Ann. Hum. Genet., Lond. (1963), 27, 189 With 6 plate8 Printed in &eat Britain 189 A small autosomal ring chromosome in a female infant with congenital malformations BY MARY LUCAS AND N. H. KEMP Haematology Department, St George’s Hospital, London, S. W. 1 J. R. ELLIS AND RUTH MARSHALL* Galton Laboratory, University College London, W.C. 1 Human karyotypes with ring chromosomes were first observed by Levan (1956) in cells from two tumours-a finding which confers on the ring chromosome the distinction of being the first structural aberration of a chromosome to have been recognized in man. Subsequently, ring chromosomes have been found in leucocytes from a patient with leukaemia (Baikie, Court Brown, Jacobs & Milne, 1959), and as one of the many aberrations resulting from exposure to X-irradiation (Tough, Buckton, Baikie & Court Brown, 1960; Buckton, Jacobs, Court Brown & Doll, 1962), or gamma-ray/fission neutron irradiation (Bender & Gooch, 1962). The association of ring chromosomes with congenital malformations has also been observed on four occasions. In each instance, the ring chromosome occurred in a complement of 46 chromosomes and in three of them the rings were derived f m different chromosomes. In one case an X chromosome was involved (Lindsten & Tillinger, 1962), two others involved small autosomes (Wang, Melnyk, McDonald, Uchida, Carr & Goldberg, ’1962), while in the fourth (Smith-White, Peacock, Turner & Den Dulk, 1963) it was uncertain whether the ring was derived from an X chromosome or one of the larger autosomes of the 6-12 group (Denver classification). The ring X chromosome (Lindsten & Tillinger, 1962) occurred in a female with gonadal dysgenesis. The ring was moderately large and rather unstable as many cells had just one normal X chromosome, these cells having presumably arisen after elimination of the ring chromosome. A few cells were also observed with two ring chromosomes. Of the two identified autosomal ring chromosomes (Wang et al. 1962), one was derived from a large acrocentric and the other from a no. 17 or 18. The former occurred in a male infant with multiple congenital abnormalities of the type usually found associated with trisomy 17 or 18. The ring chromosome was small and moderately stable for no cells were observed without it and only a few cells were recorded with abnormally large rings. The significance of this ring chromosome in relation to the malformations was uncertain, as the mother had been exposed to X-rays prior to conception and the authors considered that as a result of other structural changes, the karyotype of the affected child also showed partial trisomy for nos. 17 or 18. In contrast, the mother of the child with the ring chromosome derived from nos. 17 or 18 had never been exposed to X-irradiation. This child had minor multiple malformations and was mentally retarded. The ring chromosome was again small and, although no cells were observed without it, some instability was apparent, as approximately one-sixth of the cells were recorded with large dicentric rings. The fourth instance of a ring chromosome (Smith-White et al. 1963) occurred in a mentally and physically retarded pubertal male. The ring was moderately large, but it varied in size as a result of * Present address : Haematology Dept., King’s College Hospital Medical School, London, S.E. 6. 13-2
Transcript
Ann. Hum. Genet., Lond. (1963), 27, 189 With 6 plate8 Printed in &eat Britain
189
A small autosomal ring chromosome in a female infant with congenital malformations
BY MARY LUCAS AND N. H. KEMP Haematology Department, S t George’s Hospital, London, S. W . 1
J. R. ELLIS AND RUTH MARSHALL* Galton Laboratory, University College London, W.C. 1
Human karyotypes with ring chromosomes were first observed by Levan (1956) in cells from two tumours-a finding which confers on the ring chromosome the distinction of being the first structural aberration of a chromosome to have been recognized in man. Subsequently, ring chromosomes have been found in leucocytes from a patient with leukaemia (Baikie, Court Brown, Jacobs & Milne, 1959), and as one of the many aberrations resulting from exposure to X-irradiation (Tough, Buckton, Baikie & Court Brown, 1960; Buckton, Jacobs, Court Brown & Doll, 1962), or gamma-ray/fission neutron irradiation (Bender & Gooch, 1962). The association of ring chromosomes with congenital malformations has also been observed on four occasions. In each instance, the ring chromosome occurred in a complement of 46 chromosomes and in three of them the rings were derived f m different chromosomes. In one case an X chromosome was involved (Lindsten & Tillinger, 1962), two others involved small autosomes (Wang, Melnyk, McDonald, Uchida, Carr & Goldberg, ’1962), while in the fourth (Smith-White, Peacock, Turner & Den Dulk, 1963) it was uncertain whether the ring was derived from an X chromosome or one of the larger autosomes of the 6-12 group (Denver classification).
The ring X chromosome (Lindsten & Tillinger, 1962) occurred in a female with gonadal dysgenesis. The ring was moderately large and rather unstable as many cells had just one normal X chromosome, these cells having presumably arisen after elimination of the ring chromosome. A few cells were also observed with two ring chromosomes. Of the two identified autosomal ring chromosomes (Wang et al. 1962), one was derived from a large acrocentric and the other from a no. 17 or 18. The former occurred in a male infant with multiple congenital abnormalities of the type usually found associated with trisomy 17 or 18. The ring chromosome was small and moderately stable for no cells were observed without it and only a few cells were recorded with abnormally large rings. The significance of this ring chromosome in relation to the malformations was uncertain, as the mother had been exposed to X-rays prior to conception and the authors considered that as a result of other structural changes, the karyotype of the affected child also showed partial trisomy for nos. 17 or 18. In contrast, the mother of the child with the ring chromosome derived from nos. 17 or 18 had never been exposed to X-irradiation. This child had minor multiple malformations and was mentally retarded. The ring chromosome was again small and, although no cells were observed without it, some instability was apparent, as approximately one-sixth of the cells were recorded with large dicentric rings. The fourth instance of a ring chromosome (Smith-White et al. 1963) occurred in a mentally and physically retarded pubertal male. The ring was moderately large, but it varied in size as a result of
* Present address : Haematology Dept., King’s College Hospital Medical School, London, S.E. 6. 13-2
190 MARY LUCAS, N. H. KEMP, J. R. ELLIS AND RUTH MARSHALL breakage and reunion. Interlocked and dicentric rings were also noted. No cells were observed with two rings, which to the authors suggested that the trisomic condition was lethal, and that the origin was from an autosome of the 6-12 group rather than an X chromosome. However, this explanation may be invalidated by the presence of monosomic cells lacking the ring chromosome; monosomy for a large autosome would be expected to be as lethal as trisomy.
The ring chromosome reported in the present communication was found in a female infant aged 1 year (Fig. 1). Clinical examination at 9 months of age had revealed that she was mentally subnormal; she was irritable when handled, she did not respond to social overtures and did not play with toys. Muscle tone was normal although, when pulled to the sitting position, there was a slight head lag. Her head was small (circumference 14$ in,), was flattened over the occipital region and the anterior fontanelle was only just palpable. The lower jaw was rather small and there was mid-line cleft palate; there was no hare-lip and the ears were normal. There were no obvious dermatoglyphic abnormalities (Fig. 2). Apart from a congenital dislocation of the left hip, clinical examination revealed no further abnormalities.
The patient had been delivered normally at term after an uneventful pregnancy. At birth of the patient, the mother was 25 years old and the father 24 years old. The mother had not been X-rayed nor had she suffered from pre-eclamptic toxaemia, rubella or any other viral illness during the pregnancy. There were two previous pregnancies; the first resulted in male dizygotic twins who are healthy and appear normal, whilst the second terminated in spontaneous abortion at 4 months.
No other congenital abnormalities are known in the family although two paternal aunts died in infancy.
CYTOLOGY
The chromosomes of the patient were examined in cells from peripheral blood cult'ured by a modification of the method described by Moorhead, Nowell, Mellman, Battips & Hungerford (1960) and from fibroblast tissue cultures established from skin biopsies using the technique described by Harnden (1960). The results of these studies from two separate specimens of both skin and blood are shown in Table 1.
Both tissues showed chromosome mosaicism with principally two cell lines. In the minority were cells with 46 chromosomes and a normal female karyotype (Pl. 2a). The other cell line, which predominated, also had 46 chromosomes (Pl. l), but one small autosome, identified as no. 18 (Pl. 3) was represented by a small aberrant highly contracted chromosome, approxi- mately the size of a small acrocentric. The morphology of this chromosome at metaphase varied considerably from one cell to another, having either a quadri-partite, a bipartite or horseshoe- shaped structure while in some cells, detailed morphology was not discernible (Pl. 5a-e). In prophase, a ring structure was frequently observed (Pl. 4). The highly variable appearance of this aberrant chromosome a t metaphase is most probably the result of observing an excessively contracted ring at different angles as illustrated in Fig. 3. Further observations of the aberrant chromosome in cells undergoing endo-reduplication (PI. 6) are consistent with the postulated ring structure.
As both skin cultures aged, the proportion of cells with ring karyotypes decreased and those with normal karyotypes increased (Table 1). A similar tendency was shown in the blood cultures, which, although derived from separate specimens, were examined after different periods in culture. These results suggest that the cell line containing the ring chromosome was less well
Ring chromosome in a female infant with congenital malformations 191
m
192 MARY LUCAS, N. H. KEMP, J. R. ELLIS AND RUTH MARSHALL
adapted to survival in culture than those with normal karyotypes. The ring chromosome appeared to be remarkably stable for only a very few cells were observed with large rings (Pl. Sf) or with two rings. Some cells with 45 chromosomes, monosomic for no. 18 (PI. 2b) were observed in the skin cultures and presumably had arisen through loss of the ring. As the cultures aged, the proportion of these monosomic cells rapidly decreased, which suggests that they were even less well adapted to the cultural conditions than those containing a ring. If this is so, then i t is conceivable that a significant proportion of monosomic cells could have been present in the original biopsy material, and that the patient is a triple mosaic.
Table 1 . Karyotype analysis of cells in cultures of jibroblasts and leucocytes Karyotype and chromosome number
Days in
culture
Skin (I) 22
5 0
Skin (2) '4 68
Blood 2 3
zn = 46, normal 9
(%I 18 32
9 39 8 32
nn = 46, with ring (%I 72 66
76
76 57
66
2n = 45, zn = 46, mono- with somic large
forno. 18 ring (%) (%I 9 I
2
I3 I 2 2
I 2 2
zn = 47, with two No. rings cells (yo) examined
77
I 108 I I 0
I 0 0
4 24 41
Fig. 3. Diagram representing the morphological appearances of contracted ring chromosome when viewed from different angles.
Chromosome studies on peripheral blood leucocytes of the mother, father and two sibs showed no variation from the normal human karyotype with the sex chromosome patterns in agreement with the phenotypic sex in each instance.
Ai~r~,aI.s of Human Genetics, Vol. 27, Part 2 Plate 1
Mitotic coll and chromosomc analysis showing trhc abcrrant ring chromosomc.
M A R Y LUCAH, N. H. KEhfP, J. R. ELLIS AND RUTH MARSHALL (Facit%g $3. 192)
Annals of Human Genetics, Vol. 27, Part 2
Karyotypes showing (u) normal female, ( b ) monosomy for chromosome no. 18.
MARY LUCAS, N. H. ICEMP, J. R. ELLIS AND RUTH MARSHALL
Plate 2
Annals of Human Genetics, Viol. 27, Purt 2 Plate 3
Chromosomes of 16-18 group from normal colls (loft), from cells with the ring chromosome (centre) and from oells rnoriosomic for no. 18 (right). (S-from fibroblast skin cultures ; B-from periphorul blood loucocytc oultures.)
MARY LUCAS, N. H. KEMP, J . It. EI,JAIS AND RUTH MARSHALL
Annuls oJ Human Genetics, Vol. 27, Part 2
The aberrant ring chromosome shown in prophase and prornetapliase.
Plate 4
MARY LUCAS, N. H. KEMP, J. R. ELLIS AND RUTH MARSHALL
Arw,alx of Human Genetics, Vol. 2'7, Pwrt 2 Plate 5
Mitotic metaphases showing the different morphological appearances of the ring chromosome (inset). (u, b, G, d, j, from skin fibroblasts; e, from blood leucocytes.)
MARY LUCAS, N. H. KEMP, J . R. ELLIS AND RUTH MARSHALL
Annals of Human Genetics, Vol. 27, Part 2 Plate 6
Chroinosonim from fibroblasts showing the morphology of tho abcrrant ring chromosome (rtrrowed) after endo-reduplication.
MARY LUCAS, N. H. KEMP, J. R. ELLIS AND RTJTH MARSHALL
Ring chromosome in a female infant with congenital malformations 193
SEROLOQY
The blood groups of the patient, her parents and twin brothers are shown in Table 2. Since about 70 % of the patient's cells showed deficiency in the chromosome material on one no. 18, a number of quantitative tests, using the titration method, were performed to search for hemizygous loci. The assumption wm made that quantitative differences between the hemi- zygote and the homozygote should be as great as those between the homozygote and hetero- zygote, although it was realized that the occurrence of some normal cells might decrease any differences.
The results from the tests on ABO, Rhesus, PI, Lutheran, Lewis, Kell, Duffy, Kidd and haptoglobin systems gave no information regarding the position of these loci. However, since the patient is doubly heterozygous for the MNSs locus, the absence of any 'mixed field ' appear- ance of the agglutination, expected if all of the cells did not contain antigens M S and Ns, suggests that the MNSs locus is not situated on the missing portion of chromosome 18.
Key to Table 2 Genotype Genotype in table System Phenotype in table
A1 0 Pl 00 Same as the
phenotypes Kell Lutheran Kidd
CDelCDe Duffy CDelcDE LeLe or Lele Haptoglobin
+ - K -Kp(a- b + ) (a-b+) Jk(a+b-) Fy(a+)
2-2 2' I
PlPl? PlP2
or PIP p2p, or P,P K-1 -3.4
LdJLub .JkaJka FyaFya or
FyaFyb Hp2Hp2 HpaHpl
DISCUSSION
Ring chromosomes, which arise through loss of both distal ends of the chromosome following breakage or faulty replication, are genetioally representative of the type of structural changes classified as deletions, though deletions are more frequently encountered in a linear form. Instances of both these morphological types of deletion have been described for different chromosomes in the human karyotype. The aberrations have predominantly occurred in diploid cells and have involved those chromosomes for which trisomy occasionally occurs, namely the autosomes 13-16, 17-18, 21-22 and the sex chromosomes X and Y . The first chromosome to be noted with a linear deletion (Jacobs, Harnden, Court Brown, Goldstein, Close, MacGregor, Maclean & Strong, 1960) was an X and so was the first chromosome to be
194 MARY K. LUCAS, N. H. KEMP, J. R. ELLIS AND RUTH MARSHALL
identified as a ring aberration (Lindsten & Tillinger, 1962). One of the large acrocentrics has similarly been observed in the two deleted forms: the linear deletion was reported by Lele, Penrose & Stallard (1963), the ring aberration being reported by Wang et al. (1962). Deletions in chromosome 17/18 have only been observed in the ring configuration (Wang et aE. 1962 and present communication), whereas the deletions in the small acrocentrics (Nowell & Hungerford, 1960) and the Y chromosome (Muldal & Ockey, 1961) were found only in the linear forms.
Autosomal deletions have frequently been used in some experimental organisms to assign genes to particular chromosomes, but no satisfactory information has been obtained from the human cases investigated. A study of the blood groups in the present case showed no evidence for hemizygosity for any of the blood group genes.
With the exception of ring X chromosomes in certain stocks of Drosophila melanogaster (Morgan, 1933), ring chromosomes have always been regarded as rather unstable aberrations undergoing either numerical and structural changes or elimination during mitotic cycles. The observations on spontaneously occurring ring chromosomes in human subjects show that such aberrations must have survived a considerable number of cell cycles. This persistence of human ring chromosomes is also reflected in the occurrence of rings, three years after irradiation, in tissues characterized by high mitotic activity (Bender & Gooch, 1962).
In maize, where ring chromosomes were extensively studied by McClintock (1938), large and small rings displayed different patterns of behaviour. Small rings tended to be eliminated more rapidly than larger ones though changes in size of the ring occurred more frequently with larger rings. The data available on spontaneous ring chromosomes in man conflict with these findings of McClintock. The relatively large ring X chromosome reported by Lindsten & Tillinger (1962) appears to have been eliminated from many adult somatic cells, although this interpretation assumes that the XO cells had originated through loss of the ring chromosome. In contrast, the three small autosomal ring chromosomes which have been observed (Wang et al. 1962, and present communication) are moderately stable structures, having persisted for a considerable number of cell cycles. It is possible that, in the tissues of the present case, the proportion of cells containing the ring may decrease as the patient ages. Variation in the size of the ring has also been noted for each of the spontaneous human ring chromosomes but the available informa- tion is insufficient for comparative studies to be made between the large and the small rings. It appears, however, that small ring chromosomes show differences in respect to size variation, even between rings of similar sizes and probably originating from the same chromosome. Thus in the case of the ring derived from chromosome 17-18, described by Wang et al. (1962), approxi- mately one-sixth of the cells had abnormally large rings, whereas in the present case very few large rings were seen. Increase in the number of rings has been noted in some of the ring karyo- types reported, but this phenomenon apparently occurs less frequently than variation in ring size. In the present instance, the low incidence of cells with either two rings or enlarged rings reflects on the stability of the ring structure.
The clinical abnormalities of the present case are less severe than those in the patient described by Wang et al. (1962) in whom all the cells contained a ring chromosome, probably also derived from no. 18. Mental subnormality is the only feature known to be common to the two cases. The clinical differences may be the result of the mosaicism in the present case; the few normal cells may have helped to minimize the somatic malformations or the gene content of the chromatin inevitably deleted in the formation of the ring may vary in the two cases.
Ring chromosome in a female infant with congenital malformations 195
SUMMARY
A mentally retarded child with congenital malformations is described in whom a high proportion of cells contained a small ring chromosome derived from a no. 18 (Denver classifica- tion). The variability in the morphological appearance of the ring chromosome is illustrated and its stability is discussed in relation to other instances of ring chromosomes in both man and other organisms.
The writers wish to thank Dr Margaret D. Baber and Dr Ursula James for their co-operation during this investigation, Mr A. J. Lee for preparing the figures and Miss Penelope M. Cam, Miss Linda Z. Gorman and Miss Barbara J. Warland for technical assistance.
Research grants from the British Empire Cancer Campaign (N.H.K.), from St George’s Hospital (M.K.L.) and from the United States Public Health Service (RG-6984) (J.R.E. and R. M.) made this study possible.
REFERENCES
BAIKIE, A. G., COURT BROWN, W. M,, JACOBS, P. A. & MILNE, J. S. (1959). Chromosome studies in human leukaemia. Lancet, 2, 425.
BENDER, M. A. & GOOCH, P. C. (1962). Persistent chromosome aberrations in irradiated human subjects. Radiation Res. 16, 44.
BUCKTON, K. E., JACOBS, P. A., COURT BROWN, W. M. & DOLL, R. (1962). A study of chromosome damage persisting after X-ray therapy for ankylostng spondylitis. Lancet, 2, 676.
HARNDEN, D. G. (1960). A human skin culture technique used for cytological examination. Brit. J . Exp. Pathol. 41, 31.
JACOBS, P. A., HARNDEN, D. G., COURT BROWN, W. M., GOLDGTEIN, J., CLOSE, M. G., MACGREGOR, T. N., MACLEAN, N. & STRONG, J. A. (1960). Abnormalities involving the X chromosome in women. Lancet, 1, 1213.
LELE, K. P., PENROSE, L. S. & STALLARD, H. B. (1963). A deletion in a case of retino-blastoma. Ann. Hum. Genet., Lond., 27, 171.
LINDSTEN, J. & TILLINOER, K. G. (1962). Self perpetuating ring chromosome in a patient with gonadal dysgenesis. Lancet, 1, 593.
LEVAN, A. (1956). Self perpetuating ring chromosomes in two human tumours. Hereditas, 42, 366. MCCLINTOCK, B. (1938). The production of homozygous deficient tissues with mulant characteristics by
MOORHEAD, P. S., NOWELL, P. C., MELLMAN, W. J., BATTIPS, D. M. & HTTNGERFORD, D. A. (1960). Chromo-
MORGAN, L. V. (1933). A closed X chromosome in Drosophila melanogaster. Genetics, 18, 250. MULDAL, S. & OCKEY, C. H. (1961). Muscular dystrophy and deletion of Y chromosome. Lancet, 2, 601. NOWELL, P.C. & HUNGERFORD, D . A . (1960). Chromosome studies on normal and loulraemic human
SMITH-WHITE, S., PEACOCK, W. J., TURNER, B. & DEN DULK, G. M. (1963). A ring chromosome in man.
TOUGH, I. M., BUCKTON, K. E., BAIKIE, A. G. & COURT BROWN, W. M. (1960). X-ray-induced chromosome
WANG, H. C., MELNYK, J., MCDONALD, L. T., UCHIDA, I,, CARR, D. H. & GOLDBERG, B. (1962). Ring chromo-
means of tho aberrant mitotic behaviour of ring shaped chromosomes. Genetics, 23, 315.
some preparations of leukocytes cultured from human peripheral blood. E q e r . Cell Res. 20, 613.
