- are actually detected has led to the almost universal http://jmg.bmj.com/ enters division. adoption of a descriptive terminology. From early work with ionizing radiations in plants (Mather, 1934; Riley, 1936; Sax, 1938, 1940), it was established that, when cells are ex- 2.1 Definitions of terms amined at the first metaphase after exposure, two Since structural chromosomal changes can be broad categories of permanent structural change produced by a very wide variety of agents, the non- committal term lesion will be used for the damage or were distinguishable in chromosomes, chromosome- on September 24, 2021 by guest. Protected type and chromatid-type. event within the chromosomes which is a necessary In the former, the changes induced always appear prerequisite for aberration formation. to affect both sister chromatids of a chromosome at The following basic terms apply to all conven- the same location, whereas in the latter-with one tional structural chromosome-type or chromatid- notable exception (the isochromatid deletion)-only type aberrations. one of the sister chromatids is affected at a given location. 2.1.1 Exchanges When there is an exchange of Chromosome-types were thought to arise from chromosome parts after the occurrence and inter- damage of the chromatid thread in its unduplicated action of two lesions: or 'unsplit' stage (corresponding to the bulk of G1, Interchange: when the interacting lesions occur in the pre-DNA synthesis stage of the cell cycle), and the arms of different (homologous or non-homo- this damage was duplicated along with the chromo- logous) chromosomes, some. Damage which affected only one of the pair Intrachange: when the lesions are within one Classification and relationships of induced chromosomal structural changes 105 J Med Genet: first published as 10.1136/jmg.13.2.103 on 1 April 1976. Downloaded from chromosome. Intrachanges may be further sub- simple severance of the chromosome or chromatid divided into: to give an acentric fragment, and which is not Inter-arm intrachanges where the lesions are in clearly associated with any exchange process. opposite arms with respect to the centromere. This category will always comprise a hetero- Intra-arm intrachanges where the lesions are geneous mixture, for whatever hypothesis is adopted within one arm. In duplicated chromosomes there for the origin of aberrations (see Section 4) this class is the possibility of interaction between lesions in- will always be augmented by certain incomplete duced in sister chromatids. Chromatid-type intra- interchange and intrachange events which simulate changes can therefore be further subdivided into: (and in many cases are indistinguishable from) Inter-chromatid intrachanges where the exchange simple breaks. involves both sister chromatids, and Intra-chromatid intrachanges where the exchange 2.2 Aberration names and symbols occurs within one chromatid while its sister remains The terms defined in Section 2.1 form a con- unaffected in the exchange region. venient basis for a systematic descriptive classifica- Interaction of lesions in chromatids or undupli- tion of structural changes, and will be used as such cated chromosomes may lead to an asymmetrical in this annotation. exchange, which always gives rise to one or more However, over the course of the 50 or so years of acentric fragments (symbol ace Chicago Conference, research into radiation-induced chromosomal aber- 1966, Paris Conference, 1971)-that is, pieces of rations, certain names or symbols have become chromosome without a centromere and which there- assigned to particular aberration kinds and are fre- fore tend to be left behind and excluded from quently used in the literature. The more important daughter nuclei (with consequent loss of genetic ofthese (and their synonyms) are given in the follow- information) when the chromosomes move to the ing sections, but it must be remembered that the spindle poles at anaphase. Asymmetrical exchanges same name may well be used differently by dif- are invariably scorable. Interaction which pro- ferent authors. Similarly, several different kinds of duces a symmetrical exchange does not lead to an aberration may be included under one term, such as acentric fragment (except when the exchange process 'break' (see above). is incomplete, see below) and, unless there is some copyright. very obvious disparity produced in the lengths or 3. Achromatic lesions ('gaps') arm-ratios of the participating chromosomes, such Before embarking on detailed descriptions it will exchanges pass undetected in the case of chromo- be as well to consider an area of controversy which some-type aberrations with the usual staining can be confusing to a new worker. techniques. In marked contrast, some forms of When chromosomes are examined at the first

symmetrical exchange can be scored with an metaphase after a dose of radiation, or of chemical http://jmg.bmj.com/ efficiency approaching 100% in the case of chrom- (and even in the absence of any aberration inducing atid-type aberrations. agent), some of the chromatids may have, in addition More than two lesions may be involved in ex- to obvious structural changes, small non-staining change and so give rise to some very complex con- regions at various positions in their arms. These figurations. regions show considerable variation in size and In all kinds of exchange aberrations, the actual clarity, ranging from a small 'nick' only partially exchange process may be complete (C)-that is, all traversing the chromatid to quite long sharply the chromosome parts have joined up in their new bordered regions. There may also be indications of on September 24, 2021 by guest. Protected configuration leaving no free or 'broken' ends, or it slight dislocation, bending or stretching at the same may be incomplete (I), in which case the exchange location. of parts may be potential rather than actual. In Most of the gaps are confined to one of the sister the case of asymmetrical forms, it is possible to chromatids that make up the chromosome arm designate the incompleteness as proximal (p) or (chromatid gap or achromatic lesion, g) but occa- distal (d) with respect to the centromere (see Figs. 3 sionally they are found paired at the same location and 5). In all organisms so far investigated, chro- on the arm (chromosome gap, isolocus gap, paired matid-type exchanges always show a much higher achromatic lesion, G). frequency of incompleteness than chromosome- They are visible with nearly all staining methods type exchanges. (in particular they are Feulgen negative) under bright-field illumination, and with phase contrast. 2.1.2 Breaks Any descriptive classification must With ultraviolet illumination and with scanning include a category for the break or discontinuity, a electron microscopy they can be seen to be traversed 106 John R. K. Savage J Med Genet: first published as 10.1136/jmg.13.2.103 on 1 April 1976. Downloaded from by twin strands in the chromosomes of some organ- c. The majority are not associated with any ob- isms (Scheid and Traut, 1970, 1971). vious structural change, or noticeable elongation of Larger gaps are very easily (and frequently) the arm in which they occur. However, they are scored as breaks at metaphase, and were, in fact, sometimes found in exchange aberrations located in included in this category by a number of early the presumptive region of the actual exchange, workers. This can lead to very big discrepancies in rather like 'scars'. break frequencies between different laboratories d. Very occasionally, achromatic lesions on (for information see Evans, 1962, 1963; Revell, different chromatids are associated in pairs by 1974). Nevertheless, when chromatids carrying 'stickiness' and accompanying deformation of them are observed at anaphase, it is seen that acen- chromatids, almost as if the process of interchange tric fragments are not released, and therefore these had been initiated but was not completed. achromatic lesions cannot constitute a true dis- e. Radiation-induced chromatid gaps show a re- continuity. duced frequency if the dose is given in the absence It must be admitted straightaway that there is of oxygen-that is, a classical oxygen effect-and considerable difficulty in distinguishing achromatic their radiation dose response curve appears to be lesions from true breaks when chromosomes are ob- linear, suggesting that they are predominantly 'one- served at metaphase, and this difficulty is aggravated track' events (Neary and Evans, 1958). However, by cytological methods like air drying which exert arguments based on linearity are very insecure tension on chromosomes during spreading. The (Savage and Papworth, 1973). accepted criterion for a real chromatid disconti- Several suggestions have been made as to the nuity is that a very distinct dislocation and mis- nature of achromatic lesions. Early workers alignment must be present between the parts of the thought that they might represent partial or sub- arm on either side of the lesion. Even so, there is chromatid breakage (Section 7.4), or 'scars' marking still an element of subjectivity involved, and each sites of rejoining or points at which exchange was worker must establish by experience (and then realized. Alternatively, from the observation that rigidly adhere to) his own criterion. It cannot be they are Feulgen negative regions, it has been sug- emphasized too strongly that, in critical work, gested that they may represent a loss or depoly- scores obtained at metaphase must be checked merization of DNA, analogous to the DNA steresis copyright. against scores at anaphase. Such checking is nor- observed after ultraviolet microbeam irradiation of mally precluded by the usual cytological methods chromosome (Zirkle et al, 1956). To be visible, which involve pretreatment of cells with a spindle such loss would involve millions of nucleotides, and inhibitor like Colcemid to accumulate metaphases it is difficult to see how this could be without geneti- before sampling. cal significance and leave a viable cell. Yet another suggestion is that they represent errors in chromo- 3.1 Nature of achromatic lesions some coiling and condensation, particularly as their http://jmg.bmj.com/ In spite of the fact that achromatic lesions have frequency is maximal when this process is most been recognized for a very long time (Sax, 1938), active. This would perhaps account for their re- their precise nature still remains a mystery. Al- parability and apparent lack of genetic significance. most certainly they must represent a mixture of dif- ferent types of induced damage, but they are not 4. 'Breakage-and-reunion' and 'exchange' infrequent in untreated cells in culture (spon- theories for origin of chromosomal taneous frequency - 0.022 per cell in cultured aberrations on September 24, 2021 by guest. Protected blood lymphocytes (Evans, 1970)). As far as classification for scoring purposes is con- In addition to the facts already mentioned the cerned, the origin or mechanics of aberration forma- following are pertinent in any discussion: tion is of minimal importance. Nevertheless, it is a. The frequency is highest in cells closest to impossible to go far in the literature, or into the division at the time of irradiation-that is, in cells discussion of aberration types, without some that were in early prophase or late G2-and declines reference or allusion to the two major opposing as the frequency of true structural changes increases theories. For the guidance of new workers I have but not in a simple reciprocal manner. They are attempted in this Section a simple summary of the rare in cells irradiated while in G1. salient points of the controversy which I hope will b. They appear to be completely reparable, since provide a basic framework. For fuller treatment, corresponding structural aberrations are not seen the reviews of Evans (1962) and Revell (1974) should when cells which would have carried gaps at first be consulted. division appear in their second division. The first theory (often referred to as the Classical Classification and relationships of induced chromosomal structural changes 107 J Med Genet: first published as 10.1136/jmg.13.2.103 on 1 April 1976. Downloaded from Theory) is that of breakage-and-reunion, proposed The diagrams in this annotation illustrate most of by Sax (1938, 1940, 1941) and developed in detailed the ways in which breaks can be derived from ex- mathematical mode by Lea and his colleagues (see change failure. Lea, 1946). It is still the most widely held theory. The radiation energy-loss event is thought to result 5. Chromosome-type structural in a prmary breathat is, a severance of the un- aberrations duplicated chromosome or chromatid thread pro- ducing a pair of broken ends. These broken ends These arise from the irradiation of unsplit or un- have one of three fates. They may join up again to duplicated chromatids in the early pre-DNA syn- reform the original configuration (restitution), and thesis period (early G1) of active cells, or from non- therefore no structural aberration is detectable at dividing cells with unduplicated chromosomes-for metaphase. It is estimated that >90% of breaks example unstimulated blood lymphocytes, liver follow this pathway. Secondly, if two primary cells. They are very rarely found after chemical or breaks happen to be in close spatial and temporal drug exposure when cells are examined at the first proximity, the four broken ends may rejoin in a post-exposure metaphase. However some (modi- variety ofways to produce the exchange aberrations, fied) kinds may be found at the second or subse- some of which will be seen at metaphase. Thirdly, quent divisions, but these are secondary forms de- the residium of primary breaks which have neither rived from primary chromatid aberrations. restituted nor rejoined remains to be seen at meta- Most of the structural aberrations encountered in phase as simple breaks. Thus, on this theory the clinical work are of the chromosome-type, but again break is primary; everything else is derived from it. these are secondary forms (Section 10) and may not In 1959 Revell formalized and advanced an al- conform in detail to the primary types encountered ternative hypothesis, known today as the Exchange at first post-exposure division, and which are Theory. Basically, this states that the initial described in the following sections. damage is not a break (as defined above) but an un- stable lesion-a primary event of damage, which 5.1 Exchanges: interchanges (Fig. 1). tends to decay towards a normal or undetectable 5.1.1 Asymmetrical interchange (Synonyms: state. If, however, such events occur, or are dicentric, C/C, Paris Conference (1971): copyright. brought together, in pairs, they may take part in an dicentric dic) When complete, this interchange exchange process, and so give rise to the aberrations results in a chromosome with two centromeres seen at metaphase. In a proportion of these ex- accompanied by a single acentric fragment, which change processes, the actual exchange of parts may however is actually compound, being made up fail or be incomplete, and a discontinuity in the from the terminal pieces of the chromosomes in-

chromosomal thread will result. As the chromo- volved. http://jmg.bmj.com/ somes proceed towards mitosis, coiling and con- The distance between the centromeres can vary traction tend to disrupt the original configuration from being undetectable up to almost the total ofthe arms that existed at the time when the exchange length of the two arms involved. Some human was established, so that many of these discontinu- Robertsonian translocations may in fact be dicen- ities when seen at metaphase will be indistinguish- trics with an extremely small intercentromeric able from simple breaks. All breaks are therefore region (Mikkelsen, 1973). secondary, being the result of failed exchanges. The proximally incomplete form gives rise to two A primary event which is not involved in an ex- chromosomes with shortened arms and a single on September 24, 2021 by guest. Protected change process cannot give rise to a break at meta- acentric fragment (Fig. 1). Unless the arm phase, and it decays to a stable, undetectable state. shortening is noticeable, the aberration will pro- Thus, strictly speaking there is no such thing as bably be scored as a simple break or terminal dele- 'restitution' as defined for the Classical Theory, for tion (Section 5.3). The distally incomplete form the 'decay' process may be quite different bio- is a dicentric with two fragments. The frequency chemically from the exchange process. with which this occurs suggests that incompleteness All workers nowadays are agreed that some of the is relatively rare, less than 2% in most organisms breaks observed at metaphase arise from failed examined. exchanges; the remaining controversy turns on the The dicentric is probably the most easily recog- frequency of those that do so. Protagonists of the nized and unambiguous aberration. The com- Exchange Theory believe all do, while those of the monest scoring category in aberration literature is Classical Theory believe that only a small proportion the combined group of 'dicentrics plus centric- do so. rings' (for the latter see Section 5.2.1 below). 108 John R. K. Savage J Med Genet: first published as 10.1136/jmg.13.2.103 on 1 April 1976. Downloaded from CHROMOSOME INTERCHANGES lesions in different chromosomes I pre-duplication disposition

ASYMMETRICAL SYMMETRICAL wwI- (c/c) H

0du dicentricritranslocotion

w J -Ja. 0 ("co) (c/c+C) °0 (c) h4(C) z incomplete proximal incomplete distal terminal deletions

COMPLEX INTERCHANGES more than two chromosomes involved copyright. pre-du p lcat ion disposition

asymmetrical exchanges in opposite arms asymmetrical exchanges in a common arm http://jmg.bmj.com/

examples- dicentric with unrelated acentric fragment tricent ric + complex translocation

FIG. 1. Chromosome-type interchanges. on September 24, 2021 by guest. Protected

At the anaphase immediately following the meta- not form, and dicentrics may persist and be trans- phase at which they are observed, the acentric frag- mitted. The Robertsonian translocations cited ment is usually excluded from the daughter nuclei above are examples, and there are several cases of and the dicentric portion may form a double or dicentric Y chromosomes known in man (Cohen interlocked bridge with concomitant mechanical et al, 1973). difficulties in cell separation. Both daughter nuclei Complex exchanges The opposite arms (with re- will be genetically deficient, so that cells containing spect to the centromere) of a given chromosome may dicentrics are rapidly lost from a cell population. In take part in different asymmetrical interchanges cultured human lymphocytes the rate of loss is esti- leading to a range of polycentrics (tricentric, tetra- mated at 40-50% per division (Sasaki and Norman, centric, etc.), each exchange event contributing a 1967; Garrano and Heddle, 1973). However, if the single compound fragment. intercentromeric distance is very small, bridges may The same arm can also take part in more than one Classification and relationships of induced chromosomal structural changes 109 J Med Genet: first published as 10.1136/jmg.13.2.103 on 1 April 1976. Downloaded from asymmetrical interchange, but never more than one helpful to think of the two lesions involved as being of these can be observed at metaphase. In such brought into proximity by a loop (see Fig. 2), and in cases, as is illustrated in Fig. 1, the acentric frag- this case the loop contains the centromere. The ment which accompanies the dicentric is unrelated consequence of the exchange is a ring-shaped cen- to it, in the sense that it contains a terminal region tric chromosome and a compound acentric fragment from a chromosome not participating in the ob- formed from the two terminal regions of the chro- served dicentric. There will also be present in the mosome beyond the point of exchange. cell a chromosome with a complex, non-reciprocal The ring portion may include almost all the chro- translocation. mosome, when the acentric fragment will be very very small, or it may be little more than the centro- 5.1.2 Symmetrical interchange (Synonyms: meric region. In this latter case, unless centro- reciprocal translocation, Paris Conference (1971): meres in the cell are counted very carefully, a small reciprocal translocation t or rcp) The symmetrical centric ring may be scored as an interstitial deletion counterpart of the dicentric which, when complete, (see below 5.2.2). leads to the reciprocal transfer of terminal portions Proximal and distal incompleteness is infrequent, of two separate (homologous or non-homologous) the latter (a ring and two fragments) is usually chromosomes (Fig. 1). Any length up to an entire recognizable with care. If banding techniques are arm may be involved and the lengths are not neces- not used, proximal incompleteness is likely to be sarily the same in both chromosomes. classed as a terminal deletion. Unless the exchange results in chromosomes with At anaphase the ring portion may separate freely, anomalous arm lengths, it will go undetected with or may form a variety of loops or interlocking rings, conventional staining techniques. It is estimated leading to bridges. This, together with the loss of that efficiency of detection of this form and the peri- the acentric fragment, usually leads to the death of centric inversion (see below) is not greater than 20% the cell which contains it. However, persistent in irradiated human cells (UNSCEAR, 1969). The ring chromosomes occur in many organisms includ- advent of banding techniques has greatly increased ing man, where examples are known in each of the seven chromosomal groups. The problems of

the efficiency of detection but it is still nowhere copyright. near 100% (Seabright, 1973). mechanical separation encountered at each succes- Reciprocal translocations can be (and frequently sive anaphase lead to considerable variation in size are) transmitted to subsequent cell generations be- and morphology of a persistent ring in different cause no genetic loss is involved, and there are no cells (Smith-White et al, 1963; Kistenmacher and mechanical separation difficulties at anaphase. This Punnett, 1970; Moore et al, 1973). has obvious genetic implications. Just as with interchanges, an arm of the chromo-

Incompleteness is infrequent, and the resulting some involved in the ring may participate in a http://jmg.bmj.com/ acentric fragment will be indistinguishable from a second exchange. Either an A or an S interchange terminal deletion, although it will be unrelated to will convert the centric ring to a dicentric with a the chromosome from which it will be judged to very complicated arrangement of its component have arisen (see Fig. 1). parts, and once again the observed acentric frag- Because of the inefficiency of detection, recipro- ment will not be related to the dicentric. cal translocations are commonly omitted when Symmetrical inter-arm intrachanges (Synonyms: scoring for aberrations. pericentric inversion, Paris Conference (1971), peri- As with asymmetrical interchanges, a chromo- centric inversion, inv) lead, when complete, to the on September 24, 2021 by guest. Protected some or chromosome arm can participate in more reversal or inversion of a region of chromosome than one interchange leading to some very complex containing the centromere. Unless this results in a rearrangements. very obvious change in the centromere position (a change in arm ratio, or centromeric index) the aberration will be undetectable with normal stain- 5.2 Exchanges: intrachanges ing methods. With chromosome banding tech- 5.2.1 Inter-arm intrachange (Fig. 2) The niques the larger inversions can be seen, but in prac- complete asymmetrical inter-arm intrachange is a tice the majority will be missed with both methods. centric ring (Synonyms: C/C, Paris Conference The incomplete forms will be recorded as simple (1971), ring chromosome, r). It is analogous to an terminal deletions, but, as with incomplete sym- asymmetrical interchange where the two arms in- metrical interchanges, the acentric fragment is not volved belong to the same unduplicated chromo- derived from the observed shortened end of the some, being separated by the centromere. It is chromosome which accompanies it (Fig. 2). 110 John R. K. Savage J Med Genet: first published as 10.1136/jmg.13.2.103 on 1 April 1976. Downloaded from CHROMOSOME INTRACHANGES lesions within one chromosome INTER-ARM lesions in opposite arms ASYMMETRICAL SYMMETRICAL

pre-dupilcation disposition _ centromere in loop

I- O (C/:)Cj - u centric ring pericentric inversion

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o I t(c+c)+ terminal deletions incomplete proximal incomplete distal INTRA-ARM lesions within one arm ASYMMETRICAL |SYMMETRICAL copyright.

pre-duplication disposition ~ centromere outside loop http://jmg.bmj.com/

0. inversion uo interstitial deletion paracentric

__a 0.6tww | gf0|< (tc) t( on September 24, 2021 by guest. Protected

0 v (M C) (M terminal deletions

FIG. 2. Chromosome-type intrachanges.

5.2.2 Intra-arm intrachange (Fig. 2) Asym- The interaction of two lesions within an undupli- metrical intra-change (Synonyms: interstitial dele- cated chromosome arm can result in the removal or tion, intercalary deletion, minute, double minutes, deletion of a region between the centromere and the double dot deletions, M, Paris Conference (1971): end of the arm. The ends of the deleted portion interstitial deletion, del). almost always rejoin to form an acentric ring, while Classification and relationships of induced chromosomal structural changes ill J Med Genet: first published as 10.1136/jmg.13.2.103 on 1 April 1976. Downloaded from the terminal and centric portions of the parent largest are wholly undetectable with normal stain- chromosome rejoin to give a shortened chromosome ing methods. The larger examples are resolvable arm. with chromosome banding techniques. The ring structure can be identified by the pre- The two incomplete forms will usually be indis- sence of a distinct lumen but this is obvious only in tinguishable from simple terminal deletions. the larger deletions. Some workers classify these larger forms in a separate category, acentric rings. 5.3 Breaks However, the distinction must be arbitrary, as there (Synonyms: terminal deletions, chromosome is a continuous size distribution down to (and almost breaks, rod deletions, chromosome discontinuity, certainly below) the limits of optical resolution. fracture, C; Paris Conference (1971): terminal The majority of interstitial deletions are very small, deletion, del) and most appear as paired dots. In plants, there is The complete severance of the terminal region of a evidence of a modal size of about 1 /tm, but this may chromosome arm which gives rise to a shortened be an artefact of microscopical resolution. No chromosome and an acentric fragment, and which is quantitative information is available for size distri- not associated with any obvious exchange process. bution in human cells, but observation shows that The fragment lies free among the chromosomes of the smaller deletions are much more frequent than the metaphase spread. the larger. The size of the fragment may vary from a whole Since the deleted segments do not usually remain arm down to (and presumably below) the limits of associated with their site of origin, but lie free in the optical resolution. Very small terminal deletions metaphase spread, it is rarely possible to assign will be indistinguishable from interstitial deletions, them to the parent chromosome. For this reason so that in practice it is impossible to obtain a com- the proximally incomplete forms will appear as plete separation of these two aberration forms. Be- simple terminal deletions. Distal incompleteness cause of this, many workers lump terminal and -that is, an incomplete ring-will be scored as a interstitial deletions together in a single category of terminal deletion when large, but, since most dele- 'fragments' or 'deletions'. as tions are very small, most will still appear 'double The diagrams show that many incomplete aber- copyright. dots' and be recorded accurately. As with other rations cannot easily be distinguished from a simple aberrations, incompleteness is infrequent. terminal deletion, and in Section 4 we have con- In published data from human cells, the relative sidered whether there is a true terminal deletion frequencies of interstitial deletions show consider- apart from failed exchanges. However, for scoring able variation, much larger than that found for purposes, a category for breaks is necessary, but it dicentrics and centric-rings. In part, this must must always be remembered that it constitutes a reflect differences between observers in scoring heterogeneous mixture of aberration types. http://jmg.bmj.com/ efficiency for this category. It needs to be emphasized that, when scoring, the At anaphase, interstitial deletions, like other acentric fragments derived from asymmetrical ex- acentric fragments, are invariably excluded from the changes of the dicentric and centric-ring classes are daughter nuclei, and the larger ones may form always excluded from the category of breaks, for they micronuclei. The genetic loss occasioned by large are not simple terminal deletions, but compound deletions is probably lethal to the cell, but whether fragments made from two terminal deletions. the smaller deletions are also lethal is a matter re- are an Moreover, they integral part of an aberration, on September 24, 2021 by guest. Protected quiring investigation. There is some evidence from and to include them with the breaks would be to plants which indicates that loss of small interstitial count the aberration twice. Since incompleteness deletions does not impair cell survival. is infrequent in chromosome-type aberrations, most Larger acentric rings may, on separation, show workers adopt the principle of associating one frag- the complex looping and interlocking seen in centric ment with every 'major exchange'-that is, a dicen- rings. tric or a centric-ring (UNSCEAR, 1969). Symmetrical intra-arm intrachange (Synonyms: paracentric inversion, interstitial or intercalary in- inversion, Paris Conference (1971): paracentric 6. Chromatid-type structural aberrations inversion, inv) These arise from irradiation given at the time of, Instead of being deleted, the interstitial segment or subsequent to chromosome duplication in inter- between the two lesions may be reversed (inverted), phase. so that its proximal end is now distal and vice versa. All the kinds of chromosome changes discussed in No change in arm length is involved, so that even the Section 5 occur also at the chromatid level, but the 112 John R. K. Savage J Med Genet: first published as 10.1136/jmg.13.2.103 on 1 April 1976. Downloaded from dual nature of the duplicated chromosome opens up For a given radiation exposure, about two to three the possibility for many new kinds of exchange. times as many chromatid aberrations are found as Moreover, in marked contrast with chromosome- chromosome-types. Not all of this difference can types, the majority of symmetrical forms can be be accounted for in terms of greater detectability of observed and recorded, and this increases the num- symmetrical forms and the new interactions possible ber of categories available for scoring purposes. in duplicated chromosomes. Exact numerical com-

CHROMATID INTERCHANGES lesions in different chromosomes

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i FIG. 3. Chromatid-type interchanges. Classification and relationships of induced chromosomal structural changes 113 J Med Genet: first published as 10.1136/jmg.13.2.103 on 1 April 1976. Downloaded from parisons are difficult, however, since the radio- all clear. The predominant type varies with the or- sensitivity of the cell varies in different stages of the ganism (Savage et al, 1973), and may also vary with cell cycle. the stage of the cell cycle from which a metaphase Incompleteness is higher than in chromosome- sample may be drawn. types, amounting to about 10-25% depending on In general, U-type exchanges are favoured over organism and also with the cell stage exposed and X-types, and there is a tendency for polarized ex- the aberration inducing agent used. changes to be more frequent than non-polar ones, Nearly all chemicals which result in aberrations though this does not appear to be the case in human produce chromatid-types (Kihlman, 1966) and in- lymphocytes (Savage et al, 1973). duction appears to be via metabolic pathways at Some forms (PU=#NX, NU=PX) could be specific periods of the cell cycle-in particular, the interconvertible during the cytological process of DNA synthesis phase. The majority of spon- metaphase preparation, and this may confuse the taneous aberrations are also chromatid-type. interpretation of numerical results. Such inter- convertability will not, however, account for the 6.1 Exchanges: interchanges (c/c, Fig. 3) overall differences between frequencies of the 10 Exchanges involving two or more lesions situated sample kinds. in the chromatids of different (homologous or non- homologous) chromosomes. Asymmetrical (A) and 6.1.1 Complex interchanges (Fig. 3) A chro- Symmetrical (S) forms can be scored with equal mosome may be involved in several interchanges of facility because the pairing affinity between sister the same or different kinds. Unlike chromosome- chromatids is retained until late metaphase. type interchanges, multiple participation can be When viewed at metaphase, the centromeres of observed and recorded with considerable accuracy. the exchanged chromatids may both lie on the same The number of possible combinations is very large side of the exchange (whence they are said to be and detailed classification would be tedious. For polarized, P) or be opposed to one another (non- scoring purposes, we can group complexes into two polarized, N). Occasionally exchanges may have broad categories: obligate complexes (c/c/c) where the arms arranged in a cross-like configuration and the same lesion is participating in two different inter- are sometimes referred to as quadriradials. changes, and non-obligate complexes where each copyright. Polarized exchanges are thought to be a reflection lesion is participating in only one interchange. In of the polarized condition of the centromeres. This the latter case, each exchange counts as a separate is initiated at anaphase when the centromeres form entity for numerical purposes (2 c/c, 3 c/c etc.). the leading points of the chromosomes as they move In general, non-obligate complexes are the more to the spindle poles, and the resulting arrangement frequent type. of arms appears to be retained within the nucleus http://jmg.bmj.com/ during interphase (Evans, 1962). This centro- 6.2 Exchanges: inter-arm intrachanges meric polarization should be carefully distinguished (c/cter, Fig. 4) from intra-arm polarization which restricts the re- Since lesions may occur on the same or different joining and exchange of chromosome parts (Olivieri chromatids and a lesion pair may interact in U or X et al, 1973). exchange mode, four basic forms are possible. When the disposition of the chromatids at the However, for practical scoring purposes, these may presumptive point of exchange is examined, it can be reduced to two because it is impossible to trace a usually be assigned to one of two forms, U-shaped particular chromatid through the centromeric region on September 24, 2021 by guest. Protected or X-shaped, the latter being similar to the form of a at metaphase. The two asymmetrical forms can be chiasma at early prophase of meiosis. distinguished at anaphase, and all four forms will Utilizing these factors of centromeric polarity give quite different products if they survive to sub- (P/N), exchange form (U/X), and completeness sequent cell generations. (C/I), all chromatid interchanges can be fairly easily assigned to one of 10 categories. Twelve are 6.2.1 Inter-chromatid inter-arm intrachanges shown in Fig. 3, but the symmetrical incomplete The asymmetrical form appears at metaphase as a types of both polarized and non-polarized inter- centric ring, and is so classified for scoring purposes. changes cannot be distinguished. It is the inter- In reality, however, it is a chromatid dicentric, and play of these three factors which really determines this is clearly seen at anaphase, since the two centro- exchange symmetry. meres always go opposite poles of the spindle and The 10 categories do not occur with equal fre- form an anaphase bridge. Both daughter cells quency, though the reasons why this is so are not at therefore will be affected by the damage. The 114 John R. K. Savage J Med Genet: first published as 10.1136/jmg.13.2.103 on 1 April 1976. Downloaded from CHROMATID INTER-ARM INTRACHANGES lesions in opposite arms of one chromosome (C7/C tcr) ASYMMETRICAL SYMMETRICAL

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The proximal and distal incomplete forms (not lated. http://jmg.bmj.com/ illustrated in Fig. 4) are distinguishable if sister- chromatid pairing affinity is retained until meta- 6.2.2 Intra-chromatid inter-arm intrachanges phase, otherwise the proximal form is likely to be The asymmetrical form analogous to an X-type ex- scored as a terminal deletion. change produces a chromatid centric-ring and a com- The symmetrical form is indistinguishable from pound acentric fragment derived from the opposite the symmetrical intra-chromatid counterpart, but is ends of the same chromatid. The sister chromatid quite different from it in resultant structure. It is is unaffected. generally referred to as a double duplication-deletion, Ring size may vary, sometimes including almost on September 24, 2021 by guest. Protected and is best understood by unravelling the loop dia- the entire chromosome length. gram shown in Fig. 4. The proximal and distal incomplete forms are The complete intrachange results in two chro- usually distinguishable, although incompleteness matids whose terminal regions beyond the point of may lead to a degree of unravelling by the time the exchange are mirror-images. Thus, if the original aberration reaches metaphase. In this case, the constitution of chromosome were a b c d-e f g h i former may be scored as one, the latter as two chro- with exchange points between b c and f g, the two matid terminal deletions. derived chromatids would be a b c d-e f:: b a and The symmetrical counterpart, answering to a U- ihg:: cd-efghi. type exchange is a pericentric inversion in one chro- It is easy to see that, if the loop containing the matid, while the sister chromatid remains un- centromere were very small, an isochromosome- affected. Here again a retention of sister chroma- that is, a chromosome whose opposite arms are tid pairing to metaphase will prevent the incomplete genetically identical mirror images (Paris Con- forms from being scored as simple terminal dele- Classification and relationships of induced chromosomal structural changes 115 J Med Genet: first published as 10.1136/jmg.13.2.103 on 1 April 1976. Downloaded from CHROMATID INTRA-ARM INTRACHANGES lesions within one arm of one chromosome (CAC ) X-TYPE U-TYPE copyright.

FIG. 5. Chromatid-type intra-arm intrachanges. http://jmg.bmj.com/ tions. At anaphase, the acentric fragments from In each type, failure of the exchange process can both incomplete types will appear as terminal lead to two incomplete types, making eight in all. deletions, but it must be remembered that the Rarely, doubly incomplete types are found, sug- fragment is not directly related to the observed gesting that the whole exchange process has failed. shortened chromosome arm. This point is in dispute, however, and there are valid alternative mechanisms to account for these on September 24, 2021 by guest. Protected 6.3 Exchanges: intra-arm intrachanges (c/c, doubly incomplete forms. Fig. 5) Revell (1959), who has studied intra-arm intra- Just as with inter-arm forms, there are four basic changes in considerable detail, has numbered the types of simple-that is, single-intra-arm intra- four basic types (R1-R4 in the diagrams, Fig. 5) changes, based on lesions in the same or different and his notation is now usually employed in critical chromatids, and lesion interaction in U or X mode. work. Convention has assigned names to the more Unlike the inter-arm (centric) forms, the majority obvious forms, and these are given below. of intra-arm types are small, and seldom retain much of their original interphase conformation through to 6.3.1 Inter-chromatid intra-arm intrachanges metaphase, for this is lost as the chromosomes coil Isochromatid deletions (synonyms: isochromatid and contract in preparation for mitosis. Conse- breaks, isolocus breaks, Revell type 4, i: distinguish quently, the scoring of the four types is not equally carefully from the symbol i used by Paris Con- efficient. ference (1971) for isochromosomes) are the asym- 116 John R. K. Savage J Med Genet: first published as 10.1136/jmg.13.2.103 on 1 April 1976. Downloaded from metrical form resulting from a U-type exchange be- is involved. The smaller complete ones will tend to tween the sister chromatids. Visually it appears as be overlooked. if both sister chromatids have been severed at the same point, and that the broken ends of the sisters 6.3.2 Intra-chromatid intra-arm intrachanges have rejoined (Sister Union, SU). This aberra- The asymmetrical form is a chromatid minute tion constitutes the most obvious and least ambigu- (Synonyms: interstitial deletion, dot deletion, m, ous form of intrachange. Revell type 2). It arises from an X-type exchange The two single incomplete types are readily dis- and results in the deletion ofa small (frequently very tinguishable: non-union of the centromeric portion small) intercalary segment of one chromatid. The (non-union proximal, NUp Revell type 4a) or non- ends of the deleted portion are nearly always united union of the acentric portion (NUd Revell type 4b). to form an acentric ring-fragment, but this is obvi- Doubly incomplete types (NUpd) are indistin- ous only in the larger deletions where a distinct guishable from chromosome-type terminal deletions, lumen can be observed. The ends of the parent unless there is marked inequality in the lengths of chromatid rejoin to give a shortened chromatid. the sister chromatids of the acentric and/or the cen- The other sister chromatid is structurally unaffected, tric portion. Chromosome-type aberrations never except that, when a large piece has been deleted, show either sister union or inequalities in sister- sister chromatid pairing leads to buckling or bending chromatid lengths. The frequency of true NUpd is at the site of deletion. a matter in dispute. They appear to be rare, but The two singly incomplete types differ, proximal there may well be differences between organisms, incompleteness (type 2a) leads to a terminal deletion and between cells from different regions of inter- plus a minute. In the case of small deletions, distal phase. There are certainly differences when incompleteness (type 2b) will not be distinguishable radiations of different Linear Energy Transfer from the complete type 2 because the limits of (LET) are employed to induce chromatid aberra- resolution preclude the direct observation of a ring tions in plant cells (Lea, 1946; Savage et al, 1968). structure. Larger deletions will appear as a ter- The loop origin shown in the diagrams can often minal deletion without a very obvious source. In be inferred from eccentrically placed achromatic this case, one looks for a bent or buckled chromo- lesions which presumably mark the regions of ex- some. copyright. change, and also that the two acentric fragments of Minutes are very often dislodged from the site of NUd types are often unequal. However, the the intrachange during cytological metaphase pre- existence of true isolocus lesions giving rise to SU paration, and come to lie loose among the other without loop origin cannot be ruled out, especially chromosomes. Thus they are frequently over- for irradiation during S phase, and for chemical looked or obscured, and scoring efficiency can be aberration induction. considerably reduced. One other effect of the dis- The symmetrical form is a duplication-deletion placement of the minute is that complete types with http://jmg.bmj.com/ (Synonym: Revell type 1) derived from an X-type buckling, and proximally incomplete types (2a) can exchange between sister chromatids. It results in be mistaken for types 1 and la respectively (see Fig. the removal of an intercalary segment from one 5). sister chromatid arm, and its insertion in tandem The corresponding symmetrical form arising from fashion (without inversion) in the other. The con- a U-type exchange produces a paracentric inversion sequent inequality in lengths of the two chromatids (Revell type 3) of an intercalary segment. No dif- leads, in the complete type, to buckling or a 'dog-leg' ferences in relative lengths between the sister chro- on September 24, 2021 by guest. Protected bend at the point ofthe exchange. matids result and, unless the inverted segment is With care, both singly incomplete forms are long enough to cause breakdown in the pairing recognizable, each contributing a terminal deletion. affinity of sister chromatids (shown by a bowing-out In type la, the deleted arm is incomplete, so that of the affected region), the change will not be detec- there is an obvious separation between fragment and ted. Since most inversions are small, scoring effici- centric portion where the insertion containing arm ency for complete type 3 intra-changes is very low. has straightened out (Fig. 5). In the other type Both singly incomplete types (3a, 3b) are indis- (lb, Fig. 5) the insertion arm is incomplete, and tinguishable from one another and will be scored as there is an obvious overlap and dislocation in the simple terminal deletions. region of the breaks. In some cases, the fragment will contain the duplication. 6.4 Breaks Of course, ability to detect type 1 intrachanges Chromatid terminal deletions (Synonyms: chro- presupposes that a reasonable length of chromatid matid break, chromatid discontinuity, c). Classification and relationships of induced chromosomal structural changes 117 J Med Genet: first published as 10.1136/jmg.13.2.103 on 1 April 1976. Downloaded from For scoring purposes, any complete severance of categories, the dicentric triradial and the monocentric the chromatid thread showing clear dislocation, and triradial, and these are shown in Fig. 6. not obviously derived from an incomplete exchange Metacentric chromosomes can form a triradial process, constitutes a simple terminal deletion. between opposite arms-that is, an isochromatid- Since incompleteness is much higher for chromatid chromatid inter-arm intrachange (i/c, Fig. 6). than for chromosome-type aberrations the class of Triradials can occur together with other inter- observed terminal deletions will always be inflated changes in the formation of Complexes (Section by the inclusion of failed exchanges, particularly 6.1.6). from the intra-arm intrachange class. Protagonists of the exchange theory (Section 4) argue that this inflation is sufficient to account for 7.2 Isochromatid-isochromatid exchanges all observed breaks. (i/i, Fig. 6) The situation is, however, much more complex The joining of the centric portions of two iso- than was at first thought. The contribution of the chromatid deletions to give what appears to be a various incomplete intrachange types appears to chromosome-type dicentric. It is only distinguish- vary throughout the cell cycle, and also with the able from a true dicentric by the fact that there are agent used to induce aberrations. There may also always two fragments both of which (very rarely be real differences between organisms. Add to this only one) show sister union. Inter-arm isochroma- the existence of more complicated types of multi- tid-isochromatid intrachanges (i/i) also occur, and lesion intrachanges (some of which are discussed these will simulate chromosome-type centric rings. below, Section 7) many of which can simulate the simple types, and it can be seen that tests of aberra- 7.3 Insertion intrachanges tion theories based on the numerical relationships of These comprise certain members of a whole class the various simple types, as most tests are, are likely of intrachanges, some kinds of which occasionally to be subject to considerable error. figured in early literature, but which were first de- scribed in detail by Fox (1967) for G2 induced

7. Additional chromatid aberration types aberrations in the locust Schistocerca. copyright. So far we have dealt with the types most fre- They involve the interaction of three or four quently encountered in aberration experiments lesions variously situated in the sister chromatids of where radiation is used as the inducing agent. a single chromosome arm. There are, however, other types which occur For the three lesion forms, Fox's derivation relatively infrequently after radiation, but which are makes them equivalent to isochromatid/chromatid likely to become important as experiments with intra-arm intrachanges-that is, the interaction of a chemical mutagens increase. Many of these muta- chromatid and isochromatid deletion within an arm http://jmg.bmj.com/ gens act at the time of, or require the processes of, (cf. 7.2 above). DNA synthesis to form structural aberrations, and The whole family can also be derived from two the possibility of multi-lesion aberrations is greatly intrachanges of the same or different types occurring increased. There is plenty of evidence in the litera- very close together in the neck of a Revell loop of the ture that the forms discussed below are much more kind shown in Fig. 5 (Revell, 1974). common among chemically induced aberrations. When viewed at metaphase, the majority are in- distinguishable from simple intrachanges. There 7.1 Triradials are, however, some very characteristic types, a few on September 24, 2021 by guest. Protected (Synonyms: isochromatid-chromatid inter- of which are drawn in Fig. 6, and careful scoring of changes, i/c) (Fig. 6) these in good preparations of plant chromosomes As the name suggests, these appear as a three- suggests that the frequency of insertion intrachanges armed configuration involving two chromosomes, and related types is much greater than has hitherto and are normally accompanied by an isochromatid been realized. This is particularly true when deletion fragment which shows sister union. radiations of high LET are used. The simplest explanation on classical theory is If this finding be generally true, then, as already that they arise from the interaction of an isochroma- mentioned, numerical scores of 'simple' intra- tid break and a chromatid break-that is, they are changes are going to be very difficult to obtain. three-break aberrations. For exchange theory Moreover, since there is an increased chance of derivation, see Revell (1966). incompleteness when two intrachanges are involved, Since the isochromatid portion involved in ex- the 'contamination' of the terminal deletion change may be centric or acentric, there are two category will be augmented. In several cases, 118 John R. K. Savage J Med Genet: first published as 10.1136/jmg.13.2.103 on 1 April 1976. Downloaded from

TRIRADIALS isochromatid/chromatid interchange (i/c)

DICENTRIC FORM MONOCENTRIC FORM

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INTERCHANGE (i/i) INTER-ARM INTRACHANG ( j) on September 24, 2021 by guest. Protected

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e"dicentric" A "centric ring"

FIG. 6. Additional chromatid-type structural changes. incompleteness in both intrachanges leads to a which appears to involve only part of the thickness NUpd configuration (Section 6.3, and Fig. 6). of a chromatid at any one point. Since there is always some degree of stickiness and chromosome 7.4 Sub-chromatid aberrations clumping produced by radiation at this time (Sec- Irradiation of cells very close to mitosis (very late tion 8.3), usually only the exchange forms are seen G2, early prophase) results in a class of aberrations when cells enter anaphase. The most characteristic Classification and relationships of induced chromosomal structural changes 119 J Med Genet: first published as 10.1136/jmg.13.2.103 on 1 April 1976. Downloaded from type is a single bridge, with fragments attached to it but the term has since been redefined and applied to ('side-arm' bridges). mammalian cells for an effect often noted after cer- The structure of those bridges can be seen with tain virus infections-for example, Nichols et al particular clarity in some plant materials (Wilson (1965); Henry et al (1971). et al, 1959; Wilson and Sparrow, 1960) and from The chromosomes are reduced to masses of very such studies it has been inferred that the exchange small, thin fragments, and individual chromosomes involves a longitudinal sub-unit of the chromatid. are seldom recognized. Occasionally longer frag- Kihiman and Hartley (1967) have challenged this ments ofuncoiled chromosome lie among the debris. concept of the sub-unit and come to the conclusion The phenomenon appears to be confined to cells that these aberrations are in reality true chromatid that have fused in culture or formed syncytial aberrations disguised by the processes of chromo- masses. In this respect, one cannot help but be some contraction which precedes mitosis. struck by the similarity of the published photo- The recent demonstration (Wolff, 1970) that G1 graphs of pulverization, and the phenomenon of (unduplicated) chromosomes in human lympho- precocious chromosome condensation (PCC) seen cytes treated with such compounds as urea before when interphase cells, particularly those in S phase, irradiation can yield unambiguous chromatid aberra- are fused with cells in division under the action of tions at first post-treatment mitosis indicates, how- virus fusing factors (Johnson and Rao, 1970; ever, that the question of chromosome sub-units is Sperling and Rao, 1974). Such 'pulverization' does by no means settled. not constitute a degradative effect of virus infection, but corresponds with the visualization of chromo- 8. Unconventional aberrations somes in the process of duplication. From time to time, various chromosomal changes are reported which cannot be classified within the 8.3 Physiological effects (stickiness) framework outlined above. Some of these have This is a transient phenomenon found for a short been given descriptive names, but the exact nature time immediately after the exposure of cells to of the changes is unknown and their occurrence radiation, and affecting primarily those cells actually in stages of mitosis, especially late prophase. The serves to show how much more remains to be learnt copyright. about chromosome structure and the mechanisms of chromosomes appear to be sticky, often joined to aberration formation. one another by fine threads, and tending to clump into a mass at division. 8.1 Shattering In some materials, the effect may be present for a This was first described in detail for G2 pollen number of hours and makes it almost impossible to cells irradiated with particular wavelengths (- 265 obtain accurate aberration scores at this time. As nm) of ultraviolet light (Lovelace, 1954). How- noted above, it is concomitant with the period when http://jmg.bmj.com/ ever, the phenomenon occurs in the same material sub-chromatid aberrations occur. exposed to ionizing radiations of high LET, and is Early workers suggested that the effect might be occasionally encountered in mammalian cells in due to degradation or depolymerization of DNA. culture even with low LET radiations. However, the radiation doses which produce sticki- The affected chromosomes, as seen at metaphase, ness are very low for extensive damage ofthis sort. appear to have broken up into many small pieces of Electron micrographs of condensed chromosomes varying length. Some of them can be seen to be coupled with the limits of resolution of the optical minute acentric rings, and other pieces may show microscope make it unwise at present to press on September 24, 2021 by guest. Protected partial exchange and isochromatid-type changes. simple solutions to this problem. Sometimes all the chromosomes in a cell are affected, but frequently only one or two are shat- 8.4 Agglomerated mitotic divisions tered, the rest appearing normal, or showing con- It is now generally believed that ultrasound of the ventional chromatid-type structural changes. frequencies employed for diagnostic purposes does In the case of high LET radiations, the pheno- not induce structural aberrations ofthe conventional menon may reflect the highly localized deposition of types described in earlier sections. In plant cells, energy along the particle tracks, but this suggestion however, it does appear to have an effect very simi- will not suffice for UV. lar to the physiological effects (8.3) acting on cells in or very near division at the time of sonication. Pro- 8.2 Pulverization phases are 'bridged' by strands between chromo- The original descriptions for plant cells suggest somes and collapse into a sticky mass. Affected that the phenomenon was very similar to shattering, cells fail to complete division. Such cells have been 120 John R. K. Savage J Med Genet: first published as 10.1136/jmg.13.2.103 on 1 April 1976. Downloaded from termed agglomerated mitotic divisions (Gregory et al, cells that are well spread, or with minimal chromo- 1974). some overlapping. Cells carrying aberrations of As with physiological effects, the phenomenon is the more complicated types seldom have their transitory, and cells appearing in division at later chromosomes laid out neatly. As far as possible, times are normal. an attempt should be made to score every complete cell in a sample, and only to reject when it really 9. Scoring and scoring categories proves impossible to untangle a configuration. c. It is usually assumed for statistical purposes The method used for scoring aberrations will that the distribution of aberrations between nuclei depend, of course, upon the purpose for which the conforms to the terms of a Poisson distribution. If data are required. However, 'there can be no ques- this is valid in a particular case (and it is wise to test tion that the maximum information can only be the assumption, Savage, 1970), then the width of obtained when aberrations are categorized on the the standard error will depend simply upon the basis of their detailed structure. The Committee, number of aberrations scored. At low aberration therefore, strongly recommends that the detailed yields, therefore, it is better (though more laborious) system of scoring be employed, particularly in those to score a fixed number of aberrations than a fixed cases where attempts are made to obtain information number of cells. on dose-response relationships' (UNSCEAR, 1969, d. In any experiment, tests should always be p. 103, para. 50). made for variation in aberration yield with sampling Suggested procedures for scoring and recording time after treatment. If this be present, as is aberrations have been discussed in several publica- usually the case with chromatid aberrations, the tions-for example, Buckton and Evans (1973). interpretation ofquantitative data becomes extremely The aberration symbols given in the text and figures complicated, and its usefulness for such purposes as of this annotation (derived primarily from the work dose-response curves rather limited (Savage and of Catcheside et al (1946) have proved to be a Papworth, 1973). valuable shorthand for scoring purposes, and are widely used in radiation cytogenetics. Set out as

column headings (Table I), they enable accurate 10. Secondary or derived aberrations copyright. recording of all types of structural change on a cell All the structural changes discussed so far are by cell basis. Chromatid intrachanges can be those seen at thefirst metaphase after exposure to the assigned Revell numbers and chromatid inter- aberration inducing agent. changes designated by the factors outlined in The majority of these are lethal to the cell that Section 6.1. More complicated or unusual aber- carries them, for they occasion mechanical diffi- rations should be drawn and co-ordinate slide

culties and chromosome loss at anaphase with con- http://jmg.bmj.com/ references given to allow rechecking. sequent genetic imbalance in the daughter cells. In the case of chromosome-type aberrations, since both TABLE I chromatids are affected at any one locus, both COLUMN HEADINGS FOR SCORE SHEETS daughter cells will be subject to equal genetic loss, some Chromosome-type g, G C M C/C C/C Complexes, and to death. In contrast, forms of chroma- etc. tid-type aberrations, since they affect only one sister may lead to loss and death Chromatid-type g, G c c/c c/cter chromatid, genetic c/c Complexes, on September 24, 2021 by guest. Protected i/c, etc. of only one ofthe daughter cells. Some aberrations of both chromosome and chro- tid-types, however (mostly symmetrical forms), can Before leaving this topic, a few cautionary com- survive division and may be transmitted to future ments may not be out of place. cell generations, either intact or in some modified a. All slides should be scored under a code, so form. Acentric portions tend to be lost during that the scorer is unaware of the dose or treatment transmission. which the cells have received. The bias that know- Chromatid-type aberrations which survive the ledge of such facts can introduce can altogether first division are converted by duplication into spoil deductions from observation. The subjec- secondary chromosome types, so that all stable and tive element is very real. semi-stable forms observed in later cell generations b. Beware of cell selection. It is right to select appear as chromosome-type changes, whatever complete (unbroken) cells having a full complement their primary origin. Moreover, the same secon- of centromeres, but it is dangerous to select only dary change can arise from a variety of different Classification and relationskips of induced chromosomal structural changes 121 J Med Genet: first published as 10.1136/jmg.13.2.103 on 1 April 1976. Downloaded from TABLE II

Secondary or Derived (Transmissable) Potential Primary Source Structural Change Name Symbol Chromosome-type | Chromatid-type Reciprocal translocation t, rcp S interchange S interchange (PXC, NUC) Pericentric inversion inv Inter-arm S intrachange Intra-chromatid interarm S intrachange Paracentric inversion inv Intra-arm S intrachange Type R3 intrachange Terminal deletion del Incomplete forms of: A and S Incomplete interchange (PUIp, NXIp, interchange PXI, NUI): incomplete inter- chromatid inter-arm A and S intrachange; type Rla, R2a, R3a, and R4a intrachanges Interstitial deletion del Intra-arm A intrachange Type RI, Rlb, R2 and R2b mtrachanges Insertion (within chromosome): direct ins inverted inv ins - Complex multiple interchange Duplication dup _ Type RI interchange; insertion intrachanges Insertion (between chromosomes): direct ins 1 inverted inv ins 1lnterchange + itrachange complex Interchange + intrachange complex Isochromosome i S interchange (opposite arms of Inter-chromatid inter-arm S homologous chromosomes) intrachange; type R4, R4b intrachanges Ring r Inter-arm A intrachange Intra-chromatid inter-arm A intrachange Dicentric (within chromosome) dic _ Inter-chromatid inter-arm A intrachange: type 4 and 4b intrachanges Dicentric (between chromosomes) tdic A interchange A interchange (PUC, PUId, NXC,

NXId); dicentric triradial; copyright. isochromatid/isochromatid interchange

aberration types, so that it is seldom possible to must therefore look for the simplest origins as those infer the origin of any particular example. being the most likely.

The nomenclature for stable derived changes has In this respect, it will be noted from Table II that http://jmg.bmj.com/ been standardized (Chicago Conference, 1966; Paris chromatid-type aberrations are a much more fruit- Conference, 1971). The terminology employed is ful source of change than chromosome-types, descriptive, and concentrates on the resultant effect especially for the more complicated secondary types. of a structural change in the surviving chromosome In mammals, the majority of cells in those tissues of or chromosome part, rather than the aberration the body in active mitosis (bone marrow, gut and type. It is therefore not readily applicable to the skin epithelium, male germ cells) will yield chro- classification ofprimary changes. matid-type damage on exposure to aberration induc- In the above table (Table II), an attempt has ing agents. on September 24, 2021 by guest. Protected been made to show the relationship of the more Structural changes that are inherited may be frequently encountered secondary types (as deter- subject to further modifications by the process of mined by banding techniques), and their potential crossing-over at meiosis in the germ cells. Dis- primary progenitors. Ifbanding is not used, then a cussion of these changes is beyond the scope of this number of the derived changes listed will not be de- annotation, but an example of the origin of such tectable cytologically in mitotic cells. recombinant (rec) chromosomes arising from cross- The origins given are not, of course, the only pos- ing-over in a pericentric inversion is given in Paris sible ones. All kinds of bizarre multi-break com- Conference (1971). binations can be invoked to account for observed rearrangements, but the fact remains that the com- I would like to thank Dr R. H. Mole, Director of the MRC Radiobiology Unit, and Dr V. Hulse who pain- plex aberration configurations required to satisfy stakingly read the manuscript and offered many valuable these combinations are seldom encountered except suggestions for improving its clarity for non-specialist after very high radiation or chemical dosages. We readers. 122 John R. K. Savage J Med Genet: first published as 10.1136/jmg.13.2.103 on 1 April 1976. Downloaded from REFERENCES metric) in the formation of chromosomal aberrations. Mutation Bateman, A. J. (1968). Non-disjunction and isochromosomes from Research, 20, 101-106. irradiation of chromosome 2 in Drosophila. In Effects of radiation Paris Conference (1971). Standardization in human cytogenetics. on Meiotic Systems, pp. 63-70. IAEA, Vienna. Birth Defects: Original Article Series, 8, No. 7, 1972. The Buckton, K. E. and Evans, H. J. (eds) (1973). Methods for the National Foundation-March of Dimes, New York. Analyses of Human Chromosome Aberrations. WHO, Geneva. Revell, S. H. (1959). 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